WO2019084307A1 - Anti-mertk antibodies and methods of use thereof - Google Patents

Anti-mertk antibodies and methods of use thereof

Info

Publication number
WO2019084307A1
WO2019084307A1 PCT/US2018/057571 US2018057571W WO2019084307A1 WO 2019084307 A1 WO2019084307 A1 WO 2019084307A1 US 2018057571 W US2018057571 W US 2018057571W WO 2019084307 A1 WO2019084307 A1 WO 2019084307A1
Authority
WO
WIPO (PCT)
Prior art keywords
antibody
seq
amino acid
antigen
binding fragment
Prior art date
Application number
PCT/US2018/057571
Other languages
French (fr)
Inventor
Eduardo Sergio TROMBETTA
Richard W. GEDRICH
Diego ALVARADO
Michael B. Murphy
Tibor Keler
Laura Vitale
Tom O'NEILL
Sachin SURADE
Michael Richard Dyson
John Mccafferty
Valerie DUVA
Original Assignee
Celldex Therapeutics, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Celldex Therapeutics, Inc. filed Critical Celldex Therapeutics, Inc.
Publication of WO2019084307A1 publication Critical patent/WO2019084307A1/en

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • 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
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70514—CD4
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • 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
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/55—Fusion polypeptide containing a fusion with a toxin, e.g. diphteria toxin

Definitions

  • MerTK a transmembrane receptor
  • TAM Tyro3, Axl and MerTK
  • the extracellular domain of MerTK has two
  • TAM signaling cascades In tumors, activation of TAM signaling cascades appears to promote an anti-inflammatory and immunosuppressive microenvironment, in turn driving tumor growth.
  • Overexpression and activation of MerTK has been implicated in lymphoid leukemia, lymphoma, adenoma, melanoma, gastric, prostate and breast cancers.
  • antibodies including antigen-binding fragments, which specifically bind to MerTK, for example, the extracellular domain (ECD) of MerTK.
  • ECD extracellular domain
  • antibodies and antigen binding fragments presented herein specifically bind the ECD of human MerTK.
  • polynucleotides and vectors comprising sequences encoding such antibodies, cells comprising such polynucleotides and vectors, and compositions, reagents and kits comprising such antibodies.
  • methods for modulating MerTK activity e.g., inhibiting MerTK activity, diagnostic methods and uses, and therapeutic methods and uses of such anti-MerTK antibodies.
  • an isolated antibody, or an antigen- binding fragment thereof, which specifically binds to human MerTK comprising: (i) a light chain variable region (VL) comprising SEQ ID NO: 2; and/or (ii) a heavy chain variable region (VH) comprising SEQ ID NO: 1.
  • an isolated antibody, or an antigen-binding fragment thereof, which specifically binds to human MerTK comprising: (i) a light chain variable region (VL) comprising SEQ ID NO: 2; and (ii) a heavy chain variable region (VH) comprising SEQ ID NO: 1.
  • antibody or an antigen binding fragment thereof which specifically binds to human MerTK, comprising a VL that comprises SEQ ID NO: 2 or sequences having at least 80%, 85%, 90%, 95% or 98% amino acid sequence identity to SEQ ID NO: 2.
  • an antibody or an antigen binding fragment thereof which specifically binds to human MerTK comprising a VL that comprises a VL complementarity determining region 1 (CDRl), VL CDR2, and VL CDR3 that comprise the amino acid sequences of SEQ ID NOS: 6, 7, and 8, respectively, or conservative sequence modifications thereof.
  • VL that comprises VL CDRl, VL CDR2, and VL CDR3 that comprise the amino acid sequences of SEQ ID NOS: 6, 7, and 8,
  • an antibody or an antigen binding fragment thereof which specifically binds to human MerTK comprising a VL that comprises a VL complementarity determining region 1 (CDRl), VL CDR2, and VL CDR3 that comprise the amino acid sequences of SEQ ID NOS: 6, 7, and 8, respectively.
  • the VL that comprises VL CDRl, VL CDR2, and VL CDR3 comprises at least 80%, 85%, 90%. 95%, or 98% identity to SEQ ID NO: 2.
  • an antibody described herein or an antigen binding fragment thereof which specifically binds to human MerTK comprising a VH that comprises SEQ ID NO: 1 or sequences having at least 80%, 85%, 90%, 95% or 98% amino acid sequence identity to SEQ ID NO: 1.
  • an antibody or an antigen binding fragment thereof which specifically binds to human MerTK comprising a VH that comprises a VH complementarity determining region 1 (CDRl), VH CDR2, and VH CDR3 that comprise the amino acid sequences of SEQ ID NOS: 3, 4, and 5, respectively, or conservative sequence modifications thereof.
  • VH that comprises VH CDRl VH CDR2, and VH CDR3 that comprise the amino acid sequences of SEQ ID NOS: 3, 4, and 5,
  • SEQ ID NO: 1 comprises at least 80%,85, 90%, 95%, 98% amino acid sequence identity to SEQ ID NO: 1.
  • an antibody or an antigen binding fragment thereof which specifically binds to human MerTK, comprising a VH that comprises a VH complementarity determining region 1 (CDRl), VH CDR2, and VH CDR3 that comprise the amino acid sequences of SEQ ID NOS: 3, 4, and 5, respectively, or conservative sequence modifications thereof.
  • CDRl VH complementarity determining region 1
  • VH CDR2 VH CDR2
  • VH CDR3 that comprise the amino acid sequences of SEQ ID NOS: 3, 4, and 5, respectively, or conservative sequence modifications thereof.
  • the VH comprises at least 80% amino acid sequence identity to SEQ ID NO: 1, the VH comprises at least 85% amino acid sequence identity to SEQ ID NO: 1; the VH comprises at least 90% amino acid sequence identity to SEQ ID NO: 1; the VH comprises at least 95% amino acid sequence identity to SEQ ID NO: 1; or at least 98% amino acid sequence identity to SEQ ID NO: 1.
  • an antibody or an antigen binding fragment thereof which specifically binds to human MerTK, comprising a VL that comprises SEQ ID NO: 2 or sequences having at least 80%, 85%, 90%, 95% or 98% amino acid sequence identity to SEQ ID NO: 2, and a VH that comprises SEQ ID NO: 1 or sequences having at least 80%, 85%, 90%, 95% or 98% amino acid sequence identity to SEQ ID NO: 1.
  • an isolated antibody e.g., monoclonal antibody
  • an antigen-binding fragment thereof which specifically binds to human MerTK, comprising:
  • VL light chain variable region
  • CDRl VL complementarity determining region 1
  • VL CDR2 VL complementarity determining region 2
  • VL CDR3 VL complementarity determining region 3
  • VH heavy chain variable region
  • CDRl VH complementarity determining region 1
  • VH CDR2 VH complementarity determining region 2
  • VH CDR3 VH complementarity determining region 3
  • VL CDRl VL CDR2
  • VL CDR3 comprise the amino acid sequences of SEQ ID NOS: 6, 7, and 8, respectively or sequences having at least 80% amino acid sequence identity thereto.
  • an antibody or an antigen binding fragment thereof which specifically binds to human MerTK comprising a VH CDRl, VH CDR2, and VH CDR3 that comprises the amino acid sequences of SEQ ID NOS: 3, 4, and 5, respectively, or sequences having at least 80% amino acid sequence identity thereto.
  • the VL CDRl, VL CDR2, and VL CDR3 comprise the amino acid sequences of SEQ ID NOS: 6, 7, and 8, respectively, or sequences having at least 80% amino acid sequence identity thereto
  • the VH CDRl, VH CDR2, and VH CDR3 comprise the amino acid sequences of SEQ ID NOS: 3, 4, and 5, respectively, or sequences having at least 80% amino acid sequence identity thereto.
  • an isolated antibody e.g., monoclonal antibody
  • an antigen-binding fragment thereof which specifically binds to human MerTK, comprising:
  • VL light chain variable region
  • CDRl VL complementarity determining region 1
  • VL CDR2 VL complementarity determining region 2
  • VL CDR3 VL complementarity determining region 3
  • VH heavy chain variable region
  • CDRl VH complementarity determining region 1
  • VH CDR2 VH complementarity determining region 2
  • VH CDR3 VH complementarity determining region 3
  • an antibody or an antigen binding fragment thereof which specifically binds to human MerTK, comprising a VL that comprises VL CDRl, VL CDR2, and VL CDR3 that comprise the amino acid sequences of SEQ ID NOS: 16, 17, and 18, respectively, or sequences having at least 80% amino acid sequence identity thereto.
  • VH CDRl In a certain embodiment provided herein is an antibody or an antigen binding fragment thereof which specifically binds to human MerTK, the VH CDRl, VH CDR2, and VH CDR3 comprise the amino acid sequences of SEQ ID NOS: 13, 14, and 15, respectively or sequences having at least 80% amino acid sequence identity thereto.
  • an antibody or an antigen binding fragment thereof which specifically binds to human MerTK comprising a VL that comprises a VL CDRl, VL CDR2, and VL CDR3 comprising the amino acid sequences of SEQ ID NOS: 16, 17, and 18, respectively, or sequences having at least 80% amino acid sequence identity thereto, and comprises a VH that comprises VH CDRl, VH CDR2, and VH CDR3 comprising the amino acid sequences of SEQ ID NOS: 13, 14, and 15, respectively or sequences having at least 80% amino acid sequence identity thereto.
  • an isolated antibody e.g., monoclonal antibody
  • an antigen-binding fragment thereof which specifically binds to human MerTK, comprising:
  • VL light chain variable region
  • CDRl VL complementarity determining region 1
  • VL CDR2 VL complementarity determining region 2
  • VL CDR3 VL complementarity determining region 3
  • VH heavy chain variable region
  • CDRl VH complementarity determining region 1
  • VH CDR2 VH complementarity determining region 2
  • VH CDR3 VH complementarity determining region 3
  • an antibody or an antigen binding fragment thereof which specifically binds to human MerTK, comprising a VL that comprises a VL CDRl, VL CDR2, and VL CDR3 comprising the amino acid sequences of SEQ ID NOS: 22, 23, and 24, respectively, or sequences having at least 80% amino acid sequence identity thereto.
  • an antibody or an antigen binding fragment thereof which specifically binds to human MerTK comprising a VH that comprises a VH CDRl, VH CDR2, and VH CDR3 comprising the amino acid sequences of SEQ ID NOS: 19, 20, and 21, respectively, or sequences having at least 80% amino acid sequence identity thereto.
  • an antibody or an antigen binding fragment thereof which specifically binds to human MerTK, comprising a VL that comprise a VL CDRl, VL CDR2, and VL CDR3 comprising the amino acid sequences of SEQ ID NOS: 22, 23, and 24, respectively, or sequences having at least 80% amino acid sequence identity thereto, and comprise a VH that comprises a VH CDRl, VH CDR2, and VH CDR3 comprising the amino acid sequences of SEQ ID NOS: 19, 20, and 21, respectively, or sequences having at least 80%) amino acid sequence identity thereto.
  • an isolated antibody, or an antigen-binding fragment thereof, which specifically binds to human MerTK comprising a light chain variable region (VL) comprising VL complementary determining region 1 (CDRl), VL CDR2, and VL CDR3, wherein the VL CDRl, VL CDR2, and VL CDR3 comprise the amino acid sequences of SEQ ID NOS: 36, 37 and 38, respectively or conservative sequence modifications thereof or SEQ ID NOS: 40, 37 and 38, respectively, or conservative sequence modifications thereof; and/or a heavy chain variable region (VH) comprising VH complementary determining region 1 (CDRl), VH CDR2, and VH CDR3, wherein the VH CDRl, VH CDR2, and VH CDR3 comprise the amino acid sequences of SEQ ID NOS: 33, 34 and 35 respectively or conservative sequence modifications thereof or SEQ ID NOS: 33, 34 and 39, respectively, or conservative sequence modifications thereof.
  • VL light chain variable region
  • CDRl complementary determining region 1
  • VL CDR2
  • the antibody or antigen-binding fragment provided herein comprises a VL CDRl, VL CDR2, and VL CDR3 that comprise the amino acid sequences of SEQ ID NOS: 36, 37, and 38, respectively or sequences having at least 80% amino acid sequence identity thereto.
  • the antibody or antigen-binding fragment provided herein comprises a VH CDRl, VH CDR2, and VH CDR3 that comprise the amino acid sequences of SEQ ID NOS: 33, 34, and 35, respectively or sequences having at least 80% amino acid sequence identity thereto.
  • the VL CDRl, VL CDR2, and VL CDR3 can comprise the amino acid sequences of SEQ ID NOS: 36, 37, and 38, respectively or sequences having at least 80% amino acid sequence identity thereto, and the VH CDRl, VH CDR2, and VH CDR3 comprise the amino acid sequences of SEQ ID NOS: 33, 34, and 35, respectively or sequences having at least 80% amino acid sequence identity thereto.
  • an isolated antibody, or an antigen-binding fragment thereof, which specifically binds to human MerTK comprising a light chain variable region (VL) comprising VL complementary determining region 1 (CDRl), VL CDR2, and VL CDR3, wherein the VL CDRl, VL CDR2, and VL CDR3 comprise the amino acid sequences of SEQ ID NOS: 44, 45 and 46, respectively or conservative sequence modifications thereof or SEQ ID NOS: 48, 45 and 46, respectively, or conservative sequence modifications thereof; and/or a heavy chain variable region (VH) comprising VH complementary determining region 1 (CDRl), VH CDR2, and VH CDR3, wherein the VH CDRl, VH CDR2, and VH CDR3 comprise the amino acid sequences of SEQ ID NOS: 41, 42, 43 respectively or conservative sequence modifications thereof or SEQ ID NOS: 41, 42, 47, respectively, or conservative sequence modifications thereof.
  • VL light chain variable region
  • CDRl complementary determining region 1
  • VL CDR2
  • the antibody or antigen-binding fragment can comprise a VL CDRl, VL CDR2, and VL CDR3 that comprise the amino acid sequences of SEQ ID NOS: 44, 45 and 46, respectively or sequences having at least 80% amino acid sequence identity thereto.
  • the antibody or antigen-binding fragment can comprise a VH CDRl, VH CDR2, and VH CDR3 that comprise the amino acid sequences of SEQ ID NOS: 41, 42, 43, respectively or sequences having at least 80% amino acid sequence identity thereto.
  • the antibody or an antigen-binding fragment disclosed herein can comprise a VL CDRl, VL CDR2, and VL CDR3 that comprise the amino acid sequences of SEQ ID NOS: 44, 45 and 46, respectively or sequences having at least 80% amino acid sequence identity thereto, and the VH CDRl, VH CDR2, and VH CDR3 comprise the amino acid sequences of SEQ ID NOS: 41, 42, 43, respectively or sequences having at least 80% amino acid sequence identity thereto.
  • an antibody or an antigen binding fragment thereof which specifically binds to human MerTK that comprises a VL CDRl, VL CDR2, and VL CDR3 present in one polypeptide, and a comprises a VH CDRl, VH CDR2, and VH CDR3 on a second polypeptide.
  • an antibody or antigen binding fragment described herein which specifically binds to human MerTK comprises a VL and a VH, wherein the VL and VH are present in the same polypeptide.
  • provided herein is an isolated antibody, or an antigen-binding fragment thereof, which specifically binds to Domain 1 of human MerTK.
  • an isolated antibody, or an antigen-binding fragment thereof which binds to the same epitope of human MerTK as an antibody described herein or antigen- binding fragment thereof.
  • an isolated antibody, or an antigen-binding fragment thereof which competes for binding to human MerTK with an antibody described herein or antigen-binding fragment thereof.
  • provided herein is an isolated antibody, or an antigen-binding fragment thereof, which binds to Domain 1 of human MerTK and inhibits binding of Gas6 to MerTK and/or, inhibits MerTK phosphorylation.
  • an antibody or antigen binding fragment presented herein is a MerTK receptor antagonist.
  • an antibody or antigen binding fragment described herein, which specifically binds to human MerTK comprises a human heavy chain constant region.
  • an antibody or antigen-binding fragment described herein, which specifically binds to human MerTK comprises a human heavy chain comprising a VH and a human heavy chain constant region.
  • an antibody or antigen binding fragment described herein, which specifically binds to human MerTK comprises a human light chain constant region.
  • an antibody or antigen-binding fragment described herein, which specifically binds to human MerTK comprises a human light chain comprising a VL and a human light chain constant region.
  • an antibody or antigen binding fragment described herein which specifically binds to human MerTK comprises a human heavy chain constant region and a human light chain constant region.
  • such an antibody or antigen- binding fragment comprises a human heavy chain comprising a VH and a human heavy chain constant region and a human light chain comprising a VL and a human light chain constant region.
  • an antibody or antigen binding fragment described herein, which specifically binds to human MerTK is an IgGl antibody.
  • an antibody or antigen binding fragment described herein, which specifically binds to human MerTK is a human IgGl antibody.
  • the antibody or antigen-binding fragment comprises a heavy chain constant region or a light chain constant region. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain constant region and a light chain constant region. In certain embodiments, the antibody or antigen-binding fragment comprises a human heavy chain constant region or a human light chain constant region. In some embodiments, the antibody or antigen-binding fragment comprises a human heavy chain constant region and a human light chain constant region.
  • the antibody or antigen-binding fragment is an IgGl antibody or antigen-binding fragment. In certain embodiments, the antibody or antigen-binding fragment is an IgG2 antibody or antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment is a human IgGl antibody or antigen-binding fragment or a human IgG2 antibody or antigen-binding fragment. In some embodiments, the antibody or antigen- binding fragment comprises a kappa light chain constant region or a lambda light chain constant region. In some embodiments, the antibody or antigen-binding fragment comprises a human kappa light chain constant region or a human lambda light chain constant region.
  • the antibody or antigen-binding fragment comprises the amino acid sequence of SEQ ID NO: 49 or the amino acid sequence of SEQ ID NO: 50. In some embodiments, the antibody or antigen-binding fragment comprises the amino acid sequence of SEQ ID NO: 49 and the amino acid sequence of SEQ ID NO: 50.
  • the antibody or antigen-binding fragment disclosed herein comprises the amino acid sequence of SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53 SEQ ID NO: 55, SEQ ID NO: 56, or SEQ ID NO: 57
  • the antibody or antigen-binding fragment disclosed herein comprises the amino acid sequence of SEQ ID NO: 49 and the amino acid sequence of SEQ ID NO: 50; the amino acid sequence of SEQ ID NO: 49 and the amino acid sequence of SEQ ID NO: 51; the amino acid sequence of SEQ ID NO: 54 and the amino acid sequence of SEQ ID NO: 52; the amino acid sequence of SEQ ID NO: 54 and the amino acid sequence of SEQ ID NO: 53; the amino acid sequence of SEQ ID NO: 54 and the amino acid sequence of SEQ ID NO: 55; the amino acid sequence of SEQ ID NO: 54 and the amino acid sequence of SEQ ID NO: 56; the amino acid sequence of SEQ ID NO: 57 and the amino acid sequence of SEQ ID NO: 52; the amino acid sequence of SEQ ID NO: 57 and the amino acid sequence of SEQ ID NO: 53; the amino acid sequence of SEQ ID NO: 57 and the amino acid sequence of SEQ ID NO: 55; or the amino acid sequence of SEQ ID NO: 57 and
  • an antibody or antigen binding fragment described herein which specifically binds to human MerTK comprises a human kappa light chain constant region or a human gamma heavy chain constant region.
  • an isolated antibody described herein, or an antigen-binding fragment thereof, which specifically binds to human MerTK is a monoclonal antibody.
  • an antibody e.g., monoclonal antibody described herein, or an antigen-binding fragment thereof, which specifically binds to human MerTK, is a human antibody.
  • an antibody (e.g., monoclonal antibody) described herein, or an antigen-binding fragment thereof, which specifically binds to human MerTK is a chimeric antibody.
  • an isolated antibody e.g., monoclonal antibody described herein, or an antigen-binding fragment thereof, which specifically binds to human MerTK, is a humanized antibody.
  • an isolated antibody e.g., monoclonal antibody described herein, or an antigen-binding fragment thereof, which specifically binds to human MerTK, is a single chain antibody.
  • an isolated antibody e.g., monoclonal antibody described herein, or an antigen-binding fragment thereof, which specifically binds to human MerTK, is a multispecific, e.g., bispecific, antibody.
  • an isolated antibody e.g., monoclonal antibody described herein, or an antigen-binding fragment thereof, which specifically binds to human MerTK, is a monovalent antibody.
  • an isolated antibody e.g., monoclonal antibody described herein, or an antigen-binding fragment thereof, which specifically binds to human MerTK, is fused to a heterologous polypeptide.
  • an isolated antibody e.g., monoclonal antibody described herein, or an antigen-binding fragment thereof, which specifically binds to human MerTK, is conjugated to an agent.
  • the agent is a toxin.
  • the toxin is abrin, ricin A, pseudomonas exotoxin, cholera toxin, or diphtheria toxin.
  • composition comprising a
  • a pharmaceutical composition comprising an antibody described herein or antigen-binding fragment thereof, which specifically binds to human MerTK and a pharmaceutically acceptable carrier.
  • a polynucleotide comprising nucleotide sequences encoding a VH, a VL, or both a VL and a VH, of an antibody described herein or antigen-binding fragment thereof, which specifically binds to human MerTK.
  • a polynucleotide comprising nucleotide sequences encoding a heavy chain, a light chain, or both heavy chain and a light chain of an antibody described herein or antigen-binding fragment thereof, which specifically binds to human MerTK.
  • a population of polynucleotides comprising (i) a first polynucleotide comprising nucleotide sequences encoding a VH or a heavy chain of an antibody described herein or antigen-binding fragment thereof, which specifically binds to human MerTK, and (ii) a second polypeptide comprising nucleotide sequences encoding a VL or a light chain of the antibody.
  • a vector comprising a polynucleotide described herein comprising nucleotide sequences encoding a VH, or a VL, or a VH and VL of an antibody described herein or antigen-binding fragment thereof, which specifically binds to human MerTK.
  • a vector comprising a polynucleotide described herein comprising nucleotide sequences encoding a heavy chain, a light chain, or both heavy chain and a light chain of an antibody described herein or antigen-binding fragment thereof, which specifically binds to human MerTK.
  • a population of vectors comprising (i) a first vector comprising nucleotide sequences encoding a VH or a heavy chain of an anti-MerTK antibody or antigen-binding fragment described herein, and (ii) a second vector comprising nucleotide sequences encoding a VL or a light chain of an anti-MerTK antibody or antigen- binding fragment described herein.
  • an isolated cell comprising a
  • polynucleotide comprising nucleotide sequences encoding a VH, a VL, or both a VH and a VL of an antibody described herein or antigen-binding fragment thereof, which specifically binds to human MerTK.
  • an isolated cell comprising a
  • polynucleotide comprising nucleotide sequences encoding a heavy chain, a light chain, or both heavy chain and a light chain of an antibody described herein or antigen-binding fragment thereof, which specifically binds to human MerTK.
  • an isolated cell comprising a population of polynucleotides described herein.
  • a population of cells comprising (i) a first host cell comprising a polynucleotide described herein comprising nucleotide sequences encoding a VH or a heavy chain an anti-MerTK antibody or antigen-binding fragment described herein, and (ii) a second host cell comprising a polynucleotide comprising nucleotide sequences encoding a VL or a light chain of an anti-MerTK antibody or antigen-binding fragment described herein.
  • an isolated cell producing an antibody described herein or antigen-binding fragment thereof, which specifically binds to human MerTK.
  • kits comprising an antibody described herein or antigen-binding fragment thereof, which specifically binds to human MerTK.
  • a method of managing, protecting against, or treating cancer for example, breast cancer, prostate cancer, gastric cancer, lung cancer, e.g., non- small cell lung cancer, adenoma, melanoma, lymphoma, or leukemia, or infection, for example, bacterial, e.g., gram-negative or gram-positive bacteria, fungal, viral, or parasitic infection in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of an antibody or antigen binding fragment described herein, for example, Ab2000, or antigen- binding fragment thereof, or Ab2000-A7, or antigen-binding fragment thereof, which
  • a method of modulating an immune response comprising administering to a subject in need thereof an effective amount of an antibody described herein, for example, Ab2000, or antigen-binding fragment thereof, or Ab2000-A7, or antigen-binding fragment thereof, which specifically binds to human MerTK.
  • the subject is a subject suffering from an infection, a subject having cancer, or an immunocompromised subject such as, for example, a subject who is undergoing, or had undergone treatment with, an anticancer therapy, is HIV positive, or who has AIDS or SCID, has diabetes, or has had a transplant and is taking an immunosuppressant.
  • the subject has been treated with an immunosuppressant.
  • proinflammatory response in a subject comprising administering to a subject in need thereof an effective amount of an antibody described herein, for example, Ab2000, or antigen-binding fragment thereof, or Ab2000-A7, or antigen-binding fragment thereof, which specifically binds to human MerTK.
  • an antibody described herein for example, Ab2000, or antigen-binding fragment thereof, or Ab2000-A7, or antigen-binding fragment thereof, which specifically binds to human MerTK.
  • such a method of enhancing a proinflammatory response in a subject results in an increase in T F- ⁇ secretion.
  • a method of enhancing an immune response to a vaccine in a subject comprising administering to a subject in need thereof, who is or has been administered the vaccine, an effective amount of an antibody described herein or antigen-binding fragment thereof, which specifically binds to human MerTK.
  • the vaccine is a cancer or tumor vaccine
  • a vaccine antigen that can also be targeted, for example, to particular cell types or to particular tissues.
  • the vaccine antigen can be targeted to Antigen Presenting Cells (APCs), for example by use of agents such as antibodies targeted to APC-surface receptors such as DEC-205, for example as discussed in WO 2009/061996 (Celldex Therapeutics, Inc.), or the Mannose Receptor (CD206) for example as discussed in WO 03040169 (Medarex, Inc.).
  • APCs Antigen Presenting Cells
  • provided herein is a method of managing, preventing, protecting against, or treating metastasis in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of an antibody described herein or antigen-binding fragment thereof, which specifically binds to human MerTK.
  • the subject has cancer and the method is effective in treating or managing the subject's cancer.
  • a method for stimulating cytotoxicity of K cells in a subject comprising administering to a subject in need thereof an effective amount of an antibody described herein, for example, Ab2000, or antigen-binding fragment thereof, or Ab2000-A7, or antigen-binding fragment thereof, which specifically binds to human MerTK.
  • the subject has cancer, or is being treated for cancer with an anti-cancer therapeutic, or the subject has an infection.
  • an antibody or an antigen-binding fragment thereof which specifically binds to human MerTK, comprising culturing a cell, host cell, or population of cells described herein to express an anti-MerTK antibody or antigen-binding fragment thereof.
  • provided herein is a method of making an antibody or an antigen-binding fragment thereof which specifically binds to human MerTK, comprising expressing a polynucleotide described herein or a population of polynucleotides described herein.
  • a method of increasing proinflammatory cytokine production in a subject in need thereof comprising administering to the subject an antibody or an antigen-binding fragment thereof that specifically binds to human MerTK such that the cytokine production is increased.
  • the cytokine secretion can be increase by about 5%, 10%, 15%), 20%, 30, or more relative to secretion in the absence of the antibody.
  • the proinflammatory cytokine is T Fa (tumor necrosis factor a).
  • the proinflammatory cytokine is IL-IRA (interleukin-1 receptor antagonist).
  • the antibody or antigen-binding fragment does not substantially induce phosphorylation of MerTK.
  • the antibody or antigen-binding fragment induces phosphorylation of MerTK at least 50%, 60%>, 70%, 80%>, 90% or less than the phosphorylation induced by Gas6 binding to MerTK.
  • the antibody or antigen-binding fragment is one that specifically binds to Domain 1 of human MerTK.
  • the antibody or antigen-binding fragment is one of the antibodies or antigen-binding fragments described herein.
  • a method of increasing proinflammatory cytokine production in a subject in need thereof comprising administering to the subject an antibody or an antigen-binding fragment thereof disclsoed herein such that the production of one or more cytokines is increased.
  • the production of proinflammatory cytokine TNFa tumor necrosis factor a
  • the production of proinflammatory cytokine IL-IRA is increased.
  • provided herein is a method wherein the production of proinflammatory cytokines TNFa and IL- IRA are increased.
  • a method of increasing proinflammatory cytokine production in a subject in need thereof comprising administering to the subject an antibody or an antigen-binding fragment thereof disclosed herein such that the production of one or more cytokines is increased.
  • the production of proinflammatory cytokine TNFa tumor necrosis factor a
  • the production of proinflammatory cytokine IL-1RA is increased.
  • provided herein is a method wherein the production of proinflammatory cytokines TNFa and IL-IRA are increased.
  • provided herein is a method of increasing proinflammatory secretory factor production in a subject in need thereof, comprising administering to the subject an antibody or an antigen-binding fragment thereof disclosed herein such that the production of one or more proinflammatory secretory factors is increased.
  • provided herein is a method of increasing proinflammatory secretory factor production in a subject in need thereof, comprising administering to the subject an antibody or an antigen-binding fragment thereof disclosed herein such that the production of one or more proinflammatory secretory factors is increased.
  • a method of increasing proinflammatory secretory factor production in a subject in need thereof comprising administering to the subject an antibody or an antigen-binding fragment thereof disclosed herein such that the production of one or more proinflammatory secretory factors is increased.
  • RANTES Activation Normal T cell Expressed and Secreted
  • VEGF-A vascular endothelial growth factor A
  • IL-18 Activation Normal T cell Expressed and Secreted
  • Fig. 1 depicts the binding of anti-MerTK antibody Ab2000 to cellularly expressed full length human MerTK (hMerTK) in a dose responsive manner, with EC50 values in the picomolar to nanomolar range.
  • the mean fluorescent intensity is plotted as a function of the log- transformed molar concentration of the antibody.
  • Fig. 2 shows that the anti-MerTK antibody Ab2000 blocked Gas6 binding to recombinant MerTK extracellular domain (ECD) in a dose responsive manner, with an IC50 of 1 nM.
  • FIG. 3A depicts inhibition of Gas6-dependent MerTK phosphorylation by the anti- MerTK antibody Ab2000. Addition of 100 nM Gas6 alone was used as a reference.
  • Fig. 3B shows that titration of Ab2000 on MerTK expressing THPl cells inhibited Gas6-dependent MerTK phosphorylation with an IC50 value of 0.2 nM.
  • Fig. 4 shows that addition of the anti-MerTK antibody Ab2000 elicited only very weak MerTK phosphorylation in the absence of exogenously added Gas6 (as compared to independently-added Gas6) in MerTK expressing THPl cells. Addition of 100 nM Gas6 alone was used as a reference.
  • Fig. 5 shows that the anti-MerTK antibody Ab2000 enhanced TNF-a secretion from differentiated U937 monocyte-derived macrophages upon stimulation of different Toll-like receptors with LPS.
  • Figs. 6A and 6B show that the anti-MerTK antibody Ab2000 enhanced TNF- ⁇ secretion from primary human macrophages upon stimulation of different Toll-like receptors with LPS (Fig. 6A) or Resiquimod (R848) (Fig. 6B).
  • Figs. 7A and 7B show that the anti-MerTK antibody Ab2000 enhanced TNF- ⁇ secretion from primary human dendritic cells upon stimulation of different Toll-like receptors with LPS (Fig. 7A) or Resiquimod (R848) (Fig. 7B) as compared to a control IgGl antibody.
  • Fig. 8 demonstrates that titration of the anti-MerTK antibody Ab2000 resulted in a dose dependent increase in TNF-a secretion in dendritic cells with nanomolar potency.
  • Fig. 9 depicts the binding of anti-MerTK antibody Ab2000 and affinity matured antibodies A2000-A7, A2000-F1 and A2000-G6 to recombinant full length human MerTK (hMerTK) as determined by ELISA.
  • the absorbance at OD450 is plotted as a function of the concentration of the antibody.
  • Fig. 10A and Fig. 10B depict the binding of the anti-MerTK antibody Ab2000 and affinity matured antibodies to huMerTK-L cells and SK-mel-5 cells, respectively.
  • the mean fluorescence intesity is plotted as a function of the concentration of the antibody.
  • Fig. 11 depicts representative binding curves of the A2000 antibody and the A2000- A7 affinity matured antibody to cellularly expressed full length human MerTK (hMerTK) as determined by ELISA.
  • the absorbance at OD450 is plotted as a function of the concentration of the antibody.
  • Fig. 12 A and Fig. 12B depict the binding of the anti-MerTK affinity matured antibody Ab2000-A7 to huMerTK-L cells and SK-mel-5 cells, respectively.
  • the mean fluorescence intensity (MFI) is plotted as a function of the concentration of the antibodies.
  • Fig. 13A and Fig. 13B shows that the anti-MerTK antibodies Ab2000 and Ab2000- A7 enhance IL-IRA production by dendritic cells. Fold increase over isotype control is plotted.
  • Fig. 14 shows that the anti-MerTK antibodies Ab2000 and Ab2000-A7 enhance TNF- alpha production by dendritic cells. Fold increase over isotype control is plotted.
  • Fig. 15 depicts multiplex analysis of production of 30 different cytokines
  • Fig. 16 depicts multiplex analysis of production of 30 different cytokines
  • chemokines and growth factors in response to Ab2000-A7 in PBMCs Fold increase in protein production in response to Ab2000-A7 over isotype control is plotted.
  • Fig. 17 shows IL-IRA production (pg/ml) in PBMCs (black bar) and CD14+ cell- depleted PBMCs (grey bars) in response to Ab2000-A7 antibody.
  • Fig. 18 shows that the anti-MerTK antibody Ab2000-A7 enhances TNF-a production by macrophages. Fold increase over isotype control is plotted.
  • MerTK is a receptor tyrosine kinase of the TAM group (including Tyro-3, Axl, and MerTK) (Graham et al. (2014) Nat Rev Cancer ;14: 769-785; Lemke G. (2013) Cold Spring Harb Perspect Biol.; 5).
  • MerTK is human MerTK.
  • UniProKB accession number Q 12866 provides an exemplary human MerTK amino acid sequence.
  • Native MerTK comprises two extracellular immunoglobulin (Ig)-like domains, two extracellular fibronectin (FN) type III motifs, and a intracellular tyrosine kinase domain (see, e.g., Lemke G. (2013) Cold Spring Harb Perspect Biol.; 5).
  • GenBankTM accession number NM 006343.2 provides an exemplary human MerTK nucleic acid coding sequence.
  • antibodies which specifically bind to human MerTK, for example, an extracellular domain (ECD) of human MerTK, and modulate MerTK expression and/or MerTK activity.
  • ECD extracellular domain
  • antibody and “immunoglobulin” and “Ig” are terms of art and can be used interchangeably herein and refer to a molecule with an antigen binding site that specifically binds an antigen.
  • Antibodies can include, for example, monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, synthetic antibodies, tetrameric antibodies comprising two heavy chain and two light chain molecules, an antibody light chain monomer, an antibody heavy chain monomer, an antibody light chain dimer, an antibody heavy chain dimer, an antibody light chain/antibody heavy chain pair, an antibody with two light chain/heavy chain pairs (e.g., identical pairs), intrabodies, heteroconjugate antibodies, single domain antibodies, monovalent antibodies, bivalent antibodies (including monospecific or bispecific bivalent antibodies), single chain antibodies, or single-chain Fvs (scFv), camelized antibodies, affybodies, Fab fragments, F(ab') fragments, F(ab') 2 fragments, disulfide-linked Fvs (sdFv), anti -idiotypic (anti-Id) antibodies (including, e.g., anti-
  • an isolated antibody e.g., monoclonal antibody described herein, or an antigen-binding fragment thereof, which specifically binds to human MerTK
  • a heterologous polypeptide e.g., an isolated antibody (e.g., monoclonal antibody) described herein, or an antigen-binding fragment thereof, which specifically binds to human MerTK
  • an agent is a toxin.
  • the toxin is abrin, ricin A, pseudomonas exotoxin, cholera toxin, or diphtheria toxin.
  • Antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA or IgY), any class, (e.g., IgGl, IgG2, IgG3, IgG4, IgAl or IgA2), or any subclass (e.g., IgG2a or IgG2b) of any type (e.g., IgG, IgE, IgM, IgD, IgA or IgY), any class, (e.g., IgGl, IgG2, IgG3, IgG4, IgAl or IgA2), or any subclass (e.g., IgG2a or IgG2b) of
  • antibodies described herein are IgG antibodies (e.g., human IgG), or a class (e.g., human IgGl, IgG2, IgG3 or IgG4) or subclass thereof.
  • an antibody is a 4-chain antibody unit comprising two heavy (H) chain / light (L) chain pairs, wherein the amino acid sequences of the H chains are identical and the amino acid sequences of the L chains are identical.
  • the H and L chains comprise constant regions, for example, human constant regions.
  • the L chain constant region of such antibodies is a kappa or lambda light chain constant region, for example, a human kappa or lambda light chain constant region.
  • the H chain constant region of such antibodies comprise a gamma heavy chain constant region, for example, a human gamma heavy chain constant region.
  • such antibodies comprise IgG constant regions, for example, human IgG constant regions.
  • an "antigen" is a moiety or molecule that contains an epitope to which an antibody can specifically bind. As such, an antigen is also is specifically bound by an antibody.
  • the antigen, to which an antibody described herein binds is MerTK (e.g., human MerTK), or a fragment thereof, for example, an extracellular domain of MerTK (e.g., human MerTK).
  • an epitope is a term in the art and refers to a localized region of an antigen to which an antibody can specifically bind.
  • An epitope can be a linear epitope or a conformational, non-linear, or discontinuous, epitope.
  • an epitope can be contiguous amino acids of the polypeptide (a "linear” epitope) or an epitope can comprise amino acids from two or more non-contiguous regions of the polypeptide (a "conformational,” “non-linear” or “discontinuous” epitope).
  • a linear epitope may or may not be dependent on secondary, tertiary, or quaternary structure.
  • an antibody binds to a group of amino acids regardless of whether they are folded in a natural three dimensional protein structure.
  • an antibody requires amino acid residues making up the epitope to exhibit a particular conformation (e.g., bend, twist, turn or fold) in order to recognize and bind the epitope.
  • immunospecifically binds are analogous terms in the context of antibodies and refer to molecules that bind to an antigen (e.g., epitope) as such binding is understood by one skilled in the art.
  • an antigen e.g., epitope
  • a molecule that specifically binds to an antigen may bind to other peptides or polypeptides, generally with lower affinity as determined by, e.g., immunoassays, BiacoreTM, KinExA 3000 instrument (Sapidyne Instruments, Boise, ID), or other assays known in the art.
  • molecules that specifically bind to an antigen bind to the antigen with a K a that is at least 2 logs, 2.5 logs, 3 logs, 4 logs or greater than the K a when the molecules bind to another antigen.
  • molecules that specifically bind to an antigen do not cross react with other proteins.
  • molecules that specifically bind to an antigen do not cross react with other non-MerTK proteins.
  • the term “constant region” or “constant domain” is a well-known antibody term of art (sometimes referred to as "Fc"), and refers to an antibody portion, e.g., a carboxyl terminal portion of a light and/or heavy chain which is not directly involved in binding of an antibody to antigen but which can exhibit various effector functions, such as interaction with the Fc receptor.
  • the terms refer to a portion of an immunoglobulin molecule having a generally more conserved amino acid sequence relative to an immunoglobulin variable domain.
  • the term "heavy chain” when used in reference to an antibody can refer to any distinct types, e.g., alpha (a), delta ( ⁇ ), epsilon ( ⁇ ), gamma ( ⁇ ) and mu ( ⁇ ), based on the amino acid sequence of the constant domain, which give rise to IgA, IgD, IgE, IgG and IgM classes of antibodies, respectively, including subclasses of IgG, e.g., IgGi, IgG 2 , IgG 3 and IgG 4 .
  • an “isolated” or “purified” antibody or antigen binding fragment is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the antibody or antigen binding fragment is derived, or substantially free of chemical precursors or other chemicals when the antibody or antigen binding fragment is chemically synthesized.
  • the term "light chain” when used in reference to an antibody can refer to any distinct types, e.g., kappa ( ⁇ ) of lambda ( ⁇ ) based on the amino acid sequence of the constant domains. Light chain amino acid sequences are well known in the art. In specific embodiment is a human light chain.
  • the term "monoclonal antibody” is a well-known term of art that refers to an antibody obtained from a population of homogenous or substantially homogeneous antibodies.
  • the term “monoclonal” is not limited to any particular method for making the antibody.
  • a population of monoclonal antibodies can be generated by cells, a population of cells, or a cell line.
  • a "monoclonal antibody,” as used herein is an antibody produced by a single cell (e.g., hybridoma or host cell producing a recombinant antibody), wherein the antibody specifically binds to a MerTK epitope (e.g., an epitope of the extracellular domain of human MerTK) as determined, e.g., by ELISA or other antigen-binding or competitive binding assay known in the art or in the Examples provided herein.
  • a monoclonal antibody can be a chimeric antibody or a humanized antibody.
  • a monoclonal antibody is a monovalent antibody or multivalent (e.g., bivalent) antibody.
  • a monoclonal antibody is a monospecific or multispecific antibody (e.g., bispecific antibody).
  • polyclonal antibodies refers to an antibody population that includes a variety of different antibodies that specifically to the same and/or to different epitopes within an antigen or antigens.
  • variable region refers to a portion of an antibody, generally, a portion of a light or heavy chain, typically about the amino-terminal 110 to 120 amino acids in the mature heavy chain and about 90 to 100 amino acids in the mature light chain.
  • Variable regions comprise complementarity determining regions (CDRs) flanked by framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • the spatial orientation of CDRs and FRs are as follows, in an N-terminal to C-terminal direction: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • variable region is a human variable region.
  • the CDRs of an antibody can be determined according to (i) the Kabat numbering system (Kabat et al. (1971) Ann. NY Acad. Sci. 190:382-391 and, Kabat et al. (1991) Sequences of Proteins of Immunological Interest Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242); or (ii) the Chothia numbering scheme, which will be referred to herein as the "Chothia CDRs" (see, e.g., Chothia and Lesk, 1987, J. Mol. Biol., 196:901-917; Al-Lazikani et al., 1997, J. Mol.
  • an antibody or an antigen-binding fragment thereof which specifically binds to human MerTK comprises VL and VH CDR as set forth in Table 1, which utilizes standard one letter amino acid abbreviations.
  • an antibody or an antigen-binding fragment thereof which specifically binds to human MerTK and comprises VL CDRl (SEQ ID NO: 6), VL CDR2 (SEQ ID NO: 7), and VL CDR3 (SEQ ID NO: 8), and VH CDRl (SEQ ID NO: 3), VH CDR2 (SEQ ID NO: 4), and VH CDR3 (SEQ ID NO: 5).
  • VH CDR1 (SEQ ID NO: 3)
  • VH CDR2 (SEQ ID NO: 4)
  • VH CDR3 (SEQ ID NO: 5)
  • an antibody or an antigen-binding fragment thereof which specifically binds to human MerTK and comprises a VL and/or a VH as set forth in Table 2, which utilizes standard one letter amino acid abbreviations.
  • an antibody or an antigen-binding fragment thereof which specifically binds to human MerTK, and comprises VL (SEQ ID NO: 2) and VH (SEQ ID NO: 1).
  • an antibody that comprises the VL and VH amino acid sequences of Table 2 is referred to herein as "Ab2000.”
  • such an antibody or antigen- binding fragment comprises a separate light chain comprising the VL amino acid sequence and a separate heavy chain comprising the VH amino acid sequence.
  • such an antibody or antigen-binding fragment comprises a single chain comprising the VL amino acid sequence and the VH amino acid sequence.
  • an antibody or an antigen-binding fragment described herein comprises the VL CDR1, VL CDR2, and VL CDR3 of Ab2000.
  • the VL CDR1, VL CDR2, and VL CDR3 are as set forth in Table 1 (SEQ ID NOS: 6, 7, and 8, respectively).
  • the antibody or antigen-binding fragment further comprises a VH as set forth in Table 2.
  • an antibody or an antigen-binding fragment described herein, which specifically binds to human MerTK comprises a VL as set forth in Table 2.
  • the antibody or antigen-binding fragment further comprises VH CDRl, VH CDR2 and/or VH CDR3 as set forth in Table 1.
  • an antibody described herein, or an antigen-binding fragment thereof comprises the VH CDRl, VH CDR2, and VH CDR3 of Ab2000.
  • the VH CDRl, VH CDR2, and VH CDR3 are as set forth in Table 1.
  • the antibody or antigen-binding fragment further comprises a VL as set forth in Table 2.
  • an antibody described herein, or an antigen-binding fragment thereof, which specifically binds to human MerTK comprises a VH as set forth in Table 2.
  • the antibody or antigen-binding fragment further comprises VL CDRl, VL CDR2 and/or VL CDR3 as set forth in Table 1.
  • an antibody described herein, or an antigen-binding fragment thereof comprises the VL CDRl, VL CDR2, and VL CDR3 of Ab2000, and the VH CDRl, VH CDR2, and VH CDR3 of Ab2000.
  • the VL CDRl, VL CDR2, and VL CDR3 are as set forth in Table 1 (SEQ ID NOS: 6, 7, and 8, respectively), and the VH CDRl, VH CDR2, and VH CDR3 are as set forth in Table (SEQ ID NOS: 3, 4, and 5, respectively).
  • an antibody described herein, or an antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO: 54. In some embodiments, an antibody described herein, or an antigen-binding fragment thereof, comprises the amino acid sequence of SEQ ID NO: 52. In some embodiments, an antibody described herein, or an antigen-binding fragment thereof, comprises the amino acid sequence of SEQ ID NO: 53. In some embodiments, an antibody described herein, or an antigen-binding fragment thereof, comprises the amino acid sequence of SEQ ID NO: 54 and the amino acid sequence of SEQ ID NO: 52. In some embodiments, an antibody described herein, or an antigen-binding fragment thereof, comprises the amino acid sequence of SEQ ID NO: 54 and the amino acid sequence of SEQ ID NO: 52.
  • the CDRs of an antibody described herein are Chothia CDRs (see, e.g., Chothia and Lesk, 1987, J. Mol. Biol., 196:901-917; and U.S. Patent No. 7,709,226 and Martin, A., "Protein Sequence and Structure Analysis of Antibody Variable Domains," in Antibody Engineering, Kontermann and Diibel, eds., Chapter 31, pp. 422-439, Springer- Verlag, Berlin (2001)).
  • Chothia CDRs and like terms are recognized in the art and refer to antibody CDR sequences as determined according to the method of Chothia and Lesk, 1987, J. Mol.
  • an antibody or an antigen-binding fragment thereof which specifically binds to human MerTK comprises VL and VH CDR as set forth in Table 3.
  • an antibody or an antigen-binding fragment thereof which specifically binds to human MerTK, and comprises VL CDR1 (SEQ ID NO: 16), VL CDR2 (SEQ ID NO: 17), and VL CDR3 (SEQ ID NO: 18), and VH CDR1 (SEQ ID NO: 13), VH CDR2 (SEQ ID NO: 14), and VH CDR3 (SEQ ID NO: 15).
  • VL CDR1, VL CDR2, and VL CDR3 are as set forth in Table 3 (SEQ ID NOS: 16, 17, and 18, respectively).
  • VH CDR1, VH CDR2, and VH CDR3 are as set forth in Table 3 (SEQ ID NOS: 13, 14, and 15, respectively).
  • the VL CDR1 , VL CDR2, and VL CDR3 are as set forth in Table 3 (SEQ ID NOS: 16, 17, and 18, respectively), and the VH CDR1, VH CDR2, and VH CDR3 are as set forth in Table 3 (SEQ ID NOS: 13, 14, and 15, respectively).
  • the CDRs of an antibody described herein are AbM.
  • an antibody or an antigen-binding fragment thereof which specifically binds to human MerTK comprises VL and VH CDR as set forth in Table 4.
  • an antibody or an antigen-binding fragment thereof which specifically binds to human MerTK, and comprises VL CDRl (SEQ ID NO: 22), VL CDR2 (SEQ ID NO: 23), and VL CDR3 (SEQ ID NO: 24), and VH CDRl (SEQ ID NO: 19), VH CDR2 (SEQ ID NO: 20), and VH CDR3 (SEQ ID NO: 21).
  • VL CDRl, VL CDR2, and VL CDR3 are as set forth in Table 4 (SEQ ID NOS: 22, 23, and 24, respectively).
  • VH CDRl, VH CDR2, and VH CDR3 are as set forth in Table 4 (SEQ ID NOS: 19, 20, and 21, respectively).
  • VL CDRl, VL CDR2, and VLCDR3 are as set forth in Table 4 (SEQ ID NOS: 22, 23, and 24, respectively), and the VH CDRl, VH CDR2, and VH CDR3 are as set forth in Table 4 (SEQ ID NOS: 19, 20, and 21, respectively).
  • a MerTK-binding antibody, or antigen-binding fragment thereof, described herein comprises CDRs of antibody Ab2000, as determined by the IMGT (Immunogenetics) numbering system; see, e.g., Lefranc, M.-P., 1999, The Immunologist, 7: 132- 136 and Lefranc, M.-P. et al., 1999, Nucleic Acids Res., 27:209-212, both of which are incorporated herein by reference in their entirety (Table 5).
  • IMGT Immunogenetics
  • VL CDRl, VL CDR2, and VL CDR3 are as set forth in Table 5 (SEQ ID NOS: 44, 45, and 46, respectively).
  • VH CDRl, VH CDR2, and VH CDR3 are as set forth in Table 5 (SEQ ID NOS: 41, 42, and 43, respectively).
  • VL CDRl, VL CDR2, and VL CDR3 are as set forth in Table 5 (SEQ ID NOS: 44, 45, and 46, respectively), and the VH CDRl, VH CDR2, and VH CDR3 are as set forth in Table 5 (SEQ ID NOS: 41, 42, and 43, respectively).
  • a MerTK-binding antibody, or antigen-binding fragment thereof, described herein comprises CDRs of antibody Ab2000, as determined by the Contact numbering system; see, e.g., see MacCallum RM et al., 1996, J Mol Biol 5: 732-745, which is incorporated herein by reference in its entirety (Table 6).
  • VL CDRl, VL CDR2, and VL CDR3 are as set forth in Table 6 (SEQ ID NOS: 36, 37, and 38, respectively).
  • VH CDRl, VH CDR2, and VH CDR3 are as set forth in Table 6 (SEQ ID NOS: 33, 34, and 35, respectively).
  • VL CDRl, VL CDR2, and VLCDR3 are as set forth in Table 6 (SEQ ID NOS: 36, 37 ,and 38, respectively), and the VH CDRl, VH CDR2, and VH CDR3 are as set forth in Table 6 (SEQ ID NOS: 33, 34, and 35, respectively).
  • an antibody described herein may be described by its VL alone, or its VH alone, or by its 3 VL CDRs alone, or its 3 VH CDRs alone. See, for example, Rader et al., 1998, Proc. Natl. Acad. Sci. USA, 95: 8910-8915, which is incorporated herein by reference in its entirety, describing the humanization of the mouse anti-avP3 antibody by identifying a complementing light chain or heavy chain, respectively, from a human light chain or heavy chain library, resulting in humanized antibody variants having affinities as high or higher than the affinity of the original antibody.
  • affinity maturation techniques can be used to alter one or more CDR region(s), followed by screening of the resultant binding molecules for the desired change in binding.
  • Any affinity maturation techniques known in the art and/or described herein can be used. See also, Holland et al., 2013, J. Immun. Methods 394:55-61, which is incorporated herein by reference in its entirety, describing methods of producing libraries for directed evolution of proteins (e.g., affinity matured antibodies).
  • a CDR region altered e.g., by affinity maturation
  • can result in changes in binding affinity e.g., on-rate of binding and/or off-rate of binding.
  • methods known in the art and/or disclosed herein can be used to analyze binding affinity and
  • an antibody or an antigen-binding fragment thereof which specifically binds to human MerTK comprises VL and VH CDR as set forth in Table 7, which utilizes standard one letter amino acid abbreviations.
  • an antibody or an antigen-binding fragment thereof which specifically binds to human MerTK and comprises VL CDRl (SEQ ID NO: 30), VL CDR2 (SEQ ID NO: 7), and VL CDR3 (SEQ ID NO: 8), and VH CDRl (SEQ ID NO: 3), VH CDR2 (SEQ ID NO: 4), and VH CDR3 (SEQ ID NO: 27).
  • VL CDR1 (SEQ ID NO: 30)
  • VL CDR2 (SEQ ID NO: 7)
  • VL CDR3 (SEQ ID NO: 8)
  • VH CDR1 (SEQ ID NO: 3)
  • VH CDR2 (SEQ ID NO: 4)
  • VH CDR3 (SEQ ID NO: 27)
  • provided herein is an antibody or an antigen-binding fragment thereof developed using affinity maturation methods disclosed herein or known in the art.
  • an affinity matured antibody an antigen-binding fragment thereof which specifically binds to human MerTK and comprises a VL and/or a VH as set forth in Table 8, which utilizes standard one letter amino acid abbreviations.
  • an affinity matured antibody or an antigen-binding fragment thereof which specifically binds to human MerTK, and comprises VL (SEQ ID NO: 26) and VH (SEQ ID NO: 25).
  • an antibody that comprises the VL and VH amino acid sequences of Table 8 is referred to herein as "Ab2000-A7.”
  • such an antibody or antigen-binding fragment comprises a separate light chain comprising the VL amino acid sequence and a separate heavy chain comprising the VH amino acid sequence.
  • such an antibody or antigen-binding fragment comprises a single chain comprising the VL amino acid sequence and the VH amino acid sequence.
  • an antibody or an antigen-binding fragment described herein comprises the VL CDR1, VL CDR2, and VL CDR3 of Ab2000-A7.
  • the VL CDR1, VL CDR2, and VL CDR3 are as set forth in Table 7 (SEQ ID NOS: 30, 7, and 8, respectively).
  • the antibody or antigen-binding fragment further comprises a VH as set forth in Table 8.
  • the antibody or antigen-binding fragment further comprises a VH as set forth in Table 1.
  • an antibody or an antigen-binding fragment described herein, which specifically binds to human MerTK comprises a VH CDRl, VH CDR2 and/or VH CDR3 as set forth in Table 7.
  • the antibody or antigen-binding fragment further comprises a VL as set forth in Table 8.
  • the antibody or antigen-binding fragment further comprises a VL as set forth in Table 1.
  • an antibody described herein, or an antigen-binding fragment thereof comprises the VH CDRl, VH CDR2, and VH CDR3 of Ab2000-A7.
  • the VH CDRl, VH CDR2, and VH CDR3 are as set forth in Table 7.
  • the antibody or antigen-binding fragment further comprises a VL as set forth in Table 8.
  • an antibody described herein, or an antigen-binding fragment thereof, which specifically binds to human MerTK comprises a VH as set forth in Table 8.
  • the antibody or antigen-binding fragment further comprises VL CDRl, VL CDR2 and/or VL CDR3 as set forth in Table 7.
  • an antibody described herein, or an antigen-binding fragment thereof comprises the VL CDRl, VL CDR2, and VL CDR3 of Ab2000-A7, and the VH CDRl, VH CDR2, and VH CDR3 of Ab2000-A7.
  • the VL CDRl, VL CDR2, and VL CDR3 are as set forth in Table 7 (SEQ ID NOS: 30, 7, and 8, respectively), and the VH CDRl, VH CDR2, and VH CDR3 are as set forth in Table (SEQ ID NOS: 3, 4, and 27, respectively).
  • an antibody described herein, or an antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO: 57. In some embodiments, an antibody described herein, or an antigen-binding fragment thereof, comprises the amino acid sequence of SEQ ID NO: 55. In some embodiments, an antibody described herein, or an antigen-binding fragment thereof, comprises the amino acid sequence of SEQ ID NO: 56. In some embodiments, an antibody described herein, or an antigen-binding fragment thereof, comprises the amino acid sequence of SEQ ID NO: 57 and the amino acid sequence of SEQ ID NO: 55. In some embodiments, an antibody described herein, or an antigen-binding fragment thereof, comprises the amino acid sequence of SEQ ID NO: 57 and the amino acid sequence of SEQ ID NO: 56.
  • a MerTK-binding antibody, or antigen-binding fragment thereof, described herein comprises a VH of SEQ ID NO: 25 and a VL of SEQ ID NO: 2, wherein the MerTK-binding antibody, or antigen-binding fragment thereof is Ab2000-Fl (Table 9).
  • a MerTK-binding antibody, or antigen-binding fragment thereof, described herein comprises a VH of SEQ ID NO: 1 and a VL of SEQ ID NO: 26, wherein the MerTK-binding antibody, or antigen-binding fragment thereof is Ab2000-G6 (Table 10).
  • the CDRs of an antibody described herein are Chothia CDRs (see, e.g., Chothia and Lesk, 1987, J. Mol. Biol., 196:901-917; and U.S. Patent No. 7,709,226 and Martin, A., "Protein Sequence and Structure Analysis of Antibody Variable Domains," in Antibody Engineering, Kontermann and Diibel, eds., Chapter 31, pp. 422-439, Springer- Verlag, Berlin (2001)).
  • an antibody or an antigen-binding fragment thereof which specifically binds to human MerTK comprises VL and VH CDR as set forth in Table 11.
  • an antibody or an antigen- binding fragment thereof which specifically binds to human MerTK, and comprises VL CDRl (SEQ ID NO: 31), VL CDR2 (SEQ ID NO: 17), and VL CDR3 (SEQ ID NO: 18), and VH CDRl (SEQ ID NO: 13), VH CDR2 (SEQ ID NO: 14), and VH CDR3 (SEQ ID NO: 28).
  • VL CDRl, VL CDR2, and VL CDR3 are as set forth in Table 11 (SEQ ID NOS: 31, 17, and 18, respectively).
  • VH CDRl, VH CDR2, and VH CDR3 are as set forth in Table 11 (SEQ ID NOS: 13, 14, and 28, respectively).
  • VL CDRl , VL CDR2, and VL CDR3 are as set forth in Table 11 (SEQ ID NOS: 31, 17, and 18, respectively), and the VH CDRl, VH CDR2, and VH CDR3 are as set forth in Table 3 (SEQ ID NOS: 13, 14, and 28, respectively).
  • the CDRs of an affinity matured antibody described herein e.g., Ab2000-A7 are AbM.
  • an antibody or an antigen-binding fragment thereof which specifically binds to human MerTK comprises VL and VH CDR as set forth in Table 12.
  • an antibody or an antigen- binding fragment thereof which specifically binds to human MerTK comprises VL CDRl (SEQ ID NO: 32), VL CDR2 (SEQ ID NO: 23), and VL CDR3 (SEQ ID NO: 24), and VH CDRl (SEQ ID NO: 19), VH CDR2 (SEQ ID NO: 20), and VH CDR3 (SEQ ID NO: 29).
  • VH CDRl (SEQ ID NO: 19)
  • VH CDR2 (SEQ ID NO: 20)
  • VH CDR3 (SEQ ID NO: 29)
  • VL CDRl VL CDR2, and VL CDR3 are as set forth in Table 12 (SEQ ID NOS: 32, 23, and 24, respectively).
  • VH CDRl, VH CDR2, and VH CDR3 are as set forth in Table 12 (SEQ ID NOS: 19, 20, and 29, respectively).
  • VL CDRl, VL CDR2, and VLCDR3 are as set forth in Table 12 (SEQ ID NOS: 32, 23, and 24, respectively), and the VH CDRl, VH CDR2, and VH CDR3 are as set forth in Table 12 (SEQ ID NOS: 19, 20, and 29, respectively).
  • a MerTK-binding antibody, or antigen-binding fragment thereof, described herein comprises CDRs of antibody Ab2000-A7, as determined by the IMGT (Immunogenetics) numbering system; (see, e.g., Lefranc, M.-P., 1999, The Immunologist, 7: 132-136 and Lefranc, M.-P. et al., 1999, Nucleic Acids Res., 27:209-212), both
  • VL CDRl, VL CDR2, and VL CDR3 are as set forth in Table 13 (SEQ ID NOS: 48, 45, and 46, respectively).
  • VH CDRl, VH CDR2, and VH CDR3 are as set forth in Table 13 (SEQ ID NOS: 41, 42, and 47, respectively).
  • VL CDR1, VL CDR2, and VLCDR3 are as set forth in Table 13 (SEQ ID NOS: 48, 45, and 46, respectively)
  • VH CDR1, VH CDR2, and VH CDR3 are as set forth in Table 13 (SEQ ID NOS: 41, 42, and 47, respectively).
  • a MerTK-binding antibody, or antigen-binding fragment thereof, described herein comprises CDRs of antibody Ab2000-A7, as determined by the Contact numbering system; see, e.g., see MacCallum RM et al., 1996, J Mol Biol 5: 732-745, which is incorporated herein by reference in its entirety (Table 14).
  • VL CDR1 , VL CDR2, and VL CDR3 are as set forth in Table 14 (SEQ ID NOS: 40, 37, and 38, respectively).
  • VH CDR1, VH CDR2, and VH CDR3 are as set forth in Table 14 (SEQ ID NOS: 33, 34, and 39, respectively).
  • VL CDR1, VL CDR2, and VLCDR3 are as set forth in Table 14 (SEQ ID NOS: 44, 45, and 46, respectively), and the VH CDR1, VH CDR2, and VH CDR3 are as set forth in Table 14 (SEQ ID NOS: 41, 42, and 43, respectively).
  • the position of one or more CDRs along the VH (e.g., CDR1, CDR2, or CDR3) and/or VL (e.g., CDR1, CDR2, or CDR3) region of an antibody described herein may vary by one, two, three, four, five, or six amino acid positions so long as immunospecific binding to MerTK (e.g., human MerTK ECD) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%>, at least 70%>, at least 80%>, at least 90%>, at least 95%)).
  • MerTK e.g., human MerTK ECD
  • the position defining a CDR of any of Table 1, Table 3-7, or Table 11-14 may vary by shifting the N-terminal and/or C-terminal boundary of the CDR by one, two, three, four, five, or six amino acids, relative to the current CDR position, so long as immunospecific binding to MerTK (e.g., human MerTK) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%), at least 95%).
  • MerTK e.g., human MerTK
  • the length of one or more CDRs along the VH (e.g., CDR1, CDR2, or CDR3) and/or VL (e.g., CDR1, CDR2, or CDR3) region of an antibody described herein may vary (e.g., be shorter or longer) by one, two, three, four, five, or more amino acids, so long as immunospecific binding to MerTK (e.g., human MerTK ECD) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%).
  • MerTK e.g., human MerTK ECD
  • a VH and/or VL CDR1, CDR2, and/or CDR3 described herein may be one, two, three, four, five or more amino acids shorter than one or more of the CDRs described by SEQ ID NOS: 3-8, 13-24, and 27-48 , so long as immunospecific binding to MerTK (e.g., human MerTK ECD) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%).
