Activable protein constructs and their uses

Abstract

This text describes protein molecules that exhibit activatable target binding in diseased tissues, as well as related nucleic acid molecules, vectors, and host cells. This text also describes the medical uses of such protein molecules.

Description

[0001] Cross - reference to related applications

[0002] This application claims the benefit of UK Patent Application No. 2001196.1, filed on January 28, 2020, UK Patent Application No. 1917678.3, filed on December 4, 2019, UK Patent Application No. 1910254.0, filed on July 17, 2019, and UK Patent Application No. 1906685.1, filed on May 13, 2019. The disclosure of each of these patent applications is hereby incorporated by reference in its entirety.

[0003] Description of a text file submitted electronically

[0004] The content of the text file submitted electronically with this application is hereby incorporated by reference in its entirety: a computer - readable form copy of the sequence listing (file name: ULSL_002_04WO_SeqList_ST25.txt, recording date: May 11, 2020, file size approximately 390 kb). Technical field

[0005] The present invention relates to protein molecules that exhibit activatable target binding in diseased tissue and their medical uses. Background art

[0006] In the treatment of diseases using antibodies and other binding proteins, many potential drug targets have been described, but few are expressed only in diseased tissue. In fact, most potential targets in this space are also expressed in non - diseased tissue. Additionally, most mechanisms of action of drugs employed in challenging therapeutic areas such as cancer utilize highly effective cell - killing mechanisms. As a result, the engagement of drugs with targets in non - diseased tissue often causes unwanted side effects. There is a need for engineered forms of binding proteins that are partially or even completely inactive in healthy tissue but become highly activated in diseased tissue. Summary of the invention

[0007] The present disclosure provides a protein comprising a first portion, a second portion, and a peptide linker between the first portion and the second portion, wherein the peptide linker comprises an amino acid sequence from a human immunoglobulin hinge region or an amino acid sequence having 1 to about 7 amino acid substitutions compared to a human immunoglobulin hinge region or an amino acid sequence; wherein the peptide linker is cleavable by a protease expressed in diseased tissue; wherein the second portion is capable of specifically binding to a molecule expressed in the diseased tissue; and wherein binding of the second portion to the molecule expressed in the diseased tissue is reduced or inhibited when the peptide linker is uncleaved. In some embodiments, the peptide linker has a length between about 5 and about 15 amino acids. In some embodiments, the peptide linker comprises or consists of the following amino acid sequences: 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.

[0008] In some embodiments, the protease is human matrix metalloproteinase (MMP), human cathepsin, human enterokinase, human thrombin, human tPA, human granzyme B, human uPA, or human ADAMTs-5. In some embodiments, the peptide linker comprises a human MMP cleavage site, a human cathepsin, a human enterokinase, a human thrombin, a human tPA, a human granzyme B, a human uPA, or a human ADAMTs-5 cleavage site. In some embodiments, the human MMP is MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, MMP-12, MMP-13, or MMP14. In some embodiments, the level or activity of human MMP in diseased tissue is elevated compared to the level or activity of human MMP in non-diseased tissue. In some embodiments, the human cathepsin is cathepsin A, cathepsin C, cathepsin D, cathepsin G, cathepsin L, or cathepsin K. In some embodiments, the level or activity of human cathepsin in diseased tissue is elevated compared to the level or activity of human cathepsin in non-diseased tissue.

[0009] In some embodiments, the first moiety comprises an antibody, an antigen-binding portion of an antibody, or an extracellular domain of a receptor. In some embodiments, the first moiety is a Fab, single-chain Fab, VH domain, VL domain, immunoglobulin new antigen receptor (IgNAR), single-chain variable fragment (scFv), diabody, or T cell receptor domain. In some embodiments, the first moiety specifically binds to a molecule expressed in diseased tissue.

[0010] In some embodiments, the first moiety specifically binds to a first molecule expressed in diseased tissue, and the second moiety is capable of specifically binding to a second molecule expressed in diseased tissue, wherein the first molecule expressed in diseased tissue and the second molecule expressed in diseased tissue are different molecules. In some embodiments, the first molecule expressed in diseased tissue and the second molecule expressed in diseased tissue are expressed by the same cell. In some embodiments, the first molecule expressed in diseased tissue and the second molecule expressed in diseased tissue are expressed by different cells. In some embodiments, the first molecule expressed in diseased tissue and / or the second molecule expressed in diseased tissue are expressed on the cell surface. In some embodiments, the first molecule expressed in diseased tissue and / or the second molecule expressed in diseased tissue are soluble molecules.

[0011] In some embodiments, the first moiety specifically binds to human EGFR, human HER2, human HER3, human CD105, human C-KIT, human PD1, human PD-L1, human PSMA, human EpCAM, human Trop2, human EphA2, human CD20, human BCMA, human GITR, human OX40, human CSF1R, human Lag3, or human cMET.

[0012] In some embodiments, the second moiety specifically binds to a molecule expressed by a human immune cell. In some embodiments, the molecule expressed by a human immune cell is human CD3, human CD16A, human CD16B, human CD28, human CD89, human CTLA4, human NKG2D, human SIRPα, human SIRPγ, human PD1, human Lag3, human 4-1BB, human OX40, or human GITR.

[0013] In some embodiments, the first portion comprises a variable heavy (VH) region and a variable light (VL) region. In some embodiments, the first portion comprises an immunoglobulin constant region or a portion of an immunoglobulin constant region. In some embodiments, the immunoglobulin constant region is IgG, IgE, IgM, IgD, IgA, or IgY. In some embodiments, the immunoglobulin constant region is IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2. In some embodiments, the immunoglobulin constant region is immunologically inert. In some embodiments, the immunoglobulin constant region is a wild-type human IgG4 constant region, a human IgG4 constant region comprising the amino acid substitution S228P, a wild-type human IgG1 constant region, a human IgG1 constant region comprising the amino acid substitutions L234A and L235A, a human IgG1 constant region comprising the amino acid substitutions L234A, L235A, and G237A, a human IgG1 constant region comprising the amino acid substitutions L234A, L235A, G237A, and P331S, or a wild-type human IgG2 constant region.

[0014] In some embodiments, the second portion comprises an antibody, an antigen-binding portion of an antibody, or an extracellular domain of a receptor. In some embodiments, the second portion is a Fab, a single-chain Fab, a VH domain, a VL domain, an immunoglobulin new antigen receptor (IgNAR), a single-chain variable fragment (scFv), or a T cell receptor domain. In some embodiments, the second portion specifically binds to human CD47. In some embodiments, the second portion specifically binds to human CD3 or human PD-L1.

[0015] In some embodiments, the second portion comprises a variable heavy (VH) region and a variable light (VL) region. In some embodiments, the second portion comprises an immunoglobulin constant region or a portion of an immunoglobulin constant region. In some embodiments, the immunoglobulin constant region is IgG, IgE, IgM, IgD, IgA, or IgY. In some embodiments, the immunoglobulin constant region is IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2. In some embodiments, the immunoglobulin constant region is immunologically inert. In some embodiments, the immunoglobulin constant region is a wild-type human IgG4 constant region, a human IgG4 constant region comprising the amino acid substitution S228P, a wild-type human IgG1 constant region, a human IgG1 constant region comprising the amino acid substitutions L234A and L235A, a human IgG1 constant region comprising the amino acid substitutions L234A, L235A, and G237A, a human IgG1 constant region comprising the amino acid substitutions L234A, L235A, G237A, and P331S, or a wild-type human IgG2 constant region.

[0016] In some embodiments, the protein has one immune effector function, or two, three, or more immune effector functions. In some embodiments, the immune effector function is ADCC, CDC, or ADCP.

[0017] In some embodiments, the first part prevents or reduces the specific binding of the second part to a molecule expressed in diseased tissue. In some embodiments, the peptide linker is cleaved near or within the diseased tissue. In some embodiments, the peptide linker is cleaved near or within the diseased tissue, wherein the first part dissociates from the second part near or within the diseased tissue, and wherein the second part specifically binds to a molecule expressed in the diseased tissue near or within the diseased tissue. In some embodiments, the diseased tissue is a tumor or inflamed tissue.

[0018] In some embodiments, the first part specifically binds to human cMET, wherein the second part specifically binds to human CD47, and wherein the protein comprises a first polypeptide chain and a second polypeptide chain, the first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 16 or consisting of the same, and the second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 17 or consisting of the same.

[0019] In some embodiments, the first part specifically binds to human HER2, wherein the second part specifically binds to human CD3, and wherein the protein comprises a first polypeptide chain and a second polypeptide chain, the first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 26 or consisting of the same, and the second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 27 or consisting of the same.

[0020] In some embodiments, the first part specifically binds to human HER2, wherein the second part specifically binds to human CD47, and wherein the protein comprises a first polypeptide chain and a second polypeptide chain, the first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 34 or consisting of the same, and the second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 35 or consisting of the same.

[0021] In some embodiments, the first part specifically binds to human cMET, wherein the second part specifically binds to human CD47, and wherein the protein comprises a first polypeptide chain and a second polypeptide chain, the first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 36 or consisting of the same, and the second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 37 or consisting of the same.

[0022] In some embodiments, the first part specifically binds to human Her2, wherein the second part specifically binds to human CD3, and wherein the protein comprises a first polypeptide chain and a second polypeptide chain, the first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 38 or consisting of the same, and the second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 39 or consisting of the same.

[0023] In some embodiments, the first part specifically binds to human Her2, wherein the second part specifically binds to human CD3, and wherein the protein comprises a first polypeptide chain and a second polypeptide chain, the first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 40 or consisting of the same, and the second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 41 or consisting of the same.

[0024] In some embodiments, the first part specifically binds to human Her2, wherein the second part specifically binds to human CD47, and wherein the protein comprises a first polypeptide chain and a second polypeptide chain, wherein:

[0025] (a) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 42 or consists of the same, and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 43 or consists of the same; or

[0026] (b) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 44 or consists of the same, and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 45 or consists of the same; or

[0027] (c) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 46 or consists of the same, and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 47 or consists of the same; or

[0028] (d) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 48 or consists of the same, and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 49 or consists of the same; or

[0029] (e) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 50 or consists of the same, and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 51 or consists of the same; or

[0030] (f) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 52 or consists of the same, and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 53 or consists of the same; or

[0031] (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; or

[0032] (h) The first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 88, and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 89; or

[0033] (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; or

[0034] (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: 92; or

[0035] (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; or

[0036] (1) 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; or

[0037] (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; or

[0038] (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; or

[0039] (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;

[0040] In some embodiments, the first portion specifically binds to human Her2, wherein the second portion specifically binds to human CD3, and wherein the protein comprises a first polypeptide chain and a second polypeptide chain, the first polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO: 73, and the second polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO: 74.

[0041] In some embodiments, the first portion specifically binds to human cMET, wherein the second portion specifically binds to human cMET, and wherein the protein comprises a first polypeptide chain and a second polypeptide chain, the first polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO: 75, and the second polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO: 76.

[0042] In some embodiments, the first portion specifically binds to human Her2, wherein the second portion specifically binds to human CD3, and wherein the protein comprises a first polypeptide chain and a second polypeptide chain, wherein:

[0043] (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

[0044] (b) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 100, and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 101; or

[0045] (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; or

[0046] (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;

[0047] Also provided herein are immunoconjugates comprising the proteins of the invention linked to a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxin, a radioisotope, a chemotherapeutic agent, an immunomodulator, an anti-angiogenic agent, an anti-proliferative agent, a pro-apoptotic agent, a cell growth inhibitory enzyme, a cytolytic enzyme, a therapeutic nucleic acid, an anti-angiogenic agent, an anti-proliferative agent or a pro-apoptotic agent.

[0048] The present invention also provides a pharmaceutical composition comprising the protein of the present invention or the immunoconjugate of the present invention, and a pharmaceutically acceptable carrier, diluent or excipient.

[0049] The present invention also provides a nucleic acid molecule encoding the protein of the present invention or a part of the protein. The present invention also provides a nucleic acid molecule encoding the first polypeptide chain, the second polypeptide chain or both the first polypeptide chain and the second polypeptide chain of the protein of the present invention.

[0050] The present invention also provides an expression vector comprising the nucleic acid molecule of the present invention.

[0051] The present invention also provides a recombinant host cell comprising the nucleic acid molecule of the present invention or the expression vector of the present invention.

[0052] The present invention also provides a method for producing a protein, the method comprising culturing a recombinant host cell comprising the expression vector of the present invention under conditions in which the nucleic acid molecule is expressed, thereby producing the protein; and isolating the protein from the host cell or the culture.

[0053] The present invention also provides a method for enhancing an anti-cancer immune response in a subject, the method comprising administering to the subject 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.

[0054] The present invention also provides 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 of the present invention, the immunoconjugate of the present invention or the 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, gastric cancer, ovarian cancer, bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, cancer of the oral cavity or pharynx, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small intestine or appendiceal cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma or cancer of the blood tissue. In some embodiments, the autoimmune disease or inflammatory disease is arthritis, asthma, multiple sclerosis, psoriasis, Crohn's disease, inflammatory bowel disease, lupus, Graves' disease, Hashimoto's thyroiditis or ankylosing spondylitis. In some embodiments, the cardiovascular disease is coronary heart disease, or atherosclerosis or stroke. In some embodiments, the fibrotic disease is myocardial infarction, angina, osteoarthritis, pulmonary fibrosis, cystic fibrosis, bronchitis or asthma.

[0055] The present invention also provides the protein of the present invention, the immunoconjugate of the present invention, or the pharmaceutical composition of the present invention for treating cancer, autoimmune diseases, inflammatory diseases, cardiovascular diseases or fibrotic diseases. In some embodiments, the cancer is gastrointestinal stromal cancer (GIST), pancreatic cancer, skin cancer, melanoma, breast cancer, lung cancer, bronchial cancer, colorectal cancer, prostate cancer, gastric cancer, ovarian cancer, bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, cancer of the oral cavity or pharynx, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small intestine or appendiceal cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma or cancer of the blood tissue. In some embodiments, the autoimmune disease or inflammatory disease is arthritis, asthma, multiple sclerosis, psoriasis, Crohn's disease, inflammatory bowel disease, lupus, Graves' disease, Hashimoto's thyroiditis or ankylosing spondylitis. In some embodiments, the cardiovascular disease is coronary heart disease, atherosclerosis or stroke. In some embodiments, the fibrotic disease is myocardial infarction, angina pectoris, osteoarthritis, pulmonary fibrosis, cystic fibrosis, bronchitis or asthma.

[0056] The present invention also provides the protein of the present invention, the immunoconjugate of the present invention, or the pharmaceutical composition of the present invention for use as a medicament. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figures 1A - 1B . Challenges in Delivering Systemically Active Antibody Drugs to Solid Tumors - Taking Anti-CD47 as an Example. The CD47 antibody ( Figure 1A ) has significant challenges, such as the high expression of CD47 in the bloodstream. Red blood cells and platelets particularly form a "sink" and pose a toxicity risk problem. Tumors are also typically an "unfavorable" environment with high expression of enzymes (such as MMPs that accelerate IgG degradation). The anti-CD47 protein construct of the present invention ( Figure 1B ) is designed to eliminate CD47 binding in the native protein, which eliminates peripheral activity. Then the tumor-targeting domain drives high concentrations in the tumor environment, and the peptide linker system utilizes MMP activity in the tumor to activate CD47 binding activity in the tumor rather than peripherally.

[0058] Figures 2A - 2B . Protein Construct IgG 2 Design and Activation Principle. Protein Construct IgG 2 Design ( Figure 2A) It can 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, four polypeptide chains encode four Fab domains (2x Fab A, 2X Fab B), two linker sequences, and may or may not have an immunoglobulin hinge region and an Fc domain. Each Fab A linker domain blocks the binding activity of Fab B. The choice of linker sequence (such as the lower hinge peptide sequence from an immunoglobulin) results in a structure that will be locked in non-diseased tissue but may be rapidly cleaved and unlocked in the presence of high concentrations of proteases in the tumor environment ( Figure 2B ). The linker can be cleaved sequentially, resulting in an intermediate unlocked active state that allows Fab A and B from a single protein construct to bind their cognate targets. The secondary, potentially slower cleavage of the second linker in each FabA-Fab B protein construct unit can completely release the Fab A domain from this structure, resulting in a dissociated form. The cleaved linker based on the immunoglobulin hinge sequence can also recruit increased immune effector function on the cell membrane through endogenous anti-hinge antibodies. The variable regions are shown in white. The constant regions are shown in gray.

[0059] Figures 3A - 3B . Protein construct Fab 2 Design and activation principle. Protein construct Fab 2 The design can 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 ( Figure 3A ) or three ( Figure 3B ) polypeptide chains can encode two Fab domains (1xFabA, 1X Fab B), two or more linker sequences, and may or may not have an immunoglobulin hinge region and an Fc domain, where the pairing of the heterodimer may or may not be driven by mutations in the Fc. Each Fab A linker domain blocks the binding activity of Fab B. The choice of linker sequence (such as the lower hinge peptide sequence) results in a structure that will be locked in non-diseased tissue but is rapidly cleaved and unlocked in the presence of high concentrations of proteases in the tumor environment ( Figure 3A)。The linker can be sequentially cleaved to generate an intermediate unlocked active state that allows Fab A and B from a single protein to bind their cognate targets. A secondary, potentially slower cleavage of the second linker in each Fab A-Fab B protein unit can fully release the Fab A domain from the structure. The cleaved linker based on the immunoglobulin hinge sequence can also recruit increased immune effector function on the cell membrane by endogenous anti-hinge antibodies. The variable regions are shown in white. The constant regions are shown in gray.

[0060] Figure 4 . Protein constructs IgG 2 and Fab 2 purified by Protein A from clone 1-15. SDS-PAGE analysis of the protein constructs. Exemplary proteins of multiple constructs were expressed in CHO cells and purified using Protein A affinity chromatography. The purified proteins were then analyzed by SDS-PAGE in the non-reduced state and the reduced (r) state, along with molecular weight standards (M). It was found that clones 6, 10, and 14 (all containing the LHL linker) contained the highest proportion of products of the expected size and the lowest higher and lower molecular weight content.

[0061] Figures 5A - 5I . Protein constructs IgG 2 and Fab 2 protein size exclusion chromatography. The exemplary proteins of the selected constructs 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 the highest proportion of products of the expected size (e.g., the highlighted peaks, Figure 5I ) and the lowest higher / lower molecular weight content were found in samples from clones 6, 10, and 14 (all containing the LHL linker).

[0062] Figures 6A - 6B . SDS-PAGE analysis of SEC-purified protein constructs IgG 2 and Fab 2 protein. Finally, SEC was used to purify the key leading protein constructs clones purified by Protein A affinity. Then, the purified proteins from clones 1, 2, 4, 5, 6 and non-SEC purified 15 ( Figure 6A)SDS-PAGE analysis was performed in the non-reduced state. The purified proteins from clones 7, 8, 10, 11, 12, 13, and 14 ( Figure 6B ) were also analyzed by SDS-PAGE in the non-reduced and reduced (r) states. All proteins were loaded at approximately 1 μg / lane. Clones 6, 10, and 14 (all containing the LHL linker) were found to contain the highest proportion of the product of the expected size and the lowest higher and lower molecular weight content.

[0063] Figures 7A - 7C . Direct titration ELISA of purified full-length protein constructs and control antibodies binding to human target proteins. Control antibodies A-D5 anti-CD47, A-D5 Fab-Fc (the monovalent form of the A-D5 antibody), MH7.1 anti-C-MET, and anti-Her2 trastuzumab (all in the human IgG1 form) were titrated (in μg / ml) in direct binding ELISA against human CD47, C-MET, and Her2 proteins ( Figure 7A ). Her2CD47-LH-LH and Her2CD47-LHL-LHL in the IgG 2 form and cMETCD47-L2-L2 and cMETCD47-LHL-LHL in the Fab Figure 7B form and cMETCD47-L2-L2 and cMETCD47-LHL-LHL in the Fab 2 form were also analyzed in the same manner. Figure 7C ) were also analyzed in the same way.

[0064] Figures 8A - 8C . Hemagglutination assay of purified full-length protein constructs and control antibodies with human red blood cells. Control antibodies anti-CD235a (mouse) and A-D5 anti-CD47, A-D5 Fab-Fc (the monovalent form of the A-D5 antibody, labeled 'only FabCD47'), MH7.1 anti-C-MET, anti-Her2 trastuzumab, Her2CD47-LH-LH and Her2CD47-LHL-LHL in the IgG 2 form, and cMETCD47-L2-L2 and cMETCD47-LHL-LHL in the Fab 2 form were titrated (in nM) in a hemagglutination assay of human red blood cells from donors 1 ( Figure 8A ), donor 2 ( Figure 8B ), and donor 3 ( Figure 8C ).

[0065] Figures 9A - 9C. Direct ELISA of purified intact and MMP-digested protein constructs binding to human target proteins. Protein constructs were enzymatically digested using human MMP3, MMP7, and MMP12 during incubation for 2, 4, 8, and 24 hours, plus incubation for 24 hours in enzyme-free buffer as a negative control. Samples from these digestion time courses were then applied to direct binding ELISA against human Her2 and CD47 ( Figure 9A , 9B ) or human C-MET and human CD47 ( Figure 9C ).

[0066] Figures 10A - 10C . Functional analysis of purified intact and MMP-digested Her2CD3 Fab 2 protein constructs binding to human target proteins. Analysis of antibodies Her2CD3-L 1-LH, Her2CD3-L2-L2, and Her2CD3-LHL-LHL in Fab Figure 10A form by ELISA ( Figure 10B ), by flow cytometry ( Figure 10C ) with or without MMP digestion, and by CD3 reporter assay ( 2 ).

[0067] Figure 11 . Alternative structures based on the design and activation principles of protein constructs. Protein construct module (1), which is found in both the Fab 2 and IgG 2 designs in Figures 2 and 3, can be modified and the functional characteristics of the final molecule can be altered. 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, extracellular domains of receptors, binding domains, and especially other dimerizing immunorecognition receptors such as T cell receptors. These constructs can form many forms and examples are provided herein, such as: Four polypeptide chains (2) can encode an IgG 2 -like structure containing two complete protein construct modules with 4 binding domain units (1x A, 1X B, or 2xA or B), two or more linker sequences, and may or may not have an immunoglobulin hinge region and Fc domain, where the pairing of the heterodimer can be or not be driven by mutations in the Fc. In the expression of 3 polypeptides, the Fab 2 design can be enhanced by adding a binding domain (3) or peptide that makes the structure potentially trispecific or has an altered valence. In Fab 2In the design, trispecificity or altered valency can also be achieved by adding an additional one (4) or two (5) protein construct-linker-Fab / receptor structures at the C-terminus. It should also be noted that any structure outlined in this figure or in Figures 2 and 3 can be further functionalized by adding C-terminal or N-terminal fusions of any kind of polypeptide chain or by chemical conjugation. The variable regions are shown in white. The constant regions are shown in grey.

[0068] Figure 12 .Fab-based 2 ‘Passive’ structures based on the protein construct principle. In this case, the upper binding unit of the protein construct module can be placed at the C-terminus of the Fc domain. These constructs may or may not have an immunoglobulin hinge region and an Fc domain, and the pairing of the heterodimer may or may not be driven by mutations in the Fc. In this construct, the binding of both the Fab or receptor domain to their cognate targets should become fully active only after cleavage of at least one linker. It should also be noted that any structure outlined in this figure can be further functionalized by adding C-terminal or N-terminal fusions of any kind of polypeptide chain or by chemical conjugation. The variable regions are shown in white. The constant regions are shown in grey.

[0069] Figure 13 .Fab-based 2 ‘Activable’ antibody-drug conjugate (ADC) strategy based on the protein construct principle. In this case, the upper and lower binding units of the protein construct module can contain antibodies against the same internalizing receptor target or antibodies against two different targets found on the same cell surface. These constructs can be chemically conjugated or fused with a ‘payload’ moiety such as a toxin or other active molecule to form an ADC, and may or may not have an immunoglobulin hinge region and an Fc domain, and the pairing of the heterodimer may or may not be driven by mutations in the Fc. In this construct, the binding of the upper Fab or receptor domain to its cognate target is constitutively active, resulting in the accumulation of the antibody in the tissue where its cognate target is expressed. The construct does not initially drive internalization into the target cell because the binding is monovalent, and it is known that receptors are significantly internalized only when two or more receptor domains are cross-linked by bivalent antibody binding. The activity of the second (lower) Fab or receptor domain should become operative only after cleavage of the linker by a disease-related enzyme, which then drives multivalent receptor binding and internalization of the ADC, thus allowing delivery of the (e.g., cytotoxic or inflammatory) payload moiety. It should also be noted that any structure outlined in this figure can be further functionalized by adding C-terminal or N-terminal fusions of any kind of polypeptide chain.

[0070] Figure 14. Direct ELISA of the binding of purified full-length protein constructs to human and murine target proteins. Samples were applied to direct binding ELISAs against human Her2 and human and murine CD47.

[0071] Figures 15A - 15F . Direct ELISA of the binding of purified full-length and MMP-digested protein constructs to human target proteins. The protein constructs were enzymatically digested using human MMP7( Figure 15A ), MMP8( Figure 15B ), MMP10( Figure 15C ), MMP12( Figure 15D ), MMP13( Figure 15E ) and cathepsin S( Figure 15F ) in a time course of 2, 4, 8 and 24 hours of incubation, plus incubation in enzyme-free buffer for 24 hours as a negative control (time 0). Samples from these digestion time courses were then applied to direct binding ELISAs against human Her2 and CD47.

[0072] Figure 16 A- Figure 16 B. Biacore SPR assay of the binding of purified full-length and MMP-digested Her47-LHL-LHLF to human target proteins. Her47-LHL-LHLF was enzymatically digested using human MMP 12 in a time course of 2, 4, 8 and 24 hours of incubation, plus incubation in enzyme-free buffer for 24 hours as a negative control (undigested). Samples from these digestion time courses were then captured on an anti-Fc antibody-coated Biacore chip and human Her2( Figure 16 A) or human CD47( Figure 16 B) was flowed into the solution. Rmax values were plotted to indicate the maximum binding observed at the highest concentration of the analyte protein.

[0073] Figure 17 . Biacore SPR assay of the binding of purified full-length and 24-hour MMP-digested Her47-LHL-LHLF to human target proteins. Her47-LHL-LHLF was enzymatically digested using human MMP 12 for 24 hours 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 flowed into the solution at multiple concentrations. The binding curves showed that the undigested (full-length) Her47-LHL-LHLF protein did not interact with huCD47 even at 400 nM huCD47, while strong binding of the same protein was evident after MMP 12 activation at all tested concentrations.

[0074] Figures 18A - 18B.Full Her47-LHL-LHL IgG 2 Structural modeling of the structure. Figure 18A , IgG 2 Molecular modeling of the complete structure of the IgG molecule, showing the upper (trastuzumab) Fab domain contacting its Her2 epitope (grey). Figure 18B , IgG 2 Molecular modeling of the complete structure of the IgG molecule, showing the upper (trastuzumab) Fab domain contacting its Her2 epitope (grey), but with the CD47 extracellular domain also superimposed on its possible binding location on the lower Fab of A-D5. This analysis demonstrates that when both linkers are intact, the CD47 epitope cannot bind.

[0075] Figure 19 A- Figure 19 I. Her47 Fab with different linkers 2 Structural dynamics of the structure. Solvent accessible surface area (SASA) results were obtained for 3 linkers (LHL, LHLF, L2). Figure 19 A, Figure 19 D, and Figure 19 G show the absolute SASA values for 9 kinetic runs of the LHL and LHLF linkers and 10 runs using L2 (all over 6 ns). Figure 19 B, Figure 19 E, Figure 19 H, Figure 19 C, Figure 19 F, and Figure 19 I show the normalized results representing the difference from the starting SASA value during the 6 ns dynamic run time and during the first 2.5 ns of the 6 ns dynamic run. Figure 19 A, Figure 19 B, and Figure 19 C depict SEQ ID NO: 2. Figure 19 D, Figure 19 E, and Figure 19 F depict SEQ ID NO: 3. Figure 19 G, Figure 19 H, and Figure 19 I depict SEQ ID NO: 32.

[0076] Figures 20A - 20B .Full and activated Her47 LHL-LHL Fab 2 Structural dynamics of the structure. ( Figure 20A ) Overlapping Fab in both the full linker and activated (single linker protease-cleaved) forms 2 Structure. ( Figure 20B ) Fab from which one of the LHL or LHLF linkers has been cleaved 2Two poses obtained from molecular dynamics simulations of the region. The anti-CD47 Fab is shown in black and the anti-HER2 Fab is shown in grey. The figure shows the maximum movement of the Her2 domain and the final exposure of the anti-CD47 Fab CDRs.

[0077] Figure 21 . Flow cytometry analysis of protein binding to 'Tg32' mouse red blood cells. A-D5 IgG1, IgG 2 Her47 LHL-LHL, IgG 2 Her47 LHL-LHLF, and Fab 2 Met47 LHL-LHL were used for flow cytometry analysis of binding to red blood cells. An anti-human PE-conjugated secondary antibody was used to measure binding. A-D5 IgG1 was tested at 0.1 μg / ml, 1 μg / ml, and 10 μg / ml. IgG 2 Her47 LHL-LHL was tested at 0.1 μg / ml, 1 μg / ml, and 10 μg / ml. IgG 2 Her47 LHL-LHLF was tested at 0.1 μg / ml, 1 μg / ml, and 10 μg / ml. Fab 2 Met47 LHL-LHL was tested at 0.1 μg / ml, 1 μg / ml, and 10 μg / ml.

[0078] Figure 22 . Hemagglutination assay of 'Tg32' mouse red blood cells. A-D5 IgG1, IgG 2 Her47 LHL-LHL, IgG 2 Her47 LHL-LHLF, and Fab 2 Met47 LHL-LHL were used to agglutinate red blood cells. Fresh red blood cells pooled from multiple donor mice were used to titrate the proteins (in nM).

[0079] Figure 23 . Tolerance study in 'Tg32' mice: body weight analysis. A-D5 IgG1, IgG 2 Her47 LHL-LHL, IgG 2 Her47 LHL-LHLF, and Fab 2 Met47 LHL-LHL were used for tolerance studies in Tg32 mice. The body weight was then monitored for 60 days. The 10 mg / kg dose of A-D5 IgG1 was intolerant and the group was terminated on day 1.

[0080] Figure 24. Tolerance study in 'Tg32' mice: Reticulocyte analysis on day 5 after dosing. All proteins were dosed at 2 mg / kg and 10 mg / kg concentrations in Tg32 mice to conduct the tolerance study using A-D5 IgG1, IgG 2 Her47 LHL-LHL, IgG 2 Her47 LHL-LHLF and Fab 2 Met47 LHL-LHL. Blood samples were collected and reticulocyte levels were measured. A-D5 IgG1 at a dose of 2 mg / kg showed a significant increase in reticulocyte levels.

