Anti-mertk agonistic antibody-drug conjugates and uses thereof

Abstract

To provide antibody drug conjugates and pharmaceutical compositions for use in methods of treating cancer.SOLUTION: The present disclosure provides antibody-drug conjugates comprising (i) antibodies that specifically bind to Mer Tyrosine Kinase (MERTK) (e.g., human MERTK, or both human and mouse MERTK), and (ii) cytotoxic agents conjugated directly to the antibodies or conjugated to the antibodies via linkers, and compositions comprising such antibody-drug conjugates, the antibodies contained in the antibody-drug conjugates activates MERTK signaling of endothelial cells.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 62 / 525,993, filed June 28, 2017.

[0002] Reference to Electronically Submitted Sequence Listing The application discloses a sequence listing filed with the application as a text file entitled "SequenceListing.txt" having a size of 29 kilobytes created on June 16, 2017. The table is incorporated by reference.

[0003] 1. Field According to the present disclosure, there are provided an antibody drug conjugate comprising (i) an antibody that specifically binds to Mer tyrosine kinase (MERTK) (e.g., human MERTK, or both human and mouse MERTK) and (ii) a cytotoxic agent conjugated to the antibody directly or via a linker, and a composition comprising the antibody drug conjugate, wherein the antibody contained in the antibody drug conjugate agonizes MERTK signaling in endothelial cells. The present disclosure also provides a method of treating cancer by administering an antibody drug conjugate comprising (i) an antibody that specifically binds to MERTK and agonizes MERTK signaling in endothelial cells and (ii) a cytotoxic agent conjugated to the antibody directly or via a linker.

[0004] 2.Background Mer tyrosine kinase (MERTK) (also known as c-mer, MER, proto-oncogene c-Mer, receptor tyrosine kinase MerTK, tyrosine protein kinase Mer, STK kinase, RP38, or MGC133349) is a member of the TAM family of receptor tyrosine kinases, which also includes AXL and TYRO3 kinases. MERTK transduces signals from the extracellular space through activation by binding of ligands, most notably the soluble protein Gas-6. Binding of Gas-6 to MERTK induces autophosphorylation of MERTK at its intracellular domain, resulting in downstream signal activation (Cummings CT et al.,(2013)Clin Cancer Res 19:5275-5280; Verma A et al.,(2011)Mol Cancer Ther 10:1763-1773).

[0005] MERTK receptors exist in both membrane-bound and soluble forms. The extracellular domain can be cleaved to generate a soluble extracellular domain, which is hypothesized to act as a decoy receptor that negatively regulates activation of MERTK receptors on cells by reducing the ability and / or availability of soluble Gas-6 ligands to bind to membrane-bound MERTK (Sather S et al., (2007) Blood 109:1026-1033). As a result, MERTK has dual roles related to cancer progression, angiogenesis, and metastasis. On the one hand, Gas-6 activation of MERTK on endothelial cells inhibits endothelial cell recruitment by cancer cells in a co-culture system. Endothelial recruitment is a key feature of cancer cells that enables tumor angiogenesis, tumor growth, and metastasis. However, on the other hand, MERTK plays an opposite role in cancer cells, whose overexpression leads to increased metastasis, possibly by releasing truncated MERTK to generate soluble MERTK ectodomain protein as a decoy receptor. Thus, tumor cells secrete a soluble form of extracellular MERTK receptor that acts as a decoy receptor, blocking the ability of soluble Gas-6 ligand to activate MERTK on endothelial cells. (and / or availability), ultimately leading to endothelial recruitment, angiogenesis, and cancer progression (Png KJ et al., (2012) Nature 481:190-194).

[0006] Historically, efforts have been made to generate inhibitors, rather than activators, of MERTK for the treatment of cancer (e.g., compound UNC1062, a potent small molecule MERTK inhibitor developed as an anticancer compound). This is because MERTK was thought to function solely as an oncogene (Liu J et al. (2013) Eur J Med Chem 65:83-93; Cummings CT et al. (2013) Clin Cancer Res 19:5275-5280; Verma A et al. al.(2011)Mol Cancer Ther 10:1763-1773). Given the dual role of MERTK in cancer and endothelial cells, treatment with molecules that generally result in MERTK activation (e.g., in both endothelial and cancer cells) may increase endothelial cell recruitment and metastasis. However, compounds that activate MERTK signaling in endothelial cells but not in cancer cells may be attractive therapeutic agents for tumor angiogenesis and metastasis.

[0007] Therefore, there is a need for therapeutic options using antibodies that specifically bind to MERTK and agonize MERTK signaling in endothelial cells.

[0008] In recent years, antibody-drug conjugates (ADCs) have become one of the fastest growing classes of cancer therapeutics (Beck A et al. (2017) Nat Rev Drug Discov 16:315-337; Peters C and Brown S, (2015 ) Biosci Rep 35:art:e00225).

[0009] Citation of a reference herein should not be construed as an admission that it is prior art to the present disclosure. Summary of the Invention

[0010] 3. Overview The present invention provides antibody drug conjugates comprising (a) an antibody moiety that is an antibody or antigen-binding fragment thereof that specifically binds to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonizes human MERTK signaling in endothelial cells; (b) one or more drug moieties, each of which is a cytotoxic agent; and (c) an optional linker, wherein the cytotoxic agent is conjugated directly to the antibody moiety or via the linker to the antibody moiety.

[0011] Non-limiting exemplary anti-MERTK antibodies or antigen-binding fragments thereof that can be the antibody portion of the antibody drug conjugates of the invention are described below.

[0012] In certain embodiments, the anti-MERTK antibody or antigen-binding fragment thereof described herein specifically recognizes the extracellular domain of human MERTK. In another specific embodiment, the anti-MERTK antibody or antigen-binding fragment thereof described herein specifically recognizes the extracellular domain of mouse MERTK. In another specific embodiment, the anti-MERTK antibody or antigen-binding fragment thereof described herein competes with Gas-6 for binding to MERTK (e.g., human MERTK, or both human and mouse MERTK). In certain embodiments, the anti-MERTK antibody or antigen-binding fragment thereof described herein is monoclonal. In another specific embodiment, the anti-MERTK antibody or antigen-binding fragment thereof described herein is an immunoglobulin comprising two identical light chains and two identical heavy chains.

[0013] In certain embodiments, the anti-MERTK antibodies or antigen-binding fragments thereof described herein that specifically bind to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonize MERTK signaling in endothelial cells are: (a) the VH CDR1 of NYGMN (SEQ ID NO: 1); and / or (b) a VH CDR2 of WINTYTGEPTYADDFKG (SEQ ID NO: 6); and / or (c) the VH CDR3 of KSTVVSRYFDV (SEQ ID NO: 11); The heavy chain variable region (VH) comprises:

[0014] In certain embodiments, the anti-MERTK antibodies or antigen-binding fragments thereof described herein that specifically bind to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonize MERTK signaling in endothelial cells are: (a) the VH CDR1 of GYTFTNY (SEQ ID NO: 2); and / or (b) the VH CDR2 of (SEQ ID NO: 7); and / or (c) a VH CDR3 of STVVSRYFD (SEQ ID NO: 12); The heavy chain variable region (VH) comprises:

[0015] In certain embodiments, the anti-MERTK antibodies or antigen-binding fragments thereof described herein that specifically bind to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonize MERTK signaling in endothelial cells are: (a) a VH CDR1 of GYTFTNYGMN (SEQ ID NO: 3); and / or (b) the VH CDR2 of WINTYTGEPT (SEQ ID NO: 8); and / or (c) the VH CDR3 of KSTVVSRYFDV (SEQ ID NO: 11); The heavy chain variable region (VH) comprises:

[0016] In certain embodiments, the anti-MERTK antibodies or antigen-binding fragments thereof described herein that specifically bind to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonize MERTK signaling in endothelial cells are: (a) the VH CDR1 of TNYGMN (SEQ ID NO: 4); and / or (b) a VH CDR2 of WMGWINTYTGEPT (SEQ ID NO: 9); and / or (c) the VH CDR3 of ARKSTVVSRYFD (SEQ ID NO: 13); The heavy chain variable region (VH) comprises:

[0017] In certain embodiments, the anti-MERTK antibodies or antigen-binding fragments thereof described herein that specifically bind to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonize MERTK signaling in endothelial cells are: (a) a VH CDR1 of GYTFTNYG (SEQ ID NO: 5); and / or (b) the VH CDR2 of INTYTGEP (SEQ ID NO: 10); and / or (c) the VH CDR3 of ARKSTVVSRYFDV (SEQ ID NO: 14); The heavy chain variable region (VH) comprises:

[0018] In certain embodiments, the anti-MERTK antibodies or antigen-binding fragments thereof described herein that specifically bind to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonize MERTK signaling in endothelial cells are: (a) a VH CDR1 of GYTFTNYGMN (SEQ ID NO: 3); and / or (b) a VH CDR2 of WINTYTGEPTYADDFKG (SEQ ID NO: 6); and / or (c) the VH CDR3 of KSTVVSRYFDV (SEQ ID NO: 11); The heavy chain variable region (VH) comprises:

[0019] In another embodiment, the anti-MERTK antibody or antigen-binding fragment thereof described herein that specifically binds to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTK signaling in endothelial cells is: (a) a VL CDR1 of KASQDVGDAVT (SEQ ID NO: 15); and / or (b) the VL CDR2 of WASTRHT (SEQ ID NO: 19); and / or (c) the VL CDR3 of QQYRSYPLT (SEQ ID NO: 22); The light chain variable region (VL) comprises:

[0020] In another embodiment, the anti-MERTK antibody or antigen-binding fragment thereof described herein that specifically binds to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTK signaling in endothelial cells is: (a) a VL CDR1 of SQDVGDA (SEQ ID NO: 16); and / or (b) the VL CDR2 of WAS (SEQ ID NO: 20); and / or (c) the VL CDR3 of YRSYPL (SEQ ID NO: 23); The light chain variable region (VL) comprises:

[0021] In another embodiment, the anti-MERTK antibody or antigen-binding fragment thereof described herein that specifically binds to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTK signaling in endothelial cells is: (a) the VL CDR1 of GDAVTWC (SEQ ID NO: 17); and / or (b) the VL CDR2 of LLIYWASTRH (SEQ ID NO: 21); and / or (c) the VL CDR3 of QQYRSYPL (SEQ ID NO: 24); The light chain variable region (VL) comprises:

[0022] In another embodiment, the anti-MERTK antibody or antigen-binding fragment thereof described herein that specifically binds to MERTK (e.g., human MERTK, or both human and mouse MERTK) and stimulates MERTK signaling in endothelial cells is: (a) the VL CDR1 of QDVGDA (SEQ ID NO: 18); and / or (b) the VL CDR2 of WAS (SEQ ID NO: 20); and / or (c) the VL CDR3 of QQYRSYPLT (SEQ ID NO: 22); The light chain variable region (VL) comprises:

[0023] In another embodiment, the anti-MERTK antibody or antigen-binding fragment thereof described herein that specifically binds to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTK signaling in endothelial cells is: (a) the VH CDR1 of DYSMH (SEQ ID NO: 25); and / or (b) a VH CDR2 of WINTDTGTPEPTYA DDFKG (SEQ ID NO: 30); and / or (c) the VH CDR3 of WFGAMDY (SEQ ID NO: 35); The heavy chain variable region (VH) comprises:

[0024] In certain embodiments, the anti-MERTK antibodies or antigen-binding fragments thereof described herein that specifically bind to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonize MERTK signaling in endothelial cells are: (a) the VH CDR1 of NYTFTDY (SEQ ID NO: 26); and / or (b) the VH CDR2 of TDTG (SEQ ID NO: 31); and / or (c) the VH CDR3 of FGAMD (SEQ ID NO: 36); The heavy chain variable region (VH) comprises:

[0025] In certain embodiments, MERTK (e.g., human MERTK, or human and mouse MERTK) is The anti-MERTK antibodies or antigen-binding fragments thereof described herein that specifically bind to both MERTK and MERTK (both IL-1 and IL-2) and agonize MERTK signaling in endothelial cells are: (a) the VH CDR1 of NYTFTDYSMH (SEQ ID NO: 27); and / or (b) the VH CDR2 of WINTDTGEPT (SEQ ID NO: 32); and / or (c) the VH CDR3 of WFGAMDY (SEQ ID NO: 35); The heavy chain variable region (VH) comprises:

[0026] In certain embodiments, the anti-MERTK antibodies or antigen-binding fragments thereof described herein that specifically bind to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonize MERTK signaling in endothelial cells are: (a) a VH CDR1 of TDYSMH (SEQ ID NO: 28); and / or (b) the VH CDR2 of WVGWINTDTGEPT (SEQ ID NO: 33); and / or (c) VH CDR3 of ARWFGAMD (SEQ ID NO: 37); The heavy chain variable region (VH) comprises:

[0027] In certain embodiments, the anti-MERTK antibodies or antigen-binding fragments thereof described herein that specifically bind to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonize MERTK signaling in endothelial cells are: (a) the VH CDR1 of NYTFTDYS (SEQ ID NO: 29); and / or (b) the VH CDR2 of INTDTGEP (SEQ ID NO: 34); and / or (c) a VH CDR3 of ARWFGAMDY (SEQ ID NO: 38); The heavy chain variable region (VH) comprises:

[0028] In certain embodiments, the anti-MERTK antibodies or antigen-binding fragments thereof described herein that specifically bind to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonize MERTK signaling in endothelial cells are: (a) the VH CDR1 of NYTFTDYSMH (SEQ ID NO: 27); and / or (b) a VH CDR2 of WINTDTGTPEPTYA DDFKG (SEQ ID NO: 30); and / or (c) the VH CDR3 of WFGAMDY (SEQ ID NO: 35); The heavy chain variable region (VH) comprises:

[0029] In certain embodiments, the anti-MERTK antibodies or antigen-binding fragments thereof described herein that specifically bind to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonize MERTK signaling in endothelial cells are: (a) the VL CDR1 of KASQDVTNVVA (SEQ ID NO: 39); and / or (b) the VL CDR2 of SASYRYT (SEQ ID NO: 43); and / or (c) the VL CDR3 of QQYYRTPRT (SEQ ID NO: 46); The light chain variable region (VL) comprises:

[0030] In certain embodiments, the anti-MERTK antibodies or antigen-binding fragments thereof described herein that specifically bind to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonize MERTK signaling in endothelial cells are: (a) the VL CDR1 of SQDVTNV (SEQ ID NO: 40); and / or (b) the VL CDR2 of SAS (SEQ ID NO: 44); and / or (c) the VL CDR3 of YYRTPR (SEQ ID NO: 47); The light chain variable region (VL) comprises:

[0031] In certain embodiments, MERTK (e.g., human MERTK, or both human and mouse MERTK) is The anti-MERTK antibody or antigen-binding fragment thereof described herein that specifically binds to MERTK (META-MERTK) and agonizes MERTK signaling in endothelial cells comprises: (a) the VL CDR1 of TNVVAWY (SEQ ID NO: 41); and / or (b) the VL CDR2 of LLIYSASYRY (SEQ ID NO: 45); and / or (c) the VL CDR3 of QQYYRTPR (SEQ ID NO: 48); The light chain variable region (VL) comprises:

[0032] In certain embodiments, the anti-MERTK antibodies or antigen-binding fragments thereof described herein that specifically bind to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonize MERTK signaling in endothelial cells are: (a) the VL CDR1 of QDVTNV (SEQ ID NO: 42); and / or (b) the VL CDR2 of SAS (SEQ ID NO: 44); and / or (c) the VL CDR3 of QQYYRTPRT (SEQ ID NO: 46); The light chain variable region (VL) comprises:

[0033] In certain embodiments, the anti-MERTK antibody or antigen-binding fragment thereof comprises one, two, or all three of the above VH CDRs. In certain embodiments, the antibody or antigen-binding fragment thereof comprises a VH CDR1 of Table 1 or Table 3. In some embodiments, the anti-MERTK antibody or antigen-binding fragment thereof comprises a VH CDR2 of Table 1 or Table 3. In certain embodiments, the anti-MERTK antibody or antigen-binding fragment thereof comprises a VH CDR3 of Table 1 or Table 3. In certain embodiments, the anti-MERTK antibody or antigen-binding fragment thereof comprises the VH CDR1, VH CDR2, and VH CDR3 of antibody M6 (Table 1). In certain embodiments, the anti-MERTK antibody or antigen-binding fragment thereof comprises the VH CDR1, VH CDR2, and VH CDR3 of antibody M19 (Table 3).

[0034] In certain embodiments, the anti-MERTK antibody or antigen-binding fragment thereof comprises one, two, or all three of the above VL CDRs. In certain embodiments, the anti-MERTK antibody or antigen-binding fragment thereof comprises the VL CDR1 of Table 2 or Table 4. In some embodiments, the anti-MERTK antibody or antigen-binding fragment thereof comprises the VL CDR2 of Table 2 or Table 4. In certain embodiments, the anti-MERTK antibody or antigen-binding fragment thereof comprises the VL CDR3 of Table 2 or Table 4. In certain embodiments, the anti-MERTK antibody or antigen-binding fragment thereof comprises the VL CDR1, VL CDR2, and VL CDR3 of antibody M6 (Table 2). In certain embodiments, the anti-MERTK antibody or antigen-binding fragment thereof comprises the VL CDR1, VL CDR2, and VL CDR3 of antibody M19 (Table 4).

[0035] In another embodiment, the anti-MERTK antibody or antigen-binding fragment thereof that specifically binds to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTK signaling in endothelial cells includes: (a) the VH CDR1 of NYGMN (SEQ ID NO: 1); and / or (b) a VH CDR2 of WINTYTGEPTYADDFKG (SEQ ID NO: 6); and / or (c) the VH CDR3 of KSTVVSRYFDV (SEQ ID NO: 11); and / or (d) the VL CDR1 of KASQDVGDAVT (SEQ ID NO: 15); and / or (e) the VL CDR2 of WASTRHT (SEQ ID NO: 19); and / or (f) VL CDR3 of QQYRSYPLT (sequence number 22).

[0036] In certain embodiments, the anti-MERTK antibodies or antigen-binding fragments thereof that specifically bind to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonize MERTK signaling in endothelial cells include: (a) the VH CDR1 of GYTFTNY (SEQ ID NO: 2); and / or (b) the VH CDR2 of (SEQ ID NO: 7); and / or (c) a VH CDR3 of STVVSRYFD (SEQ ID NO: 12); and / or (d) the VL CDR1 of SQDVGDA (SEQ ID NO: 16); and / or (e) the VL CDR2 of WAS (SEQ ID NO: 20); and / or (f) VL CDR3 of YRSYPL (sequence number 23).

[0037] In certain embodiments, the anti-MERTK antibodies or antigen-binding fragments thereof that specifically bind to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonize MERTK signaling in endothelial cells include: (a) a VH CDR1 of GYTFTNYGMN (SEQ ID NO: 3); and / or (b) the VH CDR2 of WINTYTGEPT (SEQ ID NO: 8); and / or (c) the VH CDR3 of KSTVVSRYFDV (SEQ ID NO: 11); and / or (d) the VL CDR1 of KASQDVGDAVT (SEQ ID NO: 15); and / or (e) the VL CDR2 of WASTRHT (SEQ ID NO: 19); and / or (f) VL CDR3 of QQYRSYPLT (sequence number 22).

[0038] In certain embodiments, the anti-MERTK antibodies or antigen-binding fragments thereof that specifically bind to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonize MERTK signaling in endothelial cells include: (a) the VH CDR1 of TNYGMN (SEQ ID NO: 4); and / or (b) a VH CDR2 of WMGWINTYTGEPT (SEQ ID NO: 9); and / or (c) the VH CDR3 of ARKSTVVSRYFD (SEQ ID NO: 13); and / or (d) the VL CDR1 of GDAVTWC (SEQ ID NO: 17); and / or (e) the VL CDR2 of LLIYWASTRH (SEQ ID NO: 21); and / or (f) VL CDR3 of QQYRSYPL (sequence number 24).

[0039] In certain embodiments, the anti-MERTK antibodies or antigen-binding fragments thereof that specifically bind to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonize MERTK signaling in endothelial cells include: (a) a VH CDR1 of GYTFTNYG (SEQ ID NO: 5); and / or (b) the VH CDR2 of INTYTGEP (SEQ ID NO: 10); and / or (c) the VH CDR3 of ARKSTVVSRYFDV (SEQ ID NO: 14); and / or (d) the VL CDR1 of QDVGDA (SEQ ID NO: 18); and / or (e) a VL CDR2 comprising WAS (SEQ ID NO: 20); and / or (f) VL CDR3 of QQYRSYPLT (sequence number 22).

[0040] In certain embodiments, the anti-MERTK antibodies or antigen-binding fragments thereof that specifically bind to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonize MERTK signaling in endothelial cells include: (a) a VH CDR1 of GYTFTNYGMN (SEQ ID NO: 3); and / or (b) a VH CDR2 of WINTYTGEPTYADDFKG (SEQ ID NO: 6); and / or (c) the VH CDR3 of KSTVVSRYFDV (SEQ ID NO: 11); and / or (d) the VL CDR1 of KASQDVGDAVT (SEQ ID NO: 15); and / or (e) the VL CDR2 of WASTRHT (SEQ ID NO: 19); and / or (f) VL CDR3 of QQYRSYPLT (sequence number 22).

[0041] In certain embodiments, the antibody specifically binds to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTK signaling in endothelial cells. Anti-MERTK antibodies or antigen-binding fragments thereof include: (a) the VH CDR1 of DYSMH (SEQ ID NO: 25); and / or (b) a VH CDR2 of WINTDTGTPEPTYA DDFKG (SEQ ID NO: 30); and / or (c) the VH CDR3 of WFGAMDY (SEQ ID NO: 35); and / or (d) the VL CDR1 of KASQDVTNVVA (SEQ ID NO: 39); and / or (e) the VL CDR2 of SASYRYT (SEQ ID NO: 43); and / or (f) VL CDR3 of QQYYRTPRT (sequence number 46).

[0042] In certain embodiments, the anti-MERTK antibodies or antigen-binding fragments thereof that specifically bind to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonize MERTK signaling in endothelial cells include: (a) the VH CDR1 of NYTFTDY (SEQ ID NO: 26); and / or (b) the VH CDR2 of TDTG (SEQ ID NO: 31); and / or (c) the VH CDR3 of FGAMD (SEQ ID NO: 36); and / or (d) the VL CDR1 of SQDVTNV (SEQ ID NO: 40); and / or (e) the VL CDR2 of SAS (SEQ ID NO: 44); and / or (f) VL CDR3 of YYRTPR (sequence number 47).

[0043] In certain embodiments, the anti-MERTK antibodies or antigen-binding fragments thereof that specifically bind to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonize MERTK signaling in endothelial cells include: (a) the VH CDR1 of NYTFTDYSMH (SEQ ID NO: 27); and / or (b) the VH CDR2 of WINTDTGEPT (SEQ ID NO: 32); and / or (c) the VH CDR3 of WFGAMDY (SEQ ID NO: 35); and / or (d) the VL CDR1 of KASQDVTNVVA (SEQ ID NO: 39); and / or (e) the VL CDR2 of SASYRYT (SEQ ID NO: 43); and / or (f) VL CDR3 of QQYYRTPRT (sequence number 46).

[0044] In certain embodiments, the anti-MERTK antibodies or antigen-binding fragments thereof that specifically bind to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonize MERTK signaling in endothelial cells include: (a) a VH CDR1 of TDYSMH (SEQ ID NO: 28); and / or (b) the VH CDR2 of WVGWINTDTGEPT (SEQ ID NO: 33); and / or (c) the VH CDR3 of ARWFGAMD (SEQ ID NO: 37); and / or (d) the VL CDR1 of TNVVAWY (SEQ ID NO: 41); and / or (e) (SEQ ID NO: 45) VL CDR2 of LLIYSASYRY; and / or (f) VL CDR3 of QQYYRTPR (sequence number 48).

[0045] In certain embodiments, the anti-MERTK antibodies or antigen-binding fragments thereof that specifically bind to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonize MERTK signaling in endothelial cells include: (a) the VH CDR1 of NYTFTDYS (SEQ ID NO: 29); and / or (b) the VH CDR2 of INTDTGEP (SEQ ID NO: 34); and / or (c) a VH CDR3 of ARWFGAMDY (SEQ ID NO: 38); and / or (d) the VL CDR1 of QDVTNV (SEQ ID NO: 42); and / or (e) the VL CDR2 of SAS (SEQ ID NO: 44); and / or (f) VL CDR3 of QQYYRTPRT (sequence number 46).

[0046] In certain embodiments, the anti-MERTK antibodies or antigen-binding fragments thereof that specifically bind to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonize MERTK signaling in endothelial cells include: (a) the VH CDR1 of NYTFTDYSMH (SEQ ID NO: 27); and / or (b) a VH CDR2 of WINTDTGTPEPTYA DDFKG (SEQ ID NO: 30); and / or (c) the VH CDR3 of WFGAMDY (SEQ ID NO: 35); and / or (d) the VL CDR1 of KASQDVTNVVA (SEQ ID NO: 39); and / or (e) the VL CDR2 of SASYRYT (SEQ ID NO: 43); and / or (f) VL CDR3 of QQYYRTPRT (sequence number 46).

[0047] In a particular embodiment, an anti-MERTK antibody or antigen-binding fragment thereof that specifically binds to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTK signaling in endothelial cells comprises a heavy chain variable region sequence comprising the amino acid sequence of SEQ ID NO: 49. In another particular embodiment, an anti-MERTK antibody or antigen-binding fragment thereof that specifically binds to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTK signaling in endothelium comprises a heavy chain variable region sequence comprising the amino acid sequence of SEQ ID NO: 51.

[0048] In another embodiment, an anti-MERTK antibody or antigen-binding fragment thereof that specifically binds to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTK signaling in endothelial cells comprises a light chain variable region sequence comprising the amino acid sequence of SEQ ID NO: 50. In another specific embodiment, an anti-MERTK antibody or antigen-binding fragment thereof that specifically binds to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTK signaling in endothelial cells comprises a light chain variable region sequence comprising the amino acid sequence of SEQ ID NO: 52.

[0049] In another embodiment, an anti-MERTK antibody or antigen-binding fragment thereof that specifically binds to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTK signaling in endothelial cells comprises (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 49, and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 50. In another specific embodiment, an anti-MERTK antibody or antigen-binding fragment thereof that specifically binds to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTK signaling in endothelial cells comprises (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 51, and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 52.

[0050] In certain embodiments, the antibodies described herein that specifically bind to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonize MERTK signaling in endothelial cells comprise a heavy chain constant region and / or a light chain constant region. In some embodiments, the heavy chain constant region is selected from the group consisting of IgG1, IgG 2、 IgG3, IgG 4、In certain embodiments, the light chain constant region is selected from the group of human immunoglobulins consisting of IgGκ and IgGλ. In some embodiments, the antibody comprises a constant region with increased binding affinity to one or more human Fc gamma receptors. In some embodiments, the antibody comprises a constant region with reduced binding affinity to one or more human Fc gamma receptors.

[0051] In another embodiment, the anti-MERTK antibody or antigen-binding fragment thereof that specifically binds to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTK signaling in endothelial cells is an antibody that specifically binds to the same MERTK (e.g., human MERTK, or both human and mouse MERTK) as the antibodies described herein. For example, an anti-MERTK antibody or antigen-binding fragment thereof that specifically binds to MERTK (e.g., human MERTK, or both human and mouse MERTK) and activates MERTK signaling in endothelial cells is an anti-MERTK antibody or antigen-binding fragment thereof that competes with the anti-MERTK antibody or antigen-binding fragment thereof described herein for binding to MERTK (e.g., human MERTK, or both human and mouse MERTK). In another specific embodiment, the anti-MERTK antibody or antigen-binding fragment thereof that specifically binds to MERTK (e.g., human MERTK, or both human and mouse MERTK) and stimulates MERTK signaling in endothelial cells is a first anti-MERTK antibody or antigen-binding fragment thereof that competes with an anti-MERTK antibody or antigen-binding fragment thereof described herein for binding to MERTK (e.g., human MERTK, or both human and mouse MERTK), wherein said competition is indicated by a greater than 80% (e.g., 85%, 90%, 95%, 98%, or 80%-85%, 80%-90%, 85%-90%, or 85%-95%) reduction in binding of the first anti-MERTK antibody or antigen-binding fragment thereof to MERTK (e.g., human MERTK, or both human and mouse MERTK) in the presence of an anti-MERTK antibody or antigen-binding fragment thereof described herein.

[0052] In certain embodiments, the antibodies described herein that specifically bind to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonize MERTK signaling in endothelial cells are human, humanized, murine, or chimeric antibodies. In certain embodiments, the antibodies described herein that specifically bind to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonize MERTK signaling in endothelial cells have a K in the range of about 3 pM to about 400 pM. Dand binds to MERTK (e.g., human MERTK, or both human and mouse MERTK). In certain embodiments, the antibodies described herein are isolated. In certain embodiments, the antibodies described herein are monoclonal antibodies.

[0053] In another embodiment, described herein is a nucleic acid molecule encoding the heavy and / or light chain variable regions, or the heavy and / or light chains, of the anti-MERTK antibodies described herein. In a particular embodiment, the nucleic acid molecule encodes a heavy chain variable region comprising the nucleic acid sequence of SEQ ID NO: 53 or SEQ ID NO: 55. In another particular embodiment, the nucleic acid molecule encodes a light chain variable region comprising the nucleic acid sequence of SEQ ID NO: 54 or SEQ ID NO: 56. In a particular embodiment, the nucleic acid molecule is isolated.

[0054] In certain embodiments, described herein are vectors (e.g., isolated vectors) that contain polynucleotides encoding the heavy and / or light chain variable regions, or heavy and / or light chains, of the anti-MERTK antibodies described herein. In certain embodiments, described herein are host cells that contain the polynucleotides or vectors. Examples of host cells include E. coli, Pseudomonas, Bacillus, Streptomyces, yeast, CHO, YB / 20, NS0, PER-C6, HEK-293T, NIH-3T3, HeLa, BHK, HepG2, SP2 / 0, R1.1, BW, LM, COS1, COS7, BSC1, BSC40, BMT10 cells, plant cells, insect cells, and human cells in tissue culture. In certain embodiments, described herein are methods for producing an anti-MERTK antibody or antigen-binding fragment thereof that specifically binds to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTK signaling in endothelial cells, comprising culturing a host cell such that a polynucleotide is expressed and an antibody is produced.

[0055] In another embodiment, the antibody drug conjugates described herein and pharma- ceutical Provided herein is a pharmaceutical composition comprising a carrier as described above. The pharmaceutical composition can be used in the treatment of cancer.

[0056] In certain embodiments, provided herein is a method for treating cancer in a subject, comprising administering to the subject an effective amount of an antibody drug conjugate described herein. In another embodiment, provided herein is a method for treating cancer in a subject, comprising administering to the subject a pharmaceutical composition described herein. In certain embodiments, the method for treating cancer results in inhibition of endothelial cell migration, inhibition of angiogenesis, and / or inhibition of tumor progression.

[0057] In certain embodiments, the cancer treated by the methods provided herein is lung, breast, bone, ovarian, stomach, pancreas, larynx, esophagus, testis, liver, parotid gland, biliary tract, colon, rectum, cervix, uterine cancer, endometrial, kidney, bladder, prostate, or thyroid cancer. In some embodiments, the cancer treated is a sarcoma, squamous cell carcinoma, melanoma, glioma, glioblastoma, neuroblastoma, or Kaposi's sarcoma.

[0058] In certain embodiments, the cancer treated by the methods provided herein is breast cancer. In certain embodiments, the cancer treated by the methods provided herein is triple-negative breast cancer.