  • MerTK e.g., human MerTK ECD
  • VH and/or VL CDR1, CDR2, and/or CDR3 described herein may be one, two, three, four, five or more amino acids longer than one or more of the CDRs described by SEQ ID NOS: 3-8, 13-24, 27-48, so long as immunospecific binding to MerTK (e.g., human MerTK ECD) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%).
  • MerTK e.g., human MerTK ECD
  • the amino terminus of a VH and/or VL CDR1, CDR2, and/or CDR3 described herein may be extended by one, two, three, four, five or more amino acids compared to one or more of the CDRs described by SEQ ID NOS: 3-8, 13-24, 27-48, so long as immunospecific binding to MerTK (e.g., human MerTK ECD) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%).
  • MerTK e.g., human MerTK ECD
  • carboxy terminus of a VH and/or VL CDR1 , CDR2, and/or CDR3 described herein may be extended by one, two, three, four, five or more amino acids compared to one or more of the CDRs described by SEQ ID NOS: 3-8, 13-24, 27-48, so long as
  • immunospecific binding to MerTK is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%).
  • the amino terminus of a VH and/or VL CDR1, CDR2, and/or CDR3 described herein may be shortened by one, two, three, four, five or more amino acids compared to one or more of the CDRs described by SEQ ID NOS: 3-8, 13-24, 27-48, so long as immunospecific binding to MerTK (e.g., human MerTK) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%), at least 95%).
  • the carboxy terminus of a VH and/or VL CDR1, CDR2, and/or CDR3 described herein may be shortened by one, two, three, four, five or more amino acids compared to one or more of the CDRs described by SEQ ID NOS: 3-8, 13-24, 27- 48, so long as immunospecific binding to MerTK (e.g., human MerTK ECD) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%), at least 95%).
  • MerTK e.g., human MerTK ECD
  • any method known in the art can be used to ascertain whether immunospecific binding to MerTK (e.g., human MerTK ECD) is maintained, for example, the binding assays and conditions described in the "Examples" section (Section 6) provided herein.
  • section 6.2 provided herein describes an assay for measuring binding to an ECD of human MerTK.
  • an antibody comprising an antibody light chain and heavy chain, e.g., a separate light chain and heavy chain.
  • the light chain of an antibody described herein is a kappa light chain.
  • the light chain of an antibody described herein is a lambda light chain.
  • the light chain of an antibody described herein is a human kappa light chain or a human lambda light chain.
  • an antibody described herein, which specifically binds to a MerTK polypeptide comprises a light chain wherein the amino acid sequence of the VL chain region comprises any amino acid sequence described herein (e.g., SEQ ID NOS: 2 or 26), and wherein the constant region of the light chain comprises the amino acid sequence of a human kappa light chain constant region.
  • an antibody described herein, which specifically binds a MerTK polypeptide comprises a light chain wherein the amino acid sequence of the VL chain region can comprise any amino acid sequence described herein (e.g., SEQ ID NOS: 2 or 26), and wherein the constant region of the light chain comprises the amino acid sequence of a human lambda light chain constant region.
  • a MerTK polypeptide e.g., human MerTK
  • the amino acid sequence of the VL chain region can comprise any amino acid sequence described herein (e.g., SEQ ID NOS: 2 or 26)
  • the constant region of the light chain comprises the amino acid sequence of a human lambda light chain constant region.
  • Non-limiting examples of human constant region sequences have been described in the art, e.g., see U.S. Patent No. 5,693,780 and Kabat et al. (1991) Sequences of Proteins of Immunological Interest Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242.
  • the heavy chain of an antibody described herein can be an alpha (a), delta ( ⁇ ), epsilon ( ⁇ ), gamma ( ⁇ ) or mu ( ⁇ ) heavy chain.
  • the heavy chain of an antibody described can comprise a human alpha (a), delta ( ⁇ ), epsilon ( ⁇ ), gamma ( ⁇ ) or mu ( ⁇ ) heavy chain.
  • an antibody described herein which specifically binds to a MerTK polypeptide (e.g., human MerTK), comprises a heavy chain wherein the amino acid sequence of the VH chain region can comprise any amino acid sequence described herein (e.g., any of SEQ ID NOS: 1 or 25), and wherein the constant region of the heavy chain comprises the amino acid sequence of a human gamma ( ⁇ ) heavy chain constant region.
  • a MerTK polypeptide e.g., human MerTK
  • the amino acid sequence of the VH chain region can comprise any amino acid sequence described herein (e.g., any of SEQ ID NOS: 1 or 25)
  • the constant region of the heavy chain comprises the amino acid sequence of a human gamma ( ⁇ ) heavy chain constant region.
  • Non-limiting examples of human constant region sequences have been described in the art, e.g., see U.S. Patent No. 5,693,780 and Kabat et al. (1991) Sequences of Proteins of Immunological Interest Fifth Edition, U.
  • an antibody described herein which specifically binds to a MerTK polypeptide (e.g., human MerTK), comprises a light chain constant region comprising an amino acid sequence of SEQ ID NO: 49, wherein the amino acid sequence for SEQ ID NO: 49 is RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ D SKD S T YSL S S TLTL SK AD YEKHK V Y ACE VTHQ GL S SP VTK SFNRG.
  • an antibody described herein which specifically binds to a MerTK polypeptide (e.g., human MerTK), comprises a heavy chain constant region comprising an amino acid sequence of SEQ ID NO: 50, wherein the amino acid sequence for SEQ ID NO: 50 is
  • an antibody described herein which specifically binds to a MerTK polypeptide (e.g., human MerTK), comprises a light chain constant region comprising an amino acid sequence of SEQ ID NO: 49 and a heavy chain constant region comprising an amino acid sequence of SEQ ID NO: 50.
  • an antibody described herein which specifically binds to a MerTK polypeptide (e.g., human MerTK), comprises heavy chain constant region comprising an amino acid sequence of SEQ ID NO: 51, wherein the amino acid sequence for SEQ ID NO: 51 is ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GL YSL S S V VTVP S S FGTQT YTCNVDHKP SNTKVDKT VERKCC VECPPCP APP VAGP S V FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNST FRVVSVLTVVHQDWLNGKEYKCKVS KGLPAPIEKTISKTKGQPREPQVYTLPPSREEM TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRW Q
  • an antibody described herein which specifically binds to a MerTK polypeptide (e.g., human MerTK), comprises a light chain constant region comprising an amino acid sequence of SEQ ID NO: 49 and a heavy chain constant region comprising an amino acid sequence of SEQ ID NO: 51.
  • an antibody described herein which specifically binds to a
  • MerTK polypeptide e.g., human MerTK
  • an antibody described herein which specifically binds to a
  • MerTK polypeptide e.g., human MerTK
  • an antibody described herein which specifically binds to a MerTK polypeptide (e.g., human MerTK), comprises a light chain comprising an amino acid sequence of SEQ ID NO: 54, wherein the amino acid sequence for SEQ ID NO: 54 is
  • an antibody described herein, which specifically binds to a MerTK polypeptide comprises a light chain comprising an amino acid sequence of SEQ ID NO: 54 and a heavy chain comprising an amino acid sequence of SEQ ID NO: 52.
  • an antibody described herein, which specifically binds to a MerTK polypeptide comprises a light chain comprising an amino acid sequence of SEQ ID NO: 54 and a heavy chain comprising an amino acid sequence of SEQ ID NO: 53.
  • an antibody described herein which specifically binds to a
  • MerTK polypeptide e.g., human MerTK
  • an antibody described herein which specifically binds to a MerTK polypeptide (e.g., human MerTK), comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 56, wherein the amino acid sequence for SEQ ID NO: 56 is
  • an antibody described herein which specifically binds to a MerTK polypeptide (e.g., human MerTK), comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 57, wherein the amino acid sequence for SEQ ID NO: 57 is
  • an antibody described herein, which specifically binds to a MerTK polypeptide comprises a light chain comprising an amino acid sequence of SEQ ID NO: 54 and a heavy chain comprising an amino acid sequence of SEQ ID NO: 52.
  • an antibody described herein, which specifically binds to a MerTK polypeptide comprises a light chain comprising an amino acid sequence of SEQ ID NO: 54 and a heavy chain comprising an amino acid sequence of SEQ ID NO: 53.
  • an antibody described herein, which specifically binds to a MerTK polypeptide comprises a light chain comprising an amino acid sequence of SEQ ID NO: 54 and a heavy chain comprising an amino acid sequence of SEQ ID NO: 55.
  • an antibody described herein, which specifically binds to a MerTK polypeptide comprises a light chain comprising an amino acid sequence of SEQ ID NO: 54 and a heavy chain comprising an amino acid sequence of SEQ ID NO: 56.
  • an antibody described herein, which specifically binds to a MerTK polypeptide comprises a light chain comprising an amino acid sequence of SEQ ID NO: 57 and a heavy chain comprising an amino acid sequence of SEQ ID NO: 52.
  • an antibody described herein, which specifically binds to a MerTK polypeptide comprises a light chain comprising an amino acid sequence of SEQ ID NO: 57 and a heavy chain comprising an amino acid sequence of SEQ ID NO: 53.
  • an antibody described herein, which specifically binds to a MerTK polypeptide comprises a light chain comprising an amino acid sequence of SEQ ID NO: 57 and a heavy chain comprising an amino acid sequence of SEQ ID NO: 55.
  • an antibody described herein, which specifically binds to a MerTK polypeptide comprises a light chain comprising an amino acid sequence of SEQ ID NO: 57 and a heavy chain comprising an amino acid sequence of SEQ ID NO: 56.
  • an antibody described herein, which specifically binds to a MerTK polypeptide comprises a VL chain region and a VH chain region comprising any amino acid sequences described herein, and wherein the constant regions comprise the amino acid sequences of the constant regions of an IgG, IgE, IgM, IgD, IgA or IgY immunoglobulin molecule, or a human IgG, IgE, IgM, IgD, IgA or IgY immunoglobulin molecule.
  • an antibody described herein which specifically binds to a MerTK polypeptide (e.g., human MerTK) comprises a VL chain region and a VH chain region comprising any amino acid sequences described herein, and wherein the constant regions comprise the amino acid sequences of the constant regions of an IgG, IgE, IgM, IgD, IgA or IgY immunoglobulin molecule, any class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2), or any subclass (e.g., IgG2a and IgG2b) of immunoglobulin molecule.
  • a MerTK polypeptide e.g., human MerTK
  • the constant regions comprise the amino acid sequences of the constant regions of an IgG, IgE, IgM, IgD, IgA or IgY immunoglobulin molecule, any class (e.g., IgGl, IgG2, IgG3, Ig
  • the constant regions comprise the amino acid sequences of the constant regions of a human IgG, IgE, IgM, IgD, IgA or IgY immunoglobulin molecule, any class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2), or any subclass (e.g., IgG2a and IgG2b) of immunoglobulin molecule.
  • any class e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2
  • subclass e.g., IgG2a and IgG2b
  • an antibody described herein which specifically binds to a MerTK polypeptide (e.g., human MerTK), comprises a VL chain region and a VH chain region comprising any amino acid sequences described herein, and wherein the constant regions comprise the amino acid sequences of the constant regions of a human IgGl (e.g., isotype a, z, or f), human IgG2, or human IgG4.
  • a human IgGl e.g., isotype a, z, or f
  • human IgG2 e.g., isotype a, z, or f
  • Non-limiting examples of human constant regions are described in the art, e.g., see Kabat et al. (1991) Sequences of Proteins of
  • an antibody described herein which specifically binds to a MerTK polypeptide (e.g., human MerTK), comprises a light chain and a heavy chain, wherein (i) the light chain comprises a VL chain region comprising a VL CDRl, VL CDR2, and VL CDR3 of Ab2000; (ii) the heavy chain comprises a VH chain region comprising a VH CDRl, VH CDR2, and VH CDR3 of Ab2000; (iii) the light chain further comprises a constant light chain domain comprising the amino acid sequence of the constant domain of a human kappa or lambda light chain; and (iv) the heavy chain further comprises a constant heavy chain domain comprising the amino acid sequence of the constant domain of a human IgG, e.g., IgGl, heavy chain.
  • a human IgG e.g., IgGl, heavy chain.
  • an antibody described herein which specifically binds to a MerTK polypeptide (e.g., human MerTK), comprises a light chain and a heavy chain, wherein (i) the light chain comprises a VL chain region comprising a VL CDRl, VL CDR2, and VL CDR3 of Ab2000-A7; (ii) the heavy chain comprises a VH chain region comprising a VH CDRl, VH CDR2, and VH CDR3 of Ab2000-A7; (iii) the light chain further comprises a constant light chain domain comprising the amino acid sequence of the constant domain of a human kappa or lambda light chain; and (iv) the heavy chain further comprises a constant heavy chain domain comprising the amino acid sequence of the constant domain of a human IgG, e.g., IgGl, heavy chain.
  • a human IgG e.g., IgGl
  • an antibody described herein which specifically binds to a MerTK polypeptide (e.g., human MerTK), comprises a light chain and a heavy chain, wherein (i) the light chain comprises a VL chain region comprising a VL CDRl, VL CDR2, and VL CDR3 of Ab2000; (ii) the heavy chain comprises a VH chain region comprising a VH CDRl, VH CDR2, and VH CDR3 of Ab2000-A7; (iii) the light chain further comprises a constant light chain domain comprising the amino acid sequence of the constant domain of a human kappa or lambda light chain; and (iv) the heavy chain further comprises a constant heavy chain domain comprising the amino acid sequence of the constant domain of a human IgG, e.g., IgGl, heavy chain.
  • the light chain comprises a VL chain region comprising a VL CDRl, VL CDR2, and VL CDR3 of Ab2000
  • the heavy chain comprises a
  • an antibody described herein which specifically binds to a MerTK polypeptide (e.g., human MerTK), comprises a light chain and a heavy chain, wherein (i) the light chain comprises a VL chain region comprising a VL CDRl, VL CDR2, and VL CDR3 of Ab2000-A7; (ii) the heavy chain comprises a VH chain region comprising a VH CDRl, VH CDR2, and VH CDR3 of Ab2000; (iii) the light chain further comprises a constant light chain domain comprising the amino acid sequence of the constant domain of a human kappa or lambda light chain; and (iv) the heavy chain further comprises a constant heavy chain domain
  • IgG e.g., IgGl
  • heavy chain comprising the amino acid sequence of the constant domain of a human IgG, e.g., IgGl, heavy chain.
  • an antibody described herein which specifically binds to a MerTK polypeptide (e.g., human MerTK), comprises a light chain and a heavy chain, wherein (i) the light chain comprises a VL chain region comprising a VL CDRl, VL CDR2, and VL CDR3 of Table 1; (ii) the heavy chain comprises a VH chain region comprising a VH CDRl, VH CDR2, and VH CDR3 of Table 1; (iii) the light chain further comprises a constant light chain domain comprising the amino acid sequence of the constant domain of a human kappa or lambda light chain; and (iv) the heavy chain further comprises a constant heavy chain domain comprising the amino acid sequence of the constant domain of a human IgG, e.g., IgGl, heavy chain.
  • an antibody described herein which specifically binds to a MerTK polypeptide (e.g., human MerTK), comprises a light chain and a heavy chain, wherein (i) the light chain comprises a VL chain region comprising a VL CDRl, VL CDR2, and VL CDR3 of Table 7; (ii) the heavy chain comprises a VH chain region comprising a VH CDRl, VH CDR2, and VH CDR3 of Table 7; (iii) the light chain further comprises a constant light chain domain comprising the amino acid sequence of the constant domain of a human kappa or lambda light chain; and (iv) the heavy chain further comprises a constant heavy chain domain comprising the amino acid sequence of the constant domain of a human IgG, e.g., IgGl, heavy chain.
  • a human IgG e.g., IgGl
  • an antibody described herein which specifically binds to a MerTK polypeptide (e.g., human MerTK), comprises a light chain and a heavy chain, wherein (i) the light chain comprises a VL chain region comprising a VL CDRl, VL CDR2, and VL CDR3 of Table 1; (ii) the heavy chain comprises a VH chain region comprising a VH CDRl, VH CDR2, and VH CDR3 of Table 7; (iii) the light chain further comprises a constant light chain domain comprising the amino acid sequence of the constant domain of a human kappa or lambda light chain; and (iv) the heavy chain further comprises a constant heavy chain domain comprising the amino acid sequence of the constant domain of a human IgG, e.g., IgGl, heavy chain.
  • an antibody described herein which specifically binds to a MerTK polypeptide (e.g., human MerTK), comprises a light chain and a heavy chain, wherein (i) the light chain comprises a VL chain region comprising a VL CDRl, VL CDR2, and VL CDR3 of Table 7; (ii) the heavy chain comprises a VH chain region comprising a VH CDRl, VH CDR2, and VH CDR3 of Table 1; (iii) the light chain further comprises a constant light chain domain comprising the amino acid sequence of the constant domain of a human kappa or lambda light chain; and (iv) the heavy chain further comprises a constant heavy chain domain comprising the amino acid sequence of the constant domain of a human IgG, e.g., IgGl, heavy chain.
  • a human IgG e.g., IgGl
  • an antibody described herein which specifically binds to a MerTK polypeptide (e.g., human MerTK), comprises a light chain and a heavy chain, wherein (i) the light chain comprises a VL chain region comprising a VL CDRl, VL CDR2, and VL CDR3 of Table 3; (ii) the heavy chain comprises a VH chain region comprising a VH CDRl, VH CDR2, and VH CDR3 of Table 3; (iii) the light chain further comprises a constant light chain domain comprising the amino acid sequence of the constant domain of a human kappa or lambda light chain; and (iv) the heavy chain further comprises a constant heavy chain domain comprising the amino acid sequence of the constant domain of a human IgG, e.g., IgGl, heavy chain.
  • an antibody described herein which specifically binds to a MerTK polypeptide (e.g., human MerTK), comprises a light chain and a heavy chain, wherein (i) the light chain comprises a VL chain region comprising a VL CDRl, VL CDR2, and VL CDR3 of Table 11; (ii) the heavy chain comprises a VH chain region comprising a VH CDRl, VH CDR2, and VH CDR3 of Table 11; (iii) the light chain further comprises a constant light chain domain comprising the amino acid sequence of the constant domain of a human kappa or lambda light chain; and (iv) the heavy chain further comprises a constant heavy chain domain comprising the amino acid sequence of the constant domain of a human IgG, e.g., IgGl, heavy chain.
  • an antibody described herein which specifically binds to a MerTK polypeptide (e.g., human MerTK), comprises a light chain and a heavy chain, wherein (i) the light chain comprises a VL chain region comprising a VL CDRl, VL CDR2, and VL CDR3 of Table 3; (ii) the heavy chain comprises a VH chain region comprising a VH CDRl, VH CDR2, and VH CDR3 of Table 11; (iii) the light chain further comprises a constant light chain domain comprising the amino acid sequence of the constant domain of a human kappa or lambda light chain; and (iv) the heavy chain further comprises a constant heavy chain domain comprising the amino acid sequence of the constant domain of a human IgG, e.g., IgGl, heavy chain.
  • an antibody described herein which specifically binds to a MerTK polypeptide (e.g., human MerTK), comprises a light chain and a heavy chain, wherein (i) the light chain comprises a VL chain region comprising a VL CDRl, VL CDR2, and VL CDR3 of Table 11; (ii) the heavy chain comprises a VH chain region comprising a VH CDRl, VH CDR2, and VH CDR3 of Table 3; (iii) the light chain further comprises a constant light chain domain comprising the amino acid sequence of the constant domain of a human kappa or lambda light chain; and (iv) the heavy chain further comprises a constant heavy chain domain comprising the amino acid sequence of the constant domain of a human IgG, e.g., IgGl, heavy chain.
  • an antibody described herein which specifically binds to a MerTK polypeptide (e.g., human MerTK), comprises a light chain and a heavy chain, wherein (i) the light chain comprises a VL chain region comprising a VL CDRl, VL CDR2, and VL CDR3 of Table 4; (ii) the heavy chain comprises a VH chain region comprising a VH CDRl, VH CDR2, and VH CDR3 of Table 4; (iii) the light chain further comprises a constant light chain domain comprising the amino acid sequence of the constant domain of a human kappa or lambda light chain; and (iv) the heavy chain further comprises a constant heavy chain domain comprising the amino acid sequence of the constant domain of a human IgG, e.g., IgGl, heavy chain.
  • a human IgG e.g., IgGl
  • an antibody described herein which specifically binds to a MerTK polypeptide (e.g., human MerTK), comprises a light chain and a heavy chain, wherein (i) the light chain comprises a VL chain region comprising a VL CDRl, VL CDR2, and VL CDR3 of Table 12; (ii) the heavy chain comprises a VH chain region comprising a VH CDRl, VH CDR2, and VH CDR3 of Table 12; (iii) the light chain further comprises a constant light chain domain comprising the amino acid sequence of the constant domain of a human kappa or lambda light chain; and (iv) the heavy chain further comprises a constant heavy chain domain comprising the amino acid sequence of the constant domain of a human IgG, e.g., IgGl, heavy chain.
  • a human IgG e.g., IgGl
  • an antibody described herein which specifically binds to a MerTK polypeptide (e.g., human MerTK), comprises a light chain and a heavy chain, wherein (i) the light chain comprises a VL chain region comprising a VL CDRl, VL CDR2, and VL CDR3 of Table 4; (ii) the heavy chain comprises a VH chain region comprising a VH CDRl, VH CDR2, and VH CDR3 of Table 12; (iii) the light chain further comprises a constant light chain domain comprising the amino acid sequence of the constant domain of a human kappa or lambda light chain; and (iv) the heavy chain further comprises a constant heavy chain domain comprising the amino acid sequence of the constant domain of a human IgG, e.g., IgGl, heavy chain.
  • an antibody described herein which specifically binds to a MerTK polypeptide (e.g., human MerTK), comprises a light chain and a heavy chain, wherein (i) the light chain comprises a VL chain region comprising a VL CDRl, VL CDR2, and VL CDR3 of Table 12; (ii) the heavy chain comprises a VH chain region comprising a VH CDRl, VH CDR2, and VH CDR3 of Table 4; (iii) the light chain further comprises a constant light chain domain comprising the amino acid sequence of the constant domain of a human kappa or lambda light chain; and (iv) the heavy chain further comprises a constant heavy chain domain comprising the amino acid sequence of the constant domain of a human IgG, e.g., IgGl, heavy chain.
  • an antibody described herein which specifically binds to a MerTK polypeptide (e.g., human MerTK), comprises a light chain and a heavy chain, wherein (i) the light chain comprises a VL chain region comprising a VL CDRl, VL CDR2, and VL CDR3 of Table 5; (ii) the heavy chain comprises a VH chain region comprising a VH CDRl, VH CDR2, and VH CDR3 of Table 5; (iii) the light chain further comprises a constant light chain domain comprising the amino acid sequence of the constant domain of a human kappa or lambda light chain; and (iv) the heavy chain further comprises a constant heavy chain domain comprising the amino acid sequence of the constant domain of a human IgG, e.g., IgGl, heavy chain.
  • an antibody described herein which specifically binds to a MerTK polypeptide (e.g., human MerTK), comprises a light chain and a heavy chain, wherein (i) the light chain comprises a VL chain region comprising a VL CDRl, VL CDR2, and VL CDR3 of Table 13; (ii) the heavy chain comprises a VH chain region comprising a VH CDRl, VH CDR2, and VH CDR3 of Table 13; (iii) the light chain further comprises a constant light chain domain comprising the amino acid sequence of the constant domain of a human kappa or lambda light chain; and (iv) the heavy chain further comprises a constant heavy chain domain comprising the amino acid sequence of the constant domain of a human IgG, e.g., IgGl, heavy chain.
  • a human IgG e.g., IgGl
  • an antibody described herein which specifically binds to a MerTK polypeptide (e.g., human MerTK), comprises a light chain and a heavy chain, wherein (i) the light chain comprises a VL chain region comprising a VL CDRl, VL CDR2, and VL CDR3 of Table 5; (ii) the heavy chain comprises a VH chain region comprising a VH CDRl, VH CDR2, and VH CDR3 of Table 13; (iii) the light chain further comprises a constant light chain domain comprising the amino acid sequence of the constant domain of a human kappa or lambda light chain; and (iv) the heavy chain further comprises a constant heavy chain domain comprising the amino acid sequence of the constant domain of a human IgG, e.g., IgGl, heavy chain.
  • an antibody described herein which specifically binds to a MerTK polypeptide (e.g., human MerTK), comprises a light chain and a heavy chain, wherein (i) the light chain comprises a VL chain region comprising a VL CDRl, VL CDR2, and VL CDR3 of Table 13; (ii) the heavy chain comprises a VH chain region comprising a VH CDRl, VH CDR2, and VH CDR3 of Table 5; (iii) the light chain further comprises a constant light chain domain comprising the amino acid sequence of the constant domain of a human kappa or lambda light chain; and (iv) the heavy chain further comprises a constant heavy chain domain comprising the amino acid sequence of the constant domain of a human IgG, e.g., IgGl, heavy chain.
  • an antibody described herein which specifically binds to a MerTK polypeptide (e.g., human MerTK), comprises a light chain and a heavy chain, wherein (i) the light chain comprises a VL chain region comprising a VL CDRl, VL CDR2, and VL CDR3 of Table 6; (ii) the heavy chain comprises a VH chain region comprising a VH CDRl, VH CDR2, and VH CDR3 of Table 6; (iii) the light chain further comprises a constant light chain domain comprising the amino acid sequence of the constant domain of a human kappa or lambda light chain; and (iv) the heavy chain further comprises a constant heavy chain domain comprising the amino acid sequence of the constant domain of a human IgG, e.g., IgGl, heavy chain.
  • an antibody described herein which specifically binds to a MerTK polypeptide (e.g., human MerTK), comprises a light chain and a heavy chain, wherein (i) the light chain comprises a VL chain region comprising a VL CDRl, VL CDR2, and VL CDR3 of Table 14; (ii) the heavy chain comprises a VH chain region comprising a VH CDRl, VH CDR2, and VH CDR3 of Table 14; (iii) the light chain further comprises a constant light chain domain comprising the amino acid sequence of the constant domain of a human kappa or lambda light chain; and (iv) the heavy chain further comprises a constant heavy chain domain comprising the amino acid sequence of the constant domain of a human IgG, e.g., IgGl, heavy chain.
  • an antibody described herein which specifically binds to a MerTK polypeptide (e.g., human MerTK), comprises a light chain and a heavy chain, wherein (i) the light chain comprises a VL chain region comprising a VL CDRl, VL CDR2, and VL CDR3 of Table 6; (ii) the heavy chain comprises a VH chain region comprising a VH CDRl, VH CDR2, and VH CDR3 of Table 14; (iii) the light chain further comprises a constant light chain domain comprising the amino acid sequence of the constant domain of a human kappa or lambda light chain; and (iv) the heavy chain further comprises a constant heavy chain domain comprising the amino acid sequence of the constant domain of a human IgG, e.g., IgGl, heavy chain.
  • an antibody described herein which specifically binds to a MerTK polypeptide (e.g., human MerTK), comprises a light chain and a heavy chain, wherein (i) the light chain comprises a VL chain region comprising a VL CDRl, VL CDR2, and VL CDR3 of Table 14; (ii) the heavy chain comprises a VH chain region comprising a VH CDRl, VH CDR2, and VH CDR3 of Table 6; (iii) the light chain further comprises a constant light chain domain comprising the amino acid sequence of the constant domain of a human kappa or lambda light chain; and (iv) the heavy chain further comprises a constant heavy chain domain comprising the amino acid sequence of the constant domain of a human IgG, e.g., IgGl, heavy chain.
  • an antibody described herein which specifically binds to a MerTK polypeptide (e.g., human MerTK), comprises a light chain and a heavy chain, wherein (i) the light chain comprises a VL chain region comprising the amino acid sequence of SEQ ID NO: 2; (ii) the heavy chain comprises a VH chain region comprising the amino acid sequence of SEQ ID NO: 1; (iii) the light chain further comprises a constant domain comprising the amino acid sequence of the constant domain of a human kappa or lambda light chain; and (iv) the heavy chain further comprises a constant domain comprising the amino acid sequence of the constant domain of a human IgGl, e.g., IgGl, heavy chain.
  • a human IgGl e.g., IgGl
  • an antibody described herein which specifically binds to a MerTK polypeptide (e.g., human MerTK), comprises a light chain and a heavy chain, wherein (i) the light chain comprises a VL chain region comprising the amino acid sequence of SEQ ID NO: 26; (ii) the heavy chain comprises a VH chain region comprising the amino acid sequence of SEQ ID NO: 25; (iii) the light chain further comprises a constant domain comprising the amino acid sequence of the constant domain of a human kappa or lambda light chain; and (iv) the heavy chain further comprises a constant domain comprising the amino acid sequence of the constant domain of a human IgGl, e.g., IgGl, heavy chain.
  • a human IgGl e.g., IgGl
  • an antibody described herein which specifically binds to a MerTK polypeptide (e.g., human MerTK), comprises a light chain and a heavy chain, wherein (i) the light chain comprises a VL chain region comprising the amino acid sequence of SEQ ID NO: 2; (ii) the heavy chain comprises a VH chain region comprising the amino acid sequence of SEQ ID NO: 25; (iii) the light chain further comprises a constant domain comprising the amino acid sequence of the constant domain of a human kappa or lambda light chain; and (iv) the heavy chain further comprises a constant domain comprising the amino acid sequence of the constant domain of a human IgGl, e.g., IgGl, heavy chain.
  • a human IgGl e.g., IgGl
  • an antibody described herein which specifically binds to a MerTK polypeptide (e.g., human MerTK), comprises a light chain and a heavy chain, wherein (i) the light chain comprises a VL chain region comprising the amino acid sequence of SEQ ID NO: 26; (ii) the heavy chain comprises a VH chain region comprising the amino acid sequence of SEQ ID NO: 1; (iii) the light chain further comprises a constant domain comprising the amino acid sequence of the constant domain of a human kappa or lambda light chain; and (iv) the heavy chain further comprises a constant domain comprising the amino acid sequence of the constant domain of a human IgGl, e.g., IgGl, heavy chain.
  • a human IgGl e.g., IgGl
  • one or more modifications can be made to the Fc region of an antibody or antigen-binding fragment thereof described herein.
  • an antibody or antigen-binding fragment thereof described herein.
  • modification or modifications can be introduced to alter one or more functional properities of the antibody or antigen-binding fragment, such as serum half-life, complement fixation, Fc receptor binding, and/or antibody-dependent cellular cytotoxicity.
  • Examplary modfications are described, for example, in International Patent Application Publication No. WO 2008/153926 A2.
  • an antibody described herein which specifically binds to MerTK, e.g., human MerTK ECD, comprises framework regions (e.g., framework regions of the VL domain and/or VH domain) that are human framework regions or derived from human framework regions.
  • framework regions e.g., framework regions of the VL domain and/or VH domain
  • Non-limiting examples of human framework regions are described in the art, e.g., see Kabat et al. (1991) Sequences of Proteins of Immunological Interest Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242).
  • an antibody described herein comprises framework regions (e.g., framework regions of the VL domain and/or VH domain) that are primate (e.g., non-human primate) framework regions or derived from primate (e.g., non-human primate) framework regions.
  • primate e.g., non-human primate
  • non-human primate e.g., non-human primate
  • an antibody described herein comprises a VL domain having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 2, wherein the antibody specifically binds to MerTK, e.g., human MerTK ECD.
  • an antibody described herein comprises the amino acid sequence of SEQ ID NO: 26 wherein the antibody specifically binds to MerTK, e.g., human MerTK ECD.
  • an antibody described herein comprises a VL domain having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 2, wherein the antibody specifically binds to MerTK, e.g., human MerTK ECD, and wherein the antibody comprises VL CDRs that are identical to the VL CDRs of antibody Ab2000.
  • MerTK e.g., human MerTK ECD
  • an antibody described herein comprises a VL domain having the amino acid sequence of SEQ ID NO: 26, wherein the antibody specifically binds to MerTK, e.g., human MerTK ECD, and wherein the antibody comprises VL CDRs that are identical to the VL CDRs of antibody Ab2000-A7.
  • an antibody described herein comprises a VL domain having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 2, wherein the antibody specifically binds to MerTK, e.g., human MerTK ECD, and wherein the antibody comprises VL CDRs that are identical to the VL CDRs of Table 1.
  • an antibody described herein comprises a VL domain having the amino acid sequence of SEQ ID NO: 26, wherein the antibody specifically binds to MerTK, e.g., human MerTK ECD, and wherein the antibody comprises VL CDRs that are identical to the VL CDRs of Table 7.
  • an antibody described herein comprises a VH domain having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 1, wherein the antibody specifically binds to MerTK, e.g., human MerTK ECD.
  • an antibody described herein comprises a VH domain having the amino acid sequence of SEQ ID NO: 25, wherein the antibody specifically binds to MerTK, e.g., human MerTK ECD.
  • an antibody described herein comprises a VH domain having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 1, wherein the antibody specifically binds to MerTK, e.g., human MerTK ECD, and wherein the antibody comprises VH CDRs that are identical to the VH CDRs of antibody Ab2000.
  • MerTK e.g., human MerTK ECD
  • an antibody described herein comprises a VH domain having the amino acid sequence of SEQ ID NO: 25, wherein the antibody specifically binds to MerTK, e.g., human MerTK ECD, and wherein the antibody comprises VH CDRs that are identical to the VH CDRs of antibody Ab2000-A7.
  • an antibody described herein comprises a VH domain having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 1, wherein the antibody specifically binds to MerTK, e.g., human MerTK ECD, and wherein the antibody comprises VH CDRs that are identical to the VH CDRs of Table 1.
  • an antibody described herein comprises a VH domain having the amino acid sequence of SEQ ID NO: 25, wherein the antibody specifically binds to MerTK, e.g., human MerTK ECD, and wherein the antibody comprises VH CDRs that are identical to the VH CDRs of Table 7.
  • the antibodies and antigen binding fragments thereof presented herein that specifically bind to human MerTK comprise conservative sequence modifications as described herein.
  • conservative sequence modifications include conservative amino acid substitutions that include ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art.
  • amino acids with basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine
  • beta- branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
  • a predicted nonessential amino acid residue in a human anti-MerTK antibody is replaced with another amino acid residue from the same side chain family.
  • Methods of identifying nucleotide and amino acid conservative substitutions which do not eliminate antigen binding are well-known in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10):879-884 (1999); and Burks et al. Proc. Natl. Acad. Sci. USA 94:412-417 (1997)).
  • the conservative sequence modifications described herein modify the amino acid sequences of the MerTK antibodies or antigen binding fragments thereof by 50%, or 55%, or 60%, or 65%, or 70%, or 75%, or 80%, or 85%, or 90%, or 95%, or 98%, or 99%.
  • the nucleotide and amino acid sequence modifications refer to at most 1, 2, 3, 4, 5, or 6 amino acid substitutions to the CDRs described in any of Table 1, or Table 3-7, or 11-14.
  • each such CDR may contain up to 5 conservative amino acid substitutions, for example up to (not more than) 4 conservative amino acid substitutions, for example up to (not more than) 3 conservative amino acid substitutions, for example up to (not more than) 2 conservative amino acid substitutions, or no more than 1 conservative amino acid substitution.
  • an antibody or antigen-binding fragment thereof that specifically binds to MerTK comprises: i) a VL domain having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 2, ii) a VL domain having at least 80%, at least 85%), at least 90%, at least 95%, or at least 98%> sequence identity to the amino acid sequence of SEQ ID NO: 2, and wherein the antibody comprises VL CDRs that are identical to the VL CDRs of antibody Ab2000, or iii) a VL domain having at least 80%>, at least 85%>, at least 90%), at least 95%, or at least 98%> sequence identity to the amino acid sequence of SEQ ID NO: 2, and wherein the antibody comprises VL CDRs that are identical to the VL CDRs of Table 1; and iv) a VH domain
  • an antibody that specifically binds to MerTK comprises: i) a VL domain having at least 80%, at least 85%, at least 90%), at least 95%, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 2 (e.g., a VL having the amino acid sequence of SEQ ID NO: 26), ii) a VL domain having at least 80%), at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 2, and wherein the antibody comprises VL CDRs that are identical to the VL CDRs of antibody Ab2000-A7, or iii) a VL domain having at least 80%, at least 85%), at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 2, and wherein the antibody comprises VL CDRs that are identical to the VL CDRs
  • the determination of percent identity between two sequences can also be accomplished using a mathematical algorithm.
  • a preferred, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. U.S.A.
  • Gapped BLAST can be utilized as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389 3402.
  • PSI BLAST can be used to perform an iterated search which detects distant relationships between molecules (Id.).
  • the default parameters of the respective programs e.g., of XBLAST and NBLAST
  • NCBI National Center for Biotechnology Information
  • a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, 1988, CABIOS 4: 11 17. Such an algorithm is incorporated in the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package.
  • ALIGN program version 2.0
  • a PAM120 weight residue table a gap length penalty of 12, and a gap penalty of 4 can be used.
  • the percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted.
  • a MerTK antagonist provided herein is an antibody described herein that specifically binds to a MerTK polypeptide, e.g., an ECD of human MerTK, and that inhibits (e.g., partially inhibits) a MerTK activity.
  • MerTK activity can relate to any activity of MerTK such as those known or described in the art.
  • Non-limiting examples of MerTK activity include: MerTK receptor dimerization, MerTK receptor phosphorylation (e.g., tyrosine phosphorylation or autophosphorylation in the cytoplasmic domain), signaling downstream of the MerTK receptor (e.g., P13K, PLC, GRB2, RAC1, SOCS-1, AKT, STAT1, or MAPK/ERK signaling), MerTK ligand (e.g., Gas6 and PROS1) induced enhancement of cell proliferation, or cell survival (e.g., natural killer (NK) cells), modulation of NK cells, dendritic cells or macrophages, phagocytosis, inhibition of proinflammatory cytokine production (e.g., inhibition of TLR-induced production of
  • proinflammatory cytokines e.g., TNF, IL-6, IL-12 and type I interferons.
  • antibodies described herein specifically bind to human MerTK and block or inhibit (e.g., partially inhibit) binding of MerTK ligand (e.g., Gas6 and PROS1) to MerTK by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% as assessed by methods described herein or known to one of skill in the art, e.g., ELISA assay, flow cytometry, or competition assay.
  • MerTK ligand e.g., Gas6 and PROS1
  • inhibition by anti-MerTK antibodies described herein (e.g., monoclonal antibody) of MerTK ligand (e.g., Gas6 and PROS1) binding to MerTK can be characterized by ICso values, which reflects the concentration of anti-MerTK antibodies achieving 50% inhibition of binding of MerTK ligand to MerTK.
  • an anti-MerTK antibody described herein inhibits binding of MerTK ligand to MerTK with an ICso of at most about 10,000 nM, 1,000 nM, 900 nM, 800 nM, 700 nM, 600 nM, 500 nM, 400 nM, 300 nM, 200 nM, 100 nM, 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 5 nM, 1 nM, 0.75 nM, 0.5 nM, 0.1 nM, 0.05 nM, 0.01 nM, 0.005 nM, or 0.001 nM, as assessed by methods described herein and
  • an anti- MerTK antibody described herein inhibits binding of MerTK ligand to MerTK with an ICso in the range of about 0.01 nM to 10,000 nM, 0.01 nM to 1,000 nM, 0.05 nM to 500 nM, 0.05 nM to 100 nM, or 0.05 nM to 50 nM, 0.1 nM to 50 nM as assessed by methods described herein and/or known to one of skill in the art, (e.g., ELISA assay or flow cytometry).
  • an anti-MerTK antibody described herein can inhibit (e.g., partially inhibit) MerTK activity by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% as assessed by methods described herein and/or known to one of skill in the art, relative to MerTK activity in the presence of MerTK ligand stimulation without any antibody or with an unrelated antibody (e.g., an antibody that does not specifically bind to MerTK).
  • an unrelated antibody e.g., an antibody that does not specifically bind to MerTK.
  • an anti- MerTK antibody described herein can inhibit (e.g., partially inhibit) MerTK activity by at least about 25%) to about 65% as assessed by methods described herein and/or known to one of skill in the art, relative to MerTK activity in the presence of MerTK ligand stimulation without any antibody or with an unrelated antibody (e.g., an antibody that does not specifically bind to MerTK).
  • Non-limiting examples of MerTK activity can include MerTK receptor
  • phosphorylation phosphorylation, MerTK receptor signaling, MerTK ligand (e.g., Gas6 and PROS1) mediated cell proliferation, and MerTK ligand (e.g., Gas6 and PROS1) mediated cell survival (e.g., natural killer (NK) cells), modulation of maturation of NK cells, dendritic cells or macrophages, phagocytosis, inhibition of proinflammatory cytokine production (e.g., inhibition of TLR- induced production of proinflammatory cytokines (e.g., TNF, IL-6, IL-12 and type I interferons).
  • NK natural killer
  • an antibody described herein e.g., antibody Ab2000 or an antigen-binding fragment thereof, Ab2000-A7 or an antigen- binding fragment thereof, or an antibody comprising CDRs of any of Table 1, Table 3-7, or Table 11-14
  • an antibody described herein can block (e.g., partially block) or inhibit (e.g., partially inhibit) (e.g., human MerTK), specifically tyrosine phosphorylation of one or more residues in the cytoplasmic domain of MerTK.
  • antibodies described herein specifically bind to human MerTK and block, inhibit, or reduce tyrosine (e.g., human MerTK) by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%), 95%), 98%), 99%), or 100% as assessed by methods described herein or known to one of skill in the art, e.g., as described in section 6, relative to phosphorylation in the presence of MerTK ligand stimulation without any antibody or with an unrelated antibody (e.g., an antibody that does not specifically bind to MerTK).
  • tyrosine e.g., human MerTK
  • antibodies described herein specifically bind to MerTK and block or inhibit tyrosine phosphorylation in the cytoplasmic domain of MerTK by at least about 25%, optionally to about 65%, 75%, 80% or 85%), as assessed by methods described herein or known to one of skill in the art, e.g., as described in Section 6, below.
  • antibodies described herein specifically bind to MerTK and block or inhibit tyrosine phosphorylation of MerTK by at least about 25% to about 80%) as assessed by methods described herein or known to one of skill in the art, e.g., as described in Section 6, below.
  • antibodies described herein specifically bind to MerTK and block or inhibit tyrosine (e.g., human MerTK) by at least about 50%) to about 100%) as assessed by methods described herein or known to one of skill in the art, e.g., as described in Section 6, below.
  • tyrosine e.g., human MerTK
  • antibodies described herein specifically bind to MerTK and block or inhibit tyrosine phosphorylation of MerTK with an ICso of less than about 10 nM, about 1 nM, about 500 pM, about 400 pM, about 200 pM, or about 100 pM, as assessed by methods described herein in Section 6 below or known to one of skill in the art.
  • antibodies described herein specifically bind to MerTK and block or inhibit tyrosine
  • an ICso for inhibition of tyrosine phosphorylation can be determined by assaying lysates from cells, recombinantly expressing MerTK, in ELISA which detects tyrosine phosphorylation, for example, as described in Section 6 below.
  • cells, recombinantly expressing MerTK are sorted, e.g., sorted to select for cells highly expressing MerTK, prior to use in the phosphorylation inhibition assays. In some embodiments, the cells are not sorted prior to use in the
  • antibodies described herein specifically bind to MerTK and block or inhibit phosphorylation of one or more tyrosine residues in the cytoplasmic domain of MerTK by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% as assessed by methods described herein or known to one of skill in the art, e.g., immunoblotting assay, relative to phosphorylation in the presence of MerTK ligand stimulation without any antibody or with an unrelated antibody (e.g., an antibody that does not specifically bind to MerTK).
  • blocking or inhibition e.g., partial inhibition
  • MerTK ligand e.g., Gas6 and PROS1
  • PI phosphatidylinositol
  • MAPK mitogen-activated protein kinase
  • anti-MerTK antibodies described herein which act as inhibitors of MerTK activity can inhibit signaling of a member of the PI 3-kinases or MAPK.
  • anti-MerTK antibodies described herein which act as inhibitors of MerTK activity can inhibit binding (or inhibit interaction), to the cytoplasmic domain of MerTK, of one or more of PI3K, PLC, and Grb2.
  • anti- MerTK antibodies described herein which act as inhibitors of MerTK activity can inhibit activation by MerTK of one or more of PI3K, PLC, and Grb2.
  • anti-MerTK antibodies described herein which act as inhibitors of MerTK activity can inhibit downstream signaling such as
  • an anti-MerTK antibody described herein can inhibit or reduce
  • MAPK e.g., MerTK ligand (e.g., Gas6 and PROS1) induced
  • phosphorylation of MAPK by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% as assessed by methods described herein or known to one of skill in the art, e.g., Western blot or ELISA assay as described in section 6 or immunoblotting assay, relative to phosphorylation in the presence of MerTK ligand stimulation without any antibody or with an unrelated antibody (e.g., an antibody that does not specifically bind to MerTK).
  • an unrelated antibody e.g., an antibody that does not specifically bind to MerTK
  • an anti-MerTK antibody described herein can inhibit or reduce phosphorylation of AKT (e.g., MerTK ligand (e.g., Gas6 and PROS1) induced phosphorylation of AKT) by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% as assessed by methods described herein or known to one of skill in the art, e.g., as described in section 6, relative to phosphorylation in the presence of MerTK ligand stimulation without any antibody or with an unrelated antibody (e.g., an antibody that does not specifically bind to MerTK).
  • AKT e.g., MerTK ligand (e.g., Gas6 and PROS1) induced phosphorylation of AKT) by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,
  • Cells and cell lines which are appropriate for use in the assays described herein relating to MerTK activity are readily available (e.g., ATCC) or can be readily identified using methods known in the art.
  • cells and/or cell lines that express MerTK endogenously or that possess MerTK signaling or activity are known to one of skill in the art.
  • cells or cell lines that are appropriate for use in the assays described herein can express MerTK, either endogenously or recombinantly.
  • Non-limiting examples of cells that can be used in the methods and assays described herein include primary cells, transformed cells, stem cells, mast cells, primordial germ cells, oocytes, spermatocytes, embryonic stem cells, hematopoietic cells, erythroleukemia cells (e.g., F36P and TF-1 cell lines), human monocytic cell lines, e.g., acute monocytic leukemia cell lines, human myeloid leukemia cell lines, such as M07E cells; gastrointestinal stromal tumor cell lines such as ST-882, GIST430, and GIST882; neuroblastoma cell lines such as SK-N-SH, SK-SY5Y, H-EP1, SK-N-BE(2), SK-N-BE(ZkM17), SK-N-BE(2)C, LA-N-1, or LA-N-l-5s; small cell lung carcinoma cell lines such as H526, ECC12, TMK1, MKN7, GCIY, and
  • cells that can be used in the methods and assays described herein include immune cells, such as macrophages, dendritic cells, and natural killer (NK) cells.
  • immune cells such as macrophages, dendritic cells, and natural killer (NK) cells.
  • NK cells natural killer cells.
  • cells that can be used in the methods and assay described herein include THP1 cells.
  • cells and cell lines that express MerTK can routinely be generated recombinantly.
  • Non-limiting examples of cells that can be engineered to express MerTK recombinantly include COS cells, HEK 293 cells, CHO cells, H1299 cells, fibroblasts (e.g., human fibroblasts) such as NIH3T3 cells, and MEFS.
  • fibroblasts e.g., human fibroblasts
  • cells for use in the methods described herein are HEK 293 cells expressing human MerTK ECD.
  • Antibodies described herein (or an antigen-binding fragment thereof) that specifically bind to MerTK (e.g., ECD of human MerTK) can be produced by any method known in the art for the synthesis of antibodies, for example, by chemical synthesis or by recombinant expression techniques.
  • the methods described herein employs, unless otherwise indicated, conventional techniques in molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and related fields within the skill of the art. These techniques are described, for example, in the references cited herein and are fully explained in the literature. See, e.g.,, Maniatis et al.
  • an antibody described herein is an antibody (e.g., recombinant antibody) prepared, expressed, created or isolated by any means that involves creation, e.g., via synthesis, genetic engineering of DNA sequences.
  • an antibody comprise sequences (e.g., DNA sequences or amino acid sequences) that do not naturally exist within the antibody germline repertoire of an animal or mammal (e.g., human) in vivo.
  • provided herein is a method of making an antibody or an antigen- binding fragment thereof, which specifically binds to human MerTK comprising culturing a cell or host cell described herein.
  • a method of making an antibody or an antigen-binding fragment thereof which specifically binds to human MerTK comprising expressing (e.g., recombinantly expressing) the antibody or antigen-binding fragment thereof using a cell or host cell described herein (e.g., a cell or a host cell comprising
  • the cell is an isolated cell.
  • the exogenous polynucleotides have been introduced into the cell.
  • the method further comprises the step of purifying the antibody or antigen-binding fragment thereof obtained from the cell or host cell.
  • Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof.
  • monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et al.,
  • monoclonal antibody as used herein is not limited to antibodies produced through hybridoma technology.
  • monoclonal antibodies can be produced recombinantly from host cells exogenously expressing an antibody described herein (e.g., anti- MerTK antibody comprising the CDRs of any of Table 1, Table 3-7, or Table 11-14) or a fragment thereof, for example, light chain and/or heavy chain of such antibody.
  • Methods for producing and screening for specific antibodies using hybridoma technology are routine and well known in the art.
  • a mouse or other appropriate host animal such as a sheep, goat, rabbit, rat, hamster or macaque monkey, is immunized to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein (e.g., ECD of human MerTK) used for immunization.
  • lymphocytes may be immunized in vitro.
  • Lymphocytes then are fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). Additionally, a RFMMS (repetitive immunization multiple sites) technique can be used to immunize an animal (Kilptrack et al., 1997 Hybridoma 16:381-9, incorporated by reference in its entirety).
  • a suitable fusing agent such as polyethylene glycol
  • mice can be immunized with an antigen (e.g., MerTK, for example human MerTK) and once an immune response is detected, e.g., antibodies specific for the antigen are detected in the mouse serum, the mouse spleen is harvested and splenocytes isolated. The splenocytes are then fused by well known techniques to any suitable myeloma cells, for example cells from cell line SP20 available from the American Type Culture Collection (ATCC®) (Manassas, VA), to form hybridomas. Hybridomas are selected and cloned by limited dilution.
  • lymph nodes of the immunized mice are harvested and fused with NS0 myeloma cells.
  • the hybridoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells. For example, if the parental myeloma cells lack the enzyme
  • hypoxanthine guanine phosphoribosyl transferase HGPRT or HPRT
  • the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.
  • myeloma cells that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium.
  • myeloma cell lines are murine myeloma lines, such as NS0 cell line or those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, CA, USA, and SP-2 or X63-Ag8.653 cells available from the American Type Culture Collection, Rockville, MD, USA.
  • Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133 :3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).
  • Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against human MerTK antigen (e.g., ECD of human MerTK).
  • human MerTK antigen e.g., ECD of human MerTK.
  • the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by methods known in the art, for example, immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunoabsorbent assay
  • the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, D-MEM or RPMI 1640 medium.
  • the hybridoma cells may be grown in vivo as ascites tumors in an animal.
  • the monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
  • antibody variants having an improved property such as affinity, stability, and/or expression level as compared to a parent antibody may be prepared by affinity maturation.
  • Affinity maturation techniques that are known in the art and/or described herein can be used to alter the CDR region(s), followed by screening of the resultant binding molecules for the desired change in binding.
  • libraries of antibodies can be displayed on the surface of an organism (e.g., phage, bacteria, yeast, or mammalian cell) or in association with their encoding mRNA or DNA (e.g., covalently or non- covalently). Affinity selection of the displayed antibodies allows isolation of organisms or complexes carrying the genetic information encoding the antibodies.
  • antibody affinity maturation can be performed using the methods exemplified in Example 7 below.
  • Antibodies described herein include antibody fragments, which recognize specific MerTK antigens (e.g., ECD of MerTK) and can be generated by any technique known to those of skill in the art.
  • Fab and F(ab') 2 fragments described herein can be produced by proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab') 2 fragments).
  • a Fab fragment corresponds to one of the two identical arms of an antibody molecule and contains the complete light chain paired with the VH and CHI domains of the heavy chain.
  • a F(ab')2 fragment contains the two antigen- binding arms of an antibody molecule linked by disulfide bonds in the hinge region.
  • the antibodies described herein or antigen-binding fragments thereof can also be generated using various phage display methods known in the art.
  • phage display methods functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them.
  • DNA sequences encoding VH and VL domains are amplified from animal cDNA libraries (e.g., human or murine cDNA libraries of affected tissues).
  • the DNA encoding the VH and VL domains are recombined together with an scFv linker by PCR and cloned into a phagemid vector.
  • the vector is electroporated in E. coli and the E.
  • Phage used in these methods are typically filamentous phage including fd and Ml 3, and the VH and VL domains are usually recombinantly fused to either the phage gene III or gene VIII.
  • Phage expressing an antigen binding domain that binds to a particular antigen can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead. Examples of phage display methods that can be used to make the antibodies described herein include those disclosed in Brinkman et al., 1995, J. Immunol. Methods 182:41-50; Ames et al., 1995, J. Immunol.
  • the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g., as described below.
  • Techniques to recombinantly produce antibody fragments such as Fab, Fab' and F(ab') 2 fragments can also be employed using methods known in the art such as those disclosed in PCT publication No.
  • PCR primers including VH or VL nucleotide sequences, a restriction site, and a flanking sequence to protect the restriction site can be used to amplify the VH or VL sequences from a template, e.g., scFv clones.
  • the PCR amplified VH domains can be cloned into vectors expressing a VH constant region
  • the PCR amplified VL domains can be cloned into vectors expressing a VL constant region, e.g., human kappa or lambda constant regions.
  • the VH and VL domains can also be cloned into one vector expressing the necessary constant regions.
  • the heavy chain conversion vectors and light chain conversion vectors are then co-transfected into cell lines to generate stable or transient cell lines that express full-length antibodies, e.g., IgG, using techniques known to those of skill in the art.
  • a chimeric antibody is a molecule in which different portions of the antibody are derived from different immunoglobulin molecules.
  • a chimeric antibody can contain a variable region of a mouse or rat monoclonal antibody fused to a constant region of a human antibody.
  • Methods for producing chimeric antibodies are known in the art. See, e.g., Morrison, 1985, Science 229: 1202; Oi et al., 1986, BioTechniques 4:214; Gillies et al., 1989, J. Immunol. Methods 125: 191-202; and U.S. Patent Nos. 5,807,715, 4,816,567, 4,816,397, and 6,331,415.
  • a humanized antibody is capable of binding to a predetermined antigen and which comprises a framework region having substantially the amino acid sequence of a human immunoglobulin and CDRs having substantially the amino acid sequence of a non-human immunoglobulin (e.g., a murine immunoglobulin).
  • a humanized antibody also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • the antibody also can include the CHI, hinge, CH2, CH3, and CH4 regions of the heavy chain.
  • a humanized antibody can be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype, including IgGi, IgG 2 , IgG 3 and IgG 4 .
  • Humanized antibodies can be produced using a variety of techniques known in the art, including but not limited to, CDR-grafting (European Patent No. EP 239,400;
  • Single domain antibodies for example, antibodies lacking the light chains, can be produced by methods well-known in the art. See Riechmann et al., 1999, J. Immunol. 231 :25- 38; Nuttall et al., 2000, Curr. Pharm. Biotechnol. l(3):253-263; Muylderman, 2001, J.
  • antibodies that specifically bind to a MerTK antigen can, in turn, be utilized to generate anti-idiotype antibodies that "mimic" an antigen using techniques well known to those skilled in the art. (See, e.g., Greenspan & Bona, 1989, FASEB J. 7(5):437-444; and Nissinoff, 1991, J. Immunol. 147(8):2429-2438).
  • Human antibodies which specifically bind to an ECD of human MerTK can be made by a variety of methods known in the art including phage display methods described above using antibody libraries derived from human immunoglobulin sequences. See also U.S. Patent Nos. 4,444,887 and 4,716,111; and International Publication Nos. WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741.
  • human antibodies can be produced using mouse-human hybridomas.
  • human peripheral blood lymphocytes transformed with Epstein-Barr virus (EBV) can be fused with mouse myeloma cells to produce mouse-human hybridomas secreting human monoclonal antibodies, and these mouse-human hybridomas can be screened to determine ones which secrete human monoclonal antibodies that specifically bind to a target antigen (e.g., ECD of human MerTK).
  • ECD of human MerTK target antigen
  • polynucleotides comprising a nucleotide sequence encoding an antibody described herein or a fragment thereof (e.g., a variable light chain region and/or variable heavy chain region) that specifically binds to a MerTK antigen, and vectors, e.g., vectors comprising such polynucleotides for recombinant expression in host cells (e.g., E. coli and mammalian cells).
  • host cells e.g., E. coli and mammalian cells.
  • polynucleotides comprising nucleotide sequences encoding any of the antibodies provided herein, as well as vectors comprising such polynucleotide sequences, e.g., expression vectors for their efficient expression in host cells, e.g., mammalian cells.
  • an "isolated" polynucleotide or nucleic acid molecule is one which is separated from other nucleic acid molecules which are present in the natural source (e.g., in a mouse or a human) of the nucleic acid molecule.
  • an "isolated" nucleic acid molecule such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • the language "substantially free” includes preparations of polynucleotide or nucleic acid molecule having less than about 15%, 10%, 5%, 2%), 1%), 0.5%), or 0.1%) (in particular less than about 10%>) of other material, e.g., cellular material, culture medium, other nucleic acid molecules, chemical precursors and/or other chemicals.
  • a nucleic acid molecule(s) encoding an antibody described herein is isolated or purified.
  • polynucleotides comprising nucleotide sequences encoding antibodies or antigen-binding fragments thereof, which specifically bind to a MerTK polypeptide (e.g., human MerTK, for example, human MerTK) and comprises an amino acid sequence as described herein, as well as antibodies which compete with such antibodies for binding to a MerTK polypeptide (e.g., in a dose-dependent manner), or which binds to the same epitope as that of such antibodies.
  • a MerTK polypeptide e.g., human MerTK, for example, human MerTK
  • a MerTK polypeptide e.g., human MerTK, for example, human MerTK
  • antibodies which compete with such antibodies for binding to a MerTK polypeptide (e.g., in a dose-dependent manner), or which binds to the same epitope as that of such antibodies.
  • polynucleotides comprising a nucleotide sequence encoding the light chain or heavy chain of an antibody described herein.
  • the polynucleotides can comprise nucleotide sequences encoding a light chain comprising the VL of antibodies described herein (see, e.g., Table 2 and Table 8).
  • the polynucleotides can comprise nucleotide sequences encoding a heavy chain comprising the VH of antibodies described herein (see, e.g., Table 2 and Table 8).
  • a polynucleotide described herein encodes a VL chain region comprising the amino acid sequence of any one of SEQ ID NO: 2 and SEQ ID NO: 26. In specific embodiments, a polynucleotide described herein encodes a VH chain region comprising the amino acid sequence of any one of SEQ ID NO: 1 and SEQ ID NO: 25.
  • polynucleotides comprising a nucleotide sequence encoding an anti-MerTK antibody comprising three VL chain CDRs, e.g., containing VL CDR1, VL CDR2, and VL CDR3 of Table 1.
  • polynucleotides comprising three VH chain CDRs, e.g., containing VH CDR1, VH CDR2, and VH CDR3 of Table 1.
  • polynucleotides comprising a nucleotide sequence encoding an anti-MerTK antibody comprising three VL chain CDRs, e.g., containing VL CDR1, VL CDR2, and VL CDR3 of Table 1 and three VH chain CDRs, e.g., containing VH CDR1, VH CDR2, and VH CDR3 of Table 1.
  • polynucleotides comprising a nucleotide sequence encoding an anti-MerTK antibody comprising three VL chain CDRs, e.g., containing VL CDR1, VL CDR2, and VL CDR3 of Table 7.
  • polynucleotides comprising three VH chain CDRs, e.g., containing VH CDR1, VH CDR2, and VH CDR3 of Table 7.
  • polynucleotides comprising a nucleotide sequence encoding an anti-MerTK antibody comprising three VL chain CDRs, e.g., containing VL CDR1, VL CDR2, and VL CDR3 of Table 7 and three VH chain CDRs, e.g., containing VH CDR1, VH CDR2, and VH CDR3 of Table 7.
  • polynucleotides comprising a nucleotide sequence encoding an anti-MerTK antibody comprising three VL chain CDRs, e.g., containing VL CDR1, VL CDR2, and VL CDR3 of Table 1 and three VH chain CDRs, e.g., containing VH CDR1, VH CDR2, and VH CDR3 of Table 7.
  • polynucleotides comprising a nucleotide sequence encoding an anti-MerTK antibody comprising three VL chain CDRs, e.g., containing VL CDR1, VL CDR2, and VL CDR3 of Table 7 and three VH chain CDRs, e.g., containing VH CDR1, VH CDR2, and VH CDR3 of Table 1.
  • polynucleotides comprising a nucleotide sequence encoding an anti-MerTK antibody comprising three VL chain CDRs, e.g., containing VL CDRl, VL CDR2, and VL CDR3 of Table 3.
  • polynucleotides comprising three VH chain CDRs, e.g., containing VH CDRl, VH CDR2, and VH CDR3 of Table 3.
  • polynucleotides comprising a nucleotide sequence encoding an anti-MerTK antibody comprising three VL chain CDRs, e.g., containing VL CDRl, VL CDR2, and VL CDR3 of Table 3 and three VH chain CDRs, e.g., containing VH CDRl, VH CDR2, and VH CDR3 of Table 3.
  • polynucleotides comprising a nucleotide sequence encoding an anti-MerTK antibody comprising three VL chain CDRs, e.g., containing VL CDRl, VL CDR2, and VL CDR3 of Table 11.
  • polynucleotides comprising three VH chain CDRs, e.g., containing VH CDRl, VH CDR2, and VH CDR3 of Table 11.
  • polynucleotides comprising a nucleotide sequence encoding an anti-MerTK antibody comprising three VL chain CDRs, e.g., containing VL CDRl, VL CDR2, and VL CDR3 of Table 8 and three VH chain CDRs, e.g., containing VH CDRl, VH CDR2, and VH CDR3 of Table 8.
  • polynucleotides comprising a nucleotide sequence encoding an anti-MerTK antibody comprising three VL chain CDRs, e.g., containing VL CDRl, VL CDR2, and VL CDR3 of Table 3 and three VH chain CDRs, e.g., containing VH CDRl, VH CDR2, and VH CDR3 of Table 11.
