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anti-CD 38 antibodies and combinations with anti-CD 3 and anti-CD 28 antibodies

文档序号：1078002 发布日期：2020-10-16 浏览：20次 中文

阅读说明：本技术 抗cd38抗体及与抗cd3和抗cd28抗体的组合 (anti-CD 38 antibodies and combinations with anti-CD 3 and anti-CD 28 antibodies ) 是由 L·吴 L·徐 E·承 R·魏 G·纳贝尔 杨志勇 T·达布比 B·卡梅伦 C·勒穆 于 2018-10-09 设计创作，主要内容包括：本公开提供了结合CD38多肽(例如人和食蟹猴CD38多肽)的结合蛋白。例如,结合蛋白可以是单特异性、双特异性或三特异性结合蛋白,其具有至少一个结合CD38多肽的抗原结合结构域。本公开还提供了制备结合CD38多肽的结合蛋白的方法和这样的结合蛋白的用途。(The present disclosure provides binding proteins that bind to CD38 polypeptides (e.g., human and cynomolgus monkey CD38 polypeptides). For example, the binding protein may be a monospecific, bispecific, or trispecific binding protein having at least one antigen binding domain that binds to a CD38 polypeptide. The disclosure also provides methods of making binding proteins that bind to CD38 polypeptides and uses of such binding proteins.)

1. A binding protein comprising an antigen binding site that binds a CD38 polypeptide, wherein the antigen binding site comprises:

(a) an antibody heavy chain Variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO:31) or GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO:32) or IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33); and

(b) An antibody light chain Variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO:34) or QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO:35) or GAS (SEQ ID NO:40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36).

2. The binding protein of claim 1, wherein said antigen binding site comprises:

(a) an antibody heavy chain Variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO:31), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO:32), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33); and

(b) an antibody light chain Variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO:34), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO:35), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36).

3. The binding protein of claim 1 or claim 2, wherein the VH domain comprises, from N-terminus to C-terminus, the sequence FR 1-CDR-H1-FR 2-CDR-H2-FR 3-CDR-H3-FR 4; wherein FR1 comprises sequence QVQLVQSGAEVVKPGASVKVSCKAS (SEQ ID NO:86), QVQLVQSGAEVVKSGASVKVSCKAS (SEQ ID NO:87) or QVQLVQSGAEVVKPGASVKMSCKAS (SEQ ID NO: 88); wherein FR2 comprises sequence MHWVKEAPGQRLEWIGY (SEQ ID NO:90) or MHWVKEAPGQGLEWIGY (SEQ ID NO: 91); wherein FR3 comprises sequence NYNQKFQGRATLTADTSASTAYMELSSLRSEDTAVYFC (SEQ ID NO:93) or NYNQKFQGRATLTADTSASTAYMEISSLRSEDTAVYFC (SEQ ID NO: 94); and wherein FR4 comprises the sequence WGQGTLVTVSS (SEQ ID NO: 96).

4. The binding protein of claim 1 or claim 2, wherein the VH domain comprises the amino acid sequence of SEQ ID No. 5 and the VL domain comprises the amino acid sequence of SEQ ID No. 6.

5. The binding protein of claim 4, wherein said binding protein comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO. 7 and an antibody light chain comprising the amino acid sequence of SEQ ID NO. 8.

6. The binding protein of claim 1 or claim 2, wherein the VH domain comprises the amino acid sequence of SEQ ID No. 17 and the VL domain comprises the amino acid sequence of SEQ ID No. 18.

7. The binding protein of claim 6, wherein said binding protein comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO 19 and an antibody light chain comprising the amino acid sequence of SEQ ID NO 20.

8. The binding protein of claim 1 or claim 2, wherein the VH domain comprises the amino acid sequence of SEQ ID No. 21 and the VL domain comprises the amino acid sequence of SEQ ID No. 18.

9. The binding protein of claim 8, wherein the binding protein comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO. 22 and an antibody light chain comprising the amino acid sequence of SEQ ID NO. 20.

10. The binding protein of claim 1 or claim 2, wherein said VH domain comprises the amino acid sequence of SEQ ID No. 23 and said VL domain comprises the amino acid sequence of SEQ ID No. 18.

11. The binding protein of claim 10, wherein the binding protein comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO. 24 and an antibody light chain comprising the amino acid sequence of SEQ ID NO. 20.

12. The binding protein of claim 1, wherein said antigen binding site comprises:

(a) an antibody heavy chain Variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33); and

(b) an antibody light chain Variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO:40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36).

13. The binding protein of claim 1 or claim 12, wherein the VH domain comprises, from N-terminus to C-terminus, the sequence FR 1-CDR-H1-FR 2-CDR-H2-FR 3-CDR-H3-FR 4; wherein FR1 comprises sequence QVQLVQSGAEVVKPGASVKVSCKAS (SEQ ID NO:86), QVQLVQSGAEVVKSGASVKVSCKAS (SEQ ID NO:87) or QVQLVQSGAEVVKPGASVKMSCKAS (SEQ ID NO: 88); wherein FR2 comprises sequence MHWVKEAPGQRLEWIGY (SEQ ID NO:90) or MHWVKEAPGQGLEWIGY (SEQ ID NO: 91); wherein FR3 comprises sequence NYNQKFQGRATLTADTSASTAYMELSSLRSEDTAVYFC (SEQ ID NO:93) or NYNQKFQGRATLTADTSASTAYMEISSLRSEDTAVYFC (SEQ ID NO: 94); and wherein FR4 comprises the sequence WGQGTLVTVSS (SEQ ID NO: 96).

14. The binding protein of claim 1 or claim 12, wherein said VH domain comprises the amino acid sequence of SEQ ID No. 13 and said VL domain comprises the amino acid sequence of SEQ ID No. 14.

15. The binding protein of claim 14, wherein said binding protein comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID No. 15 and an antibody light chain comprising the amino acid sequence of SEQ ID No. 16.

16. A binding protein comprising an antigen binding site that binds a CD38 polypeptide, wherein the antigen binding site comprises:

(a) an antibody heavy chain Variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO:41), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO:42), and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO: 43); and

(b) an antibody light chain Variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QGIRND (SEQ ID NO:44), a CDR-L2 sequence comprising the amino acid sequence of AAS (SEQ ID NO:45), and a CDR-L3 sequence comprising the amino acid sequence of LQDYIYYPT (SEQ ID NO: 46).

17. The binding protein of claim 16, wherein said VH domain comprises the amino acid sequence of SEQ ID No. 9 and said VL domain comprises the amino acid sequence of SEQ ID No. 10.

18. The binding protein of claim 16 or claim 17, wherein the binding protein comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID No. 11 and an antibody light chain comprising the amino acid sequence of SEQ ID No. 12.

19. The binding protein of any one of claims 1-18, wherein the antigen binding site cross-reacts with an extracellular domain of a human CD38 polypeptide and an extracellular domain of a cynomolgus monkey CD38 polypeptide.

20. The binding protein of claim 19, wherein said antigen binding site binds to a human CD38 polypeptide comprising the amino acid sequence of SEQ ID No. 1.

21. The binding protein of claim 20, wherein said antigen binding site binds to said human CD38 polypeptide comprising the amino acid sequence of SEQ ID NO 1 with an equilibrium dissociation constant (K)D) Is 2.1nM or less.

22. The binding protein according to claim 19, wherein said antigen binding site binds to a human isoform E CD38 polypeptide comprising the amino acid sequence of SEQ ID No. 105.

23. The binding protein of any one of claims 19-22, wherein the antigen binding site binds to a cynomolgus monkey CD38 polypeptide comprising the amino acid sequence of SEQ ID No. 30.

24. The binding protein of claim 23, wherein said antigen binding site binds to said cynomolgus monkey CD38 polypeptide comprising the amino acid sequence of SEQ ID NO 30 with an equilibrium dissociation constant (K) D) Is 1.3nM or less.

25. The binding protein of any one of claims 1-24, wherein said binding protein is a chimeric or humanized antibody.

26. The binding protein of any one of claims 16-24, wherein the binding protein is a human antibody.

27. The binding protein of any one of claims 1-24, wherein said binding protein is a monoclonal antibody.

28. The binding protein of any one of claims 1-24, wherein said binding protein comprises one or more full length antibody heavy chains comprising an Fc region.

29. The binding protein of claim 28, wherein said Fc region is a human Fc region comprising one or more mutations that reduce or eliminate Fc receptor binding and/or effector function of the Fc region.

30. The binding protein of claim 28, wherein said Fc region is a human IgG1 Fc region.

31. The binding protein of claim 30, wherein said human IgG1 Fc region comprises amino acid substitutions at positions corresponding to positions 234, 235 and 329 of human IgG1 according to EU Index, wherein said amino acid substitutions are L234A, L235A and P329A.

32. The binding protein of claim 30, wherein said human IgG1 Fc region comprises amino acid substitutions at positions corresponding to positions 298, 299 and 300 of human IgG1 according to EU Index, wherein said amino acid substitutions are S298N, T299A and Y300S.

33. The binding protein of claim 28, wherein the Fc region is a human IgG4 Fc region.

34. The binding protein of claim 33, wherein said human IgG4 Fc region comprises amino acid substitutions at positions corresponding to positions 228 and 409 of human IgG4 according to EU Index, wherein said amino acid substitutions are S228P and R409K.

35. The binding protein of claim 33 or claim 34, wherein said human IgG4 Fc region comprises amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG4 according to EU Index, wherein said amino acid substitutions are F234A and L235A.

36. The binding protein of claim 33 or claim 34, wherein the human IgG4 Fc region comprises amino acid substitutions at positions corresponding to position 233-236 of human IgG4 according to the EU Index, wherein the amino acid substitutions are E233P, F234V, L235A and the deletion at 236.

37. The binding protein of any one of claims 1-24, wherein the binding protein comprises an antibody F (ab), F (ab ') 2, Fab' -SH, Fv or scFv fragment.

38. The binding protein of any one of claims 1-37, wherein the binding protein is conjugated to a cytotoxic agent or label.

39. The binding protein of any one of claims 1-37, wherein said binding protein is a bispecific binding protein comprising a first antigen-binding site that binds a CD38 polypeptide, and a second antigen-binding site.

40. The binding protein of any one of claims 1-37, wherein said binding protein is a trispecific binding protein comprising a first antigen binding site that binds a CD38 polypeptide, a second antigen binding site, and a third antigen binding site.

41. The binding protein of claim 40, wherein said first antigen binding site binds to the extracellular domain of a human CD38 polypeptide, and wherein said second and third antigen binding sites each bind to a T cell surface protein.

42. The binding protein of claim 41, wherein said first antigen binding site binds to the extracellular domain of a human CD38 polypeptide, and wherein (a) said second antigen binding site binds to a human CD28 polypeptide and said third antigen binding site binds to a human CD3 polypeptide, or (b) said second antigen binding site binds to a human CD3 polypeptide and said third antigen binding site binds to a human CD28 polypeptide.

43. A binding protein comprising 3 antigen binding sites that each bind to one or more target proteins, wherein at least one of the 3 antigen binding sites cross-reacts with an extracellular domain of a human CD38 polypeptide and an extracellular domain of a cynomolgus monkey CD38 polypeptide.

44. The binding protein of claim 43, wherein said binding protein is cross-reactive with a human CD38 polypeptide comprising the amino acid sequence of SEQ ID NO. 1 or SEQ ID NO. 105.

45. The binding protein of claim 43 or claim 44, wherein said binding protein is cross-reactive with a cynomolgus monkey CD38 polypeptide comprising the amino acid sequence of SEQ ID NO 30.

46. The binding protein of any one of claims 43-45, wherein said binding protein comprises an antigen binding site that cross-reacts with the extracellular domain of a human CD38 polypeptide and the extracellular domain of a cynomolgus monkey CD38 polypeptide, and 2 antigen binding sites that each bind to a T cell surface protein.

47. The binding protein of claim 46, wherein said binding protein comprises an antigen binding site that cross-reacts with an extracellular domain of a human CD38 polypeptide and an extracellular domain of a cynomolgus monkey CD38 polypeptide, an antigen binding site that binds to a human CD28 polypeptide, and an antigen binding site that binds to a human CD3 polypeptide.

48. The binding protein according to claims 43-47, wherein said binding protein comprises 4 polypeptide chains forming said 3 antigen binding sites, wherein a first polypeptide chain comprises a structure represented by the formula:

VL2-L1-VL1-L2-CL[I]

and the second polypeptide chain comprises a structure represented by the formula:

VH1-L3-VH2-L4-CH1-hinge-CH2-CH3[II]

And the third polypeptide chain comprises a structure represented by the formula:

VH3-CH1-hinge-CH2-CH3[III]

And the fourth polypeptide chain comprises a structure represented by the formula:

VL3-CL[IV]

wherein:

VL1is a first immunoglobulin light chain variable domain;

VL2Is a second immunoglobulin light chain variable domain;

VL3is a third immunoglobulin light chain variable domain;

VH1is a first immunoglobulin heavy chain variable domain;

VH2is a second immunoglobulin heavy chain variable domain;

VH3is a third immunoglobulin heavy chain variable domain;

CLis an immunoglobulin light chain constant domain;

CH1is immunoglobulin CH1A heavy chain constant domain;

CH2is immunoglobulin CH2A heavy chain constant domain;

CH3is immunoglobulin CH3A heavy chain constant domain;

the hinge is connected with the CH1And CH2An immunoglobulin hinge region of the domain, and

L1、L2、L3and L4Is an amino acid linker;

wherein the polypeptide of formula I and the polypeptide of formula II form a cross light chain-heavy chain pair, and

wherein:

(a) the V isH1The domains comprise a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO:31) or GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO:32) or IYPGQGGT (SEQ ID NO:38), and a CDR-H2 sequence comprising ARTGGLRRAYFTY (SEQ ID NO:31)NO:33) of the amino acid sequence of CDR-H3; and said VL1The domain comprises a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO:34) or QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO:35) or GAS (SEQ ID NO:40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36);

(b) The V isH2The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO:31) or GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO:32) or IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33); and said VL2The domain comprises a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO:34) or QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO:35) or GAS (SEQ ID NO:40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36); or

(c) The V isH3The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO:31) or GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO:32) or IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33); and said VL3The domain comprises a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO:34) or QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO:35) or GAS (SEQ ID NO:40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36).

49. The binding protein of claim 48, wherein

(a) The V isH1The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO:31), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO:32), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33); and said VL1The domain comprises a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO:34), comprising LAS (SEQ ID NO:35)The CDR-L2 sequence of the amino acid sequence of (1), and the CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36);

(b) the V isH1The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33); and said VL1The domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO:40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36);

(c) the V isH2The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO:31), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO:32), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33); and said V L2The domain comprises a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO:34), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO:35), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36);

(d) the V isH2The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33); and said VL2The domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO:40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36);

(e) the V isH3The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO:31), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO:32), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33); and said VL3The domains comprise a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO:34), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO:35), and A CDR-L3 sequence having the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36); or

(h) The V isH3The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33); and said VL3The domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO:40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36).

50. The binding protein of claim 49, wherein

(a) The V isH3The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO:31), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO:32), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33); and said VL3The domain comprises a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO:34), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO:35), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36); or

(b) The V isH3The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYA (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT (SEQ ID NO:38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33); and said V L3The domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSVSSYGQGF (SEQ ID NO:39), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO:40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36).

51. The binding protein of claim 50, wherein

(a) The V isH3The structural domain comprises the amino acid sequence of SEQ ID NO 5, and said VL3The domain comprises the amino acid sequence of SEQ ID NO 6;

(b) the V isH3The structural domain comprises the amino acid sequence of SEQ ID NO 17, and said VL3The domain comprises the amino acid sequence of SEQ ID NO 18;

(c) the V isH3The structural domain comprises the amino acid sequence of SEQ ID NO 21, and said VL3The domain comprises the amino acid sequence of SEQ ID NO 18;

(d) the V isH3The structural domain comprises the amino acid sequence of SEQ ID NO 23, and said VL3The domain comprises the amino acid sequence of SEQ ID NO 18; or

(e) The V isH3The domain comprises the amino acid sequence of SEQ ID NO 13, and said VL3The domain comprises the amino acid sequence of SEQ ID NO. 14.

52. The binding protein according to claims 43-47, wherein said binding protein comprises 4 polypeptide chains forming said 3 antigen binding sites, wherein a first polypeptide chain comprises a structure represented by the formula:

VL2-L1-VL1-L2-CL[I]

and the second polypeptide chain comprises a structure represented by the formula:

VH1-L3-VH2-L4-CH1-hinge-CH2-CH3[II]

And the third polypeptide chain comprises a structure represented by the formula:

VH3-CH1-hinge-CH2-CH3[III]

And the fourth polypeptide chain comprises a structure represented by the formula:

VL3-CL[IV]

wherein:

VL1is a first immunoglobulin light chain variable domain;

VL2is a second immunoglobulin light chain variable domain;

VL3is a third immunoglobulin light chain variable domain;

VH1is a first immunoglobulin heavy chain variable domain;

VH2is a second immunoglobulin heavy chain variable domain;

VH3is a third immunoglobulin heavy chain variable domain;

CLis an immunoglobulin light chain constant domain;

CH1is immunoglobulin CH1A heavy chain constant domain;

CH2is immunoglobulin CH2A heavy chain constant domain;

CH3is immunoglobulin CH3A heavy chain constant domain;

the hinge is connected with the CH1And CH2An immunoglobulin hinge region of the domain, and

L1、L2、L3and L4Is an amino acid linker;

wherein the polypeptide of formula I and the polypeptide of formula II form a cross light chain-heavy chain pair, and

wherein:

(a) the V isH1The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO:41), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO:42), and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO: 43); and said VL1The domain comprises a CDR-L1 sequence comprising the amino acid sequence of QGIRND (SEQ ID NO:44), a CDR-L2 sequence comprising the amino acid sequence of AAS (SEQ ID NO:45), and a CDR-L3 sequence comprising the amino acid sequence of LQDYIYYPT (SEQ ID NO: 46);

(b) The V isH2The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO:41), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO:42), and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO: 43); and said VL2The domain comprises a CDR-L1 sequence comprising the amino acid sequence of QGIRND (SEQ ID NO:44), a CDR-L2 sequence comprising the amino acid sequence of AAS (SEQ ID NO:45), and a CDR-L3 sequence comprising the amino acid sequence of LQDYIYYPT (SEQ ID NO: 46); or

(c) The V isH3The structural domain comprises a GFTFSSYGA CDR-H1 sequence of the amino acid sequence of (SEQ ID NO:41), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO:42), and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO: 43); and said VL3The domain comprises a CDR-L1 sequence comprising the amino acid sequence of QGIRND (SEQ ID NO:44), a CDR-L2 sequence comprising the amino acid sequence of AAS (SEQ ID NO:45), and a CDR-L3 sequence comprising the amino acid sequence of LQDYIYYPT (SEQ ID NO: 46).

53. The binding protein of claim 52, wherein said VH3The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO:41), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO:42), and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO: 43); and said V L3The domain comprises a CDR-L1 sequence comprising the amino acid sequence of QGIRND (SEQ ID NO:44), a CDR-L2 sequence comprising the amino acid sequence of AAS (SEQ ID NO:45), and a CDR-L3 sequence comprising the amino acid sequence of LQDYIYYPT (SEQ ID NO: 46).

54. The binding protein of claim 53, wherein said VH3The structural domain comprises the amino acid sequence of SEQ ID NO 9, and said VL3The domain comprises the amino acid sequence of SEQ ID NO 10.

55. The binding protein of claims 48-54, wherein

(a) The V isH1The structural domain comprises the amino acid sequence of SEQ ID NO. 49, VL1The structural domain comprises the amino acid sequence of SEQ ID NO 50, VH2The structural domain comprises the amino acid sequence of SEQ ID NO 53, and said VL2The domain comprises the amino acid sequence of SEQ ID NO. 54;

(b) the V isH2The structural domain comprises the amino acid sequence of SEQ ID NO. 49, VL2The structural domain comprises the amino acid sequence of SEQ ID NO 50, VH1The structural domain comprises the amino acid sequence of SEQ ID NO 53, and said VL1The domain comprises the amino acid sequence of SEQ ID NO. 54;

(c) the V isH1The structural domain comprises the amino acid sequence of SEQ ID NO 51, VL1The structural domain comprises the amino acid sequence of SEQ ID NO 52, VH2The structural domain comprises the amino acid sequence of SEQ ID NO 53, and said V L2The domain comprises the amino acid sequence of SEQ ID NO. 54;

(d) the V isH2The structural domain comprises the amino acid sequence of SEQ ID NO 51, VL2The structural domain comprises the amino acid sequence of SEQ ID NO 52, VH1The structural domain comprises the amino acid sequence of SEQ ID NO 53, and said VL1The domain comprises the amino acid sequence of SEQ ID NO. 54;

(e) the V isH1The structural domain comprises the amino acid sequence of SEQ ID NO. 49, VL1The structural domain comprises the amino acid sequence of SEQ ID NO 50, VH2The domain comprises the amino acid sequence of SEQ ID NO 84, and said VL2The domain comprises the amino acid sequence of SEQ ID NO. 85;

(f) the V isH2The structural domain comprises the amino acid sequence of SEQ ID NO. 49, VL2The structural domain comprises the amino acid sequence of SEQ ID NO 50, VH1The domain comprises the amino acid sequence of SEQ ID NO 84, and said VL1The domain comprises the amino acid sequence of SEQ ID NO. 85;

(g) the V isH1The structural domain comprises the amino acid sequence of SEQ ID NO 51, VL1The structural domain comprises the amino acid sequence of SEQ ID NO 52, VH2The domain comprises the amino acid sequence of SEQ ID NO 84, and said VL2The domain comprises the amino acid sequence of SEQ ID NO. 85; or

(h) The V isH2The structural domain comprises the amino acid sequence of SEQ ID NO 51, V L2The structural domain comprises the amino acid sequence of SEQ ID NO 52, VH1The domain comprises the amino acid sequence of SEQ ID NO 84, and said VL1The domain comprises the amino acid sequence of SEQ ID NO. 85.

56. The binding protein of claim 55, wherein

(a) The V isH1The domain comprises SEQ ID NO 49Amino acid sequence of the VL1The structural domain comprises the amino acid sequence of SEQ ID NO 50, VH2The structural domain comprises the amino acid sequence of SEQ ID NO 53, VL2The structural domain comprises the amino acid sequence of SEQ ID NO 54, VH3The domain comprises the amino acid sequence of SEQ ID NO 13, and said VL3The domain comprises the amino acid sequence of SEQ ID NO. 14; or

(b) The V isH1The structural domain comprises the amino acid sequence of SEQ ID NO. 49, VL1The structural domain comprises the amino acid sequence of SEQ ID NO 50, VH2The structural domain comprises the amino acid sequence of SEQ ID NO 53, VL2The structural domain comprises the amino acid sequence of SEQ ID NO 54, VH3The structural domain comprises the amino acid sequence of SEQ ID NO 9, and said VL3The domain comprises the amino acid sequence of SEQ ID NO 10.

57. The binding protein of any one of claims 48-56, wherein L1、L2、L3Or L4Is independently 0 amino acids in length.

58. The binding protein of any one of claims 48-56, wherein L 1、L2、L3Or L4Is each independently at least one amino acid in length.

59. The binding protein of any one of claims 48-56, wherein (a) L1、L2、L3And L4Each independently is 0 amino acids in length or comprises a sequence selected from the group consisting of: GGGGSGGGGS (SEQ ID NO:55), GGGGSGGGGSGGGGS (SEQ ID NO:56), S, RT, TKGPS (SEQ ID NO:57), GQPKAAP (SEQ ID NO:58) and GGSGSSGSGG (SEQ ID NO: 59); or (b) L1、L2、L3And L4Each independently comprises a sequence selected from the group consisting of: GGGGSGGGGS (SEQ ID NO:55), GGGGSGGGGSGGGGS (SEQ ID NO:56), S, RT, TKGPS (SEQ ID NO:57), GQPKAAP (SEQ ID NO:58) and GGSGSSGSGG (SEQ ID NO: 59).

60. The binding protein of any one of claims 48-56, wherein

(a)L1Comprising the sequence GQPKAAP (SEQ ID NO:58), L2Comprising the sequence TKGPS (SEQ ID NO:57), L3Comprising the sequences S, and L4Comprises the sequence RT;

(b)L1comprising the sequence GGGGSGGGGS (SEQ ID NO:55), L2Comprising the sequence GGGGSGGGGS (SEQ ID NO:55), L3Is 0 amino acids in length, and L4Is 0 amino acids in length;

(c)L1comprising the sequence GGSGSSGSGG (SEQ ID NO:59), L2Comprising the sequence GGSGSSGSGG (SEQ ID NO:59), L3Is 0 amino acids in length, and L4Is 0 amino acids in length; or

(d)L1Comprising the sequence GGGGSGGGGSGGGS (SEQ ID NO:56), L 2Is 0 amino acids in length, L3Comprising the sequences GGGGSGGGGSGGGS (SEQ ID NO:56), and L4Is 0 amino acids in length.

61. The binding protein according to any one of claims 48 to 60, wherein the hinge-C of said second and said third polypeptide chainH2-CH3The domain is human IgG4 hinge-CH2-CH3A domain, and wherein said hinge-C is according to EU IndexH2-CH3Each domain comprises amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG4, wherein the amino acid substitutions are F234A and L235A.

62. The binding protein according to any one of claims 48 to 60, wherein the hinge-C of said second and said third polypeptide chainH2-CH3The domain is human IgG4 hinge-CH2-CH3A domain, and wherein said hinge-C is according to EU IndexH2-CH3The domains each comprise amino acid substitutions at positions corresponding to position 233-236 of human IgG4, wherein the amino acid substitutions are E233P, F234V, L235A, and a deletion at 236.

63. The binding protein according to any one of claims 48 to 60, wherein the hinge-C of said second and said third polypeptide chainH2-CH3The domain is human IgG4 hinge-CH2-CH3A domain, and wherein said hinge-C is according to EU IndexH2-CH3Each domain comprises amino acid substitutions at positions corresponding to positions 228 and 409 of human IgG4, wherein the amino acid substitutions are S228P and R409K.

64. The binding protein according to any one of claims 48 to 60, wherein the hinge-C of said second and said third polypeptide chainH2-CH3The domain is human IgG4 hinge-CH2-CH3A domain, and wherein said hinge-C is according to EU IndexH2-CH3Each domain comprises amino acid substitutions at positions corresponding to positions 234, 235 and 329 of human IgG4, wherein the amino acid substitutions are L234A, L235A and P329A.

65. The binding protein according to any one of claims 48 to 60, wherein the hinge-C of said second and said third polypeptide chainH2-CH3The domain is human IgG4 hinge-CH2-CH3A domain, and wherein said hinge-C is according to EU IndexH2-CH3Each of the domains comprises amino acid substitutions at positions corresponding to positions 298, 299 and 300 of human IgG4, wherein the amino acid substitutions are S298N, T299A and Y300S.

66. The binding protein according to any one of claims 48 to 65, wherein the hinge-C of said second polypeptide chain is according to EU IndexH2-CH3The domain comprises amino acid substitutions at positions corresponding to positions 349, 366, 368 and 407 of human IgG1 or IgG4, wherein said amino acid substitutions are Y349C, T366S, L368A and Y407V, and wherein the hinge-C of said third polypeptide chain is according to EU IndexH2-CH3The domain comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4, wherein the amino acid substitutions are S354C and T366W.

67. The binding protein according to any one of claims 48 to 65, wherein the hinge-C of said second polypeptide chain is according to EU IndexH2-CH3The domain comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4, wherein the amino acid substitutions are S354C and T366W; and wherein the hinge-C of said third polypeptide chain is according to EU IndexH2-CH3The domain comprises amino acid substitutions at positions corresponding to positions 349, 366, 368 and 407 of human IgG1 or IgG4, wherein the amino acid substitutions are Y349C, T366S, L368A and Y407V.

68. The binding protein according to claim 48 or claim 52, wherein said first polypeptide chain comprises the amino acid sequence of SEQ ID NO 61, said second polypeptide chain comprises the amino acid sequence of SEQ ID NO 60, said third polypeptide chain comprises the amino acid sequence of SEQ ID NO 62, and said fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO 63.

69. The binding protein according to claim 48 or claim 52, wherein said first polypeptide chain comprises the amino acid sequence of SEQ ID NO 61, said second polypeptide chain comprises the amino acid sequence of SEQ ID NO 64, said third polypeptide chain comprises the amino acid sequence of SEQ ID NO 65, and said fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO 63.

70. The binding protein according to claim 48 or claim 52, wherein said first polypeptide chain comprises the amino acid sequence of SEQ ID NO 61, said second polypeptide chain comprises the amino acid sequence of SEQ ID NO 66, said third polypeptide chain comprises the amino acid sequence of SEQ ID NO 67, and said fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO 63.

71. The binding protein according to claim 48 or claim 52, wherein said first polypeptide chain comprises the amino acid sequence of SEQ ID NO 61, said second polypeptide chain comprises the amino acid sequence of SEQ ID NO 60, said third polypeptide chain comprises the amino acid sequence of SEQ ID NO 68, and said fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO 69.

72. The binding protein according to claim 48 or claim 52, wherein said first polypeptide chain comprises the amino acid sequence of SEQ ID NO 61, said second polypeptide chain comprises the amino acid sequence of SEQ ID NO 64, said third polypeptide chain comprises the amino acid sequence of SEQ ID NO 70, and said fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO 69.

73. The binding protein according to claim 48 or claim 52, wherein said first polypeptide chain comprises the amino acid sequence of SEQ ID NO 61, said second polypeptide chain comprises the amino acid sequence of SEQ ID NO 66, said third polypeptide chain comprises the amino acid sequence of SEQ ID NO 71, and said fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO 69.

74. A binding protein comprising 3 antigen binding sites that each bind one or more target proteins, wherein the binding protein comprises 4 polypeptide chains that form the 3 antigen binding sites, wherein a first polypeptide chain comprises a structure represented by the formula:

VL2-L1-VL1-L2-CL[I]

and the second polypeptide chain comprises a structure represented by the formula:

VH1-L3-VH2-L4-CH1-hinge-CH2-CH3[II]

And the third polypeptide chain comprises a structure represented by the formula:

VH3-CH1-hinge-CH2-CH3[III]

And the fourth polypeptide chain comprises a structure represented by the formula:

VL3-CL[IV]

wherein:

VL1is a first immunoglobulin light chain variable domain;

VL2is a second immunoglobulin light chain variable domain;

VL3is a third immunoglobulin light chain variable domain;

VH1is a first immunoglobulin heavy chain variable domain;

VH2is a second immunoglobulin heavy chain variable domain;

VH3is a third immunoglobulin heavy chain variable domain;

CLis an immunoglobulin light chain constant domain;

CH1is immunoglobulin CH1A heavy chain constant domain;

CH2is immunoglobulin CH2A heavy chain constant domain;

CH3is immunoglobulin CH3A heavy chain constant domain;

the hinge is connected with the CH1And CH2An immunoglobulin hinge region of the domain; and

L1、L2、L3and L4Is an amino acid linker;

wherein the polypeptide of formula I and the polypeptide of formula II form a cross light-heavy chain pair; and

Wherein:

(a) hinge-C of said second and said third polypeptide chainH2-CH3The domain is human IgG1 hinge-CH2-CH3A domain, and wherein said hinge-C is according to EU IndexH2-CH3Each of the domains comprises amino acid substitutions at positions corresponding to positions 298, 299 and 300 of human IgG1, wherein the amino acid substitutions are S298N, T299A and Y300S; or

(b) hinge-C of said second and said third polypeptide chainH2-CH3The domain is human IgG 4CH3A domain, and wherein said hinge-C is according to EU IndexH2-CH3The domains each comprise amino acid substitutions at positions corresponding to position 233-236 of human IgG4, wherein the amino acid substitutions are E233P, F234V, L235A, and a deletion at 236.

75. The method ofThe binding protein of claim 74, wherein VH1And VL1、VH2And VL2And VH3And VL3Forms an antigen binding site that binds to a CD38 polypeptide.

76. The binding protein of claim 74, wherein VH1And VL1、VH2And VL2And VH3And VL3The 1, 2 or 3 pairs of polypeptides forming an antigen binding site that binds to an antigen target selected from the group consisting of A2, APRIL, ATP diphosphate hydrolase, BAFF, BAFFR, BCMA, BlyS, BTK, BTLA, B7H, B7RP, B-4, C, CCL, CCR, CD122, CD137, CD152, CD154, CD160, CD272, CD274, CD275, CD GR, CD278, CD279, chitinase, CLEC, CRTMR, CSF-1, CSF-2, CSF-CSF 3, CTP, TLR, CCL, CTP, TLR.

77. The binding protein of claim 74, wherein VH1And VL1、VH2And VL2And VH3And VL3Form an antigen binding site, V, that binds to the human CD3 polypeptideH1And VL1、VH2And VL2And VH3And VL3Forming an antigen binding site that binds to a human CD28 polypeptide, and VH1And VL1、VH2And VL2And VH3And VL3The third pair of (a) forms an antigen binding site that binds to a human antigen target selected from the group consisting of A2, APRIL, ATP diphosphate hydrolase, BAFF, BAFFR, BCMA, BlyS, BTK, BTLA, B7H, B7RP, B-4, C, CCL, PROM, CCR, CD122, CD137, CD152, CD154, CD160, CD272, CD273, CD274, CD275, CD278, CD279, CDLH, chitinase, CLEC, CSF-1, CSF-2, CXCL, TLR, CLIL, TLR, CLIL 1, TLR.