[0059] In certain embodiments, the method of treating cancer further comprises administering an additional therapeutic agent to the subject. Examples of additional therapeutic agents that can be administered to the subject in combination with the antibody drug conjugates described herein or the pharmaceutical compositions described herein are described in Sections 5.6 and 5.7 below.

[0060] In certain embodiments, the additional therapeutic agent administered to a subject in combination with an antibody drug conjugate described herein is an agent used in the treatment of breast cancer, an agent used in the treatment of melanoma, an immunotherapy, or an angiogenesis inhibitor.

[0061] In certain embodiments, the additional therapeutic agent is an agent used in the treatment of breast cancer selected from the group consisting of tamoxifen, raloxifene, paclitaxel (Taxol®), cyclophosphamide, docetaxel, vinblastine, fluorouracil, everolimus, trastuzumab, trastuzumab-emtansine, pertuzumab, and lapatinib ditrilate.

[0062] In certain embodiments, the additional therapeutic agent is an agent used in the treatment of melanoma selected from the group consisting of a BRAF inhibitor, a MEK inhibitor, and dacarbazine.

[0063] In certain embodiments, the additional therapeutic agent is an antibody that is a CTLA-4 inhibitor, a PD-1 inhibitor, or a PD-L1 inhibitor.

[0064] In certain embodiments, the additional therapeutic agent is an angiogenesis inhibitor selected from the group consisting of a VEGF inhibitor, a VEGFR2 inhibitor, sunitinib, and sorafenib.

[0065] In certain embodiments, the subject treated by the methods described herein is a human.

[0066] In one aspect, the present invention provides an antibody drug conjugate comprising: (a) an antibody moiety that is an antibody or antigen-binding fragment thereof that specifically binds to human Mer tyrosine kinase (MERTK) and agonizes human MERTK signaling in endothelial cells; (b) one or more drug moieties, each of which is a cytotoxic agent; and (c) an optional linker, wherein the cytotoxic agent is conjugated directly to the antibody moiety or via the linker. This is provided in the specification.

[0067] In certain embodiments of the foregoing aspects, the antibody or antigen-binding fragment thereof further specifically binds to mouse MERTK.

[0068] In certain embodiments of any of the foregoing aspects / embodiments, the antibody specifically recognizes the extracellular domain of human MERTK, wherein the extracellular domain comprises the amino acid sequence of SEQ ID NO:58.

[0069] In certain embodiments of any of the foregoing aspects / embodiments, the antibody competes with Gas-6 for binding to human MERTK.

[0070] In certain embodiments of any of the above aspects / embodiments, the antibody is a monoclonal antibody.

[0071] In certain embodiments of any of the foregoing aspects / embodiments, the antibody is an immunoglobulin comprising two identical light chains and two identical heavy chains.

[0072] In certain embodiments of the embodiment in which the antibody is an immunoglobulin comprising two identical light chains and two identical heavy chains, each of the heavy chains comprises a variable region (VH) comprising a complementarity determining region (CDR) 1 of SEQ ID NO: 1, a CDR2 of SEQ ID NO: 6, and a CDR3 of SEQ ID NO: 11. In a further embodiment of such a particular embodiment, each of the light chains comprises a variable region (VL) comprising a complementarity determining region (CDR) 1 of SEQ ID NO: 15, a CDR2 of SEQ ID NO: 19, and a CDR3 of SEQ ID NO: 22.

[0073] In certain embodiments in which the antibody is an immunoglobulin comprising two identical light chains and two identical heavy chains, each of the heavy chains comprises a variable region (VH), which comprises a CDR1 of SEQ ID NO: 2, a CDR2 of SEQ ID NO: 7, and a CDR3 of SEQ ID NO: 12. In a further embodiment of such a particular embodiment, each of the light chains comprises a variable region (VL), which comprises a CDR1 of SEQ ID NO: 16, a CDR2 of SEQ ID NO: 20, and a CDR3 of SEQ ID NO: 23.

[0074] In a particular embodiment of the embodiment in which the antibody is an immunoglobulin comprising two identical light chains and two identical heavy chains, each heavy chain comprises a variable region (VH) comprising a CDR1 of SEQ ID NO:3, a CDR2 of SEQ ID NO:8, and a CDR3 of SEQ ID NO:11; in a further embodiment of such a particular embodiment, each light chain comprises a variable region (VL) comprising a complementarity determining region (CDR)1 of SEQ ID NO:15, a CDR2 of SEQ ID NO:19, and a CDR3 of SEQ ID NO:22.

[0075] In certain embodiments in which the antibody is an immunoglobulin comprising two identical light chains and two identical heavy chains, each heavy chain comprises a variable region (VH), wherein the VH comprises a CDR1 of SEQ ID NO: 4, a CDR2 of SEQ ID NO: 9, and a CDR3 of SEQ ID NO: 13. In a further embodiment of such a particular embodiment, each light chain comprises a variable region (VL), wherein the VL comprises a CDR1 of SEQ ID NO: 17, a CDR2 of SEQ ID NO: 21, and a CDR3 of SEQ ID NO: 24.

[0076] In certain embodiments of the embodiment in which the antibody is an immunoglobulin comprising two identical light chains and two identical heavy chains, each heavy chain comprises a variable region (VH), said VH comprising a CDR1 of SEQ ID NO:5, a CDR2 of SEQ ID NO:10 and a CDR3 of SEQ ID NO:14. In a further embodiment of such certain embodiments, each light chain comprises a variable region (VL), said VL comprising the sequence It comprises CDR1 of number 18, CDR2 of sequence number 20, and CDR3 of sequence number 22.

[0077] In certain embodiments in which the antibody is an immunoglobulin comprising two identical light chains and two identical heavy chains, each heavy chain comprises a variable region (VH) comprising a CDR1 of SEQ ID NO: 3, a CDR2 of SEQ ID NO: 6, and a CDR3 of SEQ ID NO: 11. In a further embodiment of such a particular embodiment, each light chain comprises a variable region (VL) comprising a complementarity determining region (CDR)1 of SEQ ID NO: 15, a CDR2 of SEQ ID NO: 19, and a CDR3 of SEQ ID NO: 22.

[0078] In certain embodiments in which the antibody is an immunoglobulin comprising two identical light chains and two identical heavy chains, each of the heavy chains comprises a variable region (VH), which comprises a CDR1 of SEQ ID NO: 25, a CDR2 of SEQ ID NO: 30, and a CDR3 of SEQ ID NO: 35. In a further embodiment of such a particular embodiment, each light chain comprises a variable region (VL), which comprises a CDR1 of SEQ ID NO: 39, a CDR2 of SEQ ID NO: 43, and a CDR3 of SEQ ID NO: 46.

[0079] In certain embodiments in which the antibody is an immunoglobulin comprising two identical light chains and two identical heavy chains, each heavy chain comprises a variable region (VH) comprising a CDR1 of SEQ ID NO: 26, a CDR2 of SEQ ID NO: 31, and a CDR3 of SEQ ID NO: 36. In a further embodiment of such certain embodiments, each light chain comprises a variable region (VL) comprising a CDR1 of SEQ ID NO: 40, a CDR2 of SEQ ID NO: 44, and a CDR3 of SEQ ID NO: 47.

[0080] In a particular embodiment of the embodiment in which the antibody is an immunoglobulin comprising two identical light chains and two identical heavy chains, each heavy chain comprises a variable region (VH) comprising a CDR1 of SEQ ID NO:27, a CDR2 of SEQ ID NO:32, and a CDR3 of SEQ ID NO:35; in a further embodiment of such a particular embodiment, each light chain comprises a variable region (VL) comprising a CDR1 of SEQ ID NO:39, a CDR2 of SEQ ID NO:43, and a CDR3 of SEQ ID NO:46.

[0081] In certain embodiments in which the antibody is an immunoglobulin comprising two identical light chains and two identical heavy chains, each heavy chain comprises a variable region (VH), wherein the VH comprises a CDR1 of SEQ ID NO: 28, a CDR2 of SEQ ID NO: 33, and a CDR3 of SEQ ID NO: 37. In a further embodiment of such certain embodiments, each light chain comprises a variable region (VL), wherein the VL comprises a CDR1 of SEQ ID NO: 41, a CDR2 of SEQ ID NO: 45, and a CDR3 of SEQ ID NO: 48.

[0082] In certain embodiments in which the antibody is an immunoglobulin comprising two identical light chains and two identical heavy chains, each heavy chain comprises a variable region (VH) comprising a CDR1 of SEQ ID NO:29, a CDR2 of SEQ ID NO:34, and a CDR3 of SEQ ID NO:38.

[0083] In certain embodiments of the embodiment in which the antibody is an immunoglobulin comprising two identical light chains and two identical heavy chains, each heavy chain comprises a variable region (VH), wherein the VH comprises a CDR1 of SEQ ID NO: 27, a CDR2 of SEQ ID NO: 30, and a CDR3 of SEQ ID NO: 35. In a further embodiment of such a particular embodiment, each light chain comprises a variable region (VL), wherein the VL comprises a CDR1 of SEQ ID NO: 39, a CDR2 of SEQ ID NO: 43, and a CDR3 of SEQ ID NO: 46. In another further embodiment of such a particular embodiment, each light chain comprises a variable region (VL), wherein the VL comprises a CDR1 of SEQ ID NO: 42, a CDR2 of SEQ ID NO: 44, and a CDR3 of SEQ ID NO: 46.

[0084] In certain embodiments in which the antibody is an immunoglobulin comprising two identical light chains and two identical heavy chains, each light chain comprises a variable region (VL) comprising complementarity determining region (CDR) 1 of SEQ ID NO: 15, CDR2 of SEQ ID NO: 19, and CDR3 of SEQ ID NO: 22.

[0085] In certain embodiments in which the antibody is an immunoglobulin comprising two identical light chains and two identical heavy chains, each light chain comprises a variable region (VL) comprising a CDR1 of SEQ ID NO: 16, a CDR2 of SEQ ID NO: 20, and a CDR3 of SEQ ID NO: 23.

[0086] In certain embodiments in which the antibody is an immunoglobulin comprising two identical light chains and two identical heavy chains, each light chain comprises a variable region (VL) comprising CDR1 of SEQ ID NO: 17, CDR2 of SEQ ID NO: 21, and CDR3 of SEQ ID NO: 24.

[0087] In certain embodiments in which the antibody is an immunoglobulin comprising two identical light chains and two identical heavy chains, each light chain comprises a variable region (VL), wherein the VL comprises a CDR1 of SEQ ID NO: 18, a CDR2 of SEQ ID NO: 20, and a CDR3 of SEQ ID NO: 22.

[0088] In certain embodiments in which the antibody is an immunoglobulin comprising two identical light chains and two identical heavy chains, each light chain comprises a variable region (VL) comprising a CDR1 of SEQ ID NO:39, a CDR2 of SEQ ID NO:43, and a CDR3 of SEQ ID NO:46.

[0089] In certain embodiments in which the antibody is an immunoglobulin comprising two identical light chains and two identical heavy chains, each light chain comprises a variable region (VL) comprising a CDR1 of SEQ ID NO: 40, a CDR2 of SEQ ID NO: 44, and a CDR3 of SEQ ID NO: 47.

[0090] In certain embodiments in which the antibody is an immunoglobulin comprising two identical light chains and two identical heavy chains, each light chain comprises a variable region (VL) comprising a CDR1 of SEQ ID NO: 41, a CDR2 of SEQ ID NO: 45, and a CDR3 of SEQ ID NO: 48.

[0091] In certain embodiments in which the antibody is an immunoglobulin comprising two identical light chains and two identical heavy chains, each light chain comprises a variable region (VL) comprising a CDR1 of SEQ ID NO: 42, a CDR2 of SEQ ID NO: 44, and a CDR3 of SEQ ID NO: 46.

[0092] In a particular embodiment of the embodiment in which the antibody is an immunoglobulin comprising two identical light chains and two identical heavy chains, the heavy chain comprises a variable region (VH) that comprises SEQ ID NO: 49. In a further embodiment of such a particular embodiment, the light chain comprises a variable region (VL) that comprises SEQ ID NO: 50.

[0093] In a particular embodiment of the embodiment in which the antibody is an immunoglobulin comprising two identical light chains and two identical heavy chains, the light chain comprises a variable region (VL) comprising SEQ ID NO:50.

[0094] In a particular embodiment of the embodiment in which the antibody is an immunoglobulin comprising two identical light chains and two identical heavy chains, the heavy chain comprises a variable region (VH) comprising SEQ ID NO: 51. In a further embodiment of such an embodiment, the light chain comprises a variable region (VL) comprising SEQ ID NO: 52.

[0095] In a particular embodiment of the embodiment in which the antibody is an immunoglobulin comprising two identical light chains and two identical heavy chains, the light chain comprises a variable region (VL) comprising SEQ ID NO:52.

[0096] The antibody is an immunoglobulin that contains two identical light chains and two identical heavy chains. In certain of any of the above embodiments, the antibody or antigen-binding fragment thereof comprises a human-derived constant region. In a further embodiment of such embodiment, the heavy chain constant region has an isotype selected from the group consisting of gamma 1, gamma 2, gamma 3, and gamma 4.

[0097] In certain embodiments of any of the above aspects / embodiments, the antibody or antigen-binding fragment thereof is humanized.

[0098] In certain embodiments of any of the foregoing aspects / embodiments, the antibody has a dissociation constant K in the range of about 3 pM to about 400 pM. D Binds to human MERTK.

[0099] In certain embodiments of any of the foregoing aspects / embodiments, the antibody has a K in the range of about 3 pM to 400 pM. D Binds to mouse MERTK.

[0100] In certain embodiments of any of the above aspects / embodiments, the antibody or antigen-binding fragment inhibits endothelial cell migration in vitro in the presence of breast cancer cells, wherein said migration is inhibited by more than 30% compared to endothelial cells treated with a control antibody.

[0101] In certain embodiments of any of the foregoing aspects / embodiments, the antibody or antigen-binding fragment promotes phosphorylation of MERTK in human vascular endothelial cells in vitro.

[0102] In certain embodiments of any of the foregoing aspects / embodiments, the antibody or antigen-binding fragment does not promote phosphorylation of MERTK on cancer cells in vitro.

[0103] In certain embodiments of any of the foregoing aspects / embodiments, the antibody or antigen-binding fragment inhibits tumor angiogenesis in vivo.

[0104] In certain embodiments of any of the foregoing aspects / embodiments, the antibody or antigen-binding fragment does not inhibit migration of glioblastoma multiforme cell line A172 in an in vitro transwell migration assay in the absence of endothelial cells.

[0105] In certain embodiments of any of the foregoing aspects / embodiments, the antibody or antigen-binding fragment does not reduce the expression level of MERTK on the glioblastoma multiforme cell line A172.

[0106] In another aspect, provided herein is an antibody drug conjugate comprising: (a) an antibody moiety that is an antibody or antigen-binding fragment thereof that specifically binds to human MERTK and that comprises a heavy chain variable region (VH) that comprises complementarity determining region (CDR) 1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 6, and CDR3 of SEQ ID NO: 11; (b) one or more drug moieties, each of which is a cytotoxic agent; and (c) an optional linker, wherein the cytotoxic agent is conjugated directly to the antibody moiety or via the linker to the antibody moiety. In certain embodiments of such aspect, the antibody or antigen-binding fragment thereof further comprises a light chain variable region (VL), wherein the VL comprises complementarity determining region (CDR) 1 of SEQ ID NO: 15, CDR2 of SEQ ID NO: 19, and CDR3 of SEQ ID NO: 22.

[0107] In another aspect, provided herein is an antibody drug conjugate comprising: (a) an antibody moiety that is an antibody or antigen-binding fragment thereof, the antibody or antigen-binding fragment comprising a heavy chain variable region (VH) that specifically binds to human MERTK and that comprises CDR1 of SEQ ID NO:2, CDR2 of SEQ ID NO:7, and CDR3 of SEQ ID NO:12; (b) one or more drug moieties, each of which is a cytotoxic agent; and (c) an optional linker, wherein the cytotoxic agent is conjugated directly to the antibody moiety or via the linker to the antibody moiety. In embodiments, the antibody or antigen-binding fragment thereof further comprises a light chain variable region (VL), wherein the VL comprises a CDR1 of SEQ ID NO:16, a CDR2 of SEQ ID NO:20, and a CDR3 of SEQ ID NO:23.

[0108] In another aspect, provided herein is an antibody drug conjugate comprising: (a) an antibody moiety that is an antibody or antigen-binding fragment thereof that specifically binds to human MERTK and that comprises a heavy chain variable region (VH) comprising CDR1 of SEQ ID NO:3, CDR2 of SEQ ID NO:8, and CDR3 of SEQ ID NO:11; (b) one or more drug moieties, each of which is a cytotoxic agent; and (c) an optional linker, wherein the cytotoxic agent is conjugated directly to the antibody moiety or via the linker to the antibody moiety. In certain embodiments of such aspect, the antibody or antigen-binding fragment thereof further comprises a light chain variable region (VL), wherein the VL comprises a complementarity determining region (CDR)1 of SEQ ID NO:15, a CDR2 comprising the amino acid sequence of SEQ ID NO:19, and a CDR3 comprising the amino acid sequence of SEQ ID NO:22.

[0109] In another aspect, provided herein is an antibody drug conjugate comprising: (a) an antibody moiety that is an antibody or antigen-binding fragment thereof that specifically binds human MERTK and that comprises a heavy chain variable region (VH) that comprises CDR1 of SEQ ID NO: 4, CDR2 of SEQ ID NO: 9, and CDR3 of SEQ ID NO: 13; (b) one or more drug moieties, each of which is a cytotoxic agent; and (c) an optional linker, wherein the cytotoxic agent is conjugated directly to the antibody moiety or via the linker. In certain embodiments of such aspect, the antibody or antigen-binding fragment thereof further comprises a light chain variable region (VL), wherein the VL comprises CDR1 of SEQ ID NO: 17, CDR2 of SEQ ID NO: 21, and CDR3 of SEQ ID NO: 24.

[0110] In another aspect, provided herein is an antibody drug conjugate comprising: (a) an antibody moiety that is an antibody or antigen-binding fragment thereof that specifically binds human MERTK and that comprises a heavy chain variable region (VH) that comprises CDR1 of SEQ ID NO:5, CDR2 of SEQ ID NO:10, and CDR3 of SEQ ID NO:14; (b) one or more drug moieties, each of which is a cytotoxic agent; and (c) an optional linker, wherein the cytotoxic agent is conjugated directly to the antibody moiety or via the linker. In certain embodiments of such aspect, the antibody or antigen-binding fragment thereof further comprises a light chain variable region (VL), wherein the VL comprises CDR1 of SEQ ID NO:18, CDR2 of SEQ ID NO:20, and CDR3 of SEQ ID NO:22.

[0111] In another aspect, provided herein is an antibody drug conjugate comprising: (a) an antibody moiety that is an antibody or antigen-binding fragment thereof that specifically binds to human MERTK and that comprises a heavy chain variable region (VH) comprising CDR1 of SEQ ID NO:3, CDR2 of SEQ ID NO:6, and CDR3 of SEQ ID NO:11; (b) one or more drug moieties, each of which is a cytotoxic agent; and (c) an optional linker, wherein the cytotoxic agent is conjugated directly to the antibody moiety or via the linker. In certain embodiments of such aspect, the antibody or antigen-binding fragment thereof further comprises a light chain variable region (VL), wherein the VL comprises a complementarity determining region (CDR)1 of SEQ ID NO:15, a CDR2 comprising the amino acid sequence of SEQ ID NO:19, and a CDR3 comprising the amino acid sequence of SEQ ID NO:22.

[0112] In another aspect, provided herein is an antibody drug conjugate comprising: (a) an antibody moiety that is an antibody or antigen-binding fragment thereof that specifically binds to human MERTK and that comprises a heavy chain variable region (VH) that comprises CDR1 of SEQ ID NO:25, CDR2 of SEQ ID NO:30, and CDR3 of SEQ ID NO:35; (b) one or more drug moieties, each of which is a cytotoxic agent; and (c) an optional linker, wherein the cytotoxic agent is conjugated directly to the antibody moiety or via the linker to the antibody moiety. In one embodiment, the antibody or antigen-binding fragment thereof further comprises a light chain variable region (VL), wherein the VL comprises a CDR1 of SEQ ID NO:39, a CDR2 of SEQ ID NO:43, and a CDR3 of SEQ ID NO:46.

[0113] In another aspect, provided herein is an antibody drug conjugate comprising: (a) an antibody moiety that is an antibody or antigen-binding fragment thereof that specifically binds human MERTK and that comprises a heavy chain variable region (VH) comprising CDR1 of SEQ ID NO:26, CDR2 of SEQ ID NO:31, and CDR3 of SEQ ID NO:36; (b) one or more drug moieties, each of which is a cytotoxic agent; and (c) an optional linker, wherein the cytotoxic agent is conjugated directly to the antibody moiety or via the linker. In certain embodiments of such aspect, the antibody or antigen-binding fragment thereof further comprises a light chain variable region (VL), wherein the VL comprises CDR1 of SEQ ID NO:40, CDR2 of SEQ ID NO:44, and CDR3 of SEQ ID NO:47.

[0114] In another aspect, provided herein is an antibody drug conjugate comprising: (a) an antibody moiety that is an antibody or antigen-binding fragment thereof that specifically binds human MERTK and that comprises a heavy chain variable region (VH) comprising CDR1 of SEQ ID NO:27, CDR2 of SEQ ID NO:32, and CDR3 of SEQ ID NO:35; (b) one or more drug moieties, each of which is a cytotoxic agent; and (c) an optional linker, wherein the cytotoxic agent is conjugated directly to the antibody moiety or via the linker. In certain embodiments of such aspect, the antibody or antigen-binding fragment thereof further comprises a light chain variable region (VL), wherein the VL comprises CDR1 of SEQ ID NO:39, CDR2 of SEQ ID NO:43, and CDR3 of SEQ ID NO:46.

[0115] In another aspect, provided herein is an antibody drug conjugate comprising: (a) an antibody moiety that is an antibody or antigen-binding fragment thereof that specifically binds human MERTK and comprises a heavy chain variable region (VH) that comprises CDR1 of SEQ ID NO:28, CDR2 of SEQ ID NO:33, and CDR3 of SEQ ID NO:37; (b) one or more drug moieties, each of which is a cytotoxic agent; and (c) an optional linker, wherein the cytotoxic agent is conjugated directly to the antibody moiety or via the linker. In certain embodiments of such aspect, the antibody or antigen-binding fragment thereof further comprises a light chain variable region (VL), wherein the VL comprises CDR1 of SEQ ID NO:41, CDR2 of SEQ ID NO:45, and CDR3 of SEQ ID NO:48.

[0116] In another aspect, provided herein is an antibody drug conjugate comprising: (a) an antibody moiety that is an antibody or antigen-binding fragment thereof, the antibody or antigen-binding fragment comprising a heavy chain variable region (VH) that specifically binds to human MERTK and comprises CDR1 of SEQ ID NO:29, CDR2 of SEQ ID NO:34, and CDR3 of SEQ ID NO:38; (b) one or more drug moieties, each of which is a cytotoxic agent; and (c) an optional linker, wherein the cytotoxic agent is conjugated directly to the antibody moiety or via the linker to the antibody moiety.

[0117] In another aspect, provided herein is an antibody drug conjugate comprising: (a) an antibody moiety that is an antibody or antigen-binding fragment thereof that specifically binds human MERTK and that comprises a heavy chain variable region (VH) comprising CDR1 of SEQ ID NO:27, CDR2 of SEQ ID NO:30, and CDR3 of SEQ ID NO:35; (b) one or more drug moieties, each of which is a cytotoxic agent; and (c) an optional linker, wherein the cytotoxic agent is conjugated directly to the antibody moiety or via the linker. In a particular embodiment of such aspect, the antibody or antigen-binding fragment thereof further comprises a light chain variable region (VL), wherein the VL comprises CDR1 of SEQ ID NO:39, CDR2 of SEQ ID NO:43, and CDR3 of SEQ ID NO:46. In another particular embodiment of such aspect, the antibody or antigen-binding fragment thereof further comprises a light chain variable region (VL), wherein the VL comprises CDR1 of SEQ ID NO:39, CDR2 of SEQ ID NO:43, and CDR3 of SEQ ID NO:46. further comprises a light chain variable region (VL), which comprises a CDR1 of SEQ ID NO:42, a CDR2 of SEQ ID NO:44, and a CDR3 of SEQ ID NO:46.

[0118] In certain embodiments of any of the foregoing aspects, the antibody is an immunoglobulin and the antigen-binding fragment is a portion of an immunoglobulin.

[0119] In certain embodiments of any of the foregoing aspects, the antibody or antigen-binding fragment thereof is an immunoglobulin.

[0120] In certain embodiments of any of the above aspects / embodiments, the antibody or antigen-binding fragment thereof is humanized.

[0121] In certain embodiments of any of the foregoing aspects / embodiments, the antibody or antigen-binding fragment thereof comprises a human-derived constant region. In a further embodiment of such a particular embodiment, the antibody or antigen-binding fragment thereof is a humanized immunoglobulin.

[0122] In certain embodiments of any of the preceding forms / embodiments where the antibody is not a monoclonal antibody or an immunoglobulin comprising two identical light chains and two identical heavy chains, the antibody or antigen-binding fragment thereof is a bispecific antibody.

[0123] In another aspect, provided herein is an antibody drug conjugate comprising: (A) an antibody portion which is an immunoglobulin that specifically binds to human MERTK; (B) one or more drug portions, each of which is a cytotoxic agent; and (C) an optional linker, wherein the immunoglobulin comprises (i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 51, and (ii) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 52; and wherein the cytotoxic agent is conjugated directly to the antibody portion or via the linker.

[0124] In another aspect, provided herein is an antibody drug conjugate comprising: (A) an antibody portion that is an immunoglobulin that specifically binds to human MERTK; (B) one or more drug portions, each of which is a cytotoxic agent; and (C) an optional linker, wherein the immunoglobulin comprises (i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 49, and (ii) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 50, and wherein the cytotoxic agent is conjugated directly to the antibody portion or via the linker.

[0125] In another aspect, provided herein is an antibody drug conjugate comprising: (I) an antibody portion which is an immunoglobulin that specifically binds to human MERTK; (II) one or more drug portions, each of which is a cytotoxic agent; and (III) an optional linker, wherein said immunoglobulin comprises: (A) (i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 51, and (ii) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 52; or (B) (i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 49, and (ii) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 50; wherein said cytotoxic agent is conjugated directly to said antibody portion or via said linker.

[0126] In another aspect, the present invention provides an immunoglobulin that is an antibody that competes for binding to MERTK with a reference antibody selected from the group consisting of: (I) (a) a first immunoglobulin comprising: (i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 51, and (ii) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 52; and (b) a second immunoglobulin comprising: (i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 49, and (ii) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 50; (II) one or more drug moieties, each of which is a cytotoxic agent; and (III) an optional linker, wherein the cytotoxic agent is conjugated directly to the antibody moiety or Alternatively, provided herein is an antibody drug conjugate, which is conjugated to said antibody moiety via said linker.

[0127] In certain embodiments of any of the foregoing aspects / embodiments, the molar ratio of antibody moiety to drug moiety is from 1:1 to 1:8.

[0128] In certain embodiments of any of the foregoing aspects / embodiments, the molar ratio of antibody moiety to drug moiety is from 1:3 to 1:5.

[0129] In certain embodiments of any of the foregoing aspects / embodiments, the antibody drug conjugate comprises a linker, wherein the linker is a cleavable linker.

[0130] In certain embodiments of any of the foregoing aspects / embodiments, the antibody drug conjugate comprises a linker, wherein the linker is a non-cleavable linker.

[0131] In certain embodiments of any of the foregoing aspects / embodiments, the cytotoxic agent is a small molecule, a nucleotide, a peptide, or a non-antibody protein. In a further embodiment of such certain embodiments, the cytotoxic agent is a small molecule.

[0132] In certain embodiments of any of the foregoing forms / embodiments, the cytotoxic agent is an auristatin, a maytansinoid, a pyrrolobenzodiazepine, an indolinobenzodiazepine, a calicheamicin, a camptothecin analog, a duocarmycin, a tubulin inhibitor, a tubulysin or a tubulysin analog, an ambelstatin 269, a doxorubicin, an antibiotic, an anthracycline, a microtubule inhibitor, a spliceostatin, or a tylanstatin. In a further embodiment of such a particular embodiment, the cytotoxic agent is monomethylauristatin E (MMAE) or monomethylauristatin F (MMAF). In another further embodiment of such a particular embodiment, the cytotoxic agent is DM1 or DM4.

[0133] In another aspect, there is provided herein a method for producing an antibody drug conjugate according to any of the above aspects / embodiments in which a linker is not present, the method comprising: (a) directly conjugating the cytotoxic agent to the antibody moiety to produce an antibody drug conjugate; and (b) purifying the antibody drug conjugate.

[0134] In another aspect, there is provided herein a method for producing an antibody drug conjugate according to any of the above aspects / embodiments comprising a linker, the method comprising, in order: (a) directly conjugating the linker to the antibody moiety to produce a linker antibody moiety; (b) directly conjugating the linker of the linker antibody moiety to the cytotoxic agent to produce an antibody drug conjugate; and (c) purifying the antibody drug conjugate.

[0135] In another aspect, there is provided herein a method for producing an antibody drug conjugate according to any of the above aspects / embodiments comprising a linker, the method comprising, in order: (a) directly conjugating the linker to the cytotoxic agent to produce a linker-cytotoxic agent moiety; (b) directly conjugating the linker of the linker-cytotoxic agent moiety to the antibody moiety to produce an antibody drug conjugate; and (c) purifying the antibody drug conjugate.

[0136] In another aspect, there is provided herein a pharmaceutical composition comprising a therapeutically effective amount of an antibody drug conjugate according to any of the preceding aspects / embodiments, and a pharma- ceutically acceptable carrier. .

[0137] In another aspect, there is provided herein a method of treating cancer in a subject in need thereof, comprising administering to said subject a pharmaceutical composition as described in the preceding aspect.

[0138] In certain embodiments of the above-mentioned forms of the method of treating cancer, the cancer is lung, breast, bone, ovary, stomach, pancreas, larynx, esophagus, testis, liver, parotid gland, biliary tract, colon, rectum, cervix, uterus, endometrium, kidney, bladder, prostate or thyroid cancer.In certain embodiments of such certain embodiments, the cancer is breast cancer.In a further embodiment of such certain embodiments, the cancer is triple-negative breast cancer.

[0139] In certain embodiments of the foregoing aspects of the methods of treating cancer, the cancer is a sarcoma, squamous cell carcinoma, melanoma, glioma, glioblastoma, neuroblastoma, or Kaposi's sarcoma.

[0140] In certain embodiments of any of the above aspects / embodiments of the method of treating cancer, the method further comprises administering an additional therapeutic agent to the subject.

[0141] In certain embodiments of the embodiments where the method further comprises administering an additional therapeutic agent to the subject, the additional therapeutic agent is for treating said cancer.

[0142] In certain embodiments of the embodiment in which the additional therapeutic agent is for treating the cancer, the additional therapeutic agent is a drug used to treat breast cancer, a drug used to treat melanoma, an immunotherapy, or an angiogenesis inhibitor. In a further embodiment of such a particular embodiment, the additional therapeutic agent is a drug used to treat breast cancer selected from the group consisting of tamoxifen, raloxifene, paclitaxel, cyclophosphamide, docetaxel, vinblastine, fluorouracil, everolimus, trastuzumab, trastuzumab-emtansine, pertuzumab, and lapatinib ditosylate. In another further embodiment of such a particular embodiment, the additional therapeutic agent is a drug used to treat melanoma selected from the group consisting of a BRAF inhibitor, a MEK inhibitor, and dacarbazine. In another further embodiment of such a particular embodiment, the additional therapeutic agent is an antibody that is a CTLA-4 inhibitor, a PD-1 inhibitor, or a PD-L1 inhibitor. In another further embodiment of this particular embodiment, the additional therapeutic agent is an angiogenesis inhibitor selected from the group consisting of a VEGF inhibitor, a VEGFR2 inhibitor, sunitinib, and sorafenib.

