Activatable protein constructs and uses thereof

Abstract

Described herein are activatable protein constructs and uses thereof. In particular, described herein are protein molecules that exhibit activatable target binding in diseased tissues, as well as related nucleic acid molecules, vectors, and host cells. Also described herein are medical uses of such protein molecules.

Description

[0001] This application is a divisional application of the Chinese patent application with an application date of May 13, 2020, application number 202080034818.9, and invention name “Activatable protein constructs and their uses” (the corresponding PCT application with an application date of May 13, 2020 and application number PCT / EP2020 / 063362).

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] 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 disclosures of each of which are hereby incorporated by reference in their entirety.

[0004] Description of the text file submitted electronically

[0005] The contents of the text file submitted electronically with this article are incorporated herein by reference in their entirety: Computer readable copy of the Sequence Listing (file name: ULSL_002_04WO_SeqList_ST25.txt, record date: May 11, 2020, file size approximately 390 kb). Technical Field

[0006] The present invention relates to protein molecules that exhibit activatable target binding in diseased tissues and their medical uses. Background Art

[0007] In the use of antibodies and other binding proteins to treat diseases, many potential drug targets have been described, but rarely expressed only in diseased tissues. In fact, most of the potential targets in this space are also expressed in non-diseased tissues. In addition, most of the drug mechanisms of action adopted in challenging therapies (such as cancer) fields adopt highly effective cell killing mechanisms of action. As a result, the engagement of drugs to targets in non-diseased tissues often causes undesirable side effects. It is necessary to have an engineered form of binding protein that is partially or even completely inactive in healthy tissues but becomes highly activated in diseased tissues. Summary of the Invention

[0008] Provided herein are proteins comprising a first portion and a second portion and a peptide linker between the first portion and the second portion, wherein the peptide linker comprises an amino acid sequence from a human immunoglobulin hinge region or an amino acid sequence or an amino acid sequence having 1 to about 7 amino acid substitutions compared to a human immunoglobulin hinge region; wherein the peptide linker can be cleaved by a protease expressed in a diseased tissue; wherein the second portion is capable of specifically binding to a molecule expressed in the diseased tissue; and wherein when the peptide linker is not cleaved, the binding of the second portion to the molecule expressed in the diseased tissue is reduced or inhibited. In some embodiments, the length of the peptide linker is between about 5 and about 15 amino acids. In some embodiments, the peptide linker comprises or consists of the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, or SEQ ID NO: 87.

[0009] In some embodiments, the protease is a human matrix metalloproteinase (MMP), a human cathepsin, a human enterokinase, a human thrombin, a human tPA, a human granzyme B, a human uPA, or a human ADAMTs-5. In some embodiments, the peptide linker comprises a human MMP cleavage site, a human cathepsin, 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 the human MMP is increased in diseased tissue compared to the level or activity of the 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 the human cathepsin is increased in diseased tissue compared to the level or activity of the human cathepsin in non-diseased tissue.

[0010] In some embodiments, the first portion comprises an antibody, an antigen-binding portion of an antibody, or a receptor extracellular domain. In some embodiments, the first 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), a diabody, or a T-cell receptor domain. In some embodiments, the first portion specifically binds to a molecule expressed in a diseased tissue.

[0011] In some embodiments, the first portion specifically binds to a first molecule expressed in the diseased tissue, and the second portion is capable of specifically binding to a second molecule expressed in the diseased tissue, wherein the first molecule expressed in the diseased tissue and the second molecule expressed in the diseased tissue are different molecules. In some embodiments, the first molecule expressed in the diseased tissue and the second molecule expressed in the diseased tissue are expressed by the same cell. In some embodiments, the first molecule expressed in the diseased tissue and the second molecule expressed in the diseased tissue are expressed by different cells. In some embodiments, the first molecule expressed in the diseased tissue and / or the second molecule expressed in the diseased tissue are expressed on the cell surface. In some embodiments, the first molecule expressed in the diseased tissue and / or the second molecule expressed in the diseased tissue are soluble molecules.

[0012] In some embodiments, the first portion specifically binds 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.

[0013] In some embodiments, the second portion 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.

[0014] In some embodiments, the first portion comprises a heavy chain variable (VH) region and a light chain variable (VL) region. In some embodiments, the first portion comprises an immunoglobulin constant region or a portion of an immunoglobulin constant region. In some embodiments, the immunoglobulin constant region is IgG, IgE, IgM, IgD, IgA, or IgY. In some embodiments, the immunoglobulin constant region is IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2. In some embodiments, the immunoglobulin constant region is immunologically inert. In some embodiments, the immunoglobulin constant region is a wild-type human IgG4 constant region, a human IgG4 constant region comprising the amino acid substitution S228P, a wild-type human IgG1 constant region, a human IgG1 constant region comprising the amino acid substitutions L234A and L235A, a human IgG1 constant region comprising the amino acid substitutions L234A, L235A, and G237A, a human IgG1 constant region comprising the amino acid substitutions L234A, L235A, G237A, and P331S, or a wild-type human IgG2 constant region.

[0015] In some embodiments, the second portion comprises an antibody, an antigen-binding portion of an antibody, or a receptor extracellular domain. 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.

[0016] In some embodiments, the second portion comprises a heavy chain variable (VH) region and a light chain variable (VL) region. In some embodiments, the second portion comprises an immunoglobulin constant region or a portion of an immunoglobulin constant region. In some embodiments, the immunoglobulin constant region is IgG, IgE, IgM, IgD, IgA, or IgY. In some embodiments, the immunoglobulin constant region is IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2. In some embodiments, the immunoglobulin constant region is immunologically inert. In some embodiments, the immunoglobulin constant region is a wild-type human IgG4 constant region, a human IgG4 constant region comprising the amino acid substitution S228P, a wild-type human IgG1 constant region, a human IgG1 constant region comprising the amino acid substitutions L234A and L235A, a human IgG1 constant region comprising the amino acid substitutions L234A, L235A, and G237A, a human IgG1 constant region comprising the amino acid substitutions L234A, L235A, G237A, and P331S, or a wild-type human IgG2 constant region.

[0017] 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.

[0018] In some embodiments, the first portion prevents or reduces specific binding of the second portion to a molecule expressed in a 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 portion dissociates from the second portion near or within the diseased tissue, and wherein the second portion 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.

[0019] In some embodiments, the first portion specifically binds human cMET, wherein the second portion specifically binds human CD47, and wherein the protein comprises a first polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO: 16 and a second polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO: 17.

[0020] In some embodiments, the first portion specifically binds human HER2, wherein the second portion specifically binds human CD3, and wherein the protein comprises a first polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO: 26 and a second polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO: 27.

[0021] In some embodiments, the first portion specifically binds human HER2, wherein the second portion specifically binds human CD47, and wherein the protein comprises a first polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO: 34 and a second polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO: 35.

[0022] In some embodiments, the first portion specifically binds human cMET, wherein the second portion specifically binds human CD47, and wherein the protein comprises a first polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO: 36 and a second polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO: 37.

[0023] In some embodiments, the first portion specifically binds to human Her2, wherein the second portion specifically binds to human CD3, and wherein the protein comprises a first polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO: 38 and a second polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO: 39.

[0024] In some embodiments, the first portion specifically binds to human Her2, wherein the second portion specifically binds to human CD3, and wherein the protein comprises a first polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO: 40 and a second polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO: 41.

[0025] In some embodiments, the first portion specifically binds human Her2, wherein the second portion specifically binds human CD47, and wherein the protein comprises a first polypeptide chain and a second polypeptide chain, wherein:

[0026] (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

[0027] (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

[0028] (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

[0029] (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

[0030] (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

[0031] (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

[0032] (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

[0033] (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

[0034] (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

[0035] (j) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 44, and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 92; or

[0036] (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

[0037] (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

[0038] (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

[0039] (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

[0040] (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;

[0041] In some embodiments, the first portion specifically binds to human Her2, wherein the second portion specifically binds to human CD3, and wherein the protein comprises a first polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO: 73 and a second polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO: 74.

[0042] In some embodiments, the first portion specifically binds human cMET, wherein the second portion specifically binds human cMET, and wherein the protein comprises a first polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO: 75 and a second polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO: 76.

[0043] In some embodiments, the first portion specifically binds human Her2, wherein the second portion specifically binds human CD3, and wherein the protein comprises a first polypeptide chain and a second polypeptide chain, wherein:

[0044] (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

[0045] (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

[0046] (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

[0047] (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;

[0048] Also provided herein are immunoconjugates comprising a protein of the invention linked to a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxin, a radioisotope, a chemotherapeutic agent, an immunomodulatory agent, an anti-angiogenic agent, an anti-proliferative agent, a pro-apoptotic agent, a cytostatic enzyme, a lytic enzyme, a therapeutic nucleic acid, an anti-angiogenic agent, an anti-proliferative agent, or a pro-apoptotic agent.

[0049] Also provided herein are pharmaceutical compositions comprising a protein of the invention or an immunoconjugate of the invention and a pharmaceutically acceptable carrier, diluent, or excipient.

[0050] Also provided herein are nucleic acid molecules encoding a protein of the invention 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.

[0051] Also provided herein are expression vectors comprising the nucleic acid molecules of the invention.

[0052] Also provided herein are recombinant host cells comprising a nucleic acid molecule of the invention or an expression vector of the invention.

[0053] Also provided herein is a method for producing a protein, comprising culturing a recombinant host cell comprising the expression vector of the present invention under conditions whereby the nucleic acid molecule is expressed, thereby producing the protein; and isolating the protein from the host cell or culture.

[0054] Also provided herein are methods for enhancing an anti-cancer immune response in a subject, comprising administering to the subject a therapeutically effective amount of a protein of the invention, an immunoconjugate of the invention, or a pharmaceutical composition of the invention.

[0055] Also provided herein is a method for treating cancer, autoimmune disease, inflammatory disease, cardiovascular disease or fibrotic disease in a subject, comprising administering a therapeutically effective amount of a protein of the present invention, an immunoconjugate of the present invention or a pharmaceutical composition of the present invention to the subject. 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 appendix cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma or cancer of 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 pectoris, osteoarthritis, pulmonary fibrosis, cystic fibrosis, bronchitis or asthma.

[0056] Also provided herein is a protein of the present invention, an immunoconjugate of the present invention, or a pharmaceutical composition of the present invention for the treatment of cancer, autoimmune disease, inflammatory disease, cardiovascular disease, or fibrotic disease. In some embodiments, the cancer is gastrointestinal stromal cancer (GIST), pancreatic cancer, skin cancer, melanoma, breast cancer, lung cancer, bronchial cancer, colorectal cancer, prostate cancer, gastric cancer, ovarian cancer, bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, oral or pharyngeal cancer, liver cancer, kidney cancer, testicular cancer, bile duct cancer, small intestine or appendix cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma, or cancer of 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, cardiovascular disease is coronary heart disease, atherosclerosis, or stroke. In some embodiments, the fibrotic disease is myocardial infarction, angina, osteoarthritis, pulmonary fibrosis, cystic fibrosis, bronchitis, or asthma.

[0057] Also provided herein is a protein of the invention, an immunoconjugate of the invention, or a pharmaceutical composition of the invention for use as a medicament. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1A-1B Challenges in delivering systemically active antibody drugs to solid tumors – a case study of anti-CD47. Figure 1A ) presents significant challenges, such as the high expression of CD47 in the bloodstream. Red blood cells and platelets in particular form a "sink" and toxicity risk issues. Tumors are also often a "hostile" environment with high expression of enzymes such as MMPs that accelerate IgG degradation. The anti-CD47 protein constructs of the present invention ( Figure 1B ) is designed to eliminate CD47 binding in the native protein, which eliminates peripheral activity. The tumor targeting domain then 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 in the periphery.

[0059] Figure 2A-2B Protein construct IgG 2 Design and activation principles. Protein construct IgG 2 design( Figure 2A) 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, the four polypeptide chains encode four Fab domains (2x Fab A, 2X Fab B), two linker sequences, and may or may not have an immunoglobulin hinge region and 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) creates 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 to produce an intermediate unlocked active state that allows Fab A and B from a single protein construct to bind to their cognate targets. 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, resulting in a dissociated form. The cleaved linker based on the immunoglobulin hinge sequence can also recruit increased immune effector functions on the cell membrane through endogenous anti-hinge antibodies. The variable region is represented in white. The constant region is represented in gray.

[0060] Figure 3A-Figure 3B .Protein construct Fab 2 Design and activation principles. 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. Figure 3A ) or three ( Figure 3B ) polypeptide chain can encode two Fab domains (1x FabA, 1xFabB), two or more linker sequences, and may or may not have an immunoglobulin hinge region and an Fc domain, wherein the pairing of heterodimers 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) creates 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 cleaved sequentially, thereby generating an intermediate unlocked active state that allows Fab A and B from a single protein to bind to their cognate targets. A secondary, potentially slower, cleavage of the second linker in each Fab A-Fab B protein unit can completely 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 through endogenous anti-hinge antibodies. The variable region is shown in white. The constant region is shown in gray.

[0061] Figure 4 Protein A purified protein construct IgG from clone 1-15 2 and Fab 2 SDS-PAGE analysis of proteins. Exemplary proteins of various constructs were expressed in CHO cells and purified using Protein A affinity chromatography. The purified proteins were then analyzed by SDS-PAGE in both the unreduced and reduced (r) states, along with molecular weight standards (M). Clones 6, 10, and 14 (all containing an LHL linker) were found to contain the highest proportion of product of the expected size and the lowest content of both higher and lower molecular weights.

[0062] Figures 5A-5I Protein A purified protein construct IgG 2 and Fab 2 Size Exclusion Chromatography of Proteins. Selected constructs were analyzed and fully purified using SEC. Clone 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 ). The data show that the highest proportion of product of the expected size (e.g., the highlighted peak, Figure 5I ) and lowest higher / lower molecular weight content.

[0063] Figure 6A-Figure 6B SEC-purified protein construct IgG 2 and Fab 2 The protein was analyzed by SDS-PAGE. Finally, SEC was used to purify the key lead protein construct clones purified by Protein A affinity. The purified proteins from clones 1, 2, 4, 5, and 6 and the non-SEC purified 15 ( Figure 6A) were analyzed by SDS-PAGE under non-reducing conditions. Figure 6B Purified proteins from the clones were also analyzed by SDS-PAGE in both unreduced and reduced (r) conditions. All proteins were loaded at approximately 1 μg / lane. Clones 6, 10, and 14 (all containing an LHL linker) were found to contain the highest proportion of products of the expected size and the lowest content of higher and lower molecular weights.

[0064] Figures 7A-7C Direct titration ELISA of purified intact protein constructs and control antibodies binding to human target proteins. Control antibodies A-D5 anti-CD47, A-D5Fab-Fc (monovalent form of A-D5 antibody), MH7.1 anti-C-MET, and anti-Her2 trastuzumab (all in human IgG1 form) were titrated (in μg / ml) in direct binding ELISAs against human CD47, C-MET, and Her2 proteins ( Figure 7A IgG was also analyzed in the same manner. 2 The forms of Her2CD47-LH-LH and Her2CD47-LHL-LHL ( Figure 7B ) and Fab 2 The forms of cMETCD47-L2-L2 and cMETCD47-LHL-LHL ( Figure 7C ).

[0065] Figures 8A-8C Human erythrocyte hemagglutination assays were performed using purified intact protein constructs and control antibodies. Control antibodies included anti-CD235a (mouse) and A-D5 anti-CD47, A-D5Fab-Fc (monovalent form of the A-D5 antibody, labeled 'FabCD47 only'), MH7.1 anti-C-MET, anti-Her2 trastuzumab, and IgG. 2 The Her2CD47-LH-LH and Her2CD47-LHL-LHL formats and the Fab 2 cMETCD47-L2-L2 and cMETCD47-LHL-LHL were used in human erythrocyte hemagglutination assays using cells from donor 1 ( Figure 8A ), donor 2 ( Figure 8B ) and donor 3 ( Figure 8C ) of fresh red blood cells (in nM).

[0066] Figures 9A-9CDirect 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 over a time course of 2, 4, 8, and 24 hours of incubation, plus a 24-hour incubation in enzyme-free buffer as a negative control. Samples from these digestion time courses were then applied to ELISAs targeting human Her2 and CD47 ( Figure 9A 、 9B ) or human C-MET and human CD47 ( Figure 9C ) direct binding ELISA.

[0067] Figures 10A-10C Purified intact and MMP-digested Her2CD3 Fab 2 Functional analysis of protein construct binding to human target protein. Figure 10A ), flow cytometry with or without MMP digestion ( Figure 10B ) and CD3 reporter assay ( Figure 10C ) analysis showed Fab 2 The antibodies are in the form of Her2CD3-L1-LH, Her2CD3-L2-L2 and Her2CD3-LHL-LHL.

[0068] Figure 11 . Alternative structures based on protein construct design and activation principles. In Figures 2 and 3, the Fab 2 and IgG 2 The protein construct modules (1) found in both designs can be modified and the functional characteristics of the final molecule altered. In this case, the upper binding unit or the lower unit or both of the protein construct modules can be alternative structures of immunoglobulin Fab domains, thereby allowing alternative molecules based on sequences derived from peptides, receptor extracellular domains, binding domains and especially other dimerizing immune recognition receptors such as T cell receptors. These constructs can be formed in many forms and examples are provided here, such as: four polypeptide chains (2) can encode an IgG comprising two complete protein construct modules with four binding domain units (1x A, 1x B, or 2xA or B), two or more linker sequences 2 Like structure, and may or may not have an immunoglobulin hinge region and an Fc domain, wherein the pairing of heterodimers may or may not be driven by mutations in the Fc. In the expression of three polypeptides, the Fab may be enhanced by adding binding domains (3) or peptides that make the structure potentially trispecific or with altered valency. 2 Design. In Fab 2The design can also achieve trispecificity or altered valency 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 of the structures outlined in this figure or in Figures 2 and 3 can be further functionalized by adding C-terminal or N-terminal fusions of any type of polypeptide chain or by chemical conjugation. The variable regions are shown in white. The constant regions are shown in gray.

[0069] Figure 12 Fab-based 2 A 'passive' structure of 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, where heterodimer pairing may or may not be driven by mutations in the Fc. In this construct, binding of both the Fab or receptor domain to their cognate targets should only become fully active after cleavage of at least one linker. It should also be noted that any of the structures outlined in this figure can be further functionalized by adding C-terminal or N-terminal fusions of any type of polypeptide chain or by chemical conjugation. The variable regions are shown in white. The constant regions are shown in gray.

[0070] Figure 13 Fab-based 2 " activatable " antibody drug conjugate (ADC) strategy of protein construct principle.In this case, the upper and lower binding units of the protein construct module can contain antibodies for identical internalization receptor targets or antibodies for two different targets found on the same cell surface.These constructs can be chemically puted together or merged to form ADC with "payload" part such as toxin or other active molecules, and may or may not have immunoglobulin hinge region and Fc domains, wherein the pairing of heterodimer may or may not be driven by the sudden change in Fc.In this construct, the combination of upper Fab or receptor domain and its cognate target is constitutive activated, causes antibody to accumulate in the tissue where its cognate target is expressed.Described construct does not drive internalization to enter target cell initially, because in conjunction with is monovalent, and known receptor is only significantly internalized when 2 or more receptor domains are combined and cross-linked by bivalent antibodies. The activity of the second (lower) Fab or receptor domain should only be active after the linker is cleaved by a disease-associated enzyme, which then drives multivalent receptor binding and internalization of the ADC, allowing delivery of the (e.g., cytotoxic or inflammatory) payload moiety. It should also be noted that any of the structures outlined in this figure can be further functionalized by adding c-terminal or n-terminal fusions of any type of polypeptide chain.

[0071] Figure 14Direct ELISA of purified intact protein construct binding to human and murine target proteins. Samples were applied in direct binding ELISAs against human Her2 and human and murine CD47.

[0072] Figures 15A-15F Direct ELISA of purified intact and MMP-digested protein constructs binding to human target proteins. Human MMP7 ( Figure 15A )、MMP8( Figure 15B )、MMP10( Figure 15C )、MMP12( Figure 15D )、MMP13( Figure 15E ) and cathepsin S ( Figure 15F ) The protein constructs were enzymatically digested over a time course of 2, 4, 8, and 24 hours of incubation, plus a 24-hour incubation in buffer without enzyme as a negative control (time 0). Samples from these digestion time courses were then applied to direct binding ELISAs against human Her2 and CD47.

[0073] Figures 16A-16B Biacore SPR assay of purified intact and MMP-digested Her47-LHL-LHLF binding to human target proteins. Her47-LHL-LHLF was enzymatically digested using human MMP 12 over a time course of 2, 4, 8, and 24 hours of incubation, plus a 24-hour incubation in enzyme-free buffer 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 16A ) or human CD47 ( Figure 16B ) flowed into solution. Rmax values ​​were plotted to indicate the maximum binding observed at the highest concentration of analyte protein.

[0074] Figure 17 Biacore SPR assay of purified intact and 24-hour MMP-digested Her47-LHL-LHLF binding to human target proteins. Her47-LHL-LHLF was enzymatically digested with human MMP 12 for 24 hours or without enzyme as a negative control ('before protease treatment'). The samples were then captured on an anti-Fc antibody-coated Biacore chip, and human CD47 was flowed into the solution at various concentrations. The binding curves showed that even at 400 nM huCD47, the undigested (intact) Her47-LHL-LHLF protein did not interact with huCD47, while strong binding of the same protein was evident after MMP 12 activation at all tested concentrations.

[0075] Figures 18A-18B .Intact Her47-LHL-LHL IgG2 Structural modeling of structures. Figure 18A , IgG 2 Molecular modeling of the complete structure of the molecule showing the upper (trastuzumab) Fab domain in contact with its Her2 epitope (grey). Figure 18B , IgG 2 Molecular modeling of the complete structure of the molecule shows that the upper (trastuzumab) Fab domain contacts its Her2 epitope (grey), but the CD47 ectodomain is also superimposed on its potential binding site on the lower Fab of A-D5. This analysis demonstrates that the CD47 epitope cannot bind when both linkers are intact.

[0076] Figures 19A-19I .Her47Fab with different linkers 2 Structural dynamics of the structure. Solvent accessible surface area (SASA) results were obtained for three linkers (LHL, LHLF, and L2). Figure 19A 、 Figure 19D and Figure 19G Shown are absolute SASA values ​​for 9 kinetic runs for the LHL and LHLF linkers and 10 runs using L2 (all exceeding 6 ns). Figure 19B 、 Figure 19E 、 Figure 19H 、 Figure 19C 、 Figure 19F and Figure 19I Normalized results representing the difference from the starting SASA value within the 6 ns dynamic run time and within the first 2.5 ns of the 6 ns dynamic run are shown. Figure 19A 、 Figure 19B and Figure 19C SEQ ID NO: 2 is depicted. Figure 19D 、 Figure 19E and Figure 19F SEQ ID NO: 3 is depicted. Figure 19G 、 Figure 19H and Figure 19I SEQ ID NO: 32 is depicted.