78. A kit of polynucleotides comprising:

(a) a first polynucleotide comprising the sequence of SEQ ID NO. 73, a second polynucleotide comprising the sequence of SEQ ID NO. 72, a third polynucleotide comprising the sequence of SEQ ID NO. 74, and a fourth polynucleotide comprising the sequence of SEQ ID NO. 75;

(b) a first polynucleotide comprising the sequence of SEQ ID NO. 73, a second polynucleotide comprising the sequence of SEQ ID NO. 76, a third polynucleotide comprising the sequence of SEQ ID NO. 77, and a fourth polynucleotide comprising the sequence of SEQ ID NO. 75;

(c) a first polynucleotide comprising the sequence of SEQ ID NO. 73, a second polynucleotide comprising the sequence of SEQ ID NO. 78, a third polynucleotide comprising the sequence of SEQ ID NO. 79, and a fourth polynucleotide comprising the sequence of SEQ ID NO. 75;

(d) a first polynucleotide comprising the sequence of SEQ ID NO. 73, a second polynucleotide comprising the sequence of SEQ ID NO. 72, a third polynucleotide comprising the sequence of SEQ ID NO. 80, and a fourth polynucleotide comprising the sequence of SEQ ID NO. 81;

(e) a first polynucleotide comprising the sequence of SEQ ID NO. 73, a second polynucleotide comprising the sequence of SEQ ID NO. 76, a third polynucleotide comprising the sequence of SEQ ID NO. 82, and a fourth polynucleotide comprising the sequence of SEQ ID NO. 81; or

(f) A first polynucleotide comprising the sequence of SEQ ID NO. 73, a second polynucleotide comprising the sequence of SEQ ID NO. 78, a third polynucleotide comprising the sequence of SEQ ID NO. 83, and a fourth polynucleotide comprising the sequence of SEQ ID NO. 81.

79. A polynucleotide encoding the binding protein of any one of claims 1-77.

80. A vector comprising the polynucleotide of claim 79.

81. A host cell comprising a kit of polynucleotides of claim 78, a polynucleotide of claim 79, or a vector of claim 80.

82. A method of producing a binding protein, the method comprising culturing the host cell of claim 81 such that the binding protein is produced.

83. The method of claim 82, further comprising recovering said binding protein from said host cell.

84. A pharmaceutical composition comprising the binding protein of any one of claims 1-77 and a pharmaceutically acceptable carrier.

85. A method of preventing and/or treating cancer in a patient, comprising administering to the patient a therapeutically effective amount of at least one binding protein of any one of claims 1-73 or the pharmaceutical composition of claim 84.

86. The method of claim 85, wherein said binding protein comprises one antigen binding site that binds to a T cell surface protein and another antigen binding site that binds to the extracellular domain of a human CD38 polypeptide.

87. The method of claim 86, wherein the binding protein is a trispecific binding protein comprising a first antigen binding site that binds CD3, a second antigen binding site that binds CD28, and a third antigen binding site that binds the extracellular domain of a human CD38 polypeptide.

88. The method of any one of claims 85-87, wherein at least one binding protein is co-administered with a chemotherapeutic agent.

89. The method of any one of claims 85-88, wherein the cancer is multiple myeloma.

90. The method of any one of claims 85-88, wherein the cancer is Acute Myeloid Leukemia (AML), Acute Lymphoblastic Leukemia (ALL), Chronic Lymphocytic Leukemia (CLL), or B-cell lymphoma.

91. The method of any one of claims 85-90, wherein the patient is a human.

92. The method of any one of claims 85-91, wherein the patient is selected for treatment because cells of the cancer express a human CD38 isoform E polypeptide on their cell surface.

Technical Field

The present disclosure relates to binding proteins that bind to CD38 polypeptides (e.g., human and cynomolgus monkey CD38 polypeptides), including monospecific, bispecific, or trispecific binding proteins having at least one antigen-binding domain that binds to a CD38 polypeptide, as well as polynucleotides, host cells, methods of production, and methods of use related thereto.

Background

Monoclonal antibody-based biotherapeutics have become an important approach to the development of new drugs. Monoclonal antibody technology provides specific targeting, precise signal delivery and/or payload for specific cell populations and provides sustained biological effects through its Fc function. Antibody engineering efforts have allowed the development of multispecific antibodies that bind the specificity of multiple monoclonal antibodies for various biological applications, expanding the scope of antibody drug development.

CD38 is an attractive drug target because it is expressed on the cell surface of a variety of lymphoid tumor cells (see Stevenson, G.T. (2006) mol. med.12: 345-.(daratumumab) is an anti-CD 38 antibody approved for the treatment of multiple myeloma. However, there is a need for therapeutic agents that target CD38 with different modes of action and/or improved properties, including but not limited to high affinity binding to CD38, cross-reactivity between human and cynomolgus monkey CD38 polypeptides, binding to lymphoma cells (e.g., multiple myeloma large B-cell lymphoma cell line), and the ability to induce apoptosis and/or antibody-dependent cell-mediated cytotoxicity (ADCC) and T-cell mediated anti-tumor activity.

Disclosure of Invention

Provided herein are binding proteins that bind to CD38 polypeptides (e.g., human and cynomolgus monkey CD38 polypeptides), including monospecific, bispecific, or trispecific binding proteins having at least one antigen binding site that binds to a CD38 polypeptide. Advantageously, these binding proteinsHaving the ability to recruit T cells to the vicinity of cancer cells, subsequently activate T cells and facilitate the activated T cells to kill adjacent cancer cells through the granzyme/perforin mechanism, from anti-CD 38 antibodies such as(daratumab) provides different modes of action of antitumor activity. Furthermore, the ability to bind to human and cynomolgus monkey CD38 polypeptides allows binding proteins to be easily tested in preclinical toxicology studies, e.g., to evaluate their safety profile for later clinical use.

In some embodiments, provided herein are monospecific binding proteins that bind to a human CD38 polypeptide. In some embodiments, the binding protein is cross-reactive with human and cynomolgus monkey CD38 polypeptides. In some embodiments, the binding protein binds to human isoform a and isoform E CD38 polypeptides. In some embodiments, the binding protein has one or more of the following characteristics (in any combination): binds the extracellular domain of a human CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 1) as a purified protein, as determined by SPR; binding to the extracellular domain of a human CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 1) as a purified protein, K D1.5nM or less as determined by SPR; binds to an extracellular domain of a human CD38 polypeptide expressed on the surface of a cell (e.g., comprising the amino acid sequence of SEQ ID NO: 1), as determined by flow cytometry; binds to the extracellular domain of a human CD38 polypeptide expressed on the surface of a cell (e.g., comprising the amino acid sequence of SEQ ID NO: 1), which has an apparent KD20nM, 15nM, 10nM, 5nM, 1nM or less as determined by flow cytometry; binds to the extracellular domain of a cynomolgus monkey CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 30) as a purified protein, as determined by SPR; binding to the extracellular domain of a cynomolgus monkey CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 30) as a purified protein, KDIs 3.5nM or less as determined by SPR; binds to an extracellular domain of a cynomolgus monkey CD38 polypeptide expressed on the surface of a cell (e.g., comprising the amino acid sequence of SEQ ID NO: 30), as determined by flow cytometry; combined in a thin layerThe extracellular domain of a cynomolgus monkey CD38 polypeptide expressed on the surface of a cell (e.g., comprising the amino acid sequence of SEQ ID NO: 30), which has an apparent KDIs 7.5nM or less as determined by flow cytometry; binds the extracellular domain of a human isoform E CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ id no: 105) as a purified protein, as determined by ELISA; binds to an extracellular domain of a human isoform E CD38 polypeptide expressed on the surface of a cell (e.g., comprising the amino acid sequence of SEQ ID NO: 105), as determined by flow cytometry; inducing apoptosis or antibody-dependent cellular cytotoxicity (ADCC) of cells expressing CD38 at the cell surface; having one or more mutations (e.g., in the Fc region) results in reduced binding to Fc γ RI and/or Fc γ RII as compared to the same binding protein without the one or more mutations. For an exemplary assay, see examples 1, 3 and 4. In some embodiments, the KD is measured at 4 ℃ or 25 ℃.

In some embodiments, provided herein are trispecific binding proteins that bind to a human CD38 polypeptide. In some embodiments, the trispecific binding protein binds (e.g., simultaneously) a CD38 polypeptide (e.g., expressed on the surface of a cell) and one or more other target antigens expressed on the surface of a second cell, thereby recruiting the second cell in proximity to the cell expressing the CD38 polypeptide. In some embodiments, a trispecific binding protein binds (e.g., simultaneously) a CD38 polypeptide (e.g., expressed on the surface of a cell) and one or both target antigens expressed on the surface of a T cell, thereby recruiting a T cell in proximity to a cell expressing a CD38 polypeptide. In some embodiments, the trispecific binding protein activates T cells and/or provides CD 28-mediated co-stimulatory signals to T cells. In some embodiments, the trispecific binding protein is cross-reactive with human and cynomolgus monkey CD38 polypeptides. In some embodiments, the trispecific binding protein binds to human isoform a and isoform E CD38 polypeptides. In some embodiments, the trispecific binding protein has one or more of the following characteristics (in any combination): binds the extracellular domain of a human CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 1) as a purified protein, as determined by SPR; an extracellular domain that binds a human CD38 polypeptide (e.g., Comprises the amino acid sequence of SEQ ID NO: 1) as purified protein, KDIs 1.5nM or less as determined by SPR; binds to an extracellular domain of a human CD38 polypeptide expressed on the surface of a cell (e.g., comprising the amino acid sequence of SEQ id no: 1), as determined by flow cytometry; binds to the extracellular domain of a human CD38 polypeptide expressed on the surface of a cell (e.g., comprising the amino acid sequence of SEQ ID NO: 1), which has an apparent KDIs 20nM, 15nM, 10nM, 5nM, 1nM or less as determined by flow cytometry; binds to the extracellular domain of a cynomolgus monkey CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 30) as a purified protein, as determined by SPR; binding to the extracellular domain of a cynomolgus monkey CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 30) as a purified protein, KDIs 3.5nM or less as determined by SPR; binds to an extracellular domain of a cynomolgus monkey CD38 polypeptide expressed on the surface of a cell (e.g., comprising the amino acid sequence of SEQ ID NO: 30), as determined by flow cytometry; binding to the extracellular domain of a cynomolgus monkey CD38 polypeptide expressed on the surface of a cell (e.g., comprising the amino acid sequence of SEQ ID NO: 30), which has an apparent KDIs 7.5nM or less as determined by flow cytometry; binds the extracellular domain of a human isoform E CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 105) as a purified protein, as determined by ELISA; binds to an extracellular domain of a human isoform E CD38 polypeptide expressed on the surface of a cell (e.g., comprising the amino acid sequence of SEQ ID NO: 105), as determined by flow cytometry; inducing apoptosis or antibody-dependent cellular cytotoxicity (ADCC) of cells expressing CD38 at the cell surface; having one or more mutations (e.g., in the Fc region) results in reduced binding to Fc γ RI and/or Fc γ RII as compared to the same binding protein without the one or more mutations; inducing proliferation of T cells (e.g., CD4+ and/or CD8+ T cells); inducing expression of Bcl-xL by T cells (e.g., CD4+ and/or CD8+ T cells); inducing apoptosis of CD38+ cells; binding CD38 expressed on the surface of a cell and one or more T cell target antigens expressed on the surface of a T cell; binds to CD38 expressed on the surface of cells, CD28 expressed on the surface of T cells, and CD3 expressed on the surface of T cells; for stimulating T cell receptors Activation, induction of co-stimulation of T cell receptor signaling (e.g., mediated by CD 28), and reduction in induction of cytokine release (e.g., IFN- γ, IL-2, and/or TNF- α) from PBMC with one or more mutations (e.g., in the Fc region) as compared to the same binding protein without one or more mutations, induction of cytokine release (e.g., IFN- γ and/or IL-6) from PBMC in the presence of CD38+ target cells see examples 1, 3, and 4 for exemplary assays.

In some embodiments, provided herein are binding proteins comprising an antigen binding site that binds a CD38 polypeptide, wherein the antigen binding site comprises: (a) an antibody heavy chain Variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO: 31) or GYTFTSYA (SEQ ID NO: 37), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO: 32) or IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33); and/or (b) an antibody light chain Variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO: 34) or QSVSSYGQGF (SEQ ID NO: 39), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO: 35) or GAS (SEQ ID NO: 40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36). In some embodiments, the antigen binding site comprises: (a) an antibody heavy chain Variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO: 31) or GYTFTSYA (SEQ ID NO: 37), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO: 32) or IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33); and/or (b) an antibody light chain Variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO: 34) or QSVSSYGQG (SEQ ID NO: 132), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO: 35) or GAS (SEQ ID NO: 40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36). In some embodiments, the antigen binding site comprises: (a) an antibody heavy chain Variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO: 31), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO: 32), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33); and/or (b) an antibody light chain Variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO: 34), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO: 35), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36). In some embodiments, the VH domain comprises, from N-terminus to C-terminus, the sequence FR1-CDR-H1-FR2-CDR-H2-FR3-CDR-H3-FR 4; wherein FR1 comprises sequence QVQLVQSGAEVVKPGASVKVSCKAS (SEQ ID NO: 86), QVQLVQSGAEVVKSGASVKVSCKAS (SEQ ID NO: 87) or QVQLVQSGAEVVKPGASVKMSCKAS (SEQ ID NO: 88); wherein FR2 comprises sequence MHWVKEAPGQRLEWIGY (SEQ ID NO: 90) or MHWVKEAPGQGLEWIGY (SEQ ID NO: 91); wherein FR3 comprises sequence NYNQKFQGRATLTADTSASTAYMELSSLRSEDTAVYFC (SEQ ID NO: 93) or NYNQKFQGRATLTADTSASTAYMEISSLRSEDTAVYFC (SEQ ID NO: 94); and wherein FR4 comprises the sequence WGQGTLVTVSS (SEQ ID NO: 96). In some embodiments, the VH domain comprises SEQ ID NO: 5, and/or the VL domain comprises the amino acid sequence of SEQ ID NO: 6. In some embodiments, the binding protein comprises a polypeptide comprising seq id NO: 7 and an antibody heavy chain comprising the amino acid sequence of SEQ ID NO: 8, or a light chain antibody. In some embodiments, the VH domain comprises SEQ ID NO: 17, and/or the VL domain comprises the amino acid sequence of SEQ ID NO: 18. In some embodiments, the binding protein comprises a polypeptide comprising seq id NO: 19 and an antibody heavy chain comprising the amino acid sequence of seq id NO: 20, or an antibody light chain. In some embodiments, the VH domain comprises SEQ ID NO: 21, and/or the VL domain comprises the amino acid sequence of seq id NO: 18. In some embodiments, the binding protein comprises a polypeptide comprising SEQ ID NO: 22 and an antibody heavy chain comprising the amino acid sequence of SEQ ID NO: 20, or an antibody light chain. In some embodiments, the VH domain comprises SEQ ID NO: 23, and/or the VL domain comprises the amino acid sequence of SEQ ID NO: 18. In some embodiments, the binding protein comprises a polypeptide comprising SEQ ID NO: 24 and an antibody heavy chain comprising the amino acid sequence of SEQ ID NO: 20, or an antibody light chain. In some embodiments, the antigen binding site comprises: (a) an antibody heavy chain Variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYA (SEQ ID NO: 37), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33); and (b) an antibody light chain Variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSVSSYGQGF (SEQ ID NO: 39), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO: 40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36). In some embodiments, the VH domain comprises, from N-terminus to C-terminus, the sequence FR1-CDR-H1-FR2-CDR-H2-FR3-CDR-H3-FR 4; wherein FR1 comprises sequence QVQLVQSGAEVVKPGASVKVSCKAS (SEQ ID NO: 86), QVQLVQSGAEVVKSGASVKVSCKAS (SEQ ID NO: 87) or QVQLVQSGAEVVKPGASVKMSCKAS (SEQ ID NO: 88); wherein FR2 comprises sequence MHWVKEAPGQRLEWIGY (SEQ ID NO: 90) or MHWVKEAPGQGLEWIGY (SEQ ID NO: 91); wherein FR3 comprises sequence NYNQKFQGRATLTADTSASTAYMELSSLRSEDTAVYFC (SEQ ID NO: 93) or N YNQKFQGRATLTADTSASTAYMEISSLRSEDTAVYFC (SEQ ID NO: 94); and wherein FR4 comprises the sequence WGQGTLVTVSS (SEQ ID NO: 96). In some embodiments, the VH domain comprises SEQ ID NO: 13, and/or the VL domain comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, the binding protein comprises a polypeptide comprising SEQ ID NO: 15 and an antibody heavy chain comprising the amino acid sequence of SEQ ID NO: 16, or a light chain antibody.

In some embodiments, provided herein is a binding protein comprising an antigen binding site that binds a CD38 polypeptide, wherein the antigen binding site comprises: (a) an antibody heavy chain Variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO: 41), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO: 42), and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO: 43); and (b) an antagonistA light chain Variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QGIRND (SEQ ID NO: 44), a CDR-L2 sequence comprising the amino acid sequence of AAS (SEQ ID NO: 45), and a CDR-L3 sequence comprising the amino acid sequence of LQDYIYYPT (SEQ ID NO: 46). In some embodiments, the VH domain comprises SEQ ID NO: 9, and/or the VL domain comprises the amino acid sequence of SEQ ID NO: 10. In some embodiments, the binding protein comprises a polypeptide comprising SEQ ID NO: 11 and an antibody heavy chain comprising the amino acid sequence of SEQ ID NO: 12, or an antibody light chain. In some embodiments, the antigen binding site cross-reacts with an extracellular domain of a human CD38 polypeptide and an extracellular domain of a cynomolgus monkey CD38 polypeptide. In some embodiments, the antigen binding site binds to a polypeptide comprising SEQ ID NO: 1, or a human CD38 polypeptide. In some embodiments, the antigen binding site binds to the polypeptide comprising SEQ ID NO: 1, equilibrium dissociation constant (K) of a human CD38 polypeptide D) Is 2.1nM or less. In some embodiments, the antigen binding site binds to a polypeptide comprising SEQ ID NO: 105, or a human isoform E CD38 polypeptide. In some embodiments, the antigen binding site binds to a polypeptide comprising SEQ ID NO: 30, and a cynomolgus monkey CD38 polypeptide having the amino acid sequence of seq id no. In some embodiments, the antigen binding site binds to the polypeptide comprising SEQ ID NO: 30, a cynomolgus monkey CD38 polypeptide having an amino acid sequence with an equilibrium dissociation constant (K)D) Is 1.3nM or less.

In some embodiments of any of the above embodiments, the binding protein is a chimeric or humanized antibody. In some embodiments, the binding protein is a human antibody. In some embodiments, the binding protein is a monoclonal antibody. In some embodiments, the binding protein comprises one or more full length antibody heavy chains comprising an Fc region. In some embodiments, the Fc region is a human Fc region comprising one or more mutations that reduce or eliminate Fc receptor binding and/or effector function of the Fc region. In some embodiments, the Fc region is a human IgG1Fc region. In some embodiments, the human IgG1Fc region comprises amino acid substitutions at positions corresponding to positions 234, 235, and 329 of human IgG1 according to EUIndex, wherein the amino acid substitutions are L234A, L235A, and P329A. In some embodiments, the human IgG1Fc region comprises amino acid substitutions at positions corresponding to positions 298, 299, and 300 of human IgG1 according to EU Index, wherein the amino acid substitutions are S298N, T299A, and Y300S. In some embodiments, the Fc region is a human IgG4 Fc region. In some embodiments, the human IgG4 Fc region comprises amino acid substitutions at positions corresponding to positions 228 and 409 of human IgG4 according to EUIndex, wherein the amino acid substitutions are S228P and R409K. In some embodiments, the human IgG4 Fc region comprises amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG4, according to EU Index, wherein the amino acid substitutions are F234A and L235A. In some embodiments, the human IgG4 Fc region comprises amino acid substitutions at positions corresponding to position 233-236 of human IgG4 according to the EU Index, wherein the amino acid substitutions are E233P, F234V, L235A, and the deletion at 236. In some embodiments, the human IgG4 Fc region comprises an amino acid substitution at a position corresponding to position 233-237 of human IgG4, according to the EU Index, wherein the sequence EFLGG is replaced with PVAG. In some embodiments, the binding protein comprises an antibody F (ab), F (ab ') 2, Fab' -SH, Fv, or scFv fragment. In some embodiments, the binding protein is conjugated to a cytotoxic agent or label. In some embodiments, the binding protein is a bispecific binding protein comprising a first antigen-binding site that binds a CD38 polypeptide, and a second antigen-binding site. In some embodiments, the binding protein is a trispecific binding protein comprising a first antigen-binding site that binds a CD38 polypeptide, a second antigen-binding site, and a third antigen-binding site. In some embodiments, the first antigen binding site binds to an extracellular domain of a human CD38 polypeptide, and wherein the second and third antigen binding sites each bind to a T cell surface protein. In some embodiments, the first antigen binding site binds to an extracellular domain of a human CD38 polypeptide, and wherein (a) the second antigen binding site binds to a human CD28 polypeptide and the third antigen binding site binds to a human CD3 polypeptide, or (b) the second antigen binding site binds to a human CD3 polypeptide and the third antigen binding site binds to a human CD28 polypeptide.

In some embodiments, provided herein is a binding protein comprising 3 antigen binding sites that each bind one or more target proteins, wherein at least one of the 3 antigen binding sites cross-reacts with an extracellular domain of a human CD38 polypeptide and an extracellular domain of a cynomolgus monkey CD38 polypeptide. In some embodiments, the binding protein binds to a polypeptide comprising SEQ ID NO: 1 or SEQ ID NO: 105, or a human CD38 polypeptide cross-reactive therewith. In some embodiments, the binding protein binds to a polypeptide comprising SEQ ID NO: 30, and a cynomolgus monkey CD38 polypeptide cross-reactive. In some embodiments, the binding protein comprises an antigen binding site that cross-reacts with the extracellular domain of a human CD38 polypeptide and the extracellular domain of a cynomolgus monkey CD38 polypeptide, and 2 antigen binding sites that each bind to a T cell surface protein. In some embodiments, the binding protein comprises an antigen binding site that cross-reacts with an extracellular domain of a human CD38 polypeptide and an extracellular domain of a cynomolgus monkey CD38 polypeptide, an antigen binding site that binds to a human CD28 polypeptide, and an antigen binding site that binds to a human CD3 polypeptide. In some embodiments, the binding protein comprises 4 polypeptide chains forming the 3 antigen binding sites, wherein a first polypeptide chain comprises a structure represented by the formula:

VL2-L1-VL1-L2-CL[I]

And the second polypeptide chain comprises a structure represented by the formula:

VH1-L3-VH2-L4-CH1-hinge-CH2-CH3[II]

And the third polypeptide chain comprises a structure represented by the formula:

VH3-CH1-hinge-CH2-CH3[III]

And the fourth polypeptide chain comprises a structure represented by the formula:

VL3-CL[IV]

wherein:

VL1is a first immunoglobulin light chain variable domain；

VL2Is a second immunoglobulin light chain variable domain;

VL3is a third immunoglobulin light chain variable domain;

VH1is a first immunoglobulin heavy chain variable domain;

VH2is a second immunoglobulin heavy chain variable domain;

VH3is a third immunoglobulin heavy chain variable domain;

CLis an immunoglobulin light chain constant domain;

CH1is immunoglobulin CH1A heavy chain constant domain;

CH2is immunoglobulin CH2A heavy chain constant domain;

CH3is immunoglobulin CH3A heavy chain constant domain;

the hinge is connected with the C H1 and C H2 domain, and

L1、L2、L3and L4Is an amino acid linker;

wherein the polypeptide of formula I and the polypeptide of formula II form a cross light chain-heavy chain pair, and

wherein: (a) the V isH1The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO: 31) or GYTFTSYA (SEQ ID NO: 37), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO: 32) or IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33); and said V L1The domain comprises a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO: 34) or QSVSSYGQGF (SEQ ID NO: 39), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO: 35) or GAS (SEQ ID NO: 40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36);

(b) the V isH2The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO: 31) or GYTFTSYA (SEQ ID NO: 37)A CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO: 32) or IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33); and said VL2The domain comprises a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO: 34) or QSVSSYGQGF (SEQ ID NO: 39), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO: 35) or GAS (SEQ ID NO: 40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36), or

(c) The V isH3The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO: 31) or GYTFTSYA (SEQ ID NO: 37), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO: 32) or IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33); and said V L3The domain comprises a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO: 34) or QSVSSYGQGF (SEQ ID NO: 39), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO: 35) or GAS (SEQ ID NO: 40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36). In some embodiments, the binding protein comprises 4 polypeptide chains forming the 3 antigen binding sites, wherein a first polypeptide chain comprises a structure represented by the formula:

VL2-L1-VL1-L2-CL[I]

and the second polypeptide chain comprises a structure represented by the formula:

VH1-L3-VH2-L4-CH1-hinge-CH2-CH3[II]

And the third polypeptide chain comprises a structure represented by the formula:

VH3-CH1-hinge-CH2-CH3[III]

And the fourth polypeptide chain comprises a structure represented by the formula:

VL3-CL[IV]

wherein:

VL1is the first immune ballA protein light chain variable domain;

VL2is a second immunoglobulin light chain variable domain;

VL3is a third immunoglobulin light chain variable domain;

VH1is a first immunoglobulin heavy chain variable domain;

VH2is a second immunoglobulin heavy chain variable domain;

VH3is a third immunoglobulin heavy chain variable domain;

CLis an immunoglobulin light chain constant domain;

CH1is immunoglobulin CH1A heavy chain constant domain;

CH2is immunoglobulin CH2A heavy chain constant domain;

CH3Is immunoglobulin CH3A heavy chain constant domain;

the hinge is connected with the CH1And CH2An immunoglobulin hinge region of the domain, and

L1、L2、L3and L4Is an amino acid linker;

wherein the polypeptide of formula I and the polypeptide of formula II form a cross light chain-heavy chain pair, and

wherein: (a) the V isH1The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO: 31) or GYTFTSYA (SEQ ID NO: 37), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO: 32) or IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33); and said VL1The domain comprises a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO: 34) or QSVSSYGQG (SEQ ID NO: 132), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO: 35) or GAS (SEQ ID NO: 40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36);

(b) the V isH2The domain comprises ammonia comprising GYTFTSFN (SEQ ID NO: 31) or GYTFTSYA (SEQ ID NO: 37)A CDR-H1 sequence of the amino acid sequence, a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO: 32) or IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33); and said V L2The domain comprises a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO: 34) or QSVSSYGQG (SEQ ID NO: 132), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO: 35) or GAS (SEQ ID NO: 40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36); or

(c) The V isH3The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO: 31) or GYTFTSYA (SEQ ID NO: 37), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO: 32) or IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33); and said VL3The domain comprises a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO: 34) or QSVSSYGQG (SEQ ID NO: 132), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO: 35) or GAS (SEQ ID NO: 40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36). In some embodiments, the V isH1The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO: 31), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO: 32), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33); and said V L1The domain comprises a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO: 34), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO: 35), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36); the V isH1The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYA (SEQ ID NO: 37), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33); and said VL1The domains comprise a CDR-L1 sequence comprising the amino acid sequence of QSVSSYGQGF (SEQ ID NO: 39), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO: 40), and a CDR-L2 sequence comprising QQNKEDPWT (SEQ ID N: 39)O: 36) the CDR-L3 sequence of the amino acid sequence of (a); the V isH2The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO: 31), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO: 32), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33); and said VL2The domain comprises a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO: 34), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO: 35), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36); the V is H2The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYA (SEQ ID NO: 37), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID N0: 33); and said VL2The domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSVSSYGQGF (SEQ ID NO: 39), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO: 40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36); the V isH3The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO: 31), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO: 32), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33); and said VL3The domain comprises a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO: 34), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO: 35), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36); or said VH3The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYA (SEQ ID NO: 37), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33); and said V L3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSVSSYGQGF (SEQ ID NO: 39), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO: 40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36). In some embodiments, the V isH3The domain comprises a polypeptide comprising GYTFTSFN (SEQ ID NO: 31)) The CDR-H1 sequence of amino acid sequence of (a), comprising iypggt (SEQ id no: 32) and a CDR-H2 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33) the CDR-H3 sequence of the amino acid sequence of (a); and said VL3The domain comprises a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO: 34), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO: 35), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36); or said VH3The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYA (SEQ ID NO: 37), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33); and said VL3The domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSVSSYGQGF (SEQ ID NO: 39), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO: 40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36). In some embodiments, the V is H3The domain comprises SEQ ID NO: 5, and said VL3The domain comprises seq id NO: 6; the V isH3The domain comprises SEQ ID NO: 17, and said VL3The domain comprises SEQ ID NO: 18; the V isH3The domain comprises SEQ ID NO: 21, and said VL3The domain comprises SEQ ID NO: 18; the V isH3The domain comprises SEQ ID NO: 23, and said VL3The domain comprises SEQ ID NO: 18; or said VH3The domain comprises SEQ ID NO: 13, and said VL3The domain comprises SEQ ID NO: 14. In some embodiments, wherein the binding protein comprises 4 polypeptide chains forming the 3 antigen binding sites, wherein a first polypeptide chain comprises a structure represented by the formula:

VL2-L1-VL1-L2-CL[I]

and the second polypeptide chain comprises a structure represented by the formula:

VH1-L3-VH2-L4-CH1-hinge-CH2-CH3[II]

And the third polypeptide chain comprises a structure represented by the formula:

VH3-CH1-hinge-CH2-CH3[III]

And the fourth polypeptide chain comprises a structure represented by the formula:

VL3-CL[IV]

wherein:

VL1is a first immunoglobulin light chain variable domain;

VL2is a second immunoglobulin light chain variable domain;

VL3Is a third immunoglobulin light chain variable domain;

VH1is a first immunoglobulin heavy chain variable domain;

VH2is a second immunoglobulin heavy chain variable domain;

VH3is a third immunoglobulin heavy chain variable domain;

CLis an immunoglobulin light chain constant domain;

CH1is immunoglobulin CH1A heavy chain constant domain;

CH2is immunoglobulin CH2A heavy chain constant domain;

CH3is immunoglobulin CH3A heavy chain constant domain;

the hinge is connected with the CH1And CH2An immunoglobulin hinge region of the domain, and

L1、L2、L3and L4Is an amino acid linker;

wherein the polypeptide of formula I and the polypeptide of formula II form a cross light chain-heavy chain pair, and

wherein (a) said VH1The domains comprise a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO: 41), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO: 42)And a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO: 43); and said VL1The domain comprises a CDR-L1 sequence comprising the amino acid sequence of QGIRND (SEQ ID NO: 44), a CDR-L2 sequence comprising the amino acid sequence of AAS (SEQ ID NO: 45), and a CDR-L3 sequence comprising the amino acid sequence of LQDYIYYPT (SEQ ID NO: 46);

(b) the V isH2The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO: 41), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO: 42), and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO: 43); and said V L2The domain comprises a CDR-L1 sequence comprising the amino acid sequence of QGIRND (SEQ ID NO: 44), a CDR-L2 sequence comprising the amino acid sequence of AAS (SEQ ID NO: 45), and a CDR-L3 sequence comprising the amino acid sequence of LQDYIYYPT (SEQ ID NO: 46), or

