WO2022086957A1 - Anticorps anti-mertk peg-conjugués et procédés d'utilisation - Google Patents

Anticorps anti-mertk peg-conjugués et procédés d'utilisation Download PDF

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Publication number
WO2022086957A1
WO2022086957A1 PCT/US2021/055606 US2021055606W WO2022086957A1 WO 2022086957 A1 WO2022086957 A1 WO 2022086957A1 US 2021055606 W US2021055606 W US 2021055606W WO 2022086957 A1 WO2022086957 A1 WO 2022086957A1
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antibody
seq
amino acid
acid sequence
cdr
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PCT/US2021/055606
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Wei-Ching Liang
Weiyu Lin
Yan Wu
Minhong Yan
Breanna Sachiko VOLLMAR
Mingjian FEI
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Genentech, Inc.
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Publication of WO2022086957A1 publication Critical patent/WO2022086957A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/58—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation
    • A61K2039/585—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation wherein the target is cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6093—Synthetic polymers, e.g. polyethyleneglycol [PEG], Polymers or copolymers of (D) glutamate and (D) lysine
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/40—Immunoglobulins specific features characterized by post-translational modification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/522—CH1 domain
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/526—CH3 domain
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to PEGylated anti-MeiTK. antibodies and methods of making and using the same.
  • Macrophages of the innate immune system are abundant in various types of solid tumors and may contribute to the relatively low response rate to T-cell based therapy. They are versatile cells capable of cartying out various functions, including phagocytosis. Macrophages are professional phagocytes highly specialized in removal of dying or dead cells, and cellular debris. It is estimated tltat billions of cells die every day in the human body. But it is rare to find apoptotic cells in tissues under normal physiological conditions thanks to the rapid and efficient clearance by phagocytes. In homeostasis, apoptotic cells are removed at the early stage of cell death before loss of plasma membrane integrity. Therefore, in general apoptosis is immunologically silent. In solid tumors, uncontrolled tumor growth is often accompanied bv increased cell death due to hypoxia and metabolic stress. To evade immune surveillance, tumors take advantage of the non-iininunogenic nature of
  • TAMs Tumor associated macrophages
  • MerTK has been shown to play a role in clearance of apoptotic ceils. Therefore, reduction of MerTK-mediated clearance of apoptotic ceils using MerTK inhibitors is an attractive therapeutic approach in treating cancer.
  • Existing anti-MerTK antibodies have been described but may be unsuitable for therapeutic development. For example, White et al.
  • anti-MerTK antibodies having optimal binding characteristics (e.g., on and off rates) as well as desired biological effects are needed.
  • the present invention provides PEGylated anti-MerTK antibodies and methods of using the same.
  • the present disclosure provides a PEGylated antibody that binds to MerTK, wherein the antibody is conjugated to one or more polyethylene glycol (PEG) polymer(s); wherein each of the PEG polymer(s) is conjugated to a heavy chain or light chain of the antibody at an engineered cysteine residue; and wherein the antibody comprises: (1 ) a heavy chain variable domain (ATI) comprising (a) a CDR-H1 comprising the amino acid sequence of SY2AMG (SEQ ID NO: 1), (b) a CDR-H2 comprising the amino acid sequence of IINSYGNTYYANWAKG (SEQ ID NO: 2), and (c) a CDR-H3 comprising die amino acid sequence of DPGVSSNL (SEQ ID NO: 3), and a light chain variable domain (Vi.,) comprising (d) a CDR-L1 comprising the amino acid sequence of QASQNIYSGLA (SEQ ID NO: 4), (e) a heavy chain variable domain (ATI)
  • tire present disclosure provides a PEGylated antibody that binds to MerTK, wherein the antibody is conjugated to one or more polyethylene glycol (PEG) poiymer(s): wherein each of the PEG polvmer(s) is conjugated to a heavy chain or light chain of the antibody at an engineered cysteine residue; and wherein the antibody comprises: a heavy chain variable domain (VH) comprising (a) a CDR-H1 comprising the amino acid sequence of SYAMG (SEQ ID NO: 1), (b) a CDR-H2 comprising the amino acid sequence of IINSYGNTYYANWAKG (SEQ ID NO: 2), and (c) a CORES comprising the amino acid sequence of DPGVSSNL (SEQ ID NO: 3), and a light chain variable domain (VL) comprising (d) a CDR-L1 comprising tire amino acid sequence of QASQNIYSGLA (SEQ ID NO: 4), (e) a CDR-L2 comprising the
  • the present disclosure provides a PEGylated antibody that binds to MerTK, wherein the antibody is conjugated to one or more polyethylene glycol (PEG) polymer(s); wherein each of the PEG polymer(s) is conjugated to a heavy chain or light chain of the antibody at an engineered cysteine residue; and wherein the antibody comprises: a heavy chain variable domain (VH) comprising (a) a CDR-H1 comprising the amino acid sequence of ANTMN (SEQ ID NO: 7), (b) a CDR-H2 comprising the amino acid sequence of IFTATGSTYYATWVNG (SEQ ID NO: 8), and (c) a CDR-H3 comprising the amino acid sequence of SGSGSSSGAFNI (SEQ ID NO: 9), and a light drain variable domain (VL) comprising (d) a CDR-L1 comprising the amino acid sequence of QASQSISSSLA (SEQ ID NO: 10), (e) a CDR-L2 comprising
  • the VH domain comprises a sequence having at least 95% sequence identity to the amino acid sequence of EVQLVESGEGLVQPGGSLRLSCAASGFSLSSYAMGWVRQAPGKGLEYVGIINSYGNTYYAN WAKGRFTISRDNSKNTVYLQMGSLRAEDMAVYYCARDPGVSSNLWGRGTLVTVSS (SEQ ID NO: 13); and/or the VL domain comprises a sequence having at least 95% sequence identity to the amino acid sequence of DIQMTQSPSTLSASVGDRVTITCQASQNIYSGLAWYQQKPGKAPKLLIYGASKLASGVPSRFS GSGSGTEFTLTISSLQPDDFATYYCQATYYSSNSVAFGGGTKVEIK (SEQ ID NO: 14), In some embodiments, the VH domain comprises the amino acid sequence of
  • VL domain comprises the amino acid sequence of
  • the VH domain comprises a sequence having at least 95% sequence identity to the amino acid sequence of
  • VL domain comprises a sequence having at least 95% sequence identity to the amino acid sequence of
  • the VH domain comprises the amino acid sequence of
  • EQQLVESGEGLVQPGGSLRLSCAVSGFSLSSYAMGWVRQAPGKGLEWIGIINSYGNTYYAN WAKGRFTISRDSSKNTX ⁇ QMGSLRAEDMAVYFCARDPGVSSNLWGPGTLVTVSS (SEQ ID NO: 15); and the XT, domain comprises the amino acid sequence of
  • the VH domain comprises a sequence having at least 95% sequence identity to the amino acid sequence of
  • QSVEESGGRLVTPGTPLTLTCTVSGFSLSSYAMGWVRQAPGKGLEWIGnNSYGNTYYANWA KGRFTISRTSTTVDLRMPSLTTEDTATYFCARDPGVSSNLWGPGTLVTVSS (SEQ ID NO: 17); and/or the XT, domain comprises a sequence having at least 95% sequence identity to the amino acid sequence of
  • the XT-I domai n comprises the amino acid sequence of
  • VL domain comprises the amino acid sequence of
  • the VH domain comprises a sequence having at least 95% sequence identity to the amino acid sequence of QSVEESGGRLVTPGTPLTLTCWSGIDLSANTMNWVRQAPGKGLEWTGIFTATGSTA'YATWV NGRFTISKTSTTVDLKITSPTTEDTATYTCARSGSGSSSGAFNTWGPGTLVTVSL (SEQ ID NO: 19); and/or the VL domain comprises a sequence having at least 95% sequence identity to the amino acid sequence of
  • the VH domain comprises die amino acid sequence of
  • VL domain comprises the amino acid sequence of
  • the heavy chain comprises the sequence
  • the light chain comprises the sequence DIQlvfTQSPS’n.SASVGDRVTITCQASQNIYSGLAWYQQKPGKAPKLLIYGASKLASGVPSRFS GSGTEFTLTISSLQPDDFATYYCQATYYSSNSVAFGGGTKVEIKRTVAAPSVFIFPPSDEQL KSGTASVVCLLNNFYPREAKVQWKWNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY EKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 23).
  • the antibody is conjugated to one or two PEG polymers.
  • the PEG polymer(s) are linear PEG polymers.
  • the PEG polvmer(s) are branched PEG polymers.
  • the branched PEG polymer(s) comprise 2 branches.
  • the PEGylated antibody lias a hydrodynamic radius of greater than about 6 ran.
  • the PEGylated antibody has a hydrodynamic radius of greater than or equal to about 10 sun.
  • the PEGylated antibody has a hydrodynamic radius of between about 6 ran and about 10 ran, or between about 9 nm and about 11 nm.
  • the PEG poly mer(s) each have a molecular weight of between about 10 kDa and about 40 kDa, between about 20 kDa and about 40 kDa, or between about 10 kDa and about 20 kDa, e.g., about 10 kDa, about 20 kDa, about 30 kDa, or about 40 kDa.
  • each of the PEG poly inert s) is conjugated to a heavy chain or light chain of the antibody at an engineered cysteine residue via maleirmde-cy steine conjugation.
  • each of the PEG polymer(s) is conjugated to a heavy chain or light chain of the antibody at an engineered cysteine residue via iodoacetamide-cy steine conjugation.
  • 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 drain, numbering of the light chain according to Rabat and numbering of the heavy chain according to EU index.
  • the antibody comprises two heavy chains, two light chains, and two PEG polymers; and wherein both light chains of the antibody comprise a K149C engineered cysteine conjugated to one of the two PEG polymers.
  • the antibody comprises two heavy drains, two light drains, and two PEG polymers; and wherein both light chains of the antibody comprise a K183C engineered cysteine conjugated to one of the two PEG polymers. In some embodiments, the antibody comprises two heavy chains, two light chains, and two PEG poly mers; and wherein both heavy chains of the antibody comprise a T186C engineered cysteine conjugated to one of the two PEG polymers. In some embodiments, the antibody comprises two heavy drains, two light chains, and two PEG polymers; and wherein both heavy chains of the antibody comprise a Y373C engineered cysteine conjugated to one of the two PEG polymers.
  • tire present disclosure provides a method of producing a PEGylated antibody that binds to MeiTK, comprising contacting (i.e., convalently attaching) an antibody comprising one or more free engineered cysteine residues with one or more polyethylene glycol (PEG) polymer(s) comprising a maleimide moiety under conditions suitable for each of the PEG polymer(s) to be conjugated to an engineered cysteine residue of the antibody via thioether linkage.
  • PEG polyethylene glycol
  • the one or more free engineered cysteine residues of the antibody are contacted (i.e., chemically reacted) with the maleimide moiety of the one or more PEG polymer(s) at a pH between 7.0 and 7.5.
  • the present disclosure provides a method of producing a PEGylated antibody that binds to MerTK, comprising contacting (i.e., convalently attaching) an antibody comprising one or more free engineered cysteine residues with one or more polyethylene glycol (PEG) polymer(s) comprising an iodoacetamide moiety under conditions suitable for each of the PEG polymer(s) to be conjugated to an engineered cysteine residue of the antibody via thioether linkage.
  • PEG polyethylene glycol
  • the antibody comprises a heavy chain variable domain (VH) comprising (a) a CDR-H1 comprising the amino acid sequence of SYAMG (SEQ ID NO: i), (b) a CDR-H2 comprising the amino acid sequence of IINSYGNTYYANWAKG (SEQ ID NO: 2), and (c) a CDR-H3 comprising the amino acid sequence of DPGVSSNL (SEQ ID NO: 3), and a light chain variable domain (VL) comprising (d) a CDR-LJ comprising the amino acid sequence of QASQNIYSGLA (SEQ ID NO: 4), (e) a CDR-L2 comprising the amino acid sequence of GASKLAS (SEQ ID NO: 5), and (f) a CDR-L3 comprising the amino acid sequence of QATYYSSNSVA (SEQ ID NO: 6).
  • VH heavy chain variable domain
  • the antibody comprises heavy chain variable domain ( VH) comprising (a) a CDR-H1 comprising the amino acid sequence of ANTMN (SEQ ID NO: 7), (b) a CDR-H2 comprising the amino acid sequence of IFTATGSTYYATWVNG (SEQ ID NO: 8), and (c) a CDR-H3 comprising the amino acid sequence of SGSGSSSGAFNI (SEQ ID NO: 9), and a light chain variable domain (VL) comprising (d) a CDR-L1 comprising the amino acid sequence of QASQSISSSLA (SEQ ID NO: 10), (e) a CDR-L2 comprising the amino acid sequence of AASILAS (SEQ ID NO: 11), and (f) a CDR-L3 comprising the amino acid sequence of QCTSYGSLFLGP (SEQ ID NO: 12).
  • VH heavy chain variable domain
  • the PEG polymer(s) are conjugated to the antibody at a ratio of 2.0 polymers per antibody.
  • each of the free engineered cysteine residues is blocked with a cysteine or glutathione moiety; and the method further comprises deblocking the engineered cysteine residue.
  • the method further comprises, after contacting (i.e., convalently attaching) the antibody with the PEG polymer(s), purifying tire PEGylated antibody from unconjugated antibody and PEG polymer.
  • the PEGylated antibody is purified by hydrophobic interaction chromatography .
  • the VH domain comprises a sequence having at least 95% sequence identity to the amino acid sequence of EVQLVESGEGLVQPGGSLRLSCAASGFSLSSYAMGWVRQAPGKGLEYVGIINSYGNTYYAN WAKGRFTTSRDNSKNTVYLQMGSLRAEDMAVYYCARDPGVSSNLWGRGTLVTVSS (SEQ ID NO: 13); and/or the VL domain comprises a sequence having at least 95% sequence identity to the amino acid sequence of DIQMTQSPSTLSASVGDRVnTCQASQNIYSGLAWYQQKPGKAPKLLIYGASKLASGVPSRFS GSGSGTEFTLTISSIXJPDDFATYYCQATYYSSNSVAFGGGTKVEIK (SEQ ID NO: 14).
  • the VH domain comprises the amino acid sequence of EVQLVESGEGLVQPGGSLRLSCAASGFSLSSYAMGWVRQAPGKGLEYVGIINSYGNTYYAN WAKGRFTISRDNSKNTVYLQMGSLRAEDMAVYYCARDPGVSSNLWGRGTLVTVSS (SEQ ID NO: 13); and the VL domain comprises tlie amino acid sequence of DIQMTQSPSTLSASVGDRVTITCQASQNIYSGLAWYQQKPGKAPKLLIYGASKLASGVPSRFS GSGSGTEFTLTISSLQPDDFATYYCQATYYSSNSVAFGGGTKVEIK (SEQ ID NO: 14).
  • the VH domain comprises a sequence having at least 95% sequence identity to the amino acid sequence of EQQLVESGEGLVQPGGSLRLSCAVSGFSLSSYAMGWVRQAPGKGLEW1GIINSYGNTYYAN WAKGRFTISRDSSKNTVYLQMGSLRAEDMAVYFCARDPGVSSNLWGPGTLVWSS (SEQ ID NO: 15); and/or the VL domain comprises a sequence having at least 95% sequence identity to the amino acid sequence of DVQMTQSPSTLSASVGDRVTITCQASQNIYSGLAWYQQKPGKPPKLLIYGASKLASGVPSRFS GSGSGTEFTLTISSLQPDDFATYYCQATYYSSNSVAFGGGTKVEIK (SEQ ID NO: 16).
  • the VH domain comprises the amino acid sequence of EQQLVESGEGLVQPGGSLRLSCAVSGFSLSSYAMGWVRQAPGKGLEWIGIINSYGNTYYAN WAKGRFTISRDSSKNTVYLQMGSLRAEDMAVYFCARDPGVSSNLWGPGTLVTVSS (SEQ ID NO: 15); and the VL domain comprises the amino acid sequence of
  • the VH domain comprises a sequence having at least 95% sequence identity to the amino acid sequence of
  • VL domain comprises a sequence having at least 95% sequence identity to the amino acid sequence of
  • the VH domain comprises the amino acid sequence of QSVEESGGRLVTPGTPLTLTCTVSGFSLSSYAMGWVRQAPGKGLEWIGIINSYGNTYYANWA KGRFTISRTSTTVDLRMPSLTTEDTATYFCARDPGVSSNLWGPGTLVTVSS (SEQ ID NO: 17); and tiie VL domain comprises the amino acid sequence of
  • the VH domain comprises a sequence having at least 95% sequence identity to the amino acid sequence of
  • VL domain comprises a sequence having at least 95% sequence identity to the amino acid sequence of
  • the VH domain comprises the amino acid sequence of QSVEESGGRLVTPGTPLTLTCTVSGIDLSANTMNWVRQAPGKGLEW1GIFTATGSTYYATWV NGRFTISKTSTTVDLKITSPTTEDTATYTCARSGSGSSSGAFNTWGPGTLVTVSL (SEQ ID NO: 19); and the VL domain comprises the amino acid sequence of
  • the heavy chain comprises the sequence
  • the light chain comprises the sequence DIQMTQSPSTLSASVGDRVT1TCQASQNIYSGLAWYQQKPGKAPKLL1YGASKLASGVPSRFS GSGSGTEFTLTISSLQPDDFATYYCQATYYSSNSVAFGGGTKVEIKRTVAAPSVFIFPPSDEQL KSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY EKHKWACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 23).
  • the antibody is conjugated to one or two PEG polymers.
  • the PEG polymer(s) are linear PEG polymers.
  • the PEG polymer(s) are branched PEG polymers.
  • the branched PEG poly mer(s) comprise 2. branches.
  • the PEGylated antibody lias a hydrodynamic radius of greater than about 6 am. In some embodiments, the PEGylated antibody has a hydrodynamic radius of greater than or equal to about 10 nm.
  • the PEGylated antibody lias a hydrodynamic radius of between about 6 mn and about 10 mu, or between about 9 nm and about 11 nm.
  • the PEG poly mer(s) each have a molecular weight of between about 10 kDa and about 40 kDa, between about 20 kDa and about 40 kDa, or between about 10 kDa and about 20 kDa, e.g., about 10 kDa, about 20 kDa, about 30 kDa, or about 40 kDa.
  • each of the PEG polymer(s) is conjugated to a heavy chain or light chain of the antibody at an engineered cysteine residue via maleimide-cvsteine conjugation. In some embodiments, each of the PEG polymer(s) is conjugated to a heavy chain or light chain of the antibody at an engineered cysteine residue via iodoacetamide-cvsteine conjugation. In some embodiments, 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, numbering of the light chain according to Kabat and numbering of the heavy chain according to EU index.
  • the antibody comprises two heavy drains, two light chains, and two PEG polymers; and wherein both light chains of the antibody comprise a K149C engineered cysteine conjugated to one of die two PEG polymers.
  • die antibody comprises two heavy chains, two light chains, and two PEG polymers; and wherein both light chains of the antibody comprise a K183C engineered cysteine conjugated to one of the two PEG polymers.
  • the antibody comprises two heavy chains, two light chains, and two PEG polymers; and wherein both heavy chains of the antibody comprise a Ti 86C engineered cysteine conjugated to one of the two PEG polymers.
  • the antibody comprises two heavy chains, two light chains, and two PEG polymers; and wherein both heavy chains of the antibody comprise a Y373C engineered cysteine conjugated to one of the two PEG polymers.
  • the present disclosure provides a PEGylated antibody tlrat binds to MerTK produced by the method of any one of the above embodiments.
  • the present disclosure provides a pharmaceutical composition comprising the PEGy lated anti-MerTK antibody of any one of tiie above embodiments and a pharmaceutically acceptable carrier.
  • the present disclosure provides a method of treating an individual having cancer comprising administering to the individual an effective amount of the PEGylated antibody or composition of any one of the above embodiments.
  • the present disclosure provides a method of reducing MerTK-mediated clearance of apoptotic ceils in an individual comprising administering to the individual an effective amount of the PEGylated antibody or composition of any one of the above embodiments.
  • the present disclosure provides the PEGylated anti- MerTK antibody or composition of any one of the above embodiments for use as a medicament.
  • the present disclosure provides the PEGylated anti-MerTK antibody or composition of any one of the above embodiments for use in treating cancer.
