ANTI-PVRIG/ANTI-TIGIT BISPECIFIC ANTIBODIES AND METHODS OF USE

CROSS-REFERNCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Applications Nos.

62/679,703, filed June 1, 2018 and 62/773,586, filed November 30, 2018, all of which are incorporated by reference in their entireties.

[0002] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy, created on May 29, 2019, is named H4386-50l l-WO_SL.txt and is 2,631,244 bytes in size.

I. BACKGROUND OF THE INVENTION

[0003] Naive T cells must receive two independent signals from antigen-presenting cells (APC) in order to become productively activated. The first, Signal 1, is antigen-specific and occurs when T cell antigen receptors encounter the appropriate antigen-MHC complex on the APC. The fate of the immune response is determined by a second, antigen-independent signal (Signal 2) which is delivered through a T cell costimulatory molecule that engages its APC-expressed ligand. This second signal could be either stimulatory (positive costimulation) or inhibitory (negative costimulation or coinhibition). In the absence of a costimulatory signal, or in the presence of a coinhibitory signal, T-cell activation is impaired or aborted, which may lead to a state of antigen-specific unresponsiveness (known as T-cell anergy), or may result in T-cell apoptotic death.

[0004] Costimulatory molecule pairs usually consist of ligands expressed on APCs and their cognate receptors expressed on T cells. The prototype ligand/receptor pairs of costimulatory molecules are B7/CD28 and CD40/CD40L. The B7 family consists of structurally related, cell-surface protein ligands, which may provide stimulatory or inhibitory input to an immune response. Members of the B7 family are structurally related, with the extracellular domain containing at least one variable or constant immunoglobulin domain.

[0005] Both positive and negative costimulatory signals play critical roles in the regulation of cell-mediated immune responses, and molecules that mediate these signals have proven to be effective targets for immunomodulation. Based on this knowledge, several therapeutic approaches that involve targeting of costimulatory molecules have been developed, and were shown to be useful for prevention and treatment of cancer by turning on, or preventing the turning off, of immune responses in cancer patients and for prevention and treatment of autoimmune diseases and inflammatory diseases, as well as rejection of allogenic transplantation, each by turning off uncontrolled immune responses, or by induction of“off signal” by negative costimulation (or coinhibition) in subjects with these pathological conditions.

[0006] Manipulation of the signals delivered by B7 ligands has shown potential in the treatment of autoimmunity, inflammatory diseases, and transplant rejection. Therapeutic strategies include blocking of costimulation using monoclonal antibodies to the ligand or to the receptor of a costimulatory pair, or using soluble fusion proteins composed of the costimulatory receptor that may bind and block its appropriate ligand. Another approach is induction of co-inhibition using soluble fusion protein of an inhibitory ligand. These approaches rely, at least partially, on the eventual deletion of auto- or allo-reactive T cells (which are responsible for the pathogenic processes in autoimmune diseases or

transplantation, respectively), presumably because in the absence of costimulation (which induces cell survival genes) T cells become highly susceptible to induction of apoptosis.

Thus, novel agents that are capable of modulating costimulatory signals, without

compromising the immune system’s ability to defend against pathogens, are highly advantageous for treatment and prevention of such pathological conditions.

[0007] Costimulatory pathways play an important role in tumor development. Interestingly, tumors have been shown to evade immune destruction by impeding T cell activation through inhibition of co-stimulatory factors in the B7-CD28 and TNF families, as well as by attracting regulatory T cells, which inhibit anti-tumor T cell responses (see Wang (2006), “Immune Suppression by Tumor Specific CD4+ Regulatory T cells in Cancer”, Semin.

Cancer. Biol. 16:73-79; Greenwald, et al. (2005),“The B7 Family Revisited . Ann. Rev. Immunol. 23:515-48; Watts (2005),“TNF/TNFR Family Members in Co-stimulation of T Cell Responses”, Ann. Rev. Immunol. 23:23-68; Sadum, et al, (2007)“Immune Signatures of Murine and Human Cancers Reveal Unique Mechanisms of Tumor Escape and New Targets for Cancer Immunotherapy”, Clin. Cane. Res. 13(13): 4016-4025). Such tumor expressed co-

stimulatory molecules have become attractive cancer biomarkers and may serve as tumor-associated antigens (TAAs). Furthermore, costimulatory pathways have been identified as immunologic checkpoints that attenuate T cell dependent immune responses, both at the level of initiation and effector function within tumor metastases.

[0008] Over the past decade, agonists and/or antagonists to various costimulatory proteins have been developed for treating autoimmune diseases, graft rejection, allergy and cancer.

For example, CTLA4-Ig (Abatacept, Orencia®) is approved for treatment of RA, mutated CTLA4-Ig (Belatacept, Nulojix®) for prevention of acute kidney transplant rejection and by the anti-CTLA4 antibody (Ipilimumab, Yervoy®), recently approved for the treatment of melanoma. Other costimulation regulators have been approved, such as the anti-PD-l antibodies of Merck (Keytruda®) and BMS (Opdivo®), have been approved for cancer treatments and are in testing for viral infections as well.

[0009] However, while monotherapy with anti-checkpoint inhibitor antibodies have shown promise, a number of studies (Ahmadzadeh et al, Blood 114: 1537 (2009), Matsuzaki et al., PNAS 107(17):7875-7880 (2010), Fourcade et al, Cancer Res. 72(4):887-896 (2012) and Gros et al, J. Clinical Invest. 124(5):2246 (2014)) examining tumor-infiltrating lymphocytes (TILs) have shown that TILs commonly express multiple checkpoint receptors. Moreover, it is likely that TILs that express multiple checkpoints are in fact the most tumor-reactive. In contrast, non-tumor reactive T cells in the periphery are more likely to express a single checkpoint. Checkpoint blockade with monospecific full-length antibodies is likely nondiscriminatory with regards to de-repression of tumor-reactive TILs versus autoantigen-reactive single expressing T cells that are assumed to contribute to autoimmune toxi cities.

[0010] One target of interest is PVRIG. PVRIG, also called Poliovirus Receptor Related Immunoglobulin Domain Containing Protein, Q6DKI7 or C7orfl5, is a transmembrane domain protein of 326 amino acids in length, with a signal peptide (spanning from amino acid 1 to 40), an extracellular domain (spanning from amino acid 41 to 171), a transmembrane domain (spanning from amino acid 172 to 190) and a cytoplasmic domain (spanning from amino acid 191 to 326). PVRIG binds to Poliovirus receptor-related 2 protein (PVLR2, also known as nectin-2, CD112 or herpesvirus entry mediator B, (HVEB) a human plasma membrane glycoprotein), the binding partner of PVRIG.

[0011] Another target of interest is TIGIT. TIGIT is a coinhibitory receptor that is highly expressed on effector & regulatory (Treg) CD4+ T cells, effector CD8+ T cells, and NK cells.

TIGIT has been shown to attenuate immune response by (1) direct signaling, (2) inducing ligand signaling, and (3) competition with and disruption of signaling by the costimulatory receptor CD226 (also known as DNAM-l). TIGIT signaling has been the most well-studied in NK cells, where it has been demonstrated that engagement with its cognate ligand, poliovirus receptor (PVR, also known as CD 155) directly suppresses NK cell cytotoxicity through its cytoplasmic ITIM domain. Knockout of the TIGIT gene or antibody blockade of the TI GIT/PVR interaction has shown to enhance NK cell killing in vitro, as well as to exacerbate autoimmune diseases in vivo. In addition to its direct effects on T- and NK cells, TIGIT can induce PVR-mediated signaling in dendritic or tumor cells, leading to the increase in production of anti-inflammatory cytokines such as IL10. In T-cells TIGIT can also inhibit lymphocyte responses by disrupting homodimerization of the costimulatory receptor CD226, and by competing with it for binding to PVR.

[0012] TIGIT is highly expressed on lymphocytes, including Tumor Infiltrating

Lymphocytes (TILs) and Tregs, that infiltrate different types of tumors. PVR is also broadly expressed in tumors, suggesting that the TIGIT-PVR signaling axis may be a dominant immune escape mechanism for cancer. Notably, TIGIT expression is tightly correlated with the expression of another important coinhibitory receptor, PD1. TIGIT and PD1 are co expressed on the TILs of numerous human and murine tumors. Unlike TIGIT and CTLA4, PD1 inhibition of T cell responses does not involve competition for ligand binding with a costimulatory receptor.

[0013] Accordingly, PVRIG/TIGIT bispecific antibodies, capable of targeting both pathways, are an attractive target for single antibody therapy. Such antibodies will allow for targeting of multiple checkpoint receptors and provide therapeutic importance in the treatment of cancer. Also provided are anti-PVRIG and anti-TIGIT antibodies for use as described herein.

II. BRIEF SUMMARY OF THE INVENTION

[0014] Accordingly, the present invention provides an anti -PVRIG/anti -TIGIT bispecific antibody that monovalently binds a human PVRIG and monovalently binds TIGIT for use in activating T cells for the treatment of cancer.

[0015] In some embodiments, the present invention provides an anti-PVRIG/anti-TIGIT bispecific antibody that monovalently binds a human PVRIG and monovalently binds TIGIT for use in activating T cells and/or NK cells for the treatment of cancer.

[0016] In some embodiments, the anti-PVRIG/anti-TIGIT bispecific antibody comprises:

a) a first antigen binding portion comprising:

i. a first heavy chain variable domain comprising a vhCDRl, vhCDR2, and vhCDR3 from an anti-PVRIG antibody; and ii. a first light chain variable domain comprising a vlCDRl, vlCDR2, and vlCDR3 from an anti-PVRIG antibody;

wherein the anti-PVRIG antibody is selected from the group consisting of CHA.7.518.4, CHA.7.518.1, CHA.7.518, CHA.7.524 CHA.7.530, CHA.7.538_l, CHA.7.538_2, CHA.7.502, CHA.7.503, CHA.7.506, CHA.7.508, CHA.7.510, CHA.7.512, CHA.7.514, CHA.7.516, CHA.7.518, CHA.7.520.1, CHA.7.520.2, CHA.7.522, CHA.7.524, CHA.7.526, CHA.7.527, CHA.7.528, CHA.7.530, CHA.7.534, CHA.7.535, CHA.7.537, CHA.7.538.1, CHA.7.538.2, CHA.7.543, CHA.7.544, CHA.7.545, CHA.7.546, CHA.7.547, CHA.7.548, CHA.7.549, CHAV.550, CHA7.538.1.2, CPA.7.021, CPA.7.001, CPA.7.003, CPA.7.004, CPA.7.006, CPA.7.008, CPA.7.009,

CPA.7.010, CPA.7.011, CPA.7.012, CPA.7.013, CPA.7.014,

CPA.7.015, CPA.7.017, CPA.7.018, CPA.7.0l9,CPA.7.022,

CPA.7.023, CPA.7.024, CPA.7.033, CPA.7.034, CPA.7.036,

CPA.7.040, CPA.7.046, CPA.7.047, CPA.7.049, CPA.7.050, and CHAV.518; and

a) a second antigen binding portion comprising an anti-TIGIT antigen binding domain.

[0017] In some embodiments, the anti-PVRIG/anti-TIGIT bispecific antibody comprises:

a) a first antigen binding portion comprising an anti-PVRIG antigen binding domain; and

b) a second antigen binding portion comprising:

i. a second heavy chain variable domain comprising a vhCDRl , vhCDR2, and vhCDR3 from an anti-TIGIT antibody; and

ii. a second light chain variable domain comprising a vlCDRl, vlCDR2, and vlCDR3 from an anti-TIGIT antibody;

wherein the anti-TIGIT antibody is selected from the group consisting of CPA.9.086, CHA.9.547.18, CPA.9.018, CPA.9.027, CPA.9.049, CPA.9.057, CPA.9.059, CPA.9.083, CPA.9.089, CPA.9.093, CPA.9.101, CPA.9.103, CHA.9.536.1, CHA.9.536.3, CHA.9.536.4, CHA.9.536.5, CHA.9.536.6, CHA.9.536.7, CHA.9.536.8,

CHA.9.560.1, CHA.9.560.3, CHA.9.560.4, CHA.9.560.5,

CHA.9.560.6, CHA.9.560.7, CHA.9.560.8, CHA.9.546.1,

CHA.9.547.1, CHA.9.547.2, CHA.9.547.3, CHA.9.547.4,

CHA.9.547.6, CHA.9.547.7, CHA.9.547.8, CHA.9.547.9,

CHA.9.547.13, CHA.9.541.1, CHA.9.541.3, CHA.9.541.4,

CHA.9.541.5, CHA.9.541.6, CHA.9.541.7, and CHA.9.541.8.

