WO2017096276A1 - Anticorps anti-gitr et procédés d'utilisation associés - Google Patents

Anticorps anti-gitr et procédés d'utilisation associés Download PDF

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Publication number
WO2017096276A1
WO2017096276A1 PCT/US2016/064786 US2016064786W WO2017096276A1 WO 2017096276 A1 WO2017096276 A1 WO 2017096276A1 US 2016064786 W US2016064786 W US 2016064786W WO 2017096276 A1 WO2017096276 A1 WO 2017096276A1
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Prior art keywords
antibody
antigen
heavy chain
seq
gitr
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PCT/US2016/064786
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English (en)
Inventor
Nicholas S. Wilson
Jeremy D. WAIGHT
Gerd Ritter
Takemasa Tsuji
Olivier LÉGER
Volker Seibert
David SCHAER
Taha MERGHOUB
Dennis J. Underwood
Ana M. Gonzalez
Marc VAN DIJK
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Agenus Inc.
Memorial Sloan-Kettering Cancer Center
Ludwig Institute For Cancer Research Ltd.
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Application filed by Agenus Inc., Memorial Sloan-Kettering Cancer Center, Ludwig Institute For Cancer Research Ltd. filed Critical Agenus Inc.
Publication of WO2017096276A1 publication Critical patent/WO2017096276A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • 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
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • 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

Definitions

  • the present disclosure relates to antibodies that specifically bind to human glucocorticoid-induced TNFR family related receptor (GITR), compositions comprising such antibodies, and methods of producing and using antibodies that specifically bind to GITR.
  • GITR human glucocorticoid-induced TNFR family related receptor
  • Glucocorticoid-induced TNFR-r elated protein a member of the TNFR superfamily, is expressed in many components of the innate and adaptive immune system and stimulates both acquired and innate immunity (Nocentini G et al, (1994) PNAS 94: 6216-6221; Hanabuchi S et al, (2006) Blood 107:3617-3623; Nocentini G & Riccardi C (2005) Eur J Immunol 35: 1016-1022; Nocentini G et al, (2007) Eur J Immunol 37: 1165-1169).
  • GITR Glucocorticoid-induced TNFR-r elated protein
  • GITRL mainly expressed on Antigen Presenting Cells (APCs), on endothelial cells, and also in tumor cells.
  • APCs Antigen Presenting Cells
  • the GITR/GITRL system participates in the development of autoimmune/inflammatory responses and potentiates response to infection and tumors.
  • treating animals with GITR-Fc fusion protein ameliorates autoimmune/inflammatory diseases while GITR triggering is effective in treating viral, bacterial, and parasitic infections, as well in boosting immune response against tumors (Nocentini G et al, (2012) Br J Pharmacol 165: 2089-99).
  • GITR activation increases resistance to tumors and viral infections, is involved in autoimmune/inflammatory processes and regulates leukocyte extravasation (Nocentini G & Riccardi C (2005) supra; Cuzzocrea S et al, (2004) J Leukoc Biol 76: 933-940; Shevach EM & Stephens GL (2006) Nat Rev Immunol 6: 613-618; Cuzzocrea S et al, (2006) J Immunol 177: 631-641; Cuzzocrea S et al, (2007) FASEB J 21 : 117-129).
  • GITR Human GITR is expressed at very low levels in peripheral (non-activated) T cells. After T cell activation, GITR is strongly up-regulated for several days in both CD4 + and CD8 + cells (Kwon B et al, (1999) J Biol Chem 274: 6056-6061; Gurney AL et al, (1999) Curr Biol 9: 215-218; Ronchetti S et al, (2004) supra; Shimizu J et al, (2002) supra; Ji HB et al, (2004) supra; Ronchetti S et al, (2002) Blood 100: 350-352; Li Z et al, (2003) J Autoimmun 21 : 83- 92), with CD4 + cells having a higher GITR expression than CD8 + cells (Kober J et al, (2008) Eur J Immunol 38(10): 2678-88; Bianchini R et al, (2011) Eur J Immunol 41(8): 2269-78).
  • GITR Given the role of human GITR in modulating immune responses, provided herein are antibodies that specifically bind to GITR and the use of those antibodies to modulate GITR activity.
  • GITR e.g., human GITR
  • an isolated antibody that specifically binds to human GITR comprises: (A) a first antigen-binding domain that specifically binds to human GITR; comprising: (i) a first heavy chain comprising (a) a first heavy chain constant region comprising a F405L or a K409R mutation, numbered according to the EU numbering system; and (b) a first heavy chain variable domain (VH) comprising a VH complementarity determining region (CDR) 1 comprising the amino acid sequence of XiYX 2 MX 3 (SEQ ID NO: 87), wherein Xi is D, E or G; X 2 is A or V, and X 3 is Y or H; a VH-CDR2 comprising the amino acid sequence of XiIX 2 TX 3 SGX 4 X 5 X 6 YNQKFX 7 X 8 (SEQ ID NO: 88), wherein X x is V or L, X 2 is R,
  • an isolated antibody that specifically binds to human GITR comprises:(A) a first antigen-binding domain that specifically binds to the same epitope of human GITR as an antibody comprising a VH comprising the amino acid sequence of SEQ ID NO: 18 and a VL comprising the amino acid sequence of SEQ ID NO: 19, wherein the first antigen-binding domain comprises a first heavy chain constant region comprising a F405L or a K409R mutation, numbered according to the EU numbering system; and (B) a second antigen- binding domain that does not bind to an antigen expressed by a human immune cell, comprising a second heavy chain constant region comprising a F405L or a K409R mutation, numbered according to the EU numbering system; wherein the first heavy chain constant region and the second heavy chain constant region contain different mutations.
  • an isolated antibody that specifically binds to human GITR wherein the antibody comprises:(A) a first antigen-binding domain that specifically binds to an epitope of human GITR comprising at least one amino acid in residues 60-63 of SEQ ID NO:41, wherein the first antigen-binding domain comprises a first heavy chain constant region comprising a F405L or a K409R mutation, numbered according to the EU numbering system; and (B) a second antigen-binding domain that does not bind to an antigen expressed by a human immune cell, comprising a second heavy chain constant region comprising a F405L or a K409R mutation, numbered according to the EU numbering system; wherein the first heavy chain constant region and the second heavy chain constant region contain different mutations.
  • an isolated antibody that specifically binds to human GITR comprises: (A) a first antigen-binding domain that specifically binds to each of i) human GITR comprising residues 26-241 of SEQ ID NO:41 and ii) a variant of cynomolgus GITR, the variant comprising residues 26-234 of SEQ ID NO:46, wherein the antibody does not specifically bind to cynomolgus GITR comprising residues 26-234 of SEQ ID NO:44, wherein the first antigen- binding domain comprises a first heavy chain constant region comprising a F405L or a K409R mutation, numbered according to the EU numbering system; and (B) a second antigen-binding domain that does not bind to an antigen expressed by a human immune cell, comprising a second heavy chain constant region comprising a F405L or a K409R mutation, numbered according to the EU numbering system; wherein the first heavy chain
  • an isolated antibody that specifically binds to human GITR comprises:(A) a first antigen-binding domain that specifically binds to human GITR and that exhibits, as compared to binding to a human GITR sequence of residues 26 to 241 of SEQ ID NO:41, reduced or absent binding to a protein identical to residues 26 to 241 of SEQ ID NO:41 except for the presence of a D60A or G63 A amino acid substitution, numbered according to SEQ ID NO:41, wherein the first antigen-binding domain comprises a first heavy chain constant region comprising a F405L or a K409R mutation, numbered according to the EU numbering system; and (B) a second antigen-binding domain that does not bind to an antigen expressed by a human immune cell, comprising a second heavy chain constant region comprising a F405L or a K409R mutation, numbered according to the EU numbering system; wherein the first heavy chain constant region and the second heavy chain
  • an isolated antibody that specifically binds to human GITR wherein the antibody comprises:(A) a first antigen-binding domain that specifically binds to human GITR and comprises a VH and a VL, wherein the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 20, 22, 24, and 25, wherein the first antigen-binding domain comprises a first heavy chain constant region comprising a F405L or a K409R mutation, numbered according to the EU numbering system; and (B) a second antigen-binding domain that does not bind to an antigen expressed by a human immune cell, comprising a second heavy chain constant region comprising a F405L or a K409R mutation, numbered according to the EU numbering system; wherein the first heavy chain constant region and the second heavy chain constant region contain different mutations.
  • an isolated antibody that specifically binds to human GITR comprises: (A) a first antigen-binding domain that specifically binds to human GITR and comprises a VH and a VL, wherein the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 21, 23, and 26, wherein the first antigen-binding domain comprises a first heavy chain constant region comprising a F405L or a K409R mutation, numbered according to the EU numbering system; and (B) a second antigen-binding domain that does not bind to an antigen expressed by a human immune cell, comprising a second heavy chain constant region comprising a F405L or a K409R mutation, numbered according to the EU numbering system; wherein the first heavy chain constant region and the second heavy chain constant region contain different mutations.
  • the second antigen-binding domain specifically binds to a tumor-associated antigen that is not expressed by normal cells. In one aspect, the second antigen-binding domain specifically binds to a non-human antigen. In one aspect, the second antigen-binding domain specifically binds to a viral antigen. In one aspect, the viral antigen is a HIV antigen. In one aspect, the second antigen-binding domain specifically binds to chicken albumin or hen egg lysozyme.
  • an isolated antibody that specifically binds to human GITR comprises:(A) an antigen-binding domain that specifically binds to human GITR; comprising: (i) a first heavy chain comprising (a) a first heavy chain constant region comprising a F405L or a K409R mutation, numbered according to the EU numbering system; and (b) a first heavy chain variable domain (VH) comprising a VH complementarity determining region (CDR) 1 comprising the amino acid sequence of XiYX 2 MX 3 (SEQ ID NO: 87), wherein Xi is D, E or G; X 2 is A or V, and X 3 is Y or H; a VH-CDR2 comprising the amino acid sequence of XiIX 2 TX 3 SGX4X5X6YNQKFX 7 X8 (SEQ ID NO: 88), wherein Xi is V or L, X 2 is R, K or Q, X
  • the second heavy chain further comprises a second heavy chain variable domain and wherein the isolated antibody that specifically binds to human OX40 further comprises a second light chain comprising a second light chain constant region and a second light chain variable region.
  • an isolated antibody that specifically binds to human GITR comprises: (A) an antigen-binding domain that specifically binds to the same epitope of human GITR as an antibody comprising a VH comprising the amino acid sequence of SEQ ID NO: 18 and a VL comprising the amino acid sequence of SEQ ID NO: 19, wherein the antigen-binding domain comprises a first heavy chain constant region comprising a F405L or a K409R mutation, numbered according to the EU numbering system; and (B) a heavy chain comprising a second heavy chain constant region comprising a F405L or a K409R mutation, or a fragment thereof comprising the F405L or K409R mutation, numbered according to the EU numbering system; wherein the first heavy chain constant region and the second heavy chain constant region or the fragment contain different mutations.
  • an isolated antibody that specifically binds to human GITR comprises: (A) a first antigen-binding domain that specifically binds to an epitope of human GITR comprising at least one amino acid in residues 60-63 of SEQ ID NO:41, wherein the first antigen-binding domain comprises a first heavy chain constant region comprising a F405L or a K409R mutation, numbered according to the EU numbering system; and (B) a heavy chain comprising a second IgGi heavy chain constant region comprising a F405L or a K409R mutation, or a fragment thereof comprising the F405L or K409R mutation, numbered according to the EU numbering system; wherein the first heavy chain constant region and the second heavy chain constant region or the fragment contain different mutations.
  • an isolated antibody that specifically binds to human GITR comprises: (A) a first antigen-binding domain that specifically binds to each of i) human GITR comprising residues 26-241 of SEQ ID NO:41 and ii) a variant of cynomolgus GITR, the variant comprising residues 26-234 of SEQ ID NO:46, wherein the antibody does not specifically bind to cynomolgus GITR comprising residues 26-234 of SEQ ID NO:44, wherein the first antigen- binding domain comprises a first heavy chain constant region comprising a F405L or a K409R mutation, numbered according to the EU numbering system; and (B) a heavy chain comprising a second heavy chain constant region comprising a F405L or a K409R mutation, or a fragment thereof comprising the F405L or K409R mutation, numbered according to the EU numbering system; wherein the first heavy chain constant region and the
  • an isolated antibody that specifically binds to human GITR wherein the antibody comprises:(A) a first antigen-binding domain that specifically binds to human GITR and that exhibits, as compared to binding to a human GITR sequence of residues 26 to 241 of SEQ ID NO:41, reduced or absent binding to a protein identical to residues 26 to 241 of SEQ ID NO:41 except for the presence of a D60A or G63A amino acid substitution, numbered according to SEQ ID NO:41, wherein the first antigen-binding domain comprises a first heavy chain constant region comprising a F405L or a K409R mutation, numbered according to the EU numbering system; and (B) a heavy chain comprising a second heavy chain constant region comprising a F405L or a K409R mutation, or a fragment thereof comprising the F405L or K409R mutation, numbered according to the EU numbering system; wherein the first heavy chain constant region and the second heavy
  • an isolated antibody that specifically binds to human GITR comprises: (A) a first antigen-binding domain that specifically binds to human GITR and comprises a VH and a VL, wherein the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 20, 22, 24, and 25, wherein the first antigen-binding domain comprises a first heavy chain constant region comprising a F405L or a K409R mutation, numbered according to the EU numbering system; and (B) a heavy chain comprising a second heavy chain constant region comprising a F405L or a K409R mutation, or a fragment thereof comprising the F405L or K409R mutation, numbered according to the EU numbering system; wherein the first heavy chain constant region and the second heavy chain constant region or the fragment contain different mutations.
  • an isolated antibody that specifically binds to human GITR comprising: (A) a first antigen-binding domain that specifically binds to human GITR and comprises a VH and a VL, wherein the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 21, 23, and 26, wherein the first antigen- binding domain comprises a first heavy chain constant region comprising a F405L or a K409R mutation, numbered according to the EU numbering system; and (B) a heavy chain comprising a second heavy chain constant region comprising a F405L or a K409R mutation, or a fragment thereof comprising the F405L or K409R mutation, numbered according to the EU numbering system; wherein the first heavy chain constant region and the second heavy chain constant region or the fragment contain different mutations.
  • the heavy chain further comprises a second heavy chain variable domain and wherein the isolated antibody that specifically binds to human OX40 further comprises a second light chain comprising a second light chain constant region and a second light chain variable region.
  • the antibody is a bispecific antibody.
  • the fragment of the heavy chain comprising a second heavy chain constant region is an Fc fragment.
  • the heavy chain comprising a second heavy chain constant region or fragment thereof is from an antigen-binding domain that specifically binds to a tumor-associated antigen that is not expressed by normal cells.
  • the heavy chain comprising a second heavy chain constant region or fragment thereof is from an antigen-binding domain that specifically binds to a non-human antigen.
  • the heavy chain comprising a second heavy chain constant region or fragment thereof is from an antigen-binding domain that specifically binds to a viral antigen.
  • the viral antigen is a HIV antigen.
  • the heavy chain comprising a second heavy chain constant region or fragment thereof is from an antigen-binding domain that specifically binds to chicken albumin or hen egg lysozyme.
  • the antigen-binding domain that specifically binds to human GITR comprises: (i) a VH-CDR1 comprising the amino acid sequence of XiYAMX 2 (SEQ ID NO: l), wherein X 1 is D, G, or E; and X 2 is Y or H; (ii) a VH-CDR2 comprising the amino acid sequence of X 1 IRTYSGX 2 VX3YNQKFX 4 X5 (SEQ ID NO: 2), wherein X x is V or L; X 2 is D or G; X 3 is T or S; X 4 is K, R, or Q; and X 5 is D, E, or G; (iii) a VH-CDR3 comprising the amino acid sequence of SGTVRGFAY (SEQ ID NO: 3);(iv) a VL-CDRl comprising the amino acid sequence of KSSQSLLNSXiNQKNYLT (SEQ ID NO:
  • the antigen-binding domain that specifically binds to human GITR comprises a VH-CDRl comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 7-9. In one aspect, the antigen-binding domain that specifically binds to human GITR comprises a VH-CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 10-13. In one aspect, the antigen-binding domain that specifically binds to human GITR comprises a VL-CDRl comprising the amino acid sequence of SEQ ID NO: 14 or 15. In one aspect, the antigen-binding domain that specifically binds to human GITR comprises a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 16 or 17.
  • the antigen-binding domain that specifically binds to human GITR comprises VH-CDRl, VH-CDR2, and VH-CDR3 sequences set forth in SEQ ID NOs: 7, 10, and 3; SEQ ID NOs: 8, 11, and 3; SEQ ID NOs: 9, 12, and 3; or SEQ ID NOs: 9, 13, and 3, respectively; and/or VL-CDRl, VL-CDR2, and VL-CDR3 sequences set forth in SEQ ID NOs: 14, 5, and 16; or SEQ ID NOs: 15, 5, and 17, respectively.
  • the antigen-binding domain that specifically binds to human GITR comprises VH-CDRl, VH-CDR2, VH-CDR3, VL-CDRl, VL-CDR2, and VL-CDR3 sequences set forth in SEQ ID NOs: 7, 10, 3, 14, 5, and 16, respectively.
  • the antigen-binding domain that specifically binds to human GITR comprises a VH comprising the set forth in SEQ ID NO:25. In one aspect, the antigen-binding domain that specifically binds to human GITR comprises a VH comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 20, 22, and 24. In one aspect, the antigen-binding domain that specifically binds to human GITR comprises a VH comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 20, 22, and 24. In one aspect, the antigen-binding domain that specifically binds to human GITR comprises a VH comprising the amino acid sequence of SEQ ID NO: 18.
  • the antigen-binding domain that specifically binds to human GITR comprises a VH comprising an amino acid sequence derived from a human IGHV3-73 germline sequence.
  • the antigen-binding domain that specifically binds to human GITR comprises a VL comprising the amino acid sequence of SEQ ID NO: 26. In one aspect, the antigen-binding domain that specifically binds to human GITR comprises a VL comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 21, and 23. In one aspect, the antigen-binding domain that specifically binds to human GITR comprises a VL comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 21, and 23. In one aspect, the antigen-binding domain that specifically binds to human GITR comprises a VL the amino acid sequence of SEQ ID NO: 19.
  • the antigen-binding domain that specifically binds to human GITR comprises a light chain comprising the amino acid sequence of SEQ ID NO: 37. In one aspect, the antigen-binding domain that specifically binds to human GITR comprises a light chain comprising the amino acid sequence of SEQ ID NO: 38.
  • the antigen-binding domain that specifically binds to human GITR comprises a VL comprising an amino acid sequence derived from a human IGKV2-28 germline sequence.
  • the antigen-binding domain that specifically binds to human GITR comprises VH and VL sequences set forth in SEQ ID NOs: 18 and 19, SEQ ID NOs: 20 and 21, SEQ ID NOs: 22 and 23, or SEQ ID NOs: 24 and 23, respectively.
  • the antigen- binding domain that specifically binds to human GITR comprises a VH comprising the sequence set forth in SEQ ID NO: 18 and a VL comprising the sequence set forth in SEQ ID NO: 19.
  • the first heavy chain constant region comprises a F405L mutation
  • the second heavy chain constant region comprises a K409R mutation, numbered according to the EU numbering system.
  • the first heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 108 and the second heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 109.
  • the first heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 135, and the second heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 136.
  • the antigen- binding domain that specifically binds to human GITR comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:31.
  • the antigen-binding domain that specifically binds to human GITR comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:76.
  • the first heavy chain constant region comprises a K409R mutation
  • the second heavy chain constant region comprises a F405L mutation, numbered according to the EU numbering system.
  • the first heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 109 and the second heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 108.
  • the first heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 136 and the second heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 135.
  • the antigen- binding domain that specifically binds to human GITR comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:34.
  • the antigen-binding domain that specifically binds to human GITR comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:79.
  • each heavy chain constant region is selected from the group consisting of immunoglobulins IgGi, IgG 2 , IgG 3 , IgG 4 , IgAi, and IgA 2 .
  • each heavy chain constant region is immunoglobulin IgGi.
  • the first heavy chain constant region and the second heavy chain constant region further comprise an identical mutation selected from the group consisting of a N297A, N297Q, D265A, L234F/L235E/D265A, and a combination thereof, numbered according to the EU numbering system.
  • the first heavy chain constant region and the second heavy chain constant region further comprise an identical mutation selected from the group consisting of D265A, P329A, and a combination thereof, numbered according to the EU numbering system.