  • three VL chain CDRs e.g., containing VL CDRl, VL CDR2, and VL CDR3 of Table 3
  • three VH chain CDRs e.g., containing VH CDRl, VH CDR2, and VH CDR3 of Table 11.
  • polynucleotides comprising a nucleotide sequence encoding an anti-MerTK antibody comprising three VL chain CDRs, e.g., containing VL CDRl, VL CDR2, and VL CDR3 of Table 11 and three VH chain CDRs, e.g., containing VH CDRl, VH CDR2, and VH CDR3 of Table 3.
  • polynucleotides comprising a nucleotide sequence encoding an anti-MerTK antibody comprising three VL chain CDRs, e.g., containing VL CDRl, VL CDR2, and VL CDR3 of Table 4.
  • polynucleotides comprising three VH chain CDRs, e.g., containing VH CDRl, VH CDR2, and VH CDR3 of Table 4.
  • polynucleotides comprising a nucleotide sequence encoding an anti-MerTK antibody comprising three VL chain CDRs, e.g., containing VL CDRl, VL CDR2, and VL CDR3 of Table 4 and three VH chain CDRs, e.g., containing VH CDRl, VH CDR2, and VH CDR3 of Table 4.
  • polynucleotides comprising a nucleotide sequence encoding an anti-MerTK antibody comprising three VL chain CDRs, e.g., containing VL CDRl, VL CDR2, and VL CDR3 of Table 12.
  • polynucleotides comprising three VH chain CDRs, e.g., containing VH CDRl, VH CDR2, and VH CDR3 of Table 12.
  • polynucleotides comprising a nucleotide sequence encoding an anti-MerTK antibody comprising three VL chain CDRs, e.g., containing VL CDRl, VL CDR2, and VL CDR3 of Table 12 and three VH chain CDRs, e.g., containing VH CDRl, VH CDR2, and VH CDR3 of Table 12.
  • polynucleotides comprising a nucleotide sequence encoding an anti-MerTK antibody comprising three VL chain CDRs, e.g., containing VL CDRl, VL CDR2, and VL CDR3 of Table 4 and three VH chain CDRs, e.g., containing VH CDRl, VH CDR2, and VH CDR3 of Table 12.
  • three VL chain CDRs e.g., containing VL CDRl, VL CDR2, and VL CDR3 of Table 4
  • three VH chain CDRs e.g., containing VH CDRl, VH CDR2, and VH CDR3 of Table 12.
  • polynucleotides comprising a nucleotide sequence encoding an anti-MerTK antibody comprising three VL chain CDRs, e.g., containing VL CDRl, VL CDR2, and VL CDR3 of Table 12 and three VH chain CDRs, e.g., containing VH CDRl, VH CDR2, and VH CDR3 of Table 4.
  • polynucleotides comprising a nucleotide sequence encoding an anti-MerTK antibody comprising three VL chain CDRs, e.g., containing VL CDRl, VL CDR2, and VL CDR3 of Table 5.
  • polynucleotides comprising three VH chain CDRs, e.g., containing VH CDRl, VH CDR2, and VH CDR3 of Table 5.
  • polynucleotides comprising a nucleotide sequence encoding an anti-MerTK antibody comprising three VL chain CDRs, e.g., containing VL CDRl, VL CDR2, and VL CDR3 of Table 5 and three VH chain CDRs, e.g., containing VH CDRl, VH CDR2, and VH CDR3 of Table 5.
  • polynucleotides comprising a nucleotide sequence encoding an anti-MerTK antibody comprising three VL chain CDRs, e.g., containing VL CDRl, VL CDR2, and VL CDR3 of Table 13.
  • polynucleotides comprising three VH chain CDRs, e.g., containing VH CDRl, VH CDR2, and VH CDR3 of Table 13.
  • polynucleotides comprising a nucleotide sequence encoding an anti-MerTK antibody comprising three VL chain CDRs, e.g., containing VL CDRl, VL CDR2, and VL CDR3 of Table 13 and three VH chain CDRs, e.g., containing VH CDRl, VH CDR2, and VH CDR3 of Table 13.
  • polynucleotides comprising a nucleotide sequence encoding an anti-MerTK antibody comprising three VL chain CDRs, e.g., containing VL CDRl, VL CDR2, and VL CDR3 of Table 5 and three VH chain CDRs, e.g., containing VH CDRl, VH CDR2, and VH CDR3 of Table 13.
  • three VL chain CDRs e.g., containing VL CDRl, VL CDR2, and VL CDR3 of Table 5
  • three VH chain CDRs e.g., containing VH CDRl, VH CDR2, and VH CDR3 of Table 13.
  • polynucleotides comprising a nucleotide sequence encoding an anti-MerTK antibody comprising three VL chain CDRs, e.g., containing VL CDRl, VL CDR2, and VL CDR3 of Table 13 and three VH chain CDRs, e.g., containing VH CDRl, VH CDR2, and VH CDR3 of Table 5.
  • polynucleotides comprising a nucleotide sequence encoding an anti-MerTK antibody comprising three VL chain CDRs, e.g., containing VL CDRl, VL CDR2, and VL CDR3 of Table 6.
  • polynucleotides comprising three VH chain CDRs, e.g., containing VH CDRl, VH CDR2, and VH CDR3 of Table 6.
  • polynucleotides comprising a nucleotide sequence encoding an anti-MerTK antibody comprising three VL chain CDRs, e.g., containing VL CDRl, VL CDR2, and VL CDR3 of Table 6 and three VH chain CDRs, e.g., containing VH CDRl, VH CDR2, and VH CDR3 of Table 6.
  • polynucleotides comprising a nucleotide sequence encoding an anti-MerTK antibody comprising three VL chain CDRs, e.g., containing VL CDRl, VL CDR2, and VL CDR3 of Table 14.
  • polynucleotides comprising three VH chain CDRs, e.g., containing VH CDRl, VH CDR2, and VH CDR3 of Table 14.
  • polynucleotides comprising a nucleotide sequence encoding an anti-MerTK antibody comprising three VL chain CDRs, e.g., containing VL CDRl, VL CDR2, and VL CDR3 of Table 14 and three VH chain CDRs, e.g., containing VH CDRl, VH CDR2, and VH CDR3 of Table 14.
  • polynucleotides comprising a nucleotide sequence encoding an anti-MerTK antibody comprising three VL chain CDRs, e.g., containing VL CDRl, VL CDR2, and VL CDR3 of Table 6 and three VH chain CDRs, e.g., containing VH CDRl, VH CDR2, and VH CDR3 of Table 14.
  • three VL chain CDRs e.g., containing VL CDRl, VL CDR2, and VL CDR3 of Table 6
  • three VH chain CDRs e.g., containing VH CDRl, VH CDR2, and VH CDR3 of Table 14.
  • polynucleotides comprising a nucleotide sequence encoding an anti-MerTK antibody comprising three VL chain CDRs, e.g., containing VL CDRl, VL CDR2, and VL CDR3 of Table 14 and three VH chain CDRs, e.g., containing VH CDRl, VH CDR2, and VH CDR3 of Table 6.
  • a polynucleotide described herein comprises a nucleotide sequence encoding an antibody provided herein comprising a variable light (VL) chain region comprising an amino acid described herein, wherein the antibody specifically binds to a MerTK polypeptide, e.g., a human MerTK polypeptide, for example, human MerTK (e.g., human MerTK).
  • VL variable light
  • a polynucleotide described herein comprises a nucleotide sequence encoding an antibody provided herein comprising a variable heavy (VH) chain region comprising an amino acid sequence described herein, wherein the antibody specifically binds to a MerTK polypeptide, e.g., a human MerTK polypeptide, for example, human MerTK (e.g., human MerTK).
  • VH variable heavy chain region
  • a polynucleotide provided herein comprises a nucleotide sequence encoding an antibody described herein comprising: framework regions (e.g., framework regions of the VL domain and VH domain) that are human framework regions, wherein the antibody specifically binds a MerTK polypeptide, e.g., a human MerTK polypeptide, for example, human MerTK (e.g., human MerTK).
  • framework regions e.g., framework regions of the VL domain and VH domain
  • the antibody specifically binds a MerTK polypeptide, e.g., a human MerTK polypeptide, for example, human MerTK (e.g., human MerTK).
  • a polynucleotide comprising a nucleotide sequence encoding an antibody comprising a light chain and a heavy chain, e.g., a separate light chain and heavy chain.
  • a light chain in a specific embodiment, a light chain and a heavy chain.
  • polynucleotide provided herein comprises a nucleotide sequence encoding a kappa light chain.
  • a polynucleotide provided herein comprises a nucleotide sequence encoding a lambda light chain.
  • a polynucleotide provided herein comprises a nucleotide sequence encoding an antibody described herein comprising a human kappa light chain or a human lambda light chain.
  • a polynucleotide provided herein comprises a nucleotide sequence encoding an antibody described herein, which specifically binds to a MerTK polypeptide, e.g., a human MerTK polypeptide, for example, human MerTK (e.g., human MerTK), wherein the antibody comprises a light chain, and wherein the amino acid sequence of the VL chain region can comprise any amino acid sequence described herein (e.g., SEQ ID NO: 2 or SEQ ID NO: 26), and wherein the constant region of the light chain comprises the amino acid sequence of a human kappa light chain constant region.
  • a MerTK polypeptide e.g., a human MerTK polypeptide, for example, human MerTK (e.g., human MerTK)
  • the antibody comprises a light chain
  • the amino acid sequence of the VL chain region can comprise any amino acid sequence described herein (e.g., SEQ ID NO: 2 or SEQ ID NO: 26)
  • the constant region of the light chain
  • a polynucleotide provided herein comprises a nucleotide sequence encoding an antibody described herein, which specifically binds to a MerTK polypeptide, e.g., a human MerTK polypeptide, for example, human MerTK (e.g., human MerTK), and comprises a light chain, wherein the amino acid sequence of the VL chain region can comprises any amino acid sequence described herein (e.g., SEQ ID NO: 2 or SEQ ID NO: 26), and wherein the constant region of the light chain comprises the amino acid sequence of a human lambda light chain constant region.
  • human constant region sequences can be those described in U.S. Patent No. 5,693,780.
  • a polynucleotide provided herein comprises a nucleotide sequence encoding an antibody described herein, which specifically binds to a MerTK polypeptide, e.g., a human MerTK polypeptide, for example, human MerTK (e.g., human MerTK), wherein the antibody comprises a heavy chain, wherein the amino acid sequence of the VH chain region can comprise any amino acid sequence described herein (e.g., SEQ ID NO: 1 or SEQ ID NO: 25), and wherein the constant region of the heavy chain comprises the amino acid sequence of a human gamma ( ⁇ ) heavy chain constant region.
  • a MerTK polypeptide e.g., a human MerTK polypeptide, for example, human MerTK (e.g., human MerTK)
  • the antibody comprises a heavy chain
  • the amino acid sequence of the VH chain region can comprise any amino acid sequence described herein (e.g., SEQ ID NO: 1 or SEQ ID NO: 25)
  • a polynucleotide provided herein comprises a nucleotide sequence encoding an antibody described herein (or an antigen-binding fragment thereof), which specifically binds a MerTK polypeptide, e.g., a human MerTK polypeptide, for example, human MerTK (e.g., human MerTK), wherein the antibody comprises a VL chain region and a VH chain region comprising any amino acid sequences described herein, and wherein the constant regions comprise the amino acid sequences of the constant regions of a human IgGl (e.g., isotype a, z, or f), human IgG2, or human IgG4.
  • a human IgGl e.g., isotype a, z, or f
  • polynucleotides comprising a nucleotide sequence encoding an anti-MerTK antibody, or an antigen-binding fragment or domain thereof, designated herein.
  • an antibody or antigen-binding fragment described herein which specifically binds to a MerTK polypeptide (e.g., human MerTK), can be encoded by a nucleotide that comprises nucleotide sequence of SEQ ID NO: 58, wherein the amino acid sequence for SEQ ID NO: 58 is GAGGTGC AGC TGC TGGAG A GCGGAGGAGGACTGGTGCAGCCCGGAGGATCTTTAAGACTGAGCTGCGCCGCCAGC GGCTTCACCTTCAGCAGCTACGCCATGTCTTGGGTGAGACAAGCTCCCGGAAAGGG ACTGGAGTGGGTCAGCGCCATTTCCGGAAGCGGCGGCAGCACCTACTACGCCGACA GCGTGAAGGGTCGTTTTACCATCTCTCGTGACAACAGCAAGAACACTTTATATTTAC
  • an antibody or antigen-binding fragment described herein which specifically binds to a MerTK polypeptide (e.g., human
  • MerTK can be encoded by a nucleotide that comprises nucleotide sequence of SEQ ID NO: 59, wherein the amino acid sequence for SEQ ID NO: 59 is
  • an antibody or antigen-binding fragment described herein which specifically binds to a MerTK polypeptide (e.g., human MerTK), can be encoded by a nucleotide that comprises nucleotide sequence of SEQ ID NO: 60, wherein the amino acid sequence for SEQ ID NO: 60 is
  • an antibody or antigen-binding fragment described herein, which specifically binds to a MerTK polypeptide can be encoded by a nucleotide that comprising a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 58.
  • an antibody or antigen-binding fragment described herein, which specifically binds to a MerTK polypeptide can be encoded by a nucleotide that comprising a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 59.
  • an antibody or antigen-binding fragment described herein which specifically binds to a MerTK polypeptide (e.g., human MerTK), can be encoded by a nucleotide that comprising a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 60.
  • an antibody described herein which specifically binds to a MerTK polypeptide (e.g., human MerTK) can be encoded by a nucleotide that comprising a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 60 and a nucleotide that comprising a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 58.
  • an antibody described herein which specifically binds to a MerTK polypeptide (e.g., human MerTK) can be encoded by a nucleotide that comprising a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 60 and a nucleotide that comprising a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 59.
  • polynucleotides encoding an anti-MerTK antibody or a fragment thereof that are optimized, e.g., by codon/RNA optimization, replacement with heterologous signal sequences, and elimination of mRNA instability elements.
  • Methods to generate optimized nucleic acids encoding an anti-MerTK antibody or a fragment thereof (e.g., light chain, heavy chain, VH domain, or VL domain) for recombinant expression by introducing codon changes and/or eliminating inhibitory regions in the mRNA can be carried out by adapting the optimization methods described in, e.g., U.S. Patent Nos.
  • RNA potential splice sites and instability elements (e.g., A/T or A/U rich elements) within the RNA can be mutated without altering the amino acids encoded by the nucleic acid sequences to increase stability of the RNA for recombinant expression.
  • the alterations utilize the degeneracy of the genetic code, e.g., using an alternative codon for an identical amino acid.
  • Such methods can increase expression of an anti-MerTK antibody or fragment thereof by at least 1 fold, 2 fold, 3 fold, 4 fold, 5 fold, 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, or 100 fold or more relative to the expression of an anti-MerTK antibody encoded by
  • an optimized polynucleotide sequence encoding an anti- MerTK antibody described herein or a fragment thereof can hybridize to an antisense (e.g., complementarity) polynucleotide of an unoptimized polynucleotide sequence encoding an anti-MerTK antibody described herein or a fragment thereof (e.g., VL domain and/or VH domain).
  • an antisense e.g., complementarity
  • an optimized nucleotide sequence encoding an anti-MerTK antibody described herein or a fragment hybridizes under high stringency conditions to antisense polynucleotide of an unoptimized polynucleotide sequence encoding an anti-MerTK antibody described herein or a fragment thereof.
  • an optimized nucleotide sequence encoding an anti-MerTK antibody described herein or a fragment thereof hybridizes under high stringency, intermediate or lower stringency hybridization conditions to an antisense polynucleotide of an unoptimized nucleotide sequence encoding an anti-MerTK antibody described herein or a fragment thereof.
  • Information regarding hybridization conditions have been described, see, e.g., U.S. Patent Application Publication No. US 2005/0048549 (e.g., paragraphs 72-73), which is incorporated herein by reference.
  • polynucleotides can be obtained, and the nucleotide sequence of the
  • nucleotide sequences encoding antibodies described herein and modified versions of these antibodies can be determined using methods well known in the art, i.e., nucleotide codons known to encode particular amino acids are assembled in such a way to generate a nucleic acid that encodes the antibody.
  • nucleotide codons known to encode particular amino acids are assembled in such a way to generate a nucleic acid that encodes the antibody.
  • Such a polynucleotide encoding the antibody can be assembled from chemically synthesized
  • oligonucleotides (e.g., as described in Kutmeier et al., 1994, BioTechniques 17:242), which, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligating of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.
  • a polynucleotide encoding an antibody described herein can be generated from nucleic acid from a suitable source (e.g., a hybridoma) using methods well known in the art (e.g., PCR and other molecular cloning methods). For example, PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of a known sequence can be performed using genomic DNA obtained from hybridoma cells producing the antibody of interest. Such PCR amplification methods can be used to obtain nucleic acids comprising the sequence encoding the light chain and/or heavy chain of an antibody.
  • a suitable source e.g., a hybridoma
  • methods well known in the art e.g., PCR and other molecular cloning methods.
  • PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of a known sequence can be performed using genomic DNA obtained from hybridoma cells producing the antibody of interest.
  • Such PCR amplification methods can
  • Such PCR amplification methods can be used to obtain nucleic acids comprising the sequence encoding the variable light chain region and/or the variable heavy chain region of an antibody.
  • the amplified nucleic acids can be cloned into vectors for expression in host cells and for further cloning, for example, to generate chimeric and humanized antibodies.
  • immunoglobulin can be chemically synthesized or obtained from a suitable source (e.g., an antibody cDNA library or a cDNA library generated from, or nucleic acid, preferably poly A+ RNA, isolated from, any tissue or cells expressing the antibody, such as hybridoma cells selected to express an antibody described herein) by PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence to identify, e.g., a cDNA clone from a cDNA library that encodes the antibody. Amplified nucleic acids generated by PCR can then be cloned into replicable cloning vectors using any method well known in the art.
  • a suitable source e.g., an antibody cDNA library or a cDNA library generated from, or nucleic acid, preferably poly A+ RNA, isolated from, any tissue or cells expressing the antibody, such
  • DNA encoding anti-MerTK antibodies described herein can 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 anti-MerTK antibodies).
  • Hybridoma cells can serve as a source of such DNA. Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells such as E.
  • coli cells simian COS cells, Chinese hamster ovary (CHO) cells (e.g., CHO cells from the CHO GS SystemTM (Lonza)), or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of anti-MerTK antibodies in the recombinant host cells.
  • CHO Chinese hamster ovary
  • myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of anti-MerTK antibodies in the recombinant host cells.
  • PCR primers including VH or VL nucleotide sequences, a restriction site, and a flanking sequence to protect the restriction site can be used to amplify the VH or VL sequences in scFv clones.
  • the PCR amplified VH domains can be cloned into vectors expressing a heavy chain constant region, e.g., the human gamma 4 constant region, and the PCR amplified VL domains can be cloned into vectors expressing a light chain constant region, e.g., human kappa or lambda constant regions.
  • the vectors for expressing the VH or VL domains comprise an EF-la promoter, a secretion signal, a cloning site for the variable domain, constant domains, and a selection marker such as neomycin.
  • VH and VL domains can also be cloned into one vector expressing the necessary constant regions.
  • the heavy chain conversion vectors and light chain conversion vectors are then co-transfected into cell lines to generate stable or transient cell lines that express full-length antibodies, e.g., IgG, using techniques known to those of skill in the art.
  • the DNA also can be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the murine sequences, or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide.
  • polynucleotides described herein hybridize under high stringency, intermediate or lower stringency hybridization conditions to polynucleotides encoding a VH chain region and/or VL chain region provided herein.
  • Hybridization conditions have been described in the art and are known to one of skill in the art.
  • hybridization under stringent conditions can involve hybridization to filter-bound DNA in 6x sodium chloride/sodium citrate (SSC) at about 45° C followed by one or more washes in 0.2xSSC/0.1% SDS at about 50-65° C;
  • hybridization under highly stringent conditions can involve hybridization to filter-bound nucleic acid in 6xSSC at about 45° C followed by one or more washes in O. lxSSC/0.2% SDS at about 68° C.
  • Hybridization under other stringent hybridization conditions are known to those of skill in the art and have been described, see, for example, Ausubel, F.M. et al., eds., 1989, Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York at pages 6.3.1-6.3.6 and 2.10.3.
  • cells e.g., host cells
  • cells expressing (e.g., recombinantly) antibodies described herein (or an antigen-binding fragment thereof) which specifically bind to an ECD of human MerTK and related polynucleotides and expression vectors.
  • vectors e.g., expression vectors
  • host cells comprising such vectors for recombinantly expressing anti-MerTK antibodies described herein (e.g., human or humanized antibody).
  • Recombinant expression of an antibody described herein e.g., a full-length antibody, heavy and/or light chain of an antibody, or a single chain antibody described herein
  • an antibody described herein e.g., a full-length antibody, heavy and/or light chain of an antibody, or a single chain antibody described herein
  • Recombinant expression of an antibody described herein involves construction of an expression vector containing a polynucleotide that encodes the antibody.
  • a polynucleotide encoding an antibody molecule, heavy and/or light chain of an antibody, or a fragment thereof (e.g., heavy and/or light chain variable domains) described herein has been obtained, the vector for the production of the antibody molecule can be produced by recombinant DNA technology using techniques well- known in the art.
  • a polynucleotide containing an antibody or antibody fragment (e.g., light chain or heavy chain) encoding nucleotide sequence are described herein.
  • Methods which are well known to those skilled in the art can be used to construct expression vectors containing antibody or antibody fragment (e.g., light chain or heavy chain) coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination.
  • replicable vectors comprising a nucleotide sequence encoding an antibody molecule described herein, a heavy or light chain of an antibody, a heavy or light chain variable domain of an antibody or a fragment thereof, or a heavy or light chain CDR, operably linked to a promoter.
  • Such vectors can, for example, include the nucleotide sequence encoding the constant region of the antibody molecule (see, e.g., International Publication Nos. WO 86/05807 and WO 89/01036; and U.S. Patent No. 5, 122,464) and variable domains of the antibody can be cloned into such a vector for expression of the entire heavy, the entire light chain, or both the entire heavy and light chains.
  • An expression vector can be transferred to a cell (e.g., host cell) by conventional techniques and the resulting cells can then be cultured by conventional techniques to produce an antibody described herein (e.g., an antibody comprising the CDRs of any of Table 1, Table 3-7, or Table 11-14) or a fragment thereof.
  • a cell e.g., host cell
  • the resulting cells can then be cultured by conventional techniques to produce an antibody described herein (e.g., an antibody comprising the CDRs of any of Table 1, Table 3-7, or Table 11-14) or a fragment thereof.
  • an antibody described herein e.g., an antibody comprising the CDRs of any of Table 1, Table 3-7, or Table 11-14
  • a single chain antibody described herein e.g., an antibody comprising the CDRs of any of Table 1, Table 3-7, or Table 11-14
  • vectors encoding both the heavy and light chains can be co-expressed in the host cell for expression of the entire immunoglobulin molecule, as detailed below.
  • a host cell contains a vector comprising a polynucleotide encoding both the heavy chain and light chain of an antibody described herein (e.g., an antibody comprising the CDRs of any of Table 1, Table 3-7, or Table 11-14), or a fragment thereof.
  • a host cell contains two different vectors, a first vector comprising a polynucleotide encoding a heavy chain or a heavy chain variable region of an antibody described herein (e.g., an antibody comprising the CDRs of any of Table 1, Table 3- 7, or Table 11-14), or a fragment thereof, and a second vector comprising a polynucleotide encoding a light chain or a light chain variable region of an antibody described herein (e.g., an antibody comprising the CDRs of any of Table 1, Table 3-7, or Table 11-14), or a fragment thereof.
  • a first host cell comprises a first vector comprising a
  • a second host cell comprises a second vector comprising a polynucleotide encoding a light chain or a light chain variable region of an antibody described herein (e.g., an antibody comprising the CDRs of any of Table 1, Table 3-7, or Table 11-14).
  • a heavy chain/heavy chain variable region expressed by a first cell associated with a light chain/light chain variable region of a second cell to form an anti-MerTK antibody described herein (e.g., antibody comprising the CDRs of any of Table 1, Table 3-7, or Table 11- 14) or an antigen-binding fragment thereof.
  • an anti-MerTK antibody described herein e.g., antibody comprising the CDRs of any of Table 1, Table 3-7, or Table 11- 14
  • a population of host cells comprising such first host cell and such second host cell.
  • a population of vectors comprising a first vector comprising a polynucleotide encoding a light chain/light chain variable region of an anti-MerTK antibody described herein (e.g., antibody comprising the CDRs of any of Table 1, Table 3-7, or Table 11-14), and a second vector comprising a polynucleotide encoding a heavy chain/heavy chain variable region of an anti-MerTK antibody described herein (e.g., antibody comprising the CDRs of any of Table 1, Table 3-7, or Table 11-14).
  • an anti-MerTK antibody described herein e.g., antibody comprising the CDRs of any of Table 1, Table 3-7, or Table 11-14
  • a second vector comprising a polynucleotide encoding a heavy chain/heavy chain variable region of an anti-MerTK antibody described herein (e.g., antibody comprising the CDRs of any of Table 1, Table 3-7, or Table 11-14).
  • a variety of host-expression vector systems can be utilized to express antibody molecules described herein (e.g., an antibody comprising the CDRs of any of Table 1, Table 3-7, or Table 11-14) (see, e.g., U.S. Patent No. 5,807,715).
  • Such host-expression systems represent vehicles by which the coding sequences of interest can be produced and subsequently purified, but also represent cells which can, when transformed or transfected with the appropriate nucleotide coding sequences, express an antibody molecule described herein in situ. These include but are not limited to microorganisms such as bacteria (e.g., E. coli and B.
  • subtilis transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Saccharomyces Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing antibody coding sequences; plant cell systems (e.g., green algae such as Chlamydomonas reinhardtii) infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing antibody coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, MDCK, HEK 293, NS0, PER.C6, VERO, CRL7030, HsS78
  • cells for expressing antibodies described herein are CHO cells, for example CHO cells from the CHO GS SystemTM (Lonza).
  • a mammalian expression vector is pOptiVECTM or pcDNA3.3.
  • bacterial cells such as Escherichia coli, or eukaryotic cells (e.g., mammalian cells), especially for the expression of whole recombinant antibody molecule, are used for the expression of a recombinant antibody molecule.
  • mammalian cells such as Chinese hamster ovary (CHO) cells
  • CHO Chinese hamster ovary
  • a vector such as the major intermediate early gene promoter element from human cytomegalovirus
  • antibodies described herein are produced by CHO cells or NSO cells.
  • the expression of nucleotide sequences encoding antibodies described herein which specifically bind to human MerTK is regulated by a constitutive promoter, inducible promoter or tissue specific promoter.
  • a number of expression vectors can be advantageously selected depending upon the use intended for the antibody molecule being expressed.
  • vectors which direct the expression of high levels of fusion protein products that are readily purified can be desirable.
  • Such vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruther et al., 1983, EMBO 12: 1791), in which the antibody coding sequence can be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, 1985, Nucleic Acids Res.
  • pGEX vectors can also be used to express foreign polypeptides as fusion proteins with glutathione 5-transferase (GST).
  • GST glutathione 5-transferase
  • fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to matrix glutathione agarose beads followed by elution in the presence of free glutathione.
  • the pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.
  • Autographa californica nuclear polyhedrosis virus (AcNPV), for example, can be used as a vector to express foreign genes.
  • the virus grows in Spodoptera frugiperda cells.
  • the antibody coding sequence can be cloned individually into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).
  • AcNPV Autographa californica nuclear polyhedrosis virus
  • mammalian host cells a number of viral -based expression systems can be utilized.
  • the antibody coding sequence of interest can be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence.
  • This chimeric gene can then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region El or E3) will result in a recombinant virus that is viable and capable of expressing the antibody molecule in infected hosts (e.g., see Logan & Shenk, 1984, Proc. Natl. Acad. Sci. USA 8 1 :355-359).
  • Specific initiation signals can also be required for efficient translation of inserted antibody coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression can be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see, e.g., Bittner et al., 1987, Methods in Enzymol. 153 :51-544).
  • a host cell strain can be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products can be important for the function of the protein.
  • Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed.
  • eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and
  • Such mammalian host cells include but are not limited to CHO, VERO, BHK, Hela, COS, MDCK, HEK 293, NIH 3T3, W138, BT483,
  • anti-MerTK antibodies described herein are produced in mammalian cells, such as CHO cells.
  • stable expression cells For long-term, high-yield production of recombinant proteins, stable expression cells can be generated.
  • cell lines which stably express an anti-MerTK antibody described herein e.g., an antibody comprising the CDRs of any of Table 1, Table 3-7, or Table 11-14
  • an antigen-binding fragment thereof can be engineered.
  • a cell provided herein stably expresses a light chain/light chain variable domain and a heavy chain/heavy chain variable domain which associate to form an antibody described herein (e.g., an antibody comprising the CDRs of any of Table 1, Table 3-7, or Table 11-14) or an antigen- binding fragment thereof.
  • host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker.
  • appropriate expression control elements e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.
  • engineered cells can be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media.
  • the selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci, which in turn can be cloned and expanded into cell lines.
  • Such engineered cell lines can be particularly useful in screening and evaluation of compositions that interact directly or indirectly with the antibody molecule.
  • a number of selection systems can be used, including but not limited to, the herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11 :223), hypoxanthineguanine phosphoribosyltransferase (Szybalska & Szybalski, 1992, Proc. Natl. Acad. Sci. USA 48:202), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:8-17) genes can be employed in tk-, hgprt- or aprt-cells, respectively.
  • antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., 1980, Natl. Acad. Sci. USA 77:357; O'Hare et al., 1981, Proc. Natl. Acad. Sci. USA
  • the expression levels of an antibody molecule can be increased by vector
  • the host cell can be co-transfected with two or more expression vectors described herein, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide.
  • the two vectors can contain identical selectable markers which enable equal expression of heavy and light chain polypeptides.
  • the host cells can be co-transfected with different amounts of the two or more expression vectors.
  • host cells can be transfected with any one of the following ratios of a first expression vector and a second expression vector: 1 : 1, 1 :2, 1 :3, 1 :4, 1 :5, 1 :6, 1 :7, 1 :8, 1 :9, 1 : 10, 1 : 12, 1 : 15, 1 :20, 1 :25, 1 :30, 1 :35, 1 :40, 1 :45, or 1 :50.
  • a single vector can be used which encodes, and is capable of expressing, both heavy and light chain polypeptides.
  • the light chain should be placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, 1986, Nature 322:52; and Kohler, 1980, Proc. Natl. Acad. Sci. USA 77:2197-2199).
  • the coding sequences for the heavy and light chains can comprise cDNA or genomic DNA.
  • the expression vector can be monocistronic or multicistronic.
  • a multicistronic nucleic acid construct can encode 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, or in the range of 2-5, 5-10 or 10-20 genes/nucleotide sequences.
  • a bicistronic nucleic acid construct can comprise in the following order a promoter, a first gene (e.g., heavy chain of an antibody described herein), and a second gene and (e.g., light chain of an antibody described herein).
  • a promoter e.g., a promoter
  • a first gene e.g., heavy chain of an antibody described herein
  • a second gene and e.g., light chain of an antibody described herein.
  • the transcription of both genes can be driven by the promoter, whereas the translation of the mRNA from the first gene can be by a cap-dependent scanning mechanism and the translation of the mRNA from the second gene can be by a cap-independent mechanism, e.g., by an IRES.
  • an antibody molecule described herein has been produced by recombinant expression, it can be purified by any method known in the art for purification of an
  • immunoglobulin molecule for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column
  • chromatography e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column
  • antibodies described herein can be fused to heterologous polypeptide sequences described herein or otherwise known in the art to facilitate purification.
  • an antibody described herein is isolated or purified.
  • an isolated antibody is one that is substantially free of other antibodies with different antigenic specificities than the isolated antibody.
  • a preparation of an antibody described herein is substantially free of cellular material and/or chemical precursors.
  • substantially free of cellular material includes preparations of an antibody in which the antibody is separated from cellular components of the cells from which it is isolated or recombinantly produced.
  • an antibody that is substantially free of cellular material includes preparations of antibody having less than about 30%, 20%, 10%, 5%, 2%), 1%), 0.5%), or 0.1%) (by dry weight) of heterologous protein (also referred to herein as a "contaminating protein") and/or variants of an antibody, for example, different post-translational modified forms of an antibody or other different versions of an antibody (e.g., antibody fragments).
  • heterologous protein also referred to herein as a "contaminating protein”
  • variants of an antibody for example, different post-translational modified forms of an antibody or other different versions of an antibody (e.g., antibody fragments).
  • the antibody is recombinantly produced, it is also generally substantially free of culture medium, i.e., culture medium represents less than about 20%, 10%>, 2%, 1%, 0.5%, or 0.1%) of the volume of the protein preparation.
  • the antibody When the antibody is produced by chemical synthesis, it is generally substantially free of chemical precursors or other chemicals, i.e., it is separated from chemical precursors or other chemicals which are involved in the synthesis of the protein. Accordingly, such preparations of the antibody have less than about 30%, 20%, 10%, or 5%) (by dry weight) of chemical precursors or compounds other than the antibody of interest.
  • antibodies described herein are isolated or purified.
  • compositions, pharmaceutical compositions, and kits comprising one or more antibodies (e.g., anti-MerTK antibodies comprising CDRs of any of Table 1, Table 3-7, or Table 11-14, for example an antibody comprising the VL and VH sequences of Table 2, an antibody comprising the VL and VH sequences of Table 8, an antibody comprising the VL and VH sequences of Table 9, or an antibody comprising the VL and VH sequences of Table 10) described herein, or antigen-binding fragments thereof, or conjugates thereof.
  • compositions (e.g., pharmaceutical compositions) described herein can be for in vitro, in vivo, or ex vivo uses.
  • Non-limiting examples of uses include uses to modulate (e.g., inhibit) MerTK activity and uses to manage or treat a disorder, for example, cancer.
  • a pharmaceutical composition comprising an antibody (e.g., a humanized antibody) described herein (or an antigen-binding fragment thereof) and a pharmaceutically acceptable carrier or excipient.
  • the term "pharmaceutically acceptable” means being approved by a regulatory agency of the Federal or a state government, or listed in the U.S. Pharmacopeia, European Pharmacopeia or other generally recognized Pharmacopeia for use in animals, and more particularly in humans.
  • Therapeutic formulations containing one or more antibodies provided herein e.g., antibodies comprising CDRs of any of Table 1, Table 3-7, or Table 11-14, for example an antibody comprising the VL and VH sequences of Table 2, an antibody comprising the VL and VH sequences of Table 8, an antibody comprising the VL and VH sequences of Table 9, or an antibody comprising the VL and VH sequences of Table 10) or an antigen-binding fragment thereof can be prepared for storage by mixing the antibody having the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers (Remington's
  • Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; and/or non-ionic surfactants such as TWEENTM, PLURONICSTM or polyethylene glycol (PEG).
  • Formulations can also contain more than one active compounds (for example, molecules, e.g., antibody or antibodies described herein) as necessary for the particular indication being treated.
  • formulations comprise an antibody provided herein and one or more active compounds with complementarity activities that do not adversely affect each other.
  • Such molecules are suitably present in combination in amounts that are effective for the purpose intended.
  • an antibody described herein can be combined with one or more other therapeutic agents (e.g., a tyrosine kinase inhibitor such as imatinib mesylated or sunitinib, or a histone deacetylase inhibitor such as vorinostat).
  • a tyrosine kinase inhibitor such as imatinib mesylated or sunitinib
  • a histone deacetylase inhibitor such as vorinostat
  • the formulations to be used for in vivo administration can be sterile. This is readily accomplished by filtration through, e.g., sterile filtration membranes.
  • compositions provided herein contain therapeutically effective amounts of one or more of the antibodies provided herein, and optionally one or more additional prophylactic of therapeutic agents, in a pharmaceutically acceptable carrier.
  • Such pharmaceutical compositions are useful in the prevention, treatment, management or amelioration of a condition or disorder described herein or one or more symptoms thereof.
  • compositions suitable for administration of the antibodies provided herein include any such carriers known to those skilled in the art to be suitable for the particular mode of administration.
  • antibodies described herein can be formulated as the sole
  • compositions can be combined with other active ingredients (such as one or more other prophylactic or therapeutic agents).
  • compositions provided herein can contain one or more antibodies provided herein (e.g., antibodies comprising CDRs of any of Table 1, Table 3-7, or Table 11-14, for example an antibody comprising the VL and VH sequences of Table 2, an antibody comprising the VL and VH sequences of Table 8, an antibody comprising the VL and VH sequences of Table 9, or an antibody comprising the VL and VH sequences of Table 10) or an antigen-binding fragment thereof.
  • the antibodies are formulated into suitable pharmaceutical preparations, such as solutions, suspensions, powders, sustained release formulations or elixirs in sterile solutions or suspensions for parenteral administration, or as transdermal patch preparation and dry powder inhalers.
  • compositions provided herein one or more antibodies described herein is (are) mixed with a suitable pharmaceutical carrier.
  • concentrations of the antibody or antibodies in the compositions can, for example, be effective for delivery of an amount, upon administration, that treats, prevents, or ameliorates a condition or disorder described herein or a symptom thereof.
  • compositions provided herein are formulated for single dosage administration.
  • the weight fraction of compound is dissolved, suspended, dispersed or otherwise mixed in a selected carrier at an effective concentration such that the treated condition is relieved, prevented, or one or more symptoms are ameliorated.
  • an antibody provided herein e.g., antibody comprising CDRs of any of Table 1, Table 3-7, or Table 11-14, for example an antibody comprising the VL and VH sequences of Table 2, an antibody comprising the VL and VH sequences of Table 8, an antibody comprising the VL and VH sequences of Table 9, or an antibody comprising the VL and VH sequences of Table 10) is included in the pharmaceutically acceptable carrier in an effective amount sufficient to exert a therapeutically useful effect in the absence of, or with minimal or negligible, undesirable side effects on the patient treated.
  • compositions comprising anti-MerTK antibody in a pharmaceutical composition provided herein will depend on, e.g., the physicochemical characteristics of the antibody, the dosage schedule, and amount administered as well as other factors known to those of skill in the art.
  • compositions in another embodiment, provide a dosage of about 50 mg of antibody per kilogram of body weight for administration over a period of time, e.g., every day or few days, every week, every 2 weeks, or every 3 weeks.
  • Pharmaceutical dosage unit forms can be prepared to provide from about 500 mg.
  • compositions described herein are provided for administration to humans or animals (e.g., mammals) in unit dosage forms, such as sterile parenteral (e.g., intravenous) solutions or suspensions containing suitable quantities of the compounds or pharmaceutically acceptable derivatives thereof.
  • Pharmaceutical compositions are also provided for administration to humans and animals in unit dosage form, such as tablets, capsules, pills, powders, granules, and oral or nasal solutions or suspensions, and oil-water emulsions containing suitable quantities of an anti-MerTK antibody or pharmaceutically acceptable derivatives thereof.
  • the antibody is, in one embodiment, formulated and administered in unit-dosage forms or multiple-dosage forms.
  • Unit-dose forms as used herein refers to physically discrete units suitable for human or animal (e.g., mammal) subjects and packaged individually as is known in the art. Each unit-dose contains a predetermined quantity of an anti-MerTK antibody sufficient to produce the desired therapeutic effect, in association with the required pharmaceutical carrier, vehicle or diluent. Examples of unit-dose forms include ampoules and syringes and individually packaged tablets or capsules. Unit-dose forms can be administered in fractions or multiples thereof.
  • a multiple-dose form is a plurality of identical unit-dosage forms packaged in a single container to be administered in segregated unit-dose form. Examples of multiple-dose forms include vials, bottles of tablets or capsules or bottles. Hence, in specific aspects, multiple dose form is a multiple of unit-doses which are not segregated in packaging.
  • one or more anti-MerTK antibodies described herein e.g., antibodies comprising CDRs of any of Table 1, Table 3-7, or Table 11-14, for example an antibody comprising the VL and VH sequences of Table 2, an antibody comprising the VL and VH sequences of Table 8, an antibody comprising the VL and VH sequences of Table 9, or an antibody comprising the VL and VH sequences of Table 10) or an antigen-binding fragment thereof are in a liquid pharmaceutical formulation.
  • Liquid pharmaceutically administrable compositions can, for example, be prepared by dissolving, dispersing, or otherwise mixing an antibody and optional pharmaceutical adjuvants in a carrier, such as, for example, water, saline, aqueous dextrose, glycerol, glycols, and the like, to thereby form a solution or suspension.
  • a pharmaceutical composition provided herein to be administered can also contain minor amounts of nontoxic auxiliary substances such as wetting agents, emulsifying agents, solubilizing agents, and pH buffering agents and the like.
  • Parenteral administration in one embodiment, is characterized by injection, either subcutaneously, intramuscularly or intravenously is also contemplated herein.
  • injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions.
  • the injectables, solutions and emulsions also contain one or more excipients. Suitable excipients are, for example, water, saline, dextrose, glycerol or ethanol.
  • Other routes of administration may include, enteric administration, intracerebral administration, nasal administration, intraarterial administration, intracardiac administration, intraosseous infusion, intrathecal administration, and intraperitoneal administration.
  • Preparations for parenteral administration include sterile solutions ready for injection, sterile dry soluble products, such as lyophilized powders, ready to be combined with a solvent just prior to use, including hypodermic tablets, sterile suspensions ready for injection, sterile dry insoluble products ready to be combined with a vehicle just prior to use and sterile emulsions.
  • the solutions can be either aqueous or nonaqueous.
  • suitable carriers include physiological saline or phosphate buffered saline (PBS), and solutions containing thickening and solubilizing agents, such as glucose, polyethylene glycol, and polypropylene glycol and mixtures thereof.
  • PBS physiological saline or phosphate buffered saline
  • thickening and solubilizing agents such as glucose, polyethylene glycol, and polypropylene glycol and mixtures thereof.
  • Pharmaceutically acceptable carriers used in parenteral preparations include aqueous vehicles, nonaqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, sequestering or chelating agents and other pharmaceutically acceptable substances.
  • Pharmaceutical carriers also include ethyl alcohol, polyethylene glycol and propylene glycol for water miscible vehicles; and sodium hydroxide, hydrochloric acid, citric acid or lactic acid for pH adjustment.
  • intravenous or intraarterial infusion of a sterile aqueous solution containing an anti-MerTK antibody described herein is an effective mode of
  • Another embodiment is a sterile aqueous or oily solution or suspension containing an anti-MerTK antibody described herein injected as necessary to produce the desired pharmacological effect.
  • an anti-MerTK antibody described herein can be suspended in micronized or other suitable form.
  • the form of the resulting mixture depends upon a number of factors, including the intended mode of administration and the solubility of the compound in the selected carrier or vehicle.
  • the pharmaceutical formulations are lyophilized powders, which can be reconstituted for administration as solutions, emulsions and other mixtures. They can also be reconstituted and formulated as solids or gels.
  • the lyophilized powder is prepared by dissolving an anti-MerTK antibody provided herein, in a suitable solvent.
  • the lyophilized powder is sterile.
  • Suitable solvents can contain an excipient which improves the stability or other pharmacological component of the powder or reconstituted solution, prepared from the powder. Excipients that can be used include, but are not limited to, dextrose, sorbital, fructose, corn syrup, xylitol, glycerin, glucose, sucrose or other suitable agent.
  • a suitable solvent can also contain a buffer, such as citrate, sodium or potassium phosphate or other such buffer known to those of skill in the art at, in one embodiment, about neutral pH.
  • lyophilized powder can be stored under appropriate conditions, such as at about 4 °C to room temperature.
  • Reconstitution of this lyophilized powder with water for injection provides a formulation for use in parenteral administration.
  • the lyophilized powder is added to sterile water or other suitable carrier.
  • anti-MerTK antibodies provided herein can be formulated for local administration or topical application, such as for topical application to the skin and mucous membranes, such as in the eye, in the form of gels, creams, and lotions and for application to the eye or for intraci sternal or intraspinal application.
  • Topical administration is contemplated for transdermal delivery and also for administration to the eyes or mucosa, or for inhalation therapies.
  • Nasal solutions of the active compound alone or in combination with other pharmaceutically acceptable excipients can also be administered.
  • Anti-MerTK antibodies and other compositions provided herein can also be formulated to be targeted to a particular tissue, receptor, or other area of the body of the subject to be treated. Many such targeting methods are well known to those of skill in the art. All such targeting methods are contemplated herein for use in the instant compositions. For non-limiting examples of targeting methods, see, e.g., U.S. Patent Nos.
  • anti-MerTK antibodies described herein are targeted (or otherwise administered) to the visual organs, bone marrow, gastrointestinal tract, lungs, brain, or joints.
  • an anti-MerTK antibody described herein is capable of crossing the blood-brain barrier.
  • a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions described herein, such as one or more anti-MerTK antibodies provided herein.
  • Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
  • kits that can be used in the above methods.
  • a kit comprises an antibody described herein, preferably a purified antibody, in one or more containers.
  • kits described herein contain a substantially isolated MerTK antigen (e.g., ECD of human MerTK) as a control.
  • the kits described herein further comprise a control antibody which does not react with a MerTK antigen.
  • kits described herein contain one or more elements for detecting the binding of a modified antibody to a MerTK antigen (e.g., the antibody can be conjugated to a detectable substrate such as a fluorescent compound, an enzymatic substrate, a radioactive compound or a luminescent compound, or a second antibody which recognizes the first antibody can be conjugated to a detectable substrate).
  • a kit provided herein can include a recombinantly produced or chemically synthesized MerTK antigen.
  • the MerTK antigen provided in the kit can also be attached to a solid support.
  • the detecting means of the above described kit includes a solid support to which a MerTK antigen is attached.
  • Such a kit can also include a non-attached reporter-labeled anti-human antibody or anti-mouse/rat antibody. In this embodiment, binding of the antibody to the MerTK antigen can be detected by binding of the said reporter-labeled antibody.
  • an anti-MerTK antibody e.g., an antibody comprising CDRs of any of Table 1, Table 3-7, or Table 11-14, for example an antibody comprising the VL and VH sequences of Table 2, an antibody comprising the VL and VH sequences of Table 8, an antibody comprising the VL and VH sequences of Table 9, or an antibody comprising the VL and VH sequences of Table 10) described herein or an antigen-binding fragment thereof.
  • the anti- MerTK antibody is a MerTK antagonist.
  • provided herein are methods of inhibiting (e.g., partially inhibiting) MerTK activity with an anti-MerTK antibody described herein which is a MerTK antagonist. In certain embodiments, provided herein are methods of managing or treating a condition or disorder using an anti-MerTK antibody described herein which is a MerTK antagonist or inhibitor.
  • an anti-MerTK antibody e.g., an antibody comprising CDRs of any of Table 1, Table 3-7, or Table 11-14, for example an antibody comprising the VL and VH sequences of Table 2, an antibody comprising the VL and VH sequences of Table 8, an antibody comprising the VL and VH sequences of Table 9, or an antibody comprising the VL and VH sequences of Table 10.
  • an anti-MerTK antibody e.g., an antibody comprising CDRs of any of Table 1, Table 3-7, or Table 11-14, for example an antibody comprising the VL and VH sequences of Table 2, an antibody comprising the VL and VH sequences of Table 8, an antibody comprising the VL and VH sequences of Table 9, or an antibody comprising the VL and VH sequences of Table 10.
  • Non-limiting examples of a condition which can be treated or managed with a MerTK antagonist include cancer, sepsis, and infection, described in more detail below.
  • a condition which can be treated or managed with a MerTK antagonist is one which can be managed or treated by enhancing an immune response.
  • An anti- MerTK antibody described herein or an antigen-binding fragment thereof, which is a MerTK antagonist for use in the methods provided herein is capable of inhibiting (e.g., partially inhibiting) or decreasing/reducing MerTK expression and/or a MerTK activity. Activities of MerTK are known in the art.
  • an anti-MerTK antibody described herein which is a MerTK antagonist inhibits (e.g., partially inhibits) one or more of the following MerTK activities: phosphorylation (e.g., autophosphorylation) of MerTK receptor (e.g., cytoplasmic domain of MerTK, such as cytoplasmic kinase domain of MerTK), promotion of phagocytosis, modulation of maturation of NK cells, dendritic cells or macrophages, inhibition of toll-like receptor (TLR) activation of dendritic cells, inhibition or decrease in TLR-induced proinflammatory cytokine (e.g., T F, IL-6, IL-12 and type I interferon production, increase in SOCSl expression, association with interleukin-15 (IL-15) to protect cells against TNF-induced cell death (e.g., fibroblasts and DCs), and activation of Statl in dendritic cells.
  • phosphorylation e.g., autophosphorylation
  • an anti -MerTK antibody described herein which is a MerTK antagonist inhibits (e.g., partially inhibits), by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 97%, 98%, 99%, or 100%, one or more such MerTK activities.
  • an antibody described herein e.g., an antibody comprising CDRs of any of Table 1, Table 3-7, or Table 11-14, for example an antibody comprising the VL and VH sequences of Table 2, an antibody comprising the VL and VH sequences of Table 8, an antibody comprising the VL and VH sequences of Table 9, or an antibody comprising the VL and VH sequences of Table 10) or antigen-binding fragment thereof which specifically binds to human MerTK and inhibits MerTK.
  • an antibody described herein e.g., an antibody comprising CDRs of any of Table 1, Table 3-7, or Table 11-14, for example an antibody comprising the VL and VH sequences of Table 2, an antibody comprising the VL and VH sequences of Table 8, an antibody comprising the VL and VH sequences of Table 9, or an antibody comprising the VL and VH sequences of Table 10
  • an antibody described herein e.g., an antibody comprising CDRs of any of Table 1, Table 3-7, or Table 11-14, for example an antibody
  • a subject in need of enhancing an immune response include an immunocompromised subject, for example a subject suffering from an infection (e.g., bacterial, viral, fungal, or protozoan).
  • an infection e.g., bacterial, viral, fungal, or protozoan.
  • an infection e.g., bacterial, viral, fungal, or protozoan.
  • immunocompromised subject has cancer and is undergoing, or had undergone treatment with, anti-cancer therapy, such as, chemotherapy.
  • anti-cancer therapy such as, chemotherapy.
  • immunocompromised subjects include, but are not limited to, subjects who are HIV positive, subjects with AIDS or SCID, subjects with diabetes, and subjects who have undergone transplants and are taking immunosuppressants.
  • an immunocompromised subject include, but are not limited to, subjects who are HIV positive, subjects with AIDS or SCID, subjects with diabetes, and subjects who have undergone transplants and are taking immunosuppressants.
  • immunocompromised subject is taking immunosuppressants (e.g., steroids) to manage or treat asthma, arthritis (e.g., rheumatoid arthritis), or allergy or an allergic condition.
  • immunosuppressants e.g., steroids
  • arthritis e.g., rheumatoid arthritis
  • allergy e.g., allergy or an allergic condition.
  • a method of enhancing an immune response to a vaccine in a subject comprising administering to a subject in need thereof, who is or has been administered the vaccine, an effective amount of an antibody described herein (e.g., an antibody comprising CDRs of any of Table 1, Table 3-7, or Table 11-14, for example an antibody comprising the VL and VH sequences of Table 2, an antibody comprising the VL and VH sequences of Table 8, an antibody comprising the VL and VH sequences of Table 9, or an antibody comprising the VL and VH sequences of Table 10) or antigen-binding fragment thereof which specifically binds to human MerTK and inhibits MerTK.
  • the antibody is administered before, concurrently, or after administration of the vaccine.
  • An example of vaccines in the methods provides herein include cancer/tumor vaccines, such as vaccines comprising tumor cells or tumor cell lysates and vaccines comprising dendritic cells or dendritic cell lysates activated against a tumor.
  • cancer/tumor vaccines such as vaccines comprising tumor cells or tumor cell lysates and vaccines comprising dendritic cells or dendritic cell lysates activated against a tumor.
  • an antibody described herein e.g., an antibody comprising CDRs of any of Table 1, Table 3-7, or Table 11-14, for example an antibody comprising the VL and VH sequences of Table 2, an antibody comprising the VL and VH sequences of Table 8, an antibody comprising the VL and VH sequences of Table 9, or an antibody comprising the VL and VH sequences of Table 10) or antigen-binding fragment thereof which specifically binds to human MerTK and inhibits a MerTK activity or inhibits or reduces MerTK expression.
  • an antibody described herein e.g., an antibody comprising CDRs of any of Table 1, Table 3-7, or Table 11-14, for example an antibody comprising the VL and VH sequences of Table 2, an antibody comprising the VL and VH sequences of Table 8, an antibody comprising the VL and VH sequences of Table 9, or an antibody comprising the VL and VH sequences of Table 10.
  • such a method involves inhibition of tumor growth, cancer cell proliferation, cancer cell migration, inhibition of tumor-associated macrophage activity (e.g., tumor-promoting activity such as production of cytokines), and modulating stromal cells such as macrophages or dendritic cells.
  • tumor-associated macrophage activity e.g., tumor-promoting activity such as production of cytokines
  • stromal cells such as macrophages or dendritic cells.
  • Non-limiting examples of cancers to be managed, prevented, protected against, or treated in the methods provided herein include breast cancer, pancreatic cancer, leukemia, lung cancer such as non-small cell lung cancer, glioblastoma, melanoma, prostate cancer, colon cancer, gastric cancer, pituitary adenomas, ovarian cancer, renal cancer, bladder cancer, and sarcomas, including rhabdomyosarcomas.
  • a method of treating cancer in a subject comprising administering to a subject in need thereof an antibody described herein (e.g., an antibody comprising CDRs of any of Table 1, Table 3-7, or Table 11-14, for example an antibody comprising the VL and VH sequences of Table 2, an antibody comprising the VL and VH sequences of Table 8, an antibody comprising the VL and VH sequences of Table 9, or an antibody comprising the VL and VH sequences of Table 10) or antigen-binding fragment thereof which specifically binds to human MerTK and inhibits MerTK, in an effective amount to enhancing an immune response to the cancer.
  • an antibody described herein e.g., an antibody comprising CDRs of any of Table 1, Table 3-7, or Table 11-14, for example an antibody comprising the VL and VH sequences of Table 2, an antibody comprising the VL and VH sequences of Table 8, an antibody comprising the VL and VH sequences of Table 9, or an antibody comprising the VL and VH sequences of Table 10
  • a method of managing, preventing, protecting against, or treating metastasis in a subject comprising administering to a subject in need thereof a therapeutically effective amount of an antibody described herein (e.g., an antibody comprising CDRs of any of Table 1, Table 3-7, or Table 11-14, for example an antibody comprising the VL and VH sequences of Table 2, an antibody comprising the VL and VH sequences of Table 8, an antibody comprising the VL and VH sequences of Table 9, or an antibody comprising the VL and VH sequences of Table 10) or antigen-binding fragment thereof which specifically binds to human MerTK and inhibits a MerTK activity or inhibits or reduces MerTK expression.
  • an antibody described herein e.g., an antibody comprising CDRs of any of Table 1, Table 3-7, or Table 11-14, for example an antibody comprising the VL and VH sequences of Table 2, an antibody comprising the VL and VH sequences of Table 8, an antibody comprising the VL and VH sequences of Table 9, or an
  • a method of inhibiting MerTK expression and/or activity of a tumor-associated macrophage comprising contacting a tumor-associated macrophage with an effective amount of an antibody described herein (e.g., an antibody comprising CDRs of any of Table 1, Table 3-7, or Table 11-14, for example an antibody comprising the VL and VH sequences of Table 2, an antibody comprising the VL and VH sequences of Table 8, an antibody comprising the VL and VH sequences of Table 9, or an antibody comprising the VL and VH sequences of Table 10) or antigen-binding fragment thereof which specifically binds to human MerTK and inhibits a MerTK activity or inhibits or reduces MerTK expression.
  • an antibody described herein e.g., an antibody comprising CDRs of any of Table 1, Table 3-7, or Table 11-14, for example an antibody comprising the VL and VH sequences of Table 2, an antibody comprising the VL and VH sequences of Table 8, an antibody comprising the VL and VH sequences of Table 9, or an antibody
  • a method of modulating (e.g., inhibiting) stromal cell (e.g., macrophage or dendritic cell) activity comprising contacting a stromal cell with an effective amount of an antibody described herein (e.g., an antibody comprising CDRs of any of Table 1, Table 3-7, or Table 11-14, for example an antibody comprising the VL and VH sequences of Table 2, an antibody comprising the VL and VH sequences of Table 8, an antibody comprising the VL and VH sequences of Table 9, or an antibody comprising the VL and VH sequences of Table 10) or antigen-binding fragment thereof which specifically binds to human MerTK and inhibits a MerTK activity or inhibits or reduces MerTK expression in the stromal cell.
  • an antibody described herein e.g., an antibody comprising CDRs of any of Table 1, Table 3-7, or Table 11-14, for example an antibody comprising the VL and VH sequences of Table 2, an antibody comprising the VL and VH sequences of Table 8,
  • a method of modulating (e.g., inhibiting) natural killer (NK) cell activity comprising contacting an NK cell with an effective amount of an antibody described herein (e.g., an antibody comprising CDRs of any of Table 1, Table 3-7, or Table 11-14, for example an antibody comprising the VL and VH sequences of Table 2, an antibody comprising the VL and VH sequences of Table 8, an antibody comprising the VL and VH sequences of Table 9, or an antibody comprising the VL and VH sequences of Table 10) or antigen-binding fragment thereof which specifically binds to human MerTK and inhibits a MerTK activity or inhibits or reduces MerTK expression in the NK cell.
  • an antibody described herein e.g., an antibody comprising CDRs of any of Table 1, Table 3-7, or Table 11-14, for example an antibody comprising the VL and VH sequences of Table 2, an antibody comprising the VL and VH sequences of Table 8, an antibody comprising the VL and VH sequences of Table 9, or an
  • a method of modulating (e.g., inhibiting) T cell activity comprising contacting a T cell with an effective amount of an antibody described herein (e.g., an antibody comprising CDRs of any of Table 1, Table 3-7, or Table 11-14, for example an antibody comprising the VL and VH sequences of Table 2, an antibody comprising the VL and VH sequences of Table 8, an antibody comprising the VL and VH sequences of Table 9, or an antibody comprising the VL and VH sequences of Table 10) or antigen-binding fragment thereof which specifically binds to human MerTK and inhibits a MerTK activity or inhibits or reduces MerTK expression in the T cell.
  • an antibody described herein e.g., an antibody comprising CDRs of any of Table 1, Table 3-7, or Table 11-14, for example an antibody comprising the VL and VH sequences of Table 2, an antibody comprising the VL and VH sequences of Table 8, an antibody comprising the VL and VH sequences of Table 9, or an antibody comprising the V
  • a method of inhibiting or reducing tumor growth or cancer cell proliferation in a subject comprising administering to a subject in need thereof a therapeutically effective amount of an antibody described herein (e.g., an antibody comprising CDRs of any of Table 1, Table 3-7, or Table 11-14, for example an antibody comprising the VL and VH sequences of Table 2, an antibody comprising the VL and VH sequences of Table 8, an antibody comprising the VL and VH sequences of Table 9, or an antibody comprising the VL and VH sequences of Table 10) or antigen-binding fragment thereof which specifically binds to human MerTK and inhibits a MerTK activity or inhibits or reduces MerTK expression.
  • an antibody described herein e.g., an antibody comprising CDRs of any of Table 1, Table 3-7, or Table 11-14, for example an antibody comprising the VL and VH sequences of Table 2, an antibody comprising the VL and VH sequences of Table 8, an antibody comprising the VL and VH sequences of Table 9, or an antibody comprising
  • a method of treating cancer in a subject comprising administering (e.g., administering concurrently or sequentially) to a subject in need thereof (i) an antibody described herein (e.g., an antibody comprising CDRs of any of Table 1, Table 3-7, or Table 11-14, for example an antibody comprising the VL and VH sequences of Table 2, an antibody comprising the VL and VH sequences of Table 8, an antibody comprising the VL and VH sequences of Table 9, or an antibody comprising the VL and VH sequences of Table 10) or antigen-binding fragment thereof which specifically binds to human MerTK and inhibits MerTK, and (ii) another anti-cancer agent.
  • an antibody described herein e.g., an antibody comprising CDRs of any of Table 1, Table 3-7, or Table 11-14, for example an antibody comprising the VL and VH sequences of Table 2, an antibody comprising the VL and VH sequences of Table 8, an antibody comprising the VL and VH sequences of Table 9, or an antibody compris
  • a method of managing, preventing, protecting against, or treating an infection in a subject comprising administering to a subject in need thereof a therapeutically effective amount of an antibody described herein (e.g., an antibody comprising CDRs of any of Table 1, Table 3-7, or Table 11-14, for example an antibody comprising the VL and VH sequences of Table 2, an antibody comprising the VL and VH sequences of Table 8, an antibody comprising the VL and VH sequences of Table 9, or an antibody comprising the VL and VH sequences of Table 10) or antigen-binding fragment thereof which specifically binds to human MerTK and inhibits a MerTK activity.
  • an antibody described herein e.g., an antibody comprising CDRs of any of Table 1, Table 3-7, or Table 11-14, for example an antibody comprising the VL and VH sequences of Table 2, an antibody comprising the VL and VH sequences of Table 8, an antibody comprising the VL and VH sequences of Table 9, or an antibody comprising the VL and VH sequences
  • the infection is an infection caused by bacteria (gram- negative bacteria or gram-positive bacteria), fungi, viruses, or parasites.
  • a method of managing or treating sepsis in a subject comprising administering to a subject in need thereof a therapeutically effective amount of an antibody described herein (e.g., an antibody comprising CDRs of any of Table 1, Table 3-7, or Table 11-14, for example an antibody comprising the VL and VH sequences of Table 2, an antibody comprising the VL and VH sequences of Table 8, an antibody comprising the VL and VH sequences of Table 9, or an antibody comprising the VL and VH sequences of Table 10) or antigen-binding fragment thereof which specifically binds to human MerTK and inhibits a MerTK activity.
  • an antibody described herein e.g., an antibody comprising CDRs of any of Table 1, Table 3-7, or Table 11-14, for example an antibody comprising the VL and VH sequences of Table 2, an antibody comprising the VL and VH sequences of Table 8, an antibody comprising the VL and VH sequences of Table 9, or an antibody comprising the VL and VH sequences of Table 10.
  • Sepsis a systemic inflammatory response syndrome or SIRS
  • SIRS systemic inflammatory response syndrome
  • bacteria gram-negative bacteria or gram-positive bacteria
  • fungi fungi
  • viruses or parasites.
  • Stages of sepsis include, but are not limited to, onset of sepsis, severe sepsis, and septic shock. Severe sepsis can be accompanied by organ dysfunction.