[0081] Figures 25A - 25K . Tolerance study in 'Tg32' mice: Hematological analysis on days 5, 29, and 60 after dosing. All proteins were dosed at 2 mg / kg and 10 mg / kg concentrations in Tg32 mice to conduct the tolerance study using A-D 5IgG1, IgG 2 Her47 LHL-LHL, IgG 2 Her47 LHL-LHLF and Fab 2 Met47 LHL-LHL. Blood samples were collected and levels of reticulocytes ( Figure 25A ), red blood cells (RBC, Figure 25B ), hemoglobin ( Figure 25C ), mean corpuscular hemoglobin concentration (MCHC) ( Figure 25D ), mean corpuscular volume (MCV) ( Figure 25E ), white blood cells ( Figure 25F ), monocytes ( Figure 25G ), lymphocytes ( Figure 25H ), basophils ( Figure 25I ), eosinophils ( Figure 25J ) and neutrophils ( Figure 25K ) were measured.

[0082] Figure 26 . Pharmacokinetic study in 'Tg32' mice: Data for each molecule at two doses. All proteins were dosed at 2 mg / kg and 10 mg / kg concentrations in Tg32 mice to conduct the pharmacokinetic study using IgG 2 Her47 LHL-LHL, IgG 2 Her47LHL-LHLF and Fab 2 Met47 LHL-LHL. A-D5 IgG1 was dosed at 2 mg / kg. Serum samples were collected from 30 minutes to 42 days after dosing and human IgG levels (in μg / ml) were measured.

[0083] Pharmacokinetic studies in ‘Tg32’ mice: data for each dose. All proteins were dosed at 2 mg / kg and 10 mg / kg concentrations in Tg32 mice and IgG 2 Her47 LHL-LHL, IgG 2 Her47 LHL-LHLF and Fab 2 Pharmacokinetic studies of Met47 LHL-LHL. A-D5 IgG1 was dosed at 2 mg / kg. Serum samples were collected from 30 minutes post-dose to 42 days and human IgG levels (in μg / ml) were measured. Concentrations were plotted at 2 mg / kg ( Figure 27A ) and 10 mg / kg ( Figure 27B ). A-D5 IgG1 at the 2 mg / kg dose was included in both analyses as a reference.

[0084] Figure 28 .Pharmacokinetic studies in ‘Tg32’ mice: AUC data for each dose. All proteins were dosed at 2 mg / kg and 10 mg / kg concentrations in Tg32 mice and IgG 2 Her47 LHL-LHL, IgG 2 Her47 LHL-LHLF and Fab 2 Pharmacokinetic studies of Met47 LHL-LHL. A-D5 IgG1 was dosed at 2 mg / kg. Serum samples were collected from 30 minutes post-dose to 42 days and human IgG levels (in μg / ml) were measured. The area under the curve (AUC) for each dose was calculated using the concentration measurements over time.

[0085] Figures 29A - 29B .Flow cytometry analysis of binding to NHP and human red blood cells. Flow cytometry analysis of binding to red blood cells was performed using A-D5 3M (effector null) IgG1, IgG 2 Her47 LHL-LHL, IgG 2 Her47LHL-LHLF and trastuzumab. Binding was measured using an anti-human PE-conjugated secondary antibody. A-D5 IgG1 was the only protein that exhibited concentration-dependent binding to both NHP (cynomolgus monkey) red blood cells ( Figure 29A ) and human red blood cells ( Figure 29B ).

[0086] Figures 30A - 30N. Direct ELISA of purified intact and MMP-digested protein constructs (digested at pH 7.4 and pH 6.0) binding to human target proteins. The protein constructs were enzymatically digested in a time course of incubation with human MMP at pH 7.4 or pH 6.0 for 2, 4, 8, and 24 hours, plus incubation in enzyme-free buffer for 24 hours as a negative control (time 0). Samples from these digestion time courses were then applied to direct binding ELISA against human Her2 and CD47.

[0087] Figures 31A - 31B . Direct ELISA of purified intact and cathepsin-digested protein constructs (digested at pH 7.4 and pH 6.0) binding to human target proteins. The protein constructs were enzymatically digested in a time course of incubation with human cathepsin at pH 7.4 or pH 6.0 for 2, 4, 8, and 24 hours, plus incubation in enzyme-free buffer for 24 hours as a negative control (time 0). Samples from these digestion time courses were then applied to direct binding ELISA against human Her2 and CD47.

[0088] Figures 32A - 32D . Flow cytometry analysis of binding to human cancer cells. Using anti-CD47, trastuzumab, IgG1 isotype, and IgG that had been enzymatically digested with human MMP12 at pH 7.4 in a time course of incubation for 2, 4, 8, and 24 hours, plus incubation in enzyme-free buffer for 24 hours as a negative control (time 0) 2 Her47 LHL-LHL or IgG 2 Her47 LHL-LHLF was subjected to flow cytometry analysis of binding to red blood cells. Binding was measured using an anti-human PE-conjugated secondary antibody. Binding was measured on Her2-high cell line BT-474( Figure 32A 、 Figure 32B ) and Her2-low cell line MCF-7( Figure 32C 、 Figure 32D ).

[0089] Figure 33 . MMP12-digested IgG 2 Her47 LHL-LHL or IgG 2 SDS-PAGE analysis of Her47 LHL-LHL or IgG 2 Her47 LHL-LHL or IgG 2 Her47 LHL-LHLF samples that had been enzymatically digested with human MMP12 at pH 7.4 in a time course of incubation for 2, 4, 8, and 24 hours, plus incubation in enzyme-free buffer for 24 hours as a negative control (time 0) were subjected to SDS-PAGE.

[0090] Figures 34A - 34B . IgG digested with MMP12 2 Mass spectrometry of Her47 LHL-LHL. IgG that had been enzymatically digested with human MMP12 at pH 7.4 during a time course of incubation at 2, 4, 8, and 24 hours, plus incubation for 24 hours in enzyme-free buffer as a negative control (time 0) 2 Samples of Her47 LHL-LHL were subjected to mass spectrometry. The presence of peptides indicative of intact LHL linker ( Figure 34A ) and MMP12-cleaved linker ( Figure 34B ) was measured. Figure 34A Depicts SEQ ID NO: 110. Figure 34B Depicts SEQ ID NO: 111.

[0091] Figure 35 . Size-exclusion chromatography of protein A-purified Her47 LHLF-LHL IgG1-2hDAA. The Her47LHLF-LHL IgG1-2hDAA protein was expressed in CHO cells, purified by a ProA column and analyzed by SEC. Two small peaks of larger MW and a large peak of the product of the expected size (10.30, approximately 250 kDa) were observed.

[0092] Figure 36 . SDS-PAGE analysis of purified peak fractions from size-exclusion chromatography of Her47 LHLF-LHL IgG1-2hDAA. SDS-PAGE was performed on unreduced samples of Her47 LHLF-LHL IgG1-2hDAA: Lane 1 - molecular weight standards, Lane 2 - total ProA eluate protein, Lane 3 - blank, Lane 4 - peak 1, Lane 5 - peak 2, Lane 6 - peak 3 (correct product).

[0093] Figure 37 . SDS-PAGE analysis of purified peak fractions from size-exclusion chromatography of Her47 LHLF-LHL IgG1-2hDAA. SDS-PAGE was performed on reduced samples of Her47 LHLF-LHL IgG1-2hDAA: Lane 1 - molecular weight standards, Lane 2 - total ProA eluate protein, Lane 3 - blank, Lane 4 - peak 1, Lane 5 - peak 2, Lane 6 - peak 3 (correct product).

[0094] Figures 38A - 38C. Direct ELISA of purified intact and MMP12-digested Her47 IgG1-2hDAA protein. Human MMP12 was used to enzymatically digest Her47LHL-LHLF IgG1-2hDAA ( Figure 38A ) at pH 7.4 during incubation times of 2, 4, 8, and 24 hours, plus 24 hours of incubation in enzyme-free buffer as a negative control (time 0, 2-hour, 4-hour, 8-hour, 24-hour incubations). Samples from these digestion time courses were then applied to direct-binding ELISAs against human Her2 and murine EpCAM ( Figure 38A ). ELISAs were then performed on the digested (dark gray) and undigested (light gray) samples against human CD47 ( Figure 38B ). The samples were also subjected to SDS-PAGE: lane 1 - molecular weight marker, lane 2 - 0-hour digest, lane 3 - 2-hour digest, lane 4 - 8-hour digest, and lane 4 - 24-hour digest ( Figure 38C ).

[0095] Figures 39A - 39L . Direct ELISA of purified intact and MMP12-digested IgG2 Her47 proteins with alternative linker compositions. Purified proteins from clones Her47 LHL-LHL-EK, Her47-LHL-LHL-Thr, Her47-LHL-LHL-tPA, Her47-LHL-LHL-uPA, Her47-LHL-LHL-GrB, and Her47-LHL-LHL-A5 were all tested in titration ELISAs against human Her2 and CD47 targets ( Figure 39A 、 Figure 39C 、 Figure 39E 、 Figure 39G 、 Figure 39I 、 Figure 39K ). Time course enzymatic digestions and ELISA binding to Her2 and CD47 targets were then also performed on each protein ( Figure 39B 、 Figure 39D 、 Figure 39F 、 Figure 39H 、 Figure 39J 、 Figure 39L ).

[0096] Figure 40 . Multi-dose tolerance study in NOD-SCID mice: Body weight analysis. All proteins (4 doses in total) were administered once every 5 days using IgG 2 Her47 LHL-LHL, IgG 2 Her47 LHL-LHLF, Fab 2 Her47LHL-LHL and Fab2 Tolerance study of Her47 LHL-LHLF.

[0097] Figures 41A - 41D . Protein A-purified Her2CD3 Fab 2 Size-exclusion chromatography of proteins. Fab 2 Her23LHL-LHL-S( Figure 41A )、Fab 2 Her23 LHLF-LHL-S( Figure 41B )、Fab 2 Her23 LHL-LHL( Figure 41C ) and Fab 2 Her23 LHLF-LHL( Figure 41D ) were expressed in CHO cells, purified by ProA column and analyzed by SEC. Fab 2 Her23 LHL-LHL( Figure 41C ) and Fab 2 Her23 LHLF-LHL( Figure 41D ) both showed low molecular weight contaminants (peak 15.38).

[0098] Figures 42A - 42B . CD3 co-engagement bioassay analysis of purified intact and MMP-digested Her2CD3 Fab 2 proteins was performed using Her2 low MCF-7 cells. Human MMP12 was used to enzymatically digest antibody Fab 2 Her23 LHLF-LHL-S( Figure 42A )、Fab 2 Her23 LHL-LHL-S( Figure 42B ) at pH 7.4 during a time course of incubation at 2, 4, 8, and 24 hours, plus 24 hours incubation in enzyme-free buffer as a negative control (time 0). Samples from these digestion time courses were then applied to the Promega Jurkat cell CD3 reporter assay using MCF-7 cells as target cells.

[0099] Figures 43A - 43C . CD3 co-engagement bioassay analysis of purified intact and MMP-digested Her2CD3 Fab 2 proteins was performed using Her2 high BT-474 cells. Control antibody ( Figure 43A ), as well as Fab 2 Her23 LHLF-LHL-S( Figure 43B ) or Fab 2 Her2r23 LHL-LHL-S(Figure 43C ) [Both were subjected to enzymatic digestion with human MMP12 at pH 7.4 in the time course of incubation at 2, 4, 8, and 24 hours, plus incubation in enzyme-free buffer for 24 hours as a negative control (time 0)] and applied to the Promega Jurkat cell CD3 reporter assay at 0.1 μg / ml, using BT-474 cells as target cells.

[0100] Figures 44A - 44B . For IgG 2 and Fab 2 Charge variant analysis of Her47 protein - Charge heterogeneity analysis is important in the characterization of monoclonal antibodies as it provides important information about product quality and stability. Heterogeneity can be caused by enzymatic post-translational modifications (glycosylation, lysine truncation) or chemical modifications (oxidation or deamidation) during purification and storage. Charge variant profiling of the provided test article was performed by commercial Charge Variant Assay. IgG 2 Her47 LHL-LHL( Figure 44A ) and Fab 2 Her47 LHL-LHL( Figure 44B ) both showed a homogeneous profile with one major isotype (50 - 57% of the total), one major acidic isotype (40 - 48% of the total), and one minor basic isotype (approximately 3%).

[0101] Figures 45A - 45B . Size exclusion chromatography of Her47 protein after 5 freeze-thaw cycles. IgG 2 Her47 LHL-LHL( Figure 45A ) and Fab 2 Her47 LHL-LHL( Figure 45B ) were both subjected to 5 freeze-thaw cycles and then SEC was performed on samples from cycles 0 - 5. No aggregation, fragmentation, or product loss was observed for either protein.

[0102] Figure 46 . Alternative protein construct design. The figure depicts an illustrative example of a protein construct Fab 2 design. The design can be based on the following sequence, which: 1. Removes the upper variable domain. 2. Contains a "false" non-binding variable domain. 3. The upper Fab is replaced with a diabody (or two scFvs). Variable regions are shown in white. Constant regions are shown in gray.

[0103] Figures 47A - 47B. A cell proliferation assay of purified intact Her2CD47 protein was performed using Her2-high BT-474 cells. Trastuzumab, isotype control IgG1, IgG 2 Her47 LHL-LHL( Figure 47A ) and Fab 2 Her47 LHL-LHL( Figure 47B ) were applied to BT-474 cells during a 72-hour incubation period, and cell proliferation was measured. Data are represented as the percent inhibition of cell growth.

[0104] Figures 48A - 48G . In vivo efficacy analysis of Her47 molecule in NOD-SCID mice (KYSE-410 model). Trastuzumab( Figure 48A ), IgG 2 Her47 LHL-LHLF( Figure 48B ), IgG 2 Her47 LHL-LHL( Figure 48C ), Fab 2 Her47 LHL-LHLF( Figure 48D ) and Fab 2 Her47 LHL-LHL( Figure 48E ) were each administered (intravenously, on days 0, 5, 10) to NOD-SCID mice bearing KYSE-410 tumors. Tumor volumes were measured on days 4, 7, and 11 and plotted relative to vehicle. Fab 2 Her47 LHL-LHLF and Fab 2 Her47 LHL-LHL exhibited different potencies( Figure 48F ). None of the treatment groups showed any weight loss that might indicate toxicity of the administered molecule( Figure 48G ). Detailed Description

[0105] Recombinant proteins that are conditionally active in diseased human tissue are disclosed herein. In some cases, the protein comprises a binding domain that is masked by another portion of the protein in non-diseased tissue. The protein also comprises a peptide linker that is cleaved by one or more proteases expressed in diseased tissue. Cleavage of the linker unmasks the binding domain in diseased tissue, thereby allowing the protein to selectively bind and / or function in diseased tissue. The proteins of the invention are particularly useful for binding drug targets that are expressed in both diseased and non-diseased tissue.

[0106] The present disclosure provides a number of activatable protein molecules and their medical uses. In some aspects, a variety of functional properties of the molecules are considered, including target binding specificity, effective limitation of activities not desired in the native protein but full activity in the activated form, maintenance of conditional affinity for one or more targets from human and animal test species (e.g., cynomolgus monkeys (also known as cynomolgus macaques), i.e., crab-eating monkeys (Macaca fascicularis)), biophysical stability, and / or yield from protein expression platforms used in research, clinical, and commercial supply.

[0107] In some aspects, provided are protein molecules that specifically bind to one or more human drug targets and optionally also to the cynomolgus monkey orthologs of those targets, wherein the protein molecules comprise a heavy chain region and a light chain region assembled from one or more polypeptides having the following forms:

[0108] V-C-linker-V-C

[0109] V-C-linker-V-C

[0110] or

[0111] C-linker-V-C

[0112] C-linker-V-C

[0113] In some aspects, the protein molecule comprises two polypeptide chains and has the following form:

[0114] VH1-C-linker-VH2-C

[0115] VL1-C-linker-VL2-C

[0116] In some aspects, the protein molecule comprises two polypeptide chains and has the following form:

[0117] VL1-C-linker-VH2-C

[0118] VH1-C-linker-VL2-C

[0119] "V" refers to the variable region of an immunoglobulin or T cell receptor or the extracellular domain of a receptor. "VH1" and "VL1" refer to the variable heavy and variable light chains that pair with each other to bind antigen. "VH2" and "VL2" refer to the variable heavy and variable light chains that pair with each other to bind antigen. "C" refers to the constant region of an immunoglobulin or T cell receptor. In aspects of the invention, the V-C and V-C units on either side of the linker domain form upper and lower immunoglobulin Fab domains, wherein the lower Fab domain exhibits binding to its cognate target, and this binding is reduced or eliminated by the presence of a linker domain fused to the N-terminus of each V domain in the lower Fab. In another aspect, the upper or lower Fab domain can be replaced by an Fc fragment, one or two receptor extracellular domains, or any domain with or without any specific binding function.

[0120] In some aspects, provided herein are proteins comprising a first part, a second part, and a peptide linker between the first part and the second part.

[0121] Wherein the peptide linker comprises an amino acid sequence from the human immunoglobulin hinge region or an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 (e.g., 1 to about 7) amino acid substitutions compared to the human immunoglobulin hinge region or an amino acid sequence;

[0122] Wherein the peptide linker is cleavable by a protease expressed in diseased tissue;

[0123] Wherein the second part is capable of specifically binding to a molecule expressed in diseased tissue; and

[0124] Wherein when the peptide linker is not cleaved, the binding of the second part to the molecule expressed in the diseased tissue is reduced or inhibited.

[0125] The linker moiety may also comprise a peptide linker derived from the immunoglobulin hinge region having zero, one, or more mutations away from the germline.

[0126] In some aspects, the peptide linker comprises or consists of the sequences represented by GPAPELL (SEQ ID NO: 1), GPAPELLGGGS (SEQ ID NO: 2), GPAPLGLGGGS (SEQ ID NO: 3), PPCPAPELLGGGS (SEQ ID NO: 4), PPCPAPLGLGGGS (SEQ ID NO: 5), GPAPELLGGPS (SEQ ID NO: 69), GPAPLGLGGPS (SEQ ID NO: 70), PPCPAPELLGGPS (SEQ ID NO: 71), PPCPAPLGLGGPS (SEQ ID NO: 72), GPAPEAAGAGS (SEQ ID NO: 81), GPADDDDKSGS (SEQ ID NO: 82) (cleavable by enterokinase), GPALVPRGSGS (SEQ ID NO: 83) (cleavable by thrombin), GPGPFGRSAGGP (SEQ ID NO: 84) (cleavable by tPA), GPAPLEADAGS (SEQ ID NO: 85) (cleavable by granzyme B), GPAPEARRGGS (SEQ ID NO: 86) (cleavable by uPA), or GPAPEGEARGS (SEQ ID NO: 87) (cleavable by ADAMTs-5). In some aspects, the peptide linker comprises two, three, or four of the above sequences or consists of them.

[0127] Also provided are immunoconjugates comprising the protein of the invention linked to a therapeutic agent.

[0128] In another aspect, the invention provides a nucleic acid molecule encoding the protein or a portion thereof as defined herein. Also provided is a vector comprising the nucleic acid molecule of the invention. Also provided is a host cell comprising the nucleic acid molecule or vector of the invention.

[0129] In a further aspect, provided is a method for producing the conditionally active protein of the invention, the method comprising culturing the host cell of the invention under conditions that result in the expression and / or production of the protein, and isolating the protein from the host cell or the culture.

[0130] In another aspect of the invention, provided is a pharmaceutical composition comprising the protein of the invention as defined herein, or the nucleic acid molecule of the invention as defined herein, or the vector of the invention as defined herein, or the immunoconjugate of the invention as defined herein.

[0131] Also provided are methods for enhancing an immune response in a subject, the methods comprising administering an effective amount of a protein of the invention as defined herein, or an immunoconjugate of the invention as defined herein, or a nucleic acid molecule of the invention as defined herein, or a vector of the invention as defined herein, or a pharmaceutical composition of the invention as defined herein.

[0132] In a further aspect, provided are methods for treating or preventing cancer in a subject, the methods comprising administering an effective amount of a protein of the invention as defined herein, or an immunoconjugate of the invention as defined herein, or a nucleic acid molecule of the invention as defined herein, or a vector of the invention as defined herein, or a pharmaceutical composition of the invention as defined herein.

[0133] Also provided are a protein of the invention as defined herein, or an immunoconjugate of the invention as defined herein, or a nucleic acid molecule of the invention as defined herein, or a vector of the invention as defined herein, or a pharmaceutical composition of the invention as defined herein, for use as a medicament.

[0134] Also provided are a protein of the invention as defined herein, or an immunoconjugate of the invention as defined herein, or a nucleic acid molecule of the invention as defined herein, or a vector of the invention as defined herein, or a pharmaceutical composition of the invention as defined herein, for treating cancer.

[0135] Also provided are a protein of the invention as defined herein, or an immunoconjugate, or a nucleic acid molecule, or a vector, or a pharmaceutical composition, for separate, sequential or simultaneous use in combination with a second therapeutic agent (such as an anti-cancer agent).

[0136] In a further aspect, provided is the use of a protein of the invention as defined herein, or an immunoconjugate of the invention as defined herein, or a nucleic acid molecule of the invention as defined herein, or a vector of the invention as defined herein, or a pharmaceutical composition of the invention as defined herein in the manufacture of a medicament for treating cancer.

[0137] Also provided are methods for treating or preventing an autoimmune disease or an inflammatory disease in a subject, the methods comprising administering an effective amount of a protein as defined herein, or an immunoconjugate as defined herein, or a nucleic acid molecule as defined herein, or a vector as defined herein, or a pharmaceutical composition as defined herein.

[0138] Also provided are a protein as defined herein, or an immunoconjugate as defined herein, or a nucleic acid molecule as defined herein, or a vector as defined herein, or a pharmaceutical composition as defined herein, for treating an autoimmune disease or an inflammatory disease.

[0139] There is also provided the use of a protein as defined herein, or an immunoconjugate as defined herein, or a nucleic acid molecule as defined herein, or a vector as defined herein, or a pharmaceutical composition as defined herein, in the preparation of a medicament for treating an autoimmune disease or an inflammatory disease.

[0140] There is also provided a method for treating or preventing a cardiovascular disease or a fibrotic disease in a subject, the method comprising administering an effective amount of a protein as defined herein, or an immunoconjugate as defined herein, or a nucleic acid molecule as defined herein, or a vector as defined herein, or a pharmaceutical composition as defined herein.

[0141] There is also provided a protein as defined herein, or an immunoconjugate as defined herein, or a nucleic acid molecule as defined herein, or a vector as defined herein, or a pharmaceutical composition as defined herein, for use as a medicament. There is also provided an antibody molecule or an antigen-binding portion thereof as defined herein, or an immunoconjugate as defined herein, or a nucleic acid molecule as defined herein, or a vector as defined herein, or a pharmaceutical composition as defined herein, for treating a cardiovascular disease or a fibrotic disease.

[0142] There is also provided the use of a protein as defined herein, or an immunoconjugate as defined herein, or a nucleic acid molecule as defined herein, or a vector as defined herein, or a pharmaceutical composition as defined herein, in the preparation of a medicament for treating an autoimmune disease, an inflammatory disease or a fibrotic disease.

[0143] In some aspects, the present invention provides a protein comprising a first part, a second part, and a peptide linker between the first part and the second part, wherein the peptide linker comprises an amino acid sequence from a human immunoglobulin hinge region or an amino acid sequence having amino acid substitutions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 amino acid substitutions) compared to a human immunoglobulin hinge region or an amino acid sequence; wherein the peptide linker is cleavable by a protease expressed in diseased tissue; wherein the second part is capable of specifically binding to a molecule expressed in the diseased tissue; and wherein binding of the second part to the molecule expressed in the diseased tissue is reduced or inhibited when the peptide linker is not cleaved. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some aspects, the peptide linker comprises an amino acid sequence from a human immunoglobulin hinge region or an amino acid sequence having 1 to about 7 amino acid substitutions compared to a human immunoglobulin hinge region; in some aspects, the peptide linker comprises an amino acid sequence from a human immunoglobulin hinge region or an amino acid sequence having 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 2-3, 2-4, 2-5, 2-6, 2-7, 3-4, 3-5, 3-6, 3-7, 4-5, 4-6, 4-7, 5-6, 5-7, or 6-7 amino acid substitutions compared to a human immunoglobulin hinge region.

[0144] In some aspects, the peptide linker cleavable by a protease expressed in diseased tissue can be cleaved by human matrix metalloproteinase (MMP) or human cathepsin. In some cases, the peptide linker cleavable by a protease expressed in diseased tissue can be cleaved by human enterokinase (EK), human thrombin (Thr), human tPA (tissue plasminogen activator), human granzyme B (GrB), human uPA (urokinase-type plasminogen activator), or human ADAMTs-5 (a disintegrin-like and metalloprotease with thrombospondin type 1 motifs 5; A5). In some cases, the peptide linker contains a human MMP cleavage site or a human cathepsin cleavage site. In some cases, the peptide linker contains a human enterokinase, human thrombin, human tPA, human granzyme B, human uPA, or human ADAMTs-5 cleavage site. In some cases, the peptide linker contains the MMP substrate sequence PLGL (SEQ ID NO: 12). In some cases, the peptide linker contains or consists of the amino acid sequence of GPAPELL (SEQ ID NO: 1), GPAPELLGGGS (SEQ ID NO: 2), GPAPLGLGGGS (SEQ ID NO: 3), PPCPAPELLGGGS (SEQ ID NO: 4), or PPCPAPLGLGGGS (SEQ ID NO: 5), GPAPELLGGPS (SEQ ID NO: 69), GPAPLGLGGPS (SEQ ID NO: 70), PPCPAPELLGGPS (SEQ ID NO: 71), PPCPAPLGLGGPS (SEQ ID NO: 72), GPAPEAAGAGS (SEQ ID NO: 81), GPADDDDKSGS (SEQ ID NO: 82), GPALVPRGSGS (SEQ ID NO: 83), GPGPFGRSAGGP (SEQ ID NO: 84), GPAPLEADAGS (SEQ ID NO: 85), GPAPEARRGGS (SEQ ID NO: 86), or GPAPEGEARGS (SEQ ID NO: 87).

[0145] In some cases, the peptide linker contains or consists of two, three, or four amino acid sequences in Table 1 fused in a single amino acid chain by peptide bonds. In some cases, the length of the peptide linker is between about 5 amino acids and about 15 amino acids, between about 5 amino acids and about 20 amino acids, or between about 5 amino acids and about 25 amino acids.

[0146] In some cases, the peptide linker between the first part and the second part comprises the following amino acid sequence at the N-terminus of the peptide linker sequence: X1 - proline - X2. In some aspects, X1 is alanine, glycine, serine, proline, or threonine. In some aspects, X1 is alanine, glycine, serine, proline, or threonine, aspartic acid, asparagine, or valine. In some aspects, X2 is alanine, glycine, serine, proline, or threonine. In some aspects, X2 is alanine, glycine, serine, proline, or threonine, aspartic acid, asparagine, or valine. In some aspects, X1 and X2 are the same amino acid. In some aspects, X1 and X2 are different amino acids.

[0147] In some cases, the peptide linker cleavable by a protease expressed in diseased tissue can be cleaved by any one of human MMP1, MMP2, MMP3, MMP4, MMP5, MMP6, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP18, MMP19, MMP20, MMP21, MMP22, MMP23, MMP24, MMP25, MMP26, MMP27, or MMP28. In some cases, the peptide linker cleavable by a protease expressed in diseased tissue can be cleaved by any one of human MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, MMP-12, or MMP-13. In some cases, the level or activity of human MMP in diseased tissue is elevated compared to the level or activity of human MMP in non-diseased tissue.

[0148] In some cases, the peptide linker cleavable by a protease expressed in diseased tissue can be cleaved by any one of human cathepsin A, cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin F, cathepsin G, cathepsin H, cathepsin K, cathepsin L1, cathepsin V, cathepsin O, cathepsin S, cathepsin W, or cathepsin Z. In some cases, the peptide linker cleavable by a protease expressed in diseased tissue can be cleaved by any one of human cathepsin D, cathepsin G, or cathepsin K. In some cases, the level or activity of human cathepsin in diseased tissue is elevated compared to the level or activity of human cathepsin in non-diseased tissue. In some cases, the level or activity of human cathepsin in tissue with pH < 7.0 is elevated compared to the level or activity of human cathepsin in tissue with pH ≥ 7.4.

[0149] In some aspects, the first portion of any protein of the invention comprises an antibody, an antigen-binding portion of an antibody, or a receptor extracellular domain. In some cases, the first portion is a Fab, single-chain Fab, VH domain, VL domain, immunoglobulin new antigen receptor (IgNAR), single-chain variable fragment (scFv), diabody, or T cell receptor domain. IgNAR is an antibody consisting of only homodimeric heavy chains produced by sharks and other cartilaginous fish (Feige et al., PNAS, 2014, 111(22):8155-8160).

[0150] In some cases, the first portion specifically binds to a molecule expressed in diseased tissue. In some embodiments, the first portion specifically binds to a tumor-associated antigen (TAA). In some cases, the first portion specifically binds to human EGFR, human HER2, human HER3, human CD105, human C-KIT, human PD1, human PD-L1, human PSMA, human EpCAM, human Trop2, human EphA2, human CD20, human BCMA, human GITR, human OX40, human CSF 1R, human Lag3, or human cMET. In some embodiments, the first portion also binds to the cynomolgus macaque ortholog of any of these molecules.

[0151] In some aspects, the first portion comprises a heavy chain variable (VH) region and a light chain variable (VL) region. In some cases, the first portion also comprises an immunoglobulin constant region or a portion of an immunoglobulin constant region. In some cases, the immunoglobulin constant region is IgG, IgE, IgM, IgD, IgA, or IgY.

[0152] In some aspects, the anti-HER2 variable region sequence used in the protein constructs disclosed herein is the variable region sequence of trastuzumab. In some aspects, the anti-CD3 variable region sequence used in the protein constructs disclosed herein is the variable region sequence of OKT3 or SP34. In some aspects, the anti-cMET variable region sequence for use in the protein constructs disclosed herein is provided in WO 2019 / 175186. In some aspects, the anti-CD47 variable region sequence for use in the protein constructs disclosed herein is provided in WO 2019 / 034895.

