Activatable bispecific antibodies containing a linker between the two binding domains that is a human immunoglobulin hinge region or a variant thereof and uses thereof

Abstract

Described herein are protein molecules, associated nucleic acid molecules, vectors, and host cells that exhibit activatable target binding in diseased tissue. Also described herein are methods for the medical use of such protein molecules.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of UK Patent Application No. 2001196.1, filed January 28, 2020, UK Patent Application No. 1917678.3, filed December 4, 2019, UK Patent Application No. 1910254.0, filed July 17, 2019, and UK Patent Application No. 1906685.1, filed May 13, 2019, the disclosures of each of which are incorporated herein by reference in their entireties.

[0002] Instructions for electronically submitted text files The contents of the text file submitted electronically herewith are incorporated by reference in their entirety into this specification: A computer-readable copy of the sequence listing (file name: ULSL_002_04WO_SeqList_ST25.txt, data recording date: May 11, 2020, file size approximately 390 kilobytes).

[0003] The present invention relates to protein molecules that exhibit activatable target binding in diseased tissue and their medical applications. [Background technology]

[0004] In the use of antibodies and other binding proteins to treat diseases, many potential drug targets have been described, but few are expressed only in diseased tissues. In fact, most of the potential targets in this cavity are also expressed in non-diseased tissues. In addition, most of the drug action mechanisms employed in difficult-to-treat areas such as cancer employ very strong cell killing action mechanisms. As a result, target engagement by drugs in non-diseased tissues often causes undesirable side effects. There is a need for genetically engineered forms of binding proteins that are partially or completely inactive in healthy tissues, but highly active in diseased tissues. Summary of the Invention

[0005] Provided herein is 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 derived from a human immunoglobulin hinge region, or an amino acid sequence, or an amino acid sequence having from 1 to about 7 amino acid substitutions compared to a human immunoglobulin hinge region, the peptide linker is cleavable by a protease expressed in the diseased tissue, the second portion is capable of specifically binding to a molecule expressed in the diseased tissue, and when the peptide linker is not cleaved, binding of the second portion to the molecule expressed in the diseased tissue is reduced or inhibited. In some embodiments, the peptide linker is about 5 to 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.

[0006] In some embodiments, the protease is a human matrix metalloprotease (MMP), a human cathepsin, a human enterokinase, a human thrombin, a human tPA, a human granzyme B, a human uPA, or a human ADAMTS-5. In some embodiments, the peptide linker comprises a human MMP cleavage site, a human cathepsin cleavage site, a human enterokinase cleavage site, a human thrombin cleavage site, a human tPA cleavage site, a human granzyme B cleavage site, a human uPA cleavage site, 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 MMP14. In some embodiments, the level or activity of the human MMP in the diseased tissue is higher than the level or activity of the human MMP in the 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 the human cathepsin in the diseased tissue is higher than the level or activity of the human cathepsin in the non-diseased tissue.

[0007] In some embodiments, the first portion comprises an antibody, an antigen-binding portion of an antibody, or a receptor ectodomain. In some embodiments, the first portion is a Fab, a single-chain Fab, a VH domain, a VL domain, an immunoglobulin novel antigen receptor (IgNAR), a single-chain variable fragment (scFv), a diabody, or a T-cell receptor domain. In some embodiments, the first portion specifically binds to a molecule expressed in diseased tissue.

[0008] In some embodiments, the first portion is capable of specifically binding 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, where 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 in 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 in different cells. In some embodiments, the first molecule expressed in the diseased tissue and / or the second molecule expressed in the diseased tissue are expressed on the surface of a cell. In some embodiments, the first molecule expressed in the diseased tissue and / or the second molecule expressed in the diseased tissue are soluble molecules.

[0009] In some embodiments, 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.

[0010] In some embodiments, the second portion specifically binds to a molecule expressed on a human immune cell, hi some embodiments, the molecule expressed on a human immune cell is human CD3, human CD16A, human CD16B, human CD28, human CD89, human CTLA4, human NKG2D, human SIRPα, human SIRPγ, human PD1, human Lag3, human 4-1BB, human OX40, or human GITR.

[0011] 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 IgG, IgE, IgM, IgD, IgA, or IgY. In some embodiments, the immunoglobulin constant region is IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2. 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 the amino acid substitution S228P, a wild-type human IgG1 constant region, a human IgG1 constant region comprising the amino acid substitutions L234A and L235A, a human IgG1 constant region comprising the amino acid substitutions L234A, L235A and G237A, a human IgG1 constant region comprising the amino acid substitutions L234A, L235A, G237A and P331S, or a wild-type human IgG2 constant region.

[0012] In some embodiments, the second portion comprises an antibody, an antigen-binding portion of an antibody, or a receptor ectodomain. In some embodiments, the second portion is a Fab, a single-chain Fab, a VH domain, a VL domain, an immunoglobulin novel antigen receptor (IgNAR), a single-chain variable fragment (scFv), or a T-cell receptor domain. In some embodiments, the second portion specifically binds to human CD47. In some embodiments, the second portion specifically binds to human CD3 or human PD-L1.

[0013] 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 IgG, IgE, IgM, IgD, IgA, or IgY. In some embodiments, the immunoglobulin constant region is IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2. 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 the amino acid substitution S228P, a wild-type human IgG1 constant region, a human IgG1 constant region comprising the amino acid substitutions L234A and L235A, a human IgG1 constant region comprising the amino acid substitutions L234A, L235A and G237A, a human IgG1 constant region comprising the amino acid substitutions L234A, L235A, G237A and P331S, or a wild-type human IgG2 constant region.

[0014] In some embodiments, the protein has one immune effector function, or two, three or more immune effector functions, hi some embodiments, the immune effector function is ADCC, CDC or ADCP.

[0015] In some embodiments, the first portion prevents or reduces the specific binding of the second portion to a molecule expressed in the diseased tissue. In some embodiments, the peptide linker is cleaved near or inside the diseased tissue. In some embodiments, the peptide linker is cleaved near or inside the diseased tissue, where the first portion dissociates from the second portion near or inside the diseased tissue, and 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.

[0016] In some embodiments, the first portion specifically binds to human cMET, wherein the second portion specifically binds to human CD47, and 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.

[0017] In some embodiments, the first portion specifically binds to human HER2, wherein the second portion specifically binds to human CD3, and 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.

[0018] In some embodiments, the first portion specifically binds to human HER2, wherein the second portion specifically binds to human CD47, and 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.

[0019] In some embodiments, the first portion specifically binds to human cMET, wherein the second portion specifically binds to human CD47, and 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.

[0020] In some embodiments, the first portion specifically binds to human Her2, wherein the second portion specifically binds to human CD3, and the protein comprises a first polypeptide chain that comprises or consists of the amino acid sequence of SEQ ID NO:38, and a second polypeptide chain that comprises or consists of the amino acid sequence of SEQ ID NO:39.

[0021] In some embodiments, the first portion specifically binds to human Her2, the second portion specifically binds to human CD3, and the protein comprises a first polypeptide chain that comprises or consists of the amino acid sequence of SEQ ID NO:40, and a second polypeptide chain that comprises or consists of the amino acid sequence of SEQ ID NO:41.

[0022] In some embodiments, the first portion specifically binds human Her2, the second portion specifically binds human CD47, and the protein comprises a first polypeptide chain and a second polypeptide chain, wherein: (a) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 42 and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 43; (b) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 44 and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 45; (c) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 46 and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 47; (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; (e) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:50 and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:51; (f) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:52 and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:53; (g) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:54 and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:55; (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; (i) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 90 and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 91; (j) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 44 and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 92; (k) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 44 and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 93; (l) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 44 and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 94; (m) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 44 and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 95; (n) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 44 and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 96; (o) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:44, and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:97.

[0023] In some embodiments, the first portion specifically binds to human Her2, the second portion specifically binds to human CD3, and the protein comprises a first polypeptide chain that comprises or consists of the amino acid sequence of SEQ ID NO:73 and a second polypeptide chain that comprises or consists of the amino acid sequence of SEQ ID NO:74.

[0024] In some embodiments, the first portion specifically binds to human cMET, the second portion specifically binds to human cMET, and the protein comprises a first polypeptide chain that comprises or consists of the amino acid sequence of SEQ ID NO:75, and a second polypeptide chain that comprises or consists of the amino acid sequence of SEQ ID NO:76.

[0025] In some embodiments, the first portion specifically binds human Her2, the second portion specifically binds human CD3, and the protein comprises a first polypeptide chain and a second polypeptide chain, wherein: (a) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 98 and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 99; (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; (c) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 102 and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 103; (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.

[0026] Further provided herein is an immunoconjugate 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 antiangiogenic agent, an antiproliferative agent, a proapoptotic agent, a cytostatic enzyme, a cytolytic enzyme, a therapeutic nucleic acid, an antiangiogenic agent, an antiproliferative agent, or a proapoptotic agent.

[0027] Further provided herein is a pharmaceutical composition comprising a protein of the invention or an immunoconjugate of the invention and a pharma- ceutically acceptable carrier, diluent or excipient.

[0028] Further provided herein are nucleic acid molecules encoding the proteins or portions of proteins of the invention. Further provided herein are nucleic acid molecules encoding the first polypeptide chain, the second polypeptide chain, or both the first and second polypeptide chains of the proteins of the invention.

[0029] Further provided herein is an expression vector comprising the nucleic acid molecule of the invention.

[0030] Further provided herein is a recombinant host cell comprising a nucleic acid molecule of the invention or an expression vector of the invention.

[0031] Further provided herein is a method of producing a protein, comprising culturing a recombinant host cell comprising an expression vector of the invention under conditions whereby the nucleic acid molecule is expressed, thereby producing the protein, and isolating the protein from the host cell or culture.

[0032] Further provided herein is a method for enhancing an anti-cancer immune response 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.

[0033] Further provided herein is 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 the invention, an immunoconjugate of the invention, or a pharmaceutical composition of the invention. In some embodiments, 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, oral or pharyngeal cancer, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small intestine or appendix cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma, or blood tissue cancer. In some embodiments, the autoimmune disease 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.

[0034] Further provided herein is a protein of the present invention, an immunoconjugate of the present invention, or a pharmaceutical composition of the present invention for use in the treatment of cancer, autoimmune disease, inflammatory disease, cardiovascular disease, or fibrotic disease.In some embodiments, 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, oral or pharyngeal cancer, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small intestine or appendix cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma, or blood tissue cancer.In some embodiments, the autoimmune disease 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 pectoris, osteoarthritis, pulmonary fibrosis, cystic fibrosis, bronchitis or asthma.

[0035] The protein of the present invention, the immune complex of the present invention, or the medicament of the present invention for use as a pharmaceutical Compositions are further provided herein. The present invention provides, for example, the following items. (Item 1) A protein comprising a first portion and a second portion, and a peptide linker between the first portion and the second portion, the peptide linker comprises an amino acid sequence derived from a human immunoglobulin hinge region, or an amino acid sequence having from 1 to about 7 amino acid substitutions compared to a human immunoglobulin hinge region, the peptide linker is cleavable by a protease expressed in the diseased tissue; the second portion is capable of specifically binding to a molecule expressed in the diseased tissue; and When the peptide linker is not cleaved, the binding of the second portion to the molecule expressed in the diseased tissue is reduced or inhibited. (Item 2) 2. The protein according to item 1, wherein the peptide linker is about 5 to about 15 amino acids in length. (Item 3) 3. The protein according to item 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. (Item 4) 4. The protein according to any one of items 1 to 3, wherein the protease is a human matrix metalloprotease (MMP), a human cathepsin, a human enterokinase, a human thrombin, a human tPA, a human granzyme B, a human uPA, or a human ADAMTS-5. (Item 5) 5. The protein according to any one of items 1 to 4, wherein the peptide linker comprises a human MMP cleavage site, a human cathepsin cleavage site, a human enterokinase cleavage site, a human thrombin cleavage site, a human tPA cleavage site, a human granzyme B cleavage site, a human uPA cleavage site, or a human ADAMTS-5 cleavage site. (Item 6) 6. The protein according to any one of items 1 to 5, wherein the human MMP is MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, MMP-12, MMP-13 or MMP14. (Item 7) 7. The protein according to any one of items 1 to 6, wherein a level or activity of the human MMP in the diseased tissue is higher than a level or activity of the human MMP in a non-diseased tissue. (Item 8) 6. The protein according to any one of items 1 to 5, wherein the human cathepsin is cathepsin A, cathepsin C, cathepsin D, cathepsin G, cathepsin L or cathepsin K. (Item 9) 9. The protein according to any one of items 1 to 5 and 8, wherein a level or activity of the human cathepsin in the diseased tissue is higher compared to a level or activity of the human cathepsin in a non-diseased tissue. (Item 10) 10. The protein according to any one of items 1 to 9, wherein the first portion comprises an antibody, an antigen-binding portion of an antibody, or a receptor ectodomain. (Item 11) 11. The protein of item 10, wherein the first portion is a Fab, a single-chain Fab, a VH domain, a VL domain, an immunoglobulin novel antigen receptor (IgNAR), a single-chain variable fragment (scFv), a diabody, or a T cell receptor domain. (Item 12) 12. The protein according to any one of items 1 to 11, wherein the first portion specifically binds to a molecule expressed in diseased tissue. (Item 13) 13. The protein according to any one of items 1 to 12, wherein the first portion is capable of specifically binding 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, and the first molecule expressed in the diseased tissue and the second molecule expressed in the diseased tissue are different molecules. (Item 14) 14. The protein according to item 13, wherein the first molecule expressed in the diseased tissue and the second molecule expressed in the diseased tissue are expressed in the same cell. (Item 15) 14. The protein according to item 13, wherein the first molecule expressed in the diseased tissue and the second molecule expressed in the diseased tissue are expressed in different cells. (Item 16) 14. The protein according to item 13, wherein the first molecule expressed in the diseased tissue and / or the second molecule expressed in the diseased tissue are expressed on the surface of a cell. (Item 17) 14. The protein according to item 13, wherein the first molecule expressed in the diseased tissue and / or the second molecule expressed in the diseased tissue are soluble molecules. (Item 18) 18. The protein according to any one of items 1 to 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. (Item 19) 18. The protein according to any one of items 1 to 17, wherein the second portion specifically binds to a molecule expressed in a human immune cell. (Item 20) 20. The protein of item 19, wherein the molecule expressed on human immune cells is human CD3, human CD16A, human CD16B, human CD28, human CD89, human CTLA4, human NKG2D, human SIRPα, human SIRPγ, human PD1, human Lag3, human 4-1BB, human OX40 or human GITR. (Item 21) 21. The protein according to any one of items 1 to 20, wherein the first portion comprises a heavy chain variable (VH) region and a light chain variable (VL) region. (Item 22) 22. The protein according to any one of items 1 to 21, wherein the first portion comprises an immunoglobulin constant region or a portion of an immunoglobulin constant region. (Item 23) 23. The protein of item 22, wherein the immunoglobulin constant region is IgG, IgE, IgM, IgD, IgA or IgY. (Item 24) 23. The protein of item 22, wherein the immunoglobulin constant region is IgG1, IgG2, IgG3, IgG4, IgA1 or IgA2. (Item 25) 23. The protein of item 22, wherein the immunoglobulin constant region is immunologically inert. (Item 26) 23. The protein according to item 22, wherein the immunoglobulin constant region is a wild-type human IgG4 constant region, a human IgG4 constant region comprising the amino acid substitution S228P, a wild-type human IgG1 constant region, a human IgG1 constant region comprising the amino acid substitutions L234A and L235A, a human IgG1 constant region comprising the amino acid substitutions L234A, L235A and G237A, a human IgG1 constant region comprising the amino acid substitutions L234A, L235A, G237A and P331S, or a wild-type human IgG2 constant region. (Item 27) 27. The protein according to any one of items 1 to 26, wherein the second portion comprises an antibody, an antigen-binding portion of an antibody, or a receptor ectodomain. (Item 28) 28. The protein of item 27, wherein the second portion is a Fab, a single-chain Fab, a VH domain, a VL domain, an immunoglobulin novel antigen receptor (IgNAR), a single-chain variable fragment (scFv), or a T cell receptor domain. (Item 29) 29. The protein according to any one of items 1 to 28, wherein the second portion specifically binds to human CD47. (Item 30) 29. The protein according to any one of items 1 to 28, wherein the second portion specifically binds to human CD3 or human PD-L1. (Item 31) 31. The protein according to any one of items 1 to 30, wherein the second portion comprises a heavy chain variable (VH) region and a light chain variable (VL) region. (Item 32) 32. The protein according to any one of items 1 to 31, wherein the second portion comprises an immunoglobulin constant region or a part of an immunoglobulin constant region. (Item 33) 33. The protein of item 32, wherein the immunoglobulin constant region is IgG, IgE, IgM, IgD, IgA or IgY. (Item 34) 33. The protein of item 32, wherein the immunoglobulin constant region is IgG1, IgG2, IgG3, IgG4, IgA1 or IgA2. (Item 35) 33. The protein of item 32, wherein the immunoglobulin constant region is immunologically inert. (Item 36) 33. The protein according to item 32, wherein the immunoglobulin constant region is a wild-type human IgG4 constant region, a human IgG4 constant region comprising the amino acid substitution S228P, a wild-type human IgG1 constant region, a human IgG1 constant region comprising the amino acid substitutions L234A and L235A, a human IgG1 constant region comprising the amino acid substitutions L234A, L235A and G237A, a human IgG1 constant region comprising the amino acid substitutions L234A, L235A, G237A and P331S, or a wild-type human IgG2 constant region. (Item 37) 2. The protein according to claim 1, wherein the protein has one immune effector function or two, three or more immune effector functions. (Item 38) 38. The protein according to item 37, wherein the immune effector function is ADCC, CDC or ADCP. (Item 39) 39. The protein according to any one of items 1 to 38, wherein the first portion prevents or reduces specific binding of the second portion to the molecule expressed in the diseased tissue. (Item 40) 40. The protein according to any one of items 1 to 39, wherein the peptide linker is cleaved near the diseased tissue or inside the diseased tissue. (Item 41) 41. The protein according to any one of items 1 to 40, wherein the peptide linker is cleaved near or inside the diseased tissue, the first portion dissociates from the second portion near or inside the diseased tissue, and the second portion specifically binds to the molecule expressed in the diseased tissue near or inside the diseased tissue. (Item 42) 42. The protein according to any one of items 1 to 41, wherein the diseased tissue is a tumor or an inflamed tissue. (Item 43) 2. The protein according to item 1, wherein the first portion specifically binds to human cMET and the second portion specifically binds to human CD47, and 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. (Item 44) 2. The protein according to item 1, wherein the first portion specifically binds to human HER2, the second portion specifically binds to human CD3, and 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. (Item 45) 2. The protein according to item 1, wherein the first portion specifically binds to human HER2, the second portion specifically binds to human CD47, and 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. (Item 46) 2. The protein according to item 1, wherein the first portion specifically binds to human cMET and the second portion specifically binds to human CD47, and 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. (Item 47) 2. The protein according to item 1, wherein the first portion specifically binds to human Her2, the second portion specifically binds to human CD3, and 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. (Item 48) 2. The protein according to item 1, wherein the first portion specifically binds to human Her2, the second portion specifically binds to human CD3, and 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. (Item 49) the first portion specifically binds to human Her2, the second portion specifically binds to human CD47, and the protein comprises a first polypeptide chain and a second polypeptide chain; Where: (a) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 42 and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 43; (b) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 44 and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 45; or (c) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 46 and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 47; (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; (e) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:50 and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:51; (f) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:52 and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:53; (g) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:54 and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:55; (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; (i) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 90 and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 91; (j) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 44 and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 92; (k) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 44 and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 93; (l) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 44 and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 94; (m) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 44 and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 95; (n) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 44 and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 96; (o) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 44, and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 97. (Item 50) 2. The protein according to item 1, wherein the first portion specifically binds to human Her2, the second portion specifically binds to human CD3, and 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. (Item 51) 2. The protein of claim 1, wherein the first portion specifically binds to human cMET and the second portion specifically binds to human cMET, and 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. (Item 52) the first portion specifically binds to human Her2, the second portion specifically binds to human CD3, and the protein comprises a first polypeptide chain and a second polypeptide chain, (a) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 98 and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 99; (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; (c) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 102 and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 103; (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. (Item 53) 53. An immunoconjugate comprising the protein according to any one of items 1 to 52 linked to a therapeutic agent. (Item 54) 54. The immunoconjugate of item 53, wherein the therapeutic agent is a cytotoxin, a radioisotope, a chemotherapeutic agent, an immunomodulatory agent, an antiangiogenic agent, an antiproliferative agent, a proapoptotic agent, a cytostatic enzyme, a cytolytic enzyme, a therapeutic nucleic acid, an antiangiogenic agent, an antiproliferative agent, or a proapoptotic agent. (Item 55) A pharmaceutical composition comprising the protein according to any one of items 1 to 52, or the immunoconjugate according to item 53 or 54, and a pharma- ceutically acceptable carrier, diluent or excipient. (Item 56) 53. A nucleic acid molecule encoding the protein according to any one of items 1 to 52 or a part of the protein. (Item 57) 53. A nucleic acid molecule encoding the first polypeptide chain, the second polypeptide chain, or both the first polypeptide chain and the second polypeptide chain of the protein according to any one of Items 43 to 52. (Item 58) 58. An expression vector comprising the nucleic acid molecule of item 56 or 57. (Item 59) A recombinant host cell comprising the nucleic acid molecule of item 56 or 57, or the expression vector of item 58. (Item 60) 1. A method for producing a protein, comprising: Culturing a recombinant host cell comprising the expression vector according to item 57 under conditions in which the nucleic acid molecule is expressed, thereby producing the protein; and isolating said protein from said host cell or culture. (Item 61) 54. A method for enhancing an anti-cancer immune response in a subject, comprising administering to the subject a therapeutically effective amount of the protein according to any one of items 1 to 52, the immunoconjugate according to item 53 or 54, or the pharmaceutical composition according to item 55. (Item 62) A method for treating cancer, an autoimmune disease, an inflammatory disease, a cardiovascular disease, or a fibrotic disease in a subject, the method comprising administering to the subject a therapeutically effective amount of the protein according to any one of items 1 to 52, the immunoconjugate according to item 53 or 54, or the pharmaceutical composition according to item 55. (Item 63) 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, oral or pharyngeal cancer, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small intestine or appendix cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma, or blood tissue cancer. (Item 64) 63. The method of item 62, wherein the autoimmune disease or the inflammatory disease is arthritis, asthma, multiple sclerosis, psoriasis, Crohn's disease, inflammatory bowel disease, lupus, Graves' disease, Hashimoto's thyroiditis or ankylosing spondylitis. (Item 65) 63. The method of claim 62, wherein the cardiovascular disease is coronary heart disease, or atherosclerosis or stroke. (Item 66) 63. The method of claim 62, wherein the fibrotic disease is myocardial infarction, angina pectoris, osteoarthritis, pulmonary fibrosis, cystic fibrosis, bronchitis or asthma. (Item 67) The protein according to any one of items 1 to 52, the immunoconjugate according to item 53 or 54, or the pharmaceutical composition according to item 55, for use in the treatment of cancer, an autoimmune disease, an inflammatory disease, a cardiovascular disease or a fibrotic disease. (Item 68) 68. The protein or pharmaceutical composition for use according to item 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, oral or pharyngeal cancer, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small intestine or appendix cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma, or blood tissue cancer. (Item 69) 68. The protein or pharmaceutical composition for use according to item 67, wherein said autoimmune disease or said inflammatory disease is arthritis, asthma, multiple sclerosis, psoriasis, Crohn's disease, inflammatory bowel disease, lupus, Graves' disease, Hashimoto's thyroiditis or ankylosing spondylitis. (Item 70) 68. The protein or pharmaceutical composition for use according to item 67, wherein said cardiovascular disease is coronary heart disease, or atherosclerosis or stroke. (Item 71) 68. The protein or pharmaceutical composition for use according to item 67, wherein said fibrotic disease is myocardial infarction, angina pectoris, osteoarthritis, pulmonary fibrosis, cystic fibrosis, bronchitis or asthma. (Item 72) 56. The protein according to any one of items 1 to 52, the immunoconjugate according to item 53 or 54, or the pharmaceutical composition according to item 55, for use as a pharmaceutical. [Brief description of the drawings]

