EA039583B1 - 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

Background of the Invention

The glucocorticoid-inducible tumor necrosis factor receptor (GITR) is a member of the tumor necrosis factor receptor superfamily (TNFRSF). Expression is constitutively high in regulatory T cells, low/intermediate in naive T cells, NK cells, and granulocytes, and is induced upon activation. GITR interacts with its ligand GITRL, which is mainly expressed on antigen-presenting cells. Activation of the GITR receptor can enhance both proliferation and function of effector T cells, as well as attenuate the downregulation induced by regulatory T cells. Therefore, modulation of GITR activity may serve as a basis for cancer immunotherapy and immune disorders. Thus, there is a need for agents, such as antibodies, that modulate GITR activity.

Brief summary of the present invention

The present invention relates to antibodies and antigen-binding fragments thereof that bind the glucocorticoid-inducible tumor necrosis factor receptor (GITR). The antibodies of the present invention are useful, inter alia, for targeting immune cells such as effector T cells, regulatory T cells and NK cells that express GITR.

Antibodies of the present invention may be full-length (eg, an IgG1 or IgG4 antibody) or may contain only an antigen-binding portion (eg, a Fab-, F(ab')2 or scFv fragment), and may also be modified to degrade functionality. , for example to eliminate residual effector functions (Reddy et al., 2000, J. Immunol. 164:1925-1933).

Exemplary anti-GITR antibodies according to the present invention are listed in Table 1. 1 and 2 in this document. In table. 1 sets forth amino acid sequence identifiers for heavy chain variable regions (HCVR), light chain variable regions (LCVR), heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3), and light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) of exemplary anti-GITR antibodies. . In table. 2 sets out the nucleic acid sequence identifiers for HCVR, LCVR, HCDR1, HCDR2 HCDR3, LCDR1, LCDR2, and LCDR3 exemplary anti-GITR antibodies.

The present invention relates to antibodies or antigen-binding fragments that specifically bind GITR containing HCVR containing an amino acid sequence selected from any of the HCVR amino acid sequences shown in table. 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

The present invention also relates to antibodies or antigen-binding fragments that specifically bind GITR containing LCVR containing an amino acid sequence selected from any of the LCVR amino acid sequences shown in table. 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

The present invention also relates to antibodies or antigen-binding fragments thereof that specifically bind GITR containing a pair of amino acid sequences HCVR and LCVR (HCVR/LCVR) containing any of the HCVR amino acid sequences shown in table. 1, paired with any of the LCVR amino acid sequences shown in table. 1. According to some variants of implementation of the present invention relates to antibodies or their antigen-binding fragments containing a pair of amino acid sequences HCVR/LCVR contained in any of the typical antibodies against GITR shown in table. 1. In some embodiments, the HCVR/LCVR 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 relates to antibodies, or antigen-binding fragments thereof, that specifically bind a GITR containing a heavy chain CDR1 (HCDR1) containing an amino acid sequence selected from any of the HCDR1 amino acid sequences shown in Table 1. 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

The present invention also relates to antibodies, or antigen-binding fragments thereof, that specifically bind a GITR containing a heavy chain CDR2 (HCDR2) containing an amino acid sequence selected from any of the HCDR2 amino acid sequences shown in Table 1. 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

The present invention also relates to antibodies or antigen-binding fragments thereof,

- 1 039583 which specifically bind a GITR containing a heavy chain CDR3 (HCDR3) containing an amino acid sequence selected from any of the amino acid sequences

HCDR3 given in table. 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

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

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

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

The present invention also relates to antibodies or antigen-binding fragments thereof that specifically bind GITR containing a pair of amino acid sequences HCVR3 and LCVR3 (HCVR3/LCVR3) containing any of the HCVR3 amino acid sequences shown in table. 1, paired with any of the LCVR3 amino acid sequences shown in table. 1. According to some variants of implementation of the present invention relates to antibodies or their antigen-binding fragments containing a pair of amino acid sequences HCVR3/LCVR3 contained in any of the typical antibodies against GITR shown in table. 1. In some embodiments, the HCVR3/LCVR3 amino acid sequence pair is selected from the group consisting of 104/112; 168/176; 200/208; 248/256; 296/304; 344/408 and 352/408.

The present invention also relates to antibodies or antigen-binding fragments thereof that specifically bind GITR containing a series of six CDRs (i.e. HCDR1-HCDR2-HCDR3-LCDR1LCDR2-LCDR3) contained in any of the exemplary anti-GITR antibodies shown in Table . 1. In some embodiments, the range of amino acid sequences HCDR1-HCDR2HCDR3-LCDR1-LCDR2-LCDR3 is selected from the group consisting of 100-102-104-108-110-112; 164-166-168172-174-176; 196-198-200-204-206-208; 244-246-248-252-254-256; 292-294-296-300-302-304; 340-342-344404-406-408 and 348-350-352-404-406-408.

According to a related embodiment, the present invention relates to antibodies or antigen-binding fragments thereof that specifically bind a GITR containing a series of six CDRs (i.e. HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained in the HCVR amino acid sequence pair /LCVR, as determined by any typical anti-GITR antibodies shown in table. 1. For example, the present invention relates to antibodies or antigen-binding fragments thereof that specifically bind a GITR comprising the amino acid sequence HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 contained in an HCVR/LCVR amino acid sequence pair selected from the group consisting of 98/106; 162/170; 194/202; 242/250; 290/298; 338/402 and 346/102. Methods and techniques for identifying CDRs in HCVR and LCVR amino acid sequences are well known in the art and can be used to identify CDRs in certain HCVR and/or LCVR amino acid sequences disclosed herein. Typical rules that can be used to identify CDR boundaries include, for example, Kabat definition, Chothia definition, and AbM definition. In general, the Kabat definition is based on sequence variability, the Chothia definition is based on the location of the structural loop regions, and the AbM definition is a compromise between the Kabat and Chothia approaches. See, for example, Kabat, Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md. (1991); AlLazikani et al, J. Mol. Biol. 273:927-948 (1997); and Martin et al., Proc. Natl. Acad. sci. USA 86:9268-9272 (1989). Public databases also exist for identifying CDR sequences in an antibody.

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

The present invention also relates to nucleic acid molecules encoding any of the LCVR amino acid sequences shown in table. one; in some embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCVR nucleic acid sequences shown in Table 1. 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

The present invention also relates to nucleic acid molecules encoding any of the HCDR1 amino acid sequences shown in table. one; in some embodiments, the nucleic acid molecule contains a polynucleotide sequence selected from any of the HCDR1 nucleic acid sequences shown in Table 1. 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

The present invention also relates to nucleic acid molecules encoding any of the HCDR2 amino acid sequences shown in table. one; in some embodiments, the nucleic acid molecule contains a polynucleotide sequence selected from any of the HCDR2 nucleic acid sequences shown in Table 1. 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

The present invention also relates to nucleic acid molecules encoding any of the HCDR3 amino acid sequences shown in table. one; in some embodiments, the nucleic acid molecule contains a polynucleotide sequence selected from any of the HCDR3 nucleic acid sequences shown in Table 1. 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

The present invention also relates to nucleic acid molecules encoding any of the LCDR1 amino acid sequences shown in Table. one; in some embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR1 nucleic acid sequences shown in Table 1. 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

The present invention also relates to nucleic acid molecules encoding any of the LCDR2 amino acid sequences shown in table. one; in some embodiments, the nucleic acid molecule contains a polynucleotide sequence selected from any of the LCDR2 nucleic acid sequences shown in Table 1. 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

The present invention also relates to nucleic acid molecules encoding any of the LCDR3 amino acid sequences shown in Table. one; in some embodiments, the nucleic acid molecule contains a polynucleotide sequence selected from any of the LCDR3 nucleic acid sequences shown in Table 1. 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

The present invention also relates to nucleic acid molecules encoding HCVR, wherein the HCVR contains a range of three CDRs (i.e. HCDR1-HCDR2-HCDR3), the amino acid sequence range of HCDR1-HCDR2-HCDR3 being determined by any of the exemplary anti-GITR antibodies given in table. one.

The present invention also relates to nucleic acid molecules encoding an LCVR, wherein the LCVR contains a set of three CDRs (i.e. LCDR1-LCDR2-LCDR3), the amino acid sequence set of LCDR1-LCDR2-LCDR3 being determined by any of the exemplary anti-GITR antibodies given in table. one.

The present invention also relates to nucleic acid molecules encoding both HCVR and LCVR, while HCVR contains the amino acid sequence of any of the HCVR amino acid sequences shown in table. 1, and while LCVR contains the amino acid sequence of any of the amino acid sequences of the LCVR shown in table. 1. In some embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCVR nucleic acid sequences shown in Table 1. 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity, and a polynucleotide sequence selected from any of the LCVR nucleic acid sequences shown in table. 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. In some embodiments, in this aspect of the present invention, the nucleic acid molecule encodes for HCVR and LCVR, wherein both HCVR and LCVR come from the same anti-GITR antibody shown in Table 1. one.

The present invention also relates to recombinant expression vectors capable of expressing a polypeptide containing the heavy or light chain variable region of an anti-GITR antibody. For example, the present invention relates to recombinant expression vectors containing any of the nucleic acid molecules mentioned above, i. nucleic acid molecules encoding any of the HCVR, LCVR, and/or CDR sequences set forth in Table. 1. The scope of the present invention also covers host cells into which such vectors have been introduced, as well as methods for producing antibodies or parts thereof by culturing host cells under conditions that ensure the production of antibodies or antibody fragments, and recovering the antibodies and antibody fragments thus obtained. .

The present invention relates to anti-GITR antibodies having a modified glycosylation pattern. In some embodiments, modification to remove unwanted glycosylation sites or an antibody lacking a fucose moiety present on the oligosaccharide chain may be useful, for example, to enhance antibody-dependent cellular cytotoxicity (ADCC) function (see Shield et al. (2002) JBC 277:26733). For other applications, modification of galactosylation may be carried out to modify complement-dependent cytotoxicity (CDC).

In another aspect, the present invention provides a pharmaceutical composition comprising a recombinant human antibody or fragment thereof that specifically binds GITR and a pharmaceutically acceptable carrier. In a related aspect, the present invention provides a composition that is a combination of an anti-GITR antibody and a second therapeutic agent. In one embodiment, the second therapeutic agent is any agent that combines successfully with an anti-GITR antibody. The present invention also relates to antibody-drug conjugates (ADC) containing an anti-GITR antibody conjugated to a cytotoxic agent. Exemplary combination therapeutics, co-formulations, and ADCs comprising anti-GITR antibodies of the present invention are disclosed hereinafter.

In a further aspect, the present invention relates to therapeutic methods for killing tumor cells, or inhibiting or attenuating the growth of tumor cells, or otherwise treating a patient afflicted with cancer, using an anti-GITR antibody or an antigen-binding portion of an antibody in accordance with the present invention. Therapeutic methods in this aspect of the present invention provide for the introduction of a therapeutically effective amount of a pharmaceutical composition containing the antibody or antigen-binding antibody fragment in accordance with the present invention to the subject, if necessary. A treatable disorder is any disease or condition that is ameliorated, alleviated, suppressed or prevented by targeting GITR and/or by enhancing T cell proliferation or function and/or inhibiting downregulation of activity induced by regulatory T cells.

In a further aspect, the present invention relates to therapeutic methods for killing tumor cells, or inhibiting or attenuating the growth of tumor cells, or otherwise treating a patient afflicted with cancer, using a combination of an anti-GITR antibody or an antigen-binding portion of an anti-GITR antibody and an anti-PD1 antibody or the antigen-binding portion of an anti-PD1 antibody. Therapeutic methods in this aspect of the present invention provide for administering a therapeutically effective amount of a pharmaceutical composition comprising a combination of an anti-GITR antibody composition and an anti-PD1 antibody or antigen-binding fragment to a subject in need thereof. A treatable disorder is any disease or condition that is ameliorated, alleviated, suppressed, or prevented by targeting both GITR and PD1.

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

Brief description of graphic materials

In FIG. 1 shows mean tumor volumes for each treatment group (mm ³ ± SEM) plotted against days after tumor challenge as described in Example 7. Mice were treated with either isotype antibody (open circles, o), anti-PD-1 antibody (open circles). squares, □), an anti-GITR antibody (open triangles, Δ), or a combination of an anti-PD-1 antibody and an anti-GITR antibody (solid inverted triangles, ▼).

In FIG. 2 shows survival analysis of MC38-bearing mice treated with the combination of anti-mouse GITR antibody and anti-mouse PD1 antibody as described in Example 7. Mice were treated with either isotype antibody (open circles, o), anti-PD-1 antibody (open squares, □), an anti-GITR antibody (open triangles, Δ), or a combination of an anti-PD-1 antibody and an anti-GITR antibody (open inverted triangles, V).

- 4 039583

In FIG. 3 shows a single tumor growth curve in tumor free or untreated control mice challenged with MC38 or B16.F10.9 tumor cells as described in Example 7.

In FIG. 4 shows mean tumor volumes for mice treated with different depleting antibodies as described in Example 7.

In FIG. 5 shows the results of the FACS analysis of the intratumoral ratio CD8/Treg, CD4 Teff/Treg as described in Example 7.

In FIG. 6 shows the percentage and number of cells/mm ³ tumor for subgroups of T cells in the tumor, as described in example 7.

In FIG. 7 shows the mean tumor volumes for each treatment group (mm ³ ± SEM) plotted against days after tumor challenge as shown in Example 7.

In FIG. 8 shows a survival analysis of MC38-bearing humanized GITR/GIRL mice treated with the combination of anti-human GITR antibody and anti-mouse PD1 antibody as described in Example 7.

In FIG. 9 shows FACS analysis of intratumoral and splenic percentage of CD8 T cells, percentage of Treg cells and CD8/Treg ratio as described in Example 7.

In FIG. 10 shows the mean tumor volumes for each treatment group (mm ³ ± SEM) plotted against days after tumor challenge as described in Example 7.

In FIG. 10 shows a survival analysis of MC38-bearing humanized PD1/PDL1 mice treated with a combination of an anti-mouse GITR antibody and an anti-human PD1 antibody as described in Example 7.

In FIG. 12 shows the tumor growth curve of MC38 bearing mice as described in Example 7. Y-axis shows tumor volume in cubic millimeters and X-axis shows time in days after tumor challenge. Open symbols (□, o) represent mice first treated with isotype antibody (control). Filled symbols (,·) represent mice first treated with anti-CD226 antibody. Those mice that were then treated with isotype antibody are represented by circles (o, •) and solid lines. Those mice that were then treated with the combination of anti-GITR antibody and anti-PD-1 antibody are represented by squares (□, ) and dotted lines.

In FIG. 13 shows the survival curve of MC38 bearing mice as described in Example 7. The Y-axis shows the percentage of survivors and the X-axis shows the time in days after tumor challenge. Open symbols (□, o) represent mice first treated with isotype antibody (control). Filled symbols (,·) represent mice first treated with anti-CD226 antibody. Those mice that were then treated with isotype antibody are represented by circles (o, •) and solid lines. Those mice that were then treated with the combination of anti-GITR antibody and anti-PD-1 antibody are represented by squares (□, ) and dotted lines.

In FIG. 14 shows the tumor growth curve of wild-type MC38-bearing mice (represented by diamonds [^>^]) or TIGIT knockout mice (represented by triangles ΑΆ]) treated with isotype IgG as described in Example 7. Y-axis shows tumor volume in cubic millimeters, and the x-axis shows time in days after tumor challenge. Open symbols and dotted lines represent mice first treated with isotype antibody (control). Filled symbols and solid lines represent mice first treated with anti-CD226 antibody.

In FIG. 15 shows a tumor growth curve of MC38-bearing wild-type mice (represented by circles [o, ]) or TIGIT knockout mice (represented by inverted triangles [^, ▼]) treated with isotype IgG as described in Example 7. Y-axis shows tumor volume in cubic millimeters, and the x-axis shows the time in days after the tumor challenge. Open symbols and dotted lines represent mice first treated with isotype antibody (control). Filled symbols and solid lines represent mice first treated with anti-CD226 antibody.

In FIG. 16 are Cumulative Distribution Function (CDF) plots depicting activated CD226 expression by combined treatment of total (panel A), clonal expanded (panel B) or non-expanded (panel C) CD8+ T cells. The x-axis shows CD226 expression in log2(RPKM) (Reads Per Kilobase of transcript per Million mapped reads), and the y-axis shows the cumulative frequency. The red line represents anti-GITR/anti-PD1 treatment; the black line represents isotype antibody treatment; blue line represents treatment with anti-GITR antibody; and the purple line represents anti-PD1 antibody treatment.

In FIG. 17 is a Western blot showing the relative expression of phospho-CD3ζ and phospho-CD226 as a function of PD-1 concentration.

In FIG. 18 panels A-D depict bar graphs of a range of T cell types obtained from wild-type (open bars) and CD226 knockout mice (hatched bars). Panel A is a bar graph depicting a FACS analysis of T-5 039583 cell development in the thymus (Tconv, conventional T cells; DP, CD4/CD8 double positive; SP, single positive; DN, CD4/CD8 double negative ). Panel B is a bar graph depicting a FACS (cell array) validation of the T cell subgroup population in spleen and blood from wild-type and CD226-/- animals. Panel C is a bar graph depicting a FACS analysis (MFI, Mean Fluorescence Intensity) of subsets of T cells in spleen and blood that express PD1. Panel D is a bar graph depicting a FACS analysis (MFI) of subgroups of T cells in the spleen and blood that express GITR. Panels E-I are bar graphs depicting inflammatory cytokine secretion in picograms per milliliter upon ex vivo TCR stimulation of splenocytes with anti-CD3+ anti-CD28 Ab for 16 h. Splenocytes from CD226-/- mice (shaded bars) or wild-type mice ( WT) (open bars) were stimulated with anti-CD3+ anti-CD28 Ab for 16 h. Panel E=IFN-y; panel F=IL-2; panel G=TNF-a; panel H=IL-6 and panel I=IL-5.

In FIG. 19 is a line graph plotting the percentage of surviving animals as a function of time in days after tumor challenge. Panel A shows CD226 KO mice (pink lines) or WT littermates (black lines) infected with MC38 tumor cells and treated with either anti-GITR Ab + anti-PD-1 Ab (solid circles and squares) or isotype Abs (open circles and squares) . Panels B-D show the effect of antibody treatment in (B) animals treated with an antibody that blocks CD28 signaling (10 mg/kg CTLA-4-Ig; panel B, green lines); (C) animals treated with an antibody that blocks OX40 signaling (10 mg/kg OX40L blocking antibody; panel C); and (D) animals treated with an antibody that blocks 4-1BB signaling (10 mg/kg 4-1BBL blocking antibody; panel D).

In FIG. 20 is a line graph depicting tumor size (in cubic millimeters) as a function of days post-tumor challenge for mice treated with isotype antibody (open circles and black line), anti-PD1 antibody (open squares, red line), anti-GITR antibody (open bars). triangles and green line) and anti-GITR/anti-PD1 combination therapy (open inverted triangles and blue lines). Panel A represents MC38 tumors not expressing CD155. Panel B represents MC38 tumors expressing CD155.

In FIG. 21 is a bar graph depicting a range of cells expressing CD226 (panel A), 4-1BB (panel B) and IFN-γ (panel C) from animals infected with MC38 tumor cells overexpressing CD155 (hatched bars) and MC38 cells , non-expressing CD155 (open bars).

In FIG. 22 is a dot plot of RNA-seq analysis of tumor biopsies from cancer patients demonstrating CD226 RNA expression (in log2[RPKM]) as a function of anti-PD-1 Ab treatment.

Detailed disclosure of the present invention

Before describing the present invention, it should be mentioned that the present invention is not limited to the specific methods and experimental conditions described, and therefore the methods and conditions may vary. It should also be appreciated that the terminology used herein is for the sole purpose of describing particular embodiments and is not intended to be limiting, as the scope of the present invention will only be limited by the appended claims.

Unless otherwise indicated, all technical and scientific terms used in this document have the same meaning as is conventionally understood by an ordinary specialist in the field to which the present invention pertains. As used herein, approximately, when used with respect to the particular numerical value mentioned, means that the value may vary from said value by no more than 1%. For example, the expression approximately 100 as used herein includes 99 and 101 and all values in between (eg, 99.1, 99.2, 99.3, 99.4, etc.).

