NZ788539A - Anti-gitr antibodies and uses thereof - Google Patents

Abstract

Provided herein are antibodies, and antigen-binding fragments thereof that specifically bind glucocorticoid-induced tumor necrosis factor receptor (GITR) and methods of using the same, including, e.g., methods of treatment using the same.

Description

ANTI-GITR ANTIBODIES AND USES THEREOF This application is a divisional of New Zealand patent application 748619, which is the national phase entry in New d of PCT ational ation (published as WO2017/214548) filed 9 June 2017, all of which are incorporated herein in their entireties.

FIELD The present invention s to antibodies and antigen-binding fragments thereof that specifically bind glucocorticoid-induced tumor necrosis factor receptor (GITR) and methods of use thereof.

BACKGROUND Glucocorticoid-induced tumor necrosis factor receptor (GITR) is a member of the tumor is factor receptor superfamily (TNFRSF). GITR expression is constitutively high on regulatory T cells, low/intermediate on naïve T cells, NK cells and ocytes, and inducible upon activation. GITR interacts with its ligand GITRL, which is mainly expressed on antigen-presenting cells. GITR receptor activation can both augment effector T-cell eration and function as well as attenuate the suppression induced by regulatory T cells.

Consequently, the modulation of GITR activity can serve as a basis for cancer immunotherapy and immune disorders. Thus, there is a need for agents, e.g., antibodies that modulate the ty of GITR.

BRIEF SUMMARY The present invention provides antibodies and antigen-binding fragments thereof that bind glucocorticoid-induced tumor necrosis factor receptor (GITR). The antibodies of the invention are useful, inter alia, for targeting immune cells, e.g., effector T-cells, regulatory T-cells, and NK cells that s GITR.

The antibodies of the ion can be full-length (for example, an IgG1 or IgG4 antibody) or may comprise only an antigen-binding portion (for example, a Fab, F(ab’)2 or scFv fragment), and may be modified to affect functionality, e.g., to eliminate residual effector functions (Reddy et al., 2000, J. Immunol. 164:1925-1933).

Exemplary ITR antibodies of the present invention are listed in Tables 1 and 2 herein. Table 1 sets forth the amino acid sequence identifiers of the heavy chain variable regions (HCVRs), light chain variable regions (LCVRs), heavy chain mentarity determining regions (HCDR1, HCDR2 and HCDR3), and light chain complementarity determining regions , LCDR2 and LCDR3) of the exemplary anti-GITR antibodies.

Table 2 sets forth the nucleic acid sequence identifiers of the HCVRs, LCVRs, HCDR1, HCDR2 HCDR3, LCDR1, LCDR2 and LCDR3 of the exemplary anti-GITR antibodies.

The t invention provides antibodies or antigen-binding fragments f that specifically bind GITR, comprising an HCVR comprising an amino acid sequence selected from any of the HCVR amino acid sequences listed in Table 1, or a ntially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

The present invention also provides antibodies or antigen-binding fragments thereof that specifically bind GITR, comprising an LCVR sing an amino acid sequence selected from any of the LCVR amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

The present invention also provides antibodies or antigen-binding fragments thereof that specifically bind GITR, comprising an HCVR and an LCVR amino acid sequence pair (HCVR/LCVR) sing any of the HCVR amino acid sequences listed in Table 1 paired with any of the LCVR amino acid sequences listed in Table 1. According to certain embodiments, the t invention es antibodies, or antigen-binding fragments thereof, comprising an HCVR/LCVR amino acid sequence pair contained within any of the exemplary ITR antibodies listed in Table 1. In certain embodiments, the CVR amino acid sequence pair is selected from the group consisting of: 98/106; 162/170; 194/202; 242/250; 290/298; 338/402; and 346/402.

The present invention also provides antibodies or antigen-binding nts f that specifically bind GITR, comprising a heavy chain CDR1 (HCDR1) comprising an amino acid sequence selected from any of the HCDR1 amino acid sequences listed in Table 1 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

The present ion also provides antibodies or n-binding fragments thereof that specifically bind GITR, comprising a heavy chain CDR2 (HCDR2) comprising an amino acid sequence selected from any of the HCDR2 amino acid sequences listed in Table 1 or a substantially r sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

The present invention also provides antibodies or antigen-binding fragments thereof that specifically bind GITR, comprising a heavy chain CDR3 (HCDR3) comprising an amino acid sequence selected from any of the HCDR3 amino acid sequences listed in Table 1 or a substantially similar sequence f having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

The present invention also provides antibodies or antigen-binding fragments thereof that specifically bind GITR, comprising a light chain CDR1 (LCDR1) comprising an amino acid sequence selected from any of the LCDR1 amino acid sequences listed in Table 1 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% ce identity.

The present invention also provides antibodies or antigen-binding fragments thereof that specifically bind GITR, comprising a light chain CDR2 ) comprising an amino acid sequence selected from any of the LCDR2 amino acid sequences listed in Table 1 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

The present invention also es antibodies or antigen-binding fragments thereof that specifically bind GITR, comprising a light chain CDR3 (LCDR3) comprising an amino acid sequence selected from any of the LCDR3 amino acid sequences listed in Table 1 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

The present invention also es antibodies or antigen-binding fragments thereof that specifically bind GITR, comprising an HCDR3 and an LCDR3 amino acid sequence pair /LCDR3) comprising any of the HCDR3 amino acid sequences listed in Table 1 paired with any of the LCDR3 amino acid sequences listed in Table 1. According to n embodiments, the present invention provides antibodies, or antigen-binding nts thereof, comprising an HCDR3/LCDR3 amino acid sequence pair contained within any of the exemplary anti-GITR antibodies listed in Table 1. In certain embodiments, the LCDR3 amino acid sequence pair is selected from the group consisting of: 104/112; 6; 200/208; 248/256; 296/304; 344/408; and 352/408.

The present invention also provides antibodies or antigen-binding fragments thereof that ically bind GITR, comprising a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3- LCDR1-LCDR2-LCDR3) contained within any of the exemplary anti-GITR antibodies listed in Table 1. In certain embodiments, the HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequences set is selected from the group ting of: 100104110- 112; 164168174-176; 8204208; 244248254-256; 292- 294300304; 340344406-408; and 0404408.

In a related embodiment, the present invention provides antibodies, or antigen- binding fragments thereof that specifically bind GITR, comprising a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained within an HCVR/LCVR amino acid sequence pair as defined by any of the exemplary anti-GITR antibodies listed in Table 1. For example, the present ion includes antibodies or antigen-binding fragments thereof that specifically bind GITR, comprising the HCDR1-HCDR2-HCDR3-LCDR1-LCDR2- LCDR3 amino acid sequences set contained within an HCVR/LCVR amino acid sequence pair selected from the group ting of: ; 162/170; 194/202; 242/250; 290/298; 338/402; and 346/102. Methods and techniques for identifying CDRs within HCVR and LCVR amino acid sequences are well known in the art and can be used to identify CDRs within the ied HCVR and/or LCVR amino acid sequences disclosed herein. Exemplary conventions that can be used to identify the boundaries of CDRs include, e.g., the Kabat definition, the a tion, and the AbM definition. In general terms, the Kabat definition is based on sequence variability, the a definition is based on the location of the structural loop regions, and the AbM tion is a compromise between the Kabat and Chothia approaches. See, e.g., Kabat, "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1991); ikani et al., J. Mol. Biol. 273:927- 948 (1997); and Martin et al., Proc. Natl. Acad. Sci. USA 86:9268-9272 (1989). Public databases are also available for identifying CDR sequences within an antibody.

The present invention also provides nucleic acid molecules encoding anti-GITR antibodies or portions thereof. For example, the present invention provides nucleic acid molecules encoding any of the HCVR amino acid sequences listed in Table 1; in certain embodiments the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCVR nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

The present invention also provides nucleic acid molecules encoding any of the LCVR amino acid sequences listed in Table 1; in certain embodiments the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCVR nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

The present invention also provides nucleic acid molecules ng any of the HCDR1 amino acid sequences listed in Table 1; in certain embodiments the c acid molecule ses a polynucleotide sequence selected from any of the HCDR1 nucleic acid sequences listed in Table 2, or a ntially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

The t invention also provides c acid les encoding any of the HCDR2 amino acid sequences listed in Table 1; in certain embodiments the nucleic acid molecule comprises a polynucleotide ce selected from any of the HCDR2 nucleic acid sequences listed in Table 2, or a substantially similar ce thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

The present invention also es nucleic acid molecules encoding any of the HCDR3 amino acid sequences listed in Table 1; in certain embodiments the nucleic acid le comprises a cleotide sequence selected from any of the HCDR3 nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

The present invention also provides nucleic acid les encoding any of the LCDR1 amino acid sequences listed in Table 1; in certain embodiments the nucleic acid molecule comprises a polynucleotide ce selected from any of the LCDR1 nucleic acid sequences listed in Table 2, or a ntially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity o.

The t invention also es c acid molecules encoding any of the LCDR2 amino acid sequences listed in Table 1; in certain embodiments the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR2 nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

The present invention also provides nucleic acid molecules encoding any of the LCDR3 amino acid sequences listed in Table 1; in certain embodiments the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR3 nucleic acid ces listed in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

The present invention also provides nucleic acid molecules encoding an HCVR, wherein the HCVR comprises a set of three CDRs (i.e., HCDR1-HCDR2-HCDR3), wherein the HCDR1-HCDR2-HCDR3 amino acid sequence set is as defined by any of the exemplary anti-GITR antibodies listed in Table 1.

The present invention also provides nucleic acid molecules ng an LCVR, n the LCVR comprises a set of three CDRs (i.e., LCDR2-LCDR3), wherein the LCDR1-LCDR2-LCDR3 amino acid sequence set is as defined by any of the exemplary anti-GITR antibodies listed in Table 1.

The present invention also provides nucleic acid molecules encoding both an HCVR and an LCVR, wherein the HCVR comprises an amino acid ce of any of the HCVR amino acid sequences listed in Table 1, and wherein the LCVR comprises an amino acid sequence of any of the LCVR amino acid sequences listed in Table 1. In certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCVR nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto, and a polynucleotide ce selected from any of the LCVR nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto. In certain embodiments ing to this aspect of the invention, the nucleic acid molecule encodes an HCVR and LCVR, wherein the HCVR and LCVR are both derived from the same anti-GITR antibody listed in Table 1.

The present invention also provides recombinant expression vectors capable of sing a polypeptide comprising a heavy or light chain variable region of an anti-GITR dy. For example, the present invention includes recombinant expression vectors comprising any of the nucleic acid molecules mentioned above, i.e., nucleic acid molecules encoding any of the HCVR, LCVR, and/or CDR sequences as set forth in Table 1. Also included within the scope of the present invention are host cells into which such vectors have been introduced, as well as methods of producing the antibodies or portions thereof by culturing the host cells under conditions permitting production of the dies or antibody fragments, and recovering the antibodies and dy fragments so produced.

The present ion includes anti-GITR antibodies having a modified glycosylation pattern. In some embodiments, cation to remove undesirable glycosylation sites may be , or an antibody lacking a fucose moiety present on the oligosaccharide chain, for example, to increase antibody dependent cellular cytotoxicity (ADCC) function (see Shield et al. (2002) JBC 277:26733). In other applications, modification of galactosylation can be made in order to modify complement dependent cytotoxicity (CDC).

In another aspect, the invention provides a pharmaceutical composition comprising a recombinant human antibody or fragment thereof which specifically binds GITR and a pharmaceutically acceptable carrier. In a related aspect, the invention features a composition which is a combination of an anti-GITR antibody and a second therapeutic agent. In one embodiment, the second therapeutic agent is any agent that is advantageously combined with an anti-GITR dy. The present invention also provides antibody-drug conjugates (ADCs) sing an anti-GITR antibody conjugated to a cytotoxic agent. Exemplary combination therapies, co-formulations, and ADCs involving the anti-GITR antibodies of the present invention are disclosed ere herein.

In yet another aspect, the invention provides therapeutic methods for killing tumor cells or for inhibiting or attenuating tumor cell , or otherwise treating a patient afflicted with cancer, using an anti-GITR antibody or antigen-binding portion of an antibody of the invention. The therapeutic methods ing to this aspect of the invention comprise administering a eutically effective amount of a ceutical ition comprising an dy or antigen-binding nt of an antibody of the invention to a subject in need thereof. The disorder treated is any disease or condition which is improved, ameliorated, inhibited or prevented by targeting GITR and/or by sing T-cell proliferation or function and/or inhibiting suppression activity induced by regulatory T cells.

In yet another aspect, the invention provides therapeutic methods for killing tumor cells or for inhibiting or attenuating tumor cell growth, or otherwise treating a patient afflicted with , using a combination of an anti-GITR antibody or antigen-binding portion of an ITR antibody and an anti-PD1 antibody or antigen-binding portion of an anti-PD1 antibody. The eutic methods according to this aspect of the invention comprise administering a therapeutically effective amount of a pharmaceutical composition comprising a ation of an anti-GITR and anti-PD1 antibody or antigen-binding fragment composition to a subject in need thereof. The disorder treated is any e or condition which is improved, ameliorated, inhibited or prevented by targeting both GITR and PD1.

Other embodiments will become apparent from a review of the ensuing detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 s average tumor volumes for each treatment group (mm3 ± SEM) plotted against days after tumor challenge as described in Example 7. Mice were treated with either isotype antibody (open circles, ○), anti-PD-1 antibody (open squares, □), anti- GITR antibody (open pyramids, ∆), or a combination of anti-PD-1 and anti-GITR (closed inverted pyramids, ▼).

Figure 2 depicts survival analysis of MC38 bearing mice d with the combination of an anti-mouse GITR and anti-mouse- PD1 antibody as described in Example 7. Mice were treated with either isotype dy (open circles, ○), anti-PD-1 antibody (open squares, □), anti-GITR antibody (open pyramids, ∆), or a combination of anti-PD-1 and anti- GITR (open inverted pyramids,).

Figure 3 depicts the individual tumor growth curve of tumor-free or naïve control mice nged with MC38 or B16.F10.9 tumor cells as described in Example 7.

Figure 4 depicts average tumor volumes for mice treated with different depletion antibodies as described in Example 7.

Figure 5 depicts FACS analysis result of intratumoral CD8/Treg, CD4 Teff/Treg ratio as bed in e 7.

Figure 6 depicts percentage and cell /mm3 tumor of T cell subsets in tumor as described in Example 7.

Figure 7 depicts average tumor volumes for each treatment group (mm3 ± SEM) plotted against days after tumor challenge as depicted in Example 7.

Figure 8 depicts survival analysis of MC38 g GITR/GIRL humanized mice treated with the combination of an anti-human GITR- and anti-mouse PD1-antibody as described in Example 7.

Figure 9 s FACS analysis of intratumoral and spleen percentage of CD8 T cells, percentage of Treg cells, and CD8/Treg ratio as bed in e 7.

Figure 10 depicts average tumor volumes for each treatment group (mm3 ± SEM) plotted against days after tumor challenge as described in Example 7.

Figure 11 s survival analysis of MC38 g PD1/PDL1 humanized mice treated with the combination of an anti-mouse GITR- and uman PD1-antibody as described in Example 7.

Figure 12 depicts a tumor growth curve of MC38-bearing mice as described in Example 7. The Y-axis depicts tumor volume in cubic millimeters and the X-axis depicts time in days post tumor challenge. Open s (□, ○) ent mice first treated with an isotype antibody (control). Filled symbols (■, ●) ent mice first treated with an anti- CD226 antibody. Those mice then treated with the e antibody are represented by circles (○, ●) and solid lines. Those mice then treated with the anti-GITR and anti-PD-1 combination are represented by squares (□, ■) and dotted lines.

Figure 13 depicts a survival curve of MC38-bearing mice as described in Example 7. The Y-axis depicts percent survival and the X-axis depicts time in days post tumor nge. Open symbols (□, ○) represent mice first treated with an isotype antibody (control). Filled symbols (■, ●) represent mice first treated with an anti-CD226 antibody.

Those mice then treated with the isotype antibody are represented by circles (○, ●) and solid lines. Those mice then treated with the anti-GITR and anti-PD-1 combination are represented by s (□, ■) and dotted lines.

Figure 14 depicts a tumor growth curve of MC38-bearing wild type mice (represented by diamonds [ ]) or TIGIT knock-out mice (represented by triangles [ ]) treated with isotype IgGs as described in Example 7. The Y-axis depicts tumor volume in cubic millimeters and the X-axis depicts time in days post tumor challenge. Open symbols and dotted lines represent mice first treated with an isotype antibody ol). Filled symbols and solid lines represent mice first treated with an anti-CD226 antibody.

Figure 15 depicts a tumor growth curve of MC38-bearing wild type mice (represented by circles [○, ●]) or TIGIT knock-out mice (represented by inverted les [ ]) treated with isotype IgGs as described in Example 7. The Y-axis depicts tumor volume in cubic millimeters and the X-axis depicts time in days post tumor challenge. Open symbols and dotted lines represent mice first treated with an isotype antibody (control). Filled symbols and solid lines represent mice first d with an anti-CD226 antibody.

Figure 16 includes cumulative distribution function (CDF) plots depicting the upregulated expression of CD226 by combination treatment in total (Panel A), clonal expanded (Panel B), or non-expanded (Panel C) CD8+ T cells. The X-axis depicts CD226 expression in log2(RPKM) (Reads Per Kilobase of transcript per Million mapped reads) and the Y-axis depicts cumulative frequency. The red line represents the ITR / anti-PD1 ent; the black line represents isotype antibody treatment; the blue line ents anti- GITR treatment; and the purple line ents anti-PD1 treatment.

Figure 17 is a Western blot depicting the relative expression of phospho-CD3ζ and phospho-CD226 as a function of PD-1 tration.

Figure 18, Panels A-D depict bar charts of the number of T-cell types produced in wildtype (open bars) and CD226 knock out mice (solid bars). Panel A is a bar chart depicting FACS analysis (number of cells) of T cell development in thymus (Tconv, conventional T cells; DP, CD4/CD8 double positive; SP, single positive; DN, CD4/CD8 double negative).

Panel B is a bar chart depicting FACS validation (number of cells) of the population of T cell subsets in spleen and blood in wildtype and CD226-/- animals. Panel C is a bar chart depicting FACS analysis (MFI, mean fluorescence intensity) of T cell subsets in spleen and blood that express PD1. Panel D is a bar chart depicting FACS is (MFI) of T cell subsets in spleen and blood that express GITR. Panels E-I are bar charts depicting inflammatory cytokine secretion in picograms per milliliter upon ex vivo TCR ation of splenocytes with anti-CD3 + anti-CD28 Ab for 16 hours. Splenocytes from CD226-/- (solid bars) or wild type (WT) (open bars) mice were stimulated with anti-CD3 + anti-CD28 Ab for 16 hours. Panel E = IFN-γ; panel F = IL-2; panel G = TNF-α; panel H = IL-6; and panel I = IL- Figure 19 is a line graph depicting percent animal survival as a on of time in days post tumor challenge. Panel A depicts CD226 KO mice (rose lines) or WT littermates (black lines) nged with MC38 tumor cells and treated with either anti-GITR + anti-PD-1 Ab (filled s and squares) or isotype Abs (open circles and squares). Panels B-D depict the effect of antibody treatment on (B) animals treated with antibodies blocking CD28 signaling (10 mg/kg CTLAIg; panel B, green lines); (C) animals treated with antibodies blocking OX40 signaling (10 mg/kg OX40L blocking antibody; panel C); and (D) animals treated with antibodies blocking 4-1BB signaling (10 mg/kg 4-1BBL blocking antibody; panel Figure 20 is a line graph depicting tumor size (in cubic millimeters) as a on of days after tumor challenge for mice treated with isotype (open circles and black line), anti- PD1 (open squares, red line), anti-GITR (open upright pyramids and green line), and anti- GITR / D1 combination y (open inverted pyramids and blue lines). Panel A represents MC38 tumors not expressing CD155. Panel B represents MC38 tumors expressing CD155.

Figure 21 is a bar chart depicting the number of cells sing CD226 (Panel A), 4-1BB (Panel B), and IFN-γ (Panel C) from s challenged with MC38 tumor cells over expressing CD155 (filled bars) and MC38 tumor cells that do not express CD155 (open bars).

Figure 22 depicts a dot plot RNA-seq analysis of cancer t tumor biopsies showing CD226 RNA expression (in log2[RPKM]) as a function of anti-PD-1 Ab treatment.

DETAILED DESCRIPTION Before the present invention is bed, it is to be tood that this invention is not limited to particular methods and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present ion will be limited only by the ed claims.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as ly understood by one of ordinary skill in the art to which this invention belongs. As used herein, the term "about," when used in reference to a particular recited numerical value, means that the value may vary from the d value by no more than 1%. For example, as used herein, the expression "about 100" includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred s and materials are now described. All patents, applications and non-patent publications mentioned in this specification are orated herein by reference in their entireties. tions The expression glucocorticoid-induced tumor necrosis factor receptor, "GITR," and the like, as used herein, refers to the human orticoid-induced tumor necrosis factor receptor, comprising the amino acid sequence as set forth in SEQ ID NO: 413 (NCBI Accession #NP_004186.1). The sion "GITR" includes both monomeric and multimeric GITR molecules. As used herein, the expression "monomeric human GITR" means a GITR protein or portion f that does not contain or possess any multimerizing domains and that exists under normal conditions as a single GITR molecule without a direct physical connection to another GITR molecule. An exemplary monomeric GITR molecule is the molecule referred to herein as "hGITR.mmh" comprising the amino acid sequence of SEQ ID NO: 409 (see, e.g., Example 3, herein). As used herein, the expression ic human GITR" means a construct comprising two GITR molecules connected to one another through a linker, covalent bond, non-covalent bond, or through a multimerizing domain such as an antibody Fc domain. Exemplary dimeric GITR molecules include those molecules referred to herein as "hGITR.mFc" and “hGITR.hFc”, sing the amino acid sequence of SEQ ID NO: 410 and SEQ ID NO: 411 respectively (see, e.g., Example 3, herein).

All references to ns, polypeptides and protein fragments herein are intended to refer to the human version of the respective protein, polypeptide or protein fragment unless explicitly specified as being from a non-human species. Thus, the expression "GITR" means human GITR unless specified as being from a non-human species, e.g., "mouse GITR," "monkey GITR," etc.

As used herein, the expression "cell surface-expressed GITR" means one or more GITR protein(s), or the ellular domain thereof, that is/are expressed on the surface of a cell in vitro or in vivo, such that at least a portion of a GITR protein is exposed to the extracellular side of the cell membrane and is accessible to an antigen-binding portion of an antibody. A "cell e-expressed GITR" can comprise or consist of a GITR protein sed on the surface of a cell which ly expresses GITR protein. Alternatively, "cell surface-expressed GITR" can comprise or consist of GITR protein expressed on the surface of a cell that normally does not express human GITR on its surface but has been artificially engineered to express GITR on its surface.

