COMPOSITIONS AND METHODS OF USE FOR AUGMENTED IMMUNE RESPONSE AND CANCER THERAPY

Abstract

The present invention provides antibody compositions, including, e.g., antibodies, engineered antibodies and antibody fragments that bind to a tumor necrosis factor receptor superfamily member (i.e., 18), and compositions comprising one or more additional therapeutic agents. Provided compositions are useful in enhancing CD4+ and CD8+ T cell responses, and in the treatment, amelioration and prevention of diseases that can be counteracted with an augmented immune response, e.g., cancers. Also provided are methods of use of combinations that find use in treatment or prevention of cancerous or infectious conditions and disorders.

Claims

1.-75. (canceled)

76. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject an anti-GITR agonist antibody or antibody fragment thereof in combination with one or more additional therapeutic agents, wherein the anti-GITR agonist antibody or antibody fragment comprises (a) a heavy chain variable region (VH) comprising: a VHCDR1 of SEQ ID NO:22, a VHCDR2 of a sequence selected from any one of SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, or SEQ ID NO:27, and a VHCDR3 of SEQ ID NO:29 or SEQ ID NO:109; and a light chain variable region (VL) comprising: a VLCDR1 of SEQ ID NO:30 or SEQ ID NO:31, a VLCDR2 of SEQ ID NO:33, and a VLCDR3 of SEQ ID NO:34; or (b) a VH comprising: a VHCDR1 of SEQ ID NO:84, a VHCDR2 of SEQ ID NO:80, and a VHCDR3 of SEQ ID NO:29 or SEQ ID NO:109, and a VL comprising: a VLCDR1 of SEQ ID NO:85 or SEQ ID NO:86, a VLCDR2 of SEQ ID NO:82, and a VLCDR3 of SEQ ID NO:83, and wherein the one or more additional therapeutic agents are selected from any of the following: a STING agonist, a TLR agonist, an A2AR antagonist, or an oncolytic virus, a vascular endothelial growth factor receptor (VEGFR) inhibitor, a c-Met inhibitor, a TGFβ inhibitor, an IDO/TDO inhibitor, a vaccine, a bi- or tri-specific cell engager, an inhibitor of IAP (Inhibitor of Apoptosis Protein), an inhibitor of EGFR (Epidermal Growth Factor Receptor), an inhibitor of target of rapamycin (e.g., mTOR), IL-15 or a variant thereof, a CTLA-4 inhibitor, a bispecific antibody molecule that binds to CD3 and a tumor antigen, a PD-1 inhibitor, another GITR agonist, an inhibitor of an immune checkpoint molecule selected from PD-L1, PD-L2, CTLA4, TIM-3, LAG-3, CEACAM-1, CEACAM-5, VISTA, BTLA, TIGIT, LAIR1, CD 160, 2B4 or TGFR-β, a CSF-1/1R inhibitor, an IL-17 inhibitor, an IL-1β inhibitor, a CXCR2 inhibitor, an inhibitor of PI3Kγ or PI3Kδ, a BAFF-R inhibitor, a MALT-I/BTK inhibitor, a JAK inhibitor, a CRTH2 inhibitor, a PFKFB3 inhibitor, or an agonist of a costimulatory molecule selected from OX40, CD2, CD27, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, CD83 ligand, a chemotherapeutic agent, or a cytotoxin.

77. The method of claim 76, wherein the additional therapeutic agent is an inhibitor of an immune checkpoint molecule selected from PD-1, PD-L1, PD-L2, CTLA4, TIM-3, LAG-3, CEACAM-1, CEACAM-5, VISTA, BTLA, TIGIT, LAIR1, CD 160, 2B4 or TGFR-β.

78. The method of claim 76, wherein the additional therapeutic agent is an agonist of a costimulatory molecule selected from OX40, CD2, CD27, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3 or CD83 ligand.

79. The method of claim 76, wherein the additional therapeutic agent is an antagonist of CTLA4, LAG3, or TIM3, or an inhibitor of PD-1/PD-L1 interaction.

80. The method of claim 76, wherein the cancer is selected from epithelial cancers, carcinomas, sarcomas or lymphomas.

81. The method of claim 76, wherein the cancer is selected from renal cancer, lung cancer including non-small cell lung cancer (NSCLC), glioma, fibrosarcoma, pancreatic cancer, melanomas, breast cancer, lung cancer, bronchial cancer, colorectal cancer, prostate cancer, stomach cancer, ovarian cancer, urinary bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, cancer of the oral cavity or pharynx, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small bowel or appendix cancer, salivary gland cancer, thyroid gland cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, cancer of hematological tissues, or head and neck squamous cell carcinoma (HNSCC).

82. The method of claim 76, wherein the antibody or antibody fragment comprises a heavy chain variable segment that has at least 95% sequence identity to SEQ ID NO:16, and a light chain variable segment that has at least 95% sequence identity to SEQ ID NO:17.

83. The method of claim 76, wherein the antibody or antibody fragment comprises a heavy chain comprising SEQ ID NO:16 and a light chain comprising SEQ ID NO:17.

84. The method of claim 76, wherein the heavy chain framework region 4 (FR4) of the antibody or antibody fragment is a human germline FR4 comprising SEQ ID NO:42.

85. The method of claim 76, wherein the light chain FR4 of the antibody or antibody fragment is a human germline FR4 comprising SEQ ID NO:50.

86. The method of claim 76, wherein the antibody or antibody fragment comprises: (a) a VH comprising: a VHCDR1 of SEQ ID NO:22, a VHCDR2 of SEQ ID NO:23, and a VHCDR3 of SEQ ID NO:29, and a VL comprising: a VLCDR1 of SEQ ID NO:30, a VLCDR2 of SEQ ID NO:33, and a VLCDR3 of SEQ ID NO:34; (b) a VH comprising: a VHCDR1 of SEQ ID NO:84, a VHCDR2 of SEQ ID NO:80, and a VHCDR3 of SEQ ID NO:29, and a VL comprising: a VLCDR1 of SEQ ID NO:85, a VLCDR2 of SEQ ID NO:82, and a VLCDR3 of SEQ ID NO:83; (c) a VH comprising: a VHCDR1 of SEQ ID NO:22, a VHCDR2 of SEQ ID NO:24, and a VHCDR3 of SEQ ID NO:29, and a VL comprising: a VLCDR1 of SEQ ID NO:31, a VLCDR2 of SEQ ID NO:33, and a VLCDR3 of SEQ ID NO:34; (d) a VH comprising: a VHCDR1 of SEQ ID NO:84, a VHCDR2 of SEQ ID NO:80, and a VHCDR3 of SEQ ID NO:29, and a VL comprising: a VLCDR1 of SEQ ID NO:86, a VLCDR2 of SEQ ID NO:82, and a VLCDR3 of SEQ ID NO:83; (e) a VH comprising: a VHCDR1 of SEQ ID NO:22, a VHCDR2 of SEQ ID NO:25, and a VHCDR3 of SEQ ID NO:29, and a VL comprising: a VLCDR1 of SEQ ID NO:30, a VLCDR2 of SEQ ID NO:33, and a VLCDR3 of SEQ ID NO:34; (f) a VH comprising: a VHCDR1 of SEQ ID NO:22, a VHCDR2 of SEQ ID NO:26, and a VHCDR3 of SEQ ID NO:29, and a VL comprising: a VLCDR1 of SEQ ID NO:30, a VLCDR2 of SEQ ID NO:33, and a VLCDR3 of SEQ ID NO:34; (g) a VH comprising: a VHCDR1 of SEQ ID NO:84, a VHCDR2 of SEQ ID NO:80, and a VHCDR3 of SEQ ID NO:29, and a VL comprising: a VLCDR1 of SEQ ID NO:85, a VLCDR2 of SEQ ID NO:82, and a VLCDR3 of SEQ ID NO:83; (h) a VH comprising: a VHCDR1 of SEQ ID NO:22, a VHCDR2 of SEQ ID NO:27, and a VHCDR3 of SEQ ID NO:29, and a VL comprising: a VLCDR1 of SEQ ID NO:30, a VLCDR2 of SEQ ID NO:33, and a VLCDR3 of SEQ ID NO:34; (i) a VH comprising: a VHCDR1 of SEQ ID NO:22, a VHCDR2 of SEQ ID NO:25, and a VHCDR3 of SEQ ID NO:109, and a VL comprising: a VLCDR1 of SEQ ID NO:30, a VLCDR2 of SEQ ID NO:33, and a VLCDR3 of SEQ ID NO:34; or (j) a VH comprising: a VHCDR1 of SEQ ID NO:84, a VHCDR2 of SEQ ID NO:80, a VHCDR3 of SEQ ID NO:109, and a VL comprising: a VLCDR1 of SEQ ID NO:85, a VLCDR2 of SEQ ID NO:82, and a VLCDR3 of SEQ ID NO:83.

87. The method of claim 76, wherein the antibody or antibody fragment comprises a VH comprising an amino acid sequence that has at least 90% identity to any one of SEQ ID NOs: 6, 8, 10, 12, 14, 99 or 105.

88. The method of claim 76, wherein the antibody or antibody fragment comprises a VH comprising an amino acid sequence selected from any one of SEQ ID NOs: 6, 8, 10, 12, 14, 99, or 105.

89. The method of claim 76, wherein the antibody or antibody fragment comprises a VL comprising an amino acid sequence that has at least 90% identity to SEQ ID NO: 7 or SEQ ID NO: 9.

90. The method of claim 76, wherein the antibody or antibody fragment comprises a VL comprising an amino acid sequence selected from SEQ ID NO: 7 or SEQ ID NO: 9.

91. The method of claim 76, wherein the antibody or antibody fragment comprises: (a) a VH comprising SEQ ID NO:6, and a VL comprising SEQ ID NO:7; (b) a VH comprising SEQ ID NO:8, and a VL comprising SEQ ID NO:9; (c) a VH comprising SEQ ID NO:10, and a VL comprising SEQ ID NO:7; (d) a VH comprising SEQ ID NO:12, and a VL comprising SEQ ID NO:7; (e) a VH comprising SEQ ID NO:14, and a VL comprising SEQ ID NO:7; (f) a VH comprising SEQ ID NO:99, and a VL comprising SEQ ID NO:7; and (g) a VH comprising SEQ ID NO:105, and a VL comprising SEQ ID NO:7.

