Activatable bispecific antibodies comprising linker between two binding domains which is human immunoglobulin hinge region, or variant thereof, and uses thereof

Abstract

To provide activatable bispecific antibodies comprising a linker between the two binding domains which is a human immunoglobulin hinge region, or a variant thereof, and uses thereof.SOLUTION: Described herein are protein molecules that exhibit activatable target binding in diseased tissues and related nucleic acid molecules, vectors and host cells. Also described herein are medical uses for such protein molecules. In some embodiments, the protease is human matrix metalloprotease (MMP), human cathepsin, human enterokinase, human thrombin, human tPA, human granzyme B, human uPA, or human ADAMTS-5.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of UK patent application no. 2001196.1 filed on 28 January 2020. UK Patent Application No. 1917678.3 filed 4 December 2019, 17 July 2019 UK patent application no. 1910254.0 filed on 13 May 2019 This application claims the benefit of patent application Ser. No. 1906685.1, the disclosures of each of which are incorporated herein by reference. No. 6,399,433, which is incorporated herein by reference in its entirety.

[0002] Instructions for electronically submitted text files The contents of the text files submitted electronically herewith are incorporated by reference in their entirety. A computer readable copy of the sequence listing (filename: ULS L_002_04WO_SeqList_ST25.txt, Data recording date: 2020 May 11th, file size approximately 390KB).

[0003] The present invention relates to a protein that exhibits activatable target binding in diseased tissue and its medical applications. Concerning quality molecules. [Background technology]

[0004] There are many potential drug targets for the use of antibodies and other binding proteins to treat diseases. However, few of them have been described so far, and are expressed exclusively in diseased tissues. Most of the potential targets within this cavity are also expressed in non-affected tissues. The majority of drug mechanisms employed in the challenging field of medicine are highly potent cell killing mechanisms. As a result, drug target engagement in non-diseased tissues is often less than desirable. They cause unwanted side effects and are partially or completely inactive in healthy tissue. However, engineered forms of the binding protein are required that are highly active in diseased tissues. It has been done. Summary of the Invention

[0005] comprising a first portion and a second portion, and a peptide linker between the first portion and the second portion. Proteins are provided herein, wherein the peptide linker is a human immunoglobulin Amino acid sequence derived from the hinge region or amino acid sequence of a human immunoglobulin hinge The peptide comprises an amino acid sequence having 1 to about 7 amino acid substitutions compared to the peptide region. The CAR is cleavable by a protease expressed in the diseased tissue, and the second part is delivered to the diseased tissue. It has the ability to specifically bind to molecules expressed in tissues and the peptide linker is cleaved. If not, 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 is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, S 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: No. 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, or comprising or consisting of the amino acid sequence of SEQ ID NO:87.

[0006] In some embodiments, the protease is a human matrix metalloprotease (M MP), human cathepsin, human enterokinase, human thrombin, human tPA, human globulin Ranzyme B, human uPA, or human ADAMTS-5. The peptide linker may be a human MMP cleavage site, a human cathepsin cleavage site, a human enterococcus cerevisiae cleavage site, or a human enterococcus cerevisiae cleavage site. Enzyme cleavage site, human thrombin cleavage site, human tPA cleavage site, human granzyme B cleavage site The cleavage site includes the human uPA cleavage site or the human ADAMTS-5 cleavage site. In an embodiment, the human MMP is MMP-2, MMP-3, MMP-7, MMP-8, MMP -9, MMP-10, MMP-12, MMP-13 or MMP14. In embodiments, the level or activity of human MMPs in diseased tissue is greater than or equal to the level or activity of human MMPs in non-diseased tissue. In some embodiments, the level or activity of the human MMP is higher than that of the human MMP. , cathepsin A, cathepsin C, cathepsin D, cathepsin G, cathepsin L or cathepsin In some embodiments, the level of human cathepsin in diseased tissue is The level or activity of the human cathepsin is greater than the level or activity of the human cathepsin in non-diseased tissue.

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

[0008] In some embodiments, the first portion specifically binds to a first molecule expressed in the diseased tissue. and a second portion capable of specifically binding to a second molecule expressed in the diseased tissue, Here, the first molecule expressed in the affected tissue and the second molecule expressed in the affected tissue are different molecules. In some embodiments, the first molecule expressed in the diseased tissue and the In some embodiments, the second molecule expressed in the diseased tissue is expressed in the same cell. The first molecule expressed in the target tissue and the second molecule expressed in the affected tissue are expressed in different cells. In some embodiments, the first molecule expressed in the diseased tissue and / or the first molecule expressed in the diseased tissue In some embodiments, the second molecule is expressed on the surface of the cell. 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 is a human EGFR, a human HER2, a human HER3, Human CD105, human C-KIT, human PD1, human PD-L1, human PSMA, human E pCAM, human Trop2, human EphA2, human CD20, human BCMA, human GIT R, human OX40, human CSF1R, human Lag3, or human cMET. do.

[0010] In some embodiments, the second portion specifically binds to a molecule expressed on a human immune cell. In some embodiments, the molecule expressed on human immune cells is human CD3, human CD16A, human CD16B, human CD28, human CD89, human CTLA4, human NK G2D, 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 an immunoglobulin constant region. In some embodiments, the immunoglobulin constant region comprises a portion of an immunoglobulin constant region. In some embodiments, the antibody is IgG, IgE, IgM, IgD, IgA, or IgY. The immunoglobulin constant region is classified into IgG1, IgG2, IgG3, IgG4, IgA1, and In some embodiments, the immunoglobulin constant region is an immunologically unknown. In some embodiments, the immunoglobulin constant region is a wild-type human IgG4 Constant region, human IgG4 constant region containing the amino acid substitution S228P, wild-type human IgG1 constant region Human IgG1 constant region, including amino acid substitutions L234A and L235A; Human IgG1 constant region, amino acid substitutions, including substitutions L234A, L235A, and G237A a human IgG1 constant region comprising L234A, L235A, G237A and P331S, is the wild type human IgG2 constant region.

[0012] In some embodiments, the second moiety is an antibody, an antigen-binding portion of an antibody, or a receptor external domain. In some embodiments, the second portion comprises a Fab, a single chain Fab, a VH domain, VL domain, immunoglobulin novel antigen receptor (IgNAR), single chain variable fragment (sc In some embodiments, the second portion is a human Fv, or a T cell receptor domain. In some embodiments, the second portion specifically binds to human CD3 or specifically binds to 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 an immunoglobulin constant region. In some embodiments, the immunoglobulin constant region comprises a portion of an immunoglobulin constant region. In some embodiments, the antibody is IgG, IgE, IgM, IgD, IgA, or IgY. The immunoglobulin constant region is classified into IgG1, IgG2, IgG3, IgG4, IgA1, and In some embodiments, the immunoglobulin constant region is an immunologically unknown. In some embodiments, the immunoglobulin constant region is a wild-type human IgG4 Constant region, human IgG4 constant region containing the amino acid substitution S228P, wild-type human IgG1 constant region Human IgG1 constant region, including amino acid substitutions L234A and L235A; Human IgG1 constant region, amino acid substitutions, including substitutions L234A, L235A, and G237A a human IgG1 constant region comprising L234A, L235A, G237A and P331S, is the wild type human IgG2 constant region.

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

[0015] In some embodiments, the first portion is a polypeptide that is specific for a molecule expressed in diseased tissue of the second portion. In some embodiments, the peptide linker inhibits or reduces heterologous binding to the affected tissue. In some embodiments, the peptide linker is cleaved near the tissue or inside the diseased tissue. The catheter is cut near or within the affected tissue, where the first portion is The second portion dissociates from the second portion near or within the affected tissue, and the second portion dissociates from the second portion near or within the affected tissue. In some embodiments, the antibody specifically binds to a molecule expressed in the diseased tissue. In the present case, the diseased tissue is a tumor or an inflamed tissue.

[0016] In some embodiments, the first portion specifically binds human cMET, and wherein the second portion The molecule specifically binds to human CD47, and the protein has the amino acid sequence of SEQ ID NO: 16. A first polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO: 17. and a second polypeptide chain comprising or consisting of:

[0017] In some embodiments, the first portion specifically binds to human HER2, and wherein the second portion The molecule specifically binds to human CD3, and the protein comprises the amino acid sequence of SEQ ID NO:26. A first polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO: 27. The second polypeptide chain comprises or consists of the second polypeptide chain.

[0018] In some embodiments, the first portion specifically binds to human HER2, and wherein the second portion The molecule specifically binds to human CD47, and the protein has the amino acid sequence of SEQ ID NO:34. A first polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO: 35. and a second polypeptide chain comprising or consisting of:

[0019] In some embodiments, the first portion specifically binds human cMET, and wherein the second portion The molecule specifically binds to human CD47, and the protein has the amino acid sequence of SEQ ID NO:36. A first polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO: 37. and a second polypeptide chain comprising or consisting of:

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

[0021] In some embodiments, the first moiety specifically binds to human Her2 and the second moiety specifically binds to human Her2. The protein specifically binds to CD3 and comprises the amino acid sequence of SEQ ID NO: 40 or a first polypeptide chain consisting of or consisting of the amino acid sequence of SEQ ID NO: 41; or a second polypeptide chain composed thereof.

[0022] In some embodiments, the first moiety specifically binds to human Her2 and the second moiety specifically binds to human Her2. The protein specifically binds to CD47 and comprises a first polypeptide chain and a second polypeptide chain. a cyclic 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. Will it be (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. Will it be (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. Will it be (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. Will it be (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. Will it be (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. Will it be (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. Will it be (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. Will it be (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. Will it be (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. Will it be (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. Will it be (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. Will it be (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. Will it be (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. Will it be (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. will be done.

[0023] In some embodiments, the first moiety specifically binds to human Her2 and the second moiety specifically binds to human Her2. The protein specifically binds to CD3 and comprises the amino acid sequence of SEQ ID NO: 73 or a first polypeptide chain consisting of or consisting of the amino acid sequence of SEQ ID NO: 74; or a second polypeptide chain composed thereof.

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

[0025] In some embodiments, the first moiety specifically binds to human Her2 and the second moiety specifically binds to human Her2. The protein specifically binds to CD3 and comprises a first polypeptide chain and a second polypeptide chain. a 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. Or (b) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 100; and the second polypeptide chain comprises or is composed of the amino acid sequence of SEQ ID NO: 101. Configured or (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 is composed of the amino acid sequence of SEQ ID NO: 103. Configured or (d) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 104; and the second polypeptide chain comprises or has the amino acid sequence of SEQ ID NO: 105. It is composed.

[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, , antiangiogenic agents, antiproliferative agents, proapoptotic agents, cytostatic enzymes, cytolytic enzymes, therapeutic agents The therapeutic nucleic acid is an anti-angiogenic agent, an anti-proliferative agent, or a pro-apoptotic agent.

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

[0028] Nucleic acid molecules encoding the proteins or parts of proteins of the invention are further defined herein. The first polypeptide chain, the second polypeptide chain, or the first polypeptide chain of the protein of the present invention is provided. Further provided herein are nucleic acid molecules encoding both the polypeptide chain and the second polypeptide chain. Provide.

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

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

[0031] Further provided herein is a method for producing a protein, the method comprising: The recombinant vector comprising the expression vector of the present invention is expressed under conditions whereby the protein is produced. culturing the host cell and isolating the protein from the host cell or culture; Includes.

[0032] Further provided herein is a method for enhancing an anti-cancer immune response in a subject, the method comprising: a therapeutically effective amount of the protein of the present invention, the immunoconjugate of the present invention, or the pharmaceutical composition of the present invention. The method includes administering the composition to a subject.

[0033] Treating cancer, an autoimmune disease, an inflammatory disease, a cardiovascular disease, or a fibrotic disease in a subject Further provided herein is a method of administering a therapeutically effective amount of a protein of the invention to a mammalian subject. The method includes administering to a subject a protein, an immunoconjugate of the present invention, or a pharmaceutical composition of the present 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, stomach cancer, ovarian cancer, bladder cancer, brain or central nervous system cancer cervical 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 gland cancer, bone In some embodiments, the cancer is a sarcoma, chondrosarcoma, or blood tissue cancer. Inflammatory diseases include arthritis, asthma, multiple sclerosis, psoriasis, Crohn's disease, inflammatory bowel disease, and rheumatoid arthritis. In some embodiments, the disease is rheumatoid arthritis, Graves' disease, Hashimoto's thyroiditis, or ankylosing spondylitis. The cardiovascular disease may be coronary heart disease, atherosclerosis, or stroke. In some embodiments, the fibrotic disease is myocardial infarction, angina pectoris, osteoarthritis, pulmonary tuberculosis, or the like. fibrosis, cystic fibrosis, bronchitis or asthma.

[0034] The present invention relates to a method for treating cancer, an autoimmune disease, an inflammatory disease, a cardiovascular disease or a fibrotic disease. The inventive protein, the inventive immunoconjugate or the inventive pharmaceutical composition are further defined herein as In some embodiments, the cancer is gastrointestinal stromal cancer (GIST), pancreatic cancer, skin cancer, or Cancer, melanoma, breast cancer, lung cancer, bronchial cancer, colorectal cancer, prostate cancer, stomach 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 or pharyngeal cancer, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small intestine or appendix cancer, salivary gland cancer, thyroid cancer In some embodiments, the cancer is an autologous cancer, an adrenal cancer, an osteosarcoma, a chondrosarcoma, or a blood tissue cancer. Immune or inflammatory diseases include arthritis, asthma, multiple sclerosis, psoriasis, Crohn's disease, inflammatory Intestinal disease, lupus, Graves' disease, Hashimoto's thyroiditis or ankylosing spondylitis. In embodiments, the cardiovascular disease is coronary heart disease, atherosclerosis, or stroke. In some embodiments, the fibrotic disease is myocardial infarction, angina pectoris, osteoarthritis, If you have 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 method according to any one of items 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 a non-diseased tissue. Compact quality. (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 is a soluble molecule. (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) The first portion specifically binds to human Her2, and the second portion specifically binds to human CD3. 2. The protein according to item 1, wherein the protein comprises a first polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO: 38 and a second polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO: 39. (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, (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. It may consist of (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) 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. A nucleic acid molecule that encodes. (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 the protein from the host cell or culture. 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] Described herein is a recombinant protein that is conditionally active in diseased human tissue. In some cases, the protein is expressed in a manner that is similar to that of the unaffected tissue. The protein also contains a binding domain that is masked by 1 The peptide linker is cleaved by one or more proteases. The binding domain in the diseased tissue is unmasked, thereby increasing the expression of the protein in the diseased tissue. The proteins of the present invention are capable of selectively binding and / or acting on the affected tissue. These are particularly useful for binding to drug targets that are expressed in both affected and non-affected tissues.

[0038] Several activatable protein molecules and their medical uses are provided herein. In some embodiments, the target binding specificity, undesired activity in the native protein, It effectively limits sexual activity but is fully active in its activated form, and in humans and animal test species ( For example, the crab-eating macaque, also known as the cynomolgus macaque, or Maca Maintaining conditional affinity for one or more targets from both the retention, biophysical stability, and / or protein use in research, clinical and commercial supply Multiple functional properties of the molecule are considered, including yield from the expression platform.

[0039] In some embodiments, one or more human drug targets are targeted, and optionally the catalysis of those targets. Protein molecules that specifically bind to the macula gracilis orthologue are provided, wherein the protein 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 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 format : VL1-C-Linker-VH2-C VH1-C-linker-VL2-C, has.

[0042] "V" refers to the immunoglobulin or T-cell receptor variable region, or the external domain of the receptor. "VH1" and "VL1" refer to heavy chain variable regions that pair with each other and bind to an antigen. "VH2" and "VL2" refer to the variable regions of the VH1 and VL2 chains that pair with each other to bind to an antigen. "C" refers to the heavy and light chain variable regions that are associated with an immunoglobulin or T cell receptor. In an embodiment of the present invention, the V- and V- The C units form the upper and lower immunoglobulin Fab domains, where the lower Fa The b domain is responsible for binding to its cognate target, whereas the N-terminus of each of the V domains of the lower Fab The presence of a linker domain fused to the end reduces or eliminates the binding. In this embodiment, the upper or lower Fab domain is an Fc fragment, one or two extra-receptor fragments. or any domain with or without any specific binding function. It may be replaced.

[0043] In some embodiments, the first portion and the second portion, and between the first portion and the second portion Provided herein is a protein comprising a peptide linker of Here, the peptide linker is an amino acid sequence derived from a human immunoglobulin hinge region, or or amino acid sequence, or compared with the human immunoglobulin hinge region1,2,3,4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 (e.g., 1 to about 7) meshes an amino acid sequence having an amino acid substitution, 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, the second portion will bind to a molecule expressed in the diseased tissue. The case is reduced or inhibited.

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

[0045] In some embodiments, the peptide linker is GPAPELL (SEQ ID NO: 1), GPAP ELLGGGS (SEQ ID NO: 2), GPAPLGLGGGS (SEQ ID NO: 3), PPCPAP ELLGGGS (SEQ ID NO: 4), PPCPAPLGLGGGS (SEQ ID NO: 5), GPAP ELLGGPS (SEQ ID NO: 69), GPAPLGLGGPS (SEQ ID NO: 70), PPCP APELLGGPS (SEQ ID NO: 71), PPCPAPLGLGGPS (SEQ ID NO: 72), GPAPEAAGAGS (SEQ ID NO: 81), GPADDDDKSGS (SEQ ID NO: 82) enterokinase), GPALVPRGSGS (SEQ ID NO: 83) 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 Described in GPAPEGEARGS (SEQ ID NO: 87) (cleavable by ADAMTS-5) In some embodiments, the peptide linker comprises or consists of a sequence as described above. The anchor may comprise or consist of two, three or four of the above sequences. do.

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

[0047] In another aspect, the present invention relates to a protein or a part thereof as defined herein. Nucleic acid molecules encoding the nucleic acid molecules of the invention are provided. Further provided are vectors comprising the nucleic acid molecules of the invention. A host cell comprising a nucleic acid molecule or vector of the invention is also provided.

[0048] In a further aspect, a method for producing a conditionally active protein of the invention is provided. However, this method involves culturing a host cell of the invention under conditions that result in expression and / or production of the protein. Culturing the cells and isolating the protein from the host cells or culture. .

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

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

[0051] In a further aspect, a method of treating or preventing cancer in a subject is provided, comprising: The method comprises administering to the subject a therapeutically effective amount of a protein of the invention as defined herein, The immunoconjugate of the invention as defined herein, the nucleic acid molecule of the invention as defined herein, or a pharmaceutical composition of the invention as defined herein. This includes administering a substance.

[0052] A protein of the invention as defined herein for use as a pharmaceutical The immunoconjugate of the invention as defined herein, the nucleic acid molecule of the invention as defined herein, or a pharmaceutical composition of the invention as defined herein. More things are provided.

[0053] A protein of the invention as defined herein for use in the treatment of cancer. The immunoconjugate of the invention as defined herein, the nucleic acid molecule of the invention as defined herein, or a pharmaceutical composition of the invention as defined herein. More things are provided.

[0054] Used separately, sequentially, or in combination with a second therapeutic agent, e.g., an anticancer agent The proteins, immunoconjugates, nucleic acid molecules and vectors of the invention as defined herein Additionally provided are pharmaceutical compositions.

[0055] In a further aspect, a compound as defined herein in the manufacture of a medicament for the treatment of cancer. Such a protein of the invention, such an immunoconjugate of the invention as defined herein, The nucleic acid molecule of the invention as defined herein, the vector of the invention as defined herein, or the There is provided the use of a pharmaceutical composition of the invention as defined herein.

[0056] Further provided is a method of treating or preventing an autoimmune or inflammatory disease in a subject. The method comprises 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, This includes administering a vector, a pharmaceutical composition as defined herein.

[0057] A protein as defined herein for use in the treatment of an autoimmune or inflammatory disease. an immunoconjugate as defined herein, a nucleic acid molecule as defined herein, Also provided is a vector as defined in claim 1, or a pharmaceutical composition as defined herein. do.

