Antibody to tigit

Abstract

To provide an improved method for treating cancer and chronic viral infection, and a drug to be used in the method.SOLUTION: Provided are an antibody that binds to human TIGIT (T cell immune receptor having Ig and ITIM domain) or an antigen-binding fragment thereof, and use of the antibody or fragment in treatment application such as treatment of cancer or chronic viral infection. Such a treatment method includes combination therapy with an inhibitor of other immunoregulatory receptor interaction such as PD-1 / PD-L1 interaction. Further provided are a polynucleotide encoding a heavy chain and / or a light chain variable region of antibody, an expression vector including a polynucleotide encoding a heavy chain and / or a light chain variable region of antibody, a cell including the vector, and a method for producing it by expressing an antibody or fragment from the cell.SELECTED DRAWING: None

Description

[Technical Field]

[0001] TIGIT (T cell immunoreceptor with Ig and ITIM domains), also known as WUCAM, Vstm3, or Vsig9, is a co-inhibitory receptor protein. Discovered in a genomic search for proteins specifically expressed on T cells, TIGIT contains an immunoglobulin variable domain, a transmembrane domain, and an immunoreceptor tyrosine-based inhibitory motif (ITIM), and contains the signature sequence elements of the PVR protein family. It has been shown to interact with the poliovirus receptor (PVR CD155) and nectin-2 (CD112). See, e.g., Stengel et al. (2012) Proc. Nat'l Acad. Sci. (USA) 19:5399; WO2006 / 124667; WO2009 / 126688. PVR can interact with the coactivator receptor DNAM-1 (CD226) to enhance tumor killing, but the high-affinity TIGIT / PVR interaction can inhibit such killing and act to prevent the killing of normal (self) cells that also express PVR. Stanietsky et al. (2009) Proc. Nat'l Acad. Sci. (USA) 106:17858. The dominance of this inhibitory interaction may be important in suppressing anti-autoimmune responses, but in the tumor context, suppresses tumor eradication. Ibid. [Background technology]

[0002] TIGIT suppresses T cell activation by promoting the generation of mature immunoregulatory dendritic cells. Yu et al. (2009) Nat. Immunol. 10:48. TIGIT and other such co-inhibitory molecules (e.g., CTLA-4, PD-1, Lag3, and BTLA) may play a role in tumor cell evasion of immune surveillance. Experiments have shown that PVR / CD155 is overexpressed in melanoma cells (Inozume et al. (2014) J. Invest. Dermatol. 134:S121 - Abstract 693) and various other tumors. TIGIT / PVR interaction may shield such tumor cells from immune-mediated eradication by inhibiting the anti-tumor response of T and NK cells. Stanietsky et al. (2009) Proc. Nat'l Acad. Sci. (USA) 106:17858 and Lozano et al. (2012) J. Immunol. 188:3869. Other experiments have demonstrated the expression of regulatory T cells (T reg )'s TIGIT + A subset was identified (Joller et al. (2014) Immunity 40:569), suggesting an alternative mechanism by which anti-TIGIT antibodies may enhance anti-tumor immune responses.

[0003] TIGIT may act to "shut down" immune responses similarly to other co-inhibitory responses such as CTLA-4, PD-1, and BTLA. Ibid. Validating this therapeutic approach, antibodies targeting CTLA-4 (ipilimumab) and PD-1 (nivolumab, pembrolizumab) have been approved for the treatment of human cancers. Antibodies that bind human TIGIT may also be used to treat cancer. See, e.g., WO2006 / 124667. In mouse models, antibody blockade of both PD-L1 and TIGIT inhibits CD8 +This leads to synergistic enhancement of T cell-mediated tumor rejection. Grogan et al. (2014) J. Immunol. 192(1) Suppl. 203.15; Johnston et al. (2014) Cancer Cell 26:1-15. Similar results have been obtained in animal models of melanoma. Inozume et al. (2014) J. Invest. Dermatol. 134:S121 - Abstract 693. Several experiments have shown that TIGIT blockade inhibits anti-tumor CD8 T cells only in the presence of the co-activating receptor DNAM-1 / CD226, which competes with TIGIT for binding to PVR / CD155. + It has been suggested to be effective in enhancing T cell responses. Johnston et al. (2014) Cancer Cell 26:1-15.

[0004] Recent experiments have demonstrated that intratumoral bacteria expressing the Fap2 protein can inhibit NK cell-mediated tumor killing by binding to TIGIT (Gur et al. (2015) Immunity 42:344), suggesting that eliminating such bacteria, blocking the interaction of TIGIT with Fap2, or blocking the activity of TIGIT may be useful in treating cancer in general, e.g., colorectal cancer. Hampton (2015) JAMA 313:1305.

[0005] There is a need for improved methods of treating cancer and chronic viral infections, and for pharmaceuticals such as therapeutic monoclonal antibodies for use in such methods. Pharmaceuticals for use in such improved treatment methods may include antibodies or antibody fragments that specifically bind to TIGIT and reverse or partially reverse TIGIT-mediated suppression of anti-tumor or anti-viral immune responses. Summary of the Invention

[0006] The present invention provides improved pharmaceuticals and methods for treating cancer and chronic viral infections, comprising antibodies or antigen-binding fragments thereof that bind to huTIGIT. Provided herein are isolated antibodies, such as monoclonal antibodies, particularly human monoclonal antibodies, that specifically bind to huTIGIT and have desirable functional properties, such as high-affinity specific binding to huTIGIT, binding to monkey TIGIT (e.g., cynomolgus monkey TIGIT), the ability to block the binding of TIGIT to PVR and / or Nectin-2, the ability to block the interaction of TIGIT with DNAM, or any combination of these properties.

[0007] The present invention further provides improved methods and therapeutic antibodies for use in treating cancers, including cancers in which TIGIT-mediated signaling suppresses anti-tumor immune responses, tumors in which TIGIT interaction with the co-activating receptor DNAM-1 / CD226 suppresses anti-tumor immune responses, tumors in which TIGIT-expressing regulatory T cells suppress anti-tumor immune responses, or tumors in which TIGIT otherwise inhibits anti-tumor immune responses. The present invention also provides methods and therapeutic antibodies for use in treating chronic viral infections in which TIGIT suppresses anti-viral immune responses.

[0008] In another aspect, the present invention relates to antibodies that compete with antibodies having the heavy and light chain variable domain sequences disclosed herein for binding to huTIGIT and / or cross-block antibodies having the heavy and light chain variable domains disclosed herein from binding to huTIGIT.

[0009] In certain embodiments, the anti-TIGIT antibodies or antigen-binding fragments thereof of the present invention enhance anti-tumor immune responses, e.g., antigen-specific T cell responses. In other embodiments, the anti-TIGIT antibodies or antigen-binding fragments thereof block TIGIT-mediated inhibitory signaling, enabling PVR / DNAM co-stimulation of NK cells to enhance NK-mediated anti-tumor killing. In yet another embodiment, the anti-TIGIT antibodies or antigen-binding fragments thereof of the present invention deplete a population of intratumoral regulatory T cells that would otherwise suppress anti-tumor immune responses. In yet another embodiment, the anti-TIGIT antibodies of the present invention formatted as IgG1 enhance CD8 + Exhausted T cells and T reg Deplete CD8 + In other embodiments, the anti-TIGIT antibodies or antigen-binding fragments thereof of the present invention act by one or more of the mechanisms mentioned above, as the mechanisms are not necessarily mutually exclusive.

[0010] In certain embodiments, the anti-TIGIT antibodies or antigen-binding fragments thereof of the present invention do not bind to activating Fcγ receptors (FcγRs), e.g., in embodiments that rely on enhancing the anti-tumor activity of TIGIT-expressing cells. In alternative embodiments, the anti-TIGIT antibodies or antigen-binding fragments thereof of the present invention inhibit, e.g., exhausted CD8 + T cells or T reg In embodiments that rely on killing of TIGIT-expressing cells, such as those that bind one or more activating FcγRs.

[0011] The present invention also provides an isolated monoclonal antibody (15A6) or an antigen-binding fragment thereof that specifically binds to huTIGIT and comprises heavy chain CDRH1, CDRH2, and CDRH3 sequences comprising SEQ ID NOs: 14, 15, and 16, respectively, and / or light chain CDRL1, CDRL2, and CDRL3 sequences comprising SEQ ID NOs: 17, 18, and 19, respectively.

[0012] The present invention also provides an isolated monoclonal antibody (22G2) or an antigen-binding fragment thereof that specifically binds to huTIGIT and comprises heavy chain CDRH1, CDRH2, and CDRH3 sequences comprising SEQ ID NOs: 20, 21, and 22, respectively, and / or light chain CDRL1, CDRL2, and CDRL3 sequences comprising SEQ ID NOs: 23, 24, and 25, respectively.

[0013] The present invention further provides an isolated monoclonal antibody (11G11) or an antigen-binding fragment thereof that specifically binds to huTIGIT and comprises heavy chain CDRH1, CDRH2, and CDRH3 sequences comprising SEQ ID NOs: 26, 27, and 28, respectively, and / or light chain CDRL1, CDRL2, and CDRL3 sequences comprising SEQ ID NOs: 29, 30, and 31, respectively.

[0014] The present invention still further provides an isolated monoclonal antibody (10D7) or an antigen-binding fragment thereof that specifically binds to huTIGIT and comprises heavy chain CDRH1, CDRH2, and CDRH3 sequences comprising SEQ ID NOs: 32, 33, and 34, respectively, and / or light chain CDRL1, CDRL2, and CDRL3 sequences comprising SEQ ID NOs: 35, 36, and 37, respectively.

[0015] The present invention also provides an isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds to huTIGIT and comprises the heavy chain variable chain and light chain variable chain sequences disclosed in SEQ ID NO: 2 (or 3, 4, 5) and 6, SEQ ID NO: 7 (or 8) and 9, SEQ ID NO: 10 and 11, and SEQ ID NO: 12 and 13.

[0016] The present invention provides an isolated monoclonal antibody or antigen-binding fragment thereof that binds to huTIGIT and comprises a heavy chain and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence that is at least 90%, 95%, or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 7, 8, 10, and 12.

[0017] The present invention also provides an isolated monoclonal antibody or antigen-binding fragment thereof that binds to huTIGIT and comprises a heavy chain and a light chain variable region, wherein the light chain variable region comprises an amino acid sequence that is at least 90%, 95%, or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 9, 11, and 13.

[0018] In certain embodiments, an isolated monoclonal antibody or antigen-binding fragment of the present invention (a) binds to the same epitope on huTIGIT as 15A6, 22G2, 11G11 and / or 10D7, and / or (b) inhibits binding of 15A6, 22G2, 11G11 and / or 10D7 to huTIGIT, as measured, for example, by FACS or ELISA.

[0019] In specific embodiments, an anti-huTIGIT antibody or antigen-binding fragment thereof of the invention binds to an epitope comprising or consisting of one or more of residues E60, I109, L65, N70, F107, T117, I68, H76, and N58 (antibody 22G2) of huTIGIT (SEQ ID NO: 1), an epitope comprising or consisting of one or more of residues G74, N70, H76, L65, L73, Q56, I68, H111, and P114 (antibody 11G11), or an epitope comprising or consisting of one or more of residues H76, G74, L65, N58, I68, Q139, G135, L73, F107, N70, E60, H134, A132, and I109 (antibody 15A6).

[0020] Alternatively, the anti-huTIGIT antibody or antigen-binding fragment thereof of the present invention binds to an epitope comprising or consisting of one or more sequences selected from the group consisting of NWEQQDQLLAICNADLGWH (SEQ ID NO: 38) and FCIYHTYPDGT (SEQ ID NO: 39) (antibody 22G2), or from the group consisting of QVNWEQQDQLLAICNADLGWH (SEQ ID NO: 40) and HTYP (SEQ ID NO: 41) (antibody 11G11), or from the group consisting of NWEQQDQLLAICNADLGWH (SEQ ID NO: 38), FCI, and AEHGARFQ (SEQ ID NO: 43) (antibody 15A6).

[0021] In yet further embodiments, the anti-TIGIT antibody or antigen-binding fragment thereof of the present invention binds to a core epitope on huTIGIT (SEQ ID NO: 1) comprising or consisting of one or more of residues L65, I68, N70 and H76, and / or to an epitope comprising or consisting of LLAICNADLGWH (SEQ ID NO: 44).

[0022] In some embodiments, the anti-huTIGIT antibodies or antigen-binding fragments thereof of the present invention also bind to cynomolgus monkey TIGIT.

[0023] In various embodiments, the anti-TIGIT antibody or antigen-binding fragment thereof of the present invention is a human IgG1, IgG2, IgG3, or IgG4 antibody or a mutant thereof. In certain embodiments, including but not limited to, a method for blocking TIGIT signaling in "exhausted" tumor-specific T cells or blocking inhibitory signals to NK cells to enable DNAM-1 / PVR-mediated costimulation, or a method for blocking TIGIT interaction with DNAM-1 / CD226 to impair DNAM-1 homodimerization, the anti-TIGIT antibody or antigen-binding fragment thereof comprises an effector-less or nearly effector-less Fc. Such an Fc region includes, for example, human IgG2 or IgG4, or an effector-less mutant of human IgG1 with one or more of the following mutations: L234A, L235E, G237A, A330S, and P331S (EU numbering), such as IgG1.1f (SEQ ID NO: 48), which contains all five of the listed mutations.

[0024] Not limited to, but also TIGIT + In alternative embodiments, including methods for depleting regulatory T cells, the anti-TIGIT antibody or antigen-binding fragment thereof comprises an Fc that selectively binds to an activating FcγR (FcγRI, FcγRIIa, or FcγRIIIa), such as human IgG1, or a sequence variant that has enhanced binding to an activating FcγR relative to wild-type IgG1 Fc. reg In embodiments involving the use of IgG1 forms of the anti-TIGIT antibodies of the invention to drive T cell depletion, intratumoral injection may be used as appropriate to localize the effect to the tumor microenvironment and minimize potential side effects caused by activity in peripheral tissues.

[0025] In certain embodiments, a methionine residue in the CDR region of an anti-TIGIT antibody of the present invention or an antigen-binding fragment thereof (eg, M115 in CDRH3 of 10D7, SEQ ID NO: 34) is substituted with an amino acid residue that is not susceptible to oxidation.

[0026] In certain embodiments, the anti-huTIGIT antibody or antigen-binding fragment thereof that competes for binding to, cross-blocks or binds to the same epitope as 15A6, 22G2, 11G11 or 10D7 is a human or humanized antibody.

[0027] In some embodiments, the anti-huTIGIT antibodies of the invention are not or do not bind to the same epitope as the antibodies described in U.S. Patent Application Publication No. 2009 / 0258013, e.g., they do not bind to the same epitope as anti-huTIGIT mAb 10A7 or 1F4. See also Johnston et al. (2014) Cancer Cell 26:1; Yu et al. (2009) Nat. Immunol. 10:48.

[0028] In other embodiments, the anti-huTIGIT antibody comprises variable domains derived from the same human V-domain germline sequences as the antibodies disclosed herein, including heavy chain V-domains V4-39, V4-61, or V1-69. In more specific embodiments, the anti-huTIGIT antibody comprises heavy and light chain variable domains derived from the same human heavy and light chain V-domain germline sequences as the antibodies disclosed herein, such as V4-39 / VA27(15A6), V4-61 / VL6(22G2), V4-39 / VL6(11G11), and V1-69 / VL15(10D7).

[0029] In various embodiments, the anti-huTIGIT antibodies of the invention have a K of less than 10 nM, 5 nM, 2 nM, 1 nM, 300 pM, or 100 pM. D In other embodiments, the anti-huTIGIT antibodies of the invention bind to huTIGIT with a K between 2 nM and 100 pM. D It binds to huTIGIT.

[0030] In other embodiments, anti-huTIGIT antibodies of the invention consist essentially of or comprise some combination of the CDRs of antibodies 15A6, 22G2, 11G11, and 10D7, such as CDRH1 (SEQ ID NOs: 14, 20, 26, and 32), CDRH2 (SEQ ID NOs: 15, 21, 27, and 33), CDRH3 (SEQ ID NOs: 16, 22, 28, and 34), CDRL1 (SEQ ID NOs: 17, 23, 29, and 35), CDRL2 (SEQ ID NOs: 18, 24, 30, and 36), and CDRL3 (SEQ ID NOs: 19, 25, 31, and 37). In other embodiments, the antibodies consist essentially of or comprise distinct specific combinations of the CDR sequences of antibodies 15A6, 22G2, 11G11, and 10D7.

[0031] In further embodiments, the anti-huTIGIT antibodies of the invention consist essentially of or comprise the heavy and / or light chain variable domains of antibodies 15A6 (SEQ ID NOs: 2-5 and 6), 22G2 (SEQ ID NOs: 7-8 and 9), 11G11 (SEQ ID NOs: 10 and 11), and 10D7 (SEQ ID NOs: 12 and 13), or sequences sharing at least 80%, 85%, 90%, and 95% sequence identity with these disclosed sequences.

[0032] In still further embodiments, the anti-huTIGIT antibodies of the invention consist essentially of or comprise heavy and / or light chains comprising the variable domain sequences of antibodies 15A6 (SEQ ID NOs: 2-5 and 6), 22G2 (SEQ ID NOs: 7-8 and 9), 11G11 (SEQ ID NOs: 10 and 11), and 10D7 (SEQ ID NOs: 12 and 13), or sequences sharing at least 80%, 85%, 90%, and 95% sequence identity with these disclosed sequences.

[0033] In other embodiments, the antigen-binding domain of an antibody of the invention is present in a bispecific molecule that further comprises an antigen-binding domain that specifically binds to a different immunoregulatory receptor, including but not limited to PD-1, CTLA-4, or LAG3.

[0034] The present invention further provides nucleic acids encoding the heavy and / or light chain variable regions of the anti-huTIGIT antibodies or antigen-binding fragments thereof of the present invention, expression vectors containing the nucleic acid molecules, cells transformed with the expression vectors, and methods for producing antibodies by expressing cells transformed with the expression vectors and recovering the antibodies.

[0035] The present invention also provides immunoconjugates comprising an anti-huTIGIT antibody described herein linked to an agent, such as a detectable label or a cytotoxic agent.

[0036] The present invention also provides pharmaceutical compositions comprising an anti-huTIGIT antibody or antigen-binding fragment thereof of the present invention and a carrier. Also provided herein are kits comprising an anti-TIGIT antibody or antigen-binding fragment thereof and instructions for use.

[0037] In another aspect, the invention provides methods of enhancing antigen-specific T cell responses, comprising contacting T cells with an anti-huTIGIT antibody or antigen-binding fragment thereof of the invention, such that the antigen-specific T cell response is enhanced, e.g., by reducing inhibitory signals that would otherwise attenuate the anti-tumor response. In some embodiments, the antigen-specific T cells are CD8 + Enhancement of tumor antigen-specific effector T cells, such as T cells, for example, by blocking TIGIT-mediated inhibitory effects, results in increased anti-tumor activity. The anti-huTIGIT antibodies or antigen-binding fragments thereof of the present invention can also reduce inhibitory signals in NK cells, thereby increasing their anti-tumor activity. Without intending to be limited by theory, the anti-huTIGIT antibodies of the present invention block the binding of TIGIT to PVR, thereby increasing effector T cell or NK cell function by reducing or eliminating inhibitory signals that would otherwise be delivered to the cells. Alternatively, or in addition, the anti-TIGIT antibodies or antigen-binding fragments thereof of the present invention can inhibit the interaction between TIGIT and DNAM-1 / CD226, which would otherwise reduce DNAM-1-mediated immune activation.

[0038] The present invention further provides a method for increasing IL-2 and / or IFN-γ production in T cells and / or T cell proliferation, comprising contacting T cells with an effective amount of an anti-TIGIT antibody or antigen-binding fragment thereof.

[0039] In another aspect, the present invention provides a method for treating tumors in a subject in need thereof, comprising administering an effective amount of an anti-huTIGIT antibody of the present invention. reg a method for reducing or depleting T in a tumor, wherein the antibody reg The present invention provides methods for reducing the number, effector function, or having enhanced effector function of a target protein.

[0040] The present invention provides a method for enhancing an immune response in a subject, comprising administering to the subject an effective amount of an anti-huTIGIT antibody or antigen-binding fragment thereof of the present invention, such that the immune response in the subject is enhanced. In a specific embodiment, the subject has a tumor, and an immune response against the tumor is enhanced. In another embodiment, the subject has a viral infection, and an antiviral immune response is enhanced.

[0041] The present invention also provides a method of inhibiting tumor growth in a subject, comprising administering to the subject an anti-huTIGIT antibody, or antigen-binding fragment thereof, of the present invention, such that tumor growth is inhibited.

[0042] The present invention further provides a method for treating cancer, e.g., by immunotherapy, comprising administering a therapeutically effective amount of an anti-huTIGIT antibody or antigen-binding fragment thereof of the present invention, e.g., as a pharmaceutical composition, to a subject in need thereof, thereby treating the cancer. In certain embodiments, the cancer is bladder cancer, breast cancer, uterine / cervical cancer, ovarian cancer, prostate cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, pancreatic cancer, colorectal cancer, colon cancer, kidney cancer, head and neck cancer, lung cancer, gastric cancer, germ cell cancer, bone cancer, liver cancer, thyroid cancer, skin cancer, neoplasms of the central nervous system, lymphoma, leukemia, myeloma, sarcoma, and virus-associated cancer. In certain embodiments, the cancer is metastatic cancer, refractory cancer, or recurrent cancer.

[0043] In certain embodiments, methods of modulating immune function and methods of treatment described herein comprise administering an anti-huTIGIT antibody of the invention in combination with or as a bispecific reagent with one or more additional therapeutic agents, e.g., an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-LAG3 antibody, an anti-GITR antibody, an anti-OX40 antibody, an anti-CD73 antibody, an anti-CD40 antibody, an anti-CD137 mAb, an anti-CD27 mAb, an anti-CSF-1R antibody and / or an anti-CTLA-4 antibody, a TLR agonist, or a small molecule antagonist of IDO or TGFβ. In certain embodiments, the anti-huTIGIT therapy is combined with an anti-PD-1 and / or anti-PD-L1 therapy, e.g., a therapy using an antibody or antigen-binding fragment thereof that binds human PD-1 or an antibody or antigen-binding fragment thereof that binds human PD-L1.

[0044] In some embodiments, samples obtained from patients, e.g., biopsies, are screened for expression of DNAM-1 on T cells or NK cells to select patients most likely to respond to anti-TIGIT treatment, where the presence of DNAM-1 on T cells indicates that the patient will have a beneficial anti-tumor response upon anti-TIGIT treatment, e.g., treatment with an anti-huTIGIT antibody or fragment of the present invention, and the absence of DNAM-1 identifies patients who are unlikely to benefit from anti-TIGIT treatment. In other embodiments, samples obtained from patients are screened for expression of PVR and / or Nectin-2 on tumor cells or tumor-infiltrating myeloid cells to select patients most likely to respond to anti-TIGIT treatment, where the presence of PVR and / or Nectin-2 indicates that the patient will have a beneficial anti-tumor response upon anti-TIGIT treatment, e.g., treatment with an anti-huTIGIT antibody or fragment of the present invention, and the absence of PVR and / or Nectin-2 / CD112 identifies patients who are unlikely to benefit from anti-TIGIT treatment. In various embodiments, cell surface expression of TIGIT, DNAM, PVR, and / or Nectin-2 is determined by FACS, IHC, or LC-MS. In another aspect, the invention provides a method of treating a subject in need thereof, comprising determining cell surface expression of TIGIT, DNAM, PVR, and / or Nectin-2 as described herein and administering an anti-TIGIT antibody of the invention, selectively or exclusively to those most likely to provide therapeutic benefit.

[0045] In one embodiment, the levels of soluble PVR and / or soluble nectin-2 (sPVR, sNectin-2) are measured in subjects being considered for treatment with the anti-TIGIT antibodies of the present invention, and only subjects exhibiting elevated soluble PVR and / or nectin-2 are treated with the antibody. In some embodiments, sPVR and / or sNectin-2 are detected in serum by ELISA or LC-MS.

[0046] The present invention also provides a method for detecting the presence of TIGIT in a sample, in cells within a sample (e.g., FACS), or at a specific location in a cell or tissue (e.g., IHC), or for sorting cells based on the presence or absence of TIGIT on their surface (e.g., FACS), comprising contacting the sample with an anti-huTIGIT antibody or antigen-binding fragment thereof of the present invention under conditions that allow the formation of a complex between the antibody or antigen-binding fragment thereof and TIGIT, and detecting the formation of the complex. In some embodiments, the anti-TIGIT antibody used for detection is conjugated to a detectable label.

[0047] Other features and advantages of the present disclosure will become apparent from the following detailed description and examples which should not be construed as limiting. [Brief explanation of the drawings]

[0048] [Figure 1] Figure 1 shows a schematic of a "binning" experiment in which various anti-huTIGIT antibodies of the invention are tested in pairs for their ability to block the binding of other antibodies to huTIGIT. The results show that the antibodies fall into a limited number of categories or "bins." See Example 3.

[0049] [Figure 2A]Figure 2A shows yeast display data for binding of each of the huTIGIT sequence variants to antibodies 22G2, 11G11, and 15A6. The residue number of each amino acid residue in mature huTIGIT is shown along the horizontal axis. Because the sequence listing includes a signal peptide, which is not included in the figure, the residue number is 21 less than the numbering of SEQ ID NO:1. As detailed in Example 4, yeast displaying sequence variants of huTIGIT were selected based on their inability to bind to the respective antibodies (22G2, 11G11, 15A6). Thus, positions along the huTIGIT sequence that are important for antibody binding appear at high frequency (i.e., as bars / lines rising above the vertical axis) due to their over-representation in the pool of non-binding yeast clones. The frequency of occurrence of the mutant (non-wild-type) residue at each residue is represented on the vertical axis (on a logarithmic scale), with one bar (line) or each residue. Frequency data are normalized to the frequency with which the variant residue appears at each position in an unselected library, i.e., a library that has not been subjected to selection based on its inability to bind to an anti-huTIGIT antibody of the invention. See Example 4.

[0050] [Figure 2B]Figure 2B shows yeast display data for binding of each of the huTIGIT sequence variants to antibodies 22G2, 11G11, and 15A6. The residue number of each amino acid residue in mature huTIGIT is shown along the horizontal axis. Because the sequence listing includes a signal peptide, which is not included in the figure, the residue number is 21 less than the numbering of SEQ ID NO:1. As detailed in Example 4, yeast displaying sequence variants of huTIGIT were selected based on their inability to bind to the respective antibodies (22G2, 11G11, 15A6). Thus, positions along the huTIGIT sequence that are important for antibody binding appear at high frequency (i.e., as bars / lines rising above the vertical axis) due to their over-representation in the pool of non-binding yeast clones. The frequency of occurrence of the mutant (non-wild-type) residue at each residue is represented on the vertical axis (on a logarithmic scale), with one bar (line) or each residue. Frequency data are normalized to the frequency with which the variant residue appears at each position in an unselected library, i.e., a library that has not been subjected to selection based on its inability to bind to an anti-huTIGIT antibody of the invention. See Example 4.

[0051] [Figure 2C]Figure 2C shows yeast display data for binding of each of the huTIGIT sequence variants to antibodies 22G2, 11G11, and 15A6. The residue number of each amino acid residue in mature huTIGIT is shown along the horizontal axis. Because the sequence listing includes a signal peptide, which is not included in the figure, the residue number is 21 less than the numbering of SEQ ID NO:1. As detailed in Example 4, yeast displaying sequence variants of huTIGIT were selected based on their inability to bind to the respective antibodies (22G2, 11G11, 15A6). Thus, positions along the huTIGIT sequence that are important for antibody binding appear at high frequency (i.e., as bars / lines rising above the vertical axis) due to their over-representation in the pool of non-binding yeast clones. The frequency of occurrence of the mutant (non-wild-type) residue at each residue is represented on the vertical axis (on a logarithmic scale), with one bar (line) for each residue. Frequency data are normalized to the frequency with which the variant residue appears at each position in an unselected library, i.e., a library that has not been subjected to selection based on its inability to bind to an anti-huTIGIT antibody of the invention. See Example 4.

[0052] [Figure 3] Figure 3 shows the effect of anti-TIGIT mAb 22G2 on lysis, expressed as percent specific lysis, of cells expressing human PVR by human NK cells. See Example 5. For each antibody, the left bar is wild-type P815 cells, and the right bar is P815 cells expressing human PVR.

[0053] [Figure 4A] Figure 4A shows that treatment of healthy human donor blood with a cocktail of antigenic peptides (CETF = peptides derived from CMV, EBV, influenza, and tetanus) induces upregulation of PD-1 and TIGIT on CD8+ T cells. "No Stim" samples were not treated with CETF, while "Stim" samples were. See Example 6.

[0054] [Figure 4B] Figure 4B shows the effect of anti-TIGIT mAb and / or anti-PD-1 mAb on IFNγ expression from four healthy human donor blood samples stimulated with CETF (see Example 6).

[0055] [Figure 5A] Figure 5A shows the effect of anti-TIGIT antibodies, alone or in combination with other immunomodulatory therapies, on tumor growth in a mouse model. Figure 5A shows the tumor burden (in cubic millimeters) calculated by multiplying the square of the tumor width by half the length in a CT26 mouse colon cancer model for mice treated with an anti-mouse TIGIY antibody having an effector function-enabled mouse IgG2a Fc domain ("TIGIT G2a"), an anti-mouse TIGIT antibody having an effector function-defective IgG1 D265A Fc domain ("TIGIT G1 D265A"), an anti-mouse PD-1 antibody having an effector function-defective IgG1 D265A Fc domain ("PD-1 G1 D265A"), their combination, or a control IgG1 antibody. See Example 7.

[0056] [Figure 5B] Figure 5B shows the effect of anti-TIGIT antibodies, alone or in combination with other immunomodulatory therapies, on tumor growth in a mouse model. Figure 5B shows the effect of anti-TIGIT monotherapy and combination therapy with anti-PD-1 and anti-CTLA-4 antibodies. Tumor burden is provided along with the number of tumor-free (TF) mice in each group of 10 mice at the end of the experiment. Each line represents one mouse. The mIgG1 isotype control received no tumor-free mice, as did anti-TIGIT as monotherapy. Anti-PD-1 received one tumor-free mouse as monotherapy and five when combined with anti-TIGIT. Anti-CTLA-4 received three tumor-free mice as monotherapy and six when combined with anti-TIGIT. See Example 7.

[0057] [Figure 6A] Figure 6A shows elevated PVR expression in cancer tissues. Figure 6A shows PVR mRNA expression in various tumor types as detected in The Cancer Genome Atlas (TCGA) dataset. The data includes adrenocortical carcinoma (ACC), chromophobe renal cell carcinoma (KICH), liver hepatocellular carcinoma (LIHC), colon and rectal adenocarcinoma (COAD, READ), pancreatic ductal adenocarcinoma (PAAD), pheochromocytoma & paraganglioma (PCPG), papillary renal carcinoma (KIRP), lung adenocarcinoma (LUAD), head and neck squamous cell carcinoma (HNSC), prostate adenocarcinoma (PRAD), uterine endometrial carcinoma (UCEC), uterine cervical carcinoma (CESC), and cutaneous melanoma. (SKCM), mesothelioma (MESO), urothelial bladder cancer (BLCA), clear cell renal carcinoma (KIRC), lung squamous cell carcinoma (LUSC), uterine carcinosarcoma (UCS), sarcoma (SARC), ovarian serous cystadenocarcinoma (OV), papillary thyroid carcinoma (THCA), glioblastoma multiforme (GBM), breast cancer (BRCA), low-grade glioma (LGG), and diffuse large B-cell lymphoma (DLBC). The results disclosed herein are based in whole or in part on data generated by the TCGA Research Network.

[0058] [Figure 6B] Figure 6B shows elevated PVR expression in cancer tissue. Figure 6B shows human PVR in colon adenocarcinoma tissue compared to normal colon epithelium, with darker areas in the adenocarcinoma sample indicating elevated PVR expression. See Example 9.