92. The method of claim 76, wherein the antibody or antibody fragment comprises a VH comprising SEQ ID NO:99, and a VL comprising SEQ ID NO:7.

93. The method of claim 76, wherein the antibody comprises a heavy chain comprising an amino acid sequence selected from any one of SEQ ID NOs: 65, 69, 73, 75, 77, 100, or 106; and a light chain comprising an amino acid sequence selected from SEQ ID NO:66 or SEQ ID NO:70.

94. The method of claim 76, wherein the antibody comprises: (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 65, and a light chain comprising the amino acid sequence of SEQ ID NO: 66; (b) a heavy chain comprising the amino acid sequence of SEQ ID NO: 69, and a light chain comprising the amino acid sequence of SEQ ID NO: 70; (c) a heavy chain comprising the amino acid sequence of SEQ ID NO: 73, and a light chain comprising the amino acid sequence of SEQ ID NO: 66; (d) a heavy chain comprising the amino acid sequence of SEQ ID NO:75, and a light chain comprising the amino acid sequence of SEQ ID NO: 66; (e) a heavy chain comprising the amino acid sequence of SEQ ID NO: 77, and a light chain comprising the amino acid sequence of SEQ ID NO: 66; (f) a heavy chain comprising the amino acid sequence of SEQ ID NO: 100, and a light chain comprising the amino acid sequence of SEQ ID NO: 66; or (g) a heavy chain comprising the amino acid sequence of SEQ ID NO: 106, and a light chain comprising the amino acid sequence of SEQ ID NO: 66.

95. The method of claim 76, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:100; and a light chain comprising the amino acid sequence of SEQ ID NO:66.

96. The method of claim 76, wherein the antibody or antibody fragment is humanized.

97. The method of claim 76, wherein the antibody fragment is a Fab, Fab′, F(ab′)2, Fd, Fv, or a single chain Fv fragment (scFv).

98. The method of claim 76, wherein the antibody or antibody fragment comprises a heavy chain constant region of human IgG1, and a light chain constant region of human kappa chain.

99. The method of claim 76, wherein the antibody or antibody fragment is cross-linked to a second anti-GITR antibody or antibody fragment.

100. The method of claim 76, wherein the antibody or antibody fragment induces an elevated Teff:Treg ratio in vivo.

101. The method of claim 76, wherein the antibody or antibody fragment induces a potentiated immune response in vivo.

102. A method of treating an infectious disease in a subject in need thereof, the method comprising administering to the subject an anti-GITR agonist antibody or antibody fragment thereof in combination with one or more additional therapeutic agents, wherein the anti-GITR agonist antibody or antibody fragment comprises (a) a heavy chain variable region (VH) comprising: a VHCDR1 of SEQ ID NO:22, a VHCDR2 of a sequence selected from any one of SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, or SEQ ID NO:27, and a VHCDR3 of SEQ ID NO:29 or SEQ ID NO:109; and a light chain variable region (VL) comprising: a VLCDR1 of SEQ ID NO:30 or SEQ ID NO:31, a VLCDR2 of SEQ ID NO:33, and a VLCDR3 of SEQ ID NO:34; or (b) a VH comprising: a VHCDR1 of SEQ ID NO:84, a VHCDR2 of SEQ ID NO:80, and a VHCDR3 of SEQ ID NO:29 or SEQ ID NO:109, and a VL comprising: a VLCDR1 of SEQ ID NO:85 or SEQ ID NO:86, a VLCDR2 of SEQ ID NO:82, and a VLCDR3 of SEQ ID NO:83, and wherein the one or more additional therapeutic agents are selected from any of the following: a STING agonist, a TLR agonist, an A2AR antagonist, or an oncolytic virus, a vascular endothelial growth factor receptor (VEGFR) inhibitor, a c-Met inhibitor, a TGFβ inhibitor, an IDO/TDO inhibitor, a vaccine, a bi- or tri-specific cell engager, an inhibitor of IAP (Inhibitor of Apoptosis Protein), an inhibitor of EGFR (Epidermal Growth Factor Receptor), an inhibitor of target of rapamycin (e.g., mTOR), IL-15 or a variant thereof, a CTLA-4 inhibitor, a bispecific antibody molecule that binds to CD3 and a tumor antigen, a PD-1 inhibitor, another GITR agonist, an inhibitor of an immune checkpoint molecule selected from PD-L1, PD-L2, CTLA4, TIM-3, LAG-3, CEACAM-1, CEACAM-5, VISTA, BTLA, TIGIT, LAIR1, CD 160, 2B4 or TGFR-β, a CSF-1/1R inhibitor, an IL-17 inhibitor, an IL-1β inhibitor, a CXCR2 inhibitor, an inhibitor of PI3Kγ or PI3Kδ, a BAFF-R inhibitor, a MALT-I/BTK inhibitor, a JAK inhibitor, a CRTH2 inhibitor, a PFKFB3 inhibitor, or an agonist of a costimulatory molecule selected from OX40, CD2, CD27, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, or CD83 ligand.

103. The method of claim 102, wherein the additional therapeutic agent is an inhibitor of an immune checkpoint molecule selected from PD-1, PD-L1, PD-L2, CTLA4, TIM-3, LAG-3, CEACAM-1, CEACAM-5, VISTA, BTLA, TIGIT, LAIR1, CD 160, 2B4 or TGFR-β.

104. The method of claim 102, wherein the additional therapeutic agent is an agonist of a costimulatory molecule selected from OX40, CD2, CD27, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3 or CD83 ligand.

105. The method of claim 102, wherein the additional therapeutic agent is an antagonist of CTLA4, LAG3, or TIM3, or an inhibitor of PD-1/PD-L1 interaction.

106. The method of claim 102, wherein the infectious disease is selected from a bacterial infection, a fungal infection, a viral infection or a parasitic infection.

107. A method of enhancing a T cell response in a subject in need thereof, comprising administering to the subject an anti-GITR agonist antibody or antibody fragment thereof in combination with one or more additional therapeutic agents, wherein the anti-GITR agonist antibody or antibody fragment comprises (a) a heavy chain variable region (VH) comprising: a VHCDR1 of SEQ ID NO:22, a VHCDR2 of a sequence selected from any one of SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, or SEQ ID NO:27, and a VHCDR3 of SEQ ID NO:29 or SEQ ID NO:109; and a light chain variable region (VL) comprising: a VLCDR1 of SEQ ID NO:30 or SEQ ID NO:31, a VLCDR2 of SEQ ID NO:33, and a VLCDR3 of SEQ ID NO:34; or (b) a VH comprising: a VHCDR1 of SEQ ID NO:84, a VHCDR2 of SEQ ID NO:80, and a VHCDR3 of SEQ ID NO:29 or SEQ ID NO:109, and a VL comprising: a VLCDR1 of SEQ ID NO:85 or SEQ ID NO:86, a VLCDR2 of SEQ ID NO:82, and a VLCDR3 of SEQ ID NO:83, and wherein the one or more additional therapeutic agents are selected from any of the following: a STING agonist, a TLR agonist, an A2AR antagonist, or an oncolytic virus, a vascular endothelial growth factor receptor (VEGFR) inhibitor, a c-Met inhibitor, a TGFβ inhibitor, an IDO/TDO inhibitor, a vaccine, a bi- or tri-specific cell engager, an inhibitor of IAP (Inhibitor of Apoptosis Protein), an inhibitor of EGFR (Epidermal Growth Factor Receptor), an inhibitor of target of rapamycin (e.g., mTOR), IL-15 or a variant thereof, a CTLA-4 inhibitor, a bispecific antibody molecule that binds to CD3 and a tumor antigen, a PD-1 inhibitor, another GITR agonist, an inhibitor of an immune checkpoint molecule selected from PD-L1, PD-L2, CTLA4, TIM-3, LAG-3, CEACAM-1, CEACAM-5, VISTA, BTLA, TIGIT, LAIR1, CD 160, 2B4 or TGFR-β, a CSF-1/1R inhibitor, an IL-17 inhibitor, an IL-1β inhibitor, a CXCR2 inhibitor, an inhibitor of PI3Kγ or PI3Kδ, a BAFF-R inhibitor, a MALT-I/BTK inhibitor, a JAK inhibitor, a CRTH2 inhibitor, a PFKFB3 inhibitor, or an agonist of a costimulatory molecule selected from OX40, CD2, CD27, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, or CD83 ligand.

108. The method of claim 107, wherein the additional therapeutic agent is an inhibitor of an immune checkpoint molecule selected from PD-1, PD-L1, PD-L2, CTLA4, TIM-3, LAG-3, CEACAM-1, CEACAM-5, VISTA, BTLA, TIGIT, LAIR1, CD 160, 2B4 or TGFR-β.

109. The method of claim 107, wherein the additional therapeutic agent is an agonist of a costimulatory molecule selected from OX40, CD2, CD27, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3 or CD83 ligand.

110. The method of claim 107, wherein the additional therapeutic agent is an antagonist of CTLA4, LAG3, or TIM3, or an inhibitor of PD-1/PD-L1 interaction.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0639] FIG. 1A-1D illustrate epitope mapping of the GITR mAbs of the invention. FIG. 1A depicts results of hydrogen/deuterium exchange coupled to mass spectrometry (HDXMS) analyses using Fc-GITR fusion (top and middle trace) and HIS-GITR (lower trace) fusion proteins and MAB1 parental Ab. Numbering reflects removal of native GITR signal peptide (AA 1-26) sequence. FIG. 1B depicts a schematic of N-terminal deletion constructs prepared using the extracellular domain of human GITR (hGITR ECD). FIG. 1C depicts results of binding of MAB4 and MAB5 to hGITR ECD constructs. N-terminal deletion of cysteine-rich domain 1 (CRD1) from human GITR (hGITR) extracellular domain (ECD) abrogates binding of MAB4 and MAB5 to hGITR. Similar results were obtained for MAB7 (data not shown). FIG. 1D depicts results of alanine scanning mutagenesis. MAB7 bound to all mutant proteins with the exception of GITR mutant E78A. ForteBio™ binding analysis was carried out, and results also confirmed loss of MAB7 binding to hGITRE78A mutant protein (data not shown). Results implicates a region of ECD of GITR including CRD1 and including E78 (SEQ ID NO:88: RPTGGPGCGPGRLLGTGTDARCCRVHTTRCCRDYPGEECCSEWDCMCVQPEFHCGD) as a region and potential epitope involved in binding MAB1 and MAB7 (parental mAb).

