Activatable protein constructs and uses thereof

文档序号：440413 发布日期：2021-12-24 浏览：12次中文

阅读说明：本技术 可活化蛋白质构建体及其用途 (Activatable protein constructs and uses thereof ) 是由 W·J·J·芬雷于 2020-05-13 设计创作，主要内容包括：本文描述了在患病组织中表现出可活化靶标结合的蛋白质分子、以及相关的核酸分子、载体和宿主细胞。本文还描述了此类蛋白质分子的医学用途。(Protein molecules, and related nucleic acid molecules, vectors, and host cells that exhibit activatable target binding in diseased tissue are described herein. Medical uses of such protein molecules are also described herein.)

1. A protein comprising a first portion and a second portion and a peptide linker between the first portion and the second portion,

wherein the peptide linker comprises an amino acid sequence from a human immunoglobulin hinge region or an amino acid sequence or amino acid sequence having from 1 to about 7 amino acid substitutions as compared to a human immunoglobulin hinge region;

wherein the peptide linker is cleavable by a protease expressed in the diseased tissue;

wherein the second portion is capable of specifically binding to a molecule expressed in the diseased tissue; and is

Wherein binding of said second moiety to said molecule expressed in said diseased tissue is reduced or inhibited when said peptide linker is not cleaved.

2. The protein of claim 1, wherein the peptide linker is between about 5 and about 15 amino acids in length.

3. The protein of claim 1 or claim 2, wherein the peptide linker comprises or consists of the amino acid sequence of: SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 69, SEQ ID NO 70, SEQ ID NO 71, SEQ ID NO 72, SEQ ID NO 81, SEQ ID NO 82, SEQ ID NO 83, SEQ ID NO 84, SEQ ID NO 85, SEQ ID NO 86 or SEQ ID NO 87.

4. The protein of any one of claims 1-3, wherein the protease is human Matrix Metalloproteinase (MMP), human cathepsin, human enterokinase, human thrombin, human tPA, human granzyme B, human uPA, or human ADAMTs-5.

5. The protein of any one of claims 1-4, wherein the peptide linker comprises a human MMP cleavage site, a human cathepsin, a human enterokinase, human thrombin, human tPA, human granzyme B, human uPA, or a human ADAMTS-5 cleavage site.

6. The protein of any one of claims 1-5, wherein the human MMP is MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, MMP-12, MMP-13, or MMP 14.

7. The protein of any one of claims 1-6, wherein the level or activity of said human MMP is elevated in said diseased tissue compared to the level or activity of said human MMP in non-diseased tissue.

8. The protein of any one of claims 1-5, wherein the human cathepsin is cathepsin A, cathepsin C, cathepsin D, cathepsin G, cathepsin L, or cathepsin K.

9. The protein of any one of claims 1-5 and 8, wherein the level or activity of the human cathepsin in the diseased tissue is increased compared to the level or activity of the human cathepsin in non-diseased tissue.

10. The protein of any one of claims 1-9, wherein the first portion comprises an antibody, an antigen-binding portion of an antibody, or an extracellular domain of a receptor.

11. The protein of claim 10, wherein the first portion is a Fab, single chain Fab, VH domain, VL domain, immunoglobulin neo-antigen receptor (IgNAR), single chain variable fragment (scFv), diabody, or T cell receptor domain.

12. The protein of any one of claims 1-11, wherein the first portion specifically binds to a molecule expressed in a diseased tissue.

13. The protein of any one of claims 1-12, wherein the first portion specifically binds to a first molecule expressed in a diseased tissue and the second portion is capable of specifically binding to a second molecule expressed in a diseased tissue, wherein the first molecule expressed in a diseased tissue and the second molecule expressed in a diseased tissue are different molecules.

14. The protein of claim 13, wherein the first molecule expressed in the diseased tissue and the second molecule expressed in the diseased tissue are expressed by the same cell.

15. The protein of claim 13, wherein the first molecule expressed in the diseased tissue and the second molecule expressed in the diseased tissue are expressed by different cells.

16. The protein of claim 13, wherein the first molecule expressed in the diseased tissue and/or the second molecule expressed in the diseased tissue is expressed on the cell surface.

17. The protein of claim 13, wherein the first molecule expressed in a diseased tissue and/or the second molecule expressed in a diseased tissue is a soluble molecule.

18. The protein of any one of claims 1-17, wherein the first portion specifically binds to human EGFR, human HER2, human HER3, human CD105, human C-KIT, human PD1, human PD-L1, human PSMA, human EpCAM, human Trop2, human EphA2, human CD20, human BCMA, human GITR, human OX40, human CSF1R, human Lag3, or human cMET.

19. The protein of any one of claims 1-17, wherein the second moiety specifically binds to a molecule expressed by a human immune cell.

20. The protein of claim 19, wherein the molecule expressed by a human immune cell is human CD3, human CD16A, human CD16B, human CD28, human CD89, human CTLA4, human NKG2D, human sirpa, human SIRP γ, human PD1, human lang 3, human 4-1BB, human OX40, or human GITR.

21. The protein of any one of claims 1-20, wherein the first portion comprises a heavy chain Variable (VH) region and a light chain Variable (VL) region.

22. The protein of any one of claims 1-21, wherein the first portion comprises an immunoglobulin constant region or a portion of an immunoglobulin constant region.

23. The protein of claim 22, wherein the immunoglobulin constant region is an IgG, IgE, IgM, IgD, IgA, or IgY.

24. The protein of claim 22, wherein the immunoglobulin constant region is IgG1, IgG2, IgG3, IgG4, IgA1, or IgA 2.

25. The protein of claim 22, wherein the immunoglobulin constant region is immunologically inert.

26. The protein of claim 22, wherein the immunoglobulin constant region is a wild-type human IgG4 constant region, a human IgG4 constant region comprising amino acid substitution S228P, a wild-type human IgG1 constant region, a human IgG1 constant region comprising amino acid substitutions L234A and L235A, a human IgG1 constant region comprising amino acid substitutions L234A, L235A, and G237A, a human IgG1 constant region comprising amino acid substitutions L234A, L235A, G237A, and P331S, or a wild-type human IgG2 constant region.

27. The protein of any one of claims 1-26, wherein the second portion comprises an antibody, an antigen-binding portion of an antibody, or an extracellular domain of a receptor.

28. The protein of claim 27, wherein the second portion is a Fab, single chain Fab, VH domain, VL domain, immunoglobulin neo-antigen receptor (IgNAR), single chain variable fragment (scFv), or T cell receptor domain.

29. The protein of any one of claims 1-28, wherein the second portion specifically binds human CD 47.

30. The protein of any one of claims 1-28, wherein the second portion specifically binds human CD3 or human PD-L1.

31. The protein of any one of claims 1-30, wherein the second portion comprises a heavy chain Variable (VH) region and a light chain Variable (VL) region.

32. The protein of any one of claims 1-31, wherein the second portion comprises an immunoglobulin constant region or a portion of an immunoglobulin constant region.

33. The protein of claim 32, wherein the immunoglobulin constant region is an IgG, IgE, IgM, IgD, IgA, or IgY.

34. The protein of claim 32, wherein the immunoglobulin constant region is IgG1, IgG2, IgG3, IgG4, IgA1, or IgA 2.

35. The protein of claim 32, wherein the immunoglobulin constant region is immunologically inert.

36. The protein of claim 32, wherein the immunoglobulin constant region is a wild-type human IgG4 constant region, a human IgG4 constant region comprising amino acid substitution S228P, a wild-type human IgG1 constant region, a human IgG1 constant region comprising amino acid substitutions L234A and L235A, a human IgG1 constant region comprising amino acid substitutions L234A, L235A, and G237A, a human IgG1 constant region comprising amino acid substitutions L234A, L235A, G237A, and P331S, or a wild-type human IgG2 constant region.

37. The protein of claim 1, wherein the protein has one immune effector function or two, three, or more immune effector functions.

38. The protein of claim 37, wherein the immune effector function is ADCC, CDC or ADCP.

39. The protein of any one of claims 1-38, wherein the first portion prevents or reduces specific binding of the second portion to the molecule expressed in the diseased tissue.

40. The protein of any one of claims 1-39, wherein the peptide linker is cleaved near or within the diseased tissue.

41. The protein of any one of claims 1-40, wherein the peptide linker is cleaved near or inside the diseased tissue, wherein the first portion is dissociated from the second portion near or inside the diseased tissue, and wherein the second portion specifically binds to the molecule expressed in the diseased tissue near or inside the diseased tissue.

42. The protein of any one of claims 1-41, wherein the diseased tissue is a tumor or an inflamed tissue.

43. The protein of claim 1, wherein said first portion specifically binds human cMET, wherein said second portion specifically binds human CD47, and wherein said protein comprises a first polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID No. 16 and a second polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID No. 17.

44. The protein of claim 1, wherein the first portion specifically binds to human HER2, wherein the second portion specifically binds to human CD3, and wherein the protein comprises or consists of a first polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID No. 26 and a second polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID No. 27.

45. The protein of claim 1, wherein the first portion specifically binds to human HER2, wherein the second portion specifically binds to human CD47, and wherein the protein comprises or consists of a first polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID No. 34 and a second polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID No. 35.

46. The protein of claim 1, wherein said first portion specifically binds human cMET, wherein said second portion specifically binds human CD47, and wherein said protein comprises a first polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID No. 36 and a second polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID No. 37.

47. The protein of claim 1, wherein the first portion specifically binds to human Her2, wherein the second portion specifically binds to human CD3, and wherein the protein comprises a first polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID No. 38 and a second polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID No. 39.

48. The protein of claim 1, wherein the first portion specifically binds to human Her2, wherein the second portion specifically binds to human CD3, and wherein the protein comprises a first polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO:40 and a second polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO: 41.

49. The protein of claim 1, wherein the first portion specifically binds to human Her2, wherein the second portion specifically binds to human CD47, and wherein the protein comprises a first polypeptide chain and a second polypeptide chain, wherein:

(a) said first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO 42 and said second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO 43; or

(b) Said first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO 44 and said second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO 45; or

(c) Said first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO 46 and said second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO 47; or

(d) The first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID No. 48 and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID No. 49; or

(e) Said first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:50 and said second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 51; or

(f) Said first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID No. 52 and said second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID No. 53; or

(g) Said first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID No. 54 and said second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID No. 55; or

(h) The first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:88 and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 89; or

(i) Said first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:90 and said second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 91; or

(j) Said first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:44 and said second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 92; or

(k) Said first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID No. 44 and said second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID No. 93; or

(l) Said first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID No. 44 and said second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID No. 94; or

(n) said first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:44 and said second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 96; or

(o) said first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:44 and said second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 97.

50. The protein of claim 1, wherein said first portion specifically binds to human Her2, wherein said second portion specifically binds to human CD3, and wherein said protein comprises a first polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID No. 73 and a second polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID No. 74.

51. The protein of claim 1, wherein said first portion specifically binds to human cMET, wherein said second portion specifically binds to human cMET, and wherein said protein comprises or consists of a first polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO:75 and a second polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO: 76.

52. The protein of claim 1, wherein the first portion specifically binds to human Her2, wherein the second portion specifically binds to human CD3, and wherein the protein comprises a first polypeptide chain and a second polypeptide chain, wherein:

(a) said first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:98 and said second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 99; or

(b) The first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID No. 100 and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID No. 101; or

(c) Said first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID No. 102 and said second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID No. 103; or

(d) The first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID No. 104 and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID No. 105.

53. An immunoconjugate comprising the protein of any one of claims 1-52 linked to a therapeutic agent.

54. The immunoconjugate of claim 53, wherein the therapeutic agent is a cytotoxin, a radioisotope, a chemotherapeutic agent, an immunomodulatory agent, an anti-angiogenic agent, an antiproliferative agent, a pro-apoptotic agent, a cytostatic enzyme, a cytolytic enzyme, a therapeutic nucleic acid, an anti-angiogenic agent, an antiproliferative agent, or a pro-apoptotic agent.

55. A pharmaceutical composition comprising the protein of any one of claims 1-52 or the immunoconjugate of claim 53 or 54, and a pharmaceutically acceptable carrier, diluent, or excipient.

56. A nucleic acid molecule encoding the protein or a portion of the protein of any one of claims 1-52.

57. A nucleic acid molecule encoding a first polypeptide chain, a second polypeptide chain, or both of the first polypeptide chain and the second polypeptide chain of the protein of any one of claims 43-52.

58. An expression vector comprising the nucleic acid molecule of claim 56 or claim 57.

59. A recombinant host cell comprising the nucleic acid molecule of claim 56 or claim 57 or the expression vector of claim 58.

60. A method of producing a protein comprising:

culturing a recombinant host cell comprising the expression vector of claim 57 under conditions such that the nucleic acid molecule is expressed, thereby producing the protein; and

isolating the protein from the host cell or culture.

61. A method for enhancing an anti-cancer immune response in a subject, comprising administering to the subject a therapeutically effective amount of the protein of any one of claims 1-52, the immunoconjugate of claim 53 or 54, or the pharmaceutical composition of claim 55.

62. A method of treating cancer, an autoimmune disease, an inflammatory disease, a cardiovascular disease, or a fibrotic disease in a subject, comprising administering to the subject a therapeutically effective amount of the protein of any one of claims 1-52, the immunoconjugate of claim 53 or claim 54, or the pharmaceutical composition of claim 55.

63. The method of claim 62, wherein the cancer is gastrointestinal stromal cancer (GIST), pancreatic cancer, skin cancer, melanoma, breast cancer, lung cancer, bronchial cancer, colorectal cancer, prostate cancer, gastric cancer, ovarian cancer, bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, cancer of the oral cavity or pharynx, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small or appendiceal cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma, or cancer of blood tissue.

64. The method of claim 62, wherein the autoimmune or inflammatory disease is arthritis, asthma, multiple sclerosis, psoriasis, Crohn's disease, inflammatory bowel disease, lupus, Graves ' disease, Hashimoto's thyroiditis, or ankylosing spondylitis.

65. The method of claim 62, wherein the cardiovascular disease is coronary heart disease, or atherosclerosis or stroke.

66. The method of claim 62, wherein the fibrotic disease is myocardial infarction, angina, osteoarthritis, pulmonary fibrosis, cystic fibrosis, bronchitis, or asthma.

67. The protein of any one of claims 1-52, the immunoconjugate of claim 53 or claim 54, or the pharmaceutical composition of claim 55, for use in treating cancer, an autoimmune disease, an inflammatory disease, a cardiovascular disease, or a fibrotic disease.

68. The protein or pharmaceutical composition for use of claim 67, wherein the cancer is gastrointestinal stromal cancer (GIST), pancreatic cancer, skin cancer, melanoma, breast cancer, lung cancer, bronchial cancer, colorectal cancer, prostate cancer, gastric cancer, ovarian cancer, bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, cancer of the oral cavity or pharynx, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small intestine or appendix cancer, salivary gland cancer, thyroid cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, or cancer of blood tissue.

69. A protein or pharmaceutical composition for use according to claim 67, wherein the autoimmune or inflammatory disease is arthritis, asthma, multiple sclerosis, psoriasis, Crohn's disease, inflammatory bowel disease, lupus, Graves ' disease, Hashimoto's thyroiditis or ankylosing spondylitis.

70. The protein or pharmaceutical composition for use of claim 67, wherein said cardiovascular disease is coronary heart disease, atherosclerosis or stroke.

71. The protein or pharmaceutical composition for use according to claim 67, wherein the fibrotic disease is myocardial infarction, angina, osteoarthritis, pulmonary fibrosis, cystic fibrosis, bronchitis, or asthma.

72. The protein of any one of claims 1-52, the immunoconjugate of claim 53 or 54, or the pharmaceutical composition of claim 55, for use as a medicament.

Technical Field

The present invention relates to protein molecules that exhibit binding of activatable targets in diseased tissue and their medical uses.

Background

In the treatment of diseases using antibodies and other binding proteins, a number of potential drug targets have been described, but rarely are expressed only in diseased tissues. In fact, most potential targets in this space are also expressed in non-diseased tissues. Furthermore, most of the drug mechanisms of action employed in the field of challenging therapies (such as cancer) employ highly effective cell killing mechanisms of action. As a result, the engagement of the drug to the target in non-diseased tissue often causes undesirable side effects. There is a need for engineered forms of binding proteins that are partially or even completely inactive in healthy tissue but become highly activated in diseased tissue.

Disclosure of Invention

Provided herein are proteins comprising a first portion and a second portion and a peptide linker between the first portion and the second portion, wherein the peptide linker comprises an amino acid sequence from a human immunoglobulin hinge region or an amino acid sequence or amino acid sequence having from 1 to about 7 amino acid substitutions as compared to a human immunoglobulin hinge region; wherein the peptide linker is cleavable by a protease expressed in the diseased tissue; wherein the second portion is capable of specifically binding to a molecule expressed in the diseased tissue; and wherein binding of said second moiety to said molecule expressed in said diseased tissue is reduced or inhibited when said peptide linker is not cleaved. In some embodiments, the peptide linker is between about 5 and about 15 amino acids in length. In some embodiments, the peptide linker comprises or consists of the amino acid sequence of: SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 69, SEQ ID NO 70, SEQ ID NO 71, SEQ ID NO 72, SEQ ID NO 81, SEQ ID NO 82, SEQ ID NO 83, SEQ ID NO 84, SEQ ID NO 85, SEQ ID NO 86 or SEQ ID NO 87.

In some embodiments, the protease is human Matrix Metalloproteinase (MMP), human cathepsin, human enterokinase, human thrombin, human tPA, human granzyme B, human uPA, or human ADAMTs-5. In some embodiments, the peptide linker comprises a human MMP cleavage site, a human cathepsin, a human enterokinase, a human thrombin, a human tPA, a human granzyme B, a human uPA, or a human ADAMTs-5 cleavage site. In some embodiments, the human MMP is MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, MMP-12, MMP-13, or MMP 14. In some embodiments, the level or activity of human MMPs is increased in diseased tissue compared to the level or activity of human MMPs in non-diseased tissue. In some embodiments, the human cathepsin is cathepsin a, cathepsin C, cathepsin D, cathepsin G, cathepsin L or cathepsin K. In some embodiments, the level or activity of human cathepsin in diseased tissue is increased compared to the level or activity of human cathepsin in non-diseased tissue.

In some embodiments, the first portion comprises an antibody, an antigen-binding portion of an antibody, or an extracellular domain of a receptor. In some embodiments, the first portion is a Fab, single chain Fab, VH domain, VL domain, immunoglobulin neoantigen receptor (IgNAR), single chain variable fragment (scFv), diabody, or T cell receptor domain. In some embodiments, the first portion specifically binds to a molecule expressed in the diseased tissue.

In some embodiments, the first portion specifically binds to a first molecule expressed in the diseased tissue and the second portion is capable of specifically binding to a second molecule expressed in the diseased tissue, wherein the first molecule expressed in the diseased tissue and the second molecule expressed in the diseased tissue are different molecules. In some embodiments, the first molecule expressed in the diseased tissue and the second molecule expressed in the diseased tissue are expressed by the same cell. In some embodiments, the first molecule expressed in the diseased tissue and the second molecule expressed in the diseased tissue are expressed by different cells. In some embodiments, the first molecule expressed in the diseased tissue and/or the second molecule expressed in the diseased tissue is expressed on the cell surface. In some embodiments, the first molecule expressed in the diseased tissue and/or the second molecule expressed in the diseased tissue is a soluble molecule.

In some embodiments, the first moiety specifically binds to human EGFR, human HER2, human HER3, human CD105, human C-KIT, human PD1, human PD-L1, human PSMA, human EpCAM, human Trop2, human EphA2, human CD20, human BCMA, human GITR, human OX40, human CSF1R, human Lag3, or human cMET.

In some embodiments, the second moiety specifically binds to a molecule expressed by a human immune cell. In some embodiments, the molecule expressed by the human immune cell is human CD3, human CD16A, human CD16B, human CD28, human CD89, human CTLA4, human NKG2D, human sirpa, human SIRP γ, human PD1, human lang 3, human 4-1BB, human OX40, or human GITR.

In some embodiments, the first portion comprises a heavy chain Variable (VH) region and a light chain Variable (VL) region. In some embodiments, the first portion comprises an immunoglobulin constant region or a portion of an immunoglobulin constant region. In some embodiments, the immunoglobulin constant region is an IgG, IgE, IgM, IgD, IgA, or IgY. In some embodiments, the immunoglobulin constant region is IgG1, IgG2, IgG3, IgG4, IgA1, or IgA 2. In some embodiments, the immunoglobulin constant region is immunologically inert. In some embodiments, the immunoglobulin constant region is a wild-type human IgG4 constant region, a human IgG4 constant region comprising amino acid substitution S228P, a wild-type human IgG1 constant region, a human IgG1 constant region comprising amino acid substitutions L234A and L235A, a human IgG1 constant region comprising amino acid substitutions L234A, L235A, and G237A, a human IgG1 constant region comprising amino acid substitutions L234A, L235A, G237A, and P331S, or a wild-type human IgG2 constant region.

In some embodiments, the second portion comprises an antibody, an antigen-binding portion of an antibody, or an extracellular domain of a receptor. In some embodiments, the second moiety is a Fab, single chain Fab, VH domain, VL domain, immunoglobulin neoantigen receptor (IgNAR), single chain variable fragment (scFv), or T cell receptor domain. In some embodiments, the second moiety specifically binds to human CD 47. In some embodiments, the second moiety specifically binds to human CD3 or human PD-L1.

In some embodiments, the second portion comprises a heavy chain Variable (VH) region and a light chain Variable (VL) region. In some embodiments, the second portion comprises an immunoglobulin constant region or a portion of an immunoglobulin constant region. In some embodiments, the immunoglobulin constant region is an IgG, IgE, IgM, IgD, IgA, or IgY. In some embodiments, the immunoglobulin constant region is IgG1, IgG2, IgG3, IgG4, IgA1, or IgA 2. In some embodiments, the immunoglobulin constant region is immunologically inert. In some embodiments, the immunoglobulin constant region is a wild-type human IgG4 constant region, a human IgG4 constant region comprising amino acid substitution S228P, a wild-type human IgG1 constant region, a human IgG1 constant region comprising amino acid substitutions L234A and L235A, a human IgG1 constant region comprising amino acid substitutions L234A, L235A, and G237A, a human IgG1 constant region comprising amino acid substitutions L234A, L235A, G237A, and P331S, or a wild-type human IgG2 constant region.

In some embodiments, the protein has one immune effector function, or two, three, or more immune effector functions. In some embodiments, the immune effector function is ADCC, CDC or ADCP.

In some embodiments, the first portion prevents or reduces specific binding of the second portion to a molecule expressed in the diseased tissue. In some embodiments, the peptide linker is cleaved near or within the diseased tissue. In some embodiments, the peptide linker is cleaved near or inside the diseased tissue, wherein the first portion dissociates from the second portion near or inside the diseased tissue, and wherein the second portion specifically binds to a molecule expressed in the diseased tissue near or inside the diseased tissue. In some embodiments, the diseased tissue is a tumor or an inflamed tissue.

In some embodiments, the first portion specifically binds human cMET, wherein the second portion specifically binds human CD47, and wherein the protein comprises a first polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO:16 and a second polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO: 17.

In some embodiments, the first portion specifically binds to human HER2, wherein the second portion specifically binds to human CD3, and wherein the protein comprises a first polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO:26 and a second polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO: 27.

In some embodiments, the first portion specifically binds to human HER2, wherein the second portion specifically binds to human CD47, and wherein the protein comprises a first polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO:34 and a second polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO: 35.

In some embodiments, the first portion specifically binds human cMET, wherein the second portion specifically binds human CD47, and wherein the protein comprises a first polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO:36 and a second polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO: 37.

In some embodiments, the first portion specifically binds to human Her2, wherein the second portion specifically binds to human CD3, and wherein the protein comprises a first polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO:38 and a second polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO: 39.

In some embodiments, the first portion specifically binds to human Her2, wherein the second portion specifically binds to human CD3, and wherein the protein comprises a first polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO:40 and a second polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO: 41.

In some embodiments, the first portion specifically binds to human Her2, wherein the second portion specifically binds to human CD47, and wherein the protein comprises a first polypeptide chain and a second polypeptide chain, wherein:

(h) The first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:88 and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 89; or

(o) said first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:44 and said second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 97;

In some embodiments, the first portion specifically binds to human Her2, wherein the second portion specifically binds to human CD3, and wherein the protein comprises a first polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO:73 and a second polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO: 74.

In some embodiments, the first portion specifically binds to human cMET, wherein the second portion specifically binds to human cMET, and wherein the protein comprises a first polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO:75 and a second polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO: 76.

(d) Said first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:104 and said second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 105;

also provided herein are immunoconjugates comprising a protein of the invention linked to a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxin, a radioisotope, a chemotherapeutic agent, an immunomodulatory agent, an anti-angiogenic agent, an antiproliferative agent, a pro-apoptotic agent, a cytostatic enzyme, a cytolytic enzyme, a therapeutic nucleic acid, an anti-angiogenic agent, an antiproliferative agent, or a pro-apoptotic agent.

Also provided herein are pharmaceutical compositions comprising a protein of the invention or an immunoconjugate of the invention, and a pharmaceutically acceptable carrier, diluent, or excipient.

Also provided herein are nucleic acid molecules encoding a protein or a portion of a protein of the invention. Also provided herein are nucleic acid molecules encoding a first polypeptide chain, a second polypeptide chain, or both a first polypeptide chain and a second polypeptide chain of a protein of the invention.

Also provided herein are expression vectors comprising the nucleic acid molecules of the invention.

Also provided herein are recombinant host cells comprising a nucleic acid molecule of the invention or an expression vector of the invention.

Also provided herein are methods of producing a protein, comprising culturing a recombinant host cell comprising an expression vector of the invention under conditions in which the nucleic acid molecule is expressed, thereby producing the protein; and isolating the protein from the host cell or culture.

Also provided herein are methods for enhancing an anti-cancer immune response in a subject, comprising administering to the subject a therapeutically effective amount of a protein of the invention, an immunoconjugate of the invention, or a pharmaceutical composition of the invention.

Also provided herein are methods of treating cancer, autoimmune disease, inflammatory disease, cardiovascular disease, or fibrotic disease in a subject, the method comprising administering to the subject a therapeutically effective amount of a protein of the invention, an immunoconjugate of the invention, or a pharmaceutical composition of the invention. In some embodiments, the cancer is a gastrointestinal stromal cancer (GIST), pancreatic cancer, skin cancer, melanoma, breast cancer, lung cancer, bronchial cancer, colorectal cancer, prostate cancer, gastric cancer, ovarian cancer, bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, oral or pharyngeal cancer, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small or appendiceal cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma, or cancer of blood tissue. In some embodiments, the autoimmune or inflammatory disease is arthritis, asthma, multiple sclerosis, psoriasis, crohn's disease, inflammatory bowel disease, lupus, graves ' disease, hashimoto's thyroiditis, or ankylosing spondylitis. In some embodiments, the cardiovascular disease is coronary heart disease, or atherosclerosis or stroke. In some embodiments, the fibrotic disease is myocardial infarction, angina, osteoarthritis, pulmonary fibrosis, cystic fibrosis, bronchitis, or asthma.

Also provided herein is a protein of the invention, an immunoconjugate of the invention or a pharmaceutical composition of the invention for use in the treatment of cancer, autoimmune disease, inflammatory disease, cardiovascular disease or fibrotic disease. In some embodiments, the cancer is a gastrointestinal stromal cancer (GIST), pancreatic cancer, skin cancer, melanoma, breast cancer, lung cancer, bronchial cancer, colorectal cancer, prostate cancer, gastric cancer, ovarian cancer, bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, oral or pharyngeal cancer, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small or appendiceal cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma, or cancer of blood tissue. In some embodiments, the autoimmune or inflammatory disease is arthritis, asthma, multiple sclerosis, psoriasis, crohn's disease, inflammatory bowel disease, lupus, graves ' disease, hashimoto's thyroiditis, or ankylosing spondylitis. In some embodiments, the cardiovascular disease is coronary heart disease, atherosclerosis, or stroke. In some embodiments, the fibrotic disease is myocardial infarction, angina, osteoarthritis, pulmonary fibrosis, cystic fibrosis, bronchitis, or asthma.

Also provided herein is a protein of the invention, an immunoconjugate of the invention or a pharmaceutical composition of the invention for use as a medicament.

Drawings

Fig. 1A-fig. 1b. challenge of administering systemically active antibody drugs to solid tumors-exemplified by anti-CD 47. CD47 antibodies (fig. 1A) have significant challenges, such as high expression of CD47 in the bloodstream. Red blood cells and platelets in particular form "pools" and toxicity risk problems. Tumors are also often "hostile" environments with high expression of enzymes (such as MMPs that accelerate IgG degradation). The anti-CD 47 protein construct of the invention (fig. 1B) was intended to abrogate CD47 binding in the native protein, which abrogated peripheral activity. The tumor targeting domain then drives high concentrations in the tumor environment, and the peptide linker system exploits MMP activity in the tumor to activate CD47 binding activity in the tumor rather than in the periphery.

FIG. 2A-FIG. 2B protein construct IgG²Design and activation principles. Protein construct IgG²The design (fig. 2A) may be based on sequences derived from IgG1, IgG2, IgG3, IgG4, IgA, IgE, or IgM, and may or may not have effector function capability. In this construct, the four polypeptide chains encode four Fab domains (2X Fab a, 2X Fab B), two linker sequences, and may or may not have an immunoglobulin hinge region and Fc domain. Each Fab a linker domain blocks the binding activity of Fab B. The selection of linker sequences (such as the lower hinge peptide sequence from immunoglobulins) results in a structure that will lock in non-diseased tissue, but may be rapidly cleaved and unlocked in the presence of high concentrations of proteases in the tumor environment (fig. 2B). The linker can be cleaved sequentially, resulting in an intermediate unlocked active state that allows Fab a and B from a single protein construct to bind their cognate targets. The secondary, possibly slower cleavage of the second linker in each Fab a-Fab B protein construct unit may completely release the Fab a domain from the structure, thus forming a dissociated form. Cleaved linkers based on immunoglobulin hinge sequences can also recruit increased immune effector functions on the cell membrane by endogenous anti-hinge antibodies. The variable regions are indicated in white. The constant regions are indicated in grey.

FIG. 3A-FIG. 3B. protein construct Fab²Design and activation principles. Protein construct Fab²The design may be based on sequences derived from IgG1, IgG2, IgG3, IgG4, IgA, IgE, or IgM, and may or may not have effector function capability. In this construct, two (fig. 3A) or three (fig. 3B) polypeptide chains may encode two Fab domains (1X Fab a, 1X Fab B), two or more linker sequences, and may or may not have an immunoglobulin hinge region and Fc domain, with heterologous twoPairing of the mers may or may not be driven by mutations in the Fc. Each Fab a linker domain blocks the binding activity of Fab B. The selection of linker sequences (such as the lower hinge peptide sequence) results in a structure that will lock in non-diseased tissue, but is rapidly cleaved and unlocked in the presence of high concentrations of proteases in the tumor environment (fig. 3A). The linker can be cleaved sequentially, resulting in an intermediate unlocked active state that allows Fab a and B from a single protein to bind their cognate target. The secondary, possibly slower cleavage of the second linker in each Fab a-Fab B protein unit may completely release the Fab a domain from the structure. Cleaved linkers based on immunoglobulin hinge sequences can also recruit increased immune effector functions on the cell membrane by endogenous anti-hinge antibodies. The variable regions are indicated in white. The constant regions are indicated in grey.

FIG. 4 protein A purified protein construct IgG from clones 1-15²And Fab²SDS-PAGE analysis of proteins. Various constructs exemplary proteins were expressed in CHO cells and purified using protein a affinity chromatography. The purified protein was then subjected to SDS-PAGE analysis in both the unreduced and reduced (r) states, along with molecular weight standard (M) analysis. Clones 6, 10 and 14 (all containing LHL linker) were found to contain the highest proportion of product of the expected size and the lowest higher and lower molecular weight content.

FIG. 5A-FIG. 5I. protein A purified protein construct IgG²And Fab²Size exclusion chromatography of proteins. Selected constructs exemplary proteins were analyzed and fully purified using SEC. Clones 1 (fig. 5A), 2 (fig. 5B), 3 (fig. 5C), 4 (fig. 5D), 5 (fig. 5E), 6 (fig. 5F), 12 (fig. 5G), 14 (fig. 5H) and 10 (fig. 5I) were analyzed. The data shows that the highest proportion of expected size products (e.g., highlighted peaks, fig. 5I) and lowest higher/lower molecular weight content were found in samples from clones 6, 10 and 14 (all containing LHL linker).

FIG. 6A-FIG. 6B.SEC purified protein construct IgG²And Fab²SDS-PAGE analysis of proteins. Purification of protein A by SEC Key leader protein construct clones. The purified proteins from clones 1, 2, 4, 5, 6 and the non-SEC purified 15 (FIG. 6A) were then analyzed by SDS-PAGE in the unreduced state. Purified proteins from clones 7, 8, 10, 11, 12, 13 and 14 (FIG. 6B) were also analyzed by SDS-PAGE in the unreduced and reduced (r) states. All proteins were loaded at about 1. mu.g/lane. Clones 6, 10 and 14 (all containing LHL linker) were found to contain the highest proportion of product of the expected size and the lowest higher and lower molecular weight content.

Figure 7A-figure 7b direct titration ELISA of purified intact protein constructs and control antibodies binding to human target proteins. Control antibodies a-D5 anti-CD 47, a-D5 Fab-Fc (monovalent form of a-D5 antibody), MH7.1 anti-C-MET and anti-Her 2 trastuzumab (all in the form of human IgG 1) were titrated (in μ g/ml) in a direct binding ELISA against human CD47, C-MET and Her2 proteins (fig. 7A). Also analyzed in the same manner as IgG²Forms of Her2CD47-LH-LH and Her2CD47-LHL-LHL (FIG. 7B) and in Fab²Forms cMETCD47-L2-L2 and cMETCD47-LHL-LHL (FIG. 7C).

Figure 8A-figure 8c human red blood cell hemagglutination assays of purified intact protein constructs and control antibodies. Control antibodies anti-CD 235a (murine) and A-D5 anti-CD 47, A-D5 Fab-Fc (monovalent form of A-D5 antibody, labeled 'FabCD 47 only'), MH7.1 anti-C-MET, anti-Her 2 trastuzumab, in IgG ²Forms of Her2CD47-LH-LH and Her2CD47-LHL-LHL and in Fab²Forms of cMETCD47-L2-L2 and cMETCD47-LHL-LHL titrated (in nM) in a human red blood cell hemagglutination assay using fresh red blood cells from donor 1 (FIG. 8A), donor 2 (FIG. 8B) and donor 3 (FIG. 8C).

Figure 9A-figure 9c direct ELISA of purified intact and MMP digested protein constructs binding to human target proteins. Enzymatic digestion of protein constructs was performed using human MMP3, MMP7, and MMP12 over a time course of 2, 4, 8, and 24 hours of incubation, plus 24 hours of incubation in enzyme-free buffer as a negative control. Samples from these digestion schedules were then applied to direct binding ELISA against human Her2 and CD47 (fig. 9A, 9B) or human C-MET and human CD47 (fig. 9C).

FIG. 10A-FIG. 10CMethylated intact and MMP digested Her2CD3 Fab²Functional analysis of the binding of the protein construct to the human target protein. Analysis as Fab by ELISA without MMP digestion (FIG. 10A), flow cytometry with or without MMP digestion (FIG. 10B) and CD3 reporter assay (FIG. 10C)²Forms of the antibodies Her2CD3-L1-LH, Her2CD3-L2-L2 and Her2CD 3-LHL-LHL.

FIG. 11. alternative structure based on the principle of protein construct design and activation. Fab in FIGS. 2 and 3 ²And IgG²The protein construct modules (1) found in both designs can be modified and the functional characteristics of the final molecule altered. In this case, the upper binding unit or the lower unit or both of the protein construct modules may be an alternative structure to the immunoglobulin Fab domain, allowing for alternative molecules based on sequences derived from peptides, receptor ectodomains, binding domains and, inter alia, other dimerizing immune recognition receptors such as T cell receptors. These constructs can be formed into many forms and examples are provided herein, such as: the four polypeptide chains (2) can encode an IgG comprising two complete protein construct modules with 4 binding domain units (1x A, 1X B, or 2x A or B), two or more linker sequences²Like structures, and may or may not have immunoglobulin hinge and Fc domains, wherein pairing of the heterodimers may or may not be driven by mutations in the Fc. In the expression of 3 polypeptides, Fab can be enhanced by the addition of a binding domain (3) or peptide that confers the structure with potential trispecificity or altered valency²And (5) designing. In Fab²Trispecific or altered valencies may also be achieved by the addition of one (4) or two (5) additional protein construct-linker-Fab/receptor structures at the c-terminus in the design. It should also be noted that any of the structures outlined in this figure or in fig. 2 and 3 can be further functionalized by adding c-terminal or n-terminal fusions of any kind of polypeptide chain or by chemical conjugation. The variable regions are indicated in white. The constant regions are indicated in grey.

FIG. 12 Fab based²The principle 'passive' structure of the protein construct. In thatIn this case, the upper binding unit of the protein construct module may be placed c-terminal to the Fc domain. These constructs may or may not have an immunoglobulin hinge region and Fc domain, where the pairing of heterodimers may or may not be driven by mutations in the Fc. In this construct, binding of both Fab or receptor domains to their cognate targets should become fully active only after cleavage of at least one linker. It should also be noted that any of the structures outlined in this figure can be further functionalized by adding c-terminal or n-terminal fusions of any kind of polypeptide chain or by chemical conjugation. The variable regions are indicated in white. The constant regions are indicated in grey.

FIG. 13 Fab based²The protein construct principle of the "activatable" Antibody Drug Conjugate (ADC) strategy. In this case, the upper and lower binding units of the protein construct modules may contain antibodies to the same internalizing receptor target, or to two different targets found on the same cell surface. These constructs may be chemically conjugated or fused to a "payload" moiety such as a toxin or other active molecule to form an ADC, and may or may not have an immunoglobulin hinge region and Fc domain, where pairing of heterodimers may or may not be driven by mutations in the Fc. In this construct, the binding of the upper Fab or receptor domain to its cognate target is constitutively active, resulting in the accumulation of antibody in the tissue where its cognate target is expressed. The constructs do not initially drive internalization into the target cell because binding is monovalent and receptors are known to be significantly internalized only when 2 or more receptor domains are cross-linked by bivalent antibody binding. The activity of the second (lower) Fab or receptor domain should only function after cleavage of the linker by disease-associated enzymes that then drive multivalent receptor binding and internalization of the ADC, allowing delivery of (e.g., cytotoxic or inflammatory) payload moieties. It should also be noted that any of the structures outlined in this figure can be further functionalized by adding c-terminal or n-terminal fusions of any kind of polypeptide chain.

FIG. 14 direct ELISA of purified intact protein constructs binding to human and murine target proteins. Samples were applied in a direct binding ELISA against human Her2 and human and murine CD 47.

Figure 15A-figure 15f direct ELISA of purified intact and MMP digested protein constructs binding to human target proteins. Enzymatic digestion of protein constructs was performed using a time course of human MMP7 (fig. 15A), MMP8 (fig. 15B), MMP10 (fig. 15C), MMP12 (fig. 15D), MMP13 (fig. 15E), and cathepsin S (fig. 15F) incubated at 2, 4, 8, and 24 hours, plus incubation in enzyme-free buffer for 24 hours as a negative control (time 0). Samples from these digestion schedules were then applied to direct binding ELISA against human Her2 and CD 47.

Figure 16A-figure 16b Biacore SPR assay of purified intact and MMP digested Her47-LHL-LHLF binding to human target protein. Enzymatic digestion of Her47-LHL-LHLF was performed using human MMP12 over the course of 2, 4, 8 and 24 hours of incubation, plus 24 hours of incubation in enzyme-free buffer as a negative control (undigested). Samples from these digestion schedules were then captured on anti-Fc antibody coated Biacore chips and human Her2 (fig. 16A) or human CD47 (fig. 16B) were flowed into solution. Rmax values were plotted to indicate the maximum binding observed at the highest concentration of analyte protein.

FIG. 17 Biacore SPR assay of purified intact and 24 hour MMP digested Her47-LHL-LHLF binding to human target proteins. Her47-LHL-LHLF was enzymatically digested with human MMP12 incubated for 24 hours or without enzyme as a negative control ('before protease treatment'). The samples were then captured on anti-Fc antibody coated Biacore chips and human CD47 was flowed into solution at various concentrations. The binding curves show that undigested (intact) Her47-LHL-LHLF protein has no interaction with huCD47 even at 400nM huCD47, whereas strong binding of the same protein is evident after MMP12 activation at all concentrations tested.

FIGS. 18A-18B, intact Her47-LHL-LHL IgG²Structural modeling of a structure. FIG. 18A, IgG²Molecular modeling of the complete structure of the molecule shows that the upper (trastuzumab) Fab domain is in contact with its Her2 epitope (grey). FIG. 18B, IgG²Molecular modeling of the complete structure of the molecule shows that the upper (trastuzumab) Fab domain is in contact with its Her2 epitope (grey), but the CD47 ectodomain is also superimposed on its possible binding site of the a-D5 lower Fab. This analysis demonstrated that the CD47 epitope was unable to bind when both linkers were intact.

FIGS. 19A-19I Her47 Fab with different linkers ²Structural dynamics of the structure. Solvent Accessible Surface Area (SASA) results were obtained for 3 linkers (LHL, LHLF, L2). Fig. 19A, 19D and 19G show absolute SASA values for 9 dynamic runs of LHL and LHLF joints and 10 runs using L2 (all exceeding 6 ns). Fig. 19B, 19E, 19H, 19C, 19F and 19I show normalized results representing the difference from the starting SASA value over the 6ns dynamic run time and over the first 2.5ns of the 6ns dynamic run. FIG. 19A, FIG. 19B and FIG. 19C depict SEQ ID NO 2. FIG. 19D, FIG. 19E and FIG. 19F depict SEQ ID NO 3. FIG. 19G, FIG. 19H and FIG. 19I depict SEQ ID NO 32.

FIGS. 20A-20B intact and activated Her47 LHL-LHL Fab²Structural dynamics of the structure. (FIG. 20A) overlapping Fab in both intact linker and activated (single linker cleaved by protease) format²And (5) structure. (FIG. 20B) Fab cleaved from either LHL or LHLF linker²The molecular dynamics of the region simulate the two poses acquired. anti-CD 47 Fab is shown in black and anti-HER 2 Fab is shown in grey. The figure demonstrates the maximal mobility of the Her2 domain and the eventual exposure of the anti-CD 47 Fab CDRs.

FIG. 21 flow cytometry analysis of protein binding to ` Tg32 ` mouse erythrocytes. A-D5 IgG1, IgG were used ²Her47 LHL-LHL、IgG²Her47 LHL-LHLF and Fab²Met47 LHL-LHL was analyzed by flow cytometry for binding to erythrocytes. Binding was measured using an anti-human PE conjugated secondary antibody. A-D5 IgG1 was tested at 0.1. mu.g/ml, 1. mu.g/ml and 10. mu.g/ml. IgG was tested at 0.1. mu.g/ml, 1. mu.g/ml and 10. mu.g/ml²Her47 LHL-LHL. IgG was tested at 0.1. mu.g/ml, 1. mu.g/ml and 10. mu.g/ml²Her47 LHL-LHLF. Fab was tested at 0.1. mu.g/ml, 1. mu.g/ml and 10. mu.g/ml² Met47 LHL-LHL。

FIG. 22.' Tg32' mouse erythrocyte hemagglutination assay. A-D5 IgG1, IgG were used²Her47 LHL-LHL、IgG²Her47 LHL-LHLF and Fab²Met47 LHL-LHL undergoes agglutination of erythrocytes. The proteins (in nM) were titrated using pooled fresh red blood cells from multiple donor mice.