As used herein, the expression "anti-GITR antibody" includes both monovalent and monospecific bivalent antibodies with a single specificity, as well as ific dies comprising a first arm that binds GITR and a second arm that binds a second (target) antigen, wherein the anti-GITR arm comprises any of the HCVR/LCVR or CDR sequences as set forth in Table 1 herein. The expression "anti-GITR antibody" also includes antibodydrug conjugates (ADCs) comprising an anti-GITR antibody or antigen-binding portion f conjugated to a drug or toxin (i.e., cytotoxic agent). The sion "anti-GITR antibody" also includes antibody-radionuclide conjugates (ARCs) sing an anti-GITR antibody or antigen-binding portion thereof conjugated to a radionuclide.

The term "antibody", as used herein, means any antigen-binding molecule or molecular complex comprising at least one complementarity determining region (CDR) that specifically binds to or interacts with a particular antigen (e.g., GITR). The term "antibody" includes immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, as well as multimers thereof (e.g., IgM). Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain nt . The heavy chain constant region comprises three s, CH1, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region comprises one domain (CL1). The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions , interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments of the invention, the FRs of the anti-GITR antibody (or antigen-binding portion thereof) may be cal to the human germline sequences, or may be naturally or cially modified. An amino acid consensus sequence may be defined based on a side-by-side analysis of two or more CDRs.

The term ody", as used herein, also includes antigen-binding fragments of full antibody molecules. The terms "antigen-binding portion" of an antibody, "antigen-binding nt" of an antibody, and the like, as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. Antigen-binding fragments of an antibody may be derived, e.g., from full antibody molecules using any suitable standard ques such as proteolytic ion or inant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and optionally constant domains. Such DNA is known and/or is readily available from, e.g., commercial sources, DNA libraries (including, e.g., phage-antibody libraries), or can be synthesized.

The DNA may be sequenced and lated chemically or by using molecular biology techniques, for example, to arrange one or more variable and/or constant domains into a suitable configuration, or to introduce codons, create cysteine residues, modify, add or delete amino acids, etc.

Non-limiting examples of antigen-binding fragments include: (i) Fab nts; (ii) F(ab')2 fragments; (iii) Fd nts; (iv) Fv fragments; (v) -chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an dy (e.g., an isolated complementarity determining region (CDR) such as a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide. Other engineered molecules, such as -specific antibodies, single domain antibodies, domain-deleted dies, chimeric antibodies, afted antibodies, diabodies, dies, tetrabodies, minibodies, dies (e.g. monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed within the expression "antigenbinding fragment," as used herein.

An antigen-binding fragment of an antibody will typically comprise at least one variable domain. The variable domain may be of any size or amino acid composition and will generally se at least one CDR which is nt to or in frame with one or more framework sequences. In antigen-binding fragments having a VH domain associated with a VL domain, the VH and VL domains may be situated relative to one r in any suitable arrangement. For example, the variable region may be dimeric and contain VH-VH, VH-VL or VL-VL dimers. Alternatively, the antigen-binding fragment of an antibody may contain a monomeric VH or VL domain.

In certain embodiments, an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting, exemplary configurations of variable and constant domains that may be found within an antigen-binding fragment of an antibody of the present invention include: (i) VH-CH1; (ii) VH- CH2; (iii) VH-CH3; (iv) -CH2; (v) VH-CH1-CH2-CH3; (vi) VH-CH2-CH3; (vii) VH-CL; (viii) VLCH1 ; (ix) ; (x) VL-CH3; (xi) -CH2; (xii) VL-CH1-CH2-CH3; (xiii) VL-CH2-CH3; and (xiv) VL-CL. In any configuration of variable and nt domains, including any of the exemplary configurations listed above, the variable and constant domains may be either directly linked to one another or may be linked by a full or partial hinge or linker region. A hinge region may consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids which result in a flexible or semi-flexible linkage between adjacent le and/or constant domains in a single polypeptide molecule. Moreover, an n-binding fragment of an antibody of the present invention may comprise a homo-dimer or hetero-dimer (or other multimer) of any of the variable and constant domain configurations listed above in noncovalent association with one r and/or with one or more monomeric VH or VL domain (e.g., by disulfide bond(s)).

As with full antibody molecules, antigen-binding fragments may be monospecific or pecific (e.g., bispecific). A multispecific antigen-binding fragment of an antibody will typically comprise at least two different variable domains, n each variable domain is capable of specifically binding to a separate antigen or to a different epitope on the same antigen. Any multispecific antibody format, ing the exemplary bispecific antibody formats disclosed herein, may be adapted for use in the context of an antigen-binding fragment of an antibody of the t invention using routine techniques available in the art.

The antibodies of the present invention may function through ment- dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC).

"Complement-dependent cytotoxicity" (CDC) refers to lysis of antigen-expressing cells by an antibody of the invention in the ce of complement. "Antibody-dependent cellmediated cytotoxicity" (ADCC) refers to a cell-mediated reaction in which nonspecific cytotoxic cells that s Fc receptors (FcRs) (e.g., Natural Killer (NK) cells, neutrophils, and macrophages) recognize bound antibody on a target cell and thereby lead to lysis of the target cell. CDC and ADCC can be measured using assays that are well known and available in the art. (See, e.g., U.S. Patent Nos 5,500,362 and 5,821,337, and Clynes et al. (1998) Proc. Natl. Acad. Sci. (USA) 95:652-656). The nt region of an antibody is important in the ability of an antibody to fix complement and mediate cell-dependent cytotoxicity. Thus, the e of an antibody may be selected on the basis of whether it is desirable for the antibody to mediate xicity.

In certain embodiments of the invention, the anti-GITR antibodies of the invention are human antibodies. The term "human antibody", as used herein, is ed to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations uced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3. However, the term "human antibody", as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. The term “human antibody” does not include naturally ing les that normally exist without modification or human intervention/manipulation, in a naturally occurring, unmodified living organism.

The antibodies of the invention may, in some embodiments, be inant human antibodies. The term "recombinant human antibody", as used herein, is intended to e all human antibodies that are prepared, expressed, created or isolated by inant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell (described further below), antibodies isolated from a recombinant, combinatorial human antibody library (described further below), antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes (see e.g., Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295) or antibodies ed, expressed, created or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences. Such inant human antibodies have variable and constant regions derived from human germline globulin sequences. In certain embodiments, however, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo c mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while d from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.

Human antibodies can exist in two forms that are associated with hinge heterogeneity. In one form, an immunoglobulin molecule comprises a stable four chain construct of approximately 150-160 kDa in which the dimers are held together by an interchain heavy chain disulfide bond. In a second form, the dimers are not linked via interchain disulfide bonds and a le of about 75-80 kDa is formed composed of a covalently coupled light and heavy chain (half-antibody). These forms have been ely difficult to separate, even after ty purification.

The frequency of appearance of the second form in various intact IgG isotypes is due to, but not limited to, structural differences associated with the hinge region isotype of the antibody. A single amino acid substitution in the hinge region of the human IgG4 hinge can significantly reduce the appearance of the second form (Angal et al. (1993) Molecular Immunology 30:105) to levels typically observed using a human IgG1 hinge. The t invention encompasses antibodies having one or more mutations in the hinge, CH2 or CH3 region which may be desirable, for example, in production, to improve the yield of the desired antibody form.

The antibodies of the invention may be isolated dies. An "isolated antibody," as used herein, means an antibody that has been identified and separated and/or recovered from at least one ent of its natural environment. For example, an dy that has been separated or removed from at least one component of an organism, or from a tissue or cell in which the dy naturally exists or is naturally ed, is an "isolated antibody" for purposes of the present invention. An isolated antibody also includes an antibody in situ within a recombinant cell. Isolated antibodies are antibodies that have been subjected to at least one purification or isolation step. According to certain embodiments, an isolated antibody may be substantially free of other cellular material and/or chemicals.

The anti-GITR antibodies disclosed herein may comprise one or more amino acid substitutions, insertions and/or deletions in the framework and/or CDR s of the heavy and light chain variable domains as compared to the corresponding germline sequences from which the antibodies were derived. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available from, for example, public antibody sequence databases. The present invention includes antibodies, and antigen-binding fragments thereof, which are derived from any of the amino acid sequences sed herein, wherein one or more amino acids within one or more ork and/or CDR regions are mutated to the corresponding residue(s) of the germline sequence from which the antibody was derived, or to the corresponding residue(s) of another human ne sequence, or to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence s are referred to herein collectively as "germline mutations"). A person of ordinary skill in the art, starting with the heavy and light chain le region sequences disclosed herein, can easily e numerous antibodies and antigen-binding fragments which se one or more individual germline mutations or combinations thereof. In certain embodiments, all of the framework and/or CDR residues within the VH and/or VL domains are mutated back to the residues found in the original germline ce from which the antibody was derived. In other embodiments, only certain residues are mutated back to the original germline sequence, e.g., only the mutated residues found within the first 8 amino acids of FR1 or within the last 8 amino acids of FR4, or only the mutated es found within CDR1, CDR2 or CDR3. In other ments, one or more of the framework and/or CDR residue(s) are mutated to the corresponding residue(s) of a different germline sequence (i.e., a germline sequence that is different from the germline sequence from which the antibody was ally derived).

Furthermore, the antibodies of the present invention may contain any combination of two or more germline mutations within the framework and/or CDR regions, e.g., wherein certain individual es are mutated to the corresponding residue of a particular germline sequence while certain other residues that differ from the original germline sequence are maintained or are mutated to the corresponding residue of a different germline sequence.

Once obtained, dies and antigen-binding fragments that contain one or more germline mutations can be easily tested for one or more desired property such as, improved binding icity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as the case may be), reduced immunogenicity, etc. dies and n-binding fragments obtained in this general manner are encompassed within the present invention.

The present invention also includes anti-GITR antibodies comprising variants of any of the HCVR, LCVR, and/or CDR amino acid sequences disclosed herein having one or more conservative substitutions. For example, the present invention includes anti-GITR antibodies having HCVR, LCVR, and/or CDR amino acid sequences with, e.g., 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc. conservative amino acid substitutions relative to any of the HCVR, LCVR, and/or CDR amino acid sequences set forth in Table 1 herein.

The term "epitope" refers to an antigenic inant that interacts with a ic antigen-binding site in the variable region of an antibody molecule known as a paratope. A single antigen may have more than one epitope. Thus, different antibodies may bind to different areas on an antigen and may have different biological effects. Epitopes may be either conformational or linear. A conformational epitope is produced by spatially juxtaposed amino acids from different segments of the linear polypeptide chain. A linear e is one produced by adjacent amino acid residues in a polypeptide chain. In certain circumstance, an epitope may include moieties of saccharides, phosphoryl groups, or sulfonyl groups on the antigen.

The term "substantial identity" or "substantially cal," when referring to a nucleic acid or fragment thereof, indicates that, when lly aligned with appropriate nucleotide insertions or deletions with r c acid (or its mentary strand), there is nucleotide sequence identity in at least about 95%, and more ably at least about 96%, 97%, 98% or 99% of the nucleotide bases, as measured by any well-known algorithm of ce ty, such as FASTA, BLAST or Gap, as discussed below. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule may, in certain instances, encode a ptide having the same or substantially similar amino acid sequence as the polypeptide d by the reference nucleic acid molecule.

As applied to polypeptides, the term "substantial similarity" or "substantially similar" means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 95% sequence identity, even more preferably at least 98% or 99% sequence identity. Preferably, residue positions which are not identical differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In l, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by vative substitutions, the percent ce identity or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are nown to those of skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24: 307-331, herein incorporated by reference. Examples of groups of amino acids that have side chains with similar chemical properties include (1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; (2) tic-hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and phan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartate and ate, and (7) sulfur-containing side chains are cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalaninetyrosine , lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine.

Alternatively, a conservative replacement is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256: 1443-1445, herein incorporated by reference. A "moderately conservative" ement is any change having a nonnegative value in the PAM250 log-likelihood matrix.

Sequence similarity for polypeptides, which is also referred to as sequence identity, is typically ed using sequence analysis software. Protein analysis software matches similar sequences using es of similarity assigned to various substitutions, ons and other modifications, including conservative amino acid substitutions. For instance, GCG software contains programs such as Gap and Bestfit which can be used with default parameters to determine ce homology or ce identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild type protein and a mutein thereof. See, e.g., GCG Version 6.1. Polypeptide sequences also can be compared using FASTA using default or recommended parameters, a program in GCG Version 6.1. FASTA (e.g., FASTA2 and ) es alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson (2000) supra). Another preferred algorithm when comparing a sequence of the ion to a database containing a large number of sequences from different organisms is the computer m BLAST, especially BLASTP or TBLASTN, using default parameters. See, e.g., Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1997) c Acids Res. 25:3389-402, each herein incorporated by reference.

Anti-GITR Antibodies Comprising Fc Variants According to certain embodiments of the present invention, anti-GITR antibodies are provided comprising an Fc domain sing one or more mutations which e or diminish antibody binding to the FcRn receptor, e.g., at acidic pH as compared to neutral pH.

For example, the present invention includes anti-GITR antibodies comprising a mutation in the CH2 or a CH3 region of the Fc domain, n the mutation(s) increases the affinity of the Fc domain to FcRn in an acidic environment (e.g., in an endosome where pH ranges from about 5.5 to about 6.0). Such mutations may result in an increase in serum half-life of the antibody when administered to an animal. Non-limiting examples of such Fc modifications include, e.g., a modification at position 250 (e.g., E or Q); 250 and 428 (e.g., L or F); 252 (e.g., L/Y/F/W or T), 254 (e.g., S or T), and 256 (e.g., S/R/Q/E/D or T); or a cation at on 428 and/or 433 (e.g., H/L/R/S/P/Q or K) and/or 434 (e.g., H/F or Y); or a modification at position 250 and/or 428; or a modification at position 307 or 308 (e.g., 308F, V308F), and 434. In one embodiment, the modification comprises a 428L (e.g., M428L) and 434S (e.g., N434S) modification; a 428L, 259I (e.g., V259I), and 308F (e.g., V308F) modification; a 433K (e.g., H433K) and a 434 (e.g., 434Y) modification; a 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modification; a 250Q and 428L modification (e.g., T250Q and M428L); and a 307 and/or 308 cation (e.g., 308F or 308P).

For example, the t invention includes anti-GITR antibodies comprising an Fc domain comprising one or more pairs or groups of mutations selected from the group consisting of: 250Q and 248L (e.g., T250Q and M248L); 252Y, 254T and 256E (e.g., M252Y, S254T and T256E); 428L and 434S (e.g., M428L and N434S); and 433K and 434F (e.g., H433K and N434F). All possible ations of the foregoing Fc domain mutations, and other mutations within the antibody le domains disclosed herein, are contemplated within the scope of the present invention.

Biological Characteristics of the Anti-GITR Antibodies The t invention includes antibodies and antigen-binding fragments thereof that bind monomeric human GITR with high affinity. For example, the present invention includes anti-GITR antibodies that bind monomeric human GITR (e.g., hGITR.mmh) with a KD of less than about 5.0 nM as measured by surface plasmon resonance at 37ºC, e.g., using an assay format as defined in Example 3 herein, or a substantially similar assay. In some ments, anti-GITR antibodies are provided that bind monomeric human GITR at 37ºC with a KD of less than about 4 nM, less than about 3 nM, less than about 2 nM, or less than about 1.50 nM as measured by surface plasmon resonance, e.g., using an assay format as defined in Example 3 herein, or a substantially similar assay.

The present ion also includes dies and antigen-binding fragments thereof that bind monomeric human GITR (e.g., hGITR.mmh) with a dissociative ife (t½) of greater than about 12 minutes as ed by surface plasmon resonance at 37ºC, e.g., using an assay format as defined in Example 3 herein, or a substantially similar assay.

According to certain embodiments, anti-GITR antibodies are provided that bind monomeric human GITR at 37ºC with a t½ of greater than about 12 minutes, greater than about 13 minutes, greater than about 14 minutes, greater than about 15 minutes, or longer, as measured by surface plasmon resonance, e.g., using an assay format as defined in Example 3 herein, or a substantially similar assay.

The present invention also includes antibodies and antigen-binding fragments f that bind dimeric human GITR (e.g., hGITR.mFc) with high affinity. For example, the present ion includes anti-GITR dies that bind dimeric human GITR with a KD of less than about 950 pM as measured by surface plasmon resonance at 37ºC, e.g., using an assay format as defined in Example 3 herein, or a substantially similar assay. ing to certain embodiments, anti-GITR antibodies are provided that bind dimeric human GITR at 37ºC with a KD of less than about 900 pM, less than about 850 pM, less than about 800 pM, less than about 700 pM, less than about 600 pM, less than about 500 pM, less than about 400 pM, less than about 300 pM, less than about 200 pM, or less than about 100 pM as measured by surface plasmon nce, e.g., using an assay format as defined in Example 3 herein, or a substantially similar assay.

The present invention also includes dies and antigen-binding fragments thereof that bind dimeric human GITR (e.g., hGITR.mFc) with a dissociative ife (t½) of greater than about 7 minutes as measured by surface plasmon resonance at 37ºC, e.g., using an assay format as defined in Example 3 herein, or a substantially similar assay.

According to n embodiments, anti-GITR antibodies are provided that bind dimeric human GITR at 37ºC with a t½ of greater than about 10 minutes, greater than about 20 minutes, greater than about 30 minutes, greater than about 40 s, greater than about 50 minutes, greater than about 60 minutes, greater than about 70 minutes, greater than about 80 minutes, r than about 90 s, greater than about 100 minutes, or longer, as measured by e plasmon resonance, e.g., using an assay format as defined in Example 3 herein, or a substantially similar assay.

The present disclosure also includes antibodies and antigen-binding fragments thereof that bind cell-surface-expressed GITR. For example, antibodies that bind to human GITR transfected embryonic kidney 293 (HEK-293) D9 cells with high affinity are provided . For example, the instant disclosure includes anti-GITR antibodies that bind human GITR transfected embryonic kidney 293 (HEK-293) D9 cells with an EC50 of less than about 260 pM as measured by electrochemiluminescence, e.g., using an assay format as defined in Example 4 herein, or a ntially similar assay. In certain embodiments, anti-GITR antibodies are provided that bind human GITR transfected nic kidney 293 (HEK-293) D9 cells with an EC50 of less than about 250 pM, less than about 240 pM, less than about 230 pM, or less than about 220 pM as measured by ochemiluminescence, e.g., using an assay format as defined in Example 4 herein, or a substantially similar assay.

The antibodies of the present invention may possess one or more of the aforementioned biological characteristics, or any combination thereof. The foregoing list of biological characteristics of the dies of the invention is not intended to be exhaustive.

Other biological characteristics of the dies of the present invention will be evident to a person of ordinary skill in the art from a review of the present disclosure including the working Examples herein.

Fc anchoring-dependent and anchoring-independent GITR activation and GITRL blocking The present disclosure includes antibodies and antigen-binding nts f that activate human GITR, e.g., as determined in the assay s described in Example 5 and/or Example 6 herein, or in a substantially similar assay format. As used herein, ates human GITR” refers to the activation of GITR via binding to its cognate ligand, GITR Ligand (GITRL) or to the binding of agonist anti-GITR antigen binding protein(s) to GITR. With regard to activation of GITR by agonist anti-GITR binding proteins, “activation” can be in the presence or absence of antigen-binding protein anchoring to Fc gamma receptors. Human GITR activation is manifested in the exhibition of n biological activities, including but not limited to the induction or enhancement of GITR signaling in vitro or in vivo, the reduction of regulatory T cell suppression of effector T cell activity; the decrease of circulating T reg levels in vitro or in vivo, the decrease of umoral T regs in vivo, the activation of effector T cells in vitro or in vivo, the ion or enhancement of effector T cell proliferation in vitro or in vivo, or the inhibition or reduction of tumor growth in vivo.

GITR activation in the absence of Fc Anchoring In some embodiments, the dies and antigen-binding fragments thereof provided herein activate human GITR in the absence of Fc anchoring, e.g., as ined in the assay formats described in Example 5 and/or Example 6 herein, or in a substantially similar assay format. As used herein, “in the absence of Fc anchoring” refers to the activation of GITR and GITR-mediated signaling or blocking of GITRL without the clustering of anti-GITR antibodies by different forms of the Fc gamma receptor and can be determined and quantified via, e.g., the activation of y T-cells co-cultured in vitro in the absence of cell-surface bound Fc gamma receptor(s). In some embodiments, the antibody or nbinding fragment thereof activates human GITR at an tion percentage greater than about 25% at an EC50 of less than about 3 nM in the absence of Fc anchoring, as determined by NFκB reporter assay, e.g., as described in Example 5 or substantially similar assay format. In some embodiments, the antibody or n-binding fragment thereof tes human GITR at an activation percentage greater than about 30%, greater than about 40%, greater than about 50%, greater than about 60%, or greater than about 65% at an EC50 of less than about 3 nM in the absence of Fc anchoring as ined by NFκB reporter assay, e.g., as described in Example 5 or substantially r assay format. In some embodiments, the antibody or antigen-binding fragment thereof activates human GITR at an activation percentage greater than about 30%, greater than about 40%, greater than about 50%, greater than about 60%, or greater than about 65% at an EC50 of less than about 2 nM in the e of Fc ing as determined by NFκB reporter assay, e.g., as described in Example 5 or substantially similar assay format. In some embodiments, the dy or antigen-binding fragment thereof activates human GITR at an activation tage greater than about 30%, greater than about 40%, greater than about 50%, r than about 60%, or greater than about 65% at an EC50 of less than about 1.5 nM in the absence of Fc anchoring as determined by NFκB reporter assay, e.g., as described in Example 5 or substantially similar assay format. In some embodiments, the antibody or n-binding fragment thereof activates human GITR at an activation percentage greater than about 30%, r than about 40%, r than about 50%, greater than about 60%, or greater than about 65% at an EC50 of less than about 1.4 nM in the absence of Fc anchoring as determined by NFκB reporter assay, e.g., as bed in Example 5 or substantially r assay format. In some embodiments, the antibody or antigen-binding fragment thereof activates human GITR at an activation percentage greater than about 30%, greater than about 40%, greater than about 50%, r than about 60%, or greater than about 65% at an EC50 of less than about 1.3 nM in the absence of Fc anchoring as determined by NFκB reporter assay, e.g., as described in Example 5 or substantially similar assay format.

In some embodiments, the antibody or antigen-binding fragment thereof binds GITR and exhibits T-cell proliferative activity in the absence of Fc-anchoring as determined by naïve human CD4+ T-cell proliferation assay, e.g., as described in Example 6 or substantially similar assay format. In some embodiments, the antibody or n-binding fragment thereof binds GITR and exhibits T-cell proliferative activity in the absence of Fc anchoring with an EC50 of about 8 nM or less as determined by naïve human CD4+ T-cell proliferation assay, e.g., as described in Example 6 or substantially similar assay format. In some embodiments, the antibody or antigen-binding fragment thereof binds GITR and exhibits T-cell proliferative activity in the absence of Fc anchoring at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or at least 11 fold above background at about 22 nM antibody (or antigen-binding fragment) concentration as determined by naïve human CD4+ T-cell proliferation assay, e.g., as described in Example 6 or substantially similar assay format.