Although any methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention, the following describes the preferred methods and materials. All patents, applications and non-patent publications mentioned in the present description are incorporated herein by reference in their entirety.

Definitions

As used herein, the expression glucocorticoid inducible tumor necrosis factor receptor, GITR, and the like. refers to human glucocorticoid-inducible tumor necrosis factor receptor comprising the amino acid sequence set forth in SEQ ID NO: 413 (NCBI Accession No. NP_004186.1). The GITR expression includes both monomeric and multimeric GITR molecules. As used herein, monomeric human GITR means the GITR protein, or portion thereof, that does not contain or lack any

- 6 039583 timerizing domains and which exist under normal conditions as a single GITR molecule without a direct physical connection to another GITR molecule. An exemplary monomeric GITR molecule is referred to herein as hGITR.mmh, containing the amino acid sequence of SEQ ID NO: 409 (see, for example, Example 3 herein). As used herein, dimeric human GITR refers to a construct containing two GITR molecules connected to each other via a linker, covalent bond, non-covalent bond, or via a multimerizing domain such as the Fc domain of an antibody. Exemplary dimeric GITR molecules include those referred to herein as hGITR.mFc and hGITR.hFc, containing the amino acid sequence of SEQ ID NO: 410 and SEQ ID NO: 411, respectively (see, for example, Example 3 herein ).

All references to proteins, polypeptides, and protein fragments herein are intended to refer to the human version of the corresponding protein, polypeptide, or protein fragment, unless explicitly stated to be of a non-human species. Thus, the expression GITR means human GITR, unless it is explicitly stated that it refers to a non-human species, such as mouse GITR, monkey GITR, etc.

As used herein, cell surface-expressed GITR refers to one or more GITR proteins, or their extracellular domain, that are expressed on the cell surface in vitro or in vivo such that at least a portion of the GITR protein is exposed to the extracellular side of the cell membrane and antigen- binding portion of the antibody. Cell surface expressed GITR may comprise or consist of a GITR protein expressed on a cell surface that normally expresses the GITR protein. Alternatively, cell surface-expressed GITR may comprise or consist of a GITR protein expressed on the surface of a cell that does not normally express human GITR on its surface but has been artificially engineered to express GITR on its surface.

As used herein, the term anti-GITR antibody includes both monovalent and monospecific bivalent antibodies with the same specificity, as well as bispecific antibodies containing a first arm that binds GITR and a second arm that binds a second (target) antigen, wherein the antibody arm against GITR contains any of the sequences HCVR/LCVR or CDR set forth in table. 1 in this document. The term anti-GITR antibody also includes antibody-drug conjugates (ADCs) containing an anti-GITR antibody, or an antigen-binding portion thereof, conjugated to a drug or toxin (ie, a cytotoxic agent). The term anti-GITR antibody also includes antibody-radionuclide conjugates (ARCs) containing an anti-GITR antibody, or an antigen-binding portion thereof, conjugated to a radionuclide.

As used herein, the term antibody refers to any antigen-binding molecule or molecular complex containing at least one complementarity determining region (CDR) that specifically binds to or interacts with a particular antigen (eg, GITR). The term antibody includes immunoglobulin molecules containing four polypeptide chains, two heavy (H) chains and two light (L) chains connected by disulfide bonds, as well as their multimers (eg, IgM). Each heavy chain contains a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region contains three CH1, C _H 2 and C _H 3 domains. Each light chain contains a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region contains one domain (CL1). The V _H and V _L regions can be further divided into regions of hypervariability, called complementarity-determining regions (CDRs), interspersed with regions that are more conserved, called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from amino to carboxy in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. According to other embodiments of the present invention, the FR of an anti-GITR antibody (or an antigen-binding portion thereof) may be identical to human germline sequences, or may be naturally or artificially modified. Amino acid consensus sequence can be determined based on the simultaneous analysis of two or more CDRs.

As used herein, the term antibody also includes antigen-binding fragments of full length antibody molecules. As used herein, the terms antigen-binding portion of an antibody, antigen-binding fragment of an antibody, and the like. provide for a naturally occurring, enzymatically produced, synthetic or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Antigen-binding fragments of an antibody can be obtained, for example, from full-length antibody molecules using any suitable standard techniques, such as proteolytic cleavage or genetic engineering techniques to create recombinant molecules, involving the manipulation and expression of DNA encoding

- 7 039583 variable and optionally constant domains of an antibody. Such DNA is known and/or readily available, for example from commercial sources, DNA libraries (including, for example, libraries of antibodies in phages), or it can be synthesized. DNA can be sequenced and chemically manipulated or manipulated using molecular biology techniques, for example, to bring 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. d.

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

The antigen-binding fragment of an antibody typically contains at least one variable domain. The variable domain may be of any size or amino acid composition and typically contains at least one CDR region that is adjacent to or in-frame with one or more framework sequences. In antigen-binding fragments in which a VH domain is linked to a V _L domain, the VH and V _L domains may be arranged relative to each other in any suitable order. For example, the variable region may be dimeric and contain VH-VH, VH-VL, or VL-VL dimers. Alternatively, an antigen-binding fragment of an antibody may contain a VH or VL monomeric domain.

In some embodiments, an antigen-binding fragment of an antibody may comprise at least one variable domain covalently linked to at least one constant domain. Non-limiting typical configurations of variable and constant domains that can be found in the antigen-binding fragment of the antibody in accordance with the present invention include (i) V _{H -CH} 1; (ii) VH-CH2; (iii) VH-CH3; (iv) VH-CH1-CH2; (v) V _H Ch1-Ch2-Ch3; (vi) Vh-Ch2-Ch3; (vii) Vh-Cl; (viii) Vl-Ch1; (ix) Vl-Ch2; (x) Vl-Ch3; (xi) Vl-Ch1-Ch2; (xii) V _l -C _h 1-C _h 2-C _h 3; (xiii) V _L -C _H 2 -C _H 3 and (xiv) V _L -C _L . In any configuration of the variable and constant domains, including any of the exemplary configurations above, the variable and constant domains may either be directly linked to each other, or may be linked by a complete or partial hinge or linker region. The hinge region may be composed of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, resulting in a flexible or semi-flexible bond between adjacent variable and/or constant domains in a single polypeptide molecule. In addition, the antigen-binding fragment of an antibody according to the present invention may comprise a homodimer or heterodimer (or other multimer) of any of the variable and constant domain configurations listed above in non-covalent association with each other and/or with one or more monomeric domains in in accordance with the present invention (for example, due to the disulfide bond(s)).

Like full-length antibody molecules, antigen-binding fragments can be monospecific or multispecific (eg, bispecific). A multispecific antigen-binding fragment of an antibody typically contains at least two different variable domains, with each variable domain being able to specifically bind to a different antigen or to different epitopes on the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, can be adapted for use in the context of an antigen-binding fragment of an antibody of the present invention using conventional techniques available in the art.

Antibodies in accordance with the present invention can function through complement-dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC). The term complement-dependent cytotoxicity (CDC) refers to the lysis of antigen-expressing cells by an antibody of the present invention in the presence of complement. The term antibody-dependent cell-mediated cytotoxicity (ADCC) refers to a cell-mediated response in which non-specific cytotoxic cells expressing Fc receptors (FcRs) (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize the bound antibody on the surface of the target cell and thereby lead to lysis of the target cell. CDC and ADCC can be determined using assays that are well known and available in the art. (See, for example, US Pat. The constant region of an antibody is important for the ability of an antibody to fix complement and mediate cell-dependent cytotoxicity.

- 8 039583

Thus, the isotype of an antibody can be selected based on whether it is desirable for the antibody to mediate cytotoxicity.

In some embodiments, the anti-GITR antibodies of the present invention are human antibodies. As used herein, the term human antibody includes antibodies having variable and constant regions derived from human immunoglobulin germline sequences. Human antibodies of the present invention may include amino acid residues not encoded by human immunoglobulin germline sequences (e.g., by mutations introduced by in vitro random or site-directed mutagenesis or by in vivo somatic mutation), such as in CDRs and , in particular in CDR3. However, as used herein, the term human antibody should not include antibodies in which CDR sequences derived from germline sequences from other mammalian species, such as mice, have been grafted onto human framework sequences. The term human antibody does not include naturally occurring molecules that normally exist without modification or human intervention/manipulation in a naturally occurring, unmodified living organism.

The antibodies of the present invention, in some embodiments, may be recombinant human antibodies. As used herein, the term recombinant human antibody shall encompass all human antibodies produced, expressed, generated, or isolated by recombinant methods, such as antibodies expressed using a recombinant expression vector transfected into a host cell (described below), antibodies isolated from a recombinant a combinatorial library of human antibodies (described below), antibodies derived from animals (e.g., mice) that are transgenic for human immunoglobulin genes (see, e.g., Taylor et al. (1992) Nucl. Acids Res. 20: 6287-6295) , or antibodies produced, expressed, generated, or isolated by any other method that involves splicing human immunoglobulin gene sequences with other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human immunoglobulin germline sequences. In some embodiments, however, such recombinant human antibodies are subjected to in vitro mutagenesis (or, if an animal transgenic for human Ig sequences is used, to somatic mutagenesis in vivo), and thus the amino acid sequences of the VH and V _L regions of the recombinant antibodies are sequences , which, although derived from and related to human germline VH and V _L sequences, cannot exist naturally in the in vivo human germline antibody repertoire.

Human antibodies can exist in two forms that are associated with hinge heterogeneity. In one form, the immunoglobulin molecule contains a suitable four chain construct of approximately 150-160 kDa, in which the dimers are held together by an interchain heavy chain disulfide bond. In the second form, the dimers do not bind via interchain disulfide bonds, and a molecule of approximately 75-80 kDa is formed, consisting of covalently connected light and heavy chains (half-antibody). These forms were extremely difficult to separate, even after affine purification.

The frequency of appearance of the second form in various intact IgG isotypes is determined, without limitation, by structural differences associated with the isotype of the hinge region of the antibody. A single amino acid substitution at the human IgG4 hinge region can significantly reduce the appearance of the second form (Angal et al. (1993) Molecular Immunology 30:105) to levels typically seen with the human IgG1 hinge. The present invention encompasses antibodies having one or more mutations in the hinge, the CH2 or CH3 region of which may be desired, for example, upon preparation, to increase the yield of the desired antibody form.

The antibodies of the present invention may be isolated antibodies. As used herein, the term isolated antibody means an antibody that has been identified and isolated and/or separated from at least one component of its natural environment. For example, an antibody that has been isolated or separated from at least one component of an organism, tissue, or cell in which the antibody occurs naturally or is naturally produced is an isolated antibody within the scope of the present invention. An isolated antibody also includes an antibody in situ in a recombinant cell. Isolated antibodies are antibodies that have been subjected to at least one purification or isolation step. In some embodiments, the isolated antibody may be substantially free of other cellular material and/or other chemicals.

The anti-GITR antibodies disclosed herein may contain one or more amino acid substitutions, insertions and/or deletions in the framework and/or CDR regions of the heavy and light chain variable domains compared to the corresponding germline sequences,

- 9 039583 from which these antibodies are derived. Such mutations can be readily identified by comparing the amino acid sequences disclosed herein with germline sequences available, for example, from publicly available antibody sequence databases. The present invention relates to antibodies and antigen-binding fragments thereof, which are derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids in one or more framework and/or CDR regions are mutated at the corresponding residue(s) of the sequence the germline from which the antibody was derived, or at the corresponding residue(s) of another human germline sequence, or at a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are collectively referred to herein as germline mutations). One of ordinary skill in the art, based on the heavy and light chain variable region sequences disclosed herein, can easily generate numerous antibodies and antigen-binding fragments that contain one or more individual germline mutations, or combinations thereof. In some embodiments, all framework and/or CDR residues in the VH and/or V _L domains are mutated back to residues found in the original germline sequence from which the antibody was derived. In other embodiments, only certain residues are mutated back to the original germline sequence, such as only mutated residues found in the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or mutated residues found only in CDR1, CDR2, or CDR3. In other embodiments, one or more framework and/or CDR residues are mutated into corresponding residues of a different germline sequence (ie, a germline sequence that differs from the germline sequence from which the antibody was originally derived). However, antibodies of the present invention may contain any combination of two or more germline mutations in the framework and/or CDR regions, in which, for example, certain individual residues are mutated into the corresponding residue of a particular germline sequence, while how some other residues that differ from the original germline sequence are retained or mutated into the corresponding residue of a different germline sequence. Once an antibody and antigen-binding fragments that contain one or more germline mutations have been produced, they can be easily tested for one or more desired properties, such as improved binding specificity, increased binding affinity, improved or increased antagonistic or agonistic biological properties (with appropriate circumstances), reduced immunogenicity, etc. The present invention relates to antibodies and antigen-binding fragments, which are generally obtained in this way.

The present invention also provides anti-GITR antibodies containing 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 relates to anti-GITR antibodies having the amino acid sequences HCVR, LCVR and/or CDR, eg, 10 or less, 8 or less, 6 or less, 4 or less, etc. conservative amino acid substitutions relative to any of the amino acid sequences of HCVR, LCVR and/or CDR set forth in table. 1 in this document.

The term epitope refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule known as a paratope. One antigen may have more than one epitope. Thus, different antibodies may bind to different sites on the antigen and may have different biological effects. Epitopes can be either conformational or linear. A conformational epitope is produced by spatially aligned amino acids from different segments of a linear polypeptide chain. A linear epitope is an epitope formed by adjacent amino acid residues in a polypeptide chain. In some cases, the epitope may include fragments of saccharides, phosphoryl groups or sulfonyl groups on the antigen.

The term substantial identity or substantially identical with respect to a nucleic acid or fragment thereof indicates that, when optimally aligned by appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity for at least about 90 %, and more preferably at least about 95%, 96%, 97%, 98%, or 99% nucleotide bases as measured by any well-known sequence identity algorithm such as FASTA, BLAST, or GAP, as discussed below. A nucleic acid molecule that is substantially identical to a reference nucleic acid molecule may, in certain instances, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid.

- 10 039583 nucleic acid molecule.

When applied to polypeptides, the term substantial similarity or substantially similar means that two peptide sequences, when optimally aligned, such as by the GAP or BESTFIT programs using default gap penalties, have at least 95% sequence identity, even more preferably at least 98% or 99% sequence identity. Preferably, positions of residues that are not identical are distinguished by conservative amino acid substitutions. The term conservative amino acid substitution means a substitution in which an amino acid residue is replaced with another amino acid residue having a side chain (R group) with similar chemical properties (eg, charge or hydrophobicity). In general, a conservative amino acid substitution will hardly change the functional properties of the protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of similarity can be adjusted to the appropriately conservative substitution. Methods for making such adjustments are well known to those skilled in the art. See, for example, Pearson (1994) Methods Mol Biol. 24:307-331. 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) aliphatic-hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine and tryptophan; 5) main side chains: lysine, arginine and histidine; 6) acid side chains: aspartate and glutamate; and 7) sulfur-containing side chains: cysteine and methionine. Preferred groups of conservative amino acid substitutions are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine. Alternatively, a conservative substitution is any change that has a positive value in the PAM250 log-likelihood matrix disclosed by Gonnet et al. (1992) Science 256: 1443 45, incorporated herein by reference. The term moderately conservative substitution means any change that has a non-negative value in the PAM250 log-likelihood matrix.

Sequence similarity for polypeptides, also referred to as sequence identity, is commonly measured using sequence analysis software. Protein analysis software matches similar sequences by measuring the similarity assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, the GCG software contains programs such as GAP and BESTFIT that can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species, or between a wild-type protein and its mutein. See, for example, GCG version 6.1. Polypeptide sequences can also be compared using FASTA using the default parameters or recommended parameters, the program in GCG version 6.1. FASTA (eg, FASTA2 and FASTA3) provides alignments and percent sequence identity for regions with the best match between query and search sequences (Pearson (2000), supra). Another preferred algorithm for comparing a sequence in accordance with the present invention with a database containing a large number of sequences from different organisms is the BLAST computer program, especially BLASTP or TBLASTN, using default parameters. See, for example, Altschul et al. (1990) J. Mol. Biol. 215:403-410, and Altschul et al. (1997) Nucleic Acids Res. 25:3389-402, each of which is incorporated herein by reference.

Anti-GITR antibodies containing Fc variants

In some embodiments, the present invention provides anti-GITR antibodies comprising an Fc domain containing one or more mutations that increase or decrease antibody binding to an FcRn receptor, such as at acidic versus neutral pH. For example, the present invention relates to anti-GITR antibodies containing a mutation in the CH2 or CH3 region of an Fc domain, wherein the mutation(s) increases the affinity of the Fc domain for FcRn in an acidic environment (e.g., in an endosome where the pH is in the range of about 5 .5 to about 6.0). Such mutations can lead to an increase in the serum half-life of the antibody when administered to an animal. Non-limiting examples of such Fc modifications include, for example, a modification at position 250 (eg, E or Q); 250 and 428 (for example, L or F); 252 (for example, L/Y/F/W or T); 254 (for example, S or T) and 256 (for example, S/R/Q/E/D or T), or modification at position 428, and/or 433 (for example, H/L/R/S/P/Q or K), and/or 434 (eg H/F or Y); or a modification at position 250 and/or 428; or a modification at position 307 or 308 (eg, 308F, V308F) and 434. In one embodiment, the modification includes modification 428L (eg, M428L) and 434S (eg, N434S); modification 428L, 259I (for example, V 59I) and 308F (for example, V308F); modification 433K (for example, H433K) and 434 (for example, 434Y); modification 252, 254 and 256 (for example, 252Y, 254T and 256E); modification 250Q and 428L (for example, T250Q and

M428L), as well as modification 307 and / or 308 (for example, 308F or 308P).

For example, the present invention relates to antibodies against GITR containing an Fc domain containing one or more pairs or groups of mutations selected from the group consisting of 250Q and 248L (eg, T250Q and M248L); 252Y, 254T and 256E (eg M252Y, S254T and T256E); 428L and 434S (eg M428L and N434S); as well as 433K and 434F (for example, H433K and N434F). All possible combinations of the above Fc domain mutations, as well as other mutations in the variable domains of the antibodies disclosed herein, are within the scope of the present invention.

Biological characteristics of antibodies against GITR

The present invention relates to antibodies and antigen-binding fragments thereof that bind monomeric human GITR with high affinity. For example, the present invention relates to 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 37°C, e.g., using the assay format defined in example 3 in this document, or the essence of a similar analysis. In some embodiments, anti-GITR antibodies are provided that bind monomeric human GITR at 37° C. with a K _D 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 with using surface plasmon resonance, for example, using the analysis format defined in example 3 herein, or essentially a similar analysis.

The present invention also relates to antibodies and antigen-binding fragments thereof that bind monomeric human GITR (e.g., hGITR.mmh) with a dissociative half-life (t ¹ /2) of greater than about 12 minutes, as measured by surface plasmon resonance at 37 °C, for example, using the analysis format defined in Example 3 herein, or essentially a similar analysis. In some embodiments, anti-GITR antibodies are provided that bind monomeric human GITR at 37° C. with a t ¹ /2 of greater than about 12 minutes, greater than about 13 minutes, greater than about 14 minutes, greater than about 15 minutes, or greater, as measured using surface plasmon resonance, for example, using the analysis format defined in Example 3 herein, or essentially a similar analysis.

The present invention also relates to antibodies and antigen-binding fragments thereof that bind dimeric human GITR (eg, hGITR.mFc) with high affinity. For example, the present invention relates to anti-GITR antibodies that bind dimeric human GITR with a K _D of less than about 950 pM, as measured by surface plasmon resonance at 37°C, for example, using the assay format defined in Example 3 herein. , or essentially a similar analysis. In some embodiments, anti-GITR antibodies are provided that bind dimeric human GITR at 37° C. with a K _D 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 resonance, for example, using the assay format defined in Example 3 in this document, or on the substance of a similar analysis.

The present invention also provides antibodies and antigen-binding fragments thereof that bind dimeric human GITR (e.g., hGITR.mFc) with a dissociative half-life (t ¹ /2) of greater than about 7 minutes, as measured by surface plasmon resonance at 37 °C, for example using the assay format defined in Example 3 herein, or essentially a similar assay. In some embodiments, anti-GITR antibodies are provided that bind dimeric human GITR at 37°C with a t ¹ /2 of greater than about 10 minutes, greater than about 20 minutes, greater than about 30 minutes, greater than about 40 minutes, greater than about 50 minutes, more than about 60 minutes, more than about 70 minutes, more than about 80 minutes, more than about 90 minutes, more than about 100 minutes or more, as measured by surface plasmon resonance, for example, using the analysis format, defined in example 3 herein, or as such analysis.