[0036] [Figure 1A] As an example, the challenges of systemically administering an active antibody drug, anti-CD47, to a solid tumor are shown. The CD47 antibody in this figure has significant challenges, such as the high expression of CD47 in the bloodstream, particularly on red blood cells and platelets, which creates "sink" and toxicity risk issues. Additionally, tumors are typically a "hostile" environment with high expression of enzymes such as MMPs that accelerate IgG degradation. [Figure 1B] As an example, the challenges of systemically administering an active antibody drug, anti-CD47, to solid tumors are shown. The anti-CD47 protein construct of the present invention in this figure aims to eliminate CD47 binding in the native protein, thereby canceling activity in the periphery. The tumor targeting domain then promotes high concentration in the tumor environment, and the peptide linker system harnesses MMP activity in the tumor to activate CD47 binding activity in the tumor rather than in the periphery. [Figure 2A]The design and activation principle of the protein construct IgG2 is shown. The protein construct IgG2 design (A) may be based on sequences derived from IgG1, IgG2, IgG3, IgG4, IgA, IgE, or IgM and may or may not have effector function capabilities. In this construct, the four polypeptide chains code for four Fab domains (two Fab A, two Fab B), two linker sequences, and may or may not have an immunoglobulin hinge region and an Fc domain. Each Fab A-linker domain blocks the binding activity of a Fab B. The selection of a linker sequence, such as a lower hinge peptide sequence from an immunoglobulin, creates a structure that is suppressed in non-diseased tissues but is rapidly cleaved and released in the presence of high concentrations of proteases in the tumor environment (B). The linkers can be sequentially cleaved to create an intermediate open activation state, thereby allowing Fab A and B from a single protein construct to bind to their cognate targets. Potentially slower secondary cleavage of the second linker in each Fab A-Fab B protein construct unit could completely release the Fab A domain from the structure in a dissociated form. The cleaved linker, based on the immunoglobulin hinge sequence, could also recruit increased immune effector functions at the cell membrane via endogenous anti-hinge antibodies. Variable regions are shown in white. Constant regions are shown in grey. [Figure 2B]The design and activation principle of the protein construct IgG2 is shown. The protein construct IgG2 design (A) may be based on sequences derived from IgG1, IgG2, IgG3, IgG4, IgA, IgE, or IgM and may or may not have effector function capabilities. In this construct, the four polypeptide chains code for four Fab domains (two Fab A, two Fab B), two linker sequences, and may or may not have an immunoglobulin hinge region and an Fc domain. Each Fab A-linker domain blocks the binding activity of a Fab B. The selection of a linker sequence, such as a lower hinge peptide sequence from an immunoglobulin, creates a structure that is suppressed in non-diseased tissues but is rapidly cleaved and released in the presence of high concentrations of proteases in the tumor environment (B). The linkers can be sequentially cleaved to create an intermediate open activation state, thereby allowing Fab A and B from a single protein construct to bind to their cognate targets. Potentially slower secondary cleavage of the second linker in each Fab A-Fab B protein construct unit could completely release the Fab A domain from the structure in a dissociated form. The cleaved linker, based on the immunoglobulin hinge sequence, could also recruit increased immune effector functions at the cell membrane via endogenous anti-hinge antibodies. Variable regions are shown in white. Constant regions are shown in grey. [Figure 3A]The design and activation principle of the protein construct Fab2 is shown. The protein construct Fab2 design may be based on sequences derived from IgG1, IgG2, IgG3, IgG4, IgA, IgE, or IgM, and may or may not have effector function capabilities. In this construct, two polypeptide chains (A) or three polypeptide chains (B) code for two Fab domains (one Fab A, one Fab B), two or more linker sequences, and may or may not have an immunoglobulin hinge region and an Fc domain, where heterodimeric pairing may or may not be induced by mutations in the Fc. Each Fab A-linker domain blocks the binding activity of a Fab B. The selection of linker sequences, such as the lower hinge peptide sequence, creates a structure (A) that is suppressed in non-diseased tissues, but is rapidly cleaved and released in the presence of high concentrations of proteases in the tumor environment. The linkers can be sequentially cleaved, creating an intermediate open activation state, allowing Fab A and B of a single protein to bind to their cognate targets. A potentially slower secondary cleavage of a second linker in each Fab A-Fab B protein unit can completely release the Fab A domain from the construct. Cleaved linkers based on immunoglobulin hinge sequences may also recruit increased immune effector functions at the cell membrane via endogenous anti-hinge antibodies. Variable regions are shown in white. Constant regions are shown in grey. [Figure 3B]The design and activation principle of the protein construct Fab2 is shown. The protein construct Fab2 design may be based on sequences derived from IgG1, IgG2, IgG3, IgG4, IgA, IgE, or IgM, and may or may not have effector function capabilities. In this construct, two polypeptide chains (A) or three polypeptide chains (B) code for two Fab domains (one Fab A, one Fab B), two or more linker sequences, and may or may not have an immunoglobulin hinge region and an Fc domain, where heterodimeric pairing may or may not be induced by mutations in the Fc. Each Fab A-linker domain blocks the binding activity of a Fab B. The selection of linker sequences, such as the lower hinge peptide sequence, creates a structure (A) that is suppressed in non-diseased tissues, but is rapidly cleaved and released in the presence of high concentrations of proteases in the tumor environment. The linkers can be sequentially cleaved, creating an intermediate open activation state, allowing Fab A and B of a single protein to bind to their cognate targets. A potentially slower secondary cleavage of a second linker in each Fab A-Fab B protein unit can completely release the Fab A domain from the construct. Cleaved linkers based on immunoglobulin hinge sequences may also recruit increased immune effector functions at the cell membrane via endogenous anti-hinge antibodies. Variable regions are shown in white. Constant regions are shown in grey. [Figure 4] Figure 1 shows SDS-PAGE analysis of Protein A purified protein construct IgG2 and Fab2 proteins from clones 1-15. Several exemplary construct proteins were expressed in CHO cells and purified using Protein A affinity chromatography. The purified proteins were then subjected to SDS-PAGE analysis in both non-reduced and reduced (r) conditions along with molecular weight standards (M). Clones 6, 10 and 14 (all containing the LHL linker) were found to have the highest proportion of products of the expected size and the lowest proportions of high and low molecular weight products. [Figure 5A]Size exclusion chromatography of Protein A purified protein constructs IgG2 and Fab2 proteins. Selected exemplary construct proteins were analyzed and fully purified using SEC. Clones 1 (Figure 5A), 2 (Figure 5B), 3 (Figure 5C), 4 (Figure 5D), 5 (Figure 5E), 6 (Figure 5F), 12 (Figure 5G), 14 (Figure 5H) and 10 (Figure 5I) were analyzed. The data showed that samples from clones 6, 10 and 14 (all containing the LHL linker) contained the highest proportion of products of the expected size (e.g., highlighted peak, Figure 5I) and the lowest proportion of high / low molecular weight products. [Figure 5B] Size exclusion chromatography of Protein A purified protein constructs IgG2 and Fab2 proteins. Selected exemplary construct proteins were analyzed and fully purified using SEC. Clones 1 (Figure 5A), 2 (Figure 5B), 3 (Figure 5C), 4 (Figure 5D), 5 (Figure 5E), 6 (Figure 5F), 12 (Figure 5G), 14 (Figure 5H) and 10 (Figure 5I) were analyzed. The data showed that samples from clones 6, 10 and 14 (all containing the LHL linker) contained the highest proportion of products of the expected size (e.g., highlighted peak, Figure 5I) and the lowest proportion of high / low molecular weight products. [Figure 5C] Size exclusion chromatography of Protein A purified protein constructs IgG2 and Fab2 proteins. Selected exemplary construct proteins were analyzed and fully purified using SEC. Clones 1 (Figure 5A), 2 (Figure 5B), 3 (Figure 5C), 4 (Figure 5D), 5 (Figure 5E), 6 (Figure 5F), 12 (Figure 5G), 14 (Figure 5H) and 10 (Figure 5I) were analyzed. The data showed that samples from clones 6, 10 and 14 (all containing the LHL linker) contained the highest proportion of products of the expected size (e.g., highlighted peak, Figure 5I) and the lowest proportion of high / low molecular weight products. [Figure 5D]Size exclusion chromatography of Protein A purified protein constructs IgG2 and Fab2 proteins. Selected exemplary construct proteins were analyzed and fully purified using SEC. Clones 1 (Figure 5A), 2 (Figure 5B), 3 (Figure 5C), 4 (Figure 5D), 5 (Figure 5E), 6 (Figure 5F), 12 (Figure 5G), 14 (Figure 5H) and 10 (Figure 5I) were analyzed. The data showed that samples from clones 6, 10 and 14 (all containing the LHL linker) contained the highest proportion of products of the expected size (e.g., highlighted peak, Figure 5I) and the lowest proportion of high / low molecular weight products. [Figure 5E] Size exclusion chromatography of Protein A purified protein constructs IgG2 and Fab2 proteins. Selected exemplary construct proteins were analyzed and fully purified using SEC. Clones 1 (Figure 5A), 2 (Figure 5B), 3 (Figure 5C), 4 (Figure 5D), 5 (Figure 5E), 6 (Figure 5F), 12 (Figure 5G), 14 (Figure 5H) and 10 (Figure 5I) were analyzed. The data showed that samples from clones 6, 10 and 14 (all containing the LHL linker) contained the highest proportion of products of the expected size (e.g., highlighted peak, Figure 5I) and the lowest proportion of high / low molecular weight products. [Figure 5F] Size exclusion chromatography of Protein A purified protein constructs IgG2 and Fab2 proteins. Selected exemplary construct proteins were analyzed and fully purified using SEC. Clones 1 (Figure 5A), 2 (Figure 5B), 3 (Figure 5C), 4 (Figure 5D), 5 (Figure 5E), 6 (Figure 5F), 12 (Figure 5G), 14 (Figure 5H) and 10 (Figure 5I) were analyzed. The data showed that samples from clones 6, 10 and 14 (all containing the LHL linker) contained the highest proportion of products of the expected size (e.g., highlighted peak, Figure 5I) and the lowest proportion of high / low molecular weight products. [Figure 5G]Size exclusion chromatography of Protein A purified protein constructs IgG2 and Fab2 proteins. Selected exemplary construct proteins were analyzed and fully purified using SEC. Clones 1 (Figure 5A), 2 (Figure 5B), 3 (Figure 5C), 4 (Figure 5D), 5 (Figure 5E), 6 (Figure 5F), 12 (Figure 5G), 14 (Figure 5H) and 10 (Figure 5I) were analyzed. The data showed that samples from clones 6, 10 and 14 (all containing the LHL linker) contained the highest proportion of products of the expected size (e.g., highlighted peak, Figure 5I) and the lowest proportion of high / low molecular weight products. [Figure 5H] Size exclusion chromatography of Protein A purified protein constructs IgG2 and Fab2 proteins. Selected exemplary construct proteins were analyzed and fully purified using SEC. Clones 1 (Figure 5A), 2 (Figure 5B), 3 (Figure 5C), 4 (Figure 5D), 5 (Figure 5E), 6 (Figure 5F), 12 (Figure 5G), 14 (Figure 5H) and 10 (Figure 5I) were analyzed. The data showed that samples from clones 6, 10 and 14 (all containing the LHL linker) contained the highest proportion of products of the expected size (e.g., highlighted peak, Figure 5I) and the lowest proportion of high / low molecular weight products. [Figure 5I] Size exclusion chromatography of Protein A purified protein constructs IgG2 and Fab2 proteins. Selected exemplary construct proteins were analyzed and fully purified using SEC. Clones 1 (Figure 5A), 2 (Figure 5B), 3 (Figure 5C), 4 (Figure 5D), 5 (Figure 5E), 6 (Figure 5F), 12 (Figure 5G), 14 (Figure 5H) and 10 (Figure 5I) were analyzed. The data showed that samples from clones 6, 10 and 14 (all containing the LHL linker) contained the highest proportion of products of the expected size (e.g., highlighted peak, Figure 5I) and the lowest proportion of high / low molecular weight products. [Figure 6A]SDS-PAGE analysis of SEC purified protein construct IgG2 and Fab2 proteins are shown. Protein A affinity purified major lead protein construct clones were finally purified using SEC. Purified proteins from clones 1, 2, 4, 5, 6, and non-SEC purified 15 (A) were then subjected to SDS-PAGE analysis under non-reducing conditions. Purified proteins from clones 7, 8, 10, 11, 12, 13 and 14 (B) were also subjected to SDS-PAGE analysis under both non-reducing and reducing (r) conditions. All proteins were loaded at approximately 1 μg per lane. Clones 6, 10 and 14 (all containing the LHL linker) were found to have the highest proportion of products of the expected size and the lowest proportions of high and low molecular weight products. [Figure 6B] SDS-PAGE analysis of SEC purified protein construct IgG2 and Fab2 proteins are shown. Protein A affinity purified major lead protein construct clones were finally purified using SEC. Purified proteins from clones 1, 2, 4, 5, 6, and non-SEC purified 15 (A) were then subjected to SDS-PAGE analysis under non-reducing conditions. Purified proteins from clones 7, 8, 10, 11, 12, 13 and 14 (B) were also subjected to SDS-PAGE analysis under both non-reducing and reducing (r) conditions. All proteins were loaded at approximately 1 μg per lane. Clones 6, 10 and 14 (all containing the LHL linker) were found to have the highest proportion of products of the expected size and the lowest proportions of high and low molecular weight products. [Figure 7A] Direct titration ELISA of binding of purified intact protein constructs and control antibodies to human target proteins. Control antibodies A-D5 anti-CD47, A-D5 Fab-Fc (monovalent form of A-D5 antibody), MH7.1 anti-C-MET, and anti-Her2 trastuzumab, all in human IgG1 format, were titrated in a direct binding ELISA against human CD47, C-MET and Her2 proteins (in μg / ml). [Figure 7B]Direct titration ELISA of binding of purified intact protein constructs and control antibodies to human target proteins. Her2CD47-LH-LH and Her2CD47-LHL-LHL in IgG2 format were titrated in a direct binding ELISA against human CD47, C-MET and Her2 proteins (in μg / ml). [Figure 7C] Direct titration ELISA of binding of purified intact protein constructs and control antibodies to human target proteins. The Fab2 formats cMETCD47-L2-L2 and cMETCD47-LHL-LHL were titrated in a direct binding ELISA against human CD47, c-MET and Her2 proteins (in μg / ml). [Figure 8A] Figure 1 shows human hemagglutination assays of purified intact protein constructs and control antibodies. Control antibodies anti-CD235a (mouse) and A-D5 anti-CD47, A-D5 Fab-Fc (monovalent form of the A-D5 antibody, labeled "FabCD47 alone"), MH7.1 anti-C-MET, anti-Her2 trastuzumab, Her2CD47-LH-LH and Her2CD47-LHL-LHL in IgG2 format, and cMETCD47-L2-L2 and cMETCD47-LHL-LHL in Fab2 format were titrated in a human hemagglutination assay using fresh red blood cells from donor 1 (units in nM). [Figure 8B] Figure 1 shows human hemagglutination assays of purified intact protein constructs and control antibodies. Control antibodies anti-CD235a (mouse) and A-D5 anti-CD47, A-D5 Fab-Fc (monovalent form of the A-D5 antibody, labeled "FabCD47 alone"), MH7.1 anti-C-MET, anti-Her2 trastuzumab, Her2CD47-LH-LH and Her2CD47-LHL-LHL in IgG2 format, and cMETCD47-L2-L2 and cMETCD47-LHL-LHL in Fab2 format were titrated in a human hemagglutination assay using fresh red blood cells from donor 2 (units in nM). [Figure 8C]Figure 1 shows human hemagglutination assays of purified intact protein constructs and control antibodies. Control antibodies anti-CD235a (mouse) and A-D5 anti-CD47, A-D5 Fab-Fc (monovalent form of the A-D5 antibody, labeled "FabCD47 alone"), MH7.1 anti-C-MET, anti-Her2 trastuzumab, Her2CD47-LH-LH and Her2CD47-LHL-LHL in IgG2 format, and cMETCD47-L2-L2 and cMETCD47-LHL-LHL in Fab2 format were titrated (in nM) in a human hemagglutination assay using fresh red blood cells from donor 3. [Figure 9A] Direct ELISA of binding of purified intact and MMP digested protein constructs to human target proteins. Protein constructs were subjected to enzyme digestion with human MMP3, 7 and 12 for 2, 4, 8 and 24 hours of incubation, as well as a 24 hour incubation in buffer without enzyme as a negative control. These time-course digestion samples were then subjected to direct binding ELISA against human Her2 and CD47. [Figure 9B] Direct ELISA of binding of purified intact and MMP digested protein constructs to human target proteins. Protein constructs were subjected to enzyme digestion with human MMP3, 7 and 12 for 2, 4, 8 and 24 hours of incubation, as well as a 24 hour incubation in buffer without enzyme as a negative control. These time-course digestion samples were then subjected to direct binding ELISA against human Her2 and CD47. [Figure 9C] Direct ELISA of binding of purified intact and MMP digested protein constructs to human target proteins. Protein constructs were subjected to enzymatic digestion with human MMP3, 7 and 12 for 2, 4, 8 and 24 hour time course incubations, as well as a separate 24 hour incubation in buffer without enzyme as a negative control. These time course digestion samples were then subjected to direct binding ELISA against human C-MET and human CD47. [Figure 10A]Functional analysis of the binding of purified intact and MMP-digested Her2CD3 Fab2 protein constructs to human target proteins. The Fab2 format antibodies Her2CD3-L1-LH, Her2CD3-L2-L2 and Her2CD3-LHL-LHL were analyzed by ELISA without MMP digestion. [Figure 10B] Figure 1 shows functional analysis of the binding of purified intact and MMP-digested Her2CD3 Fab2 protein constructs to human target proteins. The Fab2 format antibodies Her2CD3-L1-LH, Her2CD3-L2-L2 and Her2CD3-LHL-LHL were analyzed by flow cytometry with or without MMP digestion. [Figure 10C] Figure 1 shows the functional analysis of the binding of purified intact and MMP-digested Her2CD3 Fab2 protein constructs to human target proteins. The Fab2 format antibodies Her2CD3-L1-LH, Her2CD3-L2-L2 and Her2CD3-LHL-LHL were analyzed by CD3 reporter assay with or without MMP digestion. [Figure 11-1]Alternative structures based on protein construct design and activation principles are shown. The protein construct modules (1) discussed in both the Fab2 and IgG2 designs of Figures 2 and 3 can be modified to change the final molecular functional properties. In this case, the upper binding unit or the lower unit, or both, of the protein construct module can be alternative structures of the immunoglobulin Fab domain, allowing alternative molecules based on sequences derived from peptides, receptor ectodomains, binding domains and other dimeric immune recognition receptors, such as T-cell receptors in particular. These constructs can form many formats, some examples are provided herein. For example, four polypeptide chains (2) can code for two complete protein construct modules containing four binding domain units (one A, one B, or two A or B), an IgG2-like structure containing two or more linker sequences, and may or may not have an immunoglobulin hinge region and an Fc domain, with heterodimer pairing induced by mutations in Fc or not. Note that any of the structures outlined in this figure or in Figures 2 or 3 can be further functionalized by adding the N-terminus of a C-terminal fusion of any kind of polypeptide chain or by chemical conjugation. Variable regions are shown in white. Constant regions are shown in grey. [Figure 11-2] Alternative structures based on protein construct design and activation principles are shown. In the expression of three polypeptides, the Fab2 design can be augmented by adding binding domains (3) or peptides that make the structure potentially trispecific or alter its valency. Trispecificity or altered valency can also be achieved in the Fab2 design by adding one (4) or two (5) additional protein construct linker Fab / receptor structures at the C-terminus. Note that any of the structures outlined in this figure or in Figures 2 or 3 can be further functionalized by adding N-terminus of C-terminal fusion of any kind of polypeptide chain or by chemical conjugation. Variable regions are shown in white. Constant regions are shown in grey. [Figure 12]A "passive" structure based on the Fab2 protein structure principle is shown. In this case, the top binding unit of the protein structure module may be placed at the C-terminus of the Fc domain. These structures may or may not have an immunoglobulin hinge region and an Fc domain, where heterodimer pairing may or may not be induced by mutations in the Fc. In this structure, binding of both Fab or receptor domains to their cognate targets needs to be fully activated 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 N- or C-terminal fusions of any kind of polypeptide chain or by chemical conjugation. Variable regions are shown in white. Constant regions are shown in grey. [Figure 13]We present an "activatable" antibody drug conjugate (ADC) strategy based on the Fab2 protein construct principle. In this case, the upper and lower binding units of the protein construct module may contain antibodies against the same internalizing receptor target or two different targets found on the surface of the same cell. These constructs can be chemically conjugated or fused with "payload" moieties such as toxins or other active molecules to form ADCs, and may or may not have an immunoglobulin hinge region and Fc domain, with heterodimer pairing induced by mutations in the Fc or not. In this construct, binding of the upper Fab or receptor domain to its cognate target is constitutively active, leading to accumulation of the antibody in tissues where its cognate target is expressed. The construct does not initially promote internalization into the target cell, as it is known that receptors are only significantly internalized when the binding is monovalent and two or more receptor domains are crosslinked by bivalent antibody binding. Activity of the second (lower) Fab or receptor domain need only occur following linker cleavage by a disease-associated enzyme, which then induces multivalent receptor binding and internalization of the ADC, allowing delivery of a (e.g., cytotoxic or inflammatory) payload moiety. Note also that any of the structures outlined in this figure can be further functionalized by adding N- or C-terminal fusions of any type of polypeptide chain. [Figure 14] Direct ELISA of binding of purified, intact protein constructs to human and mouse target proteins. Samples were applied in a direct binding ELISA against human Her2 and human and mouse CD47. [Figure 15A] Direct ELISA of binding of purified intact and MMP-digested protein constructs to human target proteins. Protein constructs were subjected to enzymatic digestion with human MMP7 for 2, 4, 8 and 24 hours of incubation, as well as a 24 hour incubation in buffer without enzyme as a negative control (time 0). These time-course digestion samples were then subjected to direct binding ELISA against human Her2 and CD47. [Figure 15B] Direct ELISA of binding of purified intact and MMP-digested protein constructs to human target proteins. Protein constructs were subjected to enzymatic digestion with human MMP8 for 2, 4, 8 and 24 hours of incubation, as well as a 24 hour incubation in buffer without enzyme as a negative control (time 0). These digestion-dependent samples were then subjected to direct binding ELISA against human Her2 and CD47. [Figure 15C] Direct ELISA of binding of purified intact and MMP-digested protein constructs to human target proteins. Protein constructs were subjected to enzymatic digestion with human MMP10 for 2, 4, 8 and 24 hours of incubation, as well as a 24 hour incubation in buffer without enzyme as a negative control (time 0). These time-course digestion samples were then subjected to direct binding ELISA against human Her2 and CD47. [Figure 15D] Direct ELISA of binding of purified intact and MMP-digested protein constructs to human target proteins. Protein constructs were subjected to enzymatic digestion with human MMP12 for 2, 4, 8 and 24 hours of incubation, as well as a 24 hour incubation in buffer without enzyme as a negative control (time 0). These digestion-dependent samples were then subjected to direct binding ELISA against human Her2 and CD47. [Figure 15E] Direct ELISA of binding of purified intact and MMP-digested protein constructs to human target proteins. Protein constructs were subjected to enzymatic digestion with human MMP13 for 2, 4, 8 and 24 hours of incubation, as well as a 24 hour incubation in buffer without enzyme as a negative control (time 0). These time-course digestion samples were then subjected to direct binding ELISA against human Her2 and CD47. [Figure 15F]Direct ELISA of binding of purified intact and MMP digested protein constructs to human target proteins. Protein constructs were subjected to enzymatic digestion with human cathepsin S for 2, 4, 8 and 24 hours of incubation, as well as a 24 hour incubation in buffer without enzyme as a negative control (time 0). These digestion time series samples were then subjected to direct binding ELISA against human Her2 and CD47. [Figure 16] A-B show Biacore SPR assays of binding of purified intact and MMP-digested Her47-LHL-LHLF to human target proteins. Her47-LHL-LHLF was subjected to enzymatic digestion with human MMP12 for 2, 4, 8 and 24 hours of incubation, as well as a 24 hour incubation in buffer without enzyme as a negative control (undigested). These digestion-dependent samples were then captured on an anti-Fc antibody-coated Biacore chip, and human Her2 (A) or human CD47 (B) were run through the solution. Rmax values ​​were plotted to represent the maximum binding observed at the highest concentration of analyte protein. [Figure 17] Biacore SPR assay of binding of purified intact and 24-hour MMP-digested Her47-LHL-LHLF to human target protein. Her47-LHL-LHLF was subjected to enzymatic digestion with human MMP12 for 24 hours incubation or without enzyme as a negative control ("before protease treatment"). Samples were then captured on an anti-Fc antibody-coated Biacore chip and human CD47 was run in solution at multiple concentrations. Binding curves showed no interaction of undigested (intact) Her47-LHL-LHLF protein with human CD47, even at 400 nM, whereas the same protein after MMP12 activation showed strong binding at all concentrations tested. [Figure 18A]Structural modeling of the complete Her47-LHL-LHL IgG2 construct. Molecular modeling of the complete structure of the IgG2 molecule showing the upper (Trastuzumab) Fab domain in contact with the Her2 epitope (grey). This analysis shows that the CD47 epitope cannot be bound when both linkers are intact. [Figure 18B] Structural modeling of the complete Her47-LHL-LHL IgG2 construct. Molecular modeling of the complete structure of the IgG2 molecule showing that the upper (trastuzumab) Fab domain is in contact with the Her2 epitope (grey), but the CD47 ectodomain also overlaps with the potential binding position of the A-D5 lower Fab. This analysis indicates that the CD47 epitope cannot be bound when both linkers are intact. [Figure 19A] Figure 2 shows the structural dynamics of Her47 Fab2 constructs with different linkers. Figure 3 shows the solvent accessible surface area (SASA) results obtained for the LHL linker. Absolute SASA values ​​for all 9 kinetic runs for 6 nanoseconds are shown for the LHL linker. SEQ ID NO:2 is shown. [Figure 19B] Figure 2 shows the structural dynamics of Her47 Fab2 constructs with different linkers. Figure 3 shows the resulting solvent accessible surface area (SASA) results for the LHL linker. Figure 4 shows normalized results representing the difference from the starting SASA value over a kinetics run time of 6 nanoseconds. Figure 5 shows SEQ ID NO:2. [Figure 19C] Figure 2 shows the structural dynamics of Her47 Fab2 constructs with different linkers. Figure 3 shows the resulting solvent accessible surface area (SASA) results for the LHL linker. Figure 4 shows normalized results representing the difference from the starting SASA value over the first 2.5 ns of a 6 ns kinetics run. Figure 5 shows SEQ ID NO:2. [Figure 19D] Figure 1 shows the structural dynamics of Her47 Fab2 constructs with different linkers. Figure 2 shows the solvent accessible surface area (SASA) results obtained for the LHLF linker. Figure 3 shows absolute SASA values ​​for all 9 kinetic runs for 6 nanoseconds for the LHLF linker. [Figure 19E] Figure 2 shows the structural dynamics of Her47 Fab2 constructs with different linkers. Figure 3 shows the solvent accessible surface area (SASA) results obtained for the LHLF linker. Figure 4 shows normalized results representing the difference from the starting SASA value over a 6 nanosecond kinetics run time. Figure 5 shows SEQ ID NO:3. [Figure 19F] Figure 1 shows the structural dynamics of Her47 Fab2 constructs with different linkers. Figure 2 shows the resulting solvent accessible surface area (SASA) results for the LHLF linker. Figure 3 shows normalized results representing the difference from the starting SASA value over the first 2.5 nanoseconds of a 6 nanosecond kinetics run. Figure 4 shows SEQ ID NO:3. [Figure 19G] Figure 1 shows the structural dynamics of Her47 Fab2 constructs with different linkers. Figure 2 shows the solvent accessible surface area (SASA) results obtained for the L2 linker. Absolute SASA values ​​are shown for 10 kinetic runs, all run for 6 nanoseconds with L2. SEQ ID NO: 32 is shown. [Figure 19H] Figure 1 shows the structural dynamics of Her47 Fab2 constructs with different linkers. Figure 2 shows the resulting solvent accessible surface area (SASA) results for the L2 linker. Figure 3 shows normalized results representing the difference from the starting SASA value over a 6 nanosecond kinetics run time. Figure 4 shows SEQ ID NO: 32. [Figure 19I] Figure 1 shows the structural dynamics of Her47 Fab2 constructs with different linkers. Figure 2 shows the resulting solvent accessible surface area (SASA) results for the L2 linker. Figure 3 shows normalized results representing the difference from the starting SASA value over the first 2.5 nanoseconds of a ns dynamics run. Figure 4 shows SEQ ID NO: 32. [Figure 20A] Figure 1 shows the structural dynamics of intact and activated Her47 LHL-LHL Fab2 constructs. Shown are the overlapping Fab2 constructs in both intact linker and activated (single linker cleaved by protease) forms. [Figure 20B]Structural dynamics of intact and activated Her47 LHL-LHL Fab2 constructs. Shown are two poses obtained from molecular dynamics simulations of the Fab2 region with one LHL or LHLF linker cleaved. Anti-CD47 Fab is shown in black and anti-HER2 Fab in grey. The figure shows the furthest extent of movement of the Her2 domain and the resulting exposure of the anti-CD47 Fab CDRs. [Figure 21] 1 shows flow cytometric analysis of protein binding to "Tg32" mouse erythrocytes. Flow cytometric analysis of binding to erythrocytes was performed using A-D5 IgG1, IgG2 Her47 LHL-LHL, IgG2 Her47 LHL-LHLF and Fab2 Met47 LHL-LHL. Binding was measured using an anti-human PE-conjugated secondary antibody. A-D5 IgG1 was tested at 0.1 μg / ml, 1 μg / ml and 10 μg / ml. IgG2 Her47 LHL-LHL was tested at 0.1 μg / ml, 1 μg / ml and 10 μg / ml. IgG2 Her47 LHL-LHLF was tested at 0.1 μg / ml, 1 μg / ml and 10 μg / ml. Fab2 Met47 LHL-LHL was tested at 0.1 μg / ml, 1 μg / ml and 10 μg / ml. [Figure 22] "Tg32" mouse erythrocyte agglutination analysis. Agglutination of erythrocytes was performed using A-D5 IgG1, IgG2 Her47 LHL-LHL, IgG2 Her47 LHL-LHLF and Fab2 Met47 LHL-LHL. Proteins were titrated (in nM) using pooled fresh erythrocytes from multiple donor mice. [Figure 23] Tolerance study in "Tg32" mice: Body weight analysis. A tolerability study with A-D5 IgG1, IgG2 Her47 LHL-LHL, IgG2 Her47 LHL-LHLF and Fab2 Met47 LHL-LHL was performed in Tg32 mice with all proteins administered at concentrations of 2mg / kg and 10mg / kg. Body weight was then monitored for 60 days. The 10mg / kg dose of A-D5 IgG1 was not tolerated and the cohort was terminated on day 1. [Figure 24] Tolerance study in "Tg32" mice: Reticulocyte analysis 5 days after dosing. Tolerance studies with A-D5 IgG1, IgG2 Her47 LHL-LHL, IgG2 Her47 LHL-LHLF and Fab2 Met47 LHL-LHL were performed in Tg32 mice with all proteins administered at 2mg / kg and 10mg / kg concentrations. Blood samples were taken and reticulocyte levels were measured. A-D5 IgG1 at the 2mg / kg dose showed a significant increase in reticulocyte levels. [Figure 25A] Tolerance study in "Tg32" mice: Hematological analysis at 5, 29 and 60 days post-dosing is shown. Tolerance studies with A-D5 IgG1, IgG2 Her47 LHL-LHL, IgG2 Her47 LHL-LHLF and Fab2 Met47 LHL-LHL were performed in Tg32 mice with all proteins administered at concentrations of 2mg / kg and 10mg / kg. Blood samples were taken and reticulocyte levels were measured. [Figure 25B] Tolerance study in "Tg32" mice: Hematological analysis at 5, 29 and 60 days post-dosing is shown. Tolerance studies with A-D5 IgG1, IgG2 Her47 LHL-LHL, IgG2 Her47 LHL-LHLF and Fab2 Met47 LHL-LHL were performed in Tg32 mice with all proteins administered at concentrations of 2mg / kg and 10mg / kg. Blood samples were taken and red blood cell (RBC) levels were measured. [Figure 25C] Tolerance study in "Tg32" mice: Hematological analysis at 5, 29 and 60 days post-dosing. Tolerance studies with A-D5 IgG1, IgG2 Her47 LHL-LHL, IgG2 Her47 LHL-LHLF and Fab2 Met47 LHL-LHL were performed in Tg32 mice with all proteins administered at concentrations of 2mg / kg and 10mg / kg. Blood samples were taken and hemoglobin levels were measured. [Figure 25D]Tolerance study in "Tg32" mice: Hematological analysis at 5, 29 and 60 days post-dosing is shown. Tolerance studies with A-D5 IgG1, IgG2 Her47 LHL-LHL, IgG2 Her47 LHL-LHLF and Fab2 Met47 LHL-LHL were performed in Tg32 mice with all proteins administered at concentrations of 2mg / kg and 10mg / kg. Blood samples were taken and mean hemoglobin concentration (MCHC) levels in red blood cells were measured. [Figure 25E] Tolerance study in "Tg32" mice: Hematological analysis at 5, 29 and 60 days post-dosing is shown. Tolerance studies with A-D5 IgG1, IgG2 Her47 LHL-LHL, IgG2 Her47 LHL-LHLF and Fab2 Met47 LHL-LHL were performed in Tg32 mice with all proteins administered at concentrations of 2mg / kg and 10mg / kg. Blood samples were taken and mean corpuscular volume (MCV) levels were measured. [Figure 25F] Tolerance study in "Tg32" mice: Hematological analysis at 5, 29 and 60 days post-dosing is shown. Tolerance studies with A-D5 IgG1, IgG2 Her47 LHL-LHL, IgG2 Her47 LHL-LHLF and Fab2 Met47 LHL-LHL were performed in Tg32 mice with all proteins administered at concentrations of 2mg / kg and 10mg / kg. Blood samples were taken and white blood cell levels were measured. [Figure 25G] Tolerance study in "Tg32" mice: Hematological analysis at 5, 29 and 60 days post-dosing. Tolerance studies with A-D5 IgG1, IgG2 Her47 LHL-LHL, IgG2 Her47 LHL-LHLF and Fab2 Met47 LHL-LHL were performed in Tg32 mice with all proteins administered at concentrations of 2mg / kg and 10mg / kg. Blood samples were taken and monocyte levels were measured. [Fig. 25H]Tolerance study in "Tg32" mice: Hematological analysis at 5, 29 and 60 days post-dosing. Tolerance studies with A-D5 IgG1, IgG2 Her47 LHL-LHL, IgG2 Her47 LHL-LHLF and Fab2 Met47 LHL-LHL were performed in Tg32 mice with all proteins administered at concentrations of 2mg / kg and 10mg / kg. Blood samples were taken and lymphocyte levels were measured. [Figure 25I] Tolerance study in "Tg32" mice: Hematological analysis at 5, 29 and 60 days post-dosing. Tolerance studies with A-D5 IgG1, IgG2 Her47 LHL-LHL, IgG2 Her47 LHL-LHLF and Fab2 Met47 LHL-LHL were performed in Tg32 mice with all proteins administered at concentrations of 2mg / kg and 10mg / kg. Blood samples were taken and basophil levels were measured. [Figure 25J] Tolerance study in "Tg32" mice: Hematological analysis at 5, 29 and 60 days post-treatment. Tolerance studies with A-D5 IgG1, IgG2 Her47 LHL-LHL, IgG2 Her47 LHL-LHLF and Fab2 Met47 LHL-LHL were performed in Tg32 mice with all proteins administered at concentrations of 2mg / kg and 10mg / kg. Blood samples were taken and eosinophil levels were measured. [Figure 25K] Tolerance study in "Tg32" mice: Hematological analysis at 5, 29 and 60 days post-dosing. Tolerance studies with A-D5 IgG1, IgG2 Her47 LHL-LHL, IgG2 Her47 LHL-LHLF and Fab2 Met47 LHL-LHL were performed in Tg32 mice with all proteins administered at concentrations of 2mg / kg and 10mg / kg. Blood samples were taken and neutrophil levels were measured. [Figure 26]Pharmacokinetic studies in "Tg32" mice: data per molecule at two doses are shown. Pharmacokinetic studies with IgG2 Her47 LHL-LHL, IgG2 Her47 LHL-LHLF and Fab2 Met47 LHL-LHL were performed in Tg32 mice with all proteins administered at concentrations of 2 mg / kg and 10 mg / kg. A-D5 IgG1 was administered at 2 mg / kg. Serum samples were taken to measure human IgG levels (in μg / ml) from 30 minutes up to 42 days post-dose. [Figure 27A] Pharmacokinetic studies in "Tg32" mice: data per dose are shown. Pharmacokinetic studies with IgG2 Her47 LHL-LHL, IgG2 Her47 LHL-LHLF and Fab2 Met47 LHL-LHL were performed in Tg32 mice with all proteins administered at concentrations of 2 mg / kg and 10 mg / kg. A-D5 IgG1 was administered at 2 mg / kg. Serum samples were collected to measure human IgG levels (in μg / ml) from 30 minutes up to 42 days post-dose. Concentrations at the 2 mg / kg dose are plotted respectively. For reference, a 2 mg / kg dose of A-D5 IgG1 was included in the analysis. [Figure 27B] Pharmacokinetic studies in "Tg32" mice: data per dose are shown. Pharmacokinetic studies with IgG2 Her47 LHL-LHL, IgG2 Her47 LHL-LHLF and Fab2 Met47 LHL-LHL were performed in Tg32 mice with all proteins administered at concentrations of 2 mg / kg and 10 mg / kg. A-D5 IgG1 was administered at 2 mg / kg. Serum samples were collected to measure human IgG levels (in μg / ml) from 30 minutes up to 42 days post-dose. Concentrations at the 10 mg / kg dose were plotted respectively. For reference, a 2 mg / kg dose of A-D5 IgG1 was included in the analysis. [Figure 28]Pharmacokinetic studies in "Tg32" mice: AUC data per dose are shown. Pharmacokinetic studies with IgG2 Her47 LHL-LHL, IgG2 Her47 LHL-LHLF and Fab2 Met47 LHL-LHL were performed in Tg32 mice with all proteins administered at concentrations of 2mg / kg and 10mg / kg. A-D5 IgG1 was administered at 2mg / kg. Serum samples were collected to measure human IgG levels (in μg / ml) from 30 minutes up to 42 days post-dose. Concentration measurements over time were used to calculate the area under the curve (AUC) per dose. [Figure 29A] Flow cytometric analysis of binding to NHP red blood cells. Flow cytometric analysis of binding to red blood cells was performed using A-D5 3M (effector null) IgG1, IgG2 Her47 LHL-LHL, IgG2 Her47 LHL-LHLF and trastuzumab. Binding was measured using an anti-human PE-conjugated secondary antibody. A-D5 IgG1 was the only protein that showed concentration-dependent binding to NHP (cynomolgus monkey) red blood cells. [Figure 29B] Flow cytometric analysis of binding to human red blood cells. Flow cytometric analysis of binding to red blood cells was performed using A-D5 3M (effector null) IgG1, IgG2 Her47 LHL-LHL, IgG2 Her47 LHL-LHLF and trastuzumab. Binding was measured using an anti-human PE-conjugated secondary antibody. A-D5 IgG1 was the only protein that showed concentration-dependent binding to human red blood cells. [Figure 30A] Direct ELISA of binding of purified intact and MMP digested (digested at pH 7.4) protein constructs to human target proteins. Protein constructs were subjected to enzymatic digestion with human MMP3 at pH 7.4 for 2, 4, 8 and 24 hours of incubation, as well as a 24 hour incubation in buffer without enzyme as a negative control (time 0). These digestion-dependent samples were then subjected to direct binding ELISA against human Her2 and CD47. [Figure 30B]Direct ELISA of binding of purified intact and MMP digested (digested at pH 6.0) protein constructs to human target proteins. Protein constructs were subjected to enzymatic digestion with human MMP3 at pH 6.0 for 2, 4, 8 and 24 hours of incubation, as well as a 24 hour incubation in buffer without enzyme as a negative control (time 0). These digestion-dependent samples were then subjected to direct binding ELISA against human Her2 and CD47. [Figure 30C] Direct ELISA of binding of purified intact and MMP digested (digested at pH 7.4) protein constructs to human target proteins. Protein constructs were subjected to enzymatic digestion with human MMP7 at pH 7.4 for 2, 4, 8 and 24 hours of incubation, as well as a 24 hour incubation in buffer without enzyme as a negative control (time 0). These digestion-dependent samples were then subjected to direct binding ELISA against human Her2 and CD47. [Figure 30D] Direct ELISA of binding of purified intact and MMP digested (digested at pH 6.0) protein constructs to human target proteins. Protein constructs were subjected to enzymatic digestion with human MMP7 at pH 6.0 for 2, 4, 8 and 24 hours of incubation, as well as a 24 hour incubation in buffer without enzyme as a negative control (time 0). These digestion-dependent samples were then subjected to direct binding ELISA against human Her2 and CD47. [Figure 30E] Direct ELISA of binding of purified intact and MMP digested (digested at pH 7.4) protein constructs to human target proteins. Protein constructs were subjected to enzymatic digestion with human MMP8 at pH 7.4 for 2, 4, 8 and 24 hours of incubation, as well as a 24 hour incubation in buffer without enzyme as a negative control (time 0). These digestion-dependent samples were then subjected to direct binding ELISA against human Her2 and CD47. [Figure 30F]Direct ELISA of binding of purified intact and MMP digested (digested at pH 6.0) protein constructs to human target proteins. Protein constructs were subjected to enzymatic digestion with human MMP8 at pH 6.0 for 2, 4, 8 and 24 hours of incubation, as well as a 24 hour incubation in buffer without enzyme as a negative control (time 0). These digestion-dependent samples were then subjected to direct binding ELISA against human Her2 and CD47. [Figure 30G] Direct ELISA of binding of purified intact and MMP digested (digested at pH 7.4) protein constructs to human target proteins. Protein constructs were subjected to enzymatic digestion with human MMP10 at pH 7.4 for 2, 4, 8 and 24 hours of incubation, as well as a 24 hour incubation in buffer without enzyme as a negative control (time 0). These digestion-dependent samples were then subjected to direct binding ELISA against human Her2 and CD47. [Figure 30H] Direct ELISA of binding of purified intact and MMP digested (digested at pH 6.0) protein constructs to human target proteins. Protein constructs were subjected to enzymatic digestion with human MMP10 at pH 6.0 for 2, 4, 8 and 24 hours of incubation, as well as a 24 hour incubation in buffer without enzyme as a negative control (time 0). These digestion-dependent samples were then subjected to direct binding ELISA against human Her2 and CD47. [Figure 30I] Direct ELISA of binding of purified intact and MMP digested (digested at pH 7.4) protein constructs to human target proteins. Protein constructs were subjected to enzymatic digestion with human MMP12 at pH 7.4 for 2, 4, 8 and 24 hours of incubation, as well as a 24 hour incubation in buffer without enzyme as a negative control (time 0). These digestion-dependent samples were then subjected to direct binding ELISA against human Her2 and CD47. [Figure 30J]Direct ELISA of binding of purified intact and MMP digested (digested at pH 6.0) protein constructs to human target proteins. Protein constructs were subjected to enzymatic digestion with human MMP12 at pH 6.0 for 2, 4, 8 and 24 hours of incubation, as well as a 24 hour incubation in buffer without enzyme as a negative control (time 0). These digestion-dependent samples were then subjected to direct binding ELISA against human Her2 and CD47. [Figure 30K] Direct ELISA of binding of purified intact and MMP digested (digested at pH 7.4) protein constructs to human target proteins. Protein constructs were subjected to enzymatic digestion with human MMP13 at pH 7.4 for 2, 4, 8 and 24 hours of incubation, as well as a 24 hour incubation in buffer without enzyme as a negative control (time 0). These digestion-dependent samples were then subjected to direct binding ELISA against human Her2 and CD47. [Figure 30L] Direct ELISA of binding of purified intact and MMP digested (digested at pH 6.0) protein constructs to human target proteins. Protein constructs were subjected to enzymatic digestion with human MMP13 at pH 6.0 for 2, 4, 8 and 24 hours of incubation, as well as a 24 hour incubation in buffer without enzyme as a negative control (time 0). These digestion-dependent samples were then subjected to direct binding ELISA against human Her2 and CD47. [Figure 30M] Direct ELISA of binding of purified intact and MMP digested (digested at pH 7.4) protein constructs to human target proteins. Protein constructs were subjected to enzymatic digestion with human MMP14 at pH 7.4 for 2, 4, 8 and 24 hours of incubation, as well as a 24 hour incubation in buffer without enzyme as a negative control (time 0). These digestion-dependent samples were then subjected to direct binding ELISA against human Her2 and CD47. [Figure 30N] Direct ELISA of binding of purified intact and MMP digested (digested at pH 6.0) protein constructs to human target proteins. Protein constructs were subjected to enzymatic digestion with human MMP14 at pH 6.0 for 2, 4, 8 and 24 hours of incubation, as well as a 24 hour incubation in buffer without enzyme as a negative control (time 0). These digestion-dependent samples were then subjected to direct binding ELISA against human Her2 and CD47. [Figure 31A] Direct ELISA of binding of purified intact and cathepsin digested (digested at pH 7.4 and pH 6.0) protein constructs to human target proteins. Protein constructs were subjected to enzymatic digestion with human cathepsin enzyme at pH 7.4 or pH 6.0 for 2, 4, 8 and 24 hours of incubation over time, as well as a separate 24 hour incubation in buffer without enzyme as a negative control (time 0). These digestion over time samples were then subjected to direct binding ELISA against human Her2 and CD47. [Figure 31B] Direct ELISA of binding of purified intact and cathepsin digested (digested at pH 7.4 and pH 6.0) protein constructs to human target proteins. Protein constructs were subjected to enzymatic digestion with human cathepsin enzyme at pH 7.4 or pH 6.0 for 2, 4, 8 and 24 hours of incubation over time, as well as a separate 24 hour incubation in buffer without enzyme as a negative control (time 0). These digestion over time samples were then subjected to direct binding ELISA against human Her2 and CD47. [Figure 32A]Flow cytometric analysis of binding to human cancer cells. Flow cytometric analysis of binding to red blood cells was performed using anti-CD47, trastuzumab, IgG1 isotype, and IgG2 Her47 LHL-LHLF, which were subjected to enzymatic digestion at pH 7.4 using human MMP12 for 2, 4, 8 and 24 hours of incubation over time, and an additional 24 hour incubation in buffer without enzyme as a negative control (time 0). Binding was measured using an anti-human PE-conjugated secondary antibody. Binding was measured on the high Her2 cell line BT-474. [Figure 32B] Flow cytometric analysis of binding to human cancer cells. Flow cytometric analysis of binding to red blood cells was performed using anti-CD47, trastuzumab, IgG1 isotype, and IgG2 Her47 LHL-LHL, which were subjected to enzymatic digestion at pH 7.4 using human MMP12 for 2, 4, 8 and 24 hours of incubation over time, and an additional 24 hour incubation in buffer without enzyme as a negative control (time 0). Binding was measured using an anti-human PE-conjugated secondary antibody. Binding was measured on the high Her2 cell line BT-474. [Figure 32C] Flow cytometric analysis of binding to human cancer cells. Flow cytometric analysis of binding to red blood cells was performed using anti-CD47, trastuzumab, IgG1 isotype, and IgG2 Her47 LHL-LHLF, which were subjected to enzymatic digestion at pH 7.4 using human MMP12 for 2, 4, 8 and 24 hours of incubation over time, and an additional 24 hour incubation in buffer without enzyme as a negative control (time 0). Binding was measured using an anti-human PE-conjugated secondary antibody. Binding was measured with low Her2MCF-7. [Fig. 32D]Flow cytometric analysis of binding to human cancer cells. Flow cytometric analysis of binding to red blood cells was performed using anti-CD47, trastuzumab, IgG1 isotype, and IgG2 Her47 LHL-LHL, subjected to enzymatic digestion at pH 7.4 using human MMP12 for 2, 4, 8 and 24 hours of incubation over time, and separately incubated for 24 hours in buffer without enzyme as a negative control (time 0). Binding was measured using an anti-human PE-conjugated secondary antibody. Binding was measured with low Her2MCF-7. [Diagram 33] Figure 1 shows SDS-PAGE analysis of MMP12 digested IgG2 Her47 LHL-LHL or IgG2 Her47 LHL-LHLF. SDS-PAGE was performed on samples of IgG2 Her47 LHL-LHL or IgG2 Her47 LHL-LHLF that were subjected to enzymatic digestion at pH 7.4 with human MMP12 for a time course of incubation of 2, 4, 8 and 24 hours, as well as an additional 24 hour incubation in buffer without enzyme as a negative control (time 0). [Figure 34A] 1 shows mass spectrometry of MMP12 digested IgG2 Her47 LHL-LHL. Mass spectrometry was performed on samples of IgG2 Her47 LHL-LHL that were subjected to enzymatic digestion at pH 7.4 with human MMP12 for 2, 4, 8 and 24 hours of incubation over time, as well as a separate 24 hour incubation in buffer without enzyme present as a negative control (time 0). The presence of a peptide representing an intact LHL linker was measured. This figure depicts SEQ ID NO: 110. [Figure 34B] 1 shows mass spectrometry of MMP12 digested IgG2 Her47 LHL-LHL. Mass spectrometry was performed on samples of IgG2 Her47 LHL-LHL that were subjected to enzymatic digestion at pH 7.4 with human MMP12 for 2, 4, 8 and 24 hours of incubation over time, as well as a separate 24 hour incubation in buffer without enzyme present as a negative control (time 0). The presence of a peptide representing the MMP12 cleavage linker was measured. This figure depicts SEQ ID NO: 111. [Diagram 35] Size exclusion chromatography of Protein A purified Her47 LHLF-LHL IgG1-2hDAA. Her47 LHLF-LHL IgG1-2hDAA protein was expressed in CHO cells, purified by ProA column and analyzed by SEC. Two small high MW peaks and a large peak of the expected size product (10.30, approximately 250 kDa) were observed. [Diagram 36] Figure 1 shows SDS-PAGE analysis of purified peak fractions from size exclusion chromatography of Her47 LHLF-LHL IgG1-2hDAA. SDS-PAGE was performed on a non-reduced sample of Her47 LHLF-LHL IgG1-2hDAA. 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 (desired product). [Figure 37] SDS-PAGE analysis of purified peak fractions from size exclusion chromatography of Her47 LHLF-LHL IgG1-2hDAA. SDS-PAGE was performed on a reduced sample of Her47 LHLF-LHL IgG1-2hDAA. 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 (desired product). [Figure 38A] Direct ELISA of purified intact and MMP12 digested Her47 IgG1-2hDAA proteins. Her47 LHL-LHLF IgG1-2hDAA was subjected to enzymatic digestion with human MMP12 at pH 7.4 for 2, 4, 8 and 24 hours incubation, as well as a 24 hour incubation in buffer without enzyme as a negative control (0, 2, 4, 8, 24 hours incubation). These digestion time series samples were then subjected to direct binding ELISA against human Her2 and mouse EpCAM. [Figure 38B]Direct ELISA of purified intact and MMP12 digested Her47 IgG1-2hDAA protein. Her47 LHL-LHLF IgG1-2hDAA was subjected to enzymatic digestion at pH 7.4 with human MMP12 for 2, 4, 8 and 24 hours of incubation, as well as an additional 24 hour incubation in buffer without enzyme as a negative control (0, 2, 4, 8, 24 hours of incubation). ELISA against human CD47 was then performed on digested (dark grey) and undigested (light grey) samples. [Figure 38C] SDS-PAGE analysis of purified intact and MMP12 digested Her47 IgG1-2hDAA protein. Her47 LHL-LHLF IgG1-2hDAA was subjected to enzymatic digestion at pH 7.4 with human MMP12 for 2, 4, 8 and 24 hours of incubation, as well as an additional 24 hour incubation in buffer without enzyme as a negative control (0, 2, 4, 8, 24 hour incubation). SDS-PAGE was performed for each sample. Lane 1 - molecular weight marker, lane 2 - 0 hour digest, lane 3 - 2 hour digest, lane 4 - 8 hour digest and lane 5 - 24 hour digest. [Figure 39A] Direct ELISA of purified intact and MMP12-digested IgG2 Her47 proteins with alternative linker compositions. Purified protein from clone Her47 LHL-LHL-EK was tested in a titration ELISA against human Her2 and CD47 targets. [Figure 39B] Direct ELISA of purified intact and MMP12 digested IgG2 Her47 proteins with alternative linker compositions. Purified proteins from clone Her47 LHL-LHL-EK were subjected to sequential enzymatic digestion and ELISA for binding to Her2 and CD47 targets. [Figure 39C]Direct ELISA of purified intact and MMP12-digested IgG2 Her47 proteins with alternative linker compositions. Purified protein from clone Her47 LHL-LHL-Thr was tested in a titration ELISA against human Her2 and CD47 targets. [Figure 39D] Direct ELISA of purified intact and MMP12 digested IgG2 Her47 proteins with alternative linker compositions. Purified protein from clone Her47 LHL-LHL-Thr was subjected to sequential enzymatic digestion and ELISA for binding to Her2 and CD47 targets. [Figure 39E] Direct ELISA of purified intact and MMP12 digested IgG2 Her47 proteins with alternative linker compositions. Purified protein from clone Her47 LHL-LHL-tPA was tested in a titration ELISA against human Her2 and CD47 targets. [Figure 39F] Direct ELISA of purified intact and MMP12 digested IgG2 Her47 proteins with alternative linker compositions. Purified proteins from clone Her47 LHL-LHL-tPA were subjected to sequential enzymatic digestion and ELISA for binding to Her2 and CD47 targets. [Figure 39G] Direct ELISA of purified intact and MMP12 digested IgG2 Her47 proteins with alternative linker compositions. Purified protein from clone Her47 LHL-LHL-uPA was tested in a titration ELISA against human Her2 and CD47 targets. [Fig. 39H] Direct ELISA of purified intact and MMP12 digested IgG2 Her47 proteins with alternative linker compositions. Purified proteins from clone Her47 LHL-LHL-uPA were subjected to sequential enzymatic digestion and ELISA for binding to Her2 and CD47 targets. [Figure 39I]Direct ELISA of purified intact and MMP12-digested IgG2 Her47 proteins with alternative linker compositions. Purified protein from clone Her47 LHL-LHL-GrB was tested in a titration ELISA against human Her2 and CD47 targets. [Figure 39J] Direct ELISA of purified intact and MMP12 digested IgG2 Her47 proteins with alternative linker compositions. Purified proteins from clone Her47 LHL-LHL-GrB were subjected to sequential enzymatic digestion and ELISA for binding to Her2 and CD47 targets. [Figure 39K] Direct ELISA of purified intact and MMP12 digested IgG2 Her47 proteins with alternative linker compositions. Purified protein from clone Her47 LHL-LHL-A5 was tested in a titration ELISA against human Her2 and CD47 targets. [Figure 39L] Direct ELISA of purified intact and MMP12 digested IgG2 Her47 proteins with alternative linker compositions. Purified protein from clone Her47 LHL-LHL-A5 was subjected to sequential enzymatic digestion and ELISA for binding to Her2 and CD47 targets. [Diagram 40] Repeated dose tolerance study in NOD-SCID mice: Body weight analysis. Tolerance studies with IgG2 Her47 LHL-LHL, IgG2 Her47 LHL-LHLF, Fab2 Her47 LHL-LHL and Fab2 Her47 LHL-LHLF were performed with all proteins dosed every 5 days for a total of 4 doses. [Figure 41A] Size exclusion chromatography of Protein A purified Her2CD3 Fab2 protein. Fab2 Her23 LHL-LHL-S was expressed in CHO cells, purified by ProA column and analyzed by SEC. [Figure 41B]Size exclusion chromatography of Protein A purified Her2CD3 Fab2 protein. Fab2 Her23 LHLF-LHL-S was expressed in CHO cells, purified by ProA column and analyzed by SEC. [Figure 41C] Size exclusion chromatography of protein A purified Her2CD3 Fab2 protein. Fab2 Her23 LHL-LHL was expressed in CHO cells, purified by ProA column and analyzed by SEC. Fab2 Her23 LHL-LHL showed low molecular weight impurity (peak 15.38). [Figure 41D] Size exclusion chromatography of protein A purified Her2CD3 Fab2 protein. Fab2 Her23 LHLF-LHL was expressed in CHO cells, purified by ProA column and analyzed by SEC. Fab2 Her23 LHLF-LHL showed low molecular weight impurity (peak 15.38). [Figure 42A] Figure 1 shows CD3 interaction bioassay analysis of purified intact and MMP digested Her2CD3 Fab2 proteins using low Her2 MCF-7 cells. Antibody Fab2 Her23 LHLF-LHL-S was subjected to enzymatic digestion with human MMP12 at pH 7.4 for 2, 4, 8 and 24 hours of incubation, as well as a separate 24 hour incubation in buffer without enzyme as a negative control (time 0). These digestion time series samples were then subjected to a Promega Jurkat cell CD3 reporter assay using MCF-7 cells as target cells. [Figure 42B]Figure 1 shows CD3 interaction bioassay analysis of purified intact and MMP digested Her2CD3 Fab2 proteins using low Her2 MCF-7 cells. Antibody Fab2 Her23 LHL-LHL-S was subjected to enzymatic digestion with human MMP12 at pH 7.4 for 2, 4, 8 and 24 hours of incubation, as well as a separate 24 hour incubation in buffer without enzyme as a negative control (time 0). These digestion time series samples were then subjected to a Promega Jurkat cell CD3 reporter assay using MCF-7 cells as target cells. [Figure 43A] Figure 1 shows CD3 interaction bioassay analysis of purified intact and MMP digested Her2CD3 Fab2 proteins using high Her2 BT-474 cells. 0.1 μg / ml of control antibody was subjected to a Promega Jurkat cell CD3 reporter assay using BT-474 cells as target cells. [Figure 43B] CD3 interaction bioassay analysis using high Her2 BT-474 cells of purified intact and MMP digested Her2CD3 Fab2 proteins subjected to enzymatic digestion using human MMP12 at pH 7.4 for 2, 4, 8 and 24 hours of incubation over time, as well as a separate 24 hour incubation in buffer without enzyme present as a negative control (time 0), 0.1 μg / ml of Fab2 Her23 LHLF-LHL-S was subjected to a Promega Jurkat cell CD3 reporter assay using BT-474 cells as target cells. [Figure 43C]Figure 1 shows CD3 interaction bioassay analysis using high Her2 BT-474 cells of purified intact and MMP digested Her2CD3 Fab2 proteins subjected to enzymatic digestion with human MMP12 at pH 7.4 for 2, 4, 8 and 24 hours of incubation over time, as well as a separate 24 hour incubation in buffer without enzyme present as a negative control (time 0), 0.1 μg / ml of Fab2 Her23 LHL-LHL-S was subjected to a Promega Jurkat cell CD3 reporter assay using BT-474 cells as target cells. [Figure 44A] Charge variant analysis of IgG2 and Fab2 Her47 proteins is shown. Charge heterogeneity analysis is important for monoclonal antibody characterization since it provides important information about the quality and stability of the product. Heterogeneity can occur due to enzymatic post-translational modifications (glycosylation, lysine cleavage) or chemical modifications during purification and storage (oxidation or deamidation). Charge variant profiling of the provided specimens was performed by a commercial charge variant assay. The charge variant profile of IgG2 Her47 LHL-LHL showed a homogeneous profile with one major isoform (50-57% of the total), a major acidic isoform (40-48% of the total) and one minor basic isoform (approximately 3%). [Figure 44B]Charge variant analysis of IgG2 and Fab2 Her47 proteins is shown. Charge heterogeneity analysis is important for monoclonal antibody characterization since it provides important information about the quality and stability of the product. Heterogeneity can occur due to enzymatic post-translational modifications (glycosylation, lysine cleavage) or chemical modifications during purification and storage (oxidation or deamidation). Charge variant profiling of the provided specimens was performed by a commercial charge variant assay. The charge variant profile of Fab2 Her47 LHL-LHL showed a homogeneous profile including one major isoform (50-57% of the total), a major acidic isoform (40-48% of the total) and one minor basic isoform (approximately 3%). [Figure 45A] Size exclusion chromatography of Her47 protein after 5 cycles of freeze-thaw. IgG2 Her47 LHL-LHL was subjected to 5 cycles of freeze-thaw followed by SEC on samples from 0-5. No aggregation, fragmentation, or loss of product was observed for any of the proteins. [Figure 45B] Size exclusion chromatography of Her47 protein after 5 cycles of freeze-thaw. Fab2 Her47 LHL-LHL was subjected to 5 cycles of freeze-thaw followed by SEC on samples 0-5. No aggregation, fragmentation, or loss of product was observed for any of the proteins. [Figure 46] Figure 1 shows alternative protein construct designs. This figure illustrates a representative example of a protein construct Fab2 design. The designs can be based on: 1. a sequence with the top variable domain removed; 2. a sequence with a "dummy" non-binding variable domain included; 3. a sequence with the top Fab replaced by a diabody (i.e., two scFvs). The variable regions are shown in white. The constant regions are shown in grey. [Figure 47A]Figure 1 shows cell proliferation analysis using purified intact Her2CD47 protein high Her2 BT-474 cells. Trastuzumab, isotype control IgG1, and IgG2 Her47 LHL-LHL were administered to BT-474 cells over a 72 hour incubation period and cell proliferation was measured. Data is presented as percent inhibition of cell proliferation. [Figure 47B] Figure 1 shows cell proliferation analysis using purified intact Her2CD47 protein high Her2 BT-474 cells. Trastuzumab, isotype control IgG1, and Fab2 Her47 LHL-LHL were administered to BT-474 cells over a 72 hour incubation period and cell proliferation was measured. Data was presented as percent inhibition of cell proliferation. [Figure 48A] Figure 1 shows an in vivo efficacy analysis of Her47 molecules in NOD-SCID mice (KYSE-410 model). Trastuzumab was administered (intravenously, on days 0, 5, and 10) to NOD-SCID mice bearing KYSE-410 tumors. Tumor volume measurements were performed on days 4, 7, and 11 and plotted relative to vehicle. [Figure 48B] Figure 1 shows an in vivo efficacy analysis of Her47 molecules in NOD-SCID mice (KYSE-410 model). IgG2 Her47 LHL-LHLF was administered (intravenously, on days 0, 5, 10) to NOD-SCID mice bearing KYSE-410 tumors. Tumor volume measurements were performed on days 4, 7, and 11 and plotted relative to vehicle. [Figure 48C] Figure 1 shows an in vivo efficacy analysis of Her47 molecules in NOD-SCID mice (KYSE-410 model). IgG2 Her47 LHL-LHL was administered (intravenously, on days 0, 5, 10) to NOD-SCID mice bearing KYSE-410 tumors. Tumor volume measurements were performed on days 4, 7, and 11 and plotted relative to vehicle. [Figure 48D]Figure 1 shows an in vivo efficacy analysis of Her47 molecules in NOD-SCID mice (KYSE-410 model). Fab2 Her47 LHL-LHLF was administered (intravenously, on days 0, 5, 10) to NOD-SCID mice bearing KYSE-410 tumors. Tumor volume measurements were performed on days 4, 7, and 11 and plotted relative to vehicle. [Figure 48E] Figure 1 shows an in vivo efficacy analysis of Her47 molecules in NOD-SCID mice (KYSE-410 model). Fab2 Her47 LHL-LHL was administered (intravenously, on days 0, 5, 10) to NOD-SCID mice bearing KYSE-410 tumors. Tumor volume measurements were performed on days 4, 7, and 11 and plotted relative to vehicle. [Fig.48F] Figure 1 shows an in vivo efficacy analysis of Her47 molecules in NOD-SCID mice (KYSE-410 model). Fab2 Her47 LHL-LHLF and Fab2 Her47 LHL-LHL showed different potencies. [Figure 48G] Figure 1 shows an in vivo efficacy analysis of Her47 molecules in NOD-SCID mice (KYSE-410 model). None of the treatment groups showed weight loss, which could be considered as a toxicity of the administered molecule. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0037] Disclosed herein are recombinant proteins that are conditionally active in diseased human tissues. In some cases, the proteins contain a binding domain that is masked by another portion of the protein in non-diseased tissues. The proteins also contain 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, thereby allowing selective binding and / or action of the protein in the diseased tissue. The proteins of the present invention are particularly useful for binding to drug targets that are expressed in both diseased and non-diseased tissues.