Fig. 23 tolerance studies in 'Tg 32' mice: and (5) analyzing the body weight. Use of A-D5 IgG1, IgG in Tg32 mice with all proteins administered at 2mg/kg and 10mg/kg concentrations² Her47 LHL-LHL、IgG²Her47 LHL-LHLF and Fab²Tolerance studies of Met47 LHL-LHL. Body weight was then monitored for 60 days. The A-D5 IgG 110 mg/kg dose was intolerant and the cohort was terminated on day 1.

Fig. 24 tolerance study in 'Tg 32' mice: reticulocyte analysis at day 5 post-dose. Use of A-D5 IgG1, IgG in Tg32 mice with all proteins administered at 2mg/kg and 10mg/kg concentrations ² Her47 LHL-LHL、IgG²Her47 LHL-LHLF and Fab²Tolerance studies of Met47 LHL-LHL. Blood samples were collected and reticulocyte levels were measured. A-D5 IgG1 at a dose of 2mg/kg showed a significant increase in reticulocyte levels.

Figure 25A-figure 25k. 'Tg 32' tolerance studies in mice: hematological analysis at day 5, day 29 and day 60 post-dose. Use of A-D5 IgG1, IgG in Tg32 mice with all proteins administered at 2mg/kg and 10mg/kg concentrations² Her47 LHL-LHL、IgG²Her47 LHL-LHLF and Fab²Tolerance studies of Met47 LHL-LHL. Blood samples were collected and levels of reticulocytes (fig. 25A), red blood cells (RBCs, fig. 25B), hemoglobin (fig. 25C), Mean Corpuscular Hemoglobin Concentration (MCHC) (fig. 25D), Mean Corpuscular Volume (MCV) (fig. 25E), leukocytes (fig. 25F), monocytes (fig. 25G), lymphocytes (fig. 25H), basophils (fig. 25I), eosinophils (fig. 25J), and neutrophils (fig. 25K) were measured.

Fig. 26. pharmacokinetic studies in 'Tg 32' mice: data for each molecule at two doses. Use of IgG in Tg32 mice with administration of all proteins at 2mg/kg and 10mg/kg concentrations² Her47 LHL-LHL、IgG²Her47 LHL-LHLF and Fab²Pharmacokinetic studies of Met47 LHL-LHL. A-D5 IgG1 was administered at 2 mg/kg. Serum samples were taken from 30 minutes to 42 days post-dose and human IgG levels (in μ g/ml) were measured.

Fig. 27. pharmacokinetic studies in 'Tg 32' mice: data for each dose. Administration of all proteins at 2mg/kg and 10mg/kg concentrations in Tg32 mice Using IgG² Her47 LHL-LHL、IgG²Her47 LHL-LHLF and Fab²Pharmacokinetic studies of Met47 LHL-LHL. A-D5 IgG1 was administered at 2 mg/kg. Serum samples were taken from 30 minutes to 42 days post-dose and human IgG levels (in μ g/ml) were measured. Concentrations were plotted at 2mg/kg (FIG. 27A) and 10mg/kg (FIG. 27B) doses, respectively. The A-D5 IgG 12 mg/kg dose was included as a reference in both assays.

Fig. 28. pharmacokinetic studies in 'Tg 32' mice: AUC data for each dose. Administration of all proteins at 2mg/kg and 10mg/kg concentrations in Tg32 mice Using IgG² Her47 LHL-LHL、IgG²Her47 LHL-LHLF and Fab²Pharmacokinetic studies of Met47 LHL-LHL. A-D5 IgG1 was administered at 2 mg/kg. Serum samples were taken from 30 minutes to 42 days post-dose and human IgG levels (in μ g/ml) were measured. The area under the curve (AUC) for each dose was calculated using concentration measurements over time.

Fig. 29A-fig. 29b flow cytometry analysis of binding to NHP and human erythrocytes. The use of A-D53M (Effector null) IgG1, IgG² Her47 LHL-LHL、IgG²Her47 LHL-LHLF and trastuzumab were subjected to flow cytometry analysis for binding to erythrocytes. Binding was measured using an anti-human PE conjugated secondary antibody. a-D5 IgG1 is the only protein that showed concentration-dependent binding to both NHP (cynomolgus monkey) erythrocytes (fig. 29A) and human erythrocytes (fig. 29B).

Figure 30A-figure 30n direct ELISA of binding of purified intact and MMP digested protein constructs (digested at ph7.4 and ph 6.0) to human target proteins. The protein construct was enzymatically digested during the time course of 2, 4, 8 and 24 hours incubation with human MMP at ph7.4 or ph6.0, plus 24 hours incubation in enzyme-free buffer as negative control (time 0). Samples from these digestion schedules were then applied to direct binding ELISA against human Her2 and CD 47.

Figure 31A-figure 31b direct ELISA of binding of purified intact and cathepsin-digested protein constructs (digested at ph7.4 and ph 6.0) to human target proteins. The protein construct was enzymatically digested with human cathepsin at ph7.4 or ph6.0 over the course of 2, 4, 8 and 24 hours incubation, plus 24 hours incubation in enzyme-free buffer as negative control (time 0). Samples from these digestion schedules were then applied to direct binding ELISA against human Her2 and CD 47.

Fig. 32A-fig. 32b flow cytometry analysis of binding to human cancer cells. Enzymatic digestion of IgG using human MMP12 at ph7.4 during the time course of anti-CD 47, trastuzumab, IgG1 isotype, and IgG digested at 2, 4, 8, and 24 hours all incubated in 2, 4, 8, and 24 hours plus 24 hours in enzyme-free buffer as negative control (time 0) ²Her47 LHL-LHL or IgG²Her47 LHL-LHLF was analyzed by flow cytometry for binding to erythrocytes. Binding was measured using an anti-human PE conjugated secondary antibody. Binding was measured on Her2 high cell line BT-474 (FIG. 32A, FIG. 32B) and on Her2 low cell line MCF-7 (FIG. 32C, FIG. 32D).

FIG. 33 MMP12 digested IgG²Her47 LHL-LHL or IgG²SDS-PAGE analysis of Her47 LHL-LHLF. IgG enzymatically digested at pH7.4 using human MMP12 during time periods of 2, 4, 8, and 24 hours incubation, plus 24 hours incubation in enzyme-free buffer as a negative control (time 0)²Her47 LHL-LHL or IgG²Her47 LHL-LHLF samples were subjected to SDS-PAGE.

MMP12 digested IgG²Mass spectrometric analysis of Her47 LHL-LHL. IgG enzymatically digested at pH7.4 using human MMP12 during 2, 4, 8 and 24 hours of incubation plus 24 hours of incubation in enzyme-free buffer as a negative control (time 0)²Samples from Her47 LHL-LHL were subjected to mass spectrometry. Measurements indicated the presence of peptides that were intact LHL linker (fig. 34A) and MMP12 cleaved linker (fig. 34B). FIG. 34A depicts SEQ ID NO 110. FIG. 34B depicts SEQ ID NO 111.

FIG. 35 size exclusion chromatography of protein A purified Her47 LHLF-LHL IgG1-2 hDAA. Her47 LHLF-LHL IgG1-2hDAA protein was expressed in CHO cells, purified by ProA column and analyzed by SEC. Two small larger MW peaks were observed and a large peak (10.30, approximately 250kDa) of the expected size product.

FIG. 36 SDS-PAGE analysis of purified peak fractions from size exclusion chromatography of Her47 LHLF-LHL IgG1-2 hDAA. An unreduced sample of Her47 LHLF-LHL IgG1-2hDAA was subjected to SDS-PAGE: lane 1-molecular weight standard, lane 2-total ProA eluted protein, lane 3-blank, lane 4-peak 1, lane 5-peak 2, lane 6-peak 3 (correct product).

FIG. 37 SDS-PAGE analysis of purified peak fractions from size exclusion chromatography of Her47 LHLF-LHL IgG1-2 hDAA. SDS-PAGE of reduced samples of Her47 LHLF-LHL IgG1-2 hDAA: lane 1-molecular weight standard, lane 2-total ProA eluted protein, lane 3-blank, lane 4-peak 1, lane 5-peak 2, lane 6-peak 3 (correct product).

Figure 38A-figure 38c direct ELISA of purified intact and MMP12 digested Her47IgG1-2hDAA protein. Enzymatic digestion of Her47 LHL-LHLF IgG1-2hDAA (fig. 38A) using human MMP12 at ph7.4 was performed over a time course of 2, 4, 8 and 24 hours incubation plus 24 hours incubation in enzyme-free buffer as negative control (time 0, 2 hours, 4 hours, 8 hours, 24 hours incubation). Samples from these digestion schedules were then applied in a direct binding ELISA against human Her2 and murine EpCAM (fig. 38A). Digested (dark grey) and undigested (light grey) samples were then subjected to ELISA against human CD47 (fig. 38B). The samples were also subjected to SDS-PAGE: lane 1-molecular weight marker, lane 2-0 hr digest, lane 3-2 hr digest, lane 4-8 hr digest, and lane 4-24 hr digest (FIG. 38C).

Figure 39A-figure 39l direct ELISA of purified intact and MMP12 digested IgG2 Her47 protein with alternative linker composition. Purified proteins from clones Her47 LHL-EK, Her 47-LHL-Thr, Her 47-LHL-tPA, Her 47-LHL-uPA, Her 47-LHL-GrB and Her 47-LHL-a 5 were all tested in a titration ELISA against human Her2 and CD47 targets (fig. 39A, 39C, 39E, 39G, 39I, 39K). Each protein was then also subjected to time course enzymatic digestion and ELISA binding to Her2 and CD47 targets (fig. 39B, fig. 39D, fig. 39F, fig. 39H, fig. 39J, fig. 39L).

FIG. 40 multiple dose tolerance study in NOD-SCID mice: and (5) analyzing the body weight. IgG administration was performed by administering all proteins once every 5 days (4 doses total)² Her47 LHL-LHL、IgG² Her47 LHL-LHLF、Fab²Her47LHL-LHL and Fab²Tolerance studies in Her47 LHL-LHLF.

FIG. 41A-FIG. 41D protein A purified Her2CD3 Fab²Size exclusion chromatography of proteins. Fab²Her23 LHL-LHL-S (FIG. 41A), Fab²Her23 LHLF-LHL-S (FIG. 41B), Fab²Her23 LHL-LHL (FIG. 41C) and Fab²Her23 LHLF-LHL (FIG. 41D) was expressed in CHO cells, purified by ProA column and analyzed by SEC. Fab²Her23 LHL-LHL (FIG. 41C) and Fab²Both Her23 LHLF-LHL (FIG. 41D) exhibited lower molecular weight contaminants (peak 15.38).

FIGS. 42A-42B purified intact and MMP digested Her2CD3 Fab pair using Her2 Low MCF-7 cells²The protein was subjected to CD3 co-conjugation bioassay analysis. Human MMP12 was used for antibody Fab at pH7.4 over the course of 2, 4, 8 and 24 hours of incubation plus 24 hours of incubation in enzyme-free buffer as negative control (time 0)²Her23 LHLF-LHL-S (FIG. 42A), Fab²Her23LHL-LHL-S (FIG. 42B) was subjected to enzymatic digestion. Samples from these digestion schedules were then applied to the Promega Jurkat cell CD3 reporter assay using MCF-7 cells as target cells.

FIGS. 43A-43℃ purified intact and MMP-digested Her2CD3 Fab pair using Her2 HomeBT-474 cells²The protein was subjected to CD3 co-conjugation bioassay analysis. Control antibodies (FIG. 43A), and Fab²Her23 LHLF-LHL-S (FIG. 43B) or Fab²Her23LHL-LHL-S (FIG. 43C) [ both had been incubated at 2, 4, 8 and 24 hours, plus incubation for 24 hours in enzyme-free buffer as negative control (time 0)The enzymatic digestion is carried out using human MMP12 at pH7.4]Applied to the Promega Jurkat cell CD3 reporter assay at 0.1. mu.g/ml, BT-474 cells were used as target cells.

FIG. 44A-FIG. 44B vs. IgG²And Fab²Charge isomer (charge variant) analysis of the Her47 protein-charge heterogeneity analysis is important in the characterization of monoclonal antibodies, as it provides important information about product quality and stability. Heterogeneity may be caused by enzymatic post-translational modifications (glycosylation, lysine truncation) or chemical modifications (oxidation or deamidation) during purification and storage. Analysis of the Charge isomer spectra of the provided test samples was performed by a commercial Charge isomer Assay (Charge variable Assay). IgG ²Her47 LHL-LHL (FIG. 44A) and Fab²The charge isomer profiles of both Her47 LHL-LHL (FIG. 44B) showed a uniform profile with a major isoform (50-57% of the total), a major acidic isoform (40-48% of the total) and a minor basic isoform (approximately 3%).

Fig. 45A-fig. 45b size exclusion chromatography of Her47 protein after 5 rounds of freeze-thawing. IgG²Her47 LHL-LHL (FIG. 45A) and Fab²Her47 LHL-LHL (FIG. 45B) were both subjected to 5 rounds of freeze-thawing and samples from 0-5 rounds were then subjected to SEC. For either protein, no aggregation, fragmentation or product loss was observed.

FIG. 46 alternative protein construct design. The figure depicts the protein construct Fab²Illustrative examples of designs. The design may be based on the following sequence: 1. the upper variable domain is removed. 2. Contain "pseudo" non-binding variable domains. 3. The upper Fab was replaced by a diabody (or two scfvs). The variable regions are indicated in white. The constant regions are indicated in grey.

Fig. 47A-fig. 47b cell proliferation assays were performed on purified intact Her2CD47 protein using Her2 high BT-474 cells. Trastuzumab, isotype control IgG1, IgG²Her47 LHL-LHL (FIG. 47A) and Fab ²Her47 LHL-LHL (FIG. 47B) was applied to BT-474 cells over a 72 hour incubation period and cell proliferation was measured. Data are expressed as% inhibition of cell growth.

FIG. 48A-FIG. 48G in vivo efficacy analysis of Her47 molecules in NOD-SCID mice (KYSE-410 model). Trastuzumab (FIG. 48A), IgG²Her47 LHL-LHLF (FIG. 48B), IgG²Her47 LHL-LHL (FIG. 48C), Fab²Her47 LHL-LHLF (FIG. 48D) and Fab²Her47 LHL-LHL (FIG. 48E) were each administered (intravenously, on days 0, 5, 10) in NOD-SCID mice bearing KYSE-410 tumors. Tumor volume measurements were taken on day 4, day 7, and day 11 and plotted against vehicle. Fab²Her47 LHL-LHLF and Fab²Her47 LHL-LHL showed different potencies (FIG. 48F). None of the administered groups showed any weight loss that might be indicative of toxicity of the administered molecule (fig. 48G).

Detailed Description

Recombinant proteins conditionally active in diseased human tissues are disclosed herein. In some cases, the protein comprises a binding domain masked by another portion of the protein in the non-diseased tissue. The protein further comprises a peptide linker that is cleaved by one or more proteases expressed in the diseased tissue. Linker cleavage unmasks the binding domain in the diseased tissue, allowing the protein to selectively bind and/or function in the diseased tissue. The proteins of the invention are particularly useful for binding drug targets expressed in both diseased and non-diseased tissues.

Provided herein are a number of activatable protein molecules and their medical uses. In some aspects, a variety of functional properties of the molecule are contemplated, including target binding specificity, effective limitation of undesirable activity in native proteins but full activity in activated form, maintenance of conditional affinity for one or more targets from human and animal test species (e.g., cynomolgus monkey (also known as cynomolgus monkey), i.e., cynomolgus monkey (Macaca fascicularis)), biophysical stability, and/or yield from protein expression platforms used in research, clinical, and commercial supplies.

In some aspects, protein molecules are provided that specifically bind to one or more human drug targets and optionally also to cynomolgus orthologs of those targets, wherein the protein molecules comprise a heavy chain region and a light chain region assembled from one or more polypeptides having the form:

V-C-linker-V-C

C-linker-V-C

In some aspects, the protein molecule comprises two polypeptide chains and has the form:

VH 1-C-linker-VH 2-C

VL 1-C-linker-VL 2-C

In some aspects, the protein molecule comprises two polypeptide chains and has the form:

VL 1-C-linker-VH 2-C

VH 1-C-linker-VL 2-C

"V" refers to an immunoglobulin or T cell receptor variable region or the extracellular domain of a receptor. "VH 1" and "VL 1" refer to the heavy chain variable region and the light chain variable region paired with each other to bind antigen. "VH 2" and "VL 2" refer to the heavy chain variable region and the light chain variable region paired with each other to bind antigen. "C" refers to an immunoglobulin or T cell receptor constant region. In an aspect of the invention, the V-C and V-C units on either side of the linker domain form upper and lower immunoglobulin Fab domains, wherein the lower Fab domain exhibits binding to its cognate target that is reduced or eliminated by the presence of the linker domain fused to the N-terminus of each V domain in the lower Fab. In another aspect, the upper or lower Fab domain may be replaced with an Fc fragment, one or both receptor ectodomains, or any domain with or without any specific binding function.

In some aspects, provided herein are proteins comprising a first portion and a second portion and a peptide linker between the first portion and the second portion.

Wherein the peptide linker comprises an amino acid sequence from a human immunoglobulin hinge region or an amino acid sequence or amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 (e.g., 1 to about 7) amino acid substitutions as compared to a human immunoglobulin hinge region;

Wherein the peptide linker is cleavable by a protease expressed in the diseased tissue;

wherein the second portion is capable of specifically binding to a molecule expressed in diseased tissue; and is

Wherein binding of said second moiety to said molecule expressed in said diseased tissue is reduced or inhibited when said peptide linker is not cleaved.

The linker moiety may also comprise a peptide linker derived from an immunoglobulin hinge region having zero, one or more germline-distant mutations.

In some aspects, the peptide linker comprises or consists of a sequence set forth in GPAPELL (SEQ ID NO:1), GPAPELLGGGS (SEQ ID NO:2), GPAPLGLGGGS (SEQ ID NO:3), PPCPAPELLGGGS (SEQ ID NO:4), PPCPAPLGLGGGS (SEQ ID NO:5) GPAPELLGGPS (SEQ ID NO:69), GPAPLGLGGPS (SEQ ID NO:70), PPCPAPELLGGPS (SEQ ID NO:71), PPCPAPLGLGGPS (SEQ ID NO:72), GPAPEAAGAGS (SEQ ID NO:81), GPADDDDKSGS (SEQ ID NO:82) (cleavable by enterokinase), GPALVPRGSGS (SEQ ID NO:83) (cleavable by thrombin), GPGPFGRSAGGP (SEQ ID NO:84) (cleavable by tPA), GPAPLEADAGS (SEQ ID NO:85) (cleavable by granzyme B), GPAPEARRGGS (SEQ ID NO:86) (cleavable by uPA) or GPAPEGEARGS (SEQ ID NO:87) (cleavable by ADAMTS-5). In some aspects, the peptide linker comprises or consists of two, three or four of the above sequences.

Immunoconjugates comprising the proteins of the invention linked to a therapeutic agent are also provided.

In another aspect, the invention provides a nucleic acid molecule encoding a protein or portion thereof as defined herein. Also provided are vectors comprising the nucleic acid molecules of the invention. Host cells comprising the nucleic acid molecules or vectors of the invention are also provided.

In a further aspect, there is provided a method of producing a conditionally active protein of the invention, the method comprising culturing a host cell of the invention under conditions which result in expression and/or production of the protein, and isolating the protein from the host cell or culture.

In another aspect of the invention, there is provided a pharmaceutical composition comprising a protein of the invention as defined herein, or a nucleic acid molecule of the invention as defined herein, or a vector of the invention as defined herein, or an immunoconjugate of the invention as defined herein.

Also provided are methods for enhancing an immune response in a subject, the method comprising administering an effective amount of a protein of the invention as defined herein, or an immunoconjugate of the invention as defined herein, or a nucleic acid molecule of the invention as defined herein, or a vector of the invention as defined herein, or a pharmaceutical composition of the invention as defined herein.

In a further aspect, there is provided a method for the treatment or prevention of cancer in a subject, said method comprising administering an effective amount of a protein of the invention as defined herein, or an immunoconjugate of the invention as defined herein, or a nucleic acid molecule of the invention as defined herein, or a vector of the invention as defined herein, or a pharmaceutical composition of the invention as defined herein.

Also provided is a protein of the invention as defined herein, or an immunoconjugate of the invention as defined herein, or a nucleic acid molecule of the invention as defined herein, or a vector of the invention as defined herein, or a pharmaceutical composition of the invention as defined herein, for use as a medicament.

Also provided is a protein, or an immunoconjugate, or a nucleic acid molecule, or a vector, or a pharmaceutical composition of the invention, as defined herein, for separate, sequential or simultaneous use in combination with a second therapeutic agent (e.g., an anti-cancer agent).

In a further aspect, there is provided the use of a protein of the invention as defined herein, or an immunoconjugate of the invention as defined herein, or a nucleic acid molecule of the invention as defined herein, or a vector of the invention as defined herein, or a pharmaceutical composition of the invention as defined herein, in the manufacture of a medicament for the treatment of cancer.

Also provided is a method for treating or preventing an autoimmune or inflammatory disease in a subject, the method comprising administering an effective amount of a protein as defined herein, or an immunoconjugate as defined herein, or a nucleic acid molecule as defined herein, or a vector as defined herein, or a pharmaceutical composition as defined herein.

Also provided is the use of a protein as defined herein, or an immunoconjugate as defined herein, or a nucleic acid molecule as defined herein, or a vector as defined herein, or a pharmaceutical composition as defined herein, in the manufacture of a medicament for the treatment of an autoimmune disease or an inflammatory disease.

Also provided is a method for the treatment or prevention of cardiovascular disease or fibrotic disease in a subject, the method comprising administering an effective amount of a protein as defined herein, or an immunoconjugate as defined herein, or a nucleic acid molecule as defined herein, or a vector as defined herein, or a pharmaceutical composition as defined herein.

Also provided is a protein as defined herein, or an immunoconjugate as defined herein, or a nucleic acid molecule as defined herein, or a vector as defined herein, or a pharmaceutical composition as defined herein, for use as a medicament. Also provided is an antibody molecule or antigen-binding portion thereof as defined herein, or an immunoconjugate as defined herein, or a nucleic acid molecule as defined herein, or a vector as defined herein, or a pharmaceutical composition as defined herein, for use in the treatment of a cardiovascular disease or a fibrotic disease.

In some aspects, the invention provides a protein comprising a first portion and a second portion and a peptide linker between the first portion and the second portion, wherein the peptide linker comprises an amino acid sequence from a human immunoglobulin hinge region or an amino acid sequence or amino acid sequence having amino acid substitutions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 amino acid substitutions) as compared to a human immunoglobulin hinge region; wherein the peptide linker is cleavable by a protease expressed in the diseased tissue; wherein the second portion is capable of specifically binding to a molecule expressed in the diseased tissue; and wherein binding of the second moiety to a molecule expressed in the diseased tissue is reduced or inhibited when the peptide linker is not cleaved. In some aspects, the amino acid substitution is a conservative amino acid substitution. In some aspects, the peptide linker comprises an amino acid sequence from a human immunoglobulin hinge region or an amino acid sequence or amino acid sequence having from 1 to about 7 amino acid substitutions as compared to a human immunoglobulin hinge region; in some aspects, the peptide linker comprises an amino acid sequence from a human immunoglobulin hinge region or an amino acid sequence or amino acid sequence having 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 2-3, 2-4, 2-5, 2-6, 2-7, 3-4, 3-5, 3-6, 3-7, 4-5, 4-6, 4-7, 5-6, 5-7, or 6-7 amino acid substitutions as compared to a human immunoglobulin hinge region.

In some aspects, a peptide linker that can be cleaved by a protease expressed in diseased tissue can be cleaved by human Matrix Metalloproteinases (MMPs) or human cathepsins. In some cases, a peptide linker that can be cleaved by a protease expressed in diseased tissue can be cleaved by human Enterokinase (EK), human thrombin (Thr), human tPA (tissue plasminogen activator), human granzyme B (GrB), human uPA (urokinase-type plasminogen activator), or human ADAMTS-5 (Deplosin-like metalloprotease 5 containing thrombospondin type 1 motif; A5). In some cases, the peptide linker comprises a human MMP cleavage site or a human cathepsin cleavage site. In some cases, the peptide linker comprises a human enterokinase, human thrombin, human tPA, human granzyme B, human uPA, or human ADAMTs-5 cleavage site. In some cases, the peptide linker comprises the MMP substrate sequence PLGL (SEQ ID NO: 12). In some cases, the peptide linker comprises or consists of an amino acid sequence of GPAPELL (SEQ ID NO:1), GPAPELLGGGS (SEQ ID NO:2), GPAPLGLGGGS (SEQ ID NO:3), PPCPAPELLGGGS (SEQ ID NO:4), or PPCPAPLGLGGGS (SEQ ID NO:5), GPAPELLGGPS (SEQ ID NO:69), GPAPLGLGGPS (SEQ ID NO:70), PPCPAPELLGGPS (SEQ ID NO:71), PPCPAPLGLGGPS (SEQ ID NO:72), GPAPEAAGAGS (SEQ ID NO:81), GPADDDDKSGS (SEQ ID NO:82), GPALVPRGSGS (SEQ ID NO:83), GPGPFGRSAGGP (SEQ ID NO:84), GPAPLEADAGS (SEQ ID NO:85), GPAPEARRGGS (SEQ ID NO:86), or GPAPEGEARGS (SEQ ID NO: 87).

In some cases, the peptide linker comprises or consists of two, three, or four amino acid sequences in table 1 fused by peptide bonds in a single amino acid chain. In some cases, the peptide linker is between about 5 amino acids and about 15 amino acids, between about 5 amino acids and about 20 amino acids, or between about 5 amino acids and about 25 amino acids in length.

In some cases, the peptide linker between the first portion and the second portion comprises the following amino acid sequence at the N-terminus of the peptide linker sequence: X1-proline-X2. In some aspects, X1 is alanine, glycine, serine, proline, or threonine. In some aspects, X1 is alanine, glycine, serine, proline or threonine, aspartic acid, asparagine, or valine. In some aspects, X2 is alanine, glycine, serine, proline, or threonine. In some aspects, X2 is alanine, glycine, serine, proline or threonine, aspartic acid, asparagine, or valine. In some aspects, X1 and X2 are the same amino acid. In some aspects, X1 and X2 are different amino acids.

In some cases, a peptide linker that can be cleaved by a protease expressed in the diseased tissue can be cleaved by any of human MMP1, MMP2, MMP3, MMP4, MMP5, MMP6, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP18, MMP19, MMP20, MMP21, MMP22, MMP23, MMP24, MMP25, MMP26, MMP27, or MMP 28. In some cases, a peptide linker that can be cleaved by a protease expressed in the diseased tissue can be cleaved by any of human MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, MMP-12, or MMP-13. In some cases, the level or activity of human MMPs is increased in diseased tissue compared to the level or activity of human MMPs in non-diseased tissue.

In some cases, a peptide linker that can be cleaved by a protease expressed in diseased tissue can be cleaved by any of human cathepsin a, cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin F, cathepsin G, cathepsin H, cathepsin K, cathepsin L1, cathepsin V, cathepsin O, cathepsin S, cathepsin W, or cathepsin Z. In some cases, a peptide linker that can be cleaved by a protease expressed in diseased tissue can be cleaved by any of human cathepsin D, cathepsin G, or cathepsin K. In some cases, the level or activity of human cathepsin in diseased tissue is increased compared to the level or activity of human cathepsin in non-diseased tissue. In some cases, the level or activity of human cathepsin in tissues with pH <7.0 is increased compared to the level or activity of human cathepsin in tissues with pH ≧ 7.4.

In some aspects, the first portion of any of the proteins of the invention comprises an antibody, an antigen-binding portion of an antibody, or an extracellular domain of a receptor. In some cases, the first portion is a Fab, single chain Fab, VH domain, VL domain, immunoglobulin neo-antigen receptor (IgNAR), single chain variable fragment (scFv), diabody, or T cell receptor domain. IgNAR is an antibody which is produced by sharks and other cartilaginous fish only with homodimeric heavy chains (Feige et al, PNAS,2014,111(22): 8155-.

In some cases, the first portion specifically binds to a molecule expressed in the diseased tissue. In some embodiments, the first moiety specifically binds to a Tumor Associated Antigen (TAA). In some cases, the first moiety specifically binds to human EGFR, human HER2, human HER3, human CD105, human C-KIT, human PD1, human PD-L1, human PSMA, human EpCAM, human Trop2, human EphA2, human CD20, human BCMA, human GITR, human OX40, human CSF1R, human Lag3, or human cMET. In some embodiments, the first portion also binds to a cynomolgus monkey ortholog of any of these molecules.

In some aspects, the first portion comprises a heavy chain Variable (VH) region and a light chain Variable (VL) region. In some cases, the first portion further comprises an immunoglobulin constant region or a portion of an immunoglobulin constant region. In some cases, the immunoglobulin constant region is IgG, IgE, IgM, IgD, IgA, or IgY.

In some aspects, the anti-HER 2 variable region sequence used in the protein constructs disclosed herein is the variable region sequence of trastuzumab. In some aspects, the anti-CD 3 variable region sequence used in the protein constructs disclosed herein is the variable region sequence of OKT3 or SP 34. In some aspects, anti-cMET variable region sequences for use in the protein constructs disclosed herein are provided in WO 2019/175186. In some aspects, anti-CD 47 variable region sequences for use in the protein constructs disclosed herein are provided in WO 2019/034895.

In some aspects, the second portion of any of the proteins of the invention comprises an antibody, an antigen-binding portion of an antibody, or an extracellular domain of a receptor. In some cases, the second moiety is a Fab, single chain Fab, VH domain, VL domain, immunoglobulin neoantigen receptor (IgNAR), single chain variable fragment (scFv), diabody, or T cell receptor domain.

In some cases, the second portion specifically binds to a molecule expressed in the diseased tissue. In some embodiments, the second moiety specifically binds to a Tumor Associated Antigen (TAA). In some cases, the second moiety specifically binds to human CD 47. In some cases, the second moiety specifically binds to human PD-L1. In some cases, the second moiety specifically binds to a molecule expressed by a human immune cell. In some cases, the molecule expressed by the human immune cell is human CD3, human CD16A, human CD16B, human CD28, human CD89, human CTLA4, human NKG2D, human sirpa, human SIRP γ, human PD1, human lang 3, human 4-1BB, human OX40, or human GITR. In some embodiments, the second portion also binds to a cynomolgus monkey ortholog of any of these molecules.

In some aspects, the second portion comprises a heavy chain Variable (VH) region and a light chain Variable (VL) region. In some cases, the second portion further comprises an immunoglobulin constant region or a portion of an immunoglobulin constant region. In some cases, the immunoglobulin constant region is IgG, IgE, IgM, IgD, IgA, or IgY.

In some aspects, the first portion and/or the second portion of the protein of the invention may comprise an immunoglobulin constant region. In some embodiments, the immunoglobulin constant region is IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgE, or IgM. In further embodiments, the immunoglobulin constant region is IgG1, IgG2, IgG3, IgG1null, IgG4(S228P), IgA1, IgA2, IgE, or IgM. In some embodiments, the first portion and/or the second portion of the protein of the invention may comprise an immunologically inert constant region. In some aspects, the first portion and/or the second portion of the proteins of the invention may comprise an immunoglobulin constant region comprising a wild-type human IgG1 constant region, a human IgG1 constant region comprising amino acid substitutions L234A and L235A, a human IgG1 constant region comprising amino acid substitutions L234A, L235A, and G237A, or a human IgG1 constant region comprising amino acid substitutions L234A, L235A, G237A, and P331S. In some aspects, the first portion and/or the second portion of the proteins of the invention may comprise an immunoglobulin constant region comprising a wild-type human IgG2 constant region or a wild-type human IgG4 constant region. In some aspects, the first portion and/or the second portion of a protein of the invention may comprise an immunoglobulin constant region comprising any one of the amino acid sequences in table 10. The sequence of the Fc region in table 10 begins with the CH1 domain. In some aspects, the first portion and/or the second portion of the protein of the invention may comprise an immunoglobulin constant region comprising the amino acid sequence of the Fc region of human IgG4, human IgG4(S228P), human IgG2, human IgG1, human IgG1-3M, or human IgG 1-4M. For example, the human IgG4(S228P) Fc region comprises the following substitutions compared to a wild-type human IgG4 Fc region: S228P. For example, the human IgG1-3M Fc region comprises the following substitutions compared to the wild-type human IgG1 Fc region: L234A, L235A and G237A, whereas the human IgG1-4M Fc region comprises the following substitutions compared to the wild-type human IgG1 Fc region: L234A, L235A, G237A and P331S. In some aspects, the positions of amino acid residues in the constant regions of immunoglobulin molecules are numbered according to EU nomenclature (Ward et al, 1995 Therap. Immunol.2: 77-94). In some aspects, the immunoglobulin constant region may comprise an RDELT (SEQ ID NO:65) motif or a REEM (SEQ ID NO:66) motif (underlined in Table 10). The REEM (SEQ ID NO:66) allotype is found in a smaller population than the RDELT (SEQ ID NO:65) allotype. In some aspects, the first portion and/or the second portion of the protein of an antibody of the invention may comprise an immunoglobulin constant region comprising any one of SEQ ID NOs 56-62. In some aspects, the first portion and/or the second portion of the protein of the invention may comprise any one of the cloned heavy chain amino acid sequences and light chain amino acid sequences in tables 3-9 and any one of the Fc region amino acid sequences in table 10. In some aspects, the first portion and/or the second portion of a protein of the invention may comprise an immunoglobulin heavy chain constant region comprising any one of the Fc region amino acid sequences in table 10 and an immunoglobulin light chain constant region that is a kappa light chain constant region or a lambda light chain constant region.

In some aspects, the proteins of the invention comprise an IgG1 isotype constant region. The IgG1 isotype constant region efficiently activates all Fc γ R signaling types, driving the effector function of maximal opsonizing effector function.

In some aspects, the immunoglobulin constant region comprises a hinge region or a truncated hinge region. In some embodiments, the hinge region may comprise one, two, three, four, or more amino acid substitutions as compared to the wild-type human hinge region amino acid sequence. In some embodiments, the immunoglobulin constant region does not comprise a hinge region.

In some aspects, the first portion specifically binds to a first molecule expressed in the diseased tissue and the second portion is capable of specifically binding to a second molecule expressed in the diseased tissue, wherein the first molecule expressed in the diseased tissue and the second molecule expressed in the diseased tissue are different molecules. In some embodiments, the first molecule expressed in the diseased tissue and the second molecule expressed in the diseased tissue are expressed by the same cell. In some embodiments, the first molecule expressed in the diseased tissue and the second molecule expressed in the diseased tissue are expressed by different cells. In some embodiments, the first molecule expressed in the diseased tissue, the second molecule expressed in the diseased tissue, or both the first molecule expressed in the diseased tissue and the second molecule expressed in the diseased tissue are expressed on the cell surface. In some embodiments, the first molecule expressed in the diseased tissue and/or the second molecule expressed in the diseased tissue is a soluble molecule.

In some aspects, the first moiety specifically binds human cMET and the second moiety specifically binds human CD 47. In some aspects, the first moiety specifically binds human HER2 and the second moiety specifically binds human CD 47. In some aspects, the first moiety specifically binds human cMET and the second moiety specifically binds human CD 47. In some aspects, the first moiety specifically binds human HER2 and the second moiety specifically binds human CD 3. In some aspects, the first moiety specifically binds human cMET and the second moiety specifically binds human cMET.

In some aspects, a protein of the invention has an immune effector function or two, three, or more immune effector functions. For example, the immune effector function may be antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), or antibody-dependent cellular phagocytosis (ADCP).

In some aspects, the first portion of the protein of the invention prevents or reduces specific binding of the second portion to a molecule expressed in the diseased tissue. In some embodiments, the peptide linker of the protein of the invention is cleaved near or within the diseased tissue. In some cases, the peptide linker is cleaved near or inside the diseased tissue, wherein the first portion dissociates from the second portion near or inside the diseased tissue, and wherein the second portion specifically binds to a molecule expressed in the diseased tissue near or inside the diseased tissue. In some cases, the cleaved peptide linker comprises the binding site or target site of an anti-hinge antibody (e.g., an endogenous anti-hinge antibody of the subject), whereas the uncleaved (e.g., intact) peptide linker does not comprise the binding site or target site of an anti-hinge antibody. Binding of an anti-hinge antibody to a cleaved peptide linker in the presence of an activation protein of the invention can increase ADCC, CDC and/or ADCP (see, e.g., figure 2B).

In some aspects, the proteins of the invention stimulate inflammatory signaling in diseased tissue. Increased inflammatory signaling can increase immune recruitment to diseased tissues. In some cases, the proteins of the invention increase antigen presentation in diseased tissue. In some cases, the proteins of the invention increase tumor associated antigen specific T cell proliferation.

In some aspects, the diseased tissue may be a tumor, necrotic tissue, fibrotic tissue, tissue undergoing a coagulation cascade, or inflamed tissue.

In some aspects, provided herein are proteins comprising a first portion and a second portion, wherein the first portion specifically binds to human cMET, wherein the second portion specifically binds to human CD47, and wherein the proteins comprise a first polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID No. 16 and a second polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID No. 17, and a peptide linker between the first portion and the second portion. The protein comprises only one copy of a first polypeptide chain and only one copy of a second polypeptide chain. The peptide linker of this protein comprises two copies of the LHL sequence (see table 1), each of which is fused by a peptide bond to the first moiety at the n-terminus and to the second moiety at the c-terminus. The protein is called "Fab 2cMetCD 47-LHL-LHL" or "Met 47-LHL-LHL". The amino acid sequences are provided in table 3. The structure of the protein is depicted in fig. 3A. The second part of the protein was linked to KIH IgG1-Fc via a G4S linker (SEQ ID NO:15) and a truncated hinge region.

In some aspects, provided herein are proteins comprising a first portion and a second portion, wherein the first portion specifically binds to human HER2, wherein the second portion specifically binds to human CD3, and wherein the proteins comprise a first polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID No. 26 and a second polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID No. 27, and a peptide linker between the first portion and the second portion. The protein comprises only one copy of a first polypeptide chain and only one copy of a second polypeptide chain. The peptide linker of this protein comprises two copies of the LHL sequence fused by a peptide bond (see table 1). The protein is called "Fab 2Her2CD 3-LHL-LHL" or "Her 23-LHL-LHL". The amino acid sequences are provided in table 4. The structure of the protein is depicted in fig. 3A. The second part of the protein was linked to KIH IgG1-Fc via a G4S linker (SEQ ID NO:15) and a truncated hinge region (3M).

In some aspects, provided herein are proteins comprising a first portion and a second portion, wherein the first portion specifically binds to human HER2, wherein the second portion specifically binds to human CD47, and wherein the proteins comprise a first polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID No. 34 and a second polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID No. 35, and a peptide linker between the first portion and the second portion. The protein comprises two identical copies of a first polypeptide chain and two identical copies of a second polypeptide chain. The peptide linker of this protein comprises two copies of the LHL sequence fused by a peptide bond (see table 1). The protein is called "IgG 2Her2CD 47-LHL-LHL" or "Her 47-LHL-LHL". The amino acid sequences are provided in table 5. The structure of the protein is depicted in fig. 2A. The second part of the protein may be linked to the human IgG1 Fc sequence by a hinge region or a truncated hinge region (see table 10).

In some aspects, provided herein are proteins comprising a first portion and a second portion, wherein the first portion specifically binds to human cMET, wherein the second portion specifically binds to human CD47, and wherein the proteins comprise a first polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO:36 and a second polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO:37, and a peptide linker between the first portion and the second portion. The first polypeptide chain also comprises a human IgG1 amino acid sequence (see table 10). The second polypeptide chain further comprises a human kappa light chain amino acid sequence. The peptide linker of this protein comprises two copies of the LHL sequence fused by a peptide bond (see table 1). The protein is called "Fab 2 CMET/CD47 'single-arm' type" or "Met 47-LHL-LHL". The amino acid sequences are provided in table 6. The structure of the protein is depicted in fig. 3B. The construct was a 'knob and hole structure' one-armed Fab2 construct with the Fab2 on the knob side and the hinge and hole Fc stub on the other side. The construct may contain a non-effector null human IgG1 Fc sequence (see table 10).

In some aspects, provided herein are proteins comprising a first portion and a second portion, and a peptide linker between the first portion and the second portion, wherein the first portion specifically binds human Her2, wherein the second portion specifically binds human CD47, and wherein the protein comprises a first polypeptide chain and a second polypeptide chain, wherein:

(a) said first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:42 and said second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:43 (designated "Her 47-LHLF-LHL"); or

(b) Said first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:44 and said second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:45 (designated "Her 47-LHL-LHLF"); or

(c) Said first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:46 and said second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:47 (designated "Her 47-LHLF-LHLF"); or

(d) Said first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:48 and said second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:49 (referred to as "Her 47-LHLM-LHLM"); or

(f) Said first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:52 and said second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:53 (designated "Her 47-LHLMF-LHLM"); or

(g) Said first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:54 and said second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:55 (designated "Her 47-LHLMF-LHLMF"); or

(h) Said first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:44 and said second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:92 (designated "Her 47 LHL-LHL-EK");

(i) said first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:44 and said second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:93 (designated "Her 47-LHL-LHL-Thr");

(j) said first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:44 and said second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:94 (designated "Her 47-LHL-LHL-tPA");

(k) Said first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:44 and said second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:95 (designated "Her 47-LHL-LHL-GrB");

(l) Said first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:44 and said second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:96 (designated "Her 47-LHL-LHL-uPA"); or

(m) said first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:44 and said second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:97 (referred to as "Her 47-LHL-LHL-A5"). The amino acid sequences are provided in tables 9 and 20. The structure of the protein is depicted in fig. 2A. Peptide linker sequences for LHLF, LHLM and LHLMF linkers are provided in table 1. Peptide linker sequences for EK, Thr, tPA, GrB, uPA, and a5 linkers are provided in table 21.

(a) Said first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:88 and said second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:89 (designated "Her 47 LHLF-LHL IgG1-2 hDAA"); or

(b) Said first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:90 and said second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:91 (designated "Her 47 LHL-LHLF IgG1-2 hDAA"). These proteins are commonly referred to as "IgG 2 'IgG 1-DAA' Her2/CD 47". The amino acid sequences are provided in table 19. These proteins contain stabilizing mutations in the hinge. The structure of the protein is depicted in fig. 2A. Peptide linker sequences for LHLF and LHL linkers are provided in table 1.

