Specifically bind the bispecific antibody of PD1 and LAG3

文档序号：1745533 发布日期：2019-11-26 浏览：22次中文

阅读说明：本技术 特异性结合pd1和lag3的双特异性抗体 (Specifically bind the bispecific antibody of PD1 and LAG3 ) 是由 L·科达里·迪克 J·费舍尔 S·伊姆霍夫·荣格 C·克莱恩 S·西伯 P·A·A·韦伯于 2018-04-03 设计创作，主要内容包括：本发明涉及双特异性抗体,所述双特异性抗体包含特异性结合PD1的第一抗原结合结构域和特异性结合LAG3的第二抗原结合结构域。本发明进一步涉及生产这些分子的方法和使用它们的方法。(The present invention relates to bispecific antibody, the bispecific antibody includes the first antigen-binding domains of specific binding PD1 and the second antigen-binding domains of specific binding LAG3.The invention further relates to the method for producing these molecules and use their method.)

1. a kind of bispecific antibody, the bispecific antibody includes specific binding apoptosis albumen 1 (PD1) The first antigen-binding domains and specific binding lymphocyte activation gene -3 (LAG3) the second antigen-binding domains, Wherein

Specific binding PD1 first antigen-binding domains include

VH structural domain, the VH structural domain include

(i) HVR-H1, it includes amino acid sequence shown in SEQ ID NO:1,

(ii) HVR-H2, it includes amino acid sequences shown in SEQ ID NO:2, and

(iii) HVR-H3, it includes amino acid sequences shown in SEQ ID NO:3；And

VL structural domain, the VL structural domain include

(i) HVR-L1, it includes amino acid sequence shown in SEQ ID NO:4,

(ii) HVR-L2, it includes amino acid sequences shown in SEQ ID NO:5, and

(iii) HVR-L3, it includes amino acid sequences shown in SEQ ID NO:6.

2. bispecific antibody according to claim 1, wherein the bispecific antibody includes Fc structural domain, the Fc Structural domain is IgG, especially IgG1 Fc structural domain or IgG4 Fc structural domain, and wherein the Fc structural domain include reduce with Fc receptor, especially one or more amino acid substitutions in conjunction with Fc γ receptor.

3. bispecific antibody according to claim 1 or 2, wherein second antigen binding of specific binding LAG3 Structural domain includes

(a) VH structural domain, the VH structural domain include

(i) HVR-H1, it includes amino acid sequence shown in SEQ ID NO:14,

(ii) HVR-H2, it includes amino acid sequences shown in SEQ ID NO:15, and

(iii) HVR-H3, it includes amino acid sequences shown in SEQ ID NO:16；And

VL structural domain, the VL structural domain include

(i) HVR-L1, it includes amino acid sequence shown in SEQ ID NO:17,

(ii) HVR-L2, it includes amino acid sequences shown in SEQ ID NO:18, and

(iii) HVR-L3, it includes amino acid sequences shown in SEQ ID NO:19；Or

(b) VH structural domain, the VH structural domain include

(i) HVR-H1, it includes amino acid sequence shown in SEQ ID NO:22,

(ii) HVR-H2, it includes amino acid sequences shown in SEQ ID NO:23, and

(iii) HVR-H3, it includes amino acid sequences shown in SEQ ID NO:24；And

VL structural domain, the VL structural domain include

(i) HVR-L1, it includes amino acid sequence shown in SEQ ID NO:25,

(ii) HVR-L2, it includes amino acid sequences shown in SEQ ID NO:26, and

(iii) HVR-L3, it includes amino acid sequences shown in SEQ ID NO:27；Or

(i) HVR-H1, it includes amino acid sequence shown in SEQ ID NO:30,

(ii) HVR-H2, it includes amino acid sequences shown in SEQ ID NO:31, and

(iii) HVR-H3, it includes amino acid sequences shown in SEQ ID NO:32；And

VL structural domain, the VL structural domain include

(i) HVR-L1, it includes amino acid sequence shown in SEQ ID NO:33,

(ii) HVR-L2, it includes amino acid sequences shown in SEQ ID NO:34, and

(iii) HVR-L3, it includes amino acid sequences shown in SEQ ID NO:35；Or

(d) VH structural domain, the VH structural domain include

(i) HVR-H1, it includes amino acid sequence shown in SEQ ID NO:38,

(ii) HVR-H2, it includes amino acid sequences shown in SEQ ID NO:39, and

(iii) HVR-H3, it includes amino acid sequences shown in SEQ ID NO:40；And

VL structural domain, the VL structural domain include

(i) HVR-L1, it includes amino acid sequence shown in SEQ ID NO:41,

(ii) HVR-L2, it includes amino acid sequences shown in SEQ ID NO:42, and

(iii) HVR-L3, it includes amino acid sequences shown in SEQ ID NO:43；Or

(e) VH structural domain, the VH structural domain include

(i) HVR-H1, it includes amino acid sequence shown in SEQ ID NO:46,

(ii) HVR-H2, it includes amino acid sequences shown in SEQ ID NO:47, and

(iii) HVR-H3, it includes amino acid sequences shown in SEQ ID NO:48；And

VL structural domain, the VL structural domain include

(i) HVR-L1, it includes amino acid sequence shown in SEQ ID NO:49,

(ii) HVR-L2, it includes amino acid sequences shown in SEQ ID NO:50, and

(iii) HVR-L3, it includes amino acid sequences shown in SEQ ID NO:51.

4. bispecific antibody according to any one of claim 1 to 3, wherein described the first of specific binding PD1 Antigen-binding domains include

(a) VH structural domain and VL structural domain, the VH structural domain include amino acid sequence shown in SEQ ID NO:7, the VL Structural domain includes amino acid sequence shown in SEQ ID NO:8, or

(b) VH structural domain and VL structural domain, the VH structural domain include amino acid sequence shown in SEQ ID NO:9, the VL Structural domain includes amino acid sequence shown in SEQ ID NO:10, or

(c) VH structural domain and VL structural domain, the VH structural domain include amino acid sequence shown in SEQ ID NO:9, the VL Structural domain includes amino acid sequence shown in SEQ ID NO:11, or

(d) VH structural domain and VL structural domain, the VH structural domain include amino acid sequence shown in SEQ ID NO:9, the VL Structural domain includes amino acid sequence shown in SEQ ID NO:12, or

(e) VH structural domain and VL structural domain, the VH structural domain include amino acid sequence shown in SEQ ID NO:9, the VL Structural domain includes amino acid sequence shown in SEQ ID NO:13.

5. bispecific antibody according to any one of claim 1 to 4, wherein described the first of specific binding PD1 Antigen-binding domains include VH structural domain and VL structural domain, and the VH structural domain includes amino acid shown in SEQ ID NO:9 Sequence, the VL structural domain include amino acid sequence shown in SEQ ID NO:10.

6. bispecific antibody according to any one of claim 1 to 5, wherein described the second of specific binding LAG3 Antigen-binding domains include

(a) VH structural domain and VL structural domain, the VH structural domain include amino acid sequence shown in SEQ ID NO:20, the VL Structural domain includes amino acid sequence shown in SEQ ID NO:21, or

(b) VH structural domain and VL structural domain, the VH structural domain include amino acid sequence shown in SEQ ID NO:28, the VL Structural domain includes amino acid sequence shown in SEQ ID NO:29, or

(c) VH structural domain and VL structural domain, the VH structural domain include amino acid sequence shown in SEQ ID NO:36, the VL Structural domain includes amino acid sequence shown in SEQ ID NO:37, or

(d) VH structural domain and VL structural domain, the VH structural domain include amino acid sequence shown in SEQ ID NO:44, the VL Structural domain includes amino acid sequence shown in SEQ ID NO:45, or

(e) VH structural domain and VL structural domain, the VH structural domain include amino acid sequence shown in SEQ ID NO:52, the VL Structural domain includes amino acid sequence shown in SEQ ID NO:53.

7. bispecific antibody according to any one of claim 1 to 5, wherein described the second of specific binding LAG3 Antigen-binding domains include

(a) VH structural domain and VL structural domain, the VH structural domain include amino acid sequence shown in SEQ ID NO:54, the VL Structural domain includes amino acid sequence shown in SEQ ID NO:55, or

(b) VH structural domain and VL structural domain, the VH structural domain include amino acid sequence shown in SEQ ID NO:62, the VL Structural domain includes amino acid sequence shown in SEQ ID NO:63, or

(c) VH structural domain and VL structural domain, the VH structural domain include amino acid sequence shown in SEQ ID NO:64, the VL Structural domain includes amino acid sequence shown in SEQ ID NO:65, or

(d) VH structural domain and VL structural domain, the VH structural domain include amino acid sequence shown in SEQ ID NO:66, the VL Structural domain includes amino acid sequence shown in SEQ ID NO:67.

8. bispecific antibody according to any one of claim 1 to 6, wherein

First antigen-binding domains for specifically binding PD1 include VH structural domain and VL structural domain, the VH structural domain Comprising amino acid sequence shown in SEQ ID NO:9, the VL structural domain includes amino acid sequence shown in SEQ ID NO:10,

And second antigen-binding domains for specifically binding LAG3 include VH structural domain and VL structural domain, the VH knot Structure domain includes amino acid sequence shown in SEQ ID NO:20 and the VL structural domain includes amino shown in SEQ ID NO:21 Acid sequence or the VH structural domain include amino acid sequence shown in SEQ ID NO:52 and the VL structural domain includes SEQ Amino acid sequence shown in ID NO:53.

9. bispecific antibody described in any one of according to claim 1 to 6 or 8, wherein

10. bispecific antibody described in any one of according to claim 1 to 5 or 7, wherein

11. bispecific antibody according to any one of claim 1 to 10, wherein the bispecific antibody includes people The Fc structural domain of IgG1 subclass, the Fc structural domain have amino acid mutation L234A, L235A and P329G (according to Kabat EU Index number).

12. bispecific antibody according to any one of claim 1 to 11, wherein the bispecific antibody includes Fc Structural domain, the Fc structural domain include the modification for promoting the first and second subunit associations of the Fc structural domain.

13. bispecific antibody according to any one of claim 1 to 13, wherein described the first of the Fc structural domain Subunit includes amino acid substitution S354C and T366W (according to Kabat EU index number), and described the of the Fc structural domain Two subunits include amino acid substitution Y349C, T366S and Y407V (according to Kabat EU index number).

14. bispecific antibody according to any one of claim 1 to 13, wherein the bispecific antibody includes Fc Structural domain, the first Fab segment and the 2nd Fab segment, the first Fab segment include the antigen knot of specific binding PD1 Structural domain is closed, the 2nd Fab segment includes the antigen-binding domains of specific binding LAG3.

15. according to claim 1 to bispecific antibody described in any one of 14, wherein one in the Fab segment In, the variable domains VL and VH is substituted for one another, so that the VH structural domain is a part of light chain and the VL structure Domain is a part of heavy chain.

16. bispecific antibody according to claim 14 or 15, wherein in the antigen comprising specifically binding PD1 In the first Fab segment of binding structural domain, the variable domains VL and VH is substituted for one another.

17. according to claim 1 to bispecific antibody described in any one of 16, wherein the bispecific antibody includes Fab segment, wherein the amino acid in constant domain CL at 124 is only by lysine (K), arginine (R) or histidine (H) On the spot replace (according to Kabat EU index number), and the amino acid quilt in constant domain CH1 at 147 and 213 Glutamic acid (E) or aspartic acid (D) independently replace (according to Kabat EU index number).

18. bispecific antibody described in any one of 4 to 17 according to claim 1, wherein comprising specifically binding LAG3 The antigen-binding domains the 2nd Fab segment in, the amino acid quilt in the constant domain CL at 124 Lysine (K), arginine (R) or histidine (H) independently replace (according to Kabat EU index number), and described constant Amino acid in domain C H1 at 147 and 213 is independently replaced (according to Kabat by glutamic acid (E) or aspartic acid (D) EU index number).

19. according to claim 1 to bispecific antibody described in any one of 18, the bispecific antibody includes

(a) the first heavy chain, the first light chain, the second heavy chain and the second light chain, first heavy chain include and SEQ ID NO:96 institute The sequence shown have at least 95% sequence identity amino acid sequence, first light chain include with shown in SEQ ID NO:98 Sequence have at least 95% sequence identity amino acid sequence, second heavy chain include with shown in SEQ ID NO:97 Sequence has the amino acid sequence of at least 95% sequence identity, and second light chain includes and sequence shown in SEQ ID NO:99 The amino acid sequence at least 95% sequence identity is arranged, or

(b) the first heavy chain, the first light chain, the second heavy chain and the second light chain, first heavy chain include and SEQ ID NO:96 institute The sequence shown have at least 95% sequence identity amino acid sequence, first light chain include with shown in SEQ ID NO:98 Sequence have at least 95% sequence identity amino acid sequence, second heavy chain include with shown in SEQ ID NO:100 Sequence have at least 95% sequence identity amino acid sequence, second light chain include with shown in SEQ ID NO:101 Sequence have at least 95% sequence identity amino acid sequence, or

(c) the first heavy chain, the first light chain, the second heavy chain and the second light chain, first heavy chain include and SEQ ID NO:102 institute The sequence shown has the amino acid sequence of at least 95% sequence identity, and first light chain includes and SEQ ID NO:104 institute The sequence shown has the amino acid sequence of at least 95% sequence identity, and second heavy chain includes and SEQ ID NO:103 institute The sequence shown has the amino acid sequence of at least 95% sequence identity, and second light chain includes and SEQ ID NO:105 institute The sequence shown has the amino acid sequence of at least 95% sequence identity, or

(d) the first heavy chain, the first light chain, the second heavy chain and the second light chain, first heavy chain include and SEQ ID NO:106 institute The sequence shown has the amino acid sequence of at least 95% sequence identity, and first light chain includes and SEQ ID NO:107 institute The sequence shown has the amino acid sequence of at least 95% sequence identity, and second heavy chain includes and SEQ ID NO:103 institute The sequence shown has the amino acid sequence of at least 95% sequence identity, and second light chain includes and SEQ ID NO:105 institute The sequence shown has the amino acid sequence of at least 95% sequence identity.

20. the bispecific antibody includes the first weight according to claim 1 to bispecific antibody described in any one of 19 Chain, the first light chain, the second heavy chain and the second light chain, first heavy chain include to have extremely with sequence shown in SEQ ID NO:96 The amino acid sequence of few 95% sequence identity, first light chain include to have at least with sequence shown in SEQ ID NO:98 The amino acid sequence of 95% sequence identity, second heavy chain include to have at least with sequence shown in SEQ ID NO:100 The amino acid sequence of 95% sequence identity, second light chain include to have at least with sequence shown in SEQ ID NO:101 The amino acid sequence of 95% sequence identity.

21. according to claim 1 to bispecific antibody described in any one of 18, wherein the bispecific antibody includes the Two Fab segments, the 2nd Fab segment include the antigen-binding domains of specific binding LAG3, the 2nd Fab piece Section is merged with the C-terminal of the Fc structural domain.

22. bispecific antibody described in any one of according to claim 1 to 18 or 21, the bispecific antibody includes the One heavy chain, the first light chain, the second heavy chain and the second light chain, first heavy chain include to have with sequence shown in SEQ ID NO:96 There is the amino acid sequence of at least 95% sequence identity, first light chain includes to have with sequence shown in SEQ ID NO:98 At least amino acid sequence of 95% sequence identity, second heavy chain include to have with sequence shown in SEQ ID NO:144 At least amino acid sequence of 95% sequence identity, second light chain include to have with sequence shown in SEQ ID NO:101 At least amino acid sequence of 95% sequence identity.

23. according to claim 1 to bispecific antibody described in any one of 18, wherein the bispecific antibody includes the Three Fab segments, the 3rd Fab segment include the antigen-binding domains of specific binding LAG3.

24. bispecific antibody described in any one of according to claim 1 to 18 or 22 or 23, wherein including specific binding The Fab segment of the antigen-binding domains of PD1 is merged via peptide linker with one C-terminal in the heavy chain.

25. according to claim 1 to bispecific antibody described in any one of 18 or 22 to 24, the bispecific antibody packet Contain

(a) the first heavy chain, the first light chain, the second heavy chain and two the second light chains, first heavy chain include and SEQ ID NO: Sequence shown in 118 has the amino acid sequence of at least 95% sequence identity, and first light chain includes and SEQ ID NO: Sequence shown in 115 has the amino acid sequence of at least 95% sequence identity, and second heavy chain includes and SEQ ID NO: Sequence shown in 119 has the amino acid sequence of at least 95% sequence identity, and two second light chains include and SEQ ID Sequence shown in NO:101 has the amino acid sequence of at least 95% sequence identity, or

(b) the first heavy chain, the first light chain, the second heavy chain and two the second light chains, first heavy chain include and SEQ ID NO: Sequence shown in 120 has the amino acid sequence of at least 95% sequence identity, and first light chain includes and SEQ ID NO: Sequence shown in 115 has the amino acid sequence of at least 95% sequence identity, and second heavy chain includes and SEQ ID NO: Sequence shown in 121 has the amino acid sequence of at least 95% sequence identity, and two second light chains contain and SEQ ID Sequence shown in NO:99 has the amino acid sequence of at least 95% sequence identity, or

(c) the first heavy chain, the first light chain, the second heavy chain and two the second light chains, first heavy chain include and SEQ ID NO: Sequence shown in 122 has the amino acid sequence of at least 95% sequence identity, and first light chain includes and SEQ ID NO: Sequence shown in 115 has the amino acid sequence of at least 95% sequence identity, and second heavy chain includes and SEQ ID NO: Sequence shown in 103 has the amino acid sequence of at least 95% sequence identity, and two second light chains include and SEQ ID Sequence shown in NO:105 has the amino acid sequence of at least 95% sequence identity.

26. bispecific antibody described in any one of according to claim 1 to 18 or 22 or 23, wherein including specific binding One in the Fab segment of the antigen-binding domains of LAG3 via one C in peptide linker and the heavy chain Terminal fusion.

27. bispecific antibody described in any one of according to claim 1 to 18 or 22 or 23 or 26, the bispecific is anti- Body includes (a) first heavy chain, the first light chain, the second heavy chain and two the second light chains, and first heavy chain includes and SEQ ID Sequence shown in NO:96 has the amino acid sequence of at least 95% sequence identity, and first light chain includes and SEQ ID Sequence shown in NO:98 has the amino acid sequence of at least 95% sequence identity, and second heavy chain includes and SEQ ID Sequence shown in NO:145 has the amino acid sequence of at least 95% sequence identity, and two second light chains include and SEQ Sequence shown in ID NO:101 has the amino acid sequence of at least 95% sequence identity.

28. according to claim 1 to the bispecific antibody described in any one of 11,14 to 18 or 22 to 24, wherein described double Specific antibody includes the 4th Fab segment, and the 4th Fab segment includes the antigen-binding domains of specific binding PD1.

29. according to claim 1 to the bispecific antibody described in any one of 11,14 to 18 or 22 to 24 or 28, wherein Described two Fab segments comprising specifically binding the antigen-binding domains of PD1 are identical.

30. according to claim 1 to the bispecific antibody described in any one of 11,14 to 18 or 22 to 24 or 28 or 29, In comprising specific binding PD1 antigen-binding domains described two Fab segments respectively via peptide linker and institute State one C-terminal fusion in heavy chain.

31. according to claim 1 to the bispecific antibody described in any one of 11,14 to 18 or 22 to 24 or 28 to 30, institute Stating bispecific antibody includes

(a) two heavy chains, two the first light chains and two the second light chains, two heavy chains include and SEQ ID NO:114 Shown in sequence there is the amino acid sequence of at least 95% sequence identity, two first light chains include and SEQ ID Sequence shown in NO:115 have at least 95% sequence identity amino acid sequence, two second light chains include with Sequence shown in SEQ ID NO:101 has the amino acid sequence of at least 95% sequence identity, or

(b) two heavy chains, two the first light chains and two the second light chains, two heavy chains include and SEQ ID NO:116 Shown in sequence there is the amino acid sequence of at least 95% sequence identity, two first light chains include and SEQ ID Sequence shown in NO:115 have at least 95% sequence identity amino acid sequence, two second light chains include with Sequence shown in SEQ ID NO:99 has the amino acid sequence of at least 95% sequence identity, or

(c) two heavy chains, two the first light chains and two the second light chains, two heavy chains include and SEQ ID NO:117 Shown in sequence there is the amino acid sequence of at least 95% sequence identity, two first light chains include and SEQ ID Sequence shown in NO:115 has the amino acid sequence of at least 95% sequence identity, and two second light chains include and SEQ Sequence shown in ID NO:105 has the amino acid sequence of at least 95% sequence identity.

32. bispecific antibody according to any one of claim 1 to 13, wherein the bispecific antibody includes Fc Structural domain, two Fab segments and single chain Fab (scFab), described two Fab segments include the antigen for specifically binding LAG3 Binding structural domain, the single chain Fab (scFab) include the antigen-binding domains of specific binding PD1.

33. bispecific antibody described in any one of according to claim 1 to 13 or 32, wherein including specific binding PD1 The scFab of antigen-binding domains merged via peptide linker with one C-terminal in the heavy chain.

34. bispecific antibody described in any one of according to claim 1 to 13 or 32 or 33, the bispecific antibody packet Contain

(a) the first heavy chain, the second heavy chain and two light chains, first heavy chain include and sequence shown in SEQ ID NO:123 Amino acid sequence at least 95% sequence identity, second heavy chain include and sequence shown in SEQ ID NO:119 Amino acid sequence at least 95% sequence identity, the two light chains include and sequence shown in SEQ ID NO:101 The amino acid sequence at least 95% sequence identity is arranged, or

(b) the first heavy chain, the second heavy chain and two light chains, first heavy chain include and sequence shown in SEQ ID NO:124 Amino acid sequence at least 95% sequence identity, second heavy chain include and sequence shown in SEQ ID NO:121 Amino acid sequence at least 95% sequence identity, the two light chains include and sequence shown in SEQ ID NO:99 Amino acid sequence at least 95% sequence identity, or

(c) the first heavy chain, the second heavy chain and the second light chain, first heavy chain include and sequence shown in SEQ ID NO:125 Amino acid sequence at least 95% sequence identity, second heavy chain include and sequence shown in SEQ ID NO:103 Amino acid sequence at least 95% sequence identity, second light chain include and sequence shown in SEQ ID NO:105 Amino acid sequence at least 95% sequence identity.

35. bispecific antibody according to any one of claim 1 to 13, wherein the bispecific antibody includes Fc Structural domain, two Fab segments and VH and VL structural domain, described two Fab segments include the antigen knot for specifically binding LAG3 Structural domain is closed, VH the and VL structural domain includes the antigen-binding domains of specific binding PD1.

36. bispecific antibody described in any one of according to claim 1 to 13 or 35, wherein the institute of specific binding PD1 The VH structural domain for stating antigen-binding domains is merged via peptide linker with one C-terminal in the heavy chain, and special The VL structural domain of the antigen-binding domains of opposite sex combination PD1 is via another 's in peptide linker and the heavy chain C-terminal fusion.

37. bispecific antibody described in any one of according to claim 1 to 13 or 35 or 36, the bispecific antibody packet Containing the first heavy chain, the second heavy chain and two light chains, first heavy chain includes to have extremely with sequence shown in SEQ ID NO:126 The amino acid sequence of few 95% sequence identity, second heavy chain include to have extremely with sequence shown in SEQ ID NO:127 The amino acid sequence of few 95% sequence identity, the two light chains include to have with sequence shown in SEQ ID NO:109 At least amino acid sequence of 95% sequence identity.

38. a kind of polynucleotides, the polynucleotide encoding is anti-to bispecific described in any one of 37 according to claim 1 Body.

39. a kind of protokaryon or eukaryotic host cell, the protokaryon or eukaryotic host cell include according to claim 38 Polynucleotides.

40. a kind of generation is according to claim 1 to the method for bispecific antibody described in 37, the method includes being suitable for table Host cell according to claim 42 is cultivated under conditions of up to the bispecific antibody, and from the culture Recycle the bispecific antibody.

41. a kind of pharmaceutical composition, described pharmaceutical composition includes according to claim 1 to double special described in any one of 37 Property antibody and at least one pharmaceutically acceptable excipient.

42. according to claim 1 to bispecific antibody described in any one of 37 or drug according to claim 41 Composition, the bispecific antibody or described pharmaceutical composition are used as drug.

43. according to claim 1 to bispecific antibody described in any one of 37 or drug according to claim 41 Composition, the bispecific antibody or described pharmaceutical composition are used for

I) immune response is adjusted, such as recovery T cell activity,

Ii t cell response) is stimulated,

Iii) treatment infection,

Iv) treating cancer,

V) delay cancer development,

Vi) extend the survival period of cancer patient.

44. according to claim 1 to bispecific antibody described in any one of 37 or drug according to claim 41 Composition, the bispecific antibody or described pharmaceutical composition are used for treating cancer.

45. according to claim 1 to bispecific antibody described in any one of 37 or drug according to claim 41 Composition, the bispecific antibody or described pharmaceutical composition are for treating chronic viral infection.

46. according to claim 1 to bispecific antibody described in any one of 37 or drug according to claim 41 Composition, the bispecific antibody or described pharmaceutical composition are for prevention or treating cancer, wherein the bispecific is anti- Body and chemotherapeutant, radiation and/or other reagents for immunotherapy for cancer are administered in combination.

47. according to claim 1 to bispecific antibody described in any one of 37 or drug according to claim 41 Composition, the bispecific antibody or described pharmaceutical composition are for prevention or treating cancer, wherein the bispecific is anti- Body and anti-CEA/ AntiCD3 McAb bispecific antibody are administered in combination.

48. according to claim 1 to bispecific antibody described in any one of 37 or drug according to claim 41 Composition is in preparation for the purposes in the drug for the treatment of cancer or infectious diseases.

49. a kind of method of individual for the treatment of with cancer or chronic viral infection, the method includes applying to the individual It is a effective amount of according to claim 1 to bispecific antibody described in any one of 37 or drug according to claim 41 Composition.

50. a kind of method for inhibiting growth of tumour cell in individual, the method includes to described individual application a effective amount of Inhibit the growth of the tumour cell according to bispecific antibody described in any one of claims 1 to 37.

Invention field

The present invention relates to bispecific antibodies, it includes the first antigen-binding domains of specific binding PD1 and specifically Property combination LAG3 the second antigen-binding domains, particularly further include the bispecific antibody of Fc structural domain, should Fc structural domain includes one or more amino acid substitutions, which reduces and the combination of Fc receptor, especially It is and the combination of Fc γ receptor.The invention further relates to the method for generating these molecules and use their method.

Background technique

Importance of the immune system in terms of pre- anti-cancer is the ability based on its detection and destruction abnormal cell.However, Some tumour cells can escape immune system (Zitvogel et al., Nature by causing immunosuppressive condition Reviews Immunology 6(2006),715–727).T cell has important work in antiviral and anti-tumor immune response With.The appropriate activation of T cells with antigenic specificity leads to its clonal expansion and obtains effector function, and drenches in cytotoxic T In the case where bar cell (CTL), this enables CTL specifically to crack target cell.T cell is always to manipulate endogenous on treating The principal focal point of anti-tumor immunity, this is because the protein derived peptide in all cellular compartments of the Selective recognition of T cell Ability；Direct Recognition and the ability of killing antigen-expressing cells (pass through CD8⁺Effector T cell；Also referred to as cytotoxic T drenches Bar cell (CTL)) and coordinate the abilities of various immune responses and (pass through CD4⁺T helper cell), this incorporates adaptability and congenital Property effect mechanism.T cell dysfunction occurs due to lasting antigen exposure: T cell loses to be carried out in the presence of antigen The ability of proliferation, and cannot gradually generate cell factor and cracking target cell.The T cell of dysfunction is referred to as the T exhausted Cell, cannot be proliferated and play effector function and (such as cytotoxicity and carry out cell factor point in response to antigenic stimulus It secretes).Further study show that the feature of the T cell of exhaustion is holding for inhibition molecule PD-1 (apoptosis albumen 1) Continued reaches, and blocking PD-1 and PD-L1 (PD-1 ligand) to interact can reverse T cell to exhaust and in the mouse of infection LCMV Middle recovery antigen-specific T cell response (Barber et al., Nature 439 (2006), 682-687).However, only targeting PD- 1-PD-L1 approach is not always reverse (Gehring et al., the Gastroenterology 137 for causing T cell to be exhausted (2009), 682-690), this shows that other molecules may participate in T cell and exhaust (Sakuishi, J.Experimental Med.207(2010),2187-2194)。

Lymphocyte activation gene -3 (LAG3 or CD223) is initially being designed for Selective Separation in IL-2 dependence NK (Triebel F et al., Cancer Lett.235 (2006), 147-153) is found in the experiment for the molecule expressed in cell line. LAG3 is a kind of unique transmembrane protein, and there are four extracellular immunoglobulin superfamily spline structure domains (D1-D4) with tool CD4 has structural homology.Film far end I gG structural domain contains short amino acid sequence, i.e., does not send out in other IgG superfamily proteins Existing so-called extra loop.Intracellular domain contains unique amino acid sequence (KIEELE, SEQ ID NO:75), the amino acid Sequence is necessary to LAG3 has a negative impact to T cell function.LAG3 can be by metalloproteinases at link peptide (CP) Cracking, to generate the soluble form that can be detected in serum.As CD4, LAG3 albumen in conjunction with MHC class Ⅱmolecule, but It is that there is higher affinity and (Huard et al., the Proc.Natl.Acad.Sci.USA 94 at the site different from CD4 (1997),5744-5749).LAG3 is expressed by T cell, B cell, NK cell and plasmacytoid dendritic cells (pDC), and in T It is raised after cell activation.Its regulatory T-cell function and T cell stable state.Conventional T cells Immune anergy or that function is impaired Expression of Subsets LAG3.LAG3⁺T cell is enriched with (Sierro et al., Expert during tumor locus and chronic viral infection Opin.Ther.Targets 15(2011),91-101).Existing research shows that LAG3 works in CD8 T cell exhaustion (Blackburn et al., Nature Immunol.10 (2009), 29-37).It is therefore desirable to be able to antagonism LAG3 activity and can For generating and restoring the antibody to the immune response of tumour.

The monoclonal antibody for LAG3 is for example described in WO 2004/078928, wherein claimed include Specifically bind the antibody of CD223 and the composition of anti-cancer vaccine.WO 2010/019570 discloses the human antibody in conjunction with LAG3, Such as antibody 25F7 and 26H10.US 2011/070238 is related to can be used to treat or prevent organ-graft refection and autoimmunity The anti-LAG3 antibody of the cytotoxicity of disease.WO 2014/008218 describes the functional characteristic compared with antibody 25F7 with optimization The LAG3 antibody of (i.e. the deacylation amine site of reduction).In addition, LAG3 antibody be also disclosed in WO 2015/138920 (such as BAP050), WO 2014/140180, WO 2015/116539, WO 2016/028672, WO 2016/126858, WO 2016/ In 200782 and WO 2017/015560.

Apoptosis albumen 1 (PD-1 or CD279) is the inhibition member of CD28 receptor family, further includes CD28, CTLA-4, ICOS and BTLA.PD-1 is cell surface receptor and in the B cell, T cell and bone marrow cell of activation Express (Okazaki et al. (2002) Curr.Opin.Immunol.14:391779-82；Bennett et al. (2003) J Immunol 170:711-8).The structure of PD-1 is 1 type transmembrane protein of monomer, by an immunoglobulin variable like cell outside Structural domain and cytoplasmic domain composition, the cytoplasmic domain contain immunity receptor tyrosine-based and inhibit motif (ITIM) and exempt from Epidemic disease receptor tyrosine base converts motif (ITSM).The T cell transient expression PD1 of activation, but the lasting mistake of PD1 and its ligand PDL1 Expression promotes immunodepletion, increases so as to cause persistent virus infection, tumor escape, infection and the death rate.By by T cell Receptor carries out antigen recognizing to induce PD1 to express, and its expression is mainly maintained via continuous T cell receptor signal transduction. After the exposure of lasting antigen, PD1 locus can not be methylated again, and this facilitate lasting overexpressions.Block PD1 approach can be with Restore T cell function (Sheridan, the Nature Biotechnology 30 exhausted in cancer and chronic viral infection (2012),729-730).For example, in WO 2003/042402, WO 2004/004771, WO 2004/056875, WO 2004/ 072286、WO 2004/087196、WO 2006/121168、WO 2006/133396、WO 2007/005874、WO 2008/ 083174、WO 2008/156712、WO 2009/024531、WO 2009/014708、WO 2009/101611、WO 2009/ 114335、WO 2009/154335、WO 2010/027828、WO 2010/027423、WO 2010/029434、WO 2010/ 029435、WO 2010/036959、WO 2010/063011、WO 2010/089411、WO 2011/066342、WO 2011/ 110604, WO 2011/110621, WO 2012/145493, WO 2013/014668, WO 2014/179664 and WO 2015/ The monoclonal antibody for PD-1 is described in 112900.

It is described in WO 2015/200119 with the immunoreactive bispecific Fc double antibody with PD1 and LAG3, It is used for treating cancer or disease relevant to pathogen (such as bacterium, fungi or virus).However, it is necessary to provide new double spies Thus heterogenetic antibody not only in combination with PD1 and LAG3, and is selectively targeting the cell for expressing both PD1 and LAG3, and And the blocking to the LAG3 on other cells is avoided in view of the wide expression of LAG3 spectrum.Bispecific antibody of the invention is not only The PD1 and LAG3 being overexpressed in the T cell of both PD1 and LAG3 are effectively blocked, and they have very greatly these cells Selectivity, therefore can avoid passing through application high activity LAG3 antibody generate side effect.

Summary of the invention

The present invention relates to bispecific antibody, it includes specific bindings apoptosis albumen 1 (PD1) at least At least one second antigen binding of one antigen-binding domains and specific binding lymphocyte activation gene -3 (LAG3) Structural domain.These bispecific antibodies are advantageous, because they are provided than anti-PD1 and anti-LAG3 combined strategy better choice Property and potential effect.They are further characterised as showing the sink effect (as shown in the T cell internalization as reduction) of reduction, They preferentially in conjunction with conventional T cells and can save T cell effector function object from Treg inhibition relative to Treg, it Show enhancing tumor specific T cells effector function and enhancing in-vivo tumour eradicate.

In one aspect, the present invention provides a kind of bispecific antibodies, and it includes specific binding programmed cell is dead It dies the first antigen-binding domains of albumen 1 (PD1) and the second of specific binding lymphocyte activation gene -3 (LAG3) resists Former binding structural domain, wherein

Specific binding PD1 first antigen-binding domains include

VH structural domain, it includes

(i) HVR-H1, it includes amino acid sequence shown in SEQ ID NO:1,

(ii) HVR-H2, it includes amino acid sequences shown in SEQ ID NO:2, and

(iii) HVR-H3, it includes amino acid sequences shown in SEQ ID NO:3；And

VL structural domain, it includes

(i) HVR-L1, it includes amino acid sequence shown in SEQ ID NO:4,

(ii) HVR-L2, it includes amino acid sequences shown in SEQ ID NO:5, and

(iii) HVR-L3, it includes amino acid sequences shown in SEQ ID NO:6.

Specifically, a kind of bispecific antibody is provided, it includes specific binding apoptosis albumen 1 (PD1) the second antigen binding knot of the first antigen-binding domains and specific binding lymphocyte activation gene -3 (LAG3) Structure domain, wherein the bispecific antibody includes Fc structural domain, the Fc structural domain is IgG, in particular IgG1 Fc structural domain Or IgG4 Fc structural domain, and wherein the Fc structural domain includes one or more amino acid substitutions, one or more of ammonia Base acid, which replaces, to be reduced and the combination of Fc receptor, especially and the combination of Fc γ receptor.

(a) VH structural domain, it includes

(i) HVR-H1, it includes amino acid sequence shown in SEQ ID NO:14,

(ii) HVR-H2, it includes amino acid sequences shown in SEQ ID NO:15, and

(iii) HVR-H3, it includes amino acid sequences shown in SEQ ID NO:16；And

VL structural domain, it includes

(i) HVR-L1, it includes amino acid sequence shown in SEQ ID NO:17,

(ii) HVR-L2, it includes amino acid sequences shown in SEQ ID NO:18, and

(iii) HVR-L3, it includes amino acid sequences shown in SEQ ID NO:19；Or

(b) VH structural domain, it includes

(i) HVR-H1, it includes amino acid sequence shown in SEQ ID NO:22,

(ii) HVR-H2, it includes amino acid sequences shown in SEQ ID NO:23, and

(iii) HVR-H3, it includes amino acid sequences shown in SEQ ID NO:24；And

VL structural domain, it includes

(i) HVR-L1, it includes amino acid sequence shown in SEQ ID NO:25,

(ii) HVR-L2, it includes amino acid sequences shown in SEQ ID NO:26, and

(iii) HVR-L3, it includes amino acid sequences shown in SEQ ID NO:27；Or

(i) HVR-H1, it includes amino acid sequence shown in SEQ ID NO:30,

(ii) HVR-H2, it includes amino acid sequences shown in SEQ ID NO:31, and

(iii) HVR-H3, it includes amino acid sequences shown in SEQ ID NO:32；And

VL structural domain, it includes

(i) HVR-L1, it includes amino acid sequence shown in SEQ ID NO:33,

(ii) HVR-L2, it includes amino acid sequences shown in SEQ ID NO:34, and

(iii) HVR-L3, it includes amino acid sequences shown in SEQ ID NO:35；Or

(d) VH structural domain, it includes

(i) HVR-H1, it includes amino acid sequence shown in SEQ ID NO:38,

(ii) HVR-H2, it includes amino acid sequences shown in SEQ ID NO:39, and

(iii) HVR-H3, it includes amino acid sequences shown in SEQ ID NO:40；And

VL structural domain, it includes

(i) HVR-L1, it includes amino acid sequence shown in SEQ ID NO:41,

(ii) HVR-L2, it includes amino acid sequences shown in SEQ ID NO:42, and

(iii) HVR-L3, it includes amino acid sequences shown in SEQ ID NO:43；Or

(e) VH structural domain, it includes

(i) HVR-H1, it includes amino acid sequence shown in SEQ ID NO:46,

(ii) HVR-H2, it includes amino acid sequences shown in SEQ ID NO:47, and

(iii) HVR-H3, it includes amino acid sequences shown in SEQ ID NO:48；And

VL structural domain, it includes

(i) HVR-L1, it includes amino acid sequence shown in SEQ ID NO:49,

(ii) HVR-L2, it includes amino acid sequences shown in SEQ ID NO:50, and

(iii) HVR-L3, it includes amino acid sequences shown in SEQ ID NO:51.

(a) VH structural domain and VL structural domain, the VH structural domain include amino acid sequence shown in SEQ ID NO:7, institute Stating VL structural domain includes amino acid sequence shown in SEQ ID NO:8, or

(b) VH structural domain and VL structural domain, the VH structural domain include amino acid sequence shown in SEQ ID NO:9, institute Stating VL structural domain includes amino acid sequence shown in SEQ ID NO:10, or

(c) VH structural domain and VL structural domain, the VH structural domain include amino acid sequence shown in SEQ ID NO:9, institute Stating VL structural domain includes amino acid sequence shown in SEQ ID NO:11, or

(d) VH structural domain and VL structural domain, the VH structural domain include amino acid sequence shown in SEQ ID NO:9, institute Stating VL structural domain includes amino acid sequence shown in SEQ ID NO:12, or

(e) VH structural domain and VL structural domain, the VH structural domain include amino acid sequence shown in SEQ ID NO:9, institute Stating VL structural domain includes amino acid sequence shown in SEQ ID NO:13.

In a specific aspect, a kind of bispecific antibody is provided, it includes the first antigen knots of specific binding PD1 It closes structural domain and specifically binds the second antigen-binding domains of LAG3, wherein the first antigen of the specific binding PD1 Binding structural domain includes VH structural domain and VL structural domain, and the VH structural domain includes amino acid sequence shown in SEQ ID NO:9, The VL structural domain includes amino acid sequence shown in SEQ ID NO:10.

On the other hand, a kind of bispecific antibody is provided, it includes the of specific binding PD1 as previously described Second antigen-binding domains of one antigen-binding domains and specific binding LAG3, wherein the specific binding LAG3 Second antigen-binding domains include

(a) VH structural domain and VL structural domain, the VH structural domain include amino acid sequence shown in SEQ ID NO:20, institute Stating VL structural domain includes amino acid sequence shown in SEQ ID NO:21, or

(b) VH structural domain and VL structural domain, the VH structural domain include amino acid sequence shown in SEQ ID NO:28, institute Stating VL structural domain includes amino acid sequence shown in SEQ ID NO:29, or

(c) VH structural domain and VL structural domain, the VH structural domain include amino acid sequence shown in SEQ ID NO:36, institute Stating VL structural domain includes amino acid sequence shown in SEQ ID NO:37, or

(d) VH structural domain and VL structural domain, the VH structural domain include amino acid sequence shown in SEQ ID NO:44, institute Stating VL structural domain includes amino acid sequence shown in SEQ ID NO:45, or

(e) VH structural domain and VL structural domain, the VH structural domain include amino acid sequence shown in SEQ ID NO:52, institute Stating VL structural domain includes amino acid sequence shown in SEQ ID NO:53.

(a) VH structural domain and VL structural domain, the VH structural domain include amino acid sequence shown in SEQ ID NO:54, institute Stating VL structural domain includes amino acid sequence shown in SEQ ID NO:55, or

(b) VH structural domain and VL structural domain, the VH structural domain include amino acid sequence shown in SEQ ID NO:62, institute Stating VL structural domain includes amino acid sequence shown in SEQ ID NO:63, or

(c) VH structural domain and VL structural domain, the VH structural domain include amino acid sequence shown in SEQ ID NO:64, institute Stating VL structural domain includes amino acid sequence shown in SEQ ID NO:65, or

(d) VH structural domain and VL structural domain, the VH structural domain include amino acid sequence shown in SEQ ID NO:66, institute Stating VL structural domain includes amino acid sequence shown in SEQ ID NO:67.

The first antigen-binding domains of the specific binding PD1 include VH structural domain and VL structural domain, the VH knot Structure domain includes amino acid sequence shown in SEQ ID NO:9, and the VL structural domain includes amino acid shown in SEQ ID NO:10 Sequence,

And the second antigen-binding domains of the specific binding LAG3 include VH structural domain and VL structural domain, described VH structural domain includes amino acid sequence shown in SEQ ID NO:20 and the VL structural domain includes shown in SEQ ID NO:21 Amino acid sequence or the VH structural domain include amino acid sequence shown in SEQ ID NO:52 and the VL structural domain includes Amino acid sequence shown in SEQ ID NO:53.

On the other hand, a kind of bispecific antibody is provided, it includes the of specific binding PD1 as previously described Second antigen-binding domains of one antigen-binding domains and specific binding LAG3, wherein the bispecific antibody is people Source antibody or chimeric antibody.Specifically, the bispecific antibody is humanized antibody.

In a specific aspect, a kind of bispecific antibody is provided, it includes specific binding PD1 as previously described The first antigen-binding domains and specific binding LAG3 the second antigen-binding domains, wherein the bispecific antibody Fc structural domain comprising human IgG1's subclass, the Fc structural domain have amino acid mutation L234A, L235A and P329G (according to Kabat EU index number).

Further it is provided that a kind of bispecific antibody, it includes first antigens of specific binding PD1 as previously described Second antigen-binding domains of binding structural domain and specific binding LAG3, wherein the bispecific antibody includes Fc structure Domain, the Fc structural domain include the modification for promoting its first subunit and the second subunit association.In one aspect, it provides a kind of double special Heterogenetic antibody, wherein the first subunit of the Fc structural domain includes protrusion, and the second subunit of the Fc structural domain includes basis Protrusion enters the hole of hole method (knobs into holes method).Specifically, the first subunit of the Fc structural domain includes ammonia Base acid replace S354C and T366W (EU number), and the second subunit of the Fc structural domain comprising amino acid substitution Y349C, T366S and Y407V (according to Kabat EU index number).

On the other hand, a kind of bispecific antibody is provided, it includes the first antigen binding knots of specific binding PD1 Second antigen-binding domains in structure domain and specific binding LAG3, wherein the bispecific antibody includes Fc structural domain, the One Fab segment and the 2nd Fab segment, the first Fab segment includes the antigen-binding domains of specific binding PD1, described 2nd Fab segment includes the antigen-binding domains of specific binding LAG3.

In one aspect, a kind of bispecific antibody is provided, it includes the first antigen binding knots of specific binding PD1 Second antigen-binding domains in structure domain and specific binding LAG3, wherein in one in the Fab segment, varistructure VL and VH are substituted for one another in domain, so that the VH structural domain is a part of light chain and the VL structural domain is a part of heavy chain. Specifically, a kind of bispecific antibody is provided, wherein first of the antigen-binding domains comprising specifically binding PD1 In Fab segment, variable domains VL and VH is substituted for one another.

On the other hand, a kind of bispecific antibody is provided, it includes the first antigen bindings of specific binding PD1 Second antigen-binding domains of structural domain and specific binding LAG3, wherein the bispecific antibody includes Fab segment, In in constant domain CL, the amino acid at 124 is by lysine (K), arginine (R) or histidine (H) (according to Kabat EU index number) independently replace, and in constant domain CH1, amino acid at 147 and 213 by glutamic acid (E) or Aspartic acid (D) (according to Kabat EU index number) independently replaces.Specifically, bispecific antibody is provided, wherein In the 2nd Fab segment comprising the antigen-binding domains for specifically binding LAG3, in constant domain CL, Amino acid at 124 is independently taken by lysine (K), arginine (R) or histidine (H) (according to Kabat EU index number) Generation, and in constant domain CH1, amino acid at 147 and 213 by glutamic acid (E) or aspartic acid (D) (according to Kabat EU index number) independently replace.

(a) the first heavy chain, the first light chain, the second heavy chain and the second light chain, first heavy chain include and SEQ ID NO: Sequence shown in 96 has the amino acid sequence of at least 95% sequence identity, and first light chain includes and SEQ ID NO:98 Shown in sequence have at least 95% sequence identity amino acid sequence, second heavy chain include and SEQ ID NO:97 institute The sequence shown have at least 95% sequence identity amino acid sequence, second light chain include with shown in SEQ ID NO:99 Sequence have at least 95% sequence identity amino acid sequence, or

Second heavy chain includes the amino for having at least 95% sequence identity with sequence shown in SEQ ID NO:100 Acid sequence, second light chain include the amino for having at least 95% sequence identity with sequence shown in SEQ ID NO:101 Acid sequence, or

(c) the first heavy chain, the first light chain, the second heavy chain and the second light chain, first heavy chain include and SEQ ID NO: Sequence shown in 102 has the amino acid sequence of at least 95% sequence identity, and first light chain includes and SEQ ID NO: Sequence shown in 104 has the amino acid sequence of at least 95% sequence identity, and second heavy chain includes and SEQ ID NO: Sequence shown in 103 has the amino acid sequence of at least 95% sequence identity, and second light chain includes and SEQ ID NO: Sequence shown in 105 has the amino acid sequence of at least 95% sequence identity, or

(d) the first heavy chain, the first light chain, the second heavy chain and the second light chain, first heavy chain include and SEQ ID NO: Sequence shown in 106 has the amino acid sequence of at least 95% sequence identity, and first light chain includes and SEQ ID NO: Sequence shown in 107 has the amino acid sequence of at least 95% sequence identity, and second heavy chain includes and SEQ ID NO: Sequence shown in 103 has the amino acid sequence of at least 95% sequence identity, and second light chain includes and SEQ ID NO: Sequence shown in 105 has the amino acid sequence of at least 95% sequence identity.

More specifically, providing a kind of bispecific antibody, it includes the first antigen binding structures of specific binding PD1 Domain and specific binding LAG3 the second antigen-binding domains, the bispecific antibody include the first heavy chain, the first light chain, Second heavy chain and the second light chain, first heavy chain include to have at least 95% sequence one with sequence shown in SEQ ID NO:96 The amino acid sequence of cause property, first light chain include to have at least 95% sequence consistent with sequence shown in SEQ ID NO:98 Property amino acid sequence, second heavy chain include with sequence shown in SEQ ID NO:100 have at least 95% sequence it is consistent Property amino acid sequence, second light chain include with sequence shown in SEQ ID NO:101 have at least 95% sequence it is consistent The amino acid sequence of property.

On the other hand, a kind of bispecific antibody is provided, it includes specific binding PD1's as previously described Second antigen-binding domains of the first antigen-binding domains and specific binding LAG3, wherein the bispecific antibody packet Segment containing Fab, the Fab segment include the antigen-binding domains of specific binding LAG3, the Fab segment and Fc structural domain C-terminal fusion.

Specifically, a kind of bispecific antibody is provided, it includes the first antigen-binding domains of specific binding PD1 With the second antigen-binding domains of specific binding LAG3, the bispecific antibody includes the first heavy chain, the first light chain, the Two heavy chains and the second light chain, first heavy chain include to have at least 95% sequence consistent with sequence shown in SEQ ID NO:96 Property amino acid sequence, first light chain include with sequence shown in SEQ ID NO:98 have at least 95% sequence identity Amino acid sequence, second heavy chain include with sequence shown in SEQ ID NO:144 have at least 95% sequence identity Amino acid sequence, second light chain include with sequence shown in SEQ ID NO:101 have at least 95% sequence identity Amino acid sequence.

On the other hand, a kind of bispecific antibody is provided, it includes specific binding PD1's as previously described Second antigen-binding domains of the first antigen-binding domains and specific binding LAG3, wherein the bispecific antibody packet Containing the 3rd Fab segment, the 3rd Fab segment includes the antigen-binding domains of specific binding LAG3.In one aspect, it mentions A kind of bispecific antibody is supplied, wherein two Fab segments of the antigen-binding domains comprising specific binding LAG3 are It is identical.

On the other hand, a kind of bispecific antibody is provided, it includes specific binding PD1's as previously described Second antigen-binding domains of the first antigen-binding domains and specific binding LAG3, wherein including specific binding PD1 The Fab segments of antigen-binding domains merged via peptide linker with one C-terminal in the heavy chain.

(a) the first heavy chain, the first light chain, the second heavy chain and two the second light chains,

First heavy chain includes the amino for having at least 95% sequence identity with sequence shown in SEQ ID NO:118 Acid sequence, first light chain include the amino for having at least 95% sequence identity with sequence shown in SEQ ID NO:115 Acid sequence,

Second heavy chain includes the amino for having at least 95% sequence identity with sequence shown in SEQ ID NO:119 Acid sequence, two second light chains include to have at least 95% sequence identity with sequence shown in SEQ ID NO:101 Amino acid sequence, or

(b) the first heavy chain, the first light chain, the second heavy chain and two the second light chains,

First heavy chain includes the amino for having at least 95% sequence identity with sequence shown in SEQ ID NO:120 Acid sequence, first light chain include the amino for having at least 95% sequence identity with sequence shown in SEQ ID NO:115 Acid sequence,

Second heavy chain includes the amino for having at least 95% sequence identity with sequence shown in SEQ ID NO:121 Acid sequence, two second light chains contain the ammonia for having at least 95% sequence identity with sequence shown in SEQ ID NO:99 Base acid sequence, or

First heavy chain includes the amino for having at least 95% sequence identity with sequence shown in SEQ ID NO:122 Acid sequence, first light chain include the amino for having at least 95% sequence identity with sequence shown in SEQ ID NO:115 Acid sequence,

Second heavy chain includes the amino for having at least 95% sequence identity with sequence shown in SEQ ID NO:103 Acid sequence, two second light chains include to have at least 95% sequence identity with sequence shown in SEQ ID NO:105 Amino acid sequence.

On the other hand, a kind of bispecific antibody is provided, it includes specific binding PD1's as previously described Second antigen-binding domains of the first antigen-binding domains and specific binding LAG3, wherein including specific binding LAG3 Antigen-binding domains the Fab segment in one merged via peptide linker with one C-terminal in the heavy chain.

On the other hand, a kind of bispecific antibody is provided, it includes specific binding PD1's as previously described Second antigen-binding domains of the first antigen-binding domains and specific binding LAG3, wherein the bispecific antibody packet Containing the 4th Fab segment, the 4th Fab segment includes the antigen-binding domains of specific binding PD1.In one aspect, it mentions A kind of bispecific antibody is supplied, wherein two Fab segments of the antigen-binding domains comprising specific binding PD1 are phases With.

On the other hand, a kind of bispecific antibody is provided, it includes specific binding PD1's as previously described Second antigen-binding domains of the first antigen-binding domains and specific binding LAG3, wherein comprising specific binding Two Fab segments of the antigen-binding domains of PD1 are respectively melted via peptide linker and one C-terminal in the heavy chain It closes.

(a) two heavy chains, two the first light chains and two the second light chains, two heavy chains include and SEQ ID NO: Sequence shown in 114 has the amino acid sequence of at least 95% sequence identity, and two first light chains include and SEQ Sequence shown in ID NO:115 has the amino acid sequence of at least 95% sequence identity, and two second light chains include There is the amino acid sequence of at least 95% sequence identity with sequence shown in SEQ ID NO:101, or

(b) two heavy chains, two the first light chains and two the second light chains, two heavy chains include and SEQ ID NO: Sequence shown in 116 has the amino acid sequence of at least 95% sequence identity, and two first light chains include and SEQ Sequence shown in ID NO:115 has the amino acid sequence of at least 95% sequence identity, and two second light chains include There is the amino acid sequence of at least 95% sequence identity with sequence shown in SEQ ID NO:99, or

(c) two heavy chains, two the first light chains and two the second light chains, two heavy chains include and SEQ ID NO: Sequence shown in 117 has the amino acid sequence of at least 95% sequence identity, and two first light chains include and SEQ Sequence shown in ID NO:115 have at least 95% sequence identity amino acid sequence, two second light chains include with Sequence shown in SEQ ID NO:105 has the amino acid sequence of at least 95% sequence identity.

(a) the first heavy chain, the second heavy chain and two light chains, first heavy chain include with shown in SEQ ID NO:123 Sequence have at least 95% sequence identity amino acid sequence, second heavy chain include with shown in SEQ ID NO:119 Sequence have at least 95% sequence identity amino acid sequence, the two light chains include with shown in SEQ ID NO:101 Sequence have at least 95% sequence identity amino acid sequence, or

(b) the first heavy chain, the second heavy chain and two light chains, first heavy chain include with shown in SEQ ID NO:124 Sequence have at least 95% sequence identity amino acid sequence, second heavy chain include with shown in SEQ ID NO:121 Sequence have at least 95% sequence identity amino acid sequence, the two light chains include with shown in SEQ ID NO:99 Sequence has the amino acid sequence of at least 95% sequence identity, or

(c) the first heavy chain, the second heavy chain and the second light chain, first heavy chain include with shown in SEQ ID NO:125 Sequence have at least 95% sequence identity amino acid sequence, second heavy chain include with shown in SEQ ID NO:103 Sequence have at least 95% sequence identity amino acid sequence, second light chain include with shown in SEQ ID NO:105 Sequence has the amino acid sequence of at least 95% sequence identity.

On the other hand, a kind of bispecific antibody is provided, it includes the first antigen bindings of specific binding PD1 Structural domain and specific binding LAG3 the second antigen-binding domains, wherein the bispecific antibody include Fc structural domain, Two Fab segments and VH and VL structural domain, described two Fab segments include the antigen binding structure for specifically binding LAG3 Domain, VH the and VL structural domain include the antigen-binding domains of specific binding PD1.In one aspect, PD1 is specifically bound The VH structural domains of the antigen-binding domains merged via peptide linker with one C-terminal in the heavy chain, and it is special Property combination PD1 the antigen-binding domains VL structural domain via another C-terminal in peptide linker and the heavy chain Fusion.In a particular aspects, a kind of bispecific antibody is provided, it includes the first heavy chain, the second heavy chain and two light chains, First heavy chain includes the amino acid sequence for having at least 95% sequence identity with sequence shown in SEQ ID NO:126, Second heavy chain includes the amino acid sequence for having at least 95% sequence identity with sequence shown in SEQ ID NO:127, The two light chains include the amino acid sequence for having at least 95% sequence identity with sequence shown in SEQ ID NO:109 Column.

According to another aspect of the present invention, a kind of multicore glycosides for encoding bispecific antibody as previously described is provided Acid.The present invention also provides a kind of carrier, especially expression vector, the carrier includes polynucleotides of the invention；And it provides A kind of protokaryon or eukaryotic host cell, the protokaryon or eukaryotic host cell include polynucleotides or carrier of the invention.One In a little embodiments, host cell is eukaryocyte, especially mammalian cell.

On the other hand, a kind of the first antigen knot generated comprising specific binding PD1 as described herein is provided The method closed structural domain and specifically bind the bispecific antibody of the second antigen-binding domains of LAG3, the method includes Following steps: host cell, b a) are converted with the carrier of the polynucleotides comprising encoding the bispecific antibody) it is being suitable for table The host cell is cultivated under conditions of up to the bispecific antibody and c) recycles the bispecific from culture resists Body.Present invention also contemplates that the bispecific antibody generated by means of the present invention.

The present invention also provides a kind of pharmaceutical compositions, and it includes bispecific antibody and at least one are pharmaceutically acceptable Excipient, the bispecific antibody include the first antigen-binding domains and the spy for specifically binding PD1 as described herein The opposite sex combines the second antigen-binding domains of LAG3.

Present invention also contemplates that bispecific antibody, it includes the first antigen knots of specific binding PD1 as described herein It closes structural domain and specifically binds the second antigen-binding domains of LAG3；Or cover special comprising described pair as drug The pharmaceutical composition of property antibody.

On the other hand, the present invention provides a kind of bispecific antibody, and it includes as described herein comprising specificity Bispecific in conjunction with the second antigen-binding domains of the first antigen-binding domains and specific binding LAG3 of PD1 is anti- Body；Or the pharmaceutical composition comprising the bispecific antibody is provided, it is used for

I) immune response is adjusted, such as recovery T cell activity,

Ii immune response or function) are stimulated,

Iii) treatment infection,

Iv) treating cancer,

V) delay cancer development,

Vi) extend the survival period of cancer patient.

In one aspect, bispecific antibody is provided, first it includes specific binding PD1 as described herein is anti- Second antigen-binding domains of former binding structural domain and specific binding LAG3；Or it provides comprising the bispecific antibody Pharmaceutical composition, be used to treat the disease of individual in need.In a particular aspects, the present invention provides a kind of double special Property antibody, it includes specific binding PD1 the first antigen-binding domains and specific binding LAG3 the second antigen binding Structural domain；Or provide the pharmaceutical composition comprising the bispecific antibody for being used for treating cancer.In another particular aspects, A kind of bispecific antibody is provided, it includes the first antigen-binding domains of specific binding PD1 and specific bindings The second antigen-binding domains of LAG3；Or provide the drug comprising the bispecific antibody for adjusting immune response Composition.On the other hand, a kind of bispecific antibody is provided, it includes the first antigen bindings of specific binding PD1 Second antigen-binding domains of structural domain and specific binding LAG3；Or it provides and includes for treat chronic viral infection The pharmaceutical composition of the bispecific antibody.