[0038] Several activatable protein molecules and their medical uses are provided herein. In some embodiments, the molecules take into consideration multiple functional properties, including target binding specificity, effectively limiting undesirable activity in the native protein while being fully active in the activated form, maintaining conditional affinity for one or more targets from both humans and animal test species (e.g., cynomolgus monkeys, also known as crab-eating long-tailed monkeys, i.e., Macaca fascicularis), biophysical stability, and / or yield from protein expression platforms used in research, clinical, and commercial supply.

[0039] In some embodiments, protein molecules are provided that specifically bind to one or more human drug targets, and optionally to the cynomolgus orthologues of those targets, wherein the protein molecules have the following format: VC-Linker-VC VC-Linker-VC or C-Linker-VC C-Linker-VC, The heavy and light chain regions are assembled from one or more polypeptides of the formula:

[0040] In some embodiments, the protein molecule comprises two polypeptide chains and has the following format: VH1-C-Linker-VH2-C VL1-C-linker-VL2-C, has.

[0041] In some embodiments, the protein molecule comprises two polypeptide chains and has the following format: VL1-C-Linker-VH2-C VH1-C-linker-VL2-C, has.

[0042] "V" refers to an immunoglobulin or T cell receptor variable region, or an ectodomain of a receptor. "VH1" and "VL1" refer to the heavy and light chain variable regions that pair together to bind to an antigen. "VH2" and "VL2" refer to the heavy and light chain variable regions that pair together to bind to an antigen. "C" refers to an immunoglobulin or T cell receptor constant region. In an embodiment of the invention, the VC and VC units on either side of the linker domain form upper and lower immunoglobulin Fab domains, where the lower Fab domain exhibits binding to its cognate target, but the binding is reduced or eliminated by the presence of the linker domain fused to the N-terminus of each of the V domains of the lower Fab. In another embodiment, the upper or lower Fab domains may be replaced with an Fc fragment, one or two receptor ectodomains, or any domain with or without any specific binding function.

[0043] In some embodiments, provided herein is 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 derived from a human immunoglobulin hinge region, or an 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 compared to a human immunoglobulin hinge region, the peptide linker is cleavable by a protease expressed in the diseased tissue; the second portion is capable of specifically binding to a molecule expressed in the diseased tissue; and If the peptide linker is not cleaved, binding of the second portion to the molecule expressed in the diseased tissue is reduced or inhibited.

[0044] The linker moiety may further comprise a peptide linker derived from an immunoglobulin hinge region with zero, one or more mutations away from germline.

[0045] In some embodiments, the peptide linker is selected from the group consisting of 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), and / or GPAPELLGGPS (SEQ ID NO: 83). 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 embodiments, the peptide linker comprises or consists of two, three, or four of the above sequences.

[0046] Immunoconjugates comprising a protein of the invention linked to a therapeutic agent are also provided.

[0047] In another aspect, the present invention provides a nucleic acid molecule encoding a protein or a portion thereof as defined herein. A vector comprising the nucleic acid molecule of the present invention is further provided. Also provided is a host cell comprising the nucleic acid molecule or vector of the present invention.