  • the antibody is antagonistic to human GITR. In one aspect, the antibody deactivates, reduces, or inhibits an activity of human GITR. In one aspect, the antibody inhibits or reduces binding of human GITR to human GITR ligand. In one aspect, the antibody inhibits or reduces human GITR signaling. In one aspect, the antibody inhibits or reduces human GITR signaling induced by human GITR ligand. [0041] In one aspect, the antibody decreases CD4+ T cell proliferation induced by synovial fluid from rheumatoid arthritis patients. In one aspect, the antibody increases survival of NOG mice transplanted with human PBMCs. In one aspect, the antibody increases proliferation of regulatory T cells in a GVHD model.
  • the antibody further comprises a detectable label.
  • GITR e.g., human GITR
  • a pharmaceutical composition comprising an antibody that specifically binds to GITR (e.g., human GITR) provided herein and a pharmaceutically acceptable excipient.
  • a method of modulating an immune response in a subject comprising administering to the subject an effective amount of an antibody that specifically binds to GITR (e.g., human GITR) provided herein or a pharmaceutical composition provided herein.
  • modulating an immune response comprises reducing or inhibiting the immune response in the subject.
  • GITR e.g., human GITR
  • the disease or disorder is selected from the group consisting of: transplant rejection, graft-versus-host disease, vasculitis, asthma, rheumatoid arthritis, dermatitis, inflammatory bowel disease, uveitis, lupus, colitis, diabetes, multiple sclerosis, and airway inflammation.
  • GITR e.g., human GITR
  • a pharmaceutical composition provided herein.
  • the subject is human.
  • a method for detecting GITR in a sample comprising contacting the sample with an antibody that specifically binds to GITR (e.g., human GITR) provided herein.
  • a kit comprising an antibody that specifically binds to GITR (e.g., human GITR) provided herein or a pharmaceutical composition provided herein and a) a detection reagent, b) an GITR antigen, c) a notice that reflects approval for use or sale for human administration, or d) a combination thereof. 6.
  • Figures 1A, IB and 1C are a set of graphs showing the binding of DuoBody ® (Genmab A/S) pabl876 x isotype and an isotype control antibody to Jurkat cells expressing human GITR ( Figure 1A), activated Hut 102 cells ( Figure IB) and activated primary CD4+ T cells ( Figure 1C).
  • the mean fluorescence intensity (MFI) is plotted against a range of antibody concentrations.
  • Figures 2A and 2B Figure 2A depicts F-KB-luciferase signal from Jurkat-huGITR- F-KB-luciferase reporter cells triggered by trimeric GITRL, DuoBody pabl876 x isotype, or an isotype control antibody.
  • the relative light units (RLU) are normalized to the RLU values in the samples treated with the isotype control antibody at the highest concentration tested and plotted against a dose titration of antibody or GITRL concentrations.
  • Figure 2B is the result of a reporter assay where Jurkat-huGITR-NF-KB-luciferase reporter cells were pre-incubated with DuoBody pabl876 x isotype or an isotype control antibody before activated by trimeric GITRL.
  • the % GITRL activity is plotted against a range of antibody concentrations.
  • Figure 3 is a histogram showing the loss of binding of 1624-5 pre-B cells expressing the chimeric parental 231-32-15 antibody to biotinylated GITR (GITR-bio) when GITR-bio was pre-incubated with chimeric parental 231-32-15, pabl875 or pabl876 antibodies.
  • Figure 3 right- hand profile depicts the binding of 1624-5 pre-B cells expressing the chimeric parental 231-32- 15 antibody to GITR-bio.
  • Figure 4 shows the results of an epitope competition assay measured by surface plasmon resonance (BIAcore ® T 100/200).
  • GITR antigen was immobilized on a CM5 sensor chip and the anti-GITR antibodies applied at a concentration of 300 nM.
  • Chimeric parental 231- 32-15 antibody was applied first followed by the application of the murine antibody 6C8.
  • Figures 5A and 5B are the results of an epitope mapping experiment using a cellular library expressing GITR variants generated by error prone PCR. Shown in Figures 5A and 5B is an alignment of sequences from the GITR variants that bind to a polyclonal anti-GITR antibody but do not bind to the anti-GITR chimeric parental 231-32-15 antibody.
  • Figures 6A and 6B are the result of an epitope mapping experiment using alanine scanning.
  • the following positions in human GITR (numbered according to SEQ ID NO: 41) were separately mutated to an Alanine: P28A, T29A, G30A, G31A, P32A, T54A, T55A, R56A, C57A, C58A, R59A, D60A, Y61A, P62A, G63A, E64A, E65A, C66A, C67A, S68A, E69A, W70A, D71A, C72A, M73A, C74A, V75A and Q76A.
  • Figure 6A is a table summarizing the binding of pab l 876, pab l967, pab l975, pab l979 and the reference antibody m6C8 tol624-5 cells expressing human GITR alanine mutants.
  • Figure 6B is a set of flow cytometry plots showing the staining of 1624-5 cells expressing wild type human GITR, D60A mutant, or G63A mutant using the monoclonal antibody 231-32-15, pab l 876, or m6C8, or a polyclonal antibody. The percentage of GITR positive cells is indicated in each plot.
  • Figure 7A is a sequence alignment of human GITR, VIM cynomolgus GITR, and V1M/Q62P/S63G cynomolgus GITR, highlighting the positions 62 and 63 where two amino acids from cynomolgus GITR (GlnSer) were replaced by corresponding residues in human GITR (ProGly).
  • Figure 7B is a set of flow cytometry plots showing the staining of 1624-5 cells expressing human GITR, VIM cynomolgus GITR, or V1M/Q62P/S63G cynomolgus GITR using the monoclonal antibody 231-32-15, pab l 876, or m6C8, or a polyclonal anti-GITR antibody.
  • DuoBody antibodies that specifically bind to GITR (e.g., monoclonal antibodies that contain only one anti-GITR-specific antigen-binding domain, and optionally a second antigen-binding domain that is not GITR-specific).
  • GITR e.g., monoclonal antibodies that contain only one anti-GITR-specific antigen-binding domain, and optionally a second antigen-binding domain that is not GITR-specific.
  • antibodies that specifically binds to GITR e.g., human GITR
  • the antibodies are isolated.
  • nucleic acids such as complementary DNA (cDNA)
  • vectors e.g., expression vectors
  • cells e.g., host cells
  • methods of making such antibodies are also provided.
  • methods and uses for example, for deactivating, reducing, or inhibiting an GITR activity, and treating certain conditions, such as inflammatory or autoimmune diseases and disorders.
  • Related compositions e.g., pharmaceutical compositions
  • kits, and detection methods are also provided. 7.1 Terminology
  • antibody and “antibodies” 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, human antibodies, humanized antibodies, resurfaced antibodies, chimeric antibodies, immunoglobulins, 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, intrabodies, heteroconjugate antibodies, single domain antibodies, monovalent antibodies, single chain antibodies or single-chain Fvs (scFv), camelized antibodies, affybodies, Fab fragments, F(ab') 2 fragments, disulfide-linked Fvs (sdFv), anti -idiotypic (anti-Id) antibodies (including, e.g., anti-anti-Id antibodies), bispecific antibodies, and multi-specific antibodies.
  • monoclonal antibodies recombinantly produced antibodies
  • human antibodies humanized antibodies, resurfaced antibodies, chimeric antibodies
  • antibodies described herein refer to polyclonal antibody populations.
  • Antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, or IgY), any class (e.g., IgGi, IgG 2 , IgG 3 , IgG 4 , IgAi, or IgA 2 ), or any subclass (e.g., IgG 2a or IgG 2b ) of immunoglobulin molecule.
  • antibodies described herein are IgG antibodies, or a class (e.g., human IgGi, IgG 2 , or IgG 4 ) or subclass thereof.
  • the antibody is a humanized monoclonal antibody. In another specific embodiment, the antibody is a human monoclonal antibody, e.g., that is an immunoglobulin. In certain embodiments, an antibody described herein is an IgGi, IgG 2 , or IgG 4 antibody.
  • antigen-binding domain As used herein, the terms "antigen-binding domain,” “antigen-binding region,” “antigen-binding site,” and similar terms refer to the portion of antibody molecules which comprises the amino acid residues that confer on the antibody molecule its specificity for the antigen (e.g., the complementarity determining regions (CDR).
  • the antigen-binding region can be derived from any animal species, such as rodents (e.g., mouse, rat, or hamster) and humans.
  • variable region typically refers to a portion of an antibody, generally, a portion of a light or heavy chain, typically about the amino-terminal 110 to 125 amino acids in the mature heavy chain and about 90 to 115 amino acids in the mature light chain, which differ extensively in sequence among antibodies and are used in the binding and specificity of a particular antibody for its particular antigen.
  • CDRs complementarity determining regions
  • FR framework regions
  • variable region is a human variable region.
  • variable region comprises rodent or murine CDRs and human framework regions (FRs).
  • FRs human framework regions
  • the variable region is a primate (e.g., non- human primate) variable region.
  • the variable region comprises rodent or murine CDRs and primate (e.g., non-human primate) framework regions (FRs).
  • VL and “VL domain” are used interchangeably to refer to the light chain variable region of an antibody.
  • VFT and VH domain are used interchangeably to refer to the heavy chain variable region of an antibody.
  • Kabat numbering and like terms are recognized in the art and refer to a system of numbering amino acid residues in the heavy and light chain variable regions of an antibody, or an antigen-binding portion thereof.
  • the CDRs of an antibody can be determined according to the Kabat numbering system (see, e.g., Kabat EA & Wu TT (1971) Ann NY Acad Sci 190: 382-391 and Kabat EA et al, (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242).
  • CDRs within an antibody heavy chain molecule are typically present at amino acid positions 31 to 35, which optionally can include one or two additional amino acids, following 35 (referred to in the Kabat numbering scheme as 35A and 35B) (CDR1), amino acid positions 50 to 65 (CDR2), and amino acid positions 95 to 102 (CDR3).
  • CDRs within an antibody light chain molecule are typically present at amino acid positions 24 to 34 (CDR1), amino acid positions 50 to 56 (CDR2), and amino acid positions 89 to 97 (CDR3).
  • the CDRs of the antibodies described herein have been determined according to the Kabat numbering scheme.
  • constant region or “constant domain” are interchangeable and have its meaning common in the art.
  • the constant region is 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 constant region of an immunoglobulin molecule generally has a 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 type, 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 .
  • the term "light chain” when used in reference to an antibody can refer to any distinct type, e.g., kappa ( ⁇ ) or lambda ( ⁇ ) based on the amino acid sequence of the constant domains. Light chain amino acid sequences are well known in the art. In specific embodiments, the light chain is a human light chain.
  • EU numbering system refers to the EU numbering convention for the constant regions of an antibody, as described in Edelman, G.M. et al., Proc. Natl. Acad. USA, 63, 78-85 (1969) and Kabat et al, Sequences of Proteins of Immunological Interest, U.S. Dept. Health and Human Services, 5th edition, 1991, each of which is herein incorporated by reference in its entirety.
  • Binding affinity generally refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, "binding affinity” refers to intrinsic binding affinity which reflects a 1 : 1 interaction between members of a binding pair (e.g., antibody and antigen).
  • the affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (K D ). Affinity can be measured and/or expressed in a number of ways known in the art, including, but not limited to, equilibrium dissociation constant (K D ), and equilibrium association constant (KA).
  • the K D is calculated from the quotient of k 0 ff/k on
  • KA is calculated from the quotient of k on /k 0 ff.
  • k on refers to the association rate constant of, e.g., an antibody to an antigen
  • k 0 ff refers to the dissociation of, e.g., an antibody to an antigen.
  • the k on and k off can be determined by techniques known to one of ordinary skill in the art, such as BIAcore or KinExA.
  • a "conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain.
  • Families of amino acid residues having 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,
  • 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, for example, contiguous amino acids of a polypeptide (linear or contiguous epitope) or an epitope can, for example, come together from two or more non-contiguous regions of a polypeptide or polypeptides (conformational, non-linear, discontinuous, or non-contiguous epitope).
  • the epitope to which an antibody binds can be determined by, e.g., NMR spectroscopy, X-ray diffraction crystallography studies, ELISA assays, hydrogen/deuterium exchange coupled with mass spectrometry (e.g., liquid chromatography electrospray mass spectrometry), array-based oligo-peptide scanning assays, and/or mutagenesis mapping (e.g., site-directed mutagenesis mapping).
  • NMR spectroscopy e.g., NMR spectroscopy, X-ray diffraction crystallography studies, ELISA assays, hydrogen/deuterium exchange coupled with mass spectrometry (e.g., liquid chromatography electrospray mass spectrometry), array-based oligo-peptide scanning assays, and/or mutagenesis mapping (e.g., site-directed mutagenesis mapping).
  • crystallization may be accomplished using any of the known methods in the art (e.g., Giege R et al, (1994) Acta Crystallogr D Biol Crystallogr 50(Pt 4): 339-350; McPherson A (1990) Eur J Biochem 189: 1-23; Chayen NE (1997) Structure 5: 1269-1274; McPherson A (1976) J Biol Chem 251 : 6300-6303).
  • Antibody antigen crystals can be studied using well known X-ray diffraction techniques and can be refined using computer software such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc.; see, e.g., Meth Enzymol (1985) volumes 114 & 115, eds Wyckoff HW et al., ; U.S.
  • the epitope of an antibody is determined using alanine scanning mutagenesis studies.
  • the terms “immunospecifically binds,” “immunospecifically recognizes,” “specifically binds,” and “specifically recognizes” are analogous terms in the context of antibodies and refer to molecules that bind to an antigen ⁇ e.g., epitope or immune complex) as such binding is understood by one skilled in the art.
  • a molecule that specifically binds to an antigen can 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.
  • molecules that immunospecifically 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 K A when the molecules bind non-specifically to another antigen.
  • the terms “immunospecifically binds,” “immunospecifically recognizes,” “specifically binds,” and “specifically recognizes” refer to antibodies that have distinct specificities for more than one antigen ⁇ i.e., GITR and the antigen associated with the second antigen-binding domain).
  • antigen-binding domains that immunospecifically bind to an antigen do not cross react with other proteins under similar binding conditions.
  • antigen-binding domains that immunospecifically bind to an antigen do not cross react with other non-GITR proteins.
  • provided herein is an antibody containing an antigen-binding domain that binds to GITR with higher affinity than to another unrelated antigen.
  • an antibody containing an antigen-binding domain that binds to GITR ⁇ e.g., human GITR) with a 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or higher affinity than to another, unrelated antigen as measured by, e.g., a radioimmunoassay, surface plasmon resonance, or kinetic exclusion assay.
  • the extent of binding of an anti-GITR antigen-binding domain described herein to an unrelated, non-GITR protein is less than 10%, 15%, or 20% of the binding of the antigen-binding domain to GITR protein as measured by, e.g., a radioimmunoassay.
  • an antibody containing an antigen- binding domain that binds to human GITR with higher affinity than to another species of GITR In certain embodiments, provided herein is an antibody containing an antigen-binding domain that binds to human GITR with a 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%), 65%), 70%) or higher affinity than to another species of GITR as measured by, e.g., a radioimmunoassay, surface plasmon resonance, or kinetic exclusion assay.
  • an antibody containing an antigen-binding domain described herein, which binds to human GITR will bind to another species of GITR protein with less than 10%>, 15%, or 20% of the binding of the antibody containing an antigen-binding domain to the human GITR protein as measured by, e.g., a radioimmunoassay, surface plasmon resonance, or kinetic exclusion assay.
  • GITR GITR polypeptide
  • GITR including, but not limited to, native GITR, an isoform of GITR, or an interspecies GITR homolog of GITR.
  • GITR is also known as activation- inducible TNFR family receptor (AITR), GITR-D, CD357, and tumor necrosis factor receptor superfamily member 18 (TNFRSF18).
  • AITR activation- inducible TNFR family receptor
  • BC 152381 and BC 152386 provide human GITR nucleic acid sequences.
  • Swiss-Prot accession number Q9Y5U5-1 (TNR 18 HUMAN; SEQ ID NO:41) and GenBankTM accession number NP 004186 provide exemplary human GITR amino acid sequences for isoform 1. This amino acid sequence is 241 amino acids in length with the first 25 amino acid residues encoding the signal sequence. Isoform 1 is a type I membrane protein. An exemplary mature amino acid sequence of human GITR is provided as SEQ ID NO:40. In contrast, isoform 2 is a secreted form of human GITR and is approximately 255 amino acids in length. Swiss-Prot accession number Q9Y5U5-2 and GenBankTM accession number NP 683699 provide exemplary human GITR amino acid sequences for isoform 2.
  • Isoform 3 of human GITR is approximately 234 amino acids in length.
  • Swiss-Prot accession number Q9Y5U5-3 and GenBankTM accession number NP 683700 (isoform 3 precursor) provide exemplary human GITR amino acid sequences for isoform 3.
  • the GITR is human GITR.
  • the GITR is human GITR isoform 1 (SEQ ID NO:41).
  • the GITR is human isoform 2 (SEQ ID NO:42) or isoform 3 (SEQ ID NO:43).
  • Human GITR is designated GenelD: 8784 by Entrez Gene.
  • SEQ ID NO:44 provides the cynomolgus GITR amino acid sequence, and amino acids 26-234 of SEQ ID NO:44 represent the mature form of cynomolgus GITR.
  • human GITR refers to GITR comprising the polypeptide sequence of SEQ ID NO:40.
  • GITRL glucocorticoid- induced T FR-related protein ligand. GITRL is otherwise known as activation-induced TNF- related ligand (AITRL) and tumor necrosis factor ligand superfamily member 18 (T FSF18).
  • AITRL activation-induced TNF- related ligand
  • T FSF18 tumor necrosis factor ligand superfamily member 18
  • GenBankTM accession number AF 125303 provides an exemplary human GITRL nucleic acid sequence.
  • GenBankTM accession number P 005083 and Swiss-Prot accession number Q9UNG2 provide exemplary human GITRL amino acid sequences.
  • the term "host cell” can be any type of cell, e.g., a primary cell, a cell in culture, or a cell from a cell line.
  • the term “host cell” refers to a cell transfected with a nucleic acid molecule and the progeny or potential progeny of such a cell. Progeny of such a cell may not be identical to the parent cell transfected with the nucleic acid molecule, e.g., due to mutations or environmental influences that may occur in succeeding generations or integration of the nucleic acid molecule into the host cell genome.
  • the term "effective amount" in the context of the administration of a therapy to a subject refers to the amount of a therapy that achieves a desired prophylactic or therapeutic effect. Examples of effective amounts are provided in Section 7.5, infra.
  • the terms "subject” and "patient” are used interchangeably.
  • the subject can be an animal.
  • the subject is a mammal such as a non-primate ⁇ e.g., cow, pig, horse, cat, dog, rat, etc.) or a primate ⁇ e.g., monkey or human), most preferably a human.
  • the subject is a cynomolgus monkey.
  • such terms refer to a non-human animal ⁇ e.g. , a non-human animal such as a pig, horse, cow, cat, or dog).
  • such terms refer to a pet or farm animal.
  • such terms refer to a human.
  • the binding between a test antibody and a first antigen is "substantially weakened" relative to the binding between the test antibody and a second antigen if the binding between the test antibody and the first antigen is reduced by at least 30%, 40%, 50%), 60%), 70%), or 80%> relative to the binding between the test antibody and the second antigen, e.g., in a given experiment, or using mean values from multiple experiments, as assessed by, e.g., an assay comprising the following steps: (a) expressing on the surface of cells ⁇ e.g., 1624-5 cells) the first antigen or the second antigen; (b) staining the cells expressing the first antigen or the second antigen using, e.g., 2 ⁇ g/ml of the test antibody or a polyclonal antibody in a flow cytometry analysis and recording mean fluorescence intensity (MFI) values, e.g., as the mean from more than one measurement, wherein the polyclonal antibody recognizes both the MFI.
  • MFI mean
  • the determination of "percent identity" between two sequences can also be accomplished using a mathematical algorithm.
  • a specific, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin S & Altschul SF (1990) PNAS 87: 2264-2268, modified as in Karlin S & Altschul SF (1993) PNAS 90: 5873-5877.
  • Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul SF et al, (1990) J Mol Biol 215: 403.
  • Gapped BLAST can be utilized as described in Altschul SF et al., (1997) Nuc Acids Res 25: 3389 3402.
  • PSI BLAST can be used to perform an iterated search which detects distant relationships between molecules (Id.).
  • BLAST Gapped BLAST
  • 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).
  • NCBI National Center for Biotechnology Information
  • Another specific, 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.
  • ALIGN program version 2.0
  • 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.
  • the term "antigen-binding domain that does not bind to an antigen expressed by a human immune cell” means that the antigen-binding domain does not bind to an antigen expressed by any human cell of hematopoietic origin that plays a role in the immune response.