[0143] In certain embodiments of any of the above aspects / embodiments of the method of treating cancer, the subject is a human.

[0144] 4. Brief description of the drawings [Brief description of the drawings]

[0145] [Figure 1] 1A and 1B: Sequence of MERTK. A) Schematic of the recombinant MERTK peptide used to immunize mice for antibody production. The peptide is composed of the extracellular domain of MERTK (see FIG. 1B), a short connecting peptide (IEGRMD), and a portion of human IgG1. B) Sequence of full-length human MERTK (SEQ ID NO: 57) showing the portion of MERTK used in the recombinant MERTK peptide described in FIG. 1A (bold and underlined sequence; SEQ ID NO: 58). [Diagram 2]Figure 2: MERTK monoclonal antibody screen. Diagram showing data from an antibody capture ELISA assay used to characterize the binding properties of MERTK-binding monoclonal antibodies recovered from single hybridoma clones. Antibody clones are arbitrarily named M1 through M20 in column 1. Several of the MERTK-binding monoclonal antibodies exhibited high affinity binding to MERTK, as indicated by the large (>3.5) OD values ​​in column 2. Some of these antibodies also neutralized binding of MERTK to Gas-6, as indicated by the low (<2.5) blocking OD values ​​in column 3. [Diagram 3] Figure 3: MERTK-binding antibodies M19 and M6 inhibit endothelial recruitment. Diagram showing that either monoclonal antibody M6 or M19 at physiological concentrations can significantly inhibit endothelial recruitment by metastatic breast cancer cells. 2.5 × 104 MDA-MB-231 cells were seeded in quadruplicate. Transwell migration of 5 × 104 HUVEC cells towards cancer cells was assessed in the presence of 200 ng / mL of control antibody (IgG) or MERTK-binding antibodies isolated from the screening. Images of cells migrated through the transwell insert were acquired and cells were counted using ImageJ software. N=4. Monoclonal antibodies (M6 and M19) that significantly inhibit endothelial cell recruitment are labeled in red and green, respectively. Error bars represent standard error of the mean. [Figure 4]Figures 4A, 4B and 4C: MERTK-binding antibody M19 activates MERTK on endothelial cells. A) HUVEC cells were treated with either no FBS + no M19, 10% FBS + no M19, 10% FBS + 25 μg / mL M19, or no FBS + 25 μg / mL M19 for 16 hours and Western blot analysis was performed for activated (phosphorylated) MERTK (P-MERTK) and total MERTK and AKT levels. As shown, treatment with M19 antibody increased the levels of activated MERTK. B) Quantification of MERTK activation by M19 antibody treatment was calculated as the ratio of protein expression levels of P-MERTK to MERTK isolated from M19-treated and untreated HUVEC cells based on the Western blot data in Figure 4A. C) HUVEC cells were treated with increasing concentrations of M19 antibody as indicated for 30 min prior to Western blot analysis of P-MERTK. It is noteworthy that increasing doses of M19 treatment increased MERTK activation. [Diagram 5] Figures 5A and 5B: MERTK-binding antibody M6 activates MERTK on endothelial cells. A) HUVEC cells were treated with either no FBS + no M6, 10% FBS + no M6, 10% FBS + 25 μg / mL M6, or no FBS + 25 μg / mL M6 for 16 hours prior to Western blot analysis of activated (phosphorylated) MERTK (P-MERTK) and total MERTK levels. As shown, treatment with M6 antibody increased the levels of activated MERTK. B) Quantification of MERTK activation by M6 antibody treatment, calculated as the ratio of protein expression levels of P-MERTK to MERTK isolated from M6-treated and untreated HUVEC cells based on the Western blot data in Figure 5A. [Figure 6]Figure 6: MERTK-binding antibody M19 does not activate MERTK on cancer cells. LM2 breast cancer cells were treated with either no FBS + no M19 antibody, 10% FBS + no M19 antibody, 10% FBS + 25 μg / mL M19 antibody, or no FBS + 25 μg / mL M19 antibody for 16 hours, followed by Western blot analysis for activated MERTK (P-MERTK) and total MERTK levels. As shown, no detectable levels of P-MERTK were induced in cancer cells treated with M19 antibody. [Figure 7] Figure 7: M19 binds to human MERTK with high affinity. Diagram showing antibody binding kinetics of M19 (Mer-M19) to human MERTK (hMer) using biolayer interferometry. Purified M19 was loaded onto the AMQ sensor at a concentration of 1 μg / mL and tested for binding to analytes in solution. All analytes were prepared in a 2-fold dilution series starting from 5 nM for a total of 7 concentrations. Dynamic fits were calculated using a 1:1 model, with local fits for each concentration and an overall global fit. The binding affinity (KD) of M19 binding to human MERTK calculated by the overall global fit was 326 picomolar. [Figure 8] Figure 8: M19 binds with high affinity to mouse MERTK. Diagram showing antibody binding kinetics of M19 (Mer-M19) to mouse MERTK (msMer) using biolayer interferometry. Purified M19 was loaded onto the AMQ sensor at a concentration of 1 μg / mL and tested for binding to analytes in solution. All analytes were prepared in a 2-fold dilution series starting from 5 nM for a total of 7 concentrations. Dynamic fits were calculated using a 1:1 model, with local fits for each concentration and an overall global fit. The overall calculated global fit binding affinity (KD) of M19 binding to mouse MERTK was 305 picomolar. [Figure 9]Figures 9A and 9B: Treatment with M19 inhibits primary triple-negative breast cancer tumor growth and metastasis in vivo. A) Tumor growth in mammary fat pads of 2000 MDA-MB-231 or 5000 Lm1a1 breast cancer cells in mice treated every other week with 250 μg of either control antibody (IgG) or M19 antibody. B) Lm1a1 breast cancer cells were injected bilaterally into the mammary fat pads in mice treated every other week with 250 μg of either control antibody (IgG) or M19 antibody. After 98 days, lungs were removed and processed for H&E staining, and the number of metastatic nodules was counted. N=4. Error bars represent standard error of the mean. P values ​​were obtained using Student's T-test (*p<0.05). [Figure 10] Figure 10: Treatment with M19 inhibits angiogenesis in vivo. Xenograft tumors in mammary fat pads grown for 58 days, treated twice weekly with 250 μg of control antibody (IgG) or M19 antibody, were double stained with DAPI and CD31. Vascular density was quantified using thresholded CD31 signal. N=4. Error bars represent standard error of the mean. P values ​​were obtained using Student's T-test (*p<0.05). [Figure 11] Figure 11: M6 binds to MERTK with high affinity. M6 binds to human MERTK with high affinity. Diagram showing antibody binding kinetics of M6 to human MERTK (hMer) (Mer-M6) using biolayer interferometry. Purified M6 was loaded onto the AMQ sensor at a concentration of 1 μg / mL and tested for binding to analytes in solution. All analytes were prepared in a 2-fold dilution series starting from 5 nM for a total of 7 concentrations. Dynamic fits were calculated using a 1:1 model, with local fits for each concentration and an overall global fit. The overall calculated binding affinity (KD) for M6 binding to human MERTK was 4.6 picomolar. [Figure 12]Figure 12: Treatment with M6 inhibits primary triple-negative breast cancer tumor growth in vivo. Mammary fat pad tumor growth by 2000MDA-MB-231 in mice treated with either 250 μg of control antibody or M6 antibody every other week for 21 days. N=4. Error bars represent standard error of the mean. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0146] 5. Detailed Description According to the present specification, there is provided an antibody drug conjugate comprising: (a) an antibody moiety that is an anti-MERTK antibody (e.g., a monoclonal antibody) or an antigen-binding fragment thereof that specifically binds to MERTK (e.g., human MERTK, or both human and mouse MERTK) and activates human MERTK signaling in endothelial cells; (b) one or more drug moieties, each of which is a cytotoxic agent; and (c) an optional linker, wherein the cytotoxic agent is conjugated directly to the antibody moiety or conjugated to the antibody moiety via the linker. As used in this disclosure, the term "conjugated" shall mean attached by a covalent bond, which may be direct or through an intervening covalent bond structure.

[0147] In certain embodiments, the antibody-drug conjugates provided herein have a molar ratio of antibody moiety to drug moiety of 1:1 to 1:20. In certain embodiments, the antibody-drug conjugates provided herein have a molar ratio of antibody moiety to drug moiety of 1:1 to 1:15. In certain embodiments, the antibody-drug conjugates provided herein have a molar ratio of antibody moiety to drug moiety of 1:1 to 1:12. In certain embodiments, In some embodiments, the antibody drug conjugates provided herein have a molar ratio of antibody moiety to drug moiety of 1:1 to 1:8. In a preferred embodiment, the antibody drug conjugates provided herein have a molar ratio of antibody moiety to drug moiety of 1:3 to 1:5. In a particular embodiment, the antibody drug conjugates provided herein have a molar ratio of antibody moiety to drug moiety that is 1:3. In another particular embodiment, the antibody drug conjugates provided herein have a molar ratio of antibody moiety to drug moiety that is 1:4. In another particular embodiment, the antibody drug conjugates provided herein have a molar ratio of antibody moiety to drug moiety that is 1:5.

[0148] The drug moiety is attached to one or more chains of the antibody moiety. In some embodiments, the drug moiety is attached to one chain of the antibody moiety (e.g., when the antibody moiety is an scFv or when the antibody moiety is a multi-chain antibody such as an immunoglobulin (which is a tetramer) or an antigen-binding fragment thereof). In other embodiments, the drug moiety is attached to more than one chain of the antibody moiety (when the antibody moiety is a multi-chain antibody such as an immunoglobulin or an antigen-binding fragment thereof). In certain embodiments, the drug moiety is attached to two identical chains of an immunoglobulin, e.g., a heavy chain or a light chain. In other embodiments, the drug moiety is attached to all chains of the antibody moiety (when the antibody moiety is a multi-chain antibody such as an immunoglobulin or an antigen-binding fragment thereof).

[0149] In certain embodiments, the drug moiety is attached to one or more sites in the constant region of the antibody, hi certain embodiments, the drug moiety is attached to the Fc region of an antibody that is an immunoglobulin.

[0150] In certain embodiments, the anti-MERTK antibodies or antigen-binding fragments thereof described herein specifically recognize the extracellular domain of MERTK (e.g., human MERTK, or both human and mouse MERTK). In certain embodiments, the anti-MERTK antibodies or antigen-binding fragments thereof described herein do not bind to the 185 kilodalton (kD) MERTK glycoform expressed in Jurkat cells, the 205 kD MERTK glycoform in the monocytic cell line U937, the 135-140 kD MERTK glycoform expressed in human leukemia cells (e.g., human T-cell acute lymphoblastic leukemia), and the 170-190 kD MERTK glycoform expressed in human leukemia cells (e.g., human T-cell acute lymphoblastic leukemia). In certain embodiments, the anti-MERTK antibodies or antigen-binding fragments thereof described herein do not reduce the expression level of MERTK on cancer cells. In certain embodiments, the anti-MERTK antibody or antigen-binding fragment thereof described herein does not reduce the expression level of MERTK on the glioblastoma multiforme cell line A172. In certain embodiments, the anti-MERTK antibody or antigen-binding fragment thereof described herein is isolated. In certain embodiments, the anti-MERTK antibody or antigen-binding fragment thereof described herein specifically binds to a human MERTK protein comprising the amino acid sequence of SEQ ID NO: 57. In another specific embodiment, the anti-MERTK antibody or antigen-binding fragment thereof described herein specifically binds to an extracellular domain of human MERTK comprising the amino acid sequence of SEQ ID NO: 58. In another specific embodiment, the anti-MERTK antibody or antigen-binding fragment thereof described herein specifically binds to SEQ ID NO: 58. In certain embodiments, the anti-MERTK antibody or antigen-binding fragment thereof described herein comprises two antigen-binding sites that bind to MERTK. In certain embodiments, the two antigen-binding sites bind to the same epitope on MERTK. In certain embodiments, the two antigen-binding sites comprise identical CDRs.

[0151] Examples of antibodies include monoclonal antibodies, polyclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies containing two heavy chain molecules and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-antibody heavy chain pairs, intrabodies, heteroconjugate antibodies, single domain antibodies, and the like. Examples of antibodies include antibodies, monovalent antibodies, single chain antibodies or single chain Fvs (scFvs), camelized antibodies, affibodies, disulfide-linked Fvs (sdFvs), and anti-idiotypic (anti-Id) antibodies (including, for example, anti-anti-Id antibodies). In certain embodiments, the antibodies described herein are polyclonal antibodies. In preferred embodiments, the antibodies described herein are monoclonal antibodies. Antibodies may be of any type (e.g., IgG, IgE, IgM, IgD, IgA or IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 or IgA2), or any subclass (e.g., IgG, 2a or IgG 2b ). In certain embodiments, the antibodies described herein are IgG antibodies, or classes or subclasses thereof. In certain embodiments, the antibodies are humanized monoclonal antibodies. In another particular embodiment, the antibodies are human monoclonal antibodies, preferably immunoglobulins.

[0152] As used herein, the terms "antigen-binding fragment", "antigen-binding region" and similar terms refer to a portion of an antibody molecule that contains amino acid residues that confer specificity to the antibody molecule for an antigen (e.g., complementarity determining region (CDR)). The antigen-binding region can be derived from any animal species, such as, for example, rodents (e.g., mice, rats or hamsters) and humans. By way of example, antigen-binding fragments include Fab fragments, F(ab')2 fragments, and antigen-binding fragments of any of the above antibodies.

[0153] As used herein, the terms "variable region" or "variable domain" are used interchangeably and are common in the art. A variable region usually refers to a portion of an antibody, typically a portion of the light or heavy chain, that varies widely in sequence between antibodies and is used for the binding and specificity of a particular antibody to a particular antigen. The sequence variability is concentrated in regions called complementarity determining regions (CDRs), while the more highly conserved regions of the variable domain are called framework regions (FRs). Without wishing to be bound by a particular mechanism or theory, it is believed that the CDRs of the light and heavy chains are primarily responsible for the interaction and specificity of the antibody with the antigen. In certain embodiments, the variable region is a human variable region. In certain embodiments, the variable region comprises rodent or murine CDRs and human framework regions (FRs). In certain embodiments, the variable region is a primate (e.g., non-human primate) variable region. In certain embodiments, the variable region comprises rodent or murine CDRs and primate (e.g., non-human primate) framework regions (FRs).

[0154] CDRs are defined in various ways in the art, including the Kabat, Chothia, AbM, Contact, IMGT, and Exemplary definitions. The Kabat definition is based on sequence variability and is the most commonly used definition for predicting CDR regions (Kabat, Elvin A. et al., Sequences of Proteins of Immunological Interest. Bethesda: National Institutes of Health, 1983). The Chothia definition is based on the location of structural loop regions (Chothia et al., (1987) J Mol Biol 196:901-917). The AbM definition, which is a compromise between the Kabat and Chothia definitions, is an integrated package of programs for antibody structure modeling created by the Oxford Molecular Group (bioinf.org.uk / abs) (Martin ACR et al., (1989) PNAS 86:9268-9272). The Contact definition is based on the analysis of available complex crystal structures (bioinf.org.uk / abs) (see MacCallum RM et al., (1996) J Mol Biol 5:732-745). The IMGT definition is from IMGT ("IMGT®, the International ImMunoGeneTics Information System® website imgt.org, Founder and Director: Marie-Paule Lefranc, Montpellier, France). The Exemplary definition is A A combination of bM and Kabat (Presta et al., (1997) Cancer Res 57:4593-4599).

[0155] Also described herein are isolated nucleic acids (polynucleotides), such as complementary DNA (cDNA), encoding the anti-MERTK antibodies and antigen-binding fragments thereof as described above. When the cytotoxic agent is a peptide or protein, or when the cytotoxic agent and linker (if present) are peptides or proteins, nucleic acids encoding antibody drug conjugates are also provided. Further described herein are vectors (e.g., expression vectors) and cells (e.g., host cells) comprising nucleic acids (polynucleotides) encoding such anti-MERTK antibodies or antigen-binding fragments thereof or antibody drug conjugates. Also described herein are methods of producing such antibodies or antibody drug conjugates.

[0156] In another aspect, the present specification provides a method for producing an antibody drug conjugate comprising: (i) an anti-MERTK antibody or antigen-binding fragment thereof; and (ii) a cytotoxic agent that is directly bound to the anti-MERTK antibody or antigen-binding fragment thereof or that is bound to the anti-MERTK antibody or antigen-binding fragment thereof via a linker.

[0157] In another aspect, the present disclosure provides a method for treating cancer in a subject, comprising administering to the subject an effective amount of an antibody drug conjugate comprising (i) an anti-MERTK antibody or an antigen-binding fragment thereof, and (ii) a cytotoxic agent that is directly bound to the anti-MERTK antibody or an antigen-binding fragment thereof or that is bound to the anti-MERTK antibody or an antigen-binding fragment thereof via a linker. Related compositions (e.g., pharmaceutical compositions) and kits are also provided.

[0158] 5.1 Antibody moiety Non-limiting exemplary anti-MERTK antibodies or antigen-binding fragments thereof that can be the antibody portion of the antibody drug conjugates provided herein are provided below.

[0159] 5.1.1 Sequences and variants In certain embodiments, the anti-MERTK antibodies or antigen-binding fragments thereof described herein comprise a VH CDR1 of an antibody of Table 1 or Table 3 as defined by Kabat, Chothia, AbM, Contact, IMGT, or Exemplary. In some embodiments, the anti-MERTK antibodies or antigen-binding fragments thereof described herein comprise a VH CDR2 of an antibody of Table 1 or Table 3 as defined by Kabat, Chothia, AbM, Contact, IMGT, or Exemplary. In some embodiments, the anti-MERTK antibodies or antigen-binding fragments thereof described herein comprise a VH CDR3 of an antibody of Table 1 or Table 3 as defined by Kabat, Chothia, AbM, Contact, IMGT, or Exemplary. In some embodiments, the anti-MERTK antibodies or antigen-binding fragments thereof described herein comprise one, two, or all three of the VH CDRs of an antibody of Table 1 or Table 3 (e.g., the VH CDRs of the first row of Table 1, all of the Kabat VH CDRs of antibody M6).

[0160] In certain embodiments, the anti-MERTK antibodies or antigen-binding fragments thereof described herein comprise a VL CDR1 of an antibody of Table 2 or Table 4 as defined by Kabat, Chothia, AbM, Contact, IMGT, or Exemplary. In some embodiments, the anti-MERTK antibodies or antigen-binding fragments thereof described herein comprise a VL CDR2 of an antibody of Table 2 or Table 4 as defined by Kabat, Chothia, AbM, Contact, IMGT, or Exemplary. In some embodiments, the anti-MERTK antibodies or antigen-binding fragments thereof described herein comprise a VL CDR3 of an antibody of Table 2 or Table 4 as defined by Kabat, Chothia, AbM, Contact, IMGT, or Exemplary. The antibody or antigen-binding fragment thereof described herein comprises one, two, or all three of the VL CDRs of an antibody of Table 2 or Table 4, as defined by AbM, Contact, IMGT, or Exemplary. In some embodiments, the anti-MERTK antibody or antigen-binding fragment thereof described herein comprises one, two, or all three of the VL CDRs of an antibody of Table 2 or Table 4 (e.g., the VH CDRs of the first row of Table 1, all of the Kabat VH CDRs of antibody M6).

[0161] [Table 1]

[0162] [Table 2]

[0163] [Table 3]

[0164] [Table 4]

[0165] [Table 5]

[0166] [Table 6]

[0167] [Table 7]

[0168] [Table 8]

[0169] [Table 9]

[0170] In certain embodiments, MERTK (e.g., human MERTK, or human and mouse MERTK) is The anti-MERTK antibodies or antigen-binding fragments thereof described herein that specifically bind to both MERTK and MERTK (both IL-1 and IL-2) and agonize MERTK signaling in endothelial cells are: (a) the VH CDR1 of NYGMN (SEQ ID NO: 1); and / or (b) a VH CDR2 of WINTYTGEPTYADDFKG (SEQ ID NO: 6); and / or (c) the VH CDR3 of KSTVVSRYFDV (SEQ ID NO: 11); The heavy chain variable region (VH) comprises:

[0171] In certain embodiments, the anti-MERTK antibodies or antigen-binding fragments thereof described herein that specifically bind to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonize MERTK signaling in endothelial cells are: (a) the VH CDR1 of GYTFTNY (SEQ ID NO: 2); and / or (b) a VH CDR2 of TYTG (SEQ ID NO: 7); and / or (c) a VH CDR3 of STVVSRYFD (SEQ ID NO: 12); The heavy chain variable region (VH) comprises:

[0172] In certain embodiments, the anti-MERTK antibodies or antigen-binding fragments thereof described herein that specifically bind to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonize MERTK signaling in endothelial cells are: (a) a VH CDR1 of GYTFTNYGMN (SEQ ID NO: 3); and / or (b) the VH CDR2 of WINTYTGEPT (SEQ ID NO: 8); and / or (c) the VH CDR3 of KSTVVSRYFDV (SEQ ID NO: 11); The heavy chain variable region (VH) comprises:

[0173] In certain embodiments, the anti-MERTK antibodies or antigen-binding fragments thereof described herein that specifically bind to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonize MERTK signaling in endothelial cells are: (a) the VH CDR1 of TNYGMN (SEQ ID NO: 4); and / or (b) a VH CDR2 of WMGWINTYTGEPT (SEQ ID NO: 9); and / or (c) the VH CDR3 of ARKSTVVSRYFD (SEQ ID NO: 13); The heavy chain variable region (VH) comprises:

[0174] In certain embodiments, the anti-MERTK antibodies or antigen-binding fragments thereof described herein that specifically bind to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonize MERTK signaling in endothelial cells are: (a) a VH CDR1 of GYTFTNYG (SEQ ID NO: 5); and / or (b) the VH CDR2 of INTYTGEP (SEQ ID NO: 10); and / or (c) the VH CDR3 of ARKSTVVSRYFDV (SEQ ID NO: 14); The heavy chain variable region (VH) comprises:

[0175] In certain embodiments, the anti-MERTK antibodies or antigen-binding fragments thereof described herein that specifically bind to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonize MERTK signaling in endothelial cells are: (a) a VH CDR1 of GYTFTNYGMN (SEQ ID NO: 3); and / or (b) a VH CDR2 of WINTYTGEPTYADDFKG (SEQ ID NO: 6); and / or (c) the VH CDR3 of KSTVVSRYFDV (SEQ ID NO: 11); The heavy chain variable region (VH) comprises:

[0176] In another embodiment, MERTK (e.g., human MERTK, or human and mouse MERTK) is The anti-MERTK antibodies or antigen-binding fragments thereof described herein that specifically bind to both MERTK and MERTK and agonize MERTK signaling in endothelial cells are: (a) a VL CDR1 of KASQDVGDAVT (SEQ ID NO: 15); and / or (b) the VL CDR2 of WASTRHT (SEQ ID NO: 19); and / or (c) the VL CDR3 of QQYRSYPLT (SEQ ID NO: 22); The light chain variable region (VL) comprises:

[0177] In another embodiment, the anti-MERTK antibody or antigen-binding fragment thereof described herein that specifically binds to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTK signaling in endothelial cells is: (a) a VL CDR1 of SQDVGDA (SEQ ID NO: 16); and / or (b) the VL CDR2 of WAS (SEQ ID NO: 20); and / or (c) the VL CDR3 of YRSYPL (SEQ ID NO: 23); The light chain variable region (VL) comprises:

[0178] In another embodiment, the anti-MERTK antibody or antigen-binding fragment thereof described herein that specifically binds to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTK signaling in endothelial cells is: (a) the VL CDR1 of GDAVTWC (SEQ ID NO: 17); and / or (b) the VL CDR2 of LLIYWASTRH (SEQ ID NO: 21); and / or (c) the VL CDR3 of QQYRSYPL (SEQ ID NO: 24); The light chain variable region (VL) comprises:

[0179] In another embodiment, the anti-MERTK antibody or antigen-binding fragment thereof described herein that specifically binds to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTK signaling in endothelial cells is: (a) the VL CDR1 of QDVGDA (SEQ ID NO: 18); and / or (b) the VL CDR2 of WAS (SEQ ID NO: 20); and / or (c) the VL CDR3 of QQYRSYPLT (SEQ ID NO: 22); The light chain variable region (VL) comprises:

[0180] In certain embodiments, the anti-MERTK antibodies or antigen-binding fragments thereof described herein that specifically bind to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonize MERTK signaling in endothelial cells are: (a) the VH CDR1 of DYSMH (SEQ ID NO: 25); and / or (b) a VH CDR2 of WINTDTGTPEPTYA DDFKG (SEQ ID NO: 30); and / or (c) the VH CDR3 of WFGAMDY (SEQ ID NO: 35); The heavy chain variable region (VH) comprises:

[0181] In certain embodiments, the anti-MERTK antibodies or antigen-binding fragments thereof described herein that specifically bind to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonize MERTK signaling in endothelial cells are: (a) the VH CDR1 of NYTFTDY (SEQ ID NO: 26); and / or (b) the VH CDR2 of TDTG (SEQ ID NO: 31); and / or (c) the VH CDR3 of FGAMD (SEQ ID NO: 36); The heavy chain variable region (VH) comprises:

[0182] In certain embodiments, the antibody specifically binds to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTK signaling in endothelial cells. The anti-MERTK antibodies or antigen-binding fragments thereof described herein: (a) the VH CDR1 of NYTFTDYSMH (SEQ ID NO: 27); and / or (b) the VH CDR2 of WINTDTGEPT (SEQ ID NO: 32); and / or (c) the VH CDR3 of WFGAMDY (SEQ ID NO: 35); The heavy chain variable region (VH) comprises:

[0183] In certain embodiments, the anti-MERTK antibodies or antigen-binding fragments thereof described herein that specifically bind to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonize MERTK signaling in endothelial cells are: (a) a VH CDR1 of TDYSMH (SEQ ID NO: 28); and / or (b) the VH CDR2 of WVGWINTDTGEPT (SEQ ID NO: 33); and / or (c) VH CDR3 of ARWFGAMD (SEQ ID NO: 37); The heavy chain variable region (VH) comprises:

[0184] In certain embodiments, the anti-MERTK antibodies or antigen-binding fragments thereof described herein that specifically bind to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonize MERTK signaling in endothelial cells are: (a) the VH CDR1 of NYTFTDYS (SEQ ID NO: 29); and / or (b) the VH CDR2 of INTDTGEP (SEQ ID NO: 34); and / or (c) a VH CDR3 of ARWFGAMDY (SEQ ID NO: 38); The heavy chain variable region (VH) comprises:

[0185] In certain embodiments, the anti-MERTK antibodies or antigen-binding fragments thereof described herein that specifically bind to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonize MERTK signaling in endothelial cells are: (a) the VH CDR1 of NYTFTDYSMH (SEQ ID NO: 27); and / or (b) a VH CDR2 of WINTDTGTPEPTYA DDFKG (SEQ ID NO: 30); and / or (c) the VH CDR3 of WFGAMDY (SEQ ID NO: 35); The heavy chain variable region (VH) comprises:

[0186] In certain embodiments, the anti-MERTK antibodies or antigen-binding fragments thereof described herein that specifically bind to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonize MERTK signaling in endothelial cells are: (a) the VL CDR1 of KASQDVTNVVA (SEQ ID NO: 39); and / or (b) the VL CDR2 of SASYRYT (SEQ ID NO: 43); and / or (c) the VL CDR3 of QQYYRTPRT (SEQ ID NO: 46); The light chain variable region (VL) comprises:

[0187] In certain embodiments, the anti-MERTK antibodies or antigen-binding fragments thereof described herein that specifically bind to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonize MERTK signaling in endothelial cells are: (a) the VL CDR1 of SQDVTNV (SEQ ID NO: 40); and / or (b) the VL CDR2 of SAS (SEQ ID NO: 44); and / or (c) the VL CDR3 of YYRTPR (SEQ ID NO: 47); The light chain variable region (VL) comprises:

[0188] In certain embodiments, the present invention provides a method for the treatment of endothelial cell endothelial cell endothelial cell endothelial cell infection using a method comprising the steps of: The anti-MERTK antibody or antigen-binding fragment thereof described herein: (a) the VL CDR1 of TNVVAWY (SEQ ID NO: 41); and / or (b) the VL CDR2 of LLIYSASYRY (SEQ ID NO: 45); and / or (c) the VL CDR3 of QQYYRTPR (SEQ ID NO: 48); The light chain variable region (VL) comprises:

[0189] In certain embodiments, the anti-MERTK antibodies or antigen-binding fragments thereof described herein that specifically bind to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonize MERTK signaling in endothelial cells are: (a) the VL CDR1 of QDVTNV (SEQ ID NO: 42); and / or (b) the VL CDR2 of SAS (SEQ ID NO: 44); and / or (c) the VL CDR3 of QQYYRTPRT (SEQ ID NO: 46); The light chain variable region (VL) comprises:

[0190] In certain embodiments, the anti-MERTK antibody or antigen-binding fragment thereof comprises one, two, or all three of the above VH CDRs. In certain embodiments, the antibody or antigen-binding fragment thereof comprises a VH CDR1 of Table 1 or Table 3. In some embodiments, the anti-MERTK antibody or antigen-binding fragment thereof comprises a VH CDR2 of Table 1 or Table 3. In certain embodiments, the anti-MERTK antibody or antigen-binding fragment thereof comprises a VH CDR3 of Table 1 or Table 3. In certain embodiments, the anti-MERTK antibody or antigen-binding fragment thereof comprises the VH CDR1, VH CDR2, and VH CDR3 of antibody M6 (Table 1). In certain embodiments, the anti-MERTK antibody or antigen-binding fragment thereof comprises the VH CDR1, VH CDR2, and VH CDR3 of antibody M19 (Table 3).

[0191] In certain embodiments, the anti-MERTK antibody or antigen-binding fragment thereof comprises one, two, or all three of the above VL CDRs. In certain embodiments, the anti-MERTK antibody or antigen-binding fragment thereof comprises the VL CDR1 of Table 2 or Table 4. In some embodiments, the anti-MERTK antibody or antigen-binding fragment thereof comprises the VL CDR2 of Table 2 or Table 4. In certain embodiments, the anti-MERTK antibody or antigen-binding fragment thereof comprises the VL CDR3 of Table 2 or Table 4. In certain embodiments, the anti-MERTK antibody or antigen-binding fragment thereof comprises the VL CDR1, VL CDR2, and VL CDR3 of antibody M6 (Table 2). In certain embodiments, the anti-MERTK antibody or antigen-binding fragment thereof comprises the VL CDR1, VL CDR2, and VL CDR3 of antibody M19 (Table 4).

[0192] In another embodiment, the anti-MERTK antibody or antigen-binding fragment thereof that specifically binds to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTK signaling in endothelial cells includes: (a) the VH CDR1 of NYGMN (SEQ ID NO: 1); and / or (b) a VH CDR2 of WINTYTGEPTYADDFKG (SEQ ID NO: 6); and / or (c) the VH CDR3 of KSTVVSRYFDV (SEQ ID NO: 11); and / or (d) the VL CDR1 of KASQDVGDAVT (SEQ ID NO: 15); and / or (e) the VL CDR2 of WASTRHT (SEQ ID NO: 19); and / or (f) VL CDR3 of QQYRSYPLT (sequence number 22).