(c) The V isH3The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO: 41), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO: 42), and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO: 43); and said VL3The domain comprises a CDR-L1 sequence comprising the amino acid sequence of QGIRND (SEQ ID NO: 44), a CDR-L2 sequence comprising the amino acid sequence of AAS (SEQ ID NO: 45), and a CDR-L3 sequence comprising the amino acid sequence of LQDYIYYPT (SEQ ID NO: 46). In some embodiments, the V isH3The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO: 41), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO: 42), and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO: 43); and said VL3The domain comprises a CDR-L1 sequence comprising the amino acid sequence of QGIRND (SEQ ID NO: 44), a CDR-L2 sequence comprising the amino acid sequence of AAS (SEQ ID NO: 45), and a CDR-L3 sequence comprising the amino acid sequence of LQDYIYYPT (SEQ ID NO: 46). In some embodiments, the V is H3The domain comprises SEQ ID NO: 9, and said VL3The domain comprises SEQ ID NO: 10. In some embodiments, the V isH1Structure of the productThe domain comprises SEQ ID NO: 49, said VL1The domain comprises SEQ ID NO: 50, said VH2The domain comprises SEQ ID NO: 53, and said VL2The domain comprises SEQ ID NO: 54; the V isH2The domain comprises SEQ ID NO: 49, said VL2The domain comprises SEQ ID NO: 50, said VH1The domain comprises SEQ ID NO: 53, and said VL1The domain comprises SEQ ID NO: 54; the V isH1The domain comprises SEQ ID NO: 51, said VL1The domain comprises SEQ ID NO: 52, said VH2The domain comprises SEQ ID NO: 53, and said VL2The domain comprises SEQ ID NO: 54; the V isH2 domain comprises SEQ ID NO: 51, said VL2The domain comprises SEQ ID NO: 52, said VH1The domain comprises SEQ ID NO: 53, and said V L1The domain comprises SEQ ID NO: 54; the V isH1The domain comprises SEQ id no: 49, said VL1The domain comprises SEQ ID NO: 50, said VH2The domain comprises SEQ ID NO: 84, and said VL2The domain comprises SEQ ID NO: 85; the V isH2The domain comprises SEQ ID NO: 49, said VL2The domain comprises SEQ ID NO: 50, said VH1The domain comprises SEQ ID NO: 84, and said VL1The domain comprises SEQ ID NO: 85; the V isH1The domain comprises SEQ ID NO: 51, said VL1The domain comprises SEQ ID NO: 52, said VH2The domain comprises SEQ ID NO: 84, and said VL2The domain comprises seq id NO: 85; or said VH2The domain comprises SEQ ID NO: 51, said VL2The domain comprises SEQ ID NO: 52, said VH1The domain comprises SEQ ID NO: 84, and said VL1The domain comprises SEQ ID NO: 85. In some embodiments, the V is H1The domain comprises SEQ id no: 49, said VL1The domain comprises SEQ ID NO: 50, said VH2The domain comprises SEQ ID NO: 53, and the VL2 domain comprises the amino acid sequence of SEQ ID NO: 54, said VH3The domain comprises SEQ ID NO: 13, and said VL3The domain comprises SEQ ID NO: 14; or said VH1The domain comprises SEQ ID NO: 49, said VL1The domain comprises SEQ ID NO: 50, said VH2The domain comprises SEQ ID NO: 53, said VL2The domain comprises SEQ ID NO: 54, said VH3The domain comprises SEQ ID NO: 9, and said VL3The domain comprises seq id NO: 10. In some embodiments, L is1、L2、L3Or L4Is independently 0 amino acids in length. In some embodiments, L is1、L2、L3Or L4Is each independently at least one amino acid in length. In some embodiments, (a) L1、L2、L3And L4Each independently is 0 amino acids in length or comprises a sequence selected from the group consisting of: GGGGSGGGGS (SEQ ID NO: 55), GGGGSGGGGSGGGGS (SEQ ID NO: 56), S, RT, TKGPS (SEQ ID NO: 57), GQPKAAP (SEQ ID NO: 58) and GGSGSSGSGG (SEQ ID NO: 59); or (b) L 1、L2、L3And L4Each independently comprises a sequence selected from the group consisting of: GGGGSGGGGS (SEQ ID NO: 55), GGGGSGGGGSGGGGS (SEQ ID NO: 56), S, RT, TKGPS (SEQ ID NO: 57), GQPKAAP (SEQ ID NO: 58) and GGSGSSGSGG (SEQ ID NO: 59). In some embodiments, L is1Comprising the sequence GQPKAAP (SEQ ID NO: 58), L2Comprising the sequence TKGPS (SEQ ID NO: 57), L3Comprising the sequences S, and L4Comprises the sequence RT; l is1Comprising the sequence GGGGSGGGGS (SEQ ID NO: 55), L2Comprising the sequence GGGGSGGGGS (SEQ ID NO: 55), L3Is 0 amino acids in length, and L4Is 0 amino acids in length; l is1Comprising the sequence GGSGSSGSGG (SEQ ID NO: 59), L2Comprising the sequence GGSGSSGSGG (SEQ ID NO: 59), L3Is 0 amino acids in length, and L4Is 0 amino acids in length; or L1Comprising the sequence GGGGSGGGGSGGGS (SEQ ID NO: 56), L2Is 0 amino acids in length, L3Comprising the sequences GGGGSGGGGSGGGS (SEQ ID NO: 56), and L4Is 0 amino acids in length. In some embodiments, the hinge-C of said second and said third polypeptide chainsH2-CH3The domain is human IgG4 hinge-CH2-CH3A domain, and wherein said hinge-C is according to EU IndexH2-CH3Each domain comprises amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG4, wherein the amino acid substitutions are F234A and L235A. In some embodiments, the hinge-C of said second and said third polypeptide chains H2-CH3The domain is human IgG4 hinge-CH2-CH3A domain, and wherein said hinge-C is according to EUIndexH2-CH3The domains each comprise amino acid substitutions at positions corresponding to position 233-236 of human IgG4, wherein the amino acid substitutions are E233P, F234V, L235A, and a deletion at 236. In some embodiments, the hinge-C of said second and said third polypeptide chainsH2-CH3The domain is human IgG4 hinge-CH2-CH3A domain, and wherein said hinge-C is according to EUIndexH2-CH3Each domain comprises amino acid substitutions at positions corresponding to positions 228 and 409 of human IgG4, wherein the amino acid substitutions are S228P and R409K. In some embodiments, the hinge-C of said second and said third polypeptide chainsH2-CH3The domain is human IgG1 hinge-CH2-CH3A domain, and wherein said hinge-C is according to EU IndexH2-CH3The domains each correspond to human IgG1234. 235 and 329 positions comprising amino acid substitutions, wherein the amino acid substitutions are L234A, L235A and P329A. In some embodiments, the hinge-C of said second and said third polypeptide chainsH2-CH3The domain is human IgG1 hinge-CH2-CH3A domain, and wherein said hinge-C is according to EU IndexH2-CH3Each of the domains comprises amino acid substitutions at positions corresponding to positions 298, 299 and 300 of human IgG1, wherein the amino acid substitutions are S298N, T299A and Y300S. In some embodiments, the hinge-C of the second polypeptide chain is according to EU Index H2-CH3The domain comprises amino acid substitutions at positions corresponding to positions 349, 366, 368 and 407 of human IgG1 or IgG4, wherein the amino acid substitutions are Y349C, T366S, L368A and Y407V; and wherein the hinge-C of said third polypeptide chain is according to EU IndexH2-CH3The domain comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4, wherein the amino acid substitutions are S354C and T366W. In some embodiments, the hinge-C of the second polypeptide chain is according to EU IndexH2-CH3The domain comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4, wherein the amino acid substitutions are S354C and T366W; and wherein the hinge-C of said third polypeptide chain is according to EU IndexH2-CH3The domain comprises amino acid substitutions at positions corresponding to positions 349, 366, 368 and 407 of human IgG1 or IgG4, wherein the amino acid substitutions are Y349C, T366S, L368A and Y407V. In some embodiments, the first polypeptide chain comprises SEQ ID NO: 61, and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 60, and said third polypeptide chain comprises the amino acid sequence of SEQ id no: 62, and said fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 63. In some embodiments, the first polypeptide chain comprises SEQ ID NO: 61, and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 64, and said third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 65, and said fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 63. In some embodiments, the first The polypeptide chain comprises SEQ ID NO: 61, and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 66, and said third polypeptide chain comprises the amino acid sequence of SEQ id no: 67, and said fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 63. In some embodiments, the first polypeptide chain comprises SEQ ID NO: 61, and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 60, and a third polypeptide chain comprising the amino acid sequence of SEQ ID NO: 68, and said fourth polypeptide chain comprises seq id NO: 69. In some embodiments, the first polypeptide chain comprises SEQ ID NO: 61, and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 64, and said third polypeptide chain comprises the amino acid sequence of SEQ id no: 70, and said fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 69. In some embodiments, the first polypeptide chain comprises SEQ ID NO: 61, and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 66, and said third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 71, and said fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 69.

In some embodiments, provided herein is a binding protein comprising 3 antigen binding sites that each bind one or more target proteins, wherein the binding protein comprises 4 polypeptide chains forming the 3 antigen binding sites, wherein a first polypeptide chain comprises a structure represented by the formula:

VL2-L1-VL1-L2-CL[I]

and the second polypeptide chain comprises a structure represented by the formula:

VH1-L3-VH2-L4-CH1-hinge-CH2-CH3[II]

And the third polypeptide chain comprises a structure represented by the formula:

VH3-CH1-hinge-CH2-CH3[III]

And the fourth polypeptide chain comprises a structure represented by the formula:

VL3-CL[IV]

wherein:

VL1is a first immunoglobulin light chain variable domain;

VL2is a second immunoglobulin light chain variable domain;

VL3is a third immunoglobulin light chain variable domain;

VH1is a first immunoglobulin heavy chain variable domain;

VH2is a second immunoglobulin heavy chain variable domain;

VH3is a third immunoglobulin heavy chain variable domain;

CLis an immunoglobulin light chain constant domain;

CH1is immunoglobulin CH1A heavy chain constant domain;

CH2is immunoglobulin CH2A heavy chain constant domain;

CH3is immunoglobulin CH3A heavy chain constant domain;

the hinge is connected with the CH1And CH2An immunoglobulin hinge region of the domain, and

L1、L2、L3and L4Is an amino acid linker;

wherein the polypeptide of formula I and the polypeptide of formula II form a cross light chain-heavy chain pair, and

Wherein (a) the hinge-C of said second and said third polypeptide chainsH2-CH3The domain is human IgG1 hinge-CH2-CH3A domain, and wherein said hinge-C is according to EU IndexH2-CH3Each of the domains comprises amino acid substitutions at positions corresponding to positions 298, 299 and 300 of human IgG1, wherein the amino acid substitutions are S298N, T299A and Y300S; or (b) the hinge-C of said second and said third polypeptide chainsH2-CH3The domain is human IgG4 hinge-CH2-CH3A domain, and wherein said hinge-C is according to EUIndexH2-CH3Each of the domainsComprises amino acid substitutions at positions corresponding to positions 233-236 of human IgG4, wherein the amino acid substitutions are E233P, F234V, L235A, and a deletion at 236. In some embodiments, the human IgG4 hinge-CH2-CH3The domain comprises amino acid substitutions at positions corresponding to positions 233-237 of human IgG4, wherein the sequence EFLGG is replaced by PVAG. In some embodiments, VH1And VL1、VH2And VL2And VH3And VL3Forms an antigen binding site that binds to a CD38 polypeptide. In some embodiments, VH1And VL1、VH2And VL2And VH3And VL3The 1, 2 or 3 pair forming an antigen binding site that binds to an antigen target selected from the group consisting of A2, APRIL, ATP diphosphate hydrolase, BAFF, BAFFR, BCMA, BlyS, BTK, BTLA, B7H, B7RP, B-4, C, CCL, CCR, CD122, CD137, CD152, CD154, CD160, CD272, CD274, CD275, CD GR, CD278, CD279, chitinase, CLEC, CREC, CSF-1, CSF-2, CSF-CSF 3, CX3, CXCL, TLR 1, TLR, T7H, B-4, C-4, C, CCL, CDR, TLR, TREM1, TSLP, TSLPR, TWEAK, VEGF, VISTA, Vstm3, WUCAM, and XCR 1. In some embodiments, VH1And VL1、VH2And VL2And VH3And VL3Form an antigen binding site, V, that binds to the human CD3 polypeptideH1And VL1、VH2And VL2And VH3And VL3Forming an antigen binding site that binds to a human CD28 polypeptide, and VH1And VL1、VH2And VL2And VH3And VL3The third pair of (a) forms an antigen binding site that binds to a human antigen target selected from the group consisting of A2, APRIL, ATP diphosphate hydrolase, BAFF, BAFFR, BCMA, BlyS, BTK, BTLA, B7H, B7RP, B-4, C, CCL, PROM, CCR, CD122, CD137, CD152, CD154, CD160, CD272, CD273, CD274, CD275, CD278, CD279, CDLH, chitinase, CLEC, CSF-1, CSF-2, CXCL, TLR, CLIL, TLR, CLIL 1, TLR.

In some embodiments, provided herein is a kit of polynucleotides comprising: (a) comprises SEQ ID NO: 73 comprising the sequence of SEQ ID NO: 72, comprising the sequence of SEQ id no: 74, and a third polynucleotide comprising the sequence of SEQ ID NO: 75; (b) comprises SEQ ID NO: 73 comprising the sequence of SEQ ID NO: 76 comprising the sequence of SEQ id no: 77, and a third polynucleotide comprising the sequence of SEQ ID NO: 75; (c) comprises SEQ ID NO: 73 comprising the sequence of SEQ ID NO: 78 comprising the sequence of SEQ id no: 79, and a third polynucleotide comprising the sequence of SEQ ID NO: 75; (d) comprises SEQ ID NO: 73 comprising the sequence of SEQ ID NO: 72, comprising the sequence of SEQ id no: 80, and a third polynucleotide comprising the sequence of SEQ ID NO: 81; (e) comprises SEQ ID NO: 73 comprising the sequence of SEQ ID NO: 76 comprising the sequence of SEQ id no: 82, and a third polynucleotide comprising the sequence of SEQ ID NO: 81; or (f) comprises SEQ ID NO: 73 comprising the sequence of SEQ ID NO: 78 comprising the sequence of SEQ id no: 83, and a third polynucleotide comprising the sequence of SEQ ID NO: 81, or a third polynucleotide of the sequence of seq id no.

In some embodiments, provided herein is a polynucleotide comprising the binding protein of any one of the above embodiments. In some embodiments, provided herein is a vector comprising the polynucleotide of any one of the above embodiments.

In some embodiments, provided herein is a host cell comprising a kit, polynucleotide or vector of polynucleotides of any one of the above embodiments. In some embodiments, provided herein is a method of producing a binding protein, the method comprising culturing a host cell of any one of the above embodiments to produce the binding protein. In some embodiments, the method further comprises recovering the binding protein from the host cell.

In some embodiments, provided herein is a pharmaceutical composition comprising the binding protein of any one of the above embodiments and a pharmaceutically acceptable carrier.

In some embodiments, provided herein is a method of preventing and/or treating cancer in a patient comprising administering to the patient a therapeutically effective amount of at least one binding protein of any one of the above embodiments or a pharmaceutical composition of any one of the above embodiments. In some embodiments, the binding protein is a trispecific binding protein comprising a first antigen-binding site that binds CD3, a second antigen-binding site that binds CD28, and a third antigen-binding site that binds the extracellular domain of a human CD38 polypeptide. In some embodiments, the at least one binding protein is co-administered with a chemotherapeutic agent. In some embodiments, the cancer is multiple myeloma. In some embodiments, the cancer is Acute Myeloid Leukemia (AML), Acute Lymphoblastic Leukemia (ALL), Chronic Lymphocytic Leukemia (CLL), or B-cell lymphoma. In some embodiments, the patient is a human. In some embodiments, the patient is selected for treatment because the cells of the cancer express a human CD38 isoform E polypeptide on their cell surface (e.g., as shown in SEQ ID NO: 105). In some embodiments, the cancer cell expresses CD38 and CD 28. In some embodiments, the cancer cells express CD38 and do not express CD 28.

In some embodiments, provided herein is at least one binding protein of any one of the above embodiments or a pharmaceutical composition of any one of the above embodiments for use in preventing and/or treating cancer in a patient (e.g., a patient in need thereof, such as a patient having cancer). In some embodiments, provided herein is at least one binding protein of any one of the above embodiments or a pharmaceutical composition of any one of the above embodiments for use in the preparation of a medicament for the prevention and/or treatment of cancer in a patient (e.g., a patient in need thereof, such as a patient having cancer). In some embodiments of any of the above embodiments, the binding protein is a trispecific binding protein comprising a first antigen-binding site that binds CD3, a second antigen-binding site that binds CD28, and a third antigen-binding site that binds the extracellular domain of a human CD38 polypeptide. In some embodiments, the at least one binding protein is co-administered with a chemotherapeutic agent. In some embodiments, the cancer is multiple myeloma. In some embodiments, the cancer is Acute Myeloid Leukemia (AML), Acute Lymphoblastic Leukemia (ALL), Chronic Lymphocytic Leukemia (CLL), or B-cell lymphoma. In some embodiments, the patient is a human. In some embodiments, the patient is selected for treatment because the cells of the cancer express a human CD38 isoform E polypeptide on their cell surface (e.g., as shown in SEQ ID NO: 105). In some embodiments, the cancer cell expresses CD38 and CD 28. In some embodiments, the cancer cells express CD38 and do not express CD 28.

It is to be understood that one, some or all of the properties of the various embodiments described herein may be combined to form further embodiments of the invention. These and other aspects of the invention will become apparent to those skilled in the art. These and other embodiments of the present invention are further described by the following detailed description.

Drawings

This patent or application document contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office (Office) upon request and payment of the necessary fee.

FIG. 1A shows the binding of anti-CD 38 antibodies mAb1 (top panel) and isatuximab (bottom panel) to SU-DHL-8 human lymphoma cells or MOLP-8 human multiple myeloma cells using flow cytometry.

Figure 1B shows the results of a flow cytometry binding assay that detects binding of anti-CD 38 antibody mAb1 or isatuximab (no binding observed) to cells expressing cynomolgus monkey CD38 on the surface.

Figures 2A-2I show the results of assays that characterize the binding of anti-CD 38 antibodies to human and cynomolgus monkey CD38 polypeptides. FIG. 2A shows that humanized anti-CD 38 antibody mAb2 binds to soluble human CD38 (top panel, "hCD 38:: Histag") or cynomolgus CD38 (top panel, "cynoCD 38:: Histag") by ELISA and mAb2 binds to the surface of cells expressing human CD83 (bottom panel, as shown) or cynomolgus CD38 (bottom panel, as shown) by flow cytometry. Figure 2B shows mAb2 binding to human CD38 (top panel) or cynomolgus monkey CD38 (bottom panel) by Surface Plasmon Resonance (SPR). FIG. 2C shows that humanized anti-CD 38 antibody mAb3 binds to soluble human CD38 (top panel, "hCD 38:: Histag") or cynomolgus CD38 (top panel, "cynoCD 38:: Histag") by ELISA and mAb3 binds to the surface of cells expressing human CD83 (bottom panel, as shown) or cynomolgus CD38 (bottom panel, as shown) by flow cytometry. Figure 2D shows mAb3 binding to human CD38 (top panel) or cynomolgus monkey CD38 (bottom panel) by SPR. FIG. 2E shows that humanized anti-CD 38 antibody mAb5 binds to soluble human CD38 (top panel, "hCD 38:: Histag") or cynomolgus CD38 (top panel, "cynoCD 38:: Histag") by ELISA and mAb5 binds to the surface of cells expressing human CD83 (bottom panel, as shown) or cynomolgus CD38 (bottom panel, as shown) by flow cytometry. Figure 2F shows mAb5 binding to human CD38 (upper panel) or cynomolgus monkey CD38 (lower panel) by SPR. FIG. 2G shows that human anti-CD 38 antibody hhy1370 was bound to soluble human CD38 (top panel, "hCD 38:: Histag") or cynomolgus CD38 (top panel, "cynoCD 38:: Histag") by ELISA, and hhy1370 was bound to the surface of cells expressing human CD83 (bottom panel, as shown) or cynomolgus CD38 (bottom panel, as shown) by flow cytometry. Figure 2H shows hhy1370 binding to human CD38 (top panel) or cynomolgus monkey CD38 (bottom panel) by SPR. Figure 2I summarizes the binding data from ELISA, SPR and FACS experiments, and the percent identity of each antibody VH ("H") or VL ("L") domain to the human V region sequence from top to bottom, corresponding to mAb2, mAb3, mAb4, mAb5 and mAb6 in the last line (1195HHKK-3 is not described below by its VL/VH amino acid sequence).

FIG. 2J shows concentration-dependent induction of apoptosis by mAb7 and mAb1 in SU-DHL-8 cells after 72 hours incubation at 37 ℃.

Figure 2K shows antibody-dependent cell-mediated cytotoxicity (ADCC) activity of isatuximab and mAb1 against SU-DHL-8 cells in the presence of NK92 cells.

FIG. 2L shows concentration-dependent antibody-dependent cell-mediated cytotoxicity (ADCC) activity of isatuximab (right) and mAb1 (left) on SU-DHL-8 cells in the presence of NK92 cells after 4 hours at 37 ℃.

FIGS. 2M-2Q show the results of an apoptosis induction assay using the anti-CD 38 antibody shown against SU-DHL-8 tumor cells. Apoptosis was quantified by measuring annexin V and propidium iodide uptake via flow cytometry. Figure 2M shows the percentage of apoptotic cells induced by each antibody. Fig. 2N-2Q show dose-dependent induction of SU-DHL-8 lymphoma cell apoptosis by anti-CD 38 antibodies mAb2 (fig. 2N), mAb3 (fig. 2O), mAb4 (fig. 2P), and mAb5 (fig. 2Q), as well as the IC50 of each antibody.

Figure 3A provides a schematic of a trispecific binding protein comprising four polypeptide chains forming 3 antigen binding sites that bind the following 3 target proteins: CD28, CD3, and CD 38. The first pair of polypeptides has double variable domains (VH1-VH2 and VL2-VL1) in a cross-orientation forming 2 antigen binding sites recognizing CD3 and CD28, and the second pair of polypeptides has single variable domains (VH3 and VL3) forming a single antigen binding site recognizing CD 38. The trispecific binding protein shown in figure 3A uses IgG4 constant regions with a "knob-to-hole" mutation, where the knob is on a second pair of polypeptides with a single variable domain.

Figure 3B provides a schematic of an SPR-based assay for examining the ability of each antigen-binding domain of the anti-CD 38/anti-CD 28/anti-CD 3 trispecific binding protein to bind to its cognate antigen.

Figure 3C shows the results of SPR-based assays used to examine the binding of CD38 to anti-CD 38/anti-CD 28/anti-CD 3 trispecific binding proteins. Binding of human CD38 to the trispecific binding protein was examined separately after pre-binding CD3 (top left), after pre-binding CD28 (top right), after pre-binding CD3 followed by CD28 (bottom left), or after pre-binding CD28 followed by CD3 (bottom right).

Figure 4 shows the sequential binding of human CD3, CD28, and CD38 polypeptides to anti-CD 38/anti-CD 28/anti-CD 3 trispecific binding proteins, as determined by SPR.

Figure 5 summarizes the binding affinities of the indicated trispecific binding proteins to their cognate antigens (human CD3, CD28, and CD38) as measured by SPR.

Figure 6A compares the apparent affinity of the isatuximab antigen-binding domain in the form of human IgG1 (second sheet) or trispecific binding protein to the isatuximab, anti-CD 28, and anti-CD 3 antigen-binding domains (according to the form of figure 3A; first sheet) for binding to a human (top panel) or cynomolgus monkey (bottom panel) CD38 polypeptide as determined by flow cytometry.

FIGS. 6B-6D compare the trispecific binding protein CD38VH1xCD28supxCD3mid、CD38VH1xCD28cvnxCD3midOr the apparent affinity of monospecific anti-CD 38 antibody mAb2 for binding to cells expressing human or cynomolgus monkey CD38 polypeptide as determined by flow cytometry. FIG. 6B shows the trispecific binding protein CD38VH1xCD28supxCD3midBinding to cells expressing human (upper panel) or cynomolgus monkey (lower panel) CD38 polypeptide. FIG. 6C shows the trispecific binding protein CD38VH1xCD28cvnxCD3midBinding to cells expressing human (upper panel) or cynomolgus monkey (lower panel) CD38 polypeptide. Figure 6D shows the binding of monospecific anti-CD 38 antibody mAb2 to cells expressing human (top panel) or cynomolgus monkey (bottom panel) CD38 polypeptide.

FIG. 6E compares the trispecific binding protein CD38HHY1370xCD28supxCD3midOr the apparent affinity of monospecific anti-CD 38 antibody mAb6 for binding to cells expressing human (top panel) or cynomolgus monkey (bottom panel) CD38 polypeptide as determined by flow cytometry.

Figure 6F summarizes the binding affinity of the indicated anti-CD 38x anti-CD 28x anti-CD 3 trispecific binding protein to human CD38 as measured by SPR or flow cytometry (FACS).

FIG. 6G shows the trispecific binding protein Δ CD38 lacking the anti-CD 38 antigen binding domainVH1xCD28supxCD3midApparent affinity for binding to cells expressing human (upper panel) or cynomolgus monkey (lower panel) CD38 polypeptide, as determined by flow cytometry.

Fig. 7A and 7B show the results of an ELISA assay to determine the binding affinity of various anti-CD 38x CD28x CD3IgG4 trispecific binding proteins or control antibodies to human and rhesus monkey CD3, CD28 and CD38 polypeptides.

FIGS. 8A-8D show antibody-mediated CD38 of PBMCs from three different human donors using the indicated anti-CD 38x CD28x CD3 trispecific binding protein and control antibody+Results of cell-specific killing. Representative results using multiple myeloma cell line RPMI8266 (FIG. 8A), NCI-H929 (FIG. 8B), KMS-26 (FIG. 8C), and KMS-11 cell line (FIG. 8D) are shown, and EC50 values are provided in Table N. EC50 values obtained by using NCI-H929, KMS-26 and KMS-11 cells are provided in Table O-Q.

FIGS. 8E and 8F show antibody-mediated CD38 of PBMCs from two different donors using the anti-CD 38x CD28x CD3 trispecific binding protein with variant Fc regions shown and a control antibody+Results of cell-specific killing. Shows the use of CD38+Representative results for the KMS-11 (FIG. 8D) and U266 (FIG. 8E) cell lines, and EC50 values are provided in tables Q2 and Q3.

FIGS. 9A, 9B and 10 show activation of human T cells treated with various anti-CD 38x CD28x CD3 trispecific binding proteins or control antibodies for 24 hours (CD 69) +). FIG. 9A shows a human CD3+Activation of T cells (CD 69)+). FIG. 9B shows a human CD3+CD4+Activation of T cells (CD 69)+). FIG. 10 shows a human CD3+CD8+Activation of T cells (CD 69)+)。

Fig. 11A-11B show the results as Stebbings, r.et al. (2007) j.immunol.179: 3325-3331 based on the results of in vitro cytokine release assessment of human PBMCs treated with the indicated anti-CD 38x CD28x CD3 trispecific binding protein or control antibody by the dry plate method. FIG. 11A shows the results using 5. mu.g/mL of the indicated antibody. FIG. 11B shows the results using 25ng/mL of the indicated antibody.

FIGS. 12A-12E show anti-CD 38 in a CD34+ cord blood cell humanized NSG mouse model implanted with RPMI-8226 multiple myeloma cells(VHI)x CD28(sup)x CD3(mid)In vivo activity of trispecific binding proteins. FIG. 12A showsanti-CD 38 at the concentrations indicated(VHI)x CD28(sup)x CD3(mid)Tumor volume changes in mice treated with the trispecific binding protein relative to mice treated with the anti-CD 3/CD38 bispecific antibody. FIG. 12B shows CD38 at the concentrations indicated(VHI)xCD28(sup)x CD3(mid)Mean tumor volume at day 18 in mice treated with the trispecific binding protein relative to mice treated with the anti-CD 3/CD38 bispecific antibody. FIG. 12C shows anti-CD 38 at the concentrations indicated(VHI)x CD28(sup)x CD3(mid)Mean terminal tumor weight of trispecific binding protein treated mice relative to mice treated with anti-CD 3/CD38 bispecific antibody. FIG. 12D shows anti-CD 38 at the concentrations indicated (VHI)x CD28(sup)x CD3(mid)Mean tumor growth curves over the length of the experiment for mice treated with the trispecific binding protein relative to mice treated with the anti-CD 3/CD38 bispecific antibody. FIG. 12E shows anti-CD 38 at the concentrations indicated(VHI)x CD28(sup)x CD3(mid)Mean change in body weight at various time points over the length of the experiment for mice treated with the trispecific binding protein relative to mice treated with the anti-CD 3/CD38 bispecific antibody.

FIGS. 13A-13F show anti-CD 38 in a PBMC humanized NSG mouse model implanted with RPMI-8226 multiple myeloma cells(VHI)x CD28(sup)x CD3(mid)In vivo activity of trispecific binding proteins. FIG. 13A shows anti-CD 38 at the concentrations indicated(VHI)x CD28(sup)x CD3(mid)Tumor volume changes in mice treated with the trispecific binding protein relative to mice treated with the anti-CD 3/CD38 bispecific antibody. FIG. 13B shows anti-CD 38 at the concentrations indicated(VHI)x CD28(sup)x CD3(mid)Tumor volume at day 4 in mice treated with the trispecific binding protein relative to mice treated with the anti-CD 3/CD38 bispecific antibody. FIG. 13C shows anti-CD 38 at the concentrations indicated(VHI)x CD28(sup)x CD3(mid)Tumor volume on day 21 of trispecific binding protein treated mice versus anti-CD 3/CD38 bispecific antibody treated mice. As shown in FIG. 13DConcentration of anti-CD 38(VHI)x CD28(sup)x CD3(mid)Mean tumor volume at day 21 in mice treated with the trispecific binding protein relative to mice treated with the anti-CD 3/CD38 bispecific antibody. FIG. 13E shows anti-CD 38 at the concentrations indicated (VHI)xCD28(sup)x CD3(mid)Mean terminal tumor weight of trispecific binding protein treated mice relative to mice treated with anti-CD 3/CD38 bispecific antibody. FIG. 13F shows anti-CD 38 at the concentrations indicated(VHI)x CD28(sup)x CD3(mid)Mean tumor volumes at various time points over the length of the experiment for mice treated with the trispecific binding protein relative to mice treated with the anti-CD 3/CD38 bispecific antibody.

FIGS. 14A-14U show anti-CD 38 in non-human primates(VHI)x CD28(sup)x CD3(mid)anti-CD 38(VHI)x CD28(cvn)x CD3(mid)anti-CD 38(hhy1370)x CD28(sup)x CD3(mid)And anti-CD 38(hhy1370)xCD28(cvn)x CD3(mid)Results of dose escalation studies of trispecific binding proteins. FIG. 14A shows anti-CD 38 at various doses administered(VHI)x CD28(sup)x CD3(mid)Trispecific binding protein post-circulating CD3+T cell activation of T cells (CD 69)+). FIG. 14B shows anti-CD 38 at various doses(VHI)x CD28(cvn)x CD3(mid)Trispecific binding protein post-circulating CD3+T cell activation of T cells (CD 69)+). FIG. 14C shows administration of different doses of anti-CD 38(hhy1370)x CD28(sup)xCD3(mid)Trispecific binding protein post-circulating CD3+T cell activation of T cells (CD 69)+). FIG. 14D shows anti-CD 38 at various doses(hhy1370)x CD28(cvn)x CD3(mid)Trispecific binding protein post-circulating CD3+T cell activation of T cells (CD 69)+). FIG. 14E shows anti-CD 38 administered at the doses indicated(VHI)x CD28(sup)x CD3(mid)Trispecific binding protein post-circulating CD4+Variation of percentage of T cellsAnd (4) transforming. FIG. 14F shows anti-CD 38 administered at the doses indicated (VHI)x CD28(sup)x CD3(mid)Trispecific binding protein post-circulating CD8+Change in percentage of T cells. FIG. 14G shows anti-CD 38 using the doses indicated(VHI)xCD28(cvn)x CD3(mid)Trispecific binding protein post-circulating CD4+Change in percentage of T cells. FIG. 14H shows anti-CD 38 administered at the doses indicated(VHI)x CD28(cvn)x CD3(mid)Trispecific binding protein post-circulating CD8+Change in percentage of T cells. FIG. 14I shows anti-CD 38 administered at the doses indicated(hhy1370)x CD28(sup)x CD3(mid)Trispecific binding protein post-circulating CD4+Change in percentage of T cells. FIG. 14J shows anti-CD 38 administered at the doses indicated(hhy1370)x CD28(sup)x CD3(mid)Trispecific binding protein post-circulating CD8+Change in percentage of T cells. FIG. 14K shows anti-CD 38 administered at the doses indicated(hhyl370)x CD28(cvn)x CD3(mid)Trispecific binding protein post-circulating CD4+Change in percentage of T cells. FIG. 14L shows anti-CD 38 administered at the indicated doses(hhy1370)x CD28(cvn)x CD3(mid)Trispecific binding protein post-circulating CD8+Change in percentage of T cells. FIG. 14M shows total CD4 at 6, 24, and 48 hours after administration of 12.5. mu.g/kg of the indicated trispecific binding protein+Change in T cells. FIG. 14N shows the change in total NK cells at 6, 24 and 48 hours after administration of 12.5. mu.g/kg of the indicated trispecific binding protein. FIG. 14O shows total CD8 at 6, 24, and 48 hours after administration of 12.5. mu.g/kg of the indicated trispecific binding protein+Change in T cells. FIG. 14P shows the change in total B cells at 6, 24, and 48 hours after administration of 12.5. mu.g/kg of the indicated trispecific binding protein. FIG. 14Q shows anti-CD 38 administered at three escalating doses (0.5, 2.5, 12.5 μ g/kg) (VH1)x CD28(sup)x CD3(mid)Cytokine levels changed 6 hours after trispecific binding protein (results from different test animals labeled "117065" and "117066"). FIG. 14R shows anti-CD 38 administered at three escalating doses (0.5, 2.5, 12.5 μ g/kg)(VH1)xCD28(cvn)x CD3(mid)Cytokine levels changed 6 hours after trispecific binding protein (results from different experimental animals labeled "117067" and "117068"). FIG. 14S shows anti-CD 38 administered at three escalating doses (0.5, 2.5, 12.5. mu.g/kg)(hhyl370)x CD28(sup)x CD3(mid)Cytokine levels changed 6 hours after trispecific binding protein (results from different test animals labeled "117069" and "117070"). FIG. 14T shows anti-CD 38 administered at three escalating doses (0.5, 2.5, 12.5 μ g/kg)(hhy1370)x CD28(cvn)x CD3(mid)Cytokine levels changed 6 hours after trispecific binding protein (results from different test animals labeled "117071" and "117072"). Figure 14U shows the change in cytokine levels 24 hours after administration of three increasing doses (0.5, 2.5, 12.5 μ g/kg) of the indicated trispecific binding protein (results shown for all experimental animals).