[0059] [Figure 7]Figure 7 shows Fcγ receptor binding, expressed as a percentage of the theoretical maximum receptor binding value (Rmax), for anti-TIGIT mAb 22G2 formatted as IgG1f (SEQ ID NO: 45) or IgG1.1f (SEQ ID NO: 48). Data are shown for two different lots of IgG1.1f antibody used at 10 μM and 1 μM as indicated for six different Fcγ receptors. Within each cluster of bars, Fcγ receptor data are presented from left to right in the following order: hCD64 (FcγRI); hCD32a-H131 (FcγRIIA-H131); hCD32a-R131 (FcγRIIA-R131); hCD32b (FcγRIIB); hCD16a-V158 (FcγRIIIA-V158); and hCD16b-NA2 (FcγRIIIB-NA2, where NA2 refers to the allotypic variant). Pairs of Fcγ receptors are represented by identical bars, but their identities are apparent from the order in which they are presented. Identical reductions in binding to the cynomolgus monkey Fcγ receptors CD64, CD32a, CD32b, and CD16 were observed (not shown). DETAILED DESCRIPTION OF THE INVENTION

[0060] The present invention discloses isolated antibodies, particularly monoclonal antibodies, e.g., human monoclonal antibodies, that specifically bind to human TIGIT ("huTIGIT") and block its binding to PVR / CD155, thereby reducing or eliminating immunosuppressive signals that would otherwise occur in TIGIT-expressing cells. The present invention also provides isolated antibodies, particularly monoclonal antibodies, e.g., human or humanized monoclonal antibodies, that specifically bind to human TIGIT and block the interaction of human TIGIT with DNAM-1 / CD226, which would otherwise prevent DNAM-1 homodimerization and thus DNAM-1-mediated costimulation. Various human anti-huTIGIT monoclonal antibody sequences are provided. In certain embodiments, the antibodies described herein are derived from heavy and light chain germline sequences and / or contain specific structural features, such as CDR regions containing specific amino acid sequences.

[0061] Further provided herein are methods for producing such antibodies, immunoconjugates and bispecific molecules comprising such antibodies or antigen-binding fragments thereof, and pharmaceutical compositions formulated to contain the antibodies or fragments. Also provided herein are methods for using the antibodies alone or in combination with other immunostimulants (e.g., antibodies) and / or cancer or anti-infective drug treatments to enhance immune responses. Thus, the anti-huTIGIT antibodies described herein can be used in a variety of therapeutic applications, including, for example, inhibiting tumor growth and treating chronic viral infections.

[0062] definition In order that the description herein may be more readily understood, certain terms are first defined. Further definitions are set forth throughout the detailed description.

[0063] TIGIT refers to a "T cell immunoreceptor with Ig and ITIM domains," a member of the PVR (poliovirus receptor) family of immunoglobulin proteins, and binds to PVR / CD155 and nectin-2 / CD112. TIGIT is also referred to as TIGIT, WUCAM, Vstm3, and Vsig9. Unless otherwise specified or clear from the context, references herein to TIGIT refer to human TIGIT ("huTIGIT"), and anti-TIGIT antibodies refer to anti-human TIGIT antibodies. Human TIGIT is further described in GENE ID No. 201633 and MIM (Mendelian Inheritance in Humans): 612859. The sequence of human TIGIT (NP_776160.2), including the 21-amino acid signal sequence, is provided in SEQ ID NO: 1. Unless otherwise specified or clear from the context, "inhibition" of TIGIT refers to blocking PVR binding and signaling. The anti-TIGIT antibodies of the present invention may act by other mechanisms, such as inhibiting TIGIT signaling, blocking TIGIT / DNAM-1 interaction and / or directing depletion of regulatory T cells.

[0064] PVR (poliovirus receptor) interacts with TIGIT to induce immunosuppressive signals. PVR is also referred to as PVS, HVED, CD155, NECL5, TAGE4, ​​and Necl-5. Unless otherwise noted or clear from the context, references herein to PVR / CD155 refer to human PVR ("huPVR"). Human PVR is further described in GENE ID No. 5817 and MIM: 173850. There are four known human PVR transcript variants: α (NP_006496.4), β (NP_001129240.1), γ (NP_001129241.1), and δ (NP_001129242.2), the sequences of which are provided in SEQ ID NOs: 50-53. Unless otherwise noted, references to PVR or human PVR refer to the α transcript polypeptide.

[0065] Unless otherwise specified or clear from the context, the term "antibody," as used herein, can include whole antibodies and any antigen-binding fragment (i.e., "antigen-binding portion") or single chains thereof. In one embodiment, an "antibody" refers to a glycoprotein or antigen-binding fragment thereof comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (herein referred to as V H In certain naturally occurring IgG, IgD, and IgA antibodies, the heavy chain constant region consists of three domains, CH1, CH2, and CH3. In certain naturally occurring antibodies, each light chain consists of a light chain variable region (herein abbreviated as V L The light chain constant region consists of one domain, CL. H and V L The regions are called complementarity-determining regions (CDRs) and can be further subdivided into regions of hypervariability interspersed with more conserved regions called framework regions (FRs). H and V Lis composed of three CDRs and four framework regions (FRs), arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, such as various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0066] Antibodies usually bind to their cognate antigens. -7 ~10 -1 The dissociation constant (K D ) specifically binds with high affinity as reflected by approximately 10 -6 Any K greater than M D As used herein, an antibody that "specifically binds" to an antigen is one that binds to the antigen and a substantially identical antigen in a specific manner. -7 M or less, preferably 10 -8 M or less, and even more preferably 5 x 10 -9 M or less, most preferably 10 -8 M to 10 -10 K between M and below D An antibody that specifically binds to human TIGIT is an antibody that binds to the antigen with high affinity, meaning that it has high affinity, but does not bind to unrelated antigens with high affinity. An antigen is "substantially identical" to a given antigen if it shows a high degree of sequence identity with the given antigen, for example, if it shows at least 80%, at least 90%, preferably at least 95%, more preferably at least 97%, or even more preferably at least 99% sequence identity with the sequence of the given antigen. For example, an antibody that specifically binds to human TIGIT may also cross-react with TIGIT from certain non-human primate species (e.g., cynomolgus monkeys), but may not cross-react with TIGIT from other species or with antigens other than TIGIT.

[0067] Antibodies may exhibit modifications at the N- and / or C-terminal amino acid residues. For example, antibodies of the present invention may be produced from a construct encoding a C-terminal lysine residue, e.g., in the heavy chain, but such C-terminal lysine may be partially or completely absent in the therapeutic antibody to be marketed or administered. Alternatively, antibodies may be produced from a construct that specifically does not encode a C-terminal lysine residue, but such lysine was present in the parent antibody from which the therapeutic antibody was derived. In another example, an N-terminal glutamine or glutamic acid residue in an antibody of the present invention may be partially or completely converted to pyroglutamic acid in the therapeutic antibody to be marketed or administered. Any form of glutamine or glutamic acid present at the N-terminus of an antibody chain, including pyroglutamic acid, is encompassed within the term "glutamine" herein. Thus, antibody chain sequences provided herein having an N-terminal glutamine or glutamic acid residue encompass antibody chains regardless of the level of pyroglutamic acid formation.

[0068] Unless otherwise specified, immunoglobulins may be derived from any of the well-known isotypes, including, but not limited to, IgA, secretory IgA, IgG, and IgM. IgG isotypes are divided into subclasses in certain species: IgG1, IgG2, IgG3, and IgG4 in humans, and IgG1, IgG2a, IgG2b, and IgG3 in mice. Immunoglobulins, such as human IgG1, exist in several allotypes that differ from each other by at most two or three amino acids. Unless otherwise specified, "antibody" can include, by way of example, monoclonal and polyclonal antibodies, chimeric and humanized antibodies, human and non-human antibodies, fully synthetic antibodies, and single-chain antibodies.

[0069] As used herein, the term "antigen-binding portion" or "antigen-binding fragment" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., human TIGIT). Examples of binding fragments encompassed within the term "antigen-binding portion / fragment" of an antibody include: (i) Fab fragment-V; L , V H(ii) F(ab')2 fragment - a bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region; (iii) V H and an Fd fragment consisting of the CH1 domain, (iv) a V of a single arm of an antibody L and V H Fv fragments and (v)V fragments consisting of domains H Examples of such fragments include dAb fragments consisting of a domain (Ward et al., (1989) Nature 341:544-546). An isolated complementarity-determining region (CDR) or a combination of two or more isolated CDRs connected by a synthetic linker can comprise the antigen-binding domain of an antibody if it is capable of binding to the antigen.

[0070] Single-chain antibody constructs are also included in the present invention. Although the two domains of an Fv fragment, VL and VH, are encoded by separate genes, they can be joined using recombinant methods with a synthetic linker, allowing the VL and VH domain pair to be produced as a single protein chain, forming a monovalent molecule known as a single-chain Fv (scFv); see, e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain antibodies are also encompassed within the term "antigen-binding portion / fragment" of an antibody. These and other potential constructs are described in Chan & Carter (2010) Nat. Rev. Immunol. 10:301. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as intact antibodies. Antigen-binding portions / fragments may be produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact immunoglobulins.

[0071] Unless otherwise specified, the term "fragment", when used in connection with antibodies, such as in the claims, refers to an antigen-binding fragment of an antibody, and thus "antibody or fragment" has the same meaning as "antibody or antigen-binding fragment thereof".

[0072] A "bispecific" or "bifunctional antibody" is an artificial hybrid antibody that has two different heavy / light chain pairs, resulting in two antigen-binding sites with specificities for different antigens. Bispecific antibodies can be produced by a variety of methods, including fusion of hybridomas or linking of Fab' fragments. See, e.g., Songsivilai & Lachmann (1990) Clin. Exp. Immunol. 79:315; Kostelny et al. (1992) J. Immunol. 148:1547.

[0073] As used herein, the term "monoclonal antibody" refers to an antibody exhibiting a single binding specificity and affinity for a particular epitope, or a composition of antibodies in which all of the antibodies exhibit a single binding specificity and affinity for a particular epitope. Typically, such monoclonal antibodies are derived from a single cell or nucleic acid encoding the antibody and can be propagated without the intentional introduction of any sequence alterations. Thus, the term "human monoclonal antibody" refers to a monoclonal antibody having variable and, optionally, constant regions derived from human germline immunoglobulin sequences. In one embodiment, human monoclonal antibodies are produced by hybridomas obtained, for example, by fusing B cells obtained from a transgenic or transchromosomal non-human animal (e.g., a transgenic mouse having a genome containing human heavy chain and light chain transgenes) with immortalized cells.

[0074] As used herein, the term "recombinant human antibody" includes all human antibodies prepared, expressed, generated, or isolated by recombinant means, such as (a) antibodies isolated from animals (e.g., mice) that are transgenic or transchromosomal for human immunoglobulin genes or hybridomas prepared therefrom, (b) antibodies isolated from host cells transformed to express the antibody, e.g., from transfectomas, (c) antibodies isolated from recombinant, combinatorial human antibody libraries, and (d) antibodies prepared, expressed, generated, or isolated by any other means, including splicing of human immunoglobulin gene sequences into other DNA sequences. Such recombinant human antibodies contain variable and constant regions that utilize specific human germline immunoglobulin sequences encoded by germline genes, but include subsequent rearrangements and mutations that occur, for example, during antibody maturation. As is known in the art (see, e.g., Lonberg (2005) Nature Biotech. 23(9):1117-1125), variable regions contain antigen-binding domains encoded by various genes that rearrange to form antibodies specific to foreign antigens. In addition to rearrangement, variable regions can be further modified by multiple single amino acid changes (also called somatic mutation or hypermutation) to increase the affinity of the antibody for the foreign antigen. The constant regions further change in response to the antigen (i.e., isotype switching). Thus, the rearranged and somatically mutated nucleic acid sequences encoding light and heavy immunoglobulin polypeptides in response to the antigen may not be identical to the original germline sequences, but instead will be substantially identical or similar (i.e., have at least 80% identity).

[0075] A "human" antibody (HuMAb) refers to an antibody having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from a human germline immunoglobulin sequence. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. The terms "human" antibody and "fully human" antibody are used interchangeably.

[0076] A "humanized" antibody refers to an antibody in which some, most, or all of the amino acids outside the CDR domains of a non-human antibody, such as a mouse antibody, have been replaced with corresponding amino acids from a human immunoglobulin. In one embodiment of a humanized form of an antibody, some, most, or all of the amino acids outside the CDR domains have been replaced with amino acids from a human immunoglobulin, while some, most, or all of the amino acids within one or more CDR regions remain unchanged. Small additions, deletions, insertions, or substitutions or modifications of amino acids are permitted as long as they do not abrogate the antibody's ability to bind to a specific antigen. A "humanized" antibody retains antigen specificity similar to that of the original antibody.

[0077] A "chimeric antibody" refers to an antibody whose variable region is derived from one species and whose constant region is derived from another species, such as an antibody whose variable region is derived from a murine antibody and whose constant region is derived from a human antibody. A "hybrid" antibody refers to an antibody having heavy and light chains from different species, such as a murine (parental) heavy chain and a humanized light chain, or vice versa.

[0078] As used herein, "isotype" refers to the antibody class (e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD and IgE antibodies) encoded by heavy chain constant region genes.

[0079] "Allotype" refers to naturally occurring variants within a particular isotype group that differ by one or a few amino acids. See, e.g., Jefferis et al. (2009) mAbs 1:1.

[0080] The phrases "antibody that recognizes an antigen" and "antibody specific for an antigen" are used herein interchangeably with the term "antibody that specifically binds to an antigen."

[0081] As used herein, an "isolated antibody" refers to an antibody that is substantially free of other antibodies with different antigen specificities (e.g., an isolated antibody that specifically binds to TIGIT is substantially free of antibodies that specifically bind to antigens other than TIGIT). However, an isolated antibody that specifically binds to an epitope of human TIGIT may have cross-reactivity with other TIGIT proteins from different species.

[0082] As used herein, an antibody that "inhibits the binding of PVR to TIGIT" refers to an antibody that inhibits the binding of PVR to TIGIT in an art-recognized manner, e.g., in a FACS-based cell binding assay, with an EC of about 1 μg / mL or less, such as about 0.9 μg / mL or less, about 0.85 μg / mL or less, about 0.8 μg / mL or less, about 0.75 μg / mL or less, about 0.7 μg / mL or less, about 0.65 μg / mL or less, about 0.6 μg / mL or less, about 0.55 μg / mL or less, about 0.5 μg / mL or less, about 0.45 μg / mL or less, about 0.4 μg / mL or less, about 0.35 μg / mL or less, about 0.3 μg / mL or less, about 0.25 μg / mL or less, about 0.2 μg / mL or less, about 0.15 μg / mL or less, or about 0.1 μg / mL or less. 50 It refers to an antibody that inhibits the binding of human PVR to human TIGIT.

[0083] The "effector functions" resulting from the interaction of an antibody Fc region with a specific Fc receptor include, but are not necessarily limited to, FcγR-mediated effector functions such as Clq binding, complement-dependent cytotoxicity (CDC), Fc receptor binding, ADCC, and antibody-dependent cell-mediated phagocytosis (ADCP), and downregulation of cell surface receptors (e.g., B cell receptors; BCRs). Such effector functions generally require the Fc region to be combined with an antigen-binding domain (e.g., an antibody variable domain).

[0084] An "Fc receptor" or "FcR" is a receptor that binds to the Fc region of an immunoglobulin. FcRs that bind IgG antibodies include receptors of the FcγR family, including allelic variants and alternatively spliced ​​forms of these receptors. The FcγR family consists of three activating (FcγRI, FcγRIII, and FcγRIV in mice; FcγRIA, FcγRIIA, and FcγRIIIA in humans) and one inhibitory (FcγRIIb or equivalently, FcγRIIB) receptors. The various properties of human FcγRs are summarized in Table 1. While the majority of innate effector cell types simultaneously express one or more activating FcγRs and the inhibitory FcγRIIb, natural killer (NK) cells selectively express one activating Fc receptor (FcγRIII in mice and FcγRIIIA in humans) but do not express the inhibitory FcγRIIb in mice or humans. Human IgG1 binds to most human Fc receptors and is considered equivalent to mouse IgG2a with respect to the types of activating Fc receptors it binds.

[0085] [Table 1]

[0086] "Fc region" (fragment crystallizable region) or "Fc domain" or "Fc" refers to the C-terminal region of an antibody heavy chain that mediates binding of immunoglobulins to host tissues or factors, including binding to Fc receptors located on various cells of the immune system (e.g., effector cells) or to the first component (C1q) of the classical complement system. Thus, the Fc region comprises the constant region of an antibody that does not include the first constant region immunoglobulin domain (e.g., CH1 or CL). In IgG, IgA, and IgD antibody isotypes, the Fc region comprises the C1q domain in each of the antibody's two heavy chains. H2 and C H3 IgM and IgE Fc regions contain three heavy chain constant domains (C H For IgG, the Fc region comprises immunoglobulin domains Cγ2 and Cγ3 and the hinge between Cγ1 and Cγ2. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy chain Fc region is usually defined to stretch from an amino acid residue at position C226 or P230 (or an amino acid between these two amino acids) to the carboxy terminus of the heavy chain, where numbering is according to the EU index as in Kabat, Kabat et al. (1991) Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, MD; see also Figures 3c-3f of U.S. Patent Application Publication No. 2008 / 0248028. The C H2 The domain spans from about amino acid 231 to about amino acid 340, but H3 The domain is C in the Fc region. H2The Fc region is located C-terminal to the Fc domain, i.e., spanning from about amino acid 341 to about amino acid 447 (including the C-terminal lysine) of IgG. As used herein, the Fc region can be native-sequence Fc, including any allotypic variant or variant Fc (e.g., a non-naturally occurring Fc). Fc can also refer to this region in isolation or in the context of a protein polypeptide that contains an Fc, such as an "Fc region-containing binding protein," also referred to as an "Fc fusion protein" (e.g., an antibody or immunoadhesin).

[0087] Unless otherwise noted or clear from the context, numbering of amino acid residues in the Fc region of an antibody follows the EU numbering convention, except when specifically referring to residues in a sequence in a sequence listing, where the numbering is not necessarily consecutive. For example, literature references to the effect of amino acid substitutions in the Fc region typically use EU numbering, which allows the same number to refer to any given residue in the Fc region of an antibody, regardless of the length of the variable domain to which it is attached. In rare cases, it may be necessary to consult the referenced document to confirm that the correct Fc residue is being referenced.

[0088] A "native sequence Fc region" or "native sequence Fc" comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature. Native sequence human Fc regions include native sequence human IgG1 Fc regions, native sequence human IgG2 Fc regions, native sequence human IgG3 Fc regions, and native sequence human IgG4 Fc regions, as well as naturally occurring variants thereof. Native sequence Fc includes various allotypes of Fc. See, e.g., Jefferis et al. (2009) mAbs 1:1.

[0089] The term "epitope" or "antigenic determinant" refers to a site on an antigen (e.g., TIGIT) to which an immunoglobulin or antibody specifically binds. Epitopes within protein antigens can be formed both from contiguous amino acids (commonly linear epitopes) or from noncontiguous amino acids juxtaposed by tertiary folding of the protein (commonly conformational epitopes). Epitopes formed from contiguous amino acids are usually, but not necessarily, retained upon exposure to denaturing solvents, whereas epitopes formed from tertiary folding are usually lost upon treatment with denaturing solvents. Epitopes usually include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in a unique spatial conformation.

[0090] The term " epitope mapping " refers to the process of identifying the molecular determinant on antigen that is involved in antibody-antigen recognition.The method of determining the epitope that is bound by a given antibody is well known in the art, and includes, for example, immunoblotting and immunoprecipitation assay, in which derived (for example, derived from TIGIT) overlapping peptides or consecutive peptides are tested for reactivity with a given antibody (for example, anti-TIGIT antibody); X-ray crystallography; 2D nuclear magnetic resonance; yeast display (see, for example, Example 4 herein); and HDX-MS (see, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GE Morris, Ed. (1996)).

[0091] With respect to two or more antibodies, the term "binds to the same epitope" means that the antibodies bind to the same segment of amino acid residues, as determined by a given method. Techniques for determining whether the antibodies bind to the "same epitope on TIGIT" using the antibodies described herein include epitope mapping methods such as X-ray analysis of crystals of antigen:antibody complexes, which provide atomic resolution of the epitope, and hydrogen / deuterium exchange mass spectrometry (HDX-MS). Other methods, such as alanine scanning mutagenesis (Cunningham & Wells (1985) Science 244:1081) or yeast display of mutant target sequence variants (see Example 4 herein), monitor the binding of antibodies to antigen fragments (e.g., proteolytic fragments) or mutated variations of the antigen, in which loss of binding due to modification of amino acid residues within the antigen sequence is often considered an indication of epitope components. Furthermore, computational combinatorial methods for epitope mapping may also be used. These methods rely on the ability of an antibody of interest to affinity isolate specific short peptides from combinatorial phage-displayed peptide libraries. Antibodies with identical or closely related VH and VL or identical CDR sequences are predicted to bind the same epitope.

[0092] An antibody that "competes with another antibody for binding to a target" refers to an antibody that inhibits (partially or completely) the binding of the other antibody to a target. Whether two antibodies compete with each other for binding to a target, i.e., whether and to what extent one antibody inhibits the binding of the other antibody to a target, can be determined using known competition experiments. In certain embodiments, an antibody competes or inhibits the binding of another antibody to a target by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. The level of inhibition or competition can vary depending on which antibody is the "blocking antibody" (i.e., the cold antibody that is first incubated with the target). Competitive assays can be performed, for example, as described in Ed Harlow and David Lane, Cold Spring Harb. Protoc.; 2006; doi:10.1101 / pdb.prot4277 or Chapter 11 of "Using Antibodies" by Ed Harlow and David Lane, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA 1999. Competitive antibodies bind to the same epitope, overlapping epitopes, or adjacent epitopes (e.g., as evidenced by steric hindrance).

[0093] Other competitive binding assays include solid-phase direct or indirect radioimmunoassays (RIA), solid-phase direct or indirect enzyme immunoassays (EIA), sandwich competition assays (see Stahli et al. (1983) Methods in Enzymology 9:242); solid-phase direct biotin-avidin EIA (see Kirkland et al. (1986) J. Immunol. 137:3614); solid-phase direct labeled assays, solid-phase direct labeled sandwich assays (see Harlow and Lane (1988), Antibodies: A Laboratory Manual, Cold Spring Harbor Press); solid-phase direct labeled RIAs using I-125 labels (see Morel et al. (1988) Mol. Immunol. 25(1):7); solid-phase direct biotin-avidin EIAs (Cheung et al. (1990) Virology 176:546); and direct labeled RIAs (Moldenhauer et al. al. (1990) Scand. J. Immunol. 32:77).

[0094] As used herein, the terms "specific binding," "selective binding," "selectively binds," and "specifically binds" refer to antibody binding to an epitope on a predetermined antigen and not to other antigens. Typically, an antibody (i) binds to an epitope of approximately 10 s, as determined, for example, by surface plasmon resonance (SPR) techniques on a BIACORE® 2000 surface plasmon resonance instrument using a predetermined antigen, e.g., recombinant human TIGIT, as the analyte and an antibody as the ligand, or by Scatchard analysis of antibody binding to antigen-positive cells. -7 Less than M, e.g., approximately 10 -8 Under M, 10 -9 M or 10 -10 M or even smaller equilibrium dissociation constant (K D) and (ii) binds to a predetermined antigen with an affinity that is at least two-fold greater than its affinity for binding to a nonspecific antigen other than the predetermined antigen or a closely related antigen (e.g., BSA, casein). Thus, an antibody that "specifically binds to human TIGIT" is one that binds to a predetermined antigen with an affinity that is at least two-fold greater than its affinity for binding to a nonspecific antigen other than the predetermined antigen or a closely related antigen (e.g., BSA, casein). -7 M or less, e.g., about 10 -8 M, 10 -9 M or 10 -10 K less than or even smaller than M D The term "antibody that cross-reacts with cynomolgus monkey TIGIT" refers to an antibody that binds to soluble or cell-bound human TIGIT. -7 M or less, e.g., about 10 -8 M, 10 -9 M or 10 -10 K below M or even lower D This refers to an antibody that binds to cynomolgus monkey TIGIT.

[0095] As used herein, the term "k assoc " or "k a " refers to the association rate constant of a particular antibody-antigen interaction, whereas the term "k dis " or "k d " refers to the dissociation rate constant of a particular antibody-antigen interaction. D " as used herein refers to the equilibrium dissociation constant, which is k a k for d The proportion of (i.e., k d / k a ) and expressed as molar concentration (M). D The K value can be determined using methods well established in the art. D Preferred methods for determining include biolayer interferometry (BLI) analysis, preferably using a Fortebio Octet RED instrument, surface plasmon resonance, preferably using a biosensor system such as a BIACORE® surface plasmon resonance system (see, e.g., Example 2), or flow cytometry and Scatchard analysis.

[0096] As used herein, the term "high affinity" for an IgG antibody refers to an antibody that binds to a target antigen with a binding affinity of 10 -8 M or less, more preferably 10 -9 M or less, and even more preferably, 10 -10 K below M D However, "high affinity" binding may vary for other antibody isotypes. For example, "high affinity" binding for an IgM isotype is considered to be 10 -7 M or less, more preferably 10 -8 K below M D It refers to an antibody having the following structure:

[0097] The term "EC" in reference to in vitro or in vivo assays using antibodies or antigen-binding fragments thereof 50 " refers to the concentration of an antibody or antigen-binding fragment thereof that induces 50% of the maximal response, i.e., a response that is halfway between the maximal response and the baseline.

[0098] The term "binds to immobilized TIGIT" refers to the ability of the antibodies described herein to bind to TIGIT that is, for example, expressed on the surface of a cell or bound to a solid support.

[0099] The term "cross-reactive" as used herein refers to the ability of an antibody described herein to bind to TIGIT from a different species. For example, an antibody described herein that binds to human TIGIT can also bind to TIGIT from another species (e.g., cynomolgus monkey TIGIT). Herein, cross-reactivity may be measured by detecting specific reactivity with purified antigens in binding assays (e.g., SPR, ELISA) or binding to or otherwise functional interaction with cells that physiologically express TIGIT. Methods for examining cross-reactivity include, for example, standard binding assays as described herein, using BIACORE® surface plasmon resonance (SPR) analysis or flow cytometry technology using a BIACORE® 2000SPR instrument (Biacore AB, Uppsala, Sweden).

[0100] The term "naturally occurring" as applied to an object herein refers to the fact that the object can be found in nature. For example, naturally occurring polypeptide or polynucleotide sequences exist in organisms (including viruses) that can be isolated from sources in nature and have not been intentionally modified by humans in a laboratory.

[0101] A "polypeptide" refers to a chain comprising at least two consecutively linked amino acid residues, with no upper limit to the length of the chain. One or more amino acid residues in a protein may contain modifications, such as, but not limited to, glycosylation, phosphorylation, or disulfide bonds. A "protein" may include one or more polypeptides.

[0102] The term "nucleic acid molecule" as used herein includes DNA molecules and RNA molecules. A nucleic acid molecule may be single-stranded or double-stranded, and may be cDNA.

[0103] Also provided herein are "conservative sequence modifications" to the antibody sequences provided herein, that is, nucleotide and amino acid sequence modifications that do not inhibit the binding of the antibody encoded by the nucleotide sequence or containing the amino acid sequence to antigen.For example, modifications can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.Conservative sequence modifications include conservative amino acid substitutions, in which amino acid residues are replaced with amino acid residues that have similar side chains.Families of amino acid residues that have similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, predicted non-essential amino acid residues in anti-TIGIT antibodies are preferably replaced with another amino acid residue from the same side chain family. Methods for identifying conservative nucleotide and amino acid substitutions that do not eliminate antigen binding are well known in the art. See, e.g., Brummel et al., Biochem. 32:1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10):879-884 (1999); and Burks et al. Proc. Natl. Acad. Sci. USA 94:412-417 (1997)).

[0104] Alternatively, in another embodiment, mutations can be introduced randomly along all or part of the anti-TIGIT antibody coding sequence, such as by saturation mutagenesis, and the resulting modified anti-TIGIT antibodies can be screened for improved binding activity.

[0105] With respect to nucleic acids, the term "substantial homology" indicates that two nucleic acids or their designated sequences, when optimally aligned and compared, are identical, with appropriate nucleotide insertions or deletions, in at least about 80% of the nucleotides, usually at least about 90% to 95% of the nucleotides, and more preferably at least about 98% to 99.5% of the nucleotides. Alternatively, substantial homology exists when the segments will hybridize under selective hybridization conditions, to the complement of the strand.

[0106] With respect to polypeptides, the term "substantial homology" indicates that two polypeptides or their designated sequences, when optimally aligned and compared, are identical in at least about 80% of the amino acids, usually at least about 90% to 95%, and more preferably at least about 98% to 99.5% of the amino acids, with appropriate amino acid insertions or deletions.

[0107] The percent identity between two sequences is a function of the number of identical positions shared by the sequences when they are optimally aligned (i.e., % homology = number of identical positions / total number of positions x 100), and optimal alignment is determined by taking into account the number of gaps, the length of each gap, that need to be introduced for optimal alignment of the two sequences. Comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the non-limiting examples below.

[0108] The percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package, using a NWSgapdna.CMP matrix and gap weights of 40, 50, 60, 70, or 80 and length weights of 1, 2, 3, 4, 5, or 6. The percent identity between two nucleotide or amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4:11-17 (1989)) as incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Furthermore, percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm incorporated into the GAP program in the GCG software package, using either a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0109] The nucleic acid and protein sequences described herein can further be used as "query sequences" to perform searches against public databases, for example, to identify related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul et al. (1990) J. Mol. Biol. 215:403-10. To obtain nucleotide sequences homologous to the nucleic acid molecules described herein, BLAST nucleotide searches can be performed using the NBLAST program, score = 100, word length = 12. To obtain amino acid sequences homologous to the protein molecules described herein, BLAST protein searches can be performed using the XBLAST program, score = 50, word length = 3. To obtain gapped alignments for comparison purposes, gapped BLAST can be used, as described in Altschul et al. (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (eg, XBLAST and NBLAST) can be used.

[0110] Nucleic acids may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form. A nucleic acid is "isolated" or "substantially purified" when it has been purified from other cellular components or other contaminants, such as other cellular nucleic acids (e.g., other parts of chromosomes) or proteins, by standard techniques, including alkaline / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and others known in the art. See F. Ausubel, et al., ed., Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987).

[0111] As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, thereby replicating along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. Because the plasmid is the most commonly used form of vector, "plasmid" and "vector" can be used interchangeably herein. However, other forms of expression vectors, such as viral vectors (eg, replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions are also included.

[0112] The term "recombinant host cell" (or simply "host cell"), as used herein, refers to a cell that contains a nucleic acid that does not naturally occur in the cell, and may be a cell into which a recombinant expression vector has been introduced. It should be understood that such terms refer not only to the particular subject cell, but also to the progeny of such a cell. Because certain modifications may occur in later generations, either due to mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein.

[0113] "Immune response" refers to a biological response in a vertebrate to foreign agents that protects the organism from these agents and the diseases they cause. Immune responses are mediated by the action of cells of the immune system (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, or neutrophils) and soluble macromolecules (including antibodies, cytokines, and complement) produced by any of these cells or the liver, which result in the selective targeting, binding, damage, destruction, and / or elimination from the vertebrate body of invading pathogens, pathogen-infected cells or tissues, cancerous or other abnormal cells, or, in the case of autoimmunity or pathological inflammation, normal human cells or tissues. Immune responses are mediated, for example, by T cells, e.g., CD8 + or CD4 + Activation or inhibition of effector T cells or Th cells, such as T cells, or T reg Inhibition or depletion of "T effector" ("T eff ") cells are T cells (e.g., CD4 T cells) with cytolytic activity that secrete cytokines and activate and direct other immune cells. + and CD8 + T cells) and T helper (Th) cells, but also regulatory T cells (T reg cells).

[0114] As used herein, the term "T cell-mediated response" refers to a response mediated by effector T cells (e.g., CD8 + cells) and helper T cells (e.g., CD4 + "T-cell response" refers to responses mediated by T cells, including T cells. T cell-mediated responses include, for example, T cell cytotoxicity and proliferation.

[0115] As used herein, the term "cytotoxic T lymphocyte (CTL) response" refers to an immune response induced by cytotoxic T cells. A CTL response is a response induced by CD8 + It is primarily mediated by T cells.