  • Immune cells include lymphocytes, such as B cells and T cells; natural killer cells; and myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.
  • lymphocytes such as B cells and T cells
  • natural killer cells such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.
  • myeloid cells such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.
  • such a binding domain would not bind to GITR, or any other members of the T F receptor superfamily that are expressed by a human immune cell.
  • the antigen-binding domain can bind to an antigen such as, but not limited to, an antigen expressed in other organisms and not humans (i.e., a non-human antigen); an antigen that is not expressed by wild-type human cells; or a viral antigen, including, but not limited to, an antigen from a virus that does not infect human cells, or a viral antigen that is absent in an uninfected human immune cell.
  • an antigen such as, but not limited to, an antigen expressed in other organisms and not humans (i.e., a non-human antigen); an antigen that is not expressed by wild-type human cells; or a viral antigen, including, but not limited to, an antigen from a virus that does not infect human cells, or a viral antigen that is absent in an uninfected human immune cell.
  • an anti-GITR agonist antibody may mediate receptor clustering through bivalent antibody arms (i.e., two antibody arms that each bind GITR antigen) and/or through Fc-Fc receptor (FcR) co-engagement on accessory myeloid or lymphoid cells. Consequently, one approach of developing an anti-GITR antagonist antibody is to select an antibody that competes with GITR ligand (GITRL) for binding to GITR, diminish or eliminate the binding of the Fc region of an antibody to Fc receptors, and/or adopt a monovalent antibody format.
  • GITRL GITR ligand
  • the monovalent antibody format can include antibodies that are structurally monovalent, such as, but not limited to, anti-GITR antibodies comprising a first antigen-binding domain that binds to GITR (e.g., human GITR) paired with, for example, an Fc region (i.e., the monovalent antibody comprises only one antigen-binding arm).
  • anti-GITR antibodies comprising a first antigen-binding domain that binds to GITR (e.g., human GITR) paired with, for example, an Fc region (i.e., the monovalent antibody comprises only one antigen-binding arm).
  • the monovalent antibody format can also include antibodies that are functionally monovalent, for example, antibodies comprising only one antigen-binding domain that binds to GITR (e.g., human GITR) that is paired with a second antigen-binding domain that does not bind to an antigen expressed by a human immune cell (i.e., the antibody comprises two antigen-binding domains, but only one antigen-binding domain binds to GITR).
  • GITR e.g., human GITR
  • the antibody comprises two antigen-binding domains, but only one antigen-binding domain binds to GITR.
  • antagonist antibodies e.g., monoclonal antibodies, such as chimeric, humanized, or human antibodies
  • GITR e.g., human GITR
  • the antibodies are DuoBody anti-GITR antagonists, each comprising one antigen-binding domain that binds to GITR.
  • an antigen-binding domain as described herein that binds to GITR contains a combination of CDRs shown in a single row of Table 1 below.
  • Table 1 CDR sequences of exemplary antigen-binding domains that bind to GITR
  • an antigen-binding domain as described herein that binds to GITR contains a combination of a heavy chain variable domain and a light chain variable domain shown in a single row of Table 2 below.
  • VH Heavy chain variable domain
  • VL light chain variable domain
  • GITR VH (SEQ ID NO:)
  • GITR VL (SEQ ID NO:)
  • an antigen-binding domain that binds to GITR comprises a light chain variable region (VL) comprising:
  • VL-CDRl comprising, consisting of, or consisting essentially of the amino acid sequence of KSSQSLLNSXiNQKNYLX 2 (SEQ ID NO: 90), wherein Xi is G or S; and X 2 is T or S;
  • VL-CDR2 comprising, consisting of, or consisting essentially of the amino acid sequence of WASTRES (SEQ ID NO: 5);
  • VL-CDR3 comprising, consisting of, or consisting essentially of the amino acid sequence of QNXiYSX 2 PYT (SEQ ID NO: 92), wherein X x is D or E; and X 2 is Y, F or S, as shown in Table 4.
  • an antigen-binding domain that binds to GITR comprises a light chain variable region (VL) comprising:
  • VL-CDRl comprising, consisting of, or consisting essentially of the amino acid sequence of KSSQSLLNSXiNQKNYLT (SEQ ID NO: 4), wherein Xi is G or S;
  • VL-CDR2 comprising, consisting of, or consisting essentially of the amino acid sequence of WASTRES (SEQ ID NO: 5);
  • VL-CDR3 comprising, consisting of, or consisting essentially of the amino acid sequence of QNXiYSX 2 PYT (SEQ ID NO: 6), wherein Xi is D or E; and X 2 is Y or F, as shown in Table 4.
  • VL CDRs in Table 4 are determined according to Kabat.
  • an antigen-binding domain that binds to GITR comprises the VL framework regions described herein.
  • an antigen-binding domain described herein which specifically binds to GITR (e.g., human GITR), comprises a heavy chain variable region (VH) comprising:
  • VH-CDR1 comprising the amino acid sequence of XiYX 2 MX 3 (SEQ ID NO: 87), wherein X 1 is D, E or G; X 2 is A or V; and X 3 is Y or H;
  • a VH-CDR2 comprising the amino acid sequence of XiIX 2 TX 3 SGX 4 X 5 X 6 YNQKFX 7 X 8 (SEQ ID NO: 88), wherein X x is V or L; X 2 is R, K or Q; X 3 is Y or F; X 4 is D, E or G; X 5 is V or L; X 6 is T or S; X 7 is K, R or Q; and X 8 is D, E or G;
  • VH-CDR3 comprising the amino acid sequence of SGTVRGFAY (SEQ ID NO: 3), as shown in Table 5.
  • an antigen-binding domain described herein which specifically binds to GITR (e.g., human GITR), comprises a heavy chain variable region (VH) comprising:
  • VH-CDRl comprising the amino acid sequence of XiYAMX 2 (SEQ ID NO: l), wherein X 1 is D, G, or E; and X 2 is Y or H;
  • VH-CDR2 comprising the amino acid sequence of XiIRTYSGX 2 VX 3 YNQKFX 4 X 5 (SEQ ID NO: 2), wherein Xi is V or L; X 2 is D or G; X 3 is T or S; X 4 is K, R, or Q; and X 5 is D, E, or G;
  • VH-CDR3 comprising the amino acid sequence of SGTVRGFAY (SEQ ID NO: 3), as shown in Table 5.
  • VH CDRs in ⁇ ' able 5 are determined according to Kabat.
  • an antigen-binding domain that binds to GITR comprises the VH frameworks described herein.
  • an antigen-binding domain which specifically binds to GITR (e.g., human GITR) and comprises light chain variable region (VL) CDRs and heavy chain variable region (VH) CDRs of pab l 876w, pab l967w, pab l975w, or pab l979w, for example as set forth in Tables 4 and 5 (i.e. , SEQ ID NOs: 87, 88, and 3; SEQ ID NOs: 1, 2, and 3; SEQ ID NOs: 7, 10, and 3; SEQ ID NOs: 8, 1 1, and 3; SEQ ID NOs: 9, 12, and 3; or SEQ ID NOs: 9, 13, and 3).
  • VL light chain variable region
  • VH heavy chain variable region
  • a GITR antigen-binding domain comprises a light chain variable framework region that is derived from human IGKV4-1 germline sequence (e.g. , IGKV4-1 *01, e.g. , having amino acid sequence of SEQ ID NO: 28).
  • the GITR antigen-binding domain comprises a heavy chain variable framework region that is derived from a human IGHV1-2 germline sequence (e.g., IGHVl-2*02, e.g. , having amino acid sequence of SEQ ID NO: 27).
  • an antigen-binding domain that specifically binds to GITR comprises a VL domain comprising the amino acid sequence of SEQ ID NO: 19, 21, 23, or 26.
  • an antigen-binding domain that specifically binds to GITR comprises a VL domain consisting of or consisting essentially of the amino acid sequence of SEQ ID NO: 19, 21, 23, or 26.
  • an antigen-binding domain that specifically binds to GITR comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 18, 20, 22, 24, or 25.
  • an antigen-binding domain that specifically binds to GITR comprises a VH domain consisting of or consisting essentially of the amino acid sequence of SEQ ID NO: 18, 20, 22, 24, or 25.
  • an antigen-binding domain that specifically binds to GITR comprises a VH domain and a VL domain, wherein the VH domain and the VL domain comprise the amino acid sequences of SEQ ID NOs: 18 and 19; SEQ ID NOs:20 and 21 ; SEQ ID NOs:22 and 23; SEQ ID NOs:24 and 23; or SEQ ID NOs:25 and 26; respectively.
  • an antigen-binding domain that specifically binds to GITR comprises a VH domain and a VL domain, wherein the VH domain and the VL domain consist of or consist essentially of the amino acid sequences of SEQ ID NOs: 18 and 19; SEQ ID NOs:20 and 21 ; SEQ ID NOs:22 and 23; SEQ ID NOs:24 and 23; or SEQ ID NOs:25 and 26; respectively.
  • an antigen-binding domain comprising an light chain and heavy chain, e.g., a separate light chain and heavy chain.
  • the light chain of an antigen-binding domain described herein is a kappa light chain.
  • the light chain of an antigen-binding domain described herein is a lambda light chain.
  • the light chain of an antigen-binding domain described herein is a human kappa light chain or a human lambda light chain.
  • an antigen-binding domain described herein which immunospecifically binds to an GITR polypeptide (e.g., human GITR) comprises a light chain wherein the amino acid sequence of the VL domain comprises the sequence set forth in SEQ ID NO: 19, 21, 23 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 antigen-binding domain described herein which immunospecifically binds to GITR (e.g., human GITR) comprises a light chain wherein the amino acid sequence of the VL domain comprises the sequence set forth in SEQ ID NO: 19, 21, 23 or 26 and wherein the constant region of the light chain comprises the amino acid sequence of a human lambda light chain constant region.
  • an antigen-binding domain described herein, which immunospecifically binds to GITR comprises a light chain wherein the amino acid sequence of the VL domain comprises the sequence set forth in SEQ ID NO: 19, 21, 23 or 26 and wherein the constant region of the light chain comprises the amino acid sequence of a human kappa or lambda light chain constant region.
  • GITR e.g., human GITR
  • the amino acid sequence of the VL domain comprises the sequence set forth in SEQ ID NO: 19, 21, 23 or 26 and wherein the constant region of the light chain comprises the amino acid sequence of a human kappa or 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 EA et a/., (1991) supra.
  • an antigen-binding domain described herein, which specifically binds to GITR comprises a light chain comprising the amino acid sequence set forth in SEQ ID NO:37 or 38.
  • the heavy chain of an antigen-binding domain described herein can be an alpha (a), delta ( ⁇ ), epsilon ( ⁇ ), gamma ( ⁇ ) or mu ( ⁇ ) heavy chain.
  • the heavy chain of an antigen-binding domain described can comprise a human alpha (a), delta ( ⁇ ), epsilon ( ⁇ ), gamma ( ⁇ ) or mu ( ⁇ ) heavy chain.
  • an antigen-binding domain described herein which immunospecifically binds to GITR (e.g., human GITR), comprises a heavy chain wherein the amino acid sequence of the VH domain can comprise the sequence set forth in SEQ ID NO: 18, 20, 22, 24, or 25 and wherein the constant region of the heavy chain comprises the amino acid sequence of a human gamma ( ⁇ ) heavy chain constant region.
  • an antigen-binding domain described herein which specifically binds to GITR (e.g., human GITR), comprises a heavy chain wherein the amino acid sequence of the VH domain comprises the sequence set forth in SEQ ID NO: 18, 20, 22, 24, or 25, and wherein the constant region of the heavy chain comprises the amino acid of a human heavy chain described herein or known in the art.
  • GITR e.g., human GITR
  • 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 EA et al, (1991) supra.
  • an antigen-binding domain described herein which specifically binds to GITR (e.g., human GITR), comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:29.
  • an antigen-binding domain described herein, which specifically binds to GITR comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:30.
  • an antigen-binding domain described herein, which specifically binds to GITR comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:36.
  • an antigen-binding domain described herein which immunospecifically binds to GITR (e.g., human GITR), comprises a VL domain and a VH domain comprising any amino acid sequences described herein, 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 antigen-binding domain described herein which immunospecifically binds to GITR (e.g., human GITR), comprises a VL domain and a VH domain comprising any amino acid sequences described herein, 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., IgGi, IgG 2 , IgG 3 , IgG 4 , IgAi, and IgA 2 ), or any subclass (e.g., IgG 2a and IgG 3 ⁇ 4 ) of immunoglobulin molecule.
  • GITR e.g., human GITR
  • 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., IgGi, IgG 2 , IgG 3 , IgG 4 , IgAi, and IgA 2 ), or any subclass (e.g., IgG 2a and IgG 3 ⁇ 4 ) of immunoglobulin molecule.
  • any class e.g., IgGi, IgG 2 , IgG 3 , IgG 4 , IgAi, and IgA 2
  • subclass e.g., IgG 2a and IgG 3 ⁇ 4
  • an antigen-binding domain described herein which immunospecifically binds to GITR (e.g., human GITR), comprises a VL domain and a VH domain comprising any amino acid sequences described herein, wherein the constant regions comprise the amino acid sequences of the constant regions of a human IgGi (e.g., allotypes Glm3, Glml7, l or Gl ml 7, 1,2), human IgG 2 , or human IgG 4 .
  • a human IgGi e.g., allotypes Glm3, Glml7, l or Gl ml 7, 1,2
  • an antigen-binding domain described herein which immunospecifically binds to GITR (e.g., human GITR), comprises a VL domain and a VH domain comprising any amino acid sequences described herein, wherein the constant regions comprise the amino acid sequences of the constant region of a human IgGi (allotype Glm3).
  • GITR e.g., human GITR
  • constant regions comprise the amino acid sequences of the constant region of a human IgGi (allotype Glm3).
  • human constant regions are described in the art, e.g., see Kabat EA et al, (1991) supra.
  • an antigen-binding domain described herein which specifically binds to GITR (e.g., human GITR), comprises a light chain comprising the amino acid sequence set forth in SEQ ID NO:37 and a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:29, 30, or 36.
  • an antigen-binding domain described herein which immunospecifically binds to GITR (e.g., human GITR), comprises a VL domain having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of the VL domain of pabl876w, pabl967w, pabl975w, or pabl979w (i.e., SEQ ID NO: 19, 21, 23, or 26), e.g., wherein the antigen-binding domain comprises VL CDRs that are identical to the VL CDRs of pabl876w, pabl967w, pabl975w, or pabl979w.
  • an antigen-binding domain described herein which immunospecifically binds to GITR (e.g., human GITR), comprises a VH domain having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of the VH domain of pabl876w, pabl967w, pabl975w, or pabl979w (i.e.., SEQ ID NO: 18, 29, 22, 24, or 25), e.g., wherein the antigen- binding domain comprises VH CDRs that are identical to the VH CDRs of pabl876w, pabl967w, pabl975w, or pabl979w.
  • an antigen-binding domain described herein which immunospecifically binds to GITR (e.g., human GITR), comprises: (i) a VL domain having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of the VL domain of pabl876w, pabl967w, pabl975w, or pabl979w (i.e., SEQ ID NO: 19, 21, 23, or 26); and (ii) a VH domain having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of the VH domain of pabl876w, pabl967w, pabl975w, or pabl979w (i.e.., SEQ ID NO: 18, 20, 22, 24, or 25), e.g., where
  • an antigen-binding domain described herein may be described by its VL domain alone, or its VH domain alone, or by its 3 VL CDRs alone, or its 3 VH CDRs alone. See, for example, Rader C et al, (1998) PNAS 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.
  • the CDRs of an antigen-binding domain can be determined according to the Chothia numbering scheme, which refers to the location of immunoglobulin structural loops (see, e.g., Chothia C & Lesk AM, (1987), J Mol Biol 196: 901-917; Al-Lazikani B et al, (1997) J Mol Biol 273 : 927-948; Chothia C 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).
  • Chothia numbering scheme refers to the location of immunoglobulin structural loops
  • the Chothia CDR-H1 loop is present at heavy chain amino acids 26 to 32, 33, or 34
  • the Chothia CDR-H2 loop is present at heavy chain amino acids 52 to 56
  • the Chothia CDR-H3 loop is present at heavy chain amino acids 95 to 102
  • the Chothia CDR-L1 loop is present at light chain amino acids 24 to 34
  • the Chothia CDR-L2 loop is present at light chain amino acids 50 to 56
  • the Chothia CDR-L3 loop is present at light chain amino acids 89 to 97.
  • the end of the Chothia CDR-H1 loop when numbered using the Kabat numbering convention varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places the insertions at H35A and H35B; if neither 35A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34).
  • antigen-binding domains that specifically bind to GITR (e.g., human GITR) and comprise the Chothia VL CDRs of a VL of pabl 876w, pabl967w, pabl975w, or pabl979w.
  • antigen-binding domains that specifically bind to GITR (e.g., human GITR) and comprise the Chothia VH CDRs of a VH of pabl876w, pabl967w, pabl975w, or pabl979w.
  • antigen-binding domains that specifically bind to GITR (e.g., human GITR) and comprise the Chothia VL CDRs of a VL of pabl876w, pabl967w, pabl975w, or pabl979wand comprise the Chothia VH CDRs of a VH of pabl876w, pabl967w, pabl975w, or pabl979w.
  • antigen-binding domains that specifically bind to GITR comprise one or more CDRs, in which the Chothia and Kabat CDRs have the same amino acid sequence.
  • antigen-binding domains that specifically bind to GITR e.g., human GITR
  • the CDRs of an antigen-binding domain can be determined according to the EVIGT numbering system 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.
  • VH-CDRl is at positions 26 to 35
  • VH-CDR2 is at positions 51 to 57
  • VH-CDR3 is at positions 93 to 102
  • VL-CDRl is at positions 27 to 32
  • VL- CDR2 is at positions 50 to 52
  • VL-CDR3 is at positions 89 to 97.
  • antigen-binding domains that specifically bind to GITR ⁇ e.g., human GITR) and comprise CDRs of pabl876w, pabl967w, pabl975w, or pabl979w as determined by the IMGT numbering system, for example, as described in Lefranc M-P (1999) supra and Lefranc M-P et al, (1999) supra).
  • the CDRs of an antigen-binding domain can be determined according to MacCallum RM 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).
  • antigen-binding domains that specifically bind to GITR ⁇ e.g., human GITR) and comprise CDRs of pabl876w, pabl967w, pabl975w, or pabl979w as determined by the method in MacCallum RM et al.
  • the CDRs of an antibody can be determined according to the AbM numbering scheme, which refers AbM hypervariable regions which represent a compromise between the Kabat CDRs and Chothia structural loops, and are used by Oxford Molecular's AbM antibody modeling software (Oxford Molecular Group, Inc.).
  • AbM numbering scheme refers AbM hypervariable regions which represent a compromise between the Kabat CDRs and Chothia structural loops, and are used by Oxford Molecular's AbM antibody modeling software (Oxford Molecular Group, Inc.).
  • antigen-binding domains that specifically bind to GITR ⁇ e.g., human GITR) and comprise CDRs of pabl876w, pabl967w, pabl975w, or pabl979w as determined by the AbM numbering scheme.
  • 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 antigen-binding domain described herein may vary by one, two, three, four, five, or six amino acid positions so long as immunospecific binding to GITR ⁇ e.g., human GITR) 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 position defining a CDR of an antigen-binding domain described herein can 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 CDR position of an antigen-binding domain described herein, so long as immunospecific binding to GITR ⁇ e.g., human GITR) 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%).
  • GITR e.g., human GITR
  • 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 antigen-binding domain 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 GITR (e.g., human GITR) 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%).
  • GITR e.g., human GITR
  • a VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and/or VH CDR3 described herein may be one, two, three, four, five or more amino acids shorter than one or more of the CDRs described herein (e.g., SEQ ID NOs: l-6, SEQ ID NOs: 87, 88, 3, 90, 5, and 92; SEQ ID NOS: 7, 10, 3, 14, 5, and 16; SEQ ID NOs: 8, 1 1, 3, 15, 5, and 17; SEQ ID NOs: 9, 12, 3, 14, 5, and 16; SEQ ID NOs: 9, 13, 3, 14, 5, and 16;) so long as immunospecific binding to GITR (e.g., human GITR) 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%).
  • GITR e.g., human GITR
  • a VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and/or VH CDR3 described herein may be one, two, three, four, five or more amino acids longer than one or more of the CDRs described herein (e.g., SEQ ID NOs: 1-6, SEQ ID NOs: 87, 88, 3, 90, 5, and 92; SEQ ID NOS: 7, 10, 3, 14, 5, and 16; SEQ ID NOs: 8, 1 1, 3, 15, 5, and 17; SEQ ID NOs: 9, 12, 3, 14, 5, and 16; SEQ ID NOs: 9, 13, 3, 14, 5, and 16) so long as immunospecific binding to GITR (e.g., human GITR) 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%).