[0193] In certain embodiments, the anti-MERTK antibodies or antigen-binding fragments thereof that specifically bind to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonize MERTK signaling in endothelial cells include: (a) the VH CDR1 of GYTFTNY (SEQ ID NO: 2); and / or (b) a VH CDR2 of TYTG (SEQ ID NO: 7); and / or (c) a VH CDR3 of STVVSRYFD (SEQ ID NO: 12); and / or (d) the VL CDR1 of SQDVGDA (SEQ ID NO: 16); and / or (e) the VL CDR2 of WAS (SEQ ID NO: 20); and / or (f) VL CDR3 of YRSYPL (sequence number 23).

[0194] In certain embodiments, the anti-MERTK antibodies or antigen-binding fragments thereof that specifically bind to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonize MERTK signaling in endothelial cells include: (a) a VH CDR1 of GYTFTNYGMN (SEQ ID NO: 3); and / or (b) the VH CDR2 of WINTYTGEPT (SEQ ID NO: 8); and / or (c) the VH CDR3 of KSTVVSRYFDV (SEQ ID NO: 11); and / or (d) the VL CDR1 of KASQDVGDAVT (SEQ ID NO: 15); and / or (e) the VL CDR2 of WASTRHT (SEQ ID NO: 19); and / or (f) VL CDR3 of QQYRSYPLT (sequence number 22).

[0195] In certain embodiments, the anti-MERTK antibodies or antigen-binding fragments thereof that specifically bind to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonize MERTK signaling in endothelial cells include: (a) the VH CDR1 of TNYGMN (SEQ ID NO: 4); and / or (b) a VH CDR2 of WMGWINTYTGEPT (SEQ ID NO: 9); and / or (c) the VH CDR3 of ARKSTVVSRYFD (SEQ ID NO: 13); and / or (d) the VL CDR1 of GDAVTWC (SEQ ID NO: 17); and / or (e) the VL CDR2 of LLIYWASTRH (SEQ ID NO: 21); and / or (f) VL CDR3 of QQYRSYPL (sequence number 24).

[0196] In certain embodiments, the anti-MERTK antibodies or antigen-binding fragments thereof that specifically bind to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonize MERTK signaling in endothelial cells include: (a) a VH CDR1 of GYTFTNYG (SEQ ID NO: 5); and / or (b) the VH CDR2 of INTYTGEP (SEQ ID NO: 10); and / or (c) the VH CDR3 of ARKSTVVSRYFDV (SEQ ID NO: 14); and / or (d) the VL CDR1 of QDVGDA (SEQ ID NO: 18); and / or (e) a VL CDR2 comprising WAS (SEQ ID NO: 20); and / or (f) VL CDR3 of QQYRSYPLT (sequence number 22).

[0197] In certain embodiments, the anti-MERTK antibodies or antigen-binding fragments thereof that specifically bind to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonize MERTK signaling in endothelial cells include: (a) a VH CDR1 of GYTFTNYGMN (SEQ ID NO: 3); and / or (b) a VH CDR2 of WINTYTGEPTYADDFKG (SEQ ID NO: 6); and / or (c) the VH CDR3 of KSTVVSRYFDV (SEQ ID NO: 11); and / or (d) the VL CDR1 of KASQDVGDAVT (SEQ ID NO: 15); and / or (e) the VL CDR2 of WASTRHT (SEQ ID NO: 19); and / or (f) VL CDR3 of QQYRSYPLT (sequence number 22).

[0198] In certain embodiments, the anti-MERTK antibodies or antigen-binding fragments thereof that specifically bind to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonize MERTK signaling in endothelial cells include: (a) the VH CDR1 of DYSMH (SEQ ID NO: 25); and / or (b) a VH CDR2 of WINTDTGTPEPTYA DDFKG (SEQ ID NO: 30); and / or (c) the VH CDR3 of WFGAMDY (SEQ ID NO: 35); and / or (d) the VL CDR1 of KASQDVTNVVA (SEQ ID NO: 39); and / or (e) the VL CDR2 of SASYRYT (SEQ ID NO: 43); and / or (f) VL CDR3 of QQYYRTPRT (sequence number 46).

[0199] In certain embodiments, the anti-MERTK antibodies or antigen-binding fragments thereof that specifically bind to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonize MERTK signaling in endothelial cells include: (a) the VH CDR1 of NYTFTDY (SEQ ID NO: 26); and / or (b) the VH CDR2 of TDTG (SEQ ID NO: 31); and / or (c) the VH CDR3 of FGAMD (SEQ ID NO: 36); and / or (d) the VL CDR1 of SQDVTNV (SEQ ID NO: 40); and / or (e) the VL CDR2 of SAS (SEQ ID NO: 44); and / or (f) VL CDR3 of YYRTPR (sequence number 47).

[0200] In certain embodiments, the anti-MERTK antibodies or antigen-binding fragments thereof that specifically bind to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonize MERTK signaling in endothelial cells include: (a) the VH CDR1 of NYTFTDYSMH (SEQ ID NO: 27); and / or (b) the VH CDR2 of WINTDTGEPT (SEQ ID NO: 32); and / or (c) the VH CDR3 of WFGAMDY (SEQ ID NO: 35); and / or (d) the VL CDR1 of KASQDVTNVVA (SEQ ID NO: 39); and / or (e) the VL CDR2 of SASYRYT (SEQ ID NO: 43); and / or (f) VL CDR3 of QQYYRTPRT (sequence number 46).

[0201] In certain embodiments, the anti-MERTK antibodies or antigen-binding fragments thereof that specifically bind to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonize MERTK signaling in endothelial cells include: (a) a VH CDR1 of TDYSMH (SEQ ID NO: 28); and / or (b) the VH CDR2 of WVGWINTDTGEPT (SEQ ID NO: 33); and / or (c) the VH CDR3 of ARWFGAMD (SEQ ID NO: 37); and / or (d) the VL CDR1 of TNVVAWY (SEQ ID NO: 41); and / or (e) (SEQ ID NO: 45) VL CDR2 of LLIYSASYRY; and / or (f) VL CDR3 of QQYYRTPR (sequence number 48).

[0202] In certain embodiments, the anti-MERTK antibodies or antigen-binding fragments thereof that specifically bind to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonize MERTK signaling in endothelial cells include: (a) the VH CDR1 of NYTFTDYS (SEQ ID NO: 29); and / or (b) the VH CDR2 of INTDTGEP (SEQ ID NO: 34); and / or (c) a VH CDR3 of ARWFGAMDY (SEQ ID NO: 38); and / or (d) the VL CDR1 of QDVTNV (SEQ ID NO: 42); and / or (e) the VL CDR2 of SAS (SEQ ID NO: 44); and / or (f) VL CDR3 of QQYYRTPRT (sequence number 46).

[0203] In certain embodiments, MERTK (e.g., human MERTK, or human and mouse MERTK) is Anti-MERTK antibodies or antigen-binding fragments thereof that specifically bind to MERTK (both IL-1 and IL-2) and agonize MERTK signaling in endothelial cells include: (a) the VH CDR1 of NYTFTDYSMH (SEQ ID NO: 27); and / or (b) a VH CDR2 of WINTDTGTPEPTYA DDFKG (SEQ ID NO: 30); and / or (c) the VH CDR3 of WFGAMDY (SEQ ID NO: 35); and / or (d) the VL CDR1 of KASQDVTNVVA (SEQ ID NO: 39); and / or (e) the VL CDR2 of SASYRYT (SEQ ID NO: 43); and / or (f) VL CDR3 of QQYYRTPRT (sequence number 46).

[0204] In certain embodiments, the antibody or antigen-binding fragment thereof comprises one, two, three, four, five, or all six of the above CDRs. In certain embodiments, the antibody or antigen-binding fragment thereof comprises a VH CDR1 of Table 1 or Table 3. In some embodiments, the antibody or antigen-binding fragment thereof comprises a VH CDR2 of Table 1 or Table 3. In some embodiments, the antibody or antigen-binding fragment thereof comprises a VH CDR3 of Table 1 or Table 3. In some embodiments, the antibody or antigen-binding fragment thereof comprises a VL CDR1 of Table 2 or Table 4. In some embodiments, the antibody or antigen-binding fragment thereof comprises a VL CDR2 of Table 2 or Table 4. In some embodiments, the antibody or antigen-binding fragment thereof comprises a VL CDR3 of Table 2 or Table 4. In some embodiments, the antibody or antigen-binding fragment thereof comprises the VH CDR1, VH CDR2, and VH CDR3 of antibody M6 (Table 1). In certain embodiments, the antibody or antigen-binding fragment thereof comprises the VH CDR1, VH CDR2, and VH CDR3 of antibody M19 (Table 3). In certain embodiments, the antibody or antigen-binding fragment thereof comprises the VL CDR1, VL CDR2, and VL CDR3 of antibody M6 (Table 2). In certain embodiments, the antibody or antigen-binding fragment thereof comprises the VL CDR1, VL CDR2, and VL CDR3 of antibody M19 (Table 4). In certain embodiments, the antibody or antigen-binding fragment thereof comprises the VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 of antibody M6 (Tables 1 and 2). In certain embodiments, the antibody or antigen-binding fragment thereof comprises the VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 of antibody M19. CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 (Tables 3 and 4).

[0205] In another embodiment, the anti-MERTK antibody or antigen-binding fragment thereof that specifically binds to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTK signaling in endothelial cells comprises a heavy chain variable region (VH) and a light chain variable region (VL): (i) the VH is: (a) the VH CDR1 of DYSMH (SEQ ID NO: 25); and / or (b) a VH CDR2 of WINTDTGTPEPTYA DDFKG (SEQ ID NO: 30); and / or (c) the VH CDR3 of WFGAMDY (SEQ ID NO: 35); and (ii) the VL is: (a) the VL CDR1 of KASQDVTNVVA (SEQ ID NO: 39); and / or (b) the VL CDR2 of SASYRYT (SEQ ID NO: 43); and / or (c) the VL CDR3 of QQYYRTPRT (SEQ ID NO: 46); Includes.

[0206] In another embodiment, an anti-MERTK antibody or antigen-binding fragment thereof that specifically binds to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTK signaling in endothelial cells comprises a heavy chain variable region (VH) and a light chain variable region (VL) that are identical to those described herein. VL), (i) the VH is: (a) the VH CDR1 of NYTFTDY (SEQ ID NO: 26); and / or (b) the VH CDR2 of TDTG (SEQ ID NO: 31); and / or (c) the VH CDR3 of FGAMD (SEQ ID NO: 36); and (ii) the VL is: (a) the VL CDR1 of SQDVTNV (SEQ ID NO: 40); and / or (b) the VL CDR2 of SAS (SEQ ID NO: 44); and / or (c) the VL CDR3 of YYRTPR (SEQ ID NO: 47); Includes.

[0207] In another embodiment, the anti-MERTK antibody or antigen-binding fragment thereof that specifically binds to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTK signaling in endothelial cells comprises a heavy chain variable region (VH) and a light chain variable region (VL): (i) the VH is: (a) the VH CDR1 of NYGMN (SEQ ID NO: 1); and / or (b) a VH CDR2 of WINTYTGEPTYADDFKG (SEQ ID NO: 6); and / or (c) the VH CDR3 of KSTVVSRYFDV (SEQ ID NO: 11), and (ii) the VL is: (a) a VL CDR1 of KASQDVGDAVT (SEQ ID NO: 15); and / or (b) the VL CDR2 of WASTRHT (SEQ ID NO: 19); and / or (c) the VL CDR3 of QQYRSYPLT (SEQ ID NO: 22); Includes.

[0208] In another embodiment, the anti-MERTK antibody or antigen-binding fragment thereof that specifically binds to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTK signaling in endothelial cells comprises a heavy chain variable region (VH) and a light chain variable region (VL): (i) the VH is: (a) the VH CDR1 of GYTFTNY (SEQ ID NO: 2); and / or (b) a VH CDR2 of TYTG (SEQ ID NO: 7); and / or (c) the VH CDR3 of STVVSRYFD (SEQ ID NO: 12); and (ii) the VL is: (a) a VL CDR1 of SQDVGDA (SEQ ID NO: 16); and / or (b) the VL CDR2 of WAS (SEQ ID NO: 20); and / or (c) the VL CDR3 of YRSYPL (SEQ ID NO: 23); Includes.

[0209] In another embodiment, the anti-MERTK antibody or antigen-binding fragment thereof that specifically binds to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTK signaling in endothelial cells comprises a heavy chain variable region (VH) and a light chain variable region (VL): (i) the VH is: (a) a VH CDR1 of GYTFTNYGMN (SEQ ID NO: 3); and / or (b) the VH CDR2 of WINTYTGEPT (SEQ ID NO: 8); and / or (c) the VH CDR3 of KSTVVSRYFDV (SEQ ID NO: 11), and (ii) the VL is: (a) a VL CDR1 of KASQDVGDAVT (SEQ ID NO: 15); and / or (b) the VL CDR2 of WASTRHT (SEQ ID NO: 19); and / or (c) the VL CDR3 of QQYRSYPLT (SEQ ID NO: 22); Includes.

[0210] In another embodiment, the anti-MERTK antibody or antigen-binding fragment thereof that specifically binds to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTK signaling in endothelial cells comprises a heavy chain variable region (VH) and a light chain variable region (VL): (i) the VH is: (a) the VH CDR1 of TNYGMN (SEQ ID NO: 4); and / or (b) a VH CDR2 of WMGWINTYTGEPT (SEQ ID NO: 9); and / or (c) the VH CDR3 of ARKSTVVSRYFD (SEQ ID NO: 13); and (ii) the VL is: (a) the VL CDR1 of GDAVTWC (SEQ ID NO: 17); and / or (b) the VL CDR2 of LLIYWASTRH (SEQ ID NO: 21); and / or (c) the VL CDR3 of QQYRSYPL (SEQ ID NO: 24); Includes.

[0211] In another embodiment, the anti-MERTK antibody or antigen-binding fragment thereof that specifically binds to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTK signaling in endothelial cells comprises a heavy chain variable region (VH) and a light chain variable region (VL): (i) the VH is: (a) a VH CDR1 of GYTFTNYG (SEQ ID NO: 5); and / or (b) the VH CDR2 of INTYTGEP (SEQ ID NO: 10); and / or (c) the VH CDR3 of ARKSTVVSRYFDV (SEQ ID NO: 14); and (ii) the VL is: (a) the VL CDR1 of QDVGDA (SEQ ID NO: 18); and / or (b) the VL CDR2 of WAS (SEQ ID NO: 20); and / or (c) the VL CDR3 of QQYRSYPLT (SEQ ID NO: 22); Includes.

[0212] In another embodiment, the anti-MERTK antibody or antigen-binding fragment thereof that specifically binds to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTK signaling in endothelial cells comprises a heavy chain variable region (VH) and a light chain variable region (VL): (i) the VH is: (a) a VH CDR1 of GYTFTNYGMN (SEQ ID NO: 3); and / or (b) a VH CDR2 of WINTYTGEPTYADDFKG (SEQ ID NO: 6); and / or (c) the VH CDR3 of KSTVVSRYFDV (SEQ ID NO: 11), and (ii) the VL is: (a) a VL CDR1 of KASQDVGDAVT (SEQ ID NO: 15); and / or (b) the VL CDR2 of WASTRHT (SEQ ID NO: 19); and / or (c) the VL CDR3 of QQYRSYPLT (SEQ ID NO: 22); Includes.

[0213] In another embodiment, the anti-MERTK antibody or antigen-binding fragment thereof that specifically binds to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTK signaling in endothelial cells comprises a heavy chain variable region (VH) and a light chain variable region (VL): (i) the VH is: (a) the VH CDR1 of NYTFTDYSMH (SEQ ID NO: 27); and / or (b) the VH CDR2 of WINTDTGEPT (SEQ ID NO: 32); and / or (c) the VH CDR3 of WFGAMDY (SEQ ID NO: 35); and (ii) the VL is: (a) the VL CDR1 of KASQDVTNVVA (SEQ ID NO: 39); and / or (b) the VL CDR2 of SASYRYT (SEQ ID NO: 43); and / or (c) the VL CDR3 of QQYYRTPRT (SEQ ID NO: 46); Includes.

[0214] In another embodiment, the anti-MERTK antibody or antigen-binding fragment thereof that specifically binds to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTK signaling in endothelial cells comprises a heavy chain variable region (VH) and a light chain variable region (VL): (i) the VH is: (a) a VH CDR1 of TDYSMH (SEQ ID NO: 28); and / or (b) the VH CDR2 of WVGWINTDTGEPT (SEQ ID NO: 33); and / or (c) the VH CDR3 of ARWFGAMD (SEQ ID NO: 37); and (ii) the VL is: (a) the VL CDR1 of TNVVAWY (SEQ ID NO: 41); and / or (b) the VL CDR2 of LLIYSASYRY (SEQ ID NO: 45); and / or (c) the VL CDR3 of QQYYRTPR (SEQ ID NO: 48); Includes.

[0215] In another embodiment, the anti-MERTK antibody or antigen-binding fragment thereof that specifically binds to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTK signaling in endothelial cells comprises a heavy chain variable region (VH) and a light chain variable region (VL): (i) the VH is: (a) the VH CDR1 of NYTFTDYS (SEQ ID NO: 29); and / or (b) the VH CDR2 of INTDTGEP (SEQ ID NO: 34); and / or (c) the VH CDR3 of ARWFGAMDY (SEQ ID NO: 38); and (ii) the VL is: (a) the VL CDR1 of QDVTNV (SEQ ID NO: 42); and / or (b) the VL CDR2 of SAS (SEQ ID NO: 44); and / or (c) the VL CDR3 of QQYYRTPRT (SEQ ID NO: 46); Includes.

[0216] In another embodiment, the anti-MERTK antibody or antigen-binding fragment thereof that specifically binds to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTK signaling in endothelial cells comprises a heavy chain variable region (VH) and a light chain variable region (VL): (i) the VH is: (a) the VH CDR1 of NYTFTDYSMH (SEQ ID NO: 27); and / or (b) a VH CDR2 of WINTDTGTPEPTYA DDFKG (SEQ ID NO: 30); and / or (c) the VH CDR3 of WFGAMDY (SEQ ID NO: 35); and (ii) the VL is: (a) the VL CDR1 of KASQDVTNVVA (SEQ ID NO: 39); and / or (b) the VL CDR2 of SASYRYT (SEQ ID NO: 43); and / or (c) the VL CDR3 of QQYYRTPRT (SEQ ID NO: 46); Includes.

[0217] In certain embodiments, the VH comprises two or all three of the VH CDRs above, and / or the VL comprises two or all three of the VL CDRs above. In certain embodiments, the VH comprises the VH CDR1 of one of the antibodies of Table 1 or Table 3. In some embodiments, the VH comprises the VH CDR2 of one of the antibodies of Table 1 or Table 3. In certain embodiments, the VH comprises the VH CDR3 of one of the antibodies of Table 1 or Table 3. In some embodiments, the VL comprises the VL CDR1 of one of the antibodies of Table 2 or Table 4. In some embodiments, the VL comprises the VL CDR2 of one of the antibodies of Table 2 or Table 4. In certain embodiments, the VL comprises the VL CDR3 of one of the antibodies of Table 2 or Table 4. In some embodiments, the VH comprises the VH CDR1, VH CDR2, and VH CDR3 of antibody M6 (Table 1). In certain embodiments, the VH comprises the VH CDR1, VH CDR2, and VH CDR3 of antibody M19 (Table 3). In certain embodiments, the VL comprises the VL CDR1, VL CDR2, and VL CDR3 of antibody M6 (Table 2). In certain embodiments, the VL comprises the VL CDR1, VL CDR2, and VL CDR3 of antibody M19 (Table 4). In some embodiments, the anti-MERTK antibody or antigen-binding fragment thereof comprises a VH and a VL, wherein the VH comprises the VH CDR1, VH CDR2, and VH CDR3 of antibody M19 (Table 3), and the VL comprises the VL CDR1, VL CDR2, and VL CDR3 of the VL CDRs of antibody M19 (Table 4). In some embodiments, the anti-MERTK antibody or antigen-binding fragment thereof comprises a VH and a VL, wherein the VH comprises the VH CDR1, VH CDR2, and VH CDR3 of antibody M6 (Table 1), and the VL comprises the VL CDR1, VL CDR2, and VL CDR3 of the CDRs of antibody M6 (Table 2).

[0218] In a particular embodiment, the anti-MERTK antibody or antigen-binding fragment thereof described herein that specifically binds to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTK signaling in endothelial cells comprises a heavy chain variable region sequence (e.g., VH of antibody M6) that comprises the amino acid sequence of SEQ ID NO: 49 (Table 5). In another particular embodiment, the anti-MERTK antibody or antigen-binding fragment thereof described herein that specifically binds to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTK signaling in endothelial cells comprises a heavy chain variable region sequence (Table 6) (e.g., VH of antibody M19) that comprises the amino acid sequence of SEQ ID NO: 51 (Table 6).

[0219] In another embodiment, MERTK (e.g., human MERTK, or human and mouse MERTK) is The antibody or antigen-binding fragment thereof described herein that specifically binds to MERTK (e.g., both human MERTK, or both human and mouse MERTK) and agonizes MERTK signaling in endothelial cells comprises a light chain variable region sequence (e.g., VL of antibody M6) that comprises the amino acid sequence of SEQ ID NO: 50 (Table 5). In another specific embodiment, the antibody or antigen-binding fragment thereof described herein that specifically binds to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTK signaling in endothelial cells comprises a light chain variable region sequence (e.g., VL of antibody M19) that comprises the amino acid sequence of SEQ ID NO: 52 (Table 6).

[0220] In a specific embodiment, the antibody or antigen-binding fragment thereof described herein that specifically binds to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTK signaling in endothelial cells comprises (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 49, and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 50 (e.g., VH and VL of antibody M6). In another specific embodiment, the antibody or antigen-binding fragment thereof described herein that specifically binds to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTK signaling in endothelial cells comprises (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 50, and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 52 (e.g., VH and VL of antibody M19).

[0221] In a preferred embodiment, the antibody specifically binds to MERTK (e.g., human MERTK, or both human and mouse MERTK); (A) VH CDR1 of SEQ ID NO: 1, VH CDR2 of SEQ ID NO: 6, VH CDR3 of SEQ ID NO: 11 CDR3, a VL CDR1 of SEQ ID NO:15, a VL CDR2 of SEQ ID NO:19, and a VL CDR3 of SEQ ID NO:22; (B) VH CDR1 of SEQ ID NO: 2, VH CDR2 of SEQ ID NO: 7, VH CDR3 of SEQ ID NO: 12 CDR3, a VL CDR1 of SEQ ID NO:16, a VL CDR2 of SEQ ID NO:20, and a VL CDR3 of SEQ ID NO:23; (C) VH CDR1 of SEQ ID NO: 3, VH CDR2 of SEQ ID NO: 8, VH CDR3 of SEQ ID NO: 11 CDR3, a VL CDR1 of SEQ ID NO:15, a VL CDR2 of SEQ ID NO:19, and a VL CDR3 of SEQ ID NO:22; (D) VH CDR1 of SEQ ID NO: 4, VH CDR2 of SEQ ID NO: 9, VH CDR3 of SEQ ID NO: 13 CDR3, a VL CDR1 of SEQ ID NO:17, a VL CDR2 of SEQ ID NO:21, and a VL CDR3 of SEQ ID NO:24; (E) a VH CDR1 of SEQ ID NO:5, a VH CDR2 of SEQ ID NO:10, a VH CDR3 of SEQ ID NO:14, a VL CDR1 of SEQ ID NO:18, a VL CDR2 of SEQ ID NO:20, and a VL CDR3 of SEQ ID NO:22; or (F) VH CDR1 of SEQ ID NO: 3, VH CDR2 of SEQ ID NO: 6, VH CDR3 of SEQ ID NO: 11 CDR3, VL CDR1 of SEQ ID NO: 15, VL CDR2 of SEQ ID NO: 19, and VL CDR3 of SEQ ID NO: 22; or a humanized antigen-binding fragment thereof.

[0222] In certain aspects, the antibodies described herein may be described by their VH domain alone, or their VL domain alone, or their three VH CDRs alone, or their three VL CDRs alone. See, for example, Rader C et al. (1998) PNAS 95:8910-8915, which describes humanizing a murine anti-αVβ3 antibody by identifying complementary light or heavy chains from a library of human light or heavy chains, respectively, resulting in humanized antibody variants with high affinity or higher affinity than that of the original antibody, and is incorporated herein by reference in its entirety. Also, Clackson T et al. (1999) PNAS 95:8910-8915, which describes how to generate antibodies that bind to a specific antigen by using a particular VH domain (or VL domain) to screen a library for complementary variable domains, and is incorporated herein by reference in its entirety. See also Kim SJ, et al., (1991) Nature 352:624-628, which is incorporated herein by reference in its entirety, describing a method for generating antibodies that bind to a specific antigen by using a particular VH domain to screen a library (e.g., a human VL library) for complementary VL domains, and then using the selected VL domain to guide the selection of additional complementary (e.g., human) VH domains. % See also Hong HJ, (2007) J Microbiol 45:572-.

[0223] In certain embodiments, one or more CDR positions along the VH (e.g., CDR1, CDR2, or CDR3) and / or VL (e.g., CDR1, CDR2, or CDR3) regions of an antibody described herein may differ at 1, 2, 3, 4, 5, or 6 amino acid positions, so long as immunospecific binding to MERTK (e.g., human MERTK, or both human and mouse MERTK) is maintained (e.g., substantially, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). In another embodiment, the length of one or more CDRs along the VH (e.g., CDR1, CDR2, or CDR3) and / or VL (e.g., CDR1, CDR2, or CDR3) regions of an antibody described herein may differ by one, two, three, four, five, six or more amino acids, so long as immunospecific binding to MERTK (e.g., human MERTK, or both human and mouse MERTK) is maintained (e.g., substantially, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). In another embodiment, the amino-terminus and / or carboxy-terminus of the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VL CDR3 described herein may be one, two, three, four, five or more amino acids longer or shorter than one or more CDRs described herein, so long as immunospecific binding to MERTK (e.g., human MERTK, or both human and mouse MERTK) is maintained (e.g., substantially, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%).As used herein, the terms "immunospecifically bind", "immunospecifically recognize", "specifically bind" and "specifically recognize" are similar terms in the context of antibodies, as understood by those of skill in the art, and refer to antibodies and antigen-binding fragments thereof that bind to an antigen (e.g., an epitope or immune complex) via an antigen-binding site, and do not exclude cross-reactivity of the antibody or antigen-binding fragment with other antigens. Any method known in the art can be used to determine whether immunospecific binding to MERTK (e.g., human MERTK, or both human and mouse MERTK) is maintained, for example, binding assays and conditions are described in Example 4 and Example 7 (Section 6) below.

[0224] In certain embodiments, the present specification describes an anti-MERTK antibody or an antigen-binding fragment thereof comprising a heavy chain and / or a light chain (e.g., only a heavy chain, only a light chain, or both a heavy chain and a light chain). With respect to the light chain, in certain embodiments, the light chain of the anti-MERTK antibody or antigen-binding fragment thereof described herein is a kappa light chain. In another specific embodiment, the light chain of the anti-MERTK antibody or antigen-binding fragment thereof described herein is a lambda light chain. In yet another specific embodiment, the light chain of the anti-MERTK antibody or antigen-binding fragment thereof described herein is a human kappa light chain or a human lambda light chain.

[0225] In certain embodiments, an anti-MERTK antibody or antigen-binding fragment thereof described herein that immunospecifically binds to MERTK (e.g., human MERTK, or both human and mouse MERTK) comprises a light chain, wherein the amino acid sequence of the variable region of the light chain comprises a VL CDR1, a VL CDR2, and a VL CDR3 having the amino acid sequence of the VL CDRs of antibody M6 or antibody M19. and VL CDR3 (i.e., those listed in Tables 2 and 4), and the constant region of the light chain comprises the amino acid sequence of a kappa light chain constant region. In another specific embodiment, an anti-MERTK antibody or antigen-binding fragment thereof described herein that immunospecifically binds to MERTK (e.g., human MERTK, or both human and mouse MERTK) comprises a light chain, the amino acid sequence of the variable region of the light chain comprises the amino acid sequence of SEQ ID NO:50 or SEQ ID NO:52, and the constant region of the light chain comprises the amino acid sequence of a kappa light chain constant region. As used herein, the terms "constant region" or "constant domain" are interchangeable and have their common meaning in the art. A constant region is a portion of an antibody, e.g., the carboxyl-terminal portion of the light and / or heavy chain that is not directly involved in binding the antibody to an antigen, but can exhibit various effector functions, such as interaction with Fc receptors. The constant regions of immunoglobulin molecules generally have a more conserved amino acid sequence compared to immunoglobulin variable domains. In a specific embodiment, the antibody or antigen-binding fragment thereof agonizes MERTK signaling.

[0226] In another specific embodiment, the anti-MERTK antibody or antigen-binding fragment thereof described herein that immunospecifically binds to MERTK (e.g., human MERTK, or both human and mouse MERTK) comprises a light chain, the amino acid sequence of the variable region of the light chain comprises VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequence of the VL CDRs of antibody M6 or antibody M19 (i.e., those listed in Tables 2 and 4), and the constant region of the light chain comprises the amino acid sequence of a lambda light chain constant region. In another specific embodiment, the anti-MERTK antibody or antigen-binding fragment thereof described herein that immunospecifically binds to MERTK (e.g., human MERTK, or both human and mouse MERTK) comprises a light chain, the amino acid sequence of the variable region of the light chain comprises the amino acid sequence of SEQ ID NO: 50 or SEQ ID NO: 52, and the constant region of the light chain comprises the amino acid sequence of a lambda light chain constant region. In a specific embodiment, the antibody or antigen-binding fragment thereof agonizes MERTK signaling.

[0227] In another specific embodiment, the anti-MERTK antibody or antigen-binding fragment thereof described herein that immunospecifically binds to MERTK (e.g., human MERTK, or both human and mouse MERTK) comprises a light chain, the amino acid sequence of the variable region of the light chain comprises VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequence of the VL CDR of antibody M6 or antibody M19 (i.e., those listed in Tables 2 and 4), and the constant region of the light chain comprises the amino acid sequence of the constant region of a human kappa light chain or a human lambda light chain. In a specific embodiment, the anti-MERTK antibody or antigen-binding fragment thereof described herein that immunospecifically binds to MERTK (e.g., human MERTK, or both human and mouse MERTK) comprises a light chain, the amino acid sequence of the variable region of the light chain comprises the amino acid sequence of SEQ ID NO: 50 or SEQ ID NO: 52, and the constant region of the light chain comprises the amino acid sequence of the constant region of a human kappa light chain or a human lambda light chain. In a specific embodiment, the antibody or antigen-binding fragment thereof agonizes MERTK signaling. Non-limiting examples of human constant region sequences are disclosed in the art, see, for example, Kabat EA et al., (1991).

[0228] With respect to the heavy chain, in certain embodiments, the heavy chain of the anti-MERTK antibody or antigen-binding fragment thereof described herein may be an alpha (α), delta (δ), epsilon (ε), gamma (γ), or mu (μ) heavy chain. In another specific embodiment, the heavy chain of the antibody described herein may comprise a human alpha (α), delta (δ), epsilon (ε), gamma (γ), or mu (μ) heavy chain.

[0229] In certain embodiments, an anti-MERTK antibody described herein that immunospecifically binds to MERTK (e.g., human MERTK, or both human and mouse MERTK) or The antigen-binding fragment thereof comprises a heavy chain, the amino acid sequence of the variable region of the heavy chain comprises VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequence of the VH CDRs of antibody M6 or antibody M19 (i.e., those listed in Tables 1 and 3), and the constant region of the heavy chain comprises the amino acid sequence of the constant region of a human gamma (γ) heavy chain. In another specific embodiment, the anti-MERTK antibody or antigen-binding fragment thereof described herein that immunospecifically binds to MERTK (e.g., human MERTK, or both human and mouse MERTK) comprises a heavy chain, the amino acid sequence of the variable region of the heavy chain comprises the amino acid sequence of SEQ ID NO: 49 or SEQ ID NO: 51, and the constant region of the heavy chain comprises the amino acid sequence of the constant region of a human gamma (γ) heavy chain. In a specific embodiment, the antibody or antigen-binding fragment thereof agonizes MERTK signaling.