[0116] An "immunomodulator" or "immunoregulator" refers to an agent, e.g., a component of a signal transduction pathway, that may be involved in the regulation, control, or modification of an immune response. "Regulation," "regulation," or "modulation" of an immune response refers to any change in cells of the immune system or in the activity of such cells (e.g., effector T cells). Such regulation includes stimulation or suppression of the immune system and may be manifested by an increase or decrease in the numbers of various cell types, an increase or decrease in the activity of these cells, or any other change that may occur within the immune system. Both inhibitory and stimulatory immunomodulators have been identified, some of which may have enhanced function in the tumor microenvironment. In a preferred embodiment, the immunomodulator is located on the surface of T cells. An "immunoregulatory target" or "immunoregulatory target" is an immunomodulator that is targeted for binding by a substance, agent, moiety, compound, or molecule, and whose activity is altered by the binding of the substance, agent, moiety, compound, or molecule. Immunomodulatory targets include, for example, cell surface receptors ("immunomodulatory receptors") and receptor ligands ("immunomodulatory ligands").

[0117] "Immunotherapy" refers to the treatment of a subject suffering from or at risk of developing or suffering from a recurrence of a disease by methods involving inducing, enhancing, suppressing or otherwise modifying the immune response.

[0118] "Immunostimulating therapy" or "immunostimulatory therapy" refers to a therapy that results in an increase (induction or enhancement) of the immune response in a subject, for example, to treat cancer.

[0119] "Enhancing an endogenous immune response" means increasing the efficacy or potency of an existing immune response in a subject. This increased efficacy and potency can be achieved, for example, by overcoming mechanisms that suppress the endogenous host immune response or by stimulating mechanisms that enhance the endogenous host immune response.

[0120] As used herein, the term "linked" refers to the association of two or more molecules. The linkage can be covalent or non-covalent. The linkage can also be genetic (i.e., recombinantly fused). Such linkage can be achieved using a variety of art-recognized techniques, such as chemical conjugation and recombinant protein production.

[0121] As used herein, "administering" refers to the physical introduction of a composition containing a therapeutic agent into a subject using any of a variety of methods and delivery systems known to those skilled in the art. Preferred routes of administration of the antibodies described herein include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal, or other parenteral routes of administration, for example, by injection or infusion. As used herein, the phrase "parenteral administration" refers to modes of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intraarticular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion, as well as in vivo electroporation. Alternatively, the antibodies described herein may be administered by non-parenteral routes, such as topical, epithelial, or mucosal administration routes, for example, intranasally, orally, vaginally, rectally, sublingually, or topically. Administering can be carried out, for example, once, multiple times and / or over one or more extended periods of time.

[0122] As used herein, the terms "inhibit" or "block" (e.g., referring to inhibition / blocking of binding of PVR to TIGIT on a cell) are used interchangeably and include both partial and complete inhibition / blocking, e.g., at least about 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100%.

[0123] As used herein, "cancer" refers to a broad group of diseases characterized by the uncontrolled growth of abnormal cells in the body. Uncontrolled cell division can lead to the formation of malignant tumors or cells that can invade adjacent tissues and metastasize to distant parts of the body via the lymphatic system or bloodstream.

[0124] "Hematologic malignancies" include lymphomas, leukemias, myelomas, or lymphatic malignancies, as well as cancers of the spleen and lymph nodes. Exemplary lymphomas include both B-cell lymphomas and T-cell lymphomas. B-cell lymphomas include both Hodgkin's lymphoma and most non-Hodgkin's lymphomas. Non-limiting examples of B-cell lymphomas include diffuse large B-cell lymphoma, follicular lymphoma, mucosa-associated lymphoid tissue lymphoma, small cell lymphocytic lymphoma (overlaps with chronic lymphocytic leukemia), mantle cell lymphoma (MCL), Burkitt's lymphoma, mediastinal large B-cell lymphoma, Waldenstrom's macroglobulinemia, nodal marginal zone B-cell lymphoma, splenic marginal zone lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, and lymphomatoid granulomatosis. Non-limiting examples of T-cell lymphoma include extranodal T-cell lymphoma, cutaneous T-cell lymphoma, anaplastic large cell lymphoma and angioimmunoblastic T-cell lymphoma.Hematological malignancies also include leukemia, such as, but not limited to, secondary leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myelogenous leukemia and acute lymphocytic leukemia.Hematological malignancies also include myeloma, such as, but not limited to, multiple myeloma and smoldering multiple myeloma.Other blood system and / or B-cell or T-cell related cancers are encompassed by the term hematological malignancies.

[0125] The terms "treat," "treating," and "treatment," as used herein, refer to any type of intervention or process performed on a subject or the administration of an active agent to a subject, with the goal of reversing, alleviating, ameliorating, inhibiting, or slowing the progression, occurrence, severity, or recurrence of symptoms, complications, conditions, or biochemical manifestations associated with a disease. Prevention refers to administration to a subject not having a disease to prevent the disease from occurring or to minimize its effects if it does occur.

[0126] The term "effective dose" or "effective dosage" is defined as an amount sufficient to achieve or at least partially achieve the desired effect. A "therapeutically effective amount" or "therapeutically effective dosage" of a drug or therapeutic agent is any amount of the drug that, when used alone or in combination with another therapeutic agent, promotes disease regression as evidenced by a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, or prevention of functional impairment or disability due to disease affliction. A "prophylactically effective amount" or "prophylactically effective dosage" of a drug is an amount of the drug that, when administered alone or in combination with another therapeutic agent to a subject at risk of developing a disease or suffering a disease recurrence, inhibits the onset or recurrence of the disease. The ability of a therapeutic or prophylactic agent to promote disease regression or inhibit the onset or recurrence of a disease can be evaluated using various methods known to those skilled in the art, such as in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by assaying the activity of the agent in in vitro assays.

[0127] For example, an anti-cancer drug is a drug that slows cancer progression or promotes cancer regression in a subject. In a preferred embodiment, a therapeutically effective amount of a drug promotes cancer regression to the point of eliminating the cancer. "Promoting cancer regression" means that administering an effective amount of a drug, alone or in combination with an anti-neoplastic agent, results in a reduction in tumor growth or size, tumor necrosis, a decrease in the severity of at least one disease symptom, an increase in the frequency and duration of disease symptom-free periods, prevention of functional or disability impairment due to disease affliction, or otherwise amelioration of disease symptoms in a patient. Pharmacological efficacy refers to the ability of a drug to promote cancer regression in a patient. Physiological safety refers to an acceptably low level of toxicity or other adverse physiological effects (adverse effects) at the cellular, organ, and / or organismal levels resulting from the administration of a drug.

[0128] As an example of tumor treatment, a therapeutically effective amount or dosage of a drug preferably inhibits cell or tumor growth by at least about 20%, more preferably at least about 40%, even more preferably at least about 60%, and even more preferably at least about 80% compared to untreated controls. In the most preferred embodiment, a therapeutically effective amount or dosage of a drug completely inhibits cell or tumor growth, i.e., preferably inhibits cell or tumor growth by 100%. The ability of a compound to inhibit tumor growth can be assessed using the assays described below. Inhibition of tumor growth may not be immediate after treatment, but may occur only after a period of time or after repeated administration. Alternatively, the properties of the composition can be assessed by examining the ability of the compound to inhibit cell growth, and such inhibition can be measured in vitro using assays known to those skilled in the art. In other preferred embodiments described herein, tumor regression can be observed and can last for a period of at least about 20 days, more preferably at least about 40 days, or even more preferably at least about 60 days.

[0129] As used herein, unless otherwise clear from the context, "combination" therapy refers to the administration of two or more therapeutic agents in a coordinated manner, including, but not limited to, simultaneous administration. Specifically, combination therapy encompasses both simultaneous administration (e.g., administration of a combined drug or simultaneous administration of separate therapeutic compositions) and sequential or sequential administration, provided that the administration of one therapeutic agent is conditioned in some way on the administration of the other therapeutic agent. For example, one therapeutic agent may be administered only after a different therapeutic agent has been administered and allowed to act for a specified period of time. See, for example, Kohrt et al. (2011) Blood 117:2423.

[0130] The terms "patient" and "subject" refer to any human or non-human animal that receives either preventive or therapeutic treatment. For example, the methods and compositions described herein can be used to treat subjects with cancer. The term "non-human animal" includes all vertebrates, such as mammals and non-mammals, including non-human primates, sheep, dogs, cows, chickens, amphibians, reptiles, etc.

[0131] The various aspects described herein are described in further detail in the following subsections.

[0132] I. Anti-TIGIT antibody The present application discloses fully human anti-huTIGIT antibodies that have desirable properties for use as therapeutic agents in the treatment of diseases such as cancer, including one or more of the ability to bind human TIGIT with high affinity, the ability to bind cynomolgus monkey TIGIT, the ability to block PVR binding (and thus signaling), and the absence of sequence preferences that may reduce the chemical stability of the antibody.

[0133] The anti-TIGIT antibodies disclosed herein by sequence bind to specific epitopes on human TIGIT determined as described in Example 4 and shown in Figures 2A-2C. The three specific antibodies for which epitopes have been determined bind to similar regions of human TIGIT but differ in which specific amino acid residues are contacted. The antibodies share the properties of binding to human TIGIT with high affinity and have the ability to block PVR binding. Therefore, other antibodies that bind to the same or closely related epitopes are likely to share these desirable properties and can be discovered by competition experiments.

[0134] Furthermore, antibody 22G2 binds to cynomolgus monkey TIGIT with substantially the same affinity as it binds to human TIGIT, which is advantageous when necessary to conduct toxicity studies to support regulatory approval for the use of the antibody as a human therapeutic. Other anti-TIGIT antibodies that bind to the same or similar epitopes as 15A6 and 22G2 are likely to share this advantageous property of binding to cyno TIGIT. Antibodies binding to similar epitopes can be discovered by performing competition experiments or by directly examining the epitope.

[0135] Anti-TIGIT antibodies that compete with the anti-huTIGIT antibodies disclosed herein Anti-huTIGIT antibodies that compete with the antibodies of the invention for binding to huTIGIT, such as 15A6 and 22G2, can be generated using immunization protocols similar to those described herein (Example 1). Antibodies that compete for binding with the anti-huTIGIT antibodies described herein can also be generated by immunizing mice with a construct comprising human TIGIT or its extracellular domain (residues 22-141 of SEQ ID NO: 1; NP_776160.2), or by immunizing with a fragment of human TIGIT that contains the epitope bound by the anti-huTIGIT antibodies disclosed herein (e.g., 15A6, 22G2, and 11G11). The resulting antibodies can be screened for their ability to block the binding of 15A6 or 22G2 to human TIGIT by methods well known in the art, such as blocking the binding of a fusion protein of the extracellular domain of TIGIT and an immunoglobulin Fc domain in ELISA, or by blocking the ability to bind to cells expressing huTIGIT on their surface, for example, by FACS. In various embodiments, the test antibody is contacted with the TIGIT-Fc fusion protein (or with cells expressing huTIGIT on their surface) prior to, simultaneously with, or after the addition of 15A6 or 22G2. For example, a "binning" experiment can be performed to determine whether an antibody falls into the same "bin" as antibody 15A6 or 22G2 (Example 3), in which antibody 15A6 or 22G2 is referred to as the "reference" antibody and the antibody to be tested is referred to as the "test" antibody. Antibodies that reduce the binding of 15A6 and / or 22G2 to TIGIT (either as Fc fusions or on cells), particularly at approximately stoichiometric concentrations, are likely to bind to identical, overlapping, or adjacent epitopes and therefore may share the desirable functional properties of 15A6 and 22G2.

[0136] Competitive antibodies can also be identified using other methods known in the art. For example, standard ELISA assays or competitive ELISA assays can be used, in which a recombinant human TIGIT protein construct is immobilized on a plate, various concentrations of unlabeled test antibody are added, the plate is washed, a labeled reference antibody is added, washed, and the amount of bound label is measured. If increasing concentrations of unlabeled test antibody inhibit the binding of the labeled reference antibody, the test antibody is said to inhibit the binding of the reference antibody to the target on the plate or to compete with the binding of the reference antibody. Additionally or alternatively, BIACORE® SPR analysis can be performed to evaluate the antibody's ability to compete. The ability of the test antibody to inhibit the binding of the anti-huTIGIT antibody described herein to TIGIT demonstrates that the test antibody can compete with the reference antibody for binding to TIGIT.

[0137] Thus, for example, binding of an anti-huTIGIT antibody described herein to TIGIT on a cell, e.g., an activated T cell, can be measured by at least 10%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1110, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151%, 152%, 153%, 154%, 155%, 156%, 157%, 158%, 159%, 160%, 161%, 162%, 163%, 164%, 165%, 166%, 167%, 168%, 169%, 170%, 171%, 172%, 173%, 174%, 175%, Provided herein are anti-TIGIT antibodies that inhibit 6%, 97%, 98%, 99% or 100% and / or whose binding to TIGIT on cells, e.g., activated T cells, is inhibited by at least 10%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0138] An exemplary competition experiment to determine whether a test antibody blocks (i.e., "competes") the binding of a reference antibody can be performed as follows: Activated human T cells are prepared as follows: Peripheral blood mononuclear cells (PBMCs) are isolated from human whole blood using a Ficoll gradient and activated with 10 μg / mL phytohemagglutinin (PHA-L) (USBiol No. P3370-30) and 200 IU / mL recombinant IL-2 (Peprotech No. 200-02) for 3 days. The activated T cells are resuspended in FACS buffer (PBS with 5% fetal bovine serum) and plated at 10 per sample well in a 96-well plate. 5 Plates are seeded with 1000 cells. Unconjugated test antibodies are added to the plate at concentrations ranging from 0 to 50 μg / mL (3-fold titration starting from a top concentration of 50 μg / mL). An irrelevant IgG may be used as an isotype control for the test antibody and added at the same concentration (3-fold titration starting from a top concentration of 50 μg / mL). A sample preincubated with 50 μg / mL of unlabeled reference antibody may be included as a positive control for competitive blocking (100% inhibition), and a sample without antibody in the primary incubation may be used as a negative control (no competition; 0% inhibition). After a 30-minute incubation, without washing, labeled, e.g., biotinylated, reference antibody is added at a concentration of 2 μg / mL per well. The samples are incubated for an additional 30 minutes. Unbound antibody is removed by washing the cells with FACS buffer. The labeled reference antibody bound to the cells is detected using a detection agent, such as PE-conjugated streptavidin (Invitrogen, Catalog No. S21388), to detect the label, for example, biotin. Samples are acquired using a FACS Calibur flow cytometer (BD, San Jose) and analyzed using Flowjo software (Tree Star, Inc, Ashland, OR). Results can be expressed as % inhibition (i.e., 100% minus the amount of label at each concentration, divided by the amount of label obtained without blocking antibody).

[0139] Typically, the same experiment is then performed in reverse, i.e., the test antibody is the reference antibody, and the reference antibody is the test antibody. In certain embodiments, an antibody at least partially (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%) or completely (100%) blocks the binding of another antibody to its target, for example, human TIGIT or a fragment thereof, regardless of whether inhibition occurs when one or the other antibody is the test antibody. The test and reference antibodies "cross-block" each other's binding to their targets when the antibodies compete with each other in both directions, i.e., in a competition experiment where the test antibody is added first, and in a competition experiment where the reference antibody is added first.

[0140] An anti-huTIGIT antibody is considered to compete with an anti-huTIGIT antibody disclosed herein if it inhibits binding of 15A6 and / or 22G2 to human TIGIT by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% when present at approximately equal concentrations in a competition experiment such as that described in Example 3. Unless otherwise indicated, an antibody is considered to compete with an antibody selected from the group consisting of the anti-huTIGIT antibodies of the present invention if, when used at approximately equimolar concentrations with the selected antibody, it reduces binding of the selected antibody to human TIGIT by at least 20%, as measured in a competition ELISA experiment such as that outlined in the preceding two paragraphs.

[0141] Anti-TIGIT antibodies that bind to the same epitope Anti-huTIGIT antibodies that bind to the same or similar epitopes as the antibodies disclosed herein can be generated using immunization protocols similar to those described herein (Example 1). The resulting antibodies can be screened for high-affinity binding to human TIGIT (Example 2). Selected antibodies can then be studied in yeast display assays in which sequence variants of huTIGIT are displayed on the surface of yeast cells to determine the exact epitope bound by the antibody (Example 4).

[0142] Epitope determination can be performed by any method known in the art. The epitopes disclosed herein were determined by yeast display as described in Example 4 and shown in Figures 2A-2C. In various embodiments, an anti-huTIGIT antibody is considered to bind the same epitope as an anti-huTIGIT mAb disclosed herein, e.g., 15A6 and / or 22G2, if it contacts one or more identical residues in at least one region of huTIGIT contacted by 15A6 or 22G2; if it contacts most residues in at least one region of huTIGIT contacted by 15A6 or 22G2; if it contacts most residues in each region of huTIGIT contacted by 15A6 or 22G2; if it contacts most contacts along the entire length of huTIGIT contacted by 15A6 or 22G2; if it contacts all of the identical residues in any one region on human TIGIT contacted by 15A6 or 22G2; or if it contacts all residues in all regions contacted by 15A6 or 22G2. An epitope "region" is a cluster of residues along the primary sequence that is contacted by antibody 15A6 or 22G2, for example, as provided in SEQ ID NOs: 38-44.

[0143] Techniques for identifying antibodies that bind to the "same epitope on TIGIT" using the antibodies described herein include X-ray analysis of crystals of the antigen:antibody complex, which provides atomic resolution of the epitope. Other methods monitor the binding of antibodies to antigen fragments or mutated variations of the antigen, where loss of binding due to modification of amino acid residues within the antigen sequence is considered to indicate epitope components. Methods may also rely on the ability of a subject antibody to affinity isolate specific short peptides (either in their native three-dimensional form or in denatured form) from combinatorial phage-display peptide libraries or from protease digests of the target protein. The peptides are then considered leads for defining the epitope corresponding to the antibody used to screen the peptide library. For epitope mapping, computational algorithms have also been developed that have been shown to map conformationally discontinuous epitopes.

[0144] Epitopes or regions containing epitopes can also be identified by screening for binding to a series of overlapping peptides spanning TIGIT. Alternatively, the method of Jespers et al. (1994) Biotechnology 12:899 can be used to guide the selection of antibodies that have the same epitope and therefore similar properties to the anti-TIGIT antibodies described herein. Using phage display, the heavy chain of an anti-TIGIT antibody is first paired with a repertoire of (preferably human) light chains to select for TIGIT-binding antibodies, and then the new light chain is paired with a repertoire of (preferably human) heavy chains to select for (preferably human) TIGIT-binding antibodies that have the same epitope or epitope region as the anti-huTIGIT antibody described herein. Alternative variants of the antibodies described herein can be obtained by mutagenesis of the cDNA encoding the heavy and light chains of the antibody.

[0145] Alanine scanning mutagenesis as described by Cunningham & Wells (1989) Science 244: 1081 or some other form of point mutagenesis of amino acid residues in TIGIT (such as the yeast display method provided in Example 4) may be used to determine the functional epitope of an anti-TIGIT antibody.

[0146] The epitope or epitope region (an "epitope region" is a region that includes or overlaps with the epitope) bound by a specific antibody may also be determined by assessing the binding of the antibody to peptides comprising fragments of TIGIT. A series of overlapping peptides encompassing the sequence of TIGIT (e.g., human TIGIT) may be synthesized and screened for binding, for example, in direct ELISA, competitive ELISA (in which peptides are assessed for their ability to prevent antibody binding to TIGIT bound to wells of a microtiter plate), or on a chip. Such peptide screening methods may not be able to detect some discontinuous functional epitopes, i.e., functional epitopes that comprise amino acid residues that are not contiguous along the primary sequence of the TIGIT polypeptide chain.

[0147] Epitopes can also be identified by MS-based protein footprinting methods, such as hydrogen / deuterium exchange mass spectrometry (HDX-MS) and fast photochemical oxidation of proteins (FPOP). HDX-MS can be performed as further described in Wei et al. (2014) Drug Discovery Today 19:95, which method is specifically incorporated herein by reference. FPOP can be performed, for example, as described in Hambley & Gross (2005) J. American Soc. Mass Spectrometry 16:2057, which method is specifically incorporated herein by reference.

[0148] The epitope bound by an anti-TIGIT antibody may also be determined by structural methods such as X-ray crystal structure determination (e.g., WO2005 / 044853), molecular modeling, and nuclear magnetic resonance (NMR) spectroscopy, including NMR determination of the HD exchange rate of the labile amide hydrogen in TIGIT when free and when bound in complex with the antibody of interest (Zinn-Justin et al. (1992) Biochemistry 31:11335; Zinn-Justin et al. (1993) Biochemistry 32:6884).

[0149] For X-ray crystallography, crystallization can be achieved using any of the methods known in the art, including microbatch (e.g., Chayen (1997) Structure 5:1269), hanging drop vapor diffusion (e.g., McPherson (1976) J. Biol. Chem. 251:6300), seeding, and dialysis (e.g., Giege et al. (1994) Acta Crystallogr. D50:339; McPherson (1990) Eur. J. Biochem. 189:1). It is desirable to use protein preparations having a concentration of at least about 1 mg / mL, preferably about 10 mg / mL to about 20 mg / mL. Crystallization is best achieved in a precipitant solution containing polyethylene glycol 1000-20,000 (PEG; average molecular weight ranging from about 1000 to about 20,000 Da), preferably about 5000 to about 7000 Da, more preferably about 6000 Da, at a concentration ranging from about 10% to about 30% (w / v). It may be desirable to include a proteolytic agent, such as glycerol, at a concentration ranging from about 0.5% to about 20%. A suitable salt, such as sodium chloride, lithium chloride, or sodium citrate, may also be desirable in the precipitant solution, preferably at a concentration ranging from about 1 mM to about 1000 mM. The precipitant is preferably buffered to a pH of about 3.0 to about 5.0, preferably about 4.0. Specific buffers useful in the precipitant solution may vary and are well known in the art (Scopes, Protein Purification: Principles and Practice, Third ed., (1994) Springer-Verlag, New York). Examples of useful buffers include, but are not limited to, HEPES, Tris, MES, and acetic acid. Crystals may be grown at a variety of temperatures, including 2°C, 4°C, 8°C, and 26°C.

[0150] Antibody:antigen crystals can be studied using well-known X-ray diffraction techniques and refined using computer software such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc.; see, e.g., Blundell & Johnson (1985) Meth. Enzymol. 114 & 115, HW Wyckoff et al., eds., Academic Press; see U.S. Patent Application Publication No. 2004 / 0014194) and BUSTER (Bricogne (1993) Acta Cryst. D49:37-60; Bricogne (1997) Meth. Enzymol. 276A:361-423, Carter & Sweet, eds.; Roversi et al. (2000) Acta Cryst. D56:1313-1323), the disclosures of which are incorporated herein by reference in their entireties.

[0151] High-affinity binding anti-TIGIT antibody In some embodiments, the anti-huTIGIT antibodies of the invention, such as the anti-huTIGIT antibodies disclosed herein, bind to huTIGIT with high affinity, enhancing their potential to be effective therapeutic agents. In various embodiments, the anti-huTIGIT antibodies of the invention bind to huTIGIT with a K of less than 10 nM, 5 nM, 2 nM, 1 nM, 300 pM, 100 pM, or 60 pM. D In other embodiments, the anti-huTIGIT antibodies of the invention bind with a K between 2 nM and 60 pM. D Standard assays for assessing the binding ability of an antibody to huTIGIT include ELISA, RIA, Western blot, biolayer interferometry (BLI), and BIACORE® SPR analysis (see Example 2).

[0152] Anti-TIGIT antibody sequence variants Some variability in the antibody sequences disclosed herein can be tolerated while still maintaining the desired properties of the antibody. CDR regions are delineated using the Kabat system (Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242). Accordingly, the present invention further provides anti-huTIGIT antibodies comprising CDR sequences that are at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the CDR sequences of the antibodies disclosed herein (e.g., 15A6, 22G2, and 11G11). The present invention also provides anti-huTIGIT antibodies comprising heavy and / or light chain variable domain sequences that are at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the heavy and / or light chain variable domain sequences of the antibodies disclosed herein (e.g., 15A6, 22G2 and 11G11).

[0153] Anti-TIGIT antibodies derived from the same germline Given that antigen-binding specificity is primarily determined by CDRs, antibodies that share CDR sequences with the antibodies disclosed herein (e.g., 15A6, 22G2, and 11G11) are likely to share their desirable properties. Furthermore, selected antibodies disclosed herein (15A6, 22G2, and 11G11) bind to similar regions along the primary sequence of huTIGIT, and some heavy and light chains are derived from the same germline sequence. Thus, antibodies that combine (mix and match) CDR regions from antibodies with 15A6, 22G2, and 11G11 can also be expected to bind to huTIGIT and retain their desirable properties. "Mix and match" antibodies with binding affinity, bioactivity, and / or other properties comparable to or superior to those of specific antibodies disclosed herein can be selected for use in the methods of the invention.

[0154] In certain embodiments, anti-huTIGIT antibodies of the present invention comprise heavy chain variable regions derived from specific human germline heavy chain immunoglobulin genes and / or light chain variable regions derived from specific human germline light chain immunoglobulin genes. Antibody 15A6 has a heavy chain derived from human germline V4-39, D6-19, and JH4b and a light chain derived from germline VA27 and JK2. Antibody 22G2 has a heavy chain derived from human germline V4-61, D3-10, and JH6b and a light chain derived from germline VL6 and JK3. Antibody 11G11 has a heavy chain derived from human germline V4-39, D3-10, and JH4b and a light chain derived from germline VL6 and JK2. Antibody 10D7 has a heavy chain derived from human germline V1-69, D6-13, and JH6b and a light chain derived from germline VL15 and JK5. Other antibodies that bind to human TIGIT and are derived from some or all of these germline sequences are likely to be closely related in sequence, particularly those derived from the same V region genes, and therefore are predicted to share the same desirable properties.

[0155] As used herein, a human antibody comprises a heavy or light chain variable region "derived from" a particular germline sequence if the antibody variable region is obtained from a system that uses human germline immunoglobulin genes and is sufficiently related to that germline that the antibody sequence is more likely to be derived from that germline than any other. Such systems include immunizing transgenic mice carrying human immunoglobulin genes with the antigen of interest or screening a human immunoglobulin gene library displayed on phage with the antigen of interest. The human germline immunoglobulin sequence(s) from which the sequence of an antibody is "derived" can be identified by comparing the amino acid sequence of the human antibody with that of human germline immunoglobulins and selecting the human germline immunoglobulin sequence that is closest in sequence to the sequence of the human antibody (i.e., the highest percent identity). A human antibody "derived from" a particular human germline immunoglobulin sequence may contain amino acid differences when compared to the germline sequence, for example, due to naturally occurring somatic mutations or deliberately induced site-specific mutations. However, a selected human antibody will usually be at least 90% identical in amino acid sequence to the amino acid sequence encoded by a human germline immunoglobulin gene (e.g., the V region) and contain amino acid residues that identify the human antibody as human when compared to the germline immunoglobulin amino acid sequence of another species (e.g., a murine germline sequence). In certain cases, a human antibody may be at least 95%, or even at least 96%, 97%, 98%, or 99% identical in amino acid sequence to the amino acid sequence encoded by the germline immunoglobulin gene (e.g., the V region). Typically, a human antibody derived from a particular human germline sequence will display no more than 10 amino acid differences from the amino acid sequence encoded by the human germline immunoglobulin gene (e.g., the V region). In certain cases, a human antibody may display no more than 5, or even no more than 4, 3, 2, or 1 amino acid differences from the amino acid sequence encoded by the germline immunoglobulin gene (e.g., the V region).

[0156] II. Engineered and Modified Antibodies VH and VL regions To engineer modified antibodies, the V disclosed herein can be used as starting material. H and / or V L Also provided are engineered and modified antibodies that can be prepared using antibodies having one or more of the sequences, which modified antibodies can have altered properties from the starting antibody. Antibodies can have one or both variable regions (i.e., V H and / or V L ), e.g., by modifying one or more residues in one or more CDR regions and / or in one or more framework regions. Additionally or alternatively, antibodies can be engineered by modifying residues in the constant region(s), e.g., to alter the effector function(s) of the antibody.

[0157] One type of variable region genetic engineering that can be performed is CDR grafting. Such grafting is particularly useful in humanizing non-human anti-TIGIT antibodies that compete for binding with the anti-huTIGIT antibodies disclosed herein and / or bind to the same epitope as the anti-huTIGIT antibodies disclosed herein. Antibodies interact with target antigens mainly through amino acid residues located in six heavy and light chain complementarity-determining regions (CDRs). Therefore, the amino acid sequences within CDRs are more diverse between individual antibodies than the sequences outside the CDRs. Because CDR sequences are involved in most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of a particular reference antibody by constructing expression vectors containing CDR sequences from that particular reference antibody grafted onto framework sequences from a different antibody with different properties (see, e.g., Riechmann, L. et al. (1998) Nature 332:323-327; Jones, P. et al. (1986) Nature 321:522-525; Queen, C. et al. (1989) Proc. Natl. Acad. See. USA 86:10029-10033; Winter, U.S. Pat. No. 5,225,539, and Queen et al., U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762, and 6,180,370).

[0158] Such framework sequences can be obtained from public DNA databases or published references that contain germline antibody gene sequences. For example, germline DNA sequences of human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database, as well as in Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242; Tomlinson, IM, et al. (1992) "The Repertoire of Human Germline V H Sequences Reveals about Fifty Groups of V H Segments with Different Hypervariable Loops” J. Mol. Biol. 227:776-798; and Cox, JPL et al. (1994) “A Directory of Human Germ-line V H and “Segments Reveal a Strong Bias in Their Usage,” Eur. J. Immunol. 24:827-836, the contents of each of which are expressly incorporated herein by reference.

[0159] Preferred framework sequences for use in the antibodies described herein are those that are structurally similar to the framework sequences used by the antibodies described herein. H CDR1, 2 and 3 sequences and V LCDR1, 2, and 3 sequences can be grafted onto framework regions that have the same sequence as found in the germline immunoglobulin gene from which the framework sequences are derived, or the CDR sequences can be grafted onto framework regions that contain up to 20 preferably conservative amino acid substitutions compared to the germline sequences. For example, in certain cases, it has been found to be beneficial to mutate residues within framework regions to maintain or enhance the antigen-binding ability of antibodies (see, for example, U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,762, and 6,180,370 to Queen et al.).

[0160] The engineered antibodies described herein may be modified, for example, by modifying the V H and / or V L These include antibodies in which modifications have been made to framework residues within the framework region. Such framework modifications are often made to reduce the immunogenicity of the antibody. For example, one approach is to "backmutate" one or more framework residues to the corresponding germline sequence. More specifically, antibodies that have undergone somatic mutation may contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody framework sequence to the germline sequence from which the antibody is derived. To return the framework region sequences to their germline configuration, somatic mutations can be "backmutated" to the germline sequence, for example, by site-directed mutagenesis or PCR-mediated mutagenesis. Such "backmutated" antibodies are also intended to be encompassed.

[0161] Another type of framework modification involves mutating one or more residues within the framework regions, or even within one or more CDR regions, to remove T cell epitopes and thereby reduce the immunogenic potential of the antibody. This approach is also referred to as "deimmunization" and is described in further detail in U.S. Patent Publication No. 20030153043 by Carr et al.

[0162] Another type of variable region modification is to mutate amino acid residues in the CDR regions to improve one or more binding characteristics (e.g., affinity) of the antibody of interest. Site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce mutation(s) and effect on antibody binding or other functional properties of interest. Conservative modifications are preferably introduced. Mutations can be amino acid additions, deletions, or, preferably, substitutions. Furthermore, typically, no more than 1, 2, 3, 4, or 5 residues in the CDR regions are changed.

[0163] Methionine residues in the CDRs of antibodies can be oxidized, resulting in the potential for chemical degradation and a consequent reduction in antibody potency. Thus, anti-TIGIT antibodies having one or more methionine residues in the heavy and / or light chain CDRs replaced with amino acid residues that are not susceptible to oxidative degradation are also provided.

[0164] Similarly, deamidated sites may also be removed from anti-TIGIT antibodies, particularly in the CDRs.

[0165] To prevent glycosylation that may interfere with antigen binding, it is preferred to eliminate potential glycosylation sites within the antigen-binding domain. See, e.g., U.S. Patent No. 5,714,350.