[0018] In some embodiments, the anti -PVRIG/anti-TI GIT bispecific antibody comprises:

a) a first antigen binding portion comprising:

i. a first heavy chain variable domain comprising a vhCDRl, vhCDR2, and vhCDR3 from an anti-PVRIG antibody; and ii. a first light chain variable domain comprising a vlCDRl, vlCDR2, and vlCDR3 from an anti-PVRIG antibody

wherein the anti-PVRIG antibody is selected from the group consisting of CHA.7.518.4, CHA.7.518.1, humanized CHA.7.518, humanized CHA.7.524, humanized CHA.7.530, humanized CHA.7.538 1, humanized CHA.7.538_2, CHA.7.502, CHA.7.503, CHA.7.506, CHA.7.508, CHA.7.510, CHA.7.512, CHA.7.514, CHA.7.516,

CHA.7.518, CHA.7.520.1, CHA.7.520.2, CHA.7.522, CHA.7.524, CHA.7.526, CHA.7.527, CHA.7.528, CHA.7.530, CHA.7.534, CHA.7.535, CHA.7.537, CHA.7.538.1, CHA.7.538.2, CHA.7.543, CHA.7.544, CHA.7.545, CHA.7.546, CHA.7.547, CHA.7.548, CHA.7.549, CHAV.550, CHA7.538.1.2, CPA.7.021, CPA.7.001, CPA.7.003, CPA.7.004, CPA.7.006, CPA.7.008, CPA.7.009,

CPA.7.010, CPA.7.011, CPA.7.012, CPA.7.013, CPA.7.014,

CPA.7.015, CPA.7.017, CPA.7.018, CPA.7.0l9,CPA.7.022,

CPA.7.023, CPA.7.024, CPA.7.033, CPA.7.034, CPA.7.036,

CPA.7.040, CPA.7.046, CPA.7.047, CPA.7.049, and

CPA.7.050CHA.7.518; and

b) a second antigen binding portion comprising:

i. a second heavy chain variable domain comprising a vhCDRl, vhCDR2, and vhCDR3 from an anti -TI GIT antibody; and ii. a second light chain variable domain comprising a vlCDRl, vlCDR2, and vlCDR3 from an anti-TIGIT antibody;

wherein the anti-TIGIT antibody is selected from the group consisting of CPA.9.086, CHA.9.547.18, CPA.9.018, CPA.9.027, CPA.9.049, CPA.9.057, CPA.9.059, CPA.9.083, CPA.9.086, CPA.9.089,

CPA.9.093, CPA.9.101, CPA.9.103, CHA.9.536.1, CHA.9.536.3, CHA.9.536.4, CHA.9.536.5, CHA.9.536.6, CHA.9.536.7,

CHA.9.536.8, CHA.9.560.1, CHA.9.560.3, CHA.9.560.4,

CHA.9.560.5, CHA.9.560.6, CHA.9.560.7, CHA.9.560.8,

CHA.9.546.1, CHA.9.547.1, CHA.9.547.2, CHA.9.547.3,

CHA.9.547.4, CHA.9.547.6, CHA.9.547.7, CHA.9.547.8,

CHA.9.547.9, CHA.9.547.13, CHA.9.541.1, CHA.9.541.3,

CHA.9.541.4, CHA.9.541.5, CHA.9.541.6, CHA.9.541.7, and

CHA.9.541.8.

[0019] In some embodiments, the first antigen binding portion comprises:

i. a first heavy chain comprising VH-CHl-hinge-CH2-CH3; and ii. a first light chain comprising VL-CL, wherein the CL is the constant domain of either a kappa or lambda antibody.

[0020] In some embodiments, the first heavy chain CH3 comprises the amino acid substitutions S354C, E356D, M358L, and T366W.

[0021] In some embodiments, the CL is kappa.

[0022] In some embodiments, the second antigen binding portion comprises:

1 a second heavy chain comprising HC-CL-hinge-CH2-CH3, wherein the CL is either kappa or lambda; and

ii. a second light chain comprising VL-CH1.

[0023] In some embodiments, the second heavy chain CH3 comprises the amino acid substitutions Y349C, E356D, M358L, T366S, L368A, and Y407V.

[0024] In some embodiments, the CL is lambda.

[0025] In some embodiments, the CL is kappa.

[0026] In some embodiments, the anti -PVRIG/anti-TI GIT bispecific antibody comprises:

a) a first antigen binding portion comprising an anti-PVRIG antigen binding domain; and

b) a second anti-TIGIT antigen binding portion comprising:

i. a second heavy chain variable region comprising CPA.9.086 VH

(EVQLVETGGGLIQPGRSLRLSCAASGFTFSSYAMHWVRQAPG KGLEWVAVISYAGEVKYYADSVKGRFTISRDNSKNTLYLQMN S LRAEDT AV YY C ARDPLPLHYY GMD V W GQGTTVTV S S ; SEQ ID NO: 1634); and

ii. a second light chain variable region comprising CPA.9.086 VL

(QSALTQPRSASGNPGQRVTISCSGSSSNMGRRPVNWYQQIPGT APKLLIYSQNQRPSGVPDRFSGSQSGTSASLTISGLQSEDEAEYF CAVWDDIGRVLQLGGGTQLAVL; SEQ ID NO: 1639).

[0027] In some embodiments, the anti -PVRIG/anti-TI GIT bispecific antibody comprises:

c) a first antigen binding portion comprising an anti-PVRIG antigen binding domain; and

d) a second anti-TIGIT antigen binding portion comprising:

i. a second heavy chain variable region comprising CPA.9.547.18 VH

(EVOLVESGGGLVOPGGSLRLSCAASGFTFSSYIMSWVROAPGK GLEWVATISGGSKGOYYADSVKGRFTISRDNSKNTLYLOMNSL RAEDT AV YY C AKWLL S YY AMD YW GOGTL VTV S S : SEQ ID NO: 1664); and

ii. a second light chain variable region comprising CPA.9.547.18 VL

(DIOMTOSPSSLSASVGDRITITCRASOSMAIWLSWYOOKPGKA PKLLI YKASKSHT GVP SRF S GS GS GTDFTLTIS SLQPEDF ATYY C OOGOSYP YTF GQGTKLEIK: SEQ ID NO: 1668).

[0028] In some embodiments, the anti -PVRIG/anti-TI GIT bispecific antibody comprises:

a) a first antigen binding portion comprising an anti-PVRIG binding doming comprising:

i. a first heavy chain variable region comprising CHA.7.518.1 VH

(QV QLVQSGAEVKKPGAS VKV SCKASGYTFTDYNINWVRQAP GQGLEWMGYIYPYIGGSGYAQKFQGRVTMTRDTSTSTVYMEL S SLRS EDT AV YY C AREDKT ARN AMDYW GQGTL VTV S S ; SEQ ID NO: 1539); and

ii. a first light chain comprising CHA.7.518.1 VL

(DIQMTQSPSSLSASVGDRVTITCRVSENIYSNLAWYQQKPGKA PKLLI YEATNL AEGVP SRF S GS GS GTDFTLTI S SLQPEDF ATYY C QHF W GTP YTF GQGTKLEIK; SEQ ID NO: 1544); and

b) a second antigen binding portion comprising an anti-TIGIT antigen binding domain.

[0029] In some embodiments, the anti -PVRIG/anti-TI GIT bispecific antibody comprises:

a) a first antigen binding portion comprising an anti-PVRIG binding doming comprising:

i. a first heavy chain variable region comprising CHA.7.518.4 VH

(QV QLVQSGAEVKKPGAS VKV SCKASGYTFTDYNINWVRQAP GQGLEWMGYIYPYIGGSGYAQKFQGRVTMTRDTSTSTVYMEL S SLRS EDTAVYYC AREDKT ARN AMDYWGQGTLVTVS S ; SEQ ID NO: 3179); and

ii. a first light chain comprising CHA.7.518.4 VL

(DIQMTQSPSSLSASVGDRVTITCRVSENIYDVLAWYQQKPGKA PKLLI YEATNL AEGVP SRF S GS GS GTDFTLTI S SLQPEDF ATYY C QHF W GTP YTF GQGTKLEIK; SEQ ID NO:3l80); and

b) a second antigen binding portion comprising an anti-TIGIT antigen binding domain.

[0030] In some embodiments, the anti -PVRIG/anti-TI GIT bispecific antibody comprises:

a) a first antigen binding portion comprising an anti-PVRIG binding doming comprising:

i. a first heavy chain variable region comprising CHA.7.518.1 VH

(QV QLVQSGAEVKKPGAS VKV SCKASGYTFTDYNINWVRQAP GQGLEWMGYIYPYIGGSGYAQKFQGRVTMTRDTSTSTVYMEL S SLRS EDT AV YY C AREDKT ARN AMDYW GQGTL VTV S S ; SEQ ID NO: 1539); and

ii. a first light chain variable region comprising CHA.7.518.1

(DIQMTQSPSSLSASVGDRVTITCRVSENIYSNLAWYQQKPGKA PKLLI YEATNL AEGVP SRF S GS GS GTDFTLTI S SLQPEDF ATYY C QHF W GTP YTF GQGTKLEIK; SEQ ID NO: 1544); and

b) a second antigen binding portion comprising an anti-TIGIT binding doming comprising

i. a first heavy chain variable region comprising CPA.9.086 VH

(EVQLVETGGGLIQPGRSLRLSCAASGFTFSSYAMHWVRQAPG KGLEWVAVISYAGEVKYYADSVKGRFTISRDNSKNTLYLQMN SLRAEDTAVYY C ARDPLPLHYY GMDVW GQGTTVTV S S ; SEQ ID NO: 1634); and

ii. a first light chain variable region comprising CPA.9.086 VL

(QSALTQPRSASGNPGQRVTISCSGSSSNMGRRPVNWYQQIPGT APKLLIYSQNQRPSGVPDRFSGSQSGTSASLTISGLQSEDEAEYF CAVWDDIGRVLQLGGGTQLAVL; SEQ ID NO: 1639).

[0031] In some embodiments, the anti -PVRIG/anti-TI GIT bispecific antibody comprises:

a) a first antigen binding portion comprising an anti-PVRIG binding doming comprising:

i. a first heavy chain variable region comprising CHA.7.518.4 VH

(QV QLVQSGAEVKKPGAS VKV SCKASGYTFTDYNINWVRQAP

GQGLEWMGYIYPYIGGSGYAQKFQGRVTMTRDTSTSTVYMEL S SLRS EDTAVYYC AREDKT ARN AMDYWGQGTLVTVS S ; SEQ ID NO: 3179); and

ii. a first light chain comprising CHA.7.518.4 VL

(DIQMTQSPSSLSASVGDRVTITCRVSENIYDVLAWYQQKPGKA PKLLI YEATNL AEGVP SRF S GS GS GTDFTLTI S SLQPEDF ATYY C QHF W GTP YTF GQGTKLEIK; SEQ ID NO:3l80); and

b) a second antigen binding portion comprising an anti-TIGIT binding doming comprising:

i. a first heavy chain variable region comprising CPA.9.086 VH

(EV QLVET GGGLIQPGRSLRL S C AAS GFTF S S Y AMHWVRQ APG KGLEWVAVISYAGEVKYYADSVKGRFTISRDNSKNTLYLQMN SLRAEDTAVYY C ARDPLPLHYY GMDVW GQGTTVTV S S ; SEQ ID NO: 1634); and

ii. a first light chain variable region comprising CPA.9.086 VL

(QSALTQPRSASGNPGQRVTISCSGSSSNMGRRPVNWYQQIPGT APKLLIYSQNQRPSGVPDRFSGSQSGTSASLTISGLQSEDEAEYF CAVWDDIGRVLQLGGGTQLAVL; SEQ ID NO: 1639).

[0032] In some embodiments, the anti -PVRIG/anti-TI GIT bispecific antibody comprises:

a) a first antigen binding portion comprising an anti-PVRIG binding doming comprising:

i. a first heavy chain variable region comprising CHA.7.518.1

(QV QLVQSGAEVKKPGASVKV SCKASGYTFTDYNINWVRQAP GQGLEWMGYIYPYIGGSGYAQKFQGRVTMTRDTSTSTVYMEL S SLRS EDT AV YY C AREDKT ARN AMDYW GQGTL VTV S S ; SEQ ID NO: 1539); and

ii. a first light chain variable region comprising CHA.7.518.1 VL

(DIQMTQSPSSLSASVGDRVTITCRVSENIYSNLAWYQQKPGKA PKLLI YEATNL AEGVP SRF S GS GS GTDFTLTI S SLQPEDF ATYY C QHF W GTP YTF GQGTKLEIK; SEQ ID NO: 1544); and

b) a second antigen binding portion comprising an anti-TIGIT binding doming comprising:

i. a first heavy chain variable region comprising CHA.9.547.18 HC

(EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYIMSWVRQAPGK GLEWVATISGGSKGQYYADSVKGRFTISRDNSKNTLYLQMNSL RAEDT AV YY C AKWLL S YY AMDYW GQGTL VTV S S ; SEQ ID NO: 1664); and

ii. a first light chain variable region comprising CHA.9.547.18 VL

(DIQMTQSPSSLSASVGDRITITCRASQSMAIWLSWYQQKPGKA PKLLIYKASKSHTGVPSRFSGSGSGTDFTLTIS SLQPEDF ATYYC QQGQSYP YTF GQGTKLEIK; SEQ ID NO: 1668).

[0033] In some embodiments, the anti -PVRIG/anti-TI GIT bispecific antibody comprises:

a) a first antigen binding portion comprising an anti-PVRIG binding doming comprising:

i. a first heavy chain variable region comprising CHA.7.518.4 VH

(QV QLVQSGAEVKKPGASVKV SCKASGYTFTDYNINWVRQAP GQGLEWMGYIYPYIGGSGYAQKFQGRVTMTRDTSTSTVYMEL S SLRS EDT AV YY C AREDKT ARN AMDYW GQGTL VTV S S ; SEQ ID NO: 3179); and

ii. a first light chain comprising CHA.7.518.4 VL

(DIQMTQSPSSLSASVGDRVTITCRVSENIYDVLAWYQQKPGKA PKLLI YEATNL AEGVP SRF S GS GS GTDFTLTI S SLQPEDF ATYY C QHF W GTP YTF GQGTKLEIK; SEQ ID NO:3l80); and

b) a second antigen binding portion comprising an anti-TIGIT binding doming comprising:

i. a first heavy chain variable region comprising CHA.9.547.18 HC

(EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYIMSWVRQAPGK GLEWVATISGGSKGQYYADSVKGRFTISRDNSKNTLYLQMNSL RAEDT AV YY C AKWLL S YY AMDYW GQGTL VTV S S (SEQ ID NO: 1664); and

ii. a first light chain variable region comprising CHA.9.547.18 VL

(DIQMTQSPSSLSASVGDRITITCRASQSMAIWLSWYQQKPGKA PKLLIYKASKSHTGVPSRFSGSGSGTDFTLTIS SLQPEDF ATYYC QQGQSYP YTF GQGTKLEIK; SEQ ID NO: 1668).