  • the present disclosure provides the PEGylated anti-MerTK antibody or composition of any one of the above embodiments for use in the any of the methods disclosed herein, e.g., in methods of treating an individual having cancer and/or reducing MerTK-mediated clearance of apoptotic cells in an individual.
  • the present disclosure provides the PEGylated anti-MerTK antibody or composition of any one of the above embodiments for use in the manufacture of a medicament, e.g., for treating an individual having cancer and/or reducing MerTK-mediated clearance of apoptotic ceils in an individual.
  • administration of the PEGylated antibody causes reduced retinal toxicity in the individual, as compared to administration of an antibody that binds to MerTK tha t is not PEGylated.
  • the method further comprises administering an additional therapeutic agent to the individual.
  • the additional therapeutic agent is selected from one or more of tamoxifen, letrozole, exemestane, anastrozole, irinotecan, cetuximab, fulvestrant, vinorelbine, erlotinib, bevacizumab, vincristine, imatinib mesylate, sorafenib, lapatinib, trastuzumab, cisplatin, gemcitabine, methotrexate, vinblastine, carboplatin, paclitaxel, 5-fIuorouracil, doxorubicin, bortezomib, melpbalan, prednisone, and docetaxel.
  • the additional therapeutic agent is an immune checkpoint inhibitor.
  • the immune checkpoint inhibitor is selected from the group consisting of a cytotoxic T-lymphocyte-associated protein 4 (CTLA4) inhibitor, a programmed ceil death protein 1 (PD-1) binding antagonist, and a programmed deathligand 1 (PDL1) binding antagonist.
  • CTLA4 cytotoxic T-lymphocyte-associated protein 4
  • PD-1 programmed ceil death protein 1
  • PDL1 programmed deathligand 1
  • tire immune checkpoint inhibitor is a PDL1 binding antagonist.
  • the PDL1 binding antagonist is an anti-PDLl antibody, e.g., atezolizumab.
  • the method further comprises administering an effective amount of an additional chemotherapeutic agent to the individual.
  • the cancer is colon cancer.
  • the cancer is selected from urothelial carcinoma, non-small cell lung cancer, triple negative breast cancer, small cell lung cancer, hepatocellular carcinoma, and melanoma.
  • 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,
  • FIGS. 1A-1C depict on-target ocular toxicity using an anti-MerTK antibody.
  • FIG. 1A is a schematic of the retinal pigment epithelium (RPE) depicting the expression of MerTK on the apical membrane of the RPE. Blockage of MerTK by anti-MerTK antibody in the RPE results in failure to phagocytize shedding photoreceptor outer segments (POS), leading to accumulation of debris and ultimate degeneration and loss of photoreceptors.
  • FIG. IB shows the retinal macrophage infiltration in outer photoreceptor (PR) layer in cynomolgus monkey following administration of 30 mg kg I3B4 anti-MerTK mAb once every 3 weeks for 6 weeks (total 3 doses).
  • PR photoreceptor
  • FIG. 1C shows the retinal toxicity in Balb/c mice following administration of 30 mg/kg 14C9 anti-MerTK mAb twice weekly for 4 weeks (8 total doses). Compared with the control group, 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 & Eosin (HE), 200X.
  • FIG. 2 is a schematic representation of anti-MerTK cysteine-engineered antibody -PEG polymer conjugates, comprising either 40 kDa linear PEG (left) and 40 kDa 2-arm branched PEG (right).
  • FIGs. 3 A & 3B show the analysis of 14C9 cysteine-engineered antibody -PEG conjugates binding by Biacore SPR.
  • FIG. 3A shows the results for the binding analysis of linear PEG40K ⁇ conjugated I4C9 THIOMAB, while FIG.
  • 3B shows the results for branched PEG40K-conjugated 14C9 THIOMAB.
  • FIG. 4 shows the results of binding analysis of 14C9 Fab conjugates.
  • the 14C9.C90S EC K149C series of THIOMAB conjugates (linear PEG40K and branched PEG40K) in Fabs were assessed for their binding affinity against the surface bound mouse MerTK by Biacore SPR. All variants exhibited about a 3-fold drop in binding affinity against mouse MerTK, with no difference in between the conjugation sites.
  • FIGS. 5A-5B show a comparison of the inhibitory activity of the PEG40 conjugated 14C9 anti-MerTK antibodies and parental mAb in mouse peritoneal macrophage mediated efferocytosis.
  • FIG. 5A shows the results of the efferocytosis assay with PEG-conjugated 14C9 antibodies.
  • Each panel represents the analysis for antibodies with a different conjugation site: KI49C on the light chain (top left), K183C on the light chain (top right), or T182C on the heavy chain (bottom).
  • FIG. SB summarizes the potency and efficacy of 14C9 antibody and conjugates in the efferocytosis assay.
  • FIGS. 6A-6B shoiv a comparison of the inhibitory activity of the PEG40 conjugated 14C9 LC K149C antibodies and parental mAb in mouse peritoneal roacrophage-mediated efferocytosis.
  • FIG. 6A shows the results of the efferocytosis assay with PEG-conjugated 14C9 LC K149C antibodies using commercial (left) and in-house isolated mouse peritoneal macrophages (right).
  • FIG. 6B summarizes the potency and efficacy of 14C9 antibody and 14C9 LC K149C conjugates in the efferocytosis assay.
  • FIG. 7 show's the binding of anti-murine MerTK parental antibody and THIOMAB antibody -PEG conjugates to primary murine peritoneal macrophages as measured by FACS.
  • FIGS. 8A & 8B show a comparison of the inhibitory’ activity of the PEG40 conjugated
  • FIG. 8A shows the results of the efferocytosis assay with PEG-conjugated 14C9 Fabs.
  • FIG. 88 summarizes the potency and efficacy of 14C9 mAb, Fab and Fab PEG conjugates.
  • FIGS. K1A-10C show the results of analysis MerTK occupancy in tumor-associated macrophages (TAMs) and retinal pigment epithelium cells (RPEs) by and-MerTK antibody conjugates after a single high dose (30 mg/kg) of antibody.
  • FIG. 10C shows the levels of unoccupied MerTK on free-MerTK+ RPE cells.
  • the MFI (mean fluorescence intensity) analysis suggested partial occupancy of MerTK.
  • FIGS. 11A-11D show the MerTK occupancy on RPEs after a repeated high dose treatment (45 mg/kg on Day 1 and Day 5) with anti-MerTK antibody conjugates.
  • FIG. 11A shows levels of occupancy of MerTK, while FIG. 118 summarizes the observed occupancy.
  • FIG. 11C shows the levels of unoccupied MerTK on free-MerTK+ RPE cells after incubation with anti-MerTK antibody conjugates.
  • FIG. 1 ID summarizes the molecular weight of the anti-MerTK antibody conjugates.
  • FIGS. 12A-12C show the analysis of MerTK occupancy in tumor-associated macroplrages (TAMs) after a low repeated dose treatment (2.5 mg/kg on Day 1 and Day 5) with anti- MerTK antibody conjugates.
  • FIG. 12A shows the results of the occupancy analysis on TAMs.
  • FIG. 128 summarizes the occupancy results.
  • FIG. 12C shows the levels of unoccupied MerTK on free- MerTK+ TAMs.
  • FIG. 13 shows the induction of IFNb and interferon-stimulated genes (ISGs) in tumors after low repeated dose (2.5 mg/kg) with PEG-conjugated anti-MerTK antibodies.
  • aspects and embodiments of the present disclosure include “comprising,” “consisting,” and “consisting essentially of’ aspects and embodiments.
  • an “acceptor human framework” for the purposes herein is a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain ( VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below.
  • An acceptor human framework “derived from” a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence thereof, or it may contain amino acid sequence changes. In some aspects, the number of amino acid changes are 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less.
  • the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or human consensus framework sequence.
  • Binding affinity refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen).
  • binding affinity refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g,, antibody and antigen).
  • the affinity of a molecule X for its partner ⁇ can generally be represented by the dissociation constant (K D ). Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative and exemplary methods for measuring binding affinity are described in the following.
  • An “affinity matured” antibody refers to an antibody with one or more alterations in one or more complementary' determining regions (CDRs), compared to a parent antibody which does not possess such alterations, such alterations resulting in an improvement in the affinity of the antibody for antigen.
  • CDRs complementary' determining regions
  • anti-MeiTK antibody and “an antibody that binds to MerTK” refer to an antibody that is capable of binding MerTK with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting MerTK.
  • the extent of binding of an anti-MerTK antibody to an unrelated, non-MerTK protein is less than about 10% of the binding of the antibody to MerTK as measured, e.g., by surface plasmon resonance (SPR).
  • KD dissociation constant
  • an anti-MerTK antibody binds to an epitope of MerTK that is conserved among MerTK from different species.
  • antibody herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.
  • 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.
  • antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv, and scFab); single domain antibodies (dAbs); and multispecific antibodies formed from antibody fragments.
  • 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 ty pe of constant domain or constant region possessed by its heavy drain.
  • the antibody is of the IgGi isotype.
  • the antibody is of the IgGi isotype with the P329G, L234A and L235A mutation to reduce Fc-region effector function.
  • the antibody is of the IgG2 isotype.
  • the antibody is of the IgG4 isotype with the S228P mutation in the hinge region to improve stability of IgG4 antibody .
  • the heavy chain constant domains that correspond to tire different classes of immunoglobulins are called a, 8, s, v, and p, respectively.
  • the light chain of an antibody may be assigned to one of two types, called kappa (K) and lambda (X), based on the amino acid sequence of its constant domain.
  • constant region derived from human origin denotes a constant heavy chain region of a human antibody of the subclass IgG i , IgG2, IgG3, or IgG4 and/or a constant light chain kappa or lambda region.
  • constant regions are well known in the state of the art and e.g, described by Kabat, E, A., et al,, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health Bethesda, AID (1991) (see also e.g. Johnson, G., and Wu, T.T., Nucleic Acids Res.
  • Antibody effector functions refer to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: Clq binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibodydependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor); and B cell activation.
  • an “effective amount” of an agent refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.
  • Fc region herein is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region.
  • the term includes native sequence Fc regions and variant Fc regions.
  • a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the caiboxyl-terminus of the heavy chain.
  • antibodies produced by host cells may undergo post-translational cleavage of one or more, particularly one or two, amino acids from the C-terminus of the heavy chain.
  • an antibody produced by a host cell by expression of a specific nucleic acid molecule encoding a full-length heavy chain may include the full-length heavy chain, or it may include a cleaved variant of the full-length heavy chain.
  • This may be the case where the final two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, EU numbering system). Therefore, the C-terminal lysine (Lys447), or the C-terminal glycine (Gly 446) and lysine (Lys447), of the Fc region may or may not be present.
  • a heavy chain including anFc region as specified herein, comprised m an antibody according to the invention comprises an additional C- terminal gly cine -lysine dipeptide (G446 and K447, EU numbering system).
  • a heavy chain including an Fc region as specified herein, comprised in an antibody according to the invention comprises an additional C-terminal glycine residue (G446, numbering according to EU index).
  • EU numbering system also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.
  • “Framework” or “FR” refers to variable domain residues other than complementary' determining regions (CDRs).
  • the FR of a variable domain generally consists of four FR domains: FR I , FR2, FR3, and FR4, Accordingly, the CDR and FR sequences generally appear in the following sequence in VH (or VL): FR1-CDR-H1(CDR-L1)-FR2- CDR-H2(CDR-L2)-FR3- CDR-H3(CDR- L3)-FR4.
  • full length antibody “intact antibody”, and “whole antibody” are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that contain an Fc region as defined herein.
  • host cell refers to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells.
  • Host cells include “transformants” and “transformed cells”, which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.
  • a “human antibody” is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human or a human cell or derived from a non-human source that utilizes human antibody repertoires or other human antibody -encoding sequences. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.
  • a “human consensus framework” is a framework which represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences.
  • the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences.
  • the subgroup of sequences is a subgroup as in Kabat et al, Sequences of Proteins of Immunological Interest, Fifth Edition, NTH Publication 91- 3242, Bethesda MD (1991), vols. 1-3.
  • the subgroup is subgroup kappa I as in Kabat et al., supra.
  • the subgroup is subgroup III as in Kabat et al., supra.
  • a “humanized” antibody refers to a chimeric antibody comprising amino acid residues from non-human CDRs and amino acid residues from human FRs,
  • a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in winch all or substantially all of the CDRs correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody.
  • a humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody.
  • a “humanized form” of an antibody, e.g., a non-human antibody refers to an antibody that has undergone humanization.
  • hypervariable region refers to each of the regions of an antibody variable domain which are hypeivariable in sequence and which determine antigen binding specificity, for example “complementarity determining regions” (“CDRs”).
  • CDRs complementarity determining regions
  • antibodies comprise six CDRs: three in the VH (CDR-H1, CDR-H2, CDR-H3), and three in the VL (CDR-L1, CDR-L2, CDR-L3).
  • Exemplary CDRs herein include:
  • the CDRs are determined according to Rabat et al., supra.
  • One of skill in the art will understand that the CDR designations can also be determined according io Chothia, supra, McCallum, supra, or any other scientifically accepted nomenclature system.
  • CDR residues comprise those identified in section II. A or elsewhere in the specification.
  • an “immunoconjugate” is an antibody conjugated to one or more heterologous molecule(s), including but not limited to a cytotoxic agent.
  • An “individual” or “subject” is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and nonhuman primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain aspects, the individual or subject is a human.
  • domesticated animals e.g., cows, sheep, cats, dogs, and horses
  • primates e.g., humans and nonhuman primates such as monkeys
  • rabbits e.g., mice and rats
  • rodents e.g., mice and rats
  • an “isolated” antibody is one which has been separated from a component of its natural environment.
  • an antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC) methods.
  • electrophoretic e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis
  • chromatographic e.g., ion exchange or reverse phase HPLC
  • nucleic acid molecule or “polynucleotide” includes any compound and/or substance that comprises a polymer of nucleotides.
  • Each nucleotide is composed of a base, specifically a purine- or pyrimidine base (i.e. cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e. deoxyribose or ribose), and a phosphate group.
  • cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U) a sugar (i.e. deoxyribose or ribose), and a phosphate group.
  • die nucleic acid molecule is described by the sequence of bases, whereby said bases represent the primary structure (linear structure) of a nucleic acid molecule.
  • nucleic acid molecule encompasses deoxyribonucleic acid (DNA) including e.g., complementary’ DNA (cDN A) and genomic DNA, ribonucleic acid (RNA), in particular messenger RNA (mRNA), synthetic forms of DNA or RNA, and mixed polymers comprising two or more of these molecules.
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • mRNA messenger RNA
  • the nucleic acid molecule may be linear or circular.
  • nucleic acid molecule includes both, sense and antisense strands, as well as single stranded and double stranded forms.
  • the herein described nucleic acid molecule can contain naturally occurring or non-naturally occurring nucleotides.
  • nucleic acid molecules also encompass DNA and RNA molecules which are suitable as a vector for direct expression of an antibody of the invention in vitro and/or in vivo, e.g., in a host or patient.
  • DNA e.g., cDNA
  • RNA e.g., mRNA
  • mRNA can be chemically modified to enhance the stability' of the RNA vector and/or expression of the encoded molecule so that mRNA can be injected into a subject to generate the antibody in vivo (see e.g., Stadler ert al, Nature Medicine 2017, published online 12 June 2017, doi:10.1038/nm.4356 or EP 2 101 823 Bl).
  • An “isolated” nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment.
  • An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.
  • isolated nucleic acid encoding an anti-MerTK antibody refers to one or more nucleic acid molecules encoding anti-MerTK antibody heavy and light chains (or fragments thereof), including such nucleic acid molecule(s) in a single vector or separate vectors, and such nucleic acid molecule(s) present at one or more locations in a host cell.
  • the term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts.
  • polyclonal antibody preparations typically include different antibodies directed against different determinants (epitopes)
  • each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen.
  • the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • tlie monoclonal antibodies in accordance with the present invention may be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phagedisplay methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary 7 methods for making monoclonal antibodies being described herein.
  • a “naked antibody” refers to an antibody that is not conjugated to a heterologous moiety’ (e.g., a cytotoxic moiety ) or radiolabel.
  • the naked antibody may be present in a pharmaceutical composition,
  • Native antibodies refer to naturally occurring immunoglobulin molecules w ith vary ing structures.
  • native IgG antibodies are heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light chains and two identical heavy chains that are disulfide- bonded. From N- to C-terminus, each heavy chain has a variable domain ( VI-I), also called a vanable heavy domain or a heavy chain variable region, followed by three constant heavy domains (CHI, CH2, and CH3). Similarly, from N- to C-terminus, each light chain has a variable domain (VL), also called a variable light domain or a light drain variable region, followed by a constant light (CL) domain.
  • VI-I variable domain
  • VL variable domain
  • CL constant light
  • package insert is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings concerning the use of such therapeutic products.
  • Percent (%) amino acid sequence identity with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity for the purposes of the alignment. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software or the FASTA program package.
  • the percent identity values can be generated using the sequence comparison computer program ALIGN-2.
  • the ALIGN-2 sequence comparison computer program was authored by Genentech, Inc,, and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087 and is described in WO 2001/007611.
  • percent amino acid sequence identity values are generated using the ggsearch program of the FASTA package version 36.3.8c or later with a BLOSUM50 comparison matrix.
  • the FASTA program package was authored by 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. Enzvmol. 266:227- 258; and Pearson et. al. (1997) Genomics 46:24-36 and is publicly available from www.fasta.bioch.virginia.edu/fasta www2/fasta down. shtml or www.
  • composition or “pharmaceutical formulation” refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the pharmaceutical composition would be administered.
  • a “pharmaceutically acceptable earner” refers to an ingredient in a pharmaceutical composition or formulation, other than an active ingredient, which is nontoxic to a subject.
  • a pharmaceutically acceptable earner includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.
  • MerTK refers to any native MerTK from any vertebrate source, including mammals such as primates (e.g. humans) and rodents (e.g., mice and rats), unless otherwise indicated.
  • the term encompasses “full-length,” unprocessed MerTK as well as any form of MerTK that results from processing in the cell.
  • the term also encompasses naturally occurring variants of MerTK, e.g., splice variants or allelic variants.
  • the amino acid sequence of an exemplary human MerTK is described in US 2006/0121562.
  • treatment refers to clinical intervention in an attempt to alter the natural course of a disease in the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.
  • antibodies of the invention are used to delay development of a disease or to slow the progression of a disease.
  • variable region refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen.
  • the variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three complementary determining regions (CDRs).
  • FRs conserved framework regions
  • CDRs complementary determining regions
  • antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody' that binds the antigen to screen a library' of complementary VL. or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al.. Nature 352:624-628 ( 1991),
  • vector refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked.
  • the term includes the vector as a selfreplicaring nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it lias been introduced.
  • Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors”.
  • the invention is based, in part, on the observation that MeiTK antibody treatment can lead to on-target retinal toxicity (in particular, cells in the photoreceptor and outer nuclear layers) in preclinical primate and mouse models.
  • on-target retinal toxicity in particular, cells in the photoreceptor and outer nuclear layers
  • increasing the hydrodynamic radius of anti-MerTK antibodies e.g., by conjugating one or more polyethylene glycol, PEG, poly inerts
  • PEGylated antibodies that bind to MerTK are provided. PEGylated antibodies of the invention are useful, e.g., for the treatment of cancer.
  • C-Mer proto-oncogene tyrosine kinase is a receptor tyrosine kinase which transduces extracellular signals upon binding to various ligands, such as galectin-3, Protein S, and Gas6, thus activating expression of effector genes.
  • the MerTK pathway regulates essential cellular processes, including cell survival, cytokine production, migration, differentiation, and phagocytosis (Cabernoy N., et al. J Cell Physio. 22' 1 (2012): 401-407; Wu, G., et al. Cell Death & Disease 8 (2017): e2700).
  • MerTK is found in a variety of hematopoeietic cell types, such as macropliages, dendritic cells, natural killer (NK) cells. Importantly, the MerTK receptor pathway is active in several solid and hematological cancers, including colon cancer (Wu, G., et al. Cell Death & Disease 8 (2017): e2700).
  • MerTK is also expressed in the retinal pigmented epithelium and is required for turnover of the photoreceptor outer segments (POS) critical for vision. Indeed, mutations in MerTK have been found in patients with retinal dystrophies.
  • the MerTK receptor is composed of an extracellular component, a transmembrane (TM) domain, and an intracellular component. As shown in the diagram below, the extracellular or ligandbinding region of MerTK contains two immunoglobulin (Ig)-like domains and two fibronectin (FN) type Ill-like domains.