[0166] Targeted antigen binding In various embodiments, the antibodies of the present invention are modified to selectively block antigen binding in tissues and environments where antigen binding is harmful, while allowing beneficial antigen binding. In one embodiment, a blocking peptide "mask" that specifically binds to the antigen-binding surface of the antibody and interferes with antigen binding is generated, and this mask is linked to each of the antibody's binding arms by a peptidase-cleavable linker. See, for example, U.S. Patent No. 8,518,404 to CytomX. Such constructs are useful for treating cancers in which protease levels are significantly increased in the tumor microenvironment compared to non-tumor tissues. Selective cleavage of the cleavable linker in the tumor microenvironment allows the masking / blocking peptide to be released, allowing antigen binding selectivity in the tumor rather than in peripheral tissues where antigen binding may cause unwanted side effects.

[0167] Alternatively, in a related embodiment, a bivalent binding compound ("masking ligand") comprising two antigen-binding domains is developed that binds to both antigen-binding surfaces of a (bivalent) antibody and interferes with antigen binding, in which the two binding domain masks are linked to each other (rather than to the antibody) by a cleavable linker, e.g., a linker cleavable by peptidase. See, for example, International Patent Application Publication WO 2010 / 077643 to Tegopharm Corp. The masking ligand may comprise or be derived from the antigen to which the antibody is intended to bind, or may be independently generated. Such masking ligands are useful for treating cancers in which protease levels are significantly increased in the tumor microenvironment compared to non-tumor tissues. Selective cleavage of the cleavable linker in the tumor microenvironment allows the two binding domains to dissociate from each other, reducing the avidity of the antigen-binding surface of the antibody. The resulting dissociation of the masking ligand from the antibody allows antigen binding selectivity in the tumor but not in peripheral tissues where antigen binding may cause unwanted side effects.

[0168] Fc and modified Fc In addition to the activity of therapeutic antibodies resulting from binding of the antigen-binding domain to the antigen (e.g., to a cognate ligand or receptor protein in the case of antagonist antibodies or blockade of induced signal transduction in the case of agonist antibodies), the Fc portion of the antibody interacts with the immune system as a whole in a complex manner to elicit any number of biological effects. Effector functions, such as the Fc region of immunoglobulins, are involved in many important antibody functions, such as antigen-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-dependent cell-mediated phagocytosis (ADCP), which result in target cell killing, albeit by different mechanisms. There are five major classes or isotypes of heavy chain constant regions (IgA, IgG, IgD, IgE, IgM), each with characteristic effector functions. These isotypes can be further subdivided into subclasses; for example, IgG is divided into four subclasses known as IgG1, IgG2, IgG3, and IgG4. IgG molecules interact with three classes of Fcγ receptors (FcγR), namely, FcγRI, FcγRII, and FcγRIII, which are specific for the IgG class of antibody. Sequences important for IgG binding to FcγR receptors are reported to be located in the CH2 and CH3 domains. The serum half-life of an antibody is influenced by its ability to bind to the neonatal Fc receptor (FcRn).

[0169] The antibodies of the present invention may comprise a variable domain of the present invention combined with a constant domain comprising a different Fc region selected based on the biological activity (if any) of the antibody for the intended use. Salfeld (2007) Nat. Biotechnol. 25:1369. For example, human IgG can be classified into four subclasses, IgG1, IgG2, IgG3, and IgG4, each of which contains an Fc region with a unique profile for binding to one or more Fcγ receptors (activating receptors FcγRI (CD64), FcγRIIA, FcγRIIC (CD32); FcγRIIIA and FcγRIIIB (CD16) and inhibitory receptor FcγRIIB) and the first component of complement (C1q). Human IgG1 and IgG3 bind to all Fcγ receptors; IgG2 binds to only FcγRIIA. H131 binds to FcγRIIA R131 FcγRIIIA V158 IgG4 has lower affinity for FcγRI, FcγRIIA, FcγRIIB, FcγRIIC, and FcγRIIIA V158 The inhibitory receptor FcγRIIB binds to IgG1, IgG2, and IgG3 with lower affinity than all other Fcγ receptors. Bruhns et al. (2009) Blood 113:3716. Studies have shown that FcγRI does not bind to IgG2, and FcγRIIIB does not bind to IgG2 or IgG4. Ibid. Generally, with regard to ADCC activity, human IgG1≧IgG3>>IgG4≧IgG2. As a result, for example, an IgG1 constant domain may be selected rather than IgG2 or IgG4 for use in drugs where ADCC is desired; IgG3 may be selected for activation of FcγRIIIA-expressing NK cells, macrophages, and monocytes; and IgG4 may be selected if the antibody is to be used to desensitize allergy patients. IgG4 may also be selected when it is desired that the antibody lack all effector functions.

[0170] The anti-TIGIT variable regions described herein are Fc, e.g., IgG1, IgG2, IgG3, or IgG4. It may be linked (e.g., covalently linked or fused) to Fc, which may be, for example, any allotype or isoallotype of IgG1: G1m, G1m1(a), G1m2(x), G1m3(f), G1m17(z); any allotype or isoallotype of IgG2: G2m, G2m23(n); any allotype or isoallotype of IgG3: G3m, G3m21(g1), G3m28(g5), G3m11(b0), G3m5(b1), G3m13(b3), G3m14(b4), G3m10(b5), G3m15(s), G3m16(t), G3m6(c3), G3m24(c5), G3m26(u), G3m27(v). See, e.g., Jefferis et al. (2009) mAbs 1:1. The choice of allotype may be influenced by potential immunogenicity concerns, e.g., to minimize the formation of anti-drug antibodies.

[0171] In certain embodiments, the anti-TIGIT variable regions described herein are linked to an Fc that binds one or more activating Fc receptors (FcγRI / CD64, FcγRIIa / CD32, or FcγRIIIa / CD16), thereby stimulating ADCC and causing T cell depletion. In certain embodiments, the anti-TIGIT variable regions described herein are linked to a human IgG1 or IgG3 Fc, i.e., the antibody is of the IgG1 or IgG3 isotype. In certain embodiments, the anti-TIGIT antibodies are depleting antibodies, particularly because they are capable of inhibiting T cell proliferation in the tumor microenvironment. reg Deplete cells and T eff cells (thereby enhancing antitumor activity), but outside the tumor microenvironment, e.g., the periphery, reg and T eff In certain embodiments, the anti-TIGIT antibody does not significantly deplete T cells at the tumor site. reg Cell depletion or elimination and T effIn certain embodiments, the anti-TIGIT antibody is of either a naturally occurring or non-naturally occurring isotype (e.g., containing a mutation(s)) that stimulates the coactivation of T cells at the tumor site. eff Against T reg This results in an increase in the T ratio, which indicates potent antitumor activity, preferably outside the tumor microenvironment, e.g., in the periphery. reg and T eff Does not significantly deplete cells.

[0172] In other embodiments, the anti-TIGIT antibody is reg In certain embodiments, the anti-TIGIT antibody has an Fc with reduced or eliminated FcR binding, e.g., reduced binding to activating FcRs.

[0173] The anti-TIGIT variable regions described herein are useful for, for example, inhibiting T cell proliferation in a tumor environment. reg To enhance depletion, a non-naturally occurring Fc region can be linked, e.g., an effectorless or mostly effectorless Fc (e.g., human IgG2 or IgG4), or an Fc with enhanced binding to one or more activating Fc receptors (FcγRI, FcγRIIa, or FcγRIIIa).

[0174] The variable regions described herein may be linked to an Fc region that includes one or more modifications, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cellular cytotoxicity. Additionally, the antibodies described herein may be chemically modified (e.g., one or more chemical moieties may be attached to the antibody) or modified to alter its glycosylation, thereby altering one or more functional properties of the antibody. Each of these embodiments is described in detail below. The numbering of residues in the Fc region is that of Kabat's EU index. The sequence variants disclosed herein are provided with the residue number followed by the amino acid associated with the naturally occurring amino acid substituted for it, optionally preceded by the naturally occurring residue at that position. When multiple amino acids can be present at a given position, e.g., when sequences differ between naturally occurring isotypes, or when multiple mutations can be substituted at that position, they are separated by / (e.g., "X / Y / Z").

[0175] For example, modifications may be made to the Fc region to create Fc variants that have (a) increased or decreased antibody-dependent cell-mediated cytotoxicity (ADCC), (b) increased or decreased complement-mediated cytotoxicity (CDC), (c) increased or decreased affinity for C1q, and / or (d) increased or decreased affinity for Fc receptors relative to the parent Fc. Such Fc region variants generally contain at least one amino acid modification in the Fc region. Combinations of amino acid modifications may be particularly desirable. For example, a variant Fc region may contain substitutions of two, three, four, five, etc., at the specific Fc region positions identified herein. Exemplary Fc sequence variant pairs are disclosed herein and are also provided in U.S. Patent Nos. 5,624,821, 6,277,375, 6,737,056, 6,194,551, 7,317,091, 8,101,720, PCT Patent Publications WO00 / 42072, WO01 / 58957, WO04 / 016750, WO04 / 029207, WO04 / 035752, WO04 / 074455, WO04 / 099249, WO04 / 063351, WO05 / 070963, WO05 / 040217, WO05 / 092925 and WO06 / 020114.

[0176] Reducing effector function ADCC activity can be reduced by modifying the Fc region. In certain embodiments, sites affecting Fc receptor binding, preferably sites other than the salvage receptor binding site, can be removed. In other embodiments, the Fc region can be modified to remove the ADCC site. ADCC sites are known in the art; for example, see Sarmay et al. (1992) Molec. Immunol. 29 (5): 633-9 for the ADCC site in IgG1. In one embodiment, the G236R and L328R mutants of human IgG1 effectively eliminate FcγR binding. Horton et al. (2011) J. Immunol. 186:4223 and Chu et al. (2008) Mol. Immunol. 45:3926. In other embodiments, an Fc with reduced FcγR binding contained the amino acid substitutions L234A, L235E, and G237A. Gross et al. (2001) Immunity 15:289.

[0177] CDC activity can also be reduced by modifying the Fc region. Mutations at IgG1 positions D270, K322, P329, and P331, specifically alanine mutations D270A, K322A, P329A, and P331A, significantly reduce the ability of the corresponding antibody to bind C1q and activate complement. Idusogie et al. (2000) J. Immunol. 164:4178; WO99 / 51642. Modifications at position 331 of IgG1 (e.g., P331S) have been shown to reduce complement binding. Tao et al. (1993) J. Exp. Med. 178:661 and Canfield & Morrison (1991) J. Exp. Med. 173:1483. In another example, one or more amino acid residues within amino acid positions 231-239 are altered to thereby reduce the ability of the antibody to fix complement. WO94 / 29351.

[0178] In some embodiments, the Fc with reduced complement binding has the amino acid substitutions A330S and P331S. Gross et al. (2001) Immunity 15:289.

[0179] For uses where effector functions are to be avoided entirely, e.g., where antigen binding alone is sufficient to produce the desired therapeutic benefit and where effector functions only lead to (increase the risk of) undesirable side effects, IgG4 antibodies may be used, or antibodies or fragments lacking the Fc region or a substantial portion thereof can be devised, or the Fc may be mutated to eliminate glycosylation entirely (e.g., N297A). Alternatively, hybrid constructs of human IgG2 (C) that lack effector functions, lack the ability to bind FcγR (like IgG2), and are unable to activate complement (like IgG4) may be used. H 1 domain and hinge region) and human IgG4 (C H 2 and C H Fc modifications to reduce effector function have been developed. Rother et al. (2007) Nat. Biotechnol. 25:1256. See also Mueller et al. (1997) Mol. Immunol. 34:441; Labrijn et al. (2008) Curr. Op. Immunol. 20:479 (discussing Fc modifications to generally reduce effector function).

[0180] In other embodiments, the Fc region is altered by substituting at least one amino acid residue with a different amino acid residue to reduce all effector functions of the antibody. For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320, and 322 can be substituted with a different amino acid residue so that the antibody has reduced affinity for an effector ligand but retains the antigen-binding ability of the parent antibody. The effector ligand for which affinity is altered can be, for example, an Fc receptor (residues 234, 235, 236, 237, 297) or the C1 component of complement (residues 297, 318, 320, 322). See U.S. Patent Nos. 5,624,821 and 5,648,260, both by Winter et al.

[0181] One early patent application proposed modifications in the IgG Fc region to reduce binding to FcγRI and reduce ADCC (234A; 235E; 236A; G237A) or to block binding to complement component C1q and eliminate CDC (E318A or V / K320A and K322A / Q). WO 88 / 007089. See also Duncan & Winter (1988) Nature 332:563; Chappel et al. (1991) Proc. Nat'l Acad. Sci. (USA) 88:9036; and Sondermann et al. (2000) Nature 406:267 (discussing the effect of these mutations on FcγRIII binding).

[0182] Fc modifications that reduce effector function also include substitutions, insertions and deletions at positions 234, 235, 236, 237, 267, 269, 325 and 328, e.g., 234G, 235G, 236R, 237K, 267R, 269R, 325L and 328R. An Fc variant may include 236R / 328R. Other modifications to reduce FcyR and complement interactions include substitutions 297A, 234A, 235A, 237A, 318A, 228P, 236E, 268Q, 309L, 330S, 331S, 220S, 226S, 229S, 238S, 233P and 234V. These and other modifications are reviewed in Strohl (2009) Current Opinion in Biotechnology 20:685-691. By mutating IgG residues at one or more of positions 233-236 and 327-331, such as E233P, L234V, L235A, optionally G236Δ, A327G, A330S, and P331S in IgG1; E233P, F234V, L235A, optionally G236Δ; and A330S and P331S in IgG2, effector function (both ADCC and complement activation) can be reduced while maintaining neonatal FcR binding (while maintaining half-life). See Armour et al. (1999) Eur. J. Immunol. 29:2613; WO99 / 58572. Other mutations that reduce effector function include L234A and L235A in IgG1 (Alegre et al. (1994) Transplantation 57:1537); V234A and G237A in IgG2 (Cole et al. (1997) J. Immunol. 159:3613; see also U.S. Pat. No. 5,834,597); and S228P and L235E in IgG4 (Reddy et al. (2000) J. Immunol. 164:1925). Another combination of mutations to reduce effector function in human IgG1 includes L234F, L235E, and P331S.Oganesyan et al. (2008) Acta Crystallogr. D. Biol. Crystallogr. 64:700. See generally, Labrijn et al. (2008) Curr. Op. Immunol. 20:479. Additional mutations known to reduce effector function in the context of Fc(IgG1) fusion proteins (abatacept) include C226S, C229S, and P238S (EU residue numbering). Davis et al. (2007) J. Immunol. 34:2204.

[0183] Other Fc mutants that reduced ADCC and / or CDC are described in Glaesner et al. (2010) Diabetes Metab. Res. Rev. 26:287 (F234A and L235A to reduce ADCC and ADCP in IgG4); Hutchins et al. (1995) Proc. Nat'l Acad. Sci. (USA) 92:11980 (F234A, G237A, and E318A in IgG4); An et al. (2009) MAbs 1:572 and U.S. Patent Application Publication No. 2007 / 0148167 (H268Q, V309L, A330S, and P331S in IgG2); McEarchern et al. (2007) Blood 109:1185 (C226S, C229S, E233P, L234V, L235A in IgG1); Vafa et al. (2014) Methods 65:114 (V234V, G237A, P238S, H268A, V309L, A330S, P331S in IgG2).

[0184] In certain embodiments, an Fc that has essentially no effector function, i.e., reduced FcγR binding and reduced complement binding, is selected. An exemplary effector-less Fc, for example, an IgG1 Fc, contains the following five mutations: L234A, L235E, G237A, A330S, and P331S. Gross et al. (2001) Immunity 15:289. These five substitutions can also be combined with N297A to eliminate glycosylation.

[0185] Enhancement of effector function Alternatively, ADCC activity can be increased by modifying the Fc region: with respect to ADCC activity, human IgG1≧IgG3>>IgG4≧IgG2, and therefore, for use in drugs where ADCC is desired, IgG1 constant domains may be selected rather than IgG2 or IgG4. Alternatively, the Fc region may comprise any of the following positions: 234, 235, 236, 238, 239, 240, 241, 243, 244, 245, 247, 248, 249, 252, 254, 255, 256, 258, 262, 263, 264, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 299, 301, 303, 305, 307, 309, 312, 313, 315, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 3 The polypeptides may be modified to enhance antibody-dependent cellular cytotoxicity (ADCC) and / or to enhance affinity for Fcγ receptors by modifying one or more amino acids at positions 22, 324, 325, 326, 327, 329, 330, 331, 332, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, ​​388, 389, 398, 414, 416, 419, 430, 433, 434, 435, 436, 437, 438, or 439. See WO2012 / 142515; see also WO00 / 42072. Exemplary substitutions include 236A, 239D, 239E, 268D, 267E, 268E, 268F, 324T, 332D and 332E. Exemplary variants include 239D-332E, 236A-332E, 236A-239D-332E, 268F-324T, 267E-268F, 267E-324T and 267E-268F-324T. For example, human IgG1Fc containing a G236A variant, optionally combined with I332E, has been found to increase the FcγRIIA / FcγRIIB binding affinity ratio by approximately 15-fold. Richards et al. (2008) Mol. Cancer Therap. 7:2517; Moore et al. (2010) mAbs 2:181.Other modifications for enhancing FcyR and complement interactions include, but are not limited to, substitutions 298A, 333A, 334A, 326A, 247I, 339D, 339Q, 280H, 290S, 298D, 298V, 243L, 292P, 300L, 396L, 305I, and 396L. These and other modifications are reviewed in Strohl (2009) Current Opinion in Biotechnology 20:685-691. Specifically, both ADCC and CDC can be enhanced by altering position E333 of IgG1, e.g., E333A. Shields et al. (2001) J. Biol. Chem. 276:6591. The use of P247I and A339D / Q mutations to enhance effector function in IgG1 is disclosed in WO2006 / 020114, and D280H, K290S±S298D / V is disclosed in WO2004 / 074455. K326A / W and E333A / S mutants in human IgG1 and E333S in IgG2 have been shown to increase effector function. Idusogie et al. (2001) J. Immunol. 166:2571.

[0186] Specifically, the binding sites on human IgG1 for FcγR1, FcγRII, FcγRIII, and FcRn have been mapped, and mutants with improved binding have been described. Shields et al. (2001) J. Biol. Chem. 276:6591-6604. Specific mutations at positions 256, 290, 298, 333, 334, and 339 have been shown to improve binding to FcγRIII, including the combination mutants T256A-S298A, S298A-E333A, S298A-K224A, and S298A-E333A-K334A (which have enhanced FcγRIIIa binding and ADCC activity). Other IgG1 mutants with strongly enhanced binding to FcγRIIIa have been identified, including mutants with S239D-I332E and S239D-I332E-A330L mutations, which showed the greatest increase in affinity for FcγRIIIa, reduced FcγRIIb binding, and potent cytotoxic activity in cynomolgus monkeys. Lazar et al. (2006) Proc. Nat'l Acad. Sci. (USA) 103:4005; Awan et al. (2010) Blood 115:1204; Desjarlais & Lazar (2011) Exp. Cell Res. 317:1278. Introduction of triple mutations into antibodies such as alemtuzumab (CD52-specific), trastuzumab (HER2 / neu-specific), rituximab (CD20-specific), and cetuximab (EGFR-specific) translated into greatly enhanced ADCC activity in vitro, and the S239D-I332E mutant showed an enhanced ability to deplete B cells in monkeys. (Lazar et al. (2006) Proc. Nat'l Acad. Sci. (USA) 103:4005.) Furthermore, in transgenic mice expressing human FcγRIIIa in models of B cell malignancies and breast cancer, an IgG1 mutant containing the L235V, F243L, R292P, Y300L, V305I, and P396L mutations was identified that showed enhanced binding to FcγRIIIa and concomitant enhanced ADCC activity.Stavenhagen et al. (2007) Cancer Res. 67:8882; U.S. Patent No. 8,652,466; Nordstrom et al. (2011) Breast Cancer Res. 13:R123.

[0187] Various IgG isotypes also exhibit differential CDC activity (IgG3>IgG1>>IgG2≒IgG4). Dangl et al. (1988) EMBO J. 7:1989. For uses where enhanced CDC is desired, mutations that increase C1q binding can be introduced. The ability to recruit complement (CDC) can be enhanced by mutations at K326 and / or E333 in IgG2, such as K326W (reducing ADCC activity) and E333S, to increase binding to C1q, the first component of the complement cascade. Idusogie et al. (2001) J. Immunol. 166:2571. Introduction of S267E / H268F / S324T (alone or in any combination) into human IgG1 enhances C1q binding. Moore et al. (2010) mAbs 2:181. The Fc region "113F" of the IgG1 / IgG3 hybrid isotype antibody of Natsume et al. (2008) Cancer Res. 68:3863 (Figure 1 therein) confers enhanced CDC. See also Michaelsen et al. (2009) Scand. J. Immunol. 70:553 and Redpath et al. (1998) Immunology 93:595.

[0188] Additional mutations that can increase or decrease effector function are disclosed in Dall'Acqua et al. (2006) J. Immunol. 177: 1129. See also Carter (2006) Nat. Rev. Immunol. 6:343; Presta (2008) Curr. Op. Immunol. 20:460.

[0189] Although not necessarily related to the antagonist anti-TIGIT mAbs of the present invention, Fc variants that enhance affinity for the inhibitory receptor FcyRIIb may enhance apoptosis induction or adjuvant activity. Li & Ravetch (2011) Science 333:1030; Li & Ravetch (2012) Proc. Nat'l Acad. Sci. (USA) 109:10966; U.S. Patent Application Publication No. 2014 / 0010812. Such variants may enhance affinity for FcyRIIb receptors, including, for example, B cells and monocytes. +This may provide an antibody with immunomodulatory activity associated with cells. In one embodiment, the Fc variants provide selectively enhanced affinity for FcyRIIb relative to one or more activating receptors. Modifications to alter binding to FcyRIIb include one or more modifications at positions selected from the group consisting of 234, 235, 236, 237, 239, 266, 267, 268, 325, 326, 327, 328, and 332, according to the EU index. Exemplary substitutions for enhancing FcyRIIb affinity include, but are not limited to, 234D, 234E, 234F, 234W, 235D, 235F, 235R, 235Y, 236D, 236N, 237D, 237N, 239D, 239E, 266M, 267D, 267E, 268D, 268E, 327D, 327E, 328F, 328W, 328Y, and 332E. Exemplary substitutions include 235Y, 236D, 239D, 266M, 267E, 268D, 268E, 328F, 328W, and 328Y. Other Fc mutants for enhancing binding to FcyRIIb include 235Y-267E, 236D-267E, 239D-268D, 239D-267E, 267E-268D, 267E-268E, and 267E-328F. Specifically, S267E, G236D, S239D, L328F, and I332E mutants, including the S267E-L328F double mutant of human IgG1, are particularly valuable in enhancing affinity for the inhibitory FcyRIIb receptor. Chu et al. (2008) Mol. Immunol. 45:3926; U.S. Patent Application Publication No. 2006 / 024298; WO2012 / 087928. Enhanced specificity for FcyRIIb (FcyRIIa) R131 (as distinguished from) can be obtained by adding a P238D substitution and other mutations (Mimoto et al. (2013) Protein. Eng. Des. & Selection 26:589; WO2012 / 115241) and V262E and V264E (Yu et al. (2013) J. Am. Chem. Soc. 135:9723 and WO2014 / 184545).

[0190] Half-life extension In certain embodiments, an antibody is modified to increase its biological half-life. Various approaches are possible. For example, this can be achieved by increasing the binding affinity of the Fc region to FcRn. In one embodiment, the antibody is altered in the CH1 or CL region to contain a salvage receptor binding epitope taken from two loops of the CH2 domain of the Fc region of IgG, as described by Presta et al. in U.S. Pat. Nos. 5,869,046 and 6,121,022. Other exemplary Fc variants that increase binding to FcRn and / or improve pharmacokinetic properties contain substitutions at positions 259, 308, and 434, including, for example, 259I, 308F, 428L, 428M, 434S, 434H, 434F, 434Y, and 434M. Other mutations that enhance Fc binding to FcRn include 250E, 250Q, 428L, 428F, 250Q / 428L (Hinton et al., 2004, J. Biol. Chem. 279(8): 6213-6216, Hinton et al. 2006 Journal of Immunology 176:346-356), 256A, 272A, 305A, 307A, 311A, 312A, 378Q, 380A, 382A, and 434A (Shields et al., Journal of Biological Chemistry, 2001). 276(9):6591-6604), 252F, 252Y, 252W, 254T, 256Q, 256E, 256D, 433R, 434F, 434Y, 252Y / 254T / 256E, 433K / 434F / 436H (Dall'Acqua et al. Journal of Immunology, 2002, 169:5171-5180, Dall'Acqua et al., 2006, Journal of Biological Chemistry 281:23514-23524). See U.S. Patent No. 8,367,805.

[0191] Modifications of certain conserved residues in IgG Fc (I253, H310, Q311, H433, N434), such as the N434A mutant (Yeung et al. (2009) J. Immunol. 182:7663), have been proposed as a way to increase FcRn affinity and therefore increase the half-life of antibodies in circulation. WO98 / 023289. A combined Fc mutant containing M428L and N434S has been shown to increase FcRn binding and increase serum half-life by up to five-fold. Zalevsky et al. (2010) Nat. Biotechnol. 28:157. A combined Fc mutant containing T307A, E380A, and N434A modifications also extends the half-life of IgG1 antibodies. Petkova et al. (2006) Int. Immunol. 18:1759. Furthermore, combination Fc mutants including the M252Y-M428L, M428L-N434H, M428L-N434F, M428L-N434Y, M428L-N434A, M428L-N434M and M428L-N434S mutants have also been shown to extend half-life. WO2009 / 086320

[0192] Furthermore, a combined Fc mutant containing M252Y, S254T, and T256E increases half-life by nearly fourfold. (2006) J. Biol. Chem. 281:23514. A related IgG1 modification (M252Y-S254T-T256E-H433K-N434F) that provides increased FcRn affinity but reduced pH dependence was used to generate an IgG1 construct ("MST-HN Abdeg") for use as a competitor to prevent other antibodies from binding to FcRn, thereby increasing the clearance of either endogenous IgG (e.g., in autoimmune settings) or another exogenous (therapeutic) mAb. (2005) Nat. Biotechnol. 23:1283; WO2006 / 130834.

[0193] Other modifications to increase FcRn binding are described in Yeung et al. (2010) J. Immunol. 182:7663-7671; No. 6,277,375; No. 6,821,505; WO97 / 34631; WO2002 / 060919.

[0194] In certain embodiments, hybrid IgG isotypes can be used to increase FcRn binding and potentially increase half-life. For example, IgG1 / IgG3 hybrid mutants can be constructed by substituting IgG1 positions in the CH2 and / or CH3 regions with amino acids derived from IgG3 at positions where the two isotypes differ. In this way, hybrid mutant IgG antibodies containing one or more substitutions, e.g., 274Q, 276K, 300F, 339T, 356E, 358M, 384S, 392N, 397M, 422I, 435R, and 436F, can be constructed. In other embodiments described herein, IgG1 / IgG2 hybrid mutants can be constructed by substituting IgG2 positions in the CH2 and / or CH3 regions with amino acids derived from IgG1 at positions where the two isotypes differ. In this way, hybrid mutant IgG antibodies can be constructed that contain one or more substitutions, for example, one or more of the following amino acid substitutions: 233E, 234L, 235L, -236G (referring to the insertion of glycine at position 236), and 327A. See U.S. Patent No. 8,629,113. Hybrids of IgG1 / IgG2 / IgG4 sequences have been created that are said to increase serum half-life and improve expression. U.S. Patent No. 7,867,491 (SEQ ID NO: 18 therein).

[0195] The serum half-life of antibodies of the present invention can also be increased by pegylation. Antibodies may be pegylated, for example, to increase the biological (e.g., serum) half-life of the antibody. To pegylate an antibody, typically, the antibody or fragment thereof is reacted with a polyethylene glycol (PEG) reagent, such as a reactive ester or aldehyde derivative of PEG, under conditions such that one or more PEG groups become attached to the antibody or antibody fragment. Preferably, pegylation is carried out by an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass any form of PEG used to derivatize other proteins, such as mono(C1-C10)alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certain embodiments, the antibody to be pegylated is an unglycosylated antibody. Methods for pegylating proteins are known in the art and can be applied to the antibodies described herein. See, for example, EP 0154316 by Nishimura et al. and EP 0401384 by Ishikawa et al.

[0196] Alternatively, in some situations, it may be desirable to decrease, rather than increase, the half-life of the antibodies of the present invention. Modifications such as I253A (Hornick et al. (2000) J. Nucl. Med. 41:355) and H435A / R, I253A, or H310A (Kim et al. (2000) Eur. J. Immunol. 29:2819) in the Fc of human IgG1 can decrease FcRn binding and thus decrease half-life (increase clearance) for use in situations where rapid clearance is preferable, such as medical imaging. See also Kenanova et al. (2005) Cancer Res. 65:622. Other means for enhancing clearance include formatting the antigen-binding domain of the present invention as an antibody fragment, such as a Fab fragment, that lacks the ability to bind FcRn. Such modifications can decrease the circulating half-life of the antibody from a few weeks to several hours. Selective PEGylation of the antibody fragment can then be used to fine-tune (increase) the half-life of the antibody fragment as needed. Chapman et al. (1999) Nat. Biotechnol. 17:780. To increase half-life, the antibody fragment can also be fused with human serum albumin, for example, to form a fusion protein construct. Yeh et al. (1992) Proc. Nat'l Acad. Sci. 89:1904. Alternatively, a bispecific antibody can be constructed using the first and second antigen-binding domains of the present invention that bind to human serum albumin (HSA). See International Patent Application Publication WO2009 / 127691 and the patent references cited therein. Alternatively, to increase half-life, specialized polypeptide sequences, such as "XTEN" polypeptide sequences, can be added to the antibody fragment. Schellenberger et al. (2009) Nat. Biotechnol. 27:1186; International Patent Application Publication No. WO2010 / 091122.

[0197] Further Fc variants When an IgG4 constant domain is used, it is usually preferred to include the substitution S228P, which mimics the hinge sequence in IgG1, thereby stabilizing the IgG4 molecule while reducing, for example, Fab arm exchange between the therapeutic antibody and endogenous IgG4 in the patient being treated. Labrijn et al. (2009) Nat. Biotechnol. 27:767; Reddy et al. (2000) J. Immunol. 164:1925.

[0198] Potential protease cleavage sites in the hinge of the IgG1 construct can be eliminated by D221G and K222S modifications, increasing antibody stability. WO2014 / 043344.

[0199] The affinity and binding properties of an Fc variant for its ligand (Fc receptor) may be determined by a variety of in vitro assay methods (biochemical or immunological-based assays) known in the art, including, but not limited to, equilibrium methods (e.g., enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA)) or kinetics (e.g., BIACORE® SPR analysis) and other methods such as indirect binding assays, competitive inhibition assays, fluorescence resonance energy transfer (FRET), gel electrophoresis, and chromatography (e.g., gel filtration). These and other methods may utilize a label on one or more components being examined and / or may use a variety of detection methods, including, but not limited to, chromatographic, fluorescent, luminescent, or isotopic labeling. A detailed description of binding affinity and kinetics, focusing on antibody-immunogen interactions, can be found in Paul, W. E., ed., Fundamental Immunology, 4th Ed., Lippincott-Raven, Philadelphia (1999).

[0200] In yet other embodiments, the glycosylation of an antibody is modified to increase or decrease effector function. For example, aglycosylated antibodies lacking all effector function can be generated by mutating the conserved asparagine residue at position 297 (e.g., N297A), thus abolishing complement and FcγRI binding. Bolt et al. (1993) Eur. J. Immunol. 23:403. See also Tao & Morrison (1989) J. Immunol. 143:2595 (using N297Q in IgG1 to eliminate glycosylation at position 297).

[0201] Although aglycosylated antibodies generally lack effector function, mutations can be introduced to restore this function. Aglycosylated antibodies, such as those resulting from N297A / C / D / or H mutations or those produced in a system that does not glycosylate proteins (e.g., E. coli), can be further mutated to restore FcγR binding, for example, S298G and / or T299A / G / or H (WO2009 / 079242) or E382V and M428I (Jung et al. (2010) Proc. Nat'l Acad. Sci. (USA) 107:604).