The present disclosure also relates to antibodies and antigen-binding fragments thereof that bind cell surface expressed GITR. For example, provided herein are antibodies that bind human GITR-transfected embryonic kidney 293 (HEK-293) D9 cells with high affinity. For example, the present disclosure relates to anti-GITR antibodies that bind human GITR-transfected 293 (HEK-293) D9 embryonic kidney cells with an EC _{50 of} less than about 260 pM as measured by electrochemiluminescence, e.g., using the assay format defined in Example 4 in

- 12 039583 of this document, or essentially a similar analysis. In some embodiments, anti-GITR antibodies are provided that bind human GITR-transfected embryonic kidney 293 (HEK-293) D9 cells with an EC _{50 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 electrochemiluminescence, for example, using the assay format defined in Example 4 herein, or essentially a similar assay.

Antibodies in accordance with the present invention may have one or more of the biological characteristics mentioned above, or any combination thereof. The above list of biological characteristics of antibodies in accordance with the present invention is not exhaustive. Other biological characteristics of antibodies in accordance with the present invention will become clear to one of ordinary skill in the art upon consideration of the present disclosure, including the working examples herein.

Anchoring-Dependent Fc and Anchoring-Independent GITR Activation and GITRL Blocking

The present disclosure relates to antibodies and antigen-binding fragments thereof that activate human GITR, for example, as defined in the assay formats described in Example 5 and/or Example 6 herein, or a substantially similar assay format. As used herein, the expression activates human GITR refers to the activation of GITR by binding to its cognate ligand, the GITR ligand (GITRL), or to the binding of anti-GITR agonistic antigen-binding protein(s) to GITR. With respect to GITR activation by anti-GITR agonist antigen-binding proteins, activation can occur in the presence or absence of anchoring of the antigen-binding protein to Fc-gamma receptors. Activation of human GITR is expressed in the manifestation of certain biological activities, including, without limitation, inducing or enhancing GITR signaling in vitro or in vivo, reducing regulatory T cell suppression of effector T cell activity; decrease in levels of circulating regulatory T cells in vitro or in vivo, decrease in intratumoral regulatory T cells in vivo, activation of effector T cells in vitro or in vivo, induction or increase in proliferation of effector T cells in vitro or in vivo, or inhibition or reduction tumor growth in vivo.

GITR activation in the absence of Fc anchoring

In some embodiments, the antibodies and antigen-binding fragments provided herein activate human GITR in the absence of Fc anchoring, e.g., as defined in the assay formats described in Example 5 and/or Example 6 herein, or as such similar analysis format. As used herein, in the absence of Fc anchoring refers to GITR activation and GITR-mediated signaling or blocking of GITRL without clustering of anti-GITR antibodies by various forms of the Fc gamma receptor, and this can be determined and quantified by, for example, primary T cell activation, co-cultured in vitro in the absence of cell surface-bound Fc-gamma receptor(s). In some embodiments, the antibody, or antigen-binding fragment thereof, activates human GITR at an activation percentage greater than about 25% at an EC _{50 of} less than about 3 nM in the absence of Fc anchoring, as determined by an NFkB reporter assay, e.g., as described in Example 5, or essentially a similar format of analysis. In some embodiments, the antibody, or antigen-binding fragment thereof, activates human GITR at an activation percentage of greater than about 30%, greater than about 40%, greater than about 50%, greater than about 60%, or greater than about 65% with an EC _{50 of} less than about 3 nM in the absence of Fc anchoring as determined by an NFkB reporter assay, eg as described in Example 5, or a substantially similar assay format. In some embodiments, the antibody or antigen-binding fragment thereof activates human GITR at an activation percentage of greater than about 30%, greater than about 40%, greater than about 50%, greater than about 60%, or greater than about 65% with an EC _{50 of} less than than about 2 nM in the absence of Fc anchoring as determined by an NFkB reporter assay, eg as described in Example 5, or a substantially similar assay format. In some embodiments, the antibody, or antigen-binding fragment thereof, activates human GITR at an activation percentage of greater than about 30%, greater than about 40%, greater than about 50%, greater than about 60%, or greater than about 65% with an EC _{50 of} less than than about 1.5 nM in the absence of Fc anchoring as determined by an NFkB reporter assay, eg as described in Example 5, or a substantially similar assay format. In some embodiments, the antibody, or antigen-binding fragment thereof, activates human GITR at an activation percentage of greater than about 30%, greater than about 40%, greater than about 50%, greater than about 60%, or greater than about 65% with an EC50 of less than approximately 1.4 nM in the absence of Fc anchoring, as determined by ana

- 13 039583 lysis against the NFkB reporter, for example, as described in Example 5, or a substantially similar assay format. In some embodiments, the antibody or antigen-binding fragment thereof activates human GITR at an activation percentage of greater than about 30%, greater than about 40%, greater than about 50%, greater than about 60%, or greater than about 65% with an EC _{50 of} less than than about 1.3 nM in the absence of Fc anchoring as determined by an NFkB reporter assay, eg as described in Example 5, or a 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 a naïve human CD4+ T cell proliferation assay, e.g., as described in Example 6, or substantially similar. analysis 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 with an EC ₅₀ of about 8 nM or less, as determined by a naïve human CD4+ T cell proliferation assay, e.g., as described in example 6, or essentially a similar analysis 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 times higher than the original, at a concentration of approximately 22 nm of antibody (or antigen- binding fragment) as determined by a naïve human CD4+ T cell proliferation assay, eg as described in Example 6, or a substantially similar assay format.

GITR activation in the presence of Fc anchoring

In some embodiments, the antibodies or antigen-binding fragments provided herein activate human GITR in the presence of Fc anchoring, e.g., as defined in the assay formats described in Example 5 and/or Example 6 herein, or as such similar analysis format. As used herein, in the presence of Fc anchoring refers to activation of GITR and GITR mediated signaling or blocking of GITRL through clustering of anti-GITR antibodies by interaction of the antibody Fc region with different forms of the Fc gamma receptor (FcgR), such as FcgRI, FcgRIIa or FcgRIIIa, and this can be detected and quantified by, for example, activation of T cells co-cultured in vitro in the presence of cell surface bound Fcgamma receptor(s).

In some embodiments, the antibody or antigen-binding fragment thereof exhibits T-cell proliferative activity in the presence of at least about 2-fold Fc anchoring at a concentration of about 33 nM of the antibody (or antibody-binding fragment) as determined by naïve human CD4+ T cell proliferation assay, for example as described in Example 6, or a substantially similar assay format. In some embodiments, the antibody or antigen-binding fragment thereof exhibits T cell proliferative activity in the presence of Fc anchoring at least about 2-fold, at least about 3-fold, at least about 4-fold, or at least approximately 5 times baseline at a concentration of approximately 33 nM antibody (or antigen binding fragment) as determined by a naïve human CD4+ T cell proliferation assay, e.g., as described in Example 6, or a substantially 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 EC _{50 of} less than about 34 nM, as determined by a naïve human CD4+ T cell proliferation assay, e.g., as described in Example 6, or essentially a similar analysis format. In some embodiments, the antibody or antigen binding fragment exhibits T cell proliferative activity in the presence of Fc anchoring with an EC _{50 of} less than about 30 nM, less than about 20 nM, less than about 10 nM, less than about 5 nM, or less than about 4 nM as determined by a naïve human CD4+ T cell proliferation assay, eg as described in Example 6, or a substantially similar assay format.

Antibodies that block GITR ligand-mediated stimulation of the receptor

The present disclosure relates to antibodies that block human GITR ligand (hGITRL) mediated receptor stimulation, for example, as defined in the assay format described in Example 5 herein. As used herein, the expression blocks human GITR ligand (hGITRL) mediated receptor stimulation refers to the ability of antigen-binding proteins against GITR to block GITR from binding to its cognate ligand GITRL. Blocking the GITR ligand can interfere with the downregulation of effector T cells by regulatory T cells. GITR ligand blocking can be detected and quantified using a number of methods known in the art, including, for example, decreased T cell proliferation or cytokine secretion and increased 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, for example, as defined in the assay format described in Example 5 herein.

In some embodiments, the antibody, or antibody-binding fragment thereof, blocks human GITR ligand-mediated receptor stimulation in the absence of Fc anchoring with a percent blocking greater than about 55% with an IC _{50 of} less than about 4.0 nM, as determined by an NFkB reporter assay, for example, as described in example 5, or essentially a similar analysis format. In some embodiments, the antibody, or antibody-binding fragment thereof, blocks human GITR ligand-mediated receptor stimulation in the absence of Fc anchoring with a percent blocking greater than about 60%, greater than about 70%, greater than about 80%, or greater than about 85% with IC ₅₀ 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 an NFkB reporter assay, eg as described in Example 5, or a substantially similar assay format.

In some embodiments, antibodies or antigen-binding fragments activate human GITR and block human GITR ligand-mediated stimulation of the receptor at a blocking percentage of less than about 25% in the absence of Fc anchoring, as determined by an NFkB reporter assay, e.g., in the assay described in Example 5 or a substantially similar analysis. In some embodiments, antibodies or antigen-binding fragments activate human GITR and block human GITR ligand-mediated receptor stimulation at a percent blockage of less than about 54% in the absence of Fc anchoring, as determined by an NFkB reporter assay, e.g., in the assay described in Example 5 or essentially similar analysis. In some embodiments, antibodies or antigen-binding fragments activate human GITR and block human GITR ligand-mediated stimulation of the receptor at a blocking percentage of 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 an NFkB reporter assay, such as in the assay described in Example 5 or a substantially similar assay. In some embodiments, antibodies or antigen-binding fragments activate human GITR at an activation percentage of at least about 50% and do not block hGITRL-mediated receptor stimulation at a percentage of blocking greater than about 50% in the absence of Fc anchoring, as determined by an NFkB reporter assay. , for example, in the analysis described in example 5 or essentially similar analysis.

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

In some embodiments, antibodies both activate human GITR and block human GITR ligand (hGITRL)-mediated receptor stimulation in the absence of Fc anchoring, e.g., as defined in the assay format described in Example 5 herein, or in a substantially similar assay. In some embodiments, (A) the antibody or antigen-binding fragment has at least one of the properties selected from the group consisting of

i) human GITR activation in the absence of Fc anchoring at an activation percentage of greater than about 25% with an EC _{50 of} less than about 3 nM as determined by NFkB reporter assay; and ii) human GITR activation in the absence of Fc anchoring with an EC _{50 of} less than about 1.0 nM as determined by NFkB reporter assay; and (B) the antibody or antigen-binding fragment blocks hGITRL-mediated receptor stimulation in the absence of Fc anchoring at a percentage of block greater than about 54% with an IC _{50 of} less than about 4.0 nM as determined by an NFkB reporter assay.

In some embodiments, (A) the antibody or antigen-binding fragment activates human GITR in the absence of Fc anchoring at an activation percentage greater than about 50% with an EC ₅₀ of less than about

- 15 039583 exactly 1.5 nM as determined by NFkB reporter assay; and (B) the antibody or antigen-binding fragment blocks hGITRL-mediated receptor stimulation in the absence of Fc anchoring at a percentage of block greater than about 54% c

IC ₅₀ less than about 4.0 nM as determined by NFkB reporter assay.

Epitope mapping and related technologies

The epitope to which the antibodies 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 the GITR protein. Alternatively, an epitope may be composed of a plurality of non-conjugated amino acids (or amino acid sequences) of GITR. In some embodiments, the epitope is located on or near the GITRL binding domain of GITR. In other embodiments, the epitope is located outside the GITRL binding domain of the GITR, for example, at a position on the surface of the GITR where the antibody, when bound to such an epitope, does not interfere with the binding of GITRL to the GITR.

Various techniques known to those of ordinary skill in the art can be used to determine whether an antibody interacts with one or more amino acids in a polypeptide or protein. Typical techniques include, for example, routine cross-blocking assays such as those described in Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY), alanine scan mutation analysis, peptide blotting (Reineke, 2004 , Methods Mol Biol 248:443-463) and peptide digestion analysis. In addition, methods such as epitope exclusion, epitope extraction, and chemical modification of antigens can be used (Tomer, 2000, Protein Science 9:487-496). Another method that can be used to identify the amino acids in the polypeptide with which the antibody interacts is hydrogen-deuterium exchange, detected using mass spectrometry. In general terms, the hydrogen-deuterium exchange method involves labeling a protein of interest with deuterium and then binding the antibody to the deuterium labeled protein. The protein/antibody complex is then transferred to water, allowing hydrogen-deuterium exchange to occur on all residues except for antibody-protected residues (which remain labeled with deuterium). After dissociation of the antibody, the target protein is subjected to protease digestion and mass spectrometric analysis, thereby identifying deuterium-labeled residues corresponding to the specific amino acids with which the antibody interacts. See, for example, Ehring (1999) Analytical Biochemistry 267(2):252259; Engen and Smith (2001) Anal. Chem. 73:256A-265A.

The present invention further provides 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 set forth in Table 1 herein ). Similarly, the present invention also provides anti-GITR antibodies that compete for GITR binding with any of the specific exemplary antibodies described herein (eg, antibodies comprising any of the amino acid sequences set forth in Table 1 herein).

Whether an antibody binds to or competes for the same epitope with a reference anti-GITR antibody can be readily determined using routine methods known in the art and demonstrated herein. For example, to determine if a test antibody binds to the same epitope as a reference anti-GITR antibody, the present invention provides binding of the reference antibody to the GITR protein. The ability of the test antibody to bind to the GITR molecule is then evaluated. If the test antibody is able to bind to GITR after saturation with the reference anti-GITR antibody, then it can be concluded that the test antibody binds to a different epitope than the reference anti-GITR antibody. On the other hand, if the test antibody is unable to bind to the GITR molecule after saturation with the reference anti-GITR antibody, then the test antibody can bind to the same epitope as the epitope bound by the reference anti-GITR antibody of the present invention. Additional routine experimentation (e.g., peptide mutation and binding assays) can then be performed to confirm whether the observed lack of binding of the test antibody is due to binding to the same epitope as the reference antibody, or whether spatial blocking (or other) is responsible for the observed lack of binding. phenomenon). Experiments of this kind can be performed using ELISA, RIA, Biacore, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art. According to some embodiments of the present invention, two antibodies bind to the same (or overlapping) epitope if, for example, a 1-, 5-, 10-, 20-, or 100-fold excess of one antibody inhibits binding of the other by at least 50 %, but preferably 75%, 90% or even 99% as measured in a competitive binding assay (see, for example, Junghans et al., Cancer Res. 1990:50:1495-1502). Alternatively, two antibodies are considered to bind to the same epitope if essentially all amino acid mutations in the antigen that reduce or abolish the binding of one antibody reduce or abolish the binding of the other. Two antibodies are considered to have overlapping epitopes if only a subset of amino acid mutations that reduce or eliminate the binding of one antibody reduce or eliminate the binding of the other.

To determine if an antibody competes for binding (or cross-competes for binding) with a reference anti-GITR antibody, the binding determination methods described above are performed in two directions. In the first direction, binding of the reference antibody to the GITR protein is provided under saturating conditions, followed by evaluation of the binding of the test antibody to the GITR molecule. In the second direction, the test antibody is allowed to bind to the GITR molecule under saturating conditions, followed by evaluation of the binding of the reference antibody to the GITR molecule. If in both directions only the first (saturating) antibody is able to bind to the GITR molecule, then it is concluded that the test antibody and the reference antibody compete for binding to the GITR. One of ordinary skill in the art will appreciate that an antibody that competes for binding with a reference antibody may not necessarily bind to the same epitope as the reference antibody, but may spatially block reference antibody binding by binding to an overlapping or adjacent epitope.

Obtaining a human antibody

Anti-GITR antibodies according to the present invention may be fully human antibodies. Methods for making monoclonal antibodies, including fully human monoclonal antibodies, are known in the art. Any such known methods can be used in the context of the present invention to obtain 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 anti-GITR chimeric antibodies having a human variable region and a mouse constant region are first isolated. As in the experimental section below, antibodies are characterized and selected for desired characteristics, including affinity, ligand blocking activity, selectivity, epitope, and so on. If necessary, the mouse constant regions are replaced with the desired human constant region, eg wild-type IgG1 or IgG4 or modified IgG1 or IgG4, to generate a fully human anti-GITR antibody. While the selected constant region may vary according to the particular application, the variable region contains characteristics of high affinity antigen binding and target specificity. In some cases, fully human anti-GITR antibodies are isolated directly from antigen-positive B cells.

Bioequivalents

Anti-GITR antibodies and antibody fragments of the present invention encompass proteins having amino acid sequences that differ from those of the described antibodies, but which retain the ability to bind human GITR. Such variant antibodies and antibody fragments contain one or more additions, deletions or substitutions of amino acids compared to the original sequence, but exhibit biological activity that is essentially equivalent to that of the described antibodies. Similarly, anti-GITR antibody-encoding DNA sequences of the present invention encompass sequences that contain one or more nucleotide additions, deletions, or substitutions compared to the disclosed sequence, but which encode an anti-GITR antibody or antibody fragment that is substantially bioequivalent to the antibody. against GITR or an antibody fragment according to the present invention. Examples of such variant amino acid and DNA sequences are discussed above.

Two antigen-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 in a single dose or in multiple doses. Some antibodies will be considered equivalents or pharmaceutical alternatives if they are equivalent in their extent of absorption, but not in their rate of absorption, and yet may be considered bioequivalent because such differences in absorption rate are intentional and reflected in the labeling are not essential to achieve effective concentrations of the drug in the body, for example, in chronic use, and are considered medically insignificant for a particular investigational medicinal product.

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

In one embodiment, two antigen-binding proteins are bioequivalent if the patient can be switched one or more times from a reference product to a biological product without an expected increase in the risk of side effects, including a clinically significant change in immunogenicity or a decrease in potency. compared with ongoing therapy without such a transfer.

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

Bioequivalence can be demonstrated in vivo and in vitro methods. Measures of bioequivalence include, for example, (a) an in vivo test in humans or other mammals in which the concentration of an antibody or its metabolites is measured in blood, plasma, serum, or other body fluid as a function of time; (b) an in vitro test that has been correlated with the data and reliably predicts in vivo human bioavailability data; (c) an in vivo test in humans or other mammals in which the corresponding 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 the safety, efficacy, or bioavailability or bioequivalence of an antibody.

Bioequivalent variants of anti-GITR antibodies of the present invention can be constructed, for example, by making various residue or sequence substitutions, or by deleting terminal or internal residues or sequences that are not necessary for biological activity. For example, cysteine residues that are not important for biological activity may be deleted or replaced with other amino acids to prevent the formation of excessive or incorrect intramolecular disulfide bridges during renaturation. In other contexts, bioequivalent antibodies may include anti-GITR antibody variants containing amino acid changes that modify the glycosylation characteristics of the antibodies, for example, mutations that abolish or abolish glycosylation.

Species selectivity and species cross-reactivity

The present invention, in some embodiments, provides anti-GITR antibodies that bind to human GITR but not GITR of other species. The present invention also provides anti-GITR antibodies that bind to human GITR and to GITR of one or more non-human species. For example, anti-GITR antibodies of the present invention may bind to human GITR and may or may not bind, as the case may be, to one or more of the mouse, rat, guinea pig, hamster, gerbil, porcine, cat, dog GITR. , rabbit, goat, sheep, cow, horse, camel, cynomolgus monkey, marmoset, rhesus monkey, or chimpanzee. In some exemplary embodiments, the present invention provides anti-GITR antibodies that specifically bind human GITR and cynomolgus monkey (eg, Macaca fascicularis) GITR. Other anti-GITR antibodies of the present invention bind to human GITR but do not bind or only weakly bind to cynomolgus monkey GITR.

Multispecific antibodies

Antibodies in accordance with the present invention may be monospecific or multispecific (eg bispecific). Multispecific antibodies may be specific for different epitopes of the same target polypeptide or may contain antigen-binding domains specific for more than one target polypeptide. See, for example, Tutt et al., 1991, J. Immunol. 147:60-69; Kufer et al., 2004, Trends Biotechnol. 22:238-244. Anti-GITR antibodies of the present invention may be linked to or co-expressed with another functional molecule, such as another peptide or protein. For example, an antibody or antibody fragment may be operably linked (e.g., by chemical coupling, genetic fusion, non-covalent bonding, or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment, to produce a bispecific or multispecific antibody with a second binding specificity.