[0153] In some aspects, the second portion of any protein of the invention comprises an antibody, an antigen-binding portion of an antibody, or a receptor extracellular domain. In some cases, the second portion is a Fab, single-chain Fab, VH domain, VL domain, immunoglobulin new antigen receptor (IgNAR), single-chain variable fragment (scFv), diabody, or T cell receptor domain.

[0154] In some cases, the second moiety specifically binds to a molecule expressed in diseased tissue. In some embodiments, the second moiety specifically binds to a tumor-associated antigen (TAA). In some cases, the second moiety specifically binds to human CD47. In some cases, the second moiety specifically binds to human PD-L1. In some cases, the second moiety specifically binds to a molecule expressed by a human immune cell. In some cases, the molecule expressed by a human immune cell is human CD3, human CD16A, human CD16B, human CD28, human CD89, human CTLA4, human NKG2D, human SIRPα, human SIRPγ, human PD1, human Lag3, human 4-1BB, human OX40, or human GITR. In some embodiments, the second moiety also binds to the cynomolgus monkey ortholog of any of these molecules.

[0155] In some aspects, the second moiety comprises a variable heavy (VH) region and a variable light (VL) region. In some cases, the second moiety further comprises an immunoglobulin constant region or a portion of an immunoglobulin constant region. In some cases, the immunoglobulin constant region is IgG, IgE, IgM, IgD, IgA, or IgY.

[0156] In some aspects, the first part and / or the second part of the protein of the present invention may comprise an immunoglobulin constant region. In some embodiments, the immunoglobulin constant region is IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgE or IgM. In additional embodiments, the immunoglobulin constant region is IgG1, IgG2, IgG3, IgG1null, IgG4(S228P), IgA1, IgA2, IgE or IgM. In some embodiments, the first part and / or the second part of the protein of the present invention may comprise an immunologically inert constant region. In some aspects, the first part and / or the second part of the protein of the present invention may comprise an immunoglobulin constant region, the immunoglobulin constant region comprising a wild-type human IgG1 constant region, a human IgG1 constant region containing the amino acid substitutions L234A and L235A, a human IgG1 constant region containing the amino acid substitutions L234A, L235A and G237A or a human IgG1 constant region containing the amino acid substitutions L234A, L235A, G237A and P331S. In some aspects, the first part and / or the second part of the protein of the present invention may comprise an immunoglobulin constant region, the immunoglobulin constant region comprising a wild-type human IgG2 constant region or a wild-type human IgG4 constant region. In some aspects, the first part and / or the second part of the protein of the present invention may comprise an immunoglobulin constant region, the immunoglobulin constant region comprising any one of the amino acid sequences in Table 10. The Fc region sequences in Table 10 start from the CH1 domain. In some aspects, the first part and / or the second part of the protein of the present invention may comprise an immunoglobulin constant region, the immunoglobulin constant region comprising the amino acid sequence of the Fc region of human IgG4, human IgG4(S228P), human IgG2, human IgG1, human IgG1-3M or human IgG1-4M. For example, compared with the wild-type human IgG4 Fc region, the human IgG4(S228P) Fc region contains the following substitution: S228P. For example, the human IgG1-3M Fc region contains the following substitutions compared with the wild-type human IgG1 Fc region: L234A, L235A and G237A, while the human IgG1-4M Fc region contains the following substitutions compared with the wild-type human IgG1 Fc region: L234A, L235A, G237A and P331S. In some aspects, the positions of amino acid residues in the constant region of the immunoglobulin molecule are numbered according to EU nomenclature (Ward et al., 1995 Therap. Immunol. 2:77-94). In some aspects, the immunoglobulin constant region may comprise the RDELT (SEQ ID NO:65) motif or the REEM (SEQ ID NO:66) motif (underlined in Table 10). The REEM (SEQ ID NO:66) allotype is found in a smaller population than the RDELT (SEQ ID NO:65) allotype.In some aspects, the first part and / or the second part of the protein of the present invention may comprise an immunoglobulin constant region containing any one of SEQ ID NOs: 56 - 62. In some aspects, the first part and / or the second part of the protein of the present invention may comprise any one of the cloned heavy chain amino acid sequences and light chain amino acid sequences in Tables 3 - 9 and any one of the Fc region amino acid sequences in Table 10. In some aspects, the first part and / or the second part of the protein of the present invention may comprise an immunoglobulin heavy chain constant region and an immunoglobulin light chain constant region, the immunoglobulin heavy chain constant region comprising any one of the Fc region amino acid sequences in Table 10, and the immunoglobulin light chain constant region being a kappa light chain constant region or a lambda light chain constant region.

[0157] In some aspects, the protein of the present invention comprises an IgG1 isotype constant region. The IgG1 isotype constant region effectively activates all types of FcγR signaling, thereby driving the maximum opsonizing effector function.

[0158] In some aspects, the immunoglobulin constant region comprises a hinge region or a truncated hinge region. In some embodiments, the hinge region may comprise one, two, three, four or more amino acid substitutions compared to the wild - type human hinge region amino acid sequence. In some embodiments, the immunoglobulin constant region does not comprise a hinge region.

[0159] In some aspects, the first part specifically binds to a first molecule expressed in diseased tissue, and the second part is capable of specifically binding to a second molecule expressed in diseased tissue, wherein the first molecule expressed in diseased tissue and the second molecule expressed in diseased tissue are different molecules. In some embodiments, the first molecule expressed in diseased tissue and the second molecule expressed in diseased tissue are expressed by the same cell. In some embodiments, the first molecule expressed in diseased tissue and the second molecule expressed in diseased tissue are expressed by different cells. In some embodiments, the first molecule expressed in diseased tissue, the second molecule expressed in diseased tissue, or both the first molecule expressed in diseased tissue and the second molecule expressed in diseased tissue are expressed on the cell surface. In some embodiments, the first molecule expressed in diseased tissue and / or the second molecule expressed in diseased tissue is a soluble molecule.

[0160] In some aspects, the first portion specifically binds to human cMET, and the second portion specifically binds to human CD47. In some aspects, the first portion specifically binds to human HER2, and the second portion specifically binds to human CD47. In some aspects, the first portion specifically binds to human cMET, and the second portion specifically binds to human CD47. In some aspects, the first portion specifically binds to human HER2, and the second portion specifically binds to human CD3. In some aspects, the first portion specifically binds to human cMET, and the second portion specifically binds to human cMET.

[0161] In some aspects, the protein of the invention has an immune effector function or two, three or more immune effector functions. For example, the immune effector function can be antibody-dependent cell cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) or antibody-dependent cell phagocytosis (ADCP).

[0162] In some aspects, the first portion of the protein of the invention prevents or reduces the specific binding of the second portion to a molecule expressed in diseased tissue. In some embodiments, the peptide linker of the protein of the invention is cleaved near or within the diseased tissue. In some cases, the peptide linker is cleaved near or within the diseased tissue, wherein near or within the diseased tissue the first portion dissociates from the second portion, and wherein near or within the diseased tissue the second portion specifically binds to a molecule expressed in the diseased tissue. In some cases, the cleaved peptide linker contains a binding site or target site for an anti-hinge antibody (e.g., an endogenous anti-hinge antibody of the subject), whereas the uncleaved (e.g., intact) peptide linker does not contain a binding site or target site for the anti-hinge antibody. In the presence of the activated protein of the invention, the binding of the anti-hinge antibody to the cleaved peptide linker can increase ADCC, CDC, and / or ADCP (see, for example Figure 2B ).

[0163] In some aspects, the protein of the invention stimulates inflammatory signaling in diseased tissue. The increased inflammatory signaling can increase immune recruitment to the diseased tissue. In some cases, the protein of the invention increases antigen presentation in the diseased tissue. In some cases, the protein of the invention increases tumor-associated antigen-specific T cell proliferation.

[0164] In some aspects, the diseased tissue can be a tumor, necrotic tissue, fibrotic tissue, tissue undergoing a coagulation cascade or inflamed tissue.

[0165] In some aspects, the present disclosure provides a protein comprising a first part, a second part, and a peptide linker between the first part and the second part, wherein the first part specifically binds to human cMET, wherein the second part specifically binds to human CD47, and wherein the protein comprises a first polypeptide chain and a second polypeptide chain, the first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 16 or consisting of the amino acid sequence of SEQ ID NO: 16, and the second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 17 or consisting of the amino acid sequence of SEQ ID NO: 17. The protein comprises only one copy of the first polypeptide chain and only one copy of the second polypeptide chain. The peptide linker of the protein comprises two copies of the LHL sequence (see Table 1), each copy being fused to the first part at the N-terminus and to the second part at the C-terminus by a peptide bond. The protein is referred to as "Fab2cMetCD47-LHL-LHL" or "Met47-LHL-LHL". The amino acid sequence is provided in Table 3. The structure of the protein is as depicted in Figure 3A as described therein. The second part of the protein is linked to KIH IgG1-Fc via a G4S linker (SEQ ID NO: 15) and a truncated hinge region.

[0166] In some aspects, the present disclosure provides a protein comprising a first part, a second part, and a peptide linker between the first part and the second part, wherein the first part specifically binds to human HER2, wherein the second part specifically binds to human CD3, and wherein the protein comprises a first polypeptide chain and a second polypeptide chain, the first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 26 or consisting of the amino acid sequence of SEQ ID NO: 26, and the second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 27 or consisting of the amino acid sequence of SEQ ID NO: 27. The protein comprises only one copy of the first polypeptide chain and only one copy of the second polypeptide chain. The peptide linker of the protein comprises two copies of the LHL sequence fused by a peptide bond (see Table 1). The protein is referred to as "Fab2Her2CD3-LHL-LHL" or "Her23-LHL-LHL". The amino acid sequence is provided in Table 4. The structure of the protein is as depicted in Figure 3A as described therein. The second part of the protein is linked to KIH IgG1-Fc via a G4S linker (SEQ ID NO: 15) and a truncated hinge region (3M).

[0167] In some aspects, provided herein is a protein comprising a first part and a second part and a peptide linker between the first part and the second part, wherein the first part specifically binds to human HER2, wherein the second part specifically binds to human CD47, and wherein the protein comprises a first polypeptide chain and a second polypeptide chain, the first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 34 or consisting thereof, and the second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 35 or consisting thereof. The protein comprises two identical copies of the first polypeptide chain and two identical copies of the second polypeptide chain. The peptide linker of the protein comprises two copies of the LHL sequence (see Table 1) fused by peptide bonds. The protein is referred to as "IgG2 Her2CD47-LHL-LHL" or "Her47-LHL-LHL". The amino acid sequence is provided in Table 5. The structure of the protein is as Figure 2A depicted therein. The second part of the protein can be linked to the human IgG1 Fc sequence through a hinge region or a truncated hinge region (see Table 10).

[0168] In some aspects, provided herein is a protein comprising a first part and a second part and a peptide linker between the first part and the second part, wherein the first part specifically binds to human cMET, wherein the second part specifically binds to human CD47, and wherein the protein comprises a first polypeptide chain and a second polypeptide chain, the first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 36 or consisting thereof, and the second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 37 or consisting thereof. The first polypeptide chain further comprises a human IgG1 amino acid sequence (see Table 10). The second polypeptide chain further comprises a human κ light chain amino acid sequence. The peptide linker of the protein comprises two copies of the LHL sequence (see Table 1) fused by peptide bonds. The protein is referred to as "Fab2 CMET / CD47 'one-armed' type" or "Met47-LHL-LHL". The amino acid sequence is provided in Table 6. The structure of the protein is as Figure 3B depicted therein. The construct is a 'mortar and pestle structure' one-armed Fab2 construct having Fab2 on the pestle side and a hinge mortar Fc stump on the other side. The construct can comprise a non-effector null human IgG1 Fc sequence (see Table 10).

[0169] In some aspects, provided herein is a protein comprising a first part and a second part and a peptide linker between the first part and the second part, wherein the first part specifically binds to human Her2, wherein the second part specifically binds to human CD3, and wherein the protein comprises a first polypeptide chain and a second polypeptide chain, the first polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO: 38, the second polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO: 39. The first polypeptide chain further comprises a human IgG1-3M amino acid sequence (see Table 10). The second polypeptide chain further comprises a human κ light chain amino acid sequence. The peptide linker of the protein comprises two copies of the LHL sequence (see Table 1) fused by a peptide bond. The protein is referred to as "Fab2 Her2 / CD3 'single-arm' type" or "Her23-LHL-LHL". The amino acid sequence is provided in Table 7. The structure of the protein is as Figure 3B depicted therein. The construct is a 'mortar and pestle structure' single-arm Fab2 construct having Fab2 on the pestle side and a hinge mortar Fc stump on the other side. The construct is also effector-inactive (IgG1-3M; see Table 10).

[0170] In some aspects, provided herein is a protein comprising a first part and a second part and a peptide linker between the first part and the second part, wherein the first part specifically binds to human Her2, wherein the second part specifically binds to human CD3, and wherein the protein comprises a first polypeptide chain and a second polypeptide chain, the first polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO: 40, the second polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO: 41. The first polypeptide chain further comprises a human IgG1-3M amino acid sequence (see Table 10). The second polypeptide chain further comprises a human λ light chain amino acid sequence. The peptide linker of the protein comprises two copies of the LHL sequence (see Table 1) fused by a peptide bond. The protein is referred to as "Fab2 Her2 / CD3(34) 'single-arm' type" or "Her23(34)-LHL-LHL". The amino acid sequence is provided in Table 8. The structure of the protein is as Figure 3B depicted therein. The construct is a 'mortar and pestle structure' single-arm Fab2 construct having Fab2 on the pestle side and a hinge mortar Fc stump on the other side. The construct is also effector-inactive (IgG1-3M; see Table 10).

[0171] In some aspects, provided herein is a protein comprising a first part, a second part, and a peptide linker between the first part and the second part, wherein the first part specifically binds to human Her2, wherein the second part specifically binds to human CD47, and wherein the protein comprises a first polypeptide chain and a second polypeptide chain, wherein:

[0172] (a) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 42, and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 43 (referred to as "Her47-LHLF-LHL"); or

[0173] (b) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 44, and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 45 (referred to as "Her47-LHL-LHLF"); or

[0174] (c) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 46, and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 47 (referred to as "Her47-LHLF-LHLF"); or

[0175] (d) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 48, and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 49 (referred to as "Her47-LHLM-LHLM"); or

[0176] (e) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 50, and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 51 (referred to as "Her47-LHLM-LHLMF"); or

[0177] (f) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 52, and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 53 (referred to as "Her47-LHLMF-LHLM"); or

[0178] (g) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 54, and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 55 (referred to as "Her47-LHLMF-LHLMF"); or

[0179] (h) The first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 44, and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 92 (referred to as "Her47 LHL-LHL-EK");

[0180] (i) The first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 44, and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 93 (referred to as "Her47-LHL-LHL-Thr");

[0181] (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: 94 (referred to as "Her47-LHL-LHL-tPA");

[0182] (k) The first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 44, and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 95 (referred to as "Her47-LHL-LHL-GrB");

[0183] (1) The first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 44, and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 96 (referred to as "Her47-LHL-LHL-uPA"); or

[0184] (m) The first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 44, and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 97 (referred to as "Her47-LHL-LHL-A5"). These proteins are commonly referred to as "IgG2 Her2 / CD47". The amino acid sequences are provided in Tables 9 and 20. The structure of the protein is depicted as Figure 2A shown. The peptide linker sequences of the LHLF, LHLM, and LHLMF linkers are provided in Table 1. The peptide linker sequences of the EK, Thr, tPA, GrB, uPA, and A5 linkers are provided in Table 21.

[0185] In some aspects, provided herein are proteins comprising a first portion and a second portion and a peptide linker between the first portion and the second portion, wherein the first portion specifically binds to human Her2, wherein the second portion specifically binds to human CD47, and wherein the protein comprises a first polypeptide chain and a second polypeptide chain, wherein:

[0186] (a) The first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 88, and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 89 (referred to as "Her47 LHLF-LHL IgG1-2hDAA"); or

[0187] (b) The first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 90, and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 91 (referred to as "Her47 LHL-LHLF IgG1-2hDAA"). These proteins are generally referred to as "IgG2‘I gG1-DAA’Her2 / CD47". The amino acid sequences are provided in Table 19. These proteins contain stabilizing mutations in the hinge. The structure of the protein is as Figure 2A depicted. The peptide linker sequences of the LHLF and LHL linkers are provided in Table 1.

[0188] In some aspects, provided herein is a protein comprising a first portion, a second portion, and a peptide linker between the first portion and the second portion, wherein the first portion specifically binds to human Her2, wherein the second portion specifically binds to human CD3, and wherein the protein comprises a first polypeptide chain and a second polypeptide chain, the first polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO: 73, the second polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO: 74. The first polypeptide chain further comprises a human IgG1-3M Fc amino acid sequence (e.g., containing the 'mortar' mutation to enable heterodimerization with the second polypeptide chain), followed by a linker sequence, the VH and CH1 domains of the first binding portion, another linker sequence, and then the VH and CH1 domains of the second binding portion (see Table 13). The second polypeptide chain further comprises a human IgG1-3M Fc amino acid sequence (e.g., containing the 'pestle' mutation to enable heterodimerization with the second polypeptide chain), followed by a linker sequence, the VL and CL domains of the first binding portion, another linker sequence, and then the VL and CL domains of the second binding portion (see Table 13). The peptide linker of the protein comprises four sequences fused by peptide bonds (see Table 1), which are located between the Fc and the first portion and between the first binding portion and the second binding portion. The protein is referred to as "Fc-Her2 / CD3(34)" or "Fc-Her23(34)". The amino acid sequences are provided in Table 13. The structure of the protein is as Figure 12 depicted. The construct is a "mortar and pestle structure" Fc-Fab2 construct, which has a light chain polypeptide on the pestle side or the mortar side and a heavy chain polypeptide on the other side. The construct is also effectorless (IgG1-3M; see Table 10).

[0189] In some aspects, provided herein is a protein comprising a first part and a second part and a peptide linker between the first part and the second part, wherein the first part specifically binds to human cMET, wherein the second part specifically binds to human cMET, and wherein the protein comprises a first polypeptide chain and a second polypeptide chain, the first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 75 or consisting of the same, and the second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 76 or consisting of the same. The first polypeptide chain further comprises the human IgG1 or human IgG1-3M amino acid sequence (see Table 10). The second polypeptide chain further comprises the human kappa light chain amino acid sequence. The peptide linker of the protein comprises two copies of the LHL sequence fused by peptide bonds (see Table 1). The protein is referred to as "Fab2 CMET / CMET 'single arm' type" or "MetMet-LHL-LHL". The amino acid sequence is provided in Table 14. The structure of the protein is as Figure 3B depicted therein. The construct is a 'mortar and pestle structure' single arm Fab2 construct having Fab2 on the pestle side and a hinge mortar Fc stump on the other side. The construct may comprise a human IgG1 Fc sequence that is or is not effector null (see Table 10).

[0190] In some aspects, provided herein is a protein comprising a first part and a second part and a peptide linker between the first part and the second part, wherein the first part specifically binds to human Her2, wherein the second part specifically binds to human CD3, and wherein the protein comprises a first polypeptide chain and a second polypeptide chain, wherein:

[0191] (a) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 98 or consists of the same, and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 99 or consists of the same (referred to as "Fab2 Her23 LHL-LHLF"); or

[0192] (b) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 100 or consists of the same, and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 101 or consists of the same (referred to as "Fab2 Her23 LHL-LHL"); or

[0193] (c) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 102 or consists of the same, and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 103 or consists of the same (referred to as "Fab2 Her23 LHLF-LHL-S"); or

[0194] (d) The first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 104, and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 105 (referred to as "Fab2 Her23 LHL-LHL-S"). This protein is referred to as "Fab2Her2 / CD3". The amino acid sequences are provided in Table 22. The structure of the protein is depicted as in Figure 3A depicted. The protein comprises only one copy of the first polypeptide chain and only one copy of the second polypeptide chain. The peptide linker of the protein comprises two copies of the LHL sequence (see Table 1) fused by peptide bonds, or one copy of the LHL sequence and one copy of the LHLF sequence.

[0195] In some aspects, the present disclosure provides immunoconjugates that comprise a protein of the invention as defined herein conjugated to an additional therapeutic agent.

[0196] Examples of suitable therapeutic agents include cytotoxins, radioisotopes, chemotherapeutic agents, immunomodulators, anti-angiogenic agents, anti-proliferative agents, pro-apoptotic agents, and cell growth inhibitory enzymes and cytolytic enzymes (e.g., RNase). Additional therapeutic agents include therapeutic nucleic acids, such as genes encoding immunomodulators, anti-angiogenic agents, anti-proliferative agents, or pro-apoptotic agents. These drug descriptors are not mutually exclusive, and thus one or more of the above terms may be used to describe a therapeutic agent.

[0197] Examples of suitable therapeutic agents for immunoconjugates include taxanes, maytansine, CC-1065 and duocarmycin, calicheamicin and other enediynes, and auristatin. Other examples include antifolates, vinca alkaloids, and anthracyclines. Phytotoxins, other bioactive proteins, enzymes (i.e., ADEPT), radioisotopes, photosensitizers may also be used in immunoconjugates. Additionally, conjugates can be prepared using secondary carriers as cytotoxic agents (such as liposomes or polymers). Suitable cytotoxins include agents that inhibit or prevent cell function and / or cause cell destruction. Representative cytotoxins include antibiotics, microtubule polymerization inhibitors, alkylating agents that bind to and damage DNA, and agents that disrupt protein synthesis or the function of essential cellular proteins (such as protein kinases, phosphatases, topoisomerases, and cyclins).

[0198] Representative cytotoxins include, but are not limited to, doxorubicin, daunorubicin, idarubicin, aclarubicin, daunorubicin, mitoxantrone, epirubicin, carrubicin, noramycin, menolide, pitarubicin, valrubicin, cytarabine, gemcitabine, trifluridine, ancitabine, enocitabine, azacitidine, doxifluhdine, pentostatin, broxuhdine, capecitabine, cladhbine, decitabine, floxuhdine, fludarabine, glutathione, puromycin, fludoxidine, fludarabine, puromycin, tegafur, thiazolamide nucleoside

[0013] The invention relates to tiazofuhn, adhamycin, cisplatin, carboplatin, cyclophosphamide, dacarbazine, vinblastine, vincristine, mitoxantrone, bleomycin, nitrogen mustard, prednisone, procarbazine, methotrexate, fluorouracil, etoposide, paclitaxel, paclitaxel analogs, platinums such as cisplatin and carboplatin, mitomycin, thiotepa, taxanes, vincristine, daunorubicin, epirubicin, actinomycin, authramycin, azaserine, bleomycin, tamoxifen, idarubicin, dolastatin / auristatin, hemiasterlin, esperamicins, and maytansinoids.

[0199] Suitable immunomodulators include anti-hormonal drugs that block the effects of hormones on tumors, and immunosuppressive agents that inhibit cytokine production, downregulate self-antigen expression, or mask MHC antigens.

[0200] Also provided are nucleic acid molecules encoding a protein of the invention as defined herein or a portion of a protein. Also provided herein are nucleic acid molecules encoding a first polypeptide chain, a second polypeptide chain, or both a first polypeptide chain and a second polypeptide chain of a protein of the invention, wherein the protein of the invention comprises a plurality of non-identical polypeptide chains. In some aspects, the nucleic acid molecules as defined herein may be isolated.

[0201] Also provided is a vector comprising a nucleic acid molecule of the invention as defined herein.The vector may be an expression vector.

[0202] Also provided is a host cell comprising a nucleic acid molecule or a vector of the invention as defined herein.The host cell may be a recombinant host cell.

[0203] In a further aspect, a method of producing a protein of the invention is provided, the method comprising culturing a host cell of the invention under conditions resulting in expression and / or production of the protein, and isolating the protein from the host cell or culture.

[0204] In some aspects, the present disclosure provides pharmaceutical compositions comprising a protein of the invention as defined herein, or a nucleic acid molecule of the invention as defined herein, or a vector of the invention as defined herein.

[0205] Also provided are methods for enhancing an immune response in a subject, the methods comprising administering to the subject an effective amount of a protein of the invention as defined herein, or an immunoconjugate of the invention as defined herein, or a nucleic acid molecule of the invention as defined herein, or a vector of the invention as defined herein, or a pharmaceutical composition of the invention as defined herein.

[0206] In additional aspects, provided are methods for treating or preventing cancer in a subject, or for ameliorating the symptoms of cancer in a subject, the methods comprising administering an effective amount of a protein of the invention as defined herein, or an immunoconjugate of the invention as defined herein, or a nucleic acid molecule of the invention as defined herein, or a vector of the invention as defined herein, or a pharmaceutical composition of the invention as defined herein.

[0207] In some aspects, the cancer is a solid tumor. In some cases, the cancer is a hematological malignancy. For example, the cancer can be gastrointestinal stromal cancer (GIST), pancreatic cancer, skin cancer (e.g., melanoma), breast cancer, lung cancer, bronchial cancer, colorectal cancer, prostate cancer, gastric cancer, ovarian cancer, bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, cancer of the oral cavity or pharynx, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small intestine or appendiceal cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma or cancer of the blood tissue. In some cases, the cancer of the blood tissue is lymphoma.

[0208] In some aspects, the present disclosure provides a protein of the invention as defined herein, or an immunoconjugate of the invention as defined herein, or a nucleic acid molecule of the invention as defined herein, or a vector of the invention as defined herein, or a pharmaceutical composition of the invention as defined herein, for treating cancer or for ameliorating the symptoms of cancer.

[0209] In some aspects, the present disclosure provides a protein, or an immunoconjugate, or a nucleic acid molecule, or a vector, for use or treatment methods of the invention as defined herein, for separate, sequential or simultaneous use in combination with a second therapeutic agent (e.g., an anti-cancer agent).

[0210] In another aspect, provided is the use of a protein of the invention as defined herein, or an immunoconjugate of the invention as defined herein, or a nucleic acid molecule of the invention as defined herein, or a vector of the invention as defined herein, or a pharmaceutical composition of the invention as defined herein, in the preparation of a medicament for treating cancer or for ameliorating the symptoms of cancer.

[0211] The invention also provides a method for treating or preventing an autoimmune disease or an inflammatory disease in a subject, the method comprising administering to the subject an effective amount of a protein as defined herein, or an immunoconjugate as defined herein, or a nucleic acid molecule as defined herein, or a vector as defined herein, or a pharmaceutical composition as defined herein.

[0212] For example, the autoimmune disease or inflammatory disease can be arthritis, asthma, multiple sclerosis, psoriasis, Crohn's disease, inflammatory bowel disease, lupus, Graves' disease, and Hashimoto's thyroiditis or ankylosing spondylitis.

[0213] Also provided is a protein as defined herein, or an immunoconjugate as defined herein, or a nucleic acid molecule as defined herein, or a vector as defined herein, or a pharmaceutical composition as defined herein, for treating an autoimmune disease or an inflammatory disease.

[0214] Also provided is the use of a protein as defined herein, or an immunoconjugate as defined herein, or a nucleic acid molecule as defined herein, or a vector as defined herein, or a pharmaceutical composition as defined herein, in the preparation of a medicament for treating an autoimmune disease or an inflammatory disease.

[0215] The invention also provides a method for treating or preventing a cardiovascular disease or a fibrotic disease in a subject, the method comprising administering to the subject an effective amount of a protein as defined herein, or an immunoconjugate as defined herein, or a nucleic acid molecule as defined herein, or a vector as defined herein, or a pharmaceutical composition as defined herein.

[0216] Also provided is a protein as defined herein, or an immunoconjugate as defined herein, or a nucleic acid molecule as defined herein, or a vector as defined herein, or a pharmaceutical composition as defined herein, for treating a cardiovascular disease or a fibrotic disease.

[0217] Also provided is the use of a protein as defined herein, or an immunoconjugate as defined herein, or a nucleic acid molecule as defined herein, or a vector as defined herein, or a pharmaceutical composition as defined herein, in the preparation of a medicament for treating a cardiovascular disease or a fibrotic disease.

[0218] Cardiovascular diseases in any aspect of the present invention can be, for example, coronary heart disease, atherosclerosis or stroke.

[0219] For example, fibrotic diseases in any aspect of the present invention can be myocardial infarction, angina pectoris, osteoarthritis, pulmonary fibrosis, asthma, cystic fibrosis or bronchitis.

[0220] In some aspects, provided herein are proteins comprising the amino acid sequences disclosed herein and in the forms disclosed herein for use in therapy.

[0221] In some aspects, the pharmaceutical composition can comprise a pharmaceutically acceptable excipient, carrier or diluent. A pharmaceutically acceptable excipient can be a compound or combination of compounds that does not cause secondary reactions and allows, for example, the entry into the pharmaceutical composition that promotes the administration of the protein as defined herein, an increase in its lifespan and / or its efficacy in the body or an increase in its solubility in solution. These pharmaceutically acceptable vehicles are well known and will be adjusted by those skilled in the art according to the mode of administration of the protein as defined herein.

[0222] In some aspects, the protein as defined herein can be provided in lyophilized form for reconstitution prior to administration. For example, the lyophilized protein molecules can be reconstituted in sterile water and mixed with saline prior to administration to an individual.

[0223] The protein as defined herein will generally be administered in the form of a pharmaceutical composition, which can comprise at least one component in addition to the protein molecule. Thus, in addition to the protein as defined herein, the pharmaceutical composition can also comprise pharmaceutically acceptable excipients, carriers, buffers, stabilizers or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the protein. The precise nature of the carrier or other material will depend on the route of administration, which can be bolus injection, infusion, injection or any other suitable route, as described below.

[0224] For parenteral administration (such as subcutaneous or intravenous administration, e.g., by injection), a pharmaceutical composition comprising a protein as defined herein can be in the form of a parenterally acceptable aqueous solution, which is pyrogen-free and has a suitable pH, isotonicity, and stability. A person skilled in the relevant art can prepare a suitable solution using, for example, isotonic media such as sodium chloride injection, Ringer's injection, lactated Ringer's injection. Preservatives, stabilizers, buffers, antioxidants, and / or other additives can be used as needed, including buffers such as phosphates, citrates, and other organic acids; antioxidants (such as ascorbic acid and methionine); preservatives (such as octadecyl dimethyl benzyl ammonium chloride, hexamethonium dichloride; benzalkonium chloride; benzethonium chloride, diphenol, butyl or benzyl alcohol; alkyl esters of p-hydroxybenzoic acid (such as methyl p-hydroxybenzoate or propyl p-hydroxybenzoate); catechol; resorcinol; cyclohexanol; 3'-pentanol; and m-cresol); low molecular weight polypeptides; proteins (such as serum albumin, gelatin, or immunoglobulins); hydrophilic polymers such as polyvinylpyrrolidone; amino acids (such as glycine, glutamine, asparagine, histidine, arginine, or lysine); monosaccharides, disaccharides, and other carbohydrates (including glucose, mannose, or dextrin); chelating agents (such as EDTA); sugars (such as sucrose, mannitol, trehalose, or sorbitol); salt-forming counterions (such as sodium ions); metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants (such as TWEEN TM , PLURONICS TM or polyethylene glycol (PEG)).