[0640] FIG. 2A-2E depicts results of binding experiments of anti-GITR MAB antibodies. FIGS. 2A and 2B illustrates MAB4, and MAB5 specifically bind to GITR from human and cynomolgus monkeys (2A) but not from rodent (2B), as determined by ELISA assays. FIG. 2C illustrates MAB7 shares a similar profile binding human and cyno GITR but not murine GITR by ELISA assay. FIG. 2D illustrates MAB7 competes with GITR-ligand binding as determined by FACS competition analysis. FIG. 2E illustrates results of ELISA assays showing that the anti-GITR antibodies of the invention (e.g., MAB4, MAB5) do not bind to other members of the TNF receptor superfamily (TNFRSF). Protagen™ chip assays also confirmed that the antibodies do not bind to other off-target proteins (not shown).

[0641] FIG. 3A-3D depict intracellular signaling in 293 cells that have been engineered to express GITR. FIG. 3A illustrates that recombinant human GITR ligand (GITR-L) activates intracellular signaling in 293 cells that have been stably transfected to overexpress human GITR. FIG. 3B illustrates that monoclonal antibodies MAB4 and MAB5 activate intracellular signaling in 293 cells that have been transfected to overexpress human GITR comparably to GITR-L when the antibodies are cross-linked (EC.sub.50 for GITRL is about 65 nM versus EC.sub.50 of about 2.5 nM for agonist antibodies in the presence of cross-linker). FIG. 3C illustrates that cross-linked MAB antibody activates intracellular signaling in cells, as MAB7 and MAB8 also promote NFκB activation in 293 cells stably transfected with human GITR and the NFkB-Luciferase reporter gene in a similar manner to cross-linked MAB4. FIG. 3D illustrates that Cross-linked MAB4 and MAB5 promote NFκB activation in 293 cells that have been stably transfected with cyno GITR and the NFκB-Luciferase reporter gene. Similar activation was seen with cross-linked MAB7 (data not shown).

[0642] FIG. 4A-4C depicts in vitro co-stimulatory activity of MAB7 on T cells is dependent upon T cell activation. Anti-CD3 (OKT3), anti-CD28 (CD28.2) and anti-GITR mAbs were cross-linked (at a ratio of 1:1:3) on beads and then incubated with PBMCs. FIG. 4A illustrates MAB7 is a co-activator of CD4+ T cells and stimulates T cell proliferation in CD4+ Tcells. FIG. 4B illustrates MAB7 is a co-activator of CD8+ T cells and stimulates T cell proliferation in CD8+ Tcells. FIG. 4C illustrates cytokine production, e.g. IFNγ production following TCR engagement is increased in conjunction with MAB7. Similar results were seen for MAB4 and 5 (data not shown).

[0643] FIG. 5A-D illustrates in vitro ADCC activity of MAB7 in GITR expressing cells at varying levels. FIG. 5A, FIG. 5B, FIG. 5C, and FIG. 5D each depict results of ADCC activity using control or MAB7 antibody with various levels of GITR expression. MAB7 is able to induce signaling through the FcgRllla, with increased activity upon increased levels of GITR signaling.

[0644] FIG. 6A-6D illustrates GITR is functional in hGITR-hGITRL knock-in mice. Splenocytes were isolated from hGITR-hGITRL knock-in mice and cultured either unstimulated or stimulated with aCD3 and aCD8 antibodies for 48 hours, then pulsed with controls or MAB7 at varying concentrations for 30 minutes, then fixed and stained with fluorophore-conjugated antibodies and analyzed by flow cytometry. FIG. 6A depicts results showing expression of hGITR is upregulated on stimulated CD8+ T cells. FIG. 6B depicts results of anti hGITR antibody binding to T cells by hFc staining, showing MAB7 can bind to hGITR expressed on mouse CD8+ T cells. FIGS. 6C and 6D depict MAB7 binding to stimulated CD8+ T cells correlates with increased T cell activation, as shown by intracellular pIKK staining (6C) and T cell activation (6D). *p<0.05, **p<0.005.

[0645] FIG. 7A-7C illustrates MAB7 is functional in vivo. hGITR-hGITRL double knock-in mice with established Colon26 tumors were treated with a single dose of vehicle (n=8/timepoint) or MAB7 (n=10/timepoint) antibody. FIG. 7A depicts results of tumor measurements twice per week and tumor volume calculated using the equation (L×W.sup.2)/2. Data shown is from the fifteen (15)-day time point group. FIGS. 7B and 7C depict results from whole blood and FIGS. 7C and 7D depict results from tumors were collected 3-days post-dose and analyzed by flow cytometry for cell surface hGITR expression on immune cells. (*p<0.05, ****p<0.00005).

[0646] FIG. 8A-8E illustrates MAB7 elicits an anti-tumor immune response to Colon26 tumors in vivo. hGITR-hGITRL double knock-in mice with established Colon26 tumors were treated with a single dose of vehicle (n=8/timepoint) or MAB7 (n=10/timepoint). FIG. 8A depicts results of Tregulatory cells 3-days post-dose. FIG. 8B-8C depict results of lymphocytes (8B) and activated CD8+ T cells (8C) present in tumor site following treatment levels in tumor 15-days post-dose. The absolute number of cells was normalized to tumor size to account for the significant difference in tumor size between Vehicle and MAB7 treated groups. FIG. 8D depicts Teff/Treg ratio resulting in treated animals as determined by total intratumoral activated CD8+ T cells compared to CD4+ FOXP3+ Tregs to generate T.sub.eff/T.sub.reg ratios. FIG. 8E depicts results of splenocyte assays from purified CD8+ T cells incubated with Colon26 tumor cells ex-vivo, and measuring CTL response using IFNg ELISPOT assay. (*p<0.05, ***p<0.0005).

[0647] FIG. 9A-9C illustrates PD-1 expression is upregulated on CD8+ T cells in Colon26 tumors as well as spleens after treatment with a murine surrogate GITR antibody, DTA-1. Single cell suspensions of whole tumors or whole spleens were profiled by flow cytometry following 2 doses of DTA-1. FIG. 9A depicts results of PD-1 positive cells assessed as a percentage of total CD19-CD3+CD8+ T cells. FIG. 9B depicts results of PD-1 positive cells normalized to tumor size by absolute number of PD-1+CD19-CD3+CD8+ T cells per mm.sup.3 volume of tumor. FIG. 9C depicts results of PD-1 expression is upregulated on CD8+ T cells in spleens of Colon26 tumor bearing mice after treatment with DTA-1. PD-1 positive cells were assessed as a percentage of total CD19-CD3+CD8+ T cells. (*p<0.05 and ****p<0.0005).

[0648] FIG. 10 illustrates anti-GITR and anti-PD-1 combinations confer survival advantage compared to isotype control. Depicted are results in Colon26 mice models treated with anti-GITR (IgG2a-DTA-1) and anti-PD-1 (RMP1-14) individually or in combination as compared to isotype control.

[0649] FIG. 11 illustrates expression of LAG3(first column), TIM3(middle column), and PD1(right column) after treatment with anti-GITR, anti-PD-1 and anti-GITR/anti-PD-1 in combinations in mice with established Colon26 tumors as compared to treatment with isotype control Ab. Depicted are results in Colon26 mice models treated with anti-GITR (IgG2a-DTA-1) and anti-PD-1 (RMP1-14) individually or in combination as compared to isotype control. The top row demonstrates results in tumor samples, and the lower row depicts results in spleen samples. LAG3, TIM3 and PD1 expression is upregulated on CD8+ T cells in Colon26 tumors after treatment with a-GITR, and a-PD1 PD-1 expression is upregulated on CD8+ T cells in Colon26 tumors after treatment with anti-GITR/anti-PD-1 in combination.

EXEMPLIFICATION

Creation of GITR Agonist Antibodies MAB2, MAB3, MAB4, MAB5, MAB6, MAB7 and MAB8

[0650] Human antibodies MAB2, MAB3, MAB4, MAB5, MAB6, MAB7 and MAB8 were generated by engineering a murine monoclonal GITR agonist antibody MAB1 to have greater sequence homology to a human germline antibody. MAB2, MAB3, MAB4, MAB5, MAB6, MAB7 and MAB8 retain the epitope specificity, affinity, and cynomolgus macaque GITR cross-reactivity of the parental murine antibody, MAB1. MAB2, MAB3, MAB4, MAB5, MAB6, MAB7 and MAB8 have much higher homology to the human germline sequence than the original murine antibody and should therefore be better tolerated by the human immune system.

[0651] Mouse monoclonal MAB1 was engineered to bring its protein sequence closer to a human germline sequence and decrease its immunogenicity using the Humaneered® technology platform available through KaloBios, (South San Francisco, Calif. (on the worldwide web at kalobios.com)). Humaneered® antibodies are very close to human antibodies with V-region sequences that have high homology to a human germline sequence while still retaining the specificity and affinity of the parent or reference antibody (U.S. Patent Publ. 2005/0255552 and 2006/0134098). The process first identifies the minimum antigen binding specificity determinants (BSDs) in heavy and light chain variable regions of a reference Fab (typically sequences within the heavy chain CDR3 and the light chain CDR3). As these heavy and light chain BSDs are maintained in all libraries constructed during the process, each library is epitope-focused, and the resulting Humaneered® antibodies retain the epitope specificity of the original mouse antibody.