In some aspects, provided herein are proteins comprising a first portion and a second portion, wherein the first portion specifically binds to human Her2, wherein the second portion specifically binds to human CD3, and wherein the proteins comprise a first polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID No. 73 and a second polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID No. 74, and a peptide linker between the first portion and the second portion. The first polypeptide chain also comprises a human IgG1-3M Fc amino acid sequence (e.g., containing a 'hole' mutation to enable heterodimerization with the second polypeptide chain), followed by a linker sequence, VH and CH1 domains of the first binding portion, another linker sequence, and then VH and CH1 domains of the second binding portion (see table 13). The second polypeptide chain also comprises a human IgG1-3M Fc amino acid sequence (e.g., containing a 'knob' mutation to enable heterodimerization with the second polypeptide chain), followed by a linker sequence, VL and CL domains of the first binding portion, another linker sequence, and then VL and CL domains of the second binding portion (see table 13). The peptide linker of the protein comprises four sequences fused by peptide bonds (see table 1), which are located between the Fc and the first portion and between the first binding portion and the second binding portion. The protein is referred to as "Fc-Her 2/CD3 (34)" or "Fc-Her 23 (34)". The amino acid sequences are provided in table 13. The structure of the protein is depicted in figure 12. The construct is a "knob and hole" Fc-Fab2 construct with a light chain polypeptide on the knob side or hole side and a heavy chain polypeptide on the other side. This construct was also effector null (IgG 1-3M; see Table 10).

In some aspects, provided herein is a protein comprising a first portion and a second portion, wherein the first portion specifically binds to human cMET, wherein the second portion specifically binds to human cMET, and wherein the protein comprises a first polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO:75 and a second polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO:76, and a peptide linker between the first portion and the second portion. The first polypeptide chain further comprises a human IgG1 or human IgG1-3M amino acid sequence (see table 10). The second polypeptide chain further comprises a human kappa light chain amino acid sequence. The peptide linker of this protein comprises two copies of the LHL sequence fused by a peptide bond (see table 1). This protein is called "Fab 2 CMET/CMET 'one-armed' type" or "MetMetMet-LHL-LHL". The amino acid sequences are provided in table 14. The structure of the protein is depicted in fig. 3B. The construct was a 'knob and hole structure' one-armed Fab2 construct with the Fab2 on the knob side and the hinge and hole Fc stub on the other side. The construct may contain a human IgG1 Fc sequence that may or may not be effector null (see table 10).

In some aspects, provided herein are proteins comprising a first portion and a second portion, and a peptide linker between the first portion and the second portion, wherein the first portion specifically binds human Her2, wherein the second portion specifically binds human CD3, and wherein the protein comprises a first polypeptide chain and a second polypeptide chain, wherein:

(b) Said first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:100 and said second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:101 (designated "Fab 2Her 23 LHL-LHL"); or

(c) Said first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:102 and said second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:103 (designated "Fab 2Her 23 LHLF-LHL-S"); or

(d) The first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:104 and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:105 (designated "Fab 2Her 23 LHL-LHL-S"). This protein is designated "Fab 2Her2/CD 3". The amino acid sequences are provided in table 22. The structure of the protein is depicted in fig. 3A. The protein comprises only one copy of a first polypeptide chain and only one copy of a second polypeptide chain. The peptide linker of the protein comprises two copies of the LHL sequence (see table 1), or one copy of the LHL sequence and one copy of the LHLF sequence, fused by peptide bonds.

In some aspects, provided herein are immunoconjugates comprising a protein of the invention as defined herein linked to an additional therapeutic agent.

Examples of suitable therapeutic agents include cytotoxins, radioisotopes, chemotherapeutic agents, immunomodulators, anti-angiogenic agents, antiproliferative agents, pro-apoptotic agents, and cytostatic and lytic enzymes (e.g., rnases). Additional therapeutic agents include therapeutic nucleic acids, such as genes encoding immunomodulatory agents, anti-angiogenic agents, antiproliferative agents, or pro-apoptotic agents. These drug descriptors are not mutually exclusive and thus one or more of the above terms may be used to describe a therapeutic agent.

Examples of suitable therapeutic agents for immunoconjugates include taxanes, maytansine, CC-1065 and duocarmycin, calicheamicin and other enediynes, and auristatins (auristatins). Other examples include antifolates, vinca alkaloids, and anthracyclines. Phytotoxins, other biologically active proteins, enzymes (i.e., ADEPT), radioisotopes, photosensitizers may also be used in the immunoconjugate. In addition, conjugates can be prepared using secondary carriers as cytotoxic agents (such as liposomes or polymers). Suitable cytotoxins include agents that inhibit or prevent cellular function and/or cause cellular destruction. Representative cytotoxins include antibiotics, tubulin polymerization inhibitors, alkylating agents that bind to and disrupt DNA, and agents that disrupt protein synthesis or the function of essential cellular proteins such as protein kinases, phosphatases, topoisomes, enzymes, and cyclins.

Representative cytotoxins include, but are not limited to, doxorubicin, daunorubicin, idarubicin, aclarubicin, zorubicin, mitoxantrone, epirubicin, carrubicin, noramycin, minoxidil, pirarubicin, valrubicin, cytarabine, gemcitabine, trifluridine, ancitabine, enocitabine, azacitidine, doxifluridine, pentostatin, bromoglycoside, capecitabine, cladribine, decitabine, floxuridine, fludarabine, glutelin, puromycin, flusudine, fludarabine, puromycin, tegafur, thiazolecarboxamide nucleoside (tiazofurahn), methacycline (phahamycin), cisplatin, carboplatin, cyclophilin, dacarbazine, vinblastine, vincristine, bleomycin, prednimorazine, meprobamate, meprobrazine, methotrexate, epirubicin, methotrexate, doxorabicistributyline, doxorabicistrine, meclizine, etc, Etoposide, paclitaxel analogs, platins such as cisplatin and carboplatin, mitomycin, thiotepa, taxanes, vincristine, daunorubicin, epirubicin, actinomycin, ampramycin (authramycin), azaserine, bleomycin, tamoxifen, idarubicin, dolastatin/auristatin, hemiasterlin (hemiasterlin), estomycin, and maytansinol (maytansinoid).

Suitable immunomodulators include anti-hormonal agents that block the action of hormones on tumors, and immunosuppressive agents that inhibit cytokine production, down-regulate autoantigen expression, or mask MHC antigens.

Also provided are nucleic acid molecules encoding a protein or a portion of a protein of the invention as defined herein. Also provided herein are nucleic acid molecules encoding a first polypeptide chain, a second polypeptide chain, or both a first polypeptide chain and a second polypeptide chain of a protein of the invention comprising a plurality of non-identical polypeptide chains. In some aspects, a nucleic acid molecule as defined herein may be isolated.

Also provided are vectors comprising the nucleic acid molecules of the invention as defined herein. The vector may be an expression vector.

Also provided are host cells comprising a nucleic acid molecule or vector of the invention as defined herein. The host cell may be a recombinant host cell.

In a further aspect, there is provided a method of producing a protein of the invention, the method comprising culturing a host cell of the invention under conditions which result in the expression and/or production of the protein, and isolating the protein from the host cell or culture.

In some aspects, provided herein are pharmaceutical compositions comprising a protein of the invention as defined herein, or a nucleic acid molecule of the invention as defined herein, or a vector of the invention as defined herein.

Also provided is a method for enhancing an immune response in a subject, the method comprising administering to the subject an effective amount of a protein of the invention as defined herein, or an immunoconjugate of the invention as defined herein, or a nucleic acid molecule of the invention as defined herein, or a vector of the invention as defined herein, or a pharmaceutical composition of the invention as defined herein.

In a further aspect, there is provided a method for treating or preventing cancer, or ameliorating the symptoms of cancer, in a subject, the method comprising administering an effective amount of a protein of the invention as defined herein, or an immunoconjugate of the invention as defined herein, or a nucleic acid molecule of the invention as defined herein, or a vector of the invention as defined herein, or a pharmaceutical composition of the invention as defined herein.

In some aspects, the cancer is a solid tumor. In some cases, the cancer is a hematologic malignancy. For example, the cancer can be gastrointestinal stromal cancer (GIST), pancreatic cancer, skin cancer (e.g., melanoma), breast cancer, lung cancer, bronchial cancer, colorectal cancer, prostate cancer, gastric cancer, ovarian cancer, bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, cancer of the oral cavity or pharynx, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small intestine or appendix cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma, or cancer of blood tissue. In some cases, the cancer of the blood tissue is lymphoma.

In some aspects, provided herein is a protein of the invention as defined herein, or an immunoconjugate of the invention as defined herein, or a nucleic acid molecule of the invention as defined herein, or a vector of the invention as defined herein, or a pharmaceutical composition of the invention as defined herein, for use in treating cancer or for ameliorating a symptom of cancer.

In some aspects, provided herein is a protein, or an immunoconjugate, or a nucleic acid molecule, or a vector, for use or method of treatment of the invention as defined herein, for separate, sequential or simultaneous use in combination with a second therapeutic agent (e.g., an anti-cancer agent).

The invention also provides a method for the treatment or prevention of an autoimmune or inflammatory disease in a subject, the method comprising administering to the subject an effective amount of a protein as defined herein, or an immunoconjugate as defined herein, or a nucleic acid molecule as defined herein, or a vector as defined herein, or a pharmaceutical composition as defined herein.

For example, the autoimmune or inflammatory disease may be arthritis, asthma, multiple sclerosis, psoriasis, crohn's disease, inflammatory bowel disease, lupus, graves ' disease, and hashimoto's thyroiditis or ankylosing spondylitis.

The invention also provides a method for the treatment or prevention of a cardiovascular disease or a fibrotic disease in a subject, the method comprising administering to the subject an effective amount of a protein as defined herein, or an immunoconjugate as defined herein, or a nucleic acid molecule as defined herein, or a vector as defined herein, or a pharmaceutical composition as defined herein.

The cardiovascular disease in any aspect of the invention may be, for example, coronary heart disease, atherosclerosis, or stroke.

For example, the fibrotic disease in any aspect of the invention may be myocardial infarction, angina, osteoarthritis, pulmonary fibrosis, asthma, cystic fibrosis or bronchitis.

In some aspects, provided herein are proteins comprising and in the form disclosed herein for use in therapy.

In some aspects, the pharmaceutical composition may comprise a pharmaceutically acceptable excipient, carrier or diluent. A pharmaceutically acceptable excipient may be a compound or combination of compounds that does not elicit a secondary response and allows, for example, to facilitate administration of the protein as defined herein, an increase in its lifespan and/or its efficacy in the body, or an increase in its solubility in solution into a pharmaceutical composition. These pharmaceutically acceptable vehicles are well known and will be adjusted by the skilled person according to the mode of administration of the protein as defined herein.

In some aspects, a protein as defined herein may be provided in lyophilized form for reconstitution prior to administration. For example, lyophilized protein molecules can be reconstituted in sterile water and mixed with saline prior to administration to an individual.

A protein as defined herein will typically be administered in the form of a pharmaceutical composition which may comprise at least one component in addition to the protein molecule. Thus, in addition to a protein as defined herein, a pharmaceutical composition may comprise a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other material well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the protein. The precise nature of the carrier or other material will depend on the route of administration, which may be bolus injection, infusion, injection or any other suitable route, as described below.

For parenteral administration (e.g., subcutaneous or intravenous administration, e.g., by injection), a pharmaceutical composition comprising a protein as defined herein may be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those skilled in the art will be able to prepare suitable solutions using, for example, isotonic vehicles such as sodium chloride injection, ringer's injection, lactated ringer's injection. Preservatives, stabilizers, buffers, antioxidants and/or other additives may be used as desired, including buffers such as phosphates, citrates and other organic acids; antioxidants (such as ascorbic acid and methionine); preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens (such as methyl or propyl paraben), catechol; resorcinol; cyclohexanol; 3' -pentanol; and m-cresol); a low molecular weight polypeptide; proteins (such as serum albumin, gelatin, or immunoglobulins); hydrophilic polymers such as polyvinylpyrrolidone; amino acids (such as glycine, glutamine, and, Asparagine, histidine, arginine or lysine); monosaccharides, disaccharides, and other carbohydrates (including glucose, mannose, or dextrins); chelating agents (such as EDTA); sugars (such as sucrose, mannitol, trehalose, or sorbitol); salt-forming counterions (such as sodium ions); metal complexes (e.g., Zn-protein complexes); and/or nonionic surfactants (such as TWEEN)^TM,PLURONICS^TMOr polyethylene glycol (PEG)).

The pharmaceutical composition comprising a protein as defined herein may be administered alone or in combination (simultaneously or sequentially) with other therapies depending on the condition to be treated.

The proteins as defined herein may be used in a method of treatment of the human or animal body, including prophylactic or preventative treatment (e.g. treatment to reduce the risk of developing a disorder in an individual prior to onset of the disorder in the individual; delay onset of the disorder; or to reduce the severity of the disorder after onset). A method of treatment may comprise administering a protein as defined herein to an individual in need thereof.

Administration is typically in a "therapeutically effective amount" sufficient to show benefit to the patient. The benefit may be at least an improvement in at least one symptom. The actual amount administered, as well as the rate and time course of administration, will depend on the nature and severity of the disease being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the composition, the method of administration, the timing of administration, and other factors known to the practitioner. Prescription of treatment (e.g., decisions on dosages, etc.) is under the responsibility of general practitioners and other physicians, and may depend on the severity and/or progression of the symptoms of the disease to be treated. Suitable doses of Antibody molecules are well known in the art (Ledermann J.A. et al, 1991, int.J.cancer 47: 659-. Specific dosages may be indicated herein or in the physicians' Desk Reference (2003), as dosages appropriate to the type of drug being administered may be employed. A therapeutically effective amount or suitable dose of a protein as defined herein can be determined by comparing its in vitro and in vivo activity in an animal model. Methods for extrapolating effective doses in mice and other experimental animals to humans are known. The precise dosage will depend on a number of factors, including whether the protein is for prophylaxis or treatment, the size and location of the area to be treated, the precise nature of the protein (e.g., Fab2, IgG) and the nature of any detectable label or other molecule attached to the protein.

Typical protein doses are in the range of 100 μ g to 1g for systemic administration, and 1 μ g to 1mg for topical administration. An initial higher loading dose may be administered followed by one or more lower doses. In some aspects, the protein will comprise an intact antibody, such as an IgG1 or IgG4 isotype. This is the dose for a single treatment of an adult patient, which can be scaled for children and infants, and also scaled to molecular weight for other protein construct forms. Treatment may be repeated at daily, twice weekly, weekly or monthly intervals, at the discretion of the physician. The treatment regimen for an individual may depend on the pharmacokinetic and pharmacodynamic properties of the protein composition, the route of administration, and the nature of the condition being treated.

The treatment may be periodic, and the period between administrations may be about two weeks or more, for example about three weeks or more, about four weeks or more, about once a month or more, about five weeks or more, or about six weeks or more. For example, treatment may be every 2-4 weeks or every 4-8 weeks. The treatment may be administered before and/or after surgery, and/or may be administered or applied directly at the anatomical site of the surgical treatment or invasive surgery. Suitable formulations and routes of administration are as described above.

In some aspects, a protein as defined herein may be administered as a subcutaneous injection. The auto-injector may be used for subcutaneous injection, e.g. for long-term or short-term prophylaxis/treatment.

In some aspects, the therapeutic effect of a protein as defined herein may last several times the half-life of the protein in serum, depending on the dose. For example, a therapeutic effect of a single dose of protein as defined herein may last for 1 month or more, 2 months or more, 3 months or more, 4 months or more, 5 months or more, or 6 months or more in an individual.

As used herein, the term "CD 47" refers to IAPs (integrin-associated proteins) and variants thereof that retain at least a portion of the biological activity of CD 47. As used herein, CD47 includes the native sequence CD47 of all mammalian species (including human, rat, mouse, and chicken). In some embodiments, the term "CD 47" is used to include variants, isoforms, and species homologs of human CD 47. In some cases, as used herein, CD47 includes the native sequence CD47 of all mammalian and non-mammalian species (including human, monkey, rat, mouse, and chicken). In some embodiments, the term "CD 47" refers only to wild-type CD 47. The protein of the present invention can cross-react with CD47 from a species other than human, particularly CD47 from cynomolgus monkeys (Macaca fascicularis). Examples of human and cynomolgus monkey CD47 amino acid sequences are provided in table 11. In certain embodiments, the proteins of the invention may be completely specific for human CD47 and may not exhibit non-human cross-reactivity.

As used herein, the term "cMET" refers to MET proteins and variants thereof that retain at least a portion of the biological activity of cMET. In some cases, as used herein, cMET includes the native sequence cMET of all mammalian species (including human, rat, mouse, and chicken). In some embodiments, the term "cMET" may be used to include variants, isoforms, and species homologs of human cMET. In some cases, as used herein, cMET includes the native sequence cMET of all mammalian and non-mammalian species (including human, monkey, rat, mouse, and chicken). In some embodiments, the term "cMET" refers only to wild-type cMET. The antibodies of the invention can cross-react with cMET from species other than human, in particular cMET from cynomolgus monkeys (bundled monkeys). Examples of human and cynomolgus monkey cMET amino acid sequences are provided in table 12. In certain embodiments, the antibody may be completely specific for human cMET, and may not exhibit non-human cross-reactivity.

As used herein, the term "Her 2" refers to human epidermal growth factor receptor 2 protein and variants thereof that retain at least a portion of the biological activity of Her 2. Her2 is also known as HER2/neu, ErbB2, c-erbB-2 and human EGF receptor 2. In some embodiments, the term "Her 2" may be used to include variants, isoforms, and species homologs of human Her 2. In some cases, Her2, as used herein, includes the native sequence Her2 (also known as ErbB2) of all mammalian and non-mammalian species, including human, monkey, rat, mouse, and chicken. In some embodiments, the term "Her 2" refers only to wild-type Her 2. The antibodies of the invention can cross-react with Her2 from species other than human, in particular Her2 from cynomolgus monkeys (cynomolgus monkeys). Examples of human and cynomolgus monkey Her2/ErbB2 amino acid sequences are provided in table 15. In certain embodiments, the antibody may be completely specific for human Her2, and may not exhibit non-human cross-reactivity.

As used herein, the term "CD 3" refers to "cluster of differentiation 3" multimeric protein complexes and variants thereof that retain at least a portion of the biological activity of CD 3. The CD3 complex comprises four distinct polypeptide chains; eppirone (epsilon), gamma (gamma), delta (delta), and zeta. These polypeptide chains assemble and function as three pairs of dimers (. epsilon.gamma.,. epsilon.delta., and. zeta.). In some embodiments, the term "CD 3" may be used to include variants, isoforms, and species homologs of human CD 3. In some cases, as used herein, CD3 includes the native sequence CD3 of all mammalian and non-mammalian species (including human, monkey, rat, mouse, and chicken). In some embodiments, the term "CD 3" refers only to wild-type CD 3. The antibodies of the invention can cross-react with CD3 from species other than human, in particular CD3 from cynomolgus monkeys (cynomolgus monkeys). Examples of human and cynomolgus monkey CD3 epsilon amino acid sequences are provided in table 16. In certain embodiments, the antibody may be completely specific for human CD3, and may not exhibit non-human cross-reactivity.

As used herein, an "antagonist" as used in the context of a protein of the invention refers to a protein that is capable of binding to a molecule expressed in a diseased tissue and inhibiting the biological activity of the molecule and/or downstream pathways mediated by the molecule. For example, an "anti-CD 47 antagonist protein" (interchangeably referred to as "anti-CD 47 protein") refers to a protein that is capable of binding to CD47 and inhibiting CD47 biological activity and/or downstream pathways mediated by CD47 signaling. anti-CD 47 antagonist proteins encompass proteins that can block, antagonize, suppress or reduce (including significantly reduce) CD47 biological activity, including downstream pathways mediated by CD47 signaling, such as receptor binding and/or eliciting a cellular response to CD 47. For the purposes of the present invention, it is to be expressly understood that the term "anti-CD 47 antagonist protein" encompasses all terms, names and functional states and characteristics in which CD47 itself and CD47 bioactivity (including but not limited to its ability to enhance activation by myeloid lineage cells) or the result of the activity or bioactivity is substantially ineffective, reduced or neutralized to any meaningful degree.

A protein of the invention "specifically binds", "specifically interacts", "preferentially binds", "binds" or "interacts" with a molecule (e.g., human CD47, human Her2, human CD3, human cMET, or human PD-L1) if the protein binds with greater affinity, avidity, more readily, and/or for a longer duration than it binds with other molecules.

An "antibody molecule" is an immunoglobulin molecule capable of specifically binding a target (such as a carbohydrate, polynucleotide, lipid, polypeptide, etc.) through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, the term "antibody molecule" encompasses not only intact polyclonal or monoclonal antibodies, but also any antigen-binding fragment (e.g., "antigen-binding portion") or single chain thereof, fusion proteins comprising an antibody, and any other modified configuration of an immunoglobulin molecule comprising an antigen recognition site, including, for example, but not limited to, scFv, single domain antibodies (e.g., shark and camelid antibodies), macroantibodies (maxibody), miniantibodies (minibody), intrabodies (intrabody), diabodies (diabodies), triabodies (triabodies), tetrabodies (tetrabodies), v-NAR, and bis-scFv.

An "antibody molecule" encompasses any class of antibody, such as IgG, IgA, or IgM (or subclasses thereof), and an antibody need not be of any particular class. Depending on the amino acid sequence of the constant region of the heavy chain of an antibody, immunoglobulins can be assigned to different classes. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), e.g., lgG1, lgG2, lgG3, lgG4, lgA1, and lgA 2. The heavy chain constant regions corresponding to different classes of immunoglobulins are referred to as α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

As used herein, the term "antigen-binding portion" of an antibody molecule refers to one or more fragments of an intact antibody that retain the ability to specifically bind an antigen. The antigen binding function of an antibody molecule can be performed by fragments of an intact antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody molecule include Fab; fab'; f (ab') 2; an Fd fragment consisting of the VH and CH1 domains; (ii) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; a single domain antibody (dAb) fragment; and an isolated Complementarity Determining Region (CDR).

The term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain. The "Fc region" can be a native sequence Fc region or a variant Fc region. Although the boundaries of the Fc region of immunoglobulin heavy chains may differ, the human IgG heavy chain Fc region is generally defined as an extension from position Cys226 or from the amino acid residue of Pro230 to its carboxy terminus. The numbering of residues in the Fc region is that of the EU index as described in Kabat. The Fc region of an immunoglobulin typically comprises two constant domains, CH2 and CH 3. The Fc region may exist in dimeric or monomeric form, as is known in the art.

The "variable region" of an antibody refers to the variable region of the antibody light chain or the variable region of the antibody heavy chain, alone or in combination. As is known in the art, the variable regions of the heavy and light chains each consist of four Framework Regions (FRs) connected by three Complementarity Determining Regions (CDRs), also known as hypervariable regions, and contribute to the formation of the antigen-binding site of the antibody. When selecting FR-flanked CDRs, e.g., when humanizing or optimizing an antibody, it is preferred that the FRs be from an antibody that contains CDR sequences of the same canonical class.

As used herein, the term "conservative substitution" refers to the replacement of one amino acid by another amino acid that does not significantly detrimentally alter functional activity. A preferred example of a "conservative substitution" is the substitution of one amino acid by another amino acid having a value ≧ 0 in the BLOSUM 62 substitution matrix below (see Henikoff and Henikoff,1992, PNAS 89: 10915-:

The term "monoclonal antibody" (Mab) refers to an antibody or antigen-binding portion thereof that is derived from a single copy or clone, including, for example, any eukaryotic, prokaryotic, or phage clone, regardless of the method by which it is produced. Preferably, the monoclonal antibodies of the invention are present in a homogeneous or substantially homogeneous population.

A "humanized" antibody molecule refers to a form of a non-human (e.g., murine) antibody molecule or antigen-binding portion thereof that contains minimal sequence derived from a non-human immunoglobulin, is a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof, such as the Fv, Fab ', F (ab')2, or other antigen-binding subsequence of an antibody. A humanized antibody may be a human immunoglobulin (recipient antibody) in which residues from the recipient CDR are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity.

"human antibody" or "fully human antibody" refers to an antibody molecule or antigen-binding portion thereof derived from a transgenic mouse carrying human antibody genes or from human cells.

The term "chimeric antibody" is intended to refer to an antibody molecule, or antigen-binding portion thereof, in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as an antibody molecule in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody.

The term "immunoconjugate" refers to a protein of the invention conjugated, fused, or linked to at least one cytotoxic, cytostatic, or therapeutic agent.

The proteins of the invention may be produced using techniques well known in the art, such as recombinant techniques, phage display techniques, synthetic techniques, or a combination of such techniques or other techniques readily known in the art.

The term "isolated molecule" (where the molecule is, for example, a polypeptide, polynucleotide, or antibody) is a molecule that, due to its source or derivative source, (1) is not associated with a naturally-associated component with which it naturally accompanies in its natural state, (2) is substantially free of other molecules from the same species, (3) is expressed by cells from a different species, or (4) does not occur in nature. Thus, a molecule that is chemically synthesized or expressed in a cellular system different from the cell from which it is naturally derived will be "isolated" from its naturally associated components. The molecule may also be rendered substantially free of naturally associated components by isolation using purification techniques well known in the art. Molecular purity or homogeneity can be determined by a variety of means well known in the art. For example, the purity of a polypeptide sample can be determined using polyacrylamide gel electrophoresis, and the gel stained using techniques well known in the art to visualize the polypeptide. For some purposes, higher resolution may be provided by using HPLC or other means for purification well known in the art.

The term "epitope" refers to a portion of a molecule that is capable of being recognized and bound by a protein, antibody molecule, or antigen-binding portion thereof of the invention at one or more antigen-binding regions of the protein or antibody molecule. An epitope may consist of a defined region of the primary, secondary or tertiary protein structure and includes a combination of secondary structural units or structural domains of the target that are recognized by the antigen binding region of a protein, antibody, or antigen binding portion thereof. Epitopes can likewise consist of defined chemically active surface components of molecules such as amino acids or sugar side chains and have specific three-dimensional structural characteristics as well as specific charge characteristics. As used herein, the term "epitope" is defined as the portion of a polypeptide to which a protein or antibody molecule of the invention can specifically bind, as determined by any method well known in the art, e.g., by conventional immunoassay, antibody competitive binding assay, or by x-ray crystallography or related structure determination methods (e.g., NMR).

The term "binding affinity" or "KD" refers to the off-rate of a particular antigen-binding protein interaction or antigen-antibody interaction. KD is the off-rate (also known as the "off-rate (k)) _off) ") and association rate or" association rate (k)_on) "is used in the following description. Thus, K_DIs equal to k_off/k_onAnd is expressed as molar concentration (M). Thus K_DThe smaller the binding affinity, the stronger. Thus, K of 1 μm_DIndicating a K of 1nM_DCompared to weak binding affinity. The KD value of a binding protein or antibody can be determined using methods established in the art. One method of determining the KD of a binding protein or antibody is through the use of Surface Plasmon Resonance (SPR), typically using biosensor systems such asAnd (4) preparing the system.

The term "potency" is a measure of biological activity and can be designated as IC₅₀Or the protein of the immunoconjugate of the invention inhibits its binding partner (e.g., a molecule expressed in diseased tissue) or antigen by an effective concentration of 50% of the activity of the binding partner or antigen measured in an activity assay as described herein.

As used herein, the phrase "effective amount" or "therapeutically effective amount" refers to the amount (in terms of dosage and for a period of time and with respect to the means of administration) necessary to achieve the desired therapeutic result. An effective amount is at least the minimum amount of active agent necessary to administer a therapeutic benefit to the subject, but less than a toxic amount.

The term "inhibit" or "neutralize" with respect to a biological activity of a protein of the invention as used herein means the ability of a protein to substantially antagonize, arrest, prevent, inhibit, slow, destroy, eliminate, stop, reduce, or reverse the progression or severity of, for example, an inhibited biological activity (including but not limited to, the biological activity or binding interaction of molecules expressed in diseased tissue).

"host cell" includes a single cell or cell culture that may be or has been the recipient of a vector for incorporating a polynucleotide insert. Host cells include progeny of a single host cell, and the progeny may not necessarily be identical (morphologically or in genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. Host cells include cells transfected in vivo with a polynucleotide of the invention.

As used herein, "vector" means a construct capable of delivering and preferably expressing one or more genes or sequences of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors; naked DNA or RNA expression vectors; a plasmid, cosmid, or phage vector; a DNA or RNA expression vector associated with a cationic condensing agent; a DNA or RNA expression vector encapsulated in liposomes; and certain eukaryotic cells, such as producer cells.

The term "treating" as used herein, unless otherwise indicated, means reversing, alleviating, inhibiting the progression of, delaying the onset of, or preventing the disorder or condition or one or more symptoms of the disorder or condition to which the term applies. The term "treatment", as used herein, unless otherwise indicated, refers to the act of treating as defined above. The term "treatment" also includes adjuvant and neoadjuvant treatment of a subject. For the avoidance of doubt, reference herein to "treatment" includes reference to curative, palliative and prophylactic treatment. For the avoidance of doubt, reference herein to "treatment" also includes reference to curative, palliative and prophylactic treatment.

It should be understood that whenever an embodiment is described herein by the word "comprising," other similar embodiments are also provided which are described by "consisting of … …" and/or "consisting essentially of … ….

Where aspects or embodiments of the invention are described in terms of Markush (Markush) groups or other alternative groupings, the invention encompasses not only the entire group listed as a whole, but also each member of an individual group and all possible subgroups of the major group, and also encompasses major groups in which one or more group members are not present. The present invention also contemplates one or more of any group members explicitly excluded in the claimed invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control. Throughout this specification and the claims, the word "comprise" or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. Furthermore, unless the context requires otherwise, singular terms shall include the plural and plural terms shall include the singular. Any examples following the terms "such as (e.g.)" or "such as (for example)" are not meant to be exhaustive or limiting.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art.

Specific non-limiting embodiments of the invention will now be described with reference to the accompanying drawings.

Examples

Example 1 Generation of optimized conditionally active therapeutic antibodies

Introduction to the design reside in

In this example, we successfully generated an optimized set of conditionally active antibodies. These conditionally active antibodies are well expressed, are biophysically stable, highly soluble, and have maximum amino acid sequence identity to the preferred human strain.

Materials and methods

Cloning, transient expression, purification and characterization of proteins

Cloning of DNA sequences encoding antibodies into separate plasmids by restriction ligation cloningSeparate human IgG1 heavy and light chain encoding expression cassettes in vectors to produce IgG or IgG for expression²The construct is in a form. Similar cassettes were also cloned into a "knob and hole" human IgG1 heterodimerization Fc vector to generate Fab and Fab²Constructs. Construction of Fab using a knob and hole (KIH) heavy chain expression vector (CH3 domains T366W and T366S/L368A/Y407V mutations) ²cMETCD47 and Her2CD3 protein constructs. Fab²The Her2-CD3 construct also included effector function ablation mutations L234A/L235A/G237A. Construction of IgG using "wild-type" IgG1 heavy chain and kappa light chain expression vectors²Her2CD47 protein construct. Following transient transfection with endotoxin-free IgG expression plasmid preparations, IgG was expressed in CHO cells according to the manufacturer's protocol.

The HiTrap MabSelect Sure protein A5 mL column was usedGenerated antibodies were captured from the clarified supernatant on Pure 150L FPLC system. The eluted protein peak buffer was immediately exchanged into 1x PBS pH 7.4 by loading the eluted protein a peak fraction directly onto HiPrep 26/10 desalting column. The protein concentration was determined by measuring the absorbance at 280nm and 1. mu.g of each purified protein was analyzed by SDS-PAGE under reducing and/or non-reducing conditions using 4-20% TGX polyacrylamide gradient gel (BioRad, cat. No. 456-1093) with 1 × Tris/glycine/SDS buffer, separated by a 120V field for 1 hour. To test for the presence of non-covalently bound aggregates and supplement SDS PAGE analysis, analytical size exclusion chromatography was performed. Aliquots of selected clones were analyzed by analytical Size Exclusion Chromatography (SEC) in isocratic mode (isocratic mode) using a Superdex 200 Increase 10/300 SEC column and 1x PBS pH 7.4 as running buffer.

The selected protein was further purified using preparative SEC. Up to 1ml of antibody sample was loaded onto either a Superdex 200 Increate 10/300 SEC column or a HiLoad 26/600 Superdex 200pg column equilibrated in 1 XPBS pH 7.4. Fractions of 1ml of the peak of interest were collected and the major peak fractions were pooled. After size exclusion chromatography, the samples were again analyzed by SDS-PAGE as described above.

Hemagglutination

Red Blood Cells (RBCs) were isolated from fresh, unagglutinated human blood (of a minimum of 3 different donors), diluted to 2% in PBS, and incubated with titrations of IgG or protein constructs in U-bottom 96-well plates for 60-90 minutes. Without hemagglutination, the cells settled to the bottom of the well, forming a red precipitate. Hemagglutination was observed as a non-sedimenting RBC solution. Images of each plate were recorded and data for each sample was expressed as the titer of the last well where hemagglutination was observed.

Metalloproteinase digestion

The protein construct is combined with human Matrix Metalloproteinase (MMP) enzyme alone or a mixture of active MMP3, MMP7, and MMP12 (equal parts of each component) at a ratio of 1% total MMP to protein construct (wt/wt) at 37 ℃ in the presence of 5mM CaCl ₂Is incubated in Tris-buffered saline (pH7.4) for 16 hours. The reaction was stopped by the addition of 20mM EDTA and the samples were then tested for binding or functional activity as described.

IgG titration in combination with ELISA

To coat the ELISA plates, the target protein was diluted to 1. mu.g/ml in PBS pH7.4 and added at 100. mu.l/well overnight at 4 ℃. The coated plate was washed 3 times with PBS pH7.4, blocked with 4% skim milk protein in PBS (380. mu.l/well) at room temperature for 1 hour, and then washed 5 times with PBS-Tween 20 (PBST). Antibodies (100. mu.l/well; diluted in PBST) were then added and incubated for 1 hour at room temperature. The plate was then washed 3 times with PBS and goat anti-human IgG-HRP (100. mu.l/well) was added at room temperature for 1 hour. The plates were then washed 3 times with PBST and twice with PBS, and then 100. mu.l of TMB was added per well. By adding 100. mu.l of 2M H₂SO₄The reaction was stopped/well and the OD read at 450nm on a plate reader.

Flow cytometry integration

The binding of the protein constructs (+/-predigestion by MMP 3/7/12) and control IgG to Jurkat cells and BT-474 cells was assessed by flow cytometry. Using Zombie UV^TM Fixable ViaViability dye (Biolegend) identifies viable cells. Binding of human IgG to the protein construct was detected with a FITC-conjugated goat anti-human (H + L) secondary antibody. Binding of the mouse monoclonal anti-CD 3 control antibody was detected using Alexa-Fluor-488 goat anti-mouse IgG. The results were analyzed by measuring the Mean Fluorescence Intensity (MFI) of live cells in the FITC channel detector of a BD Fortessa flow cytometer.

T cell activation bioassay

The functional activity of the Her2CD3 protein construct was assessed in a co-culture assay using BT474 cells and NFAT-RE-luciferase Jurkat reporter cell line (Promega-TCR/CD 3 effector cells NFAT). BT-474 cells (40000 cells/well) were seeded into hybrid-Care medium (ATCC) supplemented with 10% FBS in 96-well white clear-bottomed tissue culture treated plates and in CO₂Incubate overnight in an incubator at 37 ℃. The medium was removed and a control antibody or protein construct prepared in assay medium (RPMI supplemented with 10% FBS (+/-predigestion by MMP 3/7/12) was added to the cells. TCR/CD3 effector cells (NFAT) were thawed and diluted according to the manufacturer's protocol before being added to assay wells. In CO₂After incubation in the incubator at 37 ℃ for 6 hours, the plates were re-equilibrated to room temperature and luciferase activity was determined by adding the Bio-Glo reagent for 5-10 minutes and measuring the luminescence signal (RLU). Fold induction was determined by calculating the sample RLU/RLU ratio in the absence of antibody after subtracting the background luminescence signal.

Molecular dynamics simulation

Eight systems (equivalent sequences of linker sequences X1, X2, X3 and X4 and a covalent bond with a break in one of the linkers at GS sequence positions XC1, XC2, XC3 and XC 4) were modeled and optimized using the AMBER10: EHT force field in moe (chemical Computing Group inc). The histidine charge was assigned using the protonate3D tool in MOE. Three consecutive rounds of minimization were performed with a final gradient of 0.001, allowing 10,000 steps without limitation. The single system was once again constrained with CHARMM27 force field using NAMD 2.13 and gradually minimized to reduce the total potential energy in a series of three energy minimization steps. The first step keeps all heavy atoms constrained and allows only hydrogen atoms to migrate. The second step removes the constraints on the side chains, while the third step releases all atoms, allowing them to move without restriction. Minimization in CHARMM27 is easy because it is the force field that works within the NAMD molecular dynamics simulation.

For kinetic runs, the solvent effect was described using generalized born (gb) solvation. MD simulations were established and then completed using NAMD 2.13. Four balancing steps were performed to gradually relax the harmonic constraints in 125ps increments for a total balancing time of 500 ps. The first step heats the system to 310K while applying a force of 4kcal/mol to hold the scaffold in place. The remaining three steps gradually increased the skeletal restraint from 4kcal/mol to 1kcal/mol with NVT ensemble (constant (N), volume (V) and temperature (T)). In thatA transfer function is applied to shorten the van der waals potential function. According to NAMD recommendations, the electrostatic and vdW cutoff values are setPeriodic boundary conditions are not used because they are incompatible with the implicit solvent method (implict solvent approach). The constraints imposed during equilibration were removed to freely mimic the antibody complex. Production runs were run at 310K for 6, 15, 20 or 100 ns. The cleaved linker protein XC1-4 was applied a 100ns run in order to fully explore the range of motion, while shorter runs (up to 20ns) were found to be sufficient to locate the range of motion of the uncleaved antibody construct X1-4.

In vivo analysis of PK and tolerability

Tolerability study-eighteen (28) 6-8 week old males B6.Cg-Fcgrt ^tm1DcrTg (fcgrt)32DcrJ ('Tg 32' homozygous human FcRn transgene, JAX stock #014565) mice were divided into 7 groups of 4 mice each. Body weight was measured on the day of antibody administration. The test article was administered by Intravenous (IV) injection at 0 hours in dose volumes of 2mg/kg or 10mg/kg and 10 ml/kg. 200 μ L of whole blood samples were collected into EDTA on days 5, 29 and 60 (terminal bleeding). Blood for CBC/Dif/Retic analysis, including leukocyte depletion, and methods of useNeutrophils, eosinophils, basophils, lymphocytes, monocytes, hemoglobin, erythrocytes, reticulocytes, MCHC, and MCV. Body weight was then monitored weekly for the first month and then monthly until the end of the experiment.

Pharmacokinetic study-thirty-two 6-8 week old males B6.Cg-Fcgrt^tm1DcrTg (fcgrt)32DcrJ ('Tg 32' homozygous human FcRn transgene, JAX stock #014565) mice were divided into 8 groups of 4 mice each. One day prior to administration of the test article, 35 μ L blood samples from three (3) Tg32 mice were collected in EDTA to test the test article for binding to mouse red blood cells using flow cytometry. Body weight was measured on the day of antibody administration and weekly until the end of the experiment. The test article was administered IV injection at a dose volume of 2mg/kg or 10mg/kg at 0 hours, at 10 ml/kg. Blood samples were collected from each mouse according to the bleeding schedule: 30 minutes, 4 hours, 1 day, 3 days, 5 days, 7 days, 10 days, 14 days, 21 days, 28 days, and 42 days. A 25 μ L blood sample was collected from each mouse according to the bleeding schedule. Collecting blood sample to K ₃In EDTA, processed into plasma and stored at-20 ℃. Plasma samples were then evaluated in triplicate by ELISA to estimate human IgG concentrations.

Tolerance studies in NOD-SCID mice — NOD-SCID mice were divided into 4 groups of 3 mice each. Body weight was measured daily. On day 0, the test article is administered as an IV injection of 8mg/kg or 14mg/kg, with 3 subsequent doses administered at 4mg/kg or 7mg/kg 5 days apart.

Flow cytometry analysis of binding to monkey and human erythrocytes

Erythrocytes were isolated from 3 Cyno (cynomolgus monkey) and 3 human donors. For each sample, 5 × 10⁵Individual cells (diluted in DMEM + 5% FBS) were stained for one hour with: A-D5 IgG 1: 0.0032, 0.016, 0.03 and 50 mu g/mL; trastuzumab: 0.0032, 0.016, 0.03 and 50 mu g/mL; IgG²Her47 LHL-LHLF 0 hours: 0.016, 0.08, 0.4, 2, 10, 50 μ g/mL; IgG²Her47 LHL-LHL 0 hours: 0.016, 0.08, 0.4, 2, 10 and 50 mu g/mL. Binding of test samples to erythrocytes was measured using FITC AffiniPure goat anti-human IgG (1:200 dilution; 1 hour incubation time),FITC fluorescence intensity was then measured by flow cytometry (BD LSR FortessaX-20 cell analyzer).

Metalloprotease and cathepsin digestions at pH6.0 and 7.4

The protein construct was placed in TBS (containing 5mM CaCl)₂Ph6.0 or 7.4) and then incubated with human Matrix Metalloproteinase (MMP) or cathepsin alone at a ratio of 1% total enzyme to protein construct (wt/wt) at 37 ℃ for 0, 2, 4, 8 and 24 hours. The reaction was stopped by the addition of 20mM EDTA and then the samples were frozen before testing for binding or functional activity as described.

In vitro protein stability assay

Forced oxidation-for forced oxidation analysis, the test article in PBS was treated with 0.5% H2O2 for 2 hours at room temperature, then stored at-80 ℃, and then subjected to SEC and RP analysis (whole antibody and subunit, tryptic peptide) on a Dionex Ultimate 3000RS HPLC system (ThermoFisher Scientific, Hemel Hempstead, UK). For whole antibody reduction, DTT was added to a final concentration of 0.33M and the samples were incubated at 22 ℃ for 1 hour and immediately analyzed by RP.

SEC analysis-Using an Acquisty UPLC Protein BEH SEC column attached to a Dionex Ultimate 3000RS HPLC System (ThermoFisher Scientific, Hemel Hempstead, UK) ((1.7 μm, 4.6 mm. times.150 mm (Waters, Elstree, UK)) and an acquisition UPLC Protein BEH SEC column (30X 4.6mm, 1.7 μm, (Waters, Elstree, UK)) were chromatographed. The process included isocratic elution over 10 minutes and the mobile phase was 0.2M potassium phosphate pH 6.8, 0.2M potassium chloride. The flow rate was 0.35 mL/min. Detection was by UV absorption at 280 nm.

Reverse phase analysis of intact antibodies and subunits-chromatographic separation was performed using a PLRP-S1000 (5 μm, 2.1mm × 50mm) column (Agilent Technologies, Stockport, UK) attached to a Dionex Ultimate 3000RS HPLC system (ThermoFisher Scientific, Hemel Hempstead, UK). The method included a linear gradient from 75% buffer a (0.02% TFA, 7.5% acetonitrile in H2O) to 45% buffer B (0.02% TFA, 7.5% H2O in acetonitrile) over 14 minutes. The flow rate was 0.5 mL/min and the temperature was maintained at 70 ℃ throughout the analysis. Detection was by UV absorption at 280 nm.

HIC analysis-chromatographic separation was performed using a TSKgel Butyl-NPR 4.6mm x 35mm HIC column (TOSOH Bioscience ltd., Reading, UK) attached to a Dionex Ultimate 3000RS HPLC system (ThermoFisher Scientific, Hemel Hempstead, UK). The method included a linear gradient from 60% buffer a (100mM sodium phosphate pH 7.0, 2M ammonium sulfate) to 90% buffer B (100mM sodium phosphate pH 7.0) over 9 minutes. The flow rate was 1.2 mL/min. Detection was by UV absorption at 280 nm.

Charge isomer Assay-according to the manufacturer's protocol, the Charge isomer profile of the test article is determined by Protein Charge isomer Assay (Protein Charge Variant Assay) on LabChip GXII Touch HT (Perkinelmer).