[0202] Furthermore, altering glycosylation can produce antibodies with enhanced ADCC. For example, removal of fucose from the heavy chain Asn297-linked oligosaccharide has been shown to enhance ADCC due to improved binding to FcγRIIIa. Shields et al. (2002) JBC 277:26733; Niwa et al. (2005) J. Immunol. Methods 306:151; Cardarelli et al. (2009) Clin. Cancer Res.15:3376 (MDX-1401); Cardarelli et al. (2010) Cancer Immunol. Immunotherap. 59:257 (MDX-1342). Such low-fucose antibodies can be produced, for example, in knockout Chinese hamster ovary (CHO) cells lacking fucosyltransferase (FUT8) (Yamane-Ohnuki et al. (2004) Biotechnol. Bioeng. 87:614), or in other cells that produce non-fucosylated antibodies. See, for example, Zhang et al. (2011) mAbs 3:289 and Li et al. (2006) Nat. Biotechnol. 24:210 (both describing antibody production in glycoengineered Pichia pastoris); Mossner et al. (2010) Blood 115:4393; Shields et al. (2002) J. Biol. Chem. 277:26733; Shinkawa et al. (2003) J. Biol. Chem. 278:3466; EP1176195B1. ADCC can also be enhanced as described in PCT Publication WO 03 / 035835, which discloses the use of a mutant CHO cell line, Lec13, that has a reduced ability of fucose to bind to Asn(297)-linked carbohydrate, also resulting in hypofucosylation of antibodies expressed in the host cells. See also Shields, RL et al. (2002) J. Biol. Chem. 277:26733-26740.Alternatively, fucose analogs may be added to the culture medium during antibody production to inhibit incorporation of fucose into the carbohydrate on the antibody. WO2009 / 135181.

[0203] Increasing the bisecting GlcNac structure in antibody-linked oligosaccharides also enhances ADCC. PCT Publication WO 99 / 54342 by Umana et al. describes cell lines engineered to express a glycoprotein-modifying glycosyltransferase (e.g., β(1,4)-N-acetylglucosaminyltransferase III (GnTIII)), such that antibodies expressed in the engineered cell lines exhibit increased bisecting GlcNac structures, which results in increased ADCC activity of the antibody (see also Umana et al. (1999) Nat. Biotech. 17:176-180).

[0204] Additional glycosylation variants lacking galactose, sialic acid, fucose, and xylose residues (so-called GNGN glycoforms) have been developed that exhibit enhanced ADCC and ADCP but reduced CDC, and others lacking sialic acid, fucose, and xylose (so-called G1 / G2 glycoforms) that exhibit enhanced ADCC, ADCP, and CDC. U.S. Patent Application Publication No. 2013 / 0149300. Antibodies with these glycosylation patterns are optionally produced in genetically modified N. benthamiana plants in which the endogenous xylosyl- and fucosyltransferase genes have been knocked out.

[0205] Glycoengineering can also be used to modify the anti-inflammatory properties of IgG constructs by altering the α2,6 sialylated content of the carbohydrate chain attached at Asn297 of the Fc region, where increasing the proportion of α2,6 sialylated forms results in enhanced anti-inflammatory effects. See Nimmerjahn et al. (2008) Ann. Rev. Immunol. 26:513. Conversely, decreasing the proportion of antibodies with α2,6 sialylated carbohydrates can be useful when anti-inflammatory properties are not desired. For example, methods for modifying the α2,6 sialylated content of antibodies by selective purification of the α2,6 sialylated form or by enzymatic modification are provided in U.S. Patent Application Publication No. 2008 / 0206246. In other embodiments, the amino acid sequence of the Fc region can be modified to mimic the effects of α2,6 sialylation, for example, by including an F241A modification. WO2013 / 095966.

[0206] III. Antibody physical properties The antibodies described herein may contain one or more glycosylation sites in either the light chain or heavy chain variable region. Such glycosylation sites may result in an increase in the immunogenicity of the antibody or a change in the pK of the antibody, resulting in altered antigen binding (Marshall et al. (1972) Ann. Rev. Biochem. 41:673-702; Gala and Morrison (2004) J. Immunol. 172:5489-94; Wallick et al. (1988) J. Exp. Med. 168:1099-109; Spiro (2002) Glycobiology 12:43R-56R; Parekh et al. (1985) Nature 316:452-7; Mimura et al. (2000) Mol. Immunol. 37:697-706). Glycosylation is known to occur at motifs containing NXS / T sequences. In some cases, it is preferable to have an anti-TIGIT antibody that does not contain variable region glycosylation. This can be achieved by selecting an antibody that does not contain glycosylation motifs in the variable region or by mutating residues within the glycosylated region.

[0207] In certain embodiments, the antibodies described herein do not contain asparagine isomerism sites. Deamidation of asparagine can occur at NG or DG sequences, resulting in the generation of isoaspartic acid residues that introduce a kink into the polypeptide chain and reduce its stability (the isoaspartic acid effect).

[0208] Each antibody has a unique isoelectric point (pI), which generally falls within the pH range of 6 to 9.5. The pI of IgG1 antibodies typically falls within the pH range of 7 to 9.5, and the pI of IgG4 antibodies typically falls within the pH range of 6 to 8. There is speculation that antibodies with pIs outside the normal range may have some unfolding and instability under in vivo conditions. Therefore, it is preferable to have an anti-TIGIT antibody containing a pI value that falls within the normal range. This can be achieved by selecting an antibody with a pI in the normal range or by mutating charged surface residues.

[0209] Each antibody has a characteristic melting temperature, with a higher melting temperature indicating greater overall stability in vivo (Krishnamurthy R and Manning MC (2002) Curr Pharm Biotechnol 3:361-71). M1 It is preferred that the initial unfolding temperature is above 60° C., preferably above 65° C., and even more preferably above 70° C. The melting point of an antibody can be measured using differential scanning calorimetry (Chen et al (2003) Pharm Res 20:1952-60; Ghirlando et al. (1999) Immunol Lett. 68:47-52) or circular dichroism (Murray et al. (2002) J. Chromatogr. Sci. 40:343-9).

[0210] In a preferred embodiment, antibodies are selected that do not rapidly degrade. Antibody degradation can be measured using capillary electrophoresis (CE) and MALDI-MS (Alexander AJ and Hughes DE (1995) Anal Chem. 67:3626-32).

[0211] In another preferred embodiment, antibodies are selected that have minimal aggregation effects, which can lead to the induction of unwanted immune responses and / or altered or unfavorable pharmacokinetic properties. Generally, antibodies are acceptable for aggregation of 25% or less, preferably 20% or less, even more preferably 15% or less, even more preferably 10% or less, and even more preferably 5% or less. Aggregation can be measured by several techniques, including size exclusion column (SEC), high performance liquid chromatography (HPLC), and light scattering.

[0212] IV. Nucleic acid molecules Another aspect described herein pertains to nucleic acid molecules encoding the antibodies described herein. The nucleic acid may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form when purified from other cellular components or other contaminants, e.g., other cellular nucleic acids (e.g., other chromosomal DNA, e.g., chromosomal DNA naturally associated with the isolated DNA) or proteins, by standard techniques, including alkaline / SDS treatment, CsCl banding, column chromatography, restriction enzymes, agarose gel electrophoresis, and others known in the art. A nucleic acid may be "isolated" or "substantially purified." See F. Ausubel, et al., ed. (1987) Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York. The nucleic acids described herein may be, for example, DNA or RNA, and may or may not contain intronic sequences. In certain embodiments, the nucleic acid is a cDNA molecule.

[0213] The nucleic acids described herein can be obtained using standard molecular biology techniques. For antibodies expressed by hybridomas (e.g., hybridomas prepared from transgenic mice carrying human immunoglobulin genes as described further below), cDNAs encoding the light and heavy chains of the antibodies produced by the hybridomas can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from an immunoglobulin gene library (e.g., using phage display technology), nucleic acids encoding the antibodies can be recovered from the library.

[0214] Once the DNA fragments encoding the VH and VL segments are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques to, for example, convert the variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. In these manipulations, the VL- or VH-encoding DNA fragment is operably linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. As used in this context, the term "operably linked" shall mean that the two DNA fragments are joined such that the amino acid sequences encoded by the two DNA fragments remain in-frame.

[0215] The isolated DNA encoding the VH region can be converted into a full-length heavy chain gene by operably linking the VH-encoding DNA to another DNA molecule encoding a heavy chain constant region (hinge, CH1, CH2, and / or CH3). The sequences of human heavy chain constant region genes are known in the art (see, e.g., Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region, e.g., an IgG1 region. For a Fab fragment heavy chain gene, the VH-encoding DNA can be operably linked to another DNA molecule encoding only the heavy chain CH1 constant region.

[0216] The isolated DNA encoding the VL region can be converted into a full-length light chain gene (as well as a Fab light chain gene) by operably linking the VL-encoding DNA to another DNA molecule encoding a light chain constant region, CL. The sequences of human light chain constant region genes are known in the art (see, e.g., Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region.

[0217] To generate an scFv gene, a DNA fragment encoding the VH and VL is operably linked to another fragment encoding a flexible linker, for example, encoding the amino acid sequence (Gly4-Ser)3, such that the VH and VL sequences can be expressed as a contiguous single-chain protein with the VL and VH regions connected by the flexible linker (see, e.g., Bird et al. (1988) Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., (1990) Nature 348:552-554).

[0218] V. Antibody production Various antibodies of the invention, e.g., those that compete with or bind to the same epitope as the anti-human TIGIT antibodies disclosed herein, can be produced using a variety of known techniques, such as the standard somatic cell hybridization technique described by Kohler and Milstein, Nature 256: 495 (1975). Although somatic cell hybridization procedures are preferred, in principle, other techniques for producing monoclonal antibodies can also be used, e.g., viral or oncogenic transformation of B lymphocytes, phage display techniques using libraries of human antibody genes.

[0219] A preferred animal system for preparing hybridomas is the mouse system. Hybridoma production in mice is a very well-established procedure. Immunization protocols and techniques and techniques for isolating immunized splenocytes for fusion are known in the art. Fusion partners (e.g., mouse myeloma cells) and fusion procedures are also known.

[0220] The chimeric or humanized antibodies described herein can be prepared based on the sequence of the mouse monoclonal antibody prepared as described above. Using standard molecular biology techniques, DNA encoding heavy and light chain immunoglobulins can be obtained from the target mouse hybridoma and genetically engineered to contain non-mouse (e.g., human) immunoglobulin sequences. For example, to create chimeric antibodies, mouse variable regions can be linked to human constant regions using methods known in the art (see, for example, U.S. Patent No. 4,816,567 to Cabilly et al.). To create humanized antibodies, mouse CDR regions can be inserted into human frameworks using methods known in the art (see, for example, U.S. Patent No. 5,225,539 to Winter and U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et al.).

[0221] In one embodiment, the antibody described herein is a human monoclonal antibody. Such human monoclonal antibodies directed against TIGIT can be produced using transgenic or transchromosomic mice that retain parts of the human immune system rather than the mouse system. These transgenic and transchromosomic mice include mice referred to herein as HuMAb mice and KM mice, respectively, and are collectively referred to herein as "human Ig mice."

[0222] HuMAb Mice® (Medarex, Inc.) contain human immunoglobulin gene miniloci encoding unrearranged human heavy (μ and γ) and κ light chain immunoglobulin sequences, with targeted mutations that inactivate the endogenous μ and κ chain loci (see, e.g., Lonberg, et al. (1994) Nature 368(6474): 856-859). Thus, the mice exhibit reduced expression of mouse IgM or κ, and in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to generate high-affinity human IgGκ monoclonals (reviewed in Lonberg, N. et al. (1994), supra; Lonberg, N. (1994) Handbook of Experimental Pharmacology 113:49-101; Lonberg, N. and Huszar, D. (1995) Intern. Rev. Immunol. 13: 65-93; and Harding, F. and Lonberg, N. (1995) Ann. NY Acad. Sci. 764:536-546).The preparation and use of HuMab mice and the genomic modifications carried by such mice are described in Taylor, L. et al. (1992) Nucleic Acids Research 20:6287-6295; Chen, J. et al. (1993) International Immunology 5: 647-656; Tuaillon et al. (1993) Proc. Natl. Acad. Sci. USA 90:3720-3724; Choi et al. (1993) Nature Genetics 4:117-123; Chen, J. et al. (1993) EMBO J. 12: 821-830; Tuaillon et al. (1994) J. Immunol. 152:2912-2920; Taylor, L. et al. (1994) International Immunology 6: 579-591; and Fishwild, D. et al. (1996) Nature Biotechnology 14: 845-851, the disclosures of which are incorporated herein by reference in their entireties. Further, U.S. Patent Nos. 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,877,397; 5,661,016; 5,814,318; 5,874,299; and 5,770,429, all to Lonberg and Kay; U.S. Patent No. 5,545,807 to Surani et al.; PCT Publication Nos. WO92 / 03918, WO93 / 12227, WO94 / 25585, WO97 / 13852, WO98 / 24884, and WO99 / 45962, all to Lonberg and Kay; and Korman et al. See PCT Publication No. WO01 / 14424 to al.

[0223] In certain embodiments, the antibodies described herein are produced using mice carrying human immunoglobulin sequences on transgenes and transchromosomes, e.g., mice carrying a human heavy chain transgene and a human light chain transchromosome. Such mice, referred to herein as "KM mice," are described in detail in PCT Publication WO 02 / 43478 by Ishida et al.

[0224] In addition, alternative transgenic animal systems that express human immunoglobulin genes are available in the art and can be used to produce the anti-TIGIT antibodies described herein.For example, alternative transgenic systems called Xenomouse (Abgenix, Inc.) can be used, and such mice are described in, for example, U.S. Patent Nos. 5,939,598 to Kucherlapati et al.; 6,075,181; 6,114,598; 6,150,584 and 6,162,963.

[0225] In addition, alternative transchromosomic animal systems expressing human immunoglobulin genes are available in the art and can be used to produce the anti-TIGIT antibodies described herein. For example, mice carrying both a human heavy chain transchromosome and a human light chain transchromosome, referred to as "TC mice," can be used; such mice are described in Tomizuka et al. (2000) Proc. Natl. Acad. Sci. USA 97:722-727. Furthermore, cattle carrying human heavy and light chain transchromosomes have been described in the art (Kuroiwa et al. (2002) Nature Biotechnology 20:889-894) and can be used to produce the anti-TIGIT antibodies described herein.

[0226] Additional mouse systems described in the art for producing human antibodies, e.g., human anti-TIGIT antibodies, include (i) the VELOCIMMUNE® mouse (Regeneron Pharmaceuticals, Inc.), in which the endogenous mouse heavy and light chain variable regions have been replaced by homologous recombination with human heavy and light chain variable regions operably linked to endogenous mouse constant regions, resulting in the generation of a chimeric antibody in the mouse (human V / mouse C), which is then subsequently converted to a fully human antibody using standard recombinant DNA techniques; and (ii) the MeMo® mouse (Merus Biopharmaceuticals, Inc.), in which the mouse contains unrearranged human heavy chain variable regions but a single rearranged human common light chain variable region. Such mice and their use to generate antibodies are described, for example, in WO2009 / 15777, US2010 / 0069614, WO2011 / 072204, WO2011 / 097603, WO2011 / 163311, WO2011 / 163314, WO2012 / 148873, US2012 / 0070861 and US2012 / 0073004.

[0227] The human monoclonal antibody described herein can also be prepared by using phage display method to screen the library of human immunoglobulin genes.Such phage display method for isolating human antibody has been established in the art.For example, see U.S. Patent No. 5,223,409 to Ladner et al.; U.S. Patent No. 5,403,484; and U.S. Patent No. 5,571,698 to Dower et al.; U.S. Patent No. 5,427,908 and U.S. Patent No. 5,580,717 to McCafferty et al.; U.S. Patent No. 5,969,108 and U.S. Patent No. 6,172,197 to McCafferty et al.; and U.S. Patent No. 5,885,793 to Griffiths et al.; U.S. Patent No. 6,521,404; U.S. Patent No. 6,544,731; U.S. Patent No. 6,555,313; U.S. Patent No. 6,582,915 and U.S. Patent No. 6,593,081 to Griffiths et al.

[0228] The human monoclonal antibodies described herein can also be prepared using SCID mice that have been reconstituted with human immune cells so that a human antibody response can be generated upon immunization. Such mice are described, for example, in U.S. Patent Nos. 5,476,996 and 5,698,767 to Wilson et al.

[0229] Immunization To generate fully human antibodies against human TIGIT, transgenic or transchromosomal mice containing human immunoglobulin genes (e.g., HCo12, HCo7, or KM mice) can be immunized with purified or enriched preparations of TIGIT antigen and / or cells expressing TIGIT, as described for other antigens by, for example, Lonberg et al. (1994) Nature 368(6474): 856-859; Fishwild et al. (1996) Nature Biotechnology 14: 845-851, and WO98 / 24884. Alternatively, mice can be immunized with DNA encoding human TIGIT. Preferably, mice are 6-16 weeks old at the time of the first injection. For example, a purified or enriched preparation (5-50 μg) of recombinant TIGIT antigen can be used to intraperitoneally immunize HuMAb mice. In the event that immunization with purified or concentrated preparations of TIGIT antigen does not result in antibodies, mice can also be immunized with cells, e.g., cell lines, that express TIGIT to promote an immune response. Exemplary cell lines include TIGIT-overexpressing stable CHO and Raji cell lines.

[0230] Cumulative experience with various antigens has shown that HuMAb transgenic mice respond best to an initial intraperitoneal (IP) or subcutaneous (SC) immunization with antigen in Ribi adjuvant, followed by biweekly IP / SC immunizations (up to a total of 10) with antigen in Ribi adjuvant. Immune responses can be monitored over the course of the immunization protocol using plasma samples obtained by retroorbital bleeds. Plasma can be screened by ELISA and FACS (as described below), and mice with sufficient titers of anti-TIGIT human immunoglobulin can be used for fusions. Mice are boosted intravenously with antigen and sacrificed 3 days later, and spleens and lymph nodes can be harvested. It is expected that two to three fusions may need to be performed for each immunization. Between 6 and 24 mice are typically immunized for each antigen. HCo7, HCo12, and KM strains are commonly used. Furthermore, both the HCo7 and HCo12 transgenes can be bred together into a single mouse carrying two different human heavy chain transgenes (HCo7 / HCo12).

[0231] Generation of hybridomas producing monoclonal antibodies against TIGIT To generate hybridomas producing the monoclonal antibodies described herein, spleen cells and / or lymph node cells can be isolated from immunized mice and fused with a suitable immortalized cell line, such as a mouse myeloma cell line. The resulting hybridomas can be screened for the production of antigen-specific antibodies. For example, a single-cell suspension of splenic lymphocytes from immunized mice can be fused with Sp2 / 0 non-secretory mouse myeloma cells (ATCC, CRL 1581) using 50% PEG. The cells can be fused at approximately 2 x 10 5Hybridomas are plated into flat-bottom microtiter plates at 100°C for 2 weeks and subsequently incubated in selective medium containing 10% fetal bovine serum, 18% "653" conditioned medium, 5% origen (IGEN), 4 mM L-glutamine, 1 mM sodium pyruvate, 5 mM HEPES, 0.055 mM 2-mercaptoethanol, 50 units / ml penicillin, 50 mg / ml streptomycin, 50 mg / ml gentamicin, and 1X HAT (Sigma). After approximately 2 weeks, cells can be cultured in medium in which the HAT is replaced with HT. Individual wells can then be screened by ELISA for human monoclonal IgM and IgG antibodies. Extensive hybridoma growth occurs, and medium can be observed routinely after 10-14 days. Antibody-secreting hybridomas can be replated and screened again; if still positive for human IgG, monoclonal antibodies can be subcloned at least twice by limiting dilution. The stable subclones can then be cultured in vitro to generate small amounts of antibody in tissue culture medium for characterization.

[0232] To purify monoclonal antibodies, selected hybridomas can be grown in 2-liter spinner flasks for monoclonal antibody purification. Supernatants can be filtered and concentrated before affinity chromatography using Protein A-Sepharose (Pharmacia, Piscataway, NJ). Eluted IgG can be examined by gel electrophoresis and high-performance liquid chromatography to ensure purity. The buffer solution can be exchanged into PBS, and the concentration can be determined by OD280 using a 1.43 extinction coefficient. Monoclonal antibodies can be aliquoted and stored at -80°C.

[0233] VI. Antibody production Generation of transfectomas producing monoclonal antibodies against TIGIT Antibodies of the present invention, including both the specific antibodies for which sequences are provided and other related anti-TIGIT antibodies, can be produced as is well known in the art, for example, in host cell transfectomas using a combination of recombinant DNA technology and gene transfection methods (Morrison, S. (1985) Science 229:1202).

[0234] For example, to express an antibody or antibody fragment thereof, DNA encoding partial or full-length light and heavy chains can be obtained by standard molecular biology techniques (e.g., PCR amplification or cDNA cloning using a hybridoma expressing the antibody of interest), and the DNA can be inserted into an expression vector such that the gene is operably linked to transcriptional and translational control sequences. In this context, the term "operably linked" means that the antibody gene is ligated into a vector such that transcriptional and translational control sequences within the vector perform their intended function of regulating the transcription and translation of the antibody gene. Expression vectors and expression control sequences are selected to be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene can be inserted into separate vectors, or both genes are inserted into the same expression vector. The antibody genes are inserted into the expression vector(s) by standard methods (e.g., ligation of complementary restriction sites on the antibody gene fragment and vector, or blunt-end ligation if no restriction sites are present). Using the light and heavy chain variable regions of the antibodies described herein, V H The segment is C in the vector H operably linked to the V segment(s); L The segment is located at C LFull-length antibody genes of any antibody isotype can be produced by inserting the segments operably linked into an expression vector already encoding the heavy and light chain constant regions of the desired isotype. Additionally, or alternatively, the recombinant expression vector may encode a signal peptide that facilitates secretion of the antibody chain from the host cell. The antibody chain gene can be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).

[0235] In addition to the antibody chain genes, the recombinant expression vector can carry regulatory sequences that control the expression of the antibody chain genes in a host cell. The term "regulatory sequence" is intended to include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody chain genes. Such regulatory sequences are described, for example, in Goeddel (Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, CA (1990)). It will be apparent to those skilled in the art that the design of the expression vector, including the selection of regulatory sequences, can vary depending on factors such as the choice of the host cell to be transformed, the level of expression of the desired protein, etc. Preferred regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and / or enhancers from cytomegalovirus (CMV), simian virus 40 (SV40), adenovirus (e.g., the adenovirus major late promoter (AdMLP)), and polyoma. Alternatively, non-viral regulatory sequences, such as the ubiquitin promoter or β-globin promoter, may be used. Furthermore, regulatory elements can consist of sequences derived from different sources, such as the SRα promoter system, which contains sequences from the SV40 early promoter and the long terminal repeat of the human T-cell leukemia virus type 1 (Takebe, Y. et al. (1988) Mol. Cell. Biol. 8:466-472).

[0236] In addition to the antibody chain genes and regulatory sequences, the recombinant expression vector may carry additional sequences, such as sequences regulating replication of the vector in host cells (e.g., origins of replication), and a selectable marker gene. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see, e.g., U.S. Patent Nos. 4,399,216, 4,634,665, and 5,179,017, all by Axel et al.). For example, the selectable marker gene usually confers resistance to drugs such as G418, hygromycin, or methotrexate on the host cells into which the vector has been introduced. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr-host cells with methotrexate selection / amplification) and the neo gene (for G418 selection).

[0237] For expression of the light and heavy chains, the expression vector(s) encoding the heavy and light chains are transfected into a host cell by standard techniques. The various forms of the term "transfection" are intended to encompass a variety of techniques commonly used for the introduction of exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, and the like. While it is theoretically possible to express the antibodies described herein in either prokaryotic or eukaryotic host cells, expression of the antibodies in eukaryotic cells, most preferably mammalian host cells, is most preferred, as such eukaryotic cells, particularly mammalian cells, are more likely than prokaryotic cells to assemble and secrete properly folded, immunologically active antibodies. Prokaryotic expression of antibody genes has been reported to be ineffective for highly efficient production of active antibodies (Boss, MA and Wood, CR (1985) Immunology Today 6:12-13). The antibodies of the invention can also be produced in glycoengineered strains of the yeast Pichia pastoris. Li et al. (2006) Nat. Biotechnol. 24:210.

[0238] Preferred mammalian host cells for expressing the recombinant antibodies described herein include Chinese hamster ovary (CHO) cells (e.g., including the dhfr-CHO cells described in Urlaub and Chasin (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, used with the DHFR selection marker, as described in RJ Kaufman and PA Sharp (1982) Mol. Biol. 159:601-621), NSO myeloma cells, COS cells, and SP2 cells. Another preferred expression system, particularly for use with NSO myeloma cells, is the GS gene expression system disclosed in WO 87 / 04462, WO 89 / 01036, and EP 338,841. A recombinant expression vector encoding an antibody gene is introduced into the mammalian host cells, and the antibody is produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells, or more preferably, secretion of the antibody into the culture medium in which the host cells are grown. Antibodies can be recovered from the culture medium using standard protein purification methods.

[0239] The N- and C-termini of the antibody polypeptide chains of the present invention may differ from the predicted sequences due to commonly observed post-translational modifications. For example, C-terminal lysine residues are often missing from antibody heavy chains. Dick et al. (2008) Biotechnol. Bioeng. 100:1132. N-terminal glutamine residues, and to a lesser extent glutamic acid residues, are frequently converted to pyroglutamic acid residues in both the light and heavy chains of therapeutic antibodies. Dick et al. (2007) Biotechnol. Bioeng. 97:544; Liu et al. (2011) JBC 28611211; Liu et al. (2011) J. Biol. Chem. 286:11211.

[0240] The amino acid sequences of various agonist anti-huTIGIT antibodies of the invention are provided in the sequence listing summarized in Table 5. For the reasons stated above, a C-terminal lysine is not included in any of the sequences in the sequence listing for the heavy chain or heavy chain constant domain. However, in alternative embodiments, each heavy chain of an anti-huTIGIT antibody of the invention and / or a genetic construct encoding such an antibody or its heavy or light chain contains this additional lysine residue at the C-terminus of the heavy chain(s).

[0241] VII. Assay The antibodies described herein can be tested for binding to TIGIT, for example, by standard ELISA. Briefly, microtiter plates are coated with purified TIGIT at 1-2 μg / mL in PBS and then blocked with 5% bovine serum albumin in PBS. Dilutions of antibody (e.g., dilutions of plasma from TIGIT-immunized mice) are added to each well and incubated for 1-2 hours at 37°C. The plates are washed with PBS / Tween and then incubated for 1 hour at 37°C with a secondary reagent, e.g., a goat anti-human IgG Fc-specific polyclonal reagent conjugated with horseradish peroxidase (HRP), for human antibodies or antibodies otherwise containing a human heavy chain constant region. After washing, the plates are developed with ABTS substrate (Moss Inc., product: ABTS-1000) and analyzed by spectrophotometer at OD 415-495. Sera from immunized mice are then further screened by flow cytometry for binding to cell lines expressing human TIGIT, but not to control cell lines that do not express TIGIT. Briefly, binding of anti-TIGIT antibodies is assessed by incubating TIGIT-expressing CHO cells with anti-TIGIT antibodies at a 1:20 dilution. The cells are washed, and binding is detected using a PE-labeled anti-human IgG Ab. Flow cytometry analysis is performed using a FACScan flow cytometer (Becton Dickinson, San Jose, CA). Mice that generate the highest titers are preferably used for fusion. If mouse anti-huTIGIT antibodies are to be detected, similar experiments can be performed using anti-mouse detection antibodies.

[0242] ELISA assays such as those described above can be used to screen for antibodies, and thus hybridomas that produce antibodies that show positive reactivity with the TIGIT immunogen. Preferably, hybridomas that produce antibodies that bind to TIGIT with high affinity can be subcloned and further characterized. To create a cell bank, one clone from each hybridoma that retains the reactivity of the parent cell (by ELISA) can be selected for antibody purification.

[0243] To purify anti-TIGIT antibodies, selected hybridomas can be grown in 2-liter spinner flasks for monoclonal antibody purification. Supernatants can be filtered and concentrated before affinity chromatography using Protein A-Sepharose (Pharmacia, Piscataway, NJ). Eluted IgG can be examined by gel electrophoresis and high-performance liquid chromatography to ensure purity. The buffer solution can be exchanged into PBS, and OD values ​​can be measured using a 1.43 extinction coefficient. 280 The concentration can be determined by: The monoclonal antibody can be aliquoted and stored at -80°C.

[0244] To determine whether selected anti-TIGIT monoclonal antibodies bind to unique epitopes, each antibody can be biotinylated using commercially available reagents (Pierce, Rockford, IL). Biotinylated MAb binding can be detected using a streptavidin-labeled probe. As described above, TIGIT-coated ELISA plates can be used to perform competition studies using unlabeled and biotinylated monoclonal antibodies.

[0245] To determine the isotype of purified antibodies, an isotype ELISA can be performed using reagents specific for antibodies of a particular isotype. For example, to determine the isotype of a human monoclonal antibody, the wells of a microtiter plate can be coated overnight at 4°C with 1 μg / mL of anti-human immunoglobulin. After blocking with 1% BSA, the plate is reacted with up to 1 μg / mL of the test monoclonal antibody or a purified isotype control for 1-2 hours at ambient temperature. The wells are then reacted with either a human IgG1- or human IgM-specific alkaline phosphatase-conjugated probe. The plate is developed and analyzed as described above.

[0246] To examine the binding of monoclonal antibodies to live cells expressing TIGIT, flow cytometry can be used as described in the Examples. Briefly, cell lines expressing membrane-bound TIGIT (grown under standard growth conditions) are mixed with various concentrations of monoclonal antibodies in PBS containing 0.1% BSA for 1 hour at 4°C. After washing, the cells are reacted with phycoerythrin (PE)-labeled anti-IgG antibodies under the same conditions as primary antibody staining. Samples are analyzed using a FACScan instrument using light and side scatter properties to gate on single cells and examine the binding of the labeled antibody. An alternative assay using a fluorescent microscope may be used (in addition to or instead of) the flow cytometry assay. Cells can be precisely stained as described above and examined by fluorescent microscopy. This method allows visualization of individual cells but may have reduced sensitivity depending on the density of the antigen.

[0247] Anti-TIGIT antibodies can be further tested for reactivity with the TIGIT antigen by Western blotting. Briefly, cell extracts are prepared from TIGIT-expressing cells and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. After electrophoresis, the separated antigens are transferred to a nitrocellulose membrane, blocked with 20% mouse serum, and probed with the monoclonal antibody to be tested. IgG binding is detected using anti-IgG alkaline phosphatase and developed with BCIP / NBT substrate tablets (Sigma Chem. Co., St. Louis, MO).

[0248] Methods for analyzing the binding affinity, cross-reactivity, and binding kinetics of various anti-TIGIT antibodies include standard assays known in the art, such as biolayer interferometry (BLI) analysis and BIACORE® surface plasmon resonance (SPR) analysis using a BIACORE® 2000 SPR instrument (Biacore AB, Uppsala, Sweden).

[0249] In one embodiment, the antibody specifically binds to the extracellular domain of human TIGIT. The antibody may specifically bind to a specific domain (e.g., a functional domain) within the extracellular domain of TIGIT. In a specific embodiment, the antibody specifically binds to the site on TIGIT to which PVR binds. In a specific embodiment, the antibody specifically binds to the extracellular domain of human TIGIT and the extracellular domain of cynomolgus monkey TIGIT. Preferably, the antibody binds to human TIGIT with high affinity.

[0250] VIII. Bispecific molecules The antibodies described herein can be used to form bispecific molecules. An anti-TIGIT antibody or its antigen-binding fragment can be derivatized or linked to another functional molecule, such as another peptide or protein (e.g., another antibody or receptor ligand), to generate a bispecific molecule that binds to at least two different binding sites or target molecules. The antibodies described herein can actually be derivatized or linked to two or more other functional molecules to generate a multispecific molecule that binds to two or more different binding sites and / or target molecules; such multispecific molecules are also encompassed by the term "bispecific molecule" herein. To generate the bispecific molecules described herein, the antibodies described herein can be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent binding, or otherwise) to one or more other binding molecules, such as another antibody, antibody fragment, peptide, or binding mimic, resulting in a bispecific molecule.

[0251] Thus, provided herein are bispecific molecules comprising at least one first binding specificity for TIGIT and a second binding specificity for a second target epitope. In one embodiment described herein in which the bispecific molecule is multispecific, the molecule may further comprise a third binding specificity.