[0077] Figure 20A-Figure 20B .Intact and activated Her47LHL-LHL Fab 2 Structural dynamics of structures. ( Figure 20A ) Overlapping Fab in both intact linker and activated (single linker cleaved by protease) 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. Anti-CD47 Fab is shown in black, and anti-HER2 Fab is shown in gray. This figure shows the maximum movement of the Her2 domain and the final exposure of the anti-CD47 Fab CDRs.

[0078] Figure 21 Flow cytometric analysis of protein binding to 'Tg32' mouse erythrocytes. Using A-D5IgG1, IgG 2 Her47LHL-LHL, IgG 2 Her47LHL-LHLF and Fab 2 Met47LHL-LHL was subjected to flow cytometric analysis of binding to erythrocytes. Binding was measured using an anti-human PE-conjugated secondary antibody. A-D5 IgG1 was tested at 0.1 μg / ml, 1 μg / ml, and 10 μg / ml. IgG was tested at 0.1 μg / ml, 1 μg / ml, and 10 μg / ml. 2 Her47LHL-LHL. IgG tested at 0.1μg / ml, 1μg / ml and 10μg / ml 2 Her47LHL-LHLF. Fab was tested at 0.1 μg / ml, 1 μg / ml and 10 μg / ml 2 Met47LHL-LHL.

[0079] Figure 22 .'Tg32' mouse erythrocyte hemagglutination assay. Using A-D5IgG1, IgG 2 Her47LHL-LHL, IgG 2 Her47LHL-LHLF and Fab 2 Met47LHL-LHL performs erythrocyte agglutination. Protein (in nM) is titrated using fresh erythrocytes pooled from multiple donor mice.

[0080] Figure 23 Tolerability studies in 'Tg32' mice: Body weight analysis. All proteins were administered at 2 mg / kg and 10 mg / kg in Tg32 mice using A-D5IgG1, IgG 2 Her47LHL-LHL, IgG 2 Her47LHL-LHLF and Fab 2 Tolerability study of Met47LHL-LHL. Body weight was then monitored for 60 days. The A-D5IgG1 10 mg / kg dose was not tolerated and the cohort was terminated on day 1.

[0081] Figure 24Tolerance study in 'Tg32' mice: Reticulocyte analysis on day 5 after administration. All proteins were administered at 2 mg / kg and 10 mg / kg in Tg32 mice using A-D5IgG1, IgG 2 Her47LHL-LHL, IgG 2 Her47LHL-LHLF and Fab 2 Tolerability study of Met47LHL-LHL. Blood samples were collected and reticulocyte levels were measured. A 2 mg / kg dose of A-D5IgG1 showed a significant increase in reticulocyte levels.

[0082] Figures 25A-25K Tolerability study in 'Tg32' mice: Hematological analysis on days 5, 29 and 60 after administration. All proteins were administered at 2 mg / kg and 10 mg / kg in Tg32 mice using A-D5IgG1, IgG 2 Her47LHL-LHL, IgG 2 Her47LHL-LHLF and Fab 2 Study on the tolerance of Met47LHL-LHL. Blood samples were collected and 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 ),leukocyte( Figure 25F ), monocytes ( Figure 25G ), lymphocytes ( Figure 25H ), basophils ( Figure 25I ), eosinophils ( Figure 25J ) and neutrophils ( Figure 25K )level.

[0083] Figure 26 Pharmacokinetic studies in 'Tg32' mice: data for each molecule at two doses. All proteins were dosed at 2 mg / kg and 10 mg / kg in Tg32 mice using IgG 2 Her47LHL-LHL, IgG 2 Her47LHL-LHLF and Fab 2 Pharmacokinetic study of Met47LHL-LHL. A-D5IgG1 was administered at 2 mg / kg. Serum samples were collected from 30 minutes to 42 days after administration and human IgG levels (in μg / ml) were measured.

[0084] Figures 27A-27BPharmacokinetic studies in 'Tg32' mice: data per dose. All proteins were dosed at 2 mg / kg and 10 mg / kg in Tg32 mice using IgG 2 Her47LHL-LHL, IgG 2 Her47LHL-LHLF and Fab 2 Pharmacokinetic study of Met47LHL-LHL. A-D5IgG1 was administered at 2 mg / kg. Serum samples were collected from 30 minutes to 42 days after administration and human IgG levels (in μg / ml) were measured. Figure 27A ) and 10mg / kg( Figure 27B The A-D5IgG1 2 mg / kg dose was included in both analyses as a reference.

[0085] Figure 28 Pharmacokinetic studies in 'Tg32' mice: AUC data for each dose. All proteins were dosed at 2 mg / kg and 10 mg / kg in Tg32 mice using IgG 2 Her47LHL-LHL, IgG 2 Her47LHL-LHLF and Fab 2 Pharmacokinetic study of Met47LHL-LHL. A-D5IgG1 was administered at 2 mg / kg. Serum samples were collected from 30 minutes to 42 days after administration and human IgG levels (in μg / ml) were measured. Concentration measurements over time were used to calculate the area under the curve (AUC) for each dose.

[0086] Figure 29A-29B Flow cytometric analysis of binding to NHP and human erythrocytes. Using A-D5 3M (effector null) IgG1, IgG 2 Her47LHL-LHL, IgG 2 Flow cytometric analysis of Her47LHL-LHLF and trastuzumab binding to erythrocytes was performed. Binding was measured using an anti-human PE-conjugated secondary antibody. A-D5IgG1 was the only antibody that showed binding to NHP (cynomolgus monkey) erythrocytes ( Figure 29A ) and human erythrocytes ( Figure 29B ) proteins that bind in a concentration-dependent manner.

[0087] Figures 30A-30NDirect ELISA of purified intact and MMP-digested protein constructs (digested at pH 7.4 and pH 6.0) binding to human target proteins. Protein constructs were enzymatically digested with human MMPs at pH 7.4 or pH 6.0 over a time course of 2, 4, 8, and 24 hours of incubation, plus a 24-hour incubation in enzyme-free buffer as a negative control (time 0). Samples from these digestion time courses were then used in direct binding ELISAs against human Her2 and CD47.

[0088] Figure 31A-Figure 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. Protein constructs were enzymatically digested using human cathepsins at pH 7.4 or pH 6.0 over a time course of 2, 4, 8, and 24 hours of incubation, plus a 24-hour incubation in enzyme-free buffer as a negative control (time 0). Samples from these digestion time courses were then applied to direct binding ELISAs against human Her2 and CD47.

[0089] Figures 32A-32D Flow cytometric analysis of binding to human cancer cells. Anti-CD47, trastuzumab, IgG1 isotype, and IgG enzymatically digested with human MMP12 at pH 7.4 were incubated at 2, 4, 8, and 24 hours, plus a 24-hour incubation in enzyme-free buffer as a negative control (time 0). 2 Her47LHL-LHL or IgG 2 Flow cytometric analysis of Her47LHL-LHLF binding to erythrocytes was performed. Binding was measured using an anti-human PE-conjugated secondary antibody. Figure 32A 、 Figure 32B ) and Her2 low cell line MCF-7 ( Figure 32C 、 Figure 32D ) is measured in conjunction with.

[0090] Figure 33 IgG digested with MMP12 2 Her47LHL-LHL or IgG 2 SDS-PAGE analysis of Her47LHL-LHLF. IgG enzymatically digested with human MMP12 at pH 7.4 at 2, 4, 8, and 24 hours of incubation, plus a 24-hour incubation in buffer without enzyme as a negative control (time 0). 2 Her47LHL-LHL or IgG 2 Her47LHL-LHLF samples were subjected to SDS-PAGE.

[0091] Figure 34A-Figure 34B IgG digested with MMP12 2 Mass spectrometric analysis of Her47LHL-LHL. IgG enzymatically digested with human MMP12 at pH 7.4 was analyzed over a time course of 2, 4, 8, and 24 hours of incubation, plus a 24-hour incubation in buffer without enzyme as a negative control (time 0). 2 The Her47LHL-LHL sample was subjected to mass spectrometry analysis. The measurement indicated that the LHL linker was intact ( Figure 34A ) and MMP12-cleaved linker ( Figure 34B ) of the peptide. Figure 34A SEQ ID NO: 110 is depicted. Figure 34B SEQ ID NO: 111 is depicted.

[0092] Figure 35 Size Exclusion Chromatography of Protein A-Purified Her47LHLF-LHL IgG1-2hDAA. The Her47LHLF-LHL IgG1-2hDAA protein was expressed in CHO cells, purified using a ProA column, and analyzed by SEC. Two small, larger MW peaks were observed, along with a large peak of the expected size (10.30, approximately 250 kDa).

[0093] Figure 36 SDS-PAGE analysis of peak fractions from size exclusion chromatography purification of Her47LHLF-LHL IgG1-2hDAA. SDS-PAGE was performed on a non-reduced sample of Her47LHLF-LHL IgG1-2hDAA: lane 1 - molecular weight standard, lane 2 - total ProA eluted protein, lane 3 - blank, lane 4 - peak 1, lane 5 - peak 2, lane 6 - peak 3 (correct product).

[0094] Figure 37 SDS-PAGE analysis of peak fractions from size exclusion chromatography purification of Her47LHLF-LHL IgG1-2hDAA. SDS-PAGE was performed on a reduced sample of Her47LHLF-LHL IgG1-2hDAA: lane 1 - molecular weight standard, lane 2 - total ProA eluted protein, lane 3 - blank, lane 4 - peak 1, lane 5 - peak 2, lane 6 - peak 3 (correct product).

[0095] Figures 38A-38CDirect ELISA of purified intact and MMP12-digested Her47IgG1-2hDAA proteins. Her47LHL-LHLF IgG1-2hDAA ( Figure 38A ) were enzymatically digested. Samples from these digestion time courses were then applied to direct binding ELISAs against human Her2 and mouse EpCAM ( Figure 38A ). Digested (dark grey) and undigested (light grey) samples were then subjected to ELISA for 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 ).

[0096] Figures 39A-39L Direct ELISA of purified intact and MMP12 digested IgG2 Her47 proteins with alternative linker compositions. Purified proteins from clones Her47LHL-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 Each protein was then subjected to time course enzymatic digestion and ELISA binding to Her2 and CD47 targets ( Figure 39B 、 Figure 39D 、 Figure 39F 、 Figure 39H 、 Figure 39J 、 Figure 39L ).

[0097] Figure 40 Multiple-dose tolerance study in NOD-SCID mice: Body weight analysis. All proteins were administered once every 5 days (4 doses total) using IgG. 2 Her47LHL-LHL, IgG 2 Her47LHL-LHLF、Fab 2 Her47LHL-LHL and Fab 2Tolerability study of Her47LHL-LHLF.

[0098] Figures 41A-41D Protein A purified Her2CD3Fab 2 Size Exclusion Chromatography of Proteins. Fab 2 Her23LHL-LHL-S( Figure 41A ), Fab 2 Her23LHLF-LHL-S( Figure 41B ), Fab 2 Her23LHL-LHL( Figure 41C ) and Fab 2 Her23LHLF-LHL( Figure 41D ) were expressed in CHO cells, purified by ProA column and analyzed by SEC. 2 Her23LHL-LHL( Figure 41C ) and Fab 2 Her23LHLF-LHL( Figure 41D ) Both showed a low molecular weight contaminant (peak 15.38).

[0099] Figures 42A-42B Purified intact and MMP-digested Her2CD3Fab were detected using Her2 low MCF-7 cells. 2 The proteins were subjected to CD3 co-engagement bioassay analysis. Antibody Fab was incubated with human MMP12 at pH 7.4 over a time course of 2, 4, 8, and 24 hours, plus a 24-hour incubation in enzyme-free buffer as a negative control (time 0). 2 Her23LHLF-LHL-S( Figure 42A ), Fab 2 Her23LHL-LHL-S( Figure 42B Samples from these digestion time courses were then applied to a Promega Jurkat cell CD3 reporter assay using MCF-7 cells as target cells.

[0100] Figures 43A-43C Purified intact and MMP-digested Her2CD3Fab were detected using Her2 high BT-474 cells. 2 Protein was subjected to CD3 co-binding bioassay analysis. Figure 43A ), and Fab 2 Her23LHLF-LHL-S( Figure 43B ) or Fab 2 Her23LHL-LHL-S( Figure 43C) [both enzymatically digested with human MMP12 at pH 7.4 over a time course of 2, 4, 8, and 24 hours of incubation, plus a 24-hour incubation in enzyme-free buffer as a negative control (time 0)] were used at 0.1 μg / ml in a Promega Jurkat cell CD3 reporter assay using BT-474 cells as target cells.

[0101] Figures 44A-44B IgG 2 and Fab 2 Charge variant analysis of Her47 protein - Charge heterogeneity analysis is important in the characterization of monoclonal antibodies because 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 a commercial Charge Variant Assay. IgG 2 Her47LHL-LHL( Figure 44A ) and Fab 2 Her47LHL-LHL( Figure 44B ) The charge variant profiles of both showed a homogeneous profile with one major isoform (50-57% of the total), one major acidic isoform (40-48% of the total) and one minor basic isoform (approximately 3%).

[0102] Figures 45A-45B Size exclusion chromatography of Her47 protein after 5 cycles of freeze-thaw. IgG 2 Her47LHL-LHL( Figure 45A ) and Fab 2 Her47LHL-LHL( Figure 45B ) Both were subjected to 5 cycles of freeze-thaw, and SEC was then performed on samples from cycles 0 to 5. No aggregation, fragmentation, or product loss was observed for either protein.

[0103] Figure 46 .Alternative protein construct designs. This figure depicts the protein construct Fab 2 Illustrative example of a design. This design can be based on a sequence that: 1. Removes the upper variable domain. 2. Contains a "dummy" non-binding variable domain. 3. Replaces the upper Fab with a diabody (or two scFvs). The variable regions are shown in white. The constant regions are shown in gray.

[0104] Figures 47A-47B. Cell proliferation assay was performed on purified intact Her2CD47 protein using Her2 high BT-474 cells. Trastuzumab, isotype control IgG1, IgG 2 Her47LHL-LHL( Figure 47A ) and Fab 2 Her47LHL-LHL( Figure 47B ) were applied to BT-474 cells during a 72-hour incubation period and cell proliferation was measured. Data are expressed as % inhibition of cell growth.

[0105] Figures 48A-48G In vivo efficacy analysis of Her47 molecules in NOD-SCID mice (KYSE-410 model). Figure 48A ), IgG 2 Her47LHL-LHLF( Figure 48B ), IgG 2 Her47LHL-LHL( Figure 48C ), Fab 2 Her47LHL-LHLF( Figure 48D ) and Fab 2 Her47LHL-LHL( Figure 48E ) were each administered (intravenously on days 0, 5, and 10) to NOD-SCID mice bearing KYSE-410 tumors. Tumor volumes were measured on days 4, 7, and 11 and plotted relative to vehicle. 2 Her47LHL-LHLF and Fab 2 Her47LHL-LHL showed different potency ( Figure 48F None of the dosing groups showed any weight loss that could indicate toxicity of the administered molecule ( Figure 48G ). DETAILED DESCRIPTION

[0106] Disclosed herein are recombinant proteins that are conditionally active in diseased human tissues. In some cases, the protein comprises a binding domain that is masked by another portion of the protein in non-diseased tissue. The protein further comprises a peptide linker that is cleaved by one or more proteases expressed in the diseased tissue. Linker cleavage unmasks the binding domain in the diseased tissue, thereby allowing the protein to selectively bind and / or function in the diseased tissue. The proteins of the present invention are particularly useful for binding to drug targets expressed in both diseased and non-diseased tissues.

[0107] Many activatable protein molecules and their medical uses are provided herein. In some aspects, a variety of functional properties of the molecules are considered, including target binding specificity, effective restriction of undesirable activity in the native protein but full activity in the activated form, maintaining conditional affinity for one or more targets from human and animal test species (e.g., cynomolgus macaques (also known as macaques), i.e., macaque fascicularis), biophysical stability, and / or yield from the protein expression platforms used in research, clinical, and commercial supply.

[0108] In some aspects, protein molecules are provided that specifically bind to one or more human drug targets and, optionally, also to cynomolgus monkey orthologs of those targets, wherein the protein molecules comprise heavy and light chain regions assembled from one or more polypeptides having the following format:

[0109] VC-Connector-VC

[0110] VC-Connector-VC

[0111] or

[0112] C-Connector-VC

[0113] C-Connector-VC

[0114] In some aspects, the protein molecule comprises two polypeptide chains and has the form:

[0115] VH1-C-Connector-VH2-C

[0116] VL1-C-Connector-VL2-C

[0117] In some aspects, the protein molecule comprises two polypeptide chains and has the form:

[0118] VL1-C-Connector-VH2-C

[0119] VH1-C-Connector-VL2-C

[0120] "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 heavy chain variable region and light chain variable region that are paired with each other to bind to an antigen. "VH2" and "VL2" refer to the heavy chain variable region and light chain variable region that are paired with each other to bind to an antigen. "C" refers to the constant region of an immunoglobulin or T cell receptor. In aspects of the present invention, the VC and VC 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, which 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 that has or lacks any specific binding function.

[0121] 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.

[0122] wherein the peptide linker comprises an amino acid sequence from a human immunoglobulin hinge region or an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 (e.g., 1 to about 7) amino acid substitutions compared to a human immunoglobulin hinge region;

[0123] wherein the peptide linker is cleavable by a protease expressed in diseased tissue;

[0124] wherein the second portion is capable of specifically binding to a molecule expressed in diseased tissue; and

[0125] wherein when the peptide linker is not cleaved, binding of the second portion to the molecule expressed in the diseased tissue is reduced or inhibited.

[0126] The linker moiety may also comprise a peptide linker derived from an immunoglobulin hinge region with zero, one or more mutations away from germline.

[0127] In some aspects, the peptide linker comprises 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). NO:87) (can be cleaved by ADAMTs-5) or consists of the sequence shown therein. In some aspects, the peptide linker comprises or consists of two, three or four of the above sequences.

[0128] Also provided are immunoconjugates comprising a protein of the invention linked to a therapeutic agent.

[0129] In another aspect, the present invention provides nucleic acid molecules encoding proteins as defined herein or portions thereof. Also provided are vectors comprising the nucleic acid molecules of the present invention. Also provided are host cells comprising the nucleic acid molecules or vectors of the present invention.

[0130] In a further aspect, there is provided a method of producing a conditionally active protein of the invention, the method comprising culturing a host cell of the invention under conditions resulting in expression and / or production of the protein, and isolating the protein from the host cell or culture.

[0131] In another aspect of the invention, a pharmaceutical composition is provided, comprising a protein of the invention as defined herein, or a nucleic acid molecule of the invention as defined herein, or a vector of the invention as defined herein, or an immunoconjugate of the invention as defined herein.

[0132] Also provided is a method for enhancing an immune response in a subject, the method comprising administering an effective amount of a protein of the invention as defined herein, or an immunoconjugate of the invention as defined herein, or a nucleic acid molecule of the invention as defined herein, or a vector of the invention as defined herein, or a pharmaceutical composition of the invention as defined herein.

[0133] In a further aspect, a method for treating or preventing cancer in a subject is provided, the method comprising administering an effective amount of a protein of the invention as defined herein, or an immunoconjugate of the invention as defined herein, or a nucleic acid molecule of the invention as defined herein, or a vector of the invention as defined herein, or a pharmaceutical composition of the invention as defined herein.

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

[0135] Also provided is a protein of the invention as defined herein, or an immunoconjugate of the invention as defined herein, or a nucleic acid molecule of the invention as defined herein, or a vector of the invention as defined herein, or a pharmaceutical composition of the invention as defined herein, for use in treating cancer.

[0136] Also provided is a protein, or immunoconjugate, or nucleic acid molecule, or vector, or pharmaceutical composition of the invention as defined herein for separate, sequential or simultaneous use in combination with a second therapeutic agent (eg, an anticancer agent).

[0137] In a further aspect, there is provided 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, for the preparation of a medicament for the treatment of cancer.

[0138] Also provided is a method for treating or preventing an autoimmune disease or an inflammatory disease in a subject, the method comprising administering an effective amount of a protein as defined herein, or an immunoconjugate as defined herein, or a nucleic acid molecule as defined herein, or a vector as defined herein, or a pharmaceutical composition as defined herein.

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

[0140] 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.

[0141] Also provided is a method for treating or preventing cardiovascular disease or fibrotic disease in a subject, the method comprising administering an effective amount of a protein as defined herein, or an immunoconjugate as defined herein, or a nucleic acid molecule as defined herein, or a vector as defined herein, or a pharmaceutical composition as defined herein.

[0142] Also provided are proteins as defined herein, or immunoconjugates as defined herein, or nucleic acid molecules as defined herein, or vectors as defined herein, or pharmaceutical compositions as defined herein for use as medicaments. Also provided are antibody molecules as defined herein, or antigen-binding portions thereof, or immunoconjugates as defined herein, or nucleic acid molecules as defined herein, or vectors as defined herein, or pharmaceutical compositions as defined herein for use in treating cardiovascular disease or fibrotic disease.

[0143] 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, an inflammatory disease, or a fibrotic disease.

[0144] In some aspects, the present invention provides a protein comprising a first portion and a second portion and a peptide linker between the first portion and the second portion, wherein the peptide linker comprises an amino acid sequence from a human immunoglobulin hinge region or an amino acid sequence or 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; wherein the peptide linker can be cleaved by a protease expressed in a diseased tissue; wherein the second portion is capable of specifically binding to a molecule expressed in the diseased tissue; and wherein when the peptide linker is not cleaved, the binding of the second portion to the molecule expressed in the diseased tissue is reduced or inhibited. 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 or 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 or amino acid sequence having 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 2-3, 2-4, 2-5, 2-6, 2-7, 3-4, 3-5, 3-6, 3-7, 4-5, 4-6, 4-7, 5-6, 5-7 or 6-7 amino acid substitutions compared to a human immunoglobulin hinge region.

[0145] In some aspects, the peptide linker that can be cleaved by a protease expressed in diseased tissue can be cleaved by a human matrix metalloproteinase (MMP) or a human cathepsin. In some cases, the peptide linker that can be cleaved by a protease expressed in diseased tissue can be 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 metalloproteinase 5 containing a type 1 thrombospondin motif; A5). In some cases, the peptide linker comprises a human MMP cleavage site or a human cathepsin cleavage site. In some cases, the peptide linker comprises a human enterokinase, human thrombin, human tPA, human granzyme B, human uPA or human ADAMTs-5 cleavage site. In some cases, the peptide linker comprises a MMP substrate sequence PLGL (SEQ ID NO: 12). In some cases, the peptide linker comprises or consists of the amino acid sequence of GPAPELL (SEQ ID NO: 1), GPAPELLGGGS (SEQ ID NO: 2), GPAPLGLGGGS (SEQ ID NO: 3), PPCPAPELLGGGS (SEQ ID NO: 4), or PPCPAPLGLGGGS (SEQ ID NO: 5), GPAPELLGGPS (SEQ ID NO: 69), GPAPLGLGGPS (SEQ ID NO: 70), PPCPAPELLGGPS (SEQ ID NO: 71), PPCPAPLGLGGPS (SEQ ID NO: 72), GPAPEAAGAGS (SEQ ID NO: 81), GPADDDDKSGS (SEQ ID NO: 82), GPALVPRGSGS (SEQ ID NO: 83), GPGPFGRSAGGP (SEQ ID NO: 84), GPAPLEADAGS (SEQ ID NO: 85), GPAPEARRGGS (SEQ ID NO: 86), or GPAPEGEARGS (SEQ ID NO: 87).