[0230] In certain embodiments, the anti-MERTK antibodies or antigen-binding fragments described herein that immunospecifically bind to MERTK (e.g., human MERTK, or both human and mouse MERTK) comprise a heavy chain, the amino acid sequence of the variable region of the heavy chain comprises VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequence of the VH CDRs of antibody M6 or antibody M19 (i.e., those listed in Table 1 and Table 3), and the constant region of the heavy chain comprises amino acids of a human heavy chain described herein or known in the art. In certain embodiments, the anti-MERTK antibodies or antigen-binding fragments described herein that specifically bind to MERTK (e.g., human MERTK, or both human and mouse MERTK) comprise a heavy chain, the amino acid sequence of the variable region of the heavy chain comprises the amino acid sequence of SEQ ID NO: 49 or SEQ ID NO: 51, and the constant region of the heavy chain comprises amino acids of a human heavy chain described herein or known in the art. In certain embodiments, the antibodies or antigen-binding fragments agonize MERTK signaling. Non-limiting examples of human constant region sequences are disclosed in the art, see, for example, Kabat EA et al., (1991), supra.

[0231] In certain embodiments, the anti-MERTK antibodies or antigen-binding fragments described herein that immunospecifically bind to MERTK (e.g., human MERTK, or both human and mouse MERTK) comprise a heavy chain variable region (VH) and a light chain variable region (VL) comprising any of the amino acid sequences described herein, and the constant region comprises an amino acid sequence of an IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule, or the constant region of a human IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule. In another specific embodiment, an anti-MERTK antibody or antigen-binding fragment thereof described herein that immunospecifically binds to MERTK (e.g., human MERTK, or both human and mouse MERTK) comprises a VH and a VL comprising any amino acid sequence described herein, and the constant region comprises an amino acid sequence of an IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule, including any class of immunoglobulin molecule (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or any subclass. (e.g., IgG 2a and IgG 2b In certain embodiments, the constant region is The amino acid sequence of a human IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule can be any class of immunoglobulin molecule (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or any subclass (e.g., For example, IgG 2a and IgG 2b ).

[0232] In certain embodiments, one, two or more mutations (e.g., amino acid substitutions) are introduced into the Fc region of an anti-MERTK antibody or antigen-binding fragment thereof described herein to alter one or more functional properties of the antibody.

[0233] In some embodiments, the Fc region (e.g., according to the Kabat numbering system (e.g., Kabat's EU numbering system) of an anti-MERTK antibody or antigen-binding fragment thereof described herein is One, two or more mutations (e.g., amino acid substitutions) are introduced into the CH2 domain (residues 231-340 of human IgG1) and / or the CH3 domain (residues 341-447 of human IgG1) and / or the hinge region, numbered according to the ELISA kit (Fragment Numbering System). These mutations increase or decrease the affinity of the antibody or antigen-binding fragment thereof for an Fc receptor (e.g., an activating Fc receptor) on the surface of an effector cell. Mutations in the Fc region of an anti-MERTK antibody or antigen-binding fragment thereof that decrease or increase the affinity of the antibody for an Fc receptor, and techniques for introducing such mutations into an Fc receptor or fragment thereof, are known to those of skill in the art. Examples of mutations in the Fc receptor of an anti-MERTK antibody or antigen-binding fragment thereof that can change the affinity of the antibody or antigen-binding fragment thereof for the Fc receptor are described, for example, in Smith P et al. 2012) PNAS 109:6181-6186, U.S. Patent No. 6,737,056, and International Publication Nos. WO02 / 060919; WO98 / 23289; and WO97 / 34631, which are incorporated herein by reference.

[0234] In some embodiments, the antibody or antigen-binding fragment thereof comprises a glycosylated constant region. In some embodiments, the antibody or antigen-binding fragment thereof comprises an unglycosylated constant region. Antibodies with reduced fucose content have been reported to have increased affinity for Fc receptors, such as FcγRIIIa. Thus, in certain embodiments, the antibodies or antigen-binding fragments thereof described herein have low or no fucose content. Such antibodies or antigen-binding fragments thereof can be made using techniques known to those of skill in the art. For example, the antibodies or antigen-binding fragments thereof can be expressed in cells that are defective or lack the ability to fucosylate. In certain examples, a cell line with a knockout of both alleles of α1,6-fucosyltransferase can be used to produce antibodies with reduced fucose content. The Potelligent® System (Lonza) is an example of a system that can make antibodies or antigen-binding fragments thereof with reduced fucose content. Alternatively, antibodies or antigen-binding fragments with reduced or no fucose content can be produced, for example, by: (i) culturing cells under conditions that prevent or reduce fucosylation; (ii) post-translational removal of fucose (e.g., by fucosidase enzymes); (iii) post-translational addition of desired carbohydrates, for example, after recombinant expression of non-glycosylated glycoproteins; or (iv) purification of glycoproteins to select for antibodies or antigen-binding fragments thereof that are not fucosylated. For methods of producing antibodies or antigen-binding fragments thereof that are no fucose or have reduced fucose content, see, for example, Longmore GD & Schachter H (1982) Carbohydr Res 100:365-92, and Imai-Nishiya H et al. (2007) BMC Biotechnol. 7:84.

[0235] In another specific embodiment, the anti-MERTK antibody or antigen-binding fragment thereof described herein that immunospecifically binds to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTK signaling in endothelial cells comprises a heavy chain and / or a light chain, wherein (i) the heavy chain comprises a variable region comprising (a) a VH CDR1, a VH CDR2, and a VH CDR3 having the amino acid sequence of the VH CDR of antibody M6 or antibody M19 (i.e., those listed in Tables 1 and 3), and (b) a constant heavy chain domain comprising the amino acid sequence of the constant domain of a human IgG heavy chain, and / or (ii) the light chain comprises (a) a variable region comprising a VL CDR1, a VL CDR2, and a VL CDR3 having the amino acid sequence of the VL CDR of antibody M6 or antibody M19 (i.e., those listed in Tables 2 and 4), and (b) a constant light chain domain comprising the amino acid sequence of the constant domain of a human IgG.

[0236] In another specific embodiment, MERTK (e.g., human MERTK, or human and mouse MERTK) is The anti-MERTK antibodies or antigen-binding fragments thereof described herein immunospecifically bind to MERTK in both human and mouse (MERTK) and agonize MERTK signaling in endothelial cells, and comprise a heavy chain and / or a light chain, wherein (i) the heavy chain comprises (a) a variable region comprising the amino acid sequence of SEQ ID NO:49 or SEQ ID NO:51, and (b) a constant heavy chain domain comprising the amino acid sequence of the constant domain of human IgG, and / or (ii) the light chain comprises a variable region comprising the amino acid sequence of SEQ ID NO:50 or SEQ ID NO:52, and (b) a constant light chain domain comprising the amino acid sequence of the constant domain of human kappa light chain.

[0237] The determination of percent identity between two sequences (e.g., amino acid or nucleic acid sequences) can also be performed using a mathematical algorithm. A specific, non-limiting example of a mathematical algorithm used to compare two sequences is the algorithm described in Karlin S, as modified by Karlin S & Altschul SF (1990) PNAS 90:5873-5877. & Altschul SF (1990) PNAS 87:2264-2268. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul SF et al., (1990) J Mol Biol 215:403. To obtain nucleotide sequences homologous to the nucleic acid molecules described herein, BLAST nucleotide searches can be performed with the NBLAST nucleotide program parameters set, for example, score=100, wordlength=12. To obtain amino acid sequences homologous to the protein molecules described herein, BLAST protein searches can be performed with the XBLAST program parameters set, for example, to score 50, wordlength=3. To obtain gapped alignments for comparison purposes, gapped BLAST can be used as described in Altschul SF et al., (1997) Nuc Acids Res 25:3389 3402. Alternatively, PSI BLAST can be used to perform an iterative search that detects distant relationships between molecules (see above). When utilizing BLAST, Gapped BLAST, and PSI BLAST programs, the default parameters of each program (e.g., XBLAST and NBLAST) can be used (e.g., National Center for Biotechnology Information (NCBI) on the World Wide Web, Ncbi.nlm.nih.gov). Another specific, non-limiting example of a mathematical algorithm used to compare sequences is the algorithm of Myers and Miller, 1988, CABIOS 4:11 17. Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When utilizing the ALIGN program to compare amino acid sequences, a PAM120 weight residual table, gap length penalty of 12, and gap penalty of 4 can be used.

[0238] The percentage identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating the percentage identity, typically only exact matches are counted.

[0239] In certain embodiments, an anti-MERTK antibody or antigen-binding fragment thereof that immunospecifically binds to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTK signaling in endothelial cells comprises a VH having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of the VH of SEQ ID NO: 49 or SEQ ID NO: 51. In certain embodiments, an anti-MERTK antibody or antigen-binding fragment thereof that immunospecifically binds to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTK signaling in endothelial cells comprises a VH domain having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of the VH of SEQ ID NO: 49 or SEQ ID NO: 51, and the antibody or antigen-binding fragment thereof comprises a CDR (e.g., VH CDR and / or VL CDR) as set forth in Tables 1-4. The VH and / or VL CDRs may be identical to each other.

[0240] In certain embodiments, an anti-MERTK antibody or antigen-binding fragment thereof that immunospecifically binds to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTK signaling in endothelial cells comprises a VL having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of the VL of SEQ ID NO:50 or SEQ ID NO:52. In certain embodiments, an anti-MERTK antibody or antigen-binding fragment thereof that immunospecifically binds to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTK signaling in endothelial cells comprises a VL having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of the VL of SEQ ID NO: 50 or SEQ ID NO: 52, and the antibody or antigen-binding fragment comprises CDRs (e.g., VH CDRs and / or VL CDRs) identical to the CDRs (e.g., VH CDRs and / or VL CDRs) listed in Tables 1 to 4.

[0241] In certain embodiments, an anti-MERTK antibody or antigen-binding fragment thereof that immunospecifically binds to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTK signaling in endothelial cells comprises (i) a VH domain having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of the VH domain of SEQ ID NO: 49 or SEQ ID NO: 51, and (ii) a VL domain having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of the VL domain of SEQ ID NO: 50 or SEQ ID NO: 52. In certain embodiments, an anti-MERTK antibody or antigen-binding fragment thereof that immunospecifically binds to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTK signaling in endothelial cells comprises (i) a VH domain having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of the VH domain of SEQ ID NO: 49 or SEQ ID NO: 51, and (ii) a VL domain having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of the VL domain of SEQ ID NO: 50 or SEQ ID NO: 52, and the antibody or antigen-binding fragment comprises CDRs (e.g., VH CDRs and / or VL CDRs) identical to the CDRs (e.g., VH CDRs and / or VL CDRs) set forth in Tables 1 to 4.

[0242] In another aspect, disclosed herein are antibodies that bind to the same or overlapping epitopes of MERTK (e.g., human MERTK, or both human and mouse MERTK) as the antibodies described herein (e.g., antibody M6 or antibody M19). As used herein, "epitope" is a term in the art and refers to a localized region of an antigen to which an antibody can specifically bind. An epitope may be, for example, contiguous amino acids of a polypeptide (linear or contiguous epitope) or may be composed of two or more non-contiguous regions of one or more polypeptides (conformational, non-linear, discontinuous, or non-contiguous epitope). In certain embodiments, the epitope of an antibody may be determined, for example, by NMR spectroscopy, X-ray diffraction crystallography studies, ELISA assays, hydrogen / deuterium exchange coupled with mass spectrometry (e.g., MALDI mass spectrometry), array-based oligopeptide scanning assays, and / or mutagenesis mapping (e.g., site-directed mutagenesis mapping). In the case of X-ray crystallography, crystallization is carried out according to methods known in the art (e.g., Giege R et al., (1994) Acta Crystallogr D Biol Crystallogr 50(Pt 4):339-350; McPherson A(1990)). Eur J Biochem. m 189:1-23; Chayen NE (1997) Structure 5:1269-1274; McPherson A (1976) J Biol Chem 251:6300-6303). Antibody:antigen crystals can be studied using well-known X-ray diffraction techniques, including X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc.; see, e.g., Meth Enzymol (1985) volumes 114 &115, eds Wyckoff HW et al.; US Patent Application No. 2004 / 0014194), and BUSTER (Bricogne G (1993) Acta Crystallogr D Biol Crystallogr 49(Pt 1):37-60; Bricogne G (1997) Meth Enzymol 276A:361-423, ed Carter The method can be modified using computer software such as ELISA kits, ... Competitive binding may be determined in an assay in which the immunoglobulin under test inhibits specific binding of a reference antibody to a common antigen, such as MERTK.For example, direct or indirect solid-phase radioimmunoassays (RIA), direct or indirect solid-phase enzyme immunoassays (EIA), sandwich competition assays (see Stahli C et al., (1983) Methods Enzymol 9:242-253); direct solid-phase biotin-avidin EIA (see Kirkland TN et al., (1986) J Immunol 137:3614-9); direct label assays on solid phase, direct label sandwich assays on solid phase (see Harlow E & Lane D, (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Press); direct label RIA on solid phase using I-125 label (see Morel GA et al., (1988) Mol Immunol 25(1):7-15); direct solid-phase biotin-avidin EIA (Cheung RC et al., (1990) Virology Many types of competitive binding assays are known, such as: direct label RIA (Moldenhauer G et al., (1990) Scand J Immunol 32:77-82); direct label RIA (Moldenhauer G et al., (1990) Scand J Immunol 32:77-82). Typically, such assays involve the use of purified antigen (e.g., MERTK, such as human MERTK) bound to a solid surface or cells bearing either of these, an unlabeled test immunoglobulin and a labeled reference immunoglobulin. Competitive inhibition can be measured by measuring the amount of label bound to the solid surface or cells in the presence of the test immunoglobulin. Typically, the test immunoglobulin is present in excess. Typically, the competing antibody, when present in excess, inhibits specific binding of the reference antibody to the common antigen by at least 50-55%, 55-60%, 60-65%, 65-70%, 70-75% or more. Competitive binding assays can be configured in a number of different formats, using either labeled antigen or labeled antibody. In a common version of this assay, the antigen is immobilized on a 96-well plate. Radioactive or enzymatic labels are then used to measure the ability of unlabeled antibodies to inhibit the binding of the labeled antibody to the antigen.For further details, see, e.g., Wagener C et al., (1983) J Immunol 130:2308-2315; Wagener C et al. ,(1984)J Immunol Methods 68:269-274;Kuroki M et al.,(1990)Cancer Res 50:4872-4879;Kuroki M et al.,(1992)Immunol Invest 21:523-538;Kuroki M et al.,(1992)Hybridoma 11:391-407, and Antibodies: A Laboratory Manual, Ed Harlow E&Lane D editors supra, pp. 386-389.

[0243] In certain configurations, where two antibodies recognize the same or sterically overlapping epitopes in a competitive binding assay, such as a competitive ELISA assay (which can be configured in any number of different formats using labeled antigen or labeled antibody), a competitive binding assay can be used to determine whether an antibody is competitively inhibited, e.g., in a dose-dependent manner, by another antibody (e.g., an antibody that binds to substantially the same or overlapping epitope as the reference antibody). In certain embodiments, antibodies can be tested in competitive binding assays with antibodies described herein (e.g., antibody M6 or antibody M19).

[0244] In another aspect, disclosed herein are antibodies that compete (e.g., in a dose-dependent manner) with an antibody described herein (e.g., M6 or M19) for binding to MERTK (e.g., human MERTK or both human and mouse MERTK) as determined using an assay known to one of skill in the art or described herein (e.g., ELISA competition assay or surface plasmon resonance) and agonize MERTK signaling in endothelial cells. In another aspect, disclosed herein are antibodies that competitively inhibit (e.g., in a dose-dependent manner) the binding of an antibody described herein (e.g., M6 or M19) to MERTK (e.g., human MERTK or both human and mouse MERTK) as determined using an assay known to one of skill in the art or described herein (e.g., ELISA competition assay or suspension array or surface plasmon resonance).

[0245] In certain embodiments, disclosed herein are antibodies that compete with the antibodies described herein for binding to MERTK (e.g., human MERTK or both human and mouse MERTK) to the same extent that the antibodies described herein self-compete for binding to MERTK (e.g., human MERTK or both human and mouse MERTK). In certain embodiments, disclosed herein are first antibodies that compete with the antibodies described herein for binding to MERTK (e.g., human MERTK or both human and mouse MERTK), where the competition is indicated by a greater than 80% (e.g., 85%, 90%, 95%, 98%, or 80%-85%, 80%-90%, 85%-90%, or 85%-95%) reduction in binding of the first antibody to MERTK.

[0246] In a particular aspect, disclosed herein is an antibody that competes (e.g., in a dose-dependent manner) for specific binding to MERTK (e.g., human MERTK, or both human and mouse MERTK) with an antibody comprising a VH domain having the amino acid sequence of SEQ ID NO: 49 or SEQ ID NO: 51, and a VL domain having the amino acid sequence of SEQ ID NO: 50 or SEQ ID NO: 52.

[0247] In a particular embodiment, disclosed herein are antibodies that compete (e.g., in a dose-dependent manner) for specific binding to MERTK (e.g., human MERTK, or both human and mouse MERTK) with an antibody that comprises (i) a VH domain comprising VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of the CDRs of an antibody listed in Table 1 or Table 3, and (ii) a VL domain comprising VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of the VL CDRs of an antibody listed in Table 2 or Table 4. In certain embodiments, disclosed herein are antibodies that compete (e.g., in a dose-dependent manner) with an antibody comprising the VH and VL CDRs of antibody M6 (Tables 1 and 2) for specific binding to MERTK (e.g., human MERTK, or both human and mouse MERTK).

[0248] In certain embodiments, disclosed herein are antibodies that compete (e.g., in a dose-dependent manner) with an antibody comprising the VH and VL CDRs of antibody M19 (Tables 3 and 4) for specific binding to MERTK (e.g., human MERTK, or both human and mouse MERTK).

[0249] In another specific embodiment, the anti-MERTK antibody or antigen-binding fragment thereof described herein immunospecifically binds to the same or overlapping epitope as an antibody comprising (i) a VH domain comprising a VH CDR1, a VH CDR2, and a VH CDR3 having the amino acid sequences of the CDRs of an antibody listed in Table 1 or Table 3, and (ii) a VL domain comprising a VL CDR1, a VL CDR2, and a VL CDR3 having the amino acid sequences of the CDRs of an antibody listed in Table 2 or Table 4.

[0250] In certain embodiments, the anti-MERTK antibodies or antigen-binding fragments thereof described herein immunospecifically bind to the same or overlapping epitope as antibody M6 comprising a VH domain having the amino acid sequence of SEQ ID NO: 49 and a VL domain having the amino acid sequence of SEQ ID NO: 50. In another specific embodiment, the anti-MERTK antibodies or antigen-binding fragments thereof described herein immunospecifically bind to the same or overlapping epitope as antibody M6 comprising a VH domain having the amino acid sequence of SEQ ID NO: 51 and a VL domain having the amino acid sequence of SEQ ID NO: 52. Assays known to those of skill in the art or described herein (e.g., x-ray crystallography, ELISA assays, etc.) can be used to determine whether two antibodies bind to the same epitope.

[0251] The affinity is expressed as the equilibrium dissociation constant (K D ) and the equilibrium association constant (K A ) can be measured and / or expressed in a number of ways known in the art, including, but not limited to, K D can be determined by techniques known to those of skill in the art, such as biolayer interferometry. D is determined as described in Example 4 or Example 7 in Section 6, infra.

[0252] In certain embodiments, an anti-MERTK antibody or antigen-binding fragment thereof described herein, an anti-MERTK antibody or antigen-binding fragment thereof that competes with an antibody described herein for binding to MERTK, or an anti-MERTK antibody or antigen-binding fragment thereof that binds to the same or overlapping epitope as an antibody described herein, has a K of less than 7 nM, less than 6 nM, less than 5 nM, less than 4.5 nM, less than 4 nM, less than 3.5 nM, less than 3 nM, less than 2.5 nM, less than 2 nM, less than 1.5 nM, less than 1 nM, less than 0.75 nM, less than 0.5 nM, less than 0.25 nM, less than 0.1 nM, less than 0.05 nM, less than 0.025 nM, less than 0.01 nM, or less than 0.005 nM. D MERTK (e.g., human MERTK, or human and In some embodiments, the anti-MERTK antibodies or antigen-binding fragments thereof described herein, that compete with the antibodies described herein for binding to MERTK, or that bind to the same or overlapping epitope as the antibodies described herein, have a K of approximately 7 nM, 6 nM, 5 nM, 4.5 nM, 4 nM, 3.5 nM, 3 nM, 2.5 nM, 2 nM, 1.5 nM, 1 nM, 0.75 nM, 0.5 nM, 0.25 nM, 0.1 nM, 0.05 nM, 0.025 nM, 0.01 nM, or 0.005 nM. D MERTK (e.g., human MERTK, or both human and mouse MERTK) In some embodiments, the anti-MERTK antibody or its Antigen-binding fragments, anti-MERTK antibodies or antigen-binding fragments thereof that compete with the antibodies described herein for binding to MERTK, or that bind to the same or overlapping epitope as the antibodies described herein, have a K of about 3 pM to about 400 pM. D MERTK (e.g., human MERTK, or both human and mouse MERTK) In certain embodiments, the anti-MERTK antibodies or antigen-binding fragments thereof described herein bind to ERTK. D In another specific embodiment, the anti-MERTK antibody or antigen-binding fragment thereof described herein binds to MERTK (e.g., human MERTK, or both human and mouse MERTK) with a K of about 4.6 pM. D As used herein, the term "about," when used to modify a numerical value or numerical range, means that a deviation of 5% to 10% more and a deviation of 5% to 10% less of the value or range remains within the intended meaning of the stated value or range.

[0253] In certain embodiments, epitopes of the antibodies described herein are used as immunogens to produce antibodies (see, e.g., Section 5.2, infra, for methods of producing antibodies).

[0254] In certain embodiments, the anti-MERTK antibodies or antigen-binding fragments thereof described herein are engineered or modified by methods known in the art to facilitate conjugation with a drug moiety, particularly to facilitate site-specific conjugation, e.g., by methods described in Beck A et al., (2017) Nat Rev Drug Discov 16:315-337; Peters C and Brown S, (2015) Biosci Rep 35:art:e00225; McCombs JR and Owen SC (2015) The AAPS Journal 17:339-351; Jackson DY (2016) Org Proces Res Dev 20:852-866; or Olivier KJ and Hurvitz SA ed. (2016) Antibody-Drug Conjugates: Fundamentals, Drug Development, and Clinical, Wiley. Non-limiting exemplary methods that can be used to engineer or modify an anti-MERTK antibody or antigen-binding fragment thereof to facilitate conjugation with a drug moiety (see Beck A et al., (2017) Nat Rev Drug Discov 16:315-337) include the addition of one or more additional cysteines or selenocysteines, non-natural amino acid engineering, the addition of one or more amino acid tags recognizable by specific enzymes that can aid in conjugation, glycan remodeling, the addition of amino-terminal serine, and native cysteine ​​cross-linking.

[0255] 5.1.2 Functional characteristics of antibody moieties In some forms, the anti-MERTK antibodies or antigen-binding fragments thereof described herein that immunospecifically bind to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonize MERTK signaling in endothelial cells increase the activity of MERTK (e.g., human MERTK, or both human and mouse MERTK) on endothelial cells by at least about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold. The activity of MERTK can be measured by methods described herein or known to one of skill in the art, for example, by measuring the amount of phosphorylation of MERTK. In another specific embodiment, the increase in MERTK activity, as measured by increased phosphorylation of MERTK, is at least 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, or 30-fold over MERTK activity (e.g., human MERTK, or both human and mouse MERTK activity) in endothelial cells without antibody or with an irrelevant antibody (e.g., an antibody that does not immunospecifically bind to MERTK). In some embodiments, the increase in MERTK activity is at least 50%, 60%, 70%, 80%, 90%, or 100%. In certain embodiments, the increase in MERTK activity is assessed as described in Example 3 below.

[0256] In certain embodiments, the anti-MERTK antibodies or antigen-binding fragments thereof described herein that immunospecifically bind to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonize MERTK signaling in endothelial cells compete with Gas-6 (e.g., human Gas-6) for binding to MERTK. In some embodiments, the antibodies or antigen-binding fragments thereof described herein inhibit (e.g., completely inhibit or only partially inhibit) the binding of Gas-6 to MERTK (e.g., human MERTK, or both human and mouse MERTK). In some embodiments, the anti-MERTK antibodies or antigen-binding fragments thereof described herein that immunospecifically bind to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonize MERTK signaling in endothelial cells inhibit Gas-6 (e.g., human or mouse Gas-6) binding to MERTK (e.g., human MERTK, or both human and mouse MERTK) by more than 85%, 80%, 75%, 70%, 65%, 60% or more, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20% or 10% on endothelial cells as assessed by an assay known to one of skill in the art or described herein. In certain embodiments, the assay used to assess inhibition of Gas-6 (e.g., human Gas-6) binding to MERTK (e.g., human MERTK, or both human and mouse MERTK) in the presence of the anti-MERTK antibodies or antigen-binding fragments thereof described herein is an antibody capture ELISA as described in Example 1 below.

[0257] In a particular embodiment, the level of MERTK phosphorylation in endothelial cells is measured by phospho-MERTK-specific Western blotting as described in Example 3 below.

[0258] In certain embodiments, the anti-MERTK antibody or antigen-binding fragment thereof described herein that promotes (i.e., increases) the level of MERTK phosphorylation on endothelial cells does not promote (i.e., increases) MERTK phosphorylation on cancer cells (e.g., breast cancer cells) in vitro. In certain embodiments, the level of MERTK phosphorylation in cancer cells is measured by phospho-MERTK-specific Western blotting as described in Example 3 below.

[0259] In certain embodiments, the anti-MERTK antibodies or antigen-binding fragments thereof described herein that immunospecifically bind to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonize MERTK signaling in endothelial cells inhibit migration of endothelial cells expressing MERTK in vitro in the presence of cancer cells (e.g., breast cancer cells). In some specific embodiments, migration of endothelial cells is inhibited by at least 10%, 20%, 30%, 35%, 40%, 50%, or 60%, as assessed by methods described herein and / or known to those of skill in the art. In some specific embodiments, migration of endothelial cells is inhibited by at least 40%, 45%, 50%, 55%, or 60%, as assessed by methods described herein and / or known to those of skill in the art. The extent of migration can be assessed by methods described herein and / or known to those of skill in the art. Inhibition may be relative to the extent of migration of MERTK-expressing endothelial cells without the antibody or with an unrelated antibody (e.g., an antibody that does not immunospecifically bind to MERTK). In a particular embodiment, the assay used to assess endothelial cell migration is a transwell migration assay, as described in Example 2 below.

[0260] In certain embodiments, MERTK (e.g., human MERTK, or human and mouse MERTK) is The antibodies or antigen-binding fragments thereof described herein that immunospecifically bind to MERTK (both MERTK and MERTK receptor agonists) and agonize MERTK signaling in endothelial cells do not inhibit migration of the glioblastoma multiforme cell line A172 in an in vitro transwell migration assay in the absence of endothelial cells.

[0261] In certain embodiments, the anti-MERTK antibodies or antigen-binding fragments thereof described herein that immunospecifically bind to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonize MERTK signaling in endothelial cells inhibit angiogenesis in tumors. In some embodiments, the inhibition of angiogenesis is at least 10%, 20%, 30%, 40%, 50%, 55, 60%, 65%, 70%, or 75%. In certain embodiments, the inhibition of angiogenesis is at least 50%, 55%, 60%, 65%, or 70%. The inhibition of angiogenesis can be assessed by methods described herein and / or known to those of skill in the art. The inhibition may be relative to the level of angiogenesis without the antibody or with an irrelevant antibody (e.g., an antibody that does not immunospecifically bind to MERTK). In certain embodiments, the assay used to assess angiogenesis in vivo is as described in Example 6, below.

[0262] In certain embodiments, the anti-MERTK antibodies or antigen-binding fragments thereof described herein that immunospecifically bind to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonize MERTK signaling in endothelial cells inhibit tumor (e.g., human breast cancer tumor) progression. The inhibition of tumor progression is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80%. Tumor progression can be assessed by methods described herein and / or known to those of skill in the art. Tumor progression may be relative to the state of the tumor without the antibody or with an irrelevant antibody (e.g., an antibody that does not immunospecifically bind to MERTK). In certain embodiments, the assay used to assess tumor progression is a mouse tumor transplant model described in Examples 5 and 8, below.

[0263] 5.2. Antibody Production 5.2.1. Antibody Production and Screening Also described herein are methods for producing antibodies or antigen-binding fragments thereof that specifically bind to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonize MERTK signaling in endothelial cells.

[0264] The antibody or antigen-binding fragment thereof described herein can be produced by any method known in the art for the synthesis of antibodies, for example, chemical synthesis or recombinant expression technology. The methods described herein use conventional techniques in molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and related fields within the scope of the art, unless otherwise specified. These techniques are described, for example, in the references cited herein and are fully explained in the literature. See, for example, Maniatis T et al., (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; Sambrook J et al., (1989), Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press; Sambrook J et al., (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; Press,Cold Spring Harbor,NY;Ausubel FM et al.,Current Protocols in Molecular Bi Current Protocols in Immunology, John Wiley & Sons (1987 and annually updated); Gait (ed.) (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein (ed.) (1991) Oligonucleotides and Analogues: A Practical Approach, IRL Press; Birren B et al., (eds.) (1999) Genome Analysis: A Laboratory Manual, Cold Spring Harbor Laboratory Press.

[0265] In certain embodiments, the antibodies described herein are antibodies (e.g., recombinant antibodies) that are prepared, expressed, generated, or isolated by any means involving the creation of DNA sequences, for example, synthesis, via genetic engineering. In certain embodiments, such antibodies comprise sequences encoded by DNA sequences that do not naturally occur within the in vivo antibody germline repertoire of an animal or mammal (e.g., human). In certain embodiments, the antibodies described herein are produced by a method that includes using human MERTK (SEQ ID NO: 57) or its extracellular domain (SEQ ID NO: 58) as an immunogen.

[0266] In certain embodiments, described herein is a method for making an anti-MERTK antibody or antigen-binding fragment thereof that immunospecifically binds to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTK signaling in endothelial cells, comprising culturing a cell or host cell described herein. In certain embodiments, described herein is a method for making an anti-MERTK antibody or antigen-binding fragment thereof that immunospecifically binds to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTK signaling in endothelial cells, comprising expressing (e.g., recombinantly expressing) an antibody or antigen-binding fragment thereof described herein using a cell or host cell described herein (e.g., a cell or host cell comprising a polynucleotide encoding an antibody described herein). In certain embodiments, the cell is an isolated cell. In certain embodiments, an exogenous polynucleotide has been introduced into the cell. In certain embodiments, the method further comprises purifying the antibody or antigen-binding fragment thereof obtained from the cell or host cell.

[0267] Methods for producing polyclonal antibodies are known in the art (see, e.g., Chapter 11: Short Protocols in Molecular Biology, (2002) 5th ed., Ausubel FM et al., eds., John Wiley and Sons, New York).