[0652] Next, full chain libraries (in which an entire light or heavy chain variable region is replaced with a library of human sequences) and/or cassette libraries (in which a portion of the heavy or light chain variable region of the mouse Fab is replaced with a library of human sequences) are generated. A bacterial secretion system is used to express members of the library as antibody Fab fragments, and the library is screened for Fabs that bind antigen using a colony lift binding assay (CLBA). Positive clones are further characterized to identify those with the highest affinity. Identified human cassettes supporting binding in the context of residual murine sequences are the combined in a final library screen to generate completely human V-regions.

[0653] Resulting Humaneered® antibody Fabs have V-segment sequences derived from human libraries, retain the short BSD sequences identified within the CDR3 regions, and have human germline Framework 4 regions. These Fabs are converted to full IgGs by cloning variable regions of the heavy and light chains into IgG expression vectors. Humaneered® antibodies generated in this process retain the binding specificity of the parent, murine antibody, typically having equivalent or higher affinity for antigen that the parent antibody, and have V-regions with a high degree of sequence identity compared with human germline antibody genes at the protein level.

Methods

Generation of Murine Anti-GITR mAb MAB1

[0654] Bcl-2 transgenic mice (C57BL/6-Tgn (bcl-2) 22 wehi strain) were immunized with the N-terminal region of human GITR (aa 26-161) using a procedure that calls for Repetitive Immunization at Multiple Sites (RIMMS) (McIntyre G D. Hybridoma 1997) followed by hybridoma generation from high titer mice. A hybridoma secreting MAB1 was identified and selected using a sandwich ELISA against hGITR and an NFκB Reporter Gene Assay to confirm hGITR binding and agonist activity.

Cloning of Murine V-Regions

[0655] Variable region DNA from murine monoclonal MAB1 was amplified by RT-PCR from RNA obtained from the hybridoma cell line using standard methods. Heavy chain variable region was amplified from MAB1 cDNA with HV3 (5′-GGGTCTAGACACCATGGCTGTCTTGGGGCTGCTCTTC-3′ (SEQ ID NO:95)) and HCconstant (5′-GCGTCTAGAAYCTCCACACACAGGRRCCAGTGGATAGAC-3′ (SEQ ID NO:96)). Light chain variable region was amplified from the same cDNA with LV3 (5′-GGGTCTAGACACCATGGAGWCACAKWCTCAGGTCTTTRTA-3′ (SEQ ID NO:97)) and LCconstant (5′-GCGTCTAGAACTGGATGGTGGGAAGATGG-3′ (SEQ ID NO:19)). Variable heavy and light chain products were inserted into a pcDNA3.1 vector and sequence verified. The heavy and light vectors were used as templates for PCR incorporating restriction enzyme sites for cloning into KaloBios vectors: Vh into KB1292-His (modified version of KB1292 that encodes a C-terminal flexible linker and 6-His tag (SEQ ID NO:11) of amino acid sequence AAGASHHHHHH (SEQ ID NO:13) on CH1) at NcoI (5′) and Nhel (3′); Vk into KB1296 at NcoI (5′) and BsiWI (3′). These separate heavy and light chain vectors were then combined into a single dicistronic KaloBios Fab expression vector by restriction digest with BssHII and ClaI and ligation. Fab fragments were expressed in E. coli from this vector. This Fab was tested for hGITR-antigen binding and is referred to as MAB1rFab.

Fab Purification

[0656] Fab fragments were expressed by secretion from E. coli using KaloBios expression vectors. Cells were grown in 2×YT medium to an OD500 of ˜0.6. Expression was induced by adding IPTG to 100 μM and shaking for 4 hours at 33° C. Assembled Fab was obtained from periplasmic fractions by osmotic lysis and purification by affinity chromatography using Ni-NTA columns HisTrap HP columns; GE Healthcare catalog #17-5247-01) according to standard methods. Fabs were eluted in buffer containing 500 mM imidazole and thoroughly dialyzed against PBS pH7.4 without calcium and magnesium.

Library Construction

[0657] To limit the complexity to identify complimentary human CDRs that support BSD-FR4 in human GITR binding, a cassette library approach, in which only part of the parent murine V-segment is replaced by a library of human sequences, was taken. The original murine MAB1 Vk is closest to human germline VkIII, so a mixture of two KaloBios libraries that contains VkIII germlines (KB1423 and KB1424) was used in making the Vk cassette libraries. KaloBios libraries that contains VH3 germlines (KB1413, KB1414) were used to construct Vh cassette libraries. Two types of cassettes were constructed by overlap PCR: front-end cassettes (8C1VK3FE-01, and MAB1VH3FE-01) containing human sequences in FR1, CDR1, and FR2, and FR3 cassettes (MAB1VK3FR3-01, and MAB1VH3FR3-01) containing human sequences in the FR3 were amplified using the above mentioned germline restricted KaloBios libraries. Each Vh cassette library was cloned into vector KB1292-His at NcoI (5′) and KpnI (3′); each Vk cassette library was cloned into vector KB1296-B (modified version of KaloBios vector KB1296 which has a silent HindIII site added in FR4) at NcoI (5′) and HindIII (3′). Resultant Vh or Vk plasmid libraries were then combined with the complementary chain from the optimized reference Fab (MAB1opVK or MAB1 opVH (e.g., the Vh front-end library was combined with the optimized reference Vk vector) by digestion with BssHII and ClaI and subsequent ligation to create libraries of dicistronic vectors expressing full Fabs.

[0658] No VH3 front-end clones bound the human GITR with high affinity, thus, a second VH3 front-end library (MAB1VH3FE-02) was constructed. This library contains human sequences in FR1, FR2, and a collection of CDR2s encoding either the parental murine residue or the selected human germline residue at all positions. The FR3 region sequences of this library were from six clones selected from the VH3FR3 library (MAB1VH3FR3-01).

[0659] The final Vk full-chain library (MAB1VK3FCL-01) was constructed by combining clones from VK front-end and VKFR3 cassette libraries with mutagenic VK CDR2s that encodes either the parental murine or the selected human germline residue at all positions. The resulting Vk full-chain library was cloned into KB1296b at NcoI and HindIII sites. This VK full-chain library was paired with a number of selected VH3FR3 library clones to allow functional Fab expression and screened by CLBS. The antigen specific clones were confirmed by human GITR specific ELISA and ranked by antigen affinity titration ELISA. The VH3 full-chain library (MAB1VH3FcL-01) was generated using the selected clones from the second VH3 front end library (MAB1VH3FE-02) with a collection of CDR2 sequences containing either the parental murine or human residue at each position. This VH full-chain library was cloned into KB1292-his at NcoI and KpnI sites. To yield the final full-chain human Fab expression library, selected VK full-chain clones were combined with VH full-chain library at BssHII and ClaI sites.

General ELISA

[0660] Recombinant human GITR and human Fc fusion protein (hGITR-hFc) was used for all ELISA assays. Typically, hGITR-hFc antigen diluted in PBS pH 7.4 was bound to a 96-well microtiter plate at 200 ng/well by overnight incubation at 4° C. After being rinsed three times with PBST, the plate was blocked with a solution of 1% BSA in PBS for one hour at 37° C., and then rinsed once with PBST. Fab-containing cell medium or diluted, purified Fab (50 μL) was then added to each well. After a one-hour incubation at 37° C., or overnight incubation at 4° C., the plate was rinsed three times with PBST. Anti-human-kappa chain HRP conjugate (Sigma #A7164) diluted 1:5000 in PBST (50 μL) was added to each well, and the plate was incubated for 45 min at room temperature. The plate was washed three times with PBST, then 100 μL of SureBlue TMB substrate (KPL #52-00-03) was added to each well and the plate was incubated for about 10 min at room temperature. The plate was read at 650 nm in a spectrophotometer.

Affinity Titration ELISA

[0661] In order to evaluate antigen binding of the selected Fab producing clones, an affinity titration ELISA was developed. This assay combines two consecutive ELISA steps: the first one, using goat anti-human Fab (Jackson ImmunoResearch Lab #109-005-097) capture and goat anti-human Kappa (Sigma #A7164) detection, measures Fab concentrations in cell culture medium to normalize the amount of Fab used in the second antigen titration ELISA; the second ELISA, a normal antigen specific ELISA, generates an antigen binding dilution curve with the same amount of starting Fab. By comparing the dilution curves of different clones the high affinity clones are identified.

Colony Lift Binding ELISA (CLBA)

[0662] Screening of Humaneered® antibody libraries of Fab fragments was carried out essentially as described in (U.S. Patent Publ. 2005/0255552 and 2006/0134098) using nitrocellulose filters coated with hGITR-hFC at 2.0 μg/mL in PBS pH7.4. Fabs bound to antigen-coated filter were detected using goat anti-human Kappa chain HRP conjugate (Sigma #A7164) diluted 1:5000 in PBST, and blots were developed with ECL plus Western Blotting Detection System (GE Healthcare #RPN2132).

Removal of Glycation Site in MAB4

[0663] A glycation site “KH” in the junction of FR3 and CDR3 of MAB4 heavy chain was removed by replacing the lysine with an arginine, or replacing the lysine with an arginine and the histidine with an Asparagine. The KH to RH and KH to RN conversions were accomplished by PCR based mutagenesis using the p50H plasmid as the DNA template. The reverse primer (TCTGGCGCAGTAATACACGGCC, SEQ ID NO:110) incorporated an arginine in place of the lysine; the forward primer (NNKGCCTATGGCCATGATGGCG, SEQ ID NO:111) had the degenerate NNK trinucleotide at the histidine site. PCR reactions were performed with 100 ng of template, 0.2 μM of each primer, 200 μM dNTPs, and 2.5 U of pfuUltraII DNA polymerase (Strategene) in a 50 μl reaction volume. The PCR conditions were 94° C. for 3 min for 1 cycle; 94° C. for 15 seconds, 52° C. for 20 seconds, and 65° C. for 5 minutes for 30 cycles; and finally, 1 cycle at 72° C. for 5 minutes. Dpnl (2 U) was added to the PCR reaction and incubated at 37° C. for 30 minutes to remove the template p50H. Amplified MAB4 heavy chain variants were separated by a 1% SYBR gel and purified using a Qiagen Gel Purification Kit. The gel purified PCR product was treated with T4 DNA polynucleotide kinase, ligated and transformed into DH5a chemically competent cells (Invitrogen) under ampicillin selection.