[0146] In some cases, the peptide linker comprises or consists of two, three, or four amino acid sequences in Table 1 fused by peptide bonds in a single amino acid chain. In some cases, the peptide linker is between about 5 and about 15 amino acids, between about 5 and about 20 amino acids, or between about 5 and about 25 amino acids in length.

[0147] In some cases, the peptide linker between the first and second moieties 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.

[0148] In some cases, the peptide linker cleavable by a protease expressed in diseased tissue can be cleaved by any of human MMP1, MMP2, MMP3, MMP4, MMP5, MMP6, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP18, MMP19, MMP20, MMP21, MMP22, MMP23, MMP24, MMP25, MMP26, MMP27, or MMP28. In some cases, the peptide linker cleavable by a protease expressed in diseased tissue can be cleaved by any of human MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, MMP-12, or MMP-13. In some cases, the level or activity of a human MMP is elevated in the diseased tissue compared to the level or activity of the human MMP in the non-diseased tissue.

[0149] In some cases, the peptide linker that can be cleaved by a protease expressed in a 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 that can be cleaved by a protease expressed in a 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 cathepsins in diseased tissue is increased compared to the level or activity of human cathepsins in non-diseased tissue. In some cases, the level or activity of human cathepsins in tissues with a pH < 7.0 is increased compared to the level or activity of human cathepsins in tissues with a pH ≥ 7.4.

[0150] In some aspects, the first portion of any protein of the present 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, 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. IgNAR is a homodimeric heavy chain-only antibody produced by sharks and other cartilaginous fish (Feige et al., PNAS, 2014, 111(22):8155-8160).

[0151] In some cases, the first portion specifically binds to a molecule expressed in diseased tissue. In some embodiments, the first portion specifically binds to a tumor-associated antigen (TAA). In some cases, the first portion specifically binds to human EGFR, human HER2, human HER3, human CD105, human C-KIT, human PD1, human PD-L1, human PSMA, human EpCAM, human Trop2, human EphA2, human CD20, human BCMA, human GITR, human OX40, human CSF1R, human Lag3, or human cMET. In some embodiments, the first portion also binds to the cynomolgus monkey ortholog of any of these molecules.

[0152] In some aspects, the first portion comprises a heavy chain variable (VH) region and a light chain variable (VL) region. In some cases, the first portion further comprises an immunoglobulin constant region or a portion of an immunoglobulin constant region. In some cases, the immunoglobulin constant region is IgG, IgE, IgM, IgD, IgA, or IgY.

[0153] 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 used in the protein constructs disclosed herein is provided in WO 2019 / 175186. In some aspects, the anti-CD47 variable region sequence used in the protein constructs disclosed herein is provided in WO 2019 / 034895.

[0154] In some aspects, the second portion of any protein of the present 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, 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.

[0155] In some cases, the second portion specifically binds to molecules expressed in diseased tissues. In some embodiments, the second portion specifically binds to tumor-associated antigens (TAA). In some cases, the second portion specifically binds to human CD47. In some cases, the second portion specifically binds to human PD-L1. In some cases, the second portion specifically binds to molecules expressed by human immune cells. In some cases, the molecules expressed by human immune cells are human CD3, human CD16A, human CD16B, human CD28, human CD89, human CTLA4, human NKG2D, human SIRPα, human SIRPγ, human PD1, human Lag3, human 4-1BB, human OX40 or human GITR. In some embodiments, the second portion also binds to the cynomolgus monkey orthologs of any of these molecules.

[0156] In some aspects, the second portion comprises a heavy chain variable (VH) region and a light chain variable (VL) region. In some cases, the second portion further comprises an immunoglobulin constant region or a portion of an immunoglobulin constant region. In some cases, the immunoglobulin constant region is IgG, IgE, IgM, IgD, IgA or IgY.

[0157] In some aspects, the first portion and / or the second portion 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 other embodiments, the immunoglobulin constant region is IgG1, IgG2, IgG3, IgG1null, IgG4(S228P), IgA1, IgA2, IgE, or IgM. In some embodiments, the first portion and / or the second portion of the protein of the present invention may comprise an immunologically inert constant region. In some aspects, the first and / or second portions of a protein of the invention may comprise an immunoglobulin constant region comprising a wild-type human IgG1 constant region, a human IgG1 constant region comprising amino acid substitutions L234A and L235A, a human IgG1 constant region comprising amino acid substitutions L234A, L235A, and G237A, or a human IgG1 constant region comprising amino acid substitutions L234A, L235A, G237A, and P331S. In some aspects, the first and / or second portions of a protein of the invention may comprise an immunoglobulin constant region comprising a wild-type human IgG2 constant region or a wild-type human IgG4 constant region. In some aspects, the first and / or second portions of a protein of the invention may comprise an immunoglobulin constant region comprising any one of the amino acid sequences in Table 10. The Fc region sequences in Table 10 begin at the CH1 domain. In some aspects, the first and / or second portions of the proteins of the invention can comprise an immunoglobulin constant region comprising the amino acid sequence of the Fc region of human IgG4, human IgG4(S228P), human IgG2, human IgG1, human IgG1-3M, or human IgG1-4M. For example, compared to a wild-type human IgG4 Fc region, the human IgG4(S228P) Fc region comprises the following substitution: S228P. For example, compared to a wild-type human IgG1 Fc region, the human IgG1-3M Fc region comprises the following substitutions: L234A, L235A, and G237A, while the human IgG1-4M Fc region comprises the following substitutions: L234A, L235A, G237A, and P331S, compared to a wild-type human IgG1 Fc region. In some aspects, the positions of amino acid residues in the constant region of an immunoglobulin molecule are numbered according to EU nomenclature (Ward et al., 1995 Therap. Immunol. 2:77-94). In some aspects, the immunoglobulin constant region can comprise an RDELT (SEQ ID NO: 65) motif or a REEM (SEQ ID NO: 66) motif (underlined in Table 10). The REEM (SEQ ID NO: 66) allotype is found in a smaller population than the RDELT (SEQ ID NO: 65) allotype.In some aspects, the first portion and / or the second portion of the protein of the antibody of the present invention may comprise an immunoglobulin constant region comprising any one of SEQ ID NOs: 56 to 62. In some aspects, the first portion and / or the second portion of the protein of the present invention may comprise a heavy chain amino acid sequence and a light chain amino acid sequence of any one of the clones in Tables 3 to 9 and any one of the Fc region amino acid sequences in Table 10. In some aspects, the first portion and / or the second portion of 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.

[0158] In some aspects, the proteins of the invention comprise an IgG1 isotype constant region. IgG1 isotype constant regions efficiently activate all FcγR signaling types, driving maximal opsonizing effector function.

[0159] 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.

[0160] In some aspects, the first portion specifically binds to the first molecule expressed in the diseased tissue, and the second portion can specifically bind to the second molecule expressed in the diseased tissue, wherein the first molecule expressed in the diseased tissue and the second molecule expressed in the diseased tissue are different molecules. In some embodiments, the first molecule expressed in the diseased tissue and the second molecule expressed in the diseased tissue are expressed by identical cells. In some embodiments, the first molecule expressed in the diseased tissue and the second molecule expressed in the diseased tissue are expressed by different cells. In some embodiments, the first molecule expressed in the diseased tissue, the second molecule expressed in the diseased tissue or the first molecule expressed in the diseased tissue and the second molecule expressed in the diseased tissue are expressed on the cell surface. In some embodiments, the first molecule expressed in the diseased tissue and / or the second molecule expressed in the diseased tissue are soluble molecules.

[0161] 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.

[0162] In some aspects, the protein of the present invention has an immune effector function or two, three or more immune effector functions. For example, the immune effector function can be antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) or antibody-dependent cellular phagocytosis (ADCP).

[0163] In some aspects, the first portion of a protein of the present invention prevents or reduces specific binding of the second portion to a molecule expressed in a diseased tissue. In some embodiments, the peptide linker of a protein of the present invention is cut near or inside a diseased tissue. In some cases, the peptide linker is cut near or inside a diseased tissue, wherein the first portion dissociates from the second portion near or inside a diseased tissue, and wherein the second portion specifically binds to a molecule expressed in a diseased tissue near or inside a diseased tissue. In some cases, the cleaved peptide linker comprises a binding site or target site for an anti-hinge antibody (e.g., an endogenous anti-hinge antibody of a subject), whereas the uncleaved (e.g., complete) peptide linker does not comprise a binding site or target site for an anti-hinge antibody. In the presence of an activated protein of the present invention, the binding of the anti-hinge antibody to the cleaved peptide linker can increase ADCC, CDC, and / or ADCP (see, e.g., Figure 2B ).

[0164] In some aspects, the proteins of the invention stimulate inflammatory signaling in diseased tissues. Increased inflammatory signaling can increase immune recruitment to diseased tissues. In some cases, the proteins of the invention increase antigen presentation in diseased tissues. In some cases, the proteins of the invention increase tumor-associated antigen-specific T cell proliferation.

[0165] In some aspects, the diseased tissue can be a tumor, necrotic tissue, fibrotic tissue, tissue undergoing the coagulation cascade, or inflamed tissue.

[0166] 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 or consisting of the amino acid sequence of SEQ ID NO: 16, and the second polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO: 17. The protein comprises only one copy of the first polypeptide chain and only one copy of the second polypeptide chain. The peptide linker of the protein comprises two copies of the LHL sequence (see Table 1), each copy 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 shown in FIG. Figure 3A The second part of the protein is connected to KIH IgG1-Fc via a G4S linker (SEQ ID NO: 15) and a truncated hinge region.

[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 CD3, and wherein the protein comprises a first polypeptide chain and a second polypeptide chain, the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 26, and the second polypeptide chain comprises or consists 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 shown in FIG. Figure 3A The second part of the protein is connected to KIH IgG1-Fc via a G4S linker (SEQ ID NO: 15) and a truncated hinge region (3M).

[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 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 comprises or consists of the amino acid sequence of SEQ ID NO: 34, and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 35. The protein comprises two identical copies of the first polypeptide chain and two identical copies of the second polypeptide chain. The peptide linker of the protein comprises two copies of the LHL sequence fused by a peptide bond (see Table 1). The protein is referred to as "IgG2Her2CD47-LHL-LHL" or "Her47-LHL-LHL". The amino acid sequence is provided in Table 5. The structure of the protein is as shown in FIG. Figure 2A The second part of the protein can be linked to the human IgG1 Fc sequence via the hinge region or a truncated hinge region (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 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 or consisting of the amino acid sequence of SEQ ID NO: 36, and the second polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO: 37. The first polypeptide chain also comprises a human IgG1 amino acid sequence (see Table 10). The second polypeptide chain also comprises a human κ light chain amino acid sequence. 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 "Fab2CMET / CD47 'one-armed' type" or "Met47-LHL-LHL". The amino acid sequence is provided in Table 6. The structure of the protein is shown in FIG. Figure 3B The construct is a 'knob-in-hole' one-armed Fab2 construct with Fab2 on the knob side and a hinge-hole Fc stump on the other side. The construct may comprise a non-effector null human IgG1 Fc sequence (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: 38, and the second polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO: 39. The first polypeptide chain also comprises a human IgG1-3M amino acid sequence (see Table 10). The second polypeptide chain also comprises a human κ light chain amino acid sequence. 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 "Fab2Her2 / CD3 'one-armed' type" or "Her23-LHL-LHL". The amino acid sequence is provided in Table 7. The structure of the protein is as shown Figure 3B The construct is a 'knob-in-hole' one-armed Fab2 construct with the Fab2 on the knob side and a hinge-hole Fc stump on the other side. This construct is also effector-null (IgG1-3M; see Table 10).

[0171] 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, and the second polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO: 41. The first polypeptide chain also 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 fused by a peptide bond (see Table 1). The protein is referred to as "Fab2Her2 / CD3 (34) 'one-armed' type" or "Her23 (34)-LHL-LHL". The amino acid sequence is provided in Table 8. The structure of the protein is as shown in FIG. Figure 3B The construct is a 'knob-in-hole' one-armed Fab2 construct with the Fab2 on the knob side and a hinge-hole Fc stump on the other side. This construct is also effector-null (IgG1-3M; see Table 10).

[0172] In some aspects, provided herein is a protein comprising a first portion and a second portion and a peptide linker between the first portion and the second portion, wherein the first portion specifically binds 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:

[0173] (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

[0174] (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

[0175] (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

[0176] (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

[0177] (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

[0178] (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

[0179] (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

[0180] (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 "Her47LHL-LHL-EK");

[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: 93 (referred to as "Her47-LHL-LHL-Thr");

[0182] (j) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 44, and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 94 (referred to as "Her47-LHL-LHL-tPA");

[0183] (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");

[0184] (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

[0185] (m) The first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 44, and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 97 (referred to as "Her47-LHL-LHL-A5"). These proteins are generally referred to as "IgG2Her2 / CD47". The amino acid sequences are provided in Tables 9 and 20. The structure of the protein is shown in FIG. Figure 2A The peptide linker sequences for the LHLF, LHLM, and LHLMF linkers are provided in Table 1. The peptide linker sequences for the EK, Thr, tPA, GrB, uPA, and A5 linkers are provided in Table 21.

[0186] In some aspects, provided herein is a protein comprising a first portion and a second portion and a peptide linker between the first portion and the second portion, wherein the first portion specifically binds 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:

[0187] (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 "Her47LHLF-LHL IgG1-2hDAA"); or

[0188] (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 "Her47LHL-LHLF IgG1-2hDAA"). These proteins are often referred to as "IgG2'IgG1-DAA'Her2 / CD47". The amino acid sequences are provided in Table 19. These proteins contain stabilizing mutations in the hinge. The structure of the protein is as shown Figure 2A The peptide linker sequences of the LHLF and LHL linkers are provided in Table 1.

[0189] In some aspects, provided herein is a protein comprising a first portion and a second portion and a peptide linker between the first portion and the second portion, wherein the first portion specifically binds 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 a 'hole' mutation to enable heterodimerization with the second polypeptide chain), followed by a linker sequence, the VH and CH1 domains of the first binding moiety, another linker sequence, and then the VH and CH1 domains of the second binding moiety (see Table 13). The second polypeptide chain further comprises a human IgG1-3M Fc amino acid sequence (e.g., containing a 'knob' mutation to enable heterodimerization with the second polypeptide chain), followed by a linker sequence, the VL and CL domains of the first binding moiety, another linker sequence, and then the VL and CL domains of the second binding moiety (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 part and between the first binding part and the second binding part. The protein is referred to as "Fc-Her2 / CD3 (34)" or "Fc-Her23 (34)". The amino acid sequence is provided in Table 13. The structure of the protein is shown in FIG. Figure 12 The construct is a "knob-in-hole" Fc-Fab2 construct with a light chain polypeptide on either the knob or hole side and a heavy chain polypeptide on the other side. This construct is also effector-null (IgG1-3M; see Table 10).

[0190] In some aspects, provided herein is a protein comprising a first portion and a second portion and a peptide linker between the first portion and the second portion, wherein the first portion specifically binds 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. The first polypeptide chain also comprises a human IgG1 or human IgG1-3M amino acid sequence (see Table 10). The second polypeptide chain also comprises a human κ light chain amino acid sequence. 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 "Fab2CMET / CMET 'one-armed' type" or "MetMet-LHL-LHL". The amino acid sequence is provided in Table 14. The structure of the protein is shown in FIG. Figure 3B The construct is a 'knob-in-hole' one-armed Fab2 construct with the Fab2 on the knob side and a hinge-hole Fc stump on the other side. The construct may or may not contain an effector-null human IgG1 Fc sequence (see Table 10).

[0191] In some aspects, provided herein is a protein comprising a first portion and a second portion and a peptide linker between the first portion and the second portion, wherein the first portion specifically binds 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:

[0192] (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 (referred to as "Fab2Her23LHL-LHLF"); or

[0193] (b) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 100, and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 101 (referred to as "Fab2Her23LHL-LHL"); or

[0194] (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 (referred to as "Fab2Her23LHLF-LHL-S"); or

[0195] (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 "Fab2Her23LHL-LHL-S"). This protein is referred to as "Fab2Her2 / CD3". The amino acid sequence is provided in Table 22. The structure of this protein is shown in FIG. Figure 3A 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 a peptide bond, or one copy of the LHL sequence and one copy of the LHLF sequence.

[0196] In some aspects, provided herein are immunoconjugates comprising a protein of the invention as defined herein linked to an additional therapeutic agent.

[0197] Examples of suitable therapeutic agents include cytotoxins, radioisotopes, chemotherapeutic agents, immunomodulators, anti-angiogenic agents, antiproliferative agents, pro-apoptotic agents, and cell growth inhibitory enzymes and lytic enzymes (e.g., RNA enzymes). Additional therapeutic agents include therapeutic nucleic acids, such as genes encoding immunomodulators, anti-angiogenic agents, antiproliferative agents, or pro-apoptotic agents. These drug descriptors are not mutually exclusive, and therefore one or more of the above terms can be used to describe a therapeutic agent.

[0198] Examples of suitable therapeutic agents for use in immunoconjugates include taxanes, maytansine, CC-1065 and duocarmycin, calicheamicin and other enediynes, and auristatins. Other examples include antifolates, vinca alkaloids, and anthracyclines. Plant toxins, other biologically active proteins, enzymes (i.e., ADEPT), radioisotopes, and photosensitizers can also be used in immunoconjugates. In addition, conjugates can be prepared using secondary carriers as cytotoxic agents (such as liposomes or polymers). Suitable cytotoxins include agents that inhibit or prevent cell function and / or cause cell destruction. Representative cytotoxins include antibiotics, tubulin polymerization inhibitors, alkylating agents that bind to and destroy DNA, and agents that disrupt protein synthesis or the function of essential cellular proteins (such as protein kinases, phosphatases, topoisomers, enzymes, and cell cycle proteins).

[0199] Representative cytotoxins include, but are not limited to, doxorubicin, daunorubicin, idarubicin, aclarubicin, daunorubicin, mitoxantrone, epirubicin, carrubicin, noramycin, menocryl, pitarubicin, valrubicin, cytarabine, gemcitabine, trifluridine, ancitabine, enocitabine, azacitidine, doxifluhdine, pentostatin, broxuhdine, capecitabine, cladhbine, decitabine, floxuhdine, fludarabine, glutathione, puromycin, flusudine, fludarabine, puromycin, tegafur, thiazolamide nucleoside

[00135] In some embodiments, the present invention includes but is not limited 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.

[0200] Suitable immunomodulators include antihormonal drugs that block the effects of hormones on tumors, and immunosuppressive agents that inhibit cytokine production, downregulate self-antigen expression, or mask MHC antigens.

[0201] Also provided are nucleic acid molecules encoding a protein of the invention as defined herein, or a portion of a protein thereof. 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 can be isolated.

[0202] Also provided is a vector comprising a nucleic acid molecule of the invention as defined herein.The vector may be an expression vector.

[0203] Also provided is a host cell comprising a nucleic acid molecule or vector of the invention as defined herein.The host cell may be a recombinant host cell.

[0204] In a further aspect, there is provided a method of producing a protein of the invention, the method comprising culturing a host cell of the invention under conditions resulting in expression and / or production of the protein, and isolating the protein from the host cell or culture.

[0205] In some aspects, provided herein are pharmaceutical compositions comprising a protein of the invention as defined herein, or a nucleic acid molecule of the invention as defined herein, or a vector of the invention as defined herein.

[0206] Also provided is a method for enhancing an immune response in a subject, the method comprising administering to the subject an effective amount of a protein of the invention as defined herein, or an immunoconjugate of the invention as defined herein, or a nucleic acid molecule of the invention as defined herein, or a vector of the invention as defined herein, or a pharmaceutical composition of the invention as defined herein.

[0207] In a further aspect, a method for treating or preventing cancer in a subject, or ameliorating a symptom of cancer in a subject, is provided, the method comprising administering an effective amount of a protein of the invention as defined herein, or an immunoconjugate of the invention as defined herein, or a nucleic acid molecule of the invention as defined herein, or a vector of the invention as defined herein, or a pharmaceutical composition of the invention as defined herein.

[0208] In some aspects, cancer is a solid tumor. In some cases, cancer is a hematological malignancy. For example, cancer can be gastrointestinal stromal cancer (GIST), pancreatic cancer, skin cancer (for example, 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, uterus or endometrial cancer, cancer of the oral cavity or pharynx, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small intestine or appendix cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma or the cancer of blood tissue. In some cases, the cancer of blood tissue is lymphoma.

[0209] In some aspects, provided herein is a protein of the invention as defined herein, or an immunoconjugate of the invention as defined herein, or a nucleic acid molecule of the invention as defined herein, or a vector of the invention as defined herein, or a pharmaceutical composition of the invention as defined herein, for use in treating cancer or for ameliorating a symptom of cancer.

[0210] In some aspects, provided herein are proteins, or immunoconjugates, or nucleic acid molecules, or vectors for use in the uses 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 anticancer agent).

[0211] In a further aspect, there is provided 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 a symptom of cancer.

[0212] The present 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.

[0213] For example, the autoimmune or inflammatory disease may be arthritis, asthma, multiple sclerosis, psoriasis, Crohn's disease, inflammatory bowel disease, lupus, Graves' disease, and Hashimoto's thyroiditis or ankylosing spondylitis.

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

[0215] 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.

[0216] The present invention also provides a method for treating or preventing cardiovascular disease or 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.

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

[0218] 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 cardiovascular disease or fibrotic disease.

[0219] The cardiovascular disease in any aspect of the invention may be, for example, coronary heart disease, atherosclerosis or stroke.

[0220] For example, the fibrotic disease in any aspect of the invention may be myocardial infarction, angina pectoris, osteoarthritis, pulmonary fibrosis, asthma, cystic fibrosis or bronchitis.

[0221] In some aspects, provided herein are proteins comprising the amino acid sequences disclosed herein and in the forms disclosed herein for use in therapy.

[0222] In some aspects, the pharmaceutical composition may comprise a pharmaceutically acceptable excipient, carrier or diluent. A pharmaceutically acceptable excipient may be a compound or combination of compounds that does not cause secondary reactions and allows, for example, the administration of a protein as defined herein, an increase in its lifespan and / or its efficacy in the body, or an increase in its solubility in solution into the pharmaceutical composition. 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.

[0223] In some aspects, the protein as defined herein can be provided in lyophilized form for reconstitution prior to administration. For example, the lyophilized protein molecule can be reconstituted in sterile water and mixed with saline before administration to an individual.