[0225] A pharmaceutical composition comprising a protein as defined herein can be administered alone or in combination with other therapies (simultaneously or sequentially) depending on the condition to be treated.

[0226] A protein as defined herein can be used in a method for treating a human or animal body, the treatment including prophylactic or preventive treatment (e.g., reducing the risk of developing a condition in an individual before the onset of the condition in the individual; delaying the onset of the condition; or treating to reduce the severity of the condition after onset). The treatment method can include administering a protein as defined herein to an individual in need thereof.

[0227] The administration is typically at a "therapeutically effective amount" which is sufficient to show benefit to the patient. Such benefit can be at least improvement of at least one symptom. The actual amount administered, as well as the rate and duration of administration, will depend on the nature and severity of the disease to be treated, the particular mammal to be treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the composition, the method of administration, the timing of administration and other factors known to the practicing physician. Prescription of treatment (e.g., decisions regarding dosage, etc.) is the responsibility of the general practitioner and other physicians and can depend on the severity and / or progression of the symptoms of the disease to be treated. Appropriate doses of antibody molecules are well known in the art (Ledermann J.A. et al., 1991, Int. J. Cancer 47:659-664; Bagshawe K.D. et al., 1991, Antibody, Immunoconjugates and Radiopharmaceuticals 4:915-922). Specific doses can be indicated herein or in the Physician's Desk Reference (2003) as appropriate for the type of drug being administered. The therapeutically effective amount or appropriate dose of a protein as defined herein can be determined by comparing its in vitro activity and in vivo activity in animal models. Methods for extrapolating effective doses in mice and other test animals to humans are known. The exact dose will depend on many factors including whether the protein is for prophylaxis or treatment, the size and location of the area to be treated, the exact nature of the protein (e.g., Fab2, IgG) and the nature of any detectable label or other molecule attached to the protein.

[0228] For systemic administration, typical protein doses are in the range of 100 μg to 1 g, and for local administration, typical protein doses are in the range of 1 μg to 1 mg. An initial higher loading dose can be administered, followed by one or more lower doses. In some aspects, the protein will comprise a full antibody, such as an IgG1 or IgG4 isotype. This is the dose for a single treatment in an adult patient, which can be adjusted proportionally for children and infants and also proportionally for other protein construct forms based on molecular weight. Treatment can be repeated at intervals of daily, twice weekly, weekly or monthly, at the discretion of the physician. The individual treatment regimen can depend on the pharmacokinetic and pharmacodynamic properties of the protein composition, the route of administration and the nature of the disorder being treated.

[0229] Treatment can be periodic, and the period between administrations can be about two weeks or longer, such as about three weeks or longer, about four weeks or longer, about once a month or longer, about five weeks or longer, or about six weeks or longer. For example, treatment can be every 2 - 4 weeks or every 4 - 8 weeks. Treatment can be given before and / or after surgery, and / or can be administered or applied directly at the anatomical site of surgical treatment or invasive procedure. Suitable formulations and routes of administration are as described above.

[0230] In some aspects, the protein as defined herein can be administered as a subcutaneous injection. An autoinjector can be used for subcutaneous injection, for example, for long - term or short - term prophylaxis / therapy.

[0231] In some aspects, the therapeutic effect of the protein as defined herein can persist in serum for several times the protein half - life, depending on the dose. For example, the therapeutic effect of a single dose of the protein as defined herein can persist in an individual for 1 month or longer, 2 months or longer, 3 months or longer, 4 months or longer, 5 months or longer, or 6 months or longer.

[0232] As used herein, the term "CD47" refers to IAP (Integrin - associated protein) and its variants that retain at least part of the biological activity of CD47. As used herein, CD47 includes the native sequence CD47 of all mammalian species (including human, rat, mouse, and chicken). In some embodiments, the term "CD47" is used to include variants, isotypes, and species homologs of human CD47. In some cases, as used herein, CD47 includes the native sequence CD47 of all mammalian and non - mammalian species (including human, monkey, rat, mouse, and chicken). In some embodiments, the term "CD47" refers only to wild - type CD47. The proteins of the present invention can cross - react with CD47 from species other than human (in particular, CD47 from cynomolgus monkey (Macaca fascicularis)). Examples of the amino acid sequences of human and cynomolgus monkey CD47 are provided in Table 11. In certain embodiments, the proteins of the present invention can be completely specific for human CD47 and can not exhibit non - human cross - reactivity.

[0233] As used herein, the term "cMET" refers to the MET protein and its variants that retain at least a portion of the biological activity of cMET. In some cases, as used herein, cMET includes the native sequence cMET of all mammalian species (including human, rat, mouse, and chicken). In some embodiments, the term "cMET" can be used to include variants, isotypes, and species homologs of human cMET. In some cases, as used herein, cMET includes the native sequence cMET of all mammalian and non-mammalian species (including human, monkey, rat, mouse, and chicken). In some embodiments, the term "cMET" refers only to wild-type cMET. The antibodies of the present invention can cross-react with cMET from species other than human (particularly cMET from cynomolgus monkey (Macaca fascicularis)). Examples of human and cynomolgus monkey cMET amino acid sequences are provided in Table 12. In certain embodiments, the antibody can be fully specific for human cMET and can not exhibit non-human cross-reactivity.

[0234] As used herein, the term "Her2" refers to the human epidermal growth factor receptor 2 protein and its variants that retain at least a portion of the biological activity of Her2. Her2 is also known as HER2 / neu, ErbB2, c-erbB-2, and human EGF receptor 2. In some embodiments, the term "Her2" can be used to include variants, isotypes, and species homologs of human Her2. In some cases, as used herein, Her2 includes the native sequence Her2 (also known as ErbB2) of all mammalian and non-mammalian species (including human, monkey, rat, mouse, and chicken). In some embodiments, the term "Her2" refers only to wild-type Her2. The antibodies of the present invention can cross-react with Her2 from species other than human (particularly Her2 from cynomolgus monkey (Macaca fascicularis)). Examples of human and cynomolgus monkey Her2 / ErbB2 amino acid sequences are provided in Table 15. In certain embodiments, the antibody can be fully specific for human Her2 and can not exhibit non-human cross-reactivity.

[0235] As used herein, the term "CD3" refers to "cluster of differentiation 3" multimeric protein complexes and variants thereof that retain at least a portion of the biological activity of CD3. The CD3 complex comprises four distinct polypeptide chains; epsilon (ε), gamma (γ), delta (δ), and zeta (ζ). These polypeptide chains assemble and function as three pairs of dimers ( εγ, εδ, ζζ). In some embodiments, the term "CD3" may be used to include variants, isotypes, and species homologs of human CD3. In some instances, as used herein, CD3 includes the native sequence CD3 of all mammalian and non-mammalian species (including human, monkey, rat, mouse, and chicken). In some embodiments, the term "CD3" refers only to wild-type CD3. The antibodies of the present invention may cross-react with CD3 from species other than human (particularly CD3 from cynomolgus monkeys (Macaca fascicularis)). Examples of human and cynomolgus monkey CD3ε amino acid sequences are provided in Table 16. In certain embodiments, the antibodies may be fully specific for human CD3 and may not exhibit non-human cross-reactivity.

[0236] As used herein, an "antagonist", as used in the context of the proteins of the present invention, refers to a protein that is capable of binding to a molecule expressed in diseased tissue and inhibiting the biological activity of the molecule and / or downstream pathways mediated by the molecule. For example, an "anti-CD47 antagonist protein" (interchangeably referred to as an "anti-CD47 protein") refers to a protein that is capable of binding to CD47 and inhibiting the biological activity of CD47 and / or downstream pathways mediated by CD47 signaling. Anti-CD47 antagonist proteins encompass proteins that can block, antagonize, inhibit, or reduce (including significantly reduce) the biological activity of CD47 (including downstream pathways mediated by CD47 signaling, such as receptor binding and / or eliciting a cellular response to CD47). For the purposes of the present invention, it should be clearly understood that the term "anti-CD47 antagonist protein" encompasses all terms, names, and functional states and characteristics in which CD47 itself and the biological activity of CD47 (including but not limited to its ability to enhance phagocytosis by myeloid lineage cells) or activity or the result of the biological activity is substantially ineffective, reduced, or neutralized to any meaningful extent.

[0237] A protein of the present invention "specifically binds", "specifically interacts", "preferentially binds", "binds", or "interacts" with a molecule (e.g., human CD47, human Her2, human CD3, human cMET, or human PD-L1) if the protein of the present invention binds to the molecule with greater affinity, avidity, more readily, and / or for a longer duration than it binds to other molecules.

[0238] "Antibody molecule" refers to an immunoglobulin molecule that can specifically bind to a target (such as carbohydrates, polynucleotides, lipids, polypeptides, etc.) through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, the term "antibody molecule" encompasses not only intact polyclonal or monoclonal antibodies, but also any antigen-binding fragment (e.g., "antigen-binding portion"), or its single chain, fusion proteins containing an antibody, and any other modified configuration of an immunoglobulin molecule containing an antigen recognition site, including, for example, but not limited to scFv, single-domain antibodies (e.g., shark and camelid antibodies), maxibodies, minibodies, intrabodies, diabodies, triabodies, tetra-bodies, v-NAR, and bis-scFv.

[0239] "Antibody molecule" encompasses any type of antibody, such as IgG, IgA, or IgM (or its subclass), and the antibody does not need to be of any particular type. Depending on the amino acid sequence of the constant region of the antibody heavy chain, immunoglobulins can be divided into different classes. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The constant regions of the heavy chains corresponding to the different classes of immunoglobulins are referred to as α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

[0240] As used herein, the "antigen-binding portion" of an antibody molecule refers to one or more fragments of an intact antibody that retain the ability to specifically bind an antigen. The antigen-binding function of an antibody molecule can be performed by fragments of the intact antibody. Examples of binding fragments that are encompassed by the term "antigen-binding portion" of an antibody molecule include Fab; Fab'; F(ab')2; the Fd fragment consisting of the VH and CH1 domains; the Fv fragment consisting of the VL and VH domains of a single arm of an antibody; single-domain antibody (dAb) fragments; and isolated complementarity-determining regions (CDRs).

[0241] The term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain. The "Fc region" can be a native sequence Fc region or a variant Fc region. Although the boundaries of the Fc region of an immunoglobulin heavy chain may vary, the human IgG heavy chain Fc region is generally defined as the extension from the amino acid residue at position Cys226 or from Pro230 to its carboxyl terminus. The numbering of residues in the Fc region is that of the EU index as described in Kabat. The Fc region of an immunoglobulin generally contains two constant domains, CH2 and CH3. As is known in the art, the Fc region can exist in dimer or monomer form.

[0242] The "variable region" of an antibody refers to the variable region of an antibody light chain or the variable region of an antibody heavy chain, either alone or in combination. As is known in the art, the variable regions of the heavy and light chains each consist of four framework regions (FRs) connected by three complementarity-determining regions (CDRs) (also known as hypervariable regions), and contribute to the formation of the antigen-binding site of the antibody. When selecting FRs flanking the CDRs, for example when humanizing or optimizing an antibody, it is preferred that the FRs are from an antibody containing the same canonical class CDR sequences.

[0243] As used herein, the term "conservative substitution" refers to the replacement of one amino acid with another amino acid that does not significantly and detrimentally alter the functional activity. Preferred examples of "conservative substitutions" are the replacement of one amino acid with another amino acid having a value ≥0 in the following BLOSUM 62 substitution matrix (see Henikoff and Henikoff, 1992, PNAS 89:10915-10919):

[0244]

[0245] The term "monoclonal antibody" (Mab) refers to an antibody or an antigen-binding portion thereof derived from a single copy or clone (including, for example, any eukaryotic, prokaryotic, or phage clone), regardless of the method by which it is produced. Preferably, the monoclonal antibodies of the present invention are present in a homogeneous or substantially homogeneous population.

[0246] A "humanized" antibody molecule refers to a form of an antibody molecule or an antigen-binding portion thereof that contains a minimal sequence derived from a non-human immunoglobulin and is a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof (such as an Fv, Fab, Fab', F(ab′)2, or other antigen-binding sequence of a non-human (e.g., murine) antibody molecule). A humanized antibody can be a human immunoglobulin (recipient antibody) in which the residues of the recipient CDRs are replaced with the residues of the CDRs of a non-human species (donor antibody) such as a mouse, rat, or rabbit having the desired specificity, affinity, and capacity.

[0247] A "human antibody" or "fully human antibody" refers to an antibody molecule or an antigen-binding portion thereof derived from a transgenic mouse carrying human antibody genes or from human cells.

[0248] The term "chimeric antibody" is intended to refer to an antibody molecule or an antigen-binding portion thereof in which the variable region sequence is derived from one species and the constant region sequence is derived from another species, such as an antibody molecule in which the variable region sequence is derived from a murine antibody and the constant region sequence is derived from a human antibody.

[0249] The term "immunoconjugate" refers to a protein of the present invention conjugated, fused or linked to at least one cytotoxic agent, cell growth inhibitor or therapeutic agent.

[0250] The proteins of the present invention can be produced using techniques well known in the art, such as recombinant techniques, phage display techniques, synthetic techniques or combinations of such techniques or other techniques readily known in the art.

[0251] The term "isolated molecule" (wherein the molecule is, for example, a polypeptide, polynucleotide or antibody) is a molecule which, by virtue of its origin or source of derivation, (1) is not associated with the natural associated components which accompany it in its natural state, (2) is substantially free of other molecules from the same species, (3) is expressed by cells from a different species, or (4) does not exist in nature. Thus, a molecule chemically synthesized or expressed in a cell system other than the cell of its natural origin will be "isolated" from its natural associated components. Molecules can also be rendered substantially free of natural associated components by isolation using purification techniques well known in the art. The purity or homogeneity of a molecule can be determined by a variety of means well known in the art. For example, the purity of a polypeptide sample can be determined using polyacrylamide gel electrophoresis and the gel stained using techniques well known in the art to visualize the polypeptide. For certain purposes, higher resolution can be provided by using HPLC or other means well known in the art for purification.

[0252] The term "epitope" refers to a molecular moiety that can be recognized and bound by a protein, antibody molecule or an antigen-binding portion thereof of the present invention at one or more antigen-binding regions of the protein or antibody molecule. An epitope can consist of a defined region of a primary, secondary or tertiary protein structure and includes combinations of secondary structure units or structural domains of a target recognized by an antigen-binding region of a protein, antibody or an antigen-binding portion thereof. An epitope can equally consist of defined chemically reactive surface groupings of molecules such as amino acids or sugar side chains and has specific three-dimensional structural features as well as specific charge characteristics. As used herein, the term "antigenic epitope" is defined as a polypeptide moiety to which a protein or antibody molecule of the present invention can specifically bind, as determined by any method well known in the art, such as by conventional immunoassays, antibody competitive binding assays or by x-ray crystallography or related structure determination methods (such as NMR).

[0253] The term "binding affinity" or "KD" refers to the dissociation rate of a specific antigen-binding protein interaction or antigen-antibody interaction. KD is the ratio of the dissociation rate (also referred to as "dissociation rate (k off )") to the association rate or "binding rate (k on )". Thus, K D equals k off / k on and is expressed as molar concentration (M). Thus, the smaller the K D , the stronger the binding affinity. Thus, a KD of 1 μM D indicates a weaker binding affinity compared to a KD of 1 nM D . The KD value of a binding protein or antibody can be determined using established methods in the art. One method for determining the KD of a binding protein or antibody is by using surface plasmon resonance (SPR), typically using a biosensor system such as system.

[0254] The term "potency" is a measure of biological activity and can be designated as IC 50 or the effective concentration at which the protein of the immunoconjugate of the present invention inhibits the activity of its binding partner (e.g., a molecule expressed in diseased tissue) or antigen by 50% in an activity assay as described herein.

[0255] As used herein, the phrase "effective amount" or "therapeutically effective amount" refers to the amount (in terms of dosage and duration of time and with respect to the means of administration) necessary to achieve a desired therapeutic outcome. An effective amount is at least the minimum amount of the active agent necessary to confer a therapeutic benefit to a subject, but less than a toxic amount.

[0256] As used herein, the terms "inhibit" or "neutralize" with respect to the biological activity of the proteins of the present invention mean the ability of the protein to substantially antagonize, prevent, inhibit, slow down, disrupt, eliminate, stop, reduce, or reverse the progression or severity of, for example, the biological activity being inhibited (including but not limited to the biological activity or binding interaction of a molecule expressed in diseased tissue).

[0257] "Host cell" includes a single cell or cell culture that can be or has been the recipient of a vector for incorporation of a polynucleotide insert. Host cells include the progeny of a single host cell, and due to natural, accidental, or deliberate mutations, such progeny may not necessarily be identical to the original parental cell (either morphologically or in genomic DNA complementarity). Host cells include cells transfected in vivo with the polynucleotides of the present invention.

[0258] As used herein, "vector" means a construct capable of delivering and preferably expressing one or more genes or sequences of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors; naked DNA or RNA expression vectors; plasmid, cosmid or phage vectors; DNA or RNA expression vectors associated with cationic condensing agents; DNA or RNA expression vectors encapsulated in liposomes; and certain eukaryotic cells, such as producer cells.

[0259] Unless otherwise specified, the term "treatment" as used herein means reversing, alleviating, inhibiting the progression of, delaying the progression of, delaying the onset of, or preventing the disorder or condition to which the term applies or one or more symptoms of such disorder or condition. Unless otherwise specified, the term "treatment" as used herein refers to the treatment act as defined above. The term "treatment" also includes adjuvant and neoadjuvant treatment of a subject. For the avoidance of doubt, reference to "treatment" herein includes reference to curative, palliative and prophylactic treatment. For the avoidance of doubt, reference to "treatment" herein also includes reference to curative, palliative and prophylactic treatment.

[0260] It should be understood that whenever an embodiment is described herein using the language "comprising", other similar embodiments described in terms of "consisting of" and / or "consisting essentially of" are also provided.

[0261] In cases where aspects or embodiments of the invention are described in terms of Markush groups or other alternatives, the invention covers not only the entire group listed as a whole, but also each member of the individual groups and all possible subgroups of the larger group, and also covers the larger group in the absence of one or more of the constituent members. The invention also contemplates the express exclusion of one or more of any of the constituent members from the claimed invention.

[0262] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control. Throughout this specification and the claims, the word "comprise" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of the stated integer or group of integers, but not the exclusion of any other integer or group of integers. Further, unless the context otherwise requires, singular terms shall include the plural and plural terms shall include the singular. Any examples following the terms "e.g." or "for example" are not meant to be exhaustive or limiting.

[0263] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art.

[0264] Specific non-limiting embodiments of the present invention will now be described with reference to the accompanying drawings.

[0265] Example

[0266] Example 1. Generation of optimized conditionally active therapeutic antibodies

[0267] Introduction

[0268] In this example, we have successfully generated a panel of optimized conditionally active antibodies that are well expressed, biophysically stable, highly soluble, and have maximum amino acid sequence identity to a preferred human germline.

[0269] Materials and Methods

[0270] Protein cloning, transient expression, purification and characterization

[0271] The antibody encoding DNA sequence was cloned into separate human IgG1 heavy and light chain encoding expression cassettes in separate plasmid vectors by restriction ligation cloning to produce IgG or IgG for expression. 2 Similar cassettes were also cloned into the "knob-in-hole" human IgG1 heterodimerization Fc vector to generate Fab and Fab 2 Constructs. Knob-in-hole (KIH) heavy chain expression vector (CH3 domain T366W and T366S / L368A / Y407V mutations) was used to construct Fab 2 cMETCD47 and Her2CD3 protein constructs. Fab 2 The Her2-CD3 construct also includes the effector function ablating mutations L234A / L235A / G237A. IgG was constructed using a “wild-type” IgG1 heavy chain and kappa light chain expression vector. 2 Her2CD47 protein constructs. IgG was expressed in CHO cells following transient transfection with an endotoxin-free IgG expression plasmid preparation according to the manufacturer's protocol.

[0272] Using HiTrap MabSelect Sure Protein A 5mL columns Capture the produced antibody from the clarified supernatant on a Pure 150L FPLC system. Immediately exchange the eluted protein peak buffer into 1x PBS pH 7.4 by directly loading the eluted Protein A peak fractions onto a HiPrep 26 / 10 desalting column. Determine the protein concentration by measuring the absorbance at 280 nm, and analyze 1 μg of each purified protein by SDS-PAGE under reducing and / or non-reducing conditions using a 4-20% TGX polyacrylamide gradient gel (BioRad, catalog number 456-1093) with 1x Tris / glycine / SDS buffer, separating for 1 hour at 120 V field. To test for the presence of non-covalently bound aggregates and complement the SDS-PAGE analysis, perform analytical size exclusion chromatography. Analyze aliquots of the selected clones by analytical size exclusion chromatography (SEC) using a Superdex 200 Increase 10 / 300 SEC column and 1x PBS pH 7.4 as the running buffer in isocratic mode.

[0273] Further purify the selected proteins using preparative SEC. Load up to 1 ml of the antibody sample onto a Superdex 200 Increase 10 / 300 SEC column or a HiLoad 26 / 600 Superdex 200 pg column equilibrated in 1x PBS pH 7.4. Collect 1 ml fractions of the peaks of interest and pool the main peak fractions. After size exclusion chromatography, analyze the samples again by SDS-PAGE as described above.

[0274] Hemagglutination

[0275] Isolate red blood cells (RBC) from fresh, non-agglutinated human blood (from at least 3 different donors), dilute to 2% in PBS, and incubate for 60 - 90 minutes in a U-bottom 96-well plate with titrations of IgG or protein constructs. In the absence of hemagglutination, the cells sediment to the bottom of the well, forming a red precipitate. Observe hemagglutination as a non-sedimenting RBC solution. Record images of each plate and represent the data for each sample as the titer of the last well in which hemagglutination was observed.

[0276] Metalloprotease digestion

[0277] The protein construct was incubated with either individual human matrix metalloproteinase (MMP) enzymes or a mixture of active MMP3, MMP7, and MMP12 (equal parts of each component) at a ratio of 1% total MMP to protein construct (wt / wt) in Tris-buffered saline (pH 7.4) containing 5 mM CaCl2 at 37 °C for 16 hours. The reaction was terminated by adding 20 mM EDTA, and then the binding or functional activity of the test sample was assayed as described.

[0278] IgG titration binding ELISA

[0279] To coat ELISA plates, the target protein was diluted to 1 μg / ml in PBS pH 7.4 and added at 100 μl / well and incubated overnight at 4 °C. The coated plates were washed three times with PBS pH 7.4, blocked with 4% non-fat milk protein in PBS (380 μl / well) for 1 hour at room temperature, and then washed five times with PBS-Tween 20 (PBST). Then the antibody (100 μl / well; diluted in PBST) was added and incubated for 1 hour at room temperature. Then the plates were washed three times with PBS and goat anti-human IgG-HRP (100 μl / well) was added at room temperature for 1 hour. Then the plates were washed three times with PBST and twice with PBS, and then 100 μl TMB was added to each well. The reaction was terminated by adding 100 μl 2M H2SO4 / well and the OD was read at 450 nm on a plate reader.

[0280] Flow cytometry binding

[0281] Binding of the protein construct (+ / - pre-digested with MMP3 / 7 / 12) and control IgG to Jurkat cells and BT-474 cells was evaluated by flow cytometry. Live cells were identified using Zombie UV TM Fixable Viability Dye (Biolegend). Binding of human IgG and the protein construct was detected with an FITC-conjugated goat anti-human (H+L) secondary antibody. Binding of the mouse monoclonal anti-CD3 control antibody was detected using Alexa-Fluor-488 goat anti-mouse IgG. Results were analyzed by measuring the mean fluorescence intensity (MFI) of live cells in the FITC channel detector of a BD Fortessa flow cytometer.

[0282] T cell activation bioassay

[0283] The functional activity of the Her2CD3 protein construct was evaluated using BT474 cells and the NFAT-RE-luciferase Jurkat reporter cell line (Promega - TCR / CD3 effector cell NFAT) in a co - culture assay. BT - 474 cells (40,000 cells / well) were seeded into Hybri - Care medium (ATCC) supplemented with 10% FBS in a 96 - well white clear - bottom tissue culture - treated plate and incubated overnight at 37 °C in a CO2 incubator. The medium was removed, and control antibody or protein construct (+ / - pre - digestion by MMP3 / 7 / 12) prepared in assay medium (RPMI supplemented with 10% FBS) was added to the cells. The TCR / CD3 effector cells (NFAT) were thawed and diluted according to the manufacturer's protocol and then added to the assay wells. After incubation at 37 °C in a CO2 incubator for 6 hours, the plates were re - equilibrated to room temperature, and luciferase activity was determined by adding Bio - Glo reagent for 5 - 10 minutes and measuring the luminescence signal (RLU). The fold induction was determined by calculating the ratio of sample RLU / RLU in the absence of antibody after subtracting the background luminescence signal.

[0284] Molecular dynamics simulations

[0285] Eight systems (linker sequences X1, X2, X3, and X4 and equivalent sequences with a broken covalent bond in one of the linkers at GS sequence positions XC1, XC2, XC3, and XC4) were modeled and optimized using the AMBER10:EHT force field in MOE (Chemical Computing Group Inc). The histidine charges were assigned using the protonate3D tool in MOE. Three consecutive minimizations were performed with a final gradient of 0.001, allowing 10,000 steps without restraint. The individual system was re - constrained and gradually minimized using NAMD2.13 with the CHARMM27 force field to reduce the total potential energy in a series of three energy - minimization steps. The first step kept all heavy atoms constrained and only allowed hydrogen atoms to migrate. The second step removed the constraints on the side chains, while the third step released all atoms, allowing them to move unrestricted. Minimization within CHARMM27 was straightforward as it is the force field working within the NAMD molecular dynamics simulation.

[0286] For the kinetic runs, generalized Born (GB) solvation was used to describe the solvent effect. MD simulations were set up and then completed using NAMD 2.13. Four equilibration steps were carried out, gradually relaxing the harmonic restraints in 125 ps increments for a total equilibration time of 500 ps. In the second step, the system was heated to 310 K while applying a force of 4 kcal / mol to hold the backbone in place. The remaining three steps gradually increased the backbone restraint force from 4 kcal / mol to 1 kcal / mol using the NVT ensemble (constant (N), volume (V), and temperature (T)). At the switching function was applied to truncate the van der Waals potential function. According to the recommendations of NAMD, the electrostatic and vdW cutoffs were set to Periodic boundary conditions were not used as they are incompatible with the implicit solvent approach. The restraints applied during equilibration were removed to freely simulate the antibody complex. The production runs were carried out at 310 K for 6, 15, 20, or 100 ns. A 100 ns run was applied to the cleaved linker protein XC1-4 in order to fully explore the range of motion, while it was found that shorter runs (up to 20 ns) were sufficient to localize the range of motion of the uncleaved antibody construct X1-4.

[0287] In Vivo Analysis of PK and Tolerance

[0288] Tolerance Study - Twenty-eight (28) 6-8-week-old male B6.Cg-Fcgrt tm1Dce Tg(FCGRT)32DcrJ (homozygous human FcRn transgenic, JAX stock #014565) mice were divided into 7 groups of 4 mice each. Body weights were measured on the day of antibody administration. At 0 h, the test article was administered intravenously (IV) at a dose of 2 mg / kg or 10 mg / kg and a dose volume of 10 ml / kg. Two-hundred microliters of whole blood samples were collected into EDTA on days 5, 29, and 60 (terminal bleed). The blood was used for CBC / Diff / Retic analysis, including white blood cells, neutrophils, eosinophils, basophils, lymphocytes, monocytes, hemoglobin, red blood cells, reticulocytes, MCHC, and MCV. Body weights were then monitored weekly for the first month and then monthly until the end of the experiment.

[0289] Pharmacokinetics Study - Thirty-two 6-8-week-old male B6.Cg-Fcgrttm 1DceTg(FCGRT)32DcrJ ('Tg32' homozygous human FcRn transgenic, JAX stock #014565) mice were divided into 8 groups of 4 mice each. One day before dosing, 35 μL blood samples from three (3) Tg32 mice were collected into EDTA for testing the binding of the test article to mouse red blood cells using flow cytometry. Body weights were measured on the day of antibody administration and weekly until the end of the experiment. At 0 hours, the test article was administered by IV injection at a dose volume of 10 mL / kg at 2 mg / kg or 10 mg / kg. Blood samples were collected from each mouse according to the bleeding schedule: 30 minutes, 4 hours, 1 day, 3 days, 5 days, 7 days, 10 days, 14 days, 21 days, 28 days, and 42 days. 25 μL blood samples were collected from each mouse according to the bleeding schedule. Blood samples were collected into K3EDTA, processed into plasma, and stored at -20 °C. Plasma samples were then evaluated in triplicate by ELISA to estimate human IgG concentration.

[0290] Tolerance study in NOD-SCID mice - NOD-SCID mice were divided into 4 groups of 3 mice each. Body weights were measured daily. On Day 0, the test article was administered by IV injection at 8 mg / kg or 14 mg / kg, with 3 subsequent doses administered at 4 mg / kg or 7 mg / kg at 5-day intervals.

[0291] Flow cytometry analysis of binding to monkey and human red blood cells

[0292] Red blood cells were isolated from 3 Cyno (cynomolgus monkeys) and 3 human donors. For each sample, 5 x 10 5 cells (diluted in DMEM + 5% FBS) were stained for one hour with: A-D5 IgG1: 0.0032, 0.016, 0.03, 50 μg / mL; trastuzumab: 0.0032, 0.016, 0.03, 50 μg / mL; IgG 2 Her47 LHL-LHLF 0 hours: 0.016, 0.08, 0.4, 2, 10, 50 μg / mL; IgG 2 Her47 LHL-LHL 0 hours: 0.016, 0.08, 0.4, 2, 10, 50 μg / mL. Binding of the test samples to red blood cells was measured using FITCAffiniPure goat anti-human IgG (1:200 dilution; 1-hour incubation time), followed by measurement of FITC fluorescence intensity by flow cytometry (BD LSR Fortessa X-20 cell analyzer).

[0293] Metalloprotease and cathepsin digestion at pH 6.0 and 7.4

[0294] The protein construct was placed in TBS (containing 5 mM CaCl2, pH 6.0 or 7.4), and then incubated with individual human matrix metalloproteinase (MMP) or cathepsin at a ratio of 1% total enzyme to protein construct (wt / wt) at 37 °C for 0 h, 2 h, 4 h, 8 h, and 24 h. The reaction was terminated by adding 20 mM EDTA, and then the samples were frozen before testing for binding or functional activity as described.