Of Fc affinity of human Fc receptorAnalysis of

Use ofThe T200 instrument determines the interaction affinity of antibody proteins by surface plasmon resonance. For most analyses, His 6-labeled Fc γ RI, Fc γ RIIa (167R and 167H variants), Fc γ RIIb, Fc γ RIIIa (176F and 176V variants), and Fc γ RIIIb receptors (all from nano Biological) were captured by standard amine coupling on CM5 sensor chips coated with anti-His antibodies. The receptor specific assay format was then used as described below.

Fc γ RI is a high affinity receptor for IgG1 monomer, so 1:1 kinetic analysis was performed under the following conditions: the "single cycle" assay uses a flow rate of 30. mu.l/min, with the receptor protein loaded at 10. mu.l/min to approximately 30RU (0.25. mu.g/ml diluted in HBS-P +), 5-point three-fold dilution of purified antibody titrated between 0.411nM and 33.33nM, with an applied binding time of 200s and a dissociation time of 300 s. Regeneration was performed with 2x injection of glycine pH 1.5 and analyzed using a 1:1 fit.

The interaction between monomeric IgG and Fc γ RII and Fc γ RIII receptors is a relatively low affinity interaction, and therefore a 'steady state' affinity assay is performed under the following conditions: the receptor protein was loaded at a flow rate of 30. mu.l/min to approximately 60RU (0.25. mu.g/ml diluted in HBS-P +) at 10. mu.l/min, 5-point three-fold dilution series of titrated purified antibody between 33nM and 24000nM, applied for an association time of 30s, and an dissociation time of 25 s. Regeneration was performed with 2x injection of glycine pH 1.5 and analyzed using steady state affinity calculations.

Results and discussion

Principle of protein construct design

Standard anti-cancer antigen antibodies are subject to significant pharmacological challenges in the treatment of solid tumors. A key problem limiting efficacy in this type of potential drug is that the antigen targeted by the antibody is not completely found in the tumor, but is only highly overexpressed in the tumor. This de-tumor target expression often leads to a dose-limiting side effect risk and an antigen "pool" effect, where large doses of antibody must be administered to ensure that sufficient antibody penetrates the tumor to have a therapeutic effect. One such example is the class of antibodies targeting the antigen CD47 (fig. 1A), where the challenge includes: high expression of CD47 in the bloodstream (e.g., particularly on red blood cells and platelets) is the "pool" bound by intravenously administered antibodies, minimizing the amount of drug that penetrates the tumor (even when large doses of IgG are administered). Binding of blood cells by anti-CD 47 is also a significant risk of toxicity. Indeed, it is known that anti-CD 47 antibodies are capable of causing anemia, and even cross-linking of human red blood cells, creating a risk of hemagglutination in patients. Furthermore, tumors are often "hostile" environments with high expression rates of enzymes such as MMPs that can accelerate IgG degradation. These factors all help to minimize the potential safety and efficacy of anti-CD 47 antibodies and many other types of anti-tumor target antibodies, where target expression is not limited solely to the tumor environment.

The anti-CD 47 protein construct (fig. 1B) was intended to overcome the problems of peripheral pool and toxicity experienced by anti-tumor antigen IgG by eliminating the binding of high-risk (but potentially strong mechanism of action) targets in the native protein. This effect is achieved by adding a low risk upper domain (e.g., a Fab domain targeted to another tumor antigen such as Her2) and a linker above (n-terminal to) the binding domain of a high risk lower domain such as CD 47. The use of an appropriate upper domain/linker combination results in a configuration that completely blocks the binding activity in the lower CD47 domain. The tumor targeting domain (e.g. Her2) then drives high concentrations in the tumor environment and the protein construct linker system exploits the elevated MMP activity in the tumor to cleave the linker peptide, thereby exposing the CD47 binding domain and thereby conditionally activating CD47 binding activity in the tumor but not in the periphery. This design principle has the potential to be applied to many different structural forms, examples of which are outlined below.

Protein construct IgG²The design (fig. 2A) may be based on sequences derived from IgG1, IgG2, IgG3, IgG4, IgE, IgM, or IgA, and may or may not have effector function capability. In this construct, the four polypeptide chains encode four Fab domains (2X Fab a, 2X Fab B), four linker sequences, and may or may not have an immunoglobulin hinge region and Fc domain. Each Fab a-linker domain (upper) blocks the binding activity of Fab B (lower). The target binding specificity of the upper and lower domains can be different to drive bispecific function, or the same to drive multivalent target interactions. The selection of linker sequences (such as the lower hinge peptide sequence) results in a structure that will lock in non-diseased tissue, but is rapidly cleaved and unlocked in the presence of high concentrations of proteases in the tumor environment (fig. 2B). The linkers in the protein construct design are all proteolytically cleavable, and can be cleaved sequentially, with the first 'fast' cleavage taking on the 'locked' intact structure and creating an intermediate 'unlocked' active state, which allows Fab a and B from a single protein construct to bind their cognate targets. Secondary, potentially slower cleavage of the second linker in each Fab A-Fab B protein construct unit may be Complete release of the Fab a domain from this structure results in a "dissociated" form in which the lower Fab domain is completely released for non-targeted (but possibly still limited) activity. Cleavage linkers based on immunoglobulin hinge sequences may also recruit increased immune effector functions (ADCC, CDC and ADCP) on the cell membrane by endogenous anti-hinge antibodies, a phenomenon known in human patients with (even without) potentially auto-reactive diseases.

Protein construct Fab²The design may be based on sequences derived from IgG1, IgG2, IgG3, IgG4, IgE, IgM, or IgA, and may or may not have effector function capability. In this construct, two (fig. 3A) or three (fig. 3B) polypeptide chains encode two Fab domains (1X Fab a, 1X Fab B), two or more linker sequences, and may or may not have an immunoglobulin hinge region and an Fc domain, where pairing of heterodimers may or may not be driven by mutations in the Fc. Each Fab a linker domain again blocks Fab B binding activity, and the choice of linker sequence (such as the lower hinge peptide sequence) results in a structure that will lock in non-diseased tissue, but is rapidly cleaved and unlocked in the presence of high concentrations of linker cleaving protease in the tumor environment, eventually becoming dissociated (fig. 3A).

Protein construct cloning and expression

For the production and purification of 15 Fab species presenting different linker domains²Or IgG²Bispecific, conditionally active protein constructs in molecular form (table 1), DNA cassettes for each construct type (table 2) were synthesized and cloned into expression vectors encoding human IgG1 heavy and light chains or "knob and hole" heterodimer Fc. The nomenclature (format) - (target name [ upper domain/lower domain) - (heavy chain linker type) - (light chain linker type) was used to name the proteins. All proteins were produced by transient transfection of CHO cells and then purified by protein a affinity chromatography.

The anti-HER 2 variable region sequence used in the protein constructs disclosed herein is the variable region sequence of trastuzumab. The anti-CD 3 variable region sequence used in the protein constructs disclosed herein is the variable region sequence of OKT3 or SP 34. The anti-cMET variable region sequences for use in the protein constructs disclosed herein are provided in WO 2019/175186. anti-CD 47 variable region sequences for use in the protein constructs disclosed herein are provided in WO 2019/034895.

Analysis of protein construct expression and purification characteristics

Quantification of protein a purified protein showed that the use of different linker types affected the expression yield (table 2). Protein preparations in 1x PBS pH 7.4 were also tested in analytical size exclusion chromatography to quantify the percentage of desired product. Among all 3 classes of bispecific proteins produced (cMETCD47, Her2CD47, and Her2CD3), the constructs containing LHL linkers yielded the best combination of highest yield as determined by analytical SEC and% yield of the main peak of the desired product (table 2). SDS-PAGE analysis of protein a purified protein (fig. 4) also demonstrated that clones containing short linker domains L1 and LH plus long linker domain L3 produced highly heterogeneous products with significant HMW and LMW content in the unreduced samples and significant LMW content in the reduced samples. This suggests that suboptimal linker types may lead to undesirable multimers and significant formation of decomposition products. For all constructs, the HMW impurities were essentially absent in the reduced lanes, indicating that these HMW impurities are disulfide-bound dimers or higher order polymers that are not reduced by SDS alone. For IgG²Both clones 12 and 14 were designed to show high yields (table 2), but clone 14 (containing the LHL linker) demonstrated the highest yield and high uniformity of the desired product as measured by SEC (90%) and SDS-PAGE (fig. 4).

The remaining protein samples from the clone subset were then subjected to SEC chromatography in an attempt to produce fully purified monomeric protein. Chromatograms of clones 1, 2, 3, 4, 5, 6, 10, 12 and 14 in preparative SEC are shown in fig. 5. These analyses further demonstrated that clones 1 (fig. 5A), 3 (fig. 5C), 4 (fig. 5D), 5 (fig. 5E) and 12 (fig. 5G) contained a large percentage of higher molecular weight products and generally lower molecular weight products (eluting at a CV of about 0.5-0.55 in fig. 5A-5H) near the major peak of the desired product. CompareIn contrast, clones 6 (fig. 5F), 14 (fig. 5H) and 10 (fig. 5I) showed significant, well-defined main product peaks. For clones 2 and 6, this allowed efficient SEC purification of the predominantly monomeric protein as demonstrated by SDS-PAGE of the unreduced samples (fig. 6A). Attempted SEC purification of clones 1, 4 and 5 failed, resulting in a still heterogeneous sample (fig. 6A). Fab from Her2CD3²SEC-purified proteins of clones 7, 8 and 10 showed improved homogeneity, while IgG was present at Her2CD47²Of the clones, only clone 14 achieved complete homogeneity and monomer status as confirmed in SDS-PAGE of unreduced and reduced samples (fig. 6B). Furthermore, these findings demonstrate that clones 6, 10 and 14, which all contain LHL linkers on both strands, exhibit the most reproducible beneficial characteristics.

Functional characterization of protein constructs containing the v domain of the CD47 antibody in the lower Fab

Control IgG antibodies A-D5 anti-CD 47, MH7.1 anti-C-MET, anti-Her 2 trastuzumab and A-D5 Fab-Fc (monovalent forms of A-D5 antibody containing a single Fab domain) were titrated (in μ g/ml) in a direct binding ELISA against human CD47, C-MET and Her2 proteins (FIG. 7A). Also analyzed in the same manner as IgG²Forms of Her2CD47-LH-LH and Her2CD47-LHL-LHL clones (FIG. 7B) and in Fab²Forms cMETCD47-L2-L2 and cMETCD47-LHL-LHL (FIG. 7C). The control antibody demonstrated the expected strong binding activity against its cognate target, even at the highest concentration, with little or no background to any other target (fig. 7A).

Importantly, the very strong monovalent binding of a-D5 Fab-Fc (fig. 7A) demonstrates the intrinsic affinity of a-D5 anti-CD 47 domain and the significant potency required to lock into the protein construct format of the invention making it successful. The protein constructs Her2CD47-LHL (fig. 7B) and cMETCD47-LHL (fig. 7C) also showed a similarly strong highly specific binding to the homologous target of its upper Fab domain, but no binding signal to CD47, indicating that the binding activity of the CD47 v-domain was indeed completely inhibited when using this linker combination. Importantly, a high background signal to human CD47 was observed for Her2CD47-LH-LH and to a lesser extent for cMETCD47-L2-L2, indicating that the lower part The elimination of fab binding affinity is tightly controlled by linker selection. These findings indicate that the protein constructs Her2CD47-LHL-LHL (FIG. 7B) and cMETCD47-LHL-LHL (FIG. 7C) containing the anti-CD 47 binding domain of antibody A-D5 tested in this assay have a reduced ability to bind CD47 compared to A-D5IgG1>1000 times, wherein the concentration is about 1 × 10^-2A binding signal OD of 1.0 was achieved at μ g/ml, whereas at the highest tested concentration of 10 μ g/ml neither protein construct showed a signal above 0.2.

Since hemagglutination is the major toxicity risk of anti-CD 47 antibodies, the preferential protein construct was then detected in a human red blood cell based hemagglutination assay. Control antibodies anti-CD 235a (murine) and A-D5 anti-CD 47, A-D5 Fab-Fc, MH7.1 anti-C-MET, anti-Her 2 trastuzumab, in IgG²Forms of Her2CD47-LH-LH and Her2CD47-LHL-LHL and in Fab²Forms of cMETCD47-L2-L2 and cMETCD47-LHL, titrated (in nM) in a human red blood cell hemagglutination assay using fresh red blood cells from healthy donor 1 (fig. 8A), donor 2 (fig. 8B) and donor 3 (fig. 8C). The control antibodies anti-CD 235a, A-D5 anti-CD 47 and A-D5 Fab-Fc showed effective concentration-dependent hemagglutination for all 3 donors due to cross-linking of their respective surface antigens on adjacent erythrocytes. The low potency hemagglutination observed for the A-D5 Fab-Fc (clone 15) was probably due to the presence of a small amount of functional dimer in the protein preparation, as it was purified only by the protein A column and not completely into the monomeric state by SEC. Importantly, even at the highest concentration of 140nM, no protein construct samples exhibited any ability to induce hemagglutination. This finding indicates that IgG containing the anti-CD 47 binding domain of antibody A-D5 tested in this assay compared to A-D5IgG1, which showed a titer of 0.58nM ²And Fab²Reduced ability of the form protein construct to induce agglutination>241 times.

The preferential protein constructs were enzymatically digested using human MMP3, MMP7, and MMP12 over a time course of 2, 4, 8, and 24 hours of incubation, plus 24 hours of incubation in enzyme-free buffer as a negative control. Samples from these digestion schedules were then applied against human Her2 and CD47 (fig. 9A, 9B) orDirect binding ELISA of human C-MET and CD47 (FIG. 9C). In each case, no loss of binding to Her2 or C-MET was observed over time, indicating that the addition of protease did not reduce the functional binding capacity of the upper Fab domain. In contrast, the CD47 binding capacity of all three protein constructs increased significantly over time in an MMP dependent manner. Cloning of IgG²Her2CD47-LH (fig. 9A) again showed high background binding to CD47 and minimal increase in CD47 binding over time. In contrast, cloned IgG²Her2CD47-LHL-LHL (FIG. 9B) and Fab²cMETCD47-LHL-LHL (FIG. 9C) again showed low background binding to CD47 (OD 450 nM)<0.2) and rapidly increased binding to CD47 starting at 2 hours of incubation with both MMP7 and MMP12, reaching complete saturation at 24 hours of incubation (OD 450nM of about 4.0). MMP3 appeared to be the slowest activating among the 3 MMPs, showing that all 3 protein construct examples had increased CD47 binding signal after 24 hours.

Functional characterization of protein constructs containing the v domain of the CD3 antibody in the lower Fab

Antibody Fab² Her2CD3-L1-LH、Fab²Her2CD3-L2-L2 and Fab²Her2CD3-LHL was analyzed by ELISA (fig. 10A), flow cytometry (fig. 10B) and CD3 reporter assay (fig. 10C). In the ELISA assay, all 3 proteins showed the expected strong binding activity against Her2 (upper Fab domain), even at the highest concentration, with little or no background binding to any other target (fig. 10A). All 3 protein constructs Fab were then used before further analysis²The proteins were incubated overnight with or without mixing MMP3, MMP7, and MMP 12. anti-HER 2 trastuzumab showed strong binding, Fab, in flow cytometric binding to HER2+ human cell line BT474²Her2CD3-L1-LH and Fab²Her2CD3-L2-L2 showed similarly strong binding before and after MMP digestion, while Fab²Her2CD3-LHL showed partially reduced binding after MMP digestion, indicating that a portion of the protein lost the upper fab in the 'off' state (fig. 10B). Since this finding suggests that the MMP digestion process is in Fab² Her2CD3-LHL-LHL is active, so the same sample is used in a reporter assay in which Her2+ BT474 cells are mixed with human CD3+ Jurkat cells engineered to provide a measurable signal when CD3 is activated. Data from this assay demonstrate that the bivalent anti-CD 3 antibody OKT3 directly activates CD3 signal in reporter cells, as expected (fig. 10C). The protein constructs each exhibited different characteristics: the Her2CD3-L2-L2 protein containing a 2xG4S linker (not cleavable by MMP proteases) showed high background in the assay, while the CD3 activation signal did not increase after MMP digestion, indicating that the flexible linker of this construct allows Her2 to be bound by the upper Fab, while the partial activity of the lower Fab allows some background but does not activate CD3 co-ligation (fig. 10C). Fab ²The Her2CD3-L1-LH protein showed lower background activity in the assay with a modest increase in signal following MMP digestion. Fab without MMP digestion²Her2CD3-LHL showed no measurable background signaling, showed minimal CD3 activation similar to the negative controls trastuzumab and IgG1 isotype control antibody in undigested samples, but showed effective activation in MMP digested samples. Thus, the data in FIGS. 10A-C indicate Her2CD3-LHL-LHL Fab²The format has the best combination of properties because it is simply expressed and purified, has high intrinsic Her2 binding activity, low background CD3 ligation activity, and high CD3 co-ligation activation only when activated by MMP cleavage of the LHL linker.

Cloning and expression of second generation constructs

The aforementioned multispecific Fab containing LHL linker²And IgG²The performance of the clones prompted the second series of constructs to be used to experimentally explore the potential functional sequence space in both tertiary structure and linker sequence content. The following clones were synthesized and assembled to sample these parameters:

1. being 'single-arm' type Fab²Clone of protein 'Met 47-LHL-LHL' (Table 6, FIG. 3B).

2. Re-modelled 'clone 10' Fab as 'single arm' type ²Clone of protein ` Her23-LHL-LHL ` (Table 7, FIG. 3B).

3. Being 'single-arm' type Fab²Clone of protein ` Her23(34) -LHL-LHL ` (Table 8, FIG. 3B).

4. A series of IgG 2 binding domains incorporating trastuzumab in the upper Fab and CD47 binding domain of clone A-D5 in the lower Fab²Type clones (Table 9). These clones contain mutated LHL-based linker sequences that may be more susceptible to enzymatic cleavage of a broad family of MMPs (LHLF linker), may be moderately increased length linkers with the addition of a portion of the human IgG1 intermediate hinge sequence (LHLM linker), or both (LHLMF linker).

5. In which Fab is²Fab with module at the terminus of KIH-Fcc²Cloning of the protein ` Fc-Her23 (34)' (Table 13, FIG. 12).

6. Being a 'single-arm' type Fab containing two copies of a C-Met Fab²Clone 'MetMetMetMet-LHL-LHL' of the protein (Table 14, FIG. 3B, FIG. 13). In this structural form, binding to the bivalent form of Met receptor may only occur after cleavage of a single LHL linker by a protease.

As described above, these constructs were successfully expressed and purified by protein a and size exclusion chromatography.

Second generation IgG²Analysis of constructs

The purified IgG was examined in a series of further analyses ²Performance of the clone. First, clones A-D5 IgG1 and IgG of the examples were examined² Her2CD47-LHL-LHL、IgG² Her47-LHLF-LHL、IgG²Her47-LHL-LHLF and IgG²Her47-LHLF (table 9) binding to human Her2 and human and mouse CD47 (figure 14). This analysis demonstrated that: 1) the a-D5 anti-CD 47 IgG1 protein showed high binding signals to both hCD47 and mCD 47. 2) All 4 IgG' s²The protein retained a high binding signal to hhr 2, but no/background binding signal to hCD47 or mCD47 (fig. 14).

Detection of cloned IgG²Her2CD47-LHL-LHL and IgG²Sensitivity of Her47-LHL-LHLF to enzymatic activation (at pH 7.4) of various proteases known to be overexpressed in human tumors (FIG. 15): incubation with MMP7 (FIG. 15A), MMP8 (FIG. 15B), and MMP10 (FIG. 15C) demonstratedBoth proteins can be activated by each enzyme to bind hCD47, but in each case IgG²Her47-LHL-LHLF is activated more rapidly. Incubation with MMP12 demonstrated that both proteins can be activated at equal rates for the enzyme to bind hCD47 (fig. 15D). Unexpectedly, incubation with MMP13 demonstrated IgG²Her47-LHL-LHLF can be activated by this enzyme to bind hCD47, while IgG²Her2CD47-LHL-LHL failed (FIG. 15E). Importantly, incubation with the cysteine protease cathepsin S also demonstrated that both proteins can be activated at equal rates for this enzyme to bind hCD47 (figure 15F). These findings demonstrate that in IgG ²Fab contained in protein²The peptide linker content of the module can be "tuned" to broaden the number of potentially active enzymes and even make the proteolytic activation process faster. To sample this possibility, additional linker designs are envisioned with appropriate length and content to drive sensitivity to specific classes of enzymes, where amino acid sequences that exhibit proteolytic sensitivity to the activity of other disease-associated metalloproteinases such as ADAMS, cysteine, aspartate, and serine proteases would be used to provide broader or more selective activation.

For detection of activated versus IgG²Effect of binding affinity, a Biacore assay was established that can sample both Her2 and CD47 binding. In this assay, control antibodies and IgG²Her47-LHL-LHLF protein (undigested or activated with MMP12 for 2, 4, 8 or 24 hours) was captured on the chip surface by anti-Fc antibodies and then binding affinity was measured for soluble Her2 and CD47 ectodomain proteins. Repeated binding assays at the beginning and end of the experimental run were performed for trastuzumab IgG1 and a-D5 IgG1 (without enzymatic digestion), with the data shown in table 17. These analyses showed that the calculated KD values for each antibody were highly similar in each run. Importantly, however, Rmax values (maximum binding signal at maximum analyte concentration) dropped significantly between runs at the beginning and end of the experiment (e.g., trastuzumab Rmax at 265.80RU at the beginning and 144.13RU at the end), indicating that the activity of the anti-Fc antibody capture surface was over many rounds of regeneration inherent to the Biacore method And then decreases. To examine the effect of MMP12 activation on target reactivity, Rmax and KD values were calculated for both Her2 (fig. 16A, table 18) and CD47 (fig. 16B, table 18). This analysis shows that Her2 binding and affinity are maintained within 24 hours of MMP12 activation. Importantly, no CD47 binding was observed in the 0 hour sample (without MMP12 digestion) (Rmax ═ 0), but both Rmax and apparent affinity for CD47 increased rapidly over the course of the activation time, starting from 2 hours of incubation (fig. 16B, table 18). These findings demonstrate that Fab²The lower middle Fab of the module is indeed inert and is unable to target interactions in the intact molecule until protease activation occurs. This observation is further illustrated in fig. 17, where no binding activity was observed even at a concentration of 400nM of CD47 analyte, but high binding to CD47 was evident after 24 hours of MMP12 treatment.

The above findings also demonstrate that in Fab²Cleavage of the linker peptide in the module (and thus lower Fab activation) is transient. This may be Fab containing²The pharmacological benefit of the modular molecule, since activation may be strongly biased towards diseased tissues where both the target antigen of the upper Fab and the enzyme capable of activating the lower Fab are highly overexpressed. This will result in rapid drug accumulation, long residence time and high levels of activation in such diseased tissue.

Fab²Computer modeling of modular structure and molecular dynamics

To understand Fab²The mechanism by which the module might function, we performed structural modeling and molecular dynamics analysis. A model for anti-CD 47 a-D5 Fab binding to CD47 ECD was generated using the crystal structure of human CD47 ECD binding to the Fab domain of IgG 1C 47B161 (protein database identifier 5TZT), IgG 1C 47B222 (protein database identifier 5TZ2) and IgG1 b6h12.2 (protein database identifier 5TZU) as a template using the protein modeling suite of MOE software. The structure of trastuzumab Fab complexed with Her2 extracellular domain was taken from PDB structure 1N 8Z.

The LHL and LHLF linkers between the upper trastuzumab Fab and the lower anti-CD 47 Fab were then modeled using the protein modeling suite of MOE software. To aid in the modeling of the linker, the C-terminus of Fab structures available in the protein database was examined to help define the conformation in which the LB linker can leave each of the heavy and light chain domains of trastuzumab Fab. Modeling predicts that trastuzumab Fab heavy and light chain C-termini can optionally have native interchain disulfide bonds that are typically present in the IgG1 Fab domain.

A full-length IgG 1-based model incorporating the anti-CD 47 Fab modeled above was constructed using the structure of IgG1 b12 (protein database identifier 1HZH) as a template. Structural errors in the 1HZH, such as missing structural regions, are re-modeled and corrected. The IgG1 b12 Fab was replaced with the anti-CD 47 Fab and the Fc-hinge was attached using the protein modeling suite of MOE software. This model illustrates IgG based on Her2 and CD47 and IgG1 ²Possible tertiary structure of the molecule (FIG. 18A). In this model, binding of Her2 epitope was constitutively active (fig. 18A), whereas binding of CD47 ECD was completely occluded by the linker itself and the proximity of anti-CD 47 Fab to anti-Her 2 Fab (fig. 18B). The model and Fab²The modules are identical, the Fab²Modules can bind HER2 via the upper fab without observed hindrance, but prevent binding to CD47 via the lower fab until the linker is degraded (fig. 16A, 16B).

To give further insight to Fab again²How the modules moved in solution, we performed molecular dynamics simulations using a variety of linker compositions, including LHL-LHL, LHL-LHLF, and L2L2(G4SG4S (SEQ ID NO:32) amino acid sequence, Table 1). The RMSD of each residue was calculated for each run. Furthermore, a custom descriptor dSASA was written in the SVL language (MOE) which calculated the change in Solvent Accessible Surface Area (SASA) per residue, and in each case also the set of linker-only sequences. Thus, it provides the basis for comparing how different uncleaved linkers behave with respect to conformational changes and the associated effect on the solvent accessibility of the linker, which is expected to influence the sequence-specific cleavage potential (fig. 18).

Fig. 19A-19I show 9 graphs corresponding to Solvent Accessible Surface Area (SASA) results obtained for the 3 linkers tested. First analysis for each of LHL and LHLF linkersOne for 9 thermodynamic runs and 10 runs using L2 (fig. 19A, 19D and 19G, respectively) sampled absolute SASA values (within 6 ns). This data demonstrates that the L2 linker clearly has the greatest propensity for structural heterogeneity over time (fig. 8G). Since the starting SASA values for each run were not the same, the second analysis normalized the results, calculated by subtracting all SASA values from the starting SASA values, representing the difference from the starting SASA values. The results in the 6ns kinetic run time (fig. 19B, 19E and 19H, respectively) and the first 2.5ns of the 6ns kinetic run (fig. 19C, 19F and 19I, respectively) illustrate that the structure kinetics of the LHL and LHLF linkers are significantly less than the L2 linkers. In particular, the 6ns molecular dynamics run showed that L2 exhibited the highest flexibility, thus emphasizing that different linker sequences would yield different flexibilities and thus for Fab²Concept of CDR solvent exposure profile of lower Fab in the module.

Comparison between the cleaved and uncleaved linkers shows a significant increase in flexibility for the cleaved linkers compared to the uncleaved linkers, consistent with the biological observations in fig. 14-17. For example, FIG. 20A shows Fab when both linkers are intact ²Limited movement in the module is followed by significant movement of the upper Fab domain in the context of a single cleaved LHL linker (second linker intact) during a 100ns kinetic run. This analysis shows that the degree of freedom of the upper fab domain is significantly increased, resulting in multiple positions in which it can move completely out of the way of the lower fab domain, completely exposing its CDRs to allow unconstrained interaction with, for example, CD47 (fig. 20B).

In vitro and in vivo analysis of tolerability and pharmacokinetics

To test the tolerance and pharmacokinetics of IgG2 and Fab2 in the context of CD47 as the lower Fab domain, males between 6 and 8 weeks of age were B6.Cg-Fcgrt^tm1DcrA number of exemplary molecules (A-D5 IgG1, IgG) were studied in Tg (FCGRT)32DcrJ mice² Her47 LHL-LHL、IgG²Her47 LHL-LHLF and Fab²Met47 LHL-LHL). These 'Tg 32' mice are human IgG pharmacokinetics with pharmacokinetic characteristics that mimic those of humans and primatesA human FcRn homozygous transgenic animal of mathematical characteristics. Since it is known that the neutralization is carried out in A-D5 IgG1 and in IgG² Her47 LHL-LHL、IgG²Her47 LHL-LHLF and Fab²The CD47 binding domain contained in the lower Fab domain of Met47 LHL-LHL was all able to bind to recombinant mouse CD47 protein, so they were first tested for their reactivity to mouse membrane-presented CD47 on erythrocytes by flow cytometry (figure 21). This study showed that A-D5 IgG1 showed no signal on mouse erythrocytes at 0.1. mu.g/ml, but clear concentration-dependent binding signals at both 1 and 10. mu.g/ml (FIG. 21). Importantly, binding to mouse erythrocytes was sub-saturated at 1 μ g/ml (63% binding, FIG. 21) and fully saturated at 10 μ g/ml (98% binding, FIG. 21). In contrast, protein IgG ² Her47 LHL-LHL、IgG²Her47 LHL-LHLF or Fab²Met47 LHL-LHL did not show any binding at any concentration, indicating that the anti-CD 47 variable domain of the lower Fab domain was unable to interact with CD47 on mouse erythrocytes (figure 21).

Hemagglutination assays were also performed using erythrocytes isolated from Tg32 mice. This analysis demonstrated that only A-D5 IgG1 was able to drive concentration-dependent agglutination of mouse erythrocytes (in the presence of>3.12. mu.g/ml) and protein IgG² Her47 LHL-LHL、IgG²Her47 LHL-LHLF or Fab²Met47 LHL-LHL showed no agglutination at any concentration up to 200. mu.g/ml (FIG. 22). These findings demonstrate that the in vitro detection of human CD47 protein and erythrocyte binding outlined above is reproduced in the mouse system (i.e., a-D5 IgG1 binds fully to human CD47, but for IgG 47)²Or Fab²No measurable binding of the protein) made mice a viable model to study the effects of CD47 binding on both pharmacokinetics and tolerance.

A-D5 IgG1, IgG in vivo tolerability studies² Her47 LHL-LHL、IgG²Her47 LHL-LHLF and Fab²Met47 LHL-LHL was administered once (intravenously) at a concentration of 2mg/kg or 10mg/kg, respectively, in Tg32 mice. A-D5 IgG was tolerated at the 2mg/kg dose, whereas the 10mg/kg dose was poorly tolerated, causing significant toxicity at day 0, which resulted in significant toxicity in this dosing group The study was terminated. In contrast, IgG at 2mg/kg and 10mg/kg doses² Her47 LHL-LHL、IgG²Her47 LHL-LHLF and Fab²Met47 LHL-LHL was all well tolerated in a 60 day observation of body weight (figure 23). Although the a-D5IgG at the 2mg/kg dose was generally tolerated, significant upregulation of reticulocytes was observed in this group 5 days after dosing (fig. 24). In contrast, IgG at 2mg/kg or 10mg/kg doses² Her47 LHL-LHL、IgG²Her47 LHL-LHLF or Fab²Met47 LHL-LHL was not associated with reticulocyte upregulation at day 5 or later (fig. 24, fig. 25). Reticulocyte upregulation is a known response to rapid red blood cell clearance, suggesting that IV administration of a-D5IgG1 antibody results in accelerated clearance of CD 47-high red blood cells.

To more broadly sample the effect of the administered protein, the whole hematology group was examined on day 5, day 29 and day 60 post-administration (fig. 25A-fig. 25K). These analyses demonstrated that the reticulocyte upregulation of A-D5IgG1 was transient, returning to baseline on day 29 (FIG. 25A). No A-D5IgG1, IgG were observed on days 5, 29 and 60² Her47 LHL-LHL、IgG²Her47 LHL-LHLF and Fab²Significant effect of Met47 LHL-LHL on Red Blood Cell (RBC) count, hemoglobin, Mean Corpuscular Hemoglobin Concentration (MCHC), Mean Corpuscular Volume (MCV), white blood cells, monocytes, lymphocytes, basophils, eosinophils, or neutrophil levels (fig. 25B-fig. 25K).

In vivo pharmacokinetic studies, IgG in Tg32 mice² Her47 LHL-LHL、IgG²Her47 LHL-LHLF and Fab²Met47 LHL-LHL were each administered once (intravenously) at a concentration of 2 or 10mg/kg, and A-D5 IgG1 was administered at a previously tolerated concentration of 2 mg/kg. Blood samples were collected from each mouse according to the bleeding schedule: 30 minutes, 4 hours, 1 day, 3 days, 5 days, 7 days, 10 days, 14 days, 21 days, 28 days, and 42 days. Analysis of serum antibody concentrations demonstrated that 2mg/kg of A-D5 IgG1 was very rapidly removed from circulation, reaching an average concentration within 5 days<0.5. mu.g/ml (FIG. 26). This rapid drug clearance (referred to as tissue-mediated drug treatment or TMDD) may be due to previous attempts atStrong binding to mouse erythrocytes was observed, which were then rapidly cleared from the system by phagocytosis. This finding further explained the return of reticulocyte levels to normal on day 29 of A-D5 IgG1 administration (FIG. 25A) because the molecule was essentially eliminated on day 10 (FIG. 26).

In contrast, IgG was administered at 2 and 10mg/kg doses² Her47 LHL-LHL、IgG²Her47 LHL-LHLF and Fab²Met47 LHL-LHL all showed slow clearance (fig. 26). Importantly, a 2mg/kg dose of each protein is required>Can be reached in 25 days <Concentration of 1.0. mu.g/ml (FIG. 27A), and 10mg/kg dose of each protein was maintained over 42 days>Concentration of 1.0. mu.g/ml (FIG. 27B). A single sample taken from mice given 10mg/kg of A-D5 IgG1 during the tolerability study on day 0 was also analyzed, showing the maximum serum IgG concentration reached (but not tolerated) and fully tolerated IgG²And Fab²Protein to a concentration of: (>50. mu.g/ml) were similar.

IgG 2mg/kg² Her47 LHL-LHL、IgG²Her47 LHL-LHLF and Fab²Met47 LHL-LHL all showed a normal 'alpha' phase (when IV doses of immunoglobulin were rapidly distributed from the bloodstream into tissues) followed by a long "beta" phase of antibodies circulating in the serum (fig. 26, 27A). These proteins also showed normal profiles at 10mg/kg, but with even longer circulation (fig. 26, 27B). Importantly, the β phase of each protein showed linear and parallel curves at both concentrations, indicating that the CD47 domain of these proteins did not result in the TMDD observed for a-D5 IgG1 (fig. 26). If IgG is² Her47 LHL-LHL、IgG²Her47 LHL-LHLF or Fab²Met47 LHL-LHL protein undergoes activation in the periphery, then TMDD is expected to show strongly, as activation will result in high affinity red blood cell, endothelial and platelet binding, leading to clearance of the β -phase transition to a sharply decreasing trajectory, as seen with a-D5 IgG1 (fig. 26). These observations, plus IgG ² Her47 LHL-LHL、IgG²Her47 LHL-LHLF and Fab²Lack of reticulocyte expansion in the dose of Met47 LHL-LHL, indicating peripheral activationThe level is very low. Although the pharmacokinetics of these molecules are long, this means that they are already present>Multiple recirculations were performed by FcRn in 25 day cycles.

IgG as outlined above² Her47 LHL-LHL、IgG²Her47 LHL-LHLF and Fab²Pharmacokinetic and tolerability findings for Met47 LHL-LHL indicated a normal, FcRn-mediated, antibody-like half-life extension by the Fc domain of human IgG 1. This effect resulted in a significant increase in area under the curve (AUC) values for these 3 proteins compared to a-D5 IgG1 (fig. 28). At 2mg/kg, IgG² Her47 LHL-LHL、IgG²Her47 LHL-LHLF and Fab²AUC of Met47 LHL-LHL was improved by 25-40 fold compared to A-D5 IgG 1. IgG1 was not safely achievable at 10mg/kg for A-D5 IgG1²Her47 LHL-LHL、IgG²Her47 LHL-LHLF and Fab²AUC of Met47 LHL-LHL was improved by approximately 100-fold and 250-fold over A-D5 IgG1 (FIG. 28). These improvements in AUC were significant as they were demonstrated in IgG²And Fab²The use of the CD47 binding domain within the structure can be used at high concentrations, starting from the first dose, to maximize distribution to the tumor tissue.

Flow cytometry analysis of binding to monkey and human erythrocytes

Erythrocytes were isolated from 3 cynomolgus monkey NHP (monkey) and 3 human donors and treated with A-D5 IgG1, trastuzumab, IgG ²Her47 LHL-LHLF or IgG²Her47 LHL-LHL staining. Both sets of analyses showed that only a-D5 IgG1 showed concentration-dependent binding to cynomolgus monkey (fig. 29A) or human (fig. 29B) erythrocytes. These findings confirm the above observation, namely IgG²(and thus Fab)²) The CD 47-binding domain within the structure is restricted to binding to mouse, monkey, and human CD 47.

Activation by MMP and cathepsin at pH6.0 and 7.4

MMPs and cathepsins have been shown to have the potential to enzymatically cleave peptide sequences found in LHL or LHLF linkers outlined above. Importantly, however, both classes of enzymes exhibit sensitivity to changes in pH conditions that increase or decrease their enzymatic activity. This can be critical, as it is often observed that the pH of solid tumors deviates from the normal physiological pH of humans, pH 7.4. In particular, solid tumors (and highly inflamed tissues) can develop acidic pH conditions as low as pH 6.0.

Having demonstrated the ability of various MMP enzymes to activate lower fab binding at ph7.4, we tested various MMPs and cathepsins (all of which are associated with increased activity in solid tumors) for activity at ph6.0 and 7.4. MMP3 (fig. 30A, B), MMP7 (fig. 30C, D), MMP8 (fig. 30E, F), MMP10 (fig. 30G, H), MMP12 (fig. 30I, J), MMP13 (fig. 30K, L), and MMP14 (fig. 30M, N) were detected for IgG ²Her47 LHL-LHL or IgG²Activation of Her47 LHL-LHLF. The IgG is then²Her47 LHL-LHL and IgG²MMP treated samples of Her47 LHL-LHLF were applied to bind both human Her2 and human CD47 directly. These analyses demonstrated that both proteins showed measurable activation of CD47 binding by MMP8, MMP10, and MMP12 at ph7.4 and ph 6.0. In contrast, only IgG²Her47 LHL-LHLF is activated by MMP13 (FIG. 30K, L) at pH7.4 and pH6.0 and by MMP7 (FIG. 30D) at pH 6.0. IgG²Her47 LHL-LHLF also compares to IgG at most time points²Her47 LHL-LHL showed relatively higher levels of activation by most of the tested MMPs (FIGS. 30A-N). These findings confirm that inclusion of LHLF linker at ph6.0 and/or ph7.4 results in faster activation of lower Fab binding by a wider range of MMP enzymes.

Cathepsins were also tested as potential activating enzymes. For these enzymes, a clear relationship between pH and activity was observed. IgG²Her47 LHL-LHL (FIG. 31A) or IgG²Activation of Her47 LHL-LHLF (FIG. 31B) all demonstrated that cathepsin S alone is able to activate CD47 binding at pH7.4 and 6.0. In contrast, treatment with cathepsin A, C, G, K and L showed little or no activation of CD47 binding even after 24 hours at ph7.4, but rapidly generated a strong activation signal at ph6.0 (fig. 31A, 31B). These findings indicate that the LHL and LHLF linkers can allow accelerated activation in acidified tissues by more rapid activation of MMPs and pH selective activation by a range of cathepsins than those at pH 7.4. This can be achieved by using a pH of 7.4 (non-diseased) Tissue pH, where active extracellular MMP and cathepsin levels are low) minimizes activation of lower Fab binding and maximizes it at pH6.0 (pH of diseased tissue, where MMP and cathepsin levels are high and cathepsin activity is enhanced) to further improve IgG²And Fab²Therapeutic index of the protein.

Activation of binding to lower Fab of Her2/CD47+ cells

Detection of IgG by flow cytometry²Her47 LHL-LHL and IgG²Binding characteristics of Her47 LHL-LHLF to cells expressing different levels of CD47 and Her2 on their cell surface. Staining with trastuzumab, anti-CD 47 and isotype control IgG demonstrated that BT474 cells expressed high levels of Her2 and lower levels of CD47 (fig. 32A, B), whereas MCF7 cells expressed higher levels of CD47 and lower levels of Her2 (fig. 32C, D). Activation with MMP12 was also performed after 0, 2, 8, and 24 hours²Her47 LHL-LHLF (FIG. 32A, C) and IgG²Her47 LHL-LHL (FIG. 32B, D) stained both cell types. IgG²Her47 LHL-LHLF and IgG²Her47 LHL-LHL showed similar binding profiles to trastuzumab on BT474 cells at 0, 2 and 8 hour time points, but binding was slightly reduced after 24 hours (figure 32A, B). In contrast, IgG ²Her47 LHL-LHLF and IgG²Her47 LHL-LHL all showed a low-level binding profile similar to trastuzumab on MCF7 cells at 0 hour activation, but binding was significantly higher at 2, 8 and 24 hour activation time points, mimicking an anti-CD 47 control (fig. 32C, D). These findings confirm experimentally that the activation model proposed in fig. 2B is valid, where trastuzumab-like binding of Her2 is preserved after activation and the CD47 binding profile becomes highly active only after activation. Importantly, these findings also indicate IgG²(and Fab)²) The protein may have the beneficial ability to drive the function of the lower Fab binding domain (e.g., CD47) to cells expressing low levels of the upper Fab target (e.g., Her 2).

MMP12 activation by SDS-PAGE and mass spectrometry

Treatment of IgG with MMP12 as described above²Her47 LHL-LHLF and IgG²Her47 LHL-LHL protein 0, 2, 8 and 24 hours. These protein samples were then analyzed by SDS-PAGE (FIG. 33). This analysis yielded findings clearly related to the observations of activation ELISA (fig. 15-7, fig. 30) and flow cytometry (fig. 32): at 0 hours-two intact chains were observed, with the heavy chain running at 75kDa and the light chain running just below the 50kDa marker (below 50 kDa). At the 2 hour time point, for IgG ²Her47 LHL-LHLF and IgG²Her47 LHL-LHL, a new-25 kDa product was observed, plus a faint band slightly greater than 25kDa and at about-50 kDa (over the intact light chain). The molecular weights of these new fragments correspond to the upper Fab Fd or light chain fragment (1 v domain +1 c domain ═ 25kDa), Fc (2 x c domain +1 n-linked glycosylation ═ 28-30kDa) and the complete Fd-hinge-Fc (standard IgG1 heavy chain ═ 50kDa), respectively. At the 8 and 24 hour time points, a progressive decrease in the intact strand and an increase in fragments approximately 25kDa smaller than the intact strand were observed. In particular, at 8 hours, the 25kDa (upper fab chain) product became dominant, with significant amounts of both intact heavy (75kDa) and intact light chains (below 50kDa) being retained (but reduced compared to the 0 hour sample). Thus, this SDS-PAGE analysis (fig. 33) demonstrated that the linker between the upper and lower fabs was cleaved by MMP12 enzymatic activity, whether LHL or LHLF sequences.

For accurate detection at Fab²Where in the structure the MMP12 enzyme activates IgG²Molecules, using from IgG²Her47 LHL-LHL 0, 2, 8 and 24 hour samples were subjected to mass spectrometry. Peptide samples were prepared by reduction, alkylation and proteolytic digestion using a combination of trypsin and Glu-C. These samples were analyzed by LC-MS/MS. The efficiency of cleavage by MMP was determined by comparing the MS responses of a peptide derived from the intact MMP cleavage site (SCGPAPE (SEQ ID NO:110)) and a peptide derived from the cleaved protein (SCGPAP (SEQ ID NO: 111)). This analysis successfully identified peptides that localized to both the uncleaved (SCGPAPE (SEQ ID NO:110)) and cleaved (SCGPAP (SEQ ID NO:111)) linkers, demonstrating that MMP12 cleaves between the second proline (P) and glutamic acid (E) in the LHL linker. The relative signal of the uncleaved (SCGPAPE (SEQ ID NO:110)) peptide ranges from 0 to 8 hours to The signal of the cleaved (SCGPAP (SEQ ID NO:111)) peptide gradually decreased at 24 hours (FIG. 34A), whereas the signal of the cleaved (SCGPAP: 111)) peptide gradually increased (FIG. 34B).