  • GITR e.g., human GITR
  • the amino terminus of a VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and/or VH 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 herein (e.g., SEQ ID NOs: 1-6, SEQ ID NOs: 87, 88, 3, 90, 5, and 92; SEQ ID NOS: 7, 10, 3, 14, 5, and 16; SEQ ID NOs: 8, 1 1, 3, 15, 5, and 17; SEQ ID NOs: 9, 12, 3, 14, 5, and 16; SEQ ID NOs: 9, 13, 3, 14, 5, and 16;) so long as immunospecific binding to GITR (e.g., human GITR) 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%).
  • GITR e.g., human GITR
  • the carboxy terminus of a VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and/or VH 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 herein (e.g., SEQ ID NOs: 1-6, SEQ ID NOs: 87, 88, 3, 90, 5, and 92; SEQ ID NOS: 7, 10, 3, 14, 5, and 16; SEQ ID NOs: 8, 1 1, 3, 15, 5, and 17; SEQ ID NOs: 9, 12, 3, 14, 5, and 16; SEQ ID NOs: 9, 13, 3, 14, 5, and 16) so long as immunospecific binding to GITR (e.g., human GITR) 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%).
  • GITR e.g., human GITR
  • the amino terminus of a VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and/or VH 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 herein (e.g., SEQ ID NOs: 1-6, SEQ ID NOs: 87, 88, 3, 90, 5, and 92; SEQ ID NOS: 7, 10, 3, 14, 5, and 16; SEQ ID NOs: 8, 1 1, 3, 15, 5, and 17; SEQ ID NOs: 9, 12, 3, 14, 5, and 16; SEQ ID NOs: 9, 13, 3, 14, 5, and 16) so long as immunospecific binding to GITR (e.g., human GITR) 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%).
  • GITR e.g., human GITR
  • the carboxy terminus of a VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and/or VH 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 herein (e.g., SEQ ID NOs: 1-6, SEQ ID NOs: 87, 88, 3, 90, 5, and 92; SEQ ID NOS: 7, 10, 3, 14, 5, and 16; SEQ ID NOs: 8, 1 1, 3, 15, 5, and 17; SEQ ID NOs: 9, 12, 3, 14, 5, and 16; SEQ ID NOs: 9, 13, 3, 14, 5, and 16) so long as immunospecific binding to GITR (e.g., human GITR) 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%).
  • GITR e.g., human GITR
  • GITR e.g., human GITR
  • Any method known in the art can be used to ascertain whether immunospecific binding to GITR (e.g., human GITR) is maintained, for example, the binding assays and conditions described in the "Examples" section (Section 8) provided herein.
  • an antigen-binding domain described herein which immunospecifically binds to GITR (e.g., human GITR), comprises a heavy chain and a light chain, wherein (i) the heavy and light chains comprise a VH domain and a VL domain, respectively, wherein the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 of the VH and VL domains comprise the amino acid sequences set forth in SEQ ID NOs: 1-6, SEQ ID NOs: 87, 88, 3, 90, 5, and 92; SEQ ID NOS: 7, 10, 3, 14, 5, and 16; SEQ ID NOs: 8, 1 1, 3, 15, 5, and 17; SEQ ID NOs: 9, 12, 3, 14, 5, and 16; or SEQ ID NOs: 9, 13, 3, 14, 5, and 16, respectively; (ii) the light chain further comprises a constant light chain domain comprising the amino acid sequence of the constant domain of a human kappa light chain; and
  • an antigen-binding domain described herein which immunospecifically binds to GITR (e.g., human GITR), comprises a heavy chain and a light chain, wherein (i) the heavy and light chains comprise a VH domain and a VL domain, respectively comprising the amino acid sequences set forth in SEQ ID NOs: 18 and 19, SEQ ID NOs: 20 and 21, SEQ ID NOs: 22 and 23, SEQ ID NOs: 24 and 23, or SEQ ID NOs: 25 and 26, respectively; (ii) the light chain further comprises a constant domain comprising the amino acid sequence of the constant domain of a human kappa light chain; and (iii) the heavy chain further comprises a constant domain comprising the amino acid sequence of the constant domain of a human IgGi (optionally IgGi (allotype Glm3)) heavy chain.
  • the heavy and light chains comprise a VH domain and a VL domain, respectively comprising the amino acid sequences set forth in SEQ ID NOs: 18 and 19, SEQ ID
  • an antigen-binding domain described herein which immunospecifically binds to GITR (e.g., human GITR), comprises a light chain and a heavy chain, wherein (i) the heavy and light chains comprise a VH domain and a VL domain, respectively, wherein the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 of the VH and VL domains comprise the amino acid sequences set forth in SEQ ID NOs: 1-6, SEQ ID NOs: 87, 88, 3, 90, 5, and 92; SEQ ID NOS: 7, 10, 3, 14, 5, and 16; SEQ ID NOs: 8, 1 1, 3, 15, 5, and 17; SEQ ID NOs: 9, 12, 3, 14, 5, and 16; or SEQ ID NOs: 9, 13, 3, 14, 5, and 16, respectively; (ii) the light chain further comprises a constant light chain domain comprising the amino acid sequence of the constant domain of a human kappa light chain; and
  • an antigen-binding domain described herein which immunospecifically binds to GITR (e.g., human GITR), comprises a light chain and a heavy chain, wherein (i) the heavy and light chains comprise a VH domain and a VL domain, respectively comprising the amino acid sequences set forth in SEQ ID NOs: 18 and 19, SEQ ID NOs: 20 and 21, SEQ ID NOs: 22 and 23, SEQ ID NOs: 24 and 23, or SEQ ID NOs: 25 and 26, respectively; (ii) the light chain further comprises a constant domain comprising the amino acid sequence of the constant domain of a human kappa light chain; and (iii) the heavy chain further comprises a constant domain comprising the amino acid sequence of the constant domain of a human IgG 4 heavy chain.
  • an antigen-binding domain described herein which immunospecifically binds to GITR (e.g., human GITR), comprises a light chain and a heavy chain, wherein (i) the heavy and light chains comprise a VH domain and a VL domain, respectively, wherein the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 of the VH and VL domains comprise the amino acid sequences set forth in SEQ ID NOs: 1-6, SEQ ID NOs: 87, 88, 3, 90, 5, and 92; SEQ ID NOS: 7, 10, 3, 14, 5, and 16; SEQ ID NOs: 8, 1 1, 3, 15, 5, and 17; SEQ ID NOs: 9, 12, 3, 14, 5, and 16; or SEQ ID NOs: 9, 13, 3, 14, 5, and 16, respectively; (ii) the light chain further comprises a constant light chain domain comprising the amino acid sequence of the constant domain of a human kappa light chain; and
  • an antibody described herein which immunospecifically binds to GITR (e.g., human GITR), comprises a light chain and a heavy chain, wherein (i) the heavy and light chains comprise a VH domain and a VL domain, respectively comprising the amino acid sequences set forth in SEQ ID NOs: 18 and 19, SEQ ID NOs: 20 and 21, SEQ ID NOs: 22 and 23, SEQ ID NOs: 24 and 23, or SEQ ID NOs: 25 and 26, respectively; (ii) the light chain further comprises a constant domain comprising the amino acid sequence of the constant domain of a human kappa light chain; and (iii) the heavy chain further comprises a constant domain comprising the amino acid sequence of the constant domain of a human IgG 2 heavy chain.
  • the heavy and light chains comprise a VH domain and a VL domain, respectively comprising the amino acid sequences set forth in SEQ ID NOs: 18 and 19, SEQ ID NOs: 20 and 21, SEQ ID NOs: 22 and 23, SEQ ID NOs:
  • an antibody provided herein which specifically binds to GITR (e.g., human GITR), comprises (a) a heavy chain comprising the amino acid sequence of SEQ ID NO:29 with an amino acid substitution of N to A or Q at amino acid position 297, numbered according to the EU numbering system; and (b) a light chain comprising the amino acid sequence of SEQ ID NO:37.
  • an antibody provided herein which specifically binds to GITR (e.g., human GITR), comprises (a) a heavy chain comprising the amino acid sequence of SEQ ID NO:29 with an amino acid substitution selected from the group consisting of: S to E at amino acid position 267, L to F at amino acid position 328, and both S to E at amino acid position 267 and L to F at amino acid position 328, numbered according to the EU numbering system; and (b) a light chain comprising the amino acid sequence of SEQ ID NO:37.
  • an antigen-binding domain described herein which immunospecifically binds to GITR (e.g., human GITR), 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
  • an antigen-binding domain 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.
  • CDRs from antigen-specific non-human antibodies are grafted onto homologous human or non-human primate acceptor frameworks.
  • the non-human primate acceptor frameworks are from Old World apes.
  • the Old World ape acceptor framework is from Pan troglodytes, Pan paniscus or Gorilla gorilla.
  • the non-human primate acceptor frameworks are from the chimpanzee Pan troglodytes.
  • the non-human primate acceptor frameworks are Old World monkey acceptor frameworks.
  • the Old World monkey acceptor frameworks are from the genus Macaca.
  • the non-human primate acceptor frameworks are is derived from the cynomolgus monkey Macaca cynomolgus. Non-human primate framework sequences are described in U.S. Patent Application Publication No. US 2005/0208625.
  • antibodies that contain antigen-binding domains that bind the same or an overlapping epitope of GITR (e.g., an epitope of human GITR) as an antibody described herein (e.g., antibody of pabl876w, pabl967w, pabl975w, or pabl979w).
  • the epitope of an antibody can be determined by, e.g., NMR spectroscopy, X-ray diffraction crystallography studies, ELISA assays, hydrogen/deuterium exchange coupled with mass spectrometry (e.g., liquid chromatography electrospray mass spectrometry), array-based oligo-peptide scanning assays, and/or mutagenesis mapping (e.g., site-directed mutagenesis mapping).
  • crystallization may be accomplished using any of the known methods in the art (e.g., Giege R et al, (1994) Acta Crystallogr D Biol Crystallogr 50(Pt 4): 339-350; McPherson A (1990) Eur J Biochem 189: 1-23; Chayen NE (1997) Structure 5: 1269-1274; McPherson A (1976) J Biol Chem 251 : 6300- 6303).
  • Antibody antigen crystals may be studied using well known X-ray diffraction techniques and may be refined using computer software such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc.; see, e.g., Meth Enzymol (1985) volumes 114 & 115, eds Wyckoff HW et al; U.S. Patent Application No.
  • the epitope of an antigen-binding domain is determined using alanine scanning mutagenesis studies.
  • antigen-binding domains that recognize and bind to the same or overlapping epitopes of GITR can be identified using routine techniques such as an immunoassay, for example, by showing the ability of one antibody to block the binding of another antibody to a target antigen, i.e., a competitive binding assay.
  • Competition binding assays also can be used to determine whether two antibodies have similar binding specificity for an epitope.
  • Competitive binding can be determined in an assay in which the immunoglobulin under test inhibits specific binding of a reference antibody to a common antigen, such as GITR.
  • RIA solid phase direct or indirect radioimmunoassay
  • EIA solid phase direct or indirect enzyme immunoassay
  • sandwich competition assay see Stahli C et al, (1983) Methods Enzymol 9: 242-253
  • solid phase direct biotin-avidin EIA see Kirkland TN et al, (1986) J Immunol 137: 3614-9
  • solid phase direct labeled assay solid phase direct labeled sandwich assay
  • solid phase direct label RIA using 1-125 label (see Morel GA et al, (1988) Mol Immunol 25(1): 7-15); solid phase direct biotin-avidin EIA (Cheung RC et al, (1990) Virology 176: 546- 52); and direct labeled RIA.
  • such an assay involves the use of purified antigen (e.g., GITR, such as human GITR) bound to a solid surface or cells bearing either of these, an unlabeled test immunoglobulin and a labeled reference immunoglobulin.
  • GITR purified antigen
  • Competitive inhibition can be measured by determining the amount of label bound to the solid surface or cells in the presence of the test immunoglobulin.
  • the test immunoglobulin is present in excess.
  • a competing antibody is present in excess, it will inhibit specific binding of a reference antibody to a common antigen by at least 50-55%, 55-60%, 60-65%, 65-70%), 70-75%) or more.
  • a competition binding assay can be configured in a large number of different formats using either labeled antigen or labeled antibody.
  • the antigen is immobilized on a 96-well plate.
  • the ability of unlabeled antibodies to block the binding of labeled antibodies to the antigen is then measured using radioactive or enzyme labels.
  • a competition assay is performed using surface plasmon resonance (BIAcore ® ), e.g., by an 'in tandem approach' such as that described by Abdiche YN et al, (2009) Analytical Biochem 386: 172-180, whereby GITR antigen is immobilized on the chip surface, for example, a CM5 sensor chip and the anti-GITR antibodies are then run over the chip.
  • GITR antigen is immobilized on the chip surface, for example, a CM5 sensor chip and the anti-GITR antibodies are then run over the chip.
  • the antibody containing the anti-GITR antigen-binding domain is first run over the chip surface to achieve saturation and then the potential, competing antibody is added. Binding of the competing antibody can then be determined and quantified relative to a non-competing control.
  • competition binding assays can be used to determine whether an antibody is competitively blocked, e.g., in a dose dependent manner, by another antibody for example, an antibody binds essentially the same epitope, or overlapping epitopes, as a reference antibody, when the two antibodies recognize identical or sterically overlapping epitopes in competition binding assays such as competition ELISA assays, which can be configured in all number of different formats, using either labeled antigen or labeled antibody.
  • an antibody can be tested in competition binding assays with an antibody described herein ⁇ e.g., antibody of pabl876w, pabl967w, pabl975w, or pabl979w), or a chimeric or Fab antibody thereof, or an antibody comprising VH CDRs and VL CDRs of an antibody described herein ⁇ e.g., antibody of pabl876w, pabl967w, pabl975w, or pabl979w).
  • antigen-binding domains that compete ⁇ e.g., in a dose dependent manner) for binding to GITR ⁇ e.g., human GITR) with an antigen-binding domain described herein, as determined using assays known to one of skill in the art or described herein (e.g., ELISA competitive assays or surface plasmon resonance).
  • antigen-binding domains that competitively inhibit (e.g., in a dose dependent manner) an antigen-binding domain described herein (e.g., an antigen-binding domain of pab l 876w, pab l967w, pab l975w, or pab l979w) from binding to GITR (e.g., human GITR), as determined using assays known to one of skill in the art or described herein (e.g., ELISA competitive assays, or suspension array or surface plasmon resonance assay).
  • GITR e.g., human GITR
  • an antigen-binding domain which competes (e.g., in a dose dependent manner) for specific binding to GITR (e.g., human GITR), with an antigen-binding domain comprising the amino acid sequences described herein (e.g., VL and/or VH amino acid sequences of pab l 876w, pab l967w, pab l975w, or pab l979w), as determined using assays known to one of skill in the art or described herein (e.g., ELISA competitive assays, or suspension array or surface plasmon resonance assay).
  • GITR e.g., human GITR
  • an antigen-binding domain comprising the amino acid sequences described herein (e.g., VL and/or VH amino acid sequences of pab l 876w, pab l967w, pab l975w, or pab l979w), as determined using assays known to one of skill in the art or
  • an antigen-binding domain that competes with an antigen-binding domain described herein for binding to GITR (e.g., human GITR) to the same extent that the antigen-binding domain described herein self-competes for binding to GITR (e.g., human GITR).
  • a first antigen-binding domain that competes with an antigen-binding domain described herein for binding to GITR (e.g., human GITR), wherein the first antigen-binding domain competes for binding in an assay comprising the following steps: (a) incubating GITR-transfected cells with the first antigen-binding domain in unlabeled form in a container; and (b) adding an antigen-binding domain described herein in labeled form in the container and incubating the cells in the container; and (c) detecting the binding of the antigen-binding domain described herein in labeled form to the cells.
  • GITR e.g., human GITR
  • a first antigen-binding domain that competes with an antigen- binding domain described herein for binding to GITR (e.g., human GITR), wherein the competition is exhibited as reduced binding of the first antigen-binding domain to GITR by more than 80% (e.g., 85%, 90%, 95%, or 98%, or between 80% to 85%, 80% to 90%, 85% to 90%, or 85% to 95%).
  • an antigen-binding domain which competes (e.g., in a dose dependent manner) for specific binding to GITR (e.g., human GITR), with an antigen-binding domain comprising a VH domain and a VL domain, respectively comprising the amino acid sequences set forth in SEQ ID NOs: 18 and 19, SEQ ID NOs: 20 and 21, SEQ ID NOs: 22 and 23, SEQ ID NOs: 24 and 23, or SEQ ID NOs: 25 and 26, respectively.
  • an antigen-binding domain which competes (e.g., in a dose dependent manner) for specific binding to GITR (e.g., human GITR), with an antigen-binding domain comprising (i) a VL domain comprising a VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of the VL CDRs listed in Table 4; and (ii) a VH domain comprising a VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of the CDRs listed in Table 5.
  • an antigen-binding domain described herein is one that is competitively blocked (e.g., in a dose dependent manner) by an antigen-binding domain comprising a VH domain and a VL domain, respectively comprising the amino acid sequences set forth in SEQ ID NOs: 18 and 19, SEQ ID NOs: 20 and 21, SEQ ID NOs: 22 and 23, SEQ ID NOs: 24 and 23, or SEQ ID NOs: 25 and 26, respectively, for specific binding to GITR (e.g., human GITR).
  • GITR e.g., human GITR
  • an antigen-binding domain described herein is one that is competitively blocked (e.g., in a dose dependent manner) by an antigen-binding domain comprising (i) a VL domain comprising a VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of the CDRs listed in Table 4; and (ii) a VH domain comprising a VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of the CDRs listed in Table 5.
  • an antigen-binding domain which immunospecifically binds to the same epitope as that of pab l 876w, pab l967w, pab l975w, or pab l979wfor specific binding to GITR (e.g., human GITR).
  • GITR e.g., human GITR.
  • Assays known to one of skill in the art or described herein e.g., X-ray crystallography, hydrogen/deuterium exchange coupled with mass spectrometry (e.g., liquid chromatography electrospray mass spectrometry), alanine scanning, ELISA assays, etc.
  • mass spectrometry e.g., liquid chromatography electrospray mass spectrometry
  • alanine scanning e.g., ELISA assays, etc.
  • an antigen-binding domain described herein immunospecifically binds to the same epitope as that bound by pab l 876w, pab l967w, pab l975w, or pab l979wor an epitope that overlaps the epitope.
  • an antigen-binding domain described herein immunospecifically binds to the same epitope as that of an antigen-binding domain comprising (i) a VL domain comprising a VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of the CDRs listed in Table 4 and (ii) a VH domain comprising a VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of the CDRs listed in Table 5.
  • the binding between an antigen-binding domain described herein and a variant GITR is substantially weakened relative to the binding between the antigen-binding domain and a human GITR sequence of residues 26 to 241 of SEQ ID NO: 41, wherein the variant GITR comprises the sequence of residues 26 to 241 of SEQ ID NO: 41 except for an amino acid mutation (e.g., substitution) selected from the group consisting of: D60A and G63A, numbered according to SEQ ID NO: 41.
  • an amino acid mutation e.g., substitution
  • an antigen-binding domain described herein binds to an epitope of human GITR comprising, consisting essentially of, or consisting of a residue selected from the group consisting of residues 60-63 of SEQ ID NO: 41.
  • an antigen-binding domain described herein binds to at least one residue of SEQ ID NO: 41 selected from the group consisting of residues 60-63 of SEQ ID NO:41. In some embodiments, an antigen-binding domain described herein binds to at least one residues selected from the group consisting of residues 60, 62, and 63 of SEQ ID NO:41.
  • an antigen-binding domain described herein exhibits, as compared to binding to a human GITR sequence of residues 26 to 241 of SEQ ID NO: 41, reduced or absent binding to a protein identical to residues 26 to 241 of SEQ ID NO: 41 except for the presence of an amino acid mutation (e.g., substitution) selected from the group consisting of: D60A and G63A, numbered according to SEQ ID NO: 41.
  • an amino acid mutation e.g., substitution
  • one, two, or more mutations are introduced into the Fc region of an antibody described herein (e.g., CH2 domain (residues 231-340 of human IgGi) and/or CH3 domain (residues 341-447 of human IgGi) and/or the hinge region, with numbering according to the EU numbering system to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding and/or antigen-dependent cellular cytotoxicity.
  • an antibody described herein e.g., CH2 domain (residues 231-340 of human IgGi) and/or CH3 domain (residues 341-447 of human IgGi) and/or the hinge region, with numbering according to the EU numbering system to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding and/or antigen-dependent cellular cytotoxicity.
