TW202233671A - Peg-conjugated anti-mertk antibodies and methods of use - Google Patents

Abstract

The invention provides anti-MerTK antibodies (e.g., PEGylated anti-MerTK antibodies) and methods of making and using the same.

Description

PEG-conjugated anti-MerTK antibodies and methods of using the same

The present invention relates to PEGylated anti-MerTK antibodies and methods for their preparation and use.

Most current cancer immuno-oncology (IO) therapies focus on modulating the activity of T cells, the adaptive arm of the immune system, by blocking inhibitory pathways that act as immune checkpoints. However, long-lasting responses triggered by these therapies are limited to subpopulations of cancer patients. Various immunosuppressive mechanisms in the tumor microenvironment result in relatively low response rates. The innate immune system is an integral part of an effective immune response. The innate immune system plays a key part in the initiation and subsequent direction of the adaptive immune response. Targeted innate immune systems can be paired with adaptive tumor immunotherapy (Mullard, A., Nat. Rev. Drug Discov., 17: 3-5 (2018)).

Macrophages of the innate immune system are quite abundant in various types of solid tumors and can contribute relatively low response rates to T cell-based therapies. They are multifunctional cells that perform various functions, including phagocytosis. Macrophages are professional phagocytes that are highly specialized in removing dying or dying cells and cellular debris. It is estimated that billions of cells die in the human body every day. However, apoptotic cells are rarely found in tissues under normal physiological conditions, thanks to the rapid and efficient clearance of phagocytes. In homeostasis, apoptotic cells are removed at an early stage of cell death before the integrity of the plasma membrane is lost. Therefore, apoptosis is usually immunologically silent. In solid tumors, uncontrolled tumor growth is often accompanied by increased cell death due to hypoxia and metabolic stress. To evade immune surveillance, tumors exploit the non-immune nature of apoptosis. Tumor-associated macrophages (TAMs) actively remove apoptotic tumor cells to avoid altering the immune system.

MerTK has been shown to play a role in the clearance of apoptotic cells. Therefore, reducing MerTK-mediated clearance of apoptotic cells using MerTK inhibitors is an interesting therapeutic approach in cancer therapy. Existing anti-MerTK antibodies have been described but may not be suitable for therapeutic development. For example, White et al. ("MERTK-Specific Antibodies That Have Therapeutic Antitumor Activity in Mice Disrupt the Integrity of the Retinal Pigmented Epithelium in Cynomolgus Monkeys”, presented at the American Association for Cancer Research Annual Meeting, March 31, 2019, Atlanta, GA) describing secondary MerTK antibodies: a A high affinity (8.7x10 ^-11 M) human MerTK binds (SRF1), one binds a lower affinity (4.4x10 ⁹ ) human MerTK but cross-reacts with a murine MerTK (SRF2). These antibodies have been shown in mouse models to inhibit various MerTK functions and inhibit tumor growth when combined with anti-PD-L1 antibodies. However, secondary antibodies have been found to cause retinal toxicity in cynomolgus monkeys. As such, none of the secondary antibodies are acceptable as therapeutic candidates. These findings underscore the importance of examining multiple factors, not just antibody affinity, in developing effective therapeutic candidates with acceptable safety profiles.

Accordingly, there remains a need for optimal therapies for treating, stabilizing, preventing and/or delaying the development of various cancers. In particular, anti-MerTK antibodies with optimal binding properties ( eg , on and off rates) and desired biological effects are needed. For example, there is a need for anti-MerTK antibodies that have efficacy but reduce or eliminate retinal toxicity.

All references cited herein (including patent applications, patent publications, and UniProtKB/Swiss-Prot access numbers) are incorporated by reference in their entirety as if each individual reference was specifically and individually Indicates that it is incorporated by reference.

The present invention provides a PEGylated anti-MerTK antibody and a method for using the same.

In one aspect, the present disclosure provides a PEGylated antibody that binds to MerTK, wherein the antibody binds to one or more polyethylene glycol (PEG) polymers; wherein one of the PEG polymer(s) is each binds an engineered cysteine residue to a heavy or light chain of the antibody; and wherein the antibody comprises: (1) a heavy chain variable domain (VH) comprising (a) a SYAMG (SEQ ID NO: 1) CDR-H1 of the amino acid sequence, (b) CDR-H2 containing the amino acid sequence of IINSYGNTYYANWAKG (SEQ ID NO: 2) and (c) CDR-H2 containing the amino acid sequence of DPGVSSNL (SEQ ID NO: 3) CDR-H3 of amino acid sequence, and light chain variable domain (VL) comprising (d) CDR-L1 containing the amino acid sequence of QASQNIYSGLA (SEQ ID NO: 4), (e) CDR-L1 containing GASKLAS (SEQ ID NO: 4) ID NO: 5) CDR-L2 of the amino acid sequence and (f) CDR-L3 containing the amino acid sequence of QATYYSSNSVA (SEQ ID NO: 6); or (2) the heavy chain variable domain (VH), It comprises (a) CDR-H1 containing the amino acid sequence of ANTMN (SEQ ID NO: 7), (b) CDR-H2 containing the amino acid sequence of IFTATGSTYYATWVNG (SEQ ID NO: 8) and (c) containing CDR-H3 of the amino acid sequence of SGSGSSSGAFNI (SEQ ID NO: 9), and a light chain variable domain (VL) comprising (d) CDR-H3 containing the amino acid sequence of QASQSISSSLA (SEQ ID NO: 10) L1, (e) CDR-L2 containing the amino acid sequence of AASILAS (SEQ ID NO: 11) and (f) CDR-L3 containing the amino acid sequence of QCTSYGSLFLGP (SEQ ID NO: 12). In one aspect, the present disclosure provides a PEGylated antibody that binds to MerTK, wherein the antibody binds to one or more polyethylene glycol (PEG) polymers; wherein each of the PEG polymer(s) is engineered and wherein the antibody comprises: a heavy chain variable domain (VH) comprising (a) an amine group comprising SYAMG (SEQ ID NO: 1) CDR-H1 of the acid sequence, (b) CDR-H2 comprising the amino acid sequence of IINSYGNTYYANWAKG (SEQ ID NO: 2), and (c) CDR-H2 comprising the amino acid sequence of DPGVSSNL (SEQ ID NO: 3) H3; and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of QASQNIYSGLA (SEQ ID NO: 4), (e) an amine comprising GASKLAS (SEQ ID NO: 5) CDR-L2 of the amino acid sequence, and (f) CDR-L3 comprising the amino acid sequence of QATYYSSNSVA (SEQ ID NO: 6). In one aspect, the present disclosure provides a PEGylated antibody that binds to MerTK, wherein the antibody binds to one or more polyethylene glycol (PEG) polymers; wherein each of the PEG polymer(s) is engineered and wherein the antibody comprises: a heavy chain variable domain (VH) comprising (a) an amine comprising ANTMN (SEQ ID NO: 7) CDR-H1 of the amino acid sequence, (b) CDR-H2 comprising the amino acid sequence of IFTATGSTYYATWVNG (SEQ ID NO: 8), and (c) CDRs comprising the amino acid sequence of SGSGSSSGAFNI (SEQ ID NO: 9) -H3; and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of QASQSISSSLA (SEQ ID NO: 10), (e) comprising AASILAS (SEQ ID NO: 11) CDR-L2 of the amino acid sequence, and (f) CDR-L3 comprising the amino acid sequence of QCTSYGSLFLGP (SEQ ID NO: 12).

在一些實施例中，該VH 域包含與EVQLVESGEGLVQPGGSLRLSCAASGFSLSSYAMGWVRQAPGKGLEYVGIINSYGNTYYANWAKGRFTISRDNSKNTVYLQMGSLRAEDMAVYYCARDPGVSSNLWGRGTLVTVSS (SEQ ID NO: 13) 之胺基酸序列具有至少95% 序列同一性的序列；以及/或該VL 域包含與DIQMTQSPSTLSASVGDRVTITCQASQNIYSGLAWYQQKPGKAPKLLIYGASKLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQATYYSSNSVAFGGGTKVEIK (SEQ ID NO: 14) The amino acid sequence has at least 95% sequence identity.在一些實施例中，該 VH 域包含 EVQLVESGEGLVQPGGSLRLSCAASGFSLSSYAMGWVRQAPGKGLEYVGIINSYGNTYYANWAKGRFTISRDNSKNTVYLQMGSLRAEDMAVYYCARDPGVSSNLWGRGTLVTVSS (SEQ ID NO: 13) 之胺基酸序列；以及該 VL 域包含 DIQMTQSPSTLSASVGDRVTITCQASQNIYSGLAWYQQKPGKAPKLLIYGASKLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQATYYSSNSVAFGGGTKVEIK (SEQ ID NO: 14) 之胺基酸序列。在一些實施例中，該VH 域包含與EQQLVESGEGLVQPGGSLRLSCAVSGFSLSSYAMGWVRQAPGKGLEWIGIINSYGNTYYANWAKGRFTISRDSSKNTVYLQMGSLRAEDMAVYFCARDPGVSSNLWGPGTLVTVSS (SEQ ID NO: 15) 之胺基酸序列具有至少95% 序列同一性的序列；以及/或該VL 域包含與DVQMTQSPSTLSASVGDRVTITCQASQNIYSGLAWYQQKPGKPPKLLIYGASKLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQATYYSSNSVAFGGGTKVEIK (SEQ ID NO: 16) The amino acid sequence has at least 95% sequence identity.在一些實施例中，該 VH 域包含 EQQLVESGEGLVQPGGSLRLSCAVSGFSLSSYAMGWVRQAPGKGLEWIGIINSYGNTYYANWAKGRFTISRDSSKNTVYLQMGSLRAEDMAVYFCARDPGVSSNLWGPGTLVTVSS (SEQ ID NO: 15) 之胺基酸序列；以及該 VL 域包含 DVQMTQSPSTLSASVGDRVTITCQASQNIYSGLAWYQQKPGKPPKLLIYGASKLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQATYYSSNSVAFGGGTKVEIK (SEQ ID NO: 16) 之胺基酸序列。在一些實施例中，該VH 域包含與QSVEESGGRLVTPGTPLTLTCTVSGFSLSSYAMGWVRQAPGKGLEWIGIINSYGNTYYANWAKGRFTISRTSTTVDLRMPSLTTEDTATYFCARDPGVSSNLWGPGTLVTVSS (SEQ ID NO: 17) 之胺基酸序列具有至少95% 序列同一性的序列；以及/或該VL 域包含與DVVMTQTPASVSEPVGGTVTIKCQASQNIYSGLAWYQQKPGQPPKLLIYGASKLASGVSSRFKGSGSGTEFTLTISDLECADAATYYCQATYYSSNSVAFGGGTEVVVK (SEQ ID NO: 18) The amino acid sequence has at least 95% sequence identity.在一些實施例中，該 VH 域包含 QSVEESGGRLVTPGTPLTLTCTVSGFSLSSYAMGWVRQAPGKGLEWIGIINSYGNTYYANWAKGRFTISRTSTTVDLRMPSLTTEDTATYFCARDPGVSSNLWGPGTLVTVSS (SEQ ID NO: 17) 之胺基酸序列；以及該 VL 域包含 DVVMTQTPASVSEPVGGTVTIKCQASQNIYSGLAWYQQKPGQPPKLLIYGASKLASGVSSRFKGSGSGTEFTLTISDLECADAATYYCQATYYSSNSVAFGGGTEVVVK (SEQ ID NO: 18) 之胺基酸序列。在一些實施例中，該VH 域包含與QSVEESGGRLVTPGTPLTLTCTVSGIDLSANTMNWVRQAPGKGLEWIGIFTATGSTYYATWVNGRFTISKTSTTVDLKITSPTTEDTATYFCARSGSGSSSGAFNIWGPGTLVTVSL (SEQ ID NO: 19) 之胺基酸序列具有至少95% 序列同一性的序列；以及/或該VL 域包含與DPVLTQTPASVSEPVGGTVTIKCQASQSISSSLAWYQQKPGQPPKLLIYAASILASEISSRFKGSRSGTEFTLTISDLECADAATYYCQCTSYGSLFLGPFGGGTEVVVK (SEQ ID NO: 20) The amino acid sequence has at least 95% sequence identity.在一些實施例中，該 VH 域包含 QSVEESGGRLVTPGTPLTLTCTVSGIDLSANTMNWVRQAPGKGLEWIGIFTATGSTYYATWVNGRFTISKTSTTVDLKITSPTTEDTATYFCARSGSGSSSGAFNIWGPGTLVTVSL (SEQ ID NO: 19) 之胺基酸序列；以及該 VL 域包含 DPVLTQTPASVSEPVGGTVTIKCQASQSISSSLAWYQQKPGQPPKLLIYAASILASEISSRFKGSRSGTEFTLTISDLECADAATYYCQCTSYGSLFLGPFGGGTEVVVK (SEQ ID NO: 20) 之胺基酸序列。

在一些實施例中，該重鏈包含 EVQLVESGEGLVQPGGSLRLSCAASGFSLSSYAMGWVRQAPGKGLEYVGIINSYGNTYYANWAKGRFTISRDNSKNTVYLQMGSLRAEDMAVYYCARDPGVSSNLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 21) 之序列。在一些實施例中，該重鏈包含 EVQLVESGEGLVQPGGSLRLSCAASGFSLSSYAMGWVRQAPGKGLEYVGIINSYGNTYYANWAKGRFTISRDNSKNTVYLQMGSLRAEDMAVYYCARDPGVSSNLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:22) 之序列。 In some embodiments, the light chain comprises the sequence of DIQMTQSPSTLSASVGDRVTITCQASQNIYSGLAWYQQKPGKAPKLLIYGASKLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQATYYSSNSVAFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY SEQ IDSF NRCTHQGLSSPVTK3GE.

In some embodiments, the antibody is conjugated to one or two PEG polymers. In some embodiments, the PEG polymer(s) are linear PEG polymers. In some embodiments, the PEG polymer(s) are branched PEG polymers. In some embodiments, the branched PEG polymer(s) comprises 2 branches. In some embodiments, the pegylated antibody has a hydrodynamic radius greater than about 6 nm. In some embodiments, the pegylated antibody has a hydrodynamic radius of greater than or equal to about 10 nm. In some embodiments, the hydrodynamic radius of the PEGylated antibody is between about 6 nm and about 10 nm, or between about 9 nm and about 11 nm. In some embodiments, the PEG polymer(s) each have between about 10 kDa to about 40 kDa, between about 20 kDa to about 40 kDa, or between about 10 kDa to about 20 kDa time, such as about Molecular weights of 10 kDa, about 20 kDa, about 30 kDa, or about 40 kDa. In some embodiments, each of the PEG polymer(s) is bound to the heavy or light chain of the antibody at the engineered cysteine residue via maleimide-cysteine conjugation. In some embodiments, each of the PEG polymer(s) is bound to the heavy or light chain of the antibody at the engineered cysteine residue via iodoacetamide-cysteine conjugation. In some embodiments, the engineered cysteine residues are selected from the group consisting of K149C for light chains, K183C for light chains, T186C for heavy chains, and Y373C for heavy chains, numbering for light chains according to Kabat , and the numbering of heavy chains is according to the EU index. In some embodiments, the antibody comprises two heavy chains, two light chains, and two PEG polymers; and wherein both light chains of the antibody comprise K149C engineered bound to one of the two PEG polymers cysteine. In some embodiments, the antibody comprises two heavy chains, two light chains, and two PEG polymers; and wherein both light chains of the antibody comprise K183C engineered bound to one of the two PEG polymers cysteine. In some embodiments, the antibody comprises two heavy chains, two light chains, and two PEG polymers; and wherein both heavy chains of the antibody comprise a T186C engineered bound to one of the two PEG polymers cysteine. In some embodiments, the antibody comprises two heavy chains, two light chains, and two PEG polymers; and wherein both heavy chains of the antibody comprise Y373C engineered bound to one of the two PEG polymers cysteine.

In one aspect, the present disclosure provides a method for manufacturing and MerTK A method of conjugating a pegylated antibody comprising combining an antibody comprising one or more free engineered cysteine residues with one or more polyethylene glycol (PEG) comprising a maleimide moiety The polymers are contacted (ie, covalently linked) under conditions suitable for each of the PEG polymer(s) to bind to the engineered cysteine residues of the antibody via thioether linkages. In some embodiments, the one or more free engineered cysteine residues of the antibody react with one or more free engineered cysteine residues at a pH between 7.0 and 7.5 The maleimide moieties of the PEG polymer are contacted (ie, chemically reacted). In one aspect, the present disclosure provides a method for manufacturing and MerTK A method of conjugating a PEGylated antibody comprising combining an antibody comprising one or more free engineered cysteine residues with one or more polyethylene glycol (PEG) polymers comprising an iodoacetamide moiety Contact (ie, covalently link) under conditions suitable for each of the PEG polymer(s) to be bound to the engineered cysteine residues of the antibody via thioether linkages. In some embodiments, the antibody comprises a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SYAMG (SEQ ID NO: 1), (b) IINSYGNTYYANWAKG (SEQ ID NO: 1) NO: 2) CDR-H2 of the amino acid sequence, and (c) CDR-H3 comprising the amino acid sequence of DPGVSSNL (SEQ ID NO: 3); and a light chain variable domain (VL) comprising ( d) CDR-L1 comprising the amino acid sequence of QASQNIYSGLA (SEQ ID NO: 4), (e) CDR-L2 comprising the amino acid sequence of GASKLAS (SEQ ID NO: 5), and (f) comprising QATYYSSNSVA ( CDR-L3 of the amino acid sequence of SEQ ID NO: 6). In some embodiments, the antibody comprises a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of ANTMN (SEQ ID NO: 7), (b) IFTATGSTYYATWVNG (SEQ ID NO: 7) NO: 8) CDR-H2 of the amino acid sequence, and (c) CDR-H3 comprising the amino acid sequence of SGSGSSSGAFNI (SEQ ID NO: 9); and a light chain variable domain (VL) comprising ( d) CDR-L1 comprising the amino acid sequence of QASQSISSSLA (SEQ ID NO: 10), (e) CDR-L2 comprising the amino acid sequence of AASILAS (SEQ ID NO: 11), and (f) comprising QCTSYGSLFLGP ( CDR-L3 of the amino acid sequence of SEQ ID NO: 12).

In some embodiments, the PEG polymer(s) are bound to the antibody at a ratio of 2.0 polymer per antibody. In some embodiments, the antibody is Each of the free engineered cysteine residues is blocked with a cysteine or glutathione moiety prior to contacting (ie, covalently linking) the PEG polymer; and the method further comprises applying the Engineered cysteine residues are deblocked. In some embodiments, the method further comprises, after combining the antibody with one or more After contacting (ie, covalently linking) the PEG polymer, the PEGylated antibody is purified from unbound antibody and PEG polymer. In some embodiments, pegylated antibodies are purified by hydrophobic interaction chromatography.

在一些實施例中，該VH 域包含與EVQLVESGEGLVQPGGSLRLSCAASGFSLSSYAMGWVRQAPGKGLEYVGIINSYGNTYYANWAKGRFTISRDNSKNTVYLQMGSLRAEDMAVYYCARDPGVSSNLWGRGTLVTVSS (SEQ ID NO: 13) 之胺基酸序列具有至少95% 序列同一性的序列；以及/或該VL 域包含與DIQMTQSPSTLSASVGDRVTITCQASQNIYSGLAWYQQKPGKAPKLLIYGASKLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQATYYSSNSVAFGGGTKVEIK (SEQ ID NO: 14) The amino acid sequence has at least 95% sequence identity.在一些實施例中，該 VH 域包含 EVQLVESGEGLVQPGGSLRLSCAASGFSLSSYAMGWVRQAPGKGLEYVGIINSYGNTYYANWAKGRFTISRDNSKNTVYLQMGSLRAEDMAVYYCARDPGVSSNLWGRGTLVTVSS (SEQ ID NO: 13) 之胺基酸序列；以及該 VL 域包含 DIQMTQSPSTLSASVGDRVTITCQASQNIYSGLAWYQQKPGKAPKLLIYGASKLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQATYYSSNSVAFGGGTKVEIK (SEQ ID NO: 14) 之胺基酸序列。在一些實施例中，該VH 域包含與EQQLVESGEGLVQPGGSLRLSCAVSGFSLSSYAMGWVRQAPGKGLEWIGIINSYGNTYYANWAKGRFTISRDSSKNTVYLQMGSLRAEDMAVYFCARDPGVSSNLWGPGTLVTVSS (SEQ ID NO: 15) 之胺基酸序列具有至少95% 序列同一性的序列；以及/或該VL 域包含與DVQMTQSPSTLSASVGDRVTITCQASQNIYSGLAWYQQKPGKPPKLLIYGASKLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQATYYSSNSVAFGGGTKVEIK (SEQ ID NO: 16) The amino acid sequence has at least 95% sequence identity.在一些實施例中，該 VH 域包含 EQQLVESGEGLVQPGGSLRLSCAVSGFSLSSYAMGWVRQAPGKGLEWIGIINSYGNTYYANWAKGRFTISRDSSKNTVYLQMGSLRAEDMAVYFCARDPGVSSNLWGPGTLVTVSS (SEQ ID NO: 15) 之胺基酸序列；以及該 VL 域包含 DVQMTQSPSTLSASVGDRVTITCQASQNIYSGLAWYQQKPGKPPKLLIYGASKLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQATYYSSNSVAFGGGTKVEIK (SEQ ID NO: 16) 之胺基酸序列。在一些實施例中，該VH 域包含與QSVEESGGRLVTPGTPLTLTCTVSGFSLSSYAMGWVRQAPGKGLEWIGIINSYGNTYYANWAKGRFTISRTSTTVDLRMPSLTTEDTATYFCARDPGVSSNLWGPGTLVTVSS (SEQ ID NO: 17) 之胺基酸序列具有至少95% 序列同一性的序列；以及/或該VL 域包含與DVVMTQTPASVSEPVGGTVTIKCQASQNIYSGLAWYQQKPGQPPKLLIYGASKLASGVSSRFKGSGSGTEFTLTISDLECADAATYYCQATYYSSNSVAFGGGTEVVVK (SEQ ID NO: 18) The amino acid sequence has at least 95% sequence identity.在一些實施例中，該 VH 域包含 QSVEESGGRLVTPGTPLTLTCTVSGFSLSSYAMGWVRQAPGKGLEWIGIINSYGNTYYANWAKGRFTISRTSTTVDLRMPSLTTEDTATYFCARDPGVSSNLWGPGTLVTVSS (SEQ ID NO: 17) 之胺基酸序列；以及該 VL 域包含 DVVMTQTPASVSEPVGGTVTIKCQASQNIYSGLAWYQQKPGQPPKLLIYGASKLASGVSSRFKGSGSGTEFTLTISDLECADAATYYCQATYYSSNSVAFGGGTEVVVK (SEQ ID NO: 18) 之胺基酸序列。在一些實施例中，該VH 域包含與QSVEESGGRLVTPGTPLTLTCTVSGIDLSANTMNWVRQAPGKGLEWIGIFTATGSTYYATWVNGRFTISKTSTTVDLKITSPTTEDTATYFCARSGSGSSSGAFNIWGPGTLVTVSL (SEQ ID NO: 19) 之胺基酸序列具有至少95% 序列同一性的序列；以及/或該VL 域包含與DPVLTQTPASVSEPVGGTVTIKCQASQSISSSLAWYQQKPGQPPKLLIYAASILASEISSRFKGSRSGTEFTLTISDLECADAATYYCQCTSYGSLFLGPFGGGTEVVVK (SEQ ID NO: 20) The amino acid sequence has at least 95% sequence identity.在一些實施例中，該 VH 域包含 QSVEESGGRLVTPGTPLTLTCTVSGIDLSANTMNWVRQAPGKGLEWIGIFTATGSTYYATWVNGRFTISKTSTTVDLKITSPTTEDTATYFCARSGSGSSSGAFNIWGPGTLVTVSL (SEQ ID NO: 19) 之胺基酸序列；以及該 VL 域包含 DPVLTQTPASVSEPVGGTVTIKCQASQSISSSLAWYQQKPGQPPKLLIYAASILASEISSRFKGSRSGTEFTLTISDLECADAATYYCQCTSYGSLFLGPFGGGTEVVVK (SEQ ID NO: 20) 之胺基酸序列。

在一些實施例中，該重鏈包含 EVQLVESGEGLVQPGGSLRLSCAASGFSLSSYAMGWVRQAPGKGLEYVGIINSYGNTYYANWAKGRFTISRDNSKNTVYLQMGSLRAEDMAVYYCARDPGVSSNLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 21) 之序列。在一些實施例中，該重鏈包含 EVQLVESGEGLVQPGGSLRLSCAASGFSLSSYAMGWVRQAPGKGLEYVGIINSYGNTYYANWAKGRFTISRDNSKNTVYLQMGSLRAEDMAVYYCARDPGVSSNLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:22) 之序列。 In some embodiments, the light chain comprises the sequence of DIQMTQSPSTLSASVGDRVTITCQASQNIYSGLAWYQQKPGKAPKLLIYGASKLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQATYYSSNSVAFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY SEQ IDSF NRCTHQGLSSPVTK3GE.

In some embodiments, the antibody is conjugated to one or two PEG polymers. In some embodiments, the PEG polymer(s) are linear PEG polymers. In some embodiments, the PEG polymer(s) are branched PEG polymers. In some embodiments, the branched PEG polymer(s) comprises 2 branches. In some embodiments, the pegylated antibody has a hydrodynamic radius greater than about 6 nm. In some embodiments, the pegylated antibody has a hydrodynamic radius of greater than or equal to about 10 nm. In some embodiments, the hydrodynamic radius of the PEGylated antibody is between about 6 nm and about 10 nm, or between about 9 nm and about 11 nm. In some embodiments, the PEG polymer(s) each have between about 10 kDa to about 40 kDa, between about 20 kDa to about 40 kDa, or between about 10 kDa to about 20 kDa time, such as about Molecular weights of 10 kDa, about 20 kDa, about 30 kDa, or about 40 kDa. In some embodiments, each of the PEG polymer(s) is bound to the heavy or light chain of the antibody at the engineered cysteine residue via maleimide-cysteine conjugation. In some embodiments, each of the PEG polymer(s) is bound to the heavy or light chain of the antibody at the engineered cysteine residue via iodoacetamide-cysteine conjugation. In some embodiments, the engineered cysteine residues are selected from the group consisting of K149C for light chains, K183C for light chains, T186C for heavy chains, and Y373C for heavy chains, numbering for light chains according to Kabat , and the numbering of heavy chains is according to the EU index. In some embodiments, the antibody comprises two heavy chains, two light chains, and two PEG polymers; and wherein both light chains of the antibody comprise K149C engineered bound to one of the two PEG polymers cysteine. In some embodiments, the antibody comprises two heavy chains, two light chains, and two PEG polymers; and wherein both light chains of the antibody comprise K183C engineered bound to one of the two PEG polymers cysteine. In some embodiments, the antibody comprises two heavy chains, two light chains, and two PEG polymers; and wherein both heavy chains of the antibody comprise a T186C engineered bound to one of the two PEG polymers cysteine. In some embodiments, the antibody comprises two heavy chains, two light chains, and two PEG polymers; and wherein both heavy chains of the antibody comprise Y373C engineered bound to one of the two PEG polymers cysteine.

In one aspect, the present disclosure provides a pegylated antibody that binds to MerTK produced by the method of any of the above embodiments. In one aspect, the present disclosure provides a pharmaceutical composition comprising the PEGylated anti-MerTK antibody of any of the above embodiments and a pharmaceutically acceptable carrier.

In one aspect, the present disclosure provides a method of treating an individual suffering from cancer comprising administering to the individual an effective amount of the PEGylated antibody or composition of any one of the above embodiments. In one aspect, the present disclosure provides a method of reducing MerTK-mediated clearance of apoptotic cells in an individual, comprising administering to the individual an effective amount of the pegylated antibody or composition of any of the above embodiments. In one aspect, the present disclosure provides the PEGylated anti-MerTK antibody or composition of any one of the above embodiments for use as a medicament. In one aspect, the present disclosure provides the PEGylated anti-MerTK antibody or composition of any of the above embodiments for use in the treatment of cancer. In one aspect, the present disclosure provides the PEGylated anti-MerTK of any of the foregoing embodiments An antibody or composition for use in any of the methods disclosed herein, eg, in a method of treating an individual suffering from cancer and/or reducing MerTK-mediated clearance of apoptotic cells in an individual. In one aspect, the present disclosure provides the PEGylated anti-MerTK of any of the foregoing embodiments Antibodies or compositions for use in the manufacture of, for example, a medicament for treating an individual suffering from cancer and/or reducing MerTK-mediated clearance of apoptotic cells in an individual.

In some embodiments, administration of the pegylated antibody results in a reduction in retinal toxicity in the individual compared to administration of the MerTK-conjugated non-pegylated antibody. In some embodiments, the method further comprises administering to the individual an additional therapeutic agent. In some embodiments, the additional therapeutic agent is selected from one or more of the following: tamoxifen, letrozole, exemestane, anastrozole ( anastrozole), irinotecan, cetuximab, fulvestrant, vinorelbine, erlotinib, bevacizumab, periwinkle Vincristine, imatinib mesylate, sorafenib, lapatinib, trastuzumab, cisplatin, gemcitabine ( gemcitabine, methotrexate, vinblastine, carboplatin, paclitaxel, 5-fluorouracil, doxorubicin, bortezomib ), melphalan, prednisone and docetaxel. In some embodiments, the additional therapeutic agent is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is selected from the group consisting of: a cytotoxic T lymphocyte-associated protein 4 (CTLA4) inhibitor, a programmed cell death protein 1 (PD-1) binding antagonist and planned death ligand 1 (PDL1) binding antagonists. In some embodiments, the immune checkpoint inhibitor is an anti-PDL1 binding antagonist. In some embodiments, the PDL1 binding antagonist is an anti-PDL1 antibody, such as atezolizumab (atezolizumab). In some embodiments, the method further comprises administering to the individual an effective amount of an additional chemotherapeutic agent. In some embodiments, the cancer is colorectal cancer. In some embodiments, the cancer line is selected from urothelial carcinoma, non-small cell lung cancer, triple negative breast cancer, small cell lung cancer, hepatocellular carcinoma, and melanoma. In some embodiments, the clearance of apoptotic cells is reduced by about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9 , 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4 , 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8.0 times.

It should be understood that one, some, or all of the features of the various embodiments disclosed herein may be combined to form other embodiments of the present disclosure. These and other aspects of the present disclosure will become apparent to those skilled in the art. These and other embodiments of the present disclosure are further described by the following description.

CROSS-REFERENCE TO RELATED APPLICATIONS

This case claims priority to US Provisional Application Serial No. 63/094,197, filed on October 20, 2020, the disclosure of which is incorporated herein by reference in its entirety. I. Definitions

It is to be understood that the present disclosure is not limited to particular compositions or biological systems, which may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular specific examples only and is not intended to be limiting.

As used in this specification and the appended claims, the singular forms "a (a/an)" and "the (the)" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a molecule" includes, as appropriate, combinations of two or more such molecules, and the like.

As used herein, the term "about" refers to the usual error range for each value readily known to those skilled in the art. Reference herein to "about" a value or parameter includes (and describes) embodiments directed to the value or parameter itself.

It is to be understood that aspects and embodiments of the present disclosure include "comprising" aspects and embodiments, "consisting of" aspects and embodiments, and "consisting essentially of" aspects and embodiments.

For purposes herein, an "acceptor human framework" is a framework comprising a light chain variable domain (VL) or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human common framework The backbone of the amino acid sequence is defined below. An acceptor human scaffold "derived from" a human immunoglobulin scaffold or a human common scaffold may comprise the same amino acid sequence as these, or it may contain amino acid sequence alterations. In some aspects, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or more less, or 2 or less. In some aspects, the VL acceptor human backbone is the same sequence as the VL human immunoglobulin backbone sequence or the human consensus backbone sequence.

"Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen). Unless otherwise stated, "binding affinity" as used herein refers to the intrinsic binding affinity that reflects the 1:1 interaction between members of a binding pair (eg, antibody and antigen). The affinity of a molecule X for its partner Y can generally be expressed by the dissociation constant (K _D ). Affinity can be measured by conventional methods known in the art, including those described herein. Specific illustrative and exemplary methods for measuring binding affinity are described below.

The term "affinity matured" antibody refers to an antibody that has one or more changes in one or more complementarity determining regions (CDRs) that cause the antibody to respond to an antigen compared to a parent antibody that does not have such changes. improvement in affinity.

The terms "anti-MerTK antibody" and "antibody that binds MerTK" refer to an antibody that is capable of binding to MerTK with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent targeting MerTK. In one aspect, the anti-MerTK antibody binds to an unrelated, non-MerTK protein to less than about 10% of the extent to which the antibody binds to MerTK, as measured, for example, by surface plasmon resonance (SPR). In certain aspects, antibodies that bind MerTK have a dissociation constant (K _D ) of ≤ 1 μM, ≤ 100 nM, ≤ 10 nM, ≤ 1 nM, ≤ 0.1 nM, ≤ 0.01 nM, or ≤ 0.001 nM (eg, 10 ⁻⁸ M or lower, such as ^10-8 M to ^10-13 M, such as ^10-9 to ^10-13 M). An antibody is said to "specifically bind" to _MerTK when the KD of the antibody is 1 μM or less. In certain aspects, the anti-MerTK antibody binds to an epitope of MerTK that is conserved among MerTKs of different species.

The term "antibody" herein is used in the broadest sense and encompasses a variety of antibody structures including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), and antibody fragments, so long as they display Antigen-binding activity is expected.

An "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') ₂ ; diabodies, linear antibodies formed from antibody fragments; single chain antibody molecules ( such as scFv and scFab); single domain antibodies (dAbs); and multispecific antibodies. For a review of certain antibody fragments, see Holliger and Hudson, Nature Biotechnology 23:1126-1136 (2005).

The term "chimeric" antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species.

The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these classes can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. In certain aspects, the antibody is of the IgG ₁ isotype. In certain aspects, the antibody is of the IgGl isotype with _P329G , L234A and L235A mutations to reduce Fc region effector function. In other aspects, the antibody is of the IgG2 isotype. In certain aspects, the antibody is of the IgG4 isotype with a S228P mutation in the hinge region to improve the stability of the IgG4 antibody. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The light chains of antibodies can be classified into one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domains.