GITR tion in the presence of Fc anchoring In some embodiments, the antibodies or n-binding fragments thereof provided herein activate human GITR in the presence of Fc anchoring, e.g., as ined in the assay formats described in Example 5 and/or Example 6 herein, or in a substantially similar assay . As used herein, “in the presence of Fc anchoring” refers to the activation of GITR and GITR mediated signaling or blocking of GITRL through the clustering of anti-GITR antibodies via the interaction of the Fc region of the antibodies with different forms of the Fc gamma receptor (FcgR), such as FcgRI, FcgRIIa or Ia and can be determined and quantified via, e.g., the activation of T-cells co-cultured in vitro in the presence of cell-surface bound Fc gamma receptor(s).

In some embodiments, the antibody or n-binding fragment thereof exhibits T-cell proliferative ty in the presence of Fc anchoring at least about 2 fold above background at about 33 nM dy (or antibody-binding fragment) concentration as determined by naïve human CD4+ T-cell proliferation assay, e.g., as described in Example 6 or ntially similar assay format. In some embodiments, the antibody or antigen-binding nt thereof exhibits T-cell proliferative activity in the ce of Fc anchoring at least about 2 fold, at least about 3 fold, at least about 4 fold, or at least about 5 fold above background at about 33 nM antibody (or antigen-binding fragment) concentration as determined by naïve human CD4+ T-cell proliferation assay, e.g., as described in Example 6 or ntially similar assay format. In some embodiments, the antibody or antigen-binding fragment exhibits T-cell proliferative activity in the presence of Fc anchoring with an EC50 of less than about 34 nM as determined by naïve human CD4+ T-cell proliferation assay, e.g., as described in Example 6 or substantially similar assay format. In some embodiments, the antibody or n-binding fragment exhibits T-cell proliferative activity in the presence of Fc anchoring with an EC50 of less than about 30 nM, less than about 20 nM, less than about nM, less than about 5 nM, or less than about 4 nM as determined by naïve human CD4+ T-cell proliferation assay, e.g., as described in Example 6 or substantially similar assay format.

Antibodies that block GITR ligand ed receptor stimulation The present disclosure includes antibodies that block human GITR ligand (hGITRL)-mediated receptor stimulation, e.g., as determined in the assay format described in Example 5 herein. As used herein, “blocks human GITR ligand (hGITRL)-mediated receptor stimulation” refers to the ability of anti-GITR antigen binding proteins to block the binding of GITR to its cognate ligand, GITRL. The blocking of GITR ligand can restore the suppression of effector T-cell ty by regulatory T cells. The ng of GITR ligand can be determined and quantified via a variety of methods known in the art, including, for e, the reduction in T-cell proliferation or cytokine secretion and an increase in the levels of circulating regulatory T cells.

In some embodiments, the antibodies provided herein block human GITR ligand (hGITRL)-mediated receptor stimulation in the absence of GITR anchoring, e.g., as ined in the assay format described in Example 5 herein. In some embodiments, the antibody or dy-binding fragment thereof blocks human GITR ligand-mediated receptor stimulation in the absence of Fc anchoring with a blocking percentage greater than about 55% at an IC50 less than about 4.0 nM as determined by NFκB reporter assay, e.g., as described in Example 5 or ntially similar assay format. In some embodiments, the antibody or antibody-binding fragment thereof blocks human GITR -mediated receptor stimulation in the absence of Fc anchoring with a blocking tage greater than about 60%, greater than about 70%, greater than about 80%, or greater than about 85% at an IC50 less than about 4.0 nM, less than about 3.0 nM, less than about 2.0 nM, less than about 1.0 nM, less than about 0.9 nM, less than about 0.8 nM, or less than about 0.7 nM as determined by NFκB er assay, e.g., as bed in Example 5 or substantially similar assay format.

In some embodiments, the antibodies or antigen binding fragments activates human GITR and blocks human GITR ligand-mediated or stimulation at a blocking percentage less than about 25% in the absence of Fc anchoring as ined by NFκB reporter assay, e.g., in the assay described in Example 5 or substantially similar assay. In some embodiments, the antibodies or n binding fragments activates human GITR and blocks human GITR ligand-mediated receptor stimulation at a blocking percentage less than about 54% in the absence of Fc anchoring as determined by NFκB reporter assay, e.g., in the assay described in e 5 or substantially similar assay. In some embodiments, the antibodies or antigen binding fragments activates human GITR and blocks human GITR ligand-mediated receptor stimulation at a blocking percentage less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, or less than about 1% in the absence of Fc anchoring as determined by NFκB reporter assay, e.g., in the assay described in Example 5 or substantially similar assay. In some embodiments, the antibodies or antigen binding fragments activates human GITR at an activation percentage of at least about 50% and does not block hGITRL-mediated receptor stimulation at a blocking percentage of greater than about 50% in the absence of Fc anchoring as determined by NFκB reporter assay, e.g., in the assay described in Example 5 or ntially similar assay.

In some embodiments, the antibodies or antigen binding fragments both activate human GITR and block human GITR ligand (hGITRL)-mediated receptor stimulation.

In some embodiments, the antibodies both activate human GITR and block human GITR ligand (hGITRL)-mediated or stimulation in the absence of Fc anchoring, e.g., as determined in the assay format described in e 5 herein, or a substantially r assay. In some embodiments, (A) the antibody or antigen-binding fragment possesses at least one of the properties selected from the group consisting of: i. activates human GITR in the absence of Fc anchoring at an activation percentage greater than about 25% at an EC50 less than about 3 nM as determined by NFκB reporter assay and ii. tes human GITR in the absence of Fc anchoring with an EC50 of less than about 1.0 nM as determined by NFκB reporter assay; and (B) the antibody or antigen-binding fragment blocks hGITRL-mediated receptor stimulation in the absence of Fc anchoring at a blocking percentage greater than about 54% at an IC50 of less than about 4.0 nM as determined by NFκB reporter assay.

In some embodiments, (A) the antibody or n-binding fragment activates human GITR in the absence of Fc anchoring at an activation percentage greater than about 50% at an EC50 less than about 1.5 nM as determined by NFκB reporter assay; and (B) the antibody or n-binding fragment blocks hGITRL-mediated receptor stimulation in the absence of Fc anchoring at a blocking percentage greater than about 54% at an IC50 of less than about 4.0 nM as determined by NFκB reporter assay; and Epitope Mapping and Related Technologies The epitope to which the dies of the present invention bind may consist of a single contiguous sequence of 3 or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) amino acids of a GITR protein. atively, the epitope may consist of a plurality of non-contiguous amino acids (or amino acid sequences) of GITR. In some embodiments, the e is located on or near the GITRL-binding domain of GITR. In other embodiments, the epitope is located outside of the GITRL-binding domain of GITR, e.g., at a location on the surface of GITR at which an antibody, when bound to such an epitope, does not interfere with GITRL binding to GITR.

Various techniques known to s of ordinary skill in the art can be used to determine r an antibody "interacts with one or more amino acids" within a polypeptide or n. ary techniques include, e.g., routine cross-blocking assay such as that described Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY), alanine scanning mutational analysis, peptide blots analysis ke, 2004, Methods Mol Biol 248:443-463), and peptide cleavage analysis. In on, s such as epitope excision, epitope extraction and chemical modification of antigens can be ed (Tomer, 2000, Protein Science 496). Another method that can be used to identify the amino acids within a polypeptide with which an antibody interacts is hydrogen/deuterium exchange detected by mass spectrometry. In general terms, the hydrogen/deuterium exchange method involves deuterium-labeling the protein of interest, followed by binding the antibody to the ium-labeled protein. Next, the protein/antibody complex is transferred to water to allow hydrogen-deuterium exchange to occur at all residues except for the residues protected by the antibody (which remain deuterium-labeled). After dissociation of the antibody, the target protein is subjected to protease cleavage and mass spectrometry analysis, thereby revealing the deuterium-labeled residues which correspond to the specific amino acids with which the antibody interacts. See, e.g., Ehring (1999) Analytical Biochemistry 267(2):252-259; Engen and Smith (2001) Anal. Chem. A-265A.

The present invention further includes anti-GITR antibodies that bind to the same epitope as any of the specific exemplary antibodies described herein (e.g. antibodies comprising any of the amino acid sequences as set forth in Table 1 herein). Likewise, the present invention also includes anti-GITR antibodies that compete for binding to GITR with any of the specific ary antibodies described herein (e.g. antibodies comprising any of the amino acid sequences as set forth in Table 1 herein).

One can easily determine r an dy binds to the same epitope as, or competes for g with, a reference anti-GITR antibody by using routine methods known in the art and exemplified herein. For example, to determine if a test antibody binds to the same epitope as a reference anti-GITR antibody of the invention, the reference antibody is allowed to bind to a GITR protein. Next, the ability of a test antibody to bind to the GITR molecule is ed. If the test antibody is able to bind to GITR following saturation binding with the reference anti-GITR antibody, it can be concluded that the test antibody binds to a different e than the reference anti-GITR antibody. On the other hand, if the test antibody is not able to bind to the GITR molecule ing saturation binding with the reference anti-GITR antibody, then the test dy may bind to the same epitope as the epitope bound by the reference anti-GITR antibody of the invention. Additional routine experimentation (e.g., peptide mutation and binding analyses) can then be carried out to confirm whether the observed lack of binding of the test antibody is in fact due to binding to the same epitope as the reference antibody or if steric blocking (or another phenomenon) is responsible for the lack of observed binding. Experiments of this sort can be med using ELISA, RIA, Biacore, flow try or any other quantitative or qualitative antibodybinding assay available in the art. In accordance with certain embodiments of the present invention, two antibodies bind to the same (or overlapping) epitope if, e.g., a 1-, 5-, 10-, 20- or 100-fold excess of one dy inhibits binding of the other by at least 50% but ably 75%, 90% or even 99% as measured in a competitive binding assay (see, e.g., ns et al., Cancer Res. 1990:50:1495-1502). Alternatively, two antibodies are deemed to bind to the same e if essentially all amino acid mutations in the n that reduce or eliminate binding of one antibody reduce or eliminate g of the other. Two dies are deemed to have "overlapping epitopes" if only a subset of the amino acid mutations that reduce or eliminate g of one antibody reduce or eliminate binding of the other.

To determine if an antibody competes for binding (or cross-competes for binding) with a reference anti-GITR antibody, the above-described binding methodology is performed in two orientations: In a first orientation, the reference antibody is allowed to bind to a GITR protein under saturating conditions followed by assessment of binding of the test antibody to the GITR molecule. In a second orientation, the test antibody is d to bind to a GITR molecule under saturating conditions followed by assessment of binding of the reference antibody to the GITR molecule. If, in both orientations, only the first (saturating) antibody is capable of binding to the GITR molecule, then it is concluded that the test antibody and the reference dy compete for binding to GITR. As will be appreciated by a person of ordinary skill in the art, an antibody that es for binding with a reference antibody may not necessarily bind to the same epitope as the reference antibody, but may sterically block binding of the reference antibody by binding an overlapping or adjacent epitope.

Preparation of Human Antibodies The anti-GITR antibodies of the present invention can be fully human antibodies.

Methods for generating onal antibodies, including fully human monoclonal antibodies are known in the art. Any such known s can be used in the context of the present invention to make human antibodies that specifically bind to human GITR.

Using VELOCIMMUNE™ technology, for example, or any other similar known method for generating fully human monoclonal antibodies, high affinity chimeric antibodies to GITR are initially isolated having a human le region and a mouse constant . As in the experimental section below, the antibodies are characterized and selected for desirable characteristics, including affinity, ligand blocking activity, selectivity, epitope, etc. If necessary, mouse constant regions are replaced with a d human constant region, for example wild-type or modified IgG1 or IgG4, to generate a fully human anti-GITR antibody.

While the constant region selected may vary according to specific use, high affinity antigenbinding and target specificity characteristics reside in the variable region. In certain instances, fully human anti-GITR antibodies are isolated directly from antigen-positive B cells.

Bioequivalents The anti-GITR antibodies and antibody fragments of the present invention encompass proteins having amino acid sequences that vary from those of the described antibodies but that retain the ability to bind human GITR. Such variant antibodies and dy fragments comprise one or more additions, deletions, or substitutions of amino acids when compared to parent sequence, but exhibit biological activity that is ially equivalent to that of the bed antibodies. Likewise, the anti-GITR dy-encoding DNA sequences of the present invention encompass ces that comprise one or more additions, deletions, or substitutions of nucleotides when compared to the disclosed ce, but that encode an anti-GITR antibody or antibody fragment that is ially ivalent to an anti-GITR antibody or dy fragment of the invention. Examples of such variant amino acid and DNA ces are discussed above.

Two n-binding proteins, or antibodies, are considered bioequivalent if, for example, they are pharmaceutical equivalents or pharmaceutical alternatives whose rate and extent of absorption do not show a significant difference when administered at the same molar dose under similar experimental conditions, either single dose or multiple dose. Some antibodies will be considered equivalents or pharmaceutical alternatives if they are equivalent in the extent of their absorption but not in their rate of absorption and yet may be considered bioequivalent because such differences in the rate of absorption are intentional and are reflected in the labeling, are not essential to the attainment of effective body drug concentrations on, e.g., chronic use, and are considered medically insignificant for the particular drug product d.

In one embodiment, two antigen-binding proteins are bioequivalent if there are no ally meaningful differences in their safety, purity, and potency.

In one embodiment, two antigen-binding proteins are bioequivalent if a patient can be switched one or more times between the reference product and the biological product without an expected increase in the risk of adverse effects, including a clinically icant change in genicity, or diminished effectiveness, as ed to continued therapy without such switching.

In one embodiment, two antigen-binding proteins are bioequivalent if they both act by a common mechanism or mechanisms of action for the condition or conditions of use, to the extent that such mechanisms are known.

Bioequivalence may be demonstrated by in vivo and in vitro methods.

Bioequivalence measures include, e.g., (a) an in vivo test in humans or other mammals, in which the tration of the antibody or its metabolites is measured in blood, plasma, serum, or other biological fluid as a function of time; (b) an in vitro test that has been ated with and is ably predictive of human in vivo bioavailability data; (c) an in vivo test in humans or other mammals in which the appropriate acute pharmacological effect of the antibody (or its target) is measured as a function of time; and (d) in a well-controlled clinical trial that establishes safety, efficacy, or ilability or bioequivalence of an Bioequivalent variants of anti-GITR antibodies of the invention may be constructed by, for example, making various substitutions of residues or sequences or deleting terminal or internal residues or sequences not needed for ical activity. For example, cysteine residues not essential for biological activity can be deleted or ed with other amino acids to prevent formation of unnecessary or incorrect olecular disulfide bridges upon renaturation. In other contexts, bioequivalent antibodies may include anti-GITR antibody variants comprising amino acid changes which modify the glycosylation characteristics of the antibodies, e.g., mutations which eliminate or remove glycosylation.

Species Selectivity and Species Cross-Reactivity The present invention, according to certain ments, provides anti-GITR antibodies that bind to human GITR but not to GITR from other species. The present invention also includes anti-GITR antibodies that bind to human GITR and to GITR from one or more non-human species. For example, the anti-GITR antibodies of the invention may bind to human GITR and may bind or not bind, as the case may be, to one or more of mouse, rat, guinea pig, hamster, gerbil, pig, cat, dog, rabbit, goat, sheep, cow, horse, camel, cynomologous, et, rhesus or chimpanzee GITR. According to certain exemplary embodiments of the present ion, anti-GITR antibodies are provided which specifically bind human GITR and cynomolgus monkey (e.g., Macaca fascicularis) GITR. Other anti- GITR antibodies of the invention bind human GITR but do not bind, or bind only weakly, to cynomolgus monkey GITR. pecific Antibodies The antibodies of the present invention may be monospecific or multispecific (e.g., bispecific). Multispecific antibodies may be specific for different epitopes of one target ptide or may contain antigen-binding domains specific for more than one target polypeptide. See, e.g., Tutt et al., 1991, J. l. 147:60-69; Kufer et al., 2004, Trends Biotechnol. 22:238-244. The anti-GITR antibodies of the present invention can be linked to or co-expressed with another functional molecule, e.g., another peptide or protein. For e, an antibody or fragment thereof can be functionally linked (e.g., by chemical coupling, genetic , noncovalent association or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment to produce a bispecific or a multispecific antibody with a second binding specificity.

The present invention includes bispecific antibodies wherein one arm of an immunoglobulin binds human GITR, and the other arm of the immunoglobulin is specific for a second antigen. The GITR-binding arm can comprise any of the HCVR/LCVR or CDR amino acid ces as set forth in Table 1 herein. In certain embodiments, the GITR- binding arm binds human GITR and blocks GITRL binding to GITR. In other embodiments, the GITR-binding arm binds human GITR but does not block GITRL binding to GITR. In some embodiments, the GITR binding arm binds human GITR and activates GITR signaling.

In other embodiments, the GITR binding arm blocks GITRL mediated receptor ation.

The present invention also includes bispecific dies wherein one arm of an antibody binds a first epitope of human GITR, and the other arm of said antibody binds a second distinct epitope of human GITR.

An exemplary bispecific antibody format that can be used in the context of the present invention involves the use of a first immunoglobulin (Ig) CH3 domain and a second Ig CH3 domain, wherein the first and second Ig CH3 domains differ from one another by at least one amino acid, and wherein at least one amino acid difference reduces binding of the bispecific antibody to Protein A as compared to a ific antibody lacking the amino acid difference. In one ment, the first Ig CH3 domain binds Protein A and the second Ig CH3 domain contains a mutation that reduces or hes Protein A binding such as an H95R modification (by IMGT exon numbering; H435R by EU numbering). The second CH3 may further comprise a Y96F modification (by IMGT; Y436F by EU). Further modifications that may be found within the second CH3 include: D16E, L18M, N44S, K52N, V57M, and V82I (by IMGT; D356E, L358M, N384S, K392N, V397M, and V422I by EU) in the case of IgG1 dies; N44S, K52N, and V82I (IMGT; N384S, K392N, and V422I by EU) in the case of IgG2 dies; and Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (by IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I by EU) in the case of IgG4 antibodies. Variations on the bispecific antibody format described above are contemplated within the scope of the t invention.

Other exemplary ific formats that can be used in the context of the present invention include, without tion, e.g., scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-Ig, Quadroma, knobs-into-holes, common light chain (e.g., common light chain with knobs-into-holes, etc.), CrossMab, CrossFab, (SEED)body, leucine , Duobody, IgG1/IgG2, dual acting Fab (DAF)-IgG, and Mab2 bispecific formats (see, e.g., Klein et al. 2012, mAbs 4:6, 1-11, and nces cited therein, for a review of the foregoing s). Bispecific antibodies can also be constructed using peptide/nucleic acid conjugation, e.g., wherein unnatural amino acids with onal chemical reactivity are used to generate pecific antibody-oligonucleotide conjugates which then self-assemble into multimeric complexes with defined composition, valency and geometry. (See, e.g., Kazane et al., J. Am. Chem. Soc. [Epub: Dec. 4, 2012]). eutic Formulation and Administration The invention provides pharmaceutical compositions comprising the anti-GITR antibodies or antigen-binding fragments thereof of the present ion. The pharmaceutical compositions of the invention are formulated with suitable carriers, excipients, and other agents that provide improved transfer, delivery, tolerance, and the like.

A multitude of appropriate ations can be found in the ary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, nts, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as LIPOFECTIN™, Life Technologies, Carlsbad, CA), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil ons, emulsions carbowax (polyethylene glycols of various lar weights), semi-solid gels, and semi-solid mixtures containing carbowax.

See also Powell et al. "Compendium of excipients for parenteral formulations" PDA (1998) J Pharm Sci Technol 52:238-311.

The dose of antibody administered to a patient may vary depending upon the age and the size of the t, target disease, conditions, route of administration, and the like.

The preferred dose is typically calculated according to body weight or body surface area. In an adult patient, it may be advantageous to intravenously administer the dy of the present invention normally at a single dose of about 0.01 to about 20 mg/kg body weight, more preferably about 0.02 to about 7, about 0.03 to about 5, or about 0.05 to about 3 mg/kg body weight. Depending on the severity of the condition, the frequency and the on of the treatment can be adjusted. Effective dosages and schedules for administering anti-GITR antibodies may be determined empirically; for example, patient progress can be monitored by periodic assessment, and the dose adjusted accordingly. Moreover, interspecies scaling of dosages can be performed using well-known methods in the art (e.g., Mordenti et al., 1991, Pharmaceut. Res. 8:1351).

Various delivery systems are known and can be used to administer the ceutical ition of the invention, e.g., encapsulation in liposomes, articles, microcapsules, recombinant cells capable of expressing the mutant viruses, receptor mediated endocytosis (see, e.g., Wu et al., 1987, J. Biol. Chem. 262:4429-4432).

Methods of introduction include, but are not d to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The composition may be administered by any convenient route, for example by infusion or bolus ion, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal , etc.) and may be administered together with other ically active agents. Administration can be systemic or local.

A pharmaceutical composition of the present invention can be delivered subcutaneously or intravenously with a standard needle and syringe. In addition, with respect to aneous delivery, a pen delivery device readily has applications in delivering a pharmaceutical composition of the present invention. Such a pen delivery device can be reusable or disposable. A reusable pen delivery device generally utilizes a replaceable cartridge that contains a pharmaceutical composition. Once all of the pharmaceutical ition within the cartridge has been administered and the cartridge is empty, the empty cartridge can readily be discarded and replaced with a new cartridge that contains the pharmaceutical composition. The pen delivery device can then be reused. In a disposable pen delivery device, there is no eable cartridge. Rather, the disposable pen ry device comes prefilled with the pharmaceutical composition held in a oir within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.

Numerous reusable pen and autoinjector ry devices have applications in the subcutaneous delivery of a pharmaceutical ition of the present invention. Examples include, but are not limited to AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), ONIC™ pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75/25™ pen, HUMALOG™ pen, HUMALIN 70/30™ pen (Eli Lilly and Co., Indianapolis, IN), NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen n Dickinson, Franklin Lakes, NJ), OPTIPEN™, OPTIPEN PRO™, OPTIPEN STARLET™, and OPTICLIK™ (sanofi-aventis, Frankfurt, Germany), to name only a few. Examples of disposable pen delivery devices having applications in subcutaneous delivery of a pharmaceutical composition of the present invention include, but are not limited to the SOLOSTAR™ pen i-aventis), the FLEXPEN™ (Novo k), and the KWIKPEN™ (Eli Lilly), the SURECLICKTM Autoinjector (Amgen, nd Oaks, CA), the PENLETTM (Haselmeier, Stuttgart, Germany), the EPIPEN (Dey, L.P.), and the HUMIRATM Pen (Abbott Labs, Abbott Park IL), to name only a In certain situations, the pharmaceutical composition can be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201). In another embodiment, polymeric materials can be used; see, Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Florida. In yet another embodiment, a controlled release system can be placed in proximity of the composition’s target, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, 1984, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138). Other controlled release systems are discussed in the review by Langer, 1990, Science 27-1533.