  • one, two, or more mutations are introduced into the hinge region of the Fc region (CHI domain) such that the number of cysteine residues in the hinge region are altered (e.g., increased or decreased) as described in, e.g., U.S. Patent No. 5,677,425.
  • the number of cysteine residues in the hinge region of the CHI domain may be altered to, e.g., facilitate assembly of the light and heavy chains, or to alter (e.g., increase or decrease) the stability of the antibody.
  • one, two, or more mutations are introduced into the Fc region of an antibody described herein (e.g., CH2 domain (residues 231-340 of human IgGi) and/or CH3 domain (residues 341-447 of human IgGi) and/or the hinge region, with numbering according to the EU numbering system to increase or decrease the affinity of the antibody for an Fc receptor (e.g., an activated Fc receptor) on the surface of an effector cell.
  • an Fc receptor e.g., an activated Fc receptor
  • Mutations in the Fc region of an antibody that decrease or increase the affinity of an antibody for an Fc receptor and techniques for introducing such mutations into the Fc receptor or fragment thereof are known to one of skill in the art. Examples of mutations in the Fc receptor of an antibody that can be made to alter the affinity of the antibody for an Fc receptor are described in, e.g., Smith P et al, (2012) PNAS 109: 6181-6186, U.S. Patent No. 6,737,056, and International Publication Nos. WO 02/060919; WO 98/23289; and WO 97/34631, which are incorporated herein by reference.
  • one, two, or more amino acid mutations are introduced into an IgG constant domain, or FcRn-binding fragment thereof (preferably an Fc or hinge-Fc domain fragment) to alter (e.g., decrease or increase) half- life of the antibody in vivo.
  • an IgG constant domain, or FcRn-binding fragment thereof preferably an Fc or hinge-Fc domain fragment
  • one, two or more amino acid mutations are introduced into an IgG constant domain, or FcRn- binding fragment thereof (preferably an Fc or hinge-Fc domain fragment) to decrease the half- life of the antibody in vivo.
  • one, two or more amino acid mutations are introduced into an IgG constant domain, or FcRn- binding fragment thereof (preferably an Fc or hinge-Fc domain fragment) to increase the half-life of the antibody in vivo.
  • the antibodies may have one or more amino acid mutations (e.g., substitutions) in the second constant (CH2) domain (residues 231-340 of human IgGi) and/or the third constant (CH3) domain (residues 341-447 of human IgGi), with numbering according to the EU numbering system.
  • the constant region of the IgGi of an antibody described herein comprises a methionine (M) to tyrosine (Y) substitution in position 252, a serine (S) to threonine (T) substitution in position 254, and a threonine (T) to glutamic acid (E) substitution in position 256, numbered according to the EU numbering system.
  • M methionine
  • Y tyrosine
  • S serine
  • T threonine
  • E glutamic acid
  • an antibody comprises an IgG constant domain comprising one, two, three or more amino acid substitutions of amino acid residues at positions 251-257, 285-290, 308-314, 385-389, and 428-436, numbered according to the EU numbering system.
  • one, two, or more amino acid substitutions are introduced into an IgG constant domain Fc region to alter the effector function(s) of the antibody.
  • one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320 and 322, numbered according to the EU numbering system can be replaced with a different amino acid residue such that the antibody has an altered affinity for an effector ligand but retains the antigen-binding ability of the parent antibody.
  • the effector ligand to which affinity is altered can be, for example, an Fc receptor or the CI component of complement. This approach is described in further detail in U.S. Patent Nos. 5,624,821 and 5,648,260.
  • the deletion or inactivation (through point mutations or other means) of a constant region domain may reduce Fc receptor binding of the circulating antibody. See, e.g., U.S. Patent Nos. 5,585,097 and 8,591,886 for a description of mutations that delete or inactivate the constant domain.
  • one or more amino acid substitutions may be introduced into the Fc region of an antibody described herein to remove potential glycosylation sites on Fc region, which may reduce Fc receptor binding ⁇ see, e.g., Shields RL et al, (2001) J Biol Chem 276: 6591-604).
  • one or more of the following mutations in the constant region of an antibody described herein may be made: an N297A substitution; an N297Q substitution; a L235A substitution and a L237A substitution; a L234A substitution and a L235A substitution; a E233P substitution; a L234V substitution; a L235A substitution; a C236 deletion; a P238A substitution; a D265A substitution; a A327Q substitution; or a P329A substitution, numbered according to the EU numbering system.
  • an antibody described herein comprises the constant domain of an IgGi with an N297Q or N297A amino acid substitution, numbered according to the EU numbering system.
  • an antibody described herein comprises the constant domain of an IgGi with D265A and P329A amino acid substitutions, numbered according to the EU numbering system.
  • one or more amino acids selected from amino acid residues 329, 331, and 322 in the constant region of an antibody described herein, numbered according to the EU numbering system can be replaced with a different amino acid residue such that the antibody has altered Clq binding and/or reduced or abolished complement dependent cytotoxicity (CDC).
  • CDC complement dependent cytotoxicity
  • one or more amino acid residues within amino acid positions 231 to 238, numbered according to the EU numbering system, in the N-terminal region of the CH2 domain of an antibody described herein are altered to thereby alter the ability of the antibody to fix complement. This approach is described further in International Publication No.
  • the Fc region of an antibody described herein is modified to increase the ability of the antibody to mediate antibody dependent cellular cytotoxicity (ADCC) and/or to increase the affinity of the antibody for an Fey receptor by mutating one or more amino acids (e.g., introducing amino acid substitutions) at the following positions: 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 328, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414, 416, 419, 430,
  • an antibody described herein comprises the constant domain of an IgGi with a mutation (e.g., substitution) at position 267, 328, or a combination thereof, numbered according to the EU numbering system.
  • an antibody described herein comprises the constant domain of an IgGi with a mutation (e.g., substitution) selected from the group consisting of S267E, L328F, and a combination thereof, numbered according to the EU numbering system.
  • an antibody described herein comprises the constant domain of an IgGi with a S267E/L328F mutation (e.g., substitution), numbered according to the EU numbering system.
  • an antibody described herein comprising the constant domain of an IgGi with a S267E/L328F mutation (e.g., substitution) has an increased binding affinity for FcyRIIA, FcyRIIB, or FcyRIIA and FcyRIIB, numbered according to the EU numbering system.
  • an antibody described herein comprises the constant region of an IgG 4 antibody and the serine at amino acid residue 228 of the heavy chain, numbered according to the EU numbering system, is substituted for proline.
  • an antibody described herein comprises the constant region of an IgG 2 antibody and the cysteine at amino acid residue 127 of the heavy chain, numbered according to Kabat, is substituted for serine.
  • Antibodies with reduced fucose content have been reported to have an increased affinity for Fc receptors, such as, e.g., FcyRIIIa. Accordingly, in certain embodiments, the antibodies described herein have reduced fucose content or no fucose content.
  • Such antibodies can be produced using techniques known to one skilled in the art. For example, the antibodies can be expressed in cells deficient or lacking the ability of fucosylation. In a specific example, cell lines with a knockout of both alleles of al,6-fucosyltransferase can be used to produce antibodies with reduced fucose content.
  • the Potelligent ® system (Lonza) is an example of such a system that can be used to produce antibodies with reduced fucose content.
  • antibodies with reduced fucose content or no fucose content can be produced by, e.g. : (i) culturing cells under conditions which prevent or reduce fucosylation; (ii) posttranslational removal of fucose (e.g., with a fucosidase enzyme); (iii) post-translational addition of the desired carbohydrate, e.g., after recombinant expression of a non-glycosylated glycoprotein; or (iv) purification of the glycoprotein so as to select for antibodies thereof which are not fucsoylated.
  • fucose e.g., with a fucosidase enzyme
  • post-translational addition of the desired carbohydrate e.g., after recombinant expression of a non-glycosylated glycoprotein
  • purification of the glycoprotein so as to select for antibodies thereof which are not fucsoylated.
  • Engineered glycoforms may be useful for a variety of purposes, including but not limited to enhancing or reducing effector function.
  • Methods for generating engineered glycoforms in an antibody described herein include but are not limited to those disclosed, e.g., in Umana P et al, (1999) Nat Biotechnol 17: 176-180; Davies J et al, (2001) Biotechnol Bioeng 74: 288-294; Shields RL et al, (2002) J Biol Chem 277: 26733-26740; Shinkawa T et al, (2003) J Biol Chem 278: 3466-3473; Niwa R et a/., (2004) Clin Cancer Res 1 : 6248-6255; Presta LG et al, (2002) Biochem Soc Trans 30: 487-490; Kanda Y et al, (2007) Glycobiology 17: 104-118; U.S.
  • the technology used to engineer the Fc domain of an antibody described herein is the Xmab ® Technology of Xencor (Monrovia, CA). See, e.g., U.S. Patent Nos. 8,367,805; 8,039,592; 8, 124,731; 8, 188,231; U.S. Patent Publication No. 2006/0235208; International Publication Nos. WO 05/077981; WO 11/097527; and Richards JO et al, (2008) Mol Cancer Ther 7: 2517-2527.
  • amino acid residues in the constant region of an antibody described herein in the positions corresponding to positions L234, L235, and D265 in a human IgGl heavy chain, numbered according to the EU numbering system are not L, L, and D, respectively. This approach is described in detail in International Publication No. WO 14/108483.
  • the amino acids corresponding to positions L234, L235, and D265 in a human IgGl heavy chain are F, E, and A; or A, A, and A, respectively, numbered according to the EU numbering system.
  • any of the constant region mutations or modifications described herein can be introduced into one or both heavy chain constant regions of an antibody described herein having two heavy chain constant regions.
  • the DuoBody (Genmab A/S) technology can be used to produce a heterodimeric protein by combining one half of a first homodimeric protein ⁇ e.g., one heavy and light chain pair of a first antibody) with one half of a second homodimeric protein ⁇ e.g., one heavy and light chain pair of a second antibody, or one polypeptide of a homodimeric fragment of the second antibody containing a constant region with CH3 domain residues).
  • a first homodimeric protein ⁇ e.g., one heavy and light chain pair of a first antibody
  • a second homodimeric protein e.g., one heavy and light chain pair of a second antibody
  • polypeptide of a homodimeric fragment of the second antibody containing a constant region with CH3 domain residues e.g., International Publication Nos. WO 2011/131746, WO 2011/147986, WO 2008/119353, and WO 2013/060867, and Labrijn AF
  • an antibody as described herein which immunospecifically binds to GITR ⁇ e.g., human GITR, comprises: (a) a first antigen-binding domain that specifically binds to GITR ⁇ e.g., human GITR), as described herein, comprising a first heavy chain constant region; and (b) a second antigen-binding domain that does not bind to an antigen expressed by a human immune cell ⁇ i.e., the second antigen-binding domain does not bind to GITR or any other antigen expressed by a human immune cell), as described herein, comprising a second heavy chain constant region; wherein each heavy chain constant region comprises a mutation at a residue selected from the group consisting of residues 366, 368, 370, 399, 405, 407, and 409, numbered according to the EU numbering system, and wherein the first heavy chain constant region and the second heavy
  • the antigen to which the second antigen-binding domain specifically binds is not naturally expressed by a human immune cell.
  • the human immune cell is selected from the group consisting of a T cell ⁇ e.g., a CD4+ T cell or a CD8+ T cell), a B cell, a natural killer cell, a dendritic cell, a macrophage, and an eosinophil.
  • the antigen-binding domain that specifically binds to GITR ⁇ e.g., human GITR
  • the second antigen-binding domain comprises a second VH and a second VL.
  • the antigen-binding domain that specifically binds to GITR ⁇ e.g., human GITR) comprises a first heavy chain and a first light chain
  • the second antigen-binding domain comprises a second heavy chain and a second light chain.
  • the antibody is for administration to a sample or subject in which the second antigen-binding domain is non-reactive ⁇ i.e., the antigen to which the second antigen-binding domain binds is not present in the sample or subject).
  • the second antigen-binding domain does not specifically bind to an antigen on a cell expressing GITR ⁇ e.g., human GITR).
  • the second antigen-binding domain does not specifically bind to an antigen that is naturally expressed by a cell that expresses GITR ⁇ e.g., human GITR).
  • the antibody functions as a monovalent antibody in a sample or subject, wherein the first antigen-binding domain of the antibody specifically binds to GITR ⁇ e.g., human GITR), while the second antigen-binding domain is non-reactive in the sample or subject ⁇ e.g., due to the absence of antigen to which the second antigen-binding domain specifically binds in the sample or subject).
  • the second antigen-binding domain specifically binds to a non-human antigen (i.e., an antigen expressed in other organisms and not humans). In certain embodiments, the second antigen-binding domain specifically binds to a viral antigen. In certain embodiments, the viral antigen is from a virus that does not infect humans (i.e., a non-human virus). In certain embodiments, the viral antigen is absent in a human immune cell (e.g., the human immune cell is uninfected with the virus associated with the viral antigen). In certain embodiments, the viral antigen is a HIV antigen. In certain embodiments, the second antigen-binding domain specifically binds to chicken albumin or hen egg lysozyme.
  • the second antigen-binding domain specifically binds to an antigen that is not expressed by (i.e., is absent from) wild-type cells (e.g., wild-type human cells). In certain embodiments, the second antigen- binding domain specifically binds to a tumor-associated antigen that is not expressed by (i.e., is absent from) normal cells (e.g., wild-type cells, e.g., wild-type human cells). In certain embodiments, the tumor-associated antigen is not expressed by (i.e., is absent from) human cells.
  • the second antigen-binding domain comprises a heavy chain comprising a mutation selected from the group consisting of: N297A, D265A, L234F, L235E, N297Q, P331 S, and a combination thereof, numbered according to the EU numbering system.
  • the mutation is N297A or D265A, numbered according to the EU numbering system.
  • the mutation is L234F and L235E, numbered according to the EU numbering system.
  • the mutation is L234F, L234E, and D265A, numbered according to the EU numbering system.
  • the mutation is L234F, L234E, and N297Q, numbered according to the EU numbering system. In certain embodiments, the mutation is L234F, L235E, and P331 S, numbered according to the EU numbering system. In certain embodiments, the mutation is D265A and N297Q, numbered according to the EU numbering system. In certain embodiments, the mutation is L234F, L235E, D265A, N297Q, and P331 S, numbered according to the EU numbering system.
  • the second antigen-binding domain comprises a heavy chain comprising a mutation selected from the group consisting of: D265A, P329A, and a combination thereof, numbered according to the EU numbering system.
  • the heavy chain constant regions of the first and second antigen-binding domains are selected from the group consisting of immunoglobulins IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2.
  • the immunoblobulins are human immunoglobulins. Human immunoglobulins containing mutations (e.g., substitutions) are also referred to as human immunoglobulins herein.
  • the heavy chain constant regions of the first and second antigen-binding domains are the same isotype.
  • the sequences associated with the second antigen-binding domain are also described herein as "isotype" sequences (e.g., isotype VH or isotype HC).
  • the heavy chain constant regions of the first and second antigen-binding domains are IgGl .
  • the heavy chain constant regions of the first and second antigen-binding domains are human IgGl .
  • the first antigen-binding domain comprises a first IgGl heavy chain constant region and the second antigen-binding domain comprises a second IgGl heavy chain constant region, wherein the first and second heavy chains comprise an identical mutation selected from the group consisting of N297A, N297Q, D265A, L234F/L235E/D265A, and a combination thereof, numbered according to the EU numbering system.
  • the first antigen-binding domain comprises a first IgGl heavy chain constant region and the second antigen-binding domain comprises a second IgGl heavy chain constant region, wherein the first and second heavy chains comprise an identical mutation selected from the group consisting of D265A, P329A, and a combination thereof, numbered according to the EU numbering system.
  • the first antigen-binding domain comprises a first IgG2 heavy chain constant region and the second antigen-binding domain comprises a second IgG2 heavy chain constant region, wherein the first and second heavy chain constant regions comprise a C127S mutation, numbered according to Kabat.
  • the first antigen-binding domain comprises a first IgG4 heavy chain constant region and the second antigen-binding domain comprises a second IgG4 heavy chain constant region, wherein the first and second heavy chain constant regions comprise a S228P mutation, numbered according to the EU numbering system.
  • an antibody as described herein which immunospecifically binds to GITR (e.g., human GITR), comprises: (a) an antigen-binding domain that specifically binds to GITR, as described herein, wherein the antigen-binding domain comprises a first heavy chain constant region; and (b) a heavy chain or fragment thereof, as described herein, wherein the heavy chain or fragment thereof comprises a second heavy chain constant region; wherein each of the first and second heavy chain constant regions comprises a mutation at a residue selected from the group consisting of residues 366, 368, 370, 399, 405, 407, and 409, numbered according to the EU numbering system, and wherein the first heavy chain constant region and the second heavy chain constant region contain different mutations.
  • Such an antibody can optionally comprise a first light chain or fragment thereof and a second light chain or fragment thereof.
  • the first light chain can comprise a first light chain constant domain and a first light chain variable domain.
  • the second light chain can comprise a second light chain constant domain and a second light chain variable domain.
  • the fragment of the second heavy chain is an Fc fragment.
  • the heavy chain or second heavy chain comprises a constant domain and a variable domain.
  • the second heavy chain or fragment thereof is from an antigen-binding domain that specifically binds to a non-human antigen (i.e., an antigen expressed in other organisms and not humans).
  • the second heavy chain or fragment thereof is from an antigen-binding domain that specifically binds to a viral antigen.
  • the viral antigen is from a virus that does not infect humans (i.e., a non-human virus).
  • the viral antigen is absent in a human immune cell (e.g., the human immune cell is uninfected with the virus associated with the viral antigen).
  • the viral antigen is a HIV antigen.
  • the second heavy chain or fragment thereof is from an antigen-binding domain that specifically binds to chicken albumin or hen egg lysozyme.
  • the second heavy chain or fragment thereof is from an antigen-binding domain that specifically binds to an antigen that is not expressed by (i.e., is absent from) wild- type cells (e.g., wild-type human cells).
  • the second heavy chain or fragment thereof is from an antigen-binding domain that specifically binds to a tumor-associated antigen that is not expressed by (i.e., is absent from) normal cells (e.g., wild-type cells, e.g., wild-type human cells).
  • the tumor-associated antigen is not expressed by (i.e., is absent from) human cells.
  • the second heavy chain comprises a mutation selected from the group consisting of: N297A, D265A, L234F, L235E, N297Q, P331 S, and a combination thereof, numbered according to the EU numbering system.
  • the mutation is N297A or D265A, numbered according to the EU numbering system.
  • the mutation is L234F and L235E, numbered according to the EU numbering system.
  • the mutation is L234F, L234E, and D265A, numbered according to the EU numbering system.
  • the mutation is L234F, L234E, and N297Q, numbered according to the EU numbering system.
  • the mutation is L234F, L235E, and P331 S, numbered according to the EU numbering system. In certain embodiments, the mutation is D265A and N297Q, numbered according to the EU numbering system. In certain embodiments, the mutation is L234F, L235E, D265A, N297Q, and P331 S, numbered according to the EU numbering system. In certain embodiments, the second heavy chain comprises a mutation selected from the group consisting of: D265A, P329A, and a combination thereof, numbered according to the EU numbering system.
  • the first and second heavy chain constant regions are selected from the group consisting of immunoglobulins IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2.
  • the immunoblobulins are human immunoglobulins.
  • the first and second heavy chain constant regions are the same isotype.
  • the sequences associated with the second heavy chain are also described herein as "isotype" sequences (e.g., isotype VH or isotype HC).
  • the first and second heavy chain constant regions are IgGl .
  • the first and second heavy chain constant regions are human IgGl .
  • the first antigen-binding domain comprises a first IgGl heavy chain constant region and the second antigen-binding domain comprises a second IgGl heavy chain constant region, wherein the first and second heavy chains comprise an identical mutation selected from the group consisting of N297A, N297Q, D265A, L234F/L235E/D265A, and a combination thereof, numbered according to the EU numbering system.
  • the first antigen-binding domain comprises a first IgGl heavy chain constant region and the second antigen-binding domain comprises a second IgGl heavy chain constant region, wherein the first and second heavy chains comprise an identical mutation selected from the group consisting of D265A, P329A, and a combination thereof, numbered according to the EU numbering system.
  • the first antigen-binding domain comprises a first IgG2 heavy chain constant region and the second antigen-binding domain comprises a second IgG2 heavy chain constant region, wherein the first and second heavy chain constant regions comprise a C127S mutation, numbered according to Kabat.
  • the first antigen-binding domain comprises a first IgG4 heavy chain constant region and the second antigen-binding domain comprises a second IgG4 heavy chain constant region, wherein the first and second heavy chain constant regions comprise a S228P mutation, numbered according to the EU numbering system.