The term "constant region derived from human origin" or "human constant region" as used in this application refers to the constant heavy chain region and/or the constant light chain kappa or lambda region of a human antibody of subclass IgG1, IgG2, IgG3 or IgG4. Such constant regions are known in the art, for example, in Kabat, E.A., et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991) Disclosed (see also e.g. Johnson, G. and Wu, T.T., Nucleic Acids Res. 28 (2000) 214-218; Kabat, E.A. et al., Proc. Natl. Acad. Sci. USA 72 (1975) 2785-2788) . Unless otherwise indicated herein, the numbering of amino acid residues in the constant region is according to the EU numbering system (also known as the Kabat EU index), as in Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th ed., Public As disclosed in Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242.

"Effector function" refers to those biological activities attributable to the Fc region of an antibody, which vary with antibody isotype. Examples of antibody effector functions include: Clq binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; cell surface receptors (eg, B cell receptors) downregulation of ; and B cell activation.

A "therapeutically effective amount" of an agent, such as a pharmaceutical composition, refers to an amount effective to achieve the desired therapeutic or prophylactic effect at the dose and time period required for administration.

The term "Fc region" herein is used to define the C-terminal region of an immunoglobulin heavy chain comprising at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In one aspect, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxy terminus of the heavy chain. However, antibodies produced by host cells may undergo post-translational cleavage of one or more, particularly one or both, amino acids at the C-terminus of the heavy chain. Thus, an antibody produced by a host cell by expressing a particular nucleic acid molecule encoding a full-length heavy chain may include the full-length heavy chain, or may include cleavage variants of the full-length heavy chain. This may be the case when the last two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, EU numbering system). Thus, the C-terminal lysine (Lys447) or the C-terminal glycine (Gly446) and lysine (Lys447) of the Fc region may or may not be present. Unless otherwise stated, the amino acid sequence of the heavy chain including the Fc region is expressed herein without the C-terminal glycine-lysine dipeptide. In one aspect, the heavy chain comprising an Fc region as described herein comprised in an antibody according to the invention comprises an additional C-terminal glycine-lysine dipeptide (G446 and K447, EU numbering system). In one aspect, the heavy chain comprising an Fc region as described herein, comprised in an antibody according to the invention, comprises an additional C-terminal glycine residue (G446, numbered according to the EU index). Unless otherwise stated herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system (also known as the EU index), as described by Kabat et al. (Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991) (see also above).

"Backbone" or "FR" refers to variable domain residues outside the complementarity determining regions (CDRs). The FRs of the variable domains generally consist of four FR domains: FR1, FR2, FR3, and FR4. Therefore, CDR and FR sequences usually appear in VH (or VL) in the following order: FR1-CDR-H1(CDR-L1)-FR2-CDR-H2(CDR-L2)-FR3-CDR-H3(CDR-L3) )-FR4.

The terms "full-length antibody", "intact antibody" and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to that of a native antibody or having a heavy chain comprising an Fc region as defined herein.

The terms "host cell", "host cell strain" and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including progeny cells of such cells. Host cells include "transformants" and "transformed cells," which include primary transformed cells and progeny cells derived therefrom, regardless of the number of passages. The nucleic acid content of the daughter cells may not be exactly the same as the parent cells, but may contain mutations. Mutant progeny cells that have the same function or biological activity as screened or selected from the original transformed cells are included herein.

A "human antibody" is an antibody having an amino acid sequence corresponding to that produced by a human or human cell or from a non-human source utilizing the human antibody repertoire or other human antibody coding sequences The amino acid sequence of the derived antibody. This definition of human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues.

A "human common backbone" is a backbone that represents the most common amino acid residues in a series of human immunoglobulin VL or VH backbone sequences. Typically, human immunoglobulin VL or VH sequences are selected from a subgroup of variable domain sequences. Typically, the subset of sequences is as described by Kabat et al. in Sequences of Proteins of Immunological Interest (Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3) . In one aspect, for VL, the subgroup is subgroup κI as described by Kabat et al, supra . In one aspect, for VH, the subgroup is subgroup III as described by Kabat et al, supra .

A "humanized" antibody system refers to a chimeric antibody comprising amino acid residues from non-human CDRs and amino acid residues from human FRs. In certain aspects, a humanized antibody will include substantially all of at least one (and usually two) variable domains, wherein all or substantially all CDRs correspond to non-human antibodies, and the like, and all or substantially all FRs correspond to For human antibodies and the like. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody (eg, a non-human antibody) refers to an antibody that has undergone humanization.

As used herein, the term "hypervariable region" or "HVR" refers to the various regions in the variable domain of an antibody that are hypervariable in sequence and determine antigen-binding specificity, eg, "complementarity determining regions"("CDRs"). Typically, an antibody includes six CDRs: three in the VH (CDR-H1, CDR-H2, CDR-H3), and three in the VL (CDR-L1, CDR-L2, CDR-L3). Herein, exemplary CDRs include: (a) hypervariable loops present at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1) , 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) CDRs are present at amino acid residues 24- 34(L1), 50-56(L2), 89-97(L3), 31-35b(H1), 50-65(H2), and 95-102(H3) (Kabat et al., Sequences of Proteins of Immunological Interest , 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); and (c) antigenic contacts are present at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al . J. Mol. Biol. 262: 732-745 (1996)). Unless otherwise stated, CDRs were determined according to the method described by Kabat et al., supra. Those skilled in the art will appreciate that CDR names may also be determined according to the methods described in Chothia, supra, McCallum, supra, or any other scientifically accepted nomenclature system.

In one aspect, the CDR residues comprise residues identified in Section II.A or elsewhere in this specification.

An "immunoconjugate" is an antibody that binds to one or more heterologous molecules, including but not limited to cytotoxic agents.

An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (eg, cattle, sheep, cats, dogs, and horses), primates (eg, humans and non-human primates such as monkeys), rabbits, and rodents (eg, mice and large animals). mouse). In certain aspects, the subject or individual is a human.

An "isolated" antibody is one that has been separated from components of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity by, eg, electrophoresis (eg, SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (eg, ion exchange or reverse reaction). phase HPLC) method. For a review of methods for assessing antibody purity, see, eg, Flatman et al., J. Chromatogr. B 848:79-87 (2007).

The term "nucleic acid molecule" or "polynucleotide" includes any compound and/or substance comprising a polymer of nucleotides. Each nucleotide consists of a base, specifically a purine or pyrimidine base (ie, cytosine (C), guanine (G), adenine (A), thymine (T), or uracil (U)), Sugar (ie, deoxyribose or ribose) and phosphate groups. Generally, nucleic acid molecules are described by the sequence of bases, wherein the bases represent the primary structure (linear structure) of the nucleic acid molecule. The base sequence is usually represented by 5' to 3'. As used herein, the term nucleic acid molecule includes: deoxyribonucleic acid (DNA), which includes, for example, complementary DNA (cDNA) and genomic DNA; ribonucleic acid (RNA), in particular messenger RNA (mRNA); synthesis of DNA or RNA forms; and mixed polymers comprising two or more of these molecules. Nucleic acid molecules can be linear or circular. In addition, the term nucleic acid molecule includes sense and antisense strands, as well as single- and double-stranded forms. In addition, the nucleic acid molecules described herein may comprise naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases with derivatized sugars, phosphate linkages, or chemically modified residues. Nucleic acid molecules also include vectors suitable for direct expression of the antibodies of the invention in vitro and/or in vivo, eg, in a host or patient. DNA and RNA molecules. Such DNA (eg, cDNA) or RNA (eg, mRNA) vectors can be unmodified or modified. For example, mRNA Can be chemically modified to enhance the stability of the RNA vector and/or the expression of the encoding molecule, allowing the mRNA to be injected into an individual to generate antibodies (see eg, Stadler et al., Nature Medicine 2017, published online June 12, 2017, doi : 10.1038/nm.4356 or EP 2 101 823 B1).

An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from components of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location different from the natural chromosomal location.

"Isolated anti-MerTK antibody-encoding nucleic acid" refers to one or more nucleic acid molecules encoding anti-MerTK antibody heavy and light chains (or fragments thereof), including such nucleic acid molecules in a single vector or a separate antibody, and which Nucleic acid molecules are present at one or more locations in the host cell.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, ie, the individual antibodies comprising the population are identical and/or bind the same epitope, except, for example, containing naturally occurring mutations or These variants are usually present in small amounts, in addition to possible variant antibodies produced during the production of monoclonal antibody preparations. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different epitopes (epitopes), each monoclonal antibody system of a monoclonal antibody preparation is directed against a single epitope on an antigen. Thus, the modifier "monoclonal" indicates that the antibody is characterized as being obtained from a substantially homogeneous population of antibodies, and should not be construed as requiring the production of the antibody by any particular method. For example, monoclonal antibodies intended for use in accordance with the present invention can be made by a variety of techniques including, but not limited to, fusionoma methods, recombinant DNA methods, phage display methods, and the use of transfection comprising all or part of human immunoglobulin loci Methods of transgenic animals, such methods and other exemplary methods for making monoclonal antibodies are described herein.

"Naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (eg, a cytotoxic moiety) or radiolabel. Naked antibodies can be present in pharmaceutical compositions.

"Native antibody" refers to naturally occurring immunoglobulin molecules with different structures. For example, an Ig native IgG antibody is a heterotetrameric glycoprotein of approximately 150,000 Daltons consisting of two identical light chains and two identical heavy chains that are disulfide-bonded. From the N-terminus to the C-terminus, each heavy chain has a variable domain (VH), also known as a variable heavy chain domain or heavy chain variable region, followed by three heavy chain constant domains (CH1, CH2 and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable domain (VL), also known as a variable light chain domain or light chain variable region, followed by a light chain constant (CL) domain.

The term "package insert" is used to refer to instructions usually contained in commercial packaging of therapeutic products, the instructions including indications, usage, dosage, route of administration, combination therapy, contraindications for the use of such therapeutic products and/or warnings.

"Percent amino acid sequence identity (%)" relative to the reference polypeptide sequence refers to the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence. After introducing differences (if necessary), the maximum percent sequence identity is achieved and any conservative substitutions are not considered as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be accomplished by various means within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR). ) software or FASTA program suite. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. Alternatively, percent identity values can be generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was developed by Jiannandek Corporation and its source code has been filed with the user documentation in the United States Copyright Office, Washington, DC 20559, USA, where it is registered (US Copyright Registration No. TXU510087) and published in WO 2001 /007611.

Unless otherwise stated, for the purposes of this article, the FASTA suite version 36.3.8c or later of the ggsearch program and the BLOSUM50 comparison matrix were used to generate percent amino acid sequence identity values. The FASTA suite of programs was developed by the following authors: W. R. Pearson and D. J. Lipman (1988) (“Improved Tools for Biological Sequence Analysis”, PNAS 85:2444-2448); W. R. Pearson (1996) (“Effective protein sequence comparison” Meth. Enzymol. 266:227-258); and Pearson et al. (1997) (Genomics 46:24-36), and are publicly accessible at www.fasta.bioch.virginia.edu/fasta_www2/fasta_down.shtml or www.fasta.bioch.virginia.edu/fasta_www2/fasta_down.shtml. ebi.ac.uk/Tools/sss/fasta. Alternatively, use the ggsearch (global protein:protein) program and default options (BLOSUM50; open: -10; ext: -2; Ktup = 2) Compare sequences to ensure global rather than local alignments are performed. The percent amino acid identity is provided in the output alignment header.

The term "pharmaceutical composition" or "pharmaceutical formulation" refers to a formulation that is in a form that allows the biological activity of the active ingredient contained therein to be effective and is free of other substances that would have unacceptable toxicity to the individual to which the composition is to be administered. components.

"Pharmaceutically acceptable carrier" refers to ingredients in a pharmaceutical composition or formulation other than active ingredients that are not toxic to the individual. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.

Unless otherwise specified, the term "MerTK" as used herein refers to any native MerTK from any vertebrate source, including mammals, such as primates (eg, humans) and rodents (eg, mice and rats). The term covers "full-length" unprocessed MerTK as well as any form of MerTK produced by processing in a cell. The term also encompasses natural MerTK variants, such as splice variants or allelic variants. An exemplary human MerTK amino acid sequence is disclosed in US 2006/0121562.

As used herein, "treatment" (and grammatical variants thereof, such as "in the course of treatment" or "in treatment"), refers to clinical interventions that attempt to alter the natural course of disease in the subject being treated, and may be prophylactic or in the course of clinical pathology in execution. Desired therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, alleviating any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or lessening the disease state, alleviating or improving the prognosis. In some aspects, the antibodies of the invention are used to delay the development of a disease or slow the progression of a disease.

The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in antibody binding to an antigen. The variable domains of the heavy and light chains (VH and VL, respectively) of native antibodies generally have similar structures, and each domain comprises four conserved framework regions (FRs) and three complementarity determining regions (CDRs). (See, eg, Kindt et al. Kuby Immunology , 6 ^th ed., WH Freeman and Co., page 91 (2007).) A single VH or VL domain may be sufficient to confer antigen-binding specificity. In addition, the VH or VL domains can be used to separate antibodies that bind a particular antigen from those that bind the antigen to screen libraries of complementary VL or VH domains, respectively. See, eg, Portolano et al, J. Immunol. 150:880-887 (1993); Clarkson et al, Nature 352:624-628 (1991).

As used herein, the term "vector" refers to a nucleic acid molecule capable of delivering another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of the host cell. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. These vectors are referred to herein as "expression vectors". II. COMPOSITIONS AND METHODS

In one aspect, the present invention is based in part on the observation that MerTK antibody treatment results in targeted retinal toxicity (particularly photoreceptors and cells in the outer nuclear layer) in preclinical primate and mouse models. As disclosed herein, adding anti-MerTK The hydrodynamic radius of the antibody (e.g., by conjugating one or more polyethylene glycol (PEG) polymers to the antibody) can reduce ocular distribution after systemic administration, thereby reducing distribution to RPE cells and resulting retinal toxicity. In certain aspects, PEGylated antibodies that bind to MerTK are provided. The PEGylated antibodies of the present invention are useful, for example, in the treatment of cancer.

C-Mer proto-oncogene tyrosine kinase (MerTK) is a receptor tyrosine kinase that transduces extracellular signals upon binding to various ligands, such as Galectin 3, protein S, and Gas6, thereby Expression of activated effector genes. The MerTK pathway regulates fundamental cellular processes, including cell survival, cytokine production, migration, differentiation, and phagocytosis (Cabernoy N., et al. JCell Physio. 227 (2012): 401-407; Wu, G., et al. Cell Death & Disease 8 (2017): e2700). Expression of MerTK is found in a variety of hematopoietic cell types, such as macrophages, dendritic cells, and natural killer (NK) cells. Importantly, the MerTK receptor pathway is active in a variety of solid and hematological cancers, including colorectal cancer (Wu, G., et al. Cell Death & Disease 8 (2017): e2700).

However, MerTK is also expressed in the retinal pigment epithelium and is required for the turnover of the outer segment of photoreceptors (POS), which are critical for vision. In fact, mutations in MerTK have been identified in patients with retinal dystrophic atrophy.

MerTK receptors are composed of extracellular components, transmembrane (TM) domains, and intracellular components. As shown in the figure below, the extracellular or ligand-binding region of MerTK contains two immunoglobulin-like (Ig)-like domains and two fibronectin-like (FN) type III domains.

For example, in human MerTK, the two Ig-like domains are bounded by amino acid residues 76-195 and amino acid residues 199-283, respectively. In addition, the two fibronectin-like domains of human MerTK are bounded by amino acid residues 286-384 and 388-480, respectively. The intracellular domain of MerTK contains a tyrosine kinase (TK) domain that, upon ligand binding to the extracellular domain, autophosphorylates specific tyrosine residues and promotes MerTK receptor dimerization, thereby activating downstream effectors Gene expression (Toledo, RA, et al. Clin Can. Res . 22 (2016): 2301-2312).人MerTK 包含胺基酸序列： MGPAPLPLLLGLFLPALWRRAITEAREEAKPYPLFPGPFPGSLQTDHTPLLSLPHASGYQPALMFSPTQPGRPHTGNVAIPQVTSVESKPLPPLAFKHTVGHIILSEHKGVKFNCSISVPNIYQDTTISWWKDGKELLGAHHAITQFYPDDEVTAIIASFSITSVQRSDNGSYICKMKINNEEIVSDPIYIEVQGLPHFTKQPESMNVTRNTAFNLTCQAVGPPEPVNIFWVQNSSRVNEQPEKSPSVLTVPGLTEMAVFSCEAHNDKGLTVSKGVQINIKAIPSPPTEVSIRNSTAHSILISWVPGFDGYSPFRNCSIQVKEADPLSNGSVMIFNTSALPHLYQIKQLQALANYSIGVSCMNEIGWSAVSPWILASTTEGAPSVAPLNVTVFLNESSDNVDIRWMKPPTKQQDGELVGYRISHVWQSAGISKELLEEVGQNGSRARISVQVHNATCTVRIAAVTRGGVGPFSDPVKIFIPAHGWVDYAPSSTPAPGNADPVLIIFGCFCGFILIGLILYISLAIRKRVQETKFGNAFTEEDSELVVNYIAKKSFCRRAIELTLHSLGVSEELQNKLEDVVIDRNLLILGKILGEGEFGSVMEGNLKQEDGTSLKVAVKTMKLDNSSQREIEEFLSEAACMKDFSHPNVIRLLGVCIEMSSQGIPKPMVILPFMKYGDLHTYLLYSRLETGPKHIPLQTLLKFMVDIALGMEYLSNRNFLHRDLAARNCMLRDDMTVCVADFGLSKKIYSGDYYRQGRIAKMPVKWIAIESLADRVYTSKSDVWAFGVTMWEIATRGMTPYPGVQNHEMYDYLLHGHRLKQPEDCLDELYEIMYSCWRTDPLDRPTFSVLRLQLEKLLESLPDVRNQADVIYVNTQLLESSEGLAQGSTLAPLDLNIDPDSIIASCTPRAAISVVTAEVHDSKPHEGRYILNGGSEEWEDLTSAPSAAVTAEKNSVLPGERLVRNGVSWSHSSMLPLGSSLPDE LLFADDSSEGSEVLM (SEQ ID NO: 42). A. Exemplary Anti- MerTK Antibodies

In one aspect, the present invention provides and MerTK Conjugated antibody (eg, pegylated antibody). In one aspect, isolated antibodies that bind to MerTK are provided. In one aspect, the invention provides antibodies that specifically bind MerTK. In some ways, anti-MerTK The antibody is pegylated, that is, conjugated to one or more (eg, 1 or 2) PEG polymers.

In one aspect, the invention provides an anti-MerTK antibody comprising at least one, at least two, at least three, at least four, at least five or all six CDRs selected from the group consisting of: (a) comprising SYAMG (SEQ ID NO: CDR-H1 of the amino acid sequence of 1); (b) CDR-H2 of the amino acid sequence of IINSYGNTYYANWAKG (SEQ ID NO: 2); (c) Amine of DPGVSSNL (SEQ ID NO: 3) (d) CDR-L1 comprising the amino acid sequence of QASQNIYSGLA (SEQ ID NO: 4); (e) CDR-L1 comprising the amino acid sequence of GASKLAS (SEQ ID NO: 5) L2; and (f) CDR-L3 comprising the amino acid sequence of QATYYSSNSVA (SEQ ID NO: 6).

In one aspect, the present invention provides an antibody comprising at least one, at least two or all three VH CDR sequences selected from the group consisting of: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1; ( b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3. In one aspect, the antibody comprises CDR-H3 comprising the amino acid sequence of SEQ ID NO:3. In another aspect, the antibody comprises: CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6. In yet another aspect, the antibody comprises: CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3, CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6, and an amino group comprising SEQ ID NO: 2 CDR-H2 of the acid sequence. In yet another aspect, the antibody comprises: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3.

In another aspect, the present invention provides an antibody comprising at least one, at least two or all three VL CDR sequences selected from: (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO:5; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO:6. In one aspect, the antibody comprises (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (c) comprising SEQ ID NO: 5 CDR-L3 of the amino acid sequence of ID NO: 6.

In another aspect, the present invention provides an anti-MerTK antibody comprising at least one, at least two, at least three, at least four, at least five or all six CDRs selected from the group consisting of: (a) comprising ANTMN (SEQ ID NO: 7) CDR-H1 of the amino acid sequence; (b) CDR-H2 comprising the amino acid sequence of IFTATGSTYYATWVNG (SEQ ID NO: 8); (c) CDR-H2 comprising the amino acid sequence of SGSGSSSGAFNI (SEQ ID NO: 9) CDR-H3 of the amino acid sequence; (d) CDR-L1 comprising the amino acid sequence of QASQSISSSLA (SEQ ID NO: 10); (e) CDRs comprising the amino acid sequence of AASILAS (SEQ ID NO: 11) -L2; and (f) CDR-L3 comprising the amino acid sequence of QCTSYGSLFLGP (SEQ ID NO: 12).

In one aspect, the present invention provides an antibody comprising at least one, at least two or all three VH CDR sequences selected from the group consisting of: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 7; ( b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 8; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 9. In one aspect, the antibody comprises CDR-H3 comprising the amino acid sequence of SEQ ID NO:9. In another aspect, the antibody comprises: CDR-H3 comprising the amino acid sequence of SEQ ID NO: 9; and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 12. In yet another aspect, the antibody comprises: CDR-H3 comprising the amino acid sequence of SEQ ID NO: 9, CDR-L3 comprising the amino acid sequence of SEQ ID NO: 12, and an amino group comprising SEQ ID NO: 8 CDR-H2 of the acid sequence. In yet another aspect, the antibody comprises: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 7; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 8; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 9.

In another aspect, the present invention provides an antibody comprising at least one, at least two or all three VL CDR sequences selected from the group consisting of: (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 10; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 11; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 12. In one aspect, the antibody comprises (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 10; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 11; and (c) comprising SEQ ID NO: 11 CDR-L3 of the amino acid sequence of ID NO: 12.

In any of the aspects provided herein, the anti-MerTK antibody is a humanized antibody. In one aspect, the anti-MerTK antibody further comprises a recipient human backbone, eg, a human immunoglobulin backbone or a human common backbone.

In another aspect, the anti-MerTK antibody further comprises a VH or VL comprising the FR1, FR2, FR3 or FR4 sequence of SEQ ID NO: 13 or SEQ ID NO: 14, respectively. EVQLVESGEGLVQPGGSLRLSCAASGFSLSSYAMGWVRQAPGKGLEYVGIINSYGNTYYANWAKGRFTISRDNSKNTVYLQMGSLRAEDMAVYYCARDPGVSSNLWGRGTLVTVSS (SEQ ID NO: 13) DIQMTQSPSTLSASVGDRVTITCQASQNIYSGLAWYQQKPGKAPKLLIYGASKLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQATYYSSNSVAFGGGTKVEIK (SEQ ID NO: 14)

In another aspect, the anti-MerTK antibody comprises one or more CDR sequences of VH of SEQ ID NO: 13. In another embodiment, the anti-MerTK antibody comprises one or more CDR sequences of VL of SEQ ID NO: 14. In another embodiment, the anti-MerTK antibody comprises the CDR sequence of VH of SEQ ID NO: 13 and the CDR sequence of VL of SEQ ID NO: 14.

In yet another aspect, the anti-MerTK antibody comprises the CDR-H1, CDR-H2 and CDR-H3 amino acid sequences of the VH domain of SEQ ID NO:13 and CDR-L1, CDR-L2 and CDR-L2 of the VL domain of SEQ ID NO:14 CDR-L3 amino acid sequence.

In one aspect, the anti-MerTK antibody comprises: one or more heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 13; and a backbone having the same backbone amino acid sequence of the VH domain of SEQ ID NO: 13 At least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity. In one aspect, the anti-MerTK antibody comprises: the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 13; and a backbone having at least 85% of the backbone amino acid sequence of the VH domain of SEQ ID NO: 13 %, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity. In one aspect, the anti-MerTK antibody comprises: the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 13; and a backbone having at least 95% of the backbone amino acid sequence of the VH domain of SEQ ID NO: 13 % sequence identity. In another aspect, the anti-MerTK antibody comprises: the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 13; and a backbone having at least the backbone amino acid sequence of the VH domain of SEQ ID NO: 13 98% sequence identity.

In one aspect, the anti-MerTK antibody comprises: one or more light chain CDR amino acid sequences of the VL domain of SEQ ID NO: 14; and a backbone having the same backbone amino acid sequence of the VL domain of SEQ ID NO: 14 At least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity. In one aspect, the anti-MerTK antibody comprises: the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO: 14; and a backbone having at least 85% of the backbone amino acid sequence of the VL domain of SEQ ID NO: 14 %, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity. In one aspect, the anti-MerTK antibody comprises: the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO: 14; and a backbone having at least 95% of the backbone amino acid sequence of the VL domain of SEQ ID NO: 14 % sequence identity. In another aspect, the anti-MerTK antibody comprises: the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO: 14; and a backbone having at least the backbone amino acid sequence of the VH domain of SEQ ID NO: 13 98% sequence identity.

In one aspect, the anti-MerTK antibody comprises: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; (c) comprising CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (e) CDR comprising the amino acid sequence of SEQ ID NO: 5 -L2; and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6; and a VH domain having at least 90%, 91%, 92%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity; and a VL domain having at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity. In one aspect, the VH domain has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 13. In one aspect, the VL domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 14.

In one aspect, the anti-MerTK antibody comprises: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; (c) comprising CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (e) CDR comprising the amino acid sequence of SEQ ID NO: 5 -L2; and (f) comprising the amino acid sequence CDR-L3 of SEQ ID NO: 6; and a VH domain having at least 90%, 91%, 92%, 93% with the amino acid sequence of SEQ ID NO: 13 %, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity; and a VL domain having at least 90%, 91% with the amino acid sequence of SEQ ID NO: 14 , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity; wherein the antibody specifically binds MerTK. In one aspect, the VH domain has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 13. In one aspect, the VL domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 14.

In another aspect, the anti-MerTK antibody comprises a heavy chain variable domain (VH) sequence that is at least 90%, 91%, 92%, 93%, 94%, 95% identical to the amino acid sequence of SEQ ID NO: 13 %, 96%, 97%, 98%, 99%, or 100% sequence identity. In one aspect, the anti-MerTK antibody comprises a heavy chain variable domain (VH) sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 13. In certain aspects, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity comprises substitutions relative to a reference sequence ( For example, conservative substitutions), insertions or deletions, but anti-MerTK antibodies comprising this sequence retain the ability to bind MerTK. In certain aspects, in SEQ ID NO: 13, a total of 1 to 10 amino acids are substituted, inserted and/or deleted. In certain aspects, substitutions, insertions or deletions occur in regions other than CDRs (ie, in FRs). Optionally, the anti-MerTK antibody comprises the VH sequence in SEQ ID NO: 13, which includes post-translational modifications of this sequence. In a specific aspect, the VH comprises one, two or three CDRs selected from: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (b) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 2 CDR-H2 of the amino acid sequence; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:3. In another aspect, an anti-MerTK antibody is provided, wherein the antibody comprises a light chain variable domain (VL) sequence, the VL sequence having at least 90%, 91%, 92%, 93% with the amino acid sequence of SEQ ID NO: 14 %, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity. In one aspect, the anti-MerTK antibody comprises a light chain variable domain (VL) sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 14. In certain aspects, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity comprises substitutions relative to the reference sequence ( For example, conservative substitutions), insertions or deletions, but anti-MerTK antibodies comprising this sequence retain the ability to bind MerTK. In certain aspects, in SEQ ID NO: 14, a total of 1 to 10 amino acids are substituted, inserted and/or deleted. In certain aspects, substitutions, insertions or deletions occur in regions other than CDRs (ie, in FRs). Optionally, the anti-MerTK antibody comprises the VL sequence in SEQ ID NO: 14, which includes post-translational modifications of this sequence. In a particular aspect, VL comprises one, two or three CDRs selected from: (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (b) comprising one of SEQ ID NO: 5 CDR-L2 of the amino acid sequence; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO:6.

In another aspect, an anti-MerTK antibody is provided, wherein the antibody comprises the VH sequence of any of the aspects provided above and the VL sequence of any of the aspects provided above. In one aspect, the antibody comprises the VH and VL sequences of SEQ ID NO: 13 and SEQ ID NO: 14, respectively, including post-translational modifications of those sequences.

In another aspect, the anti-MerTK antibody further comprises a VH or VL comprising the FR1, FR2, FR3 or FR4 sequence of SEQ ID NO: 15 or SEQ ID NO: 16, respectively. EQQLVESGEGLVQPGGSLRLSCAVSGFSLSSYAMGWVRQAPGKGLEWIGIINSYGNTYYANWAKGRFTISRDSSKNTVYLQMGSLRAEDMAVYFCARDPGVSSNLWGPGTLVTVSS (SEQ ID NO: 15) DVQMTQSPSTLSASVGDRVTITCQASQNIYSGLAWYQQKPGKPPKLLIYGASKLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQATYYSSNSVAFGGGTKVEIK (SEQ ID NO: 16)

In another aspect, the anti-MerTK antibody comprises one or more CDR sequences of VH of SEQ ID NO: 15. In another embodiment, the anti-MerTK antibody comprises one or more CDR sequences of VL of SEQ ID NO: 16. In another embodiment, the anti-MerTK antibody comprises the CDR sequence of VH of SEQ ID NO: 15 and the CDR sequence of VL of SEQ ID NO: 16.

In yet another aspect, the anti-MerTK antibody comprises the CDR-H1, CDR-H2 and CDR-H3 amino acid sequences of the VH domain of SEQ ID NO: 15 and CDR-L1, CDR-L2 and CDR-L2 of the VL domain of SEQ ID NO: 16 CDR-L3 amino acid sequence.

In one aspect, the anti-MerTK antibody comprises: one or more heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 15; and a backbone having the same backbone amino acid sequence of the VH domain of SEQ ID NO: 15 At least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity. In one aspect, the anti-MerTK antibody comprises: the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 15; and a backbone having at least 85% of the backbone amino acid sequence of the VH domain of SEQ ID NO: 15 %, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity. In one aspect, the anti-MerTK antibody comprises: the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 15; and a backbone having at least 95% of the backbone amino acid sequence of the VH domain of SEQ ID NO: 15 % sequence identity. In another aspect, the anti-MerTK antibody comprises: the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 15; and a backbone having at least the backbone amino acid sequence of the VH domain of SEQ ID NO: 15 98% sequence identity.

In one aspect, the anti-MerTK antibody comprises: one or more light chain CDR amino acid sequences of the VL domain of SEQ ID NO: 16; and a backbone having the same backbone amino acid sequence of the VL domain of SEQ ID NO: 16 At least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity. In one aspect, the anti-MerTK antibody comprises: the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO: 16; and a backbone having at least 85% of the backbone amino acid sequence of the VL domain of SEQ ID NO: 16 %, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity. In one aspect, the anti-MerTK antibody comprises: the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO: 16; and a backbone having at least 95% of the backbone amino acid sequence of the VL domain of SEQ ID NO: 16 % sequence identity. In another aspect, the anti-MerTK antibody comprises: the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO: 16; and a backbone having at least the backbone amino acid sequence of the VH domain of SEQ ID NO: 15 98% sequence identity.

In one aspect, the anti-MerTK antibody comprises: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; (c) comprising CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (e) CDR comprising the amino acid sequence of SEQ ID NO: 5 -L2; and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6; and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity; and a VL domain having at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity. In one aspect, the VH domain has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 15. In one aspect, the VL domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 16.

In one aspect, the anti-MerTK antibody comprises: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; (c) comprising CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (e) CDR comprising the amino acid sequence of SEQ ID NO: 5 -L2; and (f) comprising the amino acid sequence CDR-L3 of SEQ ID NO: 6; and a VH domain having at least 90%, 91%, 92%, 93% with the amino acid sequence of SEQ ID NO: 15 %, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity; and a VL domain having at least 90%, 91% with the amino acid sequence of SEQ ID NO: 16 , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity; wherein the antibody specifically binds MerTK. In one aspect, the VH domain has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 15. In one aspect, the VL domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 16.

In another aspect, the anti-MerTK antibody comprises a heavy chain variable domain (VH) sequence that is at least 90%, 91%, 92%, 93%, 94%, 95% identical to the amino acid sequence of SEQ ID NO: 15 %, 96%, 97%, 98%, 99%, or 100% sequence identity. In one aspect, the anti-MerTK antibody comprises a heavy chain variable domain (VH) sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 15. In certain aspects, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity comprises substitutions relative to a reference sequence ( For example, conservative substitutions), insertions or deletions, but anti-MerTK antibodies comprising this sequence retain the ability to bind MerTK. In certain aspects, in SEQ ID NO: 15, a total of 1 to 10 amino acids are substituted, inserted and/or deleted. In certain aspects, substitutions, insertions or deletions occur in regions other than CDRs (ie, in FRs). Optionally, the anti-MerTK antibody comprises the VH sequence in SEQ ID NO: 13, which includes post-translational modifications of this sequence. In a specific aspect, the VH comprises one, two or three CDRs selected from: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (b) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 2 CDR-H2 of the amino acid sequence; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:3. In another aspect, an anti-MerTK antibody is provided, wherein the antibody comprises a light chain variable domain (VL) sequence, the VL sequence having at least 90%, 91%, 92%, 93% with the amino acid sequence of SEQ ID NO: 16 %, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity. In one aspect, the anti-MerTK antibody comprises a light chain variable domain (VL) sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 16. In certain aspects, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity comprises substitutions relative to the reference sequence ( For example, conservative substitutions), insertions or deletions, but anti-MerTK antibodies comprising this sequence retain the ability to bind MerTK. In certain aspects, in SEQ ID NO: 16, a total of 1 to 10 amino acids are substituted, inserted and/or deleted. In certain aspects, substitutions, insertions or deletions occur in regions other than CDRs (ie, in FRs). Optionally, the anti-MerTK antibody comprises the VL sequence in SEQ ID NO: 16, which includes post-translational modifications of this sequence. In a particular aspect, VL comprises one, two or three CDRs selected from: (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (b) comprising one of SEQ ID NO: 5 CDR-L2 of the amino acid sequence; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO:6.

In another aspect, an anti-MerTK antibody is provided, wherein the antibody comprises the VH sequence of any of the aspects provided above and the VL sequence of any of the aspects provided above. In one aspect, the antibody comprises the VH and VL sequences of SEQ ID NO: 15 and SEQ ID NO: 16, respectively, including post-translational modifications of those sequences.