FIGS. 14V and 14W show anti-CD 38(VHI)x CD28(sup)x CD3(mid)And anti-CD 38(VHI)x CD28(cvn)xCD3(mid)Trispecific binding proteins induce depletion of T cells in vivo (6 hours post-dose) in the blood of higher doses of non-human primates.

FIGS. 14X and 14Y show anti-CD 38(HHY1370)x CD28(sup)x CD3(mid)And anti-CD 38(HHY1370x CD28(cvn)xCD3(mid)Trispecific binding proteins induce depletion of T cells in vivo (6 hours post-dose) in the blood of higher doses of non-human primates.

FIGS. 14Z and 14AA show administration of anti-CD 38(VHI)x CD28(sup)x CD3(mid)Or anti-CD 38(VHI)x CD28(cvn)xCD3(mid)Amount of blood T cells in non-human primates over time after trispecific binding protein.

FIGS. 14AB and 14AC show administration of anti-CD 38(HHY1370)x CD28(sup)x CD3(mid)Or anti-CD 38(HHY1370)xCD28(cvn)x CD3(mid)Blood T-cells in non-human primates after trispecific binding of proteinsAmount of cells that change over time.

Figures 14AD and 14AE show the amount of CD4+ T cells with bound trispecific binding protein after administration of a 100 μ g/kg dose in a non-human primate.

FIGS. 14AF and 14AG show the amount of CD8+ T cells with bound trispecific binding protein after administration of a 100 μ g/kg dose in a non-human primate.

Figures 15A-15C show binding or lack of binding of various Fc variants to the human Fc receptors Fc γ R I (figure 15A), Fc γ R IIa (figure 15B), and Fc γ R IIIb/C (figure 15C). The variants tested were human IgG1, human IgG4, and human IgG4 with a FALA mutation.

Figure 16 shows binding of human IgG4 with or without the fata mutation to FcRn.

FIG. 17 summarizes the trispecific binding proteins (CD 38) shown in NSG miceVH1xCD28supxCD3midIgG4、CD38VH1xCD28supxCD3midIgG4 FALA、CD38vH1xCD28supxCD3midIgG1 LALA P329A and CD38HHY1370xCD28supxCD3midIgG4 FALA).

FIGS. 18A-18C show Fc/FcR interaction mediated (non-specific) release of IFN- γ (FIG. 18A), IL-2 (FIG. 18B), or TNF- α (FIG. 18C) from human PBMCs incubated with a trispecific binding protein having a wild-type or FALA variant Fc region.

FIG. 18D shows CD38VH1xCD28supxCD3midAnd CD38HHY1370xCD28supxCD3midIn vitro activation of human PBMCs by trispecific binding proteins and IgG1 and IgG4 Fc variants thereof.

FIGS. 19A and 19B show binding by the trispecific binding protein CD38VH1xCD28supxCD3midThe CD3 and CD28 antigen binding domains are required for induction of Bcl-xL in CD4+ (fig. 19A) or CD8+ (fig. 19B) T cells. Bar graph mean sum s.d. from 3 PBMC donors. And p ≦ 0.009.

FIGS. 19C and 19D show CD38 with IgG4 FALA variant FcVH1xCD28xCD3 upregulated Bcl-xL in CD4+ (fig. 19C) or CD8+ (fig. 19D) T cells greater than the baseline bispecific antibody. Bar shapeMean sum s.d. from 3 PBMC donors. P ≦ 0.045

Figure 19E shows that T cell activation of anti-CD 38x anti-CD 28x anti-CD 3 trispecific binding protein as measured by IL-2 expression in Jurkat T cell reporter cell line is dependent on the anti-CD 3 antigen binding domain.

FIG. 19F shows CD38 compared to anti-CD 28, anti-CD 38 or anti-CD 28, anti-CD 38 and anti-CD 3 with mutations and baseline binding proteins VH1xCD28supxCD3midThe trispecific binding proteins release the cytokines TNF, IFNg, IL-2, IL-6 and IL-10.

Figure 19G shows proliferation of T cells activated by anti-CD 38x anti-CD 28x anti-CD 3 trispecific binding protein with IgG4 FALA variant Fc, benchmark anti-CD 38x anti-CD 3 bispecific antibody, or isotype control (trispecific binding protein with IgG4 FALA variant Fc with a mutated binding domain).

Figure 20 shows proliferation of T cells activated by anti-CD 38x anti-CD 28x anti-CD 3 trispecific binding protein with IgG4 FALA variant Fc, anti-CD 38x anti-CD 28x anti-CD 3 trispecific binding protein with mutations in IgG4 FALA variant Fc and CD38, CD28, or CD3 antigen binding domains, or isotype control (trispecific binding protein with IgG4 FALA variant Fc with three mutated binding domains).

FIG. 21 shows CD38 administered at the indicated doses in the NCI-H929-Luc disseminated tumor model of PBMC humanized NSG miceVH1xCD28supxCD3midIn vivo anti-tumor activity of IgG4 FALA trispecific binding protein.

FIG. 22 shows CD38 administered at the indicated doses in the NCI-H929-Luc disseminated tumor model of PBMC humanized NSG miceHHY1370xCD28supxCD3midIn vivo anti-tumor activity of IgG4 FALA trispecific binding protein.

FIGS. 23A and 23B show CD38 with human PBMCs used in vivo studies VH1xCD28supxCD3midAnd CD38HHY1370xCD28supxCD3midEfficient in vitro tumor of NCI-929-Luc cells with trispecific binding protein and benchmark anti-CD 38x anti-CD 3 bispecific antibodyKilling activity. Effector at 10: 1: PBMC ratio human PBMC from 2 donor humanized NSG mice were used after 24 hours of incubation.

FIG. 23C shows CD38 administered at the indicated doses in the NCI-H929-Luc disseminated tumor model of PBMC humanized NSG mice as compared to the baseline anti-CD 38x anti-CD 3 bispecific antibodyVH1xCD28supxCD3midAnd CD38hhy1370xCD28supxCD3midExcellent in vivo antitumor activity of trispecific binding proteins. The binding protein was administered by Intraperitoneal (IP) injection at 30. mu.g/kg weekly.

FIG. 24A shows the use of GloResponseTMIL2-luc2P Jurkat cells (Promega) in CD38VH1/CD28supxCD3midAnd single binding site KO thereof and results of luciferase reporter gene assay after stimulation with a 10nM concentration of triple KO mutant.

FIG. 24B shows the optimization of anti-CD 3xCD28CODV-Fab antibodies. The optimal configuration of α -CD3 and α -CD28 in alternative positions of CODV bispecific fabs was evaluated by cytokine release assay using human PBMC in vitro. Based on secretion of IFN-. gamma.and IL-2 in the supernatant after 24 hours, the distal CD28x and proximal CD3 were determined to be the best localisation.

FIG. 25 shows CD38VH1/CD28supxCD3midInduction of upregulation of the Bcl-2 family member Bcl-xL in primary T cells is CD28 dependent.

Fig. 26 shows that anti-CD 28 in the trispecific Ab provides the secondary signaling necessary to support primary T cell proliferation in vitro.

Figure 27 shows the configuration of the trispecific antibody color-coded by the parent antibody (left). Dark hue (purple or green) indicates heavy chain peptide; light color indicates light chain peptide. It is also shown to be based on anti-CD 38VH1Fab and CD28sup/CD3midCD38 of crystal structure of CODDVFabVH1/CD28supxCD3midStructural model of trispecific antibody (right).

Figures 28A and 28B show that Multiple Myeloma (MM) cells with high (RPMI-8226; figure 28A) and low (KMS-11; figure 28B) CD38 surface expression were efficiently lysed by human PBMCs incubated with trispecific abs at various concentrations (E: T ═ 10: 1). The contribution of each binding site to killing activity was demonstrated by binding site KO mutation, as shown.

FIGS. 29A-29C show anti-CD 28 of trispecific AbsupKO mutant in vitro against CD38high、CD38midAnd CD38lowMM cells showed significantly reduced antitumor activity. Assays using RPMI-8226 (FIG. 29A), U266 (FIG. 29B) or KMS-11 (FIG. 29C) cells are shown.

FIG. 30 shows CD38VH1/CD28supxCD3mid_The reduction of tumor burden in the FALA trispecific antibody treated group was dose dependent and statistically different in a disseminated human multiple myeloma cell line model using NSG mice reconstituted with in vitro expanded human primary T cells.

Fig. 31A and 31B show important microscopic analysis of myeloma cell lysis by CD38 trispecific abs in vitro in the presence of primary human T cells. Use and use of CellTrackerTMDark red dye-labeled RPMI-8226 myeloma cell lines human PBMCs incubated with negative controls (triple KO trispecificity; fig. 31A) or CD38/CD28xCD3 trispecificity Ab (fig. 31B) were used for time-lapse photography of microscopic images. The images presented were collected after 24 hours of incubation. Scale bar: 50 μm.

Figure 32 shows substitution mutations in the Fc region of IgG4 prepared for analysis in an Fc receptor binding assay. The sequences shown in SEQ ID NO: 111-116 (from top to bottom, respectively).

Figure 33 shows the results of SPR assays used to measure the affinity of a given IgG4 Fc variant to the indicated human Fc receptor.

Figure 34 shows that CD38 trispecific binding protein with minimal FcR binding reduces nonspecific cytokine release from human PBMC in vitro. Different FcR inactivating mutations (as shown) were analyzed for pro-inflammatory effects on the human IgG4 isotype. Human PBMCs were incubated in culture medium (unstimulated) or in the presence of myeloma cells RPMI-8226 (stimulated), and the bar graphs represent supernatant IFN- γ levels measured by ELISA.

Figure 35 shows that CD38 trispecific binding proteins with minimal FcR binding cleaved human multiple myeloma cells with different levels of CD38 expression. Cytolysis of myeloma cells with IgG4 or the indicated Fc mutations was assessed in vitro using human PBMCs with the indicated tumor targets.

FIG. 36 shows a comparison of the in vitro cytolytic activity of the trispecific anti-CD 38/CD28/CD3Ab and anti-CD 38 antibody daratumab, measured using human PBMC as effector cells, against cell lines RPMI-8226, U266 and KMS-11 (E: T ═ 10: 1).

FIGS. 37A-37D show the characterization of in vitro T cell subset expansion in response to CD38/CD3xCD 28. Assessment of expansion of T cell subsets was performed by coating wells with 350 ng/well of CD38 trispecific Ab in the absence of exogenous cytokines. T cell populations were measured at the indicated time points. Triple mutant trispecific ab was used as negative control. Flow cytometry was used to determine central and effector memory CD4T cells (fig. 37A), helper T cells (fig. 37B), central and effector memory CD8T cells (fig. 37C), and CMV pp 65-specific CD8 cells (fig. 37D) as described in example 12. CMV-specific pp65 effector cells were analyzed by pentameric staining of PBMC from HLA-A2CMV + donors treated with CD38 trispecificity or triple negative controls.

Figure 38 shows the contribution of CD28 expression on target cells to the susceptibility of cytolysis by CD38/CD3xCD 28. CD28 was knocked out in KMS-11 cells using CRISPR/Cas 9 gene targeting and used as a cytolytic target in vitro. CD38 expression was retained compared to the parent KMS-11, whereas CD28 was eliminated as confirmed by flow cytometry (top panel, KMS-11 vs KMS-11(CD28 KO)). Cytolysis of CD28KO cells was checked trispecifically with WT or CD28 null (lower panel; trispecificity vs trispecificity (CD28 KO)).

FIG. 39 shows the CD38/CD28xCD3 trispecific FALA mutant Ab vs indicated CD38+CD28-Cytolytic activity of a line, said CD38+CD28-The lineages include acute myeloid leukemia (AML (KG-1)), B-cell lymphoma (OCI-Ly19), acute T-lymphocytic leukemia (ALL (KOPN8)) and chronic lymphocytic lymphoma (CLL (Z-138)).

Figure 40 shows that in vitro activation of human PBMCs by α -CD28 superagonists requires bivalent antibody.

Detailed Description

The present disclosure provides binding proteins comprising at least one antigen binding site that binds to a CD38 polypeptide.

I. General definitions

As used in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings. Unless the context requires otherwise, singular terms shall include the plural and plural terms shall include the singular. As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a molecule" optionally includes combinations of two or more such molecules, and the like.

It is to be understood that the aspects and embodiments of the present disclosure described herein include "comprising" aspects and embodiments, "consisting of (aspects and embodiments), and" consisting essentially of (aspects and embodiments).

The term "polynucleotide" as used herein refers to a single-or double-stranded nucleic acid polymer of at least 10 nucleotides in length. In certain embodiments, the nucleotides comprising the polynucleotide may be ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide. Such modifications include base modifications, such as bromouridine; ribose modifications such as arabinoside and 2 ', 3' -dideoxyribose; and internucleotide linkage modifications such as phosphorothioate (phosphorothioate), phosphorodithioate (phosphorodithioate), phosphoroselenoate (phosphoroselenoate), phosphorodiselenoate (phosphorodiselenoate), phosphoroanilothioate, phosphoranilidate, and phosphoroamidate (phosphoroamidate). The term "polynucleotide" specifically includes DNA in single-and double-stranded form.

An "isolated polynucleotide" is a polynucleotide of genomic, cDNA, or synthetic origin, or some combination thereof, which: (1) not associated with all or part of a polynucleotide, wherein the isolated polynucleotide is found in nature, (2) associated with a polynucleotide that is not associated with nature, or (3) not occurring in nature as part of a larger sequence.

An "isolated polypeptide" is such that: (1) free of at least some other polypeptides that would normally be found, (2) substantially free of other polypeptides from the same source (e.g., from the same species), (3) expressed by cells from a different species, (4) has been isolated from at least about 50% of the polynucleotide, lipid, carbohydrate, or other material with which it is naturally associated, (5) is not associated with a portion of a polypeptide with which the "isolated polypeptide" is naturally associated (by covalent or non-covalent interactions), (6) is operably associated with a polypeptide with which it is not naturally associated (by covalent or non-covalent interactions), or (7) is not present in nature. Such isolated polypeptides may be encoded by genomic DNA, cDNA, mRNA, or other RNA of synthetic origin, or any combination thereof. Preferably, an isolated polypeptide is substantially free of polypeptides or other contaminants found in its natural environment that would interfere with its use (therapeutic, diagnostic, prophylactic, research or otherwise).

Naturally occurring antibodies typically comprise tetramers. Each such tetramer is typically composed of two identical pairs of polypeptide chains, each pair having one full-length "light chain" (typically having a molecular weight of about 25 kDa) and one full-length "heavy chain" (typically having a molecular weight of about 50-70 kDa). The terms "heavy chain" and "light chain" as used herein refer to any immunoglobulin polypeptide having sufficient variable domain sequence to confer specificity to a target antigen. The amino-terminal portion of each light and heavy chain typically includes a variable domain of about 100 to 110 or more amino acids, which is typically responsible for antigen recognition. The carboxy-terminal portion of each chain typically defines a constant domain responsible for effector function. Thus, in naturally occurring antibodies, a full-length heavy chain immunoglobulin polypeptide comprises a variable domain (V) H) And 3 constant domains (C)H1、CH2And CH3) In which V isHThe domains are located at the amino terminus and C of the polypeptideH3The domains are carboxy-terminal and the full-length light chain immunoglobulin polypeptide includes a variable domain (V)L) And constant Domain (C)L) In which V isLThe domains being located in polypeptidesAmino terminal and CLThe domain is located at the carboxy terminus.

Human light chains are generally classified as kappa and lambda light chains, and human heavy chains are generally classified as mu, gamma, α, or, and the isotype of the antibody is defined as IgM, IgD, IgG, IgA, and ige, respectively, of several subclasses, including but not limited to IgG1, IgG2, IgG3, and IgG 4. IgM of subclasses, including but not limited to IgM1 and IgM 2. IgA are similarly subdivided into subclasses, including but not limited to IgA1 and IgA 2. within full-length light and heavy chains, the variable and constant domains are typically joined by a "J" region of about 12 or more amino acids, and the heavy chains also include a "D" region of about 10 or more amino acidsUNDAMENTALIMMUNOLOGY(Paul, W., ed., Raven Press, 2nd ed., 1989), which is incorporated by reference in its entirety for all purposes. The variable region of each light/heavy chain pair typically forms an antigen binding site. The variable domains of naturally occurring antibodies typically exhibit the same general structure of relatively conserved Framework Regions (FRs), also referred to as complementarity determining regions or CDRs, joined by three hypervariable regions. The CDRs from the two chains of each pair are typically aligned by framework regions, which may enable binding of a particular epitope. From amino-terminus to carboxy-terminus, light and heavy chain variable domains typically comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR 4.

The term "set of CDRs" refers to a set of three CDRs present in a single variable region capable of binding antigen. The exact boundaries of these CDRs have been defined differently depending on the system. System described by Kabat (Kabat et al., S)EQUENCESOF PROTEINSoF IMMUNOLOGICALINTEREST(National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides a clear residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries defining the three CDRs. These CDRs may be referred to as Kabat CDRs. Chothia and colleagues (Chothia and Lesk, 1987, J.mol.biol.196: 901-17; Chothia et al, 1989, Nature 342: 877-83) found that despite great diversity at the amino acid sequence level, certain sub-portions of the Kabat CDRs were employed with almost no loss of diversityThese subsections are designated as L1, L2 and L3, or H1, H2 and H3, where "L" and "H" represent light and heavy chain regions, respectively, these regions may be referred to as Chothia CDRs having boundaries that overlap with Kabat CDRs the other boundaries defining CDRs that overlap with Kabat CDRs are described by Padlan, 1995, fasebj.9: 133-39; callum, 1996, j.mol.biol.262 (5): 732-45; and Lefranc, 2003, dev.com.immunol.27: 55-77. other CDR boundary definitions may not strictly follow one of the systems herein but still overlap with Kabat CDRs, although they may not significantly affect antigen binding despite the predicted or experimental results according to a particular residue or residue set or even the entire CDR, they may be shortened or lengthened, although they may not significantly affect antigen binding, the methods used herein may utilize CDRs defined according to any of these systems, although certain embodiments use the predicted amino acid sequences of the CDR sequences identified by the conventional numbering scheme or other CDR sequences such as the amino acid sequences identified by the light chain variable region mapping procedures described by the eye strain seq id-seq id, seq id No. 20.

In some embodiments, CDR/FR definitions in an immunoglobulin light or heavy chain are determined based on IMGT definitions (Lefranc et al. Dev. Comp. Immunol., 2003, 27 (1): 55-77; www.imgt.org).

The term "Fc" as used herein refers to a molecule that comprises, in monomeric or multimeric form, the sequence of a non-antigen-binding fragment produced by antibody digestion or otherwise, and may comprise a hinge region. The original immunoglobulin source of the native Fc is preferably of human origin and may be any immunoglobulin. Fc molecules consist of monomeric polypeptides that can be joined into dimeric or multimeric forms by covalent (i.e., disulfide) and non-covalent bonding. The number of intermolecular disulfide bonds between the monomeric subunits of a native Fc molecule ranges from 1 to 4, depending on the class (e.g., IgG, IgA, and IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, IgGA2, and IgG 4). An example of an Fc is a disulfide-bonded dimer resulting from papain digestion of IgG. The term "native Fc" as used herein is generic to monomeric, dimeric and multimeric forms.

F (ab) fragments generally include one light chain and one heavy chain V HAnd CH1Domain wherein V of the F (ab) fragmentH-CH1The heavy chain portion cannot form a disulfide bond with another heavy chain polypeptide. As used herein, a F (ab) fragment may also include a light chain comprising two variable domains separated by an amino acid linker and a light chain comprising two variable domains and C separated by an amino acid linkerH1Heavy chain of domain.

F (ab') fragments typically comprise a light chain and a portion of a heavy chain, which contain more constant regions (at C)H1And CH2Between domains) so that interchain disulfide bonds can be formed between the two heavy chains to form F (ab')2A molecule.

The term "binding protein" as used herein refers to a non-naturally occurring (or recombinant or engineered) molecule that specifically binds at least one target antigen, e.g., a CD38 polypeptide of the present disclosure.

A "recombinant" molecule is one that is prepared, expressed, produced, or isolated by recombinant means.

One embodiment of the present disclosure provides binding proteins having biological and immunological specificity for 1 to 3 target antigens. Another embodiment of the present disclosure provides a nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide chain forming such a binding protein. Another embodiment of the present disclosure provides an expression vector comprising a nucleic acid molecule comprising a polypeptide chain encoding such a binding protein. Yet another embodiment of the present disclosure provides a host cell (i.e., a nucleic acid molecule or vector comprising a polypeptide chain encoding such a binding protein) that expresses such a binding protein.

The term "interchangeability" as used herein refers to the interchangeability of variable domains within binding protein formats and having retained folding and ultimate binding affinity. "completely interchangeable" means that V is exchanged in a polypeptide chain of formula I or a polypeptide chain of formula IIH1And VH2Order of the domains and thus exchange VL1And VL2The order of the domains (i.e., reversing the order) while retaining the ability to bind the full function of the protein as demonstrated by binding affinity. Further, it should be noted that the name VHAnd VLOnly the domain positions on the particular protein chain in the final form. For example, VH1And VH2May be derived from V in a parent antibodyL1And VL2Domain, juxtaposed in a binding proteinH1And VH2Location. Likewise, VL1And VL2May be derived from V in a parent antibodyH1And VH2Domain, juxtaposed in a binding proteinH1And VH2Location. Thus, VnAnd VLThe name refers to the current position rather than the original position in the parent antibody. Thus, VHAnd VLThe domains are "swappable".

The term "antigen" or "target antigen" or "antigen target" as used herein refers to a molecule or a portion of a molecule that is capable of being bound by a binding protein and, additionally, is capable of being used in an animal to produce an antibody that is capable of binding to an epitope of the antigen. The target antigen may have one or more epitopes. For each target antigen recognized by a binding protein, the binding protein is able to compete with the intact antibody that recognizes the target antigen.

"CD 38" is a cluster of differentiation 38 polypeptide and is a glycoprotein found on the surface of many immune cells. In some embodiments, the binding proteins of the present disclosure bind to the extracellular domain of one or more CD38 polypeptides. Exemplary CD38 ectodomain polypeptide sequences include, but are not limited to, the ectodomain of human CD38 (e.g., as set forth in SEQ ID NO: 1) and the ectodomain of cynomolgus monkey CD38 (e.g., as set forth in SEQ ID NO: 30).

The term "T cell conjugate" refers to a binding protein directed against the cytotoxic activity of the host immune system, more specifically T cells, as well as to a tumor target protein.

The term "monospecific binding protein" refers to a binding protein that specifically binds to one antigen target.

The term "monovalent binding protein" refers to a binding protein having one antigen binding site.

The term "bispecific binding protein" refers to a binding protein that specifically binds to two different antigen targets. In some embodiments, a bispecific binding protein binds two different antigens. In some embodiments, bispecific binding proteins bind to two different epitopes on the same antigen.

The term "bivalent binding protein" refers to a binding protein having two binding sites.

The term "trispecific binding protein" refers to a binding protein that specifically binds three different antigen targets. In some embodiments, the trispecific binding protein binds three different antigens. In some embodiments, the trispecific binding protein binds to one, two or three different epitopes on the same antigen.

The term "trivalent binding protein" refers to a binding protein having three binding sites. In particular embodiments, a trivalent binding protein may bind to one antigen target. In other embodiments, a trivalent binding protein may bind two antigen targets. In other embodiments, a trivalent binding protein may bind three antigen targets.

An "isolated" binding protein is a protein that has been identified and isolated and/or recovered from a component of its natural environment. Contaminating components of their natural environment are materials that would interfere with diagnostic or therapeutic uses of the binding protein, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In some embodiments, the binding protein will be purified: (1) greater than 95% by weight of the antibody, and most preferably greater than 99% by weight, as determined by the Lowry method, (2) by using a rotary cup sequencer sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence, or (3) by SDS-PAGE under reducing or non-reducing conditions using coomassie blue or preferably silver staining to homogeneity. Isolated binding proteins include binding proteins in situ within recombinant cells, as at least one component of the natural environment of the binding protein is not present.

The term "substantially pure" or "substantially purified" as used herein refers to a compound or substance that is the predominant substance present (i.e., it is more abundant on a molar basis than any other individual substance in the composition). In some embodiments, the substantially purified fraction is a composition wherein the substance comprises at least about 50% (on a molar basis) of all macromolecular substances present. In other embodiments, a substantially pure composition will comprise greater than about 80%, 85%, 90%, 95%, or 99% of all macromolecular species present in the composition. In other embodiments, the substance is purified to be substantially homogeneous (contaminant substances cannot be detected in the composition by conventional detection methods), wherein the composition consists essentially of a single macromolecular substance.

The term "epitope" includes any determinant, preferably a polypeptide determinant, capable of specifically binding to an immunoglobulin or T cell receptor. In certain embodiments, epitope determinants include chemically active surface groups of a molecule, such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in certain embodiments, may have specific three-dimensional structural characteristics and/or specific charge characteristics. An epitope is a region of an antigen or antigen to which a binding protein binds. In certain embodiments, a binding protein is said to specifically bind an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and/or macromolecules. In some embodiments, when the equilibrium dissociation constant ≦ 10 -8When M is larger, more preferably, the equilibrium dissociation constant is 10 or less-9M, and most preferably when the dissociation constant is ≦ 10-10M, the binding protein is said to specifically bind to the antigen.

Dissociation constant (K) of binding proteinsD) Can be determined, for example, by surface plasmon resonance. Generally, watchesSurface plasmon resonance analysis real-time binding interactions between ligands (target antigens on a Biosensor matrix) and analytes (binding proteins in solution) were measured by Surface Plasmon Resonance (SPR) using the BIAcore system (Pharmacia Biosensor; Piscataway, NJ). Surface plasmon analysis can also be performed by immobilizing the analyte (binding protein on the biosensor matrix) and presenting the ligand (target antigen). The term "K" as used hereinD"refers to the dissociation constant of the interaction between a particular binding protein and a target antigen.

The term "binding" as used herein in reference to a binding protein refers to the ability of the binding protein or antigen binding fragment thereof to bind to an epitope-containing antigen with a Kd of at least about 1x10-6M、1x10-7M、1x10-8M、1x10-9M、1x10-10M、1x10- 11M、1x10-12M or higher, and/or binds to an epitope with an affinity that is at least two times greater than its affinity for a non-specific antigen. In some embodiments, the binding proteins of the present disclosure bind to two or more antigens, such as human and cynomolgus monkey CD38 polypeptides.

In some embodiments, the antigen binding domains and/or binding proteins of the present disclosure are "cross-reactive" with human and cynomolgus monkey CD38 polypeptides (such as CD38 extracellular domain, such as SEQ ID NO: 1 (human CD38 isoform a), SEQ ID NO: 105 (human CD38 isoform E), and SEQ ID NO: 30 (cynomolgus monkey CD 38)). Binding proteins that bind to antigen 1(Ag1) "cross-react" with antigen 2(Ag2) when EC50 is within a similar range for both antigens. In the present application, when the ratio of the affinity of Ag2 to the affinity of Ag1 is equal to or less than 10 (e.g., 5, 2, 1, or 0.5), the binding protein that binds to Ag1 cross-reacts with Ag2, the affinities being detected in the same way for both antigens.

Binding proteins that bind to Ag1 "do not cross-react significantly" with Ag2 when the affinities of the two antigens are very different. If the binding reaction is too low, the affinity of Ag2 may not be measurable. In this application, the binding protein that binds to Ag1 does not significantly cross-react with Ag2 when the binding reaction of the binding protein with Ag2 is less than 5% of the binding reaction of the same binding protein with Ag1 in the same experimental setting and the same antibody concentration. In practice, the concentration of binding protein used may be EC50 or the concentration required to reach the saturation plateau obtained with Ag 1.

The precise location of a domain transition can be determined by locating peptide segments that do not form secondary structural elements (such as β -sheets or α -helices), as evidenced by empirical data, or can be hypothesized by modeling techniques or secondary structure predictions1At V, it is located atL2C terminal and VL1On the light chain between the N-termini of the domains; and L2At V, it is located atL1C terminal and CLOn the light chain between the N-termini of the domains. The heavy chain linker is designated L3At V, it is located atH1C terminal and VH2Between the N-termini of the domains; and L4At V, it is located atH2C terminal and CH1Between the N-termini of the domains.

The term "vector" as used herein refers to any molecule (e.g., nucleic acid, plasmid, or virus) used to transfer encoded information to a host cell. The term "vector" includes a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA molecule into which additional DNA segments can be inserted. Another type of vector is a viral vector, wherein additional DNA segments can be inserted into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. In addition, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. The terms "plasmid" and "vector" are used interchangeably herein, as plasmids are the most commonly used form of vector. However, the present disclosure is intended to include other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses, and adeno-associated viruses), which serve equivalent functions.

The phrase "recombinant host cell" (or "host cell"), as used herein, refers to a cell into which a recombinant expression vector has been introduced. Recombinant host cells or host cells refer not only to the particular subject cell but also to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but such cells are still included within the scope of the term "host cell" as used herein. A variety of host cell expression systems are available for expression of binding proteins, including bacterial, yeast, baculovirus and mammalian expression systems (as well as phage display expression systems). An example of a suitable bacterial expression vector is pUC 19. For recombinant expression of the binding protein, a host cell is transformed or transfected with one or more recombinant expression vectors carrying DNA segments encoding polypeptide chains of the binding protein, whereby the polypeptide chains are expressed in the host cell and preferably secreted into the medium in which the host cell is cultured, from which the binding protein can be recovered.

The term "transformation" as used herein refers to a change in the genetic characteristics of a cell and has been transformed when the cell is modified to contain new DNA. For example, cells are transformed in which genetic modifications are made from their native state. After transformation, the transforming DNA may recombine with the DNA of the cell by physically integrating into the chromosome of the cell, or may be transiently maintained without being replicated as an episomal element, or may replicate independently as a plasmid. When the DNA replicates with cell division, the cell is considered to have been stably transformed. The term "transfection" as used herein refers to the uptake of foreign or exogenous DNA by a cell, and when exogenous DNA has been introduced into the cell membrane, the cell has been "transfected". Many transfection techniques are known in the art. Such techniques may be used to introduce one or more exogenous DNA molecules into a suitable host cell.

The term "naturally occurring" as used herein and applied to an object refers to the fact that an object can be found in nature and is not manipulated by a person. For example, a polynucleotide or polypeptide present in an organism (including viruses) that can be isolated from a natural source and not intentionally modified by man is naturally occurring. Similarly, as used herein, "non-naturally occurring" refers to an object that is not found in nature or that has been structurally modified or synthesized by humans.

As used herein, the twenty conventional amino acids and their abbreviations follow conventional usage. Stereoisomers of twenty conventional amino acids (e.g., D-amino acids); unnatural amino acids and analogs, such as α -, α -disubstituted amino acids, N-alkyl amino acids, lactic acid, and other non-conventional amino acids can also be suitable components of binding protein polypeptide chains. Examples of unconventional amino acids include: 4-hydroxyproline, gamma-carboxyglutamic acid, g-N, N, N-trimethyllysine, -N-acetyl lysine, O-phosphoserine, N-acetyl serine, N-formyl methionine, 3-methylhistidine, 5-hydroxylysine, sigma-N-methyl arginine and other similar amino acids and imino acids (e.g., 4-hydroxyproline). As used herein, in polypeptide notation, the left-hand direction is the amino-terminal direction and the right-hand direction is the carboxy-terminal direction, according to standard usage and convention.

Naturally occurring residues can be classified into the following categories based on common side chain properties:

(1) hydrophobicity: met, Ala, Val, Leu, Ile, Phe, Trp, Tyr, Pro;

(2) polar hydrophilicity: arg, Asn, Asp, Gln, Glu, His, Lys, Ser, Thr;

(3) aliphatic: ala, Gly, Ile, Leu, Val, Pro;

(4) aliphatic hydrophobicity: ala, Ile, Leu, Val, Pro;

(5) neutral hydrophilicity: cys, Ser, Thr, Asn, Gln;

(6) acidity: asp and Glu;

(7) alkalinity: his, Lys, Arg;

(8) residues that influence chain orientation: gly, Pro;

(9) aromatic: his, Trp, Tyr, Phe; and

(10) aromatic hydrophobicity: phe, Trp, Tyr.

Conservative amino acid substitutions may involve exchanging a member of one of these classes for another member of the same class. Non-conservative substitutions may involve swapping members of one of these classes for members of another class.

One skilled in the art will be able to determine suitable variants of the polypeptide chains of the binding protein using well known techniques. For example, one skilled in the art can identify suitable regions of a polypeptide chain that can be altered without disrupting activity by targeting regions not considered important for activity. Alternatively, one skilled in the art can identify residues and portions of the molecule that are conserved among similar polypeptides. In addition, even regions that may be important to biological activity or structure may be conservatively substituted without destroying biological activity or adversely affecting polypeptide structure.