The injectable preparations may include dosage forms for intravenous, subcutaneous, intracutaneous and intramuscular injections, drip infusions, etc. These injectable preparations may be prepared by methods publicly known. For e, the injectable preparations may be prepared, e.g., by dissolving, suspending or emulsifying the antibody or its salt described above in a e aqueous medium or an oily medium conventionally used for injections. As the aqueous medium for injections, there are, for example, physiological , an isotonic solution containing glucose and other auxiliary agents, etc., which may be used in combination with an appropriate solubilizing agent such as an alcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol, polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], etc. As the oily medium, there are employed, e.g., sesame oil, soybean oil, etc., which may be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc. The ion thus prepared is preferably filled in an appropriate ampoule. ageously, the pharmaceutical compositions for oral or parenteral use described above are prepared into dosage forms in a unit dose suited to fit a dose of the active ients. Such dosage forms in a unit dose include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of the aforesaid antibody contained is generally about 5 to about 500 mg per dosage form in a unit dose; especially in the form of injection, it is preferred that the aforesaid antibody is contained in about 5 to about 100 mg and in about 10 to about 250 mg for the other dosage forms.

Therapeutic Uses of the Antibodies The t invention includes s sing administering to a subject in need f a therapeutic composition comprising an anti-GITR antibody (e.g., an anti-GITR antibody comprising any of the HCVR/LCVR or CDR sequences as set forth in Table 1 herein). The eutic composition can comprise any of the anti-GITR antibodies, antigenbinding fragments f, or ADCs disclosed herein, and a pharmaceutically able carrier or t.

The antibodies of the invention are useful, inter alia, for the treatment, prevention and/or amelioration of any disease or disorder associated with or mediated by GITR expression or activity, or treatable by blocking the interaction n GITR and GITRL, and/or inhibiting or stimulating GITR activity and/or signaling. For example, the antibodies and antigen-binding fragments of the present disclosure can be used to treat immune and proliferative diseases or disorders, e.g., cancer, by modulating the immune response, though, e.g., GITR activation.

The antibodies and antigen-binding fragments of the instant disclosure can be used to treat a disease or disorder by enhancing an immune response. The instant disclosure includes s of modulating anti-tumor immune response in a subject comprising administering to the subject an anti-GITR antibody or antigen-binding fragment described herein. In certain embodiments, the antibody or antigen-binding nt reduces the suppressive activity of T or cells by T regulatory cells. In some embodiments, the antibody or antigen-binding fragment of the instant disclosure enhances intra-tumoral T or/T regulatory cell ratio conducive for therapeutic benefit. In some embodiments, the antibody or antigen-binding fragment of the instant disclosure promotes T cell survival.

Exemplary diseases or disorders that can be d by the antibodies and antigen- binding fragments include immune and proliferative diseases or ers, e.g., cancer. The antibodies and antigen-binding fragments of the present ion can be used to treat primary and/or metastatic tumors arising in the brain and meninges, oropharynx, lung and bronchial tree, gastrointestinal tract, male and female uctive tract, muscle, bone, skin and appendages, connective tissue, , immune system, blood forming cells and bone marrow, liver and y tract, and special sensory organs such as the eye. In some embodiments, the antibodies and antigen-binding fragments of the instant sure are used to treat solid or blood-borne tumors. In n embodiments, the antibodies of the instant disclosure are used to treat one or more of the following cancers: renal cell carcinoma, pancreatic carcinoma, head and neck cancer, prostate cancer, malignant gliomas, arcoma, colorectal cancer, gastric cancer (e.g., gastric cancer with MET amplification), malignant mesothelioma, le myeloma, ovarian cancer, cervical cancer, small cell lung cancer, non-small cell lung cancer, synovial sarcoma, thyroid cancer, breast cancer, melanoma, testicular, kidney, esophageal cancer, uterine , endometrial cancer, or liver .

In certain embodiments, the antibodies of the invention are useful for ng an autoimmune disease, including but not limited to, alopecia areata, autoimmune hepatitis, celiac disease, Graves’ disease, Guillain-Barre syndrome, Hashimoto’s disease, hemolytic , inflammatory bowel disease, inflammatory myopathies, multiple sclerosis, y y cirrhosis, psoriasis, rheumatoid arthritis, scleroderma, Sjögren’s syndrome, systemic lupus erthyematosus, go, autoimmune pancreatitis, autoimmune urticaria, autoimmune thrombocytopenic purpura, Crohn’s disease, diabetes type I, eosinophilic fasciitis, eosinophilic enterogastritis, Goodpasture’s syndrome, enia gravis, psoriatic tis, rheumatic fever, ulcerative colitis, vasculitis and Wegener’s granulomatosis.

In the context of the methods of treatment described herein, the anti-GITR antibody may be administered as a monotherapy (i.e., as the only therapeutic agent) or in combination with one or more additional eutic agents (examples of which are described ere herein).

Combination Therapies and Formulations Provided herein are also combination therapies ing an anti-GITR antibody of the present disclosure and any additional therapeutic agent that may be advantageously combined with an antibody of the instant disclosure or antigen-binding fragment thereof.

The present invention includes compositions and therapeutic formulations comprising any of the anti-GITR antibodies described herein in combination with one or more additional eutically active components, and methods of treatment comprising administering such combinations to subjects in need thereof.

The antibodies of the present invention may be combined istically with one or more anti-cancer drugs or therapy used to treat cancer, including, for e, renal cell carcinoma, colorectal cancer, glioblastoma multiforme, squamous cell carcinoma of head and neck, non-small-cell lung cancer, colon cancer, ovarian cancer, adenocarcinoma, prostate , glioma, and melanoma. It is contemplated herein to use anti-GITR antibodies of the invention in combination with immunostimulatory and/or supportive therapies to inhibit tumor growth, and/or enhance survival of cancer patients. The immunostimulatory therapies e direct immunostimulatory therapies to augment immune cell ty by either “releasing the brake” on suppressed immune cells or “stepping on the gas” to activate an immune response. Examples include targeting other checkpoint receptors, vaccination and adjuvants. The immunosupportive modalities may increase antigenicity of the tumor by promoting immunogenic cell death, inflammation or have other ct effects that promote an anti-tumor immune response. Examples include ion, chemotherapy, anti-angiogenic agents, and surgery.

The instant disclosure includes s of modulating anti-tumor immune se in a subject comprising administering to the subject an anti-GITR antibody in combination with one or more agonistic dies against activating receptors and one or more blocking dies against inhibitory receptors that enhance T-cell ation to promote tumor destruction.

The instant disclosure includes methods of modulating anti-tumor immune response in a subject comprising administering to the subject an anti-GITR antibody or antigen-binding fragment described herein in combination with one or more isolated antibody or antigenbinding fragment thereof that binds to a second T-cell activating receptor (i.e., other than GITR). In some embodiments, the second T-cell activating receptor is CD28, OX40, CD137, CD27, or VEM. The instant disclosure also includes formulations comprising an anti-GITR antibody or antigen binding fragment thereof provided herein and an dy or antigenbinding fragment that binds said second T-cell activating or.

In various embodiments, one or more antibodies of the present invention may be used in combination with an antibody to PD-L1, an antibody to PD-1 (e.g., nivolumab), a LAG-3 inhibitor, a CTLA-4 inhibitor (e.g., ipilimumab), a TIM3 inhibitor, a BTLA inhibitor, a TIGIT inhibitor, a CD47 inhibitor, an antagonist of another T-cell co-inhibitor or ligand (e.g., an antibody to CD-28, 2B4, LY108, LAIR1, ICOS, CD160 or VISTA), an indoleamine-2,3- dioxygenase (IDO) inhibitor, a vascular endothelial growth factor (VEGF) nist [e.g., a “VEGF-Trap” such as aflibercept or other VEGF-inhibiting fusion protein as set forth in US 7,087,411, or an anti-VEGF antibody or antigen binding fragment thereof (e.g., bevacizumab, or ranibizumab) or a small molecule kinase inhibitor of VEGF receptor (e.g., sunitinib, sorafenib, or pazopanib)], an Ang2 inhibitor (e.g., nesvacumab), a transforming growth factor beta (TGFβ) tor, an epidermal growth factor receptor (EGFR) inhibitor (e.g., erlotinib, cetuximab), an agonist to a co-stimulatory or (e.g., an agonist to glucocorticoid-induced elated protein), an dy to a tumor-specific n (e.g., CA9, CA125, melanoma-associated antigen 3 (MAGE3), carcinoembryonic antigen (CEA), vimentin, tumor-M2-PK, prostate-specific antigen (PSA), mucin-1, , and CA19-9), a vaccine (e.g., Bacillus Calmette-Guerin, a cancer vaccine), an adjuvant to increase antigen presentation (e.g., granulocyte-macrophage colony-stimulating factor), a bispecific antibody (e.g., CD3xCD20 bispecific antibody, PSMAxCD3 bispecific antibody), a xin, a chemotherapeutic agent (e.g., dacarbazine, temozolomide, cyclophosphamide, docetaxel, doxorubicin, daunorubicin, cisplatin, carboplatin, gemcitabine, rexate, ntrone, latin, paclitaxel, and vincristine), cyclophosphamide, radiotherapy, an IL-6R inhibitor (e.g., sarilumab), an IL-4R inhibitor (e.g., dupilumab), an IL-10 inhibitor, a cytokine such as IL-2, IL-7, IL-21, and IL-15, an antibody-drug ate (ADC) (e.g., anti-CD19-DM4 ADC, and anti-DS6-DM4 ADC), an anti-inflammatory drug (e.g., corticosteroids, and non-steroidal nflammatory drugs), a dietary supplement such as anti-oxidants or any palliative care to treat cancer. In certain embodiments, the anti-GITR antibodies of the present invention may be used in combination with cancer es including dendritic cell vaccines, tic viruses, tumor cell vaccines, etc. to augment the anti-tumor response. Examples of cancer vaccines that can be used in combination with anti-GITR antibodies of the present invention include MAGE3 e for melanoma and bladder cancer, MUC1 e for breast cancer, EGFRv3 (e.g., Rindopepimut) for brain cancer (including glioblastoma multiforme), or ALVAC-CEA (for CEA+ s).

In some embodiments, one or more anti-GITR antibodies described herein are administered in combination with one or more anti-PD1 antibodies, including but not limited to those described in U.S. Patent Publication No. 203579, which is orated herein by reference in its entirety. In some embodiments, the anti-GITR antibody is H1H14536P2 or H2aM14536P2. In some embodiments, the D1 antibody is REGN 2810 (also known as H4H7798N as disclosed in U.S. Patent Publication No. 2015/0203579), pembrolizumab, or nivolumab..

In certain embodiments, the anti-GITR dies of the invention may be administered in combination with radiation therapy in s to generate long-term durable anti-tumor responses and/or enhance survival of patients with cancer. In some embodiments, the anti-GITR antibodies of the invention may be administered prior to, concomitantly or after administering radiation therapy to a cancer patient. For example, radiation therapy may be administered in one or more doses to tumor lesions followed by administration of one or more doses of anti-GITR antibodies of the invention. In some embodiments, radiation therapy may be administered locally to a tumor lesion to enhance the local immunogenicity of a patient’s tumor inating radiation) and/or to kill tumor cells (ablative radiation) followed by systemic administration of an anti-GITR antibody of the invention. For example, intracranial radiation may be stered to a patient with brain cancer (e.g., glioblastoma multiforme) in combination with systemic administration of an anti- GITR dy of the invention. In certain embodiments, the anti-GITR antibodies of the invention may be stered in combination with radiation therapy and a chemotherapeutic agent (e.g., temozolomide) or a VEGF antagonist (e.g., aflibercept).

In certain embodiments, the anti-GITR antibodies of the invention may be administered in combination with one or more anti-viral drugs to treat chronic viral infection caused by LCMV, HIV, HPV, HBV or HCV. Examples of anti-viral drugs include, but are not limited to, zidovudine, lamivudine, abacavir, ribavirin, lopinavir, efavirenz, cobicistat, tenofovir, rilpivirine and corticosteroids. In some ments, the anti-GITR antibodies of the invention may be administered in ation with a LAG3 inhibitor, a CTLA-4 inhibitor or any antagonist of another T-cell co-inhibitor to treat chronic viral infection.

In certain embodiments, the anti-GITR antibodies of the invention may be combined with an antibody to a Fc receptor on immune cells for the treatment of an autoimmune disease. In one embodiment, an antibody or fragment thereof of the invention is administered in combination with an antibody or n-binding protein targeted to an antigen specific to autoimmune tissue. In n embodiments, an antibody or antigenbinding fragment f of the invention is administered in ation with an antibody or antigen-binding protein targeted to a T-cell or or a B-cell receptor, including but not limited to, Fcα (e.g., CD89), Fc gamma (e.g., CD64, CD32, CD16a, and CD16b), CD19, etc.

The antibodies of fragments thereof of the ion may be used in combination with any drug or therapy known in the art (e.g., corticosteroids and other suppressants) to treat an autoimmune disease or disorder including, but not limited to alopecia areata, autoimmune hepatitis, celiac disease, Graves’ disease, Guillain-Barre syndrome, Hashimoto’s disease, hemolytic anemia, inflammatory bowel disease, inflammatory myopathies, multiple sclerosis, primary biliary cirrhosis, psoriasis, toid arthritis, scleroderma, Sjögren’s syndrome, ic lupus erthyematosus, vitiligo, autoimmune pancreatitis, autoimmune urticaria, autoimmune ocytopenic purpura, Crohn’s disease, diabetes type I, eosinophilic fasciitis, eosinophilic enterogastritis, Goodpasture’s syndrome, myasthenia gravis, psoriatic arthritis, rheumatic fever, ulcerative colitis, itis and Wegener’s granulomatosis.

The instant disclosure also includes methods of modulating anti-tumor immune se in a t comprising administering to the subject an ITR dy or antigen-binding fragment bed herein in combination with one or more ed antibody or antigen-binding fragment thereof that binds to a T-cell inhibitory receptor. In some embodiments, the T-cell inhibitory receptor is , PD-1, TIM-3, BTLA, VISTA, or LAG-3.

The instant disclosure also includes formulations comprising an anti-GITR antibody or antigen-binding fragment thereof provided herein and an antibody or antigen-binding fragment that binds said T-cell inhibitory receptor.

The instant disclosure also includes methods of treating cancer by administering an antibody or n-binding fragment thereof or formulation described herein to a subject in ction with radiation or chemotherapy.

In some embodiments, the anti-GITR dies of the present invention are co- formulated with and/or administered in combination with one or more additional eutically active component(s) selected from the group consisting of: an EGFR antagonist (e.g., an anti-EGFR antibody [e.g., cetuximab or mumab] or small molecule inhibitor of EGFR [e.g., gefitinib or erlotinib]), an antagonist of another EGFR family member such as Her2/ErbB2, ErbB3 or ErbB4 (e.g., anti-ErbB2 [e.g., trastuzumab or T-DM1 {KADCYLA®}], anti-ErbB3 or anti-ErbB4 antibody or small molecule inhibitor of ErbB2, ErbB3 or ErbB4 activity), an antagonist of EGFRvIII (e.g., an antibody that specifically binds EGFRvIII), a cMET anagonist (e.g., an anti-cMET antibody), an IGF1R antagonist (e.g., an anti-IGF1R antibody), a B-raf inhibitor (e.g., vemurafenib, sorafenib, GDC-0879, PLX-4720), a PDGFR-α inhibitor (e.g., an anti-PDGFR-α antibody), a PDGFR-β inhibitor (e.g., an anti- PDGFR-β antibody or small molecule kinase inhibitor such as, e.g., imatinib mesylate or sunitinib malate), a PDGF ligand inhibitor (e.g., anti-PDGF-A, -B, -C, or -D antibody, r, siRNA, etc.), a VEGF antagonist (e.g., a VEGF-Trap such as aflibercept, see, e.g., US 7,087,411 (also ed to herein as a "VEGF-inhibiting fusion protein"), anti-VEGF antibody (e.g., bevacizumab), a small molecule kinase inhibitor of VEGF receptor (e.g., sunitinib, sorafenib or pazopanib)), a DLL4 antagonist (e.g., an anti-DLL4 antibody disclosed in US 2009/0142354 such as REGN421), an Ang2 antagonist (e.g., an anti-Ang2 antibody disclosed in US 027286 such as H1H685P), a FOLH1 antagonist (e.g., an anti- FOLH1 antibody), a STEAP1 or STEAP2 antagonist (e.g., an anti-STEAP1 antibody or an anti-STEAP2 antibody), a TMPRSS2 antagonist (e.g., an anti-TMPRSS2 antibody), a MSLN antagonist (e.g., an anti-MSLN antibody), a CA9 antagonist (e.g., an anti-CA9 antibody), a uroplakin antagonist (e.g., an anti-uroplakin [e.g., anti-UPK3A] antibody), a MUC16 antagonist (e.g., an UC16 antibody), a Tn antigen antagonist (e.g., an anti-Tn antibody), a CLEC12A antagonist (e.g., an anti- A antibody), a TNFRSF17 antagonist (e.g., an anti-TNFRSF17 antibody), a LGR5 antagonist (e.g., an anti-LGR5 antibody), a lent CD20 antagonist (e.g., a lent D20 antibody such as rituximab), etc. Other agents that may be beneficially administered in combination with antibodies of the invention include, e.g., tamoxifen, aromatase inhibitors, and cytokine inhibitors, including small-molecule cytokine inhibitors and antibodies that bind to cytokines such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-11, IL-12, IL-13, IL-17, IL-18, or to their respective receptors.

The present invention includes compositions and therapeutic formulations comprising any of the anti-GITR antibodies described herein in combination with one or more chemotherapeutic agents. Examples of herapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide (Cytoxan™); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, dopa, and uredopa; ethylenimines and methylamelamines including altretamine, ylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide and trimethylolomelamine; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, ichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, omycin, calicheamicin, carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin, ubicin, detorubicin, 6-diazooxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; tabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, rexate, pteropterin, trimetrexate; purine s such as fludarabine, 6- mercaptopurine, thiamiprine, thioguanine; pyrimidine s such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone nate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; bicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK™; razoxane; ran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2''- trichlorotriethylamine; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxanes, e.g. axel (Taxol™, Bristol-Myers Squibb Oncology, Princeton, N.J.) and docetaxel ere™; Aventis Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum s such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; ine; novantrone; side; daunomycin; aminopterin; ; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid; esperamicins; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included in this definition are ormonal agents that act to regulate or inhibit e action on tumors such as strogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY 117018, onapristone, and toremifene (Fareston); and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above.

The anti-GITR antibodies of the invention may also be administered and/or co- ated in combination with antivirals, antibiotics, analgesics, corticosteroids, ds, oxygen, antioxidants, COX inhibitors, cardioprotectants, metal chelators, IFN-gamma, and/or NSAIDs.

The additional therapeutically active component(s), e.g., any of the agents listed above or derivatives thereof, may be administered just prior to, concurrent with, or shortly after the administration of an anti-GITR dy of the present invention; (for purposes of the present disclosure, such administration ns are considered the administration of an anti-GITR antibody "in combination with" an additional eutically active component).

The present invention includes pharmaceutical compositions in which an anti-GITR antibody of the present invention is co-formulated with one or more of the additional therapeutically active component(s) as bed elsewhere herein.

The additional therapeutically active component(s) may be administered to a subject prior to administration of an anti-GITR antibody of the present invention. For example, a first component may be deemed to be administered "prior to" a second component if the first component is administered 1 week before, 72 hours before, 60 hours before, 48 hours before, 36 hours before, 24 hours before, 12 hours before, 6 hours before, hours before, 4 hours before, 3 hours , 2 hours , 1 hour before, 30 minutes before, 15 minutes before, 10 minutes , 5 minutes before, or less than 1 minute before administration of the second ent. In other embodiments, the additional therapeutically active component(s) may be administered to a subject after administration of an anti-GITR antibody of the present invention. For e, a first component may be deemed to be administered "after" a second component if the first component is administered 1 minute after, 5 minutes after, 10 minutes after, 15 minutes after, 30 minutes after, 1 hour after, 2 hours after, 3 hours after, 4 hours after, 5 hours after, 6 hours after, 12 hours after, 24 hours after, 36 hours after, 48 hours after, 60 hours after, 72 hours after administration of the second component. In yet other embodiments, the additional therapeutically active component(s) may be administered to a subject concurrent with administration of an anti-GITR antibody of the present invention. "Concurrent" administration, for purposes of the t invention, includes, e.g., administration of an anti- GITR antibody and an additional therapeutically active component to a subject in a single dosage form (e.g., co-formulated), or in separate dosage forms stered to the subject within about 30 minutes or less of each other. If administered in separate dosage forms, each dosage form may be administered via the same route (e.g., both the anti-GITR antibody and the additional therapeutically active component may be administered intravenously, subcutaneously, etc.); alternatively, each dosage form may be administered via a different route (e.g., the anti-GITR antibody may be stered intravenously, and the additional therapeutically active component may be stered subcutaneously). In any event, administering the components in a single dosage from, in separate dosage forms by the same route, or in separate dosage forms by different routes are all considered "concurrent administration," for purposes of the present disclosure. For purposes of the present disclosure, administration of an anti-GITR antibody "prior to", "concurrent with," or "after" (as those terms are defined herein above) stration of an onal therapeutically active component is ered administration of an anti-GITR dy "in ation with" an additional eutically active component).

The present invention includes pharmaceutical itions in which an anti-GITR antibody of the present invention is co-formulated with one or more of the additional therapeutically active component(s) as described elsewhere herein using a variety of dosage combinations.

In exemplary embodiments in which an anti-GITR antibody of the invention is stered in combination with a VEGF antagonist (e.g., a VEGF trap such as aflibercept), ing administration of co-formulations comprising an anti-GITR antibody and a VEGF antagonist, the dual components may be administered to a subject and/or coformulated using a variety of dosage combinations. For example, the anti-GITR antibody may be administered to a subject and/or contained in a co-formulation in an amount ed from the group consisting of 0.01 mg, 0.02 mg, 0.03 mg, 0.04 mg, 0.05 mg, 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1.0 mg, 1.5 mg, 2.0 mg, 2.5 mg, 3.0 mg, 3.5 mg, 4.0 mg, 4.5 mg, 5.0 mg, 6.0 mg, 7.0 mg, 8.0 mg, 9.0 mg, and 10.0 mg; and the VEGF antagonist (e.g., a VEGF trap such as aflibercept) may be stered to the t and/or contained in a co-formulation in an amount selected from the group consisting of 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1.0 mg, 1.1 mg, 1.2 mg, 1.3 mg, 1.4 mg, 1.5 mg, 1.6 mg, 1.7 mg, 1.8 mg, 1.9 mg, 2.0 mg, 2.1 mg, 2.2 mg, 2.3 mg, 2.4 mg, 2.5 mg, 2.6 mg, 2.7 mg, 2.8 mg, 2.9 mg and 3.0 mg. The combinations/coformulations may be administered to a subject according to any of the administration regimens disclosed elsewhere herein, including, e.g., twice a week, once every week, once every 2 weeks, once every 3 weeks, once every month, once every 2 months, once every 3 months, once every 4 months, once every 5 months, once every 6 months, etc.