[0295] In vitro protein stability analysis

[0296] Forced oxidation - For forced oxidation analysis, the test article in PBS was treated with 0.5% H2O2 at room temperature for 2 h, then stored at -80 °C, and then SEC and RP analyses (intact antibody and subunits, tryptic peptides) were performed on a Dionex Ultimate 3000RS HPLC system (ThermoFisher Scientific, Hemel Hempstead, UK). For intact antibody reduction, DTT was added to a final concentration of 0.33 M, and the samples were incubated at 22 °C for 1 h and immediately analyzed by RP.

[0297] SEC analysis - Chromatographic separation was performed using an Acquity UPLC Protein BEH SEC column ( 1.7 μm, 4.6 mm × 150 mm (Waters, Elstree, UK)) and an Acquity UPLC Protein BEH SEC guard column (30 × 4.6 mm, 1.7 μm, (Waters, Elstree, UK)) connected to a Dionex Ultimate 3000RS HPLC system (ThermoFisher Scientific, Hemel Hempstead, UK). The method included an isocratic elution within 10 min, and the mobile phase was 0.2 M potassium phosphate pH 6.8, 0.2 M potassium chloride. The flow rate was 0.35 mL / min. Detection was by UV absorption at 280 nm.

[0298] RP analysis of full antibodies and subunits - Chromatographic separation was performed using a PLRP-S 1000 (5 μm, 2.1 mm × 50 mm) column (Agilent Technologies, Stockport, UK) connected to a Dionex Ultimate 3000RS HPLC system (ThermoFisher Scientific, Hemel Hempstead, UK). The method included a linear gradient from 75% buffer A (H2O solution of 0.02% TFA, 7.5% acetonitrile) to 45% buffer B (acetonitrile solution of 0.02% TFA, 7.5% H2O) within 14 minutes. The flow rate was 0.5 mL / minute, and the temperature was maintained at 70 °C throughout the analysis. Detection was carried out by UV absorption at 280 nm.

[0299] HIC analysis - Chromatographic separation was performed using a TSKgel Butyl-NPR 4.6 mm × 35 mm HIC column (TOSOH Bioscience Ltd., Reading, UK) connected to a Dionex Ultimate 3000RS HPLC system (ThermoFisher Scientific, Hemel Hempstead, UK). The method included a linear gradient from 60% buffer A (100 mM sodium phosphate pH 7.0, 2 M ammonium sulfate) to 90% buffer B (100 mM sodium phosphate pH 7.0) within 9 minutes. The flow rate was 1.2 mL / minute. Detection was carried out by UV absorption at 280 nm.

[0300] Charge isomer determination - The charge isomer profile of the test article was determined by Protein Charge Variant Assay on a LabChip GXII Touch HT (PerkinElmer) according to the manufacturer's protocol.

[0301] Fc affinity of human Fc receptors Analysis

[0302] Using The T200 instrument was used to determine the interaction affinity of antibody proteins by surface plasmon resonance. For most analyses, His6-tagged FcγRI, FcγRIIa (167R and 167H variants), FcγRIIb, FcγRIIIa (176F and 176V variants), and FcγRIIIb receptors (all from Sino Biological) were captured on a CM5 sensor chip coated with anti-HIS antibody by standard amine coupling. Then the receptor-specific assay format was applied as described below.

[0303] FcγRI is a high-affinity receptor for monomeric IgG1, so 1:1 kinetic analysis was performed under the following conditions: For "single-cycle" analysis, a flow rate of 30 μl / min was used, the receptor protein was loaded at 10 μl / min to approximately 30 RU (diluted at 0.25 μg / ml in HBS-P+), a 5-point three-fold dilution series of purified antibody titrated from 0.411 nM to 33.33 nM, a binding time of 200 s was applied, and a dissociation time of 300 s. It was regenerated with 2x injection of glycine pH 1.5 and analyzed using a 1:1 fit.

[0304] The interaction between monomeric IgG and FcγRII and FcγRIII receptors is a relatively low-affinity interaction, so "steady-state" affinity analysis was performed under the following conditions: Flow rate 30 μl / min, receptor protein loaded at 10 μl / min to approximately 60 RU (diluted at 0.25 μg / ml in HBS-P+), a 5-point three-fold dilution series of purified antibody titrated between 33 nM and 24000 nM, a binding time of 30 s was applied, and a dissociation time of 25 s. It was regenerated with 2x injection of glycine pH 1.5 and analyzed using steady-state affinity calculations.

[0305] Results and Discussion

[0306] Protein construct design principles

[0307] Standard anti-cancer antigen antibodies face significant pharmacological challenges in the treatment of solid tumors. The key problem limiting efficacy in this type of potential drug is that the antigen targeted by the antibody is not completely found in the tumor but is only highly overexpressed in the tumor. This off-tumor target expression usually leads to the risk of dose-limiting side effects and the antigen "pool" effect, where large doses of the antibody must be administered to ensure sufficient antibody penetration into the tumor to have a therapeutic effect. One such example is the class of antibodies targeting the antigen CD47 ( Figure 1A ), where the challenges include: The high expression of CD47 in the bloodstream (e.g., especially on red blood cells and platelets) is a "pool" that binds the antibody administered intravenously, minimizing the amount of drug that penetrates the tumor (even when large doses of IgG are administered). The binding of anti-CD47 to blood cells is also a significant toxicity risk. In fact, anti-CD47 antibodies are known to cause anemia and even cross-link human red blood cells, creating a risk of blood clotting in patients. In addition, tumors are usually an "unfavorable" environment with a high expression rate of enzymes such as MMP that can accelerate IgG degradation. These factors all contribute to minimizing the potential safety and efficacy of anti-CD47 antibodies and many other types of anti-tumor target antibodies where target expression is not limited to the tumor environment.

[0308] Anti-CD47 protein construct ( Figure 1B)Aims to overcome the peripheral pool and toxicity issues experienced by anti-tumor antigen IgG by eliminating the binding of high-risk (but potentially strong mechanism of action) targets in natural proteins. This effect is achieved by adding a low-risk upper domain (e.g., a Fab domain targeting another tumor antigen such as Her2) and a linker above (n-terminally) the binding domain of the high-risk lower domain (such as CD47). The use of an appropriate upper domain / linker combination results in a configuration that completely blocks the binding activity in the lower CD47 domain. Then the tumor-targeting domain (e.g., Her2) drives high concentrations in the tumor environment, and the protein construct linker system utilizes the elevated MMP activity in the tumor to cleave the linker peptide, thereby exposing the CD47 binding domain and thus conditionally activating the CD47 binding activity in the tumor rather than in the periphery. This design principle has the potential to be applicable to many different structural forms, examples of which are outlined below.

[0309] Protein construct IgG 2 Design( Figure 2A ) can be based on sequences derived from IgG1, IgG2, IgG3, IgG4, IgE, IgM, or IgA and may or may not have effector function capabilities. In this construct, four polypeptide chains encode four Fab domains (2x Fab A, 2X Fab B), four linker sequences, and may or may not have an immunoglobulin hinge region and an Fc domain. Each Fab A-linker domain (upper) blocks the binding activity of Fab B (lower). The target binding specificities of the upper and lower domains can be different to drive bispecific function or the same to drive multivalent target interactions. The choice of linker sequence (such as the lower hinge peptide sequence) results in a structure that is locked in non-diseased tissues but is rapidly cleaved and unlocked in the presence of high concentrations of proteases in the tumor environment( Figure 2B ). The linkers in the protein construct design are all proteolytically cleavable and can be cleaved sequentially, where the first 'fast' cleavage takes the 'locked' intact structure and generates an intermediate 'unlocked' active state, which allows Fab A and B from a single protein construct to bind their cognate targets. The secondary, potentially slower cleavage of the second linker in each Fab A-Fab B protein construct unit can completely release the Fab A domain from the structure, generating a 'dissociated' form where the lower Fab domain is fully released for non-targeted (but potentially still restricted) activity. Cleavable linkers based on immunoglobulin hinge sequences can also increase immune effector functions (ADCC, CDC, and ADCP) on the cell membrane through the recruitment of endogenous anti-hinge antibodies, which is a known phenomenon in human patients with (or even without) potential autoimmune diseases.

[0310] Protein construct Fab 2 The design can be based on sequences derived from IgG1, IgG2, IgG3, IgG4, IgE, IgM or IgA and may or may not have effector function capabilities. In this construct, two ( Figure 3A ) or three ( Figure 3B ) polypeptide chains encode two Fab domains (1x Fab A, 1X Fab B), two or more linker sequences, and may or may not have an immunoglobulin hinge region and an Fc domain, where the pairing of the heterodimer may or may not be driven by mutations in the Fc. Each Fab A linker domain blocks the binding activity of Fab B again, and the choice of linker sequence (such as the lower hinge peptide sequence) results in a structure that will be locked in non-diseased tissue but is rapidly cleaved and unlocked in the presence of high concentrations of linker-cleaving proteases in the tumor environment, ultimately becoming dissociated ( Figure 3A ).

[0311] Protein construct cloning and expression

[0312] To generate and purify 15 bispecific conditional activity protein constructs in the form of Fab 2 or IgG 2 molecules with different linker domains (Table 1), DNA cassettes of each construct type (Table 2) were synthesized and cloned into expression vectors encoding the human IgG1 heavy and light chains or the "stapled" heterodimeric Fc. The proteins were named using the nomenclature (form)-(target name [upper domain / lower domain])-(heavy chain linker type)-(light chain linker type). All proteins were produced by transient transfection of CHO cells and then purified by protein A affinity chromatography.

[0313] The anti-HER2 variable region sequence used in the protein constructs disclosed herein is the variable region sequence of trastuzumab. The anti-CD3 variable region sequence used in the protein constructs disclosed herein is the variable region sequence of OKT3 or SP34. The anti-cMET variable region sequence for use in the protein constructs disclosed herein was provided in WO 2019 / 175186. The anti-CD47 variable region sequence for use in the protein constructs disclosed herein was provided in WO2019 / 034895.

[0314] Analysis of protein construct expression and purification characteristics

[0315] Protein quantification of protein A-purified proteins showed that the use of different linker types affected the expression yield (Table 2). Protein preparations in 1x PBS pH 7.4 were also examined by analytical size exclusion chromatography to quantify the percentage of the desired product. Among all three classes of bispecific proteins generated (cMETCD47, Her2CD47, and Her2CD3), constructs containing the LHL linker produced the best combination of the highest yield measured by analytical SEC and the % yield of the main peak of the desired product (Table 2). SDS-PAGE analysis of protein A-purified proteins ( Figure 4 ) also demonstrated that clones containing the short linker domain L1 and LH plus the long linker domain L3 produced highly heterogeneous products, with significant HMW and LMW content in the unreduced samples and significant LMW content in the reduced samples. This suggests that suboptimal linker types may lead to significant formation of undesired multimers and degradation products. For all constructs, HMW impurities were essentially absent in the reduced lanes, indicating that these HMW impurities are disulfide-linked dimers or higher-order polymers that are not reduced by SDS alone. For the IgG 2 design, both clones 12 and 14 showed high yields (Table 2), but clone 14 (containing the LHL linker) confirmed the highest yield and high homogeneity of the desired product by SEC (90%) and SDS-PAGE ( Figure 4 ).

[0316] The remaining protein samples from the clone subset were then subjected to SEC chromatography to attempt to produce fully purified monomeric proteins. Chromatograms of clones 1, 2, 3, 4, 5, 6, 10, 12, and 14 in preparative SEC are shown in Figure 5. These analyses further demonstrated that clones 1 ( Figure 5A ), 3 ( Figure 5C ), 4 ( Figure 5D ), 5 ( Figure 5E ), and 12 ( Figure 5G ) contained a large percentage of higher molecular weight products and generally lower molecular weight products near the main peak of the desired product (eluting with a CV of approximately 0.5 - 0.55 in Figures 5A - 5H ). In contrast, clones 6 ( Figure 5F ), 14 ( Figure 5H ), and 10 ( Figure 5I ) showed distinct, well-defined main product peaks. For clones 2 and 6, this allowed for effective SEC purification of predominantly monomeric proteins, as demonstrated by SDS-PAGE of the unreduced samples ( Figure 6A ). Attempted SEC purification of clones 1, 4, and 5 failed, producing still heterogeneous samples ( Figure 6A ). From Her2CD3 Fab 2The SEC-purified proteins of clones 7, 8, and 10 showed improved homogeneity, while in the Her2CD47IgG 2 clone, only clone 14 achieved complete homogeneity and monomeric status, as confirmed by SDS-PAGE of non-reduced and reduced samples ( Figure 6B ). Additionally, these findings demonstrated that clones 6, 10, and 14, which all contain the LHL linker on both chains, exhibited the most reproducible beneficial characteristics.

[0317] Functional characteristics of protein constructs containing the CD47 antibody v-domain in the lower Fab

[0318] Titration (in μg / ml) of control IgG antibodies A-D5 anti-CD47, MH7.1 anti-C-MET, anti-Her2 trastuzumab, and A-D5 Fab-Fc (monovalent form of the A-D5 antibody containing a single Fab domain) in direct binding ELISA against human CD47, C-MET, and Her2 proteins ( Figure 7A ). Her2CD47-LH-LH and Her2CD47-LHL-LHL clones in IgG 2 form ( Figure 7B ) and cMETCD47-L2-L2 and cMETCD47-LHL-LHL in Fab 2 form ( Figure 7C ) were also analyzed in the same manner. Control antibodies demonstrated the expected strong binding activity against their cognate targets, with little or no background to any other target, even at the highest concentrations ( Figure 7A ).

[0319] Importantly, the very strong monovalent binding of A-D5 Fab-Fc ( Figure 7A ) demonstrated the intrinsic affinity of the A-D5 anti-CD47 domain and the significant potency required to lock it into the protein construct form of the present invention for its success. The protein constructs Her2CD47-LHL-LHL ( Figure 7B ) and cMETCD47-LHL-LHL ( Figure 7C ) also showed similar strong and highly specific binding to the cognate targets of their upper Fab domains, but no binding signal to CD47, indicating that the binding activity of the CD47 v-domain was indeed completely inhibited when using this linker combination. Importantly, high background signals to human CD47 were observed for Her2CD47-LH-LH and to a lesser extent for cMETCD47-L2-L2, indicating that the elimination of the lower fab binding affinity is strictly controlled by linker selection. These findings suggest that the protein construct Her2CD47-LHL-LHL containing the anti-CD47 binding domain of antibody A-D5 tested in this assayFigure 7B ) and cMETCD47-LHL-LHL( Figure 7C ) has a >1000-fold reduced ability to bind CD47 compared to A-D5 IgG1, where a binding signal OD of 1.0 is achieved at approximately 1×10 -2 μg / ml, and at the highest tested concentration of 10 μg / ml, neither protein construct shows a signal higher than 0.2.

[0320] Since hemagglutination is a major toxicity risk of anti-CD47 antibodies, the preferred protein constructs were then tested in a hemagglutination assay based on human red blood cells. Control antibodies anti-CD235a (mouse) and A-D5 anti-CD47, A-D5 Fab-Fc, MH7.1 anti-C-MET, anti-Her2 trastuzumab, Her2CD47-LH-LH and Her2CD47-LHL-LHL in IgG 2 form and cMETCD47-L2-L2 and cMETCD47-LHL-LHL in Fab 2 form were titrated (in nM) using fresh red blood cells from healthy donors 1( Figure 8A ), donor 2( Figure 8B ), and donor 3( Figure 8C ). For all 3 donors, the control antibodies anti-CD235a, A-D5 anti-CD47, and A-D5 Fab-Fc showed potent concentration-dependent hemagglutination due to cross-linking of their respective surface antigens on adjacent red blood cells. The low-potency hemagglutination observed for A-D5 Fab-Fc (clone 15) may be due to the presence of a small amount of functional dimer in this protein preparation, as it was purified only by Protein A column and not fully purified to the monomer state by SEC. Importantly, even at the highest concentration of 140 nM, no protein construct sample showed any ability to induce hemagglutination. This finding indicates that the IgG 2 and Fab 2 form protein constructs containing the anti-CD47 binding domain of antibody A-D5 tested in this assay have a >241-fold reduced ability to induce agglutination compared to A-D5 IgG1, which showed a titer of 0.58 nM.

[0321] The preferred protein constructs were enzymatically digested using human MMP3, MMP7, and MMP12 over a time course of 2, 4, 8, and 24 hours of incubation, plus 24 hours of incubation in enzyme-free buffer as a negative control. Samples from these digestion time courses were then applied to sandwich ELISA assays targeting human Her2 and CD47( Figure 9A , 9B ) or human C-MET and CD47( Figure 9C)'s direct binding ELISA. In each case, no loss of binding to Her2 or C-MET was observed over time, indicating that the addition of protease did not reduce the functional binding ability of the upper Fab domain. In contrast, the CD47 binding ability of all three protein constructs increased significantly over time in a MMP-dependent manner. Clonal IgG 2 Her2CD47-LH-LH( Figure 9A ) again showed high background binding to CD47 and minimal increase in CD47 binding over time. In contrast, clonal IgG 2 Her2CD47-LHL-LHL( Figure 9B ) and Fab 2 cMETCD47-LHL-LHL( Figure 9C ) again showed low background binding to CD47 (OD 450 nM < 0.2), and binding to CD47 increased rapidly starting from incubation with both MMP7 and MMP12 for 2 hours, reaching full saturation at 24 hours of incubation (OD 450 nM was approximately 4.0). MMP3 seems to be the slowest to activate among the 3 MMPs, showing an increase in the CD47 binding signal for all 3 protein construct examples after 24 hours.

[0322] Functional characteristics of protein constructs containing the CD3 antibody v domain in the lower Fab

[0323] Antibody Fab 2 Her2CD3-L 1-LH, Fab 2 Her2CD3-L2-L2 and Fab 2 Her2CD3-LHL-LHL were analyzed by ELISA( Figure 10A ), flow cytometry( Figure 10B ), and CD3 reporter assay( Figure 10C ). In the ELISA analysis, all 3 proteins showed the expected strong binding activity to Her2 (upper Fab domain), with little or no background binding to any other target even at the highest concentration( Figure 10A ). Then all 3 protein construct Fabs 2 proteins were incubated overnight in the presence or absence of a mixture of MMP3, MMP7, and MMP12. In the flow cytometry binding to the HER2+ human cell line BT474, anti-HER2 trastuzumab showed strong binding, and Fabs 2 Her2CD3-L1-LH and Fabs 2 Her2CD3-L2-L2 showed similar strong binding before and after MMP digestion, while Fab2 Her2CD3-LHL-LHL showed a partially reduced binding after MMP digestion, indicating that a portion of the protein was losing the upper fab in the 'dissociated' state( Figure 10B ). Since this finding indicated that the MMP digestion process was active in the case of Fab 2 Her2CD3-LHL-LHL, the same sample was applied in the reporter assay, in which Her2+BT474 cells were mixed with human CD3+Jurkat cells, and the human CD3+Jurkat cells were engineered to provide a measurable signal when CD3 was activated. Data from this assay demonstrated that the bivalent anti-CD3 antibody OKT3 directly activated the CD3 signal in the reporter cells, as expected( Figure 10C ). Each of the protein constructs showed different characteristics: The Her2CD3-L2-L2 protein containing a 2xG4S linker (not cleavable by MMP proteases) showed a high background in the assay, and there was no increase in the CD3 activation signal after MMP digestion, indicating that the flexible linker of this construct allowed Her2 to be bound by the upper Fab, while the partial activity of the lower fab allowed some background but did not activate CD3 co-linking( Figure 10C ). Fab 2 The Her2CD3-L1-LH protein showed a lower background activity in the assay, and the signal increased moderately after MMP digestion. In the absence of MMP digestion, Fab 2 Her2CD3-LHL-LHL did not show a measurable background signal transduction, showing minimal CD3 activation similar to the negative control trastuzumab and IgG1 isotype control antibody in the undigested sample, but showed effective activation in the MMP-digested sample. Therefore, Figure 10A -C data indicated that the Her2CD3-LHL-LHL Fab 2 form had the best combination of properties, as it was simply expressed and purified, had high intrinsic Her2 binding activity, low background CD3 ligation activity, and high CD3 co-ligation activation only when activated by MMP cleavage of the LHL linker.

[0324] Second-generation construct cloning and expression

[0325] The above-mentioned multispecific Fab containing the LHL linker 2 and IgG 2 The performance of the clones prompted the use of a second series of constructs to experimentally explore the potential functional sequence space in both the tertiary structure and the linker sequence content. The following clones were synthesized and assembled to sample these parameters:

[0326] 1. For the'single-arm' type Fab 2Cloning of the protein 'Met47-LHL-LHL' (Table 6, Figure 3B ).

[0327] 2. Remodeled 'Clone 10' Fab of the'single-arm' type 2 Cloning of the protein 'Her23-LHL-LHL' (Table 7, Figure 3B ).

[0328] 3. Fab of the'single-arm' type 2 Cloning of the protein 'Her23(34)-LHL-LHL' (Table 8, Figure 3B ).

[0329] 4. A series of IgG 2 clones incorporating the Her2-binding domain of trastuzumab in the upper fab and the CD47-binding domain of Clone A-D5 in the lower Fab. These clones contain mutant LHL-based linker sequences that may be more sensitive to enzymatic cleavage by a broad family of MMPs (LHLF linker), a linker of moderately increased length with a portion of the human IgG1 intervening hinge sequence added (LHLM linker), or both (LHLMF linker).

[0330] 5. Cloning of the protein 'Fc-Her23(34)' (Table 13, 2 ) where the Fab 2 module is located at the KIH-Fcc terminus. Figure 12 ).

[0331] 6. Cloning of the protein 'MetMet-LHL-LHL' (Table 14, 2 ) for a'single-arm' type Fab containing two copies of the C-Met Fab. In this structural form, binding to the bivalent form of the Met receptor can only occur after proteolytic cleavage of a single LHL linker. Figure 3B , Figure 13 ).

[0332] As described above, these constructs were successfully expressed and purified by Protein A and size exclusion chromatography.

[0333] Second-generation IgG 2 Construct analysis

[0334] The performance of the above-purified IgG 2 clones was examined in a series of further analyses. First, the Example Clone A-D5 IgG1, IgG 2 Her2CD47-LHL-LHL, IgG 2 Her47-LHLF-LHL, IgG 2Her47-LHL-LHLF and IgG 2 Binding of Her47-LHLF-LHLF (Table 9) to human Her2 and to human and murine CD47 ( Figure 14 ). This analysis demonstrated that: 1) The A-D5 anti-CD47 IgG1 protein showed high binding signals to both hCD47 and mCD47. 2) All 4 IgG 2 proteins showed high binding signals to hHer2, but no / background binding signals to hCD47 or mCD47 ( Figure 14 ).

[0335] Detection of cloned IgG 2 Her2CD47-LHL-LHL and IgG 2 Sensitivity of Her47-LHL-LHLF to enzymatic activation (at pH 7.4) by various proteases known to be overexpressed in human tumors (Figure 15): Incubation with MMP7 ( Figure 15A ), MMP8 ( Figure 15B ), and MMP10 ( Figure 15C ) demonstrated that both proteins could be activated by each enzyme to bind hCD47, but in each case IgG 2 Her47-LHL-LHLF was activated more rapidly. Incubation with MMP 12 demonstrated that both proteins could be activated by this enzyme at equal rates to bind hCD47 ( Figure 15D ). Unexpectedly, incubation with MMP 13 demonstrated that IgG 2 Her47-LHL-LHLF could be activated by this enzyme to bind hCD47, while IgG 2 Her2CD47-LHL-LHL could not ( Figure 15E ). Importantly, incubation with the cysteine protease cathepsin S also demonstrated that both proteins could be activated by this enzyme at equal rates to bind hCD47 ( Figure 15F ). These findings demonstrate that the peptide linker content of the Fab 2 module contained in the IgG 2 protein can be "regulated" to broaden the number of potential activating enzymes and even make the proteolytic activation process more rapid. To sample this possibility, additional linker designs with appropriate lengths and contents to drive sensitivity to specific classes of enzymes were envisioned, where amino acid sequences showing proteolytic sensitivity to the activities of other disease-related metalloproteases such as ADAMS, cysteine, aspartic, and serine proteases, etc. would be used to provide broader or more selective activation.

[0336] To detect activation on IgG 2In conjunction with the effect of binding affinity, a Biacore assay was established that could sample both the binding of Her2 and CD47. In this assay, control antibodies and IgG 2 Her47-LHL-LHLF protein (undigested or activated with MMP12 for 2, 4, 8, or 24 hours) was captured on the chip surface via an anti-Fc antibody, and then the binding affinities for soluble Her2 and CD47 extracellular domain proteins were measured. Binding assays were repeated for trastuzumab IgG1 and A-D5 IgG1 (without enzymatic digestion) at the start and end of the experimental runs, and the data are shown in Table 17. These assays showed that the calculated KD values for each antibody were highly similar in each run. However, importantly, the Rmax values (the maximum binding signal at the maximum analyte concentration) decreased significantly between runs at the start and end of the experiment (e.g., the trastuzumab Rmax was 265.80 RU at the start and 144.13 RU at the end), indicating a decrease in the activity of the anti-Fc antibody capture surface after many rounds of regeneration inherent in the Biacore method. To examine the effect of MMP12 activation on target reactivity, the Rmax and KD values for both Her2 ( Figure 16 A, Table 18) and CD47 ( Figure 16 B, Table 18) were calculated. This analysis showed that Her2 binding and affinity were maintained within 24 hours of MMP12 activation. Importantly, no CD47 binding (Rmax = 0) was observed in the 0-hour sample (without MMP12 digestion), but both Rmax and the apparent affinity for CD47 increased rapidly during the activation time course, starting from 2 hours of incubation ( Figure 16 B, Table 18). These findings demonstrate that the lower Fabs in the Fab 2 module are indeed inert and cannot engage in target interactions in the intact molecule until protease activation occurs. This observation was further illustrated in Figure 17 where no binding activity was observed even at a CD47 analyte concentration of 400 nM, but high binding to CD47 was evident after 24 hours of MMP12 treatment.

[0337] The above findings also demonstrate that cleavage of the linker peptide in the Fab 2 module (and thus activation of the lower Fabs) is transient. This may be a pharmacological benefit of molecules containing the Fab 2 module, as activation may be strongly biased towards diseased tissues where both the target antigen of the upper Fabs and the enzymes capable of activating the lower Fabs are highly overexpressed. This would result in rapid drug accumulation, long residence times, and high levels of activation in such diseased tissues.

[0338] Fab 2Module structure and computational; computer modeling of molecular dynamics

[0339] To understand the mechanism by which the Fab 2 module may function, we performed structural modeling and molecular dynamics analysis. Using the protein modeling suite of the MOE software, the crystal structures of human CD47 ECD bound to the Fab domains of IgG1 C47B 161 (Protein Data Bank identifier 5TZT), IgG1 C47B222 (Protein Data Bank identifier 5TZ2), and IgG1 B6H12.2 (Protein Data Bank identifier 5TZU) were used as templates to generate a model of anti-CD47 A-D5Fab that binds to CD47 ECD. The structure of the trastuzumab Fab complexed with the Her2 extracellular domain was taken from the PDB structure 1N8Z.

[0340] Then, the LHL and LHLF linkers between the upper trastuzumab Fab and the lower anti-CD47 Fab were modeled using the protein modeling suite of the MOE software. To assist in the modeling of the linker, the C-termini of the Fab structures available in the Protein Data Bank were examined to help define the conformations in which the LB linker could depart from each heavy and light chain domain of the trastuzumab Fab. The modeling predicted that the C-termini of the heavy and light chains of the trastuzumab Fab could optionally have the native interchain disulfide bonds commonly present in the IgG1 Fab domain.

[0341] A full-length IgG1-based model incorporating the above-described modeled anti-CD47 Fab was constructed using the structure of IgG1 b12 (Protein Data Bank identifier 1HZH) as a template. The structure errors in 1HZH, such as missing structural regions, were remodeled and corrected. The IgG1 b12 Fab was replaced with the anti-CD47 Fab, and the Fc-hinge was attached using the protein modeling suite of the MOE software. This model illustrates the possible tertiary structure of the IgG 2 molecule based on Her2 and CD47 and IgG1( Figure 18A ). In this model, the binding of the Her2 epitope is constitutively active( Figure 18A ), while the binding of the CD47 ECD is completely occluded by the linker itself and the proximity of the anti-CD47 Fab to the anti-HER2 Fab( Figure 18B ). This model is consistent with the Fab 2 module, in which the Fab 2 module can bind to HER2 via the upper fab without any observed hindrance, but blocks the binding to CD47 via the lower fab until the linker is degraded( Figure 16 A, Figure 16 B).

[0342] To gain further insight into the Fab2 To study how the module moves in solution, we performed molecular dynamics simulations using multiple linker compositions, including LHL-LHL, LHL-LHLF, and L2L2 (amino acid sequence G4SG4S (SEQ ID NO: 32), Table 1). The root mean square deviation (RMSD) of each residue was calculated for each run. Additionally, a custom descriptor dSASA was written in the SVL language (MOE) to calculate the change in solvent-accessible surface area (SASA) of each residue and, in each case, also calculate the ensemble of only the linker sequences. This provides a basis for comparing how different uncut linkers perform in terms of conformational changes and their associated effects on linker solvent accessibility, which is expected to affect sequence-specific cleavage potential (Figure 18).

[0343] Figure 19 A- Figure 19 I shows nine graphs corresponding to the solvent-accessible surface area (SASA) results obtained for the three tested linkers. The first analysis sampled the absolute SASA values (over 6 ns) for each of the nine kinetic runs for each of the LHL and LHLF linkers and ten runs using L2 (at Figure 19 A, Figure 19 D, and Figure 19 G). This data demonstrated that the L2 linker had the greatest tendency for structural heterogeneity over time ( Figure 19 G). Since the starting SASA values for each run were not the same, a second analysis normalized the results representing the difference from the starting SASA value by subtracting all SASA values from the starting SASA value. The results over the 6 ns kinetic run time (at Figure 19 B, Figure 19 E, and Figure 19 H) and over the first 2.5 ns of the 6 ns kinetic run (at Figure 19 C, Figure 19 F, and Figure 19 I) illustrate that the structural dynamics of the LHL and LHLF linkers are significantly less than those of the L2 linker. In particular, the 6 ns molecular dynamics run showed that L2 exhibited the highest flexibility, thus highlighting the concept that different linker sequences will produce different flexibilities and, therefore, different CDR solvent exposure profiles for the lower Fab in the Fab 2 module.