  • Ig immunoglobulin
  • FN fibronectin
  • the two Ig-like domains are defined by amino acid residues 76-195 and amino acid residues 199-283, respectively.
  • the two fibronectin-Eke domains of human MerTK are defined by amino acid residues 286-384 and amino acid residues 388- 480, respectively.
  • the intracellular region of MerTK contains a tyrosine kinase (TK) domain, which autophosphorylates specific ty rosine residues following ligand binding to the extracellular region and facilitates MerTK receptor dimerization, thus activating downstream effector gene expression (Toledo, R. A, et al. Clin Can. Res. 22 (2016): 2301-2.312).
  • Human MerTK comprises the amino acid sequence: MGPAPLPLLLGLFLPALWRRAITEAREEAKPYPLFPGPFPGSLQTDHTPLLSLPHASGYQPAL MFSPTQPGRPHTGNVAIPQVTSVESKPLPPLAFKHTVGHIILSEHKGVKFNCSISVPNIYQDTTI SWWKDGKELLGAHHAITQFYPDDEVTAITASFSITSVQRSDNGSYICKMKINNEEIVSDPIYTEV QGLPHFTKQPESMNVTRNTAFNIYCQAVGPPEPVNIFWVQNSSRVNEQPEKSPSVT.TVPGLTE MAVFSCEAHNDKGLTVSKGVQINIKAIPSPPTEVSIRNSTAHSILISWVPGFDGYSPFRNCSIQV KE ADPLSNG SVMIFNTS ALPHLYQIKQLQ AL ANYSIGVSCMNEIGWS A VSPWIL A STTEG APS V /WLNVTVFLNESSDNVDIRWMKPPT 'KQQDG
  • the invention provides antibodies (e.g., PEGylated antibodies) that bind to
  • RNA molecules that bind to MerTK.
  • the invention provides antibodies that specifically bind to MerTK.
  • the anti-MerTK antibodies are PEGylated, i.e., are conjugated to one or more (e.g., i or 2) PEG polymers.
  • 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 (a) CDR-H1 comprising the amino acid sequence of SYAMG (SEQ ID NO: 1); (b) CDR-H2 comprising the amino acid sequence of IINSYGNTYYANWAKG (SEQ ID NO: 2); (c) CDR-H3 comprising the amino acid sequence of DPGVSSNL (SEQ ID NO: 3); (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) CDR-L3 comprising the amino acid sequence of QATYYSSNSVA (SEQ ID NO: 6).
  • the invention provides an antibody comprising at least one, at least two, or all three VH CDR sequences selected from (a) CDR-HI comprising the amino acid sequence of SEQ ID NO: I; (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 NOG.
  • the antibody comprises CDR- H3 comprising the amino acid sequence of SEQ ID NO:3.
  • the antibody comprises CDR-H3 comprising the amino acid sequence of SEQ ID NOG and CDR-L3 comprising the amino acid sequence of SEQ ID NOE.
  • the antibody comprises CDR-H3 comprising the amino acid sequence of SEQ ID NOG, CDR-L3 comprising the amino acid sequence of SEQ ID NOE, and CDR-H2 comprising the amino acid sequence of SEQ ID NO:2.
  • 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 tire amino acid sequence of SEQ ID NOG.
  • the 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 NOE.
  • 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) CDR-L3 comprising the amino acid sequence of SEQ ID NOE.
  • the invention provides an anti-MerTK antibody' comprising at least one, at least two, at least three, at least four, at least five, or ah six CDRs selected from (a) CDR-HI comprising the amino acid sequence of ANTMN (SEQ ID NO: 7); (b) CDR-H2 comprising the amino acid sequence of IFTATGSTYYATWVNG (SEQ ID NO: 8); (c) CDR-I-I3 comprising the amino acid sequence of SGSGSSSGAFNI (SEQ ID NO: 9); (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) CDR-L3 comprising the amino acid sequence of QCTSYGSLFLGP (SEQ ID NO: 12).
  • CDR-HI comprising the amino acid sequence of ANTMN
  • CDR-H2 comprising the amino acid sequence of I
  • the invention provides an antibody comprising at least one, at least two, or all three VH CDR sequences selected from (a) CDR-HI 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 NON.
  • the antibody comprises CDR- H3 comprising the amino acid sequence of SEQ ID NON.
  • 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.
  • the antibody' comprises CDR-H3 comprising the amino acid sequence of SEQ ID NON, CDR-L3 comprising the amino acid sequence of SEQ ID NO: 12, and CDR-H2 comprising the amino acid sequence of SEQ ID NOG.
  • the antibody comprises (a) CDR-HI comprising the amino acid sequence of SEQ ID NO:7; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NOG; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NON.
  • the invention provides an antibody comprising at least one, at least two, or ail three VL CDR sequences selected from (a) CDR-LI comprising the amino acid sequence of SEQ ID NO: 10; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO:I 1; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 12.
  • 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: i 1; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 12.
  • an anti-MerTK antibody is humanized.
  • an anti-MerTK antibody further comprises an acceptor human framework, e.g. a human immunoglobulin framework or a human consensus framework,
  • an anti-MerTK antibody further comprises a VII or VL comprising an FR1, FR2, FR3, or FR4 sequence of SEQ ID NOs:13 or 14, respectively.
  • an anti-MerTK antibody comprises one or more of the CDR sequences of the VII of SEQ ID NO: 13. In another embodiment, an anti-MerTK antibody comprises one or more of tire CDR sequences of the VL of SEQ ID NO: 14. In another embodiment, an anti-MerTK antibody comprises the CDR sequences of the VH of SEQ ID NO: 13 and the CDR sequences of the VL of SEQ ID NO: 14.
  • an anti-MerTK antibody comprises the CDR-H1, CDR-H2 and CDR-
  • an anti-MerTK antibody comprises one or more of the heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 13 and a framework of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO: 13.
  • the anti-MerTK antibody comprises the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 13 and a framework of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework ammo acid sequence of the VH domain of SEQ ID NO: 13.
  • the anti-MerTK antibody comprises the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 13 and a framework of at least 95% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO: 13.
  • the anti-MerTK antibody comprises the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 13 and a framework of at least of at least 98% sequence identity’ to the framework amino acid sequence of the VH domain of SEQ ID NO: 13.
  • an anti-MerTK antibody comprises one or more of the light drain CDR amino acid sequences of the VL domain of SEQ ID NO: 14 and a framework of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NO: 14.
  • the anti-MerTK antibody comprises the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO: 14 and a framework of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NO: 14.
  • the anti-MerTK antibody comprises the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO: 14 and a framework of at least 95% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NO: 14.
  • the anti-MerTK antibody comprises the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO: 14 and a framework of at least particularly of at least 98% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO: 13.
  • 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;
  • CDR-H3 comprising the amino acid sequence of SEQ ID NO:3
  • CDR -LI comprising the amino acid sequence of SEQ ID NO:4
  • CDR-L2 comprising the amino acid sequence of SEQ ID NO:5
  • 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 to the amino acid sequence of SEQ ID NO: 13, and a VL domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 14.
  • the VII domain lias at least 95% sequence identity to the amino acid sequence of SEQ ID NO:13.
  • the VL domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 14.
  • 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) 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-L2 comprising the amino acid sequence of SEQ ID NO:5; and (f) 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 to the amino acid sequence of SEQ ID NO: 13, and a VL domain having at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:
  • an anti-MerTK antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 13.
  • an 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.
  • a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti- MerTK antibody comprising that sequence retains the ability to bind to MeiTK.
  • a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 13.
  • substitutions, insertions, or deletions occur in regions outside the CDRs (i.e. , in the FRs).
  • the anti-MerTK antibody comprises the VH sequence in SEQ ID NO: 13, including post-translational modifications of that sequence.
  • the VH comprises one, two or three CDRs selected from: (a) CDR-H1, comprising die 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.
  • an anti-MerTK antibody is provided, wherein the antibody comprises a light chain variable domain (VL) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 14.
  • VL light chain variable domain
  • an 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.
  • VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-MerTK antibody comprising that sequence retains the ability to bind to MerTK.
  • a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 14.
  • the anti-MerTK antibody comprises the VL sequence in SEQ ID NO: 14, including post-translational modifications of that sequence.
  • the VL comprises one, two or three CDRs selected from: (a) CDR- Ll, 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.
  • an anti-MerTK antibody is provided, wherein the antibody comprises a
  • an anti-MerTK antibody further comprises a VH or VL comprising an
  • an anti-MerTK antibody comprises one or more of the CDR sequences of the VH of SEQ ID NO: 15.
  • an anti-MerTK antibody comprises one or more of the CDR sequences of the VL of SEQ ID NO: 16.
  • an anti-MerTK antibody comprises the CDR sequences of the VH of SEQ ID NO: 15 and the CDR sequences of the VL of SEQ ID NO: 16.
  • an 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 the CDR-L1, CDR-L2 and CDR- L3 amino acid sequences of the VL domain of SEQ ID NO: 16.
  • an anti-MerTK antibody comprises one or more of the heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 15 and a framework of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO: 15.
  • the anti-MerTK antibody comprises the three heavy drain CDR amino acid sequences of the VH domain of SEQ ID NO: 15 and a framework of at least 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%,, 98%,, 99% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO: 15.
  • the anti-MerTK antibody comprises the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 15 and a framework of at least 95%> sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO: 15.
  • the anti-MerTK antibody comprises the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 15 and a framework of at least of at least 98% sequence identity to the framework amino acid sequence of die VII domain of SEQ ID NO: 15.
  • an anti-MerTK antibody comprises one or more of the light chain CDR amino acid sequences of the VL domain of SEQ ID NO: 16 and a framework of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93% 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NO: 16.
  • the anti-MerTK antibody comprises the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO: 16 and a framework of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NO: 16.
  • the anti-MerTK antibody comprises the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO: 16 and a framework of at least 95% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NO: 16.
  • the anti-MerTK antibody comprises the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO: 16 and a framework of at least particularly of at least 98% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO: 15.
  • 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) 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-L2 comprising the amino acid sequence of SEQ ID NO:5; and (f) 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 to the amino acid sequence of SEQ ID NO: 15, and a VL domain having at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:
  • the anti-MeiTK 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) 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-L2 comprising the amino acid sequence of SEQ ID NO:5; and (f) 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 to the amino acid sequence of SEQ ID NO: 15, and a VL.
  • the VH domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 15.
  • the VI. domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 16.
  • an anti-MerTK antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 15.
  • an 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.
  • a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti- MerTK antibody comprising that sequence retains the ability to bind to MerTK.
  • a total of I to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 15.
  • substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs).
  • the anti-MerTK antibody comprises the VH sequence in SEQ ID NO: 13, including post-translational modifications of that sequence.
  • the VH comprises one, two or three CDRs selected from: (a) CDR-H1, comprising the amino acid sequence of SEQ ID NO:1, (b) CDR-H2, comprising the amino acid sequence of SEQ ID NOV, and (c) CDR-H3, comprising the amino acid sequence of SEQ ID NO:3.
  • an anti-MerTK antibody is provided, wherein the antibody comprises a light chain variable domain (VL) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 16.
  • VL light chain variable domain
  • an 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.
  • VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-MerTK antibody comprising that sequence retains the ability to bind to MerTK.
  • a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 16.
  • the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs).
  • the anti-MerTK antibody comprises the VL sequence in SEQ ID NO: 16, including post-translational modifications of that sequence.
  • the VL comprises one, two or three CDRs selected from: (a) CDR- Ll, 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.
  • an anti-MerTK antibody comprising a VH sequence as in any of the aspects provided above, and a VL. sequence as in any of the aspects provided above.
  • the antibody comprises the VH and VL sequences in SEQ ID NO: 15 and SEQ ID NO: 16, respectively, including post-translational modifications of those sequences.
  • an anti-MerTK antibody comprises one or more of the CDR sequences of the VH of SEQ ID NO: 17. In another embodiment, an anti-MerTK antibody comprises one or more of the CDR sequences of the VL of SEQ ID NO: 18. In another embodiment, an anti-MerTK antibody comprises the CDR sequences of the VH of SEQ ID NO: 17 and the CDR sequences of the VL of SEQ ID NO: 18.
  • an 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 the CDR-I., 1 , CDR-L2 and CDR- L3 amino acid sequences of the VL domain of SEQ ID NO: 18.
  • an anti-MerTK antibody comprises one or more of the heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 17 and one, two, three, or four human framework sequences (e.g., HC-FR1, HC-FR2, HC-FR3, and/or HC-FR4).
  • an anti- MerTK antibody comprises one or more of the light chain CDR amino acid sequences of the VL domain of SEQ ID NO: 18 and one, two, three, or four human framework sequences (e.g., LC-FRI, LC-FR2, LC-FR3, and/or LC-FR4).
  • an anti-MerTK antibody comprising a VH sequence as in any of the aspects provided above, and a VL sequence as in any of the aspects provided above.
  • the antibody comprises the VH and VL sequences in SEQ ID NO: 17 and SEQ ID NO: 18, respectively, including post-translational modifications of those sequences.
  • an anti-MerTK antibody comprises one or more of the CDR sequences of the VH of SEQ ID NO: 19. In another embodiment, an anti-MerTK antibody comprises one or more of the CDR sequences of the VL of SEQ ID NO:20, In another embodiment, an anti-MerTK antibody comprises the CDR sequences of the VH of SEQ ID NO: 19 and the CDR sequences of the VL of SEQ ID NO:20.
  • an 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 the CDR-L1, CDR-L2 and CDR- L3 amino acid sequences of the VL domain of SEQ ID NO: 20.
  • an anti-MerTK antibody comprises one or more of the heavy drain CDR amino acid sequences of the VH domain of SEQ ID NO: 19 and one, two, three, or four human framework sequences (e.g., HC-FR1, HC-FR2, HC-FR3, and/or HC-FR4).
  • an anti- MerTK antibody comprises one or more of the light chain CDR amino acid sequences of the VL domain of SEQ ID NQ:20 and one, two, three, or four human framework sequences (e.g., LC-FRl, LC-FR2, LC-FR3, and/or LC-FR4).
  • an anti-MerTK antibody comprising a VH sequence as in any of the aspects provided above, and a VL sequence as in any of the aspects provided above.
  • the antibody comprises the VH and VL sequences in SEQ ID NO: 19 and SEQ ID NO:20, respectively, including post-translational modifications of those sequences.
  • an anti-MerTK antibody according to any of the above aspects is a monoclonal antibody, including a chimeric, humanized or human antibody.
  • an anti-MerTK antibody is an antibody fragment, e.g., a Fv, Fab, Fab’, scFv, diabody, or F(ab’)2 fragment.
  • the antibody is a full length antibody, e.g., an intact IgGl antibody or other antibody class or isotype as defined herein.
  • the C-terminal glycine (Gly446) is present. In one aspect, additional! ⁇ ' the C-terminal lysine (Lys447) is present. In one aspect, additionally the C -terminal glycine (Gly446) and the C-terminal lysine (Lys447) is present.
  • the antibody comprises at least one mutation in the Fc region that reduces binding to Fc receptors and/or complement.
  • the antibody comprises a set of mutations in the Fc region referred to as LALAPG (L234A, L235 A, and P329G, numbering according to EU index). LALAPG and other Fc mutations are described further below and, for example, in U.S. Patent No. 8,969,526.
  • the anti-MerTK antibody comprises a heavy chain comprising the sequence EVQIAT’SGEGLVQPGGSLRLSCAASGFSLSSYAMGWVRQAPGKGLEYVGnNSYGNTYYAN WAKGRFTISRDNSKNrVYLQMGSLRAEDMAVYYCARDPGVSSNLWGRGTLVTVSSASTKG PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVK GFYPSD1AVEWESNGQPENNY
  • the anti-MerTK antibody comprises an Fc region (e.g., a human IgGl Fc region) comprising an N297G mutation, numbering according to EU index.
  • Fc region e.g., a human IgGl Fc region
  • the anti-MerTK antibody comprises a heavy chain comprising the sequence EVQLVESGEGLVQPGGSLRLSCAASGFSLSSYAMGWVRQAPGKGLEYVGIINSYGNTYYAN WAKGRFTISRDNSKNTVAG,QMGSLRAEDMAVYYCARDPGVSSNLWGRGTLVTVSSASTKG PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTWSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVL I-IQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPV
  • the anti-MerTK antibody comprises a light chain comprising the sequence DIQMTQSPSTLSASVGDRVTITCQASQNIYSGLAWYQQKPGKAPKLLIYGASKLASGVPSRFS GSGSGTEFTLTISSLQPDDFATYYCQATYYSSNSVAFGGGTKVEIKRTVAAPSVFIFPPSDEQL KSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY EKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 23).
  • the anti-MerTK antibody comprises a heavy chain comprising the sequence of SEQ ID NO:21 and a light chain comprising the sequence of SEQ ID NO:23. In one aspect, the anti-MerTK antibody comprises a heavy chain comprising the sequence of SEQ ID NO:22 and a light chain comprising the sequence of SEQ ID NO: 23.
  • PEG Polymers [0126] Certain aspects of the present disclosure relate to PEGylated antibodies that bind to MerTK. Any of the anti-MerTK antibodies of the present disclosure (e.g, as described supra ⁇ may be PEGylated.
  • PEGylation of an antibody refers to conjugation (e.g. , chemical coupling) of a polymer of PEG monomers to the antibody.
  • the structure of a PEG monomer is provided below, and PEG polymers are typically expressed as (O-CH2-CH2) confront-OCH3, with /? referring to the number of PEG monomers.
  • a variety of PEG polymers suitable for use herein are known in the art. See, e.g., Jevsevar, S. el al. (2010) Biolechnol. J. 5:113-128.
  • the PEG polymer(s) are linear PEG polymers. In some embodiments, the PEG poiymer(s) are branched PEG polymers. In some embodiments, the branched PEG polymer(s) comprise 2 or more branches. In some embodiments, the branched PEG poly mer(s) comprise 2 branches.
  • the PEGylated antibody lia s a hydrodynamic radius of greater than about 6 nm. In some embodiments, the PEGylated antibody lias a hydrodynamic radius of greater than or equal to about 10 mu. In some embodiments, the PEGylated antibody has a hydrodynamic radius of between about 9 mu and about 11 nm, between about 6 nm and about 11 nm, or between about 6 nm and about 10 nm.
  • an anti-MerTK antibody of the present disclosure is conjugated to one or more PEG polymer(s). In some embodiments, an anti-MerTK antibody of the present disclosure is conjugated to one or two PEG polymer] s).
  • the PEG polymer(s) each have a molecular weight of between about 10 kDa and about 40 kDa. In some embodiments, the PEG polymer(s) each have a molecular weight of between about 20 kDa and about 40 kDa. In some embodiments, the PEG polymer(s) each have a molecular weight of between about 10 kDa and about 20 kDa. In some embodiments, the PEG poly mer(s) each have a molecular weight of about 10 kDa, about 20 kDa, about 30 kDa, or about 40 kDa.
  • the PEG polymer(s) are conjugated to an anti-MerTK antibody of the present disclosure at the heavy and/or light chain(s).
  • the PEG polymer(s) are conjugated to an anti- MerTK antibody of the present disclosure at an engineered cysteine residue of the heavy and/or light chain(s). Engineered cysteine residues are described in greater detail infra.
  • the engineered cysteine residue is selected from the group consisting of K149C of the light chain, K I83C of the light chain, T186C of the heavy chain, and Y373C of the heavy chain (numbering of the light chain according to Kabat and numbering of the heavy chain according to EU index).
  • the antibody comprises two heavy chains, two light chains, and two PEG polymers; and both light chains of the antibody comprise a K149C engineered cysteine conjugated to one of the two PEG polymers.
  • the antibody comprises two heavy chains, two light chains, and two PEG poly mers; and both light chains of the antibody comprise a K183C engineered cysteine conjugated to one of the two PEG poly mers. 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 T186C engineered cysteine conjugated to one of the two PEG polymers. 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 cysteine conjugated to one of the two PEG polymers.
  • PEG polvmer(s) are conjugated to a heavy chain or light chain of an anti-MerTK antibody of the present disclosure at an engineered cysteine residue via maleimide-cysteine conjugation.
  • PEG polymer(s) are conjugated to a heavy chain or light chain of an anti-MerTK antibody of the present disclosure at an engineered cysteine residue via iodoacetamide-cysteine conjugation.