The term "patient" as used herein includes human and animal subjects (e.g., mammals such as dogs, pigs, horses, cats, cows, etc.).

The terms "treatment" or "treating" as used herein refer to both therapeutic treatment and prophylactic or preventative measures. Patients in need of treatment include patients with the disorder as well as patients susceptible to the disorder or patients in whom the disorder is to be prevented. In particular embodiments, the binding proteins can be used to treat a human suffering from cancer, or a human predisposed to cancer, or to ameliorate cancer in a human subject. Binding proteins may also be used to prevent cancer in human patients. In particular embodiments, the cancer is multiple myeloma, acute lymphoblastic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, lymphoma, breast cancer such as Her2+ breast cancer, prostate cancer, germinal center B cell lymphoma, or B cell acute lymphoblastic leukemia.

The term "pharmaceutical composition" or "therapeutic composition" as used herein refers to a compound or composition that is capable of inducing a desired therapeutic effect when properly administered to a patient.

The term "pharmaceutically acceptable carrier" or "physiologically acceptable carrier" as used herein refers to one or more formulation materials suitable for effecting or enhancing delivery of a binding protein.

The terms "effective amount" and "therapeutically effective amount" when used in reference to a pharmaceutical composition comprising one or more binding proteins, refer to an amount or dose sufficient to produce the desired therapeutic result. More specifically, a therapeutically effective amount is an amount of binding protein sufficient to inhibit over a period of time one or more clinically defined pathological processes associated with the condition being treated. The effective amount may vary depending on the specific binding protein used and also on a variety of factors and conditions associated with the patient being treated and the severity of the disorder. For example, if the binding protein is to be administered in vivo, factors obtained in preclinical animal work, such as age, weight, and health of the patient, as well as dose response curves and toxicity data, may be one of the factors considered. Determining an effective or therapeutically effective amount of a given pharmaceutical composition is well within the ability of those skilled in the art.

One embodiment of the present disclosure provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of a binding protein.

anti-CD 38 binding proteins

Certain aspects of the present disclosure relate to binding proteins comprising an antigen binding site that binds to a CD38 polypeptide (e.g., human and cynomolgus monkey CD38 polypeptides). In some embodiments, the binding protein is monospecific and/or monovalent, bispecific and/or bivalent, trispecific and/or trivalent or multispecific and/or multivalent.

Various features of exemplary monospecific, bispecific, or trispecific binding proteins are described herein. For example, in some embodiments, the binding protein or antigen-binding fragment thereof cross-reacts with human CD38 (e.g., human CD38 isoform a and/or isoform E polypeptides) and cynomolgus monkey CD 38. In some embodiments, the binding protein induces apoptosis of CD38+ cells. In some embodiments, the binding protein recruits T cells to CD38+ cells and optionally activates T cells (e.g., via TCR stimulation and/or co-stimulation).

In some embodiments, the binding protein comprises an antigen binding site comprising: an antibody heavy chain Variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO: 31) or GYTFTSYA (SEQ ID NO: 37), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO: 32) or IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33); or an antibody light chain Variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO: 34) or QSVSSYGQGF (SEQ ID NO: 39), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO: 35) or GAS (SEQ ID NO: 40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36). In some embodiments, the binding protein comprises an antigen binding site comprising: an antibody heavy chain Variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO: 31) or GYTFTSYA (SEQ ID NO: 37), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO: 32) or IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33); or an antibody light chain Variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO: 34) or QSVSSYGQG (SEQ ID NO: 132), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO: 35) or GAS (SEQ ID NO: 40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36). In some embodiments, the binding protein comprises 1, 2, 3, 4, 5, or 6 CDRs of an antibody VH and/or VL domain sequence from mAb1, mAb2, mAb3, mAb4, mAb5, mAb6, mAb2xCD28supxCD3midIgG4 FALA, mAb2xCD28supxCD3mid IgG1LALA P329A, mAb2xCD28supxCD3mid IgG1NNSA, mAb6xCD28supxCD3mid IgG4 FALA, mAb6xCD28supxCD3mid IgG1LALA P329A, or mAb6xCD28supxCD3mid IgG1NNSA, as shown in table G, H or I.

In some embodiments, the binding protein comprises an antigen binding site comprising: an antibody heavy chain Variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO: 31) or GYTFTSYA (SEQ ID NO: 37), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO: 32) or IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33); and an antibody light chain Variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO: 34) or QSVSSYGQGF (SEQ ID NO: 39), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO: 35) or GAS (SEQ ID NO: 40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36).

In some embodiments, the binding protein comprises an antigen binding site comprising: an antibody heavy chain Variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO: 31), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO: 32), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33); or an antibody light chain Variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO: 34), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO: 35), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36). In some embodiments, the binding protein comprises an antigen binding site comprising: an antibody heavy chain Variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO: 31), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO: 32), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33); and an antibody light chain Variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO: 34), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO: 35), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36). In other embodiments, the binding protein comprises an antigen binding site comprising: an antibody heavy chain Variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYA (SEQ ID NO: 37), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33); or an antibody light chain Variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSVSSYGQGF (SEQ ID NO: 39), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO: 40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36). In some embodiments, the binding protein comprises an antigen binding site comprising: an antibody heavy chain Variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYA (SEQ ID NO: 37), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33); and an antibody light chain Variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSVSSYGQGF (SEQ ID NO: 39), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO: 40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36).

In some embodiments, the VH domain comprises, from N-terminus to C-terminus, the sequence FR1-CDR-H1-FR2-CDR-H2-FR3-CDR-H3-FR 4; wherein FR1 comprises sequence QVQLVQSGAEVVKPGASVKVSCKAS (SEQ ID NO: 86), QVQLVQSGAEVVKSGASVKVSCKAS (SEQ ID NO: 87) or QVQLVQSGAEVVKPGASVKMSCKAS (SEQ ID NO: 88); wherein FR2 comprises sequence MHWVKEAPGQRLEWIGY (SEQ ID NO: 90) or MHWVKEAPGQGLEWIGY (SEQ ID NO: 91); wherein FR3 comprises sequence NYNQKFQGRATLTADTSASTAYMELSSLRSEDTAVYFC (SEQ ID NO: 93) or NYNQKFQGRATLTADTSASTAYMEISSLRSEDTAVYFC (SEQ ID NO: 94); and wherein FR4 comprises the sequence WGQGTLVTVSS (SEQ ID NO: 96). In some embodiments, the VL domain comprises, from N-terminus to C-terminus, the sequence FR1-CDR-L1-FR2-CDR-L2-FR3-CDR-L3-FR 4; wherein FR1 comprises sequence DIVLTQSPATLSLSPGERATISCRAS (SEQ ID NO: 97); wherein FR2 comprises sequence MHWYQQKPGQPPRLLIY (SEQ ID NO: 99); wherein FR3 comprises sequence SRATGIPARFSGSGSGTDFTLTISPLEPEDFAVYYC (SEQ ID NO: 101); wherein FR4 comprises sequence FGGGTKLEIK (SEQ ID NO: 103).

In some embodiments, the VH domain comprises a VH sequence identical to SEQ ID NO: 5, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical; and/or the VL domain comprises a sequence identical to SEQ ID NO: 6, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical. In some embodiments, the VH domain comprises a VH sequence identical to seq id NO: 17, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical; and/or the VL domain comprises a sequence identical to SEQ ID NO: 18, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical. In some embodiments, the VH domain comprises a VH sequence identical to SEQ ID NO: 21, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical; and/or the VL domain comprises a sequence identical to SEQ ID NO: 18, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical. In some embodiments, the VH domain comprises a VH domain identical to SEQ id no: 23, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical; and/or the VL domain comprises a sequence identical to SEQ ID NO: 18, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical. In some embodiments, the VH domain comprises a VH sequence identical to SEQ ID NO: 13, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical; and/or the VL domain comprises a sequence identical to SEQ ID NO: 14, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical.

In some embodiments, the VH domain comprises a VH sequence identical to SEQ ID NO: 5, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical; and the VL domain comprises a sequence identical to SEQ ID NO: 6, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical. In some embodiments, the VH domain comprises a VH sequence identical to seq id NO: 17, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical; and the VL domain comprises a sequence identical to SEQ ID NO: 18, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical. In some embodiments, the VH domain comprises a VH sequence identical to SEQ ID NO: 21, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical; and the VL domain comprises a sequence identical to SEQ ID NO: 18, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical. In some embodiments, the VH domain comprises a VH domain identical to SEQ id no: 23, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical; and the VL domain comprises a sequence identical to SEQ ID NO: 18, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical. In some embodiments, the VH domain comprises a VH sequence identical to SEQ ID NO: 13, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical; and the VL domain comprises a sequence identical to SEQ ID NO: 14, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical.

In some embodiments, the VH domain comprises SEQ ID NO: 5; and the VL domain comprises SEQ ID NO: 6. In some embodiments, the VH domain comprises SEQ ID NO: 17; and the VL domain comprises SEQ ID NO: 18. In some embodiments, the VH domain comprises SEQ id no: 21; and the VL domain comprises SEQ ID NO: 18. In some embodiments, the VH domain comprises SEQ ID NO: 23; and the VL domain comprises SEQ ID NO: 18. In some embodiments, the VH domain comprises SEQ ID NO: 13; and the VL domain comprises SEQ ID NO: 14.

In some embodiments, a binding protein of the present disclosure comprises a polypeptide comprising SEQ ID NO: 7 and/or an antibody heavy chain comprising the amino acid sequence of SEQ ID NO: 8, or a light chain antibody. In some embodiments, a binding protein of the present disclosure comprises a polypeptide comprising SEQ ID NO: 19 and/or an antibody heavy chain comprising the amino acid sequence of SEQ ID NO: 20, or an antibody light chain. In some embodiments, a binding protein of the present disclosure comprises a polypeptide comprising SEQ ID NO: 22 and/or an antibody heavy chain comprising the amino acid sequence of SEQ ID NO: 20, or an antibody light chain. In some embodiments, a binding protein of the present disclosure comprises a polypeptide comprising SEQ ID NO: 24 and/or an antibody heavy chain comprising the amino acid sequence of SEQ ID NO: 20, or an antibody light chain. In some embodiments, a binding protein of the present disclosure comprises a polypeptide comprising SEQ ID NO: 15 and/or an antibody heavy chain comprising the amino acid sequence of SEQ ID NO: 16, or a light chain antibody.

In some embodiments, a binding protein of the present disclosure comprises a polypeptide comprising SEQ ID NO: 7 and an antibody heavy chain comprising the amino acid sequence of SEQ ID NO: 8, or a light chain antibody. In some embodiments, a binding protein of the present disclosure comprises a polypeptide comprising SEQ ID NO: 19 and an antibody heavy chain comprising the amino acid sequence of SEQ ID NO: 20, or an antibody light chain. In some embodiments, a binding protein of the present disclosure comprises a polypeptide comprising SEQ ID NO: 22 and an antibody heavy chain comprising the amino acid sequence of SEQ ID NO: 20, or an antibody light chain. In some embodiments, a binding protein of the present disclosure comprises a polypeptide comprising SEQ ID NO: 24 and an antibody heavy chain comprising the amino acid sequence of SEQ ID NO: 20, or an antibody light chain. In some embodiments, a binding protein of the present disclosure comprises a polypeptide comprising SEQ ID NO: 15 and an antibody heavy chain comprising the amino acid sequence of SEQ ID NO: 16, or a light chain antibody.

In some embodiments, the binding protein comprises an antigen binding site comprising: an antibody heavy chain Variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO: 41), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO: 42), and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO: 43); or an antibody light chain Variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QGIRND (SEQ ID NO: 44), a CDR-L2 sequence comprising the amino acid sequence of AAS (SEQ ID NO: 45), and a CDR-L3 sequence comprising the amino acid sequence of LQDYIYYPT (SEQ ID NO: 46). In some embodiments, the binding protein comprises an antigen binding site comprising: an antibody heavy chain Variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO: 41), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO: 42), and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO: 43); and an antibody light chain Variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QGIRND (SEQ ID NO: 44), a CDR-L2 sequence comprising the amino acid sequence of AAS (SEQ ID NO: 45), and a CDR-L3 sequence comprising the amino acid sequence of LQDYIYYPT (SEQ ID NO: 46).

In some embodiments, the VH domain comprises, from N-terminus to C-terminus, the sequence FR1-CDR-H1-FR2-CDR-H2-FR3-CDR-H3-FR 4; wherein FR1 comprises sequence QVQLVESGGGVVQPGRSLRLSCAAS (SEQ ID NO: 89); wherein FR2 comprises sequence MHWVRQAPGKGLEWVAV (SEQ ID NO: 92); wherein FR3 comprises sequence YYADSVKGRFTISGDNSKNTLYLQMNSLRAEDTAVYYC (SEQ ID NO: 95); and wherein FR4 comprises the sequence WGQGTLVTVSS (SEQ ID NO: 96). In some embodiments, the VL domain comprises, from N-terminus to C-terminus, the sequence FR1-CDR-L1-FR 2-CDR-L2-FR 3-CDR-L3-FR 4; wherein FR1 comprises sequence AIQMTQSPSSLSASVGDRVTITCRAS (SEQ ID NO: 98); wherein FR2 comprises sequence GWYQQKPGKAPKLLIY (SEQ ID NO: 100); wherein FR3 comprises sequence SLQSGVPSRFSGSGSGTDFTLTISGLQPEDSATYYC (SEQ ID NO: 102); and wherein FR4 comprises the sequence WGQGTLVTVSS (SEQ ID NO: 104).

In some embodiments, the VH domain comprises a VH sequence identical to SEQ ID NO: 9, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical; and/or the VL domain comprises a sequence identical to SEQ ID NO: 10, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical.

In some embodiments, the VH domain comprises a VH sequence identical to SEQ ID NO: 9, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical; and the VL domain comprises a sequence identical to SEQ ID NO: 10, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical. In some embodiments, the VH domain comprises seq id NO: 9; and the VL domain comprises SEQ ID NO: 10.

In some embodiments, a binding protein of the present disclosure comprises a polypeptide comprising SEQ ID NO: 11 or an antibody heavy chain comprising the amino acid sequence of SEQ ID NO: 12, or an antibody light chain. In some embodiments, a binding protein of the present disclosure comprises a polypeptide comprising SEQ ID NO: 11 and an antibody heavy chain comprising the amino acid sequence of SEQ ID NO: 12, or an antibody light chain.

In some embodiments, a binding protein of the present disclosure comprises 1, 2, 3, 4, 5, or 6 CDR sequences of an antibody sequence shown in table G. In some embodiments, a binding protein of the present disclosure comprises 1, 2, 3, 4, 5, or 6 CDR sequences, VH domain sequences, and/or VL domain sequences of an antibody sequence set forth in table H. In some embodiments, a binding protein of the invention comprises 1, 2, 3, 4, 5, or 6 CDR sequences, VH domain sequences, and/or VL domain sequences of an antibody sequence set forth in table I. In some embodiments, a binding protein of the invention comprises 1, 2, 3, or 4 polypeptide sequences shown in table I.

Table h variable domain sequences of anti-CD 38(mAb1-7) and other binding proteins.

Note that: the CDR sequences are bolded and underlined in the above amino acid sequences.

Table i full length sequence of binding proteins.

Figure BDA0002526587380000571

Figure BDA0002526587380000581

Figure BDA0002526587380000641

Figure BDA0002526587380000651

Figure BDA0002526587380000671

Figure BDA0002526587380000691

Figure BDA0002526587380000701

Table j. full-length polynucleotide sequences of binding proteins.

Figure BDA0002526587380000712

Figure BDA0002526587380000741

Figure BDA0002526587380000751

Figure BDA0002526587380000761

Figure BDA0002526587380000771

Figure BDA0002526587380000811

Figure BDA0002526587380000831

CD38 polypeptide

In some embodiments, a binding protein of the present disclosure comprises an antigen binding site that binds to the extracellular domain of a human CD38 polypeptide and the extracellular domain of a cynomolgus monkey CD38 polypeptide. Exemplary assays for determining whether an antigen binding site binds an antigen are described herein and are known in the art. In some embodiments, binding is determined by an ELISA assay, e.g., as described below. In some embodiments, binding is determined by SPR assays, e.g., as described below. In some embodiments, binding is determined by flow cytometry using cells expressing a CD38 polypeptide on their cell surface, e.g., as described below. See, for example, examples 1, 3 and 4.

In some embodiments, a binding protein of the present disclosure binds to a polypeptide comprising SEQ ID NO: 1 and/or 30 or a fragment thereof (e.g., as measured by ELISA or SPR). In some embodiments, a binding protein of the present disclosure binds a polypeptide or polypeptide comprising SEQ ID NO: 1 and/or 30.

In some embodiments, a binding protein of the present disclosure binds to a CD38 isoform a polypeptide (e.g., comprising the amino acid sequence of SEQ id no: 1). In some embodiments, a binding protein of the present disclosure binds to a CD38 isoform E polypeptide (e.g., comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO: 105, and does not comprise, consists of, or consists of the entire amino acid sequence of SEQ ID NO: 1). In some embodiments, a binding protein of the present disclosure binds to a CD38 isoform a polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 1) and a CD38 isoform E polypeptide (e.g., comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 105, and not comprising the entire amino acid sequence of SEQ ID NO: 1). Without wishing to be bound by theory, it is believed that binding to a CD38 isoform E polypeptide may be advantageous, for example, to target a binding protein of the disclosure to a cell expressing a CD38 isoform E polypeptide.

Human CD38 isoform A ectodomain polypeptide sequence

Human CD38 isoform E polypeptide sequence

Figure BDA0002526587380000922

In some embodiments, the extracellular domain of a human CD38 polypeptide comprises SEQ ID NO: 1. In some embodiments, the extracellular domain of the cynomolgus monkey CD38 polypeptide comprises SEQ ID NO: 30.

Cynomolgus monkey CD38 polypeptide sequence

Multispecific (e.g., bispecific, trispecific, or multispecific) binding proteins that bind to CD38 polypeptides

In some embodiments, the binding proteins of the present disclosure are bispecific binding proteins comprising an antigen-binding site that binds one or more CD38 polypeptides and a second antigen-binding site that binds a different target antigen. In some embodiments, the binding proteins of the present disclosure are bispecific binding proteins comprising an antigen-binding site that binds to one or more CD38 polypeptides and a second antigen-binding site that binds to one or more CD38 polypeptides.

In some embodiments, the binding proteins of the present disclosure are trispecific binding proteins comprising an antigen binding site that binds one or more CD38 polypeptides, a second antigen binding site, and a third antigen binding site. For example, in some embodiments, one of the antigen binding sites binds to one or more CD38 polypeptides (e.g., the extracellular domain of a human and/or cynomolgus monkey CD38 polypeptide), and one or both antigen binding sites bind to a T cell surface protein. In some embodiments, one of the antigen binding sites binds to one or more CD38 polypeptides (e.g., the extracellular domain of a human and/or cynomolgus monkey CD38 polypeptide), one antigen binding site binds to a human CD3 polypeptide, and one antigen binding site binds to a human CD28 polypeptide. Human CD3 and CD28 polypeptides are known in the art. Provided herein are amino acid sequences of exemplary and non-limiting antibody variable domains that bind to human CD3 and CD28 polypeptides.

In some embodiments, provided herein are binding proteins comprising three antigen binding sites, each antigen binding site binding to one or more target proteins. In some embodiments, at least one of the three antigen binding sites binds the extracellular domain of a human CD38 polypeptide and the extracellular domain of a cynomolgus monkey CD38 polypeptide. In some embodiments, the human CD38 polypeptide comprises seq id NO: 1, and/or the cynomolgus monkey CD38 polypeptide comprises the amino acid sequence of SEQ ID NO: 30. In some embodiments, the binding protein comprises an antigen binding site that binds to the extracellular domain of a human CD38 polypeptide and the extracellular domain of a cynomolgus monkey CD38 polypeptide and two antigen binding sites that each bind to a T cell surface protein (e.g., a human CD28 polypeptide and/or a human CD3 polypeptide).

In some embodiments, a binding protein of the present disclosure binds to one or more tumor target proteins (e.g., one or more CD38 polypeptides) and one or more T cell target proteins. In some embodiments, the binding protein is capable of binding two different epitopes on one tumor target protein (e.g., one or more CD38 polypeptides) and a single T cell target protein. In some embodiments, the binding protein is capable of binding one tumor target protein (e.g., one or more CD38 polypeptides) and two different T cell target proteins (e.g., CD28 and CD 3). In some embodiments, the binding protein is capable of binding two different epitopes on one T cell target protein and a single tumor target protein (e.g., one or more CD38 polypeptides). In some embodiments, the binding protein is capable of binding one T cell target protein and two different tumor target proteins (e.g., one or more CD38 polypeptides and another tumor target protein). In some embodiments, the first and second polypeptide chains of the binding protein form two antigen binding sites that target two T cell target proteins, and the third and fourth polypeptide chains of the binding protein form an antigen binding site that binds one or more CD38 polypeptides. In some embodiments, the first and second polypeptide chains of the binding protein form two antigen binding sites that target two tumor target proteins (e.g., one or more CD38 polypeptides and another tumor target protein), and the third and fourth polypeptide chains of the binding protein form an antigen binding site that binds a T cell target protein. In some embodiments, the one or more T cell target proteins are one or more of CD3 and CD 28.

In some embodiments, the binding protein specifically binds to one or more CD38 polypeptides and one or more target proteins on T cells, including T cell receptor complexes. These T cell engager binding proteins are capable of transiently recruiting T cells to target cells while activating cytolytic activity of the T cells. Examples of target proteins on T cells include, but are not limited to, CD3 and CD28, and the like. Other examples of such antigen targets or target proteins are provided above. In some embodiments, a trispecific binding protein may be produced by combining the antigen binding domains of two or more monospecific antibodies (parent antibodies) into one antibody.

Bispecific binding protein forms

In some embodiments, the binding proteins of the present disclosure are bispecific and/or bivalent binding proteins comprising four polypeptide chains that form four antigen binding sites (e.g., having the structure described in international publication No. WO 2012/135345) that bind one or more (e.g., two) different antigen targets or target proteins. In some embodiments, the binding protein is bivalent and/or bispecific. In some embodiments, the binding protein is tetravalent and/or tetraspecific. In some embodiments, the binding protein is tetravalent and/or bispecific. In some embodiments, at least one antigen binding site binds to a CD38 polypeptide (e.g., the extracellular domain of a human and/or cynomolgus monkey CD38 polypeptide).

In some embodiments, the binding protein comprises two polypeptide chains having a structure represented by the formula:

VL1-L1-VL2-L2-CL[I]

and the two polypeptide chains have a structure represented by the formula:

VH2-L3-VH1-L4-CH1-Fc [II]

wherein:

VL1is a first immunoglobulin light chain variable domain;

VL2is a second immunoglobulin light chain variable domain;

VH1is a first immunoglobulin heavy chain variable domain;

VH2is a second immunoglobulin heavy chain variable domain;

CLis an immunoglobulin light chain constant domain;

CH1is immunoglobulin CH1A heavy chain constant domain;

fc comprises an immunoglobulin hinge region and CH2、CH3An immunoglobulin heavy chain constant region;

L1、L2、L3and L4Is an amino acid linker;

and wherein the polypeptide of formula I and the polypeptide of formula II form a cross light-heavy chain pair. In some embodiments, VH1And VL1Forming an antigen binding domain that binds to a CD38 polypeptide, and VH2And VL2Forming an antigen binding domain that binds to another antigen target. In some embodiments, VH2And VL2Forming an antigen binding domain that binds to a CD38 polypeptide, and VH1And VL1Forming an antigen binding domain that binds to another antigen target.

In some embodiments, the binding protein comprises two polypeptide chains forming two antigen binding sites, wherein the first polypeptide chain comprises

VL1-L1-VL2-L2-CL-Fc

And the second polypeptide chain comprises

VH2-L3-VHI-L4-CH1-Fc

Wherein:

VL1is a first immunoglobulin light chain variable domain;

VL2is a second immunoglobulin light chain variable domain;

VH1is a first immunoglobulin heavy chain variable domain;

VH2is a second immunoglobulin heavy chain variable domain;

CL is an immunoglobulin light chain constant domain;

CH1is immunoglobulin CH1A heavy chain constant domain;

CH2is immunoglobulin CH2A heavy chain constant domain;

CH3is immunoglobulin CH3A heavy chain constant domain;

fc comprises an immunoglobulin hinge region and CH2、CH3An immunoglobulin heavy chain constant region; and

L1、L2、L3and L4Is an amino acid linker;

wherein the first and second polypeptides form a cross light-heavy chain pair. In some embodiments, VH1And VL1Forming an antigen binding domain that binds to a CD38 polypeptide, and VH2And VL2Forming an antigen binding domain that binds to another antigen target. In some embodiments, VH2And VL2Forming an antigen binding domain that binds to a CD38 polypeptide, and VH1And VL1Forming an antigen binding domain that binds to another antigen target.

In some embodiments, the binding protein comprises three polypeptide chains forming two antigen binding sites, wherein a first polypeptide chain comprises

VL1-L1-VL2-L2-CL

The second polypeptide chain comprises

VH2-L3-VHI-L4-CH1-Fc

The third polypeptide chain comprises an antibody Fc region

Wherein:

VL1is a first immunoglobulin light chain variable domain;

VL2is a second immunoglobulin light chain variable domain;

VH1is a first immunoglobulin heavy chain variable domain;

VH2is a second immunoglobulin heavy chain variable domain;

CL is an immunoglobulin light chain constant domain;

CH1is immunoglobulin CH1A heavy chain constant domain;

CH2is immunoglobulin CH2A heavy chain constant domain;

CH3is immunoglobulin CH3A heavy chain constant domain;

fc comprises an immunoglobulin hinge region and CH2、CH3An immunoglobulin heavy chain constant region; and

L1、L2、L3and L4Is an amino acid linker;

wherein the first and second polypeptides form a cross light-heavy chain pair. In some embodiments, VH1And VL1Forming an antigen binding domain that binds to a CD38 polypeptide, and VH2And VL2Forming an antigen binding domain that binds to another antigen target. In some embodiments, VH2And VL2Forming an antigen binding domain that binds to a CD38 polypeptide, and VH1And VL1Forming an antigen binding domain that binds to another antigen target.

In some embodiments, the binding protein comprises a first polypeptide chain comprising a structure represented by the formula:

VL1-L1-VL2-L2-CL[I]

and a second polypeptide chain comprising a structure represented by the formula:

VH2-L3-VH1-L4-CH1[II]

wherein:

VL1is a first immunoglobulin light chain variable domain;

VL2Is a second immunoglobulin light chain variable domain;

VH1is a first immunoglobulin heavy chain variable domain;

VH2is a second immunoglobulin heavy chain variable domain;

CLis an immunoglobulin light chain constant domain;

CH1is immunoglobulin CH1A heavy chain constant domain; and

L1、L2、L3and L4Is an amino acid linker;

wherein the polypeptide of formula I and the polypeptide of formula II form a cross light-heavy chain pair. In some embodiments, VH1And VL1Forming an antigen binding domain that binds to a CD38 polypeptide, and VH2And VL2Forming an antigen binding domain that binds to another antigen target. In some embodiments, VH2And VL2Forming an antigen binding domain that binds to a CD38 polypeptide, and VH1And VL1Forming an antigen binding domain that binds to another antigen target.

In any of the bispecific binding proteins described above, the target antigen other than CD38 may be any of the following exemplary antigen targets: a2AR, APRIL, ATP diphosphohydrolase, BAFF, BAFFR, BCMA, BlyS, BTK, BTLA, B7DC, B7H1, B7H4 (also known as VTCN1), B7H5, B7H6, B7H7, B7RP1, B7-4, C3, C5, CCL2 (also known as MCP-1), CCL 53 (also known as MIP-1a), CCL4 (also known as MIP-1B), CCL5 (also known as TES), CCL7 (also known as MCP-3), CCL8 (also known as MCP-2), CCL11 (also known as eotaxin), CCL11 (also known as MIP-1d), CCL11 (also known as TARC), CCL11 (also known as MIP-3B), CCL11 (also known as MPCK-3 a), CCL11 (also known as eosinophil-72), CCL11 (also known as eosinophil-11) and CTF-11 (also known as eosinophil-11/or eotaxin-11), or CTF-11 (also known as CCL-11) or eotaxin-11, or MCK, CCR, CD (also known as FCER, an IgE receptor), CD272, CD273 (also known as PDL), CD274 (also known as PDL), CD275 (also known as B7H), CD276 (also known as B7H), CD278 (also known as ICOS), CD279 (also known as PD-1), CDH (also known as E-cadherin), chitinase, CLEC, CRTH, CSF-1 (also known as M-CSF), DNCSF-2 (also known as GM-GR), GCSF-3 (also known as GCSF), CX3CL (also known as SCYD), CXCL (also known as SDF), CXCL, ecto-1, CXCR1, and CXCR1, EGFR, ENTPD, FCER1, FCER, FLAP, FOLH, Gi, GITR, GITRL, GM-CSF, Her, HHLA, HMGB, HVEM, ICOSLG, IDO, IFN α, IgE, IGF1, IL2 β, IL1F, IL4, IL5, IL7, IL9, IL, rhIL, IL13Ra, IL17 (also known as IL receptor), IL, PROM, IL, ITGB (also known as b integrin), ITK, KIR, LAG, LAMP, leptin, LPFS, MHC class II, NCR3LG, NKG2, NTP diphosphatase-1, Vs40, PD-1H, platelet receptor, SIS, Tps 152, SLC, TSST (also known as TSK, TSCI, TLR, IL1, IL7, IL1, IL7, IL, WUCAM and XCR1 (also known as GPR5/CCXCR 1). In some embodiments, one or more of the above antigen targets is a human antigen target.

In any of the bispecific binding proteins described above, any linker or combination of linkers described herein can be used. For example, in some embodiments, L1、L2、L3Or L4Is independently 0 amino acids in length. In some embodimentsIn a table, L1、L2、L3Or L4Is each independently at least 1 amino acid in length. In some embodiments, L is1、L2、L3And L4Each independently is 0 amino acids in length or comprises a sequence selected from the group consisting of: GGGGSGGGGS (SEQ ID NO: 55), GGGGSGGGGSGGGGS (SEQ ID NO: 56), S, RT, TKGPS (SEQ ID NO: 57), GQPKAAP (SEQ ID NO: 58) and GGSGSSGSGG (SEQ ID NO: 59). In some embodiments, L is1、L2、L3And L4Each independently comprises a sequence selected from the group consisting of: GGGGSGGGGS (SEQ ID NO: 55), GGGGSGGGGSGGGGS (SEQ ID NO: 56), S, RT, TKGPS (SEQ ID NO: 57), GQPKAAP (SEQ ID NO: 58) and GGSGSSGSGG (SEQ ID NO: 59). In some embodiments, L is1Comprising the sequence GQPKAAP (SEQ ID NO: 58), L2Comprising the sequence TKGPS (SEQ ID NO: 57), L3Comprising the sequence S, L4Comprising the sequence RT. In some embodiments, L is1Comprising the sequence GGGGSGGGGS (SEQ ID NO: 55), L2Comprising the sequence GGGGSGGGGS (SEQ ID NO: 55), L 3Is 0 amino acids in length, L4Is 0 amino acids in length. In some embodiments, L is1Comprising the sequence GGSGSSGSGG (SEQ ID NO: 59), L2Comprising the sequence GGSGSSGSGG (SEQ ID NO: 59), L3Is 0 amino acids in length, L4Is 0 amino acids in length. In some embodiments, L is1Comprising the sequence GGGGSGGGGSGGGS (SEQ ID NO: 56), L2Is 0 amino acids in length, L3Comprising the sequence GGGGSGGGGSGGGS (SEQ ID NO: 56), L4Is 0 amino acids in length.

Trispecific binding proteins binding to CD38 polypeptides

In some embodiments, a binding protein of the present disclosure is a trispecific and/or trivalent binding protein comprising four polypeptide chains that form three antigen binding sites that bind one or more (e.g., three) different antigen targets or target proteins. In some embodiments, at least one antigen binding site binds to a CD38 polypeptide (e.g., the extracellular domain of a human and/or cynomolgus monkey CD38 polypeptide). In some embodiments, the first polypeptide chain comprises a structure represented by the formula:

VL2-L1-VL1-L2-CL[I]

and the second polypeptide chain comprises a structure represented by the formula:

VH1-L3-VH2-L4-CH1-hinge-CH2-CH3[II]

And the third polypeptide chain comprises a structure represented by the formula:

VH3-CH1-hinge-CH2-CH3[III]

And the fourth polypeptide chain comprises a structure represented by the formula:

VL3-CL[IV]

Wherein:

VL1is a first immunoglobulin light chain variable domain;

VL2is a second immunoglobulin light chain variable domain;

VL3is a third immunoglobulin light chain variable domain;

VH1is a first immunoglobulin heavy chain variable domain;

VH2is a second immunoglobulin heavy chain variable domain;

VH3is a third immunoglobulin heavy chain variable domain;

CLis an immunoglobulin light chain constant domain;

CH1is immunoglobulin CH1A heavy chain constant domain;

CH2is immunoglobulin CH2A heavy chain constant domain;

CH3is immunoglobulin CH3A heavy chain constant domain;

the hinge being a connection CH1And CH2An immunoglobulin hinge region of the domain, and

L1、L2、L3and L4Is an amino acid linker;

wherein the polypeptide of formula I and the polypeptide of formula II form a cross light-heavy chain pair.