In another aspect, the anti-MerTK antibody comprises one or more CDR sequences of VH of SEQ ID NO: 17. In another embodiment, the anti-MerTK antibody comprises one or more CDR sequences of VL of SEQ ID NO: 18. In another embodiment, the anti-MerTK antibody comprises the CDR sequence of VH of SEQ ID NO: 17 and the CDR sequence of VL of SEQ ID NO: 18. QSVEESGGRLVTPGTPLTLTCTVSGFSLSSYAMGWVRQAPGKGLEWIGIINSYGNTYYANWAKGRFTISRTSTTVDLRMPSLTTEDTATYFCARDPGVSSNLWGPGTLVTVSS (SEQ ID NO: 17) DVVMTQTPASVSEPVGGTVTIKCQASQNIYSGLAWYQQKPGQPPKLLIYGASKLASGVSSRFKGSGSGTEFTLTISDLECADAATYYCQATYYSSNSVAFGGGTEVVVK (SEQ ID NO: 18)

In yet another aspect, the anti-MerTK antibody comprises the CDR-H1, CDR-H2 and CDR-H3 amino acid sequences of the VH domain of SEQ ID NO: 17 and CDR-L1, CDR-L2 and CDR-L2 of the VL domain of SEQ ID NO: 18 CDR-L3 amino acid sequence.

In one aspect, the anti-MerTK antibody comprises one or more heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 17 and one, two, three or four human backbone sequences (eg, HC-FR1, HC-FR2 , HC-FR3 and/or HC-FR4). In one aspect, the anti-MerTK antibody comprises one or more light chain CDR amino acid sequences of the VL domain of SEQ ID NO: 18 and one, two, three or four human backbone sequences (eg, LC-FR1, LC-FR2 , LC-FR3 and/or LC-FR4).

In another aspect, an anti-MerTK antibody is provided, wherein the antibody comprises the VH sequence of any of the aspects provided above and the VL sequence of any of the aspects provided above. In one aspect, the antibody comprises the VH and VL sequences of SEQ ID NO: 17 and SEQ ID NO: 18, respectively, including post-translational modifications of those sequences.

In another aspect, the anti-MerTK antibody comprises one or more CDR sequences of VH of SEQ ID NO: 19. In another embodiment, the anti-MerTK antibody comprises one or more CDR sequences of VL of SEQ ID NO:20. In another embodiment, the anti-MerTK antibody comprises the CDR sequence of VH of SEQ ID NO: 19 and the CDR sequence of VL of SEQ ID NO: 20. QSVEESGGRLVTPGTPLTLTCTVSGIDLSANTMNWVRQAPGKGLEWIGIFTATGSTYYATWVNGRFTISKTSTTVDLKITSPTTEDTATYFCARSGSGSSSGAFNIWGPGTLVTVSL(SEQ ID NO: 19) DPVLTQTPASVSEPVGGTVTIKCQASQSISSSLAWYQQKPGQPPKLLIYAASILASEISSRFKGSRSGTEFTLTISDLECADAATYYCQCTSYGSLFLGPFGGGTEVVVK (SEQ ID NO: 20)

In yet another aspect, the anti-MerTK antibody comprises the CDR-H1, CDR-H2 and CDR-H3 amino acid sequences of the VH domain of SEQ ID NO: 19 and CDR-L1, CDR-L2 and CDR-L2 of the VL domain of SEQ ID NO: 20 CDR-L3 amino acid sequence.

In one aspect, the anti-MerTK antibody comprises one or more heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 19 and one, two, three or four human backbone sequences (eg, HC-FR1, HC-FR2 , HC-FR3 and/or HC-FR4). In one aspect, the anti-MerTK antibody comprises one or more light chain CDR amino acid sequences of the VL domain of SEQ ID NO: 20 and one, two, three or four human backbone sequences (eg, LC-FR1, LC-FR2 , LC-FR3 and/or LC-FR4).

In another aspect, an anti-MerTK antibody is provided, wherein the antibody comprises the VH sequence of any of the aspects provided above and the VL sequence of any of the aspects provided above. In one aspect, the antibody comprises the VH and VL sequences of SEQ ID NO: 19 and SEQ ID NO: 20, respectively, including post-translational modifications of those sequences.

In yet another aspect of the invention, the anti-MerTK antibody according to any of the above aspects is a monoclonal antibody, including a chimeric antibody, a humanized antibody or a human antibody. In one aspect, the anti-MerTK antibody is an antibody fragment, eg, a Fv, Fab, Fab', scFv, diabody or F(ab') ₂ fragment.

In another aspect, the antibody is a full-length antibody, such as an intact IgG1 antibody or other antibody class or isotype as defined herein.

In one aspect, a C-terminal glycine (Gly446) is additionally present. On the one hand, a C-terminal lysine (Lys447) is additionally present. In one aspect, a C-terminal glycine (Gly446) and a C-terminal lysine (Lys447) are additionally present.

In certain embodiments, the antibody comprises at least one mutation in the Fc region that reduces binding to Fc receptors and/or complement. In one embodiment, the antibody comprises a set of mutations in the Fc region called LALAPG (L234A, L235A and P329G, numbered according to the EU index). LALAPG and other Fc mutations are further disclosed below and, for example, in US Pat. No. 8,969,526.

一方面，抗 MerTK 抗體包含重鏈，該重鏈包含序列 EVQLVESGEGLVQPGGSLRLSCAASGFSLSSYAMGWVRQAPGKGLEYVGIINSYGNTYYANWAKGRFTISRDNSKNTVYLQMGSLRAEDMAVYYCARDPGVSSNLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 21)。

In one aspect, the anti-MerTK antibody comprises an Fc region (eg, human IgG1 Fc region), the Fc region contains the N297G mutation, numbered according to the EU index.

一方面，抗 MerTK 抗體包含重鏈，該重鏈包含序列 EVQLVESGEGLVQPGGSLRLSCAASGFSLSSYAMGWVRQAPGKGLEYVGIINSYGNTYYANWAKGRFTISRDNSKNTVYLQMGSLRAEDMAVYYCARDPGVSSNLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:22)。

In one aspect, the anti-MerTK antibody comprises a light chain comprising the sequence DIQMTQSPSTLSASVGDRVTITCQASQNIYSGLAWYQQKPGKAPKLLIYGASKLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQATYYSSNSVAFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGACESVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY:2 SEQSSTLTLSKADYEKHKVYVSSTKSFEKHKVY.

In one aspect, the anti-MerTK antibody comprises a heavy chain comprising the sequence of SEQ ID NO:21 and a light chain, the heavy chain comprising the sequence of SEQ ID NO:23. In one aspect, an anti-MerTK antibody comprises a heavy chain comprising the sequence of SEQ ID NO:22 and a light chain, the heavy chain comprising the sequence of SEQ ID NO:23, and the light chain comprising the sequence of SEQ ID NO:23. B. PEG polymers

Certain aspects of the present disclosure relate to pegylated antibodies that bind to MerTK. any objection to this disclosure MerTK Antibodies (eg, as described above) can be pegylated.

As is known in the art, PEGylation of an antibody refers to the binding (eg, chemical coupling) of a polymer of PEG monomers to the antibody. The structures of PEG monomers are provided below, and PEG polymers are generally represented as (O-CH2-CH2) _n -OCH3, where n represents the number of PEG monomers. Various PEG polymers suitable for use herein are known in the art. See, eg, Jevsevar, S. et al. (2010) Biotechnol. J. 5:113-128.

In some embodiments, the PEG polymer(s) are linear PEG polymers. In some embodiments, the PEG polymer(s) are branched PEG polymers. In some embodiments, the branched PEG polymer(s) comprises 2 or more branches. In some embodiments, the branched PEG polymer(s) comprises 2 branches.

In some embodiments, the pegylated antibody has a hydrodynamic radius greater than about 6 nm. In some embodiments, the pegylated antibody has a hydrodynamic radius of greater than or equal to about 10 nm. In some embodiments, the pegylated antibody has a hydrodynamics between about 9 nm and about 11 nm, between about 6 nm and about 11 nm, or between about 6 nm and about 10 nm radius.

In some embodiments, the anti-MerTK antibodies of the present disclosure are conjugated to one or more PEG polymers. In some embodiments, the anti-MerTK antibodies of the present disclosure are conjugated to one or two PEG polymers.

In some embodiments, the PEG polymer(s) each have a molecular weight between about 10 kDa and about 40 kDa. In some embodiments, the PEG polymer(s) each have a molecular weight between about 20 kDa and about 40 kDa. In some embodiments, the PEG polymer(s) each have a molecular weight between about 10 kDa and about 20 kDa. In some embodiments, the PEG polymer(s) each have a molecular weight of about 10 kDa, about 20 kDa, about 30 kDa, or about 40 kDa.

In some embodiments, the PEG(s) Polymers (eg, one or two PEG polymers) are conjugated to the anti-MerTK antibodies of the present disclosure in the heavy and/or light chains. In some embodiments, the PEG(s) Polymers (eg, one or two PEG polymers) at the engineered cysteine residues of the heavy and/or light chains bind to the anti-MerTK antibodies of the present disclosure. Engineered cysteine residues are disclosed in more detail below.

In some embodiments, the engineered cysteine residues are selected from the group consisting of K149C for light chains, K183C for light chains, T186C for heavy chains, and Y373C for heavy chains (light chains are numbered according to Kabat). , and the numbering of the heavy chain is according to the EU index). For example, in some embodiments, the antibody comprises two heavy chains, two light chains, and two PEG polymers; and both light chains of the antibody comprise K149C engineered to one of the two PEG polymers Cysteine. In some embodiments, the antibody comprises two heavy chains, two light chains, and two PEG polymers; and both light chains of the antibody comprise a K183C engineered half-chain conjugated to one of the two PEG polymers cystine. In some embodiments, the antibody comprises two heavy chains, two light chains, and two PEG polymers; and both heavy chains of the antibody comprise T186C engineered half-chains bound to one of the two PEG polymers cystine. In some embodiments, the antibody comprises two heavy chains, two light chains, and two PEG polymers; and both heavy chains of the antibody comprise a Y373C engineered half-chain bound to one of the two PEG polymers cystine.

Certain aspects of the present disclosure relate to methods of making pegylated antibodies that bind to MerTK. any objection to this disclosure MerTK Antibodies (eg, as described above) can be used in the methods disclosed herein. In some embodiments, one or more PEG polymers are conjugated to the heavy or light chain of an anti-MerTK antibody of the present disclosure via maleimide-cysteine conjugation at the engineered cysteine residues . In some embodiments, one or more PEG polymers are bound to the heavy or light chain of an anti-MerTK antibody of the present disclosure via iodoacetamide-cysteine conjugation at the engineered cysteine residue.

In some embodiments, the methods include combining an antibody comprising one or more free engineered cysteine residues with one or more polyethylene glycol (PEG) comprising a maleimide moiety The polymers are contacted (ie, covalently linked) under conditions suitable for each of the PEG polymer(s) to bind to the engineered cysteine residues of the antibody via thioether linkages. In some embodiments, conjugation is performed at a pH suitable to avoid conjugation with lysine and/or opening of the maleimide ring. In some embodiments, the binding is performed at pH 7 to pH 7.5. Non-limiting reaction conditions are exemplified below. In some embodiments, the binding reaction is monitored, eg, using HPLC and particle size sieve chromatography (SEC) to resolve antibody species with different polymer ratios.

In some embodiments, the methods include combining an antibody comprising one or more free engineered cysteine residues with one or more polyethylene glycol (PEG) comprising an iodoacetamide moiety The polymers are contacted (ie, covalently linked) under conditions suitable for each of the PEG polymer(s) to bind to the engineered cysteine residues of the antibody via thioether linkages.

In some embodiments, the PEG polymer is bound to the antibody at a ratio of 2.0 polymer per antibody. The ratio of PEG:antibody can, for example, be used SEC and HPLC to determine.

In some embodiments, each of the free engineered cysteine residues is blocked with a cysteine or glutathione moiety prior to binding. In some embodiments, the methods further comprise, prior to conjugation, deblocking free engineered cysteine residues. In some embodiments, by pH 8.5 Deblocking of free engineered cysteine residues by down reduction, for example, especially for sites with higher thiol pKa (including, but not limited to, K149C of the light chain). In some embodiments, for sites with higher thiol pKa (including, but not limited to, K149C of the light chain), a more reducing reducing agent is used for removal of free engineered cysteine residues block. In some embodiments, the reducing agent is DTT. Non-limiting reaction conditions are exemplified below. In some embodiments, after deblocking, the antibody is re-oxidized. In some embodiments, after deblocking, the antibody is purified, eg, using cation exchange chromatography to remove reducing agents and reduced cysteine and glutathione.

In some embodiments, the methods further comprise purifying the PEGylated antibody from the unconjugated antibody and the PEG polymer after conjugation. Non-limiting purification methods are exemplified below. For example, in some embodiments, pegylated antibodies can be purified by hydrophobic interaction chromatography (HIC). 1. MerTK biological activity

In some embodiments, the antibody (eg, a pegylated anti-MerTK antibody) reduces MerTK-mediated clearance of apoptotic cells by phagocytes, eg, a 1-10 fold reduction in clearance of apoptotic cells, 1-8 times, 1-5 times, 1-4 times, 1-3 times, 1-2 times, 2-10 times, 2-8 times, 2-5 times, 2-4 times, 2-3 times, 3-10x, 3-8x, 3-5x, 3-4x, or about 1.1x, 1.2x, 1.3x, 1.4x, 1.5x, 1.6x, 1.7x, 1.8x, 1.9x, 2.0 times, 2.1 times, 2.2 times, 2.3 times, 2.4 times, 2.5 times, 2.6 times, 2.7 times, 2.8 times, 2.9 times, 3.0 times, 3.1 times, 3.2 times, 3.3 times, 3.4 times, 3.5 times, 3.6 times , 3.7 times, 3.8 times, 3.9 times, 4.0 times, 4.1 times, 4.2 times, 4.3 times, 4.4 times, 4.5 times, 4.6 times, 4.7 times, 4.8 times, 4.9 times, 5.0 times, 5.1 times, 5.2 times, 5.3 times times, 5.4 times, 5.5 times, 5.6 times, 5.7 times, 5.8 times, 5.9 times, 6.0 times, 6.1 times, 6.2 times, 6.3 times, 6.4 times, 6.5 times, 6.6 times, 6.7 times, 6.8 times, 6.9 times, 7.0x, 7.1x, 7.2x, 7.3x, 7.4x, 7.5x, 7.6x, 7.7x, 7.8x, 7.9x or 8.0x. In some embodiments, the phagocytic cells are macrophages. In some such embodiments, the macrophage is a tumor-associated macrophage (TAM). In humans, TAMs can be identified based on the expression of various cell surface markers, including CD14, HLA-DR (MHC class II), CD312, CD115, CD16, CD163, CD204, CD206, and CD301. In addition, the manufacture of specific functional biomarkers, such as matrix metalloproteinases, IL-10, inducible nitric oxide synthase (iNOS), TNF-α, or IL-12, can be combined with cell surface biomarkers to accurately Identification of TAM populations (Quatromoni, J., et al., Am J Transl Res. 4 (2012): 376-389.) Depletion of apoptotic cells can be performed by any assay known to those skilled in the art for this purpose Measure. For example, for in vitro apoptotic cell clearance assays, phagocytic cells such as mouse peritoneal macrophages or human monocyte-derived macrophages are used. Apoptotic cells were generated by dexamethasone treatment and labeled with detection probes. Phagocytosis can be analyzed by microscopy or flow cytometry after culturing apoptotic cells with phagocytes. In some embodiments, the clearance of apoptotic cells is reduced, as measured in such apoptotic cell clearance assays at room temperature. For example, for in vivo apoptotic clearance assays, mice are injected with dexamethasone to induce thymocyte death. Resident macrophages in the thymus recognize and phagocytose dying/dead cells (Seitz, HM J Immunol. 178(9) 5635-5642 (2007)). In some embodiments, clearance of apoptotic cells is reduced, as measured in such apoptotic cell clearance assays in vivo. In some embodiments, the antibody reduces ligand-mediated MerTK signaling. In some embodiments, the ligand is hGAS6-Fc (EC50=˜84 pM). In some embodiments, the antibody induces a pro-inflammatory response. In some embodiments, the antibody induces a type I IFN response.

In some embodiments, an anti-MerTK antibody of the present disclosure (eg, a pegylated anti- MerTK antibody) reduces the phagocytic activity of apoptotic cells by about 10-100%, 20-100%, 30-100%, 40-100%, 50-100%, 60-100%, 70-100%, 75-100 %, 80-100%, 85-100%, 90-100%, 95-100%, 10-95%, 20-95%, 30-95%, 40-95%, 50-95%, 60-95 %, 70-95%, 75-95%, 80-95%, 85-95%, 90-95%, 10-90%, 20-90%, 30-90%, 40-90%, 50-90 %, 60-90%, 70-90%, 75-90%, 80-90%, 85-90%, 10-85%, 20-85%, 30-85%, 40-85%, 50-85 %, 60-85%, 70-85%, 75-85%, 80-85%, 10-80%, 20-80%, 30-80%, 40-80%, 50-80%, 60-80 %, 70-80%, 75-80%, 10-75%, 20-75%, 30-75%, 40-75%, 50-75%, 60-75%, 70-75%, 10-70 %, 20-70%, 30-70%, 40-70%, 50-70%, 60-70%, 10-65%, 20-65%, 30-65%, 40-65%, 50-65 %, 60-65%, 10-60%, 20-60%, 30-60%, 40-60%, 50-60%, 10-55%, 20-55%, 30-55%, 40-55 %, 50-55%, 10-40%, 20-40%, or 30-40%, or a reduction of at least about 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%. In some embodiments, the half maximal inhibitory concentration (IC50) of the anti-MerTK antibody for reducing the phagocytic activity of apoptotic cells is about 1 pM - 50 pM, 1 pM - 100 pM, 1 pM - 500 pM, 1 pM - 1 nM, 1 pM – 1.5 nM, 5 pM – 50 pM, 5 pM – 100 pM, 5 pM – 500 pM, 5 pM – 1 nM, 5 pM – 1.5 nM, 10 pM – 50 pM, 10 pM – 100 pM , 10 pM – 500 pM, 10 pM – 1 nM, 10 pM – 1.5 nM, 50 pM – 100 pM, 50 pM – 500 pM, 50 pM – 1 nM, 50 pM – 1.5 nM, 100 pM – 500 pM, 100 pM – 1 nM or 100 pM – 1.5 nM. Exemplary methods for determining phagocytic activity and IC50 are disclosed in the Examples below.

In some embodiments, an anti-MerTK antibody of the present disclosure (eg, a pegylated anti-MerTK Antibodies) enhance the activity of checkpoint inhibitors by about 1-2 times, 1-5 times, 1-10 times, 1-15 times, 1-20 times, 1-25 times, 1-30 times, 1-50 times , 1-75 times, 1-100 times, 1-150 times, 1-200 times, 1-250 times, 1.5-2 times, 1.5-5 times, 1.5-10 times, 1.5-15 times, 1.5-20 times , 1.5-25 times, 1.5-30 times, 1.5-50 times, 1.5-75 times, 1.5-100 times, 1.5-150 times, 1.5-200 times, 1.5-250 times, 2-5 times, 2-10 times , 2-15 times, 2-20 times, 2-25 times, 2-30 times, 2-50 times, 2-75 times, 2-100 times, 2-150 times, 2-200 times, 2-250 times , 2.5-5 times, 2.5-10 times, 2.5-15 times, 2.5-20 times, 2.5-25 times, 2.5-30 times, 2.5-50 times, 2.5-75 times, 2.5-100 times, 2.5-150 times , 2.5-200 times, 2.5-250 times, 5-10 times, 5-15 times, 5-20 times, 5-25 times, 5-30 times, 5-50 times, 5-75 times, 5-100 times , 5-150 times, 5-200 times, 5-250 times, 10-15 times, 10-20 times, 10-25 times, 10-30 times, 10-50 times, 10-75 times, 10-100 times , 10-150 times, 10-200 times, 10-250 times, 20-25 times, 20-30 times, 20-50 times, 20-75 times, 20-100 times, 20-150 times, 20-200 times , 20-250 times, 25-30 times, 25-50 times, 25-75 times, 25-100 times, 25-150 times, 25-200 times, or 25-250 times, or enhanced at least about 1 times, 2 times , 5 times, 10 times, 15 times, 20 times, 25 times, 30 times, 40 times, 50 times, 60 times, 70 times, 75 times, 80 times, 90 times, 100 times, 125 times, 150 times, 200 times , 225 times or 250 times. In certain embodiments, an anti-MerTK antibody of the present disclosure enhances the activity of a checkpoint inhibitor, as determined using the assays disclosed in the Examples below, eg, by using a combination of an anti-MerTK antibody and a checkpoint inhibitor in mice The reduction in tumor volume in the tumor model was compared to the reduction in tumor volume with the checkpoint inhibitor alone. In certain embodiments, the reduction in tumor volume is determined after at least 10 days, 14 days, 20 days, 21 days, or 30 days after treatment with the therapeutic agent. In certain embodiments, the checkpoint inhibitor is an anti-PD1 axis antagonist. In an exemplary embodiment, the checkpoint inhibitor is an anti-PD-L1 antibody. In another embodiment, the checkpoint inhibitor is an anti-PD1 antibody.

In some embodiments, an anti-MerTK antibody (eg, a pegylated anti-MerTK antibody) of the present disclosure increases cell-free DNA (cfDNA) and/or circulating tumor DNA (ctDNA) in, eg, a blood or plasma sample by about 1- 2x, 1-3x, 1-4x, 1-5x, 1-10x, 1.5-2x, 1.5-3x, 1.5-4x, 1.5-5x, 1.5-10x, 2- 3 times, 2-4 times, 2-5 times, 2-10 times, 3-5 times, 3-10 times, 4-5 times, 4-10 times, 5-10 times, or at least about 1 times more, 2x, 3x, 4x, 5x or 10x. In certain embodiments, the anti-MerTK antibodies of the present disclosure increase cell-free DNA (cfDNA) and/or circulating tumor DNA (ctDNA), as determined using the assays disclosed in the Examples below, such as by, for example, from blood or plasma samples Isolate cfDNA and/or ctDNA in the medium, and detect the level of cfDNA and/or ctDNA using PCR and quantitative DNA electrophoresis. 2. Antibody Affinity

In certain aspects, the antibodies provided herein have a dissociation constant (K _D ) of ≤ 1 μM, ≤ 100 nM, ≤ 10 nM, ≤ 1 nM, ≤ 0.1 nM, ≤ 0.01 nM, or ≤ 0.001 nM (eg, 10 ⁻⁸ M or less, eg 10 ^-13 M of 10 ^-8 M, eg 10 ^-13 M of 10 ^-9 M).

In one aspect, K _D is measured using the BIACORE ^® surface plasmon resonance assay. For example, assays are performed with immobilized antigen CM5 wafers at ~10 reaction units (RU) using a BIACORE ^® -2000 or BIACORE ^® -3000 (BIAcore, Inc., Piscataway, NJ) at 25°C. In one aspect, N -ethyl- N' -(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N -hydroxysuccinimide were used according to the supplier's instructions Amine (NHS) activated carboxymethylated dextran biosensor chip (CM5, BIACORE, Inc.). Antigen was diluted to 5 μg/ml (approximately 0.2 μM) with 10 mM sodium acetate (pH 4.8) and injected at a flow rate of 5 μl/min to obtain approximately 10 reaction units (RU) of coupled protein. After injection of antigen, 1 M ethanolamine was injected to block unreacted groups. In kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) were injected with 0.05% polysorbate 20 (TWEEN-20 ^™ ) at a flow rate of approximately 25 μl/min at 25°C Surfactant (PBST) in PBS. Association rates ( _kon ) and dissociation rates ( _koff ) were calculated using a simple one-to-one Langmuir binding model ( ^BIACORE® Evaluation Software Version 3.2) by simultaneously fitting the association and dissociation sensorgrams. The equilibrium dissociation constant (K _D ) was calculated from the k _off /k _on ratio. See, eg, Chen et al., J. Mol. Biol. 293:865-881 (1999). If the on-rate measured by the surface plasmon resonance assay described above exceeds 10 ⁶ M ^-1 s ^-1 , the on-rate can be determined using fluorescence quenching techniques, which measure Increase or decrease in fluorescence emission intensity of 20 nM antigen-antibody (Fab format) in PBS (pH 7.2) at 25°C in the presence of increasing concentrations of antigen (excitation = 295 nm; emission = 340 nm, bandpass 16 nm), the fluorescence emission intensity can be measured by a spectrophotometer such as a stopped-flow spectrophotometer (Aviv Instruments) or an 8000 series SLM-AMINCO ^™ spectrophotometer (ThermoSpectronic) with stirring cuvettes.

In another method, _KD is measured by a radiolabeled antigen binding assay (RIA). In one aspect, RIA is performed using a Fab version of the antibody of interest and its antigen. For example, solution binding of Fab to antigen is measured by equilibrating the Fab with a minimal concentration of ( ¹²⁵ I)-labeled antigen in the presence of a titration series of unlabeled antigen, followed by capture of bound antigen with anti-Fab antibody-coated plates Affinity (see, eg, Chen et al., J. Mol. Biol. 293:865-881 (1999)). To determine the conditions of the assay, ^MICROTITER® multi-well plates (Thermo Scientific) were coated overnight with 5 μg/ml capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6) and incubated at room temperature (approximately 23 Block with 2% (w/v) bovine serum albumin in PBS for two to five hours at °C). In non-adsorbent plates (Nunc #269620), 100 pM or 26 pM [ ¹²⁵ I]-antigen was mixed with serial dilutions of the Fab of interest (eg, as in Presta et al . Cancer Res. 57:4593-4599 (1997) The evaluation results of the anti-VEGF antibody Fab-12 described in ). The target Fab is then incubated overnight; however, incubation can be continued for longer (eg, about 65 hours) to ensure equilibrium is reached. Thereafter, the mixture is transferred to a capture plate and incubated at room temperature (eg, for one hour). The solution was then removed and the plate was washed eight times with 0.1% polysorbate 20 (TWEEN- ^20® ) in PBS. When the plates were dry, scintillation reagent (MICROSCINT-20 ^™ ; Packard) was added at 150 μl/well and counted for ten minutes using a TOPCOUNT ^™ gamma counter (Packard). The concentration of each Fab that provided less than or equal to 20% of maximal binding was selected for use in the competitive binding assay. 3. Antibody Fragments

In certain aspects, the antibodies provided herein are antibody fragments.

In one aspect, the antibody fragment is a Fab, Fab', Fab'-SH or F(ab') ₂ fragment, particularly a Fab fragment. Papain digestion of an intact antibody yields two identical antigen-binding fragments, termed "Fab" fragments, each comprising the variable domains of the heavy and light chains (VH and VL, respectively) and the constant domain (CL) of the light chain and the first constant domain (CH1) of the heavy chain. Thus, the term "Fab fragment" refers to an antibody fragment comprising a light chain (comprising VL and CL domains) and a heavy chain fragment (comprising VH and CH1 domains). "Fab'fragments" are distinguished from Fab fragments by the addition of residues to the carboxy-terminus of the CH1 domain, which include one or more cysteines from the antibody hinge region. Fab'-SH is a Fab' fragment in which the cysteine residue of the constant domain bears a free thiol group. Pepsin treatment produces an F(ab') ₂ fragment with two antigen binding sites (two Fab fragments) and a portion of the Fc region. See U.S. Patent No. 5,869,046 for a discussion of Fab and F(ab') ₂ fragments comprising salvage receptor binding epitope residues and having increased in vivo half-life.

In another embodiment, the antibody fragment is a diabody, triabody, or tetrabody. Diabodies are antibody fragments that have two antigen-binding sites, which may be bivalent or bispecific. See, e.g., EP 404,097; WO 1993/01161; Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993). Tri- and tetra-antibodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

In yet another aspect, the antibody fragment is a single chain Fab fragment. "Single-chain Fab fragment" or "scFab" is composed of antibody heavy chain variable domain (VH), antibody heavy chain constant domain 1 (CH1), antibody light chain variable domain (VL), antibody light chain constant domain (CL) and a polypeptide consisting of a linker, wherein the antibody domain and the linker have one of the following sequences in the N-terminal to C-terminal direction: a) VH-CH1-linker-VL-CL, b) VL-CL-linker- VH-CH1, c) VH-CL-Linker-VL-CH1 or d) VL-CH1-Linker-VH-CL. In particular, the linker is a polypeptide consisting of at least 30 amino acids and preferably 32 to 50 amino acids. This single-chain Fab fragment is stabilized by natural disulfide bonds between the CL and CH1 domains. In addition, these single chain Fab fragments can be further stabilized by insertion of cysteine residues to create interchain disulfide bonds (eg, insertion at position 44 of the variant heavy chain and position 100 of the variant light chain according to Kabat numbering) .

In another aspect, the antibody fragment is a single chain variable fragment (scFv). A "single-chain variant fragment" or "scFv" is a fusion protein of the variable domains of the heavy chain (VH) and light chain (VL) of an antibody, linked by a linker. In particular, linkers are short polypeptides of 10 to 25 amino acids, and are often rich in glycine for flexibility, serine or threonine for solubility, and can be The N-terminus of VH is connected to the C-terminus of VL, or vice versa. Despite the removal of the constant region and the introduction of a linker, the protein retains the specificity of the original antibody. For a review of scFv fragments see eg Plückthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, eds. Rosenburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994); see also WO 93/16185 ; and US Patent Nos. 5,571,894 and 5,587,458.

In another aspect, the antibody fragment is a single domain antibody. A single domain antibody is an antibody fragment comprising all or a portion of the heavy chain variable domain of an antibody or all or a portion of the light chain variable domain of an antibody. In certain aspects, the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516 B1).

Antibody fragments can be produced by various techniques including, but not limited to, proteolytic digestion of intact antibodies as described herein and recombinant production in recombinant host cells (eg, E. coli). 4. Chimeric and Humanized Antibodies

In certain aspects, the antibodies provided herein are chimeric antibodies. Certain chimeric antibodies are described, for example, in US Pat. No. 4,816,567; and Morrison et al ., Proc. Natl. Acad. Sci. USA , 81:6851-6855 (1984). In one example, a chimeric antibody comprises non-human variable regions (eg, variable regions derived from mouse, rat, hamster, rabbit, or non-human primates such as monkeys) and human constant regions. In yet another example, a chimeric antibody is a "class-switched" antibody, wherein the class or subclass has been changed compared to its parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

In certain aspects, the chimeric antibody is a humanized antibody. Typically, non-human antibodies are humanized antibodies to reduce immunogenicity to humans while retaining the specificity and affinity of the parental non-human antibody. Typically, a humanized antibody comprises one or more variable domains, wherein the CDRs (or portions thereof) are derived from non-human antibodies, and the FRs (or portions thereof) are derived from human antibody sequences. A humanized antibody will optionally contain at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (eg, an antibody from which the CDR residues are derived), eg, to restore or improve antibody specificity or affinity.

Humanized antibodies and methods for their preparation are reviewed, for example, in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further described in, for example: Riechmann et al ., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); US Pat. Nos. 5,821,337, 7,527,791, 6,982,321 and 7,087,409; Kashmiri et al ., Methods 36:25-34 (2005) (specifically Describes Determining Region (SDR) Grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (Describes "Surface Remodeling");Dall'Acqua et al, Methods 36:43-60 (2005) (describes "FR shuffling"); Osbourn et al., Methods 36:61-68 (2005); and Klimka et al, Br. J. Cancer , 83:252-260 (2000) (describes "guidelines for FR shuffling"option" method).

Human antibody framework regions that can be used for humanization include, but are not limited to: framework regions selected using a "best match" approach (see, eg, Sims et al . J. Immunol. 151:2296 (1993)); derived from light chains or Framework regions of common sequences of human antibodies of a particular subgroup of heavy chain variable regions (see, e.g., Carter et al . Proc. Natl. Acad. Sci. USA , 89:4285 (1992); and Presta et al . J. Immunol. , 151:2623 (1993)); human mature (somatic mutation) framework regions or human germline framework regions (see, eg, Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and derived from screening Framework regions of FR libraries (see eg: Baca et al, J. Biol. Chem. 272:10678-10684 (1997); and Rosok et al, J. Biol. Chem. 271:22611-22618 (1996)). 5. Multispecific Antibodies

In certain embodiments, the antibodies provided herein are multispecific antibodies, eg, bispecific antibodies. Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites (ie, different epitopes on different antigens or different epitopes on the same antigen). In certain embodiments, the multispecific antibody has three or more binding specificities. In certain aspects, one of the binding specificities is for MerTK and the other specificities are for any other antigen. In certain aspects, bispecific antibodies can bind to two (or more) different epitopes of MerTK. Multispecific (eg, bispecific) antibodies can also be used to localize cytotoxic agents or cells to cells expressing MerTK. Multispecific antibodies can be prepared as full-length antibodies or antibody fragments.

Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein and Cuello, Nature 305: 537 (1983)) and "knob and hole" (knob-in-hole) engineering (see, eg, US Pat. No. 5,731,168, and Atwell et al. J. Mol. Biol. 270:26 (1997)). Multispecific antibodies can also be made by engineering electrostatic steering effects for making antibody Fc-heterodimeric molecules (see eg WO 2009/089004); cross-linking two or more antibodies or fragments (see For example, US Patent No. 4,676,980; and Brennan et al., Science , 229: 81 (1985)); use of leucine zippers to generate bispecific antibodies (see, eg, Kostelny et al., J. Immunol. , 148(5): 1547 -1553 (1992); and WO 2011/034605); use common light chain technology to circumvent light chain mismatch problems (see eg WO 98/50431); use "diabody" technology to prepare bispecific antibody fragments (see eg Hollinger et al ., Proc. Natl. Acad. Sci. USA , 90:6444-6448 (1993)); and the use of single-chain Fv (sFv) dimers (see, eg, Gruber et al. , J. Immunol. , 152:5368 ( 1994)); and trispecific antibodies were prepared as described, for example, by Tutt et al . J. Immunol. 147: 60 (1991).

Also included herein are engineered antibodies with three or more antigen binding sites, including, for example, "Octopus antibodies" or DVD-Ig (see, eg, WO 2001/77342 and WO 2008/024715). Further examples of multispecific antibodies with three or more antigen binding sites can be found in WO 2010/115589, WO 2010/112193, WO 2010/136172, WO 2010/145792 and WO 2013/026831. Bispecific antibodies or antigen-binding fragments thereof also include "dual-acting FAbs" or "DAFs" that comprise binding to MerTK and another different antigen or to the antigen-binding site of two different epitopes of MerTK (see e.g. US 2008/0069820 and WO 2015/095539).