  • the methods comprise contacting (i.e., convalently attaching) an antibody comprising one or more free engineered cysteine residues with one or more polyethylene glycol (PEG) polymer(s) comprising a maleimide moiety under conditions suitable for each of the PEG polymer(s) to be conjugated to an engineered cysteine residue of the antibody via thioether linkage.
  • the conjugation is performed at a pH suitable to avoid conjugation to lysines and/or opening of the maleimide ring.
  • the conjugation is performed at pH 7 to pH 7.5.
  • Non-limiting reaction conditions are exemplified infra.
  • the conjugation reaction is monitored, e.g., using HPLC and size exclusion chromatography (SEC) to resolve antibody species with different polymer ratios.
  • the methods comprise contacting (i.e., convalently attaching) an antibody comprising one or more free engineered cysteine residues with one or more polyethylene glycol (PEG) polymer(s) comprising an iodoacetamide moiety under conditions suitable for each of the PEG polymer(s) to be conjugated to an engineered cysteine residue of the antibody via thioether linkage.
  • PEG polyethylene glycol
  • PEG polymers are conjugated to the antibody at a ratio of 2.0 polymers per antibody.
  • Ratio of PEGtantibody can be determined, e.g., using SEC and HPLC.
  • each of the free engineered cysteine residues is blocked with a cysteine or glutathione moiety prior to the conjugation.
  • the methods further comprise, prior to conjugation, deblocking the free engineered cysteine residues.
  • deblocking the free engineered cysteine residues is performed by reduction at pH 8.5, e.g., particularly for sites with a higher thiol pKa (including but not limited to K149C of the light chain).
  • deblocking the free engineered cysteine residues is performed with more reductant for sites with a higher thiol pKa (including but not limited to K149C of the light drain).
  • the reductant is DTT.
  • Non-limiting reaction conditions are exemplified infra.
  • the antibody after deblocking, the antibody is re-oxidized.
  • the antibody after deblocking, the antibody is purified, e.g. , using cationic exchange chromatography to remove the reductant and the reduced cysteine & glutathione.
  • the methods further comprise purifying the PEGylated antibody from unconjugated antibody and PEG polymer after conjugation.
  • Non-limiting purification methods are exemplified infra.
  • the PEGylated antibody can be purified hydrophobic interaction chromatography (HIC).
  • the antibodies reduce MerTK mediated clearance of apoptotic cells by phagocytes, e.g., the clearance of apoptotic cells is reduced by 1-10 fold, 1-8 foid, 1-5 fold, 1-4 fold, 1-3 fold, 1-2 fold, 2-10 fold, 2-8 fold, 2-5 fold, 2-4 fold, 2-3 fold, 3-10 fold, 3-8 fold, 3-5 fold, 3-4 fold, or by about 1.1 fold, 1.2 fold, 1.3 fold, 1.4 fold,
  • the phagocytes are macrophages.
  • the macrophages are tumor-associated macrophages (TAMs).
  • TAMs may be identified based on expression of various cell-surface markers, including CD 14, HLA-DR (MHC class II), CD312, CD 1 I 5, CD 16, CD163, CD2.04, CD206, and C ID 301.
  • IL-12 matrix metalloproteinases, IL- 10, inducible nitric oxide synthase (iNOS), TNF -alpha, or IL-12 may be combined with cell-surface biomarkers to accurately identify TAM populations (Quatromoni,
  • the clearance of apoptotic cells may be measured by any assay known to one of skill in the art for such purpose.
  • phagocytes such as mouse peritoneal macrophages or human monocyte derived macrophages are used.
  • Apoptotic cells are generated by treatment with dexamethasone and labeled with a detection probe.
  • Phagocytosis can be analyzed by microscopy or flow cytometry after incubation apoptotic cells with phagocytes.
  • the clearance of apoptotic cells is reduced as measured in such an apoptotic cell clearance assay at room temperature.
  • mice are injected with dexamethasone to induce thymocyte death. Resident macrophages in the thymus recognize and engulf the dying/dead cells (Seitz, H. M. J Immunol. 178(9) 5635-5642 (2007).
  • the clearance of apoptotic cells is reduced as measured in such an apoptotic cell clearance assay in vivo.
  • the antibodies reduce ligand-mediated MerTK signaling.
  • the antibodies induce a pro-inflammatory response.
  • lite antibodies induce a type I IFN response.
  • an anti-MerTK antibody of the present disclosure reduces 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%,
  • tire anti-MerTK antibody has a half maximal inhibitory concentration (IC50) for reducing phagocytic activity of apoptotic cells of 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, 10 pM - 1 nM, 10 pM - 1.5 nM, 50 pM - 100
  • an anti-MerTK antibody of the present disclosure enhances the activity of a checkpoint inhibitor by about 1-2 fold, 1- 5 fold, 1-10 fold, 1-15 fold, 1-20 fold, 1-25 fold, 1-30 fold, 1-50 fold, 1-75 fold, 1-100 fold, 1-150 fold, 1-200 fold, 1-250 fold, 1.5-2.
  • an anti-MerTK antibody of the present disclosure enhances the activity of a checkpoint inhibitor as determined using an assay as described in the Examples herein below, such as, for example, by determining a reduction in tumor volume in a mouse tumor model using a combmation of an anti-MerTK antibody plus a checkpoint inhibitor as compared to the reduction in tumor volume using the checkpoint inhibitor alone.
  • 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 agents.
  • the checkpoint inhibitor is a anti-PDl axis antagonist.
  • the checkpoint inhibitor is an anti-PD-Ll antibody.
  • the checkpoint inhibitor is an anti-PDl antibody.
  • an anti-MerTK antibody of the present disclosure increases cell-free DNA (ciDNA) and/or circulating tumor DNA (ctDNA), e.g. , in a blood or plasma sample, by about 1-2 fold, 1 -3 fold, 1-4 fold, 1-5 fold, 1-10 fold, 1.5-2 fold, 1.5-3 fold, 1.5-4 fold, 1.5-5 fold, 1.5-10 fold, 2-3 fold, 2-4 fold, 2-5 fold, 2-10 fold, 3-5 fold, 3-10 fold, 4-5 fold, 4-10 fold, 5-10 fold, or by at least about 1 fold, 2 fold, 3 fold, 4 fold, 5 fold, or 10 fold.
  • ciDNA cell-free DNA
  • ctDNA circulating tumor DNA
  • an anti-MerTK antibody of the present disclosure increases cell- free DNA (cfDNA) and/or circulating tumor DNA (ctDNA) as determined using an assay as described in the Examples herein below, such as, for example, by isolating cfDNA and/or ctDNA from a blood or plasma sample and detecting levels of cfDNA and/or ctDNA using PCR and quantitative DNA electrophoresis.
  • cfDNA cell- free DNA
  • ctDNA circulating tumor DNA
  • an antibody provided herein has a dissociation constant (K D ) of ⁇ IpM, ⁇ 100 nM, ⁇ 10 nM, ⁇ 1 nM, ⁇ 0.1 nM, ⁇ 0.01 nM, or ⁇ 0.001 nM (e.g., 10' 8 M or less, e.g., from 10“ 8 M to 10' 13 M, e.g., from 10' 9 M to 10’ i3 M).
  • K D dissociation constant
  • K D is measured using a BIACORE® surface plasmon resonance assay.
  • a BIACORE®-2000 or a BIACORE ®-3000 (BIAcore, Inc., Piscataway, N.I) is performed at 25°C with immobilized antigen CM5 chips at -10 response units (RU).
  • CM5 chips a carboxy methylated dextran biosensor chips
  • EDC AAnhyl-T ’- (3- dimethylaminopropyl)-carbodiimide hydrochloride
  • NTIS 2v-hydro xysuccinimide
  • Antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 pg/ ml (—0.2 uM) before injection at a flow rate of 5 pl/minute to achieve approximately 10 response units (RU) of coupled protein. Following the injection of antigen, 1 M ethanolamine is injected to block unreacted groups. For kinetics measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS with 0.05% polysorbate 20 (TWEEN-20TM) surfactant (PBST) at 25°C at a flow rate of approximately 25 pl/min.
  • TWEEN-20TM polysorbate 20
  • association rates (k on ) and dissociation rates (k o n) are calculated using a simple one-to-one Langmuir binding model (BI ACORE® Evaluation Software version 3.2) by simultaneously fitting the association and dissociation sensorgrams.
  • the equilibrium dissociation constant (K D ) is calculated as the ratio k O ff/k O n. See, e.g., Chen et al., J. Mol. Biol. 293:865-881 ( 1999).
  • KD is measured by a radiolabeled antigen binding assay (RIA).
  • RIA radiolabeled antigen binding assay
  • an RIA is performed with the Fab version of an antibody of interest and its antigen.
  • solution binding affinity’ of Fabs for antigen is measured by equilibrating Fab with a minimal concentration of ( 125 I)-labeled antigen in the presence of a titration series of unlabeled antigen, then capturing bound antigen with an anti-Fab antibody -coated plate (see, e.g., Chen et al,, J. Mol. Biol. 293:865-881(1999)).
  • MICROTITER® multi-well plates (Thermo Scientific) are coated overnight with 5 pg/ml of a capturing anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), and subsequently blocked with 2% (w/v) bovine serum albumin in PBS for two to five hours at room temperature (approximately 23°C).
  • a non-adsorbent plate (Nunc #269620)
  • 100 pM or 26 pM [ 125 I] -antigen are mixed with serial dilutions of a Fab of interest (e.g., consistent with assessment of the anti-VEGF antibody, Fab-12, in Presta et ah, Cancer Res. 57:4593-4599 (1997)).
  • the Fab of interest is then incubated overnight: however, the incubation may’ continue for a longer period (e.g., about 65 hours) to ensure that equilibrium is reached.
  • an antibody provided herein is an antibody fragment.
  • the antibody fragment is a Fab, Fab’, Fab’-SH, or F(ab’)?. fragment, in particular a Fab fragment.
  • Papain digestion of intact antibodies produces two identical antigenbinding fragments, called “Fab” fragments containing each the heavy- and light-chain variable domains ( VH and VL, respectively) and also the constant domain of the light chain (CL) and the first constant domain of the heavy chain (CHI).
  • Fab fragment thus refers to an antibody fragment comprising a light chain comprising a VL domain and a CL domain, and a heavy chain fragment comprising a VH domain and a CHI domain.
  • Fab' 1 fragments differ from Fab fragments by the addition of residues at the carboxy terminus of the CHI domain including one or more cysteines from the antibody hinge region.
  • Fab’-SH are Fab’ fragments in which the cysteine residue(s) of the constant domains bear a free thiol group. Pepsin treatment yields an F(ab’ty fragment that has two antigen-binding sites (two Fab fragments) and a part of the Fc region.
  • Fab and F(ab’)2 fragments comprising salvage receptor binding epitope residues and having increased in vivo half-life, see U.S. Patent No. 5,869,046.
  • the antibody fragment is a diabody, a triabody or a tetrabody.
  • “Diabodies” are antibody fragments with two antigen-binding sites that may be bivalent or bispecific. See, for example, 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). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).
  • the antibody fragment is a single chain Fab fragment.
  • a “single chain Fab fragment” or “scFab” is a polypeptide consisting of an antibody heavy chain variable domain (VH), an antibody heavy chain constant domain 1 (CHI), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL) and a linker, wherein said antibody domains and said linker have one of the following orders in N-termriial to C -terminal direction: a) VH-CHl -linker- VL-CL, b) VL-CL-linker-VH-CHl, c) VH-CL-hnker-VL-CHl or d) VL-CHl-linlver-VFI-CL.
  • said linker is a polypeptide of at least 30 ammo acids, preferably between 32 and 50 amino acids.
  • Said single chain Fab fragments are stabilized via the natural disulfide bond between the CL domain and the CHI domain.
  • these single chain Fab fragments might be further stabilized by generation of interchain disulfide bonds via insertion of cysteine residues (e.g., position 44 in the variable heavy chain and position 100 in the variable light chain according to Rabat numbering).
  • the antibody fragment is single-chain variable fragment (scFv).
  • scFv single-chain variable fragment
  • a “single-chain variable fragment” or “scFv” is a fusion protein of the variable domains of the heavy (VH) and light drains (VL) of an antibody, connected by a linker.
  • the linker is a short polypeptide of 10 to 25 amino acids and is usually rich in glycine for flexibility, as well as serine or threonine for solubility, and can either connect the N-terminus of the VH with the C -terminus of the VL, or vice versa. This protein retains the specificity of the original antibody, despite removal of the constant regions and the introduction of the linker.
  • the antibody fragment is a single-domain antibody .
  • Single-domain antibodies are antibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody.
  • a single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516 Bl).
  • Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as recombinant production by recombinant host cells (e.g,, E. coli), as described herein.
  • recombinant host cells e.g, E. coli
  • an antibody provided herein is a chimeric antibody.
  • Certain chimeric antibodies are described, e.g., in U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).
  • a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region.
  • a chimeric antibody is a “class switched” antibody in which the class or subclass has been changed from tlrat of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.
  • a chimeric antibody is a humanized antibody.
  • a non-human antibody is humanized to reduce immunogenicity to humans, while retaining die specificity and affinity of the parental non-human antibody.
  • a humanized antibody comprises one or more variable domains in which the CDRs (or portions thereof) are derived from a non-human antibody, and FRs (or portions thereof) are derived from human antibody sequences.
  • a humanized antibody optionally will also comprise at least a portion of a human constant region.
  • some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived), e.g., to restore or improve antibody specificity or affinity.
  • a non-human antibody e.g., the antibody from which the CDR residues are derived
  • Human framework regions that may be used for humanization include but are not limited to: framework regions selected using the “best-fit” method (see, e.g., Sims et al. J. Immunol.
  • framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or 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 (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions derived from screening FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272: 10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611 -22618 (1996)).
  • an antibody provided herein is a multispecific antibody, e.g., a bispecific antibody.
  • Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites, i.e., different epitopes on different antigens or different epitopes on the same antigen.
  • the multispecific antibody has three or more binding specificities.
  • one of the binding specificities is for MerTK and the other specificity is for any other antigen.
  • bispecific antibodies may bind to two (or more) different epitopes of MerTK.
  • Multispecific (e.g., bispecific) antibodies may also be used to localize cytotoxic agents or cells to cells which express MerTK. Multispecific antibodies may be prepared as full length antibodies or antibody fragments.
  • Multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs having different specificities (see Milstein and Cuello, Nature 305: 537 (1983)) and “knob-in-hole” engineering (see, e.g., U.S. Patent No. 5,731,168, and Atwell et al., J. Mol. Biol. 270:26 (1997)).
  • Multi-specific antibodies may also be made by engineering electrostatic steering effects for making antibody Fc- heterodimeric molecules (see, e.g., WO 2009/089004); cross-linking two or more antibodies or fragments (see, e.g., US Patent No.
  • Engineered antibodies witli three or more antigen binding sites including for example, “Octopus antibodies”, orDVD-Ig are also included herein (see, e.g., WO 2001/77342 and WO 2008/024715).
  • Other 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.
  • the bispecific antibody or antigen binding fragment thereof also includes a “Dual Acting FAb” or “DAF” comprising an antigen binding site that binds to MerTK as well as another different antigen, or two different epitopes of MerTK (see, e.g., US 2008/0069820 and WO 2015/095539).
  • DAF Double Acting FAb
  • Multi-specific antibodies may also be provided in an asymmetric form with a domain crossover in one or more binding arms of the same antigen specificity, i.e, by exchanging the VH/VL domains (see e.g., WO 2009/080252 and WO 2015/150447), the CH1/CL domains (see e.g., WO 2009/080253) or the complete Fab arms (see e.g., W r O 2009/080251, WO 2016/016299, also see Schaefer et al, PNAS, 108 (2011) 1187-1191, and Klein at al., MAbs 8 (2016) 1010-20).
  • the multispecific antibody comprises a cross-Fab fragment.
  • cross-Fab fragment or “xFab fragment” or “crossover Fab fragment” refers to a Fab fragment, wherein either the vanable regions or the constant regions of the heavy and light chain are exchanged.
  • a cross-Fab fragment comprises a polypeptide chain composed of the light chain variable region (VL) and the heavy chain constant region 1 (CH 1), and a polypeptide chain composed of the heavy chain variable region ( VH) and the light drain constant region (CL).
  • Asymmetrical Fab arms can also be engineered by introducing charged or non-charged amino acid mutations into domain interfaces to direct comet Fab pairing. See e.g., WO 2016/172485.
  • ammo acid sequence variants of the antibodies provided herein are contemplated.
  • Amino acid sequence variants of an antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen-binding. a) Substitution, Insertion, and Deletion Variants
  • antibody variants having one or more amino acid substitutions are provided.
  • Sites of interest for substitutional mutagenesis include the CDRs and FRs.
  • Conservative substitutions are shown in Table 1 under the heading of “preferred substitutions”. More substantial changes are provided in Table 1 under the heading of “exemplary substitutions”, and as further described below in reference to amino acid side chain classes.
  • Amino acid substitutions may be introduced into an antibody of interest and the products screened for a desired activity, e.g., retained/! mproved antigen binding, decreased immunogenicity, or improved ADCC or CDC. TABLE 1
  • Amino acids may be grouped according io common side-chain properties:
  • Non-conseivative substitutions will entail exchanging a member of one of these classes for a member of another class.
  • One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody).
  • a parent antibody e.g., a humanized or human antibody.
  • the resulting variant(s) selected for further study will have modifications (e.g., improvements) in certain biological properties (e.g., increased affinity, reduced immunogenicity) relative to the parent antibody and/or will have substantially retained certain biological properties of the parent antibody.
  • An exemplary substitutional variant is an affinity matured antibody, which may be conveniently generated, e.g., using phage display -based affinity maturation techniques such as those described herein. Briefly, one or more. CDR residues are mutated and the variant antibodies displayed on phage and screened for a particular biological activity (e.g., binding affinity).
  • Alterations may be made in CDRs, e.g., to improve antibody affinity.
  • Such alterations may be made in CDR “hotspots”, i.e., residues encoded by codons tliat undergo mutation at high frequency during the somatic maturation process (see, e.g., Chowdhury', Methods Mol. Biol. 207:179-196 (2008)), and/or residues that contact antigen, with the resulting variant VH or VL being tested for binding affinity.
  • Affinity maturation by constructing and reselecting from secondary libraries has been described, e.g., in Hoogenboom et al.
  • affinity maturation diversity is introduced into the variable genes chosen for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis).
  • a secondary library' is then created. The library' is then screened to identify any antibody variants with die desired affinity.
  • Another method to introduce diversity involves CDR-directed approaches, in which several CDR residues (e.g., 4-6 residues at a time) are randomized.
  • CDR residues involved in antigen binding may be specifically identified, e.g., using alanine scanning mutagenesis or modeling.
  • CDR-H3 and CDR-L3 in particular are often targeted.
  • substitutions, insertions, or deletions may occur within one or more CDRs so long as such alterations do not substantially reduce the ability of the antibody to bind antigen.
  • conservative alterations e.g., conservative substitutions as provided herein
  • Such alterations may, for example, be outside of antigen contacting residues in the CDRs.
  • each CDR either is unaltered, or contains no more than one, two or three amino acid substitutions.
  • a useful method for identification of residues or regions of an antibody tliat may be targeted for mutagenesis is called “alanine scanning mutagenesis” as described by Cunningham and Wells (1989) Science, 244: 1081-1085.
  • a residue or group of target residues e.g., charged residues such as arg, asp, his, ly s, and glu
  • a neutral or negatively charged amino acid e.g., alanine or poly alanine
  • Further substitutions may be introduced at the amino acid locations demonstrating functional sensitivity to the initial substitutions.
  • a crystal structure of an antigen-antibody complex may be used to identify contact points between the antibody and antigen. Such contact residues and neighboring residues may be targeted or eliminated as candidates for substitution. Variants may be screened to determine whether they contain the desired properties.
  • Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues.
  • terminal insertions include an antibody with an N-temunal methionyl residue.
  • Other insertional variants of the antibodymolecule include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g., for ADEPT (antibody directed enzyme prodrug therapy)) or a polypeptide which increases the serum half-life of the antibody.
  • ADEPT antibody directed enzyme prodrug therapy
  • an antibody provided herein is altered to increase or decrease the extent to which the antibody is glycosylated.
  • Addition or deletion of glycosylation sites to an antibody may be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites is created or removed.
  • the oligosaccharide attached thereto may be altered.
  • Native antibodies produced by mammalian ceils typically comprise a branched, biantennary oligosaccharide that is generally attached by an N-Iinkage to Asn297 of tire CH2 domain of the Fc region. See, e.g., Wright et al. TIBTECH 15:26-32 (1997).