[0034] In some embodiments, the anti -PVRIG/anti-TI GIT bispecific antibody is a humanized antibody.

[0035] In some embodiments, the present invention provides a composition comprising an anti-PVRIG/anti-TIGIT bispecific antibody as described herein.

[0036] A nucleic acid composition comprising:

a) a first nucleic acid encoding a first heavy chain or heavy chain variable domain as described herein;

b) a second nucleic acid encoding a first light chain or light chain variable domain as described herein;

c) a third nucleic acid encoding a second heavy chain or heavy chain variable domain as described herein; and

d) a fourth nucleic acid encoding a second light chain or light chain variable domain as described herein.

[0037] In some embodiments, the present invention provides an expression vector composition comprising:

a) a first expression vector comprising the first nucleic acid as described herein;

b) a second expression vector comprising the second nucleic acid as described herein;

c) a third expression vector comprising the third nucleic acid as described herein; and

d) a fourth expression vector comprising the fourth nucleic acid as described herein.

[0038] In some embodiments, the present invention provides an expression vector composition comprising:

a) a first expression vector comprising the first and second nucleic acids as described herein; and

b) a second expression vector comprising the third and fourth nucleic acids as described herein.

[0039] In some embodiments, the present invention provides a host cell comprising the expression vector composition as described herein.

[0040] In some embodiments, the present invention provides a method of making an anti-PVRIG/anti-TIGIT bispecific antibody comprising:

a) culturing the host cell as described herein under conditions wherein the antibody is expressed; and

b) recovering the antibody.

[0041] In some embodiments, the present invention provides a method of activating T cells of a patient comprising administering the anti-PVRIG/anti-TIGIT bispecific antibody as described herein to the patient, wherein a subset of the T cells of the patient are activated.

[0042] In some embodiments, the present invention provides a method of activating cytotoxic T cells (CTLs) of a patient comprising administering the anti-PVRIG/anti-TIGIT bispecific antibody as described herein to the patient, wherein a subset of the CTLs of the patient are activated.

[0043] In some embodiments, the present invention provides a method of activating NK cells of a patient comprising administering the anti-PVRIG/anti-TIGIT bispecific antibody as described herein to the patient, wherein a subset of the NK cells of the patient are activated.

[0044] In some embodiments, the present invention provides a method of activating gd T cells of a patient comprising administering the anti-PVRIG/anti-TIGIT bispecific antibody as described herein to the patient, wherein a subset of the gd T cells of the patient are activated.

[0045] In some embodiments, the present invention provides a method of activating Thl cells of a patient comprising administering the anti-PVRIG/anti-TIGIT bispecific antibody as described herein to the patient, wherein a subset of the Thl cells of the patient are activated.

[0046] In some embodiments, the present invention provides a method of decreasing or eliminating cell number and/or activity of at least one of regulatory T cells (Tregs) in a patient comprising administering the anti-PVRIG/anti-TIGIT bispecific antibody as described herein to the patient.

[0047] In some embodiments, the present invention provides a method of increasing interferon-g production and/or pro-inflammatory cytokine secretion in a patient comprising administering the anti-PVRIG/anti-TIGIT bispecific antibody as described herein to the patient.

[0048] In some embodiments, the present invention provides a method of treating cancer in a patient comprising administering the anti-PVRIG/anti-TIGIT antibody as described herein to the patient.

[0049] In some embodiments, the cancer is selected from the group consisting of prostate cancer, liver cancer (HCC), colorectal cancer, ovarian cancer, endometrial cancer, breast cancer, triple negative breast cancer, pancreatic cancer, stomach (gastric) cancer, cervical cancer, head and neck cancer, thyroid cancer, testis cancer, urothelial cancer, lung cancer (small cell lung, non-small cell lung), melanoma, non melanoma skin cancer (squamous and basal cell carcinoma), glioma, renal cancer (RCC), lymphoma (NHL or HL), Acute myeloid leukemia (AML), T cell Acute Lymphoblastic Leukemia (T-ALL), Diffuse Large B cell lymphoma, testicular germ cell tumors, mesothelioma, esophageal cancer, Merkel Cells cancer, MSI-high cancer, KRAS mutant tumors, adult T-cell leukemia/lymphoma, myeloma, and Myelodysplastic syndromes (MDS).

[0050] In some embodiments, the cancer is selected from the group consisting of triple negative breast cancer, stomach (gastric) cancer, lung cancer (small cell lung, non-small cell lung), Merkel Cells cancer, MSI-high cancer, KRAS mutant tumors, adult T-cell

leukemia/lymphoma, myeloma, and Myelodysplastic syndromes (MDS).

[0051] In some embodiments, the present invention provides an anti-PVRIG/anti-TIGIT bispecific antibody comprising:

a) a first antigen binding portion comprising an anti-PVRIG antigen binding portion comprising:

i. a first heavy chain variable domain comprising a vhCDRl, vhCDR2, and vhCDR3 from an anti-PVRIG antibody; and ii. a first light chain variable domain comprising a vlCDRl, vlCDR2, and vlCDR3 from an anti-PVRIG antibody;

wherein the anti-PVRIG antigen binding portion is selected from the group consisting of CPA.7.021, CPA.7.001, CPA.7.003, CPA.7.004, CPA.7.006, CPA.7.008, CPA.7.009, CPA.7.010, CPA.7.011,

CPA.7.012, CPA.7.013, CPA.7.014, CPA.7.015, CPA.7.017,

CPA.7.018, CPA.7.0l9,CPA.7.022, CPA.7.023, CPA.7.024,

CPA.7.033, CPA.7.034, CPA.7.036, CPA.7.040, CPA.7.046,

CPA.7.047, CPA.7.049, CPA.7.050, CHA.7.502, CHA.7.503, CHA.7.506, CHA.7.508, CHA.7.510, CHA.7.512, CHA.7.514, CHA.7.516, CHA.7.518, CHA.7.520.1, CHA.7.520.2, CHA.7.522, CHA.7.524, CHA.7.526, CHA.7.527, CHA.7.528, CHA.7.530, CHA.7.534, CHA.7.535, CHA.7.537, CHA.7.538.1, CHA.7.538.2, CHA.7.543, CHA.7.544, CHA.7.545, CHA.7.546, CHA.7.547, CHA.7.548, CHA.7.549, CHA.7.550, CHA.7.518.1; CHA7.538.1.2 and CHAV.518.4; and

b) a second antigen binding portion comprising an anti-TIGIT antigen binding domain, wherein the anti-TIGIT antigen binding domain is from an antibody as provided in Figures 24 and 41, and in particular Figure 24A-24EE.

[0052] In some embodiments, the present invention provides an anti-PVRIG/anti-TIGIT bispecific antibody comprising:

a) a first antigen binding portion comprising an anti-PVRIG antigen binding domain wherein the anti-PVRIG antigen binding domain is from an antibody as provided in Figure 35; and

b) a second antigen binding portion comprising an anti-TIGIT antigen binding domain comprising:

i. a second heavy chain variable domain comprising a vhCDRl , vhCDR2, and vhCDR3 from an anti-TIGIT antibody; and ii. a second light chain variable domain comprising a vlCDRl, vlCDR2, and vlCDR3 from an anti-TIGIT antibody;

wherein the anti-TIGIT antigen binding domain is selected from the group consisting of CP A.9.018, CPA.9.027, CPA.9.049, CPA.9.057, CPA.9.059, CPA.9.083, CPA.9.086, CPA.9.089, CPA.9.093, CPA.9.101, CPA.9.103, CHA.9.536.1, CHA.9.536.3, CHA.9.536.4, CHA.9.536.5, CHA.9.536.6, CHA.9.536.7, CHA.9.536.8,

CHA.9.560.1, CHA.9.560.3, CHA.9.560.4, CHA.9.560.5,

CHA.9.560.6, CHA.9.560.7, CHA.9.560.8, CHA.9.546.1,

CHA.9.547.1, CHA.9.547.2, CHA.9.547.3, CHA.9.547.4,

CHA.9.547.6, CHA.9.547.7, CHA.9.547.8, CHA.9.547.9,

CHA.9.547.13, CHA.9.541.1, CHA.9.541.3, CHA.9.541.4,

CHA.9.541.5, CHA.9.541.6, CHA.9.541.7, CHA.9.541.8, and CHA.9.547.18.

[0053] In some embodiments, the present invention provides an anti-PVRIG antibody comprising:

i) a heavy chain or heavy chain variable domain comprising the vhCDRl, vhCDR2, and vhCDR3 from the following sequence:

QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNINWVRQAPGQGLEWMGYIYPYIGGSGYAQKFQG RVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREDKTARNAMDYWGQGTLVTVSS (SEQ ID NO: 3179) ,

and

ii) a light chain or light chain variable domain comprisingthe vlCDRl, vlCDR2, vlCDR3 from the following sequence:

DIQMTQSPSSLSASVGDRVTITCRVSENIYDVLAWYQQKPGKAPKLLIYEATNLAEGVPSRFSGSG SGTDFTLTISSLQPEDFATYYCQHFWGTPYTFGQGTKLEIK (SEQ ID NO:3180).

[0054] In some embodiments, the present invention provides an anti-TIGIT antibody comprising:

i) a heavy chain or heavy chain variable domain comprising the vhCDRl, vhCDR2, and vhCDR3 from the following sequence:

EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYIMSWVRQAPGKGLEWVATISGGSKGQYYADSVKG RFTI SRDNSKNTLYLQMNSLRAEDTAVYYCAKWLLSYYAMDYWGQGTLVTVSS (SEQ ID NO: 1664) ,

ii) a light chain or light chain variable domain comprising the vlCDRl, vlCDR2, and vlCDR3 from the following sequence:

DIQMTQSPSSLSASVGDRITITCRASQSMAIWLSWYQQKPGKAPKLLIYKASKSHTGVPSRFSGSG SGTDFTLTISSLQPEDFATYYCQQGQSYPYTFGQGTKLEIK (SEQ ID NO:1668).

[0055] In some embodiments, the anti-PVRIG antibody comprises:

a) a heavy chain comprising CHA.7.518.4 VH

(QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNINWVRQAPGQGLE

WMGYIYPYIGGSGYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAV YYCAREDKTARNAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSE STAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTYRVV SVLTVLHQDWLNGKEYKCKV SNKGLPSS IEKTISKAKGQPREPQVYTLPPCQDELTKNQVSLWCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCS VMHEALHNHYTQKSLSLSPGK; SEQ ID NO:3l75); and

b) a light chain comprising CHA.7.518.4 VL

(DIQMTQSPSSLSASVGDRVTITCRVSENIYDVLAWYQQKPGKAPKLLI YE ATNL AEGVP S RF S GS GS GTDFTLTI S SLQPEDF ATYY C QHF W GTP YT F GQGTKLEIKRTV AAP SVFIFPPS DEQLKS GT AS V V CLLNNF YPRE AKV QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA CEVTHQGLSSPVTKSFNRGEC ; SEQ ID NO: 3362).

[0056] In some embodiments, the present invention provides a composition comprising an anti-PVRIG antibody according as described herein.

[0057] In some embodiments, the present invention provides a nucleic acid composition comprising:

i) a first nucleic acid encoding a heavy chain or heavy chain variable domain as described herein; and

ii) a second nucleic acid encoding a light chain or light chain variable domain as described herein.

[0058] In some embodiments, the present invention provides a composition comprising an anti-TIGIT antibody as described herein.

[0059] In some embodiments, the present invention provides a nucleic acid composition comprising:

i) a first nucleic acid encoding a heavy chain or heavy chain variable domain as described herein; and

ii) a second nucleic acid encoding a light chain or light chain variable domain as described herein.

[0060] In some embodiments, the present invention provides an expression vector composition comprising:

i) a first expression vector comprising the first nucleic acid as described herein; and ii) a second expression vector comprising the second nucleic acid as described herein.

[0061] An expression vector comprising:

i) the first nucleic acid as described herein; and

ii) the second nucleic acid as described herein.

[0062] In some embodiments, the present invention provides an expression vector composition comprising:

i) a first expression vector comprising the first nucleic acid as described herein; and ii) a second expression vector comprising the second nucleic acid as described herein.

[0063] In some embodiments, the present invention provides an expression vector comprising:

i) the first nucleic acid as described herein; and

ii) the second nucleic acid as described herein.

[0064] In some embodiments, the present invention provides a host cell comprising the expression vector or vector composition as described herein.

[0065] In some embodiments, the present invention provides a method of making an anti-PVRIG or anti-TIGIT antibody comprising:

i) culturing the host cell as described herein under conditions wherein the antibody is expressed; and

ii) recovering the antibody.

[0066] In some embodiments, the present invention provides a method of activating T cells of a patient comprising administering the anti-PVRIG or anti-TIGIT antibody as described herein to the patient, wherein a subset of the T cells of the patient are activated.

[0067] In some embodiments, the present invention provides a method of activating cytotoxic T cells (CTLs) of a patient comprising administering the anti-PVRIG or anti-TIGIT antibody as described herein to the patient, wherein a subset of the CTLs of the patient are activated. [0068] In some embodiments, the present invention provides a method of activating NK cells of a patient comprising administering the anti-PVRIG or anti-TIGIT antibody as described herein to the patient, wherein a subset of the NK cells of the patient are activated.

[0069] In some embodiments, the present invention provides a method of activating gd T cells of a patient comprising administering the anti-PVRIG or anti-TIGIT antibody as described herein to the patient, wherein a subset of the gd T cells of the patient are activated.

[0070] In some embodiments, the present invention provides a method of activating Thl cells of a patient comprising administering the anti-PVRIG or anti-TIGIT antibody as described herein to the patient, wherein a subset of the Thl cells of the patient are activated.