  • the sepsis is advanced sepsis.
  • the sepsis is characterized with increased expression of SOCS, for example during the stage of severe sepsis.
  • the sepsis is characterized with elevated levels of circulating Gas6 in a subject with sepsis.
  • Levels or concentrations of circulating Gas6 can be measured from blood samples using conventional methods, such as ELISAs.
  • Levels or concentrations of Gas6 can be compared with a reference value to determine whether Gas6 levels or concentrations are elevated in a subject with sepsis.
  • the reference values can be a set standard range of levels and concentrations of Gas6 accepted in the art as correlating with those of a population of healthy subjects not suffering from sepsis.
  • the reference values can be levels of concentrations of Gas6 in a subject prior to the onset of sepsis or at the early stages of sepsis.
  • Symptoms of sepsis include, but are not limited to, high fevers, hot, flushed skin, elevated heart rate, hyperventilation, altered mental status, swelling, and low blood pressure.
  • a method of managing, alleviating or treating one or more symptoms of sepsis in a subject comprising administering to a subject in need thereof a therapeutically effective amount of an antibody described herein (e.g., an antibody comprising CDRs of any of Table 1, Table 3-7, or Table 11-14, for example an antibody comprising the VL and VH sequences of Table 2, an antibody comprising the VL and VH sequences of Table 8, an antibody comprising the VL and VH sequences of Table 9, or an antibody comprising the VL and VH sequences of Table 10) or antigen-binding fragment thereof which specifically binds to human MerTK and inhibits a MerTK activity.
  • an antibody described herein e.g., an antibody comprising CDRs of any of Table 1, Table 3-7, or Table 11-14, for example an antibody comprising the VL and VH sequences of Table 2, an antibody comprising the VL and VH sequences of Table 8, an antibody comprising the VL and VH sequences of Table 9, or an antibody comprising the VL and V
  • the method of managing or treating sepsis provided herein further comprises the step of administering an antibiotic to a subject in need thereof, for example, prior to, concurrently with, or subsequent to administering an anti-MerTK antibody described herein.
  • an antibody described herein e.g., an antibody comprising CDRs of any of Table 1, Table 3-7, or Table 11-14, for example an antibody comprising the VL and VH sequences of Table 2, an antibody comprising the VL and VH sequences of Table 8, an antibody comprising the VL and VH sequences of Table 9, or an antibody comprising the VL and VH sequences of Table 10) or antigen-binding fragment thereof which specifically binds to human MerTK and inhibits a MerTK activity.
  • an antibody described herein e.g., an antibody comprising CDRs of any of Table 1, Table 3-7, or Table 11-14, for example an antibody comprising the VL and VH sequences of Table 2, an antibody comprising the VL and VH sequences of Table 8, an antibody comprising the VL and VH sequences of Table 9, or an antibody comprising the VL and VH sequences of Table 10.
  • the infection is caused by microbes, e.g., bacteria, viruses or parasites.
  • the viral infection is HIV, herpes, shingles, influenza, common cold, and encephalitis.
  • provided herein are methods for prevent or inhibit entry of a viruses by contacting a cell to an effective amount of an antibody described herein (e.g., an antibody comprising CDRs of any of Table 1, Table 3-7, or Table 11-14, for example an antibody comprising the VL and VH sequences of Table 2, an antibody comprising the VL and VH sequences of Table 8, an antibody comprising the VL and VH sequences of Table 9, or an antibody comprising the VL and VH sequences of Table 10) or antigen-binding fragment thereof which specifically binds to human MerTK and inhibits a MerTK activity.
  • the method provided herein prevents or inhibits entry of lymphocytic
  • choriomeningitis virus HIV
  • herpes virus e.g., herpes simplex virus or herpes zoster
  • influenza virus or common cold virus.
  • the anti -MerTK antibody or antigen-binding fragment thereof for use in the methods provided herein is an antibody (e.g., monoclonal antibody, such as a humanized monoclonal antibody) comprising CDRs of any of Table 1, Table 3-7, or Table 11- 14, for example an antibody comprising the VL and VH sequences of Table 2, an antibody comprising the VL and VH sequences of Table 8, an antibody comprising the VL and VH sequences of Table 9, or an antibody comprising the VL and VH sequences of Table 10.
  • the anti-MerTK antibody inhibits a MerTK activity.
  • kits for enhancing a type I interferon (IFN) response against a pathogen in a subject comprising administering to a subject in need thereof an effective amount of an anti-MerTK antibody described herein, such as a MerTK antagonist antibody.
  • an anti-MerTK antibody described herein such as a MerTK antagonist antibody.
  • administer refers to the act of injecting or otherwise physically delivering a substance (e.g., a humanized anti-MerTK antibody provided herein or an antigen-binding fragment thereof) to a subject or a patient (e.g., human), such as by mucosal, topical, intradermal, parenteral, intravenous, intramuscular delivery and/or any other method of physical delivery described herein or known in the art.
  • a substance e.g., a humanized anti-MerTK antibody provided herein or an antigen-binding fragment thereof
  • the terms "effective amount” or “therapeutically effective amount” refer to an amount of a therapy (e.g., an antibody or pharmaceutical composition provided herein) which is sufficient to reduce and/or ameliorate the severity and/or duration of a given condition, disorder or disease and/or a symptom related thereto. These terms also encompass an amount necessary for the reduction, slowing, or amelioration of the advancement or progression of a given disease, reduction, slowing, or amelioration of the recurrence, development or onset of a given disease, and/or to improve or enhance the prophylactic or therapeutic effect(s) of another therapy (e.g., a therapy other than an anti-MerTK antibody provided herein). In some embodiments, "effective amount” as used herein also refers to the amount of an antibody described herein to achieve a specified result.
  • a therapy e.g., an antibody or pharmaceutical composition provided herein
  • the term "in combination” in the context of the administration of other therapies refers to the use of more than one therapy.
  • the use of the term “in combination” does not restrict the order in which therapies are administered.
  • the therapies may be
  • the terms “manage,” “managing,” and “management” refer to the beneficial effects that a subject derives from a therapy (e.g., a prophylactic or therapeutic agent), which does not result in a cure of a condition associated with MerTK.
  • a subject is administered one or more therapies (e.g., prophylactic or therapeutic agents, such as an antibody described herein) to "manage” a condition or disorder described herein, one or more symptoms thereof, so as to prevent the progression or worsening of the condition or disorder.
  • therapies e.g., prophylactic or therapeutic agents, such as an antibody described herein
  • the terms "impede” or “impeding” in the context of a condition or disorder provided herein refer to the total or partial inhibition (e.g., less than 100%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or 5%) or blockage of the development, recurrence, onset or spread of a condition or disorder provided herein and/or symptom related thereto, resulting from the administration of a therapy or combination of therapies provided herein (e.g., a combination of prophylactic or therapeutic agents, such as an antibody described herein).
  • a therapy or combination of therapies provided herein (e.g., a combination of prophylactic or therapeutic agents, such as an antibody described herein).
  • prophylactic agent refers to any agent that can totally or partially inhibit the development, recurrence, onset or spread of a condition or disorder provided herein, and/or symptom related thereto in a subject.
  • prophylactic agent refers to any agent that can totally or partially inhibit the development, recurrence, onset or spread of a condition or disorder provided herein, and/or symptom related thereto in a subject.
  • the term “prophylactic agent” refers to any agent that can totally or partially inhibit the development, recurrence, onset or spread of a condition or disorder provided herein, and/or symptom related thereto in a subject.
  • the term “prophylactic agent” refers to any agent that can totally or partially inhibit the development, recurrence, onset or spread of a condition or disorder provided herein, and/or symptom related thereto in a subject.
  • the term “prophylactic agent” refers to any agent that can totally or partially inhibit the development, recurrence, onset or spread of
  • prophylactic agent refers to an antibody described herein. In certain other embodiments, the term “prophylactic agent” refers to an agent other than an antibody described herein.
  • a prophylactic agent is an agent which is known to be useful to or has been or is currently being used to prevent a condition or disorder provided herein, and/or a symptom related thereto or impede the onset, development, progression and/or severity of a condition or disorder provided herein, and/or a symptom related thereto.
  • the prophylactic agent is an anti-MerTK antibody, as described herein.
  • a subject is a mammal such as a non-primate (e.g., cows, pigs, horses, cats, dogs, goats, rabbits, rats, mice, etc.) or a primate (e.g., monkey and human), for example a human.
  • the subject is a mammal, e.g., a human, diagnosed with a condition or disorder provided herein.
  • the subject is a mammal, e.g., a human, at risk of developing a condition or disorder provided herein.
  • the subject is human.
  • the terms “therapies” and “therapy” can refer to any protocol(s), method(s), compositions, formulations, and/or agent(s) that can be used in the prevention, treatment, management, or amelioration of a condition or disorder or symptom thereof (e.g., a condition or disorder provided herein or one or more symptoms or condition associated therewith).
  • the terms “therapies” and “therapy” refer to drug therapy, adjuvant therapy, radiation, surgery, biological therapy, supportive therapy, and/or other therapies useful in treatment, management, prevention, or amelioration of a condition or disorder or one or more symptoms thereof.
  • the term “therapy” refers to a therapy other than an anti-MerTK antibody described herein or pharmaceutical composition thereof.
  • an “additional therapy” and “additional therapies” refer to a therapy other than a treatment using an anti-MerTK antibody described herein or pharmaceutical composition thereof.
  • a therapy includes the use of an anti-MerTK antibody described herein as an adjuvant therapy. For example, using an anti-MerTK antibody described herein in conjunction with a drug therapy, biological therapy, surgery, and/or supportive therapy.
  • the term “therapeutic agent” refers to any agent that can be used in the treatment, management or amelioration of a condition or disorder or symptom thereof.
  • the term “therapeutic agent” refers to an anti-MerTK antibody described herein or an antigen-binding fragment thereof.
  • the term “MerTK antibody” refers to an anti-MerTK antibody described herein or an antigen-binding fragment thereof.
  • therapeutic agent refers to an agent other than an antibody described herein.
  • a therapeutic agent is an agent which is known to be useful for, or has been or is currently being used for the treatment, management or amelioration of a condition or disorder provided herein or one or more symptoms or condition associated therewith or one or more symptoms related thereto.
  • an antibody or antigen-binding fragment thereof that specifically binds to human MerTK as described herein can be administered in combination with another therapeutic for addressing one or more of the indications described herein.
  • a therapeutic can be a stimulator of T cell responses.
  • such a therapeutic can be an immune checkpoint inhibitor, e.g., a PD-1, PD-L1, or CTLA-4 inhibitor.
  • Such exemplary agents can include, e.g., YervoyTM (ipilimumab) or tremelimumab (to CTLA-4), BMS-936558/nivolumab (to PD-1), MK-3475/pembrolizumab (to PD-1).
  • a therapeutic can be, for example, CDX-1127 (to CD27), CP- 870893 (to CD40), lucatumumab (to CD40), or dacetuzumab (to CD40).
  • anti-MerTK antibodies described herein and antigen-binding fragments thereof, which specifically bind to an ECD of human MerTK can be used for diagnostic purposes to detect, diagnose, or monitor a condition described herein (e.g., a condition involving MerTK and/or abnormal MerTK signaling and/or abnormal MerTK expression).
  • a condition described herein e.g., a condition involving MerTK and/or abnormal MerTK signaling and/or abnormal MerTK expression.
  • anti-MerTK antibodies described herein or an antigen-binding fragment thereof for use in diagnostic purposes are labeled.
  • kits for the detection of a condition described herein comprising: (a) assaying the expression of MerTK in cells or a tissue sample of a subject using one or more antibodies described herein or an antigen-binding fragment thereof; and (b) comparing the level of MerTK expression with a control level, e.g., levels in normal tissue samples (e.g., from a patient not having a condition described herein, or from the same patient before onset of the condition), whereby an increase or decrease in the assayed level of MerTK expression compared to the control level of MerTK expression is indicative of a condition described herein.
  • a control level e.g., levels in normal tissue samples (e.g., from a patient not having a condition described herein, or from the same patient before onset of the condition)
  • Antibodies described herein can be used to assay MerTK levels in a biological sample using classical immunohistological methods as described herein or as known to those of skill in the art (e.g., see Jalkanen et al., 1985, J. Cell. Biol. 101 :976-985; and Jalkanen et al., 1987, J. Cell. Biol. 105:3087-3096).
  • Other antibody-based methods useful for detecting protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA).
  • Suitable antibody assay labels include enzyme labels, such as, glucose oxidase; radioisotopes, such as iodine (1251, 1211), carbon (14C), sulfur (35S), tritium (3H), indium (121In), and technetium (99Tc); luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, and biotin.
  • enzyme labels such as, glucose oxidase
  • radioisotopes such as iodine (1251, 1211), carbon (14C), sulfur (35S), tritium (3H), indium (121In), and technetium (99Tc)
  • luminescent labels such as luminol
  • fluorescent labels such as fluorescein and rhodamine, and biotin.
  • monitoring of a condition described herein is carried out by repeating the method for diagnosing for a period of time after initial diagnosis.
  • Presence of the labeled molecule can be detected in the subject using methods known in the art for in vivo scanning. Skilled artisans will be able to determine the appropriate method for detecting a particular label. Methods and devices that may be used in the diagnostic methods include, but are not limited to, computed tomography (CT), whole body scan such as position emission tomography (PET), magnetic resonance imaging (MRI), and sonography.
  • CT computed tomography
  • PET position emission tomography
  • MRI magnetic resonance imaging
  • sonography sonography
  • a phage display screen was employed to identify single-chain antibodies (scFvs) that bind the extracellular domain of human and/or mouse MerTK.
  • a phage-display selection strategy aimed at discovering specific binders to human or mouse MerTK was employed. Two separate sets of selections were performed: solid-phase and solution-phase. For solid-phase selections, the tubes were coated at 10 ⁇ g/mL with antigen while for solution-phase selections, 100 nM of biotinylated antigen was used. Also, antigens with two separate tags (rCD4 and Fc) were used for selections.
  • polyclonal phage ELISA was carried out to confirm the generation of binding antibodies to the selected antigens. All of the selected populations were tested against all of the antigens. As a positive control, an unrelated antigen and its selected population were used. In addition, a commercially sourced Fc-tagged human MerTK antigen was employed. In order to assess the level of tag binders, two irrelevant proteins, one with Fc tag and the other with rCD4 tag were used. Rescued phages were incubated with coated antigens in ELISA plates and the binding detected by mouse anti-M13 antibody and europium labeled anti- mouse antibody. The signals were read as time resolved fluorescence (TRF) measurements.
  • TRF time resolved fluorescence
  • Selected antibody populations were ligated into a vector enabling expression of soluble single chain antibody (scFv) fused with a C-terminal FLAG tag and expressed downstream of a leader sequence directing its secretion into the bacterial periplasm.
  • the ligated products were transformed into E. coli BL21 (DE3) cells.
  • the design facilitates rapid screening of antibodies for their ability to bind a given antigen directly from the expression media. Post-transformation, colonies were tested using PCR for the presence of an scFv insert.
  • clones were first expressed in auto-induction media. Bacterial supernatant was used to test binding to the antigen on which they were selected. From primary screening a number of hits were obtained. Clones with a signal of more than 20X of background were cherry picked and subjected to a secondary ELISA binder screen in order to identify tag binders and eliminate them from future analysis. For the tag-binder ELISA, Fc-hexa-his and rCD4-hexa-his, purified in-house, were used as the antigen.
  • DNA sequences of both the heavy and light chains of scFv were used to identify unique antibody sequences. See Table 1 and Table 2 (VL and VH CDR amino acid sequences (Table 1) and VL and VH amino acid sequences (Table 2) of Ab2000).
  • Transfection quality DNA was prepared, and HEK293 transfections was performed at 50 mL culture volume for the unique clones.
  • the anti-MerTK antibody was titrated on mouse L cells that were engineered to express full length human MerTK in a stable manner, and further incubated with a fluorescent anti-human secondary antibody as follows.
  • Mouse L cells originally derived from subcutaneous connective tissue from male C3H/An mice (ATCC CRL-2648), and their derivatives engineered to stably express full-length, wild-type human MerTK, were maintained in complete DMEM containing 10% heat inactivated fetal bovine serum (Gibco), 2 mM GlutaMax (Gibco), Penicillin/Streptomycin (Gibco), and 3 ⁇ g/mL puromycin (Invivogen).
  • L cells stably expressing full length human or mouse MerTK were transduced with retroviruses based on the pLCPX vector (Clontech) carrying the gene of interest.
  • L cells expressing full length human MerTK ( M_006343.2) were obtained by transduction with a codon optimized MerTK cDNA cloned into pLCPX vector.
  • Stably transduced L cells were selected in media containing 3 ⁇ g/mL puromycin, sorted to purity, and maintained in growth media containing 3 ⁇ g/ml puromycin.
  • L cells were resuspended at lxlO 6 cell/mL in lx FACS buffer (PBS, 0.5% BSA, 0.09% sodium azide). L cells were plated at 100 ⁇ /well or lxlO 5 cells/well. Unlabeled antibodies were added to the cells, concentrations ranging between 6 pM to 500 nM. After 30 min on ice, cells were washed and resuspended in 100 ⁇ of lx FACS buffer containing PE- labeled donkey anti-human IgG, cells were washed and resuspended in 100 ⁇ of lx FACS buffer containing live/dead stain (7-AAD).
  • lx FACS buffer PBS, 0.5% BSA, 0.09% sodium azide
  • the software that operates the HyperCyt autosampler calculates and reports the mean fluorescent intensity values (MFI).
  • MFI mean fluorescent intensity values
  • Figure 1 shows the mean fluorescent intensity plotted as a function of the log- transformed molar concentration of the antibody.
  • Ab2000 binds to cellularly expressed full length human MerTK in a dose responsive manner, with EC50 values in the picomolar to nanomolar range.
  • Biotinylated human Gas6 was captured to a streptavidin microtiter plate (Pierce) at 5 ⁇ g/mL.
  • Goat anti-mouse IgG (Fc specific) antibody was added.
  • MerTK binding was then detected with a horseradish peroxidase conjugated donkey anti -goat IgG (H&L) antibody and TMB substrate (KPL).
  • the MerTK extracellular domain contains two N-terminal immunoglobulin- like domains (here named Dl and D2, respectively) followed by two Fibronectin-III domains (here named D3 and D4, respectively).
  • Table 15 utilizing standard one letter amino acid codes, shows the amino acid sequences of the proteins encoding each of the 4 domains that comprise the human MerTK extracellular domain.
  • Table 15 Amino acid sequences of the proteins encoding each of the 4 domains that comprise the human MerTK extracellular domain
  • ECD extracellular domain
  • biotinylation (Avi) tag and a hexahistidine tag were engineered at the 5' end of each construct.
  • Expression vectors encoding these constructs were transfected in Expi293 cells, and conditioned media containing each fusion protein was purified by nickel-affinity chromatography and buffer exchanged to phosphate buffer saline (PBS).
  • PBS phosphate buffer saline
  • ECD extracellular domain
  • THP-1 is a suspension human monocytic cell line derived from an acute monocytic leukemia patient.
  • THP-1 cells in exponential growth phase were seeded at a density of 40,000 cells/well in a 24-well plate in complete media and differentiated into macrophages with 100 ng/ml phorbol 12-myristate 13-acetate (PMA) (Abeam) for 72 hours, followed by a 24 hour recovery period during which PMA is removed.
  • PMA phorbol 12-myristate 13-acetate
  • THP-1 cells were starved for 3 hours.
  • Anti-MerTK mAb was added to a final concentration of 100 nM for 1 hour at 37°C.
  • Figures 3 A and 3B show inhibition of Gas6-dependent MerTK phosphorylation by the antibody Ab2000. Titration of Ab2000 on MerTK expressing THP1 cells inhibited Gas6- dependent MerTK phosphorylation with an IC50 value of approximately 0.2 nM ( Figure 3B). Addition of Ab2000 to concentrations as high as 10000 nM elicited only very weak MerTK phosphorylation in the absence of exogenously added Gas6 (as compared to independently- added Gas6) as shown in Figure 4.
  • Example 6 Modulation of pro-inflammatory cytokine release from human macrophages and dendritic cells by anti-MerTK monoclonal antibodies
  • Ab2000 was tested for its ability to modulate TNF-a secretion from human macrophages stimulated with various TLR agonists, e.g., LPS and resiquimod (R848) as shown below. These assays were performed on primary human macrophages, or U937 monocyte- derived macrophages.
  • TLR agonists e.g., LPS and resiquimod (R848) as shown below.
  • U937 cells Human U937 monocytes (ATCC CRL-1593.2), were fully differentiated into monocyte-derived macrophages with Phorbol 12-myristate 13-acetate (PMA) (Abeam) at 10 ng/ml for 72 hours. U937 macrophages were serum starved for 3 hours, and anti-MerTK mAbs added at 100 nM for 1 hour prior to treatment with 100 ng/mL ultra-pure lipopoly saccharide (LPS) (Invivogen) for 24 hours. Cell-free supernatants were assayed for the presence of TNF-a using an ELISA-based method as described by the manufacturer (Biolegend).
  • PMA Phorbol 12-myristate 13-acetate
  • LPS ultra-pure lipopoly saccharide
  • Macrophages were derived from human monocytes as follows: peripheral blood mononuclear cells (PBMCs) were added to a tissue culture plate and monocytes allowed to adhere for ⁇ 2 hours at 37°C and 6% CO2. The non-adherent cells were removed and the monocytes differentiated for 3 days in RPMI containing 10% FBS and 5 ng/mL PMA (Sigma). After differentiation, the serum containing media was removed from the wells, and the wells were washed with RPMI only.
  • PBMCs peripheral blood mononuclear cells
  • the cells were then incubated in the presence of anti-MerTK antibody or appropriate isotype control and LPS (InvivoGen) or resiquimod (R848) at 100 ng/mL overnight at 37°C and 6% CO2. All reagents were prepared in RPMI (no serum). The supernatant was collected, clarified and either used immediately or stored frozen for cytokine analysis. Production of TNF- ⁇ was evaluated by ELISA (R&D Systems).
  • Anti-MerTK Ab2000 was also tested for its ability to modulate TNF-a secretion from human dendritic cells stimulated with various TLR agonists. FACS analysis was performed to demonstrate that human primary dendritic cells express MerTK on their surface.
  • Dendritic cell differentiation and cytokine release Dendritic cells were derived from human monocytes as follows: peripheral blood mononuclear cells (PBMCs) were added to a T175 cm 2 flasks and monocytes allowed to adhere for 2 hours at 37°C in 6% CO2. The nonadherent cells were removed and the monocytes cultured for 7 days in RPMI containing 10% FBS, 10 ng/mL IL-4 (R&D Systems) and 100 ng/mL GM-CSF (R&D Systems). The cells were harvested, washed in RPMI (no serum) and counted.
  • PBMCs peripheral blood mononuclear cells
  • the cells were then incubated in the presence of anti-MerTK mAbs or appropriate isotype control and LPS (InvivoGen) at 5 ng/mL overnight at 37°C and 6% CO2. TNF-a production from cell free supernatants was measured by ELISA (R&D Systems).
  • the Ab2000 antibody was affinity matured using methods as described in Holland et al., 2013, J. Immun. Methods 394:55-61.
  • Ab2000-A7, Ab2000-Fl and Ab2000-G6 were assayed for their ability to bind to purified MerTK-ECD by ELISA.
  • Microtiter plates were coated with recombinant human MerTK-msFc in PBS, and then blocked with 5% bovine serum albumin in PBS.
  • Protein A purified human mAbs and an isotype control (hulgGl) were added at indicated concentrations and incubated at 37°C (Figure 9).
  • Protein A purified human mAbs were incubated with MerTK-L cells and SK-mel-5 cells at room temperature on a plate shaker. After 20 minutes, the cells were washed with PBS containing 0.1% BSA and 0.05% NaN 3 (PBA) and the bound antibodies were detected by incubating the cells with a PE labeled goat anti-human IgG Fc-specific probe. The excess probe was washed from the cells with PBA and the cell associated fluorescence was determined by analysis using a FACSCanto IITM instrument (BD Biosciences, NJ, USA) according to the manufacturer's directions.
  • PBA PBS containing 0.1% BSA and 0.05% NaN 3
  • Affinity matured antibodies showed increased binding to huMerTK-L cells (Figure 10A) and SK-mel-5 cells (Figure 10B) compared to parental antibody.
  • the human mAbs demonstrated high level binding to cells expressing human MerTK as a function of antibody concentration.
  • Figure 12A and 12B depict representative binding curves of the A2000-A7 affinity matured antibody in these cells. Similar results were observed for Ab2000-A7 IgGl and IgG2 antibodies ("Gl" and "G2,” respectively). Controls were hulgGl and hulgG2 isotype control antibodies.
  • Example 9 Modulation of pro-inflammatory cytokine release from human dendritic cells by affinity matured anti-MerTK monoclonal antibodies
  • Dendritic cell differentiation and cytokine release Ab2000-A7 was tested for its ability to modulate cytokine production in dendritic cells.
  • PBMCs peripheral blood mononuclear cells
  • monocytes were allowed to adhere for approximately 2 hours at 37°C, 6% CO2.
  • the non-adherent cells were removed and the monocytes were cultured for 7 days in RPMI containing 10% FBS, 10 ng/mL IL-4 (R&D Systems) and 100 ng/mL GM-CSF (R&D Systems).
  • the cells were harvested, washed in RPMI (no serum) and counted.
  • IL-1RA interleukin 1 receptor antagonist
  • TNF-alpha TNF-alpha
  • Dendritic cells were analyzed by Eve Technologies (Alberta, Canada) using multiplex assay for 30 different cytokines, chemokines and growth factors, including fibroblast growth factor 2 (FGF-2), eotaxin-1 (CCL11), transforming growth factor alpha (TGF-a) granulocyte- colony stimulating factor (G-CSF), Fms-related tyrosine kinase 3 ligand (Flt-3L), granulocyte macrophage-colony stimulating factor (GM-CSF), fractalkine (CX3CL1), interferon alpha-2 (IFN-a2), interferon-gamma (IFN- ⁇ ), growth-regulated oncogene alpha (GRO alpha), interleukin-10 (IL-10), monocyte chemotactic protein 3 (MCP-3), interleukin-12 p40 (IL-12P40), macrophage-derived chemokine (MDC), platelet-derived growth factor AA homodimer (FGF-2),
  • PBMCs peripheral blood mononuclear cells
  • PBMCs peripheral blood mononuclear cells
  • Purified PBMCs were plated at l .OxlO 6 cells/well in a 24-well plate and treated with 100 nM monoclonal antibody for 24 hours.
  • the lower cytokine activity observed in PBMCs relative to DCs may relate to the relatively low abundance of CD14+ cells in PBMCs, as well as their undifferentiated state, suggesting that MerTK may play a more prominent role in DCs and macrophages than in undifferentiated monocytes.
  • PBMCs Peripheral blood mononuclear cells
  • PBMCs depleted of CD14+ monocytes were plated at l .OxlO 6 cells/well in a 24-well plate and treated with 100 nM monoclonal antibody for 24 hours. Following 24 hour treatment, cell-free supernatants were collected and stored at - 80°C prior to assessing cytokine IL-IRA secretion by ELISA (R&D Systems). Mean IL-IRA levels in intact or CD14+ depleted PBMCs from 3 different donors are shown in Figure 17. CD14+ cell depletion abolishes IL-IRA secretion by Ab2000-A7 ( Figure 17). These data demonstrate that IL-IRA is produced chiefly by CD14+ cells, which also express higher MerTK levels than T, B or K cells in PBMCs from the same donors.
  • Macrophages were derived from human monocytes as follows: PBMCs were added to a T175 cm 2 flasks and monocytes allowed to adhere for approximately 2 hours at 37°C, 6% CO2. The non-adherent cells were removed and the monocytes cultured for 7 days in RPMI containing 10% FBS and 50ng/mL MCSF (R&D Systems). The cells were harvested and frozen for future use.
  • Binding affinity and binding kinetics of various human anti-MerTK antibodies were examined by bio-layer interferometry (BLI) using an OctetTM QK e instrument (Pall ForteBio, Menlo Park, CA) according to the manufacturer's guidelines.
  • AHC Anti -Human Fc Capture
  • Binding was determined by exposing six of the antibody -loaded biosensors to soluble human MerTK-CD4-Biotin-HIS analyte (Celldex, 77.5kD by SDS-PAGE). Affinity

Abstract

Provided herein are compositions, methods and uses involving antibodies that specifically bind to MerTK, a receptor tyrosine kinase, and modulate the expression and/or activity of MerTK. Also provided are uses and methods for managing, treating, or preventing disorders, such as cancer.

Description

ANTI-MERTK ANTIBODIES AND METHODS OF USE THEREOF
Cross-Reference to Related Applications
[0001] This application claims benefit of U.S. Provisional Application No. 62/577,601, filed October 26, 2017, the entire contents of which is incorporated herein by reference.
Reference to Sequence Listing Submitted Electronically
[0002] This application incorporates by reference a Sequence Listing submitted with this application as text file entitled "12638-146-228_SEQLISTING_Final.txt" created on October 25, 2018 and having a size of 50, 192 bytes.
1. FIELD
[0003] Provided herein are compositions, methods and uses involving antibodies that specifically bind to MerTK, a receptor tyrosine kinase, and modulate the expression and/or activity of MerTK for managing, treating, or preventing disorders, such as cancer.
2. BACKGROUND
[0004] MerTK, a transmembrane receptor, belongs to the TAM (Tyro3, Axl and MerTK) family of receptor tyrosine kinases. The extracellular domain of MerTK has two
immunoglobulin (Ig) and two fibronectin (FN) type III motifs (Graham et al. (2014) Nat Rev Cancer. ;14: 769-785; Rothlin et al. (2015) Annu Rev Immunol. 33 : 355-391; Lemke G. (2013) Cold Spring Harb. Perspect Biol. 5).
[0005] Two closely related ligands, Protein S (PROS1) and Growth arrest specific gene 6 (Gas6) bind and activate MerTK. Activation of PI3K and its downstream target, serine/threonine protein kinase Akt, is an important aspect of MerTK-dependent signal transduction. MerTK stimulation of the PI3K/Akt pathway in dendritic cells and macrophages leads to deactivation of NFKB, thus decreasing TNF-a production. Thus, MerTK plays an essential role in attenuation of proinflammatory cytokine responses. [0006] The TAM family of receptor tyrosine kinases also display ectopic or overexpression in numerous cancers. In tumors, activation of TAM signaling cascades appears to promote an anti-inflammatory and immunosuppressive microenvironment, in turn driving tumor growth. Overexpression and activation of MerTK has been implicated in lymphoid leukemia, lymphoma, adenoma, melanoma, gastric, prostate and breast cancers.
[0007] There is a need for therapies modulating MerTK to manage, treat or prevent conditions involving MerTK and/or abnormal MerTK signaling or abnormal MerTK expression.
3. SUMMARY
[0008] In one aspect, provided herein are antibodies, including antigen-binding fragments, which specifically bind to MerTK, for example, the extracellular domain (ECD) of MerTK. In one embodiment, antibodies and antigen binding fragments presented herein specifically bind the ECD of human MerTK. Also provided herein are polynucleotides and vectors comprising sequences encoding such antibodies, cells comprising such polynucleotides and vectors, and compositions, reagents and kits comprising such antibodies. In another aspect, provided herein are methods for modulating MerTK activity, e.g., inhibiting MerTK activity, diagnostic methods and uses, and therapeutic methods and uses of such anti-MerTK antibodies.
[0009] In a particular embodiment, provided herein is an isolated antibody, or an antigen- binding fragment thereof, which specifically binds to human MerTK, comprising: (i) a light chain variable region (VL) comprising SEQ ID NO: 2; and/or (ii) a heavy chain variable region (VH) comprising SEQ ID NO: 1.
[0010] In another particular embodiment, provided here is an isolated antibody, or an antigen-binding fragment thereof, which specifically binds to human MerTK, comprising: (i) a light chain variable region (VL) comprising SEQ ID NO: 2; and (ii) a heavy chain variable region (VH) comprising SEQ ID NO: 1.
[0011] In one embodiment, provided herein is antibody or an antigen binding fragment thereof which specifically binds to human MerTK, comprising a VL that comprises SEQ ID NO: 2 or sequences having at least 80%, 85%, 90%, 95% or 98% amino acid sequence identity to SEQ ID NO: 2.
[0012] In a certain embodiment, provided herein is an antibody or an antigen binding fragment thereof which specifically binds to human MerTK, comprising a VL that comprises a VL complementarity determining region 1 (CDRl), VL CDR2, and VL CDR3 that comprise the amino acid sequences of SEQ ID NOS: 6, 7, and 8, respectively, or conservative sequence modifications thereof. In a specific embodiment, the VL that comprises VL CDRl, VL CDR2, and VL CDR3 (that comprise the amino acid sequences of SEQ ID NOS: 6, 7, and 8,
respectively, or conservative sequence modifications thereof) comprises at least 80%, 85%, 90%. 95%, or 98% identity to SEQ ID NO: 2.
[0013] In another certain embodiment, provided herein is an antibody or an antigen binding fragment thereof which specifically binds to human MerTK, comprising a VL that comprises a VL complementarity determining region 1 (CDRl), VL CDR2, and VL CDR3 that comprise the amino acid sequences of SEQ ID NOS: 6, 7, and 8, respectively. In a specific embodiment, the VL that comprises VL CDRl, VL CDR2, and VL CDR3 (that comprise the amino acid sequences of SEQ ID NOS: 6, 7, and 8, respectively) comprises at least 80%, 85%, 90%. 95%, or 98% identity to SEQ ID NO: 2.
[0014] In one embodiment with respect to an antibody described herein or an antigen binding fragment thereof which specifically binds to human MerTK, comprising a VH that comprises SEQ ID NO: 1 or sequences having at least 80%, 85%, 90%, 95% or 98% amino acid sequence identity to SEQ ID NO: 1.
[0015] In a certain embodiment, provided herein is an antibody or an antigen binding fragment thereof which specifically binds to human MerTK, comprising a VH that comprises a VH complementarity determining region 1 (CDRl), VH CDR2, and VH CDR3 that comprise the amino acid sequences of SEQ ID NOS: 3, 4, and 5, respectively, or conservative sequence modifications thereof. In a specific embodiment, the VH that comprises VH CDRl VH CDR2, and VH CDR3 (that comprise the amino acid sequences of SEQ ID NOS: 3, 4, and 5,
respectively, or conservative sequence modifications thereof) comprises at least 80%, 85%, 90%. 95%, or 98% identity to SEQ ID NO: 1 comprises at least 80%,85, 90%, 95%, 98% amino acid sequence identity to SEQ ID NO: 1.
[0016] In a certain embodiment, provided herein is an antibody or an antigen binding fragment thereof which specifically binds to human MerTK, comprising a VH that comprises a VH complementarity determining region 1 (CDRl), VH CDR2, and VH CDR3 that comprise the amino acid sequences of SEQ ID NOS: 3, 4, and 5, respectively, or conservative sequence modifications thereof. In a specific embodiment, the VH comprises at least 80% amino acid sequence identity to SEQ ID NO: 1, the VH comprises at least 85% amino acid sequence identity to SEQ ID NO: 1; the VH comprises at least 90% amino acid sequence identity to SEQ ID NO: 1; the VH comprises at least 95% amino acid sequence identity to SEQ ID NO: 1; or at least 98% amino acid sequence identity to SEQ ID NO: 1.
[0017] In another embodiment, provide herein is an antibody or an antigen binding fragment thereof which specifically binds to human MerTK, comprising a VL that comprises SEQ ID NO: 2 or sequences having at least 80%, 85%, 90%, 95% or 98% amino acid sequence identity to SEQ ID NO: 2, and a VH that comprises SEQ ID NO: 1 or sequences having at least 80%, 85%, 90%, 95% or 98% amino acid sequence identity to SEQ ID NO: 1.
[0018] In a particular embodiment, provided herein is an isolated antibody (e.g., monoclonal antibody), or an antigen-binding fragment thereof, which specifically binds to human MerTK, comprising:
(i) a light chain variable region (VL) comprising VL complementarity determining region 1 (CDRl), VL CDR2, and VL CDR3, wherein the VL CDRl, VL CDR2, and VL CDR3 comprise the amino acid sequences of SEQ ID NOS: 6, 7 and 8, respectively or conservative sequence modifications thereof; and/or
(ii) a heavy chain variable region (VH) comprising VH complementarity determining region 1 (CDRl), VH CDR2, and VH CDR3, wherein the VH CDRl, VH CDR2, and VH CDR3 comprise the amino acid sequences of SEQ ID NOS: 3, 4 and 5 respectively or conservative sequence modifications thereof.
[0019] In one embodiment, presented herein is an antibody or an antigen binding fragment thereof which specifically binds to human MerTK, the VL CDRl, VL CDR2, and VL CDR3 comprise the amino acid sequences of SEQ ID NOS: 6, 7, and 8, respectively or sequences having at least 80% amino acid sequence identity thereto.
[0020] In a certain embodiment is an antibody or an antigen binding fragment thereof which specifically binds to human MerTK, comprising a VH CDRl, VH CDR2, and VH CDR3 that comprises the amino acid sequences of SEQ ID NOS: 3, 4, and 5, respectively, or sequences having at least 80% amino acid sequence identity thereto.
[0021] In a particular embodiment of an antibody described or an antigen binding fragment thereof which specifically binds to human MerTK, the VL CDRl, VL CDR2, and VL CDR3 comprise the amino acid sequences of SEQ ID NOS: 6, 7, and 8, respectively, or sequences having at least 80% amino acid sequence identity thereto, and the VH CDRl, VH CDR2, and VH CDR3 comprise the amino acid sequences of SEQ ID NOS: 3, 4, and 5, respectively, or sequences having at least 80% amino acid sequence identity thereto.
[0022] In a particular embodiment, provided herein is an isolated antibody (e.g., monoclonal antibody), or an antigen-binding fragment thereof, which specifically binds to human MerTK, comprising:
(i) a light chain variable region (VL) comprising VL complementarity determining region 1 (CDRl), VL CDR2, and VL CDR3, wherein the VL CDRl, VL CDR2, and VL CDR3 comprise the amino acid sequences of SEQ ID NOS: 16, 17 and 18, respectively or conservative sequence modifications thereof; and/or
(ii) a heavy chain variable region (VH) comprising VH complementarity determining region 1 (CDRl), VH CDR2, and VH CDR3, wherein the VH CDRl, VH CDR2, and VH CDR3 comprise the amino acid sequences of SEQ ID NOS: 13, 14 and 15 respectively or conservative sequence modifications thereof.
[0023] In one embodiment provided herein is an antibody or an antigen binding fragment thereof which specifically binds to human MerTK, comprising a VL that comprises VL CDRl, VL CDR2, and VL CDR3 that comprise the amino acid sequences of SEQ ID NOS: 16, 17, and 18, respectively, or sequences having at least 80% amino acid sequence identity thereto.
[0024] In a certain embodiment provided herein is an antibody or an antigen binding fragment thereof which specifically binds to human MerTK, the VH CDRl, VH CDR2, and VH CDR3 comprise the amino acid sequences of SEQ ID NOS: 13, 14, and 15, respectively or sequences having at least 80% amino acid sequence identity thereto.
[0025] In a particular embodiment presented herein is an antibody or an antigen binding fragment thereof which specifically binds to human MerTK, comprising a VL that comprises a VL CDRl, VL CDR2, and VL CDR3 comprising the amino acid sequences of SEQ ID NOS: 16, 17, and 18, respectively, or sequences having at least 80% amino acid sequence identity thereto, and comprises a VH that comprises VH CDRl, VH CDR2, and VH CDR3 comprising the amino acid sequences of SEQ ID NOS: 13, 14, and 15, respectively or sequences having at least 80% amino acid sequence identity thereto. [0026] In a particular embodiment, provided herein is an isolated antibody (e.g., monoclonal antibody), or an antigen-binding fragment thereof, which specifically binds to human MerTK, comprising:
(i) a light chain variable region (VL) comprising VL complementarity determining region 1 (CDRl), VL CDR2, and VL CDR3, wherein the VL CDRl, VL CDR2, and VL CDR3 comprise the amino acid sequences of SEQ ID NOS: 22, 23 and 24, respectively or conservative sequence modifications thereof; and/or
(ii) a heavy chain variable region (VH) comprising VH complementarity determining region 1 (CDRl), VH CDR2, and VH CDR3, wherein the VH CDRl, VH CDR2, and VH CDR3 comprise the amino acid sequences of SEQ ID NOS: 19, 20 and 21 respectively or conservative sequence modifications thereof.
[0027] In one embodiment provided herein is an antibody or an antigen binding fragment thereof which specifically binds to human MerTK, comprising a VL that comprises a VL CDRl, VL CDR2, and VL CDR3 comprising the amino acid sequences of SEQ ID NOS: 22, 23, and 24, respectively, or sequences having at least 80% amino acid sequence identity thereto.
[0028] In a certain embodiment provided herein is an antibody or an antigen binding fragment thereof which specifically binds to human MerTK, comprising a VH that comprises a VH CDRl, VH CDR2, and VH CDR3 comprising the amino acid sequences of SEQ ID NOS: 19, 20, and 21, respectively, or sequences having at least 80% amino acid sequence identity thereto.
[0029] In a particular embodiment provided herein is an antibody or an antigen binding fragment thereof which specifically binds to human MerTK, comprising a VL that comprise a VL CDRl, VL CDR2, and VL CDR3 comprising the amino acid sequences of SEQ ID NOS: 22, 23, and 24, respectively, or sequences having at least 80% amino acid sequence identity thereto, and comprise a VH that comprises a VH CDRl, VH CDR2, and VH CDR3 comprising the amino acid sequences of SEQ ID NOS: 19, 20, and 21, respectively, or sequences having at least 80%) amino acid sequence identity thereto.
[0030] In one aspect, provided herein is an isolated antibody, or an antigen-binding fragment thereof, which specifically binds to human MerTK, comprising a light chain variable region (VL) comprising VL complementary determining region 1 (CDRl), VL CDR2, and VL CDR3, wherein the VL CDRl, VL CDR2, and VL CDR3 comprise the amino acid sequences of SEQ ID NOS: 36, 37 and 38, respectively or conservative sequence modifications thereof or SEQ ID NOS: 40, 37 and 38, respectively, or conservative sequence modifications thereof; and/or a heavy chain variable region (VH) comprising VH complementary determining region 1 (CDRl), VH CDR2, and VH CDR3, wherein the VH CDRl, VH CDR2, and VH CDR3 comprise the amino acid sequences of SEQ ID NOS: 33, 34 and 35 respectively or conservative sequence modifications thereof or SEQ ID NOS: 33, 34 and 39, respectively, or conservative sequence modifications thereof.
[0031] In certain embodiments, the antibody or antigen-binding fragment provided herein comprises a VL CDRl, VL CDR2, and VL CDR3 that comprise the amino acid sequences of SEQ ID NOS: 36, 37, and 38, respectively or sequences having at least 80% amino acid sequence identity thereto. In some embodiments, the antibody or antigen-binding fragment provided herein comprises a VH CDRl, VH CDR2, and VH CDR3 that comprise the amino acid sequences of SEQ ID NOS: 33, 34, and 35, respectively or sequences having at least 80% amino acid sequence identity thereto. In some embodiments, the VL CDRl, VL CDR2, and VL CDR3 can comprise the amino acid sequences of SEQ ID NOS: 36, 37, and 38, respectively or sequences having at least 80% amino acid sequence identity thereto, and the VH CDRl, VH CDR2, and VH CDR3 comprise the amino acid sequences of SEQ ID NOS: 33, 34, and 35, respectively or sequences having at least 80% amino acid sequence identity thereto.
[0032] In one aspect, provided herein is an isolated antibody, or an antigen-binding fragment thereof, which specifically binds to human MerTK, comprising a light chain variable region (VL) comprising VL complementary determining region 1 (CDRl), VL CDR2, and VL CDR3, wherein the VL CDRl, VL CDR2, and VL CDR3 comprise the amino acid sequences of SEQ ID NOS: 44, 45 and 46, respectively or conservative sequence modifications thereof or SEQ ID NOS: 48, 45 and 46, respectively, or conservative sequence modifications thereof; and/or a heavy chain variable region (VH) comprising VH complementary determining region 1 (CDRl), VH CDR2, and VH CDR3, wherein the VH CDRl, VH CDR2, and VH CDR3 comprise the amino acid sequences of SEQ ID NOS: 41, 42, 43 respectively or conservative sequence modifications thereof or SEQ ID NOS: 41, 42, 47, respectively, or conservative sequence modifications thereof.
[0033] In certain embodiments, The antibody or antigen-binding fragment can comprise a VL CDRl, VL CDR2, and VL CDR3 that comprise the amino acid sequences of SEQ ID NOS: 44, 45 and 46, respectively or sequences having at least 80% amino acid sequence identity thereto. In some embodiments, the antibody or antigen-binding fragment can comprise a VH CDRl, VH CDR2, and VH CDR3 that comprise the amino acid sequences of SEQ ID NOS: 41, 42, 43, respectively or sequences having at least 80% amino acid sequence identity thereto.
[0034] In a particular embodiment, the antibody or an antigen-binding fragment disclosed herein can comprise a VL CDRl, VL CDR2, and VL CDR3 that comprise the amino acid sequences of SEQ ID NOS: 44, 45 and 46, respectively or sequences having at least 80% amino acid sequence identity thereto, and the VH CDRl, VH CDR2, and VH CDR3 comprise the amino acid sequences of SEQ ID NOS: 41, 42, 43, respectively or sequences having at least 80% amino acid sequence identity thereto.
[0035] In one embodiment, provided herein is an antibody or an antigen binding fragment thereof which specifically binds to human MerTK, that comprises a VL CDRl, VL CDR2, and VL CDR3 present in one polypeptide, and a comprises a VH CDRl, VH CDR2, and VH CDR3 on a second polypeptide. In a specific embodiment, an antibody or antigen binding fragment described herein which specifically binds to human MerTK comprises a VL and a VH, wherein the VL and VH are present in the same polypeptide.
[0036] In another embodiment, provided herein is an isolated antibody, or an antigen-binding fragment thereof, which specifically binds to Domain 1 of human MerTK. In a certain embodiment, provided herein is an isolated antibody, or an antigen-binding fragment thereof, which binds to the same epitope of human MerTK as an antibody described herein or antigen- binding fragment thereof. In another embodiment, provided herein is an isolated antibody, or an antigen-binding fragment thereof, which competes for binding to human MerTK with an antibody described herein or antigen-binding fragment thereof. In another embodiment, provided herein is an isolated antibody, or an antigen-binding fragment thereof, which binds to Domain 1 of human MerTK and inhibits binding of Gas6 to MerTK and/or, inhibits MerTK phosphorylation.
[0037] In one embodiment, an antibody or antigen binding fragment presented herein is a MerTK receptor antagonist.
[0038] In one embodiment, an antibody or antigen binding fragment described herein, which specifically binds to human MerTK, comprises a human heavy chain constant region. In a specific embodiment, an antibody or antigen-binding fragment described herein, which specifically binds to human MerTK, comprises a human heavy chain comprising a VH and a human heavy chain constant region.
[0039] In another embodiment, an antibody or antigen binding fragment described herein, which specifically binds to human MerTK, comprises a human light chain constant region. In a specific embodiment, an antibody or antigen-binding fragment described herein, which specifically binds to human MerTK, comprises a human light chain comprising a VL and a human light chain constant region.
[0040] In yet another embodiment, an antibody or antigen binding fragment described herein which specifically binds to human MerTK comprises a human heavy chain constant region and a human light chain constant region. In a specific embodiment, such an antibody or antigen- binding fragment comprises a human heavy chain comprising a VH and a human heavy chain constant region and a human light chain comprising a VL and a human light chain constant region.
[0041] In a certain embodiment, an antibody or antigen binding fragment described herein, which specifically binds to human MerTK, is an IgGl antibody. In a specific embodiment, an antibody or antigen binding fragment described herein, which specifically binds to human MerTK, is a human IgGl antibody.
[0042] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain constant region or a light chain constant region. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain constant region and a light chain constant region. In certain embodiments, the antibody or antigen-binding fragment comprises a human heavy chain constant region or a human light chain constant region. In some embodiments, the antibody or antigen-binding fragment comprises a human heavy chain constant region and a human light chain constant region.
[0043] In certain embodiments, the antibody or antigen-binding fragment is an IgGl antibody or antigen-binding fragment. In certain embodiments, the antibody or antigen-binding fragment is an IgG2 antibody or antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment is a human IgGl antibody or antigen-binding fragment or a human IgG2 antibody or antigen-binding fragment. In some embodiments, the antibody or antigen- binding fragment comprises a kappa light chain constant region or a lambda light chain constant region. In some embodiments, the antibody or antigen-binding fragment comprises a human kappa light chain constant region or a human lambda light chain constant region.
[0044] In certain embodiments, the antibody or antigen-binding fragment comprises the amino acid sequence of SEQ ID NO: 49 or the amino acid sequence of SEQ ID NO: 50. In some embodiments, the antibody or antigen-binding fragment comprises the amino acid sequence of SEQ ID NO: 49 and the amino acid sequence of SEQ ID NO: 50.
[0045] In some embodiments, the antibody or antigen-binding fragment disclosed herein comprises the amino acid sequence of SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53 SEQ ID NO: 55, SEQ ID NO: 56, or SEQ ID NO: 57
[0046] In some embodiments, the antibody or antigen-binding fragment disclosed herein comprises the amino acid sequence of SEQ ID NO: 49 and the amino acid sequence of SEQ ID NO: 50; the amino acid sequence of SEQ ID NO: 49 and the amino acid sequence of SEQ ID NO: 51; the amino acid sequence of SEQ ID NO: 54 and the amino acid sequence of SEQ ID NO: 52; the amino acid sequence of SEQ ID NO: 54 and the amino acid sequence of SEQ ID NO: 53; the amino acid sequence of SEQ ID NO: 54 and the amino acid sequence of SEQ ID NO: 55; the amino acid sequence of SEQ ID NO: 54 and the amino acid sequence of SEQ ID NO: 56; the amino acid sequence of SEQ ID NO: 57 and the amino acid sequence of SEQ ID NO: 52; the amino acid sequence of SEQ ID NO: 57 and the amino acid sequence of SEQ ID NO: 53; the amino acid sequence of SEQ ID NO: 57 and the amino acid sequence of SEQ ID NO: 55; or the amino acid sequence of SEQ ID NO: 57 and the amino acid sequence of SEQ ID NO: 56.
[0047] In a certain embodiment, an antibody or antigen binding fragment described herein which specifically binds to human MerTK comprises a human kappa light chain constant region or a human gamma heavy chain constant region.
[0048] In a certain embodiment, an isolated antibody described herein, or an antigen-binding fragment thereof, which specifically binds to human MerTK, is a monoclonal antibody.
[0049] In a certain embodiment, an antibody (e.g., monoclonal antibody) described herein, or an antigen-binding fragment thereof, which specifically binds to human MerTK, is a human antibody. [0050] In a certain embodiment, an antibody (e.g., monoclonal antibody) described herein, or an antigen-binding fragment thereof, which specifically binds to human MerTK, is a chimeric antibody.
[0051] In a certain embodiment, an isolated antibody (e.g., monoclonal antibody) described herein, or an antigen-binding fragment thereof, which specifically binds to human MerTK, is a humanized antibody.
[0052] In a certain embodiment, an isolated antibody (e.g., monoclonal antibody) described herein, or an antigen-binding fragment thereof, which specifically binds to human MerTK, is a single chain antibody.
[0053] In a certain embodiment, an isolated antibody (e.g., monoclonal antibody) described herein, or an antigen-binding fragment thereof, which specifically binds to human MerTK, is a multispecific, e.g., bispecific, antibody.
[0054] In a certain embodiment, an isolated antibody (e.g., monoclonal antibody) described herein, or an antigen-binding fragment thereof, which specifically binds to human MerTK, is a monovalent antibody.
[0055] In a certain embodiment, an isolated antibody (e.g., monoclonal antibody) described herein, or an antigen-binding fragment thereof, which specifically binds to human MerTK, is fused to a heterologous polypeptide.
[0056] In a certain embodiment, an isolated antibody (e.g., monoclonal antibody) described herein, or an antigen-binding fragment thereof, which specifically binds to human MerTK, is conjugated to an agent. In a particular embodiment, the agent is a toxin. In one embodiment, the toxin is abrin, ricin A, pseudomonas exotoxin, cholera toxin, or diphtheria toxin.
[0057] In a certain embodiment, provided herein is a composition comprising a
therapeutically effective amount of an antibody described herein or antigen-binding fragment thereof, which specifically binds to human MerTK.
[0058] In a certain embodiment, provided herein is a pharmaceutical composition comprising an antibody described herein or antigen-binding fragment thereof, which specifically binds to human MerTK and a pharmaceutically acceptable carrier.
[0059] In a certain embodiment, provided herein is a polynucleotide comprising nucleotide sequences encoding a VH, a VL, or both a VL and a VH, of an antibody described herein or antigen-binding fragment thereof, which specifically binds to human MerTK. [0060] In a certain embodiment, provided herein is a polynucleotide comprising nucleotide sequences encoding a heavy chain, a light chain, or both heavy chain and a light chain of an antibody described herein or antigen-binding fragment thereof, which specifically binds to human MerTK.
[0061] In a certain embodiment, provided herein is a population of polynucleotides comprising (i) a first polynucleotide comprising nucleotide sequences encoding a VH or a heavy chain of an antibody described herein or antigen-binding fragment thereof, which specifically binds to human MerTK, and (ii) a second polypeptide comprising nucleotide sequences encoding a VL or a light chain of the antibody.
[0062] In a certain embodiment, provided herein is a vector comprising a polynucleotide described herein comprising nucleotide sequences encoding a VH, or a VL, or a VH and VL of an antibody described herein or antigen-binding fragment thereof, which specifically binds to human MerTK.
[0063] In a certain embodiment, provided herein is a vector comprising a polynucleotide described herein comprising nucleotide sequences encoding a heavy chain, a light chain, or both heavy chain and a light chain of an antibody described herein or antigen-binding fragment thereof, which specifically binds to human MerTK.
[0064] In a certain embodiment, provided herein is a population of vectors comprising (i) a first vector comprising nucleotide sequences encoding a VH or a heavy chain of an anti-MerTK antibody or antigen-binding fragment described herein, and (ii) a second vector comprising nucleotide sequences encoding a VL or a light chain of an anti-MerTK antibody or antigen- binding fragment described herein.
[0065] In a certain embodiment, provided herein is an isolated cell comprising a
polynucleotide comprising nucleotide sequences encoding a VH, a VL, or both a VH and a VL of an antibody described herein or antigen-binding fragment thereof, which specifically binds to human MerTK.
[0066] In a certain embodiment, provided herein is an isolated cell comprising a
polynucleotide comprising nucleotide sequences encoding a heavy chain, a light chain, or both heavy chain and a light chain of an antibody described herein or antigen-binding fragment thereof, which specifically binds to human MerTK. [0067] In a certain embodiment, provided herein is an isolated cell comprising a population of polynucleotides described herein.
[0068] In a certain embodiment, provided herein is a population of cells comprising (i) a first host cell comprising a polynucleotide described herein comprising nucleotide sequences encoding a VH or a heavy chain an anti-MerTK antibody or antigen-binding fragment described herein, and (ii) a second host cell comprising a polynucleotide comprising nucleotide sequences encoding a VL or a light chain of an anti-MerTK antibody or antigen-binding fragment described herein.
[0069] In a certain embodiment, provided herein is an isolated cell producing an antibody described herein or antigen-binding fragment thereof, which specifically binds to human MerTK.
[0070] In a certain embodiment, provided herein is a kit comprising an antibody described herein or antigen-binding fragment thereof, which specifically binds to human MerTK.
[0071] In a particular aspect, provided herein is a method of managing, protecting against, or treating cancer, for example, breast cancer, prostate cancer, gastric cancer, lung cancer, e.g., non- small cell lung cancer, adenoma, melanoma, lymphoma, or leukemia, or infection, for example, bacterial, e.g., gram-negative or gram-positive bacteria, fungal, viral, or parasitic infection in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of an antibody or antigen binding fragment described herein, for example, Ab2000, or antigen- binding fragment thereof, or Ab2000-A7, or antigen-binding fragment thereof, which
specifically binds to human MerTK.
[0072] In a certain embodiment, provided herein is a method of modulating an immune response, e.g., enhancing an immune response, in a subject, comprising administering to a subject in need thereof an effective amount of an antibody described herein, for example, Ab2000, or antigen-binding fragment thereof, or Ab2000-A7, or antigen-binding fragment thereof, which specifically binds to human MerTK. In a particular embodiment, the subject is a subject suffering from an infection, a subject having cancer, or an immunocompromised subject such as, for example, a subject who is undergoing, or had undergone treatment with, an anticancer therapy, is HIV positive, or who has AIDS or SCID, has diabetes, or has had a transplant and is taking an immunosuppressant. In a particular embodiment, the subject has been treated with an immunosuppressant. [0073] In a certain embodiment, provided herein is a method of enhancing a
proinflammatory response in a subject, comprising administering to a subject in need thereof an effective amount of an antibody described herein, for example, Ab2000, or antigen-binding fragment thereof, or Ab2000-A7, or antigen-binding fragment thereof, which specifically binds to human MerTK. In a particular embodiment, such a method of enhancing a proinflammatory response in a subject results in an increase in T F-α secretion.
[0074] In a specific embodiment, provided herein is a method of enhancing an immune response to a vaccine in a subject, comprising administering to a subject in need thereof, who is or has been administered the vaccine, an effective amount of an antibody described herein or antigen-binding fragment thereof, which specifically binds to human MerTK. In a certain embodiment, the vaccine is a cancer or tumor vaccine
[0075] In one embodiment, provided herein is a vaccine antigen that can also be targeted, for example, to particular cell types or to particular tissues. In a specific embodiment, the vaccine antigen can be targeted to Antigen Presenting Cells (APCs), for example by use of agents such as antibodies targeted to APC-surface receptors such as DEC-205, for example as discussed in WO 2009/061996 (Celldex Therapeutics, Inc.), or the Mannose Receptor (CD206) for example as discussed in WO 03040169 (Medarex, Inc.).
[0076] In one embodiment, provided herein is a method of managing, preventing, protecting against, or treating metastasis in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of an antibody described herein or antigen-binding fragment thereof, which specifically binds to human MerTK.
[0077] In a certain embodiment, provided herein is a method for activating or
increasing/enhancing an innate immune response in a subject, comprising administering to a subject in need thereof an effective amount of an antibody described herein or antigen-binding fragment thereof, which specifically binds to human MerTK. In a particular embodiment, the subject has cancer and the method is effective in treating or managing the subject's cancer.
[0078] In a certain embodiment, provided herein is a method for stimulating cytotoxicity of K cells in a subject, comprising administering to a subject in need thereof an effective amount of an antibody described herein, for example, Ab2000, or antigen-binding fragment thereof, or Ab2000-A7, or antigen-binding fragment thereof, which specifically binds to human MerTK. In one embodiment, the subject has cancer, or is being treated for cancer with an anti-cancer therapeutic, or the subject has an infection.
[0079] In a specific embodiment, provided herein is a method of making an antibody or an antigen-binding fragment thereof which specifically binds to human MerTK, comprising culturing a cell, host cell, or population of cells described herein to express an anti-MerTK antibody or antigen-binding fragment thereof.
[0080] In a certain embodiment, provided herein is a method of making an antibody or an antigen-binding fragment thereof which specifically binds to human MerTK, comprising expressing a polynucleotide described herein or a population of polynucleotides described herein.
[0081] In another embodiment, presented herein is a method of increasing proinflammatory cytokine production in a subject in need thereof, comprising administering to the subject an antibody or an antigen-binding fragment thereof that specifically binds to human MerTK such that the cytokine production is increased. For example, the cytokine secretion can be increase by about 5%, 10%, 15%), 20%, 30, or more relative to secretion in the absence of the antibody. In a specific embodiment, the proinflammatory cytokine is T Fa (tumor necrosis factor a). In a specific embodiment, the proinflammatory cytokine is IL-IRA (interleukin-1 receptor antagonist). In yet another embodiment, the antibody or antigen-binding fragment does not substantially induce phosphorylation of MerTK. For example, the antibody or antigen-binding fragment induces phosphorylation of MerTK at least 50%, 60%>, 70%, 80%>, 90% or less than the phosphorylation induced by Gas6 binding to MerTK. In a particular embodiment, the antibody or antigen-binding fragment is one that specifically binds to Domain 1 of human MerTK. In yet another embodiment, the antibody or antigen-binding fragment is one of the antibodies or antigen-binding fragments described herein.
[0082] In a certain aspect, further provided herein is a method of increasing proinflammatory cytokine production in a subject in need thereof, comprising administering to the subject an antibody or an antigen-binding fragment thereof disclsoed herein such that the production of one or more cytokines is increased. In some embodiments, the production of proinflammatory cytokine TNFa (tumor necrosis factor a) is increased. In certain embodiments, the production of proinflammatory cytokine IL-IRA (interleukin-1 receptor antagonist) is increased. In some embodiments, provided herein is a method wherein the production of proinflammatory cytokines TNFa and IL- IRA are increased.
[0083] In some embodiments, provided herein is a method of increasing proinflammatory cytokine production in a subject in need thereof, comprising administering to the subject an antibody or an antigen-binding fragment thereof disclosed herein such that the production of one or more cytokines is increased. In some embodiments, the production of proinflammatory cytokine TNFa (tumor necrosis factor a) is increased. In certain embodiments, the production of proinflammatory cytokine IL-1RA (interleukin-1 receptor antagonist) is increased. In some embodiments, provided herein is a method wherein the production of proinflammatory cytokines TNFa and IL-IRA are increased.
[0084] In certain embodiments, provided herein is a method of increasing proinflammatory secretory factor production in a subject in need thereof, comprising administering to the subject an antibody or an antigen-binding fragment thereof disclosed herein such that the production of one or more proinflammatory secretory factors is increased.