Administration Regimens According to certain embodiments of the present invention, multiple doses of an anti-GITR antibody (or a pharmaceutical composition comprising a combination of an anti- GITR antibody and any of the additional therapeutically active agents mentioned herein) may be administered to a subject over a defined time course. The methods according to this aspect of the ion comprise sequentially administering to a subject multiple doses of an anti-GITR dy of the invention. As used herein, "sequentially administering" means that each dose of anti-GITR antibody is administered to the subject at a different point in time, e.g., on different days separated by a ermined interval (e.g., hours, days, weeks or months). The present ion includes methods which comprise sequentially administering to the t a single initial dose of an anti-GITR antibody, ed by one or more secondary doses of the anti-GITR antibody, and optionally followed by one or more tertiary doses of the anti-GITR antibody.

The terms "initial dose," "secondary doses," and "tertiary doses," refer to the temporal sequence of stration of the anti-GITR antibody of the invention. Thus, the "initial dose" is the dose which is administered at the beginning of the treatment regimen (also referred to as the "baseline dose"); the "secondary doses" are the doses which are administered after the l dose; and the "tertiary doses" are the doses which are administered after the secondary doses. The initial, secondary, and tertiary doses may all contain the same amount of anti-GITR antibody, but generally may differ from one r in terms of frequency of administration. In certain embodiments, however, the amount of anti- GITR antibody contained in the initial, secondary and/or tertiary doses varies from one another (e.g., adjusted up or down as appropriate) during the course of treatment. In certain embodiments, two or more (e.g., 2, 3, 4, or 5) doses are administered at the beginning of the treatment n as "loading doses" followed by subsequent doses that are administered on a less frequent basis (e.g., "maintenance doses").

In certain exemplary ments of the present invention, each secondary and/or tertiary dose is administered 1 to 26 (e.g., 1, 1½, 2, 2½, 3, 3½, 4, 4½, 5, 5½, 6, 6½, 7, 7½, 8, 8½, 9, 9½, 10, 10½, 11, 11½, 12, 12½, 13, 13½, 14, 14½, 15, 15½, 16, 16½, 17, 17½, 18, 18½, 19, 19½, 20, 20½, 21, 21½, 22, 22½, 23, 23½, 24, 24½, 25, 25½, 26, 26½, or more) weeks after the immediately preceding dose. The phrase "the immediately preceding dose," as used herein, means, in a sequence of le administrations, the dose of anti-GITR antibody which is administered to a patient prior to the administration of the very next dose in the sequence with no ening doses.

The methods according to this aspect of the invention may comprise administering to a patient any number of secondary and/or tertiary doses of an anti-GITR dy. For example, in certain embodiments, only a single secondary dose is stered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) secondary doses are administered to the patient. Likewise, in certain embodiments, only a single tertiary dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, , 6, 7, 8, or more) tertiary doses are administered to the patient. The administration regimen may be carried out indefinitely over the lifetime of a particular subject, or until such treatment is no longer therapeutically needed or advantageous.

In embodiments involving multiple ary doses, each ary dose may be administered at the same frequency as the other secondary doses. For example, each secondary dose may be administered to the patient 1 to 2 weeks or 1 to 2 months after the immediately preceding dose. rly, in embodiments involving multiple tertiary doses, each ry dose may be administered at the same ncy as the other tertiary doses.

For e, each tertiary dose may be administered to the patient 2 to 12 weeks after the immediately ing dose. In certain embodiments of the invention, the frequency at which the secondary and/or tertiary doses are administered to a patient can vary over the course of the treatment regimen. The frequency of administration may also be adjusted during the course of treatment by a physician depending on the needs of the individual patient following clinical examination.

The present invention includes administration ns in which 2 to 6 loading doses are administered to a patient at a first frequency (e.g., once a week, once every two weeks, once every three weeks, once a month, once every two months, etc.), followed by administration of two or more maintenance doses to the patient on a less frequent basis.

For example, according to this aspect of the invention, if the loading doses are administered at a frequency of once a month, then the maintenance doses may be administered to the patient once every six weeks, once every two , once every three months, etc.

Diagnostic Uses of the Antibodies The anti-GITR antibodies of the present invention may also be used to detect and/or measure GITR, or GITR-expressing cells in a sample, e.g., for diagnostic purposes.

For example, an anti-GITR antibody, or fragment f, may be used to diagnose a condition or disease characterized by aberrant expression (e.g., over-expression, underexpression , lack of expression, etc.) of GITR. Exemplary diagnostic assays for GITR may comprise, e.g., contacting a sample, obtained from a patient, with an anti-GITR antibody of the invention, wherein the anti-GITR antibody is d with a detectable label or reporter molecule. atively, an unlabeled anti-GITR antibody can be used in diagnostic ations in combination with a secondary antibody which is itself detectably d. The detectable label or reporter molecule can be a sotope, such as 3H, 14C, 32P, 35S, or 125I; a fluorescent or uminescent moiety such as fluorescein, or rhodamine; or an enzyme such as alkaline atase, beta-galactosidase, horseradish peroxidase, or rase. ic exemplary assays that can be used to detect or measure GITR in a sample include enzyme-linked immunosorbent assay ), radioimmunoassay (RIA), immuno-PET (e.g., 89Zr, 64Cu, etc.), and fluorescence-activated cell sorting (FACS).

Samples that can be used in GITR diagnostic assays according to the present invention include any tissue or fluid sample obtainable from a patient which contains detectable quantities of GITR protein, or fragments thereof, under normal or pathological conditions. Generally, levels of GITR in a particular sample obtained from a healthy patient (e.g., a patient not afflicted with a disease or condition associated with abnormal GITR levels or activity) will be measured to initially establish a baseline, or standard, level of GITR. This baseline level of GITR can then be ed against the levels of GITR measured in samples ed from individuals suspected of having a GITR related disease or condition.

EXAMPLES The following examples are put forth so as to provide those of ry skill in the art with a complete sure and description of how to make and use the methods and compositions of the invention, and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is average molecular , temperature is in degrees Centigrade, and pressure is at or near atmospheric.

Example 1. Generation of Anti-GITR Antibodies Anti-GITR dies were obtained by immunizing a VELOCIMMUNE® mouse (i.e., an engineered mouse comprising DNA encoding human globulin heavy and kappa light chain variable regions) with an immunogen comprising a soluble dimeric ecto domain of human GITR. The dy immune response was monitored by a GITR-specific immunoassay. Several fully human anti-GITR antibodies were isolated ly from antigenpositive B cells without fusion to myeloma cells, as described in US 2007/0280945A1.

Certain ical properties of the exemplary anti-GITR antibodies generated in accordance with the methods of this Example are described in detail in the Examples set forth below.

Example 2. Heavy and Light Chain Variable Region Amino Acid and Nucleic Acid Sequences Table 1 sets forth the amino acid sequence identifiers of the heavy and light chain variable regions and CDRs of selected anti-GITR antibodies of the invention. The ponding nucleic acid sequence identifiers are set forth in Table 2.

Table 1: Amino Acid Sequence Identifiers SEQ ID NOs: Antibody Designation HCVR HCDR1 HCDR2 HCDR3 LCVR LCDR1 LCDR2 LCDR3 74P 2 4 6 8 10 12 14 16 H1H14486P 18 20 22 24 26 28 30 32 H1H14491P 34 36 38 40 42 44 46 48 H1H14493P 50 52 54 56 58 60 62 64 H1H14495P 66 68 70 72 74 76 78 80 H1H14503P 82 84 86 88 90 92 94 96 H1H14512P 98 100 102 104 106 108 110 112 20P 114 116 118 120 122 124 126 128 H1H14523P 130 132 134 136 138 140 142 144 H1H14524P 146 148 150 152 154 156 158 160 H4H14469P 162 164 166 168 170 172 174 176 H4H14470P 178 180 182 184 186 188 190 192 H4H14475P 194 196 198 200 202 204 206 208 H4H14476P 210 212 214 216 218 220 222 224 H4H14508P 226 228 230 232 234 236 238 240 H4H14516P 242 244 246 248 250 252 254 256 H4H14521P 258 260 262 264 266 268 270 272 25P 274 276 278 280 282 284 286 288 H4H14528P 290 292 294 296 298 300 302 304 H4H14530P 306 308 310 312 314 316 318 320 H4H14531P2 322 324 326 328 402 404 406 408 H4H14532P2 330 332 334 336 402 404 406 408 H4H14536P2 338 340 342 344 402 404 406 408 H4H14539P2 346 348 350 352 402 404 406 408 H4H14541P2 354 356 358 360 402 404 406 408 H4H15736P2 362 364 366 368 402 404 406 408 H4H15740P2 370 372 374 376 402 404 406 408 H4H15744P2 378 380 382 384 402 404 406 408 H4H15745P2 386 388 390 392 402 404 406 408 H4H15753P2 394 396 398 400 402 404 406 408 Table 2: Nucleic Acid Sequence Identifiers SEQ ID NOs: Antibody Designation HCVR HCDR1 HCDR2 HCDR3 LCVR LCDR1 LCDR2 LCDR3 H1H14474P 1 3 5 7 9 11 13 15 86P 17 19 21 23 25 27 29 31 H1H14491P 33 35 37 39 41 43 45 47 H1H14493P 49 51 53 55 57 59 61 63 H1H14495P 65 67 69 71 73 75 77 79 H1H14503P 81 83 85 87 89 91 93 95 H1H14512P 97 99 101 103 105 107 109 111 H1H14520P 113 115 117 119 121 123 125 127 H1H14523P 129 131 133 135 137 139 141 143 H1H14524P 145 147 149 151 153 155 157 159 H4H14469P 161 163 165 167 169 171 173 175 H4H14470P 177 179 181 183 185 187 189 191 H4H14475P 193 195 197 199 201 203 205 207 H4H14476P 209 211 213 215 217 219 221 223 H4H14508P 225 227 229 231 233 235 237 239 H4H14516P 241 243 245 247 249 251 253 255 H4H14521P 257 259 261 263 265 267 269 271 H4H14525P 273 275 277 279 281 283 285 287 H4H14528P 289 291 293 295 297 299 301 303 H4H14530P 305 307 309 311 313 315 317 319 H4H14531P2 321 323 325 327 401 403 405 407 H4H14532P2 329 331 333 335 401 403 405 407 36P2 337 339 341 343 401 403 405 407 H4H14539P2 345 347 349 351 401 403 405 407 H4H14541P2 353 355 357 359 401 403 405 407 H4H15736P2 361 363 365 367 401 403 405 407 H4H15740P2 369 371 373 375 401 403 405 407 H4H15744P2 377 379 381 383 401 403 405 407 H4H15745P2 385 387 389 391 401 403 405 407 H4H15753P2 393 395 397 399 401 403 405 407 Antibodies are typically referred to herein according to the following nomenclature: Fc prefix (e.g. "H1H," "H4H," etc.), followed by a numerical identifier (e.g. "14493," "14495," etc.), followed by a "P" or "P2" suffix, as shown in Tables 1 and 2. Thus, according to this nomenclature, an antibody may be referred to herein as, e.g., " 86P," " H4H14531P2," etc. The H1H, and H4H prefixes on the antibody designations used herein indicate the particular Fc region isotype of the antibody. For e, an "H1H" antibody has a human IgG1 Fc, an "H4H" antibody has a human IgG4 Fc, and an H2M has a mouse IgG2 Fc (all variable regions are fully human as denoted by the first 'H' in the antibody ation). As will be appreciated by a person of ordinary skill in the art, an antibody having a ular Fc isotype can be converted to an antibody with a different Fc e, but in any event, the le domains (including the CDRs) – which are indicated by the numerical identifiers shown in Tables 1 and 2 – will remain the same, and the binding properties are expected to be cal or substantially similar regardless of the nature of the Fc domain.

Control Constructs Used in the Following Examples Control constructs were included in the following experiments for comparative purposes: Anti-GITR Control Ab I: a mouse anti-human GITR hybridoma with variable heavy and light chain domains having the amino acid sequences of the ponding domains of “clone 6C8” as set forth in WO 2006/ 105021 A2; produced with mIgG1 and mIgG2a constant regions in the following examples; and Anti-GITR Control Ab II: a human anti-GITR antibody with variable heavy and light chain domains having the amino acid sequences of the corresponding s of “36E5” as set forth in US 8709424 B2.

Example 3. Surface Plasmon Resonance Derived Binding Affinities and c nts of Human Monoclonal anti-TNFRSF18 (GITR) antibodies Binding affinities and kinetic constants of human anti-GITR antibodies were determined by surface plasmon resonance (Biacore 4000 or T-200) at 37°C (Table 3).

Antibodies, expressed as human IgG1 or IgG4 (i.e., “H1H” or “H4H” designations), were captured onto a mouse anti-human Fc CM5 Biacore sensor surface (mAb-capture format) and soluble monomeric (human (h) GITR.mmh; SEQ ID NO: 409 and Macaca fasicularis (mf) GITR.mmh; SEQ ID NO: 412) or dimeric (hGITR.hFc; SEQ ID NO: 411 and hGITRmFc; SEQ ID NO: 410). GITR proteins were injected over the sensor surface at a flow rate of 30 ute. All Biacore g studies were med in a buffer composed of 0.01M HEPES pH 7.4, 0.15M NaCl, 3mM EDTA, 0.05% v/v Surfactant P20 (HBS-ET running buffer). Antibody-reagent association was monitored for 4 minutes while dissociation in HBS-ET running buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% (v/v) Surfactant P20, pH 7.4) was monitored for 10 minutes. Kinetic ation (ka) and dissociation (kd) rate constants were determined by fitting the real-time sensorgrams to a 1:1 binding model using Scrubber 2.0c curve fitting re. g dissociation equilibrium constants (KD) and dissociative half-lives (t½) were ated from the kinetic rate nts as: KD [M] = kd / ka; and t1/2 (min) = (ln2/(60*kd). Results are summarized in Table 3.

Table 3: Biacore Binding Affinities of Human Fc mAbs at 37 ºC Binding at 37 °C/ Antibody Capture Format Antibody Analyte ka (Ms-1) Kd (s-1) KD (Molar) t½ (min) hGITR.mmh 5.32E+05 7.39E-04 1.39E-09 15.6 H1H14503P hGITR.mFc 1.12E+06 1.54E-04 1.37E-10 75.0 mfGITR.mmh 2.68E+05 5.60E-03 2.09E-08 2.1 hGITR.mmh 05 1.16E-03 09 9.9 H1H14474P hGITR.mFc 1.21E+06 1.30E-04 10 89.2 mfGITR.mmh 3.39E+05 9.51E-03 2.80E-08 1.2 hGITR.mmh 5.17E+05 1.27E-03 2.45E-09 9.1 H1H14495P hGITR.mFc 1.14E+06 1.10E-04 9.68E-11 105.0 mfGITR.mmh 2.96E+05 7.23E-03 2.45E-08 1.6 hGITR.mmh 05 03 2.79E-09 9.4 H1H14486P hGITR.mFc 9.65E+05 1.53E-04 1.59E-10 75.5 mfGITR.mmh 05 1.66E-02 1.22E-07 0.7 hGITR.mmh 4.05E+05 1.47E-03 3.62E-09 7.9 H1H14524P hGITR.mFc 9.22E+05 1.35E-04 1.46E-10 85.6 mfGITR.mmh 1.20E+05 1.89E-02 1.58E-07 0.6 hGITR.mmh 05 1.38E-03 5.06E-09 8.4 H4H14530P hGITR.mFc 2.93E+05 1.85E-04 6.30E-10 62.6 mfGITR.mmh 2.40E+05 6.11E-04 09 18.9 hGITR.mmh 3.19E+05 1.62E-03 5.06E-09 7.2 H1H14491P hGITR.mFc 8.42E+05 1.69E-04 2.01E-10 68.3 mfGITR.mmh 1.18E+05 8.89E-03 7.53E-08 1.3 hGITR.mmh 2.16E+05 1.86E-03 09 6.2 H1H14523P hGITR.mFc 5.55E+05 1.20E-04 2.16E-10 96.3 mfGITR.mmh 1.23E+05 1.09E-02 8.85E-08 1.1 Binding at 37 °C/ Antibody Capture Format Antibody Analyte ka (Ms-1 ) Kd (s -1) K D (Molar) t½ (min) hGITR.mmh 1.86E+05 2.50E-03 1.34E-08 4.6 93P hGITR.mFc 5.24E+05 1.91E-04 3.65E-10 60.4 mfGITR.mmh 1.00E+04 1.40E-02 1.40E-06 0.8 hGITR.mmh 3.48E+05 6.45E-03 1.85E-08 1.8 H4H14532P2 hGITR.mFc 7.20E+05 2.69E-04 3.73E-10 42.9 mfGITR.mmh 2.60E+05 6.48E-03 2.49E-08 1.8 hGITR.mmh 3.45E+05 7.84E-03 2.27E-08 1.5 21P hGITR.mFc 1.23E+06 4.79E-04 3.89E-10 24.1 mfGITR.mmh 1.66E+05 3.34E-03 2.01E-08 3.5 hGITR.mmh 4.24E+05 9.76E-03 2.30E-08 1.2 H4H14536P2 hGITR.mFc 1.26E+06 2.19E-04 1.73E-10 52.7 mfGITR.mmh 1.04E+05 1.47E-02 1.42E-07 0.8 hGITR.mmh 3.92E+05 1.23E-02 3.14E-08 0.9 H4H14476P hGITR.mFc 9.06E+05 2.69E-04 2.97E-10 42.9 mfGITR.mmh 1.91E+05 1.07E-02 5.58E-08 1.1 hGITR.mmh 2.25E+05 7.38E-03 3.27E-08 1.6 H4H14516P hGITR.mFc 1.68E+06 1.81E-03 1.08E-09 6.4 mfGITR.mmh 1.90E+05 1.12E-02 5.87E-08 1.0 hGITR.mmh 2.55E+05 9.35E-03 3.66E-08 1.2 H4H14508P hGITR.mFc 06 7.19E-04 5.97E-10 16.1 mfGITR.mmh 1.29E+05 03 08 2.3 mmh 2.84E+05 1.22E-02 4.30E-08 0.9 H4H14469P hGITR.mFc 1.20E+06 4.01E-04 3.35E-10 28.8 .mmh 5.91E+04 2.43E-03 4.11E-08 4.7 hGITR.mmh 3.07E+05 1.40E-02 4.57E-08 0.8 H4H14475P hGITR.mFc 1.60E+06 1.35E-03 8.47E-10 8.5 mfGITR.mmh 1.83E+05 03 4.17E-08 1.5 mmh 1.02E+05 5.23E-03 5.13E-08 2.2 H4H14528P hGITR.mFc 1.58E+06 1.77E-03 09 6.5 mfGITR.mmh NB NB NB NB hGITR.mmh 05 1.66E-02 5.24E-08 0.7 H4H14525P hGITR.mFc 7.91E+05 3.38E-04 4.27E-10 34.2 mfGITR.mmh 05 02 1.55E-07 0.6 hGITR.mmh 2.66E+05 1.67E-02 6.30E-08 0.7 H1H14520P hGITR.mFc 06 5.83E-04 5.37E-10 19.8 mfGITR.mmh 1.99E+05 1.71E-02 8.59E-08 0.7 hGITR.mmh 2.21E+05 1.43E-02 6.47E-08 0.8 H4H14470P hGITR.mFc 9.04E+05 1.04E-03 1.15E-09 11.1 mfGITR.mmh NB NB NB NB hGITR.mmh 2.14E+05 1.77E-02 8.25E-08 0.7 H4H14539P2 hGITR.mFc 8.53E+05 4.72E-04 5.54E-10 24.5 mfGITR.mmh 7.23E+04 1.65E-03 2.28E-08 7.0 Anti-GITR mmh 2.16E+05 02 1.22E-07 0.4 Control Ab I- hFc 3.82E+05 03 2.04E-08 1.5 mIgG1 mfGITR.mmh 2.18E+05 4.64E-02 2.13E-07 0.2 Anti-GITR hGITR.mmh 1.94E+05 9.67E-04 4.99E-09 11.9 Binding at 37 °C/ Antibody e Format Antibody Analyte ka (Ms-1 ) Kd (s -1) K D (Molar) t½ (min) Control Ab II- hGITR.mFc 1.83E+06 1.73E-03 9.48E-10 6.7 hIgG1 mfGITR.mmh 2.31E+05 03 3.74E-08 1.3 NB= No binding observed under conditions used As shown in Table 3, all the anti-GITR antibodies of this invention bound to human GITR, with several antibodies ying sub-nanomolar affinities to dimeric human GITR n. Additionally, a ty of the anti-GITR antibodies also displayed cross reactivity to cynomolgus GITR protein. Cross reactivity to rodent GITR proteins was not observed (data not shown).

Example 4. Anti-GITR antibodies bind specifically and potently to human GITR expressing cells In this example, the y of anti-GITR antibodies to bind specifically to a human xpressing cell line was determined using electrochemiluminescence (ECL) based detection.

Briefly, human embryonic kidney (HEK)D9 cells were stably transfected with human GITR (amino acids M1-V241, NCBI Accession #NP_004186.1, SEQ ID: 413) via Lipofectamine 2000-mediated methodology. Transfectants were ed for at least two weeks in te growth media + G418.

For cell binding studies, approximately 1x105 hGITR/HEK293-D9 or parental HEK293-D9 cells, which do not express human GITR, were seeded onto 96-well carbon electrode plates (MULTI-ARRAY, MSD) for 1 h at 37°C. Nonspecific binding sites were blocked with 2% BSA (w/v) + PBS for 1 h at room temperature (RT). Next, serial dilutions of anti-GITR antibodies, ranging from 1.7 pM to 100nM, were added to cells for 1 h at RT.

Plates were then washed to remove unbound antibodies (AquaMax2000 plate washer, MSD Analytical Technologies) and plate-bound antibodies were detected with a SULFO-TAG™ ated anti-human kappa light chain IgG antibody (Jackson Immunoresearch) for 1 h at Following washes, luminescent signals were recorded with a SECTOR Imager 6000 (MSD) instrument. Direct g signals (relative light units, RLU) were analyzed as a function of the antibody concentration and data were fitted with a sigmoidal (four-parameter logistic) dose-response model using GraphPad Prism™ software. The EC50 for binding hGITR/HEK293-D9 cells, defined as the concentration of dy at which 50% of the maximal g signal is detected, was determined to indicate g potency of each antibody. The signal detected with 100nM antibody binding to the hGITR expressing cells versus parental cells was recorded as an indication of intensity and specificity of GITR binding. Results are summarized in Table 4.

As summarized in Table 4, most of the anti-GITR antibodies of this invention bound specifically to human GITR expressing cells versus parental HEK293 with EC50s ranging from 210 pM to 85 nM. A majority of the antibodies bound to human GITR-expressing cells with sub-nanomolar EC50 values. The e control antibody did not display binding to expressing or parental cell lines.