[0058] In the manufacture of a medicament for the treatment of an autoimmune or inflammatory disease, a protein as defined herein, an immune complex as defined herein, a nuclear an acid molecule, a vector as defined herein, or a pharmaceutical composition as defined herein Further uses of the object are provided.

[0059] Further provided is a method of treating or preventing a cardiovascular or fibrotic disease in a subject. The method comprises 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, This includes administering a vector, a pharmaceutical composition as defined herein.

[0060] A protein as defined herein for use as a pharmaceutical agent, a nucleic acid molecule as defined herein, a vector as defined herein. or a pharmaceutical composition as defined herein. An antibody molecule or an antigen-binding portion thereof as defined herein for use in the treatment of a fibrotic disease. a nucleic acid molecule as defined herein; Also provided is a vector as defined herein, or a pharmaceutical composition as defined herein. can be.

[0061] In the manufacture of a medicament for the treatment of an autoimmune disease, an inflammatory disease or a fibrotic disease, a protein as defined herein, an immunoconjugate as defined herein, A nucleic acid molecule as defined herein, a vector as defined herein, or Use of such pharmaceutical compositions is further provided.

[0062] In some embodiments, the present invention provides a method for producing a first portion and a second portion, and a method for producing a first portion and a second portion. The present invention provides a protein comprising a peptide linker between the moieties, wherein the peptide linker The amino acid sequence is derived from a human immunoglobulin hinge region, or contains amino acid substitutions (e.g., 1, 2, 3, 4, 5 , 6, 7, 8, 9, 10, 11, 12 or 13 amino acid substitutions) The peptide linker comprises a sequence that is cleavable by a protease expressed in the diseased tissue. and a second portion capable of specifically binding to a molecule expressed in the diseased tissue and If the peptide linker is not cleaved, the second moiety will bind to the molecule expressed in the diseased tissue. In some embodiments, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the peptide linker comprises an amino acid sequence derived from a human immunoglobulin hinge region. The amino acid sequence of the human immunoglobulin hinge region is In some embodiments, the peptide comprises an amino acid sequence having about seven amino acid substitutions. The car is an amino acid sequence derived from a human immunoglobulin hinge region, or or 1-2, 1-3, 1-4, or 1-5 compared to the human immunoglobulin hinge region. , 1~6 pieces, 1~7 pieces, 2~3 pieces, 2~4 pieces, 2~5 pieces, 2~6 pieces, 2~7 pieces, 3~4 pieces , 3~5 pieces, 3~6 pieces, 3~7 pieces, 4~5 pieces, 4~6 pieces, 4~7 pieces, 5~6 pieces, 5~7 pieces Or it comprises an amino acid sequence having 6 to 7 amino acid substitutions.

[0063] In some embodiments, the peptide is cleavable by a protease expressed in the affected tissue. The drinker is a human matrix metalloproteinase (MMP) or human cathepsin. In some cases, it can be cleaved by proteases expressed in the affected tissue. The cleavable peptide linker is selected from human enterokinase (EK), human thrombin (Th r), 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 is cleavable by a histone dehydrogenase (HSA). Optionally, the peptide comprises a human MMP cleavage site or a human cathepsin cleavage site. Carr contains the human enterokinase cleavage site, the human thrombin cleavage site, and the human tPA cleavage site. , human granzyme B cleavage site, human uPA cleavage site, or human ADAMTS-5 cleavage site Optionally, the peptide linker comprises the MMP substrate sequence PLGL (SEQ ID NO:1). 2). In some cases, the peptide linker includes GPAPELL (SEQ ID NO: 1), G PAPELLGGGS (SEQ ID NO: 2), GPAPLGLGGGS (SEQ ID NO: 3), PPC PAPELLGGGS (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: No. 72), GPAPEAAGAGS (sequence number 81), GPADDDDKSGS (sequence number No. 82), GPALVPRGSGS (SEQ ID NO: 83), GPGPFGRSAGGP (SEQ ID NO: Sequence number 84), GPAPLEADAGS (sequence number 85), GPAPEARRGGS (sequence number 86) or the amino acid sequence of GPAPEGEARGS (SEQ ID NO: 87) , or consisting of them.

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

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

[0066] In some cases, peptide fragments that can be cleaved by proteases expressed in the affected tissue are The anchors are human MMP1, MMP2, MMP3, MMP4, MMP5, MMP6, MMP 7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP1 4, MMP15, MMP16, MMP17, MMP18, MMP19, MMP20, MM P21, MMP22, MMP23, MMP24, MMP25, MMP26, MMP27, Alternatively, it can be cleaved by any one of MMP28. The peptide linker, which is cleavable by tissue-expressed proteases, binds to human MMP-2. , MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, MMP-12 or can be cleaved by any one of MMP-13. The level or activity of human MMPs in non-diseased tissue may be compared to the level or activity of human MMPs in non-diseased tissue. is higher than the activity.

[0067] In some cases, peptide fragments that can be cleaved by proteases expressed in the affected tissue are The anchors are human cathepsin A, cathepsin B, cathepsin C, cathepsin D, and cathepsin E, cathepsin F, cathepsin G, cathepsin H, cathepsin K, cathepsin L1, cathepsin Any of cathepsin V, cathepsin O, cathepsin S, cathepsin W, or cathepsin Z In some cases, proteases expressed in the affected tissue can cleave the IL-1 protein. The peptide linker cleavable by is selected from human cathepsin D, cathepsin G, or cathepsin B. In some cases, the diseased tissue may be cleaved by any one of the following: The level or activity of human cathepsins was determined by comparing the level or activity of human cathepsins in non-diseased tissue. In some cases, the levels of human cathepsins in tissues with a pH below 7.0 were higher than their activity. The level or activity of human cathepsins in tissues with a pH of 7.4 or higher is higher than that in tissues with a pH of 7.4 or higher. It is more expensive.

[0068] In some embodiments, the first portion of any of the proteins of the invention is an antibody, Optionally, the first portion comprises a Fab, an antigen-binding portion, or a receptor ectodomain. Single chain Fab, VH domain, VL domain, immunoglobulin novel antigen receptor (IgNAR ), single chain variable fragment (scFv), diabody, or T cell receptor domain. gNARs are homodimeric heavy-chain single antibodies produced by sharks and other cartilaginous fishes (Feige et al.,PNAS,2014,111(22):8155-816 0).

[0069] In some cases, the first portion specifically binds to a molecule expressed in the diseased tissue. In some embodiments, the first portion specifically binds to a tumor-associated antigen (TAA). Thus, the first portion comprises human EGFR, human HER2, human HER3, human CD105, Human C-KIT, human PD1, human PD-L1, human PSMA, human EpCAM, human T rop2, human EphA2, human CD20, human BCMA, human GITR, human OX40 , which specifically binds to human CSF1R, human Lag3, or human cMET. In embodiments, the first portion also binds to the cynomolgus orthologue of any of these molecules. do.

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

[0071] In some embodiments, the anti-HER2 The variable region sequence is a variable region sequence of trastuzumab. The anti-CD3 variable region sequences used in the protein constructs disclosed in In some embodiments, the protein structures disclosed herein are The anti-cMET variable region sequences used in the constructs are provided in WO2019 / 175186. In some embodiments, the protein constructs disclosed herein are The anti-CD47 variable region sequences used are those provided in WO2019 / 034895. It is.

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

[0073] In some cases, the second portion specifically binds to a molecule expressed in the diseased tissue. In some embodiments, the second portion specifically binds to a tumor-associated antigen (TAA). Thus, the second portion specifically binds to human CD47. The second moiety specifically binds to human PD-L1. Optionally, the second moiety is expressed in a human immune cell. In some cases, molecules expressed on human immune cells are human CD3, human CD16A, human CD16B, human CD28, human CD89, human CTLA4, human NKG2D, human SIRPα, human SIRPγ, human PD1, human La g3, human 4-1BB, human OX40, or human GITR. The second portion also binds to the cynomolgus orthologues of either of these molecules.

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

[0075] In some embodiments, the first and / or second portions of the protein of the invention are In some embodiments, the immunoglobulin constant region may comprise an immunoglobulin constant region. , IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgE, or Ig In another embodiment, the immunoglobulin constant region is IgG1, IgG2, IgG 3, IgG1 null, IgG4 (S228P), IgA1, IgA2, IgE, or Ig In some embodiments, the first and / or second portions of the protein of the invention are The polypeptide may comprise a constant region that is immunologically inert. The first and / or second portions of the protein may comprise a wild-type human IgG1 constant region, an amino acid substitution Human IgG1 constant region containing amino acid substitutions L234A and L235A. A human IgG1 constant region containing the amino acid substitutions L234A, L35A and G237A. An immunoglobulin comprising a human IgG1 constant region comprising 235A, G237A and P331S. In some embodiments, the first portion and / or the second portion of the protein of the invention may comprise a polypeptide constant region. Alternatively, the second portion comprises a wild-type human IgG2 constant region or a wild-type human IgG4 constant region. In some embodiments, the proteins of the invention may comprise an immunoglobulin constant region, including an immunoglobulin constant region. The first and / or second portions of the immunoglobulin include any one of the amino acid sequences in Table 10. The Fc region sequences in Table 10 may include a globulin constant region. In some embodiments, the first and / or second portions of the protein of the invention are human. IgG4, human IgG4(S228P), human IgG2, human IgG1, human IgG1- 3M, or an immunoglobulin constant region containing the amino acid sequence of the Fc region of human IgG1-4M For example, the human IgG4(S228P) Fc region may comprise a region similar to that of wild-type human IgG 4Fc region contains the substitution: S228P. For example, the human IgG1-3M Fc region The substitutions compared to the wild-type human IgG1 Fc region are: L234A, L235A and G23 7A, whereas the human IgG1-4M Fc region contains the wild-type human IgG1 Fc The region contains the following substitutions: L234A, L235A, G237A, and P331S. In some embodiments, the amino acid residue positions in the constant region of the immunoglobulin molecule are The nomenclature is numbered according to the nomenclature (Ward et al., 1995 Therapy .Immunol. 2:77-94). In some embodiments, the immunoglobulin constant region contains an RDELT (SEQ ID NO: 65) motif or a REEM (SEQ ID NO: 66) motif. The REEM (SEQ ID NO: 66) allotype may be SEQ ID NO: 65) allotype is found in fewer human populations than the The first and / or second portions of the protein of the antibody of the present invention may be any of SEQ ID NOs: 56 to 62. In some embodiments, the antibody of the present invention may comprise an immunoglobulin constant region comprising any one of the following: The first and / or second portions of the protein may be any one of the clones in Tables 3 to 9. Among the heavy chain amino acid sequences and light chain amino acid sequences, and the Fc region amino acid sequences in Table 10 In some embodiments, the first portion and / or the second portion of the protein of the invention may be or the second portion is an immunoglobulin comprising any one of the Fc region amino acid sequences of Table 10. and an immunoglobulin heavy chain constant region, and a kappa light chain constant region or a lambda light chain constant region. The antibody may comprise a light chain constant region.

[0076] In some embodiments, the proteins of the invention comprise an IgG1 isotype constant region. The IgG1 isotype constant region potently activates all FcγR signaling types. , thereby maximally promoting 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 comprises a wild-type human hinge region amino acid sequence: It may contain one, two, three, four or more amino acid substitutions compared to the In an embodiment, the immunoglobulin constant region does not include a hinge region.

[0078] In some embodiments, the first portion specifically binds to a first molecule expressed in the diseased tissue. and a second portion capable of specifically binding to a second molecule expressed in the diseased tissue, 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 In some embodiments, the second molecule is expressed in the diseased tissue. The first molecule, which is expressed in the target tissue, and the second molecule, which is expressed in the affected tissue, are expressed in different cells. In an embodiment, 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 In some embodiments, the first molecule is expressed on the surface of a cell. And / or the second molecule expressed in the affected tissue is a soluble molecule.

[0079] In some embodiments, the first moiety specifically binds human cMET and the second moiety specifically binds human CD 47. In some embodiments, the first moiety specifically binds to human HER2. and the second portion specifically binds human CD47. In some embodiments, the first portion specifically binds human CD47. The second portion specifically binds to human CD47. In the present study, the first moiety specifically binds to human HER2 and the second moiety specifically binds to human CD3. In some embodiments, the first moiety specifically binds to human cMET and the second moiety specifically binds to human cMET. It specifically binds to cMET.

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

[0081] In some embodiments, a first portion of the protein of the invention is expressed in the affected tissue of a second portion. In some embodiments, the target molecule of the present invention is a target of the present invention. The peptide linker of the protein is cleaved near or within the affected tissue. In some cases, the peptide linker is cleaved near or within the diseased tissue, wherein the first portion dissociates from the second portion near or within the affected tissue, and The two parts are targeted to molecules expressed in or near the affected tissue. In some cases, the cleaved peptide linker is bound to an anti-hinge antibody (e.g., It contains a binding or targeting site for the endogenous anti-hinge antibody of the elephant, while The unmodified (e.g., intact) peptide linker does not provide a binding site for an anti-hinge antibody or or does not contain the target site. Binding of an anti-hinge antibody to the peptide linker that is cleaved Increased ADCC, CDC and / or ADCP in the presence of activated proteins of the present invention (See, for example, FIG. 2B).

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

[0083] In some embodiments, the diseased tissue is a tumor, necrotic tissue, fibrotic tissue, tissue that has undergone the coagulation cascade, or The tissue may be swollen or inflamed.

[0084] In some embodiments, the first portion and the second portion, and between the first portion and the second portion Provided herein is a protein comprising a peptide linker of the formula: a second portion that specifically binds to human CD47 and a second portion that specifically binds to the protein a first polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO:16; and a second polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO:17. The protein contains only one copy of the first polypeptide chain and one copy of the second polypeptide chain. The peptide linker of this protein contains only one copy of the LHL sequence. copies (see Table 1), each of which is connected via a peptide bond to the first moiety at its N-terminus The protein is called "Fab2 cMetCD4 The amino acid sequence is called "Met47-LHL-LHL" or "Met47-LHL-LHL". The structure of this protein is shown in Figure 3A. The second part of this protein is linked to KIH IgG1- via a G4S linker (SEQ ID NO: 15) and a truncated hinge region. Linked to Fc.

[0085] In some embodiments, the first portion and the second portion, and between the first portion and the second portion Provided herein is a protein comprising a peptide linker of the formula: a first portion that specifically binds to HER2, a second portion that specifically binds to human CD3, and the protein 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 contains only one copy of the first polypeptide chain and one copy of the second polypeptide chain. The peptide linker of this protein is connected via peptide bonds. The protein contains two copies of the LHL sequence fused together (see Table 1). "Fab2 Her2CD3-LHL-LHL" or "Her23-LHL-LHL" The amino acid sequence is presented in Table 4. The structure of the protein is shown in Figure 3A. The second part of the protein contains a G4S linker (SEQ ID NO: 15) and a truncated hinge region ( 3M) to KIH IgG1-Fc.

[0086] In some embodiments, the first portion and the second portion, and between the first portion and the second portion Provided herein is a protein comprising a peptide linker of the formula: a second portion that specifically binds to human CD47 and a protein a first polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO:34 and a second polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO:35. The protein contains two identical copies of a first polypeptide chain and a second polypeptide chain. The peptide linker of this protein is a peptide bond. This protein contains two copies of the LHL sequence fused via the are "IgG2 Her2CD47-LHL-LHL" or "Her47-LHL-L The amino acid sequence is given in Table 5. The structure of this protein is shown in Figure 2A. The second part of the protein is connected to the human I through a hinge region or a truncated hinge region. linked to the gG1 Fc sequence (see Table 10).

[0087] In some embodiments, the first portion and the second portion, and between the first portion and the second portion Provided herein is a protein comprising a peptide linker of the formula: a second portion that specifically binds to human CD47 and a second portion that specifically binds to the protein a first polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO:36; and a second polypeptide 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 interest consists of two copies of the LHL sequence fused together via a peptide bond. This protein contains the Fab2 CMET / CD47 “OneArg” gene (see Table 1). The amino acid sequence is shown in Table 6. The structure of this protein is shown in Figure 3B. The construct is a "knobs-into-holes" one. This is an arm Fab2 construct, with Fab2 on the knob side and stem hinge hole Fc on the opposite side. The construct may contain a non-effector null human IgG1 Fc sequence. (See Table 10.)

[0088] In some embodiments, the first portion and the second portion, and between the first portion and the second portion Provided herein is a protein comprising a peptide linker of the formula: a second portion that specifically binds to human CD3, and the protein 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 (Table 10 The second polypeptide chain further comprises a human kappa light chain amino acid sequence. The peptide linker of the protein consists of two copies of the LHL sequence fused together via a peptide bond. This protein contains the Fab2 Her2 / CD3 “one-agent” peptide (see Table 1). The amino acid sequence is given in Table 7. The structure of this protein is shown in Figure 3B. The construct is a "knobs-into-holes" This is a two-arm Fab2 construct with Fab2 on the knob side and stem hinge hole Fc on the opposite side. This construct is also effector null (IgG1-3M; see Table 10).

[0089] In some embodiments, the first portion and the second portion, and between the first portion and the second portion Provided herein is a protein comprising a peptide linker of the formula: a second portion that specifically binds to human CD3, and the protein 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 (Table 10 The second polypeptide chain further comprises a human lambda light chain amino acid sequence. The peptide linker of the protein consists of two copies of the LHL sequence fused together via a peptide bond. The protein contains the Fab2 Her2 / CD3 (34) This is called the "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. This is a one-arm Fab2 construct with Fab2 on the knob side and stem helix on the opposite side. This construct is also effector null (IgG1-3M; (See Table 10).

[0090] In some embodiments, the first portion and the second portion, and between the first portion and the second portion Provided herein is a protein comprising a peptide linker of the formula: a second portion that specifically binds to human CD47, and the protein a 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. (called "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. (called "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. (called "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. (called "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. (called "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. (called "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. (called "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. (called "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. (called "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. (called "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. (called "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. (called "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. (called "Her47-LHL-LHL-A5"). These proteins are It is generally referred to as "IgG2 Her2 / CD47". The amino acid sequence is shown in Tables 9 and 20. The structure of this protein is shown in Figure 2A. LHLF, LHLM and LHLMF The peptide linker sequences of the linkers are presented in Table 1. EK, Thr, tPA, GrB, u The peptide linker sequences for the PA and A5 linkers are provided in Table 21.

[0091] In some embodiments, the first portion and the second portion, and between the first portion and the second portion Provided herein is a protein comprising a peptide linker of the formula: a second portion that specifically binds to human CD47, and the protein a polypeptide chain and a second polypeptide chain, (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. (called "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. (called "Her47 LHL-LHLF IgG1-2hDAA"). These proteins are generally referred to as "IgG2 IgG1-DAA Her2 / CD47." The amino acid sequences are presented in Table 19. These proteins are stabilized at the hinge. The structure of this protein is shown in Figure 2A. The petide linker sequences are presented in Table 1.

[0092] In some embodiments, the first portion and the second portion, and between the first portion and the second portion Provided herein is a protein comprising a peptide linker of the formula: a second portion that specifically binds to human CD3, and the protein 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 comprises a human IgG1-3M Fc amino acid sequence (e.g., (containing a "hole" mutation that allows heterodimerization with the polypeptide chain), followed by linker alignment. Array, the VH and CH1 domains of the first binding moiety, another linker sequence, and a second binding moiety The second polypeptide chain further comprises a VH and a CH1 domain (see Table 13). gG1-3M Fc amino acid sequence (e.g., capable of heterodimerization with a second polypeptide chain) The first binding moiety contains a "knob" mutation that allows the first binding moiety to bind to the VL and CL domains of the first binding moiety. The VL and CL domains of the second binding moiety are further included in the nucleotide sequence (Table 1). 13). The peptide linker of this protein is fused via a peptide bond. The four sequences are shown in Table 1, and are located between the Fc and the first portion and between the first binding portion and the second binding portion. This protein is also called "Fc-Her2 / CD3(34)" or The amino acid sequence is shown in Table 13. The structure of the protein is shown in Figure 12. The construct has a "knobs-into-holes" Fc-Fab2 structure. The construct has 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; Table 10 (see ).