[0085] In one aspect, provided herein is a method of increasing proinflammatory secretory factor production in a subject in need thereof, comprising administering to the subject an antibody or an antigen-binding fragment thereof disclosed herein such that the production of one or more proinflammatory secretory factors is increased. As a non-limiting example, in certain embodiments, provided herein are methods wherein the production of one or more of
proinflammatory secretory factors TNFa, IL-IRA, fibroblast growth factor 2 (FGF-2), eotaxin-1 (CCL11), transforming growth factor alpha (TGF-a) granulocyte-colony stimulating factor (G- CSF), Fms-related tyrosine kinase 3 ligand (Flt-3L), granulocyte macrophage-colony stimulating factor (GM-CSF), fractalkine (CX3CL1), interferon alpha-2 (IFN-a2), interferon-gamma (IFN- γ), growth-regulated oncogene alpha (GRO alpha), interleukin-10 (IL-10), monocyte chemotactic protein 3 (MCP-3), interleukin-12 p40 (IL-12P40), macrophage-derived chemokine (MDC), platelet-derived growth factor AA homodimer (PDGF-AA), interleukin-13 (IL-13), platelet- derived growth factor BB homodimer (PDGF-BB), soluble CD40 ligand (sCD40L), interleukin- 1B (IL-IB), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-8 (IL-8), interferon γ-induced protein 10 (IP- 10), macrophage inflammatory protein (ΜΙΡ)-Ια, ΜΙΡ-Ιβ, Regulated on
Activation Normal T cell Expressed and Secreted (RANTES), vascular endothelial growth factor A (VEGF-A) or IL-18 is increased. 4. BRIEF DESCRIPTION OF THE FIGURES
[0086] Fig. 1 depicts the binding of anti-MerTK antibody Ab2000 to cellularly expressed full length human MerTK (hMerTK) in a dose responsive manner, with EC50 values in the picomolar to nanomolar range. The mean fluorescent intensity is plotted as a function of the log- transformed molar concentration of the antibody.
[0087] Fig. 2 shows that the anti-MerTK antibody Ab2000 blocked Gas6 binding to recombinant MerTK extracellular domain (ECD) in a dose responsive manner, with an IC50 of 1 nM.
[0088] Fig. 3A depicts inhibition of Gas6-dependent MerTK phosphorylation by the anti- MerTK antibody Ab2000. Addition of 100 nM Gas6 alone was used as a reference. Fig. 3B shows that titration of Ab2000 on MerTK expressing THPl cells inhibited Gas6-dependent MerTK phosphorylation with an IC50 value of 0.2 nM.
[0089] Fig. 4 shows that addition of the anti-MerTK antibody Ab2000 elicited only very weak MerTK phosphorylation in the absence of exogenously added Gas6 (as compared to independently-added Gas6) in MerTK expressing THPl cells. Addition of 100 nM Gas6 alone was used as a reference.
[0090] Fig. 5 shows that the anti-MerTK antibody Ab2000 enhanced TNF-a secretion from differentiated U937 monocyte-derived macrophages upon stimulation of different Toll-like receptors with LPS.
[0091] Figs. 6A and 6B show that the anti-MerTK antibody Ab2000 enhanced TNF- α secretion from primary human macrophages upon stimulation of different Toll-like receptors with LPS (Fig. 6A) or Resiquimod (R848) (Fig. 6B).
[0092] Figs. 7A and 7B show that the anti-MerTK antibody Ab2000 enhanced TNF- α secretion from primary human dendritic cells upon stimulation of different Toll-like receptors with LPS (Fig. 7A) or Resiquimod (R848) (Fig. 7B) as compared to a control IgGl antibody.
[0093] Fig. 8 demonstrates that titration of the anti-MerTK antibody Ab2000 resulted in a dose dependent increase in TNF-a secretion in dendritic cells with nanomolar potency.
[0094] Fig. 9 depicts the binding of anti-MerTK antibody Ab2000 and affinity matured antibodies A2000-A7, A2000-F1 and A2000-G6 to recombinant full length human MerTK (hMerTK) as determined by ELISA. The absorbance at OD450 is plotted as a function of the concentration of the antibody.
[0095] Fig. 10A and Fig. 10B depict the binding of the anti-MerTK antibody Ab2000 and affinity matured antibodies to huMerTK-L cells and SK-mel-5 cells, respectively. The mean fluorescence intesity is plotted as a function of the concentration of the antibody.
[0096] Fig. 11 depicts representative binding curves of the A2000 antibody and the A2000- A7 affinity matured antibody to cellularly expressed full length human MerTK (hMerTK) as determined by ELISA. The absorbance at OD450 is plotted as a function of the concentration of the antibody.
[0097] Fig. 12 A and Fig. 12B depict the binding of the anti-MerTK affinity matured antibody Ab2000-A7 to huMerTK-L cells and SK-mel-5 cells, respectively. The mean fluorescence intensity (MFI) is plotted as a function of the concentration of the antibodies.
[0098] Fig. 13A and Fig. 13B shows that the anti-MerTK antibodies Ab2000 and Ab2000- A7 enhance IL-IRA production by dendritic cells. Fold increase over isotype control is plotted.
[0099] Fig. 14 shows that the anti-MerTK antibodies Ab2000 and Ab2000-A7 enhance TNF- alpha production by dendritic cells. Fold increase over isotype control is plotted.
[00100] Fig. 15 depicts multiplex analysis of production of 30 different cytokines,
chemokines and growth factors in response to Ab2000-A7 in dendritic cells. Fold increase in protein production in response to Ab2000-A7 over isotype control is plotted.
[00101] Fig. 16 depicts multiplex analysis of production of 30 different cytokines,
chemokines and growth factors in response to Ab2000-A7 in PBMCs. Fold increase in protein production in response to Ab2000-A7 over isotype control is plotted.
[00102] Fig. 17 shows IL-IRA production (pg/ml) in PBMCs (black bar) and CD14+ cell- depleted PBMCs (grey bars) in response to Ab2000-A7 antibody.
Fig. 18 shows that the anti-MerTK antibody Ab2000-A7 enhances TNF-a production by macrophages. Fold increase over isotype control is plotted.
5. DETAILED DESCRIPTION
[00103] MerTK is a receptor tyrosine kinase of the TAM group (including Tyro-3, Axl, and MerTK) (Graham et al. (2014) Nat Rev Cancer ;14: 769-785; Lemke G. (2013) Cold Spring Harb Perspect Biol.; 5). In a specific embodiment, MerTK is human MerTK. UniProKB accession number Q 12866 provides an exemplary human MerTK amino acid sequence. Native MerTK comprises two extracellular immunoglobulin (Ig)-like domains, two extracellular fibronectin (FN) type III motifs, and a intracellular tyrosine kinase domain (see, e.g., Lemke G. (2013) Cold Spring Harb Perspect Biol.; 5). GenBank™ accession number NM 006343.2 provides an exemplary human MerTK nucleic acid coding sequence.
5.1 Antibodies
[00104] In a specific aspect, provided herein are antibodies which specifically bind to human MerTK, for example, an extracellular domain (ECD) of human MerTK, and modulate MerTK expression and/or MerTK activity.
[00105] As used herein, the terms "antibody" and "immunoglobulin" and "Ig" are terms of art and can be used interchangeably herein and refer to a molecule with an antigen binding site that specifically binds an antigen.
[00106] Antibodies can include, for example, monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, synthetic antibodies, tetrameric antibodies comprising two heavy chain and two light chain molecules, an antibody light chain monomer, an antibody heavy chain monomer, an antibody light chain dimer, an antibody heavy chain dimer, an antibody light chain/antibody heavy chain pair, an antibody with two light chain/heavy chain pairs (e.g., identical pairs), intrabodies, heteroconjugate antibodies, single domain antibodies, monovalent antibodies, bivalent antibodies (including monospecific or bispecific bivalent antibodies), single chain antibodies, or single-chain Fvs (scFv), camelized antibodies, affybodies, Fab fragments, F(ab') fragments, F(ab')2 fragments, disulfide-linked Fvs (sdFv), anti -idiotypic (anti-Id) antibodies (including, e.g., anti-anti-Id antibodies), and epitope- binding fragments of any of the above. In certain embodiments, antibodies described herein refer to polyclonal antibody populations.
[00107] In a certain embodiment, an isolated antibody (e.g., monoclonal antibody) described herein, or an antigen-binding fragment thereof, which specifically binds to human MerTK, is fused to a heterologous polypeptide. In a certain embodiment, an isolated antibody (e.g., monoclonal antibody) described herein, or an antigen-binding fragment thereof, which specifically binds to human MerTK, is conjugated to an agent. In a particular embodiment, the agent is a toxin. In one embodiment, the toxin is abrin, ricin A, pseudomonas exotoxin, cholera toxin, or diphtheria toxin.
[00108] Antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA or IgY), any class, (e.g., IgGl, IgG2, IgG3, IgG4, IgAl or IgA2), or any subclass (e.g., IgG2a or IgG2b) of
immunoglobulin molecule. In certain embodiments, antibodies described herein are IgG antibodies (e.g., human IgG), or a class (e.g., human IgGl, IgG2, IgG3 or IgG4) or subclass thereof.
[00109] In a particular embodiment, an antibody is a 4-chain antibody unit comprising two heavy (H) chain / light (L) chain pairs, wherein the amino acid sequences of the H chains are identical and the amino acid sequences of the L chains are identical. In a specific embodiment, the H and L chains comprise constant regions, for example, human constant regions. In a yet more specific embodiment, the L chain constant region of such antibodies is a kappa or lambda light chain constant region, for example, a human kappa or lambda light chain constant region. In another specific embodiment, the H chain constant region of such antibodies comprise a gamma heavy chain constant region, for example, a human gamma heavy chain constant region. In a particular embodiment, such antibodies comprise IgG constant regions, for example, human IgG constant regions.
[00110] As used herein, an "antigen" is a moiety or molecule that contains an epitope to which an antibody can specifically bind. As such, an antigen is also is specifically bound by an antibody. In a specific embodiment, the antigen, to which an antibody described herein binds is MerTK (e.g., human MerTK), or a fragment thereof, for example, an extracellular domain of MerTK (e.g., human MerTK).
[00111] As used herein, an "epitope" is a term in the art and refers to a localized region of an antigen to which an antibody can specifically bind. An epitope can be a linear epitope or a conformational, non-linear, or discontinuous, epitope. In the case of a polypeptide antigen, for example, an epitope can be contiguous amino acids of the polypeptide (a "linear" epitope) or an epitope can comprise amino acids from two or more non-contiguous regions of the polypeptide (a "conformational," "non-linear" or "discontinuous" epitope). It will be appreciated by one of skill in the art that, in general, a linear epitope may or may not be dependent on secondary, tertiary, or quaternary structure. For example, in some embodiments, an antibody binds to a group of amino acids regardless of whether they are folded in a natural three dimensional protein structure. In other embodiments, an antibody requires amino acid residues making up the epitope to exhibit a particular conformation (e.g., bend, twist, turn or fold) in order to recognize and bind the epitope.
[00112] As used herein, the terms "specifically binds," "specifically recognizes,"
"immunospecifically binds," "immunospecifically recognizes" and "immunospecific" are analogous terms in the context of antibodies and refer to molecules that bind to an antigen (e.g., epitope) as such binding is understood by one skilled in the art. For example, a molecule that specifically binds to an antigen may bind to other peptides or polypeptides, generally with lower affinity as determined by, e.g., immunoassays, Biacore™, KinExA 3000 instrument (Sapidyne Instruments, Boise, ID), or other assays known in the art. In a specific embodiment, molecules that specifically bind to an antigen bind to the antigen with a Ka that is at least 2 logs, 2.5 logs, 3 logs, 4 logs or greater than the Ka when the molecules bind to another antigen. In another specific embodiment, molecules that specifically bind to an antigen do not cross react with other proteins. In another specific embodiment, molecules that specifically bind to an antigen do not cross react with other non-MerTK proteins.
[00113] As used herein, the term "constant region" or "constant domain" is a well-known antibody term of art (sometimes referred to as "Fc"), and refers to an antibody portion, e.g., a carboxyl terminal portion of a light and/or heavy chain which is not directly involved in binding of an antibody to antigen but which can exhibit various effector functions, such as interaction with the Fc receptor. The terms refer to a portion of an immunoglobulin molecule having a generally more conserved amino acid sequence relative to an immunoglobulin variable domain.
[00114] As used herein, the term "heavy chain" when used in reference to an antibody can refer to any distinct types, e.g., alpha (a), delta (δ), epsilon (ε), gamma (γ) and mu (μ), based on the amino acid sequence of the constant domain, which give rise to IgA, IgD, IgE, IgG and IgM classes of antibodies, respectively, including subclasses of IgG, e.g., IgGi, IgG2, IgG3 and IgG4.
[00115] As used herein, an "isolated" or "purified" antibody or antigen binding fragment is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the antibody or antigen binding fragment is derived, or substantially free of chemical precursors or other chemicals when the antibody or antigen binding fragment is chemically synthesized. [00116] As used herein, the term "light chain" when used in reference to an antibody can refer to any distinct types, e.g., kappa (κ) of lambda (λ) based on the amino acid sequence of the constant domains. Light chain amino acid sequences are well known in the art. In specific embodiment is a human light chain.
[00117] The term "monoclonal antibody" is a well-known term of art that refers to an antibody obtained from a population of homogenous or substantially homogeneous antibodies. The term "monoclonal" is not limited to any particular method for making the antibody.
Generally, a population of monoclonal antibodies can be generated by cells, a population of cells, or a cell line. In specific embodiments, a "monoclonal antibody," as used herein, is an antibody produced by a single cell (e.g., hybridoma or host cell producing a recombinant antibody), wherein the antibody specifically binds to a MerTK epitope (e.g., an epitope of the extracellular domain of human MerTK) as determined, e.g., by ELISA or other antigen-binding or competitive binding assay known in the art or in the Examples provided herein. In particular embodiments, a monoclonal antibody can be a chimeric antibody or a humanized antibody. In certain embodiments, a monoclonal antibody is a monovalent antibody or multivalent (e.g., bivalent) antibody. In particular embodiments, a monoclonal antibody is a monospecific or multispecific antibody (e.g., bispecific antibody).
[00118] As used herein, the term "polyclonal antibodies" refers to an antibody population that includes a variety of different antibodies that specifically to the same and/or to different epitopes within an antigen or antigens.
[00119] As used herein, the terms "variable region" or "variable domain" refer to a portion of an antibody, generally, a portion of a light or heavy chain, typically about the amino-terminal 110 to 120 amino acids in the mature heavy chain and about 90 to 100 amino acids in the mature light chain. Variable regions comprise complementarity determining regions (CDRs) flanked by framework regions (FRs). Generally, the spatial orientation of CDRs and FRs are as follows, in an N-terminal to C-terminal direction: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Without wishing to be bound by any particular mechanism or theory, it is believed that the CDRs of the light and heavy chains are primarily responsible for the interaction of the antibody with antigen and for the specificity of the antibody for an epitope. In a specific embodiment, numbering of amino acid positions of antibodies described herein is according to the EU Index, as in Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242. In certain embodiments, the variable region is a human variable region.
[00120] In certain aspects, the CDRs of an antibody can be determined according to (i) the Kabat numbering system (Kabat et al. (1971) Ann. NY Acad. Sci. 190:382-391 and, Kabat et al. (1991) Sequences of Proteins of Immunological Interest Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242); or (ii) the Chothia numbering scheme, which will be referred to herein as the "Chothia CDRs" (see, e.g., Chothia and Lesk, 1987, J. Mol. Biol., 196:901-917; Al-Lazikani et al., 1997, J. Mol. Biol., 273 :927-948; Chothia et al., 1992, J. Mol. Biol., 227:799-817; Tramontano A et al., 1990, J. Mol. Biol. 215(1): 175-82; and U.S. Patent No. 7,709,226); or (iii) the ImMunoGeneTics (FMGT) numbering system, for example, as described in Lefranc, M.-P., 1999, The Immunologist, 7: 132-136 and Lefranc, M.-P. et al., 1999, Nucleic Acids Res., 27:209-212 ("IMGT CDRs"); or (iv) the AbM numbering system, , which will be referred to herein as the "AbM CDRs", for example as described in MacCallum et al., 1996, J. Mol. Biol., 262:732-745. See also, e.g., Martin, A., "Protein
Sequence and Structure Analysis of Antibody Variable Domains," in Antibody Engineering, Kontermann and Diibel, eds., Chapter 31, pp. 422-439, Springer- Verlag, Berlin (2001).
[00121] In certain embodiments, provided herein is an antibody or an antigen-binding fragment thereof which specifically binds to human MerTK comprises VL and VH CDR as set forth in Table 1, which utilizes standard one letter amino acid abbreviations. In a particular embodiment, provided herein is an antibody or an antigen-binding fragment thereof which specifically binds to human MerTK, and comprises VL CDRl (SEQ ID NO: 6), VL CDR2 (SEQ ID NO: 7), and VL CDR3 (SEQ ID NO: 8), and VH CDRl (SEQ ID NO: 3), VH CDR2 (SEQ ID NO: 4), and VH CDR3 (SEQ ID NO: 5).
Table 1. Ab2000 VL and VH CDR Amino Acid Sequences (Kabat)
Figure imgf000025_0001
VH CDR1 (SEQ ID NO: 3) VH CDR2 (SEQ ID NO: 4) VH CDR3 (SEQ ID NO: 5)
SYAMS AISGSGGSTYYADSVKG DRQWLLPRAFDI
[00122] In certain embodiments, provided herein is an antibody or an antigen-binding fragment thereof which specifically binds to human MerTK and comprises a VL and/or a VH as set forth in Table 2, which utilizes standard one letter amino acid abbreviations. In a particular embodiment, provided herein is an antibody or an antigen-binding fragment thereof which specifically binds to human MerTK, and comprises VL (SEQ ID NO: 2) and VH (SEQ ID NO: 1). For ease of description, an antibody that comprises the VL and VH amino acid sequences of Table 2 is referred to herein as "Ab2000." In one embodiment, such an antibody or antigen- binding fragment comprises a separate light chain comprising the VL amino acid sequence and a separate heavy chain comprising the VH amino acid sequence. In another embodiment, such an antibody or antigen-binding fragment comprises a single chain comprising the VL amino acid sequence and the VH amino acid sequence.
Table 2: Ab2000 VL and VH Amino Acid Sequences
Figure imgf000026_0001
[00123] In a particular embodiment, an antibody or an antigen-binding fragment described herein, comprises the VL CDR1, VL CDR2, and VL CDR3 of Ab2000. In a specific embodiment, the VL CDR1, VL CDR2, and VL CDR3 are as set forth in Table 1 (SEQ ID NOS: 6, 7, and 8, respectively). In another embodiment, the antibody or antigen-binding fragment further comprises a VH as set forth in Table 2. [00124] In a particular embodiment, an antibody or an antigen-binding fragment described herein, which specifically binds to human MerTK, comprises a VL as set forth in Table 2. In another embodiment, the antibody or antigen-binding fragment further comprises VH CDRl, VH CDR2 and/or VH CDR3 as set forth in Table 1.
[00125] In a particular embodiment, an antibody described herein, or an antigen-binding fragment thereof, comprises the VH CDRl, VH CDR2, and VH CDR3 of Ab2000. In a specific embodiment, the VH CDRl, VH CDR2, and VH CDR3 are as set forth in Table 1. In another embodiment, the antibody or antigen-binding fragment further comprises a VL as set forth in Table 2.
[00126] In a particular embodiment, an antibody described herein, or an antigen-binding fragment thereof, which specifically binds to human MerTK, comprises a VH as set forth in Table 2. In another embodiment, the antibody or antigen-binding fragment further comprises VL CDRl, VL CDR2 and/or VL CDR3 as set forth in Table 1.
[00127] In a particular embodiment, an antibody described herein, or an antigen-binding fragment thereof, comprises the VL CDRl, VL CDR2, and VL CDR3 of Ab2000, and the VH CDRl, VH CDR2, and VH CDR3 of Ab2000. In a particular embodiment, for example, the VL CDRl, VL CDR2, and VL CDR3 are as set forth in Table 1 (SEQ ID NOS: 6, 7, and 8, respectively), and the VH CDRl, VH CDR2, and VH CDR3 are as set forth in Table (SEQ ID NOS: 3, 4, and 5, respectively).
[00128] In some embodiments, an antibody described herein, or an antigen-binding fragment thereof, comprises the amino acid sequence of SEQ ID NO: 54. In some embodiments, an antibody described herein, or an antigen-binding fragment thereof, comprises the amino acid sequence of SEQ ID NO: 52. In some embodiments, an antibody described herein, or an antigen-binding fragment thereof, comprises the amino acid sequence of SEQ ID NO: 53. In some embodiments, an antibody described herein, or an antigen-binding fragment thereof, comprises the amino acid sequence of SEQ ID NO: 54 and the amino acid sequence of SEQ ID NO: 52. In some embodiments, an antibody described herein, or an antigen-binding fragment thereof, comprises the amino acid sequence of SEQ ID NO: 54 and the amino acid sequence of SEQ ID NO: 52.
In certain aspects, the CDRs of an antibody described herein are Chothia CDRs (see, e.g., Chothia and Lesk, 1987, J. Mol. Biol., 196:901-917; and U.S. Patent No. 7,709,226 and Martin, A., "Protein Sequence and Structure Analysis of Antibody Variable Domains," in Antibody Engineering, Kontermann and Diibel, eds., Chapter 31, pp. 422-439, Springer- Verlag, Berlin (2001)). The term "Chothia CDRs," and like terms are recognized in the art and refer to antibody CDR sequences as determined according to the method of Chothia and Lesk, 1987, J. Mol. Biol., 196:901-917, which will be referred to herein as the "Chothia CDRs" (see also, e.g., U.S. Patent No. 7,709,226 and Martin, A., "Protein Sequence and Structure Analysis of
Antibody Variable Domains," in Antibody Engineering, Kontermann and Diibel, eds., Chapter 31, pp. 422-439, Springer- Verlag, Berlin (2001)). In certain embodiments, provided herein is an antibody or an antigen-binding fragment thereof which specifically binds to human MerTK comprises VL and VH CDR as set forth in Table 3. In a particular embodiment, provided herein is an antibody or an antigen-binding fragment thereof which specifically binds to human MerTK, and comprises VL CDR1 (SEQ ID NO: 16), VL CDR2 (SEQ ID NO: 17), and VL CDR3 (SEQ ID NO: 18), and VH CDR1 (SEQ ID NO: 13), VH CDR2 (SEQ ID NO: 14), and VH CDR3 (SEQ ID NO: 15).
Table 3. Ab2000 VL and VH CDR Amino Acid Sequences (Chothia)
Figure imgf000028_0001
[00129] In a specific embodiment, the VL CDR1, VL CDR2, and VL CDR3 are as set forth in Table 3 (SEQ ID NOS: 16, 17, and 18, respectively).
[00130] In a specific embodiment, the VH CDR1, VH CDR2, and VH CDR3 are as set forth in Table 3 (SEQ ID NOS: 13, 14, and 15, respectively).
[00131 ] In a particular embodiment, for example, the VL CDR1 , VL CDR2, and VL CDR3 are as set forth in Table 3 (SEQ ID NOS: 16, 17, and 18, respectively), and the VH CDR1, VH CDR2, and VH CDR3 are as set forth in Table 3 (SEQ ID NOS: 13, 14, and 15, respectively). [00132] In certain aspects, the CDRs of an antibody described herein are AbM. In certain embodiments, provided herein is an antibody or an antigen-binding fragment thereof which specifically binds to human MerTK comprises VL and VH CDR as set forth in Table 4. In a particular embodiment, provided herein is an antibody or an antigen-binding fragment thereof which specifically binds to human MerTK, and comprises VL CDRl (SEQ ID NO: 22), VL CDR2 (SEQ ID NO: 23), and VL CDR3 (SEQ ID NO: 24), and VH CDRl (SEQ ID NO: 19), VH CDR2 (SEQ ID NO: 20), and VH CDR3 (SEQ ID NO: 21).
Table 4. Ab2000 VL and VH CDR Amino Acid Sequences (AbM)
Figure imgf000030_0001
[00133] In a specific embodiment, the VL CDRl, VL CDR2, and VL CDR3 are as set forth in Table 4 (SEQ ID NOS: 22, 23, and 24, respectively).
[00134] In a specific embodiment, the VH CDRl, VH CDR2, and VH CDR3 are as set forth in Table 4 (SEQ ID NOS: 19, 20, and 21, respectively).
[00135] In a particular embodiment, for example, the VL CDRl, VL CDR2, and VLCDR3 are as set forth in Table 4 (SEQ ID NOS: 22, 23, and 24, respectively), and the VH CDRl, VH CDR2, and VH CDR3 are as set forth in Table 4 (SEQ ID NOS: 19, 20, and 21, respectively).
[00136] In certain embodiments, a MerTK-binding antibody, or antigen-binding fragment thereof, described herein comprises CDRs of antibody Ab2000, as determined by the IMGT (Immunogenetics) numbering system; see, e.g., Lefranc, M.-P., 1999, The Immunologist, 7: 132- 136 and Lefranc, M.-P. et al., 1999, Nucleic Acids Res., 27:209-212, both of which are incorporated herein by reference in their entirety (Table 5).
Table 5. Ab2000 VL and VH CDR Amino Acid Sequences (IMGT)
Figure imgf000030_0002
[00137] In a specific embodiment, the VL CDRl, VL CDR2, and VL CDR3 are as set forth in Table 5 (SEQ ID NOS: 44, 45, and 46, respectively).
[00138] In a specific embodiment, the VH CDRl, VH CDR2, and VH CDR3 are as set forth in Table 5 (SEQ ID NOS: 41, 42, and 43, respectively).
[00139] In a particular embodiment, for example, the VL CDRl, VL CDR2, and VL CDR3 are as set forth in Table 5 (SEQ ID NOS: 44, 45, and 46, respectively), and the VH CDRl, VH CDR2, and VH CDR3 are as set forth in Table 5 (SEQ ID NOS: 41, 42, and 43, respectively).
[00140] In certain embodiments, a MerTK-binding antibody, or antigen-binding fragment thereof, described herein comprises CDRs of antibody Ab2000, as determined by the Contact numbering system; see, e.g., see MacCallum RM et al., 1996, J Mol Biol 5: 732-745, which is incorporated herein by reference in its entirety (Table 6).
Table 6. Ab2000 VL and VH CDR Amino Acid Sequences (Contact)
Figure imgf000031_0001
[00141] In a specific embodiment, the VL CDRl, VL CDR2, and VL CDR3 are as set forth in Table 6 (SEQ ID NOS: 36, 37, and 38, respectively).
[00142] In a specific embodiment, the VH CDRl, VH CDR2, and VH CDR3 are as set forth in Table 6 (SEQ ID NOS: 33, 34, and 35, respectively).
[00143] In a particular embodiment, for example, the VL CDRl, VL CDR2, and VLCDR3 are as set forth in Table 6 (SEQ ID NOS: 36, 37 ,and 38, respectively), and the VH CDRl, VH CDR2, and VH CDR3 are as set forth in Table 6 (SEQ ID NOS: 33, 34, and 35, respectively).
[00144] In certain aspects, an antibody described herein may be described by its VL alone, or its VH alone, or by its 3 VL CDRs alone, or its 3 VH CDRs alone. See, for example, Rader et al., 1998, Proc. Natl. Acad. Sci. USA, 95: 8910-8915, which is incorporated herein by reference in its entirety, describing the humanization of the mouse anti-avP3 antibody by identifying a complementing light chain or heavy chain, respectively, from a human light chain or heavy chain library, resulting in humanized antibody variants having affinities as high or higher than the affinity of the original antibody. See also, Clackson et al., 1991, Nature 352:624-628, which is incorporated herein by reference in its entirety, describing methods of producing antibodies that bind a specific antigen by using a specific VL domain (or VH domain) and screening a library for the complementarity variable domains. The screen produced 14 new partners for a specific VH domain and 13 new partners for a specific VL domain, which were strong binders, as determined by ELISA. See also, Kim & Hong, 2007, J. Microbiol. 45:572-577, which is incorporated herein by reference in its entirety, describing methods of producing antibodies that bind a specific antigen by using a specific VH domain and screening a library (e.g., human VL library) for complementarity VL domains; the selected VL domains in turn could be used to guide selection of additional complementarity (e.g., human) VH domains.
[00145] In another embodiment, affinity maturation techniques can be used to alter one or more CDR region(s), followed by screening of the resultant binding molecules for the desired change in binding. Any affinity maturation techniques known in the art and/or described herein (see Example 7) can be used. See also, Holland et al., 2013, J. Immun. Methods 394:55-61, which is incorporated herein by reference in its entirety, describing methods of producing libraries for directed evolution of proteins (e.g., affinity matured antibodies). In certain embodiments, a CDR region altered (e.g., by affinity maturation) can result in changes in binding affinity (e.g., on-rate of binding and/or off-rate of binding). In some embodiments, methods known in the art and/or disclosed herein can be used to analyze binding affinity and
immunogenicity of affinity matured antibodies. Any method known in the art can be used to ascertain immunospecific binding to MerTK (e.g., human MerTK ECD), for example, the binding assays and conditions described in the "Examples" section (Section 6) provided herein.
[00146] In certain embodiments, provided herein is an antibody or an antigen-binding fragment thereof which specifically binds to human MerTK comprises VL and VH CDR as set forth in Table 7, which utilizes standard one letter amino acid abbreviations. In a particular embodiment, provided herein is an antibody or an antigen-binding fragment thereof which specifically binds to human MerTK, and comprises VL CDRl (SEQ ID NO: 30), VL CDR2 (SEQ ID NO: 7), and VL CDR3 (SEQ ID NO: 8), and VH CDRl (SEQ ID NO: 3), VH CDR2 (SEQ ID NO: 4), and VH CDR3 (SEQ ID NO: 27).
Table 7. Ab2000-A7 VL and VH CDR Amino Acid Sequences (Kabat) VL CDR1 (SEQ ID NO: 30) VL CDR2 (SEQ ID NO: 7) VL CDR3 (SEQ ID NO: 8)
KSSQSVLYS GNNKNYLA WASTREF QQYYTTPLT
VH CDR1 (SEQ ID NO: 3) VH CDR2 (SEQ ID NO: 4) VH CDR3 (SEQ ID NO: 27)
SYAMS AISGSGGSTYYADSVKG DRQWLLPRVFDI
00147] In certain embodiments, provided herein is an antibody or an antigen-binding fragment thereof developed using affinity maturation methods disclosed herein or known in the art. In some embodiments, provided herein is an affinity matured antibody an antigen-binding fragment thereof which specifically binds to human MerTK and comprises a VL and/or a VH as set forth in Table 8, which utilizes standard one letter amino acid abbreviations. In a particular embodiment, provided herein is an affinity matured antibody or an antigen-binding fragment thereof which specifically binds to human MerTK, and comprises VL (SEQ ID NO: 26) and VH (SEQ ID NO: 25). For ease of description, an antibody that comprises the VL and VH amino acid sequences of Table 8 is referred to herein as "Ab2000-A7." In one embodiment, such an antibody or antigen-binding fragment comprises a separate light chain comprising the VL amino acid sequence and a separate heavy chain comprising the VH amino acid sequence. In another embodiment, such an antibody or antigen-binding fragment comprises a single chain comprising the VL amino acid sequence and the VH amino acid sequence.
Table 8 Ab2000-A7 VL and VH Amino Acid Sequences
Figure imgf000033_0001
[00148] In a particular embodiment, an antibody or an antigen-binding fragment described herein, comprises the VL CDR1, VL CDR2, and VL CDR3 of Ab2000-A7. In a specific embodiment, the VL CDR1, VL CDR2, and VL CDR3 are as set forth in Table 7 (SEQ ID NOS: 30, 7, and 8, respectively). In another embodiment, the antibody or antigen-binding fragment further comprises a VH as set forth in Table 8. In yet another embodiment, the antibody or antigen-binding fragment further comprises a VH as set forth in Table 1.
[00149] In a particular embodiment, an antibody or an antigen-binding fragment described herein, which specifically binds to human MerTK, comprises a VH CDRl, VH CDR2 and/or VH CDR3 as set forth in Table 7. In another embodiment, the antibody or antigen-binding fragment further comprises a VL as set forth in Table 8. . In yet another embodiment, the antibody or antigen-binding fragment further comprises a VL as set forth in Table 1.
[00150] In a particular embodiment, an antibody described herein, or an antigen-binding fragment thereof, comprises the VH CDRl, VH CDR2, and VH CDR3 of Ab2000-A7. In a specific embodiment, the VH CDRl, VH CDR2, and VH CDR3 are as set forth in Table 7. In another embodiment, the antibody or antigen-binding fragment further comprises a VL as set forth in Table 8.
[00151] In a particular embodiment, an antibody described herein, or an antigen-binding fragment thereof, which specifically binds to human MerTK, comprises a VH as set forth in Table 8. In another embodiment, the antibody or antigen-binding fragment further comprises VL CDRl, VL CDR2 and/or VL CDR3 as set forth in Table 7.
[00152] In a particular embodiment, an antibody described herein, or an antigen-binding fragment thereof, comprises the VL CDRl, VL CDR2, and VL CDR3 of Ab2000-A7, and the VH CDRl, VH CDR2, and VH CDR3 of Ab2000-A7. In a particular embodiment, for example, the VL CDRl, VL CDR2, and VL CDR3 are as set forth in Table 7 (SEQ ID NOS: 30, 7, and 8, respectively), and the VH CDRl, VH CDR2, and VH CDR3 are as set forth in Table (SEQ ID NOS: 3, 4, and 27, respectively).
[00153] In some embodiments, an antibody described herein, or an antigen-binding fragment thereof, comprises the amino acid sequence of SEQ ID NO: 57. In some embodiments, an antibody described herein, or an antigen-binding fragment thereof, comprises the amino acid sequence of SEQ ID NO: 55. In some embodiments, an antibody described herein, or an antigen-binding fragment thereof, comprises the amino acid sequence of SEQ ID NO: 56. In some embodiments, an antibody described herein, or an antigen-binding fragment thereof, comprises the amino acid sequence of SEQ ID NO: 57 and the amino acid sequence of SEQ ID NO: 55. In some embodiments, an antibody described herein, or an antigen-binding fragment thereof, comprises the amino acid sequence of SEQ ID NO: 57 and the amino acid sequence of SEQ ID NO: 56.
[00154] In certain embodiments, a MerTK-binding antibody, or antigen-binding fragment thereof, described herein comprises a VH of SEQ ID NO: 25 and a VL of SEQ ID NO: 2, wherein the MerTK-binding antibody, or antigen-binding fragment thereof is Ab2000-Fl (Table 9). In certain embodiments, a MerTK-binding antibody, or antigen-binding fragment thereof, described herein comprises a VH of SEQ ID NO: 1 and a VL of SEQ ID NO: 26, wherein the MerTK-binding antibody, or antigen-binding fragment thereof is Ab2000-G6 (Table 10).
Table 9 Ab2000-Fl VL and VH Amino Acid Sequences
Figure imgf000035_0001
[00155] In certain aspects, the CDRs of an antibody described herein are Chothia CDRs (see, e.g., Chothia and Lesk, 1987, J. Mol. Biol., 196:901-917; and U.S. Patent No. 7,709,226 and Martin, A., "Protein Sequence and Structure Analysis of Antibody Variable Domains," in Antibody Engineering, Kontermann and Diibel, eds., Chapter 31, pp. 422-439, Springer- Verlag, Berlin (2001)). [00156] In certain embodiments, provided herein is an antibody or an antigen-binding fragment thereof which specifically binds to human MerTK comprises VL and VH CDR as set forth in Table 11. In a particular embodiment, provided herein is an antibody or an antigen- binding fragment thereof which specifically binds to human MerTK, and comprises VL CDRl (SEQ ID NO: 31), VL CDR2 (SEQ ID NO: 17), and VL CDR3 (SEQ ID NO: 18), and VH CDRl (SEQ ID NO: 13), VH CDR2 (SEQ ID NO: 14), and VH CDR3 (SEQ ID NO: 28).
Table 11. Ab2000-A7 VL and VH CDR Amino Acid Sequences (Chothia)
Figure imgf000036_0001
[00157] In a specific embodiment, the VL CDRl, VL CDR2, and VL CDR3 are as set forth in Table 11 (SEQ ID NOS: 31, 17, and 18, respectively).
[00158] In a specific embodiment, the VH CDRl, VH CDR2, and VH CDR3 are as set forth in Table 11 (SEQ ID NOS: 13, 14, and 28, respectively).
[00159] In a particular embodiment, for example, the VL CDRl , VL CDR2, and VL CDR3 are as set forth in Table 11 (SEQ ID NOS: 31, 17, and 18, respectively), and the VH CDRl, VH CDR2, and VH CDR3 are as set forth in Table 3 (SEQ ID NOS: 13, 14, and 28, respectively).In certain aspects, the CDRs of an affinity matured antibody described herein (e.g., Ab2000-A7) are AbM.
[00160] In certain embodiments, provided herein is an antibody or an antigen-binding fragment thereof which specifically binds to human MerTK comprises VL and VH CDR as set forth in Table 12. In a particular embodiment, provided herein is an antibody or an antigen- binding fragment thereof which specifically binds to human MerTK, and comprises VL CDRl (SEQ ID NO: 32), VL CDR2 (SEQ ID NO: 23), and VL CDR3 (SEQ ID NO: 24), and VH CDRl (SEQ ID NO: 19), VH CDR2 (SEQ ID NO: 20), and VH CDR3 (SEQ ID NO: 29).
Table 12. Ab2000-A7 VL and VH CDR Amino Acid Sequences (AbM) VL CDRl (SEQ ID NO: 32) VL CDR2 (SEQ ID NO: 23) VL CDR3 (SEQ ID NO: 24)
KS SQSVLYS GNNKN Y LA WAS TRE F QQYYTTPLT
VH CDRl (SEQ ID NO: 19) VH CDR2 (SEQ ID NO: 20) VH CDR3 (SEQ ID NO: 29)
GFT FS SYAMS AI SGSGGS TY DRQWLLPRVFDI
[00161] In a specific embodiment, the VL CDRl , VL CDR2, and VL CDR3 are as set forth in Table 12 (SEQ ID NOS: 32, 23, and 24, respectively).
[00162] In a specific embodiment, the VH CDRl, VH CDR2, and VH CDR3 are as set forth in Table 12 (SEQ ID NOS: 19, 20, and 29, respectively). In a particular embodiment, for example, the VL CDRl, VL CDR2, and VLCDR3 are as set forth in Table 12 (SEQ ID NOS: 32, 23, and 24, respectively), and the VH CDRl, VH CDR2, and VH CDR3 are as set forth in Table 12 (SEQ ID NOS: 19, 20, and 29, respectively).In certain embodiments, a MerTK-binding antibody, or antigen-binding fragment thereof, described herein comprises CDRs of antibody Ab2000-A7, as determined by the IMGT (Immunogenetics) numbering system; (see, e.g., Lefranc, M.-P., 1999, The Immunologist, 7: 132-136 and Lefranc, M.-P. et al., 1999, Nucleic Acids Res., 27:209-212), both of which are incorporated herein by reference in their entirety (Table 13).
Table 13. Ab2000-A7 VL and VH CDR Amino Acid Sequences (IMGT)
Figure imgf000037_0001
[00163] In a specific embodiment, the VL CDRl, VL CDR2, and VL CDR3 are as set forth in Table 13 (SEQ ID NOS: 48, 45, and 46, respectively).
[00164] In a specific embodiment, the VH CDRl, VH CDR2, and VH CDR3 are as set forth in Table 13 (SEQ ID NOS: 41, 42, and 47, respectively). [00165] In a particular embodiment, for example, the VL CDR1, VL CDR2, and VLCDR3 are as set forth in Table 13 (SEQ ID NOS: 48, 45, and 46, respectively), and the VH CDR1, VH CDR2, and VH CDR3 are as set forth in Table 13 (SEQ ID NOS: 41, 42, and 47, respectively).
[00166] In certain embodiments, a MerTK-binding antibody, or antigen-binding fragment thereof, described herein comprises CDRs of antibody Ab2000-A7, as determined by the Contact numbering system; see, e.g., see MacCallum RM et al., 1996, J Mol Biol 5: 732-745, which is incorporated herein by reference in its entirety (Table 14).
Table 14. Ab2000-A7 VL and VH CDR Amino Acid Sequences (Contact)
Figure imgf000038_0001
[00167] In a specific embodiment, the VL CDR1 , VL CDR2, and VL CDR3 are as set forth in Table 14 (SEQ ID NOS: 40, 37, and 38, respectively).
[00168] In a specific embodiment, the VH CDR1, VH CDR2, and VH CDR3 are as set forth in Table 14 (SEQ ID NOS: 33, 34, and 39, respectively).
[00169] In a particular embodiment, for example, the VL CDR1, VL CDR2, and VLCDR3 are as set forth in Table 14 (SEQ ID NOS: 44, 45, and 46, respectively), and the VH CDR1, VH CDR2, and VH CDR3 are as set forth in Table 14 (SEQ ID NOS: 41, 42, and 43, respectively).
[00170] In a specific embodiment, the position of one or more CDRs along the VH (e.g., CDR1, CDR2, or CDR3) and/or VL (e.g., CDR1, CDR2, or CDR3) region of an antibody described herein may vary by one, two, three, four, five, or six amino acid positions so long as immunospecific binding to MerTK (e.g., human MerTK ECD) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%>, at least 70%>, at least 80%>, at least 90%>, at least 95%)). For example, in one embodiment, the position defining a CDR of any of Table 1, Table 3-7, or Table 11-14 may vary by shifting the N-terminal and/or C-terminal boundary of the CDR by one, two, three, four, five, or six amino acids, relative to the current CDR position, so long as immunospecific binding to MerTK (e.g., human MerTK) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%), at least 95%). In another embodiment, the length of one or more CDRs along the VH (e.g., CDR1, CDR2, or CDR3) and/or VL (e.g., CDR1, CDR2, or CDR3) region of an antibody described herein may vary (e.g., be shorter or longer) by one, two, three, four, five, or more amino acids, so long as immunospecific binding to MerTK (e.g., human MerTK ECD) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). For example, in one embodiment, a VH and/or VL CDR1, CDR2, and/or CDR3 described herein may be one, two, three, four, five or more amino acids shorter than one or more of the CDRs described by SEQ ID NOS: 3-8, 13-24, and 27-48 , so long as immunospecific binding to MerTK (e.g., human MerTK ECD) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). In another embodiment, a VH and/or VL CDR1, CDR2, and/or CDR3 described herein may be one, two, three, four, five or more amino acids longer than one or more of the CDRs described by SEQ ID NOS: 3-8, 13-24, 27-48, so long as immunospecific binding to MerTK (e.g., human MerTK ECD) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). In another embodiment, the amino terminus of a VH and/or VL CDR1, CDR2, and/or CDR3 described herein may be extended by one, two, three, four, five or more amino acids compared to one or more of the CDRs described by SEQ ID NOS: 3-8, 13-24, 27-48, so long as immunospecific binding to MerTK (e.g., human MerTK ECD) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). In another embodiment, the carboxy terminus of a VH and/or VL CDR1 , CDR2, and/or CDR3 described herein may be extended by one, two, three, four, five or more amino acids compared to one or more of the CDRs described by SEQ ID NOS: 3-8, 13-24, 27-48, so long as
immunospecific binding to MerTK (e.g., human MerTK ECD) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). In another embodiment, the amino terminus of a VH and/or VL CDR1, CDR2, and/or CDR3 described herein may be shortened by one, two, three, four, five or more amino acids compared to one or more of the CDRs described by SEQ ID NOS: 3-8, 13-24, 27-48, so long as immunospecific binding to MerTK (e.g., human MerTK) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%), at least 95%). In one embodiment, the carboxy terminus of a VH and/or VL CDR1, CDR2, and/or CDR3 described herein may be shortened by one, two, three, four, five or more amino acids compared to one or more of the CDRs described by SEQ ID NOS: 3-8, 13-24, 27- 48, so long as immunospecific binding to MerTK (e.g., human MerTK ECD) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%), at least 95%). Any method known in the art can be used to ascertain whether immunospecific binding to MerTK (e.g., human MerTK ECD) is maintained, for example, the binding assays and conditions described in the "Examples" section (Section 6) provided herein. For example, section 6.2 provided herein describes an assay for measuring binding to an ECD of human MerTK.
[00171] In specific aspects, provided herein is an antibody comprising an antibody light chain and heavy chain, e.g., a separate light chain and heavy chain. With respect to the light chain, in a specific embodiment, the light chain of an antibody described herein is a kappa light chain. In another specific embodiment, the light chain of an antibody described herein is a lambda light chain. In yet another specific embodiment, the light chain of an antibody described herein is a human kappa light chain or a human lambda light chain. In a particular embodiment, an antibody described herein, which specifically binds to a MerTK polypeptide (e.g., human MerTK) comprises a light chain wherein the amino acid sequence of the VL chain region comprises any amino acid sequence described herein (e.g., SEQ ID NOS: 2 or 26), and wherein the constant region of the light chain comprises the amino acid sequence of a human kappa light chain constant region. In another particular embodiment, an antibody described herein, which specifically binds a MerTK polypeptide (e.g., human MerTK) comprises a light chain wherein the amino acid sequence of the VL chain region can comprise any amino acid sequence described herein (e.g., SEQ ID NOS: 2 or 26), and wherein the constant region of the light chain comprises the amino acid sequence of a human lambda light chain constant region. Non-limiting examples of human constant region sequences have been described in the art, e.g., see U.S. Patent No. 5,693,780 and Kabat et al. (1991) Sequences of Proteins of Immunological Interest Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242.
[00172] With respect to the heavy chain, in a specific embodiment, the heavy chain of an antibody described herein can be an alpha (a), delta (δ), epsilon (ε), gamma (γ) or mu (μ) heavy chain. In another specific embodiment, the heavy chain of an antibody described can comprise a human alpha (a), delta (δ), epsilon (ε), gamma (γ) or mu (μ) heavy chain. In a particular embodiment, an antibody described herein, which specifically binds to a MerTK polypeptide (e.g., human MerTK), comprises a heavy chain wherein the amino acid sequence of the VH chain region can comprise any amino acid sequence described herein (e.g., any of SEQ ID NOS: 1 or 25), and wherein the constant region of the heavy chain comprises the amino acid sequence of a human gamma (γ) heavy chain constant region. Non-limiting examples of human constant region sequences have been described in the art, e.g., see U.S. Patent No. 5,693,780 and Kabat et al. (1991) Sequences of Proteins of Immunological Interest Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242.
[00173] In certain embodiments, an antibody described herein, which specifically binds to a MerTK polypeptide (e.g., human MerTK), comprises a light chain constant region comprising an amino acid sequence of SEQ ID NO: 49, wherein the amino acid sequence for SEQ ID NO: 49 is RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ D SKD S T YSL S S TLTL SK AD YEKHK V Y ACE VTHQ GL S SP VTK SFNRG. In certain embodiments, an antibody described herein, which specifically binds to a MerTK polypeptide (e.g., human MerTK), comprises a heavy chain constant region comprising an amino acid sequence of SEQ ID NO: 50, wherein the amino acid sequence for SEQ ID NO: 50 is
AS TKGP S VFPL AP S SK S T S GGT A ALGCL VKD YFPEP VT V S WNS GALT S GVHTFP A VLQ S S GL YSL S S VVTVP S S SLGTQT YICNVNHKP SNTKVDKKVEPKSCDKTHTCPPCP APELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG. In certain embodiments, an antibody described herein, which specifically binds to a MerTK polypeptide (e.g., human MerTK), comprises a light chain constant region comprising an amino acid sequence of SEQ ID NO: 49 and a heavy chain constant region comprising an amino acid sequence of SEQ ID NO: 50.
[00174] In certain embodiments, an antibody described herein, which specifically binds to a MerTK polypeptide (e.g., human MerTK), comprises heavy chain constant region comprising an amino acid sequence of SEQ ID NO: 51, wherein the amino acid sequence for SEQ ID NO: 51 is ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GL YSL S S V VTVP S S FGTQT YTCNVDHKP SNTKVDKT VERKCC VECPPCP APP VAGP S V FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNST FRVVSVLTVVHQDWLNGKEYKCKVS KGLPAPIEKTISKTKGQPREPQVYTLPPSREEM TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPG. In certain embodiments, an antibody described herein, which specifically binds to a MerTK polypeptide (e.g., human MerTK), comprises a light chain constant region comprising an amino acid sequence of SEQ ID NO: 49 and a heavy chain constant region comprising an amino acid sequence of SEQ ID NO: 51.
[00175] In certain embodiments, an antibody described herein, which specifically binds to a
MerTK polypeptide (e.g., human MerTK), comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 52, wherein the amino acid sequence for SEQ ID NO: 52 is
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTY
YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDRQWLLPRAFDIWGQGTTV
TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV
LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE
LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR
EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL
PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT
VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG.
[00176] In certain embodiments, an antibody described herein, which specifically binds to a
MerTK polypeptide (e.g., human MerTK), comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 53, wherein the amino acid sequence for SEQ ID NO: 53 is
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTY
YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDRQWLLPRAFDIWGQGTTV
TVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV
LQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVA
GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQ
FNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVD
KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG. [00177] In certain embodiments, an antibody described herein, which specifically binds to a MerTK polypeptide (e.g., human MerTK), comprises a light chain comprising an amino acid sequence of SEQ ID NO: 54, wherein the amino acid sequence for SEQ ID NO: 54 is
DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWAST REFGVPDRF SGSGSGTDFTLTIS SLQ AED VAVYFCQQ YYTTPLTFGQGTKLEIKRT VAAP S VFIFPP SDEQLK SGT AS VVCLLNNF YPREAK VQWKVDNALQ SGNS QES VTEQD SKD ST YSL S S TLTL SK AD YEKHK V Y ACE VTHQGL S SP VTK SFNRGEC .
[00178] In certain embodiments, an antibody described herein, which specifically binds to a MerTK polypeptide (e.g., human MerTK), comprises a light chain comprising an amino acid sequence of SEQ ID NO: 54 and a heavy chain comprising an amino acid sequence of SEQ ID NO: 52. In certain embodiments, an antibody described herein, which specifically binds to a MerTK polypeptide (e.g., human MerTK), comprises a light chain comprising an amino acid sequence of SEQ ID NO: 54 and a heavy chain comprising an amino acid sequence of SEQ ID NO: 53.
[00179] In certain embodiments, an antibody described herein, which specifically binds to a
MerTK polypeptide (e.g., human MerTK), comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 55, wherein the amino acid sequence for SEQ ID NO: 55 is
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTY
YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDRQWLLPRVFDIWGQGTTV
TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV
LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE
LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR
EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL
PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT
VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG.
[00180] In certain embodiments, an antibody described herein, which specifically binds to a MerTK polypeptide (e.g., human MerTK), comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 56, wherein the amino acid sequence for SEQ ID NO: 56 is
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTY YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDRQWLLPRVFDIWGQGTTV TVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSS FGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVA
GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQ
FNSTFRVVSVLTVVHQDWLNGKEYKCKVS KGLPAPIEKTISKTKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVD
KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG.
[00181] In certain embodiments, an antibody described herein, which specifically binds to a MerTK polypeptide (e.g., human MerTK), comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 57, wherein the amino acid sequence for SEQ ID NO: 57 is
DIVMTQSPDSLAVSLGERATINCKSSQSVLYSGNNKNYLAWYQQKPGQPPKLLIYWAST REFGVPDRF SGSGSGTDFTLTIS SLQ AED VAVYFCQQ YYTTPLTFGQGTKLEIKRT VAAP S VFIFPP SDEQLK SGT AS VVCLLNNF YPREAK VQWKVDNALQ SGNS QES VTEQD SKD ST YSL S S TLTL SK AD YEKHK V Y ACE VTHQGL S SP VTK SFNRGEC .
[00182] In certain embodiments, an antibody described herein, which specifically binds to a MerTK polypeptide (e.g., human MerTK), comprises a light chain comprising an amino acid sequence of SEQ ID NO: 54 and a heavy chain comprising an amino acid sequence of SEQ ID NO: 52. In certain embodiments, an antibody described herein, which specifically binds to a MerTK polypeptide (e.g., human MerTK), comprises a light chain comprising an amino acid sequence of SEQ ID NO: 54 and a heavy chain comprising an amino acid sequence of SEQ ID NO: 53.
[00183] In certain embodiments, an antibody described herein, which specifically binds to a MerTK polypeptide (e.g., human MerTK), comprises a light chain comprising an amino acid sequence of SEQ ID NO: 54 and a heavy chain comprising an amino acid sequence of SEQ ID NO: 55. In certain embodiments, an antibody described herein, which specifically binds to a MerTK polypeptide (e.g., human MerTK), comprises a light chain comprising an amino acid sequence of SEQ ID NO: 54 and a heavy chain comprising an amino acid sequence of SEQ ID NO: 56.
[00184] In certain embodiments, an antibody described herein, which specifically binds to a MerTK polypeptide (e.g., human MerTK), comprises a light chain comprising an amino acid sequence of SEQ ID NO: 57 and a heavy chain comprising an amino acid sequence of SEQ ID NO: 52. In certain embodiments, an antibody described herein, which specifically binds to a MerTK polypeptide (e.g., human MerTK), comprises a light chain comprising an amino acid sequence of SEQ ID NO: 57 and a heavy chain comprising an amino acid sequence of SEQ ID NO: 53.
[00185] In certain embodiments, an antibody described herein, which specifically binds to a MerTK polypeptide (e.g., human MerTK), comprises a light chain comprising an amino acid sequence of SEQ ID NO: 57 and a heavy chain comprising an amino acid sequence of SEQ ID NO: 55. In certain embodiments, an antibody described herein, which specifically binds to a MerTK polypeptide (e.g., human MerTK), comprises a light chain comprising an amino acid sequence of SEQ ID NO: 57 and a heavy chain comprising an amino acid sequence of SEQ ID NO: 56.
[00186] In a specific embodiment, an antibody described herein, which specifically binds to a MerTK polypeptide (e.g., human MerTK) comprises a VL chain region and a VH chain region comprising any amino acid sequences described herein, and wherein the constant regions comprise the amino acid sequences of the constant regions of an IgG, IgE, IgM, IgD, IgA or IgY immunoglobulin molecule, or a human IgG, IgE, IgM, IgD, IgA or IgY immunoglobulin molecule. In another specific embodiment, an antibody described herein, which specifically binds to a MerTK polypeptide (e.g., human MerTK) comprises a VL chain region and a VH chain region comprising any amino acid sequences described herein, and wherein the constant regions comprise the amino acid sequences of the constant regions of an IgG, IgE, IgM, IgD, IgA or IgY immunoglobulin molecule, any class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2), or any subclass (e.g., IgG2a and IgG2b) of immunoglobulin molecule. In a particular embodiment, the constant regions comprise the amino acid sequences of the constant regions of a human IgG, IgE, IgM, IgD, IgA or IgY immunoglobulin molecule, any class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2), or any subclass (e.g., IgG2a and IgG2b) of immunoglobulin molecule.
[00187] In yet another specific embodiment, an antibody described herein, which specifically binds to a MerTK polypeptide (e.g., human MerTK), comprises a VL chain region and a VH chain region comprising any amino acid sequences described herein, and wherein the constant regions comprise the amino acid sequences of the constant regions of a human IgGl (e.g., isotype a, z, or f), human IgG2, or human IgG4. Non-limiting examples of human constant regions are described in the art, e.g., see Kabat et al. (1991) Sequences of Proteins of
Immunological Interest Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242. [00188] In another particular embodiment, an antibody described herein, which specifically binds to a MerTK polypeptide (e.g., human MerTK), comprises a light chain and a heavy chain, wherein (i) the light chain comprises a VL chain region comprising a VL CDRl, VL CDR2, and VL CDR3 of Ab2000; (ii) the heavy chain comprises a VH chain region comprising a VH CDRl, VH CDR2, and VH CDR3 of Ab2000; (iii) the light chain further comprises a constant light chain domain comprising the amino acid sequence of the constant domain of a human kappa or lambda light chain; and (iv) the heavy chain further comprises a constant heavy chain domain comprising the amino acid sequence of the constant domain of a human IgG, e.g., IgGl, heavy chain.
[00189] In another embodiment, an antibody described herein, which specifically binds to a MerTK polypeptide (e.g., human MerTK), comprises a light chain and a heavy chain, wherein (i) the light chain comprises a VL chain region comprising a VL CDRl, VL CDR2, and VL CDR3 of Ab2000-A7; (ii) the heavy chain comprises a VH chain region comprising a VH CDRl, VH CDR2, and VH CDR3 of Ab2000-A7; (iii) the light chain further comprises a constant light chain domain comprising the amino acid sequence of the constant domain of a human kappa or lambda light chain; and (iv) the heavy chain further comprises a constant heavy chain domain comprising the amino acid sequence of the constant domain of a human IgG, e.g., IgGl, heavy chain.
[00190] In an alternative particular embodiment, an antibody described herein, which specifically binds to a MerTK polypeptide (e.g., human MerTK), comprises a light chain and a heavy chain, wherein (i) the light chain comprises a VL chain region comprising a VL CDRl, VL CDR2, and VL CDR3 of Ab2000; (ii) the heavy chain comprises a VH chain region comprising a VH CDRl, VH CDR2, and VH CDR3 of Ab2000-A7; (iii) the light chain further comprises a constant light chain domain comprising the amino acid sequence of the constant domain of a human kappa or lambda light chain; and (iv) the heavy chain further comprises a constant heavy chain domain comprising the amino acid sequence of the constant domain of a human IgG, e.g., IgGl, heavy chain.
[00191] In another embodiment, an antibody described herein, which specifically binds to a MerTK polypeptide (e.g., human MerTK), comprises a light chain and a heavy chain, wherein (i) the light chain comprises a VL chain region comprising a VL CDRl, VL CDR2, and VL CDR3 of Ab2000-A7; (ii) the heavy chain comprises a VH chain region comprising a VH CDRl, VH CDR2, and VH CDR3 of Ab2000; (iii) the light chain further comprises a constant light chain domain comprising the amino acid sequence of the constant domain of a human kappa or lambda light chain; and (iv) the heavy chain further comprises a constant heavy chain domain
comprising the amino acid sequence of the constant domain of a human IgG, e.g., IgGl, heavy chain.
[00192] In another particular embodiment, an antibody described herein, which specifically binds to a MerTK polypeptide (e.g., human MerTK), comprises a light chain and a heavy chain, wherein (i) the light chain comprises a VL chain region comprising a VL CDRl, VL CDR2, and VL CDR3 of Table 1; (ii) the heavy chain comprises a VH chain region comprising a VH CDRl, VH CDR2, and VH CDR3 of Table 1; (iii) the light chain further comprises a constant light chain domain comprising the amino acid sequence of the constant domain of a human kappa or lambda light chain; and (iv) the heavy chain further comprises a constant heavy chain domain comprising the amino acid sequence of the constant domain of a human IgG, e.g., IgGl, heavy chain.
[00193] In another particular embodiment, an antibody described herein, which specifically binds to a MerTK polypeptide (e.g., human MerTK), comprises a light chain and a heavy chain, wherein (i) the light chain comprises a VL chain region comprising a VL CDRl, VL CDR2, and VL CDR3 of Table 7; (ii) the heavy chain comprises a VH chain region comprising a VH CDRl, VH CDR2, and VH CDR3 of Table 7; (iii) the light chain further comprises a constant light chain domain comprising the amino acid sequence of the constant domain of a human kappa or lambda light chain; and (iv) the heavy chain further comprises a constant heavy chain domain comprising the amino acid sequence of the constant domain of a human IgG, e.g., IgGl, heavy chain.
[00194] In another particular embodiment, an antibody described herein, which specifically binds to a MerTK polypeptide (e.g., human MerTK), comprises a light chain and a heavy chain, wherein (i) the light chain comprises a VL chain region comprising a VL CDRl, VL CDR2, and VL CDR3 of Table 1; (ii) the heavy chain comprises a VH chain region comprising a VH CDRl, VH CDR2, and VH CDR3 of Table 7; (iii) the light chain further comprises a constant light chain domain comprising the amino acid sequence of the constant domain of a human kappa or lambda light chain; and (iv) the heavy chain further comprises a constant heavy chain domain comprising the amino acid sequence of the constant domain of a human IgG, e.g., IgGl, heavy chain.
[00195] In another particular embodiment, an antibody described herein, which specifically binds to a MerTK polypeptide (e.g., human MerTK), comprises a light chain and a heavy chain, wherein (i) the light chain comprises a VL chain region comprising a VL CDRl, VL CDR2, and VL CDR3 of Table 7; (ii) the heavy chain comprises a VH chain region comprising a VH CDRl, VH CDR2, and VH CDR3 of Table 1; (iii) the light chain further comprises a constant light chain domain comprising the amino acid sequence of the constant domain of a human kappa or lambda light chain; and (iv) the heavy chain further comprises a constant heavy chain domain comprising the amino acid sequence of the constant domain of a human IgG, e.g., IgGl, heavy chain.
[00196] In another particular embodiment, an antibody described herein, which specifically binds to a MerTK polypeptide (e.g., human MerTK), comprises a light chain and a heavy chain, wherein (i) the light chain comprises a VL chain region comprising a VL CDRl, VL CDR2, and VL CDR3 of Table 3; (ii) the heavy chain comprises a VH chain region comprising a VH CDRl, VH CDR2, and VH CDR3 of Table 3; (iii) the light chain further comprises a constant light chain domain comprising the amino acid sequence of the constant domain of a human kappa or lambda light chain; and (iv) the heavy chain further comprises a constant heavy chain domain comprising the amino acid sequence of the constant domain of a human IgG, e.g., IgGl, heavy chain.
[00197] In another particular embodiment, an antibody described herein, which specifically binds to a MerTK polypeptide (e.g., human MerTK), comprises a light chain and a heavy chain, wherein (i) the light chain comprises a VL chain region comprising a VL CDRl, VL CDR2, and VL CDR3 of Table 11; (ii) the heavy chain comprises a VH chain region comprising a VH CDRl, VH CDR2, and VH CDR3 of Table 11; (iii) the light chain further comprises a constant light chain domain comprising the amino acid sequence of the constant domain of a human kappa or lambda light chain; and (iv) the heavy chain further comprises a constant heavy chain domain comprising the amino acid sequence of the constant domain of a human IgG, e.g., IgGl, heavy chain.
[00198] In another particular embodiment, an antibody described herein, which specifically binds to a MerTK polypeptide (e.g., human MerTK), comprises a light chain and a heavy chain, wherein (i) the light chain comprises a VL chain region comprising a VL CDRl, VL CDR2, and VL CDR3 of Table 3; (ii) the heavy chain comprises a VH chain region comprising a VH CDRl, VH CDR2, and VH CDR3 of Table 11; (iii) the light chain further comprises a constant light chain domain comprising the amino acid sequence of the constant domain of a human kappa or lambda light chain; and (iv) the heavy chain further comprises a constant heavy chain domain comprising the amino acid sequence of the constant domain of a human IgG, e.g., IgGl, heavy chain.