  • the antigen-binding domain that specifically binds to GITR can comprise any of the anti-GITR sequences described herein.
  • the antigen-binding domain that specifically binds to GITR comprises: (a) a first heavy chain variable domain (VH) comprising a VH- complementarity determining region (CDR) 1 comprising the amino acid sequence of SEQ ID NO:7; a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 10; and a VH-CDR3 comprising the amino acid sequence of SEQ ID NO:3; and (b) a first light chain variable domain (VL) comprising a VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 14; a VL- CDR2 comprising the amino acid sequence of SEQ ID NO:5; and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 16).
  • VH first heavy chain variable domain
  • CDR complementarity determining region
  • the antigen-binding domain that specifically binds to GITR (e.g., human GITR) specifically binds to the same epitope of GITR as an antibody comprising a VH comprising the amino acid sequence of SEQ ID NO: 18 and a VL comprising the amino acid sequence of SEQ ID NO: 19.
  • the antigen-binding domain that specifically binds to GITR exhibits, as compared to binding to a human GITR sequence of residues 26 to 241 of SEQ ID NO: 41, reduced or absent binding to a protein identical to residues 26 to 241 of SEQ ID NO: 41 except for the presence of an amino acid mutation selected from the group consisting of: D60A or G63A, and a combination thereof, numbered according to SEQ ID NO: 41.
  • the antigen-binding domain that specifically binds to GITR comprises a VH and a VL, wherein the VH comprises the amino acid sequence of SEQ ID NO: 18.
  • the antigen-binding domain that binds to GITR comprises a VH and a VL, wherein the VL comprises the amino acid sequence SEQ ID NO: 19.
  • the antigen-binding domain that specifically binds to GITR comprises a VH comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence of SEQ ID NO: 18.
  • the antigen-binding domain that specifically binds to GITR comprises a VH comprising the amino acid sequence of SEQ ID NO: 18.
  • the antigen- binding domain that specifically binds to GITR comprises a VH comprising an amino acid sequence derived from a human IGHV1-2 germline sequence (e.g., IGHVl-2*02, e.g., having amino acid sequence of SEQ ID NO: 27).
  • the antigen- binding domain that specifically binds to GITR comprises a VL comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence of SEQ ID NO: 19.
  • the antigen-binding domain that specifically binds to GITR comprises a VL comprising the amino acid sequence of SEQ ID NO: 19. In certain embodiments, the antigen-binding domain that specifically binds to GITR (e.g., human GITR) comprises a light chain comprising the amino acid sequence of SEQ ID NO:37. In certain embodiments, the antigen-binding domain that specifically binds to GITR (e.g., human GITR) comprises a light chain comprising the amino acid sequence of SEQ ID NO:38.
  • the antigen-binding domain that specifically binds to GITR comprises a VL comprising an amino acid sequence derived from a human IGKV4-1 germline sequence (e.g., IGKV4-1 *01, e.g., having amino acid sequence of SEQ ID NO: 28).
  • the antigen-binding domain that specifically binds to GITR comprises the VH and VL sequences set forth in SEQ ID NOs: 18 and 19, 20 and 21, 22 and 23, or 24 and 23, respectively.
  • the antigen-binding domain that specifically binds to GITR comprises a constant region containing a mutation selected from the group consisting of: N297A, D265A, L234F, L235E, N297Q, P331 S, and a combination thereof, numbered according to the EU numbering system.
  • the mutation is N297A or D265A, numbered according to the EU numbering system.
  • the mutation is L234F and L235E, numbered according to the EU numbering system.
  • the mutation is L234F, L234E, and D265A, numbered according to the EU numbering system.
  • the mutation is L234F, L234E, and N297Q, numbered according to the EU numbering system. In certain embodiments, the mutation is L234F, L235E, and P331 S, numbered according to the EU numbering system. In certain embodiments, the mutation is D265A and N297Q, numbered according to the EU numbering system. In certain embodiments, the mutation is L234F, L235E, D265A, N297Q, and P331 S, numbered according to the EU numbering system.
  • the antigen-binding domain that specifically binds to GITR comprises a constant region containing a mutation selected from the group consisting of: D265A, P329A, and a combination thereof, numbered according to the EU numbering system.
  • the first heavy chain constant region comprises a F405L or a K409R mutation
  • the second heavy chain constant region comprises a F405L or a K409R mutation, numbered according to the EU numbering system
  • the first heavy chain constant region and the second heavy chain constant region contain different mutations.
  • the first heavy chain constant region comprises a F405L mutation
  • the second heavy chain constant region comprises a K409R mutation, numbered according to the EU numbering system.
  • the first heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 108
  • the second heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 109
  • the antigen-binding domain that specifically binds to GITR comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 31.
  • the first heavy chain constant region comprises a K409R mutation
  • the second heavy chain constant region comprises a F405L mutation, numbered according to the EU numbering system.
  • the first heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 109
  • the second heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 108.
  • the antigen-binding domain that specifically binds to GITR comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:34.
  • an antibody provided herein that immunospecifically binds to GITR contains a combination of a heavy chains and a light chain for the anti-GITR antigen- binding domain as shown in a single row of Table 3 below.
  • an antibody described herein is antagonistic to GITR (e.g., human GITR).
  • the antibody deactivates, reduces, or inhibits an activity of GITR (e.g., human GITR).
  • the antibody inhibits or reduces binding of human GITR to GITR ligand (e.g., human GITR ligand).
  • the antibody inhibits or reduces GITR (e.g., human GITR) signaling.
  • the antibody inhibits or reduces GITR (e.g., human GITR) activity (e.g., GITR signaling) induced by GITR ligand (e.g., human GITR ligand).
  • GITR e.g., human GITR
  • an antagonistic antibody described herein inhibits or reduces T cell proliferation.
  • an antagonistic antibody described herein inhibits or reduces T cell proliferation.
  • an antagonistic antibody described herein inhibits or reduces production of cytokines (e.g., inhibits or reduces production of IL-2, TNFa, IFNy, IL-4, IL-10, IL-13, or a combination thereof) by stimulated T cells.
  • an antagonistic antibody described herein blocks the interaction of GITR and GITRL (e.g., blocks the binding of GITRL and GITR to one another, e.g., blocks the binding of human GITR ligand and human GITR)).
  • an antagonistic antibody described herein which immunospecifically binds to GITR (e.g., human GITR), decreases GITR (e.g., human GITR) activity by at least about 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 2 fold, 2.5 fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, or 100 fold as assessed by methods described herein and/or known to one of skill in the art, relative to GITR (e.g., human GITR) activity without any antibody or with an unrelated antibody (e.g., an antibody that does not immunospecifically bind to GITR).
  • GITR e.g., human GITR
  • an antagonistic antibody described herein which immunospecifically binds to GITR (e.g., human GITR), decreases GITR (e.g., human GITR) activity by at least 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 and/or known to one of skill in the art, relative to GITR (e.g., human GITR) activity without any antibody or with an unrelated antibody (e.g., an antibody that does not immunospecifically bind to GITR).
  • GITR e.g., human GITR
  • Non-limiting examples of GITR (e.g., human GITR) activity can include GITR (e.g., human GITR) signaling, cell proliferation, cell survival, and cytokine production (e.g., IL-2, TNF-a, IFN- ⁇ , IL-4, IL-10, and/or IL-13).
  • GITR e.g., human GITR
  • an antagonistic antibody described herein, which immunospecifically binds to GITR e.g., human GITR
  • inhibits, reduces, or inactivates a GITR (e.g., human GITR) activity is assessed as described in the Examples, infra.
  • an antagonistic antibody described herein which immunospecifically binds to GITR (e.g., human GITR), inhibits, reduces, or deactivates the cellular proliferation of cells that express GITR and that respond to GITR signaling (e.g., cells that proliferate in response to GITR stimulation and GITR signaling, such as T cells).
  • GITR e.g., human GITR
  • Cell proliferation assays are described in the art, such as a 3H-thymidine incorporation assay, BrdU incorporation assay, or CFSE assay, and can be readily carried out by one of skill in the art.
  • T cells e.g., CD4+ or CD8+ effector T cells
  • a T cell mitogen or T cell receptor complex stimulating agent e.g., phytohaemagglutinin (PHA) and/or phorbol myristate acetate (PMA), or a TCR complex stimulating antibody, such as an anti-CD3 antibody and anti-CD28 antibody
  • PHA phytohaemagglutinin
  • PMA phorbol myristate acetate
  • a TCR complex stimulating antibody such as an anti-CD3 antibody and anti-CD28 antibody
  • an antagonistic antibody described herein which immunospecifically binds to GITR (e.g., human GITR), decreases the survival of cells (e.g., T cells, such as CD4 and CD8 effector T cells).
  • T cells e.g., CD4+ or CD8+ effector T cells
  • a T cell mitogen or T cell receptor complex stimulating agent e.g., phytohaemagglutinin (PHA) and/or phorbol myristate acetate (PMA), or a TCR complex stimulating antibody, such as an anti-CD3 antibody and anti-CD28 antibody
  • PHA phytohaemagglutinin
  • PMA phorbol myristate acetate
  • TCR complex stimulating antibody such as an anti-CD3 antibody and anti-CD28 antibody
  • an antagonistic antibody described herein which immunospecifically binds to GITR (e.g., human GITR)
  • GITR e.g., human GITR
  • Cell survival assays are described in the art (e.g., a trypan blue exclusion assay) and can be readily carried out by one of skill in the art.
  • an antagonistic antibody described herein which immunospecifically binds to GITR (e.g., human GITR), decreases cell survival (e.g., T cells, such as CD4 and CD 8 effector T cells) by at least about 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 2 fold, 2.5 fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, or 100 fold, as assessed by methods described herein or known to one of skill in the art (e.g., a trypan blue exclusion assay), without any antibody or with an unrelated antibody (e.g., an antibody that does not immunospecifically bind to GITR).
  • cell survival e.g., T cells, such as CD4 and CD 8 effector T cells
  • an antagonistic antibody described herein which immunospecifically binds to GITR (e.g., human GITR), decreases cell survival (e.g., T cells, such as CD4 and CD8 effector T cells) 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., a trypan blue exclusion assay), relative to GITR (e.g., human GITR) activity without any antibody or with an unrelated antibody (e.g., an antibody that does not immunospecifically bind to GITR).
  • cell survival e.g., T cells, such as CD4 and CD8 effector T cells
  • T cells e.g., CD4 + or CD8 + effector T cells
  • a T cell mitogen e.g., an anti-CD3 antibody or phorbol ester
  • an antagonistic antibody described herein which immunospecifically binds to GITR (e.g., human GITR)
  • GITR e.g., human GITR
  • an antagonistic antibody described herein which immunospecifically binds to GITR (e.g., human GITR)
  • GITR e.g., human GITR
  • an antagonistic antibody described herein which immunospecifically binds to GITR (e.g., human GITR)
  • T cells e.g., CD4 + or CD8 + effector T cells
  • a T cell mitogen or T cell receptor complex stimulating agent e.g., phytohaemagglutinin (PHA) and/or phorbol myristate acetate (PMA), or a TCR complex stimulating antibody, such as an anti-CD3 antibody and anti-CD28 antibody
  • PHA phytohaemagglutinin
  • PMA phorbol myristate acetate
  • TCR complex stimulating antibody such as an anti-CD3 antibody and anti-CD28 antibody
  • an antagonistic antibody described herein which immunospecifically binds to GITR (e.g., human GITR)
  • an antagonistic antibody described herein which immunospecifically binds to GITR (e.g., human GITR), does not protect effector T cells (e.g., CD4+ and CD8+ effector T cells) from activation-induced cell death.
  • an antagonistic antibody described herein which immunospecifically binds to GITR (e.g., human GITR), inhibits, reduces, or deactivates cytokine production (e.g., IL-2, T F-a, IFN- ⁇ , IL-4, IL-10, and/or IL-13) 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, relative to cytokine production in the presence or absence of GITRL (e.g., human GITRL) stimulation without any antibody or with an unrelated antibody (e.g., an antibody that does not immunospecifically bind to GITR).
  • cytokine production e.g., IL-2, T F-a, IFN- ⁇ , IL-4, IL-10, and/or IL-13
  • an antagonistic antibody described herein which immunospecifically binds to GITR (e.g., human GITR), inhibits or reduces cytokine production (e.g., IL-2, TNF-a, IFN- ⁇ , IL-4, IL-10, and/or IL-13) by at least about 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 2 fold, 2.5 fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, or 100 fold, as assessed by methods described herein or known to one of skill in the art, relative to cytokine production in the presence or absence of GITRL (e.g., human GITRL) stimulation without any antibody or with an unrelated antibody (e.g., an antibody that does not immunospecifically bind to GITR).
  • cytokine production e.g., IL-2,
  • T cells e.g., CD4+ or CD8+ effector T cells
  • a T cell mitogen or T cell receptor complex stimulating agent e.g., phytohaemagglutinin (PHA) and/or phorbol myristate acetate (PMA), or a TCR complex stimulating antibody, such as an anti-CD3 antibody and anti-CD28 antibody
  • PHA phytohaemagglutinin
  • PMA phorbol myristate acetate
  • a TCR complex stimulating antibody such as an anti-CD3 antibody and anti-CD28 antibody
  • an antagonistic antibody described herein which immunospecifically binds to GITR (e.g., human GITR)
  • cytokine production e.g., IL-2, TNF-a, IFN- ⁇ , IL-4, IL-10, and/or IL-13
  • cytokine production e.g., IL-2, TNF-a, IFN- ⁇ , IL-4, IL-10, and/or IL-13
  • T cells e.g., CD4+ or CD8+ effector T cells
  • a T cell mitogen or T cell receptor complex stimulating agent e.g., phytohaemagglutinin (PHA) and/or phorbol myristate acetate (PMA), or a TCR complex stimulating antibody, such as an anti-CD3 antibody and anti-CD28 antibody
  • PHA phytohaemagglutinin
  • PMA phorbol myristate acetate
  • a TCR complex stimulating antibody such as an anti-CD3 antibody and anti-CD28 antibody
  • an antagonistic antibody described herein which immunospecifically binds to GITR (e.g., human GITR)
  • cytokine production e.g., IL-2, TNF-a, IFN- ⁇ , IL-4, IL-10, and/or IL-13
  • cytokine production e.g., IL-2, TNF-a, IFN- ⁇ , IL-4, IL-10, and/or IL-13
  • An antibody provided herein that binds to GITR can be fused or conjugated (e.g., covalently or noncovalently linked) to a detectable label or substance.
  • detectable labels or substances include enzyme labels, such as, glucose oxidase; radioisotopes, such as iodine ( 125 I, 121 I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( 121 In), and technetium ( 99 Tc); luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, and biotin.
  • detectable labels or substances include enzyme labels, such as, glucose oxidase; radioisotopes, such as iodine ( 125 I, 121 I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( 121 In), and technetium ( 99 Tc); luminescent labels, such as luminol; and fluorescent labels, such as fluor
  • An antibody as described herein is generated according to the DuoBody technology platform (Genmab A/S) as described, e.g., in International Publication Nos. WO 2011/131746, WO 2011/147986, WO 2008/119353, and WO 2013/060867 and in Labrijn AF et al, (2013) PNAS 110(13): 5145-5150.
  • the DuoBody technology is used to create a heterodimeric protein from two homodimeric proteins, and requires that each of the homodimeric proteins includes a heavy chain constant region with a single point mutation in the CH3 domain.
  • the point mutations allow for a stronger interaction between the CH3 domains in the resultant heterodimeric protein than between the CH3 domains in either of the homodimeric proteins.
  • the single point mutation in each homodimeric protein is at residue 366, 368, 370, 399, 405, 407, or 409 in the CH3 domain of the heavy chain constant region, numbered according to the EU numbering system, as described, e.g., in International Publication No. WO 2011/131746.
  • the single point mutation is located at a different residue in one homodimeric protein as compared to the other homodimeric protein.
  • one homodimeric protein can comprise the mutation F405L (i.e., a mutation from phenylalanine to leucine at residue 405), while the other homodimeric protein can comprise the mutation K409R (i.e., a mutation from lysine to arginine at residue 409), numbered according to the EU numbering system.
  • the heavy chain constant regions of the homodimeric proteins can be an IgGi, IgG 2 , IgG 3 , or IgG 4 IgAi, or IgA 2 isotype (e.g., a human IgGi isotype).
  • Antibodies that bind GITR can, in some instances contain, IgG4 and IgGl, IgG4 and IgG2, IgG4 and IgG2, IgG4 and IgG3, or IgGl and IgG3 chain heterodimers.
  • Such heterodimeric heavy chain antibodies can routinely be engineered by, for example, modifying selected amino acids forming the interface of the CH3 domains in human IgG4 and the IgGl or IgG3 so as to favor heterodimeric heavy chain formation.
  • an antibody can be a chimeric antibody or a humanized antibody.
  • an antibody can be an F(ab') 2 fragment.
  • a F(ab') 2 fragment contains the two antigen-binding arms of a tetrameric antibody molecule linked by disulfide bonds in the hinge region.
  • provided herein is a method of making an antibody or an antigen- binding fragment which immunospecifically binds to GITR ⁇ e.g., human GITR) comprising culturing a cell or cells described herein.
  • a method of making an antibody or antigen-binding fragment which immunospecifically binds to GITR ⁇ e.g., human GITR) comprising expressing ⁇ e.g., recombinantly expressing) the antibody or antigen- binding fragment using a cell or host cell described herein ⁇ e.g., a cell or a host cell comprising polynucleotides encoding an antibody described herein).
  • 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 obtained from the cell or host cell.
  • Antigen-binding fragments of antibodies can be prepared, e.g., from monoclonal antibodies, 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 E & Lane D, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling GJ 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.
  • monoclonal antibodies can be produced recombinantly from host cells exogenously expressing an antibody described herein.
  • Monoclonal antibodies described herein can, for example, be made by the hybridoma method as described in Kohler G & Milstein C (1975) Nature 256: 495 or can, e.g., be isolated from phage libraries using the techniques as described herein, for example. Other methods for the preparation of clonal cell lines and of monoclonal antibodies expressed thereby are well known in the art (see, for example, Chapter 11 in: Short Protocols in Molecular Biology, (2002) 5th Ed., Ausubel FM et al, supra).
  • the antibodies or antigen-binding fragments thereof described herein can also be generated using various phage display methods known in the art.
  • phage display methods proteins 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 a 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 antibody or fragment 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.
  • phage display methods that can be used to make the antibodies described herein include those disclosed in Brinkman U et al, (1995) J Immunol Methods 182: 41-50; Ames RS et al, (1995) J Immunol Methods 184: 177-186; Kettleborough CA et al, (1994) Eur J Immunol 24: 952-958; Persic L et al, (1997) Gene 187: 9-18; Burton DR & Barbas CF (1994) Advan Immunol 57: 191-280; PCT Application No. PCT/GB91/001134; International Publication Nos.
  • the antibody coding regions from the phage can be isolated and used to generate antibodies, including human antibodies, 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 antibodies 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.
  • 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.
  • 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 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 SL (1985) Science 229: 1202-7; Oi VT & Morrison SL (1986) BioTechniques 4: 214- 221; Gillies SD et al, (1989) J Immunol Methods 125: 191-202; and U.S. Patent Nos. 5,807,715, 4,816,567, 4,816,397, and 6,331,415.
  • 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 239400; 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.
  • a human antibody comprises an antigen-binding domain described herein which binds to the same epitope of GITR ⁇ e.g., human GITR) as an anti-GITR antigen-binding fragment thereof described herein.
  • a human antibody comprises an antigen-binding domain which competitively blocks ⁇ e.g., in a dose- dependent manner) any one of the antigen-binding domains described herein, ⁇ e.g., pabl876w, pabl967w, pabl975w, or pabl979w) from binding to GITR ⁇ e.g., human GITR).
  • Human antibodies can be produced using any method known in the art.
  • transgenic mice which are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes
  • the human heavy and light chain immunoglobulin gene complexes can be introduced randomly or by homologous recombination into mouse embryonic stem cells.
  • the human variable region, constant region, and diversity region can be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes.
  • the mouse heavy and light chain immunoglobulin genes can be rendered non-functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletion of the 1 ⁇ 2 region prevents endogenous antibody production.
  • the modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice.
  • the chimeric mice are then bred to produce homozygous offspring which express human antibodies.
  • the transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of an antigen (e.g., GITR).
  • Monoclonal antibodies directed against the antigen can be obtained from the immunized, transgenic mice using conventional hybridoma technology.
  • the human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation.