[0048] 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.

[0049] In another aspect of the present invention there is provided a pharmaceutical composition comprising a protein of the invention as defined herein, a nucleic acid molecule of the invention as defined herein, a vector of the invention as defined herein, or an immunoconjugate of the invention as defined herein.

[0050] Further provided is a method of enhancing an immune response in a subject, the method comprising administering an effective amount of a protein of the invention as defined herein, an immunoconjugate of the invention as defined herein, a nucleic acid molecule of the invention as defined herein, a vector of the invention as defined herein, or a pharmaceutical composition of the invention as defined herein.

[0051] In a further aspect there is provided a method of treating or preventing cancer in a subject, the method comprising administering an effective amount of a protein of the invention as defined herein, an immunoconjugate of the invention as defined herein, a nucleic acid molecule of the invention as defined herein, a vector of the invention as defined herein, or a pharmaceutical composition of the invention as defined herein.

[0052] Further provided is a protein of the invention as defined herein, an immunoconjugate of the invention as defined herein, a nucleic acid molecule of the invention as defined herein, a vector of the invention as defined herein, or a pharmaceutical composition of the invention as defined herein for use as a medicament.

[0053] Further provided is a protein of the invention as defined herein, an immunoconjugate of the invention as defined herein, a nucleic acid molecule of the invention as defined herein, a vector of the invention as defined herein, or a pharmaceutical composition of the invention as defined herein for use in the treatment of cancer.

[0054] Further provided are the proteins, immunoconjugates, nucleic acid molecules, vectors, pharmaceutical compositions of the invention as defined herein for use separately, sequentially or simultaneously in combination with a second therapeutic agent, such as an anti-cancer agent.

[0055] In a further aspect there is provided the use of a protein of the invention as defined herein, an immunoconjugate of the invention as defined herein, a nucleic acid molecule of the invention as defined herein, 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.

[0056] Further provided is a method of treating or preventing an autoimmune or inflammatory disease in a subject, the method comprising administering an effective amount of a protein as defined herein, an immunoconjugate as defined herein, a nucleic acid molecule as defined herein, a vector as defined herein, a pharmaceutical composition as defined herein.

[0057] Also provided is a protein as defined herein, an immunoconjugate as defined herein, a nucleic acid molecule as defined herein, a vector as defined herein, or a pharmaceutical composition as defined herein for use in the treatment of an autoimmune or inflammatory disease.

[0058] There is further provided the use of a protein as defined herein, an immunoconjugate as defined herein, a nucleic acid molecule as defined herein, a vector as defined herein, or a pharmaceutical composition as defined herein in the manufacture of a medicament for the treatment of an autoimmune or inflammatory disease.

[0059] Further provided is a method of treating or preventing a cardiovascular or fibrotic disease in a subject, the method comprising administering an effective amount of a protein as defined herein, an immunoconjugate as defined herein, a nucleic acid molecule as defined herein, a vector as defined herein, a pharmaceutical composition as defined herein.

[0060] Further provided is a protein as defined herein, an immunoconjugate as defined herein, a nucleic acid molecule as defined herein, 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, an immunoconjugate as defined herein, a nucleic acid molecule as defined herein, 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.

[0061] There is further provided the use of a protein as defined herein, an immunoconjugate as defined herein, a nucleic acid molecule as defined herein, 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, an inflammatory disease or a fibrotic disease.

[0062] In some embodiments, 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, or an amino acid sequence derived from a human immunoglobulin hinge region, or an 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) compared to a human immunoglobulin hinge region, the peptide linker is cleavable by a protease expressed in the diseased tissue, the second portion is capable of specifically binding to a molecule expressed in the diseased tissue, and when the peptide linker is not cleaved, binding of the second portion to a molecule expressed in the diseased tissue is reduced or inhibited. In some embodiments, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the peptide linker comprises an amino acid sequence, or an amino acid sequence derived from a human immunoglobulin hinge region, or an amino acid sequence having 1 to about 7 amino acid substitutions compared to a human immunoglobulin hinge region. In some embodiments, the peptide linker comprises an amino acid sequence derived from a human immunoglobulin hinge region, or an amino acid sequence or an 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 compared to a human immunoglobulin hinge region.

[0063] In some embodiments, the peptide linker cleavable by a protease expressed in the diseased tissue is cleavable by a human matrix metalloprotease (MMP) or a human cathepsin. In some cases, the peptide linker cleavable by a protease expressed in the diseased tissue is cleavable 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 (a disintegrin-like and metalloproteinase with thrombospondin type 1 motif 5; 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 cleavage site, a human thrombin cleavage site, a human tPA cleavage site, a human granzyme B cleavage site, a human uPA cleavage site, or a 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 the 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).

[0064] In some cases, the peptide linker comprises or consists of two, three, or four of the amino acid sequences in Table 1 fused together in a single amino acid chain via peptide bonds. In some cases, the peptide linker is about 5 amino acids to about 15 amino acids, about 5 amino acids to about 20 amino acids, or about 5 amino acids to about 25 amino acids in length.

[0065] In some cases, the peptide linker between the first and second moieties comprises an amino acid sequence of X1-proline-X2 at the N-terminus of the peptide linker sequence. In some embodiments, X1 is alanine, glycine, serine, proline, or threonine. In some embodiments, X1 is alanine, glycine, serine, proline, threonine, aspartic acid, asparagine, or valine. In some embodiments, X2 is alanine, glycine, serine, proline, or threonine. In some embodiments, X2 is alanine, glycine, serine, proline, threonine, aspartic acid, asparagine, or valine. In some embodiments, X1 and X2 are the same amino acid. In some embodiments, X1 and X2 are different amino acids.

[0066] In some cases, the peptide linker that is cleavable by a protease expressed in the diseased tissue is cleavable by any one 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 MMP28. In some cases, the peptide linker that is cleavable by a protease expressed in the diseased tissue is cleavable by any one 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 the human MMP in the diseased tissue is increased as compared to the level or activity of the human MMP in the non-diseased tissue.

[0067] In some cases, the peptide linker cleavable by a protease expressed in the diseased tissue is cleavable by any one 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, the peptide linker cleavable by a protease expressed in the diseased tissue is cleavable by any one of human cathepsin D, cathepsin G, or cathepsin K. In some cases, the level or activity of human cathepsin in the diseased tissue is higher than the level or activity of human cathepsin in non-diseased tissue. In some cases, the level or activity of human cathepsin in tissue with a pH of less than 7.0 is higher than the level or activity of human cathepsin in tissue with a pH of 7.4 or higher.

[0068] In some embodiments, the first portion of any of the proteins of the invention comprises an antibody, an antigen-binding portion of an antibody, or a receptor ectodomain. In some cases, the first portion is a Fab, a single-chain Fab, a VH domain, a VL domain, an immunoglobulin novel antigen receptor (IgNAR), a single-chain variable fragment (scFv), a diabody, or a T-cell receptor domain. IgNARs are homodimeric heavy-chain single antibodies produced by sharks and other cartilaginous fishes (Feige et al., PNAS, 2014, 111(22):8155-8160).

[0069] In some cases, the first portion specifically binds to a molecule expressed in diseased tissue. In some embodiments, the first portion specifically binds to a tumor-associated antigen (TAA). In some cases, 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. In some embodiments, the first portion also binds to a cynomolgus orthologue of any of these molecules.

[0070] In some aspects, the first portion comprises a heavy chain variable (VH) region and a light chain variable (VL) region. Optionally, the first portion further comprises an immunoglobulin constant region or a portion of an immunoglobulin constant region. Optionally, the immunoglobulin constant region is IgG, IgE, IgM, IgD, IgA, or IgY.

[0071] In some embodiments, the anti-HER2 variable region sequence used in the protein constructs disclosed herein is the variable region sequence of trastuzumab. In some embodiments, the anti-CD3 variable region sequence used in the protein constructs disclosed herein is the variable region sequence of OKT3 or SP34. In some embodiments, the anti-cMET variable region sequence used in the protein constructs disclosed herein is that provided in WO2019 / 175186. In some embodiments, the anti-CD47 variable region sequence used in the protein constructs disclosed herein is that provided in WO2019 / 034895.

[0072] In some embodiments, the second portion of any of the proteins of the invention comprises an antibody, an antigen-binding portion of an antibody, or a receptor ectodomain. In some cases, the second portion is a Fab, a single-chain Fab, a VH domain, a VL domain, an immunoglobulin novel antigen receptor (IgNAR), a single-chain variable fragment (scFv), a diabody, or a T-cell receptor domain.

[0073] In some embodiments, the second portion specifically binds to a molecule expressed on diseased tissue. In some embodiments, the second portion specifically binds to a tumor associated antigen (TAA). In some embodiments, the second portion specifically binds to human CD47. In some embodiments, the second portion specifically binds to human PD-L1. In some embodiments, the second portion specifically binds to a molecule expressed on a human immune cell. In some embodiments, the molecule expressed on a human immune cell is human CD3, human CD16A, human CD16B, human CD28, human CD89, human CTLA4, human NKG2D, human SIRPα, human SIRPγ, human PD1, human Lag3, human 4-1BB, human OX40, or human GITR. In some embodiments, the second portion also binds to a cynomolgus orthologue of any of these molecules.

[0074] In some aspects, the second portion comprises a heavy chain variable (VH) region and a light chain variable (VL) region. Optionally, the second portion further comprises an immunoglobulin constant region or a portion of an immunoglobulin constant region. Optionally, the immunoglobulin constant region is IgG, IgE, IgM, IgD, IgA, or IgY.

[0075] In some aspects, the first and / or second portions 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 another embodiment, the immunoglobulin constant region is IgG1, IgG2, IgG3, IgG1 null, IgG4(S228P), IgA1, IgA2, IgE, or IgM. In some embodiments, the first and / or second portions of the protein of the invention may comprise an immunologically inert constant region. In some embodiments, the first and / or second portions of the protein 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 embodiments, the first and / or second portions of the protein 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 embodiments, the first and / or second portions of the protein of the invention may comprise an immunoglobulin constant region comprising any one of the amino acid sequences in Table 10. The Fc region sequences in Table 10 begin with the CH1 domain. In some embodiments, the first and / or second portions of the protein of the invention may comprise an immunoglobulin constant region comprising the amino acid sequence of a human IgG4, human IgG4(S228P), human IgG2, human IgG1, human IgG1-3M, or human IgG1-4M Fc region. For example, a human IgG4(S228P) Fc region comprises the substitution S228P compared to a wild-type human IgG4 Fc region. For example, a human IgG1-3M Fc region comprises the substitutions L234A, L235A, and G237A compared to a wild-type human IgG1 Fc region, while a human IgG1-4M Fc region comprises the substitutions L234A, L235A, G237A, and P331S compared to a wild-type human IgG1 Fc region.In some embodiments, the positions of amino acid residues in the constant region of an immunoglobulin molecule are numbered according to the EU nomenclature (Ward et al., 1995 Therap.Immunol. 2:77-94). In some embodiments, 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 fewer human populations than the RDELT (SEQ ID NO: 65) allotype. In some embodiments, the first and / or second portions of the protein of the antibody of the invention may comprise an immunoglobulin constant region comprising any one of SEQ ID NOs: 56-62. In some embodiments, the first and / or second portions of the protein of the invention may comprise the heavy and light chain amino acid sequences of any one of the clones in Tables 3-9, and any one of the Fc region amino acid sequences in Table 10. In some embodiments, the first and / or second portions of the 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.

[0076] In some embodiments, the proteins of the invention comprise an IgG1 isotype constant region, which potently activates all FcγR signaling types, thereby promoting maximal opsonization effector function.

[0077] 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 compared to a wild-type human hinge region amino acid sequence. In some embodiments, the immunoglobulin constant region does not comprise a hinge region.

[0078] In some aspects, the first portion is capable of specifically binding 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, where 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 in 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 in 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 surface of a cell. In some embodiments, the first molecule expressed in the diseased tissue and / or the second molecule expressed in the diseased tissue are soluble molecules.

[0079] In some embodiments, the first portion specifically binds human cMET and the second portion specifically binds human CD47. In some embodiments, the first portion specifically binds human HER2 and the second portion specifically binds human CD47. In some embodiments, the first portion specifically binds human cMET and the second portion specifically binds human CD47. In some embodiments, the first portion specifically binds human HER2 and the second portion specifically binds human CD3. In some embodiments, the first portion specifically binds human cMET and the second portion specifically binds human cMET.

[0080] In some embodiments, the proteins of the invention have one immune effector function, or two, three or more immune effector functions, For example, the immune effector functions can be antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), or antibody-dependent cellular phagocytosis (ADCP).

[0081] In some aspects, the first portion of the protein of the invention prevents or reduces the 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 within the diseased tissue, where the first portion dissociates from the second portion near or within the diseased tissue, and the second portion specifically binds to a molecule expressed in the diseased tissue near or within the diseased tissue. In some cases, the cleaved peptide linker contains a binding or targeting site for an anti-hinge antibody (e.g., an endogenous anti-hinge antibody of the subject), whereas the uncleaved (e.g., intact) peptide linker does not contain a binding or targeting site for an anti-hinge antibody. Binding of the anti-hinge antibody to the cleaved peptide linker can increase ADCC, CDC and / or ADCP in the presence of the activated protein of the invention (see, e.g., FIG. 2B).

[0082] In some embodiments, the protein of the present invention stimulates inflammatory signaling in diseased tissue.Increased inflammatory signaling can increase immune recruitment to diseased tissue.In some cases, the protein of the present invention increases antigen presentation in diseased tissue.In some cases, the protein of the present invention increases tumor-associated antigen-specific T cell proliferation.

[0083] In some embodiments, the diseased tissue may be a tumor, necrotic tissue, fibrotic tissue, tissue undergoing the coagulation cascade, or inflamed tissue.

[0084] In some embodiments, provided herein is a protein 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 cMET and the second portion specifically binds human CD47, and 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. The protein comprises only one copy of the first polypeptide chain and only one copy of the second polypeptide chain. The peptide linker of this protein comprises two copies of the LHL sequence (see Table 1), each fused via a peptide bond at the N-terminus to the first portion and at the C-terminus to the second portion. This protein is referred to as "Fab2 cMetCD47-LHL-LHL" or "Met47-LHL-LHL". The amino acid sequence is presented in Table 3. The structure of this protein is shown in Figure 3A. The second part of the protein is linked to the KIH IgG1-Fc via a G4S linker (SEQ ID NO: 15) and a truncated hinge region.

[0085] In some embodiments, provided herein is a protein 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, the second portion specifically binds human CD3, and 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. The protein comprises only one copy of the first polypeptide chain and only one copy of the second polypeptide chain. The peptide linker of this protein comprises two copies of the LHL sequence fused via a peptide bond (see Table 1). This protein is referred to as "Fab2 Her2CD3-LHL-LHL" or "Her23-LHL-LHL". The amino acid sequence is presented in Table 4. The structure of this protein is shown in FIG. 3A. The second portion of this protein is linked to KIH IgG1-Fc via a G4S linker (SEQ ID NO:15) and a truncated hinge region (3M).

[0086] In some embodiments, provided herein is a protein 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 and the second portion specifically binds human CD47, and 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. The protein comprises two identical copies of the first polypeptide chain and two identical copies of the second polypeptide chain. The peptide linker of this protein comprises two copies of the LHL sequence fused via a peptide bond (see Table 1). This protein is referred to as "IgG2 Her2CD47-LHL-LHL" or "Her47-LHL-LHL". The amino acid sequence is presented in Table 5. The structure of this protein is shown in FIG. 2A. The second portion of this protein is linked to a human IgG1 Fc sequence via a hinge region or a truncated hinge region (see Table 10).

[0087] In some embodiments, provided herein is a protein 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 cMET and the second portion specifically binds human CD47, and 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. The first polypeptide chain further 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 via a peptide bond (see Table 1). This protein is referred to as "Fab2 CMET / CD47 'one arm' type" or "Met47-LHL-LHL". The amino acid sequence is presented in Table 6. The structure of this protein is shown in FIG. 3B. The construct is a "knob-into-hole" one arm Fab2 construct, with the Fab2 on the knob side and the stem hinge hole Fc on the opposite side. This construct may contain a non-effector null human IgG1 Fc sequence (see Table 10).

[0088] In some embodiments, provided herein is a protein 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 and the second portion specifically binds human CD3, and 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. The first polypeptide chain further comprises a 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 via a peptide bond (see Table 1). This protein is referred to as "Fab2 Her2 / CD3 'one arm' type" or "Her23-LHL-LHL". The amino acid sequence is presented in Table 7. The structure of this protein is shown in FIG. 3B. The construct is a "knob-into-hole" one arm Fab2 construct, with the Fab2 on the knob side and the stem hinge hole Fc on the opposite side. This construct is also effector null (IgG1-3M; see Table 10).

[0089] In some embodiments, provided herein is a protein 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 and the second portion specifically binds human CD3, and 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. The first polypeptide chain further comprises a human IgG1-3M amino acid sequence (see Table 10). The second polypeptide chain further comprises a human lambda light chain amino acid sequence. The peptide linker of this protein comprises two copies of the LHL sequence fused via a peptide bond (see Table 1). This protein is referred to as "Fab2 Her2 / CD3(34) 'one-arm' type" or "Her23(34)-LHL-LHL". The amino acid sequence is presented in Table 8. The structure of this protein is shown in Figure 3B. The construct is a "knob-into-hole" one-arm Fab2 construct, with the Fab2 on the knob side and the stem hinge hole Fc on the opposite side. This construct is also effector null (IgG1-3M; see Table 10).

[0090] In some embodiments, provided herein is a protein 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 and the second portion specifically binds human CD47, and the protein comprises a first polypeptide chain and a second polypeptide chain, wherein: (a) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 42 and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 43 (referred to as "Her47-LHLF-LHL"); (b) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:44 and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:45 (referred to as "Her47-LHL-LHLF"); (c) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:46 and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:47 (referred to as "Her47-LHLF-LHLF"); (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 (referred to as "Her47-LHLM-LHLM"); (e) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:50 and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:51 (referred to as "Her47-LHLM-LHLMF"); (f) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:52 and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:53 (referred to as "Her47-LHLMF-LHLM"); (g) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:54 and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:55 (referred to as "Her47-LHLMF-LHLMF"); (h) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:44 and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:92 (referred to as "Her47 LHL-LHL-EK"); (i) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:44 and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:93 (referred to as "Her47-LHL-LHL-Thr"); (j) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:44 and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:94 (referred to as "Her47-LHL-LHL-tPA"); (k) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 44 and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 95 (referred to as "Her47-LHL-LHL-GrB"); (l) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:44 and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:96 (referred to as "Her47-LHL-LHL-uPA"); (m) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 44 and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 97 (referred to as "Her47-LHL-LHL-A5"). These proteins are generally referred to as "IgG2 Her2 / CD47". The amino acid sequences are provided in Tables 9 and 20. The structure of this protein is shown in Figure 2A. The peptide linker sequences for the LHLF, LHLM and LHLMF linkers are provided in Table 1. The peptide linker sequences for the EK, Thr, tPA, GrB, uPA and A5 linkers are provided in Table 21.

[0091] In some embodiments, provided herein is a protein 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 and the second portion specifically binds human CD47, and the protein comprises a first polypeptide chain and a second polypeptide chain, wherein: (a) 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 (referred to as "Her47 LHLF-LHL IgG1-2hDAA"); (b) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 90 and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 91 (referred to as "Her47 LHL-LHLF IgG1-2hDAA"). These proteins are generally referred to as "IgG2 'IgG1-DAA' Her2 / CD47". The amino acid sequences are provided in Table 19. These proteins contain stabilizing mutations in the hinge. The structure of this protein is shown in Figure 2A. The peptide linker sequences of the LHLF and LHL linkers are provided in Table 1.

[0092] In some embodiments, provided herein is a protein 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 and the second portion specifically binds human CD3, and 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. The first polypeptide chain further comprises a human IgG1-3M Fc amino acid sequence (e.g., comprising a "hole" mutation that allows heterodimerization with the second polypeptide chain), followed by a linker sequence, the VH and CH1 domains of the first binding portion, another linker sequence, and the VH and CH1 domains of the second binding portion (see Table 13). The second polypeptide chain further comprises a human IgG1-3M Fc amino acid sequence (e.g., including a "knob" mutation that allows heterodimerization with the second polypeptide chain), followed by a linker sequence, the VL and CL domains of the first binding moiety, another linker sequence, and the VL and CL domains of the second binding moiety (see Table 13). The peptide linker of this protein comprises four sequences fused via peptide bonds (see Table 1) between the Fc and the first moiety, and between the first binding moiety and the second binding moiety. This protein is called "Fc-Her2 / CD3(34)" or "Fc-Her23(34)". The amino acid sequence is presented in Table 13. The structure of this protein is shown in Figure 12. The construct is a "knob-into-hole" Fc-Fab2 construct, with a light chain polypeptide on either the knob or hole side, and a heavy chain polypeptide on the opposite side. This construct is also effector null (IgG1-3M; see Table 10).

[0093] In some embodiments, provided herein is a protein 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 cMET and the second portion specifically binds human cMET, and 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. 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 via a peptide bond (see Table 1). This protein is referred to as "Fab2 CMET / CMET 'one-arm'" or "MetMet-LHL-LHL". The amino acid sequence is presented in Table 14. The structure of this protein is shown in Figure 3B. The constructs are "knob-into-hole" one-arm Fab2 constructs with the Fab2 on the knob side and the stem hinge hole Fc on the opposite side. The constructs may contain effector null or non-effector null human IgG1 Fc sequences (see Table 10).

[0094] In some embodiments, provided herein is a protein 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 and the second portion specifically binds human CD3, and the protein comprises a first polypeptide chain and a second polypeptide chain, wherein: (a) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:98 and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:99 (designated "Fab2 Her23 LHL-LHLF"); (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 (referred to as "Fab2 Her23 LHL-LHL"); (c) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 102 and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 103 (designated "Fab2 Her23 LHLF-LHL-S"); (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 (referred to as "Fab2 Her23 LHL-LHL-S"). This protein is referred to as "Fab2 Her2 / CD3". The amino acid sequence is presented in Table 22. The structure of this protein is shown in Figure 3A. The protein contains only one copy of the first polypeptide chain and only one copy of the second polypeptide chain. The peptide linker of this protein contains two copies of the LHL sequence (see Table 1) fused via peptide bonds or one copy of the LHL sequence and one copy of the LHLF sequence.

[0095] In some embodiments, provided herein is an immunoconjugate comprising a protein of the invention as defined herein linked to an additional therapeutic agent.

[0096] Examples of suitable therapeutic agents include cytotoxins, radioisotopes, chemotherapeutic agents, immunomodulatory agents, antiangiogenic agents, antiproliferative agents, proapoptotic agents, and cytostatic and cytolytic enzymes (e.g., ribonucleases). Further therapeutic agents include therapeutic nucleic acids, such as genes encoding immunomodulatory agents, antiangiogenic agents, antiproliferative agents, or proapoptotic agents. These drug descriptors are not mutually exclusive, and thus a therapeutic agent may be described using one or more of the above terms.

[0097] Examples of suitable therapeutic agents for use in immunoconjugates include taxanes, meitaisin, CC-1065, duocarmycins, calicheamicins, other enediynes, and auristatins. Other examples include antifolates, vinca alkaloids, and anthracyclines. Plant toxins, other biologically active proteins, enzymes (i.e., ADEPT), radioisotopes, and photosensitizers may also be used in immunoconjugates. In addition, conjugates can be formed using secondary carriers as cytotoxic agents, such as liposomes or polymers. Suitable cytotoxins include agents that inhibit or block the function of cells and / or cause the destruction of cells. Representative cytotoxins include antibiotics, inhibitors of tubulin polymerization, alkylating agents that bind to and destroy DNA, and agents that disrupt protein synthesis or the function of essential cellular proteins, such as protein kinases, phosphatases, topoisomerases, enzymes, and cyclins.

[0098] Representative cytotoxins include doxorubicin, daunorubicin, idarubicin, aclarubicin, zorubicin, mitoxantrone, epirubicin, carubicin, nogalamycin, menogaril, pitarubicin, valrubicin, cytarabine, gemcitabine, trifluridine, ancitabine, enocitabine, azacytidine, doxifluridine, pentostatin, broxuhdine, capecitabine, cladhbine, decitabine, floxuhdine, fludarabine, gougerotin, puromycin, tegafur, thiazofuhn, adamycin, cisplatin, carbo These include, but are not limited to, platins, cyclophosphamide, dacarbazine, vinblastine, vincristine, mitoxantrone, bleomycin, mechlorethamine, prednisone, procarbazine, methotrexate, flurouracil, etoposide, taxol, taxol analogs, platins such as cisplatin and carboplatin, mitomycin, thiotepa, taxanes, vincristine, daunorubicin, epirubicin, actinomycin, authramicin, azaserine, bleomycin, tamoxifen, idarubicin, dolastatins / auristatins, hemiasterlin, esperamicin, and maytansinoids.

[0099] Suitable immunomodulatory agents include antihormones that block hormone action on the tumor, and immunosuppressants that inhibit cytokine production, downregulate self-antigen expression, or mask MHC antigens.

[0100] Also provided are nucleic acid molecules encoding the proteins of the invention as defined herein or portions thereof.Further provided herein are nucleic acid molecules encoding the first polypeptide chain, the second polypeptide chain, or both the first and second polypeptide chains of the proteins of the invention comprising multiple non-identical polypeptide chains.In some embodiments, the nucleic acid molecules as defined herein may be isolated.

[0101] There is further provided a vector comprising a nucleic acid molecule of the invention as defined herein. The vector may be an expression vector.

[0102] Also provided is a host cell comprising a nucleic acid molecule or vector of the invention as defined herein. The host cell may be a recombinant host cell.

[0103] 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 expression and / or production of the protein, and isolating the protein from the host cell or culture.

[0104] In some embodiments, provided herein is a pharmaceutical composition comprising a protein of the invention as defined herein, a nucleic acid molecule of the invention as defined herein, or a vector of the invention as defined herein.

[0105] Further provided is a method of 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, an immunoconjugate of the invention as defined herein, a nucleic acid molecule of the invention as defined herein, a vector of the invention as defined herein, or a pharmaceutical composition of the invention as defined herein.

[0106] In a further aspect there is provided a method of treating or preventing cancer in a subject or alleviating a symptom of cancer in a subject, the method comprising administering to the subject an effective amount of a protein of the invention as defined herein, an immunoconjugate of the invention as defined herein, a nucleic acid molecule of the invention as defined herein, a vector of the invention as defined herein, or a pharmaceutical composition of the invention as defined herein.

[0107] In some embodiments, the cancer is a solid tumor.In some cases, the cancer is a hematological 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, oral or pharyngeal cancer, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small intestine or appendix cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma, or blood tissue cancer.In some cases, the blood tissue cancer is lymphoma.

[0108] In some embodiments, provided herein is a protein of the invention as defined herein, an immunoconjugate of the invention as defined herein, a nucleic acid molecule of the invention as defined herein, a vector of the invention as defined herein, or a pharmaceutical composition of the invention as defined herein for use in the treatment of cancer or for use in or alleviating the symptoms of cancer.

[0109] In some embodiments, provided herein is a protein, immunoconjugate, nucleic acid molecule, or vector of the invention as defined herein, or a method of treatment thereof, used separately, sequentially, or simultaneously in combination with a second therapeutic agent, e.g., an anti-cancer agent.

[0110] In a further aspect there is provided the use of a protein of the invention as defined herein, an immunoconjugate of the invention as defined herein, a nucleic acid molecule of the invention as defined herein, 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 or alleviating a symptom of cancer.

[0111] The present invention also provides a method of treating or preventing an autoimmune or inflammatory disease in a subject, the method comprising administering to the subject an effective amount of a protein as defined herein, an immunoconjugate as defined herein, a nucleic acid molecule as defined herein, a vector as defined herein, or a pharmaceutical composition as defined herein.

[0112] For example, the autoimmune or inflammatory disease can be arthritis, asthma, multiple sclerosis, psoriasis, Crohn's disease, inflammatory bowel disease, lupus, Graves' disease, Hashimoto's thyroiditis or ankylosing spondylitis.

[0113] Also provided is a protein as defined herein, an immunoconjugate as defined herein, a nucleic acid molecule as defined herein, a vector as defined herein, or a pharmaceutical composition as defined herein for use in the treatment of an autoimmune or inflammatory disease.

[0114] There is further provided the use of a protein as defined herein, an immunoconjugate as defined herein, a nucleic acid molecule as defined herein, a vector as defined herein, or a pharmaceutical composition as defined herein in the manufacture of a medicament for the treatment of an autoimmune or inflammatory disease.

[0115] The present invention also provides a method of treating or preventing a cardiovascular or fibrotic disease in a subject, the method comprising administering to the subject an effective amount of a protein as defined herein, an immunoconjugate as defined herein, a nucleic acid molecule as defined herein, a vector as defined herein, or a pharmaceutical composition as defined herein.

[0116] Also provided is a protein as defined herein, an immunoconjugate as defined herein, a nucleic acid molecule as defined herein, a vector as defined herein, or a pharmaceutical composition as defined herein for use in the treatment of a cardiovascular or fibrotic disease.

[0117] There is further provided the use of a protein as defined herein, an immunoconjugate as defined herein, a nucleic acid molecule as defined herein, a vector as defined herein, or a pharmaceutical composition as defined herein in the manufacture of a medicament for the treatment of a cardiovascular disease or a fibrotic disease.

[0118] The cardiovascular disease in any aspect of the invention may be, for example, coronary heart disease, atherosclerosis, or stroke.

[0119] For example, the fibrotic disease in any aspect of the invention may be myocardial infarction, angina pectoris, osteoarthritis, pulmonary fibrosis, asthma, cystic fibrosis or bronchitis.

[0120] In some aspects, provided herein are proteins for use in therapy, comprising the amino acid sequences disclosed herein and in the formats disclosed herein.

[0121] In some embodiments, the pharmaceutical composition may comprise a pharma- ceutically acceptable excipient, carrier or diluent. A pharma-ceutically acceptable excipient may be a compound or a combination of compounds that enters the pharmaceutical composition, which does not induce secondary reactions and allows, for example, to facilitate the administration of a protein as defined herein, to improve its life span in the body and / or its effectiveness, or to increase its solubility in solution. These pharma-ceutically acceptable solvents are well known and will be adapted by the skilled artisan depending on the mode of administration of a protein as defined herein.

[0122] In some embodiments, proteins as defined herein may be provided in a lyophilized form that is reconstituted in a solution prior to administration, for example, lyophilized protein molecules may be reconstituted in sterile water and mixed with saline prior to administration to an individual.