[0664] Clones hosting the MAB7 and MAB8 heavy chain were selected by colony PCR using the forward (GCCTTTCTCTCCACAGG, SEQ ID NO:112) and reverse (GGCAAACAACAGATGGCTGG, SEQ ID NO:113) primers following GoTaqClear protocol (Promega). The PCR conditions were 94° C. for 3 minutes for 1 cycle; 94° C. for 10 seconds, 55° C. for 30 seconds, 72° C. for 45 seconds for 25 times; and finally, one cycle at 72° C. for 5 minutes. PCR reactions were cleaned up for sequencing by incubating the samples at 37° C. for 30 minutes and 80° C. for 15 minutes with Exonuclease I and Shrimp Alkaline Phosphotase. PCR samples were sequenced and the results were analyzed using Clone Manager software.

Antibody Production and Purification

[0665] Generated antibodies MAB2, MAB3, MAB4, MAB5, MAB6, MAB7 and MAB8 (IgG1 kappa) were produced by co-transfection of vectors as follows into 293 Freestyle cells using 293fectin transfection reagent (Invitrogen #51-0031) according to the manufacturer's protocol. [0666] MAB2—p35H+p35kappa [0667] MAB3—p38H+p38kappa [0668] MAB4—p50H+p35kappa [0669] MAB5—p51H+p35kappa [0670] MAB6—p56H+p35kappa [0671] MAB7—pMAB7H+p35kappa [0672] MAB8—pMAB8H+p35kappa

[0673] Antibody was purified from 293 Freestyle cells supernatants using a 5-mL HiTrap Protein A HP column (GE Healthcare #17-0403-03). Antibody was eluted using IgG Elution Buffer (Pierce #21004), and buffer exchanged into PBS by dialysis. Protein A affinity chromatography was performed on an AKTA-FPLC liquid chromatography system (GE Healthcare).

Specificity ELISA

[0674] For the specificity ELISA, a crude cell lysate was made from bacteria expressing members of the TNFRSF family. To prevent nonspecific binding to the plate 50 μL of 5% BSA was added per mL of bacterial lysate. A HisGrab Nickel 96-well plate (Pierce #15142) was coated with TNFRSF containing bacterial lysate at 100 μL of lysate/BSA per well and incubated for 1 hour at room temperature. The plate was then rinsed three times with PBST, then MAB was diluted to 0.5 μg/mL in 10% FBS in PBS and 100 μL was added to each well. The plate was incubated for 1 hour at room temperature and then rinsed three times with PBST. Anti-human kappa antibody (Sigma #A7164) conjugated to HRP was diluted 1:5000 in 1:1 PBST: 10% FBS in PBS and 100 μL added to each well. The plate was incubated for 1 hour at room temperature, and then washed three times with PBST. 100 μL of SureBlue TMB substrate was added to each well and the plate was incubated for about 10 min at room temperature before stopping the reaction with 100 μL/well of 2N H.sub.2SO.sub.4. The plate was read at 450 nm in a spectrophotometer.

ELISA (GITR Binind, Species Cross-Reactivity, Alanine Scanning)

[0675] Binding of the MABs to GITR from various species, various alanine mutant constructs or GITR extracellular domain—was assessed using a 384-well plate was coated with rat, mouse, human or cyno GITR extracellular domain (ECD) at 50 ng per well and incubated overnight at 4° C. The plate was blocked with a solution of 1% BSA in PBS for one hour at room temperature and then rinsed three times with PBST. The MAB was then diluted to 0.5 μg/mL or 1 μg/mL in PBS and 20 μL was added to each well. The plate was incubated for 1 hour at room temperature and then washed three times with PBST. Anti-human kappa antibody (Sigma #A7164), anti-human gamma antibody (Jackson Immunoresearch 109-036-098), goat anti-mouse antibody (Jackson ImmunoResearch 115-035-071) conjugated to HRP was diluted 1:5000 in Blocking Buffer (25 μL) and added to each well, or a hrp conjugated HIS antibody (QIAGEN 1014992) diluted 1:1000 in Blocking Buffer was added. The plate was incubated for 1 hour at room temperature, and then washed six times with PBST. 25 μL of SureBlue TMB (KPL 52-00-02) substrate was added to each well and the plate was incubated for about 10 min at room temperature. Plates were read at 650 nm in a spectrophotometer.

Cell Lines, Cells

Cell Lines

[0676] To generate the 293-hGITR-NFκB reporter gene cells line, 293 cells were stably transfected with an NFκB-Luciferase reporter gene and human GITR (or cyno GITR). Activation of the GITR signaling pathway in these cells was determined by measuring the levels of luciferase induced within the cells after a 24 hour incubation with GITR-L or agonistic antibody. To assess the effects of cross-linking Abs, they were incubated with an excess of a F(ab′).sub.2 goat anti-human Fcγ fragment specific antibody or protein A before using in the reporter gene assay.

[0677] Clonal Daudi cell lines were generated that express levels of GITR seen on human immune cells.

[0678] Cynomolgus monkey PBMCs were prepared and GITR binding determined using MABs. Briefly, cynomolgus blood was transferred into 50 mL conical tubes (Falcon, #352098), then diluted 1:2 in PBS (HyClone, #SH30256.01) and mixed. Diluted blood was carefully layered on top of 18 mL of 90% Ficoll Paque PLUS (GE Healthcare #17-1440-03 diluted in PBS), and tubes were spun at 2,000×g in a bench top centrifuge for 30 minutes at room temperature, with no brake. The plasma layer was carefully removed without disturbing the diffuse PBMC layer on top of the Ficoll. PBMCs were then carefully harvested and PBS was added to the isolated PBMCs until the volume in the conical tube was 45 mL, mixed, and then spun at 300×g in a bench top centrifuge for 15 minutes at room temperature. Supernatant was carefully aspirated and 4 mL of 1×BD Lysing solution (BD #555899) was added and the samples were gently vortexed. After incubating at room temperature in the dark for 3 minutes, 40 mL of PBS was added to each sample and they were spun at 200×g in a bench top centrifuge for 10 minutes at room temperature. Supernatant was carefully aspirated and the pellet was washed two times in 45 mL of PBS before being spun at 200×g in a bench top centrifuge for 10 minutes at room temperature. Resulting pellet was filtered and resuspended at 1×10.sup.6 cells/mL in CTL Test media (CTLT-005) supplemented with penicillin/streptomycin/glutamine (Hyclone #SV30082.01). 100 μL of purified cynomolgus PBMCs were placed in a 96 well round bottom plate (Corning, #3799). To activate the PBMCs, 100 μL of M-280 Tosylactivated dynabeads (Life Technologies #142.04) conjugated with SP34-2/CD28.2 antibodies was added to each well. A ratio of 3:1 CD3/CD28 Beads to PBMCs was used and the plates were incubated in a 37° C. tissue culture incubator for 48 hours. For day 0 staining 200 μL of PBMCs was placed in a 96-well round bottom plate (Corning, #3799). For samples that were stimulated for 48 hours 100 μL of supernatant was carefully removed and the remaining content of the well was carefully resuspended and 200 μL transferred to the FACS staining plate.

FACS

[0679] Plates were prepared with cells resuspended in 200 μL of cold PBS. LIVE/DEAD fixable stain (Life Technologies #L23105) was reconstituted in 50 μL of DMSO and 1 μL of reconstituted stain was added/mL of cold PBS, and cell pellets were immediately resuspended in 100 μL of the LIVE/DEAD PBS solution, incubated for 30 minutes on ice protected from light, then washed and resuspended in 100 μL of cold FACS Buffer containing 2 μg/mL of MAB7 or an Isotype Human IgG1 control antibody and plates incubated for 30 minutes on ice protected from light. Wash and resuspension in 100 μL of antibody cocktail (PerCP Cγ5.5 anti-human CD3 (BD #552852), Alexa Fluor 700 anti-human CD4 (BD #560836), V450 anti-human CD8 (BD #561426), PE-Cγ7 anti-human CD25 (BD #561405) and PE anti-human in FACS Buffer (Jackson Immuno #109-116-098)) followed and plates were then incubated for 30 minutes on ice protected from light and then spun in a bench top centrifuge at 3,200 RPM for 1 minute at 4° C. Cells were washed in FACS Buffer then resuspended in 100 μL of BD CytoFix (BD #554655) and plates were incubated for 15 minutes at room temperature protected from light then washed twice and resuspended in 100 μL of FACS Buffer. Plates were covered with foil (Beckman Coulter, #538619) and stored at 4° C. until ready to read. On the day of FACS read the plate was spun in a bench top centrifuge at 3,200 RPM for 1 minute and 50 μL of CML latex beads (Life Technologies #C37259), 4×10.sup.5/mL in FACS Buffer, was added to each well. The plates were read on a BD Fortessa flow cytometer and data analyzed using FlowJo.

Transgenic Mice

[0680] hGITR knock-in mice were generated by replacing the entire coding sequence (exons and introns) of mouse GITR with the human GITR cDNA sequence. Untranslated sequences upstream of the start codon and downstream of the stop codon are from mouse genome. Gene targeting was done by standard techniques in BALB/c ES cells with targeting vectors bearing BALB/c derived homology arms. Several ES cell clones were identified by PCR and confirmed by Southern blotting to contain the exact human cDNA knockin. Following standard mouse embryology techniques, positive ES cell clones were injected into blastocysts, which were transferred into pseudopregnant recipient foster mothers to derive chimeric offspring. Male chimeric mice were crossed with BALB/c females expressing Cre recombinase in their germline to excise the loxP flanked neomycin resistance cassette. One clone resulted in white offspring indicating germline transmission of the targeted ES cells. Excision of the loxP-flanked cassette was confirmed by PCR genotyping. A subsequent breeding step with BALB/c wt mice removed the Cre recombinase.

[0681] hGITRL knock-in mice were generated by replacing mouse the coding portion of exon 1 with the human GITRL cDNA sequence followed by a bovine growth hormone poly-A signal. All ES cell work and mouse embryology were done similar to the procedures described above. hGITR-hGITRL double knock-in mice were generated by intercrossing the two founder lines for 2 generations to produce homozygous double knock-in mice.