IgG in the form of hinge-stabilized' IgG1-DAA²Generation and analysis of

U.S. patent No. 8871204B2 teaches protease resistant IgG1 antibody variants that retain low hinge and Fc stability in the presence of MMP enzymes. In these mutant Ig G1 antibodies, the sequence of E233-L234-L235-G236(SEQ ID NO:112) in the hinge was replaced with P233-V234-A235 (where G236 was deleted); and the CH2 domain comprises at least one substitution selected from the group consisting of S239D/1332E, K326A/E333A, H268F/S324T/1332E, F243L/R292P/Y300L, S239D/H268F/S324T/1332E, S267E/H268F/S324T/1332E, K326A/1332E/E333A, S239D/K326A/E333A, S267E/I332E and G237X/S239D/E592E, wherein X is A, D, P, Q or S; wherein the amino acid residues are numbered according to EU numbering.

For detection of Fab²The use of the constructs in the context of this protease-stabilized IgG1 Fc resulted in two exemplary constructs (table 19). Both constructs (Her47 LHLF-LHL IgG1-2hDAA and Her47 LHL-LHLF IgG1-2hDAA) will Her47 Fab²IgG structure placed on the IgG1 constructed on the '2 hDAA' structure (IgG 1 containing the mutations P233-V234-A235- Δ G236 and S239D/K326A/E333A) ²In (1). Both constructs were readily expressed in transient CHO cell transfections where protein A purified protein was shown>80% of the expected molecular weight product. Exemplary analytical SEC data for Her47 LHLF-LHL IgG1-2hDAA showed 80% product at 10.30ml (FIG. 35). SDS-PAGE analysis and SEC peaks 8.47, 9.03, and 10.30ml (fig. 36) of the unreduced protein a purified protein demonstrated that peaks 8.47 and 9.03 contained higher molecular weight aggregates, while peak 10.30 contained the expected size product (approximately 250 kDa). SDS-PAGE analysis and SEC peaks 8.47, 9.03, and 10.30ml (fig. 37) of the reduced protein a purified protein demonstrated that all peaks 8.47, 9.03, and 10.30ml contained the expected size of the heavy and light chain products (approximately 80 and 50kDa, respectively). The intact monomeric (250kDa) product of Her47 LHL-LHLF IgG1-2hDAA was purified by SEC and incubated at 2, 4, 8 and 24 hours plus 24 hours in enzyme-free buffer as negativeThe time course of the sexual control (time 0, 2 hours, 4 hours, 8 hours, 24 hours incubation) was enzymatically digested with human MMP12 at ph 7.4. In the ELISA assay, all samples showed strong binding signals to human Her2, but no measurable binding to the control protein murine EpCAM (fig. 38A). In the CD47 binding ELISA, binding signal increased after 2, 4, 8, and 24 hours of incubation at 37 ℃ in the presence or absence of MMP12 enzyme, but no signal above background was observed at 0, 2, 4, 8, or 24 hours in the absence of MMP12 (fig. 38B).

Analysis of the 0 hour (undigested) and 2, 8 and 24 hour MMP12 digested samples confirmed by SDS-PAGE that the purified protein contained heavy and light chains of the expected size. MMP12 digestion over 2, 8, and 24 hours produced a 25kDa band (FIG. 38C), indicating that the linker peptide cleaved, but was conjugated to IgG²Her47LHL-LHL or IgG²Significantly less breakdown of the heavy chain was observed compared to Her47 LHL-LHLF (fig. 33), indicating that the '2 hDAA' mutation does stabilize the Fc region against MMP12 digestion.

Generation and analysis of IgG with alternative enzyme activation

Since the LHL and LHLF linkers are shown above to be readily cleaved by a variety of MMPs and cathepsins, they are present as IgG²Formal detection of six fabs²Construct type (table 20). These constructs use a series of linker designs (table 21) designed to be readily cleaved by several classes of enzymes associated with upregulation of activity in solid tumors and highly inflamed tissues, such as: enterokinase (EK), thrombin (Thr), tPA, granzyme B (GrB), uPA and ADAMTs-5 (A5). All 6 constructs were readily expressed by CHO cells and purified by ProA chromatography.

Purified proteins from clones Her47LHL-LHL-EK, Her47-LHL-LHL-Thr, Her 47-LHL-tPA, Her47-LHL-LHL-uPA, Her47-LHL-LHL-GrB and Her 47-LHL-A5 were all tested in titration ELISAs against human Her2 and CD47 targets (FIG. 39A, FIG. 39C, FIG. 39E, FIG. 39G, FIG. 39I, FIG. 39K). In all cases, these proteins showed low/background binding to CD47 at all concentrations tested (white bars), but concentration-dependent binding to Her2 (grey bars). Each protein was then also subjected to time course enzymatic digestion with MMP12 and ELISA binding to Her2 and CD47 targets (fig. 39B, 39D, 39F, 39H, 39J, 39L). These findings indicate that all proteins retained Her2 binding and exhibited increased CD47 binding activation throughout the time course of enzymatic activation. The functional activity of clones Her47 LHL-EK, Her 47-LHL-Thr, Her 47-LHL-tPA, Her 47-LHL-uPA, Her 47-LHL-GrB and Her 47-LHL-a 5 demonstrates that the Fab2 structure can be activated specifically for a given application using linker peptide sequences with multiple protease recognition sites. For example, in this case, the retention of MMP activation potential in the LHL linker in combination with any of the 6 different disease-associated enzyme cleavage motifs in the accompanying linker would allow tailoring to the environment in which MMPs and/or cathepsins are active, with increased activation potential when enterokinase, thrombin, tPA, granzyme B, uPA, ADAMTs-5 or other proteases are associated with the disease state.

In vivo multiple dose tolerance of Her47 molecule in NOD-SCID mice

In vivo multidose tolerance studies, IgG was used² Her47 LHL-LHL、IgG² Her47 LHL-LHLF、Fab²Her47 LHL-LHL and Fab²Her47 LHL-LHLF was administered four times each (intravenously) in NOD-SCID mice. IgG²Protein was administered at 14mg/kg on day 0 and 7mg/kg on days 5, 10 and 15. Fab²Protein was administered at 8mg/kg on day 0 and 4mg/kg on days 5, 10 and 15. All proteins were fully tolerated, with no clinical signs of toxicity and no weight loss in any individual animal (figure 40). These findings confirm and extend the Her 47-based Fab outlined above²Was found to be tolerant in Tg32 mice as a single dose. In the Tg32 mouse study, even a single dose of A-D5 CD47 IgG was not tolerated at 10 mg/kg. All four doses of IgG in this NOD-SCID mouse study² Her47 LHL-LHL、IgG² Her47 LHL-LHLF、Fab²Her47 LHL-LHL and Fab²Her47 LHL-LHLF is fully tolerated. This finding confirms the Her47 moleculeThe CD47 binding domain of (a) is not activated in circulation even after repeated dosing, as significant activation will induce the CD47 driven toxic signal observed for a-D5 IgG 1.

Additional 2-chain Her2CD3 Fab ²Generation and analysis of ` Single arm ` constructs

Additional Her2CD3 constructs were generated to test the ability of the new sequences to improve product uniformity and activity (table 22). All Her2CD3 constructs were expressed by CHO cells, purified from the supernatant by ProA, and then analyzed by SEC. These analyses showed that Fab²Her23 LHL-LHL-S and Fab²Her23 LHLF-LHL-S all showed a ratio to Fab²Her23 LHL-LHL and Fab²Her23 LHL-LHLF, both of which showed higher ratios of higher and lower molecular weight products (fig. 41C, 41D), had higher main product homogeneity (fig. 41A, 41B). These findings indicate that the exchange is in the construct Fab²Her23 LHL-LHL-S and Fab²The upwardly directed fab in Her23 LHLF-LHL-S renders the chain [ VL-CL-linker-VH-CH-Fc]Adding [ VH-CH-linker-VL-CL-Fc]Resulting in improved uniformity. This is in conjunction with Fab²Her23 LHL-LHL and Fab²Her23 LHL-LHLF in contrast, wherein both chains are [ VL-CL-linker-VL-CL-Fc]Adding [ VH-CH-linker-VH-CH-Fc]。

The control protein Fab was then also designed and expressed²mEpcam3 LHLF-LHL-S and Fab²mEpCam3 LHL-LHL-S (upper Fab contains the v domain of anti-murine EpCAM antibody G8.8) (Table 22). Isolation of cloned Fab by preparative SEC²Her23 LHL-LHL-S、Fab² Her23 LHLF-LHL-S、Fab²mEpcam3 LHLF-LHL-S and Fab ²Correct MW product of mEpCam3 LHL-S and analyzed in a Promega Jurkat cell-based CD3 linked reporter bioassay using MCF-7 or BT-474 as human Her2+ target cells (according to manufacturer' S instructions). Preliminary analysis using MCF-7 as target cells (expressing very low Her 2) demonstrated that both the positive control Her2-CD3 BITE protein and OKT3 IgG1 produced strong positive concentration-dependent CD3 activation signals. Cloned Fab treated with MMP12 for 2 hours²mEpcam3 LHLF-LHL-S and Fab²mEpcam3 LHL-LHL-S at any concentrationNo signal is generated in the lower (fig. 42A, B). In contrast, cloning Fab²Her23 LHLF-LHL-S (FIG. 42A) and Fab²Her23 LHL-LHL-S (FIG. 42B) showed no measurable signal at 0 hours in a similar concentration range as Her2-CD3 BITE, but the signal gradually increased at 2, 8 and 24 hours, but the maximum signal was higher in both cases than that obtained for the positive control.

All the above test samples and controls were assayed at 0.1 μ g/ml in a secondary assay using Her2 high expressing BT-474 cells as target cells (fig. 43A-C). The positive control proteins Her2-CD3 BITE and OKT3 IgG1 both induced a strong CD3 activation signal, while the negative control Fab²mEpCam3 LHLF-LHL-S and trastuzumab did not induce. Cloning of Fab ²Her23 LHLF-LHL-S (FIG. 43B) and Fab²Her23 LHL-LHL-S (FIG. 43C) showed no measurable signal at 0 hours, but the signal gradually increased at 2 hours and reached a maximum at 8 hours. Importantly, the signal at 24 hours was significantly reduced compared to 8 hours. This finding suggests that progressive enzymatic activity that can cleave both linkers (fig. 33) may lead to final separation of the upper and lower fabs. This may be a beneficial feature of molecules targeting CD3, as such molecules are preferentially activated in the highly proteolytic tumor environment, but are also gradually inactivated there, so that it would be desirable to minimize the risk of leakage of the molecule in active form into healthy tissue.

Her2-CD47 IgG²And Fab²In vitro measurement of molecular stability of proteins

Historically, engineered antibody formats often suffer from structural heterogeneity, leading to manufacturing problems. For detection of IgG²Her47 LHL-LHL and Fab²Her47 LHL-LHL protein stability, a series of in vitro measurements were performed:

forced oxidation-oxidation of exposed amino acid residues (such as tryptophan and methionine) is a common degradation pathway for mabs that affects their biological activity. In this study, forced oxidation with 0.5% H2O2 in PBS at room temperature for 2 hours was applied to IgG ²Her47 LHL-LHL and Fab²Her47 LHL-LHL protein. By increasing the oxidation form due to oxidationThe overall hydrophobicity of the antibody is changed either by polarity or by conformational changes, so the potential changes induced by forced oxidation are analyzed by Reverse Phase (RP) and Size Exclusion (SEC) chromatography. SEC analysis revealed no change in the proportion of monomer species in either test sample (IgG)²Her47 LHL-LHL and Fab²Her47 LHL-LHL both showed 99.5% and 97.4% monomer species before and after oxidation, respectively). In the RP analysis of intact antibodies, IgG was observed after forced oxidation with 0.5% H2O2 for intact (non-reducing) antibodies²Retention time reduction of Her47 LHL-LHL of 0.5 min and Fab²The retention time of Her47 LHL-LHL was reduced by 0.4 minutes. In RP analysis of subunits, for IgG²Her47 LHL-LHL, a 0.5 minute reduction in the retention time of the heavy chain and no shift in the retention time of the light chain was observed after forced oxidation. Reduced Fab in RP analysis²Her47 LHL-LHL showed different characteristics, 3 peaks were observed, representing the light chain, heavy chain and truncated hinge-Fc stub. After oxidation, shifts of 0.4-0.5 min were observed for the heavy chain and truncated hinge-Fc stub, but no shifts were observed for the light chain. Reduced IgG was observed after H2O2 treatment ²Her47 LHL-LHL and Fab²The change in retention time of Her47 LHL-LHL samples, indicating less oxidation of exposed amino acids (restricted to the Fc region of the protein).

Charge isomer analysis-charge heterogeneity analysis is important in the characterization of monoclonal antibodies because it provides important information about product quality and stability. Heterogeneity may be caused by enzymatic post-translational modifications (glycosylation, lysine truncation) or chemical modifications (oxidation or deamidation) during purification and storage. Analysis of the Charge isomer spectra of the provided test samples was performed by a commercial Charge isomer Assay (Charge variable Assay). IgG²Her47 LHL-LHL (FIG. 44A) and Fab²The charge isomer profiles of both Her47 LHL-LHL (FIG. 44B) showed a uniform profile with a major isoform (50-57% of the total), a major acidic isoform (40-48% of the total) and a minor basic isoform (approximately 3%).

Retention on HIC-Total hydrophobicity is protein fromAn indicator of association propensity, protein self-association propensity, may be a significant risk factor for aggregation and viscosity during bioprocessing. Proteins have hydrophobic 'plaques' on their surface, which are produced by the presence of side chains of hydrophobic or non-polar amino acids. Depending on their number, size and distribution, the resulting surface hydrophobicity will be a specific characteristic of each protein. HIC separates proteins based on differences in their surface hydrophobicity by utilizing reversible binding between the proteins and the hydrophobic surface of the HIC resin. IgG ²Her47 LHL-LHL and Fab²Her47 LHL-LHL exhibited HIC column retention times of 5.4 and 5.0 minutes, respectively. These values are towards a lower hydrophobic range compared to clinical monoclonal antibodies such as adalimumab (4.5 min), cetuximab (5.9 min), Brentuximab (6.3 min) and golimumab (8.1 min). Indeed, these values indicate that IgG was derived therefrom²Her47 LHL-LHL and Fab²anti-Her 2 trastuzumab similar aggregation propensity and stability of the Her2 binding domain in Her47 LHL-LHL (5.4 min retention).

Freeze-thaw stability analysis-protein instability during the freeze-thaw step is an indicator of difficulty in manufacturing and bioprocessing, as increased protein aggregation or fragmentation is a risk of reduced product quality. To evaluate the presence of Fab²This risk for structural proteins, IgG²Her47 LHL-LHL and Fab²Her47 LHL-LHL protein was subjected to 5 rounds of freeze thawing followed by SEC analysis after each round. These analyses showed IgG²Her47 LHL-LH L (FIG. 45A) and Fab²Her47 LHL-LH L (FIG. 45B) protein did not show any change in monodispersity by 5 rounds of freezing (no aggregation or decomposition products were observed).

Taken together, these findings indicate that IgG ²Her47 LHL-LHL and Fab²Her47 LHL-LHL proteins all have low risk of aggregation, low hydrophobicity, low charge heterogeneity and low propensity for oxidation.

Of the affinity of IgG variants for human Fc receptorsAnalysis of

Antibodies that target receptors on diseased cells must bind Fc γ receptors if they mediate ADCC and ADCP activity. To test whether these binding functions were retained in the Fab 2-based constructs, IgG was detected by surface plasmon resonance analysis²Her47 LHL-LHL and Fab²Binding affinity of Her47 LHL-LHL protein to all human and mouse Fc receptors. These analyses demonstrated that isotype control human IgG1 and IgG4 both exhibited the expected strong and weak binding affinities for all human Fc γ receptors (including high and low affinity variants of Fc γ RIIA and Fc γ RIIIA) (respectively) (table 23). Similarly, isotype control mice IgG2a and IgG1 exhibited the expected strong and weak binding affinities for mouse Fc γ RI, Fc γ RIII, and Fc γ RIV receptors (respectively). For IgG²Her47 LHL-LHL and Fab²Her47 LHL-LHL, the binding to each of the human and mouse Fc receptors tested was highly similar to that observed with isotype control human IgG 1. These data indicate that IgG constructed on IgG1 Fc was present in both humans and mice ²And Fab²Both proteins should be able to bind Fc receptors when bound on the surface of diseased cells.

Additional protein construct design

Additional protein constructs are envisaged (figure 46). The construct may contain: 1. a constant domain only in the 'upper Fab' position means that the activity of the 'lower Fab' is prevented from binding to its target, but may become active in the appropriate proteolytic environment. A pseudo 'non-binding' variable domain in an 'upper fab'. These pseudo variable domains will prove not to bind to any known target in vivo, so only the 'lower fab' exhibits potential drug target binding capacity, and only after proteolytic activation by one of the linker domains. The 'upper Fab' is replaced by a 'diabody' structure containing 4 variable domains. The diabody structure may or may not contain disulfide bonds, as found in 'DART' proteins. The diabody structure can facilitate the binding of 2 copies of the same target or 2 separate targets, whereinThe 'lower Fab' activity is prevented from binding to its target until activity is present in an appropriate proteolytic environment. In any of the constructs envisaged herein or shown above, Fab ²The structure may be free or fused to another functional structure such as an Fc fragment, a small domain or a half-life extending peptide such as an albumin binding moiety. They may also be chemically conjugated to small molecules, peptides or other proteins that mediate additional biological functions.

Cell proliferation assay for Her2CD47 protein using Her2 high BT-474 cells

Due to IgG²Her47 and Fab²Cell proliferation assays were performed because the Her2 binding domain of the upper Fab of the Her47 protein mediates inhibition of the kinase activity of this receptor. BT-474 cells were used for this assay as they are cell lines with known sensitive responses to Her2 inhibition. Trastuzumab, isotype control IgG1, IgG²Her47 LHL-LHL (FIG. 47A) and Fab²Her47 LHL-LHL (FIG. 47B) was applied to BT-474 cells over a 72 hour incubation period and cell proliferation was measured. Data are expressed as percent inhibition of cell growth (figure 47). These analyses indicate that while trastuzumab exhibits strong concentration-dependent inhibition of BT-474 cell proliferation, IgG²Her47 LHL-LHL (FIG. 47A) showed slightly lower potency and Fab²Her47 LHL-LHL (FIG. 47B) was again less potent (reflecting its monovalent binding capacity).

In vivo efficacy analysis of Her47 molecule in NOD-SCID mice bearing tumor xenografts (KYSE-410 model)

Trastuzumab, IgG in vivo multiple dose efficacy studies² Her47 LHL-LHL、IgG² Her47 LHL-LHLF、Fab²Her47 LHL-LHL and Fab²Her47 LHL-LHLF (Fab containing human IgG1 Fc)²Structure, as shown in figure 3B) were administered four times each (intravenously, on days 0, 5, 10, 15) in NOD-SCID mice bearing tumors generated by subcutaneous inoculation of the Her2 expressing esophageal cancer cell line KYSE-410. Once the cover is closed>125mm²The administration is started as soon as the tumor of (a) is established. IgG²Protein was administered at 14mg/kg on day 0 and 7mg/kg on days 5, 10 and 15. Fab²Protein was administered at 8mg/kg on day 0 and 4mg/kg on days 5, 10 and 15. Trastuzumab was administered at 8mg/kg on day 0 and 4mg/kg on days 5, 10 and 15. Tumor volume was measured by caliper measurement.

Trastuzumab (fig. 48A), IgG after 3 doses on day 11²Her47 LHL-LHLF (FIG. 48B), IgG²Her47 LHL-LHL (FIG. 48C) and Fab²Her47 LHL-LHLF (fig. 48D) all showed significant reduction in tumor growth compared to vehicle (two-factor ANOVA: p ═ 0.005, 0.019, and 0.003, respectively). In contrast, Fab ²Her47 LHL-LHL (fig. 48E) did not significantly reduce tumor growth by day 11 (two-factor ANOVA: p ═ 0.66). Importantly, Fab²Her47 LHL-LHL and Fab²The sequences of Her47 LHL-LHLF were identical (except for two point mutations in the LHLF linker which accelerate and amplify protease sensitivity relative to the LHL linker, as shown above), but resulted in significant differences in potency (fig. 48F). Furthermore, the data in fig. 47 demonstrate 1-arm Fab²The constructs resulted in weaker Her2 driven inhibition of cell proliferation than observed for trastuzumab, which contained the same peptide as in Fab²Her47 LHL-LHL and Fab²The same Her2 binding VH and VL domain sequences found in both Her47 LHL-LHLF. As a result, the following conclusions were made: 1. on day 11 for trastuzumab (FIG. 48A) and Fab²The roughly equivalent potency observed with Her47 LHL-LHLF (FIG. 48D) cannot be determined by Fab²High Her2 kinase activity driving in Her47 LHL-LHLF. Fab²The high potency of Her47 LHL-LHLF is therefore most likely driven by protease activation in the KYSE-410 tumor microenvironment, leading to CD47 blockade and innate immune engagement.

In summary, the findings outlined above thus demonstrate Fab²The structure allows for effective elimination of 'lower Fab' activity, exemplified by both CD47 and CD3 binding domains. The binding capacity of the ` upper fab ` domain is fully retained, but significant activity in the lower fab domain is only associated with the disease High activity-associated proteases such as MMPs and histoproteinases in diseased tissues such as tumors and fibrotic tissues are observed only after activation. Fab²Modulation of the linker sequence in the structure allows modulation of lower fab activation to be maximized in the disease microenvironment and avoids peripheral pool problems and toxicity as exemplified by the in vitro and in vivo performance of the Her47 molecule.

In vivo pharmacokinetics of Her47 molecules in NOD-SCID mice

IgG in vivo Multi-dose PK Studies² Her47 LHL-LHL、IgG² Her47 LHL-LHLF、Fab²Her47 LHL-LHL and Fab²Her47 LHL-LHLF will be administered once each (intravenously) in NOD-SCID mice. IgG²The protein will be administered at 14mg/kg, and Fab²The protein will be administered at 8 mg/kg. Blood samples were taken at 15 minutes, 30 minutes, 1 hour, 3 hours, 6 hours, and 24 hours, and serum antibody levels were measured using an anti-human IgG1 ELISA.

In vivo efficacy analysis of Her47 molecule in NOD-SCID mice bearing tumor xenografts (SKOV-3, JIMT-1 and NUGC-4 models)

IgG in vivo multidose efficacy studies² Her47 LHL-LHL、IgG² Her47 LHL-LHLF、Fab²Her47 LHL-LHL and Fab²Her47 LHL-LHLF will be administered four times each (intravenously) in NOD-SCID mice bearing tumors produced by subcutaneous inoculation of the cell lines SKOV-3, JIMT-1 and NUGC-4. IgG ²The protein will be administered at 14mg/kg on day 0 and 7mg/kg on days 5, 10 and 15. Fab²The protein will be administered at 8mg/kg on day 0 and 4mg/kg on days 5, 10 and 15. Trastuzumab will be administered at 8mg/kg on day 0 and 4mg/kg on days 5, 10 and 15. Tumor volume will be measured by caliper measurement.

In vivo analysis of tolerance and pharmacokinetics in cynomolgus monkeys

For detection of IgG²And/or Fab²Tolerance and pharmacokinetics with CD47 or CD3 as the lower Fab domain will be studied in cynomolgus monkeysA variety of exemplary molecules. For example, IgG²And/or Fab²Can be administered once, twice or three times (intravenously) each at a concentration of 2mg/kg or more. Blood samples were collected from each animal according to the bleeding schedule. Analysis of serum antibody concentrations will be measured to calculate PK and assess the risk of TMDD). To more broadly sample the effect of the administered protein, the whole hematology group will also be tested over a series of days after administration. These assays will measure reticulocyte, Red Blood Cell (RBC), hemoglobin, Mean Corpuscular Hemoglobin Concentration (MCHC), Mean Corpuscular Volume (MCV), leukocyte, monocyte, lymphocyte, basophil, eosinophil, and/or neutrophil levels.

Target co-engaged biosensor measurements

To detect activated versus Fab²The effect of binding affinity to both Her2 and CD47 (or CD3) will create a biosensor assay, such as a dynamic biosensor instrument, that can sample Her2 and CD47 (or CD3) binding on the same chip surface. In this assay, control antibody and IgG are combined²Or Fab²The proteins (undigested or activated with MMP or cathepsin, e.g. 2, 4, 8 or 24 hours) are applied individually or together at different densities on the sensor chip surface that has been differentially labelled with purified Her2 and CD47 (or CD3) ectodomain proteins. The affinity to Her2 and CD47 (or CD3) will be measured to determine the effect of multivalent interactions on functional affinity to both targets alone and on the same surface.

While the present invention has been described with reference to preferred or exemplary embodiments, those skilled in the art will recognize that various modifications and changes may be made to the present invention without departing from the spirit and scope of the invention, and that such modifications are explicitly contemplated herein. No limitations are intended to the specific embodiments disclosed herein or recited in the claims below, nor should any limitations be inferred.

No limitations are intended to the specific embodiments disclosed herein or recited in the claims below, nor should any limitations be inferred. All documents, or portions of documents, cited herein, including but not limited to patents, patent applications, articles, books, and treatises, are hereby expressly incorporated by reference in their entirety for any purpose. In the event that one or more of the incorporated documents or portions of documents define terms that conflict with the definitions of terms in this application, the definitions appearing in this application control. However, any references, articles, publications, patents, patent publications and patent applications cited herein are not, and should not be taken as, an acknowledgment or any form of suggestion that they form part of the common general knowledge in any country in the world.

Numbered embodiments

While having the appended claims, the present disclosure sets forth the following numbered embodiments:

1. a protein comprising a first portion and a second portion and a peptide linker between the first portion and the second portion,

Wherein the peptide linker is cleavable by a protease expressed in the diseased tissue;

wherein the second portion is capable of specifically binding to a molecule expressed in the diseased tissue; and is

Wherein binding of said second moiety to said molecule expressed in said diseased tissue is reduced or inhibited when said peptide linker is not cleaved.

2. The protein of embodiment 1, wherein the peptide linker is between about 5 and about 15 amino acids in length.

3. The protein of embodiment 1 or 2, wherein the peptide linker comprises or consists of the amino acid sequence of: SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 69, SEQ ID NO 70, SEQ ID NO 71, SEQ ID NO 72, SEQ ID NO 81, SEQ ID NO 82, SEQ ID NO 83, SEQ ID NO 84, SEQ ID NO 85, SEQ ID NO 86 or SEQ ID NO 87.

4. The protein of any one of embodiments 1-3, wherein the protease is human Matrix Metalloproteinase (MMP), human cathepsin, human enterokinase, human thrombin, human tPA, human granzyme B, human uPA, or human ADAMTs-5.

5. The protein of any one of embodiments 1-4, wherein the peptide linker comprises a human MMP cleavage site, a human cathepsin, a human enterokinase, human thrombin, human tPA, human granzyme B, human uPA, or a human ADAMTS-5 cleavage site.

6. The protein of any one of embodiments 1-5, wherein the human MMP is MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, MMP-12, MMP-13, or MMP 14.

7. The protein of any one of embodiments 1-6, wherein the level or activity of said human MMPs is increased in said diseased tissue compared to the level or activity of said human MMPs in non-diseased tissue.

8. The protein of any one of embodiments 1-5, wherein said human cathepsin is cathepsin A, cathepsin C, cathepsin D, cathepsin G, cathepsin L or cathepsin K.

9. The protein of any one of embodiments 1-5 and 8, wherein the level or activity of the human cathepsin in the diseased tissue is increased as compared to the level or activity of the human cathepsin in non-diseased tissue.

10. The protein of any one of embodiments 1-9, wherein the first portion comprises an antibody, an antigen-binding portion of an antibody, or an extracellular domain of a receptor.

11. The protein of embodiment 10, wherein the first portion is a Fab, single chain Fab, VH domain, VL domain, immunoglobulin neo-antigen receptor (IgNAR), single chain variable fragment (scFv), diabody, or T cell receptor domain.

12. The protein of any one of embodiments 1-11, wherein the first portion specifically binds to a molecule expressed in a diseased tissue.

13. The protein of any one of embodiments 1-12, wherein the first portion specifically binds to a first molecule expressed in a diseased tissue and the second portion is capable of specifically binding to a second molecule expressed in a diseased tissue, wherein the first molecule expressed in a diseased tissue and the second molecule expressed in a diseased tissue are different molecules.

14. The protein of embodiment 13, wherein said first molecule expressed in a diseased tissue and said second molecule expressed in a diseased tissue are expressed by the same cell.

15. The protein of embodiment 13, wherein said first molecule expressed in a diseased tissue and said second molecule expressed in a diseased tissue are expressed by different cells.

16. The protein of embodiment 13, wherein the first molecule expressed in the diseased tissue and/or the second molecule expressed in the diseased tissue is expressed on the cell surface.

17. The protein of embodiment 13, wherein said first molecule expressed in a diseased tissue and/or said second molecule expressed in a diseased tissue is a soluble molecule.

18. The protein of any one of embodiments 1-17, wherein the first portion specifically binds to human EGFR, human HER2, human HER3, human CD105, human C-KIT, human PD1, human PD-L1, human PSMA, human EpCAM, human Trop2, human EphA2, human CD20, human BCMA, human GITR, human OX40, human CSF1R, human Lag3, or human cMET.

19. The protein of any one of embodiments 1-17, wherein the second moiety specifically binds to a molecule expressed by a human immune cell.

20. The protein of embodiment 19, wherein the molecule expressed by a human immune cell is human CD3, human CD16A, human CD16B, human CD28, human CD89, human CTLA4, human NKG2D, human sirpa, human SIRP γ, human PD1, human lang 3, human 4-1BB, human OX40, or human GITR.

21. The protein of any one of embodiments 1-20, wherein said first portion comprises a heavy chain Variable (VH) region and a light chain Variable (VL) region.

22. The protein of any one of embodiments 1-21, wherein said first portion comprises an immunoglobulin constant region or a portion of an immunoglobulin constant region.

23. The protein of embodiment 22, wherein the immunoglobulin constant region is an IgG, IgE, IgM, IgD, IgA, or IgY.

24. The protein of embodiment 22, wherein the immunoglobulin constant region is IgG1, IgG2, IgG3, IgG4, IgA1, or IgA 2.

25. The protein of embodiment 22, wherein the immunoglobulin constant region is immunologically inert.

26. The protein of embodiment 22, wherein the immunoglobulin constant region is a wild-type human IgG4 constant region, a human IgG4 constant region comprising amino acid substitution S228P, a wild-type human IgG1 constant region, a human IgG1 constant region comprising amino acid substitutions L234A and L235A, a human IgG1 constant region comprising amino acid substitutions L234A, L235A, and G237A, a human IgG1 constant region comprising amino acid substitutions L234A, L235A, G237A, and P331S, or a wild-type human IgG2 constant region.

27. The protein of any one of embodiments 1-26, wherein the second portion comprises an antibody, an antigen-binding portion of an antibody, or an extracellular domain of a receptor.

28. The protein of embodiment 27, wherein the second portion is a Fab, single chain Fab, VH domain, VL domain, immunoglobulin neo-antigen receptor (IgNAR), single chain variable fragment (scFv), or T cell receptor domain.

29. The protein of any one of embodiments 1-28, wherein the second portion specifically binds human CD 47.

30. The protein of any one of embodiments 1-28, wherein the second moiety specifically binds to human CD3 or human PD-L1.

31. The protein of any one of embodiments 1-30, wherein said second portion comprises a heavy chain Variable (VH) region and a light chain Variable (VL) region.

32. The protein of any one of embodiments 1-31, wherein said second portion comprises an immunoglobulin constant region or a portion of an immunoglobulin constant region.

33. The protein of embodiment 32, wherein the immunoglobulin constant region is an IgG, IgE, IgM, IgD, IgA, or IgY.

34. The protein of embodiment 32, wherein the immunoglobulin constant region is IgG1, IgG2, IgG3, IgG4, IgA1, or IgA 2.

35. The protein of embodiment 32, wherein the immunoglobulin constant region is immunologically inert.

36. The protein of embodiment 32, wherein the immunoglobulin constant region is a wild-type human IgG4 constant region, a human IgG4 constant region comprising amino acid substitution S228P, a wild-type human IgG1 constant region, a human IgG1 constant region comprising amino acid substitutions L234A and L235A, a human IgG1 constant region comprising amino acid substitutions L234A, L235A, and G237A, a human IgG1 constant region comprising amino acid substitutions L234A, L235A, G237A, and P331S, or a wild-type human IgG2 constant region.

37. The protein of embodiment 1, wherein the protein has one immune effector function or two, three, or more immune effector functions.

38. The protein of embodiment 37, wherein said immune effector function is ADCC, CDC or ADCP.

39. The protein of any one of embodiments 1-38, wherein said first portion prevents or reduces specific binding of said second portion to said molecule expressed in said diseased tissue.

40. The protein of any one of embodiments 1-39, wherein said peptide linker is cleaved near or within said diseased tissue.

41. The protein of any one of embodiments 1-40, wherein the peptide linker is cleaved near or inside the diseased tissue, wherein the first portion is dissociated from the second portion near or inside the diseased tissue, and wherein the second portion specifically binds to a molecule expressed in the diseased tissue near or inside the diseased tissue.

42. The protein of any one of embodiments 1-41, wherein the diseased tissue is a tumor or an inflamed tissue.

43. The protein of embodiment 1, wherein said first portion specifically binds human cMET, wherein said second portion specifically binds human CD47, and wherein said protein comprises a first polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID No. 16 and a second polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID No. 17.

44. The protein of embodiment 1, wherein said first portion specifically binds to human HER2, wherein said second portion specifically binds to human CD3, and wherein said protein comprises a first polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID No. 26 and a second polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID No. 27.

45. The protein of embodiment 1, wherein said first portion specifically binds to human HER2, wherein said second portion specifically binds to human CD47, and wherein said protein comprises a first polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO:34 and a second polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO: 35.

46. The protein of embodiment 1, wherein said first portion specifically binds human cMET, wherein said second portion specifically binds human CD47, and wherein said protein comprises a first polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO:36 and a second polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO: 37.

47. The protein of embodiment 1, wherein said first portion specifically binds to human Her2, wherein said second portion specifically binds to human CD3, and wherein said protein comprises a first polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO:38 and a second polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO: 39.

48. The protein of embodiment 1, wherein said first portion specifically binds to human Her2, wherein said second portion specifically binds to human CD3, and wherein said protein comprises a first polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO:40 and a second polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO: 41.

49. The protein of embodiment 1, wherein said first portion specifically binds to human Her2, wherein said second portion specifically binds to human CD47, and wherein said protein comprises a first polypeptide chain and a second polypeptide chain, wherein:

(h) The first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:88 and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 89; or

(o) said first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:44 and said second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 97.

50. The protein of embodiment 1, wherein said first portion specifically binds to human Her2, wherein said second portion specifically binds to human CD3, and wherein said protein comprises a first polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO:73 and a second polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO: 74.

51. The protein of embodiment 1, wherein said first portion specifically binds to human cMET, wherein said second portion specifically binds to human cMET, and wherein said protein comprises a first polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO:75 and a second polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO: 76.

52. The protein of embodiment 1, wherein said first portion specifically binds to human Her2, wherein said second portion specifically binds to human CD3, and wherein said protein comprises a first polypeptide chain and a second polypeptide chain, wherein:

(d) The first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID No. 104 and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID No. 105.

53. An immunoconjugate comprising a protein of any one of embodiments 1-52 linked to a therapeutic agent.

54. The immunoconjugate of embodiment 53, wherein the therapeutic agent is a cytotoxin, a radioisotope, a chemotherapeutic agent, an immunomodulator, an anti-angiogenic agent, an antiproliferative agent, a pro-apoptotic agent, a cytostatic enzyme, a cytolytic enzyme, a therapeutic nucleic acid, an anti-angiogenic agent, an antiproliferative agent, or a pro-apoptotic agent.

55. A pharmaceutical composition comprising a protein according to any one of embodiments 1-52 or an immunoconjugate according to embodiment 53 or 54, and a pharmaceutically acceptable carrier, diluent, or excipient.

56. A nucleic acid molecule encoding a protein or a portion thereof according to any one of embodiments 1-52.

57. A nucleic acid molecule encoding a first polypeptide chain, a second polypeptide chain, or both a first polypeptide chain and a second polypeptide chain of a protein according to any one of embodiments 43-52.

58. An expression vector comprising the nucleic acid molecule of embodiment 56 or 57.

59. A recombinant host cell comprising the nucleic acid molecule of embodiment 56 or 57 or the expression vector of embodiment 58.

60. A method of producing a protein comprising:

culturing a recombinant host cell comprising the expression vector of embodiment 57 under conditions such that the nucleic acid molecule is expressed, thereby producing the protein; and

isolating the protein from the host cell or culture.

61. A method for enhancing an anti-cancer immune response in a subject comprising administering to the subject a therapeutically effective amount of a protein of any one of embodiments 1-52, an immunoconjugate of embodiment 53 or 54, or a pharmaceutical composition of embodiment 55.

62. A method of treating cancer, an autoimmune disease, an inflammatory disease, a cardiovascular disease, or a fibrotic disease in a subject comprising administering to the subject a therapeutically effective amount of a protein of any one of embodiments 1-52, an immunoconjugate of embodiment 53 or 54, or a pharmaceutical composition of embodiment 55.

63. The method of embodiment 62, wherein the cancer is gastrointestinal stromal cancer (GIST), pancreatic cancer, skin cancer, melanoma, breast cancer, lung cancer, bronchial cancer, colorectal cancer, prostate cancer, gastric cancer, ovarian cancer, bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, cancer of the oral cavity or pharynx, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small or appendiceal cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma, or cancer of blood tissue.

64. The method of embodiment 62, wherein the autoimmune or inflammatory disease is arthritis, asthma, multiple sclerosis, psoriasis, crohn's disease, inflammatory bowel disease, lupus, graves ' disease, hashimoto's thyroiditis, or ankylosing spondylitis.

65. The method of embodiment 62, wherein the cardiovascular disease is coronary heart disease, or atherosclerosis or stroke.

66. The method of embodiment 62, wherein the fibrotic disease is myocardial infarction, angina, osteoarthritis, pulmonary fibrosis, cystic fibrosis, bronchitis, or asthma.

67. The protein of any one of embodiments 1-52, the immunoconjugate of embodiment 53 or 54, or the pharmaceutical composition of embodiment 55, for use in treating cancer, an autoimmune disease, an inflammatory disease, a cardiovascular disease, or a fibrotic disease.

68. The protein or pharmaceutical composition for use according to embodiment 67, wherein the cancer is gastrointestinal stromal cancer (GIST), pancreatic cancer, skin cancer, melanoma, breast cancer, lung cancer, bronchial cancer, colorectal cancer, prostate cancer, gastric cancer, ovarian cancer, bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, cancer of the oral cavity or pharynx, liver cancer, kidney cancer, testicular cancer, biliary, small intestine or appendix cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma, or cancer of blood tissue.

69. The protein or pharmaceutical composition for use according to embodiment 67, wherein the autoimmune or inflammatory disease is arthritis, asthma, multiple sclerosis, psoriasis, Crohn's disease, inflammatory bowel disease, lupus, Graves ' disease, Hashimoto's thyroiditis or ankylosing spondylitis.

70. The protein or pharmaceutical composition for use according to embodiment 67, wherein the cardiovascular disease is coronary heart disease, atherosclerosis or stroke.

71. The protein or pharmaceutical composition for use according to embodiment 67, wherein the fibrotic disease is myocardial infarction, angina, osteoarthritis, pulmonary fibrosis, cystic fibrosis, bronchitis, or asthma.

72. The protein of any one of embodiments 1-52, the immunoconjugate of embodiment 53 or 54, or the pharmaceutical composition of embodiment 55, for use as a medicament.

Table 1 peptide linker sequences.

The underlined linker peptide sequence is germline for human IgG 1.

The bold, non-underlined peptide sequence (LG) is a mutation that places the rapidly digested MMP peptide substrate sequence 'PLGL' (SEQ ID NO: 12).

TABLE 2 protein clone number, name (ID) and expression profile observed.

Protein a column affinity purified total protein

Based on amino acid sequence only

Not performing ND-

TABLE 3 sequence of bispecific protein binding to cMet and CD 47.

TABLE 4 sequences of bispecific proteins that bind to Her2 and CD 3.

TABLE 5 sequences of bispecific proteins that bind to Her2 and CD 47.

TABLE 6 sequences of bispecific proteins binding to cMET and CD 47.

TABLE 7 sequences of bispecific proteins that bind to Her2 and CD 3.

TABLE 8 sequences of bispecific proteins that bind to Her2 and CD 3.

TABLE 9 sequences of bispecific proteins that bind to Her2 and CD 47.

TABLE 10 examples of amino acid sequences of immunoglobulin Fc regions.

Human IgG4 wild type

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQED PEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:56)

Human IgG4(S228P)

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQED PEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:57)

Human IgG1 wild type

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:58)

Human IgG1-3M

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:59)

Human IgG2 wild type

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:60)

Human IgG1 wild-type "REEM" allotype

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:61)

Human IgG1-3M "REEM" allotype

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:62)

Table 11 examples of amino acid sequences of CD47 proteins.

Human CD47 sequence

MWPLVAALLLGSACCGSAQLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKSTVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRVVSWFSPNENILIVIFPIFAILLFWGQFGIKTLKYRSGGMDEKTIALLVAGLVITVIVIVGAILFVPGEYSLKNATGLGLIVTSTGILILLHYYVFSTAIGLTSFVIAILVIQVIAYILAVVGLSLCIAACIPMHGPLLISGLSILALAQLLGLVYMKFVASNQKTIQPPRKAVEEPLNAFKESKGMMNDE(SEQ ID NO:63)

Cynomolgus monkey CD47 sequence

MWPLVAALLLGSACCGSAQLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKSTAPANFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRVVSWFSPNENILIVIFPIFAILLFWGQFGIKTLKYRSGGMDEKTIALLVAGLMITVIVIVGAILFVPGEYSLKNATGLGLIVTSTGILILLHYYVFSTAIGLTSFVIAILVIQVIAYILAVVGLSLCIAACIPMHGPLLISGLSILALAQLLGLVYMKFVASNQKTIQPPRKAVEEPLNAFKESKGMMNDE(SEQ ID NO:64)

Table 12. examples of cMET protein amino acid sequences.

Human cMET sequence

Cynomolgus monkey cMET sequence

TABLE 13 sequences of bispecific proteins that bind to Her2 and CD 3.

Table 14 sequence of Fab 2-based proteins binding to cMET and cMET.

Table 15. examples of Her2 protein amino acid sequences.