  • the oligosaccharide may include various carbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the “stem" of the biantennary oligosaccharide structure.
  • modifications of the oligosaccharide in an antibody of the invention may be made in order to create antibody variants with certain improved properties.
  • antibody variants having a non-fucosylated oligosaccharide, i.e. an oligosaccharide structure that lacks fucose attached (directly or indirectly) to an Fc region.
  • non-fucosylated oligosaccharide particularly is an N-linked oligosaccharide which lacks a fucose residue attached to the first GlcNAc in the stem of the biantennary oligosaccharide structure.
  • antibody variants are provided having an increased proportion of non-fucosylated oligosaccharides in the Fc region as compared to a native or parent antibody.
  • the proportion of non-fucosylated oligosaccharides may be at least about 20%, at least about 40%, at least about 60%, at least about 80%, or even about 100% (i.e.
  • the percentage of non-fucosylated oligosaccharides is the (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) as measured by MALDI-TOF mass spectrometry, as described in WO 2006/082515, for example.
  • Asn297 refers to the asparagine residue located at about position 297 in the Fc region (EU numbering of Fc region residues); however, Asn297 may also be located about i 3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies.
  • Such antibodies having an increased proportion of non-fucosylated oligosaccharides in the Fc region may have improved FcyRIIIa receptor binding and/or improved effector function, in particular improved ADCC function. See, e.g., US 2003/0157108; US 2004/0093621.
  • Examples of cell lines capable of producing antibodies with reduced fucosy lation include Lee 13 CHO cells deficient in protein fucosy lation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US 2003/0157108; and WO 2004/056312, especially at Example 11), and knockout cell lines, such as alpha- 1, 6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87:614-622 (2004): Kanda, Y. et al., Biotechnol.
  • antibody variants are provided with bisected oligosaccharides, e.g., in which a 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, e.g., in Umana et al., Nat Biotechnol 17, 176-180 (1999); Ferrara et al., Biotechn Bioeng 93, 851-861 (2006); W r O 99/54342; WO 2004/065540, WO 2003/011878.
  • Antibody variants with at least one galactose residue in the oligosaccharide attached to tlie Fc region aie also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, e.g., in WO 1997/30087; WO 1998/58964: and WO 1999/22764. c) Fc region variants
  • one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant.
  • the Fc region variant may comprise a human Fc region sequence (e.g., a human IgGl, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g., a substitution) at one or more amino acid positions.
  • the invention contemplates an antibody vanant that possesses some but not all effector functions, which make it a desirable candidate for applications in which the half life of tlie antibody in vivo is important yet certain effector functions (such as complement-dependent cytotoxicity (CDC) and antibody -dependent cell-mediated cytotoxicity (ADCC)) are unnecessary or deleterious.
  • CDC complement-dependent cytotoxicity
  • ADCC antibody -dependent cell-mediated cytotoxicity
  • In vitro and/or in vivo cytotoxicity assays can be conducted to confirm the reduction/depletion of CDC and/or ADCC activities.
  • Fc receptor (FcR) binding assays can be conducted to ensure that the antibody lacks FcvR binding (hence likely lacking ADCC activity), but retains FcRn binding ability’.
  • NK cells express FcyRIII only, whereas monocytes express FcyRI, FcyRU and FcyRIII.
  • FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet,z1nww. Rev. Immunol. 9:457-492 (1991).
  • Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest is described in U.S. Patent No. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc. Nat ’I Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al.. Proc.
  • nonradioactive assays methods may be employed (see, for example, ACT!'TM non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA; and CytoTox 96® nonradioactive cytotoxicity assay (Promega, Madison, Wl).
  • Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells.
  • ADCC activity’ of the molecule of interest may be assessed in vivo, e.g., in a animal model such as that, disclosed in Clynes et al. Proc. Nat ’i Acad. Sci. USA 95:652-656 (1998).
  • Clq binding assays may also be carried out to confirm that the antibody is unable to bind Clq and hence lacks CDC activity. See, e.g., Clq and C3c binding ELISA in WO 2006/029879 and
  • a CDC assay may be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996); Cragg, M.S. et al., Blood 101: 1045-1052 (2003); and Cragg, M.S. and MJ. Glennie, Blood 103:2738-2743 (2004)).
  • FcRn binding and in vivo clearance/italf life determinations can also be performed using methods known in the art (see, e.g., Petkova, S.B. et al., for 7. Immunol. 18(12): 1759-1769 (2006); WO 2013/120929 Al).
  • Antibodies with reduced effector function include those with substitution of one or more of Fc region residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Patent No. 6,737,056).
  • Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called “DANA” Fc mutant with substitution of residues 265 and 297 to alanine (US Patent No. 7,332,581).
  • an antibody variant comprises an Fc region with one or more amino acid substitutions which improve ADCC, e.g., substitutions at positions 298, 333, and/or 334 of the Fc region (EU numbering of residues).
  • an antibody variant comprises an Fc region with one or more amino acid substitutions which diminish FcyR binding, e.g., substitutions at positions 234 and 235 of the Fc region (EU numbering of residues).
  • the substitutions are L234A and L235A (LALA).
  • the antibody variant further comprises D265A and/or P329G in an Fc region derived from a human IgGl Fc region.
  • the substitutions are L234A, L235A and P329G (LALA-PG) in an Fc region derived from a human IgGl Fc region. (See, e.g., WO 2012/130831).
  • the substitutions are L234A, L235A and D265 A (LALA-DA) in an Fc region derived from a human IgGl Fc region.
  • alterations are made in the Fc region that result in altered (i.e., either improved or diminished) Clq binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as described in US Patent No. 6,194,551 , WO 99/51642, and Idusogie et al. J. Immunol. 164: 4178-4184 (2000).
  • CDC Complement Dependent Cytotoxicity
  • 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, e.g., substitution of Fc region residue 434 (See, e.g., US Patent No. 7,371,826; Dall'Acqua, W.F., et al. J. Biol. Chem. 281 (2006) 23514-23524).
  • Fc region residues critical to the mouse Fc-monse 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 12.53, 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 were found to be critical for the interaction of human Fc with murine FcRn (Kim, J.K., et al., Eur. J. Immunol. 29 (1999) 2819).
  • Studies of the human Fc-human FcRn complex have shown that residues 1253, S254, H435, and Y436 are crucial for the interaction (Firan, M., et al., Int. Immunol. 13 (2001) 993; Shields, R.L., et al., J. Biol. Chem. 276 (2001) 6591-6604).
  • Yeung, Y.A., et al. J. Immunol.
  • an antibody variant comprises an Fc region with one or more amino acid substitutions, which reduce FcRn binding, e.g., substitutions at positions 253, and/or 310, and/or
  • the antibody variant comprises an Fc region with the amino acid substitutions at positions 253, 310 and 435.
  • the substitutions are 1253 A, H310A and H435A in an Fc region derived from a human IgGl Fc-region, See, e.g., Grevys, A,, et al., J. Immunol. 194 (2015) 5497-5508.
  • an antibody variant comprises an Fc region with one or more amino acid substitutions, which reduce FcRn binding, e.g., substitutions at positions 310, and/or 433, and/or
  • the antibody variant comprises an Fc region with the amino acid substitutions at positions 310, 433 and 436.
  • the substitutions are H310A, H433A and Y436A in an Fc region derived from a human IgGl Fc-region. (See, e.g., WO 2014/177460 Al).
  • an antibody variant comprises an Fc region with one or more amino acid substitutions which increase FcRn binding, e.g., substitutions at positions 252, and/or 254, and/or 256 of the Fc region (EU numbering of residues).
  • the antibody variant comprises an Fc region with amino acid substitutions at positions 2.52, 254, and 256.
  • the substitutions are M252Y, S2.54T and T256E in an Fc region derived from a human IgGl Fc-region. See also Duncan & Winter, Nature 32.2:738-40 (1988); U.S. Patent No. 5,648,2.60; U.S. Patent No. 5,62.4,821 ; and WO 94/29351 concerning oilier examples of Fc region variants.
  • the C-terminus of the heavy chain can be a shortened C-terminus in which one or two of the C terminal amino acid residues have been removed.
  • the C-terminus of the heavy chain is a shortened C-terminus ending PG.
  • an antibody comprising a heavy chain including a C-terminal CH3 domain as specified herein comprises the C-terminal glycine-lysine dipeptide (G446 and K447, EU index numbering of amino acid positions).
  • an antibody comprising a heavy drain including a C-terminal CH3 domain comprises a C-terminal glycine residue (G446, EU index numbering of amino acid positions).
  • cysteine engineered antibodies e.g., TH1OMABTM antibodies
  • the substituted residues occur at accessible sites of the antibody.
  • reactive thiol groups are thereby positioned at accessible sites of the antibody and may be used to conjugate the antibody to other moieties, such as PEG poiymer(s), drug moieties, or linker-drug moieties, to create an immunoconjugate or PEGylated antibody, as described further herein.
  • Cysteine engineered antibodies may be generated as described, e.g., in U.S. Patent No. 7,521,541, 8,30,930, 7,855,275, 9,000,130, WO 2016040856, or WO 201 1/156328.
  • an antibody provided herein may be further modified to contain additional nonproteinaceous moieties that are known in the art and readily available.
  • the moieties suitable for derivatization of die antibody include but are not limited to water soluble polymers.
  • water soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1, 3 -dioxolane, poly- 1,3, 6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or polypvinyl pyrrolidone)poly ethylene glycol, propropylene glycol homopolymers, proly propylene oxide/etbylene oxide co-polymers, polyoxy ethylated polyols (e.
  • PEG polyethylene glyco
  • Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water.
  • the polymer may be of any molecular weight, and may be branched or unbranched.
  • the number of polymers attached to the antibody may vary’, and if more than one polymer are attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, whether the antibody derivative will be used in a therapy under defined conditions, etc.
  • the invention also provides immunoconjugates comprising an anti-MerTK antibody herein conjugated (chemically bonded) to one or more therapeutic agents such as cytotoxic agents, chemotherapeutic agents, drags, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically' active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), or radioactive isotopes.
  • therapeutic agents such as cytotoxic agents, chemotherapeutic agents, drags, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically' active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), or radioactive isotopes.
  • an immunoconjugate is an antibody -drug conjugate (ADC) in which an antibody is conjugated to one or more of the therapeutic agents mentioned above.
  • the antibody is typically connected to one or more of the therapeutic agents using linkers.
  • an immunoconjugate comprises an antibody as described herein conjugated to an enzymatically active toxin or fragment thereof, including but not limited to diphtheria A drain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aemginosa), ricin A chain, abrin A drain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and P AP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.
  • an enzymatically active toxin or fragment thereof including but not limited to diphtheria A drain, nonbinding active fragments of diphtheria toxin, exotoxin
  • an immunoconjugate comprises an antibody as described herein conjugated to a radioactive atom to form a radioconjugate.
  • a variety of radioactive isotopes are available for the production of radioconjugates. Examples include At 21 ‘, I 13 , 1 125 , Y 90 , Re 186 , Re 188 , Sm 15 ’, Bi 212 , P !? , Pb 2 12 and radioactive isotopes of Lu.
  • the radioconjugate When used for detection, it may comprise a radioactive atom for scintigraphic studies, for example tc99m or 1123, or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, mri), such as iodine-123 again, iodine-131, indium-i l l, fluorine-19, carbon-13, nitrogen-15, oxy gen- 17, gadolinium, manganese or iron.
  • NMR nuclear magnetic resonance
  • Conjugates of an antibody and cytotoxic agent may be made 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), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HC1), active esters (such as disuccinimidvl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p- azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazomumbenzoyl)- ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluor
  • a ricin immunotoxin can be prepared as described in Vitetta et al.. Science 238: 1098 (1987).
  • Carbon-14-labeled 1- isotluocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to die antibody. See WO 94/11026.
  • the linker may be a “cleavable linker” facilitating release of a cytotoxic drug in the ceil.
  • an acid- labile linker, peptidase-sensitive linker, photolabile linker, dimethyl linker or disulfide-containing linker (Chari et al., Cancer Res. 52:127-131 (1992); U.S. Patent No. 5,208,020) may be used.
  • the immunuoconjugates or ADCs herein expressly contemplate, but are not limited to such conjugates prepared with cross-linker reagents including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, STAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate) which are commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, IL,, U.S.A).
  • cross-linker reagents including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC
  • Antibodies may be produced using recombinant methods and compositions, e.g., as described in US 4,816,567. For these methods one or more isolated nucleic acid(s) encoding an antibody are provided.
  • nucleic acids In case of a native antibody or native antibody fragment two nucleic acids are required, one for the light chain or a fragment thereof and one for the heavy chain or a fragment thereof.
  • Such nucleic acid(s) encode an amino acid sequence comprising the VL and/or an amino acid sequence comprising the VH of the antibody (e.g., the light and/or heavy chain(s) of the antibody).
  • These nucleic acids can be on the same expression vector or on different expression vectors.
  • nucleic acids are required, one for the first light chain, one for the first heavy chain comprising the first heteromonomeric Fc -region polypeptide, one for the second light chain, and one for the second heavy chain comprising the second heteromonomeric Fc -region polypeptide.
  • the four nucleic acids can be comprised in one or more nucleic acid molecules or expression vectors.
  • nucleic acid(s) encode an amino acid sequence comprising the first VL and/or an amino acid sequence comprising the first VH including the first heteromonomeric Fc-region and/or an amino acid sequence comprising the second VL and/or an amino acid sequence comprising the second VH including the second heteromonomeric Fc-reglon of the antibody (e.g., the first and/or second light and/or the first and/or second heavy chains of the antibody).
  • These nucleic acids can be on the same expression vector or on different expression vectors, normally these nucleic acids are located on two or three expression vectors, i.e. one vector can comprise more than one of these nucleic acids. Examples of these bispecific antibodies are CrossMabs (see, e.g., Schaefer, W.
  • one of the heteromonomeric heavy chain compri ses the so-called “knob mutations” (T366W and optionally one of S354C or Y349C) and the other comprises the so-called “hole mutations” (T366S, L368A and Y407V and optionally Y349C or S354C) (see, e.g.. Carter, P. et al,, Iimnunotechnol. 2 (1996) 73) according to EU index numbering.
  • isolated nucleic acids encoding an antibody as used in the methods as reported herein are provided.
  • a method of making an anti-MerTK antibody comprises culturing a host ceil comprising nucleic acld(s) encoding the antibody, as provided above, under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).
  • nucleic acids encoding the antibody are isolated and inserted into one or more vectors for further cloning and/or expression in a host cell.
  • nucleic acids may be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody ) or produced by recombinant methods or obtained by chemical sy nthesis.
  • Suitable host cells for cloning or expression of antibody -encoding vectors include prokaryotic or eukaryotic cells described herein.
  • antibodies may be produced in bacteria, in particular when glycosylation and Fc effector function are not needed.
  • For expression of antibody fragments and polypeptides in bacteria see, e.g., US 5,648,237, US 5,789,199, and US 5,840,523. (See also Chariton, K.A., In: Methods in Molecular Biology, Vol. 248, Lo, B.K.C. (ed.), Humana Press, Totowa, NJ (2003), pp. 245-254, describing expression of antibody fragments in E. coli.)
  • the antibody may be isolated from the bacterial cell paste in a soluble fraction and can be further purified.
  • eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody -encoding vectors, including fungi and yeast strains whose glycosylation pathway s have been “humanized”, resulting in the production of an antibody with a partially or fully human glycosylation pattern. 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 the expression of (glycosylated) antibody are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous bacuioviral strains have been identified which may be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells.
  • Plant cell cultures can also be utilized as hosts. See, e.g., US 5,959,177, US 6,040,498, US 6,420,548, US 7, 125,978, and US 6,417,42.9 (describing PLANTIBODIESTM technology for producing antibodies in transgenic plants),
  • Vertebrate cells may also be used as hosts.
  • mammalian cell lines that are adapted to grow in suspension may be useful.
  • Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293T cells as described, e.g., in Graham, F.L. et al., J. Gen Virol. 36 (1977) 59-74); baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells as described, e.g., in Mather, IP., Biol. Reprod.
  • C VI monkey kidney cells
  • VEO-76 African green monkey kidney cells
  • HELA human cervical carcinoma cells
  • canine kidney cells MDCK; buffalo rat liver cells (BRL 3A); human lung ceils (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells (as described, e.g., in Mather, J.P. et al.. Annals N.Y. Acad. Sci. 383 (1982) 44-68); MRC 5 cells; and FS4 cells.
  • Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR- CHO cells (Urlaub, G. et al., Proc. Natl.
  • the host cell is eukaryotic, e.g., a Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., Y0, NS0, Sp20 cell).
  • a Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., Y0, NS0, Sp20 cell).
  • Anti-MerTK antibodies provided herein may be identified, screened for, or characterized for their phy sical/chemicai properties and/or biological activities by various assays known in the art.
  • an antibody of the invention is tested for its antigen binding activity, e.g., by known methods such as ELISA, Western blot, etc.
  • competition assays may be used to identify an antibody that competes with any of the anti-MerTK antibodies described supra for binding to MerTK.
  • a competing antibody binds to the same epitope (e.g., a linear or a conformational epitope) that is bound by any of the anti-MerTK antibodies described supra.
  • epitope e.g., a linear or a conformational epitope
  • mapping an epitope to which an antibody binds are provided in Morris (1996) “Epitope Mapping Protocols”, in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, N.I).
  • immobilized MerTK is incubated in a solution comprising a first labeled antibody’ that binds to MerTK (e.g., any’ of the anti-MerTK antibodies described supra) and a second unlabeled antibody that is being tested for its ability' to compete with the first antibody for binding to MerTK, The second antibody may be present in a hybridoma supernatant.
  • a control immobilized MerTK is incubated in a solution comprising the first labeled antibody but not the second unlabeled antibody. After incubation under conditions permissive for binding of the first antibody to MerTK, excess unbound antibody is removed, and tire amount of label associated with immobilized MerTK is measured.
  • assays are provided for identifying anti-MerTK antibodies thereof having biological activity.
  • Biological activity' may include, e.g., reducing MeiTK-mediated phagocytic activity, reducing MerTK-mediated clearance of apoptotic cells, and/or enhancing tumor immunogenicity’ of a checkpoint inhibitor.
  • Antibodies having such biological activity' in vivo and/or in vitro are also provided.
  • an antibody of the invention is tested for such biological activity.
  • assays suitable for measuring such biological activity are described further herein, including the Exemplification section below.
  • compositions compri sing any of the antibodies provided herein, e.g., for use in any of the below therapeutic methods.
  • a pharmaceutical composition comprises any of the antibodies provided herein and a pharmaceutically acceptable carrier.
  • a pharmaceutical composition comprises any of the antibodies provided herein and at least one additional therapeutic agent, e.g., as described below.
  • compositions of an anti-MerTK antibody as described herein are prepared by mixing such antibody having the desired degree of purity witli one or more optional pharmaceutically acceptable earners (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized compositions or aqueous solutions.
  • Pharmaceutically acceptable earners are generally nontoxic to recipients 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 octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl 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, glutamine, aspara
  • Exemplary pharmaceutically acceptable carriers herein further include insterstitial drug dispersion agents such as soluble neutral-active hyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®', Halozyme, Inc.).
  • sHASEGP soluble neutral-active hyaluronidase glycoproteins
  • rHuPH20 HYLENEX®', Halozyme, Inc.
  • Certain exemplary sHASEGPs and methods of use, including rHuPH20 are described in US Patent Publication Nos. 2005/0260186 and 2006/0104968.
  • a sHASEGP is combined with one or more additional glycosaminoglv canases such as chondroitinases.
  • Exemplary lyophilized antibody compositions are described in US Patent No. 6,267,958.
  • Aqueous antibody compositions include those described in US Patent No, 6,171,586 and WO 2006/044908, the latter compositions including a histidine-acetate buffer,
  • the plrarmaceutical composition herein may also contain more than one active ingredients as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other.
  • active ingredients e.g., an anti-PDL 1 antibody.
  • Such active ingredients are suitably present in combination in amounts that are effective for the purpose intended.
  • Active ingredients may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal ding delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions.
  • colloidal ding delivery systems for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules
  • compositions for sustained-release may be prepared.
  • suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules.
  • compositions to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes.
  • any of the anti-MeiTK antibodies provided herein may be used in therapeutic methods.