The present invention also provides a kind of bispecific antibody, first it includes specific binding PD1 as described herein is anti- Second antigen-binding domains of former binding structural domain and specific binding LAG3；Or offer is used to prevent or the packet for the treatment of cancer Pharmaceutical composition containing the bispecific antibody, wherein the bispecific antibody and chemotherapeutant, radiating and/or being used for Other drug combinations of immunotherapy for cancer are applied.In a particular aspects, a kind of bispecific antibody is provided, it includes such as The first antigen-binding domains of specific binding PD1 as described herein and the second antigen binding structure of specific binding LAG3 Domain；Or provide for preventing or the pharmaceutical composition comprising the bispecific antibody for the treatment of cancer, wherein described double special Heterogenetic antibody and anti-CEA/ AntiCD3 McAb bispecific antibody are administered in combination.

It additionally provides comprising the first antigen-binding domains of specific binding PD1 and specific binding as described herein The purposes of the bispecific antibody of the second antigen-binding domains of LAG3 is used to prepare the disease for treating individual in need The drug of disease, is especially used to prepare the drug for treating cancer；And a kind of method of disease for treating individual is provided, It includes to the composition of the individual application therapeutically effective amount, and the composition includes the double special of pharmaceutically acceptable form Property antibody, the bispecific antibody include as described herein specific binding PD1 the first antigen-binding domains and spy The opposite sex combines the second antigen-binding domains of LAG3.In a particular aspects, the disease is cancer.In another certain party Face, the disease are chronic viral infections.On the other hand, a kind of method for adjusting immune response in individual is provided, Including the composition to the individual application therapeutically effective amount, the composition includes the bispecific of pharmaceutically acceptable form Antibody, the bispecific antibody include the first antigen-binding domains and specific binding LAG3 of specific binding PD1 Second antigen-binding domains.Any aspect in above-mentioned aspect, the individual are preferably mammal, especially people.

Further it is provided that a kind of method for inhibiting growth of tumour cell in individual comprising effective to the individual application For the bispecific antibody of amount to inhibit the growth of tumour cell, the bispecific antibody includes specificity knot as described herein Close the first antigen-binding domains of PD1 and the second antigen-binding domains of specific binding LAG3.The individual is preferably Mammal, especially people.

Brief Description Of Drawings

Fig. 1: the schematic diagram of various forms of anti-LAG3 antibody of the anti-PD1/ of bispecific described herein.Figure 1A shows double Specific 1+1 form, wherein the PD1 binding structural domain includes crossFab (having VH/VL Domain swapping), and LAG3 is tied Closing structural domain includes to have CH1 structural domain and the CK structural domain of amino acid mutation to support correctly to match (" electrification variant ").Fc Part enters hole mutation (being shown by black arrow) and amino acid mutation L234A, L235A and P329G, the amino comprising protrusion The Fc γ receptor that acid mutation L234A, L235A and P329G almost eliminate human IgG1's Fc structural domain combines (such as by white area Shown in domain).Figure 1B shows 2+1 form, and the anti-LAG3 combination Fab structural domain of two of them includes the mutation in CH1/CK, and PD1 combination Fab structural domain merges at the C-terminal of a heavy chain.Fig. 1 C shows similar 2+1 form, the anti-LAG3 of two of them In conjunction with the mutation that Fab structural domain includes in CH1/CK, and PD1 combines single-stranded scFab structural domain to melt at the C-terminal of a heavy chain It closes.2+1 form is shown in Fig. 1 D, the anti-LAG3 combination Fab structural domain of two of them and PD1 combination VH and VL are respectively fused to One in the C-terminal of the heavy chain.Fig. 1 E shows the construct similar to above-mentioned Fig. 1 D, however has between VH and VL Disulfide bond through being engineered, and the variant with furin site is shown in figure 1f.Fig. 1 G shows double special Property 2+2 form, the anti-LAG3 combination Fab structural domain of two of them includes the mutation in CH1/CK, and two PD1 are combined CrossFab structural domain merges at the C-terminal of each heavy chain.It is (referred to as " anti-that bispecific 1+1 form is shown in Fig. 1 H Formula "), wherein PD1 binding structural domain includes crossFab (have VH/VL Domain swapping), and LAG3 binding structural domain and its VH structural domain merges at the C-terminal of Fc pore chain.Lag3 structural domain includes CH1 the and CK structural domain with amino acid mutation to prop up Hold correctly pairing (" electrification variant ").Fig. 1 I shows 2+1 trans forms, wherein the PD1 binding structural domain includes CrossFab (has VH/VL Domain swapping), and a LAG3 binding structural domain includes CH1 and CK with amino acid mutation Structural domain with support correctly to match (" electrification variant ") and the 2nd LAG3 binding structural domain and its VH structural domain Fc pore chain C End fusion.

The cytotoxicity for people's CD4 T cell that Fig. 2: aLAG-3 antibody pair is co-cultured with allogeneic mature dendritic cell The influence of granzyme B release and IL-2 secretion.Show what aLAG3 antibody as described herein secreted granzyme B in fig. 2 It influences, and shows the influence that aLAG3 antibody secretes IL-2 in fig. 2b.

The combination of Fig. 3: aLAG3 antibody and aPD1 antibody (0376) is to total with B cell-class into lymphoid cell lines (ARH77) The influence of the cytotoxicity granzyme B release of people's CD4 T cell of culture.Show different aLAG3 antibody and aPD1 antibody (0376) comparison of combination and individual aPD1 antibody (0376).

The combination of Fig. 4: aLAG3 antibody and aPD1 antibody (0376) is to the people with irradiated allogeneic PBMC co-cultivation Granzyme B and the IFN- release of CD4 T cell carry out the influence of Treg inhibition.Fig. 4 A is shown and individual aPD1 (0376) phase The granzyme B of ratio discharges, and Fig. 4 B shows the release of the IFN- compared with individual aPD1 (0376).

Fig. 5: by the anti-LAG3 antibody of the anti-PD1/ of bispecific and recombination PD1⁺Lag3⁺Caused by the combination of cell in combination with And Receptor dimerization.What is drawn is curve graph of the chemiluminescence (being measured as unit of RU) relative to antibody concentration.Fig. 5 A and figure 5B shows the anti-LAG3 antibody of the anti-PD1/ of bispecific compared with the anti-LAG3 antibody of monospecific.Only bispecific form It can induce chemiluminescence.Competitive assay is shown in figure 5 c.If in aLAG3 antibody (0156, MDX25F7) or anti-PD1 antibody (0376) identical bispecific antibody is provided in the presence of, then signal is almost suppressed (since PD1 is competed) or at least significant It reduces (Lag3).Further competitive assay is shown in figure 5d.The anti-LAG3 antibody of the anti-PD1/ of bispecific and identical anti-PD1 Antibody (0376) and the competition for recombinating LAG3:Fc albumen (0160) almost eliminate signal, and identical aLAG3 bonding agent (0156) presence only results in part inhibition, and two kinds of other not significant adjustment signals of anti-LAG3 antibody 0414 and 0416.

The different form (1+1 compare 2+1) of Fig. 6: the anti-PD1/ of bispecific anti-LAG3 antibody and from different aLAG3 conjugates While the comparison that combines.Fig. 6 A shows the knot of construct 0799 (the anti-PD1 (0376) of 1+1 form/anti-LAG3 (0416)) Close curve.The binding curve of construct 8311 (the anti-PD1 (0376) of 1+2 form/anti-LAG3 (0416)) is illustrated in Fig. 6 B.Figure 6C shows the binding curve of construct 0927 (the anti-PD1 (0376) of 1+1 form/anti-LAG3 (0414)).8310 (1+ of construct The anti-PD1 (0376) of 2 forms/anti-LAG3 (0414)) binding curve be illustrated in Fig. 6 D.

The different form (2+1 compare 2+2) of Fig. 7: the anti-PD1/ of bispecific anti-LAG3 antibody and from different aLAG3 conjugates While the comparison that combines.Fig. 7 A shows the knot of construct 8310 (the anti-PD1 (0376) of 1+2 form/anti-LAG3 (0414)) Close curve.The binding curve of construct 8970 (the anti-PD1 (0376) of 2+2 form/anti-LAG3 (0414)) is illustrated in Fig. 7 B.Figure 7C shows the binding curve of construct 8311 (the anti-PD1 (0376) of 1+2 form/anti-LAG3 (0416)).8984 (2+ of construct The anti-PD1 (0376) of 2 forms/anti-LAG3 (0416)) binding curve be illustrated in Fig. 7 D.(the trans- 1+1 form of construct 0725 Anti- PD1 (0376)/anti-LAG3 (0414)) and (anti-PD1 (the 0376)/anti-LAG3 of trans- 1+2 form of construct 0750 (0414)) binding curve of binding curve and construct 0927 (the anti-PD1 (0376) of 1+1 form/anti-LAG3 (0414)) Compare and is illustrated in Fig. 7 E.This 3 kinds of constructs also are compared in commercially available PD1/LAG3 combo Reporter measurement, and Corresponding binding curve is illustrated in Fig. 7 F.

Fig. 8: as using measured by flow cytometry, after adding T cell 3 hours to be applied to activation, not similar shape The internalization of the anti-LAG3 antibody of the anti-PD1/ of the bispecific of formula and the anti-LAG3 antibody of parent.Fig. 8 A shows the representative histogram of experiment Figure, various forms of internalization percentages are illustrated in Fig. 8 B.

Fig. 9: as time goes by analysis shows that, when compared with showing the other forms of internalization of higher degree, 1 The film positioning of the anti-LAG3 antibody (0927) of the anti-PD1/ of the bispecific of+1 form is higher.Fig. 9 A is shown 15 minutes, 1 hour and 3 After hour, the fluorescent image that is detected by confocal microscope.The cd4 cell of activation is shown as black ball.The fluorescence of TIM3 antibody Image is shown as the example being internalized by by force.The quantitative analysis of image is illustrated in Fig. 9 B.

Figure 10: and the combination of traditional T cell relative to and Treg combination.Figure 10 A to Figure 10 C was shown from a generation The data of table donor, it is shown that and the combination of traditional T cell (black curve) and Treg (gray area).Anti- LAG3 antibody 0414 (hu IgG1 PGLALA's) is shown in conjunction in Figure 10 A, Figure 10 B and Figure 10 C, respectively illustrates anti-PD1 antibody 0376 and double The combination of the anti-anti- LAG3 antibody (0927) of PD1/ of specificity.Given molecule is shown in figure 10d is incorporated in phase in traditional T cell For combining the δ of the geometry fluorescence mean intensity on Treg in same sample.As a result (median), which comes from, has 3 differences 3 independent experiments of donor.

Figure 11: PD1 and Treg is blocked altogether has saved Tconv effector function from Treg inhibition.Show co-cultivation 5 days Afterwards, percentage of the Treg to the inhibition of the Tconv granzyme B secreted.As a result (median) is from 10 different donors 10 independent experiments.P is calculated using two-way analysis of variance.

Figure 12: after arousing memory with immunogenicity melanoma-Antigenic Peptide pond, PD-1 and LAG-3 are blocked to from melanocyte The influence of granzyme B and the IFN- secretion of the CD4 T cell of tumor patient PBMC.Figure 12 is shown to by individual anti-PD1 (0376), the combination of anti-PD1 (0376) and aLAG3 (0414) and bispecific antibody 0927 (the anti-PD1 (0376) of 1+1 form/ Anti- LAG3 (0414)) caused by comparison to granzyme B and the IFN- influence discharged.It shows relative to from 12 kinds of melanoma What the CD4 T cell of patient PBMC peptide pond stimulation, granzyme B and IFN were generated is multiplied.

The people that Figure 13: aPD1/aLAG3 bispecific antibody pair is co-cultured with B cell-class into lymphoid cell lines (ARH77) The influence of the cytotoxicity granzyme B release of CD4 T cell.The anti-LAG3 of the difference anti-PD1/ of bispecific as described herein is resisted Body is compared with the antibody used in standard care or clinical test.

Figure 14: the effect of humanization mouse attacked with cancer of pancreas BxPC3, is studied.It is combined with CEACAM CD3 TCB When, only aPD1/aLAG3 bispecific antibody provides tumor protection statistically significant compared with traditional PD1 antibody.It shows It is with BxPC3 cell subcutaneous challenge and raw with the tumour in the humanization mouse of specified molecule and the combined therapy of CEACAM5-TCB Long curve.

Figure 15: tumor volume measurement value (mm of each individual animals within 47 days periods is shown³+/- SEM), display The homogeneity of the antitumor reaction of group.The tumor growth curve of vehicle group is shown in Figure 15 A, shows list in Figure 15 B The tumor growth curve of only CEACAM5 CD3 TCB (2.5mg/kg), show in figure 15 c CEACAM5 CD3 TCB with Receive Wu Dankang (Nivolumab) combination (1.5mg/kg) tumor growth curve, show CEACAM5 CD3 in Figure 15 D The tumor growth curve of the combination (1.5mg/kg) of TCB and pyridine aldoxime methyliodide (PAM) monoclonal antibody (Pembrolizumab), shows in Figure 15 E The tumor growth curve of the combination (1.5mg/kg) of CEACAM5 CD3 TCB and PD1/LAG3 0927, and show in Figure 15 F The tumor growth curve of (3mg/kg bispecific antibody) out.

Specific embodiment

Definition

Unless otherwise defined, all technical terms used herein and scientific and technical terminology all have as belonging to the present invention Usually used identical meanings in field.For the purpose for explaining this specification, will using defined below, and in due course, Term used in the singular also will include plural number, and vice versa.

As used herein, term " antigen binding molecules " refers to molecule of the antigen binding determinant in its largest sense Molecule.The example of antigen binding molecules is antibody, antibody fragment and bracket antigen-binding proteins.

The term " antibody " of this paper in the broadest sense using and cover various antibody structures, including but not limited to singly Clonal antibody, polyclonal antibody, monospecific and multi-specificity antibody (for example, bispecific antibody) and antibody fragment, only Them are wanted to show required antigen-binding activity.

Term " monospecific " antibody as used herein indicates such antibody, has one or more bound sites Point, each binding site are bound to the same epitope of same antigen.Term " bispecific " means that antibody can be specifically bound At least two different antigenic determinants, such as each freely a pair of of heavy chain of antibody variable domains (VH) and the variable knot of antibody light chain The two basic change site that structure domain (VL) is formed is in conjunction with the different epitopes on not synantigen or same antigen.Such bispecific is anti- Body is 1+1 form.Other bispecific antibody forms be 2+1 form (the two basic change site comprising the first antigen or epitope and One binding site of the second antigen or epitope) or 2+2 form (the two basic change site and second comprising the first antigen or epitope The two basic change site of antigen or epitope).In general, bispecific antibody includes two antigen binding sites, each antigen binding Site-specific is in different antigenic determinants.

As the term used in current application " valence " indicates that antigen binding molecules have the binding structural domain specified number. Therefore, term " divalent " " tetravalence " and " sexavalence " respectively indicate in antigen binding molecules there are two basic change structural domain, four knot Close structural domain and six binding structural domains.Bispecific antibody according to the present invention is at least " divalent ", and to can be " three Valence " or " multivalence " (such as " tetravalence " or " sexavalence ").In a particular aspects, antibody tool of the invention there are two or More binding sites and be bispecific.That is, even if there are more than two binding site, (i.e. antibody is three It is valence or multivalence) in the case where, antibody is also possible to bispecific.

Term " full length antibody " and " complete antibody " refer to being used interchangeably herein to be had and native antibody structure The antibody of essentially similar structure." natural antibody " refers to the naturally occurring immunoglobulin molecules with different structure. For example, natural IgG class antibody is about 150, the heterotetrameric glycoproteins of 000 dalton, by disulfide bonding two light chains and Two heavy chain compositions.From N-terminal to C-terminal, each heavy chain has (the also referred to as variable heavy chain domain or heavy chain variable region (VH) Variable domains), it is three constant domains (CH1, CH2 and CH3) (also referred to as heavy chain constant region) later.Similarly, from the end N C-terminal is held, every light chain has variable region (VL) (also referred to as variable light chain domain or light variable domains), Zhi Houshi Light chain constant domain (CL) (also referred to as constant region of light chain).The heavy chain of antibody can be assigned as one of five seed types, institute Stating five seed types and being referred to as α (IgA), δ (IgD), ε (IgE), γ (IgG) or μ (IgM), some of which further to divide For hypotype, such as γ 1 (IgG1), γ 2 (IgG2), γ 3 (IgG3), γ 4 (IgG4), α 1 (IgA1) and α 2 (IgA2).Antibody Amino acid sequence of the light chain based on its constant domain, can belong to one of two types, and described two types are known as Kappa (κ) and lambda (λ).

" antibody fragment " refers to the molecule other than complete antibody, and it includes a part of complete antibody, the part is combined The antigen that complete antibody is combined.The example of antibody fragment includes but is not limited to Fv, Fab, Fab ', Fab '-SH, F (ab ')₂；It is double Body antibody, four body antibody, intersects Fab segment at three body antibody；Linear antibodies；Single-chain antibody molecules (such as scFv)；By antibody piece The multi-specificity antibody that section and single domain antibody are formed.About the summary of certain antibody fragments, referring to Hudson et al., Nat Med 9,129-134(2003).About the summary of scFv segment, see, for example, Pl ü ckthun in The harmacology of Monoclonal Antibodies,vol.113,Rosenburg and Moore eds.,Springer-Verlag,New Described in York, pp.269-315 (1994)；Referring also to WO 93/16185；And United States Patent (USP) No.5,571,894 and No.5,587,458.For comprising saving receptor binding domain residue and with the Fab segment and F of extended Half-life in vivo (ab’)₂The discussion of segment, referring to United States Patent (USP) No.5,869,046.Diabody is that there are two antigen-binding domains for tool Antibody fragment, the Diabody can be it is divalent or bispecific, see, for example, EP 404,097；WO 1993/ 01161；Hudson et al., Nat Med 9,129-134 (2003)；And Hollinger et al., Proc Natl Acad Sci USA 90,6444-6448(1993).Three body antibody is also illustrated in (2003) in Hudson et al., Nat Med 9,129-134 And four body antibody.Single domain antibody is that all or part of heavy-chain variable domains comprising antibody or all or part of light chain can The antibody fragment in structure changes domain.In certain embodiments, single domain antibody be people's single domain antibody (Domantis, Inc., Waltham, MA；See, for example, United States Patent (USP) No.6,248,516B1).In addition, antibody fragment includes single chain polypeptide, it is described single-stranded Polypeptide has the feature of VH structural domain, and functional antigen binding site can be assembled to together with VL structural domain；Or there is VL The feature of structural domain can be assembled to functional antigen binding site, to provide full length antibody together with VH structural domain Antigenic binding property.Antibody fragment can be prepared by various technologies, including but not limited to the proteolytic digestion of complete antibody And generated by recombinant host cell (such as Escherichia coli or bacteriophage), as described herein.

Papain digestion complete antibody generates two identical antigen-binding fragments for being known as " Fab " segment, each " Fab " segment contains the constant domain of heavy-chain variable domains and light variable domains and light chain and the first perseverance of heavy chain Constant domain (CH1).Therefore, as used herein, term " Fab segment " refers to such antibody fragment, and it includes tie containing VL The VH structural domain and the first constant domain (CH1) of the light chain segments and heavy chain of structure domain and light chain constant domain (CL). Fab ' segment with Fab segment the difference is that Fab ' segment be added in the carboxyl terminal of heavy chain CH1 structural domain it is some residual Base, these residues include one or more cysteines from antibody hinge region.Fab '-SH is Fab ' segment, wherein constant The cysteine residues of structural domain have free sulfhydryl groups.Pepsin generates F (ab')₂Segment, there are two anti-for segment tool A part of former binding site (two Fab segments) and the area Fc.

Term " intersecting Fab segment " or " xFab segment " or " crossover Fab segment " refer to such Fab segment, wherein The variable region or constant region of heavy chain and light chain are exchanged.The different chain compositions of two kinds of crossover Fab molecule are possible, and are wrapped Be contained in bispecific antibody of the invention: in one aspect, the variable region of Fab heavy chain and light chain is exchanged, i.e. crossover Fab Molecule includes the peptide chain being made of light chain variable region (VL) and heavy chain constant region (CH1), and by heavy chain variable region (VH) and gently The peptide chain of chain constant region (CL) composition.Crossover Fab molecule is also referred to as CrossFab_(VLVH).On the other hand, when Fab weight When the constant region of chain and light chain is exchanged, crossover Fab molecule includes by heavy chain variable region (VH) and constant region of light chain (CL) group At peptide chain, and the peptide chain being made of light chain variable region (VL) and heavy chain constant region (CH1).Crossover Fab molecule is also referred to as For CrossFab_(CLCH1)。

" single chain Fab segment " or " scFab " are by heavy chain of antibody variable domains (VH), antibody constant domain 1 (CH1), the polypeptide of antibody light chain variable domains (VL), antibody light chain constant domain (CL) and connector composition, wherein described Antibody domain and the connector have one of following sequence: a) VH-CH1- connector-on N-terminal to C-terminal direction VL-CL, b) VL-CL- connector-VH-CH1, c) VH-CL- connector-VL-CH1 or d) VL-CH1- connector-VH-CL；And wherein The connector is at least 30 amino acid, the preferably polypeptide between 32 and 50 amino acid.The single chain Fab segment via Natural disulphide bonds between CL structural domain and CH1 structural domain and stabilize.In addition, these single chain Fab molecules can by via Cysteine residues (such as 44 in the variable heavy chain numbered according to Kabat and 100 in variable light) are inserted into generate Interchain disulfide bond, and further stabilize.

" crossover single chain Fab segment " or " x-scFab " are by heavy chain of antibody variable domains (VH), antibody constant structure Domain 1 (CH1), antibody light chain variable domains (VL), antibody light chain constant domain (CL) and connector composition polypeptide, wherein institute State antibody domain and the connector has one of following sequence: a) VH-CL- connector-on N-terminal to C-terminal direction VL-CH1 and b) VL-CH1- connector-VH-CL；Wherein VH and VL is formed together the antigen binding structure in conjunction with antigentic specificity Domain, and wherein the connector is the polypeptide of at least 30 amino acid.In addition, these x-scFab molecules can be by via slotting Enter cysteine residues (such as 44 in the variable heavy chain numbered according to Kabat and 100 in variable light) and generates chain Between disulfide bond, and further stabilize.

" single chain variable fragment (scFv) " is the heavy chain variable region (V of antibody_H) and light chain variable region (V_L) fusion protein, It is connect with the short circuit head peptide of 10 to about 25 amino acid.Connector is generally rich in glycine to obtain flexibility, and is rich in silk ammonia Acid or threonine, and can be by V to obtain solubility_HN-terminal and V_LC-terminal connection, or vice versa.Although removal Constant region simultaneously introduces connector, but the albumen remains the specificity of original antibodies.ScFv antibody is for example described in Houston, J.S., Methods in Enzymol.203 (1991) 46-96) in.In addition, antibody fragment includes single chain polypeptide, The single chain polypeptide has the feature of VH structural domain, and functional antigen binding site can be assembled to together with VL structural domain； Or the feature with VL structural domain, functional antigen binding site can be assembled to together with VH structural domain, to provide complete The antigenic binding property of long antibody.

" bracket antigen-binding proteins " are known in the art, such as the ankyrin repeat eggs of fibronectin and design White (DARPin) has been used as the substitution bracket of antigen-binding domains, see, for example, Gebauer and Skerra, Engineered protein scaffolds as next-generation antibody therapeutics.Curr Opin Chem Biol 13:245-255 (2009) and Stumpp et al., Darpins:A new generation of protein therapeutics.Drug Discovery Today 13:695-701(2008).In one aspect of the invention, bracket Antigen-binding proteins are selected from the group being made of following item: CTLA-4 (Evibody), lipocalin protein (Anticalin), albumen Molecule derived from A (the Z structural domain (affine body) of such as albumin A), A structural domain (the huge antibody of Avimer/), serum transferrin (trans- body)；Variable domains (the single domain of the ankyrin repeat albumen (DARPin) of design, antibody light chain or heavy chain Antibody, sdAb), the variable domains (nano antibody, aVH) of heavy chain of antibody, V_NARSegment, fibronectin (AdNectin), c-type Lectin domain (tetranectin)；Variable domains (the V of neoantigen receptor beta-lactamase_NARSegment), people γ-crystal egg White or ubiquitin protein (Affilin molecule)；The kunitz type structural domain of human protease inhibitors, miniature body (such as from The protein of knottin family), peptide aptamer and fibronectin (adnectin).(cytotoxic T lymphocyte is related by CTLA-4 Antigen 4) it is the CD28 family receptors mainly expressed in CD4+ T cell.Its extracellular domain has variable domains sample Ig It folds.Ring corresponding to antibody CDR can be replaced with heterologous sequence, to assign different binding properties.Have through being engineered The CTLA-4 molecule of different binding specificities is also referred to as Evibody (such as US7166697B1).Evibody and antibody (such as tie Structure domain antibodies) isolated variable region size it is roughly the same.About further details, referring to Journal of Immunological Methods 248(1-2),31-45(2001).Lipocalin protein is extracellular protein family, Transport small hydrophobic molecule, such as steroids, Choline, biostearin and lipid.They have rigidity β-sheet secondary knot Structure has many rings at the open end of pyramidal structure, can be designed in conjunction with different target antigens.Anticalin's is big It is small between 160-180 amino acid, and derive from lipocalin protein.About further details, referring to Biochim Biophys Acta 1482:337-350 (2000), US7250297B1 and US20070224633.Affine body is derived from golden yellow The bracket of the albumin A of color staphylococcus pyogenes (Staphylococcus aureus) can be engineered to combine antigen. The structural domain is made of three helical bundles of about 58 amino acid.Library is produced by the randomization of surface residue.About Further details, referring to Protein Eng.Des.Sel.2004,17,455-462 and EP 1641818A1.Avimer is source In the Multidomain albumen of A structural domain bracket family.The native domain of about 35 amino acid is using determining disulfide bonding Structure.Diversity is generated by changing the natural variation that A structural domain family shows.About further details, ginseng See Nature Biotechnology 23 (12), 1556-1561 (2005) and Expert Opinion on Investigational Drugs 16 (6), 909-917 (in June, 2007).Transferrins is monomer serum transport glycoprotein. It can be engineered transferrins by being inserted into peptide sequence in the surface loop of permission, to combine different target antigens.Engineering The example of transferrins bracket include trans- body.About further details, referring to J.Biol.Chem 274,24066- 24073(1999).The ankyrin repeat albumen (DARPin) of design derives from ankyrin, and ankyrin is mediated integration film egg The protein families of white and cytoskeleton attachment.Single ankyrin repeat is by two alpha-helixes and a β-corner group At 33 residue motifs.They can by the residue in the first alpha-helix and β-corner that are randomized in each repetitive sequence, And it is designed to combine different target antigens.Their combination interface can by increase module quantity come increase (affinity at Ripe method).About further details, referring to J.Mol.Biol.332,489-503 (2003), PNAS 100 (4), 1700- 1705 (2003) and J.Mol.Biol.369,1015-1028 (2007) and US20040132028A1.

Single domain antibody can be changed the antibody fragment that antibody domain forms by single monomer.First single domain source In variable domains (nano antibody or the V of the heavy chain of antibody of camellid_HH segment).In addition, term single domain antibody packet Containing self people's heavy-chain variable domains (aVH) or from the V of shark_NARSegment.Fibronectin can be engineered anti-to combine Former bracket.Adnectin by III type people fibronectin (FN3) 15 repetitive units the 10th structural domain natural amino acid The main chain of sequence forms.Three rings of one end of β-interlayer can be through being engineered, so that Adnectin being capable of specificity Identify interested therapeutic targets.About further details, referring to Protein Eng.Des.Sel.18,435-444 (2005), US20080139791, WO2005056764 and US6818418B1.Peptide aptamer is combined identification molecule, the identification point of the combination Son is made of constant scaffolding protein, usually thioredoxin (TrxA), and the constant scaffolding protein contains in active sites The controlled variable peptide loop being inserted at point.About further details, referring to Expert Opin.Biol.Ther.5,783-797 (2005).Microbody is derived from containing 3-4 cysteine bridge, the naturally occurring micro protein that length is 25-50 amino acid, institute The example for stating micro protein includes KalataBI and conotoxin and knottin.Micro protein has ring, which can be through being engineered It is the foldable integral for including up to 25 amino acid without influencing micro protein.Knottin structural domain about engineering into one Details is walked, referring to WO2008098796.

Refer to such antigen binding molecules " in conjunction with the antigen binding molecules of same epitope " as reference molecule, competing Striving antigen binding molecules described in measurement makes the combination of reference molecule and its antigen be blocked 50% or more, and on the contrary, Reference molecule makes the combination of antigen binding molecules and its antigen be blocked 50% or more in competition assay.

As used herein, term " antigen-binding domains " or " antigen binding site " refer to special in antigen binding molecules The part of property combination antigenic determinant.More specifically, term " antigen-binding domains " refers to a part of antibody, the part Include part or all of specific binding with antigen and complementary region.In the case where antigen is very big, antigen binding point Son can be known as epitope only in conjunction with the specific part of antigen, the part.Antigen-binding domains can be by for example one or more Variable domains (also referred to as variable region) provide.Preferably, antigen-binding domains include antibody's light chain variable region (VL) and resist Body heavy chain variable region (VH).In one aspect, antigen-binding domains can be in conjunction with described in its antigen and blocking or part blocks The function of antigen.The antigen-binding domains of specific binding PD1 or LAG3 include antibody as further defined herein and its Segment.In addition, antigen-binding domains may include bracket antigen-binding proteins, such as the repeat sequence protein based on design or set The binding structural domain of the repetitive sequence structural domain of meter (see, for example, WO 2002/020565).

As used herein, term " antigenic determinant " is synonymous with " antigen " and " epitope ", and refers on polypeptide macromolecule Site (such as one section of continuous amino acid or the conformation configuration being made of the different zones of non-contiguous amino acids), antigen binding Part is in conjunction with the site, to form antigen-binding portion thereof-antigenic compound.Useful antigenic determinant can be for example On the surface of tumour cell, on the surface of virus infected cell, on the surface of other diseased cells, on the surface of immunocyte, It is found in educt and/or extracellular matrix (ECM) in serum.Unless otherwise stated, being used herein as the egg of antigen White matter can be the protein of any native form from any vertebrate origin, and the vertebrate origin includes such as The mammals such as primate (such as mankind) and rodent (such as mouse and rat).In a specific embodiment, Antigen is human protein.When referring to specific protein herein, which covers " overall length ", unprocessed protein, with And any type of protein generated by processing into the cell.The term also covers naturally occurring protein variant, such as Splice variant or allelic variant.

" specific binding " refer in conjunction with for antigen have selectivity, and can with it is unwanted or nonspecific Interaction distinguishes.Ability of the antigen binding molecules in conjunction with specific antigen can pass through enzyme linked immunosorbent assay (ELISA) (ELISA) or other technologies familiar to those skilled in the art (such as surface plasma body resonant vibration (SPR) technology is (in BIAcore Analyzed on instrument) (Liljeblad et al., Glyco J 17,323-329 (2000)) and traditional binding assay (Heeley, Endocr Res 28,217-229 (2002)) it measures.In one embodiment, such as measured by through SPR, antigen Binding molecule is less than about the 10% of the combination degree of the antigen binding molecules and antigen to the combination degree of uncorrelated albumen.In In some embodiments, with the dissociation constant (Kd) of the molecule of antigen binding be≤1 μM ,≤100nM ,≤10nM ,≤1nM ,≤ 0.1nM ,≤0.01nM or≤0.001nM (such as 10^-7M or lower, such as 10^-7M to 10^-13M, such as 10^-9M to 10^-13M)。

" affinity " or " binding affinity " refers to the single binding site gametophyte in connection of molecule (such as antibody) The intensity of the summation of noncovalent interaction between (such as antigen).Unless otherwise stated, it is as used herein, " in conjunction with Affinity " refers to inherent binding affinity, and that reflects the 1:1 phase interactions between member's (such as antibody and antigen) of combination pair With.Molecule X usually can indicate that dissociation constant (Kd) is dissociation rate with dissociation constant (Kd) to the affinity of its gametophyte Y The ratio of constant and association rate constants (respectively koff and kon).Therefore, equivalent affinity may include that different rates is normal Number, as long as the ratio of rate constant keeps identical.Affinity can be measured by conventional method known in the art, including Those described herein method.The ad hoc approach for measuring affinity is surface plasma body resonant vibration (SPR).

As used herein, " high-affinity " of term antibody refers to that antibody is 10 to the Kd of target antigen^-9M or lower, even More particularly 10^-10M or lower." low-affinity " of term antibody refers to that the Kd of antibody is 10^-8M or higher.

The antibody of " affinity maturation " refers to has one or more changes in one or more hypervariable regions (HVR) Antibody, compared with the parental antibody for not having such change, such change results in improvement of the antibody to the affinity of antigen.

Term is " comprising specifically binding the first antigen-binding domains of PD1 and the second antigen of specific binding LAG3 The bispecific antibody of binding structural domain " " bispecific antibody of specific binding PD1 and LAG3 " " is specific to PD1 and LAG3 Bispecific antigen binding molecules " or " the anti-anti- LAG3 antibody of PD1/ " be used interchangeably herein, and be refer to Enough affinity combination PD1 and LAG3, so that the antibody can be used as targeting pair for diagnosing and/or treating agent of PD1 and LAG3 Specific antibody.

Term " PD1 ", also referred to as apoptosis albumen 1 are a kind of I type film eggs being made of 288 amino acid It is white, initially in description (Ishida et al., EMBO J., 11 (1992), 3887-3895) in 1992.PD-1 be T cell adjust because The member of the extension CD28/CTLA-4 family of son, and have there are two ligand PD-L1 (B7-H1, CD274) and PD-L2 (B7-DC, CD273).Protein structure includes extracellular IgV structural domain, is transmembrane region and intracellular tail portion later.Contain intracellular tail portion Inhibit two phosphorylation sites in motif and immunity receptor tyrosine-based conversion motif, this table positioned at immunity receptor tyrosine-based Bright PD-1 negative regulator TCR signal.The cytoplasmic tail of this and SHP-1 phosphatase and SHP-2 phosphatase and PD-1 after ligand binding Combination it is consistent.Although PD-1 is not expressed on Naive T cells, it is raised after the activation that T cell receptor (TCR) is mediated, And (Agata et al., Int.Immunology 8 (1996), 765- are all observed in activation and exhaustion T cell 772).These T cells exhausted have the phenotype of dysfunction and cannot suitably react.Although PD-1 has opposite Wider expression pattern, but its it is most important effect probably as in T cell co-suppression receptor (Chinai et al., Trends in Pharmacological Sciences 36(2015),587-595).Therefore, current treatment method focuses In block the interaction of PD-1 and its ligand to enhance t cell response.Term " programmed death 1 " " apoptosis 1 " " protein PD-1 " " PD-1 " " PD1 " " PDCD1 " " hPD-1 " and " hPD-I " are used interchangeably, and the change including people PD-1 Body, isotype, species homologue, and the analog with PD-1 at least one common epitope.The amino acid sequence of people PD1 It is illustrated in UniProt (www.uniprot.org) accession number Q15116 (SEQ ID NO:128).

Term " anti-PD1 antibody " and " antibody comprising combining the antigen-binding domains of PD1 " refer to such antibody, Can be in conjunction with PD1, the PD1 polypeptide especially expressed on cell surface, and have enough affinity so that the antibody Can be used as targeting PD1 diagnoses and/or treats agent.In one aspect, such as it is as thin in passed through radiommunoassay (RIA) or streaming Born of the same parents' art (FACS) uses bio-sensor system (such asSystem) institute measured by surface plasma body resonant vibration It measures, the combination degree of anti-PD1 antibody and incoherent non-PD1 albumen is less than the pact of the antibody and the combination degree of PD1 10%.In some aspects, there is K in conjunction with the antigen-binding proteins of people PD1_DValue for≤1 μM ,≤100nM ,≤10nM ,≤1nM, ≤ 0.1nM ,≤0.01nM or≤0.001nM (such as 10^-8M or lower, such as 10^-8M to 10^-13M, such as 10^-9M to 10^-13M) The binding affinity in conjunction with people PD1.In a preferred embodiment, in surface plasma body resonant vibration measurement, user The corresponding K of extracellular domain (ECD) the measurement binding affinity of PD1 (PD1-ECD)_DValue, to obtain PD1 binding affinity. Term " anti-PD1 antibody " also covers can be in conjunction with the bispecific antibody of PD1 and the second antigen.

Unless otherwise stated, term " LAG3 " as used herein or " Lag-3 " or " lymphocyte activator gene- 3 " or " CD223 " refer to any natural LAG3 from any vertebrate origin, the vertebrate origin includes such as clever The mammals such as long class animal (such as mankind) and rodent (such as mouse and rat).The term is covered " overall length ", is not added The LAG3 of work, and any type of LAG3 generated by processing into the cell.The term also covers the naturally occurring change of LAG3 Body, such as splice variant or allelic variant.In a preferred embodiment, term " LAG3 " refers to people LAG3.It is exemplary The amino acid sequence of the LAG3 of processed (signal-sequenceless) is as shown in SEQ ID NO:73.Exemplary cells extracellular portion (ECD) amino acid sequence of LAG3 is as shown in SEQ ID NO:74.

Term " anti-LAG3 antibody " and refer to such antibody " in conjunction with the antibody of LAG3 ", it can be with enough affinity In conjunction with LAG3 so that the antibody can be used as targeting LAG3 diagnose and/or treat agent.In one aspect, such as by putting It penetrates measured by immunoassays (RIA), the combination degree of anti-LAG3 antibody and incoherent non-LAG3 albumen is less than the antibody With about the 10% of LAG3 combination degree.Be in certain embodiments ,≤1 μM in conjunction with the dissociation constant (Kd) of the antibody of LAG3 ,≤ 100nM ,≤10nM ,≤1nM ,≤0.1nM ,≤0.01nM or≤0.001nM (such as 10^-8M or lower, such as 10^-8M to 10^- ¹³M, such as 10^-9M to 10^-13M).In some aspects, the epitope of anti-LAG3 antibody combination LAG3, the epitope is from not jljl It is conservative in the LAG3 of kind.In a preferred embodiment, " anti-LAG3 antibody " antibody of people LAG3 " specific binding " and " in conjunction with the antibody of people LAG3 " refers to specific binding people LAG3 antigen or the antibody of its extracellular domain (ECD), has K_DValue is 1.0 × 10^-8Mol/l or lower, in one embodiment K_DValue is 1.0 × 10^-9Mol/l or lower is implemented at one K in example_DValue is 1.0 × 10^-9Mol/l to 1.0 × 10^-13The binding affinity of mol/l.It is thin in the situation, such as using LAG3 Extracellular domain, using standard binding assay (such as Applications of surface plasmon resonance (GE-Healthcare Uppsala, Sweden)) determine binding affinity.Term " anti-LAG3 antibody " also covers can be in conjunction with LAG3 and the second antigen Bispecific antibody.

" block property " antibody or " antagonist " antibody be inhibit or reduce the antigen that it is combined biological activity it is anti- Body.In some embodiments, blocking antibody or antagonist antibodies substantially or entirely inhibit the bioactivity of antigen.For example, Bispecific antibody of the invention blocks the signal transduction for passing through PD-1 and LAG3, to answer the functionality carried out by T cell It answers (such as proliferation, cell factor generate, target cell killing) and is restored to antigenic stimulus from dysfunction state.

Term " variable region " or " variable domains " refer to the heavy chain of antibody for participating in antigen binding molecules and antigen binding or The structural domain of light chain.The heavy chain of natural antibody and the variable domains (respectively VH and VL) of light chain usually have similar knot Structure, wherein each structural domain includes four conserved framework regions (FR) and three hypervariable regions (HVR).See, for example, Kindt et al., Kuby Immunology, the 6th edition, W.H.Freeman and Co., page 91 (2007).Single VH or VL structural domain can be enough Assign antigen-binding specificity.

Term " hypervariable region " or " HVR " as used herein refer to that high change and/or formation ceiling structure are fixed in sequence Each of the constant region for immunoglobulin sequence region of ring (" hypervariable loop ").In general, natural four chain antibody includes six HVR: three In VH (H1, H2, H3), three in VL (L1, L2, L3).HVR is generally comprised from hypervariable loop and/or from " complementation determines The amino acid residue in area " (CDR), from " complementary determining region " (CDR) amino acid residue have highest sequence variability and/ Or participate in antigen recognizing.Exemplary hypervariable loop occurs in amino acid residue 26-32 (L1), 50-52 (L2), 91-96 (L3), 26- At 32 (H1), 53-55 (H2) and 96-101 (H3).(Chothia and Lesk, J.Mol.Biol.196:901-917 (1987).) Amino acid residue 24- in L1 occurs for exemplary CDR (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and CDR-H3) 34, the amino acid of amino acid residue 31-35B, H2 of amino acid residue 89-97, H1 of amino acid residue 50-56, L3 of L2 are residual At the amino acid residue 95-102 of base 50-65 and H3.(Kabat et al., Sequences of Proteins of Immunological Interest, the 5th edition, Public Health Service, National Institutes of Health,Bethesda,MD(1991).) hypervariable region (HVR) is also referred to as complementary determining region (CDR), and these terms are herein In be interchangeably used for finger-type into the variable region portion of antigen binding domain.The specific region by Kabat et al., U.S.Dept.of Health and Human Services,"Sequences of Proteins of Immunological Interest " (1983) and Chothia et al., J.Mol.Biol.196:901-917 (1987) description, wherein the definition Overlapping or subset including the amino acid residue when being compared to each other.However, referring to antibody using any definition in the two CDR or its variant should be in the range of term as defined and used herein.As a comparison, listing packet in lower Table A Corresponding amino acid residue containing the CDR as defined in each of references cited herein before.Include the definite of specific CDR Residue number will change according to the sequence and size of CDR.In the case where the variable region amino acid sequence of given antibody, this field Technical staff can routinely determine which residue includes specific CDR.

Table A .CDR definition¹

CDR	Kabat	Chothia	AbM²
				V_H CDR1	31-35	26-32	26-35
V_H CDR2	50-65	52-58	50-58
				V_H CDR3	95-102	95-102	95-102
V_L CDR1	24-34	26-32	24-34
				V_L CDR2	50-56	50-52	50-56
V_L CDR3	89-97	91-96	89-97

¹The number that all CDR are defined in Table A is the numbering convention (see below) proposed according to Kabat et al..

²" AbM " as used in Table A with lowercase " b " refers to " AbM " antibody by Oxford Molecular The CDR that modeling software defines.

Kabat et al. also defines the numbering system of the variable region sequences suitable for any antibody.Ordinary skill Personnel can clearly by should " Kabat number " system distribute to any variable region sequences, and independent of sequence itself except Any experimental data.As used herein, " Kabat number " refers to by Kabat et al., U.S.Dept.of Health and Human Services, volume described in " Sequence of Proteins of Immunological Interest " (1983) Number system.Unless otherwise stated, referring to that the number of particular amino acid residue position in antibody variable region is compiled according to Kabat Number system.

In addition to the CDR1 in VH, CDR generally comprises the amino acid residue to form hypervariable loop.CDR also includes that " specificity determines Residue " or " SDR " are the residues with antigen contact.SDR is included in the CDR region domain for being referred to as and shortening CDR or a-CDR.Show Ammonia in L1 occurs for example property a-CDR (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2 and a-CDR-H3) Amino acid residue 31-35B, H2 of amino acid residue 89-96, H1 of amino acid residue 50-55, L3 of base acid residue 31-34, L2 Amino acid residue 50-58 and H3 amino acid residue 95-102 at.(referring to Almagro and Fransson, Front.Biosci.13:1619-1633(2008).) unless otherwise stated, HVR residue in variable domains and other Residue (such as FR residue) is numbered according to Kabat et al. herein.

" frame " or " FR " refers to the variable domains residue in addition to hypervariable region (HVR) residue.The FR of variable domains It is usually made of following four FR structural domain: FR1, FR2, FR3 and FR4.Therefore, HVR and FR sequence is usually in VH (or VL) With the appearance of following sequence: FR1-H1 (L1)-FR2-H2 (L2)-FR3-H3 (L3)-FR4.

" receptor people frame " for this paper purpose is such frame, and it includes immune from people as defined below Globulin frame or people share light variable domains (VL) frame of frame or the amino of heavy-chain variable domains (VH) frame Acid sequence.Receptor people's frame that " deriving from " human immunoglobulin(HIg) frame or people share frame may include and people's immune globulin White edge frame or people share the identical amino acid sequence of frame or it may include amino acid sequence variation.In some embodiments In, amino acid variation quantity be 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or Less, 4 or less, 3 or less or 2 or less.In some embodiments, VL receptor people frame in sequence with VL Human immunoglobulin(HIg) Frame sequence or human consensus framework sequence are identical.

Term " chimeric " antibody refers to such antibody, and a part of heavy chain and/or light chain derives from the antibody Particular source or species, and the rest part of heavy chain and/or light chain derives from different sources or species.

" classification " of antibody refers to the type of constant domain or constant region possessed by the heavy chain of antibody.There are five major class Antibody: IgA, IgD, IgE, IgG and IgM, and several in these classifications can be further divided into subclass (isotype), such as IgG₁、IgG₂、IgG₃、IgG₄、IgA₁And IgA₂.Heavy chain constant domain corresponding to different classes of immunoglobulin claims respectively For α, δ, ε, γ and μ.

" humanization " antibody refers to such chimeric antibody, it includes the amino acid residue from inhuman HVR and comes from people The amino acid residue of FR.In certain embodiments, humanized antibody will basically comprise it is all at least one, usual two are variable Structural domain, wherein all or substantially all HVR (such as CDR) correspond to the HVR of non-human antibody, and all or substantially institute Some FR correspond to the FR of human antibody.Humanized antibody optionally may include from human antibody antibody constant region at least A part.The antibody of " humanization form ", such as non-human antibody refer to the antibody for having been subjected to humanization.What the present invention covered " humanized antibody " of other forms is such antibody, and relative to original antibodies, the constant region in the antibody is had already passed through Other modifications and changes combine especially with regard to C1q to generate property according to the present invention and/or Fc receptor (FcR) combine Property.

" people " antibody is such antibody, the amino acid sequence having with it is being generated by people or people's cell or from benefit The amino acid sequence of the antibody in the inhuman source of employment antibody library or other people antibody coding sequences is corresponding.This of human antibody is fixed Justice particularly eliminates the humanized antibody comprising non-human antigen-binding residues.

Term " monoclonal antibody " as used herein refers to the antibody that the antibody population of basically homogeneity obtains, that is, In addition to possible antibody variants (such as generate containing naturally occurring mutation or in the production process of monoclonal antibody formulation, Such variant usually exists in a small amount) except, each antibody comprising the group is identical and/or in conjunction with identical table Position.From the polyclonal antibody preparations for the different antibodies for generally including to be directed to different determinants (epitope) on the contrary, monoclonal antibody system Every kind of monoclonal antibody in agent is for the single determinant on antigen.Therefore, modifier " monoclonal " indicates the feature of antibody It is that the antibody population of basically homogeneity obtains, and should not be construed as needing to generate antibody by any ad hoc approach.Example Such as, monoclonal antibody used according to the invention can be prepared by multiple technologies, including but not limited to hybridoma method, recombination DNA method, phage display method, and utilize the transgenic animals containing all or part of human immunoglobulin gene's seat The such method and other illustrative methods for being used to prepare monoclonal antibody is described herein in method.

Term " Fc structural domain " herein or " area Fc " are for defining at least part of antibody weight containing constant region Chain C-terminal region.The term includes the area native sequences Fc and the area variant Fc.Specifically, the area human IgG heavy chain Fc from Cys226 or from Pro230 extends to the carboxyl terminal of heavy chain.However, the C-terminal lysine (Lys447) in the area Fc may exist or be not present.Weight The amino acid sequence of chain is always rendered as with C-terminal lysine, however in the present invention includes the change of no C-terminal lysine Body.

The area IgG Fc includes IgG CH2 structural domain and IgG CH3 structural domain." the CH2 structural domain " in the area human IgG Fc is usual Substantially 340 amino acid residues are extended to from substantially 231 amino acid residues.In one embodiment, carbohydrate Chain is attached to CH2 structural domain.The CH2 structural domain of this paper can be native sequences CH2 structural domain or variant CH2 structural domain."CH3 Structural domain " includes one section of residue of CH2 domain C end in the area Fc (that is, from about 341 amino acid residues of IgG to about 447 amino acid residues).The area CH3 of this paper can be native sequences CH3 structural domain or variant CH3 structural domain (such as one There is in another chain in chain with " protrusion " (" protrusion ") being introduced into the CH3 in " cavity " (" hole ") that introduces accordingly Structural domain；Referring to the United States Patent (USP) No.5,821,333 being clearly incorporated herein by reference).Such variant CH3 structural domain can For promoting the Heterodimerization of two different heavy chain of antibody as described herein.Unless dictate otherwise herein, otherwise Fc The number of amino acid residue is according to EU numbering system in area or constant region, and EU numbering system is also referred to as EU index, such as in Kabat Et al., Sequences of Proteins of Immunological Interest, the 5th edition, Public Health Described in Service, National Institutes of Health, Bethesda, MD, 1991.

" protrusion enters hole (knob-into-hole) " technical description is in such as US 5,731,168；US7,695,936； Ridgway et al., Prot Eng 9,617-621 (1996) and Carter, J Immunol Meth 248, in 7-15 (2001). In general, this method is related to being introduced into raised (" protrusion ") in the interface of the first polypeptide and introduce in the interface of the second polypeptide corresponding Cavity (" hole ") so that the protrusion can be positioned in the cavity, to promote formation and the obstruction of heterodimer The formation of homodimer.Protrusion is by replacing the boundary from the first polypeptide with larger side chain (such as tyrosine or tryptophan) The small amino acid side chains in face and construct.It is by with lesser amino with the compensation cavity with the same or similar size of protrusion Sour side chain (such as alanine or threonine) replaces big amino acid side chains and creates in the interface of the second polypeptide.Protrusion and sky Chamber can be prepared by changing the nucleic acid of coding polypeptide, such as by site-specific mutagenesis or pass through peptide synthesis.At one In specific embodiment, the amino acid substitution T366W in one in projection-decorated two subunits comprising Fc structural domain, and hole is repaired Decorations comprising Fc structural domain two subunits in another in amino acid substitution T366S, L368A and Y407V.In another tool In body embodiment, the subunit comprising projection-decorated Fc structural domain additionally comprises amino acid substitution S354C, and includes what hole was modified The subunit of Fc structural domain additionally comprises amino acid substitution Y349C.Introducing the two cysteine residues leads to two in the area Fc Disulphide bridges are formed between subunit, to further stabilize dimer (Carter, J Immunol Methods 248,7-15 (2001))。

" region being equal with the area Fc of immunoglobulin " is intended to include the naturally occurring etc. of the area Fc of immunoglobulin Position genetic mutation, and have to generate and replace, add or delete but do not reduce immunoglobulin-mediated effector function substantially The variant of the modification of the ability of (such as antibody-dependent cytotoxicity).For example, one or more amino acid can be made from immune The N-terminal or C-terminal in the area Fc of globulin lack, and do not lose biological function substantially.It can be according to as is generally known in the art General rule select such variant, so as on activity have it is the smallest influence (see, for example, Bowie, J.U. et al., Science 247:1306-10(1990))。

Term " effector function " refers to that for being attributable to the area Fc of antibody, changing with the variation of antibody isotype A little bioactivity.The example of antibody mediated effect subfunction includes: that C1q is combined and complement-dependent cytotoxicity (CDC), Fc receptor knot Conjunction, the cytotoxicity (ADCC) of antibody dependent cellular mediation, antibody dependent cellular phagocytosis (ADCP), cell factor point It secretes, the antigen uptake for the antigen presenting cell that immune complex mediates, lower cell surface receptor (such as B-cell receptor), with And B cell activation.

" activation Fc receptor " is such Fc receptor, after the engagement of the area Fc of antibody, stimulation is caused to carry the thin of receptor The signal transduction event of born of the same parents' execution effector function.Activating Fc receptor includes III a (CD16a) of Fc γ R, Fc γ R I (CD64), Fc II a of γ R (CD32) and Fc α R I (CD89).Specific activation Fc receptor is III a of people Fc γ R (referring to UniProt accession number P08637, version 141).

Term " peptide linker " refers to comprising one or more amino acid, the peptide of normally about 2 to 20 amino acid.Peptide linker is It is as known in the art or be described herein.Suitable non-immunogenic link peptide is such as (G₄S)_n、(SG₄)_nOr G₄ (SG₄)_nPeptide linker, wherein " n " be usually between 1 and 10, generally between the number between 2 and 4, in particular 2, that is, select The free peptide of the group of following item composition: GGGGS (SEQ ID NO:129), GGGGSGGGGS (SEQ ID NO:130), SGGGGSGGGG (SEQ ID NO:131) and GGGGSGGGGSGGGG (SEQ ID NO:132), but also include following sequence: GSPGSSSSGS(SEQ ID NO:133)、(G4S)₃(SEQ ID NO:134)、(G4S)₄(SEQ ID NO:135)、GSGSGSGS (SEQ ID NO:136)、GSGSGNGS(SEQ ID NO:137)、GGSGSGSG(SEQ ID NO:138)、GGSGSG(SEQ ID NO:139), GGSG (SEQ ID NO:140), GGSGNGSG (SEQ ID NO:141), GGNGSGSG (SEQ ID NO:142) and GGNGSG(SEQ ID NO:143).Peptide linker of special interest is (G4S) (SEQ ID NO:129), (G₄S)₂Or GGGGSGGGGS(SEQ ID NO:130)、(G4S)₃(SEQ ID NO:134) and (G₄S)₄(SEQ ID NO:135), and more Specifically (G₄S)₂Or GGGGSGGGGS (SEQ ID NO:130).

" being fused to " or " being connected to " be finger (such as antigen-binding domains and FC structural domain) directly or via One or more peptide linkers and pass through peptide be keyed.

Term " amino acid " as used in this application indicates the naturally occurring carboxyl a-amino acid including following item Group: alanine (three-letter codes: ala, single letter code: A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine (cys, C), glutamine (gln, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, ) and valine (val, V) Y.

" Amino acid sequence identity percentage (%) " relative to reference polypeptide (protein) sequence be defined as than To the amino acid residue in candidate sequence and the amino acid residue in reference polypeptide sequence and introduce notch (if necessary) After realizing maximum percentage of sequence identity, and any conservative substitution be not thought of as the one of the sequence identity Amino acid residue in the case where part, in the candidate sequence identical with the amino acid residue in the reference polypeptide sequence Percentage.For determining that comparing for Amino acid sequence identity percentage can be in a manner of various within the scope of art technology It realizes, such as uses publicly available computer software, such as BLAST, BLAST-2, ALIGN.SAWI or Megalign (DNASTAR) software.Those skilled in the art can determine the suitable parameter for aligned sequences, including in the sequence compared Realize high specific to required any algorithm in overall length.However, For the purpose of this paper, comparing computer program using sequence ALIGN-2 generates the value of Amino acid sequence identity %.ALIGN-2 sequence compares computer program by Genentech, Inc. It writes, and source code is submitted to U.S.Copyright Office, Washington D.C. together with customer documentation, 20559, there with S. Copyright registration number TXU510087 registration.ALIGN-2 program can be from Genentech, Inc., South San Francisco, California disclose acquisition, or can be from the compilation of source code.ALIGN-2 program is answered compiled To use in UNIX operating system, the UNIX operating system includes number UNIX V4.0D.It is equal that all sequences compare parameter It is arranged by ALIGN-2 program and constant.In the case where carrying out amino acid sequence comparison using ALIGN-2, amino acid is given (it can alternatively be expressed as given amino acid sequence A to the Amino acid sequence identity % of sequence A and given amino acid sequence B Have or comprising a certain Amino acid sequence identity % with given amino acid sequence B) calculate it is as follows:

100 multiplied by score X/Y

Wherein X is the ammonia to be scored in comparison of the program to A and B by alignment programs ALIGN-2 as identical match The number of base acid residue, and wherein Y is the sum of amino acid residue in B.It should be appreciated that the length in amino acid sequence A differs In the case where the length of amino acid sequence B, the Amino acid sequence identity % of A and B will be equal to the amino acid sequence of B and A Consistency %.Unless otherwise specified, otherwise the value of all Amino acid sequence identity % used herein is as previous It is obtained described in section using ALIGN-2 computer program.

In certain aspects, it is contemplated to the amino acid sequence variation of the bispecific antibody of present invention provided herein.Example Such as, it may be necessary to improve the binding affinity and/or other biological property of bispecific antibody.The amino of bispecific antibody Sequence variants can be prepared by introducing modification appropriate in the nucleotide sequence to coding molecule or by peptide synthesis.This Class modification includes the missing of residue, and/or insertion and/or substitution in such as antibody amino acids sequence.It can be lacked, be inserted into With substituted any combination to realize that final construct, precondition are that final construct has required feature, such as antigen knot It closes.Interested site for replacing mutagenesis includes HVR and frame (FR).Conservative substitution gauge outfit in table B " preferably takes It provides under generation ", and is further described hereinafter with reference to amino acid side chain classification (1) to (6).Amino acid substitution can be introduced In interested molecule, and activity needed for being carried out to product (such as the immunogenicity of reservation/improvement antigen binding, reduction, or Improved ADCC or CDC) screening.

Table B

Amino acid can be grouped according to common side chain properties:

(1) hydrophobicity: nor-leucine, Met, Ala, Val, Leu, Ile；

(2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln；

(3) acid: Asp, Glu；

(4) alkaline: His, Lys, Arg；

(5) residue of chain orientation: Gly, Pro is influenced；

(6) aromatic series: Trp, Tyr, Phe.

Non-conservation, which replaces, will need to exchange another category with one member in these classifications.

Term " amino acid sequence variation " includes substantive variant, wherein in parent's antigen binding molecules (such as humanization Or human antibody) one or more some hypervariable region residues in there are amino acid substitutions.In general, relative to parent's antigen binding molecules, Be selected as one or more gained variants for further studying will in terms of certain biological characteristics (for example, affinity increase, Immunogenicity reduces) there is change (for example, improvement) and/or will substantially retain certain biological characteristics of parent's antigen binding molecules Property.Exemplary substitution variant is affinity maturation antibody, for example can be based on phage display technology as those described herein using all The affinity maturation technology shown is conveniently generated.In brief, by one or more HVR residue mutations and by variant antigen knot Molecular display is closed on bacteriophage and is screened for particular organisms active (such as binding affinity).In some embodiments In, replacing, be inserted into or lacking can occur in one or more HVR, as long as such change does not reduce antigen binding point substantially The antigen binding capacity of son.For example, substantially can not be reduced the conservative of binding affinity in HVR sexually revises (example Such as, conservative replaces as provided herein).Can be used for identifying can be targeted the antibody residue of mutagenesis or the method in region is referred to as " alanine scanning mutagenesis ", as described in Cunningham and Wells (1989) Science, 244:1081-1085.In the method In, identify residue or one group of target residue (for example, charged residues, such as Arg, Asp, His, Lys and Glu) and with neutrality or with negative Whether amino acid (for example, the alanine or more alanine) replacement of electricity is affected with the interaction for determining antibody and antigen. Other substitutions can introduced to the initial amino acid position for replacing displaying function sensitive.Alternatively or in addition, antigen-is utilized The crystal structure of antigen binding molecules compound identifies the contact point between antibody and antigen.It can target or eliminate as substituted Candidate such contact residues and adjacent residues.Variant can be screened to determine whether they have desired characteristic.