[0224] Proteins as defined herein will typically be administered in the form of a pharmaceutical composition that may comprise at least one component in addition to the protein molecule. Thus, in addition to the protein as defined herein, the pharmaceutical composition may also comprise a pharmaceutically acceptable excipient, carrier, buffer, stabilizer, or other material well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the protein. The precise nature of the carrier or other material will depend on the route of administration, which may be a bolus, infusion, injection, or any other suitable route, as described below.

[0225] For parenteral administration (e.g., subcutaneous or intravenous administration, e.g., by injection), pharmaceutical compositions comprising a protein as defined herein may be in the form of a parenterally acceptable aqueous solution that is pyrogen-free and has a suitable pH, isotonicity, and stability. One skilled in the art will be able to prepare suitable solutions using, for example, isotonic vehicles such as sodium chloride injection, Ringer's injection, lactated Ringer's injection. Preservatives, stabilizers, buffers, antioxidants, and / or other additives may be used as needed, including buffers such as phosphates, citrates, and other organic acids; antioxidants (such as ascorbic acid and methionine); preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens (such as methyl paraben or propyl paraben); catechol; resorcinol; cyclohexanol; 3'-pentanol; and m-cresol); low molecular weight polypeptides; proteins 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 dextrins); chelating agents (such as EDTA); sugars (such as sucrose, mannitol, trehalose, or sorbitol); salt-forming counterions (such as sodium ions); metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants (such as TWEEN®). TM ,PLURONICS TM or polyethylene glycol (PEG)).

[0226] Pharmaceutical compositions comprising a protein as defined herein may be administered alone or in combination with other treatments (simultaneously or sequentially), depending on the condition to be treated.

[0227] The proteins as defined herein can be used in methods of treating the human or animal body, including prophylactic or preventative treatment (e.g., treatment that reduces the risk of developing a disorder in an individual before the onset of the disorder in the individual; delays the onset of the disorder; or reduces the severity of the disorder after onset). The methods of treatment may comprise administering a protein as defined herein to an individual in need thereof.

[0228] Administration is generally a "therapeutically effective amount", which is sufficient to show benefits to the patient. This benefit can be at least to improve at least one symptom. The actual amount administered and the rate and time course of administration will depend on the nature and severity of the disease to be treated, the specific mammal to be treated, the clinical condition of the individual patient, the cause of the disorder, the delivery site of the composition, the method of administration, the time schedule of administration and other factors known to the practitioner. The prescription of treatment (such as the decision on dosage, etc.) belongs to the responsibility of the general practitioner and other doctors and may depend on the severity and / or progression of the symptoms of the disease to be treated. The appropriate dosage of the antibody molecule is well known in the art (Ledermann JA et al., 1991, Int. J. Cancer 47: 659-664; Bagshawe KD et al., 1991, Antibody, Immunoconjugates and Radiopharmaceuticals 4: 915-922). Specific dosage can be indicated herein or in the Physician's Desk Reference (2003) because a dosage suitable for the type of drug administered can be used. A therapeutically effective amount or suitable dose of a protein as defined herein can be determined by comparing its in vitro and in vivo activity in animal models. Methods for extrapolating effective doses in mice and other experimental animals to humans are known. The precise dose will depend on many factors, including whether the protein is used for prevention or treatment, the size and location of the area to be treated, the precise nature of the protein (e.g., Fab2, IgG), and the nature of any detectable label or other molecule attached to the protein.

[0229] For systemic administration, typical protein dosage is in the range of 100 μ g to 1 g, and for local administration, typical protein dosage is in the range of 1 μ g to 1 mg. An initial higher loading dose can be used, followed by one or more lower dosages. In some aspects, the protein will comprise a complete antibody, such as an IgG1 or IgG4 isotype. This is a dosage for a single treatment of adult patients, which can be adjusted proportionally for children and infants, and also adjusted proportionally with molecular weight for other protein construct forms. Depending on the doctor's judgment, treatment can be repeated at intervals every day, twice a week, weekly or monthly. Individual treatment regimens can depend on the pharmacokinetic and pharmacodynamic properties, route of administration and the nature of the disease being treated of the protein composition.

[0230] Treatment can be cyclical, and the period between administrations can be about two weeks or longer, for example, 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 directly administered or applied to the anatomical site of surgical treatment or invasive surgery. Suitable formulations and routes of administration are as described above.

[0231] In some aspects, a protein as defined herein may be administered as a subcutaneous injection. Subcutaneous injections may be performed using an autoinjector, for example for long-term or short-term prophylaxis / treatment.

[0232] In some aspects, the therapeutic effect of a protein as defined herein can last for several times the half-life of the protein in serum, depending on the dosage. For example, the therapeutic effect of a single dose of a protein as defined herein can last 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 in an individual.

[0233] As used herein, the term "CD47" refers to IAP (integrin-associated protein) and variants thereof that retain at least some of the biological activity of CD47. As used herein, CD47 includes native sequence CD47 from all mammalian species, including humans, rats, mice, and chickens. In some embodiments, the term "CD47" is used to include variants, isoforms, and species homologs of human CD47. In some cases, as used herein, CD47 includes native sequence CD47 from all mammalian and non-mammalian species, including humans, monkeys, rats, mice, and chickens. In some embodiments, the term "CD47" refers only to wild-type CD47. The proteins of the present invention may cross-react with CD47 from species other than humans, particularly CD47 from cynomolgus monkeys (Macaca fascicularis). Examples of human and cynomolgus monkey CD47 amino acid sequences are provided in Table 11. In certain embodiments, the proteins of the present invention may be completely specific for human CD47 and may not exhibit non-human cross-reactivity.

[0234] As used herein, the term "cMET" refers to MET proteins and variants thereof that retain at least some of the biological activity of cMET. In some cases, as used herein, cMET includes native sequence cMET of all mammalian species (including humans, rats, mice, and chickens). In some embodiments, the term "cMET" can be used to include variants, isoforms, and species homologs of human cMET. In some cases, as used herein, cMET includes native sequence cMET of all mammalian and non-mammalian species (including humans, monkeys, rats, mice, and chickens). In some embodiments, the term "cMET" refers only to wild-type cMET. The antibodies of the present invention may cross-react with cMET from species other than humans, particularly cMET from cynomolgus monkeys (branch monkeys). Examples of human and cynomolgus monkey cMET amino acid sequences are provided in Table 12. In certain embodiments, the antibodies may be completely specific for human cMET and may not exhibit non-human cross-reactivity.

[0235] As used herein, the term "Her2" refers to human epidermal growth factor receptor 2 proteins and variants thereof that retain at least some of the biological activity of Her2. Her2 is also known as HER2 / neu, ErbB2, c-erbB-2, and human EGF receptor 2. In some embodiments, the term "Her2" can be used to include variants, isoforms, and species homologs of human Her2. In some cases, as used herein, Her2 includes the native sequence Her2 (also referred to as ErbB2) of all mammalian and non-mammalian species (including humans, monkeys, rats, mice, and chickens). In some embodiments, the term "Her2" refers only to wild-type Her2. The antibodies of the present invention can cross-react with Her2 (particularly Her2 from cynomolgus monkeys (branch monkeys)) from species other than humans. Examples of human and cynomolgus monkey Her2 / ErbB2 amino acid sequences are provided in Table 15. In certain embodiments, antibodies can be completely specific to human Her2 and may not exhibit non-human cross-reactivity.

[0236] As used herein, the term "CD3" refers to a "cluster of differentiation 3" multimeric protein complex and variants thereof that retain at least some of the biological activity of CD3. The CD3 complex comprises four different 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" can be used to include variants, isoforms, and species homologs of human CD3. In some cases, as used herein, CD3 includes natural sequence CD3 of all mammalian and non-mammalian species (including humans, monkeys, rats, mice, and chickens). 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 humans, particularly CD3 from cynomolgus monkeys (branching monkeys). Examples of human and cynomolgus monkey CD3ε amino acid sequences are provided in Table 16. In certain embodiments, the antibodies may be completely specific to human CD3 and may not exhibit non-human cross-reactivity.

[0237] As used herein, an "antagonist," as used in the context of a protein of the present invention, refers to a protein that is capable of binding to a molecule expressed in a diseased tissue and inhibiting the biological activity of the molecule and / or downstream pathways mediated by the molecule. For example, an "anti-CD47 antagonist protein" (interchangeably referred to as an "anti-CD47 protein") refers to a protein that is capable of binding to CD47 and inhibiting CD47 biological activity and / or downstream pathways mediated by CD47 signaling. Anti-CD47 antagonists encompass proteins that can block, antagonize, suppress, or reduce (including significantly reduce) CD47 biological activity (including downstream pathways mediated by CD47 signaling, such as receptor binding and / or eliciting a cellular response to CD47). For the purposes of the present invention, it should be clearly understood that the term "anti-CD47 antagonist protein" encompasses all terms, names, functional states, and characteristics in which CD47 itself and CD47 biological activity (including but not limited to its ability to enhance activation by myeloid lineage cells) or the results of that activity or biological activity are substantially ineffective, reduced, or neutralized to any meaningful extent.

[0238] A protein of the invention "specifically binds," "specifically interacts," "preferentially binds," "binds," or "interacts" with a molecule (e.g., human CD47, human Her2, human CD3, human cMET, or human PD-L1) if the protein of the invention binds with greater affinity, avidity, more readily, and / or for a longer duration than it binds to other molecules.

[0239] An "antibody molecule" is an immunoglobulin molecule that can specifically bind to a target (such as a carbohydrate, polynucleotide, lipid, polypeptide, etc.) through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, the term "antibody molecule" encompasses not only complete polyclonal or monoclonal antibodies, but also any antigen-binding fragment (e.g., "antigen-binding portion") or a single chain thereof, a fusion protein comprising an antibody, and any other modified configuration of an immunoglobulin molecule comprising an antigen recognition site, including, for example, but not limited to, scFv, single domain antibodies (e.g., shark and camelid antibodies), maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFv.

[0240] " Antibody molecule " encompasses any kind of antibody, such as IgG, IgA or IgM (or its subclass), and the antibody does not need to be any specific kind. Depending on the amino acid sequence of the antibody heavy chain constant region, 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), for example, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy chain constant region corresponding to the immunoglobulins of different classes is referred to as α, δ, ε, γ and μ respectively. The subunit structure and the three-dimensional configuration of different classes of immunoglobulins are well known.

[0241] As used herein, the term "antigen-binding portion" of an antibody molecule refers to one or more fragments of an intact antibody that retains the ability to specifically bind to an antigen. The antigen-binding function of an antibody molecule can be performed by fragments of intact antibodies. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody molecule include Fab; Fab'; F(ab')2; an Fd fragment consisting of a VH and CH1 domain; an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; a single domain antibody (dAb) fragment; and isolated complementary determining regions (CDRs).

[0242] 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 extending from the amino acid residue at position Cys226 or from Pro230 to its carboxyl terminus. The numbering of the residues in the Fc region is that of the EU index as described in Kabat. The Fc region of an immunoglobulin generally comprises two constant domains, CH2 and CH3. As is known in the art, the Fc region can exist in dimer or monomer form.

[0243] The "variable region" of an antibody refers to the variable region of the antibody light chain or the variable region of the antibody heavy chain, either alone or in combination. As known in the art, the variable regions of the heavy and light chains are each composed of four framework regions (FRs) connected by three complementary determining regions (CDRs) (also referred to as hypervariable regions), and contribute to the formation of the antigen binding site of the antibody. When selecting FR flanking CDRs, for example, when humanizing or optimizing an antibody, it is preferred that the FRs be from antibodies containing the same canonical class CDR sequences.

[0244] As used herein, the term "conservative substitution" refers to the replacement of an amino acid by another amino acid that does not significantly deleteriously alter the functional activity. A preferred example of a "conservative substitution" is the replacement of an amino acid by another amino acid that has a value ≥ 0 in the following BLOSUM 62 substitution matrix (see Henikoff and Henikoff, 1992, PNAS 89: 10915-10919):

[0245]

[0246] The term "monoclonal antibody" (Mab) refers to an antibody or antigen-binding portion thereof that is derived from a single copy or clone (including, for example, any eukaryotic, prokaryotic, or phage clone), regardless of the method by which it is produced. Preferably, the monoclonal antibodies of the invention are present in a homogeneous or substantially homogeneous population.

[0247] A "humanized" antibody molecule refers to a form of a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof (such as a non-human (e.g., murine) antibody molecule or antigen-binding portion thereof) that contains minimal sequence derived from a non-human immunoglobulin, immunoglobulin chain, or fragment thereof (such as an antibody's Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequence). A humanized antibody can be a human immunoglobulin (recipient antibody) in which residues from a recipient CDR are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity.

[0248] "Human antibody" or "fully human antibody" refers to an antibody molecule or antigen-binding portion thereof derived from a transgenic mouse carrying human antibody genes or from human cells.

[0249] The term "chimeric antibody" is intended to refer to antibody molecules, or antigen-binding portions thereof, in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as antibody molecules in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody.

[0250] The term "immunoconjugate" refers to a protein of the invention conjugated, fused or linked to at least one cytotoxic, cytostatic or therapeutic agent.

[0251] The proteins of the present invention can be produced using techniques well known in the art, such as recombinant technology, phage display technology, synthetic technology, or a combination of such technologies or other technologies readily known in the art.

[0252] The term "isolated molecule" (wherein the molecule is, for example, a polypeptide, polynucleotide, or antibody) is a molecule that, by reason of its source or derivation, is (1) not associated with naturally associated components that accompany it in its native state, (2) substantially free of other molecules from the same species, (3) expressed by cells from a different species, or (4) not found in nature. Thus, a molecule that is chemically synthesized or expressed in a cellular system different from the cells from which it naturally originates will be "isolated" from its naturally associated components. A molecule can also be rendered substantially free of naturally associated components by separation using purification techniques well known in the art. Molecular purity or homogeneity can be determined by a variety of means well known in the art. For example, the purity of a polypeptide sample can be determined using polyacrylamide gel electrophoresis and staining the gel to visualize the polypeptide using techniques well known in the art. For some purposes, higher resolution can be provided by using HPLC or other means well known in the art for purification.

[0253] The term "epitope" refers to a molecular moiety that can be recognized and bound by a protein, antibody molecule, or its antigen-binding portion thereof of the present invention at one or more antigen-binding regions of a protein or antibody molecule. An epitope can be composed of a limited region of primary, secondary, or tertiary protein structure, and includes a combination of secondary structural units or structural domains of a target identified by the antigen-binding region of a protein, antibody, or its antigen-binding portion thereof. An epitope can also be composed of a limited chemically active surface grouping of molecules such as amino acids or sugar side chains, and has specific three-dimensional structural characteristics and specific charge characteristics. As used herein, the term "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 (e.g., NMR).

[0254] The term "binding affinity" or "KD" refers to the dissociation rate of a particular antigen-binding protein interaction or antigen-antibody interaction. KD is the dissociation rate (also known as the "dissociation rate (k off )") and the association rate or "binding rate (k on )”. Therefore, K D Equal to k off / k on And it is expressed as molar concentration (M). So K D The smaller the K, the stronger the binding affinity. Therefore, a K of 1 μM D showed a K of 1 nM D The KD value of a binding protein or antibody can be determined using methods established 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.

[0255] The term "potency" is a measure of biological activity and may be assigned as IC 50 or an immunoconjugate of the invention is an effective concentration of a protein to its binding partner (e.g., a molecule expressed in a diseased tissue) or antigen that inhibits 50% of the activity of the binding partner or antigen measured in an activity assay as described herein.

[0256] As used herein, the phrase "effective amount" or "therapeutically effective amount" refers to the amount (in dosage form and for a sustained period of time and with respect to the means of administration) necessary to achieve the desired therapeutic result. An effective amount is at least the minimum amount of an active agent necessary to confer a therapeutic benefit on a subject, but less than a toxic amount.

[0257] As used herein, the term "inhibit" or "neutralize" with respect to a biological activity of a protein of the invention means the ability of the protein to substantially antagonize, inhibit, prevent, inhibit, slow, destroy, eliminate, stop, reduce or reverse, for example, the progression or severity of the inhibited biological activity (including but not limited to the biological activity of a molecule expressed in a diseased tissue or a binding interaction).

[0258] "Host cell" includes a single cell or cell culture that can be or has been a recipient of a vector for incorporating a polynucleotide insert. Host cells include the progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. Host cells include cells transfected in vivo with a polynucleotide of the invention.

[0259] 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 a cationic condensing agent; DNA or RNA expression vectors encapsulated in liposomes; and certain eukaryotic cells, such as producer cells.

[0260] Unless otherwise indicated, the term "treat" as used herein means reversing, alleviating, inhibiting the progression of the disorder or condition to which the term applies, or one or more symptoms of such disorder or condition, delaying its progression, delaying its onset, or preventing the disorder or condition, or one or more symptoms of the disorder or condition. Unless otherwise indicated, the term "treat" as used herein refers to the act of treating as defined above. The term "treatment" also includes adjuvant and neoadjuvant treatment of the subject. For the avoidance of doubt, references to "treatment" herein include references to curative, palliative, and preventive treatments. For the avoidance of doubt, references to "treatment" herein also include references to curative, palliative, and preventive treatments.

[0261] It should be understood that whenever an embodiment is described herein with the wording "comprising," other similar embodiments described with "consisting of" and / or "consisting essentially of" are also provided.

[0262] Where aspects or embodiments of the invention are described in terms of Markush groups or other alternative groupings, the invention encompasses not only the entire group listed as a whole, but also each member of the individual group and all possible subgroups of the larger group, and also encompasses the larger group without one or more of the group members. The invention also contemplates the specific exclusion of one or more of any group members from the claimed invention.

[0263] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention pertains. In the event of conflict, the present specification (including definitions) will prevail. Throughout this specification and claims, the word "comprise" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a recited integer or group of integers, but not the exclusion of any other integer or group of integers. Furthermore, unless the context requires otherwise, singular terms shall include the plural and plural terms shall include the singular. Any examples following the term "e.g." or "for example" are not intended to be exhaustive or limiting.

[0264] 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.

[0265] Specific non-limiting embodiments of the present invention will now be described with reference to the accompanying drawings.

[0266] Example

[0267] Example 1. Generation of optimized conditionally active therapeutic antibodies

[0268] introduction

[0269] In this example, we 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.

[0270] Materials and methods

[0271] Protein cloning, transient expression, purification, and characterization

[0272] The antibody-encoding DNA sequences were 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. Fab was constructed using a knob-in-hole (KIH) heavy chain expression vector (CH3 domain T366W and T366S / L368A / Y407V mutations). 2 cMETCD47 and Her2CD3 protein constructs. Fab 2 The Her2-CD3 construct also includes the effector function ablation 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.

[0273] HiTrap MabSelect Sure Protein A 5mL column was used in The antibody of generation is captured from the supernatant of clarification on Pure 150LF PLC system.By directly loading the protein A peak fraction of wash-out on HiPrep 26 / 10 desalting column, immediately the protein peak buffer of wash-out is exchanged in 1x PBS pH 7.4.Determine protein concentration by measuring the absorbance at 280nm, and the protein of 1 μ g often kind of purification uses 4-20% TGX polyacrylamide gradient gel (BioRad, catalog number (Cat. No.) 456-1093) to analyze with 1x Tris / glycine / SDS buffer under reducing and / or non-reducing conditions by SDS-PAGE, by 120V field separation 1 hour.In order to test the existence of the aggregation of non-covalent bond and to supplement SDS PAGE analysis, carried out analytical size exclusion chromatography. Aliquots of selected clones were analyzed by analytical size exclusion chromatography (SEC) in isocratic mode using a Superdex200 Increase 10 / 300 SEC column and 1 x PBS pH 7.4 as running buffer.

[0274] The selected proteins were further purified using preparative SEC. Up to 1 ml of the antibody sample was loaded 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. 1 ml fractions of the peak of interest were collected and the main peak fractions were combined. Following size exclusion chromatography, the samples were analyzed again by SDS-PAGE as described above.

[0275] Hemagglutination

[0276] Red blood cells (RBCs) were isolated from fresh, non-agglutinated human blood (minimum 3 different donors), diluted to 2% in PBS, and incubated for 60-90 minutes in U-bottom 96-well plates with titrations of IgG or protein constructs. In the absence of hemagglutination, cells settled to the bottom of the wells, forming a red precipitate. Hemagglutination was observed as a non-settling RBC solution. Images of each plate were recorded, and the data for each sample were expressed as the titer of the last well in which hemagglutination was observed.

[0277] Metalloproteinase digestion

[0278] The protein constructs were 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 for 16 hours at 37° C. The reaction was terminated by the addition of 20 mM EDTA, and the samples were tested for binding or functional activity as described.

[0279] IgG titration binding ELISA

[0280] In order to coat the ELISA plate, the target protein was diluted to 1 μg / ml in PBS pH 7.4 and added at 100 μl / well, overnight at 4°C. The coated plate was washed 3 times with PBS pH 7.4, blocked with 4% skim milk protein (380 μl / well) in PBS for 1 hour at room temperature, and then washed 5 times with PBS-Tween 20 (PBST). Antibodies were then added (100 μl / well; diluted in PBST) and incubated at room temperature for 1 hour. The plate was then washed 3 times with PBS and goat anti-human IgG-HRP (100 μl / well) was added at room temperature for 1 hour. The plate was then washed 3 times with PBST and washed twice with PBS, and then 100 μl TMB was added to each well. The reaction was terminated by adding 100 μl 2MH2SO4 / well, and OD was read at 450nm on a plate reader.

[0281] Flow cytometry combined

[0282] Binding of protein constructs (+ / - pre-digestion with MMP3 / 7 / 12) and control IgG to Jurkat and BT-474 cells was assessed by flow cytometry. TM Fixable Viability dye (Biolegend) was used to identify viable cells. Binding of human IgG to the protein construct was detected using a FITC-conjugated goat anti-human (H+L) secondary antibody. Binding of a 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 viable cells in the FITC channel of a BD Fortessa flow cytometer.

[0283] T cell activation bioassay

[0284] The functional activity of Her2CD3 protein constructs was assessed in a co-culture assay using BT474 cells and the NFAT-RE-luciferase Jurkat reporter cell line (Promega – TCR / CD3 Effector Cells NFAT). BT-474 cells (40,000 cells / well) were seeded in 96-well, white, clear-bottom tissue culture-treated plates in Hybri-Care medium (ATCC) supplemented with 10% FBS and incubated overnight at 37°C in a CO2 incubator. The medium was removed, and control antibodies or protein constructs (+ / - pre-digested with MMP3 / 7 / 12) prepared in assay medium (RPMI supplemented with 10% FBS) were added to the cells. TCR / CD3 effector cells (NFAT) were thawed and diluted according to the manufacturer's protocol before being added to the assay wells. After incubation at 37°C for 6 hours in a CO2 incubator, 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). Fold induction was determined by calculating the ratio of RLU / RLU of samples in the absence of antibody after subtracting the background luminescence signal.