Multispecific antibodies can also be provided in asymmetric formats comprising domains that intersect in one or more binding arms with the same antigen specificity, i.e. by exchanging VH/VL domains (see eg WO 2009/080252 and WO 2015/ 150447), CH1/CL domains (see eg WO 2009/080253) or complete Fab arms (see eg WO 2009/080251, WO 2016/016299, see also Schaefer et al, PNAS, 108 (2011) 1187-1191, and Klein et al, MAbs 8 (2016) 1010-20) implementation. In one aspect, the multispecific antibody comprises a cross-Fab fragment. The term "cross-Fab fragment" or "xFab fragment" or "cross-Fab fragment" refers to a Fab fragment in which the variable or constant regions of the heavy and light chains are exchanged. A cross-Fab fragment contains a polypeptide chain consisting of a light chain variable region (VL) and a heavy chain constant region 1 (CH1) and a polypeptide chain consisting of a heavy chain variable region (VH) and a light chain constant region (CL). Asymmetric Fab arms can also be designed by introducing mutations of charged or uncharged amino acids into the domain interface to direct correct Fab pairing. See eg WO 2016/172485.

Various other molecular formats for multispecific antibodies are known in the art and are included herein (see eg, Spiess et al., Mol Immunol 67 (2015) 95-106). 6. Antibody Variants

In certain aspects, amino acid sequence variants of the antibodies provided herein are contemplated. For example, it may be desirable to alter the binding affinity and/or other biological properties of the antibody. Amino acid sequence variants of the antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions and/or insertions and/or substitutions of residues in the amino acid sequence of the antibody. Any combination of deletions, insertions and substitutions can be performed to obtain the final construct, provided that the final construct has the desired characteristics, eg, antigen binding characteristics. a) Substitution, insertion and deletion variants

In certain aspects, antibody variants with one or more amino acid substitutions are provided. Targeted sites for substitutional mutagenesis include CDRs and FRs. Conservative substitutions are listed in Table 1 under the heading "Preferred Substitutions". More substantial changes are provided in Table 1 under the heading "Exemplary Substitutions" and are further described below with reference to amino acid side chain classes. Amino acid substitutions can be introduced into the antibody of interest and the products screened for the desired activity, eg, retained/improved antigen binding characteristics, reduced immunogenicity, or improved ADCC or CDC. Table 1 original residue Exemplary substitution better replacement Ala (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His; Asp; Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn;Glu Asn Glu (E) Asp;Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val; Met; Ala; Phe; Leu Leu (L) norleucine; Ile; Val; Met; Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr; Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Leu

Amino acids can be grouped according to common side chain characteristics: (1) Hydrophobicity: n-leucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Alkaline: His, Lys, Arg; (5) Residues affecting chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe.

Non-conservative substitutions require exchanging members of one of these classes for members of the other class.

One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (eg, a humanized or human antibody). Typically, the resulting variant selected for further study will have a modification (eg, improve) relative to the parent antibody in certain biological properties (eg, increase affinity, decrease immunogenicity) and/or substantially retain the parent antibody certain biological properties. Exemplary substitutional variants are affinity matured antibodies, which can be conveniently produced, eg, using phage display-based affinity maturation techniques, such as those described herein. In short, replace one or more. CDR residues are mutated, and variant antibodies are displayed on phage and screened for specific biological activities (eg, binding affinity).

Changes (eg, substitutions) can be made in the CDRs to improve antibody affinity. Such modifications can be made in CDR "hot spots," ie, residues encoded by codons that undergo high frequency mutation during somatic maturation (see, eg, Chowdhury, Methods Mol. Biol. 207:179-196 (2008)) and and/or residues in contact with the antigen and the resulting variant VH or VL tested for binding affinity. Affinity maturation is achieved by construction and reselection from secondary libraries, such as Hoogenboom et al. in Methods in Molecular Biology 178: 1-37 (O'Brien et al. ed., Human Press, Totowa, NJ, (2001)) described. In certain aspects of affinity maturation, diversity is introduced into variant genes selected for maturation by various methods (eg, error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). Then create the second library. The library is then screened to identify any antibody variants with the desired affinity. Another approach to introducing diversity is a CDR-directed approach, in which several CDR residues (eg, 4-6 residues at a time) are randomized. CDR residues involved in antigen binding can be specifically identified by, eg, alanine scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are frequently targeted.

In certain aspects, substitutions, insertions or deletions may occur within one or more of the CDRs, so long as such modifications do not significantly reduce the ability of the antibody to bind antigen. For example, conservative modifications (eg, conservative substitutions provided herein) can be implemented in the CDRs that do not substantially reduce binding affinity. For example, such modifications may be outside of antigen-contacting residues in the CDRs. In certain VH and VL sequence variants provided above, each CDR is unchanged, or contains no more than one, two or three amino acid substitutions.

A useful method for identifying potentially mutagenizable antibody residues or regions is called "alanine scanning mutagenesis" as described by Cunningham and Wells (1989) ( Science , 244:1081-1085). In this method, residues or groups of target residues (eg, charged residues such as arg, asp, his, lys, and glu) are identified, and neutral or negatively charged amino acids (eg, alanine or polyalanine) substitution to determine whether antibody-antigen interactions are affected. More substitutions can be introduced at amino acid positions, indicating good functional sensitivity to the initial substitution. Alternatively or additionally, the crystal structure of the antigen-antibody complex can be used to identify contact points between the antibody and the antigen. Such contact residues and adjacent residues can be targeted or eliminated as candidates for substitution. Variants can be screened to determine whether they contain desired properties.

Amino acid sequence insertions include the length of amino and/or carboxy-terminal fusions, from one residue to polypeptides comprising a hundred or more residues, and intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionine residue. Other insertional variants of antibody molecules include enzymes fused to the N-terminus or C-terminus of the antibody (eg, for ADEPT (Enzyme Prodrug Therapy for Antibodies)) or polypeptides that increase the serum half-life of the antibody. b) Glycosylation variants

In certain embodiments, the antibodies provided herein are altered to increase or decrease the degree to which the antibody is glycosylated. The addition or deletion of glycosylation sites in an antibody is conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites are created or removed.

When the antibody contains an Fc region, the oligosaccharide to which it is attached can be altered. Natural antibodies produced by mammalian cells typically contain branched biantennary oligosaccharides, usually N-linked to Asn297 of the CH2 domain of the Fc region. See, eg, Wright et al., TIBTECH 15:26-32 (1997). Oligosaccharides can include various carbohydrates such as mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid as well as fucose attached to GlcNAc in the "stem" of the biantennary oligosaccharide structure . In some embodiments, the oligosaccharides in the antibodies of the invention can be modified to generate antibody variants with certain improved properties.

In one aspect, antibody variants are provided that have afucosylated oligosaccharides, i.e., oligosaccharide structures lacking (directly or indirectly) fucose linked to the Fc region. These afucosylated oligosaccharides (also known as "defucosylated" oligosaccharides) specifically lack a fucose residue linked to the first GlcNAc in the stem of the biantennary oligosaccharide structure of N-linked oligosaccharides. In one aspect, antibody variants are provided that have an increased proportion of afucosylated oligosaccharides in the Fc region as compared to the native or parent antibody. For example, the proportion of afucosylated oligosaccharides can be at least about 20%, at least about 40%, at least about 60%, at least about 80%, or even about 100% (ie, no fucosylated oligosaccharides are present) . The percentage of afucosylated oligosaccharides is the sum (average) amount of oligosaccharides lacking fucose residues relative to the sum of all oligosaccharides attached to Asn 297 (e.g. complex, hybrid and high mannose structures) , this percentage is determined by MALDI-TOF mass spectrometry, eg as described in WO 2006/082515. Asn297 refers to the asparagine residue located near position 297 in the Fc region (EU numbering of Fc region residues); however, Asn297 may also be located approximately ±3 amino acids upstream or downstream of position 297, i.e. due to the Minor sequence variation between positions 294 and 300. Such antibodies with increased proportions of afucosylated oligosaccharides in the Fc region may have improved FcγRIIIa receptor binding and/or improved effector function, in particular improved ADCC function. See eg US 2003/0157108; US 2004/0093621.

Examples of cell lines capable of producing antibodies with reduced fucosylation include Lec13 CHO cells lacking protein fucosylation (Ripka et al., Arch. Biochem. Biophys. 249:533-545 (1986); US 2003 /0157108; and WO 2004/056312, especially in Example 11); and knockout cell lines, such as CHO cells knockout the alpha-1,6-fucosyltransferase gene FUT8 (see, eg, Yamane-Ohnuki et al. Human Biotech. Bioeng. 87:614-622 (2004); Kanda, Y. et al ., Biotechnol. Bioeng ., 94(4):680-688 (2006); and WO 2003/085107); or GDP-fucoid Cells with reduced or absent carbohydrate synthesis or transporter activity (see eg US2004259150, US2005031613, US2004132140, US2004110282).

In another embodiment, the antibody variant is provided with bisected oligosaccharides, eg, wherein the biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function as described above. Examples of such antibody variants are described, for example, in: Umana et al., Nat Biotechnol 17, 176-180 (1999); Ferrara et al., Biotechn Bioeng 93, 851-861 (2006); WO 99/54342; WO 2004/065540 , WO 2003/011878.

Antibody variants having at least one galactose residue on the oligosaccharide linked to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997/30087, WO 1998/58964 and WO 1999/22764. c) Fc region variants

In certain aspects, one or more amino acid modifications can be introduced into the Fc region of the antibodies provided herein, resulting in Fc region variants. Fc region variants may comprise human Fc region sequences (eg, human IgGl, IgG2, IgG3, or IgG4 Fc regions) that contain amino acid modifications (eg, substitutions) at one or more amino acid positions.

In certain aspects, the present invention contemplates an antibody variant having some, but not all, effector functions, making it a desirable candidate antibody for applications in which antibody in vivo half-life is important, but some effector functions (eg complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC)) are unnecessary or detrimental. In vitro and/or in vivo cytotoxicity assays can be performed to confirm the reduction/depletion of CDC and/or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcR binding (and thus likely lacks ADCC activity), but retains FcRn binding ability. Primary cells that mediate ADCC, NK cells express Fc RIII only, while monocytes express Fc RI, Fc RII and Fc RIII. The expression of FcRs on hematopoietic cells is summarized in Table 3 on page 464 of the paper by Ravetch and Kinet ( Annu. Rev. Immunol. 9:457-492 (1991)). Non-limiting examples of in vitro assays for assessing ADCC activity of target molecules are described in U.S. Patent No. 5,500,362 (see, e.g., Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al, Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (see Bruggemann, M. et al, J. Exp. Med. 166:1351- 1361 (1987)). Alternatively, non-radioactive assays can be employed (see eg: ACTI ^™ Non-Radioactive Cytotoxicity Assay for Flow Cytometry (CellTechnology, Inc. Mountain View, CA); and CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega , Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, ADCC activity of target molecules can be assessed in vivo in animal models such as those disclosed by Clynes et al. in Proc. Natl Acad. Sci. USA 95:652-656 (1998). A C1q binding assay can also be performed to confirm that the antibody is unable to bind C1q and thus lacks CDC activity. See eg WO 2006/029879 and WO 2005/100402 for C1q and C3c binding ELISAs. To assess complement activation, a CDC assay can be performed (see eg: Gazzano-Santoro et al ., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance/half-life assays can also be performed using methods well known in the art (see eg Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006); WO 2013/ 120929).

Antibodies with reduced effector function include those in which one or more of the Fc region residues 238, 265, 269, 270, 297, 327, and 329 are substituted (US Pat. No. 6,737,056). Such Fc variants include Fc variants with two or more substitutions in amino acid positions 265, 269, 270, 297 and 327, including the so-called "DANA" Fc variant, in which residues 265 and 297 Substituted with alanine (US Patent No. 7,332,581).

Certain antibody variants with improved or reduced binding to FcRs are described therein. (See eg, US Patent No. 6,737,056; WO 2004/056312 and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).)

In certain aspects, the antibody variant comprises an Fc region with one or more amino acid substitutions that improve ADCC, such as substitutions at positions 298, 333 and/or 334 (EU numbering of residues) of the Fc region .

In certain aspects, the antibody variant comprises an Fc region with one or more amino acid substitutions that attenuate FcγR binding, such as substitutions at positions 234 and 235 (EU numbering of residues) of the Fc region. In one aspect, the substitutions are L234A and L235A (LALA). In certain aspects, the antibody variant further comprises D265A and/or P329G in the Fc region, which are derived from a human IgGl Fc region. In one aspect, the substitutions are L234A, L235A and P329G (LALA-PG) in the Fc region, which is derived from the human IgG1 Fc region. See eg WO 2012/130831. On the other hand, the substitutions were L234A, L235A and D265A (LALA-DA) in the Fc region, which was derived from the human IgG1 Fc region.

In certain aspects, modifications in the Fc region result in modified (ie, improved or reduced) C1q binding and/or complement-dependent cytotoxicity (CDC), eg, US Pat. No. 6,194,551, WO 99/51642, and Idusogie et al. J . Immunol. 164: 4178-4184 (2000).

Has longer half-life and improved interaction with the neonatal Fc receptor (FcRn) responsible for the transfer of maternal IgG to the fetus, see Guyer et al J. Immunol. 117:587 (1976) and Kim et al J. Immunol. 24: 249 (1994)) are described in US2005/0014934 (Hinton et al.). Those antibodies comprise an Fc region with one or more substitutions therein that improve binding of the Fc region to FcRn. Such Fc variants include Fc variants with substitutions at one or more Fc region residues: 238, 252, 254, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340 , 356, 360, 362, 376, 378, 380, 382, 413, 424, or 434, eg, substitution of Fc region residue 434 (see eg, US Pat. No. 7,371,826; Dall'Acqua, WF et al., J. Biol. Chem . 281 (2006) 23514-23524).

Fc region residues critical for mouse Fc-mouse FcRn interaction have been identified by site-directed mutagenesis (see, e.g., Dall'Acqua, W.F. et al. J. Immunol 169 (2002) 5171-5180). Residues I253, H310, H433, N434 and H435 (EU numbering of residues) are involved in the interaction (Medesan, C. et al., Eur. J. Immunol. 26 (1996) 2533; Firan, M. et al., Int. Immunol. 13 (2001) 993; Kim, J.K. et al., Eur. J. Immunol. 24 (1994) 542). Residues 1253, H310 and H435 have been found to be critical for the interaction of human Fc with mouse FcRn (Kim, J.K. et al., Eur. J. Immunol. 29 (1999) 2819). Studies of the human Fc-human FcRn complex indicate that residues I253, S254, H435 and Y436 are critical for interaction (Firan, M. et al., Int. Immunol. 13 (2001) 993; Shields, R.L. et al. , J. Biol. Chem. 276 (2001) 6591-6604). In Yeung, Y.A. et al. (J. Immunol. 182 (2009) 7667-7671), various mutants of residues 248 to 259 and 301 to 317 and 376 to 382 and 424 to 437 have been reported and studied.

In certain aspects, the antibody variant comprises an Fc region with one or more amino acid substitutions that reduce FcRn binding, such as positions 253, and/or 310, and/or 435 (EU numbering of residues in the Fc region) ) is replaced. In certain aspects, the antibody variant comprises an Fc region with amino acid substitutions at positions 253, 310, and 435. In one aspect, the substitutions are I253A, H310A and H435A in the Fc region, which are derived from the human IgGl Fc region. See, eg, Grevys, A et al, J. Immunol. 194 (2015) 5497-5508.

In certain aspects, the antibody variant comprises an Fc region with one or more amino acid substitutions that reduce FcRn binding, such as positions 310, and/or 433, and/or 436 (EU numbering of residues in the Fc region) ) is replaced. In certain aspects, the antibody variant comprises an Fc region with amino acid substitutions at positions 310, 433, and 436. In one aspect, the substitutions are H310A, H433A and Y436A in the Fc region, which are derived from the human IgGl Fc region. (See e.g. WO 2014/177460 Al.)

In certain aspects, the antibody variant comprises an Fc region with one or more amino acid substitutions that increase FcRn binding, such as positions 252, and/or 254, and/or 256 (EU numbering of residues in the Fc region) ) is replaced. In certain aspects, the antibody variant comprises an Fc region with amino acid substitutions at positions 252, 254, and 256. In one aspect, the substitutions are M252Y, S254T and T256E in the Fc region, which are derived from the human IgGl Fc region. See also Duncan & Winter, Nature 322:738-40 (1988); US Patent No. 5,648,260; US Patent No. 5,624,821; and WO 94/29351 for additional examples of Fc region variants.

The C-terminus of the heavy chain may be a shortened C-terminus in which one or both of the C-terminal amino acid residues have been removed. In a preferred aspect, the C-terminus of the heavy chain is a shortened C-terminal terminated PG. In one of all aspects reported herein, an antibody comprising a heavy chain comprises a C-terminal CH3 domain as specified herein comprising a C-terminal glycine-lysine dipeptide (G446 and K447, amino acid positions the EU index number). In one of all aspects reported herein, an antibody comprising a heavy chain comprises a C-terminal CH3 domain as specified herein comprising a C-terminal glycine residue (G446, EU index numbering of amino acid positions). d) Cysteine engineered antibody variants

In certain aspects, it may be desirable to create cysteine-engineered antibodies, such as THIOMAB ^™ antibodies, in which one or more residues of the antibody are replaced with cysteine residues. In certain embodiments, the substituted residues occur at sites accessible to the antibody. By substituting cysteine for those residues, reactive thiol groups are thereby positioned at accessible sites for the antibody and can be used to bind the antibody to other moieties such as one or more PEG polymers, drug moieties or linker-drug moieties) to form immunoconjugates or pegylated antibodies, as further disclosed herein. Cysteine engineered antibodies can be made as disclosed in, eg, US Pat. Nos. 7,521,541, 8,30,930, 7,855,275, 9,000,130, WO 2016040856, or WO 2011/156328. e) Antibody Derivatives

In certain embodiments, the antibodies provided herein can be further modified to include additional non-proteinaceous moieties known in the art and readily available. Moieties suitable for derivatization of antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, Poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly (n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (eg, glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymers can be of any molecular weight, and can be branched or unbranched. The number of polymers attached to the antibody can vary, and if more than one polymer is attached, they can be the same or different molecules. In general, the amount and/or type of polymer used for derivatization can be determined based on considerations including, but not limited to, the particular property or function of the antibody to be improved, whether the antibody derivative will be used in a given condition The treatment below is moderate. 7. Immunoconjugates

The present invention also provides immunoconjugates comprising an anti-MerTK antibody herein that bind (chemically bond) to one or more therapeutic agents such as cytotoxic agents, chemotherapeutic agents, drugs, growth inhibitors, toxins (eg, derived from bacteria, fungi, plant or animal protein toxins, enzymatically active toxins or fragments thereof) or radioisotopes.

In one aspect, the immunoconjugate is an antibody-drug conjugate (ADC), wherein the antibody is conjugated to one or more of the therapeutic agents described above. Linkers are typically used to link antibodies to one or more therapeutic agents. An overview of ADC technology, including examples of therapeutics, drugs, and linkers, is presented in Pharmacol Review 68:3-19 (2016).

In another embodiment, the immune complex comprises an antibody described herein conjugated to an enzymatically active toxin or fragment thereof including, but not limited to, diphtheria A chain, a non-binding active fragment of diphtheria toxin, Exotoxin A chain (derived from Pseudomonas aeruginosa), Ricin A chain, Acacia toxin A chain, Modine A chain, α-sarcinus, oleoresin, carnation toxin, American business Terrestrial proteins (PAPI, PAPII and PAP-S), bitter melon inhibitor, curcumin, crotontoxin, saponin inhibitor, gelonin, mitoxanthin, aspergillus constrictor, phenomycin, inoxomycin and Trichothecenes toxins.

In another embodiment, the immune complex comprises an antibody described herein conjugated to a radioactive atom to form a radioactive complex. A variety of radioisotopes are available for the production of radioconjugates. Examples include radioisotopes of At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² , Pb ²¹² , and Lu. When a radioconjugate is used for detection, it may contain radioactive atoms such as tc99m or I123 for scintigraphic studies, or spin for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, mri) Labels, for example, iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

Conjugates of antibody and cytotoxic agent can be prepared using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithio)propionate ( SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminosulfane (IT), iminoester bifunctional derivatives of (eg, dimethyl adipate hydrochloride, HCl), active esters (eg, disuccinimidyl suberic acid), aldehydes (eg, glutaraldehyde), bisazides ( For example, bis(p-azidobenzyl)hexamethylenediamine), double nitrogen derivatives (e.g., bis-(p-diazobenzyl)-ethylenediamine), diisocyanates (e.g., toluene 2, 6-diisocyanate) and dual reactive fluorine compounds (eg, 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxin can be prepared as described in Vitetta et al., Science 238:1098 (1987). An exemplary chelating agent for binding radionucleotides to antibodies is carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA). See WO 94/11026. The linker may be a "cleavable linker" that facilitates the release of the cytotoxic drug in the cell. For example, acid-labile linkers, peptidase-sensitive linkers, photolabile linkers, dimethyl linkers, or disulfide-containing linkers can be used (Chari et al., Cancer Res. 52:127 -131 (1992); US Patent No. 5,208,020).

Immunoconjugates or ADCs herein specifically contemplate, but are not limited to, such conjugates made with cross-linking agents including, but not limited to, commercially available (eg, from Pierce Biotechnology, Inc. (Rockford, IL., USA) commercially available) of BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, Sulfo-EMCS, Sulfo-GMBS, Sulfo-KMUS, Sulfo-MBS, Sulfo-SIAB, Sulfo-SMCC and Sulfo-SMPB and SVSB (succinimidyl-(4-vinyl sulfo)benzoate). C. Reconstitution Methods and Compositions

Antibodies can be produced using recombinant methods and compositions, eg, as described in US 4,816,567. For these methods, one or more isolated nucleic acids encoding antibodies are provided.

In the case of a native antibody or native antibody fragment, two nucleic acids are required, one for the light chain or fragment thereof and the other for the heavy chain or fragment thereof. These nucleic acids encode amino acid sequences comprising VL and/or amino acid sequences comprising the VH of an antibody (eg, the light and/or heavy chains of an antibody). These nucleic acids can be on the same expression vector or on different expression vectors.

In the case of a bispecific antibody with a heterodimeric heavy chain, four nucleic acids are required, one for the first light chain, one for the first heavy chain (which contains the first heteromeric Fc region polypeptide), one for the second light chain and one for the second heavy chain (which comprises a second heterologous monomeric Fc region polypeptide). These four nucleic acids can be contained in one or more nucleic acid molecules or expression vectors. These nucleic acids encode the amino acid sequence comprising the first VL, and/or the amino acid sequence comprising the first VH (which comprises the first heteromeric Fc region), and/or the amino acid sequence comprising the second VL sequence, and/or amino acid sequence (eg, the first and/or second light chain of the antibody, and/or the first and/or or second heavy chain). These nucleic acids can be on the same expression vector or on different expression vectors, usually these nucleic acids are located on two or three expression vectors, that is, one vector may contain more than one of these nucleic acids. Examples of these bispecific antibodies are CrossMabs (see, eg, Schaefer, W. et al., PNAS, 108 (2011) 11187-1191). For example, one of the heterologous monomer heavy chains contains a so-called "knob mutation" (T366W, one of S354C or Y349C as appropriate), and the other contains a so-called "hole mutation" (T366S, L368A and Y407V, and optionally Y349C or S354C) (see eg Carter, P. et al., Immunotechnol. 2 (1996) 73) (numbered according to the EU index).

In one aspect, an isolated nucleic acid encoding an antibody for use in a method as reported herein is provided.

In one aspect, there is provided a method of making an anti-MerTK antibody, wherein the method comprises culturing a host cell comprising one or more antibody-encoding nucleic acids as provided above under conditions suitable for expressing the antibody, and obtaining an antibody from the host cell (or host cell culture medium) to recover antibodies.

For recombinant production of anti-MerTK antibodies, nucleic acids encoding antibodies such as those disclosed above are isolated and inserted into one or more vectors for further colonization and/or expression in host cells. Such nucleic acids can be readily isolated and sequenced by conventional methods (eg, using oligonucleotide probes capable of binding specifically to genes encoding antibody heavy and light chains), or produced by recombinant methods or chemical synthesis.

Suitable host cells for the colonization or expression of antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies may be produced in bacteria, especially without the need for glycosylation and Fc utility functions. See, eg, US 5,648,237, US 5,789,199 and US 5,840,523 for the expression of antibody fragments and polypeptides in bacteria. (See also Charlton, K.A., in: Methods in Molecular Biology, Vol. 248, Lo, B.K.C. (eds.), Humana Press, Totowa, NJ (2003), pp. 245-254, which describe the use of antibody fragments in E. coli expression in.) After expression, the antibody can be separated from the bacterial cell paste in the soluble fraction and can be further purified.

In addition to prokaryotes, eukaryotic microorganisms (such as filamentous fungi or yeast) are also suitable hosts for colonization or expression of antibody-encoding vectors, including fungal and yeast strains whose glycosylation pathways have been "humanized", This results in the production of antibodies with partially or fully human glycosylation patterns. See Gerngross, T.U., Nat. Biotech. 22 (2004) 1409-1414; and Li, H. et al., Nat. Biotech. 24 (2006) 210-215.

Suitable host cells for expression (glycosylated) antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. A number of baculovirus strains have been identified that can be used in combination with insect cells, particularly for transfection of Spodoptera frugiperda cells.

Plant cell cultures can also be used as hosts. See, eg, US 5,959,177, US 6,040,498, US 6,420,548, US 7,125,978 and US 6,417,429 (which describe the PLANTIBODIES™ technology for producing antibodies in transgenic plants).

Vertebrate cells can also be used as hosts. For example, mammalian cell lines suitable for growth in suspension can be used. Other examples of useful mammalian host cell lines include: monkey kidney CV1 line transformed with SV40 (COS-7); human embryonic kidney line (eg, Graham, F.L. et al., J. Gen Virol. 36 (1977) 59-74 293 or 293T cells as described in); baby hamster kidney cells (BHK); mouse Sertoli cells (eg, TM4 cells as described in Mather, J.P., Biol. Reprod. 23 (1980) 243-252); monkeys Kidney cells (CV1); African green monkey kidney cells (VERO-76); Human cervical cancer cells (HELA); Canine kidney cells (MDCK); Buffalo rat hepatocytes (BRL 3A); Human lung cells (W138); Human hepatocytes (Hep G2); mouse mammary tumor cells (MMT 060562); TRI cells (as described in Mather, J.P. et al., Annals N.Y.Acad. Sci. 383 (1982) 44-68); MRC5 cells; and FS4 cell. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub, G. et al., Proc. Natl. Acad. Sci. USA 77 (1980) 4216-4220); and bone marrow Tumor cell lines such as Y0, NSO and Sp2/0. For a review of some suitable mammalian host cell lines for antibody production see, e.g.: Yazaki, P. and Wu, A.M., Methods in Molecular Biology, Vol. 248, ed. Lo, B.K.C., Humana Press, Totowa, NJ (2004 ), pp. 255-268.

In one aspect, the host cell is a eukaryotic cell, such as a Chinese hamster ovary (CHO) cell or a lymphoid cell (eg, Y0, NSO, Sp20 cells). D. to analyze

The anti-MerTK antibodies provided herein can be identified, screened or characterized for their physical/chemical properties and/or biological activity by various assays known in the art. 1. Binding Assays and Other Assays

In one aspect, the antigen-binding activity of the antibodies of the invention is detected using known methods such as ELISA, Western blot, and the like.

On the other hand, competition assays can be used to identify antibodies that compete with any of the anti-MerTK antibodies disclosed above for binding to MerTK. In certain aspects, such competing antibodies bind to the same epitope (eg, a linear or conformational epitope) that is bound by any of the anti-MerTK antibodies disclosed herein. Detailed exemplary methods for mapping epitopes to antibody binding are provided in Morris (1996) "Epitope Mapping Protocols" (in Methods in Molecular Biology Vol. 66 (Humana Press, Totowa, NJ)).

In an exemplary competition assay, immobilized MerTK is incubated in a solution comprising a first labeled antibody (eg, any of the anti-MerTK antibodies disclosed above) that binds to MerTK and a second unlabeled antibody (being tested). its ability to compete with the primary antibody for binding to MerTK). The secondary antibody can be present in the supernatant of the fusion tumor. As a control, immobilized MerTK was incubated in a solution containing the first labeled antibody but not the second unlabeled antibody. After incubation under conditions that allow binding of the primary antibody to MerTK, excess unbound antibody is removed and the amount of label associated with the immobilized MerTK is measured. If the amount of label associated with the immobilized MerTK in the test sample is significantly reduced relative to the control sample, this indicates that the secondary antibody is competing with the primary antibody for binding to MerTK. See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). 2. Activity Assay

In one aspect, assays for identifying the biological activity of anti-MerTK antibodies are provided. Biological activities may include, for example, reducing MerTK-mediated phagocytic activity, reducing MerTK-mediated clearance of apoptotic cells, and/or enhancing tumor immunogenicity of checkpoint inhibitors. Antibodies having such biological activities in vivo and/or in vitro are also provided.

In certain aspects, such biological activity assays are performed on the antibodies of the invention. Examples of assays suitable for measuring such biological activities are disclosed further herein, including the Examples section below. E. Pharmaceutical composition

In yet another aspect, pharmaceutical compositions comprising any of the antibodies provided herein are provided, eg, for use in any of the following methods of treatment. In one aspect, a pharmaceutical composition comprises any of the antibodies provided herein and a pharmaceutically acceptable carrier. In another aspect, a pharmaceutical composition comprises any of the antibodies provided herein and at least one additional therapeutic agent (as described below).

Prepared as herein by admixing such antibodies of the desired degree of purity with one or more optional pharmaceutically acceptable carriers ( Remington's Pharmaceutical Sciences 16th edition, Osol, A. ed. (1980)) Pharmaceutical compositions of the disclosed anti-MerTK antibodies in the form of lyophilized compositions or aqueous solutions. Pharmaceutically acceptable carriers are generally non-toxic to the recipient at the dosages and concentrations employed and include, but are not limited to: buffers such as histidine, phosphate, citrate, acetate and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethylammonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butanol, or benzyl alcohol) ; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol and m-cresol); low molecular weight ( less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamic acid, asparagine Acids, histidines, arginines, or lysines; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents (eg, EDTA); sugars, such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions, such as sodium; metal complexes (eg, zinc protein complexes); and/or nonionic surfactants, such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersants, such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), eg, human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 ( ^HYLENEX® , Halozyme, Inc.). Certain exemplary sHASEGPs and uses, including rHuPH20, are described in US Patent Publication Nos. 2005/0260186 and 2006/0104968. In one aspect, sHASEGP is associated with one or more additional glycosaminoglycanase enzymes such as chondroitinase.

Exemplary lyophilized antibody compositions are disclosed in U.S. Patent No. 6,267,958. Water-soluble antibody compositions include those described in US Pat. No. 6,171,586 and WO 2006/044908, the latter of which includes a histidine-acetate buffer.

The pharmaceutical compositions described herein may also contain more than one active ingredient suitable for the particular indication being treated, preferably, those having complementary active ingredients that do not adversely affect each other. For example, it may be necessary to further provide one or more immune checkpoint inhibitors, e.g., anti-PDL1 Antibody. These active ingredients are suitably present in combination in amounts effective for the intended purpose.

The active ingredient may be entrapped, for example, in microcapsules prepared by coacervation techniques or by interfacial polymerization (eg, hydroxymethylcellulose microcapsules or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively), colloidal drugs In delivery systems such as liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules or in macroemulsions. These techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

Pharmaceutical compositions can be prepared for sustained release. Suitable examples of sustained release formulations include semipermeable substrates of solid hydrophobic polymers containing antibodies in the form of shaped articles such as membranes or microcapsules.

Pharmaceutical compositions for in vivo administration are generally sterile. Sterility can be readily achieved, for example, by filtration through sterile membranes. F. METHODS OF TREATMENT AND ROUTE OF ADMINISTRATION

Any of the anti-MerTK antibodies provided herein can be used in therapeutic methods. In any of the methods disclosed herein, in some embodiments, the methods (eg, using the present disclosure to pegylated anti-MerTK antibodies) compare to similar methods using non-pegylated anti-MerTK antibodies. Antibodies) reduced retinal toxicity.

In one aspect, an anti-MerTK antibody for use as a drug is provided. In other aspects, anti-MerTK antibodies for the treatment of cancer are provided. In certain aspects, anti-MerTK antibodies for use in methods of treatment are provided. In certain aspects, the invention provides an anti-MerTK antibody for use in a method of treating an individual suffering from cancer, the method comprising administering to the individual an effective amount of an anti-MerTK antibody. In one such aspect, the method further comprises administering an effective amount of at least one additional therapeutic agent (as described below) (eg, one, two, three, four, five or six additional therapeutic agents) the individual. In other aspects, the invention provides anti-MerTK antibodies for enhancing immune function and/or reducing MerTK-mediated clearance of apoptotic cells. In certain aspects, the invention provides anti-MerTK antibodies for use in a method of enhancing immune function and/or reducing MerTK-mediated clearance of apoptotic cells in an individual, the method comprising administering to the individual an effective amount of an anti-MerTK antibody to Enhance immune function and/or reduce MerTK-mediated clearance of apoptotic cells. An "individual" according to any of the above aspects is preferably a human.

In yet another aspect, the present invention provides the use of an anti-MerTK antibody in the manufacture or preparation of a medicament. In one aspect, the drug is used to treat cancer. In yet another aspect, the medicament is for use in a method of treating cancer, the method comprising administering to a subject suffering from cancer an effective amount of the medicament. In one such aspect, the method further comprises administering to the subject an effective amount of at least one additional therapeutic agent (eg, as described below). In yet another aspect, the drug is used to enhance immune function and/or reduce MerTK-mediated clearance of apoptotic cells. In yet another aspect, the medicament is used in a method of enhancing immune function and/or reducing MerTK-mediated clearance of apoptotic cells in an individual, the method comprising administering to the individual an effective amount of the medicament to enhance immune function and/or reduce MerTK-mediated clearance of apoptotic cells. An "individual" according to any of the above aspects may be a human.