[0252] In one embodiment, the bispecific molecules described herein comprise as binding specificities at least one antibody or antibody fragment thereof, including, for example, Fab, Fab', F(ab'), Fv, or single-chain Fv. An antibody can also be a light or heavy chain dimer or any smallest fragment thereof, such as an Fv or single-chain construct, as described in Ladner et al., U.S. Patent No. 4,946,778, the disclosure of which is expressly incorporated by reference.

[0253] Although human monoclonal antibodies are preferred, other antibodies that may be used in the bispecific molecules described herein include murine, chimeric, and humanized monoclonal antibodies.

[0254] The bispecific molecules described herein can be prepared by conjugating the component binding specificities using methods known in the art. For example, each binding specificity of the bispecific molecule can be generated separately and then conjugated to each other. When the binding specificities are proteins or peptides, various coupling or cross-linking agents can be used for covalent conjugation. Examples of cross-linking agents include protein A, carbodiimide, N-succinimidyl-S-acetyl-thioacetate (SATA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide (oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), and sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) (see, e.g., Karpovsky et al. (1984) J. Exp. Med. 160:1686; Liu, MA et al. (1985) Proc. Natl. Acad. Sci. USA 82:8648). Other methods include those described in Paulus (1985) Behring Ins. Mitt. No. 78, 118-132; Brennan et al. (1985) Science 229:81-83) and Glennie et al. (1987) J. Immunol. 139: 2367-2375. Preferred conjugating agents include SATA and sulfo-SMCC, both available from Pierce Chemical Co. (Rockford, IL).

[0255] When the binding specificities are antibodies, they can be conjugated via sulfhydryl bonding of the C-terminal hinge regions of the two heavy chains. In a particularly preferred embodiment, the hinge region is modified to contain an odd number of sulfhydryl residues, preferably one, prior to conjugation.

[0256] Alternatively, both binding specificities can be encoded in the same vector and expressed and assembled in the same host cell. This method is particularly useful when the bispecific molecule is a mAb x mAb, mAb x Fab, Fab x F(ab')2, or ligand x Fab fusion protein. The bispecific molecules described herein can be single-chain molecules containing one single-chain antibody and a binding determinant, or single-chain bispecific molecules containing two binding determinants. A bispecific molecule can contain at least two single-chain molecules. Methods for preparing bispecific molecules are described, for example, in U.S. Patent Nos. 5,260,203; 5,455,030; 4,881,175; 5,132,405; 5,091,513; 5,476,786; 5,013,653; 5,258,498, and 5,482,858.

[0257] Binding of a bispecific molecule to its specific target can be confirmed using art-recognized methods such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassays (e.g., growth inhibition), or Western blot assays. Each of these assays generally detects the presence of a protein-antibody complex of particular interest by employing a labeled reagent (e.g., an antibody) specific for the complex of interest.

[0258] IX. Composition Further provided are compositions, e.g., pharmaceutical compositions, containing the anti-TIGIT antibody or antigen-binding fragment(s) described herein, formulated together with a pharmaceutically acceptable carrier. Such compositions may contain one or a combination of (e.g., two or more different) antibodies, immunoconjugates, or bispecific molecules described herein. For example, the pharmaceutical compositions described herein may contain a combination of antibodies (or immunoconjugates or bispecifics) that bind to different epitopes on the target antigen or have complementary activities.

[0259] In certain embodiments, the composition comprises an anti-TIGIT antibody at a concentration of at least 1 mg / ml, 5 mg / ml, 10 mg / ml, 50 mg / ml, 100 mg / ml, 150 mg / ml, 200 mg / ml, or at 1-300 mg / ml or 100-300 mg / ml.

[0260] The pharmaceutical compositions described herein can also be administered in combination therapy, i.e., in combination with other agents. For example, the combination therapy can include the anti-TIGIT antibody described herein in combination with at least one other anti-cancer agent and / or T cell stimulating (e.g., activating) agent. Examples of therapeutic agents that can be used in combination therapy are described in more detail below in the section on the use of the antibodies described herein.

[0261] In some embodiments, the therapeutic compositions disclosed herein may contain other compounds, drugs, and / or agents used for the treatment of cancer. Such compounds, drugs, and / or agents include, for example, chemotherapeutic agents, small molecule drugs, or antibodies that stimulate an immune response to a given cancer. In some cases, the therapeutic compositions may include, for example, one or more of an anti-CTLA-4 antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CD40 antibody, an anti-OX40 (also known as CD134, TNFRSF4, ACT35, and / or TXGP1L) antibody, an anti-LAG-3 antibody, an anti-CD73 antibody, an anti-CD137 antibody, an anti-CD27 antibody, an anti-CSF-1R antibody, a TLR agonist, or a small molecule antagonist of IDO or TGFβ.

[0262] As used herein, "pharmaceutically acceptable carriers" include any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound, i.e., antibody, immunoconjugate, or bispecific molecule, may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

[0263] The pharmaceutical compounds described herein may include one or more pharmaceutically acceptable salts. A "pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects (see, e.g., Berge, SM, et al. (1977) J. Pharm. Sci. 66:1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from non-toxic inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphorous acid, and the like, as well as those derived from non-toxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids, and the like. Base addition salts include those derived from alkaline earth metals such as sodium, potassium, magnesium, calcium, and the like, as well as those derived from non-toxic organic amines such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine, and the like.

[0264] The pharmaceutical compositions described herein may also contain pharmaceutically acceptable antioxidants. Examples of pharmaceutically acceptable antioxidants include (1) water-soluble antioxidants such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc., (2) oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, etc., and (3) metal chelators such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0265] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions described herein include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.Proper fluidity can be maintained, for example, by using coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by using surfactants.

[0266] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the presence of microorganisms can be ensured both by sterilization procedures, as described above, and by the inclusion of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol sorbic acid, etc. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, etc., in the compositions. Furthermore, prolonged absorption of injectable pharmaceutical forms can be brought about by the inclusion of agents that delay absorption, such as aluminum monostearate and gelatin.

[0267] Pharmaceutically acceptable carriers include sterile aqueous solution or dispersion and sterile powder for the immediate preparation of sterile injectable solution or dispersion.The use of such media and agents for pharmaceutically active substances is known in the art.Except when any conventional media or agent is incompatible with active compound, it can be considered for use in the pharmaceutical compositions described herein.Supplementary active compounds can also be incorporated into compositions.

[0268] Therapeutic compositions must typically be sterile and stable under the conditions of manufacture and storage. The compositions can be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentrations. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride, in the composition. Prolonged absorption of injectable compositions can be achieved by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

[0269] Sterile injectable solution can be prepared by incorporating the active compound in the required amount in suitable solvent with one or combination of the above-listed components as needed, and then by sterilization microfiltration.Generally, dispersion is prepared by incorporating active compound in a sterile vehicle that contains basic dispersion medium and other components that are required from above-listed.For the sterile powder that is used to prepare sterile injectable solution, the preferred method of preparation is vacuum drying and freeze-drying (lyophilization), which can obtain the powder of active ingredient and any other desired components from the solution that has been previously sterilized and filtered.

[0270] The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending on the subject being treated and the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the composition that produces a therapeutic effect. Generally, in combination with a pharmaceutically acceptable carrier, this amount will range from about 0.01 percent to about 99 percent of the active ingredient, preferably from about 0.1 percent to about 70 percent, and most preferably from about 1 percent to about 30 percent of the active ingredient, out of one hundred percent.

[0271] The dosage regimen is adjusted to provide the optimum desired response (e.g., therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is particularly advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. As used herein, dosage unit form refers to physically discrete units suitable as unitary dosages for the subject being treated, each unit containing a predetermined amount of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specifications for the dosage unit forms described herein are determined by and directly depend on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the technical field of compounding such active compounds for the treatment of susceptibility in individuals.

[0272] For antibody administration, the dosage ranges from about 0.0001 to 100 mg / kg of host body weight, more usually 0.01 to 5 mg / kg of host body weight. For example, dosages can be 0.3 mg / kg, 1 mg / kg, 3 mg / kg, 5 mg / kg, or 10 mg / kg, or within the range of 1 to 10 mg / kg. Exemplary treatment regimens involve administration once weekly, once every two weeks, once every three weeks, once every four weeks, once monthly, or once every three to six months. In a preferred embodiment, the anti-TIGIT antibody of the present invention is administered every two weeks. Other preferred dosing regimens for the anti-TIGIT antibodies described herein include 1 mg / kg, 3 mg / kg, or 5 mg / kg of body weight administered intravenously, with the antibodies given using one of the following dosing schedules: (i) every 4 weeks for 6 doses, then every 3 months; (ii) every 3 weeks; or (iii) 3 mg / kg once followed by 1 mg / kg every 3 weeks.

[0273] In some methods, two or more monoclonal antibodies with different binding specificities are administered simultaneously, in which case the dosage of each antibody administered falls within the ranges indicated. Therapeutic antibodies are usually administered on multiple occasions. The interval between single doses can be, for example, weekly, monthly, every three months, or yearly. Intervals can also be irregular, as indicated by measuring blood levels of antibody against the target antigen in the patient. In some methods, dosage is adjusted to achieve a plasma antibody concentration of about 1-1000 μg / mL, and in some methods, about 25-300 μg / mL.

[0274] The antibody can be administered as a sustained-release formulation, in which case less frequent administration is required. The dosage and frequency vary depending on the half-life of the antibody in the patient. Generally, human antibodies exhibit the longest half-life, followed by humanized antibodies, chimeric antibodies, and non-human antibodies. The dosage and frequency of administration can vary depending on whether the treatment is preventive or therapeutic. In preventive applications, relatively low dosages are administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, relatively high dosages at relatively short intervals are sometimes required until the progression of the disease is reduced or terminated, preferably until the patient shows partial or complete remission of the symptoms of the disease. Thereafter, the patient may optionally receive a preventive administration regimen, although in many immuno-oncology indications, continued treatment is not required.

[0275] The actual dosage level of the active ingredient in the pharmaceutical compositions described herein may be varied to obtain an amount of the active ingredient that is not toxic to the patient and is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration. The selected dosage level will depend on various pharmacokinetic factors, including the activity of the particular composition described herein or its ester, salt, or amide used, the route of administration, the time of administration, the excretion rate of the particular compound used, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular composition used, the age, sex, weight, condition, general health, and previous medical history of the patient being treated, and similar factors well known in the pharmaceutical arts.

[0276] A "therapeutically effective dose" of an anti-TIGIT antibody described herein preferably results in a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, or prevention of functional impairment or disability due to disease affliction. In the context of cancer, a therapeutically effective dose preferably prevents further exacerbation of physical symptoms associated with the cancer. Symptoms of cancer are well known in the art and include, for example, unusual lentigo features, changes in the appearance of lentigines including asymmetry, border, color and / or diameter, newly pigmented skin areas, abnormal lentigines, dark areas under the fingernails, breast lumps, nipple changes, breast cysts, breast pain, death, weight loss, weakness, excessive fatigue, eating disorders, loss of appetite, chronic cough, worsening shortness of breath, hemoptysis, blood in the urine, blood in the stool, nausea, vomiting, liver metastasis, lung metastasis, bone metastasis, abdominal bloating, flatulence, fluid in the peritoneal cavity, vaginal bleeding, constipation, abdominal distension, perforation of the colon, acute peritonitis (infection, fever, pain), pain, vomiting blood, profuse sweating, fever, hypertension, anemia, diarrhea, jaundice, dizziness, chills, muscle cramps, colon metastasis, lung metastasis, bladder metastasis, liver metastasis, bone metastasis, kidney metastasis and pancreatic metastasis, difficulty swallowing, etc. Therapeutic efficacy may be observable immediately after the first administration of the anti-huTIGIT mAb of the present invention, or may be observed only after a period of time and / or a series of doses. Such delayed efficacy may only be observed after several months of treatment, up to 6, 9, or 12 months. Given the delayed efficacy exhibited by some immuno-oncology agents, it is important not to prematurely conclude that the anti-huTIGIT mAb of the present invention lacks therapeutic efficacy.

[0277] Therapeutically effective dose, such as may be desired when there is an early or preceding symptom of disease, can prevent or delay the occurrence of cancer.The laboratory tests used in diagnosing cancer include chemistry (including measuring TIGIT level), hematology, serology and radiology.Therefore, any clinical or biochemical assay that monitors any of the above may be used to determine whether a particular treatment is a therapeutically effective dose for treating cancer.Those skilled in the art will be able to determine such amount based on factors such as the size of the subject, the severity of the subject's symptoms and the specific composition or selected route of administration.

[0278] The compositions described herein can be administered by one or more routes of administration using one or more of a variety of methods known in the art. As will be apparent to those skilled in the art, the route and / or mode of administration will vary depending on the desired results. Preferred routes of administration for the antibodies described herein include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, or other parenteral routes of administration, for example, by injection or infusion. As used herein, the term "parenteral administration" refers to modes of administration other than enteral and topical administration, usually by injection, including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intraarticular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion.

[0279] Alternatively, the antibodies described herein can be administered by non-parenteral routes such as topical, epithelial or mucosal routes of administration, for example, intranasal, oral, vaginal, rectal, sublingual or topical.

[0280] Active compound can be prepared with carriers that can protect the compound from rapid release, such as sustained release preparations, including implants, transdermal patches and microencapsulated delivery systems.Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid can be used.Many methods for preparing such preparations are patented or generally known to those skilled in the art.For example, see Sustained and Controlled Release Drug Delivery Systems, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0281] Therapeutic compositions can be administered using medical devices known in the art.For example, in preferred embodiments, therapeutic compositions described herein can be administered using needleless hypodermic injection devices, such as the devices disclosed in United States Patent (USP) No. 5,399,163; United States Patent No. 5,383,851; United States Patent No. 5,312,335; United States Patent No. 5,064,413; United States Patent No. 4,941,880; United States Patent No. 4,790,824; or United States Patent No. 4,596,556.The well-known example of implantable agent and module for use with anti-TIGIT antibody described herein includes United States Patent (USP) No. 4,487,603, which discloses an implantable microinfusion pump for dispensing medicine at a controlled rate. No. 4,486,194, which discloses a therapeutic device for administering medication through the skin; No. 4,447,233, which discloses a medication infusion pump for delivering medication at precise infusion rates; No. 4,447,224, which discloses a variable flow implantable infusion device for continuous drug delivery; No. 4,439,196, which discloses an osmotic drug delivery system having a multi-chamber compartment; and No. 4,475,196, which discloses an osmotic drug delivery system. These patents are incorporated herein by reference. Many other such implants, delivery systems, and modules are known to those skilled in the art.

[0282] In certain embodiments, the anti-TIGIT antibodies described herein can be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) ​​excludes many highly hydrophilic compounds. To ensure that the therapeutic compounds described herein cross the BBB (if necessary), they can be formulated, for example, in liposomes. For methods of producing liposomes, see, for example, U.S. Patent Nos. 4,522,811; 5,374,548; and 5,399,331. Liposomes can contain one or more moieties that selectively transport into specific cells or organs, thus enhancing targeted drug delivery (see, for example, VV Ranade (1989) J. Clin. Pharmacol. 29:685). Exemplary targeting moieties include folate or biotin (see, e.g., U.S. Patent No. 5,416,016 to Low et al.); mannosides (Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153:1038); antibodies (P.G. Bloeman et al. (1995) FEBS Lett. 357:140; M. Owais et al. (1995) Antimicrob. Agents Chemother. 39:180); surfactant protein A receptor (Briscoe et al. (1995) Am. J. Physiol. 1233:134); p120 (Schreier et al. (1994) J. Biol. Chem. 269:9090), and K. Keinanen; M.L. Laukkanen (1994) FEBS Lett. 346:123; see also JJ Killion; IJ Fidler (1994) Immunomethods 4:273.

[0283] X. Uses and Methods The antibodies, antibody compositions, and methods described herein have numerous in vitro and in vivo uses, for example, involving enhancing immune responses by blocking TIGIT signaling or detecting TIGIT. In preferred embodiments, the antibodies described herein are human or humanized antibodies. For example, the anti-TIGIT antibodies described herein can be administered to cultured cells in vitro or ex vivo, or to human subjects in vivo, for example, to enhance immunity in various diseases. Thus, provided herein are methods for modifying immune responses in a subject, comprising administering to the subject an antibody or antigen-binding fragment thereof described herein, such that the immune response in the subject is enhanced, stimulated, or upregulated.

[0284] Preferred subjects include human patients for whom an enhanced immune response would be desirable. The method is particularly suitable for treating human patients with disorders that can be treated by increasing the immune response (e.g., T cell-mediated immune response). In certain embodiments, the method is particularly suitable for in vivo cancer treatment. To achieve antigen-specific enhancement of immunity, the anti-TIGIT antibody described herein can be administered together with the antigen of interest, or the antigen may already be present in the subject to be treated (e.g., a subject with a tumor or a virus). When an antibody against TIGIT is administered together with another agent, the two can be administered separately or simultaneously.

[0285] Also encompassed is a method for detecting the presence or measuring the amount of human TIGIT antigen in a sample, comprising contacting a sample and a control sample with a human monoclonal antibody or antigen-binding fragment thereof that specifically binds to human TIGIT under conditions that allow the formation of a complex between the antibody or fragment and human TIGIT. The formation of the complex is then detected, wherein a difference in complex formation between the sample and the control sample indicates the presence of human TIGIT antigen in the sample. Furthermore, the anti-TIGIT antibodies described herein can be used to purify human TIGIT by immunoaffinity purification.

[0286] Given the ability of the anti-TIGIT antibodies described herein to block the inhibition or co-inhibition of T cell responses, e.g., antigen-specific T cell responses, in vitro and in vivo methods are provided herein using the antibodies described herein to stimulate, enhance, or up-regulate antigen-specific T cell responses, e.g., anti-tumor T cell responses. In certain embodiments, CD3 stimulation is also provided (e.g., by co-incubation with cells expressing membrane CD3), which may be provided simultaneously with, before, or after treatment with the anti-TIGIT antibody. For example, provided herein is a method for enhancing antigen-specific T cell responses, comprising contacting T cells with an anti-TIGIT antibody described herein and, optionally, CD3, such that the antigen-specific T cell response is enhanced, e.g., by removing the TIGIT-mediated inhibitory effect. Any suitable indicator of antigen-specific T cell responses can be used to measure antigen-specific T cell responses. Non-limiting examples of such suitable indicators include increased T cell proliferation in the presence of the antibody and / or increased cytokine production in the presence of the antibody. In a preferred embodiment, interleukin-2 and / or interferon-γ production by antigen-specific T cells is enhanced.

[0287] Further encompassed is a method of enhancing an immune response (e.g., an antigen-specific T cell response) in a subject, comprising administering an anti-TIGIT antibody described herein to the subject, such that the immune response (e.g., an antigen-specific T cell response) in the subject is enhanced. In a preferred embodiment, the subject is a subject with a tumor, and the immune response against the tumor is enhanced. The tumor may be a solid tumor or a liquid tumor, e.g., a hematological malignancy. In certain embodiments, the tumor is an immunogenic tumor. In certain embodiments, the tumor is non-immunogenic. In certain embodiments, the tumor is PD-L1 positive. In certain embodiments, the tumor is PD-L1 negative. The subject may also be a subject with a virus, and the immune response against the virus is enhanced.

[0288] Further provided are methods for inhibiting tumor cell growth in a subject, comprising administering to the subject an anti-TIGIT antibody described herein, such that tumor growth is inhibited in the subject. Also provided are methods for treating a chronic viral infection in a subject, comprising administering to the subject an anti-TIGIT antibody described herein, such that the chronic viral infection is treated in the subject.

[0289] The subject is also administered a therapeutically effective amount of T reg and administering an anti-TIGIT antibody described herein comprising an Fc that stimulates depletion of T cells from the tumor microenvironment of a subject having a tumor, e.g., a cancerous tumor. reg Methods for depleting cells are also encompassed herein. The Fc can be an Fc with effector function or enhanced effector function, e.g., binding or enhanced binding to one or more activating Fc receptors. In a preferred embodiment, T reg Depletion of T cells in the tumor microenvironment eff without significant depletion or inhibition of T and outside the tumor microenvironment eff Cells and T reg In certain embodiments, the subject is, for example, experiencing a significant depletion or inhibition of T cells in the tumor microenvironment. eff T cells regIn certain embodiments, anti-TIGIT antibodies inhibit Tregs in tumors and / or TILs in tumor-infiltrating lymphocytes (TILs). reg For example, in the CT26 tumor model, anti-mouse TIGIT antibodies formatted as mouse IgG2a (which exhibits effector function) can deplete T reg and CD8 + It partially depleted both T cells but CD4 + T cells were not depleted. The effector-less counterpart anti-TIGIT antibody, formatted as mouse IgG1 D265A, did not deplete T cells. Considering whether to use an anti-TIGIT antibody with effector function or an effector-less anti-TIGIT antibody, it is important to consider T cells that may enhance anti-tumor immune responses. reg depletion of CD8, which eliminates some of the cells needed to actually kill tumor cells + Due consideration must be given to the trade-off between T cell depletion. reg Depletion of TIGIT may be predicted to enhance antitumor activity, but recent studies have shown that TIGIT + T reg The connection of TIGIT in eff T cell proliferation reg It has been demonstrated that TIGIT signaling promotes cell-mediated suppression (Joller et al. (2014) Immunity 40:569), suggesting that blocking TIGIT signaling (e.g., using the antagonist anti-TIGIT antibodies of the present invention) may also enhance anti-tumor activity. reg ii) blocking TIGIT signaling in tumors, thus reducing its immunosuppressive activity, and ii) inhibiting anti-tumor CD8 +It may be most effective to use an antagonist anti-TIGIT antibody lacking effector function to activate T cells while simultaneously avoiding effector function-mediated depletion, and iii) enhancing DNAM-mediated activation by allowing DNAM to bind to PVR that would otherwise be bound by TIGIT (and by reducing direct TIGIT-DNAM interaction) (Johnston et al. (2014) Cancer Cell 26:923).

[0290] In certain embodiments, anti-TIGIT antibody is given to the subject as adjuvant therapy.Compared with current standard treatment, the treatment of the subject with cancer using anti-TIGIT antibody can lead to long-term durable response, at least 1, 2, 3, 4, 5, 10 years or more long-term survival, at least 1, 2, 3, 4, 5, or 10 years or more recurrence-free survival.In certain embodiments, the treatment of the subject with cancer using anti-TIGIT antibody prevents cancer recurrence or delays cancer recurrence, for example, for 1, 2, 3, 4, 5, or 10 years or more.Anti-TIGIT treatment can be used as first-line treatment or second-line treatment.

[0291] These and other methods described herein are described in further detail below.

[0292] cancer Blocking TIGIT-mediated PVR / Nectin-2 signaling with an anti-TIGIT antibody can enhance the immune response against cancerous cells in a patient. Provided herein are methods for treating a subject with cancer, comprising administering to the subject an anti-TIGIT antibody described herein, such that the subject is treated, e.g., as a result of which cancerous tumor growth is inhibited or reduced and / or tumor regression occurs. An anti-TIGIT antibody can be used alone to inhibit cancerous tumor growth. Alternatively, an anti-TIGIT antibody can be used with another agent, e.g., other immunogens, standard cancer therapies, or other antibodies, as described below. Combinations with PD-1 inhibitors, such as anti-PD-1 or anti-PD-L1 antibodies, are also provided.

[0293] Thus, provided herein is a method for treating cancer, for example, by inhibiting the growth of tumor cells in a subject, comprising administering to the subject a therapeutically effective amount of an anti-TIGIT antibody described herein, for example, 15A6, 22G2, 11G11, or 10D7, or an antigen-binding fragment thereof. The antibody may be a human anti-TIGIT antibody (such as any of the human anti-huTIGIT antibodies described herein), or a chimeric or humanized non-human anti-huTIGIT antibody, for example, a chimeric or humanized anti-TIGIT antibody that competes for binding with or binds to the same epitope as at least one anti-TIGIT antibody described herein.

[0294] Cancers whose growth can be inhibited using the antibodies of the present invention include cancers that are normally responsive to immunotherapy. Non-limiting examples of cancers for treatment include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, squamous non-small cell lung cancer (NSCLC) and NSCLC, glioma, gastrointestinal cancer, renal cancer (e.g., clear cell carcinoma), ovarian cancer, liver cancer, colorectal cancer, endometrial cancer, kidney cancer (e.g., renal cell carcinoma (RCC)), prostate cancer (e.g., hormone-refractory prostate adenocarcinoma), thyroid cancer, neuroblastoma, pancreatic cancer, glioblastoma (glioblastoma multiforme), cervical cancer, gastric cancer, bladder cancer, hepatoma, breast cancer, colon carcinoma, and head and neck cancer (or carcinoma), gastric cancer cancer), germ cell tumors, childhood sarcoma, sinonasal natural killer, melanoma (e.g., metastatic melanoma such as cutaneous or intraocular melanoma), bone cancer, skin cancer, uterine cancer, cancer of the anal region, testicular cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, solid tumors of childhood , cancer of the ureter, carcinoma of the renal pelvis, neoplasms of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, tumors of the spinal axis, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers including those induced by asbestos, virus-associated cancers (e.g., human papillomavirus (HPV)-associated tumors), and hematological malignancies derived from either of the two major blood cell lineages, i.e., myeloid cell lines (producing granulocytes, erythrocytes, platelets, macrophages, and mast cells) or lymphoid cell lines (producing B, T, NK, and plasma cells), e.g., all types of leukemia, lymphoma, and myeloma, e.g., acute myeloid leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), and chronic myeloid leukemia (CML), undifferentiated AML ( acute, chronic, lymphocytic and / or myeloid leukemias, such as M0), myeloblastic leukemia (M1), myeloblastic leukemia (M2; with cellular maturation), promyelocytic leukemia (M3 or M3 variant [M3V]), myelomonocytic leukemia (M4 or M4 variant with eosinophilia [M4E]), monocytic leukemia (M5), erythroleukemia (M6), megakaryoblastic leukemia (M7), isolated granulocytic sarcoma and chloroma;Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), B-cell lymphoma, T-cell lymphoma, lymphoplasmacytic lymphoma, monocytoid B-cell lymphoma, mucosa-associated lymphoid tissue (MALT) lymphoma, anaplastic (e.g., Ki 1+) large cell lymphoma, adult T-cell lymphoma / leukemia, mantle cell lymphoma, angioimmunoblastic T-cell lymphoma, angiocentric lymphoma, intestinal T-cell lymphoma, primary mediastinal B-cell lymphoma, precursor T-lymphoblastic lymphoma, T-lymphoblastic and lymphoma / leukemia (T-Lbly / T-ALL), peripheral T-cell lymphoma, lymphoblastic lymphoma, post-transplant lymphoproliferative disorder, true histiocytic lymphoma, primary central nervous system lymphoma, primary effusion lymphoma, lymphoma Lymphomas such as lymphoblastic lymphoma (LBL), hematopoietic neoplasms of lymphoid lineage, acute lymphoblastic leukemia, diffuse large B-cell lymphoma, Burkitt lymphoma, follicular lymphoma, diffuse histiocytic lymphoma (DHL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, cutaneous T-cell lymphoma (CTLC) (also called mycosis fungoides or Sézary syndrome) and lymphoplasmacytic lymphoma with Waldenstrom hypergammaglobulinemia (LPL) myelomas, including IgG myeloma, light-chain myeloma, non-secretory myeloma, smoldering myeloma (also called low-grade myeloma), solitary plasmacytoma and multiple myeloma, chronic lymphocytic leukemia (CLL), and hairy cell lymphoma; hematopoietic tumors of myeloid lineage, tumors of mesenchymal origin, including fibrosarcoma and rhabdomyosarcoma; tumors of the central and peripheral nerves, including seminoma, teratocarcinoma, astrocytoma, and schwannoma; fibrosarcoma, rhabdomyosarcoma, and rhabdomyosarcoma. tumors of mesenchymal origin, including thyroid carcinoma (cystic sarcoma) and osteosarcoma; and other tumors, including melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, thyroid follicular carcinoma and teratocarcinoma, hematopoietic tumors of lymphoid lineage, such as T-cell and B-cell tumors, including T-cell disorders, such as, but not limited to, T-prolymphocytic leukemia (T-PLL), including the small cell and cerebriform cell types; preferably, large granular lymphocytic leukemia (LGL) of the T-cell type; a / d T-NHL hepatosplenic lymphoma; peripheral / mature T-cell lymphoma (pleomorphic and immunoblastic subtypes); angiocentric (nasal) T-cell lymphoma; cancer of the head and neck, kidney, rectum, thyroid;These cancers include acute myeloid lymphoma and any combination of the above cancers. The methods described herein may also be used to treat metastatic cancers, refractory cancers (e.g., cancers refractory to previous immunotherapy, e.g., using blocking CTLA-4 or PD-1 antibodies), and recurrent cancers.

[0295] Anti-TIGIT antibodies can be administered as monotherapy or as the sole immunostimulatory therapy, or can be combined with immunogens such as cancerous cells, purified tumor antigens (including recombinant proteins, peptides, and carbohydrate molecules), or cells transfected with genes encoding immunostimulatory cytokines in cancer vaccine strategies (He et al. (2004) J. Immunol. 173:4919-28). Non-limiting examples of tumor vaccines that can be used include tumor cells transfected to express peptides of melanoma antigens, such as gp100, MAGE antigens, Trp-2, MART1, and / or tyrosinase, or the cytokine GM-CSF.

[0296] Numerous experimental strategies have been devised for tumor vaccination (see Rosenberg, S., 2000, Development of Cancer Vaccines, ASCO Educational Book Spring: 60-62; Logothetis, C., 2000, ASCO Educational Book Spring: 300-302; Khayat, D. 2000, ASCO Educational Book Spring: 414-428; Foon, K. 2000, ASCO Educational Book Spring: 730-738; also see Restifo, N. and Sznol, M., Cancer Vaccines, Ch. 61, pp. 3023-3043 in DeVita et al. (eds.), 1997, Cancer: Principles and Practice of Oncology, Fifth Edition). In one of these strategies, vaccines are prepared using autologous or allogeneic tumor cells. These cellular vaccines have been shown to be most effective when tumor cells are transduced to express GM-CSF, which has been shown to be a potent activator of antigen presentation for tumor vaccination (Dranoff et al. (1993) Proc. Natl. Acad. Sci. USA 90: 3539-43).

[0297] Studies of gene expression and large-scale gene expression patterns in various tumors have led to the definition of so-called tumor-specific antigens (Rosenberg, SA (1999) Immunity 10: 281-7). In many cases, these tumor-specific antigens are differentiation antigens expressed in tumors and in tumor-derived cells, such as the melanocyte antigen gp100, MAGE antigens, and Trp-2. More importantly, many of these antigens can be shown to be targets of tumor-specific T cells in the host. To generate an immune response against these proteins, TIGIT inhibition can be used in conjunction with a collection of recombinant proteins and / or peptides expressed in tumors. These proteins are normally viewed by the immune system as self-antigens and are therefore tolerant to them. Tumor antigens can include the protein telomerase, which is required for the synthesis of chromosomal telomeres and is expressed in more than 85% of human cancers and in only a limited number of somatic tissues (Kim et al. (1994) Science 266: 2011-2013). Tumor antigens can also be "neoantigens" expressed in cancer cells due to somatic mutations that alter the protein sequence or create a fusion protein between two unrelated sequences (i.e., bcr-abl in the Philadelphia chromosome) or idiotypes from B-cell tumors.

[0298] Other tumor vaccines may contain proteins derived from viruses involved in human cancer, such as human papillomavirus (HPV), hepatitis viruses (HBV and HCV), and Kaposi's herpes sarcoma virus (KHSV). Another form of tumor-specific antigen that can be used with TIGIT inhibition is purified heat shock proteins (HSPs) isolated from the tumor tissue itself. These heat shock proteins contain fragments of proteins derived from tumor cells, and these HSPs are highly efficient in delivering them to antigen-presenting cells to induce tumor immunity (Suot & Srivastava (1995) Science 269:1585-1588; Tamura et al. (1997) Science 278:117-120).

[0299] Dendritic cells (DCs) are potent antigen-presenting cells that can be used to prime antigen-specific responses. DCs can be produced ex vivo and loaded with various protein and peptide antigens as well as tumor cell extracts (Nestle et al. (1998) Nature Medicine 4: 328-332). DCs can also be genetically transduced to express these tumor antigens. DCs have also been directly fused with tumor cells for immunization purposes (Kugler et al. (2000) Nature Medicine 6:332-336). As a vaccination method, DC immunization can be effectively combined with TIGIT blockade to activate (deregulate) more potent antitumor responses.