[0093] In some embodiments, the first portion and the second portion, and between the first portion and the second portion Provided herein is a protein comprising a peptide linker of the formula: a second portion that specifically binds to human cMET and a third portion that specifically binds to the protein a first polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO:75 and a second polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO:76. The first polypeptide chain 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 the protein further comprises The protein contains two copies of the LHL sequence (see Table 1). This is called the "ET / CMET 'one-arm' type" or "MetMet-LHL-LHL" The amino acid sequence is presented in Table 14. The structure of this protein is shown in Figure 3B. The construct "knob-into-hole" one-arm Fab2 construct, with Fab2 on the knob side and The construct has a stem hinge hole Fc on the side. This construct is either effector null or May contain a non-effector null human IgG1 Fc sequence (see Table 10).

[0094] In some embodiments, the first portion and the second portion, and between the first portion and the second portion Provided herein is a protein comprising a peptide linker of the formula: the first portion specifically binds to human CD3, the second portion specifically binds to human CD3, and the protein a 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. (called "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 is composed 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 is composed of the amino acid sequence of SEQ ID NO: 103. (referred to as "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 has the amino acid sequence of SEQ ID NO: 105. This protein is composed of Fab2 Her23 LHL-LHL-S. The protein is called "Fab2 Her2 / CD3." The amino acid sequence is presented in Table 22. The structure of this protein is shown in Figure 3A. The protein consists of one amino acid sequence of the first polypeptide chain. The peptide of this protein contains only one copy of the second polypeptide chain and only one copy of the third polypeptide chain. The tidolinker consists of two copies of the LHL sequence (see Table 1) fused via a peptide bond. ), or one copy of the LHL sequence and one copy of the LHLF sequence.

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

[0096] Examples of suitable therapeutic agents include cytotoxins, radioisotopes, chemotherapeutic agents, immunomodulatory agents, anti-vascular Neoplastic agents, antiproliferative agents, proapoptotic agents, and cytostatic and cytolytic enzymes (e.g. Further therapeutic agents include immunomodulatory agents, anti-angiogenic agents, and the like. Therapeutic nucleic acids include genes encoding biologics, antiproliferative agents, or proapoptotic agents. These drug descriptors are not mutually exclusive; therefore, a therapeutic agent may be any of the above terms. may be described using one or more of the following:

[0097] Examples of suitable therapeutic agents for use in immunoconjugates include taxanes, meitaisin, CC-10, 65, duocarmycins, calicheamicins, other enediynes, and auristatins Other examples include antifolates, vinca alkaloids, and anthracyclines. Plant toxins, other bioactive proteins, enzymes (i.e., ADEPT ), radioisotopes, photosensitizers may also be used in the immunoconjugates. Formulation using secondary carriers such as liposomes or polymers as cytotoxic agents. Suitable cytotoxins include those that inhibit or block the function of cells and / or Examples of cytotoxic agents include antibiotics, tubulin, and erythropoietin. inhibitors of ribonucleic acid polymerization, alkylating agents that bind to and destroy DNA, and inhibitors of protein synthesis or Agents that disrupt the function of essential cellular proteins, e.g., protein kinases, phosphatases, These include topoisomerases, enzymes and cyclins.

[0098] Representative cytotoxins include doxorubicin, daunorubicin, idarubicin, and acurar. Bicine, Zorubicin, Mitoxantrone, Epirubicin, Carubicin, Nogalamycin, Menogaril, pitarubicin, valrubicin, cytarabine, gemcitabine, trifluridine , ancitabine, enocitabine, azacitidine, doxifluridine ne), pentostatin, broxuhdine, capecitabine, cla Cladhbine, decitabine, floxuhdine, Fludarabine, gougerotin, puromycin, tegafur, thiazofin fuhn), adamycin, cisplatin, carboplatin, Clophosphamide, dacarbazine, vinblastine, vincristine, mitoxantrone , bleomycin, mechlorethamine, prednisone, procarbazine, methotrexate , flurouracil, etoposide, taxol, taxol derivatives analogs, platins such as cisplatin and carboplatin, mitomycin, thiotepa, oxalate, vincristine, daunorubicin, epirubicin, actinomycin, australis authramicin, azaserine, bleomycin, tamoxifen, Idarubicin, dolastatin / auristatin, hemiasterlin, esperamicin and These include, but are not limited to, maytansinoids.

[0099] Suitable immunomodulatory agents include antihormones that block hormone action on tumors; and inhibiting cytokine production or downregulating self-antigen expression, or These include immunosuppressants that mask MHC antigens.

[0100] A nucleic acid sequence encoding a protein of the invention as defined herein or a part of the protein. Nucleic acid molecules that encode the polypeptides of the invention are also provided. A first polypeptide chain, a second polypeptide chain, or a first polypeptide chain and a second polypeptide chain Further provided herein is a nucleic acid molecule that encodes both the ribosome and the ribosome strand. A nucleic acid molecule as defined herein may be isolated.

[0101] Further provided is a vector comprising a nucleic acid molecule of the invention as defined herein. The target 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, a method for producing a protein of the invention is provided, the method comprising the steps of: The method comprises culturing a host cell of the invention under conditions that result in expression and / or production of the protein. and isolating the protein from the host cell or culture.

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

[0105] Further provided is a method of enhancing an immune response in a subject, comprising administering an effective amount of The protein of the invention as defined herein, the immunoglobulin of the invention as defined herein A complex, a nucleic acid molecule of the invention as defined herein, a or a pharmaceutical composition of the invention as defined herein to a subject. Includes.

[0106] In a further embodiment, the method comprises treating or preventing cancer in a subject or administering to a subject a therapeutically effective amount of the compound of formula (I). In one embodiment, a method for alleviating the symptoms of a pulmonary artery disease is provided, the method comprising administering to the patient an effective amount of a pulmonary artery disease, as defined herein. a protein of the invention as defined herein, an immunoconjugate of the invention as defined herein, The nucleic acid molecule of the invention as defined herein, the vector of the invention as defined herein, or the This involves administering to a subject 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 may be gastrointestinal stromal cancer (GIST), pancreatic cancer, or 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 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, It may be osteosarcoma, chondrosarcoma, or blood tissue cancer. In some cases, the blood tissue cancer is It is a malignant lymphoma.

[0108] In some embodiments, the method may be used in the treatment of cancer or for the symptoms of cancer. The protein of the invention as defined herein, The immunoconjugate of the invention as defined herein, the 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. Compositions are provided herein.

[0109] In some embodiments, the second therapeutic agent, e.g., an anti-cancer agent, may be administered separately, sequentially, or in combination with the second therapeutic agent. The proteins of the invention as defined herein, used simultaneously in combination with The combinations, nucleic acid molecules, or vectors, or methods of treatment thereof, are provided herein.

[0110] In a further embodiment, the manufacture of a medicament for treating cancer or alleviating a symptom of cancer. The protein of the invention as defined herein, The immunoconjugate of the invention, the nucleic acid molecule of the invention as defined herein, The use of such a vector of the invention or a pharmaceutical composition of the invention as defined herein is provided. It will be offered.

[0111] The present invention also relates to a method of treating or preventing an autoimmune or inflammatory disease in a subject. The method further comprises 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, The method includes administering to a subject such a vector, or a pharmaceutical composition as defined herein. nothing.

[0112] For example, autoimmune or inflammatory diseases include arthritis, asthma, multiple sclerosis, psoriasis, clotting syndrome, and the like. If you have chronic kidney disease, inflammatory bowel disease, lupus, Graves' disease, Hashimoto's thyroiditis, or ankylosing spondylitis, There may be cases where this occurs.

[0113] A protein as defined herein for use in the treatment of an autoimmune or inflammatory disease. an immunoconjugate as defined herein, a nucleic acid molecule as defined herein, Also provided is a vector as defined in claim 1, or a pharmaceutical composition as defined herein. do.

[0114] In the manufacture of a medicament for the treatment of an autoimmune or inflammatory disease, a protein as defined herein, an immune complex as defined herein, a nuclear an acid molecule, a vector as defined herein, or a pharmaceutical composition as defined herein Further uses of the object are provided.

[0115] The present invention also relates to a method of treating or preventing a cardiovascular disease or a fibrotic disease in a subject. The method further comprises 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, The method includes administering to a subject such a vector, or a pharmaceutical composition as defined herein. nothing.

[0116] A protein as defined herein for use in the treatment of a cardiovascular or fibrotic disease. an immunoconjugate as defined herein, a nucleic acid molecule as defined herein, Also provided is a vector as defined in claim 1, or a pharmaceutical composition as defined herein. do.

[0117] In the manufacture of a medicament for the treatment of a cardiovascular disease or a fibrotic disease, a protein as defined herein, an immune complex as defined herein, a nuclear an acid molecule, a vector as defined herein, or a pharmaceutical composition as defined herein Further uses of the object are provided.

[0118] In any aspect of the present invention, the cardiovascular disease may be, for example, coronary heart disease, atherosclerosis, or pulmonary embolism. The cause may be vascular arteriosclerosis, or stroke.

[0119] For example, the fibrotic disease in any of the embodiments of the present invention is a myocardial infarction, angina pectoris, degenerative joint disease, or the like. The condition may be arthritis, pulmonary fibrosis, asthma, cystic fibrosis or bronchitis.

[0120] In some embodiments, the compounds disclosed herein and in the form disclosed herein are Provided herein are proteins for therapeutic use that contain the amino acid sequences.

[0121] In some embodiments, the pharmaceutical composition comprises a pharma- ceutically acceptable excipient, carrier, or diluent. Pharmaceutically acceptable excipients are those that do not induce secondary reactions and that, for example, are suitable for use in the preparation of pharmaceutical compositions according to the present invention. Facilitating the administration of a protein as defined herein, its life span in the body and / or its Adding an active ingredient to a compound or pharmaceutical composition that allows for increased efficacy or increased solubility in solution These pharma- ceutically acceptable solvents are well known in the art. and can be adapted by the skilled artisan depending on the mode of administration of the protein as defined herein. It will be.

[0122] In some embodiments, the protein as defined herein is formulated into a solution prior to administration. For example, the lyophilized protein molecule may be provided in a lyophilized form that is suitable for administration to an individual. The solution may be prepared in sterile water and mixed with physiological saline beforehand.

[0123] Proteins as defined herein are usually administered in the form of pharmaceutical compositions. It may contain at least one component in addition to the protein molecule. The compositions may comprise, in addition to a protein as defined herein, a pharma- ceutically acceptable excipient, The composition may include carriers, buffers, stabilizers, or other materials known to those of skill in the art. The carrier or other agent must be non-toxic and must not interfere with the efficacy of the protein. The exact nature of the material will depend on the route of administration, which may be bolus, infusion, or both, as discussed below. , injection or any other suitable route.

[0124] For administration parenterally, e.g., by injection, e.g., subcutaneously or intravenously, as defined herein Pharmaceutical compositions containing such proteins are pyrogen-free, pH-, isotonicity- and stability-controlled. The composition may be in the form of a parenterally acceptable aqueous solution having appropriate properties. Use an isotonic vehicle such as sodium chloride injection, Ringer's injection, or lactated Ringer's injection, Solutions such as phosphates, citrates and other organic acids can be prepared appropriately. buffering agents; antioxidants such as ascorbic acid and methionine; preservatives (e.g., octadecyl Dimethylbenzyl ammonium chloride;Hexamethonium chloride;Benzalkonium chloride;Salt benzethonium chloride; phenol, butyl or benzyl alcohol; methyl or Alkylparabens such as propylparaben; catechol; resorcinol; cyclohexanoate 3'-pentanol; and m-cresol; low molecular weight polypeptides; serum albumin Proteins such as amino acids, gelatins, or immunoglobulins; polyvinylpyrrolidone Hydrophilic polymers; glycine, glutamine, asparagine, histidine, arginine or Amino acids such as lysine; monosaccharides including glucose, mannose, or dextrin , disaccharides and other carbohydrates; chelating agents such as EDTA; sucrose, mannitol, trehalose A sugar such as halo or sorbitol; a salt-forming counterion such as sodium; a metal complex ( Zn-protein complex); and / or TWEEN™, PLURONICS (trademark) or polyethylene glycol (PEG) Preservatives, stabilizers, buffers, antioxidants and / or other additives may be used as required. It is okay to do so.

[0125] Pharmaceutical compositions comprising a protein as defined herein may be used alone or in combination with other therapeutic agents. may be administered simultaneously or sequentially depending on the condition being treated.

[0126] Proteins as defined herein may be used in prophylactic or preventative treatments (e.g., to treat a disease caused by an infection in an individual). to reduce the risk of developing a condition; to delay its onset; or to reduce its severity after onset; The present invention may be used in methods of treatment of the human or animal body, including the treatment of a disease condition prior to its onset. The therapeutic method comprises administering a protein as defined herein to an individual in need thereof. may include:

[0127] Administration is typically in a "therapeutically effective amount", which is sufficient to show benefit to a patient. Such effect may be at least amelioration of at least one symptom. The actual amount administered, and 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 dosage will depend on the method of administration, the schedule of administration, and other factors known to those skilled in the art. Prescription, for example determining dosage, is the responsibility of general practitioners and other medical doctors. The suitability of the antibody molecule may depend on the severity of the symptoms and / or progression of the disease being treated. Appropriate doses are well known in the art (Ledermann JA et al. l.,1991,Int.J.Cancer 47:659-664;Bagshawe KDet al.,1991,Antibody,Immunoconjugat es and Radiopharmaceuticals 4:915-922). throw Depending on the type of drug being administered, Specific dosages may be used as set forth in the 2003 International Journal of Clinical Chemistry. The therapeutically effective amount or preferred dose of a protein as defined herein can be determined in animal models. The results were determined by comparing their in vitro and in vivo activities in Methods are known for extrapolating effective doses in mice and other test animals to humans. The exact dose will depend on whether the protein is for prophylactic or therapeutic use and the area to be treated. the size and location of the region, the exact nature of the protein (e.g., Fab2, IgG), and Several properties, including any detectable labels or other molecules attached to the protein, may be used. It depends on factors such as:

[0128] Typical protein doses range from 100 μg to 1 g for systemic administration and 100 μg to 1 g for local administration. The range is 1 μg to 1 mg. A high loading dose is administered first, followed by smaller doses. More than one administration may be given. In some embodiments, the protein is a whole antibody, e.g., an I It contains either the IgG1 or IgG4 isotype. This is the dose for single treatment of adult patients, It can be proportioned for children and infants and can be adjusted to other protein structures in proportion to the molecular weight. Treatment can be daily or twice a week, at the discretion of the doctor. The treatment schedule for an individual may be repeated at weekly or monthly intervals. The pharmacokinetic and pharmacodynamic properties of the protein composition, the route of administration, and the nature of the condition being treated. may depend on.

[0129] The treatment may be cyclical and the period between doses may be about 2 weeks or more, e.g. , about 3 weeks or more, about 4 weeks or more, about once a month or more, about 5 weeks or more, or about 6 weeks or more. For example, treatment may be every 2-4 weeks, or every 4-8 weeks. May be given pre-operatively and / or post-operatively and / or following a surgical or invasive procedure. The composition may be administered or applied directly to the anatomical site of the affected area. Suitable formulations and routes of administration are described in , as mentioned above.

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

[0131] In some embodiments, the therapeutic effect of a protein as defined herein is dose-dependent. For example, as defined herein, The therapeutic effect of a single administration of a protein lasts for more than one month, two months, or three months in an individual. The symptoms may last for more than four months, more than five months, or more than six months.

[0132] As used herein, the term "CD47" refers to an IAP (integrin-associated protein This specification refers to CD47 and its variants that retain at least some of the biological activity of CD47. As used herein, CD47 is a marker for all mammalian cells, including humans, rats, mice, and chickens. In some embodiments, the term "CD47" refers to It is used to include variants, isoforms and species homologues of human CD47. In some cases, as used herein, CD47 may be used in human, monkey, rat, mouse, and the native sequence CD47 of all mammalian and non-mammalian species, including chicken. In some embodiments, the term "CD47" refers only to wild-type CD47. Proteins that express CD47 from species other than humans, particularly cynomolgus monkeys (Macaca fascicularis), It may cross-react with CD47 derived from humans and cynomolgus monkeys. Examples of CD47 amino acid sequences of the polypeptides of the present invention are provided in Table 11. The protein may be completely specific for human CD47 and exhibit no cross-reactivity with non-human It is not necessary.

[0133] As used herein, the term "cMET" refers to the MET protein and the biosynthesis of cMET. In some cases, the term "variant" as used herein refers to a variant thereof that retains at least a portion of the activity of the compound. When used, cMET is used in all mammalian species, including humans, rats, mice, and chickens. In some embodiments, the term "cMET" refers to the native sequence cMET of human May be used to include cMET variants, isoforms, and species homologs In some cases, as used herein, cMET can be used in humans, monkeys, rats, Contains native cMET sequences from all mammalian and non-mammalian species, including mouse and chicken. In some embodiments, the term "cMET" refers only to wild-type cMET. The antibodies are directed against cMET from species other than humans, particularly cynomolgus monkeys (Macaca fascicularis). cMET from humans and cynomolgus monkeys may cross-react with cMET from humans and cynomolgus monkeys. Examples of cMET amino acid sequences for antibodies are provided in Table 12. In certain embodiments, the antibody May be completely specific for human cMET and may not exhibit cross-reactivity with non-human .

[0134] As used herein, the term "Her2" refers to the human epidermal growth factor receptor 2 protein. It refers to variants thereof that retain at least some of the quality and biological activity of Her2. Also known as HER2 / neu, ErbB2, c-erbB-2 and human EGF receptor 2 In some embodiments, the term "Her2" refers to a variant of human Her2. Sometimes used to include species, isoforms, and species homologs. As used herein, Her2 refers to human, monkey, rat, mouse and chicken Native sequences of Her2 (also known as ErbB2) from all mammalian and non-mammalian species, including In some embodiments, the term "Her2" refers to wild-type Her2. The antibody of the present invention is directed to Her2 from species other than humans, particularly cynomolgus monkeys (Ma It may cross-react with Her2 derived from the caca fascicularis. Examples of human and cynomolgus Her2 / ErbB2 amino acid sequences are provided in Table 15. In certain embodiments, the antibody may be fully specific for human Her2 and may be compatible with non-human Her2. It may not exhibit cross-reactivity.

[0135] As used herein, the term "CD3" refers to the "cluster of differentiation 3" multimeric protein complex. It refers to a complex of CD3 and variants thereof that retain at least some of the biological activity of CD3. It combines into four different polypeptide chains; epsilon (ε), gamma (γ), and delta (δ). These polypeptide chains assemble into three pairs of dimers (εγ, εδ In some embodiments, the term "CD3" refers to the human CD3 antigen. In some cases, the term may be used to include variants, isoforms, and species homologs. Thus, as used herein, CD3 is a marker for human, monkey, rat, mouse and chicken. It contains the native sequence CD3 of all mammalian and non-mammalian species, including birds. In certain embodiments, the term "CD3" refers only to wild-type CD3. The antibodies of the present invention are directed to non-human CD3 from species, especially cynomolgus monkeys (Macaca fascicularis) It may cross-react with the CD3 from which it is derived. Examples of amino acid sequences are provided in Table 16. In certain embodiments, the antibody is fully encoded by human CD3. The antibody may be specific for the target antigen and may not exhibit cross-reactivity with non-human antigens.

[0136] As used herein, the term "antagonism" used in reference to the proteins of the invention. The drug “antibody” binds to a molecule expressed in the affected tissue and enhances the biological activity and / or function of that molecule. A protein that can inhibit downstream pathway(s) mediated by a factor(s). Examples For example, "anti-CD47 antagonist protein" (synonymous with "anti-CD47 protein" ) binds to CD47 and is involved in the biological activity of CD47 and / or CD47 signaling. Anti-CD47 refers to a protein that can inhibit downstream pathway(s) mediated by CD47. Antagonist proteins inhibit receptor binding and / or induce cellular responses to CD47 CD47 biological activities, including downstream pathways mediated by CD47 signaling, such as A protein that can be blocked, antagonized, inhibited, or reduced (in some cases significantly) For purposes of the present invention, the term "anti-CD47 antagonist protein" includes CD47 itself and CD47 biological activities (including but not limited to phagocytosis by cells of the myeloid lineage) (including its ability to enhance activity) or the result of activity or biological activity is significant All terms, headings, and functional states and characteristics that are substantially ineffective, diminished, or neutralized at any one time. It will be clearly understood that this encompasses:

[0137] The proteins of the present invention bind with greater affinity than they bind to other molecules. If the molecule ( For example, human CD47, human Her2, human CD3, human cMET, or human PD-L 1) and "specifically binds," "specifically interacts," "preferentially binds," "binds," or "Interact."