In yet another aspect, the present invention provides methods of treating cancer. In one aspect, the method comprises administering to an individual suffering from the cancer an effective amount of an anti-MerTK antibody. In one such aspect, the method further comprises administering to the subject an effective amount of at least one additional therapeutic agent (as described below).

An "individual" according to any of the above aspects may be a human.

In yet another aspect, the present invention provides methods for enhancing immune function and/or reducing MerTK in an individual (eg, an individual suffering from cancer) Methods of mediated clearance of apoptotic cells. In one aspect, the method comprises administering to the individual an effective amount of an anti-MerTK antibody to enhance immune function and/or reduce MerTK-mediated clearance of apoptotic cells. In one aspect, a "subject" is a human.

In yet another aspect, the present invention provides pharmaceutical compositions comprising any of the anti-MerTK antibodies provided herein, eg, for use in any of the above-described methods of treatment. In one aspect, a pharmaceutical composition comprises any of the anti-MerTK antibodies provided herein and a pharmaceutically acceptable carrier. In another aspect, a pharmaceutical composition comprises any of the anti-MerTK antibodies provided herein and at least one additional therapeutic agent, eg, as disclosed below. 1. Monotherapy

In some embodiments, an anti-MerTK antibody of the present disclosure (eg, a pegylated anti-MerTK antibody) is administered as monotherapy to treat an individual suffering from cancer. As used herein, "cancer" refers to or discloses the physiological condition in mammals that is typically characterized by unregulated cell growth/proliferation. In certain embodiments, the cancer can be a solid cancer or a hematological cancer. Solid cancers are usually characterized by the formation of tumor masses in specific tissues. As used herein, the term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues. Non-limiting examples of solid cancers to be treated using the anti-MerTK antibodies of the present disclosure include carcinomas, lymphomas, blastomas, and sarcomas. More specific examples of such cancers include, but are not limited to, squamous cell carcinoma (eg, epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), peritoneal carcinoma, liver Cell, gastric or stomach cancer (including gastrointestinal and gastrointestinal stromal cancers), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urinary tract cancer, liver cancer, breast cancer , colorectal cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer or kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, melanoma, superficial cancer Diffuse melanoma, malignant freckled melanoma, acral freckled melanoma, nodular melanoma, and abnormal vascular proliferation associated with macular hamartomatosis, edema (eg, associated with brain tumors), Meigs Syndrome, brain cancer, head and neck cancer and related metastases. In certain embodiments, cancers suitable for treatment by the anti-MerTK antibodies of the present disclosure include breast cancer, colorectal cancer, rectal cancer, non-small cell lung cancer, glioblastoma, renal cell carcinoma, prostate cancer, liver cancer, pancreas Carcinoma, soft tissue sarcoma, Kaposi's sarcoma, carcinoid, head and neck cancer, ovarian cancer and mesothelioma. In some embodiments, the cancer line is selected from the group consisting of small cell lung cancer, glioblastoma, neuroblastoma, melanoma, breast cancer, gastric cancer, colorectal cancer (CRC), and hepatocellular carcinoma. Also, in some embodiments, the cancer is selected from the group consisting of non-small cell lung cancer, colorectal cancer, glioblastoma, and breast cancer, including metastatic forms of these cancers. In some embodiments, the cancer is colorectal cancer, including colorectal cancer and rectal cancer. In some embodiments, the cancer is urothelial carcinoma, non-small cell lung cancer, triple negative breast cancer, small cell lung cancer, hepatocellular carcinoma, or melanoma.

By contrast, blood cancers originate in the blood or bone marrow. In some embodiments, the blood cancer to be treated using the anti-MerTK antibodies of the present disclosure is leukemia. Examples of leukemias include, but are not limited to, chronic lymphocytic leukemia (CLL); acute lymphocytic leukemia (ALL); hairy cell leukemia; chronic myeloid leukemia and acute myeloid leukemia. In some embodiments, the blood cancer to be treated using the anti-MerTK antibodies of the present disclosure is lymphoma. Non-limiting examples of lymphomas include T cell lymphomas (such as adult T cell leukemia/lymphoma; hepatosplenic T cell lymphoma; peripheral T cell lymphoma, anaplastic large cell lymphoma; and angioimmunoblastic T cell lymphomas) lymphoma), B-cell lymphomas (including low-grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate/follicular NHL; intermediate-grade diffuse NHL; cellular NHL; high-grade lymphoblastic NHL; high-grade small non-lysing cell NHL; bulky NHL; diffuse large B-cell lymphoma; mantle cell lymphoma; Birch's lymphoma; AIDS-related lymphoma; globulinemia), Hodgkin's lymphoma, and post-transplant lymphoproliferative disease (PTLD). In some embodiments, the blood cancer to be treated using the anti-MerTK antibodies of the present disclosure is myeloma. In a specific embodiment, the myeloma is plasmacytoma or multiple myeloma. In certain embodiments, cancers suitable for treatment by the anti-MerTK antibodies of the present disclosure include non-Hodgkin's lymphoma and multiple myeloma.

In another aspect, provided herein are methods for treating or delaying the progression of cancer in an individual comprising administering to the individual an effective amount of an anti-MerTK antibody as disclosed in the present disclosure. In some embodiments, the treatment results in a persistent response in the subject after the treatment is discontinued. The methods disclosed herein can be used to treat conditions that require enhanced immunogenicity, such as increased tumor immunogenicity for the treatment of cancer. Also provided herein are methods of enhancing immune function in an individual suffering from cancer, comprising administering to the individual an effective amount of an anti-MerTK antibody as disclosed in the present disclosure. In some embodiments, the cancer expresses functional STING, functional Cx43, and functional cGAS polypeptides. A functional protein is one that is capable of performing its normal function in a cell. Examples of functional proteins can include wild-type proteins, marker proteins, and mutant proteins that retain or improve protein function compared to wild-type proteins. Protein function can be measured by any method known to those of skill in the art, including assaying protein or mRNA expression and sequencing genomic DNA or mRNA. In some embodiments, the cancer comprises tumor-associated macrophages expressing functional STING polypeptides. In some embodiments, the cancer comprises tumor cells expressing functional cGAS polypeptides. In some embodiments, the cancer comprises tumor cells expressing functional Cx43 polypeptides. In some embodiments, the cancer is colorectal cancer, including colorectal cancer and rectal cancer. In some embodiments, the cancer is urothelial carcinoma, non-small cell lung cancer, triple negative breast cancer, small cell lung cancer, hepatocellular carcinoma, or melanoma.

Also provided herein are methods of reducing MerTK-mediated clearance of apoptotic cells in an individual comprising administering to the individual an effective amount of an anti-MerTK antibody as disclosed in the present disclosure to reduce MerTK-mediated clearance of apoptotic cells. In some embodiments, the clearance of apoptotic cells is reduced by 1-10 times, 1-8 times, 1-5 times, 1-4 times, 1-3 times, 1-2 times, 2-10 times, 2- 8 times, 2-5 times, 2-4 times, 2-3 times, 3-10 times, 3-8 times, 3-5 times, 3-4 times, or at least about 1.1 times, 1.2 times, 1.3 times less , 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2.0 times, 2.1 times, 2.2 times, 2.3 times, 2.4 times, 2.5 times, 2.6 times, 2.7 times, 2.8 times, 2.9 times, 3.0 times times, 3.1 times, 3.2 times, 3.3 times, 3.4 times, 3.5 times, 3.6 times, 3.7 times, 3.8 times, 3.9 times, 4.0 times, 4.1 times, 4.2 times, 4.3 times, 4.4 times, 4.5 times, 4.6 times, 4.7 times, 4.8 times, 4.9 times, 5.0 times, 5.1 times, 5.2 times, 5.3 times, 5.4 times, 5.5 times, 5.6 times, 5.7 times, 5.8 times, 5.9 times, 6.0 times, 6.1 times, 6.2 times, 6.3 times , 6.4 times, 6.5 times, 6.6 times, 6.7 times, 6.8 times, 6.9 times, 7.0 times, 7.1 times, 7.2 times, 7.3 times, 7.4 times, 7.5 times, 7.6 times, 7.7 times, 7.8 times, 7.9 times, or 8.0 times times. The reduction of MerTK-mediated clearance of apoptotic cells can be determined by comparing the level of MerTK-mediated clearance of apoptotic cells with MerTK-mediated clearance of apoptotic cells in a sample from an individual after administration of an effective amount of an anti-MerTK antibody or immunoconjugate thereof. The induced clearance of apoptotic cells is determined by comparison with a reference level. In some embodiments, the reference level is the level of MerTK-mediated clearance of apoptotic cells in the reference sample. In some embodiments, the reference sample is taken from a subject prior to administration of an effective amount of an anti-MerTK antibody or immunoconjugate thereof. In some embodiments, the sample comprises tumor tissue or tumor cells.

In some embodiments, the anti-MerTK antibodies of the present disclosure reduce the phagocytic activity of apoptotic cells by about 10-100%, 20-100%, 30-100%, 40-100%, 50-100%, 60-100% , 70-100%, 75-100%, 80-100%, 85-100%, 90-100%, 95-100%, 10-95%, 20-95%, 30-95%, 40-95% , 50-95%, 60-95%, 70-95%, 75-95%, 80-95%, 85-95%, 90-95%, 10-90%, 20-90%, 30-90% , 40-90%, 50-90%, 60-90%, 70-90%, 75-90%, 80-90%, 85-90%, 10-85%, 20-85%, 30-85% , 40-85%, 50-85%, 60-85%, 70-85%, 75-85%, 80-85%, 10-80%, 20-80%, 30-80%, 40-80% , 50-80%, 60-80%, 70-80%, 75-80%, 10-75%, 20-75%, 30-75%, 40-75%, 50-75%, 60-75% , 70-75%, 10-70%, 20-70%, 30-70%, 40-70%, 50-70%, 60-70%, 10-65%, 20-65%, 30-65% , 40-65%, 50-65%, 60-65%, 10-60%, 20-60%, 30-60%, 40-60%, 50-60%, 10-55%, 20-55% , 30-55%, 40-55%, 50-55%, 10-40%, 20-40%, or 30-40%, or a reduction of at least about 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%. In some embodiments, the half maximal inhibitory concentration (IC50) of the anti-MerTK antibody for reducing the phagocytic activity of apoptotic cells is about 1 pM - 50 pM, 1 pM - 100 pM, 1 pM - 500 pM, 1 pM - 1 nM, 1 pM – 1.5 nM, 5 pM – 50 pM, 5 pM – 100 pM, 5 pM – 500 pM, 5 pM – 1 nM, 5 pM – 1.5 nM, 10 pM – 50 pM, 10 pM – 100 pM , 10 pM – 500 pM, 10 pM – 1 nM, 10 pM – 1.5 nM, 50 pM – 100 pM, 50 pM – 500 pM, 50 pM – 1 nM, 50 pM – 1.5 nM, 100 pM – 500 pM, 100 pM – 1 nM or 100 pM – 1.5 nM.

Anti-MerTK antibodies can be administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecal cavity, intraventricularly, or intranasally. Appropriate doses of anti-MerTK antibodies can be determined based on the type of disease to be treated, the severity and course of the disease, the individual's clinical condition, the individual's clinical history and response to treatment, and the discretion of the attending physician. 2. Combination with another therapy

The antibodies of the invention can be administered alone or in combination therapy. For example, combination therapy includes administration of an antibody of the invention and administration of at least one additional therapeutic agent (eg, one, two, three, four, five, or six additional therapeutic agents). In certain aspects, combination therapy comprises administration of an antibody of the invention and administration of at least one additional therapeutic agent such as an immune checkpoint inhibitor.

In some embodiments, the uses and methods may further comprise additional therapy or administration of an effective amount of an additional therapeutic agent. Other treatments may be radiation therapy, surgery (eg lumpectomy and mastectomy), chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, or a combination of the foregoing. Additional therapy may take the form of adjuvant therapy or neoadjuvant therapy. In some embodiments, the additional therapy is the administration of a small molecule enzyme inhibitor or an anti-metastatic agent. In some embodiments, the other therapy is the administration of side-effect-limiting agents (eg, agents intended to reduce the occurrence and/or severity of side effects of treatment, such as anti-nausea agents, etc.). In some embodiments, the additional therapy is radiation therapy. In some embodiments, the additional therapy is surgery. In some embodiments, the additional therapy is a combination of radiation therapy and surgery. In some embodiments, the additional therapy is gamma radiation. In some embodiments, the additional therapy is therapy targeting the PI3K/AKT/mTOR pathway, HSP90 inhibitors, tubulin inhibitors, apoptosis inhibitors, and/or chemopreventive agents.

In some embodiments, the additional therapy is an antagonist against B7-H3 (also known as CD276), eg, the blocking antibody MGA271, an antagonist against TGFβ, eg, metelimumab (also known as CAT-192), fresolimumab (also known as GC1008), or LY2157299, including treatment of adoptive transfer of chimeric antigen receptor (CAR) expressing T cells (eg, cytotoxic T cells or CTLs) , including adoptive transfer of T cells harboring a dominant-negative TGFβ receptor (eg, a dominant-negative TGFβ receptor type II), including treatment with the HERCREEM regimen (see, eg, ClinicalTrials.gov identifier NCT00889954) for CD137 (also known as TNFRSF9, 4-1BB, or ILA) agonists, eg, the activating antibody urrelumab (also known as BMS-663513), agonists against CD40, eg, the activating antibody CP-870893, Agonists against OX40 (also known as CD134), eg, activating antibody administered in combination with another anti-OX40 antibody (eg, AgonOX), agonists against CD27, eg, activating antibody CDX-1127, indole Amine-2,3-dioxygenase (IDO), 1-methyl-D-tryptophan (also known as 1-D-MT), antibody-drug conjugates (in some embodiments, maytansine or monomethyl auristatin E (MMAE), anti-NaPi2b antibody-MMAE conjugate (also known as DNIB0600A or RG7599), trastuzumab-emtansine (also known as T-DM1, ado-trastuzumab-maytansine or KADCYLA®, Jiannandec), DMUC5754A, an antibody-drug conjugate targeting the endothelin B receptor (EDNBR), e.g., an antibody against EDNBR binds to MMAE (an angiogenesis inhibitor), antibodies against VEGF, e.g., VEGF-A, bevacizumab (also known as AVASTIN®, Jiannandec), directed against angiopoietin 2 (also known as Ang2) antibody, MEDI3617, an antineoplastic drug that targets CSF-1R (also known as M-CSFR or CD115), anti-CSF-1R (also known as IMC-CS4), interferons (eg, interferon alpha or Interferon gamma), Roferon-A, GM-CSF (also known as recombinant human granulosa macrophage line stimulating factor, rhu GM-CSF, sargrastim, or Leukine®), IL-2 (also known as adrenaline aldesleukin or Proleukin®), IL-12, target Antibodies to CD20 (in some embodiments, the CD20-targeted antibody is obinutuzumab (also known as GA101 or Gazyva®) or rituximab), GITR-targeted Antibody (in some embodiments, the antibody targeting GITR is TRX518), combination cancer vaccine (in some embodiments, the cancer vaccine is a peptide cancer vaccine, in some embodiments a personalized peptide vaccine; in some embodiments , peptide cancer vaccines are multivalent long peptides, polypeptides, peptide mixtures, hybrid peptides, or peptide-pulsed dendritic cell vaccines (see e.g., Yamada et al., Cancer Sci, 104:14-21, 2013)), in combination with adjuvants , TLR agonists, e.g., Poly-ICLC (also known as Hiltonol®), LPS, MPL or CpG ODN, tumor necrosis factor (TNF) alpha, IL-1, HMGB1, IL-10 antagonists, IL-4 antagonists , IL-13 antagonists, HVEM antagonists, ICOS agonists, e.g., by administration of ICOS-L or an agonist antibody against ICOS, therapy targeting CX3CL1, therapy targeting CXCL10, therapy targeting CCL5, LFA-1 or ICAM1 agonists, selectin agonists, targeted therapies, B-Raf inhibitors, vemurafenib (also known as Zelboraf®, dabrafenib (also known as Tafinlar®) ), erlotinib (also known as Tarceva®), inhibitors of MEK such as MEK1 (also known as MAP2K1) or MEK2 (also known as MAP2K2), cobimetinib (also known as GDC- 0973 or Xl-518), trametinib (also known as Mekinist®), K-Ras inhibitors, c-Met inhibitors, onartuzumab (also known as MetMAb), Alk inhibitor, AF802 (also known as CH5424802 or alectinib), phosphatidylinositol 3-kinase (PI3K) inhibitor, BKM120, idelalisib (also known as GS-1101 or CAL) -101), perifosine (also known as KRX-0401), Akt, MK2206, GSK690693, GDC-0941, mTOR inhibitor, sirolimus (also known as rapamycin) , temsirolimus (also known as CCI-779 or Torisel®), everolimus s) (also known as RAD001), ridaforolimus (also known as AP-23573, MK-8669 or rapamycin), OSI-027, AZD8055, INK128, dual PI3K/mTOR inhibitor, XL765 , GDC-0980, BEZ235 (also known as NVP-BEZ235), BGT226, GSK2126458, PF-04691502 or PF-05212384 (also known as PKI-587). In some embodiments, the additional therapeutic agent is CT-011 (also known as Pidilizumab or MDV9300; CAS Registry No. 1036730-42-3; CureTech/Medivation). CT-011, also known as hBAT or hBAT-1, is an antibody disclosed in WO2009/101611. 3. In combination with checkpoint inhibitors

In some embodiments, the additional therapeutic agent is an immune checkpoint inhibitor. In certain aspects, the present application provides methods for enhancing immune function in an individual suffering from cancer comprising administering an effective amount of an anti-MerTK antibody in combination with an immune checkpoint inhibitor. In certain embodiments, the anti-MERTK antibody increases the immune effect of an immune checkpoint inhibitor by about 2-fold, 3-fold, 5-fold, 8-fold, 10-fold, 15-fold, or 20-fold. In certain embodiments, the anti-MERTK antibody increases the immune effect of an immune checkpoint inhibitor by about 1-2-fold, 1-5-fold, 1-10-fold, 1-15-fold, 1-20-fold, 1-25-fold , 1-30 times, 1-50 times, 1-75 times, 1-100 times, 1-150 times, 1-200 times, 1-250 times, 1.5-2 times, 1.5-5 times, 1.5-10 times , 1.5-15 times, 1.5-20 times, 1.5-25 times, 1.5-30 times, 1.5-50 times, 1.5-75 times, 1.5-100 times, 1.5-150 times, 1.5-200 times, 1.5-250 times , 2-5 times, 2-10 times, 2-15 times, 2-20 times, 2-25 times, 2-30 times, 2-50 times, 2-75 times, 2-100 times, 2-150 times , 2-200 times, 2-250 times, 2.5-5 times, 2.5-10 times, 2.5-15 times, 2.5-20 times, 2.5-25 times, 2.5-30 times, 2.5-50 times, 2.5-75 times , 2.5-100 times, 2.5-150 times, 2.5-200 times, 2.5-250 times, 5-10 times, 5-15 times, 5-20 times, 5-25 times, 5-30 times, 5-50 times , 5-75 times, 5-100 times, 5-150 times, 5-200 times, 5-250 times, 10-15 times, 10-20 times, 10-25 times, 10-30 times, 10-50 times , 10-75 times, 10-100 times, 10-150 times, 10-200 times, 10-250 times, 20-25 times, 20-30 times, 20-50 times, 20-75 times, 20-100 times , 20-150 times, 20-200 times, 20-250 times, 25-30 times, 25-50 times, 25-75 times, 25-100 times, 25-150 times, 25-200 times, or 25-250 times , or an increase of at least about 1, 2, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 75, 80, 90, 100x, 125x, 150x, 200x, 225x or 250x.

In some embodiments, the individual suffers from a cancer that is resistant (proven to be resistant) to one or more immune checkpoint inhibitors. In some embodiments, resistance to an immune checkpoint inhibitor includes cancer recurrence or refractory cancer. Relapse can refer to the recurrence of cancer at the original site or a new site after treatment. In some embodiments, resistance to an immune checkpoint inhibitor includes progression of cancer during treatment with an immune checkpoint inhibitor. In some embodiments, resistance to immune checkpoint inhibitors includes cancers that do not respond to treatment. The cancer may be drug-resistant at the start of treatment or it may become drug-resistant during treatment. In some embodiments, the cancer is in an early or advanced stage.

More details on therapeutic immune checkpoint inhibitors are provided in: e.g. Byun et al, (2017) Nat Rev Endocrinol . 13: 195-207; La-Beck et al, (2015) Pharmacotherapy .35(10 ): 963-976; Buchbinder et al, (2016) Am J Clin Oncol. 39(1): 98-106; Michot et al, (2016) Eur J Cancer. 54: 139-148; and Topalian et al, ( 2016) Nat Rev Cancer. 16: 275-287.

In some embodiments, the immune checkpoint inhibitor is a cytotoxic T lymphocyte-associated protein 4 (CTLA4) (also known as CD152) inhibitor. In some embodiments, CTLA-4 Inhibitors are blocking antibodies, ipilimumab (also known as MDX-010, MDX-101 or Yervoy®), tremelimumab (also known as ticilimumab) or CP-675, 206).

In some embodiments, the immune checkpoint inhibitor is a PD-1 axis binding antagonist.

Provided herein are methods of treating cancer in a subject comprising administering to the subject an effective amount of a PD-1 axis binding antagonist and an anti-MerTK antibody of the present disclosure (eg, pegylated anti-MerTK) Antibody). Also provided herein are methods of enhancing immune function or response in an individual (eg, an individual suffering from cancer) comprising administering to the individual an effective amount of a PD-1 axis binding antagonist of the present disclosure and an anti-MerTK antibody (eg, a pegylated anti-MerTK antibody). MerTK Antibody).

In such methods, PD-1 axis binding antagonists include PD-1 binding antagonists, PDL1 binding antagonists and/or PDL2 binding antagonists. Alternative names for "PD-1" include CD279 and SLEB2. Alternative names for "PDL1" include B7-H1, B7-4, CD274 and B7-H. Alternative names for "PDL2" include B7-DC, Btdc and CD273. In some embodiments, PD-1, PDL1 and PDL2 are human PD-1, PDL1 and PDL2.

In some embodiments, a PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand binding partner. In a specific aspect, the PD-1 ligand binding partner is PDL1 and/or PDL2. In another embodiment, a PDL1 binding antagonist is a molecule that inhibits the binding of PDL1 to its binding partner. In a specific aspect, the PDL1 binding partner is PD-1 and/or B7-1. In another embodiment, a PDL2 binding antagonist is a molecule that inhibits the binding of PDL2 to its binding partner. In a specific aspect, the PDL2 binding partner is PD-1. The antagonist may be an antibody, antigen-binding fragment thereof, immunoadhesin, fusion protein, oligopeptide or small molecule. If the antagonist is an antibody, in some embodiments, the antibody comprises a human constant region selected from the group consisting of IgGl, IgG2, IgG3, and IgG4.

In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody (eg, a human antibody, humanized antibody, or chimeric antibody). A variety of anti-PD-1 antibodies can be used in the methods disclosed herein. In any of the embodiments herein, the PD-1 antibody can bind to human PD-1 or a variant thereof. In some embodiments, the anti-PD-1 antibody is a monoclonal antibody. In some embodiments, the anti-PD-1 antibody is an antibody fragment selected from the group consisting of Fab, Fab', Fab'-SH, Fv, scFv, and (Fab') ₂ fragments. In some embodiments, the anti-PD-1 antibody is a chimeric antibody or a humanized antibody. In other embodiments, the anti-PD-1 antibody is a human antibody.

In some specific examples, the anti-PD-1 antibody is nivolumab (CAS Registry Number: 946414-94-4). Nivolumab (Bristol-Myers Squibb/Ono), also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558 and OPDIVO®, is an anti-PD-1 antibody described in WO2006/121168 . In some embodiments, the anti-PD-1 antibody comprises heavy and light chain sequences, wherein: (a) The heavy chain contains the amino acid sequence: QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWY DGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO: 24)，且 (b) the light chain comprises the amino acid sequence: EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO: 25).

In some embodiments, the anti-PD-1 antibody comprises six HVR sequences from SEQ ID NO: 24 and SEQ ID NO: 25 (e.g., three from heavy chain HVR of SEQ ID NO: 24 and three light chain HVRs from SEQ ID NO: 25). In some embodiments, the anti-PD-1 antibody comprises a heavy chain variable domain from SEQ ID NO:24 and a light chain variable domain from SEQ ID NO:25.

In some specific examples, the anti-PD-1 antibody is pelivizumab (CAS Registry No. 1374853-91-4). Pembrolizumab (Merck), also known as MK-3475, Merck 3475, lambrolizumab, SCH-900475 and KEYTRUDA®, is an anti-PD-1 antibody disclosed in WO2009/114335 . In some embodiments, the anti-PD-1 antibody comprises heavy and light chain sequences, wherein: (a) The heavy chain contains the amino acid sequence: QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGG INPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYW GQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCP APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTK PREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 26)，且 (b) the light chain comprises the amino acid sequence: EIVLTQSPAT LSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLES GVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKRTVAAPSVF IFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL2SSPVTKSFNRGEC (SEQ) NO.

In some embodiments, the anti-PD-1 antibody comprises six HVR sequences from SEQ ID NO: 26 and SEQ ID NO: 27 (e.g., three from Heavy chain HVR of SEQ ID NO: 26 and three light chain HVRs from SEQ ID NO: 27). In some embodiments, the anti-PD-1 antibody comprises a heavy chain variable domain from SEQ ID NO:26 and a light chain variable domain from SEQ ID NO:27.

In some specific examples, the anti-PD-1 antibody is MEDI-0680 (AMP-514; AstraZeneca). MEDI-0680 is a humanized IgG4 anti-PD-1 antibody.

In some specific examples, the anti-PD-1 antibody is PDR001 (CAS Registry No. 1859072-53-9; Novartis). PDR001 is a humanized IgG4 anti-PD-1 antibody that blocks the binding of PDL1 and PDL2 to PD-1.

In some specific examples, the anti-PD-1 antibody is REGN2810 (Regeneron). REGN2810 is a human anti-PD-1 antibody.

In some specific examples, the anti-PD-1 antibody is BGB-108 (BeiGene). In some specific examples, the anti-PD-1 antibody is BGB-A317 (BeiGene).

In some specific examples, the anti-PD-1 antibody is JS-001 (Shanghai Junshi). JS-001 is a humanized anti-PD-1 antibody.

In some specific examples, the anti-PD-1 antibody is STI-A1110 (Sorrento). STI-A1110 is a human anti-PD-1 antibody.

In some specific examples, the anti-PD-1 antibody is INCSHR-1210 (Incyte). INCSHR-1210 is a human IgG4 anti-PD-1 antibody.

In some specific examples, the anti-PD-1 antibody is PF-06801591 (Pfizer).

In some specific examples, the anti-PD-1 antibody is TSR-042 (also known as ANB011; Tesaro/AnaptysBio).

In some specific examples, the anti-PD-1 antibody is AM0001 (ARMO Biosciences).

In some specific examples, the anti-PD-1 antibody is ENUM 244C8 (Enumeral Biomedical Holdings). ENUM 244C8 is an anti-PD-1 antibody that inhibits PD-1 function without blocking the binding of PDL1 to PD-1.

In some specific examples, the anti-PD-1 antibody is ENUM 388D4 (Enumeral Biomedical Holdings). ENUM 388D4 is an anti-PD-1 antibody that competitively inhibits the binding of PDL1 to PD-1.

In some embodiments, the PD-1 antibody comprises six HVRs Sequences (eg, three heavy chain HVRs and three light chain HVRs) and/or heavy and light chain variable domains from the PD-1 antibody disclosed in: WO2015/112800 (Applicant: Regeneron ), WO2015/112805 (applicant: Regeneron), WO2015/112900 (applicant: Novartis), US20150210769 (assigned to Novartis), WO2016/089873 (applicant: Celgene), WO2015/035606 (applicant: Baekje Shenzhou Company), WO2015/085847 (Applicant: Shanghai Hengrui Pharmaceutical/Jiangsu Hengrui Pharmaceutical), WO2014/206107 (Applicant: Shanghai Junshi Bio/Junmeng Bio), WO2012/145493 (Applicant: Amplimmune), US9205148 (assigned to MedImmune), WO2015/119930 (applicant: Pfizer/Merck), WO2015/119923 (applicant: Pfizer/Merck), WO2016/032927 (applicant: Pfizer/Merck) ), WO2014/179664 (Applicant: AnaptysBio), WO2016/106160 (Applicant: Enumeral) and WO2014/194302 (Applicant: Sorrento Corporation).

In some embodiments, the PD-1 axis binding antagonist is an anti-PDL1 antibody. A variety of anti-PDL1 antibodies are encompassed and described herein. In any of the specific examples herein, the isolated anti-PDL1 antibody can bind to human PDL1, eg, human PDL1 as set forth in UniProtKB/Swiss-Prot Accession No. Q9NZQ7.1, or a variant thereof. In some embodiments, the anti-PDL1 antibody is capable of inhibiting the binding between PDL1 and PD-1 and/or between PDL1 and B7-1. In some embodiments, the anti-PDL1 antibody is a monoclonal antibody. In some embodiments, the anti-PDLl antibody is an antibody fragment selected from the group consisting of Fab, Fab'-SH, Fv, scFv, and (Fab') ₂ fragments. In some embodiments, the anti-PDL1 antibody is a humanized antibody. In some embodiments, the anti-PDL1 antibody is a human antibody. Examples of anti-PDL1 antibodies useful in the methods of the present disclosure and methods of making them are disclosed in PCT patent application WO 2010/077634 Al and US Pat. No. 8,217,149, which are incorporated herein by reference.

In some embodiments, the anti-PDL1 antibody is atezolizumab (CAS Registry Number: 1422185-06-5). Atezolizumab (Jannandek), also known as MPDL3280A, is an anti-PDL1 antibody.

In some embodiments, the anti-PDL1 antibody comprises a heavy chain variable region and a light chain variable region, wherein: (a) The heavy chain variable region comprises HVR-H1, HVR-H2 and HVR-H3 of GFTFSDSWIH (SEQ ID NO: 28), AWISPYGGSTYYADSVKG (SEQ ID NO: 29) and RHWPGGFDY (SEQ ID NO: 30), respectively sequence, and (b) the light chain variable region comprises HVR-L1, HVR-L2 and HVR-L3 of RASQDVSTAVA (SEQ ID NO: 31), SASFLYS (SEQ ID NO: 32) and QQYLYHPAT (SEQ ID NO: 33), respectively sequence.

In some embodiments, the anti-PDL1 antibody is MPDL3280A, also known as atezolizumab and TECENTRIQ® (CAS Registry No: 1422185-06-5). In some embodiments, the anti-PDL1 antibody comprises heavy and light chain sequences, wherein: (a) The heavy chain variable region sequence comprises the amino acid sequence: EVQLVESGGGLVQPGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS (SEQ ID NO: 34), and (b) the light chain variable region sequence comprises the amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIY SASF LYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR (SEQ ID NO: 35).

In some embodiments, the anti-PDL1 antibody comprises heavy and light chain sequences, wherein: (a) The heavy chain contains the amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 36)，且 (b) the light chain comprises the amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 37)

In some specific examples, the anti-PDL1 antibody is avelumab (CAS Registry No: 1537032-82-8). Avelumab, also known as MSB0010718C, is a human monoclonal IgG1 anti-PDL1 antibody (Merck KGaA, Pfizer). In some embodiments, the anti-PDL1 antibody comprises heavy and light chain sequences, wherein: (a) The heavy chain contains the amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYIMMWVRQAPGKGLEWVSSIYPSGGITFYADTVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARIKLGTVTTVDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 38)，且 (b) the light chain comprises the amino acid sequence: QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTRVFGTGTKVTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 39).

In some embodiments, the anti-PDL1 antibody comprises six HVR sequences from SEQ ID NO: 38 and SEQ ID NO: 39 (e.g., three from heavy chain HVR of SEQ ID NO: 38 and three light chain HVRs from SEQ ID NO: 39). In some embodiments, the anti-PDL1 antibody comprises a heavy chain variable domain from SEQ ID NO:38 and a light chain variable domain from SEQ ID NO:39.

In some embodiments, the anti-PDL1 antibody is durvalumab (CAS Accession No: 1428935-60-7). Durvalumab, also known as MEDI4736, is an Fc-optimized human monoclonal IgGl kappa anti-PDLl antibody (MedImmune, AstraZeneca) described in WO2011/066389 and US2013/034559. In some embodiments, the anti-PDL1 antibody comprises heavy and light chain sequences, wherein: (a) The heavy chain contains the amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWMSWVRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGGWFGELAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 40)，且 (b) the light chain comprises the amino acid sequence: EIVLTQSPGTLSLSPGERATLSCRASQRVSSSYLAWYQQKPGQAPRLLIYDASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSLPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS1QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 4).

In some embodiments, the anti-PDL1 antibody comprises six HVR sequences from SEQ ID NO: 40 and SEQ ID NO: 41 (e.g., three from heavy chain HVR of SEQ ID NO: 40 and three light chain HVRs from SEQ ID NO: 41). In some embodiments, the anti-PDL1 antibody comprises a heavy chain variable domain from SEQ ID NO:40 and a light chain variable domain from SEQ ID NO:41.

In some specific examples, the anti-PDL1 antibody is MDX-1105 (Bristol Myers Squibb). MDX-1105, also known as BMS-936559, is an anti-PDL1 antibody described in WO2007/005874.

In some specific examples, the anti-PDL1 antibody is LY3300054 (Eli Lilly).

In some specific examples, the anti-PDL1 antibody is STI-A1014 (Sorrento). STI-A1014 is a human anti-PDL1 antibody.

In some specific examples, the anti-PDL1 antibody is KN035 (Suzhou Alphamab). KN035 is a single domain antibody (dAB) generated from a camelid phage display library.

In some specific instances, anti-PDL1 Antibodies comprise a cleavable moiety or linker, which upon cleavage (eg, by a protease in the tumor microenvironment) activates the antibody antigen-binding domain to allow it to bind its antigen, eg, by removing non-binding space moieties. In some specific examples, the anti-PDL1 antibody is CX-072 (CytomX Therapeutics).

In some embodiments, the PDL1 antibody comprises six HVRs Sequences (eg, three heavy chain HVRs and three light chain HVRs) and/or heavy and light chain variable domains from the PDL1 antibodies disclosed in: US20160108123 (assigned to Novartis), WO2016/ 000619 (applicant: BeiGene), WO2012/145493 (applicant: Amplimmune), US9205148 (assigned to MedImmune), WO2013/181634 (applicant: Sorrento) and WO2016/061142 (applicant: Novartis ).