The present invention relates to bispecific antibodies, wherein one arm of the immunoglobulin binds human GITR and the other arm of the immunoglobulin is specific for a second antigen. The GITR binding arm may comprise any of the HCVR/LCVR or CDR amino acid sequences set forth in Table 1. 1 in this document. In some embodiments, the GITR binding arm binds human GITR and blocks the binding of GITRL 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 stimulation of the receptor. The present invention also relates to bispecific antibodies, wherein one arm of the antibody binds a first human GITR epitope and the other arm of said antibody binds a second distinct human GITR epitope.

A typical bispecific antibody format that can be used in the context of the present invention involves the use of a first C _H 3 domain of an immunoglobulin (Ig) and a second CH3 domain of an Ig, wherein the first and second CH3 domains of the Ig differ from each other by at least one amino acid and at least one amino acid difference reduces the binding of the bispecific antibody to protein A compared to a bispecific antibody without an amino acid difference. In one embodiment, the first Ig CH3 domain binds protein A and the second Ig CH3 domain contains a mutation that reduces or abolishes protein A binding, such as an H95R modification (according to IMGT exon numbering; H435R according to EU numbering). The second CH3 may further comprise the modification Y96F (as per IMGT; Y436F as per EU). Other modifications that may be in the second CH3 include D16E, L18M, N44S, K52N, V57M and V82I (as per IMGT; D356E, L358M, N384S, K392N, V397M and V422I as per EU) in the case of IgG1 antibodies; N44S, K52N and V82I (IMGT; N384S, K392N and V422I according to EU) in the case of IgG2 antibodies; and Q15R, N44S, K52N, V57M, R69K, E79Q and V82I (as per IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q and V422I as per EU) for IgG4 antibodies. Variations in the format of the bispecific antibody described above are contemplated within the scope of the present invention.

Other exemplary bispecific formats that may be used in the context of the present invention include, without limitation, for example, scFv or diabody based bispecific formats, IgG-scFv fusions, double variable domain (DVD)-Ig, quadroma, ridges to troughs, common light chain (e.g., common light chain in ridges on troughs, etc.), CrossMab, CrossFab, (SEED)body, leucine zippers, Duobody, IgG1/IgG2, Fab (DAF)-IgG dual action and Mab ² bispecific formats (see, for example, Klein et al. 2012, mAbs 4:6, 1-11, and references therein for an overview of the formats set forth above). Bispecific antibodies can also be constructed using peptide/nucleic acid conjugation, for example, when non-natural amino acids with orthogonal chemical reactivity are used to prepare site-specific antibody-oligonucleotide conjugates, which then self-assemble to form multimeric complexes with defined composition, valency, and geometry. (See, for example, Kazane et al., J. Am. Chem. Soc. Epub: Dec. 4, 2012).

Therapeutic composition and administration

The present invention relates to pharmaceutical compositions containing antibodies against GITR or antigen-binding fragments in accordance with the present invention. Pharmaceutical compositions according to the present invention are formulated with suitable carriers, adjuvants, and other means to provide suitable transfer, delivery, tolerability, and the like. Many suitable formulations can be found in the collection known to all pharmaceutical chemists, Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipid containing lipid (cationic or anionic) vesicles (such as LIPOFECTIN™, Life Technologies, Carlsbad, CA), DNA conjugates, anhydrous absorbable pastes, oil-in-water and water-in-oil emulsions, Carbowax emulsions (polyethylene glycols of various molecular 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 on the age and size of the patient, the target disease, condition, route of administration, and the like. The preferred dose is generally calculated according to body weight or body surface area. An adult patient may benefit from intravenous administration of an antibody of the present invention, typically at a single dose of from about 0.01 to about 20 mg/kg body weight, more preferably from about 0.02 to about 7, from 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 duration of treatment can be adjusted. Effective dosages and regimens for administering anti-GITR antibodies can be determined empirically; for example, the progress of the patient can be monitored by periodic evaluation and dose adjusted accordingly. Moreover, interspecies dose scaling can be performed using methods well known in the art (eg, Mordenti et al., 1991, Pharmaceut. Res. 5:1351).

Various delivery systems are known and can be used to administer the pharmaceutical composition of the present invention, e.g., liposome encapsulation, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, receptor-mediated endocytosis (see, e.g., Wu et al. , 1987, J Biol Chem 262:4429-4432). Routes of administration include, without limitation, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The composition may be administered by any suitable route, for example by infusion or bolus injection of the substance, by absorption through epithelial or mucosal surfaces (eg, oral mucosa, rectal or small intestinal mucosa, etc.), and may be administered together with other biologically active agents. The introduction can be systemic or local.

The pharmaceutical composition in accordance with the present invention can be delivered subcutaneously or intravenously using a standard needle and syringe. In addition, with regard to the subcutaneous

- 19 039583 delivery, the delivery of the pharmaceutical composition in accordance with the present invention can easily be used with a device for delivery type syringe-pen. Such a pen-type delivery device may be reusable or disposable. A reusable pen-type delivery device typically uses a replaceable cartridge that contains a pharmaceutical composition. Once all of the pharmaceutical composition from the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and a new cartridge containing the pharmaceutical composition can be placed. The pen-type delivery device can then be reused. In the case of a disposable delivery device, there is no replacement cartridge. Rather, a disposable pen-type delivery device is filled with a pharmaceutical composition held in a reservoir of the device. Once the pharmaceutical composition is removed from the reservoir, the empty device is discarded.

For subcutaneous delivery of a pharmaceutical composition in accordance with the present invention, a variety of reusable pens and autoinjector delivery devices are used. Examples include, without limitation, AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75/25™ pen, HUMALOG™ pen, Syringe - 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), syringe pen BD™ (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN™, OPTIPEN PRO™, OPTIPEN STARLET™ and OPTICLIK™ (Sanofi-Aventis, Frankfurt, Germany) among others. Examples of disposable pen-type delivery devices used for subcutaneous delivery of a pharmaceutical composition in accordance with the present invention include, without limitation, the SOLOSTAR™ pen (Sanofi-Aventis), FLEXPEN™ (Novo Nordisk), and KWIKPEN™ (Eli Lilly), the SURECLICK™ autoinjector device (Amgen, Thousand Oaks, CA), PENLET™ (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, L.P.), and the HUMIRA™ pen (Abbott Labs, Abbott Park IL), among others.

In some situations, the pharmaceutical composition may be delivered in a controlled release system. According to one embodiment, a pump may be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201). According to another embodiment, polymeric materials can be used; see, Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Florida. In a further embodiment, the controlled release system can be placed near the target of the composition, in which case only a fraction of the systemic dose is required (see, for example, Goodson, 1984, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 ). Other controlled release systems are reviewed by Langer, 1990, Science 249:1527-1533.

Injectable preparations may include dosage forms for intravenous, subcutaneous, intradermal and intramuscular injections, drip infusions, and the like. These injectable preparations can be manufactured by conventional methods. For example, injectable preparations can be made, for example, by dissolving, suspending or emulsifying an antibody or salt thereof, as described above, in a sterile aqueous or oily medium commonly used for injection. As the aqueous injection medium, there can be mentioned, for example, physiological saline solution, isotonic solution containing glucose, other excipients, etc., which can be used in combination with a suitable solubilizing agent such as an alcohol (for example, ethanol), polyhydric an alcohol (eg, propylene glycol, polyethylene glycol), a non-ionic surfactant (eg, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) hydrogenated castor oil adduct)) and the like. As the oil medium, for example, sesame oil, soybean oil, etc. are used, which can be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc. The injection thus prepared is preferably filled into a suitable ampoule.

Preferably, the pharmaceutical compositions for oral or parenteral administration described above are formulated into dosage forms whose unit dose corresponds to that of the active ingredients. Such unit dosage forms include, for example, tablets, pills, capsules, injections (ampoules), suppositories, and the like. The amount contained above antibodies, as a rule, is from about 5 to about 500 mg per dosage form in a unit dose; especially in the form of injection, it is preferred that the above antibody is from about 5 to about 100 mg and from about 10 to about 250 mg for other dosage forms.

Therapeutic applications of antibodies

The present invention relates to methods comprising administering to a subject, in need thereof, a therapeutic composition comprising an anti-GITR antibody (eg, an anti-GITR antibody comprising any of the HCVR/LCVR or CDR sequences set forth in Table 1 herein). The therapeutic composition may contain any of the anti-GITR antibodies,

- 20 039583 of their antigen-binding fragments or ADC disclosed herein, and a pharmaceutically acceptable carrier or diluent.

The antibodies of the present 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 between GITR and GITRL and/or by inhibiting or stimulating activity and/or signaling GITR. For example, antibodies and antigen-binding fragments according to the present disclosure can be used to treat immune and proliferative diseases or disorders, such as cancer, by modulating the immune response through, for example, GITR activation.

The antibodies and antigen-binding fragments of the present disclosure may be used to treat a disease or disorder by enhancing an immune response. The present disclosure relates to methods for modulating an anti-tumor immune response in a subject, comprising administering to the subject an anti-GITR antibody or antigen-binding fragment as described herein. In some embodiments, the antibody or antigen-binding fragment reduces the suppressive activity of effector T cells by regulatory T cells. In some embodiments, an antibody or antigen-binding fragment according to the present disclosure enhances a favorable intratumoral ratio of effector T cells/regulatory T cells for therapeutic benefit. In some embodiments, an antibody or antigen-binding fragment of the present disclosure promotes T cell survival.

Typical diseases or disorders that can be treated with antibodies and antigen binding fragments include immune and proliferative diseases or disorders such as cancer. Antibodies and antigen-binding fragments according to the present invention can be used to treat primary and/or metastatic tumors arising in the brain and its meninges, oropharynx, lung and bronchial tree, gastrointestinal tract, male and female reproductive organs, muscles , bones, skin and appendages, connective tissue, spleen, immune system, hematopoietic cells and bone marrow, liver and urinary tract, and special sensory organs such as the eye. In some embodiments, the antibodies and antigen-binding fragments of the present disclosure are used to treat solid or blood-borne tumors. In some embodiments, the antibodies of the present disclosure are used to treat one or more of the following cancers: renal cell carcinoma, pancreatic carcinoma, head and neck cancer, prostate cancer, malignant glioma, osteosarcoma, colon and rectal cancer, cancer gastric (eg, MET-amplifying gastric cancer), malignant mesothelioma, multiple myeloma, ovarian cancer, cervical cancer, small cell lung cancer, non-small cell lung cancer, synovial sarcoma, thyroid cancer, breast cancer, melanoma, testicular, renal, esophageal cancer , uterine cancer, endometrial cancer, or liver cancer.

In some embodiments, the antibodies of the present invention are useful in the treatment of an autoimmune disease, including, but not limited to, alopecia areata, autoimmune hepatitis, celiac disease, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, hemolytic anemia, inflammatory bowel disease, inflammatory myopathies, multiple sclerosis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, scleroderma, Sjögren's syndrome, systemic lupus erythematosus, vitiligo, autoimmune pancreatitis, autoimmune urticaria, autoimmune thrombocytopenic purpura, Crohn's disease, type I diabetes mellitus, eosinophilic fasciitis, eoscherogadosinophilic syndrome myasthenia gravis, psoriatic arthritis, rheumatic fever, ulcerative colitis, vasculitis, and Wegener's granulomatosis.

In the context of the treatments described herein, an anti-GITR antibody may be administered as monotherapy (i.e., as the sole therapeutic agent) or in combination with one or more additional therapeutic agents (examples of which are described elsewhere herein) .

Combination Therapeutics and Formulations

Also provided herein are combination therapeutics using an anti-GITR antibody of the present disclosure and any additional therapeutic that can be successfully combined with an antibody of the present disclosure or an antigen-binding fragment thereof.

The present invention relates to compositions and therapeutic formulations containing any of the anti-GITR antibodies described herein in combination with one or more additional therapeutically active components, and to methods of treatment involving the introduction of such combinations to subjects in need of it.

Antibodies of the present invention may be combined synergistically with one or more anti-cancer drugs or therapy used to treat cancer, including, for example, renal cell carcinoma, colon and rectal cancer, glioblastoma multiforme, squamous cell carcinoma of the region head and neck, non-small cell lung cancer, colon cancer, ovarian cancer, adenocarcinoma, prostate cancer, glioma and melanoma. The present document contemplates the use of anti-GITR antibodies of the present invention in combination with immunostimulatory and/or immunosuppressive therapies to suppress tumor growth and/or increase survival in cancer patients. Immunostimulatory therapeutics include direct immunostimulatory therapeutics to enhance the activity of immune cells, either by releasing the brake on suppressed immune cells or by pushing the gas to activate an immune response. Examples include targeting other checkpoint receptors, vaccination, and adjuvants. Immune-supporting methods can enhance tumor antigenicity by providing immunogenic cell death, inflammation, or other indirect effects that promote an anti-tumor immune response. Examples include radiation therapy, chemotherapy, antiangiogenic agents, and surgery.

The present disclosure relates to methods for modulating an anti-tumor immune response in a subject, comprising administering to the subject an anti-GITR antibody in combination with one or more anti-activating receptor agonist antibodies and one or more inhibitory receptor blocking antibodies that enhance T-cell stimulation to promote tumor destruction.

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

In various embodiments, one or more antibodies of the present invention may be used in combination with an anti-PD-L1 antibody, an anti-PD-1 antibody (e.g., nivolumab), a LAG-3 inhibitor, a CTLA-4 inhibitor (e.g., ipilimumab) , TIM3 inhibitor, BTLA inhibitor, TIGIT inhibitor, CD47 inhibitor, other T-cell co-inhibitor or ligand antagonist (eg, anti-CD-28, 2B4, LY108, LAIR1, ICOS, CD160, or VISTA), indolamine-2,3 inhibitor β-dioxygenase (IDO), a vascular endothelial growth factor (VEGF) antagonist (e.g., VEGF-Trap such as aflibercept or other VEGF-inhibiting fusion protein mentioned in US Pat. No. 7,087,411, or an anti-VEGF antibody or antigen-binding fragment thereof (e.g. , bevacizumab, or ranibizumab) or a small molecule VEGF receptor kinase inhibitor (eg, sunitinib, sorafenib, or pazopanib)), an Ang2 inhibitor (eg, nesvacumab), a trans inhibitor forming growth factor beta (TGFP), epidermal growth factor receptor (EGFR) inhibitor (eg, erlotinib, cetuximab), costimulatory receptor agonist (eg, glucocorticoid-induced TNFR-related protein agonist), tumor-specific antigen antibody (eg, CA9 , CA125, melanoma-associated antigen 3 (MAGE3), carcinoembryonic antigen (CEA), vimentin, tumor-M2-PK, prostate-specific antigen (PSA), mucin-1, MART-1 and CA19-9), vaccine (eg, Bacillus Calmette-Guerin, cancer vaccine), adjuvant to increase antigen presentation (eg, granulocyte-macrophage colony-stimulating factor), bispecific antibody (eg, CD3xCD20 bispecific antibody, PSMAxCD3 bispecific antibody), cytotoxin, chemotherapeutic agent (eg, dacarbazine, temozolomide , cyclophosphamide, docetaxel, doxorubicin, daunorubicin, cisplatin, carb platinum, gemcitabine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, and vincristine), cyclophosphamide, radiation therapy, IL-6R inhibitor (eg, sarilumab), IL-4R inhibitor (eg, dupilumab), IL-10 inhibitor, cytokine such as IL -2, IL-7, IL-21 and IL-15, antibody drug conjugate (ADC) (eg, anti-CD19-DM4 ADC and anti-DS6-DM4 ADC), anti-inflammatory drug (eg, corticosteroids and non-steroidal anti-inflammatory drugs), nutritional supplements such as antioxidants, or any palliative care for cancer. In certain embodiments, anti-GITR antibodies of the present invention can be used in combination with cancer vaccines, including dendritic cell vaccines, oncolytic viruses, tumor cell vaccines, etc., to enhance the antitumor response. Examples of cancer vaccines that can be used in combination with anti-GITR antibodies of the present invention include the MAGE3 vaccine for melanoma and bladder cancer, the MUC1 vaccine for breast cancer, EGFRv3 (eg, Rindopepimut) for cancer. brain (including glioblastoma multiforme) or ALVAC-CEA (for CEA+ cancers).

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 US Patent Publication No. 2015/0203579, which is incorporated herein by reference in in all its fullness. In some embodiments, the anti-GITR antibody is H1H14536P2 or H2aM14536P2. In some embodiments, the anti-GITR antibody is REGN 2810 (also known as H4H7798N, disclosed in US Patent Publication No. 2015/0203579), pembrolizumab, or nivolumab.

In certain embodiments, the anti-GITR antibodies of the present invention may be administered in combination with radiation therapy in methods for producing long-term sustained anti-tumor responses and/or enhancing the survival of cancer patients. In certain embodiments, the anti-GITR antibodies of the present invention may be administered prior to, simultaneously with, or after the administration of radiation therapy to a cancer patient. For example, radiation therapy can be administered in one or more doses to tumor lesions followed by one or more doses of anti-GITR antibodies in accordance with the present invention. In certain embodiments, radiation therapy can be administered locally to a tumor lesion to enhance the local immunogenicity of the patient's tumor (adjuvant radiation) and/or to kill tumor cells (destructive radiation) followed by systemic administration of an anti-GITR antibody in accordance with the present invention. For example, intracranial irradiation can be administered to a patient with brain cancer (eg, glioblastoma multiforme) in combination with systemic administration of an anti-GITR antibody in accordance with the present invention. In certain embodiments, the anti-GITR antibodies of the present invention may be administered in combination with radiation therapy and a chemotherapeutic agent (eg, temozolomide) or a VEGF antagonist (eg, aflibercept).

In some embodiments, the anti-GITR antibodies of the present invention may be administered in combination with one or more antiviral drugs for the treatment of a chronic LCMV, HIV, HPV, HBV, or HCV viral infection. Examples of antiviral drugs include, without limitation, zidovudine, lamivudine, abacavir, ribavirin, lopinavir, efavirenz, cobicistat, tenofovir, rilpivirine, and corticosteroids. In certain embodiments, the anti-GITR antibodies of the present invention may be administered in combination with a LAG3 inhibitor, a CTLA-4 inhibitor, or any other T cell co-inhibitor antagonist to treat a chronic viral infection.

In some embodiments, anti-GITR antibodies of the present invention can be combined with an anti-Fc receptor antibody on immune cells to treat an autoimmune disease. In one embodiment, an antibody or fragment thereof according to the present invention is administered in combination with an antibody or antigen-binding protein that targets an antigen specific for an autoimmune tissue. In some embodiments, an antibody or antigen-binding fragment thereof according to the present invention is administered in combination with an antibody or antigen-binding protein that targets a T cell receptor or a B cell receptor, including but not limited to Fca (e.g., CD89), Fcgamma (eg, CD64, CD32, CD16a and CD16b), CD19, etc. Antibodies and fragments thereof according to the present invention may be used in combination with any drug or therapy known in the art (e.g., corticosteroids and other immunosuppressants) to treat an autoimmune disease or disorder, including, but not limited to, alopecia areata, autoimmune hepatitis , celiac disease, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, hemolytic anemia, inflammatory bowel disease, inflammatory myopathies, multiple sclerosis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, scleroderma, Sjögren's syndrome, systemic lupus erythematosus, vitiligo, autoimmune pancreatitis, autoimmune urticaria, autoimmune thrombocytopenic purpura, Crohn's disease, type I diabetes mellitus, eosinophilic fasciitis, eosinophilic enterogastritis, Goodpasture's syndrome, myasthenia gravis, psoriatic arthritis, rheumatic fever, ulcerative colitis, vasculitis, and Wegener's granulomatosis.

The present disclosure also provides methods for modulating an antitumor immune response in a subject, comprising administering to the subject an anti-GITR antibody or antigen-binding fragment as described herein in combination with one or more isolated antibodies or antigen-binding fragments thereof that bind to an inhibitory T cell. receptor. In some embodiments, the T cell inhibitory receptor is CTLA-4, PD-1, TIM-3, BTLA, VISTA, or LAG-3. The present disclosure also relates to compositions comprising an anti-GITR antibody or antigen-binding fragment provided herein and an antibody or antigen-binding fragment that binds to said inhibitory T cell receptor.