[0138] An "antibody molecule" refers to at least one antigen-recognizing polypeptide located in the variable region of an immunoglobulin molecule. Target specificity for carbohydrates, polynucleotides, lipids, polypeptides, etc. via the site As used herein, the term "antibody" refers to an immunoglobulin molecule capable of binding to a target antigen. A "molecule" refers to any antibody, not just an intact polyclonal or monoclonal antibody. Fusion proteins comprising an antibody or an antigen-binding fragment (e.g., an "antigen-binding portion") or a single chain thereof and, for example, but not limited to, scFv, single domain antibodies (e.g., shark and lambda da antibody), maxibody, minibody, intrabody, diabody, triabody Immunoglobulins that contain antigen recognition sites, such as tetrabodies, v-NARs, and bis-scFvs. Any other modified configuration of the purine molecule is included.

[0139] An "antibody molecule" refers to any antibody molecule, such as IgG, IgA, or IgM (or a subclass thereof). The present invention encompasses antibodies of any class, and the antibody need not be of any particular class. Depending on the antibody amino acid sequence of the constant region of the chains, immunoglobulins are assigned to different classes. There are five main classes of immunoglobulins: IgA, IgD, IgE, and Ig G and IgM, some of which are subclasses (isotypes), e.g. It can be further divided into lgG1, lgG2, lgG3, lgG4, lgA1 and lgA2. The heavy chain constant regions corresponding to different classes of immunoglobulins are The different classes of immunoglobulins are called a, delta, epsilon, gamma, and mu. The subunit structures and three-dimensional organization of ribonucleic acid are well known.

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

[0141] The term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain. The "Fc region" may be a native sequence Fc region or a variant Fc region. Although the boundaries of the Fc region of an immunoglobulin heavy chain can vary, the human IgG heavy chain Fc region It usually consists of a carboxyl group from the amino acid residue at Cys226 or from Pro230. The Fc region is defined as extending to the C1 terminus. The numbering of residues in the Fc region is according to Kabat. The Fc region of an immunoglobulin generally consists of two constant domains. As is known in the art, the Fc region may be a dimer or may exist in monomeric form.

[0142] A "variable region" of an antibody refers to the variable region of the antibody light chain or the variable region of the antibody heavy chain, either alone or in combination. As known in the art, the variable regions of heavy and light chains are Each is connected by three complementarity determining regions (CDRs), also known as hypervariable regions. It is composed of four framework regions (FR) that play a key role in the formation of the antigen-binding site of an antibody. When selecting FRs flanking the CDRs, for example, the antibody may be humanized or optimized. In such cases, FRs derived from an antibody containing CDR sequences of the same reference class are preferred.

[0143] As used herein, the term "conservative substitution" refers to a substitution that significantly alters the functional activity of an amino acid. A conservative substitution refers to a substitution of one amino acid with another that does not result in a deleterious change. The amino acid of the following BLOSUM 62 substitution matrix has a value of 0 or more. (Henikoff & Henikoff, 1992, PNA (see S 89: 10915-10919). [Table 1]

[0144] The term "monoclonal antibody" (Mab) refers to any eukaryotic, prokaryotic, or Antibodies derived from a single copy or clone, including a phage clone, or their antigen-binding Preferably, the monoclonal antibody of the present invention is The antibodies are present in a homogeneous or substantially homogeneous population.

[0145] "Humanized" antibody molecules are chimeric immunoglobulin molecules that contain minimal sequence derived from non-human immunoglobulin. Immunoglobulins, immunoglobulin chains, or fragments thereof (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequence of an antibody), ) refers to a form of an antibody molecule or an antigen-binding portion thereof. A humanized antibody is one that does not resemble the recipient's CD4 Residues derived from R are selected from mouse, rat, or rabbit antibodies with the desired specificity, affinity, and potency. The human immunoglobulin A (HIN) is replaced by residues from the CDRs of a non-human species (donor antibody), such as a human IgG. It may be a globulin (recipient antibody).

[0146] A "human antibody" or "fully human antibody" is an antibody produced by a transgenic mouse carrying human antibody genes. The term refers to an antibody molecule or antigen-binding portion thereof derived from mouse or human cells.

[0147] The term "chimeric antibody" refers to an antibody whose variable region sequences are derived from one species and whose constant region sequences are derived from another. is intended to refer to an antibody molecule or antigen-binding portion thereof derived from any species, including, for example, An antibody molecule whose variable region sequence is derived from a mouse antibody and whose constant region sequence is derived from a human antibody. It is intended that

[0148] The term "immunoconjugate" refers to a compound that contains at least one cytotoxic agent, cytostatic agent or therapeutic agent. It refers to a protein of the invention complexed, fused, or linked to an agent.

[0149] The proteins of the present invention can be produced by techniques well known in the art, for example, recombinant techniques, phagocytosis, and the like. display technology, synthesis technology, or any other technology or field thereof It can be readily produced using combinations with other well known techniques.

[0150] The term "isolated molecule" (e.g., whether the molecule is a polypeptide, polynucleotide, or antigen (i) a plant or organism that, by reason of its origin or its source of derivation, is associated with (2) a molecule that is not associated with components with which it is naturally associated; (3) a molecule expressed in cells from a different species, or (4) a molecule found in nature They are molecules that do not exist in the human body and therefore can be chemically synthesized or synthesized from naturally occurring cells. Molecules expressed in different cell systems are "isolated" from the components with which they are naturally associated. The molecule may also be purified by isolation using purification techniques well known in the art. It can be made substantially free of associated components. Uniformity can be assessed by several methods well known in the art. For example, the purity of a polypeptide sample can be determined using polyacrylamide gel electrophoresis and staining of the gel. The polypeptides are visualized and evaluated using techniques well known in the art. For certain purposes, purification may be performed using HPLC or other purification methods well known in the art. It is possible to obtain higher resolution by using other methods for fabrication.

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

[0152] The term "binding affinity" or "KD" refers to the affinity of a particular antigen-binding protein interaction or antigen KD refers to the dissociation rate of an antibody interaction. off ) also known as dissociation The rate of association, 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 The KD value of a binding protein or antibody is the binding affinity of the Bound tannins can be calculated using methods well established in the art. One method for calculating the KD of a protein or antibody is typically the Biacore (registered trademark) Surface plasmon resonance (SPR) using biosensor systems such as the NIRS (Nippon Imaging Technology) system There are some things that depend on it.

[0153] The term "potency" is a measure of biological activity and is also known as IC 50 , i.e., as described herein Binding partners (e.g., those expressed in diseased tissues) that are measured in activity assays such as those described herein. 50% inhibition of the activity of an antigen or a molecule capable of binding to an antigen (a molecule capable of binding to an antigen) or a binding partner thereof The concentrations are expressed as effective concentrations of proteins in the immunocomplexes.

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

[0155] The term "inhibition" or "inhibition" as used herein in reference to the biological activity of a protein of the invention "Neutralization" refers to, for example but not limited to, the abrogation of the biological activity or binding of a molecule expressed in affected tissue. substantially antagonizes or impedes the progression or severity of whatever is to be inhibited, including a synthetic interaction , refers to the ability of a protein to prevent, inhibit, slow down, destroy, eliminate, stop, decrease or reverse do.

[0156] A "host cell" is a cell that contains a vector(s) for incorporating a polynucleotide insert. An individual cell or cell that can be or has been a recipient. Host cells include the progeny of a single host cell, which progeny may be naturally, accidentally, or otherwise occur. or completely identical to the original parent cell (either morphologically or genomic DNA) due to deliberate mutations. A host cell may be capable of expressing the polynucleotide(s) of the present invention. This includes cells transfected in vivo with

[0157] As used herein, a "vector" refers to a vector that is capable of transmitting one or more genes of interest in a host cell. Constructs capable of delivering, and preferably expressing, the gene(s) or sequence(s) Examples of vectors include viral vectors, naked DNA or RNA expression vectors. vector, plasmid, cosmid or phage vector, bound to a cationic condensing agent DNA or RNA expression vectors, DNA or RNA encapsulated in liposomes Certain eukaryotic cells, such as NA expression vectors, and producer cells, Not limited to these.

[0158] The term "treating" as used herein, unless otherwise specified, includes such A disease or condition to which a given term applies, or one or more symptoms of such a disease or condition To reverse, improve, inhibit, retard, or delay the onset of a condition The term "treatment" as used herein means to cause, prevent, or Unless otherwise specified, the term "treating" refers to the act of treating as defined above. " 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 curative, Contains multiple references to palliative and preventative treatments.

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

[0160] Aspects or embodiments of the invention may be described in terms of Markush groups or other alternative groups. When a compound is used, the invention encompasses not only the entire group recited as a whole, but also each and every member of the group. , and all possible subgroups of the main group, as well as the main group in which one or more group elements are missing. The invention also encompasses any one or more of the group elements in the claimed invention. Explicit exclusion is envisaged.

[0161] Unless otherwise defined, all technical and scientific terms used herein are within the meaning of the present disclosure. In the event of conflict, In all cases, the present specification, including definitions, will take precedence. , the term "comprises" or "comprises" or Variations such as "comprising" refer to the inclusion of a recited integer or group of integers. It will be understood that the term refers to any integer or group of integers, but does not exclude any other integer or group of integers. Unless otherwise required by context, singular terms shall include plurals and plural terms shall refer to the singular. The terms "eg" or "for example" are used interchangeably. Any example(s) following "example" are not meant to be exhaustive or exclusive. It is not.

[0162] The techniques of the present invention are directed to, but not limited to, molecular biology (including recombinant techniques), microbiology, cellular biology, and the like. Conventional techniques of cell biology, biochemistry and immunology will be employed, which techniques are within the skill of the art. It is within the skill range.

[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, the inventors succeeded in generating a panel of optimized conditionally active antibodies. These conditionally active antibodies were successfully expressed, biophysically stable, and soluble. There is high and more preferred maximal amino acid sequence identity with human germline.

[0165] Materials and Methods Protein cloning, transient expression, purification and characterization The DNA sequences encoding the antibodies are isolated via restriction enzyme-ligation cloning. The clones were cloned into separate human IgG1 heavy and light chain encoding expression cassettes in a plasmid vector. Cloning and expression of IgG or IgG 2 A similar cassette was created. The mice were also cloned into a "knobs-into-holes" human IgG1 heterodimerized Fc vector. Fab and Fab 2 The construct was made using the knob-into-hole (KIH) heavy chain expression vector. vector (CH3 domain T366W and T366S / L368A / Y407V mutations) Using Fab 2 cMETCD47 and Her2CD3 protein constructs were generated. Also, Fab 2 The Her2-CD3 construct contained the effector function truncation mutation L234A / L2 35A / G237A. The "wild type" IgG1 heavy chain and kappa light chain expression vectors were used. Using IgG 2 Her2CD47 protein constructs were generated using manufacturer's protocol. Transient transfection with endotoxin-free IgG-expressing plasmid preparations according to After transfection, IgG was expressed in CHO cells.

[0166] HiTrap MabSele on AKTA Pure 150 L FPLC system Clarify generated antibodies using ct Sure Protein A 5mL columns The peak fraction of eluted protein A was collected from the supernatant. The eluted protein peak was immediately purified with 1xPBMC by loading directly onto a 1x PBS desalting column. The buffer was exchanged into 100% ethanol (pH 7.4). Protein concentrations were determined and diluted to 4-20% with 1x Tris / glycine / SDS buffer. TGX polyacrylamide gradient gel (BioRad, catalog number 456-1093) The purified proteins were separated for 1 h at 120 V electric field using reducing and / or non-reducing conditions. One microgram of the protein was analyzed by SDS-PAGE. To test and complement the SDS PAGE analysis, analytical size exclusion chromatography was performed. Fee was implemented. Superdex 200 Increase 10 / 300 SE Analytical analysis was performed using a C column and 1x PBS (pH 7.4) as the running buffer. Selected eluates were analyzed by size exclusion chromatography (SEC) in isocratic mode. Aliquots of the clones were analyzed.

[0167] Preparative SEC was used to further purify selected proteins. Antibody samples up to 1 ml 1×PBS (pH 7.4) equilibrated Superdex 200 Increase e 10 / 300 SEC column or HiLoad 26 / 600 Superdex The column was loaded with 200 pg of 1 ml of the peak of interest. After size exclusion chromatography, the fractions were analyzed by SDS-PAGE as described above. The samples were then reanalyzed.

[0168] red blood cell agglutination Fresh, unclotted red blood cells (RBCs) from human blood (minimum three different donors) The cells were isolated from the IgG and diluted to 2% in PBS and then inoculated into a U-bottom 96 well plate with IgG or protein constructs. The plate was incubated for 60-90 minutes and titrated. The cells settled to the bottom of the well and formed a red pellet. Hemagglutination was observed in the absence of precipitated R The images of each plate were recorded and the last plate where red blood cell aggregation was observed was Data was expressed per sample as well titer.

[0169] Metalloprotease digestion 5 mM CaCl 2 Protein assembly was performed in Tris-buffered saline (pH 7.4) containing The compounds were administered together with individual human matrix metalloproteinase (MMP) enzymes or with active MMP A mixture of P3, MMP7, and MMP12 (equal amounts of each) was used to stimulate the protein constructs. The total MMPs were mixed at a ratio of 1% (wt / wt) and incubated at 37°C for 16 hours. The reaction is stopped by adding 1 mM EDTA and then the samples are digested as described Binding or functional activity was tested.

[0170] IgG titration binding ELISA To coat the ELISA plate, the target protein was diluted in PBS (pH 7.4 ) to 1 μg / ml and added 100 μl per well for overnight at 4°C. The plate was washed three times with PBS (pH 7.4) and then diluted with PBS (380 μl / well). The cells were blocked with 4% skim milk protein at room temperature for 1 hour, and then with PBS-Tween The plate was washed five times with n20 (PBST). Antibodies (100 μl / well; diluted in PBST) were then added. The plate was then washed three times with PBS and incubated at room temperature for 1 hour. Goat anti-human IgG-HRP was added (100 μl / well) for 1 hour at room temperature. After washing the plates three times with PBST and twice with PBS, 100 μl of TMB was added per well. Add 100 μl of 2M H 2 SO 4 The reaction was stopped by adding The OD was read at 450 nm on a rate reader.

[0171] Flow cytometry binding assay Protein constructs (with / without predigestion with MMP3 / 7 / 12) and control I Binding of gG to Jurkat and BT-474 cells was measured by flow cytometry. Zombie UV™ Fixable Viability Dye ( Live cells were identified using a ELISA kit (Biolegend). Binding was detected using a FITC-conjugated goat anti-human (H+L) secondary antibody. Clonal anti-CD3 control antibody binding was determined using Alexa-Fluor-488 goat anti-mouse Ig Detection was performed using the FITC channel of a BD Fortessa flow cytometer. The products were evaluated by measuring the mean fluorescence intensity (MFI) of live cells with a fluorometer detector.

[0172] T cell activation bioassay BT474 cells and the NFAT-RE-luciferase Jurkat reporter cell line ( Promega-TCR / CD3 effector cells (NFAT) were used to The functional activity of the three protein constructs was assessed in a co-culture assay. BT-474 cells (40,000 cells / well) were cultured in Hybri-Care medium (ATCC). (Cells) were seeded into 96-well white clear-bottom tissue culture-treated plates and incubated with CO 2 In the incubator The plates were then incubated at 37°C overnight. The medium was removed and replaced with assay medium (supplemented with 10% FBS). Control antibodies or protein constructs (MMP3 / 7 / 12) were prepared in RPMI supplemented with 100 mM NaCl. The TCR / TCR+ ... CD3 effector cells (NFAT) were thawed, diluted, and then added to the assay wells. At 37°C, CO 2 After 6 hours of incubation in the incubator, the plate was Re-equilibrate to room temperature, add Bio-Glo reagent for 5-10 minutes, and measure the luminescence signal ( Luciferase activity was determined by RLU (reduced unit per unit time). The RLU / RLU ratio of the samples was calculated after subtraction of the end luminescence signal. The induction factor was calculated.

[0173] Molecular dynamics simulation MOE's AMBER10: EHT force field (Chemical Computing Group oup Inc., eight systems (linker sequences X1, X2, X3, and X4, and one of the linkers at GS sequence positions XC1, XC2, XC3, and XC4 is covalently cleaved. The nucleotide sequence (equivalent to the sequence of the nucleotide sequence that was previously cut) was modeled and optimized using Protonate3D in MOE. The histidine charges were assigned using the tool, which was allowed 10,000 steps without constraints. Three successive minimizations were performed with a final gradient of 0.001, which allowed for the NAMD 2.13 was used with the HARMM27 force field to perform incremental minimization and to obtain three successive The total potential energy was reduced by the energy minimization of the first step. In the second step, all the heavy atoms were constrained, allowing only the hydrogen atoms to move. The constraints are removed, and in the third step all atoms are released, allowing them to move without constraints. CHARMM27 is the force field used in NAMD molecular dynamics simulations , which made minimization within CHARMM27 easier.

[0174] To perform the dynamics simulation, the Generalised Born method was used to describe the solvent effects. (GB) solvation was used. MD simulations were set up and then run with NAMD2.13. A four-step equilibration was performed, with 12 h for a total equilibration time of 500 ps. The harmonic constraint was gradually weakened in steps of 5 picoseconds. In the first step, a force of 4 kcal / mol was applied. The system was heated to 310 K while adding the main chain, fixing it in place. , using the NVT ensemble (constant number (N), volume (V) and temperature (T)) at 4 kca The main chain was gradually released from l / mol to 1 kcal / mol. To reduce the Waals potential function, the switching function is set to 10 Å. The electrostatic and vdW cutoffs were set to 14 Å as recommended by NAMD. Periodic boundary conditions were not used because they are incompatible with the implicit solvent method. The constraints imposed during equilibration were lifted to allow for free simulation of coalescence. 310K Production runs were performed at either 6, 15, 20 or 100 ns. To fully probe the area, a 100 ns run was performed on the cleaved linker protein XC1-4. However, in order to map the migration range for the uncleaved antibody construct X1-4, Shorter runs (up to 20 nanoseconds) have proven sufficient.

[0175] In vivo analysis of PK and tolerability Tolerance Study - 28 6-8 week old male B6.Cg-Fcgrt tm1Dcr Tg( FCGRT)32DcrJ ("Tg32" homozygous human FcRn gene transfer, JAXA Stock number 014565) mice were divided into 7 groups, with 4 mice in each group. Body weights were measured on the day of antibody administration. At time 0, 2 mg / kg was administered as an IV injection. The test article was administered at 10 mg / kg or 10 ml / kg. L of whole blood samples were collected into EDTA on days 5, 29 and 60 (last bleed). White blood cells, neutrophils, eosinophils, basophils, lymphocytes, monocytes, hemoglobin, red blood cells, reticulocytes The serum was then used for CBC / Dif / Retic analysis, including erythrocytes, MCHC and MCV. Body weight was monitored once a week during the first month and then once a month until the end of the study.

[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 placed in eight groups, with four mice in each group. On the day before administration of the test article, 35 μl blood samples were collected from three Tg32 mice and then transferred to E The samples were collected during DTA and flow cytometry was used to test the binding of the test article to mouse red blood cells. Body weights were measured on the day of antibody administration and once a week until the end of the experiment. Into the eye, at 2 mg / kg or 10 mg / kg as an IV injection in a volume of 10 ml / kg Test articles were administered. Bleeding schedule: 30 min, 4 h, 1, 3, 5, 7, 10, 14, On days 21, 28 and 42, blood samples were taken from each mouse according to the bleeding schedule. A 25 μl blood sample was taken from each mouse according to the protocol. 3 Taken in EDTA The plasma samples were then collected, processed to plasma, and stored at -20°C. was assessed by ELISA in triplicate.