Amino acid sequence insertion includes that length range is a residue extremely polypeptide containing 100 or more residues Insertion in amino and/or carboxyl-terminal fusion, and the sequence of one or more amino acid residues.End insertion example include Bispecific antibody with N-terminal methionyl residue.Other insertion variants of molecule include and increase bispecific antibody Serum half-life polypeptide N-terminal or C-terminal fusion.

In some aspects, change bispecific antibody provided herein to increase or decrease the degree of antibody glycosylation.It can By changing amino acid sequence, so that generating or removing one or more glycosylation sites, such as it can be changed and attach to Fc structure The carbohydrate in domain, advantageously to obtain the glycosylation variants of molecule.The natural antibody generated by mammalian cell is usual Comprising double antennary oligosaccharides with branch, double antennary oligosaccharides usually attach to the CH2 structural domain in the area Fc by N- connection Asn297.See, e.g., Wright et al. TIBTECH 15:26-32 (1997).Oligosaccharides may include various carbohydrate, example Such as, mannose, N- acetyl glucosamine (GlcNAc), galactolipin and sialic acid, and attach to the " main of double antennary oligosaccharide structures It is dry " in GlcNAc fucose.In some embodiments, the oligosaccharides in bispecific antibody of the invention can be repaired Decorations, to generate the variant with certain improved characteristics.In one aspect, the variant of bispecific antibody is provided, is had Lack attachment (direct or indirect) in the carbohydrate structure of the fucose in the area Fc.Such fucosylation variant, which can have, to be changed Kind ADCC function, see, for example, U.S. Patent Publication text No.US 2003/0157108 (Presta, L.) or US 2004/ 0093621 (Kyowa Hakkokogyo Co., Ltd (Kyowa Hakko Kogyo Co., Ltd)).Bispecific antibody of the invention Other variants include the variant with two parting oligosaccharides, such as wherein attaching to double antennary oligosaccharides in the area Fc is to pass through GlcNAc Two points.Such variant can have the function of reduced fucosylation and/or improved ADCC, see, for example, WO 2003/ 011878 (Jean-Mairet et al.)；United States Patent (USP) No.6,602,684 (Umana et al.)；And US 2005/0123546 (Umana et al.).Additionally provide the variant in the oligosaccharides for attaching to the area Fc at least one galactose residue.Such antibody Variant can have the function of improved CDC and be described in such as WO 1997/30087 (Patel et al.)；WO 1998/58964 (Raju,S.)；And in WO 1999/22764 (Raju, S.).

In some aspects, it may be desirable to the engineered variant of the cysteine of bispecific antibody of the invention is generated, Such as " thioMAb ", wherein one or more residues of molecule are replaced by cysteine residues.In a particular embodiment, replace Residue be present in the accessible site of molecule.By replacing those residues with cysteine, reactive thiol group group is thus fixed Positioned at the accessible site of antibody, and it can be used for antibody and other parts, such as drug moiety or linker-drug part are sewed It closes, to generate immunoconjugates.In certain embodiments, any one of following residue or more can be replaced with cysteine It is a: the V205 (Kabat number) of light chain；The A118 (EU number) of heavy chain；And the S400 (EU number) in the area heavy chain Fc.It can be such as example The engineered antigen binding molecules of generation cysteine as described in United States Patent (USP) No.7,521,541.

In some aspects, can further modify bispecific antibody provided herein so that its contain it is known in the art and easy In the other non-protein portion of acquisition.The part for being suitable for antibody derivatization includes but is not limited to water-soluble polymer.Water The non-limiting example of soluble polymer includes but is not limited to polyethylene glycol (PEG), the copolymer of ethylene glycol/propylene glycol, carboxylic first Base cellulose, glucan, polyvinyl alcohol, polyvinylpyrrolidone, poly- 1,3- dioxolanes, poly- 1,3,6- trioxane, ethylene/ Copolymer-maleic anhydride, polyaminoacid (homopolymer or randomcopolymer) and glucan or poly- (n-VP) poly- second two Alcohol, propropylene glycol homopolymers, polypropylene oxide/ethylene oxide copolymer, polyoxyethylated polyols (such as glycerol), polyvinyl alcohol And their mixture.Due to its stability in water, methoxy PEG-propionaldehyde can have advantage during manufacturing.Polymer can With any molecular weight, and it can have branch or do not have branch.The number for attaching to the polymer of antibody is alterable, and And if more than one polymer is attached, they can be identical or different molecule.In general, can be based on considered below Factor determine for derivatization polymer number and/or type, including but not limited to antibody specific feature to be improved or The therapy etc. whether function, bispecific antibody derivatives will be used under qualifications.

On the other hand, the non-proteinaceous portion that provides antibody and can be selectively heated and being exposed to radiation The conjugate divided.In one embodiment, non-proteinaceous part be carbon nanotube (Kam, N.W. et al., Proc.Natl.Acad.Sci.USA 102(2005)11600-11605).Radiation can have any wavelength, and including but not It is limited to not injure ordinary cells, but non-proteinaceous part is heated to thin near antibody-non-proteinaceous part The wavelength of the killed temperature of born of the same parents.

" immunoconjugates " are and one or more heterologous molecules, the including but not limited to antibody of cytotoxic agent conjugation.

Term " polynucleotides " refers to the nucleic acid molecules or construct of separation, such as mRNA (mRNA), viral source RNA or Plasmid DNA (pDNA).Polynucleotides may include conventional phosphoric acid diester linkage or unconventional key (such as amido bond, such as in peptide Present in nucleic acid (PNA)).Term " nucleic acid molecules ", which refers to, is present in any one or more of polynucleotides nucleic acid area Section, such as DNA or RNA segment.

About " separation " nucleic acid molecules or polynucleotides, refer to the nucleic acid molecules removed from its natural surroundings, DNA or RNA.For example, coding is considered as separating for purposes of the present invention comprising the recombination of polynucleotide of polypeptide in the carrier 's.The other example of isolated polynucleotides includes maintaining recombination of polynucleotide in Heterologous Host Cells or in solution In purifying (partially or substantially purifying) polynucleotides.Isolated polynucleotides include being included in generally comprise multicore In the cell of thuja acid molecule, but polynucleotide molecule is present in outside chromosome or is present in different from its native chromosomal sites Polynucleotide molecule on chromosome location.Isolated RNA molecule includes internal or external RNA transcript of the invention, and Normal chain and minus strand form and double-stranded form.Isolated polynucleotides or nucleic acid according to the present invention further include by being synthetically produced Such molecule.In addition, polynucleotides or nucleic acid can be or may include controlling element, such as promoter, ribosome bind site Or transcription terminator.

About the nucleic acid with reference nucleotide sequence of the invention at least nucleotide sequence of such as 95% " consistent " Or polynucleotides, refer in addition to polynucleotide sequence may include every 100 nucleotide at most five points of reference nucleotide sequence Except mutation, nucleotide sequence and the reference sequences of polynucleotides are consistent.In other words, have and reference core to obtain The polynucleotides of the consistent nucleotide sequence of nucleotide sequence at least 95%, in reference sequences at most 5% nucleotide can lack or By other nucleotide replace or reference sequences in the nucleotide of at most 5% quantity of total nucleotide can be plugged into reference to sequence In column.These changes of reference sequences can occur 5 ' or 3 ' terminal positions of reference nucleotide sequence or those terminal positions it Between any position, or be individually dispersed among the residue of reference sequences, or be dispersed in ginseng with one or more continuous groups It examines in sequence.As a kind of actual conditions, known computer program, the journey such as discussed above with respect to polypeptide can be used Sequence (such as ALIGN-2), it is conventional determine any specific polynucleotide sequence whether with nucleotide sequence of the invention at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% is consistent.

Term " expression cassette " refers to the polynucleotides by recombinantly or synthetically generating, and having allows specific nucleic acid thin in target A series of specific nucleic acid elements transcribed in born of the same parents.Recombinant expression cassettes can mix plasmid, chromosome, mitochondrial DNA, Plasmid DNA, In virus or nucleic acid fragment.Typically, the recombinant expression cassettes part of expression vector further includes to be transcribed in addition to other sequences Nucleic acid sequence and promoter.In certain embodiments, expression cassette of the invention includes to encode bispecific antigen knot of the invention Close molecule or the polynucleotide sequence of its segment.

Term " carrier " or " expression cassette " are synonymous with " expression construct " and refer to for introducing specific gene The DNA molecular of the expression of the gene is and guided in the target cell being operably associated with it.The term includes multiple as self The carrier of nucleic acid structure processed, and the carrier being integrated into the genome for the host cell that it has been introduced into.Expression of the invention Carrier includes expression cassette.Expression vector allows the transcription of a large amount of stable mRNAs.Once expression vector is in target cell interior, i.e., logical It crosses cell transcription and/or translating mechanism generates the ribonucleic acid molecule or protein encoded by the gene.In one embodiment, Expression vector of the invention includes expression cassette, the expression cassette include encode bispecific antigen binding molecules of the invention or its The polynucleotide sequence of segment.

Term " host cell " " host cell line " and " host cell cultures " are used interchangeably, and refer to external source core The cell that acid has been introduced, the filial generation including such cell.Host cell includes " transformant " and " transformed cells ", packet Primary transformed cell and the filial generation from the primary transformed cell are included, does not consider passage number.Filial generation may not be with parent The nucleic acid content of cell is completely the same, but may contain mutation.It herein include screening or selecting such as in original transformation cell The muton generation with identical function or bioactivity selected.It is anti-that host cell can be used for generating bispecific of the invention Any kind of cell system of former binding molecule.Specifically, host cell is protokaryon or eukaryotic host cell.Host cell packet The cell for including culture, gives some instances, such as the mammalian cell of culture, such as Chinese hamster ovary celI, bhk cell, NS0 cell, SP2/0 cell, YO myeloma cell, P3X63 murine myeloma cell, PER cell, PER.C6 cell or hybridoma, ferment Mother cell, insect cell and plant cell, and include the plant in transgenic animals, genetically modified plants or culture or animal groups Cell in knitting.

" effective quantity " of medicament refers to generates the required amount of physiology variation in the cell or tissue that it is applied.

" therapeutically effective amount " of medicament (such as pharmaceutical composition) refers in required dosage and effectively realizes the phase on the period The amount of the treatment or prevention result of prestige.Disease is for example eliminated, reduces, postpones, minimizes or prevent to the medicament of therapeutically effective amount Adverse effect.

" individual " or " subject " is mammal.Mammal includes but is not limited to animal (such as the ox, silk floss raised and train Sheep, cat, dog and horse), primate (such as people and non-human primate, such as monkey), rabbit and rodent (such as Mouse and rat).Specifically, individual or subject are people.

Term " pharmaceutical composition " refers to the effective form of bioactivity in the active constituent for allowing to be included in, And the preparation without the other component for the subject that will be administered preparaton with unacceptable toxicity.

" pharmaceutically acceptable excipient " refers to the ingredient in pharmaceutical composition in addition to effective component, to subject It is nontoxic.Pharmaceutically acceptable excipient includes but is not limited to buffer, stabilizer or preservative.

Term " package insert " is used to refer to the specification generally included in the commercial packing for the treatment of product, contains and relates to this The information of the related indication, usage, dosage, application, conjoint therapy, contraindication and/or the warning that use of class treatment product.

As used herein, " treat (treatment) " (and its grammatical variants, such as " treatment (treat) " or " treatment (treating) ") refer to and attempt to change the clinical intervention for treating individual nature process, and can be in order to prevent or It is carried out in the process of clinicopathologia.The desired effects for the treatment of include but is not limited to prevent the generation or recurrence, mitigation disease of disease Shape, any direct or indirect pathological consequence for weakening disease, prevention transfer, rate, improvement or the mitigation for reducing progression of disease Morbid state, and alleviate or improve prognosis.In some embodiments, molecule of the invention is used to postpone the development or use of disease In the progress for slowing down disease.

Term " cancer " as used herein refers to proliferative disease, such as lymthoma, lymphocytic leukemia, lung Cancer, non-small cell lung (NSCL) cancer, bronchial alveolar cells lung cancer, osteocarcinoma, cancer of pancreas, cutaneum carcinoma, head or neck cancer, skin or eye Interior melanoma, uterine cancer, oophoroma, the carcinoma of the rectum, cancer of the anal region, gastric cancer (stomach cancer), gastric cancer (gastric Cancer), colon cancer, breast cancer, uterine cancer, carcinoma of fallopian tube, carcinoma of endometrium, cervix cancer, carcinoma of vagina, carcinoma of vulva, Huo Qi Golden disease, the cancer of the esophagus, carcinoma of small intestine, internal system cancer, thyroid cancer, parathyroid carcinoma, adrenal, soft tissue sarcoma, urethra Cancer, carcinoma of penis, prostate cancer, bladder cancer, kidney or carcinoma of ureter, clear-cell carcinoma, carcinoma of renal pelvis, celiothelioma, hepatocellular carcinoma, bile duct Cancer, central nervous system (CNS) tumour, vertebra axis tumour, brain stem glioma, glioblastoma multiforme, astrocytoma, Neurinoma, ependymoma, medulloblastoma, meningioma, squamous cell carcinoma, pituitary adenoma and ewing's sarcoma, including it is above The combination of the intractable pattern of any one of cancer or one or more above cancers.

Bispecific antibody of the invention

The present invention provides novel bispecific antibodies, and it includes specific bindings apoptosis albumen 1 (PD1) The first antigen-binding domains and specific binding lymphocyte activation gene -3 (LAG3) the second antigen-binding domains, Its with particularly advantageous characteristic, such as productibility, stability, binding affinity, bioactivity, certain T cells it is special Property targeting, targeting efficiency and reduced toxicity.

In some aspects, a kind of bispecific antibody is provided, it includes the first antigen binding knots of specific binding PD1 Second antigen-binding domains in structure domain and specific binding LAG3, show in reduction when in conjunction with T cell surface Change.Internalization indicates the important sinking of molecule, and the molecule can degrade within a few hours, while receptor targeted is on cell surface It rapidly expresses again, inhibits TCR signal transduction to be ready for use on.In a further aspect, a kind of bispecific antibody is provided, Comprising specifically binding the first antigen-binding domains of PD1 and the second antigen-binding domains of specific binding LAG3, Preferentially combine conventional T cells, rather than Treg.This is favourable, because can be led to using the LAG-3 on blocking antibody targeting Treg It crosses the inhibition sexual function for increasing them and finally covers the positive blocking effect in other T cells but harmful.In another side Face provides a kind of bispecific antibody, and it includes the first antigen-binding domains of specific binding PD1 and specific bindings The second antigen-binding domains of LAG3 can save T cell effector function from Treg inhibition.On the other hand, A kind of bispecific antibody is provided, it includes the first antigen-binding domains of specific binding PD1 and specific bindings The second antigen-binding domains of LAG3 can induce CD4 T cell when with tumor cell line ARH77 co-incubation Granzyme B secretion, as provided herein shown in measurement.On the other hand, a kind of bispecific antibody is provided, it includes Specifically bind the first antigen-binding domains of PD1 and the second antigen-binding domains of specific binding LAG3, display Increased tumor specific T cells effector function and/or the cytotoxic effect of enhancing T cell out.On the other hand, it mentions A kind of bispecific antibody is supplied, it includes the first antigen-binding domains of specific binding PD1 and specific binding LAG3 The second antigen-binding domains, show enhancing in-vivo tumour eradicate.

A. the Exemplary bispecific antibodies of PD1 and LAG3 are combined

In one aspect, the present invention provides a kind of bispecific antibodies, and it includes the first antigens of specific binding PD1 Second antigen-binding domains of binding structural domain and specific binding LAG3, wherein described the first of specific binding PD1 is anti- Former binding structural domain includes

VH structural domain, it includes

(i) HVR-H1, it includes amino acid sequence shown in SEQ ID NO:1,

(ii) HVR-H2, it includes amino acid sequences shown in SEQ ID NO:2, and

(iii) HVR-H3, it includes amino acid sequences shown in SEQ ID NO:3；And

VL structural domain, it includes

(i) HVR-L1, it includes amino acid sequence shown in SEQ ID NO:4,

(ii) HVR-L2, it includes amino acid sequences shown in SEQ ID NO:5, and

(iii) HVR-L3, it includes amino acid sequences shown in SEQ ID NO:6.

In one aspect, bispecific antibody includes Fc structural domain, and the Fc structural domain is IgG, specifically IgG1 Fc Structural domain or IgG4 Fc structural domain, and wherein Fc structural domain has the effector function for reducing or even being eliminated.Specifically, Fc structural domain includes one or more amino acid substitutions, and one or more of amino acid substitutions reduce and the combination of Fc receptor, Specifically and the combination of Fc γ receptor.

On the other hand, a kind of bispecific antibody is provided, it includes the first antigen bindings of specific binding PD1 Second antigen-binding domains of structural domain and specific binding LAG3, wherein bispecific antibody includes Fc structural domain, described Fc structural domain is IgG, specifically IgG1 Fc structural domain or IgG4 Fc structural domain, and wherein Fc structural domain include one or Multiple amino acid substitutions, one or more of amino acid substitutions reduce and the combination of Fc receptor, specifically with Fc γ receptor Combination.

(a) VH structural domain, it includes

(i) HVR-H1, it includes amino acid sequence shown in SEQ ID NO:14,

(ii) HVR-H2, it includes amino acid sequences shown in SEQ ID NO:15, and

(iii) HVR-H3, it includes amino acid sequences shown in SEQ ID NO:16；And

VL structural domain, it includes

(i) HVR-L1, it includes amino acid sequence shown in SEQ ID NO:17,

(ii) HVR-L2, it includes amino acid sequences shown in SEQ ID NO:18, and

(iii) HVR-L3, it includes amino acid sequences shown in SEQ ID NO:19；Or

(b) VH structural domain, it includes

(i) HVR-H1, it includes amino acid sequence shown in SEQ ID NO:22,

(ii) HVR-H2, it includes amino acid sequences shown in SEQ ID NO:23, and

(iii) HVR-H3, it includes amino acid sequences shown in SEQ ID NO:24；And

VL structural domain, it includes

(i) HVR-L1, it includes amino acid sequence shown in SEQ ID NO:25,

(ii) HVR-L2, it includes amino acid sequences shown in SEQ ID NO:26, and

(iii) HVR-L3, it includes amino acid sequences shown in SEQ ID NO:27；Or

(i) HVR-H1, it includes amino acid sequence shown in SEQ ID NO:30,

(ii) HVR-H2, it includes amino acid sequences shown in SEQ ID NO:31, and

(iii) HVR-H3, it includes amino acid sequences shown in SEQ ID NO:32；And

VL structural domain, it includes

(i) HVR-L1, it includes amino acid sequence shown in SEQ ID NO:33,

(ii) HVR-L2, it includes amino acid sequences shown in SEQ ID NO:34, and

(iii) HVR-L3, it includes amino acid sequences shown in SEQ ID NO:35；Or

(d) VH structural domain, it includes

(i) HVR-H1, it includes amino acid sequence shown in SEQ ID NO:38,

(ii) HVR-H2, it includes amino acid sequences shown in SEQ ID NO:39, and

(iii) HVR-H3, it includes amino acid sequences shown in SEQ ID NO:40；And

VL structural domain, it includes

(i) HVR-L1, it includes amino acid sequence shown in SEQ ID NO:41,

(ii) HVR-L2, it includes amino acid sequences shown in SEQ ID NO:42, and

(iii) HVR-L3, it includes amino acid sequences shown in SEQ ID NO:43；Or

(e) VH structural domain, it includes

(i) HVR-H1, it includes amino acid sequence shown in SEQ ID NO:46,

(ii) HVR-H2, it includes amino acid sequences shown in SEQ ID NO:47, and

(iii) HVR-H3, it includes amino acid sequences shown in SEQ ID NO:48；And

VL structural domain, it includes

(i) HVR-L1, it includes amino acid sequence shown in SEQ ID NO:49,

(ii) HVR-L2, it includes amino acid sequences shown in SEQ ID NO:50, and

(iii) HVR-L3, it includes amino acid sequences shown in SEQ ID NO:51.

(a) VH structural domain, it includes

(i) HVR-H1, it includes amino acid sequence shown in SEQ ID NO:80,

(ii) HVR-H2, it includes amino acid sequences shown in SEQ ID NO:81, and

(iii) HVR-H3, it includes amino acid sequences shown in SEQ ID NO:82；And

VL structural domain, it includes

(i) HVR-L1, it includes amino acid sequence shown in SEQ ID NO:83,

(ii) HVR-L2, it includes amino acid sequences shown in SEQ ID NO:84, and

(iii) HVR-L3, it includes amino acid sequences shown in SEQ ID NO:85；Or

(b) VH structural domain, it includes

(i) HVR-H1, it includes amino acid sequence shown in SEQ ID NO:88,

(ii) HVR-H2, it includes amino acid sequences shown in SEQ ID NO:89, and

(iii) HVR-H3, it includes amino acid sequences shown in SEQ ID NO:90；And

VL structural domain, it includes

(i) HVR-L1, it includes amino acid sequence shown in SEQ ID NO:91,

(ii) HVR-L2, it includes amino acid sequences shown in SEQ ID NO:92, and

(iii) HVR-L3, it includes amino acid sequences shown in SEQ ID NO:93.

(a) VH structural domain and VL structural domain, the VH structural domain include amino acid sequence shown in SEQ ID NO:86, institute Stating VL structural domain includes amino acid sequence shown in SEQ ID NO:87, or

(b) VH structural domain and VL structural domain, the VH structural domain include amino acid sequence shown in SEQ ID NO:94, institute Stating VL structural domain includes amino acid sequence shown in SEQ ID NO:95.

On the other hand, a kind of bispecific antibody is provided, it includes the first antigen bindings of specific binding PD1 Second antigen-binding domains of structural domain and specific binding LAG3, wherein the second antigen binding knot of specific binding LAG3 Structure domain includes VH structural domain, and the VH structural domain includes

(i) HVR-H1, it includes amino acid sequence shown in SEQ ID NO:56,

(ii) HVR-H2, it includes amino acid sequences shown in SEQ ID NO:57, and

(iii) HVR-H3, it includes amino acid sequences shown in SEQ ID NO:58；And

VL structural domain, it includes

(i) HVR-L1, it includes amino acid sequence shown in SEQ ID NO:59,

(ii) HVR-L2, it includes amino acid sequences shown in SEQ ID NO:60, and

(iii) HVR-L3, it includes amino acid sequences shown in SEQ ID NO:61.

In one aspect, bispecific antibody of the invention includes the first antigen-binding domains of specific binding PD1 With the second antigen-binding domains of specific binding LAG3, first antigen-binding domains include that VH structural domain and VL are tied Structure domain, the VH structural domain includes amino acid sequence shown in SEQ ID NO:9 and the VL structural domain includes SEQ ID NO: Amino acid sequence shown in 10；Second antigen-binding domains include VH structural domain and VL structural domain, the VH structural domain Comprising amino acid sequence shown in SEQ ID NO:20 and the VL structural domain includes amino acid sequence shown in SEQ ID NO:21 Column.

On the other hand, bispecific antibody of the invention includes the first antigen binding structure of specific binding PD1 Second antigen-binding domains in domain and specific binding LAG3, first antigen-binding domains include VH structural domain and VL Structural domain, the VH structural domain includes amino acid sequence shown in SEQ ID NO:9 and the VL structural domain includes SEQ ID Amino acid sequence shown in NO:10；Second antigen-binding domains include VH structural domain and VL structural domain, the VH structure Domain includes amino acid sequence shown in SEQ ID NO:52 and the VL structural domain includes amino acid shown in SEQ ID NO:53 Sequence.

On the other hand, bispecific antibody of the invention includes the first antigen binding structure of specific binding PD1 Second antigen-binding domains in domain and specific binding LAG3, first antigen-binding domains include VH structural domain and VL Structural domain, the VH structural domain includes amino acid sequence shown in SEQ ID NO:9 and the VL structural domain includes SEQ ID Amino acid sequence shown in NO:10；Second antigen-binding domains include VH structural domain and VL structural domain, the VH structure Domain includes amino acid sequence shown in SEQ ID NO:62 and the VL structural domain includes amino acid shown in SEQ ID NO:63 Sequence.

On the other hand, bispecific antibody of the invention includes the first antigen binding structure of specific binding PD1 Second antigen-binding domains in domain and specific binding LAG3, first antigen-binding domains include VH structural domain and VL Structural domain, the VH structural domain includes amino acid sequence shown in SEQ ID NO:86 and the VL structural domain includes SEQ ID Amino acid sequence shown in NO:87；Second antigen-binding domains include VH structural domain and VL structural domain, the VH structure Domain includes amino acid sequence shown in SEQ ID NO:62 and the VL structural domain includes amino acid shown in SEQ ID NO:63 Sequence.

On the other hand, bispecific antibody of the invention includes the first antigen binding structure of specific binding PD1 Second antigen-binding domains in domain and specific binding LAG3, first antigen-binding domains include VH structural domain and VL Structural domain, the VH structural domain includes amino acid sequence shown in SEQ ID NO:94 and the VL structural domain includes SEQ ID Amino acid sequence shown in NO:95；Second antigen-binding domains include VH structural domain and VL structural domain, the VH structure Domain includes amino acid sequence shown in SEQ ID NO:62 and the VL structural domain includes amino acid shown in SEQ ID NO:63 Sequence.

On the other hand, comprising specifically binding the first antigen-binding domains of PD1 and specifically binding LAG3's The bispecific antibody of second antigen-binding domains is people, humanization or chimeric antibody.Specifically, the antibody is humanization Or chimeric antibody.

In one aspect, comprising specifically bind PD1 the first antigen-binding domains and specific binding LAG3 the The bispecific antibody of two antigen-binding domains is divalent.This means that bispecific antibody includes specific binding PD1 An antigen-binding domains (1+1 form) for one antigen-binding domains and specific binding LAG3.

In one aspect, a kind of bispecific antibody is provided, it includes the first antigen binding knots of specific binding PD1 Second antigen-binding domains in structure domain and specific binding LAG3, wherein bispecific antibody includes Fc structural domain, the first Fab Segment and the 2nd Fab segment, antigen-binding domains of the first Fab segment comprising specific binding PD1, described second Fab segment includes the antigen-binding domains of specific binding LAG3.In a particular aspects, in one in Fab segment, Variable domains VL and VH is substituted for one another, so that VH structural domain is a part of light chain and VL structural domain is a part of heavy chain. In a particular aspects, in the first Fab segment of the antigen-binding domains comprising specifically binding PD1, variable domains VL and VH are substituted for one another.

(b) the first heavy chain, the first light chain, the second heavy chain and the second light chain, first heavy chain include and SEQ ID NO: Sequence shown in 96 has the amino acid sequence of at least 95% sequence identity, and first light chain includes and SEQ ID NO:98 Shown in sequence have at least 95% sequence identity amino acid sequence, second heavy chain include and SEQ ID NO:100 Shown in sequence have at least 95% sequence identity amino acid sequence, second light chain include and SEQ ID NO:101 Shown in sequence have at least 95% sequence identity amino acid sequence, or

More specifically, bispecific antibody includes

(a) the first heavy chain, the first light chain, the second heavy chain and the second light chain, first heavy chain include SEQ ID NO:96 Shown in amino acid sequence, first light chain include SEQ ID NO:98 shown in amino acid sequence, the second heavy chain packet Amino acid sequence shown in the NO:97 of ID containing SEQ, second light chain include amino acid sequence shown in SEQ ID NO:99, Or

(b) the first heavy chain, the first light chain, the second heavy chain and the second light chain, first heavy chain include SEQ ID NO:96 Shown in amino acid sequence, first light chain include SEQ ID NO:98 shown in amino acid sequence, the second heavy chain packet Amino acid sequence shown in the NO:100 of ID containing SEQ, second light chain include amino acid sequence shown in SEQ ID NO:101 Column, or

(c) the first heavy chain, the first light chain, the second heavy chain and the second light chain, first heavy chain include SEQ ID NO:102 Shown in amino acid sequence, first light chain include SEQ ID NO:104 shown in amino acid sequence, the second heavy chain packet Amino acid sequence shown in the NO:103 of ID containing SEQ, second light chain include amino acid sequence shown in SEQ ID NO:105 Column, or

(d) the first heavy chain, the first light chain, the second heavy chain and the second light chain, first heavy chain include SEQ ID NO:106 Shown in amino acid sequence, first light chain include SEQ ID NO:107 shown in amino acid sequence, the second heavy chain packet Amino acid sequence shown in the NO:103 of ID containing SEQ, second light chain include amino acid sequence shown in SEQ ID NO:105 Column.

More specifically, bispecific antibody includes the first heavy chain, the first light chain, the second heavy chain and the second light chain, described the One heavy chain includes amino acid sequence shown in SEQ ID NO:96, and first light chain includes amino shown in SEQ ID NO:98 Acid sequence, second heavy chain include amino acid sequence shown in SEQ ID NO:100, and second light chain includes SEQ ID Amino acid sequence shown in NO:101.

On the other hand, a kind of bispecific antibody is provided, it includes the first antigen bindings of specific binding PD1 Second antigen-binding domains of structural domain and specific binding LAG3, wherein bispecific antibody includes Fc structural domain, first Fab segment and the 2nd Fab segment, the first Fab segment include the antigen-binding domains of specific binding PD1, and described the Two Fab segments include the antigen-binding domains of specific binding LAG3, the C-terminal of the 2nd the Fab segment and Fc structural domain Fusion.Specifically, the Fab segment of the antigen-binding domains comprising specific binding LAG3 is via its VH structural domain and Fc structure The C-terminal in domain merges (trans- 1+1 form).

In a particular aspects, bispecific antibody includes the first heavy chain, the first light chain, the second heavy chain and the second light chain, First heavy chain includes the amino acid sequence for having at least 95% sequence identity with sequence shown in SEQ ID NO:96, institute Stating the first light chain includes the amino acid sequence for having at least 95% sequence identity with sequence shown in SEQ ID NO:98, described Second heavy chain includes the amino acid sequence for having at least 95% sequence identity with sequence shown in SEQ ID NO:144, described Second light chain includes the amino acid sequence for having at least 95% sequence identity with sequence shown in SEQ ID NO:101.More Body, bispecific antibody includes the first heavy chain, the first light chain, the second heavy chain and the second light chain, and first heavy chain includes SEQ Amino acid sequence shown in ID NO:96, first light chain include amino acid sequence shown in SEQ ID NO:98, and described the Two heavy chains include amino acid sequence shown in SEQ ID NO:144, and second light chain includes ammonia shown in SEQ ID NO:101 Base acid sequence.

On the other hand, a kind of bispecific antibody is provided, it includes the first antigen bindings of specific binding PD1 Second antigen-binding domains of structural domain and specific binding LAG3, wherein bispecific antibody includes Fc structural domain, first Fab segment, the 2nd Fab segment and the 3rd Fab segment, the first Fab segment include the antigen binding knot of specific binding PD1 Structure domain, the 2nd Fab segment include the antigen-binding domains of specific binding LAG3, and the 3rd Fab segment includes spy The opposite sex combines the antigen-binding domains of LAG3.In a particular aspects, the antigen-binding domains comprising specifically binding PD1 Fab segment merged via peptide linker with one C-terminal in heavy chain.

In this regard, bispecific antibody is trivalent, wherein divalent combination LAG3 and monovalence combination PD1.This means double Specific antibody includes two antigen bindings of an antigen-binding domains and specific binding LAG3 of specific binding PD1 Structural domain (2+1 form).

(a) the first heavy chain, the first light chain, the second heavy chain and two the second light chains,

(b) the first heavy chain, the first light chain, the second heavy chain and two the second light chains,

More specifically, bispecific antibody includes

(a) the first heavy chain, the first light chain, the second heavy chain and two the second light chains, first heavy chain include SEQ ID Amino acid sequence shown in NO:118, first light chain include SEQ ID NO:115 shown in amino acid sequence, described second Heavy chain includes amino acid sequence shown in SEQ ID NO:119, and two second light chains include shown in SEQ ID NO:101 Amino acid sequence, or

(b) the first heavy chain, the first light chain, the second heavy chain and two the second light chains, first heavy chain include SEQ ID Amino acid sequence shown in NO:120, first light chain include SEQ ID NO:115 shown in amino acid sequence, described second Heavy chain includes amino acid sequence shown in SEQ ID NO:121, and two second light chains include shown in SEQ ID NO:99 Amino acid sequence, or

(c) the first heavy chain, the first light chain, the second heavy chain and two the second light chains, first heavy chain include SEQ ID Amino acid sequence shown in NO:122, first light chain include SEQ ID NO:115 shown in amino acid sequence, described second Heavy chain includes amino acid sequence shown in SEQ ID NO:103, and two second light chains include shown in SEQ ID NO:105 Amino acid sequence.

On the other hand, a kind of bispecific antibody is provided, it includes the first antigen bindings of specific binding PD1 Second antigen-binding domains of structural domain and specific binding LAG3, wherein bispecific antibody includes Fc structural domain, first Fab segment, the 2nd Fab segment and the 3rd Fab segment, the first Fab segment include the antigen binding knot of specific binding PD1 Structure domain, the 2nd Fab segment include the antigen-binding domains of specific binding LAG3, and the 3rd Fab segment includes spy The opposite sex combines the antigen-binding domains of LAG3, wherein the Fab segment of the antigen-binding domains comprising specific binding LAG3 In one (trans- 2+1 form) is merged with one C-terminal in heavy chain via peptide linker.

In a particular aspects, a kind of bispecific antibody is provided, it includes the first antigen knots of specific binding PD1 It closes structural domain and specifically binds the second antigen-binding domains of LAG3, wherein bispecific antibody includes the first heavy chain, the One light chain, the second heavy chain and two the second light chains, first heavy chain include to have extremely with sequence shown in SEQ ID NO:96 The amino acid sequence of few 95% sequence identity, first light chain include to have at least with sequence shown in SEQ ID NO:98 The amino acid sequence of 95% sequence identity, second heavy chain include to have at least with sequence shown in SEQ ID NO:145 The amino acid sequence of 95% sequence identity, two second light chains include to have with sequence shown in SEQ ID NO:101 At least amino acid sequence of 95% sequence identity.More specifically, bispecific antibody includes the first heavy chain, the first light chain, the Two heavy chains and two the second light chains, first heavy chain include to have at least 95% sequence with sequence shown in SEQ ID NO:96 The amino acid sequence of consistency, first light chain include to have at least 95% sequence one with sequence shown in SEQ ID NO:98 The amino acid sequence of cause property, second heavy chain include to have at least 95% sequence one with sequence shown in SEQ ID NO:145 The amino acid sequence of cause property, two second light chains include to have at least 95% sequence with sequence shown in SEQ ID NO:101 The amino acid sequence of column consistency.

On the other hand, a kind of bispecific antibody is provided, it includes the first antigen bindings of specific binding PD1 Second antigen-binding domains of structural domain and specific binding LAG3, wherein bispecific antibody includes Fc structural domain, two Fab segment and single chain Fab (scFab), described two Fab segments respectively contain the antigen binding structure of specific binding LAG3 Domain, the single chain Fab (scFab) include the antigen-binding domains of specific binding PD1.Specifically, comprising specific binding The scFab of the antigen-binding domains of PD1 is merged via peptide linker with one C-terminal in the heavy chain.

More specifically, bispecific antibody includes

(a) the first heavy chain, the second heavy chain and two light chains, first heavy chain include ammonia shown in SEQ ID NO:123 Base acid sequence, second heavy chain include amino acid sequence shown in SEQ ID NO:119, and the two light chains include SEQ Amino acid sequence shown in ID NO:101, or

(b) the first heavy chain, the second heavy chain and two light chains, first heavy chain include ammonia shown in SEQ ID NO:124 Base acid sequence, second heavy chain include amino acid sequence shown in SEQ ID NO:121, and the two light chains include SEQ Amino acid sequence shown in ID NO:99, or

(c) the first heavy chain, the second heavy chain and two light chains, first heavy chain include ammonia shown in SEQ ID NO:125 Base acid sequence, second heavy chain include amino acid sequence shown in SEQ ID NO:103, and the two light chains include SEQ Amino acid sequence shown in ID NO:105.

In this regard, bispecific antibody is tetravalence, wherein divalent combination LAG3 and divalent combination PD1.This means double Specific antibody includes two antigen bindings of two antigen-binding domains and specific binding LAG3 of specific binding PD1 Structural domain (2+2 form).

In one aspect, bispecific antibody of the invention includes

(a) two light chains and two heavy chains of the antibody comprising two Fab segments, the Fab segment include specificity knot The antigen-binding domains of LAG3 are closed, and

(b) two other Fab segments of the antigen-binding domains comprising specific binding PD1, wherein described two Other Fab segment is respectively connect via peptide linker with the C-terminal of the heavy chain of (a).

In a particular aspects, peptide linker is (G₄S)₄.On the other hand, the antigen binding comprising specific binding PD1 The other Fab segment of two of structural domain is crossover Fab segment, and wherein variable domains VL and VH is substituted for one another and VL- CH chain is respectively connect via peptide linker with the C-terminal of the heavy chain of (a).

In one aspect, a kind of bispecific antibody is provided, it includes the first antigen binding knots of specific binding PD1 Second antigen-binding domains in structure domain and specific binding LAG3, wherein respectively containing the antigen binding of specific binding PD1 Two Fab segments of structural domain are respectively merged via peptide linker with one C-terminal in heavy chain.

More specifically, bispecific antibody includes

(a) two heavy chains, two the first light chains and two the second light chains, two heavy chains include SEQ ID NO: Amino acid sequence shown in 114, two first light chains include amino acid sequence shown in SEQ ID NO:115, described Two the second light chains include amino acid sequence shown in SEQ ID NO:101, or

(b) two heavy chains, two the first light chains and two the second light chains, two heavy chains include SEQ ID NO: Amino acid sequence shown in 116, two first light chains include amino acid sequence shown in SEQ ID NO:115, described Two the second light chains include amino acid sequence shown in SEQ ID NO:99, or

(c) two heavy chains, two the first light chains and two the second light chains, two heavy chains include SEQ ID NO: Amino acid sequence shown in 117, two first light chains include amino acid sequence shown in SEQ ID NO:115, described Two the second light chains include amino acid sequence shown in SEQ ID NO:105.

Reduce the combination of Fc receptor and/or the Fc structural domain modification of effector function

In some aspects, a kind of bispecific antibody is provided, it includes the first antigen binding knots of specific binding PD1 Second antigen-binding domains in structure domain and specific binding LAG3, wherein bispecific antibody includes Fc structural domain, the Fc Structural domain includes one or more amino acid modifications, and one or more of amino acid modifications reduce and the combination of Fc receptor, tool It is and the combination of Fc γ receptor to body, and reduces or eliminates effector function.

In some aspects, one or more amino acid modifications can be incorporated herein in the area Fc of the antibody of offer, to generate Fc region variants.Fc region variants may include people Fc region sequence (such as human IgG1, the area IgG2, IgG3 or IgG4 Fc), at one or Include amino acid modification (such as substitution) on multiple amino acid positions.

Following sections description is modified of the invention comprising the Fc structural domain of the combination of reduction Fc receptor and/or effector function The preferred aspect of bispecific antigen binding molecules.In one aspect, anti-the present invention relates to first comprising specifically binding PD1 The bispecific antibody of the second antigen-binding domains of former binding structural domain and specific binding LAG3, wherein Fc structural domain packet Containing one or more amino acid substitutions, one or more of amino acid substitutions reduce and the combination of Fc receptor, specifically with The combination of Fc γ receptor.Specifically, Fc structural domain belongs to human IgG1's subclass, has amino acid mutation L234A, L235A and P329G (according to Kabat EU index number).

Fc structural domain assigns advantageous pharmacokinetic properties to bispecific antibody of the invention, including facilitates target group The long serum half-life well gathered and advantageous tissue-blood distribution ratio in knitting.However, at the same time, may cause this The cell of the bispecific antibody of invention undesirably targeted expression Fc receptor, rather than preferred antigen bearing cell.Therefore, In a particular embodiment, with natural IgG Fc structural domain, specifically IgG1 Fc structural domain or IgG4 Fc structural domain are compared, this The Fc structural domain of the bispecific antibody of invention shows the binding affinity and/or reduced effector to Fc receptor reduced Function.More specifically, Fc structural domain is IgG1 Fc structural domain.

At a this aspect, (or include the of the invention of natural IgG1 Fc structural domain with natural IgG1 Fc structural domain Bispecific antigen binding molecules) it compares, the Fc structural domain (or the bispecific of the invention comprising the Fc structural domain is anti- Former binding molecule) show to the binding affinity of Fc receptor less than 50%, preferably less than 20%, more preferably less than 10% and most preferably less than 5%；And/or with natural IgG1 Fc structural domain (or this hair comprising natural IgG1 Fc structural domain Bright bispecific antigen binding molecules) it compares, the Fc structural domain (or it is of the invention double special comprising the Fc structural domain Property antigen binding molecules) show effector function less than 50%, preferably less than 20%, more preferably less than 10% and most Preferably less than 5%.In one aspect, Fc structural domain (or the bispecific antigen knot of the invention comprising the Fc structural domain Close molecule) Fc receptor and/or inductive effect subfunction are not combined significantly.In a particular aspects, Fc receptor is Fc γ receptor.In On one side, Fc receptor is people's Fc receptor.In one aspect, Fc receptor is the Fc receptor of activation.In a specific aspect, Fc by Body is the human Fc gamma receptor of activation, and more specifically people Fc γ RIIIa, Fc γ RI or Fc γ RIIa, most specifically are people Fc γ RIIIa.In one aspect, Fc receptor is inhibition Fc receptor.In a specific aspect, Fc receptor be inhibition people Fc γ by Body, more specifically people Fc γ RIIB.In one aspect, effector function is in CDC, ADCC, ADCP and cytokine secretion One or more.In a particular aspects, effector function is ADCC.In one aspect, with natural IgG1 Fc structural domain phase Than Fc structural domain shows the substantially similar binding affinity to neonatal Fc receptor.When Fc structural domain is (or comprising described The bispecific antigen binding molecules of the invention of Fc structural domain) natural IgG1 Fc structural domain is shown (or comprising natural IgG1 The bispecific antigen binding molecules of the invention of Fc structural domain) to greater than about the 70% of the binding affinity of FcRn, specifically Greater than about 80%, when being more specifically greater than about 90%, substantially similar and FcRn combination is realized.

In a particular aspects, Fc structural domain is by engineered, compared with non-engineered Fc structural domain The binding affinity and/or reduced effector function to Fc receptor with reduction.In a particular aspects, of the invention is double The Fc structural domain of specific antigen binding molecule includes to reduce Fc structural domain to the binding affinity and/or effector function of Fc receptor One or more amino acid mutations of energy.Typically, identical one or more amino acid mutations are present in the two of Fc structural domain In each of a subunit.In one aspect, amino acid mutation reduces Fc structural domain to the binding affinity of Fc receptor.Another On one side, binding affinity of the Fc structural domain to Fc receptor is reduced at least 2 times, at least 5 times or at least 10 by amino acid mutation Times.It in one aspect, include engineering compared with the bispecific antibody of the invention comprising non-engineered Fc structural domain The bispecific antigen binding molecules of the invention for changing the Fc structural domain of transformation show lacking to the binding affinity of Fc receptor In 20%, more specifically less than 10%, more specifically less than 5%.In a particular aspects, Fc receptor is Fc γ receptor.At other Aspect, Fc receptor are people's Fc receptors.In one aspect, Fc receptor is inhibition Fc receptor.In a specific aspect, Fc receptor is Inhibition human Fc gamma receptor, more specifically people Fc γ RIIB.In some respects, Fc receptor is the Fc receptor of activation.At one Specific aspect, Fc receptor be activation human Fc gamma receptor, more specifically people Fc γ RIIIa, Fc γ RI or Fc γ RIIa, most It is body people Fc γ RIIIa.Preferably, it is reduced with the combination of each of these receptors.In some respects, to complement components Binding affinity, specifically the binding affinity of C1q is also reduced.In one aspect, to neonatal Fc receptor (FcRn) Binding affinity does not reduce.When Fc structural domain (or bispecific antigen binding molecules of the invention comprising the Fc structural domain) Show non-engineered form (or the present invention of the non-engineered form comprising Fc structural domain of Fc structural domain Bispecific antigen binding molecules) to greater than about the 70% of the binding affinity of FcRn when, realize it is substantially similar with The combination of FcRn realizes reservation of the Fc structural domain to the binding affinity of the receptor.Fc structural domain includes the Fc The bispecific antigen binding molecules of the invention of structural domain can express greater than about the 80% of this affinity or be even greater than About 90%.In certain embodiments, the Fc structural domain of bispecific antigen binding molecules of the invention is engineered transformation, with phase Than there is reduced effector function in non-engineered Fc structural domain.Reduced effector function may include but unlimited In one of the following or multiple: reduced complement-dependent cytotoxicity (CDC), the antibody dependent cellular mediation that reduces Cytotoxicity (ADCC), reduce antibody dependent cellular phagocytosis (ADCP), the cytokine secretion of reduction, reduction it is immune multiple Close object mediate antigen presenting cell antigen uptake, reduce and NK cell combination, reduce and macrophage combination, Reduce and monocyte combination, reduce and polymorphonuclear cell combination, reduction direct signal conduct Inducing Cells The T cell of apoptosis, the dendritic cell maturation of reduction or reduction causes.

Antibody with reduced effector function include have the one or more area Fc residue 238,265,269,270, 297, those of 327 and 329 substitution (United States Patent (USP) No.6,737,056).Such Fc mutant is included in two or more 265, there is the Fc mutant replaced, including so-called " DANA " Fc mutant at 269,270,297 and 327 amino acids, Residue 265 and 297 is substituted by alanine (United States Patent (USP) No.7,332,581).Describe with improve or reduction with Certain antibody variants of the combination of FcR.(such as United States Patent (USP) No.6,737,056；WO 2004/056312 and Shields, R.L. et al., J.Biol.Chem.276 (2001) 6591-6604).

In one aspect of the invention, Fc structural domain includes ammonia at the position E233, L234, L235, N297, P331 and P329 Base acid replaces.In some respects, Fc structural domain includes amino acid substitution L234A and L235A (" LALA ").It is real as one It applies in example, Fc structural domain is IgG1 Fc structural domain, in particular human IgG1 Fc structural domain.In one aspect, Fc structural domain exists It include amino acid substitution at P329.At a more specific aspect, amino acid substitution is P329A or P329G, in particular P329G.In one embodiment, Fc structural domain at the P329 of position comprising amino acid substitution and include selected from by E233P, The other amino acid substitution of the group of L234A, L235A, L235E, N297A, N297D or P331S composition.In implementation particularly In example, Fc structural domain includes amino acid mutation L234A, L235A and P329G (" P329G LALA ").Amino acid substitution The Fc γ receptor that human IgG1 Fc structural domain is almost eliminated in " P329G LALA " combination combines, such as PCT Patent Application No.WO Described in 2012/130831 A1.The document also describes the method for preparing such mutation Fc structural domain and for determining its spy Property (such as Fc receptor combine or effector function) method.This antibody is that with mutation L234A and L235A or have mutation The IgG1 of L234A, L235A and P329G are (according to the EU index number of Kabat et al., Kabat et al., Sequences of Proteins of Immunological Interest, the 5th edition Public Health Service, National Institutes of Health,Bethesda,MD,1991)。

In one aspect, bispecific antibody of the invention is appointed comprising (EU index of all positions all in accordance with Kabat) (i) Selection of land has the area homodimer Fc of human IgG1's subclass of mutation P329G, L234A and L235A, or (ii) optionally has mutation The area homodimer Fc of 4 subclass of human IgG of P329G, S228P and L235E, or (iii) optionally have mutation P329G, L234A, L235A, I253A, H310A and H435A, or optionally there is mutation P329G, L234A, L235A, H310A, H433A With the area homodimer Fc of human IgG1's subclass of Y436A, or the area (iv) heterodimer Fc, one of area Fc polypeptide includes mutation T366W and another area Fc polypeptide include mutation T 366S, L368A and Y407V or one of area Fc polypeptide includes prominent Become T366W and Y349C and another area Fc polypeptide include mutation T 366S, L368A, Y407V and S354C or one of them The area Fc polypeptide include mutation T 366W and S354C and another area Fc polypeptide include mutation T 366S, L368A, Y407V and Y349C, or the area heterodimer Fc of human IgG1's subclass (v), the area two of them Fc polypeptide include mutation P329G, L234A and L235A, and the area Fc polypeptide includes mutation T 366W, and another area Fc polypeptide include mutation T 366S, L368A and Y407V；Or in which the area Fc polypeptide includes mutation T 366W and Y349C, and another area Fc polypeptide include mutation T 366S, L368A, Y407V and S354C；Or in which the area a Fc polypeptide includes mutation T 366W and S354C, and another area Fc polypeptide Include mutation T 366S, L368A, Y407V and Y349C.

In one aspect, Fc structural domain is IgG4 Fc structural domain.In a more specific embodiment, Fc structural domain is (Kabat number) includes amino acid substitution at the position S228, specifically the IgG4 Fc structural domain of amino acid substitution S228P.In In one more specific embodiment, Fc structural domain is that the IgG4 Fc comprising amino acid substitution L235E and S228P and P329G is tied Structure domain.The internal Fab arm that this amino acid substitution reduces IgG4 antibody exchanges (referring to Stubenrauch et al., Drug Metabolism and Disposition 38,84-91(2010)).Therefore, in one aspect, a kind of bispecific is provided Antibody, it includes the area heterodimer Fc of 4 subclass of (EU index of all positions all in accordance with Kabat) human IgG, the areas two of them Fc Polypeptide includes mutation P329G, S228P and L235E and the area Fc polypeptide includes mutation T 366W and another area Fc polypeptide Include mutation T 366W and Y349C and another area Fc comprising mutation T 366S, L368A and Y407V or in which the area Fc polypeptide Polypeptide includes mutation T 366S, L368A, Y407V and S354C or in which the area Fc polypeptide includes mutation T 366W and S354C and Another area Fc polypeptide includes mutation T 366S, L368A, Y407V and Y349C.

It combined with extended half-life period and improved neonatal Fc receptor (FcRn), be responsible for Maternal immunoglobulin G being transferred to tire Youngster (Guyer, R.L. et al., J.Immunol.117 (1976) 587-593 and Kim, J.K. et al., J.Immunol.24 (1994) 2429-2434) antibody be described in US 2005/0014934.Those antibody include such area Fc, the area Fc In there are the one or more of the combination for improving the area Fc and FcRn to replace.Such Fc variant includes one in the following area Fc residue Place or many places have the Fc variant replaced: 238,256,265,272,286,303,305,307,311,312,317,340,356, 360,362,376,378,380,382,413,424 or 434, for example, to the substitution of the area Fc residue 434 (United States Patent (USP) No.7, 371,826).About other examples of Fc region variants, Duncan, A.R. and Winter, G., Nature 322 (1988) are seen also 738-740；US 5,648,260；US 5,624,821；And WO 94/29351.

And the combination of Fc receptor can be led to for example using reference instrument (such as BIAcore instrument (GE Healthcare)) It crosses ELISA or is readily determined by surface plasma body resonant vibration (SPR), and Fc receptor can such as be obtained by recombinant expression .This document describes suitable such binding assays.Alternatively, the cell line of the known specific Fc receptor of expression can be used (such as Express the NK cells of human beings of III a receptor of Fc γ) assess Fc structural domain or the cell-stimulating bispecific antigen comprising Fc structural domain Binding affinity of the binding molecule to Fc receptor.The effector function of Fc structural domain or pair comprising Fc structural domain of the invention Specific antibody can be measured by methods known in the art.This document describes the suitable measurements for measuring ADCC.With United States Patent (USP) No 5,500,362 is described in other examples of the external test for the ADCC activity for evaluating interested molecule； Hellstrom et al., Proc Natl Acad Sci USA 83,7059-7063 (1986) and Hellstrom et al., Proc Natl Acad Sci USA 82,1499-1502(1985)；United States Patent (USP) No 5,821,337；Bruggemann et al., J Exp Med 166,1351-1361(1987).Alternatively, on-radiation measuring method can be used (see, for example, thin for streaming The ACTI of born of the same parents' art^TMNon-radioactive cell toxicity test (CellTechnology, Inc.Mountain View, CA)；And CytoToxNon-radioactive cell toxicity test (state of Wisconsin, Madison, Pu Luomaige company (Promega, Madison,WI)).Useful effector cell for such measurement includes peripheral blood mononuclear cells (PBMC) and natural kill (NK) Cell.It alternately or in addition, can be for example such as in Clynes et al., Proc Natl Acad Sci USA 95,652- The ADCC activity of interested molecule is evaluated in animal model disclosed in 656 (1998) in vivo.

Following section describes comprising reducing the present invention that Fc receptor combines and/or the Fc structural domain of effector function is modified Bispecific antibody preferred aspect.In an aspect, the present invention relates to bispecific antibodies, and it includes specific bindings The first antigen-binding domains of PD1 and the second antigen-binding domains of specific binding LAG3, wherein the Fc structural domain Comprising one or more amino acid substitutions, one or more of amino acid substitutions reduce antibody and the combination of Fc receptor is affine Power, the especially binding affinity to Fc γ receptor.In another aspect, the present invention relates to bispecific antibody, it includes The first antigen-binding domains of PD1 and the second antigen-binding domains of specific binding LAG3 are specifically bound, wherein institute Stating Fc structural domain includes the one or more amino acid substitutions for reducing effector function.In a particular aspect, the Fc structural domain is Human IgG1's subclass, have amino acid mutation L234A, L235A and P329G (according to Kabat EU index number).

Promote the Fc structural domain modification of Heterodimerization

Bispecific antigen binding molecules of the invention include and one or the other in two subunits of Fc structural domain The different antigen-binding domains of fusion, therefore two subunits of the Fc structural domain may include in two different polypeptide chains In.The recombinant co-expression of these polypeptides and subsequent dimerization result in several possible combinations of two kinds of polypeptides.In order in weight The yield and purity of bispecific antibody of the invention are improved in group production, therefore in bispecific antigen binding of the invention point The modification that the association of polypeptide needed for promoting is introduced in the Fc structural domain of son will be advantageous.

Therefore, at specific aspect, the present invention relates to a kind of bispecific antibody, it includes the of specific binding PD1 Second antigen-binding domains of one antigen-binding domains and specific binding LAG3, wherein the Fc structural domain includes to promote The modification of the association of first and second subunits of the Fc structural domain.It is widest between two subunits of human IgG Fc structural domain Protein-protein interaction site is in the CH3 structural domain of Fc structural domain.Therefore, in an aspect, the modification is tied in Fc In the CH3 structural domain in structure domain.

In a specific aspect, the modification is so-called " protrusion enters hole " modification, and the modification includes Fc structural domain " hole " modification in another in two subunits of " protrusion " modification and Fc structural domain in one in two subunits.Cause This, the present invention relates to a kind of bispecific antibodies, and it includes the first antigen-binding domains and specificity of specific binding PD1 In conjunction with the second antigen binding site of LAG3, wherein entering hole method according to protrusion, the first subunit of the Fc structural domain includes prominent Rise and the second subunit of the Fc structural domain include hole.In a particular aspects, the first subunit of Fc structural domain includes amino Acid replace S354C and T366W (EU number) and the second subunit of Fc structural domain comprising amino acid substitution Y349C, T366S and Y407V (according to Kabat EU index number).

Protrusion enters hole technical description in such as US 5,731,168；US 7,695,936；Ridgway et al., Prot Eng In 9,617-621 (1996) and Carter, J Immunol Meth 248,7-15 (2001).In general, this method is related to first The interface of polypeptide is introduced into raised (" protrusion ") and introduces corresponding cavity (" hole ") in the interface of the second polypeptide, so that described Protrusion can be positioned in the cavity, to promote the formation of heterodimer and to hinder the formation of homodimer.Protrusion It is the structure and small amino acid side chains with interface of larger side chain (such as the tyrosine or tryptophan) substitution from the first polypeptide It builds.With the compensation cavity with the same or similar size of protrusion be by with lesser amino acid side chain (such as alanine or Threonine) replace big amino acid side chains and creates in the interface of the second polypeptide.

Therefore, in one aspect, in the first subunit of the Fc structural domain of bispecific antigen binding molecules of the invention In CH3 structural domain, certain amino acid residue is replaced by the amino acid residue with larger side-chain bulk, thus in the first subunit Protrusion is generated in CH3 structural domain, the protrusion can be positioned in the cavity in the CH3 structural domain of the second subunit；And in Fc structure In the CH3 structural domain of second subunit in domain, certain amino acid residue is replaced by the amino acid residue with smaller side chain volume, from And cavity is generated in the CH3 structural domain of the second subunit, the protrusion in the CH3 structural domain of the first subunit can be positioned on described In cavity.Protrusion and cavity can be prepared by changing the nucleic acid of coding polypeptide, such as by site-specific mutagenesis or be led to Cross peptide synthesis.In In a specific aspect, in the CH3 structural domain of the first subunit of Fc structural domain, the threonine at 366 is residual Base is replaced by trp residue (T366W), and the tyrosine in the CH3 structural domain of the second subunit of Fc structural domain, at 407 Residue is replaced by valine residue (Y407V).In an aspect, in addition in the second subunit of Fc structural domain, at 366 Threonine residues are replaced by serine residue (T366S), and the leucine residue at 368 is by alanine residue (L368A) Replacement.

In another aspect, in addition in the first subunit of Fc structural domain, the serine residue at 354 is by half Guang ammonia Sour residue (S354C) replacement, and in addition in the second subunit of Fc structural domain, 349 tyrosine residues are by cysteine Residue (Y349C) replacement.Introducing the two cysteine residues causes to form disulphide bridges between two subunits of Fc structural domain, To further stabilize dimer (Carter (2001), J Immunol Methods 248,7-15).In a certain party In face, the first subunit of Fc structural domain second subunit of Fc structural domain comprising amino acid substitution S354C and T366W (EU number) Include amino acid substitution Y349C, T366S and Y407V (according to Kabat EU index number).

But it is also possible to alternately or in addition enter hole technology using other protrusions as described in EP 1 870 459.In In one embodiment, multi-specificity antibody includes the mutation R409D and K370E in the CH3 structural domain of " protrusion chain ", and " hole Mutation D399K and E357K in the CH3 structural domain of chain " (according to Kabat EU index number).