[0344] Comparison between the cut and uncut linkers showed a significant increase in flexibility for the cut linker compared to the uncut linker, which is consistent with the Figures 14 - 17 biological observations in Figure 20A . For example, 2Limited movement within the module, followed by significant movement of the upper Fab domain in the context of a single cleaved LHL junction (second junction intact) during a 100 ns dynamics run. This analysis shows a significant increase in the degrees of freedom of the upper fab domain, resulting in multiple positions where it can move completely out of the path of the lower fab domain, thereby fully exposing its CDRs to allow unconstrained interaction with, for example, CD47 ( Figure 20B )

[0345] In vitro and in vivo analysis of tolerance and pharmacokinetics

[0346] To detect the tolerance and pharmacokinetics of IgG2 and Fab2 in the context of CD47 as the lower Fab domain, multiple exemplary molecules (A-D5 IgG1, IgG Dce Tg(FCGRT)32DcrJ mice were studied. These 'Tg32' mice are homozygous transgenic animals for human FcRn with human IgG pharmacokinetic characteristics that mimic those of humans and primates. Since it is known that the CD47-binding domains contained in the lower Fab domains of A-D 5IgG1 and in IgG 2 Her47 LHL-LHL, IgG 2 Her47 LHL-LHLF and Fab 2 Met47 LHL-LHL are all capable of binding recombinant murine CD47 protein, their reactivity to murine membrane-presented CD47 on red blood cells was first tested by flow cytometry ( 2 Her47 LHL-LHL, IgG 2 Her47 LHL-LHLF and Fab 2 Met47 LHL-LHL). This study showed that A-D5 IgG1 did not show a signal on murine red blood cells at 0.1 μg / ml, but showed clear concentration-dependent binding signals at both 1 and 10 μg / ml ( Figure 21 ) Figure 21 ) Figure 21 ) and was fully saturated at 10 μg / ml (98% binding, Figure 21 ) 2 Her47 LHL-LHL, IgG 2 Her47 LHL-LHLF or Fab 2Met47 LHL-LHL did not show any binding at any concentration, indicating that the anti-CD47 variable domain of the lower Fab domain could not interact with CD47 on mouse red blood cells( Figure 21 ).

[0347] Using red blood cells isolated from Tg32 mice, hemagglutination assays were also performed. This analysis demonstrated that only A-D5 IgG1 was able to drive concentration-dependent agglutination of mouse red blood cells (at >3.12 μg / ml), while the protein IgG 2 Her47 LHL-LHL, IgG 2 Her47 LHL-LHLF or Fab 2 Met47 LHL-LHL did not show agglutination at any concentration up to 200 μg / ml( Figure 22 ). These findings indicate that the in vitro assay of human CD47 protein and red blood cell binding outlined above was reproduced in the mouse system (i.e., A-D5 IgG1 binds fully to human CD47, but there was no measurable binding for IgG 2 or Fab 2 proteins), making the mouse a viable model for studying the effects of CD47 binding on both pharmacokinetics and tolerance.

[0348] In the in vivo tolerance study, A-D5 IgG1, IgG 2 Her47 LHL-LHL, IgG 2 Her47 LHL-LHLF and Fab 2 Met47 LHL-LHL were each administered once (intravenously) at a concentration of 2 mg / kg or 10 mg / kg in Tg32 mice. The 2 mg / kg dose of A-D5 IgG was tolerated, while the 10 mg / kg dose was poorly tolerated, causing significant toxicity on day 0, which led to the termination of the study in this dosing group. In contrast, the 2 mg / kg and 10 mg / kg doses of IgG 2 Her47 LHL-LHL, IgG 2 Her47 LHL-LHLF and Fab 2 Met47 LHL-LHL were all well tolerated during the 60-day body weight observation( Figure 23 ). Although the 2 mg / kg dose of A-D5 IgG was generally tolerated, a significant upregulation of reticulocytes was observed in this group 5 days after dosing( Figure 24 ). In contrast, the 2 mg / kg or 10 mg / kg doses of IgG 2 Her47 LHL-LHL, IgG 2Her47LHL-LHL or Fab 2 Met47 LHL-LHL was not associated with reticulocyte upregulation at day 5 or later ( Figure 24 , Figure 25). Reticulocyte upregulation is a known response to rapid red blood cell clearance, suggesting that IV administration of the A-D5 IgG1 antibody results in accelerated clearance of CD47-high red blood cells.

[0349] To more broadly sample the effects of the administered protein, a complete hematology panel was assayed at days 5, 29, and 60 post-dose ( Figures 25A - 25K ). These analyses demonstrated that the reticulocyte upregulating effect of A-D5 IgG1 was transient, returning to baseline by day 29 ( Figure 25A ). No significant effects of A-D5 IgG1, IgG 2 Her47LHL-LHL, IgG 2 Her47 LHL-LHLF, and Fab 2 Met47 LHL-LHL on red blood cell (RBC) count, hemoglobin, mean corpuscular hemoglobin concentration (MCHC), mean corpuscular volume (MCV), white blood cells, monocytes, lymphocytes, basophils, eosinophils, or neutrophil levels were observed ( Figures 25B - 25K ).

[0350] In an in vivo pharmacokinetic study, in Tg32 mice, IgG 2 Her47 LHL-LHL, IgG 2 Her47 LHL-LHLF, and Fab 2 Met47 LHL-LHL were each administered once (intravenously) at a concentration of 2 or 10 mg / kg, and A-D5 IgG1 was administered at a previously tolerated concentration of 2 mg / kg. Blood samples were collected from each mouse according to a bleeding schedule: 30 minutes, 4 hours, 1 day, 3 days, 5 days, 7 days, 10 days, 14 days, 21 days, 28 days, and 42 days. Analysis of serum antibody concentrations demonstrated that 2 mg / kg of A-D5 IgG1 was very rapidly removed from the circulation, reaching an average concentration < 0.5 μg / ml within 5 days ( Figure 26 ). This rapid drug clearance (termed tissue-mediated drug disposition or TMDD) may be due to the previously observed strong binding to mouse red blood cells, which are then rapidly cleared from the system by phagocytosis. This finding further explains the return of reticulocyte levels to normal by day 29 following A-D5 IgG1 administration ( Figure 25A ), as the molecule was essentially eliminated by day 10 ( Figure 26 ).

[0351] In contrast, IgG at 2 and 10 mg / kg doses 2 Her47 LHL-LHL, IgG 2 Her47 LHL-LHLF and Fab 2 Met47 LHL-LHL all showed slow clearance ( Figure 26 ). Importantly, each protein at 2 mg / kg dose took >25 days to reach a concentration <1.0 μg / ml ( Figure 27A ), and each protein at 10 mg / kg dose remained >1.0 μg / ml at day 42 ( Figure 27B ). Individual samples collected from mice given 10 mg / kg of A-D5 IgG1 during the tolerance study on day 0 were also analyzed, showing that the maximum serum IgG concentration reached (but not tolerated) was similar to that reached by the fully tolerated IgG 2 and Fab 2 proteins (>50 μg / ml).

[0352] IgG at 2 mg / kg 2 Her47 LHL-LHL, IgG 2 Her47 LHL-LHLF and Fab 2 Met47 LHL-LHL all showed a normal 'α' phase (when the IV dose of immunoglobulin rapidly distributes from the bloodstream to tissues), followed by a long 'β' phase of the antibody circulating in the serum ( Figure 26 , 27A ). These proteins also showed a normal distribution curve at 10 mg / kg, but with an even longer circulation ( Figure 26 , 27B ). Importantly, the β phase of each protein showed linear and parallel curves at both concentrations, indicating that the CD47 domain of these proteins does not result in the TMDD observed for A-D5 IgG1 ( Figure 26 ). If IgG 2 Her47LHL-LHL, IgG 2 Her47 LHL-LHLF or Fab 2 Met47 LHL-LHL protein undergoes activation in the periphery, TMDD is expected to be strongly manifested because activation will lead to high-affinity binding to red blood cells, endothelium, and platelets, resulting in clearance that changes the β phase to a sharp decline trajectory, as seen for A-D5 IgG1 ( Figure 26 ). These observations, together with IgG 2 Her47LHL-LHL, IgG2 Her47 LHL-LHL and Fab 2 Lack of reticulocyte expansion in the dose of Met47 LHL-LHL indicates a very low level of peripheral activation. Although the pharmacokinetics of these molecules is long, which means they have been recycled multiple times through FcRn in a circulation of >25 days.

[0353] IgG outlined above 2 Her47 LHL-LHL, IgG 2 Her47 LHL-LHLF and Fab 2 The pharmacokinetic and tolerability findings of Met47 LHL-LHL indicate a normal, FcRn-mediated, antibody-like half-life extension through the human IgG1 Fc domain. Compared with A-D5 IgG1, this effect results in a significant increase in the area under the curve (AUC) values of these 3 proteins ( Figure 28 ). At 2 mg / kg, IgG 2 Her47 LHL-LHL, IgG 2 Her47 LHL-LHLF and Fab 2 The AUC of Met47 LHL-LHL is improved by 25 - 40 times compared with A-D5 IgG1. At 10 mg / kg, a dose not safely achievable for A-D5 IgG1, IgG 2 Her47 LHL-LHL, IgG 2 Her47 LHL-LHLF and Fab 2 The AUC of Met47 LHL-LHL is improved by approximately 100 - 250 times compared with A-D5 IgG1 ( Figure 28 ). These improvements in AUC are significant because they indicate that the use of the CD47-binding domain within the IgG 2 and Fab 2 structure can be used at high concentrations starting from the first dose, maximizing the distribution to tumor tissues.

[0354] Flow cytometry analysis of binding to cynomolgus monkey and human red blood cells

[0355] Red blood cells were isolated from 3 cynomolgus non-human primates (NHPs, monkeys) and 3 human donors and stained with A-D5 IgG1, trastuzumab, IgG 2 Her47 LHL-LHLF or IgG 2 Her47 LHL-LHL. Both sets of analyses showed that only A-D5 IgG1 exhibited binding to cynomolgus monkeys ( Figure 29A ) or humans ( Figure 29B)Concentration-dependent binding of red blood cells. These findings confirm the above observation that IgG 2 (and thus Fab 2 ) The CD47 binding domain within the structure is restricted to binding mouse, monkey, and human CD47.

[0356] Activation by MMP and cathepsin at pH 6.0 and 7.4

[0357] MMPs and cathepsins have been shown to have the potential to enzymatically cleave peptide sequences found in the LHL or LHLF linkers outlined above. However, importantly, both classes of enzymes exhibit sensitivity to changes in pH conditions that increase or decrease their enzymatic activity. This can be crucial because it is often observed that the pH of solid tumors deviates from the normal physiological pH of humans, pH 7.4. In particular, solid tumors (and highly inflamed tissues) can develop acidic pH conditions as low as pH 6.0.

[0358] As demonstrated above, multiple MMP enzymes have the ability to activate lower fab binding at pH 7.4. We examined the activities of multiple MMPs and cathepsins (all of which are associated with increased activity in solid tumors) at pH 6.0 and 7.4. MMP3 was examined ( Figure 30A 、B), MMP7 ( Figure 30C 、D), MMP8 ( Figure 30E 、F), MMP10 ( Figure 30G 、H), MMP12 ( Figure 30I 、J), MMP13 ( Figure 30K 、L) and MMP14 ( Figure 30M 、N) for the activation of IgG 2 Her47 LHL-LHL or IgG 2 Her47 LHL-LHLF. The IgG 2 Her47 LHL-LHL and IgG 2 Her47 LHL-LHLF MMP-treated samples were applied to directly bind both human Her2 and human CD47. These analyses demonstrated that both proteins exhibited measurable activation of CD47 binding by MMP8, MMP10, and MMP12 at both pH 7.4 and pH 6.0. In contrast, only IgG 2 Her47 LHL-LHLF was activated by MMP13 ( Figure 30K 、L) at both pH 7.4 and pH 6.0 and by MMP7 ( Figure 30D ) at pH 6.0. IgG 2 Her47 LHL-LHLF was also more active than IgG 2Her47 LHL-LHL exhibited a relatively higher level of activation by most of the tested MMPs ( Figure 30A -N). These findings confirm that inclusion of the LHLF linker at pH 6.0 and / or pH 7.4 results in faster activation of the binding of the lower Fab by a broader range of MMP enzymes.

[0359] Cathepsins were also tested as potential activating enzymes. For these enzymes, a clear relationship between pH and activity was observed. IgG 2 Her47 LHL-LHL ( Figure 31A ) or IgG 2 Her47 LHL-LHLF ( Figure 31B ) activation demonstrated that only cathepsin S was able to activate CD47 binding at pH 7.4 and 6.0. In contrast, treatment with cathepsins A, C, G, K, and L showed little or no activation of CD47 binding even after 24 hours at pH 7.4, but rapidly produced strong activation signals at pH 6.0 ( Figure 31A 、 31B ). These findings suggest that, compared to those at pH 7.4, the LHL and LHLF linkers may allow for accelerated activation in acidified tissues through more rapid activation by MMPs and pH-selective activation by a range of cathepsins. This can further improve the therapeutic index of IgG 2 and Fab 2 proteins by minimizing activation of the binding of the lower Fab at pH 7.4 (the pH of non-diseased tissues, where active extracellular MMP and cathepsin levels are low) and maximizing it at pH 6.0 (the pH of diseased tissues, where MMP and cathepsin levels are high and cathepsin activity is enhanced).

[0360] Activation of the binding of the lower Fab to Her2 / CD47+ cells

[0361] Flow cytometry was performed to examine the binding characteristics of IgG 2 Her47 LHL-LHL and IgG 2 Her47 LHL-LHLF to cells expressing different levels of CD47 and Her2 on their cell surfaces. Staining with trastuzumab, anti-CD47, and isotype control IgG demonstrated that BT474 cells express high levels of Her2 and lower levels of CD47 ( Figure 32A 、B), whereas MCF7 cells express higher levels of CD47 and low levels of Her2 ( Figure 32C 、D). After activation with MMP12 for 0, 2, 8, and 24 hours, binding with IgG 2 Her47 LHL-LHLF was also performedFigure 32A , C), and IgG 2 Her47 LHL-LHL( Figure 32B , D) stained two cell types. IgG 2 Her47 LHL-LHLF and IgG 2 Her47 LHL-LHL showed a binding profile similar to that of trastuzumab on BT474 cells at 0, 2, and 8 h time points, but binding decreased slightly after 24 h( Figure 32A , B). In contrast, IgG 2 Her47 LHL-LHLF and IgG 2 Her47 LHL-LHL showed a low level of binding profile similar to that of trastuzumab on MCF7 cells at 0 h activation, but binding was significantly higher at 2, 8, and 24 h activation time points, mimicking the anti-CD47 control( Figure 32C , D). These findings experimentally confirmed that Figure 2B the activation model proposed in 2 (and Fab 2 ) proteins may have the beneficial ability to drive the function of the lower fab binding domain (such as CD47) towards cells expressing low levels of the upper Fab target (such as Her2).

[0362] Activation of MMP12 by SDS-PAGE and mass spectrometry

[0363] IgG was treated with MMP12 as described above 2 Her47 LHL-LHLF and IgG 2 Her47LHL-LHL proteins for 0, 2, 8, and 24 h. Then these protein samples were analyzed by SDS-PAGE( Figure 33 ). This analysis yielded findings clearly related to the observations of activation ELISA( Figure 15D , Figure 30) and flow cytometry (Figure 32): At 0 h - two intact chains were observed, where the heavy chain ran at 75 kDa and the light chain just ran below the 50 kDa marker (less than 50 kDa). At the 2 h time point, for IgG 2 Her47LHL-LHLF and IgG 2For both Her47 LHL-LHL, a new ~25 kDa product was observed, plus a faint band slightly larger than 25 kDa and at approximately ~50 kDa (above the intact light chain). The molecular weights of these new fragments corresponded to the upper Fab Fd or light chain fragments (1 v domain + 1 c domain = ~25 kDa), Fc (2x c domains + 1 N-linked glycosylation = 28 - 30 kDa), and intact Fd-hinge-Fc (standard IgG1 heavy chain = 50 kDa), respectively. At the 8- and 24-hour time points, a progressive decrease in the intact chains and an increase in fragments approximately 25 kDa smaller than the intact chains were observed. In particular, at 8 hours, the 25 kDa (upper fab chain) product became dominant, and significant amounts of both the intact heavy chain (75 kDa) and intact light chain (below 50 kDa) were maintained (but decreased compared to the 0-hour sample). Thus, this SDS-PAGE analysis ( Figure 33 ) demonstrated that for both the LHL and LHLF sequences, the linker between the upper and lower Fabs was cleaved by MMP12 enzymatic activity.

[0364] To precisely detect where the MMP12 enzyme activates the IgG 2 molecule within the Fab 2 structure, mass spectrometry was performed using 0-, 2-, 8-, and 24-hour samples from IgG 2 Her47LHL-LHL. Peptide samples were prepared by reduction, alkylation, and proteolytic digestion using a combination of trypsin and Glu-C. These samples were analyzed by LC-MS / MS. The efficiency of MMP cleavage was determined by comparing the MS responses of peptides derived from the intact MMP cleavage site (SCGPAPE (SEQ ID NO: 110)) with peptides derived from the cleaved protein (SCGPAP (SEQ ID NO: 111)). This analysis successfully identified peptides localized to both the uncleaved (SCGPAPE (SEQ ID NO: 110)) and cleaved (SCGPAP (SEQ ID NO: 111)) linkers, demonstrating that in the LHL linker, MMP 12 cleaves between the second proline (P) and glutamate (E). The relative signal of the uncleaved (SCGPAPE (SEQ ID NO: 110)) peptide gradually decreased from 0 hours to 8 hours to 24 hours ( Figure 34A ), whereas the signal of the cleaved (SCGPAP (SEQ ID NO: 111)) peptide gradually increased ( Figure 34B ).

[0365] Generation and analysis of IgG 2 in the hinge-stabilized 'IgG1-DAA' form

[0366] U.S. Patent No. 8,871,204 B2 teaches protease-resistant IgG1 antibody variants that maintain low hinge and Fc stability in the presence of MMP enzymes. In these mutant IgG1 antibodies, the sequence of E233-L234-L235-G236 (SEQ ID NO: 112) in the hinge is replaced with P233-V234-A235 (where G236 is deleted); and the CH2 domain contains at least one substitution selected from S239D / I332E, K326A / E333A, H268F / S324T / I332E, F243L / R292P / Y300L, S239D / H268F / S324T / I332E, S267E / H268F / S324T / I332E, K326A / I332E / E333A, S239D / K326A / E333A, S267E / I332E, and G237X / S239D / I332E, where X is A, D, P, Q, or S; wherein the amino acid residues are numbered according to EU numbering.

[0367] For detecting the use of the Fab 2 structure in the context of this protease-stable IgG1 Fc, two exemplary constructs (Table 19) were generated. These two constructs (Her47 LHLF-LHL IgG1-2hDAA and Her47 LHL-LHLF IgG1-2hDAA) placed the Her47 Fab 2 structure on an IgG 2 constructed on the '2hDAA' structure (IgG1 containing the P233-V234-A235-ΔG236 and S239D / K326A / E333A mutations). Both constructs were readily expressed in transient CHO cell transfection, and protein A-purified proteins exhibited >80% of the expected molecular weight product. Exemplary analytical SEC data for Her47 LHLF-LHL IgG1-2hDAA showed 80% of the product at 10.30 ml ( Figure 35 ). SDS-PAGE analysis of the protein A-purified protein without reduction and SEC peaks at 8.47, 9.03, and 10.30 ml ( Figure 36 ) demonstrated that peaks 8.47 and 9.03 contained higher molecular weight aggregates, while peak 10.30 contained the product of the expected size (approximately 250 kDa). SDS-PAGE analysis of the protein A-purified protein with reduction and SEC peaks at 8.47, 9.03, and 10.30 ml ( Figure 37)Demonstrate that all peaks at 8.47, 9.03, and 10.30 ml contain heavy and light chain products of the expected sizes (approximately 80 and 50 kDa, respectively). The intact monomer (250 kDa) product of Her47 LHL-LHLF IgG1-2hDAA was purified by SEC and subjected to enzymatic digestion with human MMP12 at pH 7.4 in a time course of incubation at 2, 4, 8, and 24 hours, plus 24 hours incubation in enzyme-free buffer as a negative control (time 0, 2-hour, 4-hour, 8-hour, 24-hour incubation). In ELISA assays, all samples showed strong binding signals to human Her2 but no measurable binding to the control protein murine EpCAM( Figure 38A ). In CD47-binding ELISA, the binding signal increased after incubation at 37 °C for 2, 4, 8, and 24 hours in the presence or absence of MMP12 enzyme, but no signal above background was observed at 0, 2, 4, 8, or 24 hours in the absence of MMP12( Figure 38B ).

[0368] Analysis of 0-hour (undigested) and 2-, 8-, and 24-hour MMP12-digested samples by SDS-PAGE confirmed that the purified protein contained heavy and light chains of the expected sizes. Digestion with MMP12 for 2, 8, and 24 hours produced a 25 kDa band( Figure 38C ), indicating linker peptide cleavage, but significantly less heavy chain degradation was observed compared to IgG 2 Her47 LHL-LHL or IgG 2 Her47 LHL-LHLF( Figure 33 ), indicating that the '2hDAA' mutation does stabilize the Fc region against MMP12 digestion.

[0369] Generation and analysis of IgG with alternative enzyme activation

[0370] Since the LHL and LHLF linkers were shown above to be susceptible to cleavage by multiple MMPs and cathepsins, six Fab 2 construct types were detected in IgG 2 form (Table 20). These constructs used a series of linker designs (Table 21) that were designed to be susceptible to cleavage by several classes of enzymes associated with upregulated activity in solid tumors and highly inflamed tissues, such as enterokinase (EK), thrombin (Thr), tPA, granzyme B (GrB), uPA, and ADAMTs-5 (A5). All 6 constructs were readily expressed by CHO cells and purified by ProA chromatography.

[0371] Purified proteins from cloned Her47 LHL-LHL-EK, Her47-LHL-LHL-Thr, Her47-LHL-LHL-tPA, Her47-LHL-LHL-uPA, Her47-LHL-LHL-GrB, and Her47-LHL-LHL-A5 were all tested in titration ELISA against human Her2 and CD47 targets( Figure 39A , Figure 39C , Figure 39E , Figure 39G , Figure 39I , Figure 39K ). In all cases, these proteins showed low / background binding to CD47 at all tested concentrations (white bars), but concentration-dependent binding to Her2 (gray bars). Each protein was then also subjected to time-course enzymatic digestion with MMP 12 and ELISA binding to Her2 and CD47 targets( Figure 39B , Figure 39D , Figure 39F , Figure 39H , Figure 39J , Figure 39L ). These findings indicate that all proteins retained Her2 binding and showed increased CD47 binding activation throughout the time-course of enzymatic activation. The functional activities of cloned Her47 LHL-LHL-EK, Her47-LHL-LHL-Thr, Her47-LHL-LHL-tPA, Her47-LHL-LHL-uPA, Her47-LHL-LHL-GrB, and Her47-LHL-LHL-A5 demonstrate that the Fab2 structure is capable of using a linker peptide sequence with multiple protease recognition sites to customize activation for a given application. For example, in this case, the retention of MMP activation potential in the LHL linker in combination with any one of six different disease-associated enzyme cleavage motifs in the accompanying linker would allow customization for environments where MMP and / or cathepsin are active, with increased activation potential when enterokinase, thrombin, tPA, granzyme B, uPA, ADAMTs-5, or other proteases are associated with the disease state.

[0372] In vivo multi-dose tolerance of Her47 molecule in NOD-SCID mice

[0373] In the in vivo multi-dose tolerance study, IgG 2 Her47 LHL-LHL, IgG 2 Her47LHL-LHLF, Fab 2 Her47 LHL-LHL, and Fab 2Her47 LHL-LHLF was administered four times (intravenously) to NOD-SCID mice. IgG 2 protein was administered at 14 mg / kg on day 0 and at 7 mg / kg on days 5, 10, and 15. Fab 2 protein was administered at 8 mg / kg on day 0 and at 4 mg / kg on days 5, 10, and 15. All proteins were well tolerated, with no clinical signs of toxicity and no weight loss for any individual animal ( Figure 40 ). These findings confirm and extend the tolerance findings of the Her47 Fab 2 -based molecules at a single dose in Tg32 mice. In the Tg32 mouse study, even a single dose of A-D5 CD47 IgG at 10 mg / kg was not tolerated. In this NOD-SCID mouse study, all four doses of IgG 2 Her47 LHL-LHL, IgG 2 Her47 LHL-LHLF, Fab 2 Her47 LHL-LHL and Fab 2 Her47 LHL-LHLF were well tolerated. This finding confirms that the CD47-binding domain of the Her47 molecule is not activated in the circulation even after repeated dosing, as significant activation would induce the CD47-driven toxicity signals observed for A-D5 IgG1.

[0374] Generation and analysis of additional 2-chain Her2CD3 Fab 2 ‘single-arm’ constructs

[0375] Additional Her2CD3 constructs were generated to examine the ability of the new sequences to improve product homogeneity and activity (Table 22). All Her2CD3 constructs were expressed by CHO cells, purified from the supernatant by ProA, and then analyzed by SEC. These analyses showed that Fab 2 Her23 LHL-LHL-S and Fab 2 Her23 LHLF-LHL-S both exhibited higher main product homogeneity ( 2 Her23LHL-LHL and Fab 2 Her23 LHL-LHLF (both of which exhibited a higher proportion of higher and lower molecular weight products ( Figure 41C 、 Figure 41D )) than Figure 41A 、 Figure 41B ). These findings suggest that by swapping in the construct Fab 2Her23 LHL-LHL-S and Fab 2 In Her23 LHLF-LHL-S, the upwardly directed fabs result in the chains being [VL-CL-linker-VH-CH-Fc] plus [VH-CH-linker-VL-CL-Fc], leading to improved uniformity. This is in contrast to Fab 2 Her23 LHL-LHL and Fab 2 Her23 LHL-LHLF, in which both chains are [VL-CL-linker-VL-CL-Fc] plus [VH-CH-linker-VH-CH-Fc].

[0376] Control proteins Fab were then also designed and expressed 2 mEpCam3 LHLF-LHL-S and Fab 2 mEpCam3 LHL-LHL-S (the upper Fab contains the v domain of the anti-mouse EpCAM antibody G8.8) (Table 22). Cloned Fabs were separated by preparative SEC 2 Her23 LHL-LHL-S, Fab 2 Her23 LHLF-LHL-S, Fab 2 mEpCam3 LHLF-LHL-S and Fab 2 The correct MW products of mEpCam3LHL-LHL-S were obtained and analyzed in a Promega Jurkat cell-based CD3 ligation reporter cell bioassay using MCF-7 or BT-474 as human Her2+ target cells (according to the manufacturer's instructions). Preliminary analysis using MCF-7 as the target cell (which expresses very low levels of Her2) demonstrated that both the positive control Her2-CD3 BITE protein and OKT3 IgG1 produced strong positive concentration-dependent CD3 activation signals. Cloned Fabs treated with MMP 12 for 2 hours 2 mEpCam3 LHLF-LHL-S and Fab 2 mEpCam3 LHL-LHL-S did not produce a signal at any concentration ( Figure 42A , B). In contrast, cloned Fabs 2 Her23 LHLF-LHL-S ( Figure 42A ) and Fab 2 Her23 LHL-LHL-S ( Figure 42B ) did not show measurable signals at 0 hours in a concentration range similar to Her2-CD3 BITE, but the signals gradually increased at 2, 8, and 24 hours, and in both cases the maximum signal was higher than that obtained with the positive control.

[0377] In a secondary assay using Her2-overexpressing BT-474 cells as target cells, all of the above test samples and controls were assayed at 0.1 μg / ml ( Figure 43A -C). The positive control proteins Her2-CD3 BITE and OKT3 IgG1 both induced strong CD3 activation signals, while the negative control Fab 2 mEpCam3 LHLF-LHL-S and trastuzumab did not. The cloned Fab 2 Her23 LHLF-LHL-S ( Figure 43B ) and Fab 2 Her23 LHL-LHL-S ( Figure 43C ) showed no measurable signal at 0 h, but the signal gradually increased at 2 h and reached a maximum at 8 h. Importantly, the signal at 24 h was significantly lower compared to 8 h. This finding suggests that the progressive enzymatic activity that can cleave both linkers ( Figure 33 ) may lead to the eventual separation of the upper and lower Fabs. This could be a beneficial feature for CD3-targeting molecules, as such molecules are preferentially activated in the highly proteolytic tumor environment but also gradually inactivated there, thus minimizing the risk of the molecule in its active form leaking into healthy tissues would be desirable.

[0378] Her2-CD47 IgG 2 and Fab 2 In vitro measurement of the molecular stability of proteins

[0379] Historically, engineered antibody formats have often suffered from structural heterogeneity, leading to manufacturing problems. To examine the stability of the IgG 2 Her47 LHL-LHL and Fab 2 Her47 LHL-LHL proteins, a series of in vitro measurements were performed:

[0380] Forced oxidation - Oxidation of exposed amino acid residues such as tryptophan and methionine is a common degradation pathway for mAbs, which affects their biological activity. In this study, forced oxidation with 0.5% H2O2 in PBS for 2 h at room temperature was applied to the IgG 2 Her47LHL-LHL and Fab 2 Her47 LHL-LHL proteins. Since oxidation can alter the overall hydrophobicity of the antibody either by increasing the polarity of the oxidized form or through conformational changes, the potential changes induced by forced oxidation were analyzed by reverse-phase (RP) and size-exclusion (SEC) chromatography. SEC analysis revealed no change in the proportion of monomeric species in either test sample (IgG 2Her47 LHL-LHL and Fab 2 Both Her47 LHL-LHL showed 99.5% and 97.4% monomer species before and after oxidation, respectively. In the RP analysis of the intact antibody, for the intact (non-reduced) antibody, after forced oxidation with 0.5% H2O2, IgG was observed 2 The retention time of Her47 LHL-LHL decreased by 0.5 minutes and that of Fab 2 The retention time of Her47 LHL-LHL decreased by 0.4 minutes. In the RP analysis of the subunits, for IgG 2 Her47 LHL-LHL, after forced oxidation, the retention time of the heavy chain decreased by 0.5 minutes and that of the light chain showed no displacement. In the RP analysis, the reduced Fab 2 Her47 LHL-LHL showed different characteristics, and 3 peaks were observed, representing the light chain, heavy chain, and truncated hinge-Fc stump. After oxidation, a displacement of 0.4 - 0.5 minutes was observed for the heavy chain and truncated hinge-Fc stump, but no displacement was observed for the light chain. After treatment with H2O2, reduced IgG 2 Her47LHL-LHL and Fab 2 The change in the retention time of the Her47 LHL-LHL sample indicated less oxidation of the exposed amino acids (limited to the Fc region of the protein).