Functional Assays

[0682] Functional activity of MABs were tested in an NFkB reporter gene assay for agonist activity. MAB was diluted to 6 μg/mL in PBS and incubated for 30 minutes at room temperature in the presence/absence of a 3 fold excess of F(ab′).sub.2 fragment goat anti-human Fcγ specific crosslinker. Alternatively, MAB was diluted to 6 μg/mL in PBS and incubated for 30 minutes at room temperature in the presence/absence of a 2 fold excess of Protein A. 10 μL of incubated MAB was then added to a 384 well white clear bottomed assay plate. A HEK-293 cell line stably transfected with hGITR and a NFκB reporter gene was diluted to 5×10.sup.5 cells/mL and 20 μL of the cell suspension was added to each well. The plate was incubated for 24 hours in a 37° C. tissue culture incubator. 30 μL of Cell Bright Glo was added to each well and the plate was read for luminescence on the Acquest.

[0683] The ability of MAB to block ligand binding was assessed using HEK293 NFκB reporter parental cells and hGITR stable cells were used in competition binding assays and FACS analysis. Briefly, harvested cells were plated 1×10.sup.6 cells per mL, 100 μL per well to a 96 well round bottom FACS plate (Corning), then resuspended in 200 μL of cold FACS buffer (1×PBS+1% FBS-HI+0.1% sodium azide) per well. Human GITR ligand titration was prepared from 270 nM to 1.52 pM in FACS buffer at 100 μL per well. Plates were incubated for 1 hour on ice protected from light, cells washed, then prepared 4 nM isotype control or MAB solutions were prepared and added to appropriate wells at 100 μL per well and plates were incubated for 1 hour on ice protected from light, cells washed, then PE-conjugated goat-anti-human detection antibody (Jackson ImmunoResearch) prepared at 1:100 dilution in FACS buffer was added at 100 μL per well and plates were incubated for 30 minutes on ice protected from light. Cells were washed in FACS buffer then cells were fixed with 100 μL per well of BD CytoFix (BD Biosciences) and incubated for further 15 minutes on ice protected from light. Fixed cells were washed twice, resuspeded at a final volume of 150 μL per well of FACS buffer, and samples analyzed within 1 week on a BD Fortessa flow cytometer (BD Bioscience).

[0684] Agonist activity of MABs could also be seen on primary T cells that express endogenous levels of GITR via proliferation and cytokine secretion from primary T cells. MABs were conjugated on to M-280 Tosylactivated beads (Invitrogen #142.04) following the manufacturer's instructions. In some experiments agonist CD3 (OKT3) and CD28 (CD28.2) antibodies were also conjugated to beads. 1×10.sup.5 freshly purified CFSE-labelled human PBMCs were plated in 10 uL of CTL Test media (CTL #CTLW-010) into a 96 well round bottomed tissue culture plate. 100 uL of MAB conjugated beads was then added at a ratio of 1 T cell: 1 beads. Plates were then incubated for 3 days in a 37° C. tissue culture incubator. Levels of secreted cytokines in the media were measured using a MSD multiplex assay according to manufacturer's instructions. Cells were stained with anti-CD4, -CD8a, -CD25, -GITR antibodies and a LIVE/DEAD stain after staining cells were fixed and read on the flow cytometer. Proliferation of each CD4 and CD8 cells was assessed by CFSE staining and counting beads were added prior to the FACS read to allow normalization of the samples.

[0685] Co-stimulatory activity of MABs on T cells was also measured using an ELISpot method for detection of IFNg. Briefly, ELISPOT plates (Millipore MSIPS4510) were prepared by coating with 70% ethanol for 2 minutes followed by PBS wash and incubation overnight with 50 ug IFNg monoclonal antibody in PBS (Mabtech 3321-3). Purified CD8+ T cells were isolated from spleens of Vehicle or MAB7-treated mice 15-days post-dose. T cells were plated into the coated ELISPOT plates at 0.25×10̂6 cells per well in CTL media (CTL Test-medium (CTL CTLT-005), 1 mM Hepes (Mediatech MT25-060-C1), 2 mM L-glutamine (Mediatech MT25-005-C1), 1 mM sodium pyruvate (Mediatech MT25-000-C1), 100 uM MEM Non-essential amino acids (Mediatech MT25-025-C1), 66 uM 2-Mercaptoethanol (Gibco 21985-023), 100 U/mL Pten/Strep (Gibco 10378016). Colon26 cells were treated with 10% ConA sup (BD Biosciences 354115) at 37° C. for 48 hours to upregulate MHC Class II expression and washed with CTL media prior to addition to T-cells. Colon26 tumor cells (20,000/well) were added to CTLs and incubated at 37° C. for 24-48 hours. Plates were then washed with 0.05% Tween-20/PBS and 10 ug of biotinylated anti-IFNg Mab (Mabtech R4-6A2-biotin) was added to each well and incubated for 2 hours at 37° C. Plates were then washed with 0.05% Tween-20/PBS and Vectastain ABC solution (Vector Labs PK6100) was added to each well and incubated for 1 hour at room temperature. Cells were then washed with 0.05% Tween-20/PBS and AEC substrate, prepared according to manufacturer's protocol (Sigma A6926) was added to each well and incubated for 4 minutes at room temperature. Plates were then rinsed with tap water, dried and stored in the dark for 24 hours prior to reading.

[0686] The ability of MABs to induce ADCC was measured using a reporter assay. In a 96 well white plate (Perkin Elmer F6178) 2×10.sup.3 hGITR-Daudi cells were incubated with 4×10.sup.4 Jurkat-V158 cells (stably expresses the V158 variant of the human FcgRIIIa and an NFAT reporter) at a ratio of 1 Daudi cell to 20 Jurkat) cells in 50 uL of RPMI+10% FBS. An equal volume of MAB was added to the well and the plates was incubated for 3 hours in a 37° C. tissue culture incubator. After the incubation 60 uL of Bright-Glo was added to each well and the plate was read on a luminometer.

[0687] In vitro splenocyte assays were carried out using spleens isolated from mice. Briefly, spleens from mice were dissociated by automated homogenization in 5 mL AutoMACS Rinsing Solution (Miltenyi Biotech 130-091-222) containing 5% BSA (Miltenyi Biotech 130-091-376) using gentleMACS C tubes (Miltenyi Biotech 130-096-334) on a gentleMACS Octo Dissociator (Miltenyi Biotech 130-095-937). Homogenates were strained through a 0.70 uM pore size cell strainer (Fisher Scientific 22363548) and washed with 10 mL AutoMACS buffer. Splenocytes were resuspended and plated at 100,000 cells/well in RPMI (HyClone SH30027.02)+10% human serum (Cellgro 35-060.C1)+1× Pen/Strep/L-Glut (Gibco 15 140-112) in a 96-well tissue culture plate (Costar 3799). For T-cell stimulation, 0.4 ug/mL of anti-mouse CD3 (eBioscience 16-0031-86) and 0.8 ug/mL of anti-mouse CD28 (eBioscience 13-0281-86) antibodies were added to appropriate wells. After 48 hours, cells were either analyzed immediately or pulsed with control or therapeutic antibody for 30 minutes to 96 hours, stained with fluorophore-conjugated antibodies and analyzed by flow cytometry.

[0688] Flow cytometry: For surface markers, cells were stained with anti-CD19 (BD Biosciences 562291), anti-CD8 (Biolegend 100725), anti-CD4 (eBioscience 25-0041), anti-CD69 (BD Biosciences 561238), anti-hGITR (Miltenyi Biotech 130-092-895) and anti-hIgG (Life Sciences A-10631) antibodies for 30 minutes at 4C. For intracellular staining, following incubation with cell surface antibodies, cells were washed, fixed and permeabliized with FOXP3 Fix/Perm buffer (Biolegend 421403) according to manufacture's protocol and incubated with anti-phospho-IKKa/b antibody (Cell Signaling 2697) or anti-FOXP3 antibody (eBioscience 50-4774-42) for 30 minutes at 4C. Cells were read on a BD LSRFortessa cytometer using BD FACSDiva software (BD Biosciences) and flow data was analyzed using FlowJo software (TreeStar Inc.).

[0689] Single cell suspensions were generated from tumors and spleens and stained for analysis by flow cytometry. For example, cell markers were assessed using the following antibodies: a-CD8-BUV395 (clone 53-6.7, BD Biosciences 563786), a-CD19-APC-Cy7 (clone 6D5, BioLegend 115530), a-CD3-PerCp (clone 145-2C11, BD Bioscience 553067), a-PD-1-PE-Cy7 (clone RMP1-30, Biolegend 109110). Flow cytometry was run on BD LSRFortessa cytometers using BD FACSDiva software (BD Biosciences). Cytometry data was analyzed using FlowJo Software (FlowJo LLC). Graphs were generated and statistics run using Prism software (GraphPad Software). All data were shown as mean±standard deviation (SD). Group comparisons were carried out using student's T-test with two-tailed 95% confidence interval. For all statistical evaluations the level of significance was set at p<0.05. Significance compared to the vehicle control group is reported unless otherwise stated.

[0690] In vivo tumor models. The Colon26 murine colon carcinoma cell line was obtained from the Division of Cancer Treatment and Diagnosis at the National Cancer Institute (vial: 0507238). Murine Colon26 carcinoma cells were cultured in RPMI 1640 medium (HyClone SH30027.02) supplemented with 10% FBS (Gibco 10099-141), 10 mM HEPES (Gibco 15630-080) and 1 mM sodium pyruvate (Gibco 11360-070). 8-10 week old female hGITR-hGITRL knock-in mice were injected subcutaneously with 0.5×10̂6 Colon26 cells in 100 uL of RPMI in the flank. Tumors were measured using digital calipers and tumor volume calculated using the equation (L×W.sup.2)/2. When tumors reached an average size of 180 mm.sup.3, mice were randomized and dosed with a single intraperitoneal injection of vehicle (PBS) or therapeutic antibody (15 mg/kg) in 200 uL PBS. Mice were sacrificed and tumors collected for analysis by flow cytometry 7 days after dosing with therapeutic antibodies. All animal experiments were performed in an AAALAC accredited facility using IACUC approved protocols. Statistical analysis was performed in Prism software using student's t-test with two-tailed 95% confidence interval or One-way ANOVA with Tukey correction.