Human Her2(erbB-2) sequence

Cynomolgus monkey Her2(erbB2) sequence

MAESPASAFRDSLRKSVRTAAGNPGVPELGGTHPGLREEREKVKLGVATPRLVGMQLEGASWERACSQSQEEEEVEEEGCLRKYKNEVVELRFPSIGTGETRGAPWAAVRPFPRGSFRRRAPGPHPSPHPAPHALPAGSSRSHGAGAAVSTMELAAWYRWGLLLALLPPGATGTQVCTGTDMKLRLPASPETHLDMLRHLYQGCQVVQGNLELTYLPTNASLSFLQDIQEVQGYVLIAHNQVRQVPLQRLRIVRGTQLFEDNYALAVLDNGDPLNNTTPVTGASPGGLRELQLRSLTEILKGGVLIQRNPQLCYQDTILWKDIFHKNNQLALTLIDTNRSRACHPCSPVCKGSRCWGESSEDCQSLTRTVCAGGCARCKGPLPTDCCHEQCAAGCTGPKHSDCLACLHFNHSGICELHCPALVTYNTDTFESMPNPEGRYTFGASCVTACPYNYLSTDVGSCTLVCPLHNQEVTAEDGTQRCEKCSKPCARVCYGLGMEHLREVRAVTSANIQEFAGCKKIFGSLAFLPESFDGDPASNTAPLQPEQLRVFETLEEITGYLYISAWPDSLPDLSVLQNLQVIRGRILHNGAYSLTLQGLGISWLGLRSLRELGSGLALIHHNTRLCFVHTVPWDQLFRNPHQALLHTANRPEDECVGEGLACHQLCARGHCWGPGPTQCVNCSQFLRGQECVEECRVLQGLPREYVNARHCLPCHPECQPQNGSVTCFGPEADQCVACAHYKDPPFCVARCPSGVKPDLSYMPIWKFPDEEGTCQSCPINCTHSCVDLDDKGCPAEQRASPLTSIISAVVGILLVVVLGVVFGILIKRRQQKIRKYTMRRLLQETELVEPLTPSGAMPNQAQMRILKETELRKVKVLGSGAFGTVYKGIWIPDGENVKIPVAIKVLRENTSPKANKEILDEAYVMAGVGSPYVSRLLGICLTSTVQLVTQLMPYGCLLDHVRENRGRLGSQDLLNWCMQIAKGMSYLEDVRLVHRDLAARNVLVKSPNHVKITDFGLARLLDIDETEYHADGGKVPIKWMALESILRRRFTHQSDVWSYGVTVWELMTFGAKPYDGIPAREIPDLLEKGERLPQPPICTIDVYMIMVKCWMIDSECRPRFRELVSEFSRMARDPQRFVVIQNEDLGPASPLDSTFYRSLLEDDDMGDLVDAEEYLVPQQGFFCPDPAPGTGGMVHHRHRSSSTRSGGGDLTLGLEPSEEEAPRSPRAPSEGTGSDVFDGDLGMGAAKGLQSLPAHDPSPLQRYSEDPTVPLPSETDGYVAPLTCSPQPEYVNQPDVRPQPPSPQEGPLSPARPTGATLERPKTLSPGKNGVVKDVFAFGGAVENPEYLAPRGGAAPQPHLPPAFSPAFDNLYYWDQDPSERGAPPSTFKGTPTAENPEYLGLDVPV(SEQ ID NO:78)

Table 16. examples of amino acid sequences of the epsilon domain of CD 3.

Human CD3 epsilon sequence

MQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI(SEQ ID NO:79)

Cynomolgus monkey CD3 epsilon sequence

MQSGTRWRVLGLCLLSIGVWGQDGNEEMGSITQTPYQVSISGTTVILTCSQHLGSEAQWQHNGKNKEDSGDRLFLPEFSEMEQSGYYVCYPRGSNPEDASHHLYLKARVCENCMEMDVMAVATIVIVDICITLGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQQDLYSGLNQRRI(SEQ ID NO:80)

TABLE 17 Biacore binding values of control anti-Her 2 and anti-CD 47 antibodies

TABLE 18 in IgG²Biacore binding to Her2 and CD47 during MMP12 activation of Her47-LHL-LHLF proteins.

N/A is not applicable. No binding signal was observed.

TABLE 19 sequences of bispecific proteins that bind to Her2 and CD 47.

TABLE 20 sequences of bispecific proteins that bind to Her2 and CD 47.

TABLE 21 sequence of linker peptide containing protease cleavage motif.

Table 22 sequences of bispecific proteins binding to Her2 and CD3 or to EpCAM and CD 3.

Table 23. binding affinity to human and murine Fc receptors as determined by Biacore.

Not completed ND

NB — no binding.

Sequence listing

<110> ULTRAHUMAN SIX Limited (ULTRAHUMAN SIX LIMITED)

<120> activatable protein construct and use thereof

<130> ULSL-002/04WO 332949-2010

<150> GB 2001196.1

<151> 2020-01-28

<150> GB 1917678.3

<151> 2019-12-04

<150> GB 1910254.0

<151> 2019-07-17

<150> GB 1906685.1

<151> 2019-05-13

<160> 112

<170> PatentIn 3.5 edition

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<213> Artificial Sequence (Artificial Sequence)

<220>

<223> peptide linker

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Gly Pro Ala Pro Glu Leu Leu

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<213> Artificial Sequence (Artificial Sequence)

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Gly Pro Ala Pro Glu Leu Leu Gly Gly Gly Ser

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<213> Artificial Sequence (Artificial Sequence)

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Gly Pro Ala Pro Leu Gly Leu Gly Gly Gly Ser

1 5 10

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<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Gly Ser

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Pro Pro Cys Pro Ala Pro Leu Gly Leu Gly Gly Gly Ser

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<220>

<223> Fab2 CMET/CD47 heavy chain

<400> 6

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Thr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gln Glu Ile Thr Thr Glu Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Gly Gly Gly

210 215 220

Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro

225 230 235 240

Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr

245 250 255

Asn Tyr Tyr Ile Phe Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu

260 265 270

Trp Met Gly Ile Ile Asn Pro Val Asp Gly Asp Thr Asn Tyr Asn Pro

275 280 285

Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr

290 295 300

Ala Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr

305 310 315 320

Tyr Cys Ala Arg Gly Gly Tyr Thr Met Asp Arg Trp Gly Gln Gly Thr

325 330 335

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

340 345 350

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

355 360 365

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

370 375 380

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

385 390 395 400

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

405 410 415

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

420 425 430

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Gly

435 440 445

Gly Gly Gly Ser Gly Gly Gly Gly Ser Cys Pro Pro Cys Pro Ala Pro

450 455 460

Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

465 470 475 480

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

485 490 495

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

500 505 510

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

515 520 525

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

530 535 540

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

545 550 555 560

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

565 570 575

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys

580 585 590

Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

595 600 605

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

610 615 620

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

625 630 635 640

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

645 650 655

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

660 665 670

Leu Ser Leu Ser Pro Gly

675

<210> 7

<211> 673

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Fab2 CMET/CD47 light chain

<400> 7

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

Ala Asn Ser Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

35 40 45

Arg Leu Leu Ile Tyr Arg Gly Ser Thr Arg Glu Ser Gly Ile Pro Asp

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Lys

85 90 95

Ser Glu Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Gly Gly Gly Gly Ser Asp

210 215 220

Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly Glu

225 230 235 240

Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser Asn

245 250 255

Gly Tyr Asn Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro

260 265 270

Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Leu Ser Gly Val Pro Asp

275 280 285

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser

290 295 300

Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Asn Thr

305 310 315 320

His Thr Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg

325 330 335

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

340 345 350

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

355 360 365

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

370 375 380

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

385 390 395 400

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

405 410 415

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

420 425 430

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Gly Gly Gly Gly Ser Gly

435 440 445

Gly Gly Gly Ser Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

450 455 460

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

465 470 475 480

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

485 490 495

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

500 505 510

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

515 520 525

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

530 535 540

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

545 550 555 560

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

565 570 575

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

580 585 590

Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

595 600 605

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

610 615 620

Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp

625 630 635 640

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

645 650 655

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

660 665 670

Gly

<210> 8

<211> 683

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Fab2 CMET/CD47 heavy chain

<400> 8

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Thr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gln Glu Ile Thr Thr Glu Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Gly Gly Gly

210 215 220

Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala

225 230 235 240

Glu Val Lys Lys Pro Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser

245 250 255

Gly Tyr Thr Phe Thr Asn Tyr Tyr Ile Phe Trp Val Arg Gln Met Pro

260 265 270

Gly Lys Gly Leu Glu Trp Met Gly Ile Ile Asn Pro Val Asp Gly Asp

275 280 285

Thr Asn Tyr Asn Pro Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp

290 295 300

Lys Ser Ile Ser Thr Ala Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser

305 310 315 320

Asp Thr Ala Met Tyr Tyr Cys Ala Arg Gly Gly Tyr Thr Met Asp Arg

325 330 335

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly

340 345 350

Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly

355 360 365

Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

370 375 380

Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

385 390 395 400

Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

405 410 415

Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val

420 425 430

Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys

435 440 445

Ser Cys Asp Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Cys Pro

450 455 460

Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe

465 470 475 480

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

485 490 495

Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe

500 505 510

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

515 520 525

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

530 535 540

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

545 550 555 560

Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

565 570 575

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

580 585 590

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly

595 600 605

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

610 615 620

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

625 630 635 640

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

645 650 655

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

660 665 670

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

675 680

<210> 9

<211> 678

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Fab2 CMET/CD47 light chain

<400> 9

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

Ala Asn Ser Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

35 40 45

Arg Leu Leu Ile Tyr Arg Gly Ser Thr Arg Glu Ser Gly Ile Pro Asp

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Lys

85 90 95

Ser Glu Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Gly Gly Gly Gly Ser Gly

210 215 220

Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro

225 230 235 240

Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser

245 250 255

Leu Leu His Ser Asn Gly Tyr Asn Tyr Leu His Trp Tyr Leu Gln Lys

260 265 270

Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Leu

275 280 285

Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe

290 295 300

Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr

305 310 315 320

Cys Phe Gln Asn Thr His Thr Pro Arg Thr Phe Gly Gly Gly Thr Lys

325 330 335

Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro

340 345 350

Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu

355 360 365

Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp

370 375 380

Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp

385 390 395 400

Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys

405 410 415

Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln

420 425 430

Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Gly

435 440 445

Gly Gly Gly Ser Gly Gly Gly Gly Ser Cys Pro Pro Cys Pro Ala Pro

450 455 460

Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

465 470 475 480

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

485 490 495

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

500 505 510

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

515 520 525

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

530 535 540

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

545 550 555 560

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

565 570 575

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys

580 585 590

Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp

595 600 605

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

610 615 620

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser

625 630 635 640

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

645 650 655

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

660 665 670

Leu Ser Leu Ser Pro Gly

675

<210> 10

<211> 688

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Fab2 CMET/CD47 heavy chain

<400> 10

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Thr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gln Glu Ile Thr Thr Glu Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Gly Gly Gly

210 215 220

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu

225 230 235 240

Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu Ser Leu Lys Ile

245 250 255

Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asn Tyr Tyr Ile Phe Trp

260 265 270

Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met Gly Ile Ile Asn

275 280 285

Pro Val Asp Gly Asp Thr Asn Tyr Asn Pro Ser Phe Gln Gly Gln Val

290 295 300

Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr Leu Gln Trp Ser

305 310 315 320

Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys Ala Arg Gly Gly

325 330 335

Tyr Thr Met Asp Arg Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

340 345 350

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

355 360 365

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

370 375 380

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

385 390 395 400

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

405 410 415

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

420 425 430

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

435 440 445

Lys Val Glu Pro Lys Ser Cys Asp Lys Gly Gly Gly Gly Ser Gly Gly

450 455 460

Gly Gly Ser Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

465 470 475 480

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

485 490 495

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

500 505 510

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

515 520 525

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

530 535 540

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

545 550 555 560

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

565 570 575

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

580 585 590

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp

595 600 605

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

610 615 620

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

625 630 635 640

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

645 650 655

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

660 665 670

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

675 680 685

<210> 11

<211> 680

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Fab2 CMET/CD47 light chain

<400> 11

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

Ala Asn Ser Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

35 40 45

Arg Leu Leu Ile Tyr Arg Gly Ser Thr Arg Glu Ser Gly Ile Pro Asp

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Lys

85 90 95

Ser Glu Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Gly Gly Gly Gly Ser Gly

210 215 220

Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser

225 230 235 240

Pro Leu Ser Leu Pro Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys

245 250 255

Arg Ser Ser Gln Ser Leu Leu His Ser Asn Gly Tyr Asn Tyr Leu His

260 265 270

Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Lys

275 280 285

Val Ser Asn Arg Leu Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly

290 295 300

Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp

305 310 315 320

Val Gly Val Tyr Tyr Cys Phe Gln Asn Thr His Thr Pro Arg Thr Phe

325 330 335

Gly Gly Gly Thr Lys Val Arg Thr Val Ala Ala Pro Ser Val Phe Ile

340 345 350

Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val

355 360 365

Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys

370 375 380

Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu

385 390 395 400

Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu

405 410 415

Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr

420 425 430

His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu

435 440 445

Cys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Cys Pro Pro Cys Pro

450 455 460

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

465 470 475 480

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

485 490 495

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

500 505 510

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

515 520 525

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

530 535 540

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

545 550 555 560

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

565 570 575

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu

580 585 590

Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro

595 600 605

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

610 615 620

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

625 630 635 640

Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

645 650 655

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

660 665 670

Lys Ser Leu Ser Leu Ser Pro Gly

675 680

<210> 12

<211> 4

<212> PRT

<213> unknown

<220>

<223> MMP peptide substrate sequence

<400> 12

Pro Leu Gly Leu

<210> 13

<211> 675

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Fab2 CMET/CD47 light chain

<400> 13

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

Ala Asn Ser Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

35 40 45

Arg Leu Leu Ile Tyr Arg Gly Ser Thr Arg Glu Ser Gly Ile Pro Asp

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Lys

85 90 95

Ser Glu Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Gly Pro Ala Pro Glu Leu

210 215 220

Leu Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro

225 230 235 240

Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His

245 250 255

Ser Asn Gly Tyr Asn Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln

260 265 270

Ser Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Leu Ser Gly Val

275 280 285

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys

290 295 300

Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln

305 310 315 320

Asn Thr His Thr Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

325 330 335

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

340 345 350

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

355 360 365

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

370 375 380

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

385 390 395 400

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

405 410 415

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

420 425 430

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Gly Gly Gly Gly

435 440 445

Ser Gly Gly Gly Gly Ser Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

450 455 460

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

465 470 475 480

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

485 490 495

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

500 505 510

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

515 520 525

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

530 535 540

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

545 550 555 560

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

565 570 575

Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val

580 585 590

Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

595 600 605

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

610 615 620

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr

625 630 635 640

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

645 650 655

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

660 665 670

Ser Pro Gly

675

<210> 14

<211> 680

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Fab2 CMET/CD47 heavy chain

<400> 14

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Thr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gln Glu Ile Thr Thr Glu Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Gly Pro Ala

210 215 220

Pro Glu Leu Leu Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys

225 230 235 240

Lys Pro Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Thr

245 250 255

Phe Thr Asn Tyr Tyr Ile Phe Trp Val Arg Gln Met Pro Gly Lys Gly

260 265 270

Leu Glu Trp Met Gly Ile Ile Asn Pro Val Asp Gly Asp Thr Asn Tyr

275 280 285

Asn Pro Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile

290 295 300

Ser Thr Ala Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala

305 310 315 320

Met Tyr Tyr Cys Ala Arg Gly Gly Tyr Thr Met Asp Arg Trp Gly Gln

325 330 335

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

340 345 350

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

355 360 365

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

370 375 380

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

385 390 395 400

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

405 410 415

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

420 425 430

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

435 440 445

Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Cys Pro Pro Cys Pro

450 455 460

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

465 470 475 480

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

485 490 495

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

500 505 510

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

515 520 525

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

530 535 540

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

545 550 555 560

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

565 570 575

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu

580 585 590

Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro

595 600 605

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

610 615 620

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

625 630 635 640

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

645 650 655

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

660 665 670

Lys Ser Leu Ser Leu Ser Pro Gly

675 680

<210> 15

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> peptide linker

<400> 15

Gly Gly Gly Gly Ser

1 5

<210> 16

<211> 684

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Fab2 CMET/CD47 heavy chain

<400> 16

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Thr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gln Glu Ile Thr Thr Glu Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Gly Pro Ala

210 215 220

Pro Glu Leu Leu Gly Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly

225 230 235 240

Ala Glu Val Lys Lys Pro Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly

245 250 255

Ser Gly Tyr Thr Phe Thr Asn Tyr Tyr Ile Phe Trp Val Arg Gln Met

260 265 270

Pro Gly Lys Gly Leu Glu Trp Met Gly Ile Ile Asn Pro Val Asp Gly

275 280 285

Asp Thr Asn Tyr Asn Pro Ser Phe Gln Gly Gln Val Thr Ile Ser Ala

290 295 300

Asp Lys Ser Ile Ser Thr Ala Tyr Leu Gln Trp Ser Ser Leu Lys Ala

305 310 315 320

Ser Asp Thr Ala Met Tyr Tyr Cys Ala Arg Gly Gly Tyr Thr Met Asp

325 330 335

Arg Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys

340 345 350

Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly

355 360 365

Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

370 375 380

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

385 390 395 400

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

405 410 415

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn

420 425 430

Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro

435 440 445

Lys Ser Cys Asp Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Cys

450 455 460

Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu

465 470 475 480

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

485 490 495

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys

500 505 510

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

515 520 525

Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu

530 535 540

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

545 550 555 560

Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

565 570 575

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

580 585 590

Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys

595 600 605

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

610 615 620

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

625 630 635 640

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

645 650 655

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

660 665 670

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

675 680

<210> 17

<211> 679

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Fab2 CMET/CD47 light chain

<400> 17

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

Ala Asn Ser Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

35 40 45

Arg Leu Leu Ile Tyr Arg Gly Ser Thr Arg Glu Ser Gly Ile Pro Asp

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Lys

85 90 95

Ser Glu Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Gly Pro Ala Pro Glu Leu

210 215 220

Leu Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu

225 230 235 240

Pro Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln

245 250 255

Ser Leu Leu His Ser Asn Gly Tyr Asn Tyr Leu His Trp Tyr Leu Gln

260 265 270

Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg

275 280 285

Leu Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp

290 295 300

Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr

305 310 315 320

Tyr Cys Phe Gln Asn Thr His Thr Pro Arg Thr Phe Gly Gly Gly Thr

325 330 335

Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe

340 345 350

Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys

355 360 365

Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val

370 375 380

Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln

385 390 395 400

Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser

405 410 415

Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His

420 425 430

Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

435 440 445

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Cys Pro Pro Cys Pro Ala

450 455 460

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

465 470 475 480

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

485 490 495

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

500 505 510

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

515 520 525

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

530 535 540

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

545 550 555 560

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

565 570 575

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr

580 585 590

Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser

595 600 605

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

610 615 620

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val

625 630 635 640

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

645 650 655

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

660 665 670

Ser Leu Ser Leu Ser Pro Gly

675

<210> 18

<211> 452

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Fab2 CMET/CD47 heavy chain

<400> 18

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu

1 5 10 15

Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Tyr Ile Phe Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile Asn Pro Val Asp Gly Asp Thr Asn Tyr Asn Pro Ser Phe

50 55 60

Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Tyr Thr Met Asp Arg Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

115 120 125

Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu

130 135 140

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

145 150 155 160

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser

165 170 175

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

180 185 190

Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr

195 200 205

Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Gly Gly Gly

210 215 220

Gly Ser Gly Gly Gly Gly Ser Cys Pro Pro Cys Pro Ala Pro Glu Leu

225 230 235 240

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

245 250 255

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

260 265 270

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

275 280 285

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

290 295 300

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

305 310 315 320

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala

325 330 335

Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

340 345 350

Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln

355 360 365

Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

370 375 380

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

385 390 395 400

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

405 410 415

Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser

420 425 430

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

435 440 445

Leu Ser Pro Gly

450

<210> 19

<211> 450

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Fab2 CMET/CD47 light chain

<400> 19

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser

20 25 30

Asn Gly Tyr Asn Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Leu Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Asn

85 90 95

Thr His Thr Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Gly Gly Gly Gly Ser

210 215 220

Gly Gly Gly Gly Ser Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly

450

<210> 20

<211> 688

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Fab2 Her2/CD3 heavy chain

<400> 20

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Gly

210 215 220

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Gln Ser

225 230 235 240

Gly Ala Glu Leu Ala Arg Pro Gly Ala Ser Val Lys Met Ser Cys Lys

245 250 255

Ala Ser Gly Tyr Thr Phe Thr Arg Tyr Thr Met His Trp Val Lys Gln

260 265 270

Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser Arg

275 280 285

Gly Tyr Thr Asn Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr

290 295 300

Thr Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr

305 310 315 320

Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp His

325 330 335

Tyr Cys Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser

340 345 350

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

355 360 365

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

370 375 380

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

385 390 395 400

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

405 410 415

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

420 425 430

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

435 440 445

Lys Val Glu Pro Lys Ser Cys Asp Lys Gly Gly Gly Gly Ser Gly Gly

450 455 460

Gly Gly Ser Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro

465 470 475 480

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

485 490 495

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

500 505 510

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

515 520 525

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

530 535 540

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

545 550 555 560

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

565 570 575

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

580 585 590

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp

595 600 605

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

610 615 620

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

625 630 635 640

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

645 650 655

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

660 665 670

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

675 680 685

<210> 21

<211> 668

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Fab2 Her2/CD3 light chain

<400> 21

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

210 215 220

Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly

225 230 235 240

Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met

245 250 255

Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr

260 265 270

Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala His Phe Arg Gly Ser

275 280 285

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Gly Met Glu Ala Glu

290 295 300

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Phe Thr

305 310 315 320

Phe Gly Ser Gly Thr Lys Leu Glu Ile Asn Arg Thr Val Ala Ala Pro

325 330 335

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

340 345 350

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

355 360 365

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

370 375 380

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

385 390 395 400

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

405 410 415

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

420 425 430

Asn Arg Gly Glu Cys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Cys

435 440 445

Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu

450 455 460

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

465 470 475 480

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys

485 490 495

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

500 505 510

Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu

515 520 525

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

530 535 540

Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

545 550 555 560

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

565 570 575

Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys

580 585 590

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

595 600 605

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

610 615 620

Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

625 630 635 640

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

645 650 655

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

660 665

<210> 22

<211> 683

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Fab2 Her2/CD3 heavy chain

<400> 22

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Gly

210 215 220

Gly Gly Gly Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala

225 230 235 240

Arg Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr

245 250 255

Phe Thr Arg Tyr Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly

260 265 270

Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr

275 280 285

Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser

290 295 300

Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala

305 310 315 320

Val Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr

325 330 335

Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly

340 345 350

Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly

355 360 365

Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

370 375 380

Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

385 390 395 400

Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

405 410 415

Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val

420 425 430

Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys

435 440 445

Ser Cys Asp Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Cys Pro

450 455 460

Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe

465 470 475 480

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

485 490 495

Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe

500 505 510

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

515 520 525

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

530 535 540

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

545 550 555 560

Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

565 570 575

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

580 585 590

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly

595 600 605

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

610 615 620

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

625 630 635 640

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

645 650 655

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

660 665 670

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

675 680

<210> 23

<211> 665

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Fab2 Her2/CD3 light chain

<400> 23

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys Gly Pro Ala Pro Glu Leu Leu Gln Ile Val

210 215 220

Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val

225 230 235 240

Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr

245 250 255

Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser

260 265 270

Lys Leu Ala Ser Gly Val Pro Ala His Phe Arg Gly Ser Gly Ser Gly

275 280 285

Thr Ser Tyr Ser Leu Thr Ile Ser Gly Met Glu Ala Glu Asp Ala Ala

290 295 300

Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Phe Thr Phe Gly Ser

305 310 315 320

Gly Thr Lys Leu Glu Ile Asn Arg Thr Val Ala Ala Pro Ser Val Phe

325 330 335

Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val

340 345 350

Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp

355 360 365

Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr

370 375 380

Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr

385 390 395 400

Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val

405 410 415

Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly

420 425 430

Glu Cys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Cys Pro Pro Cys

435 440 445

Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro

450 455 460

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

465 470 475 480

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

485 490 495

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

500 505 510

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

515 520 525

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

530 535 540

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

545 550 555 560

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

565 570 575

Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr

580 585 590

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

595 600 605

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

610 615 620

Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

625 630 635 640

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

645 650 655

Gln Lys Ser Leu Ser Leu Ser Pro Gly

660 665

<210> 24

<211> 685

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Fab2 Her2/CD3 heavy chain

<400> 24

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Gly

210 215 220

Pro Ala Pro Glu Leu Leu Gln Val Gln Leu Gln Gln Ser Gly Ala Glu

225 230 235 240

Leu Ala Arg Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly

245 250 255

Tyr Thr Phe Thr Arg Tyr Thr Met His Trp Val Lys Gln Arg Pro Gly

260 265 270

Gln Gly Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr

275 280 285

Asn Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys

290 295 300

Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp

305 310 315 320

Ser Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu

325 330 335

Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Ala Ser Thr

340 345 350

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser

355 360 365

Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

370 375 380

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

385 390 395 400

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

405 410 415

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys

420 425 430

Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu

435 440 445

Pro Lys Ser Cys Asp Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

450 455 460

Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe

465 470 475 480

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

485 490 495

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

500 505 510

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

515 520 525

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

530 535 540

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

545 550 555 560

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

565 570 575

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

580 585 590

Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val

595 600 605

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

610 615 620

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

625 630 635 640

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

645 650 655

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

660 665 670

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

675 680 685

<210> 25

<211> 663

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Fab2 Her2/CD3 light chain

<400> 25

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys Gly Gly Gly Gly Ser Gln Ile Val Leu Thr

210 215 220

Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met

225 230 235 240

Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln

245 250 255

Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Leu

260 265 270

Ala Ser Gly Val Pro Ala His Phe Arg Gly Ser Gly Ser Gly Thr Ser

275 280 285

Tyr Ser Leu Thr Ile Ser Gly Met Glu Ala Glu Asp Ala Ala Thr Tyr

290 295 300

Tyr Cys Gln Gln Trp Ser Ser Asn Pro Phe Thr Phe Gly Ser Gly Thr

305 310 315 320

Lys Leu Glu Ile Asn Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe

325 330 335

Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys

340 345 350

Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val

355 360 365

Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln

370 375 380

Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser

385 390 395 400

Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His

405 410 415

Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

420 425 430

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Cys Pro Pro Cys Pro Ala

435 440 445

Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

450 455 460

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

465 470 475 480

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

485 490 495

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

500 505 510

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

515 520 525

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

530 535 540

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

545 550 555 560

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr

565 570 575

Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser

580 585 590

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

595 600 605

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val

610 615 620

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

625 630 635 640

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

645 650 655

Ser Leu Ser Leu Ser Pro Gly

660

<210> 26

<211> 689

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Fab2 Her2/CD3 heavy chain

<400> 26

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Gly

210 215 220

Pro Ala Pro Glu Leu Leu Gly Gly Gly Ser Gln Val Gln Leu Gln Gln

225 230 235 240

Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala Ser Val Lys Met Ser Cys

245 250 255

Lys Ala Ser Gly Tyr Thr Phe Thr Arg Tyr Thr Met His Trp Val Lys

260 265 270

Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser

275 280 285

Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu

290 295 300

Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu

305 310 315 320

Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp

325 330 335

His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser

340 345 350

Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser

355 360 365

Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp

370 375 380

Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr

385 390 395 400

Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr

405 410 415

Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln

420 425 430

Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp

435 440 445

Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Gly Gly Gly Gly Ser Gly

450 455 460

Gly Gly Gly Ser Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala

465 470 475 480

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

485 490 495

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

500 505 510

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

515 520 525

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

530 535 540

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

545 550 555 560

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

565 570 575

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

580 585 590

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

595 600 605

Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

610 615 620

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

625 630 635 640

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

645 650 655

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

660 665 670

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

675 680 685

Gly

<210> 27

<211> 669

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Fab2 Her2/CD3 light chain

<400> 27

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys Gly Pro Ala Pro Glu Leu Leu Gly Gly Gly

210 215 220

Ser Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro

225 230 235 240

Gly Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr

245 250 255

Met Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile

260 265 270

Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala His Phe Arg Gly

275 280 285

Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Gly Met Glu Ala

290 295 300

Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Phe

305 310 315 320

Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Asn Arg Thr Val Ala Ala

325 330 335

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

340 345 350

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

355 360 365

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

370 375 380

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

385 390 395 400

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

405 410 415

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

420 425 430

Phe Asn Arg Gly Glu Cys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

435 440 445

Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe

450 455 460

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

465 470 475 480

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

485 490 495

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

500 505 510

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

515 520 525

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

530 535 540

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

545 550 555 560

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

565 570 575

Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val

580 585 590

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

595 600 605

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

610 615 620

Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

625 630 635 640

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

645 650 655

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

660 665

<210> 28

<211> 344

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> IgG Her2/CD47 heavy chain

<400> 28

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Gly

210 215 220

Gly Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys

225 230 235 240

Lys Pro Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Thr

245 250 255

Phe Thr Asn Tyr Tyr Ile Phe Trp Val Arg Gln Met Pro Gly Lys Gly

260 265 270

Leu Glu Trp Met Gly Ile Ile Asn Pro Val Asp Gly Asp Thr Asn Tyr

275 280 285

Asn Pro Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile

290 295 300

Ser Thr Ala Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala

305 310 315 320

Met Tyr Tyr Cys Ala Arg Gly Gly Tyr Thr Met Asp Arg Trp Gly Gln

325 330 335

Gly Thr Leu Val Thr Val Ser Ser

340

<210> 29

<211> 333

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> IgG Her2/CD47 light chain

<400> 29

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys Gly Pro Ala Pro Glu Leu Leu Asp Ile Val

210 215 220

Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly Glu Pro Ala

225 230 235 240

Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser Asn Gly Tyr

245 250 255

Asn Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu

260 265 270

Leu Ile Tyr Lys Val Ser Asn Arg Leu Ser Gly Val Pro Asp Arg Phe

275 280 285

Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val

290 295 300

Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Asn Thr His Thr

305 310 315 320

Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

325 330

<210> 30

<211> 346

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> IgG Her2/CD47 heavy chain

<400> 30

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Gly

210 215 220

Pro Ala Pro Glu Leu Leu Gln Val Gln Leu Val Gln Ser Gly Ala Glu

225 230 235 240

Val Lys Lys Pro Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly

245 250 255

Tyr Thr Phe Thr Asn Tyr Tyr Ile Phe Trp Val Arg Gln Met Pro Gly

260 265 270

Lys Gly Leu Glu Trp Met Gly Ile Ile Asn Pro Val Asp Gly Asp Thr

275 280 285

Asn Tyr Asn Pro Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Lys

290 295 300

Ser Ile Ser Thr Ala Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp

305 310 315 320

Thr Ala Met Tyr Tyr Cys Ala Arg Gly Gly Tyr Thr Met Asp Arg Trp

325 330 335

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

340 345

<210> 31

<211> 330

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> IgG Her2/CD47 light chain

<400> 31

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys Gly Gly Gly Ser Asp Ile Val Met Thr Gln

210 215 220

Ser Pro Leu Ser Leu Pro Val Thr Pro Gly Glu Pro Ala Ser Ile Ser

225 230 235 240

Cys Arg Ser Ser Gln Ser Leu Leu His Ser Asn Gly Tyr Asn Tyr Leu

245 250 255

His Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr

260 265 270

Lys Val Ser Asn Arg Leu Ser Gly Val Pro Asp Arg Phe Ser Gly Ser

275 280 285

Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu

290 295 300

Asp Val Gly Val Tyr Tyr Cys Phe Gln Asn Thr His Thr Pro Arg Thr

305 310 315 320

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

325 330

<210> 32

<211> 10

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> peptide linker

<400> 32

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10

<210> 33

<211> 15

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> peptide linker

<400> 33

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 34

<211> 350

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> IgG Her2/CD47 heavy chain

<400> 34

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Gly

210 215 220

Pro Ala Pro Glu Leu Leu Gly Gly Gly Ser Gln Val Gln Leu Val Gln

225 230 235 240

Ser Gly Ala Glu Val Lys Lys Pro Gly Glu Ser Leu Lys Ile Ser Cys

245 250 255

Lys Gly Ser Gly Tyr Thr Phe Thr Asn Tyr Tyr Ile Phe Trp Val Arg

260 265 270

Gln Met Pro Gly Lys Gly Leu Glu Trp Met Gly Ile Ile Asn Pro Val

275 280 285

Asp Gly Asp Thr Asn Tyr Asn Pro Ser Phe Gln Gly Gln Val Thr Ile

290 295 300

Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr Leu Gln Trp Ser Ser Leu

305 310 315 320

Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys Ala Arg Gly Gly Tyr Thr

325 330 335

Met Asp Arg Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

340 345 350

<210> 35

<211> 337

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> IgG Her2/CD47 light chain

<400> 35

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys Gly Pro Ala Pro Glu Leu Leu Gly Gly Gly

210 215 220

Ser Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro

225 230 235 240

Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His

245 250 255

Ser Asn Gly Tyr Asn Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln

260 265 270

Ser Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Leu Ser Gly Val

275 280 285

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys

290 295 300

Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln

305 310 315 320

Asn Thr His Thr Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

325 330 335

Lys

<210> 36

<211> 348

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Fab2 CMET/CD47 single-arm heavy chain

<400> 36

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Thr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gln Glu Ile Thr Thr Glu Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Gly Pro Ala

210 215 220

Pro Glu Leu Leu Gly Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly

225 230 235 240

Ala Glu Val Lys Lys Pro Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly

245 250 255

Ser Gly Tyr Thr Phe Thr Asn Tyr Tyr Ile Phe Trp Val Arg Gln Met

260 265 270

Pro Gly Lys Gly Leu Glu Trp Met Gly Ile Ile Asn Pro Val Asp Gly

275 280 285

Asp Thr Asn Tyr Asn Pro Ser Phe Gln Gly Gln Val Thr Ile Ser Ala

290 295 300

Asp Lys Ser Ile Ser Thr Ala Tyr Leu Gln Trp Ser Ser Leu Lys Ala

305 310 315 320

Ser Asp Thr Ala Met Tyr Tyr Cys Ala Arg Gly Gly Tyr Thr Met Asp

325 330 335

Arg Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

340 345

<210> 37

<211> 341

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Fab2 CMET/CD47 single-arm light chain

<400> 37

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

Ala Asn Ser Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

35 40 45

Arg Leu Leu Ile Tyr Arg Gly Ser Thr Arg Glu Ser Gly Ile Pro Asp

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Lys

85 90 95

Ser Glu Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Gly Pro Ala Pro Glu Leu

210 215 220

Leu Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu

225 230 235 240

Pro Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln

245 250 255

Ser Leu Leu His Ser Asn Gly Tyr Asn Tyr Leu His Trp Tyr Leu Gln

260 265 270

Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg

275 280 285

Leu Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp

290 295 300

Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr

305 310 315 320

Tyr Cys Phe Gln Asn Thr His Thr Pro Arg Thr Phe Gly Gly Gly Thr

325 330 335

Lys Val Glu Ile Lys

340

<210> 38

<211> 353

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Fab2 Her2/CD3 single-arm heavy chain

<400> 38

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Gly

210 215 220

Pro Ala Pro Glu Leu Leu Gly Gly Gly Ser Gln Val Gln Leu Gln Gln

225 230 235 240

Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala Ser Val Lys Met Ser Cys

245 250 255

Lys Ala Ser Gly Tyr Thr Phe Thr Arg Tyr Thr Met His Trp Val Lys

260 265 270

Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser

275 280 285

Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu

290 295 300

Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu

305 310 315 320

Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp

325 330 335

His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser

340 345 350

Ser

<210> 39

<211> 331

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Fab2 Her2/CD3 single-arm light chain

<400> 39

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys Gly Pro Ala Pro Glu Leu Leu Gly Gly Gly

210 215 220

Ser Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro

225 230 235 240

Gly Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr

245 250 255

Met Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile

260 265 270

Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala His Phe Arg Gly

275 280 285

Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Gly Met Glu Ala

290 295 300

Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Phe

305 310 315 320

Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

325 330

<210> 40

<211> 359

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Fab2 Her2/CD3(34) single-arm heavy chain

<400> 40

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Gly

210 215 220

Pro Ala Pro Glu Leu Leu Gly Gly Gly Ser Glu Val Lys Leu Leu Glu

225 230 235 240

Ser Gly Gly Gly Leu Val Gln Pro Lys Gly Ser Leu Lys Leu Ser Cys

245 250 255

Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr Ala Met Asn Trp Val Arg

260 265 270

Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Arg Ser Lys

275 280 285

Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser Val Lys Asp Arg Phe

290 295 300

Thr Ile Ser Arg Asp Asp Ser Gln Ser Ile Leu Tyr Leu Gln Met Asn

305 310 315 320

Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr Tyr Cys Val Arg His Gly

325 330 335

Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr Trp Gly Gln Gly

340 345 350

Thr Leu Val Thr Val Ser Ser

355

<210> 41

<211> 334

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Fab2 Her2/CD3(34) one-armed light chain

<400> 41

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys Gly Pro Ala Pro Glu Leu Leu Gly Gly Gly

210 215 220

Ser Gln Ala Val Val Thr Gln Glu Ser Ala Leu Thr Thr Ser Pro Gly

225 230 235 240

Glu Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr

245 250 255

Ser Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Asp His Leu Phe Thr

260 265 270

Gly Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala Arg

275 280 285

Phe Ser Gly Ser Leu Ile Gly Asp Lys Ala Ala Leu Thr Ile Thr Gly

290 295 300

Ala Gln Thr Glu Asp Glu Ala Ile Tyr Phe Cys Ala Leu Trp Tyr Ser

305 310 315 320

Asn Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

325 330

<210> 42

<211> 350

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> IgG2 Her2/CD47 heavy chain

<400> 42

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Gly

210 215 220

Pro Ala Pro Leu Gly Leu Gly Gly Gly Ser Gln Val Gln Leu Val Gln

225 230 235 240

Ser Gly Ala Glu Val Lys Lys Pro Gly Glu Ser Leu Lys Ile Ser Cys

245 250 255

Lys Gly Ser Gly Tyr Thr Phe Thr Asn Tyr Tyr Ile Phe Trp Val Arg

260 265 270

Gln Met Pro Gly Lys Gly Leu Glu Trp Met Gly Ile Ile Asn Pro Val

275 280 285

Asp Gly Asp Thr Asn Tyr Asn Pro Ser Phe Gln Gly Gln Val Thr Ile

290 295 300

Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr Leu Gln Trp Ser Ser Leu

305 310 315 320

Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys Ala Arg Gly Gly Tyr Thr

325 330 335

Met Asp Arg Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

340 345 350

<210> 43

<211> 337

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> IgG2 Her2/CD47 light chain

<400> 43

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys Gly Pro Ala Pro Glu Leu Leu Gly Gly Gly

210 215 220

Ser Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro

225 230 235 240

Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His

245 250 255

Ser Asn Gly Tyr Asn Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln

260 265 270

Ser Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Leu Ser Gly Val

275 280 285

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys

290 295 300

Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln

305 310 315 320

Asn Thr His Thr Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

325 330 335

Lys

<210> 44

<211> 350

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> IgG2 Her2/CD47 heavy chain

<400> 44

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Gly

210 215 220

Pro Ala Pro Glu Leu Leu Gly Gly Gly Ser Gln Val Gln Leu Val Gln

225 230 235 240

Ser Gly Ala Glu Val Lys Lys Pro Gly Glu Ser Leu Lys Ile Ser Cys

245 250 255

Lys Gly Ser Gly Tyr Thr Phe Thr Asn Tyr Tyr Ile Phe Trp Val Arg

260 265 270

Gln Met Pro Gly Lys Gly Leu Glu Trp Met Gly Ile Ile Asn Pro Val

275 280 285

Asp Gly Asp Thr Asn Tyr Asn Pro Ser Phe Gln Gly Gln Val Thr Ile

290 295 300

Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr Leu Gln Trp Ser Ser Leu

305 310 315 320

Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys Ala Arg Gly Gly Tyr Thr

325 330 335

Met Asp Arg Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

340 345 350

<210> 45

<211> 337

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> IgG2 Her2/CD47 light chain

<400> 45

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys Gly Pro Ala Pro Leu Gly Leu Gly Gly Gly

210 215 220

Ser Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro

225 230 235 240

Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His

245 250 255

Ser Asn Gly Tyr Asn Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln

260 265 270

Ser Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Leu Ser Gly Val

275 280 285

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys

290 295 300

Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln

305 310 315 320

Asn Thr His Thr Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

325 330 335

Lys

<210> 46

<211> 350

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> IgG2 Her2/CD47 heavy chain

<400> 46

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Gly

210 215 220

Pro Ala Pro Leu Gly Leu Gly Gly Gly Ser Gln Val Gln Leu Val Gln

225 230 235 240

Ser Gly Ala Glu Val Lys Lys Pro Gly Glu Ser Leu Lys Ile Ser Cys

245 250 255

Lys Gly Ser Gly Tyr Thr Phe Thr Asn Tyr Tyr Ile Phe Trp Val Arg

260 265 270

Gln Met Pro Gly Lys Gly Leu Glu Trp Met Gly Ile Ile Asn Pro Val

275 280 285

Asp Gly Asp Thr Asn Tyr Asn Pro Ser Phe Gln Gly Gln Val Thr Ile

290 295 300

Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr Leu Gln Trp Ser Ser Leu

305 310 315 320

Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys Ala Arg Gly Gly Tyr Thr

325 330 335

Met Asp Arg Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

340 345 350

<210> 47

<211> 337

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> IgG2 Her2/CD47 light chain

<400> 47

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys Gly Pro Ala Pro Leu Gly Leu Gly Gly Gly

210 215 220

Ser Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro

225 230 235 240

Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His

245 250 255

Ser Asn Gly Tyr Asn Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln

260 265 270

Ser Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Leu Ser Gly Val

275 280 285

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys

290 295 300

Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln

305 310 315 320

Asn Thr His Thr Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

325 330 335

Lys

<210> 48

<211> 352

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> IgG2 Her2/CD47 heavy chain

<400> 48

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Pro

210 215 220

Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Gly Ser Gln Val Gln Leu

225 230 235 240

Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu Ser Leu Lys Ile

245 250 255

Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asn Tyr Tyr Ile Phe Trp

260 265 270

Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met Gly Ile Ile Asn

275 280 285

Pro Val Asp Gly Asp Thr Asn Tyr Asn Pro Ser Phe Gln Gly Gln Val

290 295 300

Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr Leu Gln Trp Ser

305 310 315 320

Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys Ala Arg Gly Gly

325 330 335

Tyr Thr Met Asp Arg Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

340 345 350

<210> 49

<211> 339

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> IgG2 Her2/CD47 light chain

<400> 49

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

210 215 220

Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val

225 230 235 240

Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu

245 250 255

Leu His Ser Asn Gly Tyr Asn Tyr Leu His Trp Tyr Leu Gln Lys Pro

260 265 270

Gly Gln Ser Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Leu Ser

275 280 285

Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

290 295 300

Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys

305 310 315 320

Phe Gln Asn Thr His Thr Pro Arg Thr Phe Gly Gly Gly Thr Lys Val

325 330 335

Glu Ile Lys

<210> 50

<211> 352

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> IgG2 Her2/CD47 heavy chain

<400> 50

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Pro

210 215 220

Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Gly Ser Gln Val Gln Leu

225 230 235 240

Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu Ser Leu Lys Ile

245 250 255

Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asn Tyr Tyr Ile Phe Trp

260 265 270

Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met Gly Ile Ile Asn

275 280 285

Pro Val Asp Gly Asp Thr Asn Tyr Asn Pro Ser Phe Gln Gly Gln Val

290 295 300

Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr Leu Gln Trp Ser

305 310 315 320

Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys Ala Arg Gly Gly

325 330 335

Tyr Thr Met Asp Arg Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

340 345 350

<210> 51

<211> 339

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> IgG2 Her2/CD47 light chain

<400> 51

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys Pro Pro Cys Pro Ala Pro Leu Gly Leu Gly