[0285] Molecular dynamics simulation

[0286] The AMBER10:EHT force field in MOE (Chemical Computing Group Inc) was used to model and optimize eight systems (equivalent sequences with a broken covalent bond in one of the joints of the GS sequence positions XC1, XC2, XC3 and XC4). The histidine charge was assigned using the protonate3D tool in MOE. Three rounds of continuous minimization were performed with a final gradient of 0.001, allowing 10,000 steps without restrictions. The single system was constrained once again using the CHARMM27 force field in NAMD 2.13 and gradually minimized 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, and the third step released all atoms, allowing them to move without restriction. Minimization in CHARMM27 is easy because it is a force field that works in NAMD molecular dynamics simulations.

[0287] For the kinetic runs, generalized Born (GB) solvation was used to describe solvent effects. MD simulations were set up and then completed using NAMD 2.13. Four equilibration steps were performed to gradually relax the harmonic restraints in 125 ps increments with a total equilibration time of 500 ps. The first step heated the system 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 (constants (N), volume (V) and temperature (T)). A transfer function is applied at to shorten the van der Waals potential function. Following the recommendations of NAMD, the electrostatic and vdW cutoffs are set to Periodic boundary conditions were not used because they were incompatible with the implicit solvent approach. Constraints imposed during equilibrium were removed to freely simulate the antibody complex. Production runs were performed at 310K for 6, 15, 20, or 100 ns. 100 ns runs were applied to the cleaved adaptor protein XC1-4 to fully explore the range of motion, while shorter runs (up to 20 ns) were found to be sufficient to locate the range of motion for the uncleaved antibody construct X1-4.

[0288] In vivo PK and tolerability analysis

[0289] Tolerance Study - Twenty-eight (28) 6-8 week old male B6.Cg-Fcgrt tm1Dcr Tg(FCGRT)32DcrJ ('Tg32' homozygous human FcRn transgenic, JAX reserve #014565) mice were divided into 7 groups of 4 mice each. Body weight was measured on the day of antibody administration. At 0 hours, the test article was administered by intravenous (IV) injection at a dose volume of 2 mg / kg or 10 mg / kg and 10 ml / kg. 200 μL whole blood samples were collected in EDTA on days 5, 29, and 60 (terminal bleed). Blood was analyzed for CBC / Dif / Retic, including white blood cells, neutrophils, eosinophils, basophils, lymphocytes, monocytes, hemoglobin, red blood cells, reticulocytes, MCHC, and MCV. Body weight was then monitored weekly for the first month and then monthly until the end of the experiment.

[0290] Pharmacokinetic study - Thirty-two 6-8 week old male B6.Cg-Fcgrt tm1DcrTg(FCGRT)32DcrJ ('Tg32' homozygous human FcRn transgenic, JAX reserve #014565) mice were divided into 8 groups of 4 mice each. One day before administration of the test article, 35 μL blood samples were collected from three (3) Tg32 mice into EDTA to test the binding of the test article to mouse red blood cells using flow cytometry. Body weight was measured on the day of antibody administration and weekly until the end of the experiment. 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 concentrations.

[0291] Tolerability Study in NOD-SCID Mice - NOD-SCID mice were divided into four groups of three 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, followed by three subsequent doses at 4 mg / kg or 7 mg / kg at 5-day intervals.

[0292] Flow cytometric analysis of binding to monkey and human erythrocytes

[0293] Erythrocytes were isolated from 3 Cynomolgus monkeys and 3 human donors. For each sample, 5x10 5 Cells (diluted in DMEM + 5% FBS) were stained for one hour with the following substances: A-D5IgG1: 0.0032, 0.016, 0.03, 50 μg / mL; trastuzumab: 0.0032, 0.016, 0.03, 50 μg / mL; IgG 2 Her47LHL-LHLF 0 hour: 0.016, 0.08, 0.4, 2, 10, 50 μg / mL; IgG 2 Her47LHL-LHL 0 hour: 0.016, 0.08, 0.4, 2, 10, 50 μg / mL. Binding of test samples to erythrocytes was measured using FITCAffiniPure goat anti-human IgG (1:200 dilution; 1 hour incubation time), followed by flow cytometry measurement of FITC fluorescence intensity (BD LSR Fortessa X-20 cell analyzer).

[0294] Metalloproteinase and cathepsin digestion at pH 6.0 and 7.4

[0295] The protein constructs were placed in TBS (containing 5 mM CaCl2, pH 6.0 or 7.4) and then incubated with individual human matrix metalloproteinases (MMPs) or cathepsins at a ratio of 1% total enzyme to protein construct (wt / wt) at 37°C for 0 hours, 2 hours, 4 hours, 8 hours, and 24 hours. The reaction was stopped by adding 20 mM EDTA, and the samples were frozen before testing for binding or functional activity as described.

[0296] In vitro protein stability analysis

[0297] Forced Oxidation - For forced oxidation analysis, the test articles in PBS were treated with 0.5% H2O2 at room temperature for 2 hours and then stored at -80°C before SEC and RP analysis (intact antibodies and subunits, tryptic peptides) on a Dionex Ultimate 3000RS HPLC system (ThermoFisher Scientific, Hemel Hempstead, UK). For intact antibody reduction, DTT was added to a final concentration of 0.33M and the samples were incubated at 22°C for 1 hour and immediately analyzed by RP.

[0298] SEC analysis—The HPLC was performed using an Acquity UPLC Protein BEH SEC column ( 1.7 μm, 4.6 mm × 150 mm (Waters, Elstree, UK)) and Acquity UPLC Protein BEH SEC guard column (30 × 4.6 mm, 1.7 μm, Chromatographic separation was performed using a HPLC-MS / MS analyzer (Waters, Elstree, UK). The method involved isocratic elution over 10 minutes with a mobile phase of 0.2 M potassium phosphate, pH 6.8, and 0.2 M potassium chloride. The flow rate was 0.35 mL / min. Detection was by UV absorption at 280 nm.

[0299] Reverse-Phase Analysis of Intact Antibodies and Subunits - Chromatographic separations were performed using a PLRP-S1000 (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 consisted of a linear gradient from 75% buffer A (0.02% TFA, 7.5% acetonitrile in H₂O) to 45% buffer B (0.02% TFA, 7.5% H₂O in acetonitrile) over 14 minutes. The flow rate was 0.5 mL / min, and the temperature was maintained at 70°C throughout the analysis. Detection was by UV absorption at 280 nm.

[0300] HIC Analysis - Chromatographic separation was performed using a TSKgel Butyl-NPR 4.6 mm × 35 mm HIC column (TOSOHBioscience Ltd., Reading, UK) connected to a Dionex Ultimate 3000RS HPLC system (ThermoFisher Scientific, Hemel Hempstead, UK). The method consisted of 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) over 9 minutes. The flow rate was 1.2 mL / min. Detection was by UV absorption at 280 nm.

[0301] Charge Variant Assay - The charge variant profile of the test articles was determined by Protein Charge Variant Assay on a LabChip GXII Touch HT (PerkinElmer) according to the manufacturer's protocol.

[0302] Fc affinity of human Fc receptors analyze

[0303] use The T200 instrument determines 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 antibodies via standard amine coupling. Receptor-specific assay formats were then applied as described below.

[0304] FcγRI is a high-affinity receptor for IgG1 monomers, so 1:1 kinetic analysis was performed under the following conditions: "Single-cycle" analysis used a flow rate of 30 μl / min, receptor protein was loaded at 10 μl / min to approximately 30 RU (diluted to 0.25 μg / ml in HBS-P+), a five-point three-fold dilution of purified antibody titrated from 0.411 nM to 33.33 nM, an association time of 200 s, and a dissociation time of 300 s. Regeneration was performed with 2x injections of glycine pH 1.5, and analysis was performed using a 1:1 fit.

[0305] 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 loading at 10 μl / min to approximately 60 RU (0.25 μg / ml diluted in HBS-P+), a 5-point three-fold dilution series of purified antibody titrated between 33 nM and 24000 nM, an association time of 30 s and a dissociation time of 25 s. Regeneration was performed with 2x injections of glycine pH 1.5 and analysis was performed using steady-state affinity calculations.

[0306] Results and discussion

[0307] Principles of protein construct design

[0308] Standard anti-cancer antigen antibodies are subject to significant pharmacological challenges in the treatment of solid tumors. A key issue limiting efficacy in this type of potential drug is that the antigen targeted by the antibody is not entirely found in the tumor, but is only highly overexpressed in the tumor. This off-tumor target expression often leads to dose-limiting side effects and antigen "pooling" effects, in which large doses of antibody must be given to ensure that enough antibody penetrates the tumor to have a therapeutic effect. One such example is the class of antibodies targeting the antigen CD47 ( Figure 1A ), among other challenges: the high expression of CD47 in the bloodstream (e.g., particularly on red blood cells and platelets) is a "pool" bound by intravenously administered antibodies, minimizing the amount of drug that penetrates the tumor (even when large doses of IgG are given). Binding of blood cells by anti-CD47 is also a significant toxicity risk. In fact, anti-CD47 antibodies are known to cause anemia and even cross-linking of human red blood cells, creating a risk of hemagglutination in patients. In addition, tumors are often "hostile" environments with high expression rates of enzymes such as MMPs 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.

[0309] Anti-CD47 protein constructs ( Figure 1B ) aims to overcome the peripheral pool and toxicity problems 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 the binding domain of a high-risk lower domain (such as CD47) (n-terminus). The use of an appropriate upper domain / linker combination results in a configuration that completely blocks binding activity in the lower CD47 domain. The tumor targeting domain (e.g., Her2) then drives high concentrations in the tumor environment, and the protein construct linker system utilizes elevated MMP activity in the tumor to cut the linker peptide, thereby exposing the CD47 binding domain and thereby conditionally activating 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.

[0310] 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 immunoglobulin hinge region and Fc domain. Each Fab A-linker domain (top) blocks the binding activity of Fab B (bottom). The target binding specificity of the upper and lower domains can be different to drive bispecific function, or the same to drive multivalent target interactions. The selection of linker sequences (such as lower hinge peptide sequences) produces a structure that will be locked in non-diseased tissues, but is rapidly cut 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, with the first 'fast' cleavage taking the 'locked' intact structure and generating an intermediate 'unlocked' active state, which allows Fab A and B from a single protein construct to bind to their cognate targets. A 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 an "dissociated" form in which the lower Fab domain is completely released for non-targeted (but potentially still localized) activity. Cleavage linkers based on immunoglobulin hinge sequences can also recruit increased immune effector functions (ADCC, CDC, and ADCP) on the cell membrane via endogenous anti-hinge antibodies, a known phenomenon in human patients with (or even without) underlying autoreactive diseases.

[0311] 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. Figure 3A ) or three ( Figure 3B ) polypeptide chain encodes two Fab domains (1xFab A, 1X Fab B), two or more linker sequences, and may or may not have an immunoglobulin hinge region and an Fc domain, wherein the pairing of heterodimers may or may not be driven by mutations in the Fc. Each Fab A linker domain again blocks the binding activity of Fab B, and the choice of linker sequence (such as the lower hinge peptide sequence) creates a structure that will be locked in non-diseased tissue, but in the presence of high concentrations of linker-cleaving proteases in the tumor environment, it is rapidly cleaved and unlocked, ultimately becoming dissociated ( Figure 3A ).

[0312] Protein construct cloning and expression

[0313] To generate and purify 15 Fabs with different linker domains 2 or IgG 2 The bispecific conditionally active protein constructs (Table 1) were synthesized for each construct type (Table 2) and cloned into expression vectors encoding either human IgG1 heavy and light chains or "knob-in-hole" heterodimer Fc. Proteins were named using the nomenclature (format)-(target name [upper domain / lower domain])-(heavy chain linker type)-(light chain linker type). All proteins were produced by transient transfection of CHO cells and then purified by protein A affinity chromatography.

[0314] The anti-HER2 variable region sequences used in the protein constructs disclosed herein are those of trastuzumab. The anti-CD3 variable region sequences used in the protein constructs disclosed herein are those of OKT3 or SP34. Anti-cMET variable region sequences for use in the protein constructs disclosed herein are provided in WO 2019 / 175186. Anti-CD47 variable region sequences for use in the protein constructs disclosed herein are provided in WO 2019 / 034895.

[0315] Analysis of protein construct expression and purification characteristics

[0316] Protein A purified proteins were quantified to show that the use of different linker types affected the expression yield (Table 2). Protein preparations in 1x PBS pH 7.4 were also tested in analytical size exclusion chromatography to quantify the percentage of desired product. Of all three types of bispecific proteins produced (cMETCD47, Her2CD47, and Her2CD3), the construct containing the LHL linker produced the best combination of the highest yield and % yield of the desired product main peak as measured by analytical SEC (Table 2). SDS-PAGE analysis of protein A purified proteins ( Figure 4 ) also demonstrated that clones containing the short linker domains 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 undesirable multimers and degradation products. For all constructs, HMW impurities were essentially absent in the reduced lanes, indicating that these HMW impurities are disulfide-bonded dimers or higher order aggregates that are not reduced by SDS alone. For IgG 2 Design, clones 12 and 14 showed high yields (Table 2), but clone 14 (containing the LHL linker) was significantly lower than clone 14 by SEC (90%) and SDS-PAGE ( Figure 4 ) measurements confirmed the highest yield and high homogeneity of the desired product.

[0317] The remaining protein samples from the subset of clones were then subjected to SEC chromatography in an attempt to produce completely purified monomeric protein. The 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 clone 1 ( Figure 5A )、3( Figure 5C )、4( Figure 5D )、5( Figure 5E ) and 12( Figure 5G ) contains a large percentage of higher molecular weight products and generally lower molecular weight products (at Figures 5A-5H In contrast, clone 6 ( Figure 5F )、14( Figure 5H ) and 10( Figure 5I ) showed a prominent, well-defined major product peak. For clones 2 and 6, this allowed efficient SEC purification of proteins that were primarily monomeric, as demonstrated by SDS-PAGE of unreduced samples ( Figure 6A Attempted SEC purification of clones 1, 4, and 5 failed, yielding samples that remained heterogeneous ( Figure 6A ). From Her2CD3Fab 2SEC-purified proteins from clones 7, 8, and 10 showed improved homogeneity, whereas Her2CD47IgG 2 Of the clones, only clone 14 achieved complete homogeneity and monomeric state, as confirmed by SDS-PAGE of unreduced and reduced samples ( Figure 6B ). Furthermore, these findings demonstrate that clones 6, 10, and 14, which all contained LHL linkers on both chains, exhibited the most reproducible beneficial characteristics.

[0318] Functional characterization of a protein construct containing the CD47 antibody v domain in the lower Fab

[0319] Control IgG antibodies A-D5 anti-CD47, MH7.1 anti-C-MET, anti-Her2 trastuzumab, and A-D5Fab-Fc (a monovalent form of the A-D5 antibody containing a single Fab domain) were titrated (in μg / ml) in direct binding ELISAs against human CD47, C-MET, and Her2 proteins. Figure 7A IgG was also analyzed in the same manner. 2 The Her2CD47-LH-LH and Her2CD47-LHL-LHL clones ( Figure 7B ) and Fab 2 The forms of cMETCD47-L2-L2 and cMETCD47-LHL-LHL ( Figure 7C The control antibody demonstrated the expected strong binding activity against its cognate target, even at the highest concentration, with little or no background against any other target ( Figure 7A ).

[0320] Importantly, the very strong monovalent binding of A-D5Fab-Fc ( Figure 7A ) demonstrated the intrinsic affinity of the A-D5 anti-CD47 domain and the remarkable efficacy required to lock into the protein construct format of the present invention to make it successful. Protein construct Her2CD47-LHL-LHL ( Figure 7B ) and cMETCD47-LHL-LHL( Figure 7C ) also showed similar strong, highly specific binding to its cognate target of the upper Fab domain, but no binding signal to CD47, indicating that the binding activity of the CD47v-domain was indeed completely inhibited when this linker combination was used. Importantly, a high background signal to human CD47 was observed for Her2CD47-LH-LH and to a lesser extent for cMETCD47-L2-L2, indicating that the abolition of the lower fab binding affinity was strictly controlled by the linker choice. These findings indicate that the protein construct Her2CD47-LHL-LHL ( containing the anti-CD47 binding domain of antibody A-D5 tested in this assay Figure 7B ) and cMETCD47-LHL-LHL( Figure 7C ) was less able to bind CD47 than A-D5IgG1 by >1000-fold, with approximately 1×10 -2 A binding signal OD of 1.0 was achieved at 10 μg / ml, whereas at the highest tested concentration of 10 μg / ml, neither protein construct showed a signal higher than 0.2.

[0321] Since hemagglutination is the main toxicity risk of anti-CD47 antibodies, the preferred protein constructs were then tested in a hemagglutination assay based on human erythrocytes. Control antibodies anti-CD235a (mouse) and A-D5 anti-CD47, A-D5Fab-Fc, MH7.1 anti-C-MET, anti-Her2 trastuzumab, IgG 2 The Her2CD47-LH-LH and Her2CD47-LHL-LHL formats and the Fab 2 cMETCD47-L2-L2 and cMETCD47-LHL-LHL were used in human erythrocyte hemagglutination assays from healthy donor 1 ( Figure 8A ), Donor 2( Figure 8B ) and donor 3 ( Figure 8C ) were titrated (in nM) with fresh red blood cells of 140 nM. For all 3 donors, the control antibodies anti-CD235a, A-D5 anti-CD47 and A-D5Fab-Fc showed effective concentration-dependent hemagglutination due to cross-linking of their respective surface antigens on adjacent red blood cells. The low efficacy hemagglutination observed for A-D5Fab-Fc (clone 15) may be due to the presence of a small amount of functional dimer in this protein preparation, as it was only purified by protein A column and not completely purified to a monomeric state by SEC. Importantly, even at the highest concentration of 140 nM, none of the protein construct samples showed any ability to induce hemagglutination. This finding indicates that the IgG containing the anti-CD47 binding domain of antibody A-D5 tested in this assay is superior to the A-D5IgG1 that showed a titer of 0.58 nM. 2 and Fab 2 The ability of the formal protein construct to induce aggregation was reduced >241-fold.

[0322] The prioritized 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 a 24-hour incubation in buffer without enzyme as a negative control. Samples from these digestion time courses were then applied to a PCR reaction targeting human Her2 and CD47 ( Figure 9A 、 9B ) or human C-MET and CD47 ( Figure 9C) direct binding ELISA. In each case, no loss of binding to Her2 or C-MET was observed over time, indicating that the addition of the protease did not reduce the functional binding capacity of the upper Fab domain. In contrast, the CD47 binding capacity of all three protein constructs increased significantly over time in an MMP-dependent manner. 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, cloned IgG 2 Her2CD47-LHL-LHL( Figure 9B ) and Fab 2 cMETCD47-LHL-LHL( Figure 9C ) again exhibited low background binding to CD47 (OD 450 nM <0.2), and binding to CD47 increased rapidly starting at 2 hours of incubation with both MMP7 and MMP12, reaching complete saturation (OD 450 nM of approximately 4.0) at 24 hours of incubation. MMP3 appeared to be the slowest to activate of the three MMPs, showing an increase in CD47 binding signal after 24 hours for all three protein construct examples.

[0323] Functional characterization of a protein construct containing the CD3 antibody v domain in the lower Fab

[0324] Antibody Fab 2 Her2CD3-L1-LH、Fab 2 Her2CD3-L2-L2 and Fab 2 Her2CD3-LHL-LHL was detected by ELISA ( Figure 10A ), flow cytometry ( Figure 10B ) and CD3 reporter assay ( Figure 10C In the ELISA assay, all three proteins showed the expected strong binding activity to Her2 (upper Fab domain), with little or no background binding to any other targets even at the highest concentrations ( Figure 10A ). All three protein constructs Fab were then 2 Proteins were incubated overnight in the presence or absence of mixed MMP3, MMP7, and MMP12. In flow cytometric binding to the HER2+ human cell line BT474, anti-HER2 trastuzumab showed strong binding, and Fab 2 Her2CD3-L1-LH and Fab 2 Her2CD3-L2-L2 showed similar strong binding before and after MMP digestion, while Fab2 Her2CD3-LHL-LHL showed partially reduced binding after MMP digestion, indicating that a portion of the protein lost its upper fab in the 'dissociated' state ( Figure 10B ). This finding suggests that MMP digestion is involved in the Fab 2 Since Her2+ BT474 cells were active in the presence of CD3-LHL-LHL, the same samples were used in a reporter assay in which Her2+ BT474 cells were mixed with human CD3+ Jurkat cells 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 reporter cells, as expected ( Figure 10C Each protein construct exhibited different characteristics: the Her2CD3-L2-L2 protein containing a 2xG4S linker (not cleavable by MMP proteases) showed high background in the assay, while no increase in CD3 activation signal after MMP digestion indicated 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 not CD3 activation, to co-ligate ( Figure 10C ). Fab 2 The Her2CD3-L1-LH protein showed low background activity in the assay, with a modest increase in signal after MMP digestion. 2 Her2CD3-LHL-LHL exhibited no measurable background signaling, showed minimal CD3 activation similar to the negative control trastuzumab and the IgG1 isotype control antibody in undigested samples, but showed potent activation in MMP-digested samples. Figure 10A The data in -C show that Her2CD3-LHL-LHL Fab 2 The format has an optimal combination of properties in that it is simple to express and purify, has high intrinsic Her2 binding activity, low background CD3 ligation activity, and high CD3 co-ligation activation only upon activation by MMP cleavage of the LHL linker.

[0325] Second-generation construct cloning and expression

[0326] The above multispecific Fab containing LHL linker 2 and IgG 2 The performance of the clones prompted a second series of constructs to be used to experimentally explore the potential functional sequence space in both tertiary structure and linker sequence content. The following clones were synthesized and assembled to sample these parameters:

[0327] 1. Single-arm Fab 2The protein was cloned into 'Met47-LHL-LHL' (Table 6, Figure 3B ).

[0328] 2. Remodeling of the 'Clone 10' Fab for the 'single-arm' type 2 Protein cloning 'Her23-LHL-LHL' (Table 7, Figure 3B ).

[0329] 3. Single-arm Fab 2 The protein cloning 'Her23(34)-LHL-LHL' (Table 8, Figure 3B ).

[0330] 4. A series of IgGs incorporating the Her2 binding domain of trastuzumab in the upper Fab and the CD47 binding domain of clone A-D5 in the lower Fab 2 These clones contained a mutated LHL-based linker sequence that may be more sensitive to enzymatic cleavage by a broad family of MMPs (LHLF linker), a linker of modestly increased length that may have a portion of the human IgG1 middle hinge sequence added (LHLM linker), or both (LHLMF linker).

[0331] 5. For which Fab 2 Fab module located at the end of KIH-Fcc 2 Cloning of protein 'Fc-Her23(34)' (Table 13, Figure 12 ).