In yet another specific aspect, the anti-PD-1 or PDL1 antibody has reduced or minimal effector function. In yet another specific aspect, the minimal effector function results from a "less effector Fc mutation" or glycosylation mutation. In another specific example, the effectorless Fc is mutated to an N297A or D265A/N297A substitution in the constant region. In some embodiments, the isolated anti-PDL1 antibody is deglycosylated. Glycosylation of antibodies is typically N-linked or O-linked. N-linked means that the carbohydrate moiety is attached to the side chain of the asparagine residue. The tripeptide sequences, aspartic acid-X-serine and aspartic acid-X-threonine, where X is any amino acid except proline, are the Recognition sequence for enzymatic association of amino acid side chains. Thus, the presence of any of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose or xylose to a hydroxylamine acid, most commonly serine or threonine, but 5 can also be used -Hydroxyproline or 5-hydroxylysine. Removal of the glycosylation site form antibody is preferably accomplished by altering the amino acid sequence such that one of the tripeptide sequences described above (for N-linked glycosylation sites) is removed. Can be done by substituting an asparagine, serine, or threonine residue within a glycosylation site with another amino acid residue (eg, glycine, alanine, or conservative substitutions) Change.

In some embodiments, the anti-MERTK antibody increases the immune effect of the anti-PDL1 antibody by about 3-fold after 20 days of combination therapy. In some embodiments, the anti-MERTK antibody increases the immune effect of the anti-PDL1 antibody about 10-fold after 30 days of treatment.

In some embodiments, the PD-1 binding antagonist is an immunoadhesin (eg, an immunoadhesin comprising the extracellular or PD-1 binding portion of PDL1 or PDL2 fused to a constant region (eg, the Fc region of an immunoglobulin sequence) ). In some specific examples, the PD-1 binding antagonist is AMP-224. AMP-224 (CAS Registry No. 1422184-00-6; GlaxoSmithKline/MedImmune), also known as B7-DCIg, is a PDL2-Fc fusion soluble receptor described in WO2010/027827 and WO2011/066342.

In some embodiments, the PD-1 binding antagonist is a peptide or small molecule compound. In some specific examples, the PD-1 binding antagonist is AUNP-12 (Pierre Fabre/Aurigene). See, eg, WO2012/168944, WO2015/036927, WO2015/044900, WO2015/033303, WO2013/144704, WO2013/132317 and WO2011/161699.

In some embodiments, the PDL1 binding antagonist is a small molecule that inhibits PD-1. In some embodiments, the PDL1 binding antagonist is a small molecule that inhibits PDL1. In some embodiments, the PDL1 binding antagonist is a small molecule that inhibits PDL1 and VISTA. In some specific examples, the PDL1 binding antagonist is CA-170 (also known as AUPM-170). In some embodiments, the PDL1 binding antagonist is a small molecule that inhibits PDL1 and TIM3. In some embodiments, the small molecule is a compound described in WO2015/033301 and WO2015/033299.

In another aspect, provided herein are methods for enhancing immune function in an individual suffering from cancer comprising administering an effective amount of an anti-MerTK antibody (eg, pegylated anti-MerTK) antibody) in combination with an immune checkpoint inhibitor. Various aspects of immune function that can be enhanced by the anti-MerTK antibodies disclosed herein, as well as methods for measuring such enhancement, are disclosed below.

In some embodiments of the methods of the present disclosure, the cancer (in some embodiments, a cancer sample of a patient examined using a diagnostic test) has elevated levels of T cell infiltration. As used herein, T cell infiltration of cancer can refer to the presence of T cells, such as tumor-infiltrating lymphocytes (TILs), within or otherwise associated with cancer tissue. It is known in the art that T cell infiltration may be associated with improved clinical outcomes in certain cancers (see eg Zhang et al., N. Engl. J. Med. 348(3):203-213 (2003)).

However, T cell exhaustion is also a major immunological feature of cancer, with many tumor-infiltrating lymphocytes (TILs) exhibiting high levels of inhibitory co-receptors and a lack of ability to make effector cytokines (Wherry, EJ Nature immunology 12: 492- 499 (2011); Rabinovich, GA, et al., Annual review of immunology 25:267-296 (2007)). In some embodiments of the methods of the present disclosure, the individual suffers from T cell dysfunction. In some embodiments of the methods of the present disclosure, T cell dysfunction is characterized by T cell anergy or a reduced ability to secrete cytokines, proliferate, or perform cytolytic activity. In some embodiments of the methods of the present disclosure, the T cell dysfunction is characterized by T cell depletion. In some embodiments of the methods of the present disclosure, the T cells are CD4+ and CD8+ T cells. In some embodiments, the T cells are CD4+ and/or CD8+ T cells.

In some embodiments, CD8+ T cells are characterized, eg, by the presence of CD8b expression (eg, by rtPCR using eg Fluidigm) (Cd8b is also known as T cell surface glycoprotein CD8 beta chain; CD8 antigen, alpha polypeptide p37; Accession number is NM_172213). In some embodiments, the CD8+ T cells are from peripheral blood. In some embodiments, the CD8+ T cells are derived from a tumor.

In some embodiments, Treg cells are characterized, eg, by the presence of Fox3p expression (eg, by rtPCR using eg Fluidigm) (Foxp3 also known as forkhead box protein P3; scurfin; FOXP3δ7; immunodeficiency, polyendocrine disease , enteropathy, X-linked; accession number NM_014009). In some embodiments, the Tregs are from peripheral blood. In some embodiments, the Treg cells are derived from a tumor.

In some embodiments, inflammatory T cells are characterized, e.g., by the presence of Tbet and/or CXCR3 expression (e.g., by rtPCR using, e.g., Fluidigm). In some embodiments, the inflammatory T cells are from peripheral blood. In some embodiments, the inflammatory T cells are derived from a tumor.

In some embodiments of the methods of the present disclosure, the CD4 and/or CD8 T cells exhibit increased release of cytokines selected from the group consisting of IFN-γ, TNF-α, and interleukin. Cytokine release can be measured by any means known in the art, eg, using Western blotting, ELISA or immunohistochemical assay to detect the presence of cytokines released in samples containing CD4 and/or CD8 T cells.

In some embodiments of the methods of the present disclosure, the CD4 and/or CD8 T cells are effector memory T cells. In some embodiments of the methods of the present disclosure, the CD4 and/or CD8 ^effector memory T cells are characterized by the expression of ^{CD44highCD62Llow} . The expression of CD44 ^high CD62L ^low can be detected by any means known in the art, such as by preparing a single cell suspension of tissue (eg, cancer tissue) and performing surface staining and flow cytometry using commercially available antibodies against CD44 and CD62L. Cell Analysis Technology. In some embodiments of the methods of the present disclosure, CD4 and/or CD8 effector memory T cells are characterized as having CXCR3 (also known as CXC chemokine receptor type 3; Mig receptor; IP10 receptor; G protein coupled Expression of co-receptor 9; interferon-inducible protein 10 receptor; accession number NM_001504). In some embodiments, the CD4 and/or CD8 effector memory T cells are derived from peripheral blood. In some embodiments, the CD4 and/or CD8 effector memory T cells are derived from a tumor.

In some embodiments of the methods of the present disclosure, Treg function is inhibited relative to prior to administration of the combination. In some embodiments, T cells are decreased relative to prior to administration of the combination.

In some embodiments, the number of Tregs is reduced relative to prior to administration of the combination. In some embodiments, plasma interferon gamma is increased relative to before administration of the combination. For example, Treg numbers can be assessed by determining the percentage of CD4+Fox3p+CD45+ cells (eg, by FACS analysis). In some embodiments, the absolute number of Tregs in the sample is determined, for example. In some embodiments, the Tregs are from peripheral blood. In some embodiments, the Treg are from a tumor.

In some embodiments, T cell priming, activation and/or proliferation is increased relative to prior to administration of the combination. In some embodiments, the T cells are CD4+ and/or CD8+ T cells. In some embodiments, T cell proliferation is detected by determining the percentage of Ki67+CD8+ T cells (eg, by FACS analysis). In some embodiments, T cell proliferation is detected by determining the percentage of Ki67+CD4+ T cells (eg, by FACS analysis). In some embodiments, the T cells are from peripheral blood. In some embodiments, the T cells are derived from a tumor.

Such combination therapies mentioned above encompass combined administration (in which two or more therapeutic agents are contained in the same or separate pharmaceutical compositions), as well as separate administration, in which case the administration of the antibodies of the invention may be administered Occurs before, concurrently with, and/or after the additional therapeutic agent(s). In one aspect, the administration of the anti-MerTK antibody and the administration of the additional therapeutic agent occur within about one month of each other, or within about one, two, or three weeks, or within about one, two, three, four, within five or six days. In one aspect, the antibody and additional therapeutic agent are administered to the patient on Day 1 of treatment. The antibodies of the invention can also be used in combination with radiation therapy.

The antibodies of the invention (and any additional therapeutic agents) can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, and if local treatment is desired, intralesional administration can be employed. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration can be by any suitable route, eg, by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is transient or chronic. Various dosing regimens are contemplated herein including, but not limited to, single or multiple administrations at various time points, bolus administration, and pulse infusion.

The antibodies of the invention will be formulated, administered and administered in a manner consistent with good medical practice. In this case, factors to consider include the specific disorder to be treated, the specific mammal to be treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the drug, the method of administration, the schedule of administration, and what is known to the medical practitioner. other factors. The antibody is not required, but can optionally be formulated with one or more agents currently used to prevent or treat the disease. The effective amount of these other agents depends on the amount of antibody present in the pharmaceutical composition, the type of disease or treatment, and other factors discussed above. These are generally used at the same dose and route of administration as described herein, or about 1% to 99% of the dose described herein, or at any dose and by any route determined empirically/clinically as appropriate.

For the prevention or treatment of disease, the appropriate dose of the antibodies of the invention (either alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, Administration of the antibody for prophylactic or therapeutic purposes, previous treatment, the patient's clinical history and response to the antibody, and the judgment of the attending physician. The antibody is suitably administered to the patient in one or a series of treatments. Depending on the type and severity of the disease, about 1 µg/kg to 15 mg/kg (eg, 0.1 mg/kg – -10 mg/kg) of antibody may be administered, for example, by one or more divided administrations or by continuous infusion. The initial candidate dose administered to the patient. A typical daily dose may range from about 1 µg/kg to 100 mg/kg or more, depending on the factors above. For repeated administrations over several days or longer, depending on the condition, treatment will generally continue until the desired suppression of disease symptoms occurs. An exemplary dose of the antibody will range from 0.05 mg/kg to about 10 mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg, or 10 mg/kg (or any combination thereof) may be administered to the patient. Such doses may be administered intermittently, eg, weekly or every three weeks (eg, such that the patient receives from about two to about twenty, or eg, about six doses of the antibody). An initial higher loading dose can be administered, followed by one or more lower doses.

Any anti-MerTK antibody disclosed herein and any immune checkpoint inhibitor known in the art or disclosed herein can be used in the methods of the present disclosure.

In some embodiments, the combination therapy of the present disclosure includes administration of an anti-MerTK antibody and an immune checkpoint inhibitor. Anti-MerTK antibodies and immune checkpoint inhibitors can be administered in any suitable manner known in the art. For example, the anti-MerTK antibody and the immune checkpoint inhibitor can be administered sequentially (at different times) or simultaneously (at the same time). In some embodiments, the immune checkpoint inhibitor and the anti-MerTK antibody are in separate compositions. In some embodiments, the immune checkpoint inhibitor is in the same composition as the anti-MerTK antibody.

The anti-MerTK antibody and the immune checkpoint inhibitor can be administered by the same route of administration or by different routes of administration. In some embodiments, the immune checkpoint inhibitor is intravenous, intramuscular, subcutaneous, topical, oral, transdermal, intraperitoneal, intraorbital, by implantation, by inhalation, intrathecal, intraventricular, or nasal Inject. In some embodiments, the anti-MerTK antibody is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecal, intraventricular, or intranasal give. Immune checkpoint inhibitors and anti-MerTK antibodies can be administered in effective amounts to prevent or treat disease. Appropriate doses of anti-MerTK antibody and/or immune checkpoint inhibitor may be based on the type of disease being treated, the type of immune checkpoint inhibitor and anti-MerTK antibody, the severity and course of the disease, the individual's clinical condition, the individual's clinical history and response to treatment and at the discretion of the attending physician. In some embodiments, combination therapy with an anti-MerTK antibody and an immune checkpoint inhibitor (eg, an anti-PD-1 or anti-PDL1 antibody) is synergistic, whereby the effective dose of the anti-MerTK antibody in the combination is relative to the anti-MerTK The effective dose of the antibody as a single agent is reduced.

As a general recommendation, a therapeutically effective amount of antibody administered to humans will be in the range of about 0.01 to about 50 mg/kg of patient body weight, whether by single administration or multiple administrations. In some embodiments, the antibody is used at about 0.01 to about 45 mg/kg, about 0.01 to about 40 mg/kg, about 0.01 to about 35 mg/kg, about 0.01 to about 30 mg/kg, about 0.01 to about 0.01 to about 25 mg/kg, about 0.01 to about 20 mg/kg, about 0.01 to about 15 mg/kg, about 0.01 to about 10 mg/kg, about 0.01 to about 5 mg/kg, or about 0.01 to about 1 mg/kg, For example. In some embodiments, the antibody is administered at 15 mg/kg. However, other dosage regimens can be used. In one embodiment, on day 1 of a 21 day cycle, at about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg A dose of mg, about 1000 mg, about 1100 mg, about 1200 mg, about 1300 mg, or about 1400 mg is administered to a human of an anti-MerTK antibody disclosed herein or an anti-PDL1 antibody disclosed herein. The dose may be administered in a single dose or in multiple doses (eg, 2 or 3 doses), eg, by infusion. The antibody dose administered in combination therapy can be reduced compared to monotherapy. The progress of this therapy is easily monitored by conventional techniques.

The progress of this therapy is easily monitored by conventional techniques and tests.

In one aspect, the present disclosure provides an anti-MerTK antibody as disclosed above (eg, a pegylated anti-MerTK antibody) for use as a medicament Antibody). In some embodiments, the use is the treatment of cancer. In some embodiments, the use is to reduce MerTK-mediated clearance of apoptotic cells. Further provided herein is the use of an anti-MerTK antibody as disclosed above in the manufacture of a medicament. In some embodiments, the medicament is used to treat cancer. In some embodiments, the cancer expresses a functional cGAS-STING cytosolic DNA sensing pathway protein. These proteins are part of the cGAS-STING signaling pathway and function in innate immunity to detect the presence of cytosolic DNA to trigger the expression of inflammatory genes. Examples of cGAS-STING cytosolic DNA sensing pathway proteins include, but are not limited to, cGAS, STING, TBK-1, IRF3, p50, p60, p65, NF-κΒ, ISRE, IKK, and STAT6. In some embodiments, the cancer expresses functional STING, functional Cx43, and functional cGAS polypeptides. A functional protein is one that is capable of performing its normal function in a cell. Examples of functional proteins can include wild-type proteins, marker proteins, and mutant proteins that retain or improve protein function compared to wild-type proteins. Protein function can be measured by any method known to those of skill in the art, including assaying protein or mRNA expression and sequencing genomic DNA or mRNA. In some embodiments, the cancer comprises tumor-associated macrophages expressing functional STING polypeptides. In some embodiments, the cancer comprises tumor cells expressing functional cGAS polypeptides. In some embodiments, the cancer comprises tumor cells expressing functional Cx43 polypeptides. In certain embodiments, the cancer is colorectal cancer. In some embodiments, the cancer is urothelial carcinoma, non-small cell lung cancer, triple negative breast cancer, small cell lung cancer, hepatocellular carcinoma, or melanoma. In some embodiments, the drug is used to reduce MerTK-mediated clearance of apoptotic cells.

On the other hand, the individual suffers from a cancer that exhibits (eg, has been shown to exhibit on a diagnostic test) the PDL1 biomarker. In some embodiments, the patient's cancer exhibits a low PDL1 biomarker. In some embodiments, the patient's cancer expresses a high PDL1 biomarker. In some embodiments of any method, assay and/or kit, when the PDL1 biomarker is in 0% of the sample, no PDL1 biomarker is present in the sample.

In some embodiments of any method, assay and/or kit, the PDL1 biomarker is present in the sample when the PDL1 biomarker makes up more than 0% of the sample. In some embodiments, the PDL1 biomarker is present in at least 1% of the sample. In some embodiments, the PDL1 biomarker is present in at least 5% of the sample. In some embodiments, the PDL1 biomarker is present in at least 10% of the sample.

In some embodiments of any method, assay and/or kit, the PDL1 biomarker in the sample is detected using a method selected from the group consisting of: FACS, Western blotting, ELISA, immunoprecipitation, immunoprecipitation Histochemistry, immunofluorescence, radioimmunoassay, dot blot, immunodetection methods, HPLC, surface plasmon resonance, spectroscopy, mass spectrometry, HPLC, qPCR, RT-qPCR, multiplex qPCR or RT-qPCR, RNA- seq, microarray analysis, SAGE, MassARRAY technology and FISH, and combinations thereof.

In some embodiments of any method, assay and/or kit, the PDL1 biomarker in a sample is detected by protein expression. In some embodiments, protein expression is determined by immunohistochemistry (IHC). In some embodiments, the PDL1 biomarker is detected using an anti-PDL1 antibody. In some embodiments, the PDL1 biomarker is detected by IHC as weak staining intensity. In some embodiments, the PDL1 biomarker is detected by IHC as moderate staining intensity. In some embodiments, the PDL1 biomarker is detected by IHC as strong staining intensity. In some embodiments, the PDL1 biomarker is detected on tumor cells, tumor-infiltrating immune cells, stromal cells, and any combination thereof. In some embodiments, the staining is membrane staining, cytoplasmic staining, or a combination thereof.

In some embodiments of any method, assay and/or kit, the absence of the PDL1 biomarker is detected as the absence or absence of staining in the sample. In some embodiments of any method, assay and/or kit, the presence of the PDL1 biomarker is detected as any staining in the sample. G. Products

In another aspect of the present invention, there is provided an article of manufacture containing materials useful in the treatment, prevention and/or diagnosis of the above-mentioned disorders. Manufactured products include containers and labels or drug inserts on or associated with containers. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. The container can be formed from a variety of materials such as glass or plastic. The container may contain a composition, by itself or in combination with another composition effective for treating, preventing, and/or diagnosing a condition, and may have a sterile access port (eg, the container may be a stopper with a perforation through a hypodermic needle) intravenous solution bag or vial). At least one active agent in the composition is an antibody of the invention. The label or package insert indicates that the composition can be used to treat the selected condition. In addition, the article of manufacture can include (a) a first container with a composition contained therein, wherein the composition comprises an antibody of the invention; and (b) a second container with a composition contained therein, wherein the composition comprises other Cytotoxic or other therapeutic agents. The article of manufacture of this embodiment of the invention may further comprise a package insert indicating that the composition can be used to treat a particular disease. Alternatively or additionally, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution and dextrose solution. From a commercial and user standpoint, it may further contain other materials including other buffers, diluents, filters, needles and syringes.

This description is believed to be sufficient to enable those skilled in the art to practice the compositions and methods of the present disclosure. Various modifications, in addition to those shown and disclosed herein, will be apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. All publications, patents, and patent applications cited herein are incorporated by reference in their entirety for all purposes. Example

The present invention will be more fully understood with reference to the following examples. However, it should not be construed as limiting the scope of the present disclosure. It should be understood that the examples and embodiments set forth herein are for illustrative purposes only and that various modifications or changes in view of them will occur to those skilled in the art and are to be included in the spirit and scope of this application and the scope of the appended claims within the range. Example 1 : Targeted Ocular Toxicity of Anti- MerTK Antibody Therapy

MerTK is expressed on the apical membrane of RPE cells and plays a key role in the phagocytosis of shed photoreceptor cells. The RPE of MerTK loss-of-function and constitutive knockout rodents is unable to remove detached photoreceptor outer segments by phagocytosis, resulting in retinal degeneration. People with defective MerTK function develop night blindness in early childhood, followed by a rapid loss of vision in early adulthood. Retinal toxicity has been observed when MerTK is blocked with small molecule MerTK inhibitors and anti-MERTK monoclonal antibodies (mAbs).

This example discloses an assessment of the potential retinal toxicity associated with administration of anti-MerTK monoclonal antibodies. Materials and methods First pilot toxicology study

In the first pilot toxicology study (Toxicology Study 1) in C57BL/6N mice, 14C9 anti-MerTK mAb was administered intravenously (IV) at doses of 0 (control), 10 and 30 mg/kg Administered twice weekly (BIW) for 4 weeks (9 doses total). Males and females (n = 4/sex/group) were assigned to use multiple toxicity groups and necropsies were scheduled at the end of the dosing period (Study Day 32). Blood from other mice in the 14C9 mAb-treated group (n = 6/sex/group) was collected for toxicokinetic assessment. Evaluation criteria included the following parameters: clinical observations, body weight, clinical pathology (hematology and clinical chemistry), anatomical pathology of the organ of major concern, and toxicokinetics. Second pilot toxicology study

To determine whether the retinopathy observed in Toxicology Study 1 is related to impaired retinal function, a follow-up pilot toxicology study (Toxicology Study 2) was performed in BALB/c mice, including full-field electrooculography (ffERG). In this study, 14C9 mAb was administered IV at 0, 5, or 30 mg/kg twice weekly (BIW; total 8 doses) or 45 mg/kg three times weekly (TIW; total 12 doses) for continuous 4 weeks, followed by a 6-week recovery period. Male and female mice (n = 4/sex/group) were assigned to toxicity groups at all dose levels, and terminal and convalescent necropsies were performed on study days 30 and 72, respectively. Blood was collected from other mice in the 14C9 mAb-treated group (n=6/sex/group) for toxicokinetic assessment. Evaluation criteria included the following parameters: clinical observations, body weight, clinical pathology (hematology and clinical chemistry), anatomical pathology of eyes and testes, ffERG and toxicokinetics. Third pilot toxicology study

A third pilot toxicology study was conducted in cynomolgus monkeys. The 13B4 anti-MerTK mAb was administered intravenously (IV) as a single dose or repeated doses every 3 weeks (Q3W) for 6 weeks. Female monkeys (n = 3/group) were assigned to dose groups, and terminal and convalescent necropsies were performed on study day 45. Animals receiving a single dose were dosed at 10 mg/kg, while Q3W dosed animals were dosed at 10 or 30 mg/kg (3 doses total). Evaluation criteria included the following parameters: clinical observation, food consumption, body weight, physical examination, ophthalmological examination, fundus photography, intraocular pressure, ERG (full field of view [ffERG] and multifocal [mfERG]), optical coherence tomography (OCT), clinical Pathology (eg hematology, clinical chemistry and urinalysis), anatomical pathology, toxicokinetics and anti-drug antibody measurements. result

MerTK is required for cell turnover in the outer segment of photoreceptors (POS), and sustained blockade of MerTK caused retinal toxicity ( Figure 1A ). To assess potential retinal toxicity associated with administration of anti-MerTK mAbs, three toxicology studies were performed.

The first of these toxicology studies, Toxicology Study 1, was conducted in C57BL/6N mice using mouse surrogate anti-MerTK mAb 14C9. In the first study, mice were administered 14C9 mAb at doses of 0, 10 and 30 mg/kg twice weekly for 4 weeks. An increase in the area under the curve (AUC) was observed in the toxicokinetic curve of 14C9 exposure. This increase was dose proportional as the increase was observed as the dose was increased from 10 to 30 mg/kg. One male mouse in the 30 mg/kg dose group was found dead on Study Day 5, and the cause of death was not considered to be related to 14C9 treatment. Two male mice in the 10 mg/kg dose group and one male mouse in the 30 mg/kg dose group were moribund and euthanized earlier on study days 22 and 18, respectively. The cause of death of these animals was considered procedure related and not related to 14C9 administration. For the remaining animals that survived to terminal necropsy, analysis of necropsy samples revealed 14C9-related toxicity in the eye and testis. 14C9-related outer retinal degeneration, consisting of minimal to mild photoreceptor (PR) vacuolation, increased PR layer cellularity, and shedding of the outer nuclear layer, was observed in these mice, and the incidence and severity increased in a dose-dependent manner. In addition, in the testis, moderate to marked seminiferous tubule atrophy and degeneration (associated with decreased testicular weight) was observed in all 14C9-treated male mice without any dose dependence. Based on these findings, it was concluded that administration of 14C9 mAb to C57BL/6N mice at 10 and 30 mg/kg BIW for 4 weeks caused retinal and testicular toxicity.

To determine whether the retinopathy observed in Toxicology Study 1 was related to impaired retinal function, a follow-up pilot toxicology study (Toxicology Study 2) was conducted. This second toxicology study was performed in BALB/c mice and combined full-field electrooculography (ffERG). In this study, 14C9 mAb was administered at 0, 5, or 30 mg/kg twice weekly or 45 mg/kg three times weekly for 4 weeks, followed by a 6-week recovery period. Toxicokinetic analysis showed that the AUC of 14C9 exposure in this study increased proportionally with doses as doses increased from 10 to 30 mg/kg BIW and 45 mg/kg TIW.

One female mouse in the 30 mg/kg dose group was found dead on Study Day 15, a cause not thought to be related to 14C9 treatment. One male mouse in the 30 mg/kg dose group died on Study Day 21 and was euthanized earlier, with a cause of death believed to be anesthesia-related. For the remaining animals in Toxicology Study 2 that survived to terminal necropsy, clinicopathological findings were limited to reversible, minimally reversible, minimal alanine aminotransferase (ALT)-based aspartate aminotransferase (AST) levels in the animals tested There was a minimal increase in globulin levels and a decrease in the albumin/globulin ratio at the 45 mg/kg dose. As shown in Figure 1C , 14C9-related degeneration of the outer retina was also observed, ranging from minimal to marked PR vacuolization, increased PR layer cellularity, shedding of the outer nuclear layer, and outer nuclear layer with degeneration or necrosis The cellularity decreased, and the severity increased in a dose-dependent manner. Similar findings were observed in one control male. Retinopathy was associated with reduced PR function as assessed by ffERG, neither of which resolved during recovery. Significant atrophy and degeneration of seminiferous tubules were observed in the testes of all male mice administered 14C9 without any dose dependence.

Based on the results of toxicology study 2, it was concluded that administration of 14C9 mAb to BALB/c mice at 10 or 30 mg/kg BIW or 45 mg/kg TIW for 4 weeks resulted in irreversible retinal and testicular lesions and PR function is impaired. During Toxicology Study 1, the retinopathy observed in BALB/c mice (albino strain) was significantly more extensive than that observed in C57BL6/N mice (pigmented strain), indicating that anti-MerTK mAb retinal There may be strain-related differences in the sensitivity of the effect.

Finally, a third toxicology study (Toxicology Study 3) was performed in cynomolgus monkeys using the unconjugated 13B4 anti-MerTK mAb of the hIgG1 isotype carrying the LALAPG mutation. Anti-MerTK mAb was administered intravenously (IV) as a single dose of 10 mg/kg or repeated doses of 10 or 30 mg/kg every 3 weeks (Q3W) for 6 weeks. Female monkeys (n = 3/group) were assigned to dose groups, and terminal and convalescent necropsies were performed on study day 45.

All animals survived until necropsy scheduled for study day 45. In all dose groups, changes in clinicopathological parameters were limited to minimally increased AST and slightly increased ALT levels (no histological correlation), which returned to baseline in animals that received a single 10 mg/kg 13B4 administration . Microscopic findings associated with 13B4 in lymphoid tissue were observed in animals dosed with 13B4 ≥ 10 mg/kg Q3W, including minimal or slight reduction of lymphocytes in the thymus (compared with reduced thymus weight in animals given 30 mg/kg). associated), and minimally increased nuclear fragmentation in follicular germinal centers in the spleen, mesenteric, and mandibular lymph nodes, and in the GALT/Peyer's plexus.

As shown in Figure 1B , at 30 mg/kg Q3W, minimal macrophage infiltration was observed in the photoreceptor layer of one animal. For this group, no evidence of photoreceptor damage was observed, nor changes in ffERG, mfERG, and OCT. In addition, no ERG changes were observed in the 10 mg/kg Q3W group. Evidence for a pharmacodynamic (PD) effect was observed at both dose levels, presented as increased cell-free DN and increased apoptotic debris in follicular germinal centers, likely due to impaired clearance by resident macrophages. Based on the results of toxicology study 3, it can be concluded that 13B4 mAb was administered to cynomolgus monkeys at a single 10 mg/kg dose or at repeated doses of 10 or 30 mg/kg Q3W (3 doses in total). It was well tolerated, and 13B4-related microscopic findings were found in the lymphoid tissue and eyes of animals that received multiple doses of 30 mg/kg 13B4.

Based on the results of these toxicology studies, the key safety risk of retinal toxicity is believed to be due to the targeted inhibition of MerTK-mediated RPE phagocytosis of detached photoreceptor outer segments. Retinal toxicity may also be driven by time above threshold, and based on available data, ERG or OCT assays lack detectability in cynomolgus monkeys and mice. Example 2 : Binding of large PEG polymers to anti- MerTK antibodies via engineered cysteines

This example discloses the manufacture of polymer-conjugated anti-MerTK antibodies. Materials and Methods Preparation of THIOMAB Antibodies for Conjugation

Antibodies with engineered cysteines at various positions are produced, expressed in CHO cells and purified by standard methods including protein A affinity chromatography followed by particle size sieve chromatography. Bindable PEG polymers with reactive maleimide moieties and a nominal molecular weight of 40 kDa were purchased from NOFAmerica under the catalog numbers GL2-400MA (2-arm branched) and ME-400MA (linear).

Engineered cysteines in the THIOMAB antibody format are typically blocked by cysteine or glutathione that occurs during mammalian cell expression. Standard procedures for deblocking are performed to remove cysteine or glutathione from the engineered cysteine to bind the desired moiety. Briefly, a 50-100 molar excess of the reducing agent DTT was added to 10 mg/mL antibody at basic pH 7.5-8.5 in 100 mM Tris pH 8.5, 150 mM NaCl, 2 mM EDTA, The mixture was incubated at room temperature for about 18 hours. The antibody is then purified using cation exchange to remove DTT as well as reduced cysteine and glutathione. The partially reduced antibody was re-oxidized with a 15 molar excess of DHAA in 20 mM Tris pH 7 at room temperature for 2-3 hours. Oxidation status was assessed using LC-MS to check the quality of intact oxidized antibodies and to monitor the presence of free light chains. The reoxidized antibody was then purified by cation exchange chromatography and formulated with 10 mM succinate pH 5, 150 mM NaCl, 2 mM EDTA. Preparation of PEG conjugates

The conjugation of the PEG polymer to the THIOMAB antibody was produced as follows. 4-6 molar equivalents of PEG dissolved in 10 mM succinate pH 5 was added to a 10 mg/mL antibody solution whose pH was adjusted with 1 M Hepes pH 7.2 to a final concentration of 100 mM Hepes pH 7.2. The binding reaction is carried out at room temperature for approximately 3 to 18 hours until the maximal antibody to polymer ratio is achieved. The progress of the binding reaction was monitored using HPLC equipped with a YARRA SEC-4000 column, which can resolve antibody to polymer ratios of 0, 1, and 2. The binding reaction was then purified using hydrophobic interaction chromatography. Adjust the pH of the binding reaction to pH 6.5 with 1 M sodium acetate, pH 5.0, and add ammonium sulfate to a final concentration of 800 mM. Conjugates were then purified using a ProPAC HIC-10 bonded silica 10x150 mm column and formulated using dialysis in 20 mM histidine acetate pH 5.5, 240 mM sucrose, 0.02% Tween-20. Taking into account that PEG does not absorb at 280 nm, the extinction coefficient of the conjugated antibody component was used to determine the antibody-based concentration of the formulated conjugate using UV absorbance at 280 nm.

Ratio and percent aggregation of polymer to antibody was characterized using HPLC equipped with a YARRA SEC-4000 and 0.2 M potassium phosphate, 0.25 M potassium chloride, pH 6.2, 15% isopropanol. Endotoxin levels were determined using Charles River EndoSafe cartridges. The hydrodynamic radii of the conjugates were determined using a UPLC SEC-MALS/QELS Wyatt system equipped with an Acclaim-1000 column, and the resulting data were processed and analyzed using the Astra 7.1.2 software package. Binding affinity determination

For binding affinity determination of 14C9.C90S THIOMAB conjugates in IgG, surface plasmon resonance (SRP) measurements were performed using a Biacore™-8K instrument (GE Healthcare). Briefly, each IgG variant was captured by a Series S sensor chip Protein A (29127555, GE Healthcare) to approximately 100RU, followed by a 3-fold serial dilution of mouse MerTK at a flow rate of 50 μl/min at 25°C Compounds (0.6 nM to 50 nM) were injected into HBS-EP buffer (100 mM 4-(2-hydroxyethyl)-1-piperidinesulfonic acid (HEPES) pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% (v/v) surfactant P20). Association rates ( _kon ) and dissociation rates ( _koff ) were calculated using a simple one-to-one Langmuir binding model (Biacore Insight software version 2.0). The equilibrium dissociation constant (K _D ) was calculated from the k _off /k _on ratio.

For binding affinity determination of THIOMAB conjugates of the 14C9.C90S_LC_K149C series in Fab, surface plasmon resonance (SRP) measurements were performed using a Biacore™-T200 instrument (GE Healthcare). Briefly, human Fc-labeled mouse MerTK (R&D 591-MR) was captured by a protein A sensor chip to achieve approximately 50RU, and then 3-fold serialization of each Fab variant at a flow rate of 50 μl/min at 25°C Dilutions (0.6 nM to 50 nM) were injected into HBS-EP buffer (100 mM 4-(2-hydroxyethyl)-1-piperidinesulfonic acid (HEPES) pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05 % (v/v) surfactant P20). Association rates ( _kon ) and dissociation rates ( _koff ) were calculated using a simple one-to-one Langmuir binding model (BIAcore T200 evaluation software version 2.0). The equilibrium dissociation constant (K _D ) was calculated from the k _off /k _on ratio. result

It is hypothesized that increasing antibody size (eg, hydrodynamic radius) through polymer conjugation may reduce ocular uptake of anti-MerTK antibodies and reduce on-target ocular toxicity observed with the parental antibody. Hydrophilic polymer infusion PEG has traditionally been used to increase the systemic half-life of small molecules and antibody fragments, and more recently to increase the vitreous half-life of intravitreal administered biologics. The rationale is to increase the size of the drug through conjugation with PEG, which is significantly reduced due to bypassing the glomerular filtration cutoff for systemic administration and bypassing the retinal clearance mechanism of IVT. Increasing the size of the anti-MerTK antibody may reduce distribution at the eye by preventing the antibody from crossing the blood-retinal barrier, while maintaining the ability of the antibody to bind to MerTK on tumor-associated macrophages in the tumor microenvironment, but has not been systematically studied Addressing the effect of size on blood-retinal barrier penetration (del Amo et al. (2018) Progress in Retinal and Eye Research 57 :134–185).