In an aspect, bispecific antibody includes T366W mutation and " pore chain " in the CH3 structural domain of " protrusion chain " CH3 structural domain in mutation T 366S, L368A and Y407V, and the in addition mutation in the CH3 structural domain of " protrusion chain " Mutation D399K and E357K in the CH3 structural domain of R409D and K370E and " pore chain " (according to Kabat EU index number).

In an aspect, bispecific antibody includes the mutation Y349C in one in described two CH3 structural domains Mutation S354C, T366S, L368A and Y407V in another with T366W and in described two CH3 structural domains, Huo Zheduo Specific antibody includes mutation Y349C and T366W in one in described two CH3 structural domains and in described two CH3 Mutation S354C, T366S, L368A and Y407V in another in structural domain and in addition in the CH3 structural domain of " protrusion chain " In mutation R409D and K370E and mutation D399K and E357K in the CH3 structural domain of " pore chain " (according to Kabat EU rope Draw number).

In another aspect, the modification for promoting the first subunit and the second subunit association of Fc structural domain includes mediating electrostatic The modification of steering effect, such as described in PCT Publication WO 2009/089004.In general, this method is related to electrically charged Amino acid residue replaces one or more amino acid residues of the interface of two Fc structural domain subunits, so that the homologous dimerization bodily form At becoming unfavorable on electrostatic, but Heterodimerization is advantageous on electrostatic.

It is different to implement for modifying the CH3 structural domain of heavy chain of multi-specificity antibody other than " protrusion enters hole technology " The other technologies of source dimerization are also as known in the art.These technologies, especially in WO 96/27011, WO 98/ 050431、EP 1870459、WO 2007/110205、WO 2007/147901、WO 2009/089004、WO 2010/ 129304, WO 2011/90754, WO 2011/143545, WO 2012/058768, WO 2013/157954 and WO 2013/ Technology described in 096291 is considered herein as the combined alternative of " protrusion enters hole technology " Yu bispecific antibody Case.

In an aspect, it in bispecific antibody, is supported using method described in EP 1870459 mostly special Property the first heavy chain of antibody and the Heterodimerization of the second heavy chain.This method is based on the CH3/ between the first and second heavy chains The Charged acids of oppositely charged are introduced at specific amino acids position in CH3- structural domain-interface.

Therefore, in this aspect of the tertiary structure of multi-specificity antibody, the CH3 structural domain and the second heavy chain of the first heavy chain CH3 structural domain form interface between corresponding antibody CH3 structural domain, wherein the phase of the CH3 structural domain of the first heavy chain The amino acid sequence of the CH3 structural domain of amino acid sequence and the second heavy chain is answered to respectively contain in the tertiary structure of the antibody The interface in one group of amino acid, wherein from be located at a heavy chain CH3 structural domain in interface in one group of ammonia Base acid, the first amino acid is by positively charged amino acid substitution, and the interface from positioned at the CH3 structural domain of another heavy chain In one group of amino acid, the second amino acid is by negatively charged amino acid substitution.According to the bispecific antibody of this aspect Referred to herein as " bispecific antibody of (+/-) engineering of CH3 " (" +/- " represent in corresponding CH3 structural domain of wherein abridging The amino acid of the oppositely charged of middle introducing).

In an aspect, in the bispecific antibody of (+/-) engineering of CH3, positively charged amino acid is selected from K, R And H, and negatively charged amino acid is selected from E or D.

In an aspect, in the bispecific antibody of (+/-) engineering of CH3, positively charged amino acid be selected from K and R, and negatively charged amino acid is selected from E or D.

In an aspect, in the bispecific antibody of (+/-) engineering of CH3, positively charged amino acid is K, and Negatively charged amino acid is E.

In an aspect, in the bispecific antibody of (+/-) engineering of the CH3 in the CH3 structural domain of a heavy chain, Amino acid R at 409 is replaced by D and the amino acid K at position is replaced by E, and in the CH3 structural domain of another heavy chain, 399 amino acid D are replaced by K and the amino acid E at 357 is replaced by K (according to Kabat EU index number).

In an aspect, the first weight of multi-specificity antibody is supported using method described in WO 2013/157953 The Heterodimerization of chain and the second heavy chain.In one embodiment, the amino in the CH3 structural domain of a heavy chain, at 366 Sour T is replaced by K, and in the CH3 structural domain of another heavy chain, the amino acid L at 351 is replaced by D (according to Kabat EU rope Draw number).In another embodiment, in the CH3 structural domain of a heavy chain, amino acid T at 366 replaced by K and Amino acid L at 351 is replaced by K, and in the CH3 structural domain of another heavy chain, the amino acid L at 351 is replaced by D (according to Kabat EU index number).

In another aspect, in the CH3 structural domain of a heavy chain, amino acid T at 366 is replaced and 351 by K Amino acid L at position is replaced by K, and in the CH3 structural domain of another heavy chain, amino acid L at 351 replaced by D (according to Kabat EU index number).It is included in the CH3 structural domain of another heavy chain in addition, at least one of following substitution replaces: Amino acid Y at 349 is replaced by E, and the amino acid Y at 349 is replaced by D, and the amino acid L at 368 is replaced by E (according to Kabat EU index number).In one embodiment, the amino acid L at 368 is replaced by E (according to Kabat EU rope Draw number).

In an aspect, the first weight of multi-specificity antibody is supported using method described in WO 2012/058768 The Heterodimerization of chain and the second heavy chain.In an aspect, the amino acid in the CH3 structural domain of a heavy chain, at 351 L is replaced by Y and the amino acid Y at 407 is replaced by A, and the amino in the CH3 structural domain of another heavy chain, at 366 Sour T is replaced by A and the amino acid K at 409 is replaced by F (according to Kabat EU index number).In another embodiment, Other than above-mentioned substitution, in the CH3 structural domain of another heavy chain, amino acid (being initially T) at 411, at 399 Amino acid (being initially D), the amino acid (being initially S) at 400, the amino acid (being initially F) at 405, the ammonia at 390 At least one amino acid in amino acid (being initially K) at base acid (being initially N) and 392 is substituted (according to Kabat EU Index number).Preferred substitution is:

It (is compiled according to Kabat EU index with the amino acid T at amino acid substitution 411 selected from N, R, Q, K, D, E and W Number),

The amino acid D (according to Kabat EU index number) at amino acid substitution 399 selected from R, W, Y and K is used,

The amino acid S (according to Kabat EU index number) at amino acid substitution 400 selected from E, D, R and K is used,

It (is compiled according to Kabat EU index with the amino acid F at amino acid substitution 405 selected from I, M, T, S, V and W Number)；

With the amino acid N at amino acid substitution 390 selected from R, K and D (according to Kabat EU index number)；And

It (is compiled according to Kabat EU index with the amino acid K at amino acid substitution 392 selected from V, M, R, L, F and E Number).

In another aspect, bispecific antibody is engineered according to WO 2012/058768, i.e., in a weight In the CH3 structural domain of chain, the amino acid L at 351 is replaced by Y and the amino acid Y at 407 is replaced by A, and at another In the CH3 structural domain of heavy chain, the amino acid T at 366 is replaced by V and the amino acid K at 409 replaced by F (according to Kabat EU index number).In another embodiment of multi-specificity antibody, in the CH3 structural domain of a heavy chain, 407 Amino acid Y at position is replaced by A, and in the CH3 structural domain of another heavy chain, amino acid T at 366 replaced by A and Amino acid K at 409 is replaced by F (according to Kabat EU index number).In the last one above-described embodiment, at another In the CH3 structural domain of heavy chain, the amino acid K at 392 is replaced by E, and the amino acid T at 411 is replaced by E, the ammonia at 399 Base acid D is replaced by R, and the amino acid S at 400 is replaced by R (according to Kabat EU index number).

In an aspect, the first weight of multi-specificity antibody is supported using method described in WO 2011/143545 The Heterodimerization of chain and the second heavy chain.In an aspect, at 368 and/or 409 in the CH3 structural domain of two heavy chains Place introduces amino acid modification (according to Kabat EU index number).

In an aspect, the first weight of bispecific antibody is supported using method described in WO 2011/090762 The Heterodimerization of chain and the second heavy chain.WO 2011/090762 is related to being repaired according to the amino acid of " protrusion enters hole " (KiH) technology Decorations.In one embodiment, in the CH3 structural domain of a heavy chain, the amino acid T at 366 is replaced by W, and at another In the CH3 structural domain of heavy chain, the amino acid Y at 407 is replaced by A (according to Kabat EU index number).In another implementation In example, in the CH3 structural domain of a heavy chain, the amino acid T at 366 is replaced by Y, and in the CH3 structure of another heavy chain In domain, the amino acid Y at 407 is replaced by T (according to Kabat EU index number).

In an aspect, the first weight of bispecific antibody is supported using method described in WO 2009/089004 The Heterodimerization of chain and the second heavy chain.In one embodiment, the amino in the CH3 structural domain of a heavy chain, at 392 Sour K or N (is replaced by E or D in one embodiment, is taken in a preferred embodiment by D by negatively charged amino acid substitution Generation), and in the CH3 structural domain of another heavy chain, at amino acid D at 399, the amino acid E at 356 or D or 357 Amino acid E by positively charged amino acid substitution (replaced in one embodiment by K or R, in a preferred embodiment by K replaces, and in a preferred embodiment, the amino acid at 399 or 356 is replaced by K) it (is compiled according to Kabat EU index Number).In another embodiment, the amino acid other than above-mentioned substitution, in the CH3 structural domain of a heavy chain, at 409 K or R (is replaced by E or D in one embodiment, is taken in a preferred embodiment by D by negatively charged amino acid substitution Generation) (according to Kabat EU index number).In another aspect, it is replaced as above-mentioned substituted supplement or as above-mentioned substituted Generation, in the CH3 structural domain of a heavy chain, amino acid K at 439 and/or the amino acid K at 370 independently of one another by Negatively charged amino acid substitution (being replaced in one embodiment by E or D, replaced in a preferred embodiment by D) (according to Kabat EU index number).

In an aspect, the first weight of multi-specificity antibody is supported using method described in WO 2007/147901 The Heterodimerization of chain and the second heavy chain.In one embodiment, the amino in the CH3 structural domain of a heavy chain, at 253 Sour K is replaced by E, and the amino acid D at 282 is replaced by K, and 322 amino acid K are replaced by D, and in another heavy chain In CH3 structural domain, the amino acid D at 239 is replaced by K, and the amino acid E at 240 is replaced by K, and the amino at 292 Sour K is replaced by D (according to Kabat EU index number).

As the C-terminal of the heavy chain for the bispecific antibody reported herein can be the complete C terminated with amino acid residue PGK End.The C-terminal of heavy chain can be the C-terminal of shortening, and the end one or two C has been had been removed in the C-terminal of the shortening Terminal amino acid residue.In a preferred aspect, the C-terminal of heavy chain is the C-terminal of the shortening terminated with PG.

The one aspect in all aspects reported herein, fixed includes to contain C-terminal CH3 structural domain as mentioned in this article Heavy chain bispecific antibody, comprising C-terminal glycine-lysine dipeptides (G446 and K447, according to Kabat EU index volume Number).In the one embodiment in all aspects reported herein, fixed includes to contain C-terminal CH3 structural domain as mentioned in this article The bispecific antibody of heavy chain, comprising C-terminal glycine residue (G446, according to Kabat EU index number).

Modification in Fab structural domain

In an aspect, the present invention relates to a kind of bispecific antibodies, and it includes the first Fab of specific binding PD1 2nd Fab segment of segment and specific binding LAG3, wherein in one of the Fab segment, variable domains VH and VL Or constant domain CH1 and CL is exchanged.Bispecific antibody is prepared according to Crossmab technology.

In WO2009/080252, WO2009/080253 and Schaefer, W. et al., PNAS, 108 (2011) 11187- Being described in detail in a combination arm in 1191 has domain substitute/exchange multi-specificity antibody (CrossMabVH-VL Or CrossMabCH-CL).They considerably reduce the mispairing of the wrong heavy chain of light chain and anti-second antigen by resisting the first antigen Caused by-product (compared with the method for no such Domain swapping).

In In a specific aspect, the present invention relates to a kind of bispecific antibodies, and it includes the first of specific binding PD1 2nd Fab segment of Fab segment and specific binding LAG3, wherein in one in the Fab segment, variable domains VL Substituted for one another with VH, so that the VH structural domain is a part of light chain and the VL structural domain is a part of heavy chain.One In a specific aspect, bispecific antibody is such bispecific antibody, wherein in the antigen knot comprising specifically binding PD1 In the first Fab segment for closing structural domain, variable domains VL and VH is substituted for one another.

In another aspect, and in order to further improve correct pairing, the first Fab comprising specifically binding PD1 The bispecific antibody of segment and the 2nd Fab segment of specific binding LAG3 replaces (institute containing different Charged acids " charged residues " of meaning).These modifications are introduced into intersection or non-crossing CH1 and CL structural domain.Such as in WO2015/ 150447, such modification is described in WO2016/020309 and PCT/EP2016/073408.

In a particular aspects, the present invention relates to a kind of bispecific antibodies, and it includes the first of specific binding PD1 2nd Fab segment of Fab segment and specific binding LAG3, wherein in one of the Fab segment in constant domain CL, Amino acid at 124 is independently replaced by lysine (K), arginine (R) or histidine (H) (to be compiled according to Kabat EU index Number), and the amino acid in constant domain CH1 at 147 and 213 is independently taken by glutamic acid (E) or aspartic acid (D) Generation (according to Kabat EU index number).In a particular aspects, bispecific antibody is such bispecific antibody, In comprising specifically bind TIM3 antigen-binding domains the 2nd Fab segment in, in constant domain CL at 124 Amino acid independently replaced (according to Kabat EU index number) by lysine (K), arginine (R) or histidine (H), and In constant domain CH1, the amino acid at 147 and 213 independently replaces (root by glutamic acid (E) or aspartic acid (D) According to Kabat EU index number).

In a particular aspects, the present invention relates to a kind of bispecific antibodies, and it includes the first of specific binding PD1 2nd Fab segment of Fab segment and specific binding LAG3, wherein in one in CL structural domain, at 123 (EU numbers) Amino acid by arginine (R) replace and 124 (EU number) at amino acid by lysine (K) substitution, and Wherein in one of CH1 structural domain, the amino acid at 147 (EU numbers) and 213 (EU number) is by glutamic acid (E) Replace.In a particular aspects, bispecific antibody is such bispecific antibody, wherein including specific binding In 2nd Fab segment of the antigen-binding domains of LAG3, the amino acid at 123 (EU number) is replaced by arginine (R) And the amino acid at 124 (EU number) is replaced by lysine (K), and wherein in one in CH1 structural domain, 147 Position (EU number) and 213 (EU number) at amino acid by glutamic acid (E) substitution.

In another aspect, bispecific antibody is bivalent antibody, it includes

A) the first light chain and the first heavy chain of the antibody of the first antigen are specifically bound, and

B) the second light chain and the second heavy chain for specifically binding the antibody of the second antigen, wherein second light chain and described The variable domains VL and VH of second heavy chain are substituted for one another.

A) antibody under is free of just like the modification reported under b), and a) under heavy chain and light chain be isolated chain.

In the antibody under b), variable light chain domain VL is replaced by the variable heavy chain domain VH of the antibody in light chain It changes, and variable heavy chain domain VH is replaced by the variable light chain domain VL of the antibody in heavy chain.

In one aspect, (i) in the constant domain CL of the first light chain under a), at 124 (being numbered according to Kabat) Amino acid by positively charged amino acid substitution, and wherein in the constant domain CH1 of the first heavy chain under a), 147 Amino acid (according to Kabat EU index number) at the amino acid at place or 213 is by negatively charged amino acid substitution；Or (ii) in b) under the second light chain constant domain CL in, the amino acid at 124 (being numbered according to Kabat) is positively charged Amino acid substitution, and wherein in the constant domain CH1 of the second heavy chain under b), at amino acid at 147 or 213 Amino acid (according to Kabat EU index number) by negatively charged amino acid substitution.

In another aspect, (i) in the constant domain CL of the first light chain under a), the amino acid at 124 is relied Propylhomoserin (K), arginine (R) or histidine (H) independently replace (numbering according to Kabat) (in a preferred embodiment, quilt Lysine (K) or arginine (R) independently replace), and wherein in the constant domain CH1 of the first heavy chain under a), 147 Amino acid at the amino acid at place or 213 is independently replaced (according to Kabat EU rope by glutamic acid (E) or aspartic acid (D) Draw number)；Or (ii) in b) under the second light chain constant domain CL in, amino acid at 124 by lysine (K), Arginine (R) or histidine (H) independently replace (being numbered according to Kabat) (in a preferred embodiment, by lysine (K) or arginine (R) independently replaces), and the ammonia wherein in the constant domain CH1 of the second heavy chain under b), at 147 Base acid or the amino acid at 213 places are independently replaced by glutamic acid (E) or aspartic acid (D) (according to Kabat EU index volume Number).

In an aspect, in the constant domain CL of the second heavy chain, the amino acid at 124 and 123 is replaced by K (according to Kabat EU index number).

In an aspect, in the constant domain CL of the second heavy chain, amino acid at 123 is replaced and 124 by R Amino acid at position is replaced by K (according to Kabat EU index number).

In an aspect, in the constant domain CH1 of the second light chain, the amino acid at 147 and 213 is taken by E Generation (according to Kabat EU index number).

In an aspect, in the constant domain CL of the first light chain, the amino acid at 124 and 123 is taken by K Generation, and in the constant domain CH1 of the first heavy chain, the amino acid at 147 and 213 is replaced by E (according to Kabat EU rope Draw number).

In an aspect, in the constant domain CL of the first light chain, amino acid at 123 is replaced and 124 by R Amino acid at position is replaced by K, and in the constant domain CH1 of the first heavy chain, 147 and 213 amino acid is all taken by E Generation (according to Kabat EU index number).

In an aspect, in the constant domain CL of the second heavy chain, the amino acid at 124 and 123 is taken by K Generation；In the constant domain CH1 of the second light chain, the amino acid at 147 and 213 is replaced by E；In the variable of the first light chain In structural domain VL, the amino acid at 38 is replaced by K；In the variable domains VH of the first heavy chain, the amino acid at 39 is by E Replace；In the variable domains VL of the second heavy chain, the amino acid at 38 is replaced by K；And in the variable knot of the second light chain In the VH of structure domain, the amino acid at 39 is replaced by E (according to Kabat EU index number).

In an aspect, bispecific antibody is bivalent antibody, it includes

A) the first light chain and the first heavy chain of the antibody of the first antigen are specifically bound, and

B) the second light chain and the second heavy chain for specifically binding the antibody of the second antigen, wherein the second light chain and the second heavy chain Variable domains VL and VH it is substituted for one another, and wherein the constant domain CL and CH1 of the second light chain and the second heavy chain are replaced each other It changes.

A) antibody under is free of just like the modification reported under b), and a) under heavy chain and light chain be isolated chain.B) Under antibody in, in light chain, variable light chain domain VL is replaced by the variable heavy chain domain VH of the antibody, and constant light Hinge domain CL is replaced by the constant heavy domain C H1 of the antibody；In heavy chain, variable heavy chain domain VH is by described anti- The variable light chain domain VL of body is replaced, and constant heavy domain C H1 is replaced by the constant light domain C L of the antibody.

In an aspect, bispecific antibody is bivalent antibody, it includes

A) the first light chain and the first heavy chain of the antibody of the first antigen are specifically bound, and

B) the second light chain and the second heavy chain for specifically binding the antibody of the second antigen, wherein second light chain and described The constant domain CL and CH1 of second heavy chain are substituted for one another.

A) antibody under is free of just like the modification reported under b), and a) under heavy chain and light chain be isolated chain.B) Under antibody in, in light chain, constant light domain C L by the antibody constant heavy domain C H1 replace；And in weight In chain, constant heavy domain C H1 is replaced by the constant light domain C L of the antibody.

In an aspect, bispecific antibody is the bispecific antibody comprising the following terms

A) full length antibody specifically binds the first antigen and is made of two heavy chain of antibody and two antibody light chains；With And

B) one, two, three or four single chain Fab segment of the second antigen is specifically bound,

Wherein b) under the single chain Fab segment via in the heavy chain of the full length antibody or the C-terminal of light chain or N-terminal The peptide linker at place with a) under the full length antibody merge.

In an aspect, in conjunction with one or two identical single chain Fab segment of the second antigen via in the overall length Peptide linker at the heavy chain of antibody or the C-terminal of light chain is merged with the full length antibody.

In an aspect, in conjunction with one or two identical single chain Fab (scFab) segment of the second antigen via in institute The peptide linker at the C-terminal of the heavy chain of full length antibody is stated to merge with the full length antibody.

In an aspect, in conjunction with one or two identical single chain Fab (scFab) segment of the second antigen via in institute The peptide linker at the C-terminal of the light chain of full length antibody is stated to merge with the full length antibody.

In an aspect, in conjunction with two identical single chain Fab (scFab) segments of the second antigen via in the overall length Peptide linker at each heavy chain of antibody or the C-terminal of light chain is merged with the full length antibody.

In an aspect, in conjunction with two identical single chain Fab (scFab) segments of the second antigen via in the overall length Peptide linker at the C-terminal of each heavy chain of antibody is merged with the full length antibody.

In an aspect, in conjunction with two identical single chain Fab (scFab) segments of the second antigen via in the overall length Peptide linker at the C-terminal of every light chain of antibody is merged with the full length antibody.

In an aspect, bispecific antibody is trivalent antibodies, it includes

A) full length antibody, full length antibody first antigen of specific binding are simultaneously light by two heavy chain of antibody and two antibody Chain composition,

B) the first polypeptide is made of following item:

Ba) heavy chain of antibody variable domains (VH), or

Bb) heavy chain of antibody variable domains (VH) and antibody constant domain 1 (CH1),

Wherein first polypeptide is with the N-terminal of its VH structural domain via two heavy chains of peptide linker and the full length antibody In one C-terminal fusion,

C) the second polypeptide is made of following item:

Ca) antibody light chain variable domains (VL), or

Cb) antibody light chain variable domains (VL) and antibody light chain constant domain (CL),

Wherein second polypeptide is with the N-terminal of VL structural domain via in two heavy chains of peptide linker and the full length antibody Another C-terminal fusion, and

Wherein the heavy chain of antibody variable domains (VH) of the first polypeptide and the antibody light chain variable domains of the second polypeptide (VL) antigen-binding domains of the second antigen of specific binding are formed together.

In an aspect, b) under polypeptide heavy chain of antibody variable domains (VH) and c) under polypeptide antibody light chain Variable domains (VL) are connected and introducing disulfide bond between following position via interchain disulfide bridge and stabilisation:

(i) heavy-chain variable domains 44 to light variable domains 100, or

(ii) heavy-chain variable domains 105 to light variable domains 43, or

(iii) it (is always compiled according to Kabat EU index for heavy-chain variable domains 101 to light variable domains 100 Number).

Such as in WO 94/029350, Rajagopal, V. et al., Prot.Eng. (1997) 1453-1459； Kobayashi, H. et al., Nucl.Med.Biol.25 (1998) 387-393；And Schmidt, M. et al., Oncogene 18 (1999) it is described in 1711-1721 and introduces non-natural disulphide bridges for stabilized technology.In one embodiment, b) and C) the optional disulfide bond under between the variable domains of polypeptide is at heavy-chain variable domains 44 and light variable domains 100 Between.In one embodiment, b) and c) under polypeptide variable domains between optional disulfide bond in heavy-chain variable domains It (is always numbered according to Kabat) between 105 and light variable domains 43.In one embodiment, in single chain Fab segment Variable domains VH and VL between there is no the optional disulfide-stabilized trivalent bispecific antibody to be preferred.

In an aspect, bispecific antibody is tri-specific or four specific antibodies comprising following item

A) the first light chain and the first heavy chain of the full length antibody of the first antigen are specifically bound, and

B) the second (modification) light chain and second (modification) heavy chain of the full length antibody of the second antigen are specifically bound, Middle variable domains VL and VH is substituted for one another, and/or wherein constant domain CL and CH1 are substituted for one another, and

C) wherein one to four of other one or two kinds of antigens of specific binding (i.e. the third and/or the 4th kind of antigen) Antigen-binding domains are merged via peptide linker with the C-terminal of light chain or heavy chain a) and/or b) or N-terminal.

A) antibody under is free of just like the modification reported under b), and a) under heavy chain and light chain be isolated chain.

In an aspect, under c) tri-specific or four specific antibodies include specific binding it is one or two kinds of other One or two antigen-binding domains of antigen.

In an aspect, antigen-binding domains are selected from the group being made of scFv segment and scFab segment.

In an aspect, antigen-binding domains are scFv segments.

In an aspect, antigen-binding domains are scFab segments.

In an aspect, antigen-binding domains with a) and/or b) under the C-terminal of heavy chain merge.

In an aspect, tri-specific or four specific antibodies include specifically bind another antigen one under c) A or two antigen-binding domains.

In an aspect, tri-specific or four specific antibodies include two for specifically binding third antigen under c) Identical antigen-binding domains.In a preferred embodiment, such two identical antigen-binding domains via Identical peptide linker with a) and b) under the C-terminal of heavy chain merge.In a preferred embodiment, two identical antigen knots Closing structural domain is scFv segment or scFab segment.

In an aspect, tri-specific or four specific antibodies include the third and fourth antigen of specific binding under c) Two antigen-binding domains.In one embodiment, described two antigen-binding domains via identical peptide linker with A) the C-terminal fusion of the heavy chain under and b).In a preferred embodiment, described two antigen-binding domains are scFv pieces Section or scFab segment.

In an aspect, bispecific antibody is the tetravalent antibody of bispecific, it includes

A) two light chains and two heavy chains of the antibody of the first antigen (and including two Fab segments) are specifically bound,

B) other two Fab segment for specifically binding the antibody of the second antigen, wherein the other Fab segment passes through It is merged by peptide linker with the C-terminal of heavy chain a) or N-terminal, and

Following modification is wherein executed in Fab segment

(i) in two Fab segments a), or in two Fab segments b), variable domains VL and VH are replaced each other It changes and/or constant domain CL and CH1 is substituted for one another, or

(ii) in a) two Fab segments in, variable domains VL and VH is substituted for one another, and constant domain CL and CH1 is substituted for one another, and in two Fab segments b), variable domains VL and VH be substituted for one another or constant domain CL and CH1 is substituted for one another, or

(iii) in a) two Fab segments in, variable domains VL and VH be substituted for one another or constant domain CL and CH1 It is substituted for one another, and in two Fab segments b), variable domains VL and VH is substituted for one another, and constant domain CL and CH1 It is substituted for one another, or

(iv) in a) two Fab segments in, variable domains VL and VH is substituted for one another, and in two Fab segments b) In, constant domain CL and CH1 is substituted for one another, or

(v) in two Fab segments a), constant domain CL and CH1 is substituted for one another, and in two Fab segments b) In, variable domains VL and VH is substituted for one another.

In an aspect, the C-terminal of the heavy chain of the other Fab segment via peptide linker and a) or heavy chain a) N-terminal fusion.

In an aspect, the other Fab segment is merged via peptide linker with the C-terminal of heavy chain a).

In an aspect, the other Fab segment is merged via peptide linker with the N-terminal of heavy chain a).

In an aspect, in Fab segment, following modification is executed: in two Fab segments a), or in two b) In a Fab segment, variable domains VL and VH is substituted for one another and/or constant domain CL and CH1 is substituted for one another.

In an aspect, bispecific antibody is tetravalent antibody, it includes:

A) it specifically binds the first antigen and includes (modified) of the first antibody of the first VH-CH1 structural domain pair Heavy chain, wherein the N-terminal of the 2nd VH-CH1 structural domain pair of the first antibody melts via the C-terminal of peptide linker and the heavy chain It closes,

B) two light chains of first antibody a),

C) it specifically binds the second antigen and includes (modified) weight of the secondary antibody of the first VH-CL structural domain pair Chain, wherein the N-terminal of the 2nd VH-CL structural domain pair of the secondary antibody is merged via peptide linker with the C-terminal of the heavy chain, And

D) two (modified) light chains of secondary antibody c), every light chain include CL-CH1 structural domain pair.

In an aspect, bispecific antibody includes

A) heavy chain and light chain of the first full length antibody of the first antigen are specifically bound, and

B) heavy chain and light chain for specifically binding the second full length antibody of the second antigen, wherein the N-terminal of the heavy chain passes through The C-terminal of the light chain is connected to by peptide linker.

A) antibody under is free of just like the modification reported under b), and heavy chain and light chain are isolated chains.

In an aspect, bispecific antibody includes

A) full length antibody specifically binds the first antigen and is made of two heavy chain of antibody and two antibody light chains；With And

B) the Fv segment of the second antigen is specifically bound, the Fv segment includes VH2 structural domain and VL2 structural domain, wherein Described two structural domains are connected with each other via disulphide bridges,

Wherein only have one of VH2 structural domain or VL2 structural domain via the first antigen of peptide linker and specific binding The heavy chain or light chain of full length antibody merge.

In bispecific antibody, a) under heavy chain and light chain be isolated chain.

In an aspect, another in VH2 structural domain or VL2 structural domain be not via peptide linker and specific binding the The heavy chain or light chain of the full length antibody of one antigen merge.

In all aspects reported herein, the first light chain includes VL structural domain and CL structural domain, and the first heavy chain packet Structural domain containing VH, CH1 structural domain, hinge area, CH2 structural domain and CH3 structural domain.

In an aspect, bispecific antibody is trivalent antibodies, it includes

A) two Fab segments of the first antigen are specifically bound,

B) a CrossFab segment for specifically binding the second antigen, the CH1 and CL knot in the CrossFab segment Structure domain exchanges each other,

C) area Fc, it includes the first area Fc heavy chain and the 2nd area Fc heavy chain,

The C-terminal of the CH1 structural domain of two of them Fab segment is connected to the N-terminal of the area heavy chain Fc polypeptide, and wherein The C-terminal of the CL structural domain of CrossFab segment is connected to the N-terminal of one VH structural domain in Fab segment.

In an aspect, bispecific antibody is trivalent antibodies, it includes

A) two Fab segments of the first antigen are specifically bound,

B) a CrossFab segment for specifically binding the second antigen, the CH1 and CL knot in the CrossFab segment Structure domain exchanges each other,

C) area Fc, it includes the first area Fc heavy chain and the 2nd area Fc heavy chain,

Wherein the C-terminal of the CH1 structural domain of the first Fab segment is connected to one N-terminal in the area heavy chain Fc polypeptide, and And the C-terminal of the CL structural domain of CrossFab segment is connected to the N-terminal of the area another heavy chain Fc polypeptide, and wherein the 2nd Fab piece The C-terminal of the CH1 structural domain of section is connected to the N-terminal of the VH structural domain of the first Fab segment or is connected to CrossFab segment The N-terminal of VH structural domain.

In an aspect, bispecific antibody includes

A) full length antibody specifically binds the first antigen and is made of two heavy chain of antibody and two antibody light chains；With And

Wherein heavy Fab fragment is inserted in one CH1 structural domain in the heavy chain of the full length antibody and the overall length Between the corresponding area Fc of antibody, and the N-terminal of light chain Fab segment is conjugated to the C-terminal of the light chain of the full length antibody, described The light chain of full length antibody and the heavy chain for the full length antibody for being inserted into the heavy Fab fragment match.

In an aspect, bispecific antibody includes

A) full length antibody specifically binds the first antigen and is made of two heavy chain of antibody and two antibody light chains；With And

B) the Fab segment of the second antigen is specifically bound, the Fab segment includes to constitute heavy chain fragment and light chain segments VH2 structural domain and VL2 structural domain, wherein variable light chain domain VL2 is by the Weight variable of the antibody in the light chain segments Hinge domain VH2 replacement, and in the heavy chain fragment, variable heavy chain domain VH2 is by the variable light structure of the antibody Domain VL2 replacement, and wherein the C-terminal of the heavy chain fragment of the Fab segment is conjugated in the heavy chain of the full length antibody One N-terminal, and the C-terminal of the light chain segments of the Fab segment is conjugated to the described light of the full length antibody The heavy chain for the full length antibody that the heavy chain fragment of the N-terminal of chain, the light chain of the full length antibody and the Fab segment is conjugated to Pairing.

Polynucleotides

The present invention also provides the isolated polynucleotides for encoding bispecific antibody as described herein or its segment.

Term " nucleic acid molecules " or " polynucleotides " include any compound and/or substance comprising nucleotide polymer. Each nucleotide is by base composition, especially purine or pyrimidine bases (i.e. cytimidine (C), guanine (G), adenine (A), chest Gland pyrimidine (T) or uracil (U)), sugared (i.e. deoxyribose or ribose) and phosphate group.In general, nucleic acid molecules pass through base sequence Column are described, and thus the base represents the primary structure (linear structure) of nucleic acid molecules.Base sequence be typically expressed as from 5' to 3'.Herein, term nucleic acid molecules cover DNA (DNA) (including such as complementary DNA (cDNA) and gene Group DNA), ribonucleic acid (RNA) (especially mRNA (mRNA)), DNA or RNA synthesized form, and include these molecules In the mixed polymer of two or more.Nucleic acid molecules can be linear or cricoid.In addition, term nucleic acid molecules packet Include sense strand and antisense strand and single-stranded and double-stranded form.In addition, nucleic acid molecules described herein can contain it is naturally occurring Or non-naturally occurring nucleotide.The example of non-naturally occurring nucleotide include have derivatization sugar or phosphate backbone key or The modified nucleotide base of residue through chemical modification.Nucleic acid molecules, which also cover, to be suitable as antibody of the invention The DNA and RNA molecule of carrier external and/or that (for example, in host or patient's body) is directly expressed in vivo.Such DNA (such as CDNA) or RNA (such as mRNA) carrier can be it is unmodified or modified.For example, can be chemically modified to mRNA To enhance the stability of RNA carrier and/or the expression of coding molecule, allow to for mRNA being injected into subject's body to generate Internal antibody (see, for example, Stadler et al., Nature Medicine is delivered on June 12nd, 2017,2017 online, doi: 2 101 823 B1 of 10.1038/nm.4356 or EP).

" separation " polynucleotides refer to the nucleic acid molecules separated with the component of its natural surroundings.Isolated multicore glycosides Acid includes such nucleic acid molecules, and it includes in the cell for usually containing the nucleic acid molecules, but the nucleic acid molecules exist In outside chromosome or at the chromosome location different from its native chromosomal sites.

The isolated polynucleotides for encoding bispecific antibody of the present invention can be expressed as coding intact antigen binding molecule Single polynucleotides, or be expressed as coexpression multiple (such as two or more) polynucleotides.By the multicore co-expressed The polypeptide of thuja acid coding can associate via such as disulfide bond or other means to form functional antigen binding molecule.For example, The chain moiety of immunoglobulin can be with origin from the independent polynucleotide encoding of the heavy chain moiety of immunoglobulin.Work as coexpression When, heavy chain polypeptide will associate with light chain polypeptide to form immunoglobulin.

In certain aspects, isolated polynucleotide encoding is included in bispecific according to the present invention as described herein Polypeptide in antibody.

In one aspect, the present invention relates to the isolated polynucleotides of encoding bispecific antibody, the bispecific is anti- Body includes the first antigen-binding domains and the specific binding leaching of specific binding apoptosis protein 1 (PD1) Second antigen-binding domains of bar cell activation gene 3 (LAG3), wherein first antigen binding of specific binding PD1 Structural domain includes VH structural domain, and the VH structural domain includes (i) HVR-H1, and it includes amino acid sequences shown in SEQ ID NO:1 Column, (ii) HVR-H2, it includes amino acid sequences shown in SEQ ID NO:2, and (iii) HVR-H3, and it includes SEQ ID Amino acid sequence shown in NO:3；And VL structural domain, the VL structural domain include (i) HVR-L1, it includes SEQ ID NO:4 Shown in amino acid sequence；(ii) HVR-L2, it includes amino acid sequences shown in SEQ ID NO:5, and (iii) HVR- L3, it includes amino acid sequences shown in SEQ ID NO:6.

B. recombination method

Recombination method and composition can be used to generate antibody, for example, such as in US 4, described in 816,567.For this A little methods provide one or more isolated nucleic acid of encoding antibody.

In the case where natural antibody or natural antibody segment, two kinds of nucleic acid are needed, one kind is used for light chain or its segment, and one Kind is used for heavy chain or its segment.Such nucleic acid encode constitutes the amino acid sequence of the VL of antibody and/or constitutes the ammonia of the VH of antibody Base acid sequence (such as light chain and/or heavy chain of antibody).These nucleic acid can be on identical expression vector or different expression On carrier.With certain bispecific antibodies of heterodimeric heavy chain, four kinds of nucleic acid are needed, one kind being used for first Light chain, a kind of the first heavy chain to contain the first area different monomer (heteromonomeric) Fc polypeptide, one kind are light for second Chain, and a kind of the second heavy chain to contain the second area different monomer Fc polypeptide.Four kinds of nucleic acid may include in one or more cores In acid molecule or expression vector.For example, such nucleic acid encode constitutes the amino acid sequence of the first VL of antibody and/or constitutes antibody The first VH comprising the first area different monomer Fc amino acid sequence and/or constitute antibody the 2nd VL amino acid sequence and/ Or constitute amino acid sequence (such as the first light chain and/or the of antibody of the 2nd VH comprising the second area different monomer Fc of antibody Two light chains and/or the first heavy chain and/or the second heavy chain).These nucleic acid can be on identical expression vector or in different tables Up on carrier, these usual nucleic acid are located on two or three expression vectors, i.e., a carrier may include in these nucleic acid It is more than one.The example of these bispecific antibodies is CrossMabs and T cell bispecific (see, for example, Schaefer, W. Et al., PNAS, 108 (2011) 11187-1191).For example, one in the different monomer heavy chain " protrusion is prominent comprising so-called Become " (T366W, and optionally one of S354C or Y349C), and another in the different monomer heavy chain includes institute " hole mutation " (T366S, L368A and Y407V, and optionally Y349C or S354C) of meaning is (see, for example, Carter, P. etc. People, Immunotechnol.2 (1996) 73).

In an aspect, the isolated nucleic acid for encoding bispecific antibody as described herein is provided.Such nucleic acid can To encode the amino acid sequence for the VL for being constituted the antigen-binding domains for specifically binding PD1 and LAG3 respectively and/or be constituted institute State the amino acid sequence (such as in light chain and/or heavy chain of antibody) of the VH of antigen-binding domains.In another aspect, it mentions One or more carriers (such as expression vector) comprising such nucleic acid are supplied.In another aspect, it provides comprising this nucleoid The host cell of acid.At such aspect, host cell includes following item (such as being used to lower conversion): (1) comprising compiling The first vector of first pair of nucleic acid of code amino acid sequence and the Second support of second pair of nucleic acid comprising encoding amino acid sequence, A nucleic acid in first pair of nucleic acid includes the first VL of antibody, and another nucleic acid includes the first VH of antibody；Institute The 2nd VL that a nucleic acid in second pair of nucleic acid includes antibody is stated, and another nucleic acid includes the 2nd VH of antibody, or (2) first vector of the first nucleic acid comprising encoding amino acid sequence, a pair of of nucleic acid comprising encoding amino acid sequence second The third carrier of carrier and a pair of of nucleic acid comprising encoding amino acid sequence, the coding amino that the first vector is included First nucleic acid of acid sequence includes one of variable domains (preferably light variable domains)；The Second support included Encoding amino acid sequence a pair of of nucleic acid in a nucleic acid include light variable domains, and another nucleic acid includes the One heavy-chain variable domains；A nucleic acid in a pair of of the nucleic acid for the encoding amino acid sequence that the third carrier is included such as exists Equally comprising corresponding another light variable domains in Second support, and another nucleic acid includes the second weight chain variable structure Domain, or (3) include that the first vector of nucleic acid of encoding amino acid sequence, nucleic acid comprising encoding amino acid sequence second carry 4th carrier of body, the third carrier of nucleic acid comprising encoding amino acid sequence and the nucleic acid comprising encoding amino acid sequence, The nucleic acid for the encoding amino acid sequence that the first vector is included includes the first VL of antibody, and the Second support is included The nucleic acid of encoding amino acid sequence includes the first VH of antibody, the nucleic acid for the encoding amino acid sequence that the third carrier is included The 2nd VL comprising antibody, the nucleic acid for the encoding amino acid sequence that the 4th carrier is included include the 2nd VH of antibody.In In one aspect, host cell is eukaryocyte, such as Chinese hamster ovary (CHO) cell or lymphocyte (such as Y0, NS0, Sp20 cell).In an aspect, a kind of method for preparing bispecific antibody is provided, wherein the method includes suitable Under conditions of expressing antibody culture comprising encoding said antibody as provided above nucleic acid host cell, and optionally from The recycling antibody in host cell (or host cell culture medium).

For the first antigen-binding domains and specific binding LAG3 as described herein comprising specific binding PD1 The second antigen-binding domains bispecific antibody recombination generate, will for example encode the bispecific as described above The nucleic acid of antibody is separated and is inserted into one or more carriers further to be cloned and/or be expressed in host cell. Conventional program can be used easily such nucleic acid is separated and is sequenced (for example, by using can be with encoding antibody Heavy chain and light chain gene specific combine oligonucleotide probe).

The Suitable host cells of carrier for cloning or expressing encoding antibody include that protokaryon or eukaryon as described herein are thin Born of the same parents.For example, antibody can be generated in bacterium, especially when not needing glycosylation and Fc effector function.About in bacterium Middle expression antibody fragment and polypeptide (are seen also see, for example, US 5,648,237, US 5,789,199 and US 5,840,523 Charlton, K.A.: Methods in Molecular Biology, volume 248, Lo, B.K.C. (ed.), Humana Press, Totowa, NJ (2003), in the 245-254 pages, expression of the antibody fragment of description in Escherichia coli.) antibody can It separates, and can be further purified from bacterial cell paste in soluble fraction after expression.

Other than prokaryotes, the eukaryotic microorganisms such as filamentous fungi or yeast are also the carrier for encoding antibody Suitable clones or expressive host, the eukaryotic microorganisms include such fungi and yeasts strain, glycosylate approach " people Source ", so as to cause the antibody with partially or completely people's glycosylation pattern is generated.Referring to Gerngross, T.U., Nat.Biotech.22(2004)1409-1414；And Li, H. et al., Nat.Biotech.24 (2006) 210-215.

Suitable host cells for expressing glycosylated antibodies also derive from multicellular organism, and (invertebrate and vertebra are dynamic Object).The example of invertebral zooblast includes plant cell and insect cell.Having identified can be in conjunction with insect cell It uses, especially for transfecting many baculovirals strain of fall army worm (Spodoptera frugiperda) cell.

Plant cell cultures also are used as host.See, for example, United States Patent (USP) No.5,959,177,6,040,498,6, 420,548,7,125,978 and 6,417,429 (PLANTIBODIESTM for generating antibody in transgenic plants is described Technology).

Vertebrate cells also are used as host.For example, the mammal cell line for being suitable for growing in suspension may It is useful.Other examples of useful mammalian host cell line are the monkey kidney CV1 systems (COS-7) converted by SV40；People Embryonic kidney cells system is (such as in such as Graham, F.L. et al., 293 or 293 described in J.Gen Virol.36 (1977) 59-74 Cell)；Small hamster kidney cell (BHK)；Mouse foot-cells (such as in Mather, J.P., Biol.Reprod.23 (1980) TM4 cell described in 243-252)；MK cells (CV1)；African green monkey kidney cell (VERO-76)；Human cervical carcinoma Cell (HELA)；Canine kidney cells (MDCK)；Buffalo rats liver (BRL 3A)；Human pneumonocyte (W138)；Human liver cell (Hep G2)；Mouse mammary tumor (MMT 060562)；TRI cell, such as example in Mather, J.P. et al., Annals Described in N.Y.Acad.Sci.383 (1982) 44-68；5 cell of MRC；And FS4 cell.Other useful mammal places Chief cell system includes Chinese hamster ovary (CHO) cell, including DHFR-CHO cell (Urlaub, G. et al., Proc.Natl.Acad.Sci.USA 77(1980)4216-4220)；And myeloma cell line, such as Y0, NS0 and Sp2/0. About the summary for being suitable for certain mammalian host cell lines that antibody generates, see, for example, Yazaki, P. and Wu, A.M., Methods in Molecular Biology, volume 248, Lo, B.K.C. (editor), Humana Press, Totowa, NJ (2004), the 255-268 pages.

C. it measures

The second of the first antigen-binding domains comprising specific binding PD1 and specific binding LAG3 provided herein Its object can be identified, be screened or be characterized to the bispecific antibody of antigen-binding domains by various measurements known in the art Reason/chemical property and/or bioactivity.

1. affinity determination

Bispecific antigen binding molecules, antibody and antibody fragment provided herein can make the affinity of corresponding antigens With such asThe reference instruments such as instrument (GE Healthcare) and such as can by recombinant expression obtain receptor or Target protein is determined according to method described in example by surface plasma body resonant vibration (SPR).For measuring binding affinity Certain illustrative and exemplary embodiment be described in example 2,8 or 11.According on one side, used at 25 DEG CT100 machine (GE Healthcare) measures K by surface plasma body resonant vibration_D。

2. binding assay and other measurements

In an aspect, such as by known methods such as ELISA, Western blottings double spies of the invention are tested The antigen-binding activity of heterogenetic antibody.Bispecific antibody provided herein and corresponding recombinant antigen or and antigen-expressing cells Combination can be assessed by the ELISA as described in example 8 or 11.

In a further aspect, shown in binding assay using fresh peripheral blood mononuclear cells (PBMC) with such as The combination of the difference peripheral blood mononuclear cells such as monocyte, NK cell and T cell (PBMC).

In another aspect, the dimerization to prove two different receptor PD1 and LAG3 is measured using cell dimerization Change or last combination/interaction, described two different receptor PD1 and LAG3 with the bispecific antibody that resists both targets It is merged in cytoplasm after connection or crosslinking with two segments of enzyme.Therefore, individually only a kind of receptor is shown as no enzymatic activity. It interacts for this species specificity, the cytoplasm C-terminal of two kinds of receptors is individually merged with the heterologous subunit of reporter enzyme.Individually Single enzyme subunit do not show reporter activity.It is contemplated, however, that will lead to both receptors in conjunction with while two kinds of receptors Local cytoplasm aggregation, the complementation of two heterologous enzyme subunits, and may eventually lead to the formation of specificity and functional enzyme, the enzyme hydrolysis bottom Object is to generate chemiluminescence signal (example 11).

3. determination of activity

In an aspect, it provides for identifying biologically active the first antigen comprising specific binding PD1 The measurement of the bispecific antibody of the second antigen-binding domains of binding structural domain and specific binding LAG3.Biological activity May include for example enhance different immunocytes (especially T cell) activation and/or proliferation, immunomodulating cytokines it is (all Such as IFN γ or TNF-α) secretion, the blocking to PD1 approach, the blocking to LAG3 approach, the killing to tumour cell energy Power.It additionally provides in vivo and/or in vitro with the antibody of such bioactivity.

In certain aspects, such bioactivity of antibody of the invention is tested.In an aspect, it provides immune thin Born of the same parents' measurement, work of lymphocyte of the measurement from an individual (donor X) to the lymphocyte from another individual (donor Y) Change.Mixed lymphocyte reaction (MLP) (MLR) can prove the influence for blocking PD1 approach to lymphocyte effector cell.Exist or There is no in the case where bispecific antibody of the invention, the activation of the T cell in measurements determination and its IFN-γ secretion.In reality The measurement is described in further detail in example 9.

D. immunoconjugates

The present invention also provides immunoconjugates, and it includes the double spies of the invention being conjugated with one or more cytotoxic agents Heterogenetic antibody, one or more cytotoxic agents be such as chemotherapeutant or drug, growth inhibitor, toxin (such as Bacterium, fungi, plant or animal origin archon, enzyme activity toxin or their segment) or radioactive isotope.

E. the method and composition for diagnosing and detecting

In certain aspects, the first antigen-binding domains and specificity provided herein comprising specific binding PD1 In conjunction with the second antigen-binding domains of LAG3 any one of bispecific antibody can be used for detecting in biological sample PD1 and The presence of both LAG3.Term " detection " as used herein covers quantitative or qualitative detection.In certain embodiments, biological sample Product include cell or tissue, such as AML cancer stem cell.

In an aspect, it provides for diagnosing or the bispecific antibody of detection method.In another aspect, it mentions The existing method of PD1 and LAG3 in detection biological sample is supplied.In certain embodiments, this method is included in permission as herein The bispecific antibody connects biological sample with the bispecific antibody with both PD1 and LAG3 Touching, and detect whether to form compound between bispecific antibody and both antigens.Such method can be in vitro Or vivo approaches.In one embodiment, qualified carry out using comprising specific binding is selected using bispecific antibody The bispecific antibody of second antigen-binding domains of the first antigen-binding domains and specific binding LAG3 of PD1 is controlled The subject for the treatment of, such as wherein PD1 and LAG3 is the biomarker for selecting patient.

In certain aspects, the bispecific antibody of label is provided.Mark the label including but not limited to directly detected Or part (such as fluorescent marker, chromogenic label, electron dense label, chemiluminescent labeling and radioactive label), and Connect the part (such as enzyme or ligand) of (such as by enzymatic reaction or interaction of molecules) detection.Exemplary indicia includes but not It is limited to radioactive isotope 32P, 14C, 125I, 3H and 131I；Fluorogen, such as Rare Earth Chelate or fluorescein (fluorescein) and its derivative, rhodamine and its derivative, dansyl, umbelliferone；Luciferase (luciferase), example Such as Fluc and bacteriofluorescein enzyme (United States Patent (USP) No.4,737,456)；Luciferin (luciferin)；2,3- bis- Hydrogen benzodiazine diketone (2,3-dihydrophthalazinedione)；Horseradish peroxidase (HRP)；Alkaline phosphatase；β- Galactosidase；Glucoamylase；Lysozyme；Carbohydrate oxidase, such as glucose oxidase, galactose oxidase and glucose- 6- phosphate dehydrogenase；Heterocyclic oxidases, such as urate oxidase and xanthine oxidase；With using hydrogen peroxide come oxidation dye The enzyme (such as HRP, lactoperoxidase or microperoxisome) of precursor is coupled；Biotin/avidin；Spinning mark Note；Bacteriophage labels or stabilized radical.

F. pharmaceutical composition, preparation and administration method

In another aspect, the present invention provides pharmaceutical compositions, and it includes any provided herein comprising specific Bispecific in conjunction with the second antigen-binding domains of the first antigen-binding domains and specific binding LAG3 of PD1 is anti- Body, described pharmaceutical composition is for example for any following treatment method.In one embodiment, described pharmaceutical composition includes Any bispecific antibody provided herein and at least one pharmaceutically acceptable excipient.In another embodiment, medicine Compositions include any bispecific antibody provided herein and at least one other therapeutic agent for example as described below.

Pharmaceutical composition of the invention includes the therapeutically effective amount being dissolved or dispersed in pharmaceutically acceptable excipient One or more bispecific antibodies.Term " pharmaceutically or pharmacologically acceptable " refers to that molecular entity and composition exist It is usually nontoxic to recipient under used dosage and concentration, i.e., it is not generated when depending on the circumstances and being applied to animal (such as people) Unfavorable, allergy or other adverse reactions.Medicine containing at least one bispecific antibody and optional additional active ingredients The preparation of compositions will be in view of the disclosure be it is known to those skilled in the art, such as by Remington's Pharmaceutical Sciences, the 18th edition, Mack Printing Company, exemplified by 1990, the document is to draw It is incorporated herein with mode.Specifically, the composition is lyophilized preparation or aqueous solution.As used herein, " pharmaceutically acceptable tax Shape agent " include any and all solvents, buffer, decentralized medium, coating, surfactant, antioxidant, preservative (such as Antibacterial agent, antifungal agent), isotonic agent, salt, stabilizer and their combination, as those of ordinary skill in the art For it is known.

Parenteral composition includes designed for the parenteral composition by injection application, and the injection is such as skin Under, intradermal, intralesional, intravenous, intra-arterial, intramuscular, intrathecal or intraperitoneal injection.For injection, bispecific of the invention Antibody can be prepared in aqueous solution, preferably in the buffer of PHYSIOLOGICALLY COMPATIBLE (such as Hanks solution, Ringer's solution or physiology salt Water buffer) in prepare.Solution can contain preparaton (formulatory agent), such as suspending agent, stabilizer and/or point Powder.Alternatively, bispecific antibody can be powder type, for using preceding with suitable medium (such as sterile no heat Raw water) building.As needed, by by fusion protein of the invention with required amount and various other ingredients one for being set forth below It rises in incorporation solvent appropriate, to prepare sterile injectable solution.For example, sterile can be filtered by aseptic filter membrane is easy It realizes on ground.In general, by the way that the active constituent of various sterilizings is mixed the sterile matchmaker containing basic dispersion medium and/or other compositions Dispersion is prepared in Jie's object.It is excellent in the case where being used to prepare the aseptic powdery of sterile injectable solution, suspension or lotion The preparation method of choosing is vacuum drying or freeze drying technology, and the vacuum drying or freeze drying technology are generated from being previously sterile filtered Powder of the active constituent of liquid medium plus any additional required ingredient.If necessary, liquid medium should be buffered suitably, And liquid diluent should be kept isotonic using enough salt water or glucose first before injection.The composition must be It is stable under manufacture and storage requirement, and save as the contamination of the anti-microorganisms such as bacterium and fungi.It should be appreciated that Contaminated with endotoxins should keep minimum with the level of security of such as less than 0.5ng/mg protein.Suitable pharmaceutically acceptable tax Shape agent includes but is not limited to: buffer, such as phosphate, citrate and other organic acids；Antioxidant, including ascorbic acid And methionine；Preservative (such as stearyl dimethyl benzyl ammonium chloride；Chloor-hexaviet；Benzalkonium chloride；Benzethonium chloride； Phenol, butanol or benzylalcohol；Alkyl paraben, such as methyl p-hydroxybenzoate or propylparaben；Youngster Tea phenol；Resorcinol；Cyclohexanol；3- amylalcohol；Metacresol)；Low molecular weight (less than about 10 residues) polypeptide；Protein, such as Seralbumin, gelatin or immunoglobulin；Hydrophilic polymer, such as polyvinylpyrrolidone；Amino acid, such as sweet ammonia Acid, glutamine, asparagine, histidine, arginine or lysine；Monosaccharide, disaccharides and other carbohydrate, including grape Sugar, mannose or dextrin；Chelating agent, such as EDTA；Sugar, such as sucrose, mannitol, trehalose or D-sorbite；It contends at salt Ion, such as sodium；Metal complex (such as zinc protein complex)；And/or nonionic surfactant, such as polyethylene glycol (PEG).Water injection suspension liquid containing increase suspension viscosity compound, such as sodium carboxymethylcellulose, D-sorbite, Glucan etc..Optionally, the suspension also reagent containing suitable stabilizer or increase compound solubility, to allow to prepare Highly concentrated solution.In addition, the suspension of reactive compound can be prepared into oily injection suspensions appropriate.Suitable lipophilicity Solvent or carrier include fat oil, such as sesame oil；Or Acrawax, such as ethyl oleate or triglycerides；Or lipid Body.

Active constituent, which can be embedded in, for example (such as to be divided by condensation technique or the microcapsules prepared by interfacial polymerization Wei hydroxymethyl cellulose or gelatin-microcapsule and poly- (methyl methacrylate) microcapsules) in；In colloid drug delivery systems In (for example, liposome, albumin, microballoon, microemulsion, nanoparticle and Nano capsule)；Or in thick lotion.Such technology exists It is disclosed in Remington's Pharmaceutical Sciences (the 18th edition, Mack Printing Company, 1990). Sustained-release preparations can be prepared.The suitable examples of Sustained-release preparations include the semi permeability base of the solid hydrophobic polymers containing polypeptide Matter, the matrix are the forms of the moulded products such as film or microcapsules.In a particular embodiment, the extension of Injectable composition Absorbing can be by the composition using the reagent (such as aluminum monostearate, gelatin or their combination) of delay absorption To realize.

The exemplary pharmaceutically acceptable excipient of this paper further includes interstitial drug dispersing agent, such as solvable neutral active Hyaluronidase glycoprotein (sHASEGP), such as human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 (Baxter International,Inc.).Certain exemplary sHASEGP and application method, including RHuPH20 is described in U.S. Patent Publication No.2005/0260186 and 2006/0104968.In an aspect, will SHASEGP is combined with one or more other glycosaminoglycan enzymes (such as chondroitinase).

Illustrative lyophilized antibodies preparation is described in United States Patent (USP) No.6,267,958.Aqueous antibody formulation is included in beauty Described in state patent No.6,171,586 and WO2006/044908 those, preparation in rear one includes histidine-acetate Buffer.

Other than previously described composition, bispecific antibody can also be configured to long-acting prepared product.It is such long-acting Preparation can be applied by implantation (such as being subcutaneously or intramuscularly implanted into) or by intramuscular injection.Thus, for example, bispecific antibody It can be prepared with suitable polymerization or hydrophobic material (such as the emulsion being configured in acceptable oil) or ion exchange resin, or It is configured to sparing soluble derivative (such as being configured to slightly soluble salt).

Pharmaceutical composition comprising bispecific antibody of the invention can by means of it is conventional mixing, dissolution, emulsification, encapsulating, Embedding or freeze-drying process manufacture.One or more physiologically acceptable carriers, diluent, tax can be used in pharmaceutical composition Shape agent or auxiliary agent are prepared in a usual manner, and the carrier, diluent, excipient or auxiliary agent help for protein to be processed into can be with The prepared product pharmaceutically used.Preparation appropriate depends on selected administration method.

The bispecific antibody can be configured to the composition with free acid or alkali, neutrality or salt form.Pharmaceutically may be used The salt of receiving is the salt for substantially retaining the bioactivity of free acid or alkali.These pharmaceutically acceptable salts include sour addition Salt, such as the acid-addition salts formed with the free amine group of protein compositions, or with inorganic acid (such as hydrochloric acid or phosphoric acid) or have The acid-addition salts that machine acid (such as acetic acid, oxalic acid, tartaric acid or mandelic acid) is formed.It can also be derived with the salt that free carboxy is formed From inorganic base, such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide or iron hydroxide；Or organic base, it is such as different Propylamine, trimethylamine, histidine or procaine.Compared with corresponding free alkali form, pharmaceutical salts are tended to be more soluble in aqueous In other proton solvents.

It is required active constituent for the specific adaptations disease treated that the composition of this paper, which also contains more than one, excellent Choosing is the active constituent with the complementary activity that will not be negatively affected each other.This active component is suitably to expected purpose Effective amount combination exists.

It is usually sterile for being ready to use in the preparation applied in vivo.For example, sterile can be filtered by aseptic filter membrane is held It changes places realization.

G. Treatment and composition for

Any the first antigen-binding domains provided herein comprising specific binding PD1 and specific binding LAG3 The bispecific antibodies of the second antigen-binding domains can be used in treatment method.