[0123] Proteins as defined herein are usually administered in the form of pharmaceutical compositions, which may contain at least one component in addition to protein molecules. Thus, in addition to proteins as defined herein, pharmaceutical compositions may contain pharma- ceutically acceptable excipients, carriers, buffers, stabilizers, or other materials known to those skilled in the art. Such materials must be non-toxic and must not inhibit the efficacy of the protein. The exact nature of the carrier or other materials depends on the route of administration, which may be by bolus, infusion, injection, or any other suitable route, as discussed below.

[0124] For parenteral administration, e.g., by injection, e.g., subcutaneously or intravenously, pharmaceutical compositions comprising a protein as defined herein may be in the form of a parenterally acceptable aqueous solution that is pyrogen-free and of suitable pH, isotonicity and stability. Those skilled in the art will be able to prepare suitable solutions using isotonic solvents, e.g., sodium chloride injection, Ringer's injection, lactated Ringer's injection, etc. Buffers such as phosphates, citrates and other organic acids; antioxidants such as ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl 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); low molecular weight polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; glycols; and the like. Preservatives, stabilizers, buffers, antioxidants and / or other additives may be used as needed, including amino acids such as synthase, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn protein complexes); and / or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

[0125] Pharmaceutical compositions containing the proteins as defined herein may be administered alone or in combination with other therapeutic agents, either simultaneously or sequentially depending on the condition to be treated.

[0126] The proteins as defined herein may be used in methods of treatment of the human or animal body, including prophylactic or preventative treatment (e.g., reducing the risk of a condition developing in an individual; delaying onset; treating before the onset of a condition in an individual to reduce its severity after onset). The methods of treatment may comprise administering a protein as defined herein to an individual in need thereof.

[0127] Administration is usually in a "therapeutically effective amount", which is an amount sufficient to show an effect on the patient. Such effect may be at least the amelioration of 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 what is being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disease, the site of delivery of the composition, the method of administration, the administration schedule, and other factors known to medical practitioners. Prescription of therapeutic agents, such as determining dosage, is within the responsibility of general practitioners and other physicians, and may depend on the severity of the symptoms and / or progression of the disease being treated. Appropriate doses of antibody molecules are well known in the art (Ledermann JA et al., 1991, Int. J. Cancer 47:659-664; Bagshawe KD et al., 1991, Antibody, Immunoconjugates and Radiopharmaceuticals 4:915-922). Depending on the type of pharmaceutical agent administered, specific dosages may be used as may be provided herein or in the Physician's Desk Reference (2003). A therapeutically effective amount or suitable dose of a protein as defined herein may be determined by comparing its in vitro activity and in vivo activity in animal models. Methods are known for extrapolating effective dosages in mice and other test animals to humans. The exact dosage will depend on several factors, including whether the protein is prophylactic or therapeutic, the size and location of the area to be treated, the exact nature of the protein (e.g., Fab2, IgG), and the nature of any detectable label or other molecule attached to the protein.

[0128] Typical protein doses range from 100 μg to 1 g for systemic administration and 1 μg to 1 mg for local administration. An initial high loading dose may be administered, followed by one or more administrations of smaller doses. In some embodiments, the protein comprises a whole antibody, e.g., IgG1 or IgG4 isotype. This is a single treatment dose for adult patients and can be adjusted proportionally for children and infants, and also for other protein construct formats proportional to molecular weight. Treatment may be repeated at daily, twice-weekly, weekly or monthly intervals, at the discretion of the physician. An individual's treatment schedule may depend on the pharmacokinetic and pharmacodynamic properties of the protein composition, the route of administration, and the nature of the condition being treated.

[0129] Treatment may be periodic and the period between administrations may be about 2 weeks or more, e.g., about 3 weeks or more, about 4 weeks or more, about monthly or more, about 5 weeks or more, about 6 weeks or more. For example, treatment may be every 2-4 weeks, or every 4-8 weeks. Therapeutic agents may be given pre-operatively and / or post-operatively, and / or may be administered or applied directly to the anatomical site of surgical or invasive treatment. Suitable formulations and routes of administration are described above.

[0130] In some embodiments, the protein as defined herein may be administered as a subcutaneous injection, for example using an autoinjector during long-term or short-term prophylaxis / treatment.

[0131] In some embodiments, the therapeutic effect of a protein as defined herein may last for several protein half-lives in serum, depending on the dose. For example, the therapeutic effect of a single administration of a 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.

[0132] As used herein, the term "CD47" refers to IAP (integrin associated protein) and variants thereof that retain at least some of the biological activity of CD47. As used herein, CD47 includes native sequence CD47 of all mammalian species, including human, rat, mouse, and chicken. In some embodiments, the term "CD47" is used to include variants, isoforms, and species homologs of human CD47. In some cases, as used herein, CD47 includes native sequence CD47 of all mammalian and non-mammalian species, including human, monkey, rat, mouse, and chicken. In some embodiments, the term "CD47" refers only to wild-type CD47. The proteins of the invention may cross-react with CD47 from 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 cross-reactivity with non-humans.

[0133] As used herein, the term "cMET" refers to MET protein and variants thereof that retain at least some of the biological activity of cMET. In some cases, as used herein, cMET includes 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 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 present invention may cross-react with cMET from species other than human, particularly cMET from cynomolgus monkeys (Macaca fascicularis). Examples of human and cynomolgus monkey cMET amino acid sequences are provided in Table 12. In certain embodiments, the antibodies may be completely specific for human cMET and may not exhibit cross-reactivity with non-humans.

[0134] As used herein, the term "Her2" refers to human epidermal growth factor receptor 2 protein and variants thereof that retain at least some of the biological activity of Her2. Her2 is also known as HER2 / neu, ErbB2, c-erbB-2, and human EGF receptor 2. In some embodiments, the term "Her2" may be used to include variants, isoforms, and species homologs of human Her2. In some cases, as used herein, Her2 includes 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 "Her2" refers only to wild-type Her2. The antibodies of the present invention may cross-react with Her2 from species other than human, particularly Her2 from cynomolgus monkeys (Macaca fascicularis). Examples of human and cynomolgus monkey Her2 / ErbB2 amino acid sequences are provided in Table 15. In certain embodiments, the antibody may be fully specific for human Her2 and may exhibit no cross-reactivity with non-humans.

[0135] As used herein, the term "CD3" refers to the "cluster of differentiation 3" multimeric protein complex and variants thereof that retain at least some of the biological activity of CD3. The CD3 complex comprises four distinct polypeptide chains; epsilon (ε), gamma (γ), delta (δ) and zeta (ζ). These polypeptide chains assemble and function as three pairs of dimers (εγ, εδ, ζζ). In some embodiments, the term "CD3" may be used to include variants, isoforms and species homologs of human CD3. In some cases, as used herein, CD3 includes native sequence CD3 of all mammalian and non-mammalian species, including human, monkey, rat, mouse and chicken. In some embodiments, the term "CD3" refers only to wild-type CD3. The antibodies of the present invention may cross-react with CD3 from species other than human, particularly CD3 from cynomolgus monkeys (Macaca fascicularis). Examples of human and cynomolgus monkey CD3 epsilon amino acid sequences are provided in Table 16. In certain embodiments, the antibodies may be fully specific for human CD3 and may exhibit no cross-reactivity with non-humans.

[0136] As used herein, "antagonist" used in relation to the proteins of the invention refers to a protein capable of binding to a molecule expressed in diseased tissue and inhibiting the biological activity of that molecule and / or downstream pathway(s) mediated by that molecule. For example, "anti-CD47 antagonist protein" (synonymous term "anti-CD47 protein") refers to a protein capable of binding to CD47 and inhibiting the biological activity of CD47 and / or downstream pathway(s) mediated by CD47 signaling. Anti-CD47 antagonist proteins encompass proteins capable of blocking, antagonizing, suppressing or reducing (in some cases significantly) CD47 biological activity, including downstream pathways mediated by CD47 signaling, such as receptor binding and / or eliciting a cellular response to CD47. For purposes of the present invention, the term "anti-CD47 antagonist protein" will be clearly understood to encompass CD47 itself, and all terms, titles, and functional states and properties in which a CD47 biological activity (including but not limited to its ability to enhance activation of phagocytosis by cells of the myeloid lineage), or the consequence of an activity or biological activity, is substantially abolished, reduced or neutralized to any meaningful extent.

[0137] 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 it binds with greater affinity, avidity, more readily and / or with longer duration than the protein binds to another molecule.

[0138] An "antibody molecule" is an immunoglobulin molecule capable of specifically binding to 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 intact polyclonal or monoclonal antibodies, as well as any antigen-binding fragment (e.g., "antigen-binding portion") or single chains thereof, fusion proteins comprising antibodies, and any other modified configurations of immunoglobulin molecules that contain an antigen recognition site, such as, but not limited to, scFvs, single domain antibodies (e.g., shark and camel antibodies), maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR, and bis-scFvs.

[0139] An "antibody molecule" encompasses an antibody of any class, such as IgG, IgA, or IgM (or subclasses thereof), and an antibody need not be of any particular class. Depending on the antibody amino acid sequence of the constant region of its heavy chain, immunoglobulins may be assigned to different classes. There are five main classes of immunoglobulins, IgA, IgD, IgE, IgG, and IgM, some of which may be further divided into subclasses (isotypes), e.g., lgG1, lgG2, lgG3, lgG4, lgA1, and lgA2. The heavy chain constant regions corresponding to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known.

[0140] The term "antigen-binding portion" of an antibody molecule, as used herein, refers to one or more fragments of an intact antibody that retain the ability to specifically bind to an antigen. The antigen-binding function of an antibody molecule may be performed by a fragment of an intact antibody. Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody molecule include Fab; Fab'; F(ab')2; Fd fragment consisting of the VH and CH1 domains; Fv fragment consisting of the VL and VH domains of a single arm of an antibody; single domain antibody (dAb) fragments, and isolated complementarity determining regions (CDRs).

[0141] The term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain. An "Fc region" may be a native sequence Fc region or a variant Fc region. Although the boundaries of an immunoglobulin heavy chain Fc region may vary, the human IgG heavy chain Fc region is usually defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxyl-terminus thereof. The numbering of residues in the Fc region is that of the EU index in Kabat. The Fc region of an immunoglobulin generally comprises two constant domains, CH2 and CH3. As is known in the art, the Fc region may exist in a dimeric or monomeric form.

[0142] The "variable region" of an antibody refers to the variable region of an antibody light chain or the variable region of an antibody heavy chain, either alone or in combination. As known in the art, the variable regions of the heavy and light chains are each composed 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 FRs adjacent to CDRs, for example, when humanizing or optimizing an antibody, FRs from an antibody containing CDR sequences of the same canonical class are preferred.

[0143] As used herein, the term "conservative substitution" refers to the substitution of an amino acid with another amino acid that does not significantly and adversely change the functional activity. A preferred example of a "conservative substitution" is the substitution of one amino acid with another amino acid having a value of 0 or greater in the BLOSUM 62 substitution matrix below (see Henikoff & Henikoff, 1992, PNAS 89: 10915-10919). [Table 1]

[0144] 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, and not the method by which it is produced. Preferably, the monoclonal antibodies of the invention are present in a homogeneous or substantially homogeneous population.

[0145] A "humanized" antibody molecule refers to a form of a non-human (e.g., murine) antibody molecule or antigen-binding portion thereof that is a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequence of an antibody) that contains minimal sequence derived from the non-human immunoglobulin. Humanized antibodies can be human immunoglobulins (recipient antibody) in which residues from the CDRs of the recipient are replaced by residues from the CDRs of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity.

[0146] A "human antibody" or "fully human antibody" refers to an antibody molecule, or antigen-binding portion thereof, derived from a transgenic mouse or a human cell harboring human antibody genes.

[0147] The term "chimeric antibody" is intended to refer to an antibody molecule, or antigen-binding portion thereof, whose variable region sequences are derived from one species and whose constant region sequences are derived from another species, such as an antibody molecule whose variable region sequences are derived from a murine antibody and whose constant region sequences are derived from a human antibody.

[0148] The term "immunoconjugate" refers to a protein of the invention conjugated, fused or linked to at least one cytotoxic, cytostatic or therapeutic agent.

[0149] The proteins of the present invention can 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.

[0150] The term "isolated molecule" (e.g., when the molecule is a polypeptide, polynucleotide, or antibody) refers to a molecule that, by virtue of its origin or the source of its derivation, (1) is not associated with naturally associated components associated with it in its natural state, (2) is substantially free of other molecules from the same species, (3) is expressed in cells from a different species, or (4) is not found in nature. Thus, a molecule that is chemically synthesized or expressed in a cellular system different from the cell in which it naturally occurs is "isolated" from naturally associated components. A molecule can also be rendered substantially free of naturally associated components by isolation using purification techniques well known in the art. The purity or homogeneity of a molecule can be assessed by several methods well known in the art. For example, the purity of a polypeptide sample can be assessed using polyacrylamide gel electrophoresis and staining of the gel to visualize the polypeptide using techniques well known in the art. For certain purposes, it is possible to obtain higher resolution by using HPLC or other methods for purification well known in the art.

[0151] The term "epitope" refers to a portion of a molecule capable of being recognized and bound by a protein, antibody molecule, or antigen-binding portion thereof of the present invention at one or more of the antigen-binding regions of the protein or antibody molecule. An epitope may be composed of a defined region of primary, secondary, or tertiary protein structure, and includes a combination of secondary structural units or structural domains of a target that are recognized by the antigen-binding region of a protein, an antibody, or an antigen-binding portion thereof. In addition, an epitope is composed of a defined collection of chemically active surface molecules, such as amino acids or sugar side chains, and has unique three-dimensional structural characteristics, as well as unique charge characteristics. The term "antigenic epitope" as used herein is defined as a portion of a polypeptide to which a protein or antibody molecule of the present invention can specifically bind, as identified by any method known in the art, for example, by conventional immunoassays, antibody competitive binding assays, or by X-ray crystallography or related structure determination methods (e.g., NMR).

[0152] The term "binding affinity" or "KD" refers to the dissociation rate of a particular antigen-binding protein interaction or antigen-antibody interaction. KD is defined as the "off rate (k off ) and the association rate or on-rate (k on )" Therefore, K D is k off / k on and is expressed as a molar concentration (M). Therefore, K D The smaller the K, the higher the affinity of binding. Thus, a K of 1 μM D has a K of 1 nM D It means that the binding affinity is lower than that of the antibody. The KD value of a binding protein or antibody can be calculated using methods well established in the art. One method for calculating the KD of a binding protein or antibody is typically by surface plasmon resonance (SPR) using a biosensor system such as the Biacore® system.

[0153] The term "potency" is a measure of biological activity and is also known as IC50 i.e., the effective concentration of an antigen or a protein of an immune complex of the invention against an antigen that inhibits by 50% the activity of a binding partner (e.g., a molecule expressed in diseased tissue) or binding partner as measured in an activity assay as described herein.

[0154] The phrase "effective amount" or "therapeutically effective amount" as used herein refers to the amount necessary (relative to dosage and duration and method of administration) to obtain the desired therapeutic result. An effective amount is at least the minimum amount of active agent required to provide a therapeutic benefit to a subject, and is less than a toxic amount.

[0155] The terms "inhibit" or "neutralize" as used herein with respect to the biological activity of a protein of the invention refer to the ability of the protein to substantially antagonize, impede, prevent, suppress, slow down, destroy, eliminate, stop, reduce or reverse the progression or severity of whatever is being inhibited, including, for example, but not limited to, the biological activity or binding interaction of a molecule expressed in affected tissue.

[0156] A "host cell" includes an individual cell or cell culture that can be or has been the recipient of a vector(s) for incorporating a polynucleotide insert. A host cell includes the progeny of a single host cell, which may not be completely identical (in morphology or genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. A host cell includes cells transfected in vivo with a polynucleotide(s) of the invention.

[0157] As used herein, "vector" refers to a construct capable of delivering, and preferably expressing, one or more gene(s) or sequence(s) of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA or RNA expression vectors bound to cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells such as producer cells.

[0158] The term "treating" as used herein means, unless otherwise specified, to reverse, ameliorate, inhibit the progression of, slow the progression of, delay the onset of, or prevent the disease or condition to which such term applies, or one or more symptoms of such disease or condition. The term "treatment" as used herein means the act of treating as described above, unless otherwise specified. The term "treating" also includes adjuvant and neoadjuvant treatment of a subject. For the avoidance of doubt, references herein to "treatment" include references to curative, palliative, and prophylactic treatment. For the avoidance of doubt, references herein to "treatment" also include references to curative, palliative, and prophylactic treatment.

[0159] Whenever an embodiment is described herein using the phrase "comprising," it is understood that other similar embodiments described with the terms "consisting of" and / or "consisting essentially of" are also provided.

[0160] When aspects or embodiments of the invention are described in terms of a Markush group or other alternative group, the invention encompasses not only the entire group recited as a whole, but each and every member of the group and all possible subgroups of the main group, as well as the main group lacking one or more of the group elements. The invention also envisions the explicit exclusion of any one or more of the group elements in the claimed invention.

[0161] Unless otherwise defined, 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 disclosure belongs. In case of conflict, the present specification, including definitions, will control. Throughout this specification and claims, the term "comprise" or variations such as "comprises" or "comprising" will be understood to mean the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers. Unless otherwise required by context, singular terms shall include the plural, and plural terms shall include the singular. Any example(s) following the term "eg" or "for example" are not meant to be exhaustive or limiting.

[0162] The techniques 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.

[0163] Certain non-limiting embodiments of the present invention will now be described with reference to the accompanying drawings. EXAMPLES

[0164] Example 1. Generation of optimized conditionally active therapeutic antibodies Overview In this example, we have successfully generated a panel of optimized conditionally active antibodies that are well expressed, biophysically stable, highly soluble, and have maximal amino acid sequence identity with the preferred human germline.

[0165] Materials and Methods Protein cloning, transient expression, purification and characterization The DNA sequences encoding the antibodies are cloned into separate human IgG1 heavy and light chain encoding expression cassettes in separate plasmid vectors via restriction enzyme-ligation cloning to produce IgG or IgG1 heavy chains for expression. 2 A similar cassette was also cloned into the "knobs-into-holes" human IgG1 heterodimerization Fc vector to generate Fab and Fab 2 Constructs were made using the knobs-into-holes (KIH) heavy chain expression vector (CH3 domain T366W and T366S / L368A / Y407V mutations) to express Fab 2 cMETCD47 and Her2CD3 protein constructs were generated. 2 The Her2-CD3 construct contained the effector function truncation mutations L234A / L235A / G237A. IgG1 heavy chain and kappa light chain expression vectors were used to express 2 The Her2CD47 protein construct was generated and IgG was expressed in CHO cells following transient transfection with an endotoxin-free IgG expression plasmid preparation according to the manufacturer's protocol.

[0166] Purified antibodies were harvested from the clarified supernatant using a HiTrap MabSelect Sure Protein A 5mL column on an AKTA Pure 150 L FPLC system. The eluted protein peak was immediately buffer exchanged into 1x PBS (pH 7.4) by directly loading the eluted Protein A peak fraction onto a HiPrep 26 / 10 desalting column. Protein concentrations were determined by measuring absorbance at 280 nm, and 1 μg of each purified protein was analyzed by SDS-PAGE under reducing and / or non-reducing conditions using 4-20% TGX polyacrylamide gradient gels (BioRad, Cat. No. 456-1093) with 1x Tris / Glycine / SDS buffer, separated at 120 V electric field for 1 h. Analytical size-exclusion chromatography was performed to test for the presence of non-covalent aggregates and to complement the SDS PAGE analysis. Aliquots of selected clones were analyzed by analytical size-exclusion chromatography (SEC) in isocratic mode using a Superdex 200 Increase 10 / 300 SEC column and 1× PBS (pH 7.4) as running buffer.

[0167] Selected proteins were further purified using preparative SEC. Up to 1 ml of antibody sample was loaded onto a Superdex 200 Increase 10 / 300 SEC column or a HiLoad 26 / 600 Superdex 200pg column equilibrated with 1x PBS (pH 7.4). 1 ml fractions of the peak of interest were collected and the main peak fractions were pooled. After size exclusion chromatography, samples were analyzed again by SDS-PAGE as described above.

[0168] red blood cell agglutination Red blood cells (RBCs) were isolated from fresh, unclotted human blood (minimum of three different donors), diluted to 2% in PBS, and titrated by incubation with IgG or protein constructs for 60-90 min in U-bottom 96-well plates. In the absence of hemagglutination, cells settled to the bottom of the well and formed a red pellet. Hemagglutination was observed as an unsettled RBC solution. Images of each plate were recorded and data were expressed per sample as the titer of the last well in which hemagglutination was observed.

[0169] Metalloprotease digestion Protein constructs were incubated with individual human matrix metalloproteinase (MMP) enzymes or with a mixture of active MMP3, MMP7, and MMP12 (equal parts of each) in a ratio of 1% total MMP to protein construct (wt / wt) in Tris-buffered saline (pH 7.4) containing 5 mM CaCl2 for 16 hours at 37° C. The reactions were stopped by adding 20 mM EDTA, and samples were then tested for binding or functional activity as described.

[0170] IgG titration binding ELISA To coat the ELISA plates, the target protein was diluted to 1 μg / ml in PBS (pH 7.4) and added 100 μl per well overnight at 4° C. The coated plates were washed three times with PBS (pH 7.4) and blocked with 4% skim milk protein in PBS (380 μl / well) for 1 h at room temperature, then washed five times with PBS-Tween 20 (PBST). Antibody (100 μl / well; diluted in PBST) was then added and incubated for 1 h at room temperature. Subsequently, the plates were washed three times with PBS and goat anti-human IgG-HRP was added (100 μl / well) for 1 h at room temperature. The plates were then washed three times with PBST and twice with PBS, after which 100 μl of TMB was added per well. The reaction was stopped by adding 100 μl of 2M H2SO4 per well and the OD was read at 450 nm on a plate reader.

[0171] Flow cytometry binding assay Binding of protein constructs (with and without predigestion with MMP3 / 7 / 12) and control IgG to Jurkat and BT-474 cells was assessed by flow cytometry. Zombie UV™ Fixable Viability Dye (Biolegend) was used to identify live cells. Binding of human IgG and protein constructs was detected using a FITC-conjugated goat anti-human (H+L) secondary antibody. Mouse monoclonal anti-CD3 control antibody binding was detected using Alexa-Fluor-488 goat anti-mouse IgG. Products were assessed by measuring the mean fluorescence intensity (MFI) of live cells in the FITC channel detector of a BD Fortessa flow cytometer.

[0172] T cell activation bioassay The functional activity of Her2CD3 protein constructs was assessed in a co-culture assay using BT474 cells and the NFAT-RE-luciferase Jurkat reporter cell line (Promega-TCR / CD3 effector cells NFAT). BT-474 cells (40000 cells / well) were seeded in 96-well white clear bottom tissue culture treated plates in Hybri-Care medium (ATCC) supplemented with 10% FBS and incubated overnight at 37°C in a CO2 incubator. The medium was removed and control antibodies or protein constructs (with or without predigestion with MMP3 / 7 / 12) made in assay medium (RPMI supplemented with 10% FBS) were added to the cells. TCR / CD3 effector cells (NFAT) were thawed, diluted and then added to the assay wells according to the manufacturer's protocol. After 6 h of incubation in a CO2 incubator at 37 °C, the plates were re-equilibrated to room temperature and luciferase activity was determined by adding Bio-Glo reagent for 5-10 min and measuring the luminescence signal (RLU). Fold induction was determined by calculating the ratio of RLU / RLU of the samples after subtracting the background luminescence signal in the absence of antibody.

[0173] Molecular dynamics simulation Eight systems (linker sequences X1, X2, X3, and X4, and equivalent sequences with a covalent bond broken in one of the linkers at GS sequence positions XC1, XC2, XC3, and XC4) were modeled and optimized using the AMBER10:EHT force field in MOE (Chemical Computing Group Inc). Histidine charges were assigned using the Protonate3D tool in MOE. Three successive minimizations were performed with a final gradient of 0.001 allowing 10,000 steps without constraints. Individual systems were reconstrained and gradually minimized using NAMD2.13 with the CHARMM27 force field to reduce the total potential energy in three successive steps of energy minimization. In the first step, all heavy atoms were constrained, allowing only hydrogen atom movement. In the second step, side chain constraints were removed, and in the third step, all atoms were released, allowing unconstrained movement. Minimization within CHARMM27 was facilitated as this is the force field used within the NAMD molecular dynamics simulations.

[0174] Dynamics methods were performed using Generalised Born (GB) solvation to describe the solvent effects. MD simulations were set up and then run using NAMD2.13. A four-step equilibration was performed, gradually weakening the harmonic constraints in 125 ps increments for a total equilibration time of 500 ps. In the first step, the system was heated to 310 K while applying a force of 4 kcal / mol to fix the backbone in place. In the remaining three steps, the backbone constraints were gradually removed from 4 kcal / mol to 1 kcal / mol using the NVT ensemble (constant number (N), volume (V) and temperature (T)). A switching function was applied at 10 Å to reduce the van der Waals potential function. Electrostatic and vdW cutoffs were set to 14 Å as recommended by NAMD. Periodic boundary conditions were not used as they are not compatible with the implicit solvent method. The constraints added during equilibration were removed for free simulation of the antibody complex. Production runs were performed for either 6, 15, 20 or 100 ns at 310 K. A 100 ns run was applied to the cleaved linker protein XC1-4 to fully explore the migration range, whereas shorter runs (up to 20 ns) proved sufficient to map the migration range for the uncleaved antibody construct X1-4.

[0175] In vivo analysis of PK and tolerability Tolerance Study - 28 6-8 week old male B6.Cg-Fcgrt tm1DcrTg(FCGRT)32DcrJ ("Tg32" homozygous human FcRn transgenic, JAX stock number 014565) mice were divided into seven groups with four mice in each group. Body weights were measured on the day of antibody administration. Test articles were administered at 2 mg / kg or 10 mg / kg as an IV injection at time 0, in a dose volume of 10 ml / kg. A total of 200 μL of whole blood samples were collected in EDTA on days 5, 29 and 60 (last bleed). Blood was used for CBC / Dif / Retic analysis including white blood cells, neutrophils, eosinophils, basophils, lymphocytes, monocytes, hemoglobin, red blood cells, reticulocytes, MCHC and MCV. Body weights were subsequently monitored weekly for the first month and monthly thereafter until the end of the experiment.

[0176] Pharmacokinetic study of B6.Cg-Fcgrt in 32 6- to 8-week-old male rats tm1Dcr Tg(FCGRT)32DcrJ ("Tg32" homozygous human FcRn transgenic, JAX stock number 014565) mice were divided into eight groups with four mice in each group. The day before test article administration, 35 μl blood samples were collected in EDTA from three Tg32 mice and tested for binding of the test article to mouse red blood cells using flow cytometry. Body weights were measured on the day of antibody administration and once a week until the end of the experiment. Test articles were administered at 2 mg / kg or 10 mg / kg as an IV injection at a dose of 10 ml / kg at time 0. Blood samples were collected from each mouse according to the bleeding schedule: 30 min, 4 h, 1, 3, 5, 7, 10, 14, 21, 28 and 42 days. 25 μl blood samples were collected from each mouse according to the bleeding schedule. Blood samples were collected in K3EDTA, processed to plasma, and stored at −20° C. Plasma samples were then assessed by ELISA in triplicate for estimation of human IgG concentrations.

[0177] Tolerance Study in NOD-SCID Mice - NOD-SCID mice were divided into 4 groups with 3 mice in each group. Body weights were measured daily. Test article was administered as an IV injection at 8 mg / kg or 14 mg / kg on day 0, followed by 3 doses at 4 or 7 mg / kg at 5 day intervals.

[0178] Flow cytometric analysis of binding to monkey and human erythrocytes Erythrocytes were isolated from three Cyno (Macaca fascicularis) monkeys and three human donors. For each sample, 5 × 10 5 Cells (diluted in DMEM + 5% FBS) were incubated with A-D5 IgG1: 0.0032; 0.016; 0.03; 50 μg / mL for 1 h, trastuzumab: 0.0032; 0.016; 0.03; 50 μg / mL, IgG 2 Her47 LHL-LHLF: 0.016; 0.08; 0.4; 2; 10; 50 μg / mL at 0 hours, IgG 2 Her47 LHL-LHL: 0.016; 0.08; 0.4; 2; 10; 50 μg / mL for 0 h. Binding of test samples to erythrocytes was measured using FITC AffiniPure Goat Anti-Human IgG (1:200 dilution; 1 h incubation time) followed by flow cytometric measurement of FITC fluorescence intensity (BD LSR FortessaX-20 cell analyzer).

[0179] Metalloprotease and cathepsin digestion at pH 6.0 and 7.4 Protein constructs were incubated in TBS (containing 5 mM CaCl2, pH 6.0 or 7.4) with individual human matrix metalloproteinase (MMP) or cathepsin enzymes at a ratio of 1% total enzyme to protein construct (wt / wt) for 0, 2, 4, 8 and 24 hours at 37° C. The reactions were stopped by adding 20 mM EDTA and the samples were then frozen before testing for binding or functional activity as described.

[0180] In vitro protein stability analysis Forced oxidation - For forced oxidation analysis, test articles in PBS were treated with 0.5% H2O2 for 2 h at room temperature and then stored at -80°C before SEC and RP analysis (intact antibody and subunits, tryptic peptides) on a Dionex Ultimate 3000RS HPLC system (ThermoFisher Scientific, Hemel Hempstead, UK). For intact antibody reduction, DTT was added to a final concentration of 0.33 M and samples were incubated for 1 h at 22°C and immediately analyzed by RP.

[0181] SEC Analysis - Chromatographic separation was performed using an Acquity UPLC Protein BEH SEC column, 200 Angstroms, 1.7 μm, 4.6 mm×150 mm (Waters, Elstree, UK) and an Acquity UPLC Protein BEH SEC guard column 30×4.6 mm, 1.7 μm, 200 Angstroms (Waters, Elstree, UK) coupled to a Dionex Ultimate 3000RS HPLC system (ThermoFisher Scientific, Hemel Hempstead, UK). The method consisted of an isocratic elution over 10 minutes with a mobile phase of 0.2 M potassium phosphate pH 6.8, 0.2 M potassium chloride. The flow rate was 0.35 mL / min. Detection was by UV absorption at 280 nm.

[0182] Reversed-phase analysis of intact antibodies and subunits - Chromatographic separation was performed using a PLRP-S 1000, 5 μm, 2.1 mm × 50 mm column (Agilent Technologies, Stockport, UK) coupled to a Dionex Ultimate 3000RS HPLC system (ThermoFisher Scientific, Hemel Hempstead, UK). The method consisted of 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 min. The flow rate was 0.5 mL / min and the temperature was maintained at 70 °C during the analysis. Detection was by UV absorbance at 280 nm.