In some embodiments, a binding protein of the present disclosure is a trispecific and/or trivalent binding protein comprising four polypeptide chains that form three antigen binding sites that bind one or more (e.g., three) different antigen targets or target proteins. In some embodiments, at least one antigen binding site binds to a CD38 polypeptide (e.g., the extracellular domain of a human and/or cynomolgus monkey CD38 polypeptide). In some embodiments, the first polypeptide chain comprises a structure represented by the formula:

VL2-L1-VL1-L2-CL[I]

And the second polypeptide chain comprises a structure represented by the formula:

VH1-L3-VH2-L4-CH1[II]

and the third polypeptide chain comprises a structure represented by the formula:

VH3-CH1[III]

and the fourth polypeptide chain comprises a structure represented by the formula:

VL3-CL[IV]

wherein:

VL1is a first immunoglobulin light chain variable domain;

VL2is a second immunoglobulin light chain variable domain;

VL3is a third immunoglobulin light chain variable domain;

VH1is a first immunoglobulin heavy chain variable domain;

VH2is a second immunoglobulin heavy chain variable domain;

VH3is a third immunoglobulin heavy chain variable domain;

CLis an immunoglobulin light chain constant domain;

CH1is immunoglobulin CH1A heavy chain constant domain; and

L1、L2、L3and L4Is an amino acid linker;

and wherein the polypeptide of formula I and the polypeptide of formula II form a cross light-heavy chain pair. In some embodiments, the second and third polypeptide chains further comprise a peptide with CH1A linked Fc region comprising an immunoglobulin hinge region and CH2And CH3An immunoglobulin heavy chain constant domain.

In some embodiments, the first polypeptide chain and the second polypeptide chain have a cross-orientation that forms two different antigen binding sites. In some embodiments, VH1 and VL1 form a binding pair and form a first antigen binding site. In some embodiments, VH2 and VL2 form a binding pair and form a second antigen binding site. In some embodiments, the first antigen-binding site binds to a CD3 polypeptide (e.g., human CD3) and the second antigen-binding site binds to a CD28 polypeptide (e.g., human CD 28). In some embodiments, the second antigen-binding site binds to a CD3 polypeptide (e.g., human CD3) and the first antigen-binding site binds to a CD28 polypeptide (e.g., human CD 28). In some embodiments, the third polypeptide and the fourth polypeptide form a third antigen binding site. In some embodiments, VH3 and VL3 form a binding pair and form a third antigen binding site. In some embodiments, the third antigen-binding site binds to a CD38 polypeptide (e.g., human and optionally cynomolgus monkey CD 38). Exemplary binding protein forms having cross-orientations contemplated for use herein are also described in U.S. patent application No. 15/487,243 and international application No. PCT/US 2017/027488.

In some embodiments of any of the bispecific, trispecific, or multispecific binding proteins described herein, the antigen-binding site binds a CD38 polypeptide (e.g., human and optionally cynomolgus monkey CD 38). In some embodiments, the other (e.g., not binding to CD38) antigen binding site of any of the bispecific, trispecific, or multispecific binding proteins described herein binds to CD28 or CD 3. In some embodiments, VH1The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO: 31) or GYTFTSYA (SEQ ID NO: 37), comprising IYPGNGGT (SEQ ID NO: 37): 32) or IYPGQGGT (SEQ ID NO: 38) and a CDR-H2 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33) the CDR-H3 sequence of the amino acid sequence of (1), and VL1The domain comprises a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO: 34) or QSVSSYGQGF (SEQ ID NO: 39), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO: 35) or GAS (SEQ ID NO: 40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36). In some embodiments, VH2The domains comprise a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO: 31) or GYTFTSYA (SEQ ID NO: 37), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO: 32) or IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33), and V L2The domain comprises a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO: 34) or QSVSSYGQGF (SEQ ID NO: 39), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO: 35) or GAS (SEQ ID NO: 40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36). In some embodiments, VH3The domains comprise a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO: 31) or GYTFTSYA (SEQ ID NO: 37), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO: 32) or IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33), and VL3The domain comprises a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO: 34) or QSVSSYGQGF (SEQ ID NO: 39), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO: 35) or GAS (SEQ ID NO: 40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36). In some embodiments, VH1The domains comprise a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO: 31) or GYTFTSYA (SEQ ID NO: 37), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO: 32) or IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33), V L1The domain comprises an amino acid sequence comprising ESVDSYGNGF (SEQ ID NO: 34) or QSVSSYGQG (SEQ ID NO: 132)A CDR-L1 sequence, a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO: 35) or GAS (SEQ ID NO: 40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36). In some embodiments, VH2The domains comprise a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO: 31) or GYTFTSYA (SEQ ID NO: 37), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO: 32) or IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33), VL2The domain comprises a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO: 34) or QSVSSYGQG (SEQ ID NO: 132), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO: 35) or GAS (SEQ ID NO: 40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36). In some embodiments, VH3The domains comprise a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO: 31) or GYTFTSYA (SEQ ID NO: 37), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO: 32) or IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33), V L3The domain comprises a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO: 34) or QSVSSYGQG (SEQ ID NO: 132), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO: 35) or GAS (SEQ ID NO: 40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36).

In some embodiments, VH1The domains comprise a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO: 41), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO: 42), and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO: 43), and/or VL1The domain comprises a CDR-L1 sequence comprising the amino acid sequence of QGIRND (SEQ ID NO: 44), a CDR-L2 sequence comprising the amino acid sequence of AAS (SEQ ID NO: 45), and a CDR-L3 sequence comprising the amino acid sequence of LQDYIYYPT (SEQ ID NO: 46). In some embodiments, VH2The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO: 41), an amino acid comprising IWYDGSNK (SEQ ID NO: 42)CDR-H2 sequence of the sequence, and CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO: 43), and/or VL2The domain comprises a CDR-L1 sequence comprising the amino acid sequence of QGIRND (SEQ ID NO: 44), a CDR-L2 sequence comprising the amino acid sequence of AAS (SEQ ID NO: 45), and a CDR-L3 sequence comprising the amino acid sequence of LQDYIYYPT (SEQ ID NO: 46). In some embodiments, V H3The domains comprise a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO: 41), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO: 42), and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO: 43), and/or VL3The domain comprises a CDR-L1 sequence comprising the amino acid sequence of QGIRND (SEQ ID NO: 44), a CDR-L2 sequence comprising the amino acid sequence of AAS (SEQ ID NO: 45), and a CDR-L3 sequence comprising the amino acid sequence of LQDYIYYPT (SEQ ID NO: 46).

In some embodiments, VH1The domains comprise a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO: 41), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO: 42), and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO: 43), and VL1The domain comprises a CDR-L1 sequence comprising the amino acid sequence of QGIRND (SEQ ID NO: 44), a CDR-L2 sequence comprising the amino acid sequence of AAS (SEQ ID NO: 45), and a CDR-L3 sequence comprising the amino acid sequence of LQDYIYYPT (SEQ ID NO: 46). In some embodiments, VH2The domains comprise a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO: 41), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO: 42), and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO: 43), and V L2The domain comprises a CDR-L1 sequence comprising the amino acid sequence of QGIRND (SEQ ID NO: 44), a CDR-L2 sequence comprising the amino acid sequence of AAS (SEQ ID NO: 45), and a CDR-L3 sequence comprising the amino acid sequence of LQDYIYYPT (SEQ ID NO: 46). In some embodiments, VH3The domains comprise a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO: 41), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO: 42), and a CDR-H2 sequence comprising ARMFRGAFDY (SE)Q ID NO: 43) the CDR-H3 sequence of the amino acid sequence of (1), and VL3The domain comprises a CDR-L1 sequence comprising the amino acid sequence of QGIRND (SEQ ID NO: 44), a CDR-L2 sequence comprising the amino acid sequence of AAS (SEQ ID NO: 45), and a CDR-L3 sequence comprising the amino acid sequence of LQDYIYYPT (SEQ ID NO: 46).

In some embodiments, VH1The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO: 31), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO: 32), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33), and/or VL1The domain comprises a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO: 34), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO: 35), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36). In some embodiments, V H1The domains comprise a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYA (SEQ ID NO: 37), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33), and/or VL1The domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSVSSYGQGF (SEQ ID NO: 39), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO: 40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36). In some embodiments, VH2The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO: 31), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO: 32), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33), and/or VL2The domain comprises a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO: 34), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO: 35), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36). In some embodiments, VH2The domains comprise a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYA (SEQ ID NO: 37), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT (SEQ ID NO: 38), and an amino acid sequence comprising ARTGGLRRAYFTY (SEQ ID NO: 33) CDR-H3 sequences of, and/or VL2The domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSVSSYGQGF (SEQ ID NO: 39), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO: 40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36). In some embodiments, VH3The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO: 31), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO: 32), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33), and/or VL3The domain comprises a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO: 34), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO: 35), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36). In some embodiments, VH3The domains comprise a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYA (SEQ ID NO: 37), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33), and/or VL3The domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSVSSYGQGF (SEQ ID NO: 39), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO: 40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36).

In some embodiments, VH1The domains comprise a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO: 31), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO: 32), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33), and VL1The domain comprises a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO: 34), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO: 35), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36). In some embodiments, VH1The domains comprise a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYA (SEQ ID NO: 37), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33), and VL1The domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSVSSYGQGF (SEQ ID NO: 39), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO: 40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36). In some embodiments, VH2The domains comprise a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO: 31), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO: 32), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33), and V L2The domain comprises a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO: 34), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO: 35), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36). In some embodiments, VH2The domains comprise a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYA (SEQ ID NO: 37), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33), and VL2The domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSVSSYGQGF (SEQ ID NO: 39), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO: 40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36). In some embodiments, VH3The domains comprise a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO: 31), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO: 32), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33), and VL3The domain comprises a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO: 34), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO: 35), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36). In some embodiments, V H3The domains comprise a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYA (SEQ ID NO: 37), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33), and VL3The domain comprises a CDR-L comprising the amino acid sequence of QSVSSYGQGF (SEQ ID NO: 39)1, a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO: 40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36).

In some embodiments, VH3The domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSFN (SEQ ID NO: 31), a CDR-H2 sequence comprising the amino acid sequence of IYPGNGGT (SEQ ID NO: 32), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33), and/or VL3The domain comprises a CDR-L1 sequence comprising the amino acid sequence of ESVDSYGNGF (SEQ ID NO: 34), a CDR-L2 sequence comprising the amino acid sequence of LAS (SEQ ID NO: 35), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36). In some embodiments, VH3The domains comprise a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYA (SEQ ID NO: 37), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 33), and/or V L3The domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSVSSYGQGF (SEQ ID NO: 39), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO: 40), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 36).

In some embodiments, VH3The domain comprises SEQ ID NO: 5, or VL3The domain comprises SEQ ID NO: 6. In some embodiments, VH3The domain comprises SEQ ID NO: 17, or VL3The domain comprises SEQ ID NO: 18. In some embodiments, VH3The domain comprises seq id NO: 21, or VL3The domain comprises SEQ ID NO: 18. In some embodiments, V H3 domain comprises SEQ ID NO: 23, or VL3The domain comprises SEQ ID NO: 18. In some embodiments, VH3The domain comprises SEQ ID NO: 13, or VL3The domain comprises SEQ id no: 14.

In some embodimentsIn, VH3The domain comprises SEQ ID NO: 5, and/or VL3The domain comprises SEQ ID NO: 6. In some embodiments, V H3The domain comprises SEQ ID NO: 17, and VL3The domain comprises SEQ ID NO: 18. In some embodiments, VH3The domain comprises seq id NO: 21, and VL3The domain comprises SEQ ID NO: 18. In some embodiments, VH3The domain comprises SEQ ID NO: 23, and VL3The domain comprises SEQ ID NO: 18. In some embodiments, VH3The domain comprises SEQ ID NO: 13, and VL3The domain comprises SEQ id no: 14.

In some embodiments, VH3The domains comprise a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO: 41), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO: 42), and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO: 43), and/or VL3The domain comprises a CDR-L1 sequence comprising the amino acid sequence of QGIRND (SEQ ID NO: 44), a CDR-L2 sequence comprising the amino acid sequence of AAS (SEQ ID NO: 45), and a CDR-L3 sequence comprising the amino acid sequence of LQDYIYYPT (SEQ ID NO: 46).

In some embodiments, VH3The domain comprises SEQ ID NO: 9, and/or V L3The domain comprises SEQ id no: 10.

In some embodiments of any of the trispecific binding proteins of the present disclosure, one antigen-binding domain binds to a CD3 polypeptide (e.g., human CD3) and one antigen-binding domain binds to a CD28 polypeptide (e.g., human CD 28). In some embodiments, VH1The domain comprises a sequence from SEQ ID NO: three CDRs of 49 or 51, as shown in Table H, and VL1The domain comprises a sequence from SEQ ID NO: 50 or 52, as shown in table H. In some embodiments, VH2The domain comprises a sequence from SEQ ID NO: three CDRs of 49 or 51, as shown in Table HAnd V isL2The domain comprises a sequence from SEQ ID NO: 50 or 52, as shown in table H. In some embodiments, VH1The domain comprises a sequence from SEQ ID NO: 53 or 84, as shown in Table H, and VL1The domain comprises a sequence from SEQ ID NO: 54 or 85, as shown in table H. In some embodiments, VH2The domain comprises a sequence from SEQ ID NO: 53 or 84, as shown in Table H, and VL2The domain comprises a sequence from SEQ ID NO: 54 or 85, as shown in table H.

In some embodiments, VH1The domain comprises SEQ ID NO: 49 amino acid sequence, V L1The domain comprises SEQ ID NO: 50, VH2The domain comprises SEQ ID NO: 53, and VL2The domain comprises SEQ ID NO: 54, or a pharmaceutically acceptable salt thereof. In some embodiments, VH2The domain comprises SEQ ID NO: 49 amino acid sequence, VL2The domain comprises SEQ ID NO: 50, VH1The domain comprises SEQ ID NO: 53, and VL1The domain comprises SEQ ID NO: 54, or a pharmaceutically acceptable salt thereof. In some embodiments, VH1The domain comprises seq id NO: 51, VL1The domain comprises SEQ ID NO: 52, VH2The domain comprises SEQ id no: 53, and VL2The domain comprises SEQ ID NO: 54, or a pharmaceutically acceptable salt thereof. In some embodiments, VH2The domain comprises SEQ ID NO: 51, VL2The domain comprises SEQ ID NO: 52, VH1The domain comprises SEQ ID NO: 53, and VL1The domain comprises SEQ ID NO: 54, or a pharmaceutically acceptable salt thereof.

In some embodiments, VH1The domain comprises SEQ ID NO: 49 amino acid sequence, VL1The domain comprises SEQ ID NO: 50, VH2The domain comprises SEQ ID NO: 53 amino acid sequence, V L2The domain comprises SEQ ID NO: 54, VH3The domain comprises SEQ IDNO: 13, and VL3The domain comprises SEQ ID NO: 14. In some embodiments, VH1The domain comprises SEQ ID NO: 49 amino acid sequence, VL1The domain comprises SEQ ID NO: 50, VH2The domain comprises SEQ ID NO: 53 amino acid sequence, VL2The domain comprises SEQ ID NO: 54, VH3The domain comprises SEQ ID NO: 9, and VL3The domain comprises SEQ ID NO: 10.

In certain embodiments, the first polypeptide chain comprises a sequence identical to SEQ ID NO: 61, and a second polypeptide chain comprising a polypeptide sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 60, and a third polypeptide chain comprising a polypeptide sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 62, and a fourth polypeptide chain comprising a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 63, and a polypeptide sequence which is at least 95% identical in amino acid sequence. In certain embodiments, the first polypeptide chain comprises a sequence identical to SEQ ID NO: 61, and a second polypeptide chain comprising a polypeptide sequence at least 95% identical to the amino acid sequence of SEQ id no: 64, and a third polypeptide chain comprising a polypeptide sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 65, and a fourth polypeptide chain comprising a polypeptide sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 63, and a polypeptide sequence which is at least 95% identical in amino acid sequence. In certain embodiments, the first polypeptide chain comprises a sequence identical to SEQ ID NO: 61, and a second polypeptide chain comprising a polypeptide sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 66, and a third polypeptide chain comprising a polypeptide sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 67, and a fourth polypeptide chain comprising a polypeptide sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 63, and a polypeptide sequence which is at least 95% identical in amino acid sequence. In certain embodiments, the first polypeptide chain comprises a sequence identical to SEQ ID NO: 61, and a second polypeptide chain comprising a polypeptide sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 60, and a third polypeptide chain comprising a polypeptide sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 68, and a fourth polypeptide chain comprising a polypeptide sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 69 is at least 95% identical in amino acid sequence. In certain embodiments, the first polypeptide chain comprises a sequence identical to SEQ ID NO: 61, and a second polypeptide chain comprising a polypeptide sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 64, and a third polypeptide chain comprising a polypeptide sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 70, and a fourth polypeptide chain comprising a polypeptide sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 69 is at least 95% identical in amino acid sequence. In certain embodiments, the first polypeptide chain comprises a sequence identical to SEQ ID NO: 61, and a second polypeptide chain comprising a polypeptide sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 66, and a third polypeptide chain comprising a polypeptide sequence at least 95% identical to the amino acid sequence of SEQ id no: 71, and a fourth polypeptide chain comprising a polypeptide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 69 is at least 95% identical in amino acid sequence.

In certain embodiments, the first polypeptide chain comprises SEQ ID NO: 61, and a second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 60, and a third polypeptide chain comprising the amino acid sequence of SEQ ID NO: 62, and a fourth polypeptide chain comprising the amino acid sequence of SEQ ID NO: 63. In certain embodiments, the first polypeptide chain comprises SEQ ID NO: 61, and a second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 64 and a third polypeptide chain comprising the amino acid sequence of SEQ ID NO: 65, and a fourth polypeptide chain comprising the amino acid sequence of SEQ ID NO: 63. In certain embodiments, the first polypeptide chain comprises SEQ ID NO: 61, and a second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 66, and a third polypeptide chain comprising the amino acid sequence of SEQ ID NO: 67, and a fourth polypeptide chain comprising the amino acid sequence of SEQ ID NO: 63. In certain embodiments, the first polypeptide chain comprises SEQ ID NO: 61, and a second polypeptide chain comprising the amino acid sequence of SEQ id no: 60, and a third polypeptide chain comprising the amino acid sequence of SEQ ID NO: 68, and a fourth polypeptide chain comprising the amino acid sequence of SEQ ID NO: 69. In certain embodiments, the first polypeptide chain comprises SEQ ID NO: 61, and a second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 64 and a third polypeptide chain comprising the amino acid sequence of SEQ ID NO: 70, and a fourth polypeptide chain comprising the amino acid sequence of SEQ ID NO: 69. In certain embodiments, the first polypeptide chain comprises SEQ ID NO: 61, and a second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 66, and a third polypeptide chain comprising the amino acid sequence of SEQ ID NO: 71, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 69.

In any of the trispecific binding proteins described above, the target antigen other than CD38 may be any of the following exemplary antigen targets: a2AR, APRIL, ATP diphosphohydrolase, BAFF, BAFFR, BCMA, BlyS, BTK, BTLA, B7DC, B7H1, B7H4 (also known as VTCN1), B7H5, B7H6, B7H7, B7RP1, B7-4, C3, C5, CCL2 (also known as MCP-1), CCL 53 (also known as MIP-1a), CCL4 (also known as MIP-1B), CCL5 (also known as TES), CCL7 (also known as MCP-3), CCL8 (also known as MCP-2), CCL11 (also known as eotaxin), CCL11 (also known as MIP-1d), CCL11 (also known as TARC), CCL11 (also known as MIP-3B), CCL11 (also known as MPCK-3 a), CCL11 (also known as eosinophil-72), CCL11 (also known as eosinophil-11) and CTF-11 (also known as eosinophil-11/or eotaxin-11), or CTF-11 (also known as CCL-11) or eotaxin-11, or MCK, CCR, CD (also known as FCER, an IgE receptor), CD272, CD273 (also known as PDL), CD274 (also known as PDL), CD275 (also known as B7H), CD276 (also known as B7H), CD278 (also known as ICOS), CD279 (also known as PD-1), CDH (also known as E-cadherin), chitinase, CLEC, CRTH, CSF-1 (also known as M-CSF), DNCSF-2 (also known as GM-GR), GCSF-3 (also known as GCSF), CX3CL (also known as SCYD), CXCL (also known as SDF), CXCL, ecto-1, CXCR1, and CXCR1, EGFR, ENTPD, FCER1, FCER, FLAP, FOLH, Gi, GITR, GITRL, GM-CSF, Her, HHLA, HMGB, HVEM, ICOSLG, IDO, IFN α, IgE, IGF1, IL2 β, IL1F, IL4, IL5, IL7, IL9, IL, rhIL, IL13Ra, IL17 (also known as IL receptor), IL, PROM, IL, ITGB (also known as b integrin), ITK, KIR, LAG, LAMP, leptin, LPFS, MHC class II, NCR3LG, NKG2, NTP diphosphatase-1, Vs40, PD-1H, platelet receptor, SIS, Tps 152, SLC, TSST (also known as TSK, TSCI, TLR, IL1, IL7, IL1, IL7, IL, WUCAM and XCR1 (also known as GPR5/CCXCR 1). In some embodiments, one or more of the above antigen targets is a human antigen target.

In some embodiments, the binding protein of the present disclosure is an antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a chimeric, humanized, or human antibody.

Binding proteins of the present disclosure can be made using domains or sequences obtained or derived from any human or non-human antibody, including, for example, human, murine, or humanized antibodies.

Joint

In some embodiments, linker L1、L2、L3And L4Ranges of (d) are no amino acids (length ═ 0) to about 100 amino acids long, or less than 100, 50, 40, 30, 20, or 15 amino acids or less. The linker may also be 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid long. Binding of L in protein1、L2、L3And L4May all have the same amino acid sequence, or may all have different amino acid sequences.

Examples of suitable linkers include a single glycine (Gly) residue; diglycine peptide (Gly-Gly); tripeptide (Gly-Gly); a peptide having four glycine residues; a peptide having five glycine residues; a peptide having six glycine residues; a peptide having seven glycine residues; and peptides having eight glycine residues. Other combinations of amino acid residues may be used, such as the peptide GGGGSGGGGS (SEQ ID NO: 55), the peptide GGGGSGGGGSGGS (SEQ ID NO: 56), the peptide TKGPS (SEQ ID NO: 57), the peptide GQPKAAP (SEQ ID NO: 58), and the peptide GGSGSSGSGG (SEQ ID NO: 59). The examples listed above are not intended to limit the scope of the present disclosure in any way, and show that linkers comprising randomly selected amino acids selected from the group consisting of: valine, leucine, isoleucine, serine, threonine, lysine, arginine, histidine, aspartic acid, glutamic acid, asparagine, glutamine, glycine and proline. For additional descriptions of linker sequences see, e.g., WO2012135345 and international application number PCT/US 2017/027488.

The identity and sequence of the amino acid residues in the linker may vary depending on the type of secondary structural element required to be achieved in the linker. For example, glycine, serine and alanine are most suitable for linkers with the greatest flexibility. Certain combinations of glycine, proline, threonine, and serine are useful if a more rigid and extended linker is desired. Depending on the desired properties, any amino acid residue may be considered a linker in combination with other amino acid residues to construct a desired larger peptide linker.

In some embodiments, L is1、L2、L3Or L4Is independently 0 amino acids in length. In some embodiments, L is1、L2、L3Or L4Is each independently at least 1 amino acid in length. In some embodiments, L1Is of length L3Is at least twice as long. In some embodiments, L is2Is of length L4Is at least twice as long. In some embodiments, L1Is of length L3Is at least twice as long, and L2Is of length L4Is at least twice as long. In some embodiments, L is1Is 3 to 12 amino acid residues in length, L2Is 3 to 14 amino acid residues in length, L3Is 1 to 8 amino acid residues in length, L 4Is 1 to 3 amino acid residues in length. In some embodiments，L1Is 5 to 10 amino acid residues in length, L2Is 5 to 8 amino acid residues in length, L3Is 1 to 5 amino acid residues in length, L4Is 1 to 2 amino acid residues in length. In some embodiments, L is1Is 7 amino acid residues in length, L2Is 5 amino acid residues in length, L3Is 1 amino acid residue in length, L4Is 2 amino acid residues in length. In some embodiments, L is1Is 10 amino acid residues in length, L2Is 10 amino acid residues in length, L3Is 0 amino acid residues in length, L4Is 0 amino acid residues in length. In some embodiments, L is1、L2、L3And L4Each having an independently selected length of 0 to 15 amino acids (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids), wherein at least two linkers have a length of 1 to 15 amino acids (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids). In some embodiments, L is1、L2、L3And L4Each of which is 0 amino acids in length.

In some embodiments, L is1、L2、L3And/or L4Comprising a sequence derived from a naturally occurring sequence at the junction between an antibody variable domain and an antibody constant domain (e.g., as described in WO 2012/135345). For example, in some embodiments, the linker comprises an endogenous V HAnd CH1Between domains or at endogenous VLAnd CLSequences found at transitions between domains (e.g., κ or λ). In some embodiments, the linker is comprised in endogenous human VHAnd CH1Between domains or in endogenous human VLAnd CLSequences found at transitions between domains (e.g., human κ or λ).

In some embodiments, L is1、L2、L3And L4Each independently is 0 amino acids in length or comprises a sequence selected from the group consisting of:GGGGSGGGGS (SEQ ID NO: 55), GGGGSGGGGSGGGGS (SEQ ID NO: 56), S, RT, TKGPS (SEQ ID NO: 57), GQPKAAP (SEQ ID NO: 58) and GGSGSSGSGG (SEQ ID NO: 59). In some embodiments, L is1、L2、L3And L4Each independently comprises a sequence selected from the group consisting of: GGGGSGGGGS (SEQ ID NO: 55), GGGGSGGGGSGGGGS (SEQ ID NO: 56), S, RT, TKGPS (SEQ ID NO: 57), GQPKAAP (SEQ ID NO: 58) and GGSGSSGSGG (SEQ ID NO: 59).

In some embodiments, L is1Comprising the sequence GQPKAAP (SEQ ID NO: 58), L2Comprising the sequence TKGPS (SEQID NO: 57), L3Comprising the sequences S, and L4Comprising the sequence RT. In some embodiments, L is1Comprising the sequence GGGGSGGGGS (SEQ ID NO: 55), L2Comprising the sequence GGGGSGGGGS (SEQ ID NO: 55), L3Is 0 amino acids in length, and L 4Is 0 amino acids in length. In some embodiments, L is1Comprising the sequence GGSGSSGSGG (SEQ ID NO: 59), L2Comprising the sequence GGSGSSGSGG (SEQ ID NO: 59), L3Is 0 amino acids in length, and L4Is 0 amino acids in length. In some embodiments, L is1Comprising the sequence GGGGSGGGGSGGGS (SEQ ID NO: 56), L2Is 0 amino acids in length, L3Comprising the sequences GGGGSGGGGSGGGS (SEQ ID NO: 56), and L4Is 0 amino acids in length.

Fc region and constant Domain

In some embodiments, a binding protein of the present disclosure comprises a full-length antibody heavy chain or a polypeptide chain comprising an Fc region. In some embodiments, the Fc region is a human Fc region, for example a human IgG1, IgG2, IgG3, or IgG4Fc region. In some embodiments, the Fc region comprises an antibody hinge, CH1、CH2、CH3And optionally CH4A domain. In some embodiments, the Fc region is a human IgG1Fc region. In some embodiments, the Fc region is a human IgG4Fc region. In some embodiments, the Fc region comprises one or more mutations described below.

In some embodiments, a binding protein of the present disclosure includes one or two Fc variants. The term "Fc variant" as used herein refers to a molecule or sequence that is modified from a native Fc but still comprises a binding site for the salvage receptor (salvaging receptor) FcRn (neonatal Fc receptor). Exemplary Fc variants and their interaction with salvage receptors are known in the art. Thus, the term "Fc variant" can comprise a molecule or sequence of a humanized non-human native Fc. In addition, native Fc contains regions that can be removed because they provide undesirable structural features or biological activity of the antibody-like binding proteins of the invention. Thus, the term "Fc variant" encompasses a molecule or sequence that lacks one or more native Fc sites or residues, or in which one or more Fc sites or residues are modified, affected or involved: (1) disulfide bond formation, (2) incompatibility with the selected host cell, (3) N-terminal heterogeneity upon expression in the selected host cell, (4) glycosylation, (5) interaction with complement, (6) binding to Fc receptors other than salvage receptors, or (7) antibody-dependent cellular cytotoxicity (ADCC).

In some embodiments, the Fc region comprises one or more mutations that reduce or eliminate Fc receptor binding and/or effector function of the Fc region (e.g., Fc receptor-mediated antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), and/or antibody-dependent cellular cytotoxicity (ADCC)).

In some embodiments, the Fc region is a human IgG1 Fc region comprising one or more amino acid substitutions according to EU Index at positions corresponding to positions 234, 235, and/or 329 of human IgG 1. In some embodiments, the amino acid substitution is L234A, L235A, and/or P329A. In some embodiments, the Fc region is a human IgG1 Fc region comprising amino acid substitutions according to EU Index at positions corresponding to positions 298, 299, and/or 300 of human IgG 1. In some embodiments, the amino acid substitution is S298N, T299A, and/or Y300S.

In some embodiments, the Fc region is a human IgG4Fc region comprising one or more mutations that reduce or eliminate fcyi and/or fcyi II binding. In some embodiments, the Fc region is a human IgG4Fc region comprising one or more mutations that reduce or eliminate fcyi and/or fcyi II binding but do not affect FcRn binding. In some embodiments, the Fc region is a human IgG4Fc region comprising amino acid substitutions according to EU Index at positions corresponding to positions 228 and/or 409 of human IgG 4. In some embodiments, the amino acid substitution is S228P and/or R409K. In some embodiments, the Fc region is a human IgG4Fc region comprising amino acid substitutions according to EU Index at positions corresponding to positions 234 and/or 235 of human IgG 4. In some embodiments, the amino acid substitution is F234A and/or L235A. In some embodiments, the Fc region is a human IgG4Fc region comprising amino acid substitutions according to EU Index at positions corresponding to positions 228, 234, 235, and/or 409 of human IgG 4. In some embodiments, the amino acid substitution is S228P, F234A, L235A, and/or R409K. In some embodiments, the Fc region is a human IgG4Fc region comprising amino acid substitutions at positions corresponding to position 233-236 of human IgG4 according to the EU Index. In some embodiments, the amino acid substitution is E233P, F234V, L235A, and the deletion at 236. In some embodiments, the Fc region is a human IgG4Fc region comprising amino acid mutations at substitutions corresponding to positions 228, 233-236, and/or 409 of human IgG4, according to the EU Index. In some embodiments, the amino acid mutation is S228P; E233P, F234V, L235A and the deletion at 236; and/or R409K.

In some embodiments, a binding protein of the present disclosure comprises one or more mutations to improve purification, e.g., by modulating affinity for a purification reagent. For example, it is well known that heterodimeric binding proteins can be selectively purified from their homodimeric forms if one of the two Fc regions of the heterodimeric form contains a mutation that reduces or eliminates binding to protein a, because the heterodimeric form will have an intermediate affinity based on purification of protein a compared to either homodimeric form, and can be selectively eluted from protein a, e.g., by using a different pH (see, e.g., Smith, e.j.et al. (2015) sci.rep.5: 17943). In some embodiments, the mutation comprises a substitution at positions corresponding to positions 435 and 436 of human IgG1 or IgG4 according to the EU Index, wherein the amino acid substitutions are H435R and Y436F. In some embodiments, the binding protein comprises a second polypeptide chain further comprising a second polypeptide chain with CH1Is connected to the firstAn Fc region, the first Fc region comprising an immunoglobulin hinge region and CH2And CH3An immunoglobulin heavy chain constant region, and the third polypeptide chain further comprises a second amino acid sequence substantially identical to CH1A linked second Fc region comprising an immunoglobulin hinge region and C H2And CH3An immunoglobulin heavy chain constant region; and wherein only one of the first and second Fc regions comprises amino acid substitutions at positions corresponding to positions 435 and 436 of human IgG1 or IgG4 according to EU Index, wherein the amino acid substitutions are H435R and Y436F. In some embodiments, a binding protein of the present disclosure comprises a knob and hole mutation and one or more mutations to improve purification. In some embodiments, the first and/or second Fc region is a human IgG1 Fc region. In some embodiments, the first and/or second Fc region is a human IgG4 Fc region.

To improve the yield of some binding proteins (e.g., bispecific or trispecific binding proteins), the CH3 domain can be altered by a "knob and hole" technique, as described, for example, in international publication nos. WO 96/027011, Ridgway et al, 1996, Protein eng.9: 617-21 and merchat et al, 1998, nat. biotechnol.16: 677-81 in several instances. Specifically, two C's are changedH3Interaction surface of the Domain to increase the content of these two CsH3Heterodimerization of the two heavy chains of the domains. Two CH3Each of the domains (two heavy chains) may be a "knob" and the other a "hole". The introduction of disulfide bridges further stabilizes the heterodimer (Merchant et al, 1998; Atwellet al, 1997, J.mol.biol.270: 26-35) and increases the yield. In particular embodiments, the knob is located on a second pair of polypeptides having a single variable domain. In other embodiments, the knob is located on the first pair of polypeptides in a crossed orientation. In other embodiments, C H3The domains do not include a knob in the mortar.