[0071] In some embodiments, the present invention provides a method of decreasing or eliminating cell number and/or activity of at least one of regulatory T cells (Tregs) in a patient comprising administering the anti-PVRIG or anti-TIGIT antibody as described herein to the patient.

[0072] In some embodiments, the present invention provides a method of increasing interferon-g production and/or pro-inflammatory cytokine secretion in a patient comprising administering the anti-PVRIG or anti-TIGIT antibody as described herein to the patient.

[0073] In some embodiments, the present invention provides a method of treating cancer in a patient comprising administering the anti-PVRIG or anti-TIGIT antibody as described herein to the patient.

[0074] In some embodiments, the cancer is selected from the group consisting of prostate cancer, liver cancer (HCC), colorectal cancer, ovarian cancer, endometrial cancer, breast cancer, triple negative breast cancer, pancreatic cancer, stomach (gastric) cancer, cervical cancer, head and neck cancer, thyroid cancer, testis cancer, urothelial cancer, lung cancer (small cell lung, non-small cell lung), melanoma, non melanoma skin cancer (squamous and basal cell carcinoma), glioma, renal cancer (RCC), lymphoma (NHL or HL), Acute myeloid leukemia (AML), T cell Acute Lymphoblastic Leukemia (T-ALL), Diffuse Large B cell lymphoma, testicular germ cell tumors, mesothelioma, esophageal cancer, Merkel Cells cancer, MSI-high cancer, KRAS mutant tumors, adult T-cell leukemia/lymphoma, myeloma, and Myelodysplastic syndromes (MDS).

[0075] In some embodiments, the cancer is selected from the group consisting of triple negative breast cancer, stomach (gastric) cancer, lung cancer (small cell lung, non-small cell lung), Merkel Cells cancer, MSI-high cancer, KRAS mutant tumors, adult T-cell leukemia/lymphoma, myeloma, and Myelodysplastic syndromes (MDS).

[0076] In some embodiments, the present invention provides a method of treating cancer in a patient comprising administering a combination therapy comprising an anti-PVRIG/anti-TIGIT bispecific antibody according to as described herein.

[0077] In some embodiments, the present invention provides a method of treating cancer in a patient comprising administering a combination therapy comprising an anti-PVRIG antibody as described herein and an anti-PD-l antibody.

[0078] In some embodiments, the anti-PD-l antibody is an antibody selected from the group consisting of pembrolizumab and nivolumab.

[0079] In some embodiments, the present invention provides a method of treating cancer in a patient comprising administering a combination therapy comprising an anti-TIGIT antibody as described herein and an anti-PD-l antibody.

[0080] In some embodiments, the anti-PD-l antibody is an antibody selected from the group consisting of pembrolizumab and nivolumab.

[0081] In some embodiments, the present invention provides a method of treating cancer in a patient comprising administering a combination therapy comprising an anti-TIGIT antibody as described herein and an anti-PVRIG antibody as described herein.

[0082] In some embodiments, the present invention provides a method of treating cancer in a patient comprising administering a triple combination therapy comprising an anti-TIGIT antibody as described herein, an anti-PVRIG antibody, and an anti-PD-l antibody.

[0083] In some embodiments, the present invention provides a method of treating cancer in a patient comprising administering a triple combination therapy comprising an anti-TIGIT, an anti-PVRIG antibody as described herein, and an anti-PD-l antibody.

[0084] In some embodiments, the present invention provides a method of treating cancer in a patient comprising administering a triple combination therapy comprising an anti-TIGIT as described herein, an anti-PVRIG antibody as described herein, and an anti-PD-l antibody.

[0085] In some embodiments, the anti-PD-l antibody is an antibody selected from the group consisting of pembrolizumab and nivolumab.

[0086] In some embodiments, the present invention provides a method of activating T cells of a patient comprising administering the anti-PVRIG or anti-TIGIT antibody as described herein to the patient, wherein a subset of the T cells of the patient are activated.

[0087] In some embodiments, the present invention provides a method of activating cytotoxic T cells (CTLs) of a patient comprising administering the anti-PVRIG or anti-TIGIT antibody as described herein to the patient, wherein a subset of the CTLs of the patient are activated.

[0088] In some embodiments, the present invention provides a method of activating gd T cells of a patient comprising administering the anti-PVRIG or anti-TIGIT antibody as described herein to the patient, wherein a subset of the gd T cells of the patient are activated.

[0089] In some embodiments, the present invention provides a method of activating Thl cells of a patient comprising administering the anti-PVRIG or anti-TIGIT antibody as described herein to the patient, wherein a subset of the Thl cells of the patient are activated.

[0090] In some embodiments, the present invention provides a method of decreasing or eliminating cell number and/or activity of at least one of regulatory T cells (Tregs) in a patient comprising administering the anti-PVRIG or anti-TIGIT antibody as described herein to the patient.

[0091] In some embodiments, the present invention provides a method of increasing interferon-g production and/or pro-inflammatory cytokine secretion in a patient comprising administering the anti-PVRIG or anti-TIGIT as described herein to the patient.

[0092] In some embodiments, the present invention provides a method of treating cancer in a patient comprising administering the anti-PVRIG or anti-TIGIT antibody as described herein to the patient.

[0093] In some embodiments, the cancer is selected from the group consisting of prostate cancer, liver cancer (HCC), colorectal cancer, ovarian cancer, endometrial cancer, breast cancer, triple negative breast cancer, pancreatic cancer, stomach (gastric) cancer, cervical cancer, head and neck cancer, thyroid cancer, testis cancer, urothelial cancer, lung cancer (small cell lung, non-small cell lung), melanoma, non melanoma skin cancer (squamous and basal cell carcinoma), glioma, renal cancer (RCC), lymphoma (NHL or HL), Acute myeloid leukemia (AML), T cell Acute Lymphoblastic Leukemia (T-ALL), Diffuse Large B cell lymphoma, testicular germ cell tumors, mesothelioma, esophageal cancer, Merkel Cells

cancer, MSI-high cancer, KRAS mutant tumors, adult T-cell leukemia/lymphoma, myeloma, and Myelodysplastic syndromes (MDS).

[0094] In some embodiments, the cancer is selected from the group consisting of triple negative breast cancer, stomach (gastric) cancer, lung cancer (small cell lung, non-small cell lung), Merkel Cells cancer, MSI-high cancer, KRAS mutant tumors, adult T-cell

leukemia/lymphoma, myeloma, and Myelodysplastic syndromes (MDS).

III. BRIEF DESCRIPTION OF THE DRAWINGS

[0095] Figure 1 depicts the sequences of human PVRIG (showing two different methionine starting points as well as the full length sequence). The signal peptide is underlined, the ECD is double underlined. PVRIG, also called Poliovirus Receptor Related Immunoglobulin Domain Containing Protein, Q6DKI7 or C7orfl5, relates to amino acid and nucleic acid sequences shown in RefSeq accession identifier NP_076975, shown in Figure 1.

[0096] Figure 2 depicts the sequence of the human Poliovirus receptor-related 2 protein (PVLR2, also known as nectin-2, CD112 or herpesvirus entry mediator B, (HVEB)), the binding partner of PVRIG. PVLR2 is a human plasma membrane glycoprotein.

[0097] Figure 3A-3C shows the CDR sequences for Fabs that were determined to successfully block interaction of the PVRIG with its counterpart PVRL2.

[0098] Figures 4A-4AA shows the amino acid sequences of the variable heavy and light domains, the full length heavy and light chains, and the variable heavy and variable light CDRs for the enumerated human CPA anti-PVRIG sequences of the invention that both bind PVRIG and block binding of PVRIG and PVLR2.

[0099] Figures 5A-5H depicts the amino acid sequences of the variable heavy and light domains, the full length heavy and light chains, and the variable heavy and variable light CDRs for eight human CPA anti-PVRIG sequences of the invention that bind PVRIG and but do not block binding of PVRIG and PVLR2.

[00100] Figures 6A-6G depicts the CDRs for all CPA anti-PVRIG antibody sequences that were generated that bind PVRIG, including those that do not block binding of PVRIG and PVLR2.

[00101] Figures 7A-7AE depicts the variable heavy and light chains as well as the vhCDRl, vhCDR2, vhCDR3, vlCDRl, vlCDR2 and vlCDR3 sequences of each of the enumerated CHA antibodies of the invention, CHA.7.502, CHA.7.503, CHA.7.506, CHA.7.508, CHA.7.510, CHA.7.512, CHA.7.514, CHA.7.516, CHA.7.518, CHA.7.520.1, CHA.7.520.2, CHA.7.522, CHA.7.524, CHA.7.526, CHA.7.527, CHA.7.528, CHA.7.530, CHA.7.534, CHA.7.535, CHA.7.537, CHA.7.538.1, CHA.7.538.2, CHA.7.543, CHA.7.544,

CHA.7.545, CHA.7.546, CHA.7.547, CHA.7.548, CHA.7.549,CHA.7.550, and

CHA.7.518.4 (these include the variable heavy and light sequences from mouse sequences (from Hybridomas).

[00102] Figures 8A-8D depicts the vhCDRl, vhCDR2, vhCDR3, vlCDRl, vlCDR2 and vlCDR3 sequences of each of the enumerated CPA antibodies of the invention, CPA.7.001 to CPA.7.050 are human sequences (from Phage display).

[00103] Figures 9A-9C depicts the sequences of human IgGl, IgG2, IgG3 and IgG4.

[00104] Figure 10 depicts a number of human PVRIG ECD fragments.

[0100] Figures 11 A-l II depicts a collation of the humanized sequences of five CHA antibodies.

[0101] Figures 12A-12E depicts a collation of the humanized sequences of five CHA antibodies.

[0102] Figure 13 depicts schemes for combining the humanized VH and VL CHA antibodies of Figures 11 A-l II and Figures 12A-12E. The“chimVH” and“chimVL” are the mouse variable heavy and light sequences attached to a human IgG constant domain.

[0103] Figures 14A-14BX show a number of PVRIG sequences and other sequences that find use in the invention.

[0104] Figure 15 A and 15B depict the variable heavy and light chains as well as the vhCDRl, vhCDR2, vhCDR3, vlCDRl, vlCDR2 and vlCDR3 sequences of each of the enumerated CHA antibodies of the invention, CHA.7.5l8. l.H4(S24lP), and

CHA.7.538. l.2.H4(S24lP).

[00105] Figure 16A-16E depict four humanized sequences for each of CHAV.518,

CHA.7.524, CHA.7.530, CHA.7.538_l and CHA.7.538_2. All humanized antibodies comprise the H4(S24lP) substitution. Note that the light chain for CHA.7.538_2 is the same as for CHA.7.538 1. The“Hl” of each is a“CDR swap” with no changes to the human

framework. Subsequent sequences alter framework changes shown in larger bold font. CDR sequences are noted in bold. CDR definitions are AbM from website www. bioinf. org. uk/abs/. Human germline and joining sequences from IMGT® the international ImMunoGeneTics® information system www.imgt.org (founder and director: Marie-Paule Lefranc, Montpellier, France). Residue numbering shown as sequential (seq) or according to Chothia from website www.bioinf.org.uk/abs/ (AbM). “b” notes buried sidechain;“p” notes partially buried;“i” notes sidechain at interface between VH and VL domains. Sequence differences between human and murine germlines noted by asterisk (*). Potential additional mutations in frameworks are noted below sequence. Potential changes in CDR sequences noted below each CDR sequence as noted on the figure (# deamidation substitutions: Q/S/A; these may prevent asparagine (N) deamidation. @ tryptophan oxidation substitutions: Y/F/H; these may prevent tryptophan oxidation; @ methionine oxidation substitutions: L/F/A).

[00106] Figure 17A to 17C depicts a collation of the humanized sequences of three CHA antibodies: CHA.7.518, CHA.7.538.1, and CHA.7.538.2.

[00107] Figure 18 depicts schemes for combining the humanized VH and VL CHA antibodies. The“chimVH” and“chimVL” are the mouse variable heavy and light sequences attached to a human IgG constant domain.

[00108] Figure 19. Sequence alignment of PVRIG orthologs. Aligned sequences of the human, cynomolgus, marmoset, and rhesus PVRIG extra-cellular domain. The differences between human and cynomolgus are highlighted in yellow.

[00109] Figure 20A-20D depict the amino acid sequences and the nucleic acid sequence for the variable heavy chain (A and B, respectfully) and the amino acid sequences and the nucleic acid sequence for the variable light chain (C and D, respectfully) for AB-407 (BOJ-5G4-F4).

[00110] Figures 21A and 21B depicts the amino acid sequences of the constant domains of human IgGl (with some useful amino acid substitutions), IgG2, IgG3, IgG4, IgG4 with a hinge variant that finds particular use in the present invention, and the constant domains of the kappa and lambda light chains.

[00111] Figure 22 depicts the sequences of human and cynomolgus macaque (referred to as cyno) TIGIT ECD and of the human PVR ECD proteins.

[00112] Figure 23A-23D depict the sequences of anti-TIGIT antibodies. Unless otherwise noted, the CDRs utilize the IMGT numbering (including the antibodies of the sequence listing).

[00113] Figure 24A-24SSSS depict the sequences of numerous anti-TIGIT antibodies for use in the bispecifc antibodies of the present invention.

[00114] Figure 25 depicts a schematic of an exemplary bispecific antibody

emboidment using the CrossMab Technology (MAbs. 2016 Aug-Sep; 8(6): 1010-1020) and knobs in hole technology (Nat Biotechnol. 1998 Jul;l6(7):677-8l).

[00115] Figure 26 provides the sequences of the heavy and light chains for each arm of the CHA.7.5l8. l.H4(S24lP)/CPA.9.086 bispecific antibody. The variable domains are shown in italics. The CDRs are colored in red. The mutations in the Fc domains are shown in bold and underlined.