[0183] HIC analysis - Chromatographic separation was performed using a TSKgel Butyl-NPR 4.6 mm x 35 mm HIC column (TOSOH Bioscience Ltd., Reading, UK) coupled to a Dionex Ultimate 3000RS HPLC system (ThermoFisher Scientific, Hemel Hempstead, UK). The method consisted of a linear gradient of 60% buffer A (100 mM sodium phosphate pH 7.0, 2 M ammonium sulfate) to 90% buffer B (100 mM sodium phosphate pH 7.0) over 9 min. The flow rate was 1.2 mL / min. Detection was by UV absorption at 280 nm.

[0184] Charge variant analysis - Charge variant profiling of test articles was determined by protein charge variant analysis on a LabChip GXII Touch HT (PerkinElmer) according to the manufacturer's protocol.

[0185] BIACORE® Analysis of Fc Affinity to Human Fc Receptors The interaction affinity of antibody proteins was measured by surface plasmon resonance using a BIACORE® T200 instrument. In most assays, His6-tagged FcγRI, FcγRIIa (167R and 167H variants), FcγRIIb, FcγRIIIa (176F and 176V variants), and FcγRIIIb receptors (all Sino Biological) were captured on a CM5 sensor chip coated with anti-HIS antibody by standard amine coupling method. Receptor-specific assay formats were then applied as follows:

[0186] Since FcγRI is a high affinity receptor for IgG1 monomers, a 1:1 kinetic analysis was performed under the following conditions: "single cycle" analysis using a flow rate of 30 μl / min, loading receptor protein to approximately 30 RU at 10 μl / min (diluted to 0.25 μg / ml in HBS-P+), applying a five-point 3-fold dilution of purified antibody titrated from 0.411 nM to 33.33 nM with an association time of 200 s and a dissociation time of 300 s, regenerating with two injections of glycine pH 1.5, and analyzing using a 1:1 fit.

[0187] Since the interaction between monomeric IgG and the FcγRII and FcγRIII receptors is a relatively low affinity interaction, a "steady state" affinity analysis was performed under the following conditions: flow rate 30 μl / min, receptor protein loaded to approximately 60 RU at 10 μl / min (diluted to 0.25 μg / ml in HBS-P+), association time 30 s, dissociation time 25 s, application of 5-point 3-fold dilutions of purified antibody titrated from 33 nM to 24000 nM, regenerated with 2 injections of glycine pH 1.5, and analyzed using steady state affinity calculations.

[0188] Results and Discussion Protein construct design principles Standard anti-cancer antigen antibodies pose significant pharmacological challenges in the treatment of solid tumors. The main problem limiting the efficacy of this class of potential drugs is that the antigens targeted by the antibodies are not found exclusively in tumors, but are only highly overexpressed in tumors. This extratumoral target expression often results in dose-limiting side effects risks and antigen "sink" effects when large amounts of the antibody must be administered to ensure that sufficient antibody penetrates the tumor to exert a therapeutic effect. One such example is the class of antibodies targeting the antigen CD47 (Figure 1A), where challenges include the fact that the highly expressed CD47 in the bloodstream (e.g., especially on red blood cells and platelets) is a "sink" that is 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-CD47 is also a significant toxicity risk. Indeed, anti-CD47 antibodies are known to cause anemia and even cross-linking of human red blood cells, resulting in a risk of red blood cell aggregation in patients. In addition, tumors are typically "hostile" environments with high expression of enzymes such as MMPs that can accelerate IgG degradation. All of these factors work together to minimize the potential safety and efficacy of anti-CD47 antibodies, and many other types of anti-tumor targeted antibodies, when target expression is not limited to the tumor environment.

[0189] The anti-CD47 protein construct (Figure 1B) aims to overcome the peripheral sink and toxicity issues encountered with anti-tumor antigen IgG by eliminating the binding of high-risk (but potentially potent mechanism of action) targets in the native protein. This effect is achieved by adding a linker (N-terminal) onto the binding domain of a low-risk top domain (e.g., a Fab domain targeting another tumor antigen such as Her2) and a high-risk bottom domain (e.g., CD47). Using the appropriate top domain / linker combination, a configuration is created that completely blocks the binding activity of the bottom CD47 domain. The tumor targeting domain (e.g., Her2) then promotes enrichment in the tumor environment, and the protein construct linker system exploits the elevated MMP activity in the tumor to cleave the linker peptide, exposing the CD47 binding domain, thereby conditionally activating CD47 binding activity in the tumor rather than in the periphery. This design principle can potentially be applied to many different structural formats, examples of which are outlined below.

[0190] Protein Construct IgG 2The design (Figure 2A) may be based on sequences derived from IgG1, IgG2, IgG3, IgG4, IgE, IgM or IgA and may or may not have effector function capabilities. In this construct, the four polypeptide chains encode four Fab domains (two Fab A, two Fab B), four linker sequences, and may or may not have an immunoglobulin hinge region and an Fc domain. Each Fab A-linker domain (top) blocks the binding activity of Fab B (bottom). The target binding specificities of the top and bottom domains may be different to facilitate bispecific function or the same to facilitate multivalent target interactions. Selection of linker sequences, such as the bottom hinge peptide sequence, creates a construct that is suppressed in non-diseased tissues but is rapidly cleaved and released in the presence of high concentrations of proteases in the tumor environment (Figure 2B). The linkers in the protein construct design are all proteolytically cleavable and can be cleaved in sequence with an initial "fast" cleavage of the "contained" intact construct creating an intermediate "open" activation state, allowing Fab A and B from a single protein construct to bind to their cognate targets. A potentially slower secondary cleavage of the second linker in each Fab A-Fab B protein construct unit can create a "dissociated" form in which the Fab A domain is completely released from the construct and the lower Fab domain is completely free for off-target (but still localized) activity. Cleaved linkers based on immunoglobulin hinge sequences may also recruit increased immune effector functions (ADCC, CDC and ADCP) at the cell membrane via endogenous anti-hinge antibodies, a known phenomenon in human patients with (and sometimes without) underlying autoreactive diseases.

[0191] Protein construct Fab 2The design may be based on sequences derived from IgG1, IgG2, IgG3, IgG4, IgE, IgM or IgA and may or may not have effector function capabilities. In this construct, two (Figure 3A) or three (Figure 3B) polypeptide chains encode two Fab domains (one Fab A, one Fab B), two or more linker sequences, and may or may not have an immunoglobulin hinge region and an Fc domain, with heterodimeric pairing induced by mutations in the Fc. Again, each Fab A-linker domain blocks the binding activity of Fab B, and the choice of linker sequence, such as the lower hinge peptide sequence, creates a structure that is suppressed in non-diseased tissues but is rapidly cleaved, released and ultimately dissociated in the presence of high concentrations of linker-cleaving proteases in the tumor environment (Figure 3A).

[0192] Cloning and expression of protein constructs Fabs with different linker domains (Table 1) 2 or IgG 2 To generate and purify the 15 bispecific, conditionally active protein constructs formed as molecules, DNA cassettes for each construct type (Table 2) were synthesized and cloned into expression vectors encoding human IgG1 heavy and light chains, or "knobs-into-holes" heterodimeric Fc. Proteins were named using the following format: scientific name (format)-(target name [upper domain / lower domain)-(heavy chain linker type)-(light chain linker type). All proteins were produced by transient transfection of CHO cells and then purified by protein A affinity chromatography.

[0193] The anti-HER2 variable region sequence used in the protein constructs disclosed herein is the variable region sequence of trastuzumab. The anti-CD3 variable region sequence used in the protein constructs disclosed herein is the variable region sequence of OKT3 or SP34. The anti-cMET variable region sequence used in the protein constructs disclosed herein is that provided in WO2019 / 175186. The anti-CD47 variable region sequence used in the protein constructs disclosed herein is that provided in WO2019 / 034895.

[0194] Analysis of protein construct expression and purification characteristics Quantification of Protein A purified proteins showed that using different linker types affected expression yields (Table 2). Protein preparations in 1x PBS (pH 7.4) were also examined by analytical size exclusion chromatography to quantify the percentage of desired product. Analytical SEC for all three classes of generated bispecific proteins (cMETCD47, Her2CD47 and Her2CD3) showed that constructs containing LHL linkers gave the most optimal combination of highest yield of desired product and percentage of main peak generation (Table 2). SDS-PAGE analysis of Protein A purified proteins (Figure 4) also showed that clones containing the long linker domain L3, in addition to the short linker domains L1 and LH, produced highly heterogeneous products with a high percentage of HMW and LMW products in non-reduced samples and a high percentage of LMW products in reduced samples. This suggests that suboptimal linker types may result in significant formation of undesired multimers and damaged products. For all constructs, HMW impurities were nearly absent in the reduced lanes, suggesting that these HMW impurities were either disulfide-linked dimers or larger that could not be reduced by SDS alone. 2 By design, both clones 12 and 14 showed high yields (Table 2), but clone 14 (containing the LHL linker) showed the highest yield and high homogeneity of the desired product by SEC (90%) and SDS-PAGE (Figure 4).

[0195] SEC chromatography was then performed on the remaining protein samples from a subset of clones to generate fully purified monomeric proteins. Chromatography traces of clones 1, 2, 3, 4, 5, 6, 10, 12 and 14 on preparative SEC are shown in Figure 5. These analyses further showed that clones 1 (Figure 5A), 3 (Figure 5C), 4 (Figure 5D), 5 (Figure 5E) and 12 (Figure 5G) contained a significant proportion of high molecular weight, and often low molecular weight products, around the main peak of the desired product (eluting at approximately 0.5-0.55 CV in Figures 5A-5H). In contrast, clones 6 (Figure 5F), 14 (Figure 5H) and 10 (Figure 5I) exhibited a predominantly clear main product peak. In the case of clones 2 and 6, effective SEC purification of the predominantly monomeric protein was possible, as shown by SDS-PAGE of the non-reduced samples (Figure 6A). Attempts at SEC purification of clones 1, 4, and 5 were unsuccessful and still produced heterogeneous samples (Figure 6A). Her2CD3 Fab was highly purified as evidenced by SDS-PAGE of non-reduced and reduced samples. 2 SEC-purified proteins from clones 7, 8 and 10 showed improved homogeneity, but Her2CD47 IgG 2 Of the clones, only clone 14 achieved complete homogeneity and monomeric state (Figure 6B). Again, these findings demonstrate that clones 6, 10, and 14, all of which contain LHL linkers on both chains, exhibited the most reproducible and beneficial properties.

[0196] Functional characterization of protein constructs containing CD47 antibody V domains in the lower Fab Control IgG antibodies A-D5 anti-CD47, MH7.1 anti-C-MET, anti-Her2 trastuzumab, and A-D5 Fab-Fc (a monovalent form of the A-D5 antibody containing a single Fab domain) were titrated (in μg / ml) in a direct binding ELISA against human CD47, C-MET, and Her2 proteins (Figure 7A). 2 Her2CD47-LH-LH and Her2CD47-LHL-LHL clones of the format (Figure 7B), as well as Fab 2The formats cMETCD47-L2-L2 and cMETCD47-LHL-LHL (Figure 7C) were also analyzed in the same manner. The control antibodies showed the expected strong binding activity to their cognate targets, but little or no background to other targets, even at the highest concentrations (Figure 7A).

[0197] Importantly, the very strong monovalent binding of A-D5 Fab-Fc indicated the intrinsic affinity and important potency of the A-D5 anti-CD47 domain (Figure 7A), which must be countered to successfully format the protein construct of the present invention. Protein constructs Her2CD47-LHL-LHL (Figure 7B) and cMETCD47-LHL-LHL (Figure 7C) also showed similar strong and highly specific binding of their upper Fab domains to their cognate targets, but when using this linker combination, there was no binding signal for CD47, demonstrating indeed a complete inhibition of the binding activity of the CD47V domain. Importantly, in the case of Her2CD47-LH-LH, a high background signal of human CD47 was observed, and to a lesser extent in cMETCD47-L2-L2, suggesting that the loss of binding affinity of the lower Fab can be precisely controlled by the linker selection. These findings suggest that the protein constructs tested in this assay, Her2CD47-LHL-LHL (Figure 7B) and cMETCD47-LHL-LHL (Figure 7C), which contain the anti-CD47 binding domain of antibody A-D5, have >1000-fold reduced ability to bind CD47 compared to A-D5 IgG1, with a binding signal OD of 1.0 being approximately 1 × 10 -2 μg / ml, while neither protein construct showed a signal above 0.2 at the highest concentration tested, 10 μg / ml.

[0198] Because hemagglutination is the primary toxic risk of anti-CD47 antibodies, prioritized protein constructs were tested in a human erythrocyte-based hemagglutination assay. Control antibodies anti-CD235a (mouse) and A-D5 anti-CD47, A-D5 Fab-Fc, MH7.1 anti-c-MET, anti-Her2 trastuzumab, IgG 2 Her2CD47-LH-LH and Her2CD47-LHL-LHL in the format, as well as Fab 2 The formats cMETCD47-L2-L2 and cMETCD47-LHL-LHL were titrated (in nM) in a human hemagglutination assay using fresh erythrocytes from healthy donor 1 (Figure 8A), donor 2 (Figure 8B), and donor 3 (Figure 8C). In all three donors, the control antibodies anti-CD235a, A-D5 anti-CD47, and A-D5 Fab-Fc showed strong concentration-dependent hemagglutination due to cross-linking of their corresponding surface antigens on adjacent erythrocytes. The low potency hemagglutination effect observed with A-D5 Fab-Fc (clone 15) could be due to the presence of a small amount of functional dimer in this protein preparation, since it was only purified on a protein A column and not fully purified to the monomeric state by SEC. Importantly, none of the protein construct samples showed the ability to produce a hemagglutination effect, even at the highest concentration of 140 nM. This finding was supported by the IgG antibody A-D5, which contains the anti-CD47 binding domain tested in this assay. 2 and Fab 2 These results suggest that this protein construct has over 241-fold reduced ability to cause aggregation compared to A-D5 IgG1, which showed a potency of 0.58 nM.

[0199] The prioritized protein constructs were subjected to enzymatic digestion with human MMP3, 7 and 12 for 2, 4, 8 and 24 hours of incubation over time, as well as a separate 24 hour incubation in buffer without enzyme as a negative control. These time-course digestion samples were then subjected to direct binding ELISA against human Her2 and CD47 (Figure 9A,B) or human C-MET and CD47 (Figure 9C). In both cases, no loss of binding was observed over the time course for either Her2 or C-MET, suggesting that the addition of proteases does not reduce the functional binding capacity of the upper Fab domain. In contrast, the CD47-binding capacity of all three protein constructs was clearly increased over time in an MMP-dependent manner. Clone IgG 2 Her2CD47-LH-LH (Figure 9A) again showed high background binding to CD47 and the lowest increase in CD47 binding over time. In contrast, clone IgG 2 Her2CD47-LHL-LHL (Figure 9B) and Fab 2 cMETCD47-LHL-LHL (Figure 9C) again showed low background binding to CD47 (OD450nM less than 0.2) and a rapid increase in CD47 binding from 2 hours incubation with both MMP7 and 12, reaching full saturation (OD450nM approximately 4.0) by 24 hours incubation. MMP3 appears to be the slowest active of the three MMPs, but showed an increased CD47 binding signal after 24 hours in all three protein constructs.

[0200] Functional characterization of protein constructs containing CD3 antibody V domains in the lower Fab Antibody Fab 2 Her2CD3-L1-LH, Fab 2 Her2CD3-L2-L2 and Fab 2Her2CD3-LHL-LHL was analyzed by ELISA (Figure 10A), flow cytometry (Figure 10B) and CD3 reporter assay (Figure 10C). In ELISA analysis, all three proteins showed the expected strong binding activity to Her2 (upper Fab domain), with little or no background binding to other targets, even at the highest concentration (Figure 10A). The three protein constructs Fab 2 All proteins were incubated overnight with or without a mixture of MMP3, 7 and 12 and then further analyzed. Flow cytometry of binding to the HER2+ human cell line BT474 showed that anti-HER2 trastuzumab showed strong binding, with Fab 2 Her2CD3-L1-LH and Fab 2 Her2CD3-L2-L2 similarly showed strong binding before and after MMP digestion, while Fab 2 Her2CD3-LHL-LHL showed a partial reduction in binding after MMP digestion, suggesting a proportion of the protein in the "dissociated" state that had lost the upper Fab (Figure 10B). 2Since this finding suggests that the MMP digestion process is activated in the case of Her2CD3-LHL-LHL, the same samples were applied to a reporter assay, but this time Her2+BT474 cells were mixed with human CD3+Jurkat cells genetically engineered to provide a measurable signal when CD3 is activated. Data from this assay showed that, as expected, the bivalent anti-CD3 antibody OKT3 directly activated the CD3 signal in the reporter cells (Figure 10C). Each of the protein constructs showed a different profile. The Her2CD3-L2-L2 protein, which contains two G4S linkers (not cleavable by MMP proteases), showed high background in the assay, but no increase in CD3 activation signal was observed after MMP digestion, suggesting that the flexible linker of this construct allows binding of Her2 by the upper Fab and partial activation of the lower Fab allowing some background, but not activating CD3 interligation (Figure 10C). Fab 2 The Her2CD3-L1-LH protein showed lower background activity in the assay, but a modest increase in signal was seen after MMP digestion. 2 Her2CD3-LHL-LHL demonstrated no measurable background signaling in the absence of MMP digestion and showed minimal activation of CD3 similar to the negative control trastuzumab and the IgG1 isotype control antibody in the undigested samples, whereas robust activation was seen in the MMP digested samples. Thus, the data in Figures 10A-C are consistent with the results of simply expressing and purifying Her2CD3-LHL-LHL Fab. 2 This format is suggested to have the best combination of properties, including high intrinsic Her2 binding activity, low background CD3 ligation activity, and high CD3 cross-ligation activity only when activated by MMP cleavage of the LHL linker.

[0201] Second generation construct cloning and expression The above multispecific Fab containing an LHL linker 2 and IgG 2The performance of the clones prompted another series of constructs to experimentally examine 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. "One-arm" Fab 2 protein, clone “Met47-LHL-LHL” (Table 6 , Figure 3B ). 2. Modified "One-Arm" Type "Clone 10" Fab 2 protein, clone “Her23-LHL-LHL” (Table 7 , Figure 3B ). 3. "One-arm" Fab 2 protein, clone “Her23(34)-LHL-LHL” (Table 8 , Figure 3B ). 4. IgG containing the Her2-binding domain of trastuzumab in the upper Fab and the CD47-binding domain of clone A-D5 in the lower Fab 2 A series of clones of this type (Table 9) were synthesized that contained a mutated LHL-based linker sequence (LHLF linker), a moderately extended linker with a portion of the human IgG1 middle hinge sequence (LHLM linker), or both (LHLMF linker), which may be sensitive to enzymatic cleavage by a broad panel of MMPs. 5.Fab 2 Fab in which the module is located at the C-terminus of KIH-Fc 2 The protein clone “Fc-Her23(34)” (Table 13, Figure 12). 6. "One-arm" Fab containing two copies of C-Met Fab 2 The protein clone "MetMet-LHL-LHL" (Table 14, Figure 3B, Figure 13). In this structural form, binding to the Met receptor in a bivalent form can only occur after the single LHL linker is cleaved by a protease. These constructs were successfully expressed and purified by protein A and size exclusion chromatography as previously described.

[0202] Second generation IgG 2 Analysis of constructs The above purified IgG 2 The performance of the clones was examined in a series of further analyses. Exemplary clone A-D5 IgG1, IgG 2 Her2CD47-LHL-LHL, IgG 2 Her47-LHLF-LHL, IgG 2 Her47-LHL-LHLF and IgG 2 We first investigated the binding of Her47-LHLF-LHLF (Table 9) to human Her2, as well as human and mouse CD47 (Figure 14). This analysis showed two things: 1) the A-D5 anti-CD47 IgG1 protein showed high binding signals to both hCD47 and mCD47. 2) the four IgG 2 All of the proteins maintained high binding signals to hHer2, but had no background binding signals to hCD47 or mCD47 (Figure 14).

[0203] Clone IgG 2 Her2CD47-LHL-LHL and IgG 2 Her47-LHL-LHLF was tested for sensitivity to enzyme activation (at pH 7.4) by incubation with multiple proteases known to be overexpressed in human tumors, namely MMP7 (Fig. 15A), MMP8 (Fig. 15B), and MMP10 (Fig. 15C) (Fig. 15). Both proteins were able to activate hCD47 binding by each enzyme, but IgG 2 Her47-LHL-LHLF showed more rapid activation in both cases. Incubation with MMP12 showed that in the case of this enzyme, both proteins were able to activate hCD47 binding at equal rates (Figure 15D). Unexpectedly, incubation with MMP13 reduced the activation of IgG 2 Her47-LHL-LHLF can activate hCD47 binding by this enzyme, whereas IgG 2Her2CD47-LHL-LHL could not be activated (Figure 15E). Importantly, incubation with the cysteine ​​protease cathepsin S also showed that, in the case of this enzyme, both proteins could activate hCD47 binding at equal rates (Figure 15F). These findings support the conclusion that IgG 2 Fab in proteins 2 It was demonstrated that the peptide linker content of the modules could be "tuned" to make the proteolytic activation process even faster in order to increase the number of potentially activating enzymes. To sample this potential, additional linker designs of appropriate length and content were envisioned to promote sensitivity to specific classes of enzymes, such as those using amino acid sequences that exhibit proteolytic sensitivity to the activity of other disease-related metalloproteinases, such as ADAMS, cysteine, aspartic acid, and serine proteases, providing either broader or more selective activation.

[0204] IgG 2 To examine the effect of activation on binding affinity, a Biacore assay was performed that allows sampling of both Her2 and CD47 binding. 2Her47-LHL-LHLF proteins (undigested or activated by MMP12 for 2, 4, 8 or 24 hours) were captured on the chip surface via anti-Fc antibodies, and then the binding affinity to soluble Her2 and CD47 ectodomain proteins was measured. Binding assays for trastuzumab IgG1 and A-D5 IgG1 (without enzymatic digestion) were repeated at the beginning and end of the experiment. The data are shown in Table 17. These assays showed that the KD values ​​calculated for each antibody were very similar in each assay. However, importantly, the Rmax values ​​(maximum binding signal at maximum analyte concentration) were significantly decreased between assays at the beginning and end of the experiment (e.g., Rmax 265.80 RU at the beginning and 144.13 RU at the end for trastuzumab), suggesting that the activity of the capture surface for anti-Fc antibodies decreases after multiple regenerations inherent in the Biacore methodology. To examine the effect of MMP12 activation on target reactivity, Rmax and KD values ​​for both Her2 (Figure 16A, Table 18) and CD47 (Figure 16B, Table 18) were calculated. This analysis showed that Her2 binding and affinity was maintained over 24 hours of MMP12 activation. Importantly, no CD47 binding (Rmax=0) was observed in samples at 0 hours (no MMP12 digestion), but both the Rmax and apparent affinity for CD47 increased rapidly over the time course of activation from 2 hours of incubation onwards (Figure 16B, Table 18). These findings support the conclusion that Fab 2 The bottom Fab of the module was demonstrated to be completely inactive and incapable of target interactions on the intact molecule until protease activation occurred. This observation is further illustrated in Figure 17, where no binding activity was observed even at a 400 nM concentration of CD47 analyte, yet high binding to CD47 after 24 hours of MMP12 treatment was evident.

[0205] Based on the above findings, Fab 2It was also demonstrated that cleavage of the linker peptide in the module (and therefore activation of the lower Fab) is transient, as activation is 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. 2 There may be pharmacological benefits for molecules containing the module, resulting in rapid drug accumulation, prolonged residence, and high levels of activity in such affected tissues.

[0206] Fab 2 In silico modeling of modular structure and molecular dynamics Fab 2 To understand the mechanism by which the module may function, we performed structural modeling and molecular dynamics analysis. Using the crystal structures of human CD47 ECD bound to the Fab domains of IgG1 C47B161 (Protein Data Bank Identifier 5TZT), IgG1 C47B222 (Protein Data Bank Identifier 5TZ2) and IgG1 B6H12.2 (Protein Data Bank Identifier 5TZU) as templates, we generated a model of anti-CD47 A-D5 Fab bound to CD47 ECD using the protein modeling suite of MOE software. The structure of trastuzumab Fab in complex with Her2 extracellular domain was taken from PDB structure 1N8Z.

[0207] The LHL and LHLF linkers between the upper Trastuzumab Fab and the lower anti-CD47 Fab were then modeled using the protein modeling suite of MOE software. To aid in modeling the linkers, the C-termini of Fab structures available in the Protein Data Bank were examined to help define structures in which the LB linkers could be removed from each of the heavy and light domains of the Trastuzumab Fab. Modeling predicted that the Trastuzumab Fab heavy and light chain C-termini could optionally have the native interchain disulfide bonds normally present in IgG1 Fab domains.

[0208] A full-length IgG1-based model incorporating the modeled anti-CD47 Fab described above was constructed using the structure of IgG1 b12 (Protein Data Bank identifier 1HZH) as a template. Structural errors in 1HZH, such as missing structural regions, were remodeled and corrected. The protein modeling suite of MOE software was used to replace the IgG1 b12 Fab with the anti-CD47 Fab and to attach the Fc hinge. This model was based on Her2 and CD47. 2 The model shows a possible tertiary structure of the molecule with IgG1 (Figure 18A). In the model, binding of the Her2 epitope is constitutively active (Figure 18A), while binding of the CD47 ECD is completely blocked by both the linker and the proximity of the anti-CD47 Fab to the anti-HER2 Fab (Figure 18B). The model shows that the Fab can bind HER2 through the upper Fab without any observed hindrance, but inhibits binding to CD47 through the lower Fab until the linker is degraded. 2 It is consistent with the module (Figure 16A, Figure 16B).

[0209] Fab 2 To gain further insight into how the modules move in solution, we performed molecular dynamics simulations using multiple 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. In addition, a custom descriptor dSASA was written in the SVL language (MOE) to calculate the change in the solvent accessible surface area (SASA) for each residue and in each case the aggregate of only the linker sequence. This provides a basis for comparison of how different uncleaved linkers behave in terms of structural changes and the associated impact on the solvent accessibility of the linker, which is expected to affect the sequence-specific cleavage potential (Figure 18).

[0210] Figures 19A-I show nine graphs of the corresponding solvent accessible surface area (SASA) results obtained for the three linkers tested. In a first analysis, absolute SASA values ​​were sampled for nine kinetic runs (6 ns) for each of the LHL and LHLF linkers, and ten with L2 (Figures 19A, 19D, and 19G, respectively). The data clearly showed that the L2 linker had the greatest propensity for structural heterogeneity over time (Figure 8G). Since the starting SASA values ​​were not the same for each run, a secondary analysis normalized the results to represent the difference from the starting SASA value, calculated by subtracting all SASA values ​​from the starting SASA value. Results over the 6 ns dynamics run time (Figures 19B, 19E, and 19H, respectively) and the first 2.5 ns of the 6 ns dynamics run (Figures 19C, 19F, and 19I, respectively) indicate that the LHL and LHLF linkers have significantly less conformational dynamics than the L2 linker. In particular, the 6 ns molecular dynamics run indicates that L2 is the most mobile, so the different linker sequences may have different mobility and therefore Fab 2 Emphasis is placed on concepts that will improve the CDR solvent exposure profile for the lower Fab of the module.

[0211] Comparison of the cleaved and uncleaved linkers shows a dramatic increase in mobility of the cleaved linker compared to the uncleaved linker, consistent with the biological observations in Figures 14-17. For example, Figure 20A shows the mobility of Fab when both linkers are intact during a 100 ns kinetic run. 2 The analysis shows restricted movement of the modules followed by a dramatic movement of the upper Fab domain in the context of a single cleavage of the LHL linker (secondary linker intact). This analysis shows that the upper Fab domain gains significantly increased freedom, resulting in a complex configuration in which it can move completely away from the lower Fab domain and fully expose its CDRs allowing free interaction with, for example, CD47 (Figure 20B).

[0212] In vitro and in vivo analysis of tolerability and pharmacokinetics To investigate the tolerability and pharmacokinetics of IgG2 and Fab2 in the context of CD47 like lower Fab domains, several exemplary molecules (A-D5 IgG1, IgG 2 Her47 LHL-LHL, IgG 2 Her47 LHL-LHLF and Fab 2 Met47 LHL-LHL) were isolated from 6-8 week-old male B6.Cg-Fcgrt mice. tm1Dcr These "Tg32" mice are human FcRn homozygous transgenic animals and have the pharmacokinetic properties of human IgG that mimic those of humans and primates. 2 Her47 LHL-LHL, IgG 2 Her47 LHL-LHLF and Fab 2 Since all CD47-binding domains contained in the lower Fab domain of Met47 LHL-LHL were confirmed to be capable of binding recombinant mouse CD47 protein, their reactivity to mouse membrane-presented CD47 on erythrocytes was first tested by flow cytometry (Figure 21). This study demonstrated that A-D5 IgG1 shows no signal on mouse erythrocytes at 0.1 μg / ml, but a clear concentration-dependent binding signal at both 1 μg / ml and 10 μg / ml (Figure 21). Importantly, binding to mouse erythrocytes was nearly saturated at 1 μg / ml (63% binding, Figure 21) and completely saturated at 10 μg / ml (98% binding, Figure 21). In contrast, the IgG protein 2 Her47 LHL-LHL, IgG 2 Her47 LHL-LHLF, or Fab 2 Neither Met47 LHL-LHL showed binding at any concentration, indicating that the anti-CD47 variable domain of the lower Fab domain is incapable of interacting with CD47 on mouse erythrocytes (FIG. 21).

[0213] A hemagglutination assay was also performed using red blood cells isolated from Tg32 mice. This analysis showed that only A-D5 IgG1 was capable of promoting concentration-dependent agglutination of mouse red blood cells (above 3.12 μg / ml), whereas protein IgG 2 Her47 LHL-LHL, IgG 2 Her47 LHL-LHLF, or Fab 2 We demonstrated that neither Met47 LHL-LHL showed aggregation at any concentration up to 200 μg / ml (FIG. 22). These findings support the human CD47 protein and erythrocyte binding outlined above (i.e., complete binding to human CD47 by A-D5 IgG1, but not IgG 2 Or Fab 2 In vitro assays of CD47 binding (no measurable binding to the protein) were demonstrated to be recapitulated in a mouse system, making the mouse a viable model to study the effects of CD47 binding on both pharmacokinetics and tolerability.