  • the methods e. g. , using a PEGylated anti-MerTK antibody of the present disclosure
  • the methods result in decreased retinal toxicity, as compared to simitar methods using a non-PEGylated anti-MerTK antibody.
  • an anti-MerTK antibody for use as a medicament is provided.
  • an anti-MerTK antibody for use in treating cancer is provided.
  • an anti- MerTK antibody for use in a method of treatment is provided.
  • the invention provides an anti-MerTK antibody for use in a method of treating an individual having cancer comprising administering to the individual an effective amount of the anti-MerTK antibody .
  • the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent (e.g., one, two, three, four, five, or six additional therapeutic agents), e.g., as described below.
  • the invention provides an anti-MerTK antibody for use in enhancing immune function and/or reducing Me rTK -mediated clearance of apoptotic cells.
  • the invention provides an anti-MerTK antibody for use in a method of enhancing immune function and/or reducing MerTK-mediated clearance of apoptotic cells in an individual comprising administering to the individual an effective amount of the 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.
  • the invention provides for the use of an anti-MerTK antibody in the manufacture or preparation of a medicament.
  • the medicament is for treatment of cancer.
  • the medicament is for use in a method of treating cancer comprising administering to an individual having cancer an effective amount of the medicament.
  • the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, e.g., as described below.
  • the medicament is for enhancing immune function and/or reducing MerTK-mediated clearance of apoptotic cells.
  • the medicament is for use in a method of enhancing immune function and/or reducing MerTK-mediated clearance of apoptotic cells in an individual comprising administering to the individual an effective amount of the medicament to entrance immune function and/or reduce MerTK- mediated clearance of apoptotic cells.
  • An “individual” according to any of the above aspects may be a human,
  • the invention provides a method for treating a cancer.
  • the method comprises administering to an individual having such cancer an effective amount of an anti-MerTK antibody .
  • the method further comprises administering to the individual 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.
  • the invention provides a method for enhancing immune function and/or reducing MerTK-mediated clearance of apoptotic cells in an individual, e.g. , an individual having cancer.
  • 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.
  • an “individual” is a human.
  • the invention provides pharmaceutical compositions comprising any of the anti-MerTK antibodies provided herein, e.g., for use in any of the above therapeutic methods.
  • a pharmaceutical composition comprises any of the anti-MerTK antibodies provided herein and a pharmaceutically acceptable carrier.
  • a pharmaceutical composition comprises any of the anti-MerTK antibodies provided herein and at least one additional therapeutic agent, e.g., as described below.
  • an anti-MerTK antibody of the present disclosure is administered as a monotherapy to treat an individual having cancer.
  • cancer refers to or describes the physiological condition in mammals that is typical ly characterized by unregulated cell growth.
  • the cancer may be a solid cancer or a hematologic cancer. Solid cancers are generally characterized by tumor mass formation in specific tissues. “Tumor,” as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
  • Non-limiting examples of solid cancers to be treated with an anti-MerTK antibody of the present disclosure include carcinoma, lymphoma, blastoma, and sarcoma. More particular examples of such cancers include, but not limited to, squamous cell cancer (e.g, epithelial squamous cell cancer), lung cancer including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer and gastrointestinal stromal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer of the urinary tract, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, mel
  • cancers that are amenable to treatment by anti-MerTK antibodies of the present disclosure include breast cancer, colorectal cancer, rectal cancer, non-small cell lung cancer, glioblastoma, renal cell cancer, prostate cancer, liver cancer, pancreatic cancer, soft-tissue sarcoma, kaposi’s sarcoma, carcinoid carcinoma, head and neck cancer, ovarian cancer, and mesothelioma.
  • the cancer is selected from: small cell lung cancer, glioblastoma, neuroblastomas, melanoma, breast carcinoma, gastric cancer, colorectal cancer (CRC), and hepatocellular carcinoma.
  • the cancer is selected from: non-small cell lung cancer, colorectal cancer, glioblastoma and breast carcinoma, including metastatic forms of those cancers.
  • the cancer is colorectal cancer, including coion cancer and rectal cancer.
  • the cancer is urothelial carcinoma, non-small cell lung cancer, triple negative breast cancer, small cell lung cancer, hepatocellular carcinoma, or melanoma.
  • hematologic cancers originate in the blood or bone marrow.
  • the hematologic cancer to be treated with an anti-MerTK antibody of the present disclosure is leukemia.
  • leukemias include, without limitation, chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; and acute myeloblastic leukemia.
  • CLL chronic lymphocytic leukemia
  • ALL acute lymphoblastic leukemia
  • hairy cell leukemia chronic myeloblastic leukemia
  • acute myeloblastic leukemia acute myeloblastic leukemia
  • the hematologic cancer to be treated with an anti-MerTK antibody of the present disclosure is lymphoma.
  • lymphoma examples include T-cell lymphoma (such as adult T-cell leukemia/lymphoma; hepatosplenic T- cell lymphoma; peripheral T-cell lymphoma, anaplastic large ceil lymphoma; and angioimmunoblastic T ceil lymphoma), B-ceil lymphoma (including low' grade/follicular nonHodgkin’s lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; diffuse large B-cell lymphoma; mantle cell lymphoma; Burkitt lymphoma; AIDS-related lymphoma; and Waldenstrom’s Macroglobulinemia), Hodgkin’s lymphoma, and post-transplant lymphoproliferative disorder (PTLD).
  • T-cell lymphoma
  • the hematologic cancer to be treated with an anti-MerTK antibody of die present disclosure is myeloma.
  • the myeloma is plasmacytoma or multiple myeloma.
  • cancers that are amenable to treatment by anti-MerTK antibodies of the present disclosure include non-Hodgkin’s lymphoma and multiple myeloma.
  • kits for treating or delaying progression of cancer in an individual comprising administering to the individual an effective amount of an anti- MerTK antibody as described in the present disclosure.
  • the treatment results in a sustained response in the individual after cessation of the treatment.
  • the methods described herein may find use in treating conditions where enhanced immunogenicity' is desired such as increasing tumor immunogenicity for the treatment of cancer.
  • methods of enhancing immune function in an individual having cancer comprising administering to the individual an effective amount of an anti-MerTK antibody as described in the present disclosure.
  • the cancer expresses functional STING, functional Cx43, and functional cGAS polypeptides.
  • Functional proteins are proteins that are able to carry out their regular functions in a cell. Examples of functional proteins may include wild-type proteins, tagged proteins, and mutated proteins that retain or improve protein function as compared to a wild-type protein. Protein function can be measured by any method known to those of skill in the art, including assaying for protein or mRNA expression and sequencing genomic DNA or rnRN A.
  • the cancer comprises tumor-associated macrophages that express functional STING polypeptides.
  • the cancer comprises tumor cells that express functional cGAS polypeptides.
  • the cancer comprises tumor cells that express functional Cx43 polypeptides.
  • the cancer is colorectal cancer, including colon cancer and rectal cancer.
  • 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 described in the present disclosure to reduce MerTK-mediated clearance of apoptotic cells.
  • the clearance of apoptotic cells is reduced by 1-10 fold, 1-8 fold, 1-5 fold, 1-4 fold, 1-3 fold, 1-2 fold, 2-10 fold, 2-8 fold, 2-5 fold, 2-4 fold, 2-3 fold, 3-10 fold, 3- 8 fold, 3-5 fold, 3-4 fold, or by at least about 1.1 fold, 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 1.6 fold, 1.7 fold, 1.8 fold, 1.9 fold, 2.0 fold, 2.1 fold, 2.2 fold, 2.3 fold, 2.4 fold, 2.5 fold, 2.6 fold, 2.7 fold, 2.8 fold, 2.9 fold, 3.0 fold, 3.1 fold, 3.2 fold, 3.3 fold, 3.4 fold, 3.5 fold, 3.6 fold, 3.7 fold, 3.8 fold, 3.9 fold, 4.0 fold, 4.1 fold, 4.2 fold, 4.3 fold, 4.4 fold, 4.5 fold, 4.6 fold, 4.7 fold, 4.8 fold, 4.9 fold, 5.0 fold, 5.
  • Reduction of MerTK-mediated clearance of apoptotic cells may be determined by comparing the level of MerTK- mediated clearance of apoptotic cells in a sample from an individual after administration of an effective amount of an anti-MerTK antibody or an immunoconjugate t hereof to a reference level of MerTK-mediated clearance of apoptotic cells.
  • the reference level is the level of MerTK-mediated clearance of apoptotic cells a reference sample.
  • the reference sample is taken from the subject taken prior to administration of an effective amount of an anti-MerTK antibody or an immunoconjugate thereof.
  • the sample comprises tumor tissue or tumor cells.
  • an anti-MerTK antibody of the present disclosure reduces 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-
  • the anti-MerTK antibody has a half maximal inhibitory’ concentration (IC50) for reducing phagocytic activity of apoptotic ceils of 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 nM, 10
  • the anti-MerTK antibody may be administered intravenously , intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitaily, by implantation, by inhalation, intrathecaily, intraventncularly, or intranasally.
  • the appropriate dosage of the anti-MerTK antibody may be determined based on the type of disease to be treated, the severity and course of the disease, the clinical condition of the individual, the individual’s clinical history' and response to the treatment, and the discretion of the attending physician.
  • Antibodies of the invention can be administered alone or used in a combination therapy.
  • the combination therapy includes administering an antibody of the invention and administering at least one additional therapeutic agent (e.g. one, two, three, four, five, or six additional therapeutic agents).
  • the combination therapy comprises administering an antibody of the invention and administering at least one additional therapeutic agent, such as an immune checkpoint inhibitor.
  • the uses and methods may further comprise an additional therapy or administration of an effective amount of an additional therapeutic agent.
  • the additional therapy may be radiation therapy, surgery (e.g., lumpectomy and a mastectomy), chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, or a combination of the foregoing.
  • the additional therapy may be in the form of adjuvant or neoadjuvant therapy.
  • the additional therapy is the administration of small molecule enzymatic inhibitor or anti -meta static agent.
  • the additional therapy is the administration of side-effect limiting agents (e.g., agents intended to lessen the occurrence and/or severity of side effects of treatment, such as anti -nausea agents, etc.).
  • the additional therapy is radiation therapy.
  • the additional therapy is surgery.
  • the additional therapy is a combination of radiation therapy and surgery.
  • the additional therapy is gamma irradiation.
  • the additional therapy is therapy targeting PI3K/AKT/mTOR pathway, I-ISP90 inhibitor, tubulin inhibitor, apoptosis inhibitor, and/or chemopreventative agent.
  • the additional therapy is an antagonist directed against B7-H3 (also known as CD276), e.g., a blocking antibody, MGA271, an antagonist directed against a TGF beta, e.g., metelimumab (also known as CAT-192), fresolimumab (also known as GCI008), or LY2157299, a treatment comprising adoptive transfer of a T cell (e.g., a cytotoxic T cell or CTL) expressing a chimeric antigen receptor (CAR), a treatment comprising adoptive transfer of a T cell comprising a dominant-negative TGF beta receptor, e.g, a dominant-negative TGF beta type II receptor, a treatment comprising a HERCREEM protocol (see, e.g., ClinicalTrials.gov Identifier NCT00889954), an agonist directed against CD137 (also known as TNFRSF9, 4- IBB, or ILA), e.g., an antagonist directed against CD137 (also known
  • cobimetinib also known as GDC-0973 or XL-518
  • trametinib also known as Mekinist®
  • K-Ras an inhibitor of K-Ras
  • an inhibitor of c-Met an inhibitor of c-Met, onartuzumab (also known as MetMAb)
  • Aik an inhibitor of Aik
  • AF802 also known as CH5424802 or alectinib
  • an inhibitor of a phosphatidylinositol 3-kinase PI3K
  • BKM120 idelalisib
  • perifosine also known as KRX-0401
  • an Akt Akt
  • MK2206 GSK690693
  • GDC-0941 an inhibitor of mTOR
  • sirolimus also known as rapamycin
  • temsirolimus also known as CCI-779 or Torisel®
  • everolimus also known as RAD001
  • ridaforolimus also known as
  • the additional therapeutic agent is CT- Oil (also known as Pidilizumab orMDV9300; CAS Registry' No. 1036730-42-3; CureTeclv'Medivation).
  • CT-011 also known as IiBAT or IiBAT-1, is an antibody described in W02009/101611.
  • the additional therapeutic agent is an immune checkpoint inhibitor.
  • the application provides methods for enhancing immune function in an individual having cancer comprising administering an effective amount of a combination of an anti- MerTK antibody and an immune checkpoint inhibitor.
  • the anti-AffiRTK antibody increases die immune effect of an immune checkpoint inhibitor by about 2 fold, 3 fold, 5 fold, 8 fold, 10 fold, 15 fold or 20 fold.
  • the anti-AffiRTK 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 fold, 1-50 Ibid, 1-75 Ibid, 1-100 fold, 1-150 fold, 1-200 fold, 1-250 fold, 1.5-2 fold, 1.5-5 fold, 1.5-10 fold, 1.5-15 fold, 1.5-20 fold, 1.5-25 fold, 1.5-30 fold, 1.5-50 fold, 1.5- 75 fold, 1.5-100 fold, 1.5-150 fold, 1.5-200 fold, 1.5-250 fold, 2-5 fold, 2-10 fold, 2-15 fold, 2-20 fold, 2-25 fold, 2-30 fold, 2-50 fold, 2-75 fold, 2-100 fold, 2-150 fold, 2-200 fold, 2-250 fold, 2.5-5 fold, 2.5-10 fold, 2.5-15 fold, 2.5-20 fold, 2.5-25 fold, 2.5-30 fold, 2.5-50 fold, 2.5-75 fold, 2.5-100 fold, 2.5-150 fold, 2.5-200 fold, 2.5-250 fold, 5-10 fold, 5-15 fold, 5-20 fold, 5-25 fold, 5-25 fold,
  • the individual has cancer that is resistant (has been demonstrated to be resistant) to one or more immune checkpoint inhibitors.
  • resistance to immune checkpoint inhibitors includes recurrence of cancer or refractory' cancer. Recurrence may refer to the reappearance of cancer, in the original site or a new site, after treatment.
  • resistance to immune checkpoint inhibitors includes progression of the cancer during treatment with the immune checkpoint inhibitors.
  • resistance to immune checkpoint inhibitors includes cancer that does not respond to treatment. The cancer may be resistant at the beginning of treatment or it may become resistant during treatment. In some embodiments, the cancer is at early stage or at late stage.
  • the immune checkpoint inhibitor is a cytotoxic T-lymphocyte-associated protein 4 (CTLA4) (also known as CD 152) inhibitor.
  • CTL4 cytotoxic T-lymphocyte-associated protein 4
  • the C'TLA-4 inhibitor is a blocking antibody, ipilimumab (also known as MDX-010, MDX-101, or Yen'oy®), tremelimumab (also known as ticilimumab or CP-675, 206).
  • ipilimumab also known as MDX-010, MDX-101, or Yen'oy®
  • tremelimumab also known as ticilimumab or CP-675, 206.
  • the immune checkpoint inhibitor is a PD-1 axis binding antagonist.
  • kits for treating cancer in an individual comprising administering to the individual an effective amount of a PD-1 axis binding antagonist and an anti-MerTK antibody of the present disclosure (e.g., a PEGylated anti-MerTK antibody).
  • an anti-MerTK antibody of the present disclosure e.g., a PEGylated anti-MerTK antibody.
  • methods of enhancing immune function or response in an individual comprising administering to the individual an effective amount of a PD-1 axis binding antagonist and an anti-MerTK antibody of the present disclosure (e.g., a PEGylated anti-MerTK antibody).
  • the PD-1 axis binding antagonist includes a PD-1 binding antagonist, a PDL1 binding antagonist, and/or a PDL2 binding antagonist.
  • 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.
  • PD-1, PDL1, and PDL2 are human PD-1, PDL1 and PDL2.
  • the PD-1 binding antagonist is a molecule tliat inhibits the binding of PD-1 to its ligand binding partner(s).
  • the PD-1 ligand binding partners are PDL1 and/or PDL2.
  • a PDL1 binding antagonist is a molecule that inhibits the binding of PDL1 to its binding partner(s).
  • PDL1 binding partner(s) are PD-1 and/or B7- 1 .
  • the PDL2 binding antagonist is a molecule that inhibits the binding of PDL2 to its binding partner(s).
  • a PDL2 binding partner is PD-1.
  • the antagonist may be an antibody, an antigen binding fragment thereof, an immunoadhesin, a fusion protein, an oligopeptide or a small molecule. If the antagonist is an antibody, in some embodiments the antibody comprises a human constant region selected from die group consisting of IgGl, IgG2, IgG3 and IgG4.
  • the PD-1 binding antagonist is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody).
  • an anti-PD-1 antibody e.g., a human antibody, a humanized antibody, or a chimeric antibody.
  • the PD-I antibody can bind to a human PD-1 or a variant thereof.
  • the anti-PD- i antibody is a monoclonal antibody.
  • the anti-PD-1 antibody is an antibody fragment selected from the group consisting of Fab, Fab’, Fab’-SH, Fv, scFv, and (Fab’)i fragments.
  • the anti-PD-1 antibody is a chimeric or humanized antibody.
  • the anti-PD- 1 antibody is a human antibody.
  • the anti-PD-1 antibody is nivolumab (CAS Regisby Number: 946414-94-4).
  • Nivolumab also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO®, is an anti-PD-1 antibody described in W02006/121168.
  • the anti-PD-1 antibody comprises a heavy chain and a light chain sequence, wherein:
  • the heavy chain comprises the amino acid sequence:
  • ilie light chain comprises the amino acid sequence:
  • the anti-PD-1 antibody comprises the six HVR sequences from SEQ ID NO: 24 and SEQ ID NO: 25 (e.g., the three heavy chain HVRs from SEQ ID NO:24 and the three light chain HVRs from SEQ ID NO: 25). In some embodiments, the anti-PD-1 antibody comprises the heavy chain variable domain from SEQ ID NO: 24 and the light chain variable domain from SEQ ID NO: 25.
  • the anti-PD-1 antibody is pembrolizumab (CAS Registry Number: 1374853-91-4).
  • Pembrolizumab (Merck), also known as MK-3475, Merck 3475, lambrolizumab, SCH-900475, and KEYTRUDA®, is an anti-PD-1 antibody described in W02009/114335.
  • die anti-PD-1 antibody comprises a heavy chain and a light chain sequence, wherein:
  • the heavy chain comprises the amino acid sequence:
  • the light chain comprises the amino acid sequence: EIVLTQSPAT LSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLrYLASYLES GVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKRTVAAPSVF IFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVl’EQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 27).
  • the anti-PD-1 antibody comprises the six HVR sequences from SEQ ID NO: 26 and SEQ ID NO: 27 (e.g., the three heavy chain HVRs from SEQ ID NO: 26 and the three light chain HVRs from SEQ ID NO:27). In some embodiments, the anti-PD-1 antibody comprises the heavy chain variable domain from SEQ ID NO: 26 and the light chain variable domain from SEQ ID NO: 27.
  • the anti-PD-1 antibody is MEDI-0680 (AMP-514: AstraZeneca).
  • MEDI-0680 is a humanized IgG4 anti-PD-1 antibody.
  • 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.
  • the anti-PD-1 antibody is REGN2810 (Regeneron).
  • REGN28I0 is a human anti-PD-1 antibody.
  • the anti-PD-1 antibody is BGB-108 (BeiGene). In some embodiments, the anti-PD-1 antibody is BGB-A317 (BeiGene).
  • the anti-PD-1 antibody is JS-001 (Shanghai Junshi).
  • JS-001 is a humanized anti-PD-1 antibody.
  • the anti-PD-1 antibody is STI-A1110 (Sorrento).
  • STI-Ali lO is a human anti-PD- 1 antibody ,
  • the anti-PD-1 antibody is INCSHR-1210 (Incyte).
  • INCSHR-1210 is a human lgG4 anti-PD-1 antibody.
  • the anti-PD-1 antibody is PF-06801591 (Pfizer).
  • the anti-PD- 1 antibody is TSR-042 (also known as ANB011;
  • the anti-PD-1 antibody is AM0001 (ARMO Biosciences). [0263] In some embodiments, 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 binding of PDL1 to PD-1.
  • the anti-PD-1 antibody is ENUM 388D4 (Enumeral Biomedical Holdings)
  • ENUM 388D4 is an anti-PD-1 antibody that competitively inhibits binding of PDL1 to PD-1.