[0381] Charge isomer analysis - Charge heterogeneity analysis is important in the characterization of monoclonal antibodies because it provides important information about product quality and stability. Heterogeneity may be caused by enzymatic post-translational modifications (glycosylation, lysine truncation) or chemical modifications (oxidation or deamidation) during purification and storage. The charge isomer spectrum of the test article provided was analyzed by a commercial Charge Variant Assay. IgG 2 Her47 LHL-LHL( Figure 44A ) and Fab 2 Her47 LHL-LHL( Figure 44B ) Both showed a homogeneous spectrum with one major isotype (50 - 57% of the total), one major acidic isotype (40 - 48% of the total), and one minor basic isotype (about 3%).

[0382] Retention on HIC - Overall hydrophobicity is an indicator of the protein's tendency to self - associate, which can be a significant risk factor for aggregation and viscosity during bioprocessing. Proteins have hydrophobic 'patches' on their surface, which arise from the presence of side chains of hydrophobic or non - polar amino acids. Depending on their number, size, and distribution, the resulting surface hydrophobicity will be a specific characteristic of each protein. HIC separates proteins based on differences in their surface hydrophobicity, using the reversible binding between the protein and the hydrophobic surface of the HIC resin. IgG 2 Her47 LHL - LHL and Fab 2 Her47 LHL - LHL exhibited HIC column retention times of 5.4 and 5.0 minutes, respectively. Compared to clinical monoclonal antibodies such as adalimumab (4.5 minutes), cetuximab (5.9 minutes), brentuximab (6.3 minutes), and golimumab (8.1 minutes), these values are towards the lower hydrophobicity range. In fact, these values indicate a similar aggregation tendency and stability (5.4 - minute retention) to trastuzumab, an anti - Her2 antibody with the Her2 - binding domain in IgG 2 Her47LHL - LHL and Fab 2 Her47 LHL - LHL.

[0383] Freeze - thaw stability analysis - The instability of proteins during freeze - thaw steps is an indicator of difficulties in manufacturing and bioprocessing, as increased protein aggregation or fragmentation is a risk for reduced product quality. To evaluate this risk for proteins containing the Fab 2 structure, IgG 2 Her47 LHL - LHL and Fab 2 Her47 LHL - LHL proteins were subjected to 5 rounds of freeze - thaw, followed by SEC analysis after each round. These analyses showed that IgG 2 Her47 LHL - LH L( Figure 45A ) and Fab 2 Her47LHL - LH L( Figure 45B ) proteins did not show any change in monodispersity (no aggregated or degraded products were observed) after 5 rounds of freezing.

[0384] In summary, these findings indicate that IgG 2 Her47 LHL - LHL and Fab 2 Her47 LHL - LHL proteins all have low aggregation risk, low hydrophobicity, low charge heterogeneity, and low oxidation tendency.

[0385] The affinity of IgG variants for human Fc receptors Analysis

[0386] If antibodies that target receptors on diseased cells mediate ADCC and ADCP activities, they must bind to Fcγ receptors. To detect whether these binding functions are retained in Fab2-based constructs, IgG was assayed by surface plasmon resonance analysis 2 Her47 LHL-LHL and Fab 2 Her47 LHL-LHL protein for binding affinities to all human and murine Fc receptors. These assays demonstrated that isotype control human IgG1 and IgG4 both exhibited the expected strong and weak binding affinities (respectively) to all human Fcγ receptors, including the high- and low-affinity variants of FcγRIIA and FcγRIIIA (Table 23). Similarly, isotype control murine IgG2a and IgG1 exhibited the expected strong and weak binding affinities (respectively) to murine FcγRI, FcγRIII, and FcγRIV receptors. For IgG 2 Her47 LHL-LHL and Fab 2 Her47 LHL-LHL, binding to each of the human and murine Fc receptors tested was highly similar to that observed with isotype control human IgG1. These data suggest that in both humans and mice, IgG 2 and Fab 2 proteins both should be able to bind to Fc receptors when bound to the surface of diseased cells.

[0387] Additional protein construct designs

[0388] Additional protein constructs ( Figure 46 ) were envisioned. The constructs may contain: 1. Constant domains only in the 'upper fab' position, meaning that the activity of the 'lower Fab' is blocked from binding its target, but may become active in an appropriate proteolytic environment. 2. False 'non-binding' variable domains in the 'upper fab'. These false variable domains will be shown not to bind any known target in vivo, so that only the 'lower fab' exhibits potential drug target binding ability, and only after proteolytic activation through one of the linker domains. 3. The 'upper Fab' is replaced by a 'diabody' structure containing 4 variable domains. This diabody structure may or may not contain disulfide bonds, as found in the 'DART' protein. The diabody structure can facilitate binding of 2 copies of the same target or 2 separate targets, where the 'lower Fab' activity is blocked from binding its target until it becomes active in an appropriate proteolytic environment. In any of the constructs envisioned herein or shown above, Fab 2The structures can be free or fused to another functionalized structure such as an Fc fragment, a small domain, or a peptide that extends the half-life such as an albumin-binding moiety. They can also be chemically conjugated to small molecules, peptides, or other proteins that mediate additional biological functions.

[0389] Cell proliferation assay of Her2CD47 protein using Her2-high BT-474 cells

[0390] Due to IgG 2 Her47 and Fab 2 Since the Her2-binding domain of the upper Fab of Her47 protein is able to mediate inhibition of the kinase activity of the receptor, a cell proliferation assay was performed. This assay used BT-474 cells because they are a cell line known to be responsive to Her2 inhibition. Trastuzumab, isotype control IgG1, IgG 2 Her47LHL-LHL( Figure 47A ) and Fab 2 Her47 LHL-LHL( Figure 47B ) were applied to BT-474 cells during a 72-hour incubation period, and cell proliferation was measured. The data are presented as the percentage inhibition of cell growth (Figure 47). These analyses showed that while trastuzumab exhibited strong concentration-dependent inhibition of BT-474 cell proliferation, IgG 2 Her47 LHL-LHL( Figure 47A ) exhibited slightly lower potency and Fab 2 Her47 LHL-LHL( Figure 47B ) had even lower potency (reflecting its monovalent binding capacity).

[0391] In vivo efficacy analysis of Her47 molecule in NOD-SCID mice bearing tumor xenografts (KYSE-410 model)

[0392] In an in vivo multi-dose efficacy study, trastuzumab, IgG 2 Her47 LHL-LHL, IgG 2 Her47 LHL-LHLF, Fab 2 Her47 LHL-LHL and Fab 2 Her47 LHL-LHLF (Fab 2 structure containing human IgG1 Fc, as Figure 3BAs shown in (administered four times intravenously on days 0, 5, 10, and 15) in tumor-bearing NOD-SCID mice, the tumors were generated by subcutaneous inoculation of the esophageal cancer cell line KYSE-410 expressing Her2. Once tumors > 125 mm 2 were established, dosing was initiated. IgG 2 protein was administered at 14 mg / kg on day 0 and at 7 mg / kg on days 5, 10, and 15. Fab 2 protein was administered at 8 mg / kg on day 0 and at 4 mg / kg on days 5, 10, and 15. Trastuzumab was administered at 8 mg / kg on day 0 and at 4 mg / kg on days 5, 10, and 15. Tumor volume was measured by caliper measurement.

[0393] After 3 doses, on day 11, trastuzumab ( Figure 48A ), IgG 2 Her47LHL-LHLF ( Figure 48B ), IgG 2 Her47 LHL-LHL ( Figure 48C ), and Fab 2 Her47LHL-LHLF ( Figure 48D ) all showed a significant reduction in tumor growth compared to the vehicle (two-way ANOVA: p = 0.005, 0.005, 0.019, and 0.003 respectively). In contrast, Fab 2 Her47LHL-LHL ( Figure 48E ) did not significantly reduce tumor growth by day 11 (two-way ANOVA: p = 0.66). Importantly, the sequences of Fab 2 Her47 LHL-LHL and Fab 2 Her47LHL-LHLF are identical (except for two point mutations in the LHLF linker, which accelerate and expand protease sensitivity relative to the LHL linker as shown above), but result in a significant difference in potency ( Figure 48F ). In addition, the data in Figure 47 demonstrate that the 1-armed Fab 2 structure results in weaker Her2-driven cell proliferation inhibition than that observed for trastuzumab, which contains the same Her2-binding VH and VL domain sequences found in both Fab 2 Her47 LHL-LHL and Fab 2 Her47 LHL-LHLF. As a result, the following conclusions were drawn: 1. On day 11 for trastuzumab ( Figure 48A ) and Fab 2Her47 LHL-LHLF( Figure 48D ) The roughly equal potencies observed cannot be driven by high Her2 kinase activity in Fab 2 Her47LHL-LHLF. 2. The high potency of Fab 2 Her47 LHL-LHLF is thus most likely driven by protease activation in the KYSE-410 tumor microenvironment, leading to CD47 blockade and innate immune engagement.

[0394] In summary, the findings outlined above thus demonstrate that the Fab 2 structure allows for the effective elimination of 'lower Fab' activity, exemplified by both the CD47 and CD3 binding domains. The binding ability of the 'upper fab' domain is fully maintained, but significant activity in the lower fab domain is only observed after activation by proteases such as MMP and cathepsin, which are associated with high activity in diseased tissues such as tumors and fibrotic tissues. The Fab 2 structure allows for the modulation of the linker sequence to maximize lower fab activation in the disease microenvironment and avoid peripheral pool issues and toxicity, as exemplified by the performance of the Her47 molecule in vitro and in vivo.

[0395] In vivo pharmacokinetics of the Her47 molecule in NOD-SCID mice

[0396] In an in vivo multi-dose PK study, IgG 2 Her47 LHL-LHL, IgG 2 Her47 LHL-LHLF, Fab 2 Her47LHL-LHL and Fab 2 Her47 LHL-LHLF will each be administered once (intravenously) to NOD-SCID mice. IgG 2 proteins will be administered at 14 mg / kg, and Fab 2 proteins will be administered at 8 mg / kg. Blood samples will be collected at 15 minutes, 30 minutes, 1 hour, 3 hours, 6 hours, and 24 hours, and serum antibody levels will be measured using anti-human IgG1 ELISA.

[0397] In vivo efficacy analysis of the Her47 molecule in NOD-SCID mice bearing tumor xenografts (SKOV-3, JIMT-1, and NUGC-4 models)

[0398] In an in vivo multi-dose efficacy study, IgG 2 Her47 LHL-LHL, IgG 2 Her47LHL-LHLF, Fab2 Her47 LHL-LHL and Fab 2 Her47 LHL-LHLF will be administered four times (intravenously) to tumor-bearing NOD-SCID mice, and the tumors are generated by subcutaneous inoculation of cell lines SKOV-3, JIMT-1, and NUGC-4. IgG 2 The protein will be administered at 14 mg / kg on day 0 and at 7 mg / kg on days 5, 10, and 15. Fab 2 The protein will be administered at 8 mg / kg on day 0 and at 4 mg / kg on days 5, 10, and 15. Trastuzumab will be administered at 8 mg / kg on day 0 and at 4 mg / kg on days 5, 10, and 15. Tumor volume will be measured by caliper measurement.

[0399] In vivo analysis of tolerance and pharmacokinetics in cynomolgus monkeys

[0400] To detect IgG 2 and / or Fab 2 The tolerance and pharmacokinetics in the case of CD47 or CD3 as the lower Fab domain will be studied for multiple exemplary molecules in cynomolgus monkeys. For example, IgG 2 and / or Fab 2 can each be administered one, two, or three times (intravenously) at a concentration of 2 mg / kg or higher. Blood samples will be collected from each animal according to the bleeding schedule. Analyses measuring serum antibody concentration will be performed to calculate PK and assess the risk of TMDD). To sample the effects of the administered protein more extensively, a complete hematology panel will also be detected on a series of days after dosing. These analyses will measure reticulocytes, red blood cells (RBC), hemoglobin, mean corpuscular hemoglobin concentration (MCHC), mean corpuscular volume (MCV), white blood cells, monocytes, lymphocytes, basophils, eosinophils, and / or neutrophil levels.

[0401] Target co-engagement biosensor measurement

[0402] To detect the effect of activation on the binding affinity of Fab 2 to both Her2 and CD47 (or CD3), a biosensor assay that can sample the binding of Her2 and CD47 (or CD3) on the same chip surface, such as a kinetic biosensor instrument, will be established. In this assay, control antibodies and IgG 2 or Fab 2Proteins (undigested or activated with MMP or cathepsin for, e.g., 2, 4, 8, or 24 hours) are applied alone or together at different densities to the surface of a sensor chip differentially labeled with purified extracellular domain proteins of Her2 and CD47 (or CD3). The affinities for Her2 and CD47 (or CD3) will be measured to determine the effect of multivalent interactions on the individual functional affinities for the two targets and on the same surface.

[0403] Although the invention has been described with reference to preferred or exemplary embodiments, those skilled in the art will recognize that various modifications and variations can be made without departing from the spirit and scope of the invention, and such modifications are clearly contemplated herein. It is not intended to limit the specific embodiments set forth in this disclosure and the appended claims, nor should any limitations be inferred therefrom.

[0404] It is not intended to limit the specific embodiments set forth in this disclosure and the appended claims, nor should any limitations be inferred therefrom. All documents or portions of documents cited herein, including but not limited to patents, patent applications, articles, books, and papers, are hereby expressly incorporated by reference in their entirety for any purpose. In the event that the definition of a term in one or more of the incorporated documents or portions of documents conflicts with the definition of that term in this application, the definition set forth in this application shall control. However, any reference, article, publication, patent, patent publication, and patent application cited herein does not and should not be taken as an admission or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world.

[0405] Numbered embodiments

[0406] Notwithstanding the appended claims, the present disclosure sets forth the following numbered embodiments:

[0407] 1. A protein comprising a first part, a second part, and a peptide linker between the first part and the second part,

[0408] wherein the peptide linker comprises an amino acid sequence from a human immunoglobulin hinge region or an amino acid sequence having 1 to about 7 amino acid substitutions compared to a human immunoglobulin hinge region or an amino acid sequence;

[0409] wherein the peptide linker is cleavable by a protease expressed in diseased tissue;

[0410] wherein the second part is capable of specifically binding to a molecule expressed in the diseased tissue; and

[0411] wherein when the peptide linker is not cleaved, the binding of the second part to the molecule expressed in the diseased tissue is reduced or inhibited.

[0412] 2. The protein according to embodiment 1, wherein the length of the peptide linker is between about 5 and about 15 amino acids.

[0413] 3. The protein according to embodiment 1 or 2, wherein the peptide linker comprises or consists of the following amino acid sequences: 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.

[0414] 4. The protein according to any one of embodiments 1-3, wherein the protease is human matrix metalloproteinase (MMP), human cathepsin, human enterokinase, human thrombin, human tPA, human granzyme B, human uPA or human ADAMTs-5.

[0415] 5. The protein according to any one of embodiments 1-4, wherein the peptide linker comprises a human MMP cleavage site, human cathepsin, human enterokinase, human thrombin, human tPA, human granzyme B, human uPA or human ADAMTs-5 cleavage site.

[0416] 6. The protein according to any one of embodiments 1-5, wherein the human MMP is MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, MMP-12, MMP-13 or MMP14.

[0417] 7. The protein according to any one of embodiments 1-6, wherein the level or activity of the human MMP in the diseased tissue is increased compared to the level or activity of the human MMP in the non-diseased tissue.

[0418] 8. The protein according to any one of embodiments 1-5, wherein the human cathepsin is cathepsin A, cathepsin C, cathepsin D, cathepsin G, cathepsin L or cathepsin K.

[0419] 9. The protein according to any one of embodiments 1-5 and 8, wherein the level or activity of the human cathepsin in the diseased tissue is increased compared to the level or activity of the human cathepsin in the non-diseased tissue.

[0420] 10. The protein according to any one of embodiments 1-9, wherein the first part comprises an antibody, an antigen-binding part of an antibody, or an extracellular domain of a receptor.

[0421] 11. The protein according to embodiment 10, wherein the first part is a Fab, a single-chain Fab, a VH domain, a VL domain, an immunoglobulin new antigen receptor (IgNAR), a single-chain variable fragment (scFv), a diabody, or a T cell receptor domain.

[0422] 12. The protein according to any one of embodiments 1-11, wherein the first part specifically binds to a molecule expressed in diseased tissue.

[0423] 13. The protein according to any one of embodiments 1-12, wherein the first part specifically binds to a first molecule expressed in diseased tissue, and the second part is capable of specifically binding to a second molecule expressed in diseased tissue, wherein the first molecule expressed in diseased tissue and the second molecule expressed in diseased tissue are different molecules.

[0424] 14. The protein according to embodiment 13, wherein the first molecule expressed in diseased tissue and the second molecule expressed in diseased tissue are expressed by the same cell.

[0425] 15. The protein according to embodiment 13, wherein the first molecule expressed in diseased tissue and the second molecule expressed in diseased tissue are expressed by different cells.

[0426] 16. The protein according to embodiment 13, wherein the first molecule expressed in diseased tissue and / or the second molecule expressed in diseased tissue are expressed on the cell surface.

[0427] 17. The protein according to embodiment 13, wherein the first molecule expressed in diseased tissue and / or the second molecule expressed in diseased tissue are soluble molecules.

[0428] 18. The protein according to any one of embodiments 1-17, wherein the first part 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.

[0429] 19. The protein according to any one of embodiments 1-17, wherein the second part specifically binds to a molecule expressed by a human immune cell.

[0430] 20. The protein according to embodiment 19, wherein the molecule expressed by the human immune cell is human CD3, human CD16A, human CD16B, human CD28, human CD89, human CTLA4, human NKG2D, human SIRPa, human SIRPγ, human PD1, human Lag3, human 4-1BB, human OX40 or human GITR.

[0431] 21. The protein according to any one of embodiments 1-20, wherein the first part comprises a heavy chain variable (VH) region and a light chain variable (VL) region.

[0432] 22. The protein according to any one of embodiments 1-21, wherein the first part comprises an immunoglobulin constant region or a part of an immunoglobulin constant region.

[0433] 23. The protein according to embodiment 22, wherein the immunoglobulin constant region is IgG, IgE, IgM, IgD, IgA or IgY.

[0434] 24. The protein according to embodiment 22, wherein the immunoglobulin constant region is IgG1, IgG2, IgG3, IgG4, IgA1 or IgA2.

[0435] 25. The protein according to embodiment 22, wherein the immunoglobulin constant region is immunologically inert.

[0436] 26. The protein according to embodiment 22, wherein the immunoglobulin constant region is a wild-type human IgG4 constant region, a human IgG4 constant region comprising the amino acid substitution S228P, a wild-type human IgG1 constant region, a human IgG1 constant region comprising the amino acid substitutions L234A and L235A, a human IgG1 constant region comprising the amino acid substitutions L234A, L235A and G237A, a human IgG1 constant region comprising the amino acid substitutions L234A, L235A, G237A and P331S, or a wild-type human IgG2 constant region.

[0437] 27. The protein according to any one of embodiments 1-26, wherein the second part comprises an antibody, an antigen-binding part of an antibody or an extracellular domain of a receptor.

[0438] 28. The protein according to embodiment 27, wherein the second part is Fab, single-chain Fab, VH domain, VL domain, immunoglobulin new antigen receptor (IgNAR), single-chain variable fragment (scFv) or T cell receptor domain.

[0439] 29. The protein according to any one of embodiments 1-28, wherein the second part specifically binds to human CD47.

[0440] 30. The protein according to any one of embodiments 1-28, wherein the second part specifically binds to human CD3 or human PD-L1.

[0441] 31. The protein according to any one of embodiments 1-30, wherein the second part comprises a heavy chain variable (VH) region and a light chain variable (VL) region.

[0442] 32. The protein according to any one of embodiments 1-31, wherein the second part comprises an immunoglobulin constant region or a portion of an immunoglobulin constant region.

[0443] 33. The protein according to embodiment 32, wherein the immunoglobulin constant region is IgG, IgE, IgM, IgD, IgA or IgY.

[0444] 34. The protein according to embodiment 32, wherein the immunoglobulin constant region is IgG1, IgG2, IgG3, IgG4, IgA1 or IgA2.

[0445] 35. The protein according to embodiment 32, wherein the immunoglobulin constant region is immunologically inert.

[0446] 36. The protein according to embodiment 32, wherein the immunoglobulin constant region is a wild-type human IgG4 constant region, a human IgG4 constant region comprising the amino acid substitution S228P, a wild-type human IgG1 constant region, a human IgG1 constant region comprising the amino acid substitutions L234A and L235A, a human IgG1 constant region comprising the amino acid substitutions L234A, L235A and G237A, a human IgG1 constant region comprising the amino acid substitutions L234A, L235A, G237A and P331S, or a wild-type human IgG2 constant region.

[0447] 37. The protein according to embodiment 1, wherein the protein has one immune effector function or two, three or more immune effector functions.

[0448] 38. The protein according to embodiment 37, wherein the immune effector function is ADCC, CDC or ADCP.

[0449] 39. The protein according to any one of embodiments 1-38, wherein the first part prevents or reduces the specific binding of the second part to the molecule expressed in the diseased tissue.

[0450] 40. The protein according to any one of embodiments 1-39, wherein the peptide linker is cleaved near or within the diseased tissue.

[0451] 41. The protein according to any one of embodiments 1-40, wherein the peptide linker is cleaved near or within the diseased tissue, wherein the first part dissociates from the second part near or within the diseased tissue, and wherein the second part specifically binds to a molecule expressed in the diseased tissue.

[0452] 42. The protein according to any one of embodiments 1-41, wherein the diseased tissue is a tumor or an inflamed tissue.

[0453] 43. The protein according to embodiment 1, wherein the first part specifically binds to human cMET, wherein the second part specifically binds to human CD47, and wherein the protein comprises a first polypeptide chain and a second polypeptide chain, the first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 16 or consisting of the same, and the second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 17 or consisting of the same.

[0454] 44. The protein according to embodiment 1, wherein the first part specifically binds to human HER2, wherein the second part specifically binds to human CD3, and wherein the protein comprises a first polypeptide chain and a second polypeptide chain, the first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 26 or consisting of the same, and the second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 27 or consisting of the same.

[0455] 45. The protein according to embodiment 1, wherein the first part specifically binds to human HER2, wherein the second part specifically binds to human CD47, and wherein the protein comprises a first polypeptide chain and a second polypeptide chain, the first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 34 or consisting of the same, and the second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 35 or consisting of the same.

[0456] 46. The protein according to embodiment 1, wherein the first part specifically binds to human cMET, wherein the second part specifically binds to human CD47, and wherein the protein comprises a first polypeptide chain and a second polypeptide chain, the first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 36 or consisting of the same, and the second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 37 or consisting of the same.

[0457] 47. The protein according to embodiment 1, wherein the first part specifically binds to human Her2, wherein the second part specifically binds to human CD3, and wherein the protein comprises a first polypeptide chain and a second polypeptide chain, the first polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO: 38, and the second polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO: 39.

[0458] 48. The protein according to embodiment 1, wherein the first part specifically binds to human Her2, wherein the second part specifically binds to human CD3, and wherein the protein comprises a first polypeptide chain and a second polypeptide chain, the first polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO: 40, and the second polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO: 41.

[0459] 49. The protein according to embodiment 1, wherein the first part specifically binds to human Her2, wherein the second part specifically binds to human CD47, and wherein the protein comprises a first polypeptide chain and a second polypeptide chain, wherein:

[0460] (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; or

[0461] (b) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 44, and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 45; or

[0462] (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; or

[0463] (d) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 48, and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 49; or

[0464] (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; or

[0465] (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; or

[0466] (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; or

[0467] (h) The first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 88, and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 89; or

[0468] (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; or

[0469] (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: 92; or

[0470] (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; or

[0471] (1) 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; or

[0472] (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; or

[0473] (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; or

[0474] (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.

[0475] 50. The protein according to embodiment 1, wherein the first part specifically binds to human Her2, the second part specifically binds to human CD3, and the protein comprises a first polypeptide chain and a second polypeptide chain, the first polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO: 73, and the second polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO: 74.

[0476] 51. The protein according to embodiment 1, wherein the first part specifically binds to human cMET, the second part specifically binds to human cMET, and the protein comprises a first polypeptide chain and a second polypeptide chain, the first polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO: 75, and the second polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO: 76.

[0477] 52. The protein according to embodiment 1, wherein the first part specifically binds to human Her2, the second part specifically binds to human CD3, and the protein comprises a first polypeptide chain and a second polypeptide chain, wherein:

[0478] (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

[0479] (b) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 100, and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 101; or

[0480] (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; or

[0481] (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.

[0482] 53. An immunoconjugate comprising the protein according to any one of embodiments 1-52 linked to a therapeutic agent.

[0483] 54. The immunoconjugate according to embodiment 53, wherein the therapeutic agent is a cytotoxin, a radioisotope, a chemotherapeutic agent, an immunomodulator, an anti-angiogenic agent, an anti-proliferative agent, a pro-apoptotic agent, a cell growth inhibitory enzyme, a cytolytic enzyme, a therapeutic nucleic acid, an anti-angiogenic agent, an anti-proliferative agent or a pro-apoptotic agent.

[0484] 55. A pharmaceutical composition comprising the protein according to any one of embodiments 1-52 or the immunoconjugate according to embodiment 53 or 54, and a pharmaceutically acceptable carrier, diluent or excipient.

[0485] 56. A nucleic acid molecule encoding the protein according to any one of embodiments 1-52 or a portion of the protein.

[0486] 57. A nucleic acid molecule encoding the first polypeptide chain, the second polypeptide chain, or both the first polypeptide chain and the second polypeptide chain of the protein according to any one of embodiments 43-52.

[0487] 58. An expression vector comprising the nucleic acid molecule according to embodiment 56 or 57.

[0488] 59. A recombinant host cell comprising the nucleic acid molecule according to embodiment 56 or 57 or the expression vector according to embodiment 58.

[0489] 60. A method for producing a protein, comprising:

[0490] culturing a recombinant host cell comprising the expression vector according to embodiment 57 under conditions such that the nucleic acid molecule is expressed, thereby producing the protein; and

[0491] isolating the protein from the host cell or the culture.

[0492] 61. A method for enhancing an anti-cancer immune response in a subject, comprising administering to the subject a therapeutically effective amount of the protein according to any one of embodiments 1-52, the immunoconjugate according to embodiment 53 or 54, or the pharmaceutical composition according to embodiment 55.

[0493] 62. A method for treating cancer, an autoimmune disease, an inflammatory disease, a cardiovascular disease or a fibrotic disease in a subject, comprising administering to the subject a therapeutically effective amount of the protein according to any one of embodiments 1-52, the immunoconjugate according to embodiment 53 or 54, or the pharmaceutical composition according to embodiment 55.

[0494] 63. The method according to embodiment 62, wherein the cancer is gastrointestinal stromal cancer (GIST), pancreatic cancer, skin cancer, melanoma, breast cancer, lung cancer, bronchial cancer, colorectal cancer, prostate cancer, gastric cancer, ovarian cancer, bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, cancer of the oral cavity or pharynx, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small intestine or appendiceal cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma or cancer of the blood tissue.

[0495] 64. The method according to embodiment 62, wherein the autoimmune or inflammatory disease is arthritis, asthma, multiple sclerosis, psoriasis, Crohn's disease, inflammatory bowel disease, lupus, Graves' disease, Hashimoto's thyroiditis or ankylosing spondylitis.

[0496] 65. The method according to embodiment 62, wherein the cardiovascular disease is coronary heart disease, or atherosclerosis or stroke.

[0497] 66. The method according to embodiment 62, wherein the fibrotic disease is myocardial infarction, angina, osteoarthritis, pulmonary fibrosis, cystic fibrosis, bronchitis or asthma.

[0498] 67. The protein according to any one of embodiments 1-52, the immunoconjugate according to embodiment 53 or 54, or the pharmaceutical composition according to embodiment 55, for the treatment of cancer, autoimmune disease, inflammatory disease, cardiovascular disease or fibrotic disease.

[0499] 68. The protein or pharmaceutical composition for use according to embodiment 67, wherein the cancer is gastrointestinal stromal cancer (GIST), pancreatic cancer, skin cancer, melanoma, breast cancer, lung cancer, bronchial cancer, colorectal cancer, prostate cancer, gastric cancer, ovarian cancer, bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, cancer of the oral cavity or pharynx, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small intestine or appendiceal cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma or cancer of the blood tissue.

[0500] 69. The protein or pharmaceutical composition for use according to embodiment 67, wherein the autoimmune or inflammatory disease is arthritis, asthma, multiple sclerosis, psoriasis, Crohn's disease, inflammatory bowel disease, lupus, Graves' disease, Hashimoto's thyroiditis or ankylosing spondylitis.

[0501] 70. The protein or pharmaceutical composition for use according to embodiment 67, wherein the cardiovascular disease is coronary heart disease, atherosclerosis or stroke.

[0502] 71. A protein or pharmaceutical composition for use according to the use described in embodiment 67, wherein the fibrotic disease is myocardial infarction, angina pectoris, osteoarthritis, pulmonary fibrosis, cystic fibrosis, bronchitis or asthma.

[0503] 72. A protein according to any one of embodiments 1 - 52, an immunoconjugate according to embodiment 53 or 54, or a pharmaceutical composition according to embodiment 55, for use as a medicament.

[0504] Table 1. Peptide linker sequences.

[0505]

[0506]

[0507] The underlined linker peptide sequence is of the human IgG1 germline.

[0508] The bold non - underlined peptide sequence (LG) is a mutation of the MMP peptide substrate sequence 'PLGL' (SEQ ID NO: 12) for rapid digestion.

[0509] Table 2. Protein clone numbers, names (ID), and observed expression characteristics.

[0510]

[0511] * Total protein after affinity purification on a Protein A column

[0512] ** Based only on the amino acid sequence

[0513] ND = Not done

[0514] Table 3. Sequences of bispecific proteins that bind to cMet and CD47.

[0515]

[0516]

[0517]

[0518]

[0519]

[0520]

[0521]

[0522]

[0523]

[0524]

[0525]

[0526] Table 4. Sequences of bispecific proteins that bind to Her2 and CD3.

[0527]

[0528]

[0529]

[0530]

[0531]

[0532]

[0533]

[0534] Table 5. Sequences of bispecific proteins that bind to Her2 and CD47.

[0535]

[0536]

[0537]

[0538]

[0539] Table 6. Sequences of bispecific proteins that bind to cMET and CD47.