[0214] In vivo tolerability studies showed that A-D5 IgG1, IgG 2 Her47 LHL-LHL, IgG 2 Her47 LHL-LHLF and Fab 2 Met47 LHL-LHL were administered once (intravenously) to Tg32 mice at a concentration of 2 mg / kg or 10 mg / kg, respectively. The 2 mg / kg dose of A-D5 IgG was well tolerated, whereas the 10 mg / kg dose was poorly tolerated and caused overt toxicity on day 0, terminating the study in this treatment cohort. In contrast, IgG 2 Her47 LHL-LHL, IgG 2 Her47 LHL-LHLF and Fab 2 The 2 mg / kg and 10 mg / kg doses of Met47 LHL-LHL were all well tolerated over 60 days of body weight monitoring (Figure 23). The 2 mg / kg dose of A-D5 IgG was generally well tolerated, although a significant upregulation of reticulocytes was observed in this group at 5 days post-treatment (Figure 24). In contrast, IgG 2 Her47 LHL-LHL, IgG2 Her47 LHL-LHLF or Fab 2 The 2 mg / kg or 10 mg / kg doses of Met47 LHL-LHL were not associated with reticulocyte upregulation on day 5 or later (Figure 24, Figure 25). Reticulocyte upregulation is a known response to rapid red blood cell clearance, suggesting that IV administration of A-D5 IgG1 antibody leads to accelerated clearance of high CD47 red blood cells.

[0215] To sample the broader effects of the administered protein, a full hematology panel was examined at 5, 29, and 60 days after administration (Figures 25A-K). These analyses showed that the reticulocyte upregulation effect of A-D5 IgG1 was transient, returning to baseline by day 29 (Figure 25A). A-D5 IgG1, IgG 2 Her47 LHL-LHL, IgG 2 Her47 LHL-LHLF and Fab 2 In Met47 LHL-LHL, no significant effects were observed on red blood cell (RBC) counts, hemoglobin, mean hemoglobin concentration in red blood cells (MCHC), mean corpuscular volume (MCV), white blood cells, monocytes, lymphocytes, basophils, eosinophils, or neutrophil levels at days 5, 29, or 60 (Figures 25B-K).

[0216] In an in vivo pharmacokinetic study, IgG 2 Her47 LHL-LHL, IgG 2 Her47 LHL-LHLF and Fab 2Met47 LHL-LHL was administered once (intravenously) to Tg32 mice at a concentration of 2 or 10 mg / kg, respectively, and A-D5 IgG1 was administered at a previously tolerated concentration of 2 mg / kg. Blood samples were taken from each mouse according to the bleeding schedule: 30 min, 4 h, 1, 3, 5, 7, 10, 14, 21, 28 and 42 days. Analysis of serum antibody concentrations showed that A-D5 IgG1 at 2 mg / kg was very rapidly cleared from the blood circulation, reaching a mean concentration below 0.5 μg / ml within 5 days (Figure 26). This rapid drug clearance (known as tissue-mediated pharmacokinetics or TMDD) may be due to the previously observed strong binding to mouse red blood cells, which allows it to be rapidly removed from the system by phagocytosis. This finding was further explained by the fact that administration of A-D5 IgG1 resulted in reticulocyte levels returning to normal by day 29 (Figure 25A), as the molecule was essentially cleared by day 10 (Figure 26).

[0217] In contrast, IgG 2 Her47 LHL-LHL, IgG 2 Her47 LHL-LHLF and Fab 2 All of the Met47 LHL-LHLs showed slow clearance at both the 2 mg / kg and 10 mg / kg doses (Figure 26). Importantly, the 2 mg / kg dose of each protein took more than 25 days to reach concentrations below 1.0 μg / ml (Figure 27A), and the 10 mg / kg dose of each protein maintained concentrations above 1.0 μg / ml at day 42 (Figure 27B). A single sample taken from a mouse administered 10 mg / kg A-D5 IgG1 on day 0 of the tolerability study was also analyzed, and the maximum serum IgG concentration achieved (but not tolerated) was consistent with the fully tolerated IgG1. 2 and Fab 2 Concentrations were shown to be similar to those achieved with the protein (>50 μg / ml).

[0218] 2mg / kg IgG 2 Her47 LHL-LHL, IgG 2 Her47 LHL-LHLF and Fab2 All of the Met47 LHL-LHL proteins showed a standard "alpha" phase (when IV immunoglobulin is rapidly distributed from the bloodstream to tissues) followed by a long "beta" phase in which the antibody circulates in the serum (Figures 26, 27A). These proteins also showed a standard distribution profile at 10 mg / kg, but with a longer period in the blood circulation (Figures 26, 27B). Importantly, the beta phase of each protein followed linear and parallel curves at both concentrations, suggesting that the CD47 domain of these proteins does not lead to the TMDD observed with A-D5 IgG1 (Figure 26). IgG 2 Her47 LHL-LHL, IgG 2 Her47 LHL-LHLF or Fab 2 If the Met47 LHL-LHL protein were activated in the periphery, TMDD would be expected to be more pronounced, as activation would result in high affinity erythrocyte, endothelial, and platelet binding, resulting in clearance that changes the beta phase to a sharp downward trajectory as seen with A-D5 IgG1 (Figure 26). These observations are consistent with the IgG 2 Her47 LHL-LHL, IgG 2 Her47 LHL-LHLF and Fab 2 Administration of Met47 LHL-LHL suggests low levels of peripheral activation, together with the lack of reticulocyte amplification, implying that despite the long pharmacokinetics of these molecules, they underwent multiple FcRn recycling in circulation for >25 days.

[0219] IgG 2 Her47 LHL-LHL, IgG 2 Her47 LHL-LHLF and Fab 2 The pharmacokinetic and tolerability findings outlined above for Met47 LHL-LHL demonstrated standard FcRn-mediated antibody-like half-life extension via the human IgG1 Fc domain. This effect resulted in significantly increased area under the curve (AUC) values ​​for these three proteins compared to A-D5 IgG1 (Figure 28). At 2 mg / kg, the AUC was 1.0-fold higher than that of IgG1. 2Her47 LHL-LHL, IgG 2 Her47 LHL-LHLF and Fab 2 Met47 LHL-LHL was increased 25-40 times more than A-D5 IgG1. At 10 mg / kg, AUC was 25-40 times higher than IgG1, although this was not a safely achievable dose for A-D5 IgG1. 2 Her47 LHL-LHL, IgG 2 Her47 LHL-LHLF and Fab 2 Met47 LHL-LHL was increased approximately 100-250 times more than A-D5 IgG1 (Figure 28). IgG 2 and Fab 2 These AUC improvements are highly significant as they suggest that the use of the CD47 binding domain within the construct allows for the use of high concentrations from the first dose, maximizing distribution towards the tumor tissue.

[0220] Flow cytometric analysis of binding to monkey and human erythrocytes Red blood cells were isolated from three cyno NHPs and three human donors and were immunoreactive with A-D5 IgG1, trastuzumab, and IgG. 2 Her47 LHL-LHLF or IgG 2 Both sets of analyses demonstrated that only A-D5 IgG1 exhibited concentration-dependent binding to either cyno (Figure 29A) or human (Figure 29B) erythrocytes. These findings support the conclusion that IgG 2 (Therefore, Fab 2 ) The CD47 binding domain within the construct confirmed the above observations, with restricted binding to mouse, monkey and human CD47.

[0221] Activation by MMPs and cathepsins at pH 6.0 and 7.4 It has already been shown that MMPs and cathepsins may enzymatically cleave peptide sequences found in either the LHL or LHLF linkers outlined above. Importantly, however, both of these classes of enzymes may be sensitive to changes in pH conditions that increase or decrease their enzymatic activity. This may be of great importance, since the pH of solid tumors is frequently observed to deviate from the standard physiological pH in humans, which is pH 7.4. In particular, solid tumors (and highly inflamed tissues) may develop acidic pH conditions as low as pH 6.0.

[0222] Having demonstrated above that multiple MMP enzymes have the ability to activate the lower Fab bond at pH 7.4, we examined the activity of multiple MMP and cathepsin enzymes, all of which are associated with increased activity in solid tumors, at both pH 6.0 and 7.4. IgG against MMP3 (Figure 30A,B), MMP7 (Figure 30C,D), MMP8 (Figure 30E,F), MMP10 (Figure 30G,H), MMP12 (Figure 30I,J), MMP13 (Figure 30K,L) and MMP14 (Figure 30M,N) 2 Her47 LHL-LHL, or IgG 2 We then examined the activation of Her47 LHL-LHLF. 2 Her47 LHL-LHL and IgG 2 MMP-treated samples of Her47 LHL-LHLF were subjected to direct binding to both human Her2 and human CD47. These analyses demonstrated that at both pH 7.4 and pH 6.0, both proteins showed measurable activation of CD47 binding by MMPs 8, 10, and 12. In contrast, IgG 2 Only Her47 LHL-LHLF was activated by MMP13 at both pH 7.4 and pH 6.0 (Fig. 30K, L) and by MMP7 at pH 6.0 (Fig. 30D). 2 Her47 LHL-LHLF also inhibits IgG 2Her47 LHL-LHL showed relatively higher levels of activation by most of the MMPs tested at most time points (Figures 30A-N). These findings confirm that inclusion of the LHLF linker results in more rapid activation of the lower Fab linkage by a broader range of MMP enzymes at both pH 6.0 and / or pH 7.4.

[0223] Cathepsins were also examined as potential activating enzymes. For these enzymes, a clear relationship between pH and activity was observed. IgG 2 Her47 LHL-LHL (Figure 31A) or IgG 2 Activation of both Her47 LHL-LHLF (Figure 31B) showed that only cathepsin S was capable of activating CD47 binding at both pH 7.4 and pH 6.0. In contrast, treatment with cathepsins A, C, G, K, and L showed little or no activation of CD47 binding at pH 7.4 even after 24 hours, but at pH 6.0 a strong activation signal was rapidly generated (Figures 31A, 31B). These findings suggest that the LHL and LHLF linkers may allow accelerated activation in acidified tissues compared to those at pH 7.4 through more rapid activation by MMPs and by pH-selective activation by a range of cathepsins. This minimizes activation of the lower Fab bond at pH 7.4 (the pH of non-diseased tissue where active extracellular MMP and cathepsin levels are low) and maximizes it at pH 6.0 (the pH of diseased tissue where MMP and cathepsin levels are high and cathepsin activity is enhanced), thereby enhancing IgG 2 and Fab 2 The therapeutic index of the protein can be further improved.

[0224] Activation of lower Fab binding to Her2 / CD47+ cells IgG against cells expressing different levels of CD47 and Her2 on the cell surface 2 Her47 LHL-LHL and IgG 2Flow cytometry was performed to examine the binding properties of Her47 LHL-LHLF. Staining with trastuzumab, anti-CD47 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). IgG after activation with MMP12 for 0, 2, 8 and 24 hours 2 Her47 LHL-LHLF (Figure 32A, C) and IgG 2 Staining of both cell types with Her47 LHL-LHL (Figure 32B, D) was also performed. IgG 2 Her47 LHL-LHLF and IgG 2 Both Her47 LHL-LHL showed similar binding profiles to trastuzumab on BT474 cells at 0, 2, and 8 hours, with a slight decrease in binding after 24 hours (Figure 32A,B). In contrast, IgG 2 Her47 LHL-LHLF and IgG 2 Both Her47 LHL-LHL showed a similar low-level binding profile to trastuzumab on MCF7 cells at 0 h activation, but binding was significantly higher at 2, 8, and 24 h activation time points, mimicking the anti-CD47 control (Figure 32C,D). These findings experimentally validated the activation model presented in Figure 2B, where trastuzumab-like binding of Her2 was maintained after activation, and the CD47 binding profile only became highly activating after activation. Importantly, these findings also supported the IgG 2 (And Fab 2 ) proteins may have the beneficial ability to promote the function of the lower Fab binding domain (e.g., CD47) on cells expressing low levels of the upper Fab target (e.g., Her2).

[0225] Analysis of MMP12 activation by SDS-PAGE and mass spectrometry IgG 2 Her47 LHL-LHLF and IgG 2Her47 LHL-LHL protein was treated with MMP12 as described above for 0, 2, 8 and 24 hours. These protein samples were then analyzed by SDS-PAGE (Figure 33). This analysis yielded findings that clearly correlated with the activation ELISA (Figures 15-17, 30) and flow cytometry (Figure 32) observations: at 0 hours, two intact chains were observed, a heavy chain at the 75 kDa marker and a light chain at just below 50 kDa (below 50 kDa) marker. At 2 hours, IgG 2 Her47 LHL-LHLF and IgG 2 For both Her47 LHL-LHL, a new product of approximately 25 kDa was observed, as well as faint bands slightly greater than 25 kDa and approximately 50 kDa (above 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 = approximately 25 kDa), Fc (2 × C domain + 1 n-linked glycosylation = 28-30 kDa), and intact Fd-hinge-Fc (canonical IgG1 heavy chain = 50 kDa), respectively. At 8 and 24 hours, a gradual decrease in intact chains and an increase in fragments approximately 25 kDa smaller than the intact chains were observed. Notably, at the 8 hour time point, the 25 kDa (upper Fab chain) product was predominant in amount, while significant amounts of both the intact heavy chain (75 kDa) and the intact light chain (below 50 kDa) remained (although reduced compared to the 0 hour sample). Thus, this SDS-PAGE analysis (Figure 33) demonstrated that both linkers between the upper and lower Fab were actively cleaved by the MMP12 enzyme, regardless of the LHL or LHLF sequence.

[0226] Fab 2 In the structure, the MMP12 enzyme is IgG 2 To find out exactly where to activate the molecule, IgG 2Mass spectrometry was performed on 0, 2, 8 and 24 hour samples of Her47 LHL-LHL. Peptide samples were generated 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 MMP cleavage was determined by comparing the MS response of peptides derived from the intact MMP cleavage site (SCGPAPE (SEQ ID NO: 110)) with those derived from the cleaved protein (SCGPAP (SEQ ID NO: 111)). This analysis successfully identified peptides mapping to both the uncleaved (SCGPAPE (SEQ ID NO: 110)) and cleaved (SCGPAP (SEQ ID NO: 111)) linkers, demonstrating that in the LHL linker, MMP12 cleaves between the second proline (P) and glutamic acid (E). The relative signal of the uncleaved (SCGPAPE (SEQ ID NO: 110)) peptide decreased stepwise from 0 to 8 and from 8 to 24 hours (Figure 34A), while the signal of the cleaved (SCGPAP (SEQ ID NO: 111)) peptide increased stepwise (Figure 34B).

[0227] Hinge-stabilized "IgG1-DAA" form of IgG 2 Generation and Analysis US Patent No. 8,871,204 B2 teaches protease-resistant IgG1 antibody variants that maintain lower hinge and Fc stability in the presence of MMP enzymes. In these mutant IgG1 antibodies, the sequence E233-L234-L235-G236 (SEQ ID NO: 112) in the hinge is replaced by P233-V234-A235 (G236 deleted); and at least one substitution selected from S324T / 1332E, S267E / H268F / S324T / 1332E, K326A / 1332E / E333A, S239D / K326A / E333A, S267E / I332E and G237X / S239D / 1332E (X is A, D, P, Q or S); amino acid residues are numbered according to EU numbering.

[0228] Fab related to protease-stable IgG1 Fc etc. 2 To investigate the use of the construct, two exemplary constructs were generated (Table 19). These two constructs (Her47 LHLF-LHL IgG1-2hDAA and Her47 LHL-LHLF IgG1-2hDAA) are IgG1-specific IgG1-specific IgG2-specific IgG3-specific IgG4 ... 2 Her47 Fab 2The constructs are arranged as follows (IgG1 contains P233-V234-A235-ΔG236 and S239D / K326A / E333A mutations). Both constructs were readily expressed in transient CHO cell transfections and Protein A purified protein showed product of greater than 80% of the expected molecular weight. Example analytical SEC data for Her47 LHLF-LHL IgG1-2hDAA showed 80% product at 10.30 ml (Figure 35). SDS-PAGE analysis of non-reduced Protein A purified protein and SEC peaks 8.47, 9.03 and 10.30 ml (Figure 36) showed that peaks 8.47 and 9.03 contained high molecular weight aggregates, while peak 10.30 contained the expected size product (approximately 250 kDa). SDS-PAGE analysis of reduced protein A purified protein and SEC peaks 8.47, 9.03 and 10.30 ml (Figure 37) showed that all peaks 8.47, 9.03 and 10.30 ml contained the expected size heavy and light chain products (approximately 80 and 50 kDa, respectively). The intact monomer (250 kDa) product of Her47 LHL-LHLF IgG1-2hDAA was purified by SEC and subjected to enzymatic digestion at pH 7.4 using human MMP12 for 2, 4, 8 and 24 hours of incubation over time, with an additional 24 hour incubation in buffer without enzyme as a negative control (0, 2, 4, 8, 24 hours of incubation). In ELISA analysis, all samples showed strong binding signals to human Her2, but no measurable binding to the control protein mouse EpCAM (Figure 38A). In the CD47 binding ELISA, the binding signal increased after 2, 4, 8 and 24 hours of incubation at 37°C in the presence or absence of MMP12 enzyme, but no signal above background was observed at 0, 2, 4, 8 or 24 hours without MMP12 (Figure 38B).

[0229] Analysis by SDS-PAGE of 0 hr (undigested) and 2, 8, and 24 hr MMP12 digested samples confirmed that the purified protein contained heavy and light chains of the expected sizes. MMP12 digestion for 2, 8, and 24 hr produced a 25 kD band (Figure 38C), indicating linker peptide cleavage, whereas damage in the heavy chain was consistent with that of the IgG 2 Her47 LHL-LHL or IgG 2 It was confirmed that the amount of Fc-related protein was clearly less than that of Her47 LHL-LHLF (FIG. 33), indicating that the "2hDAA" mutation indeed stabilizes the Fc region and resists MMP12 digestion.

[0230] Alternative enzyme-activated IgG 2 Generation and Analysis Since the LHL and LHLF linkers were shown above to be susceptible to cleavage by multiple MMPs and cathepsin enzymes, the six Fab 2 Construct type IgG 2 The constructs were tested in a variety of formats (Table 20). These constructs used a range of linker designs (Table 21) that were designed to be susceptible to cleavage by several classes of enzymes associated with upregulated activity in solid tumors and highly inflamed tissues, including enterokinase (EK), thrombin (Thr), tPA, granzyme B (GrB), uPA, and ADAMTS-5 (A5). All six constructs were readily expressed in CHO cells and purified by ProA chromatography.

[0231] All purified proteins from clones Her47-LHL-LHL-EK, Her47-LHL-LHL-Thr, Her47-LHL-LHL-tPA, Her47-LHL-LHL-uPA, Her47-LHL-LHL-GrB and Her47-LHL-LHL-A5 were tested in a titration ELISA against human Her2 and CD47 targets (Figures 39A, 39C, 39E, 39G, 39I, 39K). In all cases, the proteins showed low / background binding to CD47 (white bars) at all concentrations tested, but concentration-dependent binding to Her2 (grey bars). Each protein was then subjected to a time-dependent enzymatic digestion with MMP12 and ELISA for binding to Her2 and CD47 targets (Figures 39B, 39D, 39F, 39H, 39J, 39L). These findings demonstrated that all proteins maintained Her2 binding over time with enzyme activation and showed increased activation of CD47 binding. The functional activity of clones Her47-LHL-LHL-EK, Her47-LHL-LHL-Thr, Her47-LHL-LHL-tPA, Her47-LHL-LHL-uPA, Her47-LHL-LHL-GrB and Her47-LHL-LHL-A5 demonstrated that linker peptide sequences with a wide variety of protease recognition sites in the Fab2 construct can be utilized to tailor activation to specific applications. For example, in this case, retention of MMP activation potential in the LHL linker can be tailored to the environment in which MMP and / or cathepsin enzymes are active, in combination with any of six different disease-associated enzyme cleavage motifs in the associated linker, resulting in increased activation potential when enterokinase, thrombin, tPA, granzyme B, uPA, ADAMTS-5 or other protease enzymes are involved in the disease state.

[0232] In vivo tolerance to repeated administration of Her47 molecules in NOD-SCID mice In an in vivo repeated dose tolerability study, IgG 2 Her47 LHL-LHL, IgG 2 Her47 LHL-LHLF, Fab 2Her47 LHL-LHL and Fab 2 Her47 LHL-LHLF were each administered (intravenously) four times to NOD-SCID mice. IgG 2 The protein was administered at 14 mg / kg on day 0 and 7 mg / kg on days 5, 10 and 15. Fab 2 Proteins were administered at 8 mg / kg on day 0 and 4 mg / kg on days 5, 10 and 15. All proteins were fully tolerated, with no clinical signs of toxicity or weight loss in any individual animal (Figure 40). These findings support the previously outlined efficacy of Her47 Fab in Tg32 mice at a single dose. 2 This confirms and elaborates on the findings of tolerability to the base molecule. In the Tg32 mouse study, even a single dose of A-D5 CD47 IgG was not tolerated at 10 mg / kg. In this NOD-SCID mouse study, IgG 2 Her47 LHL-LHL, IgG 2 Her47 LHL-LHLF, Fab 2 Her47 LHL-LHL and Fab 2 All four doses of Her47 LHL-LHLF were fully tolerated. This finding confirmed that the CD47-binding domain of the Her47 molecule was not activated during circulation, even after repeated dosing, whereas significant activation would have induced the CD47-promoted toxic signal observed with A-D5 IgG1.

[0233] Additional double-stranded Her2CD3 Fab 2 Generation and analysis of "one-arm" constructs Additionally, novel sequences were examined for their ability to generate Her2CD3 constructs and improve product homogeneity and activity (Table 22). All Her2CD3 constructs were expressed in CHO cells, purified from the supernatant by ProA, and analyzed by SEC. These analyses were performed using Fab 2 Her23 LHL-LHL-S and Fab 2 Both Her23 LHLF-LHL-S (Fig. 41A,B) Fab fragments showed a high ratio of high and low molecular weight products.2 Her23 LHL-LHL and Fab 2 These findings demonstrate that the primary product is more homogeneous than both Her23 LHL-LHLF (Figures 41C, 41D). These findings support the conclusion that the construct Fab is a Fab fragment with chains [VL-CL-linker-VH-CH-Fc] and [VH-CH-linker-VL-CL-Fc]. 2 Her23 LHL-LHL-S and Fab 2 This suggests that the homogeneity was improved by swapping the orientation of the upper Fab of Her23 LHLF-LHL-S, which is a Fab with both chains being [VL-CL-linker-VL-CL-Fc] and [VH-CH-linker-VH-CH-Fc]. 2 Her23 LHL-LHL and Fab 2 In contrast to Her23 LHL-LHLF.

[0234] Next, the control protein Fab 2 mEpCam3 LHLF-LHL-S and Fab 2 The clone mEpCam3 LHL-LHL-S (top Fab contains the V domain of the anti-mouse EpCAM antibody G8.8) was also designed and expressed (Table 22). 2 Her23 LHL-LHL-S, Fab 2 Her23 LHLF-LHL-S, Fab 2 mEpCam3 LHLF-LHL-S and Fab 2 The desired MW product of mEpCam3 LHL-LHL-S was isolated by preparative SEC and analyzed (according to the manufacturer's instructions) in Promega's Jurkat cell-based CD3 ligation reporter cell bioassay using MCF-7 or BT-474 as human Her2+ target cells. Primary analysis using MCF-7 (which has very low Her2 expression) as target cells showed that both the positive control Her2-CD3 BITE protein and OKT3 IgG1 generated a strong positive concentration-dependent CD3 activation signal. Clonal Fab treated with MMP12 for 2 hours 2 mEpCam3 LHLF-LHL-S and Fab 2No signal was generated at any concentration with mEpCam3 LHL-LHL-S (Figure 42A, B). In contrast, clone Fab 2 Her23 LHLF-LHL-S (Figure 42A) and Fab 2 Both Her23 LHL-LHL-S (Figure 42B) had no measurable signal at 0 hours, but showed a gradual increase in signal at 2, 8 and 24 hours in a concentration range similar to Her2-CD3 BITE, and in both cases a higher maximum signal than that reached with the positive control.

[0235] In a secondary assay using Her2-highly expressing BT-474 cells as target cells, all of the above test samples and controls were evaluated at 0.1 μg / ml (Figure 43A-C). Both the positive control proteins Her2-CD3 BITE and OKT3 IgG1 induced strong CD3 activation signaling, whereas the negative control Fab 2 There was no signaling with mEpCam3 LHLF-LHL-S and trastuzumab. Clone Fab 2 Her23 LHLF-LHL-S (Figure 43B) and Fab 2 Both Her23 LHL-LHL-S (Figure 43C) showed no measurable signal at 0 h, but gradually increased signal at 2 h, reaching a maximum at 8 h. Importantly, at 24 h, the signal was significantly reduced compared to 8 h. This finding suggests that stepwise enzymatic activity that could cleave both linkers (Figure 33) could ultimately result in the separation of the upper and lower Fabs. This could be a beneficial property for CD3 targeting molecules, as ideally such molecules would be preferentially active in the highly proteolytic tumor environment, but would be gradually inactivated there, minimizing the risk of molecules leaking out in an active form and entering healthy tissues.

[0236] Her2-CD47 IgG 2 and Fab 2 In vitro measurement of protein molecular stability Historically, engineered antibody formats have often faced structural heterogeneity, causing manufacturing problems. IgG 2 Her47 LHL-LHL and Fab 2 To examine the stability of the Her47 LHL-LHL protein, a series of in vitro measurements were performed.

[0237] Forced oxidation - Oxidation of exposed amino acid residues such as tryptophan and methionine is a common degradation pathway for mAbs and affects their biological activity. In this study, forced oxidation with 0.5% H2O2 in PBS for 2 h at room temperature was performed on IgG 2 Her47 LHL-LHL and Fab 2 The Her47 LHL-LHL protein was subjected to oxidation. Since oxidation may alter the overall hydrophobicity of the antibody by increasing the polarity of the oxidized form or through structural changes, potential changes caused by forced oxidation were analyzed by reversed-phase (RP) and size-exclusion (SEC) chromatography. SEC analysis revealed that the ratio of monomeric species was unchanged in both test samples (IgG 2 Her47 LHL-LHL and Fab 2 Both Her47 LHL-LHL showed 99.5 and 97.4% monomer species before and after oxidation, respectively. RP analysis of intact antibodies showed that for intact (non-reduced) antibodies after forced oxidation with 0.5% H2O2, IgG 2 Her47 LHL-LHL retention time was 0.5 min, Fab 2 The retention time of Her47 LHL-LHL was observed to be shorter by 0.4 min. Subunit RP analysis showed that IgG after forced oxidation 2 For Her47 LHL-LHL, a 0.5 min decrease in the retention time of the heavy chain and no shift in the retention time of the light chain were observed. 2Her47 LHL-LHL showed a distinct profile in RP analysis, with three peaks observed, representing the light chain, the heavy chain, and the truncated hinge Fc of the stem. After oxidation, a shift of 0.4-0.5 min was observed for the heavy chain and the truncated hinge Fc of the stem, but not for the light chain. The retention time shift was greater after H2O2 treatment than for reduced IgG. 2 Her47 LHL-LHL and Fab 2 This was observed in both Her47 LHL-LHL samples, suggesting minor oxidation of exposed amino acids (restricted to the Fc region of the protein).

[0238] Charge variant analysis - Charge heterogeneity analysis is important for the characterization of monoclonal antibodies since it provides important information about the quality and stability of the product. Heterogeneity can occur due to enzymatic post-translational modifications (glycosylation, lysine cleavage) or chemical modifications during purification and storage (oxidation or deamidation). Charge variant profiling of the provided specimens was performed by commercial charge variant assays. IgG 2 Her47 LHL-LHL (Figure 44A) and Fab 2 Charge variant profiles of both Her47 LHL-LHL (Figure 44B) showed homogeneous profiles, including one major isoform (50-57% of total), a major acidic isoform (40-48% of total) and one minor basic isoform (approximately 3%).

[0239] Retention in HIC - Overall hydrophobicity is an indicator of a protein's tendency to self-associate, which can be a significant risk factor for aggregation and viscosity during bioprocessing. Proteins have hydrophobic "patches" on their surface, which are generated by the presence of hydrophobic or non-polar amino acid side chains. Depending on their number, size and distribution, the resulting surface hydrophobicity is a unique property for each protein. HIC separates proteins based on differences in their surface hydrophobicity, taking advantage of the reversible binding between proteins and the hydrophobic surface of the HIC resin. IgG 2 Her47 LHL-LHL and Fab 2Her47 LHL-LHL showed HIC column retention times of 5.4 and 5.0 min, respectively. These values ​​indicate a lower range of hydrophobicity compared to those obtained with 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 the aggregation tendency and stability are similar to those of IgG. 2 Her47 LHL-LHL and Fab 2 These results suggest that both Her2-binding domains of Her47 LHL-LHL are induced, similar to the value of anti-Her2 trastuzumab (5.4 min retention).

[0240] Freeze-thaw stability analysis - Protein instability during freeze-thaw procedures is an indicator of failure in manufacturing and bioprocessing, as increased protein aggregation or fragmentation poses the risk of reduced product quality. 2 To assess this risk for proteins containing structures, IgG 2 Her47 LHL-LHL and Fab 2 The Her47 LHL-LHL protein was subjected to five freeze-thaw cycles with subsequent SEC analysis performed after each cycle. 2 Her47 LHL-LHL (Figure 45A) or Fab 2 None of the Her47 LHL-LHL (FIG. 45B) proteins demonstrated any change in monodispersity over the five freezes (no aggregation or damage products were observed).

[0241] Taken together, these findings suggest that IgG 2 Her47 LHL-LHL and Fab 2 Both Her47 LHL-LHL proteins were suggested to have low aggregation risk, low hydrophobicity, low charge heterogeneity and low propensity towards oxidation.

[0242] BIACORE® Analysis of Affinity of IgG Variants to Human Fc Receptors Antibodies targeting receptors on diseased cells must bind to Fcγ receptors in order to mediate ADCC and ADCP activity. To determine whether these binding functions are maintained with Fab2-based constructs, IgG 2 Her47 LHL-LHL and Fab 2 The Her47 LHL-LHL protein was tested for binding affinity to all human and mouse Fc receptors via surface plasmon resonance analysis. These analyses demonstrated that both isotype control human IgG1 and IgG4 exhibited the expected strong and weak binding affinity (respectively) to all human Fcγ receptors, including both high and low affinity variants of FcγRIIA and FcγRIIIA (Table 23). Similarly, isotype control mouse IgG2 and IgG1 exhibited the expected strong and weak binding affinity (respectively) to mouse FcγRI, FcγRIII and FcγRIV receptors. IgG 2 Her47 LHL-LHL and Fab 2 In the case of Her47 LHL-LHL, binding to each of the human and mouse Fc receptors tested was very similar to that observed with the isotype control human IgG1. These data support the conclusion that IgG1 Fc-based 2 and Fab 2 Both of the proteins suggest that they are capable of binding to Fc receptors upon binding on the surface of diseased cells in both humans and mice.