210 215 220

Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val

225 230 235 240

Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu

245 250 255

Leu His Ser Asn Gly Tyr Asn Tyr Leu His Trp Tyr Leu Gln Lys Pro

260 265 270

Gly Gln Ser Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Leu Ser

275 280 285

Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

290 295 300

Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys

305 310 315 320

Phe Gln Asn Thr His Thr Pro Arg Thr Phe Gly Gly Gly Thr Lys Val

325 330 335

Glu Ile Lys

<210> 52

<211> 352

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> IgG2 Her2/CD47 heavy chain

<400> 52

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Pro

210 215 220

Pro Cys Pro Ala Pro Leu Gly Leu Gly Gly Gly Ser Gln Val Gln Leu

225 230 235 240

Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu Ser Leu Lys Ile

245 250 255

Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asn Tyr Tyr Ile Phe Trp

260 265 270

Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met Gly Ile Ile Asn

275 280 285

Pro Val Asp Gly Asp Thr Asn Tyr Asn Pro Ser Phe Gln Gly Gln Val

290 295 300

Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr Leu Gln Trp Ser

305 310 315 320

Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys Ala Arg Gly Gly

325 330 335

Tyr Thr Met Asp Arg Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

340 345 350

<210> 53

<211> 339

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> IgG2 Her2/CD47 light chain

<400> 53

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

210 215 220

Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val

225 230 235 240

Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu

245 250 255

Leu His Ser Asn Gly Tyr Asn Tyr Leu His Trp Tyr Leu Gln Lys Pro

260 265 270

Gly Gln Ser Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Leu Ser

275 280 285

Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

290 295 300

Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys

305 310 315 320

Phe Gln Asn Thr His Thr Pro Arg Thr Phe Gly Gly Gly Thr Lys Val

325 330 335

Glu Ile Lys

<210> 54

<211> 352

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> IgG2 Her2/CD47 heavy chain

<400> 54

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Pro

210 215 220

Pro Cys Pro Ala Pro Leu Gly Leu Gly Gly Gly Ser Gln Val Gln Leu

225 230 235 240

Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu Ser Leu Lys Ile

245 250 255

Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asn Tyr Tyr Ile Phe Trp

260 265 270

Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met Gly Ile Ile Asn

275 280 285

Pro Val Asp Gly Asp Thr Asn Tyr Asn Pro Ser Phe Gln Gly Gln Val

290 295 300

Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr Leu Gln Trp Ser

305 310 315 320

Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys Ala Arg Gly Gly

325 330 335

Tyr Thr Met Asp Arg Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

340 345 350

<210> 55

<211> 339

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> IgG2 Her2/CD47 light chain

<400> 55

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys Pro Pro Cys Pro Ala Pro Leu Gly Leu Gly

210 215 220

Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val

225 230 235 240

Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu

245 250 255

Leu His Ser Asn Gly Tyr Asn Tyr Leu His Trp Tyr Leu Gln Lys Pro

260 265 270

Gly Gln Ser Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Leu Ser

275 280 285

Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

290 295 300

Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys

305 310 315 320

Phe Gln Asn Thr His Thr Pro Arg Thr Phe Gly Gly Gly Thr Lys Val

325 330 335

Glu Ile Lys

<210> 56

<211> 327

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 56

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro

100 105 110

Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

305 310 315 320

Leu Ser Leu Ser Leu Gly Lys

325

<210> 57

<211> 327

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> human IgG4(S228P) immunoglobulin Fc region

<400> 57

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

100 105 110

Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

305 310 315 320

Leu Ser Leu Ser Leu Gly Lys

325

<210> 58

<211> 330

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 58

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 59

<211> 330

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> human IgG1-3M immunoglobulin Fc region

<400> 59

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 60

<211> 326

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 60

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro

100 105 110

Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

115 120 125

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

130 135 140

Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly

145 150 155 160

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn

165 170 175

Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp

180 185 190

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro

195 200 205

Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu

210 215 220

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn

225 230 235 240

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

245 250 255

Ser Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

260 265 270

Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

275 280 285

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

290 295 300

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

305 310 315 320

Ser Leu Ser Pro Gly Lys

325

<210> 61

<211> 330

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 61

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 62

<211> 329

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> human IgG1-3M REEM allotype immunoglobulin Fc region

<400> 62

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

245 250 255

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

260 265 270

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

275 280 285

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

290 295 300

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

305 310 315 320

Lys Ser Leu Ser Leu Ser Pro Gly Lys

325

<210> 63

<211> 323

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 63

Met Trp Pro Leu Val Ala Ala Leu Leu Leu Gly Ser Ala Cys Cys Gly

1 5 10 15

Ser Ala Gln Leu Leu Phe Asn Lys Thr Lys Ser Val Glu Phe Thr Phe

20 25 30

Cys Asn Asp Thr Val Val Ile Pro Cys Phe Val Thr Asn Met Glu Ala

35 40 45

Gln Asn Thr Thr Glu Val Tyr Val Lys Trp Lys Phe Lys Gly Arg Asp

50 55 60

Ile Tyr Thr Phe Asp Gly Ala Leu Asn Lys Ser Thr Val Pro Thr Asp

65 70 75 80

Phe Ser Ser Ala Lys Ile Glu Val Ser Gln Leu Leu Lys Gly Asp Ala

85 90 95

Ser Leu Lys Met Asp Lys Ser Asp Ala Val Ser His Thr Gly Asn Tyr

100 105 110

Thr Cys Glu Val Thr Glu Leu Thr Arg Glu Gly Glu Thr Ile Ile Glu

115 120 125

Leu Lys Tyr Arg Val Val Ser Trp Phe Ser Pro Asn Glu Asn Ile Leu

130 135 140

Ile Val Ile Phe Pro Ile Phe Ala Ile Leu Leu Phe Trp Gly Gln Phe

145 150 155 160

Gly Ile Lys Thr Leu Lys Tyr Arg Ser Gly Gly Met Asp Glu Lys Thr

165 170 175

Ile Ala Leu Leu Val Ala Gly Leu Val Ile Thr Val Ile Val Ile Val

180 185 190

Gly Ala Ile Leu Phe Val Pro Gly Glu Tyr Ser Leu Lys Asn Ala Thr

195 200 205

Gly Leu Gly Leu Ile Val Thr Ser Thr Gly Ile Leu Ile Leu Leu His

210 215 220

Tyr Tyr Val Phe Ser Thr Ala Ile Gly Leu Thr Ser Phe Val Ile Ala

225 230 235 240

Ile Leu Val Ile Gln Val Ile Ala Tyr Ile Leu Ala Val Val Gly Leu

245 250 255

Ser Leu Cys Ile Ala Ala Cys Ile Pro Met His Gly Pro Leu Leu Ile

260 265 270

Ser Gly Leu Ser Ile Leu Ala Leu Ala Gln Leu Leu Gly Leu Val Tyr

275 280 285

Met Lys Phe Val Ala Ser Asn Gln Lys Thr Ile Gln Pro Pro Arg Lys

290 295 300

Ala Val Glu Glu Pro Leu Asn Ala Phe Lys Glu Ser Lys Gly Met Met

305 310 315 320

Asn Asp Glu

<210> 64

<211> 323

<212> PRT

<213> monkey fasciculation (Macaca fascicularis)

<400> 64

Met Trp Pro Leu Val Ala Ala Leu Leu Leu Gly Ser Ala Cys Cys Gly

1 5 10 15

Ser Ala Gln Leu Leu Phe Asn Lys Thr Lys Ser Val Glu Phe Thr Phe

20 25 30

Cys Asn Asp Thr Val Val Ile Pro Cys Phe Val Thr Asn Met Glu Ala

35 40 45

Gln Asn Thr Thr Glu Val Tyr Val Lys Trp Lys Phe Lys Gly Arg Asp

50 55 60

Ile Tyr Thr Phe Asp Gly Ala Leu Asn Lys Ser Thr Ala Pro Ala Asn

65 70 75 80

Phe Ser Ser Ala Lys Ile Glu Val Ser Gln Leu Leu Lys Gly Asp Ala

85 90 95

Ser Leu Lys Met Asp Lys Ser Asp Ala Val Ser His Thr Gly Asn Tyr

100 105 110

Thr Cys Glu Val Thr Glu Leu Thr Arg Glu Gly Glu Thr Ile Ile Glu

115 120 125

Leu Lys Tyr Arg Val Val Ser Trp Phe Ser Pro Asn Glu Asn Ile Leu

130 135 140

Ile Val Ile Phe Pro Ile Phe Ala Ile Leu Leu Phe Trp Gly Gln Phe

145 150 155 160

Gly Ile Lys Thr Leu Lys Tyr Arg Ser Gly Gly Met Asp Glu Lys Thr

165 170 175

Ile Ala Leu Leu Val Ala Gly Leu Met Ile Thr Val Ile Val Ile Val

180 185 190

Gly Ala Ile Leu Phe Val Pro Gly Glu Tyr Ser Leu Lys Asn Ala Thr

195 200 205

Gly Leu Gly Leu Ile Val Thr Ser Thr Gly Ile Leu Ile Leu Leu His

210 215 220

Tyr Tyr Val Phe Ser Thr Ala Ile Gly Leu Thr Ser Phe Val Ile Ala

225 230 235 240

Ile Leu Val Ile Gln Val Ile Ala Tyr Ile Leu Ala Val Val Gly Leu

245 250 255

Ser Leu Cys Ile Ala Ala Cys Ile Pro Met His Gly Pro Leu Leu Ile

260 265 270

Ser Gly Leu Ser Ile Leu Ala Leu Ala Gln Leu Leu Gly Leu Val Tyr

275 280 285

Met Lys Phe Val Ala Ser Asn Gln Lys Thr Ile Gln Pro Pro Arg Lys

290 295 300

Ala Val Glu Glu Pro Leu Asn Ala Phe Lys Glu Ser Lys Gly Met Met

305 310 315 320

Asn Asp Glu

<210> 65

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> immunoglobulin constant region

<400> 65

Arg Asp Glu Leu Thr

1 5

<210> 66

<211> 4

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> immunoglobulin constant region

<400> 66

Arg Glu Glu Met

<210> 67

<211> 1390

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 67

Met Lys Ala Pro Ala Val Leu Ala Pro Gly Ile Leu Val Leu Leu Phe

1 5 10 15

Thr Leu Val Gln Arg Ser Asn Gly Glu Cys Lys Glu Ala Leu Ala Lys

20 25 30

Ser Glu Met Asn Val Asn Met Lys Tyr Gln Leu Pro Asn Phe Thr Ala

35 40 45

Glu Thr Pro Ile Gln Asn Val Ile Leu His Glu His His Ile Phe Leu

50 55 60

Gly Ala Thr Asn Tyr Ile Tyr Val Leu Asn Glu Glu Asp Leu Gln Lys

65 70 75 80

Val Ala Glu Tyr Lys Thr Gly Pro Val Leu Glu His Pro Asp Cys Phe

85 90 95

Pro Cys Gln Asp Cys Ser Ser Lys Ala Asn Leu Ser Gly Gly Val Trp

100 105 110

Lys Asp Asn Ile Asn Met Ala Leu Val Val Asp Thr Tyr Tyr Asp Asp

115 120 125

Gln Leu Ile Ser Cys Gly Ser Val Asn Arg Gly Thr Cys Gln Arg His

130 135 140

Val Phe Pro His Asn His Thr Ala Asp Ile Gln Ser Glu Val His Cys

145 150 155 160

Ile Phe Ser Pro Gln Ile Glu Glu Pro Ser Gln Cys Pro Asp Cys Val

165 170 175

Val Ser Ala Leu Gly Ala Lys Val Leu Ser Ser Val Lys Asp Arg Phe

180 185 190

Ile Asn Phe Phe Val Gly Asn Thr Ile Asn Ser Ser Tyr Phe Pro Asp

195 200 205

His Pro Leu His Ser Ile Ser Val Arg Arg Leu Lys Glu Thr Lys Asp

210 215 220

Gly Phe Met Phe Leu Thr Asp Gln Ser Tyr Ile Asp Val Leu Pro Glu

225 230 235 240

Phe Arg Asp Ser Tyr Pro Ile Lys Tyr Val His Ala Phe Glu Ser Asn

245 250 255

Asn Phe Ile Tyr Phe Leu Thr Val Gln Arg Glu Thr Leu Asp Ala Gln

260 265 270

Thr Phe His Thr Arg Ile Ile Arg Phe Cys Ser Ile Asn Ser Gly Leu

275 280 285

His Ser Tyr Met Glu Met Pro Leu Glu Cys Ile Leu Thr Glu Lys Arg

290 295 300

Lys Lys Arg Ser Thr Lys Lys Glu Val Phe Asn Ile Leu Gln Ala Ala

305 310 315 320

Tyr Val Ser Lys Pro Gly Ala Gln Leu Ala Arg Gln Ile Gly Ala Ser

325 330 335

Leu Asn Asp Asp Ile Leu Phe Gly Val Phe Ala Gln Ser Lys Pro Asp

340 345 350

Ser Ala Glu Pro Met Asp Arg Ser Ala Met Cys Ala Phe Pro Ile Lys

355 360 365

Tyr Val Asn Asp Phe Phe Asn Lys Ile Val Asn Lys Asn Asn Val Arg

370 375 380

Cys Leu Gln His Phe Tyr Gly Pro Asn His Glu His Cys Phe Asn Arg

385 390 395 400

Thr Leu Leu Arg Asn Ser Ser Gly Cys Glu Ala Arg Arg Asp Glu Tyr

405 410 415

Arg Thr Glu Phe Thr Thr Ala Leu Gln Arg Val Asp Leu Phe Met Gly

420 425 430

Gln Phe Ser Glu Val Leu Leu Thr Ser Ile Ser Thr Phe Ile Lys Gly

435 440 445

Asp Leu Thr Ile Ala Asn Leu Gly Thr Ser Glu Gly Arg Phe Met Gln

450 455 460

Val Val Val Ser Arg Ser Gly Pro Ser Thr Pro His Val Asn Phe Leu

465 470 475 480

Leu Asp Ser His Pro Val Ser Pro Glu Val Ile Val Glu His Thr Leu

485 490 495

Asn Gln Asn Gly Tyr Thr Leu Val Ile Thr Gly Lys Lys Ile Thr Lys

500 505 510

Ile Pro Leu Asn Gly Leu Gly Cys Arg His Phe Gln Ser Cys Ser Gln

515 520 525

Cys Leu Ser Ala Pro Pro Phe Val Gln Cys Gly Trp Cys His Asp Lys

530 535 540

Cys Val Arg Ser Glu Glu Cys Leu Ser Gly Thr Trp Thr Gln Gln Ile

545 550 555 560

Cys Leu Pro Ala Ile Tyr Lys Val Phe Pro Asn Ser Ala Pro Leu Glu

565 570 575

Gly Gly Thr Arg Leu Thr Ile Cys Gly Trp Asp Phe Gly Phe Arg Arg

580 585 590

Asn Asn Lys Phe Asp Leu Lys Lys Thr Arg Val Leu Leu Gly Asn Glu

595 600 605

Ser Cys Thr Leu Thr Leu Ser Glu Ser Thr Met Asn Thr Leu Lys Cys

610 615 620

Thr Val Gly Pro Ala Met Asn Lys His Phe Asn Met Ser Ile Ile Ile

625 630 635 640

Ser Asn Gly His Gly Thr Thr Gln Tyr Ser Thr Phe Ser Tyr Val Asp

645 650 655

Pro Val Ile Thr Ser Ile Ser Pro Lys Tyr Gly Pro Met Ala Gly Gly

660 665 670

Thr Leu Leu Thr Leu Thr Gly Asn Tyr Leu Asn Ser Gly Asn Ser Arg

675 680 685

His Ile Ser Ile Gly Gly Lys Thr Cys Thr Leu Lys Ser Val Ser Asn

690 695 700

Ser Ile Leu Glu Cys Tyr Thr Pro Ala Gln Thr Ile Ser Thr Glu Phe

705 710 715 720

Ala Val Lys Leu Lys Ile Asp Leu Ala Asn Arg Glu Thr Ser Ile Phe

725 730 735

Ser Tyr Arg Glu Asp Pro Ile Val Tyr Glu Ile His Pro Thr Lys Ser

740 745 750

Phe Ile Ser Gly Gly Ser Thr Ile Thr Gly Val Gly Lys Asn Leu Asn

755 760 765

Ser Val Ser Val Pro Arg Met Val Ile Asn Val His Glu Ala Gly Arg

770 775 780

Asn Phe Thr Val Ala Cys Gln His Arg Ser Asn Ser Glu Ile Ile Cys

785 790 795 800

Cys Thr Thr Pro Ser Leu Gln Gln Leu Asn Leu Gln Leu Pro Leu Lys

805 810 815

Thr Lys Ala Phe Phe Met Leu Asp Gly Ile Leu Ser Lys Tyr Phe Asp

820 825 830

Leu Ile Tyr Val His Asn Pro Val Phe Lys Pro Phe Glu Lys Pro Val

835 840 845

Met Ile Ser Met Gly Asn Glu Asn Val Leu Glu Ile Lys Gly Asn Asp

850 855 860

Ile Asp Pro Glu Ala Val Lys Gly Glu Val Leu Lys Val Gly Asn Lys

865 870 875 880

Ser Cys Glu Asn Ile His Leu His Ser Glu Ala Val Leu Cys Thr Val

885 890 895

Pro Asn Asp Leu Leu Lys Leu Asn Ser Glu Leu Asn Ile Glu Trp Lys

900 905 910

Gln Ala Ile Ser Ser Thr Val Leu Gly Lys Val Ile Val Gln Pro Asp

915 920 925

Gln Asn Phe Thr Gly Leu Ile Ala Gly Val Val Ser Ile Ser Thr Ala

930 935 940

Leu Leu Leu Leu Leu Gly Phe Phe Leu Trp Leu Lys Lys Arg Lys Gln

945 950 955 960

Ile Lys Asp Leu Gly Ser Glu Leu Val Arg Tyr Asp Ala Arg Val His

965 970 975

Thr Pro His Leu Asp Arg Leu Val Ser Ala Arg Ser Val Ser Pro Thr

980 985 990

Thr Glu Met Val Ser Asn Glu Ser Val Asp Tyr Arg Ala Thr Phe Pro

995 1000 1005

Glu Asp Gln Phe Pro Asn Ser Ser Gln Asn Gly Ser Cys Arg Gln

1010 1015 1020

Val Gln Tyr Pro Leu Thr Asp Met Ser Pro Ile Leu Thr Ser Gly

1025 1030 1035

Asp Ser Asp Ile Ser Ser Pro Leu Leu Gln Asn Thr Val His Ile

1040 1045 1050

Asp Leu Ser Ala Leu Asn Pro Glu Leu Val Gln Ala Val Gln His

1055 1060 1065

Val Val Ile Gly Pro Ser Ser Leu Ile Val His Phe Asn Glu Val

1070 1075 1080

Ile Gly Arg Gly His Phe Gly Cys Val Tyr His Gly Thr Leu Leu

1085 1090 1095

Asp Asn Asp Gly Lys Lys Ile His Cys Ala Val Lys Ser Leu Asn

1100 1105 1110

Arg Ile Thr Asp Ile Gly Glu Val Ser Gln Phe Leu Thr Glu Gly

1115 1120 1125

Ile Ile Met Lys Asp Phe Ser His Pro Asn Val Leu Ser Leu Leu

1130 1135 1140

Gly Ile Cys Leu Arg Ser Glu Gly Ser Pro Leu Val Val Leu Pro

1145 1150 1155

Tyr Met Lys His Gly Asp Leu Arg Asn Phe Ile Arg Asn Glu Thr

1160 1165 1170

His Asn Pro Thr Val Lys Asp Leu Ile Gly Phe Gly Leu Gln Val

1175 1180 1185

Ala Lys Gly Met Lys Tyr Leu Ala Ser Lys Lys Phe Val His Arg

1190 1195 1200

Asp Leu Ala Ala Arg Asn Cys Met Leu Asp Glu Lys Phe Thr Val

1205 1210 1215

Lys Val Ala Asp Phe Gly Leu Ala Arg Asp Met Tyr Asp Lys Glu

1220 1225 1230

Tyr Tyr Ser Val His Asn Lys Thr Gly Ala Lys Leu Pro Val Lys

1235 1240 1245

Trp Met Ala Leu Glu Ser Leu Gln Thr Gln Lys Phe Thr Thr Lys

1250 1255 1260

Ser Asp Val Trp Ser Phe Gly Val Leu Leu Trp Glu Leu Met Thr

1265 1270 1275

Arg Gly Ala Pro Pro Tyr Pro Asp Val Asn Thr Phe Asp Ile Thr

1280 1285 1290

Val Tyr Leu Leu Gln Gly Arg Arg Leu Leu Gln Pro Glu Tyr Cys

1295 1300 1305

Pro Asp Pro Leu Tyr Glu Val Met Leu Lys Cys Trp His Pro Lys

1310 1315 1320

Ala Glu Met Arg Pro Ser Phe Ser Glu Leu Val Ser Arg Ile Ser

1325 1330 1335

Ala Ile Phe Ser Thr Phe Ile Gly Glu His Tyr Val His Val Asn

1340 1345 1350

Ala Thr Tyr Val Asn Val Lys Cys Val Ala Pro Tyr Pro Ser Leu

1355 1360 1365

Leu Ser Ser Glu Asp Asn Ala Asp Asp Glu Val Asp Thr Arg Pro

1370 1375 1380

Ala Ser Phe Trp Glu Thr Ser

1385 1390

<210> 68

<211> 1381

<212> PRT

<213> monkey fasciculation (Macaca fascicularis)

<400> 68

Met Lys Ala Pro Ala Val Leu Val Pro Gly Ile Leu Val Leu Leu Phe

1 5 10 15

Thr Leu Val Gln Arg Ser Asn Gly Glu Cys Lys Glu Ala Leu Ala Lys

20 25 30

Ser Glu Met Asn Val Asn Met Lys Tyr Gln Leu Pro Asn Phe Thr Ala

35 40 45

Glu Thr Ala Ile Gln Asn Val Ile Leu His Glu His His Ile Phe Leu

50 55 60

Gly Ala Thr Asn Tyr Ile Tyr Val Leu Asn Glu Glu Asp Leu Gln Lys

65 70 75 80

Val Ala Glu Tyr Lys Thr Gly Pro Val Leu Glu His Pro Asp Cys Phe

85 90 95

Pro Cys Gln Asp Cys Ser Ser Lys Ala Asn Leu Ser Gly Gly Val Trp

100 105 110

Lys Asp Asn Ile Asn Met Ala Leu Val Val Asp Thr Tyr Tyr Asp Asp

115 120 125

Gln Leu Ile Ser Cys Gly Ser Val Asn Arg Gly Thr Cys Gln Arg His

130 135 140

Val Phe Pro His Asn His Thr Ala Asp Ile Gln Ser Glu Val His Cys

145 150 155 160

Ile Phe Ser Pro Gln Ile Glu Glu Pro Asn Gln Cys Pro Asp Cys Val

165 170 175

Val Ser Ala Leu Gly Ala Lys Val Leu Ser Ser Val Lys Asp Arg Phe

180 185 190

Ile Asn Phe Phe Val Gly Asn Thr Ile Asn Ser Ser Tyr Phe Pro His

195 200 205

His Pro Leu His Ser Ile Ser Val Arg Arg Leu Lys Glu Thr Lys Asp

210 215 220

Gly Phe Met Phe Leu Thr Asp Gln Ser Tyr Ile Asp Val Leu Pro Glu

225 230 235 240

Phe Arg Asp Ser Tyr Pro Ile Lys Tyr Ile His Ala Phe Glu Ser Asn

245 250 255

Asn Phe Ile Tyr Phe Leu Thr Val Gln Arg Glu Thr Leu Asn Ala Gln

260 265 270

Thr Phe His Thr Arg Ile Ile Arg Phe Cys Ser Leu Asn Ser Gly Leu

275 280 285

His Ser Tyr Met Glu Met Pro Leu Glu Cys Ile Leu Thr Glu Lys Arg

290 295 300

Lys Lys Arg Ser Thr Lys Lys Glu Val Phe Asn Ile Leu Gln Ala Ala

305 310 315 320

Tyr Val Ser Lys Pro Gly Ala Gln Leu Ala Arg Gln Ile Gly Ala Ser

325 330 335

Leu Asn Asp Asp Ile Leu Phe Gly Val Phe Ala Gln Ser Lys Pro Asp

340 345 350

Ser Ala Glu Pro Met Asp Arg Ser Ala Met Cys Ala Phe Pro Ile Lys

355 360 365

Tyr Val Asn Asp Phe Phe Asn Lys Ile Val Asn Lys Asn Asn Val Arg

370 375 380

Cys Leu Gln His Phe Tyr Gly Pro Asn His Glu His Cys Phe Asn Arg

385 390 395 400

Thr Leu Leu Arg Asn Ser Ser Gly Cys Glu Ala Arg Arg Asp Glu Tyr

405 410 415

Arg Ala Glu Phe Thr Thr Ala Leu Gln Arg Val Asp Leu Phe Met Gly

420 425 430

Gln Phe Ser Glu Val Leu Leu Thr Ser Ile Ser Thr Phe Val Lys Gly

435 440 445

Asp Leu Thr Ile Ala Asn Leu Gly Thr Ser Glu Gly Arg Phe Met Gln

450 455 460

Val Val Val Ser Arg Ser Gly Pro Ser Thr Pro His Val Asn Phe Leu

465 470 475 480

Leu Asp Ser His Pro Val Ser Pro Glu Val Ile Val Glu His Pro Leu

485 490 495

Asn Gln Asn Gly Tyr Thr Leu Val Val Thr Gly Lys Lys Ile Thr Lys

500 505 510

Ile Pro Leu Asn Gly Leu Gly Cys Arg His Phe Gln Ser Cys Ser Gln

515 520 525

Cys Leu Ser Ala Pro Pro Phe Val Gln Cys Gly Trp Cys His Asp Lys

530 535 540

Cys Val Arg Ser Glu Glu Cys Pro Ser Gly Thr Trp Thr Gln Gln Ile

545 550 555 560

Cys Leu Pro Ala Ile Tyr Lys Val Phe Pro Thr Ser Ala Pro Leu Glu

565 570 575

Gly Gly Thr Arg Leu Thr Ile Cys Gly Trp Asp Phe Gly Phe Arg Arg

580 585 590

Asn Asn Lys Phe Asp Leu Lys Lys Thr Arg Val Leu Leu Gly Asn Glu

595 600 605

Ser Cys Thr Leu Thr Leu Ser Glu Ser Thr Met Asn Thr Leu Lys Cys

610 615 620

Thr Val Gly Pro Ala Met Asn Lys His Phe Asn Met Ser Ile Ile Ile

625 630 635 640

Ser Asn Gly His Gly Thr Thr Gln Tyr Ser Thr Phe Ser Tyr Val Asp

645 650 655

Pro Ile Ile Thr Ser Ile Ser Pro Lys Tyr Gly Pro Met Ala Gly Gly

660 665 670

Thr Leu Leu Thr Leu Thr Gly Asn Tyr Leu Asn Ser Gly Asn Ser Arg

675 680 685

His Ile Ser Ile Gly Gly Lys Thr Cys Thr Leu Lys Ser Val Ser Asn

690 695 700

Ser Ile Leu Glu Cys Tyr Thr Pro Ala Gln Thr Ile Ser Thr Glu Phe

705 710 715 720

Ala Val Lys Leu Lys Ile Asp Leu Ala Asn Arg Glu Thr Ser Ile Phe

725 730 735

Ser Tyr Arg Glu Asp Pro Ile Val Tyr Glu Ile His Pro Thr Lys Ser

740 745 750

Phe Ile Ser Gly Gly Ser Thr Ile Thr Gly Val Gly Lys Asn Leu His

755 760 765

Ser Val Ser Val Pro Arg Met Val Ile Asn Val His Glu Ala Gly Arg

770 775 780

Asn Phe Thr Val Ala Cys Gln His Arg Ser Asn Ser Glu Ile Ile Cys

785 790 795 800

Cys Thr Thr Pro Ser Leu Gln Gln Leu Asn Leu Gln Leu Pro Leu Lys

805 810 815

Thr Lys Ala Phe Phe Met Leu Asp Gly Ile Leu Ser Lys Tyr Phe Asp

820 825 830

Leu Ile Tyr Val His Asn Pro Val Phe Lys Pro Phe Glu Lys Pro Val

835 840 845

Met Ile Ser Met Gly Asn Glu Asn Val Leu Glu Ile Lys Gly Asn Asp

850 855 860

Ile Asp Pro Glu Ala Val Lys Gly Glu Val Leu Lys Val Gly Asn Lys

865 870 875 880

Ser Cys Glu Asn Ile His Leu His Ser Glu Ala Val Leu Cys Thr Val

885 890 895

Pro Asn Asp Leu Leu Lys Leu Asn Ser Glu Leu Asn Ile Glu Trp Lys

900 905 910

Gln Ala Ile Ser Ser Thr Val Leu Gly Lys Val Ile Val Gln Pro Asp

915 920 925

Gln Asn Phe Thr Gly Leu Ile Ala Gly Val Val Ser Ile Ser Ile Ala

930 935 940

Leu Leu Leu Leu Leu Gly Leu Phe Leu Trp Leu Lys Lys Arg Lys Gln

945 950 955 960

Ile Lys Asp Leu Gly Ser Glu Leu Val Arg Tyr Asp Ala Arg Val His

965 970 975

Thr Pro His Leu Asp Arg Leu Val Ser Ala Arg Ser Val Ser Pro Thr

980 985 990

Thr Glu Met Val Ser Asn Glu Ser Val Asp Tyr Arg Ala Thr Phe Pro

995 1000 1005

Glu Asp Gln Phe Pro Asn Ser Ser Gln Asn Gly Ser Cys Arg Gln

1010 1015 1020

Val Gln Tyr Pro Leu Thr Asp Met Ser Pro Ile Leu Thr Ser Gly

1025 1030 1035

Asp Ser Asp Ile Ser Ser Pro Leu Leu Gln Asn Thr Val His Ile

1040 1045 1050

Asp Leu Ser Ala Leu Asn Pro Glu Leu Val Gln Ala Val Gln His

1055 1060 1065

Val Val Ile Gly Pro Ser Ser Leu Ile Val His Phe Asn Glu Val

1070 1075 1080

Ile Gly Arg Gly His Phe Gly Cys Val Tyr His Gly Thr Leu Leu

1085 1090 1095

Asp Asn Asp Gly Lys Lys Ile His Cys Ala Val Lys Ser Leu Asn

1100 1105 1110

Arg Ile Thr Asp Ile Gly Glu Val Ser Gln Phe Leu Thr Glu Gly

1115 1120 1125

Ile Ile Met Lys Asp Phe Ser His Pro Asn Val Leu Ser Leu Leu

1130 1135 1140

Gly Ile Cys Leu Arg Ser Glu Gly Ser Pro Leu Val Val Leu Pro

1145 1150 1155

Tyr Met Lys His Gly Asp Leu Arg Asn Phe Ile Arg Asn Glu Thr

1160 1165 1170

His Asn Pro Thr Val Lys Asp Leu Ile Gly Phe Gly Leu Gln Val

1175 1180 1185

Ala Lys Gly Met Lys Tyr Leu Ala Ser Lys Lys Phe Val His Arg

1190 1195 1200

Asp Leu Ala Ala Arg Asn Cys Met Leu Asp Glu Lys Phe Thr Val

1205 1210 1215

Lys Val Ala Asp Phe Gly Leu Ala Arg Asp Met Tyr Asp Lys Glu

1220 1225 1230

Tyr Tyr Ser Val His Asn Lys Thr Gly Ala Lys Leu Pro Val Lys

1235 1240 1245

Trp Met Ala Leu Glu Ser Leu Gln Thr Gln Lys Phe Thr Thr Lys

1250 1255 1260

Ser Asp Val Trp Ser Phe Gly Val Leu Leu Trp Glu Leu Met Thr

1265 1270 1275

Arg Gly Ala Pro Pro Tyr Pro Asp Val Asn Thr Phe Asp Ile Thr

1280 1285 1290

Val Tyr Leu Leu Gln Gly Arg Arg Leu Leu Gln Pro Glu Tyr Cys

1295 1300 1305

Pro Asp Pro Leu Tyr Glu Val Met Leu Lys Cys Trp His Pro Lys

1310 1315 1320

Ala Glu Met Arg Pro Ser Phe Ser Glu Leu Val Ser Arg Ile Ser

1325 1330 1335

Ala Ile Phe Ser Thr Phe Ile Gly Glu His Tyr Val His Val Asn

1340 1345 1350

Ala Thr Tyr Val Asn Val Lys Cys Val Ala Pro Tyr Pro Ser Leu

1355 1360 1365

Leu Ser Ser Glu Asp Asn Ala Asp Asp Glu Val Asp Thr

1370 1375 1380

<210> 69

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> peptide linker

<400> 69

Gly Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

1 5 10

<210> 70

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> peptide linker

<400> 70

Gly Pro Ala Pro Leu Gly Leu Gly Gly Pro Ser

1 5 10

<210> 71

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> peptide linker

<400> 71

Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

1 5 10

<210> 72

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> peptide linker

<400> 72

Pro Pro Cys Pro Ala Pro Leu Gly Leu Gly Gly Pro Ser

1 5 10

<210> 73

<211> 694

<212> PRT

<213> Artificial sequence Fc-Her2/CD3(34) heavy chain

<400> 73

Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe

1 5 10 15

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

20 25 30

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

35 40 45

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

50 55 60

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

65 70 75 80

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

85 90 95

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

100 105 110

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

115 120 125

Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val

130 135 140

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

145 150 155 160

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

165 170 175

Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

180 185 190

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

195 200 205

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Pro Ala

210 215 220

Pro Leu Gly Leu Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly

225 230 235 240

Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala

245 250 255

Ser Gly Phe Asn Ile Lys Asp Thr Tyr Ile His Trp Val Arg Gln Ala

260 265 270

Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Tyr Pro Thr Asn Gly

275 280 285

Tyr Thr Arg Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala

290 295 300

Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala

305 310 315 320

Glu Asp Thr Ala Val Tyr Tyr Cys Ser Arg Trp Gly Gly Asp Gly Phe

325 330 335

Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

340 345 350

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

355 360 365

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

370 375 380

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

385 390 395 400

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

405 410 415

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

420 425 430

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

435 440 445

Lys Val Glu Pro Lys Ser Cys Gly Pro Ala Pro Leu Gly Leu Gly Gly

450 455 460

Gly Ser Glu Val Lys Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro

465 470 475 480

Lys Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn

485 490 495

Thr Tyr Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu

500 505 510

Trp Val Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr

515 520 525

Ala Asp Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Gln

530 535 540

Ser Ile Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala

545 550 555 560

Met Tyr Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser

565 570 575

Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala

580 585 590

Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser

595 600 605

Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe

610 615 620

Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly

625 630 635 640

Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu

645 650 655

Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr

660 665 670

Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys

675 680 685

Val Glu Pro Lys Ser Cys

690

<210> 74

<211> 672

<212> PRT

<213> Artificial sequence Fc-Her2/CD3(34) light chain

<400> 74

Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe

1 5 10 15

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

20 25 30

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

35 40 45

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

50 55 60

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

65 70 75 80

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

85 90 95

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

100 105 110

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

115 120 125

Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val

130 135 140

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

145 150 155 160

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

165 170 175

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

180 185 190

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

195 200 205

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Pro Ala

210 215 220

Pro Leu Gly Leu Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro

225 230 235 240

Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Gln

245 250 255

Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro

260 265 270

Gly Lys Ala Pro Lys Leu Leu Ile Tyr Asp Ala Ser Asn Leu Glu Thr

275 280 285

Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

290 295 300

Phe Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Phe Cys

305 310 315 320

Gln His Phe Asp His Leu Pro Leu Ala Phe Gly Gly Gly Thr Lys Val

325 330 335

Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro

340 345 350

Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu

355 360 365

Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn

370 375 380

Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser

385 390 395 400

Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala

405 410 415

Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly

420 425 430

Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Gly Pro

435 440 445

Ala Pro Glu Leu Leu Gly Gly Gly Ser Gln Ala Val Val Thr Gln Glu

450 455 460

Ser Ala Leu Thr Thr Ser Pro Gly Glu Thr Val Thr Leu Thr Cys Arg

465 470 475 480

Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn Trp Val Gln

485 490 495

Glu Lys Pro Asp His Leu Phe Thr Gly Leu Ile Gly Gly Thr Asn Lys

500 505 510

Arg Ala Pro Gly Val Pro Ala Arg Phe Ser Gly Ser Leu Ile Gly Asp

515 520 525

Lys Ala Ala Leu Thr Ile Thr Gly Ala Gln Thr Glu Asp Glu Ala Ile

530 535 540

Tyr Phe Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly Gly Gly

545 550 555 560

Thr Lys Leu Thr Val Leu Gly Gln Pro Lys Ala Ala Pro Ser Val Thr

565 570 575

Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu

580 585 590

Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly Ala Val Thr Val Ala Trp

595 600 605

Lys Ala Asp Ser Ser Pro Val Lys Ala Gly Val Glu Thr Thr Thr Pro

610 615 620

Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu

625 630 635 640

Thr Pro Glu Gln Trp Lys Ser His Arg Ser Tyr Ser Cys Gln Val Thr

645 650 655

His Glu Gly Ser Thr Val Glu Lys Thr Val Ala Pro Thr Glu Cys Ser

660 665 670

<210> 75

<211> 350

<212> PRT

<213> Artificial sequence Fab2 CMET/CMET Single-arm heavy chain

<400> 75

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Thr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gln Glu Ile Thr Thr Glu Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Gly Pro Ala

210 215 220

Pro Glu Leu Leu Gly Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly

225 230 235 240

Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala

245 250 255

Ser Gly Tyr Thr Phe Thr Ser Tyr Thr Met His Trp Val Arg Gln Ala

260 265 270

Pro Gly Gln Gly Leu Glu Trp Met Gly Ile Ile Asn Pro Ser Gly Gly

275 280 285

Ser Thr Ser Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Met Thr Arg

290 295 300

Asp Thr Ser Thr Ser Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser

305 310 315 320

Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gln Glu Ile Thr Thr Glu

325 330 335

Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

340 345 350

<210> 76

<211> 340

<212> PRT

<213> Artificial sequence Fab2 CMET/CMET Single-arm light chain

<400> 76

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

Ala Asn Ser Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

35 40 45

Arg Leu Leu Ile Tyr Arg Gly Ser Thr Arg Glu Ser Gly Ile Pro Asp

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Lys

85 90 95

Ser Glu Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Gly Pro Ala Pro Glu Leu