In order to use in the treatment method, previously defined first comprising specific binding PD1 of such as this paper resists The bispecific antibody of the second antigen-binding domains of former binding structural domain and specific binding LAG3 can meet good doctor The mode for treating practice is prepared, determines dosage and apply.In this case factor in need of consideration include the specific illness treated, The reason of clinical disease of the specific mammal, individual patient treated, illness, applies the site of delivery of medicament, method of administration Other factors known to arrangement of time and medical practitioner.

In an aspect, bispecific antibody is provided, as defined herein, the bispecific antibody includes special Property the first antigen-binding domains of combination PD1 and the second antigen-binding domains of specific binding LAG3, it is described double special Property antibody be used as drug.In in other respects, bispecific antibody is provided, as defined herein, the bispecific antibody Comprising specifically binding the first antigen-binding domains of PD1 and the second antigen-binding domains of specific binding LAG3, institute Bispecific antibody is stated for treating disease, is especially used for treating cancer.In certain embodiments, it is anti-to provide bispecific Body, the bispecific antibody include specific binding PD1 the first antigen-binding domains and specific binding LAG3 the Two antigen-binding domains, the bispecific antibody is in treatment method.In one embodiment, the present invention provides double special Heterogenetic antibody, as described herein, the bispecific antibody include the first antigen-binding domains and the spy of specific binding PD1 The opposite sex combines the second antigen-binding domains of LAG3, and the bispecific antibody is used to treat the disease of individual in need. In certain embodiments, the present invention provides bispecific antibody, and it includes the first antigen-binding domains of specific binding PD1 With the second antigen-binding domains of specific binding LAG3, the bispecific antibody is used to treat the individual with disease In method, the method includes the bispecific antibodies to the individual application therapeutically effective amount.In certain embodiments, wait control The disease for the treatment of is cancer.On the other hand, disease to be treated is communicable disease, especially chronic viral infection, such as HIV (human immunodeficiency virus), HBV (hepatitis type B virus), HCV (hepatitis C), HSV1 (herpes simplex virus 1 class), CMV (cytomegalovirus), LCMV (lymphocytic meningitis virus) or EBV (Epstein epstein-Barr virus (Epstein-Barr virus)).Subject in need for the treatment of, patient or " individual " are usually mammal, more particularly people.

In another aspect, the present invention provides previously defined first comprising specific binding PD1 of such as this paper to resist The bispecific antibody of the second antigen-binding domains of former binding structural domain and specific binding LAG3 is manufacturing or is preparing use Purposes in the drug of disease for treating individual in need.In one embodiment, the drug is for treating disease In method, the method includes the drugs to the individual application therapeutically effective amount with the disease.

In certain aspects, disease to be treated is proliferative disorder, in particular cancer.The example of cancer includes bladder Cancer, the cancer of the brain, head and neck cancer, cancer of pancreas, lung cancer, breast cancer, oophoroma, uterine cancer, cervical carcinoma, carcinoma of endometrium, cancer of the esophagus, colon Cancer, colorectal cancer, the carcinoma of the rectum, gastric cancer, prostate cancer, leukemia, cutaneum carcinoma, squamous cell carcinoma, osteocarcinoma and kidney.It can be used Second antigen knot of the first antigen-binding domains comprising specific binding PD1 and specific binding LAG3 according to the present invention Close structural domain bispecific antibody treatment other cell proliferation disorders include but is not limited to be located at abdomen, bone, breast, Digestive system, liver, pancreas, peritonaeum, endocrine gland (adrenal gland, parathyroid gland, hypophysis, testis, ovary, thymus gland, thyroid gland), Eyes, incidence, nervous system (central nervous system and peripheral neverous system), lymphatic system, pelvis, skin, soft tissue, spleen Tumour in dirty, chest and urogenital system.It further include precancerosis disease or lesion and metastasis of cancer.In certain aspects, cancer Selected from the group being made of following item: clear-cell carcinoma, cutaneum carcinoma, lung cancer, colorectal cancer, breast cancer, the cancer of the brain, head and neck cancer.At other In aspect, cancer is selected from cancer, lymthoma (for example, hodgkin's lymphomas and non Hodgkin lymphom), blastoma, sarcoma And leukaemia.In another aspect, cancer to be treated is selected from squamous cell carcinoma, Small Cell Lung Cancer, non-small cell lung cancer, lung gland Cancer, squamous cell lung carcinoma, peritoneal cancer, hepatocellular carcinoma, human primary gastrointestinal cancers, cancer of pancreas, glioma, cervical carcinoma, oophoroma, liver cancer (liver Cancer), bladder cancer, liver cancer (hepatoma), breast cancer, colon cancer, colorectal cancer, carcinoma of endometrium or uterine cancer, saliva Gland cancer, kidney, prostate cancer, carcinoma of vulva, thyroid cancer, liver cancer (hepatic carcinoma), leukaemia and other lymph groups Knit Hypertrophic illness and various types of head and neck cancers.

In another aspect, disease to be treated is infectious diseases, especially chronic viral infection.Term " chronic disease Poison infection " refers to that subject bears slow virus or by chronic viral infection.The example of chronic viral infection is human immune deficiency It is viral (HIV), hepatitis b virus infected (HBV), infection with hepatitis C virus (HCV), herpes simplex virus 1 (HSV1), huge Cell virus (CMV), lymphocytic choriomeningitis virus (LCMV) or Epstein epstein-Barr virus (EBV).

Skilled in the art will readily recognize that in many cases, bispecific molecule possibly can not provide healing, And it can only provide Partial benefits.In some embodiments, the physiological change with some benefits is also considered as in treatment and has Benefit.Therefore, in some embodiments, the amount for providing the bispecific antibody of physiological change is considered as " effective quantity " or " controls Treat effective quantity ".

In another aspect, the present invention provides it is a kind of for treat individual disease method, the method includes to The first antigen-binding domains and specificity comprising specific binding PD1 of the invention of the individual application therapeutically effective amount In conjunction with the bispecific antibody of the second antigen-binding domains of LAG3.In one embodiment, to the individual application combination Object, the composition include the bispecific antibody of the invention of pharmaceutically acceptable form.In certain embodiments, to be treated Disease be proliferative diseases.In a particular embodiment, the disease is cancer.In certain embodiments, if wait control The disease for the treatment of is cancer, then the method also includes at least one other treatments to the individual application therapeutically effective amount Agent, such as anticancer agent.In another aspect, the disease is chronic viral infection.According to any reality in above-described embodiment " individual " for applying example can be mammal, preferably people.

In order to prevent or treat disease, the first antigen-binding domains comprising specific binding PD1 of the invention and spy The opposite sex (is controlled in conjunction with the bispecific antibody of the second antigen-binding domains of LAG3 when exclusive use or with one or more add Agent is treated when being applied in combination) suitable dosage will depend on type, administration method, the patient's weight, fusion protein of disease to be treated Type, the severity of disease and process, bispecific antibody be to apply for prevention or therapeutic purposes, is previously or same The therapeutic intervention of Shi Jinhang, the clinical medical history of patient and to the reaction of fusion protein and the judgment of attending physician.It is in office In the case of what, the practitioner that is responsible for application will determine the concentration of active constituent and appropriate agent in composition for individual subjects Amount.Various dosing arrangements of time are contemplated herein, single or multiple applications including but not limited at various time points are injected Application and pulse infusion.

The first antigen-binding domains as herein defined comprising specific binding PD1 are with specific binding LAG3's The bispecific antibody of second antigen-binding domains is suitably applied to patient a moment or in a series of treatments. Depending on the type and seriousness of disease, the bispecific of about 1 μ g/kg to 15mg/kg (such as 0.1mg/kg-10mg/kg) is anti- Body can be for example through one or many initial candidate dosages being administered alone or be applied to patient by continuous infusion.It takes Certainly in above-mentioned factor, a kind of typical daily dose can range from about 1 μ g/kg to 100mg/kg or more.For a couple of days or The repetitive administration of longer time, depends on illness, and treatment would generally last up to the disease symptoms needed for occurring and inhibit.It is double special A kind of range of exemplary dose of property antibody is about 0.005mg/kg to about 10mg/kg.In other examples, dosage can also wrap Include application about 1 μ g/kg weight, about 5 μ g/kg weight, about 10 μ g/kg weight, about 50 μ g/kg weight, about 100 μ g/kg bodies every time Weight, about 200 μ g/kg weight, about 350 μ g/kg weight, about 500 μ g/kg weight, about 1mg/kg weight, about 5mg/kg weight, about 10mg/kg weight, about 50mg/kg weight, about 100mg/kg weight, about 200mg/kg weight, about 350mg/kg weight, about 500mg/kg weight is to about 1000mg/kg weight or more, and any range that can wherein derive.From this paper institute columns It, can be based on above-mentioned number application about 5mg/kg weight to about 100mg/kg weight, about 5 in the example for the range that word can be derived μ g/kg weight to about 500mg/kg weight etc. range.Therefore, can to patient apply about 0.5mg/kg, 2.0mg/kg, One or more dosage of 5.0mg/kg or 10mg/kg (or any combination of them).Such dosage can be applied intermittently, example As weekly or every three weeks application (for example, make patient receiving about 2 to about 20 or the fusion protein of for example, about 6 dosage).It can apply With initial higher load dosage, one or more relatively low-doses are then applied.However, other dosages come in handy.The treatment The progress of method is easily monitored by routine techniques and measurement.

The first antigen-binding domains as herein defined comprising specific binding PD1 are with specific binding LAG3's The bispecific antibody of second antigen-binding domains is usually effectively to realize that the amount of expected purpose uses.In order to for treat or Prevent disease symptom, bispecific antibody of the invention or its pharmaceutical composition to apply or apply with therapeutically effective amount.Treatment has The determination of effect amount is completely in the limit of power of those skilled in the art, in particular according to detailed disclosures provided herein.

For systemic administration, can initially be estimated to treat effective agent according to external test (such as cell culture measurement) Amount.Dosage can be then prepared in animal model, include the IC such as measured in cell culture to realize₅₀Circulation composition model It encloses.This type of information can be used for more accurately determining the useful dosage to the mankind.

Predose can also be estimated according to intra-body data (such as animal model) using technology well known in the art.This Field those of ordinary skill can easily optimize the application to people based on animal data.

Dosage and interval can individually be adjusted, and be enough to maintain the blood plasma bispecific antibody of curative effect horizontal to provide.It is logical The range for crossing the common patient dose of injection application is about 0.1 to 50mg/kg/ days, normally about 0.5 to 1mg/kg/ days.By every It is applied multiple dosage and may be implemented to treat effective blood plasma level.The level in blood plasma can be for example measured by HPLC.

In the case where local application or selectivity intake, effective local concentration of bispecific antibody may be dense with blood plasma It spends unrelated.Those skilled in the art optimization will treat effective local dose without excessive experiment.

The bispecific antibody for the treatment of effective dose described herein usually will be the case where will not cause significant toxicity Lower offer treatment benefit.The toxicity and therapeutic efficiency of fusion protein can pass through the standard pharmaceutical journey in cell culture or experimental animal Sequence measures.Cell culture measurement and zooscopy can be used for measuring LD₅₀(50% dosage of lethal group) and ED₅₀(In Effective dosage is treated in the 50% of group) dose ratio between toxicity and curative effect is therapeutic index, the therapeutic index can be with It is expressed as ratio LD₅₀/ED₅₀.It is preferred for showing the bispecific antibody of big therapeutic index.In one embodiment, according to Bispecific antibody of the invention shows high therapeutic index.The data obtained from cell culture measurement and zooscopy can be used for Prepare a series of dosage for being suitable for the mankind.Dosage is preferably in the ED including the very little or none toxicity of toxicity₅₀Circulation composition model In enclosing.Dosage can depend on many factors and change in the range, and many factors are for example used dosage form, institute The illness etc. of the administration method, subject that utilize.Exact formula, administration method and dosage can be by individual physicians according to patient Illness come select (see, for example, Fingl et al., 1975, in The Pharmacological Basis of Therapeutics, the 1st chapter, in page 1, the full content of the document is incorporated herein by reference).

The attending physician of the patient treated with bispecific antibody of the invention will know how and when due to toxicity, Organ dysfunction etc. terminates, interrupts or adjusts application.On the contrary, being cured mainly if clinical response is insufficient (excluding toxicity) Doctor, which also will appreciate that, adjusts treatment to higher level.The size of applied dose will be in the management of interested illness Severity, administration method of illness to be treated etc. and change.For example, can be commented partially by standard prognostic evaluation method Estimate the seriousness of illness.In addition, dosage and possible dose frequency will also become according to the age of individual patient, weight and reaction Change.

Other medicaments and treatment

The first antigen-binding domains comprising specific binding PD1 and specific binding LAG3 as described earlier in this article The second antigen-binding domains bispecific antibody can in the treatment with other one or more pharmaceutical agent combinations apply.Example Such as, bispecific antibody of the invention can be co-administered at least one additional therapeutic agent.Term " therapeutic agent " includes that can apply With any medicament for treating the symptom or disease that need the individual of such treatment.Such additional therapeutic agent may include being suitable for Any active constituent for the specific indication treated preferably has the work for the complementary activity that will not be negatively affected each other Property ingredient.In certain embodiments, additional therapeutic agent is another anticancer agent.

In one aspect of the invention, the first antigen binding structure as described herein comprising specific binding PD1 The bispecific antibody of the second antigen-binding domains of domain and specific binding LAG3 includes the bispecific antibody Pharmaceutical composition is for prevention or treating cancer, wherein the bispecific antibody is with chemotherapeutant, radiation and/or for cancer Other drug combinations of disease immunotherapy are applied.

The first antigen binding in a particular aspects of the invention, as described herein comprising specific binding PD1 The bispecific antibody of the second antigen-binding domains of structural domain and specific binding LAG3 is anti-comprising the bispecific The pharmaceutical composition of body is for prevention or treating cancer, wherein the bispecific antibody and T cell activation AntiCD3 McAb bispecific Antibody, especially anti-CEA/ AntiCD3 McAb bispecific antibody are administered in combination.In an aspect, anti-CEA/ AntiCD3 McAb bispecific is anti- Body is T cell activation AntiCD3 McAb bispecific antibody, it includes the second antigen-binding domains, second antigen binding structure Domain includes (a) heavy chain variable region (V_H) and/or light chain variable region (V CEA_LCEA), the heavy chain variable region includes CDR-H1 (SEQ Sequence shown in ID NO:154), CDR-H2 (sequence shown in SEQ ID NO:155) and CDR-H3 (SEQ ID NO:156 institute The sequence shown), the light chain variable region include CDR-L1 (sequence shown in SEQ ID NO:157), CDR-L2 (SEQ ID NO: Sequence shown in 158) and CDR-L3 (sequence shown in SEQ ID NO:159)；Or (b) heavy chain variable region (V_HCEA) and/or Light chain variable region (V_LCEA), the heavy chain variable region includes CDR-H1 (sequence shown in SEQ ID NO:162), CDR-H2 (sequence shown in SEQ ID NO:163) and CDR-H3 (sequence shown in SEQ ID NO:164), the light chain variable region includes CDR-L1 (sequence shown in SEQ ID NO:165), CDR-L2 (sequence shown in SEQ ID NO:166) and CDR-L3 (SEQ Sequence shown in ID NO:167).In an aspect, anti-CEA/ AntiCD3 McAb bispecific antibody is that T cell activation AntiCD3 McAb is double special Heterogenetic antibody, it includes the heavy chain variable region (V containing amino acid sequence shown in SEQ ID NO:160_HCEA), and/or contain Light chain variable region (the V of amino acid sequence shown in SEQ ID NO:161_LCEA), or contain the second antigen-binding domains, it is described Second antigen-binding domains include the heavy chain variable region (V containing amino acid sequence shown in SEQ ID NO:168_HCEA) and/or Light chain variable region (V containing amino acid sequence shown in SEQ ID NO:169_LCEA)。

In another aspect, anti-CEA/ AntiCD3 McAb bispecific antibody includes Fc structural domain, and the Fc structural domain includes to reduce And the combination of Fc receptor and/or the one or more amino acid substitutions for reducing effector function.Specifically, anti-CEA/ AntiCD3 McAb is double Specific antibody include IgG1 Fc structural domain, the IgG1 Fc structural domain include amino acid substitution L234A, L235A and P329G。

In a particular aspects, anti-CEA/ AntiCD3 McAb bispecific antibody includes and sequence shown in SEQ ID NO:146 The consistent polypeptide of at least 95%, 96%, 97%, 98% or 99%, with sequence at least 95% shown in SEQ ID NO:147, 96%, the consistent polypeptide of 97%, 98% or 99%, with sequence at least 95% shown in SEQ ID NO:148,96%, 97%, 98% or 99% consistent polypeptide, and with sequence at least 95%, 96%, 97%, 98% shown in SEQ ID NO:149 or 99% consistent polypeptide.In still another embodiment, bispecific antibody includes polypeptide sequence shown in SEQ ID NO:146 Shown in polypeptide sequence shown in column, polypeptide sequence, SEQ ID NO:148 shown in SEQ ID NO:147 and SEQ ID NO:149 Polypeptide sequence (CEA CD3 TCB).

In another particular aspects, anti-CEA/ AntiCD3 McAb bispecific antibody includes and sequence shown in SEQ ID NO:150 Arrange at least 95%, 96%, 97%, 98% or 99% consistent polypeptide, with sequence at least 95% shown in SEQ ID NO:151, 96%, the consistent polypeptide of 97%, 98% or 99%, with sequence at least 95% shown in SEQ ID NO:152,96%, 97%, 98% or 99% consistent polypeptide, and with sequence at least 95%, 96%, 97%, 98% shown in SEQ ID NO:153 or 99% consistent polypeptide.In still another embodiment, bispecific antibody includes polypeptide sequence shown in SEQ ID NO:150 Shown in polypeptide sequence shown in column, polypeptide sequence, SEQ ID NO:152 shown in SEQ ID NO:151 and SEQ ID NO:153 Polypeptide sequence (CEACAM5CD3 TCB).

In another aspect, a kind of pharmaceutical composition is provided, it includes as described herein comprising specific binding The bispecific antibody of second antigen-binding domains of the first antigen-binding domains and specific binding LAG3 of PD1, with And T cell activation AntiCD3 McAb bispecific antibody, especially anti-CEA/ AntiCD3 McAb bispecific antibody.In a particular aspects, The pharmaceutical composition is especially used for treating cancer for combining, serially or simultaneously treating disease.More specifically, the composition For treating solid tumor.

In another aspect, the present invention provides the sides of a kind of cancer for treating individual or the cancer progression for delaying individual Method, the method includes applying a effective amount of the first antigen knot as described herein comprising specific binding PD1 to subject It closes structural domain and specifically binds the bispecific antibody and T cell activation AntiCD3 McAb pair of the second antigen-binding domains of LAG3 Specific antibody, the combination of especially anti-CEA/ AntiCD3 McAb bispecific antibody or anti-FolR1/ AntiCD3 McAb bispecific antibody.

Other such medicaments suitably exist effectively to measure combination to expected purpose.Effective measurement of other such medicaments Certainly in the amount of fusion protein used, illness or the type for the treatment of and other factors discussed above.The bispecific antibody Usually with about 1% to 99% with identical dosage described herein and administration method or dosage described herein, or with any dosage And it is used by the suitable approach of experience/be clinically determined as.

Above-mentioned such conjoint therapy covers combined administration, and (two of them or more therapeutic agent is comprised in same composition Or in separated composition) and separate administration, in the case where separate administration, the application of bispecific antibody can applied With before additional therapeutic agent and/or adjuvant, occur simultaneously and/or later.

H. product

In another aspect of the present invention, a kind of product is provided, containing, which can be used for, treats, prevents and/or diagnoses The substance of above-mentioned illness.The product includes container and on the container or label relevant to the container or package insert (package insert).Suitable container includes such as bottle, bottle, syringe, intravenous injection (IV) solution bag.It is described Container can be formed by multiple materials such as glass or plastics.The container accommodates composition, the composition individually or with Another kind effective for treatment, prevention and/or the diagnosis illness combination of compositions, and the container can have it is sterile enter Mouth (such as the container can be parenteral solutions bag or the bottle with the available plug that needle-penetration is subcutaneously injected).It is described At least one of composition activating agent be as herein previously defined in comprising specifically bind PD1 the first antigen binding knot The bispecific antibody of the second antigen-binding domains of structure domain and specific binding LAG3.

Label or package insert indicate the composition for treating selected illness.In addition, the product may include (a) The first container contains the composition comprising bispecific antibody of the invention in the first container；And (b) second container, Contain in the second container and includes other cytotoxic agent or combination with other therapeutic agents object.System in this embodiment of the invention Product also may include package insert, and the package insert indicates that the composition can be used for treating particular condition.

Alternately or in addition, the product may also include second (or third) container, described second (or third) container Including pharmaceutically acceptable buffer, such as water for injection,bacteriostatic (BWFI), phosphate buffered saline (PBS), Ringer's solution and Glucose solution.The product may also include from other substances needed for business and user perspective, including other buffers, dilution Agent, filter, syringe needle and syringe.

Table C (sequence):

The paragraph numbered below describes each aspect of the present invention:

1. a kind of bispecific antibody, it includes the first antigens of specific binding apoptosis albumen 1 (PD1) Second antigen-binding domains of binding structural domain and specific binding lymphocyte activation gene -3 (LAG3), wherein

Specific binding PD1 first antigen-binding domains include

VH structural domain, it includes

(i) HVR-H1, it includes amino acid sequence shown in SEQ ID NO:1,

(ii) HVR-H2, it includes amino acid sequences shown in SEQ ID NO:2, and

(iii) HVR-H3, it includes amino acid sequences shown in SEQ ID NO:3；And

VL structural domain, it includes

(i) HVR-L1, it includes amino acid sequence shown in SEQ ID NO:4,

(ii) HVR-L2, it includes amino acid sequences shown in SEQ ID NO:5, and

(iii) HVR-L3, it includes amino acid sequences shown in SEQ ID NO:6.

2. according to bispecific antibody described in paragraph 1, wherein the bispecific antibody includes Fc structural domain, the Fc Structural domain is IgG, especially IgG1 Fc structural domain or IgG4 Fc structural domain, and wherein the Fc structural domain include reduce with Fc receptor, especially one or more amino acid substitutions in conjunction with Fc γ receptor.

3. the bispecific antibody according to paragraph 1 or 2, wherein second antigen binding of specific binding LAG3 Structural domain includes

(a) VH structural domain, it includes

(i) HVR-H1, it includes amino acid sequence shown in SEQ ID NO:14,

(ii) HVR-H2, it includes amino acid sequences shown in SEQ ID NO:15, and

(iii) HVR-H3, it includes amino acid sequences shown in SEQ ID NO:16；And

VL structural domain, it includes

(i) HVR-L1, it includes amino acid sequence shown in SEQ ID NO:17,

(ii) HVR-L2, it includes amino acid sequences shown in SEQ ID NO:18, and

(iii) HVR-L3, it includes amino acid sequences shown in SEQ ID NO:19；Or

(b) VH structural domain, it includes

(i) HVR-H1, it includes amino acid sequence shown in SEQ ID NO:22,

(ii) HVR-H2, it includes amino acid sequences shown in SEQ ID NO:23, and

(iii) HVR-H3, it includes amino acid sequences shown in SEQ ID NO:24；And

VL structural domain, it includes

(i) HVR-L1, it includes amino acid sequence shown in SEQ ID NO:25,

(ii) HVR-L2, it includes amino acid sequences shown in SEQ ID NO:26, and

(iii) HVR-L3, it includes amino acid sequences shown in SEQ ID NO:27；Or

(i) HVR-H1, it includes amino acid sequence shown in SEQ ID NO:30,

(ii) HVR-H2, it includes amino acid sequences shown in SEQ ID NO:31, and

(iii) HVR-H3, it includes amino acid sequences shown in SEQ ID NO:32；And

VL structural domain, it includes

(i) HVR-L1, it includes amino acid sequence shown in SEQ ID NO:33,

(ii) HVR-L2, it includes amino acid sequences shown in SEQ ID NO:34, and

(iii) HVR-L3, it includes amino acid sequences shown in SEQ ID NO:35；Or

(d) VH structural domain, it includes

(i) HVR-H1, it includes amino acid sequence shown in SEQ ID NO:38,

(ii) HVR-H2, it includes amino acid sequences shown in SEQ ID NO:39, and

(iii) HVR-H3, it includes amino acid sequences shown in SEQ ID NO:40；And

VL structural domain, it includes

(i) HVR-L1, it includes amino acid sequence shown in SEQ ID NO:41,

(ii) HVR-L2, it includes amino acid sequences shown in SEQ ID NO:42, and

(iii) HVR-L3, it includes amino acid sequences shown in SEQ ID NO:43；Or

(e) VH structural domain, it includes

(i) HVR-H1, it includes amino acid sequence shown in SEQ ID NO:46,

(ii) HVR-H2, it includes amino acid sequences shown in SEQ ID NO:47, and

(iii) HVR-H3, it includes amino acid sequences shown in SEQ ID NO:48；And

VL structural domain, it includes

(i) HVR-L1, it includes amino acid sequence shown in SEQ ID NO:49,

(ii) HVR-L2, it includes amino acid sequences shown in SEQ ID NO:50, and

(iii) HVR-L3, it includes amino acid sequences shown in SEQ ID NO:51.

4. the bispecific antibody according to any one of paragraph 1 to 3, wherein described the first of specific binding PD1 Antigen-binding domains include

5. the bispecific antibody according to any one of paragraph 1 to 4, wherein described the second of specific binding LAG3 Antigen-binding domains include

6. the bispecific antibody according to any one of paragraph 1 to 4, wherein described the second of specific binding LAG3 Antigen-binding domains include

7. the bispecific antibody according to any one of paragraph 1 to 5, wherein

8. the bispecific antibody according to any one of paragraph 1 to 5, wherein

9. the bispecific antibody according to any one of paragraph 1 to 4 or 6, wherein

10. the bispecific antibody according to any one of paragraph 1 to 5, wherein the bispecific antibody is source of people Change antibody or chimeric antibody.

11. the bispecific antibody according to any one of paragraph 1 to 10, wherein the bispecific antibody includes people The Fc structural domain of IgG1 subclass, the Fc structural domain have amino acid mutation L234A, L235A and P329G (according to Kabat EU Index number).

12. the bispecific antibody according to any one of paragraph 1 to 11, wherein the bispecific antibody includes Fc Structural domain, the Fc structural domain include the modification for promoting the first and second subunit associations of the Fc structural domain.

13. the bispecific antibody according to any one of paragraph 1 to 12, wherein the first subunit of the Fc structural domain Comprising protrusion, and the second subunit of the Fc structural domain includes the hole for entering hole method according to protrusion.

14. the bispecific antibody according to any one of paragraph 1 to 13, wherein the first subunit of the Fc structural domain Comprising amino acid substitution S354C and T366W (EU number), and the second subunit of the Fc structural domain includes amino acid substitution Y349C, T366S and Y407V (according to Kabat EU index number).

15. the bispecific antibody according to any one of paragraph 1 to 14, wherein the bispecific antibody includes Fc Structural domain, the first Fab segment and the 2nd Fab segment, the first Fab segment include the antigen knot of specific binding PD1 Structural domain is closed, the 2nd Fab segment includes the antigen-binding domains of specific binding LAG3.

16. the bispecific antibody according to any one of paragraph 1 to 15, wherein one in the Fab segment In, the variable domains VL and VH is substituted for one another so that the VH structural domain is a part of the light chain and the VL is tied Structure domain is a part of the heavy chain.

17. the bispecific antibody according to paragraph 15 or 16, wherein in the antigen comprising specifically binding PD1 In the first Fab segment of binding structural domain, the variable domains VL and VH is substituted for one another.

18. the bispecific antibody according to any one of paragraph 1 to 17, wherein the bispecific antibody includes Fab segment, wherein the amino acid at 124 is by lysine (K), arginine (R) or histidine in the constant domain CL (H) independently replace (according to Kabat EU index number), and in the constant domain CH1, at 147 and 213 Amino acid is independently replaced by glutamic acid (E) or aspartic acid (D) (according to Kabat EU index number).

19. the bispecific antibody according to any one of paragraph 15 to 18, wherein comprising specifically binding LAG3 The antigen-binding domains the 2nd Fab segment in, the amino acid in the constant domain CL, at 124 Independently replaced (according to Kabat EU index number) by lysine (K), arginine (R) or histidine (H), and described constant In domain C H1, amino acid at 147 and 213 independently replaced by glutamic acid (E) or aspartic acid (D) (according to Kabat EU index number).

20. the bispecific antibody according to any one of paragraph 1 to 19, the bispecific antibody include

21. the bispecific antibody according to any one of paragraph 1 to 19, wherein the bispecific antibody includes the Three Fab segments, the 3rd Fab segment include the antigen-binding domains of specific binding LAG3.

22. the bispecific antibody according to any one of paragraph 1 to 19 or 21, wherein respectively containing specific binding Described two Fab segments of the antigen-binding domains of LAG3 are identical.

23. the bispecific antibody according to any one of paragraph 1 to 19 or 21 or 22, wherein including specific binding The Fab segment of the antigen-binding domains of PD1 is merged via peptide linker with one C-terminal in the heavy chain.

24. the bispecific antibody according to any one of paragraph 1 to 19 or 21 to 23, the bispecific antibody packet Contain

(a) the first heavy chain, the first light chain, the second heavy chain and the second light chain, first heavy chain include and SEQ ID NO: Sequence shown in 118 has the amino acid sequence of at least 95% sequence identity, and first light chain includes and SEQ ID NO: Sequence shown in 115 has the amino acid sequence of at least 95% sequence identity, and second heavy chain includes and SEQ ID NO: Sequence shown in 119 has the amino acid sequence of at least 95% sequence identity, and second light chain includes and SEQ ID NO: Sequence shown in 101 has the amino acid sequence of at least 95% sequence identity, or

(b) the first heavy chain, the first light chain, the second heavy chain and the second light chain, first heavy chain include and SEQ ID NO: Sequence shown in 120 has the amino acid sequence of at least 95% sequence identity, and first light chain includes and SEQ ID NO: Sequence shown in 115 has the amino acid sequence of at least 95% sequence identity, and second heavy chain includes and SEQ ID NO: Sequence shown in 121 has the amino acid sequence of at least 95% sequence identity, and second light chain includes and SEQ ID NO: Sequence shown in 99 has the amino acid sequence of at least 95% sequence identity, or

(c) the first heavy chain, the first light chain, the second heavy chain and the second light chain, first heavy chain include and SEQ ID NO: Sequence shown in 122 has the amino acid sequence of at least 95% sequence identity, and first light chain includes and SEQ ID NO: Sequence shown in 115 has the amino acid sequence of at least 95% sequence identity, and second heavy chain includes and SEQ ID NO: Sequence shown in 103 has the amino acid sequence of at least 95% sequence identity, and second light chain includes and SEQ ID NO: Sequence shown in 105 has the amino acid sequence of at least 95% sequence identity.

25. the bispecific antibody according to any one of paragraph 1 to 19 or 21 to 23, wherein the bispecific is anti- Body includes the 4th Fab segment, and the 4th Fab segment includes the antigen-binding domains of specific binding PD1.

26. the bispecific antibody according to any one of paragraph 1 to 19 or 21 to 23 or 25, wherein respectively containing spy The opposite sex is identical in conjunction with described two Fab segments of the antigen-binding domains of PD1.

27. the bispecific antibody according to any one of paragraph 1 to 19 or 21 to 23 or 25 or 26, wherein respectively packet Described two Fab segments of the antigen-binding domains of the PD1 containing specific binding are respectively via peptide linker and the heavy chain In one C-terminal fusion.

28. the bispecific antibody according to any one of paragraph 1 to 19 or 21 to 23 or 25 to 27, described double special Property antibody includes

29. the bispecific antibody according to any one of paragraph 1 to 14, wherein the bispecific antibody includes Fc Structural domain, two Fab segments and single chain Fab (scFab), described two Fab segments respectively contain the anti-of specific binding LAG3 Former binding structural domain, the single chain Fab (scFab) include the antigen-binding domains of specific binding PD1.

30. the bispecific antibody according to any one of paragraph 1 to 14 or 29, wherein including specific binding PD1 The scFab of antigen-binding domains merged via peptide linker with one C-terminal in the heavy chain.

31. the bispecific antibody according to any one of paragraph 1 to 14 or 29 or 30, the bispecific antibody packet Contain

32. the bispecific antibody according to any one of paragraph 1 to 14, wherein the bispecific antibody includes Fc Structural domain, two Fab segments and VH and VL structural domain, described two Fab segments respectively contain the antigen of specific binding LAG3 Binding structural domain, VH the and VL structural domain include the antigen-binding domains of specific binding PD1.

33. the bispecific antibody according to any one of paragraph 1 to 14 or 32, wherein specific binding PD1's is anti- The VH structural domain of former binding structural domain is merged via peptide linker with one C-terminal in the heavy chain, and specificity knot The VL structural domain for closing the antigen-binding domains of PD1 is merged via peptide linker with another C-terminal in the heavy chain.

34. the bispecific antibody according to any one of paragraph 1 to 14 or 32 or 33, the bispecific antibody packet Containing the first heavy chain, the second heavy chain and two light chains, first heavy chain includes to have extremely with sequence shown in SEQ ID NO:126 The amino acid sequence of few 95% sequence identity, second heavy chain include to have extremely with sequence shown in SEQ ID NO:127 The amino acid sequence of few 95% sequence identity, the two light chains are respectively contained to be had with sequence shown in SEQ ID NO:109 There is the amino acid sequence of at least 95% sequence identity.

35. a kind of polynucleotides encode the bispecific antibody according to any one of paragraph 1 to 34.

36. a kind of carrier, especially expression vector, it includes the polynucleotides according to paragraph 35.

37. a kind of protokaryon or eukaryotic host cell, it includes the polynucleotides according to paragraph 35 or according to paragraph 36 The carrier.

38. a kind of method for generating the bispecific antibody according to paragraph 1 to 34, the method includes being suitable for table The host cell according to paragraph 37 is cultivated under conditions of up to the bispecific antibody, and is recycled from the culture The bispecific antibody.

39. a kind of pharmaceutical composition, it includes the bispecific antibody according to any one of paragraph 1 to 34 and at least A kind of pharmaceutically acceptable excipient.

40. the bispecific antibody according to any one of paragraph 1 to 34 or the pharmaceutical composition according to paragraph 39 Object, the bispecific antibody or described pharmaceutical composition are used as drug.

41. the bispecific antibody according to any one of paragraph 1 to 34 or the pharmaceutical composition according to paragraph 39 Object, the bispecific antibody or described pharmaceutical composition are used for

I) immune response is adjusted, such as recovery T cell activity,

Ii t cell response) is stimulated,

Iii) treatment infection,

Iv) treating cancer,

V) delay cancer development,

Vi) extend the survival period of cancer patient.

42. the bispecific antibody according to any one of paragraph 1 to 34 or the pharmaceutical composition according to paragraph 39 Object, the bispecific antibody or described pharmaceutical composition are for prevention or treating cancer.

43. the bispecific antibody according to any one of paragraph 1 to 34 or the pharmaceutical composition according to paragraph 39 Object, the bispecific antibody or described pharmaceutical composition are for treating chronic viral infection.

44. the bispecific antibody according to any one of paragraph 1 to 34 or the pharmaceutical composition according to paragraph 39 Object, the bispecific antibody or described pharmaceutical composition for prevent or treating cancer, wherein the bispecific antibody with Chemotherapeutant, radiation and/or other reagents for immunotherapy for cancer are administered in combination.

45. a kind of method for inhibiting growth of tumour cell in individual comprising apply a effective amount of basis to the individual Bispecific antibody described in any one of paragraph 1 to 34 is to inhibit the growth of the tumour cell.

Example

It is the example of method and composition of the invention below.It should be appreciated that providing general description provided above In the case of, various other embodiments can be practiced.

Material and conventional method

General information about human immunoglobulin(HIg) light chain and the nucleotide sequence of heavy chain is given in: Kabat, E.A. etc. People, Sequences of Proteins of Immunological Interest, the 5th edition, Public Health In Service, National Institutes of Health, Bethesda, MD (1991).According to root as defined above According to Kabat (Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, the 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991)) number System to the amino acid of antibody chain is numbered and quotes.

Recombinant DNA technology

DNA is manipulated using standard method, such as in Sambrook, J. et al., Molecular Cloning:A laboratory manual；Cold Spring Harbor Laboratory Press,Cold Spring Harbor,New York, described in 1989.Molecular biology reagents are used according to the explanation of manufacturer.

Gene chemical synthesis

Required constant gene segment C is prepared from the oligonucleotides as made from chemical synthesis.Flank is single restriction enzyme The constant gene segment C of the 600-1800bp long of nucleic acid cleavage sites, and annealing and connecting oligonucleotides (including PCR amplification) Assembly, and it is then based on pGA4 cloning vector, via specified restriction site (such as KpnI/SacI or AscI/PacI) gram It is grand to arrive in pPCRScript (Stratagene).The DNA sequence dna of subcloning gene fragments is confirmed by DNA sequencing.According to The given specification of Geneart (Regensburg, Germany) is ranked up gene chemical synthesis segment.

Determined dna sequence

By in MediGenomix GmbH (Martinsried, Germany) or Sequiserve GmbH The double-strand executed at (Vaterstetten, Germany) is sequenced to determine DNA sequence dna.

DNA and protein sequence analysis and sequence data management

Using GCG (Genetics Computer Group, Madison, Wisconsin) software package 10.2 editions and The Vector NT1 Advance external member 8.0 editions of Infomax come carry out sequence creation, map is drawn, analysis, annotation and explanation.

Expression vector

In order to express the antibody, apply based on or without CMV- intron A promoter cDNA tissue or The change of the expression plasmid for transient expression (such as in HEK293) cell based on the genomic organization with CMV promoter Body.

In addition to antibody expression box, carrier also includes:

Replication orgin allows to carry out the duplication of the plasmid in Escherichia coli, and

Beta-lactam enzyme gene assigns the amicillin resistance in Escherichia coli.

The transcript unit of antibody gene is made of following elements:

The unique restriction sites at -5 ' ends,

The enhancer of early stage at once and promoter from human cytomegalovirus,

It is intron A sequence later in the case where cDNA is organized,

The 5' non-translational region of human immunoglobulin gene,

Heavy chain immunoglobulin signal sequence,

Human antibody chain (wild type or having Domain swapping), ball is immunized as cDNA or as having in the people's antibody chain The genomic organization of albumen exon: intron tissue

3' non-translational region with polyadenylation signal sequence, and

The unique restriction sites at the end -3'.

Generated by PCR and/or gene chemical synthesis include antibody chain as described below fusion, and by the fusion base Because of recombination method and technology known to passing through, corresponding core is connected by using the unique restriction sites in respective carrier Sour section is assembled.The nucleic acid sequence of subclone is verified by DNA sequencing.In order to be transiently transfected, pass through plasmid Preparation prepares larger amount of plasmid (Nucleobond AX, Macherey-Nagel) from the culture of Escherichia coli of conversion.

Cell culture technology

Using such as in Current Protocols in Cell Biology (2000), Bonifacino, J.S., Dasso, M., Harford, J.B., Lippincott-Schwartz, J. and Yamada, K.M. (editor), John Wiley& Standard cell culture techniques described in Sons, Inc.

As described below, by the HEK293-EBNA cell of adherent growth or in the HEK29-F cell of suspension growth Transient cotransfection corresponding expression plasmid expresses multi-specificity antibody.

Transient transfection in HEK293 system

293F cell is transiently transfected by using Freestyle system (ThermoFisher) to generate all antibody and double Specific antibody.293F cell is cultivated in F17 culture medium herein, is transfected with 293Free (Novagene), and at 4 hours After raise with VPA 4mM and Feed 7, and after 16h raise with 0.6% glucose.In addition, using Expi293F^TMExpression system examination Agent box (ThermoFisher).Here, by Expi293F^TMCell is in Expi293^TMIt is cultivated in expression culture medium, and according to manufacture The explanation of quotient uses ExpiFectamine^TM293 transfection reagent boxes are transfected.Additionally draw due to having in the interface CH1/CL CrossMAb of the Charged acids entered to (about further details, referring to the position in corresponding sequence)^Vh-VLBispecific is anti- The improved stability and purity of body and reduced aggregation tendency, so plasmid ratio is not adjusted.Therefore, using needle Relative plasmid ratio 1:1:1:1 to 1+1 CrossM or for 2+2 CrossMab relative plasmid ratio 1:1:1 come cotransfection LC Plasmid, HC plasmid intersect LC plasmid and intersect HC plasmid.Cell supernatant is harvested after 7 days, and is purified by standard method.

Protein determination

By using according to Pace et al., Protein Science, 1995,4,2411-1423 based on amino acid sequence Optical density (OD) at the molar extinction coefficient measurement 280nm of calculating, to determine the antibody of purifying and the protein compression of derivative Degree.

Antibody concentration measurement in supernatant

Cell culture supernatant is estimated by carrying out immunoprecipitation with protein A agarose beads (Roche (Roche)) The concentration of middle antibody and derivative.By 60 μ L protein A agarose beads in TBS-NP40 (50mM Tris, pH 7.5,150mM NaCl, 1%Nonidet-P40) in washing three times.Then, the cell culture supernatant of 1-15mL is applied in TBS-NP40 The protein A agarose beads of middle pre-equilibration.After being incubated at room temperature 1 hour, by bead in Ultrafree-MC- filter column (Amicon) it washed once on 0.5mL TBS-NP40, washed with 2 × phosphate buffered saline (PBS) of 0.5mL (2 × PBS, Roche) Twice, and with the 100mM sodium citrate of 0.5mL pH 5.0 is of short duration it washs four times.Pass through 35 μ l's of addition LDS sample buffer (Invitrogen) carrys out the antibody of elution of bound.By half sample respectively withSample reduction Agent mixing keeps not restoring, and heats 10 minutes at 70 DEG C.5-30 μ l is applied to 4-12% as a result, Bis-Tris SDS-PAGE (Invitrogen) (carries out non-reduced SDS-PAGE using MOPS buffer, and use hasThe MES buffer of antioxidant running buffer solution additive (Invitrogen) carries out reduction SDS-PAGE), And use Coomassie blue stain.

The concentration of the antibody and derivative in cell culture supernatant is measured by affine HPLC chromatogram standard measure.Letter and Yan Zhi, by the cell culture supernatant containing antibody and derivative in conjunction with a-protein be applied to 200mM KH2PO4, 100mM sodium citrate, the Applied Biosystems Poros A/20 column in pH 7.4, and in Agilent HPLC 1100 200mM NaCl, 100mM citric acid are used in system, pH 2.5 is eluted from matrix.By UV absorbance and integrating peak areas come The protein of quantitative elution.The standard IgG1 antibody of purifying is used as standard items.

Alternatively, measuring the concentration of antibody and derivative in cell culture supernatant by Sandwich-IgG-ELISA. In brief, by StreptaWell High Bind Strepatavidin A-96 hole microtiter plate (Roche) with 100 μ L/ <h-Fc γ>BI (Dianova) is coated with the biotinylated anti-human igg capture molecule F (ab ') 2 of the 0.1 μ g/mL in hole at room temperature It 1 hour or is coated at 4 DEG C overnight, is then washed three times with the tween (PBST, Sigma) of the PBS in 200 holes μ L/, 0.05%. The cell culture supernatant of PBS (Sigma) dilution series containing corresponding antibodies in 100 holes μ L/ is added in hole, and in room It is incubated for 1-2 hours on microtiter plate shaker under temperature.Hole is washed three times with the PBST in 200 holes μ L/, and with 100 μ l The F (ab ') 2<hFc γ>POD (Dianova) of 0.1 μ g/mL is as detection antibody, at room temperature on microtiter plate shaker Detect the antibody of combination within 1-2 hours.Unbonded detection antibody is washed away three times with the PBST in 200 holes μ L/, and passes through addition The detection antibody of combination is detected in 100 holes L ABTS/ μ.On Tecan Fluor spectrometer, in the measurement wavelength (ginseng of 405nm Wavelength is examined to execute absorbance measurement under 492nm).

Protein purification

Reference standard scheme protein purification from the cell culture supernatant of filtering.In short, antibody is applied to Protein A Sepharose column (GE healthcare) is simultaneously washed with PBS.At pH 2.8 realize antibody elution, later immediately in And sample.By size exclusion chromatography (Superdex 200, GE Healthcare) in PBS or 20mM histidine, The protein of aggregation is separated with monomeric igg in 150mM NaCl pH 6.0.Monomeric igg fraction is merged, using for example (if desired) is concentrated in MILLIPORE Amicon Ultra (30MWCO) Centrifugal concentrators, freezes and is stored in -20 DEG C or -80 DEG C Under.The part of sample is provided for example to carry out subsequent albumen by SDS-PAGE, size exclusion chromatography (SEC) or mass spectrography Matter analysis and analysis and characterization.

SDS-PAGE

It is used according to the explanation of manufacturerPrecast gel system (Invitrogen).Specifically, it uses 10% or 4-12%Bis-TRIS precast gel (pH 6.4) and MES (reduction gel, hasAntioxidant running buffer additive) or MOPS (nonreducing gel) electrophoretic buffer.

Analyze size exclusion chromatography

It is executed by HPLC chromatogram method for measuring the aggregation of antibody and the size exclusion chromatography (SEC) of oligomeric state.Letter For it, Protein A purified antibody is applied to 300mM NaCl, the 50mM in 1100 system of Agilent HPLC KH₂PO₄/K₂HPO₄, Tosoh TSKgel G3000SW column in pH 7.5, or be applied on Dionex HPLC system 2 × 200 column of Superdex (GE Healthcare) in PBS.By UV absorbance and integrating peak areas come the albumen of quantitative elution Matter.BioRad gel filtration standards 151-1901 is used as standard items.

Mass spectrum

This section describes the characterization to the multi-specificity antibody for exchanging (VH/VL CrossMab) with VH/VL, with emphasis on In the correct assembly of the multi-specificity antibody.Disappeared by complete CrossMab to de-glycosylation and de-glycosylation/fibrinolysin Change or de-glycosylation/restricted LysC digestion CrossMab carries out electrospray ionization mass spectrometry (ESI-MS), to analyze expection Primary structure.

It will be in VH/VL CrossMab phosphate or Tris buffer with the protein concentration of 1mg/ml at 37 DEG C N- glycosidase F de-glycosylation at most 17h.Plasmin digestion or limited LysC (Roche) digestion are in the Tris buffer of pH 8 The deglycosylated VH/VL CrossMab of 100 μ g execute at room temperature 120 hours and execute 40min at 37 DEG C respectively.In Before mass spectral analysis, by sample via HPLC desalination on Sephadex G25 column (GE Healthcare).Equipped with In the maXis 4G UHR-QTOF MS system (Bruker Daltonik) in the source TriVersa NanoMate (Advion) via ESI-MS measures gross mass.

Using surface plasma body resonant vibration (SPR) measurement multi-specificity antibody to the binding affinity of corresponding antigens (BIACORE)

It is carried out by using BIACORE instrument (GE Healthcare Biosciences AB, Uppsala, Sweden) Surface plasma body resonant vibration studies the combination of the antibody and corresponding antigens of generation.Software is assessed using corresponding Biacore Analysis sensing figure and calculating affinity data.

Example 1

The generation of anti-PD-1 antibody

The immunity inoculation of mouse

NMRI mouse is to pass through first through inherent immunity using the plasmid expression vector of encoding full leng people PD-1 Subcutaneous administration 100ug carrier DNA (plasmid15300_hPD1-fl), then using electroporation, (2 1000V/cm's is rectangular Pulse continues 0.1ms, is spaced 0.125s；It is followed by the square pulse of 4 287.5V/cm, continues 10ms, is spaced 0.125s). Mouse received continuous immunity inoculation for any 6 days in the 0th, 14,28,42,56,70 and 84 day.It took a blood sample at the 36th, 78 and 92 day And serum is prepared, for carrying out titration (seeing below) by ELISA method.At the 96th day, the highest mouse of potency is selected, is led to It crosses and is injected intravenously 50ug recombined human PD1 people Fc chimera to it to enhance.3 days after enhancing, by splenocyte and myeloma Cell line is merged, and separates monoclonal antibody by hybridoma technology.

The measurement (ELISA) of serum titer

People's recombination PD1 people Fc chimera (hole 0.3ug/ml, 100ul/) is fixed on 96 hole NUNC in PBS It on Maxisorp plate, then carries out the following processing: closing the plate with the 2%Crotein C (hole 200ul/) in PBS；It applies With serial dilution antiserum (in duplicate, in the 0.5%Crotein C in PBS, the hole 100ul/)；The goat-anti being conjugated with HRP (Jackson Immunoresearch/Dianova 115-036-071,000) 1/16 detects mouse antibody.For all of above Step, plate are incubated for 1 hour at 37 DEG C.Between all steps, washed 3 times with the 0.05%Tween20 in PBS.By adding The BM indigo plant POD soluble substrate (Roche) in the hole 100ul/ is added to generate signal；By adding 1M HCl (hole 100ul/) stop signal. Using 690nm as reference, the light absorption value at 450nm is read.Potency is defined as the half peak signal that dilution antiserum generates.

Example 2

Characterize the combination of anti-PD1 antibody/anti-PD1 antibody and people PD1

ELISA method detection hu PD1

The coated plate of Nunc maxisorp Streptavidin (MicroCoat#11974998001) is with 25 holes μ l/ Biotinylated PD1-ECD-AviHis is coated, and is incubated overnight at 4 DEG C.It washs in (PBST- buffer, 3 × 90 holes μ l/) Afterwards, the anti-PD1 sample of 25 μ l or reference antibody (the anti-PD1 of people is added；The anti-PD1 of Roche/mouse；Biolegen；Catalog number (Cat.No.) 329912), and It is incubated for 1 hour at room temperature.After washing (PBST- buffer, 3 × 90 holes μ l/), with the dilution of 1:2000/1:1000, it is added Goat-anti people H+L-POD (JIR, the JIR109-036-088)/sheep anti mouse-POD (GE Healthcare, NA9310) in 25 holes μ l/, And it is incubated at room temperature on shaking table 1 hour.After washing (PBST- buffer, 3 × 90 holes μ l/), the bottom TMB in 25 holes μ l/ is added Object (Roche, catalog number (Cat.No.) 11835033001), and being incubated for OD value is 2-3.It is measured at 370/492nm.

The result of ELISA is with EC₅₀Value [ng/ml] is listed in the summary sheet 1 and summary sheet 2 of lower section.

Cell LISA method detects PD1

Adherency CHO-K1 cell line is steadily transfected with the plasmid 15311_hPD1-fl_pUC_Neo of encoding full leng people PD1, And selected with G418 (neomycin resistance marker on plasmid), then connect with the concentration of 0.01 × 10E6 cells/well Kind is grown overnight in 384 hole plates.

The PD1 sample or the anti-PD1 of people (Roche)/mouse anti-PD1 (Biolegend, catalog number (Cat.No.) in 25 holes μ l/ is added in next day 329912) reference antibody, and be incubated at 4 DEG C 2 hours (to avoid internalization).Carefully after washing (PBST, 1 × 90 hole μ l/), lead to It crosses and 0.05% glutaraldehyde for being diluted in 1 × PBS buffer solution (Sigma, catalog number (Cat.No.) G5882,25%) (30 hole μ l/) is added by cell It is fixed, and be incubated for 10 minutes at room temperature.After washing (PBST, 3 × 90 holes μ l/), the secondary antibody in 25 holes μ l/: goat-anti people H+ is added L-POD (JIR, JIR109-036-088)/sheep anti mouse-POD (GE NA9310) is for detecting, then on shaking table at room temperature It is incubated for 1 hour.After washing (PBST, 3 × 90 holes μ l/), the tmb substrate solution (Roche 11835033001) in 25 holes μ l/ is added, And being incubated for OD value is 1.0-2.0.At 370/492nm, plate is measured.

The result of cell ELISA is with " EC₅₀CHO-PD1 " value [ng/ml] is listed in the table 2 of lower section.

ELISA method detection cyno PD1

The coated plate of Nunc maxisorp Streptavidin (MicroCoat#11974998001) is with 25 holes μ l/ Biotinylated cynoPD1-ECD- biotin is coated, and is incubated overnight at 4 DEG C.Wash (PBST- buffer, 3 × 90 μ The hole l/) after, the anti-PD1 sample of 25 μ l or reference antibody (the anti-PD1 of people, Roche) is added, and it is small to be incubated on shaking table 1 at room temperature When.After washing (PBST- buffer, 3 × 90 holes μ l/), with the dilution of 1:1000, the goat-anti people H+L-POD in 25 holes μ l/ is added (JIR, JIR109-036-088), and be incubated at room temperature on shaking table 1 hour.It washs in (PBST- buffer, 3 × 90 holes μ l/) Afterwards, the tmb substrate (Roche, 11835033001) in 25 holes μ l/ is added, and being incubated for OD value is 2-3.It is carried out at 370/492nm Measurement.

The result of ELISA is with EC₅₀Value [ng/ml] is listed in the summary sheet 1 and summary sheet 2 of lower section.

PD ligand 1 alternate test

The coated plate of Nunc maxisorp Streptavidin (MicroCoat#11974998001) is with 25 holes μ l/ Biotinylated PD1-ECD-AviHis is coated, and is incubated overnight at 4 DEG C.It washs in (PBST- buffer, 3 × 90 holes μ l/) Afterwards, the anti-PD1 sample of 25 μ l or reference antibody (mouse anti-PD1, Biolegen, catalog number (Cat.No.) 329912) is added, and in room on shaking table Temperature is lower to be incubated for 1 hour.After washing (PBST- buffer, 3 × 90 holes μ l/), PD-L1 (the recombinant human B 7-H1/ in 25 holes μ l/ is added PD-L1 Fc chimera, 156-B7, R&D), and be incubated at room temperature on shaking table 1 hour.Washing (PBST- buffer, 3 × 90 The hole μ l/) after, with the dilution of 1:1000, the goat-anti people H+L-POD (JIR, 109-036-088) in 25 holes μ l/ is added, and shaking It is incubated at room temperature on bed 1 hour.After washing (PBST- buffer, 3 × 90 holes μ l/), tmb substrate (sieve in 25 holes μ l/ is added Family name, 11835033001), and being incubated for OD value is 2-3.It is measured at 370/492nm.

The result of ELISA is with IC₅₀Value [ng/ml] is listed in the summary sheet 1 of lower section.

2 alternate test of PD ligand

The coated plate of Nunc maxisorp Streptavidin (MicroCoat#11974998001) is with 25 holes μ l/ Biotinylated PD1-ECD-AviHis is coated, and is incubated overnight at 4 DEG C.It washs in (PBST- buffer, 3 × 90 holes μ l/) Afterwards, the anti-PD1 sample of 25 μ l or reference antibody (the anti-huPD1 of mouse, Roche) is added, and is incubated at room temperature on shaking table 1 hour.It washes After washing (PBST- buffer, 3 × 90 holes μ l/), be added 25 holes μ l/ PD-L2 (recombinant human B 7-DC/PD-L2Fc chimera, 1224-PL-100, R&D), and be incubated at room temperature on shaking table 1 hour.After washing (PBST- buffer, 3 × 90 holes μ l/), With the dilution of 1:2000, the goat-anti people H+L-POD (JIR, 109-036-088) in 25 holes μ l/ is added, and in room temperature on shaking table It is lower to be incubated for 1 hour.After washing (PBST- buffer, 3 × 90 holes μ l/), TMB (tetramethyl benzidine) substrate in 25 holes μ l/ is added (Roche, #11835033001), and being incubated for OD value is 2-3.It is measured at 370/492nm.

The result of ELISA is with IC₅₀Value [ng/ml] is listed in the summary sheet 1 of lower section.

The measurement of epitope mapping ELISA/ binding competition

Nunc maxisorp plate (Nunc#464718) be with the capture antibody in 25 holes μ l/ (sheep anti-mouse igg, JIR, It is 115-006-071) coated, and be incubated at room temperature on shaking table 1 hour.It washs in (PBST- buffer, 3 × 90 holes μ l/) Afterwards, it is closed at room temperature on shaking table 1 hour with the 2%BSA containing PBS buffer solution.Wash (PBST- buffer, 3 × 90 μ l/ Hole) after, the 25 anti-PD1 samples of μ l mouse are added, and be incubated at room temperature on shaking table 1 hour.Wash (PBST- buffer, 3 × 90 μ The hole l/) after, with the mouse IgG (JIR, 015-000-003) in 30 holes μ l/ closing capture antibody 1 hour at room temperature on shaking table.With This simultaneously, with the second sample antibody the biotinylated PD1-ECD-AviHis of preincubate 1 hour at room temperature on shaking table.It washes After washing assay plate (PBST- buffer, 3 × 90 holes μ l/), PD1 antibody mixture is transferred to assay plate, and in room on shaking table Temperature is lower to be incubated for 1 hour.After washing (PBST- buffer, 3 × 90 holes μ l/), with the dilution of 1:4000, the chain in 25 holes μ l/ is added Mould Avidin POD (Roche, #11089153001), and be incubated at room temperature on shaking table 1 hour.Washing (PBST- buffer, 3 × 90 holes μ l/) after, the tmb substrate (Roche, #11089153001) in 25 holes μ l/ is added, and being incubated for OD value is 1.5-2.5.In It is measured at 370/492nm.Epitope group is defined by the hierarchical clustering of control reference antibody.

Table 1: in conjunction with PD-L1 inhibits and the epitope district's groups (ELISA) of exemplary antibodies

Table 2: the biochemistry and cell combination of the humanization PD1 antibody from parent mouse antibody PD1-0103 (ELISA)

The Biacore of the anti-PD-1 antibody of humanization is characterized

Using measuring several mouse PD1 bonding agents and commercialization based on the test of surface plasma body resonant vibration (SPR) Binding kinetics parameter between people's PD1 combination reference substance.Therefore, by being coupled to (Biacore) CM5 sensor chip surface The fixed anti-human igg of amine.Then sample is captured, and makes hu PD1-ECD in conjunction with them.After each analysis circulation, regeneration is passed Sensor chip surface.By the way that data are fitted to 1:1 Lang Gemiaoer (langmuir) interaction model, average out often Several and Kinetics Rate Constants By Using.

By using the amine coupling kit provided by GE Healthcare, will about 2000 response units (RU) 20 μ G/ml anti-human igg (GE Healthcare#BR-1008-39) is coupled to the CM5 sensor in the Biacore T200 of pH 5.0 The flow cell 1 and 2 (or 3 and 4) of chip.

Sample and running buffer are HBS-EP+ (0.01M HEPES, 0.15M NaCl, 3mM EDTA, 0.05%v/v table Face activating agent P20, pH 7.4).Flow cell temperature is set as 25 DEG C, and sample compartment temperature is set as 12 DEG C.It is loaded with running buffer System.