  • the PD-1 antibody comprises the six H VR sequences (e.g. , the three heavy chain H VRs and the three light chain HVRs) and/or the heavy chain variable domain and light chain variable domain from a PD-1 antibody described in W02015/112800 (Applicant: Regeneron), W02015/112805 (Applicant: Regeneron), WO2015/112900 (Applicant: Novartis), US20150210769 (Assigned to Novartis), WO2016/089873 (Applicant: Celgene), W02015/035606 (Applicant: Beigene), WO2015/085847 (Applicants: Shanghai Hengrui Pharmaceutical/Jiangsu Hengrui Medicine), W02014/206107 (Applicants: Shanghai Junshi Biosciences/Junmeng Biosciences), WO2012/145493 (Applicant: Amplimmune), US9205148 (Assigned to Medlmmune), W02015/119930 (Applicants: Pfizer, '
  • the PD-1 axis binding antagonist is an anti-PDLl antibody.
  • anti-PDLl antibodies are contemplated and described herein.
  • the isolated anti-PDLl antibody can bind to a human PDL1, for example a human PDL1 as shown in UniProtKB/Swiss-Prol Accession No.Q9NZQ7.1, or a variant thereof.
  • the anti-PDLl antibody is capable of inhibiting binding between PDL1 and PD-1 and/or between PDL1 and B7-1.
  • the anti-PDLl antibody is a monoclonal antibody.
  • the anti-PDLl antibody is an antibody fragment selected from the group consisting of Fab, Fab’-SH, Fv, scFv, and (Fab’)j fragments.
  • the anti- PDLl antibody is a humanized antibody.
  • the anti-PDLl antibody is a human antibody. Examples of anti-PDLl antibodies useful for the methods of the present disclosure, and methods for making thereof are described in PCT patent application WO 2010/077634 A 1 and US Patent No. 8,217, 149, which are incorporated herein by reference.
  • the anti-PDLl antibody is atezolizumab (CAS Registry Number: 1422185-06-5). Atezolizumab (Genentech), also known as MPDL3280A, is an anti-PDLl antibody, [0268] In some embodiments, the anti-PDLl antibody comprises a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region comprises an l-IVR-Hl, HVR-H2, and HVR-H3 sequence of GFTFSDSW1H (SEQ ID NO: 28), AWISPYGGSTYYADSVKG (SEQ ID NO: 29) and RHWPGGFDY (SEQ ID NO: 30), respectively
  • the light chain variable region comprises an HVR-L 1 , HVR-L2, and HVR-L3 sequence of RASQDVSTA VA (SEQ ID NO: 31), SASFLYS (SEQ TD NO: 32) and QQYLYHPAT (SEQ ID NO: 33), respective! ⁇ '.
  • the anti-PDLl antibody is MPDL3280A, also known as atezolizumab and TECENTRIQ® (CAS Regisriy Number: 1422185-06-5).
  • the anti-PDLl antibody comprises a heavy chain and a light drain sequence, wherein:
  • the heavy drain variable region sequence comprises the amino acid sequence:
  • the light chain variable region sequence comprises the amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLL1Y SASF LYSGVPSRFSGSGSGTOFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR (SEQ ID NO: 35).
  • the anti-PDLl antibody comprises a heavy drain and a light drain sequence, wherein:
  • the heavy chain comprises the amino acid sequence:
  • the light chain comprises the amino acid sequence:
  • the anti-PDLl antibody is avelumab (CAS Registry Number:
  • Avelumab also known as MSB0010718C, is a human monoclonal IgGl anti-PDLl antibody (Merck KGaA, Pfizer).
  • the anti-PDLl antibody comprises a heavy drain and a light chain sequence, wherein:
  • the heavy chain comprises the amino acid sequence:
  • Hie light chain comprises the amino acid sequence:
  • the anti-PDLl antibody comprises the six HVR sequences from SEQ ID NO: 38 and SEQ ID NO: 39 (e.g., the three heavy chain HVRs from SEQ ID NO:38 and the three light chain HVRs from SEQ ID NO: 39). In some embodiments, the anti-PDLl antibody comprises the heavy chain variable domain from SEQ ID NO: 38 and the light chain variable domain from SEQ ID NO: 39.
  • the anti-PDLl antibody is durvalumab (CAS Registry Number: 1428935-60-7), Durvalumab, also known as MEDI4736, is an Fc optimized human monoclonal IgGl kappa anti-PDLl antibody (Medlmmune, AstraZeneca) described in WO2011/066389 and US2013/034559.
  • the anti-PDLl antibody comprises a heavy chain and a light chain sequence, wherein:
  • the heavy chain comprises the amino acid sequence:
  • the light chain comprises the amino acid sequence: EIVLTQSPGTLSLSPGERATLSCRASQRVSSSYLAWYQQKPGQAPRLLIYDASSRATGIPDRFS GSGSGTOFTLTISRLEPEDFAVYYCQQYGSLPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKS GTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 41).
  • the anti-PDLl antibody comprises the six HVR sequences from SEQ ID NO:40 and SEQ ID NO:41 (e.g., the three heavy chain HVRs from SEQ ID NO:40 and the three light chain HVRs from SEQ ID N0:41). In some embodiments, the anti-PDLl antibody comprises the heavy chain variable domain from SEQ ID NO: 40 and the light chain variable domain from SEQ ID NO: 41.
  • the anti-PDLl antibody is MDX-1105 (Bristol Myers Squibb). MDX-1105, also known as BMS-936559, is an anti-PDLl antibody described in W02007/005874. [0276] In some embodiments, the anti-PDLl antibody is LY3300054 (Eli Lilly).
  • the anti-PDLl antibody is STI-A1014 (Sorrento).
  • STI-A1014 is a human anti-PDLl antibody.
  • the anti-PDLl antibody is KN035 (Suzhou Alphamab).
  • KN035 is single-domain antibody (dAB) generated from a camel phage display libraiy.
  • the anti-PDLl antibody comprises a cleavable moiety or linker that, when cleaved (e.g. , by a protease in the tumor microenvironment), activates an antibody antigen binding domain to allow it to bind its antigen, e.g., by removing a non-binding steric moiety.
  • the anti-PDLl antibody is CX-072 (CytomX Therapeutics).
  • the PDL1 antibody comprises the six HVR sequences (e.g., the three heavy drain H VRs and the three light chain HVRs) and/or the heavy chain variable domain and light chain variable domain from a PDL 1 antibody described in US20160108123 (Assigned to Novartis), W02016/000619 (Applicant: Beigene), WO2012/145493 (Applicant: Amplimmune), US9205148 (Assigned to Medlmmune), WO2013/181634 (Applicant: Sorrento), and W02016/061142 (Applicant: Novartis).
  • HVR sequences e.g., the three heavy drain H VRs and the three light chain HVRs
  • the minimal effector function results from an “effector-less Fc mutation” or a glycosylation mutation.
  • the effectorless Fc mutation is an N297A or D265A/N297A substitution in the constant region.
  • the isolated anti-PDLl antibody is aglycosvlated. Glycosylation of antibodies is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue.
  • the tripeptide sequences asparagine-X-serine and asparagine- X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side drain.
  • O-linked glycosylation refers to the attachment of one of the sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5 -hydroxy proline or 5- hydroxylysine may also be used.
  • the anti-MERTK antibody increases the immune effect of the anti- PDL1 antibody about 3 fold after 20 days of combination treatment. In some embodiments, the anti- MERTK antibody increases the immune effect of the anti-PDLl antibody about 10 fold after 30 days of treatment.
  • the PD-1 binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD- 1 binding portion of PDL 1 or PDL2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence).
  • the PD-1 binding antagonist is AMP-224.
  • AMP-224 (CAS Registry No. 1422184-00-6;
  • GlaxoSnrithKline/Medlimnune also known as B7-DCIg, is a PDL2-Fc fusion soluble receptor described in W02010/027827 and WO2011/066342.
  • the PD-1 binding antagonist is a peptide or small molecule compound.
  • the PD-1 binding antagonist is AUNP-I2 (PierreFabre/Aurigene). See, e.g., WO2012/ 168944, WO2015/036927, WO2015/044900, W02015/033303, WO2013/144704, WO2013/132317, and WO2011/161699.
  • the PDL1 binding antagonist is a small molecule that inhibits PD- 1 .
  • the PDL I binding antagonist is a small molecule that inhibits PDL1 .
  • the PDL1 binding antagonist is a small molecule that inhibits PDL1 and VISTA.
  • the PDL1 binding antagonist is CA-170 (also known as AUPM-170).
  • the PDL1 binding antagonist is a small molecule tliat inhibits PDL1 and TIM3, In some embodiments, the small molecule is a compound described in W02015/033301 and WO2015/033299.
  • an anti- MerTK antibody e.g., a PEGylated anti-MerTK antibody
  • an immune checkpoint inhibitor e.g., a PEGylated anti-MerTK antibody
  • the cancer in some embodiments, a sample of the patient’s cancer as examined using a diagnostic test
  • T cell infiltration of a cancer may refer to the presence of T cells, such as tumor-infiltrating lymphocytes (TILs), within or otherwise associated with the cancer tissue. It is known in the art that T cell infiltration may be associated with improved clinical outcome in certain cancers (see, e.g., Zhang etal., N. Engl. J. Med. 348(3):203-213 (2003)).
  • T cell exhaustion is aiso a major immunological feature of cancer, with many tumor-infiltrating lymphocytes (TILs) expressing high levels of inhibitory co-receptors and lacking the capacity to produce effector cytokines (Wherry', E.J. Nature immunology 12: 492-499 (2011); Rabinovich, G.A., et al., Annual review of immunology 25:267-296 (2007)).
  • TILs tumor-infiltrating lymphocytes
  • the individual has a T cell dysfunctional disorder.
  • the T ceil dysfunctional disorder is characterized by T cell anergy or decreased ability’ to secrete cytokines, proliferate or execute cytolytic activity’.
  • the T cell dysfunctional disorder is characterized by T ceil exhaustion.
  • the T cells are CD4+ and CD8+ T cells.
  • the T cells are CD4-1- and/or CD8+ T cells.
  • CD8+ T cells are characterized, e.g., by presence of CD8b expression (e.g., by rtPCR using e.g., Fluidigm) (Cd8b is also known as T-celi surface glycoprotein CD8 beta chain; CD8 antigen, alpha polypeptide p37; Accession No. is NM_172213).
  • CD8+ T ceils aie from peripheral blood.
  • CD8+ T cells are from tumor.
  • Treg cells are characterized, e.g., by presence of Fox3p expression (e.g., by rtPCR e.g., using Fluidigm)
  • Fox3 is also known as forkhead box protein P3; scurfin;
  • Treg are from peripheral blood.
  • Treg cells are from tumor.
  • inflammatory T cells are characterized, e.g,, by presence of Tbet and/or CXCR3 expression (e.g., by rtPCR using, e.g., Fluidigm).
  • inflammatory' T cells are from peripheral blood.
  • inflammatojy T ceils are from tumor.
  • CD4 and/or CD8 T cells exhibit increased release of cy tokines selected from the group consisting of IFN- y, TNF-a and interleukins.
  • Cytokine release may be measured by any means known in the art, e.g., using Western blot, ELISA, or immunohistochemical assays to detect the presence of released cytokines in a sample containing CD4 and/or CD8 T cells.
  • the CD4 and/or CD8 T ceils are effector memoiy T cells. In some embodiments of the methods of the present disclosure, the CD4 and/or CD8 effector memoiy T ceils are characterized by having the expression of CD44 hlgh CD62L
  • CD44 mgh CD62L !OB may be detected by any means known in the art, e.g., by preparing single cell suspensions of tissue (e.g., a cancer tissue) and performing surface staining and flow cytometry using commercial antibodies against CD44 and CD62L,
  • the CD4 and/or CD8 effector memory T cells are characterized by having expression of CXCR3 (also known as C-X-C chemokine receptor type 3; Mig receptor; IP10 receptor; G protein-coupled receptor 9; interferon-inducible protein 10 receptor; Accession No.
  • the CD4 and/or CD8 effector memory’ T cells are from peripheral blood.
  • the CD4 and/or CD8 effector memoiy T cells are from tumor.
  • Treg function is suppressed relative to prior to the administration of the combination.
  • T cell exhaustion is decreased relative to prior to the administration of the combination.
  • number of Treg is decreased relative to prior to the administration of the combination.
  • plasma interferon gamma is increased relative to prior to the administration of the combination, Treg number may be assessed, e.g., by determining percentage of CD4+Fox3p-t- CD454- cells (e.g., by FACS analysis).
  • absolute number of Treg e.g., in a sample, is determined.
  • Treg are from peripheral blood.
  • Treg are from tumor.
  • T cell priming, activation and/or proliferation is increased relative to prior to the administration of the combination.
  • the T cells are CD4+ and/or CD8+ T cells.
  • T cell proliferation is detected by determining percentage of K167+ CD8+ T cells (e.g., by FACS analysis).
  • T cell proliferation is detected by determining percentage of Ki67+ CD4+ T cells (e.g., by FACS analysis).
  • the T cells are from peripheral blood. In some embodiments, the T cells are from tumor.
  • Such combination therapies noted above encompass combined administration (where two or more therapeutic agents are included in the same or separate pharmaceutical compositions), and separate administration, in which case, administration of the antibody of the invention can occur prior to, simultaneously, and/or following, administration of the additional therapeutic agent or agents.
  • administration of the anti-MerTK antibody and administration of an additional therapeutic agent occur within about one month, or within about one, two or three weeks, or within about one, two, three, four, five, or six days, of each other.
  • the antibody and additional therapeutic agent are administered to the patient on Day 1 of the treatment.
  • Antibodies of the invention can also be used in combination with radiation therapy.
  • An antibody of the invention can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration.
  • Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, e.g., by injections, such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic.
  • Various dosing schedules including but not limited to single or multiple administrations over various time-points, bolus administration, and pulse infusion are contemplated herein.
  • Armbodies of the invention would be formulated, dosed, and administered in a fashion consistent with good medical practice.
  • Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, die method of administration, the scheduling of administration, and other factors known to medical practitioners.
  • the antibody need not be, but is optionally formulated with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents depends on the amount of antibody present in the pharmaceutical composition, the type of disorder or treatment, and other factors discussed above. These are generally used in the same dosages and with administration routes as described herein, or about from 1 io 99% of the dosages described herein, or in any dosage and by any route that is empirically /clinically determined to be appropriate.
  • an antibody of the invention when used 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 seventy and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, previous therapy, the patient’s clinical history' and response to the antibody, and the discretion of the attending physician.
  • the antibody is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 p.g/kg to 15 mg/kg (e.g., 0.
  • i mg/kg- lOmg/kg can be an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion.
  • One typical daily dosage might range from about 1 pg/kg to 100 mg/kg or more, depending on the factors mentioned above.
  • the treatment would generally be sustained until a desired suppression of disease sy mptoms occurs.
  • One exemplary' dosage of the antibody would be in the range from about 0.05 mg/kg to about 10 mg/kg.
  • 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, e.g., every' week or every three weeks (e.g., such that the patient receives from about tw'O to about twenty, or, e.g., about six doses of the antibody).
  • An initial higher loading dose, followed by one or more lower doses may be administered.
  • the combination therapy of the present disclosure comprises administration of an anti-MerTK antibody and an immune checkpoint inhibitor.
  • the anti-MerTK antibody and the immune checkpoint inhibitor may be administered in any suitable manner known in the art.
  • the anti-MerTK antibody and the immune checkpoint inhibitor may be administered sequentially (at different times) or concurrently (at the same time).
  • the immune checkpoint inhibitor is in a separate composition as the anti-MerTK antibody.
  • the immune checkpoint inhibitor is in the same composition as the anti-MerTK antibody .
  • the anti-MerTK antibody and the immune checkpoint inhibitor may be administered by the same route of administration or by different routes of administration.
  • the immune checkpoint inhibitor is administered intravenously , intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorb itallv, by implantation, by inhalation, intrathecally, intraventricuiarly, or intranasally.
  • the anti-MerTK antibody is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricnlarly, or intranasally.
  • An effective amount of the immune checkpoint inhibitor and the anti-MerTK antibody may be administered for prevention or treatment of disease.
  • the appropriate dosage of the anti- MerTK antibody and/or the immune checkpoint inhibitor may be determined based on the type of disease to be treated, the type of the immune checkpoint inhibitor and the anti-MerTK antibody, the severity and course of the disease, the clinical condition of the individual, the individual’s clinical histoiy and response to the treatment, and the discretion of the attending physician.
  • combination treatment with anti-MerTK antibody and an immune checkpoint inhibitor are synergistic, whereby an efficacious dose of an anti- MerTK antibody in the combination is reduced relative to efficacious dose of the anti-MerTK antibody as a single agent.
  • the therapeutically effective amount of the antibody administered to human will be in the range of about 0.01 to about 50 mg/kg of patient body weight whether by one or more administrations.
  • the antibody used is 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 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 administered daily, for example.
  • the antibody is administered at 15 mg/kg. However, other dosage regimens may be useful.
  • an anti-MerTK antibody described herein or an anti-PDLl antibody described herein is administered to a human at a dose of 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, about 1000 mg, about 1100 mg, about 1200 mg, about 1300 mg or about 1400 mg on day 1 of 21 -day cycles.
  • the dose may be administered as a single dose or as multiple doses (e.g., 2 or 3 doses), such as infusions.
  • the dose of the antibody administered in a combination treatment may be reduced as compared to a single treatment. The progress of this therapy is easily monitored by conventional techniques.
  • the present disclosure provides the anti-MerTK antibodies as described above (e.g. , PEGylated anti-MerTK antibodies) for use as a medicament.
  • the use is in treating cancer.
  • the use is in reducing MerTK-mediated clearance of apoptolic cells.
  • the anti-MerTK antibodies as described above in the manufacture of a medicament.
  • the medicament is for treatment of cancer.
  • the cancer expresses functional cGAS-STING cytosolic DNA sensing pathway proteins.
  • cGAS-STTNG cytosolic DNA sensing pathway proteins include but are not limited to cGAS, STING, TBK-1, IRF3, p50, p60, p65, NF-KB, ISRE, IKK, and STAT6.
  • the cancer expresses functional STING, functional Cx43, and functional cGAS polypeptides.
  • Functional proteins are proteins that are able to carry out their regular functions in a cell.
  • the cancer comprises tumor-associated macrophages that express functional STING polypeptides.
  • the cancer comprises tumor cells that express functional cGAS polypeptides.
  • the cancer comprises tumor cells that express functional Cx43 polypeptides.
  • the cancer is colon cancer.
  • the cancer is urothelial carcinoma, non-small cell lung cancer, triple negative breast cancer, small cell lung cancer, hepatocellular carcinoma, or melanoma.
  • the medicament is for reducing MerTK-mediated clearance of apoptotic ceils.
  • the individual has cancer that expresses (has been shown to express e.g., in a diagnostic test) PDL1 biomarker.
  • the patient’s cancer expresses low PDLI biomarker.
  • the patient’s cancer expresses high PDLI biomarker.
  • the PDL1 biomarker is absent from the sample when it comprises 0% of the sample.
  • the PDL1 bio marker is present in the sample when it comprises more than 0% of the sample. In some embodiments, the PDLI biomarker is present in at least 1% of the sample. In some embodiments, the PDLI biomarker is present in at least 5% of the sample. In some embodiments, the PDLI biomarker is present in at least 10% oftbe sample.
  • the PDLI biomarker is detected in the sample using a method selected from the group consisting of FACS, Western biot, El, ISA, immunoprecipitation, immunohistochemistty, immunofluorescence, radioimmunoassay, dot blotting, immunodetection methods, HPLC, surface plasmon resonance, optical spectroscopy, mass spectre metery, HPLC, qPCR, RT-qPCR, multiplex qPCR or RT-qPCR, RNA-seq, microarray analysis, SAGE, MassARRAY technique, and FISH, and combinations thereof.
  • the PDLI bio marker is detected in the sample by protein expression.
  • protein expression is determined by immunohistochemistty (IHC).
  • the PDLI biomarker is detected using an anti-PDLl antibody.
  • the PDLI biomarker is detected as a weak staining intensity by IHC,
  • the PDL 1 biomarker is detected as a moderate staining intensity by IHC.
  • the PDL1 biomarker is detected as a strong staining intensity by IHC.
  • the PDL1 biomarker is detected on tumor cells, tumor infiltrating immune cells, stromal cells and any combinations thereof.
  • the staining is membrane staining, cytoplasmic staining or combinations thereof.
  • the absence of the PDL1 biomarker is detected as absent or no staining in the sample. In some embodiments of any of the methods, assays and/or kits, the presence of the PDL1 biomarker is detected as any staining in the sample.