[0540]

[0541]

[0542] Table 7. Sequences of bispecific proteins that bind to Her2 and CD3.

[0543]

[0544]

[0545] Table 8. Sequences of bispecific proteins that bind to Her2 and CD3.

[0546]

[0547]

[0548] Table 9. Sequences of bispecific proteins that bind to Her2 and CD47.

[0549]

[0550]

[0551]

[0552]

[0553]

[0554]

[0555]

[0556] Table 10. Examples of amino acid sequences of the immunoglobulin Fc region.

[0557] Human IgG4 wild type

[0558]

[0559] Human IgG4 (S228P)

[0560]

[0561] Human IgG1 wild type

[0562]

[0563] Human IgG1-3M

[0564]

[0565] Human IgG2 wild type

[0566]

[0567] Human IgG1 wild type "REEM" allotype

[0568]

[0569] Human IgG1-3M "REEM" allotype

[0570]

[0571] Table 11. Examples of amino acid sequences of the CD47 protein.

[0572] Human CD47 sequence

[0573]

[0574] Cynomolgus monkey CD47 sequence

[0575]

[0576] Table 12. Examples of cMET protein amino acid sequences.

[0577] Human cMET sequence

[0578]

[0579] Cynomolgus monkey cMET sequence

[0580]

[0581] Table 13. Sequences of bispecific proteins that bind to Her2 and CD3.

[0582]

[0583]

[0584]

[0585] Table 14. Sequences of Fab2-based proteins that bind to cMET and cMET.

[0586]

[0587] Table 15. Examples of Her2 protein amino acid sequences. Human Her2 (erbB-2) sequence

[0588]

[0589] Cynomolgus monkey Her2 (erbB2) sequence

[0590]

[0591]

[0592] Table 16. Examples of CD3ε domain amino acid sequences.

[0593] Human CD3ε sequence

[0594]

[0595] Cynomolgus monkey CD3ε sequence

[0596]

[0597] Table 17. Biacore binding values of control anti-Her2 and anti-CD47 antibodies

[0598]

[0599] Table 18. During the MMP 12 activation of Her47-LHL-LHLF protein, Biacore binding to Her2 and CD47. 2 Her47-LHL-LHLF protein's Biacore binding to Her2 and CD47 during MMP 12 activation.

[0600]

[0601] N / A = Not applicable. No binding signal was observed.

[0602] Table 19. Sequences of bispecific proteins that bind to Her2 and CD47.

[0603]

[0604]

[0605]

[0606]

[0607] Table 20. Sequences of bispecific proteins that bind to Her2 and CD47.

[0608]

[0609]

[0610]

[0611]

[0612]

[0613]

[0614]

[0615] Table 21. Sequences of linker peptides containing protease cleavage motifs.

[0616]

[0617] Table 22. Sequences of bispecific proteins that bind to Her2 and CD3 or to EpCAM and CD3.

[0618]

[0619]

[0620]

[0621]

[0622]

[0623]

[0624]

[0625]

[0626]

[0627]

[0628] Table 23. Binding affinities to human and murine Fc receptors determined by Biacore.

[0629]

[0630] ND = Not done

[0631] NB = No binding. Sequence Listing <110> ULTRAHUMAN SIX LIMITED <120> Activable protein constructs and uses thereof <130> ULSL-002 / 04WO 332949-2010 <150> GB 2001196.1 <151> 2020-01-28 <150> GB 1917678.3 <151> 2019-12-04 <150> GB 1910254.0 <151> 2019-07-17 <150> GB 1906685.1 <151> 2019-05-13 <160> 112 <170> PatentIn version 3.5 <210> 1 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Peptide linker <400> 1 Gly Pro Ala Pro Glu Leu Leu 1 5 <210> 2 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Peptide linker <400> 2 Gly Pro Ala Pro Glu Leu Leu Gly Gly Gly Ser 1 5 10 <210> 3 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Peptide linker <400> 3 Gly Pro Ala Pro Leu Gly Leu Gly Gly Gly Ser 1 5 10 <210> 4 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Peptide linker <400> 4 Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Gly Ser 1 5 10 <210> 5 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Peptide linker <400> 5 Pro Pro Cys Pro Ala Pro Leu Gly Leu Gly Gly Gly Ser 1 5 10 <210> 6 <211> 678 <212> PRT <213> Artificial Sequence <220> <223> Fab2 CMET / CD47 heavy chain <400> 6 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Thr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gln Glu Ile Thr Thr Glu Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Gly Gly Gly 210 215 220 Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro 225 230 235 240 Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr 245 250 255 Asn Tyr Tyr Ile Phe Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu 260 265 270 Trp Met Gly Ile Ile Asn Pro Val Asp Gly Asp Thr Asn Tyr Asn Pro 275 280 285 Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr 290 295 300 Ala Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr 305 310 315 320 Tyr Cys Ala Arg Gly Gly Tyr Thr Met Asp Arg Trp Gly Gln Gly Thr 325 330 335 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 340 345 350 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 355 360 365 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 370 375 380 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 385 390 395 400 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 405 410 415 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 420 425 430 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Gly 435 440 445 Gly Gly Gly Ser Gly Gly Gly Gly Ser Cys Pro Pro Cys Pro Ala Pro 450 455 460 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 465 470 475 480 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 485 490 495 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 500 505 510 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 515 520 525 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 530 535 540 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 545 550 555 560 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 565 570 575 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 580 585 590 Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 595 600 605 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 610 615 620 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 625 630 635 640 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 645 650 655 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 660 665 670 Leu Ser Leu Ser Pro Gly 675 <210> 7 <211> 673 <212> PRT <213> Artificial Sequence <220> <223> Fab2 CMET / CD47 light chain <400> 7 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Ala Asn Ser Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro 35 40 45 Arg Leu Leu Ile Tyr Arg Gly Ser Thr Arg Glu Ser Gly Ile Pro Asp 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Lys 85 90 95 Ser Glu Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 100 105 110 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155 160 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200 205 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Gly Gly Gly Gly Ser Asp 210 215 220 Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly Glu 225 230 235 240 Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser Asn 245 250 255 Gly Tyr Asn Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro 260 265 270 Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Leu Ser Gly Val Pro Asp 275 280 285 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser 290 295 300 Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Asn Thr 305 310 315 320 His Thr Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 325 330 335 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 340 345 350 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 355 360 365 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 370 375 380 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 385 390 395 400 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 405 410 415 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 420 425 430 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Gly Gly Gly Gly Ser Gly 435 440 445 Gly Gly Gly Ser Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 450 455 460 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 465 470 475 480 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 485 490 495 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 500 505 510 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 515 520 525 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 530 535 540 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 545 550 555 560 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 565 570 575 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 580 585 590 Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 595 600 605 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 610 615 620 Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp 625 630 635 640 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 645 650 655 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 660 665 670 Gly <210> 8 <211> 683 <212> PRT <213> Artificial Sequence <220> <223> Fab2 CMET / CD47 heavy chain <400> 8 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Thr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gln Glu Ile Thr Thr Glu Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Gly Gly Gly 210 215 220 Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala 225 230 235 240 Glu Val Lys Lys Pro Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser 245 250 255 Gly Tyr Thr Phe Thr Asn Tyr Tyr Ile Phe Trp Val Arg Gln Met Pro 260 265 270 Gly Lys Gly Leu Glu Trp Met Gly Ile Ile Asn Pro Val Asp Gly Asp 275 280 285 Thr Asn Tyr Asn Pro Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp 290 295 300 Lys Ser Ile Ser Thr Ala Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser 305 310 315 320 Asp Thr Ala Met Tyr Tyr Cys Ala Arg Gly Gly Tyr Thr Met Asp Arg 325 330 335 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 340 345 350 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 355 360 365 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 370 375 380 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 385 390 395 400 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 405 410 415 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 420 425 430 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys 435 440 445 Ser Cys Asp Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Cys Pro 450 455 460 Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 465 470 475 480 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 485 490 495 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 500 505 510 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 515 520 525 Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 530 535 540 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 545 550 555 560 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 565 570 575 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 580 585 590 Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly 595 600 605 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 610 615 620 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 625 630 635 640 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 645 650 655 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 660 665 670 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 675 680 <210> 9 <211> 678 <212> PRT <213> Artificial Sequence <220> <223> Fab2 CMET / CD47 light chain <400> 9 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Ala Asn Ser Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro 35 40 45 Arg Leu Leu Ile Tyr Arg Gly Ser Thr Arg Glu Ser Gly Ile Pro Asp 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Lys 85 90 95 Ser Glu Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 100 105 110 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155 160 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200 205 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Gly Gly Gly Gly Ser Gly 210 215 220 Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro 225 230 235 240 Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser 245 250 255 Leu Leu His Ser Asn Gly Tyr Asn Tyr Leu His Trp Tyr Leu Gln Lys 260 265 270 Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Leu 275 280 285 Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 290 295 300 Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr 305 310 315 320 Cys Phe Gln Asn Thr His Thr Pro Arg Thr Phe Gly Gly Gly Thr Lys 325 330 335 Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro 340 345 350 Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu 355 360 365 Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp 370 375 380 Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp 385 390 395 400 Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys 405 410 415 Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln 420 425 430 Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Gly 435 440 445 Gly Gly Gly Ser Gly Gly Gly Gly Ser Cys Pro Pro Cys Pro Ala Pro 450 455 460 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 465 470 475 480 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 485 490 495 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 500 505 510 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 515 520 525 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 530 535 540 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 545 550 555 560 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 565 570 575 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 580 585 590 Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp 595 600 605 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 610 615 620 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser 625 630 635 640 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 645 650 655 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 660 665 670 Leu Ser Leu Ser Pro Gly 675 <210> 10 <211> 688 <212> PRT <213> Artificial Sequence <220> <223> Fab2 CMET / CD47 heavy chain <400> 10 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Thr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gln Glu Ile Thr Thr Glu Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Gly Gly Gly 210 215 220 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu 225 230 235 240 Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu Ser Leu Lys Ile 245 250 255 Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asn Tyr Tyr Ile Phe Trp 260 265 270 Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met Gly Ile Ile Asn 275 280 285 Pro Val Asp Gly Asp Thr Asn Tyr Asn Pro Ser Phe Gln Gly Gln Val 290 295 300 Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr Leu Gln Trp Ser 305 310 315 320 Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys Ala Arg Gly Gly 325 330 335 Tyr Thr Met Asp Arg Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 340 345 350 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 355 360 365 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 370 375 380 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 385 390 395 400 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 405 410 415 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 420 425 430 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 435 440 445 Lys Val Glu Pro Lys Ser Cys Asp Lys Gly Gly Gly Gly Ser Gly Gly 450 455 460 Gly Gly Ser Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 465 470 475 480 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 485 490 495 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 500 505 510 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 515 520 525 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 530 535 540 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 545 550 555 560 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 565 570 575 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 580 585 590 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp 595 600 605 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 610 615 620 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 625 630 635 640 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 645 650 655 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 660 665 670 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 675 680 685 <210> 11 <211> 680 <212> PRT <213> Artificial Sequence <220> <223> Fab2 CMET / CD47 light chain <400> 11 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Ala Asn Ser Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro 35 40 45 Arg Leu Leu Ile Tyr Arg Gly Ser Thr Arg Glu Ser Gly Ile Pro Asp 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Lys 85 90 95 Ser Glu Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 100 105 110 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155 160 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200 205 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Gly Gly Gly Gly Ser Gly 210 215 220 Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser 225 230 235 240 Pro Leu Ser Leu Pro Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys 245 250 255 Arg Ser Ser Gln Ser Leu Leu His Ser Asn Gly Tyr Asn Tyr Leu His 260 265 270 Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Lys 275 280 285 Val Ser Asn Arg Leu Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly 290 295 300 Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp 305 310 315 320 Val Gly Val Tyr Tyr Cys Phe Gln Asn Thr His Thr Pro Arg Thr Phe 325 330 335 Gly Gly Gly Thr Lys Val Arg Thr Val Ala Ala Pro Ser Val Phe Ile 340 345 350 Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val 355 360 365 Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys 370 375 380 Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu 385 390 395 400 Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu 405 410 415 Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr 420 425 430 His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu 435 440 445 Cys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Cys Pro Pro Cys Pro 450 455 460 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 465 470 475 480 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 485 490 495 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 500 505 510 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 515 520 525 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 530 535 540 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 545 550 555 560 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 565 570 575 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu 580 585 590 Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro 595 600 605 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 610 615 620 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 625 630 635 640 Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 645 650 655 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 660 665 670 Lys Ser Leu Ser Leu Ser Pro Gly 675 680 <210> 12 <211> 4 <212> PRT <213> Unknown <220> <223> MMP Peptide Substrate Sequence <400> 12 Pro Leu Gly Leu 1 <210> 13 <211> 675 <212> PRT <213> Artificial Sequence <220> <223> Fab2 CMET / CD47 Light Chain <400> 13 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Ala Asn Ser Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro 35 40 45 Arg Leu Leu Ile Tyr Arg Gly Ser Thr Arg Glu Ser Gly Ile Pro Asp 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Lys 85 90 95 Ser Glu Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 100 105 110 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155 160 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200 205 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Gly Pro Ala Pro Glu Leu 210 215 220 Leu Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro 225 230 235 240 Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His 245 250 255 Ser Asn Gly Tyr Asn Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln 260 265 270 Ser Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Leu Ser Gly Val 275 280 285 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys 290 295 300 Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln 305 310 315 320 Asn Thr His Thr Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile 325 330 335 Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 340 345 350 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn 355 360 365 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 370 375 380 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 385 390 395 400 Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 405 410 415 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 420 425 430 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Gly Gly Gly Gly 435 440 445 Ser Gly Gly Gly Gly Ser Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 450 455 460 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 465 470 475 480 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 485 490 495 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 500 505 510 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 515 520 525 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 530 535 540 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 545 550 555 560 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 565 570 575 Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val 580 585 590 Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 595 600 605 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 610 615 620 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr 625 630 635 640 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 645 650 655 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 660 665 670 Ser Pro Gly 675 <210> 14 <211> 680 <212> PRT <213> Artificial Sequence <220> <223> Fab2 CMET / CD47 heavy chain <400> 14 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Thr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gln Glu Ile Thr Thr Glu Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Gly Pro Ala 210 215 220 Pro Glu Leu Leu Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys 225 230 235 240 Lys Pro Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Thr 245 250 255 Phe Thr Asn Tyr Tyr Ile Phe Trp Val Arg Gln Met Pro Gly Lys Gly 260 265 270 Leu Glu Trp Met Gly Ile Ile Asn Pro Val Asp Gly Asp Thr Asn Tyr 275 280 285 Asn Pro Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile 290 295 300 Ser Thr Ala Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala 305 310 315 320 Met Tyr Tyr Cys Ala Arg Gly Gly Tyr Thr Met Asp Arg Trp Gly Gln 325 330 335 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 340 345 350 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 355 360 365 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 370 375 380 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 385 390 395 400 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 405 410 415 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 420 425 430 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 435 440 445 Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Cys Pro Pro Cys Pro 450 455 460 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 465 470 475 480 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 485 490 495 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 500 505 510 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 515 520 525 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 530 535 540 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 545 550 555 560 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 565 570 575 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu 580 585 590 Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro 595 600 605 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 610 615 620 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 625 630 635 640 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 645 650 655 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 660 665 670 Lys Ser Leu Ser Leu Ser Pro Gly 675 680 <210> 15 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Peptide linker <400> 15 Gly Gly Gly Gly Ser 1 5 <210> 16 <211> 684 <212> PRT <213> Artificial Sequence <220> <223> Fab2 CMET / CD47 heavy chain <400> 16 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Thr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gln Glu Ile Thr Thr Glu Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Gly Pro Ala 210 215 220 Pro Glu Leu Leu Gly Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly 225 230 235 240 Ala Glu Val Lys Lys Pro Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly 245 250 255 Ser Gly Tyr Thr Phe Thr Asn Tyr Tyr Ile Phe Trp Val Arg Gln Met 260 265 270 Pro Gly Lys Gly Leu Glu Trp Met Gly Ile Ile Asn Pro Val Asp Gly 275 280 285 Asp Thr Asn Tyr Asn Pro Ser Phe Gln Gly Gln Val Thr Ile Ser Ala 290 295 300 Asp Lys Ser Ile Ser Thr Ala Tyr Leu Gln Trp Ser Ser Leu Lys Ala 305 310 315 320 Ser Asp Thr Ala Met Tyr Tyr Cys Ala Arg Gly Gly Tyr Thr Met Asp 325 330 335 Arg Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys 340 345 350 Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly 355 360 365 Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 370 375 380 Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 385 390 395 400 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 405 410 415 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn 420 425 430 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro 435 440 445 Lys Ser Cys Asp Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Cys 450 455 460 Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu 465 470 475 480 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 485 490 495 Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys 500 505 510 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 515 520 525 Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 530 535 540 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 545 550 555 560 Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys 565 570 575 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 580 585 590 Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys 595 600 605 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 610 615 620 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 625 630 635 640 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 645 650 655 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 660 665 670 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 675 680 <210> 17 <211> 679 <212> PRT <213> Artificial Sequence <220> <223> Fab2 CMET / CD47 light chain <400> 17 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Ala Asn Ser Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro 35 40 45 Arg Leu Leu Ile Tyr Arg Gly Ser Thr Arg Glu Ser Gly Ile Pro Asp 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Lys 85 90 95 Ser Glu Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 100 105 110 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155 160 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200 205 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Gly Pro Ala Pro Glu Leu 210 215 220 Leu Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu 225 230 235 240 Pro Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln 245 250 255 Ser Leu Leu His Ser Asn Gly Tyr Asn Tyr Leu His Trp Tyr Leu Gln 260 265 270 Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg 275 280 285 Leu Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp 290 295 300 Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr 305 310 315 320 Tyr Cys Phe Gln Asn Thr His Thr Pro Arg Thr Phe Gly Gly Gly Thr 325 330 335 Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe 340 345 350 Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys 355 360 365 Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val 370 375 380 Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln 385 390 395 400 Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser 405 410 415 Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His 420 425 430 Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 435 440 445 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Cys Pro Pro Cys Pro Ala 450 455 460 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 465 470 475 480 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 485 490 495 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 500 505 510 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 515 520 525 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 530 535 540 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 545 550 555 560 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 565 570 575 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr 580 585 590 Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser 595 600 605 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 610 615 620 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val 625 630 635 640 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 645 650 655 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 660 665 670 Ser Leu Ser Leu Ser Pro Gly 675 <210> 18 <211> 452 <212> PRT <213> Artificial Sequence <220> <223> Fab2 CMET / CD47 heavy chain <400> 18 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Tyr Ile Phe Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Ile Ile Asn Pro Val Asp Gly Asp Thr Asn Tyr Asn Pro Ser Phe 50 55 60 Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Tyr Thr Met Asp Arg Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Gly Gly Gly 210 215 220 Gly Ser Gly Gly Gly Gly Ser Cys Pro Pro Cys Pro Ala Pro Glu Leu 225 230 235 240 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 245 250 255 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 260 265 270 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 275 280 285 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 290 295 300 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 305 310 315 320 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 325 330 335 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 340 345 350 Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln 355 360 365 Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 370 375 380 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 385 390 395 400 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 405 410 415 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 420 425 430 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 435 440 445 Leu Ser Pro Gly 450 <210> 19 <211> 450 <212> PRT <213> Artificial Sequence <220> <223> Fab2 CMET / CD47 light chain <400> 19 Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser 20 25 30 Asn Gly Tyr Asn Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Leu Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Asn 85 90 95 Thr His Thr Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 145 150 155 160 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 190 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 195 200 205 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Gly Gly Gly Gly Ser 210 215 220 Gly Gly Gly Gly Ser Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360 365 Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Pro Gly 450 <210> 20 <211> 688 <212> PRT <213> Artificial Sequence <220> <223> Fab2 Her2 / CD3 heavy chain <400> 20 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Gly 210 215 220 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Gln Ser 225 230 235 240 Gly Ala Glu Leu Ala Arg Pro Gly Ala Ser Val Lys Met Ser Cys Lys 245 250 255 Ala Ser Gly Tyr Thr Phe Thr Arg Tyr Thr Met His Trp Val Lys Gln 260 265 270 Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser Arg 275 280 285 Gly Tyr Thr Asn Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr 290 295 300 Thr Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr 305 310 315 320 Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp His 325 330 335 Tyr Cys Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser 340 345 350 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 355 360 365 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 370 375 380 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 385 390 395 400 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 405 410 415 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 420 425 430 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 435 440 445 Lys Val Glu Pro Lys Ser Cys Asp Lys Gly Gly Gly Gly Ser Gly Gly 450 455 460 Gly Gly Ser Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro 465 470 475 480 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 485 490 495 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 500 505 510 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 515 520 525 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 530 535 540 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 545 550 555 560 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 565 570 575 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 580 585 590 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp 595 600 605 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 610 615 620 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 625 630 635 640 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 645 650 655 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 660 665 670 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 675 680 685 <210> 21 <211> 668 <212> PRT <213> Artificial Sequence <220> <223> Fab2 Her2 / CD3 light chain <400> 21 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 210 215 220 Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 225 230 235 240 Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met 245 250 255 Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg T...

Claims

1. A protein comprising a first part and a second part, and a first peptide linker and a second peptide linker between the first part and the second part, wherein the first part comprises an antibody, an antigen-binding part of an antibody, or an extracellular domain of a receptor, and wherein the first part is capable of specifically binding to a first molecule expressed in diseased tissue; wherein each of the first peptide linker and the second peptide linker comprises an amino acid sequence from the human immunoglobulin hinge region or an amino acid sequence having 1 to 7 amino acid substitutions compared to the human immunoglobulin hinge region; wherein the first peptide linker and the second peptide linker are cleavable by a protease expressed in diseased tissue; wherein the second part comprises an antibody, an antigen-binding part of an antibody, or an extracellular domain of a receptor, and wherein the second part is capable of specifically binding to a second molecule expressed in the diseased tissue; wherein the first molecule and the second molecule are each independently selected from: (A) Her2 and CD47; (B) Her2 and CD3; or (C) cMET and CD47; wherein: (a) when the first molecule is human Her2 and the second molecule is human CD47, then (i) the first peptide linker consists of SEQ ID NO:2 and the second peptide linker consists of SEQ ID NO:3; (ii) the first peptide linker consists of SEQ ID NO:3 and the second peptide linker consists of SEQ ID NO:3; (iii) the first peptide linker consists of SEQ ID NO:4 and the second peptide linker consists of SEQ ID NO:4; (iv) the first peptide linker consists of SEQ ID NO:4 and the second peptide linker consists of SEQ ID NO:5; or (v) the first peptide linker consists of SEQ ID NO:5 and the second peptide linker consists of SEQ ID NO:5; (b) when the first molecule is human Her2 and the second molecule is human CD3, then (i) the first peptide linker consists of SEQ ID NO:2 and the second peptide linker consists of SEQ ID NO:2; or (ii) the first peptide linker consists of SEQ ID NO:2 and the second peptide linker consists of SEQ ID NO:3; or (c) when the first molecule is human cMET and the second molecule is human CD47, then the first peptide linker consists of SEQ ID NO:2 and the second peptide linker consists of SEQ ID NO:2; and wherein when the first peptide linker and the second peptide linker are not cleaved, the binding of the second part to the second molecule expressed in the diseased tissue is reduced or inhibited.

2. The protein according to claim 1, wherein the protease is human matrix metalloproteinase (MMP), human cathepsin, human enterokinase, human thrombin, human tPA, human granzyme B, human uPA, or human ADAMTs-5.

3. The protein according to claim 1, wherein the first peptide linker or the second peptide linker comprises a human MMP cleavage site, human cathepsin, human enterokinase, human thrombin, human tPA, human granzyme B, human uPA or human ADAMTs-5 cleavage site.

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

5. The protein according to claim 3, wherein the level or activity of the human MMP in the diseased tissue is elevated compared to the level or activity of the human MMP in the non-diseased tissue.

6. The protein according to claim 3, wherein the human cathepsin is cathepsin A, cathepsin C, cathepsin D, cathepsin G, cathepsin L or cathepsin K.

7. The protein according to claim 3, wherein the level or activity of the human cathepsin in the diseased tissue is elevated compared to the level or activity of the human cathepsin in the non-diseased tissue.

8. The protein according to claim 1, wherein the first portion is a Fab, single-chain Fab, immunoglobulin new antigen receptor (IgNAR), single-chain variable fragment (scFv), diabody or T cell receptor domain.

9. The protein according to claim 1, wherein the first molecule and the second molecule are expressed by the same cell.

10. The protein according to claim 1, wherein the first molecule and the second molecule are expressed by different cells.

11. The protein according to claim 1, wherein the first molecule and / or the second molecule is expressed on the cell surface.

12. The protein according to claim 1, wherein the first molecule and / or the second molecule is a soluble molecule.

13. The protein according to claim 1, wherein the first portion comprises a heavy chain variable (VH) region and a light chain variable (VL) region.

14. The protein according to claim 1, wherein the first portion comprises an immunoglobulin constant region or a portion of an immunoglobulin constant region.

15. The protein according to claim 14, wherein the immunoglobulin constant region is IgG, IgE, IgM, IgD, IgA or IgY.

16. The protein according to claim 14, wherein the immunoglobulin constant region is IgG1, IgG2, IgG3, IgG4, IgA1 or IgA2.

17. The protein according to claim 14, wherein the immunoglobulin constant region is immunologically inert.

18. The protein according to claim 14, 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.

19. The protein according to claim 1, wherein the second part is a Fab, a single-chain Fab, an immunoglobulin new antigen receptor (IgNAR), a single-chain variable fragment (scFv), or a T cell receptor domain.

20. The protein according to claim 1, wherein the second part comprises a heavy chain variable (VH) region and a light chain variable (VL) region.

21. The protein according to claim 1, wherein the second part comprises an immunoglobulin constant region or a portion of an immunoglobulin constant region.

22. The protein according to claim 21, wherein the immunoglobulin constant region is IgG, IgE, IgM, IgD, IgA, or IgY.

23. The protein according to claim 21, wherein the immunoglobulin constant region is IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2.

24. The protein according to claim 21, wherein the immunoglobulin constant region is immunologically inert.

25. The protein according to claim 21, 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.

26. The protein according to claim 1, wherein the protein has one immune effector function or two, three, or more immune effector functions.

27. The protein according to claim 26, wherein the immune effector function is ADCC, CDC, or ADCP.

28. The protein according to claim 1, wherein the first part prevents or reduces the specific binding of the second part to the second molecule expressed in the diseased tissue.

29. The protein according to claim 1, wherein the first peptide linker or the second peptide linker is cleaved near or within the diseased tissue.

30. The protein according to claim 1, wherein the first peptide linker or the second peptide linker is cleaved near or within the diseased tissue, wherein the first part dissociates from the second part near or within the diseased tissue, and wherein the second part specifically binds to the second molecule expressed in the diseased tissue near or within the diseased tissue.

31. The protein according to claim 1, wherein the diseased tissue is a tumor or an inflamed tissue.

32. The protein according to claim 1, wherein the first part specifically binds to human cMET, wherein the second part specifically binds to human CD47, and wherein the protein comprises a first polypeptide chain having the amino acid sequence of SEQ ID NO:16 and a second polypeptide chain having the amino acid sequence of SEQ ID NO:

17.

33. The protein according to claim 1, wherein the first part specifically binds to human HER2, wherein the second part specifically binds to human CD3, and wherein the protein comprises a first polypeptide chain having the amino acid sequence of SEQ ID NO:26 and a second polypeptide chain having the amino acid sequence of SEQ ID NO:

27.

34. The protein according to claim 1, wherein the first part specifically binds to human Her2, wherein the second part specifically binds to human CD3, and wherein the protein comprises a first polypeptide chain having the amino acid sequence of SEQ ID NO:40 and a second polypeptide chain having the amino acid sequence of SEQ ID NO:

41.

35. The protein according to claim 1, wherein the first part specifically binds to human Her2, wherein the second part specifically binds to human CD47, and wherein the protein comprises: (a) a first polypeptide chain having the amino acid sequence of SEQ ID NO:42 and a second polypeptide chain having the amino acid sequence of SEQ ID NO:43; or (b) a first polypeptide chain having the amino acid sequence of SEQ ID NO:44 and a second polypeptide chain having the amino acid sequence of SEQ ID NO:45; or (c) a first polypeptide chain having the amino acid sequence of SEQ ID NO:46 and a second polypeptide chain having the amino acid sequence of SEQ ID NO:47; or (d) the first polypeptide chain having the amino acid sequence of SEQ ID NO:48 and a second polypeptide chain having the amino acid sequence of SEQ ID NO:49; or (e) a first polypeptide chain having the amino acid sequence of SEQ ID NO:50 and a second polypeptide chain having the amino acid sequence of SEQ ID NO:51; or (f) a first polypeptide chain having the amino acid sequence of SEQ ID NO:52 and a second polypeptide chain having the amino acid sequence of SEQ ID NO:53; or (g) a first polypeptide chain having the amino acid sequence of SEQ ID NO:54 and a second polypeptide chain having the amino acid sequence of SEQ ID NO:55; or (h) a first polypeptide chain having the amino acid sequence of SEQ ID NO:88 and a second polypeptide chain having the amino acid sequence of SEQ ID NO:89; or (i) a first polypeptide chain having the amino acid sequence of SEQ ID NO:90 and a second polypeptide chain having the amino acid sequence of SEQ ID NO:

91.

36. The protein according to claim 1, wherein the first part specifically binds to human Her2, the second part specifically binds to human CD3, and the protein comprises: (a) a first polypeptide chain having the amino acid sequence of SEQ ID NO:98 and a second polypeptide chain having the amino acid sequence of SEQ ID NO:99; or (b) a first polypeptide chain having the amino acid sequence of SEQ ID NO:100 and a second polypeptide chain having the amino acid sequence of SEQ ID NO:101; or (c) a first polypeptide chain having the amino acid sequence of SEQ ID NO:102 and a second polypeptide chain having the amino acid sequence of SEQ ID NO:103; or (d) a first polypeptide chain having the amino acid sequence of SEQ ID NO:104 and a second polypeptide chain having the amino acid sequence of SEQ ID NO:

105.

37. A pharmaceutical composition comprising the protein according to any one of claims 1-36, and a pharmaceutically acceptable carrier, diluent or excipient.

38. A nucleic acid molecule encoding the protein according to any one of claims 1-36.

39. An expression vector comprising the nucleic acid molecule according to claim 38.

40. A recombinant host cell comprising the nucleic acid molecule according to claim 38 or the expression vector according to claim 39.

41. A method for producing a protein, the method comprising: culturing a recombinant host cell comprising the expression vector according to claim 39 under conditions under which the nucleic acid molecule is expressed, thereby producing the protein; and isolating the protein from the host cell or the culture.

42. Use of the protein according to claim 35 in the preparation of a medicament for the treatment of cancer, wherein the cancer is breast cancer or esophageal cancer.

Images