Sample is injected 20 seconds with the concentration of 10nM, and is bound to the second flow cell.Then by concentration (144nM, 48nM, 16nM, 5.33nM, 1.78nM, 0.59nM, 0.20nM and 0nM) it is injected into above each sample for a whole set of people PD1-ECD, Continue 120s.It is followed by the Dissociation time of 30/300s, and with 3M MgCl₂The 20s regeneration step twice carried out, wherein finally It include once " the additional washing after injection " carried out with running buffer.

Finally, double reference data is fitted to 1:1Langmuir phase interaction using Biacore T200 assessment software Use model.It obtains_D、k_aAnd k_dValue is as shown in table 3.

Table 3: normal by the Kinetics Rate Constants By Using and balance of the chimeric PD1-0103 and humanization PD1-Ab of Biacore measurement Number

Ligand	k_a[M^-1s^-1]	k_d[s^-1]	K_D[nM]
				Chimeric PD1-0103	3.86E+05	3.07E-04	0.8
PD1-0103-0312	1.95E+05	3.45E-04	1.8
				PD1-0103-0313	1.60E+05	3.67E-04	2.3
PD1-0103-0314	1.87E+05	2.79E-04	1.5
				PD1-0103-0315	1.89E+05	2.91E-04	1.5

As shown in table 3, all humanization versions for being fitted into PD1-0103 (it is generated referring to example 6) are shown and parental antibody (chimeric PD1-0103) similar kinetic characteristics.

Dynamics

CM5 transducer series S is installed to 4000 system of Biacore, test point is carried out according to the explanation of manufacturer Fluid dynamics processing.

By multi-clone rabbit IgG antibody<IgGFC γ M>R (Jackson ImmunoResearch Laboratories Inc. it) is fixed on the test point 1 and 5 in flow cell 1,2,3,4 with 10000RU.Pass through EDC/NHS according to the explanation of manufacturer Chemical process completes coupling.Remaining test point in flow cell is as reference.Sample buffer is supplement 1mg/ml carboxymethyl Portugal The system buffer liquid of polysaccharide gel.

In one embodiment, which drives at 25 DEG C.In another embodiment, which drives at 37 DEG C It is dynamic.By capturing each mouse monoclonal antibody of 50nM on the sensor surface with the speed injection 1min of 10 μ l/min.Then, By each antigen with 100nM, 2 × 33nM, the series of concentrations of 11nM, 4nM, 1nM and system buffer liquid 0nM with the speed of 30 μ l/min Injection, association phases-time are 4min.Dissociation 4min is monitored again.It utilizes with the speed injection 10mM pH's 1.5 of 30 μ l/min Glycine 3min regenerates capture systems.Software is assessed to calculate dependent dynamics using Biacore according to the explanation of manufacturer Data.

Epitope mapping

As manufacturer is recommended, Biacore CAP sensor is installed to 4000 instrument of Biacore, and prepare It is good.Buffering instrument liquid is HBS-ET (HEPES, 150mM NaCl, 3mM EDTA, the 0.005%w/v Tween of 10mM pH 7.4 20).Instrument is run at 25 DEG C.

All samples dilute in system buffer liquid.By in the flow cell 1,2,3 and 4 in test point 1 and 5 with 30 μ 35kDa biotinylated antigen PD1-ECD-AviHis is trapped in CAP sensor sheet with 200RU by the speed injection 1min of l/min Face.Test point 2,3 and 4 is used as reference.In another embodiment, in the same way by 35kDa biotinylated antigen PD1- ECD-AviHis is trapped in CAP sensor surface with 200RU.

Then, with the primary antibody 3min of the speed injection 100nM of 30 μ l/min, then with the speed injection of 30 μ l/min The secondary antibody 3min of 100nM.Primary antibody is injected until the surface for presenting antigen is fully saturated.Terminate in primary antibody and secondary antibody injection stage When, " advanced stage combination " (BL) reporting point is set to monitor the combining response of each antibody.Calculate molar ratio, i.e. secondary antibody combining response Quotient between " BL2 " and primary antibody combining response " BL1 ".Molar ratio is used as when antigen can by the antigen of primary antibody compound tense secondary antibody And property index.

By with 30 μ l/min inject manufacturer recommend 2M guanidine hydrochloride and 250mM NaOH regeneration buffer 2min by Compound is removed completely from sensor surface, then with the speed injection system buffer liquid 1min of 30 μ l/min.

Example 3

The influence that the different anti-PD-1 antibody on cell factors generate in mixed lymphocyte reaction (MLP) (MLR)

3A) mixed lymphocyte reaction (MLP) (MLR) is immunocyte test, and measurement comes from the leaching of an individual (donor X) The activation of bar cell to the lymphocyte from another individual (donor Y).Mixed lymphocyte reaction (MLP) blocks PD1 for showing Influence of the access to lymphocyte effector cell.In test, in the case that detection is presence or absence of anti-PD1 monoclonal antibody The activation of T cell and their IFN γ secretion.

In order to carry out allogeneic MLR, using Leukosep, (Greiner Bio One, 288) 227 pass through density gradient The peripheral blood mononuclear cells (PBMC) of healthy donors of the centrifugal process separation from least four unknown HLA types.In brief, liver Elementization blood sample is diluted with the PBS of three times volume, and the 25ml aliquot of dilute blood is in 50ml Leukosep It is layered in test tube.After being centrifuged 15 minutes (no to interrupt) at room temperature with 800g, the fraction containing lymphocyte, In are collected Functional trial is washed and be directly used in PBS or refrigerant (10%DMSO, 90% are resuspended in 1.0E+07 cell/ml FCS it in), and is stored in liquid nitrogen.By with the stimulation cell/responsive cell ratio mixing of 1:1 from two different donors PBMC reacts to establish the two-way MLR of individual, and in 37 DEG C, 5%CO₂, presence or absence of various concentration range the anti-PD1 of purifying Monoclonal antibody (PD1-0050, PD1-0069, PD1-0073, PD1-0078, PD1-0098, PD1-0102 and PD1-0103) Under the conditions of, at least co-cultured 6 days in flat 96 orifice plate in duplicate.As anti-PD1 antibody is referred to, (have prominent with human IgG1 Become L234A, L235A and P329G (the EU index of Kabat)) main chain synthesize and clone including receiving Wu Dankang (also referred to as MDX- 5C4 or MDX-1106) or pyridine aldoxime methyliodide (PAM) monoclonal antibody (also referred to as MK-3475 or Org 1.09A) in any one VH and VL structural domain it is anti- Body.It without using antibody or uses Isotype control antibodies as negative control, uses rec hu IL-2 (20Eu/ml) as sun Property control.After the 6th day, 100 μ l culture mediums are taken out from each culture for cytokine measurements.Use OptEIA The level of ELISA kit (BD Biosciences) measurement IFN γ.

The results are shown in Table 4 (IFN γ secretion/release).Anti- PD1 monoclonal antibody promotes T thin with concentration dependant manner Born of the same parents' activation and IFN γ secretion.Relative to do not add any blocking mAb (the IFN γ value E-c of basic allogeneic stimulation induction) and The IFN γ for adding the MLR of 20EU/ml rec hu IL-2 (positive control=100%IFNg value E+c) generates, according to following public affairs Formula calculates the % value added of IFN γ secretion: opposite stimulation [%]=((sample-E-c)/(E+c-E-c) * 100

Table 4: IFN γ is secreted after allogeneic stimulates and anti-PD-1 antibody use to handle percentage and as positive control Recombinant human il-2 handles the comparison of (20EU/ml) (=100% increases) effect

By enhancing the secretion of interferon gamma (IFN γ), several PD1 blocking antibody PD1-0050, PD1-0069, PD1- 0073, PD1-0078, PD1-0098, PD1-0102 and PD1-0103 show strong immunoregulatory activities and (are not shown all The data of antibody).

3B) in further experiment, to chimeric PD1-0103 (have mutation L234A, L235A and P329G (Kabat's EU index) human IgG1's isotype) evaluate.PD1 is blocked consumingly to enhance allogeneic stimulation with chimeric PD1-0103 The IFN γ of primary human T-Cells is secreted.Chimeric PD1-0103 ratio acts on stronger with reference to anti-PD1 antibody.In order to compare, human IgG1 is used The main chain of (with L234A, L235A and P329G mutation (the EU index of Kabat)) is synthesized and is cloned including receiving Wu Dankang (also referred to as For VH the and VL structural domain of any one in MDX5C4 or MDX-1106) and pyridine aldoxime methyliodide (PAM) monoclonal antibody (also referred to as MK-3475 or Org 1.09A) The anti-PD1 antibody of reference.

3C) in other experiment, as described above, fighting the humanization variants (figure of PD-1 antibody PD1-0103 in MLR Humanized antibody PD1-0103-0312 and PD1-0103-0314 in 2 and Fig. 3, separately please refer to lower section example 9) immunoregulation Active a) IFN γ discharges (secretion) b) TTNF- α discharges (secretion) and evaluated.Compare chimeric PD1-0103 antibody and its people The effect of source version and the anti-PD1 antibody of reference.(there is L234A, L235A and P329G with human IgG1 with reference to anti-PD1 antibody Be mutated (the EU index of Kabat)) main chain, including receive Wu Dankang (also referred to as MDX5C4 or MDX-1106) and pyridine aldoxime methyliodide (PAM) monoclonal antibody ( Referred to as MK-3475 or Org1.09A) in any one VH and VL structural domain.After MLR is cultivated 6 days, 50 μ l supernatants are obtained And use Bio-Plex Pro^TMHuman cell factor Th1/Th2 measuring method (Bio-Rad Laboratories Inc.) is in single culture The middle multiple cell factors of measurement.(data that all cell factors are not shown).Compared to anti-PD1 antibody is referred to, it is fitted into PD1- 0103 antibody and its humanization pattern (PD1-0103_0312 and PD1-0103_0314) more effectively enhance T cell activation and IFN-γ secretion.In addition, chimeric PD1-0103 antibody and its humanization variants increase the tumor necrosis factor α of antigen presenting cell (TNF α) and IL-12 secrete and enhance the ability of monocyte/macrophage or antigen presenting cell stimulation T cell.

Example 4

Anti- PD-1 blocks the cytotoxicity granzyme to the people's CD4 T cell co-cultured with allogeneic mature dendritic cell The influence of B release and IFN-γ secretion

The influence in allogeneic environment is handled in order to further study anti-PD-1, we have developed a kind of measuring method, It is wherein that the CD4 T cell of fresh purifying is total in the presence of allogeneic mature dendritic cell (mDC) of cells of monocytic origin Culture 5 days.Monocyte was separated from fresh PBMC by plastic adherence before one week, removes non-adherent cell later.Then I Generated over 5 days by monocyte by being cultivated in the culture medium containing GM-CSF (50ng/ml) and IL-4 (100ng/ml) Immature DC.In order to induce iDC mature, TNF-α, IL-1 β and IL-6 (each 50ng/ml) are added to culture medium, then trained by us It supports 2 days.Then we are by measuring II class Main Tissues using flow cytometry (LSRFortessa, BD Biosciences) Histocompatibility complex (MHC II), the surface expression of CD80, CD83 and CD86 are mature to assess DC.

On the day of the mixed lymphocyte reaction (MLP) (mMLR) of minimum, via microballon kit (Miltenyi Biotec) from 10 obtained from irrelevant donor⁸CD4 T cell is enriched in a PBMC.Before culture, with 5 μM of carboxy-fluorescein Element-succinimide ester (CFSE) marks CD4 T cell.Then by 10⁵A CD4 T cell is together with mature allogeneic DC (5:1) is plated in 96 hole plates, wherein being 10 μ g/ml (if do not indicated differently in figure presence or absence of concentration Words) the anti-PD1 antibody of blocking (PD1-0103, chimeric PD1-0103 or humanized antibody PD1-0103-0312, PD1-0103- 0313, PD1-0103-0314, PD1-0103-0315, be abbreviated as 0312,0313,0314,0315).

After five days, cell culture supernatant is collected, and horizontal for measuring IFN-γ by ELISA (R&D system).Make thin It is small that born of the same parents place 5 again in the presence of Golgi Plug (brefeldin A) and Golgi Stop (coban) at 37 DEG C When.Be washed out cell, with anti-human CD4 antibody and Live/Dead can fixed dye Aqua (Invitrogen) carry out surface dye / permeabilization is fixed with Fix/Perm buffer (BD Bioscience) later in color.By granzyme B (BD Bioscience), IFN-γ and IL-2 (then both come from eBioscience) carry out cell inner dyeing.

It was found that all humanization variants PD1-0103 (humanized antibody PD1-0103-0312, PD1-0103-0313, PD1- 0103-0314, PD1-0103-0315, be abbreviated as 0312,0313,0314,0315) in terms of enhancing granzyme B and interferon gamma Equally good (data are not shown).

Example 5

Chimeric PD1 antibody derivatives

Pass through the variable weight district and variable light via the anti-PD1 mouse antibodies PD1-0098 and PD1-0103 of PCR amplification Area, and they are cloned into heavy chain expression vector and light chain expression vector and generates chimeric PD1 antibody, the heavy chain expression Carrier is with mutation L234A, L235A and P329G (the EU index of Kabat) (leucine 234 comprising eliminating effector function Sport alanine, leucine 235 sports alanine, proline 3 29 sports glycine) human IgG1's skeleton/people CH1- The form of the fusion protein of hinge-CH2-CH3, the light chain expression vector are the form with the fusion protein of people C- κ.Then will LC and HC plasmid co-transfection is purified from supernatant into HEK293, and after 7 days by the standard method of antibody purification. Chimeric PD1 antibody renames to be fitted into chiPD1-0098 (chiPD1-0098) and chimeric PD1-0103 (chiPD1-0103). In order to compare, is synthesized and cloned with the main chain of human IgG1's (with L234A, L235A and P329G mutation (the EU index of Kabat)) Appoint in Wu Dankang (also referred to as MDX-5C4 or MDX-1106) and pyridine aldoxime methyliodide (PAM) monoclonal antibody (also referred to as MK-3475 or Org 1.09A) including receiving The anti-PD1 antibody of the reference of the VH and VL structural domain of one.

Example 6

Generation, expression and the purifying and characterization of the humanization variants (huMab PD-0103) of anti-PD1 antibody PD-0103

The humanization of the VH and VL structural domain of the anti-PD1 antibody 0103 of mouse

The amino acid sequence (SEQ ID NO:7 and 8) of mouse VH and VL structural domain based on the anti-PD1 antibody PD1-0103 of mouse, Generate the anti-PD1 antibody variants of humanization.

The VH variant of humanization is based on the ethnic group system combined with people's J- element germline IGHJ5-01 with several mutation IMGT_hVH_3_23.(generating SEQ ID NO:9).

The humanization variants of VL based on the ethnic group system IMGT_hVK_4_1 combined with people's J- element germline IGKJ1-01, IMGT_hVK_2_30, IMGT_hVK_3_11 and IMGT_hVK_1_39.Different mutation generates SEQ ID NO:10 to SEQ ID The humanization variants of NO:13.

DNA is translated by the humanization amino acid sequence of the heavy chain variable region of PD1-0103 and light chain variable region is counter, synthesis Resulting cNDA (GenArt), and be then cloned into heavy chain expression vector or light chain expression vector, the heavy chain table It is that (leucine 234 sports alanine, leucine 235 with LALA and the PG mutation comprising eliminating effector function up to carrier Sport alanine, proline 3 29 sports glycine) human IgG1's skeleton/people's CH1- hinge-CH2-CH3 fusion protein Form, the light chain expression vector is form with the fusion protein of people C- κ.Then LC and HC plasmid co-transfection is arrived In HEK293, and purified from supernatant by the standard method of antibody purification after 7 days.Resulting humanization PD1 is anti- Body name is as follows:

VH the and VL sequence of the humanization variants antibody of table 5:PD1-0103

Humanization PD1-0103 antibody variants and parent chimeric PD1-0103 are characterized as described above.As a result in table 6 It shows.

Table 6: the result of humanization PD1-0103 antibody variants and parent chimeric PD1-0103 summarize

Humanization variants PD-0103-0312 is hereinafter referred to as PD1 antibody cloning PD1-0376.

Example 7

The generation of anti-LAG3 antibody

Rabbit is immunized

The Roche proprietary (Roche proprietary) of expression humanized antibody spectrum is turned with the Plasmid DNA of expression LAG3 Gene rabbit is immunized.

Using the plasmid expression vector of encoding full leng people LAG3 (15352_pIntronA_fl-hLag3_DNA-IMS), lead to Intradermal application 400ug carrier DNA is crossed, electroporation (5 rectangular pulses of 750V/cm, duration 10ms, interval are then carried out 1s), inherent immunity is carried out to one group of 3 rabbit.Rabbit was at the 0th day, the 14th day, the 28th day, the 49th day, the 70th day, the 98th day With 7 continuous immunities of receiving in the 126th day.In the 35th day, the 77th day, the 105th day and the 133rd day acquisition blood, (estimated was total The 10% of blood volume).Preparation separates peripheral blood for carrying out the serum of titer determination (see below) by ELISA Monocyte, the peripheral blood mononuclear cells are used as the source of the antigen-specific b cells in hereafter B cell cloning procedure.

The measurement (ELISA) of serum titer

People is recombinated into LAG3 albumen, 96 hole NUNC Maxisorp plates are immobilized onto PBS with the hole 2ug/ml, 100ul/ On, then: plate is used in the 2%Crotein C in PBS and is closed, the hole 200ul/；By sero-fast one formula two of serial dilutions Part is applied in the 0.5%Crotein C in PBS, the hole 100ul/；Use the donkey anti-rabbit IgG antibody of (1) HRP conjugation (Jackson Immunoresearch/Dianova 711-036-152；1/16000), or (2) HRP conjugation rabbit anti-human igg Antibody (Pierce/Thermo Scientific 31423；, or (3) biotinylated Goat anti-Human κ antibody 1/5000) (Southern Biotech/Biozol 2063-08,1/5000) and streptavidin-HRP are detected；Respectively dilution In 0.5%Crotein C in PBS, the hole 100ul/.For all steps, plate is incubated into 1h at 37 DEG C.In all steps Between rapid, plate is used in the 0.05%Tween 20 in PBS and is washed 3 times.It is soluble by the BM indigo plant POD for adding the hole 100ul/ Substrate (Roche) generates signal；By adding 1M HCl (hole 100ul/) stop signal.Using 690nm as reference, read at 450nm Light absorption value.Potency is defined as the half peak signal that dilution antiserum generates.

The separation of Peripheral Blood in Rabbits monocyte (PBMC)

Obtain the blood sample by immune transgenic rabbits.By 1 × PBS (the Austrian pa of the EDTA containing whole blood The PAA company (PAA, Pasching, Austria) in emerging city) twice of dilution, mammalian lymphocytes cell (Ontario is used later The laboratory Sai Dalan (Cedarlane Laboratories, Burlington, Ontario, Canada) in Burlinton city, province) root Density centrifugation is carried out according to the specification of manufacturer.PBMC is washed twice with 1 × PBS.

EL-4 B5 medium

Using RPMI 1640 (Germany Chinese mugwort step on Bach Pan Biotech company (PAN Biotech, Aidenbach, Germany)), and add 10%FCS (the extra large cloning companies (Hyclone, Logan, UT, USA) in the city Utah, USA Luo Gen), 2mM Glutamin, 1% penicillin/Streptomycin Solution (the PAA company in Pasing, Austria city), 2mM Sodium Pyruvate, 10mM HEPES (the PAN Biotech company that Germany Chinese mugwort steps on Bach) and the 0.05mM b- mercaptoethanol (Ji Bo of Scotland Paisley Company (Gibco, Paisley, Scotland)).

Plate is coated with proteantigen

By 6 hole plate of steril cell culture carbonate buffer solution (0.1M sodium bicarbonate, 34mM disodium bicarbonate, pH 9.55) the people LAG3 ECD (2 μ g/ml) for being conjugated in the part people Fc in is coated with overnight at 4 DEG C.Using preceding in sterile PBS Washing flat board is three times.

Cell exhausts

(A) using sterile 6 hole plate (cell culture grade) for being covered with the single layer Chinese hamster ovary celI being paved with, by non-specific Property adherency and non-specifically bind lymphocytes exhaust monocytes/macrophages.

(B) sterile 6 hole plate of blank (cell culture grade) is used, to exhaust macrophage and list by non-specific adhesion Nucleus and other cells.

The half of PBMC sample is used for (a) and half is used for (b).

Each hole is at most equipped with 4ml culture medium and at most 6 × 10⁶It is a from the PBMC through immunizing rabbit, and make its 1h is combined in incubator at 37 DEG C.Cell (peripheral blood lymphocytes (PBL)) in supernatant washes in a pan sieve step for antigen.

Enrichment of the B cell on LAG3 antigen

Proteantigen: every 4ml culture medium is inoculated with to the 6 hole tissue culture plates for being coated with LAG3-ECD-huFc albumen At most 6 × 10⁶It is a to exhaust the PBL of step from use 6 hole plate of blank, and combine it at 37 DEG C in the incubator 1h.By the way that with 1 × PBS, carefully washing hole 1-2 times removes non-adherent cell.Continue 10min at 37 DEG C in the incubator Make remaining sticky cell detachment by trypsase.Stop trypsinized using EL-4 B5 medium.Cell is maintained at On ice, until carrying out immunofluorescence dyeing.

Cell surface antigen: every 4ml is inoculated with to the 6 hole tissue culture plates for being covered with single layer people's LAG3 positive CHO cells Culture medium at most 6 × 10⁶It is a to exhaust the PBL of step from 6 hole plates for using CHO to cover, and make it in the incubator 1h is combined at 37 DEG C.By the way that with 1 × PBS, carefully washing hole 1-2 times removes non-adherent cell.In the incubator at 37 DEG C Continuing 10min down makes remaining sticky cell detachment by trypsase.Stop trypsinized using EL-4 B5 medium. Cell is kept on ice, until carrying out immunofluorescence dyeing.

Immunofluorescence dyeing and flow cytometry

Anti-igg FITC (Dusseldorf ,Germany AbD Serotec company (AbD Serotec, D ü sseldorf, Germany it)) is used for anti-huCk PE (the Dianova company (Dianova, Hamburg, Germany) of Hamburg, Germany) antibody Unicellular sorting.It, will be from exhausting and the cell of enriching step and the anti-igg FITC in PBS and resist for padding HuCk PE antibody incubates together, and incubates 45min at 4 DEG C in the dark.After dyeing, by PBMC with ice-cold PBS It washes twice.Finally, PBMC being resuspended in ice-cold PBS and carrying out facs analysis immediately.It is added before facs analysis dense Degree be 5 μ g/ml propidium iodide (California, USA San Diego BD Pharmingen company (BD Pharmingen, San Diego, CA, USA)) to distinguish dead cell and living cells.Equipped with the Becton Dickinson of computer FACSAria and FACSDiva software (BD Biosciences, the U.S.) is used for unicellular sorting.

B cell culture

The culture of rabbit B cell passes through Seeber et al. (S Seeber et al. PLoS One 9 (2), e86184.2014 4 days 2 months) described in method carry out.In brief, the EL- by the rabbit B cell singly sorted in 96 hole plates with 200 holes μ l/ 4 B5 mediums incubate 7 days at 37 DEG C in the incubator together, the EL-4 B5 medium contain Pansorbin cell (1: 100000) (the Calbiochem company (Merck) of Darmstadt, Germany (Calbiochem (Merck), Darmstadt, Deutschland)), 5% rabbit thymocyte supernatant (German Bell's grace Reed MicroCoat company (MicroCoat, Bernried, Germany)) and through γ irradiate mouse EL-4 B5 thymoma (5 × 10e5 cells/well).Remove B cell The supernatant of culture collects remaining cell immediately and it is frozen in 100 μ l RLT buffering at -80 DEG C to be used to screen In liquid (the Kai Jie company (Qiagen, Hilden, Germany) of Heerden, Germany).

The separation in the V structure domain of LAG3 antibody

The PCR amplification in V structure domain

Use NucleoSpin 8/96RNA kit (Macherey&Nagel；740709.4,740698) according to manufacture The scheme of quotient prepares total serum IgE by B cell lysate (being resuspended in RLT buffer-Qiagen- catalog number (Cat.No.) 79216).With 60 μ l without RNA enzyme water elution RNA.The RNA of 6 μ l is used to use III First-Strand Synthesis SuperMix of Superscript (Invitrogen 18080-400) and oligo dT primer generate cDNA by reverse transcriptase reaction according to the explanation of manufacturer. All steps carry out on Hamilton ML Star System.With the final volume of 50 μ l, AccuPrime is utilized Supermix (Invitrogen 12344-040) expands heavy chain immunoglobulin and light chain variable region (VH using the cDNA of 4 μ l And VL), primer BcPCR_FHLC_leader.fw is used for light chain using primer rbHC.up and rbHC.do for heavy chain With BcPCR_huC κ .rev (table 7).All forward primers (respectively VH and VL) have specificity to signal peptide, and all anti- There is specificity to constant region to primer (respectively VH and VL).PCR condition for RbVH is as follows: thermal starting, and 94 DEG C, 5min；94 DEG C of 20s, 70 DEG C of 20s, 68 DEG C of 45s, 35 circulations；And finally extend 7min at 68 DEG C.PCR for HuVL Condition is as follows: thermal starting, and 94 DEG C, 5min；94 DEG C of 20s, 52 DEG C of 20s, 68 DEG C of 45s, 40 circulations；And it is final at 68 DEG C Extend 7min.

Table 7

8 μ l in 50 μ l PCR solution are loaded on 48E-Gel 2% (Invitrogen G8008-02).Positive PCR Reaction solution uses II kit (Macherey&Nagel of NucleoSpin Extract；740609250) according to the side of manufacturer Case is purified, and is eluted in 50 μ l elution buffers.All purifying steps are in Hamilton ML Starlet It is carried out on System.

The recombinant expression of family's rabbit monoclonal bivalent antibody

In order to recombinantly express a rabbit monoclonal bivalent antibody, by jag PCR cloning PCR (RS Haun et al., Biotechniques(1992)13,515-518；MZ Li et al. people, Nature Methods (2007) 4,251-256) it will coding The PCR product of VH or VL is as cDNA clone into expression vector.Expression vector contains expression cassette, and the expression cassette is by including interior 5'CMV promoter and 3'BGH polyadenylation sequence composition containing sub- A.Other than expression cassette, plasmid, which also contains, to be derived from The replication orgin of pUC18 and the beta-lactam enzyme gene for assigning amicillin resistance, at Escherichia coli (E.coli) Middle carry out plasmid amplification.Three kinds of variants of basic plasmid, the basic plasmid are used are as follows: containing being designed to receive the area VH Family's rabbit igg constant region, simultaneously containing a kind of plasmid to receive people's κ LC constant region in the area VL.Pass through PCR using overlapping primers The linearisation expression plasmid of amplification coding κ or γ constant region and VL/VH insetion sequence.The PCR product of purifying is polymerize with T4DNA Enzyme incubates together, this generates single-stranded overhang.Stop reaction by addition dCTP.

In the next step, plasmid and insetion sequence merged and warm together with the recA of inductive site specificity recombination It educates.By recombinant plasmid transformed into Escherichia coli.Second day, picking grew bacterium colony and by plasmid preparation, restricted digestion Analysis and DNA sequencing test the plasmid correctly recombinated.

For antibody expression, by isolated HC and LC plasmid transient cotransfection into HEK293 cell, and harvested after 1 week Supernatant.

Example 8

The characterization of anti-LAG3 antibody

Table 8: different anti-LAG3 antibody characterizations summarize

The ELISA of people Lag3

The recombination for being 800ng/ml with the protein concentration in 25 holes μ l/ by Nunc maxisorp plate (Nunc 464718) People LAG-3Fc chimeric protein (R&D system, 2319-L3) coating, and be incubated overnight at 4 DEG C or incubate 1h at room temperature.It washes After washing (PBST buffer, 3 × 90 holes μ l/), by each hole with 90 μ l Block buffer (PBS+2%BSA+0.05%Tween 20) 1h is incubated at room temperature.After washing (PBST buffer, 3 × 90 holes μ l/), the concentration of 25 μ l of addition is the anti-of 1-9 μ g/ml Lag3 sample (the 1:3 dilution in OSEP buffer), and 1h is incubated at room temperature.Wash (PBST buffer, 3 × 90 μ The hole l/) after, with 1:2000 dilution add 25 holes μ l/ Goat anti-Human Ig κ chain antibody-HRP conjugate (Milipore, AP502P 1h), and is at room temperature incubated.After washing (3 × 90 holes μ l/, use PBST buffer), the TMB in 25 holes μ l/ is added Substrate (Roche, 11835033001), and incubate 2-10min.It is carried out on 2 instrument of Tecan Safire at 370/492nm Measurement.

Cell surface Lag3 combination ELISA

The Lag3 cell (recombinaant CHO cell of expression Lag3,10000 cells/wells) in 25 holes μ l/ is inoculated into through organizing In 384 hole plates (Corning, 3701) for cultivating processing, and incubated one or two day at 37 DEG C.It second day, is trained removing After supporting base, the anti-Lag3 sample (the 1:3 dilution in OSEP buffer, start with the concentration of 6-40nM) of 25 μ l is added, And 2h is incubated at 4 DEG C.After washing (1 × 90 μ l, in PBST), by adding the glutaraldehyde in 30 holes μ l/ to 0.05% Cell is fixed 10min by ultimate density (Sigma, catalog number (Cat.No.): G5882) at room temperature.Wash (PBST buffer, 3 × 90 μ l/ Hole) after, with 1:1000 dilution add 25 holes μ l/ Goat anti-Human Ig κ chain antibody-HRP conjugate (Milipore, AP502P 1h), and is at room temperature incubated.After washing (PBST buffer, 3 × 90 holes μ l/), the tmb substrate in 25 holes μ l/ is added (Roche, 11835033001), and incubate 6-10min.It is measured on 2 instrument of Tecan Safire at 370/492nm.

The SPR (Biacore) of anti-LAG3 antibody is characterized

The measuring method based on surface plasma body resonant vibration (SPR) is used to determine at 25 DEG C in bivalent form or as one The dynamics of combination between the anti-Lag3 antibody of valence Fab segment and the people Lag3 extracellular domain (ECD) of people's Fc labeling Parameter.

Therefore, two flow cells of C1 biologic sensor chip are by using " immobilization guide (immobilization Wizard the neutravidin of 25 μ g/ml) " will be diluted in the acetate buffer of pH 4.5 in Biacore T200 Albumen (neutravidin) is fixed thereon and prepares.The immobilization that this generates about 1900RU is horizontal.Then, using transporting 20 μ g/ml dilutions in row buffering liquid (HBS-EP+, GE Healthcare) make CaptureSelect^TMBiotin anti-igg- Fc (people) conjugate is in conjunction with neutravidin.

The each circulation of this method itself is made of four orders.First order: capture about 46RU huLag3-Fc (20s, 10μl/min).Second order: sample injection 120s, followed by the dissociation of long 1200s, flow velocity is 30 μ l/min.Third and fourth Order: 30s is regenerated by injection glycine-HCl (pH 1.5).Then each monoclonal antibody is measured using previously described method The dilution series (3.13nM-200nM, twice of dilution in running buffer) of segment and other blank cycle.Due to Capture level can not reproduce completely, therefore Rmax fitting parameter application 1:1 youth lattice Miao of " part " is then set as by application You are fitted (Langmuir fit), obtain dynamics using Biacore T200 assessment software.As a result (K_DValue and kd value) In It is shown in table 8.

Epitope mapping

Epitope is carried out using the measurement based on surface plasma body resonant vibration (SPR) to open a position.Therefore, aLag3 bonding agent (binder) on Biacore T200 instrument in conjunction with huLag3.Then, the aLag3 bonding agent-being previously formed is assessed Accessibility of the huLag3 compound to other bonding agents.

This measurement is carried out using SA CAP kit (GE Healthcare).If not in addition description, is tried according to SA CAP Agent box handbook carries out the measurement.Operation only includes a cyclical patterns.After hybridization, make biotinylated, huFc labeling HuLag3 10nM dilution in sensor core on piece the 20s in conjunction with streptavidin, flow rate be 10 μ l/min. Then 180s is continued with the flow rate of 30 μ l/min and injects the first 200nM sample being diluted in running buffer, and it Inject the second sample under the same conditions immediately afterwards.Then make surface regeneration.

Then sample is distributed into the different epitopes group with similar competitive mode.Using the threshold value of 6.1RU, based on the The relative response of two injections carries out the first rude classification, and the threshold value is just above when using bonding agent as the first and second samples Observed peak when injection.All values and judgement are finally verified by the visual inspection of sensing figure.

As a result it is shown in table 8.It authenticated three Primary epitope modes (E1, E2 and E3).Due to aLag3-0416 and people Source BAP 050 shares identical group, but not exclusively inhibits each other, therefore can distribute them to subgroup E2b and E2c.

The combination of anti-Lag3 antibody and recombination cyno Lag3 positive HEK cell from tg rabbit

In addition to use on the surface recombinantly express people Lag3 HEK cell be combined analysis other than, also have evaluated with The combination of machin Lag3 positive HEK cell.It, will be previously with the cyno-LAG-3 frost transiently transfected for this experiment HEK293F cell thaws, is centrifuged and is supplemented in PBS/2%FBS.By 1.5 × 10⁵It is flat that a cells/well is inoculated into 96 holes In plate.Anti- Lag3 antibody is added, until the ultimate criterion concentration of 10 μ g/ml.To be referred to and as control, make in an experiment Standby and measurement autofluorescence and positive control (Medarex 25F7) and isotype controls (huIgG1 from Sigma, catalogue Number #I5154, data are not shown) antibody.HEK cell incubates 45min together with instruction antibody on ice, with containing 2%FBS's 200 μ l ice-cold PBS buffer solution washes twice, later add secondary antibody (APC label Goat anti-Human IgG-κ, Invitrogen, Catalog number (Cat.No.) #MH10515) (1:50 is diluted in the hole FACS-Puffer/), and incubate 30min again on ice.Cell is used again The ice-cold PBS/2%FBS buffer of 200 μ l washes twice, and is then finally resuspended in sample in 150 μ l FACS buffer solutions, and And it measures and combines in II HTS module of FACS CANTO-.

As a result: under represent the combination of different anti-Lag3 antibody and the HEK293 cell of expression cynoLAG3 and intersect instead Ying Xing is provided in conjunction with the geometrical mean (GeoMean) of % positive cell or signal strength.

Table 9: anti-Lag3 antibody and the combination for recombinating cyno Lag3 positive HEK cell

LAG3 antibody	% positive cell	Geometrical mean
			With reference to LAG3 antibody MDX25F7	41.2	3062
aLAG3(0411)	88.6	11007
			aLAG3(0414)	81.6	9169
aLAG3(0416)	67.9	4221
			aLAG3(0417)	75.9	7115
aLAG3(0403)	82.0	7457

The combination of (activation) machin PBMC/T cell of anti-Lag3 antibody and expression Lag3 from tg rabbit

After the combination for the Lag3 for having evaluated and recombinating Lag3 albumen and recombinantly expressed on mammalian cell, also comment The combination of Lag3 estimated and expressed in the machin T cell of activation.

By facs analysis, newly-generated anti-Lag3 antibody (from the transgenic rabbits of Roche) is confirmed and in machin The binding characteristic of the Lag3 expressed on the cell surface of T cell or PBMC.Although Lag3 is not expressed on nave T cell, It is raised in activation and/or in the T cell of exhaustion.Therefore, machin peripheral blood is prepared by fresh machin blood Monocyte (PBMC) is then activated by CD3/CD28 pretreatment (1 μ g/ml) for 2-3 days.Then to activating cell Lag3 expression is analyzed: in brief, by 1-3 × 10⁵It is the meaning of 10 μ g/ml that a activating cell uses ultimate density on ice The anti-Lag3 antibody shown and corresponding control antibodies dye 30-60min.Via the anti-human igg or anti-rabbit IgG bis- of fluorescent dye conjugation It is anti-, detect the anti-Lag3 antibody combined.After dyeing, cell is washed twice with PBS/2%FCS, and in FACS It is analyzed on Fortessa (BD).

As a result: following table summarizes the percentage of the Lag3 positive cell in the machin PBMC of activation.

Table 10: the combination of different anti-LAG3 antibody and (activation) machin PBMC/T cell for expressing Lag3

In the machin T cell of activation, all rabbit-anti Lag3 antibody are shown and Lag3⁺The significant combination of cell.By This, Lag3 reference antibody (such as MDX25F7, BMS-986016) anti-compared to people, all newly-generated antibody show increase Positive cell percentage.

The inhibition (passing through ELISA) of the combination of LAG-3 and the MHC- II expressed on people's A375 tumour cell

The A375 cell (10000 cells/wells) in 25 holes μ l/ is inoculated into 384 hole plates through tissue culture treated In (Corning, 3701), and it is incubated overnight at 37 DEG C.Anti- Lag3 antibody with 1:3 dilution in cell culture medium Biotinylated Lag3 (250ng/ml) precincubation 1h together, is started with the antibody concentration of 3 μ g/ml.From with inoculating cell After removing culture medium in hole, 25 μ l antibody-Lag3 Preincubation mix are transferred in hole and incubate 2h at 4 DEG C.It washes After washing (1 × 90 μ l, in PBST), by add 30 μ l/ hole glutaraldehydes to 0.05% ultimate density (Sigma, catalog number (Cat.No.): G5882), cell is fixed into 10min at room temperature.After washing (PBST buffer, 3 × 90 holes μ l/), with 1:2000 or 1:8000 Dilution adds the poly- HRP40- streptavidin (Fitzgerald, 65R-S104PHRPx) in 25 holes μ l/, and in room Temperature is lower to incubate 1h.After washing (PBST buffer, 3 × 90 holes μ l/), tmb substrate (Roche, the # in 25 holes μ l/ are added 11835033001) and 2 to 10min are incubated.It is measured on 2 instrument of Tecan Safire at 370/492nm.

The inhibition (passing through facs analysis) of the combination of LAG-3 and the MHC- II expressed on people's A375 tumour cell

Measuring principle: the antagonism function in order to study anti-Lag3 antibody carries out II: Lag3 competition assay of MHC.With or not In the case where anti-Lag3 antibody precincubation, the biotinylated Lag3:Fc fusion protein generated with inside is to MHC II⁺People A375 cell is dyed.This analysis carries out in FACS competitive assay: A375 cell (ATCC, #CRL-1619) is being added Added with EBSS (PAN, catalog number (Cat.No.) #P04-00509), 10%FBS, 2mM L-Glutamin, 1 × NEAA and 1 × Sodium Pyruvate EM Eagle culture medium in cultivate 2-3 generation.The 20 μ g/ml that all antibody are diluted to 25 μ l in FACS buffer solution are finally dense It spends (in 96 hole U base plates).The biotinylated recombination LAG-3:Fc fusion protein generated inside 25 μ l is added to culture Base or anti-Lag3 antibody or control, until the ultimate density of 10 μ g/ml, and precincubation 30min at room temperature.A375 cell is used PBS is washed and is adjusted in PBS to 3 × 10⁶A cell/ml.Every hole is inoculated with 100 μ l in 96 hole V base plates.Plate is centrifuged And remove supernatant.Then LAG-3:Fc fusion protein/antibody mixture of precincubation (50 hole μ l/) is added to cell, and And 1h is incubated at room temperature.Hereafter, cell is washed with 200 μ l FACS buffer solutions.For the biotin combined with cell MHC II The detection of the Lag3:Fc albumen of change uses the goat anti-biotin antibodies (Miltenyi of APC conjugation with 3 μ l/ samples Biotec, catalog number (Cat.No.) #130-090-856), and 10-15min is incubated again.After dyeing, cell is washed again, then It is transferred in U base plate in 150 μ l FACS buffer solutions (PBS/2%FBS), and using HTS module in FACS Canto- It is analyzed on II.

Two anti-Lag3 antibody (clone 25F7 and 26H10；Medarex) it is used as positive control, and human IgG1 (Sigma, mesh Record #I5154) it is used as isotype controls appropriate.All antibody are used with 10 μ g/ml ultimate densities.

As a result: under represent facs analysis as a result, illustrating the suppression of the combination of the MHC- II on Lag3 albumen and cell Percentage (being calculated as with reference to there is no the binding signals that maximum value when blocking antibody reduces) processed.

Table 11: the combination of different anti-LAG3 antibody and (activation) machin PBMC/T cell for expressing Lag3

These data support the resistance to influence each other with the functionality of Lag3 and the cell of all test antibodies interacts It is disconnected.

Standard LAG3 blocks the neutralization effect of novel anti-Lag3 antibody in Bio/Reporter measurement

In order to test the neutralization effect that novel anti-Lag3 antibody restores repressed t cell response in vitro, using can quotient Purchase the reporting system obtained.This system is by Lag3⁺NFAT Jurkat effector cell (Pu Luomaige company, catalog number (Cat.No.) # CS194801)、MHC-Ⅱ⁺Raji cell (ATCC, #CLL-86) and superantigen composition.In brief, reporting system is based on Three steps: (1) the NFAT cell activation of superantigen induction, (2) are by inhibiting MHC II (Raji cell) and Lag3⁺ NFAT The inhibition for the activation signal that interaction between Jurkat effector cell mediates, and (3) pass through Lag3- antagonism/neutralization VH- Fc fusion constructs and restore NFAT activation signal.

For this experiment, Raji and Lag-3 is cultivated as described in supplier⁺Jurkat/NFAT-luc2 effector T cell.In In 96 hole culture plate (Costar, catalog number (Cat.No.) #3917) of white flat bottom, in measurement culture medium (1640 (PAN of RPMI Biotech, catalog number (Cat.No.) #P04-18047), 1%FCS) in prepare the serial dilutions (40pg/ of several anti-Lag3 and reference antibody ml-50μg/ml).By 1 × 10⁵A Lag3⁺NFAT-Jurkat cells/well is added to antibody-solutions.After this step, will 2.5×10⁴A Raji cells/well is added to Jurakt cell/antibody mixture and the SED of ultimate density 50ng/ml is super Antigen (Toxin technology, catalog number (Cat.No.) DT303).In 37 DEG C and 5%CO₂Lower incubation is after six hours, by the bottom Bio-Glo Object (Pu Luomaige company, #G7940) is warming up to room temperature and 75 μ l are added in every hole, 5-10min is incubated, then according to kit The recommendation of manufacturer measure overall luminous in Tecan Infinite plate reader.

The NFAT fluorescein that II/Lag3 of MHC as caused by different anti-Lag3 antibody is mediated in SED stimulation is shown in table The recovery that enzyme signal inhibits is (as EC₅₀Value provides):

Table 12: the result when standard LAG3 is blocked in Bio/Reporter measurement using different anti-LAG3 antibody

N.t. the molecule that do not tested in this experiment

Example 9

The functional characterization of anti-LAG3 antibody

Table 13 summarizes different anti-LAG3 antibody in different measurements as described herein (individually or with anti-PD1 antibody combination) Bioactivity and effect.

Table 13: the bioactivity of different anti-LAG3 antibody (individually or with anti-PD1 antibody combination) summarizes

PD-1 and LAG-3 blocks the cytotoxicity to the people's CD4 T cell co-cultured with allogeneic mature dendritic cell The influence of granzyme B release and IL-2 secretion

In order to screen the anti-lag-3 blocking antibody combined in allogeneic environment with anti-PD-1, a kind of measuring method is developed, It is wherein that the CD4 T cell of fresh purifying is total in the presence of allogeneic mature dendritic cell (mDC) of cells of monocytic origin Culture 5 days.Monocyte was separated from fresh PBMC by plastic adherence before one week, removes non-adherent cell later.Then lead to It crosses in the culture medium containing GM-CSF (50ng/ml) and IL-4 (100ng/ml) to cultivate 5 days and prematurity is generated by monocyte DC.In order to induce iDC mature, TNF-α, IL-1 β and IL-6 (each 50ng/ml) are added to culture medium, are further cultured for 2 days.Then By measuring II class major histocompatibility complex using flow cytometry (LSRFortessa, BD Biosciences) The surface expression of (MHC II), CD80, CD83 and CD86 are mature to assess DC.

On the day of the mixed lymphocyte reaction (MLP) (mMLR) of minimum, via microballon kit (Miltenyi Biotec) from 10 obtained from irrelevant donor⁸CD4 T cell is enriched in a PBMC.Before culture, with 5 μM of carboxy-fluorescein Element-succinimide ester (CFSE) marks CD4 T cell.Then by 10⁵A CD4 T cell and mature allogeneic DC (5: 1) it is plated in 96 hole plates together, wherein being the individual of 10 μ g/ml presence or absence of concentration or resisting with inosculating antibody LAG-3 What body (aLAG3 (0403) to aLAG (0418)) or reference antibody (humanization BAP050 (LAG525) and BMS 986016) combined Block anti-PD-1 antibody aPD1 (0376) (=PD1-0103-0312, such as previously herein or in PCT application PCT/EP2016/ Described in 073248).DP47 is the uncombined people having in the part Fc with to avoid the LALA mutation identified by Fc γ R IgG, and it is used as negative control.

After five days, collects cell culture supernatant and measure IL-2 level for passing through ELISA (R&D system), and make Cell places 5 at 37 DEG C in the presence of Golgi Plug (brefeldin A) and Golgi Stop (coban) again Hour.Be washed out cell, with anti-human CD4 antibody and Live/Dead can fixed dye Aqua (Invitrogen) carry out surface Dyeing, is fixed/permeabilization with Fix/Perm buffer (BD Bioscience) later.We are to granzyme B (BD Bioscience) and IFN-γ (eBioscience) has carried out cell inner dyeing.As a result it is shown in Fig. 2A and Fig. 2 B.

PD-1 and LAG-3 is blocked to the thin of the people's CD4 T cell co-cultured with B cell-class into lymphoid cell lines (ARH77) The influence of cellular toxicity granzyme B release.

In functional study, by CD4 T cell and the tumor cell line ARH77 (B of expression PDL-1 level more lower than mDC Cell lymphoblastoid cell lines) it co-cultures together, preferably to characterize the contribution that LAG-3 antagonism blocks PD-1.Experimental setup Keep constant with mMLR with the rest part of reading.With reference anti-lag-3 antibody (humanization BAP050 (LAG525) and BMS 986016) (P < 0.05) is compared with individual anti-PD-1 (P < 0.01), anti-PD-1 antibody and anti-lag-3 antibody (aLAG3 (0414) With aLAG3 (0416), the ability selection of IL-2 and granzyme B is co-secreted in mMLR based on it) combination lead to CD4 T cell The secretion of granzyme B increase more significantly, as shown in Figure 3.

PD-1 and LAG-3 block to the granzyme B of people's CD4 T cell with irradiated allogeneic PBMC co-cultivation and The influence that the Treg of IFN-γ release inhibits.

Inhibit in the functional study measured being related to regulatory T cells (Treg), via microballon kit (Miltenyi Biotec) PBMC from identical donor is divided in two samples: an example enrichment CD4 T cell and another sample is rich Collection is defined as CD4⁺CD25^{It is high}CD127^{It is low}The Treg of T cell.Once Liang Ge group is purified, i.e., with 5 μM of carboxy-fluorescein- Succinimide ester (CFSE) marks CD4 T cell, while marking Treg with 5 μM of Cell-Trace-Violet (CTV), so as to The CD4 T cell and the Treg can be distinguished on subsequent FACS.

Then by CD4 T cell (10⁵) and Treg (10⁵) in 96 orifice plates with 1:1 ratio with from irrelevant donor through spoke According to the PBMC (10 that exhausts of CD4⁵) co-culture together, wherein presence or absence of concentration be 10g/ml with anti-PD-1 antibody (aLAG3 (0414) and aLAG3 (0416) refer to anti-lag-3 antibody (humanization to the anti-lag-3 antibody of aPD1 (0376) combination BAP050 (LAG525) and BMS 986016).As estimation Treg to the amplitude of the inhibition of CD4 T cell effector function Control, also by CD4 T cell (10⁵) in the presence of Treg with irradiated PBMC (10⁵) co-culture together.

After five days, cell culture supernatant is collected, and is used subsequently to measure IFN-γ level by ELISA (R&D system), And make cell in the presence of Golgi Plug (brefeldin A) and Golgi Stop (coban) at 37 DEG C again It places 5 hours.Be washed out cell, with anti-human CD4 antibody and Live/Dead can fixed dye Aqua (Invitrogen) carry out / permeabilization is fixed with Fix/Perm buffer (BD Bioscience) later in padding.To granzyme B (BD Bioscience) and IFN-γ (eBioscience) carries out cell inner dyeing.As a result it is illustrated in figures 4A and 4 B.

Anti- PD-1 antibody aPD1 (0376) (=PD1-0103-0312 comes from PCT application PCT/EP2016/073248) with The combination of anti-lag-3 antibody (aLAG3 (0414) and aLAG3 (0416)) causes Tconv from the strict control of regulatory T cells Escape, such as by with there is (P < 0.05) in the case where individual anti-PD-1 or checkpoint inhibitor be not present in the case where (P < 0.001) secretion of the Tconv compared to significant a greater amount of granzyme B is confirmed.The reference anti-lag-3 combined with anti-PD-1 Significantly rescue Tconv effector function does not inhibit antibody (humanization BAP050 (LAG525) and BMS 986016) from Treg. Although IFN-γ obtains similar as a result, 4 donor differences, which are used only, is not up to significance,statistical.

After arousing memory with immunogenicity melanoma-Antigenic Peptide pond, PD-1 and LAG-3 are blocked to from melanoma patient The influence of the granzyme B and IFN-γ secretion of the CD4 T cell of PBMC.

It previously has been described, melanoma patient PBMC contains the specific for tumour antigen T cell of detectable frequency.Therefore, For POC purpose, we stimulate overnight melanoma patient collecting object again with immunogenicity melanoma associated antigen peptide Anti-lag-3 antibody (0414) is tested on PBMC plus anti-PD-1 compares individually anti-PD-1.

10 from melanoma patient⁵To 10⁶A PBMC is presence or absence of individual and anti-lag-3 (aLAG3 (0414)=(0414), 10 μ g/ml) antibody combination saturated concentration (10g/ml) anti-PD-1 (0376) in the case where in room temperature Lower incubation.Then in protein transport inhibitor Golgi Plug (brefeldin A) and Golgi Stop (coban) In the presence of, by T cell immunogenic cancer related antigen such as MAGEA1, MAGEA3, MAGEA4, Melan-A/MART-1, NYESO-1, melanocyte albumen Pmel 17gp100, tyrosinase, tyrosinase related protein1 the object that collects stimulated again Night.

Be washed out cell, with anti-human CD4 antibody and Live/Dead can fixed dye Aqua (Invitrogen) carry out table Face dyeing, is fixed/permeabilization with Fix/Perm buffer (BD Bioscience) later.To granzyme B (BD Bioscience) and IFN-γ (eBioscience) carries out cell inner dyeing.

The combination (P < 0.01 and P < 0.001) of anti-lag-3 and anti-PD-1 antibody significant (P < 0.01 and P < 0.0001) enhancing Specific for tumour antigen T cell effector function (i.e. granzyme B and IFN-γ secretion), and individually PD-1 blocking is not shown Any effect (data are not shown).

Example 10

The generation and generation of the anti-LAG3 antibody of the anti-PD1/ of bispecific

10.1 have VH/VL Domain swapping/replacement (CrossMAb in a combination arm^Vh-VL) and at the interface CH1/CL In have single electrically charged amino acid substitution combination PD1 and LAG3 bispecific antibody generation and expression

Pass through classical molecular biology as described in conventional method chapters and sections in conjunction with the multi-specificity antibody of people PD1 and people LAG3 Technology generates, and the transient expression in the 293F of Expi293F as described above.Polyspecific 1+1 CrossMAb^Vh-VlAntibody It is also described in WO 2009/080252.Multi-specificity antibody uses the nucleic acid for the amino acid sequence described containing coding schedule 14 Expression plasmid expression.1+1 CrossMAb^Vh-VlThe schematic structure of bispecific antibody is shown in figure 1A.

Table 14: the amino acid sequence (1+1 with VH/VL Domain swapping/replacement light chain (LC) and heavy chain (HC) CrossMAb^Vh-Vl)

All constructs have used protrusion to enter hole heterodimerization technology, with the typical protrusion in the first CH3 structural domain (T366W) replace and the 2nd CH3 structural domain in corresponding aperture replace (T366S, L368A and Y410V) (and two in addition introduce Cysteine residues S354C/Y349 ' C) (be included in above-mentioned respective heavy chain (HC) sequence in).

10.2 have CH1/Ck Domain swapping/replacement (2+2 CrossMab in two basic change arm^CH1/Ck) and another The production of the multi-specificity antibody of combination PD1 and LAG3 in the interface CH1/CL of a combination arm with electrically charged amino acid substitution Raw and expression

In this example, pass through as described in conventional method chapters and sections in conjunction with the multi-specificity antibody of people PD1 and people TIM3 through Allusion quotation Protocols in Molecular Biology generates, and the transient expression in the 293F of Expi293F as described above.Polyspecific 2+2 CrossMAb^CH1/CkAntibody is also described in WO 2010/145792.Multi-specificity antibody uses the ammonia described containing coding schedule 15 The expression plasmid of the nucleic acid of base acid sequence is expressed.2+2 CrossMAb^CH1/CkThe schematic structure of bispecific antibody is in figure 1A It shows.

Table 15: the amino acid sequence (2+2 with VH/VL Domain swapping/replacement light chain (LC) and heavy chain (HC) CrossMAb^CH1/Ck)

10.3 have CH1/Ck knot in a combination arm (the PD1 crossFab merged with the C-terminal of Fc protrusion heavy chain) Structure domain exchange/replacement (2+1 CrossMab^CH1/Ck) and in the interface CH1/CL of another combination arm have electrically charged amino The generation and expression of the multi-specificity antibody of combination PD1 and LAG3 that acid replaces

In this example, pass through as described in conventional method chapters and sections in conjunction with the multi-specificity antibody of people PD1 and people TIM3 through Allusion quotation Protocols in Molecular Biology generates, and the transient expression in the 293F of Expi293F as described above.Polyspecific 2+1 CrossMAb^CH1/CkAntibody is also described in WO2013/026831.Multi-specificity antibody uses the ammonia described containing coding schedule 16 The expression plasmid of the nucleic acid of base acid sequence is expressed.2+1 CrossMAb^CH1/CkThe schematic structure of bispecific antibody is in fig. ib It shows.

Table 16: the amino acid sequence (2+1 with CH1/Ck Domain swapping/replacement light chain (LC) and heavy chain (HC) CrossMab^CH1/Ck)

Alternatively, the PD1 crossFab merged with the C-terminal of Fc protrusion heavy chain can be substituted for single chain Fab (scFab).Such polyspecific 2+1 antibody comprising scFab is also described in WO2010/136172, and be can be used and contained There is the expression plasmid of the nucleic acid for the amino acid sequence described in coding schedule 17 to express.With the C-terminal merged in Fc protrusion heavy chain The schematic structure of 2+1 bispecific antibody of scFab be shown in Figure 1C.

Table 17: the amino acid sequence of light chain (LC) and heavy chain (HC) with PD1 scFab

10.4 have respectively fusion in the production of the multi-specificity antibody of the combination PD1 and LAG3 of the VH/VL of the C-terminal of heavy chain Raw and expression (2+1 PRIT form)

In this example, pass through as described in conventional method chapters and sections in conjunction with the multi-specificity antibody of people PD1 and people TIM3 through Allusion quotation Protocols in Molecular Biology generates, and the transient expression in the 293F of Expi293F as described above.It is such mostly special Anisotropic 2+1 antibody is also described in WO 2010/115589.Multi-specificity antibody uses the amino acid described containing coding schedule 18 The expression plasmid of the nucleic acid of sequence is expressed.The schematic structure of the bispecific antibody of 2+1 PRIT- type is shown in Fig. 1 D.

Table 18: there is the amino acid of light chain (LC) and heavy chain (HC) that VH the and VL structural domain of heavy chain is blended in C-terminal Sequence

10.5 have VH/VL Domain swapping/replacement (1+1 CrossMab in a combination arm^VH/VLTrans forms) and And the interface CH1/CL of LAG3 Fab merged of C-terminal of the hole Fc heavy chain in the combination with electrically charged amino acid substitution The generation and expression of the multi-specificity antibody of PD1 and LAG3

The multi-specificity antibody of monovalence combination people PD1 and people LAG3 are generated, wherein LAG3 Fab is chained via its Weight variable Structure domain is merged with one C-terminal in heavy chain, is preferably merged with the C-terminal of the hole Fc heavy chain.The molecule such as conventional method It is generated described in chapters and sections by classical Protocols in Molecular Biology, and the instantaneous table in the 293F of Expi293F as described above It reaches.Multi-specificity antibody uses the expression plasmid of the nucleic acid for the amino acid sequence described containing coding schedule 19 to express.1+1 CrossMab^VH/VLThe schematic structure of the bispecific antibody of trans--type is shown in Fig. 1 H.

Table 19: there is the amino acid sequence of light chain (LC) and heavy chain (HC) that the aLAG3 Fab of heavy chain is blended in C-terminal

10.6 have VH/VL Domain swapping/replacement (2+1 CrossMab in a combination arm^VH/VLTrans forms) and There is electrically charged amino in the interface CH1/CL of two LAG3 Fab (one of those is merged with the C-terminal of the hole Fc heavy chain) The generation and expression of the multi-specificity antibody of combination PD1 and LAG3 that acid replaces

The multi-specificity antibody of monovalence combination people PD1 and divalent combination people LAG3 are generated, wherein LAG3 Fab is via it Variable heavy chain domain is merged with one C-terminal in heavy chain, is preferably merged with the C-terminal of the hole Fc heavy chain.The molecule It is generated as described in conventional method chapters and sections by classical Protocols in Molecular Biology, and in the 293F of Expi293F as described above Middle transient expression.Multi-specificity antibody uses the expression plasmid of the nucleic acid for the amino acid sequence described containing coding schedule 20 to express.2 +1 CrossMab^VH/VLThe schematic structure of the bispecific antibody of trans--type is shown in Fig. 1 I.

Table 20: the amino acid sequence of light chain (LC) and heavy chain (HC), wherein one in aLAG3 Fab is merged in C-terminal In heavy chain

10.7 combine the purifying and characterization of the multi-specificity antibody of PD1 and TIM3

By the combination of a-protein affinity chromatography and size exclusion chromatography, the mostly special of above-mentioned expression is purified from supernatant Heterogenetic antibody.All multi-specificity antibodies can be generated with good yield and be stable.For identity (passing through mass spectrography) The analytical characteristicses such as purity (passing through SDS-PAGE), content of monomer and stability characterize product obtained.

Mass spectrum

By complete CrossMab and de-glycosylation/Plasmin digestion to de-glycosylation or de-glycosylation/restricted The CrossMab of LysC digestion carries out electrospray ionization mass spectrometry (ESI-MS), to analyze expected primary structure.