[00116] Figure 27 provides the SPR sensorgrams (black lines) of (A) human TIGIT binding to captured TIGIT-PVRIG Bispecific antibody over two independent surfaces and of (B) human PVRIG binding to captured TIGIT-PVRIG Bispecific antibody over two independent surfaces. The red lines in both (A) and (B) are the global fit of the sensorgrams to a simple 1 : 1 kinetic binding model. The concentration range of TIGIT was 362pM -88nM and the concentration range of PVRIG was 460pM-l 12hM. Because there wasn’t sufficient dissociation signal decay data after 15 minutes with the TIGIT antigen, the kd was arbitrarily held constant at le 5 sec 1 for the global fit. When holding kd constant for the TIGIT-bispecific interaction, the ka was estimated to be 3.9e6 IVT'sec 1 with the kd/ka ratio calculating KD = 2.6pM. The binding constants for the PVRIG-Bispecific antibody interaction were ka = l.3e6 iWsec 1. kd = 2.4e 4 sec 1, and KD = 187rM.

[00117] Figure 28 depicts SPR“sandwich” assay demonstrating simultaneous binding of human TIGIT and human PVRIG to the anti-TIGIT-PVRIG bispecific antibody. The bispecific antibody is first injected (A) over human TIGIT covalently immobilized to the biosensor chip. Human PVRIG is then injected (B) over the TIGIT-bound bispecific antibody.

[00118] Figure 29 depicts characterization of pp65 specific CD8+ T cells. A) PBMCs were activated with pp65(495 - 503) peptide, IL-2 and IL-7 for 11 days. Flow cytometry was performed to assess the percentage of pp65 reactive T cells and the expression of PVRIG, TIGIT, and PD-l. Representative gating hierarchy from 1 donor and representative tetramer staining from two donors are shown. From left to right, lymphocytes were gated by forward scatter (FSC)/side scatter (SSC) (upper left), and live CD3+CD8+ positive cells. Within the CD3+CD8+ positive population, the percentage of cells that bind HLA-A201 pp65 tetramer is determined and data from 2 donors are shown. B. The expression of PVRIG, TIGIT and PD-l on the pp65 reactive T cells was evaluated by flow cytometry. The number in the upper right-hand comer denotes the MFIr of the target of interest relative to isotype control.

[00119] Figure 30 depicts the effect of inhibitory receptor blockade on CMV pp65 reactive CD8 T cells in co-culture with Mel-624 pp65 cancer cell line. CMV pp65 reactive T cells for 2 donors, Donor 4 (A) and Donor 72 (B) were co-cultured with a modified Mel-624 tumor cell line ectopically expressing pp65 in the presence of CHA.7.5l8. l.H4(S24lP) and CPA.9.086 either alone or in combination, CHA.7.5l8. l.H4(S24lP)/CPA.9.086 BsAb or a human IgG isotype control for l8hrs. Conditioned media were assayed for cytokine secretion. The numbers above the bars indicate the % change relative to isotype control. The bar graphs show the average + standard deviation for IFN-g (N=2).

[00120] Figure 31 provides dose-dependent titration of inhibitory receptor blockade on CMV pp65 reactive CD8 T cells in co-culture with Mel-624 pp65 cancer cell line. CMV pp65 reactive T cells for 2 donors, Donor 4 (A) and Donor 72 (B) were co-cultured with a modified Mel-624 tumor cell line ectopically expressing pp65 in the presence of

CHAV.518. l.H4(S24lP) and CPA.9.086 either alone or in combination,

CHAV.518. l.H4(S24lP)/CPA.9.086 BsAb or a human IgG isotype control for l8hrs. A 10 point, 4-fold dilution series starting at 20 pg/ml was used for each antibody. Conditioned media were assayed for cytokine secretion. ECso (nM) values for combined blockade using CHAV.518. l.H4(S24lP) and CPA.9.086 and for the bispecific antibody treatments are reported. Representative data (N=2).

[00121] Figure 32 provides an overview of Fc heterodimerzation strategies for use in bispecific antibody generation applicable to the bispecific antibodies of the invention. (See, Godar, et. al., Expert Opinion on Therapeutic Patents, 28(3):25l-276 (2018).)

[00122] Figure 33 provides an overview of asymmetric bispecific antibody formats applicable to the bispecific antibodies of the invention. (See, Brinkmann and Kontermann, The making of bispecific antibodies, MAbs, 9(2): 182-212 (2017).)

[00123] Figure 34 provides an overview of symmetric bispecific antibody formats applicable to the bispecific antibodies of the invention. (See. Brinkmann and Kontermann, The making of bispecific antibodies, MAbs, 9(2): 182-212 (2017).)

[00124] Figure 35 provides additional anti-PVRIG antibodies for use in the bispecifc antibodies of the present invention.

[00125] Figure 36 provides the SPR sensorgrams (black lines) of human PVRIg binding to captured PVRIg bispecific and monospecific antibodies. The red lines in both are the global fit of the sensorgrams to a simple 1 : 1 kinetic binding model. The concentration range of PVRIG was 460pM-l 12hM. (A) CHA.7.518.4-H4 hole+CHA.9.547. l8-H4 CrossMab knob, (B) CHA.7.518.4-H4 hole+CPA.9.086-CrossMab H4 knob, (C)

CHA.7.518.4-H4 hole+CPA.9.086 scFv (VL-VH)-H4 knob, (D) CHA.7.518.4-H4 hole+CPA.9.086 scFv (VH-VL)-H4 knob, (E) CPA.9.086 scFv (VH-VL) H4

varl+CHA.7.518.1 Fab H4 var2, (F) CHA.7.518.1 scFv (VL-VH) H4 varl+CPA.9.086 Fab H4 var2, (G) CHA.9.547.18 scFv (VH-VL) H4 varl+CHA.7.5l8.4 Fab H4 var2, (H)

CHAV.518.4 scFv (VL-VH) H4 varl+CHA.9.547 8 Fab H4 var2, (I) CHA.7.518.1 H4, (I) (J) Synagis H4. The binding affinities were estimated from the kd/ka ratio (KD ) calculation for each antibody and are presented in Figure 38.

[00126] Figure 37 provides the SPR sensorgrams (black lines) of human TIGIT binding to captured TIGIT bispecific and monospecific antibodies. The red lines are the global fit of the sensorgrams to a simple 1 : 1 kinetic binding model. The concentration range of TIGIT was 362pM-88nM. (A) CHA.7.518.4-H4 hole+CHA.9.547. l8-H4 CrossMab knob, (B) CHA.7.518.4-H4 hole+CPA.9.086-CrossMab H4 knob, (C) CHA.7.518.4-H4 hole+CPA.9.086 scFv (VL-VH)-H4 knob, (D) CHA.7.518.4-H4 hole+CPA.9.086 scFv (VH-VL)-H4 knob, (E) CPA.9.086 scFv (VH-VL) H4 varl+CHA.7.518.1 Fab H4 var2, (F) CHAV.518.1 scFv (VL-VH) H4 varl+CPA.9.086 Fab H4 var2, (G) CHA.9.547.18 scFv (VH-VL) H4 varl+CHA.7.518.4 Fab H4 var2, (H) CHA.7.518.4 scFv (VL-VH) H4 varl+CHA.9.547. l8 Fab H4 var2, (I) CPA.9.086 H4, (J) Synagis H4. The binding affinities were estimated from the kd/ka ratio (KD ) calculation for each antibody and are presented in Figure 39.

[00127] Figure 38 provides the PVRIG binding affinities of bispecific and

monospecific antibodies determined by SPR.

[00128] Figure 39 provides the TIGIT binding affinities of bispecific and monospecific antibodies determined by SPR.

[00129] Figure 40 provides additional anti-PVRIG antibodies for use in the bispecifc antibodies of the present invention. Red font text indicates amino acid substitutions and () indicates a deletion relative to the reference human IgG4 amino acid sequence. Underlined text indicates CDRs. Grey highlighted text indicates the Fc domains.

[00130] Figure 41 provides additional anti-TIGIT antibodies for use in the bispecifc antibodies of the present invention. Red font text indicates amino acid substitutions and () indicates a deletion relative to the reference human IgG4 amino acid sequence. Underlined text indicates CDRs. Grey highlighted text indicates the Fc domains.

[00131] Figure 42 provides a diagram of an exemplary CrossMab bispecific antibody format that has one traditional antibody arm and one in which the heavy chain and light chain constant domains are swapped. Amino acid substitutions for the“knob into hole” format are indicated according to human IgGl Eu numbering.

[00132] Figure 43 provides a diagram of an exemplary“bottle opener” bispecific antibody format that has one traditional antibody arm and one scFv-Fc fusion arm. Amino acid substitutions for the“knob into hole” format are indicated according to human IgGl Eu numbering.

[00133] Figure 44 provides a diagram of an exemplary“bottle opener” bispecific antibody format that has one traditional antibody arm and one scFv-Fc fusion arm. Amino acid substitutions for the“isovolumetric heterodimerization” format are indicated according to human IgGl Eu numbering. Additional substitutions that contribute to reduced FcgR binding are indicated.

[00134] Figure 45 provides data regarding the expression and purification of anti-PVRIG-TIGIT bispecific antibodies, monomer content and % correct assembled bispecific antibody as determined by LC-MS after MabSelect Sure affinity chromatography step and size exclusion chromatography (if % monomer was less than 95% from affinity

chromatography).

[00135] Figure 46 provides data regarding CMV screening assays for anti-PVRIG-TIGIT bispecific antibody screening on three different T-cell donors and performed at 10 ug/ml total antibody concentration per treatment

[00136] Figure 47 provides flow cytometry examples of PVRIG, TIGIT and PD-l ligand (PVR, PVRL2 and PD-L1, respectively) expression on recombinant CHO cell lines used in the cell assays.

[00137] Figure 48 provides flow cytometry examples of PVRIG, TIGIT and PD-l expression levels in T-cells from three different donors.

[00138] Figure 49 provides ELISA binding data demonstrating simultaneous binding of both PVRIG and TIGIT for varaious anti-PVRIG-TIGIT antibodies using a PVRIG coated plate and detecting bound antibodies using biotinylated TIGIT-His and Steptavidin- HRP.

[00139] Figure 50 provides ELISA binding data demonstrating simultaneous binding of both PVRIG and TIGIT for varaious anti-PVRIG-TIGIT antibodies using a TIGIT coated plate and detecting bound antibodies using biotinylated PVRIG-His and Steptavidin- HRP.

[00140] Figure 51 provides EC-50 data for the ELISA assay data provided in Figures 49 and 50.

[00141] Figure 52 provides example stability data of bispecific antibodies in multiple formats as assessed by Differential Scanning Fluorimetry (DSF) and aggregate formation after a low pH hold and 3 cycles of freezing and thaw.

[00142] Figure 53 provides stability assessment of the different bispecific formats. Tests were performed to simulate viral inactivation by holding the antibodies at pH 3 for various times and to stress test by performing 3 serial freeze/thaw cycles. Changes in the relative amount of heavy chain (scFv or Fab-containing) (A), light chain (B) under reducing conditions or intact molecule (C) in non-reducing conditions (CE-SDS) and in low molecular weight, high molecular weigh, and monomer species content (SEC-UPLC) (D). Data are reported as a change from T=0.

[00143] Figure 54A provides an in vitro co-culture assay with human CMV-specific CD8+ T cells from three donors (Donor 4, Donor 210, and Donor 234) were utilized to assess the effect of anti-TIGIT antibodies (CPA.9.086 and CHA.9.547.18) and anti-PVRIG antibodies (CHA.7.518.1, CHA.7.518.4) on antigen-specific cytokine secretion either alone, in combination or as bispecific antibodies. The target cell line used in the assay was the HLA-A2 + PVRL2 + PVR + expressing melanoma 624 (Mel-624) cell line which has been modified to ectopically express pp65. Cells were plated at 75,000 cells/well in 96-well round- bottom tissue culture treated plates. 15,000 human CD8+ T cells were added to each well.

The indicated anti-human PVRIG, TIGIT or isotype control hIgG4 antibodies were added at a concentration of 1 pg/ml. Co-cultures were incubated at 37 °C with 5% C02 for 24 hours.

The amount of human IFNy in the co-culture supernatant was measured by flow cytometry using a LEGENDplex IFN bead assay (BD Biosciences). The percent increase of IFNy secretion for each antibody over the hIgG4 isotype is indicated (n=2 experiments, representative results from 1 experiment shown). (A) CHA.7.518.4-H4 hole+CPA.9.086-CrossMab H4 knob, (B) CHA.7.518.4-H4 hole+CPA.9.086 scFv (VH-VL)-H4 knob, (C) CPA.9.086 scFv (VH-VL) H4 varl+CHA.7.5l8.l Fab H4 var2, (D) CHA.7.518.1 scFv (VL-VH) H4 varl+CPA.9.086 Fab H4 var2, (E) CHA.7.518.1 H4, (F) CPA.9.086 H4, (G)

CHAV.518.1 H4, (H) CPA.9.086 H4, (I) Synagis H4.

[00144] Figure 54B provides a summary of the two CMV assay replicates performed with different expansions of the donor cells.