  • mice capable of producing human antibodies include the XenomouseTM (Abgenix, Inc.; U.S. Patent Nos. 6,075, 181 and 6,150, 184), the HuAb- MouseTM (Mederex, Inc./Gen Pharm; U.S. Patent Nos. 5,545,806 and 5,569, 825), the Trans Chromo MouseTM (Kirin) and the KM MouseTM (Medarex/Kirin).
  • Human antibodies or antigen-binding fragments which specifically bind to GITR 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, 4,716, 111, and 5,885,793; 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 immunospecifically bind to a target antigen (e.g., GITR (e.g., human GITR)).
  • GITR e.g., human GITR
  • 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 immunospecifically binds to a GITR (e.g., human GITR) antigen, and vectors, e.g., vectors comprising such polynucleotides for recombinant expression in host cells (e.g., E. coli and mammalian cells).
  • GITR e.g., human GITR
  • vectors e.g., vectors comprising such polynucleotides for recombinant expression in 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, which immunospecifically bind to an GITR polypeptide (e.g., human GITR) and comprises an amino acid sequence as described herein, as well as antibodies that compete with such antibodies for binding to an GITR polypeptide (e.g., in a dose-dependent manner), or which binds to the same epitope as that of such antibodies.
  • an GITR polypeptide e.g., human GITR
  • antibodies that compete with such antibodies for binding to an GITR 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 or light chain variable domain comprising the VL CDRs of antibodies described herein (see, e.g., Table 4).
  • the polynucleotides can comprise nucleotide sequences encoding a heavy chain or heavy chain variable domain comprising the VH CDRs of antibodies described herein (see, e.g., Table 5).
  • a polynucleotide described herein encodes a VL domain comprising the amino acid sequence set forth in SEQ ID NO: 19, 21, 23, and 26. In specific embodiments, a polynucleotide described herein encodes a VH domain comprising the amino acid sequence set forth in SEQ ID NO: 18, 20, 22, 24, or 25.
  • polynucleotides comprising a nucleotide sequence encoding an anti-GITR antigen-binding domain comprising three VL chain CDRs, e.g., containing VL CDRl, VL CDR2, and VL CDR3 of any one of antibodies described herein (e.g., see Table 4).
  • polynucleotides comprising three VH chain CDRs, e.g., containing VH CDRl, VH CDR2, and VH CDR3 of any one of antibodies described herein (e.g., see Table 5).
  • polynucleotides comprising a nucleotide sequence encoding an anti-GITR antigen-binding domain comprising three VL chain CDRs, e.g., containing VL CDRl, VL CDR2, and VL CDR3 of any one of antibodies described herein (e.g., see Table 4) and three VH chain CDRs, e.g., containing VH CDRl, VH CDR2, and VH CDR3 of any one of antibodies described herein (e.g., see Table 5).
  • a polynucleotide described herein comprises a nucleotide sequence encoding an antibody or antigen-binding domain provided herein comprising a light chain variable region comprising an amino acid sequence described herein (e.g., 19, 21, 23, or 26), wherein the antibody or antigen-binding domain immunospecifically binds to GITR (e.g., human GITR).
  • GITR e.g., human GITR
  • a polynucleotide described herein comprises a nucleotide sequence encoding an antibody or antigen-binding domain provided herein comprising a heavy chain variable region comprising an amino acid sequence described herein (e.g., SEQ ID NO: 18, 20, 22, 24, or 25), wherein the antibody or antigen-binding domain immunospecifically binds to GITR (e.g., human GITR).
  • GITR e.g., human GITR
  • 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 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, which immunospecifically binds to GITR (e.g., human GITR), wherein the antibody comprises a light chain, wherein the amino acid sequence of the VL domain can comprise the amino acid sequence set forth in SEQ ID NO: 19, 21 , 23, or 26 and wherein the constant region of the light chain comprises the amino acid sequence of a human kappa light chain constant region.
  • GITR e.g., human GITR
  • a polynucleotide provided herein comprises a nucleotide sequence encoding an antibody, which immunospecifically binds to GITR (e.g., human GITR), and comprises a light chain, wherein the amino acid sequence of the VL domain can comprise the amino acid sequence set forth in SEQ ID NO: 19, 21, 23, or 26, and wherein the constant region of the light chain comprises the amino acid sequence of a human lambda light chain constant region.
  • GITR e.g., human GITR
  • the amino acid sequence of the VL domain can comprise the amino acid sequence set forth in SEQ ID NO: 19, 21, 23, or 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 immunospecifically binds to GITR (e.g., human GITR), wherein the antibody comprises a heavy chain, wherein the amino acid sequence of the VH domain can comprise the amino acid sequence set forth in SEQ ID NO: 18, 20, 22, 24, or 25, and wherein the constant region of the heavy chain comprises the amino acid sequence of a human gamma ( ⁇ ) heavy chain constant region.
  • GITR e.g., human GITR
  • a polynucleotide provided herein comprises a nucleotide sequence(s) encoding a VH domain and/or a VL domain of an antibody described herein (such as SEQ ID NO: 18, 20, 22, 24, or 25 for the VH domain and/or SEQ ID NO: 19, 21, 23, or 26 for the VL domain), which immunospecifically binds to GITR (e.g., human GITR).
  • GITR e.g., human GITR
  • a polynucleotide provided herein comprises a nucleotide sequence encoding an antibody described herein, which immunospecifically binds GITR (e.g., human GITR), wherein the antibody comprises a VL domain and a VH domain comprising any amino acid sequences described herein, and wherein the constant regions comprise the amino acid sequences of the constant regions of a human IgGi (e.g., allotype 1, 17, or 3), human IgG 2 , or human IgG 4 .
  • GITR e.g., human GITR
  • the constant regions comprise the amino acid sequences of the constant regions of a human IgGi (e.g., allotype 1, 17, or 3), human IgG 2 , or human IgG 4 .
  • polynucleotides comprising a nucleotide sequence encoding an anti-GITR antibody or domain thereof, designated herein, see, e.g., Tables 1-5.
  • polynucleotides encoding an anti-GITR 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-GITR 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.
  • potential splice sites and instability 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.
  • an optimized polynucleotide sequence encoding an anti- GITR antibody described herein or a fragment thereof can hybridize to an antisense (e.g., complementary) polynucleotide of an unoptimized polynucleotide sequence encoding an anti-GITR antibody described herein or a fragment thereof (e.g., VL domain or VH domain).
  • an optimized nucleotide sequence encoding an anti-GITR antibody described herein or a fragment hybridizes under high stringency conditions to antisense polynucleotide of an unoptimized polynucleotide sequence encoding an anti-GITR antibody described herein or a fragment thereof.
  • an optimized nucleotide sequence encoding an anti-GITR antibody described herein or a fragment thereof hybridizes under high stringency, intermediate stringency, or lower stringency hybridization conditions to an antisense polynucleotide of an unoptimized nucleotide sequence encoding an anti-GITR antibody described herein or a fragment thereof.
  • Information regarding hybridization conditions has 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.
  • 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 that hybridize under high stringency, intermediate or lower stringency hybridization conditions to polynucleotides that encode an antibody described herein hybridize under high stringency, intermediate or lower stringency hybridization conditions to polynucleotides encoding a VH domain (e.g., SEQ ID NO: 18, 20, 22, 24, or 25) and/or VL domain (e.g., SEQ ID NO: 19, 21, 23, or 26) provided herein.
  • VH domain e.g., SEQ ID NO: 18, 20, 22, 24, or 25
  • VL domain e.g., SEQ ID NO: 19, 21, 23, or 26
  • 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 FM 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
  • expressing e.g., recombinantly
  • antibodies described herein which specifically bind to GITR (e.g., human GITR) and related polynucleotides and expression vectors.
  • vectors e.g., expression vectors
  • host cells comprising such vectors for recombinantly expressing anti- GITR antibodies described herein (e.g., human or humanized antibody).
  • methods for producing an antibody described herein, comprising expressing such antibody in a host cell.
  • Recombinant expression of an antibody or fragment thereof described herein involves construction of an expression vector containing a polynucleotide that encodes the antibody or fragment.
  • GITR e.g., human GITR
  • 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 pabl876w, pabl967w, pabl975w, or pabl979w) or a fragment thereof.
  • a cell e.g., host cell
  • an antibody described herein e.g., an antibody comprising the CDRs of pabl876w, pabl967w, pabl975w, or pabl979w
  • host cells containing a polynucleotide encoding an antibody described herein (e.g., an antibody comprising the CDRs of pabl876w, pabl967w, pabl975w, or pabl979w) or fragments thereof (e.g., a heavy or light chain thereof, or fragment thereof), operably linked to a promoter for expression of such sequences in the host cell.
  • an antibody described herein e.g., an antibody comprising the CDRs of pabl876w, pabl967w, pabl975w, or pabl979w
  • fragments thereof e.g., a heavy or light chain thereof, or fragment thereof
  • 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.
  • 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 pab l 876w, pab l967w, pab l975w, or pab l979w), or a fragment thereof.
  • an antibody comprising the CDRs of pab l 876w, pab l967w, pab l975w, or pab l979w
  • 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 pab l 876w, pab l967w, pab l975w, or pab l979w), 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 pab l 876w, pab l967w, pab l975w, or pab l979w), or a fragment thereof.
  • 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 pab l 876w, pab l96
  • 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 pab l 876w, pab l967w, pab l975w, or pab l979w), or a fragment thereof
  • 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 pab l 876w, pab l967w, pab l975w, or pab l979w).
  • 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-GITR antibody described herein e.g., antibody comprising the CDRs pab l 876w, pab l967w, pab l975w, or pab l979w.
  • an anti-GITR antibody described herein e.g., antibody comprising the CDRs pab l 876w, pab l967w, pab l975w, or pab l979w.
  • a population of vectors comprising a first vector comprising a polynucleotide encoding a light chain/light chain variable region of an anti-GITR antibody described herein (e.g., antibody comprising the CDRs of pab l 876w, pab l967w, pab l975w, or pab l979w), and a second vector comprising a polynucleotide encoding a heavy chain/heavy chain variable region of an anti-GITR antibody described herein (e.g., antibody comprising the CDRs of pab l 876w, pab l967w, pab l975w, or pab l979w).
  • an anti-GITR antibody described herein e.g., antibody comprising the CDRs of pab l 876w, pab l967w, pab l975w, or pab l979w
  • a variety of host-expression vector systems can be utilized to express antibody molecules described herein (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 ⁇ e.g., COS1 or COS), CHO, BHK, MDCK, HEK 293, NSO,
  • cells for expressing antibodies described herein are CHO cells, for example CHO cells from the CHO GS SystemTM (Lonza).
  • cells for expressing antibodies described herein are human cells, e.g., human cell lines.
  • 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 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 MK & Hofstetter H (1986) Gene 45: 101- 105; and Cockett MI et al, (1990) Biotechnology 8: 662-667).
  • antibodies described herein are produced by CHO cells or NSO cells.
  • the expression of nucleotide sequences encoding antibodies described herein which immunospecifically bind GITR is regulated by a constitutive promoter, inducible promoter or tissue specific promoter.
  • 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 phosphorylation of the gene product can be used.
  • Such mammalian host cells include but are not limited to CHO, VERO, BHK, Hela, MDCK, HEK 293, NIH 3T3, W138, BT483, Hs578T, HTB2, BT20 and T47D, NSO (a murine myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7030, COS (e.g., COS 1 or COS), PER.C6, VERO, HsS78Bst, HEK-293T, HepG2, SP210, Rl . l, B-W, L-M, BSC l, BSC40, YB/20, BMT10 and HsS78Bst cells.
  • anti-GITR antibodies described herein are produced in mammalian cells, such as CHO cells.
  • the antibodies described herein have reduced fucose content or no fucose content.
  • Such antibodies can be produced using techniques known one skilled in the art.
  • the antibodies can be expressed in cells deficient or lacking the ability of to fucosylate.
  • cell lines with a knockout of both alleles of al,6- fucosyltransferase can be used to produce antibodies with reduced fucose content.
  • the Potelligent® system (Lonza) is an example of such a system that can be used to produce antibodies with reduced fucose content.
  • 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-GITR antibody described herein can be engineered.
  • an antibody molecule described herein 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.
  • chromatography e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography
  • centrifugation e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography
  • differential solubility e.g., differential solubility, or by any other standard technique for the purification of proteins.
  • the 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.
  • heterologous protein also referred to herein as a "contaminating protein”
  • variants of an antibody for example, different post-translational modified forms of an antibody.
  • the antibody or fragment 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 or fragment When the antibody or fragment 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 or fragment have less than about 30%>, 20%, 10%>, or 5% (by dry weight) of chemical precursors or compounds other than the antibody or fragment of interest.
  • antibodies described herein are isolated or purified.
  • compositions comprising an antibody described herein having the desired degree of purity in a physiologically acceptable carrier, excipient or stabilizer (Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA). Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed.
  • compositions as described herein that comprise an antibody described herein can be useful in reducing, inhibiting, or deactivating an GITR activity and treating a condition, such as an inflammatory or autoimmune disease or disorder or an infectious disease.
  • Pharmaceutical compositions as described herein that comprise an antibody described herein can be useful in reducing, inhibiting, or deactivating an GITR activity and treating a condition selected from the group consisting of infections (viral, bacterial, fungal and parasitic), endotoxic shock associated with infection, arthritis, rheumatoid arthritis, asthma, chronic obstructive pulmonary disease (COPD), pelvic inflammatory disease, Alzheimer's Disease, inflammatory bowel disease, Crohn's disease, ulcerative colitis, Peyronie's Disease, coeliac disease, gallbladder disease, Pilonidal disease, peritonitis, psoriasis, vasculitis, surgical adhesions, stroke, Type I Diabetes, lyme disease, arthritis, meningoencephalitis, uve
  • compositions to be used for in vivo administration can be sterile. This is readily accomplished by filtration through, e.g., sterile filtration membranes.
  • GITR GITR-specific binds to GITR described herein ⁇ e.g., an anti-GITR antagonistic antibody, e.g., an anti-GITR-monovalent antibody), or a composition comprising such an antibody.
  • an antibody that binds to GITR described herein e.g., an anti-GITR antagonistic antibody, e.g., an anti-GITR-monovalent antibody
  • the methods for modulating one or more immune functions or responses in a subject as presented herein are methods for deactivating, reducing, or inhibiting one or more immune functions or responses in a subject, comprising to a subject in need thereof administering an anti-GITR antagonistic antibody or a composition thereof as described herein.
  • presented herein are methods for preventing and/or treating diseases in which it is desirable to deactivate, reduce, or inhibit one or more immune functions or responses, comprising administering to a subject in need thereof an anti-GITR antagonistic antibody described herein or a composition thereof.
  • presented herein are methods of treating an autoimmune or inflammatory disease or disorder comprising administering to a subject in need thereof an effective amount of an anti-GITR antagonistic antibody or a composition thereof as described herein.
  • methods of treating an infectious disease comprising administering to a subject in need thereof an effective amount of an anti-GITR antagonistic antibody or a composition thereof as described herein.
  • the subject is a human.
  • the disease or disorder is selected from the group consisting of: infections (viral, bacterial, fungal and parasitic), endotoxic shock associated with infection, arthritis, rheumatoid arthritis, asthma, chronic obstructive pulmonary disease (COPD), pelvic inflammatory disease, Alzheimer's Disease, inflammatory bowel disease, Crohn's disease, ulcerative colitis, Peyronie's Disease, coeliac disease, gallbladder disease, Pilonidal disease, peritonitis, psoriasis, vasculitis, surgical adhesions, stroke, Type I Diabetes, lyme disease, arthritis, meningoencephalitis, uveitis, autoimmune uveitis, immune mediated inflammatory disorders of the central and peripheral nervous system such as multiple sclerosis, lupus (such as systemic lupus erythematosus) and Guillain-Barr syndrome, dermatitis, Atopic dermatitis, autoimmune hepatitis, fibrosing
  • infections viral
  • the disease or disorder is selected from the group consisting of: transplant rejection, graft-versus-host disease, vasculitis, asthma, rheumatoid arthritis, dermatitis, inflammatory bowel disease, uveitis, lupus, colitis, diabetes, multiple sclerosis, and airway inflammation.
  • an anti-GITR antagonistic antibody is administered to a patient diagnosed with an autoimmune or inflammatory disease or disorder to decrease the proliferation and/or effector function of one or more immune cell populations (e.g., T cell effector cells, such as CD4 + and CD8 + T cells) in the patient.
  • immune cell populations e.g., T cell effector cells, such as CD4 + and CD8 + T cells
  • an anti-GITR antagonistic antibody described herein deactivates or reduces or inhibits one or more immune functions or responses in a subject by at least 99%, at least 98%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, at least 50%, at least 45%, at least 40%, at least 45%, at least 35%, at least 30%, at least 25%, at least 20%, or at least 10%, or in the range of between 10% to 25%, 25%) to 50%), 50% to 75%), or 75% to 95% relative to the immune function in a subject not administered the anti-GITR antagonistic antibody described herein using assays well known in the art, e.g., ELISPOT, ELISA, and cell proliferation assays.
  • assays well known in the art, e.g., ELISPOT, ELISA, and cell proliferation assays.
  • the immune function is cytokine production (e.g., IL-2, T F-a, IFN- ⁇ , IL-4, IL-10, and/or IL-13 production).
  • the immune function is T cell proliferation/expansion, which can be assayed, e.g., by flow cytometry to detect the number of cells expressing markers of T cells (e.g., CD3, CD4, or CD8).
  • the immune function is antibody production, which can be assayed, e.g., by ELISA.
  • the immune function is effector function, which can be assayed, e.g., by a cytotoxicity assay or other assays well known in the art.
  • the immune function is a Thl response.
  • the immune function is a Th2 response.
  • the immune function is a memory response.
  • non-limiting examples of immune functions that can be reduced or inhibited by an anti-GITR antagonistic antibody or composition thereof as described herein are proliferation/expansion of effector lymphocytes (e.g., decrease in the number of effector T lymphocytes), and stimulation of apoptosis of effector lymphocytes (e.g., effector T lymphocytes).
  • an immune function reduced or inhibited by an anti- GITR antagonistic antibody or composition thereof as described herein is proliferation/expansion in the number of or activation of CD4 + T cells (e.g., Thl and Th2 helper T cells), CD8 + T cells (e.g., cytotoxic T lymphocytes, alpha/beta T cells, and gamma/delta T cells), B cells (e.g., plasma cells), memory T cells, memory B cells, tumor-resident T cells, CD122 + T cells, natural killer (NK) cells), macrophages, monocytes, dendritic cells, mast cells, eosinophils, basophils or polymorphonucleated leukocytes.
  • CD4 + T cells e.g., Thl and Th2 helper T cells
  • CD8 + T cells e.g., cytotoxic T lymphocytes, alpha/beta T cells, and gamma/delta T cells
  • B cells e.g., plasma cells
  • an anti-GITR antagonistic antibody or composition thereof as described herein deactivates or reduces or inhibits the proliferation/expansion or number of lymphocyte progenitors.
  • an anti- GITR antagonistic antibody or composition thereof as described herein decreases the number of CD4 + T cells (e.g., Thl and Th2 helper T cells), CD8 + T cells (e.g., cytotoxic T lymphocytes, alpha/beta T cells, and gamma/delta T cells), B cells (e.g., plasma cells), memory T cells, memory B cells, tumor-resident T cells, CD122 + T cells, natural killer cells (NK cells), macrophages, monocytes, dendritic cells, mast cells, eosinophils, basophils or polymorphonucleated leukocytes by approximately at least 99%, at least 98%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, at least 50%
  • any of the methods herein comprise administration to a subject of an antibody as described herein and a checkpoint targeting agent.
  • the checkpoint targeting agent is an antibody (e.g., an anti-PD-1 antibody, an anti-PD-Ll antibody, an anti-PD-L2 antibody, an anti-CTLA-4 antibody, an anti-TIM-3 antibody, an anti-LAG-3 antibody, an anti-CEACAMl antibody, an anti-GITR antibody, or an anti-OX40 antibody).
  • the checkpoint targeting agent is an antagonist or agonist antibody.
  • An antibody or composition described herein can be delivered to a subject by a variety of routes.
  • an antibody or composition which will be effective in the treatment and/or prevention of a condition will depend on the nature of the disease, and can be determined by standard clinical techniques.
  • the precise dose to be employed in a composition will also depend on the route of administration, and the seriousness of the disease, and should be decided according to the judgment of the practitioner and each subject's circumstances.
  • effective doses may also vary depending upon means of administration, target site, physiological state of the patient (including age, body weight and health), whether the patient is human or an animal, other medications administered, or whether treatment is prophylactic or therapeutic.
  • the patient is a human but non-human mammals including transgenic mammals can also be treated. Treatment dosages are optimally titrated to optimize safety and efficacy.
  • an in vitro assay is employed to help identify optimal dosage ranges.