CH1/Ck CrossMab deglycosylation is held at 37 DEG C with N- glycosidase F in phosphate or Tris buffer Continuous at most 17h, protein concentration 1mg/ml.With the 100 deglycosylated CH1/Ck of μ g in Tris pH of buffer 8 CrossMab carries out fibrinolysin or restricted LysC (Roche) digestion, continues 120 hours at room temperature respectively and holds at 37 DEG C Continuous 40min.Before mass spectral analysis, by sample via HPLC desalination on Sephadex G25 column (GE Healthcare).In Equipped with the maXis 4G UHR-QTOF MS system (Bruker Daltonik) in the source TriVersa NanoMate (Advion) On via ESI-MS measure gross mass.

The stability of multi-specificity antibody

In order to assess the stability of antibody construct, thermal stability and aggregation start temperature are assessed according to following procedure. The sample of indicated antibody is made in 20mM histidine/histidine chloride, 140mM NaCl, pH 6.0 with the concentration of 1mg/mL It is standby, when being transferred in 10 μ L micro tube arrays, and using 266nm laser excitation with Optim1000 instrument (Avacta Inc.) record Static light scattering data and fluorescence data, while sample is heated to 90 DEG C from 25 DEG C with the rate of 0.1 DEG C/min.

Assemble start temperature (T_agg) it is defined as temperature when scattered light intensity starts increase.Melting temperature (T_m) be defined as Fluorescence intensity is relative to the inflection point in the curve graph of wavelength.

Example 11

The characterization of the anti-LAG3 antibody of the anti-PD1/ of bispecific

11.1 in conjunction with Elisa

ELISA method detection hu PD1

The coated plate of Nunc maxisorp streptavidin (MicroCoat#11974998001) is used into concentration Biotinylated PD1-ECD-AviHis for 25 holes μ l/ of 500ng/ml is coated with, and is incubated overnight at 4 DEG C.Washing After (PBST buffer, 3 × 90 holes μ l/), the anti-PD1 antibody samples of 25 μ l are added with the concentration gradually increased, and at room temperature Incubate 1h.After washing (PBST buffer, 3 × 90 holes μ l/), the Goat anti-Human H+ in 25 holes μ l/ is added with the dilution of 1:5000 L-POD (JIR, JIR109-036-098), and incubate 1h at room temperature on the oscillator.Wash (PBST buffer, 3 × 90 μ The hole l/) after, the tmb substrate (Roche, 11835033001) in 25 holes μ l/ is added, and incubate until OD is 2-3.In 370/492nm Place measures.

The cell ELISA of people PD1

Cell ELISA result is in the following table 21 with " EC₅₀CHO-PD1 " value [nM] is listed.

The ELISA of people Lag3

Cell surface Lag3 combination ELISA

The inhibition (passing through ELISA) of the combination of LAG-3 and the MHC- II expressed on people's A375 tumour cell

The A375 cell (10000 cells/wells) in 25 holes μ l/ is inoculated into 384 hole plates through tissue culture treated In (Corning, 3701), and it is incubated overnight at 37 DEG C.Anti- Lag3 antibody with 1:3 dilution in cell culture medium Biotinylated Lag3 (250ng/ml) precincubation 1h together, is started with the antibody concentration of 3 μ g/ml.From with inoculating cell After removing culture medium in hole, 25 μ l antibody-Lag3 Preincubation mix are transferred in hole, and incubate 2h at 4 DEG C.It washes After washing (1 × 90 μ l, in PBST), ultimate density (Sigma, the catalogue of the glutaraldehyde to 0.05% by adding 30 holes μ l/ Number: G5882), cell is fixed into 10min at room temperature.After washing (PBST buffer, 3 × 90 holes μ l/), with 1:2000 or 1: 8000 dilutions add the poly- HRP40- streptavidin (Fitzgerald, 65R-S104PHRPx) in 25 holes μ l/, and 1h is incubated at room temperature.Wash (PBST buffer, 3 × 90 holes μ l/) after, add 25 holes μ l/ tmb substrate (Roche, 11835033001) and 2 to 10min are incubated.It is measured on 2 instrument of Tecan Safire at 370/492nm.Inhibit ELISA result is in the following table 21 with " IC₅₀II/ELISA " of MHC value [nM] is listed.

Table 21: the combination of the anti-LAG3 antibody of the anti-PD1/ of different bispecifics summarizes

11.2 in conjunction with Biacore

In conjunction with the antigenic binding property of the multi-specificity antibody of PD1 and LAG3

Pass throughAssess the combination of multi-specificity antibody and its corresponding target antigen (i.e. PD1 and TIM3).

PD1 is combined and can be assessed according to following procedure:

Anti-human Fc IgG is immobilized into the surface of (Biacore) CM5 sensor chip by amine coupling.Then sample is captured This, and make hu PD1-ECD in conjunction with them.After each analysis circulation, reg sensor chip surface.By the way that data are intended It is bonded to 1:1 Lang Gemiaoer (Langmuir) interaction model, it is final to obtain the equilibrium constant and Kinetics Rate Constants By Using.

The amine coupling kit that is there is provided using GE Healthcare will about 10,000 response units (RU) 20 μ g/ml Anti-human igg (GE Healthcare#BR-1008-39) is coupled to all streams of the CM5 sensor chip in Biacore T200 On dynamic pond.Sample and running buffer are HBS-EP+ (0.01M HEPES, 0.15M NaCl, 3mM EDTA, 0.05%v/v table Face activating agent P20, pH 7.4).Flow cell temperature is set as 25 DEG C, and sample compartment temperature is set as 12 DEG C.It is loaded with running buffer System.

Different samples is injected 15 seconds with the concentration of 10nM and is continuously incorporated into flow cell 2,3 and 4.Then by a whole group People PD1-ECD concentration (300nM, 100nM, 2 × 33.3nM, 11.1nM, 3.7nM, 1.2nM and 2 × 0nM) is injected at each sample On, continue 300s, followed by the Dissociation time of 10/600s and with 3M MgCl₂The two 30s regeneration steps carried out, wherein most " the additional washing after injection " that the latter is carried out containing useful running buffer.Finally, assessing software using Biacore T200 Double reference data is fitted to 1:1Langmuir interaction model.

LAG3 is combined and is assessed according to following procedure:

This measurement is carried out using the Biacore SA CAP kit provided by GE Healthcare.Dynamics are 25 It is obtained in HBS-EP+ (GE Healthcare) buffer at DEG C.

Such as defined in the handbook of CAP kit, SA CAP chip is accessed into Biacore T200.Operation method contains Four orders.Firstly, " General " is used to order, 300s injection CAP reagent is continued with the flow rate of 10 μ l/min, with miscellaneous Hand over the single stranded DNA of immobilization.It is the long 15 seconds biotinylated Fc labeling in running buffer after the order The injection of 1 μ g/ml dilution of people's Lag3 extracellular domain.This causes the capture of about 50RU horizontal.Use single cycle order Five different sample concentrations (100nM -6.25nM, 2 times of dilutions) are injected, followed by long 1200 seconds dissociation stages.Then As defined regenerates chip in SA CAP kits manuals.

Finally, assessing the software 3.0 editions resulting curves of assessment using Biacore T200.

As a result: interaction not being fitted to 1:1 Lang Gemiaoer binding model, because all in addition to 0799 and 0927 There are two Lag3 bound fraction and thus it is shown that affinities for sample tool.Since 0799 and 0927 containing small mispairing (miss-paired) divalent sample group, they also show that some affinities.

Therefore sensing figure sorts according only to their dissociation rate.This by the visual comparison of single cycle kinetic curve into Row.By doing so, showing that 0416-Lag3 ECD compound is more stable than any other sample in sample sets.Monovalence < Lag3 > Crossmab form (0799) still shows the dissociation rate lower than any other sample in this experiment.

It was furthermore observed that Lag3-0414/Lag3-0927-Lag3 ECD the compound of homologous (affine) is in this experiment Those of it is most weak in sample.0414 and 0416 homologous bound fraction and they monovalence Crossmab corresponding part 0927 and 0799 analogous parts are roughly the same.As a result it is shown in table 22.

Table 22: pass through the bond quality of the determining PD1-LAG3 bispecific antibody of SPR measurement

Sample	Bond quality
		aLAG3(0414)	++
aLAG3(0416)	+++
		PD1/LAG3 0927(1+1)	+
PD1/LAG3 0799(1+1)	+++
		aLAG3(25F7)	++
aLAG3(MDX26H10)	++
		aLAG3(BMS986016)	++
aLAG3(BAP050)	+

Compared to 1+1 bispecific antibody 0927 and 0799, the affinity force estimation of trans forms:

Bispecific molecule (sample) and their independent target and PD1 and the combination (analyte) of LAG3 has been determined Dissociation constant, to assess the avidity gain by providing in combination with all potency.

Before being measured on Biacore 8K, the standard amine coupling reagent kit system that is provided using GE Healthcare Standby CM5 sensor chip.Therefore by the specific mutation in the part Fc (i.e. the part Fc of carrying PGLALA mutation) for sample Inside preparation antibody, herein referred as anti-PGLALA antibody (such antibody is described in WO 2017/072210) is in pH 5.0 The concentration of 50 μ g/ml is diluted in acetate buffer.The antibody is coupled to all streams through 1200s with the flow velocity of 8 μ l/min Dynamic pond and channel, to obtain the combining response of about 19000RU.

HBS-EP⁺Buffer (GE HC) is used as the running buffer of sensor chip preparation and main operation itself.By Start to analyze after the starting of the sample injection composition of three 17s long, carries out regeneration step followed by 10mM NaOH solution. In the first step, by injecting 17s with the flow rate of 10 μ l/min, different analyte captures are arrived by anti-PGLALA antibody On the flow cell two of individual passage in sensor chip surface.Second step, continuing 200s with the flow rate of 50 μ l/min will One of three kinds of analytes (PD1, LAG3-Fc, 2+2PD1/LAG3-Fc fusion) are injected into two flow cells, followed by The dissociation stage of long 1000s (being 600s in the case where LAG3-Fc).Finally, by continuously injecting (long 30s) 10mM twice NaOH dissolves anti-PGLALA antibody/sample composites.Each individually kinetic determination is by with different analyte concentrations Four circulations of (0nM, 5nM, 25nM and 100nM) form.

Software evaluation the data obtained is assessed using Biacore 8K.Fitting is dissociated using 1:1 and turns resulting kd value Change compound half-life period (in minutes) into.PD1/Lag3-Fc fused antigen binding molecule and its main independent contributor Difference between the affinity combination of (PD1 or Lag3) is calculated and is sorted into one in three classifications, and description passes through monovalence And bispecific combines the stability gain (table 23) obtained.

Table 23: the stable composite provided by the determining affinity by PD1-LAG3 bispecific antibody of SPR measurement The increase of property

11.3 after being simultaneously engaged with via the anti-LAG3 bispecific antibody of the anti-PD1/ of bispecific cell PD1 and LAG3 Dimerization

Generate the anti-LAG3 antibody of the anti-PD1/ of bispecific in a variety of manners as described in example 10.This raji cell assay Raji is for opening up Show two kinds of not dimerization of isoacceptor or last combination/interactions, the receptor with the bispecific that is directed to two kinds of targets It is merged in cytoplasm when antibody connection or crosslinking with two segments of enzyme.Therefore, individually only a kind of receptor is shown as no enzyme activity Property.It interacts for this species specificity, the cytoplasm C-terminal of two kinds of receptors is individually merged with the heterologous subunit of reporter enzyme. Individually single enzyme subunit does not show reporter activity.It is contemplated, however, that the anti-LAG3 bispecific antibody constructs of anti-PD1/ and two Assemble while kind of receptor in conjunction with the local cytoplasm that will lead to two kinds of receptors, the complementation of two heterologous enzyme subunits, and eventually leads to Specificity and functional enzyme are formed, the enzyme hydrolysis substrate is to generate chemiluminescence signal.

In order to analyze the crosslinked action of the anti-LAG3 antibody of the anti-PD1/ of bispecific, by 10,000 PD1⁺LAG3⁺People U2OS is thin Born of the same parents/hole is inoculated into 96 hole plate of white flat bottom (Costar, catalog number (Cat.No.) #3917), and the overnight incubation in measurement culture medium. Second day, cell culture medium is discarded and replaces with fresh culture.It prepares antibody or ligand dilution and adds titration Instruction (bispecific) antibody of amount, and incubated 2 hours at 37 DEG C.Then, add substrate/buffer solution mixture (such as PathHunterFlash detection reagent), and 1h is incubated again.In order to measure the change for combining and inducing at the same time when dimerization It learns and shines, use Tecan infinite plate reader.

As a result it shows in Fig. 5 A and 5B.What is drawn is chemiluminescence (being measured as unit of RU) relative to antibody concentration Curve graph.The anti-LAG3 antibody of monospecific (divalent) cannot cause chemiluminescence signal, and the anti-PD1/ of all bispecifics is anti- LAG3 antibody is with concentration dependant manner induced chemical luminous signal.

In order to show the specificity in combination with (and induction of luminous signal), be at war with experiment: it is as indicated previously, it uses Bispecific antibody (1252) is handled, with dosage-dependent manner induced luminescence signal (Fig. 5 C).If in aLAG3 antibody Identical bispecific antibody is provided in the presence of (0156, MDX25F7) or anti-PD1 antibody (0376), then signal is almost suppressed It (since PD1 is competed) or is at least significantly reduced (LAG3).Two kinds of parental antibodies and bispecific antibody (1252,2+1LAG3/ PD1 form) contained in bonding agent be identical.Competitive antibody is respectively provided with the constant density of 20 μ g/ml.

The result of another experiment is shown in figure 5d.It is similar with previous competitive assay, with parent aLAG3 (0156) or PD1 antibody (0376) (respectively constant to be in 10 μ g/ml) is incubated together to bispecific antibody (1252, double special aLAG3- The 2+1 form of 0156 and PD1-0376) have with the binding characteristic of PD1 Lag3 double expression cell and influences, such as pass through luminous signal Measured.Signal is almost eliminated with anti-PD1 antibody (0376) and the competition for recombinating LAG3:Fc albumen (0160), and it is single The presence of aLAG3 bonding agent (0156) only results in part inhibition.Other two kinds of anti-LAG3 in conjunction with the epitope different from 0156 Antibody 0414 and 0416 not with the bispecific antibody competitive binding comprising aLAG3 bonding agent (0156) because they are not significant Adjustment signal.

In another experiment, to including different aLAG3 bonding agents (0414 relative to 0416) and different form (1+1 phase For 2+1) the anti-LAG3/ of bispecific anti-PD1 antibody while combine and be compared (Fig. 6 A to 6D).As it was earlier mentioned, surveying The anti-PD1 bispecific antibody of several anti-LAG3/ is tried, the antibody is 1+1 CrossMab form (0799 and 0927) or 2+1 Form (C-terminal of two Lag3 combination arms and a PD1 crossFab segment composition: 8311 and 8310).In figures 6 a and 6b The curve (absorbance is relative to concentration) of construct with bonding agent aLAG3-0416 is shown, and tool is shown in Fig. 6 C and 6D There is the curve of the corresponding construct of aLAG4-0414.All constructs tested can be with cell combination and induced chemical is sent out Light.For binding curve EC calculated₅₀Value is shown in lower section table 24.

Table 24: the EC measured in dimerization binding assay₅₀Value

Bispecific antibody	Form	MW[kD]	EC₅₀[pM]
				0927(PD1-0376/LAG3-0414)	1+1	145	41
0799(PD1-0376/LAG3-0416)	1+1	145	76
				8310(PD1-0376/LAG3-0414)	1+2	193	28
8311(PD1-0376/LAG3-0416)	1+2	193	119

In another experiment, to including different aLAG3 bonding agents (0414 relative to 0416) and different form (2+1 phase For 2+2) the anti-LAG3/ of bispecific anti-PD1 antibody while combine and be compared (Fig. 7 A to 7D).Test anti-LAG3/ Anti- PD1 bispecific antibody, the antibody are 2+1 form (two LAG3 combination arms and a PD1 crossFab segment composition C-terminal: 8310) or 2+2crossmab form (two LAG3 combination arms and two PD1 crossFab segment compositions 8311 and C-terminal: 8970 and 8984).Curve (the absorbance of construct with bonding agent aLAG3-0414 is shown in Fig. 7 A and 7B Relative to concentration), and the curve of the corresponding construct with aLAG4-0416 is shown in Fig. 7 C and 7D.All structures tested Building body can be with cell combination and induced chemical shines.For binding curve EC calculated₅₀Value is shown in lower section table 25.

Table 25: the EC measured in dimerization binding assay₅₀Value

Bispecific antibody	Form	MW[kD]	EC₅₀[pM]
				8310(PD1-0376/LAG3-0414)	2+1	193	114
8311(PD1-0376/LAG3-0416)	2+1	193	124
				8970(PD1-376/LAG3-0414)	2+2	242	83
8984(PD1-0376/LAG3-0416)	2+2	242	91

In another experiment, by classical 1+1 CrossMAb^Vh-VLThe anti-PD1 antibody of the anti-LAG3/ of the bispecific of form It is combined and 1+1 trans forms (PD1/LAG3 0725) and 2+1 trans forms (PD1/LAG3 while 0927 PD1/LAG3 0750) the anti-PD1 antibody of the anti-LAG3/ of bispecific is compared.For this experiment, the following change to this method is applied. In order to analyze the cross-linking effect of the anti-PD1 antibody formation of different anti-LAG3/, by 7500 PD1⁺LAG3⁺People U2OS cells/well with Antibody non-serial dilutions (ultimate density be 0.29pM to 5484pM) be inoculated into 96 hole plate of white flat bottom together, and In CO₂20h is incubated at 37 DEG C in incubator.Then, assay plate is balanced to room temperature, and it is mixed to add substrate/buffer It closes object (PathHunterFlash detection reagent, Discoverx), and incubates 4h again.It is combined at the same time and dimerization to measure The chemiluminescence induced when change uses SpectraMax L plate reader (Molecular Devices).

In figure 7e, depict PD1-LAG3 bispecific antibody 1+1 CrossMab (0927), have N-terminal aPD1 and The trans- CrossMab of the 1+1 of C-terminal aLAG3 (0725) and add the 2+1 of C-terminal aLAG3 trans- with N-terminal aPD1 and N The dose-response curve (shining relative to concentration) of CrossMab (0750).Compared with PD1/LAG3 0927, PD1/LAG3 0725 PD1 LAG3 receptor cross-linking effect is significantly higher, and the effect of PD1/LAG3 0750 is then slightly lower.

The trans- CrossMab variant of the anti-PD1 of the anti-LAG3/ of 11.4 bispecifics is in PD-1 and LAG-3 combo Reporter Measurement in measurement

In order to test the neutralization in terms of different anti-PD1-LAG3 antibody formations restores repressed t cell response in vitro Effect uses commercially available reporting system.PD1 and LAG3 combo bioassay by expression PD1, LAG3 and T cell by The reporter cell of body (TCR), the tumour cell for expressing MHC- II and PDL1 and TCR active antigen composition.

Effector cell is to express people PD1, people LAG3, people TCR and the fluorescein driven by NFAT response element (NFAT-RE) The Jurkat T cell of enzyme reporter gene.Target cell is the A375 cell for expressing human PD-L 1.In brief, reporting system is based on Three steps: (1) the NFAT cell-stimulating of TCR active antigen induction, (2) are by MHC II (A375 cell) and LAG3⁺(Jurkat Cell) and PD-L1 (A375 cell) and PD1 (Jurkat cell) between interaction mediate activation signal inhibition, And the recovery of (3) NFAT activation signal as caused by PD1 and LAG3 antagonism/neutralizing antibody.

For this experiment, by every hole 1 × 10⁴A A375 cell and TCR active antigen (Pu Luomaige company) are together 96 In CO in the flat assay plate in hole₂It is incubated overnight at 37 DEG C in incubator.Then, culture medium is removed from plate, and is added The serial dilutions of the anti-PD1 antibody of anti-LAG3/ are added (to measure concentration finally as 0.01nM to 857nM) and every hole 5 × 10⁴It is a Jurkat effector cell.In CO₂After being incubated 6 hours at 37 DEG C in incubator, assay plate is balanced to room temperature, and to 80 μ l ONE-Glo Ex substrates (Pu Luomaige company) are added in each hole.After incubating 10min, in SpectraMax L Measurement shines in plate reader (Molecular Devices).It is expected that the anti-LAG3 bispecific antibody constructs of anti-PD1/ and two kinds Assemble while receptor in conjunction with the local cytoplasm that will lead to two kinds of receptors, the complementation of two heterologous enzyme subunits, and eventually leads to shape At specificity and functional enzyme, the enzyme hydrolysis substrate is to generate chemiluminescence signal.In figure 7f, it is anti-to depict anti-PD1/ LAG3 bispecific antibody 1+1 CrossMab (0927), the trans- CrossMab of the 1+1 with N-terminal aPD1 and C-terminal aLAG3 (0725) and with N-terminal aPD1 and N add the dose-response curve of the trans- CrossMab of 2+1 (0750) of C-terminal aLAG3 (shining relative to concentration).0927 and 0725 by blocking the interaction of PD1 and LAG3 and its respective ligand to restore to report The ability of cell activation is comparable, but is higher than 0750.This passes through the EC listed in table 26₅₀Value further demonstrates that.

Table 26: the EC measured in PD-1 and LAG-3combo report measurement₅₀Value

Example 12

The functional characterization of the anti-LAG3 antibody of the anti-PD1/ of bispecific

12.1 in conjunction with T cell surface when reduction internalization

Pass through flow cytometry measure receptor internalisation

Receptor internalisation represents the important sinking of molecule, and the molecule can degrade within a few hours, while receptor targeted exists It is rapidly expressed again on cell surface, inhibits TCR signal transduction to be ready for use on.Therefore we pass through hybridoma supematant assesse Receptor internalisation when our construct combines, wherein being used as at 4 DEG C with the sample that different bispecific forms dyes With reference to be compared with the sample for incubating 3 hours after the dyeing at 4 DEG C at 37 DEG C.

It is more by being cultivated together with the soluble anti-CD28 antibody of AntiCD3 McAb and 1 μ g/ml previously in conjunction with the plate of 1 μ g/ml Clone activates three days CD4 T cells in the presence of anti-LAG3 or anti-PD1/ anti-LAG3 bispecific antibody (duplicate) 4 It is incubated 30 minutes at DEG C.Then cell is washed, is divided into two groups, one group therein incubates 3 hours and another group again at 37 DEG C It is dyed with the secondary antibody (eBioscience) of label, is then fixed with BD Cell Fix immediately.After incubating 3 hours, second Group cell is also before fixing with two anti-dye of label.After dyeing, cell is washed twice with PBS/2%FCS, is then adopted Collection.

Cell is acquired on LSRFortessa (BD Biosciences), and is compared between two groups on cell surface The expression of detectable antibody.As a result it shows in the fig. 8b.It is observed that after 3 hours, all bispecific forms And monospecific divalent aLAG-3 antibody is internalized by, however the anti-LAG3 antibody (PD1/ of the anti-PD1/ of bispecific of 1+1 form LAG3 0799 and PD1/LAG3 0927) it is minimum by internalization.

Antibody positioning and internalization are visualized by fluorescent confocal microscopy

The CD4 positive cell of activation is dyed with CMFDA (Invitrogen), and is plated in and uses Retronectin On the round coverslip of (Takara Bio) processing.Make cell adherent 4 hours at 37 DEG C, then by the antibody of fluorescence labels (1g/mL:a-LAG3 (1256), 1+1 PD1/LAG3 Bispec (0927), PD1-LAG3 1+2 Bispec (8310) and use The PD1-LAG3 2+2 Bispec (8970) that Alexa 647 is marked) it is directly appended in growth medium, when cultivating different Between (15min, 1 hour and 3 hours).With cold PBS (Lonza) quenching reaction and wash off unbonded antibody.Then by cell It mixes at 4 DEG C 20 minutes with Cytofix (BD) and is washed twice with PBS (Lonza).Then coverslip is shifted and is mounted on In glass slide with Fluoromount G (eBioscience), and kept in 4 DEG C, dark before imaging Night.A) fluorescent image is shown in figure 9 a.White signal represents the positioning of labelled antibody.B the cytotropic film of height target will) be come from The intensity of the fluorescence signal of ROI obtains counting in box divided by the intensity of the fluorescence signal of the cytoplasm ROI from same cell Scheme the ratio shown in (Box Chart).In order to be compared to sample, one-way analysis of variance method (One Way is used ANOVA (*=p < 0.05 uncorrected Fisher LSD) is analyzed；*=p < 0.01).As a result it shows in figures 9 b and 9.It pushes away at any time It is moving analysis shows that, when compared with the intracellular gathering of TIM3 antibody (be used as control), bispecific antibody and LAG3 are anti- Film positioning in body is higher.It is observed that after 3 hours, other than 1+1 PD1/LAG3 Bispec (0927), institute There are bispecific form and monospecific divalent aLAG-3 antibody to be internalized by (Fig. 9 A).

Using 60 × oil-immersion objective, fluorescent confocal microscopy is carried out using the inversion LSM 700 from Zeiss.Use connection Image is collected to microscopical Zen software (Zeiss).The analysis of image uses Imaris software (Bitplane；Oxford Instrument it) carries out, and statisticallys analyze and carried out by GraphPad Prism (Graphpad Software).

12.2 and conventional T cells combination relative to and Treg combination

The desired characteristic of leading PD1-LAG3 BsAb be preferentially combine conventional T cells rather than the ability of Treg because LAG3 on Treg shows as their inhibition function of negative regulator.Therefore, using the LAG3 on blocking antibody targeting Treg, can increase Add their inhibition function and finally cover the positive blocking effect in other T cells, to have an adverse effect.Therefore, we Have evaluated the conventional T cells and regulatory T of activation of the different anti-anti- LAG3 bispecific antibody forms of PD1/ together with gathering The competitive binding of cell.

The sorting from healthy donors PBMC (Miltenyi) of regulatory T cells (Treg) and conventional T cells (Tconv), point Not Yong 5mM Cell Trace Violet or CFSE film dye marker, and the AntiCD3 McAb with 1:1 ratio in conjunction with 1g/ml plate It is incubated together 3 days with 1g/ml solubility anti-CD28 antibody.On day 3, by cell and the anti-PD1, the anti-LAG3 or double that directly mark Specific antibody incubates 30min at 4 DEG C together, is fixed with BD Cell Fix, and in LSRFortessa (BD Biosciences it is acquired on).

Although the anti-LAG3 parental antibody of monospecific equally well combines Treg and conventional T cells (Figure 10 A), resist PD1 counterpart preferentially combines conventional T cells (figure since PD1 is higher than the expression on Treg on effector T cell 10B).It is interesting that the 1+1 form (0927) of PD1/LAG3 bispecific antibody also keeps preferentially combining routine compared to Treg The ability (Figure 10 C) of T cell.It can also be opposite by describing the signal on conventional T cells with this preferential combination of conventional T cells In the signal on Treg difference (δ) and visualize (Figure 10 D).2+1 and 2+2 form does not show the affinity of pairing effect T cell The selectivity of driving, and and the combination of the anti-LAG3 antibody of monospecific in terms of be comparable.

12.3PD-1 and LAG-3 blocks the particle to people's CD4 T cell with irradiated allogeneic PBMC co-cultivation The influence that enzyme B and the Treg of IFN-γ release inhibit

Whether the difference for further testing the binding characteristic of bispecific antibody form will provide for Tconv better than Treg Any functionality advantage.Inhibit in the functional study measured being related to regulatory T cells (Treg), via microballon kit PBMC from identical donor is divided in two samples by (Miltenyi Biotec): an example enrichment CD4 T cell and it is another One example enrichment is defined as CD4⁺CD25^{It is high}CD127^{It is low}The Treg of T cell.Once Liang Ge group is purified, that is, use the carboxylic of 5M Base-fluorescein-succinimide ester (CFSE) marks CD4 T cell, while being marked with 5M Cell-Trace-Violet (CTV) Treg, so as to then distinguish the CD4 T cell and the Treg on FACS.

Then by CD4 T cell (10⁵) and Treg (10⁵) in 96 orifice plates with 1:1 ratio with from irrelevant donor through spoke According to PBMC (10⁵) co-culture together, wherein being the anti-with anti-PD-1 antibody combination of 10g/ml presence or absence of concentration LAG3 antibody (leading (lead) 0414 and candidate (backup) 0416) or competitive anti-LAG3 antibody (BMS-986016 and source of people Change BAP050).Control as estimation Treg to the amplitude of the inhibition of CD4 T cell effector function, also by CD4 T cell (10⁵) in the presence of Treg with irradiated PBMC (10⁵) co-culture together.

After five days, we collect cell culture supernatant, are used subsequently to measure IFN γ water by ELISA (R&D system) It is flat, and make cell in the presence of Golgi Plug (brefeldin A) and Golgi Stop (coban) at 37 DEG C Under place again 5 hours.It is washed out cell, it can fixed dye Aqua (Invitrogen) with anti-human CD4 antibody and Live/Dead Padding is carried out ,/permeabilization is fixed with Fix/Perm buffer (BD Bioscience) later.We are to granzyme B (BD Bioscience) and IFN γ (eBioscience) carry out cell inner dyeing.As a result it is shown in FIG. 11.

Our PD1/LAG-3 bispecific antibody (0927) causes Tconv and escapes from the strict control of regulatory T cells Ease, such as by with there are (P < 0.05) in the case of the anti-PD1 antibody of individual parent or pyridine aldoxime methyliodide (PAM) monoclonal antibody or there is no checkpoint inhibitor In the case of the secretion of the Tconv compared to significant a greater amount of granzyme B of (P < 0.001) confirmed.With receive what military monoclonal antibody combined Significantly rescue Tconv effector function does not inhibit competitive anti-LAG3 antibody BMS-986016 from Treg.

12.4PD-1/LAG-3 the people that bispecific antibody pair is co-cultured with B cell-class into lymphoid cell lines (ARH77) The influence of the cytotoxicity granzyme B release of CD4 T cell

We have evaluated and the combination of anti-PD-1 and anti-lag-3 parental antibody or the anti-PD1 antibody used with nursing standard It compares, when being co-cultured with tumor cell line ARH77, our different bispecific antibody forms induction CD4 T cell secretion The ability of granzyme B.

6 kinds of forms in total are tested, 3 kinds of forms are by anti-PD1 (0376) and anti-LAG3 (hu 1256, chi 0414) antibody Combination producing and another 3 kinds of forms by anti-PD1 (0376) and anti-lag-3 (hu1257, chi 0416) antibody tormation.

As in Figure 13 as can be seen that by anti-LAG3 (hu 1256, anti-LAG3 0414) as IgG1 PGLALA and resisting The combination of two kinds of bispecific form 1+1 (0927) and 2+2 (8970) and parental antibody that PD1 (0376) is generated is worked as and is not located The CD4 T cell of reason significantly increases granzyme B secretion (respectively P=0.0005, P=0.01 and P of CD4 T cell when comparing =0.0001).Corresponding 2+1 form (8310) shows similar trend, but not up to significance,statistical (P=0.07).

About by by anti-lag-3 (hu 1257, anti-LAG3 0416) as IgG1 PGLALA and anti-PD1 (0376) The bispecific antibody of combination producing, when compared with untreated cd4 cell, 1+1 form (0799) and 2+2 (8984) are significant Increase the frequency (respectively P=0.0032 and P=0.0064) of granzyme B positive CD4 T cell.

When compared with the cell cultivated in the case where checkpoint inhibitor is not present, receive military monoclonal antibody and pyridine aldoxime methyliodide (PAM) monoclonal antibody it is equal The higher granzyme B secretion of CD4 T cell is not significantly promoted.(Figure 13).

Table 27: the influence that the PD1-LAG3 bispecific antibody tested discharges cytotoxicity granzyme B

Sample	It influences
		PD1/LAG3 0927(1+1)	+++
PD1/LAG3 8970(2+2)	+
		PD1/LAG3 8310(1+2)	+/-
PD1/LAG3 0799(1+1)	++
		PD1/LAG3 8984(2+2)	++
PD1/LAG3 8311(1+2)	+/-
		aLAG3(BMS986016)	++

12.5 after collecting object with immunogenicity melanoma-Antigenic Peptide and arousing memory, and PD-1 and LAG-3 are blocked to from black The influence of the granzyme B and IFN-γ secretion of the CD4 T cell of plain tumor patient PBMC

It previously has been described, melanoma patient PBMC contains the specific for tumour antigen T cell of detectable frequency.Therefore, For the purpose that concept proves, overnight melanoma patient is stimulated collecting object again with immunogenicity melanoma associated antigen peptide The upper combination to anti-lag-3 antibody (0414) plus anti-PD-1 (0376) of PBMC is double special relative to the derivative of 1+1 (0927) form Property antibody or individually anti-PD-1 tested.

10 from melanoma patient⁵To 10⁶A PBMC presence or absence of saturated concentration (10g/ml) it is individual, With anti-lag-3 (0414,10g/ml) antibody combination or as the anti-of bispecific 1+1 form (0927,20g/ml) antibody PD-1 is incubated at room temperature in the case where (0376).Then in protein transport inhibitor Golgi Plug (brefeldin A) and in the presence of Golgi Stop (coban), by T cell immunogenic cancer related antigen such as MAGEA1, MAGEA3, MAGEA4, Melan-A/MART-1, NYESO-1, melanocyte albumen Pmel 17gp100, tyrosinase, tyrosine Enzyme GAP-associated protein GAP 2 collects object again and stimulates overnight.

The combination (P < 0.01 and P < 0.001) of anti-lag-3 and anti-PD-1 antibody and significant (P < 0.01 of bispecific antibody With P < 0.0001) enhancing specific for tumour antigen T cell effector function (i.e. granzyme B and IFN-γ secretion), and it is individual PD-1 blocking does not show any effect (Figure 12).

Example 13

The internal effective antitumor effect of the combination treatment of PD1/LAG3 bispecific antibody and CEACAM5 CD3 TCB

TCB molecule is prepared for according to method described in 2016/079076 A1 of WO 2014/131712 A1 or WO.In The preparation of anti-CEA/ AntiCD3 McAb bispecific antibody (CEA CD3 TCB or CEA TCB) used in experiment is described in WO 2014/ In the example 3 of 131712 A1.CEA CD3 TCB is " 2+1 IgG CrossFab " antibody and including two different heavy chains The light chain different with two.The point mutation (" protrusion enters hole ") in CH3 structural domain is introduced into promote the assembling of two different heavy chains. The exchange of the VH and VL structural domain in CD3 combination Fab is carried out, to promote the correct assembling of two different light chains.2+1 means point There are two the antigen bindings with specific antigen-binding domains and one for CD3 for CEA with specificity for son tool Structural domain.CEA CD3 TCB includes SEQ ID NO:146, SEQ ID NO:147, SEQ ID NO:148 and SEQ ID NO: Amino acid sequence shown in 149.CEACAM5 CD3 TCB form having the same, but include another CEA bonding agent and wrap Point mutation in the CH and CL structural domain of the bonding agent containing CD3, to support the correct pairing of light chain.CEACAM5 CD TCB includes SEQ ID NO:150, SEQ ID NO:151, amino acid sequence shown in SEQ ID NO:152 and SEQ ID NO:153.

A) experimental material and method

PD1/LAG3 bispecific antibody 0927 is with the concentration of 1.5mg/kg or 3mg/kg with people CEACAM5 CD3 TCB's Combination is tested in human pancreas' BXPC3 cancer model.BXPC3 cell and Apoptosis (3T3) is subcutaneous together Co-transplantation is into NSG humanization mouse.

The preparation of BXPC3 cell line: BXPC3 cell (human pancreatic cancer cell) is initially obtained from ECACC (European cell culture Collection (European Collection of Cell Culture)), and be deposited in Glycart after spreading cultivation In portion's cell bank.Containing 10%FCS (the Austrian laboratory PAA (PAA Laboratories, Austria)), 1% BXPC3 cell is cultivated in the RPMI of Glutamax.In 5%CO₂Under, cell is cultivated at 37 DEG C in water saturated atmosphere.

The generation of full-length human mouse: guide (GV-Solas according to the rules；Felasa；TierschG), experiment is opened Female NSG mouse (laboratory Jackson) when the beginning for 4-5 week old maintains without under conditions of special pathogen, and day recycles It is dark for 12h illumination/12h.Experimental study scheme passes through the examination and approval (P 2011/128) of local government.After arrival, By animal maintenance one week to shake down and observe.Periodically carry out continuous health status monitoring.NSG mouse i.p. is infused The busulfan of 15mg/kg is penetrated, then i.v. injects 1 × 10 separated from Cord blood after one day⁵A human hematopoietic stem cell.In The 14-16 weeks after stem cell injection, sublingual bloodletting is carried out to mouse and successful humanization pair is directed to by flow cytometry Blood is analyzed.The mouse effectively transplanted is turned into different processing groups according to their human T-cell's frequency accidental.

Efficacy experiments: at the 0th day, in the presence of the substrate glue of 1:1 ratio, by the HSC-NSG mouse of full-length human With 1 × 10⁶A BXPC3 cell (human pancreatic cancer cell expresses CEACAM5) is subcutaneously excited.Lead to during entire experiment Cross slide calliper rule 2 to 3 measurement tumours weekly.At the 15th day, mouse is turned at random with 250mm for tumor size³Be averaged Tumor size, and start arrange weekly therapy (medium (histidine buffering liquid), anti-PD1 (0376), receive Wu Dankang, group Nurse monoclonal antibody or anti-PD1-LAG3 0927) and given with 400 μ l of maximum by intraperitoneal injection.2-3 use slide calliper rule are surveyed weekly It measures tumour growth and calculates gross tumor volume as follows:

T_v: (W²/ 2) × L (W: width, L: length)

Research was terminated at the 47th day.

B) result

Tumor volume measurement result (mm within 47 day period³+/- SEM) in Figure 14 with being averaged in respective handling group Volume is shown.It is handled with CEACAM5-TCB and only shows progression of disease identical with untreated medium group.On the contrary, receiving force Monoclonal antibody and pyridine aldoxime methyliodide (PAM) monoclonal antibody reduce tumour growth, however, not up to tumour growth controls., it is surprising that concentration is 3mg/Kg PD1/LAG3 bispecific antibody 0927 completely inhibit tumour growth in all processing animals, show in addition to PD-1 The synergistic effect that LAG-3 is blocked altogether.

In Figure 15 A into 15F, show every solitary animal in the period of 47 days in tumor volume measurement result (mm³ +/- SEM), it is shown that the homogeneity of antitumor reaction in every group.

It is aobvious using Deng Nite method (Dunnett ' s Method) computational statistics for the mono- processing of CEACAM5 CD3 TCB Work property.In order to test multiple comparisons cell mean significant difference, using Deng Nite method automatically generate standard variance analyze (ANOVA).Whether Deng Nite method testing mean is different from the average value of control group.

Resulting TGI and TCR value shown in table 28 (TGI means Tumor growth inhibition, and TGI > 100 mean tumor regression, And TGI=100 is defined as tumor stasis；TCR means processing and the ratio that compares, and TCR=1 means no effect, and TCR=0 It is defined as subsiding completely).

Tumor growth inhibition (TGI) at table 28: the 46 days and processing and the ratio (TCR) compareed

It is further shown using Deng Nite method with p value compared with control.

Being handled using CEACAM5 CD3 TCB not can control tumour growth in the background of cancer of pancreas.However, its with The combination of the anti-PD1/LAG3 0927 of bispecific antibody generates strong influence to tumour control with dose specific manner.Statistics Learn analysis shows that, when compared with singly processing, there are two types of the combinations of the anti-PD1/LAG3 0927 of concentration for tool, but be not have it is anti- PD1 antibody receives the statistically significant difference that the combination of military monoclonal antibody and pyridine aldoxime methyliodide (PAM) monoclonal antibody causes tumour growth to control, and shows double The anti-PD1/LAG3 antibody of specificity is to the superiority of the inhibition of independent PD1, to make tumor growth stagnation.

Sequence table

<110> F. Hoffmann-La Roche AG

<120>bispecific antibody of PD1 and LAG3 is specifically bound

<130> P34215-WO

<150> EP17165125.0

<151> 2017-04-05

<160> 169

<170>PatentIn version 3 .5

<210> 1

<211> 7

<212> PRT

<213>artificial sequence

<220>

<223>heavy chain HVR-H1, PD1-0103

<400> 1

Gly Phe Ser Phe Ser Ser Tyr

1 5

<210> 2

<211> 3

<212> PRT

<213>artificial sequence

<220>

<223>heavy chain HVR-H2, PD1-0103

<400> 2

Gly Gly Arg

<210> 3

<211> 9

<212> PRT

<213>artificial sequence

<220>

<223>heavy chain HVR-H3, PD1-0103

<400> 3

Thr Gly Arg Val Tyr Phe Ala Leu Asp

1 5

<210> 4

<211> 11

<212> PRT

<213>artificial sequence

<220>

<223>light chain HVR-L1, PD1-0103

<400> 4

Ser Glu Ser Val Asp Thr Ser Asp Asn Ser Phe

1 5 10

<210> 5

<211> 3

<212> PRT

<213>artificial sequence

<220>

<223>light chain HVR-L2, PD1-0103

<400> 5

Arg Ser Ser

<210> 6

<211> 6

<212> PRT

<213>artificial sequence

<220>

<223>light chain HVR-L3, PD1-0103

<400> 6

Asn Tyr Asp Val Pro Trp

1 5

<210> 7

<211> 120

<212> PRT

<213>artificial sequence

<220>

<223>heavy-chain variable domains VH, PD1-0103

<400> 7

Glu Val Ile Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Ser Ser Tyr

20 25 30

Thr Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Asp Trp Val

35 40 45

Ala Thr Ile Ser Gly Gly Gly Arg Asp Ile Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Glu Met Ser Ser Leu Met Ser Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Val Leu Leu Thr Gly Arg Val Tyr Phe Ala Leu Asp Ser Trp Gly Gln

100 105 110

Gly Thr Ser Val Thr Val Ser Ser

115 120

<210> 8

<211> 111

<212> PRT

<213>artificial sequence

<220>

<223>light variable domains VL, PD1-0103

<400> 8

Lys Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Pro Val Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Thr Ser

20 25 30

Asp Asn Ser Phe Ile His Trp Tyr Gln Gln Arg Pro Gly Gln Ser Pro

35 40 45

Lys Leu Leu Ile Tyr Arg Ser Ser Thr Leu Glu Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asp

65 70 75 80

Pro Val Glu Ala Asp Asp Val Ala Thr Tyr Tyr Cys Gln Gln Asn Tyr

85 90 95

Asp Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 9

<211> 120

<212> PRT

<213>artificial sequence

<220>

<223>humanization variants of PD1-0103_01-heavy-chain variable domains VH

(PD1 0376)

<400> 9

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Ser Ser Tyr

20 25 30

Thr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Gly Gly Gly Arg Asp Ile Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Val Leu Leu Thr Gly Arg Val Tyr Phe Ala Leu Asp Ser Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 10

<211> 111

<212> PRT

<213>artificial sequence

<220>

<223>humanization variants of PD1-0103_01-light variable domains VL

(PD1 0376)

<400> 10

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ala Ser Glu Ser Val Asp Thr Ser

20 25 30

Asp Asn Ser Phe Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro

35 40 45

Lys Leu Leu Ile Tyr Arg Ser Ser Thr Leu Glu Ser Gly Val Pro Asp

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Asn Tyr

85 90 95

Asp Val Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 11

<211> 111

<212> PRT

<213>artificial sequence

<220>

<223>humanization variants of PD1-0103_02-light variable domains VL

<400> 11

Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Thr Ser

20 25 30

Asp Asn Ser Phe Ile His Trp Tyr Gln Gln Arg Pro Gly Gln Ser Pro

35 40 45

Arg Leu Leu Ile Tyr Arg Ser Ser Thr Leu Glu Ser Gly Val Pro Asp

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser

65 70 75 80

Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gln Gln Asn Tyr

85 90 95

Asp Val Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 12

<211> 111

<212> PRT

<213>artificial sequence

<220>

<223>humanization variants of PD1-0103_03-light variable domains VL

<400> 12

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Glu Ser Val Asp Thr Ser

20 25 30

Asp Asn Ser Phe Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro

35 40 45

Arg Leu Leu Ile Tyr Arg Ser Ser Thr Leu Glu Ser Gly Ile Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Asn Tyr

85 90 95

Asp Val Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 13

<211> 111

<212> PRT

<213>artificial sequence

<220>

<223>humanization variants of PD1-0103_04-light variable domains VL

<400> 13

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Glu Ser Val Asp Thr Ser

20 25 30

Asp Asn Ser Phe Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro

35 40 45

Arg Leu Leu Ile Tyr Arg Ser Ser Thr Leu Glu Ser Gly Ile Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Asn Tyr

85 90 95

Asp Val Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 14

<211> 5

<212> PRT

<213>artificial sequence

<220>

<223>heavy chain HVR-H1, aLAG3 (0414)

<400> 14

Asp Tyr Thr Met Asn

1 5

<210> 15

<211> 17

<212> PRT

<213>artificial sequence

<220>

<223>heavy chain HVR-H2, aLAG3 (0414)

<400> 15

Val Ile Ser Trp Asp Gly Gly Gly Thr Tyr Tyr Thr Asp Ser Val Lys

1 5 10 15

Gly

<210> 16

<211> 12

<212> PRT

<213>artificial sequence

<220>

<223>heavy chain HVR-H3, aLAG3 (0414)

<400> 16

Gly Leu Thr Asp Thr Thr Leu Tyr Gly Ser Asp Tyr

1 5 10

<210> 17

<211> 11

<212> PRT

<213>artificial sequence

<220>

<223>light chain HVR-L1, aLAG3 (0414)

<400> 17

Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn

1 5 10

<210> 18

<211> 7

<212> PRT

<213>artificial sequence

<220>

<223>light chain HVR-L2, aLAG3 (0414)

<400> 18

Ala Ala Ser Thr Leu Gln Ser

1 5

<210> 19

<211> 9

<212> PRT

<213>artificial sequence

<220>

<223>light chain HVR-L3, aLAG3 (0414)

<400> 19

Gln Gln Thr Tyr Ser Ser Pro Leu Thr

1 5

<210> 20

<211> 121

<212> PRT

<213>artificial sequence

<220>

<223>heavy-chain variable domains VH, aLAG3 (0414)

<400> 20

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Asp Asp Tyr

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Ser Trp Asp Gly Gly Gly Thr Tyr Tyr Thr Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Phe Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Gly Leu Thr Asp Thr Thr Leu Tyr Gly Ser Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 21

<211> 107

<212> PRT

<213>artificial sequence

<220>

<223>light variable domains VL, aLAG3 (0414)

<400> 21

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Tyr Ser Ser Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 22

<211> 5

<212> PRT

<213>artificial sequence

<220>

<223>heavy chain HVR-H1, aLAG3 (0403)

<400> 22

Asp Tyr Thr Met His

1 5

<210> 23

<211> 17

<212> PRT

<213>artificial sequence

<220>

<223>heavy chain HVR-H2, aLAG3 (0403)

<400> 23

Leu Val Ser Trp Asp Gly Gly Gly Thr Tyr Tyr Thr Asn Ser Val Lys

1 5 10 15

Gly

<210> 24

<211> 12

<212> PRT

<213>artificial sequence

<220>

<223>heavy chain HVR-H3, aLAG3 (0403)

<400> 24

Ala Ile Thr Asp Thr Ser Leu Tyr Gly Tyr Asp Tyr

1 5 10

<210> 25

<211> 11

<212> PRT

<213>artificial sequence

<220>

<223>light chain HVR-L1, aLAG3 (0403)

<400> 25

Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn

1 5 10

<210> 26

<211> 7

<212> PRT

<213>artificial sequence

<220>

<223>light chain HVR-L2, aLAG3 (0403)

<400> 26

Ala Ala Ser Ser Leu Gln Ser

1 5

<210> 27

<211> 9

<212> PRT

<213>artificial sequence

<220>

<223>light chain HVR-L3, aLAG3 (0403)

<400> 27

Gln Gln Thr Tyr Ser Thr Pro Leu Thr

1 5

<210> 28

<211> 121

<212> PRT

<213>artificial sequence

<220>

<223>heavy-chain variable domains VH, aLAG3 (0403)

<400> 28

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr

20 25 30

Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Leu Val Ser Trp Asp Gly Gly Gly Thr Tyr Tyr Thr Asn Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Lys Ala Ile Thr Asp Thr Ser Leu Tyr Gly Tyr Asp Tyr Trp Gly

100 105 110

Gln Gly Ile Leu Val Thr Val Ser Ser

115 120

<210> 29

<211> 107

<212> PRT

<213>artificial sequence

<220>

<223>light variable domains VL, aLAG3 (0403)

<400> 29

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Asn Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Tyr Ser Thr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 30

<211> 5

<212> PRT

<213>artificial sequence

<220>

<223>heavy chain HVR-H1, aLAG3 (0411)

<400> 30

Asp Tyr Thr Met Asn

1 5

<210> 31

<211> 17

<212> PRT

<213>artificial sequence

<220>

<223>heavy chain HVR-H2, aLAG3 (0411)

<400> 31

Val Ile Ser Trp Asp Gly Gly Ala Thr Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 32

<211> 12

<212> PRT

<213>artificial sequence

<220>

<223>heavy chain HVR-H3, aLAG3 (0411)

<400> 32

Gly Leu Thr Asp Asp Thr Leu Tyr Gly Ser Asp Tyr

1 5 10

<210> 33

<211> 11

<212> PRT

<213>artificial sequence

<220>

<223>light chain HVR-L1, aLAG3 (0411)

<400> 33

Arg Ala Ser Gln Ser Ile Val Ser Tyr Leu Asn

1 5 10

<210> 34

<211> 7

<212> PRT

<213>artificial sequence

<220>

<223>light chain HVR-L2, aLAG3 (0411)

<400> 34

Ala Ser Ser Ser Leu Gln Ser

1 5

<210> 35

<211> 9

<212> PRT

<213>artificial sequence

<220>

<223>light chain HVR-L3, aLAG3 (0411)

<400> 35

Gln Gln Thr Tyr Ser Thr Pro Leu Thr

1 5

<210> 36

<211> 121

<212> PRT

<213>artificial sequence

<220>

<223>heavy-chain variable domains VH, aLAG3 (0411)

<400> 36

Glu Val His Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Val Asp Asp Tyr

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Val Ile Ser Trp Asp Gly Gly Ala Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Phe Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Gly Leu Thr Asp Asp Thr Leu Tyr Gly Ser Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 37

<211> 107

<212> PRT

<213>artificial sequence

<220>

<223>light variable domains VL, aLAG3 (0411)

<400> 37

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Val Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ser Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Tyr Ser Thr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 38

<211> 5

<212> PRT

<213>artificial sequence

<220>

<223>heavy chain HVR-H1, aLAG3 (0417)

<400> 38

Asp Tyr Ala Met Ser

1 5

<210> 39

<211> 17

<212> PRT

<213>artificial sequence

<220>

<223>heavy chain HVR-H2, aLAG3 (0417)

<400> 39

Gly Ile Asp Asn Ser Gly Tyr Tyr Thr Tyr Tyr Thr Asp Ser Val Lys

1 5 10 15

Gly

<210> 40

<211> 13

<212> PRT

<213>artificial sequence

<220>

<223>heavy chain HVR-H3, aLAG3 (0417)

<400> 40

Thr His Ser Gly Leu Ile Val Asn Asp Ala Phe Asp Ile

1 5 10

<210> 41

<211> 11

<212> PRT

<213>artificial sequence

<220>

<223>light chain HVR-L1, aLAG3 (0417)

<400> 41

Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn

1 5 10

<210> 42

<211> 7

<212> PRT

<213>artificial sequence

<220>

<223>light chain HVR-L2, aLAG3 (0417)

<400> 42

Ala Ala Ser Ser Leu Gln Ser

1 5

<210> 43

<211> 9

<212> PRT

<213>artificial sequence

<220>

<223>light chain HVR-L3, aLAG3 (0417)

<400> 43

Gln Gln Thr Tyr Ser Thr Pro Leu Thr

1 5

<210> 44

<211> 122

<212> PRT

<213>artificial sequence

<220>

<223>heavy-chain variable domains VH, aLAG3 (0417)

<400> 44

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ala Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Gly Ile Asp Asn Ser Gly Tyr Tyr Thr Tyr Tyr Thr Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Val Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Leu Cys

85 90 95

Thr Lys Thr His Ser Gly Leu Ile Val Asn Asp Ala Phe Asp Ile Trp

100 105 110

Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120

<210> 45

<211> 107

<212> PRT

<213>artificial sequence

<220>

<223>light variable domains VL, aLAG3 (0417)

<400> 45

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Tyr Ser Thr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 46

<211> 5

<212> PRT

<213>artificial sequence

<220>

<223>heavy chain HVR-H1, aLAG3 (0416)

<400> 46

Asp Tyr Ala Met Ser

1 5

<210> 47

<211> 17

<212> PRT

<213>artificial sequence

<220>

<223>heavy chain HVR-H2, aLAG3 (0416)

<400> 47

Gly Ile Asp Asn Ser Gly Tyr Tyr Thr Tyr Tyr Thr Asp Ser Val Lys

1 5 10 15

Gly

<210> 48

<211> 13

<212> PRT

<213>artificial sequence

<220>

<223>heavy chain HVR-H3, aLAG3 (0416)

<400> 48

Thr His Ser Gly Leu Ile Val Asn Asp Ala Phe Asp Ile

1 5 10

<210> 49

<211> 11

<212> PRT

<213>artificial sequence

<220>

<223>light chain HVR-L1, aLAG3 (0416)

<400> 49

Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn

1 5 10

<210> 50

<211> 7

<212> PRT

<213>artificial sequence

<220>

<223>light chain HVR-L2, aLAG3 (0416)

<400> 50

Asp Ala Ser Ser Leu Glu Ser

1 5

<210> 51

<211> 9

<212> PRT

<213>artificial sequence

<220>

<223>light chain HVR-L3, aLAG3 (0416)

<400> 51

Gln Gln Ser Tyr Ser Thr Pro Leu Thr

1 5

<210> 52

<211> 122

<212> PRT

<213>artificial sequence

<220>

<223>heavy-chain variable domains VH, aLAG3 (0416)

<400> 52

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ala Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Gly Ile Asp Asn Ser Gly Tyr Tyr Thr Tyr Tyr Thr Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Val Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Leu Cys

85 90 95

Thr Lys Thr His Ser Gly Leu Ile Val Asn Asp Ala Phe Asp Ile Trp

100 105 110

Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120

<210> 53

<211> 107

<212> PRT

<213>artificial sequence

<220>

<223>light variable domains VL, aLAG3 (0416)

<400> 53

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Ala Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 54

<211> 120

<212> PRT

<213>artificial sequence

<220>

<223>heavy-chain variable domains VH, BMS-986016

<400> 54

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Asp Tyr

20 25 30

Tyr Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asn His Arg Gly Ser Thr Asn Ser Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Leu Ser Leu Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Arg Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Phe Gly Tyr Ser Asp Tyr Glu Tyr Asn Trp Phe Asp Pro Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 55

<211> 107

<212> PRT

<213>artificial sequence

<220>

<223>light variable domains VL BMS-986016

<400> 55

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Asn Leu Glu Ile Lys

100 105

<210> 56

<211> 5

<212> PRT

<213>artificial sequence

<220>

<223>heavy chain HVR-H1, MDX25F7 (25F7)

<400> 56

Asp Tyr Tyr Trp Asn

1 5

<210> 57

<211> 16

<212> PRT

<213>artificial sequence

<220>

<223>heavy chain HVR-H2, MDX25F7 (25F7)

<400> 57

Glu Ile Asn His Asn Gly Asn Thr Asn Ser Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 58

<211> 10

<212> PRT

<213>artificial sequence

<220>

<223>heavy chain HVR-H3, MDX25F7 (25F7)

<400> 58

Gly Tyr Ser Asp Tyr Glu Tyr Asn Trp Phe

1 5 10

<210> 59

<211> 11

<212> PRT

<213>artificial sequence

<220>

<223>light chain HVR-L1, MDX25F7 (25F7)

<400> 59

Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Ala

1 5 10

<210> 60

<211> 7

<212> PRT

<213>artificial sequence

<220>

<223>light chain HVR-L2, MDX25F7 (25F7)

<400> 60

Asp Ala Ser Asn Arg Ala Thr

1 5

<210> 61

<211> 9

<212> PRT

<213>artificial sequence

<220>

<223>light chain HVR-L3, MDX25F7 (25F7)

<400> 61

Gln Gln Arg Ser Asn Trp Pro Leu Thr

1 5

<210> 62

<211> 120

<212> PRT

<213>artificial sequence

<220>

<223>heavy-chain variable domains VH, MDX25F7 (25F7)

<400> 62

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Asp Tyr

20 25 30

Tyr Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asn His Asn Gly Asn Thr Asn Ser Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Leu Ser Leu Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Arg Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Phe Gly Tyr Ser Asp Tyr Glu Tyr Asn Trp Phe Asp Pro Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 63

<211> 107

<212> PRT

<213>artificial sequence

<220>

<223>light variable domains VL, MDX25F7 (25F7)

<400> 63

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Asn Leu Glu Ile Lys

100 105

<210> 64

<211> 125

<212> PRT

<213>artificial sequence

<220>

<223>heavy-chain variable domains VH, humanization BAP050 (LAG525)

<400> 64

Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu

1 5 10 15

Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Phe Thr Leu Thr Asn Tyr

20 25 30

Gly Met Asn Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Lys Trp Met

35 40 45

Gly Trp Ile Asn Thr Asp Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe

50 55 60

Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Ser

65 70 75 80

Leu Gln Ile Asn Asn Leu Lys Asn Ala Asp Thr Ala Thr Tyr Phe Cys

85 90 95

Ala Arg Asn Pro Pro Tyr Tyr Tyr Gly Thr Asn Asn Ala Glu Ala Met

100 105 110

Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210> 65

<211> 107

<212> PRT

<213>artificial sequence

<220>

<223>light variable domains VL, humanization BAP050 (LAG525)

<400> 65

Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Ser Ser Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Met Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Val Leu Ile

35 40 45

Tyr Tyr Thr Ser Thr Leu His Leu Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Leu

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Asn Leu Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 66

<211> 122

<212> PRT

<213>artificial sequence

<220>

<223>heavy-chain variable domains VH, MDX26H10 (26H10)

<400> 66

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Trp Ala Val Ala Ser Trp Asp Tyr Gly Met Asp Val Trp

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 67

<211> 108

<212> PRT

<213>artificial sequence

<220>

<223>light variable domains VL, MDX26H10 (26H10)

<400> 67

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro

85 90 95

Phe Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys

100 105

<210> 68

<211> 107

<212> PRT

<213>artificial sequence

<220>

<223>human kappa light chain constant region

<400> 68

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1 5 10 15

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 69

<211> 105

<212> PRT

<213>artificial sequence

<220>

<223>people's lambda light chain constant region

<400> 69

Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu

1 5 10 15

Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe

20 25 30

Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val

35 40 45

Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys

50 55 60

Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser

65 70 75 80

His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu

85 90 95

Lys Thr Val Ala Pro Thr Glu Cys Ser

100 105

<210> 70

<211> 329

<212> PRT

<213>artificial sequence

<220>

<223>people's heavy chain constant region of IgG1 is derived from

<400> 70

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly

325

<210> 71

<211> 329

<212> PRT

<213>artificial sequence

<220>

<223>from people's heavy chain constant region of the IgG1 with mutation L234A, L235A and P329G