IV. DETAILED DESCRIPTION OF THE INVENTION

A. Overview

[00145] The present invention provides a number of useful anti-PVRIG, anti-TIGIT, and/or anti-PVRIG/anti-TIGIT bispecific antibodies, for use in particular in the treatment of cancer. Cancer can be considered as an inability of the patient to recognize and eliminate cancerous cells. In many instances, these transformed (e.g. cancerous) cells counteract immunosurveillance. There are natural control mechanisms that limit T-cell activation in the body to prevent unrestrained T-cell activity, which can be exploited by cancerous cells to evade or suppress the immune response. Restoring the capacity of immune effector cells-especially T cells-to recognize and eliminate cancer is the goal of immunotherapy. The field of immuno-oncology, sometimes referred to as“immunotherapy” is rapidly evolving, with several recent approvals of T cell checkpoint inhibitory antibodies such as Yervoy, Keytruda and Opdivo. These antibodies are generally referred to as“checkpoint inhibitors” because they block normally negative regulators of T cell immunity. It is generally understood that a variety of immunomodulatory signals, both costimulatory and coinhibitory, can be used to orchestrate an optimal antigen-specific immune response. Generally, these antibodies bind to checkpoint inhibitor proteins such as CTLA-4 or PD-l, which under normal circumstances prevent or suppress activation of cytotoxic T cells (CTLs). By inhibiting the checkpoint protein, for example through the use of antibodies that bind these proteins, an increased T cell response against tumors can be achieved. That is, these cancer checkpoint proteins suppress the immune response; when the proteins are blocked, for example using antibodies to the checkpoint protein, the immune system is activated, leading to immune stimulation, resulting in treatment of conditions such as cancer and infectious disease.

[00146] The present invention is directed to the use of bispecific antibodies to additional checkpoint proteins, PVRIG and TIGIT. PVRIG is expressed on the cell surface of NK and T-cells and shares several similarities to other known immune checkpoints. The identification and methods used to show that PVRIG is a checkpoint receptor are discussed in WO2016/134333, expressly incorporated herein by reference. Anti-PVRIG, anti-TIGIT, and/or anti-PVRIG/anti -TIGIT bispecific antibodies to human PVRIG that block the interaction and/or binding of PVLR2 are provided herein. When PVRIG is bound by its ligand (PVRL2), an inhibitory signal is elicited which acts to attenuate the immune response of NK and T-cells against a target cell (i.e. analogous to PD-1/PDL1). Blocking the binding of PVRL2 to PVRIG shuts-off this inhibitory signal of PVRIG and as a result modulates the immune response of NK and T-cells. Utilizing an antibody against PVRIG that blocks binding to PVRL2 is a therapeutic approach that enhances the killing of cancer cells by NK and T-cells. Blocking antibodies have been generated which bind PVRIG and block the binding of its ligand, PVRL2.

[00147] Similarly, TIGIT has been shown to also have attributes of a checkpoint receptor, and the present invention provides anti-PVRIG, anti-TIGIT, and/or anti-PVRIG/anti -TIGIT bispecific antibodies that block the interaction and/or binding of TIGIT to PVR are provided. When TIGIT is bound by its ligand (PVR), an inhibitory signal is elicited which acts to attenuate the immune response of NK and T-cells against a target cell (i.e. analogous to PD-1/PDL1). Blocking the binding of PVR to TIGIT shuts-off this inhibitory signal of TIGIT and as a result modulates the immune response of NK and T-cells. Utilizing an antibody against TIGIT that blocks binding to PVR is a therapeutic approach that enhances the killing of cancer cells by NK and T-cells. Blocking antibodies have been generated which bind TIGIT and block the binding of its ligand, PVR.

[00148] Additionally, the invention provides anti-PVRIG, anti-TIGIT, and/or anti- PVRIG/ anti -TIGIT bispecific antibodiesfor use in the treatment of cancer.

B. Definitions

[00149] In order that the application may be more completely understood, several definitions are set forth below. Such definitions are meant to encompass grammatical equivalents.

[00150] IgG domain definitions used herein are in accordance with IMGT reference sequences (www.IMGT.org)

What is claimed:

1. An anti-PVRIG/anti-TIGIT bispecific antibody that monovalently binds a human PVRIG and monovalently binds TIGIT for use in activating T cells and/or NK cells for the treatment of cancer.

2. An anti-PVRIG/anti-TIGIT bispecific antibody comprising:

b) a first antigen binding portion comprising:

iii. a first heavy chain variable domain comprising a vhCDRl, vhCDR2, and vhCDR3 from an anti-PVRIG antibody; and iv. a first light chain variable domain comprising a vlCDRl, vlCDR2, and vlCDR3 from an anti-PVRIG antibody;

wherein the anti-PVRIG antibody is selected from the group consisting of CHA.7.518.4, CHA.7.518.1, CHA.7.518, CHA.7.524 CHA.7.530, CHA.7.538_l, CHA.7.538_2, CHA.7.502, CHA.7.503, CHA.7.506, CHA.7.508, CHA.7.510, CHA.7.512, CHA.7.514, CHA.7.516, CHA.7.518, CHA.7.520.1, CHA.7.520.2, CHA.7.522, CHA.7.524, CHA.7.526, CHA.7.527, CHA.7.528, CHA.7.530, CHA.7.534, CHA.7.535, CHA.7.537, CHA.7.538.1, CHA.7.538.2, CHA.7.543, CHA.7.544, CHA.7.545, CHA.7.546, CHA.7.547, CHA.7.548, CHA.7.549, CHAV.550, CHA7.538.1.2, CPA.7.021, CPA.7.001, CPA.7.003, CPA.7.004, CPA.7.006, CPA.7.008, CPA.7.009,

CPA.7.010, CPA.7.011, CPA.7.012, CPA.7.013, CPA.7.014,

CPA.7.015, CPA.7.017, CPA.7.018, CPA.7.0l9,CPA.7.022,

CP A.7.023, CPA.7.024, CPA.7.033, CPA.7.034, CPA.7.036,

CPA.7.040, CPA.7.046, CPA.7.047, CPA.7.049, CPA.7.050, and CHAV.518; and

b) a second antigen binding portion comprising an anti-TIGIT antigen binding domain.

3. An anti-PVRIG/anti-TIGIT bispecific antibody comprising:

c) a first antigen binding portion comprising an anti-PVRIG antigen binding domain; and

d) a second antigen binding portion comprising:

iii. a second heavy chain variable domain comprising a vhCDRl, vhCDR2, and vhCDR3 from an anti -TI GIT antibody; and

iv. a second light chain variable domain comprising a vlCDRl, vlCDR2, and vlCDR3 from an anti-TIGIT antibody;

wherein the anti-TIGIT antibody is selected from the group consisting of CPA.9.086, CHA.9.547.18, CPA.9.018, CPA.9.027, CPA.9.049, CPA.9.057, CPA.9.059, CPA.9.083, CPA.9.089, CPA.9.093, CPA.9.101, CPA.9.103, CHA.9.536.1, CHA.9.536.3, CHA.9.536.4, CHA.9.536.5, CHA.9.536.6, CHA.9.536.7, CHA.9.536.8,

CHA.9.560.1, CHA.9.560.3, CHA.9.560.4, CHA.9.560.5,

CHA.9.560.6, CHA.9.560.7, CHA.9.560.8, CHA.9.546.1,

CHA.9.547.1, CHA.9.547.2, CHA.9.547.3, CHA.9.547.4,

CHA.9.547.6, CHA.9.547.7, CHA.9.547.8, CHA.9.547.9,

CHA.9.547.13, CHA.9.541.1, CHA.9.541.3, CHA.9.541.4,

CHA.9.541.5, CHA.9.541.6, CHA.9.541.7, and CHA.9.541.8.

4. An anti-PVRIG/anti-TIGIT bispecific antibody comprising:

c) a first antigen binding portion comprising:

iii. a first heavy chain variable domain comprising a vhCDRl, vhCDR2, and vhCDR3 from an anti-PVRIG antibody; and

iv. a first light chain variable domain comprising a vlCDRl, vlCDR2, and vlCDR3 from an anti-PVRIG antibody

wherein the anti-PVRIG antibody is selected from the group consisting of CHA.7.518.4, CHA.7.518.1, CHA.7.518, CHA.7.524, CHA.7.530, CHA.7.538_l, CHA.7.538_2, CHA.7.502, CHA.7.503, CHA.7.506, CHA.7.508, CHA.7.510, CHA.7.512, CHA.7.514, CHA.7.516, CHA.7.518, CHA.7.520.1, CHA.7.520.2, CHA.7.522, CHA.7.524, CHA.7.526, CHA.7.527, CHA.7.528, CHA.7.530, CHA.7.534, CHA.7.535, CHA.7.537, CHA.7.538.1, CHA.7.538.2, CHA.7.543, CHA.7.544, CHA.7.545, CHA.7.546, CHA.7.547, CHA.7.548, CHA.7.549, CHAV.550, CHA7.538.1.2, CPA.7.021, CPA.7.001, CPA.7.003, CPA.7.004, CPA.7.006, CPA.7.008, CPA.7.009,

CPA.7.010, CPA.7.011, CPA.7.012, CPA.7.013, CPA.7.014,

CPA.7.015, CPA.7.017, CPA.7.018, CPA.7.0l9,CPA.7.022,

CP A.7.023, CPA.7.024, CPA.7.033, CPA.7.034, CPA.7.036,

CPA.7.040, CPA.7.046, CPA.7.047, CPA.7.049, and CPA.7.050CHA.7.518; and

d) a second antigen binding portion comprising:

iii. a second heavy chain variable domain comprising a vhCDRl, vhCDR2, and vhCDR3 from an anti -TI GIT antibody; and

iv. a second light chain variable domain comprising a vlCDRl, vlCDR2, and vlCDR3 from an anti-TIGIT antibody;

wherein the anti-TIGIT antibody is selected from the group consisting of CPA.9.086, CHA.9.547.18, CPA.9.018, CPA.9.027, CPA.9.049, CPA.9.057, CPA.9.059, CPA.9.083, CPA.9.086, CPA.9.089,

CPA.9.093, CPA.9.101, CPA.9.103, CHA.9.536.1, CHA.9.536.3, CHA.9.536.4, CHA.9.536.5, CHA.9.536.6, CHA.9.536.7,

CHA.9.536.8, CHA.9.560.1, CHA.9.560.3, CHA.9.560.4,

CHA.9.560.5, CHA.9.560.6, CHA.9.560.7, CHA.9.560.8,

CHA.9.546.1, CHA.9.547.1, CHA.9.547.2, CHA.9.547.3,

CHA.9.547.4, CHA.9.547.6, CHA.9.547.7, CHA.9.547.8,

CHA.9.547.9, CHA.9.547.13, CHA.9.541.1, CHA.9.541.3,

CHA.9.541.4, CHA.9.541.5, CHA.9.541.6, CHA.9.541.7, and

CHA.9.541.8.

5. The anti-PVRIG/anti-TIGIT bispecific antibody of claim 4, wherein first antigen

binding portion comprises:

iii. a first heavy chain comprising VH-CHl-hinge-CH2-CH3; and iv. a first light chain comprising VL-CL, wherein the CL is the constant domain of either a kappa or lambda antibody.

6. The anti-PVRIG/anti-TIGIT bispecific antibody of claim 5, wherein the first heavy chain CH3 comprises the amino acid substitutions S354C, E356D, M358L, and T366W.

7. The anti-PVRIG/anti-TIGIT bispecific antibody of claim 5, wherein the CL is kappa.

8. The anti-PVRIG/anti-TIGIT bispecific antibody of claim 4, wherein the second antigen binding portion comprises:

iii. a second heavy chain comprising HC-CL-hinge-CH2-CH3, wherein the CL is either kappa or lambda; and

iv. a second light chain comprising VL-CH1.

9. The anti-PVRIG/anti-TIGIT bispecific antibody of claim 8, wherein the second heavy chain CH3 comprises the amino acid substitutions Y349C, E356D, M358L, T366S, L368A, and Y407V.

10. The anti-PVRIG/anti-TIGIT bispecific antibody of claim 8, wherein the CL is

lambda.

11. The anti-PVRIG/anti-TIGIT bispecific antibody of claim 8, wherein the CL is kappa.

12. An anti-PVRIG/anti-TIGIT bispecific antibody comprising:

e) a first antigen binding portion comprising an anti-PVRIG antigen binding domain; and

f) a second anti-TIGIT antigen binding portion comprising:

iii. a second heavy chain variable region comprising CPA.9.086 VH

(EV QLVET GGGLIQPGRSLRL S C AAS GFTF S S Y AMHWVRQ APG KGLEWVAVISYAGEVKYYADSVKGRFTISRDNSKNTLYLQMN SLRAEDTAVYY C ARDPLPLHYY GMDVW GQGTTVTV S S ; SEQ ID NO: 1634); and

iv. a second light chain variable region comprising CPA.9.086 VL

(QSALTQPRSASGNPGQRVTISCSGSSSNMGRRPVNWYQQIPGT APKLLIYSQNQRPSGVPDRFSGSQSGTSASLTISGLQSEDEAEYF CAVWDDIGRVLQLGGGTQLAVL; SEQ ID NO: 1639).

13. An anti-PVRIG/anti-TIGIT bispecific antibody comprising:

g) a first antigen binding portion comprising an anti-PVRIG antigen binding domain; and

h) a second anti-TIGIT antigen binding portion comprising:

iii. a second heavy chain variable region comprising CPA.9.547.18 VH

(EVOLVESGGGLVOPGGSLRLSCAASGFTFSSYIMSWVROAPGK GLEWV ATISGGSKGQYY ADS VKGRFTISRDNSKNTLYLQMNSL

RAEDT AY YY C AKWLL S YY AMD YW GOGTL VTV S S ; SEQ ID NO: 1664); and

iv. a second light chain variable region comprising CPA.9.547.18 VL

(DIOMTOSPSSLSASVGDRITITCRASOSMAIWLSWYOOKPGKA PKLLI YKASKSHT GVP SRF S GS GS GTDFTLTIS SLQPEDF ATYY C OOGOSYP YTF GQGTKLEIK; SEQ ID NO: 1668).