[0268] The term "monoclonal antibody" as used herein is not limited to antibodies produced by hybridoma technology. Monoclonal antibodies can be produced using a wide variety of techniques known in the art, such as the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, monoclonal antibodies can be produced recombinantly from host cells exogenously expressing an antibody or fragment thereof described herein, such as the light chain and / or heavy chain of such an antibody. Methods for preparing clonal cell lines and the monoclonal antibodies expressed thereby are well known in the art (see, e.g., Ausubel FM et al., Chapter 11:: Short Protocols in Molecular Biology, (2002) 5th ed., supra). For example, monoclonal antibodies can be produced by the methods described in Harlow E & Lane D, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling GJ et al., in: Monoclonal Antibodies and d T-Cell Hybridomas 563 681 (Elsevier, NY, 1981); and Kohler G & Milstein C (1975) Nature 256:495. Methods for producing and screening specific antibodies using hybridoma technology are known in the art. In some embodiments, a mouse (or other animal such as a rat, monkey, donkey, pig, sheep, hamster, or dog) can be immunized with an antigen (e.g., MERTK (e.g., human MERTK, or both human and mouse MERTK)). Then, when an immune response is detected, e.g., when antibodies specific for the antigen are detected in the mouse serum, the mouse spleen is harvested and the spleen cells are isolated. The spleen cells are then fused to any suitable myeloma cells, e.g., cells from cell line SP20 available from the American Type Culture Collection (ATCC®) (Manassas, VA), by well-known techniques, to form a hybridoma. Hybridomas are selected and cloned by limiting dilution.

[0269] The hybridoma cells thus prepared are seeded and grown in a suitable medium, preferably containing one or more substances that inhibit the growth or survival of the unfused parent myeloma cells. The medium in which the hybridoma cells are growing is assayed for the production of monoclonal antibodies against MERTK (e.g., human MERTK, or both human and mouse MERTK). After hybridoma cells that produce antibodies of the desired specificity, affinity, and / or activity are identified, the clones can be subcloned, grown, and isolated from the medium by standard methods (Goding JW (Ed), Monoclonal Antibodies: Principles and Practice, supra). The binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by methods known in the art, such as immunoprecipitation or in vitro binding assays, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

[0270] In certain embodiments, described herein are monoclonal antibodies produced by a single cell (e.g., a hybridoma or host cell producing recombinant antibodies) that immunospecifically bind to MERTK (e.g., human MERTK, or both human and mouse MERTK) as measured, for example, by ELISA or other antigen-binding or competitive binding assays known in the art or as described in Example 1 provided herein. In certain embodiments, the monoclonal antibody may be a chimeric or humanized antibody. In certain embodiments, the monoclonal antibody is a monovalent antibody or a multivalent (e.g., bivalent) antibody. In certain embodiments, the monoclonal antibody is a monospecific antibody or a multispecific antibody (e.g., a bispecific antibody).

[0271] The antibodies described herein include antibody fragments that recognize MERTK (e.g., human MERTK, or both human and mouse MERTK) and can be generated by any technique known to one of skill in the art. For example, the Fab and F(ab')2 fragments described herein can be produced by proteolysis of immunoglobulin molecules using enzymes such as papain (to generate Fab fragments) or pepsin (to generate F(ab')2 fragments). The Fab fragment corresponds to one of the two identical arms of the antibody molecule and contains the complete light chain paired with the VH and CH1 domains of the heavy chain. The F(ab')2 fragment contains the entire light chain that is separated from the hinge region by a cleavage reaction. It comprises the two antigen-binding arms of an antibody molecule linked by disulfide bonds in the region.

[0272] Moreover, the antibodies or antigen-binding fragments thereof described herein can also be produced using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them. In particular, the DNA sequences encoding the VH and VL domains can be The VH and VL domains are amplified from an animal cDNA library (e.g., a human or mouse cDNA library of diseased tissue). DNA encoding the VH and VL domains are recombined with an scFv linker by PCR and cloned into a phagemid vector. The vector is electroporated into E. coli, which is infected with helper phage. The phages used in these methods are typically filamentous phages, such as fd and M13, and the VH and VL domains are usually recombinantly fused to either gene III or gene VIII of the phage. Phage expressing antigen-binding domains that bind to a particular antigen can be selected or identified with the antigen, for example, using labeled antigen or antigen bound or captured to a solid surface or bead. Examples of phage display methods that can be used to generate the antibodies described herein include those described in Brinkman U et al., (1995) J Immunol Methods 182:41-50; Ames RS et al., (1995) J Immunol Methods 184:177-186; Kettleborough CA et al., (1994) Eur J Immunol 24:952-958; Persic L et al., (1997) Gene 187:9-18; Burton DR & Barbas CF (1994) Advan Immunol 57:191-280; PCT Application No. PCT / GB91 / 001134; International Publication Nos. WO90 / 02809, WO91 / 10737, WO92 / 01047, WO92 / 18619, WO93 / 11236, WO95 / 15982, WO95 / 20401, and WO97 / 13844, and U.S. Pat. 426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727, 5,733,743 and 5,969,108.

[0273] After phage selection, as described in the above references, the antibody coding region from the phage can be isolated and used to generate whole antibodies, such as human antibodies, or other desired antigen-binding fragments, and expressed in any desired host, such as mammalian cells, insect cells, plant cells, yeast, and bacteria, as described below. Techniques for recombinantly producing antibody fragments, such as Fab, Fab', and F(ab')2 fragments, can also be performed using methods known in the art, such as those disclosed in PCT Publication No. WO92 / 22324; Mullinax RL et al., (1992) BioTechniques 12(6):864-9; Sawai H et al., (1995) Am J Reprod Immunol 34:26-34; and Better M et al., (1988) Science 240:1041-1043.

[0274] In one form, to generate whole antibodies, PCR primers containing VH or VL nucleotide sequences, restriction sites, and adjacent sequences that protect the restriction sites can be used to amplify VH or VL sequences from templates, such as scFv clones. Using cloning techniques known to those skilled in the art, PCR amplified VH domains can be cloned into vectors expressing VH constant regions, and PCR amplified VL domains can be cloned into vectors expressing VL constant regions, such as human kappa or lambda constant regions. VH and VL domains can be cloned into one vector expressing the required constant regions. Then, using techniques known to those skilled in the art, heavy chain conversion vectors and light chain conversion vectors are co-transfected into cell lines to generate stable or transient cell lines expressing full-length antibodies, such as IgG.

[0275] A chimeric antibody is a molecule in which different portions of the antibody are derived from different immunoglobulin molecules. For example, a chimeric antibody may contain the variable region of a mouse or rat monoclonal antibody fused to the constant region of a human antibody. Methods for producing chimeric antibodies are known in the art. See, for example, Morrison SL (1985) Science 229:1 202-7; Oi VT & Morrison SL (1986) BioTechniques 4:214-221; Gillies SD et al., (1989) J Immunol Methods 125:191-202; and U.S. Patent Nos. 5,807,715, 4,816,567, 4,816,397, and 6,331,415.

[0276] A humanized antibody can bind to a given antigen and comprises a framework region having substantially the amino acid sequence of a human immunoglobulin and a CDR having substantially the amino acid sequence of a non-human immunoglobulin (e.g., a mouse immunoglobulin). In certain embodiments, the humanized antibody also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. The antibody may also include the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. The humanized antibody may be selected from any class of immunoglobulins, such as IgM, IgG, IgD, IgA, and IgE, and any isotype, such as IgG1, IgG2, IgG3, and IgG4. Humanized antibodies can be produced by a variety of techniques, including CDR grafting (EP 239400, WO 91 / 09967, and U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089), veneering or resurfacing (EP 592106 and EP 519596; Padlan EA (1991) Mol Immunol 28(4 / 5):489-498; Studnicka GM et al., (1994) Prot Engineering 7(6)):805-814; and Roguska MA et al., (1997) Prot Engineering 7(6)):805-814. al., (1994) PNAS 91:969-973), chain shuffling (U.S. Pat. No. 5,565,332), and techniques disclosed in, for example, U.S. Pat. No. 6,407,213, U.S. Pat. No. 5,766,886, International Publication No. WO 93 / 17105; Tan P et al., (2002) J Immunol 169:1119-25; Caldas C et al., (2000) Protein Eng. 13(5):353-60; Morea V et al., (2000) Methods 20(3):267-79; Baca M et al., (1997) J Biol Chem 272(16):10678-84; Roguska MA et al., (1996) Protein Eng 9(10):895 904; Couto JR et al., (1995) Cancer Res. 55(23 Supp):5973s-5977s; Couto JR et al., (1995) Cancer Res 55(8):1717-22; Sandhu JS (1994) Gene 150(2):409-10 and Pedersen JT et al., (1994) J Mol Biol 235(3):959-73. See also U.S. Application Publication No. US 2005 / 0042664A1 (February 24, 2005), which is incorporated herein by reference in its entirety.

[0277] Methods for making multispecific (e.g., bispecific) antibodies have been disclosed, see, e.g., U.S. Patent Nos. 7,951,917; 7,183,076; 8,227,577; 5,837,242; 5,989,830; 5,869,620; 6,132,992 and 8,586,713.

[0278] Single domain antibodies, e.g., antibodies lacking light chains, can be produced by methods well known in the art. See Riechmann L & Muyldermans S (1999) J Immunol 231:25-38; Nuttall SD et al., (2000) Curr Pharm Biotechnol 1(3):253-263; Muyldermans S, (2001) J Biotechnol 74(4):277-302; U.S. Patent No. 6,005,079; and International Publication Nos. WO94 / 04678, WO94 / 25591 and WO01 / 44301.

[0279] Additionally, antibodies that immunospecifically bind to a MERTK antigen can be used to generate anti-idiotypic antibodies that "mimic" the antigen using techniques well known to those of skill in the art (see, e.g., Greenspan NS & Bona CA (1989) FASEB J 7(5):437-444; and Nissinoff A (1991) J Immunol 147(8):2429-2438).

[0280] In certain embodiments, the antibody described herein that binds to the same or overlapping epitope of MERTK (e.g., human MERTK, or both human and mouse MERTK) as the MERTK agonist antibody described herein is a human anti-MERTK antibody or an antigen-binding fragment thereof. In certain embodiments, the antibody described herein that competitively inhibits the binding of any of the antibodies described herein (e.g., M6 or M19) to MERTK (e.g., human MERTK, or both human and mouse MERTK) is a human anti-MERTK antibody or an antigen-binding fragment thereof. Human antibodies can be produced using any method known in the art. For example, transgenic mice that cannot express functional endogenous immunoglobulins but can express human immunoglobulin genes can be used. In particular, complexes of human heavy and light chain immunoglobulin genes can be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, human variable regions, constant regions, and diversity regions can be introduced into mouse embryonic stem cells in addition to human heavy and light chain genes. The mouse heavy and light chain immunoglobulin genes can be rendered non-functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. HHomozygous deletion of the region inhibits endogenous antibody production. The modified embryonic stem cells are cultured and microinjected into blastocysts to generate chimeric mice. The chimeric mice are then bred to generate homozygous offspring expressing human antibodies. The transgenic mice are immunized in the usual manner with a selected antigen, such as all or part of an antigen (e.g., MERTK). Monoclonal antibodies against the antigen can be obtained from the immunized transgenic mice using conventional hybridoma technology. The human immunoglobulin transgenes carried by the transgenic mice rearrange during B cell differentiation and subsequently undergo class switching and somatic mutation. Thus, using such technology, it is possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies. For an overview of this technology for producing human antibodies, see, for example, Lonberg N & Huszar D (1995) Int Rev Immunol 13:65-93. For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., International Publication Nos. WO 98 / 24893, WO 96 / 34096, and WO 96 / 33735 and U.S. Pat. Nos. 5,413,923, 5,625,126, 5,633,425, 5,569,825, 5,661,016, 5,545,806, 5,814,318, and 5,939,598. Examples of mice capable of producing human antibodies include the Xenomouse™ (Abgenix, Inc.; U.S. Patents 6,075,181 and 6,150,184), HuAb-Mouse™ (Mederex, Inc. / Gen Pharm; U.S. Patents 5,545,806 and 5,569,825), Trans Chromo Mouse™ (Kirin), and KM Mouse™ (Medarex / Kirin).

[0281] Human antibodies that specifically bind to MERTK (e.g., human MERTK, or both human and mouse MERTK) can be generated by a variety of methods known in the art, such as the phage display methods described above using antibody libraries derived from human immunoglobulin sequences. See U.S. Patent Nos. 4,444,887, 4,716,111, and 5,885,793; and International Publication Nos. WO98 / 46645, WO98 / 50433, WO98 / 24893, WO98 / 16654, WO96 / 34096, WO96 / 34097, WO96 / 34099, WO96 / 34098, WO96 / 3409 ... 3735, and WO91 / 10741.

[0282] In some embodiments, mouse-human hybridomas can be used to produce human antibodies. For example, Epstein-Barr Virus (EBV) transformed human peripheral blood lymphocytes can be fused with mouse myeloma cells to generate mouse-human hybridomas that secrete human monoclonal antibodies, and these mouse-human hybridomas can be screened to determine which secrete human monoclonal antibodies that immunospecifically bind to a target antigen (e.g., MERTK (e.g., human MERTK, or both human and mouse MERTK)). Such methods are known and disclosed in the art. See, for example, Shinmoto H et al., (2004) Cytotechnology 46:19-23; Naganawa Y et al., (2005) Human Antibodies 14:27-31.

[0283] In certain embodiments, methods for producing an antibody or antigen-binding fragment thereof that specifically binds to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTK signaling in endothelial cells are described in Example 1, below.

[0284] In a specific embodiment, methods for screening and selecting antibodies or antigen-binding fragments thereof that specifically bind to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonize MERTK signaling in endothelial cells are described in Example 1 below.

[0285] Once the anti-MERTK antibodies or antigen-binding fragments thereof described herein have been produced, they can be purified by any method known in the art for purifying immunoglobulin molecules, such as chromatography (e.g., ion exchange, affinity, particularly affinity for a particular antigen following Protein A, and size column chromatography), centrifugation, differential solubility, or other standard techniques for protein purification. Additionally, the antibodies described herein can be fused to heterologous polypeptide sequences described herein or known in the art to facilitate purification.

[0286] In certain embodiments, the anti-MERTK antibodies or antigen-binding fragments thereof described herein are isolated or purified. Generally, an isolated antibody is an antibody that is substantially free of other antibodies having antigen specificity different from the isolated antibody. For example, in certain embodiments, preparations of the antibodies described herein are substantially free of cellular material and / or chemical precursors. The term "substantially free of cellular material" includes preparations of antibodies in which the antibody is separated from cellular components of the cells from which the antibody is isolated or recombinantly produced. Thus, antibodies that are substantially free of cellular material include preparations of antibodies having less than approximately 30%, 20%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% (by dry weight) of heterologous proteins (also referred to herein as "contaminating proteins") and / or variants of the antibody, such as different forms of post-translational modifications of the antibody or other different versions of the antibody (e.g., antibody fragments). When the antibody is recombinantly produced, it is also generally substantially free of culture medium, i.e., culture medium accounts for less than approximately 20%, 10%, 2%, 1%, 0.5%, or 0.1% of the volume of the protein preparation. When the antibody is produced by chemical synthesis, it is generally substantially free of chemical precursors or other chemicals, i.e., separated from chemical precursors or other chemicals involved in the synthesis of the protein. Thus, such preparations of the antibody have less than approximately 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors or compounds other than the antibody of interest. In certain embodiments, the antibodies described herein are isolated or purified.

[0287] 5.2.2 Polynucleotides Also disclosed herein are polynucleotides comprising nucleotide sequences encoding the antibodies or antigen-binding fragments thereof (e.g., variable light and / or variable heavy chain regions) described herein that immunospecifically bind to an antigen of MERTK (e.g., human MERTK, or both human and mouse MERTK), as well as vectors, e.g., vectors comprising the polynucleotides for efficient expression of the polynucleotides in host cells (e.g., E. coli and mammalian cells). In some embodiments, the polynucleotides are isolated or purified.

[0288] As used herein, an "isolated" polynucleotide or nucleic acid molecule is one that is separated from other nucleic acid molecules present in the natural source of the nucleic acid molecule (e.g., in a mouse or human). Furthermore, an "isolated" nucleic acid molecule, such as a cDNA molecule, is substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. For example, the term "substantially free" includes preparations of polynucleotides or nucleic acid molecules that have less than about 15%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% of other materials, such as cellular material, culture medium, other nucleic acid molecules, chemical precursors, and / or other chemicals.

[0289] In a particular aspect, disclosed herein are antibodies or antigen-binding fragments thereof that immunospecifically bind to a MERTK polypeptide (e.g., human MERTK) and comprise an amino acid sequence described herein, as well as polynucleotides comprising nucleotide sequences encoding antibodies that compete (e.g., in a dose-dependent manner) with such antibodies for binding to a MERTK polypeptide or that bind to an epitope that is the same as or overlaps with the epitope of such antibodies.

[0290] In a particular embodiment, described herein is a polynucleotide comprising a nucleotide sequence encoding a light or heavy chain of an antibody described herein. The polynucleotide can comprise a nucleotide sequence encoding a heavy chain comprising a VH CDR described herein (see, e.g., Tables 1 and 3). The polynucleotide can comprise a nucleotide sequence encoding a light chain comprising a VL CDR described herein (see, e.g., Tables 2 and 4).

[0291] In certain embodiments, disclosed herein is a polynucleotide comprising a nucleotide sequence encoding a MERTK agonist antibody comprising three VH chain CDRs, including VH CDR1, VH CDR2, and VH CDR3, e.g., as set forth in Table 1 or Table 3. In certain embodiments, disclosed herein is a polynucleotide comprising a nucleotide sequence encoding a MERTK agonist antibody comprising three VL chain CDRs, including VL CDR1, VL CDR2, and VL CDR3, e.g., as set forth in Table 2 or Table 4. In certain embodiments, disclosed herein is a polynucleotide comprising a nucleotide sequence encoding a MERTK agonist antibody comprising three VH chain CDRs, including VH CDR1, VH CDR2, and VH CDR3, e.g., as set forth in Table 1 or Table 3, and three VL chain CDRs, including VL CDR1, VL CDR2, and VL CDR3, e.g., as set forth in Table 2 and Table 4. In certain embodiments, the polynucleotides described herein encode the VH CDR1, VH CDR2, and VH CDR3 of M6 (i.e., SEQ ID NOs: 1, 6, and 11; SEQ ID NOs: 2, 7, and 12; SEQ ID NOs: 3, 8, and 11; SEQ ID NOs: 4, 9, and 13; SEQ ID NOs: 5, 10, and 14; or SEQ ID NOs: 3, 6, and 11). In certain embodiments, the polynucleotides described herein encode the VL CDR1, VL CDR2, and VL CDR3 of M6 (i.e., SEQ ID NOs: 15, 19, and 22; SEQ ID NOs: 16, 20, and 23; SEQ ID NOs: 17, 21, and 24; or SEQ ID NOs: 18, 20, and 22). In certain embodiments, the polynucleotides described herein encode the VH CDR1, VH CDR2, and VH CDR3 of M6 (i.e., SEQ ID NOs:1, 6, and 11; SEQ ID NOs:2, 7, and 12; SEQ ID NOs:3, 8, and 11; SEQ ID NOs:4, 9, and 13; SEQ ID NOs:5, 10, and 14; or SEQ ID NOs:3, 6, and 11), and three M6 VL CDRs (i.e., SEQ ID NOs:15, 19, and 22; SEQ ID NOs:16, 20, and 23; SEQ ID NOs:17, 21, and 24; or SEQ ID NOs:18, 20, and 22).

[0292] In certain embodiments, the polynucleotides described herein encode the VH CDR1, VH CDR2, and VH CDR3 of M19 (i.e., SEQ ID NOs:25, 30, and 35; SEQ ID NOs:26, 31, and 36; SEQ ID NOs:27, 32, and 35; SEQ ID NOs:28, 33, and 37; SEQ ID NOs:29, 34, and 38; or SEQ ID NOs:27, 30, and 35). In certain embodiments, the polynucleotides described herein encode the VL CDR1, VL CDR2, and VL CDR3 of M19 (e.g., SEQ ID NOs:39, 43, and 46; SEQ ID NOs:40, 44, and 47; SEQ ID NOs:41, 45, and 48; or SEQ ID NOs:42, 44, and 46). In certain embodiments, the polynucleotides described herein encode the three VH CDRs of M19 (i.e., SEQ ID NOs:25, 30, and 35; SEQ ID NOs:26, 31, and 36; SEQ ID NOs:27, 32, and 35; SEQ ID NOs:28, 33, or 37; SEQ ID NOs:29, 34, and 38; or SEQ ID NOs:27, 30, and 35) and the three VL CDRs of M19 (i.e., SEQ ID NOs:39, 43, and 46; SEQ ID NOs:40, 44, and 47; SEQ ID NOs:41, 45, and 48; or SEQ ID NOs:42, 44, and 46).

[0293] In certain embodiments, the polynucleotides described herein comprise a nucleotide sequence encoding an anti-MERTK antibody described herein comprising a heavy chain variable region comprising an amino acid sequence described herein (SEQ ID NO: 49 or 51), which immunospecifically binds to MERTK (e.g., human MERTK, or both human and mouse MERTK). In certain embodiments, the polynucleotides described herein comprise a nucleotide sequence encoding an anti-MERTK antibody described herein comprising a light chain variable region comprising an amino acid sequence described herein (SEQ ID NO: 50 or 52), which immunospecifically binds to MERTK (e.g., human MERTK, or both human and mouse MERTK). In certain embodiments, the polynucleotides described herein comprise a nucleotide sequence encoding an anti-MERTK antibody described herein comprising a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 49 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 50, which immunospecifically binds to MERTK (e.g., human MERTK, or both human and mouse MERTK). In certain embodiments, the polynucleotide described herein comprises a nucleotide sequence encoding an anti-MERTK antibody described herein comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:51 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:52, which antibody immunospecifically binds to MERTK (e.g., human MERTK, or both human and mouse MERTK).

[0294] In certain embodiments, the polynucleotides described herein encode a VH comprising the nucleic acid sequence of SEQ ID NO:53 or SEQ ID NO:55. In certain embodiments, the polynucleotides described herein encode a VL comprising the nucleic acid sequence of SEQ ID NO:54 or SEQ ID NO:56. In certain embodiments, the polynucleotides described herein encode a VH comprising the nucleic acid sequence of SEQ ID NO:53, and a VL comprising the nucleic acid sequence of SEQ ID NO:54 (e.g., M6). In another specific embodiment, the polynucleotides described herein encode a VH comprising the nucleic acid sequence of SEQ ID NO:55, and a VL comprising the nucleic acid sequence of SEQ ID NO:56 (e.g., M19).

[0295] In certain aspects, disclosed herein are polynucleotides that include nucleotide sequences that encode light and heavy chains, e.g., antibodies that include separate light and heavy chains. In certain embodiments, the polynucleotides described herein comprise a nucleotide sequence encoding a kappa light chain. In another particular embodiment, the polynucleotides described herein comprise a nucleotide sequence encoding a lambda light chain. In yet another particular embodiment, the polynucleotides described herein comprise a nucleotide sequence encoding an antibody described herein comprising a human kappa light chain or a human lambda light chain. In certain embodiments, the polynucleotides described herein comprise a nucleotide sequence encoding an antibody described herein that immunospecifically binds to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTK signaling in endothelial cells, the antibody comprising a light chain, the amino acid sequence of the variable region of the light chain can comprise the amino acid sequence of either SEQ ID NO:50 or SEQ ID NO:52, and the constant region of the light chain comprises the constant region of a human kappa light chain. In another specific embodiment, the polynucleotides described herein comprise a nucleotide sequence encoding an antibody described herein that immunospecifically binds to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTK signaling in endothelial cells, and comprises a light chain, the amino acid sequence of the variable region of the light chain can comprise the amino acid sequence of either SEQ ID NO: 50 or SEQ ID NO: 52, and the constant region of the light chain comprises the amino acid sequence of the constant region of a human lambda light chain.

[0296] In certain embodiments, an optimized polynucleotide sequence encoding a MERTK agonist antibody or antigen-binding fragment thereof (e.g., VH and / or VL domains) described herein can hybridize to an antisense (e.g., complementary) polynucleotide of a non-optimized polynucleotide sequence encoding a MERTK agonist antibody or antigen-binding fragment thereof (e.g., VH and / or VL domains) described herein. In certain embodiments, an optimized nucleotide sequence encoding a MERTK agonist antibody or antigen-binding fragment thereof described herein hybridizes under high stringency conditions to an antisense polynucleotide of a non-optimized polynucleotide sequence encoding a MERTK agonist antibody or antigen-binding fragment thereof described herein. In certain embodiments, an optimized nucleotide sequence encoding a MERTK agonist antibody or antigen-binding fragment thereof described herein hybridizes under high stringency, medium or low stringency hybridization conditions to an antisense polynucleotide of a non-optimized nucleotide sequence encoding a MERTK agonist antibody or antigen-binding fragment thereof described herein. In certain embodiments, the polynucleotides described herein are codon-optimized. Information regarding hybridization conditions has been disclosed, for example, in US Patent Application Publication No. 2005 / 0048549 (eg, paragraphs 72-73), which is incorporated herein by reference.

[0297] Polynucleotides can be obtained and the nucleotide sequence of polynucleotides can be determined by any method known in the art. The nucleotide sequence encoding the antibodies and modified versions of these antibodies described herein can be determined using methods well known in the art, i.e., nucleotide codons known to code for specific amino acids are assembled to generate nucleic acids encoding antibodies. Such polynucleotides encoding antibodies can be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier G et al., (1994), BioTechniques 17:242-6), in brief, overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligating the oligonucleotides, and amplifying the ligated oligonucleotides by PCR.

[0298] Alternatively, polynucleotides encoding the antibodies described herein can be generated from nucleic acid from a suitable source (e.g., a hybridoma) using methods well known in the art (e.g., PCR and other molecular cloning methods). For example, polynucleotides encoding the 3' and 5' ends of a known sequence can be generated. PCR amplification using synthetic primers hybridizable to the 'ends can be performed using genomic DNA obtained from hybridoma cells producing the antibody of interest. Such PCR amplification methods can be used to obtain nucleic acids containing sequences encoding the light and / or heavy chains of the antibody. Such PCR amplification methods can be used to obtain nucleic acids containing sequences encoding the variable light and / or variable heavy chain regions of the antibody. The amplified nucleic acids can be cloned into vectors for expression and further cloning in host cells, for example to generate chimeric and humanized antibodies.

[0299] If a clone containing nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, nucleic acid encoding the immunoglobulin can be chemically synthesized or obtained from an appropriate source (e.g., an antibody cDNA library or a nucleic acid, preferably polyA+ RNA, or isolated cDNA library produced from tissues or cells expressing the antibody, such as hybridoma cells selected to express an antibody described herein) by PCR amplification using synthetic primers hybridizable 3' and 5' to the sequence, or by cloning using oligonucleotide probes specific for a particular gene sequence to identify, for example, a cDNA clone from a cDNA library encoding the antibody. Amplified nucleic acids generated by PCR can then be cloned into replicable cloning vectors using any method well known in the art.

[0300] DNA encoding the MERTK agonist antibody described herein can be easily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of the MERTK agonist antibody). Hybridoma cells can serve as a source of such DNA. Once isolated, the DNA is introduced into an expression vector, which is then transfected into host cells such as E. coli cells, monkey COS cells, Chinese Hamster Ovary (CHO) cells (e.g., CHO cells from CHO GS System™ (Lonza)) that do not otherwise produce immunoglobulin proteins, or myeloma cells, to allow synthesis of the MERTK agonist antibody in the recombinant host cells.

[0301] To generate whole antibodies, the VH or VL sequences of scFv clones can be amplified using PCR primers that contain the VH or VL nucleotide sequence, a restriction site, and adjacent sequences that protect the restriction site. Using cloning techniques known to those skilled in the art, the PCR amplified VH domain can be cloned into a vector expressing a heavy chain constant region, such as the human gamma 4 constant region, and the PCR amplified VL domain can be cloned into a vector expressing a light chain constant region, such as the human kappa or lambda constant region. In certain embodiments, the vector for expressing the VH or VL domain contains a promoter, a secretion signal, a cloning site for the variable domain, a constant domain, and a selection marker. The VH and VL domains can also be cloned into one vector expressing the necessary constant regions. The heavy chain conversion vector and the light chain conversion vector are co-transfected into a cell line to generate a stable or transient cell line expressing full-length antibody (e.g., IgG) using techniques known to those skilled in the art.

[0302] The DNA can also be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains for the murine sequences, or by covalently linking all or part of the coding sequence for a non-immunoglobulin polypeptide to the immunoglobulin coding sequence.

[0303] Polynucleotides encoding the antibodies described herein can be hybridized under high stringency, medium stringency or low stringency hybridization conditions. Also disclosed herein are polynucleotides that hybridize to the polynucleotides encoding VH (SEQ ID NO: 49 or 51) and / or VL (SEQ ID NO: 50 or 52) provided herein under high stringency, medium or low stringency hybridization conditions.

[0304] Hybridization conditions are disclosed in the art and known to those skilled in the art. For example, hybridization under stringent conditions may include hybridization to filter-bound DNA in 6× sodium chloride / sodium citrate (SSC) at about 45° C., followed by one or more washes in 0.2×SSC / 0.1% SDS at about 50-65° C.; hybridization under highly stringent conditions may include hybridization to filter-bound nucleic acid in 6×SSC at about 45° C., followed by one or more washes in 0.1×SSC / 0.2% SDS at about 68° C. Hybridization under other stringent hybridization conditions is known and described in the art, see, e.g., Ausubel FM et al., eds., (1989) Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York, pages 6.3.1-6.3.6 and 2.10.3.

[0305] In certain embodiments, the drug moiety is a peptide or protein fused (directly or through a linker that is a peptide or protein) to the N-terminus or C-terminus of one or more chains of the antibody moiety. For example, if the antibody moiety is an scFv, the drug moiety can be fused (directly or through a linker that is a peptide or protein) at the N-terminus or C-terminus of the scFv. In certain embodiments, if the antibody moiety is a multi-chain antibody or antigen-binding fragment thereof, the drug moiety can be fused (directly or through a linker that is a peptide or protein) to one of the chains of the antibody moiety. In the case of an antibody that is an immunoglobulin, in certain embodiments, both heavy chains can be fused (directly or through a linker that is a peptide or protein) to the drug moiety and / or both light chains can be fused (directly or through a linker that is a peptide or protein) to the linker drug moiety. In certain such embodiments, provided herein is a polynucleotide that comprises a nucleotide sequence that encodes a fusion protein composed of a drug moiety and an antibody moiety or chain thereof, and a linker (if present) between the drug moiety and the antibody moiety. Such polynucleotides can be generated and isolated using the same methods as described above for anti-MERTK antibodies.

[0306] 5.2.3 Cells and vectors Also disclosed herein is a vector (e.g., an expression vector) comprising a polynucleotide comprising a nucleotide sequence encoding a MERTK agonist antibody or antigen-binding fragment thereof for recombinant expression in a host cell, preferably a mammalian cell. Also disclosed herein is a host cell comprising such a vector for recombinantly expressing a MERTK agonist antibody or antigen-binding fragment thereof (e.g., a human antibody or a humanized antibody) described herein.