[00199] In another particular embodiment, an antibody described herein, which specifically binds to a MerTK polypeptide (e.g., human MerTK), comprises a light chain and a heavy chain, wherein (i) the light chain comprises a VL chain region comprising a VL CDRl, VL CDR2, and VL CDR3 of Table 11; (ii) the heavy chain comprises a VH chain region comprising a VH CDRl, VH CDR2, and VH CDR3 of Table 3; (iii) the light chain further comprises a constant light chain domain comprising the amino acid sequence of the constant domain of a human kappa or lambda light chain; and (iv) the heavy chain further comprises a constant heavy chain domain comprising the amino acid sequence of the constant domain of a human IgG, e.g., IgGl, heavy chain.
[00200] In another particular embodiment, an antibody described herein, which specifically binds to a MerTK polypeptide (e.g., human MerTK), comprises a light chain and a heavy chain, wherein (i) the light chain comprises a VL chain region comprising a VL CDRl, VL CDR2, and VL CDR3 of Table 4; (ii) the heavy chain comprises a VH chain region comprising a VH CDRl, VH CDR2, and VH CDR3 of Table 4; (iii) the light chain further comprises a constant light chain domain comprising the amino acid sequence of the constant domain of a human kappa or lambda light chain; and (iv) the heavy chain further comprises a constant heavy chain domain comprising the amino acid sequence of the constant domain of a human IgG, e.g., IgGl, heavy chain.
[00201] In another particular embodiment, an antibody described herein, which specifically binds to a MerTK polypeptide (e.g., human MerTK), comprises a light chain and a heavy chain, wherein (i) the light chain comprises a VL chain region comprising a VL CDRl, VL CDR2, and VL CDR3 of Table 12; (ii) the heavy chain comprises a VH chain region comprising a VH CDRl, VH CDR2, and VH CDR3 of Table 12; (iii) the light chain further comprises a constant light chain domain comprising the amino acid sequence of the constant domain of a human kappa or lambda light chain; and (iv) the heavy chain further comprises a constant heavy chain domain comprising the amino acid sequence of the constant domain of a human IgG, e.g., IgGl, heavy chain.
[00202] In another particular embodiment, an antibody described herein, which specifically binds to a MerTK polypeptide (e.g., human MerTK), comprises a light chain and a heavy chain, wherein (i) the light chain comprises a VL chain region comprising a VL CDRl, VL CDR2, and VL CDR3 of Table 4; (ii) the heavy chain comprises a VH chain region comprising a VH CDRl, VH CDR2, and VH CDR3 of Table 12; (iii) the light chain further comprises a constant light chain domain comprising the amino acid sequence of the constant domain of a human kappa or lambda light chain; and (iv) the heavy chain further comprises a constant heavy chain domain comprising the amino acid sequence of the constant domain of a human IgG, e.g., IgGl, heavy chain.
[00203] In another particular embodiment, an antibody described herein, which specifically binds to a MerTK polypeptide (e.g., human MerTK), comprises a light chain and a heavy chain, wherein (i) the light chain comprises a VL chain region comprising a VL CDRl, VL CDR2, and VL CDR3 of Table 12; (ii) the heavy chain comprises a VH chain region comprising a VH CDRl, VH CDR2, and VH CDR3 of Table 4; (iii) the light chain further comprises a constant light chain domain comprising the amino acid sequence of the constant domain of a human kappa or lambda light chain; and (iv) the heavy chain further comprises a constant heavy chain domain comprising the amino acid sequence of the constant domain of a human IgG, e.g., IgGl, heavy chain.
[00204] In another particular embodiment, an antibody described herein, which specifically binds to a MerTK polypeptide (e.g., human MerTK), comprises a light chain and a heavy chain, wherein (i) the light chain comprises a VL chain region comprising a VL CDRl, VL CDR2, and VL CDR3 of Table 5; (ii) the heavy chain comprises a VH chain region comprising a VH CDRl, VH CDR2, and VH CDR3 of Table 5; (iii) the light chain further comprises a constant light chain domain comprising the amino acid sequence of the constant domain of a human kappa or lambda light chain; and (iv) the heavy chain further comprises a constant heavy chain domain comprising the amino acid sequence of the constant domain of a human IgG, e.g., IgGl, heavy chain. [00205] In another particular embodiment, an antibody described herein, which specifically binds to a MerTK polypeptide (e.g., human MerTK), comprises a light chain and a heavy chain, wherein (i) the light chain comprises a VL chain region comprising a VL CDRl, VL CDR2, and VL CDR3 of Table 13; (ii) the heavy chain comprises a VH chain region comprising a VH CDRl, VH CDR2, and VH CDR3 of Table 13; (iii) the light chain further comprises a constant light chain domain comprising the amino acid sequence of the constant domain of a human kappa or lambda light chain; and (iv) the heavy chain further comprises a constant heavy chain domain comprising the amino acid sequence of the constant domain of a human IgG, e.g., IgGl, heavy chain.
[00206] In another particular embodiment, an antibody described herein, which specifically binds to a MerTK polypeptide (e.g., human MerTK), comprises a light chain and a heavy chain, wherein (i) the light chain comprises a VL chain region comprising a VL CDRl, VL CDR2, and VL CDR3 of Table 5; (ii) the heavy chain comprises a VH chain region comprising a VH CDRl, VH CDR2, and VH CDR3 of Table 13; (iii) the light chain further comprises a constant light chain domain comprising the amino acid sequence of the constant domain of a human kappa or lambda light chain; and (iv) the heavy chain further comprises a constant heavy chain domain comprising the amino acid sequence of the constant domain of a human IgG, e.g., IgGl, heavy chain.
[00207] In another particular embodiment, an antibody described herein, which specifically binds to a MerTK polypeptide (e.g., human MerTK), comprises a light chain and a heavy chain, wherein (i) the light chain comprises a VL chain region comprising a VL CDRl, VL CDR2, and VL CDR3 of Table 13; (ii) the heavy chain comprises a VH chain region comprising a VH CDRl, VH CDR2, and VH CDR3 of Table 5; (iii) the light chain further comprises a constant light chain domain comprising the amino acid sequence of the constant domain of a human kappa or lambda light chain; and (iv) the heavy chain further comprises a constant heavy chain domain comprising the amino acid sequence of the constant domain of a human IgG, e.g., IgGl, heavy chain.
[00208] In another particular embodiment, an antibody described herein, which specifically binds to a MerTK polypeptide (e.g., human MerTK), comprises a light chain and a heavy chain, wherein (i) the light chain comprises a VL chain region comprising a VL CDRl, VL CDR2, and VL CDR3 of Table 6; (ii) the heavy chain comprises a VH chain region comprising a VH CDRl, VH CDR2, and VH CDR3 of Table 6; (iii) the light chain further comprises a constant light chain domain comprising the amino acid sequence of the constant domain of a human kappa or lambda light chain; and (iv) the heavy chain further comprises a constant heavy chain domain comprising the amino acid sequence of the constant domain of a human IgG, e.g., IgGl, heavy chain.
[00209] In another particular embodiment, an antibody described herein, which specifically binds to a MerTK polypeptide (e.g., human MerTK), comprises a light chain and a heavy chain, wherein (i) the light chain comprises a VL chain region comprising a VL CDRl, VL CDR2, and VL CDR3 of Table 14; (ii) the heavy chain comprises a VH chain region comprising a VH CDRl, VH CDR2, and VH CDR3 of Table 14; (iii) the light chain further comprises a constant light chain domain comprising the amino acid sequence of the constant domain of a human kappa or lambda light chain; and (iv) the heavy chain further comprises a constant heavy chain domain comprising the amino acid sequence of the constant domain of a human IgG, e.g., IgGl, heavy chain.
[00210] In another particular embodiment, an antibody described herein, which specifically binds to a MerTK polypeptide (e.g., human MerTK), comprises a light chain and a heavy chain, wherein (i) the light chain comprises a VL chain region comprising a VL CDRl, VL CDR2, and VL CDR3 of Table 6; (ii) the heavy chain comprises a VH chain region comprising a VH CDRl, VH CDR2, and VH CDR3 of Table 14; (iii) the light chain further comprises a constant light chain domain comprising the amino acid sequence of the constant domain of a human kappa or lambda light chain; and (iv) the heavy chain further comprises a constant heavy chain domain comprising the amino acid sequence of the constant domain of a human IgG, e.g., IgGl, heavy chain.
[00211] In another particular embodiment, an antibody described herein, which specifically binds to a MerTK polypeptide (e.g., human MerTK), comprises a light chain and a heavy chain, wherein (i) the light chain comprises a VL chain region comprising a VL CDRl, VL CDR2, and VL CDR3 of Table 14; (ii) the heavy chain comprises a VH chain region comprising a VH CDRl, VH CDR2, and VH CDR3 of Table 6; (iii) the light chain further comprises a constant light chain domain comprising the amino acid sequence of the constant domain of a human kappa or lambda light chain; and (iv) the heavy chain further comprises a constant heavy chain domain comprising the amino acid sequence of the constant domain of a human IgG, e.g., IgGl, heavy chain.
[00212] In another particular embodiment, an antibody described herein, which specifically binds to a MerTK polypeptide (e.g., human MerTK), comprises a light chain and a heavy chain, wherein (i) the light chain comprises a VL chain region comprising the amino acid sequence of SEQ ID NO: 2; (ii) the heavy chain comprises a VH chain region comprising the amino acid sequence of SEQ ID NO: 1; (iii) the light chain further comprises a constant domain comprising the amino acid sequence of the constant domain of a human kappa or lambda light chain; and (iv) the heavy chain further comprises a constant domain comprising the amino acid sequence of the constant domain of a human IgGl, e.g., IgGl, heavy chain.
[00213] In another particular embodiment, an antibody described herein, which specifically binds to a MerTK polypeptide (e.g., human MerTK), comprises a light chain and a heavy chain, wherein (i) the light chain comprises a VL chain region comprising the amino acid sequence of SEQ ID NO: 26; (ii) the heavy chain comprises a VH chain region comprising the amino acid sequence of SEQ ID NO: 25; (iii) the light chain further comprises a constant domain comprising the amino acid sequence of the constant domain of a human kappa or lambda light chain; and (iv) the heavy chain further comprises a constant domain comprising the amino acid sequence of the constant domain of a human IgGl, e.g., IgGl, heavy chain.
[00214] In another particular embodiment, an antibody described herein, which specifically binds to a MerTK polypeptide (e.g., human MerTK), comprises a light chain and a heavy chain, wherein (i) the light chain comprises a VL chain region comprising the amino acid sequence of SEQ ID NO: 2; (ii) the heavy chain comprises a VH chain region comprising the amino acid sequence of SEQ ID NO: 25; (iii) the light chain further comprises a constant domain comprising the amino acid sequence of the constant domain of a human kappa or lambda light chain; and (iv) the heavy chain further comprises a constant domain comprising the amino acid sequence of the constant domain of a human IgGl, e.g., IgGl, heavy chain.
[00215] In another particular embodiment, an antibody described herein, which specifically binds to a MerTK polypeptide (e.g., human MerTK), comprises a light chain and a heavy chain, wherein (i) the light chain comprises a VL chain region comprising the amino acid sequence of SEQ ID NO: 26; (ii) the heavy chain comprises a VH chain region comprising the amino acid sequence of SEQ ID NO: 1; (iii) the light chain further comprises a constant domain comprising the amino acid sequence of the constant domain of a human kappa or lambda light chain; and (iv) the heavy chain further comprises a constant domain comprising the amino acid sequence of the constant domain of a human IgGl, e.g., IgGl, heavy chain.
[00216] In certain embodiments, one or more modifications can be made to the Fc region of an antibody or antigen-binding fragment thereof described herein. Generally, such a
modification or modifications can be introduced to alter one or more functional properities of the antibody or antigen-binding fragment, such as serum half-life, complement fixation, Fc receptor binding, and/or antibody-dependent cellular cytotoxicity. Examplary modfications are described, for example, in International Patent Application Publication No. WO 2008/153926 A2.
[00217] In specific embodiments, an antibody described herein, which specifically binds to MerTK, e.g., human MerTK ECD, comprises framework regions (e.g., framework regions of the VL domain and/or VH domain) that are human framework regions or derived from human framework regions. Non-limiting examples of human framework regions are described in the art, e.g., see Kabat et al. (1991) Sequences of Proteins of Immunological Interest Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242).
[00218] In certain embodiments, an antibody described herein comprises framework regions (e.g., framework regions of the VL domain and/or VH domain) that are primate (e.g., non-human primate) framework regions or derived from primate (e.g., non-human primate) framework regions.
[00219] In certain embodiments, an antibody described herein comprises a VL domain having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 2, wherein the antibody specifically binds to MerTK, e.g., human MerTK ECD. As a non-limiting example, in some embodiments, an antibody described herein comprises the amino acid sequence of SEQ ID NO: 26 wherein the antibody specifically binds to MerTK, e.g., human MerTK ECD.
[00220] In certain embodiments, an antibody described herein comprises a VL domain having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 2, wherein the antibody specifically binds to MerTK, e.g., human MerTK ECD, and wherein the antibody comprises VL CDRs that are identical to the VL CDRs of antibody Ab2000. As a non-limiting example, in certain embodiments, an antibody described herein comprises a VL domain having the amino acid sequence of SEQ ID NO: 26, wherein the antibody specifically binds to MerTK, e.g., human MerTK ECD, and wherein the antibody comprises VL CDRs that are identical to the VL CDRs of antibody Ab2000-A7.
[00221] In certain embodiments, an antibody described herein comprises a VL domain having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 2, wherein the antibody specifically binds to MerTK, e.g., human MerTK ECD, and wherein the antibody comprises VL CDRs that are identical to the VL CDRs of Table 1. As a non-limiting example, in certain embodiments, an antibody described herein comprises a VL domain having the amino acid sequence of SEQ ID NO: 26, wherein the antibody specifically binds to MerTK, e.g., human MerTK ECD, and wherein the antibody comprises VL CDRs that are identical to the VL CDRs of Table 7.
[00222] In certain embodiments, an antibody described herein comprises a VH domain having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 1, wherein the antibody specifically binds to MerTK, e.g., human MerTK ECD. As a non-limiting example, in certain embodiments, an antibody described herein comprises a VH domain having the amino acid sequence of SEQ ID NO: 25, wherein the antibody specifically binds to MerTK, e.g., human MerTK ECD.
[00223] In certain embodiments, an antibody described herein comprises a VH domain having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 1, wherein the antibody specifically binds to MerTK, e.g., human MerTK ECD, and wherein the antibody comprises VH CDRs that are identical to the VH CDRs of antibody Ab2000. As a non-limiting example, in certain embodiments, an antibody described herein comprises a VH domain having the amino acid sequence of SEQ ID NO: 25, wherein the antibody specifically binds to MerTK, e.g., human MerTK ECD, and wherein the antibody comprises VH CDRs that are identical to the VH CDRs of antibody Ab2000-A7.
[00224] In certain embodiments, an antibody described herein comprises a VH domain having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 1, wherein the antibody specifically binds to MerTK, e.g., human MerTK ECD, and wherein the antibody comprises VH CDRs that are identical to the VH CDRs of Table 1. As a non-limiting example, in certain embodiments, an antibody described herein comprises a VH domain having the amino acid sequence of SEQ ID NO: 25, wherein the antibody specifically binds to MerTK, e.g., human MerTK ECD, and wherein the antibody comprises VH CDRs that are identical to the VH CDRs of Table 7.
[00225] In other embodiments, the antibodies and antigen binding fragments thereof presented herein that specifically bind to human MerTK comprise conservative sequence modifications as described herein. With respect to polypeptides such as antibodies or antigen binding fragment thereof, conservative sequence modifications include conservative amino acid substitutions that include ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta- branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, in a particular embodiment, a predicted nonessential amino acid residue in a human anti-MerTK antibody is replaced with another amino acid residue from the same side chain family. Methods of identifying nucleotide and amino acid conservative substitutions which do not eliminate antigen binding are well-known in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10):879-884 (1999); and Burks et al. Proc. Natl. Acad. Sci. USA 94:412-417 (1997)). In particular embodiments, the conservative sequence modifications described herein modify the amino acid sequences of the MerTK antibodies or antigen binding fragments thereof by 50%, or 55%, or 60%, or 65%, or 70%, or 75%, or 80%, or 85%, or 90%, or 95%, or 98%, or 99%. In certain embodiments, the nucleotide and amino acid sequence modifications refer to at most 1, 2, 3, 4, 5, or 6 amino acid substitutions to the CDRs described in any of Table 1, or Table 3-7, or 11-14. Thus, for example, each such CDR may contain up to 5 conservative amino acid substitutions, for example up to (not more than) 4 conservative amino acid substitutions, for example up to (not more than) 3 conservative amino acid substitutions, for example up to (not more than) 2 conservative amino acid substitutions, or no more than 1 conservative amino acid substitution.
[00226] In certain embodiments, an antibody or antigen-binding fragment thereof that specifically binds to MerTK, e.g., human MerTK ECD described herein comprises: i) a VL domain having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 2, ii) a VL domain having at least 80%, at least 85%), at least 90%, at least 95%, or at least 98%> sequence identity to the amino acid sequence of SEQ ID NO: 2, and wherein the antibody comprises VL CDRs that are identical to the VL CDRs of antibody Ab2000, or iii) a VL domain having at least 80%>, at least 85%>, at least 90%), at least 95%, or at least 98%> sequence identity to the amino acid sequence of SEQ ID NO: 2, and wherein the antibody comprises VL CDRs that are identical to the VL CDRs of Table 1; and iv) a VH domain having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%) sequence identity to the amino acid sequence of SEQ ID NO: 1, v) a VH domain having at least 80%), at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 1 and wherein the antibody comprises VH CDRs that are identical to the VH CDRs of antibody Ab2000, or vi) a VH domain having at least 80%, at least 85%), at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 1 and wherein the antibody comprises VH CDRs that are identical to the VH CDRs of Table 1.
[00227] In certain embodiments, an antibody that specifically binds to MerTK, e.g., human MerTK ECD described herein comprises: i) a VL domain having at least 80%, at least 85%, at least 90%), at least 95%, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 2 (e.g., a VL having the amino acid sequence of SEQ ID NO: 26), ii) a VL domain having at least 80%), at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 2, and wherein the antibody comprises VL CDRs that are identical to the VL CDRs of antibody Ab2000-A7, or iii) a VL domain having at least 80%, at least 85%), at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 2, and wherein the antibody comprises VL CDRs that are identical to the VL CDRs of Table 7; and iv) a VH domain having at least 80%, at least 85%, at least 90%, at least 95%), or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 1 (e.g., a VH having the amino acid sequence of SEQ ID NO: 25), v) a VH domain having at least 80%, at least 85%), at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 1 and wherein the antibody comprises VH CDRs that are identical to the VH CDRs of antibody Ab2000-A7, or vi) a VH domain having at least 80%, at least 85%, at least 90%), at least 95%, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 1 and wherein the antibody comprises VH CDRs that are identical to the VH CDRs of Table 7.
[00228] The determination of percent identity between two sequences (e.g., amino acid sequences or nucleic acid sequences) can also be accomplished using a mathematical algorithm. A preferred, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. U.S.A.
87:2264 2268, modified as in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. U.S.A. 90:5873 5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403. BLAST nucleotide searches can be performed with the NBLAST nucleotide program parameters set, e.g., for score=100, word length=12 to obtain nucleotide sequences homologous to a nucleic acid molecules described herein. BLAST protein searches can be performed with the XBLAST program parameters set, e.g., to score 50, wordlength=3 to obtain amino acid sequences homologous to a protein molecule described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389 3402. Alternatively, PSI BLAST can be used to perform an iterated search which detects distant relationships between molecules (Id.). When utilizing BLAST, Gapped BLAST, and PSI Blast programs, the default parameters of the respective programs (e.g., of XBLAST and NBLAST) can be used (see, e.g., National Center for Biotechnology Information (NCBI) on the worldwide web,
ncbi.nlm.nih.gov). Another preferred, non limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, 1988, CABIOS 4: 11 17. Such an algorithm is incorporated in the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.
[00229] The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted.
[00230] Provided herein are antibodies that specifically bind to MerTK and that can modulate MerTK activity and/or expression (e.g., inhibit MerTK activity and/or expression). In specific aspects, provided herein are antibodies which are MerTK antagonists. In certain embodiments, a MerTK antagonist provided herein is an antibody described herein that specifically binds to a MerTK polypeptide, e.g., an ECD of human MerTK, and that inhibits (e.g., partially inhibits) a MerTK activity.
[00231] MerTK activity can relate to any activity of MerTK such as those known or described in the art. Non-limiting examples of MerTK activity include: MerTK receptor dimerization, MerTK receptor phosphorylation (e.g., tyrosine phosphorylation or autophosphorylation in the cytoplasmic domain), signaling downstream of the MerTK receptor (e.g., P13K, PLC, GRB2, RAC1, SOCS-1, AKT, STAT1, or MAPK/ERK signaling), MerTK ligand (e.g., Gas6 and PROS1) induced enhancement of cell proliferation, or cell survival (e.g., natural killer (NK) cells), modulation of NK cells, dendritic cells or macrophages, phagocytosis, inhibition of proinflammatory cytokine production (e.g., inhibition of TLR-induced production of
proinflammatory cytokines (e.g., TNF, IL-6, IL-12 and type I interferons)).
[00232] In specific embodiments, antibodies described herein specifically bind to human MerTK and block or inhibit (e.g., partially inhibit) binding of MerTK ligand (e.g., Gas6 and PROS1) to MerTK by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% as assessed by methods described herein or known to one of skill in the art, e.g., ELISA assay, flow cytometry, or competition assay.
[00233] In certain aspects, inhibition by anti-MerTK antibodies described herein (e.g., monoclonal antibody) of MerTK ligand (e.g., Gas6 and PROS1) binding to MerTK can be characterized by ICso values, which reflects the concentration of anti-MerTK antibodies achieving 50% inhibition of binding of MerTK ligand to MerTK. Thus, in specific
embodiments, an anti-MerTK antibody described herein (e.g., antibody Ab2000 or an antigen- binding fragment thereof, antibody Ab2000-A7 or an antigen-binding fragment thereof, or an antibody comprising CDRs of any of Table 1, Table 3-7, or Table 11-14) inhibits binding of MerTK ligand to MerTK with an ICso of at most about 10,000 nM, 1,000 nM, 900 nM, 800 nM, 700 nM, 600 nM, 500 nM, 400 nM, 300 nM, 200 nM, 100 nM, 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 5 nM, 1 nM, 0.75 nM, 0.5 nM, 0.1 nM, 0.05 nM, 0.01 nM, 0.005 nM, or 0.001 nM, as assessed by methods described herein and/or known to one of skill in the art, (e.g., ELISA assay or flow cytometry). In particular embodiments, an anti- MerTK antibody described herein inhibits binding of MerTK ligand to MerTK with an ICso in the range of about 0.01 nM to 10,000 nM, 0.01 nM to 1,000 nM, 0.05 nM to 500 nM, 0.05 nM to 100 nM, or 0.05 nM to 50 nM, 0.1 nM to 50 nM as assessed by methods described herein and/or known to one of skill in the art, (e.g., ELISA assay or flow cytometry).
[00234] In certain embodiments, an anti-MerTK antibody described herein can inhibit (e.g., partially inhibit) MerTK activity by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% as assessed by methods described herein and/or known to one of skill in the art, relative to MerTK activity in the presence of MerTK ligand stimulation without any antibody or with an unrelated antibody (e.g., an antibody that does not specifically bind to MerTK). In certain embodiments, an anti- MerTK antibody described herein can inhibit (e.g., partially inhibit) MerTK activity by at least about 25%) to about 65% as assessed by methods described herein and/or known to one of skill in the art, relative to MerTK activity in the presence of MerTK ligand stimulation without any antibody or with an unrelated antibody (e.g., an antibody that does not specifically bind to MerTK). Non-limiting examples of MerTK activity can include MerTK receptor
phosphorylation, MerTK receptor signaling, MerTK ligand (e.g., Gas6 and PROS1) mediated cell proliferation, and MerTK ligand (e.g., Gas6 and PROS1) mediated cell survival (e.g., natural killer (NK) cells), modulation of maturation of NK cells, dendritic cells or macrophages, phagocytosis, inhibition of proinflammatory cytokine production (e.g., inhibition of TLR- induced production of proinflammatory cytokines (e.g., TNF, IL-6, IL-12 and type I interferons).
[00235] As an inhibitor of MerTK activity (e.g., MerTK antagonist), an antibody described herein (e.g., antibody Ab2000 or an antigen-binding fragment thereof, Ab2000-A7 or an antigen- binding fragment thereof, or an antibody comprising CDRs of any of Table 1, Table 3-7, or Table 11-14) can block (e.g., partially block) or inhibit (e.g., partially inhibit) (e.g., human MerTK), specifically tyrosine phosphorylation of one or more residues in the cytoplasmic domain of MerTK.
[00236] Thus, in specific embodiments, antibodies described herein (e.g., antibody Ab2000, or an antigen-binding fragment thereof, Ab2000-A7 or an antigen-binding fragment thereof, or an antibody comprising CDRs of any of Table 1, Table 3-7, or Table 11-14) specifically bind to human MerTK and block, inhibit, or reduce tyrosine (e.g., human MerTK) by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%), 95%), 98%), 99%), or 100% as assessed by methods described herein or known to one of skill in the art, e.g., as described in section 6, relative to phosphorylation in the presence of MerTK ligand stimulation without any antibody or with an unrelated antibody (e.g., an antibody that does not specifically bind to MerTK). In particular embodiments, antibodies described herein specifically bind to MerTK and block or inhibit tyrosine phosphorylation in the cytoplasmic domain of MerTK by at least about 25%, optionally to about 65%, 75%, 80% or 85%), as assessed by methods described herein or known to one of skill in the art, e.g., as described in Section 6, below. In certain embodiments, antibodies described herein specifically bind to MerTK and block or inhibit tyrosine phosphorylation of MerTK by at least about 25% to about 80%) as assessed by methods described herein or known to one of skill in the art, e.g., as described in Section 6, below. In certain embodiments, antibodies described herein (e.g., antibody Ab2000 or an antigen-binding fragment thereof, Ab2000-A7, or antigen-binding fragment thereof, or an antibody comprising CDRs of any of Table 1, Table 3-7, or Table 11-14) specifically bind to MerTK and block or inhibit tyrosine (e.g., human MerTK) by at least about 50%) to about 100%) as assessed by methods described herein or known to one of skill in the art, e.g., as described in Section 6, below.
[00237] In specific embodiments, antibodies described herein specifically bind to MerTK and block or inhibit tyrosine phosphorylation of MerTK with an ICso of less than about 10 nM, about 1 nM, about 500 pM, about 400 pM, about 200 pM, or about 100 pM, as assessed by methods described herein in Section 6 below or known to one of skill in the art. In specific embodiments, antibodies described herein specifically bind to MerTK and block or inhibit tyrosine
phosphorylation of MerTK with an ICso in the range of about 10 nM to about 100 pM, about 1 nM to 100 pM, or about 500 pM to about 100 pM. For example, an ICso for inhibition of tyrosine phosphorylation can be determined by assaying lysates from cells, recombinantly expressing MerTK, in ELISA which detects tyrosine phosphorylation, for example, as described in Section 6 below. In certain embodiments, cells, recombinantly expressing MerTK, are sorted, e.g., sorted to select for cells highly expressing MerTK, prior to use in the phosphorylation inhibition assays. In some embodiments, the cells are not sorted prior to use in the
phosphorylation inhibition assays.
[00238] In specific embodiments, antibodies described herein (e.g., antibody Ab2000 or an antigen-binding fragment thereof, antibody Ab2000-A7 or an antigen-binding fragment thereof or an antibody comprising CDRs of any of Table 1, Table 3-7, or Table 11-14) specifically bind to MerTK and block or inhibit phosphorylation of one or more tyrosine residues in the cytoplasmic domain of MerTK by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% as assessed by methods described herein or known to one of skill in the art, e.g., immunoblotting assay, relative to phosphorylation in the presence of MerTK ligand stimulation without any antibody or with an unrelated antibody (e.g., an antibody that does not specifically bind to MerTK). In specific embodiments, blocking or inhibition (e.g., partial inhibition) of phosphorylation of MerTK by antibodies described herein can be assessed upon MerTK ligand stimulation.
[00239] Signaling events downstream of MerTK receptor phosphorylation can serve as indicators of MerTK activity. For example, MerTK ligand (e.g., Gas6 and PROS1) binding to its receptor MerTK stimulates several distinct signaling pathways, including for example members of phosphatidylinositol (PI) 3 -kinases, and mitogen-activated protein kinase (MAPK) (see Korshunov, Clin. Sci. (Lond.), 2012, 122(8):361-368).
[00240] Thus, in certain aspects, anti-MerTK antibodies described herein which act as inhibitors of MerTK activity (e.g., antibody Ab2000 or an antigen-binding fragment thereof, antibody Ab2000-A7 or an antigen-binding fragment thereof or an antibody comprising CDRs of any of Table 1, Table 3-7, or Table 11-14) can inhibit signaling of a member of the PI 3-kinases or MAPK. In particular embodiments, anti-MerTK antibodies described herein which act as inhibitors of MerTK activity can inhibit binding (or inhibit interaction), to the cytoplasmic domain of MerTK, of one or more of PI3K, PLC, and Grb2. In certain embodiments, anti- MerTK antibodies described herein which act as inhibitors of MerTK activity can inhibit activation by MerTK of one or more of PI3K, PLC, and Grb2.
[00241] In particular embodiments, anti-MerTK antibodies described herein which act as inhibitors of MerTK activity (e.g., antibody Ab2000 or an antigen-binding fragment thereof, antibody Ab2000-A7 or an antigen-binding fragment thereof or an antibody comprising CDRs of any of Table 1, Table 3-7, or Table 11-14) can inhibit downstream signaling such as
phosphorylation of MAPK, phosphorylation of P13K, or phosphorylation of AKT. Thus, in certain embodiments, an anti-MerTK antibody described herein can inhibit or reduce
phosphorylation of MAPK (e.g., MerTK ligand (e.g., Gas6 and PROS1) induced
phosphorylation of MAPK) by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% as assessed by methods described herein or known to one of skill in the art, e.g., Western blot or ELISA assay as described in section 6 or immunoblotting assay, relative to phosphorylation in the presence of MerTK ligand stimulation without any antibody or with an unrelated antibody (e.g., an antibody that does not specifically bind to MerTK). In certain embodiments, an anti-MerTK antibody described herein can inhibit or reduce phosphorylation of AKT (e.g., MerTK ligand (e.g., Gas6 and PROS1) induced phosphorylation of AKT) by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% as assessed by methods described herein or known to one of skill in the art, e.g., as described in section 6, relative to phosphorylation in the presence of MerTK ligand stimulation without any antibody or with an unrelated antibody (e.g., an antibody that does not specifically bind to MerTK).
[00242] Cells and cell lines which are appropriate for use in the assays described herein relating to MerTK activity are readily available (e.g., ATCC) or can be readily identified using methods known in the art. For example, cells and/or cell lines that express MerTK endogenously or that possess MerTK signaling or activity are known to one of skill in the art. In certain embodiments, cells or cell lines that are appropriate for use in the assays described herein can express MerTK, either endogenously or recombinantly.
[00243] Non-limiting examples of cells that can be used in the methods and assays described herein include primary cells, transformed cells, stem cells, mast cells, primordial germ cells, oocytes, spermatocytes, embryonic stem cells, hematopoietic cells, erythroleukemia cells (e.g., F36P and TF-1 cell lines), human monocytic cell lines, e.g., acute monocytic leukemia cell lines, human myeloid leukemia cell lines, such as M07E cells; gastrointestinal stromal tumor cell lines such as ST-882, GIST430, and GIST882; neuroblastoma cell lines such as SK-N-SH, SK-SY5Y, H-EP1, SK-N-BE(2), SK-N-BE(ZkM17), SK-N-BE(2)C, LA-N-1, or LA-N-l-5s; small cell lung carcinoma cell lines such as H526, ECC12, TMK1, MKN7, GCIY, and HGC27; and melanoma cell lines such as SKMEL3, SKMEL5, G361, MALME-3M, HMCB. In particular embodiments, cells that can be used in the methods and assays described herein include immune cells, such as macrophages, dendritic cells, and natural killer (NK) cells. In particular embodiments, cells that can be used in the methods and assay described herein include THP1 cells.
[00244] Alternatively, cells and cell lines that express MerTK, e.g., human MerTK, can routinely be generated recombinantly. Non-limiting examples of cells that can be engineered to express MerTK recombinantly include COS cells, HEK 293 cells, CHO cells, H1299 cells, fibroblasts (e.g., human fibroblasts) such as NIH3T3 cells, and MEFS. In a specific
embodiment, cells for use in the methods described herein are HEK 293 cells expressing human MerTK ECD.
5.2 Antibody Production
[00245] Antibodies described herein (or an antigen-binding fragment thereof) that specifically bind to MerTK (e.g., ECD of human MerTK) can be produced by any method known in the art for the synthesis of antibodies, for example, by chemical synthesis or by recombinant expression techniques. The methods described herein employs, unless otherwise indicated, conventional techniques in molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and related fields within the skill of the art. These techniques are described, for example, in the references cited herein and are fully explained in the literature. See, e.g.,, Maniatis et al. (1982) Molecular Cloning: A Laboratory Manual Cold Spring Harbor Laboratory Press; Sambrook et al. (1989), Molecular Cloning: A Laboratory Manual Second Edition, Cold Spring Harbor Laboratory Press; Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel et al, Current Protocols in Molecular Biology, John Wiley & Sons (1987 and annual updates); Current Protocols in Immunology, John Wiley & Sons (1987 and annual updates) Gait (ed.) (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein (ed.) (1991)
Oligonucleotides and Analogues: A Practical Approach, IRL Press; Birren et al. (eds.) (1999) Genome Analysis: A Laboratory Manual, Cold Spring Harbor Laboratory Press.
[00246] In a specific embodiment, an antibody described herein is an antibody (e.g., recombinant antibody) prepared, expressed, created or isolated by any means that involves creation, e.g., via synthesis, genetic engineering of DNA sequences. In certain embodiments, such antibody comprise sequences (e.g., DNA sequences or amino acid sequences) that do not naturally exist within the antibody germline repertoire of an animal or mammal (e.g., human) in vivo.
[00247] In a certain aspect, provided herein is a method of making an antibody or an antigen- binding fragment thereof, which specifically binds to human MerTK comprising culturing a cell or host cell described herein. In a certain aspect, provided herein is a method of making an antibody or an antigen-binding fragment thereof which specifically binds to human MerTK comprising expressing (e.g., recombinantly expressing) the antibody or antigen-binding fragment thereof using a cell or host cell described herein (e.g., a cell or a host cell comprising
polynucleotides encoding an antibody described herein). In a particular embodiment, the cell is an isolated cell. In a particular embodiment, the exogenous polynucleotides have been introduced into the cell. In a particular embodiment, the method further comprises the step of purifying the antibody or antigen-binding fragment thereof obtained from the cell or host cell.
[00248] Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et al.,
Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2nd ed. 1988);
Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563 681 (Elsevier, N.Y., 1981). The term "monoclonal antibody" as used herein is not limited to antibodies produced through hybridoma technology. For example, monoclonal antibodies can be produced recombinantly from host cells exogenously expressing an antibody described herein (e.g., anti- MerTK antibody comprising the CDRs of any of Table 1, Table 3-7, or Table 11-14) or a fragment thereof, for example, light chain and/or heavy chain of such antibody.
[00249] Methods for producing and screening for specific antibodies using hybridoma technology are routine and well known in the art. For example, in the hybridoma method, a mouse or other appropriate host animal, such as a sheep, goat, rabbit, rat, hamster or macaque monkey, is immunized to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein (e.g., ECD of human MerTK) used for immunization. Alternatively, lymphocytes may be immunized in vitro. Lymphocytes then are fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). Additionally, a RFMMS (repetitive immunization multiple sites) technique can be used to immunize an animal (Kilptrack et al., 1997 Hybridoma 16:381-9, incorporated by reference in its entirety). [00250] In some embodiments, mice (or other animals, such as rats, monkeys, donkeys, pigs, sheep, hamster, or dogs) can be immunized with an antigen (e.g., MerTK, for example human MerTK) and once an immune response is detected, e.g., antibodies specific for the antigen are detected in the mouse serum, the mouse spleen is harvested and splenocytes isolated. The splenocytes are then fused by well known techniques to any suitable myeloma cells, for example cells from cell line SP20 available from the American Type Culture Collection (ATCC®) (Manassas, VA), to form hybridomas. Hybridomas are selected and cloned by limited dilution. In certain embodiments, lymph nodes of the immunized mice are harvested and fused with NS0 myeloma cells.
[00251] The hybridoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells. For example, if the parental myeloma cells lack the enzyme
hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.
[00252] Specific embodiments employ myeloma cells that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Among these myeloma cell lines are murine myeloma lines, such as NS0 cell line or those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, CA, USA, and SP-2 or X63-Ag8.653 cells available from the American Type Culture Collection, Rockville, MD, USA. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133 :3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).
[00253] Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against human MerTK antigen (e.g., ECD of human MerTK). The binding specificity of monoclonal antibodies produced by hybridoma cells is determined by methods known in the art, for example, immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). [00254] After hybridoma cells are identified that produce antibodies of the desired specificity, affinity, and/or activity, the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, D-MEM or RPMI 1640 medium. In addition, the hybridoma cells may be grown in vivo as ascites tumors in an animal.
[00255] The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
[00256] In some embodiments, antibody variants having an improved property such as affinity, stability, and/or expression level as compared to a parent antibody may be prepared by affinity maturation. Affinity maturation techniques that are known in the art and/or described herein can be used to alter the CDR region(s), followed by screening of the resultant binding molecules for the desired change in binding. Without being bound by theory, libraries of antibodies can be displayed on the surface of an organism (e.g., phage, bacteria, yeast, or mammalian cell) or in association with their encoding mRNA or DNA (e.g., covalently or non- covalently). Affinity selection of the displayed antibodies allows isolation of organisms or complexes carrying the genetic information encoding the antibodies. Two or three rounds of mutation and selection using display methods such as phage display usually results in antibody fragments with improved affinities (e.g., in the low nanomolar or picomolar range). Further methods that can be used to generate antibody libraries and/or antibody affinity maturation are disclosed in Holland et al., 2013, J. of Immunol. Methods 394:55-61, incorporated herein by reference in its entirety. In some specific embodiments, antibody affinity maturation can be performed using the methods exemplified in Example 7 below.
[00257] Antibodies described herein include antibody fragments, which recognize specific MerTK antigens (e.g., ECD of MerTK) and can be generated by any technique known to those of skill in the art. For example, Fab and F(ab')2 fragments described herein can be produced by proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments). A Fab fragment corresponds to one of the two identical arms of an antibody molecule and contains the complete light chain paired with the VH and CHI domains of the heavy chain. A F(ab')2 fragment contains the two antigen- binding arms of an antibody molecule linked by disulfide bonds in the hinge region.
[00258] Further, the antibodies described herein or antigen-binding fragments thereof can also be generated using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them. In particular, DNA sequences encoding VH and VL domains are amplified from animal cDNA libraries (e.g., human or murine cDNA libraries of affected tissues). The DNA encoding the VH and VL domains are recombined together with an scFv linker by PCR and cloned into a phagemid vector. The vector is electroporated in E. coli and the E. coli is infected with helper phage. Phage used in these methods are typically filamentous phage including fd and Ml 3, and the VH and VL domains are usually recombinantly fused to either the phage gene III or gene VIII. Phage expressing an antigen binding domain that binds to a particular antigen can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead. Examples of phage display methods that can be used to make the antibodies described herein include those disclosed in Brinkman et al., 1995, J. Immunol. Methods 182:41-50; Ames et al., 1995, J. Immunol. Methods 184: 177-186; Kettleborough et al., 1994, Eur. J. Immunol. 24:952-958; Persic et al., 1997, Gene 187:9-18; Burton et al., 1994, Advances in Immunology 57: 191-280; PCT Application No. PCT/GB91/01 134; International Publication Nos. WO 90/02809, WO 91/10737, WO 92/01047, WO 92/18619, WO 93/1 1236, WO 95/15982, WO 95/20401, and W097/13844; and U.S. Patent Nos.
5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727, 5,733,743 and 5,969,108.
[00259] As described in the above references, after phage selection, the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g., as described below. Techniques to recombinantly produce antibody fragments such as Fab, Fab' and F(ab')2 fragments can also be employed using methods known in the art such as those disclosed in PCT publication No. WO 92/22324; Mullinax et al., 1992, BioTechniques 12(6):864-869; Sawai et al., 1995, AJRI 34:26-34; and Better et al., 1988, Science 240: 1041-1043. [00260] In one aspect, to generate whole antibodies, PCR primers including VH or VL nucleotide sequences, a restriction site, and a flanking sequence to protect the restriction site can be used to amplify the VH or VL sequences from a template, e.g., scFv clones. Utilizing cloning techniques known to those of skill in the art, the PCR amplified VH domains can be cloned into vectors expressing a VH constant region, and the PCR amplified VL domains can be cloned into vectors expressing a VL constant region, e.g., human kappa or lambda constant regions. The VH and VL domains can also be cloned into one vector expressing the necessary constant regions. The heavy chain conversion vectors and light chain conversion vectors are then co-transfected into cell lines to generate stable or transient cell lines that express full-length antibodies, e.g., IgG, using techniques known to those of skill in the art.
[00261] A chimeric antibody is a molecule in which different portions of the antibody are derived from different immunoglobulin molecules. For example, a chimeric antibody can contain a variable region of a mouse or rat monoclonal antibody fused to a constant region of a human antibody. Methods for producing chimeric antibodies are known in the art. See, e.g., Morrison, 1985, Science 229: 1202; Oi et al., 1986, BioTechniques 4:214; Gillies et al., 1989, J. Immunol. Methods 125: 191-202; and U.S. Patent Nos. 5,807,715, 4,816,567, 4,816,397, and 6,331,415.
[00262] A humanized antibody is capable of binding to a predetermined antigen and which comprises a framework region having substantially the amino acid sequence of a human immunoglobulin and CDRs having substantially the amino acid sequence of a non-human immunoglobulin (e.g., a murine immunoglobulin). In particular embodiments, a humanized antibody also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. The antibody also can include the CHI, hinge, CH2, CH3, and CH4 regions of the heavy chain. A humanized antibody can be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype, including IgGi, IgG2, IgG3 and IgG4. Humanized antibodies can be produced using a variety of techniques known in the art, including but not limited to, CDR-grafting (European Patent No. EP 239,400;
International publication No. WO 91/09967; and U.S. Patent Nos. 5,225,539, 5,530, 101, and 5,585,089), veneering or resurfacing (European Patent Nos. EP 592, 106 and EP 519,596; Padlan, 1991, Molecular Immunology 28(4/5):489-498; Studnicka et al., 1994, Protein Engineering 7(6):805-814; and Roguska et al., 1994, PNAS 91 :969-973), chain shuffling (U.S. Patent No. 5,565,332), and techniques disclosed in, e.g., U.S. Pat. No. 6,407,213, U.S. Pat. No. 5,766,886, WO 9317105, Tan et al., J. Immunol. 169: 1119 25 (2002), Caldas et al., Protein Eng. 13(5):353- 60 (2000), Morea et al., Methods 20(3):267 79 (2000), Baca et al., J. Biol. Chem.
272(16): 10678-84 (1997), Roguska et al., Protein Eng. 9(10):895 904 (1996), Couto et al., Cancer Res. 55 (23 Supp):5973s- 5977s (1995), Couto et al., Cancer Res. 55(8): 1717-22 (1995), Sandhu JS, Gene 150(2):409-10 (1994), and Pedersen et al., J. Mol. Biol. 235(3):959-73 (1994). See also U.S. Patent Pub. No. US 2005/0042664 Al (Feb. 24, 2005), which is incorporated by reference herein in its entirety.
[00263] Methods for making multispecific (e.g., bispecific antibodies) have been described, see, for example, U.S. Patent Nos. 7951917, 7183076, 8227577, 5837242, 5989830, 5869620, 6132992, and 8586713.
[00264] Single domain antibodies, for example, antibodies lacking the light chains, can be produced by methods well-known in the art. See Riechmann et al., 1999, J. Immunol. 231 :25- 38; Nuttall et al., 2000, Curr. Pharm. Biotechnol. l(3):253-263; Muylderman, 2001, J.
Biotechnol. 74(4):277302; U.S. Patent No. 6,005,079; and International Publication Nos. WO 94/04678, WO 94/25591, and WO 01/44301.
[00265] Further, antibodies that specifically bind to a MerTK antigen can, in turn, be utilized to generate anti-idiotype antibodies that "mimic" an antigen using techniques well known to those skilled in the art. (See, e.g., Greenspan & Bona, 1989, FASEB J. 7(5):437-444; and Nissinoff, 1991, J. Immunol. 147(8):2429-2438).
[00266] Human antibodies which specifically bind to an ECD of human MerTK can be made by a variety of methods known in the art including phage display methods described above using antibody libraries derived from human immunoglobulin sequences. See also U.S. Patent Nos. 4,444,887 and 4,716,111; and International Publication Nos. WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741.
[00267] In some embodiments, human antibodies can be produced using mouse-human hybridomas. For example, human peripheral blood lymphocytes transformed with Epstein-Barr virus (EBV) can be fused with mouse myeloma cells to produce mouse-human hybridomas secreting human monoclonal antibodies, and these mouse-human hybridomas can be screened to determine ones which secrete human monoclonal antibodies that specifically bind to a target antigen (e.g., ECD of human MerTK). Such methods are known and are described in the art, see, e.g., Shinmoto et al., Cytotechnology, 2004, 46: 19-23; Naganawa et al., Human Antibodies, 2005, 14:27-31.
5.2.1 Polynucleotides
[00268] In certain aspects, provided herein are polynucleotides comprising a nucleotide sequence encoding an antibody described herein or a fragment thereof (e.g., a variable light chain region and/or variable heavy chain region) that specifically binds to a MerTK antigen, and vectors, e.g., vectors comprising such polynucleotides for recombinant expression in host cells (e.g., E. coli and mammalian cells). Provided herein are polynucleotides comprising nucleotide sequences encoding any of the antibodies provided herein, as well as vectors comprising such polynucleotide sequences, e.g., expression vectors for their efficient expression in host cells, e.g., mammalian cells.
[00269] As used herein, an "isolated" polynucleotide or nucleic acid molecule is one which is separated from other nucleic acid molecules which are present in the natural source (e.g., in a mouse or a human) of the nucleic acid molecule. Moreover, an "isolated" nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. For example, the language "substantially free" includes preparations of polynucleotide or nucleic acid molecule having less than about 15%, 10%, 5%, 2%), 1%), 0.5%), or 0.1%) (in particular less than about 10%>) of other material, e.g., cellular material, culture medium, other nucleic acid molecules, chemical precursors and/or other chemicals. In a specific embodiment, a nucleic acid molecule(s) encoding an antibody described herein is isolated or purified.
[00270] In particular aspects, provided herein are polynucleotides comprising nucleotide sequences encoding antibodies or antigen-binding fragments thereof, which specifically bind to a MerTK polypeptide (e.g., human MerTK, for example, human MerTK) and comprises an amino acid sequence as described herein, as well as antibodies which compete with such antibodies for binding to a MerTK polypeptide (e.g., in a dose-dependent manner), or which binds to the same epitope as that of such antibodies.
[00271] In certain aspects, provided herein are polynucleotides comprising a nucleotide sequence encoding the light chain or heavy chain of an antibody described herein. The polynucleotides can comprise nucleotide sequences encoding a light chain comprising the VL of antibodies described herein (see, e.g., Table 2 and Table 8). The polynucleotides can comprise nucleotide sequences encoding a heavy chain comprising the VH of antibodies described herein (see, e.g., Table 2 and Table 8). In specific embodiments, a polynucleotide described herein encodes a VL chain region comprising the amino acid sequence of any one of SEQ ID NO: 2 and SEQ ID NO: 26. In specific embodiments, a polynucleotide described herein encodes a VH chain region comprising the amino acid sequence of any one of SEQ ID NO: 1 and SEQ ID NO: 25.
[00272] In particular embodiments, provided herein are polynucleotides comprising a nucleotide sequence encoding an anti-MerTK antibody comprising three VL chain CDRs, e.g., containing VL CDR1, VL CDR2, and VL CDR3 of Table 1. In specific embodiments, provided herein are polynucleotides comprising three VH chain CDRs, e.g., containing VH CDR1, VH CDR2, and VH CDR3 of Table 1. In specific embodiments, provided herein are polynucleotides comprising a nucleotide sequence encoding an anti-MerTK antibody comprising three VL chain CDRs, e.g., containing VL CDR1, VL CDR2, and VL CDR3 of Table 1 and three VH chain CDRs, e.g., containing VH CDR1, VH CDR2, and VH CDR3 of Table 1.
[00273] In particular embodiments, provided herein are polynucleotides comprising a nucleotide sequence encoding an anti-MerTK antibody comprising three VL chain CDRs, e.g., containing VL CDR1, VL CDR2, and VL CDR3 of Table 7. In specific embodiments, provided herein are polynucleotides comprising three VH chain CDRs, e.g., containing VH CDR1, VH CDR2, and VH CDR3 of Table 7. In specific embodiments, provided herein are polynucleotides comprising a nucleotide sequence encoding an anti-MerTK antibody comprising three VL chain CDRs, e.g., containing VL CDR1, VL CDR2, and VL CDR3 of Table 7 and three VH chain CDRs, e.g., containing VH CDR1, VH CDR2, and VH CDR3 of Table 7. In specific embodiments, provided herein are polynucleotides comprising a nucleotide sequence encoding an anti-MerTK antibody comprising three VL chain CDRs, e.g., containing VL CDR1, VL CDR2, and VL CDR3 of Table 1 and three VH chain CDRs, e.g., containing VH CDR1, VH CDR2, and VH CDR3 of Table 7. In specific embodiments, provided herein are polynucleotides comprising a nucleotide sequence encoding an anti-MerTK antibody comprising three VL chain CDRs, e.g., containing VL CDR1, VL CDR2, and VL CDR3 of Table 7 and three VH chain CDRs, e.g., containing VH CDR1, VH CDR2, and VH CDR3 of Table 1. [00274] In particular embodiments, provided herein are polynucleotides comprising a nucleotide sequence encoding an anti-MerTK antibody comprising three VL chain CDRs, e.g., containing VL CDRl, VL CDR2, and VL CDR3 of Table 3. In specific embodiments, provided herein are polynucleotides comprising three VH chain CDRs, e.g., containing VH CDRl, VH CDR2, and VH CDR3 of Table 3. In specific embodiments, provided herein are polynucleotides comprising a nucleotide sequence encoding an anti-MerTK antibody comprising three VL chain CDRs, e.g., containing VL CDRl, VL CDR2, and VL CDR3 of Table 3 and three VH chain CDRs, e.g., containing VH CDRl, VH CDR2, and VH CDR3 of Table 3.
[00275] In particular embodiments, provided herein are polynucleotides comprising a nucleotide sequence encoding an anti-MerTK antibody comprising three VL chain CDRs, e.g., containing VL CDRl, VL CDR2, and VL CDR3 of Table 11. In specific embodiments, provided herein are polynucleotides comprising three VH chain CDRs, e.g., containing VH CDRl, VH CDR2, and VH CDR3 of Table 11. In specific embodiments, provided herein are polynucleotides comprising a nucleotide sequence encoding an anti-MerTK antibody comprising three VL chain CDRs, e.g., containing VL CDRl, VL CDR2, and VL CDR3 of Table 8 and three VH chain CDRs, e.g., containing VH CDRl, VH CDR2, and VH CDR3 of Table 8. In specific embodiments, provided herein are polynucleotides comprising a nucleotide sequence encoding an anti-MerTK antibody comprising three VL chain CDRs, e.g., containing VL CDRl, VL CDR2, and VL CDR3 of Table 3 and three VH chain CDRs, e.g., containing VH CDRl, VH CDR2, and VH CDR3 of Table 11. In specific embodiments, provided herein are
polynucleotides comprising a nucleotide sequence encoding an anti-MerTK antibody comprising three VL chain CDRs, e.g., containing VL CDRl, VL CDR2, and VL CDR3 of Table 11 and three VH chain CDRs, e.g., containing VH CDRl, VH CDR2, and VH CDR3 of Table 3.
[00276] In particular embodiments, provided herein are polynucleotides comprising a nucleotide sequence encoding an anti-MerTK antibody comprising three VL chain CDRs, e.g., containing VL CDRl, VL CDR2, and VL CDR3 of Table 4. In specific embodiments, provided herein are polynucleotides comprising three VH chain CDRs, e.g., containing VH CDRl, VH CDR2, and VH CDR3 of Table 4. In specific embodiments, provided herein are polynucleotides comprising a nucleotide sequence encoding an anti-MerTK antibody comprising three VL chain CDRs, e.g., containing VL CDRl, VL CDR2, and VL CDR3 of Table 4 and three VH chain CDRs, e.g., containing VH CDRl, VH CDR2, and VH CDR3 of Table 4. [00277] In particular embodiments, provided herein are polynucleotides comprising a nucleotide sequence encoding an anti-MerTK antibody comprising three VL chain CDRs, e.g., containing VL CDRl, VL CDR2, and VL CDR3 of Table 12. In specific embodiments, provided herein are polynucleotides comprising three VH chain CDRs, e.g., containing VH CDRl, VH CDR2, and VH CDR3 of Table 12. In specific embodiments, provided herein are polynucleotides comprising a nucleotide sequence encoding an anti-MerTK antibody comprising three VL chain CDRs, e.g., containing VL CDRl, VL CDR2, and VL CDR3 of Table 12 and three VH chain CDRs, e.g., containing VH CDRl, VH CDR2, and VH CDR3 of Table 12. In specific embodiments, provided herein are polynucleotides comprising a nucleotide sequence encoding an anti-MerTK antibody comprising three VL chain CDRs, e.g., containing VL CDRl, VL CDR2, and VL CDR3 of Table 4 and three VH chain CDRs, e.g., containing VH CDRl, VH CDR2, and VH CDR3 of Table 12. In specific embodiments, provided herein are
polynucleotides comprising a nucleotide sequence encoding an anti-MerTK antibody comprising three VL chain CDRs, e.g., containing VL CDRl, VL CDR2, and VL CDR3 of Table 12 and three VH chain CDRs, e.g., containing VH CDRl, VH CDR2, and VH CDR3 of Table 4.
[00278] In particular embodiments, provided herein are polynucleotides comprising a nucleotide sequence encoding an anti-MerTK antibody comprising three VL chain CDRs, e.g., containing VL CDRl, VL CDR2, and VL CDR3 of Table 5. In specific embodiments, provided herein are polynucleotides comprising three VH chain CDRs, e.g., containing VH CDRl, VH CDR2, and VH CDR3 of Table 5. In specific embodiments, provided herein are polynucleotides comprising a nucleotide sequence encoding an anti-MerTK antibody comprising three VL chain CDRs, e.g., containing VL CDRl, VL CDR2, and VL CDR3 of Table 5 and three VH chain CDRs, e.g., containing VH CDRl, VH CDR2, and VH CDR3 of Table 5.
[00279] In particular embodiments, provided herein are polynucleotides comprising a nucleotide sequence encoding an anti-MerTK antibody comprising three VL chain CDRs, e.g., containing VL CDRl, VL CDR2, and VL CDR3 of Table 13. In specific embodiments, provided herein are polynucleotides comprising three VH chain CDRs, e.g., containing VH CDRl, VH CDR2, and VH CDR3 of Table 13. In specific embodiments, provided herein are polynucleotides comprising a nucleotide sequence encoding an anti-MerTK antibody comprising three VL chain CDRs, e.g., containing VL CDRl, VL CDR2, and VL CDR3 of Table 13 and three VH chain CDRs, e.g., containing VH CDRl, VH CDR2, and VH CDR3 of Table 13. In specific embodiments, provided herein are polynucleotides comprising a nucleotide sequence encoding an anti-MerTK antibody comprising three VL chain CDRs, e.g., containing VL CDRl, VL CDR2, and VL CDR3 of Table 5 and three VH chain CDRs, e.g., containing VH CDRl, VH CDR2, and VH CDR3 of Table 13. In specific embodiments, provided herein are
polynucleotides comprising a nucleotide sequence encoding an anti-MerTK antibody comprising three VL chain CDRs, e.g., containing VL CDRl, VL CDR2, and VL CDR3 of Table 13 and three VH chain CDRs, e.g., containing VH CDRl, VH CDR2, and VH CDR3 of Table 5.
[00280] In particular embodiments, provided herein are polynucleotides comprising a nucleotide sequence encoding an anti-MerTK antibody comprising three VL chain CDRs, e.g., containing VL CDRl, VL CDR2, and VL CDR3 of Table 6. In specific embodiments, provided herein are polynucleotides comprising three VH chain CDRs, e.g., containing VH CDRl, VH CDR2, and VH CDR3 of Table 6. In specific embodiments, provided herein are polynucleotides comprising a nucleotide sequence encoding an anti-MerTK antibody comprising three VL chain CDRs, e.g., containing VL CDRl, VL CDR2, and VL CDR3 of Table 6 and three VH chain CDRs, e.g., containing VH CDRl, VH CDR2, and VH CDR3 of Table 6.
[00281] In particular embodiments, provided herein are polynucleotides comprising a nucleotide sequence encoding an anti-MerTK antibody comprising three VL chain CDRs, e.g., containing VL CDRl, VL CDR2, and VL CDR3 of Table 14. In specific embodiments, provided herein are polynucleotides comprising three VH chain CDRs, e.g., containing VH CDRl, VH CDR2, and VH CDR3 of Table 14. In specific embodiments, provided herein are polynucleotides comprising a nucleotide sequence encoding an anti-MerTK antibody comprising three VL chain CDRs, e.g., containing VL CDRl, VL CDR2, and VL CDR3 of Table 14 and three VH chain CDRs, e.g., containing VH CDRl, VH CDR2, and VH CDR3 of Table 14. In specific embodiments, provided herein are polynucleotides comprising a nucleotide sequence encoding an anti-MerTK antibody comprising three VL chain CDRs, e.g., containing VL CDRl, VL CDR2, and VL CDR3 of Table 6 and three VH chain CDRs, e.g., containing VH CDRl, VH CDR2, and VH CDR3 of Table 14. In specific embodiments, provided herein are
polynucleotides comprising a nucleotide sequence encoding an anti-MerTK antibody comprising three VL chain CDRs, e.g., containing VL CDRl, VL CDR2, and VL CDR3 of Table 14 and three VH chain CDRs, e.g., containing VH CDRl, VH CDR2, and VH CDR3 of Table 6. [00282] In certain embodiments, a polynucleotide described herein comprises a nucleotide sequence encoding an antibody provided herein comprising a variable light (VL) chain region comprising an amino acid described herein, wherein the antibody specifically binds to a MerTK polypeptide, e.g., a human MerTK polypeptide, for example, human MerTK (e.g., human MerTK).
[00283] In certain embodiments, a polynucleotide described herein comprises a nucleotide sequence encoding an antibody provided herein comprising a variable heavy (VH) chain region comprising an amino acid sequence described herein, wherein the antibody specifically binds to a MerTK polypeptide, e.g., a human MerTK polypeptide, for example, human MerTK (e.g., human MerTK).
[00284] In specific embodiments, a polynucleotide provided herein comprises a nucleotide sequence encoding an antibody described herein comprising: framework regions (e.g., framework regions of the VL domain and VH domain) that are human framework regions, wherein the antibody specifically binds a MerTK polypeptide, e.g., a human MerTK polypeptide, for example, human MerTK (e.g., human MerTK).
[00285] In specific aspects, provided herein is a polynucleotide comprising a nucleotide sequence encoding an antibody comprising a light chain and a heavy chain, e.g., a separate light chain and heavy chain. With respect to the light chain, in a specific embodiment, a
polynucleotide provided herein comprises a nucleotide sequence encoding a kappa light chain. In another specific embodiment, a polynucleotide provided herein comprises a nucleotide sequence encoding a lambda light chain. In yet another specific embodiment, a polynucleotide provided herein comprises a nucleotide sequence encoding an antibody described herein comprising a human kappa light chain or a human lambda light chain. In a particular
embodiment, a polynucleotide provided herein comprises a nucleotide sequence encoding an antibody described herein, which specifically binds to a MerTK polypeptide, e.g., a human MerTK polypeptide, for example, human MerTK (e.g., human MerTK), wherein the antibody comprises a light chain, and wherein the amino acid sequence of the VL chain region can comprise any amino acid sequence described herein (e.g., SEQ ID NO: 2 or SEQ ID NO: 26), and wherein the constant region of the light chain comprises the amino acid sequence of a human kappa light chain constant region. In another particular embodiment, a polynucleotide provided herein comprises a nucleotide sequence encoding an antibody described herein, which specifically binds to a MerTK polypeptide, e.g., a human MerTK polypeptide, for example, human MerTK (e.g., human MerTK), and comprises a light chain, wherein the amino acid sequence of the VL chain region can comprises any amino acid sequence described herein (e.g., SEQ ID NO: 2 or SEQ ID NO: 26), and wherein the constant region of the light chain comprises the amino acid sequence of a human lambda light chain constant region. For example, human constant region sequences can be those described in U.S. Patent No. 5,693,780.
[00286] In a particular embodiment, a polynucleotide provided herein comprises a nucleotide sequence encoding an antibody described herein, which specifically binds to a MerTK polypeptide, e.g., a human MerTK polypeptide, for example, human MerTK (e.g., human MerTK), wherein the antibody comprises a heavy chain, wherein the amino acid sequence of the VH chain region can comprise any amino acid sequence described herein (e.g., SEQ ID NO: 1 or SEQ ID NO: 25), and wherein the constant region of the heavy chain comprises the amino acid sequence of a human gamma (γ) heavy chain constant region.
[00287] In yet another specific embodiment, a polynucleotide provided herein comprises a nucleotide sequence encoding an antibody described herein (or an antigen-binding fragment thereof), which specifically binds a MerTK polypeptide, e.g., a human MerTK polypeptide, for example, human MerTK (e.g., human MerTK), wherein the antibody comprises a VL chain region and a VH chain region comprising any amino acid sequences described herein, and wherein the constant regions comprise the amino acid sequences of the constant regions of a human IgGl (e.g., isotype a, z, or f), human IgG2, or human IgG4.
[00288] In a specific embodiment, provided herein are polynucleotides comprising a nucleotide sequence encoding an anti-MerTK antibody, or an antigen-binding fragment or domain thereof, designated herein.