Hydrophilic polymers such as PEG are available in a variety of forms, with different available sizes, formats (linear, branched) and reactive moieties for binding to antibodies. Compared to other hydrophilic polymers, PEG has been widely used in the clinic and has received multiple approvals for PEGylated proteins and antibody fragments. Two forms of PEG with a nominal molecular weight of 40 kDa (linear and 2-arm branched form) were chosen for evaluation because previous data suggest that the form of PEG affects pharmacokinetics and biodistribution (Leong, SR et al. (2001) Cytokine 16:106-119; Vugmeyster, Y. et al. (2012) Bioconjugate chemistry 23:1452-1462).

For both murine and human anti-MerTK antibodies, engineered cysteine (THIOMAB) antibody technology was used to engineer site-specific cysteines into the antibody backbone for binding. Cysteine-maleimide conjugation chemistry in combination with THIOMAB antibodies allows for site-specific binding that enables the creation of homogeneous products with respect to antibody-to-polymer ratios and control of antibody-to-polymer linkages stability. A panel of predicted stable sites in the anti-murine MerTK mIgG2a format was screened that might translate to known stable sites on the anti-human MerTK huIgG1 format (Ohri, R. et al. (2018) Bioconjugate Chemistry 29:473 -485), where the selection of the alleles is based on the sequence and structure of the mIgG2a backbone and the human IgG1 backbone. Three sites on the constant domain of the Fab region of the antibody were chosen to engineer cysteines, LC K183, LC K149C and HC T182C.

Both linear and 2-arm branched 40 kDa PEG polymers with maleimide moieties react readily with unblocked, reduced cysteines of THIOMAB antibody for 3-18 hours after incubation at ambient temperature Polymer-antibody conjugates with a polymer-to-antibody ratio of at least 1.8 were made and less than 10% aggregation was observed during the reaction. The expected maximum polymer to antibody ratio is 2, since each single engineered cysteine appears twice in the heterodimeric antibody. Binding of 40 kDa PEG was monitored using SEC-HPLC and a YARRA SEC-4000 column capable of separating conjugates with antibody-to-polymer ratios of 2 and 1 from unbound antibody. After purification and formulation, the antibody to polymer ratio averaged 2.0 with 0% aggregation, which was frequently observed in different binding sites in both anti-mouse 14C9 mIgG2a and anti-human 13B4 huIgG1 antibodies. A schematic representation of the PEG conjugates formed according to this method is shown in Figure 2 .

After conjugation, the hydrodynamic ratios of the anti-MerTK PEG conjugates were characterized. The hydrodynamic radius and molecular weight of the anti-MerTK PEG conjugates were determined using UPLC equipped with MALS and QELS detectors, and the samples were analyzed on an ACCLAIM-1000 SEC column with phosphate buffered saline as the mobile phase. The molecular weights of the anti-MerTK 14C9 antibody and PEG conjugates were observed to be in agreement with the theoretical molecular weight values ( Table 2 ). The hydrodynamic radius of the unbound antibody was determined to be 5 nm, in good agreement with the reported value. The hydrodynamic radius of the PEGylated conjugate was determined to be twice that observed for the unconjugated antibody, and the linear PEG-40K was slightly larger at 10.8 nm compared to 10.3 nm for the 2-arm branched PEG-40K ( Table 1). 2 ). Table 2. Hydrodynamic radii of parental anti-mouse MerTK antibody and THIOMAB antibody-PEG conjugates measured by SEC-MALS/QELS. 14C9 antibody / conjugate MW (kDa) Rh (nm) Parent 14C9.C90S 149 5.1 14C9 LC K149C MC-PEG40K-Linear 251 10.8 14C9 LC K149C MC-PEG40K-branched chain 250 10.3

The binding affinity of the anti-MerTK PEG conjugates was assessed by Biacore analysis. As shown in Figures 3A-3B , PEG binding to 14C9 appears to have a modest improvement (3-5 fold) in binding affinity to the extracellular domain (ECD) of mouse MerTK. No differences were observed between the different sites where cysteines were engineered on the Fab domain. Furthermore, as shown in Figure 4 , when binding was analyzed using surface plasmon resonance (SPR), a modest increase (3-fold) in binding affinity for MerTK ECD was observed. in conclusion

Both murine anti-MerTK and human anti-MerTK THIOMAB antibodies bind readily to maleimide-linked linear and 2-arm branched 40 kDa PEG polymers, producing conjugates with an antibody-to-polymer ratio of 2.0 and in <5% aggregation in purified, formulated conjugates. The hydrodynamic radius of the conjugate is approximately two times larger than that of the unconjugated antibody, and the radius of the linear pegylated antibody is slightly larger than that of the branched form. Example 3 : In vitro cytotoxicity and macrophage binding assay of PEGylated anti- MerTK antibodies

This example reveals inhibition of macrophage-mediated efferocytosis by anti-MerTK antibody conjugates. Materials and Methods Reagents

Cytokine mouse M-CSF was obtained from PeproTech (Rocky Hill, NJ). Staurosporine from Streptomyces sp. was purchased from Sigma-Aldrich (Saint Louis, MI). pHrodo Red succinimidyl ester (pHrodo Red SE) was purchased from Thermo Scientific, (Whaltham, MA). mouse peritoneal macrophages

Mouse peritoneal macrophages (MPM) were either obtained from Cell Biologics (Cat# C57-6032TF, Chicago, IL) or freshly isolated in-house. For isolation of MPM, 8-week-old C57-BL6 mice were used. Mice were first euthanized with _CO , and then 10 mL of cold PBS (with 3% FBS) was injected into the abdominal cavity of each mouse. After withdrawing the liquid back into the same syringe, the collected peritoneal cell suspension was centrifuged at 1,400 rpm for 5 minutes, and the pelleted cells were resuspended in RPMI medium supplemented with 10% FBS. In temperature-sensitive Nunc UpCell 10 cm dishes (Thermo Scientific, Whaltham, MA), commercial MPM (6 million cells) were plated with 10% FBS and recombinant mouse M-CSF (30 ng/mL) Culture in RPMI medium for 3 days to promote recovery. After recovery, MPMs were harvested by washing with cold PBS without dissociation enzymes. Preparation of pHrodo- labeled apoptotic Jurkat cells

Jurkat cells (ATCC #TIB-152) were cultured in RPMI medium supplemented with 10% FBS. Cells from exponentially growing cultures were harvested and induced to apoptosis by treatment with 1.0 µM staurosporine for 4 hours at room temperature. Cells were then washed twice and resuspended in PBS to a density of 1.0 x ¹⁰⁶ cells/ml. Apoptotic cells were then labeled by incubating in 1.0 µM pHrodo Red succinimidyl for 1 hour at room temperature in the dark. After labeling, apoptotic cells were washed with PBS and slow centrifuged (750×g) 3 times for 10 min. Cells were then resuspended in RPMI medium + 10% FBS and used for exocytosis assays. Real-time Imaging Treasure Action Test via IncuCyte Zoom

Macrophages were seeded overnight at a density of ^4.0 x 104 cells/well on 96-well, low evaporation Nunclon Delta surface plates (Thermo Scientific) in RPMI medium supplemented with 10% FBS. Serial dilutions of antibodies were prepared in RPMI medium supplemented with 10% FBS and added to 96-well plates containing differentiated macrophages for 1 hour. After incubation with blocking antibody, freshly prepared ^pHrodo -labeled apoptotic cells were added to macrophages at a density of 8.0 x 105 cells/well. After adding apoptotic cells, the 96-well plate was placed in an IncuCyte Zoom instrument (Essen Biosciences; Ann Harbor, MI) and images were acquired every 15 minutes for 24-48 hours using a 10x objective and the red channel. Total red fluorescence intensity was quantified using the IncuCyte base software (2016B) and background noise was subtracted using the Top-Hat method. Cell fusion in each image was also quantified and used to normalize total red fluorescence intensity from different wells. The efferocytosis activity was quantified as (total red fluorescence intensity - background fluorescence intensity of apoptotic cells)/(macrophage fusion), and maximal activity (100%) was defined as in untreated macrophages + apoptotic cells the value obtained in the hole. Inhibition curves were generated using exocytosis recorded 4-8 hours after addition of apoptotic cells. The efferocytosis activity of duplicate or triplicate wells was plotted as a function of antibody concentration and the data were fitted to a sigmoidal model with Prism (Graphpad Software; La Jolla, CA). The _IC50 value was calculated as the concentration of test material required to reduce the exocytosis activity of macrophages by 50%. result

THIOMAB antibody-PEG conjugates ( FIG. 2 ) were prepared as disclosed in Example 2. The ability of the PEG-conjugated anti-MerTK antibody to inhibit mouse peritoneal macrophage-mediated burial was then assessed. All conjugated antibodies inhibited mouse peritoneal macrophage-mediated burial ( Figure 5A ). In particular, the PEG40K-branch-conjugated 14C9 antibody showed comparable potency to the parental mAb in inhibiting efferocytosis. The potency and efficacy of the 14C9 parental antibody and the PEG-conjugated antibody are summarized in Figure 5B .

The 14C9 LC K149C binder was selected for further evaluation. These 14C9 PEG antibody conjugates showed comparable inhibitory activity to the parental antibodies in mouse cytotoxicity assays ( Figures 6A-6B ). Both branched and linear PEG-conjugated antibodies inhibited mouse peritoneal macrophage-mediated efferocytosis. The PEG40K-branch-conjugated 14C9 antibody showed comparable potency to the parental mAb and was more potent than the PEG40K-linearly conjugated 14C9 antibody. The 14C9 LC K149C conjugate also exhibited an approximately 10-fold increase in EC50 cell binding as determined by flow cytometry ( Figure 7 ). The EC50s determined for binding to murine macrophages are summarized in Table 3 . Table 3. Parental anti-murine MerTK antibody and THIOMAB antibody-PEG conjugates bind to murine MerTK on primary peritoneal murine macrophages. 14C9 antibody / conjugate EC50 (nM) fold change Parent 14C9.C90S 0.2 - 14C9 LC K149C MC-PEG40K-Linear 1.8 9 14C9 LC K149C MC-PEG40K-branched chain 2.7 13.5

An exocytosis assay was also performed to evaluate 14C9 Fab conjugates. As shown in Figures 8A-8B , 14C9 Fab conjugates inhibited burial with similar potency to unconjugated 14C9 Fab. A reduction in potency was observed for all Fabs compared to the parental 14C9 antibody. Example 4 : In vivo characterization of PEGylated anti- MerTK antibodies

This example discloses the in vivo evaluation of PEG-conjugated anti-MerTK antibodies. Materials and methods Single high-dose study

For this study, female C57BL/6J mice were inoculated with 100,000 MC38 cells subcutaneously in the right lower abdomen. Animals were grouped based on body weight and tumor volume to ensure a similar distribution of pre-treatment body weight and starting tumor volume. Anti-gp120 (control antibody), anti-MerTK or antibody conjugates were administered by intravenous (i.v) injection at a dose of 30 mg/kg antibody fraction. Two days later, tumors and eyes were collected.

For the receptor occupancy assay, half of the tumor and eyes were dissociated to obtain a single cell suspension. Cells are stained with live/dead dye (L10119) and specific markers, followed by washing and fixation steps. Cells were then stained with anti-MerTK(14C9)-AF647 to assess MerTK receptor occupancy on tumor-associated macrophages (TAM) and RPE. For tumor pharmacodynamics assays, RNA was isolated from half of the tumors and gene expression was examined using a qPCR assay. Antibodies used: anti-CD45 (clone 30-F11), anti-CD11b (clone M1/70), anti-CD11c (clone HL3), anti-Ly6G (clone 1A8), anti-Ly6C (clone HK1.4), Anti-CD90.2 (clone 30-H12), class II anti-MHC (M5/114.15.1), anti-F4/80 (clone BM8), anti-CD24 (clone GK1.5), anti-CD31 (clone 390) ) and anti-RPE65 (401.8B11.3D9). Taqman probes used: IFNb (Mm00439546_s1), IFIT1 (Mm00515153_m1), USP18 (Mm01188805_m1) and IRF7 (Mm00516793_g1). Repeat high-dose study

In this study, anti-gp120 (control antibody), anti-MerTK or antibody conjugates were administered intravenously (IV) at a dose of 45 mg/kg antibody fraction on days 1 and 5 to native (non-tumor bearing). ) female C57BL/6J mice. On day 7, eyes were collected and MerTK occupancy on RPE was assessed as disclosed in the single high dose study (16-3045Y). Repeat low-dose study

Repeated low-dose studies were performed as revealed for repeated high-dose studies, except that the antibody or antibody conjugate was administered at a dose of 2.5 mg/kg antibody fraction on days 1 and 5. On day 7, receptor occupancy and tumor pharmacodynamic responses on tumor-associated macrophages (TAMs) were examined. result

During the initial study, PEG-conjugated 14C9 anti-MerTK antibody was administered intravenously to MC38 tumor-bearing mice at a single dose of 30 mg/kg. Eye and tumor samples were obtained to assess MerTK receptor occupancy, as well as to assess induction of interferon responses in tumors. As shown in Figure 9 , the parental antibody and the PEG-conjugated antibody exhibited comparable induction of interferon beta (IFNb) and interferon-stimulated genes (ISG) in tumors. The PEG-conjugated antibody slightly maintained MerTK occupancy on retinal pigment epithelial cells (RPE), but on tumor-associated macrophages (TAM) ( Figures 10A-10C ). In addition, mean fluorescence intensity (MFI) indicated partial occupancy, with approximately 70% or more of unoccupied MerTK remaining when using the antibody conjugate compared to approximately 27% when using the parent antibody. Approximately 20% of the RPE retained approximately 30% MerTK receptor occupancy when using the parental antibody, while at least 90% of the RPE retained at least 70% MerTK receptor occupancy when using the antibody conjugate.

After the initial single-dose study, a repeat high-dose study was conducted to evaluate MerTK on RPE in non-tumor-bearing mice following repeated treatment with high-dose anti-MerTK mAb (14C9) or Ab conjugate Occupancy rate. In this study, non-tumor bearing mice were administered anti-gp120 (control antibody), anti-MerTK or antibody conjugates at a dose of 45 mg/kg by intravenous injection on study days 1 and 5. On day 7, eye samples were collected to assess MerTK occupancy on the RPE.

PEG-conjugated antibodies reduced MerTK occupancy on RPE after repeated high doses of antibody were administered ( FIGS. 11A-11B ). After repeated doses of 45 mg/kg, the parent antibody almost completely occupied MerTK on the RPE. In contrast, the antibody conjugate retained at least 60% of the free MerTK+ RPE, while the PEG-40K branch (PEG-40K-B) retained approximately 90% of the free MerTK+ RPE. On RPE bearing functional or unoccupied MerTK (free MerTK+ cells), the level of unoccupied MerTK was improved when using the antibody conjugate compared to the parental antibody ( Figure 11C ). The PEG-40K-branched conjugate was relatively good, with about 90% of the RPE maintaining about 57% of the MerTK receptor occupancy. As described above, all antibody conjugates maintained at least 70% MerTK receptor occupancy in at least 90% of the RPE following a single high dose (30 mg/kg) administration.

Finally, PEG-conjugated antibodies were evaluated in repeated low-dose studies in which 2.5 mg/kg was administered to tumor-bearing mice on days 1 and 5. Both PEG40K-linear and PEG40K-branched antibodies demonstrated lower MerTK occupancy on TAMs compared to the parental Ab after repeated low dose administration ( Figures 12A-12C ). With the low dose (2.5 mg/kg x2) of the parental antibody, approximately 31% of the TAM retained approximately 35.8% of the free MerTK receptor, while approximately 69% of the TAM was fully occupied. In contrast, PEG40K-linear and PEG-40K-branched conjugated antibodies resulted in approximately 73% of the TAM retaining approximately 59% of the free MerTK receptor, while approximately 27% of the TAM was fully occupied. At low repeat doses, the parental antibody and the PEG-conjugated antibody exhibited comparable ISG induction in tumors ( Figure 13 ).

Although the disclosure has been described in some detail by way of illustrations and examples for purposes of clarity of understanding, such descriptions and examples should not be construed as limiting the scope of the disclosure. The disclosures of all patent and scientific literature cited herein are expressly incorporated by reference in their entirety.

Figures 1A-1C depict targeted ocular toxicity using anti-MerTK antibodies. Figure 1A is a schematic representation of the retinal pigment epithelium (RPE) depicting the expression of MerTK on the apical membrane of the RPE. Blockade of MerTK by anti-MerTK antibodies in the RPE results in an inability to phagocytose the shed photoreceptor outer segment (POS), leading to debris accumulation and eventual denaturation and loss of photoreceptors. Figure IB shows retinal macrophage infiltration in the outer photoreceptor (PR) layer of cynomolgus monkeys following administration of 30 mg/kg 13B4 anti-MerTK mAb every 3 weeks for 6 weeks (3 doses in total). The outer nuclear layer (ONL) is marked in the image. Hematoxylin and Eosin (HE), 200X. Figure 1C shows retinal toxicity in Balb/c mice following administration of 30 mg/kg 14C9 anti-MerTK mAb twice weekly for 4 weeks (8 doses in total). Compared to controls, animals receiving 30 mg/kg 14C9 had marked outer retinal degeneration characterized by PR vacuolization, increased cellularity of the PR layer, and decreased cellularity, degeneration, and necrosis of the ONL. Hematoxylin and Eosin (HE), 200X. Figure 2 is a schematic representation of anti-MerTK cysteine engineered antibody-PEG polymer conjugates comprising 40 kDa linear PEG (left) and 40 kDa 2 arm branched PEG (right). Figures 3A and 3B show analysis of 14C9 cysteine engineered antibody-PEG conjugate binding by Biacore SPR. A total of 6 THIOMAB conjugates on 14C9.C90S were assessed by Biacore SPR, including 2 PEGylated moieties (linear PEG40K and branched PEG40K) at 3 sites (LC_K149C, HC_T182C, LC_K183C), and untreated IgG binding affinity of treated or control treated 14C9.C90S to mouse MerTK. Figure 3A shows the results of a binding assay for linear PEG40K-bound 14C9THIOMAB, while Figure 3B shows the results for branched PEG40K-bound 14C9THIOMAB. The results show that the full PEG-conjugated 14C9.C90S IgG variant appears to have a modest improvement (3-5 fold) in binding affinity to mouse MerTK, with no difference between binding sites. Figure 4 shows the results of binding assays for 14C9 Fab conjugates. The binding affinities of the 14C9.C90S LC K149C series of THIOMAB conjugates (linear PEG40K and branched PEG40K) in Fab were assessed for surface-bound mouse MerTK by Biacore SPR. All variants exhibited about a 3-fold decrease in binding affinity for mouse MerTK, with no differences between binding sites. Figures 5A-5B show a comparison of the inhibitory activity of the PEG40-conjugated 14C9 anti-MerTK antibody and the parental mAb in mouse peritoneal macrophage-mediated efferocytosis. Figure 5A shows the results of the cytotoxicity assay with PEG-conjugated 14C9 antibody. Each figure represents the analysis of antibodies with different binding sites: K149C on the light chain (top left), K183C on the light chain (top right) or T182C on the heavy chain (bottom). Figure 5B summarizes the potency and efficacy of the 14C9 antibody and conjugates in an exocytosis assay. Figures 6A-6B show a comparison of the inhibitory activity of the PEG40-conjugated 14C9LCK149C antibody and the parental mAb in mouse peritoneal macrophage-mediated efferocytosis. Figure 6A shows the results of an exocytosis assay with PEG-conjugated 14C9LCK149C antibody using commercial (left) and in-house isolated mouse peritoneal macrophages (right). Figure 6B summarizes the potency and efficacy of the 14C9 antibody and the 14C9 LC K149C conjugate in an exocytosis assay. Figure 7 shows the binding of anti-murine MerTK parental antibody and THIOMAB antibody-PEG conjugates to primary murine peritoneal macrophages as measured by FACS. Figures 8A and 8B show a comparison of the inhibitory activity of PEG40-conjugated 14C9 with parental mAbs and Fabs in mouse peritoneal macrophage-mediated efferocytosis. Figure 8A shows the results of an exocytosis assay with PEG-conjugated 14C9 Fab. Figure 8B summarizes the potency and efficacy of 14C9 mAb, Fab and Fab PEG conjugates. Figure 9 shows the results of examining MerTK occupancy and type I IFN signaling following a single high dose (30 mg/kg) of PEG-conjugated 14C9 antibody or parental mAb (n = 8/group). Analysis of IFNb and interferon-stimulated gene (ISG) induction in tumors is shown. Figures 10A-10C show the results of analysis of MerTK occupancy in tumor-associated macrophages (TAM) and retinal pigment epithelial cells (RPE) by anti-MerTK antibody conjugates following a single high dose (30 mg/kg) of antibody. Figure 10A shows the results of the occupancy analysis on TAM and RPE (n = 8/group), while Figure 10B summarizes the occupancy results. Figure 1OC shows the level of unoccupied MerTK on free MerTK+ RPE cells. MFI (Mean Fluorescence Intensity) analysis indicated partial occupancy of MerTK. Figures 11A-11D show MerTK occupancy on RPE after repeated high dose treatment with anti-MerTK antibody conjugate (45 mg/kg on days 1 and 5). Figure 11A shows the occupancy levels of MerTK, while Figure 11B summarizes the observed occupancy. Figure 11C shows the levels of unoccupied MerTK on free MerTK+ RPE cells after incubation with anti-MerTK antibody conjugates. Figure 1 ID summarizes the molecular weights of the anti-MerTK antibody conjugates. Figures 12A-12C show analysis of MerTK occupancy in tumor-associated macrophages (TAMs) following low repeat dose treatment with anti-MerTK antibody conjugates (2.5 mg/kg on days 1 and 5). Figure 12A shows the results of an occupancy analysis of TAMs. Figure 12B summarizes the occupancy results. Figure 12C shows the level of unoccupied MerTK on free MerTK+ TAM cells. Figure 13 shows induction of IFNb and interferon-stimulated genes (ISGs) in tumors following low repeat doses (2.5 mg/kg) of PEG-conjugated anti-MerTK antibody.