The present disclosure also relates to methods of treating cancer by administering an antibody, or an antigen-binding fragment thereof, or a formulation described herein, to a subject in conjunction with radiation or chemotherapy.

In some embodiments, an anti-GITR antibody of the present invention is co-formulated with and/or administered in combination with one or more additional therapeutically active moieties selected from the group consisting of an EGFR antagonist (e.g., an anti-EGFR antibody (e.g., cetuximab or pinitumumab ) or a small molecule EGFR inhibitor (eg, gefitinib or erlotinib)), an antagonist of another member of the EGFR family, such as Her2/ErbB2, ErbB3, or ErbB4 (eg, anti-ErbB2 antibodies (eg, tarstuzumab or T-DM1 {KADCYLA®}), against ErbB3 or against ErbB4 or a small molecule inhibitor of ErbB2, ErbB3 or ErbB4 activity), an EGFRvIII antagonist (eg, an antibody that specifically binds EGFRvIII), a cMET antagonist (eg, an anti-cMET antibody), an IGF1R antagonist (eg, an anti-IGF1R antibody), a B inhibitor -raf (eg, vemurafenib, sarfenib, GDC-0879, PLX-4720), PDGFR-α inhibitor (eg, an antibodies against PDGFR-α), a PDGFR-β inhibitor (for example, an antibody against PDGFR-β or a small molecule kinase inhibitor such as, for example, imatinib mesylate or sunitinib malate), a PDGF ligand inhibitor (for example, an antibody against PDGF-A, -B , -C or -D, aptamer, siRNA, etc.), VEGF antagonist (e.g. VEGF-Trap such as aflibercept, see e.g. US Pat. No. 7,087,411 (also referred to herein as VEGF inhibitory fusion protein) , an anti-VEGF antibody (e.g., bevacizumab, a small molecule VEGF receptor kinase inhibitor (e.g., sunitinib, sorafenib, or pazopanib)), a DLL4 antagonist (e.g., an anti-DLL4 antibody disclosed in US Pat. No. 2009/0142354 such as REGN421), an Ang2 antagonist (e.g. an anti-Ang2 antibody disclosed in US Pat. AP2), TMPRSS2 antagonist (eg, anti-TMPRSS2 antibodies), MSLN antagonist (eg, anti-MSLN antibodies), CA9 antagonist (eg, anti-CA9 antibodies), uroplakin antagonist (eg, anti-uroplakin antibodies (eg, anti-UPK3A antibodies)), MUC16 antagonist (eg, anti-MUC16 antibodies), Tn antigen antagonist (eg, anti-Tn antibodies), CLEC12A antagonist (eg, anti-CLEC12A antibodies), TNFRSF17 antagonist (eg, anti-TNFRSF17 antibodies), LGR5 antagonist (eg, anti-LGR5 antibodies) , a monovalent CD20 antagonist (eg, a monovalent anti-CD20 antibody such as rituximab), etc. Other agents that can be effectively administered in combination with antibodies of the present invention include, for example, 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 with their corresponding receptors.

The present invention relates to compositions and therapeutic formulations containing any of the anti-GITR antibodies described herein in combination with one or more chemotherapeutic agents. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (Cytoxan™); alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carbokwon, meturedopa and uredopa; ethyleneimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolomelamin; nitrogen mustards such as chlorambucil, chlornaphasine, holofosfamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembihin, phenesterin, prednemustine, trofosfamide, uramustine; nitrosoureas such as carmustine, chlorzotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclacinomycins, actinomycin, autramycin, azaserine, bleomycins, sactinomycin, calicheamicin, carabicin, carminomycin, carcinophyllin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxoL-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marsellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rhodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate, irinotecan, leucovorin, and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprin, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxyfluridine, enocytabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, estolactone; adrenal depressants such as aminoglutethimide, mitotane, trilostane; agents that compensate for the lack of folic acid, such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraksat; defofamine; demecolcin; diaziquon; elfornitine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pyrarubicin; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK™; razoxane; sisofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2''-trichlorotriethylamine; urethane; vindesine; decarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; hacytosine; arabinoside (AraC); cyclophosphamide; thiotepa; taxanes, eg paclitaxel (Taxol™, Bristol-Myers Squibb Oncology, Princeton, N.J.) and docetaxel (Taxotere™; Aventis Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbin; novantron; teniposide; daunomycin; aminopterin; xeloda; ibandronate; SRT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid; esperamycins; capecitabine; as well as pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included in this definition are antihormonal agents that act to regulate or inhibit the action of hormones on tumors, e.g. antiestrogen, including e.g. 117018, onapristone and toremifene (fareston); and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; as well as pharmaceutically acceptable salts, acids or derivatives of any of the above.

Anti-GITR antibodies of the present invention can also be administered and/or co-formulated in combination with antivirals, antibiotics, analgesics, corticosteroids, steroids, oxygen, antioxidants, COX inhibitors, cardioprotectors, metal chelators, IFN-gamma, and/or NSAIDs.

The additional therapeutically active component(s), for example any of the above-listed derivative agents, may be administered prior to, simultaneously with, or immediately after administration of the anti-GITR antibody of the present invention (for the purposes of this disclosure, such administration regimens are considered to be administration of the anti-GITR antibody to combinations with an additional therapeutically active component). The present invention relates to pharmaceutical compositions wherein an anti-GITR antibody of the present invention is formulated with one or more of the additional therapeutically active ingredients described elsewhere herein.

The additional therapeutically active ingredient(s) may be administered to the subject prior to administration of the anti-GITR antibody of the present invention. For example, the first component may be considered to be administered before the second component if the first component is administered 1 week, 72 hours, 60 hours, 48 hours, 36 hours, 24 hours, 12 hours, 6 hours, 5 h, 4 h, 3 h, 2 h, 1 h, 30 min, 15 min, 10 min, 5 min or less than 1 min before the introduction of the second component. In other embodiments, the additional therapeutically active ingredient(s) may be administered to a subject following administration of an anti-GITR antibody of the present invention. For example, the first component may be considered to be administered after the 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 h after, 5 h after, 6 h after, 12 h after, 24 h after, 36 h after, 48 h after, 60 h after, 72 h after administration of the second component. In further embodiments, the additional therapeutically active ingredient(s) may be administered to a subject concurrently with administration of an anti-GITR antibody of the present invention. Simultaneous administration for purposes of the present invention includes, for example, administering an anti-GITR antibody and an additional therapeutically active component to a subject in the same dosage form (e.g., co-formulated) or in separate dosage forms administered to the subject within about 30 minutes or less of each other. . When administered in separate dosage forms, each dosage form can be administered in the same way (eg, both the anti-GITR antibody and the additional therapeutically active ingredient can be administered intravenously, subcutaneously, etc.); alternatively, each dosage form may be administered by a different route (eg, the anti-GITR antibody may be administered intravenously and the additional therapeutic active ingredient may be administered subcutaneously). In any case, the administration of the components in the same dosage form, in different dosage forms by the same route, or in different dosage forms by different routes, is considered to be simultaneous administration for the purposes of this disclosure. For purposes of this disclosure, administration of an anti-GITR antibody before, concurrently, or after (as these terms are defined herein above) administration of an additional therapeutically active ingredient is considered to be administration of an anti-GITR antibody in combination with an additional therapeutically active ingredient.

The present invention relates to pharmaceutical compositions in which an anti-GITR antibody of the present invention is formulated with one or more additional therapeutically active ingredients as described elsewhere herein using a variety of combination dosage forms.

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In exemplary embodiments in which an anti-GITR antibody of the present invention is administered in combination with a VEGF antagonist (e.g., a VEGF decoy protein such as aflibercept), including co-formulations comprising an anti-GITR antibody and a VEGF antagonist, the individual components may be administered to the subject and/or co-formulated using a variety of combination dosage forms. For example, the anti-GITR antibody may be administered to the subject and/or may be co-formulated in an amount selected from the group consisting of 0.01, 0.02, 0.03, 0.04, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3, 0, 3.5, 4.0, 4.5, 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0 mg; and a VEGF antagonist (e.g., a VEGF decoy protein such as aflibercept) may be administered to the subject and/or may be co-formulated in an amount selected from the group consisting of 0.1, 0.2, 0.3, 0 .4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6 , 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2 .9 and 3.0 mg.

Combinations/co-formulations may be administered to a subject according to any of the administration schedules disclosed elsewhere herein, including, for example, twice a week, once a week, once every 2 weeks, once every 3 weeks, once a month, once every 2 months, once every 3 months, once every 4 months, once every 5 months, once every 6 months, etc.

Modes of administration

In some 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 referred to herein) may be administered to a subject over a period of time. Methods according to this aspect of the present invention provide for sequential administration of multiple doses of an anti-GITR antibody in accordance with the present invention to a subject. As used herein, the term sequential administration means that each dose of an anti-GITR antibody is administered to a subject at a different time point, eg, on different days separated by a predetermined interval (eg, hours, days, weeks, or months). The present invention relates to methods that involve sequentially administering to a patient a single initial dose of an anti-GITR antibody followed by one or more secondary doses of an anti-GITR antibody and optionally followed by one or more tertiary doses of an anti-GITR antibody.

The terms initial dose, secondary doses and tertiary doses refer to the time sequence of administration of an anti-GITR antibody in accordance with the present invention. Thus, the initial dose is the dose that is administered at the beginning of the treatment regimen (it is also called the initial dose; secondary doses are doses that are administered after the initial dose; and tertiary doses are doses that are administered after secondary doses. Initial, secondary and tertiary doses may all contain the same amount of anti-GITR antibody, but generally may differ from each other in terms of frequency of administration.However, in certain embodiments, the amount of anti-GITR antibody contained in the initial, secondary, and/or tertiary doses , varies (e.g., up or down as needed) over the course of treatment.In certain embodiments, two or more (e.g., 2, 3, 4, or 5) doses are administered at the start of the treatment regimen as loading doses followed by subsequent doses that are administered less frequently (eg, maintenance doses).

According to some typical embodiments of the present invention, each secondary and/or tertiary dose is administered through 1-26 (for example, 1, 11/2, ₂ , 21/2, 3, 31/2, ₄ , 41/2, 5, 51/ ₂ , 6, 6 ^1/2 , 7, 71/2, 8, 81/2, 9, 91/2, 10, 101/2, 11, 111/2, 12, 121/2, 13, 131/2 , 14, 141/2, 15, 151/2, 16, 161/2, 17, 171/2, 18, 181/2, 19, 191/2, 20, 201/2, 21, 211/2, 22 , 221/2, 23, 231/2, 24, 21/2, 25, 21/2, 26, 21/2 or more) weeks immediately after the previous dose. As used herein, the phrase immediately preceding dose means, in a multiple dose sequence, the dose of the anti-GITR antibody that is administered to the patient prior to each successive dose in the sequence without any intermediate doses.

Methods according to this aspect of the present invention may involve administering any number of secondary and/or tertiary doses of anti-GITR antibody to the patient. For example, in certain embodiments, only one secondary dose is administered to the patient. In other embodiments, two or more (eg, 2, 3, 4, 5, 6, 7, 8 or more) secondary doses are administered to the patient. Similarly, in certain embodiments, only one tertiary dose is administered to the patient. In other embodiments, two or more (eg, 2, 3, 4, 5, 6, 7, 8 or more) tertiary doses are administered to a patient. The administration regimen may be administered indefinitely for the lifetime of a particular subject or until such treatment is no longer therapeutically necessary or beneficial.

In embodiments involving multiple secondary doses, each secondary dose may be administered at the same frequency as the other secondary doses. For example, each secondary dose may be administered to a patient 1-2 weeks or 1-2 months after the immediately preceding dose. Similarly, in embodiments involving multiple tertiary doses, each tertiary dose may be administered at the same frequency as the other tertiary doses. For example, each tertiary dose may be administered to a patient 2-12 weeks after the immediately preceding dose. According to certain embodiments of the present invention, the frequency with which secondary and/or tertiary doses are administered to a patient may vary during the course of a treatment regimen. The frequency of administration during the course of treatment can also be adjusted by the attending physician depending on the needs of the individual patient after a clinical experiment.

The present invention provides administration regimens in which 2-6 loading doses are administered to the 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, and etc.) followed by the introduction of two or more maintenance doses to the patient at a lower frequency. For example, according to this aspect of the present invention, if loading doses are administered at a frequency of once a month, then maintenance doses can be administered to the patient once every six weeks, once every two months, once every three months, and so on.

Diagnostic use of antibodies

Anti-GITR antibodies of the present invention can also be used to detect and/or measure GITR or GITR-expressing cells in a sample, for example, for diagnostic purposes. For example, an anti-GITR antibody, or fragment thereof, can be used to diagnose a condition or disease characterized by abnormal expression (eg, overexpression, underexpression, no expression, etc.) of GITR. Exemplary diagnostic assays for GITR may include, for example, contacting a sample obtained from a patient with an anti-GITR antibody of the present invention, wherein the anti-GITR antibody is labeled with a detectable label or reporter. Alternatively, an unlabeled anti-GITR antibody can be used in diagnostic applications in combination with a secondary antibody that is itself labeled with a detectable label. The detectable label or reporter molecule may be a radioactive isotope such as ³ H, ¹⁴ C, ³² P, ³⁵ S, or ¹²⁵ I; a fluorescent or chemiluminescent moiety such as fluorescein or rhodamine; or an enzyme such as alkaline phosphatase, beta-galactosidase, horseradish peroxidase or luciferase. Specific illustrative assays that can be used to detect or measure GITR in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), Immuno-PET (e.g., ⁸⁹ Zr, ⁶⁴ Cu, etc.) and cell sorting. with activated fluorescence (FACS).

Samples that may be used in the GITR diagnostic assays of the present invention include any tissue or fluid sample obtained from a patient that contains detectable amounts of the GITR protein or fragments thereof, under normal or pathological conditions. Typically, GITR levels in a particular sample obtained from a healthy patient (eg, a patient not affected by a disease or condition associated with abnormal GITR levels or activity) will be measured to initially establish baseline or standard GITR levels. This initial GITR content can then be compared to GITR levels measured in samples obtained from individuals suspected of having a GITR-related disease or condition.

Examples

The following examples are provided to provide those skilled in the art with a complete disclosure and description of how to make and use the methods and compositions of the present invention, and are not limited to the scope of what the present inventors refer to their invention. Attempts have been made to ensure accuracy with respect to the numbers used (eg amounts, temperature, etc.), but some experimental errors and deviations must be taken into account. Unless otherwise indicated, parts are parts by weight, molecular weight means average molecular weight, temperature is in degrees Celsius, and pressure is atmospheric or near atmospheric.

Example 1 Generating Anti-GITR Antibodies.

Anti-GITR antibodies were generated by immunizing a VELOCIMMUNE® mouse (ie, a recombinant mouse containing DNA encoding the human immunoglobulin heavy and kappa light chain variable regions) with an immunogen containing a soluble dimeric human GITR ectodomain. The immune response with the formation of antibodies was monitored using GITR-specific immunoassay. Some fully human anti-GITR antibodies were isolated directly from antigen-positive B cells without fusion with myeloma cells, as described in US Pat. No. 2007/0280945 A1.

Some of the biological properties of exemplary GITR antibodies generated according to the methods of this example are detailed in the examples below.

Example 2 Heavy and Light Chain Variable Region Amino Acid and Nucleic Acid Sequences.

In table. 1 sets forth the amino acid sequence identifiers for the heavy and light chain variable regions and CDRs of selected anti-GITR antibodies in accordance with the present invention. The corresponding nucleic acid sequence identifiers are set forth in Table 1. 2.

Table 1. Amino acid sequence identifiers

SEQID NO: Antibody designation HCVR DR1 HCDR2 HDDR3 LCVR LCDR1 LCDR2 LCDR3 H1H14474R 2 4 6 eight ten 12 fourteen sixteen H1H14486R eighteen 20 22 24 26 28 thirty 32 H1H14491R 34 36 38 40 42 44 46 48 H1H14493R fifty 52 54 56 58 60 62 64 H1H14495R 66 68 70 72 74 76 78 80 H1H14503R 82 84 86 88 90 92 94 96 H1H14512R 98 100 102 104 106 108 110 112 H1H14520R 114 116 118 120 122 124 126 128 H1H14523R 130 132 134 136 138 140 142 144 H1H14524R 146 148 150 152 154 156 158 160 H4H14469R 162 164 166 168 170 172 174 176 H4H14470R 178 180 182 184 186 188 190 192 H4H14475R 194 196 198 200 202 204 206 208 H4H14476R 210 212 214 216 218 220 222 224 H4N14508R 226 228 230 232 234 236 238 240 H4H14516R 242 244 246 248 250 252 254 256 H4H14521R 258 260 262 264 266 268 270 272 H4H14525R 274 276 278 280 282 284 286 288 H4H14528R 290 292 294 296 298 300 302 304 H4H14530R 306 308 310 312 314 316 318 320 H4H14531R2 322 324 326 328 402 404 406 408 H4H14532R2 330 332 334 336 402 404 406 408 H4H14536R2 338 340 342 344 402 404 406 408 Н4Н14539Р2 346 348 350 352 402 404 406 408 H4H14541R2 354 356 358 360 402 404 406 408 H4H15736R2 362 364 366 368 402 404 406 408 H4H15740R2 370 372 374 376 402 404 406 408 H4H15744R2 378 380 382 384 402 404 406 408 Н4Н15745Р2 386 388 390 392 402 404 406 408 H4H15753R2 394 396 398 400 402 404 406 408

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Table 2. Nucleic acid sequence identifiers

SEQID NO: Designation antibodies HCVR HCDR1 HCDR2 HDDR3 LCVR LCDR1 LCDR2 LCDR3 H1H14474R one 3 5 7 nine eleven thirteen fifteen H1H14486R 17 nineteen 21 23 25 27 29 31 H1H14491R 33 35 37 39 41 43 45 47 H1H14493R 49 51 53 55 57 59 61 63 H1H14495R 65 67 69 71 73 75 77 79 H1H14503R 81 83 85 87 89 91 93 95 H1H14512R 97 99 101 103 105 107 109 111 H1H14520R FROM 115 117 119 121 123 125 127 H1H14523R 129 131 133 135 137 139 141 143 H1H14524R 145 147 149 151 153 155 157 159 H4H14469R 161 163 165 167 169 171 173 175 H4H14470R 177 179 181 183 185 187 189 191 H4H14475R 193 195 197 199 201 203 205 207 H4H14476R 209 211 213 215 217 219 221 223 H4H14508R 225 227 229 231 233 235 237 239 H4H14516R 241 243 245 247 249 251 253 255 H4H14521R 257 259 261 263 265 267 269 271 H4H14525R 273 275 277 279 281 283 285 287 H4H14528R 289 291 293 295 297 299 301 303 H4H14530R 305 307 309 311 313 315 317 319 H4H14531R2 321 323 325 327 401 403 405 407 H4H14532R2 329 331 333 335 401 403 405 407 H4H14536R2 337 339 341 343 401 403 405 407 Н4Н14539Р2 345 347 349 351 401 403 405 407 H4H14541R2 353 355 357 359 401 403 405 407 H4H15736R2 361 363 365 367 401 403 405 407 H4H15740R2 369 371 373 375 401 403 405 407 H4H15744R2 377 379 381 383 401 403 405 407 Н4Н15745Р2 385 387 389 391 401 403 405 407 H4H15753R2 393 395 397 399 401 403 405 407

Antibodies are generally referred to herein according to the following nomenclature: the prefix Fc (e.g., SHN, H4H, etc.), then the numeric identifier (e.g., 14493, 14495, etc.), then the suffix P or P2, as shown in table. 1 and 2. Thus, according to this nomenclature, an antibody may be referred to herein, for example, as WIII4486P, H4H14531P2, etc. The prefixes H1H and H4H in antibody designations used herein indicate the specific isotype of the Fc region of an antibody. For example, antibody H1H has a human IgG1 Fc, antibody H4H has a human IgG4 Fc, and H2M has a mouse IgG2 Fc (all variable regions are fully human, as indicated by the first H in the antibody designation). One of ordinary skill in the art will understand that an antibody having a particular Fc isotype can be converted into an antibody with a different Fc isotype, but in any case, the variable domains (including CDRs) identified by the numeric identifiers shown in Table 1 and 2 will remain the same and assume that the binding properties will be identical or substantially similar, regardless of the nature of the Fc domain.