[0243] Further protein construct design Further protein constructs were considered (Figure 46). The constructs may include: 1. Only a constant domain in the "upper Fab" position. This means that the activity of the "lower Fab" is blocked from binding to its target, but can become active in the appropriate proteolytic environment. 2. A dummy "non-binding" variable domain in the "upper Fab". These dummy variable domains will not be proven to bind to any known target in the body. Thus, only the "lower Fab" shows potential drug target binding capability, and only after proteolytic activation of one of the linker domains. 3. The "upper Fab" is replaced by a "diabody" structure containing four variable domains. This diabody structure may or may not contain a disulfide bond as found in "DART" proteins. The diabody structure may facilitate binding of two copies of the same target or two separate targets, so that the activity of the "lower Fab" is blocked from binding to its target until it is activated in the appropriate proteolytic environment. In any of the constructs envisaged herein or shown above, the Fab 2 The constructs may be free or fused to another functionalized construct, e.g., small domains or peptides that extend half-life, such as Fc fragments, albumin binding moieties, etc. They may also be chemically conjugated to small molecules, peptides or other proteins that mediate additional biological functions.

[0244] Cell proliferation analysis of Her2CD47 protein using Her2-high BT-474 cells IgG 2 Her47 and Fab 2 Since the Her2-binding domain of the upper Fab of the Her47 protein is capable of mediating inhibition of the kinase activity of this receptor, a cell proliferation assay was performed. In this assay, BT-474 cells, a cell line known for its sensitive response to Her2 inhibition, were used. Trastuzumab, isotype control IgG1, IgG 2 Her47 LHL-LHL (Figure 47A) and Fab 2Her47 LHL-LHL (Figure 47B) was administered to BT-474 cells over a 72 hour incubation period and cell proliferation was measured. Data was presented as percent inhibition of cell proliferation (Figure 47). These analyses showed that trastuzumab showed a strong concentration-dependent inhibition of BT-474 cell proliferation, whereas IgG 2 Her47 LHL-LHL (Figure 47A) showed slightly lower potency and Fab 2 Her47 LHL-LHL (FIG. 47B) also demonstrated low potency (reflecting monovalent binding capacity).

[0245] In vivo efficacy analysis of Her47 molecule in tumor xenograft-bearing NOD-SCID mice (KYSE-410 model) In vivo repeated dose efficacy studies, trastuzumab, IgG 2 Her47 LHL-LHL, IgG 2 Her47 LHL-LHLF, Fab 2 Her47 LHL-LHL and Fab 2 Her47 LHL-LHLF (a Fab containing human IgG1 Fc as in Fig. 3B) 2 The 125 mm construct was administered four times (intravenously, on days 0, 5, 10, and 15) to NOD-SCID mice bearing tumors generated by subcutaneous inoculation of the Her2-expressing esophageal cancer cell line KYSE-410. 2 Treatment was started after the tumors reached > 100 μg / kg. 2 The protein was administered at 14 mg / kg on day 0 and 7 mg / kg on days 5, 10 and 15. Fab 2 Protein was administered at 8 mg / kg on day 0 and 4 mg / kg on days 5, 10 and 15. Trastuzumab was administered at 8 mg / kg on day 0 and 4 mg / kg on days 5, 10 and 15. Tumor volumes were measured by caliper measurement.

[0246] After three doses, on the 11th day, trastuzumab (Figure 48A), IgG 2 Her47 LHL-LHLF (Figure 48B), IgG 2Her47 LHL-LHL (Figure 48C) and Fab 2 All Her47 LHL-LHLFs (FIG. 48D) showed a significant reduction in tumor growth compared to vehicle (2-way ANOVA: p=0.005, 0.005, 0.019, and 0.003, respectively). In contrast, Fab 2 Her47 LHL-LHL (Figure 48E) showed no significant reduction in tumor growth by day 11 (2-way ANOVA: p=0.66). Importantly, Fab 2 Her47 LHL-LHL and Fab 2 The sequences of Her47 LHL-LHLF are identical (except for two point mutations in the LHLF linker that accelerate and enhance protease sensitivity compared to the LHL linker, as shown above), resulting in a striking difference in potency (Figure 48F). Furthermore, the data in Figure 47 show that the one-arm Fab 2 In the structure, Fab 2 Her47 LHL-LHL and Fab 2 These results indicate that the Her2-promoted inhibition of cell proliferation was weaker than that observed with trastuzumab, which contains the same Her2-binding VH and VL domain sequences as those found in both Her47 LHL-LHLF. The results are as follows: 1. Trastuzumab (Figure 48A) and Fab 2 The nearly equivalent efficacy at day 11 observed in both Her47 LHL-LHLF (Figure 48D) was due to the 2 Her47 cannot be promoted by high Her2 kinase activity in LHL-LHLF. 2. Therefore, Fab 2 The high potency of Her47 LHL-LHLF is likely driven by protease activation in the KYSE-410 tumor microenvironment, leading to CD47 inhibition and innate immune engagement.

[0247] Thus, the findings outlined above suggest that in aggregates, Fab 2The construct is demonstrated to allow efficient elimination of the "lower Fab" activity by the action of both the CD47 and CD3 binding domains as exemplified. The binding capacity of the "upper Fab" domain is fully maintained, whereas significant activity of the lower Fab domain is only observed after activation by proteases such as MMPs and cathepsins, which are associated with high activity in diseased tissues such as tumors and fibrotic tissues. 2 Modulation of the linker sequence in the construct allows tailoring of the lower Fab activation to maximize it in the disease microenvironment and avoid both peripheral sink problems and toxicity, as exemplified by measuring the potency of the Her47 molecule in vitro and in vivo.

[0248] In vivo pharmacokinetics of Her47 molecule in NOD-SCID mice In an in vivo repeated dose PK study, IgG 2 Her47 LHL-LHL, IgG 2 Her47 LHL-LHLF, Fab 2 Her47 LHL-LHL and Fab 2 Her47 LHL-LHLF were each administered once (intravenously) to NOD-SCID mice. IgG 2 Protein was administered at 14 mg / kg, and Fab 2 Protein is administered at 8 mg / kg. Blood samples are taken at 15 min, 30 min, 1 hr, 3 hr, 6 hr and 24 hr and serum antibody levels are measured using an anti-human IgG1 ELISA.

[0249] In vivo efficacy analysis of Her47 molecule in tumor xenograft-bearing NOD-SCID mice (SKOV-3, JIMT-1 and NUGC-4 models) In an in vivo repeated dose efficacy study, IgG 2 Her47 LHL-LHL, IgG 2 Her47 LHL-LHLF, Fab 2 Her47 LHL-LHL and Fab 2Her47 LHL-LHLF is administered four times (intravenously) to NOD-SCID mice bearing tumors generated by subcutaneous inoculation of the cell lines SKOV-3, JIMT-1 and NUGC-4, respectively. IgG 2 The protein is administered at 14 mg / kg on day 0 and 7 mg / kg on days 5, 10 and 15. Fab 2 Protein is administered at 8 mg / kg on day 0 and 4 mg / kg on days 5, 10 and 15. Trastuzumab is administered at 8 mg / kg on day 0 and 4 mg / kg on days 5, 10 and 15. Tumor volumes are measured by caliper measurement.

[0250] In vivo analysis of tolerability and pharmacokinetics in cynomolgus monkeys IgG in the context of CD47 or CD3, such as the lower Fab domain 2 and / or Fab 2 Several exemplary molecules will be studied in cynomolgus monkeys to determine the tolerability and pharmacokinetics of IgG. 2 and / or Fab 2 may be administered (intravenously) once, twice or three times, at concentrations of 2 mg / kg or greater, respectively. Blood samples are taken from each animal according to a bleeding schedule. Serum antibody concentration analyses are performed to calculate PK and assess risk of TMDD. A full hematology panel is also run on each successive day after dosing to sample the broader effects of the administered protein. These analyses measure reticulocytes, red blood cells (RBCs), hemoglobin, mean hemoglobin concentration in red blood cells (MCHC), mean corpuscular volume (MCV), white blood cells, monocytes, lymphocytes, basophils, eosinophils and / or neutrophil levels.

[0251] Biosensor measurements of target interaction Fab to both Her2 and CD47 (or CD3) 2To examine the effect of binding affinity activation, a biosensor assay is performed that can sample the binding of both Her2 and CD47 (or CD3) on the same chip surface, such as the Dynamic Biosensors device. In this assay, a control antibody and an IgG 2 Or Fab 2 Proteins (undigested or activated by MMP or cathepsin enzymes for 2, 4, 8 or 24 hours) are applied to a sensor chip surface specifically labeled with purified Her2 and CD47 (or CD3) ectodomain proteins, either separately or together at different densities. The affinity for Her2 and CD47 (or CD3) is measured to confirm the effect of multivalent interactions on the functional affinity for both targets separately and on the same surface.

[0252] While the present invention has been described in terms of preferred or exemplary embodiments, those skilled in the art will recognize that various modifications and variations thereon may be made without departing from the spirit and scope of the invention, and such modifications are expressly contemplated herein. No limitations with respect to the specific embodiments disclosed herein and set forth in the appended claims are intended, nor should any be inferred.

[0253] No limitations with respect to the specific embodiments disclosed herein and described in the appended claims are intended, and no inference should be made. All documents, or portions of documents, cited herein, including, but not limited to, patents, patent applications, articles, books, and papers, are expressly incorporated herein by reference in their entirety for any purpose. In the event that one or more of the incorporated documents or portions of documents defines a term in contradiction to the definition of that term in this application, the definition set forth in this application shall prevail. However, the statements of any references, articles, publications, patents, patent publications, and patent applications cited herein are not, and should not be taken as, an acknowledgment or any suggestion that they constitute valid prior art or form part of the general knowledge in any country in the world.

[0254] Numbered Embodiments Without limiting the scope of the appended claims, the present disclosure describes the following numbered embodiments.

[0255] 1. A protein comprising a first portion and a second portion, and a peptide linker between the first portion and the second portion, the peptide linker comprises an amino acid sequence derived from a human immunoglobulin hinge region, or an amino acid sequence having from 1 to about 7 amino acid substitutions compared to a human immunoglobulin hinge region, the peptide linker is cleavable by a protease expressed in the diseased tissue; the second portion is capable of specifically binding to a molecule expressed in the diseased tissue; and When the peptide linker is not cleaved, the binding of the second portion to the molecule expressed in the diseased tissue is reduced or inhibited.

[0256] 2. The protein of embodiment 1, wherein the peptide linker is about 5 to about 15 amino acids in length.

[0257] 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.

[0258] 4. A protein according to any one of embodiments 1 to 3, wherein the protease is a human matrix metalloprotease (MMP), human cathepsin, human enterokinase, human thrombin, human tPA, human granzyme B, human uPA or human ADAMTS-5.

[0259] 5. A protein described in any one of embodiments 1 to 4, wherein the peptide linker comprises a human MMP cleavage site, a human cathepsin cleavage site, a human enterokinase cleavage site, a human thrombin cleavage site, a human tPA cleavage site, a human granzyme B cleavage site, a human uPA cleavage site or a human ADAMTS-5 cleavage site.

[0260] 6. A protein described in any one of embodiments 1 to 5, wherein the human MMP is MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, MMP-12, MMP-13 or MMP14.

[0261] 7. A protein described in any one of embodiments 1 to 6, wherein the level or activity of the human MMP in the diseased tissue is higher than the level or activity of the human MMP in non-diseased tissue.

[0262] 8. A protein according to any one of embodiments 1 to 5, wherein the human cathepsin is cathepsin A, cathepsin C, cathepsin D, cathepsin G, cathepsin L or cathepsin K.

[0263] 9. A protein described in any one of embodiments 1 to 5 and 8, wherein the level or activity of the human cathepsin in the diseased tissue is higher than the level or activity of the human cathepsin in non-diseased tissue.

[0264] 10. The protein of any one of embodiments 1 to 9, wherein the first portion comprises an antibody, an antigen-binding portion of an antibody, or a receptor ectodomain.

[0265] 11. The protein of embodiment 10, wherein the first portion is a Fab, a single-chain Fab, a VH domain, a VL domain, an immunoglobulin novel antigen receptor (IgNAR), a single-chain variable fragment (scFv), a diabody, or a T-cell receptor domain.

[0266] 12. The protein of any one of embodiments 1 to 11, wherein the first portion specifically binds to a molecule expressed in diseased tissue.

[0267] 13. A protein described in any one of embodiments 1 to 12, wherein the first portion is capable of specifically binding to a first molecule expressed in diseased tissue, and the second portion is capable of specifically binding to a second molecule expressed in diseased tissue, and the first molecule expressed in diseased tissue and the second molecule expressed in diseased tissue are different molecules.

[0268] 14. The protein of embodiment 13, wherein the first molecule expressed in the diseased tissue and the second molecule expressed in the diseased tissue are expressed in the same cell.

[0269] 15. The protein of embodiment 13, wherein the first molecule expressed in the diseased tissue and the second molecule expressed in the diseased tissue are expressed in different cells.

[0270] 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 are expressed on the surface of a cell.

[0271] 17. The protein described in embodiment 13, wherein the first molecule expressed in the diseased tissue and / or the second molecule expressed in the diseased tissue are soluble molecules.

[0272] 18. The protein of any one of embodiments 1 to 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.

[0273] 19. The protein of any one of embodiments 1 to 17, wherein the second portion specifically binds to a molecule expressed in a human immune cell.

[0274] 20. The protein of embodiment 19, wherein the molecule expressed on human immune cells is human CD3, human CD16A, human CD16B, human CD28, human CD89, human CTLA4, human NKG2D, human SIRPα, human SIRPγ, human PD1, human Lag3, human 4-1BB, human OX40 or human GITR.

[0275] 21. The protein of any one of embodiments 1 to 20, wherein the first portion comprises a heavy chain variable (VH) region and a light chain variable (VL) region.

[0276] 22. The protein of any one of embodiments 1 to 21, wherein the first portion comprises an immunoglobulin constant region or a portion of an immunoglobulin constant region.

[0277] 23. The protein of embodiment 22, wherein the immunoglobulin constant region is IgG, IgE, IgM, IgD, IgA or IgY.

[0278] 24. The protein of embodiment 22, wherein the immunoglobulin constant region is IgG1, IgG2, IgG3, IgG4, IgA1 or IgA2.

[0279] 25. The protein of embodiment 22, wherein the immunoglobulin constant region is immunologically inactive.

[0280] 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 the amino acid substitution S228P, a wild-type human IgG1 constant region, a human IgG1 constant region comprising the amino acid substitutions L234A and L235A, a human IgG1 constant region comprising the amino acid substitutions L234A, L235A and G237A, a human IgG1 constant region comprising the amino acid substitutions L234A, L235A, G237A and P331S, or a wild-type human IgG2 constant region.

[0281] 27. The protein of any one of embodiments 1 to 26, wherein the second portion comprises an antibody, an antigen-binding portion of an antibody, or a receptor ectodomain.

[0282] 28. The protein of embodiment 27, wherein the second portion is a Fab, a single-chain Fab, a VH domain, a VL domain, an immunoglobulin novel antigen receptor (IgNAR), a single-chain variable fragment (scFv), or a T-cell receptor domain.

[0283] 29. The protein of any one of embodiments 1 to 28, wherein the second portion specifically binds to human CD47.

[0284] 30. The protein of any one of embodiments 1-28, wherein the second portion specifically binds to human CD3 or human PD-L1.

[0285] 31. The protein of any one of embodiments 1 to 30, wherein the second portion comprises a heavy chain variable (VH) region and a light chain variable (VL) region.

[0286] 32. The protein of any one of embodiments 1 to 31, wherein the second portion comprises an immunoglobulin constant region or a portion of an immunoglobulin constant region.

[0287] 33. The protein of embodiment 32, wherein the immunoglobulin constant region is IgG, IgE, IgM, IgD, IgA or IgY.

[0288] 34. The protein of embodiment 32, wherein the immunoglobulin constant region is IgG1, IgG2, IgG3, IgG4, IgA1 or IgA2.

[0289] 35. The protein of embodiment 32, wherein the immunoglobulin constant region is immunologically inactive.

[0290] 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 the amino acid substitution S228P, a wild-type human IgG1 constant region, a human IgG1 constant region comprising the amino acid substitutions L234A and L235A, a human IgG1 constant region comprising the amino acid substitutions L234A, L235A and G237A, a human IgG1 constant region comprising the amino acid substitutions L234A, L235A, G237A and P331S, or a wild-type human IgG2 constant region.

[0291] 37. The protein of embodiment 1, wherein the protein has one immune effector function, or two, three or more immune effector functions.

[0292] 38. The protein of embodiment 37, wherein the immune effector function is ADCC, CDC or ADCP.

[0293] 39. A protein described in any one of embodiments 1 to 38, wherein the first portion prevents or reduces specific binding of the second portion to the molecule expressed in the diseased tissue.

[0294] 40. A protein described in any one of embodiments 1 to 39, wherein the peptide linker is cleaved near or within the diseased tissue.

[0295] 41. A protein described in any one of embodiments 1 to 40, wherein the peptide linker is cleaved near or within the diseased tissue, the first portion dissociates from the second portion near or within the diseased tissue, and the second portion specifically binds to the molecule expressed in the diseased tissue near or within the diseased tissue.

[0296] 42. The protein of any one of embodiments 1 to 41, wherein the diseased tissue is a tumor or an inflamed tissue.

[0297] 43. The protein of embodiment 1, wherein the first portion specifically binds to human cMET and the second portion specifically binds to human CD47, and 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.

[0298] 44. The protein of embodiment 1, wherein the first portion specifically binds to human HER2, the second portion specifically binds to human CD3, and 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.

[0299] 45. The protein of embodiment 1, wherein the first portion specifically binds to human HER2, the second portion specifically binds to human CD47, and 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.

[0300] 46. ​​The protein of embodiment 1, wherein the first portion specifically binds to human cMET and the second portion specifically binds to human CD47, and 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.

[0301] 47. The protein of embodiment 1, wherein the first portion specifically binds to human Her2, the second portion specifically binds to human CD3, and 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.

[0302] 48. The protein of embodiment 1, wherein the first portion specifically binds to human Her2, the second portion specifically binds to human CD3, and 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.

[0303] 49. The first portion specifically binds to human Her2, the second portion specifically binds to human CD47, and the protein comprises a first polypeptide chain and a second polypeptide chain, wherein: (a) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 42 and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 43; (b) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 44 and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 45; or (c) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 46 and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 47; (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; (e) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:50 and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:51; (f) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:52 and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:53; (g) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:54 and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:55; (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; (i) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 90 and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 91; (j) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 44 and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 92; (k) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 44 and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 93; (l) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 44 and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 94; (m) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 44 and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 95; (n) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 44 and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 96; (o) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 44, and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 97.

[0304] 50. The protein of embodiment 1, wherein the first portion specifically binds to human Her2, the second portion specifically binds to human CD3, and 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.

[0305] 51. The protein of embodiment 1, wherein the first portion specifically binds to human cMET, the second portion specifically binds to human cMET, and 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.

[0306] 52. The first portion specifically binds to human Her2, the second portion specifically binds to human CD3, and the protein comprises a first polypeptide chain and a second polypeptide chain, wherein: (a) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 98 and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 99; (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; (c) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 102 and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 103; (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.

[0307] 53. An immunoconjugate comprising a protein according to any one of embodiments 1 to 52 linked to a therapeutic agent.

[0308] 54. The immunoconjugate of embodiment 53, wherein the therapeutic agent is a cytotoxin, a radioisotope, a chemotherapeutic agent, an immunomodulatory agent, an antiangiogenic agent, an antiproliferative agent, a proapoptotic agent, a cytostatic enzyme, a cytolytic enzyme, a therapeutic nucleic acid, an antiangiogenic agent, an antiproliferative agent, or a proapoptotic agent.

[0309] 55. A pharmaceutical composition comprising a protein according to any one of embodiments 1 to 52, or an immunoconjugate according to embodiment 53 or 54, and a pharma- ceutically acceptable carrier, diluent or excipient.

[0310] 56. A nucleic acid molecule encoding a protein or a part of a protein described in any one of embodiments 1 to 52.

[0311] 57. A nucleic acid molecule encoding the first polypeptide chain, the second polypeptide chain, or both the first polypeptide chain and the second polypeptide chain of the protein described in any one of embodiments 43 to 52.

[0312] 58. An expression vector comprising the nucleic acid molecule of embodiment 56 or 57.

[0313] 59. A recombinant host cell comprising a nucleic acid molecule described in embodiment 56 or 57, or an expression vector described in embodiment 58.

[0314] 60. A method for producing a protein, comprising: Culturing a recombinant hos...

Claims

1. A protein comprising a first portion and a second portion, and a peptide linker between the first portion and the second portion, the first portion comprises an antibody, an antigen-binding portion of an antibody, or a receptor ectodomain, the first portion being capable of specifically binding to a molecule expressed in the diseased tissue; the peptide linker comprises an amino acid sequence derived from a human immunoglobulin hinge region or an amino acid sequence having 1 to 7 amino acid substitutions compared to a human immunoglobulin hinge region; the peptide linker is cleavable by a protease expressed in the diseased tissue; the second portion comprises an antibody, an antigen-binding portion of an antibody, or a receptor ectodomain, the second portion being capable of specifically binding to a molecule expressed in the diseased tissue; if the peptide linker is not cleaved, the binding of the second portion to the molecule expressed in the diseased tissue is reduced or inhibited; and when the first portion contacts the diseased tissue, cleavage of the peptide linker unmasks the second portion and allows binding of the second portion to the molecule expressed in the diseased tissue; wherein the peptide linker comprises 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.

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

3. 3. The protein of claim 1 or 2, wherein the protease is a human matrix metalloprotease (MMP), a human cathepsin, a human enterokinase, a human thrombin, a human tPA, a human granzyme B, a human uPA, or a human ADAMTS-5.

4. The protein according to any one of claims 1 to 3, wherein the peptide linker comprises a human MMP cleavage site, a human cathepsin cleavage site, a human enterokinase cleavage site, a human thrombin cleavage site, a human tPA cleavage site, a human granzyme B cleavage site, a human uPA cleavage site, or a human ADAMTS-5 cleavage site.

5. The protein according to claim 3 or 4, wherein the human MMP is MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, MMP-12, MMP-13 or MMP14.

6. 5. The protein of claim 3 or 4, wherein the human cathepsin is cathepsin A, cathepsin C, cathepsin D, cathepsin G, cathepsin L or cathepsin K.

7. 7. The protein of any one of claims 1 to 6, wherein the first portion is a Fab, a single chain Fab, a VH domain, a VL domain, an immunoglobulin novel antigen receptor (IgNAR), a single chain variable fragment (scFv), a diabody, or a T cell receptor domain.

8. The protein according to any one of claims 1 to 7, wherein the first portion is capable of specifically binding to a first molecule expressed in diseased tissue, the second portion is capable of specifically binding to a second molecule expressed in diseased tissue, and the first molecule expressed in diseased tissue and the second molecule expressed in diseased tissue are different molecules.

9. The protein of claim 8 , wherein the first molecule expressed in the diseased tissue and the second molecule expressed in the diseased tissue are expressed in the same cell.

10. The protein of claim 8 , wherein the first molecule expressed in the diseased tissue and the second molecule expressed in the diseased tissue are expressed in different cells.

11. The protein according to claim 8 , wherein the first molecule expressed in the diseased tissue and / or the second molecule expressed in the diseased tissue are expressed on the surface of a cell.

12. The protein of claim 8 , wherein the first molecule expressed in the diseased tissue and / or the second molecule expressed in the diseased tissue is a soluble molecule.

13. 13. The protein of any one of claims 1 to 12, 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.

14. The protein according to any one of claims 1 to 13, wherein the second portion is capable of specifically binding to a molecule expressed on a human immune cell.

15. The protein of claim 14, wherein the molecule expressed on a human immune cell is human CD3, human CD16A, human CD16B, human CD28, human CD89, human CTLA4, human NKG2D, human SIRPα, human SIRPγ, human PD1, human Lag3, human 4-1BB, human OX40, or human GITR.

16. The protein according to any one of claims 1 to 15, wherein the first portion comprises a heavy chain variable (VH) region and a light chain variable (VL) region.

17. The protein of any one of claims 1 to 16, wherein the first portion comprises an immunoglobulin constant region or a portion of an immunoglobulin constant region.

18. The immunoglobulin constant region is (a) IgG, IgE, IgM, IgD, IgA or IgY, or (b) IgG1, IgG2, IgG3, IgG4, IgA1 or IgA2 The protein of claim 17.

19. The protein of claim 17 , wherein the immunoglobulin constant region is immunologically inert.

20. 18. The protein of claim 17, wherein the immunoglobulin constant region is a wild-type human IgG4 constant region, a human IgG4 constant region comprising the amino acid substitution S228P, a wild-type human IgG1 constant region, a human IgG1 constant region comprising the amino acid substitutions L234A and L235A, a human IgG1 constant region comprising the amino acid substitutions L234A, L235A and G237A, a human IgG1 constant region comprising the amino acid substitutions L234A, L235A, G237A and P331S, or a wild-type human IgG2 constant region.

21. 21. The protein of any one of claims 1 to 20, wherein the second portion is a Fab, a single chain Fab, a VH domain, a VL domain, an immunoglobulin novel antigen receptor (IgNAR), a single chain variable fragment (scFv), or a T cell receptor domain.

22. The protein according to any one of claims 1 to 21, wherein the second portion is capable of specifically binding to human CD47, human CD3 or human PD-L1.

23. The protein according to any one of claims 1 to 22, wherein the second portion comprises a heavy chain variable (VH) region and a light chain variable (VL) region.

24. The protein of any one of claims 1 to 23, wherein the second portion comprises an immunoglobulin constant region or a portion of an immunoglobulin constant region.

25. The immunoglobulin constant region is (a) IgG, IgE, IgM, IgD, IgA or IgY, or (b) IgG1, IgG2, IgG3, IgG4, IgA1 or IgA2 The protein of claim 24.

26. 25. The protein of claim 24, wherein the immunoglobulin constant region is immunologically inert.

27. 25. The protein of claim 24, wherein the immunoglobulin constant region is a wild-type human IgG4 constant region, a human IgG4 constant region comprising the amino acid substitution S228P, a wild-type human IgG1 constant region, a human IgG1 constant region comprising the amino acid substitutions L234A and L235A, a human IgG1 constant region comprising the amino acid substitutions L234A, L235A and G237A, a human IgG1 constant region comprising the amino acid substitutions L234A, L235A, G237A and P331S, or a wild-type human IgG2 constant region.

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

29. 29. The protein of claim 28, wherein the immune effector function is ADCC, CDC or ADCP.

30. The protein of any one of claims 1 to 29, wherein the peptide linker is cleaved near or within the diseased tissue.

31. The protein according to any one of claims 1 to 30, wherein the diseased tissue is a tumor or an inflamed tissue.

32. 2. The protein of claim 1, wherein the first portion specifically binds to human cMET, the second portion specifically binds to human CD47, and 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.

33. 2. The protein of claim 1, wherein the first portion specifically binds to human HER2, the second portion specifically binds to human CD3, and 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.

34. 2. The protein of claim 1, wherein the first portion specifically binds to human HER2, the second portion specifically binds to human CD47, and 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.

35. 2. The protein of claim 1, wherein the first portion specifically binds to human cMET, the second portion specifically binds to human CD47, and 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.

36. 2. The protein of claim 1, wherein the first portion specifically binds to human Her2, the second portion specifically binds to human CD3, and 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.

37. 2. The protein of claim 1, wherein the first portion specifically binds to human Her2, the second portion specifically binds to human CD3, and 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.

38. the first portion specifically binds to human Her2, the second portion specifically binds to human CD47, and the protein comprises a first polypeptide chain and a second polypeptide chain, (a) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 42 and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 43; (b) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 44 and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 45; (c) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 46 and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 47; (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; (e) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:50 and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:51; (f) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:52 and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:53; (g) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:54 and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:55; (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; (i) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 90 and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 91; (j) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:44 and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:92; (k) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 44 and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 93; (l) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:44 and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:94; (m) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:44 and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:95; (n) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 44, and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 96; (o) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:44, and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:

97.

39. 2. The protein of claim 1, wherein the first portion specifically binds to human Her2, the second portion specifically binds to human CD3, and 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.

40. 2. The protein of claim 1, wherein the first portion specifically binds to human cMET, the second portion specifically binds to human cMET, and 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.

41. the first portion specifically binds to human Her2, the second portion specifically binds to human CD3, and the protein comprises a first polypeptide chain and a second polypeptide chain, (a) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:98 and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:99; (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; (c) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 102 and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 103; (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.

42. An immunoconjugate comprising a protein according to any one of claims 1 to 41 linked to a therapeutic agent.

43. 43. The immunoconjugate of claim 42, wherein the therapeutic agent is a cytotoxin, a radioisotope, a chemotherapeutic agent, an immunomodulatory agent, a cytostatic enzyme, a cytolytic enzyme, a therapeutic nucleic acid, an anti-angiogenic agent, an anti-proliferative agent, or a pro-apoptotic agent.

44. A pharmaceutical composition comprising a protein according to any one of claims 1 to 41, or an immunoconjugate according to claim 42 or 43, and a pharma- ceutically acceptable carrier, diluent or excipient.

45. A nucleic acid molecule encoding the protein according to any one of claims 1 to 41.

46. A protein according to any one of claims 1 to 41, an immunoconjugate according to claim 42 or 43, or a pharmaceutical composition according to claim 44, for enhancing an anti-cancer immune response in a subject.

47. A protein according to any one of claims 1 to 41, an immunoconjugate according to claim 42 or 43, or a pharmaceutical composition according to claim 44, for use in the treatment of cancer, an autoimmune disease, an inflammatory disease, a cardiovascular disease or a fibrotic disease.

48. 48. The protein or pharmaceutical composition for use according to claim 47, 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, oral or pharyngeal cancer, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small intestine or appendix cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma, or blood tissue cancer.

49. 48. The protein, immunoconjugate or pharmaceutical composition for use according to claim 47, wherein the autoimmune disease or the inflammatory disease is arthritis, asthma, multiple sclerosis, psoriasis, Crohn's disease, inflammatory bowel disease, lupus, Graves' disease, Hashimoto's thyroiditis or ankylosing spondylitis.

50. 48. The protein, immunoconjugate or pharmaceutical composition for use according to claim 47, wherein said cardiovascular disease is coronary heart disease, or atherosclerosis or stroke.

51. 48. The protein, immunoconjugate or pharmaceutical composition for use according to claim 47, wherein said fibrotic disease is myocardial infarction, angina pectoris, osteoarthritis, pulmonary fibrosis, cystic fibrosis, bronchitis or asthma.

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