            <![CDATA[<110> GENENTECH INC.]]> <![CDATA[<120> PEG-conjugated anti-MerTK antibodies and methods for their use]]> <![CDATA[<130 > 14639-20517.42]]> <![CDATA[<140> not yet assigned]]> <![CDATA[<141> as above]]> <![CDATA[<150> US 63/094,197]]> <![ CDATA[<151> 2020-10-20]]> <![CDATA[<160> 42]]> <![CDATA[<170> FastSEQ for Windows, Version 4.0]]> <![CDATA[< 210> 1]]> <![CDATA[<211> 5]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220 > ]]> <![CDATA[<223> Synthetic Construct]]> <![CDATA[<400> 1]]> Ser Tyr Ala Met Gly 1 5 <![CDATA[<210> 2]]> < ![CDATA[<211> 16]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Manual Sequence]]> <![CDATA[<220> ]]> <![ CDATA[<223> Synthetic Construct]]> <![CDATA[<400> 2]]> Ile Ile Asn Ser Tyr Gly Asn Thr Tyr Tyr Ala Asn Trp Ala Lys Gly 1 5 10 15 <![CDATA[<210 > 3]]> <![CDATA[<211> 8]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Manual Sequence]]> <![CDATA[<220> ]]> <![CDATA[<223> Synthetic Construct]]> <![CDATA[<400> 3]]> Asp Pro Gly Val Ser Ser Asn Leu 1 5 <![CDATA[<210> 4]] > <![CDATA[<211> 11]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Manual Sequence]]> <![CDATA[<220> ]]> < ![CDATA[<223> Synthetic Constructs]]> <![ CDATA[<400> 4]]> Gln Ala Ser Gln Asn Ile Tyr Ser Gly Leu Ala 1 5 10 <![CDATA[<210> 5]]> <![CDATA[<211> 7]]> <![ CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220> ]]> <![CDATA[<223> Synthetic Construct]]> <![ CDATA[<400> 5]]> Gly Ala Ser Lys Leu Ala Ser 1 5 <![CDATA[<210> 6]]> <![CDATA[<211> 11]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220> ]]> <![CDATA[<223> Synthetic Construct]]> <![CDATA[<400> 6]]> Gln Ala Thr Tyr Tyr Ser Ser Asn Ser Val Ala 1 5 10 <![CDATA[<210> 7]]> <![CDATA[<211> 5]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220> ]]> <![CDATA[<223> Synthetic Construct]]> <![CDATA[<400> 7]]> Ala Asn Thr Met Asn 1 5 <![CDATA[<210> 8]]> <![CDATA[<211> 16]]> <![CDATA[<212> PRT]]> <![ CDATA[<213> Artificial Sequence]]> <![CDATA[<220> ]]> <![CDATA[<223> Synthetic Construct]]> <![CDATA[<400> 8]]> Ile Phe Thr Ala Thr Gly Ser Thr Tyr Tyr Ala Thr Trp Val Asn Gly 1 5 10 15 <![CDATA[<210> 9]]> <![CDATA[<211> 12]]> <![CDATA[<212> PRT ]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220> ]]> <![CDATA[<223> Synthetic Construct]]> <![CDATA[<400> 9 ]]> Ser Gly Ser Gly Ser Ser Ser Gly Ala Phe Asn Ile 1 5 10 <![CDATA[<210> 10]]> <![CDATA[<211> 11]]> <![CDATA[<212> PRT]]> < ![CDATA[<213> Artificial Sequence]]> <![CDATA[<220> ]]> <![CDATA[<223> Synthetic Construct]]> <![CDATA[<400> 10]]> Gln Ala Ser Gln Ser Ile Ser Ser Ser Leu Ala 1 5 10 <![CDATA[<210> 11]]> <![CDATA[<211> 7]]> <![CDATA[<212> PRT]]> < ![CDATA[<213> Artificial Sequence]]> <![CDATA[<220> ]]> <![CDATA[<223> Synthetic Construct]]> <![CDATA[<400> 11]]> Ala Ala Ser Ile Leu Ala Ser 1 5 <![CDATA[<210> 12]]> <![CDATA[<211> 12]]> <![CDATA[<212> PRT]]> <![CDATA[< 213> Artificial Sequence]]> <![CDATA[<220> ]]> <![CDATA[<223> Synthetic Construct]]> <![CDATA[<400> 12]]> Gln Cys Thr Ser Tyr Gly Ser Leu Phe Leu Gly Pro 1 5 10 <![CDATA[<210> 13]]> <![CDATA[<211> 116]]> <![CDATA[<212> PRT]]> <![CDATA[ <213> Artificial Sequence]]> <![CDATA[<220> ]]> <![CDATA[<223> Synthetic Construct]]> <![CDATA[<400> 13]]> Glu Val Gln Leu Val Glu Ser Gly Glu Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Ser Ser Tyr 20 25 30 Ala Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Val35 40 45 Gly Ile Ile Asn Ser Tyr Gly Asn Thr Tyr Tyr Ala Asn Trp Ala Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Gly Ser Leu Arg Ala Glu Asp Met Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Asp Pro Gly Val Ser Ser Asn Leu Trp Gly Arg Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <![CDATA[<210> 14]]> <![ CDATA[<211> 109]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Manual Sequence]]> <![CDATA[<220> ]]> <![CDATA[ <223> Synthetic Construct]]> <![CDATA[<400> 14]]> Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asn Ile Tyr Ser Gly 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Ala Thr Tyr Tyr Ser Ser Asn 85 90 95 Ser Val Ala Phe Gly Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <![CDATA[<210> 15]]> <![CDATA[<211> 116]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Manual Sequence] ]> <![CDATA[<220> ]]> <![CDATA[<223> Synthetic Construct]]> <![CDATA[<400> 15]]> Glu Gln Gln Leu Val Glu Ser Gly Glu Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Ser Leu Ser Ser Tyr 20 25 30 Ala Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Ile Ile Asn Ser Tyr Gly Asn Thr Tyr Tyr Ala Asn Trp Ala Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Ser Ser Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Gly Ser Leu Arg Ala Glu Asp Met Ala Val Tyr Phe Cys Ala 85 90 95 Arg Asp Pro Gly Val Ser Ser Asn Leu Trp Gly Pro Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <![CDATA[<210> 16]]> <![CDATA[<211> 109 ]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220> ]]> <![CDATA[<223> Synthetic Construct ]]> <![CDATA[<400> 16]]> Asp Val Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asn Ile Tyr Ser Gly 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Pro Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Ala Thr Tyr Tyr Ser Ser Asn 85 90 95 Ser Val Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <![CDATA[<210> 17]]> <![CDATA[<211> 113]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Manual Sequence]]> <![CDATA[<220> ]]> <![CDATA[<223> Synthetic Construct]]> <![CDATA[<400> 17]]> Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro 1 5 10 15 Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Tyr Ala 20 25 30 Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly 35 40 45 Ile Ile Asn Ser Tyr Gly Asn Thr Tyr Tyr Ala Asn Trp Ala Lys Gly 50 55 60 Arg Phe Thr Ile Ser Arg Thr Ser Thr Thr Val Asp Leu Arg Met Pro 65 70 75 80 Ser Leu Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg As p Pro 85 90 95 Gly Val Ser Ser Asn Leu Trp Gly Pro Gly Thr Leu Val Thr Val Ser 100 105 110 Ser <![CDATA[<210> 18]]> <![CDATA[<211> 109]]> < ![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220> ]]> <![CDATA[<223> Synthetic Construct]]> < ![CDATA[<400> 18]]> Asp Val Val Met Thr Gln Thr Pro Ala Ser Val Ser Glu Pro Val Gly 1 5 10 15 Gly Thr Val Thr Ile Lys Cys Gln Ala Ser Gln Asn Ile Tyr Ser Gly 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Lys Leu Ala Ser Gly Val Ser Ser Arg Phe Lys Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Asp Leu Glu Cys 65 70 75 80 Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Ala Thr Tyr Tyr Ser Ser Asn 85 90 95 Ser Val Ala Phe Gly Gly Gly Thr Glu Val Val Val Lys 100 105 <![CDATA[<210> 19]]> <![CDATA[<211> 117]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Manual Sequence]]> <![CDATA[<220> ] ]> <![CDATA[<223> Synthetic Construct]]> <![CDATA[<400> 19]]> Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro 1 5 10 15 Leu Thr Leu Thr Cys Thr Val Ser Gly Ile Asp Leu Ser Ala Asn Thr 20 25 30 Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly 35 40 45 Ile Phe Thr Ala Thr Gly Ser Thr Tyr Tyr Ala Thr Trp Val Asn Gly 50 55 60 Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu Lys Ile Thr 65 70 75 80 Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Ser Gly 85 90 95 Ser Gly Ser Ser Ser Gly Ala Phe Asn Ile Trp Gly Pro Gly Thr Leu 100 105 110 Val Thr Val Ser Leu 115 <![CDATA[<210> 20]]> <![CDATA[<211> 110] ]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220> ]]> <![CDATA[<223> Synthetic Construct] ]> <![CDATA[<400> 20]]> Asp Pro Val Leu Thr Gln Thr Pro Ala Ser Val Ser Glu Pro Val Gly 1 5 10 15 Gly Thr Val Thr Ile Lys Cys Gln Ala Ser Gln Ser Ile Ser Ser Ser 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ile Leu Ala Ser Glu Ile Ser Ser Arg Phe Lys Gly 50 55 60 Ser Arg Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Asp Leu Glu Cys 65 70 75 80 Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Cys Thr Ser Tyr Gly Ser Leu 85 90 95 Phe Leu Gly Pro Phe Gly Gly Gly Thr Glu Val Val Val Lys 100 105 110 <! [CDATA[<210> 21]]> <![CDATA[<211> 445]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Manual Sequence]]> <![ CDATA[<220> ]]> <![CDATA[<223> Synthetic Construct]]> <![CDATA[<400> 21]]> Glu Val Gln Leu Val Glu Ser Gly Glu Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Ser Ser Tyr 20 25 30 Ala Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Val 35 40 45 Gly Ile Ile Asn Ser Tyr Gly Asn Thr Tyr Tyr Ala Asn Trp Ala Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Gly Ser Leu Arg Ala Glu Asp Met Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Asp Pro Gly Val Ser Ser Asn Leu Trp Gly Arg Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser A rg Trp 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 <![CDATA[<210> 22] ]> <![CDATA[<211> 445]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Manual Sequence]]> <![CDATA[<220> ]]> <![CDATA[<223> Synthetic Construct]]> <![CDATA[<400> 22]]> Glu Val Gln Leu Val Glu Ser Gly Glu Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Ser Ser Tyr 20 25 30 Ala Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Val 35 40 45 Gly Ile Ile Asn Ser Tyr Gly Asn Thr Tyr Tyr Ala Asn Trp Ala Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Gly Ser Leu Arg Ala Glu Asp Met Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Asp Pro Gly Val Ser Ser Asn Leu Trp Gly Arg Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr Gly Ser Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 < ![CDATA[<210> 23]]> <![CDATA[<211> 216]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <! [CDATA[<220> ]]> <![CDATA[<223> Synthetic Construct]]> <![CDATA[<400> 23]]> Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asn Ile Tyr Ser Gly 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Ala Thr Tyr Tyr Ser Ser Asn 85 90 95 Ser Val Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val 100 105 110 Ala A la Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys 115 120 125 Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg 130 135 140 Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn 145 150 155 160 Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser 165 170 175 Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys 180 185 190 Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr 195 200 205 Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <![CDATA[<210> 24]]> <![CDATA[<211> 440]]> <! [CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![ CDATA[<220> ]]> <![CDATA[<223> Synthetic Construct]]> <![CDATA[<400> 24]]> Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser Asn Ser 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Asn Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 100 105 110 Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser 115 120 125 Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp 130 135 140 Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr 145 150 155 160 Ser Gly Val His Thr Phe Pro Ala Val Leu Gln S er Ser Gly Leu Tyr 165 170 175 Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys 180 185 190 Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp 195 200 205 Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala 210 215 220 Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 225 230 235 240 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 245 250 255 Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val 260 265 270 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 275 280 285 Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 290 295 300 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser A sn Lys Gly 305 310 315 320 Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 325 330 335 Arg Glu Pro Gln Val Tyr Thr Leu Pro Ser Gln Glu Glu Met Thr 340 345 350 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 355 360 365 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 370 375 380 Lys Thr Thr Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 385 390 395 400 Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe 405 410 415 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 420 425 430 Ser Leu Ser Leu Ser Leu Gly Lys 435 440 <![CDATA[<210> 25]]> <![CDATA[<211> 214]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Manual Sequence]]> <![CD ATA[<220> ]]> <![CDATA[<223> Synthetic Construct]]> <![CDATA[<400> 25]]> Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Ser Asn Trp Pro Arg 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <![CDATA[<210> 26]]> <![CDATA[<211> 447]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]] > <![CDATA[<220> ]]> <![CDATA[<223> Synthetic Construct]]> <![CDATA[<400> 26]]> Gln Val Gln Leu Val Gln Ser Gly Val Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe Asn Glu Lys Phe 50 55 60 Lys Asn Arg Val Thr Leu Thr Thr Asp Ser Ser Thr Thr Thr Ala Tyr 65 70 75 80 Met Glu Leu Lys Ser Leu Gln Phe Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Asp Tyr Arg Phe Asp Met Gly Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro 210 215 220 Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu 260 265 270 Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440 445 <![CDATA[<210> 27]]> <![CDATA[<211> 218]]> <![CDATA[<212> PRT]]> <![CDATA[< 213> Artificial Sequence]]> <![CDATA[<220> ]]> <![CDATA[<223> Synthetic Construct]]> <![CDATA[<400> 27]]> Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Lys Gly Val Ser Thr Ser 20 25 30 Gly Tyr Ser Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro 35 40 45 Arg Leu Leu Ile Tyr Leu Ala Ser Tyr Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Ser Arg 85 90 95 Asp Leu Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 100 105 110 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155 160 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200 205 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <![CDATA[<210> 28]]> <![CDATA[< 211> 10]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Manual Sequence]]> <![CDATA[<220> ]]> <![CDATA[<223> Synthetic Construct]]> <![CDATA[<400> 28]]> Gly Phe Thr Phe Ser Asp Ser Trp Ile His 1 5 10 <![CDATA[<210> 29 ]]> <![CDATA[<211> 18]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Manual Sequence]]> <![CDATA[<220> ]] > <![CDATA[<223> Synthetic Construct]]> <![CDATA[<400> 29]]> Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 1 5 10 15 Lys Gly < ![CDATA[<210> 30]]> <![CDATA[<211> 9]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Manual Sequence]]> <! [CDATA[<220> ]]> <![CDATA[<223> Synthetic Construct]]> <![CDATA[<400> 30]]> Arg His Trp Pro Gly Gly Phe Asp Tyr 1 5 <![CDATA [<210> 31]]> <![CDATA[<211> 11]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[ <220> ]]> <![CDATA[<223> Synthetic Construct]]> <![CDATA[<400> 31]]> Arg Ala Ser Gln Asp Val Ser Thr Ala Val Ala 1 5 10 <![CDATA [<210> 32]]> <![CDATA[<211> 7]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[ <220> ]]> <![CDATA[<223> Synthetic Construct]]> <![CDATA[<400> 32]]> Ser Ala Ser Phe Leu Tyr Ser 1 5 <![CDATA[<210> 33 ]]> <![CDATA[<211> 9]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Manual Sequence]]> <![CDATA[<220> ]] > <![CDATA[<223> Synthetic Construct]]> <![CDATA[<400> 33]]> Gln Gln Tyr Leu Tyr His Pro Ala Thr 1 5 <![CDATA[<210> 34]]> <![CDATA[<211> 118]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Manual Sequence]]> <![CDATA[<220> ]]> <! [CDATA[<223> Synthetic Construct]]> <![CDATA[<400> 34]]> Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser 20 25 30 Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <![CDATA[<210> 35]]> <![CDATA[<211> 108]]> <![CDATA[<212> PRT] ]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220> ]]> <![CDATA[<223> Synthetic Construct]]> <![CDATA[<400> 35] ]> Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Tyr His Pro Ala 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 <![CDATA[< 210> 36]]> <![CDATA[<211> 447]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220 > ]]> <![CDATA[<223> Synthetic Construct]]> <![CDATA[<400> 36]]> Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser 20 25 30 Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg His Trp Pro Gly Gl y Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pr o Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val 290 295 300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350 Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn As n Tyr Lys Thr Thr Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 <![CDATA[<210> 37]]> <![CDATA[<211> 214]]> <![CDATA[< 212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220> ]]> <![CDATA[<223> Synthetic Construct]]> <![CDATA[< 400> 37]]> Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Tyr His P ro Ala 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <![CDATA [<210> 38]]> <![CDATA[<211> 449]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[ <220> ]]> <![CDATA[<223> Synthetic Construct]]> <![ CDATA[<400> 38]]> Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ile Met Met Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Tyr Pro Ser Gly Gly Ile Thr Phe Tyr Ala Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ile Lys Leu Gly Thr Val Thr Thr Val Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly <![CDATA[<210> 39]]> <![CDATA[<211> 216]]> <![CDATA[<212> PRT]]> <![CDATA[<213>Artificial Sequence]]> <![CDATA[<220> ]]> <![CDATA[<223> Synthetic Construct]]> <![CDATA[<400> 39]]> Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr 20 25 30 Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45 Met Ile Tyr Asp Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser 85 90 95 Ser Thr Arg Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly Gln 100 105 110 Pro Lys Ala Asn Pro Thr Val Thr Leu Phe Pro Pro Ser Ser Glu Glu 115 120 125 Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr 130 135 140 Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro Val Lys 145 150 155 160 Ala Gly Val Glu Thr Thr Lys Pro Se r Lys Gln Ser Asn Asn Lys Tyr 165 170 175 Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His 180 185 190 Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys 195 200 205 Thr Val Ala Pro Thr Glu Cys Ser 210 215 <![CDATA[<210> 40]]> <![CDATA[<211> 450]]> <![CDATA[<212> PRT]]> <![CDATA[ <213> Artificial Sequence]]> <![CDATA[<220> ]]> <![CDATA[<223> Synthetic Construct]]> <![CDATA[<400> 40]]> Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr 20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Asn Ile Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly Gly Trp Phe Gly Glu Leu Ala Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys 210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Lys Val Ser Asn Lys Ala Leu Pro Ala Ser Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Pro Gly 450 <![CDATA[<210> 41]]> <![CDATA[<211> 215]]> <![CDATA[<212> PRT] ]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220> ]]> <![CDATA[<223> Synthetic Construct]]> <![CDATA[<400> 41] ]> Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Arg Val Ser Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Asp Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Leu Pro 85 90 9 5 Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala 100 105 110 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125 Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135 140 Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145 150 155 160 Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165 170 175 Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 180 185 190 Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195 200 205 Ser Phe Asn Arg Gly Glu Cys 210 215 <![CDATA[< 210> 42]]> <![CDATA[<211> 999]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<400 > 42]]> Met Gly Pro Ala Pro Leu Pro Leu Leu Leu Gly Leu Phe Leu Pro Ala 1 5 10 15 Leu Trp Arg Arg Ala Ile Thr Glu Ala Arg Glu Glu Ala Lys Pro Tyr 20 25 30 Pro Leu Phe Pro Gly Pro Phe Pro Gly Ser Leu Gln Thr Asp His Thr 35 40 45 Pro Leu Leu Ser Leu Pro His Ala Ser Gly Tyr Gln Pro Ala Leu Met 50 55 60 Phe Ser Pro Thr Gln Pro Gly Arg Pro His Thr Gly Asn Val Ala Ile 65 70 75 80 Pro Gln Val Thr Ser Val Glu Ser Lys Pro Leu Pro Pro Leu Ala Phe 85 90 95 Lys His Thr Val Gly His Ile Ile Leu Ser Glu His Lys Gly Val Lys 100 105 110 Phe Asn Cys Ser Ile Ser Val Pro Asn Ile Tyr Gln Asp Thr Thr Ile 115 120 125 Ser Trp Trp Lys Asp Gly Lys Glu Leu Leu Gly Ala His His Ala Ile 130 135 140 Thr Gln Phe Tyr Pro Asp Asp Glu Val Thr Ala Ile Ile Ala Ser Phe 145 150 155 160 Ser Ile Thr Ser Val Gln Arg Ser Asp Asn Gly Ser Tyr Ile Cys Lys 165 170 175 Met Lys Ile Asn Asn Glu Glu Ile Val Ser Asp Pro Ile Tyr Ile G lu 180 185 190 Val Gln Gly Leu Pro His Phe Thr Lys Gln Pro Glu Ser Met Asn Val 195 200 205 Thr Arg Asn Thr Ala Phe Asn Leu Thr Cys Gln Ala Val Gly Pro Pro 210 215 220 Glu Pro Val Asn Ile Phe Trp Val Gln Asn Ser Ser Arg Val Asn Glu 225 230 235 240 Gln Pro Glu Lys Ser Pro Ser Val Leu Thr Val Pro Gly Leu Thr Glu 245 250 255 Met Ala Val Phe Ser Cys Glu Ala His Asn Asp Lys Gly Leu Thr Val 260 265 270 Ser Lys Gly Val Gln Ile Asn Ile Lys Ala Ile Pro Ser Pro Pro Thr 275 280 285 Glu Val Ser Ile Arg Asn Ser Thr Ala His Ser Ile Leu Ile Ser Trp 290 295 300 Val Pro Gly Phe Asp Gly Tyr Ser Pro Phe Arg Asn Cys Ser Ile Gln 305 310 315 320 Val Lys Glu Ala Asp Pro Leu Ser Asn Gly Ser Val M et Ile Phe Asn 325 330 335 Thr Ser Ala Leu Pro His Leu Tyr Gln Ile Lys Gln Leu Gln Ala Leu 340 345 350 Ala Asn Tyr Ser Ile Gly Val Ser Cys Met Asn Glu Ile Gly Trp Ser 355 360 365 Ala Val Ser Pro Trp Ile Leu Ala Ser Thr Thr Glu Gly Ala Pro Ser 370 375 380 Val Ala Pro Leu Asn Val Thr Val Phe Leu Asn Glu Ser Ser Asp Asn 385 390 395 400 Val Asp Ile Arg Trp Met Lys Pro Pro Thr Lys Gln Gln Asp Gly Glu 405 410 415 Leu Val Gly Tyr Arg Ile Ser His Val Trp Gln Ser Ala Gly Ile Ser 420 425 430 Lys Glu Leu Leu Glu Glu Glu Val Gly Gln Asn Gly Ser Arg Ala Arg Ile 435 440 445 Ser Val Gln Val His Asn Ala Thr Cys Thr Val Arg Ile Ala Ala Val 450 455 460 Thr Arg Gly Gly Val Gly Pro Phe Ser Asp Pro Val Lys Ile P he Ile 465 470 475 480 Pro Ala His Gly Trp Val Asp Tyr Ala Pro Ser Ser Thr Pro Ala Pro 485 490 495 Gly Asn Ala Asp Pro Val Leu Ile Ile Phe Gly Cys Phe Cys Gly Phe 500 505 510 Ile Leu Ile Gly Leu Ile Leu Tyr Ile Ser Leu Ala Ile Arg Lys Arg 515 520 525 Val Gln Glu Thr Lys Phe Gly Asn Ala Phe Thr Glu Glu Asp Ser Glu 530 535 540 Leu Val Val Asn Tyr Ile Ala Lys Lys Ser Phe Cys Arg Arg Ala Ile 545 550 555 560 Glu Leu Thr Leu His Ser Leu Gly Val Ser Glu Glu Leu Gln Asn Lys 565 570 575 Leu Glu Asp Val Val Ile Asp Arg Asn Leu Leu Ile Leu Gly Lys Ile 580 585 590 Leu Gly Glu Gly Glu Phe Gly Ser Val Met Glu Gly Asn Leu Lys Gln 595 600 605 Glu Asp Gly Thr Ser Leu Lys Val Ala Val Lys T hr Met Lys Leu Asp 610 615 620 Asn Ser Ser Gln Arg Glu Ile Glu Glu Phe Leu Ser Glu Ala Ala Cys 625 630 635 640 Met Lys Asp Phe Ser His Pro Asn Val Ile Arg Leu Leu Gly Val Cys 645 650 655 Ile Glu Met Ser Ser Gln Gly Ile Pro Lys Pro Met Val Ile Leu Pro 660 665 670 Phe Met Lys Tyr Gly Asp Leu His Thr Tyr Leu Leu Tyr Ser Arg Leu 675 680 685 Glu Thr Gly Pro Lys His Ile Pro Leu Gln Thr Leu Leu Lys Phe Met 690 695 700 Val Asp Ile Ala Leu Gly Met Glu Tyr Leu Ser Asn Arg Asn Phe Leu 705 710 715 720 His Arg Asp Leu Ala Ala Arg Asn Cys Met Leu Arg Asp Asp Met Thr 725 730 735 Val Cys Val Ala Asp Phe Gly Leu Ser Lys Lys Ile Tyr Ser Gly Asp 740 745 750 Tyr Tyr Arg Gln Gly Arg Ile Ala L ys Met Pro Val Lys Trp Ile Ala 755 760 765 Ile Glu Ser Leu Ala Asp Arg Val Tyr Thr Ser Lys Ser Asp Val Trp 770 775 780 Ala Phe Gly Val Thr Met Trp Glu Ile Ala Thr Arg Gly Met Thr Pro 785 790 795 800 Tyr Pro Gly Val Gln Asn His Glu Met Tyr Asp Tyr Leu Leu His Gly 805 810 815 His Arg Leu Lys Gln Pro Glu Asp Cys Leu Asp Glu Leu Tyr Glu Ile 820 825 830 Met Tyr Ser Cys Trp Arg Thr Asp Pro Leu Asp Arg Pro Thr Phe Ser 835 840 845 Val Leu Arg Leu Gln Leu Glu Lys Leu Leu Glu Ser Leu Pro Asp Val 850 855 860 Arg Asn Gln Ala Asp Val Ile Tyr Val Asn Thr Gln Leu Leu Glu Ser 865 870 875 880 Ser Glu Gly Leu Ala Gln Gly Ser Thr Leu Ala Pro Leu Asp Leu Asn 885 890 895 Ile Asp Pro Asp Ser I le Ile Ala Ser Cys Thr Pro Arg Ala Ala Ile 900 905 910 Ser Val Val Thr Ala Glu Val His Asp Ser Lys Pro His Glu Gly Arg 915 920 925 Tyr Ile Leu Asn Gly Gly Ser Glu Glu Trp Glu Asp Leu Thr Ser Ala 930 935 940 Pro Ser Ala Ala Val Thr Ala Glu Lys Asn Ser Val Leu Pro Gly Glu 945 950 955 960 Arg Leu Val Arg Asn Gly Val Ser Trp Ser His Ser Ser Met Leu Pro 965 970 975 Leu Gly Ser Ser Leu Pro Asp Glu Leu Leu Phe Ala Asp Asp Ser Ser 980 985 990 Glu Gly Ser Glu Val Leu Met 995
      

Claims (69)

A PEGylated antibody that binds to MerTK, wherein the antibody binds to one or more polyethylene glycol (PEG) polymers; wherein each of the PEG polymer(s) is in engineered cysteine residues is bound to the heavy or light chain of the antibody; and wherein the antibody comprises: (1) A heavy chain variable domain (VH) comprising (a) CDR-H1 containing the amino acid sequence of SYAMG (SEQ ID NO: 1), (b) an amine containing IINSYGNTYYANWAKG (SEQ ID NO: 2) The amino acid sequence of CDR-H2 and (c) the CDR-H3 containing the amino acid sequence of DPGVSSNL (SEQ ID NO: 3), and the light chain variable domain (VL), which comprises (d) the amino acid sequence containing QASQNIYSGLA (SEQ ID NO: 3). NO: 4) CDR-L1 of amino acid sequence, (e) CDR-L2 containing amino acid sequence of GASKLAS (SEQ ID NO: 5) and (f) amine containing QATYYSSNSVA (SEQ ID NO: 6) CDR-L3 of the amino acid sequence; or (2) A heavy chain variable domain (VH) comprising (a) CDR-H1 containing the amino acid sequence of ANTMN (SEQ ID NO: 7), (b) an amine containing IFTATGSTYYATWVNG (SEQ ID NO: 8) CDR-H2 of the amino acid sequence and (c) CDR-H3 containing the amino acid sequence of SGSGSSSGAFNI (SEQ ID NO: 9), and a light chain variable domain (VL) comprising (d) a QASQSISSSLA (SEQ ID NO: 9) NO: 10) CDR-L1 of amino acid sequence, (e) CDR-L2 containing amino acid sequence of AASILAS (SEQ ID NO: 11) and (f) amine containing QCTSYGSLFLGP (SEQ ID NO: 12) The amino acid sequence of CDR-L3. 如請求項1 之抗體，其中該VH 域包含與EVQLVESGEGLVQPG GSLRLSCAASGFSLSSYAMGWVRQAPGKGLEYVGIINSYGNTYYANWAKGRFTISRDNSKNTVYLQMGSLRAEDMAVYYCARDPGVSSNLWGRGTLVTVSS (SEQ ID NO: 13) 之胺基酸序列具有至少95% 序列同一性的序列；及/或該VL 域包含與DIQMTQSPSTLSASVGDRVTITCQASQ NIYSGLAWYQQKPGKAPKLLIYGASKLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQATYYSSNSVAFGGGTKVEIK (SEQ ID NO: 14) amino acid sequence with at least 95% sequence identity. 如請求項 2 之抗體，其中該 VH 域包含 EVQLVESGEGLVQPGGSLR LSCAASGFSLSSYAMGWVRQAPGKGLEYVGIINSYGNTYYANWAKGRFTISRDNSKNTVYLQMGSLRAEDMAVYYCARDPGVSSNLWGRGTLVTVSS (SEQ ID NO: 13) 之胺基酸序列；且該 VL 域包含 DIQMTQSPSTLSASVGDRVTITCQASQNIYSGLAWYQQKPGKAPKLLIYGASKLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQATYYSSNSVAFGGGTKVEIK (SEQ ID NO: 14) 之胺基酸序列。 如請求項 2 或請求項 3 之抗體，其中該重鏈包含 EVQLVESGEGLVQPGGSLRLSCAASGFSLSSYAMGWVRQAPGKGLEYVGIINSYGNTYYANWAKGRFTISRDNSKNTVYLQMGSLRAEDMAVYYCARDPGVSSNLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 21) 之胺基酸序列。 如請求項 2 或請求項 3 之抗體，其中該重鏈包含 EVQLVESGEGLVQPGGSLRLSCAASGFSLSSYAMGWVRQAPGKGLEYVGIINSYGNTYYANWAKGRFTISRDNSKNTVYLQMGSLRAEDMAVYYCARDPGVSSNLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 22) 之胺基酸序列。 如請求項 2 至 5 中任一項之抗體，其中該輕鏈包含 DIQMTQSPSTLSASVGDRVTITCQASQNIYSGLAWYQQKPGKAPKLLIYGASKLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQATYYSSNSVAFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 23) 之胺基酸序列。 The antibody of any one of claims 1 to 6, wherein the antibody is conjugated to one or two PEG polymers. The antibody of any one of claims 1 to 7, wherein the PEG polymer(s) are linear PEG polymers. The antibody of any one of claims 1 to 7, wherein the PEG polymer(s) are branched PEG polymers. The antibody of claim 9, wherein the branched PEG polymer(s) comprises 2 branches. The antibody of any one of claims 1 to 10, wherein the pegylated antibody has a hydrodynamic radius greater than about 6 nm. The antibody of any one of claims 1 to 11, wherein the pegylated antibody has a hydrodynamic radius of greater than or equal to about 10 nm. The antibody of any one of claims 1 to 12, wherein the pegylated antibody has a hydrodynamic radius between about 9 nm and about 11 nm. The antibody of any one of claims 1 to 13, wherein the PEG polymer(s) each have a molecular weight between about 10 kDa and about 40 kDa. The antibody of any one of claims 1 to 14, wherein each of the PEG polymer(s) is conjugated to an engineered cysteine residue via a maleimide-cysteine conjugation The heavy or light chain of the antibody. The antibody of any one of claims 1 to 14, wherein each of the PEG polymer(s) is bound to the antibody via an iodoacetamide-cysteine conjugation at an engineered cysteine residue. heavy or light chain. The antibody of any one of claims 1 to 16, wherein the engineered cysteine residue is selected from the group consisting of: K149C of the light chain, K183C of the light chain, T186C of the heavy chain and Y373C of the heavy chain, the numbering of the light chain is according to Kabat and the numbering of the heavy chain is according to the EU index. The antibody of claim 17, wherein the antibody comprises two heavy chains, two light chains, and two PEG polymers; and wherein the two light chains of the antibody comprise conjugated to one of the two PEG polymers K149C engineered cysteine. The antibody of claim 17, wherein the antibody comprises two heavy chains, two light chains, and two PEG polymers; and wherein the two light chains of the antibody comprise conjugated to one of the two PEG polymers K183C engineered cysteine. 17. The antibody of claim 17, wherein the antibody comprises two heavy chains, two light chains, and two PEG polymers; and wherein the two heavy chains of the antibody comprise conjugated to one of the two PEG polymers T186C engineered cysteine. 17. The antibody of claim 17, wherein the antibody comprises two heavy chains, two light chains, and two PEG polymers; and wherein the two heavy chains of the antibody comprise conjugated to one of the two PEG polymers Y373C engineered cysteine. A method of making a pegylated antibody that binds to MerTK, the method comprising combining an antibody comprising one or more free engineered cysteine residues with one or more polyimide moieties comprising a maleimide moiety. an ethylene glycol (PEG) polymer, contacted under conditions suitable for each of the PEG polymer(s) to bind to the engineered cysteine residues of the antibody via a thioether linkage; wherein the antibody comprises : (1) A heavy chain variable domain (VH) comprising (a) CDR-H1 containing the amino acid sequence of SYAMG (SEQ ID NO: 1), (b) an amine containing IINSYGNTYYANWAKG (SEQ ID NO: 2) The amino acid sequence of CDR-H2 and (c) the CDR-H3 containing the amino acid sequence of DPGVSSNL (SEQ ID NO: 3), and the light chain variable domain (VL), which comprises (d) the amino acid sequence containing QASQNIYSGLA (SEQ ID NO: 3). NO: 4) CDR-L1 of amino acid sequence, (e) CDR-L2 containing amino acid sequence of GASKLAS (SEQ ID NO: 5) and (f) amine containing QATYYSSNSVA (SEQ ID NO: 6) CDR-L3 of the amino acid sequence; or (2) A heavy chain variable domain (VH) comprising (a) CDR-H1 containing the amino acid sequence of ANTMN (SEQ ID NO: 7), (b) an amine containing IFTATGSTYYATWVNG (SEQ ID NO: 8) CDR-H2 of the amino acid sequence and (c) CDR-H3 containing the amino acid sequence of SGSGSSSGAFNI (SEQ ID NO: 9), and a light chain variable domain (VL) comprising (d) a QASQSISSSLA (SEQ ID NO: 9) NO: 10) CDR-L1 of amino acid sequence, (e) CDR-L2 containing amino acid sequence of AASILAS (SEQ ID NO: 11) and (f) amine containing QCTSYGSLFLGP (SEQ ID NO: 12) The amino acid sequence of CDR-L3. The method of claim 22, wherein the one or more free engineered cysteine residues of the antibody are between pH 7.0 and 7.5 and the maleic acid of the one or more PEG polymers Amine moiety contacts. A method of making a PEGylated antibody that binds to MerTK, the method comprising combining an antibody comprising one or more free engineered cysteine residues with one or more polyethylene glycols comprising an iodoacetamide moiety (PEG) polymers, contacted under conditions suitable for each of the PEG polymer(s) to bind to the engineered cysteine residues of the antibody via thioether linkages; wherein the antibody comprises: (1) A heavy chain variable domain (VH) comprising (a) CDR-H1 containing the amino acid sequence of SYAMG (SEQ ID NO: 1), (b) an amine containing IINSYGNTYYANWAKG (SEQ ID NO: 2) The amino acid sequence of CDR-H2 and (c) the CDR-H3 containing the amino acid sequence of DPGVSSNL (SEQ ID NO: 3), and the light chain variable domain (VL), which comprises (d) the amino acid sequence containing QASQNIYSGLA (SEQ ID NO: 3). NO: 4) CDR-L1 of amino acid sequence, (e) CDR-L2 containing amino acid sequence of GASKLAS (SEQ ID NO: 5) and (f) amine containing QATYYSSNSVA (SEQ ID NO: 6) CDR-L3 of the amino acid sequence; or (2) A heavy chain variable domain (VH) comprising (a) CDR-H1 containing the amino acid sequence of ANTMN (SEQ ID NO: 7), (b) an amine containing IFTATGSTYYATWVNG (SEQ ID NO: 8) CDR-H2 of the amino acid sequence and (c) CDR-H3 containing the amino acid sequence of SGSGSSSGAFNI (SEQ ID NO: 9), and a light chain variable domain (VL) comprising (d) a QASQSISSSLA (SEQ ID NO: 9) NO: 10) CDR-L1 of amino acid sequence, (e) CDR-L2 containing amino acid sequence of AASILAS (SEQ ID NO: 11) and (f) amine containing QCTSYGSLFLGP (SEQ ID NO: 12) The amino acid sequence of CDR-L3. The method of any one of claims 22 to 24, wherein the PEG polymer(s) is bound to the antibody at a ratio of 2.0 polymer per antibody. The method of any one of claims 22 to 25, wherein each of the free engineered cysteine residues is modified with cysteine prior to contacting the antibody with the PEG polymer(s) or glutathione block; and the method further comprises deblocking the engineered cysteine residue. The method of any one of claims 22 to 26, further comprising purifying the PEGylated antibody from unbound antibody and PEG polymer after contacting the antibody with the PEG polymer(s). The method of claim 27, wherein the pegylated antibody is purified by hydrophobic interaction chromatography. The method of any one of claims 22 to 28, wherein the VH domain comprises a sequence that is at least 95% identical to the amino acid sequence of EVQLVESGEGLVQPGGSLRLSCAASGFSLSSYAMGWVRQAPGKGLEYVGIINSYGNTYYANWAKGRFTISRDNSKNTVYLQMGSLRAEDMAVYYCARDPGVSSNLWGRGTLVTVSS (SEQ ID NO: 13); and/or the VL domain comprises The amino acid sequence of DIQMTQSPSTLSASVGDRVTITCQASQNIYSGLAWYQQKPGKAPKLLIYGASKLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQATYYSSNSVAFGGGTKVEIK (SEQ ID NO: 14) is a sequence having at least 95% sequence identity. 如請求項 29 之方法，其中該 VH 域包含 EVQLVESGEGLVQPGG SLRLSCAASGFSLSSYAMGWVRQAPGKGLEYVGIINSYGNTYYANWAKGRFTISRDNSKNTVYLQMGSLRAEDMAVYYCARDPGVSSNLWGRGTLVTVSS (SEQ ID NO: 13) 之胺基酸序列；且該 VL 域包含 DIQMTQSPSTLSASVGDRVTITCQASQNIYSGLAWYQQKPGKAPKLLIYGASKLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQATYYSSNSVAFGGGTKVEIK (SEQ ID NO: 14) 之胺基酸序列。 如請求項 29 或請求項 30 之方法，其中該重鏈包含 EVQLVESGEGLVQPGGSLRLSCAASGFSLSSYAMGWVRQAPGKGLEYVGIINSYGNTYYANWAKGRFTISRDNSKNTVYLQMGSLRAEDMAVYYCARDPGVSSNLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 21) 之胺基酸序列。 如請求項 29 或請求項 30 之方法，其中該重鏈包含 EVQLVESGEGLVQPGGSLRLSCAASGFSLSSYAMGWVRQAPGKGLEYVGIINSYGNTYYANWAKGRFTISRDNSKNTVYLQMGSLRAEDMAVYYCARDPGVSSNLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 22) 之胺基酸序列。 如請求項 29 至 32 中任一項之方法，其中該輕鏈包含 DIQMTQSPSTLSASVGDRVTITCQASQNIYSGLAWYQQKPGKAPKLLIYGASKLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQATYYSSNSVAFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 23) 之胺基酸序列。 The method of any one of claims 22 to 33, wherein the PEG polymer(s) are linear PEG polymers. The method of any one of claims 22 to 33, wherein the PEG polymer(s) are branched PEG polymers. The method of claim 35, wherein the branched PEG polymer(s) comprises 2 branches. The method of any one of claims 22 to 36, wherein the pegylated antibody has a hydrodynamic radius greater than about 6 nm. The method of any one of claims 22 to 37, wherein the pegylated antibody has a hydrodynamic radius of greater than or equal to about 10 nm. The method of any one of claims 22 to 38, wherein the pegylated antibody has a hydrodynamic radius between about 9 nm and about 11 nm. The method of any one of claims 22 to 39, wherein each of the PEG polymer(s) has a molecular weight between about 10 kDa and about 40 kDa. The method of any one of claims 22 to 40, wherein the one or more free engineered cysteine residues are selected from the group consisting of: K149C of the light chain, K183C of the light chain, T186C of the heavy chain and Y373C of the heavy chain, the numbering of the light chain is according to Kabat and the numbering of the heavy chain is according to the EU index. The method of claim 41, wherein the antibody comprises two heavy chains, two light chains, and two PEG polymers; and wherein the two light chains of the antibody comprise conjugated to one of the two PEG polymers K149C engineered cysteine. The method of claim 41, wherein the antibody comprises two heavy chains, two light chains, and two PEG polymers; and wherein the two light chains of the antibody comprise conjugated to one of the two PEG polymers K183C engineered cysteine. The method of claim 41, wherein the antibody comprises two heavy chains, two light chains, and two PEG polymers; and wherein the two heavy chains of the antibody comprise conjugated to one of the two PEG polymers T186C engineered cysteine. The method of claim 41, wherein the antibody comprises two heavy chains, two light chains, and two PEG polymers; and wherein the two heavy chains of the antibody comprise conjugated to one of the two PEG polymers Y373C engineered cysteine. A PEGylated antibody that binds to MerTK produced by the method of any one of claims 22 to 45. A pharmaceutical composition comprising the PEGylated antibody of any one of claims 1 to 21 and 46 and a pharmaceutically acceptable carrier. The pharmaceutical composition of claim 47, further comprising an additional therapeutic agent. The pharmaceutical composition of claim 48, wherein the additional therapeutic agent is selected from one or more of the following: tamoxifen, letrozole, exemestane, anastrozole, irinotecan, cetuximab, fulvestrant, vinorelbine, erlotinib, bevacizumab ( bevacizumab, vincristine, imatinib mesylate, sorafenib, lapatinib, trastuzumab, cisplatin ), gemcitabine, methotrexate, vinblastine, carboplatin, paclitaxel, 5-fluorouracil, doxorubicin, boron Tezomib, melphalan, prednisone, and docetaxel. The pharmaceutical composition of claim 48, wherein the additional therapeutic agent is an immune checkpoint inhibitor. The pegylated antibody of any one of claims 1 to 21 and 46 or the pharmaceutical composition of any one of claims 47 to 50 is used as a medicine. The pegylated antibody of any one of claims 1 to 21 and 46 or the pharmaceutical composition of any one of claims 47 to 50, for use in the treatment of cancer. Use of a pegylated antibody according to any one of claims 1 to 21 and 46 or a pharmaceutical composition according to any one of claims 47 to 50 in the manufacture of a medicament for the treatment of cancer. A pegylated antibody according to any one of claims 1 to 21 and 46 or a pharmaceutical composition according to any one of claims 47 to 50, prepared for use in reducing MerTK-mediated apoptosis of apoptotic cells in an individual Use in scavenging drugs. A method of treating an individual suffering from cancer, comprising administering to the individual an effective amount of a pegylated antibody according to any one of claims 1 to 21 and 46 or a pharmaceutical composition according to any one of claims 47 to 50 thing. The method of claim 55, wherein administering the pegylated antibody reduces retinal toxicity in the subject compared to administering the unpegylated antibody that binds to MerTK. The method of claim 55 or claim 56, further comprising administering to the individual an additional therapeutic agent. The method of claim 57, wherein the additional therapeutic agent is selected from one or more of the following: tamoxifen, rituzole, isomestane, anastrozole, anticancer, cetuximab Monoclonal antibody, fulvestrant, winnopine, dexamethasone, bevacizumab, vincristine, imatinib mesylate, sorafenib, lapatinib, trastuzumab, cisplatin , Gemcitabine, Ammethopterin, Vinblastine, Carboplatin, Paclitaxel, 5-Fluorouracil, Doxorubicin, Bortezomib, Mycofuran, Prednisone and Docetaxel. The method of claim 57, wherein the additional therapeutic agent is an immune checkpoint inhibitor. The method of claim 59, wherein the immune checkpoint inhibitor is selected from the group consisting of cytotoxic T lymphocyte-associated protein 4 (CTLA4) inhibitors, programmed cell death protein 1 (PD-1) Binding antagonists and planned death ligand 1 (PDL1) binding antagonists. The method of claim 59, wherein the immune checkpoint inhibitor is a PDL1 binding antagonist. The method of claim 61, wherein the PDL1 binding antagonist is an anti-PDL1 antibody. The method of claim 62, wherein the anti-PDL1 antibody is atezolizumab. The method of any one of claims 59 to 63, further comprising administering to the individual an effective amount of an additional chemotherapeutic agent. The method of any one of claims 55 to 64, wherein the cancer is colorectal cancer. The method of any one of claims 55 to 64, wherein the cancer is selected from the group consisting of colorectal cancer, urothelial cancer, non-small cell lung cancer, triple negative breast cancer, small cell lung cancer, hepatocellular carcinoma, and Melanoma. A method of reducing MerTK-mediated clearance of apoptotic cells in an individual, comprising administering to the individual an effective amount of a pegylated antibody as claimed in any one of claims 1 to 21 and 46 or as claimed in claims 47 to 47 The pharmaceutical composition of any one of 50 to reduce MerTK-mediated clearance of apoptotic cells. The method of claim 67, wherein administering the pegylated antibody reduces retinal toxicity in the subject compared to administering the unpegylated antibody that binds to MerTK. The method of claim 67 or claim 68, wherein the clearance of apoptotic cells is reduced by about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5 , 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0 , 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5 , 7.6, 7.7, 7.8, 7.9 or 8.0 times.

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