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Control constructs used in the following examples

Control constructs were included in the following experiments for comparison purposes. Control anti-GITR Ab I: mouse anti-human GITR antibody hybridoma with heavy and light chain variable domains having the amino acid sequences of the respective domains of clone 6C8 as set forth in WO 2006/105021 A2; obtained with mIgG1 and mIgG2a constant regions in the following examples; and a control anti-GITR Ab II: a human anti-GITR antibody with heavy and light chain variable domains having the amino acid sequences of the respective 36E5 domains as set forth in US Pat. No. 8,709,424 B2.

Example 3 Surface Plasma Resonance Binding Affinities and Kinetic Constants of Human Anti-TNFRSF18 Monoclonal Antibodies (GITR).

Binding affinities and kinetic constants of human anti-GITR antibodies were determined using surface plasmon resonance (Biacore 4000 or T-200) at 37°C (Table 3). Antibodies expressed as human IgG1 or IgG4 (i.e. designations H1H or H4H) were captured onto a CM5 Biacore sensor surface coated with mouse anti-human Fc (mAb capture format) and soluble monomer (human (h) GITR.mmh; SEQ ID NO: 409 and Macaca fasicularis (mf) GITR.mmh; SEQ ID NO: 412) or dimer (hGITR.hFc; SEQ ID NO: 411 and hGITRmFc; SEQ ID NO: 410). GITR proteins were injected onto the sensory surface at a flow rate of 30 μg/min. All binding studies on Biacore were performed in a buffer consisting of 0.01 M HEPES, pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.05% v/v. Surfactant P20 (moving buffer HBS-ET). Antibody-reagent association was observed within 4 min, 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 ) were observed for 10 min. The kinetic rate constants of association (k _a ) and dissociation (kd) were determined by real-time sensogram fitting for a 1:1 binding model using Scrubber 2.0c curve fitting software. Binding dissociation equilibrium constants (KD) and dissociative half-lives (t ¹ / ₂ ) were calculated from the kinetic rate constants as: K _D [M]=k _d /k _a ; and t _1/2 (minutes)=(ln2/(60*k _d ). The results are shown in Table 3.

Table 3. Biacore-determined mAb binding affinities against human Fc at 37°C

Binding at 37°C/antibody capture format Antibody analyte ka (Me' ¹ ) Kd (c ¹ ) Kd (mol) 1½ (min.) H1H14503R hGITR.mmh 5.32E+05 7.39E-04 l,39E-09 15.6 hGITRmFc 1D2E+06 l,54E-04 l,37E-10 75.0 mfGITR.mmh 2.68E+05 5.60E-03 2.09E-08 2.1 H1H14474R hGITR.mmh 5.91E+05 1D6E-03 l,96E-09 9.9 hGITRmFc l,21E+06 l,30E-04 l,07E-10 89.2 mfGITR.mmh 3.39E+05 9.51E-03 2.80E-08 1.2 H1H14495R hGITR.mmh 5.17E+05 l,27E-03 2.45E-09 9.1 hGITRmFc 1D4E+06 Ι,ΙΟΕ-04 9.68E-11 105.0 mfGITR.mmh 2.96E+05 7.23E-03 2.45E-08 1.6 H1H14486R hGITR.mmh 4.39E+05 l,23E-03 2.79E-09 9.4 hGITRmFc 9.65E+05 l,53E-04 l,59E-10 75.5 mfGITR.mmh l,37E+05 l,66E-02 l,22E-07 0.7

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H1H14524R hGITR.mmh 4.05E+05 l,47E-03 3.62E-09 7.9 hGITRmFc 9.22E+05 l,35E-04 l,46E-10 85.6 mfGITR.mmh l,20E+05 l,89E-02 l,58E-07 0.6 H4H14530R hGITR.mmh 2.72E+05 l,38E-03 5.06E-09 8.4 hGITRmFc 2.93E+05 l,85E-04 6.30E-10 62.6 mfGITR.mmh 2.40E+05 6D1E-04 2.55E-09 18.9 H1H14491R hGITR.mmh ZD9E+05 l,62E-03 5.06E-09 7.2 hGITRmFc 8.42E+05 l,69E-04 2.01E-10 68.3 mfGITR.mmh 1D8E+05 8.89E-03 7.53E-08 1.3 H1H14523R hGITR.mmh 2.16E+05 l,86E-03 8.63E-09 6.2 hGITRmFc 5.55E+05 l,20E-04 2D6E-10 96.3 mfGITR.mmh l,23E+05 l,09E-02 8.85E-08 1.1 H1H14493R hGITR.mmh l,86E+05 2.50E-03 l,34E-08 4.6 hGITRmFc 5.24E+05 l,91E-04 3.65E-10 60.4 mfGITR.mmh l,00E+04 l,40E-02 l,40E-06 0.8 H4H14532R2 hGITR.mmh 3.48E+05 6.45E-03 l,85E-08 1.8 hGITRmFc 7.20E+05 2.69E-04 3.73E-10 42.9 mfGITR.mmh 2.60E+05 6.48E-03 2.49E-08 1.8 H4H14521R hGITR.mmh 3.45E+05 7.84E-03 2.27E-08 1.5 hGITRmFc l,23E+06 4.79E-04 3.89E-10 24.1 mfGITR.mmh l,66E+05 3.34E-03 2.01E-08 3.5 H4H14536R2 hGITR.mmh 4.24E+05 9.76E-03 2.30E-08 1.2 hGITRmFc l,26E+06 2D9E-04 l,73E-10 52.7 mfGITR.mmh l,04E+05 l,47E-02 l,42E-07 0.8 H4H14476R hGITR.mmh 3.92E+05 l,23E-02 ZD4E-08 0.9 hGITRmFc 9.06E+05 2.69E-04 2.97E-10 42.9 mfGITR.mmh l,91E+05 l,07E-02 5.58E-08 1.1 H4H14516R hGITR.mmh 2.25E+05 7.38E-03 3.27E-08 1.6 hGITRmFc l,68E+06 l,81E-03 l,08E-09 6.4 mfGITR.mmh l,90E+05 1D2E-02 5.87E-08 1.0 H4N14508R hGITR.mmh 2.55E+05 9.35E-03 3.66E-08 1.2 hGITRmFc l,21E+06 7D9E-04 5.97E-10 16.1 mfGITR.mmh l,29E+05 5.07E-03 3.93E-08 2.3 H4H14469R hGITR.mmh 2.84E+05 l,22E-02 4.30E-08 0.9 hGITRmFc l,20E+06 4.01E-04 3.35E-10 28.8 mfGITR.mmh 5.91E+04 2.43E-03 4D1E-08 4.7 H4H14475R hGITR.mmh 3.07E+05 l,40E-02 4.57E-08 0.8 hGITRmFc l,60E+06 l,35E-03 8.47E-10 8.5 mfGITR.mmh l,83E+05 7.65E-03 4D7E-08 1.5 H4H14528R hGITR.mmh l,02E+05 5.23E-03 5DZE-08 2.2 hGITRmFc l,58E+06 l,77E-03 1D2E-09 6.5 mfGITR.mmh NB NB NB NB H4H14525R hGITR.mmh ZD7E+05 l,66E-02 5.24E-08 0.7 hGITRmFc 7.91E+05 3.38E-04 4.27E-10 34.2 mfGITR.mmh l,33E+05 2.05E-02 l,55E-07 0.6 H1H14520R hGITR.mmh 2.66E+05 l,67E-02 6.30E-08 0.7 hGITRmFc l,09E+06 5.83E-04 5.37E-10 19.8 mfGITR.mmh l,99E+05 l,71E-02 8.59E-08 0.7 H4H14470R Н4Н14539Р2 Anti-GITR antibodies hGITR.mmh hGITRmFc mfGITR.mmh hGITR.mmh hGITRmFc mfGITR.mmh hGITR.mmh 2.21E+05 9.04E+05NB 2D4E+05 8.53E+05 7.23E+04 2.16E+05 l,43E-02 l,04E-03 NB l,77E-02 4.72E-04 l,65E-03 2.63E-02 6.47E-08 1D5E-09 NB 8.25E-08 5.54E-10 2.28E-08 l,22E-07 0.8 H.1 NB 0.7 24.5 7.0 0.4 hGITR.hFc 3.82E+05 7.80E-03 2.04E-08 1.5 mfGITR.mmh 2D8E+05 4.64E-02 2DZE-07 0.2 Antibodies against GITR hGITR.mmh l,94E+05 9.67E-04 4.99E-09 H.9 hGITRmFc l,83E+06 l,73E-03 9.48E-10 6.7 mfGITR.mmh 2.31E+05 8.63E-03 3.74E-08 1.3

NB = No observed binding under the conditions used

As shown in Table. 3, all anti-GITR antibodies of the present invention bound to human GITR, with some antibodies showing subnanomolar affinities for the human GITR dimeric protein. In addition, most of the antibodies against

GITR also showed cross-reactivity with the cynomolgus monkey GITR protein.

No cross-reactivity with the rodent GITR protein was observed (data not shown).

Example 4 Specific and efficient binding of anti-GITR antibodies to cells expressing human GITR.

In this example, the ability of anti-GITR antibodies to specifically bind to a human GITR expressing cell line was determined using electrochemiluminescence (ECL) detection.

Briefly, human embryonic kidney (HEK)-293-D9 cells were stably transfected with human GITR (M1-V241 amino acids, NCBINP accession no. 004186.1, SEQ ID: 413) using the Lipofectamine 2000 mediated method. Transfectants were collected for at least 2 weeks in complete growth medium + G418.

For cell binding studies, approximately 1x10 ⁵ hGITR/HEK293-D9 cells or HEK293-D9 parent cells that do not express human GITR were plated in 96-well carbon electrode plates (MULTI-ARRAY, MSD) for 1 hour at 37°C. . Non-specific binding sites were blocked with 2% BSA (w/v) + PBS for 1 h at room temperature (RT). Then, serial dilutions of anti-GITR antibodies ranging from 1.7 pM to 100 nM were added to the cells over 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 SULFOTAG™ anti-human IgG kappa light chain light chain antibody (Jackson Immunoresearch) for 1 hour at RT.

After washes, luminescent signals were recorded using a SECTOR Imager 6000 (MSD). Direct binding signals (in relative light units, RLU) were analyzed as a function of antibody concentration and data were fitted to a sigmoidal (four-parameter logistic) dose-response model using GraphPad Prism™ software. The EC ₅₀ for binding hGITR/HEK293-D9 cells, defined as the antibody concentration at which 50% of the maximum binding signal is detected, was determined to indicate the binding efficiency of each antibody. The signal detected with binding of 100 nM antibody to hGITR expressing cells relative to parental cells was recorded as an indication of the intensity and specificity of GITR binding. The results are presented in table. 4.

As shown in Table. 4, most of the anti-GITR antibodies of the present invention specifically bind to human GITR expressing cells relative to parental HEK293 cells with EC ₅₀ ranging from 210 pM to 85 nM. Most antibodies bind to human GITR expressing cells with subnanomolar EC ₅₀ values. The isotype control antibody did not show binding to hGITR expressing or parental cell lines.

Table 4 Anti-GITR Antibody Binding EC ₅₀ and Binding Intensity at 100 nM on Human GITR Expressing Cells

Cell binding hGITR/HEK293- D9 Linking with hGITR/HEK293-D9 cells (at 100 nM) Linking with HEK293D9 cells (at 100 nM) Antibody EC50 (M) Average signal (RLU) Average signal (RLU) H1H14474R 2.80E-10 6230 680 H1H14486R 4.30E-10 5830 280 H1H14491R 4.00E-10 6840 300

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H1H14493R 3.00E-10 7220 790 H1H14495R 4.30E-10 6470 340 H1H14503R 2D0E-10 5880 330 H1H14512R 2.50E-10 4620 180 H1H14520R 2.40E-10 6450 ISO H1H14523R 4.00E-10 6350 530 H1H14524R 2D0E-10 5740 500 H4H14469R 2.30E-10 5230 260 H4H14470R 1.40E-09 8390 1580 H4H14475R 8.00E-09 7500 1580 H4H14476R 5.70E-10 8120 1770 H4N14508R 4.50E-10 6870 580 H4H14516R 7.00E-10 10330 2560 H4H14521R 4.30E-10 10080 600 H4H14525R 6.00E-10 8840 1490 H4H14528R 4.50E-10 7310 420 H4H14530R 8.50Е-08 4200 520 H4H14532R2 4.30E-10 5740 300 H4H14536R2 3.60E-10 8960 330 Н4Н14539Р2 3.00E-10 4910 300 Control AB against GITR IIhlgGl 2.60E-10 13750 10240 Isotype control Ab-hIgG4 NB 750 650

In conclusion, this example demonstrates that the anti-GITR antibodies of the present invention exhibit specific and efficient binding to human GITR expressing cell lines.

Example 5 Anti-GITR antibodies are partial blockers and partial activators in the NF-kB/luciferase reporter assay with or without antibody anchoring to Fc-gamma R.

In this example, the ability of anti-GITR antibodies to activate hGITR or block hGITR ligand (hGITRL)-mediated receptor stimulation in the presence or absence of antibody anchoring of Fc-gamma receptors (Fc-raMMa-R) was assessed using luciferase reporter assays.

Briefly, a Jurkat cell line was constructed with the appropriate inclusion of hGITR and an NF-kB dependent luciferase reporter (hGITR/Jurkat/NF-KB-Luc). The NF-kB/luciferase reporter was injected into Jurkat cells using a Cignal Lenti Reporter system (SABiosciences). Lentiviruses expressing hGITR were generated in HEK293/T17 using the Lenti-X Lentiviral Expression System (Clontech). Jurkat/NF-KB-Luc cells were transfected with hGITR-expressing lentivirus by polybren-mediated transduction and harvested at 500 μg/ml G418 for 2 weeks. For antibody anchoring studies, HEK293 cells were transduced with Ec-gamma-RI expressing lentivirus as described above.

First, the activating and blocking properties of anti-hGITR antibodies were evaluated in the absence of Gs-gamma-R anchoring (non-anchored bioassay format). Approximately 4x10 ⁴ Jurkat/NFKBLuc/hGITR cells were plated overnight (ON) in PDL-coated 96-well plates in OptiMEM + 0.5% FBS.

To determine the activating ability of the antibody, the cells were incubated for 6 h at 37°C.

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0.5 pM to 100 nM. To assess blocking of hGITRL antibody-mediated receptor stimulation, cells were preincubated for 30 min with serially diluted anti-hGITR antibodies (from 0.5 pM to 100 nM) followed by a constant dose of 10 nM hGITRL for 6 h.

The activating and blocking properties of the selected anti-hGITR antibodies were then determined in the presence of Fc-raMMa-R anchoring (an anchored bioassay format). As above, 2.5 x ^{10 4} Jurkat/NfKBLuc/hGITR cells were plated in PDL-coated 96-well plates in complete growth medium.

To evaluate antibody activation, cells were pre-incubated for 1 h at 37° C. with serially diluted anti-hGITR or hGITRL mAbs (0.5 pM to 100 nM). Then immediately added 1x10 ⁴ cells hFc-gamma R1/HEK293 in the wells, followed by a 6-hour incubation. To evaluate blockage, hGITR/Jurkat/NfKBluc cells were preincubated for 1 h with serially diluted anti-hGITR antibodies (0.5 pM to 100 nM). 1x10 ⁴ hFcR1/HEK293 cells were added to the wells followed by a constant dose of 10 nM hGITRL.

For both anchored and unanchored bioassays, luciferase activity was measured with One Glow reagent (Promega) and relative light units (RLU) were measured with a Victor luminometer (Perkin Elmer). EC ₅₀ /IC5o values were determined by a four parameter logistic equation based on a 12-response curve using GraphPad Prism. The results are presented in table. 5 and tab. 6. To determine % blocking, raw RLUs (relative light units) from untreated wells were subtracted from those of treated wells and percentage blocking was calculated according to the following formula: [100 - (RLU of antibody at max. dose/RLU of ligand at constant dose)]*100] . % activation was calculated according to the following formula: (normalized RLU mAb/max. GITR ligand response)*100; mAb normalized RLUs were determined by subtracting RLUs from untreated wells from those of treated wells. The mean fold activation was calculated as follows: RLU at maximum antibody dose/original RLU from untreated wells.

Table 5 Blocking and activating properties of anti-GITR antibodies in the absence of Fc-gamma-R anchoring

Antibody IC50 (nM) % blocking ECso (nM) % activation H4H14475R ND -2 1.0 70 H1H14491R 0.60 90 2.0 60 H4H14521R 3.80 60 0.4 fifty H1H14503R 0.60 90 1.4 fifty H4H14469R 0.70 90 2.3 fifty H4H14516R 2.30 70 0.8 45 H1H14523R 0.90 70 2.5 40 H1H14524R 0.70 80 2.2 40 H4H14528R 3.40 90 1.1 thirty H1H14495R 0.70 80 1.3 thirty

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H1H14474R 0.80 80 1.2 thirty H4N14508R 0.90 40 1.2 thirty H4H14532R2 1.00 90 1.2 thirty H1H14486R 1.10 fifty 1.2 thirty H1H14493R 0.60 90 1.2 25 H1H14512R 0.70 100 1.2 20 H4H14525R 1.40 70 1.1 20 Н4Н14539Р2 1.20 thirty 1.3 20 H4H14536R2 2.00 90 1.1 20 H4H14470R 0.90 80 1.2 20 H4H14476R 2.60 90 1.0 ten H1H14520R 0.90 80 1.1 ten Control AB against GITR ImlgGl 0.10 54 1.02 25 Control isotype IgGl NB NB ΝΑ ΝΑ Control isotype IgG4 NB NB ΝΑ ΝΑ

NB = no blocking

ΝΑ = no activation

As set out in Table. 5 above and tab. 6 below, the antibodies tested exhibited partial activating and partial blocking properties in both unanchored and anchored bioassays. In the unanchored format, the antibodies mediated receptor stimulation with EC ₅₀ ranging from 0.4 nM to 2.5 nM. Some antibodies, such as H4H14475P and H4H14491P, were potent activators of the GITR receptor, showing an activation percentage of 70 and 60, respectively. Most of the antibodies tested also demonstrated blocking hGITRL-mediated receptor stimulation with IC50's ranging from 0.6 nM to 3.8 nM. Some exemplary antibodies such as H1H14512P and H4H14536P2 showed strong blocking activity of 100% and 90%, respectively. H4H14475P, the most effective activator, showed the least activity in the blocking assay (percent blocking - 2%).

Table 6 Blocking and activating properties of anti-GITR antibodies in the presence of Tc-gamma-R anchoring

Antibody IC ₅₀ (nM) % blocking _ec50 (hM) Multiplicity of activation relative to the original signal Η1Η14512Ρ 0.10 64 0.02 7.0 Η4Η14475Ρ 0.20 43 0.04 8.0 Η4Η14536Ρ2 0.20 73 0.01 5.0 Control Ab vs. GITR I-mlgGl 0.20 60 0.10 6.0 Control Ab against GITR I-mIgG2a 0.01 74 0.20 5.0 Control Ab vs. GITR Π-hIgGl 0.20 70 0.01 8.0

The selected antibodies tested in the Tc-gamma-R anchoring format also exhibited a range of activating and blocking properties. H4H14475P, the strongest activator in the non-root format, also effectively activated hGITR in the anchored bioassay with an activation fold 8.0 higher than the original signal. Strong blockers in the unanchored blocking format, such as H1N4512P and H4H14536P2, also showed effective blocking in the anchored assay (% block 60% and 70%).

In conclusion, the results show that the anti-GITR antibodies of the present invention exhibit effective GITR activating properties as well as the ability to block GITRL-mediated receptor stimulation in the absence of Fc-gamma-R anchoring in a recombinant bioassay. Exemplary antibodies such as H4H14775P and H4H1536P2 also retain their activating and blocking properties, respectively, in the presence of Fc-gamma-R anchoring.

Example 6 Anti-GITR antibody H4H14536P2 shows 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 a recombinant bioassay for their ability to activate hGITR in the presence or absence of Fc gamma receptor (Fc gamma R) anchoring. In this example, the effect of antibody anchoring on hGITR activation was assessed in a primary proliferation bioassay of naïve human CD4+ T cells. The human CD4+ T cell system has the advantage that GITR copy number remains at endogenous levels, while higher GITR copy number cells are used in the recombinant system.