[0177] Tolerance study in NOD-SCID mice: NOD-SCID mice were divided into groups Mice were divided into four groups of three each. Body weight was measured daily. On day 0, mice were administered i.v. 8 mg / kg or 14 mg / kg intravenously, followed by 4 or 7 mg / kg every 5 days. The test article was administered three times.

[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 fee, 5 x 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 hour, trastuzumab: 0.0 032;0.016;0.03;50 μg / mL, IgG 2 Her47 LHL-L HLF: 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;50μg / The binding of the test sample to red blood cells was assessed using FITC AffiniPure. Measured using Goat Anti-Human IgG (1:200 dilution; 1 h incubation time), followed by flow cytometric measurement of FITC fluorescence intensity ( Cell analysis was performed using a BD LSR FortessaX-20 cell analyzer.

[0179] Metalloprotease and cathepsin digestion at pH 6.0 and 7.4 Protein constructs were dissolved in TBS (5 mM CaCl 2 (including pH 6.0 or 7.4) and individual human matrix metalloproteinase (MMP) or cathepsin enzymes. Both were incubated at 0, 2 °C at 37 °C with a total enzyme to protein construct ratio of 1% (wt / wt). The reaction was then incubated for 4, 8, and 24 hours. The reaction is stopped and the samples are then frozen before binding or functioning of the samples as described. The activity was tested.

[0180] In vitro protein stability analysis Forced Oxidation - For forced oxidation analysis, test articles in PBS were incubated with 0.5% H2O2 at room temperature. After 2 hours of treatment, the cells were stored at -80°C and then resuspended in Dionex Ultimate 300. 0RS HPLC system (ThermoFisher Scientific, Hem El Hempstead, UK) for SEC and RP analysis (intact and subunit antibodies). For intact antibody reduction, DTT was added at 0.33 100 mM final concentration, samples were incubated at 22°C for 1 h, and immediately purified by RP. and analyzed.

[0181] SEC analysis - Dionex Ultimate 3000RS HPLC system (T hermoFisher Scientific,Hemel Hempstead,U K) Acquity UPLC Protein BEH SEC column coupled to 200 Å, 1.7 μm, 4.6 mm × 150 mm (Waters, Els tree, UK) and Acquity UPLC Protein BEH SEC A 30×4.6 mm, 1.7 μm, 200 Å (Waters, El Chromatographic separation was achieved using a cyclohexanediaminetetraacetate (Stree, UK). The elution was performed over 10 minutes with a mobile phase of 0.2M potassium phosphate pH 6. The pH was 0.8 and 0.2 M potassium chloride. The flow rate was 0.35 mL / min. Detection was at 280°C. The measurement was performed using UV absorption at 100 nm.

[0182] Reverse Phase Analysis of Intact Antibodies and Subunits - Dionex Ultimate 30 00RS HPLC system (ThermoFisher Scientific, He PLRP-S 1000, 5 μm, 2 μm, coupled to a 1000mm diameter fluorophore (Hempstead, UK) .1 mm × 50 mm column (Agilent Technologies, Stockp Chromatographic separation was performed using 75% buffer (European Chemical Industry, UK). A (0.02% TFA, 7.5% acetonitrile in H2O) to 45% buffer B (acetonitrile) A linear gradient of 0.02% TFA in 1,2-dichloroethane to 7.5% H2O in 14 min was used. The flow rate was 0.5 mL / min and the temperature was maintained at 70° C. during the analysis. Detection was by UV absorption at 280 nm.

[0183] HIC Analysis - Dionex Ultimate 3000RS HPLC System (T hermoFisher Scientific,Hemel Hempstead,U K) TSKgel Butyl-NPR 4.6mm x 35mm HIC cartridge The rhamnoid plasminogen activator (TOSOH Bioscience Ltd., Reading, UK) was used. The chromatographic separation was carried out using 60% buffer A (100 mM sodium phosphate). 100 mM phosphate buffer (pH 7.0, 2 M ammonium sulfate) to 90% buffer B (100 mM phosphate A linear gradient from 0.1 to 1000 mg sodium (pH 7.0) was run over 9 minutes. The flow rate was 1.2 mL / min. Detection was by UV absorption at 280 nm.

[0184] Charge variant analysis - LabChip GXII Touch HT (Perki Protein charge variant analysis was performed using a 3D ELISA kit (Elmer) according to the manufacturer's protocol. The charge variant profiling of the test articles was determined.

[0185] BIACORE® Analysis of Fc Affinity to Human Fc Receptors The interaction affinity of the antibody protein was measured using the BIACORE® T200 instrument. In most analyses, His6-tagged FcγR was used to measure I, FcγRIIa (167R and 167H variants), FcγRIIb, FcγRI IIa (176F and 176V variants), and FcγRIIIb receptors (all Sin o Biological) was coupled to anti-HIS antibody by standard amine coupling techniques. The receptor-specific assay format was then used to capture the IgG1- and IgG2-specific IgG1 ... It was applied as follows:

[0186] FcγRI is a high affinity receptor for IgG1 monomers, and therefore has a 1:1 kinetic The analysis was performed as a "single cycle" analysis using a flow rate of 30 μl / min, 10 μl / min (HBS Load receptor protein to approximately 30 RU with -P+ (diluted to 0.25 μg / ml) and associate Purified antibody titrated from 0.411 nM to 33.33 nM with a dissociation time of 200 s and a dissociation time of 300 s. The experiment was carried out under the conditions of 5-point 3-fold dilutions of glycine pH 1.5. The resulting data were regenerated and analyzed using a 1:1 fit.

[0187] The interaction between monomeric IgG and the FcγRII and FcγRIII receptors is relatively Because of the low affinity interactions, "steady-state" affinity analysis was performed at a flow rate of 30 μl / min and 10 μl 1 / min (diluted to 0.25 μg / ml in HBS-P+) to approximately 60 RU of receptor protein Loaded, titrated from 33 nM to 24,000 nM with an association time of 30 seconds and a dissociation time of 25 seconds. The antibody was diluted 3-fold at 5 points and the experiment was carried out under the following conditions. Glycine pH 1.5 was injected twice. The cells were regenerated by addition 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 effectiveness of this class of potential drugs is the lack of a single antigen targeted by the antibody. However, it is not found exclusively in tumors, but is merely highly overexpressed in tumors. This extratumoral target expression allows sufficient antibody penetration into the tumor to exert a therapeutic effect. Risk of dose-limiting side effects when large amounts of antibodies must be administered to ensure This often results in a sink and antigen “sink” effect. One such example is the antigen CD47 (Figure 1A), the challenge in this case is to prevent their release into the bloodstream (e.g. For example, CD47, which is highly expressed (especially on red blood cells and platelets), is bound by intravenously administered antibodies. This is a "sink" that minimizes the amount of drug that penetrates the tumor (IgG is a high dose). Anti-CD47 binding of hemocytes also has significant toxicity. In fact, anti-CD47 antibodies can cause anemia and even cross-linking of human red blood cells. It is known that tumors pose a risk of hemagglutination in patients. Typically, the expression of enzymes such as MMPs that can accelerate IgG degradation is high, making them the “preferred” All of these factors contribute to the anti-tumor effect when target expression is not restricted to the tumor environment. Minimize the potential safety and efficacy of CD47 antibodies and many other types of anti-tumor targeted antibodies They work together to keep it to a minimum.

[0189] The anti-CD47 protein construct (Figure 1B) suppresses the high-risk By eliminating the binding of a common (but potentially powerful) target, antitumor The aim is to overcome the peripheral sink and toxicity problems encountered with native IgG. The results showed that the low-risk upper domain (e.g., Fas targeting another tumor antigen such as Her2) b domain), and on top of the binding domain for the high-risk lower domain (e.g., CD47) (N-terminal This is achieved by adding a linker (at the end of the domain) to the appropriate top domain / linker combination. By using a combination of these antibodies, we were able to completely block the binding activity of the lower CD47 domain. Second, the tumor targeting domain (e.g., Her2) is highly concentrated in the tumor environment. The protein construct linker system exploits elevated MMP activity in tumors to promote The car peptide is cleaved, exposing the CD47-binding domain, which is more readily accessible than the periphery. This design principle has been applied to many different structures. These formats may be applicable, examples of which are outlined below.

[0190] Protein Construct IgG 2 The design (Figure 2A) consists of IgG1, IgG2, IgG3, IgG 4. may be based on sequences derived from IgE, IgM or IgA and have effector functions In this construct, the four polypeptide chains are It encodes the ab domains (two Fab A and two Fab B) and four linker sequences. and may or may not have an immunoglobulin hinge region and an Fc domain. The ab A-linker domain (top) blocks the binding activity of Fab B (bottom) The target binding specificities of the upper and lower domains may be different to facilitate bispecific function. or may be the same to promote multivalent target interactions. By selecting a linker sequence, such as a peptide sequence, the expression of the peptide can be suppressed in non-affected tissues but not in the tumor environment. In the presence of high concentrations of proteases in Figure 2B). All linkers in the protein construct design are proteolytically cleavable. The first "fast" cleavage of the intact structure, which is "contained", occurs, and the middle can be cut in sequence creating an "open" activation state between the This allows Fab A and B from the protein construct to bind to their cognate targets. Potentially, the second linker in each Fab A-Fab B protein construct unit A slower secondary cleavage completely releases the Fab A domain from the structure, and the lower Fab domain is The “dissociated” domain is completely freed for off-target (but still localized) activity. A cleaved linker based on the immunoglobulin hinge sequence can be used to create a "closed" form. - also mediates immune effector functions (ADCC, C DC and ADCP), which may mobilize an increase in It is a known phenomenon in human patients (sometimes not).

[0191] Protein construct Fab 2 Designed for IgG1, IgG2, IgG3, IgG4, IgE , may be based on sequences derived from IgM or IgA and have effector function capabilities In this construct, two (Figure 3A) or three (Figure 3B) poles are The peptide chain consists of two Fab domains (one Fab A, one Fab B), two or more The above linker sequence is encoded, and the immunoglobulin hinge region and Fc domain are included. In this case, heterodimer pairing is controlled by mutations in the Fc. Again, each Fab A-linker domain may be derivatized or underivatized. b Blocks the binding activity of B and enhances the binding activity of B by selecting a linker sequence such as the lower hinge peptide sequence. It is suppressed in non-affected tissues but is expressed in high concentrations of linker-cleaving proteins in the tumor environment. In the presence of ase, it produces a structure that is rapidly cleaved, released, and eventually dissociated (Figure 1). 3A).

[0192] Cloning and expression of protein constructs Fabs with different linker domains (Table 1) 2 or IgG 2 Formed as a molecule To generate and purify 15 bispecific, conditionally active protein constructs, The DNA cassettes for each type of product (Table 2) were synthesized to construct human IgG1 heavy and light chains or The antibody was cloned into an expression vector encoding the "knobs-into-holes" heterodimeric Fc. Proteins are sorted by scientific name (format)-(target name [upper domain / lower domain)-(heavy chain linker The antibody was named using the light chain linker type (-type)-(light chain linker type). All proteins were produced by transfection and then analyzed by protein A affinity chromatography. It was purified by raffin.

[0193] The anti-HER2 variable region sequences used in the protein constructs disclosed herein are Variable region sequences of stuzumab. The anti-CD3 variable region sequence is the variable region sequence of OKT3 or SP34. The anti-cMET variable region sequences used in the disclosed protein constructs are described in WO2019 / 17 5186. The anti-CD47 variable region sequences used are those provided in WO2019 / 034895. It is.

[0194] Analysis of protein construct expression and purification characteristics Protein A purified protein was quantified and showed no significant differences when different linker types were used. It was shown that the protein yield was affected by the concentration of 1× PBS (pH 7.4) (Table 2). The preparations were also subjected to analytical size exclusion chromatography to quantify the proportion of the desired product. All three classes of bispecific proteins generated (cMET Analytical SEC for Her2CD47, Her2CD47 and Her2CD3 revealed that LH Constructs containing the L linker produced the highest yields of the desired product and percentage of the main peak. The most optimal combination of protein A was obtained (Table 2). PAGE analysis (Figure 4) also revealed that in addition to the short linker domains L1 and LH, the long linker domain - The clones containing domain L3 showed a high ratio of HMW and LMW products in the non-reduced samples. In addition, the reduced sample produced very heterogeneous products with a high proportion of LMW products. Suboptimal linker types were shown to result in significant production of undesired multimers and damage products. All constructs contained HMW impurities, suggesting that this may lead to a new formation of These HMW impurities were almost absent in the reduced lane, indicating that they were not reduced by SDS alone. These results suggest that the IgG antibody is a disulfide-linked dimer or larger that cannot be bound to the IgG antibody. 2 By design, both clones 12 and 14 showed high yields (Table 2), but clone 14 (L The desired product (containing the HL linker) was confirmed by SEC (90%) and SDS-PAGE. The results showed the highest yield and high homogeneity of the product (Figure 4).

[0195] A subset of the clones was then purified to generate fully purified monomeric protein. SEC chromatography was performed on the remaining protein samples from the preparative SEC Chromatography trays of clones 1, 2, 3, 4, 5, 6, 10, 12 and 14 in These analyses were further carried out on clones 1 (Fig. 5A), 3 (Fig. 5C), and 4 (Fig. 5D), 5 (Figure 5E) and 12 (Figure 5G) are around the main peak of the desired product (Figure 5A ~5H elutes at about 0.5-0.55 CV) and contains a significant proportion of high molecular weight and often In contrast, clone 6 (Fig. 5F), 14 (Figure 5H) and 10 (Figure 5I) exhibited a prominent and distinct major product peak. In the case of 6 and 7, the monomeric form was predominantly observed as shown by SDS-PAGE of the non-reduced sample. Efficient SEC purification of the protein was possible (Figure 6A). Attempts at EC purification were unsuccessful and still produced heterogeneous samples (Figure 6A). Her2CD3 Fab was isolated from the reduced sample as evidenced by SDS-PAGE. 2 Claw SEC-purified proteins from clones 7, 8, and 10 showed improved homogeneity, but not Her2. CD47 IgG 2 Of the clones, only clone 14 was found to be completely homogeneous and monomeric. Again, clones 6, 10 and 11, which contain LHL linkers on both chains, were used. These findings demonstrate that all 14 exhibited the most reproducible and beneficial properties. It was.

[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 trasts Mab, and A-D5 Fab-Fc (a monovalent form of the A-D5 antibody containing a single Fab domain) ) were assayed against human CD47, c-MET and Her2 proteins by direct binding ELISA. Titrated (unit: μg / ml) (Figure 7A). IgG 2 Format: Her2CD47-LH-LH and Her2CD47-LHL-LHL clones (Figure 7B), as well as Fab 2 Format of c METCD47-L2-L2 and cMETCD47-LHL-LHL (Figure 7C) also The control antibodies were analyzed in the same manner and showed the expected strong binding activity against their cognate targets. However, even at the highest concentration, there was almost no background for other targets. There was no evidence of any association with oxidative stress (Figure 7A).

[0197] Importantly, the extremely strong monovalent binding of A-D5 Fab-Fc was consistent with that of A-D5 anti-CD47 The intrinsic affinity and significant potency of the domains were demonstrated (Figure 7A), which are consistent with the proteins of the present invention. This is something that needs to be contained in order for the protein construct to be successful. Protein constructs Her2CD47-LHL-LHL (Figure 7B) and cMETCD47-LHL -LHL (Fig. 7C) also showed similar potency of their upper Fab domains against their cognate targets. When using this linker combination, however, the binding to CD47 was significantly higher than that to CD47. There was no binding signal, and the binding activity of the CD47 V domain was indeed completely inhibited. Importantly, Her2CD47-LH-LH showed high levels of human CD47. A background signal was observed, but to a lesser extent in cMETCD47-L2-L2. This indicates that the loss of binding affinity in the lower Fab can be precisely controlled by the linker selection. These findings suggest that the antibody A tested in this assay A protein construct containing the anti-CD47 binding domain of Her2CD47-LHL-L HL (Fig. 7B) and cMETCD47-LHL-LHL (Fig. 7C) were significantly higher than A-D5 IgG This suggests that the ability of IL-1 to bind to CD47 is more than 1000 times lower than that of IL-1. , a binding signal OD of 1.0 is approximately 1 x 10 -2 μg / ml, while The protein construct from et al. also gave a signal exceeding 0.2 at the highest concentration tested, 10 μg / ml. did not show any.

[0198] Since hemagglutination is the major toxic risk of anti-CD47 antibodies, human erythrocyte-based erythrocytes were Prioritized protein constructs were tested in a globulin aggregation assay. Control antibody anti-CD23 5a (mouse) and A-D5 anti-CD47, A-D5 Fab-Fc, MH7.1 anti-CM ET, anti-Her2 trastuzumab, IgG 2 Her2CD47-LH-LH and H er2CD47-LHL-LHL and Fab 2 Format: cMETCD47-L2-L 2 and cMETCD47-LHL-LHL were isolated from healthy donor 1 (Figure 8A), donor 2 (Figure 8B), and donor 3 (Figure 8C). In all three donors, the control antibody anti-CD235a, A-D5 Anti-CD47 and A-D5 Fab-Fc bind to their corresponding surface antigens on adjacent red blood cells. A-D5 Fab-Fc (cloning) showed strong concentration-dependent hemagglutination due to cross-linking of the chromosome. The low hemagglutination effect observed in the study was due to the fact that the antibody was purified only on a protein A column. The chromatin in this protein preparation was not completely purified to the monomeric state by SEC. This may be due to the presence of a small amount of functional dimers in the nucleosomes. The protein construct sample also showed no ability to produce hemagglutination effects, even at the highest concentration of 140 nM. This finding is consistent with the anti-CD47 binding of antibody A-D5 tested in this assay. IgG containing domains 2 and Fab 2 The protein construct in this format had a titer of 0.58 nM. The antibody showed a 241-fold lower ability to induce aggregation compared to A-D5 IgG1. It is suggesting.

[0199] The prioritized protein constructs were incubated for 2, 4, 8 and 24 hours. In addition, the cells were subjected to enzymatic digestion with human MMP3, 7, and 12 in a negative control. As an alternative, incubation was also performed for 24 hours in the buffer without the enzyme. The samples that underwent time-dependent digestion were analyzed using human Her2 and CD47 (Figure 9A, B) or human C -MET and CD47 (Figure 9C). No loss of binding was observed over time to either Her2 or c-MET. This is because the addition of proteases does not reduce the functional binding ability of the upper Fab domain. In contrast, the CD47-binding ability of all three protein constructs was significantly higher than that of MM. The increase in the levels of IgG clones was significant over time, depending on the P level. 2 Her2CD47-LH -LH (Figure 9A) again showed high background binding to CD47 and The increase in CD47 binding over time was the smallest. In contrast, clone IgG 2 H er2CD47-LHL-LHL (Figure 9B) and Fab 2 cMETCD47-LHL- LHL (Fig. 9C) again showed low background binding to CD47 (OD <0.2 at 450 nM) and a 2-hour incubation with both MMP7 and 12 After incubation, CD47 binding increased rapidly and was completely saturated by 24 h of incubation. The results showed that MMP3 is the most abundant MMP among the three. The slowest activity was observed for all three protein constructs after 24 hours. In some cases, CD47 binding signals were increased.