In some embodiments, a binding protein of the present disclosure (e.g., a trispecific binding protein) comprises a "knob" mutation on a second polypeptide chain and a "hole" mutation on a third polypeptide chain. In some embodiments, a binding protein of the present disclosure comprises a "knob" mutation on a third polypeptide chain and a "hole" mutation on a second polypeptide chain. In some embodiments, the "knob" mutation comprises a substitution at a position corresponding to position 354 and/or 366 of human IgG1 or IgG4, according to the EU Index. In some embodiments, the amino acid substitution is S354C, T366W, T366Y, S354C and T366W, or S354C and T366Y. In some embodiments, the "knob" mutation comprises substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4, according to the EU Index. In some embodiments, the amino acid substitutions are S354C and T366W. In some embodiments, the "hole" mutation comprises a substitution at a position corresponding to position 407 and optionally positions 349, 366 and/or 368 of human IgG1 or IgG4 according to EUIndex. In some embodiments, the amino acid substitution is Y407V or Y407T, and optionally Y349C, T366S, and/or L368A. In some embodiments, the "hole" mutations comprise substitutions at positions corresponding to positions 349, 366, 368 and 407 of human IgG1 or IgG4, according to the EU Index. In some embodiments, the amino acid substitutions are Y349C, T366S, L368A, and Y407V.

In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant regions, wherein the first Fc region comprises amino acid substitutions at positions corresponding to position 366 and optionally position 354 of human IgG1 or IgG4 according to the EU Index, wherein the amino acid substitutions are T366W or T366Y, and optionally S354C; and wherein the third polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant regions, wherein the second Fc region comprises an amino acid substitution according to EU Index at a position corresponding to position 407 and optionally position 349, 366 and/or 368 of human IgG1 or IgG4, wherein the amino acid substitution is Y407V or Y407T, and optionally Y349C, T366S and/or L368A.

In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant regions, wherein the first Fc region comprises an amino acid substitution at a position corresponding to position 407 and optionally positions 349, 366, and/or 368 of human IgG1 or IgG4 according to the EU Index, wherein the amino acid substitution is Y407V or Y407T, and optionally Y349C, T366S, and/or L368A; and wherein the third polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant regions, wherein the second Fc region comprises an amino acid substitution, according to EU Index, at a position corresponding to position 366 and optionally position 354 of human IgG1 or IgG4, wherein the amino acid substitution is T366W or T366Y, and optionally S354C.

In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant regions, wherein the first Fc region comprises an amino acid substitution at a position corresponding to position 366 of human IgG1 or IgG4 according to the EU Index, wherein the amino acid substitution is T366W; and wherein the third polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant regions, wherein the second Fc region comprises an amino acid substitution, according to EU Index, at a position corresponding to position 366, 368 and/or 407 of human IgG1 or IgG4, wherein the amino acid substitution is T366S, L368A, and/or Y407V.

In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant regions, wherein the first Fc region comprises an amino acid substitution, according to EU Index, at a position corresponding to position 366, 368, and/or 407 of human IgG1 or IgG4, wherein the amino acid substitution is T366S, L368A, and/or Y407V; and wherein the third polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant regions, wherein the second Fc region comprises an amino acid substitution, according to EU Index, at a position corresponding to position 366 of human IgG1 or IgG4, wherein the amino acid substitution is T366W.

In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to the EU Index, wherein the amino acid substitutions are S354C and T366W; wherein the third polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region comprises amino acid substitutions according to EU Index at positions corresponding to positions 349, 366, 368 and 407 of human IgG1 or IgG4, wherein the amino acid substitutions are Y349C, T366S, L368A and Y407V. In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitutions according to EU Index at positions corresponding to positions 349, 366, 368, and 407 of human IgG1 or IgG4, wherein the amino acid substitutions are Y349C, T366S, L368A, and Y407V; wherein the third polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region comprises amino acid substitutions according to EU Index at positions corresponding to positions 354 and 366 of human IgG1 or IgG4, wherein the amino acid substitutions are S354C and T366W. In some embodiments, the first and/or second Fc region is a human IgG1 Fc region. In some embodiments, the first and/or second Fc region is a human IgG4 Fc region.

In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, wherein the first Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitutions at positions corresponding to positions 228, 354, 366, and 409 of human IgG4 according to the EU Index, wherein the amino acid substitutions are S228P, S354C, T366W, and R409K; and wherein the third polypeptide chain further comprises a second Fc region linked to CH1, wherein the second Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, wherein the second Fc region comprises amino acid substitutions, according to EU Index, at positions corresponding to positions 228, 349, 366, 368, 407, and 409 of human IgG4, wherein the amino acid substitutions are S228P, Y349C, T366S, L368A, Y407V, and R409K. In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, wherein the first Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitutions at positions corresponding to positions 228, 349, 366, 368, 407, and 409 of human IgG4 according to EU Index, wherein the amino acid substitutions are S228P, Y349C, T366S, L368A, Y407V, and R409K; and wherein the third polypeptide chain further comprises a second Fc region linked to CH1, wherein the second Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, wherein the second Fc region comprises amino acid substitutions at positions corresponding to positions 228, 354, 366, and 409 of human IgG4 according to the EU Index, wherein the amino acid substitutions are S228P, S354C, T366W, and R409K.

In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, wherein the first Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitutions according to EU Index at positions corresponding to positions 234, 235, 354, and 366 of human IgG4, wherein the amino acid substitutions are F234A, L235A, S354C, and T366W; and wherein the third polypeptide chain further comprises a second Fc region linked to CH1, wherein the second Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, wherein the second Fc region comprises amino acid substitutions at positions corresponding to positions 234, 235, 349, 366, 368 and 407 of human IgG4 according to EU Index, wherein the amino acid substitutions are F234A, L235A, Y349C, T366S, L368A, and Y407V. In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, wherein the first Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitutions at positions corresponding to positions 234, 235, 349, 366, 368 and 407 of human IgG4 according to EU Index, wherein the amino acid substitutions are F234A, L235A, Y349C, T366S, L368A and Y407V; and wherein the third polypeptide chain further comprises a second Fc region linked to CH1, wherein the second Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, wherein the second Fc region comprises amino acid substitutions at positions corresponding to positions 234, 235, 354, and 366 of human IgG4 according to the EU Index, wherein the amino acid substitutions are F234A, L235A, S354C, and T366W.

In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, wherein the first Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitutions at positions corresponding to positions 228, 234, 235, 354, 366, and 409 of human IgG4 according to the EU Index, wherein the amino acid substitutions are S228P, F234A, L235A, S354C, T366W, and R409K; and wherein the third polypeptide chain further comprises a second Fc region linked to CH1, wherein the second Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region comprises amino acid substitutions according to EU Index at positions corresponding to positions 228, 234, 235, 349, 366, 368, 407, and 409 of human IgG4, wherein the amino acid substitutions are S228P, F234A, L235A, Y349C, T366S, L368A, Y407V, and R409K. In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, wherein the first Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitutions according to EU Index at positions corresponding to positions 228, 234, 235, 349, 366, 368, 407, and 409 of human IgG4, wherein the amino acid substitutions are S228P, F234A, L235A, Y349C, T366S, L368A, Y407V, and R409K; and wherein the third polypeptide chain further comprises a second Fc region linked to CH1, wherein the second Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, wherein the second Fc region comprises amino acid substitutions, according to the EU Index, at positions corresponding to positions 228, 234, 235, 354, 366, and 409 of human IgG4, wherein the amino acid substitutions are S228P, F234A, L235A, S354C, T366W, and R409K.

In some embodiments, a binding protein of the present disclosure comprises one or more mutations to improve serum half-life (see, e.g., Hinton, p.r.et al (2006) j.immunol.176 (1): 346-56). In some embodiments, the mutation comprises substitutions at positions corresponding to positions 428 and 434 of human IgG1 or IgG4 according to EU Index, wherein the amino acid substitutions are M428L and N434S. In some embodiments, the binding protein comprises a second polypeptide chain further comprising a first Fc region linked to CH1, the first Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, the third polypeptide chain further comprising a second Fc region linked to CH1, the second Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first and/or second Fc region comprises amino acid substitutions according to EU Index at positions corresponding to positions 428 and 434 of human IgG1, wherein the amino acid substitutions are M428L and N434S. In some embodiments, a binding protein of the present disclosure comprises a knob and hole mutation and one or more mutations to improve serum half-life. In some embodiments, the first and/or second Fc region is a human IgG1 Fc region. In some embodiments, the first and/or second Fc region is a human IgG4 Fc region.

In some embodiments, the binding proteins of the present disclosure comprise one or more mutations to improve the stability of the hinge region and/or the dimeric interface of, for example, IgG4 (see, e.g., Spiess, c.et al (2013) j.biol.chem.288: 26583-. In some embodiments, the mutation comprises substitutions at positions corresponding to positions 228 and 409 of human IgG4 according to the EU Index, wherein the amino acid substitutions are S228P and R409K. In some embodiments, the binding protein comprises a second polypeptide chain further comprising a second polypeptide chain with CH1 a first Fc region comprising an immunoglobulin hinge region and CH2And CH3The immunoglobulin heavy chain constant domain, and the third polypeptide chain further comprises a second amino acid sequence substantially identical to CH1A linked second Fc region comprising an immunoglobulin hinge region and CH2And CH3An immunoglobulin heavy chain constant domain; wherein the first and second Fc regions are human IgG4 Fc regions; and wherein the first and second Fc regions comprise, according to the EU Index, at positions corresponding to positions 228 and 409 of human IgG4, respectivelyComprising amino acid substitutions, wherein the amino acid substitutions are S228P and R409K. In some embodiments, a binding protein of the present disclosure comprises a knob and hole mutation and one or more mutations to improve stability. In some embodiments, the first and/or second Fc region is a human IgG4 Fc region.

In some embodiments, the binding proteins of the present disclosure comprise one or more mutations to improve purification, e.g., by modulating affinity for a purification reagent. For example, it is known that heterodimeric binding proteins can be selectively purified from their homodimeric forms if one of the two Fc regions of the heterodimeric form contains a mutation that reduces or eliminates binding to protein a, because the heterodimeric form will have an intermediate affinity based on purification of protein a compared to either homodimeric form, and can be selectively eluted from protein a, e.g., by using a different pH (see, e.g., Smith, e.j.et al. (2015) sci.rep.5: 17943). In some embodiments, the mutation comprises substitutions at positions corresponding to positions 435 and 436 of human IgG1 or IgG4 according to the EU Index, wherein the amino acid substitutions are H435R and Y436F. In some embodiments, the binding protein comprises a second polypeptide chain further comprising a second polypeptide chain with CH1A linked first Fc region comprising an immunoglobulin hinge region and CH2And CH3The immunoglobulin heavy chain constant domain, and the third polypeptide chain further comprises a second amino acid sequence substantially identical to CH1A linked second Fc region comprising an immunoglobulin hinge region and C H2And CH3An immunoglobulin heavy chain constant domain; and wherein only one of the first and second Fc regions comprises amino acid substitutions at positions corresponding to positions 435 and 436 of human IgG1 or IgG4 according to the EU Index, wherein the amino acid substitutions are H435R and Y436F. In some embodiments, the binding proteins of the present disclosure comprise a knob and hole mutation and one or more mutations to improve purification. In some embodiments, the first and/or second Fc region is a human IgG1 Fc region. In some embodiments, the first and/or second Fc region is a human IgG4 Fc region.

In some embodiments, a binding protein of the present disclosure comprises one or more mutations to improve serum half-life (see, e.g., Hinton, p.r.et al (2006) j.immunol.176 (1): 346-56). In some embodiments, the mutation comprises substitutions at positions corresponding to positions 428 and 434 of human IgG1 or IgG4 according to EU Index, wherein the amino acid substitutions are M428L and N434S. In some embodiments, the binding protein comprises a second polypeptide chain further comprising a first Fc region linked to CH1, the first Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, and the third polypeptide chain further comprising a second Fc region linked to CH1, the second Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first and/or second Fc region comprises amino acid substitutions according to EU Index at positions corresponding to positions 428 and 434 of human IgG1, wherein the amino acid substitutions are M428L and N434S. In some embodiments, a binding protein of the present disclosure comprises a knob and hole mutation and one or more mutations to improve serum half-life. In some embodiments, the first and/or second Fc region is a human IgG1 Fc region. In some embodiments, the first and/or second Fc region is a human IgG4 Fc region.

In some embodiments, a binding protein of the present disclosure comprises one or more mutations to reduce effector function, such as Fc receptor-mediated antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), and/or antibody-dependent cellular cytotoxicity (ADCC). In some embodiments, the second polypeptide chain further comprises a second amino acid sequence substantially similar to CH1A linked first Fc region comprising an immunoglobulin hinge region and CH2And CH3An immunoglobulin heavy chain constant domain; wherein the third polypeptide chain further comprises a CH1A linked second Fc region comprising an immunoglobulin hinge region and CH2And CH3An immunoglobulin heavy chain constant domain; wherein the first and second Fc regions are human IgG1 Fc regions; and wherein the first and second Fc regions each comprise amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG1 according to EUIndex, wherein the amino acid substitutions are L234A and L235A. In some embodiments, the Fc regions of the second and third polypeptide chains are human IgG1 Fc regions, and wherein the Fc regions are at positions corresponding to positions 234 and 235 of human IgG1 according to EU IndexEach of (a) and (b) comprises an amino acid substitution, wherein the amino acid substitutions are L234A and L235A. In some embodiments, the second polypeptide chain further comprises a second amino acid sequence substantially similar to C H1A linked first Fc region comprising an immunoglobulin hinge region and CH2And CH3An immunoglobulin heavy chain constant domain; wherein the third polypeptide chain further comprises a CH1A linked second Fc region comprising an immunoglobulin hinge region and CH2And CH3An immunoglobulin heavy chain constant domain; wherein the first and second Fc regions are human IgG1 Fc regions; and wherein the first and second Fc regions each comprise amino acid substitutions at positions corresponding to positions 234, 235 and 329 of human IgG1 according to the EU Index, wherein the amino acid substitutions are L234A, L235A and P329A. In some embodiments, the Fc regions of the second and third polypeptide chains are human IgG1 Fc regions, and wherein the Fc regions each comprise an amino acid substitution at positions corresponding to positions 234, 235, and 329 of human IgG1 according to EU Index, wherein the amino acid substitutions are L234A, L235A, and P329A. In some embodiments, the Fc regions of the second and third polypeptide chains are human IgG4 Fc regions, and the Fc regions each comprise an amino acid substitution at positions corresponding to positions 234 and 235 of human IgG4 according to EU Index, wherein the amino acid substitutions are F234A and L235A. In some embodiments, the binding protein comprises a second polypeptide chain further comprising a second polypeptide chain with C H1A linked first Fc region comprising an immunoglobulin hinge region and CH2And CH3The immunoglobulin heavy chain constant domain, and the third polypeptide chain further comprises a second amino acid sequence substantially identical to CH1A linked second Fc region comprising an immunoglobulin hinge region and CH2And CH3An immunoglobulin heavy chain constant domain; and wherein the first and second Fc regions each comprise amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG4, according to the EU Index, wherein the amino acid substitutions are F234A and L235A.

In some embodiments, a binding protein of the present disclosure comprises a knob and hole mutation and one or more mutations to reduce effector function. In some embodiments, the first and/or second Fc region is a human IgG1 Fc region. In some embodiments, the first and/or second Fc region is a human IgG4 Fc region. For further description of the mutation at Fc position 329, see, e.g., Shields, r.l.et al (2001) j.biol. chem.276: 6591 and 6604 and WO 1999051642.

In some embodiments, the types of mutations described above may be combined in any order or combination. For example, a binding protein of the present disclosure may comprise two or more "knob" and "hole" mutations, one or more mutations to improve serum half-life, one or more mutations to improve IgG4 stability, one or more mutations to improve purification, and/or one or more mutations to reduce effector function as described above.

In some embodiments, the binding proteins of the present disclosure comprise antibody fragments, including but not limited to antibodies F (ab), F (ab ') 2, Fab' SH, Fv, or scFv fragments.

Measurement of

The present disclosure provides antigen binding proteins that bind to human and/or cynomolgus monkey CD38 polypeptides, induce proliferation of T cells (e.g., CD4+ and/or CD8+ T cells), and/or induce apoptosis of CD38+ cells. Provided herein are exemplary assays for measuring these parameters and identifying such binding proteins. For example, in some embodiments, the binding affinity between the binding protein or antigen-binding fragment thereof and the purified CD38 polypeptide is measured by SPR (e.g., as described below), and the binding affinity between the binding protein or antigen-binding fragment thereof and the CD38 polypeptide expressed on the surface of the cell is measured by flow cytometry (e.g., as described below).

In some embodiments, the antigen binding site of a binding protein of the present disclosure binds to a polypeptide comprising SEQ ID NO: 1, having an equilibrium dissociation constant (K) of human CD38 polypeptideD) Is 20nM or less, 15nM or less, 12nM or less, 10nM or less, 5nM or less, 2nM or less, 1nM or less, or 0.8nM or less, as determined by using a nucleic acid molecule comprising SEQ ID NO: 1, for example, as described below, as measured by a flow cytometric assay of cells for a human CD38 polypeptide. In some embodiments, the antigen binding site binds to a polypeptide comprising SEQ ID NO: 30, a cynomolgus monkey CD38 polypeptide having an amino acid sequence of seq id no Equilibrium dissociation constant (K)D) Is 20nM or less, 15nM or less, 12nM or less, 10nM or less, 5nM or less, 2nM or less, 1nM or less, or 0.75nM or less, as determined by using a nucleic acid molecule comprising SEQ ID NO: 30, e.g., as described below, as measured by a flow cytometric assay of cells of a cynomolgus monkey CD38 polypeptide. In some embodiments, the antigen binding site binds to a polypeptide comprising SEQ ID NO: 1, having an equilibrium dissociation constant (K) of human CD38 polypeptideD) Is 20nM or less, 15nM or less, 12nM or less, 10nM or less, 5nM or less, 2nM or less, 1nM or less, or 0.83nM or less, as determined by using a nucleic acid comprising SEQ ID NO: 1, for example, as described below. In some embodiments, the antigen binding site binds to a polypeptide comprising SEQ ID NO: 30, having an equilibrium dissociation constant (K) of a cynomolgus monkey CD38 polypeptideD) Is 20nM or less, 15nM or less, 12nM or less, 10nM or less, 5nM or less, 3.5nM or less, 1.5nM or less, or 1nM or less, as determined by using a nucleic acid comprising SEQ ID NO: 30, e.g., as described below, as measured by SPR assay of cynomolgus monkey CD38 polypeptide of amino acid sequence No. 30. As demonstrated herein, in some embodiments, the binding proteins of the present disclosure can have one or more of the exemplary binding characteristics described herein. In some embodiments, the KD is measured at 4 ℃ or 5 ℃.

In some embodiments, the monospecific binding proteins of the present disclosure have one or more of the following characteristics: binds the extracellular domain of a human CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 1) as a purified protein, as determined by SPR or ELISA; binding the extracellular domain of a human CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 1) as a purified protein, K thereofDIs 20nM or less, 15nM or less, 12nM or less, 10nM or less, 5nM or less, 2nM or less, or 1.5nM or less as determined by SPR or ELISA; binds to an extracellular domain of a human CD38 polypeptide expressed on the surface of a cell (e.g., comprising the amino acid sequence of SEQ ID NO: 1), as determined by flow cytometry; binding expressed on the surface of cellsThe extracellular domain of a human CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 1), its apparent KDIs 20nM or less, 15nM or less, 12nM or less, 10nM or less, 5nM or less, 2nM or less, or 1nM or less as determined by flow cytometry; binds to the extracellular domain of a cynomolgus monkey CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 30) as a purified protein, as determined by SPR or ELISA; k binding to the extracellular domain of a cynomolgus monkey CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 30) as a purified protein DIs 20nM or less, 15nM or less, 12nM or less, 10nM or less, 5nM or less, 2nM or less, or 1nM or less as determined by SPR or ELISA; binds to an extracellular domain of a cynomolgus monkey CD38 polypeptide expressed on the surface of a cell (e.g., comprising the amino acid sequence of SEQ ID NO: 30), as determined by flow cytometry; binding to the extracellular domain of a cynomolgus monkey CD38 polypeptide expressed on the surface of a cell (e.g., comprising the amino acid sequence of SEQ ID NO: 30), which has an apparent KDIs 20nM or less, 15nM or less, 12nM or less, 10nM or less, 5nM or less, 2nM or less, or 1nM or less as determined by flow cytometry; binds to the extracellular domain of a human isoform E CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 105) as a purified protein, as determined by SPR or ELISA; binds to an extracellular domain of a human isoform E CD38 polypeptide expressed on the surface of a cell (e.g., comprising the amino acid sequence of SEQ ID NO: 105), as determined by flow cytometry; inducing apoptosis or antibody-dependent cellular cytotoxicity (ADCC) of cells expressing CD38 on their cell surface; having one or more mutations (e.g., in the Fc region) results in reduced binding to Fc γ RI and/or Fc γ RII as compared to the same binding protein without the one or more mutations. In some embodiments, a binding protein of the disclosure binds a CD38 polypeptide (e.g., human or cynomolgus monkey) expressed on the surface of a cell with an EC50 of 20nM or less, 15nM or less, 12nM or less, 10nM or less, 5nM or less, 2nM or less, or 1nM or less as determined by flow cytometry. In some embodiments, the binding proteins of the present disclosure bind to a CD38 polypeptide (e.g., human or cynomolgus monkey) as a purification A protein having an EC50 of 20nM or less, 15nM or less, 12nM or less, 10nM or less, 5nM or less, 2nM or less, or 1nM or less as determined by ELISA. In some embodiments, the KD is measured at 4 ℃ or 25 ℃.

In some embodiments, the trispecific binding proteins of the present disclosure have one or more of the following characteristics: induction of proliferation of T cells (e.g., CD4+ and/or CD8+ T cells), induction of expression of T cells of Bcl-xL (e.g., CD4+ and/or CD8+ T cells), induction of apoptosis of CD38+ cells (e.g., as measured by annexin V staining and/or propidium iodide uptake), binding to CD38 expressed on the cell surface and one or more T cell target antigens expressed on the T cell surface, binding to CD38 expressed on the cell surface, CD28 expressed on the T cell surface and CD3 expressed on the T cell surface, stimulation of activation of T cell receptors (e.g., as measured by CD69 expression), induction of co-stimulation of T cell receptor signaling (e.g., as mediated by CD 362), compared to the same binding protein or a plurality of mutations, having one or more mutations (e.g., as measured in the same binding protein sequence of CD 69), release of one or more mutations (e.g., in the same binding protein), as a polypeptide, e.g., a polypeptide comprising IFN-gamma-IFN-binding protein, such as measured by IFN-CD-NO-or a polypeptide, such as measured by ELISA, or by the amino acid sequence of a polypeptide, or by the expression of a polypeptide, such as measured in the expression of a polypeptide, or by the expression of a polypeptide, such as measured in the cell receptor signaling of DIs 1.5nM or less as determined by SPR or ELISA; binds to an extracellular domain of a human CD38 polypeptide expressed on the surface of a cell (e.g., comprising the amino acid sequence of SEQ ID NO: 1), as determined by flow cytometry; binds to the extracellular domain of a human CD38 polypeptide expressed on the surface of a cell (e.g., comprising the amino acid sequence of SEQ ID NO: 1), which has an apparent KDIs 12nM or less as determined by flow cytometry; binds to the extracellular domain of a cynomolgus monkey CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 30) as a purified protein, as determined by SPR or ELISA; combined with cynomolgus monkey CD38The extracellular domain of a peptide (e.g., comprising the amino acid sequence of SEQ ID NO: 30) as a purified protein, K thereofDIs 3.5nM or less as determined by SPR or ELISA; binds to an extracellular domain of a cynomolgus monkey CD38 polypeptide expressed on the surface of a cell (e.g., comprising the amino acid sequence of SEQ ID NO: 30), as determined by flow cytometry; binding to the extracellular domain of a cynomolgus monkey CD38 polypeptide expressed on the surface of a cell (e.g., comprising the amino acid sequence of SEQ ID NO: 30), which has an apparent KDIs 7.5nM or less as determined by flow cytometry; binds to the extracellular domain of a human isoform E CD38 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 105) as a purified protein, as determined by SPR or ELISA; binds to an extracellular domain of a human isoform E CD38 polypeptide expressed on the surface of a cell (e.g., comprising the amino acid sequence of SEQ ID NO: 105), as determined by flow cytometry; inducing apoptosis or antibody-dependent cellular cytotoxicity (ADCC) of cells expressing CD38 on their cell surface; having one or more mutations (e.g., in the Fc region) results in reduced binding to Fc γ RI and/or Fc γ RII as compared to the same binding protein without the one or more mutations. In some embodiments, a binding protein of the disclosure binds a CD38 polypeptide (e.g., human or cynomolgus monkey) expressed on the surface of a cell with an EC50 of 20nM or less, 15nM or less, 12nM or less, 10nM or less, 5nM or less, 2nM or less, or 1nM or less as determined by flow cytometry. In some embodiments, a binding protein of the disclosure binds a CD38 polypeptide (e.g., human or cynomolgus monkey) as a purified protein with an EC50 of 20nM or less, 15nM or less, 12nM or less, 10nM or less, 5nM or less, 2nM or less, or 1nM or less, as determined by ELISA.

Nucleic acids

Polynucleotides encoding polypeptides that form binding proteins are constructed using standard recombinant DNA methods, incorporated into recombinant expression vectors, and such vectors are introduced into host cells. See, e.g., Sambrook et al, 2001, MOLECULARCLONING：A LABORATORYMANUAL(Cold Spring Harbor Laboratory Press, 3 rd.). Enzymatic reactionAnd purification techniques can be performed according to the manufacturer's instructions, as is commonly done in the art, or as described herein. Unless specific definitions are provided, nomenclature used in connection with, and laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and pharmaceutical and medicinal chemistry described herein are those well known and commonly used in the art. Similarly, conventional techniques may be used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, delivery, and patient treatment.

Other aspects of the disclosure relate to isolated nucleic acid molecules comprising a nucleotide sequence encoding any of the binding proteins described herein. In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence that is identical to SEQ ID NO: 60-83 at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91% of the sequence. At least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical and/or a sequence as set forth in table J.

Certain aspects of the present disclosure relate to kits of polynucleotides. In some embodiments, one or more polynucleotides is a vector (e.g., an expression vector). The kits, among other things, are useful for producing one or more binding proteins described herein, e.g., trispecific binding proteins of the present disclosure. In some embodiments, the kit comprises one, two, three, or four nucleotides as set forth in table J (e.g., of mAb2xCD28supxCD3mid IgG4 FALA, mAb2xCD28supxCD3mid IgG1LALA P329A, mAb2xCD28supxCD3mid IgG1 NNSA, mAb6xCD28supxCD3mid IgG4 FALA, mAb6xCD28supxCD3mid IgG1LALA P329A, or mAb6xCD28supxCD3mid IgG1 NNSA). In some embodiments, the polynucleotide kit comprises: comprises the amino acid sequence of SEQ ID NO: 73 comprising the sequence of SEQ ID NO: 72, comprising the sequence of SEQ ID NO: 74, and a third polynucleotide comprising the sequence of SEQ ID NO: 75. In some embodiments, the polynucleotide kit comprises: comprises the amino acid sequence of SEQ ID NO: 73 comprising the sequence of SEQ ID NO: 76 comprising the sequence of SEQ ID NO: 77, and a third polynucleotide comprising the sequence of SEQ ID NO: 75. In some embodiments, the polynucleotide kit comprises: comprises the amino acid sequence of SEQ ID NO: 73 comprising the sequence of SEQ ID NO: 78 comprising the sequence of SEQ ID NO: 79, and a third polynucleotide comprising the sequence of SEQ ID NO: 75. In some embodiments, the polynucleotide kit comprises: comprises the amino acid sequence of SEQ ID NO: 73 comprising the sequence of SEQ ID NO: 72, comprising the sequence of SEQ ID NO: 80, and a third polynucleotide comprising the sequence of SEQ ID NO: 81, or a third polynucleotide of the sequence of seq id no. In some embodiments, the polynucleotide kit comprises: comprises the amino acid sequence of SEQ ID NO: 73 comprising the sequence of SEQ ID NO: 76 comprising the sequence of SEQ ID NO: 82, and seq id NO: 81, or a third polynucleotide of the sequence of seq id no. In some embodiments, the polynucleotide kit comprises: comprises the amino acid sequence shown in SEQ ID NO: 73 comprising the sequence of SEQ ID NO: 78 comprising the sequence of SEQ ID NO: 83, and a third polynucleotide comprising the sequence of SEQ ID NO: 81, or a third polynucleotide of the sequence of seq id no.

In some embodiments, the isolated nucleic acid is operably linked to a heterologous promoter to direct transcription of a binding protein-encoding nucleic acid sequence. A promoter may refer to a nucleic acid control sequence that directs transcription of a nucleic acid. The first nucleic acid sequence is operably linked to the second nucleic acid sequence when the first nucleic acid sequence is in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence for a binding protein if the promoter affects the transcription or expression of the coding sequence. Examples of promoters may include, but are not limited to, promoters obtained from the genome of viruses such as polyoma virus, fowlpox virus, adenovirus (such as adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retroviruses, hepatitis B virus, simian virus 40(SV40), and the like, promoters from heterologous eukaryotic promoters (such as actin promoter, immunoglobulin promoter, from heat shock promoter, and the like), CAG promoter (Niwa et al, Gene 108 (2): 193-9, 1991), phosphoglycerate kinase (PGK) -promoter, tetracycline-inducible promoter (Masui et al, Nucleic Acids Res.33: e43, 2005), lac system, trp system, tac system, trc system, major operator and promoter regions of bacteriophage lambda, promoter of 3-phosphoglycerate kinase, promoter of yeast acid phosphatase, and a promoter for yeast α -mating factor. The polynucleotides encoding the binding proteins of the present disclosure may be under the control of a constitutive promoter, an inducible promoter, or any other suitable promoter described herein or readily recognized by one of skill in the art.

In some embodiments, the isolated nucleic acid is incorporated into a vector. In some embodiments, the vector is an expression vector. The expression vector may include one or more regulatory sequences operably linked to the polynucleotide to be expressed. The term "regulatory sequence" includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Examples of suitable enhancers may include, but are not limited to, enhancer sequences from mammalian genes (such as globin, elastase, albumin, alpha-fetoprotein, insulin, etc.), and enhancer sequences from eukaryotic viruses (such as the SV40 enhancer on the posterior side of the origin of replication (bp100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the posterior side of the origin of replication, adenovirus enhancers, etc.). Examples of suitable vectors can include, for example, plasmids, cosmids, episomes, transposons, and viral vectors (e.g., adenovirus, vaccinia virus, Sindbis-virus, measles, herpes virus, lentivirus, retrovirus, adeno-associated viral vectors, and the like). The expression vectors can be used to transfect host cells such as, for example, bacterial cells, yeast cells, insect cells, and mammalian cells. Biologically functional viral and plasmid DNA vectors capable of expression and replication in a host are known in the art and can be used to transfect any cell of interest.

Other aspects of the disclosure relate to a vector system comprising one or more vectors encoding the first, second, third, and fourth polypeptide chains of any of the binding proteins described herein. In some embodiments, the vector system comprises a first vector encoding a first polypeptide chain of a binding protein, a second vector encoding a second polypeptide chain of a binding protein, a third vector encoding a third polypeptide chain of a binding protein, and a fourth vector encoding a fourth polypeptide chain of a binding protein. In some embodiments, the vector system comprises a first vector encoding a first and second polypeptide chain of a binding protein, and a second vector encoding a third and fourth polypeptide chain of a binding protein. In some embodiments, the vector system comprises a first vector encoding a first and third polypeptide chain of a binding protein, and a second vector encoding a second and fourth polypeptide chain of a binding protein. In some embodiments, the vector system comprises a first vector encoding a first and fourth polypeptide chain of a binding protein, and a second vector encoding a second and third polypeptide chain of a binding protein. In some embodiments, the vector system comprises a first vector encoding a first, second, third, and fourth polypeptide chain of a binding protein. The one or more vectors of the vector system may be any of the vectors described herein. In some embodiments, one or more vectors are expression vectors.