14. An anti-PVRIG/anti-TIGIT bispecific antibody comprising:

c) a first antigen binding portion comprising an anti-PVRIG binding doming comprising:

i. a first heavy chain variable region comprising CHA.7.518.1 VH

(QV QLVQSGAEVKKPGAS VKV SCKASGYTFTDYNINWVRQAP GQGLEWMGYIYPYIGGSGYAQKFQGRVTMTRDTSTSTVYMEL S SLRS EDT AV YY C AREDKT ARN AMDYW GQGTL VTV S S ; SEQ ID NO: 1539); and

ii. a first light chain comprising CHA.7.518.1 VL

(DIQMTQSPSSLSASVGDRVTITCRVSENIYSNLAWYQQKPGKA PKLLI YEATNLAEGVP SRF S GS GS GTDFTLTI S SLQPEDF ATYY C QHF W GTP YTF GQGTKLEIK; SEQ ID NO: 1544); and

d) a second antigen binding portion comprising an anti-TIGIT antigen binding domain.

15. An anti-PVRIG/anti-TIGIT bispecific antibody comprising:

c) a first antigen binding portion comprising an anti-PVRIG binding doming comprising:

i. a first heavy chain variable region comprising CHA.7.518.4 VH

(QV QLVQSGAEVKKPGAS VKV SCKASGYTFTDYNINWVRQAP GQGLEWMGYIYPYIGGSGYAQKFQGRVTMTRDTSTSTVYMEL S SLRS EDTAVYYC AREDKT ARN AMDYWGQGTLVTVS S ; SEQ ID NO: 3179); and

ii. a first light chain comprising CHA.7.518.4 VL

(DIQMTQSPSSLSASVGDRVTITCRVSENIYDVLAWYQQKPGKA PKLLI YEATNLAEGVP SRF S GS GS GTDFTLTI S SLQPEDF ATYY C QHF W GTP YTF GQGTKLEIK; SEQ ID NO:3l80); and

d) a second antigen binding portion comprising an anti-TIGIT antigen binding domain.

16. An anti-PVRIG/anti-TIGIT bispecific antibody comprising:

c) a first antigen binding portion comprising an anti-PVRIG binding doming comprising:

i. a first heavy chain variable region comprising CHA.7.518.1 VH

(QV QLVQSGAEVKKPGASVKV SCKASGYTFTDYNINWVRQAP GQGLEWMGYIYPYIGGSGYAQKFQGRVTMTRDTSTSTVYMEL S SLRS EDT AV YY C AREDKT ARN AMDYW GQGTL VTV S S ; SEQ ID NO: 1539); and

ii. a first light chain variable region comprising CHA.7.518.1 (DIQMTQSPSSLSASVGDRVTITCRVSENIYSNLAWYQQKPGKA PKLLI YEATNL AEGVP SRF S GS GS GTDFTLTI S SLQPEDF ATYY C QHF W GTP YTF GQGTKLEIK; SEQ ID NO: 1544); and

d) a second antigen binding portion comprising an anti-TIGIT binding doming comprising

i. a first heavy chain variable region comprising CPA.9.086 VH

(EVQLVETGGGLIQPGRSLRLSCAASGFTFSSYAMHWVRQAPG KGLEWVAVISYAGEVKYYADSVKGRFTISRDNSKNTLYLQMN SLRAEDTAVYY C ARDPLPLHYY GMDVW GQGTTVTV S S ; SEQ ID NO: 1634); and

ii. a first light chain variable region comprising CPA.9.086 VL (QSALTQPRSASGNPGQRVTISCSGSSSNMGRRPVNWYQQIPGT APKLLIYSQNQRPSGVPDRFSGSQSGTSASLTISGLQSEDEAEYF CAVWDDIGRVLQLGGGTQLAVL; SEQ ID NO: 1639).

17. An anti-PVRIG/anti-TIGIT bispecific antibody comprising:

c) a first antigen binding portion comprising an anti-PVRIG binding doming comprising:

i. a first heavy chain variable region comprising CHA.7.518.4 VH

(QV QLVQSGAEVKKPGASVKV SCKASGYTFTDYNINWVRQAP GQGLEWMGYIYPYIGGSGYAQKFQGRVTMTRDTSTSTVYMEL

S SLRS EDT AV YY C AREDKT ARN AMDYW GQGTL VTV S S ; SEQ ID NO: 3179); and

ii. a first light chain comprising CHA.7.518.4 VL

(DIQMTQSPSSLSASVGDRVTITCRVSENIYDVLAWYQQKPGKA PKLLI YEATNL AEGVP SRF S GS GS GTDFTLTI S SLQPEDF ATYY C QHF W GTP YTF GQGTKLEIK; SEQ ID NO:3l80); and

d) a second antigen binding portion comprising an anti-TIGIT binding doming comprising:

i. a first heavy chain variable region comprising CPA.9.086 VH

(EV QLVET GGGLIQPGRSLRL S C AAS GFTF S S Y AMHWVRQ APG KGLEWVAVISYAGEVKYYADSVKGRFTISRDNSKNTLYLQMN SLRAEDTAVYY C ARDPLPLHYY GMDVW GQGTTVTV S S ; SEQ ID NO: 1634); and

ii. a first light chain variable region comprising CPA.9.086 VL (QSALTQPRSASGNPGQRVTISCSGSSSNMGRRPVNWYQQIPGT APKLLIYSQNQRPSGVPDRFSGSQSGTSASLTISGLQSEDEAEYF CAVWDDIGRVLQLGGGTQLAVL; SEQ ID NO: 1639).

18. An anti-PVRIG/anti-TIGIT bispecific antibody comprising:

c) a first antigen binding portion comprising an anti-PVRIG binding doming comprising:

i. a first heavy chain variable region comprising CHA.7.518.1

(QV QLVQSGAEVKKPGAS VKV SCKASGYTFTDYNINWVRQAP GQGLEWMGYIYPYIGGSGYAQKFQGRVTMTRDTSTSTVYMEL S SLRS EDTAVYYC AREDKT ARN AMDYWGQGTLVTVS S ; SEQ ID NO: 1539); and

ii. a first light chain variable region comprising CHA.7.518.1 VL

(DIQMTQSPSSLSASVGDRVTITCRVSENIYSNLAWYQQKPGKA PKLLI YEATNL AEGVP SRF S GS GS GTDFTLTI S SLQPEDF ATYY C QHF W GTP YTF GQGTKLEIK; SEQ ID NO: 1544); and

d) a second antigen binding portion comprising an anti-TIGIT binding doming comprising:

i. a first heavy chain variable region comprising CHA.9.547.18 HC (EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYIMSWVRQAPGK GLEWVATISGGSKGQYYADSVKGRFTISRDNSKNTLYLQMNSL RAEDT AV YY C AKWLL S YY AMD YW GQGTL VTV S S ; SEQ ID NO: 1664); and

ii. a first light chain variable region comprising CHA.9.547.18 VL

(DIQMTQSPSSLSASVGDRITITCRASQSMAIWLSWYQQKPGKA PKLLIYKASKSHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQGQSYPYTFGQGTKLEIK; SEQ ID NO: 1668).

19. An anti-PVRIG/anti-TIGIT bispecific antibody comprising:

c) a first antigen binding portion comprising an anti-PVRIG binding doming comprising:

i. a first heavy chain variable region comprising CHA.7.518.4 VH

(QV QLVQSGAEVKKPGASVKV SCKASGYTFTDYNINWVRQAP GQGLEWMGYIYPYIGGSGYAQKFQGRVTMTRDTSTSTVYMEL S SLRS EDT AV YY C AREDKT ARN AMDYW GQGTL VTV S S ; SEQ ID NO: 3179); and

ii. a first light chain comprising CHA.7.518.4 VL

(DIQMTQSPSSLSASVGDRVTITCRVSENIYDVLAWYQQKPGKA PKLLI YEATNL AEGVP SRF S GS GS GTDFTLTI S SLQPEDF ATYY C QHF W GTP YTF GQGTKLEIK; SEQ ID NO:3l80); and

d) a second antigen binding portion comprising an anti-TIGIT binding doming comprising:

i. a first heavy chain variable region comprising CHA.9.547.18 HC

(EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYIMSWVRQAPGK GLEWVATISGGSKGQYYADSVKGRFTISRDNSKNTLYLQMNSL RAEDT AV YY C AKWLL S YY AMDYW GQGTL VTV S S (SEQ ID NO: 1664); and

ii. a first light chain variable region comprising CHA.9.547.18 VL

(DIQMTQSPSSLSASVGDRITITCRASQSMAIWLSWYQQKPGKA PKLLIYKASKSHTGVPSRFSGSGSGTDFTLTIS SLQPEDF ATYYC QQGQSYPYTFGQGTKLEIK; SEQ ID NO: 1668).

20. An anti-PVRIG/anti-TIGIT bispecific antibody according to any one of claims 1-19 and 37-38 wherein said anti-PVRIG/anti-TIGIT bispecific antibody is a humanized antibody.

21. A composition comprising an anti-PVRIG/anti-TIGIT bispecific antibody according to any one of claims 1 to 20.

22. A nucleic acid composition comprising:

e) a first nucleic acid encoding a first heavy chain or heavy chain variable domain according to any one of claims 1 to 20;

f) a second nucleic acid encoding a first light chain or light chain variable domain according to any one of claims 1 to 20;

g) a third nucleic acid encoding a second heavy chain or heavy chain variable domain according to any one of claims 1 to 20; and

h) a fourth nucleic acid encoding a second light chain or light chain variable domain according to any one of claims 1 to 20.

23. An expression vector composition comprising:

e) a first expression vector comprising the first nucleic acid of claim 22; f) a second expression vector comprising the second nucleic acid of claim 22; g) a third expression vector comprising the third nucleic acid of claim 22; and h) a fourth expression vector comprising the fourth nucleic acid of claim 22.

24. An expression vector composition comprising:

c) a first expression vector comprising the first and second nucleic acids of claim

22; and

d) a second expression vector comprising the third and fourth nucleic acids of claim 22.

25. A host cell comprising the expression vector composition of claim 23 or claim 24.

26. A method of making an anti-PVRIG/anti-TIGIT bispecific antibody comprising: c) culturing the host cell of claim 25 under conditions wherein the antibody is expressed; and

d) recovering the antibody.

27. A method of activating T cells of a patient comprising administering the anti- PVRIG/anti-TIGIT bispecific antibody of any one of claims 1 to 20 to the patient, wherein a subset of the T cells of the patient are activated.

28. A method of activating cytotoxic T cells (CTLs) of a patient comprising administering the anti-PVRIG/anti-TIGIT bispecific antibody of any one of claims 1 to 20 to the patient, wherein a subset of the CTLs of the patient are activated.

29. A method of activating NK cells of a patient comprising administering the anti- PVRIG/anti-TIGIT bispecific antibody of any one of claims 1 to 20 to the patient, wherein a subset of the NK cells of the patient are activated.

30. A method of activating gd T cells of a patient comprising administering the anti- PVRIG/anti-TIGIT bispecific antibody of any one of claims 1 to 20 to the patient, wherein a subset of the gd T cells of the patient are activated.

31. A method of activating Thl cells of a patient comprising administering the anti- PVRIG/anti-TIGIT bispecific antibody of any one of claims 1 to 20 to the patient, wherein a subset of the Thl cells of the patient are activated.

32. A method of decreasing or eliminating cell number and/or activity of at least one of regulatory T cells (Tregs) in a patient comprising administering the anti-PVRIG/anti- TIGIT bispecific antibody of any one of claims 1 to 20 to the patient.

33. A method of increasing interferon-g production and/or pro-inflammatory cytokine secretion in a patient comprising administering the anti-PVRIG/anti-TIGIT bispecific antibody of any one of claims 1 to 20 to the patient.

34. A method of treating cancer in a patient comprising administering the anti- PVRIG/anti-TIGIT antibody of any one of claims 1 to 20 to the patient.

35. The method of claim 34, wherein the cancer is selected from the group consisting of prostate cancer, liver cancer (HCC), colorectal cancer, ovarian cancer, endometrial cancer, breast cancer, triple negative breast cancer, pancreatic cancer, stomach (gastric) cancer, cervical cancer, head and neck cancer, thyroid cancer, testis cancer, urothelial cancer, lung cancer (small cell lung, non-small cell lung), melanoma, non melanoma skin cancer (squamous and basal cell carcinoma), glioma, renal cancer

(RCC), lymphoma (NHL or HL), Acute myeloid leukemia (AML), T cell Acute Lymphoblastic Leukemia (T-ALL), Diffuse Large B cell lymphoma, testicular germ cell tumors, mesothelioma, esophageal cancer, Merkel Cells cancer, MSI-high cancer, KRAS mutant tumors, adult T-cell leukemia/lymphoma, myeloma, and

Myelodysplastic syndromes (MDS).

36. The method of claim 35, wherein the cancer is selected from the group consisting of triple negative breast cancer, stomach (gastric) cancer, lung cancer (small cell lung, non-small cell lung), Merkel Cells cancer, MSI-high cancer, KRAS mutant tumors, adult T-cell leukemia/lymphoma, myeloma, and Myelodysplastic syndromes (MDS).

37. An anti-PVRIG/anti-TIGIT bispecific antibody comprising:

c) a first antigen binding portion comprising an anti-PVRIG antigen binding portion comprising:

i. a first heavy chain variable domain comprising a vhCDRl, vhCDR2, and vhCDR3 from an anti-PVRIG antibody; and

ii. a first light chain variable domain comprising a vlCDRl, vlCDR2, and vlCDR3 from an anti-PVRIG antibody;

wherein the anti-PVRIG antigen binding portion is selected from the group consisting of CPA.7.021, CPA.7.001, CPA.7.003, CPA.7.004, CPA.7.006, CPA.7.008, CPA.7.009, CPA.7.010, CPA.7.011,

CPA.7.012, CPA.7.013, CPA.7.014, CPA.7.015, CPA.7.017,

CPA.7.018, CPA.7.0l9,CPA.7.022, CPA.7.023, CPA.7.024,

CPA.7.033, CPA.7.034, CPA.7.036, CPA.7.040, CPA.7.046,

CPA.7.047, CPA.7.049, CPA.7.050, CHA.7.502, CHA.7.503, CHA.7.506, CHA.7.508, CHA.7.510, CHA.7.512, CHA.7.514, CHA.7.516, CHA.7.518, CHA.7.520.1, CHA.7.520.2, CHA.7.522, CHA.7.524, CHA.7.526, CHA.7.527, CHA.7.528, CHA.7.530, CHA.7.534, CHA.7.535, CHA.7.537, CHA.7.538.1, CHA.7.538.2, CHA.7.543, CHA.7.544, CHA.7.545, CHA.7.546, CHA.7.547, CHA.7.548, CHA.7.549, CHA.7.550, CHA.7.518.1; CHA7.538.1.2 and CHAV.518.4; and

d) a second antigen binding portion comprising an anti-TIGIT antigen binding domain, wherein the anti-TIGIT antigen binding domain is from an antibody as provided in Figures 24 and 41, and in particular Figure 24A-24EE.