[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 2 Her2CD3-LHL-LHL was analyzed by ELISA (Figure 10A), flow cytometry (Figure 10B), and immunohistochemistry (Figure 10C). The results were analyzed by cytometry (Figure 10B) and CD3 reporter assay (Figure 10C). In ELISA analysis, all three proteins bound to Her2 (upper Fab domain). Although it showed the expected strong binding activity to the target, even at the highest concentration, it showed no binding activity to other targets. There was little or no background binding (Figure 10A). Protein Construct Fab 2 All of the proteins were either combined with a mixture of MMP3, 7, and 12 or The HER2+ human cell line BT47 was incubated overnight without any further analysis. In flow cytometry of binding to HER2, anti-HER2 trastuzumab showed strong binding. , Fab 2 Her2CD3-L1-LH and Fab 2 Her2CD3-L2-L2 Similarly, strong binding was observed before and after MMP digestion, while Fab 2 Her 2CD3-LHL-LHL showed a partial decrease in binding after MMP digestion, This suggests that the proportion of proteins in the “dissociated” state that have lost their upper Fab (Figure 10B). Fab 2 In the case of Her2CD3-LHL-LHL, the MMP digestion process was activated. Since this finding suggests that the IL-1 activation may be involved, the same samples were applied to a reporter assay. However, in this case, Her2+BT474 cells were found to express a measurable signal when CD3 was activated. The amyloid plaques were mixed with human CD3+ Jurkat cells that had been genetically engineered to provide a marker for the amyloid plaques. Data from the assay showed that, as expected, the bivalent anti-CD3 antibody OKT3 inhibited the expression of reporter cells. The protein construct directly activated the CD3 signal in the cells (Fig. 10C). Each showed a different profile. The Her2CD3-L2-L2 protein, which contains the cleavable nucleotide sequence ( Although it showed high background, no increase in CD3 activation signal was observed after MMP digestion. This is because the flexible linker in this construct allows for binding of Her2 by the upper Fab and Allowing partial activation of the lower Fab, but not CD3 This suggests that reciprocal ligation is not activating (Figure 10C). 2 Her 2CD3-L1-LH protein showed lower background activity in the assay However, a modest increase in signal was observed after MMP digestion. 2 Her2CD3- LHL-LHL shows measurable background signal in the absence of MMP digestion. The negative control trastuzumab and IgG1 isoforms were not detected in the undigested samples, indicating no transfer. It showed minimal activation of CD3, similar to the type control antibody, but potent activation in the MMP digested samples. Thus, the data in Figures 10A-C are consistent with the results obtained simply by expressing and purifying the protein. Her2CD3-LHL-LHL Fab 2 The format has high specific Her2 binding activity, low Background CD3 ligation activity and activity due to MMP cleavage of the LHL linker The best combination of properties, including high CD3 co-ligation activity only when This suggests that there is a combination of

[0201] Second generation construct cloning and expression The above multispecific Fab containing an LHL linker 2 and IgG 2 Depending on the performance of the clone, Another series of constructs was envisioned, with potential functional sequences in both tertiary structure and linker sequence content. The space was experimentally examined. The following clones were used to sample these parameters: was synthesized and assembled. 1. "One-arm" Fab 2 The protein clone "Met47-LHL-LH L” (Table 6, Figure 3B). 2. Modified "One-Arm" Type "Clone 10" Fab 2 The protein clone "H er23-LHL-LHL” (Table 7, Figure 3B). 3. "One-arm" Fab 2 The protein clone "Her23(34)-LH L-LHL” (Table 8, Figure 3B). 4. The Her2-binding domain of trastuzumab is used as the upper Fab, and clone A is used as the lower Fab. IgG incorporating the CD47-binding domain of -D5 2 A series of clones of type (Table 9). These clones are likely to be sensitive to enzymatic cleavage by a broad panel of MMPs. A mutant LHL-based linker sequence (LHLF linker), a human IgG1 middle hinge sequence A linker with a moderate extension (LHLM linker), or both (LH LMF linker). 5.Fab 2 Fab in which the module is located at the C-terminus of KIH-Fc 2 With protein One clone, “Fc-Her23(34)” (Table 13, Figure 12). 6. "One-arm" Fab containing two copies of C-Met Fab 2 With protein One clone, "MetMet-LHL-LHL" (Table 14, Figure 3B, Figure 13). This structure In this model, binding to the Met receptor in a bivalent form is achieved by a single LHL linker that acts as a protease inhibitor. This can only occur after being disconnected by Isolate these constructs by Protein A and size exclusion chromatography as previously described The product was successfully expressed and purified.

[0202] Second generation IgG 2 Analysis of constructs The above purified IgG2 The performance of the clones was examined in a series of further analyses. Loan A-D5 IgG1, IgG 2 Her2CD47-LHL-LHL, IgG 2 Her47-LHLF-LHL, IgG 2 Her47-LHL-LHLF and IgG 2 Human Her2, Her47-LHLF-LHLF (Table 9), and human and mouse C Binding to D47 was first examined (Figure 14). This analysis showed two things: 1) A -D5 anti-CD47 IgG1 protein shows high binding to both hCD47 and mCD47. 2) Four IgGs 2 All of the proteins showed high binding to hHer2. maintained binding signal but had no background binding to hCD47 or mCD47 There was no signal (Figure 14).

[0203] Clone IgG 2 Her2CD47-LHL-LHL and IgG 2 Her47-L HL-LHLF was compared with several proteases known to be overexpressed in human tumors, including That is, MMP7 (Fig. 15A), MMP8 (Fig. 15B), and MMP10 (Fig. 15C) The sensitivity of the enzyme to activation by incubation with 1,2-dichlorophenyl ether (pH 7.4) was examined. (Figure 15) and both proteins were able to activate hCD47 binding by each enzyme. However, IgG 2 Her47-LHL-LHLF was activated more rapidly in both cases Incubation with MMP12 showed that, in the case of this enzyme, both proteins We demonstrated that both proteins were able to activate hCD47 binding at equal rates (Figure 15D). In addition, incubation with MMP13 reduced the IgG 2 Her47-LHL-LHLF can activate hCD47 binding by this enzyme, whereas IgG 2 Her2 We demonstrated that CD47-LHL-LHL may not be activated (Figure 15E). Interestingly, incubation with the cysteine ​​protease cathepsin S also inhibited this enzyme. showed that in the case of (Figure 15F). These findings suggest that IgG 2 Fab in proteins 2 Module The peptide linker content of 100% increases the number of potentially active enzymes, resulting in an even faster It has been demonstrated that the proteolytic activation process can be "tuned" to occur quickly. To sample this potential, appropriate enzymes that promote sensitivity to specific classes of enzymes must be identified. Further linker designs of suitable length and content, e.g., ADAMS, cysteine, asparagine, on the activity of other disease-related metalloproteinases, such as phosphate and serine proteases. Using amino acid sequences that show proteolytic sensitivity, They envisioned a design that would provide either selective activation or

[0204] IgG 2 To examine the effect of activation on binding affinity, both Her2 binding and CD47 binding were We performed a Biacore assay that allows sampling of both the , control antibody and IgG 2 Her47-LHL-LHLF protein (2, 4, 8 or 24 h) were digested or activated by MMP12, and then incubated with anti-Fc antibody The soluble Her2 and CD47 ectodomain proteins were then captured on the chip surface via The binding affinity of trastuzumab IgG1 and A-D5 IgG1 ( Binding assays against 100% lysate (without enzyme digestion) were repeated at the beginning and end of the experiment. The data are shown in Table 17. These analyses showed that the KD values ​​calculated for each antibody were very high in each analysis. However, importantly, the Rmax values ​​(maximum values ​​at the highest analyte concentration) were similar. The large binding signal) was significantly reduced between the beginning and end of the experiment (e.g., trastuzumab). For mab, Rmax was 265.80 RU at the start and 144.13 RU at the end, which is anti-F c The activity of the antibody capture surface decreases after multiple regeneration cycles inherent in the Biacore method To examine the effect of MMP12 activation on target reactivity, Rmax and KD values ​​for both r2 (Figure 16A, Table 18) and CD47 (Figure 16B, Table 18) This analysis demonstrated that Her2 binding and affinity were significantly correlated with MMP12 activation over 24 hours. Importantly, the samples at 0 h (without MMP12 digestion) No CD47 binding (Rmax=0) was observed in the IgG4A-specific ... Both affinities were evident over time, starting from 2 h incubation and activation. These findings suggest that Fab 2 Bottom of the module Fab is completely inactive and remains an intact molecule until protease activation occurs. This demonstrated the inability to undergo target interactions. This observation is further illustrated in FIG. Here, no binding activity was observed even at a concentration of 400 nM for the CD47 analyte, whereas MM High binding to CD47 after 24 hours of P12 treatment was evident.

[0205] Based on the above findings, Fab 2 Cleavage of the linker peptide in the module (hence It was also demonstrated that the activation of the lower Fab was transient, which was consistent with the activation of the upper Fab. In diseased areas where both the target antigen and the enzyme capable of activating the lower Fab are highly overexpressed Fab is highly biased in activation toward the tissues. 2 Pharmacology for molecules containing modules This can provide a potential therapeutic benefit, allowing rapid drug accumulation and long retention in such affected tissues. This results in retention and high levels of activity.

[0206] Fab 2 In silico modeling of modular structure and molecular dynamics Fab 2 To understand the mechanism by which the modules may function, we carried out structural modeling and molecular dynamics analysis. Protein Structure Data Bank identifier 5TZT), IgG1 C47B222 (protein structure Databank identifier 5TZ2) and IgG1 B6H12.2 (Protein Structure Databank identifier The crystal structure of human CD47 ECD bound to the Fab domain of the 5TZU (linked nucleotide sequence identifier: 5TZU) was The protein modeling suite of MOE software was used as a template. A model was generated of anti-CD47 A-D5 Fab bound to CD47 ECD. The structure of trastuzumab Fab in complex with the two extracellular domains is shown in PDB structure 1N8 Obtained from Z.

[0207] Next, LHL and LHL between the upper trastuzumab Fab and the lower anti-CD47 Fab The F linker was modeled using the protein modeling suite of MOE software. To aid in modeling the linker, The C-terminus of the Fab construct was examined, as well as that of the heavy and light chain domains of the trastuzumab Fab. This helped define the structures from which the LB linker could be removed. The trastuzumab Fab heavy and light chain C-termini are located in the IgG1 Fab domain. It was predicted that the peptide may optionally have native interchain disulfide bonds connecting the amino acids.

[0208] The full-length IgG1-based model incorporating the anti-CD47 Fab described above was The structure of IgG1 b12 (protein data bank identifier 1HZH) was used as a template. The structural errors in 1HZH, such as missing structural regions, were corrected by The protein was remodeled and corrected using the protein modeling suite of MOE software. In this study, the IgG1 b12 Fab was replaced with an anti-CD47 Fab and an Fc hinge was added. This model is based on Her2 and CD47-based IgG 2 Molecular weight IgG1 and triglyceride The model shows that Her2 epitope binding occurs in the constitutive structure of the ribosome (Figure 18A). 18A, whereas CD47 ECD binding was related to its linker and The proximity of anti-CD47 Fab to anti-HER2 Fab completely occludes both (Figure 18B). The model was able to bind HER2 via the upper Fab without any observed hindrance. However, it does not bind to CD47 via the lower Fab until the linker is degraded. Fab 2 It is consistent with the module (Figure 16A, Figure 16B).

[0209] Fab 2 To gain further insight into how modules move in solution, The inventors have identified LHL-LHL, LHL-LHLF and L2L2(G4SG4S (SEQ ID NO: 32) Molecular dynamics simulations were performed using multiple linker compositions including the amino acid sequence (Table 1). The RMSD for each residue was calculated for each run. In addition, a custom description The dSASA is written in the SVL language (MOE), and each residue and in each case the phosphorus We calculated the change in the solvent accessible surface area (SASA) of the aggregates of only the Kerr sequence. We will investigate how different uncleaved linkers behave in terms of catalytic changes and sequence-specific cleavage potential. The associated effect on the solvent accessibility of the linker is expected to affect the potency (Figure 18). Provides a basis for comparison.

[0210] Figures 19A-19I show the corresponding solvent accessibility profiles obtained for the three linkers tested. The nine graphs of surface area (SASA) results are shown. In the first analysis, the LHL and LHLF 9 runs for each anchor and 10 runs for L2 (6 ns) The absolute SASA values ​​were sampled for each run (Figures 19A, 19D, and (Figure 19G). The data show that the L2 linker has the greatest propensity for structural heterogeneity over time. The initial SASA values ​​were not the same for each run (Figure 8G). Therefore, in a secondary analysis, we subtracted all SASA values ​​from the baseline SASA values. The results were normalized to represent the difference from the starting SASA value calculated using the 6 ns kinetics run. 19B, 19E, and 19H, respectively) and of the 6 ns kinetics run. The results over the first 2.5 ns (FIGS. 19C, 19F, and 19I, respectively) are The HL and LHLF linkers show significantly less structural dynamics than the L2 linker. In particular, a 6 nanosecond molecular dynamics run shows that L2 is the most mobile. Different linker sequences have different mobilities and hence Fab 2 For the lower Fab of the module The concept of improving the CDR solvent exposure profile of the siRNA-based siRNA will be highlighted.

[0211] Comparison of cleavable and non-cleavable linkers shows that This shows that the mobility of the linker cleaved by the cleavage is dramatically increased, and the biological viewpoints shown in Figures 14 to 17 are For example, Figure 20A shows the results of both phosphorylation reactions during a 100 ns kinetic run. Fab when car is intact 2 Limited movement of the module followed by L The upper Fab domain in the context of a single cleavage of the HL linker (secondary linker intact) This analysis shows a dramatic shift in the upper Fab domain, which is responsible for the large increase in autoantibody activity. As a result, the lower Fab domain is completely detached and free to interact with, for example, CD47. It is a complex configuration that can be moved to fully expose its CDRs, allowing for interaction. This was shown to be the case (Figure 20B).

[0212] In vitro and in vivo analysis of tolerability and pharmacokinetics Tolerance of IgG2 and Fab2 in the context of CD47 like lower Fab domains To investigate the pharmacokinetics, several exemplary molecules (A-D5 IgG1, IgG 2 He r47 LHL-LHL, IgG 2 Her47 LHL-LHLF and Fab2 Met 47 LHL-LHL) were cultured in 6-8 week old male B6.Cg-Fcgrt mice. tm1Dc r The study was conducted on Tg(FCGRT)32DcrJ mice. These "Tg32" mice Human I is a transgenic animal homozygous for human FcRn, mimicking that of humans and primates. A-D5 has the pharmacokinetic properties of IgG1 and IgG 2 Her47 LHL-LHL, IgG 2 Her47 LHL-LHLF and Fab 2 Met47 The CD47-binding domains contained in the lower Fab domain of LHL-LHL are all recombinant It has been shown to have the ability to bind to mouse CD47 protein, which is essential for the identification of mouse CD47 on red blood cells. Their reactivity to mouse membrane-presented CD47 was first examined by flow cytometry. This study demonstrated that A-D5 IgG1 inhibited the IgG1 expression in mouse erythrocytes at 0.1 μg / ml. No signal was observed at 1 μg / ml, whereas clear concentration-dependent results were observed at both 1 μg / ml and 10 μg / ml. We demonstrated that the binding to mouse erythrocytes was consistent with the IgG antibody (Figure 21). Near saturation at 1 μg / ml (63% binding, FIG. 21) and complete saturation at 10 μg / ml (98% binding, FIG. 21). In contrast, protein IgG 2 Her47 LHL-L HL, IgG 2 Her47 LHL-LHLF, or Fab 2 Met47 LHL Neither of the -LHLs showed binding at any concentration, nor did they bind to the anti-CD47 variable domain of the lower Fab domain. The domain showed no ability to interact with CD47 on mouse erythrocytes (Figure 21 ).

[0213] Hemagglutination assays were also performed using red blood cells isolated from Tg32 mice. Analysis showed that only A-D5 IgG1 was capable of promoting concentration-dependent aggregation of mouse erythrocytes. (above 3.12 μg / ml), whereas the protein IgG 2 Her47 LHL -LHL, IgG 2 Her47 LHL-LHLF, or Fab 2 Met47 L Neither HL nor LHL showed agglutination at any concentration up to 200μg / ml These findings support the previously outlined mechanism of action of the human CD47 protein. and erythrocyte binding (i.e., complete binding of A-D5 IgG1 to human CD47, However, IgG 2 Or Fab 2 (no measurable binding to proteins) o The test was demonstrated to be reproducible in the mouse system, providing insight into both pharmacokinetics and tolerability. This provides a viable model for studying the effects of CD47 binding on IL-1 and IL-2.

[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 was administered to Tg32 mice at a concentration of 2 mg / kg or 10 mg / kg for 1 h. The 2 mg / kg dose of A-D5 IgG was tolerated, but the 10 mg / kg dose was The g / kg dose was poorly tolerated and caused overt toxicity on day 0, with In contrast, IgG 2 Her47 LHL-LHL, IgG 2 He r47 LHL-LHLF and Fab2 Met47 LHL-LHL 2mg / kg and The 10 mg / kg and 10 mg / kg doses were all well tolerated over a 60-day body weight monitoring period (Figure 2). 3) The 2 mg / kg dose of A-D5 IgG was generally well tolerated, but On day 5, reticulocytes were significantly upregulated in this group. In contrast, IgG 2 Her47 LHL-LHL, IgG 2 Her47 LHL-LHLF or Fab 2 Met47 LHL-LHL 2mg / The 10 mg / kg or 10 mg / kg doses showed significant reticulocyte upregulation on day 5 or later. There was no association with the expression of erythrocytes (Figures 24 and 25). This is a known response to rapid red blood cell clearance, which is due to the A-D5 IgG1 antibody. These results suggest that IV administration of CD47+ erythrocytes leads to accelerated clearance of CD47+ erythrocytes.

[0215] To sample a broader range of effects of the administered protein, A complete hematology panel was performed on day 60 (Figures 25A-K). The reticulocyte upregulation effect of A-D5 IgG1 is transient and A-D5 IgG1, Ig G 2 Her47 LHL-LHL, IgG 2 Her47 LHL-LHLF and F ab 2 In Met47 LHL-LHL, red blood cell ( RBC) count, hemoglobin, mean red blood cell hemoglobin concentration (MCHC), mean red blood cell Regarding the MCV, leukocyte, monocyte, lymphocyte, basophil, eosinophil or neutrophil levels No significant effects were observed (Figures 25B-K).

[0216] In an in vivo pharmacokinetic study, IgG 2 Her47 LHL-LHL, IgG 2 Her47 LHL-LHLF and Fab 2 Met47 LHL-LHL, respectively The drug was administered intravenously once at a concentration of 2 or 10 mg / kg to Tg32 mice, and D5 IgG1 was administered at a previously tolerated concentration of 2 mg / kg. Le: 30 minutes, 4 hours, 1st, 3rd, 5th, 7th, 10th, 14th, 21st, 28th and 42nd days, according to Blood samples were taken from each mouse. Serum antibody concentrations were analyzed using 2 mg / kg A-D5 IgG1 is cleared very rapidly from the circulation, dropping below 0.5 μg / ml within 5 days. This rapid drug clearance (tissue-mediated drug clearance) was shown to reach an average concentration of 100 mg / kg / day (Figure 26). The mechanism by which this occurs (known as TMDD or TMDD) is due to the previously observed strong binding to mouse red blood cells. This may be due to the binding of IgG to IgG, which allows for rapid removal from the system by phagocytosis. The study further found that administration of A-D5 IgG1 resulted in normal reticulocyte levels by day 29. (Figure 25A), because the molecule was essentially eliminated by day 10 (Figure 26). This was to further explain the situation.

[0217] In contrast, IgG 2 Her47 LHL-LHL, IgG 2 Her47 LHL- LHLF and Fab 2 All of the Met47 LHL-LHL cases were treated with 2 mg / kg and 10 mg / kg doses. Both the 100 mg / kg doses showed slow clearance (Figure 26). A 2 mg / kg dose of protein required 25 min to reach a concentration below 1.0 μg / ml. It took more than 1.0 days (Figure 27A), and the 10 mg / kg dose of each protein produced 1.0 On day 0 of the tolerability study, the 10 mg / kg / mL concentration was maintained above 10 mg / kg / mL (FIG. 27B). A single sample from a mouse administered 1 g of A-D5 IgG1 was also analyzed and achieved The maximum serum IgG concentration observed (but not tolerated) was 1.0 mg / kg / day, which was fully tolerated. 2 and F ab 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 LH L-LHLF and Fab 2 All of the Met47 LHL-LHL cases were The standard "alpha" phase (when an IV dose is rapidly distributed from the bloodstream to tissues), followed by the antibody showed a long "beta" phase circulating in serum (Fig. 26, 27A). It also showed a standard distribution profile at 10 mg / kg, but with a longer blood circulation. (Figures 26, 27B). Importantly, the beta phase of each protein was The CD47 domains of these proteins form linear and parallel curves, with the A-D5 This suggests that TMDD, which was observed with IgG1, is not observed (Figure 26). 2 H er47 LHL-LHL, IgG 2 Her47 LHL-LHLF or Fab 2 If Met47 LHL-LHL proteins are activated in the periphery, TMDD is more severe. This is expected because activation results in high affinity erythrocyte, endothelial and platelet binding. This resulted in a sharp downward trajectory in the beta phase as seen with A-D5 IgG1. These observations are due to the fact that IgG clearance is replaced by IgG clearance (Figure 26). 2 H er47 LHL-LHL, IgG 2 Her47 LHL-LHLF and Fab 2 M Administration of et47 LHL-LHL did not result in reticulocyte amplification and peripheral activity This suggests that the activation levels are low despite the long pharmacokinetic profiles of these molecules. This means that they underwent multiple FcRn recycling cycles over 25 days of circulation. is.