Table 4: Anti-GITR antibody binding EC50 and binding intensity at 100 nM on human GITR expressing cells Binding to Binding to g to hGITR/HEK293- hGITR/HEK293- HEK293-D9 D9 cells D9 cells Cells (at 100nM) (at 100nM) Average Signal Average Signal dy EC50 (M) (RLU) (RLU) H1H14474P 2.80E-10 6230 680 H1H14486P 4.30E-10 5830 280 H1H14491P 4.00E-10 6840 300 H1H14493P 3.00E-10 7220 790 H1H14495P 4.30E-10 6470 340 03P 2.10E-10 5880 330 H1H14512P 2.50E-10 4620 180 H1H14520P 2.40E-10 6450 1130 H1H14523P 4.00E-10 6350 530 H1H14524P 2.10E-10 5740 500 H4H14469P 10 5230 260 H4H14470P 1.40E-09 8390 1580 H4H14475P 8.00E-09 7500 1580 H4H14476P 5.70E-10 8120 1770 H4H14508P 4.50E-10 6870 580 H4H14516P 7.00E-10 10330 2560 H4H14521P 4.30E-10 10080 600 H4H14525P 6.00E-10 8840 1490 H4H14528P 4.50E-10 7310 420 H4H14530P 8.50E-08 4200 520 H4H14532P2 4.30E-10 5740 300 H4H14536P2 3.60E-10 8960 330 H4H14539P2 3.00E-10 4910 300 Anti-GITR Control Ab II- 2.60E-10 13750 10240 hIgG1 Isotype Control Ab- NB 750 650 hIgG4 In summary, this example demonstrates that the ITR antibodies of this invention display specific and potent binding to human GITR-expressing cell lines.

Example 5. Anti-GITR dies are l blockers and partial activators in NF- κB/Luciferase reporter assay in the ce or absence of Fc gamma R antibody anchoring In this example, the ability of ITR antibodies to activate hGITR or block hGITR ligand (hGITRL)-mediated receptor stimulation in the presence or absence of antibody anchoring to Fc gamma receptors (Fc gamma Rs) was assessed via luciferasebased reporter assays.

Briefly, a Jurkat cell line with stable incorporation of hGITR and NF- κB-dependent luciferase reporter was ered (hGITR/Jurkat/NF- κBLuc). The NF- κB Luciferase reporter was introduced into Jurkat Cells using the Cignal Lenti Reporter system (SABiosciences). Lentiviruses expressing hGITR were generated in HEK293/T17 ing the Lenti-X Lentiviral Expression System (Clontech). Jurkat/NF-κB-Luc cells were transduced with the hGITR-expressing lentivirus via polybrene-mediated uction and selected in 500 ug/ml G418 for 2 weeks. For antibody anchoring studies, HEK293 cells were transduced with the Fc gamma RI-expressing lentivirus, as described above.

First, the activation and blocking properties of anti-hGITR antibodies in the absence of Fc gamma R anchoring (non-anchored bioassay format) was assessed. Approximately 4x104 Jurkat/NF-κBLuc/hGITR cells were seeded overnight (ON) in PDL coated 96 well plates in OptiMEM + 0.5% FBS.

To determine antibody activation ability, cells were ted for 6 h at 37°C with ly diluted anti hGITR dies or hGITRL with concentrations ranging from 0.5 pM to 100 nM. To assess antibody blocking of hGITRL ed receptor stimulation, cells were pre-incubated for 30 min with ly diluted anti hGITR antibodies (0.5 pM to 100 nM) followed by a constant dose of 10nM hGITRL for 6 h.

Next, the activation and ng properties of selected anti-hGITR dies in the presence of Fc gamma R anchoring (anchored bioassay format) was determined. Similar to the above, 4Jurkat/NfκBLuc/hGITR cells were seeded in PDL coated 96 well plates in complete growth media.

To assess antibody activation, cells were pre-incubated for 1 h at 37°C with serially diluted anti-hGITR mAbs or hGITRL (0.5 pM to 100 nM). Then, 1x104 hFc gamma R1/HEK293 cells were immediately added to the wells followed by a 6 h tion. To assess blocking, hGITR/Jurkat/NfκBLuc cells were pre-incubated for 1h with serially diluted anti-hGITR antibodies (0.5 pM to 100 nM). 1x104 hFcγR1/HEK293 cells were added to the wells followed by the addition of a constant dose of 10 nM hGITRL.

For both anchored and non-anchored bioassay formats, Luciferase activity was measured with One glow reagent (Promega) and relative light units (RLUs) were measured on a Victor luminometer (Perkin Elmer). The EC50/IC50 values were determined from a four- parameter logistic on over a 12-point response curve using GraphPad Prism. Results are ized in Table 5 and Table 6. To determine % blocking, background RLU (relative light units) from untreated wells are subtracted from treated wells, and the percent blocking is calculated according to the following formula: [100 - (antibody RLU at max dose/constant ligand dose 100]. % activation is calculated according the following formula: (normalized mAb RLU/max GITR ligand response)*100; normalized mAb RLU is determined by subtracting the RLU from untreated wells from treated wells. Mean fold activation is calculated as: RLU at maximum Antibody dose/ background RLU from untreated wells.

Table 5: Blocking and activation ties of anti-GITR antibodies in the absence of Fc gamma R anchoring IC50 % EC50 % Antibody (nM) Blocking- (nM) tion H4H14475P ND -2 1.0 70 H1H14491P 0.60 90 2.0 60 H4H14521P 3.80 60 0.4 50 H1H14503P 0.60 90 1.4 50 H4H14469P 0.70 90 2.3 50 H4H14516P 2.30 70 0.8 45 H1H14523P 0.90 70 2.5 40 H1H14524P 0.70 80 2.2 40 28P 3.40 90 1.1 30 H1H14495P 0.70 80 1.3 30 74P 0.80 80 1.2 30 H4H14508P 0.90 40 1.2 30 H4H14532P2 1.00 90 1.2 30 H1H14486P 1.10 50 1.2 30 93P 0.60 90 1.2 25 H1H14512P 0.70 100 1.2 20 H4H14525P 1.40 70 1.1 20 H4H14539P2 1.20 30 1.3 20 H4H14536P2 2.00 90 1.1 20 H4H14470P 0.90 80 1.2 20 H4H14476P 2.60 90 1.0 10 H1H14520P 0.90 80 1.1 10 Anti-GITR Control Ab 0.10 54 1.02 25 I-mIgG1 Isotype Control- IgG1 NB NB NA NA Isotype Control- IgG4 NB NB NA NA NB= no blocking NA = no activation As summarized in the Table 5 above and Table 6 below, the antibodies tested displayed partial activating and partial-blocking properties in both the chored and ed bioassay formats. In the non-anchored format, dies mediated receptor stimulation with EC50s ranging from 0.4 nM to 2.5 nM. Several antibodies, such as 75P and H4H14491P were potent activators of the GITR receptor displaying 70 and 60 percent activation respectively. A majority of the antibodies tested also displayed blocking of hGITRL mediated receptor stimulation, with IC50s g from 0.6 nM to 3.8 nM.

Several exemplary antibodies, such as H1H14512P and H4H14536P2 displayed potent blocking activity of 100% and 90% respectively. H4H14475P, the most potent activator, displayed the least activity in the blocking assay (percent blocking: -2%).

Table 6: Blocking and activation ties of anti-GITR dies in the presence of Fc gamma R anchoring IC50 % EC50 tion Antibody (nM) Blocking (nM) over basal signal H1H14512P 0.10 64 0.02 7.0 H4H14475P 0.20 43 0.04 8.0 H4H14536P2 0.20 73 0.01 5.0 Anti-GITR Control Ab I- 0.20 60 0.10 6.0 mIgG1 Anti-GITR Control Ab I- 0.01 74 0.20 5.0 mIgG2a Anti-GITR Control Ab II- 0.20 70 0.01 8.0 hIgG1 Selected antibodies tested in the Fc gamma R-anchoring bioassay format also displayed a range of activation and blocking properties. H4H14475P, the strongest activator in the non-anchored format also ly activated hGITR in the anchored bioassay with a fold activation of 8.0 above the basal . Strong blockers in the non-anchored blocking format, such as H1H14512P and H4H14536P2, also displayed potent blocking in the anchored assay (% ng: 60% and 70 %).

In summary, the results demonstrate that the anti-GITR antibodies of this invention display potent GITR ting properties as well as the ability to block GITRL mediated receptor stimulation in the absence of Fc gamma R anchoring in an engineered bioassay.

Exemplary antibodies, such as H4H14775P and H4H1536P2 also maintain their activating and blocking ties, respectively, in the presence of Fc gamma R anchoring.

Example 6. Anti-GITR antibody H4H14536P2 demonstrates potent activity in a Naïve Human CD4+ T-cell proliferation assay in the presence and absence of Fc gamma R anchoring As described above, anti-GITR antibodies were tested in an engineered ay for their ability to activate hGITR in the presence or absence of anchoring Fc gamma receptors (Fc gamma R). In this example, the effect of antibody anchoring on hGITR activation was ed in a naïve human CD4+ T-cell proliferation primary bioassay. The human CD4+ T-cell system has the advantage that GITR copy number is at endogenous levels, whereas the engineered system utilizes cells with a higher GITR copy number.

First, anti-GITR antibodies were tested for CD4+ T-cell proliferative ability in the presence of plate-bound anti-CD3. y, Human CD4+ T cells were isolated from y donor leukopacks using Human CD4+ T cell Enrichment Cocktail (Stemcell Technologies).

Naïve T cells were further enriched by ion of CD45RO+ cells by MACS (Miltenyi Biotech). Approximately 5x104 T cells were plated onto 96-well U-bottomed polystyrene plates pre-coated with a suboptimal amount of the D3 mAb OKT3 (30 ng/mL) and ed s of anti-GITR antibodies or controls. Three days after stimulation, tritiated thymidine (1 μCi per well, Perkin Elmer Health es NET027001) was added to each microwell and pulsed for 18 hours. Cells were harvested onto filter plates (Unifilter-96 GF/C 6005174) using a Filtermate Harvester (Perkin Elmer Health Sciences D961962).

Scintillation fluid (Perkin Elmer Health Sciences Microscint20 6013621) was added to filter plates and radioactive counts were measured using a plate reader n Elmer Health Sciences Topcount NXT). T-cell proliferation relative to control, given as the mean fold activation at 10.6 nM of antibody concentration, is presented in Table 7. In this assay format, .6 nM represented the point at which T-cell proliferation reached a plateau on the dose response curve.

Table 7: T-cell proliferative activity (Fold activation) of plate-bound anti-GITR antibodies at 10.6 nM in the presence of plate-bound anti-CD3 Ab Donor Mean Fold Antibody 1 2 3 4 5 6 7 8 Activation H4H14536P2 3 24 24 25 24 47 26 16 23 08P 10 4 4 7 4 11 3 2 5 H4H14525P 6 1 1 2 1 3 2 1 2 H4H14469P 3 12 12 15 12 17 11 11 12 H4H14532P2 2 6 6 12 6 19 7 2 8 H4H14470P 0 3 3 4 3 6 4 1 3 H4H14475P 0 2 2 2 2 12 1 0 3 H4H14528P 2 8 8 4 8 31 5 4 8 Donor Mean Fold Antibody 1 2 3 4 5 6 7 8 Activation H4H14539P2 2 12 12 13 12 39 11 6 13 H4H14516P 1 4 4 12 4 14 4 1 5 H4H14521P 1 4 4 5 4 12 3 2 4 Anti-GITR Control Ab 1- 6 23 23 40 23 66 25 11 27 mIgG1 Isotype Control 1 1 1 1 1 1 1 1 As the results in Table 7 show, the anti-GITR antibodies tested demonstrated T- cell proliferative ability when plate-bound in the presence of plate-bound anti-CD3. The Anti- GITR l Ab I demonstrated proliferative activity 27-fold above the isotype control. The majority of the anti-GITR antibodies of this invention displayed activation 2-8 fold above the isotype control, with several exemplary dies, H4H14469P, H4H14539P2, and H4H14536P2 demonstrating activation 12, 13 and 23 fold above the control, respectively. In summary, the results demonstrate that the ITR dies tested demonstrate T-cell proliferative activity in this classical format.

Next, additional assay formats were ed to test the ability of anti-GITR antibodies to activate s in the presence or absence of urface bound Fc gamma R.

To assess anti-GITR antibody ability to activate T cells in the ce of Fc gamma R1 anchoring, HEK293 cells were engineered to s the high affinity hFc gamma R1 receptor, as described above. HEK293/ Fc gamma RI cells were treated with 50 ug/mL Mitomycin C for 30 min at 37°C to inhibit proliferation. After subsequent washes to remove traces of Mitomycin C, cells were coated with 300 ng/mL D3 antibody OKT3 to stimulate T cell activation. HEK293/Fc gamma RI cells were co-cultured with human naïve CD4+ T cells in a 1:2 ratio and titrated amounts of anti-GITR antibodies or controls were added to the co-culture medium.

T cell proliferation was assessed by measurement of the levels of tritiated thymidine incorporation. 72 h after stimulation, tritiated thymidine (0.5μCi per well, Perkin Elmer Health Sciences) was added to each microwell for an additional 18 h at 37°C. Cells were harvested onto filter plates (Unifilter-96 GF/C 6005174) using a Filtermate Harvester (Perkin Elmer Health es D961962). Scintillation fluid (Perkin Elmer Health es Microscint20 6013621) was added to filter plates and radioactive counts were measured using a plate reader (Perkin Elmer Health Sciences Topcount NXT). T-cell proliferation relative to control, given as the mean fold activation at a 33 nM concentration of antibody is presented in Table 8. In this assay , 33 nM represented the point at which T-cell proliferation reached a plateau on the dose response curve.

Table 8: T-cell proliferative activity (Fold activation) of anti-GITR dies at 33nM in the presence of Fc gamma R anchoring Donor Mean Antibody Fold 1 2 3 4 5 Activation H4H14536P2 1.3 6.3 1.5 2.7 15.7 5.5 H4H14508P 1.0 0.7 1.1 1.8 1.3 1.2 H4H14525P 1.2 0.8 1.0 1.5 1.5 1.2 H4H14469P 0.70 1.0 0.9 1.5 1.5 1.1 H4H14532P2 0.80 0.9 1.0 1.7 2.1 1.3 H4H14470P 1.8 1.0 1.4 1.5 2.0 1.5 H4H14475P 1.0 1.3 1.3 1.6 1.5 1.3 H4H14528P 0.9 1.5 1.1 1.9 1.7 1.4 H4H14539P2 0.9 0.7 1.1 1.5 1.5 1.1 H4H14516P 1.2 0.9 1.2 1.3 1.1 1.1 H4H14521P 1.5 1.1 1.1 1.7 0.9 1.2 76P 0.6 0.20 0.8 0.8 0.1 0.5 Anti-GITR Control mAbs Control I-mIgG1 2.8 2.8 1.4 1.1 1.7 2.0 Control I-mIgG2a 1.1 0.1 1.0 1.0 1.2 1.3 Control II-hIgG1 0.1 0.6 1.3 0.2 1.2 0.7 Isotype Control-hIgG4 1.0 1.0 1.0 1.0 1.0 1.0 Table 9: T-cell proliferative activity (EC50) of ITR antibodies at 33nM in the presence of Fc gamma R anchoring Donor Mean Antibody 1 2 3 4 5 EC50 (nM) H4H14536P2 1.2 0.5 1.3 11.2 1.6 3.2 Control I-mIgG1 37.2 NA 42.9 3.5 52.7 34.1 As the results in Table 8 summarize, several antibodies showed activation above ls with levels ranging from 1-2 fold. However, one exemplary antibody, H4H14536P2, demonstrated potent T-cell proliferation activity in the anchored g. With a mean fold activation of 5.5, H4H14536P2 stimulated r T-cell activation compared to the anti- GITR comparator antibodies (mean fold activation range: 0.7 -2.0). H4H14536P2 had a mean EC50 of T-cell proliferation of 3.2 nM compared with 34.1 nM for the most potent anti- GITR control Ab, Control I-mIgG (Table 9). Furthermore, in this assay format, 75P, a potent activator in the engineered ay bed above, demonstrated modest proliferative activity in this primary bioassay setting.

Next, antibodies were tested for T-cell proliferation activity in the absence of Fc gamma R anchoring. Human CD4+ T cells were isolated as described above, and plated onto 96-well U-bottomed polystyrene plates pre-coated with 30 ng/mL of the anti-CD3 antibody, OKT3. Similar to above, titrated concentrations of anti-GITR antibodies or controls were added to the culture medium. T cell proliferation was measured by tritiated thymidine incorporation. T-cell proliferation observed in four donors at 22 nM dy tration is presented as the fold activation compared to isotype control in Table 10. The EC50 (nM) of H4H14536P2 is shown in Table 11.

As observed in the anchored assay , H4H14536P2 again yed potent T cell proliferative activity at 22 nM in the non-anchored format. H4H14536P2 activated T cells with a mean fold activation of 11.0 and an EC50 of 8.3 nM. In this assay format, control anti- GITR antibody I exhibited no T-cell proliferation capability.

Table 10: T-cell proliferative activity (Fold activation) of anti-GITR antibodies at 22 nM in the absence of Fc gamma R anchoring Donor Mean dy 1 2 3 4 Activation H4H14536P2 6.2 4.2 10.1 23.4 11.0 H4H14508P 0.7 0.9 0.9 1.4 1.0 H4H14525P 0.7 1.0 0.9 1.0 0.9 H4H14469P 0.9 0.8 1.0 0.9 0.9 H4H14532P2 0.7 0.8 1.0 1.1 0.9 H4H14470P 0.7 0.9 1.0 1.7 1.1 75P 0.7 1.1 0.8 0.8 0.9 H4H14528P 0.6 0.8 0.9 1.0 0.8 H4H14539P2 0.5 0.8 1.1 0.8 0.8 H4H14516P 0.7 1.2 1.0 0.9 0.9 H4H14521P 0.5 1.2 0.8 1.0 0.9 H4H14476P 0.7 0.9 0.9 0.8 0.8 Anti-GITR Control Ab Control I- mIgG1 0.7 0.8 1.0 1.0 0.9 Isotype Control 1.0 1.0 1.0 1.0 1.0 Table 11: EC50 (nM) of H4H14536P2 Donor: 1 2 3 4 Average H4H14536P2 EC50 (nM): 12.2 6.4 9.0 5.7 8.3 In summary, this example demonstrates that one exemplary anti-GITR antibody, H4H14536P2, displays potent T-cell proliferative activity in the presence and e of hFc gamma R1 anchoring, while the anti-GITR comparative antibody Control I displayed no T-cell proliferative activity in the non-anchored setting. Thus, the ability of H4H14536P2 to activate T cells in the absence of hFc gamma R1 anchoring is a unique property, implying that the antibody may not have to compete with endogenous IgG binding to Fc gamma receptors in vivo to retain activity. This unique property of 36P2 may confer an age in a therapeutic setting.

Example 7. Administration of anti-GITR antibodies in combination with anti-PD1 antibodies istically controls and eradicates tumors As assessment of the effect of administering anti-GITR antibodies in combination with anti-PD1 antibodies on tumor growth was performed using the following methods. The results of the assessment are summarized below.

Tumor implantation, treatment n and growth measurement MC38 colorectal cancer cells (obtained from ATCC) were implanted subcutaneously in C57BL/6 mice (3x105 cells/mouse) (defined as day 0). On day 6 (i.e., 6 days post tumor implantation), mice were segregated into 4 groups (5 mice per group) and each group was treated intra-peritoneally (IP) with: (1) rat IgG2a (2A3, Bio X cell, Cat.# BE0089) (isotype control) + rat IgG2b (LTF2, Bio X cell, Cat.# ) (isotype control) (2) anti-GITR onal antibody DTA1 (rat anti-mouse GITR, Bio X cell, Cat.# ) + rat IgG2a (control) (3) anti-PD-1 monoclonal antibody RPM1-14 (rat anti-mouse PD-1, Bio X cell, Cat.# BE0146) + rat IgG2b (control) or (4) anti-GITR antibody DTA1+ anti-PD-1 antibody RPM1-14. Antibody injection(s) were then administrated again on day 13. dy treatments were dosed at 5 mg/kg of each antibody. Tumors were measured two ionally (length x width) and tumor volume was calculated (length x width2 x 0.5). Mice were euthanized when the tumor reached a designated tumor end-point (tumor volume > 3 or tumor ulceration).

Tumor re-challenge assessment Mice treated with the combination of anti-PD-1 antibody and anti-GITR antibody that remained tumor free for over 80 days were re-challenged with 3 x 105 of the eic tumor (MC38) in the right flank and 2.5 x 105 of an allogeneic (B16F10.9) tumor cell line (melanoma cell line, ATCC) in the left flank. Tumors were monitored as described above.

Antibody depletion experiments Mice injected with different ing mAbs (anti-CD4, anti-CD8, anti-CD25,) starting at one day prior of tumor challenge and given at twice weekly for total eight doses, were treated with the combination therapy or the isotype control IgG. The depletion efficiency was confirmed by FACS analysis of peripheral blood s.

Flow cytometry (FACS) analysis of intratumoral lymphocytes Mice were treated as described above. Five days after antibody treatment, tumor and spleen were collected. Tumors were minced with scissors and dissociated to single cell suspension with se TL/DNAse I mix. Spleens were dissociated with MACS Octo Dissociator. Cells were stained with panels of FACS antibodies against mouse CD45, CD3, CD4, CD8, CD25 and FoxP3, as well as activation markers (PD1, GITR, Ki67, CD160, CTLA4, ICOS, TIM3, LAG3, KLRG1 and CD44). Cells were acquired on BD Fortessa X20 or LSR II and analyzed by FlowJo software.

Administration of anti-mouse GITR antibodies in ation with anti-mouse PD1 antibodies significantly induces tumor regression and provides long-term tumor remission in MC38 bearing mice Using the methods described above, the efficacy of administering an anti-mouse GITR antibody (clone DTA-1, Bio X cell, Cat.# BE0063) in combination with an ouse PD-1 antibody (clone RMP1-14, Bio X cell, Cat.# BE0146) in the control of subcutaneous MC38 tumors was assessed. As shown in Figure 1 and Tables 12 and 13, ation treatment of PD1 blockade and anti-GITR (DTA-1) antibody icantly induced tumor regression in MC38 tumor g mice, in comparison to anti-PD-1 or anti-GITR mAb alone or isotype control treated mice. Furthermore, mice d with combination therapy showed long-term tumor remission, as 100% of the mice remained tumor free for over 120 days (Figure 2, Tables 14, 15).