Isolated host cell

Other aspects of the disclosure relate to isolated host cells comprising one or more of the isolated polynucleotides, polynucleotide kits, vectors, and/or vector systems described herein. In some embodiments, the host cell is a bacterial cell (e.g., an e. In some embodiments, the host cell is a yeast cell (e.g., a saccharomyces cerevisiae (s.cerevisiae) cell). In some embodiments, the host cell is an insect cell. Examples of insect host cells can include, for example, Dropophila (Dropophila) cells (e.g., S2 cells), Trichoplusia ni (Trichoplusia ni) cells (e.g., High Five cells)TMCells) and Spodoptera frugiperda (Spodoptera frugiperda) cells (e.g., Sf21 or Sf9 cells). In some embodiments, the host cell is a mammalian cell. Examples of mammalian host cells can include, for example, human embryonic kidney cells (e.g., 293 or 293 cells subcloned for growth in suspension culture), Expi293TM cells, CHO cells, baby hamster kidney cells (e.g., BHK, ATCCCCL 10), mouse support cells (e.g., TM4 cells), monkey kidney cells (e.g., CV1 ATCC CCL 70), VERO monkey kidney cells (e.g., VERO-76, ATCC CRL-1587), human cervical cancer cells (e.g., HELA, ATCC CCL 2), canine kidney cells (e.g., MDCK, ATCC CCL 34), buffalo rat liver cells (e.g., BRL 3A, ATCC CRL 1442), human lung cells (e.g., W138, ATCC CCL 75), human liver cells (e.g., Hep G2, HB8065), mouse mammary tumor cells (e.g., MMT 060562, ATCC CCL51), TRI cells, MRC 5 cells, FS4 cells, human hepatoma lines (e.g., hepg., HepG2), and myeloma cells (e.g., NS0 and Sp2/0 cells).

Other aspects of the disclosure relate to methods of producing any of the binding proteins described herein. In some embodiments, the method comprises a) culturing a host cell (e.g., any of the host cells described herein) comprising an isolated nucleic acid, vector, and/or vector system (e.g., any of the isolated nucleic acids, vectors, and/or vector systems described herein) under conditions in which the host cell expresses the binding protein; b) isolating the binding protein from the host cell. Methods for culturing host cells under conditions for expression of the protein are well known to those of ordinary skill in the art. Methods for isolating proteins from cultured host cells are well known to those of ordinary skill in the art and include, for example, affinity chromatography (e.g., two-step affinity chromatography including protein a affinity chromatography followed by size exclusion chromatography).

In some embodiments, the binding proteins of the present disclosure are purified by protein a affinity chromatography, kappa light chain affinity chromatography (e.g., using kappasselect resin; GE Healthcare according to manufacturer's instructions), and optionally lambda light chain affinity chromatography (e.g., using LambdaFabSelect resin; GE Healthcare according to manufacturer's instructions). In some embodiments, the binding proteins of the present disclosure are purified by protein a affinity chromatography, λ light chain affinity chromatography (e.g., using LambdaFabSelect resin; GE Healthcare according to the manufacturer's instructions), and optionally κ light chain affinity chromatography (e.g., using KappaSelect resin; GE Healthcare according to the manufacturer's instructions). In some embodiments, the binding protein comprises two Fc regions, each comprising C H3A domain according toEU Index, only one CH3The domain comprises amino acid substitutions at positions corresponding to positions 435 and 436 of human IgG1 or IgG4, wherein the amino acid substitutions are H435R and Y436F. In some embodiments, the binding proteins of the present disclosure are purified sequentially by protein a affinity chromatography, followed by kappa light chain affinity chromatography (e.g., using kappa light chain affinity resin; GE Healthcare according to the manufacturer's instructions), followed optionally by lambda light chain affinity chromatography (e.g., using LambdaFabSelect resin; GE Healthcare according to the manufacturer's instructions). In some embodiments, the binding proteins of the present disclosure are purified sequentially by protein a affinity chromatography, followed by λ light chain affinity chromatography (e.g., using LambdaFabSelect resin; GE Healthcare according to the manufacturer's instructions), followed optionally by κ light chain affinity chromatography (e.g., using kappa-select resin; GE Healthcare according to the manufacturer's instructions). For example, in some embodiments, the binding protein is contacted with protein a at a temperature suitable for substituting the binding protein from a protein comprising 0 or 2C containing amino acid substitutions to H435R and Y436FH3Binding protein separation of domains from protein A, contact with kappa light chain affinity media (e.g., resins as used in KappaSelect; GE Healthcare), and separation of binding proteins from proteins comprising only lambda C LBinding proteins for the domains are eluted from the kappa light chain affinity medium under conditions for separation (e.g., according to the manufacturer's instructions). Suitable conditions for protein A elution are known in the art and include, but are not limited to, a step elution gradient at pH 4.5-2.8. In some embodiments, the use is useful for protein purification of protein a or protein a variants. In some embodiments, protein a is attached to a substrate or resin, e.g., as part of a chromatography medium. In some embodiments, after elution from the kappa light chain affinity medium, the binding protein is contacted with a lambda light chain affinity medium (e.g., as used in a LambdaFabSelect resin; GE Healthcare) and eluted from the lambda light chain affinity medium under conditions suitable to separate the binding protein from binding proteins comprising only the kappa CL domain (e.g., according to the manufacturer's instructions). In some embodiments, the binding proteins of the present disclosure are detected using HIC chromatography. In some embodiments, the binding protein comprises: a first polypeptide chainWhich comprises λ CLA domain; c of the second polypeptide chainH3A domain comprising amino acid substitutions according to EU Index at positions corresponding to positions 354 and 366 of human IgG1 or IgG4, wherein the amino acid substitutions are S354C and T366W; c of the third polypeptide chain H3A domain comprising amino acid substitutions according to EU Index at positions corresponding to positions 349, 366, 368, 407, 435 and 436 of human IgG1 or IgG4, wherein the amino acid substitutions are Y349C, T366S, L368A, Y407V, H435R and Y436F; and a fourth polypeptide chain comprising a kappa CLA domain. In some embodiments, the binding protein is produced by a host cell. In some embodiments, the binding protein is purified from a cell culture medium or host cell extract. In some embodiments, the binding protein is secreted by the host cell or produced and extracted from the host cell (e.g., prior to contact with protein a). In some embodiments, the binding protein is in a cell culture medium or host cell extract when contacted with protein a. In some embodiments, the binding protein is purified from other binding proteins, polypeptides, and/or other cellular components.

Trispecific binding proteins

In some embodiments, the binding proteins of the present disclosure are trispecific and/or trivalent binding proteins comprising four polypeptide chains forming three antigen binding sites that bind one or more (e.g., three) different antigen targets or target proteins. In some embodiments, the first polypeptide chain comprises a structure represented by the formula:

VL2-L1-VL1-L2-CL[I]

And the second polypeptide chain comprises a structure represented by the formula:

VH1-L3-VH2-L4-CH1-hinge-CH2-CH3[II]

And the third polypeptide chain comprises a structure represented by the formula:

VH3-CH1-hinge-CH2-CH3[III]

And the fourth polypeptide chain comprises a structure represented by the formula:

VL3-CL[IV]

wherein:

VL1is a first immunoglobulin light chain variable domain;

VL2is a second immunoglobulin light chain variable domain;

VL3is a third immunoglobulin light chain variable domain;

VH1is a first immunoglobulin heavy chain variable domain;

VH2is a second immunoglobulin heavy chain variable domain;

VH3is a third immunoglobulin heavy chain variable domain;

CLis an immunoglobulin light chain constant domain;

CH1is immunoglobulin CH1A heavy chain constant domain;

CH2is immunoglobulin CH2A heavy chain constant domain;

CH3is immunoglobulin CH3A heavy chain constant domain;

the hinge being a connection CH1And CH2An immunoglobulin hinge region of the domain; and

L1、L2、L3and L4Is an amino acid linker;

wherein the polypeptide of formula I and the polypeptide of formula II form a cross light-heavy chain pair. In some embodiments, the first polypeptide chain and the second polypeptide chain have a cross-orientation that forms two different antigen binding sites. As described above, the second and third polypeptide chains comprise an Fc region (e.g., comprising hinge-C)H2-CH3A domain). In some embodiments, one or both Fc regions is a human IgG4 Fc region comprising an amino acid substitution according to EU Index at a position corresponding to position 233-236 of human IgG 4. In some embodiments, the amino acid substitution is E233P, F234V, L235A, and the deletion at 236. In some embodiments, the Fc region is a human IgG4 Fc region, which corresponds to human IgG4 at 228, 233-236, and 236 according to the EU Index And/or the substitution at position 409 comprises an amino acid mutation. In some embodiments, the amino acid mutation is S228P; E233P, F234V, L235A and the deletion at 236; and/or R409K. In some embodiments, one or both Fc regions is a human IgG1Fc region comprising one or more amino acid substitutions according to EUIndex at positions corresponding to positions 234, 235, and/or 329 of human IgG 1. In some embodiments, the amino acid substitution is L234A, L235A, and/or P329A. In some embodiments, the Fc region is a human IgG1Fc region comprising amino acid substitutions according to EU Index at positions corresponding to positions 298, 299, and/or 300 of human IgG 1. In some embodiments, the amino acid substitution is S298N, T299A, and/or Y300S.

In some embodiments, the binding proteins of the present disclosure are trispecific and/or trivalent binding proteins comprising four polypeptide chains forming three antigen binding sites that bind one or more (e.g., three) different antigen targets or target proteins. In some embodiments, at least one antigen binding site binds to a CD38 polypeptide (e.g., the extracellular domain of a human and/or cynomolgus monkey CD38 polypeptide). In some embodiments, the first polypeptide chain comprises a structure represented by the formula:

VL2-L1-VL1-L2-CL[I]

And the second polypeptide chain comprises a structure represented by the formula:

VH1-L3-VH2-L4-CH1[II]

and the third polypeptide chain comprises a structure represented by the formula:

VH3-CH1[III]

and the fourth polypeptide chain comprises a structure represented by the formula:

VL3-CL[IV]

wherein:

VL1is a first immunoglobulin light chain variable domain;

VL2is a second immunoglobulin light chain variable domain;

VL3is a third immunoglobulin light chain variable structureA domain;

VH1is a first immunoglobulin heavy chain variable domain;

VH2is a second immunoglobulin heavy chain variable domain;

VH3is a third immunoglobulin heavy chain variable domain;

CLis an immunoglobulin light chain constant domain;

CH1is immunoglobulin CH1A heavy chain constant domain; and

L1、L2、L3and L4Is an amino acid linker;

and wherein the polypeptide of formula I and the polypeptide of formula II form a cross light-heavy chain pair. In some embodiments, the second and third polypeptide chains further comprise a peptide with CH1A linked Fc region comprising an immunoglobulin hinge region and CH2And CH3An immunoglobulin heavy chain constant domain. In some embodiments, one or both Fc regions is a human IgG4 Fc region comprising amino acid substitutions at positions corresponding to position 233-236 of human IgG4 according to EUIndex. In some embodiments, the amino acid substitution is E233P, F234V, L235A, and the deletion at 236. In some embodiments, the Fc region is a human IgG4 Fc region comprising amino acid mutations at substitutions corresponding to positions 228, 233-236, and/or 409 of human IgG4, according to the EU Index. In some embodiments, the amino acid mutation is S228P; E233P, F234V, L235A and the deletion at 236; and/or R409K. In some embodiments, one or both Fc regions is a human IgG1Fc region comprising one or more amino acid substitutions according to EU Index at positions corresponding to positions 234, 235, and/or 329 of human IgG 1. In some embodiments, the amino acid substitution is L234A, L235A, and/or P329A. In some embodiments, the Fc region is a human IgG1Fc region comprising amino acid substitutions according to EU Index at positions corresponding to positions 298, 299, and/or 300 of human IgG 1. In some embodiments, the amino acid substitution is S298N, T299A, and/or Y300S.

In some embodiments, VH1 and VL1 form a binding pair and form a first antigen binding site. In some embodiments, VH2 and VL2 form a binding pair and form a second antigen binding site. In some embodiments, VH3 and VL3 form a binding pair and form a third antigen binding site. Binding proteins may also be used for cell activation, tumor targeting, neutralization of cytokine activity, neutralization of viral infections, a combination of multiple signaling events to treat cancer, arthritis, and/or inflammatory disorders. For example, in some embodiments, the binding protein specifically binds to one, two, or three antigen targets selected from the group consisting of: a2AR, APRIL, ATP diphosphohydrolase, BAFF, BAFFR, BCMA, BlyS, BTK, BTLA, B7DC, B7H1, B7H4 (also known as VTCN1), B7H5, B7H6, B7H7, B7RP1, B7-4, C3, C5, CCL2 (also known as MCP-1), CCL 53 (also known as MIP-1a), CCL4 (also known as MIP-1B), CCL5 (also known as TES), CCL7 (also known as MCP-3), CCL8 (also known as MCP-2), CCL11 (also known as eotaxin), CCL11 (also known as MIP-1d), CCL11 (also known as TARC), CCL11 (also known as MIP-3B), CCL11 (also known as MPCK-3 a), CCL11 (also known as eosinophil-72), CCL11 (also known as eosinophil-11) and CTF-11 (also known as eosinophil-11/or eotaxin-11), or CTF-11 (also known as CCL-11) or eotaxin-11, or MCK, CCR, CD (also known as FCER, an IgE receptor), CD272, CD273 (also known as PDL), CD274 (also known as PDL), CD275 (also known as B7H), CD276 (also known as B7H), CD278 (also known as ICOS), CD279 (also known as PD-1), CDH (also known as E-cadherin), chitinase, CLEC, CRTH, CSF-1 (also known as M-CSF), DNCSF-2 (also known as GM-GR), GCSF-3 (also known as GCSF), CX3CL (also known as SCYD), CXCL (also known as SDF), CXCL, ecto-1, CXCR1, and CXCR1, EGFR, ENTPD, FCER1, FCER, FLAP, FOLH, Gi, GITR, GITRL, GM-CSF, Her, HHLA, HMGB, HVEM, ICOSLG, IDO, IFN α, IgE, IGF1, IL2 β, IL1F, IL4, IL5, IL7, IL9, IL, rhIL, IL13Ra, IL17 (also known as IL receptor), IL, PROM, IL, ITGB (also known as b integrin), ITK, KIR, LAG, LAMP, leptin, LPFS, MHC class II, NCR3LG, NKG2, NTP diphosphatase-1, Vs40, PD-1H, platelet receptor, SIS, Tps 152, SLC, TSST (also known as TSK, TSCI, TLR, IL1, IL7, IL1, IL7, IL, WUCAM and XCR1 (also known as GPR5/CCXCR 1). In some embodiments, one or more of the above antigen targets is a human antigen target.

In some embodiments, one of the three antigen binding sites binds to a CD3 polypeptide (e.g., a human CD3 polypeptide), one of the three antigen binding sites binds to a CD28 polypeptide (e.g., a human CD28 polypeptide), and one of the three antigen binding sites binds to a third polypeptide. In some embodiments, the antigen binding site that specifically binds to an antigen target other than CD3 or CD28 binds to an antigen target selected from the group consisting of: a2AR, APRIL, ATP diphosphohydrolase, BAFF, BAFFR, BCMA, BlyS, BTK, BTLA, B7DC, B7H1, B7H4 (also known as VTCN1), B7H5, B7H6, B7H7, B7RP1, B7-4, C3, C5, CCL2 (also known as MCP-1), CCL 53 (also known as MIP-1a), CCL4 (also known as MIP-1B), CCL5 (also known as TES), CCL7 (also known as MCP-3), CCL8 (also known as MCP-2), CCL11 (also known as eotaxin), CCL11 (also known as MIP-1d), CCL11 (also known as TARC), CCL11 (also known as MIP-3B), CCL11 (also known as MPCK-3 a), CCL11 (also known as eosinophil-72), CCL11 (also known as eosinophil-11) and CTF-11 (also known as eosinophil-11/or eotaxin-11), or CTF-11 (also known as CCL-11) or eotaxin-11, or MCK, CCR, CD (also known as FCER, an IgE receptor), CD272, CD273 (also known as PDL), CD274 (also known as PDL), CD275 (also known as B7H), CD276 (also known as B7H), CD278 (also known as ICOS), CD279 (also known as PD-1), CDH (also known as E-cadherin), chitinase, CLEC, CRTH, CSF-1 (also known as M-CSF), DNCSF-2 (also known as GM-GR), GCSF-3 (also known as GCSF), CX3CL (also known as SCYD), CXCL (also known as SDF), CXCL, ecto-1, CXCR1, and CXCR1, EGFR, ENTPD, FCER1, FCER, FLAP, FOLH, Gi, GITR, GITRL, GM-CSF, Her, HHLA, HMGB, HVEM, ICOSLG, IDO, IFN α, IgE, IGF1, IL2 β, IL1F, IL4, IL5, IL7, IL9, IL, rhIL, IL13Ral, IL13Ra, IL17 (also known as IL receptor), IL, ITGB (also known as b integrin), ITK, KIR, LAG, LAMP, leptin, LPFS, MHC class II, NCR3LG, NKG2, NTP diphosphatase-1, Vss, STE40, PD-1H, platelet receptor, TMS 152, SISP, SLC, TSST, TLR, and T, WUCAM and XCR1 (also known as GPR5/CCXCR 1). In some embodiments, one or more of the above antigen targets is a human antigen target.

In any of the trispecific binding proteins described above, any linker or combination of linkers described herein may be used. For example, in some embodiments, L1、L2、L3Or L4Is independently 0 amino acids in length. In some embodiments, L is1、L2、L3Or L4Is each independently at least 1 amino acid in length. In some embodiments, L is1、L2、L3And L4Each independently is 0 amino acids in length or comprises a sequence selected from the group consisting of: GGGGSGGGGS (SEQ ID NO: 55), GGGGSGGGGSGGGGS (SEQ ID NO: 56), S, RT, TKGPS (SEQ ID NO: 57), GQPKAAP (SEQ ID NO: 58) and GGSGSSGSGG (SEQ ID NO: 59). In some embodiments, L is1、L2、L3And L4Each independently comprises a sequence selected from the group consisting of: GGGGSGGGGS (SEQ ID NO: 55), GGGGSGGGGSGGGGS (SEQ ID NO: 56), S, RT, TKGPS (SEQ ID NO: 57), GQPKAAP (SEQ ID NO: 58) and GGSGSSGSGG (SEQ ID NO: 59). In some embodiments, L is1Comprising the sequence GQPKAAP (SEQ ID NO: 58), L2Comprising the sequence TKGPS (SEQ ID NO: 57), L3Comprising the sequence S, L4Comprising the sequence RT. In some embodiments, L is1Comprising the sequence GGGGSGGGGS (SEQ ID NO: 55), L2Comprising the sequence GGGGSGGGGS (SEQ ID NO: 55), L 3Is 0 amino acids in length, L4Is 0 amino acids in length. In some embodiments, L is1Comprising the sequence GGSGSSGSGG (SEQ ID NO: 59), L2Comprising the sequence GGSGSSGSGG (SEQ ID NO: 59), L3Is 0 amino acids in length, L4Is 0 amino acids in length. In some embodiments, L is1Comprising the sequence GGGGSGGGGSGGGS (SEQ ID NO: 56), L2Is 0 amino acids in length, L3Comprising the sequence GGGGSGGGGSGGGS (SEQ ID NO: 56), L4Is 0 amino acids in length.

Use of binding proteins

The binding proteins can be used in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays for detecting and quantifying one or more target antigens. The binding protein will bind to one or more target antigens with an affinity suitable for the assay method used.

For diagnostic applications, in certain embodiments, the binding protein may be labeled with a detectable moiety. The detectable moiety may be any moiety capable of directly or indirectly generating a detectable signal. For example, the detectable moiety may be a radioisotope, such as3H、14C、32P、35S、125I、99Tc、111In or67Ga, fluorescent or chemiluminescent compounds, such as fluorescein isothiocyanate, rhodamine or fluorescein, or enzymes, such as alkaline phosphatase, β -galactosidase or horseradish peroxidase 。

Binding proteins may also be used for in vivo imaging. The binding protein labeled with a detectable moiety can be administered to an animal, preferably in the bloodstream, and the presence and location of the labeled antibody in the host determined. The binding protein may be labeled with any moiety that is detectable in the animal by means of detection, whether by nuclear magnetic resonance, radiology, or other means of detection known in the art.

For clinical or research applications, in certain embodiments, the binding protein may be conjugated to a cytotoxic agent. Various antibodies conjugated to cytotoxic agents (i.e., antibody-drug conjugates) have been used to target cytotoxic payloads to specific tumor cells. Cytotoxic agents and linkers that conjugate the agent to an antibody are known in the art; see, e.g., Parslow, a.c.et al. (2016) Biomedicines 4: 14and Kalim, m.et al (2017) drug des.devel.ther.11: 2265-2276.

The present disclosure also relates to a kit comprising a binding protein and other reagents for detecting the level of a target antigen in a biological sample. Such reagents may include detectable labels, blocking sera, positive and negative control samples, and detection reagents. In some embodiments, the kit comprises a composition comprising any of the binding proteins, polynucleotides, vectors, vector systems, and/or host cells described herein. In some embodiments, the kit comprises a container and a label or package insert (package insert) on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. The container may be formed from a variety of materials, such as glass or plastic. The container contains the composition by itself, either by itself or in combination with another composition effective in treating, preventing and/or diagnosing a condition, and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). In some embodiments, the label or package insert indicates that the composition is for use in the prevention, diagnosis, and/or treatment of a selected condition. Alternatively, or in addition, the article of manufacture or kit may further comprise a second (or third) container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution, and dextrose solution. It may further include other materials as desired from a commercial and user perspective, including other buffers, diluents, filters, needles and syringes.

In some embodiments, the binding proteins of the present disclosure are administered to a patient in need thereof to treat or prevent cancer. In some embodiments, the present disclosure relates to methods of preventing and/or treating a proliferative disease or disorder (e.g., cancer). In some embodiments, the method comprises administering to the patient a therapeutically effective amount of at least one binding protein described herein or a pharmaceutical composition related thereto. In some embodiments, the present disclosure relates to the use of at least one binding protein described herein or a pharmaceutical composition related thereto for the prevention and/or treatment of a proliferative disease or disorder (e.g., cancer) in a patient in need thereof. In some embodiments, the present disclosure relates to at least one binding protein described herein or a pharmaceutical composition related thereto for the preparation of a medicament for the prevention and/or treatment of a proliferative disease or disorder (e.g., cancer) in a patient in need thereof. In some embodiments, the patient is a human. In some embodiments, the binding protein comprises one antigen binding site that binds to a T cell surface protein and another antigen binding site that binds to an extracellular domain of a human CD38 polypeptide, e.g., as described above in section II. In some embodiments, the binding protein comprises an antigen binding site that binds the extracellular domain of a human CD38 polypeptide, an antigen binding site that binds a human CD28 polypeptide, and an antigen binding site that binds a human CD3 polypeptide.

In some embodiments, the cancer cell expresses a human CD38 isoform a polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 1) on its cell surface. In some embodiments, the cancer cell expresses a human CD38 isoform E polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 105) on its cell surface. In some embodiments, the patient is selected for treatment based on the cancer cell expressing a human CD38 isoform E polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 105) on its cell surface. In some embodiments, the cancer cell expresses CD38 and CD 28. In some embodiments, the cancer cells express CD38 and do not express CD 28.

In some embodiments, the cancer is multiple myeloma, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, lymphoma, breast cancer such as Her2+ breast cancer, prostate cancer, germinal center B cell lymphoma, or B cell acute lymphoblastic leukemia. In certain embodiments, the cancer is multiple myeloma. In certain embodiments, the cancer is Acute Myeloid Leukemia (AML), Acute Lymphoblastic Leukemia (ALL), Chronic Lymphocytic Leukemia (CLL), or B-cell lymphoma.

In certain embodiments, the cancer is multiple myeloma. anti-CD 38 antibodies have been tested for the treatment of multiple myeloma, such as daratuzumab and isatuximab. However, although multiple myeloma is considered treatable, almost all patients inevitably relapse, leading to the development of treatment-refractory disease. In some embodiments, the cancer is relapsed or refractory multiple myeloma. In some embodiments, the patient has been treated with a previous multiple myeloma treatment. In some embodiments, the binding proteins of the present disclosure are administered to a patient as a first, second, or third line therapy for multiple myeloma. Without wishing to be bound by theory, it is believed that the anti-CD 38x anti-CD 28x anti-CD 3 binding proteins of the present disclosure are useful for treating multiple myeloma, for example, by recruiting T cells to tumor cells via anti-CD 38 (or anti-CD 28/anti-CD 38), activating participating T cells via anti-CD 3/anti-CD 28, and/or killing tumor cells via a perforin/granzyme-based mechanism. CD28 has been reported as a novel cancer marker for multiple myeloma. See Nair, j.r.et al (2011) j.immunol.187: 1243-1253.

In some embodiments, at least one binding protein is administered (or is to be administered) in combination with one or more anti-cancer therapies (e.g., any anti-cancer therapy known in the art, such as a chemotherapeutic agent or chemotherapy). In some embodiments, at least one binding protein is administered (or to be administered) prior to one or more anti-cancer therapies. In some embodiments, the at least one binding protein is administered (or is to be administered) concurrently with the one or more anti-cancer therapies. In some embodiments, the at least one binding protein is administered (or to be administered) after one or more antiretroviral therapies.

Binding protein therapeutic compositions and administration thereof

Therapeutic or pharmaceutical compositions comprising binding proteins are within the scope of the present disclosure. Such therapeutic or pharmaceutical compositions may comprise a therapeutically effective amount of a binding protein or binding protein-drug conjugate in admixture with a pharmaceutically or physiologically acceptable formulation selected for the mode of administration.

At the dosages and concentrations employed, acceptable formulation materials are preferably non-toxic to recipients.

Suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine), antimicrobials, antioxidants (such as ascorbic acid, sodium sulfite or sodium bisulfite), buffering agents (such as borates, bicarbonates, Tris-HCl, citrates, phosphates or other organic acids), bulking agents (such as mannitol or glycine), chelating agents (such as ethylenediaminetetraacetic acid (EDTA)), complexing agents (such as caffeine, polyvinylpyrrolidone, β -cyclodextrin, or hydroxypropyl- β -cyclodextrin), bulking agents, monosaccharides, disaccharides and other carbohydrates (such as glucose, mannose or dextrin), proteins (such as albumin, gelatin or immunoglobulin), coloring agents, flavoring and diluents, emulsifying agents, hydrophilic polymers (such as polyvinylpyrrolidone), low molecular weight polypeptides, salt-forming agents (such as sodium), preservatives (such as benzalkonium chloride, salicylic acid, mannitol, or mannitol), mannitol, or mannitol, solvents (such as mannitol, or mannitol), or mannitol, benzyl alcohol (such as mannitol, or mannitol), glycerol, sodium hydrogen peroxide, or sodium benzoate), sodium hydrogen peroxide, sodium benzoate, sodium chloride Alcohol or sorbitol), suspending agents, surfactants or wetting agents (such as pluronics; PEG; sorbitan esters; polysorbates such as polysorbate 20 or polysorbate 80; triton; tromethamine; lecithin; cholesterol or tyloxapal), stability enhancing agents (such as sucrose or sorbitol), tonicity enhancing agents (such as alkali metal halides-preferably sodium or potassium chloride-or mannitol sorbitol), delivery vehicles, diluents, excipients and/or pharmaceutical adjuvants (see, e.g., REMINGTON′ SPHARMACEUTICAL SCIENCES (18th ed., a.r. gennaro, ed., Mack Publishing Company1990), and subsequent versions thereof, are incorporated herein by reference for any purpose.

The optimal pharmaceutical composition will be determined by the skilled person, depending on, for example, the intended route of administration, the form of delivery and the desired dosage. Such compositions can affect the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the binding protein.

The primary vehicle or carrier in the pharmaceutical composition may be aqueous or non-aqueous. For example, a suitable vehicle or carrier for injection may be water, physiological saline solution, or artificial cerebrospinal fluid, possibly supplemented with other materials common in compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. Other exemplary pharmaceutical compositions comprise a Tris buffer at about pH 7.0-8.5 or an acetate buffer at about pH 4.0-5.5, which may further include sorbitol or a suitable substitute. In one embodiment of the present disclosure, the binding protein composition may be prepared for storage by mixing the selected composition having the desired purity with an optional formulation in the form of a lyophilized cake or an aqueous solution. In addition, the binding protein may be formulated as a lyophilizate using a suitable excipient, such as sucrose.

The pharmaceutical compositions of the present disclosure may be selected for parenteral or subcutaneous delivery. Alternatively, the composition may be selected for inhalation or delivery through the digestive tract, such as oral administration. The preparation of such pharmaceutically acceptable compositions is within the skill of the art.

The formulation components are present at concentrations acceptable to the site of administration. For example, buffering agents are used to maintain the composition at physiological pH or slightly lower pH, typically in the pH range of about 5 to about 8.

When parenteral administration is contemplated, the therapeutic composition used may be in the form of a pyrogen-free, parenterally acceptable, aqueous solution which comprises the desired binding protein in a pharmaceutically acceptable vehicle. A particularly suitable vehicle for parenteral injection is sterile distilled water, wherein the binding protein is formulated as a sterile isotonic solution which is suitably preserved. Another formulation may involve formulating the desired molecule with a pharmaceutical agent, such as injectable microspheres, bioerodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads, or liposomes, which provide controlled or sustained release of the product, which can then be delivered by depot injection (depot injection). Hyaluronic acid may also be used, which may have the effect of promoting circulation duration. Other suitable methods for introducing the desired molecule include implantable drug delivery devices.

In one embodiment, the pharmaceutical composition may be formulated for inhalation. For example, the binding protein may be formulated as a dry powder for inhalation. The binding protein inhalation solution may also be formulated with a propellant for aerosol delivery. In yet another embodiment, the solution may be atomized.

It is also contemplated that certain formulations may be administered orally. In one embodiment of the present disclosure, the binding proteins administered in this manner may be formulated with or without those carriers typically used to formulate solid dosage forms such as tablets and capsules. For example, the capsule may be designed to release the active portion of the formulation at a point in the gastrointestinal tract when bioavailability is maximized and pre-systemic degradation is minimized. Additional agents may be included to facilitate absorption of the binding protein. Diluents, flavoring agents, low melting waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binding agents may also be used.

Another pharmaceutical composition may comprise an effective amount of a binding protein in admixture with non-toxic excipients which are suitable for the preparation of tablets. Solutions can be prepared in unit dose form by dissolving the tablets in sterile water or another suitable vehicle. Suitable excipients include, but are not limited to, inert diluents such as calcium carbonate, sodium carbonate or bicarbonate, lactose or calcium phosphate; or a binder such as starch, gelatin or acacia; or a lubricant such as magnesium stearate, stearic acid or talc.

Other pharmaceutical compositions of the present disclosure will be apparent to those skilled in the art, including formulations involving sustained or controlled delivery of binding proteins in formulations. Techniques for formulating a variety of other sustained or controlled delivery methods, such as liposome carriers, bioerodible microparticles or porous beads, and depot injections, are also known to those skilled in the art. Further examples of sustained-release preparations include semipermeable polymer matrices in the form of shaped articles, e.g., films, or microcapsules. The sustained release matrix may comprise a polyester, a hydrogel, polylactide, a copolymer of L-glutamic acid and L-glutamic acid-gamma-ethyl ester, poly (2-hydroxyethyl-methacrylate), ethylene-vinyl acetate, or poly-D (-) -3-hydroxybutyric acid. Sustained release compositions may also include liposomes, which can be prepared by any of several methods known in the art.

Pharmaceutical compositions for in vivo administration must generally be sterile. This can be done by filtration through sterile filtration membranes. In the case of a lyophilized composition, sterilization using this method may be performed before or after lyophilization and reconstitution. Compositions for parenteral administration may be stored in lyophilized form or in solution form. In addition, parenteral compositions are typically placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

Once the pharmaceutical composition is formulated, it can be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder. Such formulations may be stored in a ready-to-use form or in a form that requires reconstitution prior to administration (e.g., lyophilization).

The present disclosure also encompasses kits for producing a single dose administration unit. The kits can each comprise a first container having a dried protein and a second container having an aqueous formulation. Also included within the scope of the present disclosure are kits containing single-chamber and multi-chamber pre-filled syringes (e.g., liquid syringes and lyophilized syringes).

The effective amount of the binding protein pharmaceutical composition to be used therapeutically will depend, for example, on the therapeutic context and purpose. One skilled in the art will appreciate that the appropriate dosage level for treatment will thus depend, in part, on the molecule delivered, the indication for which the binding protein is used, the route of administration, and the size (body weight, body surface or organ size) and condition (age and general health) of the patient. Thus, the clinician can titrate the dosage and vary the route of administration to achieve the optimal therapeutic effect.

The frequency of administration will depend on the pharmacokinetic parameters of the binding protein in the formulation used. Generally, the clinician will administer the composition until a dosage is reached that achieves the desired effect. Thus, the composition may be administered over time as a single dose, as two or more doses (which may or may not contain the same amount of the desired molecule), or as a continuous infusion via an implanted device or catheter. Further modification of appropriate dosages are routinely made by those of ordinary skill in the art and are within the scope of the tasks they routinely perform. Appropriate dosages may be determined by using appropriate dose response data.

The route of administration of the pharmaceutical composition is consistent with known methods, e.g., oral; injection by intravenous, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, intraportal or intralesional routes; by a sustained release system; or by an implant device. If desired, the composition may be administered continuously by bolus injection (bolus injection) or by infusion or by an implanted device.

The composition may also be administered topically via an implant membrane, sponge, or other suitable material onto which the desired molecule has been absorbed or encapsulated. Where an implanted device is used, the device may be implanted into any suitable tissue or organ and the desired molecule may be delivered via diffusion, timed release bolus, or continuous administration.

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