[00289] As a non-limiting example, in some embodiments, an antibody or antigen-binding fragment described herein, which specifically binds to a MerTK polypeptide (e.g., human MerTK), can be encoded by a nucleotide that comprises nucleotide sequence of SEQ ID NO: 58, wherein the amino acid sequence for SEQ ID NO: 58 is GAGGTGC AGC TGC TGGAG A GCGGAGGAGGACTGGTGCAGCCCGGAGGATCTTTAAGACTGAGCTGCGCCGCCAGC GGCTTCACCTTCAGCAGCTACGCCATGTCTTGGGTGAGACAAGCTCCCGGAAAGGG ACTGGAGTGGGTCAGCGCCATTTCCGGAAGCGGCGGCAGCACCTACTACGCCGACA GCGTGAAGGGTCGTTTTACCATCTCTCGTGACAACAGCAAGAACACTTTATATTTAC
AGATGAACTCTTTAAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAAGGATCG
TCAGTGGCTGCTGCCCAGAGCCTTCGACATCTGGGGCCAAGGTACAACCGTGACCGT
GAGCAGCGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAG
CACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAGCC
GGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGG
CTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCC
TCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAA
CACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCC
CACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAA
AACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTG
GACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGG
AGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCG
TGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACA
AGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAA
GCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGA
GCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCG
ACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCAC
GCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGA
CAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTC
TGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTTAATAG.
[00290] As a non-limiting example, in some embodiments, an antibody or antigen-binding fragment described herein, which specifically binds to a MerTK polypeptide (e.g., human
MerTK), can be encoded by a nucleotide that comprises nucleotide sequence of SEQ ID NO: 59, wherein the amino acid sequence for SEQ ID NO: 59 is
GAGGTGCAGCTGCTGGAGAGCGGAGGAGGACTGGTGCAGCCCGGAGGATCTTTAAG ACTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACGCCATGTCTTGGGTGAG ACAAGCTCCCGGAAAGGGACTGGAGTGGGTCAGCGCCATTTCCGGAAGCGGCGGCA GCACCTACTACGCCGACAGCGTGAAGGGTCGTTTTACCATCTCTCGTGACAACAGCA AGAACACTTTATATTTACAGATGAACTCTTTAAGGGCCGAGGACACCGCCGTGTACT ACTGCGCCAAGGATCGTCAGTGGCTGCTGCCCAGAGCCTTCGACATCTGGGGCCAA GGTACAACCGTGACCGTGAGCAGCGCTAGCACCAAGGGCCCTAGCGTGTTCCCTCTC
GCCCCTTGTTCTCGTAGCACAAGCGAGAGCACAGCCGCTCTGGGCTGTCTGGTGAAG
GACTACTTCCCCGAGCCCGTGACCGTGAGCTGGAATAGCGGCGCTTTAACATCCGGC
GTCCACACATTTCCCGCTGTGCTGCAGTCCTCCGGTTTATACTCTTTAAGCTCCGTGG
TGACCGTGCCTAGCTCCAACTTCGGCACCCAGACCTACACTTGTAACGTGGACCACA
AGCCCTCCAACACCAAGGTGGACAAGACAGTGGAGAGGAAGTGTTGCGTCGAGTGC
CCTCCTTGTCCCGCTCCTCCCGTTGCTGGACCCTCCGTGTTCCTCTTCCCCCCCAAGC
CCAAGGACACTTTAATGATCAGCAGAACCCCCGAAGTCACTTGTGTGGTGGTGGAC
GTGTCCCACGAGGACCCCGAAGTGCAGTTCAACTGGTACGTCGACGGCGTGGAGGT
GCACAACGCCAAGACCAAGCCTCGTGAGGAACAGTTCAACAGCACCTTTAGGGTGG
TGTCCGTGCTGACAGTGGTCCATCAAGATTGGCTGAACGGCAAGGAGTACAAGTGT
AAGGTGAGCAACAAGGGTTTACCCGCCCCCATCGAGAAGACCATTTCCAAGACCAA
GGGCCAGCCCAGAGAGCCTCAAGTTTACACTTTACCCCCCTCTCGTGAAGAGATGAC
CAAGAACCAAGTTTCTTTAACTTGTTTAGTGAAGGGCTTCTACCCCTCCGACATCGC
CGTGGAATGGGAGTCCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCCA
TGCTGGACTCCGACGGCAGCTTCTTTTTATACTCCAAACTGACCGTGGACAAGTCTC
GTTGGCAGCAAGGTAACGTGTTCTCTTGTTCCGTGATGCACGAGGCCCTCCACAACC
ACTACACCCAGAAGTCTTTATCTTTATCCCCCGGC.
[00291] As a non-limiting example, in some embodiments, an antibody or antigen-binding fragment described herein, which specifically binds to a MerTK polypeptide (e.g., human MerTK), can be encoded by a nucleotide that comprises nucleotide sequence of SEQ ID NO: 60, wherein the amino acid sequence for SEQ ID NO: 60 is
GACATCGTGATGACCCAGTCCCCCGATTCTTTAGCTGTGTCTTTAGGCGAAAGGGCC
ACCATCAACTGCAAGTCCTCCCAGTCCGTGCTGTACTCCTCCAACAACAAGAACTAT
TTAGCTTGGTACCAGCAGAAGCCCGGTCAGCCCCCTAAGCTGCTGATCTACTGGGCT
TCCACTCGTGAGTTCGGCGTGCCCGATCGTTTTTCCGGCAGCGGCTCCGGCACCGAC
TTTACTTTAACCATCTCCTCTTTACAAGCTGAAGACGTGGCCGTGTACTTCTGCCAGC
AGTACTACACCACCCCTCTGACCTTCGGCCAAGGTACCAAGCTGGAGATCAAGCGT
ACGGTGGCCGCTCCAAGCGTGTTCATCTTTCCCCCTTCTGACGAGCAGCTGAAGTCT
GGCACAGCCTCCGTGGTGTGCCTGCTGAACAACTTCTACCCCAGAGAGGCCAAGGT
GCAGTGGAAGGTGGATAACGCTCTGCAGTCTGGCAATTCCCAGGAGAGCGTGAC CGAGCAGGACTCTAAGGATTCCACATATAGCCTGAGCTCTACCCTGACACTGTCTAA
GGCCGATTACGAGAAGCACAAGGTGTATGCTTGCGAGGTGACCCATCAGGGCCTGT
CCAGCCCAGTGACAAAGTCCTTCAATCGCGGCGAGTGT.
[00292] In some embodiments, an antibody or antigen-binding fragment described herein, which specifically binds to a MerTK polypeptide (e.g., human MerTK), can be encoded by a nucleotide that comprising a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 58. In some embodiments, an antibody or antigen-binding fragment described herein, which specifically binds to a MerTK polypeptide (e.g., human MerTK), can be encoded by a nucleotide that comprising a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 59. In some embodiments, an antibody or antigen-binding fragment described herein, which specifically binds to a MerTK polypeptide (e.g., human MerTK), can be encoded by a nucleotide that comprising a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 60.
[00293] In certain embodiments, an antibody described herein, which specifically binds to a MerTK polypeptide (e.g., human MerTK), can be encoded by a nucleotide that comprising a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 60 and a nucleotide that comprising a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 58. In certain embodiments, an antibody described herein, which specifically binds to a MerTK polypeptide (e.g., human MerTK), can be encoded by a nucleotide that comprising a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 60 and a nucleotide that comprising a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 59.
[00294] Also provided herein are polynucleotides encoding an anti-MerTK antibody or a fragment thereof that are optimized, e.g., by codon/RNA optimization, replacement with heterologous signal sequences, and elimination of mRNA instability elements. Methods to generate optimized nucleic acids encoding an anti-MerTK antibody or a fragment thereof (e.g., light chain, heavy chain, VH domain, or VL domain) for recombinant expression by introducing codon changes and/or eliminating inhibitory regions in the mRNA can be carried out by adapting the optimization methods described in, e.g., U.S. Patent Nos. 5,965,726; 6,174,666; 6,291,664; 6,414,132; and 6,794,498, accordingly. For example, potential splice sites and instability elements (e.g., A/T or A/U rich elements) within the RNA can be mutated without altering the amino acids encoded by the nucleic acid sequences to increase stability of the RNA for recombinant expression. The alterations utilize the degeneracy of the genetic code, e.g., using an alternative codon for an identical amino acid. In some embodiments, it can be desirable to alter one or more codons to encode a conservative mutation, e.g., a similar amino acid with similar chemical structure and properties and/or function as the original amino acid. Such methods can increase expression of an anti-MerTK antibody or fragment thereof by at least 1 fold, 2 fold, 3 fold, 4 fold, 5 fold, 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, or 100 fold or more relative to the expression of an anti-MerTK antibody encoded by
polynucleotides that have not been optimized.
[00295] In certain embodiments, an optimized polynucleotide sequence encoding an anti- MerTK antibody described herein or a fragment thereof (e.g., VL domain and/or VH domain) can hybridize to an antisense (e.g., complementarity) polynucleotide of an unoptimized polynucleotide sequence encoding an anti-MerTK antibody described herein or a fragment thereof (e.g., VL domain and/or VH domain). In specific embodiments, an optimized nucleotide sequence encoding an anti-MerTK antibody described herein or a fragment hybridizes under high stringency conditions to antisense polynucleotide of an unoptimized polynucleotide sequence encoding an anti-MerTK antibody described herein or a fragment thereof. In a specific embodiment, an optimized nucleotide sequence encoding an anti-MerTK antibody described herein or a fragment thereof hybridizes under high stringency, intermediate or lower stringency hybridization conditions to an antisense polynucleotide of an unoptimized nucleotide sequence encoding an anti-MerTK antibody described herein or a fragment thereof. Information regarding hybridization conditions have been described, see, e.g., U.S. Patent Application Publication No. US 2005/0048549 (e.g., paragraphs 72-73), which is incorporated herein by reference.
[00296] The polynucleotides can be obtained, and the nucleotide sequence of the
polynucleotides determined, by any method known in the art. Nucleotide sequences encoding antibodies described herein and modified versions of these antibodies can be determined using methods well known in the art, i.e., nucleotide codons known to encode particular amino acids are assembled in such a way to generate a nucleic acid that encodes the antibody. Such a polynucleotide encoding the antibody can be assembled from chemically synthesized
oligonucleotides (e.g., as described in Kutmeier et al., 1994, BioTechniques 17:242), which, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligating of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.
[00297] Alternatively, a polynucleotide encoding an antibody described herein can be generated from nucleic acid from a suitable source (e.g., a hybridoma) using methods well known in the art (e.g., PCR and other molecular cloning methods). For example, PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of a known sequence can be performed using genomic DNA obtained from hybridoma cells producing the antibody of interest. Such PCR amplification methods can be used to obtain nucleic acids comprising the sequence encoding the light chain and/or heavy chain of an antibody. Such PCR amplification methods can be used to obtain nucleic acids comprising the sequence encoding the variable light chain region and/or the variable heavy chain region of an antibody. The amplified nucleic acids can be cloned into vectors for expression in host cells and for further cloning, for example, to generate chimeric and humanized antibodies.
[00298] If a clone containing a nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, a nucleic acid encoding the
immunoglobulin can be chemically synthesized or obtained from a suitable source (e.g., an antibody cDNA library or a cDNA library generated from, or nucleic acid, preferably poly A+ RNA, isolated from, any tissue or cells expressing the antibody, such as hybridoma cells selected to express an antibody described herein) by PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence to identify, e.g., a cDNA clone from a cDNA library that encodes the antibody. Amplified nucleic acids generated by PCR can then be cloned into replicable cloning vectors using any method well known in the art.
[00299] DNA encoding anti-MerTK antibodies described herein can 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 anti-MerTK antibodies). Hybridoma cells can serve as a source of such DNA. Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells (e.g., CHO cells from the CHO GS System™ (Lonza)), or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of anti-MerTK antibodies in the recombinant host cells. [00300] To generate whole antibodies, PCR primers including VH or VL nucleotide sequences, a restriction site, and a flanking sequence to protect the restriction site can be used to amplify the VH or VL sequences in scFv clones. Utilizing cloning techniques known to those of skill in the art, the PCR amplified VH domains can be cloned into vectors expressing a heavy chain constant region, e.g., the human gamma 4 constant region, and the PCR amplified VL domains can be cloned into vectors expressing a light chain constant region, e.g., human kappa or lambda constant regions. In certain embodiments, the vectors for expressing the VH or VL domains comprise an EF-la promoter, a secretion signal, a cloning site for the variable domain, constant domains, and a selection marker such as neomycin. The VH and VL domains can also be cloned into one vector expressing the necessary constant regions. The heavy chain conversion vectors and light chain conversion vectors are then co-transfected into cell lines to generate stable or transient cell lines that express full-length antibodies, e.g., IgG, using techniques known to those of skill in the art.
[00301] The DNA also can be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the murine sequences, or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide.
[00302] Also provided are polynucleotides that hybridize under high stringency, intermediate or lower stringency hybridization conditions to polynucleotides that encode an antibody described herein. In specific embodiments, polynucleotides described herein hybridize under high stringency, intermediate or lower stringency hybridization conditions to polynucleotides encoding a VH chain region and/or VL chain region provided herein.
[00303] Hybridization conditions have been described in the art and are known to one of skill in the art. For example, hybridization under stringent conditions can involve hybridization to filter-bound DNA in 6x sodium chloride/sodium citrate (SSC) at about 45° C followed by one or more washes in 0.2xSSC/0.1% SDS at about 50-65° C; hybridization under highly stringent conditions can involve hybridization to filter-bound nucleic acid in 6xSSC at about 45° C followed by one or more washes in O. lxSSC/0.2% SDS at about 68° C. Hybridization under other stringent hybridization conditions are known to those of skill in the art and have been described, see, for example, Ausubel, F.M. et al., eds., 1989, Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York at pages 6.3.1-6.3.6 and 2.10.3.
5.2.2 Cells and Vectors
[00304] In certain aspects, provided herein are cells (e.g., host cells) expressing (e.g., recombinantly) antibodies described herein (or an antigen-binding fragment thereof) which specifically bind to an ECD of human MerTK and related polynucleotides and expression vectors. Provided herein are vectors (e.g., expression vectors) comprising polynucleotides comprising nucleotide sequences encoding anti-MerTK antibodies or a fragment for recombinant expression in host cells, preferably in mammalian cells. Also provided herein are host cells comprising such vectors for recombinantly expressing anti-MerTK antibodies described herein (e.g., human or humanized antibody). In a particular aspect, provided herein are methods for producing an antibody described herein, comprising expressing such antibody from a host cell.
[00305] Recombinant expression of an antibody described herein (e.g., a full-length antibody, heavy and/or light chain of an antibody, or a single chain antibody described herein) that specifically binds to human MerTK involves construction of an expression vector containing a polynucleotide that encodes the antibody. Once a polynucleotide encoding an antibody molecule, heavy and/or light chain of an antibody, or a fragment thereof (e.g., heavy and/or light chain variable domains) described herein has been obtained, the vector for the production of the antibody molecule can be produced by recombinant DNA technology using techniques well- known in the art. Thus, methods for preparing a protein by expressing a polynucleotide containing an antibody or antibody fragment (e.g., light chain or heavy chain) encoding nucleotide sequence are described herein. Methods which are well known to those skilled in the art can be used to construct expression vectors containing antibody or antibody fragment (e.g., light chain or heavy chain) coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Also provided are replicable vectors comprising a nucleotide sequence encoding an antibody molecule described herein, a heavy or light chain of an antibody, a heavy or light chain variable domain of an antibody or a fragment thereof, or a heavy or light chain CDR, operably linked to a promoter. Such vectors can, for example, include the nucleotide sequence encoding the constant region of the antibody molecule (see, e.g., International Publication Nos. WO 86/05807 and WO 89/01036; and U.S. Patent No. 5, 122,464) and variable domains of the antibody can be cloned into such a vector for expression of the entire heavy, the entire light chain, or both the entire heavy and light chains.
[00306] An expression vector can be transferred to a cell (e.g., host cell) by conventional techniques and the resulting cells can then be cultured by conventional techniques to produce an antibody described herein (e.g., an antibody comprising the CDRs of any of Table 1, Table 3-7, or Table 11-14) or a fragment thereof. Thus, provided herein are host cells containing a polynucleotide encoding an antibody described herein or fragments thereof, or a heavy or light chain thereof, or fragment thereof, or a single chain antibody described herein (e.g., an antibody comprising the CDRs of any of Table 1, Table 3-7, or Table 11-14), operably linked to a promoter for expression of such sequences in the host cell. In certain embodiments, for the expression of double-chained antibodies, vectors encoding both the heavy and light chains, individually, can be co-expressed in the host cell for expression of the entire immunoglobulin molecule, as detailed below. In certain embodiments, a host cell contains a vector comprising a polynucleotide encoding both the heavy chain and light chain of an antibody described herein (e.g., an antibody comprising the CDRs of any of Table 1, Table 3-7, or Table 11-14), or a fragment thereof. In specific embodiments, a host cell contains two different vectors, a first vector comprising a polynucleotide encoding a heavy chain or a heavy chain variable region of an antibody described herein (e.g., an antibody comprising the CDRs of any of Table 1, Table 3- 7, or Table 11-14), or a fragment thereof, and a second vector comprising a polynucleotide encoding a light chain or a light chain variable region of an antibody described herein (e.g., an antibody comprising the CDRs of any of Table 1, Table 3-7, or Table 11-14), or a fragment thereof. In other embodiments, a first host cell comprises a first vector comprising a
polynucleotide encoding a heavy chain or a heavy chain variable region of an antibody described herein (e.g., an antibody comprising the CDRs of any of Table 1, Table 3-7, or Table 11-14), or a fragment thereof, and a second host cell comprises a second vector comprising a polynucleotide encoding a light chain or a light chain variable region of an antibody described herein (e.g., an antibody comprising the CDRs of any of Table 1, Table 3-7, or Table 11-14). In specific embodiments, a heavy chain/heavy chain variable region expressed by a first cell associated with a light chain/light chain variable region of a second cell to form an anti-MerTK antibody described herein (e.g., antibody comprising the CDRs of any of Table 1, Table 3-7, or Table 11- 14) or an antigen-binding fragment thereof. In certain embodiments, provided herein is a population of host cells comprising such first host cell and such second host cell.
[00307] In a particular embodiment, provided herein is a population of vectors comprising a first vector comprising a polynucleotide encoding a light chain/light chain variable region of an anti-MerTK antibody described herein (e.g., antibody comprising the CDRs of any of Table 1, Table 3-7, or Table 11-14), and a second vector comprising a polynucleotide encoding a heavy chain/heavy chain variable region of an anti-MerTK antibody described herein (e.g., antibody comprising the CDRs of any of Table 1, Table 3-7, or Table 11-14).
[00308] A variety of host-expression vector systems can be utilized to express antibody molecules described herein (e.g., an antibody comprising the CDRs of any of Table 1, Table 3-7, or Table 11-14) (see, e.g., U.S. Patent No. 5,807,715). Such host-expression systems represent vehicles by which the coding sequences of interest can be produced and subsequently purified, but also represent cells which can, when transformed or transfected with the appropriate nucleotide coding sequences, express an antibody molecule described herein in situ. These include but are not limited to microorganisms such as bacteria (e.g., E. coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Saccharomyces Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing antibody coding sequences; plant cell systems (e.g., green algae such as Chlamydomonas reinhardtii) infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing antibody coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, MDCK, HEK 293, NS0, PER.C6, VERO, CRL7030, HsS78Bst, HeLa, and NIH 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter). In a specific embodiment, cells for expressing antibodies described herein (e.g., an antibody comprising the CDRs of any of Table 1, Table 3-7, or Table 11-14) or an antigen-binding fragment thereof are CHO cells, for example CHO cells from the CHO GS System™ (Lonza). In a specific embodiment, a mammalian expression vector is pOptiVEC™ or pcDNA3.3. In a particular embodiment, bacterial cells such as Escherichia coli, or eukaryotic cells (e.g., mammalian cells), especially for the expression of whole recombinant antibody molecule, are used for the expression of a recombinant antibody molecule. For example, mammalian cells such as Chinese hamster ovary (CHO) cells, in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system for antibodies (Foecking et al., 1986, Gene 45: 101; and Cockett et al., 1990, Bio/Technology 8:2). In certain embodiments, antibodies described herein are produced by CHO cells or NSO cells. In a specific embodiment, the expression of nucleotide sequences encoding antibodies described herein which specifically bind to human MerTK is regulated by a constitutive promoter, inducible promoter or tissue specific promoter.
[00309] In bacterial systems, a number of expression vectors can be advantageously selected depending upon the use intended for the antibody molecule being expressed. For example, when a large quantity of such an antibody is to be produced, for the generation of pharmaceutical compositions of an antibody molecule, vectors which direct the expression of high levels of fusion protein products that are readily purified can be desirable. Such vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruther et al., 1983, EMBO 12: 1791), in which the antibody coding sequence can be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, 1985, Nucleic Acids Res. 13 :3101-3109; Van Heeke & Schuster, 1989, J. Biol. Chem. 24:5503-5509); and the like. For example, pGEX vectors can also be used to express foreign polypeptides as fusion proteins with glutathione 5-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to matrix glutathione agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.
[00310] In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV), for example, can be used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The antibody coding sequence can be cloned individually into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter). [00311] In mammalian host cells, a number of viral -based expression systems can be utilized. In cases where an adenovirus is used as an expression vector, the antibody coding sequence of interest can be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene can then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region El or E3) will result in a recombinant virus that is viable and capable of expressing the antibody molecule in infected hosts (e.g., see Logan & Shenk, 1984, Proc. Natl. Acad. Sci. USA 8 1 :355-359). Specific initiation signals can also be required for efficient translation of inserted antibody coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression can be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see, e.g., Bittner et al., 1987, Methods in Enzymol. 153 :51-544).
[00312] In addition, a host cell strain can be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products can be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and
phosphorylation of the gene product can be used. Such mammalian host cells include but are not limited to CHO, VERO, BHK, Hela, COS, MDCK, HEK 293, NIH 3T3, W138, BT483,
Hs578T, HTB2, BT20 and T47D, NS0 (a murine myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7030 and HsS78Bst cells. In certain embodiments, anti-MerTK antibodies described herein (e.g., an antibody comprising the CDRs of any of Table 1, Table 3-7, or Table 11-14) are produced in mammalian cells, such as CHO cells.
[00313] For long-term, high-yield production of recombinant proteins, stable expression cells can be generated. For example, cell lines which stably express an anti-MerTK antibody described herein (e.g., an antibody comprising the CDRs of any of Table 1, Table 3-7, or Table 11-14) or an antigen-binding fragment thereof can be engineered. In specific embodiments, a cell provided herein stably expresses a light chain/light chain variable domain and a heavy chain/heavy chain variable domain which associate to form an antibody described herein (e.g., an antibody comprising the CDRs of any of Table 1, Table 3-7, or Table 11-14) or an antigen- binding fragment thereof.
[00314] In certain aspects, rather than using expression vectors which contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of the foreign DNA/polynucleotide, engineered cells can be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci, which in turn can be cloned and expanded into cell lines. This method can
advantageously be used to engineer cell lines which express an anti-MerTK antibody described herein or a fragment thereof. Such engineered cell lines can be particularly useful in screening and evaluation of compositions that interact directly or indirectly with the antibody molecule.
[00315] A number of selection systems can be used, including but not limited to, the herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11 :223), hypoxanthineguanine phosphoribosyltransferase (Szybalska & Szybalski, 1992, Proc. Natl. Acad. Sci. USA 48:202), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:8-17) genes can be employed in tk-, hgprt- or aprt-cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., 1980, Natl. Acad. Sci. USA 77:357; O'Hare et al., 1981, Proc. Natl. Acad. Sci. USA
78: 1527); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, which confers resistance to the aminoglycoside G-418 (Wu and Wu, 1991, Biotherapy 3 :87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573- 596; Mulligan, 1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann. Rev.
Biochem. 62: 191-217; May, 1993, TIB TECH 11(5):155-2 15); and hygro, which confers resistance to hygromycin (Santerre et al., 1984, Gene 30: 147). Methods commonly known in the art of recombinant DNA technology can be routinely applied to select the desired recombinant clone, and such methods are described, for example, in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and in Chapters 12 and 13, Dracopoli et al. (eds.), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colberre-Garapin et al., 1981, J. Mol. Biol. 150: 1, which are incorporated by reference herein in their entireties.
[00316] The expression levels of an antibody molecule can be increased by vector
amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3 (Academic Press, New York, 1987)). When a marker in the vector system expressing antibody is amplifiable, increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the antibody gene, production of the antibody will also increase (Crouse et al., 1983, Mol. Cell. Biol. 3 :257).
[00317] The host cell can be co-transfected with two or more expression vectors described herein, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide. The two vectors can contain identical selectable markers which enable equal expression of heavy and light chain polypeptides. The host cells can be co-transfected with different amounts of the two or more expression vectors. For example, host cells can be transfected with any one of the following ratios of a first expression vector and a second expression vector: 1 : 1, 1 :2, 1 :3, 1 :4, 1 :5, 1 :6, 1 :7, 1 :8, 1 :9, 1 : 10, 1 : 12, 1 : 15, 1 :20, 1 :25, 1 :30, 1 :35, 1 :40, 1 :45, or 1 :50.
[00318] Alternatively, a single vector can be used which encodes, and is capable of expressing, both heavy and light chain polypeptides. In such situations, the light chain should be placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, 1986, Nature 322:52; and Kohler, 1980, Proc. Natl. Acad. Sci. USA 77:2197-2199). The coding sequences for the heavy and light chains can comprise cDNA or genomic DNA. The expression vector can be monocistronic or multicistronic. A multicistronic nucleic acid construct can encode 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, or in the range of 2-5, 5-10 or 10-20 genes/nucleotide sequences. For example, a bicistronic nucleic acid construct can comprise in the following order a promoter, a first gene (e.g., heavy chain of an antibody described herein), and a second gene and (e.g., light chain of an antibody described herein). In such an expression vector, the transcription of both genes can be driven by the promoter, whereas the translation of the mRNA from the first gene can be by a cap-dependent scanning mechanism and the translation of the mRNA from the second gene can be by a cap-independent mechanism, e.g., by an IRES.
[00319] Once an antibody molecule described herein has been produced by recombinant expression, it can be purified by any method known in the art for purification of an
immunoglobulin molecule, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column
chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. Further, the antibodies described herein can be fused to heterologous polypeptide sequences described herein or otherwise known in the art to facilitate purification.
[00320] In specific embodiments, an antibody described herein is isolated or purified.
Generally, an isolated antibody is one that is substantially free of other antibodies with different antigenic specificities than the isolated antibody. For example, in a particular embodiment, a preparation of an antibody described herein is substantially free of cellular material and/or chemical precursors. The language "substantially free of cellular material" includes preparations of an antibody in which the antibody is separated from cellular components of the cells from which it is isolated or recombinantly produced. Thus, an antibody that is substantially free of cellular material includes preparations of antibody having less than about 30%, 20%, 10%, 5%, 2%), 1%), 0.5%), or 0.1%) (by dry weight) of heterologous protein (also referred to herein as a "contaminating protein") and/or variants of an antibody, for example, different post-translational modified forms of an antibody or other different versions of an antibody (e.g., antibody fragments). When the antibody is recombinantly produced, it is also generally substantially free of culture medium, i.e., culture medium represents less than about 20%, 10%>, 2%, 1%, 0.5%, or 0.1%) of the volume of the protein preparation. When the antibody is produced by chemical synthesis, it is generally substantially free of chemical precursors or other chemicals, i.e., it is separated from chemical precursors or other chemicals which are involved in the synthesis of the protein. Accordingly, such preparations of the antibody have less than about 30%, 20%, 10%, or 5%) (by dry weight) of chemical precursors or compounds other than the antibody of interest. In a specific embodiment, antibodies described herein are isolated or purified.
5.3 Pharmaceutical Compositions and Kits [00321] Provided herein are compositions, pharmaceutical compositions, and kits comprising one or more antibodies (e.g., anti-MerTK antibodies comprising CDRs of any of Table 1, Table 3-7, or Table 11-14, for example an antibody comprising the VL and VH sequences of Table 2, an antibody comprising the VL and VH sequences of Table 8, an antibody comprising the VL and VH sequences of Table 9, or an antibody comprising the VL and VH sequences of Table 10) described herein, or antigen-binding fragments thereof, or conjugates thereof. In particular aspects, compositions (e.g., pharmaceutical compositions) described herein can be for in vitro, in vivo, or ex vivo uses. Non-limiting examples of uses include uses to modulate (e.g., inhibit) MerTK activity and uses to manage or treat a disorder, for example, cancer. In specific embodiments, provided herein is a pharmaceutical composition comprising an antibody (e.g., a humanized antibody) described herein (or an antigen-binding fragment thereof) and a pharmaceutically acceptable carrier or excipient.
[00322] As used herein, the term "pharmaceutically acceptable" means being approved by a regulatory agency of the Federal or a state government, or listed in the U.S. Pharmacopeia, European Pharmacopeia or other generally recognized Pharmacopeia for use in animals, and more particularly in humans.
[00323] Therapeutic formulations containing one or more antibodies provided herein (e.g., antibodies comprising CDRs of any of Table 1, Table 3-7, or Table 11-14, for example an antibody comprising the VL and VH sequences of Table 2, an antibody comprising the VL and VH sequences of Table 8, an antibody comprising the VL and VH sequences of Table 9, or an antibody comprising the VL and VH sequences of Table 10) or an antigen-binding fragment thereof can be prepared for storage by mixing the antibody having the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers (Remington's
Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA; Remington: The Science and Practice of Pharmacy, 21st ed. (2006) Lippincott Williams & Wilkins, Baltimore, MD), in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).
[00324] Formulations, such as those described herein, can also contain more than one active compounds (for example, molecules, e.g., antibody or antibodies described herein) as necessary for the particular indication being treated. In certain embodiments, formulations comprise an antibody provided herein and one or more active compounds with complementarity activities that do not adversely affect each other. Such molecules are suitably present in combination in amounts that are effective for the purpose intended. For example, an antibody described herein can be combined with one or more other therapeutic agents (e.g., a tyrosine kinase inhibitor such as imatinib mesylated or sunitinib, or a histone deacetylase inhibitor such as vorinostat). Such combination therapy can be administered to the patient serially or simultaneously or in sequence.
[00325] The formulations to be used for in vivo administration can be sterile. This is readily accomplished by filtration through, e.g., sterile filtration membranes.
[00326] In specific aspects, the pharmaceutical compositions provided herein contain therapeutically effective amounts of one or more of the antibodies provided herein, and optionally one or more additional prophylactic of therapeutic agents, in a pharmaceutically acceptable carrier. Such pharmaceutical compositions are useful in the prevention, treatment, management or amelioration of a condition or disorder described herein or one or more symptoms thereof.
[00327] Pharmaceutical carriers suitable for administration of the antibodies provided herein include any such carriers known to those skilled in the art to be suitable for the particular mode of administration.
[00328] In addition, the antibodies described herein can be formulated as the sole
pharmaceutically active ingredient in the composition or can be combined with other active ingredients (such as one or more other prophylactic or therapeutic agents).
[00329] Compositions provided herein can contain one or more antibodies provided herein (e.g., antibodies comprising CDRs of any of Table 1, Table 3-7, or Table 11-14, for example an antibody comprising the VL and VH sequences of Table 2, an antibody comprising the VL and VH sequences of Table 8, an antibody comprising the VL and VH sequences of Table 9, or an antibody comprising the VL and VH sequences of Table 10) or an antigen-binding fragment thereof. In one embodiment, the antibodies are formulated into suitable pharmaceutical preparations, such as solutions, suspensions, powders, sustained release formulations or elixirs in sterile solutions or suspensions for parenteral administration, or as transdermal patch preparation and dry powder inhalers. [00330] In compositions provided herein, one or more antibodies described herein is (are) mixed with a suitable pharmaceutical carrier. The concentrations of the antibody or antibodies in the compositions can, for example, be effective for delivery of an amount, upon administration, that treats, prevents, or ameliorates a condition or disorder described herein or a symptom thereof.
[00331] In one embodiment, compositions provided herein are formulated for single dosage administration. To formulate a composition, the weight fraction of compound is dissolved, suspended, dispersed or otherwise mixed in a selected carrier at an effective concentration such that the treated condition is relieved, prevented, or one or more symptoms are ameliorated.
[00332] In certain aspects, an antibody provided herein (e.g., antibody comprising CDRs of any of Table 1, Table 3-7, or Table 11-14, for example an antibody comprising the VL and VH sequences of Table 2, an antibody comprising the VL and VH sequences of Table 8, an antibody comprising the VL and VH sequences of Table 9, or an antibody comprising the VL and VH sequences of Table 10) is included in the pharmaceutically acceptable carrier in an effective amount sufficient to exert a therapeutically useful effect in the absence of, or with minimal or negligible, undesirable side effects on the patient treated.
[00333] Concentrations of anti-MerTK antibody in a pharmaceutical composition provided herein will depend on, e.g., the physicochemical characteristics of the antibody, the dosage schedule, and amount administered as well as other factors known to those of skill in the art.
[00334] The pharmaceutical compositions, in another embodiment, provide a dosage of about 50 mg of antibody per kilogram of body weight for administration over a period of time, e.g., every day or few days, every week, every 2 weeks, or every 3 weeks. Pharmaceutical dosage unit forms can be prepared to provide from about 500 mg.
[00335] Pharmaceutical compositions described herein are provided for administration to humans or animals (e.g., mammals) in unit dosage forms, such as sterile parenteral (e.g., intravenous) solutions or suspensions containing suitable quantities of the compounds or pharmaceutically acceptable derivatives thereof. Pharmaceutical compositions are also provided for administration to humans and animals in unit dosage form, such as tablets, capsules, pills, powders, granules, and oral or nasal solutions or suspensions, and oil-water emulsions containing suitable quantities of an anti-MerTK antibody or pharmaceutically acceptable derivatives thereof. The antibody is, in one embodiment, formulated and administered in unit-dosage forms or multiple-dosage forms. Unit-dose forms as used herein refers to physically discrete units suitable for human or animal (e.g., mammal) subjects and packaged individually as is known in the art. Each unit-dose contains a predetermined quantity of an anti-MerTK antibody sufficient to produce the desired therapeutic effect, in association with the required pharmaceutical carrier, vehicle or diluent. Examples of unit-dose forms include ampoules and syringes and individually packaged tablets or capsules. Unit-dose forms can be administered in fractions or multiples thereof. A multiple-dose form is a plurality of identical unit-dosage forms packaged in a single container to be administered in segregated unit-dose form. Examples of multiple-dose forms include vials, bottles of tablets or capsules or bottles. Hence, in specific aspects, multiple dose form is a multiple of unit-doses which are not segregated in packaging.
[00336] In certain embodiments, one or more anti-MerTK antibodies described herein (e.g., antibodies comprising CDRs of any of Table 1, Table 3-7, or Table 11-14, for example an antibody comprising the VL and VH sequences of Table 2, an antibody comprising the VL and VH sequences of Table 8, an antibody comprising the VL and VH sequences of Table 9, or an antibody comprising the VL and VH sequences of Table 10) or an antigen-binding fragment thereof are in a liquid pharmaceutical formulation. Liquid pharmaceutically administrable compositions can, for example, be prepared by dissolving, dispersing, or otherwise mixing an antibody and optional pharmaceutical adjuvants in a carrier, such as, for example, water, saline, aqueous dextrose, glycerol, glycols, and the like, to thereby form a solution or suspension. In certain embodiments, a pharmaceutical composition provided herein to be administered can also contain minor amounts of nontoxic auxiliary substances such as wetting agents, emulsifying agents, solubilizing agents, and pH buffering agents and the like.
[00337] Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, see, e.g., Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA; Remington: The Science and Practice of Pharmacy, 21st ed. (2006) Lippincott Williams & Wilkins, Baltimore, MD. Dosage forms or compositions containing antibody in the range of 0.005% to 100% with the balance made up from non-toxic carrier can be prepared.
[00338] Parenteral administration, in one embodiment, is characterized by injection, either subcutaneously, intramuscularly or intravenously is also contemplated herein. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. The injectables, solutions and emulsions also contain one or more excipients. Suitable excipients are, for example, water, saline, dextrose, glycerol or ethanol. Other routes of administration may include, enteric administration, intracerebral administration, nasal administration, intraarterial administration, intracardiac administration, intraosseous infusion, intrathecal administration, and intraperitoneal administration.
[00339] Preparations for parenteral administration include sterile solutions ready for injection, sterile dry soluble products, such as lyophilized powders, ready to be combined with a solvent just prior to use, including hypodermic tablets, sterile suspensions ready for injection, sterile dry insoluble products ready to be combined with a vehicle just prior to use and sterile emulsions. The solutions can be either aqueous or nonaqueous.
[00340] If administered intravenously, suitable carriers include physiological saline or phosphate buffered saline (PBS), and solutions containing thickening and solubilizing agents, such as glucose, polyethylene glycol, and polypropylene glycol and mixtures thereof.
[00341] Pharmaceutically acceptable carriers used in parenteral preparations include aqueous vehicles, nonaqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, sequestering or chelating agents and other pharmaceutically acceptable substances.
[00342] Pharmaceutical carriers also include ethyl alcohol, polyethylene glycol and propylene glycol for water miscible vehicles; and sodium hydroxide, hydrochloric acid, citric acid or lactic acid for pH adjustment.
[00343] In certain embodiments, intravenous or intraarterial infusion of a sterile aqueous solution containing an anti-MerTK antibody described herein is an effective mode of
administration. Another embodiment is a sterile aqueous or oily solution or suspension containing an anti-MerTK antibody described herein injected as necessary to produce the desired pharmacological effect.
[00344] In specific embodiments, an anti-MerTK antibody described herein can be suspended in micronized or other suitable form. The form of the resulting mixture depends upon a number of factors, including the intended mode of administration and the solubility of the compound in the selected carrier or vehicle. [00345] In other embodiments, the pharmaceutical formulations are lyophilized powders, which can be reconstituted for administration as solutions, emulsions and other mixtures. They can also be reconstituted and formulated as solids or gels.
[00346] The lyophilized powder is prepared by dissolving an anti-MerTK antibody provided herein, in a suitable solvent. In some embodiments, the lyophilized powder is sterile. Suitable solvents can contain an excipient which improves the stability or other pharmacological component of the powder or reconstituted solution, prepared from the powder. Excipients that can be used include, but are not limited to, dextrose, sorbital, fructose, corn syrup, xylitol, glycerin, glucose, sucrose or other suitable agent. A suitable solvent can also contain a buffer, such as citrate, sodium or potassium phosphate or other such buffer known to those of skill in the art at, in one embodiment, about neutral pH. Subsequent sterile filtration of the solution followed by lyophilization under standard conditions known to those of skill in the art provides an example of a formulation. In one embodiment, the resulting solution will be apportioned into vials for lyophilization. Lyophilized powder can be stored under appropriate conditions, such as at about 4 °C to room temperature.
[00347] Reconstitution of this lyophilized powder with water for injection provides a formulation for use in parenteral administration. For reconstitution, the lyophilized powder is added to sterile water or other suitable carrier.
[00348] In certain aspects, anti-MerTK antibodies provided herein can be formulated for local administration or topical application, such as for topical application to the skin and mucous membranes, such as in the eye, in the form of gels, creams, and lotions and for application to the eye or for intraci sternal or intraspinal application. Topical administration is contemplated for transdermal delivery and also for administration to the eyes or mucosa, or for inhalation therapies. Nasal solutions of the active compound alone or in combination with other pharmaceutically acceptable excipients can also be administered.
[00349] Anti-MerTK antibodies and other compositions provided herein can also be formulated to be targeted to a particular tissue, receptor, or other area of the body of the subject to be treated. Many such targeting methods are well known to those of skill in the art. All such targeting methods are contemplated herein for use in the instant compositions. For non-limiting examples of targeting methods, see, e.g., U.S. Patent Nos. 6,316,652, 6,274,552, 6,271,359, 6,253,872, 6, 139,865, 6, 131,570, 6, 120,751, 6,071,495, 6,060,082, 6,048,736, 6,039,975, 6,004,534, 5,985,307, 5,972,366, 5,900,252, 5,840,674, 5,759,542 and 5,709,874. In some embodiments, anti-MerTK antibodies described herein are targeted (or otherwise administered) to the visual organs, bone marrow, gastrointestinal tract, lungs, brain, or joints. In specific embodiments, an anti-MerTK antibody described herein is capable of crossing the blood-brain barrier.
[00350] Provided herein is a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions described herein, such as one or more anti-MerTK antibodies provided herein. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
[00351] Also provided herein are kits that can be used in the above methods. In one embodiment, a kit comprises an antibody described herein, preferably a purified antibody, in one or more containers. In a specific embodiment, kits described herein contain a substantially isolated MerTK antigen (e.g., ECD of human MerTK) as a control. In another specific embodiment, the kits described herein further comprise a control antibody which does not react with a MerTK antigen. In another specific embodiment, kits described herein contain one or more elements for detecting the binding of a modified antibody to a MerTK antigen (e.g., the antibody can be conjugated to a detectable substrate such as a fluorescent compound, an enzymatic substrate, a radioactive compound or a luminescent compound, or a second antibody which recognizes the first antibody can be conjugated to a detectable substrate). In specific embodiments, a kit provided herein can include a recombinantly produced or chemically synthesized MerTK antigen. The MerTK antigen provided in the kit can also be attached to a solid support. In a more specific embodiment, the detecting means of the above described kit includes a solid support to which a MerTK antigen is attached. Such a kit can also include a non-attached reporter-labeled anti-human antibody or anti-mouse/rat antibody. In this embodiment, binding of the antibody to the MerTK antigen can be detected by binding of the said reporter-labeled antibody.
5.4 Uses and Methods [00352] In particular aspects, provided herein are methods of modulating MerTK activity with an anti-MerTK antibody (e.g., an antibody comprising CDRs of any of Table 1, Table 3-7, or Table 11-14, for example an antibody comprising the VL and VH sequences of Table 2, an antibody comprising the VL and VH sequences of Table 8, an antibody comprising the VL and VH sequences of Table 9, or an antibody comprising the VL and VH sequences of Table 10) described herein or an antigen-binding fragment thereof. In certain embodiments, the anti- MerTK antibody is a MerTK antagonist.
[00353] In specific embodiments, provided herein are methods of inhibiting (e.g., partially inhibiting) MerTK activity with an anti-MerTK antibody described herein which is a MerTK antagonist. In certain embodiments, provided herein are methods of managing or treating a condition or disorder using an anti-MerTK antibody described herein which is a MerTK antagonist or inhibitor.
[00354] Provided herein are methods of managing or treating a condition by inhibiting an activity of MerTK with an anti-MerTK antibody (e.g., an antibody comprising CDRs of any of Table 1, Table 3-7, or Table 11-14, for example an antibody comprising the VL and VH sequences of Table 2, an antibody comprising the VL and VH sequences of Table 8, an antibody comprising the VL and VH sequences of Table 9, or an antibody comprising the VL and VH sequences of Table 10) described herein that acts as a MerTK antagonist. Non-limiting examples of a condition which can be treated or managed with a MerTK antagonist include cancer, sepsis, and infection, described in more detail below.
[00355] In certain embodiments, a condition which can be treated or managed with a MerTK antagonist is one which can be managed or treated by enhancing an immune response. An anti- MerTK antibody described herein or an antigen-binding fragment thereof, which is a MerTK antagonist for use in the methods provided herein is capable of inhibiting (e.g., partially inhibiting) or decreasing/reducing MerTK expression and/or a MerTK activity. Activities of MerTK are known in the art. In specific embodiments, an anti-MerTK antibody described herein which is a MerTK antagonist inhibits (e.g., partially inhibits) one or more of the following MerTK activities: phosphorylation (e.g., autophosphorylation) of MerTK receptor (e.g., cytoplasmic domain of MerTK, such as cytoplasmic kinase domain of MerTK), promotion of phagocytosis, modulation of maturation of NK cells, dendritic cells or macrophages, inhibition of toll-like receptor (TLR) activation of dendritic cells, inhibition or decrease in TLR-induced proinflammatory cytokine (e.g., T F, IL-6, IL-12 and type I interferon production, increase in SOCSl expression, association with interleukin-15 (IL-15) to protect cells against TNF-induced cell death (e.g., fibroblasts and DCs), and activation of Statl in dendritic cells. Methods for measuring these activities are known in the art.
[00356] In specific embodiments, an anti -MerTK antibody described herein which is a MerTK antagonist inhibits (e.g., partially inhibits), by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 97%, 98%, 99%, or 100%, one or more such MerTK activities.
[00357] In particular aspects, provided herein is a method of enhancing an immune response in a subject comprising administering to a subject in need thereof an effective amount of an antibody described herein (e.g., an antibody comprising CDRs of any of Table 1, Table 3-7, or Table 11-14, for example an antibody comprising the VL and VH sequences of Table 2, an antibody comprising the VL and VH sequences of Table 8, an antibody comprising the VL and VH sequences of Table 9, or an antibody comprising the VL and VH sequences of Table 10) or antigen-binding fragment thereof which specifically binds to human MerTK and inhibits MerTK.
[00358] In specific embodiments, a subject (e.g., human subject) in need of enhancing an immune response include an immunocompromised subject, for example a subject suffering from an infection (e.g., bacterial, viral, fungal, or protozoan). In certain embodiments, an
immunocompromised subject has cancer and is undergoing, or had undergone treatment with, anti-cancer therapy, such as, chemotherapy. Other non-limiting examples of
immunocompromised subjects include, but are not limited to, subjects who are HIV positive, subjects with AIDS or SCID, subjects with diabetes, and subjects who have undergone transplants and are taking immunosuppressants. In certain embodiments, an
immunocompromised subject is taking immunosuppressants (e.g., steroids) to manage or treat asthma, arthritis (e.g., rheumatoid arthritis), or allergy or an allergic condition.
[00359] In a particular embodiment, provided herein is a method of enhancing an immune response to a vaccine in a subject, comprising administering to a subject in need thereof, who is or has been administered the vaccine, an effective amount of an antibody described herein (e.g., an antibody comprising CDRs of any of Table 1, Table 3-7, or Table 11-14, for example an antibody comprising the VL and VH sequences of Table 2, an antibody comprising the VL and VH sequences of Table 8, an antibody comprising the VL and VH sequences of Table 9, or an antibody comprising the VL and VH sequences of Table 10) or antigen-binding fragment thereof which specifically binds to human MerTK and inhibits MerTK. In specific embodiments, the antibody is administered before, concurrently, or after administration of the vaccine.
[00360] An example of vaccines in the methods provides herein include cancer/tumor vaccines, such as vaccines comprising tumor cells or tumor cell lysates and vaccines comprising dendritic cells or dendritic cell lysates activated against a tumor.
[00361] Provided herein is a method of managing, preventing, protecting against, or treating cancer in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of an antibody described herein (e.g., an antibody comprising CDRs of any of Table 1, Table 3-7, or Table 11-14, for example an antibody comprising the VL and VH sequences of Table 2, an antibody comprising the VL and VH sequences of Table 8, an antibody comprising the VL and VH sequences of Table 9, or an antibody comprising the VL and VH sequences of Table 10) or antigen-binding fragment thereof which specifically binds to human MerTK and inhibits a MerTK activity or inhibits or reduces MerTK expression. In particular embodiments, such a method involves inhibition of tumor growth, cancer cell proliferation, cancer cell migration, inhibition of tumor-associated macrophage activity (e.g., tumor-promoting activity such as production of cytokines), and modulating stromal cells such as macrophages or dendritic cells.
[00362] Non-limiting examples of cancers to be managed, prevented, protected against, or treated in the methods provided herein include breast cancer, pancreatic cancer, leukemia, lung cancer such as non-small cell lung cancer, glioblastoma, melanoma, prostate cancer, colon cancer, gastric cancer, pituitary adenomas, ovarian cancer, renal cancer, bladder cancer, and sarcomas, including rhabdomyosarcomas.
[00363] In a specific embodiment, provided herein is a method of treating cancer in a subject comprising administering to a subject in need thereof an antibody described herein (e.g., an antibody comprising CDRs of any of Table 1, Table 3-7, or Table 11-14, for example an antibody comprising the VL and VH sequences of Table 2, an antibody comprising the VL and VH sequences of Table 8, an antibody comprising the VL and VH sequences of Table 9, or an antibody comprising the VL and VH sequences of Table 10) or antigen-binding fragment thereof which specifically binds to human MerTK and inhibits MerTK, in an effective amount to enhancing an immune response to the cancer. [00364] In one aspect, provided herein is a method of managing, preventing, protecting against, or treating metastasis in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of an antibody described herein (e.g., an antibody comprising CDRs of any of Table 1, Table 3-7, or Table 11-14, for example an antibody comprising the VL and VH sequences of Table 2, an antibody comprising the VL and VH sequences of Table 8, an antibody comprising the VL and VH sequences of Table 9, or an antibody comprising the VL and VH sequences of Table 10) or antigen-binding fragment thereof which specifically binds to human MerTK and inhibits a MerTK activity or inhibits or reduces MerTK expression.
[00365] In a certain aspect, provided herein is a method of inhibiting MerTK expression and/or activity of a tumor-associated macrophage comprising contacting a tumor-associated macrophage with an effective amount of an antibody described herein (e.g., an antibody comprising CDRs of any of Table 1, Table 3-7, or Table 11-14, for example an antibody comprising the VL and VH sequences of Table 2, an antibody comprising the VL and VH sequences of Table 8, an antibody comprising the VL and VH sequences of Table 9, or an antibody comprising the VL and VH sequences of Table 10) or antigen-binding fragment thereof which specifically binds to human MerTK and inhibits a MerTK activity or inhibits or reduces MerTK expression. In a specific embodiment, such a method disrupts tumor-macrophage cross talk and inhibits the ability of tumor-associated macrophages to promote cancer cell
proliferation.
[00366] In a certain aspect, provided herein is a method of modulating (e.g., inhibiting) stromal cell (e.g., macrophage or dendritic cell) activity, comprising contacting a stromal cell with an effective amount of an antibody described herein (e.g., an antibody comprising CDRs of any of Table 1, Table 3-7, or Table 11-14, for example an antibody comprising the VL and VH sequences of Table 2, an antibody comprising the VL and VH sequences of Table 8, an antibody comprising the VL and VH sequences of Table 9, or an antibody comprising the VL and VH sequences of Table 10) or antigen-binding fragment thereof which specifically binds to human MerTK and inhibits a MerTK activity or inhibits or reduces MerTK expression in the stromal cell. In a specific embodiment, such method is for modulating stromal cell activity in a subject with cancer.
[00367] In a certain aspect, provided herein is a method of modulating (e.g., inhibiting) natural killer (NK) cell activity, comprising contacting an NK cell with an effective amount of an antibody described herein (e.g., an antibody comprising CDRs of any of Table 1, Table 3-7, or Table 11-14, for example an antibody comprising the VL and VH sequences of Table 2, an antibody comprising the VL and VH sequences of Table 8, an antibody comprising the VL and VH sequences of Table 9, or an antibody comprising the VL and VH sequences of Table 10) or antigen-binding fragment thereof which specifically binds to human MerTK and inhibits a MerTK activity or inhibits or reduces MerTK expression in the NK cell. In a specific embodiment, such method is for modulating NK cell activity in a subject with cancer.
[00368] In another aspect, provided herein is a method of modulating (e.g., inhibiting) T cell activity, comprising contacting a T cell with an effective amount of an antibody described herein (e.g., an antibody comprising CDRs of any of Table 1, Table 3-7, or Table 11-14, for example an antibody comprising the VL and VH sequences of Table 2, an antibody comprising the VL and VH sequences of Table 8, an antibody comprising the VL and VH sequences of Table 9, or an antibody comprising the VL and VH sequences of Table 10) or antigen-binding fragment thereof which specifically binds to human MerTK and inhibits a MerTK activity or inhibits or reduces MerTK expression in the T cell. In a specific embodiment, such method is for modulating stromal cell activity in a subject with cancer.
[00369] In a particular embodiment, provided herein is a method of inhibiting or reducing tumor growth or cancer cell proliferation in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of an antibody described herein (e.g., an antibody comprising CDRs of any of Table 1, Table 3-7, or Table 11-14, for example an antibody comprising the VL and VH sequences of Table 2, an antibody comprising the VL and VH sequences of Table 8, an antibody comprising the VL and VH sequences of Table 9, or an antibody comprising the VL and VH sequences of Table 10) or antigen-binding fragment thereof which specifically binds to human MerTK and inhibits a MerTK activity or inhibits or reduces MerTK expression.
[00370] In a specific embodiment, provided herein is a method of treating cancer in a subject comprising administering (e.g., administering concurrently or sequentially) to a subject in need thereof (i) an antibody described herein (e.g., an antibody comprising CDRs of any of Table 1, Table 3-7, or Table 11-14, for example an antibody comprising the VL and VH sequences of Table 2, an antibody comprising the VL and VH sequences of Table 8, an antibody comprising the VL and VH sequences of Table 9, or an antibody comprising the VL and VH sequences of Table 10) or antigen-binding fragment thereof which specifically binds to human MerTK and inhibits MerTK, and (ii) another anti-cancer agent.
[00371] In one embodiment, provided herein is a method of managing, preventing, protecting against, or treating an infection in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of an antibody described herein (e.g., an antibody comprising CDRs of any of Table 1, Table 3-7, or Table 11-14, for example an antibody comprising the VL and VH sequences of Table 2, an antibody comprising the VL and VH sequences of Table 8, an antibody comprising the VL and VH sequences of Table 9, or an antibody comprising the VL and VH sequences of Table 10) or antigen-binding fragment thereof which specifically binds to human MerTK and inhibits a MerTK activity.
[00372] In a specific embodiment, the infection is an infection caused by bacteria (gram- negative bacteria or gram-positive bacteria), fungi, viruses, or parasites.
[00373] In a particular embodiment, provided herein is a method of managing or treating sepsis in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of an antibody described herein (e.g., an antibody comprising CDRs of any of Table 1, Table 3-7, or Table 11-14, for example an antibody comprising the VL and VH sequences of Table 2, an antibody comprising the VL and VH sequences of Table 8, an antibody comprising the VL and VH sequences of Table 9, or an antibody comprising the VL and VH sequences of Table 10) or antigen-binding fragment thereof which specifically binds to human MerTK and inhibits a MerTK activity.
[00374] Sepsis (a systemic inflammatory response syndrome or SIRS) is an immunological condition characterized by whole-body inflammation caused by infection, such as infection by bacteria (gram-negative bacteria or gram-positive bacteria), fungi, viruses, or parasites. Stages of sepsis include, but are not limited to, onset of sepsis, severe sepsis, and septic shock. Severe sepsis can be accompanied by organ dysfunction.
[00375] In certain embodiments, the sepsis is advanced sepsis. In a particular embodiment, the sepsis is characterized with increased expression of SOCS, for example during the stage of severe sepsis. In a particular embodiment, the sepsis is characterized with elevated levels of circulating Gas6 in a subject with sepsis. Levels or concentrations of circulating Gas6 can be measured from blood samples using conventional methods, such as ELISAs. Levels or concentrations of Gas6 can be compared with a reference value to determine whether Gas6 levels or concentrations are elevated in a subject with sepsis. For example, the reference values can be a set standard range of levels and concentrations of Gas6 accepted in the art as correlating with those of a population of healthy subjects not suffering from sepsis. As another example, the reference values can be levels of concentrations of Gas6 in a subject prior to the onset of sepsis or at the early stages of sepsis. Symptoms of sepsis include, but are not limited to, high fevers, hot, flushed skin, elevated heart rate, hyperventilation, altered mental status, swelling, and low blood pressure.
[00376] In a particular embodiment, provided herein is a method of managing, alleviating or treating one or more symptoms of sepsis in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of an antibody described herein (e.g., an antibody comprising CDRs of any of Table 1, Table 3-7, or Table 11-14, for example an antibody comprising the VL and VH sequences of Table 2, an antibody comprising the VL and VH sequences of Table 8, an antibody comprising the VL and VH sequences of Table 9, or an antibody comprising the VL and VH sequences of Table 10) or antigen-binding fragment thereof which specifically binds to human MerTK and inhibits a MerTK activity.
[00377] In certain embodiments, the method of managing or treating sepsis provided herein further comprises the step of administering an antibiotic to a subject in need thereof, for example, prior to, concurrently with, or subsequent to administering an anti-MerTK antibody described herein.
[00378] In certain aspects, provided herein are methods for managing, treating, preventing, or protecting against infection in a subject, comprising administering to a subject in need thereof an effective amount of an antibody described herein (e.g., an antibody comprising CDRs of any of Table 1, Table 3-7, or Table 11-14, for example an antibody comprising the VL and VH sequences of Table 2, an antibody comprising the VL and VH sequences of Table 8, an antibody comprising the VL and VH sequences of Table 9, or an antibody comprising the VL and VH sequences of Table 10) or antigen-binding fragment thereof which specifically binds to human MerTK and inhibits a MerTK activity.
[00379] In certain embodiments, the infection is caused by microbes, e.g., bacteria, viruses or parasites. In certain embodiments, the viral infection is HIV, herpes, shingles, influenza, common cold, and encephalitis. [00380] In certain embodiments, provided herein are methods for prevent or inhibit entry of a viruses by contacting a cell to an effective amount of an antibody described herein (e.g., an antibody comprising CDRs of any of Table 1, Table 3-7, or Table 11-14, for example an antibody comprising the VL and VH sequences of Table 2, an antibody comprising the VL and VH sequences of Table 8, an antibody comprising the VL and VH sequences of Table 9, or an antibody comprising the VL and VH sequences of Table 10) or antigen-binding fragment thereof which specifically binds to human MerTK and inhibits a MerTK activity. In particular embodiments, the method provided herein prevents or inhibits entry of lymphocytic
choriomeningitis virus, HIV, herpes virus (e.g., herpes simplex virus or herpes zoster), influenza virus, or common cold virus.
[00381] In certain embodiments, the anti -MerTK antibody or antigen-binding fragment thereof for use in the methods provided herein is an antibody (e.g., monoclonal antibody, such as a humanized monoclonal antibody) comprising CDRs of any of Table 1, Table 3-7, or Table 11- 14, for example an antibody comprising the VL and VH sequences of Table 2, an antibody comprising the VL and VH sequences of Table 8, an antibody comprising the VL and VH sequences of Table 9, or an antibody comprising the VL and VH sequences of Table 10. In particular embodiments, the anti-MerTK antibody inhibits a MerTK activity.
[00382] In other aspects, provided herein are methods of enhancing a type I interferon (IFN) response against a pathogen in a subject, comprising administering to a subject in need thereof an effective amount of an anti-MerTK antibody described herein, such as a MerTK antagonist antibody.
[00383] As used herein, "administer" or "administration" refers to the act of injecting or otherwise physically delivering a substance (e.g., a humanized anti-MerTK antibody provided herein or an antigen-binding fragment thereof) to a subject or a patient (e.g., human), such as by mucosal, topical, intradermal, parenteral, intravenous, intramuscular delivery and/or any other method of physical delivery described herein or known in the art.
[00384] As used herein, the terms "effective amount" or "therapeutically effective amount" refer to an amount of a therapy (e.g., an antibody or pharmaceutical composition provided herein) which is sufficient to reduce and/or ameliorate the severity and/or duration of a given condition, disorder or disease and/or a symptom related thereto. These terms also encompass an amount necessary for the reduction, slowing, or amelioration of the advancement or progression of a given disease, reduction, slowing, or amelioration of the recurrence, development or onset of a given disease, and/or to improve or enhance the prophylactic or therapeutic effect(s) of another therapy (e.g., a therapy other than an anti-MerTK antibody provided herein). In some embodiments, "effective amount" as used herein also refers to the amount of an antibody described herein to achieve a specified result.
[00385] As used herein, the term "in combination" in the context of the administration of other therapies refers to the use of more than one therapy. The use of the term "in combination" does not restrict the order in which therapies are administered. The therapies may be
administered, e.g., serially, sequentially, concurrently, or concomitantly.
[00386] As used herein, the terms "manage," "managing," and "management" refer to the beneficial effects that a subject derives from a therapy (e.g., a prophylactic or therapeutic agent), which does not result in a cure of a condition associated with MerTK. In certain embodiments, a subject is administered one or more therapies (e.g., prophylactic or therapeutic agents, such as an antibody described herein) to "manage" a condition or disorder described herein, one or more symptoms thereof, so as to prevent the progression or worsening of the condition or disorder.
[00387] As used herein, the terms "impede" or "impeding" in the context of a condition or disorder provided herein refer to the total or partial inhibition (e.g., less than 100%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or 5%) or blockage of the development, recurrence, onset or spread of a condition or disorder provided herein and/or symptom related thereto, resulting from the administration of a therapy or combination of therapies provided herein (e.g., a combination of prophylactic or therapeutic agents, such as an antibody described herein).
[00388] As used herein, the term "prophylactic agent" refers to any agent that can totally or partially inhibit the development, recurrence, onset or spread of a condition or disorder provided herein, and/or symptom related thereto in a subject. In certain embodiments, the term
"prophylactic agent" refers to an antibody described herein. In certain other embodiments, the term "prophylactic agent" refers to an agent other than an antibody described herein. Generally, a prophylactic agent is an agent which is known to be useful to or has been or is currently being used to prevent a condition or disorder provided herein, and/or a symptom related thereto or impede the onset, development, progression and/or severity of a condition or disorder provided herein, and/or a symptom related thereto. In specific embodiments, the prophylactic agent is an anti-MerTK antibody, as described herein.
[00389] As used herein, the terms "subject" and "patient" are used interchangeably. As used herein, a subject is a mammal such as a non-primate (e.g., cows, pigs, horses, cats, dogs, goats, rabbits, rats, mice, etc.) or a primate (e.g., monkey and human), for example a human. In one embodiment, the subject is a mammal, e.g., a human, diagnosed with a condition or disorder provided herein. In another embodiment, the subject is a mammal, e.g., a human, at risk of developing a condition or disorder provided herein. In another embodiment, the subject is human.
[00390] As used herein, the terms "therapies" and "therapy" can refer to any protocol(s), method(s), compositions, formulations, and/or agent(s) that can be used in the prevention, treatment, management, or amelioration of a condition or disorder or symptom thereof (e.g., a condition or disorder provided herein or one or more symptoms or condition associated therewith). In certain embodiments, the terms "therapies" and "therapy" refer to drug therapy, adjuvant therapy, radiation, surgery, biological therapy, supportive therapy, and/or other therapies useful in treatment, management, prevention, or amelioration of a condition or disorder or one or more symptoms thereof. In certain embodiments, the term "therapy" refers to a therapy other than an anti-MerTK antibody described herein or pharmaceutical composition thereof. In specific embodiments, an "additional therapy" and "additional therapies" refer to a therapy other than a treatment using an anti-MerTK antibody described herein or pharmaceutical composition thereof. In a specific embodiment, a therapy includes the use of an anti-MerTK antibody described herein as an adjuvant therapy. For example, using an anti-MerTK antibody described herein in conjunction with a drug therapy, biological therapy, surgery, and/or supportive therapy.