[0300] TIGIT inhibition can also be combined with standard cancer treatments (e.g., surgery, radiation, and chemotherapy). TIGIT inhibition can be effectively combined with chemotherapy treatment plans. In these cases, it may be possible to reduce the dose of the administered chemotherapy reagent (Mokyr et al. (1998) Cancer Research 58: 5301-5304). One example of such a combination is an anti-TIGIT antibody combined with decarbazine for the treatment of melanoma. Another example of such a combination is an anti-TIGIT antibody combined with interleukin-2 (IL-2) for the treatment of melanoma. The scientific rationale behind the combined use of TIGIT inhibition and chemotherapy is that cell death, which is the result of the cytotoxic effect of most chemotherapeutic compounds, should lead to increased levels of tumor antigens in the antigen presentation pathway. Other combined treatments that may synergize with TIGIT inhibition due to cell death include radiation, surgery, and hormone deprivation. Each of these protocols creates a source of tumor antigens in the host. Angiogenesis inhibitors can also be combined with TIGIT inhibition. Inhibition of angiogenesis leads to tumor cell death, which can deliver tumor antigens to the host antigen presentation pathway.

[0301] The anti-TIGIT antibodies described herein can also be used in combination with bispecific antibodies that target effector cells against tumor cells expressing Fcα or Fcγ receptors (see, e.g., U.S. Patent Nos. 5,922,845 and 5,837,243). Bispecific antibodies can be used to target two distinct antigens. For example, anti-Fc receptor / anti-tumor antigen (e.g., Her-2 / neu) bispecific antibodies have been used to target macrophages to tumor sites. This targeting can efficiently activate tumor-specific responses. The T cell arm of these responses is enhanced by inhibition of TIGIT. Alternatively, antigens can be delivered directly to DCs by using bispecific antibodies that bind to tumor antigens and dendritic cell-specific cell surface markers.

[0302] Tumors evade host immune surveillance through a variety of mechanisms. Many of these mechanisms can be overcome by inactivating immunosuppressive proteins expressed by the tumor. These include, among others, TGF-β (Kehrl et al. (1986) J. Exp. Med. 163: 1037-1050), IL-10 (Howard & O'Garra (1992) Immunology Today 13: 198-200), and Fas ligand (Hahne et al. (1996) Science 274: 1363-1365). Antibodies against each of these entities can be used in combination with anti-TIGIT antibodies to counter the effects of immunosuppressants and favor the host's tumor immune response.

[0303] Other antibodies that activate host immune responsiveness can be used in combination with anti-TIGIT antibodies. These include molecules on the surface of dendritic cells that activate DC function and antigen presentation. Anti-CD40 antibodies can effectively substitute for T cell helper activity (Ridge et al. (1998) Nature 393: 474-478) and can be used in conjunction with anti-TIGIT antibodies. Activating antibodies against T cell costimulatory molecules such as OX-40 (Weinberg et al. (2000) Immunol 164: 2160-2169), CD137 / 4-1BB (Melero et al. (1997) Nature Medicine 3: 682-685 (1997) and ICOS (Hutloff et al. (1999) Nature 397: 262-266) can also provide increased levels of T cell activation. Inhibitors of PD-1 or PD-L1 or CTLA-4 (e.g., U.S. Patent No. 5,811,097) may also be used in conjunction with anti-TIGIT antibodies.

[0304] Bone marrow transplantation is currently used to treat various tumors of hematopoietic origin. Graft-versus-host disease is a consequence of this treatment, but therapeutic benefits can be obtained from graft-versus-tumor responses. TIGIT inhibition can increase the effectiveness of tumor-specific T cells transplanted from the donor.

[0305] There are also several experimental therapeutic protocols involving ex vivo activation and expansion of antigen-specific T cells and adoptive transfer of these cells into recipients to stimulate antigen-specific T cells against tumors (Greenberg & Riddell (1999) Science 285: 546-51). These methods can also be used to activate T cell responses to infectious agents such as CMV. Ex vivo activation in the presence of anti-TIGIT antibodies can increase the frequency and activity of adoptively transferred T cells.

[0306] Chronic viral infections In another aspect, the invention described herein provides a method of treating an infectious disease in a subject, comprising administering to the subject an anti-TIGIT antibody or antigen-binding fragment thereof, such that the subject is treated for the infectious disease.

[0307] Similar to its application to tumors as discussed above, antibody-mediated TIGIT inhibition can be used alone or as an adjuvant in combination with vaccines to enhance immune responses to pathogens, toxins, and self-antigens. Examples of pathogens for which this therapeutic approach may be particularly useful include, but are not limited to, HIV, hepatitis (A, B, and C), influenza, herpes, giardia, malaria, leishmaniasis, Staphylococcus aureus, and Pseudomonas aeruginosa. TIGIT inhibition is particularly useful against established infections with agents such as HIV, which exhibit altered antigens over the course of infection. These novel epitopes are recognized as foreign at the time of administration of anti-human TIGIT antibodies, thus triggering a strong T cell response.

[0308] Some examples of pathogenic viruses that cause infections treatable by the methods described herein include HIV, hepatitis (A, B, or C), herpesviruses (e.g., VZV, HSV-1, HAV-6, HSV-II, and CMV, Epstein-Barr virus), adenovirus, influenza virus, flavivirus, echovirus, rhinovirus, coxsackievirus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papillomavirus, molluscum contagiosum virus, poliovirus, rabies virus, JC virus, and arboviral encephalitis virus.

[0309] Some examples of pathogenic bacteria that cause infections treatable by the methods described herein include chlamydia, rickettsia bacteria, mycobacteria, staphylococci, streptococci, pneumonococci, meningococci and gonococci, klebsiella, proteus, serratia, pseudomonas, legionella, diphtheria, salmonella, bacillus, cholera, tetanus, botulism, anthrax, plague, leptospirosis, and lyme disease bacteria.

[0310] Some examples of pathogenic fungi that cause infections treatable by the methods described herein include Candida (e.g., albicans, krusei, glabrata, tropicalis), Cryptococcus neoformans, Aspergillus (e.g., fumigatus, niger), genera of the Mucorales order (e.g., mucor, absidia, rhizopus), Sporothrix schenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis, and Histoplasma capsulatum.

[0311] Some examples of pathogenic parasites that cause infections treatable by the methods described herein include Entamoeba histolytica, Balantidium coli, Naegleria fowleri, Acanthamoeba species, Giardia lambia, Cryptosporidium species, Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondii, and the like. gondii) and Brazilian hookworm (Nippostrongylus brasiliensis).

[0312] In all of the above methods, TIGIT inhibition can be combined with other forms of immunotherapy, such as cytokine therapy (e.g., interferon, GM-CSF, G-CSF, IL-2) or bispecific antibody therapy that provides enhanced presentation of tumor antigens (see, e.g., Holliger (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak (1994) Structure 2:1121-1123).

[0313] Vaccine adjuvants The anti-TIGIT antibody described herein can be used to enhance antigen-specific immune responses by co-administering the anti-TIGIT antibody with a target antigen (e.g., a vaccine). Thus, provided herein is a method for enhancing an immune response to an antigen in a subject, comprising administering to the subject (i) the antigen, and (ii) an anti-TIGIT antibody or its antigen-binding fragment, such that the immune response to the antigen in the subject is enhanced. The antigen can be, for example, a tumor antigen, a viral antigen, a bacterial antigen, or an antigen derived from a pathogen. Non-limiting examples of such antigens include those discussed in the above sections, such as the tumor antigens (or tumor vaccines) discussed above or antigens derived from the above viruses, bacteria, or other pathogens.

[0314] In certain embodiments, a peptide or fusion protein comprising an epitope to which an anti-TIGIT antibody binds is used as a vaccine instead of, or in addition to, an anti-TIGIT antibody.

[0315] Suitable routes of administration of the antibody compositions (e.g., human monoclonal antibodies, multispecific and bispecific molecules, and immunoconjugates) described herein, in vivo and in vitro, are well known in the art and can be selected by one of ordinary skill in the art. For example, antibody compositions can be administered by injection (e.g., intravenously or subcutaneously). Suitable dosages of the molecules used will vary depending on the age and weight of the subject and the concentration and / or formulation of the antibody composition.

[0316] As previously described, the anti-TIGIT antibodies described herein can be co-administered with one or more therapeutic agents, such as cytotoxic agents, radiotoxic agents, or immunosuppressants. The antibody can be linked to the agent (as an immunoconjugate) or administered separately. In the latter case (separate administration), the antibody can be administered before, after, or simultaneously with the agent, or can be co-administered with other known therapies, such as anti-cancer therapies, such as radiation. Such therapeutic agents include, among others, anti-tumor and anti-neoplastic agents, such as doxorubicin (adriamycin), cisplatin, bleomycin sulfate, carmustine, chlorambucil, dacarbazine, and cyclophosphamide hydroxyurea, which are themselves effective only at levels that are toxic or subtoxic to patients. Cisplatin is administered intravenously at a dose of 100 mg / ml once every four weeks, and adriamycin is administered intravenously at a dose of 60-75 mg / ml once every 21 days. Co-administration of an anti-TIGIT antibody or its antigen-binding fragment with a chemotherapeutic drug provides two anti-cancer agents that operate by different mechanisms to produce a cytotoxic effect on human tumor cells. Such co-administration may solve problems due to the development of resistance to the drug or changes in the antigenicity of tumor cells that make them unresponsive to the antibody.

[0317] Also within the scope described herein are kits comprising the antibody compositions described herein (e.g., human antibodies, bispecific or multispecific molecules, or immunoconjugates) and instructions for use. The kits may further contain at least one additional reagent or one or more additional human antibodies described herein (e.g., human antibodies with complementary activity that bind to an epitope in the TIGIT antigen distinct from that of the first human antibody). The kits will typically include labeling indicating the intended use of the contents of the kit. The term labeling includes any writing or recorded material supplied on or with the kit, or that otherwise accompanies the kit.

[0318] Combination therapy In addition to the combination therapies provided above, the anti-TIGIT antibodies described herein can also be used in combination therapies to treat cancer, for example, as described below.

[0319] The present invention provides a method of combination therapy in which an anti-TIGIT antibody is co-administered with one or more additional agents, such as an antibody that is effective in stimulating an immune response, thereby further enhancing, stimulating, or upregulating the immune response in a subject. As shown in Example 7 and shown in Figures 5A and 5B, administration of an antagonistic anti-TIGIT antibody and an antagonistic anti-PD-1 antibody to mice had an enhanced effect in inhibiting tumor growth.

[0320] Generally, the anti-TIGIT antibodies described herein can be combined with (i) agonists of costimulatory receptors and / or (ii) antagonists of inhibitory signals in T cells, either of which results in the amplification of antigen-specific T cell responses (immune checkpoint regulators). Most costimulatory and costimulatory molecules are members of the immunoglobulin superfamily (IgSF), and the anti-TIGIT antibodies described herein may be administered with agents that target members of the IgSF family and enhance immune responses. One important family of membrane-bound ligands that bind to costimulatory or costimulatory receptors is the B7 family, which includes B7-1, B7-2, B7-H1 (PD-L1), B7-DC (PD-L2), B7-H2 (ICOS-L), B7-H3, B7-H4, B7-H5 (VISTA), and B7-H6. Another family of membrane-bound ligands that bind to costimulatory or costimulatory receptors is the TNF family of molecules that bind to cognate TNF receptor family members, including CD40 and CD40L, OX-40, OX-40L, CD70, CD27L, CD30, CD30L, 4-1BBL, CD137 / 4-1BB, TRAIL / Apo2-L, TRAILR1 / DR4, TRAILR2 / DR5, TRAILR3, TRAILR4, OPG, RANK, These include RANKL, TWEAKR / Fn14, TWEAK, BAFFR, EDAR, XEDAR, TACI, APRIL, BCMA, LTβR, LIGHT, DcR3, HVEM, VEGI / TL1A, TRAMP / DR3, EDAR, EDA1, XEDAR, EDA2, TNFR1, lymphotoxin alpha / TNFβ, TNFR2, TNFα, LTβR, lymphotoxin alpha 1β2, FAS, FASL, RELT, DR6, TROY, and NGFR (see, e.g., Tansey (2009) Drug Discovery Today 00:1).

[0321] T cell activation is also regulated by soluble cytokines. Thus, anti-TIGIT antibodies can be used in combination with (i) antagonists (or inhibitors or blockers) of proteins of the IgSF family, B7 family, or TNF family that inhibit T cell activation, or antagonists of cytokines that inhibit T cell activation (e.g., IL-6, IL-10, TGF-β, VEGF, or other immunosuppressive cytokines), and / or (ii) agonists of stimulatory receptors of the IgSF family, B7 family, or TNF family, or of cytokines that stimulate T cell activation, to stimulate an immune response, e.g., to treat proliferative diseases such as cancer.

[0322] In one aspect, the T cell response is achieved by combining an anti-TIGIT mAb of the invention with (i) CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, TIM-3, Galectin-9, CEACAM-1, BTLA, CD69, Galectin-1, CD113, GPR56, VISTA, 2B4, CD48, GARP, PD1H, LAIR1, TIM-1, CD96 (WO2015 / 024060; Bernhardt et al. (2014) Nat. Immunol. 15:406) and TIM-4, and (ii) an antagonist of a protein that inhibits T cell activation (e.g., an immune checkpoint inhibitor), and an agonist of a protein that stimulates T cell activation, such as B7-1, B7-2, CD28, 4-1BB (CD137), 4-1BBL, ICOS, CD40, ICOS-L, OX40, OX40L, GITR, GITRL, CD70, CD27, CD40, DR3, and CD28H.

[0323] Exemplary agents that modulate one of the above proteins and may be combined with agonist anti-TIGIT antibodies, such as those described herein, to treat cancer include YERVOY® / ipilimumab or tremelimumab (against CTLA-4), galiximab (against B7.1), OPDIVO® / nivolumab / BMS-936558 (against PD-1), pidilizumab / CT-011 (against PD-1), KEYTRUDA® / pembrolizumab / MK-3475 (against PD-1), AMP224 (against B7-DC / PD-L2), BMS-936559 (against B7-H1), MPDL3280A (against B7-H1), MEDI-570 (against ICO), and EGFR-1 (against ICO). S), AMG557 (against B7H2), MGA271 (against B7H3 - WO11 / 109400), IMP321 (against LAG-3), urelumab / BMS-663513 and PF-05082566 (against CD137 / 4-1BB), CDX-1127 (against CD27), MEDI-6383 and MEDI-6469 (against OX40), RG-7888 (against OX40L - WO06 / 029879), atacicept (against TACI), CP-870893 (against CD40), lucatumumab (against CD40), dacetuzumab (against CD40) and muromonab-CD3 (against CD3).

[0324] Other molecules that can be combined with antagonist anti-TIGIT antibodies to treat cancer include antagonists of inhibitory receptors on NK cells or agonists of activating receptors on NK cells. For example, antagonist anti-TIGT antibodies can be combined with KIR antagonists (e.g., lirilumab).

[0325] Still other agents for combination therapy include agents that inhibit or deplete macrophages or monocytes, CSF-1R antagonists such as CSF-1R antagonist antibodies, including, but not limited to, RG7155 (WO11 / 70024, WO11 / 107553, WO11 / 131407, WO13 / 87699, WO13 / 119716, WO13 / 132044) or FPA-008 (WO11 / 140249, WO13169264, WO14 / 036357).

[0326] In general, the antagonist anti-TIGIT antibodies described herein include one or more agonistic agents that ligate positive costimulatory receptors, blocking agents that attenuate signaling by inhibitory receptors, and one or more agents that systemically increase the frequency of anti-tumor T cells, overcome the respective immunosuppressive pathways within the tumor microenvironment (e.g., by blocking the engagement of inhibitory receptors (e.g., PD-L1 / PD-1 interactions) and inhibit T cells). reg can be used in conjunction with agents that deplete or inhibit (e.g., using anti-CD25 monoclonal antibodies (e.g., daclizumab) or by ex vivo anti-CD25 bead depletion), inhibit metabolic enzymes such as IDO, or reverse / prevent T cell anergy or exhaustion) and agents that trigger innate immune activation and / or inflammation at the tumor site.

[0327] Provided herein are methods for stimulating an immune response in a subject, comprising administering to the subject an antagonistic anti-TIGIT molecule, e.g., an antibody, and one or more additional immunostimulatory antibodies, such as a PD-1 antagonist, e.g., an antagonistic antibody, a PD-L1 antagonist, e.g., an antagonistic antibody, a CTLA-4 antagonist, e.g., an antagonistic antibody, and / or a LAG3 antagonist, e.g., an antagonistic antibody, so as to stimulate an immune response in the subject, for example, to inhibit tumor growth or stimulate an antiviral response. In one embodiment, the subject is administered an antagonistic anti-TIGIT antibody and an antagonistic anti-PD-1 antibody. In one embodiment, the subject is administered an antagonistic anti-TIGIT antibody and an antagonistic anti-PD-L1 antibody. In one embodiment, the subject is administered an antagonistic anti-TIGIT antibody and an antagonistic anti-CTLA-4 antibody. In one embodiment, at least one additional immunostimulatory antibody (e.g., anti-PD-1, anti-PD-L1, anti-CTLA-4 and / or anti-LAG3) is a human antibody. Alternatively, the at least one additional immunostimulatory antibody can be, e.g., a chimeric or humanized antibody prepared from, e.g., a murine anti-PD-1, anti-PD-L1, anti-CTLA-4 and / or anti-LAG3 antibody.

[0328] Provided herein is a method for treating a hyperproliferative disease (e.g., cancer) comprising administering to a subject an antagonistic anti-TIGIT antibody and an antagonistic PD-1 antibody. TIGIT and PD-1 are co-expressed in melanoma (Chauvin et al. (2015) J. Clin. Invest. 125:2046), and also in CD8 cells derived from non-small cell lung cancer (NSCLC) and renal cell carcinoma (RCC) patients. + Co-expressed at relatively high levels in TILS. See Table 2 (total CD3 + CD8 + TIGIT as a percentage of TILS + / PD-1 + Percentages of cells are shown).

[0329] [Table 2]

[0330] In certain embodiments, the anti-TIGIT antibody is administered at a subtherapeutic dose, the anti-PD-1 antibody is administered at a subtherapeutic dose, or both are administered at subtherapeutic doses. Also provided herein is a method for modifying adverse events associated with the treatment of a hyperproliferative disease with an immunostimulatory agent, comprising administering to a subject an anti-TIGIT antibody and an anti-PD-1 antibody at a subtherapeutic dose. In certain embodiments, the subject is a human. In certain embodiments, the anti-PD-1 antibody is a human sequence monoclonal antibody, and the anti-TIGIT antibody is a human sequence monoclonal antibody, such as an antibody comprising the CDRs or variable regions of an antibody disclosed herein.

[0331] In one embodiment, only subjects with tumors that exhibit high PVR and / or Nectin-2 expression and high PD-L1 expression are selected for combination therapy with the anti-TIGIT antibody of the present invention and a PD-1 antagonist. In another embodiment, subjects with tumors that exhibit high PVR and / or Nectin-2 expression but low PD-L1 expression are selected for monotherapy with the anti-TIGIT antibody of the present invention or combination therapy with another therapeutic agent other than a PD-1 antagonist.

[0332] In other embodiments, the present invention provides a combination therapy in which an anti-TIGIT antibody of the present invention is administered following treatment with a PD-1 / PD-L1 antagonist. In one embodiment, the anti-TIGIT antibody is administered only after treatment with a PD-1 / PD-L1 antagonist has failed, resulting in an incomplete therapeutic response, or tumor recurrence or relapse (referred to herein as "PD-1 failure"). In a further embodiment, tumors in such PD-1 failure cases are screened for PVR and / or Nectin-2 expression, and only those with high-level expression are treated with an anti-TIGIT antibody.

[0333] Suitable PD-1 antagonists for use in the methods described herein include, but are not limited to, ligands, antibodies (e.g., monoclonal antibodies and bispecific antibodies), and multivalent agents. In one embodiment, the PD-1 antagonist is a fusion protein, e.g., an Fc fusion protein such as AMP-244. In one embodiment, the PD-1 antagonist is an anti-PD-1 or anti-PD-L1 antibody.

[0334] Exemplary anti-PD-1 antibodies include OPDIVO® / nivolumab (BMS-936558) or antibodies comprising the CDRs or variable regions of one of antibodies 17D8, 2D3, 4H1, 5C4, 7D3, 5F4, and 4A11, described in WO2006 / 121168. In certain embodiments, the anti-PD-1 antibody is MK-3475 (KEYTRUDA® / pembrolizumab / formerly lambrolizumab), described in WO2012 / 145493; AMP-514 / MEDI-0680, described in WO2012 / 145493; and CT-011 (pidilizumab; formerly CT-AcTibody or BAT; see, e.g., Rosenblatt et al. (2011) J. Immunotherapy 34:409). Further known PD-1 antibodies and other PD-1 inhibitors include those described in WO2009 / 014708, WO03 / 099196, WO2009 / 114335, WO2011 / 066389, WO2011 / 161699, WO2012 / 145493, U.S. Patent Nos. 7,635,757 and 8,217,149, and U.S. Patent Publication No. 2009 / 0317368. Any of the anti-PD-1 antibodies disclosed in WO2013 / 173223 may also be used. Anti-PD-1 antibodies that compete for binding with and / or bind to the same epitope on PD-1 as one of these antibodies may also be used in combination therapy.

[0335] Provided herein are methods for treating a hyperproliferative disease (e.g., cancer), comprising administering an antagonistic anti-TIGIT antibody and an antagonistic PD-L1 antibody to a subject. In certain embodiments, the anti-TIGIT antibody is administered at a subtherapeutic dose, the anti-PD-L1 antibody is administered at a subtherapeutic dose, or both are administered at subtherapeutic doses. Provided herein are methods for modifying adverse events associated with the treatment of a hyperproliferative disease with an immunostimulatory agent, comprising administering an anti-TIGIT antibody and a subtherapeutic dose of an anti-PD-L1 antibody to a subject. In certain embodiments, the subject is a human. In certain embodiments, the anti-PD-L1 antibody is a human sequence monoclonal antibody, and the anti-TIGIT antibody is a human sequence monoclonal antibody, such as an antibody comprising the CDRs or variable regions of an antibody disclosed herein.

[0336] In one embodiment, the anti-PD-L1 antibody is BMS-936559 (referred to as 12A4 in WO 2007 / 005874 and U.S. Patent No. 7,943,743), MSB0010718C (WO 2013 / 79174), or an antibody comprising the CDRs or variable regions of 3G10, 12A4, 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7, and 13G4, as described in PCT Publication No. WO 07 / 005874 and U.S. Patent No. 7,943,743. In a specific embodiment, the anti-PD-L1 antibody is MEDI4736 (also known as anti-B7-H1) or MPDL3280A (also known as RG7446). Any of the anti-PD-L1 antibodies disclosed in WO2013 / 173223, WO2011 / 066389, WO2012 / 145493, U.S. Patent Nos. 7,635,757 and 8,217,149, and U.S. Patent Publication No. 2009 / 145493 may be used. Anti-PD-L1 antibodies that compete with and / or bind to the same epitope as any of these antibodies may also be used in combination therapy.

[0337] In yet further embodiments, the agonist anti-huCD40 antibodies of the invention are combined with an antagonist of PD-1 / PD-L1 signaling, such as a PD-1 antagonist or a PD-L1 antagonist, in combination with a third immunotherapeutic agent. In one embodiment, the third immunotherapeutic agent is a GITR antagonist or an OX-40 antagonist, such as an anti-GITR or anti-OX40 antibody disclosed herein.

[0338] In another embodiment, the immuno-oncology agent is a GITR agonist, such as an agonist GITR antibody. Suitable GITR antibodies include, for example, BMS-986153, BMS-986156, TRX-518 (WO06 / 105021, WO09 / 009116), and MK-4166 (WO11 / 028683).

[0339] In another embodiment, the immuno-oncology agent is an IDO antagonist. Suitable IDO antagonists include, for example, INCB-024360 (WO2006 / 122150, WO07 / 75598, WO08 / 36653, WO08 / 36642), indoximod, or NLG-919 (WO09 / 73620, WO09 / 1156652, WO11 / 56652, WO12 / 142237).

[0340] Provided herein is a method for treating hyperproliferative diseases (e.g., cancer), comprising administering to a subject the anti-TIGIT antibody and CTLA-4 antagonist antibody described herein.In certain embodiments, the anti-TIGIT antibody is administered at a subtherapeutic dose, the anti-CTLA-4 antibody is administered at a subtherapeutic dose, or both are administered at subtherapeutic doses.Provided herein is a method for modifying adverse events associated with the treatment of hyperproliferative diseases using immunostimulatory agents, comprising administering to a subject an anti-TIGIT antibody and a subtherapeutic dose of an anti-CTLA-4 antibody.In certain embodiments, the subject is a human. In certain embodiments, the anti-CTLA-4 antibody is YERVOY® (ipilimumab or antibody 10D1 described in PCT Publication WO 01 / 14424), tremelimumab (formerly ticilimumab, CP-675,206), and antibodies described in the following publications: WO 98 / 42752; WO 00 / 37504; U.S. Patent No. 6,207,156; Hurwitz et al. (1998) Proc. Natl. Acad. Sci. USA 95(17):10067-10071; Camacho et al. (2004) J. Clin. Oncology 22(145):Abstract No. 2505 (antibody CP-675206); and Mokyr et al. (1998) Cancer Res. 58:5301-5304. Any of the anti-CTLA-4 antibodies disclosed in WO2013 / 173223 may be used.

[0341] Provided herein is a method for treating a hyperproliferative disease (e.g., cancer) comprising administering an anti-TIGIT antibody and an anti-LAG-3 antibody to a subject. In a further embodiment, the anti-TIGIT antibody is administered at a subtherapeutic dose, the anti-LAG-3 antibody is administered at a subtherapeutic dose, or both are administered at subtherapeutic doses. Provided herein is a method for modifying adverse events associated with the treatment of a hyperproliferative disease with an immunostimulatory agent comprising administering an anti-TIGIT antibody and a subtherapeutic dose of an anti-LAG-3 antibody to a subject. In certain embodiments, the subject is a human. In certain embodiments, the anti-LAG-3 antibody is a human sequence monoclonal antibody, and the anti-TIGIT antibody is a human sequence monoclonal antibody, such as an antibody comprising the CDRs or variable regions of the antibodies disclosed herein. Examples of anti-LAG-3 antibodies include antibodies comprising the CDRs or variable regions of antibodies 25F7, 26H10, 25E3, 8B7, 11F2, or 17E5, as described in U.S. Patent Publications US2011 / 0150892 and WO2014 / 008218. In one embodiment, the anti-LAG-3 antibody is BMS-986016. Other art-recognized anti-LAG-3 antibodies that can be used include IMP731, as described in US2011 / 007023. IMP-321 may also be used. Anti-LAG-3 antibodies that compete with and / or bind to the same epitope as any of these antibodies may also be used in combination therapy.

[0342] Administration of the anti-TIGIT antibody and antagonist, for example, antagonist antibody, described herein to one or more second target antigens, such as LAG-3 and / or CTLA-4 and / or PD-1 and / or PD-L1, can enhance the immune response against cancerous cells in patients.Cancers whose growth can be inhibited using the antibodies of the present disclosure include cancers that are generally responsive to immunotherapy.Representative examples of cancers for treatment using the combination therapy of the present disclosure include the cancers specifically listed above in the discussion of monotherapy using anti-TIGIT antibodies.

[0343] In certain embodiments, the therapeutic antibody combinations discussed herein can be administered simultaneously as a single composition in a pharmaceutically acceptable carrier, or as separate compositions containing each antibody in a pharmaceutically acceptable carrier. In other embodiments, the therapeutic antibody combinations can be administered separately. For example, an anti-CTLA-4 antibody and an anti-TIGIT antibody can be administered sequentially, such as administering the anti-CTLA-4 antibody first and the anti-TIGIT antibody second, or administering the anti-TIGIT antibody first and the anti-CTLA-4 antibody second. Additionally or alternatively, an anti-PD-1 antibody and an anti-TIGIT antibody can be administered sequentially, such as administering the anti-PD-1 antibody first and the anti-TIGIT antibody second, or administering the anti-TIGIT antibody first and the anti-PD-1 antibody second. Additionally or alternatively, the anti-PD-L1 antibody and the anti-TIGIT antibody can be administered sequentially, with the anti-PD-L1 antibody being administered first and the anti-TIGIT antibody being administered second, or the anti-TIGIT antibody being administered first and the anti-PD-L1 antibody being administered second, etc. Additionally or alternatively, the anti-LAG-3 antibody and the anti-TIGIT antibody can be administered sequentially, with the anti-LAG-3 antibody being administered first and the anti-TIGIT antibody being administered second, or the anti-TIGIT antibody being administered first and the anti-LAG-3 antibody being administered second.

[0344] Furthermore, when two or more doses of a combination therapy are administered sequentially, the order of sequential administration can be reversed at each administration time point, or the same order can be maintained, or sequential administration can be combined with simultaneous administration or any combination thereof. For example, the first administration of a combined anti-CTLA-4 antibody and an anti-TIGIT antibody can be simultaneous, the second administration can be sequential with the anti-CTLA-4 antibody being the first and the anti-TIGIT antibody being the second, the third administration can be sequential with the anti-TIGIT antibody being the first and the anti-CTLA-4 antibody being the second, etc. Additionally or alternatively, the first administration of a combined anti-PD-1 antibody and an anti-TIGIT antibody can be simultaneous, the second administration can be sequential with the anti-PD-1 antibody being the first and the anti-TIGIT antibody being the second, the third administration can be sequential with the anti-TIGIT antibody being the first and the anti-PD-1 antibody being the second, etc. Additionally or alternatively, the first administration of a combined anti-PD-L1 antibody and anti-TIGIT antibody may be simultaneous, the second administration may be sequential with the anti-PD-L1 antibody being the first and the anti-TIGIT antibody being the second, the third administration may be sequential with the anti-TIGIT antibody being the first and the anti-PD-L1 antibody being the second, etc. Additionally or alternatively, the first administration of a combined anti-LAG-3 antibody and anti-TIGIT antibody may be simultaneous, the second administration may be sequential with the anti-LAG-3 antibody being the first and the anti-TIGIT antibody being the second, and the third administration may be sequential with the anti-TIGIT antibody being the first and the anti-LAG-3 antibody being the second. Another exemplary dosing scheme may include a first administration where anti-TIGIT is the first and anti-CTLA-4 antibody (and / or anti-PD-1 antibody and / or anti-PD-L1 antibody and / or anti-LAG-3 antibody) is the second and sequential, with subsequent administrations being simultaneous.

[0345] Optionally, the combination of anti-TIGIT or anti-TIGIT antibody as the sole immunotherapeutic agent and one or more additional immunotherapeutic antibodies (e.g., anti-CTLA-4 and / or anti-PD-1 and / or anti-PD-L1 and / or anti-LAG-3 blocking) can be further combined with immunogens such as cancerous cells, purified tumor antigens (including recombinant proteins, peptides, and carbohydrate molecules), cells, and cells transfected with genes encoding immunostimulatory cytokines (He et al. (2004) J. Immunol. 173:4919-28). Non-limiting examples of tumor vaccines that can be used include tumor cells transfected to express peptides of melanoma antigens, such as gp100, MAGE antigens, Trp-2, MART1, and / or tyrosinase peptides, or the cytokine GM-CSF (discussed further below). A TIGIT inhibitor and one or more additional antibodies (e.g., CTLA-4 and / or PD-1 and / or PD-L1 and / or LAG-3 blockade) can also be further combined with standard cancer treatment. For example, a TIGIT inhibitor and one or more additional antibodies (e.g., CTLA-4 and / or PD-1 and / or PD-L1 and / or LAG-3 blockade) can be effectively combined with a chemotherapy treatment plan. In these cases, it is possible to reduce the dose of other chemotherapy reagents administered together with the combination of the present disclosure (Mokyr et al. (1998) Cancer Research 58: 5301-5304). An example of such a combination is a combination of an anti-TIGIT agonist antibody with or without an additional antibody, such as an anti-CTLA-4 antibody and / or an anti-PD-1 antibody and / or an anti-PD-L1 antibody and / or an anti-LAG-3 antibody, further combined with decarbazine for the treatment of melanoma. Another example is the combination of an anti-TIGIT antibody, with or without an anti-CTLA-4 antibody and / or an anti-PD-1 antibody and / or an anti-PD-L1 antibody and / or an LAG-3 antibody, further combined with interleukin-2 (IL-2) for the treatment of melanoma.The scientific rationale behind the combined use of TIGIT inhibition and CTLA-4 and / or PD-1 and / or PD-L1 and / or LAG-3 blockade with chemotherapy is that cell death, a result of the cytotoxic effects of most chemotherapy compounds, should lead to increased levels of tumor antigens in the antigen presentation pathway. Other combination therapies that may synergize with combined TIGIT inhibition, with or without CTLA-4 and / or PD-1 and / or PD-L1 and / or LAG-3 blockade due to cell death, include radiation, surgery, or hormone deprivation. Each of these protocols creates a source of tumor antigens in the host. Angiogenesis inhibitors can also be combined with combined TIGIT inhibition and CTLA-4 and / or PD-1 and / or PD-L1 and / or LAG-3 blockade. Inhibition of angiogenesis leads to tumor cell death, which can provide a source of tumor antigens for the host's antigen presentation pathway.