[0332] 6. A 'one-arm' Fab containing two copies of C-Met Fab 2 Cloning of protein 'MetMet-LHL-LHL' (Table 14, Figure 3B 、 Figure 13 In this structural form, binding to the bivalent Met receptor can only occur after cleavage of a single LHL linker by a protease.

[0333] As described above, these constructs were successfully expressed and purified by protein A and size exclusion chromatography.

[0334] Second generation IgG 2 Construct analysis

[0335] The purified IgG was tested in a series of further analyses. 2 First, the clones A-D5IgG1 and 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 human and mouse CD47 ( Figure 14 This analysis demonstrated that: 1) A-D5 anti-CD47 IgG1 protein exhibited high binding signals to both hCD47 and mCD47. 2) All four IgG 2 The protein maintains high binding signal to hHer2 but no / background binding signal to hCD47 or mCD47 ( Figure 14 ).

[0336] 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 ( FIG. 15 ): 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 faster. Incubation with MMP12 demonstrated that both proteins can be activated at equal rates for the enzyme to bind hCD47 ( Figure 15D ). Unexpectedly, incubation with MMP13 demonstrated that IgG 2 Her47-LHL-LHLF can be activated by the enzyme to bind hCD47, while IgG 2 Her2CD47-LHL-LHL cannot ( Figure 15E Importantly, incubation with the cysteine ​​protease cathepsin S also demonstrated that both proteins can be activated at equal rates for this enzyme to bind hCD47 ( Figure 15F These findings demonstrate that in IgG 2 Fab contained in the protein 2 The peptide linker content of the module can be "tuned" to broaden the number of potential activating enzymes and even make the proteolytic activation process faster. To sample this possibility, additional linker designs with appropriate length and content to drive sensitivity to specific types of enzymes are envisioned, where amino acid sequences that show proteolytic sensitivity to the activities of other disease-related metalloproteinases such as ADAMS, cysteine, aspartic acid, and serine proteases will be used to provide broader or more selective activation.

[0337] To detect activated IgG 2To investigate the effect of binding affinity, a Biacore assay was developed that could sample both Her2 and CD47 binding. 2 The Her47-LHL-LHLF protein (undigested or activated with MMP12 for 2, 4, 8 or 24 hours) was captured on the chip surface by anti-Fc antibodies, and the binding affinity to soluble Her2 and CD47 extracellular domain proteins was then measured. Binding analysis was repeated for trastuzumab IgG1 and A-D5IgG1 (without enzymatic digestion) at the beginning and end of the experimental run, and the data are shown in Table 17. These analyses showed that the calculated KD values ​​for each antibody were highly similar in each run. However, importantly, the Rmax value (maximum binding signal at maximum analyte concentration) decreased significantly between runs at the beginning and end of the experiment (e.g., trastuzumab Rmax was 265.80RU at the beginning and 144.13RU at the end), indicating that the activity of the anti-Fc antibody capture surface decreased after many rounds of regeneration inherent to the Biacore method. In order to detect the effect of MMP12 activation on target reactivity, the Her2 ( Figure 16A , Table 18) and CD47 ( Figure 16B , Table 18) show both Rmax and KD values ​​for Her2 binding and affinity were maintained over 24 hours of MMP12 activation. Importantly, no CD47 binding (Rmax = 0) was observed in the 0 hour sample (no MMP12 digestion), but both Rmax and apparent affinity for CD47 increased rapidly over the course of activation, starting at 2 hours of incubation ( Figure 16B , Table 18). These findings demonstrate that Fab 2 The lower Fab in the module is indeed inert and cannot interact with the target in the intact molecule until protease activation occurs. This observation is supported by Figure 17 This is further illustrated in , where no binding activity was observed even at 400 nM concentration of CD47 analyte, but high binding to CD47 was evident after 24 h of MMP12 treatment.

[0338] The above findings also prove that in Fab 2 Cleavage of the linker peptide in the module (and hence activation of the lower Fab) is transient. This may be due to the presence of 2 The pharmacological benefits of modular molecules are that activation may be strongly biased towards diseased tissues where both the target antigen of the upper Fab and the enzyme capable of activating the lower Fab are highly overexpressed. This will lead to rapid drug accumulation, long residence time and high levels of activation in such diseased tissues.

[0339] Fab 2 Computer modeling of modular structure and molecular dynamics

[0340] To understand Fab 2 To understand the mechanism by which the module may function, we performed structural modeling and molecular dynamics analysis. Using the protein modeling suite of MOE software, we generated models of anti-CD47A-D5 Fab bound to CD47 ECD using the crystal structures of human CD47 ECD bound to the Fab domains of IgG1C47B161 (Protein Database identifier 5TZT), IgG1C47B222 (Protein Database identifier 5TZ2), and IgG1B6H12.2 (Protein Database identifier 5TZU) as templates. The structure of trastuzumab Fab in complex with the Her2 extracellular domain was taken from PDB structure 1N8Z.

[0341] The protein modeling suite of MOE software was then used to model the LHL and LHLF linkers between the upper trastuzumab Fab and the lower anti-CD47 Fab. To aid in linker modeling, the C-termini of available Fab structures in the Protein Data Bank were examined to help define the conformations in which the LB linker could exit from each heavy and light chain domain of the trastuzumab Fab. Modeling predicted that the C-termini of the trastuzumab Fab heavy and light chains may optionally contain native interchain disulfide bonds typically present in IgG1 Fab domains.

[0342] A full-length IgG1-based model incorporating the anti-CD47 Fab modeled above was constructed using the structure of IgG1b12 (Protein Database identifier 1HZH) as a template. Structural errors in 1HZH, such as missing structural regions, were remodeled and corrected. IgG1b12 Fab was replaced with anti-CD47 Fab, and the Fc-hinge was attached using the protein modeling suite of MOE software. This model illustrates the interaction of Her2 and CD47 with IgG1. 2 Possible tertiary structures of molecules ( Figure 18A In this model, binding of the Her2 epitope is constitutively active ( Figure 18A ), while the binding of CD47 ECD was completely blocked by the linker itself and the proximity of anti-CD47 Fab and anti-HER2 Fab ( Figure 18B ). This model is similar to Fab 2 modules are consistent, the Fab 2 The module can bind HER2 via the upper fab without observable hindrance, but is blocked from binding CD47 via the lower fab until the linker is degraded ( Figure 16A 、 Figure 16B ).

[0343] To further understand Fab 2To understand how the module moves in solution, we performed molecular dynamics simulations using a variety of linker compositions, including LHL-LHL, LHL-LHLF, and L2L2 (G4SG4S (SEQ ID NO: 32) amino acid sequence, Table 1). The RMSD of each residue was calculated for each run. In addition, a custom descriptor, dSASA, was written in the SVL language (MOE), which calculates the change in solvent accessible surface area (SASA) for each residue and, in each case, for a collection of linker sequences only. This provides a basis for comparing how different uncleaved linkers behave in terms of conformational changes and the associated effects on the solvent accessibility of the linker, which is expected to affect sequence-specific cleavage potential ( FIG. 18 ).

[0344] Figures 19A-19I Nine graphs corresponding to the solvent accessible surface area (SASA) results obtained for the three linkers tested are shown. The first analysis analyzed nine kinetic runs for each of the LHL and LHLF linkers and ten runs using L2 (respectively Figure 19A 、 Figure 19D and Figure 19G ) sampled absolute SASA values ​​(within 6 ns). This data demonstrates that the L2 linker clearly has the greatest tendency for structural heterogeneity over time ( Figure 19G Since the starting SASA values ​​were different for each run, the 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 within the 6 ns kinetic run time (respectively Figure 19B 、 Figure 19E and Figure 19H ) and the results within the first 2.5 ns of a 6 ns kinetic run (respectively Figure 19C 、 Figure 19F and Figure 19I ) indicates that the structural dynamics of LHL and LHLF linkers are significantly less than that of L2 linkers. In particular, the 6 ns molecular dynamics run showed that L2 exhibited the highest flexibility, thus emphasizing that different linker sequences will produce different flexibility and thus for Fab 2 Concept of the solvent exposure profile of the CDRs of the lower Fab in the module.

[0345] Comparison between cleaved and uncleaved linkers showed a significant increase in flexibility of the cleaved linker compared to the uncleaved linker, consistent with the Figure 14-17 For example, Figure 20A Shown is the Fab when both linkers are intact. 2Limited movement in the module is followed by significant movement of the upper Fab domain in the context of a single cleaved LHL linker (the second linker intact) during a 100 ns kinetic run. This analysis shows that the upper Fab domain has a significant increase in freedom, leading to multiple positions where it can move completely out of the path of the lower Fab domain, thereby fully exposing its CDRs to allow unconstrained interactions with, for example, CD47 ( Figure 20B ).

[0346] In vitro and in vivo analysis of tolerability and pharmacokinetics

[0347] To examine the tolerability and pharmacokinetics of IgG2 and Fab2 in the context of CD47 as the lower Fab domain, 6-8 week old male B6.Cg-Fcgrt tm1Dcr Several exemplary molecules (A-D5IgG1, IgG 2 Her47LHL-LHL, IgG 2 Her47LHL-LHLF and Fab 2 Met47LHL-LHL). These 'Tg32' mice are human FcRn homozygous transgenic animals with human IgG pharmacokinetic characteristics that mimic those of humans and primates. 2 Her47LHL-LHL, IgG 2 Her47LHL-LHLF and Fab 2 All of the CD47 binding domains contained in the lower Fab domain of Met47LHL-LHL were able to bind to recombinant mouse CD47 protein, so their reactivity to mouse membrane-presented CD47 on erythrocytes was first tested by flow cytometry ( Figure 21 ). This study showed that A-D5IgG1 did not show a signal on mouse erythrocytes at 0.1 μg / ml, but showed a clear concentration-dependent binding signal at both 1 and 10 μg / ml ( Figure 21 Importantly, binding to mouse erythrocytes was subsaturated at 1 μg / ml (63% binding, Figure 21 ) and was fully saturated at 10 μg / ml (98% bound, Figure 21 ). In contrast, the protein IgG 2 Her47LHL-LHL, IgG 2 Her47LHL-LHLF or Fab 2Met47LHL-LHL did not show any binding at any concentration, indicating that the anti-CD47 variable domain of the lower Fab domain cannot interact with CD47 on mouse erythrocytes ( Figure 21 ).

[0348] Hemagglutination assays were also performed using erythrocytes isolated from Tg32 mice. This analysis demonstrated that only A-D5IgG1 was able to drive concentration-dependent agglutination of mouse erythrocytes (at >3.12 μg / ml), while protein IgG 2 Her47LHL-LHL, IgG 2 Her47LHL-LHLF or Fab 2 Met47LHL-LHL did not show agglutination at any concentration up to 200 μg / ml ( Figure 22 These findings indicate that the in vitro assays for human CD47 protein binding to erythrocytes outlined above were reproduced in the mouse system (i.e., A-D5IgG1 fully bound to human CD47, but not to IgG1). 2 or Fab 2 The mouse has no measurable binding to the protein), making it a viable model to study the effects of CD47 binding on both pharmacokinetics and tolerability.

[0349] In the in vivo tolerance study, A-D5IgG1, IgG 2 Her47LHL-LHL, IgG 2 Her47LHL-LHLF and Fab 2 Met47LHL-LHL was administered once (intravenously) in Tg32 mice at either 2 mg / kg or 10 mg / kg. 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 Her47LHL-LHL, IgG 2 Her47LHL-LHLF and Fab 2 Met47LHL-LHL was well tolerated in all patients during the 60-day weight observation period ( Figure 23 Although the 2 mg / kg dose of A-D5IgG was generally well tolerated, a significant upregulation of reticulocytes was observed in this group 5 days after dosing ( Figure 24 ). In contrast, IgG at a dose of 2 mg / kg or 10 mg / kg 2 Her47LHL-LHL, IgG 2 Her47LHL-LHLF or Fab 2Met47LHL-LHL was not associated with reticulocyte upregulation on day 5 or later ( Figure 24 , Figure 25). Reticulocyte upregulation is a known response to rapid erythrocyte clearance, suggesting that IV administration of the A-D5IgG1 antibody results in accelerated clearance of CD47 high erythrocytes.

[0350] To more broadly sample the effects of the administered protein, a complete hematology panel was examined on days 5, 29, and 60 after dosing ( Figures 25A-25K These analyses demonstrated that the reticulocyte upregulation effect of A-D5IgG1 was transient, returning to baseline by day 29 ( Figure 25A No A-D5IgG1, IgG2, or IgG3 was observed on days 5, 29, and 60. 2 Her47LHL-LHL, IgG 2 Her47LHL-LHLF and Fab 2 Met47LHL-LHL had a significant effect on red blood cell (RBC) count, hemoglobin, mean corpuscular hemoglobin concentration (MCHC), mean corpuscular volume (MCV), leukocyte, monocyte, lymphocyte, basophil, eosinophil or neutrophil levels ( Figures 25B-25K ).

[0351] In the in vivo pharmacokinetic study, IgG 2 Her47LHL-LHL, IgG 2 Her47LHL-LHLF and Fab 2 Met47LHL-LHL was administered once (intravenously) at a concentration of 2 or 10 mg / kg, and A-D5IgG1 was administered at a previously tolerated concentration of 2 mg / kg. Blood samples were collected from each mouse according to the following 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-D5IgG1 was rapidly eliminated from the circulation, reaching an average concentration of <0.5 μg / ml within 5 days. Figure 26 This rapid drug clearance (termed tissue-mediated drug disposition or TMDD) is likely due to the previously observed strong binding to mouse erythrocytes, which are then rapidly cleared from the system by phagocytosis. This finding further explains the return of reticulocyte levels to normal on day 29 of A-D5IgG1 administration ( Figure 25A ), as the molecule was essentially eliminated by day 10 ( Figure 26 ).

[0352] In contrast, at doses of 2 and 10 mg / kg, IgG2 Her47LHL-LHL, IgG 2 Her47LHL-LHLF and Fab 2 All Met47LHL-LHL showed slow clearance ( Figure 26 Importantly, a 2 mg / kg dose of each protein required >25 days to reach concentrations <1.0 μg / ml ( Figure 27A ), and the 10 mg / kg dose of each protein maintained a concentration of >1.0 μg / ml at 42 days ( Figure 27B A single sample collected from mice given 10 mg / kg of A-D5IgG1 on day 0 during the tolerability study was also analyzed, showing that the maximum serum IgG concentrations achieved (but not tolerated) were comparable to those of the fully tolerated IgG 2 and Fab 2 The concentrations achieved by the proteins (>50 μg / ml) were similar.

[0353] 2 mg / kg IgG 2 Her47LHL-LHL, IgG 2 Her47LHL-LHLF and Fab 2 Met47LHL-LHL all showed a normal 'α' phase (when an IV dose of immunoglobulin is rapidly distributed from the bloodstream to the tissues), followed by a long 'β' phase ( Figure 26 、 27A These proteins also showed normal distribution curves at 10 mg / kg, but with even longer circulation ( Figure 26 、 27B Importantly, the β-phase of each protein exhibited linear and parallel curves at both concentrations, indicating that the CD47 domains of these proteins do not contribute to the TMDD observed for A-D5IgG1 ( Figure 26 If IgG 2 Her47LHL-LHL, IgG 2 Her47LHL-LHLF or Fab 2 If the Met47LHL-LHL protein undergoes activation in the periphery, TMDD would be expected to manifest strongly, as activation would result in high-affinity erythrocyte, endothelial, and platelet binding, leading to clearance that shifts the β phase to a steeply descending trajectory, as seen with A-D5IgG1 ( Figure 26 These observations, combined with IgG 2 Her47LHL-LHL, IgG 2 Her47LHL-LHLF and Fab 2The lack of reticulocyte expansion at the doses of Met47LHL-LHL suggests a low level of peripheral activation, despite the long pharmacokinetics of these molecules, which means they have been recycled multiple times across FcRn in the circulation for >25 days.

[0354] IgG as outlined above 2 Her47LHL-LHL, IgG 2 Her47LHL-LHLF and Fab 2 The pharmacokinetic and tolerability findings of Met47LHL-LHL indicate normal, FcRn-mediated, antibody-like half-life extension via the human IgG1 Fc domain. This effect resulted in significantly increased area under the curve (AUC) values ​​for these three proteins compared to A-D5IgG1 ( Figure 28 ). At 2 mg / kg, IgG 2 Her47LHL-LHL, IgG 2 Her47LHL-LHLF and Fab 2 The AUC of Met47LHL-LHL was improved 25-40 fold compared to A-D5IgG1. At 10 mg / kg, a dose not safely achievable for A-D5IgG1, the IgG 2 Her47LHL-LHL, IgG 2 Her47LHL-LHLF and Fab 2 The AUC of Met47LHL-LHL was improved by approximately 100-250 times compared with A-D5IgG1 ( Figure 28 These improvements in AUC are significant as they indicate that 2 and Fab 2 The intracellular use of the CD47 binding domain allows for the use of high concentrations starting with the first dose to maximize distribution to tumor tissue.

[0355] Flow cytometric analysis of binding to monkey and human erythrocytes

[0356] Erythrocytes were isolated from 3 cynomolgus macaque NHPs and 3 human donors and treated with A-D5IgG1, trastuzumab, IgG 2 Her47LHL-LHLF or IgG 2 Her47LHL-LHL staining. Both analysis groups showed that only A-D5IgG1 showed the same Figure 29A ) or people ( Figure 29B These findings confirm the above observations that IgG 2 (and therefore Fab 2) structure is restricted to binding mouse, monkey, and human CD47.

[0357] Activation of MMPs and cathepsins at pH 6.0 and 7.4

[0358] MMPs and cathepsins have been shown to have the potential to enzymatically cleave the peptide sequences found in the LHL or LHLF linkers outlined above. Importantly, however, both classes of enzymes exhibit sensitivity to changes in pH conditions that either increase or decrease their enzymatic activity. This can be crucial, as solid tumors are often observed to have pH values ​​that deviate 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.

[0359] As previously demonstrated, multiple MMP enzymes have the ability to activate lower fab binding at pH 7.4. We examined the activity of multiple MMPs and cathepsins (all of which are associated with increased activity in solid tumors) at pH 6.0 and 7.4. MMP3 ( Figure 30A , B), MMP7( Figure 30C , D), MMP8( Figure 30E , F), MMP10( Figure 30G , H), MMP12 ( Figure 30I , J), MMP13( Figure 30K , L) and MMP14 ( Figure 30M , N) for IgG 2 Her47LHL-LHL or IgG 2 Activation of Her47LHL-LHLF. Then IgG 2 Her47LHL-LHL and IgG 2 MMP-treated samples of Her47LHL-LHLF were used 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 Her47LHL-LHLF is activated by MMP13 ( Figure 30K , L) and activated by MMP7 ( Figure 30D ) activated. IgG 2 Her47LHL-LHLF was also higher than IgG at most time points 2 Her47LHL-LHL showed relatively higher levels of activation by most tested MMPs ( Figure 30A-N). These findings demonstrate that inclusion of the LHLF linker at pH 6.0 and / or pH 7.4 results in binding of the lower Fab and faster activation by a wider range of MMP enzymes.

[0360] Cathepsins were also tested as potential activating enzymes. For these enzymes, a clear relationship between pH and activity was observed. 2 Her47LHL-LHL( Figure 31A ) or IgG 2 Her47LHL-LHLF( Figure 31B ) demonstrated that only cathepsin S was able to activate CD47 binding at both 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 at pH 7.4 even after 24 hours, but rapidly generated a strong activation signal at pH 6.0 ( Figure 31A 、 31B These findings suggest that the more rapid activation of LHL and LHLF linkers by MMPs and pH-selective activation by a range of cathepsins compared to those at pH 7.4 may allow for accelerated activation in acidified tissues. This may further improve IgG by minimizing activation of lower Fab binding at pH 7.4 (the pH of non-diseased tissue, where levels of active extracellular MMPs and cathepsins are low) and maximizing it at pH 6.0 (the pH of diseased tissue, where MMP and cathepsin levels are high and cathepsin activity is enhanced). 2 and Fab 2 Therapeutic index of proteins.

[0361] Activation of the lower Fab binding to Her2 / CD47+ cells

[0362] IgG was detected by flow cytometry 2 Her47LHL-LHL and IgG 2 Binding characteristics of Her47LHL-LHLF to cells expressing different levels of CD47 and Her2 on their cell surface. Staining with trastuzumab, anti-CD47, and isotype control IgG demonstrated that BT474 cells expressed high levels of Her2 and lower levels of CD47 ( Figure 32A , B), whereas MCF7 cells expressed higher levels of CD47 and lower levels of Her2 ( Figure 32C , D). After activation with MMP12 for 0, 2, 8 and 24 hours, IgG 2 Her47LHL-LHLF( Figure 32A , C) and IgG 2 Her47LHL-LHL( Figure 32B, D) staining of two cell types. IgG 2 Her47LHL-LHLF and IgG 2 Her47LHL-LHL showed a similar binding profile to trastuzumab on BT474 cells at 0, 2, and 8 h time points, but binding was slightly reduced after 24 h ( Figure 32A , B). In contrast, IgG 2 Her47LHL-LHLF and IgG 2 Both Her47LHL and LHL showed a similar low-level binding profile to trastuzumab on MCF7 cells at 0 h activation, but binding was significantly higher at 2, 8, and 24 h activation time points, compared to the simulated anti-CD47 control ( Figure 32C , D). These findings were confirmed experimentally Figure 2B The activation model proposed in

[15] is valid, in which trastuzumab-like binding of Her2 is maintained after activation and the CD47 binding profile becomes highly active only after activation. Importantly, these findings also indicate that IgG 2 (and Fab 2 ) proteins may have the beneficial ability to drive the function of the lower Fab binding domain (e.g., CD47) toward cells that express low levels of the upper Fab target (e.g., Her2).

[0363] Analysis of MMP12 activation by SDS-PAGE and mass spectrometry

[0364] IgG was treated with MMP12 as described above 2 Her47LHL-LHLF and IgG 2 Her47LHL-LHL protein was expressed at 0, 2, 8, and 24 hours. These protein samples were then analyzed by SDS-PAGE ( Figure 33 This assay produced comparable results to the activation ELISA ( Figure 15D 30) and flow cytometry (Figure 32) observations clearly correlated findings: at 0 hours - two intact chains were observed, with the heavy chain running at 75 kDa and the light chain running just below the 50 kDa marker. At the 2 hour time point, for IgG 2 Her47LHL-LHLF and IgG 2For both Her47LHL-LHL, a new ~25 kDa product was observed, plus a faint band slightly larger than 25 kDa at approximately ~50 kDa (above the intact light chain). The molecular weights of these new fragments correspond to the upper Fab Fd or light chain fragment (1 v domain + 1 c domain = ~25 kDa), Fc (2 x c domain + 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 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 predominant, with significant amounts of both intact heavy chain (75 kDa) and intact light chain (less than 50 kDa) remaining (but reduced compared to the 0 hour sample). Therefore, the SDS-PAGE analysis ( Figure 33 ) demonstrated that, regardless of the LHL or LHLF sequence, the linker between the upper and lower Fabs was cleaved by MMP12 enzymatic activity.