210 215 220

Leu Gly Gly Gly Ser Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu

225 230 235 240

Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln

245 250 255

Ser Val Ser Ser Tyr Ala Asn Ser Tyr Leu His Trp Tyr Gln Gln Lys

260 265 270

Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Arg Gly Ser Thr Arg Glu

275 280 285

Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe

290 295 300

Thr Leu Thr Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr

305 310 315 320

Cys Gln Gln Ser Lys Ser Glu Pro Leu Thr Phe Gly Gly Gly Thr Lys

325 330 335

Val Glu Ile Lys

340

<210> 77

<211> 1255

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 77

Met Glu Leu Ala Ala Leu Cys Arg Trp Gly Leu Leu Leu Ala Leu Leu

1 5 10 15

Pro Pro Gly Ala Ala Ser Thr Gln Val Cys Thr Gly Thr Asp Met Lys

20 25 30

Leu Arg Leu Pro Ala Ser Pro Glu Thr His Leu Asp Met Leu Arg His

35 40 45

Leu Tyr Gln Gly Cys Gln Val Val Gln Gly Asn Leu Glu Leu Thr Tyr

50 55 60

Leu Pro Thr Asn Ala Ser Leu Ser Phe Leu Gln Asp Ile Gln Glu Val

65 70 75 80

Gln Gly Tyr Val Leu Ile Ala His Asn Gln Val Arg Gln Val Pro Leu

85 90 95

Gln Arg Leu Arg Ile Val Arg Gly Thr Gln Leu Phe Glu Asp Asn Tyr

100 105 110

Ala Leu Ala Val Leu Asp Asn Gly Asp Pro Leu Asn Asn Thr Thr Pro

115 120 125

Val Thr Gly Ala Ser Pro Gly Gly Leu Arg Glu Leu Gln Leu Arg Ser

130 135 140

Leu Thr Glu Ile Leu Lys Gly Gly Val Leu Ile Gln Arg Asn Pro Gln

145 150 155 160

Leu Cys Tyr Gln Asp Thr Ile Leu Trp Lys Asp Ile Phe His Lys Asn

165 170 175

Asn Gln Leu Ala Leu Thr Leu Ile Asp Thr Asn Arg Ser Arg Ala Cys

180 185 190

His Pro Cys Ser Pro Met Cys Lys Gly Ser Arg Cys Trp Gly Glu Ser

195 200 205

Ser Glu Asp Cys Gln Ser Leu Thr Arg Thr Val Cys Ala Gly Gly Cys

210 215 220

Ala Arg Cys Lys Gly Pro Leu Pro Thr Asp Cys Cys His Glu Gln Cys

225 230 235 240

Ala Ala Gly Cys Thr Gly Pro Lys His Ser Asp Cys Leu Ala Cys Leu

245 250 255

His Phe Asn His Ser Gly Ile Cys Glu Leu His Cys Pro Ala Leu Val

260 265 270

Thr Tyr Asn Thr Asp Thr Phe Glu Ser Met Pro Asn Pro Glu Gly Arg

275 280 285

Tyr Thr Phe Gly Ala Ser Cys Val Thr Ala Cys Pro Tyr Asn Tyr Leu

290 295 300

Ser Thr Asp Val Gly Ser Cys Thr Leu Val Cys Pro Leu His Asn Gln

305 310 315 320

Glu Val Thr Ala Glu Asp Gly Thr Gln Arg Cys Glu Lys Cys Ser Lys

325 330 335

Pro Cys Ala Arg Val Cys Tyr Gly Leu Gly Met Glu His Leu Arg Glu

340 345 350

Val Arg Ala Val Thr Ser Ala Asn Ile Gln Glu Phe Ala Gly Cys Lys

355 360 365

Lys Ile Phe Gly Ser Leu Ala Phe Leu Pro Glu Ser Phe Asp Gly Asp

370 375 380

Pro Ala Ser Asn Thr Ala Pro Leu Gln Pro Glu Gln Leu Gln Val Phe

385 390 395 400

Glu Thr Leu Glu Glu Ile Thr Gly Tyr Leu Tyr Ile Ser Ala Trp Pro

405 410 415

Asp Ser Leu Pro Asp Leu Ser Val Phe Gln Asn Leu Gln Val Ile Arg

420 425 430

Gly Arg Ile Leu His Asn Gly Ala Tyr Ser Leu Thr Leu Gln Gly Leu

435 440 445

Gly Ile Ser Trp Leu Gly Leu Arg Ser Leu Arg Glu Leu Gly Ser Gly

450 455 460

Leu Ala Leu Ile His His Asn Thr His Leu Cys Phe Val His Thr Val

465 470 475 480

Pro Trp Asp Gln Leu Phe Arg Asn Pro His Gln Ala Leu Leu His Thr

485 490 495

Ala Asn Arg Pro Glu Asp Glu Cys Val Gly Glu Gly Leu Ala Cys His

500 505 510

Gln Leu Cys Ala Arg Gly His Cys Trp Gly Pro Gly Pro Thr Gln Cys

515 520 525

Val Asn Cys Ser Gln Phe Leu Arg Gly Gln Glu Cys Val Glu Glu Cys

530 535 540

Arg Val Leu Gln Gly Leu Pro Arg Glu Tyr Val Asn Ala Arg His Cys

545 550 555 560

Leu Pro Cys His Pro Glu Cys Gln Pro Gln Asn Gly Ser Val Thr Cys

565 570 575

Phe Gly Pro Glu Ala Asp Gln Cys Val Ala Cys Ala His Tyr Lys Asp

580 585 590

Pro Pro Phe Cys Val Ala Arg Cys Pro Ser Gly Val Lys Pro Asp Leu

595 600 605

Ser Tyr Met Pro Ile Trp Lys Phe Pro Asp Glu Glu Gly Ala Cys Gln

610 615 620

Pro Cys Pro Ile Asn Cys Thr His Ser Cys Val Asp Leu Asp Asp Lys

625 630 635 640

Gly Cys Pro Ala Glu Gln Arg Ala Ser Pro Leu Thr Ser Ile Ile Ser

645 650 655

Ala Val Val Gly Ile Leu Leu Val Val Val Leu Gly Val Val Phe Gly

660 665 670

Ile Leu Ile Lys Arg Arg Gln Gln Lys Ile Arg Lys Tyr Thr Met Arg

675 680 685

Arg Leu Leu Gln Glu Thr Glu Leu Val Glu Pro Leu Thr Pro Ser Gly

690 695 700

Ala Met Pro Asn Gln Ala Gln Met Arg Ile Leu Lys Glu Thr Glu Leu

705 710 715 720

Arg Lys Val Lys Val Leu Gly Ser Gly Ala Phe Gly Thr Val Tyr Lys

725 730 735

Gly Ile Trp Ile Pro Asp Gly Glu Asn Val Lys Ile Pro Val Ala Ile

740 745 750

Lys Val Leu Arg Glu Asn Thr Ser Pro Lys Ala Asn Lys Glu Ile Leu

755 760 765

Asp Glu Ala Tyr Val Met Ala Gly Val Gly Ser Pro Tyr Val Ser Arg

770 775 780

Leu Leu Gly Ile Cys Leu Thr Ser Thr Val Gln Leu Val Thr Gln Leu

785 790 795 800

Met Pro Tyr Gly Cys Leu Leu Asp His Val Arg Glu Asn Arg Gly Arg

805 810 815

Leu Gly Ser Gln Asp Leu Leu Asn Trp Cys Met Gln Ile Ala Lys Gly

820 825 830

Met Ser Tyr Leu Glu Asp Val Arg Leu Val His Arg Asp Leu Ala Ala

835 840 845

Arg Asn Val Leu Val Lys Ser Pro Asn His Val Lys Ile Thr Asp Phe

850 855 860

Gly Leu Ala Arg Leu Leu Asp Ile Asp Glu Thr Glu Tyr His Ala Asp

865 870 875 880

Gly Gly Lys Val Pro Ile Lys Trp Met Ala Leu Glu Ser Ile Leu Arg

885 890 895

Arg Arg Phe Thr His Gln Ser Asp Val Trp Ser Tyr Gly Val Thr Val

900 905 910

Trp Glu Leu Met Thr Phe Gly Ala Lys Pro Tyr Asp Gly Ile Pro Ala

915 920 925

Arg Glu Ile Pro Asp Leu Leu Glu Lys Gly Glu Arg Leu Pro Gln Pro

930 935 940

Pro Ile Cys Thr Ile Asp Val Tyr Met Ile Met Val Lys Cys Trp Met

945 950 955 960

Ile Asp Ser Glu Cys Arg Pro Arg Phe Arg Glu Leu Val Ser Glu Phe

965 970 975

Ser Arg Met Ala Arg Asp Pro Gln Arg Phe Val Val Ile Gln Asn Glu

980 985 990

Asp Leu Gly Pro Ala Ser Pro Leu Asp Ser Thr Phe Tyr Arg Ser Leu

995 1000 1005

Leu Glu Asp Asp Asp Met Gly Asp Leu Val Asp Ala Glu Glu Tyr

1010 1015 1020

Leu Val Pro Gln Gln Gly Phe Phe Cys Pro Asp Pro Ala Pro Gly

1025 1030 1035

Ala Gly Gly Met Val His His Arg His Arg Ser Ser Ser Thr Arg

1040 1045 1050

Ser Gly Gly Gly Asp Leu Thr Leu Gly Leu Glu Pro Ser Glu Glu

1055 1060 1065

Glu Ala Pro Arg Ser Pro Leu Ala Pro Ser Glu Gly Ala Gly Ser

1070 1075 1080

Asp Val Phe Asp Gly Asp Leu Gly Met Gly Ala Ala Lys Gly Leu

1085 1090 1095

Gln Ser Leu Pro Thr His Asp Pro Ser Pro Leu Gln Arg Tyr Ser

1100 1105 1110

Glu Asp Pro Thr Val Pro Leu Pro Ser Glu Thr Asp Gly Tyr Val

1115 1120 1125

Ala Pro Leu Thr Cys Ser Pro Gln Pro Glu Tyr Val Asn Gln Pro

1130 1135 1140

Asp Val Arg Pro Gln Pro Pro Ser Pro Arg Glu Gly Pro Leu Pro

1145 1150 1155

Ala Ala Arg Pro Ala Gly Ala Thr Leu Glu Arg Pro Lys Thr Leu

1160 1165 1170

Ser Pro Gly Lys Asn Gly Val Val Lys Asp Val Phe Ala Phe Gly

1175 1180 1185

Gly Ala Val Glu Asn Pro Glu Tyr Leu Thr Pro Gln Gly Gly Ala

1190 1195 1200

Ala Pro Gln Pro His Pro Pro Pro Ala Phe Ser Pro Ala Phe Asp

1205 1210 1215

Asn Leu Tyr Tyr Trp Asp Gln Asp Pro Pro Glu Arg Gly Ala Pro

1220 1225 1230

Pro Ser Thr Phe Lys Gly Thr Pro Thr Ala Glu Asn Pro Glu Tyr

1235 1240 1245

Leu Gly Leu Asp Val Pro Val

1250 1255

<210> 78

<211> 1406

<212> PRT

<213> monkey fasciculation (Macaca fascicularis)

<400> 78

Met Ala Glu Ser Pro Ala Ser Ala Phe Arg Asp Ser Leu Arg Lys Ser

1 5 10 15

Val Arg Thr Ala Ala Gly Asn Pro Gly Val Pro Glu Leu Gly Gly Thr

20 25 30

His Pro Gly Leu Arg Glu Glu Arg Glu Lys Val Lys Leu Gly Val Ala

35 40 45

Thr Pro Arg Leu Val Gly Met Gln Leu Glu Gly Ala Ser Trp Glu Arg

50 55 60

Ala Cys Ser Gln Ser Gln Glu Glu Glu Glu Val Glu Glu Glu Gly Cys

65 70 75 80

Leu Arg Lys Tyr Lys Asn Glu Val Val Glu Leu Arg Phe Pro Ser Ile

85 90 95

Gly Thr Gly Glu Thr Arg Gly Ala Pro Trp Ala Ala Val Arg Pro Phe

100 105 110

Pro Arg Gly Ser Phe Arg Arg Arg Ala Pro Gly Pro His Pro Ser Pro

115 120 125

His Pro Ala Pro His Ala Leu Pro Ala Gly Ser Ser Arg Ser His Gly

130 135 140

Ala Gly Ala Ala Val Ser Thr Met Glu Leu Ala Ala Trp Tyr Arg Trp

145 150 155 160

Gly Leu Leu Leu Ala Leu Leu Pro Pro Gly Ala Thr Gly Thr Gln Val

165 170 175

Cys Thr Gly Thr Asp Met Lys Leu Arg Leu Pro Ala Ser Pro Glu Thr

180 185 190

His Leu Asp Met Leu Arg His Leu Tyr Gln Gly Cys Gln Val Val Gln

195 200 205

Gly Asn Leu Glu Leu Thr Tyr Leu Pro Thr Asn Ala Ser Leu Ser Phe

210 215 220

Leu Gln Asp Ile Gln Glu Val Gln Gly Tyr Val Leu Ile Ala His Asn

225 230 235 240

Gln Val Arg Gln Val Pro Leu Gln Arg Leu Arg Ile Val Arg Gly Thr

245 250 255

Gln Leu Phe Glu Asp Asn Tyr Ala Leu Ala Val Leu Asp Asn Gly Asp

260 265 270

Pro Leu Asn Asn Thr Thr Pro Val Thr Gly Ala Ser Pro Gly Gly Leu

275 280 285

Arg Glu Leu Gln Leu Arg Ser Leu Thr Glu Ile Leu Lys Gly Gly Val

290 295 300

Leu Ile Gln Arg Asn Pro Gln Leu Cys Tyr Gln Asp Thr Ile Leu Trp

305 310 315 320

Lys Asp Ile Phe His Lys Asn Asn Gln Leu Ala Leu Thr Leu Ile Asp

325 330 335

Thr Asn Arg Ser Arg Ala Cys His Pro Cys Ser Pro Val Cys Lys Gly

340 345 350

Ser Arg Cys Trp Gly Glu Ser Ser Glu Asp Cys Gln Ser Leu Thr Arg

355 360 365

Thr Val Cys Ala Gly Gly Cys Ala Arg Cys Lys Gly Pro Leu Pro Thr

370 375 380

Asp Cys Cys His Glu Gln Cys Ala Ala Gly Cys Thr Gly Pro Lys His

385 390 395 400

Ser Asp Cys Leu Ala Cys Leu His Phe Asn His Ser Gly Ile Cys Glu

405 410 415

Leu His Cys Pro Ala Leu Val Thr Tyr Asn Thr Asp Thr Phe Glu Ser

420 425 430

Met Pro Asn Pro Glu Gly Arg Tyr Thr Phe Gly Ala Ser Cys Val Thr

435 440 445

Ala Cys Pro Tyr Asn Tyr Leu Ser Thr Asp Val Gly Ser Cys Thr Leu

450 455 460

Val Cys Pro Leu His Asn Gln Glu Val Thr Ala Glu Asp Gly Thr Gln

465 470 475 480

Arg Cys Glu Lys Cys Ser Lys Pro Cys Ala Arg Val Cys Tyr Gly Leu

485 490 495

Gly Met Glu His Leu Arg Glu Val Arg Ala Val Thr Ser Ala Asn Ile

500 505 510

Gln Glu Phe Ala Gly Cys Lys Lys Ile Phe Gly Ser Leu Ala Phe Leu

515 520 525

Pro Glu Ser Phe Asp Gly Asp Pro Ala Ser Asn Thr Ala Pro Leu Gln

530 535 540

Pro Glu Gln Leu Arg Val Phe Glu Thr Leu Glu Glu Ile Thr Gly Tyr

545 550 555 560

Leu Tyr Ile Ser Ala Trp Pro Asp Ser Leu Pro Asp Leu Ser Val Leu

565 570 575

Gln Asn Leu Gln Val Ile Arg Gly Arg Ile Leu His Asn Gly Ala Tyr

580 585 590

Ser Leu Thr Leu Gln Gly Leu Gly Ile Ser Trp Leu Gly Leu Arg Ser

595 600 605

Leu Arg Glu Leu Gly Ser Gly Leu Ala Leu Ile His His Asn Thr Arg

610 615 620

Leu Cys Phe Val His Thr Val Pro Trp Asp Gln Leu Phe Arg Asn Pro

625 630 635 640

His Gln Ala Leu Leu His Thr Ala Asn Arg Pro Glu Asp Glu Cys Val

645 650 655

Gly Glu Gly Leu Ala Cys His Gln Leu Cys Ala Arg Gly His Cys Trp

660 665 670

Gly Pro Gly Pro Thr Gln Cys Val Asn Cys Ser Gln Phe Leu Arg Gly

675 680 685

Gln Glu Cys Val Glu Glu Cys Arg Val Leu Gln Gly Leu Pro Arg Glu

690 695 700

Tyr Val Asn Ala Arg His Cys Leu Pro Cys His Pro Glu Cys Gln Pro

705 710 715 720

Gln Asn Gly Ser Val Thr Cys Phe Gly Pro Glu Ala Asp Gln Cys Val

725 730 735

Ala Cys Ala His Tyr Lys Asp Pro Pro Phe Cys Val Ala Arg Cys Pro

740 745 750

Ser Gly Val Lys Pro Asp Leu Ser Tyr Met Pro Ile Trp Lys Phe Pro

755 760 765

Asp Glu Glu Gly Thr Cys Gln Ser Cys Pro Ile Asn Cys Thr His Ser

770 775 780

Cys Val Asp Leu Asp Asp Lys Gly Cys Pro Ala Glu Gln Arg Ala Ser

785 790 795 800

Pro Leu Thr Ser Ile Ile Ser Ala Val Val Gly Ile Leu Leu Val Val

805 810 815

Val Leu Gly Val Val Phe Gly Ile Leu Ile Lys Arg Arg Gln Gln Lys

820 825 830

Ile Arg Lys Tyr Thr Met Arg Arg Leu Leu Gln Glu Thr Glu Leu Val

835 840 845

Glu Pro Leu Thr Pro Ser Gly Ala Met Pro Asn Gln Ala Gln Met Arg

850 855 860

Ile Leu Lys Glu Thr Glu Leu Arg Lys Val Lys Val Leu Gly Ser Gly

865 870 875 880

Ala Phe Gly Thr Val Tyr Lys Gly Ile Trp Ile Pro Asp Gly Glu Asn

885 890 895

Val Lys Ile Pro Val Ala Ile Lys Val Leu Arg Glu Asn Thr Ser Pro

900 905 910

Lys Ala Asn Lys Glu Ile Leu Asp Glu Ala Tyr Val Met Ala Gly Val

915 920 925

Gly Ser Pro Tyr Val Ser Arg Leu Leu Gly Ile Cys Leu Thr Ser Thr

930 935 940

Val Gln Leu Val Thr Gln Leu Met Pro Tyr Gly Cys Leu Leu Asp His

945 950 955 960

Val Arg Glu Asn Arg Gly Arg Leu Gly Ser Gln Asp Leu Leu Asn Trp

965 970 975

Cys Met Gln Ile Ala Lys Gly Met Ser Tyr Leu Glu Asp Val Arg Leu

980 985 990

Val His Arg Asp Leu Ala Ala Arg Asn Val Leu Val Lys Ser Pro Asn

995 1000 1005

His Val Lys Ile Thr Asp Phe Gly Leu Ala Arg Leu Leu Asp Ile

1010 1015 1020

Asp Glu Thr Glu Tyr His Ala Asp Gly Gly Lys Val Pro Ile Lys

1025 1030 1035

Trp Met Ala Leu Glu Ser Ile Leu Arg Arg Arg Phe Thr His Gln

1040 1045 1050

Ser Asp Val Trp Ser Tyr Gly Val Thr Val Trp Glu Leu Met Thr

1055 1060 1065

Phe Gly Ala Lys Pro Tyr Asp Gly Ile Pro Ala Arg Glu Ile Pro

1070 1075 1080

Asp Leu Leu Glu Lys Gly Glu Arg Leu Pro Gln Pro Pro Ile Cys

1085 1090 1095

Thr Ile Asp Val Tyr Met Ile Met Val Lys Cys Trp Met Ile Asp

1100 1105 1110

Ser Glu Cys Arg Pro Arg Phe Arg Glu Leu Val Ser Glu Phe Ser

1115 1120 1125

Arg Met Ala Arg Asp Pro Gln Arg Phe Val Val Ile Gln Asn Glu

1130 1135 1140

Asp Leu Gly Pro Ala Ser Pro Leu Asp Ser Thr Phe Tyr Arg Ser

1145 1150 1155

Leu Leu Glu Asp Asp Asp Met Gly Asp Leu Val Asp Ala Glu Glu

1160 1165 1170

Tyr Leu Val Pro Gln Gln Gly Phe Phe Cys Pro Asp Pro Ala Pro

1175 1180 1185

Gly Thr Gly Gly Met Val His His Arg His Arg Ser Ser Ser Thr

1190 1195 1200

Arg Ser Gly Gly Gly Asp Leu Thr Leu Gly Leu Glu Pro Ser Glu

1205 1210 1215

Glu Glu Ala Pro Arg Ser Pro Arg Ala Pro Ser Glu Gly Thr Gly

1220 1225 1230

Ser Asp Val Phe Asp Gly Asp Leu Gly Met Gly Ala Ala Lys Gly

1235 1240 1245

Leu Gln Ser Leu Pro Ala His Asp Pro Ser Pro Leu Gln Arg Tyr

1250 1255 1260

Ser Glu Asp Pro Thr Val Pro Leu Pro Ser Glu Thr Asp Gly Tyr

1265 1270 1275

Val Ala Pro Leu Thr Cys Ser Pro Gln Pro Glu Tyr Val Asn Gln

1280 1285 1290

Pro Asp Val Arg Pro Gln Pro Pro Ser Pro Gln Glu Gly Pro Leu

1295 1300 1305

Ser Pro Ala Arg Pro Thr Gly Ala Thr Leu Glu Arg Pro Lys Thr

1310 1315 1320

Leu Ser Pro Gly Lys Asn Gly Val Val Lys Asp Val Phe Ala Phe

1325 1330 1335

Gly Gly Ala Val Glu Asn Pro Glu Tyr Leu Ala Pro Arg Gly Gly

1340 1345 1350

Ala Ala Pro Gln Pro His Leu Pro Pro Ala Phe Ser Pro Ala Phe

1355 1360 1365

Asp Asn Leu Tyr Tyr Trp Asp Gln Asp Pro Ser Glu Arg Gly Ala

1370 1375 1380

Pro Pro Ser Thr Phe Lys Gly Thr Pro Thr Ala Glu Asn Pro Glu

1385 1390 1395

Tyr Leu Gly Leu Asp Val Pro Val

1400 1405

<210> 79

<211> 206

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 79

Met Gln Ser Gly Thr His Trp Arg Val Leu Gly Leu Cys Leu Leu Ser

1 5 10 15

Val Gly Val Trp Gly Gln Asp Gly Asn Glu Glu Met Gly Gly Ile Thr

20 25 30

Gln Thr Pro Tyr Lys Val Ser Ile Ser Gly Thr Thr Val Ile Leu Thr

35 40 45

Cys Pro Gln Tyr Pro Gly Ser Glu Ile Leu Gln His Asn Asp Lys Asn

50 55 60

Ile Gly Gly Asp Glu Asp Asp Lys Asn Ile Gly Ser Asp Glu Asp His

65 70 75 80

Leu Ser Leu Lys Glu Phe Ser Glu Leu Glu Gln Ser Gly Tyr Tyr Val

85 90 95

Cys Tyr Pro Arg Gly Ser Lys Pro Glu Asp Ala Asn Phe Tyr Leu Tyr

100 105 110

Leu Arg Ala Arg Val Cys Glu Asn Cys Met Glu Met Asp Val Met Ser

115 120 125

Val Ala Thr Ile Val Ile Val Asp Ile Cys Ile Thr Gly Gly Leu Leu

130 135 140

Leu Leu Val Tyr Tyr Trp Ser Lys Asn Arg Lys Ala Lys Ala Lys Pro

145 150 155 160

Val Thr Arg Gly Ala Gly Ala Gly Gly Arg Gln Arg Gly Gln Asn Lys

165 170 175

Glu Arg Pro Pro Pro Val Pro Asn Pro Asp Tyr Glu Pro Ile Arg Lys

180 185 190

Gly Gln Arg Asp Leu Tyr Ser Gly Leu Asn Gln Arg Arg Ile

195 200 205

<210> 80

<211> 198

<212> PRT

<213> monkey fasciculation (Macaca fascicularis)

<400> 80

Met Gln Ser Gly Thr Arg Trp Arg Val Leu Gly Leu Cys Leu Leu Ser

1 5 10 15

Ile Gly Val Trp Gly Gln Asp Gly Asn Glu Glu Met Gly Ser Ile Thr

20 25 30

Gln Thr Pro Tyr Gln Val Ser Ile Ser Gly Thr Thr Val Ile Leu Thr

35 40 45

Cys Ser Gln His Leu Gly Ser Glu Ala Gln Trp Gln His Asn Gly Lys

50 55 60

Asn Lys Glu Asp Ser Gly Asp Arg Leu Phe Leu Pro Glu Phe Ser Glu

65 70 75 80

Met Glu Gln Ser Gly Tyr Tyr Val Cys Tyr Pro Arg Gly Ser Asn Pro

85 90 95

Glu Asp Ala Ser His His Leu Tyr Leu Lys Ala Arg Val Cys Glu Asn

100 105 110

Cys Met Glu Met Asp Val Met Ala Val Ala Thr Ile Val Ile Val Asp

115 120 125

Ile Cys Ile Thr Leu Gly Leu Leu Leu Leu Val Tyr Tyr Trp Ser Lys

130 135 140

Asn Arg Lys Ala Lys Ala Lys Pro Val Thr Arg Gly Ala Gly Ala Gly

145 150 155 160

Gly Arg Gln Arg Gly Gln Asn Lys Glu Arg Pro Pro Pro Val Pro Asn

165 170 175

Pro Asp Tyr Glu Pro Ile Arg Lys Gly Gln Gln Asp Leu Tyr Ser Gly

180 185 190

Leu Asn Gln Arg Arg Ile

195

<210> 81

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> peptide linker

<400> 81

Gly Pro Ala Pro Glu Ala Ala Gly Ala Gly Ser

1 5 10

<210> 82

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> peptide linker

<400> 82

Gly Pro Ala Asp Asp Asp Asp Lys Ser Gly Ser

1 5 10

<210> 83

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> peptide linker

<400> 83

Gly Pro Ala Leu Val Pro Arg Gly Ser Gly Ser

1 5 10

<210> 84

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> peptide linker

<400> 84

Gly Pro Gly Pro Phe Gly Arg Ser Ala Gly Gly Pro

1 5 10

<210> 85

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> peptide linker

<400> 85

Gly Pro Ala Pro Leu Glu Ala Asp Ala Gly Ser

1 5 10

<210> 86

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> peptide linker

<400> 86

Gly Pro Ala Pro Glu Ala Arg Arg Gly Gly Ser

1 5 10

<210> 87

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> peptide linker

<400> 87

Gly Pro Ala Pro Glu Gly Glu Ala Arg Gly Ser

1 5 10

<210> 88

<211> 678

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> IgG2 IgG1-DAA Her2/CD47 heavy chain

<400> 88

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Gly

210 215 220

Pro Ala Pro Leu Gly Leu Gly Gly Gly Ser Gln Val Gln Leu Val Gln

225 230 235 240

Ser Gly Ala Glu Val Lys Lys Pro Gly Glu Ser Leu Lys Ile Ser Cys

245 250 255

Lys Gly Ser Gly Tyr Thr Phe Thr Asn Tyr Tyr Ile Phe Trp Val Arg

260 265 270

Gln Met Pro Gly Lys Gly Leu Glu Trp Met Gly Ile Ile Asn Pro Val

275 280 285

Asp Gly Asp Thr Asn Tyr Asn Pro Ser Phe Gln Gly Gln Val Thr Ile

290 295 300

Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr Leu Gln Trp Ser Ser Leu

305 310 315 320

Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys Ala Arg Gly Gly Tyr Thr

325 330 335

Met Asp Arg Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser

340 345 350

Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr

355 360 365

Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro

370 375 380

Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val

385 390 395 400

His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser

405 410 415

Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile

420 425 430

Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val

435 440 445

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

450 455 460

Pro Pro Val Ala Gly Pro Asp Val Phe Leu Phe Pro Pro Lys Pro Lys

465 470 475 480

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

485 490 495

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

500 505 510

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

515 520 525

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

530 535 540

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Ala Ala Leu

545 550 555 560

Pro Ala Pro Ile Ala Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

565 570 575

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys

580 585 590

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

595 600 605

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

610 615 620

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

625 630 635 640

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

645 650 655

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

660 665 670

Leu Ser Leu Ser Pro Gly

675

<210> 89

<211> 444

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> IgG2 IgG1-DAA Her2/CD47 light chain

<400> 89

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys Gly Pro Ala Pro Glu Leu Leu Gly Gly Gly

210 215 220

Ser Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro

225 230 235 240

Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His

245 250 255

Ser Asn Gly Tyr Asn Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln

260 265 270

Ser Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val

275 280 285

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys

290 295 300

Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln

305 310 315 320

Asn Thr His Thr Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

325 330 335

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

340 345 350

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

355 360 365

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

370 375 380

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

385 390 395 400

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

405 410 415

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

420 425 430

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

435 440

<210> 90

<211> 678

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> IgG2 IgG1-DAA Her2/CD47 heavy chain

<400> 90

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Gly

210 215 220

Pro Ala Pro Glu Leu Leu Gly Gly Gly Ser Gln Val Gln Leu Val Gln

225 230 235 240

Ser Gly Ala Glu Val Lys Lys Pro Gly Glu Ser Leu Lys Ile Ser Cys

245 250 255

Lys Gly Ser Gly Tyr Thr Phe Thr Asn Tyr Tyr Ile Phe Trp Val Arg

260 265 270

Gln Met Pro Gly Lys Gly Leu Glu Trp Met Gly Ile Ile Asn Pro Val

275 280 285

Asp Gly Asp Thr Asn Tyr Asn Pro Ser Phe Gln Gly Gln Val Thr Ile

290 295 300

Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr Leu Gln Trp Ser Ser Leu

305 310 315 320

Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys Ala Arg Gly Gly Tyr Thr

325 330 335

Met Asp Arg Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser

340 345 350

Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr

355 360 365

Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro

370 375 380

Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val

385 390 395 400

His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser

405 410 415

Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile

420 425 430

Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val

435 440 445

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

450 455 460

Pro Pro Val Ala Gly Pro Asp Val Phe Leu Phe Pro Pro Lys Pro Lys

465 470 475 480

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

485 490 495

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

500 505 510

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

515 520 525

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

530 535 540

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Ala Ala Leu

545 550 555 560

Pro Ala Pro Ile Ala Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

565 570 575

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys

580 585 590

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

595 600 605

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

610 615 620

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

625 630 635 640

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

645 650 655

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

660 665 670

Leu Ser Leu Ser Pro Gly

675

<210> 91

<211> 444

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> IgG2 IgG1-DAA Her2/CD47 light chain

<400> 91

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys Gly Pro Ala Pro Leu Gly Leu Gly Gly Gly

210 215 220

Ser Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro

225 230 235 240

Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His

245 250 255

Ser Asn Gly Tyr Asn Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln

260 265 270

Ser Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val

275 280 285

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys

290 295 300

Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln

305 310 315 320

Asn Thr His Thr Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

325 330 335

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

340 345 350

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

355 360 365

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

370 375 380

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

385 390 395 400

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

405 410 415

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

420 425 430

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

435 440

<210> 92

<211> 337

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> IgG2 Her2/CD47 light chain

<400> 92

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys Gly Pro Ala Asp Asp Asp Asp Lys Ser Gly

210 215 220

Ser Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro

225 230 235 240

Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His

245 250 255

Ser Asn Gly Tyr Asn Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln

260 265 270

Ser Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val

275 280 285

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys

290 295 300

Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln

305 310 315 320

Asn Thr His Thr Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

325 330 335

Lys

<210> 93

<211> 337

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> IgG2 Her2/CD47 light chain

<400> 93

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys Gly Pro Ala Leu Val Pro Arg Gly Ser Gly

210 215 220

Ser Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro

225 230 235 240

Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His

245 250 255

Ser Asn Gly Tyr Asn Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln

260 265 270

Ser Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val

275 280 285

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys

290 295 300

Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln

305 310 315 320

Asn Thr His Thr Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

325 330 335

Lys

<210> 94

<211> 338

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> IgG2 Her2/CD47 light chain

<400> 94

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys Gly Pro Gly Pro Phe Gly Arg Ser Ala Gly

210 215 220

Gly Pro Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr

225 230 235 240

Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu

245 250 255

His Ser Asn Gly Tyr Asn Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly

260 265 270

Gln Ser Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly

275 280 285

Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu

290 295 300

Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe

305 310 315 320

Gln Asn Thr His Thr Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu

325 330 335

Ile Lys

<210> 95

<211> 337

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> IgG2 Her2/CD47 light chain

<400> 95

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys Gly Pro Ala Pro Leu Glu Ala Asp Ala Gly

210 215 220

Ser Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro

225 230 235 240

Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His

245 250 255

Ser Asn Gly Tyr Asn Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln

260 265 270

Ser Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val

275 280 285

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys

290 295 300

Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln

305 310 315 320

Asn Thr His Thr Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

325 330 335

Lys

<210> 96

<211> 337

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> IgG2 Her2/CD47 light chain

<400> 96

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys Gly Pro Ala Pro Glu Ala Arg Arg Gly Gly

210 215 220

Ser Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro

225 230 235 240

Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His

245 250 255

Ser Asn Gly Tyr Asn Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln

260 265 270

Ser Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val

275 280 285

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys

290 295 300

Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln

305 310 315 320

Asn Thr His Thr Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

325 330 335

Lys

<210> 97

<211> 337

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> IgG2 Her2/CD47 light chain

<400> 97

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys Gly Pro Ala Pro Glu Gly Glu Ala Arg Gly

210 215 220

Ser Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro

225 230 235 240

Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His

245 250 255

Ser Asn Gly Tyr Asn Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln

260 265 270

Ser Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val

275 280 285

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys

290 295 300

Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln

305 310 315 320

Asn Thr His Thr Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

325 330 335

Lys

<210> 98

<211> 674

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Fab2 Her2/CD3 heavy chain

<400> 98

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Gly

210 215 220

Pro Ala Pro Glu Leu Leu Gly Gly Gly Ser Gln Val Gln Leu Gln Gln

225 230 235 240

Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala Ser Val Lys Met Ser Cys

245 250 255

Lys Ala Ser Gly Tyr Thr Phe Thr Arg Tyr Thr Met His Trp Val Lys

260 265 270

Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser

275 280 285

Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu

290 295 300

Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu

305 310 315 320

Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp

325 330 335

His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser

340 345 350

Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser

355 360 365

Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp

370 375 380

Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr

385 390 395 400

Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr

405 410 415

Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln

420 425 430

Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp

435 440 445

Lys Lys Val Glu Pro Lys Ser Cys Gly Pro Ala Pro Glu Ala Ala Gly

450 455 460

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

465 470 475 480

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

485 490 495

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

500 505 510

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

515 520 525

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

530 535 540

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

545 550 555 560

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

565 570 575

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

580 585 590

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

595 600 605

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

610 615 620

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

625 630 635 640

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

645 650 655

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

660 665 670

Pro Gly

<210> 99

<211> 656

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Fab2 Her2/CD3 light chain

<400> 99

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys Gly Pro Ala Pro Leu Gly Leu Gly Gly Gly

210 215 220

Ser Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro

225 230 235 240

Gly Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr

245 250 255

Met Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile

260 265 270

Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala His Phe Arg Gly

275 280 285

Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Gly Met Glu Ala

290 295 300

Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Phe

305 310 315 320

Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

325 330 335

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

340 345 350

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

355 360 365

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

370 375 380

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

385 390 395 400

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

405 410 415

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

420 425 430

Phe Asn Arg Gly Glu Cys Gly Pro Ala Pro Glu Ala Ala Gly Ala Pro

435 440 445

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

450 455 460

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

465 470 475 480

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

485 490 495

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

500 505 510

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

515 520 525

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

530 535 540

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

545 550 555 560

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp

565 570 575

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

580 585 590

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

595 600 605

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

610 615 620

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

625 630 635 640

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

645 650 655

<210> 100

<211> 674

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Fab2 Her2/CD3 heavy chain

<400> 100

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Gly

210 215 220

Pro Ala Pro Glu Leu Leu Gly Gly Gly Ser Gln Val Gln Leu Gln Gln

225 230 235 240

Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala Ser Val Lys Met Ser Cys

245 250 255

Lys Ala Ser Gly Tyr Thr Phe Thr Arg Tyr Thr Met His Trp Val Lys

260 265 270

Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser

275 280 285

Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu

290 295 300

Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu

305 310 315 320

Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp

325 330 335

His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser

340 345 350

Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser

355 360 365

Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp

370 375 380

Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr

385 390 395 400

Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr

405 410 415

Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln

420 425 430

Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp

435 440 445

Lys Lys Val Glu Pro Lys Ser Cys Gly Pro Ala Pro Glu Ala Ala Gly

450 455 460

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

465 470 475 480

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

485 490 495

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

500 505 510

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

515 520 525

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

530 535 540

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

545 550 555 560

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

565 570 575

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

580 585 590

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

595 600 605

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

610 615 620

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

625 630 635 640

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

645 650 655

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

660 665 670

Pro Gly

<210> 101

<211> 656

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Fab2 Her2/CD3 light chain

<400> 101

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys Gly Pro Ala Pro Glu Leu Leu Gly Gly Gly

210 215 220

Ser Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro

225 230 235 240

Gly Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr

245 250 255

Met Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile

260 265 270

Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala His Phe Arg Gly

275 280 285

Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Gly Met Glu Ala

290 295 300

Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Phe

305 310 315 320

Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

325 330 335

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

340 345 350

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

355 360 365

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

370 375 380

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

385 390 395 400

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

405 410 415

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

420 425 430

Phe Asn Arg Gly Glu Cys Gly Pro Ala Pro Glu Ala Ala Gly Ala Pro

435 440 445

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

450 455 460

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

465 470 475 480

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

485 490 495

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

500 505 510

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

515 520 525

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

530 535 540

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

545 550 555 560

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp

565 570 575

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

580 585 590

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

595 600 605

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

610 615 620

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

625 630 635 640

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

645 650 655

<210> 102

<211> 665

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Fab2 Her2/CD3 heavy chain

<400> 102

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys Gly Pro Ala Pro Leu Gly Leu Gly Gly Gly

210 215 220

Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly

225 230 235 240

Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg

245 250 255

Tyr Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp

260 265 270

Ile Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys

275 280 285

Phe Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala

290 295 300

Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr

305 310 315 320

Cys Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln

325 330 335

Gly Thr Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

340 345 350

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

355 360 365

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

370 375 380

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

385 390 395 400

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

405 410 415

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

420 425 430

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Gly

435 440 445

Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro

450 455 460

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

465 470 475 480

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

485 490 495

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

500 505 510

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

515 520 525

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

530 535 540

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

545 550 555 560

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

565 570 575

Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr

580 585 590

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

595 600 605

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

610 615 620

Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

625 630 635 640

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

645 650 655

Gln Lys Ser Leu Ser Leu Ser Pro Gly

660 665

<210> 103

<211> 665

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Fab2 Her2/CD3 light chain

<400> 103

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Gly

210 215 220

Pro Ala Pro Glu Leu Leu Gly Gly Gly Ser Gln Ile Val Leu Thr Gln

225 230 235 240

Ser Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr

245 250 255

Cys Ser Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys

260 265 270

Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Leu Ala

275 280 285

Ser Gly Val Pro Ala His Phe Arg Gly Ser Gly Ser Gly Thr Ser Tyr

290 295 300

Ser Leu Thr Ile Ser Gly Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr

305 310 315 320

Cys Gln Gln Trp Ser Ser Asn Pro Phe Thr Phe Gly Ser Gly Thr Lys

325 330 335

Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro

340 345 350

Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu

355 360 365

Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp

370 375 380

Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp

385 390 395 400

Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys

405 410 415

Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln

420 425 430

Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Gly

435 440 445

Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro

450 455 460

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

465 470 475 480

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

485 490 495

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

500 505 510

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

515 520 525

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

530 535 540

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

545 550 555 560

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

565 570 575

Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr

580 585 590

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

595 600 605

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

610 615 620

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

625 630 635 640

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

645 650 655

Gln Lys Ser Leu Ser Leu Ser Pro Gly

660 665

<210> 104

<211> 665

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Fab2 Her2/CD3 heavy chain

<400> 104

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys Gly Pro Ala Pro Glu Leu Leu Gly Gly Gly

210 215 220

Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly

225 230 235 240

Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg

245 250 255

Tyr Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp

260 265 270

Ile Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys

275 280 285

Phe Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala

290 295 300

Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr

305 310 315 320

Cys Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln

325 330 335

Gly Thr Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

340 345 350

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

355 360 365

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

370 375 380

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

385 390 395 400

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

405 410 415

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

420 425 430

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Gly

435 440 445

Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro

450 455 460

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

465 470 475 480

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

485 490 495

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

500 505 510

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

515 520 525

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

530 535 540

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

545 550 555 560

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

565 570 575

Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr

580 585 590

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

595 600 605

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

610 615 620

Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

625 630 635 640

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

645 650 655

Gln Lys Ser Leu Ser Leu Ser Pro Gly

660 665

<210> 105

<211> 665

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Fab2 Her2/CD3 light chain

<400> 105

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Gly

210 215 220

Pro Ala Pro Glu Leu Leu Gly Gly Gly Ser Gln Ile Val Leu Thr Gln

225 230 235 240

Ser Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr

245 250 255

Cys Ser Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys

260 265 270

Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Leu Ala

275 280 285

Ser Gly Val Pro Ala His Phe Arg Gly Ser Gly Ser Gly Thr Ser Tyr

290 295 300

Ser Leu Thr Ile Ser Gly Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr

305 310 315 320

Cys Gln Gln Trp Ser Ser Asn Pro Phe Thr Phe Gly Ser Gly Thr Lys

325 330 335

Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro

340 345 350

Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu

355 360 365

Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp

370 375 380

Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp

385 390 395 400

Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys

405 410 415

Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln

420 425 430

Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Gly

435 440 445

Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro

450 455 460

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

465 470 475 480

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

485 490 495

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

500 505 510

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

515 520 525

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

530 535 540

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

545 550 555 560

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

565 570 575

Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr

580 585 590

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

595 600 605

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

610 615 620

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

625 630 635 640

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

645 650 655

Gln Lys Ser Leu Ser Leu Ser Pro Gly

660 665

<210> 106

<211> 665

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Fab2 Her2/CD3 heavy chain

<400> 106

Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Glu Thr Val Ser Ile Glu Cys Leu Ala Ser Glu Gly Ile Ser Asn Asp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Ser Gly Lys Ser Pro Gln Leu Leu Ile

35 40 45

Tyr Ala Thr Ser Arg Leu Gln Asp Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Arg Tyr Ser Leu Lys Ile Ser Gly Met Gln Pro

65 70 75 80

Glu Asp Glu Ala Asp Tyr Phe Cys Gln Gln Ser Tyr Lys Tyr Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys Gly Pro Ala Pro Leu Gly Leu Gly Gly Gly

210 215 220

Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly

225 230 235 240

Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg

245 250 255

Tyr Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp

260 265 270

Ile Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys

275 280 285

Phe Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala

290 295 300

Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr

305 310 315 320

Cys Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln

325 330 335

Gly Thr Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

340 345 350

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

355 360 365

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

370 375 380

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

385 390 395 400

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

405 410 415

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

420 425 430

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Gly

435 440 445

Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro

450 455 460

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

465 470 475 480

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

485 490 495

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

500 505 510

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

515 520 525

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

530 535 540

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

545 550 555 560

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

565 570 575

Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr

580 585 590

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

595 600 605

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

610 615 620

Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

625 630 635 640

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

645 650 655

Gln Lys Ser Leu Ser Leu Ser Pro Gly

660 665

<210> 107

<211> 666

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Fab2 Her2/CD3 light chain

<400> 107

Glu Val Gln Leu Ala Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Met Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Phe

20 25 30

Pro Met Ala Trp Val Arg Gln Ala Pro Thr Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Thr Ser Gly Gly Ser Thr Tyr Tyr Arg Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr Tyr Cys

85 90 95

Thr Arg Thr Leu Tyr Ile Leu Arg Val Phe Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Val Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Gly Pro Ala Pro Glu Leu Leu Gly Gly Gly Ser Gln Ile Val Leu Thr

225 230 235 240

Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met

245 250 255

Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln

260 265 270

Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Leu

275 280 285

Ala Ser Gly Val Pro Ala His Phe Arg Gly Ser Gly Ser Gly Thr Ser

290 295 300

Tyr Ser Leu Thr Ile Ser Gly Met Glu Ala Glu Asp Ala Ala Thr Tyr

305 310 315 320

Tyr Cys Gln Gln Trp Ser Ser Asn Pro Phe Thr Phe Gly Ser Gly Thr

325 330 335

Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe

340 345 350

Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys

355 360 365

Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val

370 375 380

Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln

385 390 395 400

Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser

405 410 415

Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His

420 425 430

Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

435 440 445

Gly Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro

450 455 460

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

465 470 475 480

Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn

485 490 495

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

500 505 510

Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

515 520 525

Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

530 535 540

Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys

545 550 555 560

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp

565 570 575

Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe

580 585 590

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

595 600 605

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

610 615 620

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly

625 630 635 640

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

645 650 655

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

660 665

<210> 108

<211> 665

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Fab2 Her2/CD3 heavy chain

<400> 108

Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Glu Thr Val Ser Ile Glu Cys Leu Ala Ser Glu Gly Ile Ser Asn Asp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Ser Gly Lys Ser Pro Gln Leu Leu Ile

35 40 45

Tyr Ala Thr Ser Arg Leu Gln Asp Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Arg Tyr Ser Leu Lys Ile Ser Gly Met Gln Pro

65 70 75 80

Glu Asp Glu Ala Asp Tyr Phe Cys Gln Gln Ser Tyr Lys Tyr Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys Gly Pro Ala Pro Glu Leu Leu Gly Gly Gly

210 215 220

Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly

225 230 235 240

Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg

245 250 255

Tyr Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp

260 265 270

Ile Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys

275 280 285

Phe Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala

290 295 300

Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr

305 310 315 320

Cys Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln

325 330 335

Gly Thr Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

340 345 350

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

355 360 365

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

370 375 380

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

385 390 395 400

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

405 410 415

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

420 425 430

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Gly

435 440 445

Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro

450 455 460

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

465 470 475 480

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

485 490 495

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

500 505 510

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

515 520 525

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

530 535 540

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

545 550 555 560

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

565 570 575

Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr

580 585 590

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

595 600 605

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

610 615 620

Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

625 630 635 640

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

645 650 655

Gln Lys Ser Leu Ser Leu Ser Pro Gly

660 665

<210> 109

<211> 666

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Fab2 Her2/CD3 light chain

<400> 109

Glu Val Gln Leu Ala Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Met Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Phe

20 25 30

Pro Met Ala Trp Val Arg Gln Ala Pro Thr Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Thr Ser Gly Gly Ser Thr Tyr Tyr Arg Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr Tyr Cys

85 90 95

Thr Arg Thr Leu Tyr Ile Leu Arg Val Phe Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Val Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Gly Pro Ala Pro Glu Leu Leu Gly Gly Gly Ser Gln Ile Val Leu Thr

225 230 235 240

Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met

245 250 255

Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln

260 265 270

Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Leu

275 280 285

Ala Ser Gly Val Pro Ala His Phe Arg Gly Ser Gly Ser Gly Thr Ser

290 295 300

Tyr Ser Leu Thr Ile Ser Gly Met Glu Ala Glu Asp Ala Ala Thr Tyr

305 310 315 320

Tyr Cys Gln Gln Trp Ser Ser Asn Pro Phe Thr Phe Gly Ser Gly Thr

325 330 335

Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe

340 345 350

Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys

355 360 365

Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val

370 375 380

Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln

385 390 395 400

Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser

405 410 415

Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His

420 425 430

Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

435 440 445

Gly Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro

450 455 460

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

465 470 475 480

Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn

485 490 495

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

500 505 510

Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

515 520 525

Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

530 535 540

Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys

545 550 555 560

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp

565 570 575

Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe

580 585 590

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

595 600 605

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

610 615 620

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly

625 630 635 640

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

645 650 655

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

660 665

<210> 110

<211> 7

<212> PRT

<213> unknown

<220>

<223> intact MMP cleavage site

<400> 110

Ser Cys Gly Pro Ala Pro Glu

1 5

<210> 111

<211> 6

<212> PRT

<213> unknown

<220>

<223> cleaved MMP cleavage site

<400> 111

Ser Cys Gly Pro Ala Pro

1 5

<210> 112

<211> 4

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> hinge sequence

<400> 112

Glu Leu Leu Gly

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