Table 12. Average tumor volumes for each treatment group (mm3 ± SEM) and tumor free mice following anti-GITR and/or anti-PD-1 Ab treatment Tumor Volume (mm3) Tumor Free mice Mean (SEM) Treatment Group Day 10 Day 13 Day 17 Day 19 Day 21 Day 21 Isotype (Rat IgG2a + Rat 196 (44) 232 (46) 802 (869) NA NA 0/5 IgG2b) Anti-PD1 + Rat IgG2b 181 (37) 259 (103) 551 (199) 880 (335) 1550 (616) 0/5 Anti-GITR + Rat IgG2a 172 (9) 262 (72) 407 (112) 741(269) 882 (307) 0/5 ITR + Anti-PD1 130 (29) 41 (13) 0 (0) 0 (0) 0 (0) 5/5 Table 13. Summary of tumor free mice of three independent experiments following anti-GITR and/or anti-PD1 Ab treatment Tumor Free Treatment Group Day 21 Isotype (Rat IgG2a + Rat IgG2b) 0/15 Anti-PD1 + Rat IgG2b 0/15 Anti-GITR + Rat IgG2a 1/15 Anti-GITR + D1 10/15 Table 14. Survival Proportions (percentage) Anti-PD-1 + Days Isotype Anti-PD-1 Anti-GITR Anti-GITR 0 100 100 100 100 17 80 21 40 24 0 60 26 20 20 52 0 0 123 100 Administration of anti-mouse GITR antibodies in combination with anti-mouse PD1 antibodies induces tumor/antigen-specific immunologic memory response To determine whether mice treated with the combined stration of anti-PD-1 and anti-GITR dies ped a tumor/antigen-specific memory response, survival tumor-free mice were re-challenged with 3 x 105 of syngeneic MC38 colon carcinoma cells in the right flank and 2.5 x 105 of allogeneic melanoma cell line B16F10.9 in the left flank. It was found that MC38 tumors did not grow in mice treated with the D1 antibody and ITR antibody combination, while the same tumors grew in naive control mice ut any previous treatment) (Figure 3). In contrast, the allogeneic tumor (melanoma) did not grow in both groups, demonstrating that the combined administration of anti-PD-1 and anti GITR antibodies induced tumor-antigen specific immunologic memory response capable of controlling the second challenge with the same type of tumor.

Immune population study Mice were treated with CD4, CD8 and CD25 depleting mAbs prior to anti-PD-1 antibody and anti-GITR antibody combination treatment. It was found that depletion of CD8+ cells fully abrogated the anti-tumoral effect (MC38 tumors), while depletion of CD4 or CD25 T cells showed partial inhibition e 4, Table 15). Thus, the umor effect of the combination y in MC38 tumors appears inantly dependent on CD8+ T cells.

The effect of anti-GITR and anti-PD1 combination treatment on tumor infiltrating lymphocytes (TILs) was assessed. It was found that the combination treatment induced a significant increase in the CD8/Treg ratio in comparison to mono-therapy treatment (anti-PD- 1 or anti-GITR) or isotype control (Figure 5). The effect of the ation treatment on CD4/Treg ratio was found to be less pronounced. Anti-PD-1 and anti-GITR combination treatment sed the tage of intra-tumoral Tregs while it increased the CD8 T cells (Figure 6). Further, anti-PD-1 treatment alone induced expansion of the Treg cell number, while the anti-PD-1/anti-GITR combination treatment significantly reduced it in comparison to the isotype control treated mice.

Table 15. Anti-tumor efficacy after CD4, CD8, or CD25 depletion Tumor size (mm3) Mean (SEM) Depletion Immunotherapy Day 8 Day 12 Day 16 Antibody e control 55 (12) 161 (60) 555 (224) Anti-CD4 48 (17) 60 (22) 135 (71) Isotype control Anti-CD8 49 (17) 176 (431) 825 (431) Anti-CD25 59 (16) 61 (21) 182 (68) Isotype control 43 (19) 26 (16) 11 (7) Anti-GITR + Anti- Anti-CD4 68 (21) 50 (32) 123 (122) PD1 Anti-CD8 67 (23) 222 (86) 1041 (543) Anti-CD25 14 (6) 35 (30) 80 (64) Administration of uman GITR antibodies in combination with anti-mouse PD1 antibodies significantly induces tumor regression and provides long-term tumor remission in MC38 bearing ITRL humanized mice The efficacy of administering an uman GITR antibody (H2aM14536P2) in combination with an anti-mouse PD-1 antibody (clone RMP1-14 Bio X cell, Cat.# BE0146) in the control of subcutaneous MC38 tumors was assessed in GITR/GITRL zed mice. It was found that anti-mouse PD-1 blockade synergized with the anti-human GITR antibody and significantly d tumor regression (4/6 mice) in MC38 tumor bearing mice, in comparison to anti-PD1 (1/7) or ITR (1/7) mAb alone or isotype control (0/7) treated mice, as shown in the average tumor growth curves (Figure 7, Table 16). Further, mice treated with the combination therapy showed long-term tumor remission, as over 60% of the mice remain tumor free at day 50, in comparison to 0% for the isotype control and 10% for the anti-PD-1 or the anti-GITR ent groups (Figure 8, Table 16).

Anti-human GITR dies increase intra-tumoral CD8/Treg ratio The effect of anti-human GITR antibodies on intra-tumoral and splenic T cell populations was assessed. Anti-human GITR antibodies H2aM14536P2 and H1H14536P2 were ted. It was found that both uman GITR dy isotypes (mIg2a and hIgG1) induced a significant increase in the intra-tumoral CD8/Treg ratio (Figure 9). The same treatment had no effect on peripheral spleen T cell subsets. Human IgG1 and mouse IgG2a isotype IgG were used in the assay for controls.

Table 16. Anti-tumor efficacy mediated by uman GITR antibody and anti-mouse PD1 antibody treatment Tumor size (mm3) Tumor Free Mean (SEM) Mice Treatment Group Day 9 Day 13 Day 16 Day 19 Day 51 Isotype control 146 (26) 248 (53) 402 (97) 838 (205) 0/7 (0%) PD1 120 (23) 163 (50) 275 (103)617 (257) 1/7 (14%) H2aM14536P2 134 (28) 162 (51) 194 (51) 346 (87) 1/7 (14%) H2aH14536P2 +Anti- 122 (18) 90 (54) 114 (88) 192 (165) PD-1 4/6 (67%) Administration of anti-mouse GITR antibodies in combination with anti-human PD1 dies significantly induces tumor regression and provides long-term tumor remission in MC38 bearing PD1/PDL1 humanized mice The efficacy of stering an anti-mouse GITR antibody (DTA-1) in combination with an anti-human PD-1 antibody (REGN2810, also known as H4H7798N as disclosed in US Patent Publication No. 2015/0203579) in the control of subcutaneous MC38 tumors was assessed in PD1/PDL1 humanized mice. It was found that anti-human PD-1 blockade synergized with the anti-mouse GITR antibody and induced tumor growth delay in MC38 tumor bearing mice, in comparison to anti PD1 or anti GITR mAb alone or isotype control treated mice as shown in the average tumor growth curves (Figure 10, Table 17). Further, mice treated with the combination therapy showed long-term tumor remission as over 40% of the mice remained tumor free at day 45, in comparison to 0% for the isotype l, the anti-PD-1 or the anti-GITR treatment groups (Figure 11).

Table 17. Anti-tumor efficacy mediated by anti-mouse GITR + anti-human PD1 Ab treatment Tumor size (mm3) Mean (SEM) Treatment Group Day 13 Day 17 Day 20 Day 24 Isotype control 301 (38) 742 (81) 1392 (104) 2790 (366) Anti-PD1(REGN2810) 184 (21) 354 (143) 589 (201) 937 (324) Anti-GITR 362 (99) 713 (360) 1199 (563) NA Anti-GITR + Anti-PD-1 212 (117) 120 (60) 127 (62) 167 (98) Example 8: RNA Extraction and Analysis Single-cell sorting RNA-seq analysis On day 8 and 11 post tumor challenge, single cell suspension of tumor was prepared by mouse tumor dissociation kit (Miltenyi Biotec, Bergisch Gladbach, DE) and spleens were dissociated with gentleMACS™ Octo Dissociator (Miltenyi Biotec). Tumors and s from the same treatment group were pooled and viable CD8+ T cells were sorted by FACS. FACS sorted T cells were mixed with C1 Cell Suspension Reagent (Fluidigm, South San Francisco, CA) before loading onto a 5- to 10-μm C1 Integrated Fluidic Circuit (IFC; Fluidigm). EAD staining solution was ed by adding 2.5 μL ethidium homodimer-1 and 0.625 μL calcein AM (Life Technologies, ad, CA) to 1.25 mL C1 Cell Wash Buffer (Fluidigm) and 20 μL was loaded onto the C1 IFC. Each capture site was carefully examined under a Zeiss microscope in bright field, GFP, and Texas Red channels for cell doublets and ity. Cell lysing, reverse transcription, and cDNA amplification were performed on the C1 -Cell Auto Prep IFC, as specified by the manufacturer (protocol 68 E1). The SMARTer™ Ultra Low RNA Kit (Clontech, Mountain View, CA) was used for cDNA synthesis from the single cells. Illumina NGS libraries were constructed using the NEXTERA XT DNA Sample Prep kit (Illumina), according to the manufacturer’s recommendations (protocol 100-7168 E1). A total of 2,222 single cells were sequenced on Illumina NextSeq (Illumina, San Diego, CA) by multiplexed single-read run with 75 cycles.

Raw sequence data (BCL files) were converted to FASTQ format via Illumina CASAVA 1.8.2. Reads were decoded based on their barcodes. Read quality was evaluated using FastQC (www.bioinformatics. babraham.ac.uk/projects/fastqc/).

Example 9: Role of CD226 and TIGIT in combination treatment Genetic inactivation or pharmacological inhibition of CD226 reversed the tumor regression ed by anti-GITR / anti-PD1 combination treatment in some ments, while inhibition of other TNF-receptor or B7 superfamily members had no effect.

CD226 Blocking experiment 0.5 mg of anti-CD226 (10E5, ience, San Diego, CA) or rat IgG2b isotype control IgG (LTF2, Bio X Cell, West Lebanon, NH) were injected intraperitoneally (i.p.) on day 5 post tumor challenge and one day prior to immunotherapy. Perpendicular tumor diameters were measured blindly 2-3 times per weeks using l calipers (VWR, Radnor, PA). Volume was calculated using the formula L × W × 0.5, where L is the longest dimension and W is the perpendicular dimension. Differences in survival were determined for each group by the Kaplan-Meier method and the overall P value was calculated by the log-rank testing using survival is by PRISM version 6 Pad Software Inc., La Jolla, CA).

An event was defined as death when tumor burden reached the protocol-specified size of 2000 mm3 in maximum tumor volume to minimize morbidity.

As shown in Figures 12 and 13, MC38 tumor bearing mice were treated with either CD226 blocking Ab or isotype Ab (control IgG) 1d prior to immunotherapy with anti-GITR + anti-PD-1 or isotype Abs. e tumor growth curve (Fig. 12) and survival curves (Fig. 13) are shown. Data are representative of three experiments, n = 5 mice per group, survival is by Log-rank test.

Wild type or TIGIT KO mice were challenged with MC38 tumors, treated with anti- CD226 or control IgG and either received isotype control (Fig. 14) or anti-GITR+anti-PD-1 combination therapy (Fig. 15). Data shown are average tumor growth curves representative of two experiments (n = 4-5 mice per group).

Using the CD226 blocking mAb, it was shown that co-stimulatory ing h CD226 is required for the anti-tumor immunity ed by combination treatment.

Furthermore, the CD226 signaling pathway was required for enhanced tumor surveillance in TIGIT KO mice (Figs. 14 and 15).

RNA signatures in CD8+ T cells from combination treatment samples To identify unique gene signatures in clonally expanded CD8+ T cells (tumors harvested at day 11) from combination ent samples, comprehensive comparisons across different treatment groups were performed. Genes upregulated in clonally expanded CD8+ T cells from combination therapy were compared to upregulated genes of CD8+ T cells from isotype treatment or non-expanded CD8+ T cells with ation treatment. Heat mapping analysis identified thirty genes overlapping within the comparison. An RNA signature change of ≥ 2-fold (p < 0.01) was observed within the expanded CD8 T cell population for the 30 genes after the anti-GITR/anti-PD1 combination treatment of tumors.

Those 30 genes include Id2, S100a11, Ndufb3, Serinc3, Ctsd, S100a4, Ppp1ca, Lbr, Peli1, Lcp2, Ube2h, Cd226, Mapkapk3, Racgap1, Arf3, Mki67, Ergic2, Azi2, 2, Sik1, Pde4d, Ppp3cc, Nek7, Emc4, Vav1, Dock10, Tmem173, Fam3c, Ppp1cc, and Glud1.

A four-way comparison across all five groups (i.e., (i) isotype treatment, (ii) anti- GITR expanded CD8, (iii) ITR/anti-PD1 combination expanded CD8, (iv) anti-PD1 expanded CD8, and (v) anti-GITR/anti-PD1 combination non-expanded CD8) was next performed to identify genes specifically regulated upon combination therapy versus monotherapy treatment. Two overlapping upregulated genes (p < 0.01, ≥ 2 fold change in expression) were identified in the four-way analysis. CD226, which is a costimulatory molecule that plays an important role in anti-tumor response, was identified as one of the two genes shared across different comparison pairs. sion analysis of ent subsets of intratumoral CD8+ T cells ((a) total, (b) clonally expanded, or (c) non-expanded) across ent groups (i.e., (i) e, (ii) anti-GITR, (iii) anti-PD1, and (iv) anti-GITR/anti-PD1 ation) revealed that CD226 mRNA levels were significantly increased by combination treatment on clonally expanded T cells (fold change = 10.7), while this difference was d in bulk (fold change = 3.5) and non-expanded CD8+ T cells (not icant). Further, CD226 mRNA levels were significantly increased by combination treatment on clonally ed CD8 T cells in comparison to anti-PD-1 (fold change = 6.5) and anti-GITR (fold change = 9.2) (Fig. 16).

Association between PD1 and CD226 The potential association between PD1 and CD226 les was next investigated. To examine if CD226 is a target for desphosphorylation by the PD1-Shp2 complex, we reconstituted different components involved in T cell signaling in a cell-free large unilamellar vesicle (LUV) system (i.e., CD3, CD226, cytosolic tyrosine kinase Lck, Zap70, SLP76 52, and PI3K (p85a). The sensitivity of each component in response to PD-1 titration on the LUVs was measured by phosphotyrosine (pY) immunoblotting (Fig. 17). We confirmed that TCR/CD3ζ was not a target of sphorylation by PDShp2, whereas CD226 was efficiently dephosphorylated by PD1-Shp2 in a dose dependent manner after 30 minutes of treatment (Fig. 17). This data demonstrated an ation between PD-1 and CD226.

Next, the relationship between PD-1 inhibition and CD226 expression was igated in a clinical setting. RNA-seq analysis was performed on tumor biopsies collected from 43 advanced cancer patients pre- and post-PD-1 targeted treatment. CD226 expression was significantly increased after two doses of anti-hPD-1 treatment in cancer patients (Fig. 22). Further, clinical data from The Cancer Genome Atlas (TCGA) was interrogated to examine if CD226 expression level correlates with the overall T cell activation strength and may be predictive of a better prognosis in cancer patients. Indeed, patients with high baseline CD226 expression have icantly higher al ilities in five (skin cutaneous ma, lung adenocarcinoma, head and neck squamous carcinoma, uterine corpus endometrial carcinoma and sarcoma) out of twenty different types of cancer evaluated (skin cutaneous melanoma, lung adenocarcinoma, head and neck squamous carcinoma, uterine corpus endometrial carcinoma, sarcoma, rectum adenocarcinoma, breast invasive carcinoma, kidney renal clear cell carcinoma, cervical squamous cell carcinoma and endocervical adenocarcinoma, glioblastoma multiforme, colon adenocarcinoma, stomach adenocarcinoma, bladder urothelial carcinoma, thyroid carcinoma, prostate arcinoma, pancreatic adenocarcinoma, brain lower grade glioma, lung squamous cell oma, kidney renal papillary cell carcinoma, and ovarian serous cystadenocarcinoma. l, these results t an immunotherapy strategy that boosts CD226 signaling while simultaneously blocking TIGIT (e.g., via anti-GITR treatment) for maximum T cell activation.

Genetic Inactivation of CD226 Using a CD226 blocking mAb, we showed that costimulatory signaling through CD226 was required for the anti-tumor immunity mediated by combination treatment (Figs. 12, 13). Since CD226 Ab could possibly have a potential depleting effect on subset of CD8 T cells, CD226 was genetically vated in C57BL/6 background mice to confirm that result.

CD226-/- mice showed no defect on T cell (CD4+, CD8+, Tregs) homeostasis (Fig. 18, panels A-D) and responded similarly to wild-type mice to TCR activation (Fig. 18, panels EI ). We observed that the combination treatment no longer conferred the anti-tumor effect or al benefit in /- mice, suggesting that CD226 was essential for the observed anti-tumor effects of the combination (Fig. 19, panel A). The effect of CD226 is ic, since the inhibition of other members of the TNF or superfamily (OX40L or 4-1BBL) or blockade of the B7 costimulatory molecule (CD28) using Ig preserved the anti-tumor effect mediated by the combination therapy (Fig. 19, panels B-D).

Requirement for CD226 in TIGIT Null Animals Overall -cell sorting RNA-seq and FACS phenotyping data showed that anti- PD-1 favored the expression of CD226, while anti-GITR treatment down-regulated surface expression of TIGIT, synergistically restoring the homeostatic T cell function.

We showed that the CD226 signaling pathway was required for enhanced tumor surveillance in TIGIT-/- mice (Figs. 14, 15). Additionally, mice bearing MC38 tumor cells overexpressing CD155/PVR6, which is the major ligand for CD226, showed significant delay of tumor growth upon anti-PD-1 or anti-GITR or combination therapy in comparison to MC38-empty vector (MC38-EV) tumor cells or mice treated with isotype control (Fig. 20).

Immune profiling analysis of mice lanted with MC38-CD155 confirmed ned higher CD155 expression level on MC38-CD155 cells over M38-EV (empty vector) postimplantation.

We found that CD155 over-expression on MC38 tumor cells was associated with decreased detectable CD226 expression on CD4+, CD8+ T and Tregs cells (Fig. 21A) , while it boosted T cell activation as indicated by ed IFNγ (Fig. 21B) and 4-1BB (Fig. 21C) expression on intra-tumoral T cells. No effect was observed in the periphery.

Without being bound by any theory, it is esized that CD226 expression level should correlate with the overall T cell activation th and may be predictive of a better prognosis in cancer patients. Indeed, patients with high CD226 expression have significantly higher survival ilities in three types of cancer (skin ous melanoma, lung arcinoma and sarcoma). These data support an immunotherapy strategy that boosts CD226 signaling while simultaneously blocking TIGIT for maximum T cell activation.

The forgoing experiments demonstrate the synergistic effect of administering an anti-GITR antibody in combination with an anti-PD1 antibody. In particular, among other things, the experiments above demonstrate that the combined administration of an anti-GITR antibody and anti-PD1 antibody induces tumor sion, provides long-term tumor ion, and induces tumor/antigen-specific immunologic memory response.

Example 10: TCR Analysis For TCR analysis, we developed a new bioinformatic pipeline rpsTCR for reconstructing and extracting TCR sequences, especially TCR-CDR3 sequences from random priming short RNA sequencing reads. The rpsTCR took paired- and single-end short reads and maps these reads to mouse or human genomes and transcriptomes, but not TCR gene loci and transcripts using TopHat (Bioinformatics 25, 1105–1111 (2009)) with default parameters. Mapped reads were discarded and unmapped reads are recycled for extraction of TCR sequences. Low quality nucleotides in the unmapped reads were trimmed. Then reads with length less than 35bp were filtered out using HTQC toolkit (Bioinformatics 14, 33 . QC passed short reads was assembled into longer reads using iSSAKE (Bioinformatics 25, 458–464 (2009)) default setting. TCRklass (J l 194, 446–454 (2015)) was used to identify CDR3 sequences with Scr (conserved residue support score) set from default 1.7 to 2. A targeted q data from a healthy human PBMC samples was used as a positive control to evaluate whether the extra steps introduced to the pipeline resulted in higher false positive or false negative rates comparing to TCRklass alone.

The ty of unique CDR3 sequences from TCRB (64,031) or TCRA (51,448) were detected by both rpsTCR and TCRklass. The squared correlations between rpsTCR and TCRklass were 0.9999 and 0.9365 for TCRB-CDR3 and DR3, respectively. Six TCR-negative cancer or ncer cell lines were used as negative controls. No CDR3 sequences were detected by rpsTCR, whereas some CDR sequences were extracted by TCRklass from some TCR-negative cancer cell lines.

To further validate the performance of the subject pipeline, we sequenced a heathy mouse PBMC sample using both targeted TCR-seq and random g RNA-seq approaches (200M, 2x100bp). gh the number of CDR3 sequences assembled from RNA-seq data was much smaller than that from the targeted TCR-seq approach, about 45% of the CDR3 sequences identified from RNA-seq data using rpsTCR were also observed among CDR3 sequences from targeted TCR-seq. Because of the technique limitation of targeted TCR-seq, it is not surprising that a fraction of the CDR3 sequences we extracted from RNA-seq data were not present in the TCR-seq results. For example, the ency of ’ race adapter used for ed TCR-seq is lly low and the multiply PCR tends to amplify high frequency TCRs, thus only a small portion of TCRs can be targeted. As expected, much higher percentage (~ 70%) of the CDR3 sequences identified from RNA-seq data using rpsTCR were also observed among high frequency CDR3 sequences (>= 0.1%) from targeted TCR-seq, while only about 40% extracted using ss alone. er, we cut the 100bp read length in 50bp segments and randomly selected 200M reads. Among the top 10 CDR3 ces ranked by targeted TCR-seq ch, 8 CDR3 sequences were detected by our rpsTCR, while only 3 were detected by TCRklass. We then applied our rpsTCR pipeline to extracting CDR3 sequences from the single cell RNA-seq data generated from intratumoral CD8 T cells of MC38 treated with different antibodies. Our CDR3_beta and CDR3_alpha sequence detection rates were comparable to published data using targeted TCR-seq approach to detect TCR sequences from single cell cing of T cells The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become nt to those skilled in the art from the foregoing description and the anying figures. Such modifications are intended to fall within the scope of the appended claims.

The following numbered paragraphs define particular s of the present invention:: 1. An isolated antibody or antigen-binding fragment thereof that binds glucocorticoidinduced tumor necrosis factor receptor (GITR), n the antibody or antigenbinding fragment exhibits one or more properties selected from the group consisting (i) binds monomeric human GITR at 37ºC with a KD of less than about .0 nM as measured by surface plasmon resonance; (ii) binds monomeric human GITR at 37 ºC with a t½ of r than about 12 minutes; (iii) binds dimeric human GITR at 37ºC with a KD of less than about 950 pM as measured by surface plasmon resonance; (iv) binds dimeric human GITR at 37ºC with a t½ of greater than about 7 minutes; and (v) binds human GITR transfected human embryonic kidney 293 (HEK- 293) D9 cells with an EC50 of less than about 260 pM. 2. The antibody or antigen-binding fragment of paragraph 1, wherein the antibody or antigen-binding fragment tes human GITR. 3. An isolated antibody or antigen-binding fragment thereof that binds glucocorticoid-induced tumor necrosis factor receptor (GITR), wherein the antibody or antigen-binding fragment activates human GITR in the e of Fc anchoring. 4. The antibody or n-binding fragment of paragraph 3, wherein the antibody or antigen-binding fragment activates human GITR at an activation percentage greater than about 25% at an EC50 of less than about 3 nM as determined by NFκB reporter assay . The antibody or n-binding nt of paragraph 3 or 4, wherein the antibody or antigen-binding fragment activates human GITR with an EC50 of less than about 1.0 nM as determined by NFκB reporter assay. 6. An isolated antibody or antigen-binding fragment thereof that binds orticoid-induced tumor necrosis factor or (GITR), wherein the antibody or antigen-binding fragment exhibits T-cell proliferative activity in the e of Fc anchoring as determined in naïve human CD4+ T-cell proliferation assay. 7. The antibody or antigen-binding fragment of paragraph 6, wherein the antibody or antigen-binding fragment exhibits T-cell proliferative activity in the absence of Fc anchoring with an EC50 of about 8 nM or less as determined in naïve human CD4+ T-cell proliferation assay. 8. The antibody or antigen-binding fragment of paragraph 1, wherein the antibody or antigen-binding fragment activates human GITR in the presence of Fc anchoring. 9. An isolated dy or antigen-binding fragment thereof that binds orticoid-induced tumor necrosis factor receptor (GITR), wherein the antibody or antigen-binding fragment exhibits T-cell proliferative activity in the presence of Fc ing at least about 2 fold above background as determined in naïve human CD4+ T-cell proliferation assay.