[0365] In order to accurately detect 2 Where in the structure does the MMP12 enzyme activate IgG? 2 molecules, using IgG 2 0, 2, 8 and 24 hour samples of Her47LHL-LHL were subjected to mass spectrometry analysis. Peptide samples were prepared by reduction, alkylation and proteolytic digestion using a combination of trypsin and Glu-C. These samples were analyzed by LC-MS / MS. The efficiency of cleavage by MMP was determined by comparing the MS response of a peptide derived from the intact MMP cleavage site (SCGPAPE (SEQ ID NO: 110)) with a peptide derived from the cleaved protein (SCGPAP (SEQ ID NO: 111)). This analysis successfully identified peptides that were localized to both the uncleaved (SCGPAPE (SEQ ID NO: 110)) and cleaved (SCGPAP (SEQ ID NO: 111)) linker, demonstrating that in the LHL linker, MMP12 cleaves between the second proline (P) and glutamic acid (E). The relative signal of the uncleaved (SCGPAPE (SEQ ID NO: 110)) peptide 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 ).

[0366] IgG in hinge-stabilized 'IgG1-DAA' format 2 Generation and analysis of

[0367] U.S. Patent No. 8871204B2 teaches protease-resistant IgG1 antibody variants that maintain lower hinge and Fc stability in the presence of MMP enzymes. In these mutant IgG1 antibodies, E233-L234-L235-G236 (SEQ ID NO:112) is replaced with P233-V234-A235 (wherein G236 is deleted); and the CH2 domain comprises at least one substitution selected from S239D / 1332E, K326A / E333A, H268F / S324T / 1332E, F243L / R292P / Y300L, S239D / H268F / S324T / 1332E, S267E / H268F / S324T / 1332E, K326A / 1332E / E333A, S239D / K326A / E333A, S267E / I332E, and G237X / S239D / 1332E, wherein X is A, D, P, Q, or S; and wherein the amino acid residues are numbered according to EU numbering.

[0368] To detect Fab 2 The use of the construct in the context of this protease-stabilized IgG1 Fc resulted in two exemplary constructs (Table 19). These two constructs (Her47LHLF-LHLIgG1-2hDAA and Her47LHL-LHLF IgG1-2hDAA) combined the Her47 Fab 2 The structure was placed on the IgG built on the '2hDAA' structure (IgG1 containing P233-V234-A235-ΔG236 and S239D / K326A / E333A mutations). 2 Both constructs were readily expressed in transient CHO cell transfections, with Protein A purified proteins exhibiting >80% of the expected molecular weight product. Exemplary analytical SEC data for Her47LHLF-LHL IgG1-2 hDAA showed 80% product at 10.30 ml ( Figure 35 ). SDS-PAGE analysis of the unreduced protein A purified protein and SEC peaks 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 expected size product (approximately 250 kDa). SDS-PAGE analysis of reduced protein A purified protein and SEC peaks 8.47, 9.03, and 10.30 ml ( Figure 37) demonstrated that all peaks 8.47, 9.03, and 10.30 ml contained heavy and light chain products of the expected size (approximately 80 and 50 kDa, respectively). The intact monomer (250 kDa) product of Her47LHL-LHLF IgG1-2hDAA was purified by SEC and enzymatically digested with human MMP12 at pH 7.4 over a time course of 2, 4, 8, and 24 hours of incubation, plus a 24-hour incubation in enzyme-free buffer as a negative control (time 0, 2 hours, 4 hours, 8 hours, 24 hours incubation). In ELISA analysis, all samples showed strong binding signals to human Her2, but no measurable binding to the control protein mouse EpCAM ( Figure 38A In the CD47 binding ELISA, the binding signal increased after 2, 4, 8, and 24 hours of incubation at 37°C in the presence or absence of MMP12 enzyme, but no signal above background was observed at 0, 2, 4, 8, or 24 hours in the absence of MMP12 ( Figure 38B ).

[0369] 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 size. A 25 kDa band ( Figure 38C ), indicating linker peptide cleavage, but with IgG 2 Her47LHL-LHL or IgG 2 Her47LHL-LHLF( Figure 33 ), significantly less degradation of the heavy chain was observed, indicating that the '2hDAA' mutation indeed stabilizes the Fc region against MMP12 digestion.

[0370] Generation and analysis of IgG with alternative enzyme activation

[0371] Since the LHL and LHLF linkers were shown to be easily cleaved by various MMPs and cathepsins, IgG 2 Format detection of six Fab 2 Construct types (Table 20). These constructs use a series of linker designs (Table 21) that are designed to be easily cleaved by several classes of enzymes associated with increased activity in solid tumors and highly inflamed tissues, such as enterokinase (EK), thrombin (Thr), tPA, granzyme B (GrB), uPA, and ADAMTs-5 (A5). All six constructs were easily expressed by CHO cells and purified by ProA chromatography.

[0372] Purified proteins from clones Her47LHL-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 In all cases, these proteins showed low / background binding to CD47 at all concentrations tested (white bars), but concentration-dependent binding to Her2 (grey bars). Each protein was then subjected to a time-course enzymatic digestion with MMP12 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 exhibited increased CD47 binding activation throughout the time course of enzymatic activation. The functional activity of clones Her47LHL-LHL-EK, Her47-LHL-LHL-Thr, Her47-LHL-LHL-tPA, Her47-LHL-LHL-uPA, Her47-LHL-LHL-GrB, and Her47-LHL-LHL-A5 demonstrates that the Fab2 structure can be tailored to activation for a given application using linker peptide sequences with multiple protease recognition sites. For example, in this case, the retention of MMP activation potential in the LHL linker combined with any of six different disease-associated enzyme cleavage motifs in the accompanying linker allows for tailoring to environments where MMPs and / or cathepsins are active, with increased activation potential when enterokinase, thrombin, tPA, granzyme B, uPA, ADAMTs-5, or other proteases are associated with disease states.

[0373] In vivo multi-dose tolerance of Her47 in NOD-SCID mice

[0374] In an in vivo multiple-dose tolerance study, IgG 2 Her47LHL-LHL, IgG 2 Her47LHL-LHLF、Fab 2 Her47LHL-LHL and Fab 2 Her47LHL-LHLF was administered four times (intravenously) in NOD-SCID mice.2 Protein was dosed at 14 mg / kg on day 0 and 7 mg / kg on days 5, 10, and 15. Fab 2 Protein was dosed at 8 mg / kg on day 0 and 4 mg / kg on days 5, 10, and 15. All proteins were well tolerated with no clinical signs of toxicity and no weight loss in any individual animal ( Figure 40 These findings confirm and extend the Her47Fab-based 2 The molecule was found to be tolerable in Tg32 mice at a single dose. In the Tg32 mouse study, even a single dose of A-D5CD47 IgG at 10 mg / kg was not tolerated. All four doses of IgG in this NOD-SCID mouse study were 2 Her47LHL-LHL, IgG 2 Her47LHL-LHLF、Fab 2 Her47LHL-LHL and Fab 2 Her47LHL-LHLF was completely tolerable. 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 toxic signals observed for A-D5IgG1.

[0375] Additional 2-chain Her2CD3Fab 2 Generation and analysis of 'single-arm' constructs

[0376] Additional Her2CD3 constructs were generated to test the ability of the new sequences to improve product uniformity and activity (Table 22). All Her2CD3 constructs were expressed in CHO cells, purified from the supernatant by ProA, and then analyzed by SEC. These analyses showed that the Fab 2 Her23LHL-LHL-S and Fab 2 Her23LHLF-LHL-S showed a higher 2 Her23LHL-LHL and Fab 2 Her23LHL-LHLF (both showed a higher ratio of higher and lower molecular weight products ( Figure 41C 、 Figure 41D ))Higher main product uniformity ( Figure 41A 、 Figure 41B ). These findings suggest that by exchanging 2 Her23LHL-LHL-S and Fab 2The upward oriented Fab in Her23LHLF-LHL-S makes the chains [VL-CL-Linker-VH-CH-Fc] plus [VH-CH-Linker-VL-CL-Fc], resulting in improved homogeneity. 2 Her23LHL-LHL and Fab 2 Her23LHL-LHLF is in contrast, where both chains are [VL-CL-Linker-VL-CL-Fc] plus [VH-CH-Linker-VH-CH-Fc].

[0377] Then we also designed and expressed a control protein Fab 2 mEpCam3LHLF-LHL-S and Fab 2 mEpCam3LHL-LHL-S (upper Fab contains the v domain of anti-murine EpCAM antibody G8.8) (Table 22). Cloned Fab was isolated by preparative SEC. 2 Her23LHL-LHL-S、Fab 2 Her23LHLF-LHL-S、Fab 2 mEpCam3LHLF-LHL-S and Fab 2 mEpCam3LHL-LHL-S was the correct MW product and analyzed in a Promega Jurkat cell-based CD3 ligation reporter bioassay using MCF-7 or BT-474 as human Her2+ target cells (according to the manufacturer's instructions). Preliminary analysis using MCF-7 as a target cell (which expresses very low Her2) demonstrated that both the positive control Her2-CD3BITE protein and OKT3IgG1 produced a strong positive concentration-dependent CD3 activation signal. Cloned Fab treated with MMP12 for 2 hours 2 mEpCam3LHLF-LHL-S and Fab 2 mEpCam3LHL-LHL-S did not produce signals at any concentration ( Figure 42A , B). In contrast, cloned Fab 2 Her23LHLF-LHL-S( Figure 42A ) and Fab 2 Her23LHL-LHL-S( Figure 42B ) showed no measurable signal at 0 h within a concentration range similar to that of Her2-CD3BITE, but the signal gradually increased at 2, 8, and 24 h, but in both cases the maximum signal was higher than that obtained with the positive control.

[0378] In a secondary assay using Her2-high-expressing BT-474 cells as target cells, all the above test samples and controls were assayed at 0.1 μg / ml ( Figure 43A -C). Both the positive control proteins Her2-CD3BITE and OKT3IgG1 induced strong CD3 activation signals, while the negative control Fab 2 mEpCam3LHLF-LHL-S and trastuzumab do not induce. Cloned Fab 2 Her23LHLF-LHL-S( Figure 43B ) and Fab 2 Her23LHL-LHL-S( Figure 43C ) showed no measurable signal at 0 hours, but the signal gradually increased at 2 hours and reached a maximum at 8 hours. Importantly, the signal at 24 hours was significantly reduced compared to 8 hours. This finding indicates that the progressive enzymatic activity ( Figure 33 ) may lead to the eventual separation of the upper and lower Fabs. This may be a beneficial feature of molecules targeting CD3, as such molecules are preferentially activated in the highly proteolytic tumor environment but are also gradually inactivated there, so minimizing the risk of leakage of the active form of the molecule into healthy tissue would be ideal.

[0379] Her2-CD47IgG 2 and Fab 2 In vitro measurement of protein molecular stability

[0380] Historically, engineered antibody formats have often suffered from structural heterogeneity, leading to manufacturing issues. 2 Her47LHL-LHL and Fab 2 The stability of the Her47LHL-LHL protein was assessed using a series of in vitro measurements:

[0381] 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 hours at room temperature was applied to IgG 2 Her47LHL-LHL and Fab 2 Her47LHL-LHL protein. Since oxidation can alter the overall hydrophobicity of antibodies 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 changes in the proportion of monomeric species in any of the tested samples (IgG 2 Her47LHL-LHL and Fab 2Her47LHL-LHL both showed 99.5% and 97.4% monomeric species before and after oxidation, respectively. In the RP analysis of intact antibodies, IgG was observed after forced oxidation with 0.5% H2O2 for intact (non-reduced) antibodies. 2 The retention time of Her47LHL-LHL decreased by 0.5 minutes and the Fab 2 The retention time of Her47LHL-LHL decreased by 0.4 minutes. 2 For Her47LHL-LHL, a 0.5 min decrease in the retention time of the heavy chain and no shift in the retention time of the light chain were observed after forced oxidation. 2 Her47LHL-LHL showed a different profile with three peaks observed, representing the light chain, heavy chain, and truncated hinge-Fc residue. After oxidation, a shift of 0.4-0.5 min was observed for the heavy chain and truncated hinge-Fc residue, but no shift was observed for the light chain. After H2O2 treatment, reduced IgG was observed. 2 Her47LHL-LHL and Fab 2 The shift in retention time of the Her47LHL-LHL sample indicated less oxidation of exposed amino acids limited to the Fc region of the protein.

[0382] Charge Variant Analysis - Charge heterogeneity analysis is important in the characterization of monoclonal antibodies because 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 using a commercial Charge Variant Assay. IgG 2 Her47LHL-LHL( Figure 44A ) and Fab 2 Her47LHL-LHL( Figure 44B ) The charge variant profiles of both showed a homogeneous profile with one major isoform (50-57% of the total), one major acidic isoform (40-48% of the total) and one minor basic isoform (approximately 3%).

[0383] Retention on HIC - Overall hydrophobicity is an indicator of a protein's tendency to self-associate, which can be a significant risk factor for aggregation and viscosity during bioprocessing. Proteins have hydrophobic 'patches' on their surface, which are created by the presence of side chains of hydrophobic or non-polar amino acids. Depending on their number, size and distribution, the resulting surface hydrophobicity will be specific to each protein. HIC separates proteins based on differences in their surface hydrophobicity, exploiting the reversible binding between the protein and the hydrophobic surface of the HIC resin. IgG 2 Her47LHL-LHL and Fab 2 Her47LHL-LHL showed HIC column retention times of 5.4 and 5.0 minutes, respectively. Compared with 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 hydrophobic range. In fact, these values ​​indicate that the IgG derived from it is more hydrophobic than that of the IgG. 2 Her47LHL-LHL and Fab 2 The Her2 binding domain in Her47LHL-LHL has a similar aggregation tendency and stability to anti-Her2 trastuzumab (5.4 min retention).

[0384] Freeze-thaw stability analysis - Protein instability during freeze-thaw steps is an indicator of difficulties in manufacturing and bioprocessing because increased protein aggregation or fragmentation is a risk of reduced product quality. 2 This risk of structural proteins, IgG 2 Her47LHL-LHL and Fab 2 The Her47LHL-LHL protein was subjected to five cycles of freeze-thaw followed by SEC analysis after each cycle. These analyses showed that IgG 2 Her47LHL-LH L( Figure 45A ) and Fab 2 Her47LHL-LH L( Figure 45B ) The protein did not show any change in monodispersity after 5 cycles of freezing (no aggregation or decomposition products were observed).

[0385] Together, these findings suggest that IgG 2 Her47LHL-LHL and Fab 2 The Her47LHL-LHL proteins all have low aggregation risk, low hydrophobicity, low charge heterogeneity, and low oxidation tendency.

[0386] Affinity of IgG variants for human Fc receptors analyze

[0387] If antibodies targeting receptors on diseased cells mediate ADCC and ADCP activity, they must bind to Fcγ receptors. To test whether these binding functions are retained in Fab2-based constructs, IgG was assayed by surface plasmon resonance analysis. 2 Her47LHL-LHL and Fab 2 Binding affinity of Her47LHL-LHL protein to all human and mouse Fc receptors. These analyses demonstrated that both isotype control human IgG1 and IgG4 exhibited the expected strong and weak binding affinities (respectively) to all human Fcγ receptors, including high and low affinity variants of FcγRIIA and FcγRIIIA (Table 23). Similarly, isotype control mouse IgG2a and IgG1 exhibited the expected strong and weak binding affinities (respectively) to mouse FcγRI, FcγRIII, and FcγRIV receptors. 2 Her47LHL-LHL and Fab 2 The binding of Her47LHL-LHL to each human and mouse Fc receptor tested was highly similar to that observed with the isotype control human IgG1. These data indicate that IgG constructed on IgG1 Fc is highly resistant to IgG1 in both humans and mice. 2 and Fab 2 Both proteins should be able to bind to Fc receptors when bound to the surface of diseased cells.

[0388] Additional protein construct design

[0389] Additional protein constructs are envisioned ( Figure 46 ). The construct may contain: 1. A constant domain 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. A pseudo 'non-binding' variable domain in the 'upper fab'. These pseudo variable domains will be shown not to bind any known target in vivo, so only the 'lower fab' exhibits potential drug target binding ability, and only after proteolytic activation by one of the linker domains. 3. The 'upper Fab' is replaced by a 'diabody' structure containing 4 variable domains. The diabody structure may or may not contain disulfide bonds, as found in 'DART' proteins. The diabody structure can promote the 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 construct contemplated herein or shown above, the Fab 2The constructs can be free or fused to another functionalized construct such as an Fc fragment, a small domain, or a peptide that extends 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.

[0390] Cell proliferation analysis of Her2CD47 protein using Her2 high BT-474 cells

[0391] Due to IgG 2 Her47 and Fab 2 Since the Her2 binding domain of the upper Fab of the Her47 protein can mediate inhibition of the kinase activity of this receptor, a cell proliferation assay was performed. This assay used BT-474 cells, a cell line known to be sensitive to Her2 inhibition. Trastuzumab, isotype control IgG1, and IgG 2 Her47LHL-LHL( Figure 47A ) and Fab 2 Her47LHL-LHL( Figure 47B ) were applied to BT-474 cells during a 72-hour incubation period and cell proliferation was measured. Data are presented as percentage inhibition of cell growth (Figure 47). These analyses showed that although trastuzumab exhibited a strong concentration-dependent inhibition of BT-474 cell proliferation, IgG 2 Her47LHL-LHL( Figure 47A ) showed slightly lower potency and Fab 2 Her47LHL-LHL( Figure 47B ) was again less potent (reflecting its monovalent binding capacity).

[0392] In vivo efficacy analysis of Her47 molecules in NOD-SCID mice bearing tumor xenografts (KYSE-410 model)

[0393] In an in vivo multiple-dose efficacy study, trastuzumab, IgG 2 Her47LHL-LHL, IgG 2 Her47LHL-LHLF、Fab 2 Her47LHL-LHL and Fab 2 Her47LHL-LHLF (Fab containing human IgG1Fc 2 Structure, such as Figure 3B ) were administered four times (intravenously on days 0, 5, 10, and 15) to NOD-SCID mice bearing tumors generated by subcutaneous inoculation of the Her2-expressing esophageal cancer cell line KYSE-410. 2Once the tumor is established, the drug is administered. 2 Protein was dosed at 14 mg / kg on day 0 and 7 mg / kg on days 5, 10, and 15. Fab 2 Protein was dosed at 8 mg / kg on day 0 and 4 mg / kg on days 5, 10, and 15. Trastuzumab was dosed at 8 mg / kg on day 0 and 4 mg / kg on days 5, 10, and 15. Tumor volumes were measured by caliper measurement.

[0394] After 3 doses, on day 11, trastuzumab ( Figure 48A ), IgG 2 Her47LHL-LHLF( Figure 48B ), IgG 2 Her47LHL-LHL( Figure 48C ) and Fab 2 Her47LHL-LHLF( Figure 48D ) all showed significant reduction in tumor growth compared to 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, Fab 2 Her47LHL-LHL and Fab 2 The sequences of Her47LHL-LHLF are identical (except for two point mutations in the LHLF linker that accelerate and amplify protease sensitivity relative to the LHL linker, as shown above), but result in significant differences in potency ( Figure 48F ). In addition, the data in Figure 47 demonstrate that 1-arm Fab 2 The construct resulted in weaker inhibition of Her2-driven cell proliferation than observed for trastuzumab, which contains the same Her2-binding domain as that in the Fab. 2 Her47LHL-LHL and Fab 2 The same Her2 binding VH and VL domain sequences were found in both Her47LHL-LHLF. Figure 48A ) and Fab 2 Her47LHL-LHLF( Figure 48D ) The roughly equal potency observed cannot be explained by the Fab 2 High Her2 kinase activity in Her47LHL-LHLF drives Fab 2The high potency of Her47LHL-LHLF is therefore most likely driven by protease activation in the KYSE-410 tumor microenvironment, leading to CD47 blockade and innate immune engagement.

[0395] In summary, the findings outlined above therefore demonstrate that Fab 2 The structure allows for efficient elimination of the 'lower Fab' activity, using both the CD47 and CD3 binding domains as examples. The binding capacity 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 MMPs and cathepsins, which are associated with high activity in diseased tissues such as tumors and fibrotic tissues. 2 Modulation of the linker sequence in the structure allows for maximal regulation of downregulation of fab activation in the disease microenvironment and avoids peripheral pooling issues and toxicity, as exemplified by the performance of the Her47 molecule in vitro and in vivo.

[0396] In vivo pharmacokinetics of Her47 in NOD-SCID mice

[0397] In an in vivo multiple-dose PK study, IgG 2 Her47LHL-LHL, IgG 2 Her47LHL-LHLF、Fab 2 Her47LHL-LHL and Fab 2 Her47LHL-LHLF will be administered once each (intravenously) in NOD-SCID mice. 2 Protein will be dosed at 14 mg / kg and Fab 2 Protein will be dosed 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 an anti-human IgG1 ELISA.

[0398] In vivo efficacy analysis of Her47 molecules in NOD-SCID mice bearing tumor xenografts (SKOV-3, JIMT-1, and NUGC-4 models)

[0399] In an in vivo multiple-dose efficacy study, IgG 2 Her47LHL-LHL, IgG 2 Her47LHL-LHLF、Fab 2 Her47LHL-LHL and Fab 2 Her47LHL-LHLF will be administered four times (intravenously) in NOD-SCID mice bearing tumors generated by subcutaneous inoculation of cell lines SKOV-3, JIMT-1, and NUGC-4.2 The protein will be dosed at 14 mg / kg on day 0 and 7 mg / kg on days 5, 10, and 15. Fab 2 Protein will be dosed at 8 mg / kg on day 0 and 4 mg / kg on days 5, 10, and 15. Trastuzumab will be dosed at 8 mg / kg on day 0 and 4 mg / kg on days 5, 10, and 15. Tumor volumes will be measured by caliper measurement.

[0400] In vivo analysis of tolerability and pharmacokinetics in cynomolgus monkeys

[0401] To detect IgG 2 and / or Fab 2 The tolerability and pharmacokinetics of various exemplary molecules with CD47 or CD3 as the lower Fab domain will be studied in cynomolgus monkeys. 2 and / or Fab 2 Each animal will be administered once, twice, or three times (intravenously) at a concentration of 2 mg / kg or more. Blood samples will be collected from each animal according to the bleeding schedule. Analysis of serum antibody concentration will be measured to calculate PK and assess the risk of TMDD. In order to more extensively sample the effects of the administered protein, a complete hematology panel will also be tested within a series of days after administration. These analyses will measure reticulocytes, red blood cells (RBC), hemoglobin, mean corpuscular hemoglobin concentration (MCHC), mean corpuscular volume (MCV), leukocytes, monocytes, lymphocytes, basophils, eosinophils, and / or neutrophil levels.