[0307] Recombinant expression of an antibody described herein (e.g., a full length antibody, an antibody heavy and / or light chain, or a single chain antibody described herein) that specifically binds to MERTK (e.g., human MERTK, or both human and mouse MERTK) involves construction of an expression vector containing a polynucleotide encoding the antibody. Once a polynucleotide encoding the antibody molecule described herein, the antibody heavy and / or light chain, or an antigen-binding fragment thereof (e.g., the heavy and / or light chain variable regions), can be obtained, a vector for the production of the antibody molecule can be generated by recombinant DNA technology using techniques well known in the art. Thus, disclosed herein are methods for preparing proteins by expressing polynucleotides comprising nucleotide sequences encoding antibodies or antibody fragments (e.g., light or heavy chains). Methods well known to those skilled in the art can be used to construct expression vectors comprising sequences encoding antibodies or antibody fragments (e.g., light or heavy chains) and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Also disclosed herein are replicable vectors comprising nucleotide sequences encoding the antibody molecules described herein, the heavy or light chains of the antibodies, the heavy or light chain variable domains of anti-MERTK antibodies or antigen-binding fragments thereof, or the CDRs of the heavy or light chains, operably linked to a promoter. Such vectors can, for example, comprise nucleotide sequences encoding the constant regions of the antibody molecules (see, for example, International Publication Nos. WO86 / 05807 and WO89 / 01036; and U.S. Patent No. 5,122,464), and the variable domains of the antibodies can be cloned into such vectors for expression of the entire heavy chain, the entire light chain, or both the entire heavy and light chains.

[0308] The expression vector can be transferred into a cell (e.g., a host cell) by conventional techniques, and the resulting cell can then be cultured by conventional techniques to produce an antibody or antigen-binding fragment thereof described herein. Thus, disclosed herein is a host cell comprising a polynucleotide encoding an antibody or antigen-binding fragment thereof described herein, or a heavy or light chain thereof, or a fragment thereof, or a single chain antibody described herein, operably linked to a promoter for expression of the sequence in the host cell. As used herein, the term "host cell" can be any type of cell, for example, a primary cell, a cell in culture, or a cell from a cell line. In certain embodiments, the term "host cell" refers to a cell transfected with a nucleic acid molecule and the progeny or potential progeny of such a cell. The progeny of such a cell may not be identical to the parent cell transfected with the nucleic acid molecule, for example, due to mutations or environmental influences that may occur in subsequent generations or upon integration of the nucleic acid molecule into the host cell genome.

[0309] A variety of host-expression vector systems can be utilized to express the antibody molecules described herein. By such host-expression system is meant not only the vehicle by which a coding sequence of interest can be produced and subsequently purified, but also cells which, when transformed or transfected with the appropriate nucleotide coding sequences, are capable of expressing the antibody molecules described herein in situ. These include, for example, bacteria (e.g., Escherichia coli and Bacillus subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Saccharomyces pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant virus (e.g., baculovirus) expression vectors containing antibody coding sequences; plant cell systems (e.g., plants infected with recombinant virus expression vectors (e.g., Cauliflower Mosaic Virus, CaMV; Tobacco Mosaic Virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing antibody coding sequences. or mammalian cell systems (e.g., COS (e.g., COS1 or COS), CHO, BHK, MDCK, HEK293, NS0, PER.C6, VERO, CRL7O3O, HsS78Bst, HeLa, and NIH3T3, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSC1, BSC40, YB / 20 and BMT10 cells) harboring a recombinant expression construct comprising a promoter derived from the genome of a mammalian cell (e.g., a metallothionein promoter) or a promoter derived from a mammalian virus (e.g., an adenovirus late promoter; a vaccinia virus 7.5K promoter). In certain embodiments, the cells expressing the antibodies or antigen-binding fragments thereof described herein are CHO cells, e.g., For example, CHO cells from CHO GS System™ (Lonza). In certain embodiments, the cells for expressing the antibodies described herein are human cells, e.g., human cell lines. In certain embodiments, the mammalian expression vector is pOptiVEC™ or pcDNA3.3. In certain embodiments, bacterial cells such as E. coli or eukaryotic cells (e.g., mammalian cells) are used for the expression of the recombinant antibody molecule, particularly for the expression of full-length recombinant antibody molecules. For example, mammalian cells such as Chinese hamster ovary (CHO) cells are effective expression systems for antibodies when combined with vectors such as the major intermediate-early gene promoter element from human cytomegalovirus (Foecking MK & Hofstetter H (1986) Gene 45:101-5; and Cockett MI et al., (1990) Biotechnology 8(7):662-7). In certain embodiments, the antibodies described herein are produced by CHO cells or NS0 cells. In certain embodiments, expression of a nucleotide sequence encoding an antibody described herein that immunospecifically binds to MERTK (e.g., human MERTK, or both human and mouse MERTK) and agonizes MERTK signaling in endothelial cells is regulated by a constitutive promoter, an inducible promoter, or tissue-specific.

[0310] In bacterial systems, a number of expression vectors may be advantageously selected depending on the use of the antibody molecule to be expressed. For example, when producing large quantities of such antibodies, for the manufacture of pharmaceutical compositions of the antibody molecule, vectors directing the expression of high levels of fusion protein products that are easily purified may be desirable. Such vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruether U & Mueller-Hill B (1983) EMBO J 2:1791-1794), in which the antibody coding sequence can be ligated separately into the vector in frame with the lacZ coding region, thereby producing a fusion protein; pIN vector (Inouye S & Inouye M (1985) Nuc Acids Res 13:3101-3109; Van Heeke G & Schuster SM (1989) J Biol Chem 24:5503-5509), and the like. For example, pGEX vectors can be used to express foreign polypeptides as fusion proteins with glutathione 5-transferase (GST). Generally, such fusion proteins are soluble and can be easily purified from lysed cells by adsorption and binding to matrix glutathione agarose beads followed by elution in the presence of free glutathione. pGEX vectors are designed to contain protease cleavage sites by thrombin or factor Xa, so that the cloned target gene product is released from the GST moiety.

[0311] In an insect system, for example, Autographa californica nuclear polyhedrosis virus (AcNPV) can be used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The antibody coding sequence can be cloned individually into non-essential regions (e.g., the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (e.g., the polyhedrin promoter).

[0312] In mammalian host cells, many viral-based expression systems are available. When adenovirus is used as the expression vector, sequences encoding the antibody of interest can be ligated to an adenovirus transcription / translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene can then be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion into non-essential regions of the viral genome (e.g., regions E1 or E3) results in recombinant viruses that are viable and capable of expressing the antibody molecule in an infected host (see, e.g., Logan J & Shenk T (1984) PNAS 81(12):3655-9). Specific initiation signals may also be required for efficient translation of inserted antibody coding sequences. These signals include the ATG initiation codon and adjacent sequences. In addition, the initiation codon must direct translation of the entire insert. To ensure, the signal must be in phase with the reading frame of the desired coding sequence. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression can be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see, e.g., Bitter G et al., Trans. 2002, 143:131-132). al., (1987) Methods Enzymol. 153:516-544).

[0313] In addition, a host cell line can be selected that regulates the expression of the inserted sequence or modifies and processes the gene product in a desired specific manner. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products can be important for the function of the protein. Different host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be selected to ensure the correct modification and processing of the expressed foreign protein. For this purpose, eukaryotic host cells that possess the cellular machinery for proper processing, glycosylation, and phosphorylation of the primary transcript of the gene product can be used. Such mammalian host cells include, but are not limited to, CHO, VERO, BHK, Hela, MDCK, HEK293, NIH3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NS0 (a mouse myeloma cell line that does not endogenously produce immunoglobulin chains), CRL7O3O, COS (e.g., COS1 or COS), PER.C6, VERO, HsS78Bst, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSC1, BSC40, YB / 20, BMT10 and HsS78Bst cells. In certain embodiments, the MERTK agonist antibodies described herein are produced in mammalian cells, such as CHO cells.

[0314] In certain embodiments, the antibodies or antigen-binding fragments thereof described herein have low fucose content or do not contain fucose. Such antibodies can be produced using techniques known to those skilled in the art. For example, the antibodies can be expressed in cells that are defective or lack the ability to fucosylate. In certain examples, a cell line with knockout of both alleles of α1,6-fucosyltransferase can be used to produce antibodies or antigen-binding fragments thereof with reduced fucose content. The Potelligent® System (Lonza) is an example of a system that can be used to generate antibodies or antigen-binding fragments thereof with reduced fucose content.

[0315] For long-term, high-yield production of recombinant protein, stable expression cells can be produced.For example, cell lines can be produced that stably express the MERTK agonist antibody or antigen-binding fragment thereof described herein.In certain embodiments, the cells described herein stably express the light chain / light chain variable domain and the heavy chain / heavy chain variable domain that assemble to form the antibody or antigen-binding fragment thereof described herein.

[0316] In certain forms, rather than using expression vectors containing viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. After introduction of the foreign DNA / polynucleotide, the engineered cells are grown in enriched medium for 1-2 days and then switched to selective medium. The selectable marker on the recombinant plasmid confers resistance to selection, allowing the cells to stably integrate the plasmid into their chromosomes and grow to form foci that can then be cloned and propagated in cell lines. This method can be advantageously used to generate cell lines expressing the MERTK agonist antibodies or antigen-binding fragments thereof described herein. Such engineered cell lines can be used to screen for compositions that directly or indirectly interact with the antibody molecule. This may be particularly useful for testing and evaluation.

[0317] These include, but are not limited to, herpes simplex virus thymidine kinase (Wigler M A number of selection systems, such as the genes for tk cells, hgprt cells, or aprt cells, respectively, can be used, such as the genes for tk, hgprt, or aprt cells, respectively, and the genes for tk, hgprt, or aprt cells, respectively, and the genes for tk, hgprt, or aprt cells, respectively. Antimetabolite resistance can also be used as the basis for selection of the following genes: dhfr, which confers resistance to methotrexate (Wigler M et al., (1980) PNAS 77(6):3567-70; O'Hare K et al., (1981) PNAS 78:1527-31); gpt, which confers resistance to mycophenolic acid (Mulligan RC & Berg P (1981) PNAS 78(4):2072-6); neo, which confers resistance to the aminoglycoside G-418 (Wu GY & Wu CH (1991) Biotherapy 3:87-95; Tolstoshev P (1993) Ann Rev Pharmacol Toxicol 32:573-596; Mulligan RC (1993) Science 260:926-932; and Morgan RA & Anderson WF (1993) Ann Rev Biochem 62:191-217; Nabel GJ & Felgner PL (1993) Trends Biotechnol 11(5):211-5); and hygro, which confers resistance to hygromycin (Santerre RF et al., (1984) Gene 30(1-3):147-56). Methods commonly known in the field of recombinant DNA technology can be routinely applied to select the desired recombinant clone, and such methods are disclosed, for example, in Ausubel FM et al., (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, New York (1993); Kriegler M, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); Dracopoli NC et al. (eds.), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994), Chapters 12 and 13; Colbere-Garapin F et al., (1981) J Mol Biol 150:1-14, the entireties of which are incorporated herein by reference.

[0318] The expression level of an antibody molecule can be increased by vector amplification (for review, see Bebbington CR & Hentschel CCG, Use of Gene Amplification-Based Vectors for Expression of Cloned Genes in Mammalian Cells in DNA Cloning, Vol. 3 (Academic Press, New York, 1987)). If the vector-based marker expressing the antibody is amplifiable, increasing the level of inhibitor present in the host cell culture will increase the copy number of the marker gene. Since the amplified region is associated with the antibody gene, production of the antibody will also increase (Crouse GF et al., (1983) Mol Cell Biol 3:257-66).

[0319] A host cell can be co-transfected with two or more expression vectors as described herein, a first vector encoding a heavy chain derived polypeptide and a second vector encoding a light chain derived polypeptide. The two vectors can contain identical selection markers that allow for equal expression of heavy and light chain polypeptides. A host cell can be co-transfected with different amounts of two or more expression vectors. For example, a host cell can be transfected with a first expression vector and a second expression vector in any of the following ratios: 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29, 1:30, 1:31, 1:32, 1:33, 1:34, 1:35, 1:36, 1:37, 1:38, 1:39, 1:40, 1:41, 1:42, 1:43, 1:44, 1:45, 1:46, 1:47, 1:48, 1:49, 1:50, 1:51, 1:52, 1:53, 1:54, 1:55, 1:56, 1:57, 1:58, 1:59, 1:60, 1:61, 1:62, 1:63, 1:64, 1:65, 1:66, 1:67, 1: :8, 1:9, 1:10, 1:12, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, or 1:50.

[0320] Alternatively, a single vector can be used that encodes and is capable of expressing both heavy and light chain polypeptides. In such an expression vector, transcription of both genes is driven by a common promoter, while translation of mRNA from the first gene can be by a cap-dependent scanning mechanism and translation of mRNA from the second gene can be by a cap-independent mechanism, e.g., an IRES.

[0321] In certain embodiments, the drug moiety is a peptide or protein fused (directly or through a linker that is a peptide or protein) to the N-terminus or C-terminus of one or more chains of the antibody moiety. For example, if the antibody moiety is an scFv, the drug moiety can be fused (directly or through a linker that is a peptide or protein) at the N-terminus or C-terminus of the scFv. In certain embodiments, if the antibody moiety is a multi-chain antibody or antigen-binding fragment thereof, the drug moiety can be fused (directly or through a linker that is a peptide or protein) to one of the chains of the antibody moiety. In the case of an antibody that is an immunoglobulin, in certain embodiments, both heavy chains can be fused (directly or through a linker that is a peptide or protein) to the drug moiety and / or both light chains can be fused (directly or through a linker that is a peptide or protein) to the drug moiety. In certain such embodiments, provided herein is a vector (e.g., an expression vector) comprising a polynucleotide comprising a nucleotide sequence encoding a fusion protein composed of a drug moiety and an antibody moiety or chain thereof, and a linker (if present) between the drug moiety and the antibody moiety, for recombinant expression in a host cell, preferably a mammalian cell. Also provided herein is an ex vivo host cell comprising such a vector for recombinantly expressing a fusion protein comprised of a drug moiety and an antibody moiety or chain thereof, and a linker (if any) between the drug moiety and the antibody moiety. The characteristics and methods of making such vectors and ex vivo host cells may be the same as those described above for anti-MERTK antibodies. Also provided herein is a method of producing a fusion protein comprised of a drug moiety and an antibody moiety or chain thereof, and a linker (if any) between the drug moiety and the antibody moiety, comprising culturing such an ex vivo host cell under conditions such that a polynucleotide comprising a nucleotide sequence encoding the fusion protein is expressed by the ex vivo host cell to produce the fusion protein.

[0322] 5.3. Functional properties of antibody-drug conjugates In certain embodiments, the antibody drug conjugates provided herein cause cell death of MERTK-expressing endothelial cells in vitro. In some specific embodiments, at least 10%, 20%, 30%, 35%, 40%, 50%, or 60% of MERTK-expressing endothelial cells cultured in the presence of an antibody drug conjugate described herein for a period of time are dead at the end of the period, as assessed by methods known to those of skill in the art for measuring cell death in vitro. In other specific embodiments, the percentage of MERTK-expressing endothelial cells undergoing cell death is at least 50%, 100%, 2-fold, 3-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, or 1000-fold higher when cultured in the presence of an antibody drug conjugate described herein for a period of time compared to when cultured without the antibody drug conjugate (e.g., when cultured without any antibody drug conjugate, or when cultured with an antibody drug conjugate that includes an irrelevant antibody (e.g., an antibody that does not immunospecifically bind to MERTK), or when cultured with an unconjugated anti-MERTK antibody or unconjugated anti-MERTK antigen-binding fragment described herein). The period of time may be, for example, about 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 16 hours, 1 day, or the like. , 2 days, 3 days, 4 days, 5 days, 6 days, or 1 week.

[0323] In certain embodiments, the antibody drug conjugates described herein inhibit angiogenesis in tumors. In some embodiments, the inhibition of angiogenesis is at least 10%, 20%, 30%, 40%, 50%, 55, 60%, 65%, 70%, or 75%. In certain embodiments, the inhibition of angiogenesis is at least 50%, 55%, 60%, 65%, or 70%. The inhibition of angiogenesis can be assessed by methods described herein and / or known to those of skill in the art. The inhibition can be relative to the level of angiogenesis without the antibody drug conjugate (e.g., when cultured without any antibody drug conjugate, or when cultured with an antibody drug conjugate that includes an irrelevant antibody (e.g., an antibody that does not immunospecifically bind to MERTK), or when cultured with an unconjugated anti-MERTK antibody or unconjugated anti-MERTK antigen-binding fragment described herein). In certain embodiments, the assay used to assess angiogenesis in vivo is as described in Example 6, infra.

[0324] In certain embodiments, the antibody drug conjugates described herein inhibit tumor (e.g., human breast cancer tumor) progression. Inhibition of tumor progression is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80%. Tumor progression can be assessed by methods described herein and / or known to those of skill in the art. Tumor progression can be relative to the state of the tumor without the antibody-drug conjugate (e.g., when cultured without any antibody drug conjugate, or when cultured with an antibody drug conjugate that includes an irrelevant antibody (e.g., an antibody that does not immunospecifically bind to MERTK), or when cultured with an unconjugated anti-MERTK antibody or unconjugated anti-MERTK antigen-binding fragment described herein). In certain embodiments, the assay used to assess tumor progression is a mouse tumor transplant model described in Examples 5 and 8, below.

[0325] 5.4 Drug Moieties and Linkers The cytotoxic agents used in antibody-drug conjugates are often referred to as payloads or warheads. Most of the cytotoxic agents used in antibody-drug conjugates target DNA or microtubules and have high cytotoxic potency (approximately 10 -10 ~10 -12 M IC 50 Range) (Beck A et al., (2017), Nat Rev Drug Discov 16:315-337). The cytotoxic agent can be a small molecule, a nucleotide, a peptide, or a non-antibody protein. In certain embodiments, the cytotoxic agent is a small molecule. In another specific embodiment, the cytotoxic agent is a non-antibody protein. Non-limiting exemplary cytotoxic agents that can be used in the antibody drug conjugates according to the invention are described in Beck A et al., (2017) Nat Rev Drug Discov 16:315-337; Peters C and Brown S, (2015) Biosci Rep 35:art:e00225; McCombs JR and Owen SC (2015) The AAPS Journal 17:339-351; Jackson DY (2016) Org Process Res Dev 20:852-866; Olivier KJ and Hurvitz SA ed., (2016) Antibody-Drug Conjugates: Fundamentals, Drug In certain embodiments, the cytotoxic agent used in the antibody drug conjugate is an auristatin (such as monomethylauristatin E (MMAE), monomethylauristatin F (MMAF), Aur0101, PF06380101, auristatin W, or auristatin F) or a derivative thereof, a maytansinoid (such as DM1 or DM4), a pyrrolobenzodiazepine (PBD) (such as SGD1882 or SG3199), an indolinobenzodiazepine (such as DGN462 or DGN549), a calicheamicin (Ozogamicin), or a cytotoxic agent (such as cytotoxic agent). sine (such as CM1), camptothecin analogs (such as SN38, DX-8951f, or DX-8951f derivatives), duocarmycins (such as seco-duocarmycin-hydroxy-benzamide-azaindole (seco-DUBA), minor groove binding alkylating agents (MGBA), or MED-2460), tubulin inhibitors (such as cryptophycins), tubulysin or tubulysin analogs (such as AZ13599185), amberstatin 269, doxorubicin, antibiotics (such as rifalog), anthracyclines (such as PNU-159682), microtubule inhibitors (such as rhizoxin), spliceostatin, or tylanstatin. In certain embodiments, the cytotoxic agent used in the antibody drug conjugate is MMAE or MMAF. In another particular embodiment, the cytotoxic agent used in the antibody drug conjugate is DM1 or DM4.

[0326] The term "small molecule" as used herein refers to an organic or inorganic compound (which may be, for example, a heteroorganic or organometallic compound) having a molecular weight of less than about 10,000 grams per mole. In certain embodiments, a small molecule has a molecular weight of less than about 5,000 grams per mole, or less than about 2,000 grams per mole, or less than about 1,000 grams per mole, or less than about 500 grams per mole, or less than about 100 grams per mole.

[0327] In some embodiments, the cytotoxic agent is directly attached to the antibody moiety. In other embodiments, the cytotoxic agent is attached to the antibody moiety via a linker. Suitable linkers for use in the present invention are stable in the bloodstream to limit off-target toxicity, and unstable at the site of the cancer to allow release of the cytotoxic agent (Peters C and See Brown S, (2015) Biosci Rep 35:art:e00225. Non-limiting exemplary linkers that can be used in the present invention are disclosed in Beck A et al., (2017) Nat Rev Drug Discov 16:315-337; Peters C and Brown S, (2015) Biosci Rep 35:art:e00225; McCombs JR and Owen SC (2015) The AAPS Journal 17:339-351; Jackson DY (2016) Org Process Res Dev 20:852-866; Olivier KJ and Hurvitz SA ed., (2016) Antibody-Drug Conjugates: Fundamentals, Drug Development, and Clinical, Wiley. In some embodiments, the linker is a cleavable linker, for example, having a motif sensitive to lysosomal proteases (e.g., cathepsin B), a motif sensitive to acidic pH (e.g., hydrazone), or a motif containing a disulfide bridge that can be reduced by glutathione. In other embodiments, the linker is a non-cleavable linker. By way of example and not limitation, the linker can be a small molecule, a nucleotide, a peptide, or a non-antibody protein. In certain embodiments, the linker is a small molecule. In certain embodiments, the linker used in the antibody drug conjugate is cathepsin B, hydrazone, succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), maleimidocaproic acid (mc), valine-citrulline (vc), N-hydroxysuccinimidyl 4-(2-pyridyldithio)-2-sulfobutanoate (sulfoSPDB), N-hydroxysuccinimidyl 4-(2-pyridyldithio)butanoate (SPDB), N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP), valine-alanine (va), polyethylene glycol 8-valine-citrulline (PEG8-va), mb-vc, CL2A, a cleavable vc-based linker, or a fleximer polymer linker.

[0328] Non-limiting exemplary linker-drug moiety pairs that can be used in the present invention are described in Beck A et al., (2017) Nat Rev Drug Disc. v 16:315-337; Peters C and Brown S,(2015)Biosci Rep 35:art:e00225; McCombs JR and Owen SC(2015)The AAPS Journal 17:339-351; Jackson DY(2016)Org Process Res Dev 20:852-866; Olivier KJ and Hurvitz SA ed.,(2016)Antibody-Drug Conjugates:Fundamentals, Drug Development, and Clinical,Wiley. In certain embodiments, the linker-drug moiety pair of the antibody drug conjugate is vc-MMAE, mc-MMAF, SMCC-DM1, sulfo-SPDB-DM4, SPDB-DM4, SPP-DM1, va-SGD1882, polyethylene glycol 8 (PEG8)-va-SG3199, sulfo-SPDB-DGN462, hydrazone-CM1, vc-seco-DUBA, mb-vc-MGBA, CL2A-SN38, peptide linker with DX-8951 derivative, hydrazone-doxorubicin, cleavable vc-based linker-Aur0101, vc-PF06380101, fleximer polymer linker with auristatin F, cleavable linker-tubulin inhibitor, or vc-refalogue.

[0329] 5.5. Preparation of Antibody Drug Conjugates In another aspect, provided herein is a method for producing an antibody drug conjugate as described herein, wherein a linker is absent, comprising: (a) directly conjugating the cytotoxic agent to the antibody moiety to produce an antibody drug conjugate; and (b) purifying the antibody drug conjugate.

[0330] In another aspect, provided herein is a method for producing an antibody drug conjugate as described herein that includes a linker, the method comprising, in order: (a) directly conjugating the linker to the antibody moiety to produce a linker antibody moiety; (b) directly conjugating the linker of the linker antibody moiety to the cytotoxic agent to produce an antibody drug conjugate; and (c) purifying the antibody drug conjugate.

[0331] In another aspect, provided herein is a method for producing an antibody drug conjugate described herein that includes a linker, the method comprising, in order: (a) directly conjugating the linker to the cytotoxic agent to produce a linker-cytotoxic agent moiety; (b) directly conjugating the linker of the linker-cytotoxic agent moiety to the antibody moiety to produce an antibody drug conjugate; and (c) purifying the antibody drug conjugate.

[0332] The conjugation and purification steps were performed as described in Beck A et al.,(2017) Nat Rev Drug Discov 16:315-337;Peters C and Brown S,(2015) Biosci Rep 35:art:e00225;McCombs JR and Owen SC(2015) The AAPS Journal 17:339-351;Jackson DY(2016) Org Process Res Dev 20:852-866; Olivier KJ and Hurvitz SA ed.,(2016)Antibody-Drug Conjugates: Fundamentals, Drug Development, and This can be performed by methods known in the art used for making antibody drug conjugates, such as those described in Clinical, Wiley. In some embodiments, the drug moiety is attached to one chain of the antibody moiety (e.g., an immunoglobulin (which is a tetramer) or antigen-binding fragment thereof, if the antibody moiety is an scFv, or an immunoglobulin (which is a tetramer) or antigen-binding fragment thereof, if the antibody moiety is a multi-chain antibody). In other embodiments, the drug moiety is attached to more than one chain of the antibody moiety (e.g., an immunoglobulin or antigen-binding fragment thereof, if the antibody moiety is a multi-chain antibody). In certain embodiments, the drug moiety is attached to two or more chains of the immunoglobulin. The drug moieties are attached to the same chain, e.g., a heavy or light chain. In other embodiments, the drug moieties are attached to all chains of the antibody moiety (e.g., an immunoglobulin or antigen-binding fragment thereof, when the antibody moiety is a multi-chain antibody). In certain embodiments, the antibody drug conjugates described herein are purified by chromatography. In another particular embodiment, the antibody drug conjugates described herein are purified by centrifugation. By way of example and not limitation, conjugation chemistries that can be used to generate antibody drug conjugates include thiols and maleimides, thiols and autohydrolyzed maleimides, thiols and phenyloxadiazole sulfones, oxime ligation, reaction of alkoxyamines to keto groups, strain-promoted azide-alkyne cycloaddition (SPAAC), copper-free click chemistry, cysteine ​​oxidized to formylglycine, hydrazino-iso-Picteh-Spengler (HIPS) ligation, ligation of γ-carboxyamide groups from glutamine residues to primary amines, ligation of LPETG and poly(amino)-1,1-diamino ... These include primary amines of the glycine motif, maleimides and 6-thiofucose, fucose-specific conjugation of hydrazides, periodate oxidation (aldehyde) and aminooxy payloads, strain-promoted alkyne azide cycloaddition, C2-keto-gal oximation, site-selective aldehyde oxidation and oxime ligation, oxime ligation NH2 and indole-based 5-difluoro-2,4-dinitrobenzene derivatives, thiols and bissulfones, thiols and dibromomaleimides, thiols and maleimides followed by pH 9.2 treatment (45 °C, 48 h), or thiols and arylpropionitriles (see Beck A et al., (2017) Nat Rev Drug Discov 16:315-337).

[0333] The anti-MERTK antibodies or antigen-binding fragments thereof described herein can be generated as described in section 5.2. In certain embodiments, the anti-MERTK antibodies or antigen-binding fragments thereof described herein are generated or modified by methods known in the art, for example, by methods described in Beck A et al., (2017) Nat Rev Drug Discov 16:315-337; Peters C and Brown S, (2015) Biosci Rep 35:art:e00225; McCombs JR and Owen SC (2015) The AAPS Journal 17:339-351; Jackson DY (2016) Org Process Res Dev 20:852-866; or Olivier KJ and Hurvitz SA ed., (2016) Antibody-Drug Conjugates: Fundamentals, Drug Development, and Clinical, Wiley, to facilitate conjugation with a drug moiety, particularly to facilitate site-specific conjugation with a drug moiety. Non-limiting exemplary methods that can be used to generate or modify an anti-MERTK antibody or antigen-binding fragment thereof described herein to facilitate conjugation with a drug moiety include the addition of one or more additional cysteines or selenocysteines, non-natural amino acid engineering, the addition of one or more amino acid tags recognizable by specific enzymes that can aid in conjugation, glycan remodeling, the addition of amino-terminal serine, and native cysteine ​​bridges.

[0334] The linker and cytotoxic agent can be generated by any method known in the art for generating linkers and cytotoxic agents, respectively.

[0335] Pharmaceutical Compositions Provided herein is a pharmaceutical composition comprising the antibody drug conjugate described herein and a pharma- ceutically acceptable carrier.In certain embodiments, the antibody drug conjugate is purified.In certain embodiments, the antibody drug conjugate is present in the pharmaceutical composition in a therapeutically effective amount.

[0336] In certain embodiments, the population of antibody drug conjugates included in the pharmaceutical composition is 1 to 2 In another particular embodiment, the population of antibody drug conjugates in the pharmaceutical composition has a DAR of 1-15. In another particular embodiment, the population of antibody drug conjugates in the pharmaceutical composition has a DAR of 1-12. In another particular embodiment, the population of antibody drug conjugates in the pharmaceutical composition has a DAR of 1-8. In another particular embodiment, the population of antibody drug conjugates in the pharmaceutical composition has a DAR of 3-5. In another particular embodiment, the population of antibody drug conjugates in the pharmaceutical composition has a DAR of 3.5-4.

[0337] Acceptable carriers, which may be excipients or stabilizers, are nontoxic to recipients at the dosages and concentrations employed and include buffers such as phosphate, citric acid, and other organic acids; antioxidants such as ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl alcohol or benzyl alcohol, alkyl parabens such as methyl paraben or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, m-cresol, and the like); polypeptides of low molecular weight (less than about 10 residues); serum albumin, gelatin, These include, but are not limited to, proteins such as immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates such as glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as, for example, TWEEN™, PLURONICS™, or polyethylene glycol (PEG).

[0338] In certain embodiments, the pharmaceutical composition comprises an antibody drug conjugate described herein, and optionally one or more additional prophylactic or therapeutic agents, in a pharma- ceutically acceptable carrier. In certain embodiments, the pharmaceutical composition comprises an effective amount of an antibody drug conjugate described herein, and optionally one or more additional prophylactic or therapeutic agents, in a pharma- ceutically acceptable carrier. In certain embodiments, the pharmaceutical composition and / or antibody drug conjugate described herein can be combined with a therapeutically effective amount of any of the additional therapeutic agents described herein (see Section 5.7.2 below).

[0339] In some embodiments, the antibody drug conjugate is the only active ingredient contained in the pharmaceutical composition.

[0340] The pharmaceutical compositions described herein can be used to treat cancer.

[0341] Pharmaceutically acceptable carriers used in parenteral formulations include aqueous vehicles, non-aqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, sequestering or chelating agents, and other pharma- ceutically acceptable substances. Examples of aqueous vehicles include sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, dextrose and lactated Ringer's injection. Non-aqueous parenteral vehicles include fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, and peanut oil. Antimicrobial agents in bacteriostatic or fungistatic concentrations may be added to parenteral formulations packaged in multi-dose containers, including phenol or cresol, mercury, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride, and benzethonium chloride. Isotonic agents include sodium chloride and dextrose. Buffers include phosphates and citrates. Antioxidants include sodium bisulfate. As a local anesthetic Examples of suitable carriers include procaine hydrochloride. Suspending and dispersing agents include sodium carboxymethylcellulose, hydroxypropylmethylcellulose, and polyvinylpyrrolidone. Emulsifying agents include polysorbate 80 (TWEEN® 80). Sequestering or chelating agents for metal ions include EDTA. Pharmaceutical carriers also include ethyl alcohol, polyethylene glycol, propylene glycol for water-miscible vehicles, and sodium hydroxide, hydrochloric acid, citric acid, or lactic acid for pH adjustment.

[0342] The pharmaceutical composition may be formulated for any route of administration to a subject. Specific examples of routes of administration include intranasal, oral, pulmonary, transdermal, intradermal, and parenteral. Parenteral administration, characterized by either subcutaneous, intramuscular, or intravenous injection, is also contemplated herein. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Injectables, solutions, and emulsions also include one or more excipients. Suitable excipients are, for example, water, saline, dextrose, glycerol, or ethanol. In addition, if necessary, the pharmaceutical composition to be administered can also include small amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, pH buffers, stabilizers, solubility enhancers, and other such agents, for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate, and cyclodextrins.