  • Effective doses may be extrapolated from dose response curves derived from in vitro or animal model test systems.
  • human antibodies have a longer half-life within the human body than antibodies from other species due to the immune response to the foreign polypeptides. Thus, lower dosages of human antibodies and less frequent administration is often possible.
  • An anti-GITR antibody described herein can be used to assay GITR protein levels in a biological sample using classical immunohistological methods known to those of skill in the art, including immunoassays, such as the enzyme linked immunosorbent assay (ELISA), immunoprecipitation, or Western blotting.
  • immunoassays such as the enzyme linked immunosorbent assay (ELISA), immunoprecipitation, or Western blotting.
  • Suitable antibody assay labels include enzyme labels, such as, glucose oxidase; radioisotopes, such as iodine ( 125 I, 121 I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( 121 In), and technetium ( 99 Tc); 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 ( 125 I, 121 I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( 121 In), and technetium ( 99 Tc)
  • luminescent labels such as luminol
  • fluorescent labels such as fluorescein and rhodamine, and biotin.
  • Such labels can be used to label an antibody described herein.
  • a second antibody that recognizes an anti-GITR antibody described herein can be labeled
  • Assaying for the expression level of GITR protein is intended to include qualitatively or quantitatively measuring or estimating the level of a GITR protein in a first biological sample either directly ⁇ e.g., by determining or estimating absolute protein level) or relatively ⁇ e.g., by comparing to the disease associated protein level in a second biological sample).
  • GITR polypeptide expression level in the first biological sample can be measured or estimated and compared to a standard GITR protein level, the standard being taken from a second biological sample obtained from an individual not having the disorder or being determined by averaging levels from a population of individuals not having the disorder.
  • the "standard" GITR polypeptide level is known, it can be used repeatedly as a standard for comparison.
  • biological sample refers to any biological sample obtained from a subject, cell line, tissue, or other source of cells potentially expressing GITR. Methods for obtaining tissue biopsies and body fluids from animals ⁇ e.g., humans) are well known in the art. Biological samples include peripheral mononuclear blood cells.
  • An anti-GITR antibody described herein can be used for prognostic, diagnostic, monitoring and screening applications, including in vitro and in vivo applications well known and standard to the skilled artisan and based on the present description.
  • Prognostic, diagnostic, monitoring and screening assays and kits for in vitro assessment and evaluation of immune system status and/or immune response may be utilized to predict, diagnose and monitor to evaluate patient samples including those known to have or suspected of having an immune system-dysfunction or with regard to an anticipated or desired immune system response, antigen response or vaccine response.
  • the assessment and evaluation of immune system status and/or immune response is also useful in determining the suitability of a patient for a clinical trial of a drug or for the administration of a particular chemotherapeutic agent or an antibody, including combinations thereof, versus a different agent or antibody.
  • an anti-GITR antibody can be used in immunohistochemistry of biopsy samples.
  • an anti-GITR antibody can be used to detect levels of GITR, or levels of cells which contain GITR on their membrane surface, which levels can then be linked to certain disease symptoms.
  • Anti-GITR antibodies described herein may carry a detectable or functional label.
  • fluorescence labels include, for example, reactive and conjugated probes, e.g., Aminocoumarin, Fluorescein and Texas red, Alexa Fluor dyes, Cy dyes and DyLight dyes.
  • 51 anti-GITR antibody can carry a radioactive label, such as the isotopes H, C, P, S, CI, Cr, 57 Co, 58 Co, 59 Fe, 67 Cu, 90 Y, 99 Tc, m In, 117 Lu, 121 I, 124 I, 125 I, 131 I, 198 Au, 211 At, 213 Bi, 225 Ac and 186 Re.
  • radioactive labels currently available counting procedures known in the art may be utilized to identify and quantitate the specific binding of anti-GITR antibody to GITR ⁇ e.g., human GITR).
  • detection may be accomplished by any of the presently utilized colorimetric, spectrophotometric, fluorospectrophotometric, amperometric or gasometric techniques as known in the art. This can be achieved by contacting a sample or a control sample with an anti-GITR antibody under conditions that allow for the formation of a complex between the antibody and GITR. Any complexes formed between the antibody and GITR are detected and compared in the sample and the control.
  • the antibodies described herein for GITR the antibodies thereof can be used to specifically detect GITR expression on the surface of cells.
  • the antibodies described herein can also be used to purify GITR via immunoaffinity purification.
  • an assay system which may be prepared in the form of a test kit for the quantitative analysis of the extent of the presence of, for instance, GITR or GITR/GITRL complexes.
  • the system or test kit may comprise a labeled component, e.g., a labeled antibody, and one or more additional immunochemical reagents. See, e.g., Section 7.6 below for more on kits.
  • kits comprising one or more antibodies described herein or conjugates thereof.
  • 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 antibodies provided herein.
  • the kits contain a pharmaceutical composition described herein and any prophylactic or therapeutic agent, such as those described herein.
  • the kits may contain a T cell mitogen, such as, e.g., phytohaemagglutinin (PHA) and/or phorbol myristate acetate (PMA), or a TCR complex stimulating antibody, such as an anti-CD3 antibody and anti-CD28 antibody.
  • PHA phytohaemagglutinin
  • PMA phorbol myristate acetate
  • TCR complex stimulating antibody such as an anti-CD3 antibody and anti-CD28 antibody.
  • 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
  • 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 GITR antigen (e.g., human GITR) that can be used as a control.
  • the kits described herein further comprise a control antibody which does not react with a GITR antigen.
  • kits described herein contain one or more elements for detecting the binding of an antibody to a GITR 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 GITR antigen.
  • the GITR 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 GITR 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 GITR antigen can be detected by binding of the said reporter-labeled antibody.
  • an anti-GITR-monovalent antibody was constructed using the Genmab DuoBody technology: DuoBody pabl876 x isotype which comprises a GITR-binding arm and an isotype control arm.
  • the SEQ ID NOs corresponding to the heavy chain and light chain sequences of this DuoBody are listed in Table 6.
  • Hutl02 cells human T cell lymphoma, ATCC were incubated for 72 hours in RPMI media, supplemented with 1 ⁇ g/ml phytohaemagglutinin (PHA) and 10% heat- inactivated FBS, at 37°C and 5% C0 2 to induce GITR expression.
  • PHA phytohaemagglutinin
  • FBS heat- inactivated FBS
  • test antibodies (10-point dose titration, 0.5-10,000 ng/ml) diluted in FACS buffer (PBS, 2 mM EDTA, 0.5% BSA, pH 7.2) for 30 minutes at 4°C. Samples were washed two times in FACS buffer and then incubated with APC-conjugated mouse anti-human kappa detection antibody (Life Technologies, HP6062, 1 : 100 dilution in FACS buffer) for 30 minutes at 4°C.
  • test antibodies (10-point dose titration, 0.5-10,000 ng/ml) diluted in FACS buffer (PBS, 2 mM EDTA, 0.5% BSA, pH 7.2) for 30 minutes at 4°C.
  • FACS buffer PBS, 2 mM EDTA, 0.5% BSA, pH 7.2
  • DuoBody pabl876 x isotype bound to Jurkat cells expressing human GITR ( Figure 1A), activated Hut 102 cells ( Figure IB) and activated primary CD4+ T cells ( Figure 1C) in a dose-dependent manner.
  • GITR signaling depends on receptor clustering to form higher order receptor complexes that efficiently recruit apical adapter proteins to drive intracellular signal transduction.
  • an anti-GITR agonist antibody may mediate receptor clustering through bivalent antibody arms and/or through Fc-Fc receptor (FcR) co-engagement on accessory myeloid or lymphoid cells.
  • one approach for developing an anti-GITR antagonist antibody is to select an antibody that competes with GITR ligand (GITRL) for binding to GITR, diminish or eliminate the binding of the Fc region of the antibody to Fc receptors, and/or adopt a monovalent antibody format (containing only one GITR- specific antigen-binding domain, and optionally a second antigen-binding domain that is not GITR-specific).
  • GITRL GITR ligand
  • GITR GITR ectopically expressing GITR as well as F-i B-luciferase (Nano luciferase, NanoLuc ® ) reporter were generated by transduction of lentiviral vectors (EFla promoter) into Jurkat cells. Stable clones were generated via single-cell sorting (FACS ARIA Fusion). Expression of GITR was verified by flow cytometry.
  • Jurkat- huGITR- F-KB-luciferase cells were incubated with increasing concentrations of DuoBody pabl876 x isotype or trimeric GITRL (12-point dose titration, 0.05-10,000 ng/ml) for 2 hours in RPMI media, supplemented with 10% heat-inactivated FBS, at 37°C and 5% C02.
  • samples were incubated with prepared Nano-Glo ® Luciferase Assay Substrate (Promega, 1 : 1 v/v) in passive lysis buffer for 5 minutes at room temperature. Data were collected using the En Vision ® Multilabel Plate Reader (Perkin-Elmer). Values were plotted using Graphpad Prism software.
  • DuoBody pabl876 x isotype was assessed for its ability to block GITRL- induced NF-KB signaling.
  • Jurkat-huGITR-NF-KB-luciferase cells were incubated with increasing concentrations of DuoBody pabl876 x isotype or an isotype control antibody (10- point dose titration, 0.5-10,000 ng/ml) for 30 minutes. Samples were then washed two times with RPMI, resuspended in 1 ⁇ g/ml of trimeric GITRL and incubated for additional 2 hours at 37°C. Luciferase activity was detected and analyzed as described above.
  • This example characterizes the epitope of the following anti-GITR antibodies: a chimeric parental 231-32-15 antibody and its humanized versions (pabl876, pabl875, pabl967, pabl975, and pabl979).
  • a reference anti-GITR antibody named m6C8 was also used in some studies for comparison.
  • the antibody m6C8 was generated based on the variable regions of the antibody 6C8 provided in WO 06/105021 (herein incorporated by reference).
  • the SEQ ID NOs corresponding to the heavy chain variable regions and light chain variable regions of these anti-GITR antibodies are listed in Table 7.
  • a cell binding assay was performed. 1624-5 pre-B cells expressing the chimeric parental 231-32-15 antibody were harvested and lxlO 6 cells were resuspended in 200 ⁇ FACS buffer plus: i) biotinylated GITR (GITR-bio) (1 : 1000), preincubated for 15 min with 2 ⁇ g chimeric parental 231-32-15 antibody; ii) GITR-bio (1 : 1000), preincubated for 15 min with 2 ⁇ g pabl875; iii) GITR-bio (1 : 1000), preincubated for 15 min with 2 ⁇ g pabl876; or iv) GITR-bio (1 : 1000).
  • FIG. 3 shows that the humanized variant antibodies retained the epitope specificity of the chimeric parental 231-32-15 antibody.
  • the right-hand profile shows the binding of GITR- bio to 1624-5 pre-B cells expressing the chimeric parental 231-32-25 antibody.
  • Anti-GITR antibodies 25 ⁇ were diluted to 2 ⁇ g/ml in assay buffer (Roche 11112589001) and incubated with 1500 Luminex ® beads (5 ⁇ , Luminex Corp, no 5 LC10005- 01) coupled with anti-human IgG (F(ab)2-specific, JIR, 105-006-097 overnight in 0.5 ml LoBind tubes (Eppendorf, 0030108.116) under shaking conditions, in the dark. This mixture was then transferred to pre-wetted 96-well filter plates (Millipore, MABVN1250). Plates were washed twice with 200 ⁇ /well PBS to remove unbound antibody.
  • ovalbumin (1289 RU, Pierce ThermoFisher 77120) was immobilized for reference. Immobilization was performed according to a standard protocol from the manufacturer (GE Healthcare) for amine coupling (activation of surface with 0.4 M EDC and 0.1 M NHS, GE Healthcare Amine coupling kit, BR-1000-50). Unreacted groups were inactivated with 1 M ethanol-amine-HCl, pH8.5. Afterwards anti-GITR antibodies were run through the different surfaces at a concentration of 300 nM (45 ⁇ g/ml) for 240 seconds at 5 ⁇ /min. Using these conditions saturation of the GITR surface should have been reached.
  • a dissociation time of 60 seconds was included before adding the competing antibody (300 nM, 5 ⁇ /min).
  • Regeneration of the chip surface was performed using 10 mM Glycine pH2.0 (GE Healthcare, BR- 1003 -55) for 60 seconds at 10 ⁇ /min.
  • Binning was performed using the response units (RU) of the non-competed control (100% binding, saturating conditions).
  • Error prone PCR mutagenesis was used to generate variants of human GITR with random mutations in the extracellular domain.
  • the GeneMorphll Random Mutagenesis Kit (Stratagene) was used, according to the manufacturer's instructions. In brief, 20 PCR cycles in a volume of 50 ⁇ was performed using an in-house construct as template (13 ng, construct number 4377 pMA-T-huGITR), 0.05 U/ ⁇ Mutazyme II DNA polymerase, lx Mutazyme II reaction buffer, 0.2 ⁇ of each primer and 0.2 mM of each deoxynucleoside- triphosphate (dATP, dCTP, dGTP, and dTTP).
  • dATP deoxynucleoside- triphosphate
  • the samples were amplified by PCR (Eppendorf, Germany) using the following program: 95°C for 2 min; 20 cycles of 95°C for 30 sec, 56°C for 30 sec, 72°C for 1 min; and a final extension step of 72°C for 10 min.
  • the PCR product was gel purified using 1% agarose gel, the DNA band corresponding to the expected size of 720 bp was cut out and gel extraction was done using a NucleoSpin Gel and PCR cleanup kit from Macherey&Nagel according to the product manual. Purified DNA was ligated into an in-house expression vector via Xhol / EcoRI sites using T4 DNA ligase and a ratio of 1 :3 (vectoninsert).
  • Ligation 25°C was stopped after 2 hours with a heat denaturation step for 10 min at 65°C.
  • DNA from the ligation reaction was EtOH precipitated using yeast t-RNA. Standard digestion and ligation techniques were used.
  • the ligation reaction was electroporated into DH10B cells (E.coli ElectroMax DH10B electrocompetent cells, Invitrogen; 1900V/ 5ms). Electroporated bacteria were plated onto LB-agar + 100 ⁇ g/ml ampicillin plates and approximately 1.9xl0 8 colonies were obtained.
  • retroviral particles Standard techniques of transfection followed by transduction were used to express human GITR mutants on the surface of 1624-5 cells.
  • a DNA library and vectors expressing retroviral proteins Gag, Pol and Env were transfected into a retroviral packaging cell line (HEK cells) using X-tremeGENE 9 DNA transfection reagent (Roche Diagnostics GmbH, Germany).
  • the resulting retroviral particles accumulated in the cell culture supernatant of the retroviral packaging cells.
  • Two days post transfection cell-free viral vector particle-containing supernatants were harvested and subjected to spin-infection of 1624-5 cells.
  • a transduction efficiency (% human GITR expressing cells) of roughly 4% was obtained.
  • FACS was used to select cells expressing correctly folded human GITR mutants using a polyclonal anti-GITR antibody and to subsequently select individual cells expressing human GITR variants that did not bind to the anti-GITR chimeric parental 231-32-15 antibody.
  • antibody binding cells were analyzed by FACS and cells that exhibited specific antibody binding were separated from the non-binding cell population by preparative, high-speed FACS (FACSAriall, BD Biosciences).
  • Antibody reactive or non-reactive cell pools were expanded again in tissue culture and, due to the stable expression phenotype of retrovirally transduced cells, cycles of antibody-directed cell sorting and tissue culture expansion were repeated, up to the point that a clearly detectable anti-GITR antibody (chimeric parental 231-32-15) non-reactive cell population was obtained.
  • This anti-GITR antibody (chimeric parental 231-32-15) non- reactive cell population was subjected to a final, single-cell sorting step. After several days of cell expansion, single cell sorted cells were again tested for non-binding to anti-GITR chimeric parental 231-32-15 antibody and binding to a polyclonal anti-GITR antibody using 96 well plate analysis on a FACSCalibur (BD Biosciences).
  • Figures 5A and 5B show the alignment of sequences from these variants.
  • the amino acid residues in Figures 5A and 8B are numbered according to the immature amino acid sequence of human GITR (SEQ ID NO: 41).
  • Sequencing identified regions with increased mutations or "hot spots" e.g., P62 and G63, providing an indication of the epitope on human GITR recognized by anti-GITR chimeric parental 231-32-15 antibody.
  • alanine mutants expressed on 1624-5 cells were tested in flow cytometry (FACSCalibur; BD Biosciences) for the binding of the anti-GITR humanized antibodies pabl876, pabl967, pabl975 and pabl979, and the reference antibody m6C8.
  • MFI ratio mean fluorescence intensity (MFI) value of the tested monoclonal antibody was divided by the MFI value of the polyclonal antibody, generating an MFI ratio (monoclonal antibody/polyclonal antibody) for individual GITR alanine mutants.
  • An average MFI ratio (“AMFI ratio") was calculated based on the individual MFI ratios for all the mutants.
  • Figure 6A is a table summarizing the binding of pabl876, pabl967, pabl975, pabl979 and the reference antibody m6C8 tol624-5 cells expressing human GITR alanine mutants.
  • An individual MFI ratio that is above 60% of the AMFI ratio is considered to indicate similar binding, after normalization, of that of the polyclonal antibody and is represented by "+” in Figure 6A.
  • An individual MFI ratio that is between 30% and 60% of the AMFI ratio is represented by "+/-" in Figure 6A.
  • An individual MFI ratio that is below 30% of the AMFI ratio is represented by "-" in Figure 6A.
  • the D60A mutant and the G63A mutant numbered according to SEQ ID NO: 41, specifically disrupted or weakened the binding of pabl876, pabl967, pabl975 and pabl979, but not that of the reference antibody m6C8.
  • the C58A mutant disrupted the binding of all five antibodies and is likely a structural mutation rather than an epitope- specific one.
  • the C74A mutant had weak expression and could not be used for binding comparison.
  • the anti-GITR antibodies 231-32-15, pabl876, and m6C8 were compared for their binding to wild type versus mutant human GITR. Briefly, wild type human GITR and two GITR alanine mutants (the D60A mutant and the G63A mutant, numbered according to SEQ ID NO: 41) were expressed on the surface of 1624-5 cells as described above and tested in a flow cytometry analysis as described above where cells were first stained using 2 ⁇ g/ml of the monoclonal antibodies 231-32-15, pabl876, and m6C8, or a polyclonal antibody conjugated to APC, and then stained using a secondary anti-IgG antibody if necessary for detection (APC conjugated; 1 : 1000; BD Cat No.
  • APC conjugated 1 : 1000; BD Cat No.
  • MFI mean fluorescence intensity
  • the percentages shown in Figure 6B are the percentages of GITR positive cells in each plot.
  • antibody binding was reduced by 82% and 88% for 231-32-15 and pabl876, respectively, compared with a 10% reduction for m6C8.
  • the binding of 231-32-15 and pabl876 was reduced by 37%) and 59%>, respectively, whereas the binding of m6C8 was increased by 62%>.
  • the immature protein of cynomolgus GITR comprises the amino acid sequence of SEQ ID NO: 44.
  • the first residue of the signal peptide of cynomolgus GITR was replaced by methionine, generating VIM cynomolgus GITR.
  • FIG. 7 A is a sequence alignment between human GITR, VIM cynomolgus GITR, and V1M/Q62P/S63G cynomolgus GITR.
  • the three proteins shown in Figure 7A were expressed on the surface of 1624-5 cells as described above and tested in a flow cytometry analysis as described above where cells were first stained using 2 ⁇ g/ml of the monoclonal antibodies 231-32-15, pab l 876, and m6C8, or a polyclonal antibody conjugated to APC, and then stained using a secondary anti-IgG antibody (APC conjugated; 1 : 1000; BD Cat no. 109-136-097).
  • APC conjugated 1 : 1000; BD Cat no. 109-136-097
  • the anti-GITR antibodies 231-32-15 and pab l 876 displayed binding only to the cells expressing V1M/Q62P/S63G cynomolgus GITR, but not the cells expressing VIM cynomolgus GITR.

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Abstract

La présente invention concerne des anticorps antagonistes qui se lient spécifiquement au récepteur apparenté à la famille du TNFR induit par les glucocorticoïdes (GITR) humain et des compositions comprenant ces anticorps. Selon un aspect spécifique, les anticorps se lient spécifiquement au GITR humain et modulent l'activité du GITR, par exemple, réduisent, désactivent ou inhibent l'activité du GITR. La présente invention concerne également des procédés de traitement de maladies ou de troubles auto-immuns ou inflammatoires, par administration d'un anticorps qui se lie spécifiquement au GITR humain et module l'activité du GITR, par exemple, réduit, désactive ou inhibe l'activité du GITR.
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