[0402] Biosensor measurement of target co-engagement

[0403] To detect activation of Fab 2 To investigate the effect of binding affinity to both Her2 and CD47 (or CD3), a biosensor assay, such as a dynamic biosensor instrument, would be established that can sample Her2 and CD47 (or CD3) binding on the same chip surface. In this assay, a control antibody and IgG 2 or Fab 2 Proteins (undigested or activated with MMPs or cathepsins for example for 2, 4, 8 or 24 hours) are applied alone or together at varying densities onto a sensor chip surface that has been differentially labeled with purified Her2 and CD47 (or CD3) extracellular domain proteins. Affinity for Her2 and CD47 (or CD3) will be measured to determine the impact of multivalent interactions on functional affinity for both targets individually and on the same surface.

[0404] Although the present invention has been described with reference to preferred or exemplary embodiments, those skilled in the art will recognize that various modifications and variations of the present invention may be effected without departing from the spirit and scope of the invention, and such modifications are expressly contemplated herein. No limitation is intended or should be inferred with respect to the specific embodiments disclosed herein and recited in the appended claims.

[0405] No limitation is intended to be placed on the specific embodiments disclosed herein and set forth in the appended claims, nor should any limitation be inferred. 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 one or more incorporated documents or portions of documents define a term that contradicts the definition of the term in this application, the definition appearing in this application shall prevail. However, any references, articles, publications, patents, patent publications and patent applications cited herein are 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.

[0406] Numbered implementation plan

[0407] Notwithstanding the appended claims, the present disclosure sets forth the following numbered embodiments:

[0408] 1. A protein comprising a first portion and a second portion and a peptide linker between the first portion and the second portion,

[0409] 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;

[0410] wherein the peptide linker is cleavable by a protease expressed in diseased tissue;

[0411] wherein the second portion is capable of specifically binding to a molecule expressed in the diseased tissue; and

[0412] wherein when the peptide linker is not cleaved, binding of the second portion to the molecule expressed in the diseased tissue is reduced or inhibited.

[0413] 2. The protein of embodiment 1, wherein the peptide linker is between about 5 and about 15 amino acids in length.

[0414] 3. The protein of embodiment 1 or 2, wherein the peptide linker comprises or consists of the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, or SEQ ID NO: 87.

[0415] 4. The protein of any one of embodiments 1-3, wherein the protease is human matrix metalloproteinase (MMP), human cathepsin, human enterokinase, human thrombin, human tPA, human granzyme B, human uPA, or human ADAMTs-5.

[0416] 5. A protein as described in any 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.

[0417] 6. The protein of any one of embodiments 1-5, wherein the human MMP is MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, MMP-12, MMP-13, or MMP14.

[0418] 7. The protein of any one of embodiments 1-6, wherein the level or activity of the human MMP is increased in the diseased tissue compared to the level or activity of the human MMP in non-diseased tissue.

[0419] 8. The protein of any one of embodiments 1-5, wherein the human cathepsin is cathepsin A, cathepsin C, cathepsin D, cathepsin G, cathepsin L, or cathepsin K.

[0420] 9. The protein of any one of embodiments 1-5 and 8, wherein the level or activity of the human cathepsin is increased in the diseased tissue compared to the level or activity of the human cathepsin in non-diseased tissue.

[0421] 10. The protein of any one of embodiments 1-9, wherein the first portion comprises an antibody, an antigen-binding portion of an antibody, or a receptor extracellular domain.

[0422] 11. The protein of embodiment 10, wherein the first portion is a Fab, a single-chain Fab, a VH domain, a VL domain, an immunoglobulin new antigen receptor (IgNAR), a single-chain variable fragment (scFv), a diabody, or a T cell receptor domain.

[0423] 12. The protein of any one of embodiments 1-11, wherein the first portion specifically binds to a molecule expressed in diseased tissue.

[0424] 13. The protein of any one of embodiments 1-12, wherein the first portion specifically binds to a first molecule expressed in a diseased tissue, and the second portion is capable of specifically binding to a second molecule expressed in a diseased tissue, wherein the first molecule expressed in the diseased tissue and the second molecule expressed in the diseased tissue are different molecules.

[0425] 14. The protein of embodiment 13, wherein the first molecule expressed in the diseased tissue and the second molecule expressed in the diseased tissue are expressed by the same cell.

[0426] 15. The protein of embodiment 13, wherein the first molecule expressed in the diseased tissue and the second molecule expressed in the diseased tissue are expressed by different cells.

[0427] 16. The protein of embodiment 13, wherein the first molecule expressed in the diseased tissue and / or the second molecule expressed in the diseased tissue is expressed on the surface of a cell.

[0428] 17. The protein of embodiment 13, wherein the first molecule expressed in the diseased tissue and / or the second molecule expressed in the diseased tissue is a soluble molecule.

[0429] 18. The protein of any one of embodiments 1-17, wherein the first portion specifically binds to human EGFR, human HER2, human HER3, human CD105, human C-KIT, human PD1, human PD-L1, human PSMA, human EpCAM, human Trop2, human EphA2, human CD20, human BCMA, human GITR, human OX40, human CSF1R, human Lag3, or human cMET.

[0430] 19. The protein of any one of embodiments 1-17, wherein the second portion specifically binds to a molecule expressed by a human immune cell.

[0431] 20. The protein of embodiment 19, wherein the molecule expressed by human immune cells is human CD3, human CD16A, human CD16B, human CD28, human CD89, human CTLA4, human NKG2D, human SIRPα, human SIRPγ, human PD1, human Lag3, human 4-1BB, human OX40, or human GITR.

[0432] 21. The protein of any one of embodiments 1-20, wherein the first portion comprises a heavy chain variable (VH) region and a light chain variable (VL) region.

[0433] 22. The protein of any one of embodiments 1-21, wherein the first portion comprises an immunoglobulin constant region or a portion of an immunoglobulin constant region.

[0434] 23. The protein of embodiment 22, wherein the immunoglobulin constant region is IgG, IgE, IgM, IgD, IgA, or IgY.

[0435] 24. The protein of embodiment 22, wherein the immunoglobulin constant region is IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2.

[0436] 25. The protein of embodiment 22, wherein the immunoglobulin constant region is immunologically inert.

[0437] 26. The protein of embodiment 22, wherein the immunoglobulin constant region is a wild-type human IgG4 constant region, a human IgG4 constant region comprising the amino acid substitution S228P, a wild-type human IgG1 constant region, a human IgG1 constant region comprising the amino acid substitutions L234A and L235A, a human IgG1 constant region comprising the amino acid substitutions L234A, L235A, and G237A, a human IgG1 constant region comprising the amino acid substitutions L234A, L235A, G237A, and P331S, or a wild-type human IgG2 constant region.

[0438] 27. The protein of any one of embodiments 1-26, wherein the second portion comprises an antibody, an antigen-binding portion of an antibody, or a receptor extracellular domain.

[0439] 28. The protein of embodiment 27, wherein the second portion is a Fab, a single-chain Fab, a VH domain, a VL domain, an immunoglobulin new antigen receptor (IgNAR), a single-chain variable fragment (scFv), or a T cell receptor domain.

[0440] 29. The protein of any one of embodiments 1-28, wherein the second portion specifically binds to human CD47.

[0441] 30. The protein of any one of embodiments 1-28, wherein the second portion specifically binds human CD3 or human PD-L1.

[0442] 31. The protein of any one of embodiments 1-30, wherein the second portion comprises a heavy chain variable (VH) region and a light chain variable (VL) region.

[0443] 32. The protein of any one of embodiments 1-31, wherein the second portion comprises an immunoglobulin constant region or a portion of an immunoglobulin constant region.

[0444] 33. The protein of embodiment 32, wherein the immunoglobulin constant region is IgG, IgE, IgM, IgD, IgA, or IgY.

[0445] 34. The protein of embodiment 32, wherein the immunoglobulin constant region is IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2.

[0446] 35. The protein of embodiment 32, wherein the immunoglobulin constant region is immunologically inert.

[0447] 36. The protein of embodiment 32, wherein the immunoglobulin constant region is a wild-type human IgG4 constant region, a human IgG4 constant region comprising the amino acid substitution S228P, a wild-type human IgG1 constant region, a human IgG1 constant region comprising the amino acid substitutions L234A and L235A, a human IgG1 constant region comprising the amino acid substitutions L234A, L235A, and G237A, a human IgG1 constant region comprising the amino acid substitutions L234A, L235A, G237A, and P331S, or a wild-type human IgG2 constant region.

[0448] 37. The protein of embodiment 1, wherein the protein has one immune effector function or two, three, or more immune effector functions.

[0449] 38. The protein of embodiment 37, wherein the immune effector function is ADCC, CDC, or ADCP.

[0450] 39. The protein of any one of embodiments 1-38, wherein the first portion prevents or reduces specific binding of the second portion to the molecule expressed in the diseased tissue.

[0451] 40. The protein of any one of embodiments 1-39, wherein the peptide linker is cleaved near or within the diseased tissue.

[0452] 41. The protein of any one of embodiments 1-40, wherein the peptide linker is cleaved near or within the diseased tissue, wherein the first portion dissociates from the second portion near or within the diseased tissue, and wherein the second portion specifically binds to a molecule expressed in the diseased tissue.

[0453] 42. The protein of any one of embodiments 1-41, wherein the diseased tissue is a tumor or inflamed tissue.

[0454] 43. The protein of embodiment 1, wherein the first portion specifically binds to human cMET, wherein the second portion specifically binds to human CD47, and wherein the protein comprises a first polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO: 16 and a second polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO: 17.

[0455] 44. The protein of embodiment 1, wherein the first portion specifically binds to human HER2, wherein the second portion specifically binds to human CD3, and wherein the protein comprises a first polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO: 26 and a second polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO: 27.

[0456] 45. The protein of embodiment 1, wherein the first portion specifically binds to human HER2, wherein the second portion specifically binds to human CD47, and wherein the protein comprises a first polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO: 34 and a second polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO: 35.

[0457] 46. ​​The protein of embodiment 1, wherein the first portion specifically binds to human cMET, wherein the second portion specifically binds to human CD47, and wherein the protein comprises a first polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO: 36 and a second polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO: 37.

[0458] 47. The protein of embodiment 1, wherein the first portion specifically binds to human Her2, wherein the second portion specifically binds to human CD3, and wherein the protein comprises a first polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO: 38 and a second polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO: 39.

[0459] 48. The protein of embodiment 1, wherein the first portion specifically binds to human Her2, wherein the second portion specifically binds to human CD3, and wherein the protein comprises a first polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO: 40 and a second polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO: 41.

[0460] 49. The protein of embodiment 1, wherein the first portion specifically binds to human Her2, wherein the second portion specifically binds to human CD47, and wherein the protein comprises a first polypeptide chain and a second polypeptide chain, wherein:

[0461] (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

[0462] (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

[0463] (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

[0464] (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

[0465] (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

[0466] (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

[0467] (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

[0468] (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

[0469] (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

[0470] (j) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 44, and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 92; or

[0471] (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

[0472] (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

[0473] (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

[0474] (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

[0475] (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.

[0476] 50. The protein of embodiment 1, wherein the first portion specifically binds to human Her2, wherein the second portion specifically binds to human CD3, and wherein the protein comprises a first polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO: 73 and a second polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO: 74.

[0477] 51. The protein of embodiment 1, wherein the first portion specifically binds to human cMET, wherein the second portion specifically binds to human cMET, and wherein the protein comprises a first polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO: 75 and a second polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO: 76.

[0478] 52. The protein of embodiment 1, wherein the first portion specifically binds to human Her2, wherein the second portion specifically binds to human CD3, and wherein the protein comprises a first polypeptide chain and a second polypeptide chain, wherein:

[0479] (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

[0480] (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

[0481] (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

[0482] (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.

[0483] 53. An immunoconjugate comprising the protein of any one of embodiments 1-52 linked to a therapeutic agent.

[0484] 54. The immunoconjugate of embodiment 53, wherein the therapeutic agent is a cytotoxin, a radioisotope, a chemotherapeutic agent, an immunomodulatory agent, an anti-angiogenic agent, an anti-proliferative agent, a pro-apoptotic agent, a cytostatic enzyme, a lytic enzyme, a therapeutic nucleic acid, an anti-angiogenic agent, an anti-proliferative agent, or a pro-apoptotic agent.

[0485] 55. A pharmaceutical composition comprising the protein of any one of embodiments 1-52 or the immunoconjugate of embodiment 53 or 54, and a pharmaceutically acceptable carrier, diluent, or excipient.

[0486] 56. A nucleic acid molecule encoding the protein or a portion of the protein of any one of embodiments 1-52.

[0487] 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 of any one of embodiments 43-52.

[0488] 58. An expression vector comprising the nucleic acid molecule of embodiment 56 or 57.

[0489] 59. A recombinant host cell comprising the nucleic acid molecule of embodiment 56 or 57 or the expression vector of embodiment 58.

[0490] 60. A method for producing a protein, comprising:

[0491] Culturing a recombinant host cell comprising the expression vector of embodiment 57 under conditions whereby the nucleic acid molecule is expressed, thereby producing the protein; and

[0492] The protein is isolated from the host cell or culture.

[0493] 61. A method for enhancing an anti-cancer immune response in a subject, comprising administering to the subject a therapeutically effective amount of the protein of any one of embodiments 1-52, the immunoconjugate of embodiment 53 or 54, or the pharmaceutical composition of embodiment 55.

[0494] 62. A method of treating cancer, an autoimmune disease, an inflammatory disease, a cardiovascular disease, or a fibrotic disease in a subject, comprising administering to the subject a therapeutically effective amount of a protein as described in any one of embodiments 1-52, an immunoconjugate as described in embodiment 53 or 54, or a pharmaceutical composition as described in embodiment 55.

[0495] 63. The method of embodiment 62, wherein the cancer is gastrointestinal stromal cancer (GIST), pancreatic cancer, skin cancer, melanoma, breast cancer, lung cancer, bronchial cancer, colorectal cancer, prostate cancer, stomach cancer, ovarian cancer, bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, cancer of the oral cavity or pharynx, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small intestine or appendix cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma, or cancer of blood tissue.

[0496] 64. The method of embodiment 62, wherein the autoimmune or inflammatory disease is arthritis, asthma, multiple sclerosis, psoriasis, Crohn's disease, inflammatory bowel disease, lupus, Graves' disease, Hashimoto's thyroiditis, or ankylosing spondylitis.

[0497] 65. The method of embodiment 62, wherein the cardiovascular disease is coronary heart disease, or atherosclerosis, or stroke.

[0498] 66. The method of embodiment 62, wherein the fibrotic disease is myocardial infarction, angina pectoris, osteoarthritis, pulmonary fibrosis, cystic fibrosis, bronchitis, or asthma.

[0499] 67. The protein of any one of embodiments 1-52, the immunoconjugate of embodiment 53 or 54, or the pharmaceutical composition of embodiment 55, for use in treating cancer, an autoimmune disease, an inflammatory disease, a cardiovascular disease, or a fibrotic disease.

[0500] 68. A 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, stomach 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, bile duct cancer, small intestine or appendix cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma, or cancer of blood tissue.

[0501] 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.

[0502] 70. The protein or pharmaceutical composition for use according to embodiment 67, wherein the cardiovascular disease is coronary heart disease, atherosclerosis or stroke.

[0503] 71. The protein or pharmaceutical composition for use according to embodiment 67, wherein the fibrotic disease is myocardial infarction, angina pectoris, osteoarthritis, pulmonary fibrosis, cystic fibrosis, bronchitis or asthma.

[0504] 72. The protein of any one of embodiments 1-52, the immunoconjugate of embodiment 53 or 54, or the pharmaceutical composition of embodiment 55 for use as a medicament.

[0505] Table 1. Peptide linker sequences.

[0506]

[0507]

[0508] The underlined linker peptide sequence is that of human IgG1 germline.

[0509] The bold non-underlined peptide sequence (LG) is a mutation that places the rapidly digested MMP peptide substrate sequence 'PLGL' (SEQ ID NO: 12).

[0510] Table 2. Protein clone numbers, names (IDs) and observed expression characteristics.

[0511]

[0512] *Total protein after affinity purification using Protein A column

[0513] **Based on amino acid sequence only

[0514] ND = Not Conducted

[0515] Table 3. Sequences of bispecific proteins that bind to cMet and CD47.

[0516]

[0517]

[0518]

[0519]

[0520]

[0521]

[0522]

[0523]

[0524]

[0525]

[0526]

[0527]

[0528] Table 4. Sequences of bispecific proteins that bind to Her2 and CD3.

[0529]

[0530]

[0531]

[0532]

[0533]

[0534]

[0535]

[0536]

[0537] Table 5. Sequences of bispecific proteins that bind to Her2 and CD47.

[0538]

[0539]

[0540]

[0541]

[0542] Table 6. Sequences of bispecific proteins that bind to cMET and CD47.

[0543]

[0544]

[0545] Table 7. Sequences of bispecific proteins that bind to Her2 and CD3.

[0546]

[0547]

[0548] Table 8. Sequences of bispecific proteins that bind to Her2 and CD3.

[0549]

[0550]

[0551] Table 9. Sequences of bispecific proteins that bind to Her2 and CD47.

[0552]

[0553]

[0554]

[0555]

[0556]

[0557]

[0558]

[0559] Table 10. Examples of immunoglobulin Fc region amino acid sequences.

[0560] Human IgG4 wild type

[0561] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQED PEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:56)

[0562] Human IgG4(S228P)

[0563] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQED PEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:57)

[0564] Human IgG1 wild type

[0565] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS RDELT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:58)

[0566] Human IgG1-3M

[0567] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS RDELT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:59)

[0568] Human IgG2 wild type

[0569] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:60)

[0570] Human IgG1 wild type "REEM" allotype

[0571] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS REEM TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:61)

[0572] Human IgG1-3M "REEM" allotype

[0573] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS REEM KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:62)

[0574] Table 11. Examples of the amino acid sequences of CD47 protein

[0575] Human CD47 sequence

[0576] MWPLVAALLLGSACCGSAQLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKSTVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRVVSWFSPNENILIVIFPIFAILLFWGQFGIK TLKYRSGGMDEKTIALLVAGLVITVIVIVGAILFVPGEYSLKNATGGLLIVTSTGILILLHYYVFSTAIGLTSFVIAILVIQVIAYILAVVGLSLCIAACIPMHGPLLISGLSILALAQLLGLVYMKFVASNQKTIQPPRKAVEEPLNAFKESKGMMNDE(SEQ ID NO:63) Cynomolgus monkey CD47 sequence

[0577] MWPLVAALLLGSACCGSAQLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKSTAPANFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRVVSWFSPNENILIVIFPIFAILLFWGQFGIK TLKYRSGGMDEKTIALLVAGLMITVIVIVGAILFVPGEYSLKNATGGLLIVTSTGILILLHYYVFSTAIGLTSFVIAILVIQVIAYILAVVGLSLCIAACIPMHGPLLISGLSILALAQLLGLVYMKFVASNQKTIQPPRKAVEEPLNAFKESKGMMNDE(SEQ ID NO:64)

[0578] Table 12. Examples of cMET protein amino acid sequences.

[0579] Human cMET sequence

[0580]

[0581] Cynomolgus monkey cMET sequence

[0582]

[0583] Table 13. Sequences of bispecific proteins that bind to Her2 and CD3.

[0584]

[0585]

[0586]

[0587] Table 14. Sequences of cMET and cMET-binding Fab2-based proteins.

[0588]

[0589]

[0590] Table 15. Examples of Her2 protein amino acid sequences.

[0591] Human Her2 (erbB-2) sequence

[0592]

[0593] Cynomolgus monkey Her2 (erbB2) sequence

[0594]

[0595] Table 16. Examples of CD3 epsilon domain amino acid sequences.

[0596] Human CD3ε sequence

[0597] MQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPE DANFYLYLRARVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI(SEQ ID NO:79)

[0598] Cynomolgus monkey CD3ε sequence

[0599] MQSGTRWRVLGLCLSIGVWGQDGNEEMGSITQTPYQVSISGTTVILTCSQHLGSEAQWQHNGKNKEDSGDRLFLPEFSEMEQSGYYVCYPRGSNPEDASH HLYLKARVCENCMEMDVMAVATIVIVDICITLGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQQDLYSGLNQRRI(SEQ ID NO:80)

[0600] Table 17. Biacore binding values ​​for control anti-Her2 and anti-CD47 antibodies

[0601]

[0602] Table 18. In IgG 2 Biacore binding of Her47-LHL-LHLF protein to Her2 and CD47 during MMP12 activation.

[0603]

[0604] N / A = Not Applicable. No binding signal was observed.

[0605] Table 19. Sequences of bispecific proteins that bind to Her2 and CD47.

[0606]

[0607]

[0608]

[0609]

[0610]

[0611] Table 20. Sequences of bispecific proteins that bind to Her2 and CD47.

[0612]

[0613]

[0614]

[0615]

[0616]

[0617] Table 21. Sequences of linker peptides containing protease cleavage motifs.

[0618]

[0619] Table 22. Sequences of bispecific proteins binding to Her2 and CD3 or to EpCAM and CD3.

[0620]

[0621]

[0622]

[0623]

[0624]

[0625]

[0626]

[0627]

[0628]

[0629]

[0630] Table 23. Binding affinity to human and murine Fc receptors determined by Biacore.

[0631]

[0632]

[0633] ND = Not Completed NB = No Binding.

Claims

1. A protein comprising a first portion and a second portion and a peptide linker between the first portion and the second portion, wherein the peptide linker comprises an amino acid sequence from a human immunoglobulin hinge region or an amino acid sequence having 1 to about 7 amino acid substitutions compared to a human immunoglobulin hinge region; 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 when the peptide linker is not cleaved, binding of the second portion to the molecule expressed in the diseased tissue is reduced or inhibited.

2. The protein of claim 1 , wherein the first portion specifically binds to human cMET, wherein the second portion specifically binds to human CD47, and wherein the protein comprises a first polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO: 16 and a second polypeptide chain comprising or consisting of the amino acid sequence of SEQ ID NO:

17.

3. An immunoconjugate comprising the protein of claim 1 or 2 linked to a therapeutic agent.

4. A pharmaceutical composition comprising the protein according to claim 1 or 2 or the immunoconjugate according to claim 3, and a pharmaceutically acceptable carrier, diluent or excipient.

5. A nucleic acid molecule encoding the protein according to claim 1 or 2 or a portion of the protein. 6 . 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 claim 2 .

7. An expression vector comprising the nucleic acid molecule according to claim 5 or claim 6.

8. A recombinant host cell comprising the nucleic acid molecule according to claim 5 or claim 6 or the expression vector according to claim 7.

9. A method for producing a protein, comprising: Culturing a recombinant host cell comprising the expression vector of claim 7 under conditions in which the nucleic acid molecule is expressed, thereby producing the protein; as well as The protein is isolated from the host cell or culture.

10. A method of treating cancer, autoimmune disease, inflammatory disease, cardiovascular disease or fibrotic disease in a subject, comprising administering to the subject a therapeutically effective amount of the protein of claim 1 or 2, the immunoconjugate of claim 3 or the pharmaceutical composition of claim 4.

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