[0219] IgG 2 Her47 LHL-LHL, IgG 2 Her47 LHL-LHLF and Fab 2 Pharmacokinetics and tolerability as outlined above for Met47 LHL-LHL The findings are consistent with the canonical FcRn-mediated antibody-like half-life of human IgG1 Fc domains. This effect was shown to be longer than that of A-D5 IgG1. The area under the curve (AUC) values ​​for quality were significantly increased (Figure 28). At 2 mg / kg, the AUC was , IgG 2 Her47 LHL-LHL, IgG 2 Her47 LHL-LHLF and Fab 2 Met47 LHL-LHL increased 25-40 times more than A-D5 IgG1 10 mg / kg was not a safely achievable dose for A-D5 IgG1. However, AUC is IgG 2 Her47 LHL-LHL, IgG 2Her47 LHL -LHLF and Fab 2 Met47 LHL-LHL was approximately 10 times more effective than A-D5 IgG1. The increase was 100 to 250 times (Figure 28). IgG 2 and Fab 2 CD47 binding within the construct The use of domains allows for high concentrations to be administered from the first dose, and distribution to tumor tissues is These AUC improvements are highly significant, as they suggest that maximizing This is what happens.

[0220] Flow cytometric analysis of binding to monkey and human erythrocytes Red blood cells were isolated from three cyno NHPs (monkeys) and three human donors and were analyzed using A-D5 IgG1, trastuzumab, IgG 2 Her47 LHL-LHLF or IgG 2 Both sets of analyses were stained with Her47 LHL-LHL. Only α-amyloid β-lactam induced concentration-dependent binding to either cyno (Figure 29A) or human (Figure 29B) erythrocytes. These findings demonstrated that IgG 2 (Therefore, Fab 2 ) structure The CD47-binding domain in is restricted to binding to mouse, monkey, and human CD47. The above observations were confirmed.

[0221] Activation by MMPs and cathepsins at pH 6.0 and 7.4 MMPs and cathepsins can be linked using either the LHL or LHLF linkers outlined above. We have already shown that it is possible to enzymatically cleave peptide sequences found in Importantly, both of these classes of enzymes have the ability to increase or decrease their enzymatic activity. The pH of solid tumors may be sensitive to changes in pH conditions in humans. Since deviations from the normal physiological pH of 7.4 are frequently observed, This can be very important, especially in solid tumors (and in highly inflamed tissues). ) can develop into an acidic pH state as low as pH 6.0.

[0222] Multiple MMP enzymes have the ability to activate the lower Fab bond at pH 7.4 Since the above-mentioned evidence has been provided, the present inventors have determined that multiple MMP and cathepsin enzymes (which (all of which are associated with increased activity in solid tumors) at both pH 6.0 and 7.4. MMP3 (Figure 30A, B), MMP7 (Figure 30C, D), MMP8 (Figure 30E) , F), MMP10 (Fig. 30G, H), MMP12 (Fig. 30I, J), MMP13 (Fig. 3 0K, L) and IgG against MMP14 (Fig. 30M, N) 2 Her47 LHL-L HL, or IgG 2 We then examined the activation of Her47 LHL-LHLF. G 2 Her47 LHL-LHL and IgG 2 Her47 LHL-LHLF MM P-treated samples were subjected to direct binding to both human Her2 and human CD47. Analysis showed that both proteins, MMP8, 10 and 12, were potent at both pH 7.4 and pH 6.0. We demonstrated that IgG showed measurable activation of CD47 binding. 2 H Only er47 LHL-LHLF was upregulated by MMP13 at both pH 7.4 and pH 6.0. It was activated by β-lactamase at pH 6.0 (Fig. 3K, L) and by MMP7 at pH 6.0 (Fig. 3 0D). IgG 2Her47 LHL-LHLF also inhibits IgG 2 Her47 LH Relatively higher levels of most of the MMPs tested at most time points than L-LHL. These findings suggest that the inclusion of the LHLF linker is essential for activation of the IL-1 domain. The lower Fa was activated by a broader range of MMP enzymes at both pH 6.0 and / or pH 7.4. b binding was confirmed to result in more rapid activation of the binding of

[0223] Cathepsins were also examined as potential activating enzymes. For these enzymes, pH and activity A clear relationship was observed between IgG 2 Her47 LHL-LHL (Fig. 31A) or IgG 2 Activation of Her47 LHL-LHLF (Figure 31B) was observed at pH At both pH 7.4 and 6.0, only cathepsin S is capable of activating CD47 binding In contrast, treatment with cathepsins A, C, G, K, and L resulted in However, little or no activation of CD47 binding was observed at pH 7.4. At pH 6.0, however, a strong activation signal was rapidly generated (Figure 31A, 3 1B). These findings suggest that the LHL and LHLF linkers promote more rapid activation by MMPs. at pH 7.4 through cleavage and pH-selective activation by a series of cathepsins. These results suggest that this may allow for accelerated activation in acidified tissues compared with This resulted in activation of the lower Fab bond at pH 7.4 (active extracellular MMPs and catecholamines). At pH 6.0 (where MMP and catabolism levels are low) and pH 7.0 (where MMP and catabolism levels are low) the levels were minimized. It is maximized at the pH of affected tissues where cathepsin levels are high and cathepsin activity is increased. By this, IgG2 and Fab 2 The therapeutic index of proteins can be further improved. Cut.

[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 H er47 LHL-LHL and IgG 2 Investigating the binding properties of Her47 LHL-LHLF Flow cytometry was performed to determine the expression of trastuzumab, anti-CD47 and isoforms. Staining with type control IgG demonstrated that BT474 cells express high levels of Her2 and lower levels of 32A,B), whereas MCF7 cells expressed higher levels of We demonstrated that the cells express CD47 and low levels of Her2 (Fig. 32C, D). IgG after activation with MMP12 for 8 and 24 hours 2 Her47 LHL-LHLF (Figure 32A, C) and IgG 2 Her47 LHL-LHL (Fig. 32B, D) Staining of both cell types was also performed. 2 Her47 LHL-LHLF and IgG 2 Both Her47 LHL-LHL were expressed in BT474 cells at 0, 2 and 8 hours. The binding profile was similar to that of trastuzumab, but binding was slightly reduced after 24 hours. In contrast, IgG 2 Her47 LHL-LHLF and Ig G 2 Both Her47 LHL-LHL were transfected with MCF7 cells at 0 h activation. It showed a similar low-level binding profile to rastuzumab, but at 2, 8, and 24 hours Upon activation, binding was significantly higher and mimicked the anti-CD47 control (FIG. 32C, D). These findings suggest that trastuzumab-like binding of Her2 is maintained after activation and The CD47 binding profile presented in Figure 2B appears to become highly activated only after activation. The activation model proposed here was experimentally confirmed to be valid. Importantly, these findings In addition, IgG 2 (And Fab 2 ) proteins are associated with low levels of the top Fab target (e.g., H er2) to cells expressing the lower Fab binding domain (e.g., CD47) These findings suggest that it may have beneficial abilities to promote function.

[0225] Analysis of MMP12 activation by SDS-PAGE and mass spectrometry IgG 2 Her47 LHL-LHLF and IgG 2 Her47 LHL-LHL Proteins were treated with MMP12 as described above for 0, 2, 8 and 24 hours. These protein samples were then analyzed by SDS-PAGE (Figure 33). Analysis showed that the activation of the IgG was significantly improved by ELISA (Figures 15-17, 30) and flow cytometry (Figure 32). ) were clearly correlated. The heavy chain, the 75 kDa marker, and the marker just below 50 kDa (below 50 kDa) At 2 hours, IgG light chains were observed. 2 Her47 LHL-LHLF and I gG 2 In both Her47 LHL-LHL, a new product of approximately 25 kDa was produced, and A faint band just above 25 kDa and about 50 kDa (above the intact light chain) The molecular weights of these new fragments were similar to those of the upper Fab Fd or light chain fragments (1 V domain + 1 C domain = approx. 25 kDa), Fc (2 × C domain + 1 n-linked glycoprotein) silyl = 28-30 kDa), and intact Fd-hinge-Fc (standard IgG1 heavy chain = 50 kDa), respectively. A gradual decrease in the amount of the intact chain and an increase in a fragment approximately 25 kDa smaller than the intact chain were observed. At 8 hours, intact heavy chain (75 kDa) and intact light chain (50 kDa) were (bottom) remained significant (but decreased compared to the 0-hour sample) at 25k The amount of Da (upper Fab chain) product was overwhelmingly greater. AGE analysis (Figure 33) showed that both linkers between the upper and lower Fabs were LHL or was shown to be actively cleaved by the MMP12 enzyme regardless of the LHLF sequence.

[0226] Fab 2 In the structure, the MMP12 enzyme is IgG 2 Precisely adjust the location of molecule activation In order to detect 2 Her47 LHL-LHL samples were collected at 0, 2, 8 and 24 hours. Mass spectrometry was performed using trypsin and Glu-C. These samples were prepared by reduction, alkylation, and proteolytic digestion using L The efficiency of MMP cleavage was analyzed by C-MS / MS. The MS response of the peptide derived from position (SCGPAPE (SEQ ID NO: 110)) and the cleaved By comparing it with that derived from the protein (SCGPAP (SEQ ID NO: 111)) This analysis determined that the uncleaved (SCGPAPE (SEQ ID NO: 110) ) linker and the cleaved (SCGPAP (SEQ ID NO: 111)) linker. We successfully identified the peptide that is involved in the LHL linker, and found that MMP12 is involved in the second protease. It has been shown that it cleaves between phosphorus (P) and glutamic acid (E). The relative signals of the SCGPAPE (SEQ ID NO: 110) peptide are 0 to 8, 8 to 24 The amount of cleaved (SCGPAP (SEQ ID NO: 34A)) decreased gradually with time (Fig. 34A). 111)) The signal of the peptide increased stepwise (Figure 34B).

[0227] Hinge-stabilized "IgG1-DAA" form of IgG 2 Generation and Analysis US Patent No. 8,871,204 B2 discloses lower hinge and Fc stability in the presence of MMP enzymes. These mutant types I teach protease-resistant IgG1 antibody variants that maintain For the gG1 antibody, the hinge is E233-L234-L235-G236 (SEQ ID NO: 112). The sequence is replaced by P233-V234-A235 (with G236 deleted). ; CH2 domain, S239D / 1332E, K326A / E333A, H268F / S324T / 1332E, F243L / R292P / Y300L, S239D / H268 F / S324T / 1332E, S267E / H268F / S324T / 1332E, K3 26A / 1332E / E333A, S239D / K326A / E333A, S267E / I332E and G237X / S239D / 1332E (X is A, D, P, Q or S) and the amino acid residues are selected from the group consisting of: are numbered according to

[0228] Fab related to protease-stable IgG1 Fc etc. 2 To check the use of structures In this study, two exemplary constructs were generated (Table 19). These two constructs (Her47 L HLF-LHL IgG1-2hDAA and Her47 LHL-LHLF IgG1- 2hDAA) is an IgG antibody based on the "2hDAA" structure. 2 Her47 Fab 2 The structure is arranged as follows (IgG1 is P233-V234-A235-ΔG236 and S239D / K326A / E333A mutations). Both constructs were transiently transfected with CHO cells. Protein A was readily expressed in cell transfection and purified to the expected molecular weight. The amount of the product was more than 80%. Her47 LHLF-LHL IgG1-2hD An example of analytical SEC data for AA showed 80% product in 10.30 ml (Figure 35). Non-reduced Protein A purified protein and SEC peaks 8.47, 9.03 and 10.3 SDS-PAGE analysis of 0.0 ml (Figure 36) showed that peaks 8.47 and 9.03 are high molecular weight Peak 10.30 contains aggregates, while peak 10.30 contains the expected size product (approximately 250 The purified protein and SEC peak 8.4 kDa were shown to contain reduced protein A. SDS-PAGE analysis of 7, 9.03 and 10.30 ml (Figure 37) showed that all peaks 8 0.47, 9.03, and 10.30 ml contained the expected size heavy and light chain products (respectively Her47 LHL-LHLF Ig The intact monomer (250 kDa) product of G1-2hDAA was purified by SEC. , human MMP12 was used for time-course incubations of 2, 4, 8 and 24 hours. H7.4 enzyme digestion and, separately, in buffer without enzyme as a negative control. 24-hour incubation (0, 2, 4, 8, and 24-hour incubation) In ELISA analysis, all samples showed strong binding signals to human Her2. However, there was no measurable binding of the control protein to mouse EpCAM (Figure 38A The CD47 binding ELISA was performed at 37°C in the presence or absence of MMP12 enzyme. After 2, 4, 8 and 24 h of incubation, the binding signal increased, but MMP No signal above background was observed at 0, 2, 4, 8 or 24 hours without 12. No changes were observed (Figure 38B).

[0229] SDS-PAG of 0-hour (undigested) and 2-, 8-, and 24-hour MMP12 digested samples Analysis by E showed that the purified protein contained heavy and light chains of the expected sizes. A 25 kD band was generated after MMP12 digestion for 2, 8, and 24 hours (Figure 3). 8C), showing linker peptide cleavage, while damage in the heavy chain was not observed in IgG 2 Her47 LHL-LHL or IgG 2 Her47 LHL-LHLF is significantly less than It was confirmed that the Fc region was stabilized (Figure 33), which indicates that the "2hDAA" mutation indeed stabilizes the Fc region. We demonstrated that it resists MMP12 digestion.

[0230] Alternative enzyme-activated IgG 2 Generation and Analysis The LHL and LHLF linkers are susceptible to cleavage by multiple MMP and cathepsin enzymes. As shown above, the six Fab 2 Construct type IgG 2 Inspected in the format ( These constructs include those that demonstrate anti-inflammatory properties in solid tumors and highly inflamed tissues. Several classes of enzymes are associated with upregulatory activity, e.g., enterocytes. endokinase (EK), thrombin (Thr), tPA, granzyme B (GrB), uP A series of linker designs that are easily cleaved by ADAMTS-5 (A5) (Table 21) were used. All six constructs were readily expressed by CHO cells and were Purified by oA chromatography.

[0231] Clone Her47-LHL-LHL-EK, Her47-LHL-LHL-Thr, Her47-LHL-LHL-tPA, Her47-LHL-LHL-uPA, Her4 Purified proteins from 7-LHL-LHL-GrB and Her47-LHL-LHL-A5 All proteins were tested in a titration ELISA against human Her2 and CD47 targets (Figure 1). 39A, 39C, 39E, 39G, 39I, 39K). In all cases, these The protein showed low / background binding to CD47 at all concentrations tested. The antibody showed a concentration-dependent binding to Her2 (white bars), but not to Her2 (gray bars). Each protein was subjected to sequential enzymatic digestion with MMP12 to identify Her2 and CD47 targets. ELISA for binding to 39B, 39D, 39F, 39H, 39J, 39 L). These findings suggest that all proteins bind Her2 over the time course of enzyme activation. Clone Her47 maintained the cytotoxic activity of HER2-dependent activating factor 1 (CD47) and showed increased activation of CD47 binding. -LHL-LHL-EK, Her47-LHL-LHL-Thr, Her47-LHL- LHL-tPA, Her47-LHL-LHL-uPA, Her47-LHL-LHL- The functional activity of GrB and Her47-LHL-LHL-A5 resulted in multiple binding sites in the Fab2 construct. Linker peptide sequences with a variety of protease recognition sites are available to tailor the peptide to specific applications. For example, in this case, the MM at the LHL linker was Retention of P activation potential is due to the presence of six different disease-related enzyme cleavage motifs in the associated linker. - In combination with either The effect of this is to regulate enterokinase, thrombin, tPA, Granzyme B, uPA, ADAMTS-5, or other protease enzymes are involved in disease states In such cases the activation potential is increased.

[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, I gG 2 Her47 LHL-LHLF, Fab 2 Her47 LHL-LHL and F ab 2 Her47 LHL-LHLF were injected four times into NOD-SCID mice. IgG 2 Protein was administered at 14 mg / kg on day 0, and at 5, 10 and 7 mg / kg on the 15th day. 2 Protein was administered at 8 mg / kg on day 0; and 4 mg / kg on days 5, 10, and 15. All proteins were fully tolerated. There were no clinical signs of toxicity or weight loss in any of the individual animals. These findings are consistent with the previously outlined single-dose phenotype in Tg32 mice (Figure 40). Her47 Fab 2Confirmed and elaborated on findings of tolerability to the base molecule In a Tg32 mouse study, even a single dose of A-D5 CD47 IgG was administered at 10 In this NOD-SCID mouse study, IgG 2 He r47 LHL-LHL, IgG 2 Her47 LHL-LHLF, Fab 2 Her 47 LHL-LHL and Fab 2 All four doses of Her47 LHL-LHLF This finding supports the notion that the CD47-binding domain of the Her47 molecule It was confirmed that the drug was not activated in the blood circulation even after repeated administration. If CD47 was activated, it would have induced the CD47-promoted toxic signal observed with A-D5 IgG1. There will be.

[0233] Additional two-stranded Her2CD3 Fab 2 Generation and analysis of "one-arm" constructs Additionally, the ability to generate Her2CD3 constructs and improve product uniformity and activity has been demonstrated. All Her2CD3 constructs were transformed with CHO cells. These fragments were expressed in 100% ethanol, purified from the supernatant by ProA, and analyzed by SEC. Analysis is Fab 2 Her23 LHL-LHL-S and Fab 2 Her23 LHLF- Both LHL-S (Figures 41A and 41B) have a high ratio of high and low molecular weight products. Fab showed that 2 Her23 LHL-LHL and Fab 2 Her23 LHL- The results show that the main product is more homogeneous than both LHLF (Figures 41C and 41D). These findings support the conclusion that the chains are [VL-CL-linker-VH-CH-Fc] and [V H-CH-linker-VL-CL-Fc] 2 Her23 LHL-LHL-S and Fab 2 Coordination of the upper Fab of Her23 LHLF-LHL-S This suggests that the uniformity was improved by swapping the two chains. Both are [VL-CL-linker-VL-CL-Fc] and [VH-CH-linker- VH-CH-Fc] 2 Her23 LHL-LHL and Fab 2 Her 23 Contrast with LHL-LHLF.

[0234] Next, the control protein Fab 2 mEpCam3 LHLF-LHL-S and Fab 2 mEpCam3 LHL-LHL-S (upper Fab is anti-mouse EpCAM antibody G8.8 A clone Fab (containing the V domain of 2 Her2 3 LHL-LHL-S, Fab 2 Her23 LHLF-LHL-S, Fab 2 m EpCam3 LHLF-LHL-S and Fab 2 mEpCam3 LHL-LHL- The desired MW product of S was isolated by preparative SEC and cultured as human Her2+ target cells using MCs. Promega's Jurkat cell-based CD3 The ligation reporter cells were analyzed in a bioassay (according to the manufacturer's instructions). Primary analysis using MCF-7 as control cells (which have very low Her2 expression) showed positive vs. Both the control Her2-CD3 BITE protein and OKT3 IgG1 showed strong positive concentrations The results showed that MMP12-treated clonal cells generate a CD3-dependent activation signal. Fab 2 mEpCam3 LHLF-LHL-S and Fab 2 mEpCam3 No signal was generated in LHL-LHL-S at any concentration (Figure 42A In contrast, clone Fab 2 Her23 LHLF-LHL-S (Fig. 42A) and Fab 2 Both Her23 LHL-LHL-S (Figure 42B) were measurable at 0 hours. Although there was no detectable signal, the concentration range was similar to that of Her2-CD3 BITE, and the signal was observed at 2 and 8 and 24 h, the signal gradually increased and in both cases exceeded that achieved with the positive control. The maximum signal was higher than that of the control.