[00391] As used herein, the term "therapeutic agent" refers to any agent that can be used in the treatment, management or amelioration of a condition or disorder or symptom thereof. In certain embodiments, the term "therapeutic agent" refers to an anti-MerTK antibody described herein or an antigen-binding fragment thereof. In certain other embodiments, the term
"therapeutic agent" refers to an agent other than an antibody described herein. In specific embodiments, a therapeutic agent is an agent which is known to be useful for, or has been or is currently being used for the treatment, management or amelioration of a condition or disorder provided herein or one or more symptoms or condition associated therewith or one or more symptoms related thereto.
[00392] In one embodiment, for example, an antibody or antigen-binding fragment thereof that specifically binds to human MerTK as described herein can be administered in combination with another therapeutic for addressing one or more of the indications described herein. In a specific embodiment, such a therapeutic can be a stimulator of T cell responses. For example, such a therapeutic can be an immune checkpoint inhibitor, e.g., a PD-1, PD-L1, or CTLA-4 inhibitor. Such exemplary agents can include, e.g., Yervoy™ (ipilimumab) or tremelimumab (to CTLA-4), BMS-936558/nivolumab (to PD-1), MK-3475/pembrolizumab (to PD-1). In additional embodiments, such a therapeutic can be, for example, CDX-1127 (to CD27), CP- 870893 (to CD40), lucatumumab (to CD40), or dacetuzumab (to CD40).
5.4.1 Diagnostic Uses
[00393] In one aspect, anti-MerTK antibodies described herein and antigen-binding fragments thereof, which specifically bind to an ECD of human MerTK can be used for diagnostic purposes to detect, diagnose, or monitor a condition described herein (e.g., a condition involving MerTK and/or abnormal MerTK signaling and/or abnormal MerTK expression). In specific
embodiments, anti-MerTK antibodies described herein or an antigen-binding fragment thereof for use in diagnostic purposes are labeled.
[00394] In certain embodiments, provided herein are methods for the detection of a condition described herein comprising: (a) assaying the expression of MerTK in cells or a tissue sample of a subject using one or more antibodies described herein or an antigen-binding fragment thereof; and (b) comparing the level of MerTK expression with a control level, e.g., levels in normal tissue samples (e.g., from a patient not having a condition described herein, or from the same patient before onset of the condition), whereby an increase or decrease in the assayed level of MerTK expression compared to the control level of MerTK expression is indicative of a condition described herein.
[00395] Antibodies described herein can be used to assay MerTK levels in a biological sample using classical immunohistological methods as described herein or as known to those of skill in the art (e.g., see Jalkanen et al., 1985, J. Cell. Biol. 101 :976-985; and Jalkanen et al., 1987, J. Cell. Biol. 105:3087-3096). Other antibody-based methods useful for detecting protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assay labels are known in the art and include enzyme labels, such as, glucose oxidase; radioisotopes, such as iodine (1251, 1211), carbon (14C), sulfur (35S), tritium (3H), indium (121In), and technetium (99Tc); luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, and biotin.
[00396] In one embodiment, monitoring of a condition described herein is carried out by repeating the method for diagnosing for a period of time after initial diagnosis.
[00397] Presence of the labeled molecule can be detected in the subject using methods known in the art for in vivo scanning. Skilled artisans will be able to determine the appropriate method for detecting a particular label. Methods and devices that may be used in the diagnostic methods include, but are not limited to, computed tomography (CT), whole body scan such as position emission tomography (PET), magnetic resonance imaging (MRI), and sonography.
6. EXAMPLES
[00398] The examples in this section (i.e., Section 6) are offered by way of illustration, and not by way of limitation.
6.1 Example 1: Generating anti-MerTK antibodies
[00399] A phage display screen was employed to identify single-chain antibodies (scFvs) that bind the extracellular domain of human and/or mouse MerTK.
6.1.1 Antigen cloning, production, purification, and characterization
[00400] All antigen ectodomain genes were cloned from synthetic genes, and sequence verified. All of the antigens were expressed by transient transfection of HEK293 cells and purified to homogeneity as judged by SDS-PAGE analysis of purified samples and size exclusion chromatography (SEC). Human and mouse MerTK with rat CD4 tags were biotinylated with EZ-Link Sulfo-LHS-LC-Biotin reagent (Thermo Scientific) to give a ratio of 1 : 1 to 1 :2 molecules of biotin per protein molecule.
6.1.2 Antibody-phage display selections
[00401] A phage-display selection strategy aimed at discovering specific binders to human or mouse MerTK was employed. Two separate sets of selections were performed: solid-phase and solution-phase. For solid-phase selections, the tubes were coated at 10 μg/mL with antigen while for solution-phase selections, 100 nM of biotinylated antigen was used. Also, antigens with two separate tags (rCD4 and Fc) were used for selections.
6.1.3 Polyclonal ELISA
[00402] Following antibody selection, polyclonal phage ELISA was carried out to confirm the generation of binding antibodies to the selected antigens. All of the selected populations were tested against all of the antigens. As a positive control, an unrelated antigen and its selected population were used. In addition, a commercially sourced Fc-tagged human MerTK antigen was employed. In order to assess the level of tag binders, two irrelevant proteins, one with Fc tag and the other with rCD4 tag were used. Rescued phages were incubated with coated antigens in ELISA plates and the binding detected by mouse anti-M13 antibody and europium labeled anti- mouse antibody. The signals were read as time resolved fluorescence (TRF) measurements.
6.1.4 Single chain antibody (scFv) sub-cloning
Selected antibody populations were ligated into a vector enabling expression of soluble single chain antibody (scFv) fused with a C-terminal FLAG tag and expressed downstream of a leader sequence directing its secretion into the bacterial periplasm. The ligated products were transformed into E. coli BL21 (DE3) cells. The design facilitates rapid screening of antibodies for their ability to bind a given antigen directly from the expression media. Post-transformation, colonies were tested using PCR for the presence of an scFv insert.
6.1.5 Primary Binding ELISA
[00403] For screening, clones were first expressed in auto-induction media. Bacterial supernatant was used to test binding to the antigen on which they were selected. From primary screening a number of hits were obtained. Clones with a signal of more than 20X of background were cherry picked and subjected to a secondary ELISA binder screen in order to identify tag binders and eliminate them from future analysis. For the tag-binder ELISA, Fc-hexa-his and rCD4-hexa-his, purified in-house, were used as the antigen.
6.1.6 DNA Sequencing and Sequence Analysis
[00404] DNA sequences of both the heavy and light chains of scFv were used to identify unique antibody sequences. See Table 1 and Table 2 (VL and VH CDR amino acid sequences (Table 1) and VL and VH amino acid sequences (Table 2) of Ab2000).
6.1.7 Cloning, Expression and Purification [00405] scFvs were cloned into a human IgGl framework and the identity confirmed by sequencing. Sequence analysis was performed to indicate presence of unique clones.
Transfection quality DNA was prepared, and HEK293 transfections was performed at 50 mL culture volume for the unique clones.
6.2 Example 2: Anti-MerTK monoclonal antibody binds to cellularly expressed full length human MerTK
[00406] To determine whether Ab2000 IgGl monoclonal antibody binds to cellularly expressed human MerTK, the anti-MerTK antibody was titrated on mouse L cells that were engineered to express full length human MerTK in a stable manner, and further incubated with a fluorescent anti-human secondary antibody as follows.
6.2.1 Stable Expression of Human MerTK in Mouse L Cells:
[00407] Mouse L cells, originally derived from subcutaneous connective tissue from male C3H/An mice (ATCC CRL-2648), and their derivatives engineered to stably express full-length, wild-type human MerTK, were maintained in complete DMEM containing 10% heat inactivated fetal bovine serum (Gibco), 2 mM GlutaMax (Gibco), Penicillin/Streptomycin (Gibco), and 3 μg/mL puromycin (Invivogen).
[00408] To obtain L cells stably expressing full length human or mouse MerTK, cells were transduced with retroviruses based on the pLCPX vector (Clontech) carrying the gene of interest. L cells expressing full length human MerTK ( M_006343.2) were obtained by transduction with a codon optimized MerTK cDNA cloned into pLCPX vector.
[00409] Stably transduced L cells were selected in media containing 3 μg/mL puromycin, sorted to purity, and maintained in growth media containing 3 μg/ml puromycin.
6.2.2 Binding Assays
[00410] L cells were resuspended at lxlO6 cell/mL in lx FACS buffer (PBS, 0.5% BSA, 0.09% sodium azide). L cells were plated at 100 μΐ/well or lxlO5 cells/well. Unlabeled antibodies were added to the cells, concentrations ranging between 6 pM to 500 nM. After 30 min on ice, cells were washed and resuspended in 100 μΐ of lx FACS buffer containing PE- labeled donkey anti-human IgG, cells were washed and resuspended in 100 μΐ of lx FACS buffer containing live/dead stain (7-AAD). After incubating for 20 min at room temperature, cells were washed and resuspended in ΙΟΟμΙ in Fixation/Permeabilization Solution (BD Bioscienes). After 30 min on ice, cells were washed and resuspended in 100 μΐ of wash solution for 96-well plates and analyzed by flow cytometry.
6.2.3 Data Analysis and Results
[00411] The software that operates the HyperCyt autosampler calculates and reports the mean fluorescent intensity values (MFI). The extent of antibody binding to cells was estimated by the MFI, after subtracting the MFI values obtained on cells stained with secondary antibodies alone (in the absence of primary antibodies).
[00412] Figure 1 shows the mean fluorescent intensity plotted as a function of the log- transformed molar concentration of the antibody. Ab2000 binds to cellularly expressed full length human MerTK in a dose responsive manner, with EC50 values in the picomolar to nanomolar range.
6.3 Example 3: Anti-MerTK Antibodies Block Binding Of Human MerTK to
Gas6
[00413] To characterize the effect of anti -MerTK antibody Ab2000 on MerTK activity, specifically, a plate-based assay was developed to establish whether a given antibody blocks binding of Gas6 to human MerTK (hMerTK).
[00414] Biotinylated human Gas6 (R&D Systems) was captured to a streptavidin microtiter plate (Pierce) at 5 μg/mL. The anti-MerTK antibodies ([final] = 20 μg/mL, or a titration thereof) were mixed with soluble recombinant human MerTK-msFc ([final] = 0.05 μg/mL), incubated for 10 minutes at room temperature then added to the plate. Goat anti-mouse IgG (Fc specific) antibody was added. MerTK binding was then detected with a horseradish peroxidase conjugated donkey anti -goat IgG (H&L) antibody and TMB substrate (KPL).
[00415] As shown in Figure 2, Ab2000 inhibited Gas6 binding to hMerTK in a dose responsive manner, with an IC50 of 1 nM.
6.4 Example 4: Domain Mapping
[00416] An ELISA-based method was utilized to determine to which of the four MerTK domains Ab2000 binds.
6.4.1 Domain construction and purification
[00417] The MerTK extracellular domain (ECD) contains two N-terminal immunoglobulin- like domains (here named Dl and D2, respectively) followed by two Fibronectin-III domains (here named D3 and D4, respectively). Table 15, utilizing standard one letter amino acid codes, shows the amino acid sequences of the proteins encoding each of the 4 domains that comprise the human MerTK extracellular domain.
Table 15: Amino acid sequences of the proteins encoding each of the 4 domains that comprise the human MerTK extracellular domain
Figure imgf000114_0001
[00418] Proteins encoding each of the 4 domains that comprise the human MerTK
extracellular domain (ECD) were fused in frame to a rat CD4 domain, followed by a
biotinylation (Avi) tag and a hexahistidine tag. The IL-2 signal sequence for secretion into the culture media was engineered at the 5' end of each construct. Expression vectors encoding these constructs were transfected in Expi293 cells, and conditioned media containing each fusion protein was purified by nickel-affinity chromatography and buffer exchanged to phosphate buffer saline (PBS).
6.4.2 Domain binding analysis and Results [00419] Purified domains and ECDs were coated on Nickel-coated 96 well plates (Thermo Scientific) at 100 ng/well in borate buffer. Plates were washed and incubated with 50 nM of each mAb at room temperature for 1 hour, followed by washing and incubation with an HRP- conjugated anti-human secondary antibody for 1 hour at room temperature. Plates were washed and developed using a TMB substrate as described by the manufacturer (Thermo Scientific). A BioTek plate reader was used to quantitate the TMB signal at A450.
[00420] The data show that Ab2000 binds to recombinantly purified MerTK Dl alone, and a protein comprising Dl and D2. Ab2000 did not bind to recombinantly purified MerTK D2, D3, or D4 alone. Ab2000 also did not bind to a protein comprising D2 and D3 nor did it bind to a protein comprising D3 and D4. Moreover, Ab2000 binds to the intact human MerTK
extracellular domain (ECD) and did not bind to the Axl ECD.
6.5 Example 5: Modulation of MerTK phosphorylation by anti-MerTK
monoclonal antibodies
[00421] The ability of the anti-MerTK Ab2000 IgGl to modulate tyrosine (e.g., human MerTK) naturally expressed in THP-1 monocyte-derived macrophages was evaluated. Briefly, the assay was carried out under two conditions: In the first (Figure 3 A and Figure 3B), Ab2000 IgGl was tested for its ability to inhibit Gas6-dependent MerTK tyrosine phosphorylation. In the second (Figure 4), the mAb was tested for its ability to stimulate MerTK phosphorylation on its own (using Gas6 as an independent reference).
6.5.1 MerTK phosphorylation assay:
[00422] THP-1 is a suspension human monocytic cell line derived from an acute monocytic leukemia patient. THP-1 cells in exponential growth phase were seeded at a density of 40,000 cells/well in a 24-well plate in complete media and differentiated into macrophages with 100 ng/ml phorbol 12-myristate 13-acetate (PMA) (Abeam) for 72 hours, followed by a 24 hour recovery period during which PMA is removed. On the day of the assay, THP-1 cells were starved for 3 hours. Anti-MerTK mAb was added to a final concentration of 100 nM for 1 hour at 37°C.
[00423] In assays that measure mAb-dependent MerTK phosphorylation, cells were washed and lysed. In this assay format, 100 nM recombinantly-purified carboxylated mouse Gas6 was also added for 1 hour as an independent treatment to serve as a positive control and reference. [00424] In assays that measure mAb inhibition of Gas6-dependent MerTK phosphorylation, Gas6 at 100 nM was added for 10 minutes to cells pre-incubated with mAbs for 1 hour, after which the cells were washed and lysed.
[00425] Figures 3 A and 3B show inhibition of Gas6-dependent MerTK phosphorylation by the antibody Ab2000. Titration of Ab2000 on MerTK expressing THP1 cells inhibited Gas6- dependent MerTK phosphorylation with an IC50 value of approximately 0.2 nM (Figure 3B). Addition of Ab2000 to concentrations as high as 10000 nM elicited only very weak MerTK phosphorylation in the absence of exogenously added Gas6 (as compared to independently- added Gas6) as shown in Figure 4.
6.6 Example 6: Modulation of pro-inflammatory cytokine release from human macrophages and dendritic cells by anti-MerTK monoclonal antibodies
[00426] Ab2000 was tested for its ability to modulate TNF-a secretion from human macrophages stimulated with various TLR agonists, e.g., LPS and resiquimod (R848) as shown below. These assays were performed on primary human macrophages, or U937 monocyte- derived macrophages.
[00427] U937 cells: Human U937 monocytes (ATCC CRL-1593.2), were fully differentiated into monocyte-derived macrophages with Phorbol 12-myristate 13-acetate (PMA) (Abeam) at 10 ng/ml for 72 hours. U937 macrophages were serum starved for 3 hours, and anti-MerTK mAbs added at 100 nM for 1 hour prior to treatment with 100 ng/mL ultra-pure lipopoly saccharide (LPS) (Invivogen) for 24 hours. Cell-free supernatants were assayed for the presence of TNF-a using an ELISA-based method as described by the manufacturer (Biolegend).
[00428] Human primary macrophages: Macrophages were derived from human monocytes as follows: peripheral blood mononuclear cells (PBMCs) were added to a tissue culture plate and monocytes allowed to adhere for ~2 hours at 37°C and 6% CO2. The non-adherent cells were removed and the monocytes differentiated for 3 days in RPMI containing 10% FBS and 5 ng/mL PMA (Sigma). After differentiation, the serum containing media was removed from the wells, and the wells were washed with RPMI only. The cells were then incubated in the presence of anti-MerTK antibody or appropriate isotype control and LPS (InvivoGen) or resiquimod (R848) at 100 ng/mL overnight at 37°C and 6% CO2. All reagents were prepared in RPMI (no serum). The supernatant was collected, clarified and either used immediately or stored frozen for cytokine analysis. Production of TNF-α was evaluated by ELISA (R&D Systems).
[00429] As shown in Figures 5, 6A and 6B, addition of 100 nM of the MerTK mAb Ab2000 resulted in increased TNF-a release in macrophages from several sources and donors, consistent with its role as a MerTK inhibitor as demonstrated in the phosphorylation assays.
[00430] Anti-MerTK Ab2000 was also tested for its ability to modulate TNF-a secretion from human dendritic cells stimulated with various TLR agonists. FACS analysis was performed to demonstrate that human primary dendritic cells express MerTK on their surface.
[00431] FACS Analysis: Biotinylated MerTK antibody (R&D Systems) at 5 μg/mL was incubated with dendritic cells at room temperature on a plate shaker. After 20 minutes, the cells were washed with PBS containing 0.1% BSA and 0.05% NaN3 (PBA) and the bound antibodies were detected by incubating the cells with a PE labeled strepavidin probe for an additional 20 minutes. The excess probe was washed from the cells with PBA and the cell-associated fluorescence was determined by analysis using a FACSCanto II™ instrument (BD Biosciences, NJ, USA) according to the manufacturer's directions. FACS analysis demonstrated that human primary dendritic cells express MerTK on their surface.
[00432] Dendritic cell differentiation and cytokine release: Dendritic cells were derived from human monocytes as follows: peripheral blood mononuclear cells (PBMCs) were added to a T175 cm2 flasks and monocytes allowed to adhere for 2 hours at 37°C in 6% CO2. The nonadherent cells were removed and the monocytes cultured for 7 days in RPMI containing 10% FBS, 10 ng/mL IL-4 (R&D Systems) and 100 ng/mL GM-CSF (R&D Systems). The cells were harvested, washed in RPMI (no serum) and counted. The cells were then incubated in the presence of anti-MerTK mAbs or appropriate isotype control and LPS (InvivoGen) at 5 ng/mL overnight at 37°C and 6% CO2. TNF-a production from cell free supernatants was measured by ELISA (R&D Systems).
[00433] As shown in Figures 7A and 7B, addition of Ab2000 to dendritic cells stimulated with LPS or resiquimod (R848) promoted an increase in TNF-a release relative to addition of LPS or R848 alone, consistent with the role of Ab2000 as a MerTK inhibitor as demonstrated in phosphorylation assays. Addition of Ab2000 without TLR agonists did not induce meaningful TNF-a release. Figure 8 demonstrates that titration of Ab2000 resulted in a dose dependent increase in TNF-a secretion in dendritic cells with nanomolar potency. 6.7 Example 7: Affinity maturation of Ab2000 antibody
[00434] The Ab2000 antibody was affinity matured using methods as described in Holland et al., 2013, J. Immun. Methods 394:55-61.
[00435] Purified affinity matured and parental scFvs were tested for binding to purified MerTK-ECD by ELISA, and to L cells expressing human MerTK (MerTK-L cells) by flow cytometry. MerTK-L cells are described in Example 2. Affinity matured variants that demonstrated enhanced binding in both assays were converted to full human IgGl for further analysis.
[00436] Sequences for Ab2000 and three affinity matured variants are shown in Table 16. Single point mutations were uncovered in the VH of Ab2000-Fl (SEQ ID NO: 25) and the VL of Ab2000-G6 (SEQ ID NO: 26). Ab2000-A7 encodes both the point mutation in VH (SEQ ID NO: 25) and the point mutation in VL (SEQ ID NO: 26) and was uncovered de novo from the affinity maturation.
Table 16. VL and VH Amino Acid Sequences and VL CDR and VH CDR Amino Acid
Sequences for Anti-MerTK Antibodies
Figure imgf000118_0001
AKDRQWLLPR (SEQ ID NO:
GFTFSSYA AFDI 1)
(SEQ ID NO: ISGSGGST (SEQ ID NO:
IMGT 41) (SEQ ID NO: 42) 43)
KSSQSVLYSS NNKNYLA WASTREF QQYYTTPLT
Kabat (SEQ ID NO: 6) (SEQ ID NO: 7) (SEQ ID NO: 8) DIVMTQSP
DSLAVSLG
KSSQSVLYSS
ERATINCKS NNKNYLA QQYYTTPLT
(SEQ ID NO: WASTREF SQSVLYSSN
(SEQ ID NO:
NKNYLAW
Chothia 16) (SEQ ID NO: 17) 18)
YQQKPGQP
KSSQSVLYSS
PKLLIYWAS NNKNYLA QQYYTTPLT
A2000 TREFGVPD
(SEQ ID NO: WASTREF (SEQ ID NO:
VL RFSGSGSGT
AbM 22) (SEQ ID NO: 23) 24)
DFTLTISSL
LYSSNNKNYL
QAEDVAVY AWY QQYYTTPL
FCQQYYTT
(SEQ ID NO: LLIYWASTRE (SEQ ID NO: PLTFGQGT
Contact 36) (SEQ ID NO: 37) 38)
KLEIK
QSVLYSSNNK (SEQ ID NO: NY QQYYTTPLT
2)
(SEQ ID NO: WAS (SEQ ID NO:
IMGT 44) (SEQ ID NO: 45) 46)
DRQWLLPRVF
AISGSGGSTYY DI
SYAMS ADSVKG (SEQ ID NO: EVQLLESG
Kabat (SEQ ID NO: 3) (SEQ ID NO: 4) 27) GGLVQPGG
SLRLSCAAS
DRQWLLPRVF
GFTFSSYA
GFTFSSY DI
MSWVRQAP
(SEQ ID NO: SGSGGS (SEQ ID NO:
GKGLEWVS
Chothia 13) (SEQ ID NO: 14) 28)
AISGSGGST
DRQWLLPRVF
A2000 YY ADSVKG
GFTFS SYAMS DI
-A7 RFTISRDNS
(SEQ ID NO: AISGSGGSTY (SEQ ID NO:
VH KNTLYLQM
AbM 19) (SEQ ID NO: 20) 29)
NSLRAEDT
AKDRQWLLPR
AVYYCAKD
S SYAMS WVSAISGSGGS VFD
RQWLLPRV
(SEQ ID NO: TY (SEQ ID NO:
FDIWGQGT
Contact 33) (SEQ ID NO: 34) 39)
TVTVSS
AKDRQWLLPR (SEQ ID NO:
GFTFSSYA VFDI 25)
(SEQ ID NO: ISGSGGST (SEQ ID NO:
IMGT 41) (SEQ ID NO: 42) 47)
KSSQSVLYSG WASTREF QQYYTTPLT DIVMTQSP
Kabat NNKNYLA (SEQ ID NO: 7) (SEQ ID NO: 8) DSLAVSLG
Figure imgf000120_0001
KSSQSVLYSS TREFGVPD
NNKNYLA QQYYTTPLT RFSGSGSGT
(SEQ ID NO: WASTREF (SEQ ID NO: DFTLTISSL
AbM 22) (SEQ ID NO: 23) 24) QAEDVAVY
LYSSNNKNYL FCQQYYTT
AWY QQYYTTPL PLTFGQGT
(SEQ ID NO: LLIYWASTRE (SEQ ID NO: KLEIK
Contact 36) (SEQ ID NO: 37) 38) (SEQ ID NO:
QSVLYSSNNK 2)
NY QQYYTTPLT
(SEQ ID NO: WAS (SEQ ID NO:
IMGT 44) (SEQ ID NO: 45) 46)
AISGSGGSTYY DRQWLLPRAF
SYAMS ADSVKG DI EVQLLESG
Kabat (SEQ ID NO: 3) (SEQ ID NO: 4) (SEQ ID NO: 5) GGLVQPGG
DRQWLLPRAF SLRLSCAAS
GFTFSSY DI GFTFS SYA
(SEQ ID NO: SGSGGS (SEQ ID NO: MSWVRQAP
Chothia 13) (SEQ ID NO: 14) 15) GKGLEWVS
DRQWLLPRAF AISGSGGST
A2000 GFTFS SYAMS DI YY ADSVKG
G6 (SEQ ID NO: AISGSGGSTY (SEQ ID NO: RFTISRDNS
VH AbM 19) (SEQ ID NO: 20) 21) KNTLYLQM
AKDRQWLLPR NSLRAEDT
S SYAMS WVSAISGSGGS AFD AVYYCAKD
(SEQ ID NO: TY (SEQ ID NO: RQWLLPRA
Contact 33) (SEQ ID NO: 34) 35) FDIWGQGT
AKDRQWLLPR TVTVSS
GFTFS SYA AFDI (SEQ ID NO:
(SEQ ID NO: ISGSGGST (SEQ ID NO: 1)
IMGT 41) (SEQ ID NO: 42) 43)
KSSQSVLYSG NNKNYLA DIVMTQSP
(SEQ ID NO: WASTREF QQYYTTPLT DSLAVSLG
Kabat 30) (SEQ ID NO: 7) (SEQ ID NO: 8) ERATINCKS
SQSVLYSG
KSSQSVLYSG
NNKNYLA NNKNYLA QQYYTTPLT
WYQQKPG
A2000 (SEQ ID NO: WASTREF (SEQ ID NO:
QPPKLLIYW
G6 Chothia 3 D (SEQ ID NO: 17) 18)
ASTREFGVP
VL KSSQSVLYSG
DRFSGSGSG NNKNYLA QQYYTTPLT
TDFTLTISSL
(SEQ ID NO: WASTREF (SEQ ID NO:
QAEDVAVY
AbM 32) (SEQ ID NO: 23) 24)
FCQQYYTT
QQYYTTPL PLTFGQGT
LYSGNNKNYL LLIYWASTRE (SEQ ID NO: KLEIK
Contact AWY (SEQ ID NO: 37) 38) (SEQ ID NO: (SEQ ID NO:
40) 26)
QSVLYSGNNK NY QQYYTTPLT
(SEQ ID NO: WAS (SEQ ID NO:
IMGT 48) (SEQ ID NO: 45) 46)
6.8 Example 8: Binding of affinity matured antibodies to human MerTK
6.8.1 Binding to purified MerTK-ECD
[00437] The Ab2000 parental antibody and the three affinity matured antibodies (Ab2000-A7, Ab2000-Fl and Ab2000-G6) were assayed for their ability to bind to purified MerTK-ECD by ELISA. Microtiter plates were coated with recombinant human MerTK-msFc in PBS, and then blocked with 5% bovine serum albumin in PBS. Protein A purified human mAbs and an isotype control (hulgGl) were added at indicated concentrations and incubated at 37°C (Figure 9). The plates were washed with PBS/Tween and then incubated with a goat-anti-human IgG Fc-specific polyclonal reagent conjugated to horseradish peroxidase at 37°C. After washing, the plates were developed with HRP substrate, and analyzed at OD 450-650 using a microtiter plate reader. As shown in Figures 9 and 11, the three affinity matured antibodies showed superior binding to purified MerTK-ECD compared to parental A2000 antibody. Figure 11 depicts representative binding curves of the A2000-A7 affinity matured antibody. "Gl" and "G2" signify IgGl and IgG2 antibodies. Similar results were observed for Ab2000-A7 IgGl and IgG2 antibodies ("Gl" and "G2," respectively). Unless otherwise noted, antibodies are human IgGl .
6.8.2 Binding to MerTK-expressing cells
[00438] The ability of anti -MerTK human mAbs to bind to MerTK on cells expressing human MerTK was investigated by flow cytometry. Antibodies were tested for binding to human cell lines expressing human MerTK on their surface, huMerTK-L cells and SK-mel-5 melanoma cells (ATCC HTB-70).
[00439] Protein A purified human mAbs were incubated with MerTK-L cells and SK-mel-5 cells at room temperature on a plate shaker. After 20 minutes, the cells were washed with PBS containing 0.1% BSA and 0.05% NaN3 (PBA) and the bound antibodies were detected by incubating the cells with a PE labeled goat anti-human IgG Fc-specific probe. The excess probe was washed from the cells with PBA and the cell associated fluorescence was determined by analysis using a FACSCanto II™ instrument (BD Biosciences, NJ, USA) according to the manufacturer's directions.
[00440] Affinity matured antibodies showed increased binding to huMerTK-L cells (Figure 10A) and SK-mel-5 cells (Figure 10B) compared to parental antibody. The human mAbs demonstrated high level binding to cells expressing human MerTK as a function of antibody concentration. Figure 12A and 12B depict representative binding curves of the A2000-A7 affinity matured antibody in these cells. Similar results were observed for Ab2000-A7 IgGl and IgG2 antibodies ("Gl" and "G2," respectively). Controls were hulgGl and hulgG2 isotype control antibodies.
6.9 Example 9: Modulation of pro-inflammatory cytokine release from human dendritic cells by affinity matured anti-MerTK monoclonal antibodies
[00441] Dendritic cell differentiation and cytokine release: Ab2000-A7 was tested for its ability to modulate cytokine production in dendritic cells. To derive dendritic cells from human monocytes, PBMCs were added to a T175 cm2 flasks and monocytes were allowed to adhere for approximately 2 hours at 37°C, 6% CO2. The non-adherent cells were removed and the monocytes were cultured for 7 days in RPMI containing 10% FBS, 10 ng/mL IL-4 (R&D Systems) and 100 ng/mL GM-CSF (R&D Systems). The cells were harvested, washed in RPMI (no serum) and counted.
[00442] The cells were then incubated in the presence of anti-MerTK antibody or appropriate isotype control overnight at 37°C, 6% CO2. All reagents were prepared in RPMI (no serum). The supernatant was collected, clarified and either used immediately or stored frozen for cytokine analysis.
[00443] Production of IL-1RA (interleukin 1 receptor antagonist) and TNF-alpha was evaluated by ELISA (R&D Systems). Results were expressed as fold increase in cytokine production over isotype control. Compared to parental Ab2000 antibody, Ab2000-A7 resulted in an increase in production of IL-1RA (Figure 13A and 13B) and TNF-alpha (Figure 14) in dendritic cells.
[00444] Dendritic cells were analyzed by Eve Technologies (Alberta, Canada) using multiplex assay for 30 different cytokines, chemokines and growth factors, including fibroblast growth factor 2 (FGF-2), eotaxin-1 (CCL11), transforming growth factor alpha (TGF-a) granulocyte- colony stimulating factor (G-CSF), Fms-related tyrosine kinase 3 ligand (Flt-3L), granulocyte macrophage-colony stimulating factor (GM-CSF), fractalkine (CX3CL1), interferon alpha-2 (IFN-a2), interferon-gamma (IFN-γ), growth-regulated oncogene alpha (GRO alpha), interleukin-10 (IL-10), monocyte chemotactic protein 3 (MCP-3), interleukin-12 p40 (IL-12P40), macrophage-derived chemokine (MDC), platelet-derived growth factor AA homodimer (PDGF- AA), interleukin-13, platelet-derived growth factor BB homodimer (PDGF-BB), soluble CD40 ligand (sCD40L), interleukin-1 receptor antagonist (IL-IRA), IL-IB, IL-4, IL-6, IL-8, interferon γ-induced protein 10 (IP- 10), macrophage inflammatory protein (ΜΙΡ)-Ια, ΜΙΡ-Ιβ, Regulated on Activation Normal T cell Expressed and Secreted (RANTES), tumor necrosis factor (TNF)-a, and vascular endothelial growth factor A (VEGF-A), and IL-18. The fold-increase over human IgGl is depicted in Figure 15.
[00445] Most of the screened factors (i.e., cytokines, chemokines and growth factors) showed an increase in production in response to Ab2000-A7, with IL-8, MIP-la and TNF-alpha exhibiting the greatest increase in production (Figure 15). Data were normalized to cytokine levels from control human IgGl -treated DCs. The results indicate that mAb A2000-A7 can elicit pro-inflammatory cytokine secretion in dentritic cells in a MerTK-dependent manner.
6.9.1 Cytokine Production in Human PBMCs
[00446] Ab2000-A7 was tested for its ability to modulate cytokine production in peripheral blood mononuclear cells (PBMCs). PBMCs were isolated from donor whole blood following manufacturer's protocol (StemCell Technologies). Purified PBMCs were plated at l .OxlO6 cells/well in a 24-well plate and treated with 100 nM monoclonal antibody for 24 hours.
Following 24 hour treatment, cell-free supernatants were collected and stored at -80°C prior to assessing cytokine secretion. Multiplex analysis was carried out by Eve Technologies (Alberta, CA), and data were normalized to cytokine levels from control human IgGl -treated PBMCs. Mean cytokine increases over control from 4 independent donors are shown in Figure 16. In PBMCs, IL-IRA, IP-10, and MIP-la showed the greatest fold increase in production (Figure 16). These results indicate that the mAb A2000-A7 can elicit pro-inflammatory cytokine secretion in PBMCs in a MerTK-dependent manner. The lower cytokine activity observed in PBMCs relative to DCs may relate to the relatively low abundance of CD14+ cells in PBMCs, as well as their undifferentiated state, suggesting that MerTK may play a more prominent role in DCs and macrophages than in undifferentiated monocytes.
6.9.2 CD14+ Cell depletion ablates IL-IRA Secretion in mAb-treated PBMCs
[00447] It was determined whether IL-IRA production by PBMCs was dependent on the presence of CD14+ monocytes. Peripheral blood mononuclear cells (PBMCs) were isolated from donor whole blood following manufacturer's protocol (StemCell Technologies). CD14+ monocytes were depleted from purified PBMCs using CD 14+ microbeads following
manufacturer's protocol (Miltenyi Biotec). PBMCs depleted of CD14+ monocytes were plated at l .OxlO6 cells/well in a 24-well plate and treated with 100 nM monoclonal antibody for 24 hours. Following 24 hour treatment, cell-free supernatants were collected and stored at - 80°C prior to assessing cytokine IL-IRA secretion by ELISA (R&D Systems). Mean IL-IRA levels in intact or CD14+ depleted PBMCs from 3 different donors are shown in Figure 17. CD14+ cell depletion abolishes IL-IRA secretion by Ab2000-A7 (Figure 17). These data demonstrate that IL-IRA is produced chiefly by CD14+ cells, which also express higher MerTK levels than T, B or K cells in PBMCs from the same donors.
6.9.3 Cytokine Production in Human Macrophages
[00448] Macrophages were derived from human monocytes as follows: PBMCs were added to a T175 cm2 flasks and monocytes allowed to adhere for approximately 2 hours at 37°C, 6% CO2. The non-adherent cells were removed and the monocytes cultured for 7 days in RPMI containing 10% FBS and 50ng/mL MCSF (R&D Systems). The cells were harvested and frozen for future use.
[00449] On the day of the experiment, the cells were thawed, washed twice in RPMI and counted. The cells were then incubated in the presence of anti-MerTK antibody or appropriate isotype control overnight at 37°C, 6% CO2. All reagents were prepared in RPMI (no serum). The supernatant was collected, clarified and either used immediately or stored frozen for cytokine analysis. As shown in Fig. 18, production of TNF-alpha was evaluated by ELISA (R&D Systems). A2000-A7 results in an increase in TNF-alpha in macrophages. These data indicate that targeting MerTK with mAb A2000-A7 can induce cytokine secretion to different extents from various immune cell types, indicating a differential contribution of MerTK to immune modulation. 6.10 Example 10: Determination of affinity and rate constants of human affinity matured mAbs by Bio-Layer Interferometry (BLI)
[00450] Binding affinity and binding kinetics of various human anti-MerTK antibodies were examined by bio-layer interferometry (BLI) using an Octet™ QKe instrument (Pall ForteBio, Menlo Park, CA) according to the manufacturer's guidelines.
[00451] Purified antibodies were captured on Anti -Human Fc Capture (AHC) biosensors (Fortebio Product No. 18-5060). Each antibody was prepared in dilution buffer (lOmM Ρ04, 150mM NaCl, lmg/mL BSA, 0.05% Tween 20, pH 7.2) to ^g/mL and loaded on freshly hydrated and conditioned AHC biosensors for 180 seconds at 30°C and lOOOrpm plate shake speed to achieve a target response of l .Onm. For one assay, eight biosensors were loaded with the same antibody.
[00452] Binding was determined by exposing six of the antibody -loaded biosensors to soluble human MerTK-CD4-Biotin-HIS analyte (Celldex, 77.5kD by SDS-PAGE). Affinity
measurements were determined using 2-fold serial dilutions of analyte ranging from 200 to 6.25nM in dilution buffer at 30°C and lOOOrpm plate shake speed. Association of the antibody- loaded biosensors in analyte wells was carried out for 180 seconds, the biosensors were then moved to dilution buffer wells for 900 seconds for dissociation measurements.
[00453] Corresponding controls were conducted in each case by keeping the two remaining biosensors with captured antibody in dilution buffer wells for association and dissociation steps. The data for the control biosensors was used to subtract background and account for biosensor drift and antibody dissociation from the biosensors.
[00454] Fortebio' s Data Analysis Software version 11.0.0.4 (Pall ForteBio, Menlo Park, CA) was used in each case to derive kinetic parameters from the concentration series of analyte in dilution buffer binding to captured antibody. The association and dissociation curves were fitted to a 1 : 1 binding model using the data analysis software according to the manufacturer's guidelines.
[00455] The affinity and kinetic parameters (with background subtracted) as determined are shown in Table 17, where k0n = rate of association, kdis = rate of dissociation, and KD = affinity constant, determined by the ratio kdis/k0n
[00456] The results showed that A2000-A7 antibody (both IgGl and IgG2) binds to human MerTK with a greater affinity than parental A2000 antibody. Table 17. Affinity to human MerTK by Octet
Figure imgf000127_0001

Claims

WHAT IS CLAIMED:
1. An isolated antibody, or an antigen-binding fragment thereof, which specifically binds to human MerTK, comprising:
(i) a light chain variable region (VL) comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 2; and/or
(ii) a heavy chain variable region (VH) comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 1.
2. The antibody or an antigen-binding fragment of claim 1, comprising:
(i) a light chain variable region (VL) comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 2; and
(ii) a heavy chain variable region (VH) comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 1.
3. The antibody or antigen-binding fragment of claim 1 or 2, wherein the VL comprises a VL complementarity determining region 1 (CDRl), VL CDR2, and VL CDR3, comprising the amino acid sequences of SEQ ID NOS: 6, 7 and 8, respectively, or conservative sequence modifications thereof, or SEQ ID NOS: 30, 7 and 8, respectively, or conservative sequence modifications thereof.
4. The antibody or antigen-binding fragment of any one of claims 1 to 3, wherein the VL CDRl, VL CDR2, and VL CDR3 comprise the amino acid sequences of SEQ ID NOS: 6, 7 and 8, respectively, or SEQ ID NOS: 30, 7 and 8, respectively.
5. The antibody or antigen-binding fragment of any one of claims 1 to 4, wherein the VL comprises at least 80% amino acid sequence identity to SEQ ID NO: 2.
6. The antibody or antigen-binding fragment of any one of claims 1 to 4, wherein the VL comprises at least 85% amino acid sequence identity to SEQ ID NO: 2.
7. The antibody or antigen-binding fragment of any one of claim 1 to 4, wherein the VL comprises at least 90% amino acid sequence identity to SEQ ID NO: 2.
8. The antibody or antigen-binding fragment of any one of claim 1 to 4, wherein the VL comprises at least 95% amino acid sequence identity to SEQ ID NO: 2.
9. The antibody or antigen-binding fragment of any one of claim 1 to 4, wherein the VL comprises at least 98% amino acid sequence identity to SEQ ID NO: 2.
10. The antibody or antigen-binding fragment of claim 1 or 2, wherein the VL comprises SEQ ID NO: 2 or SEQ ID NO: 26.
11. The antibody or antigen-binding fragment of any one of claims 1 to 10, wherein the VH comprises a VH complementarity determining region 1 (CDRl), VH CDR2, and VH CDR3, comprising the amino acid sequences of SEQ ID NOS: 3, 4 and 5, respectively, or conservative sequence modifications thereof, or SEQ ID NOS: 3, 4 and 27, respectively, or conservative sequence modifications thereof.
12. The antibody or antigen-binding fragment of claim 11, wherein the VH CDRl, VH CDR2, and VH CDR3 comprise the amino acid sequences of SEQ ID NOS: 3, 4 and 5, respectively, or SEQ ID NOS: 3, 4 and 27, respectively.
13. The antibody or antigen-binding fragment of any one of claims 1 to 12, wherein the VH comprises at least 80% amino acid sequence identity to SEQ ID NO: 1.
14. The antibody or antigen-binding fragment of any one of claims 1 to 12, wherein the VH comprises at least 85% amino acid sequence identity to SEQ ID NO: 1.
15. The antibody or antigen-binding fragment of any one of claims 1 to 12, wherein the VH comprises at least 90% amino acid sequence identity to SEQ ID NO: 1.
16. The antibody or antigen-binding fragment of any one of claims 1 to 12, wherein the VH comprises at least 95% amino acid sequence identity to SEQ ID NO: 1.
17. The antibody or antigen-binding fragment of any one of claims 1 to 3, wherein the VH comprises at least 98% amino acid sequence identity to SEQ ID NO: 1.
18. The antibody or antigen-binding fragment of claims 1 or 2, wherein the VH comprises SEQ ID NO: 1 or SEQ ID NO: 25.
19. An isolated antibody, or an antigen-binding fragment thereof, which specifically binds to human MerTK, comprising:
(i) a light chain variable region (VL) comprising VL complementarity determining region 1 (CDRl), VL CDR2, and VL CDR3, wherein the VL CDRl, VL CDR2, and VL CDR3 comprise the amino acid sequences of SEQ ID NOS: 6, 7 and 8, respectively, or conservative sequence modifications thereof, or SEQ ID NOS: 30, 7 and 8, respectively, or conservative sequence modifications thereof; and/or
(ii) a heavy chain variable region (VH) comprising VH complementarity determining region 1 (CDRl), VH CDR2, and VH CDR3, wherein the VH CDRl, VH CDR2, and VH CDR3 comprise the amino acid sequences of SEQ ID NOS: 3, 4 and 5 respectively or conservative sequence modifications thereof, or SEQ ID NOS: 3, 4 and 27, respectively, or conservative sequence modifications thereof.
20. The antibody or antigen-binding fragment of claim 19, comprising:
(i) a light chain variable region (VL) comprising VL complementarity determining region 1 (CDRl), VL CDR2, and VL CDR3, wherein the VL CDRl, VL CDR2, and VL CDR3 comprise the amino acid sequences of SEQ ID NOS: 6, 7 and 8, respectively, or conservative sequence modifications thereof, or SEQ ID NOS: 30, 7 and 8, respectively, or conservative sequence modifications thereof; and
(ii) a heavy chain variable region (VH) comprising VH complementarity determining region 1 (CDRl), VH CDR2, and VH CDR3, wherein the VH CDRl, VH CDR2, and VH CDR3 comprise the amino acid sequences of SEQ ID NOS: 3, 4 and 5, respectively, or conservative sequence modifications thereof, or SEQ ID NOS: 3, 4 and 27, respectively, or conservative sequence modifications thereof.
21. The antibody or antigen-binding fragment of claim 19, comprising:
(i) a light chain variable region (VL) comprising VL complementarity determining region 1 (CDRl), VL CDR2, and VL CDR3, wherein the VL CDRl, VL CDR2, and VL CDR3 comprise the amino acid sequences of SEQ ID NOS: 6, 7 and 8, respectively, or SEQ ID NOS: 30, 7 and 8, respectively; and/or
(ii) a heavy chain variable region (VH) comprising VH complementarity determining region 1 (CDRl), VH CDR2, and VH CDR3, wherein the VH CDRl, VH CDR2, and VH CDR3 comprise the amino acid sequences of SEQ ID NOS: 3, 4 and 5, respectively, or SEQ ID NOS: 3, 4 and 27, respectively.
22. The antibody or antigen-binding fragment of claim 20, comprising:
(i) a light chain variable region (VL) comprising VL complementarity determining region 1 (CDRl), VL CDR2, and VL CDR3, wherein the VL CDRl, VL CDR2, and VL CDR3 comprise the amino acid sequences of SEQ ID NOS: 6, 7 and 8, respectively, or SEQ ID NOS: 30, 7 and 8, respectively; and
(ii) a heavy chain variable region (VH) comprising VH complementarity determining region 1 (CDRl), VH CDR2, and VH CDR3, wherein the VH CDRl, VH CDR2, and VH CDR3 comprise the amino acid sequences of SEQ ID NOS: 3, 4 and 5 respectively, or SEQ ID NOS: 3, 4 and 27, respectively.
23. An isolated antibody, or an antigen-binding fragment thereof, which specifically binds to human MerTK, comprising:
(i) a light chain variable region (VL) comprising VL complementarity determining region 1 (CDRl), VL CDR2, and VL CDR3, wherein the VL CDRl, VL CDR2, and VL CDR3 comprise the amino acid sequences of SEQ ID NOS: 16, 17 and 18, respectively, or conservative sequence modifications thereof or SEQ ID NOS: 31, 17 and 18,
respectively, or conservative sequence modifications thereof; and/or
(ii) a heavy chain variable region (VH) comprising VH complementarity determining region 1 (CDRl), VH CDR2, and VH CDR3, wherein the VH CDRl, VH CDR2, and VH CDR3 comprise the amino acid sequences of SEQ ID NOS: 13, 14 and 15 respectively, or conservative sequence modifications thereof or SEQ ID NOS: 13, 14 and 28, respectively, or conservative sequence modifications thereof.
24. The antibody or antigen-binding fragment of claim 23, wherein the VL CDRl, VL CDR2, and VL CDR3 comprise the amino acid sequences of SEQ ID NOS: 16, 17, and 18, respectively, or sequences having at least 80% amino acid sequence identity thereto.
25. The antibody or antigen-binding fragment of claim 23, wherein the VH CDRl, VH CDR2, and VH CDR3 comprise the amino acid sequences of SEQ ID NOS: 13, 14, and 15, respectively, or sequences having at least 80% amino acid sequence identity thereto.
26. The antibody or an antigen-binding fragment of claim 23, wherein the VL CDRl, VL CDR2, and VL CDR3 comprise the amino acid sequences of SEQ ID NOS: 16, 17, and 18, respectively, or sequences having at least 80% amino acid sequence identity thereto, and the VH CDRl, VH CDR2, and VH CDR3 comprise the amino acid sequences of SEQ ID NOS: 13, 14, and 15, respectively, or sequences having at least 80% amino acid sequence identity thereto.
27. An isolated antibody, or an antigen-binding fragment thereof, which specifically binds to human MerTK, comprising:
(i) a light chain variable region (VL) comprising VL complementarity determining region 1 (CDRl), VL CDR2, and VL CDR3, wherein the VL CDRl, VL CDR2, and VL CDR3 comprise the amino acid sequences of SEQ ID NOS: 22, 23 and 24, respectively, or conservative sequence modifications thereof or SEQ ID NOS: 32, 23 and 24, respectively, or conservative sequence modifications thereof; and/or
(ii) a heavy chain variable region (VH) comprising VH complementarity determining region 1 (CDRl), VH CDR2, and VH CDR3, wherein the VH CDRl, VH CDR2, and VH CDR3 comprise the amino acid sequences of SEQ ID NOS: 19, 20 and 21 respectively, or conservative sequence modifications thereof or SEQ ID NOS: 19, 20 and 29, respectively, or conservative sequence modifications thereof.
28. The antibody or antigen-binding fragment of claim 27, wherein the VL CDRl, VL CDR2, and VL CDR3 comprise the amino acid sequences of SEQ ID NOS: 22, 23 and 24, respectively, or sequences having at least 80% amino acid sequence identity thereto.
29. The antibody or antigen-binding fragment of claim 27, wherein the VH CDRl, VH CDR2, and VH CDR3 comprise the amino acid sequences of SEQ ID NOS: 19, 20, and 21, respectively, or sequences having at least 80% amino acid sequence identity thereto.
30. The antibody or antigen-binding fragment of claim 27, wherein the VL CDRl, VL CDR2, and VL CDR3 comprise the amino acid sequences of SEQ ID NOS: 22, 23 and 24, respectively, or sequences having at least 80% amino acid sequence identity thereto, and the VH CDRl, VH CDR2, and VH CDR3 comprise the amino acid sequences of SEQ ID NOS: 19, 21, and 22, respectively or sequences having at least 80% amino acid sequence identity thereto.
31. An isolated antibody, or an antigen-binding fragment thereof, which specifically binds to human MerTK, comprising:
(i) a light chain variable region (VL) comprising VL complementary determining region 1 (CDRl), VL CDR2, and VL CDR3, wherein the VL CDRl, VL CDR2, and VL CDR3 comprise the amino acid sequences of SEQ ID NOS: 36, 37 and 38, respectively, or conservative sequence modifications thereof, or SEQ ID NOS: 40, 37 and 38, respectively, or conservative sequence modifications thereof; and/or
(ii) a heavy chain variable region (VH) comprising VH complementary determining region 1 (CDRl), VH CDR2, and VH CDR3, wherein the VH CDRl, VH CDR2, and VH CDR3 comprise the amino acid sequences of SEQ ID NOS: 33, 34 and 35 respectively, or conservative sequence modifications thereof or SEQ ID NOS: 33, 34 and 39, respectively, or conservative sequence modifications thereof.
32. The antibody or antigen-binding fragment of claim 32, wherein the VL CDRl, VL CDR2, and VL CDR3 comprise the amino acid sequences of SEQ ID NOS: 36, 37, and 38, respectively, or sequences having at least 80% amino acid sequence identity thereto.
33. The antibody or antigen-binding fragment of claim 32, wherein the VH CDRl, VH CDR2, and VH CDR3 comprise the amino acid sequences of SEQ ID NOS: 33, 34, and 35, respectively, or sequences having at least 80% amino acid sequence identity thereto.
34. The antibody or an antigen-binding fragment of claim 32, wherein the VL CDRl, VL CDR2, and VL CDR3 comprise the amino acid sequences of SEQ ID NOS: 36, 37, and 38, respectively, or sequences having at least 80% amino acid sequence identity thereto, and the VH CDRl, VH CDR2, and VH CDR3 comprise the amino acid sequences of SEQ ID NOS: 33, 34, and 35, respectively, or sequences having at least 80% amino acid sequence identity thereto.
35. An isolated antibody, or an antigen-binding fragment thereof, which specifically binds to human MerTK, comprising:
(i) a light chain variable region (VL) comprising VL complementary determining region 1 (CDRl), VL CDR2, and VL CDR3, wherein the VL CDRl, VL CDR2, and VL CDR3 comprise the amino acid sequences of SEQ ID NOS: 44, 45 and 46, respectively, or conservative sequence modifications thereof, or SEQ ID NOS: 48, 45 and 46, respectively, or conservative sequence modifications thereof; and/or
(ii) a heavy chain variable region (VH) comprising VH complementary determining region 1 (CDRl), VH CDR2, and VH CDR3, wherein the VH CDRl, VH CDR2, and VH CDR3 comprise the amino acid sequences of SEQ ID NOS: 41, 42, 43 respectively, or conservative sequence modifications thereof, or SEQ ID NOS: 41, 42, 47, respectively, or conservative sequence modifications thereof.
36. The antibody or antigen-binding fragment of claim 35, wherein the VL CDRl, VL CDR2, and VL CDR3 comprise the amino acid sequences of SEQ ID NOS: 44, 45 and 46, respectively, or sequences having at least 80% amino acid sequence identity thereto.
37. The antibody or antigen-binding fragment of claim 35, wherein the VH CDRl, VH CDR2, and VH CDR3 comprise the amino acid sequences of SEQ ID NOS: 41, 42, 43, respectively, or sequences having at least 80% amino acid sequence identity thereto.
38. The antibody or an antigen-binding fragment of claim 35, wherein the VL CDRl, VL CDR2, and VL CDR3 comprise the amino acid sequences of SEQ ID NOS: 44, 45 and 46, respectively, or sequences having at least 80% amino acid sequence identity thereto, and the VH CDRl, VH CDR2, and VH CDR3 comprise the amino acid sequences of SEQ ID NOS: 41, 42, 43, respectively, or sequences having at least 80% amino acid sequence identity thereto.
39. An isolated antibody, or an antigen-binding fragment thereof, which specifically binds to Domain 1 of human MerTK.
40. An isolated antibody, or an antigen-binding fragment thereof, which binds to the same epitope of human MerTK as the antibody of any one of claims 1 to 39.
41. An isolated antibody, or an antigen-binding fragment thereof, which competes for binding to human MerTK with the antibody of any one of claims 1 to 39.
42. An isolated antibody, or an antigen-binding fragment thereof, which binds to Domain 1 of human MerTK and inhibits binding of Gas6 to MerTK and/or inhibits MerTK
phosphorylation.
43. The antibody or antigen-binding fragment of any one of claims 1 to 42, wherein the antibody comprises a heavy chain constant region or a light chain constant region.
44. The antibody or antigen-binding fragment of claim 43, wherein the antibody comprises a heavy chain constant region and a light chain constant region.
45. The antibody or antigen-binding fragment of claim 43, wherein the antibody comprises a human heavy chain constant region or a human light chain constant region.
46. The antibody or antigen-binding fragment of claim 45, wherein the antibody comprises a human heavy chain constant region and a human light chain constant region.
47. The antibody or antigen-binding fragment of any one of claims 43 to 46, wherein the antibody or antigen-binding fragment is an IgGl antibody or antigen-binding fragment.
48. The antibody or antigen-binding fragment of claim 47, wherein the antibody or antigen- binding fragment is an IgGl antibody or antigen-binding fragment or a human IgG2 antibody or antigen-binding fragment.
49. The antibody or antigen-binding fragment of any one of claims 43 to 48, wherein the antibody comprises a kappa light chain constant region or a lambda light chain constant region.
50. The antibody or antigen-binding fragment of any one of claims 43 to 48, wherein the antibody comprises a human kappa light chain constant region or a human lambda light chain constant region.
51. The antibody or antigen-binding fragment of any one of claims 43 to 50, wherein the antibody comprises the amino acid sequence of SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53 SEQ ID NO: 55, SEQ ID NO: 56, or SEQ ID NO: 57.
52. The antibody or antigen-binding fragment of claim 51, wherein the antibody comprises the amino acid sequence of SEQ ID NO: 49 and the amino acid sequence of SEQ ID NO: 50; the amino acid sequence of SEQ ID NO: 49 and the amino acid sequence of SEQ ID NO: 51; the amino acid sequence of SEQ ID NO: 54 and the amino acid sequence of SEQ ID NO: 52; the amino acid sequence of SEQ ID NO: 54 and the amino acid sequence of SEQ ID NO: 53; the amino acid sequence of SEQ ID NO: 54 and the amino acid sequence of SEQ ID NO: 55; the amino acid sequence of SEQ ID NO: 54 and the amino acid sequence of SEQ ID NO: 56; the amino acid sequence of SEQ ID NO: 57 and the amino acid sequence of SEQ ID NO: 52; the amino acid sequence of SEQ ID NO: 57 and the amino acid sequence of SEQ ID NO: 53; the amino acid sequence of SEQ ID NO: 57 and the amino acid sequence of SEQ ID NO: 55; or the amino acid sequence of SEQ ID NO: 57 and the amino acid sequence of SEQ ID NO: 56.
53. The antibody or antigen-binding fragment of any one of claims 1 to 52, which is a monoclonal antibody.
54. The antibody or antigen-binding fragment of any one of claims 1 to 53, which is a bispecific antibody.
55. The antibody or antigen-binding fragment of any one of claims 1 to 54, which is fused to a heterologous polypeptide.
56. The antibody or antigen-binding fragment of any one of claims 1 to 54, which is conjugated to an agent.
57. The antibody or antigen-binding fragment of claim 56, wherein the agent is a toxin.
58. A composition comprising a therapeutically effective amount of the antibody or antigen- binding fragment of any one of claims 1 to 57.
59. A pharmaceutical composition comprising the antibody or antigen-binding fragment of any one of claims 1 to 57 and a pharmaceutically acceptable carrier.
60. A polynucleotide comprising nucleotide sequences encoding a VH chain region, a VL chain region, or both a VL chain region and a VH chain region, of an antibody or antigen- binding fragment of any one of claims 1 to 57.
61. A polynucleotide comprising nucleotide sequences encoding a heavy chain, a light chain, or both heavy chain and a light chain of an antibody or antigen-binding fragment of any one of claims 1 to 57.
62. A population of polynucleotides comprising (i) a first polynucleotide comprising nucleotide sequences encoding a VH or a heavy chain of the antibody or antigen-binding fragment of any one of claims 1 to 57 and (ii) a second polypeptide comprising nucleotide sequences encoding a VL or a light chain of the antibody or antigen-binding fragment of any one of claims 1 to 57.
63. A vector comprising the polynucleotide of claim 60 or 61.
64. A population of vectors comprising (i) a first vector comprising nucleotide sequences encoding a VH or a heavy chain of the antibody or antigen-binding fragment of any one of claims 1 to 57, and (ii) a second vector comprising nucleotide sequences encoding a VL or a light chain of the antibody or antigen-binding fragment of any one of claims 1 to 57.
65. An isolated cell comprising the polynucleotide of claim 60 or 61.
66. An isolated cell comprising the population of polynucleotides of claim 64.
67. A population of cells comprising (i) a first host cell comprising a polynucleotide comprising nucleotide sequences encoding a VH or a heavy chain of the antibody or antigen- binding fragment of any one of claims 1 to 57, and (ii) a second host cell comprising a polynucleotide comprising nucleotide sequences encoding a VL or a light chain of the antibody or antigen-binding fragment of any one of claims 1 to 57.
68. An isolated cell producing the antibody or antigen-binding fragment of any one of claims 1 to 57.
69. A kit comprising the antibody or antigen-binding fragment of any one of claims 1 to 57.
70. A method of managing, protecting against, or treating cancer in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of the antibody or antigen-binding fragment of any one of claims 1 to 57.
71. A method of enhancing an immune response in a subject comprising administering to a subject in need thereof an effective amount of the antibody or antigen-binding fragment of any one of claims 1 to 57.
72. The method of claim 71, wherein the subject is an immunocompromised subject.
73. The method of claim 72, wherein the immunocompromised subject is suffering from an infection, has cancer, is undergoing, or had undergone treatment with, an anti-cancer therapy, is HIV positive, or has AIDS or SCID, or diabetes, or has had a transplant and is taking
immunosuppressants.
74. The method of claim 73, wherein the subject has been treated with an
immunosuppressant.
75. A method of enhancing an immune response to a vaccine in a subject, comprising administering to a subject in need thereof, who is or has been administered the vaccine, an effective amount of the antibody or antigen-binding fragment of any one of claims 1 to 57.
76. The method of claim 75, wherein the vaccine is a cancer or tumor vaccine.
77. A method of managing, preventing, protecting against, or treating metastasis in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of the antibody or antigen-binding fragment of any one of claims 1 to 57.
78. A method of managing or treating sepsis in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of the antibody or antigen-binding fragment of any one of claims 1 to 57.
79. A method for activating or enhancing an innate immune response in a subject, comprising administering to a subject in need thereof an effective amount of the antibody or antigen-binding fragment of any one of claims 1 to 57.
80. The method of claim 79, wherein the subject has cancer, or is being treated for cancer with an anti-cancer therapeutic, or the subject has an infection.
81. A method of making an antibody or an antigen-binding fragment thereof which specifically binds to human MerTK, comprising culturing the cell or population of cells of any one of claims 65-68 to express the antibody or antigen-binding fragment.
82. A method of making an antibody or an antigen-binding fragment thereof, which specifically binds to human MerTK, comprising expressing the polynucleotide or population of polynucleotides of any one of claim 60-62.
83. A method of increasing proinflammatory cytokine production in a subject in need thereof, comprising administering to the subject an antibody or an antigen-binding fragment thereof that specifically binds to human MerTK such that the cytokine production is increased.
84. The method of claim 83, wherein the proinflammatory cytokine is TNFa (tumor necrosis factor a) or IL-IRA (interleukin- 1 receptor antagonist).
85. A method of increasing proinflammatory cytokine production in a subject in need thereof, comprising administering to the subject an antibody or an antigen-binding fragment thereof of any one of claims 1 to 57 such that the production of one or more cytokines is increased.
86. The method of claim 85, wherein the production of proinflammatory cytokine TNFa (tumor necrosis factor a) is increased.
87. The method of claim 86, wherein the production of proinflammatory cytokine IL-IRA (interleukin- 1 receptor antagonist) is increased.
88. The method of claim 86, wherein the production of proinflammatory cytokines TNFa and IL-IRA are increased.
89. A method of increasing proinflammatory secretory factor production in a subject in need thereof, comprising administering to the subject an antibody or an antigen-binding fragment thereof of any one of claims 1 to 49 such that the production of one or more proinflammatory secretory factors is increased.
90. The method of claim 89, wherein the production of one or more of proinflammatory secretory factors TNFa, IL-IRA, fibroblast growth factor 2 (FGF-2), eotaxin-1 (CCL11), transforming growth factor alpha (TGF-a) granulocyte-colony stimulating factor (G-CSF), Fms- related tyrosine kinase 3 ligand (Flt-3L), granulocyte macrophage-colony stimulating factor (GM-CSF), fractalkine (CX3CL1), interferon alpha-2 (IFN-a2), interferon-gamma (IFN-γ), growth-regulated oncogene alpha (GRO alpha), interleukin- 10 (IL-10), monocyte chemotactic protein 3 (MCP-3), interleukin- 12 p40 (IL-12P40), macrophage-derived chemokine (MDC), platelet-derived growth factor AA homodimer (PDGF-AA), interleukin- 13 (IL-13), platelet- derived growth factor BB homodimer (PDGF-BB), soluble CD40 ligand (sCD40L), interleukin- 1B (IL-IB), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-8 (IL-8), interferon γ-induced protein 10 (IP- 10), macrophage inflammatory protein (ΜΙΡ)-Ια, ΜΙΡ-Ιβ, Regulated on
Activation Normal T cell Expressed and Secreted (RANTES), vascular endothelial growth factor A (VEGF-A) or IL-18 is increased.
91. The method of any one of claims 70 to 90, wherein the antibody does not substantially induce phosphorylation of MerTK.
92. The method of any one of claims 70 to 91, wherein the antibody specifically binds to Domain 1 of human MerTK.
93. The method of any one of claims 70 to 92, wherein the antibody is an antibody of any one of claims 1 to 4.
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