First, anti-GITR antibodies were tested for the proliferative capacity of CD4+ T cells in the presence of plate-bound anti-CD3 antibody. Briefly, human CD4+ T cells were isolated from healthy donor leukocytes using Human CD4 ⁺ T cell Enrichment Cocktail (Stemcell Technologies). Naive T cells were then enriched by depletion of CD45RO+ cells using MACS (Miltenyi Biotech). Approximately 5× ^{10 4} T cells were seeded in 96-well U-bottom polystyrene plates pre-coated with a suboptimal amount of anti-CD3 OKT3 mAb (30 ng/ml) and titrated amounts of anti-GITR antibodies or controls. Three days after stimulation, tritiated thymidine (1 μCi per well, Perkin Elmer Health Sciences NET027001) was added to each microwell and pulses were generated for 18 h. Cells were harvested on 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 the filter plates and radioactive pulses were measured using a plate reader (Perkin Elmer Health Sciences Topcount NXT). T-cell proliferation relative to control, presented as the average fold activation at an antibody concentration of 10.6 nm, is shown in table. 7. In this assay format, the concentration of 10.6 nM represented the point at which T cell proliferation plateaued on the dose-response curve.

Table 7. T-cell proliferative activity (activation fold) of plate-bound anti-GITR antibodies at 10.6 nM in the presence of

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H4H14469R 3 12 12 fifteen 12 17 And And 12 H4H14532R2 2 6 6 12 6 nineteen 7 2 eight H4H14470R 0 3 3 4 3 6 4 one 3 H4H14475R 0 2 2 2 2 12 one 0 3 H4H14528R 2 eight eight 4 eight 31 5 4 eight Н4Н14539Р2 2 12 12 thirteen 12 39 And 6 thirteen H4H14516R one 4 4 12 4 fourteen 4 one 5 H4H14521R one 4 4 5 4 12 3 2 4 Control AB against GITR 1-mIgGl 6 23 23 40 23 66 25 And 27 isotypic the control one one one one one one one one one

The results in the table. 7 show that the anti-GITR antibodies tested exhibited T-cell proliferative capacity when bound to the plate in the presence of plate-bound anti-CD3 antibodies. Control AB against GITR I showed a proliferative activity 27 times higher than that of the isotype control. Most of the anti-GITR antibodies of the present invention exhibited 2 to 8 times the activation of the isotype control, with some exemplary H4H14469P, H4H14539P2 and H4H14536P2 antibodies showing 12, 13 and 23 times the activation of the control, respectively. In conclusion, the results show that the anti-GITR antibodies tested exhibited T-cell proliferative activity in this classic format.

Additional format assays were then used to test the ability of anti-GITR antibodies to activate T cells in the presence or absence of cell surface bound Pc-gamma-K.

To evaluate the ability of an anti-GITR antibody to activate T cells in the presence of Pc-gamma-K1 anchoring, HEK293 cells were engineered to express the high-affinity hFcgamma-Rl receptor as described above. HEK293/Fc-gamma-RI cells were treated with 50 μg/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 of anti-CD3 OKT antibody to stimulate T cell activation. HEK293/Fc-gamma-RI cells were co-cultured with human naïve CD4+ T cells at a ratio of 1:2 and titratable amounts of anti-GITR antibodies or controls were added to the co-culture medium.

T cell proliferation was assessed by measuring incorporation levels of tritiated thymidine. 72 hours after stimulation, tritiated thymidine (0.5 μCi per well, Perkin Elmer Health Sciences) was added to each microwell for an additional 18 hours at 37°C. Cells were collected on 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 the filter plates and radioactive pulses were measured using a plate reader (Perkin Elmer Health Sciences Topcount NXT). T cell proliferation relative to control, presented as the average fold activation at an antibody concentration of 33 nM, is shown in Table 1. 8. In this assay format, the 33 nM concentration represented the point at which T cell proliferation plateaued on the dose-response curve.

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Table 8. T-cell proliferative activity (activation fold) of anti-GITR antibodies at 33 nM in the presence of Fc-gamma-R anchoring

Antibody Donor Average activation multiplicity one 2 3 4 5 H4H14536R2 1.3 6.3 1.5 2.7 15.7 5.5 H4N14508R 1.0 0.7 1.1 1.8 1.3 1.2 H4H14525R 1.2 0.8 1.0 1.5 1.5 1.2 H4H14469R 0.70 1.0 0.9 1.5 1.5 1.1 H4H14532R2 0.80 0.9 1.0 1.7 2.1 1.3 H4H14470R 1.8 1.0 1.4 1.5 2.0 1.5 H4H14475R 1.0 1.3 1.3 1.6 1.5 1.3 H4H14528R 0.9 1.5 1.1 1.9 1.7 1.4 Н4Н14539Р2 0.9 0.7 1.1 1.5 1.5 1.1 H4H14516R 1.2 0.9 1.2 1.3 1.1 1.1 H4H14521R 1.5 1.1 1.1 1.7 0.9 1.2 H4H14476R 0.6 0.20 0.8 0.8 0.1 0.5 Checklists against GITR Control I-mlgGl 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-hlgGl 0.1 0.6 1.3 0.2 1.2 0.7 isotypic control hIgG4 1.0 1.0 1.0 1.0 1.0 1.0

Table 9 T-cell proliferative activity (EC50) of anti-GITR antibodies at 33 nM in the presence of Fc-gamma-R anchoring

Antibody Donor Average EC ₅ o (nM) one 2 3 4 5 H4H14536R2 1.2 0.5 1.3 AND 2 1.6 3.2 Control I-mlgGl 37.2 NA 42.9 3.5 52.7 34.1

The results in the table. 8 show that several antibodies showed activation in excess of controls, with levels varying from 1 to 2 times. However, one exemplary H4H14536P2 antibody showed effective T-cell proliferative activity under anchoring conditions. With an average activation fold of 5.5, H4H14536P2 stimulated higher T-cell activation compared to the reference antibodies against GITR (average fold range 0.7-2.0). H4H14536P2 had a mean EC ₅₀ of T cell proliferation of 3.2 nM compared to 34.1 nM for the most potent control Ab against the GITR-control I-mIgG (Table 9). In addition, in this assay format, H4H14475P, the effective activator in the recombinant bioassay described above, showed the highest proliferative activity under the conditions of this primary bioassay.

The antibodies were then tested for T-cell proliferative activity in the absence of Fc-gamma-R anchoring. Human CD4+ T cells were isolated as described above and plated in 96-well U-bottomed polystyrene plates pre-coated with 30 ng/mL antibody.

- 38 039583 against CD3 OKT3. As described above, titrated concentrations of anti-GITR antibodies or controls were added to the culture medium. T cell proliferation was measured by incorporating tritiated thymidine. T-cell proliferation observed in four donors at an antibody concentration of 22 nM is presented as the fold of activation compared to the isotype control in Table 1. 10. EC50 (nM) H4H14536P2 is shown in table. eleven.

As observed in the anchored assay format, H4H14536P2 again showed effective T cell proliferative activity at 22 nM in the unanchored format. H4H14536P2 activated T cells with an average activation fold of 11.0 and an EC50 of 8.3 nM. In this assay format, the control anti-GITR I antibody showed no T-cell proliferative capacity.

Table 10 T-cell proliferative activity (fold activation) of anti-GITR antibodies at 22 nM in the absence of Fc-gamma-R anchoring

Donor Medium multiplicity Antibody one 2 3 4 activation H4H14536R2 6.2 4.2 10.1 23.4 11.0 H4N14508R 0.7 0.9 0.9 1.4 1.0 H4H14525R 0.7 1.0 0.9 1.0 0.9 H4H14469R 0.9 0.8 1.0 0.9 0.9 H4H14532R2 0.7 0.8 1.0 1.1 0.9 H4H14470R 0.7 0.9 1.0 1.7 1.1 H4H14475R 0.7 1.1 0.8 0.8 0.9 H4H14528R 0.6 0.8 0.9 1.0 0.8 Н4Н14539Р2 0.5 0.8 1.1 0.8 0.8 H4H14516R 0.7 1.2 1.0 0.9 0.9 H4H14521R 0.5 1.2 0.8 1.0 0.9 H4H14476R 0.7 0.9 0.9 0.8 0.8 Control AB — against GITR Control I-mlgGl 0.7 0.8 1.0 1.0 0.9 Isotype control 1.0 1.0 1.0 1.0 1.0 Table 11. E C50 (nM ) H4H145: 36R2 Donor one 2 3 4 The average ESeo N4N14536R2 12.2 6.4 9.0 5.7 8.3 (nM)

In conclusion, this example demonstrates that one exemplary anti-GITR antibody H4H14536P2 exhibits effective T-cell proliferative activity in the presence and absence of hFc-gamma-R1 anchoring, while the reference anti-GITR I antibody exhibits no T-cell proliferative activity under conditions lack of anchoring. Thus, the ability of H4H14536P2 to activate T cells in the absence of hFc-gamma R1 anchoring is a unique property, suggesting that the antibody may not compete with endogenous IgG for binding to Fc-gamma receptors in vivo while maintaining activity. This unique property of H4H14536P2 may provide an advantage in therapeutic settings.

Example 7 Administration of anti-GITR antibodies in combination with anti-PD1 antibodies synergistically controls and eradicates tumors.

The effect of administering anti-GITR antibodies in combination with anti-PD1 antibodies on tumor growth was evaluated using the following methods. The evaluation results are shown below.

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Tumor implantation, treatment regimen and growth measurement

MC38 colon 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 after tumor implantation), mice were divided into 4 groups (5 mice per group) and each group was treated intraperitoneally (IP) as follows: (1) rat IgG2a (2A3, Bio X cell, cat no. BE0089) (isotype control) + rat IgG2b (LTF2, Bio X cell, cat no. BE0090) (isotype control) (2) anti-GITR monoclonal antibody DTA1 (rat anti-mouse GITR antibody, Bio X cell, no. BE0063) + 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. The antibody injection(s) were then administered again on day 13. Antibody treatments were administered at a dose of 5 mg/kg of each antibody. Tumors were measured in two directions (length x width) and the tumor volume was calculated (length x width ² x 0.5). Mice were sacrificed when the tumor reached a defined tumor endpoint (tumor volume >2000 mm ³ or tumor ulceration).

Evaluation of repeated tumor provocation

Mice treated with a combination of anti-PD-1 antibody and anti-GITR antibody that remained tumor-free for 80 days were re-challenged with 3x105 syngeneic tumor cells (MC38) in the right flank and 2.5x105 allogeneic tumor cells (B16F10.9) (lines melanoma cells, ATCC) to the left side. Tumors were monitored as described above.

Antibody Depletion Experiments

Mice were injected with different depleting mAbs (anti-CD4, anti-CD8, anti-CD25) starting one day prior to tumor challenge and given twice a week for a total of eight doses, treated with combination therapy or isotype control IgG. The effectiveness of the depletion was confirmed by FACS analysis of peripheral blood samples.

Flow cytometry (FACS) analysis of intratumoral lymphocytes

Mice were treated as described above. Tumor and spleen were harvested 5 days after antibody treatment. Tumors were minced with scissors and dissociated to a single cell suspension with a mixture of Liberase TL/DNAse I. Spleens were dissociated with a gentleMACS Octo Dissociator. Cells were stained with FACS antibody panels 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 purchased in BD Fortessa X20 or LSR II and analyzed using FlowJo software.

Administration of anti-mouse GITR antibodies in combination 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. no. BE0063) in combination with an anti-mouse PD-1 antibody (clone RMP1-14, Bio X cell, cat. no. according to cat. BE0146) in the control of subcutaneous tumors MC38. As shown in FIG. 1 and in table. 12 and 13, the combined treatment of PD1 blockade and anti-GITR antibody (DTA-1) significantly induced tumor regression in MC38 tumor-bearing mice, in contrast to treated anti-PD-1 or anti-GITR mAbs alone or with isotype control mice. In addition, mice treated with the combination therapy exhibited long-term tumor remission as 100% of mice remained tumor-free for 120 days (FIG. 2, Tables 14, 15).

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Table 12 Mean tumor volumes for each treatment group (mm ³ ± SEM) and tumor-free mice after anti-GITR and/or anti-PD-1 Ab treatment

Tumor volume (mm ³ ) Mice without

Mean (SEM) of the tumor

Processed group Day 10 Day 13 Day 17 Day 19 Day 21 Day 21 Isotype (rat IgG2a + rat IgG2b) 196 (44) 232 (46) 802(869) NA NA 0/5 Antibody against PD1 + rat IgG2b 181 (37) 259 (103) 551(199) 880 (335) 1550 (616) 0/5 Anti-GITR antibody + rat IgG2a 172(9) 262 (72) 407(112) 741(269) 882 (307) 0/5 Antibody against GITR + anti-PD1 antibody 130 (29) 41(13) θ(0) θ(0) 0(0) 5/5

Table 13 Summary of tumor-free mice from three independent experiments treated with anti-GITR and/or anti-PD1 Ab

Mice without tumor

Processed group Day 21 Isotype (rat IgG2a + rat IgG2b) 0/15 Anti-PD1 antibody + rat IgG2b 0/15 Anti-GITR antibody + rat IgG2a 1/15 Anti-GITR antibody + Anti-PD1 antibody 10/15

Table 14. Shares of survivors (percentage)

days Isotype Antibody vs. PD-1 Antibody against GITR Anti-PD1 antibody + Anti-GITR antibody 0 100 100 100 100 17 80 21 40 24 0 60 26 20 35 20 52 0 0 123 100

Administration of anti-mouse GITR antibodies in combination with anti-mouse PD1 antibodies induces a tumor/antigen specific immunological memory response.

To determine whether mice treated with combined anti-PD-1 and anti-GITR antibodies developed a tumor-/antigen-specific anamnestic response, surviving tumor-free mice were re-challenged with 3 x 10 ⁵ syngeneic colon carcinoma cells.

- 41 039583 intestine MC38 in the right side and 2.5x105 allogeneic melanoma cell line B16F10.9 in the left side. It was found that MC38 tumors did not grow in mice treated with the combination of anti-PD1 antibody and anti-GITR antibody, while the same tumors grew in untreated control mice (without any pre-treatment) (FIG. 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 induces a tumor/antigen-specific immunological memory response capable of controlling a second challenge by the same tumor type.

Immune Population Study

Mice were treated with anti-CD4, CD8 and CD25 depleting mAbs prior to combined anti-PD-1 antibody and anti-GITR antibody treatment. Depletion of CD8+ cells was found to completely abolish the antitumor effect (MC38 tumors), while depletion of CD4 or CD25 T cells showed partial inhibition (FIG. 4, Table 15). Thus, the antitumor effect of combination therapy on MC38 tumors appears predominantly in dependence on CD8 + T cells.

The effect of combined anti-GITR antibody and anti-PD1 antibody treatment on tumor infiltrating lymphocytes (TIL) was evaluated. It was found that the combination treatment induced a significant increase in the CD8/Treg ratio compared to the monotherapy treatment (anti-PD-1 antibody or anti-GITR antibody) or isotype control (FIG. 5). We found that the effect of combined treatment on the CD4/Treg ratio was less pronounced. Combined treatment with anti-PD-1 antibody and anti-GITR antibody reduced the percentage of intratumoral Tregs while it increased CD8 T cells (FIG. 6). In addition, anti-PD-1 antibody treatment alone induced an increase in the number of Treg cells, while combined anti-PD-1 antibody/anti-GITR antibody treatment significantly reduced it compared to isotype control-treated mice.

Table 15. Antitumor efficacy after exhaustion

CD4, CD8 or CD25_____________________

Tumor size (mm ³ )

Mean (SEM)

Immunotherapy Debilitating antibody Day 8 Day 12 Day 16 isotypic the control isotypic the control Antibody against CD4 Antibody against CD8 Antibody against CD25 55 (12) 48 (17) 49(17) 59(16) 161 (60) 60 (22) 176 (431) 61 (21) 555(224) 135 (71) 825 (431) 182 (68) Anti-GITR antibody + Anti-PD1 antibody isotypic the control 43 (19) 26 (16) P(7) Antibody against CD4 68 (21) 50 (32) 123 (122) Antibody against CD8 67 (23) 222 (86) 1041 (543) Antibody against CD25 14(6) 35 (30) 80 (64)

Administration of anti-human GITR antibodies in combination with anti-mouse PD1 antibodies significantly induces tumor regression and provides long-term tumor remission in MC38 GITR/ITRL-bearing humanized mice.

Efficacy of administering an anti-human GITR antibody (H2aM14536P2) in combination with an anti-mouse PD-1 antibody (clone RMP1-14 Bio X cell, Cat # BE0146) in controlling subcutaneous

- 42,039,583 MC38 tumors were evaluated in GITR/GITRL humanized mice. It was found that blockade of anti-mouse PD-1 antibody synergized with anti-human GITR antibody and significantly induced tumor regression (4/6 mice) in MC38 tumor-bearing mice compared to mice treated with anti-PD1 mAb (1/7) or anti-GITR ( 1/7) alone or as an isotype control (0/7) as shown in the mean tumor growth curves (Fig. 7, Table 16). In addition, mice treated with combination therapy exhibited sustained tumor remission as more than 60% of mice remained tumor-free at day 50, compared to 0% for isotope control treated groups and 10% for anti-PD-1 antibody treated groups. or an anti-GITR antibody (FIG. 8, Table 16).

Anti-human GITR antibodies increased intratumoral CD8/Treg ratio

The effect of anti-human GITR antibodies on T cell populations within the tumor and in the spleen was assessed. Anti-human GITR antibodies H2aM14536P2 and H1N4536P2 were evaluated. It was found that both isotypes of anti-human GITR antibody (mIg2a and hIgG1) induced a significant increase in the intratumoral CD8/Treg ratio (FIG. 9). The same treatment had no effect on subgroups of peripheral splenic T cells. Human IgG1 and mouse IgG2a IgG isotype were used in the control assay.

Table 16. Antitumor efficacy mediated by anti-human GITR and anti-mouse PD1 treatment

Tumor size (mm ³ ) Mice without

Mean (SEM) of the tumor

Processed group Day 9 Day 13 Day 16 Day 19 Day 51 Isotype control 146 (26) 248 (53) 402 (97) 838 (205) 0/7 (0%) Antibody against mPDl 120 (23) 163 (50) 275(103) 617 (257) 1/7 (14%) H2aM14536R2 134 (28) 162 (51) 194 (51) 346 (87) 1/7 (14%) H2aH14536R2 + anti-PD-1 antibody 122(18) 90 (54) 114(88) 192 (165) 4/6 (67%)

Administration of anti-mouse GITR antibodies in combination with anti-human PD1 antibodies significantly induces tumor regression and provides long-term tumor remission in MC38-bearing PD1/PDL1 humanized mice

The efficacy of administration of 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 controlling subcutaneous MC38 tumors was evaluated on PD1/PDL1 humanized mice. It was found that blockade of anti-human PD-1 antibody synergized with anti-mouse GITR antibody and induced a delay in tumor growth in MC38 tumor-bearing mice compared to mice treated with anti-PD1 or anti-GITR mAb alone or with isotype control, as shown in the mean tumor growth curves. (Fig. 10, Table 17). In addition, mice treated with the combination therapy exhibited sustained tumor remission as more than 40% of mice remained tumor-free at day 45, compared to 0% for isotype control, anti-PD-1 antibody, or anti-GITR antibody treated groups (Fig. eleven).

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Table 17 Antitumor Efficacy Mediated by Anti-Mouse GITR + Anti-Human PD1 Ab Treatment

Tumor size (mm ³ ) Mean (SEM)

Processed group Day 13 Day 17 Day 20 Day 24 Isotype control 301 (38) 742 (81) 1392 (104) 2790 (366) Antibody against PD1(REGN281O) 184 (21) 354 (143) 589 (201) 937(324) Anti-GITR antibody 362 (99) 713 (360) 1199 (563) NA Anti-GITR + Anti-PD-1 212(117) 120 (60) 127 (62) 167 (98)

Example 8 Extraction and analysis of RNA.