. The antibody or antigen-binding fragment of paragraph 9, wherein the antibody or antigen-binding fragment exhibits T-cell proliferative activity in the presence of Fc anchoring with an EC50 of less than about 34 nM as determined by naïve human CD4+ T- cell proliferation assay. 11. The antibody or antigen-binding fragment of paragraph 1, wherein the antibody or antigen-binding fragment blocks hGITR ligand (hGITRL)-mediated receptor stimulation. 12. The antibody or antigen-binding fragment of paragraph 11, wherein the antibody or n-binding fragment blocks hGITRL-mediated receptor stimulation in the absence of Fc anchoring. 13. An isolated antibody or antigen-binding fragment thereof that binds glucocorticoid-induced tumor necrosis factor receptor (GITR), wherein the antibody or antigen-binding fragment blocks hGITRL-mediated receptor stimulation in the absence of Fc anchoring at a ng percentage r than about 54% at an IC50 of less than about 4.0 nM as determined by NFκB reporter assay. 14. An isolated antibody or antigen-binding fragment f that binds glucocorticoid-induced tumor necrosis factor receptor (GITR), wherein the antibody or antigen-binding fragment ses: (a) the complementarity determining s (CDRs) of a heavy chain variable region (HCVR) having an amino acid sequence as set forth in Table 1; and (b) the CDRs of a light chain le region (LCVR) having an amino acid sequence as set forth in Table 1.

. The isolated antibody or antigen-binding fragment of paragraph 14, wherein the antibody or antigen-binding nt comprises an HCVR having an amino acid sequence as set forth in Table 1 and a LCVR having an amino acid sequence as set forth in Table 1. 16. The ed antibody or antigen-binding fragment of paragraph 14, wherein the antibody or antigen-binding fragment comprises: (i) a HCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 100, 164, 196, 244, 292, 340, and 348; (ii) a HCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 102, 166, 198, 246, 294, 342, and 350; (iii) a HCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 104, 168, 200, 248, 296, 344, 352; (iv) a LCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 108, 172, 204, 252, 300, and 404 (v) a LCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 110, 174, 206, 254, 302, and 406, and (vi) a LCDR3 domain having an amino acid sequence ed from the group consisting of SEQ ID NOs: 112, 176, 208, 256, 304, and 408. 17. The isolated antibody or antigen-binding fragment of aph 14, n the antibody or antigen-binding fragment comprises: (i) a heavy chain complementarity determining region (HCDR)-1 comprising SEQ ID NO: 100; an HCDR2 comprising SEQ ID NO: 102; an HCDR3 comprising SEQ ID NO: 104; a light chain complementarity determining region (LCDR)-1 comprising SEQ ID NO: 108; an LCDR2 comprising SEQ ID NO: 110; and an LCDR3 comprising SEQ ID NO: 112; (ii) a heavy chain complementarity ining region (HCDR)-1 comprising SEQ ID NO: 164; an HCDR2 comprising SEQ ID NO: 166; an HCDR3 comprising SEQ ID NO: 168; a light chain mentarity determining region (LCDR)-1 comprising SEQ ID NO: 172; an LCDR2 comprising SEQ ID NO: 174; and an LCDR3 comprising SEQ ID NO: 176; (iii) a heavy chain complementarity determining region (HCDR)-1 comprising SEQ ID NO: 196; an HCDR2 comprising SEQ ID NO: 198; an HCDR3 sing SEQ ID NO: 200; a light chain complementarity determining region (LCDR)-1 comprising SEQ ID NO: 204; an LCDR2 comprising SEQ ID NO: 206; and an LCDR3 sing SEQ ID NO: 208; (iv) a heavy chain complementarity determining region (HCDR)-1 comprising SEQ ID NO: 244; an HCDR2 comprising SEQ ID NO: 246; an HCDR3 comprising SEQ ID NO: 248; a light chain mentarity ining region (LCDR)-1 comprising SEQ ID NO: 252; an LCDR2 comprising SEQ ID NO: 254; and an LCDR3 comprising SEQ ID NO: 256; (v) a heavy chain complementarity determining region (HCDR)-1 comprising SEQ ID NO: 292; an HCDR2 comprising SEQ ID NO: 294; an HCDR3 comprising SEQ ID NO: 296; a light chain complementarity determining region (LCDR)-1 comprising SEQ ID NO: 300; an LCDR2 comprising SEQ ID NO: 302; and an LCDR3 comprising SEQ ID NO: 304; (vi) a heavy chain complementarity determining region (HCDR)-1 comprising SEQ ID NO: 340; an HCDR2 comprising SEQ ID NO: 342; an HCDR3 comprising SEQ ID NO: 344; a light chain complementarity determining region (LCDR)-1 comprising SEQ ID NO: 404; an LCDR2 comprising SEQ ID NO: 406; and an LCDR3 comprising SEQ ID NO: 408; or (vii) a heavy chain complementarity determining region -1 comprising SEQ ID NO: 348; an HCDR2 comprising SEQ ID NO: 350; an HCDR3 comprising SEQ ID NO: 352; a light chain complementarity determining region (LCDR)-1 comprising SEQ ID NO: 404; an LCDR2 comprising SEQ ID NO: 406; and an LCDR3 comprising SEQ ID NO: 408. 18. An isolated antibody or antigen-binding nt thereof, wherein the antibody or antigen-binding nt thereof competes for binding to GITR with a reference antibody comprising an HCVR/LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOS: 98/106; 162/170; 194/202; 242/250; 290/298; 338/402; and 346/102. 19. An isolated antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding nt thereof binds to the same e on GITR as a reference antibody comprising an CVR amino acid sequence pair selected from the group consisting of SEQ ID NOS: 98/106; 162/170; 194/202; 242/250; 290/298; 338/402; and 346/102.

. A pharmaceutical composition comprising the antibody or antigen-binding fragment of any of paragraphs 1-19 and a pharmaceutically acceptable r or diluent. 21. A method for treating cancer in a subject comprising administering the composition of paragraph 20. 22. A method of modulating anti-tumor immune response in a subject sing administering to the subject the antibody that binds GITR of any of paragraphs 1-19 or antigen-binding fragment thereof. 23. The method of paragraph 20, further comprising administering an antibody or antigen-binding fragment thereof that binds to a second T-cell activating or. 24. The method of paragraph 21, wherein the T-cell ting receptor is CD28, OX40, CD137, CD27, or HVEM.

. The method of any of aphs 19-24, further comprising administering an antibody or n-binding nt thereof that binds to a T-cell inhibitory receptor. 26. The method of paragraph 25, n the T-cell inhibitory receptor is CTLA-4, PD-1, TIM-3, BTLA, VISTA, or LAG-3. 27. The method of any of paragraphs 20-26, further comprising administering radiation therapy to said subject. 28. The method of any of paragraphs 20-27, further comprising administering one or more chemotherapeutic agent. 29. The method of paragraph 26, wherein the T-cell inhibitory receptor is PD1.

. The method of paragraph 29, wherein the antibody that binds to a T-cell receptor is REGN 2810. 31. The method of any of paragraphs 22-30, wherein the antibody that binds GITR is an antibody of paragraph 17.

Claims (29)

1. What is claimed is: 1. An isolated antibody or antigen-binding fragment f that binds glucocorticoidinduced tumor necrosis factor receptor (GITR) comprising: (i) a heavy chain variable region (HCVR) comprising the three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2 and HCDR3) contained within an HCVR amino acid sequence having 90% sequence identity to an amino acid sequence of SEQ ID NO: 338; and a light chain variable region (LCVR) comprising the three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within an LCVR amino acid sequence having 90% ce identity an amino acid ce of SEQ ID NO: 402; (ii) an HCVR comprising the three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within an HCVR amino acid sequence having 90% sequence identity to an amino acid sequence of SEQ ID NO: 98; and an LCVR comprising the three light chain CDRs , LCDR2, and LCDR3) contained within an LCVR amino acid sequence having 90% sequence identity an amino acid sequence of SEQ ID NO: 106; (iii) an HCVR comprising the three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within an HCVR amino acid sequence having 90% sequence identity to an amino acid sequence of SEQ ID NO: 162; and an LCVR comprising the three light chain CDRs , LCDR2, and LCDR3) ned within an LCVR amino acid sequence having 90% sequence identity an amino acid sequence of SEQ ID NO: 170; (iv) an HCVR comprising the three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within an HCVR amino acid ce having 90% sequence identity to an amino acid sequence of SEQ ID NO: 194; and an LCVR sing the three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within an LCVR amino acid sequence having 90% sequence identity an amino acid sequence of SEQ ID NO: 202; (v) an HCVR comprising the three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within an HCVR amino acid sequence having 90% ce identity to an amino acid sequence of SEQ ID NO: 242; and an LCVR comprising the three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within an LCVR amino acid sequence having 90% sequence identity an amino acid sequence of SEQ ID NO: 250; (vi) an HCVR comprising the three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within an HCVR amino acid sequence having 90% sequence identity to an amino acid sequence of SEQ ID NO: 290; and an LCVR comprising the three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within an LCVR amino acid sequence having 90% sequence identity an amino acid sequence of SEQ ID NO: 298.(vii) an HCVR comprising the three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within an HCVR amino acid sequence having 90% sequence identity to an amino acid sequence of SEQ ID NO: 346; and an LCVR comprising the three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within an LCVR amino acid sequence having 90% sequence identity an amino acid sequence of SEQ ID NO: 402; (viii) an HCVR comprising the three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) ned within an HCVR amino acid sequence having 90% sequence identity to an amino acid sequence of SEQ ID NO: 274; and an LCVR comprising the three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within an LCVR amino acid sequence having 90% sequence identity an amino acid sequence of SEQ ID NO: 282; (ix) an HCVR comprising the three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within an HCVR amino acid sequence having 90% sequence identity to an amino acid sequence of SEQ ID NO: 178; and an LCVR comprising the three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within an LCVR amino acid sequence having 90% ce identity an amino acid sequence of SEQ ID NO: 186; (x) an HCVR comprising the three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within an HCVR amino acid sequence having 90% sequence identity to an amino acid sequence of SEQ ID NO: 210; and an LCVR comprising the three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within an LCVR amino acid sequence having 90% ce identity an amino acid ce of SEQ ID NO: 218; or (xi) an HCVR sing the three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within an HCVR amino acid sequence having 90% sequence identity to an amino acid sequence of SEQ ID NO: 114; and an LCVR comprising the three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within an LCVR amino acid sequence having 90% sequence identity an amino acid sequence of SEQ ID NO: 122.
2. 2. The isolated antibody or antigen-binding fragment f of claim 1, wherein the antibody or antigen-binding fragment thereof ses an HCVR having 90% sequence identity to SEQ ID NO: 338 and an LCVR having 90% sequence identity to SEQ ID NO: 402.
3. 3. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or n-binding fragment f comprises an HCVR having 90% sequence identity to SEQ ID NO: 242 and an LCVR having 90% sequence identity to SEQ ID NO: 250.
4. 4. The isolated antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment f comprises: (i) a heavy chain complementarity determining region (HCDR)-1 comprising SEQ ID NO: 100; an HCDR2 comprising SEQ ID NO: 102; an HCDR3 comprising SEQ ID NO: 104; a light chain mentarity determining region (LCDR)-1 comprising SEQ ID NO: 108; an LCDR2 comprising SEQ ID NO: 110; and an LCDR3 comprising SEQ ID NO: 112; (ii) a heavy chain mentarity determining region (HCDR)-1 comprising SEQ ID NO: 164; an HCDR2 comprising SEQ ID NO: 166; an HCDR3 comprising SEQ ID NO: 168; a light chain complementarity determining region (LCDR)-1 comprising SEQ ID NO: 172; an LCDR2 comprising SEQ ID NO: 174; and an LCDR3 comprising SEQ ID NO: 176; (iii) a heavy chain complementarity determining region (HCDR)-1 comprising SEQ ID NO: 196; an HCDR2 comprising SEQ ID NO: 198; an HCDR3 comprising SEQ ID NO: 200; a light chain complementarity ining region -1 comprising SEQ ID NO: 204; an LCDR2 sing SEQ ID NO: 206; and an LCDR3 comprising SEQ ID NO: 208; (iv) a heavy chain complementarity determining region (HCDR)-1 comprising SEQ ID NO: 244; an HCDR2 comprising SEQ ID NO: 246; an HCDR3 comprising SEQ ID NO: 248; a light chain complementarity determining region (LCDR)-1 comprising SEQ ID NO: 252; an LCDR2 comprising SEQ ID NO: 254; and an LCDR3 sing SEQ ID NO: 256; (v) a heavy chain complementarity determining region (HCDR)-1 comprising SEQ ID NO: 292; an HCDR2 comprising SEQ ID NO: 294; an HCDR3 comprising SEQ ID NO: 296; a light chain complementarity determining region (LCDR)-1 comprising SEQ ID NO: 300; an LCDR2 comprising SEQ ID NO: 302; and an LCDR3 comprising SEQ ID NO: 304; (vi) a heavy chain complementarity determining region (HCDR)-1 comprising SEQ ID NO: 340; an HCDR2 comprising SEQ ID NO: 342; an HCDR3 comprising SEQ ID NO: 344; a light chain complementarity determining region (LCDR)-1 comprising SEQ ID NO: 404; an LCDR2 comprising SEQ ID NO: 406; and an LCDR3 comprising SEQ ID NO: 408; or (vii) a heavy chain complementarity determining region -1 comprising SEQ ID NO: 348; an HCDR2 comprising SEQ ID NO: 350; an HCDR3 comprising SEQ ID NO: 352; a light chain mentarity determining region (LCDR)-1 comprising SEQ ID NO: 404; an LCDR2 comprising SEQ ID NO: 406; and an LCDR3 comprising SEQ ID NO: 408.
5. 5. The antibody or antigen-binding fragment thereof of claim 1, comprising: an HCDR-1 comprising SEQ ID NO: 340; an HCDR2 comprising SEQ ID NO: 342; an HCDR3 comprising SEQ ID NO: 344; an LCDR-1 comprising SEQ ID NO: 404; an LCDR2 comprising SEQ ID NO: 406; and an LCDR3 comprising SEQ ID NO: 408.
6. 6. The antibody or antigen-binding nt thereof of claim 1, comprising: an HCDR-1 sing SEQ ID NO: 244; an HCDR2 comprising SEQ ID NO: 246; an HCDR3 comprising SEQ ID NO: 248; an LCDR-1 comprising SEQ ID NO: 252; an LCDR2 comprising SEQ ID NO: 254; and an LCDR3 comprising SEQ ID NO: 256.
7. 7. An isolated dy or antigen-binding fragment f, wherein the antibody or antigen-binding fragment thereof competes for binding to GITR with a reference dy comprising an CVR amino acid sequence pair selected from the group consisting of SEQ ID NOS: 98/106; 162/170; 194/202; 242/250; 290/298; 338/402; and 346/402.
8. 8. An isolated antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof binds to the same epitope on GITR as a reference antibody comprising an HCVR/LCVR amino acid sequence pair selected from the group ting of SEQ ID NOS: 98/106; 162/170; 194/202; 242/250; 290/298; 338/402; and 346/402.
9. 9. The isolated antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment exhibits one or more properties selected from the group consisting of: (i) binds monomeric human GITR at 37ºC with a KD of less than about 5.0 nM as measured by surface plasmon nce; (ii) binds monomeric human GITR at 37 ºC with a t½ of greater than about 12 minutes; (iii) binds dimeric human GITR at 37ºC with a KD of less than about 950 pM as measured by e plasmon resonance; (iv) binds dimeric human GITR at 37ºC with a t½ of greater than about 7 minutes; and (v) binds human GITR transfected human embryonic kidney 293 (HEK-293) D9 cells with an EC50 of less than about 260 pM.
10. 10. The isolated antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof activates human GITR.
11. 11. The ed antibody or antigen-binding nt f of claim 1, wherein the antibody or antigen-binding fragment thereof activates human GITR in the absence of Fc anchoring.
12. 12. The antibody or antigen-binding fragment thereof of claim 10, wherein the antibody or n-binding fragment thereof activates human GITR at an activation percentage greater than about 25% at an EC50 of less than about 3 nM as determined by NFκB reporter assay.
13. 13. The antibody or antigen-binding fragment thereof of claim 10, wherein the antibody or antigen-binding fragment thereof activates human GITR with an EC50 of less than about 1.0 nM as determined by NFκB reporter assay.
14. 14. The antibody or antigen-binding fragment f of claim 11, wherein the antibody or antigen-binding fragment thereof exhibits T-cell proliferative activity in the absence of Fc anchoring as determined in naïve human CD4+ T-cell proliferation assay.
15. 15. The antibody or antigen-binding fragment thereof of claim 11, wherein the antibody or antigen-binding fragment thereof exhibits T-cell erative ty in the absence of Fc anchoring with an EC50 of about 8 nM or less as determined in naïve human CD4+ T-cell proliferation assay.
16. 16. The antibody or antigen-binding nt f of claim 1, wherein the antibody or antigen-binding fragment thereof activates human GITR in the presence of Fc anchoring.
17. 17. The antibody or antigen-binding fragment thereof of claim 16, wherein the antibody or antigen-binding fragment thereof exhibits T-cell proliferative activity in the presence of Fc anchoring at least about 2 fold above ound as determined in naïve human CD4+ T-cell proliferation assay.
18. 18. The antibody or antigen-binding fragment thereof of claim 16, wherein the antibody or antigen-binding fragment thereof ts T-cell proliferative activity in the presence of Fc anchoring with an EC50 of less than about 34 nM as determined by naïve human CD4+ T-cell proliferation assay.
19. 19. An isolated antibody or antigen-binding fragment thereof that blocks human glucocorticoid-induced tumor necrosis factor or ligand L)-mediated receptor stimulation comprising: (i) a heavy chain variable region (HCVR) comprising the three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2 and HCDR3) contained within an HCVR amino acid sequence having 90% sequence identity to an amino acid sequence of SEQ ID NO: 338; and a light chain variable region (LCVR) sing the three light chain CDRs (LCDR1, LCDR2, and LCDR3) ned within an LCVR amino acid sequence having 90% sequence identity an amino acid sequence of SEQ ID NO: 402; (ii) an HCVR sing the three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) ned within an HCVR amino acid sequence having 90% sequence identity to an amino acid sequence of SEQ ID NO: 98; and an LCVR comprising the three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within an LCVR amino acid sequence having 90% sequence identity an amino acid sequence of SEQ ID NO: 106; (iii) an HCVR comprising the three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within an HCVR amino acid sequence having 90% sequence identity to an amino acid sequence of SEQ ID NO: 274; and an LCVR comprising the three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within an LCVR amino acid sequence having 90% sequence identity an amino acid ce of SEQ ID NO: 282; (iv) an HCVR comprising the three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within an HCVR amino acid sequence having 90% sequence identity to an amino acid sequence of SEQ ID NO: 178; and an LCVR comprising the three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within an LCVR amino acid sequence having 90% sequence identity an amino acid sequence of SEQ ID NO: 186; (v) an HCVR comprising the three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within an HCVR amino acid sequence having 90% sequence identity to an amino acid sequence of SEQ ID NO: 210; and an LCVR comprising the three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within an LCVR amino acid sequence having 90% sequence identity an amino acid sequence of SEQ ID NO: 218; or (vi) an HCVR comprising the three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) ned within an HCVR amino acid sequence having 90% ce identity to an amino acid sequence of SEQ ID NO: 114; and an LCVR comprising the three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within an LCVR amino acid sequence having 90% sequence identity an amino acid sequence of SEQ ID NO: 122.
20. 20. The antibody or antigen-binding fragment of claim 19, wherein the antibody or antigen-binding fragment thereof blocks hGITRL-mediated receptor stimulation in the absence of Fc ing.
21. 21. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-20 and a pharmaceutically acceptable carrier or diluent.
22. 22. Use of the ceutical composition of claim 21 in the preparation of a medicament for the treatment of cancer in a subject, wherein the treatment comprises administering the composition to the t.
23. 23. Use of the antibody or antigen-binding fragment thereof of any one of claims 1 to 20 in the preparation of a medicament for the modulation of an anti-tumor immune response in a subject, wherein the tion comprises administering to the subject the dy or antigen-binding fragment that binds GITR.
24. 24. Use of the antibody or antigen-binding fragment thereof of any one of claims 1 to 20 for the ation of a ment for the modulation of an umor immune response in a t, wherein the modulation reduces the suppressive activity of T effector cells by T regulatory cells, enhances intra-tumoral T effector/T regulatory cell ratio conducive for eutic benefit, and/or promotes T cell survival in the subject.
25. 25. The use of any one of claims 22 – 24, wherein the medicament is therapeutically combined with an antibody or n-binding fragment thereof that binds to a second T-cell activating receptor; and/or wherein the T-cell activating receptor is CD28, OX40, CD137, CD27, or HVEM.
26. 26. The use of any one of claims 22 – 24, wherein the medicament is therapeutically combined with an antibody or antigen-binding fragment thereof that binds to a T-cell inhibitory receptor; and/or wherein the T-cell inhibitory receptor is CTLA-4, PD-1, TIM- 3, BTLA, VISTA, or LAG-3.
27. 27. The use of any one of claims 22 – 26, wherein the medicament is therapeutically combined with radiation therapy to said t; and/or wherein the medicament is therapeutically combined with one or more chemotherapeutic agent.
28. 28. The use of claim 26, wherein the T-cell tory receptor is PD1; and/or wherein the antibody that binds to a T-cell receptor is REGN 2810.
29. 29. The use of any one of claims 23 – 28, wherein the antibody that binds GITR is an antibody of claim 2. om mmcmzmso om .55 or Eta $59“. 0 m>mm 00mm ooom com 000? 00m (swim) azgs Jowni 100 nge tumor 2 50 after FIGURE 000000 OCDCOVN IBAQMHS % [_ mm Warn—firm cm 3:: o 39. 3m 8m 3v SN a w>_mz om anus. 9 mum—8:20 $8 8m 0% new 8N o SEE m 35:20 on his Worn—firm om MEDGE BEE O 305. _|