[0343] Formulations for parenteral administration of antibody drug conjugates include sterile solutions ready for injection, sterile dry soluble products such as lyophilized powders, such as hypodermic tablets, ready to be mixed with a solvent immediately prior to use, sterile suspensions ready for injection, sterile dry insoluble products ready to be mixed with a vehicle immediately prior to use, and sterile emulsions. The solutions may be aqueous or non-aqueous.

[0344] If administered intravenously, suitable carriers include physiological saline or phosphate buffered saline (PBS), as well as solutions containing thickening and solubilizing agents, such as glucose, polyethylene glycol, polypropylene glycol, and the like, and mixtures thereof.

[0345] Topical mixtures containing antibody drug conjugates are prepared as described for local and systemic administration. The resulting mixtures may be solutions, suspensions, emulsions, etc., and may be formulated as creams, gels, ointments, emulsions, solutions, elixirs, lotions, suspensions, tinctures, pastes, foams, aerosols, douches, sprays, suppositories, bandages, skin patches, or other formulations suitable for topical administration.

[0346] The antibody drug conjugates described herein can be formulated as aerosols for local application, such as inhalation (see, for example, U.S. Patent Nos. 4,044,126, 4,414,209 and 4,364,923, which describe aerosols for the delivery of steroids useful in the treatment of inflammatory diseases, particularly asthma). These formulations for administration to the respiratory tract can be in the form of aerosols or solutions for nebulizers, or fine powders for insufflation, alone or in combination with an inert carrier such as lactose. In such cases, the particles of the formulation have a diameter, in one embodiment, of less than 50 microns, in one embodiment, less than 10 microns.

[0347] The antibody drug conjugates described herein can be formulated for local or topical application, such as topical application to the skin or mucous membranes, including in the eye, in the form of gels, creams, and lotions, or for application to the eye, or for intracapsular or intrathecal application. Topical administration can be for transdermal delivery, for administration to the eye or mucous membranes, or for inhalation therapy. Nasal solutions of the antibody drug conjugates can be used alone or in combination with other pharma- ceutically acceptable solutions. The compound may also be administered in combination with an excipient that is suitable for administration.

[0348] Transdermal patches, including iontophoretic and electrophoretic devices, are well known to those skilled in the art and can be used to administer antibody-drug conjugates.For example, such patches are disclosed in U.S. Patent Nos. 6,267,983, 6,261,595, 6,256,533, 6,167,301, 6,024,975, 6,010715, 5,985,317, 5,983,134, 5,948,433 and 5,860,957.

[0349] In certain embodiments, pharmaceutical compositions comprising the antibody drug conjugates described herein are lyophilized powders and can be reconstituted for administration as solutions, emulsions and other mixtures. They can also be reconstituted and formulated as solids or gels. The lyophilized powders are prepared by dissolving the antibody drug conjugates described herein or pharma- ceutically acceptable derivatives thereof in a suitable solvent. In some embodiments, the lyophilized powders are sterile. The solvent may contain excipients that improve the stability or other pharmacological components of the powder or reconstituted solutions prepared from the powder. Excipients that can be used include, but are not limited to, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerin, glucose, sucrose or other suitable agents. The solvent may also contain a buffer such as citrate, sodium or potassium phosphate, or other such buffers known to those of skill in the art, in one embodiment at about neutral pH. Subsequent sterile filtration of the solution followed by lyophilization under standard conditions known to those of skill in the art provides the desired formulation. In one embodiment, the resulting solution is apportioned into vials for lyophilization. Each vial contains a single dose or multiple doses of the compound. The lyophilized powder can be stored under appropriate conditions, such as at about 4° C. to room temperature.

[0350] This lyophilized powder is reconstituted with water for injection to provide a formulation for use in parenteral administration. For reconstitution, the lyophilized powder is added to sterile water or other suitable carrier. The exact amount varies depending on the compound selected. Such amounts can be empirically determined.

[0351] The antibody drug conjugates described herein and other compositions provided herein can also be formulated to target specific tissues, receptors, or other areas of the body to be treated.Many such targeting methods are well known to those skilled in the art.All such targeting methods are contemplated herein for use in the compositions.For non-limiting examples of targeting methods, see, for example, U.S. Patent Nos. 6,316,652, 6,274,552, 6,271,359, 6,253,872, 6,139,865, 6,131,570, 6,120,751, 6,071,495, 6,060,082, 6,048,736, 6,039,975, 6,004,534, 5,985,307, 5,972,366, 5,900,252, 5,840,5 and 5,709,874. In certain embodiments, the antibody drug conjugates described herein are targeted to tumors.

[0352] Compositions to be used for in vivo administration can be sterile, which is readily accomplished, for example, by filtration through sterile filtration membranes.

[0353] 5.7 Uses and Methods 5.7.1. Therapeutic Uses and Methods 5.7.1.1. Cancer In one aspect, provided herein is a method of treating cancer in a subject comprising administering to a subject in need thereof an antibody drug conjugate as described herein, or a pharmaceutical composition comprising an antibody drug conjugate as described herein. In certain embodiments, administering to a subject in need thereof a pharmaceutical composition comprising an antibody drug conjugate as described herein. Provided herein is a method of treating cancer in a subject, comprising administering to a subject in need thereof a pharmaceutical composition comprising an antibody drug conjugate as described herein. In certain embodiments, provided herein is a method of treating cancer in which it is desirable to agonize MERTK signaling in endothelial cells to inhibit angiogenesis, comprising administering to a subject in need thereof a pharmaceutical composition comprising an antibody drug conjugate as described herein.

[0354] In certain embodiments, administration of an antibody drug conjugate described herein or a pharmaceutical composition described herein to a subject having cancer achieves at least one, two, three, four or more of the following effects: (i) a reduction or amelioration of the severity of one or more symptoms of cancer; (ii) a reduction in the duration of one or more symptoms associated with cancer; (iii) prevention of recurrence of symptoms associated with cancer; (iv) a reduction in hospitalization of the subject; (v) a reduction in the length of hospitalization; (vi) an increase in survival rate of the subject; (vii) an enhancement or improvement in the therapeutic efficacy of another treatment; (viii) an inhibition of the progression or onset of one or more symptoms associated with cancer; (ix) a reduction in the number of symptoms associated with cancer; (x) an improvement in quality of life as assessed by methods well known in the art; (x) an inhibition of recurrence of the tumor; (xi) regression of the tumor and / or one or more symptoms associated therewith; (xii) a reduction in the severity of the tumor and / or one or more symptoms associated therewith; or inhibiting the progression of one or more symptoms associated therewith; (xiii) a reduction in tumor growth; (xiv) a reduction in tumor size (e.g., volume or diameter); (xv) a reduction in the formation of newly formed tumors; (xvi) prevention, eradication, removal, or control of primary tumors, local tumors, and / or metastatic tumors; (xvii) a reduction in the number or size of metastases; (xviii) a reduction in mortality; (xix) an increase in recurrence-free survival; (xx) a tumor that is maintained and does not increase in size or shows an increase that is less than the increase in tumor size following administration of standard treatment, as measured by conventional methods available to those of skill in the art, such as magnetic resonance imaging (MRI), dynamic contrast-enhanced MRI (DCE-MRI), X-ray, computed tomography (CT) scan, or positron emission tomography (PET) scan, and / or (xxi) an increase in the patient's duration of remission.

[0355] In some embodiments, the cancer treated according to the methods described herein is a cancer of the lung, breast, bone, ovary, stomach, pancreas, larynx, esophagus, testis, liver, parotid gland, biliary tract, colon, rectum, cervix, uterus, endometrium, kidney, bladder, prostate, or thyroid. In some embodiments, the cancer is a sarcoma, squamous cell carcinoma, melanoma, glioma, glioblastoma, neuroblastoma, or Kaposi's sarcoma. In some embodiments, the cancer treated according to these methods is metastatic.

[0356] In certain embodiments, the cancer treated according to the methods described herein is breast cancer.In certain embodiments, the cancer treated according to the methods described herein is triple-negative breast cancer.Triple-negative breast cancer refers to tumors or cells that originate from or grow in breast tissue that do not express the gene Her2 (also called Neu), estrogen receptor (also called ER), or progesterone receptor (also called PR) genes.

[0357] As used herein, the terms "subject" and "patient" are used interchangeably. In some embodiments, the subject is a mammal, such as a primate (e.g., a monkey or a human), and most preferably a human.

[0358] In certain embodiments, the antibody drug conjugate described herein is administered to the subject.In certain embodiments, two or more different antibody drug conjugates described herein are administered to the subject.In some embodiments, the antibody drug conjugate is administered to the subject in combination with one or more other treatments described herein (section 5.7.2 above).

[0359] 5.7.1.2 Route of Administration and Dose The antibody drug conjugates or compositions described herein can be delivered to a subject by a variety of routes. These routes include, but are not limited to, parenteral, intranasal, intratracheal, oral, intradermal, topical, intramuscular, intraperitoneal, transdermal, intravenous, intratumoral, conjunctival and subcutaneous routes. Pulmonary administration can also be used, for example, by use of an inhaler or nebulizer, and formulations containing an aerosolizing agent for use as a spray. In one embodiment, the antibody drug conjugate or composition described herein is parenterally administered to a subject. In certain embodiments, the parenteral administration is intravenous, intramuscular, or subcutaneous. In certain embodiments, the antibody drug conjugate is administered intravenously to a subject.

[0360] The amount of antibody drug conjugate or composition that will be effective in the treatment and / or prevention of symptoms will depend on the nature of the disease, and can be determined by standard clinical techniques.

[0361] The exact dosage used in the composition also depends on the route of administration and the type of cancer, and should be determined according to the physician's judgment and each subject's circumstances.For example, the effective dosage may vary depending on the administration means, the target site, the physiological condition of the patient (age, weight, health, etc.), whether the patient is human or animal, other drugs administered, or whether the treatment is preventive or therapeutic.The therapeutic dosage is optimally adjusted to optimize safety and effectiveness.

[0362] In certain embodiments, in vitro assays are used to help identify optimal dosage ranges. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0363] For antibody drug conjugates, the dosage may be in the range of about 0.0001-100 mg / kg, more typically 0.01-15 mg / kg, of patient body weight. For example, the dosage may be 1 mg / kg body weight, 10 mg / kg body weight, or in the range of 1-10 mg / kg, in other words 70 mg or 700 mg or in the range of 70-700 mg for a 70 kg patient, respectively. In some embodiments, the dose administered to the patient is about 1 mg to about 20 mg per kg of patient body weight. In general, human antibodies have a longer half-life in the human body than antibodies from other species due to the immune response to foreign polypeptides. Thus, it is often possible to reduce the dosage and frequency of administration of antibody-drug conjugates that include human antibodies.

[0364] Exemplary treatment regimens involve administration once every two weeks, once a month, or once every three to six months, over an interval of a year or several years or more. In some methods, two or more antibody drug conjugates are administered to a subject simultaneously. The antibody drug conjugates are typically administered multiple times. The interval between single doses is weekly, monthly, every three months, every six months, or yearly.

[0365] Combination therapy In certain embodiments, the method provided herein for treating cancer in a subject, comprising administering to a subject in need thereof a pharmaceutical composition comprising an antibody drug conjugate as described herein, further comprises administering to the subject one or more additional therapeutic agents. In certain embodiments, the additional therapeutic agent is for treating cancer. In certain embodiments, the additional therapeutic agent is for treating side effects of treatment with an antibody drug conjugate as described herein.

[0366] In certain embodiments, the additional agent is an agent used in the treatment of breast cancer, an agent used in the treatment of melanoma, an immunotherapy, or an angiogenesis inhibitor.

[0367] In certain embodiments, the additional therapeutic agent is tamoxifen, raloxifene, paclitaxel (TAXOL®), cyclophosphamide, docetaxel, vinblastine. The agent used to treat breast cancer is selected from the group consisting of fluorouracil, everolimus, trastuzumab, trastuzumab-emtansine, pertuzumab, and lapatinib ditosylate.

[0368] In certain embodiments, the additional therapeutic agent is an agent used to treat melanoma selected from the group consisting of a BRAF inhibitor, a MEK inhibitor, and dacarbazine.

[0369] In certain embodiments, the additional therapeutic agent is an antibody that is a CTLA-4 inhibitor, a PD-1 inhibitor, or a PD-L1 inhibitor.

[0370] In certain embodiments, the additional therapeutic agent is an angiogenesis inhibitor selected from the group consisting of a VEGF inhibitor, a VEGFR2 inhibitor, sunitinib, and sorafenib.

[0371] In other embodiments, the additional therapeutic agent is an agent listed in Table 10.

[0372]

Table 10-1

[0373]

Table 10-2

[0374]

Table 10-3

[0375]

Table 10-4

[0376]

Table 10-5

[0377]

Table 10-6

[0378]

Table 10-7

[0379] The antibody drug conjugates described herein can be administered simultaneously or sequentially (before and / or after) an additional therapeutic agent. The antibody drug conjugate and the additional therapeutic agent can be administered in the same or different compositions and by the same or different routes of administration. The first treatment (which is an antibody drug conjugate or additional therapeutic agent described herein) may be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), simultaneously with, or after (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of the second treatment (which is an antibody drug conjugate or additional therapeutic agent described herein) to the cancer patient. In certain embodiments, the additional therapeutic agent administered to the subject in combination with the antibody drug conjugate is administered in the same composition (pharmaceutical composition). In other embodiments, an additional therapeutic agent administered in combination with an antibody drug conjugate is administered to the subject in a composition different from the antibody drug conjugate (e.g., two or more pharmaceutical compositions are used).

[0380] 5.8.Kit Also provided herein is a kit comprising one or more antibody drug conjugates described herein.In certain embodiments, provided herein is a pharmaceutical pack or kit comprising one or more containers filled with one or more of the components of the pharmaceutical compositions described herein, such as one or more antibody drug conjugates described herein.In some embodiments, the kit comprises a pharmaceutical composition described herein and a prophylactic or therapeutic agent.

[0381] Optionally associated with such containers are notices, dosage forms, and / or instructions for their use in a form prescribed by a government agency regulating the manufacture, use, or sale of pharmaceutical or biological products. In certain embodiments, instructions included in the kit provide guidance regarding dosages and / or dosing regimens for administration of the pharmaceutical composition.

[0382] Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, packets, sachets, tubes, inhalers, pumps, bags, vials, containers, syringes, and other packaging materials appropriate for the selected pharmaceutical composition and intended method of administration and treatment.

[0383] The kits provided herein may further include a device used to administer the active ingredient. Examples of such devices include, but are not limited to, syringes, needleless injectors, drip bags, patches, and inhalers.

[0384] The kits provided herein can further include a pharma- ceutically acceptable vehicle that can be used to administer the components.For example, if the components are provided in solid form that must be reconstituted for parenteral administration, the kit can include a sealed container of a suitable vehicle in which the components can be dissolved to form a particulate-free sterile solution suitable for parenteral administration, or reconstituted as a suspension for oral administration.Examples of pharma-ceutically acceptable vehicles include aqueous vehicles, including but not limited to Water for Injection USP, Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, and Lactated Ringer's Injection; water-miscible vehicles, including but not limited to ethyl alcohol, polyethylene glycol, polypropylene glycol; non-aqueous vehicles, including but not limited to corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

[0385] The following examples are offered by way of illustration and not by way of limitation. EXAMPLES

[0386] 6. Working Example 6.1. Example 1: Generation of high affinity MERTK-binding monoclonal antibodies This example describes a monoclonal antibody that inhibits endothelial recruitment by metastatic breast cancer cells by binding to MERTK and activating its phosphorylation. The peptide antigen used to immunize mice to generate the antibodies described in this example was purchased from R&D Systems (Recombinant Human Mer Fc Chimera, #CF891-MR-100). This is a chimeric peptide consisting of a large portion of the extracellular domain of MERTK (Arg26 to Ala499; SEQ ID NO:58) fused to a portion of the Fc region of human IgG1 protein (Pro100-Lys330) with a small intervening connecting peptide (IEGRMD). See FIG. 1A for a schematic of the chimeric peptide. Mice were immunized with this chimeric peptide, and then B cells isolated from the immunized mice were fused to a myeloma cell line to generate a hybridoma library. Next, the supernatants of these hybridomas were isolated and hybridoma cells producing antibodies that bind to MERTK were identified using an antibody capture competitive ELISA assay (Figure 2), as described in Section 6.1.1 below. After this was identified, hybridomas producing antibodies with high affinity to MERTK were screened using an antibody capture competitive ELISA assay (Figure 2), as described in Section 6.1.2 below, to identify hybridomas producing antibodies that can inhibit MERTK binding to Gas-6. To isolate single-clonal (monoclonal) hybridoma cells, separation and screening were performed on a hybridoma library. 960 single-hybridoma clones (monoclonals) were screened from this library to identify clones producing monoclonal antibodies that bind to MERTK with high affinity and neutralize Gas-6 binding.

[0387] 6.1.1. ELISA capture assay for MERTK binding: To identify monoclonal antibodies that bind to MERTK, an ELISA antibody capture assay was used to screen the secreted antibodies produced from each hybridoma clone generated from mice immunized with recombinant MERTK protein. First, polystyrene plates were coated with coating antibody (goat anti-mouse IgG (Fc); Lot No.: Jackson 98959; antibody concentration 10 μg / mL). Then, supernatant containing primary antibody from each monoclonal hybridoma clone was added to each well of the plate. Next, tagged antigen (recombinant human Mer-Fc; RnD Systems #891-MR; Lot No.: CXK0211051; concentration 300 ng / mL) was added to each well. Then, detection antibody (horseradish peroxidase [HRP]-conjugated goat anti-human IgG (Fc); Lot No.: Jackson 86954; dilution: 1 / 5000) was added to each well. Following this, HRP substrate was added, followed by the addition of stop solution to complete the reaction. The signal (OD) from each well was read using a spectrophotometer. Of the 960 hybridoma clones tested, 20 showed OD values ​​greater than 1, indicating significant binding affinity to human MERTK. An OD reading of 1 indicates at least four times the signal observed in the negative control (blank medium without antibody), which had an OD value of 0.22. These 20 clones, arbitrarily named M1 through M20, are listed in FIG. 2. The second column in FIG. 2 (heading "Mer") shows the OD measurements (indicative of binding affinity) for each clone.

[0388] 6.1.2. Competitive Gas-6 Binding ELISA Assay To identify antibodies that inhibit Gas-6 binding to MERTK, a Gas-6 competitive binding ELISA assay was performed for the 20 MERTK-binding antibodies (M1–M20) listed above. First, polystyrene plates were coated with recombinant human Gas-6 (RnD Systems, rhGas6, #885-GS-050, concentration 7 μg / mL). Then, the supernatant containing the primary antibody from each monoclonal hybridoma clone was added to each well of the plate. At the same time, recombinant human MERTK (Mer-Fc RnD Systems, #891-MR; Lot No.: CXK0211051; concentration 300 ng / mL) was added to each well. Then, the detection antibody (horseradish peroxidase [HRP]-conjugated goat anti-human IgG (Fc); Lot No.: Jackson 86954; dilution: 1 / 5000) was added to each well. Following this, the reaction was completed by adding HRP substrate and stop solution. The signal (OD) was measured from each plate using a spectrophotometer. Of all hybridoma clones tested in this assay, 11 had OD values ​​below 2.4, indicating inhibition of Gas-6 binding to MERTK. An OD reading of 2.4 indicates inhibition of Gas-6 binding to MERTK, as the signal observed in the negative control (blank medium without antibody) was 2.43. These 20 clones are shown in Figure 2. The third column in Figure 2 (heading "(Mer+Clone)") shows the OD reading for each clone, indicating the amount of inhibition of Gas-6 binding.

[0389] Two monoclonal antibodies (M6 and M19) were selected for further studies, including whether they could inhibit endothelial recruitment by metastatic cells using a transwell endothelial migration assay.

[0390] 6.2. Example 2: M19 and M6 antibodies can inhibit endothelial cell recruitment by triple-negative breast cancer cells in vitro To identify monoclonal antibodies capable of inhibiting endothelial recruitment by MERTK activation, the high affinity Gas-6 competitive monoclonal antibodies generated in the screening described in Example 1 above were tested in an in vitro endothelial recruitment assay using transwells. Metastatic MDA-MB-231 human breast cancer cells were placed in the bottom of a Boyden chamber where their ability to recruit HUVECS through the porous transwell insert could be assayed. MERTK binding antibodies from the above screening (including both high and low affinity antibodies) were added individually to the transwells at physiological concentrations. Of all the antibodies tested, M19 and M6 most significantly inhibited HUVEC cell recruitment (migratory cells / field) (50% reduction in migratory cells) versus the negative control antibody IgG (Figure 3). This indicates the ability of monoclonal antibodies M19 and M6 to inhibit human endothelial cell recruitment by human metastatic cancer cells (Figure 3).

[0391] 6.3. Example 3: M19 and M6 antibodies activate phosphorylation of MERTK in endothelial cells To confirm that M19 and M6 are indeed the antibodies that activate MERTK, Western blot analysis was performed to examine the activity of endothelial cells with or without M19 or M6 antibody treatment. The levels of phosphorylated (activated) MERTK expressed by HUVEC were quantified (Figures 4 and 5).

[0392] To perform Western blot analysis, HUVEC cells (or LM2 breast cancer cells) were grown to 80% confluence in EGM-2 medium (Lonza, cat#CC-3162) containing 10% FBS. After 16 h of incubation under the given conditions, cells were washed with PBS and lysed in 1 mL RIPA buffer (G-Biosceince, Cat#786-490) containing protease and phosphatase inhibitors (Roche, Cat#11836170001 and 04906845001, respectively). Proteins from cell lysates were then separated by SDS-PAGE and transferred to PVDF membranes. The following primary antibodies were used for protein detection: phosphorylated-MERTK (FabGennix, cat#PMKT-140AP), MERTK (Abcam, cat#ab52968), phosphorylated-Akt (Santa Cruz Biotechnology, cat#sc-7985R), and Akt (Santa Cruz Biotechnology, cat#sc-1618). Primary antibodies were detected using appropriate HRP-conjugated secondary antibodies (Life Technologies).

[0393] Cultured cells treated with specific antibodies increased MERTK activation on endothelial cells in a dose-dependent manner during both chronic (16 h) and acute (30 min) antibody treatment (Figures 4 and 5). Interestingly, treatment with M19 antibody did not increase MERTK activation in LM2 breast cancer cells (Figure 6).

[0394] 6.4. Example 4: M19 binds with high affinity to both human and mouse MERTK This example shows that the M19 antibody has high affinity for both human and mouse MERTK (K of ∼0.3 nM). D ) binding. To characterize the binding affinity of the M19 antibody to recombinant human and mouse MERTK, biomolecular interaction analysis was performed using biolayer interferometry (Figures 7 and 8). Binding was observed between M19 and both mouse and human MERTK. From the overall global fit, the binding affinity (KD ) is equivalent to (K D =~0.3 nM) (Figures 7 and 8).

[0395] 6.5. Example 5: Therapeutic Administration of M19 Inhibits Tumor Progression of Human Breast Cancer In Vivo This example demonstrates that M19, an antibody that activates MERTK, can inhibit tumor progression and tumor metastasis in vivo in a mouse model of human breast cancer. To test whether monoclonal antibody M19 can reduce tumor burden and inhibit tumor progression in vivo, growth factor-reduced Matrigel was mixed with 2000 MDA-MB-231 or 5000 Lm1a1 cells in a 1:1 ratio and injected bilaterally into the mammary fat pad of NOD-SCID mice. After this surgery, an intraperitoneal injection of 250 μg of M19 antibody or control IgG antibody was administered, followed by two weekly injections. Tumor size of palpable tumors was measured weekly using a calibrator. Treatment with M19 antibody showed significant therapeutic inhibition of both primary tumor growth (Figure 9A) and metastasis (Figure 9B) of triple-negative breast cancer in vivo.

[0396] 6.6. Example 6: M19 inhibits tumor angiogenesis in vivo To determine whether therapeutic treatment with M19 antibody results in inhibition of angiogenesis in vivo, we quantified vascular density in tumors of M19-treated and untreated mice. 2000 MDA-MB-231 was injected bilaterally into the mammary gland of NOD-SCID mice. 58 days after treatment with 250 μg of M19 antibody or control IgG antibody, Tumors were excised and vascular density was quantified (Figure 10). Indeed, therapeutic treatment with M19 antibody resulted in significant inhibition (>50%) of angiogenesis in vivo (Figure 10).

[0397] 6.7. Example 7: M6 binds to both human MERTKs with high affinity This example demonstrates that the M6 ​​antibody has high affinity for recombinant human MERTK (K of ∼4.6 pmol). D) to characterize the binding affinity of the M6 ​​antibody. Biomolecular interaction analysis was performed using biolayer interferometry (Figure 11). Binding was observed between M6 and human MERTK. The overall global fit showed that the binding affinity (K D ) was calculated to be approximately 4.6 pmoles (Figure 11).

[0398] 6.8. Example 8: Therapeutic Administration of M6 Inhibits Human Breast Cancer Tumor Progression In Vivo This example shows that MERTK-activating antibody M6 can inhibit tumor progression in vivo in a mouse model of human breast cancer. To test whether monoclonal antibody M6 can reduce tumor burden and inhibit tumor progression in vivo, growth factor-reduced Matrigel (BD Biosciences) was mixed with 2000 MDA-MB-231 breast cancer cells in a 1:1 ratio and injected bilaterally into the mammary fat pad of NOD-SCID mice. After this operation, M6 antibody or control IgG antibody was injected intraperitoneally, followed by two weekly injections. Tumor size of palpable tumors was measured weekly using a calibrator. Indeed, twice weekly treatment with M6 antibody resulted in a significant reduction in tumor size (Figure 12).

[0399] 6.9. Example 9: M6 and M19 Antibody Sequences To further characterize M19 and M6, the sequences of the heavy and light chain variable regions of these antibodies were determined. The amino acid sequences of both the heavy and light chain variable regions of M6 are shown in Table 5. The nucleic acid sequences of both the heavy and light chain variable regions of M6 are shown in Table 7. The amino acid sequences of the CDRs of M6 are shown in Table 1 (for VH) and Table 2 (for VL).

[0400] The amino acid sequences of both the heavy and light chain variable regions of M19 are shown in Table 6. The nucleic acid sequences of both the heavy and light chain variable regions of M6 are shown in Table 8. The amino acid sequences of the CDRs of M19 are shown in Table 3 (for VH) and Table 4 (for VL).

[0401] The present invention is not limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to be included within the scope of the appended claims.

[0402] 6.10. Example 10: Treatment of metastatic triple-negative breast cancer with anti-MERTK agonist antibody drug conjugates Monoclonal antibody M6, described in Example 1 (section 6.1), is humanized and conjugated to the cytotoxic agent monomethylauristatin F (MMAF) via maleimidocaproic acid (mc) (i.e., the linker). The anti-MERTK antibody-drug conjugate is purified and administered intravenously to patients presenting with metastatic triple-negative breast cancer. The patients are monitored for response by clinical evaluation before, during, and after administration of the antibody drug conjugate.

[0403] All documents (e.g., publications or patents or patent applications) cited in this specification are herein incorporated by reference in their entirety for all purposes to the same extent as if each individual document (e.g., publication or patent or patent application) was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

Claims

1. (a) an antibody portion that is an antibody or antigen-binding fragment thereof that specifically binds to human Mer tyrosine kinase (MERTK); (b) one or more drug moieties, each of which is a cytotoxic agent; and (c) an optional linker; and Including, the cytotoxic agent is conjugated directly to the antibody moiety or via the linker; The antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), (i) the VH comprises a complementarity determining region (CDR) 1 of SEQ ID NO: 25, a CDR2 of SEQ ID NO: 30, and a CDR3 of SEQ ID NO: 35, and the VL comprises a CDR1 of SEQ ID NO: 39, a CDR2 of SEQ ID NO: 43, and a CDR3 of SEQ ID NO: 46; (ii) the VH comprises a CDR1 of SEQ ID NO:26, a CDR2 of SEQ ID NO:31, and a CDR3 of SEQ ID NO:36, and the VL comprises a CDR1 of SEQ ID NO:40, a CDR2 of SEQ ID NO:44, and a CDR3 of SEQ ID NO:47; (iii) the VH comprises a CDR1 of SEQ ID NO:27, a CDR2 of SEQ ID NO:32, and a CDR3 of SEQ ID NO:35, and the VL comprises a CDR1 of SEQ ID NO:39, a CDR2 of SEQ ID NO:43, and a CDR3 of SEQ ID NO:46; (iv) the VH comprises a CDR1 of SEQ ID NO:28, a CDR2 of SEQ ID NO:33, and a CDR3 of SEQ ID NO:37, and the VL comprises a CDR1 of SEQ ID NO:41, a CDR2 of SEQ ID NO:45, and a CDR3 of SEQ ID NO:48; (v) the VH comprises a CDR1 of SEQ ID NO:29, a CDR2 of SEQ ID NO:34, and a CDR3 of SEQ ID NO:38, and the VL comprises a CDR1 of SEQ ID NO:42, a CDR2 of SEQ ID NO:44, and a CDR3 of SEQ ID NO:46; or (vi) the VH comprises a CDR1 of SEQ ID NO: 27, a CDR2 of SEQ ID NO: 30, and a CDR3 of SEQ ID NO: 35, and the VL comprises a CDR1 of SEQ ID NO: 39, a CDR2 of SEQ ID NO: 43, R2, and a CDR3 of SEQ ID NO:

46.

2. The antibody drug conjugate of claim 1 , wherein the antibody is an immunoglobulin.

3. The antibody drug conjugate of claim 1 or 2, wherein the antibody or antigen-binding fragment thereof is humanized.

4. The antibody drug conjugate of any one of claims 1 to 3, wherein the antibody or antigen-binding fragment comprises a human-derived constant region.

5. The antibody-drug conjugate of any one of claims 1 to 4, wherein the antibody is a bispecific antibody.

6. The antibody drug conjugate of any one of claims 1 to 5, wherein the VH comprises the amino acid sequence of SEQ ID NO:51 and the VH comprises the amino acid sequence of SEQ ID NO:

52.

7. The antibody drug conjugate of any one of claims 1 to 6, comprising the linker, wherein the linker is a cleavable linker.

8. The antibody-drug conjugate of any one of claims 1 to 6, comprising the linker, wherein the linker is a non-cleavable linker.

9. The antibody drug conjugate of any one of claims 1 to 8, wherein the cytotoxic agent is monomethylauristatin E (MMAE) or monomethylauristatin F (MMAF).

10. The antibody drug conjugate of any one of claims 1 to 9, wherein the cytotoxic agent is SN38.

11. A method for producing an antibody-drug conjugate according to any one of claims 1 to 10, comprising the linker, comprising: (a) directly conjugating the linker to the cytotoxic agent to produce a linker-cytotoxic agent moiety; (b) directly conjugating the linker of the linker-cytotoxic agent moiety to the antibody moiety to produce an antibody drug conjugate; and (c) purifying the antibody drug conjugate.

12. A pharmaceutical composition comprising a therapeutically effective amount of the antibody drug conjugate of any one of claims 1 to 11 and a pharma- ceutically acceptable carrier.

13. 13. The pharmaceutical composition of claim 12 for use in a method for the treatment of cancer.

14. 14. The pharmaceutical composition of claim 13, wherein the cancer is cancer of the lung, breast, bone, ovary, stomach, pancreas, larynx, esophagus, testis, liver, parotid gland, biliary tract, colon, rectum, cervix, uterus, endometrium, kidney, bladder, prostate or thyroid, or the cancer is sarcoma, squamous cell carcinoma, melanoma, glioma, glioblastoma, neuroblastoma or Kaposi's sarcoma.

15. 15. The pharmaceutical composition of claim 14, wherein the cancer is breast cancer, optionally triple negative breast cancer.

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