Analysis of single cell sorted RNA-seq On days 8 and 11 after tumor challenge, tumor single cell suspension was prepared using a mouse tumor dissociation kit (Miltenyi Biotec, Bergisch Gladbach, DE) and spleens were separated using the gentleMACS™ Octo Dissociator (Miltenyi Biotec). ). Tumors and spleens 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 the 5-10 μm C1 Integrated Fluidic Circuit (IFC; Fluidigm). A LIVE/DEAD stain solution was prepared by adding 2.5 µl ethidium homodimer-1 and 0.625 µl calcein AM (Life Technologies, Carlsbad, CA) to 1.25 µl C1 Cell Wash Buffer (Fluidigm) and 20 µl loaded onto C1 IFC. Each capture site was carefully examined under a Zeiss microscope in a light field, GFP, and Texas Red channels for cellular doublets and viability. Cell lysis, reverse transcription and cDNA amplification were performed on C1 Single-Cell Auto Prep IFC as specified by the manufacturer (protocol 100-7168 E1). The Ultra Low RNA Kit SMARTer™ (Clontech, Mountain View, CA) was used to synthesize cDNA from 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 2222 individual cells were sequenced on Illumina NextSeq (Illumina, San Diego, CA) in a single-read multiplexed run at 75 cycles. Raw sequencing data (BCL files) was converted to FASTQ format using Illumina CASAVA 1.8.2. The readings were decoded based on their barcodes. Read quality was assessed using FastQC (www.bioinformatics.babraham.ac.uk/projects/fastqc/).

Example 9 Role of CD226 and TIGIT in combined processing.

Genetic inactivation or pharmacological inhibition of CD226 abolished tumor regression mediated by anti-GITR/anti-PD1 combination treatment in some experiments, while inhibition of other members of the TNF receptor superfamily or B7 had no effect.

CD226 blocking experiment

0.5 mg of anti-CD226 antibody (10E5, eBioscience, San Diego, CA) or rat IgG2b isotype control IgG (LTF2, Bio X Cell, West Lebanon, NH) was administered by intraperitoneal (ip) injection on day 5 after tumor challenge and at one days before immunotherapy. Perpendicular tumor diameters were measured blindly 2-3 times per week using digital calipers (VWR, Radnor, PA). The volume was calculated using the formula LxWx0.5 in which L is the largest 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 using a log-rank test using PRISM version 6 survival analysis (GraphPad Software Inc., La Jolla, CA). An event was defined as death if the tumor burden reached the protocol size of 2000 mm ³ in the maximum tumor volume to minimize morbidity.

As shown in FIG. 12 and 13, MC38 tumor-bearing mice were treated with either blocking anti-CD226 Ab or isotype Ab (IgG control) 1 day prior to immunotherapy with anti-GITR + anti-PD-1 or isotype Ab. The mean tumor growth curve (FIG. 12) and survival curves (FIG. 13) are shown. Data represent three experiments, n=5 mice per group, log-rank survival analysis.

WT or TIGIT KO mice were challenged with MC38 tumors, treated with anti-CD226 antibody or control IgG, and given either an isotype control (Figure 14) or a combination

- 44 039583 therapy with anti-GITR antibodies + anti-PD-1 antibodies (Fig. 15). Data shown are mean tumor growth curves representing two experiments (n=4-5 mice per group).

Using a blocking mAb against CD226, co-stimulatory signaling by CD226 was shown to be required for antitumor immunity mediated by combination therapy. In addition, the CD226 signaling pathway was required to enhance tumor surveillance in TIGIT KO mice (FIGS. 14 and 15).

RNA signatures in CD8+ T cells from samples after combined processing

To identify unique gene signatures in clonally proliferating CD8+ T cells (tumors harvested on day 11) from combined treatment samples, a full comparison was performed across all different treatment groups. Activated genes in clonally proliferating CD8+ T cells after combination therapy were compared to those in CD8+ T cells after isotype treatment or non-expandable CD8+ T cells with combination treatment. Thermal mapping analysis revealed thirty genes that overlapped in comparison. An >2-fold change in RNA signature (p < 0.01) was observed in a population of expandable CD8 T cells for 30 genes after combined anti-GITR/anti-PD1 tumor treatment. These 30 genes include Id2, S100a11, Ndufb3, Serinc3, Ctsd, S100a4, Ppp1ca, Lbr, Peli1, Lcp2, Ube2h, Cd226, Mapkapk3, Racgap1, Arf3, Mki67, Ergic2, Azi2, Dyncli2, Sik1, Pde4d, Ppp3cc, Nek7, Emc4, Vav1, Dock10, Tmem173, Fam3c, Ppp1cc and Glud1.

A four-parameter comparison was then performed across all five groups (i.e., (i) isotype treatment, (ii) anti-GITR antibody of proliferating CD8s, (iii) combination of anti-GITR antibody/anti-PD1 antibody of proliferating CD8s, (iv) anti-PD1 antibody of proliferating CD8s and (v) a combination of anti-GITR antibody/anti-PD1 antibody of non-reproducing CD8) to identify genes specifically upregulated in combination therapy relative to monotherapy treatment. Two overlapping activated genes were identified (p<0.01, >2-fold change in expression) in a four-parameter analysis. CD226, which is a co-stimulatory molecule that plays an important role in the antitumor response, was identified as one of two genes distributed across all different comparison pairs. Expression analysis of different subgroups of intratumoral CD8+ T cells ((a) all, (b) clonally propagated or (c) non-propagated) across all treated groups (i.e. (i) isotype, (ii) anti-GITR antibody, (iii) anti-PD1 antibody and (iv) anti-GITR antibody/anti-PD1 antibody combination) revealed that CD226 mRNA levels were significantly increased by the combined treatment in clonally propagated T cells (fold change = 10.7), while this difference was attenuated in mass ( fold change =3.5) and in non-reproducing CD8+ T cells (slight). In addition, CD226 mRNA levels were significantly increased by the combined treatment in clonally propagated CD8 T cells compared to those treated with anti-PD-1 antibody (fold change = 6.5) and anti-GITR antibody (fold change = 9.2) ( Fig. 16).

Association between PD1 and CD226

Next, the potential association between PD1 and CD226 molecules was investigated. To test whether CD226 is the target for dephosphorylation by the PD1-Shp2 complex, various components involved in T-cell signaling were restored in the 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 titration of PD-1 in LUV was measured by phosphotyrosine (pY) immunoblotting (FIG. 17).It was confirmed that TCR/CD3Z was not the target of dephosphorylation by the PD-1- Shp2 while CD226 was effectively dephosphorylated by the PD1Shp2 complex in a dose-dependent manner 30 min after treatment (Figure 17) These data demonstrated an association between PD-1 and CD226.

The relationship between PD-1 inhibition and CD226 expression was then investigated in a clinical setting. RNA sequencing analysis was performed on tumor biopsies collected from 43 advanced cancer patients before and after PD-1 targeted treatment. CD226 expression was significantly increased after two doses of anti-hPD-1 antibody treatment in cancer patients (FIG. 22). Next, we examined clinical data from The Cancer Genome Atlas (TCGA) to test whether the level of CD226 expression is consistent with the overall strength of T cell activation and whether it can predict a better prognosis in cancer patients. Indeed, patients with high baseline CD226 expression had significantly higher survival rates for five (skin melanoma, lung adenocarcinoma, head and neck squamous cell carcinoma, corpus endometrial carcinoma, and sarcoma) of the twenty different cancer types assessed (skin melanoma, lung adenocarcinoma, head and neck squamous cell carcinoma, uterine endometrial carcinoma, sarcoma, rectal adenocarcinoma, invasive breast carcinoma, clear cell renal cell carcinoma, cervical squamous cell carcinoma and endocervical adenocarcinoma, glioblastoma multiforme, colon adenocarcinoma, gastric adenocarcinoma, bladder urothelial carcinoma , thyroid carcinoma, prostate adenocarcinoma, pancreatic adenocarcinoma

- 45 039583 gland, poorly differentiated glioma of the brain, squamous cell carcinoma of the lung, papillary type of renal cell carcinoma of the kidney and severe cystadenocarcinoma of the ovary). Overall, these results support an immunotherapeutic strategy that enhances CD226 signaling while blocking TIGIT (eg, through treatment with an anti-GITR antibody) to maximize T cell activation.

Genetic inactivation of CD226

Using a CD226 blocking mAb, it was shown that co-stimulatory signaling by CD226 was required for combination treatment-mediated antitumor immunity (FIGS. 12, 13). Because the anti-CD226 Ab may have had a potential debilitating effect on the CD8 T cell subset, CD226 was genetically inactivated in the original C57BL/6 mice to confirm this result. CD226 -/- mice showed no defect in T cell (CD4+, CD8+, Treg) homeostasis (FIG. 18, panels A-D) and responded like wild-type mice to TCR activation (FIG. 18, panels E-I). observed that the combination treatment no longer provided antitumor effect or survival in CD226 -/- mice, indicating that CD226 is required for the observed antitumor effects of the combination (Fig. 19, panel A). The effect of CD226 is specific because inhibition of other members of the TNF receptor superfamily (OX40L or 4-1BBL) or blockade of the B7 costimulatory molecule (CD28) using CTLA4-Ig maintained the antitumor effect mediated by the combination therapy (Fig. 19, panels B-D).

CD226 requirement in TIGIT null animals

Total RNA sequencing data obtained by single cell sorting and FACS phenotyping showed that anti-PD-1 antibody favored CD226 expression, while anti-GITR antibody treatments suppressed TIGIT surface expression, synergistically impairing homeostatic T cell function.

The CD226 signaling pathway has been shown to be required to enhance tumor surveillance in TIGIT -/- mice (FIGS. 14, 15). In addition, mice harboring MC38 tumor cells overexpressing CD155/PVR6, which is the major ligand for CD226, showed a significant delay in tumor growth when treated with anti-PD-1 antibody or anti-GITR antibody or combination therapy compared with MC38-empty tumor cells. vector (MC38-EV) or mice treated with isotype control (Fig. 20). An immune profiling analysis of MC38-CD155 transplanted mice confirmed a sustained higher level of CD155 expression on MC38-CD155 cells compared to M38-EV (empty vector) after implantation. It was found that CD155 overexpression on MC38 tumor cells was associated with reduced detectable CD226 expression on CD4+, CD8+ T cells and Tregs (FIG. 21A), while it increased T cell activation as shown by upregulated IFNy expression (FIG. 21B). ) and 4-1BB (FIG. 21C) on intratumoral T cells. No effect was observed in the periphery.

Without going into any theory, it has been suggested that the level of CD226 expression should correlate with overall T cell activation and may give a better prognosis in cancer patients. Indeed, patients with high levels of CD226 expression have a significantly higher survival rate for three types of cancer (skin melanoma, lung adenocarcinoma, and sarcoma). These data support an immunotherapeutic strategy that enhances CD226 signaling while blocking TIGIT to maximize T cell activation.

The above experiments demonstrate the synergistic effect of administering an anti-GITR antibody in combination with an anti-PD1 antibody. In particular, inter alia, the above experiments show that the combined administration of an anti-GITR antibody and an anti-PD1 antibody induces tumor regression, provides long-term remission of tumors, and induces a tumor/antigen-specific immunological memory response.

Example 10 TCR Analysis.

For TCR analysis, a novel rpsTCR bioinformatics pipeline was developed to recover and extract TCR sequences, in particular TCR-CDR3 sequences, from randomly primed short RNA sequencing reads. rpsTCR takes paired- and single-end short reads and maps these reads to mouse or human genomes and transcriptomes, but not to TCR gene loci and transcripts, using TopHat (Bioinformatics 25, 1105-1111 (2009)), with default parameters . Mapped reads were discarded and non-mapped reads were reused to extract TCR sequences. Low-quality nucleotides in non-mapped reads were trimmed. Reads shorter than 35 bp were then filtered out using the HTQC instrument (Bioinformatics 14, 33 (2013). QC-passed short reads were assembled into longer reads using iSSAKE default parameters (Bioinformatics 25, 458-464 (2009)). TCRklass (J Immunol 194, 446-454 (2015)) for the identification of CDR3 sequences in Scr (assessment of evidence for residue conservation) was set to 2 instead of the default value of 1.7. Target TCR-seq data from healthy human PBMC samples were used as a positive controls to evaluate whether additional steps introduced into the pipeline resulted in higher false positive or false negative rates compared to TCRklass alone.

Most of the unique CDR3 sequences from TCRB (64031) or TCRA (51448) were detected using both rpsTCR and TCRklass. The squared correlations between rpsTCR and TCRklass were 0.9999 and 0.9365 for TCRB-CDR3 and TCRA-CDR3, respectively. Six lines of TCR-negative cancerous or non-cancerous cell lines were used as negative controls. No CDR3 sequences were detected by rpsTCR, while some CDR sequences were extracted by TCRklass from some TCR-negative cancer cell lines.

To further validate the effectiveness of the test pipeline, a sample of healthy mouse PBMCs was sequenced using both targeted TCR-seq and random priming RNA-seq (200 M, 2x100 bp) approaches. Although the number of CDR3 sequences collected from the RNA-seq data was much less than in the target TCR-seq approach, approximately 45% of the CDR3 sequences identified from the RNA-seq data using rpsTCR were also observed among the CDR3 sequences from the target TCR-seq. . Due to the limitation of the target TCR-seq methodology, it is not surprising that a portion of the CDR3 sequences that were extracted from the RNAseq data were not present in the TCR-seq results. For example, the efficiency of the 5'RACE adapter used to target a TCR-seq is generally low, and Multiply PCR tends to amplify high frequency TCRs, so only a small portion of the TCR may be targeted. As expected, a much higher percentage (~70%) of CDR3 sequences identified from RNA-seq data using rpsTCR were also observed among high-frequency CDR3 sequences (>=0.1%) from the target TCR-seq, while only approximately 40% was recovered using TCRklass alone. Furthermore, 100 bp reads were cut into 50 bp segments and 200 M reads were randomly selected. Among the top 10 CDR3 sequences ranked by the TCR-seq target approach, 8 CDR3 sequences were detected by the present inventors' rpsTCR and only 3 were detected by TCRklass. The present inventors' rpsTCR pipeline was then used to extract CDR3 sequences from single cell RNA-seq data derived from intratumoral MC38 CD8 T cells treated with different antibodies. The detection frequencies of CDR3_beta and CDR3_alpha sequences using our approach were compared to published data using the TCR-seq targeting approach to detect TCR sequences from single cell sequencing for T cells.

The scope of the present invention should not be limited to the specific embodiments described herein. Indeed, various modifications of the present invention, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are covered by the scope of the appended claims.

CLAIM

Claims (27)

CLAIM 1. An isolated antibody or antigen-binding fragment thereof that binds the glucocorticoid-inducible tumor necrosis factor receptor (GITR) containing three complementarity-determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) of the heavy chain contained within an amino acid sequence having at least at least 90% sequence identity of a heavy chain variable region (HCVR) containing the amino acid sequence of SEQ ID NO: 338 and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained within an amino acid sequence having at least 90% sequence identity of the variable region light chain region (LCVR) containing the amino acid sequence of SEQ ID NO: 402. 2. An isolated antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof contains an HCVR containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 338, and an LCVR containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 402. 3. An isolated antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof contains an HCVR containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 338, and an LCVR containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 402. 4. Selected antibody or antigen-binding fragment according to claim 1, wherein the antibody or antigen-binding fragment contains the amino acid sequence HCDR1 containing SEQ ID NO: 340; the amino acid sequence of HCDR2 containing SEQ ID NO: 342; the amino acid sequence of HCDR3 containing SEQ ID NO: 344; amino acid sequence - 47 039583 LCDR1 containing SEQ ID NO: 404; amino acid sequence LCDR2 containing SEQ ID NO: 406; and the LCDR3 amino acid sequence containing SEQ ID NO: 408. 5. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment activates human GITR. 6. An antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof activates human GITR in the absence of Fc-gamma receptor anchorage. 7. An antibody or antigen-binding fragment thereof according to claim 4, wherein the antibody or antigen-binding fragment thereof activates human GITR in the presence of Fc gamma receptor anchoring with an EC _{50 of} less than about 1.0 nM as determined by reporter assay NFkB. 8. An isolated antibody or antigen-binding fragment thereof that binds the glucocorticoid-inducible tumor necrosis factor receptor (GITR) containing three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within an amino acid sequence having at least 90% sequence identity An HCVR containing the amino acid sequence of SEQ ID NO: 338 and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained within an amino acid sequence having at least 90% sequence identity to an LCVR containing the amino acid sequence of SEQ ID NO: 402, with the antibody, or antigen-binding fragment thereof, exhibits T cell proliferative activity in the absence of Fc anchoring, as determined in a naïve human CD4+ T cell proliferation assay. 9. An antibody or antigen-binding fragment thereof according to claim 8, wherein the antibody or antigen-binding fragment thereof exhibits proliferative activity against T cells in the absence of Fc anchoring with an EC ₅₀ of approximately 8 nM or less, as determined in a naive human CD4+ proliferation assay T cells. 10. An antibody or antigen-binding fragment thereof according to claim 8, wherein the antibody or antigen-binding fragment thereof activates human GITR in the presence of Fc anchoring. 11. An isolated antibody or antigen-binding fragment thereof that binds the glucocorticoid-inducible tumor necrosis factor receptor (GITR) containing three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within an amino acid sequence having at least 90% sequence identity An HCVR containing the amino acid sequence of SEQ ID NO: 338 and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained within an amino acid sequence having at least 90% sequence identity to an LCVR containing the amino acid sequence of SEQ ID NO: 402, with the antibody or antigen-binding fragment exhibits a T cell proliferative activity in the presence of Fc anchoring at least about 2-fold higher than baseline, as determined by a naïve human CD4+ T cell proliferation assay. 12. An antibody or antigen-binding fragment according to claim 11, wherein the antibody or antigen-binding fragment exhibits proliferative activity against T cells in the presence of Fc anchoring with an EC _{50 of} less than about 34 nM, as determined using a naive human CD4+ T proliferation assay -cells. 13. The antibody or antigen-binding fragment of claim 11, wherein the antibody or antigen-binding fragment blocks hGITR ligand (hGITRL) mediated receptor stimulation. 14. The antibody or antigen-binding fragment of claim 11, wherein the antibody or antigen-binding fragment blocks hGITRL-mediated receptor stimulation in the absence of Fc anchoring. 15. An isolated antibody or antigen-binding fragment thereof that binds the glucocorticoid-inducible tumor necrosis factor receptor (GITR) containing three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within an amino acid sequence having at least 90% sequence identity An HCVR containing the amino acid sequence of SEQ ID NO: 338 and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained within an amino acid sequence having at least 90% sequence identity to an LCVR containing the amino acid sequence of SEQ ID NO: 402, with This antibody or antigen-binding fragment blocks hGITRL-mediated receptor stimulation in the absence of Fc anchoring at a percent blocking greater than about 54% with an IC _{50 of} less than about 4.0 nM as determined by NFkB reporter assay. 16. A pharmaceutical composition comprising an antibody or antigen binding fragment according to any one of claims 1 to 15 and a pharmaceutically acceptable carrier or diluent. 17. A method of treating cancer in a subject, comprising administering the composition of claim 16, wherein the cancer is selected from the group consisting of renal cell carcinoma, pancreatic carcinoma, head and neck cancer, prostate cancer, malignant glioma, osteosarcoma, colon cancer, and rectal cancer, gastric cancer, malignant mesothelioma, multiple myeloma, ovarian cancer, cervical cancer, small cell lung cancer, non-small cell lung cancer, synovial - 48 039583 sarcoma, thyroid cancer, breast cancer, melanoma, testicular cancer, kidney cancer, esophagus cancer, uterine cancer, endometrial cancer and liver cancer. 18. A method for modulating an antitumor immune response in a subject, comprising administering to the subject an antibody that binds the GITR according to any one of claims 1 to 15, or an antigen-binding fragment thereof. 19. The method of claim 17 or 18, further comprising administering an antibody or antigen-binding fragment thereof that binds to a second T-cell activating receptor. 20. The method of claim 19 wherein the T cell activating receptor is CD28, OX40, CD137, CD27, or HVEM. 21. The method of any one of claims 17-20, further comprising administering an antibody, or an antigen-binding fragment thereof, that binds to an inhibitory T-cell receptor. 22. The method of claim 21, wherein the T cell inhibitory receptor is CTLA-4, PD-1, TIM-3, BTLA, VISTA, or LAG-3. 23. A method according to any one of claims 17-22, further comprising administering radiation therapy to said subject. 24. The method according to any one of claims 17-23, further comprising the administration of one or more chemotherapeutic agents. 25. The method of claim 22 wherein the T cell inhibitory receptor is PD1. 26. The method of claim 25, wherein the antibody that binds to the T cell receptor is REGN 2810. 27. The method according to any one of claims 17-26, wherein the antibody that binds to GITR is an antibody according to claim 1.