[0691] Surrogate murine GITR Colon26 model testing. Charles River Labs female 6-8 week old BALB/c mice were used as the experimental animal. For implantation, cells were resuspended in Hank's 1× Balanced Salt Solution (Hyclone Cat# SH30030.02) and implanted with a subcutaneous injection into the right lower flank using a 28 g needle (100 ml injection volume). After implantation, mice were randomized according to tumor volume. Mice were dosed with 5 mg/kg of IgG2a-DTA-1 or mouse IgG2a isotype control by subcutaneous injection. Clone DTA-1, a rat anti-mouse GITR antibody (S. Sakaguchi, Kyoto University, Kyoto Japan) was modified to create a murine chimeric IgG2a by fusing the DTA-1 variable region sequences to murine IgG2a Fc regions to create IgG2a-DTA-1.

[0692] Combination Therapy. To assess in vivo activity of surrogate anti-GITR antibody (mouse IgG2a-DTA-1), in combination with surrogate anti-PD-1 antibody (rat, IgG2a RMP1-14, Biolegend), female 6-8 weeks old BALB/cJ mice from Jackson Laboratories (Bar Harbor, Me.) were implanted subcutaneously in the right supra-axillary region with 5×10.sup.5 Colon26 cells in a volume of 100 uL. For implantation, Colon26 cells were suspended in Dulbecco's PBS, calcium and magnesium free from Lonza (17-512F). Mice were enrolled in the study ten days post implantation with a mean±SEM tumor volume of 115 mm.sup.3±7. After being randomly assigned to one of 4 groups (n=10-16/group), mice were dosed concurrently once weekly for 2 weeks with isotype (group1), RMP1-14 (10 mg/kg, group2), IgG2a-DTA-1 (5 mg/kg, group3) or the combination of RMP1-14 and IgG2a-DTA (10 mg/kg and 5 mg/kg, respectively, group 4) as described in Table 6. Day 0 is defined as the day of randomization. The isotype control group contained mIgG2a (Biolegend) at 5 mg/kg and rat IgG2a (Biolegend) at 10 mg/kg. IgG2a-DTA and its isotype control (mIgG2a) were dosed via subcutaneous injection at 5 mg/kg. RMP1-14 and its isotype control (rIgG2a) were dosed via intraperitoneal injection. Dosing volume was 10 mg/mL for all treatments. Body weights and tumor volumes were collected two-three times per week. Individual animals were scored as achieving end point when tumor volumes equaled or exceed 1200 mm.sup.3. Anti-tumor activity was reported based on changes in the median time to endpoint (TTE), assessed by Kaplan-Meier survival analysis.

[0693] Combination Therapy. To assess expression of costimulatory molecules following administration of anti-GITR or anti-GITR in combination with anti-PD-1, single cell suspensions of whole tumors and spleens were profiled by flow cytometry following 1 dose of a GITR (clone IgG2a-DTA-1) or anti-PD1 (clone RMP1-14) or anti-GITR+PD1 in combination. mIgG2a was used as control. LAG3, TIM3 and PD-1 positive cells were assessed as a percentage of total CD3+CD8+ T cells in tumors and spleens. Pvalues are calculated with t-test. *p<0.05 and **p<0.005.

Results

Murine and Reference V-Region Amino Acid Sequences

[0694] RT-PCR products from hyrbidoma cells that express MAB1 were sequenced, and this sequence was largely (95% or greater) verified at the protein level using a ThermoElectron LTQ-Orbitrap Mass Spectrometer. The heavy and light chain variable regions of MAB1 were then cloned in KaloBios vectors in order to create the reference Fab MAB1rFab. The first amino acid in MAB1 has to be changed from an asparagine (N) to a glutamic acid (E) residue to enable cloning into KaloBios vectors for generation of the reference Fab; therefore, the MAB1rFab has glutamic acid at the first VK position. The Fab MAB1rFab has intact murine V-regions from MAB1 fused with human constant regions. In addition to MAB1rFab, an optimized Fab, MAB1opFab, was constructed. Several framework amino acid residues in MAB1rFab were changed to human germline residues in MAB1opFab.

Reference and Optimized Reference Fab Affinity Analysis

[0695] Human germline residues incorporated into the optimized reference MAB1opFab in FR1 and FR3 are those specified by the PCR primers used to amplify the human V-segment repertoire and thus are present in all members of the Humaneered® Antibody V-region libraries. The optimized reference Fab is constructed to assess whether or not any of the changes to human germline alter the properties of Fab binding. Affinity constants (Ka (1/Ms), Kd (1/s), and KD (M) of MAB1rFab, MAB1opFab was assayed using the ForteBio Octed QK system and Striptavidin High Binding Biosensors coated with biotinylated hGITR-hFc. Compared with MAB1rFab, MAB1opFab, had very similar Kd, but five-fold improvement on Ka indicating that the amino acid changes in MAB1rFab are tolerated.

Library Construction and Selection of Humaneered® Antibody Fabs

[0696] Heavy and light chain front-end and FR3 cassette libraries, germline-family restricted to VH3 and VKIII, were generated and screened by CLBA. For VK, clones that support binding to human GITR were identified from both VK front-end (MAB1VK3FE-01) and FR3 (MAB1VK3FR3-01) cassette libraries. For VH, clones that support binding to human GITR were identified from FR3 cassette library (MAB1VH3FR3-01), but not from the VH3 front-end library (MAB1VH3FE-01). Since the majority members in Vk front-end and FR3 cassette libraries were positive in CLBA, the whole repertoire of these two libraries was used to construct Vk full-chain library (MAB1VK3FcL-01) by overlapping PCR with mutagenic VK CDR2s that encodes either the parental murine or the selected human germline (VKIII L-16) residue at all positions. A number of hGITR positive clones were identified from VH3FR3 library (MAB1VH3FR3-01) through CLBA and confirmed by human GITR specific ELISA. Six of them were used to pair with VK full-chain library (MAB1Vk3FcL-01) to enable functional Fab expression and the screen of this library.

[0697] Since there were no clones that bind hGITR with high affinity were identified from VH front-end library (MAB1VH3FE-01), subsequently, a second VH3 front-end library (MAB1VH3FE-02) was constructed. This library has either the parental murine or human germline (VH3 3-30) residue at each position of CDR1 and the FR3 sequences from the six selected VHFR3 clones. Many hGITR binders were identified from both VK full-chain library (MAB1Vk3FcL-01) and the second VH front-end library (MAB1VH3FE-02). These clones were confirmed by human GITR specific ELISA assay on Fab-containing cell supernatants and rank-ordered by hGITR affinity titration ELISA.

[0698] Based on hGITR affinity titration ELISA, four VK full-chain clones were selected from VK full-chain library (MAB1VK3FcL01), and six clones were selected from MAB1VH3FE-02 library. The six VH clones were used as the backbone to construct the VH full-chain library with either MAB1 murine or the closest human germline (VH3 3-30) residue at each position in CDR2. This VH full-chain repertoire was paired with the four VK full-chain clones to form the final human full-chain Fab library. CLBA identified many hGITR binding clones, that were confirmed by ELISA using the respective culture supernatant as the Fab source. Five human full-chain Fab clones (MAB2, MAB3, MAB4, MAB5, and MAB6) were selected based on DNA sequence analysis and hGITR affinity titration ELIS A results.

Testing the Affinity of Humaneered® Antibody Fabs for GITR Antigen Using ForteBio Octet Analysis

[0699] The five human full-chain Fabs (MAB2, MAB3, MAB4, MAB5, and MAB6) were expressed and purified. The binding kinetics of these human Fabs were then compared to the kinetics of the optimized reference Fab (MAB1opFab) using the ForteBio Octet system (numerical data summarized in Table 3).

TABLE-US-00009 TABLE 3 Affinity of Fabs for human GITR Fab KD [M] MAB1opFab (a)* 1.25E−8 MAB2 (a) 6.84E−9 MAB3 (a) 2.98E−9 MAB1opFab (b)* 6.59E−9 MAB4 (b) 2.43E−9 MAB5 (b) 3.74E−9 MAB1opFab (c)* 1.47E−8 MAB6 (c) 5.94E−9 *a, b, c indicate three separate Forte experiments. The results are global fitting of two sample duplicates.

Amino Acid Sequence of Antibodies MAB2, MAB3, MAB4, MAB5, MAB6, and Percentage Identity to Human Germline Sequence

[0700] The variable region amino acid sequences of the five Fabs (MAB2, MAB3, MAB4, MAB5, MAB6,) are shown in Table 1. The percent identity to human germline sequences of the five Fabs was determined by aligning the Vh and Vk amino acid sequences against a single germline sequence (VKIII L16/A27 and VH3 3-30, respectively; Table 4). Residues in CDRH3 and CDRL3 were omitted from the calculation for each chain.

TABLE-US-00010 TABLE 4 Percent identity of the five Fabs to human germline sequences Fab VKIII L15/A27 VH3 3-30 Fv MAB2 95% 86% 90% MAB3 98% 85% 91% MAB4 95% 85% 89% MAB5 95% 83% 89% MAB6 95% 82% 88% MAB7 95% 85% 89% MAB8 95% 85% 89%

Conservation of Human GITR Antigenic Epitope

[0701] Antigen epitope conservation was evaluated by an indirect Competition ELISA. All five Fabs blocked the parental mouse antibody MAB1 binding to human GITR indicating that these human Fabs retain the epitope specificity of the original murine antibody.

Analysis of Antigen Specificity of MAB4 and MAB5 by ELISA

[0702] In order to test whether antigen specificity of the parental mouse antibody MAB1 was retained in the IgGs, MAB2, MAB3, MAB4 and MAB5, binding of the antibodies to a panel of human TNFRs was tested in an ELISA assay, The results of one such assay with MAB4 and MAB5 (FIG. 2C) show that MAB4 and MAB5 retain high specificity for GITR, similar to the murine antibody MAB1. Similar results were obtained with MAB2, MAB3 and MAB6.