[0235] In a secondary assay using Her2-highly expressing BT-474 cells as target cells, All test samples and controls were evaluated at 0.1 μg / ml (FIGS. 43A-C). Both the proteins Her2-CD3 BITE and OKT3 IgG1 have strong CD3 activation signals. induced signal transduction, whereas the negative control Fab 2 mEpCam3 LHLF- There was no signaling with LHL-S and trastuzumab. Clone Fab 2 Her 23 LHLF-LHL-S (Figure 43B) and Fab 2 Her23 LHL-LHL-S (Figure 43C) showed no measurable signal at 0 hours, but gradually increased at 2 hours. The signal gradually increased and reached a maximum at 8 hours. Importantly, at 24 hours, The signal was significantly reduced compared to that of the control. This finding may be due to the cleavage of both linkers. The stepwise enzymatic activity (Figure 33) ultimately leads to the separation of the upper and lower Fabs. This suggests that such molecules may be involved in the development of highly proteolytic tumors. It is preferentially activated in the tumor environment, where it gradually becomes inactivated and leaks out in an active form to the healthy tissue. Ideally, we would want to minimize the risk of the molecule entering healthy tissues, so we would target CD3. This can be a beneficial property for the molecule.

[0236] Her2-CD47 IgG 2 and Fab 2 Protein Molecular Stability in Vitro measurement Historically, engineered antibody formats have often faced structural heterogeneity and manufacturing challenges. IgG 2 Her47 LHL-LHL and Fab 2 He To examine the stability of the r47 LHL-LHL protein, a series of in vitro measurements were performed. The decision was made.

[0237] Forced oxidation - Oxidation of exposed amino acid residues such as tryptophan and methionine occurs at mA b, which affects their biological activity. In this study, PBS Forced oxidation of IgG with 0.5% H2O2 in 0.5% CO for 2 h at room temperature 2 Her47 LH L-LHL and Fab 2 Her47 LHL-LHL protein. The overall affinity of the antibody is increased by the increased polarity of the oxidized form or through conformational changes. Potential changes caused by forced oxidation may change the hydrophobicity of the sample, so reversed-phase (R The chromatographic analysis was performed using size exclusion (SEC) and chromatographic techniques. It was revealed that the ratio of monomeric species did not change in either test sample (IgG 2 H er47 LHL-LHL and Fab 2 Both Her47 LHL-LHL are pre-oxidative and After oxidation and precipitation, the monomer species was 99.5 and 97.4%, respectively. In the RP analysis, 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 Her47 The retention time of LHL-LHL was observed to be shorter by 0.4 min. In the analysis, IgG after forced oxidation 2 In Her47 LHL-LHL, the retention time of the heavy chain was A 0.5 min reduction in retention time and no shift in the retention time of the light chain was observed. 2 Her47 LHL-LHL showed a different profile in RP analysis, with light chain, heavy chain and After oxidation, three peaks were observed, representing the heavy chain and the truncated hinge Fc of the stem. A shift of 0.4 to 0.5 min was observed for the hinge Fc but not for the light chain. The retention time shift was due to the reduced IgG after H2O2 treatment. 2 Her47 LHL-LHL and F ab 2 This was observed in both Her47 LHL-LHL samples, which is due to the exposed amino acid This suggests minor oxidation of the Fc region of the protein.

[0238] Charge variant analysis - Charge heterogeneity analysis provides insight into product quality and stability. Heterogeneity is important in the characterization of monoclonal antibodies because it provides important information. Post-translational modifications of the enzyme (glycosylation, lysine cleavage) or chemical modifications during purification and storage (oxidation) or deamidation). Charge variants of the test article provided Profiling was performed by a commercially available charge variant assay. IgG 2 H er47 LHL-LHL (Figure 44A) and Fab 2 Her47 LHL-LHL (Fig. 4 4B) charge variant profiles of both chromosomes are consistent with one major isoform (overall The major acidic isoforms (40–48% of the total) and one It showed a homogeneous profile including the inner basic isoform (approximately 3%).

[0239] Retention in HIC – Overall hydrophobicity is a significant risk of aggregation and viscosity during bioprocessing It is an indicator of the tendency of a protein to self-associate, which can be a factor in determining whether a protein is self-associated. In addition, they have hydrophobic "patches" created by the presence of hydrophobic or nonpolar amino acid side chains. Depending on their number, size and distribution, the resulting surface hydrophobicity varies from protein to protein. HIC is a method to detect the reversible binding between proteins and the hydrophobic surface of the HIC resin. is used to separate proteins based on differences in their surface hydrophobicity. 2 Her 47 LHL-LHL and Fab 2 Her47 LHL-LHL, 5.4 These values ​​were significantly higher than those of adalimumab (4.5 min). ), cetuximab (5.9 min), brentuximab (6.3 min) and golimumab (8.1 min). This shows a lower range of hydrophobicity compared to values ​​obtained with clinical monoclonal antibodies such as In fact, these values ​​indicate that the aggregation tendency and stability are 2 Her47 LH L-LHL and Fab2 Both Her2-binding domains of Her47 LHL-LHL were induced This suggests that the retention time is similar to that of the anti-Her2 trastuzumab (5.4 min). .

[0240] Freeze-thaw stability analysis - increased protein aggregation or fragmentation poses a risk of reduced product quality Protein instability during freeze-thaw procedures is a major concern in manufacturing and bioprocessing because it can lead to It is an indicator of impairment in the 2 Assess this risk for protein containing structures For this, IgG 2 Her47 LHL-LHL and Fab 2 Her47 LHL-L The HL protein was subjected to five freeze-thaw cycles, with subsequent SEC analysis performed after each cycle. These analyses were performed for IgG 2 Her47 LHL-LHL (Figure 45A) or Fab 2 H Neither the er47 LHL-LHL (Figure 45B) protein showed any significant change over the five freeze cycles. No change in monodispersity was observed (no aggregation or damage products were observed). (It was.)

[0241] Taken together, these findings suggest that IgG 2 Her47 LHL-LHL and Fab 2 H Both er47 LHL-LHL proteins have low aggregation risk, low hydrophobicity, and low charge disulfide. It was shown to have homogeneity and low tendency to oxidation.

[0242] BIACORE® Analysis of Affinity of IgG Variants to Human Fc Receptors Antibodies targeting receptors on diseased cells are the desired targets for mediating ADCC and ADCP activity. These binding functions are maintained in Fab2-based constructs. To investigate whether IgG 2 Her47 LHL-LHL and Fab 2 Her47 LHL-LHL protein was identified in all human The isoforms were examined for their binding affinity to human and mouse Fc receptors. Both type control human IgG1 and IgG4 expressed high levels of FcγRIIA and FcγRIIIA. For all human Fcγ receptors, including both affinity and low affinity variants ( The results demonstrated that the compounds exhibited the predicted strong and weak binding affinities, respectively (Table 23). Similarly, the isotype controls mouse IgG2 and IgG1 were expressed as mouse FcγRI, Fc Predicted strong binding affinity for the γRIII and FcγRIV receptors (respectively) and It showed weak binding affinity. IgG 2 Her47 LHL-LHL and Fab 2 Her 47 In the case of LHL-LHL, each of the human and mouse Fc receptors tested Binding to IgG1 was very similar to that observed with the isotype control human IgG1. These data support the idea that IgG1 Fc-based IgG 2 and Fab 2 protein Both bind to Fc receptors upon binding on the surface of diseased cells in both humans and mice. This suggests that there is an ability to do so.

[0243] Further protein construct design Further protein constructs were considered (Figure 46). The constructs may include: 1. Only constant domains in the "upper Fab" position. This means that the activity of the "lower Fab" is determined by its target It is blocked from binding to the protein by enzymes, but can become active in the right proteolytic environment. 2. A dummy "non-binding" variable domain in the "upper Fab". These dummy variable domains have been shown not to bind to any known targets in the body. Therefore, only the "lower Fab" shows potential drug target binding ability and is It appears only after proteolytic activation of one of the anchor domains. ab" is replaced by a "diabody" structure containing four variable domains. The diabody structure of this protein does not contain disulfide bonds like those found in the "DART" proteins. The diabody construct may or may not contain a diabody targeting the same target or two separate targets. This allows the binding of two copies of the target protein in a suitable proteolytic environment. The activity of the "lower Fab" is blocked from binding to its target until activation. In any of the constructs envisaged or shown above, Fab 2 The structure is free or other functionalized structures, such as Fc fragments, albumin binding moieties, etc., that extend half-life. They may also be fused to small domains or peptides that extend the length of the Chemically conjugated to small molecules, peptides, or other proteins that mediate biological functions It is okay to do so.

[0244] Cell proliferation analysis of Her2CD47 protein using Her2-high BT-474 cells IgG 2 Her47 and Fab 2 Her2 binding to the upper Fab of Her47 protein The binding domain is capable of mediating inhibition of the kinase activity of this receptor, thus inhibiting cell proliferation. This assay uses cells known to respond sensitively to Her2 inhibition. The BT-474 cell line was used. Trastuzumab, isotype control IgG1, IgG 2 Her47 LHL-LHL (Figure 47A) and Fab 2 Her47 LHL -LHL (Figure 47B) was incubated with BT-474 cells for a 72 hour incubation period. The cells were dosed with 100 mg of 10 ... (Figure 47). These analyses demonstrated that trastuzumab significantly inhibited BT-474 cell proliferation in a concentration-dependent manner. Inhibitory effect, but IgG 2 Her47 LHL-LHL (Figure 47A) showed slightly lower efficacy. Power and Fab 2 Her47 LHL-LHL (Fig. 47B) also shows low potency ( It was shown that the binding ability of the valence

[0245] Herceptin expression in tumor xenograft-bearing NOD-SCID mice (KYSE-410 model) In vivo efficacy analysis of 47 molecules In vivo repeated dose efficacy studies, trastuzumab, IgG 2 Her47 L HL-LHL, IgG 2 Her47 LHL-LHLF, Fab 2 Her47 LH L-LHL and Fab 2 Her47 LHL-LHLF (human IgG1 as in Fig. 3B) Fab containing Fc 2 The structures were then used to express Her2 in esophageal cancer cell line KYSE-41. The tumor-bearing NOD-SCID mice were administered 4 doses (intravenously) of Intravenous injection was performed on days 0, 5, 10, and 15. 2 After the development of tumors exceeding 100 μg / kg, Administration of IgG was started. 2 Protein was administered at 14 mg / kg on day 0, and at 5, 10 and 20 mg / kg on day 1. 7mg / kg was administered on the 15th day. Fab 2 Protein, 8mg / kg on day 0, Trastuzumab was administered at 8 mg / kg on day 0, 4 mg / kg on day 5, 10, and 15. / kg, and 4 mg / kg on days 5, 10, and 15. Tumor volumes were measured using calipers. Measured using a measuring device.

[0246] After three doses, on the 11th day, trastuzumab (Figure 48A), IgG 2 Her47 L HL-LHLF (Figure 48B), IgG 2 Her47 LHL-LHL (Figure 48C) and Fab 2 All of the Her47 LHL-LHLF (Figure 48D) showed significant differences compared to vehicle. showed a decrease in tumor growth (2-way ANOVA: p=0.005, 0.005, respectively). , 0.019 and 0.003). In contrast, Fab 2 Her47 LHL-LHL (Fig. 48E) did not show a significant reduction in tumor growth by day 11 (2-way ANOV A: p=0.66). Importantly, Fab 2 Her47 LHL-LHL and Fab 2 The sequence of Her47 LHL-LHLF is Compared with 47F. Furthermore, the data in FIG. , One-Arm Fab 2 In the structure, Fab 2 Her47 LHL-LHL and Fab 2 The same Her2-binding VH and VL domains are seen in both Her47 LHL-LHLF. Her2-enhanced inhibition of cell proliferation was greater than that observed with trastuzumab containing the main sequence. The results are as follows: 1. Trastuzumab Fab (Figure 48A) 2 This was observed in both Her47 LHL-LHLF (Figure 48D). The nearly equivalent efficacy on day 11 was observed for Fab 2 High He in Her47 LHL-LHLF 2. Therefore, Fab cannot be stimulated by r2 kinase activity. 2 Her47 L High potency of HL-LHLF is due to protease activation in the KYSE-410 tumor microenvironment This is likely driven by CD47 inhibition and innate immune involvement.

[0247] Thus, the findings outlined above suggest that in aggregates, Fab 2 The structure is, for example, As shown, the effect of both the CD47 and CD3 binding domains on the activity of the “lower Fab” The binding capacity of the "upper Fab" domain is demonstrated to be completely Although the activity of the lower Fab domain is maintained in the cytoplasm, significant activity of the lower Fab domain is observed in diseased tissues such as tumors and fibrotic tissues. only after activation by proteases such as MMPs and cathepsins, which are associated with high activity in tissues. Observed. Fab 2 By adjusting the linker sequence in the construct, As exemplified by measuring the ability of the Her47 molecule in vivo, Adjusting lower Fab activation to maximize and avoid both peripheral sink problems and toxicity It will be possible to do this.

[0248] In vivo pharmacokinetics of Her47 molecule in NOD-SCID mice In an in vivo repeated dose PK study, IgG 2 Her47 LHL-LHL, Ig G 2 Her47 LHL-LHLF, Fab 2 Her47 LHL-LHL and Fa b 2 Her47 LHL and LHLF were administered once to NOD-SCID mice. (intravenous) IgG 2 Protein was administered at 14 mg / kg, and Fab 2 Protein 8 Blood samples were 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] Tumor xenograft-bearing NOD-SCID mice (SKOV-3, JIMT-1 and NUGC In vivo efficacy analysis of Her47 molecules in the -4 model In an in vivo repeated dose efficacy study, IgG 2 Her47 LHL-LHL, I gG 2 Her47 LHL-LHLF, Fab 2 Her47 LHL-LHL and F ab 2 Her47 LHL-LHLF were compared with the cell lines SKOV-3 and JIMT- Tumor-bearing NOD-SCID mice were generated by subcutaneous inoculation of NUGC-1 and NUGC-4. IgG will be administered intravenously four times. 2 Protein was administered at 14 mg / kg on day 0 and Fab was administered at 7 mg / kg on days 5, 10 and 15. 2 Protein, 8mg on day 0 10 mg / kg on days 10, 11, and 15, and 4 mg / kg on days 5, 10, and 15. 8 mg / kg is administered on day 0 and 4 mg / kg on days 5, 10 and 15. The product is measured by a caliper measuring device.

[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 Or Fab 2 To investigate the tolerability and pharmacokinetics of several exemplary molecules, For example, IgG 2 and / or Fab 2 , 2mg / kg, respectively. The above concentrations may be administered intravenously in one, two or three doses. Each animal will be harvested according to a schedule to calculate PK and assess risk of TMDD. Serum antibody concentrations will be analyzed to sample the broader effects of the administered protein. A complete hematology panel will also be performed on each successive day after dosing to monitor the , reticulocytes, red blood cells (RBCs), hemoglobin, mean hemoglobin concentration in red blood cells (M CHC), mean corpuscular volume (MCV), white blood cells, monocytes, lymphocytes, basophils, eosinophils and and / or measure neutrophil levels.

[0251] Biosensor measurements of target interaction Fab to both Her2 and CD47 (or CD3) 2 The effect of activation of binding affinity To investigate this, we have developed a system for measuring the activity of the same protein on the same chip surface, such as the Dynamic Biosensors device. A biosynthetic assay that can sample the binding of both Her2 and CD47 (or CD3) An osensor assay is performed using a control antibody and IgG 2 Or Fab 2 Ta Proteins (digested by MMP or cathepsin enzymes for 2, 4, 8 or 24 hours) The purified Her2 and CD47 (or CD3) exogenous IgG antibodies were used to detect the expression of CD47 (or CD3) in the presence of IgG. Sensor chips specifically labeled with domain proteins separately or together at different densities The affinity for Her2 and CD47 (or CD3) was measured. To determine the effect of multivalent interactions on functional affinity for both targets separately and on the same surface, I acknowledge.

[0252] While the present invention has been described in terms of preferred or exemplary embodiments, those skilled in the art may Various modifications and variations may be made to the same without departing from the spirit and scope of the present invention. It will be understood that such modifications may be made and that such modifications are expressly contemplated herein. Certain embodiments disclosed herein and claimed in the appended claims are No limitations are intended and no inference should be made.

[0253] With respect to certain embodiments disclosed herein and claimed in the appended claims, No limitation is intended, and no inference should be made. All documents, or portions of documents, including but not limited to patents, patent applications, articles, books, and and articles, all of which are expressly incorporated by reference herein in their entirety for any purpose. One or more of the incorporated documents or portions of documents may not define a term as that term in this application. In the event of a conflicting definition, the definition set forth in this application shall prevail. Any references, articles, publications, patents, patent publications, and patent applications cited herein are hereby incorporated by reference. They establish valid prior art or are part of the general knowledge in any country in the world. This document is not intended to be, or should be taken to be, a recognition or recommendation that do not have.

[0254] Numbered Embodiments Notwithstanding the claims set forth below, the present disclosure describes the following numbered embodiments: do.

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

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

[0257] 3. The peptide linker is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, Row number 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 the sequence 87. Proteins listed.

[0258] 4. The protease is a human matrix metalloprotease (MMP), a human catecholamine (Ca(2+)-1, Ca ... pusin, human enterokinase, human thrombin, human tPA, human granzyme B, human 4. The method according to any one of claims 1 to 3, wherein the antibody is human uPA or human ADAMTS-5. of protein.

[0259] 5. The peptide linker may be a human MMP cleavage site, a human cathepsin cleavage site, a human E. coli cleavage site, a human erythropoietin ... interokinase cleavage site, human thrombin cleavage site, human tPA cleavage site, human granulocyte The cleavage site may include a human uPA cleavage site, a human ADAMTS-5 cleavage site, or a human uPA cleavage site. The protein according to any one of aspects 1 to 4.

[0260] 6. The human MMP is MMP-2, MMP-3, MMP-7, MMP-8, MMP- 9. MMP-10, MMP-12, MMP-13 or MMP14, 5. A protein according to any one of claims 1 to 4.

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

[0262] 8. The human cathepsin is cathepsin A, cathepsin C, cathepsin D, cathepsin As described in any one of embodiments 1 to 5, which is cathepsin G, cathepsin L or cathepsin K. of protein.

[0263] 9. The level or activity of the human cathepsin in the diseased tissue is greater than or equal to the level or activity of the human cathepsin in non-diseased tissue. In any one of embodiments 1 to 5 and 8, the level or activity of said human cathepsin in A protein according to any one of claims 1 to 4.

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

[0265] 11. The first portion may be a Fab, a single chain Fab, a VH domain, a VL domain, an immunoglobulin, Immunoglobulin receptor (IgNAR), single-chain variable fragment (scFv), diabody, and is a T cell receptor domain.

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

[0267] 13. The first portion specifically binds to a first molecule expressed in diseased tissue, and the second portion specifically binds to a first molecule expressed in diseased tissue. the portion is capable of specifically binding to a second molecule expressed in the diseased tissue, In one embodiment, the first molecule expressed in the affected tissue and the second molecule expressed in the affected tissue are different molecules. The protein according to any one of embodiments 1 to 12.

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

[0269] 15. The first molecule expressed in the diseased tissue and the second molecule expressed in the diseased tissue 14. The protein of embodiment 13, wherein the protein is expressed in different cells.

[0270] 16. The first molecule expressed in the diseased tissue and / or the first molecule expressed in the diseased tissue 14. The protein of embodiment 13, wherein the second molecule is expressed on the surface of a cell. 【0271...

Claims

[Claim 1] The invention described in the specification.

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