38. An anti-PVRIG/anti-TIGIT bispecific antibody comprising:

c) a first antigen binding portion comprising an anti-PVRIG antigen binding domain wherein the anti-PVRIG antigen binding domain is from an antibody as provided in Figure 35; and

d) a second antigen binding portion comprising an anti-TIGIT antigen binding domain comprising:

iii. a second heavy chain variable domain comprising a vhCDRl , vhCDR2, and vhCDR3 from an anti-TIGIT antibody; and iv. a second light chain variable domain comprising a vlCDRl, vlCDR2, and vlCDR3 from an anti-TIGIT antibody;

wherein the anti-TIGIT antigen binding domain is selected from the group consisting of CP A.9.018, CPA.9.027, CPA.9.049, CPA.9.057, CPA.9.059, CPA.9.083, CPA.9.086, CPA.9.089, CPA.9.093, CPA.9.101, CPA.9.103, CHA.9.536.1, CHA.9.536.3, CHA.9.536.4, CHA.9.536.5, CHA.9.536.6, CHA.9.536.7, CHA.9.536.8,

CHA.9.560.1, CHA.9.560.3, CHA.9.560.4, CHA.9.560.5,

CHA.9.560.6, CHA.9.560.7, CHA.9.560.8, CHA.9.546.1,

CHA.9.547.1, CHA.9.547.2, CHA.9.547.3, CHA.9.547.4,

CHA.9.547.6, CHA.9.547.7, CHA.9.547.8, CHA.9.547.9,

CHA.9.547.13, CHA.9.541.1, CHA.9.541.3, CHA.9.541.4,

CHA.9.541.5, CHA.9.541.6, CHA.9.541.7, CHA.9.541.8, and

CHA.9.547.18.

39. An anti-PVRIG antibody comprising:

i) a heavy chain or heavy chain variable domain comprising the vhCDRl, vhCDR2, and vhCDR3 from the following sequence:

QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNINWVRQAPGQGLEWMGYIYPYIGGSGYAQKFQG RVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREDKTARNAMDYWGQGTLVTVSS (SEQ ID NO: 3179) ,

ii) a light chain or light chain variable domain comprisingthe vlCDRl, vlCDR2, vlCDR3 from the following sequence:

DIQMTQSPSSLSASVGDRVTITCRVSENIYDVLAWYQQKPGKAPKLLIYEATNLAEGVPSRFSGSG SGTDFTLTISSLQPEDFATYYCQHFWGTPYTFGQGTKLEIK (SEQ ID NO:3180).

40. An anti-TIGIT antibody comprising:

i) a heavy chain or heavy chain variable domain comprising the vhCDRl, vhCDR2, and vhCDR3 from the following sequence:

EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYIMSWVRQAPGKGLEWVATISGGSKGQYYADSVKG RFTI SRDNSKNTLYLQMNSLRAEDTAVYYCAKWLLSYYAMDYWGQGTLVTVSS (SEQ ID NO: 1664) ,

ii) a light chain or light chain variable domain comprising the vlCDRl, vlCDR2, and vlCDR3 from the following sequence:

DIQMTQSPSSLSASVGDRITITCRASQSMAIWLSWYQQKPGKAPKLLIYKASKSHTGVPSRFSGSG SGTDFTLTISSLQPEDFATYYCQQGQSYPYTFGQGTKLEIK (SEQ ID NO:1668).

41. The anti-PVRIG antibody of claim 38 comprising:

c) a heavy chain comprising CHA.7.518.4 VH

(QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNINWVRQAPGQGLE

WMGYIYPYIGGSGYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAV

YYCAREDKTARNAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSE

STAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV

VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLG

GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEV

HNAKTKPREEQFNSTYRVV SVLTVLHQDWLNGKEYKCKV SNKGLPSS

IEKTISKAKGQPREPQVYTLPPCQDELTKNQVSLWCLVKGFYPSDIAV

EWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCS

VMHEALHNHYTQKSLSLSPGK; SEQ ID NO:3l75); and

d) a light chain comprising CHA.7.518.4 VL

(DIQMTQSPSSLSASVGDRVTITCRVSENIYDVLAWYQQKPGKAPKLLI YE ATNL AEGVP S RF S GS GS GTDFTLTI S SLQPEDF ATYY C QHF W GTP YT F GQGTKLEIKRTV AAP SVFIFPPS DEQLKS GT AS V V CLLNNF YPRE AKV QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA CEVTHQGLSSPVTKSFNRGEC ; SEQ ID NO: 3362).

42. A composition comprising an anti-PVRIG antibody according to claim 38 or claim 40.

43. A nucleic acid composition comprising:

i) a first nucleic acid encoding a heavy chain or heavy chain variable domain according to claim 38 or claim 40; and

ii) a second nucleic acid encoding a light chain or light chain variable domain according to claim 38 or claim 40.

44. A composition comprising an anti-TIGIT antibody according to claim 39.

45. A nucleic acid composition comprising:

i) a first nucleic acid encoding a heavy chain or heavy chain variable domain according to claim 38; and

ii) a second nucleic acid encoding a light chain or light chain variable domain according to claim 38.

46. An expression vector composition comprising:

i) a first expression vector comprising the first nucleic acid of claim 41; and ii) a second expression vector comprising the second nucleic acid of claim 41.

47. An expression vector comprising:

i) the first nucleic acid of claim 41; and

ii) the second nucleic acid of claim 41.

48. An expression vector composition comprising:

i) a first expression vector comprising the first nucleic acid of claim 43; and ii) a second expression vector comprising the second nucleic acid of claim 43.

49. An expression vector comprising:

i) the first nucleic acid of claim 43; and

ii) the second nucleic acid of claim 43.

50. A host cell comprising the expression vector or vector composition of anyone of claims 44 to 47.

51. A method of making an anti-PVRIG or anti-TIGIT antibody comprising:

i) culturing the host cell of claim 48 under conditions wherein the antibody is expressed; and

ii) recovering the antibody.

52. A method of activating T cells of a patient comprising administering the anti-PVRIG or anti-TIGIT antibody of any one of claims 38 to 40 to the patient, wherein a subset of the T cells of the patient are activated.

53. A method of activating cytotoxic T cells (CTLs) of a patient comprising administering the anti-PVRIG or anti-TIGIT antibody of any one of claims 38 to 40 to the patient, wherein a subset of the CTLs of the patient are activated.

54. A method of activating NK cells of a patient comprising administering the anti- PVRIG or anti-TIGIT antibody of any one of claims 38 to 40 to the patient, wherein a subset of the NK cells of the patient are activated.

55. A method of activating gd T cells of a patient comprising administering the anti- PVRIG or anti-TIGIT antibody of any one of claims 38 to 40 to the patient, wherein a subset of the gd T cells of the patient are activated.

56. A method of activating Thl cells of a patient comprising administering the anti- PVRIG or anti-TIGIT antibody of any one of claims 38 to 40 to the patient, wherein a subset of the Thl cells of the patient are activated.

57. A method of decreasing or eliminating cell number and/or activity of at least one of regulatory T cells (Tregs) in a patient comprising administering the anti-PVRIG or anti-TIGIT antibody of any one of claims 38 to 40 to the patient.

58. A method of increasing interferon-g production and/or pro-inflammatory cytokine secretion in a patient comprising administering the anti-PVRIG or anti-TIGIT antibody of any one of claims 38 to 40 to the patient.

59. A method of treating cancer in a patient comprising administering the anti-PVRIG or anti-TIGIT antibody of any one of claims 38 to 40 to the patient.

60. The method of claim 58, wherein the cancer is selected from the group consisting of prostate cancer, liver cancer (HCC), colorectal cancer, ovarian cancer, endometrial cancer, breast cancer, triple negative breast cancer, pancreatic cancer, stomach

(gastric) cancer, cervical cancer, head and neck cancer, thyroid cancer, testis cancer, urothelial cancer, lung cancer (small cell lung, non-small cell lung), melanoma, non melanoma skin cancer (squamous and basal cell carcinoma), glioma, renal cancer (RCC), lymphoma (NHL or HL), Acute myeloid leukemia (AML), T cell Acute Lymphoblastic Leukemia (T-ALL), Diffuse Large B cell lymphoma, testicular germ cell tumors, mesothelioma, esophageal cancer, Merkel Cells cancer, MSI-high cancer, KRAS mutant tumors, adult T-cell leukemia/lymphoma, myeloma, and

Myelodysplastic syndromes (MDS).

61. The method of claim 58, wherein the cancer is selected from the group consisting of triple negative breast cancer, stomach (gastric) cancer, lung cancer (small cell lung, non-small cell lung), Merkel Cells cancer, MSI-high cancer, KRAS mutant tumors, adult T-cell leukemia/lymphoma, myeloma, and Myelodysplastic syndromes (MDS).

62. A method of treating cancer in a patient comprising administering a combination therapy comprising an anti-PVRIG/anti-TIGIT bispecific antibody according to any one of claims 1 to 20.

63. A method of treating cancer in a patient comprising administering a combination therapy comprising an anti-PVRIG antibody of claim 38 and an anti-PD-l antibody.

64. A method according to claim 62, wherein the anti-PD-l antibody is an antibody

selected from the group consisting of pembrolizumab and nivolumab.

65. A method of treating cancer in a patient comprising administering a combination therapy comprising an anti-TIGIT antibody of claim 39 and an anti-PD-l antibody.

66. A method according to claim 64, wherein the anti-PD-l antibody is an antibody

selected from the group consisting of pembrolizumab and nivolumab.

67. A method of treating cancer in a patient comprising administering a combination therapy comprising an anti-TIGIT antibody of claim 39 and an anti-PVRIG antibody of claim 38.

68. A method of treating cancer in a patient comprising administering a triple

combination therapy comprising an anti-TIGIT antibody of claim 39, an anti-PVRIG antibody, and an anti-PD-l antibody.

69. A method of treating cancer in a patient comprising administering a triple combination therapy comprising an anti-TIGIT, an anti-PVRIG antibody of claim 38, and an anti-PD-l antibody.

70. A method of treating cancer in a patient comprising administering a triple

combination therapy comprising an anti-TIGIT of claim 39, an anti-PVRIG antibody of claim 38, and an anti-PD-l antibody.

71. A method according to any one of claims 64 to 69, wherein the anti-PD-l antibody is an antibody selected from the group consisting of pembrolizumab and nivolumab.

72. A method of activating T cells of a patient comprising administering the anti-PVRIG or anti-TIGIT antibody of any one of claims 38 to 40 to the patient, wherein a subset of the T cells of the patient are activated.

73. A method of activating cytotoxic T cells (CTLs) of a patient comprising administering the anti-PVRIG or anti-TIGIT antibody of any one of claims 38 to 40 to the patient, wherein a subset of the CTLs of the patient are activated.

74. A method of activating gd T cells of a patient comprising administering the anti- PVRIG or anti-TIGIT antibody of any one of claims 38 to 40 to the patient, wherein a subset of the gd T cells of the patient are activated.

75. A method of activating Thl cells of a patient comprising administering the anti- PVRIG or anti-TIGIT antibody of any one of claims 38 to 40 to the patient, wherein a subset of the Thl cells of the patient are activated.

76. A method of decreasing or eliminating cell number and/or activity of at least one of regulatory T cells (Tregs) in a patient comprising administering the anti-PVRIG or anti-TIGIT antibody of any one of claims 38 to 40 to the patient.

77. A method of increasing interferon-g production and/or pro-inflammatory cytokine secretion in a patient comprising administering the anti-PVRIG or anti-TIGIT antibody of any one of claims 38 to 40 to the patient.

78. A method of treating cancer in a patient comprising administering the anti-PVRIG or anti-TIGIT antibody of any one of claims 38 to 40 to the patient.

79. The method of claim 77, wherein the cancer is selected from the group consisting of prostate cancer, liver cancer (HCC), colorectal cancer, ovarian cancer, endometrial cancer, breast cancer, triple negative breast cancer, pancreatic cancer, stomach (gastric) cancer, cervical cancer, head and neck cancer, thyroid cancer, testis cancer, urothelial cancer, lung cancer (small cell lung, non-small cell lung), melanoma, non melanoma skin cancer (squamous and basal cell carcinoma), glioma, renal cancer (RCC), lymphoma (NHL or HL), Acute myeloid leukemia (AML), T cell Acute Lymphoblastic Leukemia (T-ALL), Diffuse Large B cell lymphoma, testicular germ cell tumors, mesothelioma, esophageal cancer, Merkel Cells cancer, MSI-high cancer, KRAS mutant tumors, adult T-cell leukemia/lymphoma, myeloma, and

Myelodysplastic syndromes (MDS).

80. The method of claim 77, wherein the cancer is selected from the group consisting of triple negative breast cancer, stomach (gastric) cancer, lung cancer (small cell lung, non-small cell lung), Merkel Cells cancer, MSI-high cancer, KRAS mutant tumors, adult T-cell leukemia/lymphoma, myeloma, and Myelodysplastic syndromes (MDS).