  • an article of manufacture containing materials useful for the treatment and/or prevention of the disorders described above comprises a container and a label or package insert on or associated with the container.
  • Suitable containers include, for example, bottles, vials, sy ringes, IV solution bags, etc.
  • the containers may be formed from a variety of materials such as glass or plastic.
  • the container holds a composition which is by itself or combined with another composition effective for treating, preventing and/or diagnosing the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
  • At least one active agent in the composition is an antibody of the invention.
  • the label or package insert indicates that the composition is used for treating the condition of choice.
  • the article of manufacture may comprise (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 a further cytotoxic or otherwise therapeutic agent.
  • the article of manufacture in this aspect of the invention may further comprise a package insert indicating that the compositions can be used to treat a particular condition.
  • the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically -acceptable buffer, such as bacteriostatic water for injection (B WFI), phosphate- buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.
  • B WFI bacteriostatic water for injection
  • phosphate- buffered saline such as
  • Example 1 On-target ocular toxicity with anti-MerTK antibody treatment
  • MerTK is expressed on the apical membrane of RPE cells and plays a critical role in the phagocytosis of shedding photoreceptor cells.
  • the RPE of MerTK loss-of-function and constitutive knockout rodents is unable to remove shedding photoreceptor outer segments by phagocytosis resulting in retinal degeneration.
  • Humans with defective MerTK function develop night blindness in early childhood followed by a rapid decrease in visual acuity into early adulthood. Retinal toxicities have been observed with MerTK blockade with a small molecule MerTK inhibitor and an anti- MERTK monoclonal antibody (mAb).
  • This example describes the assessment of potential retinal toxicities associated with administration of an anti-MerTK monoclonal antibody.
  • Toxicology study 2 Toxicology study 2 with BALB/c mice incorporating full-field electroretinogram (ffERG) was conducted.
  • 14C9 mAh was administered IV at 0, 5 or 30 mg/kg twice weekly (BIW; total 8 doses) or 45 mg/kg thrice weekly (TIW; 12 total doses) for 4 weeks, with a 6-week recovery' period.
  • BIW bismuth-containing mice
  • TIW 45 mg/kg thrice weekly
  • the evaluation ccriteria included the following parameters: clinical observations, food consumption, body weight, physical examinations, ophthalmologic examinations, fundus ocular photography, intraocular pressure, ERG (full-field [ffERG] and mult-focal [mfERG]), optical coherence tomography (OCT), clinical pathology (e.g. hematology, clinical chemistry and urinalysis), anatomic pathology', toxicokinetics, and anti-drug antibody measurements.
  • ERG full-field [ffERG] and mult-focal [mfERG]
  • OCT optical coherence tomography
  • clinical pathology e.g. hematology, clinical chemistry and urinalysis
  • anatomic pathology' e.g. hematology, clinical chemistry and urinalysis
  • toxicokinetics e.g. hematology, clinical chemistry and urinalysis
  • Toxicology study 1 was conducted in C57BL/6N mice with the mouse surrogate anti-MerTK mAb, 14C9.
  • mice were administered 14C9 mAb was twice weekly for 4 weeks at doses of 0, 10 and 30 mg/kg.
  • An increase in the area under the curve (AUG) was observed in the toxocokinetic curve for 14C9 exposure. This increase was dose-proportional, as it was observed with an increase in dose from 10 to 30 mg/kg.
  • 14C9 ⁇ related outer retinal degeneration consisting of minimal -to -mild photoreceptor (PR) vacuolation, increased cellularity in the PR layer, and drop down of the outer nuclear layer was observed in these mice, with a dose -dependent increase in incidence and severity. Additionally, in the testes, moderate-to-marked seminiferous tubule atrophy and degeneration (correlating with decreased testis weight) was observed in all 14C9-treated male mice without any dose-dependency. Based on these findings, it was concluded tlrat administration of 14C9 mAb to C57BL/6N mice at 10 and 30 mg/kg BIW for 4-weeks resulted in retinal and testicular toxicity.
  • PR minimal -to -mild photoreceptor
  • Toxicology study 2 To determine whether the retinal lesions observed in Toxicology Study 1 were associated with functional impairment of the retina, a subsequent pilot toxicology study (Toxicology study 2) was conducted. This second toxicology study was conducted with BALB/c mice and incorporated full-field electroretinogram (ffERG). In tills study, 14C9 mAb was administered at 0, 5 or 30 mg/kg twice weekly or 45 mg/kg thrice weekly for 4 weeks, with a 6-week recovery’ period. Toxicokinetic analysis revealed a dose-proportional increase in AUC for 14C9 exposure in this study, witli an increase in dose from 10 to 30 mg/kg BIW, and 45 mg/kg TIW.
  • 14C9-reIated outer retinal degeneration consisting of minimal-to-marked PR vacuolization, increased cellularity in the PR layer, drop down of the outer nuclear layer, and decreased cellularity with degeneration or necrosis of the outer nuclear layer, was also observed, with a dose-dependent increase in severity. Similar findings were observed in one control male. The retinal lesions correlated with decreases in PR func tion as assessed by ffERG, neither of which resolved during the recovery- period. Marked seminiferous tubule atrophy and degeneration without any dose- dependency was observed in the testes of all 14C9 administered male mice.
  • a third toxicology study (Toxicology Study 3) was conducted in cymologus monkeys witli unconjugated I3B4 anti-MerTK mAb having a hlgGl isotype with LALAPG mutation.
  • Anti-MerTK mAb was intravenously (IV) administered as a single dose of 10 mg/kg or a repeat dose of 10 or 30 mg/kg every 3 weeks (Q3 W) for 6 weeks.
  • 13B4-related microscopic findings in lymphoid tissues were observed in animals administered 13B4 > 10 mg/kg Q3W and included minimal or slight decreased lymphocytes in the thymus (correlating with decreased thymus weights in animals administered 30 mg/kg), and minimally increased kaiyorrhectic debris in follicular germinal centers in the spleen, mesenteric and mandibular lymph nodes, and GALT/Peyer’s patch.
  • retinal toxicity Based on the results of these toxicology studies, the key safety risk of retinal toxicity is thought to be due to on-target inhibition of MerTK -mediated RPE engulfment of shedding photoreceptor outer segments.
  • the retinal toxicity may also be driven by time over threshold, and based on the available data, there is a lack of monitorability in cynomolgus and mouse by either ERG or OCT analysis.
  • Example 2 Conjugating large PEG polymers to anti-MerTK antibodies via engineered cysteines
  • CHO cells and purified via standard methods including Protein A affinity chromatography followed by size exclusion chromatography.
  • Conjugatable PEG polymers with a reactive maieimide moiety and nominal molecular weight of 40 kDa were purchased from NOF America, catalog numbers GL2- 400MA (2 -arm branched) and ME-400MA (linear).
  • the engineered cysteine in the THIOMAB antibody format is commonly blocked with cysteine or glutathione that occurs during the expression in mammalian cells. A standard process for deblocking was earned out to remove the cysteine or glutathione from the engineered cysteine for conjugation of the desired moiety.
  • reducing agent DTT
  • a 10 mg/mL antibody at alkaline pH of 7.5-8,5 in 100 mM Tris pH 8.5, 150 mM NaCl, 2 mM EDTA, and the mix was incubated at room temperature for approximately 18 hours.
  • the antibody was then purified using cationic exchange to remove the DTT and the reduced cysteine & glutathione.
  • the partially reduced antibody was re-oxidized using 15 molar excess of DHAA for 2-3 hours at room temperature and in 20 mM Tris pH 7.
  • the oxidation stale was assessed using LC-MS to check for the mass of intact oxidized antibody and monitor the presence of free light chain.
  • the re-oxdized antibody was then purified by cationic exchange chromatography and formulated with 10 mM succinate pH 5, 150 mM NaCl, 2 mM EDTA.
  • the pH of the conjugation reaction was adjusted to pH 6.5 with IM sodium acetate pH 5.0 and a final concentration of 800 mM ammonium sulfate was added.
  • the conjugate was then purified using a ProPAC HIC-10 bonded silica 10x150 mm column and formulated into 20 rnM histidine acetate pH 5.5, 240 mM sucrose, 0.02% Tween-20 using dialysis. UV absorbance at 280 nm was used to determine the antibody -based concentration of the formulated conjugates, given that PEG does not absorb at 280 nm, in combination with extinction coefficient of the antibody ⁇ component,
  • the polymer to antibody ratio and percent aggregation were characterized using HPLC equipped with YARRA SEC-4000 with 0.2. M Potassium phosphate, 0.25M Potassium chloride, pH 6,2, 15% ispropanol. The endotoxin levels were determined using Charles River EndoSafe cartridges. The hydrody namic radius of the conjugates were determined using an UPLC SEC-MALS/QELS Wyatt system equipped with a Acclaim-1000 column, with the resulting data being processed and analyzed using the Astra 7.1.2 software package.
  • human Fc -tagged mouse MerTK (R&D 591 -MR) was captured by protein A sensor chip to achieve approximately 50RIJ, and then 3-fold serial dilutions of each Fab variant (0.6 nM to 50 nM) were injected in HBS-EP buffer (100 mM 4-(2-hydroxyethyl)-l- piperazineethanesulfonic acid (HEPES) pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% (v/v) Surfactant P20) at 25°C with a flow rate of 50j.il/min.
  • HBS-EP buffer 100 mM 4-(2-hydroxyethyl)-l- piperazineethanesulfonic acid (HEPES) pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% (v/v) Surfactant P20) at 25°C with a flow rate of 50j.il/min.
  • Increasing the size of the anti-MerTK antibody may decrease the distribution to the eye by preventing the antibody from crossing the blood-retinal barrier, while maintaining the ability of the antibody to bind MerTK on tumor associated macrophages in the tumor micro-environment, though there has not been a systematic study addressing the effect of size on penetration of the blood-retinal barrier (del Amo el at. (2016) Progress in Retinal and Eye Research 57:134-185).
  • Hydrophilic polymers such as PEG are available in a wide variety of formats with different sizes available, formats (linear, branched) and reactive moieties for conjugation to the antibody. Compared to other hydrophilic polymers, PEG has been widely used in the clinic with several approvals forPEGylated proteins and antibody fragments. Two formats of PEG, linear and 2- arm branched format, at a nominal molecular weight of 40 kDa were chosen for assessment, as previous data has suggested that the format of the PEG can impact the pharmacokinetics and biodistribution (Leong, S.R. et al. (2001) Cytokine 16: 106- 119; Vugmeyster, Y. et al. (2012) Bioconjugate chemistry 23: 1452-1462).
  • cysteine-engineered (THIOMAB) antibody technology Site-specific cysteines were engineered into the antibody framework for conjugation for both the murine and human anti-MerTK antibodies using the cysteine-engineered (THIOMAB) antibody technology.
  • the cysteine-maleimide conjugation chemistry in combination with the THIOMAB antibody allows for site-specific conjugation that enables production of homogenous products regarding antibody -to-polymer ratio and enables control of the stability of the connection between the antibody and the polymer.
  • a set of predicted stable sites for the anti-murine MerTK m!gG2a format that would likely translate to the known stable sites on the anti-human MerTK huIgGl format were screened (Ohri, R. et al.
  • the antibody -to-polymer ratio was enriched to an average of 2.0 with 0% aggregation, routinely observed across the different conjugation sites, in both tire anti -murine 14C9 m!gG2a and the anti-human 13134 huIgGl antibodies.
  • a schematic of the PEG-conjugates formed following this approach is shown in FIG. 2.
  • the hydrodynamic ratio of the anti-MerTK PEG conjugates was characterized.
  • the hydrodynamic radius and molecular weight of the anti-MerTK PEG conjugates was determined using UPLC equipped with MATS and QELS detector, and samples were runned over an ACCLAIM-1000 SEC column with phosphate buffered saline.
  • the molecular weight of the anti-MerTK 14C9 antibody and the PEG conjugates were observed to agree with the theoretical molecular weight values (Table 2).
  • the hydrodynamic radius of the unconjugated antibody was determined to 5 nm, in good agreement with reported values.
  • the hydrodynamic radii of the PEGylated conjugates was determined to be twice tliat observed for the unconjugated antibody, with the linear PEG-40K slightly larger at 10.8 nm compared to the 2-ann branched PEG-40K at 10.3 nm (Table 2).
  • Both the murine anti-MerTK and human anti-MerTK THIOMAB antibodies readily conjugated with maleimide linked linear and 2-arm branched 40 kDa PEG polymers, producing conjugates with an antibody to polymer ratio of 2.0 and ⁇ 5% aggregation in tire purified, formulated conjugates.
  • the hydrodynamic radius of the conjugates was approximately two-times larger than the unconjugated antibody with the linear PEGvlated antibody with a marginally larger radius than the branched version.
  • Example 3 In vitro efferocytosis and macrophage binding analyses ofPEGylated anti-MerTK antibodies
  • This example describes the inhibition of macrophage-mediated efferocytosis by anti- MerTK antibody conjugates.
  • Cytokine mouse M-CSF was obtained from PeproTech (Rocky Hill, NJ). Staurosporine from Streptomyces sp. was obtained from Sigma-Aldrich (Saint Louis, MI). pHrodo Red, succinimidyl ester (pHrodo Red SE) was obtained from Thermo Scientific, (Whaltham, MA).
  • MPMs Mouse peritoneal macrophages
  • C57-BL6 mice 8 weeks old C57-BL6 mice were used. The mice were first euthanized by COr and 10 mL of chilled PBS (with 3% FBS) was injected into the peritoneal cavity of each mouse. After drawing the fluid back to the same syringe, the collected peritoneal cell suspensions were centrifuged at 1,400 rpm for 5 min and the pelleted cells were resuspended inRPMI medium, supplemented with 10% FBS.
  • MPMs (6 million cells) were cultured in RPMI medium supplemented with 10% FBS and recombinant mouse M-CSF (30 ng/mL) in a temperature-sensitive Nunc UpCell 10-cm dish (Thermo Scientific, Wbaltham, MA) for 3 days to promote recovery. After recovery, MPMs were harvested by washing with chilled-PBS without the use of dissociation enzymes.
  • Jurkat cells (ATCC #T1B-I5zs were cultured in RPMI medium supplemented with 10% FBS. Cells from an exponentially growing culture were harvested and induced to undergo apoptosis by treatment with 1.0 uM Staurosporine for a period of 4 hours at room temperature. The cells were then washed twice and re-suspended in PBS to a density of 1.0 x 10 6 cells/ml. The apoptotic cells were then labeled by incubating in 1.0 pM of pHrodo Red, succinimidyl ester in die dark, at room temperature for 1 hour.
  • the apoptotic cells were washed with PBS and a 10 min slow- spin centrifugation (750xg) 3 times. The cells were then re-suspended in RPMI medium + 10% FBS and then used in the efferocytosis assays.
  • Macrophages were seeded overnight at a density of 4.0 x 10 4 cells/well on a 96-well, low- evaporation Nunclon Delta Surface plate (Thermo Scientific) in RPMI medium supplemented with 10% FBS. Serial dilutions of antibodies were prepared in RPMI media supplemented with 10% FBS and then added to the 96-well plate containing the differentiated macrophages for 1 hr. After incubation with blocking antibodies, freshly prepared pHrodo-labeled apoptotic cells were added to the macrophages at a density of 8.0 x 10 s cells/well.
  • the 96-well plate was placed in the IncuCyte Zoom instrument (Essen Biosciences; Ann Harbor, MI) and images were obtained every 15 minutes for a period of 24-48 hours using the lOx objective lens and the red channel.
  • Total red fluorescence intensity was quantified using the IncuCyte Basic Software (2016B), with the background noise subtracted using the Top-Hat method. Cell confluency in each image was also quantified and was used to normalize the total red fluorescence intensity from different wells.
  • the efferocytosis activity was quantified as (total red fluorescence intensity’ - background fluorescence intensity’ of apoptotic cells)/(macrophage ceil confluence), and the maximum activity’ (100%) was defined as the value acquired in wells with untreated macrophages + apoptotic cells. Inhibition ciuves were generated using efferocytosis activities recorded 4-8 hours after addition of apoptotic cells. The efferocytosis activity from duplicate or triplicate wells were plotted as a function of antibody concentration and the data were fitted to a sigmoidal model with Prism (Graphpad Software; La Jolla, CA). The ICw value was calculated as die concentration of test material required to reduce the efferocytosis activity of macrophages by 50%,
  • THIOMAB antibody -PEG conjugates (FIG. 2) were prepared as described in Example 2.
  • the 14C9 LC K 149C conjugates were chosen for further evaluation. These 14C9 PEG- antibody conjugates showed comparable inhibitory activities as the parent antibody in the mouse efferocytosis assay (FIGs. 6A-6B). Both branched and linear PEG conjugated antibodies inhibited mouse pentoneal macrophage-mediated efferocytosis. PEG40K-Branched conjugated 14C9 antibody showed a potency comparable to that of the parental mAb and was more potent than the PEG40K- Linear conjugated 14C9 antibody. The 14C9 LC K149C conjugates also exhibited around a 10-fold increase in EC50 cell binding as determined by flow cytometry (FIG. 7). The EC50s determined for binding to murine macrophages are summarized in Table 3.
  • the efferocytosis assay was also performed to evaluate 14C9 Fab conjugates. As shown in FIGs. 8A-8B, the 14C9 Fab conjugates inhibited efferocytosis with similar potency as the unconjugated 14C9 Fab. A decrease in potency was observed for all Fabs as compared to the parental 14C9 antibody.
  • This example describes the in vivo evaluation of PEG-conjugated anti-MerTK antibodies.
  • mice Female C57BL/6J mice were subcutaneously inoculated with 0. 1 million MC38 cells on the lower right flank. Animals were grouped based on weight and tumor volume to ensure similar weight and starting tumor volume distribution before treatment. Anti-gpl20 (control antibody), anti-MerTK or antibody conjugates were administered via intravenous (i.v) injection at a dose of 30 mg/kg of the antibody part. Two day later, tumors and eyes were collect.
  • i.v intravenous
  • anti-gp!20 control antibody
  • anti-MerTK antibody conjugates
  • IV intravenous
  • a repeated low' dose study was earned out as described for the repeated high dose study except that the antibody or antibody’ conjugates were administered at a dose of 2.5 mg/'kg of the antibody part on both day 1 and day’ 5.
  • TAMs tumor associated macrophages
  • TAMs tumor pliarmacodynamic response
  • the PEG-conjugated 14C9 anti-MerTK antibodies were administered as a single 30 rng/kg dose intravenously to MC38-turnor bearing mice. Eye and tumor samples were obtained to evaluate MerTK receptor occupancy, as well as for evaluation of induction of an interferon response in the tumors. As shown in FIG. 9, the parental antibody and the PEG- conjugated antibodies exhibited a comparable induction of interferon beta (IFNb) and interferon- stimulated genes (ISGs) in tumors.
  • IFNb interferon beta
  • ISGs interferon- stimulated genes
  • the PEG-conjugated antibodies marginally maintained occupancy of MerTK on retinal pigment epithelium cells (RPEs), but maintained occupancy on tumor-associated macrophages (TAMs) (FIGs. 10A-10C).
  • the mean fluorescence intensity (MFI) suggested partial occupancy, with around 70% or higher unoccupied MerTK remaining with the antibody conjugates as compared to around 27% with the parental antibody.
  • MFI mean fluorescence intensity
  • the parental antibody nearly completely occupied MerTK on RPEs.
  • the antibody conjugates maintained at least 60% free-MerTK+ RPEs, with PEG-40K Branched (PEG-40K-B) maintaining around 90% free-MerTK+ RPEs.
  • PEG-40K-Branched conjugates were relatively better, with around 90% of RPEs maintaining occupancy of around 57% MerTK receptors.
  • all of the antibody conjugates resulted in at least 90% RPEs maintaining occupancy of at least 70% MerTK receptors.
  • PEG40K- Linear- and PEG-40K-Branched-conjugated antibodies resulted in around 73% of TAMs remaining with around 59% of free MerTK receptors, and in around 27% TAMs with complete occupancy.
  • the parental and PEG-conjugated antibodies exhibited comparable induction of ISGs in tumors (FIG. 13).

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Abstract

L'invention concerne des anticorps anti-MerTK pegylé et des procédés de fabrication et d'utilisation associés.
PCT/US2021/055606 2020-10-20 2021-10-19 Anticorps anti-mertk peg-conjugués et procédés d'utilisation WO2022086957A1 (fr)

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