Antibody Binding to Human and Cynomolgus Macaque but not Rodent GITR Protein in ELISA

[0703] The parental mouse antibody MAB1 binds to human and cynomolgus but not rodent GITR protein. FIG. 2A-B shows that, like MAB1, antibodies MAB4 and MAB5 were able to bind in a similar manner both human and cynomolgus GITR, but not rodent GITR. Similar results were found with MAB6, 7, and 8.

[0704] Binding affinities of GITR agonist antibodies MAB4 and MAB5 for human (hGITR) and cyno (cGITR) GITR, were determined by Biacore analysis. See Table 5. Monoclonal antibodies MAB4 and MAB5 bind to human GITR with subnanomolar binding affinities (KD). Antibodies MAB4 and MAB5 bind to cyno GITR with nanomolar binding affinities that are about 2-3 fold lower than the binding affinities for human GITR. The anti-GITR agonist antibodies of the invention bind selectively to human and cyno GITR in a number of biochemical assays, including flow cytometry, ELISA, Biacore, and Protagen™ chip assays.

TABLE-US-00011 TABLE 5 Binding affinities of MAbs to human- and cyno- GITR Antigen mAb KD (nM) hGITR MAB4 0.684 (±0.331) hGITR MAB5 0.973 (±0.167) hGITR MAB7 4.29 (±0.14) cGITR MAB4  1.78 (±0.543) cGITR MAB5  1.87 (±0.520) cGITR MAB7 3.67 (±0.09)

[0705] Monoclonal antibody MAB7 binds to human as well as cyno CD4+ Tcells. FACS analysis of isolated cyno or human PBMCs demonstrated MAB7 binds isolated CD4+ Tcells. Additionally, FACS experiments demonstrated GITR (by binding of MAB7) and CD25 upregulation following CD3/CD28 activiation of PBMCs (CD4+ Tcells). (data not shown)

Functional Activity of Antibodies and in Reporter Gene Assays and Cell Assays

[0706] Antibodies were assayed in a reporter gene assay for functional activity (FIG. 3). Each of MAB4, MAB5, MAB7 and MAB8 IgGs induce NFκB activity when crosslinked, at levels similar to GITR ligand (GITR-L). See FIG. 3A-D. Similar results were obtained with MAB2, MAB3, and MAB6 (data not shown).

[0707] MAB7 competes with human GITR ligand for binding human GITR expressing stable cell line. Competition assays were performed in triplicate sets of values, FACS competition analysis demonstrates inhibition of ligand binding. See FIG. 2D.

[0708] To confirm functional activity on endogenous GITR, antibodies were conjugated to beads and incubated with purified CFSE labelled human PBMCs. MAB7 induces an increase in proliferation of both CD4+ T cells (FIG. 4A) and CD8+ T cells (FIG. 4B) compared to an isotype control antibody. This increase in proliferation was also accompanied by an increase in the secretion of several cytokines, including IFNγ (FIG. 4C), TNFα, IL-10 and IL-13 (not shown). Similar results were found with MAB 4, MAB5 (not shown). We were able to show that the increase in proliferation and IFNγ production induced by MAB7 was dependent upon the presence of anti-CD3 and anti-CD28 agonistic antibodies on the beads. If these co-stimulatory antibodies were omitted MAB had no agonist effects on either CD4+ or CD8+ T cells. Similar results were obtained with MAB2, MAB3, MAB4; MAB5 and MAB6.

[0709] MAB7 was also found to demonstrate capability to induce FcgRIIIa signaling (shown to be correlated with ADCC activity) in an in vitro assay when high levels of GITR are present. Daudi-hGITR cells incubated with MAB7 or control Ab, and the Jurkat-V158 cell line showed MAB7 is able to induce FcgRIIIa signaling in an in vitro assay and that the ability of MAB7 to induce FcgRIIIa signaling correlates with the receptor level expressed on the surface of the Daudi cells (i.e. higher receptor levels equals greater FcgRIIIa signaling induction). See FIG. 5.

[0710] hGITR is expressed on T-cells and is functional in hGITR-hGITRL knock-in mice. Splenocytes were isolated from wild type or hGITR-hGITRL knock-in mice and cultured either without stimulation or with stimulation using a-CD3 and a-CD28 antibodies for 24, 48, 72 or 96 hours. Cells were then stained with fluorophore-conjugated antibodies and analyzed by flow cytometry, demonstrating human GITR is expressed, and costimulation results in increased GITR expression profile in wild type or transgenic mice. Splenocytes isolated from hGITR-hGITRL knock-in mice demonstrate induction of GITR expression in response to costimulation in culture (FIG. 6A). MAB7 effectively binds hGITR expressed on CD8+ cells (FIG. 6B); and MAB7 binding to stimulated Tcells correlates with increased Tcell activation as measured by pIKK staining (FIG. 6C) and T cell activation marker CD25+(FIG. 6D).

[0711] MAB7 is functional in vivo. hGITR-hGITRL double knock-in mice with established Colon26 tumors were treated with a single dose of vehicle (n=8/timepoint) or MAB7 (n=10/timepoint) antibody as described above. Tumors were measured twice per week and tumor volume calculated using the equation (L×W.sup.2)/2. MAB treated animals demonstrated delayed growth of Colon26 tumors. At three days post treatment, whole blood (FIG. 7B-7C) and tumors (FIG. 7D-7E) were collected and analyzed by flow cytometry for cell surface hGITR expression on immune cells. Results suggest hGITR occupancy and shedding resulting in decreased hGITR from treated groups for both Tregulatory cells and Thelper cells in both blood and tumors (*p<0.05, ****p<0.00005).

[0712] MAB7 elicits an anti-tumor immune response to Colon26 tumors in vivo. hGITR-hGITRL double knock-in mice with established Colon26 tumors were treated with a single dose of vehicle (n=8/timepoint) or MAB7 (n=10/timepoint). FIG. 8A depicts results 3-days post-dose, demonstrating Tregs are reduced in treated animals. FIG. 8B-8C depict results 15-days post-dose, demonstrating increased lymphocytes (8B) and increased activated CD8+ T cells (8C) present in tumor site following treatment. The absolute number of cells was normalized to tumor size to account for the significant difference in tumor size between Vehicle and MAB7 treated groups. MAB7 results suggest treatment results in increased Teff/Treg ratio in treated animals as determined by total intratumoral activated CD8+ T cells compared to CD4+ FOXP3+ Tregs. See FIG. 8D. Additionally, results of splenocyte assays from purified CD8+ T cells incubated with Colon26 tumor cells ex-vivo, and measuring CTL response using IFNg ELISPOT assay suggest increased tumor specific IFNg response in CD8+ T cells from MAB7 treated animals. (*p<0.05, ***p<0.0005). See FIG. 8E.

[0713] Treatment of mice with anti-mGITR Ab upregulates PD-1 expression in tumors and spleen. Mice with established Colon26 tumors were treated with two doses of control or murine anti mGITR antibody. FIG. 9A9C depicts results demonstrating PD-1 expression is upregulated on CD8+ T cells in Colon26 tumors as well as spleens after treatment with surrogate GITR antibody, IgG2a-DTA-1.

[0714] GITR and PD-1 combinations confer survival advantage compared to isotype control. Anti-GITR (DTA-1) and anti-PD-1 (RMP1-14) were administered alone and in combination in mice with established Colon26 tumors. See FIG. 10. Combination administration shows significant survival advantage compared to isotype control (***p<0.0005 pairwise comparison using the Gehan-Breslow-Wilcoxon test). Anti-mGITR (IgG2a-DTA-1) single agent shows significant survival advantage compared to isotype control (*p<0.05 pairwise comparison using the Gehan-Breslow-Wilcoxon test). The data indicate that the combination of IgG2a-DTA-1 and RMP1-14 confers a statistically significant survival advantage relative to isotype control treatment with a median TTE greater than 42 days (median TTE not achieved) (P<0.0005) relative to 22 days for the isotype treated group. Notably, 3/10 animals achieved a complete regression (CR), 2/10 animals achieved stable disease (SD). IgG2a-DTA-1 monotherapy resulted in a median TTE of 30.5 days (P<0.05), with 3/10 animals achieving stable disease (SD). The median survival of the RMP1-14 treated group was 24 days, which was not statistically significantly different from the isotype treated group. Kaplan Meier Graphs were generated and statistics performed using Prism software (GraphPad Software). Group comparisons were carried out as pairwise comparison using the Gehan-Breslow-Wilcoxon test. For all statistical evaluations the level of significance was set at p<0.05. Significance compared to the vehicle control group is reported. Stable disease is defined as 3 successive tumor volume measurements with 10% or less change in tumor volume.

TABLE-US-00012 TABLE 6 Combination Therapy Group Ab1 (5 mg/kg, SC) Ab2 n/group 1 mIgG2a rIgG2a (10 mg/kg, IP) n = 10 2 RMP1-14 (10 mg/kg, IP) mIgG2a (5 mg/kg, SC) n = 16 mIgG2a 3 DTA-1 rIgG2a (10 mg/kg, IP) n = 10 4 DTA-1 RMP1-14 (10 mg/kg, IP) n = 10 IP = intraperitoneal; SC = subcutaneous

[0715] Expression of costimulatory molecules was assessed in tumors following administration of anti-GITR or anti-GITR in combination with anti-PD-1. See FIG. 11. Results of single cell suspensions of whole tumors and spleens profiled by flow cytometry following 1 dose of anti-GITR or anti-PD1 or anti-GITR+PD1 in combination demonstrated increased expression of LAG3, TIM3 and PD-1 on CD8+ T cells in Colon26 tumors after treatment with GITR, PD-1 and in combination. A single combination dose demonstrated upregulated expression of PD-1 in spleen CD8+ cells.

INCORPORATION BY REFERENCE

[0716] All publications, patents, and Accession numbers mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference.

EQUIVALENTS

[0717] While specific embodiments of the subject invention have been discussed, the above specification is illustrative and not restrictive. Many variations of the invention will become apparent to those skilled in the art upon review of this specification and the claims below. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.