<400> 71

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly

325

<210> 72

<211> 326

<212> PRT

<213>artificial sequence

<220>

<223>people's heavy chain constant region of IgG4 is derived from

<400> 72

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro

100 105 110

Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

305 310 315 320

Leu Ser Leu Ser Leu Gly

325

<210> 73

<211> 497

<212> PRT

<213>homo sapiens

<400> 73

Val Pro Val Val Trp Ala Gln Glu Gly Ala Pro Ala Gln Leu Pro Cys

1 5 10 15

Ser Pro Thr Ile Pro Leu Gln Asp Leu Ser Leu Leu Arg Arg Ala Gly

20 25 30

Val Thr Trp Gln His Gln Pro Asp Ser Gly Pro Pro Ala Ala Ala Pro

35 40 45

Gly His Pro Leu Ala Pro Gly Pro His Pro Ala Ala Pro Ser Ser Trp

50 55 60

Gly Pro Arg Pro Arg Arg Tyr Thr Val Leu Ser Val Gly Pro Gly Gly

65 70 75 80

Leu Arg Ser Gly Arg Leu Pro Leu Gln Pro Arg Val Gln Leu Asp Glu

85 90 95

Arg Gly Arg Gln Arg Gly Asp Phe Ser Leu Trp Leu Arg Pro Ala Arg

100 105 110

Arg Ala Asp Ala Gly Glu Tyr Arg Ala Ala Val His Leu Arg Asp Arg

115 120 125

Ala Leu Ser Cys Arg Leu Arg Leu Arg Leu Gly Gln Ala Ser Met Thr

130 135 140

Ala Ser Pro Pro Gly Ser Leu Arg Ala Ser Asp Trp Val Ile Leu Asn

145 150 155 160

Cys Ser Phe Ser Arg Pro Asp Arg Pro Ala Ser Val His Trp Phe Arg

165 170 175

Asn Arg Gly Gln Gly Arg Val Pro Val Arg Glu Ser Pro His His His

180 185 190

Leu Ala Glu Ser Phe Leu Phe Leu Pro Gln Val Ser Pro Met Asp Ser

195 200 205

Gly Pro Trp Gly Cys Ile Leu Thr Tyr Arg Asp Gly Phe Asn Val Ser

210 215 220

Ile Met Tyr Asn Leu Thr Val Leu Gly Leu Glu Pro Pro Thr Pro Leu

225 230 235 240

Thr Val Tyr Ala Gly Ala Gly Ser Arg Val Gly Leu Pro Cys Arg Leu

245 250 255

Pro Ala Gly Val Gly Thr Arg Ser Phe Leu Thr Ala Lys Trp Thr Pro

260 265 270

Pro Gly Gly Gly Pro Asp Leu Leu Val Thr Gly Asp Asn Gly Asp Phe

275 280 285

Thr Leu Arg Leu Glu Asp Val Ser Gln Ala Gln Ala Gly Thr Tyr Thr

290 295 300

Cys His Ile His Leu Gln Glu Gln Gln Leu Asn Ala Thr Val Thr Leu

305 310 315 320

Ala Ile Ile Thr Val Thr Pro Lys Ser Phe Gly Ser Pro Gly Ser Leu

325 330 335

Gly Lys Leu Leu Cys Glu Val Thr Pro Val Ser Gly Gln Glu Arg Phe

340 345 350

Val Trp Ser Ser Leu Asp Thr Pro Ser Gln Arg Ser Phe Ser Gly Pro

355 360 365

Trp Leu Glu Ala Gln Glu Ala Gln Leu Leu Ser Gln Pro Trp Gln Cys

370 375 380

Gln Leu Tyr Gln Gly Glu Arg Leu Leu Gly Ala Ala Val Tyr Phe Thr

385 390 395 400

Glu Leu Ser Ser Pro Gly Ala Gln Arg Ser Gly Arg Ala Pro Gly Ala

405 410 415

Leu Pro Ala Gly His Leu Leu Leu Phe Leu Ile Leu Gly Val Leu Ser

420 425 430

Leu Leu Leu Leu Val Thr Gly Ala Phe Gly Phe His Leu Trp Arg Arg

435 440 445

Gln Trp Arg Pro Arg Arg Phe Ser Ala Leu Glu Gln Gly Ile His Pro

450 455 460

Pro Gln Ala Gln Ser Lys Ile Glu Glu Leu Glu Gln Glu Pro Glu Pro

465 470 475 480

Glu Pro Glu Pro Glu Pro Glu Pro Glu Pro Glu Pro Glu Pro Glu Gln

485 490 495

Leu

<210> 74

<211> 422

<212> PRT

<213>homo sapiens

<400> 74

Val Pro Val Val Trp Ala Gln Glu Gly Ala Pro Ala Gln Leu Pro Cys

1 5 10 15

Ser Pro Thr Ile Pro Leu Gln Asp Leu Ser Leu Leu Arg Arg Ala Gly

20 25 30

Val Thr Trp Gln His Gln Pro Asp Ser Gly Pro Pro Ala Ala Ala Pro

35 40 45

Gly His Pro Leu Ala Pro Gly Pro His Pro Ala Ala Pro Ser Ser Trp

50 55 60

Gly Pro Arg Pro Arg Arg Tyr Thr Val Leu Ser Val Gly Pro Gly Gly

65 70 75 80

Leu Arg Ser Gly Arg Leu Pro Leu Gln Pro Arg Val Gln Leu Asp Glu

85 90 95

Arg Gly Arg Gln Arg Gly Asp Phe Ser Leu Trp Leu Arg Pro Ala Arg

100 105 110

Arg Ala Asp Ala Gly Glu Tyr Arg Ala Ala Val His Leu Arg Asp Arg

115 120 125

Ala Leu Ser Cys Arg Leu Arg Leu Arg Leu Gly Gln Ala Ser Met Thr

130 135 140

Ala Ser Pro Pro Gly Ser Leu Arg Ala Ser Asp Trp Val Ile Leu Asn

145 150 155 160

Cys Ser Phe Ser Arg Pro Asp Arg Pro Ala Ser Val His Trp Phe Arg

165 170 175

Asn Arg Gly Gln Gly Arg Val Pro Val Arg Glu Ser Pro His His His

180 185 190

Leu Ala Glu Ser Phe Leu Phe Leu Pro Gln Val Ser Pro Met Asp Ser

195 200 205

Gly Pro Trp Gly Cys Ile Leu Thr Tyr Arg Asp Gly Phe Asn Val Ser

210 215 220

Ile Met Tyr Asn Leu Thr Val Leu Gly Leu Glu Pro Pro Thr Pro Leu

225 230 235 240

Thr Val Tyr Ala Gly Ala Gly Ser Arg Val Gly Leu Pro Cys Arg Leu

245 250 255

Pro Ala Gly Val Gly Thr Arg Ser Phe Leu Thr Ala Lys Trp Thr Pro

260 265 270

Pro Gly Gly Gly Pro Asp Leu Leu Val Thr Gly Asp Asn Gly Asp Phe

275 280 285

Thr Leu Arg Leu Glu Asp Val Ser Gln Ala Gln Ala Gly Thr Tyr Thr

290 295 300

Cys His Ile His Leu Gln Glu Gln Gln Leu Asn Ala Thr Val Thr Leu

305 310 315 320

Ala Ile Ile Thr Val Thr Pro Lys Ser Phe Gly Ser Pro Gly Ser Leu

325 330 335

Gly Lys Leu Leu Cys Glu Val Thr Pro Val Ser Gly Gln Glu Arg Phe

340 345 350

Val Trp Ser Ser Leu Asp Thr Pro Ser Gln Arg Ser Phe Ser Gly Pro

355 360 365

Trp Leu Glu Ala Gln Glu Ala Gln Leu Leu Ser Gln Pro Trp Gln Cys

370 375 380

Gln Leu Tyr Gln Gly Glu Arg Leu Leu Gly Ala Ala Val Tyr Phe Thr

385 390 395 400

Glu Leu Ser Ser Pro Gly Ala Gln Arg Ser Gly Arg Ala Pro Gly Ala

405 410 415

Leu Pro Ala Gly His Leu

420

<210> 75

<211> 6

<212> PRT

<213>homo sapiens

<400> 75

Lys Ile Glu Glu Leu Glu

1 5

<210> 76

<211> 37

<212> DNA

<213>artificial sequence

<220>

<223>primer rbHC.up

<400> 76

aagcttgcca ccatggagac tgggctgcgc tggcttc 37

<210> 77

<211> 21

<212> DNA

<213>artificial sequence

<220>

<223>primer rbHCf.do

<400> 77

ccattggtga gggtgcccga g 21

<210> 78

<211> 20

<212> DNA

<213>artificial sequence

<220>

<223>primer BcPCR_FHLC_leader.fw

<400> 78

atggacatga gggtccccgc 20

<210> 79

<211> 24

<212> DNA

<213>artificial sequence

<220>

<223>primer BcPCR_huCkappa.rev

<400> 79

gatttcaact gctcatcaga tggc 24

<210> 80

<211> 8

<212> PRT

<213>artificial sequence

<220>

<223>heavy chain HVR-H1, PD1-0098

<400> 80

Gly Tyr Ser Ile Thr Ser Asp Tyr

1 5

<210> 81

<211> 3

<212> PRT

<213>artificial sequence

<220>

<223>heavy chain HVR-H2, PD1-0098

<400> 81

Tyr Ser Gly

<210> 82

<211> 9

<212> PRT

<213>artificial sequence

<220>

<223>heavy chain HVR-H3, PD1-0098

<400> 82

His Gly Ser Ala Pro Trp Tyr Phe Asp

1 5

<210> 83

<211> 12

<212> PRT

<213>artificial sequence

<220>

<223>light chain HVR-L1, PD1-0098

<400> 83

Ser Gln Asn Ile Val His Ser Asp Gly Asn Thr Tyr

1 5 10

<210> 84

<211> 3

<212> PRT

<213>artificial sequence

<220>

<223>light chain HVR-L2, PD1-0098

<400> 84

Lys Val Ser

<210> 85

<211> 6

<212> PRT

<213>artificial sequence

<220>

<223>light chain HVR-L3, PD1-0098

<400> 85

Gly Ser His Phe Pro Leu

1 5

<210> 86

<211> 120

<212> PRT

<213>artificial sequence

<220>

<223>heavy-chain variable domains VH, PD1-0098

<400> 86

Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Asp

20 25 30

Tyr Ala Trp Asn Trp Ile Arg Gln Phe Pro Gly Asp Lys Leu Glu Trp

35 40 45

Leu Gly Tyr Ile Thr Tyr Ser Gly Phe Thr Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Ile Ser Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Phe

65 70 75 80

Leu Gln Leu Asn Ser Val Ala Thr Glu Asp Thr Ala Thr Tyr Tyr Cys

85 90 95

Ala Arg Trp His Gly Ser Ala Pro Trp Tyr Phe Asp Tyr Trp Gly Arg

100 105 110

Gly Thr Thr Leu Thr Val Ser Ser

115 120

<210> 87

<211> 112

<212> PRT

<213>artificial sequence

<220>

<223>light variable domains VL PD1-0098

<400> 87

Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Asn Ile Val His Ser

20 25 30

Asp Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Asn Leu Leu Ile Tyr Lys Val Ser Arg Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Phe Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105 110

<210> 88

<211> 7

<212> PRT

<213>artificial sequence

<220>

<223>heavy chain HVR-H1, PD1-0069

<400> 88

Gly Tyr Thr Phe Thr Asp Tyr

1 5

<210> 89

<211> 3

<212> PRT

<213>artificial sequence

<220>

<223>heavy chain HVR-H2, PD1-0069

<400> 89

Tyr Ser Gly

<210> 90

<211> 7

<212> PRT

<213>artificial sequence

<220>

<223>heavy chain HVR-H3, PD1-0069

<400> 90

Gly Ile Thr Thr Gly Phe Ala

1 5

<210> 91

<211> 11

<212> PRT

<213>artificial sequence

<220>

<223>light chain HVR-L1, PD1-0069

<400> 91

Ser Lys Gly Val Ser Thr Ser Ser Tyr Ser Phe

1 5 10

<210> 92

<211> 3

<212> PRT

<213>artificial sequence

<220>

<223>light chain HVR-L2, PD1-0069

<400> 92

Tyr Ala Ser

<210> 93

<211> 6

<212> PRT

<213>artificial sequence

<220>

<223>light chain HVR-L3, PD1-0069

<400> 93

Ser Arg Glu Phe Pro Trp

1 5

<210> 94

<211> 118

<212> PRT

<213>artificial sequence

<220>

<223>heavy-chain variable domains VH, PD1-0069

<400> 94

Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Arg Pro Gly Val

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Ala Met His Trp Val Lys Gln Ser His Ala Arg Thr Leu Glu Trp Ile

35 40 45

Gly Val Ile Ser Thr Tyr Ser Gly Asp Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Met Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Leu Glu Leu Ala Arg Met Thr Ser Glu Asp Ser Ala Ile Tyr Tyr Cys

85 90 95

Ala Arg Leu Gly Ile Thr Thr Gly Phe Ala Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ala

115

<210> 95

<211> 111

<212> PRT

<213>artificial sequence

<220>

<223>light variable domains VL PD1-0069

<400> 95

Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Lys Gly Val Ser Thr Ser

20 25 30

Ser Tyr Ser Phe Met His Trp Tyr Gln Gln Lys Pro Arg Gln Pro Pro

35 40 45

Lys Leu Leu Ile Lys Tyr Ala Ser Tyr Leu Glu Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His

65 70 75 80

Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys His His Ser Arg

85 90 95

Glu Phe Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 96

<211> 443

<212> PRT

<213>artificial sequence

<220>

<223>heavy chain 1 of 1+1 PD1/LAG3 0799

<400> 96

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ala Ser Glu Ser Val Asp Thr Ser

20 25 30

Asp Asn Ser Phe Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro

35 40 45

Lys Leu Leu Ile Tyr Arg Ser Ser Thr Leu Glu Ser Gly Val Pro Asp

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Asn Tyr

85 90 95

Asp Val Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Ser

100 105 110

Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser

115 120 125

Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp

130 135 140

Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr

145 150 155 160

Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr

165 170 175

Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln

180 185 190

Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp

195 200 205

Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro

210 215 220

Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro

225 230 235 240

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

245 250 255

Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn

260 265 270

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

275 280 285

Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

290 295 300

Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

305 310 315 320

Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys

325 330 335

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp

340 345 350

Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe

355 360 365

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

370 375 380

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

385 390 395 400

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly

405 410 415

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

420 425 430

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440

<210> 97

<211> 452

<212> PRT

<213>artificial sequence

<220>

<223>heavy chain 2 of 1+1 PD1/LAG3 0799

<400> 97

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ala Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Gly Ile Asp Asn Ser Gly Tyr Tyr Thr Tyr Tyr Thr Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Val Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Leu Cys

85 90 95

Thr Lys Thr His Ser Gly Leu Ile Val Asn Asp Ala Phe Asp Ile Trp

100 105 110

Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala

225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val

355 360 365

Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro Gly Lys

450

<210> 98

<211> 227

<212> PRT

<213>artificial sequence

<220>

<223>light chain 1 of 1+1 PD1/LAG3 0799

<400> 98

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Ser Ser Tyr

20 25 30

Thr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Gly Gly Gly Arg Asp Ile Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Val Leu Leu Thr Gly Arg Val Tyr Phe Ala Leu Asp Ser Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Val Ala Ala Pro Ser Val

115 120 125

Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser

130 135 140

Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln

145 150 155 160

Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val

165 170 175

Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu

180 185 190

Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu

195 200 205

Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg

210 215 220

Gly Glu Cys

225

<210> 99

<211> 214

<212> PRT

<213>artificial sequence

<220>

<223>light chain 2 of 1+1 PD1/LAG3 0799

<400> 99

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Ala Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Arg Lys Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 100

<211> 451

<212> PRT

<213>artificial sequence

<220>

<223>heavy chain 2 of 1+1 PD1/LAG3 0927

<400> 100

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Asp Asp Tyr

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Ser Trp Asp Gly Gly Gly Thr Tyr Tyr Thr Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Phe Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Gly Leu Thr Asp Thr Thr Leu Tyr Gly Ser Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Lys

450

<210> 101

<211> 214

<212> PRT

<213>artificial sequence

<220>

<223>light chain 2 of 1+1 PD1/LAG3 0927

<400> 101

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Tyr Ser Ser Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Arg Lys Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 102

<211> 443

<212> PRT

<213>artificial sequence

<220>

<223>heavy chain 1 of 1+1 PD1/LAG3 0222

<400> 102

Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Lys Gly Val Ser Thr Ser

20 25 30

Ser Tyr Ser Phe Met His Trp Tyr Gln Gln Lys Pro Arg Gln Pro Pro

35 40 45

Lys Leu Leu Ile Lys Tyr Ala Ser Tyr Leu Glu Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His

65 70 75 80

Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys His His Ser Arg

85 90 95

Glu Phe Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Ser

100 105 110

Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser

115 120 125

Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp

130 135 140

Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr

145 150 155 160

Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr

165 170 175

Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln

180 185 190

Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp

195 200 205

Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro

210 215 220

Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro

225 230 235 240

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

245 250 255

Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn

260 265 270

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

275 280 285

Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

290 295 300

Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

305 310 315 320

Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys

325 330 335

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp

340 345 350

Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe

355 360 365

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

370 375 380

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

385 390 395 400

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly

405 410 415

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

420 425 430

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440

<210> 103

<211> 450

<212> PRT

<213>artificial sequence

<220>

<223>heavy chain 2 of 1+1 PD1/LAG3 0222

<400> 103

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Asp Tyr

20 25 30

Tyr Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asn His Asn Gly Asn Thr Asn Ser Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Leu Ser Leu Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Arg Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Phe Gly Tyr Ser Asp Tyr Glu Tyr Asn Trp Phe Asp Pro Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210> 104

<211> 225

<212> PRT

<213>artificial sequence

<220>

<223>light chain 1 of 1+1 PD1/LAG3 0222

<400> 104

Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Arg Pro Gly Val

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Ala Met His Trp Val Lys Gln Ser His Ala Arg Thr Leu Glu Trp Ile

35 40 45

Gly Val Ile Ser Thr Tyr Ser Gly Asp Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Met Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Leu Glu Leu Ala Arg Met Thr Ser Glu Asp Ser Ala Ile Tyr Tyr Cys

85 90 95

Ala Arg Leu Gly Ile Thr Thr Gly Phe Ala Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ala Ala Ser Val Ala Ala Pro Ser Val Phe Ile

115 120 125

Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val

130 135 140

Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys

145 150 155 160

Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu

165 170 175

Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu

180 185 190

Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr

195 200 205

His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu

210 215 220

Cys

225

<210> 105

<211> 214

<212> PRT

<213>artificial sequence

<220>

<223>light chain 2 of 1+1 PD1/LAG3 0222

<400> 105

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Asn Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Arg Lys Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 106

<211> 444

<212> PRT

<213>artificial sequence

<220>

<223>heavy chain 1 of 1+1 PD1/LAG3 0224

<400> 106

Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Asn Ile Val His Ser

20 25 30

Asp Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Asn Leu Leu Ile Tyr Lys Val Ser Arg Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Phe Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105 110

Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser

115 120 125

Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys

130 135 140

Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu

145 150 155 160

Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu

165 170 175

Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr

180 185 190

Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val

195 200 205

Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

210 215 220

Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe

225 230 235 240

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

245 250 255

Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe

260 265 270

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

275 280 285

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

290 295 300

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

305 310 315 320

Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

325 330 335

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg

340 345 350

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly

355 360 365

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

370 375 380

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

385 390 395 400

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

405 410 415

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

420 425 430

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440

<210> 107

<211> 227

<212> PRT

<213>artificial sequence

<220>

<223>light chain 1 of 1+1 PD1/LAG3 0224

<400> 107

Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Asp

20 25 30

Tyr Ala Trp Asn Trp Ile Arg Gln Phe Pro Gly Asp Lys Leu Glu Trp

35 40 45

Leu Gly Tyr Ile Thr Tyr Ser Gly Phe Thr Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Ile Ser Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Phe

65 70 75 80

Leu Gln Leu Asn Ser Val Ala Thr Glu Asp Thr Ala Thr Tyr Tyr Cys

85 90 95

Ala Arg Trp His Gly Ser Ala Pro Trp Tyr Phe Asp Tyr Trp Gly Arg

100 105 110

Gly Thr Thr Leu Thr Val Ser Ser Ala Ser Val Ala Ala Pro Ser Val

115 120 125

Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser

130 135 140

Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln

145 150 155 160

Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val

165 170 175

Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu

180 185 190

Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu

195 200 205

Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg

210 215 220

Gly Glu Cys

225

<210> 108

<211> 443

<212> PRT

<213>artificial sequence

<220>

<223>aLAG3 (0156) heavy chain (MDX25F7)

<400> 108

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Asp Tyr

20 25 30

Tyr Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asn His Asn Gly Asn Thr Asn Ser Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Leu Ser Leu Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Arg Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Phe Gly Tyr Ser Asp Tyr Glu Tyr Asn Trp Phe Asp Pro Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Gly Gln Pro Lys Ala Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Cys Cys Gly Asp Thr Pro Ser Ser Thr Val Thr

130 135 140

Leu Gly Cys Leu Val Lys Gly Tyr Leu Pro Glu Pro Val Thr Val Thr

145 150 155 160

Trp Asn Ser Gly Thr Leu Thr Asn Gly Val Arg Thr Phe Pro Ser Val

165 170 175

Arg Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Ser Val Thr

180 185 190

Ser Ser Ser Gln Pro Val Thr Cys Asn Val Ala His Pro Ala Thr Asn

195 200 205

Thr Lys Val Asp Lys Thr Val Ala Pro Ser Thr Cys Ser Lys Pro Thr

210 215 220

Cys Pro Pro Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro

225 230 235 240

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

245 250 255

Cys Val Val Val Asp Val Ser Gln Asp Asp Pro Glu Val Gln Phe Thr

260 265 270

Trp Tyr Ile Asn Asn Glu Gln Val Arg Thr Ala Arg Pro Pro Leu Arg

275 280 285

Glu Gln Gln Phe Asn Ser Thr Ile Arg Val Val Ser Thr Leu Pro Ile

290 295 300

Ala His Gln Asp Trp Leu Arg Gly Lys Glu Phe Lys Cys Lys Val His

305 310 315 320

Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Arg

325 330 335

Gly Gln Pro Leu Glu Pro Lys Val Tyr Thr Met Gly Pro Pro Arg Glu

340 345 350

Glu Leu Ser Ser Arg Ser Val Ser Leu Thr Cys Met Ile Asn Gly Phe

355 360 365

Tyr Pro Ser Asp Ile Ser Val Glu Trp Glu Lys Asn Gly Lys Ala Glu

370 375 380

Asp Asn Tyr Lys Thr Thr Pro Ala Val Leu Asp Ser Asp Gly Ser Tyr

385 390 395 400

Phe Leu Tyr Asn Lys Leu Ser Val Pro Thr Ser Glu Trp Gln Arg Gly

405 410 415

Asp Val Phe Thr Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

420 425 430

Thr Gln Lys Ser Ile Ser Arg Ser Pro Gly Lys

435 440

<210> 109

<211> 214

<212> PRT

<213>artificial sequence

<220>

<223>aLAG3 (0156) light chain (MDX25F7)

<400> 109

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Asn Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 110

<211> 444

<212> PRT

<213>artificial sequence

<220>

<223>aLAG3 (0414) heavy chain

<400> 110

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Asp Asp Tyr

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Ser Trp Asp Gly Gly Gly Thr Tyr Tyr Thr Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Phe Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Gly Leu Thr Asp Thr Thr Leu Tyr Gly Ser Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gln Pro Lys Ala Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Cys Cys Gly Asp Thr Pro Ser Ser Thr Val

130 135 140

Thr Leu Gly Cys Leu Val Lys Gly Tyr Leu Pro Glu Pro Val Thr Val

145 150 155 160

Thr Trp Asn Ser Gly Thr Leu Thr Asn Gly Val Arg Thr Phe Pro Ser

165 170 175

Val Arg Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Ser Val

180 185 190

Thr Ser Ser Ser Gln Pro Val Thr Cys Asn Val Ala His Pro Ala Thr

195 200 205

Asn Thr Lys Val Asp Lys Thr Val Ala Pro Ser Thr Cys Ser Lys Pro

210 215 220

Thr Cys Pro Pro Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Ile Phe

225 230 235 240

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

245 250 255

Thr Cys Val Val Val Asp Val Ser Gln Asp Asp Pro Glu Val Gln Phe

260 265 270

Thr Trp Tyr Ile Asn Asn Glu Gln Val Arg Thr Ala Arg Pro Pro Leu

275 280 285

Arg Glu Gln Gln Phe Asn Ser Thr Ile Arg Val Val Ser Thr Leu Pro

290 295 300

Ile Ala His Gln Asp Trp Leu Arg Gly Lys Glu Phe Lys Cys Lys Val

305 310 315 320

His Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

325 330 335

Arg Gly Gln Pro Leu Glu Pro Lys Val Tyr Thr Met Gly Pro Pro Arg

340 345 350

Glu Glu Leu Ser Ser Arg Ser Val Ser Leu Thr Cys Met Ile Asn Gly

355 360 365

Phe Tyr Pro Ser Asp Ile Ser Val Glu Trp Glu Lys Asn Gly Lys Ala

370 375 380

Glu Asp Asn Tyr Lys Thr Thr Pro Ala Val Leu Asp Ser Asp Gly Ser

385 390 395 400

Tyr Phe Leu Tyr Asn Lys Leu Ser Val Pro Thr Ser Glu Trp Gln Arg

405 410 415

Gly Asp Val Phe Thr Cys Ser Val Met His Glu Ala Leu His Asn His

420 425 430

Tyr Thr Gln Lys Ser Ile Ser Arg Ser Pro Gly Lys

435 440

<210> 111

<211> 214

<212> PRT

<213>artificial sequence

<220>

<223>aLAG3 (0414) light chain

<400> 111

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Tyr Ser Ser Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 112

<211> 445

<212> PRT

<213>artificial sequence

<220>

<223>aLAG3 (0416) heavy chain

<400> 112

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ala Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Gly Ile Asp Asn Ser Gly Tyr Tyr Thr Tyr Tyr Thr Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Val Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Leu Cys

85 90 95

Thr Lys Thr His Ser Gly Leu Ile Val Asn Asp Ala Phe Asp Ile Trp

100 105 110

Gly Gln Gly Thr Met Val Thr Val Ser Ser Gly Gln Pro Lys Ala Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Cys Cys Gly Asp Thr Pro Ser Ser Thr

130 135 140

Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Leu Pro Glu Pro Val Thr

145 150 155 160

Val Thr Trp Asn Ser Gly Thr Leu Thr Asn Gly Val Arg Thr Phe Pro

165 170 175

Ser Val Arg Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Ser

180 185 190

Val Thr Ser Ser Ser Gln Pro Val Thr Cys Asn Val Ala His Pro Ala

195 200 205

Thr Asn Thr Lys Val Asp Lys Thr Val Ala Pro Ser Thr Cys Ser Lys

210 215 220

Pro Thr Cys Pro Pro Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Ile

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser Gln Asp Asp Pro Glu Val Gln

260 265 270

Phe Thr Trp Tyr Ile Asn Asn Glu Gln Val Arg Thr Ala Arg Pro Pro

275 280 285

Leu Arg Glu Gln Gln Phe Asn Ser Thr Ile Arg Val Val Ser Thr Leu

290 295 300

Pro Ile Ala His Gln Asp Trp Leu Arg Gly Lys Glu Phe Lys Cys Lys

305 310 315 320

Val His Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

325 330 335

Ala Arg Gly Gln Pro Leu Glu Pro Lys Val Tyr Thr Met Gly Pro Pro

340 345 350

Arg Glu Glu Leu Ser Ser Arg Ser Val Ser Leu Thr Cys Met Ile Asn

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ser Val Glu Trp Glu Lys Asn Gly Lys

370 375 380

Ala Glu Asp Asn Tyr Lys Thr Thr Pro Ala Val Leu Asp Ser Asp Gly

385 390 395 400

Ser Tyr Phe Leu Tyr Asn Lys Leu Ser Val Pro Thr Ser Glu Trp Gln

405 410 415

Arg Gly Asp Val Phe Thr Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Ile Ser Arg Ser Pro Gly Lys

435 440 445

<210> 113

<211> 214

<212> PRT

<213>artificial sequence

<220>

<223>aLAG3 (0416) light chain

<400> 113

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Ala Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 114

<211> 695

<212> PRT

<213>artificial sequence

<220>

<223>heavy chain of 2+2 PD1/LAG3 8970

<400> 114

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Asp Asp Tyr

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Ser Trp Asp Gly Gly Gly Thr Tyr Tyr Thr Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Phe Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Gly Leu Thr Asp Thr Thr Leu Tyr Gly Ser Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

450 455 460

Gly Gly Gly Ser Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val

465 470 475 480

Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser

485 490 495

Phe Ser Ser Tyr Thr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly

500 505 510

Leu Glu Trp Val Ala Thr Ile Ser Gly Gly Gly Arg Asp Ile Tyr Tyr

515 520 525

Pro Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys

530 535 540

Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala

545 550 555 560

Val Tyr Tyr Cys Val Leu Leu Thr Gly Arg Val Tyr Phe Ala Leu Asp

565 570 575

Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Val Ala

580 585 590

Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser

595 600 605

Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu

610 615 620

Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser

625 630 635 640

Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu

645 650 655

Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val

660 665 670

Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys

675 680 685

Ser Phe Asn Arg Gly Glu Cys

690 695

<210> 115

<211> 216

<212> PRT

<213>artificial sequence

<220>

<223>light chain 1 of 2+2 PD1/LAG3 8970

<400> 115

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ala Ser Glu Ser Val Asp Thr Ser

20 25 30

Asp Asn Ser Phe Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro

35 40 45

Lys Leu Leu Ile Tyr Arg Ser Ser Thr Leu Glu Ser Gly Val Pro Asp

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Asn Tyr

85 90 95

Asp Val Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Ser

100 105 110

Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser

115 120 125

Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp

130 135 140

Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr

145 150 155 160

Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr

165 170 175

Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln

180 185 190

Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp

195 200 205

Lys Lys Val Glu Pro Lys Ser Cys

210 215

<210> 116

<211> 696

<212> PRT

<213>artificial sequence

<220>

<223>heavy chain of 2+2 PD1/LAG3 8984

<400> 116

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ala Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Gly Ile Asp Asn Ser Gly Tyr Tyr Thr Tyr Tyr Thr Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Val Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Leu Cys

85 90 95

Thr Lys Thr His Ser Gly Leu Ile Val Asn Asp Ala Phe Asp Ile Trp

100 105 110

Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala

225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

450 455 460

Gly Gly Gly Gly Ser Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu

465 470 475 480

Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe

485 490 495

Ser Phe Ser Ser Tyr Thr Met Ser Trp Val Arg Gln Ala Pro Gly Lys

500 505 510

Gly Leu Glu Trp Val Ala Thr Ile Ser Gly Gly Gly Arg Asp Ile Tyr

515 520 525

Tyr Pro Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser

530 535 540

Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr

545 550 555 560

Ala Val Tyr Tyr Cys Val Leu Leu Thr Gly Arg Val Tyr Phe Ala Leu

565 570 575

Asp Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Val

580 585 590

Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys

595 600 605

Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg

610 615 620

Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn

625 630 635 640

Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser

645 650 655

Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys

660 665 670

Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr

675 680 685

Lys Ser Phe Asn Arg Gly Glu Cys

690 695

<210> 117

<211> 694

<212> PRT

<213>artificial sequence

<220>

<223>heavy chain of 2+2 PD1/LAG3 9010

<400> 117

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Asp Tyr

20 25 30

Tyr Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asn His Asn Gly Asn Thr Asn Ser Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Leu Ser Leu Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Arg Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Phe Gly Tyr Ser Asp Tyr Glu Tyr Asn Trp Phe Asp Pro Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

450 455 460

Gly Gly Ser Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln

465 470 475 480

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe

485 490 495

Ser Ser Tyr Thr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

500 505 510

Glu Trp Val Ala Thr Ile Ser Gly Gly Gly Arg Asp Ile Tyr Tyr Pro

515 520 525

Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn

530 535 540

Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val

545 550 555 560

Tyr Tyr Cys Val Leu Leu Thr Gly Arg Val Tyr Phe Ala Leu Asp Ser

565 570 575

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Val Ala Ala

580 585 590

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

595 600 605

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

610 615 620

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

625 630 635 640

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

645 650 655

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

660 665 670

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

675 680 685

Phe Asn Arg Gly Glu Cys

690

<210> 118

<211> 695

<212> PRT

<213>artificial sequence

<220>

<223>heavy chain 1 of 2+1 PD1/LAG3 8310

<400> 118

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Asp Asp Tyr

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Ser Trp Asp Gly Gly Gly Thr Tyr Tyr Thr Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Phe Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Gly Leu Thr Asp Thr Thr Leu Tyr Gly Ser Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

450 455 460

Gly Gly Gly Ser Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val

465 470 475 480

Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser

485 490 495

Phe Ser Ser Tyr Thr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly

500 505 510

Leu Glu Trp Val Ala Thr Ile Ser Gly Gly Gly Arg Asp Ile Tyr Tyr

515 520 525

Pro Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys

530 535 540

Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala

545 550 555 560

Val Tyr Tyr Cys Val Leu Leu Thr Gly Arg Val Tyr Phe Ala Leu Asp

565 570 575

Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Val Ala

580 585 590

Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser

595 600 605

Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu

610 615 620

Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser

625 630 635 640

Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu

645 650 655

Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val

660 665 670

Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys

675 680 685

Ser Phe Asn Arg Gly Glu Cys

690 695

<210> 119

<211> 451

<212> PRT

<213>artificial sequence

<220>

<223>heavy chain 2 of 2+1 PD1/LAG3 8310

<400> 119

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Asp Asp Tyr

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Ser Trp Asp Gly Gly Gly Thr Tyr Tyr Thr Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Phe Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Gly Leu Thr Asp Thr Thr Leu Tyr Gly Ser Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Lys

450

<210> 120

<211> 696

<212> PRT

<213>artificial sequence

<220>

<223>heavy chain 1 of 2+1 PD1/LAG3 8311

<400> 120

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ala Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Gly Ile Asp Asn Ser Gly Tyr Tyr Thr Tyr Tyr Thr Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Val Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Leu Cys

85 90 95

Thr Lys Thr His Ser Gly Leu Ile Val Asn Asp Ala Phe Asp Ile Trp

100 105 110

Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala

225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val

355 360 365

Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

450 455 460

Gly Gly Gly Gly Ser Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu

465 470 475 480

Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe

485 490 495

Ser Phe Ser Ser Tyr Thr Met Ser Trp Val Arg Gln Ala Pro Gly Lys

500 505 510

Gly Leu Glu Trp Val Ala Thr Ile Ser Gly Gly Gly Arg Asp Ile Tyr

515 520 525

Tyr Pro Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser

530 535 540

Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr

545 550 555 560

Ala Val Tyr Tyr Cys Val Leu Leu Thr Gly Arg Val Tyr Phe Ala Leu

565 570 575

Asp Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Val

580 585 590

Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys

595 600 605

Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg

610 615 620

Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn

625 630 635 640

Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser

645 650 655

Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys

660 665 670

Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr

675 680 685

Lys Ser Phe Asn Arg Gly Glu Cys

690 695

<210> 121

<211> 452

<212> PRT

<213>artificial sequence

<220>

<223>heavy chain 2 of 2+1 PD1/LAG3 8311

<400> 121

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ala Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Gly Ile Asp Asn Ser Gly Tyr Tyr Thr Tyr Tyr Thr Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Val Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Leu Cys

85 90 95

Thr Lys Thr His Ser Gly Leu Ile Val Asn Asp Ala Phe Asp Ile Trp

100 105 110

Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala

225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val

355 360 365

Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro Gly Lys

450

<210> 122

<211> 694

<212> PRT

<213>artificial sequence

<220>

<223>heavy chain 1 of 2+1 PD1/LAG3 1252

<400> 122

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Asp Tyr

20 25 30

Tyr Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asn His Asn Gly Asn Thr Asn Ser Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Leu Ser Leu Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Arg Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Phe Gly Tyr Ser Asp Tyr Glu Tyr Asn Trp Phe Asp Pro Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

450 455 460

Gly Gly Ser Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln

465 470 475 480

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe

485 490 495

Ser Ser Tyr Thr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

500 505 510

Glu Trp Val Ala Thr Ile Ser Gly Gly Gly Arg Asp Ile Tyr Tyr Pro

515 520 525

Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn

530 535 540

Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val

545 550 555 560

Tyr Tyr Cys Val Leu Leu Thr Gly Arg Val Tyr Phe Ala Leu Asp Ser

565 570 575

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Val Ala Ala

580 585 590

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

595 600 605

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

610 615 620

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

625 630 635 640

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

645 650 655

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

660 665 670

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

675 680 685

Phe Asn Arg Gly Glu Cys

690

<210> 123

<211> 941

<212> PRT

<213>artificial sequence

<220>

<223>heavy chain 1 of 2+1 PD1/LAG3 8312

<400> 123

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Asp Asp Tyr

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Ser Trp Asp Gly Gly Gly Thr Tyr Tyr Thr Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Phe Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Gly Leu Thr Asp Thr Thr Leu Tyr Gly Ser Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

450 455 460

Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Asp Ser

465 470 475 480

Leu Ala Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ala Ser

485 490 495

Glu Ser Val Asp Thr Ser Asp Asn Ser Phe Ile His Trp Tyr Gln Gln

500 505 510

Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Arg Ser Ser Thr Leu

515 520 525

Glu Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp

530 535 540

Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr

545 550 555 560

Tyr Cys Gln Gln Asn Tyr Asp Val Pro Trp Thr Phe Gly Gln Gly Thr

565 570 575

Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe

580 585 590

Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys

595 600 605

Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val

610 615 620

Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln

625 630 635 640

Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser

645 650 655

Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His

660 665 670

Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

675 680 685

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

690 695 700

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val

705 710 715 720

Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu

725 730 735

Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Ser Ser Tyr Thr Met

740 745 750

Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Thr

755 760 765

Ile Ser Gly Gly Gly Arg Asp Ile Tyr Tyr Pro Asp Ser Val Lys Gly

770 775 780

Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln

785 790 795 800

Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Val Leu

805 810 815

Leu Thr Gly Arg Val Tyr Phe Ala Leu Asp Ser Trp Gly Gln Gly Thr

820 825 830

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

835 840 845

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

850 855 860

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

865 870 875 880

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

885 890 895

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

900 905 910

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

915 920 925

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

930 935 940

<210> 124

<211> 942

<212> PRT

<213>artificial sequence

<220>

<223>heavy chain 1 of 2+1 PD1/LAG3 8313

<400> 124

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ala Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Gly Ile Asp Asn Ser Gly Tyr Tyr Thr Tyr Tyr Thr Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Val Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Leu Cys

85 90 95

Thr Lys Thr His Ser Gly Leu Ile Val Asn Asp Ala Phe Asp Ile Trp

100 105 110

Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala

225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val

355 360 365

Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

450 455 460

Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Asp

465 470 475 480

Ser Leu Ala Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ala

485 490 495

Ser Glu Ser Val Asp Thr Ser Asp Asn Ser Phe Ile His Trp Tyr Gln

500 505 510

Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Arg Ser Ser Thr

515 520 525

Leu Glu Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr

530 535 540

Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val

545 550 555 560

Tyr Tyr Cys Gln Gln Asn Tyr Asp Val Pro Trp Thr Phe Gly Gln Gly

565 570 575

Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile

580 585 590

Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val

595 600 605

Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys

610 615 620

Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu

625 630 635 640

Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu

645 650 655

Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr

660 665 670

His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu

675 680 685

Cys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

690 695 700

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu

705 710 715 720

Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser

725 730 735

Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Ser Ser Tyr Thr

740 745 750

Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala

755 760 765

Thr Ile Ser Gly Gly Gly Arg Asp Ile Tyr Tyr Pro Asp Ser Val Lys

770 775 780

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu

785 790 795 800

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Val

805 810 815

Leu Leu Thr Gly Arg Val Tyr Phe Ala Leu Asp Ser Trp Gly Gln Gly

820 825 830

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

835 840 845

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

850 855 860

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

865 870 875 880

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

885 890 895

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

900 905 910

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

915 920 925

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

930 935 940

<210> 125

<211> 940

<212> PRT

<213>artificial sequence

<220>

<223>heavy chain 1 of 2+1 PD1/LAG3 1088

<400> 125

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Asp Tyr

20 25 30

Tyr Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asn His Asn Gly Asn Thr Asn Ser Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Leu Ser Leu Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Arg Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Phe Gly Tyr Ser Asp Tyr Glu Tyr Asn Trp Phe Asp Pro Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

450 455 460

Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu

465 470 475 480

Ala Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ala Ser Glu

485 490 495

Ser Val Asp Thr Ser Asp Asn Ser Phe Ile His Trp Tyr Gln Gln Lys

500 505 510

Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Arg Ser Ser Thr Leu Glu

515 520 525

Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe

530 535 540

Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr

545 550 555 560

Cys Gln Gln Asn Tyr Asp Val Pro Trp Thr Phe Gly Gln Gly Thr Lys

565 570 575

Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro

580 585 590

Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu

595 600 605

Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp

610 615 620

Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp

625 630 635 640

Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys

645 650 655

Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln

660 665 670

Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Gly

675 680 685

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

690 695 700

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln

705 710 715 720

Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg

725 730 735

Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Ser Ser Tyr Thr Met Ser

740 745 750

Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Thr Ile

755 760 765

Ser Gly Gly Gly Arg Asp Ile Tyr Tyr Pro Asp Ser Val Lys Gly Arg

770 775 780

Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met

785 790 795 800

Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Val Leu Leu

805 810 815

Thr Gly Arg Val Tyr Phe Ala Leu Asp Ser Trp Gly Gln Gly Thr Leu

820 825 830

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

835 840 845

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

850 855 860

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

865 870 875 880

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

885 890 895

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

900 905 910

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

915 920 925

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

930 935 940

<210> 126

<211> 589

<212> PRT

<213>artificial sequence

<220>

<223>heavy chain 1 of 2+1 PD1/LAG3 0918

<400> 126

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Asp Tyr

20 25 30

Tyr Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asn His Asn Gly Asn Thr Asn Ser Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Leu Ser Leu Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Arg Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Phe Gly Tyr Ser Asp Tyr Glu Tyr Asn Trp Phe Asp Pro Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

450 455 460

Gly Gly Gly Gly Ser Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu

465 470 475 480

Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe

485 490 495

Ser Phe Ser Ser Tyr Thr Met Ser Trp Val Arg Gln Ala Pro Gly Lys

500 505 510

Gly Leu Glu Trp Val Ala Thr Ile Ser Gly Gly Gly Arg Asp Ile Tyr

515 520 525

Tyr Pro Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser

530 535 540

Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr

545 550 555 560

Ala Val Tyr Tyr Cys Val Leu Leu Thr Gly Arg Val Tyr Phe Ala Leu

565 570 575

Asp Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

580 585

<210> 127

<211> 580

<212> PRT

<213>artificial sequence

<220>

<223>heavy chain 2 of 2+1 PD1/LAG3 0918

<400> 127

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Asp Tyr

20 25 30

Tyr Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asn His Asn Gly Asn Thr Asn Ser Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Leu Ser Leu Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Arg Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Phe Gly Tyr Ser Asp Tyr Glu Tyr Asn Trp Phe Asp Pro Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

450 455 460

Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu

465 470 475 480

Ala Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ala Ser Glu

485 490 495

Ser Val Asp Thr Ser Asp Asn Ser Phe Ile His Trp Tyr Gln Gln Lys

500 505 510

Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Arg Ser Ser Thr Leu Glu

515 520 525

Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe

530 535 540

Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr

545 550 555 560

Cys Gln Gln Asn Tyr Asp Val Pro Trp Thr Phe Gly Gln Gly Thr Lys

565 570 575

Val Glu Ile Lys

580

<210> 128

<211> 288

<212> PRT

<213>homo sapiens

<400> 128

Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln

1 5 10 15

Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp

20 25 30

Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp

35 40 45

Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val

50 55 60

Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala

65 70 75 80

Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg

85 90 95

Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg

100 105 110

Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu

115 120 125

Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val

130 135 140

Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro

145 150 155 160

Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Val Gly Val Val Gly Gly

165 170 175

Leu Leu Gly Ser Leu Val Leu Leu Val Trp Val Leu Ala Val Ile Cys

180 185 190

Ser Arg Ala Ala Arg Gly Thr Ile Gly Ala Arg Arg Thr Gly Gln Pro

195 200 205

Leu Lys Glu Asp Pro Ser Ala Val Pro Val Phe Ser Val Asp Tyr Gly

210 215 220

Glu Leu Asp Phe Gln Trp Arg Glu Lys Thr Pro Glu Pro Pro Val Pro

225 230 235 240

Cys Val Pro Glu Gln Thr Glu Tyr Ala Thr Ile Val Phe Pro Ser Gly

245 250 255

Met Gly Thr Ser Ser Pro Ala Arg Arg Gly Ser Ala Asp Gly Pro Arg

260 265 270

Ser Ala Gln Pro Leu Arg Pro Glu Asp Gly His Cys Ser Trp Pro Leu

275 280 285

<210> 129

<211> 5

<212> PRT

<213>artificial sequence

<220>

<223>peptide linker G4S

<400> 129

Gly Gly Gly Gly Ser

1 5

<210> 130

<211> 10

<212> PRT

<213>artificial sequence

<220>

<223>peptide linker (G4S) 2

<400> 130

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10

<210> 131

<211> 10

<212> PRT

<213>artificial sequence

<220>

<223>peptide linker (SG4) 2

<400> 131

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

1 5 10

<210> 132

<211> 14

<212> PRT

<213>artificial sequence

<220>

<223>peptide linker G4 (SG4) 2

<400> 132

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

1 5 10

<210> 133

<211> 10

<212> PRT

<213>artificial sequence

<220>

<223>peptide linker

<400> 133

Gly Ser Pro Gly Ser Ser Ser Ser Gly Ser

1 5 10

<210> 134

<211> 15

<212> PRT

<213>artificial sequence

<220>

<223>peptide linker (G4S) 3

<400> 134

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 135

<211> 20

<212> PRT

<213>artificial sequence

<220>

<223>peptide linker (G4S) 4

<400> 135

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser

<210> 136

<211> 8

<212> PRT

<213>artificial sequence

<220>

<223>peptide linker

<400> 136

Gly Ser Gly Ser Gly Ser Gly Ser

1 5

<210> 137

<211> 8

<212> PRT

<213>artificial sequence

<220>

<223>peptide linker

<400> 137

Gly Ser Gly Ser Gly Asn Gly Ser

1 5

<210> 138

<211> 8

<212> PRT

<213>artificial sequence

<220>

<223>peptide linker

<400> 138

Gly Gly Ser Gly Ser Gly Ser Gly

1 5

<210> 139

<211> 6

<212> PRT

<213>artificial sequence

<220>

<223>peptide linker

<400> 139

Gly Gly Ser Gly Ser Gly

1 5

<210> 140

<211> 4

<212> PRT

<213>artificial sequence

<220>

<223>peptide linker

<400> 140

Gly Gly Ser Gly

<210> 141

<211> 8

<212> PRT

<213>artificial sequence

<220>

<223>peptide linker

<400> 141

Gly Gly Ser Gly Asn Gly Ser Gly

1 5

<210> 142

<211> 8

<212> PRT

<213>artificial sequence

<220>

<223>peptide linker

<400> 142

Gly Gly Asn Gly Ser Gly Ser Gly

1 5

<210> 143

<211> 6

<212> PRT

<213>artificial sequence

<220>

<223>peptide linker

<400> 143

Gly Gly Asn Gly Ser Gly

1 5

<210> 144

<211> 461

<212> PRT

<213>artificial sequence

<220>

<223>1+1 PD1/LAG3 0725(1+1 is trans-) heavy chain 2

<400> 144

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln

225 230 235 240

Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg

245 250 255

Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Asp Asp Tyr Thr Met Asn

260 265 270

Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Val Ile

275 280 285

Ser Trp Asp Gly Gly Gly Thr Tyr Tyr Thr Asp Ser Val Lys Gly Arg

290 295 300

Phe Thr Ile Ser Arg Asp Asp Phe Lys Asn Thr Leu Tyr Leu Gln Met

305 310 315 320

Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Gly

325 330 335

Leu Thr Asp Thr Thr Leu Tyr Gly Ser Asp Tyr Trp Gly Gln Gly Thr

340 345 350

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

355 360 365

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

370 375 380

Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

385 390 395 400

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

405 410 415

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

420 425 430

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

435 440 445

Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys

450 455 460

<210> 145

<211> 685

<212> PRT

<213>artificial sequence

<220>

<223>2+1 PD1/LAG3 0750(2+1 is trans-) heavy chain 2

<400> 145

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Asp Asp Tyr

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Ser Trp Asp Gly Gly Gly Thr Tyr Tyr Thr Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Phe Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Gly Leu Thr Asp Thr Thr Leu Tyr Gly Ser Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln

450 455 460

Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg

465 470 475 480

Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Asp Asp Tyr Thr Met Asn

485 490 495

Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Val Ile

500 505 510

Ser Trp Asp Gly Gly Gly Thr Tyr Tyr Thr Asp Ser Val Lys Gly Arg

515 520 525

Phe Thr Ile Ser Arg Asp Asp Phe Lys Asn Thr Leu Tyr Leu Gln Met

530 535 540

Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Gly

545 550 555 560

Leu Thr Asp Thr Thr Leu Tyr Gly Ser Asp Tyr Trp Gly Gln Gly Thr

565 570 575

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

580 585 590

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

595 600 605

Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

610 615 620

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

625 630 635 640

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

645 650 655

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

660 665 670

Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys

675 680 685

<210> 146

<211> 215

<212> PRT

<213>artificial sequence

<220>

<223>light chain CEA (CEA TCB)

<400> 146

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Ala Ala Val Gly Thr Tyr

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Arg Lys Arg Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys His Gln Tyr Tyr Thr Tyr Pro Leu

85 90 95

Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala

100 105 110

Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser

115 120 125

Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu

130 135 140

Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser

145 150 155 160

Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu

165 170 175

Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val

180 185 190

Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys

195 200 205

Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 147

<211> 214

<212> PRT

<213>artificial sequence

<220>

<223>light chain CD3 (CEA TCB)

<400> 147

Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly

1 5 10 15

Thr Val Thr Leu Thr Cys Gly Ser Ser Thr Gly Ala Val Thr Thr Ser

20 25 30

Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Gly Gln Ala Phe Arg Gly

35 40 45

Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe

50 55 60

Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Ala

65 70 75 80

Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn

85 90 95

Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Ser Ser Ala

100 105 110

Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser

115 120 125

Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe

130 135 140

Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly

145 150 155 160

Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu

165 170 175

Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr

180 185 190

Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys

195 200 205

Val Glu Pro Lys Ser Cys

210

<210> 148

<211> 694

<212> PRT

<213>artificial sequence

<220>

<223>CEA CD3 crossfab VHck fc protrusion P329GLALA (CEA TCB)

<400> 148

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Glu Phe

20 25 30

Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Thr Lys Thr Gly Glu Ala Thr Tyr Val Glu Glu Phe

50 55 60

Lys Gly Arg Val Thr Phe Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Trp Asp Phe Ala Tyr Tyr Val Glu Ala Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Leu

225 230 235 240

Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser

245 250 255

Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr Ala Met Asn Trp Val

260 265 270

Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Arg Ile Arg Ser

275 280 285

Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg

290 295 300

Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Leu Tyr Leu Gln Met

305 310 315 320

Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Val Arg His

325 330 335

Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr Trp Gly Gln

340 345 350

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Val Ala Ala Pro Ser Val

355 360 365

Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser

370 375 380

Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln

385 390 395 400

Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val

405 410 415

Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu

420 425 430

Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu

435 440 445

Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg

450 455 460

Gly Glu Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

465 470 475 480

Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

485 490 495

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

500 505 510

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

515 520 525

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn

530 535 540

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

545 550 555 560

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly

565 570 575

Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

580 585 590

Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn

595 600 605

Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

610 615 620

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

625 630 635 640

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

645 650 655

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

660 665 670

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

675 680 685

Ser Leu Ser Pro Gly Lys

690

<210> 149

<211> 451

<212> PRT

<213>artificial sequence

<220>

<223>CEA VHCH1 Fc hole P329GLALA (CEA TCB)

<400> 149

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Glu Phe

20 25 30

Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Thr Lys Thr Gly Glu Ala Thr Tyr Val Glu Glu Phe

50 55 60

Lys Gly Arg Val Thr Phe Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Trp Asp Phe Ala Tyr Tyr Val Glu Ala Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Lys

450

<210> 150

<211> 232

<212> PRT

<213>artificial sequence

<220>

<223> CD3 VH-CL (CEACAM5 TCB)

<400> 150

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr

20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe

100 105 110

Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Val

115 120 125

Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys

130 135 140

Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg

145 150 155 160

Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn

165 170 175

Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser

180 185 190

Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys

195 200 205

Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr

210 215 220

Lys Ser Phe Asn Arg Gly Glu Cys

225 230

<210> 151

<211> 449

<212> PRT

<213>artificial sequence

<220>

<223>hole CEACAM5 VH-CH1 (EE)-Fc(, P329G LALA)

<400> 151

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Asp Pro Ala Asn Gly Asn Ser Lys Tyr Val Pro Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Pro Phe Gly Tyr Tyr Val Ser Asp Tyr Ala Met Ala Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro

<210> 152

<211> 674

<212> PRT

<213>artificial sequence

<220>

<223>CEACAM5 VH-CH1 (EE)-CD3 VL-CH1-Fc(protrusion, P329G LALA)

<400> 152

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Asp Pro Ala Asn Gly Asn Ser Lys Tyr Val Pro Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Pro Phe Gly Tyr Tyr Val Ser Asp Tyr Ala Met Ala Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ala Val Val Thr

225 230 235 240

Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr

245 250 255

Cys Gly Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn Trp

260 265 270

Val Gln Glu Lys Pro Gly Gln Ala Phe Arg Gly Leu Ile Gly Gly Thr

275 280 285

Asn Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu

290 295 300

Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Ala Gln Pro Glu Asp Glu

305 310 315 320

Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly

325 330 335

Gly Gly Thr Lys Leu Thr Val Leu Ser Ser Ala Ser Thr Lys Gly Pro

340 345 350

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

355 360 365

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

370 375 380

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

385 390 395 400

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

405 410 415

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

420 425 430

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

435 440 445

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala

450 455 460

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

465 470 475 480

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

485 490 495

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

500 505 510

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

515 520 525

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

530 535 540

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro

545 550 555 560

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

565 570 575

Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val

580 585 590

Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

595 600 605

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

610 615 620

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

625 630 635 640

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

645 650 655

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

660 665 670

Ser Pro

<210> 153

<211> 218

<212> PRT

<213>artificial sequence

<220>

<223> CEACAM5 VL-CL(RK)

<400> 153

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Gly Glu Ser Val Asp Ile Phe

20 25 30

Gly Val Gly Phe Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

35 40 45

Arg Leu Leu Ile Tyr Arg Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Thr Asn

85 90 95

Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Arg Lys

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 154

<211> 5

<212> PRT

<213>artificial sequence

<220>

<223> CEA-HCDR1

<400> 154

Glu Phe Gly Met Asn

1 5

<210> 155

<211> 17

<212> PRT

<213>artificial sequence

<220>

<223> CEA-HCDR2

<400> 155

Trp Ile Asn Thr Lys Thr Gly Glu Ala Thr Tyr Val Glu Glu Phe Lys

1 5 10 15

Gly

<210> 156

<211> 12

<212> PRT

<213>artificial sequence

<220>

<223> CEA-HCDR3

<400> 156

Trp Asp Phe Ala Tyr Tyr Val Glu Ala Met Asp Tyr

1 5 10

<210> 157

<211> 11

<212> PRT

<213>artificial sequence

<220>

<223> CEA-LCDR1

<400> 157

Lys Ala Ser Ala Ala Val Gly Thr Tyr Val Ala

1 5 10

<210> 158

<211> 7

<212> PRT

<213>artificial sequence

<220>

<223> CEA-LCDR2

<400> 158

Ser Ala Ser Tyr Arg Lys Arg

1 5

<210> 159

<211> 10

<212> PRT

<213>artificial sequence

<220>

<223> CEA-LCDR3

<400> 159

His Gln Tyr Tyr Thr Tyr Pro Leu Phe Thr

1 5 10

<210> 160

<211> 121

<212> PRT

<213>artificial sequence

<220>

<223> CEA VH

<400> 160

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Glu Phe

20 25 30

Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Thr Lys Thr Gly Glu Ala Thr Tyr Val Glu Glu Phe

50 55 60

Lys Gly Arg Val Thr Phe Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Trp Asp Phe Ala Tyr Tyr Val Glu Ala Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 161

<211> 108

<212> PRT

<213>artificial sequence

<220>

<223> CEA VL

<400> 161

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Ala Ala Val Gly Thr Tyr

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Arg Lys Arg Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys His Gln Tyr Tyr Thr Tyr Pro Leu

85 90 95

Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 162

<211> 5

<212> PRT

<213>artificial sequence

<220>

<223> CEA-HCDR1 (CEACAM5)

<400> 162

Asp Thr Tyr Met His

1 5

<210> 163

<211> 17

<212> PRT

<213>artificial sequence

<220>

<223> CEA-HCDR2 (CEACAM5)

<400> 163

Arg Ile Asp Pro Ala Asn Gly Asn Ser Lys Tyr Val Pro Lys Phe Gln

1 5 10 15

Gly

<210> 164

<211> 12

<212> PRT

<213>artificial sequence

<220>

<223> CEA-HCDR3 (CEACAM5)

<400> 164

Phe Gly Tyr Tyr Val Ser Asp Tyr Ala Met Ala Tyr

1 5 10

<210> 165

<211> 15

<212> PRT

<213>artificial sequence

<220>

<223> CEA-LCDR1 (CEACAM5)

<400> 165

Arg Ala Gly Glu Ser Val Asp Ile Phe Gly Val Gly Phe Leu His

1 5 10 15

<210> 166

<211> 7

<212> PRT

<213>artificial sequence

<220>

<223> CEA-LCDR2 (CEACAM5)

<400> 166

Arg Ala Ser Asn Arg Ala Thr

1 5

<210> 167

<211> 9

<212> PRT

<213>artificial sequence

<220>

<223> CEA-LCDR3 (CEACAM5)

<400> 167

Gln Gln Thr Asn Glu Asp Pro Tyr Thr

1 5

<210> 168

<211> 121

<212> PRT

<213>artificial sequence

<220>

<223> CEA VH (CEACAM5)

<400> 168

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Asp Pro Ala Asn Gly Asn Ser Lys Tyr Val Pro Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Pro Phe Gly Tyr Tyr Val Ser Asp Tyr Ala Met Ala Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 169

<211> 111

<212> PRT

<213>artificial sequence

<220>

<223> CEA VL (CEACAM5)

<400> 169

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Gly Glu Ser Val Asp Ile Phe

20 25 30

Gly Val Gly Phe Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

35 40 45

Arg Leu Leu Ile Tyr Arg Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Thr Asn

85 90 95

Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

292页详细技术资料下载

上一篇：一种医用注射器针头装配设备

下一篇：嵌合分子及其用途

Specifically bind the bispecific antibody of PD1 and LAG3

相关技术

网友询问留言