[0346] The combination of an anti-TIGIT antagonist antibody or a TIGIT antagonist and a CTLA-4 and / or PD-1 and / or PD-L1 and / or LAG-3 blocking antibody as the sole immunotherapeutic agent can also be used in combination with a bispecific antibody that targets Fcα or Fcγ receptor-expressing effector cells against tumor cells (e.g., U.S. Patent Nos. 5,922,845 and 5,837,243). Bispecific antibodies can be used to target two distinct antigens. The T cell arm of these responses is enhanced by the use of combined TIGIT inhibition and CTLA-4 and / or PD-1 and / or PD-L1 and / or LAG-3 blockade.

[0347] In another example, a combination of an anti-TIGIT antagonist antibody or anti-TIGIT antibody as the sole immunotherapeutic agent and an additional immunostimulatory agent, e.g., an anti-CTLA-4 antibody and / or an anti-PD-1 antibody and / or an anti-PD-L1 antibody and / or an LAG-3 agent, e.g., an antibody, can be used with an anti-tumor, anti-neoplastic antibody, such as RITUXAN® (rituximab), HERCEPTIN® (trastuzumab), BEXAR® (tositumomab), ZEVALIN® (ibritumomab), CAMPA® (alemtuzumab), LYMPHOCIDE® (eprtuzumab), Avastin® (bevacizumab), and TARCEVA® (erlotinib). By way of example, and not wishing to be bound by theory, treatment with an anti-cancer antibody or an anti-cancer antibody conjugated to a toxin can lead to cancer cell death (e.g., tumor cells), which enhances the immune response mediated by an immunostimulatory agent, such as TIGIT, CTLA-4, PD-1, PD-L1, or LAG-3 agent, e.g., antibody. In an exemplary embodiment, treatment of a hyperproliferative disease (e.g., cancer tumor) can include an anti-cancer agent, e.g., an antibody, simultaneously, sequentially, or any combination thereof, in combination with anti-TIGIT and, optionally, additional immunostimulatory agents, such as anti-CTLA-4 and / or anti-PD-1 and / or anti-PD-L1 and / or anti-LAG-3 agents, e.g., antibodies, which can enhance the anti-tumor immune response by the host.

[0348] Tumors evade host immune surveillance through a variety of mechanisms. Many of these mechanisms can be overcome by inactivating proteins expressed by tumors that are immunosuppressive. These include, among others, TGF-β (Kehrl et al. (1986) J. Exp. Med. 163: 1037-1050), IL-10 (Howard & O'Garra (1992) Immunology Today 13: 198-200), and Fas ligand (Hahne et al. (1996) Science 274: 1363-1365). Antibodies to each of these entities can be further combined with anti-TIGIT antibodies, with or without additional immunostimulatory agents, e.g., anti-CTLA-4 and / or anti-PD-1 and / or anti-PD-L1 and / or anti-LAG-3 agents, e.g., antibodies, to counter the effects of immunosuppressants and favor an anti-tumor immune response by the host.

[0349] Other agents, e.g., antibodies, that can be used to activate host immune responsiveness can further be used in combination with anti-TIGIT antibodies, with or without additional immunostimulatory agents, such as anti-CTLA-4 and / or anti-PD-1 and / or anti-PD-L1 and / or anti-LAG-3 antibodies. These include molecules on the surface of dendritic cells that activate DC function and antigen presentation. Anti-CD40 antibodies (Ridge et al., supra) can be used with anti-TIGIT antibodies and, optionally, additional immunostimulatory agents, such as anti-CTLA-4 and / or anti-PD-1 and / or anti-PD-L1 and / or anti-LAG-3 agents, e.g., antibodies. Other activating antibodies against the T cell costimulatory molecules OX-40 (Weinberg et al. (2000) Immunol 164:2160-2169), CD137 / 4-1BB (Melero et al. (1997) Nature Medicine 3:682-685 (1997)), and ICOS (Hutloff et al. (1999) Nature 397:262-266) also provide increased levels of T cell activation.

[0350] As discussed above, bone marrow transplantation is currently used to treat a variety of tumors of hematopoietic origin. Anti-TIGIT immunotherapy can be used alone or in combination with CTLA-4 and / or PD-1 and / or PD-L1 and / or LAG-3 blockade to enhance the efficacy of tumor-specific T cells transplanted from the donor.

[0351] Some experimental therapeutic protocols involve ex vivo activation and expansion of antigen-specific T cells and adoptive transfer of these cells into a recipient for tumor-directed antigen-specific T cells (Greenberg & Riddell, supra). These methods can also be used to activate T cell responses to infectious pathogens such as CMV. Ex vivo activation in the presence of anti-TIGIT, with or without additional immunostimulatory therapy, e.g., anti-CTLA-4 and / or anti-PD-1 and / or anti-PD-L1 and / or anti-LAG-3 antibodies, can be expected to increase the frequency and activity of adoptively transferred T cells.

[0352] Provided herein are methods for modifying adverse events associated with the treatment of hyperproliferative diseases (e.g., cancer) with immunostimulatory agents, comprising administering an anti-TIGIT antibody to a subject with or without a subtherapeutic dose of an anti-CTLA-4 and / or anti-PD-1 and / or anti-PD-L1 and / or anti-LAG-3 agent, e.g., an antibody. For example, the methods described herein provide a method for reducing the incidence of immunostimulatory therapeutic antibody-induced colitis or diarrhea by administering a non-absorbed steroid to a patient. As used herein, a "non-absorbed steroid" refers to a glucocorticoid that exhibits extensive first-pass metabolism, resulting in low steroid bioavailability, i.e., less than about 20%, after metabolism in the liver. In one embodiment described herein, the non-absorbed steroid is budesonide. Budesonide is a locally active glucocorticoid that is extensively metabolized, primarily by the liver, after oral administration. ENTOCORT EC® (Astra-Zeneca) is a pH- and time-dependent oral formulation of budesonide developed to optimize drug delivery to the ileum and entire colon. ENTOCORT EC® is approved in the United States for the treatment of mild to moderate Crohn's disease involving the ileum and / or ascending colon. The usual oral dose of ENTOCORT EC® for the treatment of Crohn's disease is 6-9 mg / day. ENTOCORT EC® is released in the intestinal tract and subsequently absorbed and retained in the intestinal mucosa. Upon passing through the intestinal mucosal target tissue, ENTOCORT EC® is extensively metabolized in the liver by the cytochrome P450 system to metabolites with negligible glucocorticoid activity. Therefore, bioavailability is low (approximately 10%). Budesonide's low bioavailability results in an improved therapeutic ratio compared to other glucocorticoids with less extensive first-pass metabolism. Budesonide produces fewer adverse effects, including less hypothalamic-pituitary suppression, than systemically active corticosteroids.However, chronic administration of ENTOCORT EC® can result in systemic glucocorticoid effects, such as systemic hypercorticism and adrenal suppression. See PDR 58th ed. 2004; 608-610.

[0353] In a further embodiment, TIGIT inhibition, with or without CTLA-4 and / or PD-1 and / or PD-L1 and / or LAG-3 blockade (i.e., immunostimulatory therapeutic antibodies anti-TIGIT and, optionally, anti-CTLA-4 and / or anti-PD-1 and / or anti-PD-L1 and / or anti-LAG-3 antibodies), along with non-absorbable steroids, can be further combined with salicylic acid, including, for example, 5-ASA drugs such as sulfasalazine (AZULFIDINE®, Pharmacia & UpJohn); olsalazine (DIPENTUM®, Pharmacia & UpJohn); balsalazide (COLAZAL®, Salix Pharmaceuticals, Inc.); and mesalamine (ASACOL®, Procter & Gamble Pharmaceuticals; PENTASA®, Shire US; CANASA®, Axcan Scandipharm, Inc.; ROWASA®, Solvay).

[0354] According to the methods described herein, the salicylic acid administered in combination with anti-TIGIT, with or without anti-CTLA-4 and / or anti-PD-1 and / or anti-PD-L1 and / or LAG-3 antibodies and non-absorbable steroids, can include any overlapping or sequential administration of salicylic acid and non-absorbable steroids for the purpose of reducing the incidence of colitis induced by immunostimulatory antibodies. Thus, for example, the methods described herein for reducing the incidence of colitis induced by immunostimulatory antibodies include simultaneous or sequential administration of salicylic acid and non-absorbable steroids (e.g., salicylic acid is administered 6 hours after non-absorbable steroids), or any combination thereof. Furthermore, the salicylate and the non-absorbable steroid can be administered by the same route (e.g., both orally) or by different routes (e.g., the salicylate is administered orally and the non-absorbable steroid is administered rectally), which may be different from the route(s) used to administer the anti-TIGIT and anti-CTLA-4 and / or anti-PD-1 and / or anti-PD-L1 and / or anti-LAG-3 antibodies.

[0355] The anti-TIGIT antibodies and combination antibody therapies described herein may be used in conjunction with other known therapies selected for their particular usefulness for the indication (e.g., cancer) being treated. The anti-TIGIT antibody combinations described herein may also be used sequentially with known pharmaceutically acceptable drug(s).

[0356] For example, the anti-TIGIT antibodies and combination antibody therapies described herein may be administered in combination with radiation, chemotherapy (e.g., camptothecin (CPT-11), 5-fluorouracil (5-FU), cisplatin, doxorubicin, irinotecan, paclitaxel, gemcitabine, cisplatin, paclitaxel, carboplatin-paclitaxel (Taxol), doxorubicin, 5-fu, or camptothecin plus apo2l / TRAIL (6X)). combination), one or more proteasome inhibitors (e.g., bortezomib or MG132), one or more Bcl-2 inhibitors (e.g., BH3I-2' (bcl-xl inhibitor), indoleamine dioxygenase-1 (IDO1) inhibitors (e.g., INCB24360), AT-101 (R-(-)-gossypol derivative), ABT-263 (small molecule), GX-15-070 (obatoclax) or MCL-1 (myeloid leukemia cell differentiation protein-1) antagonist), iAP (inhibitor of apoptosis protein) antagonists (e.g., smac7, smac4, small molecule smac mimetics, synthetic smac peptides (Fulda et al., Nat Med 2002;8:808-15), ISIS23722 (LY2181308) or AEG-35156 (GEM-640)), HDAC (histone deacetylase) inhibitors, anti-CD20 antibodies (e.g., rituximab), angiogenesis inhibitors (e.g., bevacizumab), anti-angiogenic agents targeting VEGF and VEGFR (e.g., Avastin®), synthetic triterpenoids (Hyer et al., Cancer Research 2005;65:4799-808), c-FLIP (cellular FLICE inhibitory protein) modulators (e.g., natural and synthetic ligands of PPARγ (peroxisome proliferator-activated receptor gamma), 5809354 or 5569100), kinase inhibitors (e.g., sorafenib), mTOR inhibitors such as trastuzumab, cetuximab, temsirolimus, rapamycin and temsirolimus, bortezomib, JAK2 inhibitors, HSP90 inhibitors, PI3K-AKT inhibitors, lenalidomide, GSK3β inhibitors, IAP inhibitors and / or genotoxic drugs.

[0357] The anti-TIGIT antibodies and combination antibody therapies described herein can further be used in combination with one or more anti-proliferative cytotoxic agents. Classes of compounds that may be used as anti-proliferative cytotoxic agents include, but are not limited to:

[0358] Alkylating agents (including but not limited to nitrogen mustards, ethyleneimine derivatives, alkylsulfonates, nitrosoureas, and triazenes): uracil mustard, chlormethine, cyclophosphamide (CYTOXAN™), phosphamide, melphalan, chlorambucil, pipobroman, triethylenemelamine, triethylenethiophosphoramine, busulfan, carmustine, lomustine, streptozocin, dacarbazine, and temozolomide.

[0359] Antimetabolites (including but not limited to folate antagonists, pyrimidine analogs, purine analogs and adenosine deaminase inhibitors): methotrexate, 5-fluorouracil, floxuridine, cytarabine, 6-mercaptopurine, 6-thioguanine, fludarabine phosphate, pentostatin and gemcitabine.

[0360] In addition to other microtubulin inhibitors known in the art, antagonistic anti-TIGIT antibodies, including but not limited to taxanes, paclitaxel (paclitaxel is also known as TAXOL TM (commercially available as cyclopropyl-1,1-dimethyl-2,1-propanol), docetaxel, discodermolide (DDM), dictyostatin (DCT), peloruside A, epothilone, epothilone A, epothilone B, epothilone C, epothilone D, epothilone E, epothilone F, furanoepothilone D, desoxyepothilone B,

[17] -dehydrodesoxyepothilone B,

[18] -dehydrodesoxyepothilone B, C12,13-cyclopropyl-epothilone A, C6-C8 bridged epothilone A, trans-9, 10-Dehydroepothilone D, cis-9,10-dehydroepothilone D, 16-desmethylepothilone B, epothilone B10, discoderomolide, patupilone (EPO-906), KOS-862, KOS-1584, ZK-EPO, ABJ-789, XAA296A (discoderomolide), TZT-1027 (sobridotin), ILX-651 (tasidotin hydrochloride), halichondrin B, eribulin mesylate mesylate (E-7389), hemiasterin (HTI-286), E-7974, cryptophycin, LY-355703, maytansinoid immunoconjugate (DM-1), MKC-1, ABT-751, T1-38067, T-900607, SB-715992 (ispinesib), SB-743921, MK-0731, STA-5312 Suitable antiproliferative agents for combination with erytherobin, 17β-acetoxy-2-ethoxy-6-oxo-B-homo-estra-1,3,5(10)-trien-3-ol, cyclostreptin, isolaulimalide, laulimalide, 4-epi-7-dehydroxy-14,16-didemethyl-(+)-discodermolide and cryptothilone 1.

[0361] If it is desired to render abnormally proliferative cells quiescent prior to or in conjunction with treatment with the anti-TIGIT antibodies described herein, hormones and steroids (including synthetic analogs), such as 17a-ethynylestradiol, diethylstilbestrol, testosterone, prednisone, fluoxymesterone, dromostanolone propionate, testolactone, megestrol acetate, methylprednisolone, methyl-testosterone, prednisolone, triamcinolone, chlorotrianisene, hydroxyprogesterone, aminoglutethimide, estramustine, medroxyprogesterone acetate, leuprolide, flutamide, toremifene, ZOLADEX™, and the like, can also be administered to the patient. When using the methods or compositions described herein, other agents used in modulating tumor growth or metastasis in clinical settings, such as antiemetics, can also be administered as desired.

[0362] Methods for the safe and effective administration of chemotherapeutic agents are known to those skilled in the art. Furthermore, their administration is described in standard literature. For example, the administration of many chemotherapeutic agents is described in the Physicians' Desk Reference (PDR), e.g., the 1996 edition (Medical Economics Company, Montvale, NJ 07645-1742, USA), the disclosure of which is incorporated herein by reference.

[0363] The chemotherapeutic agent(s) and / or radiation therapy can be administered according to a treatment protocol known in the art. It will be clear to those skilled in the art that the administration of the chemotherapeutic agent(s) and / or radiation therapy can vary depending on the disease being treated and the known effects of the chemotherapeutic agent(s) and / or radiation therapy on that disease. In addition, the treatment protocol (e.g., dosage and time of administration) can be varied according to the knowledge of a skilled clinician, taking into account the observed effects of the administered therapeutic agent on the patient and the observed response of the disease to the administered therapeutic agent.

[0364] Patient Selection In various embodiments of the present invention, patients are tested to determine whether they are likely to respond to anti-TIGIT therapy before being treated with the anti-TIGIT antibodies of the present invention, and only those exhibiting traits associated with a therapeutic response are treated. Expression of proteins associated with the TIGIT pathway, including TIGIT, DNAM, PVR, nectin-2, soluble PVR (sPVR), and soluble nectin-2 (sNectin-2), or a combination thereof, may be measured. Both PVR and nectin-2 mRNA are highly expressed in the majority of human tumors. See Example 9 and Figure 6A. In one embodiment, sPVR and / or sNectin-2 are detected in human serum, e.g., by ELISA, where elevated levels of sPVR and / or sNectin-2 indicate subjects with cancer who are likely to respond to treatment with the anti-TIGIT antibodies of the present invention.

[0365] In some embodiments, to select patients most likely to respond to anti-TIGIT therapy, samples from patients are screened for expression of DNAM-1 in T cells, where the presence of DNAM-1 in T cells or NK cells suggests that the patient will have a beneficial anti-tumor response upon treatment with anti-TIGIT therapy, e.g., an anti-huTIGIT antibody or fragment of the present invention, and the absence of DNAM-1 in T cells or NK cells indicates a patient less likely to benefit from anti-TIGIT therapy. In other embodiments, samples obtained from patients are screened for expression of PVR and / or Nectin-2 / CD112 on tumor cells or tumor-infiltrating myeloid cells to select patients most likely to respond to anti-TIGIT therapy, where the presence of PVR and / or Nectin-2 / CD112 on tumor cells or tumor-infiltrating myeloid cells indicates that the patient will have a beneficial anti-tumor response upon treatment with anti-TIGIT therapy, e.g., an anti-huTIGIT antibody or fragment of the present invention, and the absence of PVR and / or Nectin-2 / CD112 on tumor cells or tumor-infiltrating myeloid cells identifies patients who are unlikely to benefit from anti-TIGIT therapy.

[0366] In one embodiment, the levels of soluble PVR and / or Nectin-2 are measured in patients being considered for treatment with the anti-TIGIT antibodies of the present invention, and only patients who exhibit elevated soluble PVR and / or Nectin-2 are treated with the antibody. For example, high soluble PVR and / or Nectin-2 may be used as patient selection biomarkers.

[0367] Tumor types and individual subjects are considered to be at risk if tumor cells express elevated levels of PVR and / or Nectin-2, and if such tumors express high levels of invasive TIGIT + CD8 + Those with T cells are most likely to respond to treatment with the anti-TIGIT antibodies of the present invention.

[0368] The present disclosure is further illustrated by the following examples, which should not be construed as further limiting. The contents of all figures and all references, Genbank sequences, patents and published patent applications cited throughout this application are expressly incorporated herein by reference. In particular, the disclosures of PCT Publications WO09 / 045957, WO09 / 073533, WO09 / 073546, WO09 / 054863 and PCT / US2013 / 072918 and US Patent Publication No. 2011 / 0150892 are expressly incorporated by reference. [Example]

[0369] Example 1 Generation of anti-huTIGIT antibodies Human anti-huTIGIT monoclonal antibodies were generated using transgenic mice expressing human antibody genes as follows.

[0370] antigen The huTIGIT soluble recombinant protein was used as an antigen for immunization. The soluble fusion protein has a MW of 40.7 kD and consists of the extracellular portion of huTIGIT linked to mouse IgG2a Fc at its C-terminus. This fusion protein is referred to herein as the "huTIGIT-muFc fusion protein." The fusion protein was produced by standard recombinant DNA methods and expressed in transfected CHO cells, which secreted the soluble fusion protein into the culture supernatant. The CHO host cells used for transfection were obtained from Invitrogen (catalog number 11619-012). The secreted soluble fusion protein was purified for use as an immunogen. The sequence of full-length human TIGIT, including the signal sequence, is provided in SEQ ID NO: 1.

[0371] transgenic mice A fully human monoclonal antibody against human TIGIT was administered to CHD patients. ** ;CKD2 **Mice were prepared using the following genotypes: ;CMD++;JKD++;KCo5(9272)+^;SC20+ (hereafter referred to as KM® mice). Individual transgene designations are in parentheses, followed by the lineage number of the randomly integrated transgene. The symbols ++ and + indicate homozygosity or hemizygosity, but because mice are routinely screened using PCR-based assays that do not allow for the distinction between heterozygosity and homozygosity for randomly integrated human Ig transgenes, the + designation may be given to mice that are actually homozygous for these elements. In this strain, the endogenous mouse kappa light chain gene is homozygously disrupted as described in Chen et al. (1993) EMBO J. 12:811-820, and the endogenous mouse heavy chain gene is homozygously disrupted as described in Example 1 of WO2001 / 09187. Additionally, this mouse strain carries a human kappa light chain transgene as described in Fishwild et al. (1996) Nature Biotechnology 14:845-851, KCo5, a yeast artificial chromosome (YAC) carrying most of the human kappa light chain locus as described in WO2000 / 026373.

[0372] Immunization of mice To generate fully human monoclonal antibodies against human TIGIT, KM mice were immunized with purified huTIGIT-muFc fusion protein. The general immunization scheme is described in Lonberg, N. et al. (1994) Nature 368(6474): 856-859; Fishwild, D. et al. (1996) Nature Biotechnology 14: 845-851 and WO98 / 24884. Mice were approximately 4 months old at the time of the first injection of antigen. Mice were immunized intraperitoneally and subcutaneously with either purified recombinant huTIGIT-muFc antigen preparation (10 μg purified from transfected mammalian cells expressing the fusion protein) or 300-19 cells transfected with human TIGIT. The immunogen was mixed 1:1 with RIBI adjuvant (Sigma catalog number M6536).

[0373] Mice were immunized five times, 5–7 days apart. The first and second immunizations were performed with recombinant protein. The third immunization was performed with cells, the fourth with protein, and the fifth with cells. One week after the final immunization, mice were bled to assess antigen-specific titers. Immune responses were monitored by retroorbital bleeding. Plasma was screened by FACS analysis using transfected 300-19 cells, and mice with the highest anti-human TIGIT human IgG titers were used for fusions. Mice received a final boost by intravenous (IV) and intraperitoneal (IP) injection of soluble antigen two days before sacrifice and transfected cells three days before spleen collection.

[0374] Generation of hybridomas producing human monoclonal antibodies against human TIGIT Mouse splenocytes isolated from high-titer KM mice and mouse myeloma fusion partners were fused using electric field-based electrofusion with a Cyto Pulse large-chamber cell fusion electroporator (Cyto Pulse Sciences, Inc., Glen Burnie, MD). Single-cell suspensions of splenic lymphocytes obtained from immunized mice were fused with an equal number of P3X63 Ag8.6.53 (ATCC CRL 1580) nonsecreting mouse myeloma cells (fusion number: 2541). The resulting cells were plated at 2.0 × 10 cells per well in flat-bottom microtiter plates in selective DMEM medium containing high glucose (Cellgro No. 10-013-CM) and 10% fetal bovine serum (Hyclone No. SH30071.03) supplemented with β-mercaptoethanol (1000×, Gibco No. 21985-023), 7 mM HEPES (Cellgro 25-060-Cl), an additional 2 mM L-glutamine (Cellgro 25-005-Cl), HAT (50×, Sigma No. H-0262), 5% hybridoma cloning factor (BioVeris No. 210001), 10% P388DI (ATCC No. CRL TIB-63) conditioned medium, and penicillin-streptomycin (100×, Cellgro No. 30-002-Cl). 4 After approximately 7 days, a portion of the medium containing HAT was replaced with medium containing HT (Cellgro #25-047-CI).

[0375] After 10–12 days, individual wells were screened for the presence of human IgG / human kappa light chain antibodies using a homogeneous HTRF assay. In this assay, supernatants from the 96-well fusion plates were mixed with europium-cryptate-labeled goat anti-human IgG (Fc fragment-specific), biotinylated goat anti-human kappa light chain (Bethyl no. A80-115B), and streptavidin-XLent and incubated for 1 hour. Plates were then read on a RUBYstar reader.

[0376] Hybridoma cells from wells positive for human IgG / human kappa light chain or human IgG / human lambda light chain antibodies were then screened by FACS using 300-19 cells transfected with human TIGIT and non-transfected 300-19 cells as a control. FACS-positive parental lines were transferred to 24-well plates. After 2-3 days, cell supernatants from individual wells were rescreened by FACS to confirm IgG specificity for human TIGIT.

[0377] Hybridomas were cloned by serial dilution and rescreened by FACS. 36 antibodies were selected for expansion and purification. Four antibodies (15A6, 22G2, 11G11, 10D7) were subsequently selected for sequencing and further analysis.

[0378] Example 2 Binding of anti-huTIGIT antibodies to soluble human TIGIT The binding of anti-huTIGIT antibodies to soluble human TIGIT was examined by BIACORE® surface plasmon resonance (SPR) analysis. Anti-huTIGIT antibodies were captured on a human kappa-coated chip (approximately 5 kRU; Southernbiotech catalog number 2060-01), and recombinant human TIGIT (rhTIGIT / Fc) was flowed across the chip at concentrations of 500 nM, 250 nM, 125 nM, 62 nM, and 31 nM. Capture concentrations of mAb / volume ranged from 2 to 40 μg / mL (5 μL at 10 μL / min). The antigen association time was 5 min at 15 μL / min, and the antigen dissociation time was 6 min. Regeneration was performed using 50 mM HCl / 50 mM NaOH (12 μL each at 100 μL / min). The results are shown in Table 3.

[0379] [Table 3]

[0380] The binding of antibodies 14B2, 19H2 and 26D8 was too weak to be reliably measured.

[0381] The previous binding constant determinations shown in Table 3 were used to help select anti-huTIGIT antibodies for further study. The binding constants of the subcloned and purified antibodies 15A6 and 22G2 were then determined to be 1.5 nM and 90 pM, respectively, using full titration curves.

[0382] Further SPR experiments were performed comparing the 15A6 and 22G2 antibodies with modified framework residues and altered human IgG1 constant regions. The human IgG1f sequence is provided in SEQ ID NO: 45, and has an allotypic variant (R97K, E239D, and M241L, differing from SEQ ID NO: 45) as provided in SEQ ID NO: 46. The human IgG1.3 sequence is provided in SEQ ID NO: 47 (which differs from SEQ ID NO: 45 at L117A, L118E, and G120A, corresponding to L234A, L235E, and G237A under EU numbering). Another effector-less human IgG1 constant region, human IgG1.1f, is provided in SEQ ID NO: 48 (which differs from SEQ ID NO: 45 at L117A, L118E, G120A, A213S, and P214S, which correspond to L234A, L235E, G237A, A330S, and P331S under EU numbering). Figure 7 shows the dramatic reduction in Fcγ receptor binding in the IgG1.1f construct. TIGIT binds to CD8 + Anti-TIGIT antibodies, highly expressed in TILs and possessing effector functions, are required for tumor eradication through their highly antitumor CD8 + The use of such an "inert" Fc region may be advisable, as it may deplete TIL and / or NK cells.

[0383] SPR experiments comparing the 15A6 antibody with framework variants A72S and / or N112T and the 22G2 antibody with framework variant H3Q to their unmodified forms demonstrated that neither the framework changes nor the isotype significantly affected binding to human TIGIT. The results are provided in Table 4.

[0384] [Table 4]

[0385] Further SPR experiments revealed that mAb 22G2 binds human TIGIT with a K of 0.06 nM. D With cynomolgus monkey TIGIT, the K value was 0.09 nM. D It was found that they bind.

[0386] The amino acid residue numbering in the Sequence Listing is 117 less than that in the literature due to the use of EU numbering and the absence of variable domains in the IgG sequences in the Sequence Listing. The C-terminal lysine (K) residue present in the genetic constructs of human antibodies is often missing from commercially produced antibodies, such as the therapeutic antibodies of the present invention. Dick et al. (2008) Biotechnol. Bioeng. 100:1132. Thus, while this lysine is not contained in any of SEQ ID NOS: 45-48, in one embodiment, the anti-huTIGIT antibodies of the present invention contain this additional lysine residue at the C-terminus of the heavy chain(s). In some embodiments, the antibodies of the present invention are in a preparation containing a mixture of heavy chains with and without a C-terminal lysine, e.g., as a result of unintentional C-terminal clipping. In some embodiments, the anti-huTIGIT antibodies of the present invention contain one or more heavy chains and one or more light chains, such as two heavy chains and two light chains.

[0387] Example 3 Anti-TIGIT antibodies are binned into multiple groups To determine which anti-human TIGIT antibodies compete with others for binding to huTIGIT and therefore bind to similar epitopes, antibody binning experiments were performed. Antibodies 14B2, 13E6, 6F9, 11G11, 10C9, 16F6, 11C9, 27A9, 10D7, 20G6, 24E8, 24G1, 27F1, 15A6, 4E4, 13D1, 9B11, 10B8, 22G2, 19H2, 8C8, 17G4, 25E7, 26D8, and 16A8 were tested.

[0388] Pairwise competition between anti-huTIGIT antibodies was determined as follows: a first antibody was bound to the surface of a sensor chip, and a second antibody was preincubated with a TIGIT polypeptide construct in a mixture. The preincubated mixture was then flowed over the sensor chip to determine the extent to which the second antibody interfered with the binding of the TIGIT polypeptide construct to the first antibody on the chip surface. Briefly, the first anti-huTIGIT antibody was immobilized on the surface of a SensorChip CM5 chip (Series S, GE Healthcare catalog number BR-1005-30). Flow cell 2, flow cell 3, and flow cell 4 (5000 RU) were used, and flow cell 1 served as a negative control. The second antibody was diluted to a starting concentration of 120 μg / mL (2X). A series of dilutions of the second antibody was performed by diluting the antibody concentration 1:3 with buffer to obtain seven different concentrations an...

Claims

1. An isolated antibody or antigen-binding fragment thereof that competes with one or more antibodies selected from the group consisting of 14B2, 13E6, 6F9, 11G11, 10C9, 16F6, 11C9, 27A9, 10D7, 20G6, 24E8, 24G1, 27F1, 15A6, 4E4, 13D1, 9B11, 10B8, 22G2, 19H2, 8C8, 17G4, 25E7, 26D8, and 16A8 for binding to human TIGIT (a T cell immunoreceptor having Ig and ITIM domains).

2. The isolated antibody or fragment of claim 1, wherein competition in a cross-blocking assay includes the ability to reduce the binding of a selected antibody to human TIGIT (SEQ ID NO: 1) by at least 20% in a competitive ELISA when used at approximately equimolar concentrations with the selected antibody.

3. a. an epitope comprising one or more of residues E60, 1109, L65, N70, F107, T117, 168, H76, and N58 of huTIGIT (SEQ ID NO: 1) (antibody 22G2); b. an epitope comprising one or more of residues G74, N70, H76, L65, L73, Q56, 168, H111, and P114 (antibody 11G11); or c. An epitope comprising one or more of residues H76, G74, L65, N58, 168, Q139, G135, L73, F107, N70, E60, H134, A132, and 1109 (antibody 15A6). An isolated antibody or antigen-binding fragment thereof, which binds to TIGIT (a human T cell immunoreceptor having Ig and ITIM domains).

4. 4. The isolated antibody or fragment of claim 3, which binds to TIGIT at an epitope comprising one or more of residues L65, I68, N70 and H76.

5. a. an epitope comprising the sequence NWEQQDQLLAICNADLGWH (SEQ ID NO: 38) and / or FCIYHTYPDGT (SEQ ID NO: 39) (antibody 22G2); b. an epitope comprising the sequence QVNWEQQDQLLAICNADLGWH (SEQ ID NO: 40) and / or HTYP (SEQ ID NO: 41) (antibody 11G11), or c. An epitope comprising the sequence NWEQQDQLLAICNADLGWH (SEQ ID NO: 38), FCI and / or AEHGARFQ (SEQ ID NO: 43) (antibody 15A6) 4. The isolated antibody or fragment of claim 3, which binds to TIGIT at

6. 6. The isolated antibody or fragment of claim 5, which binds to TIGIT at an epitope comprising the sequence LLAICNADLGWH (SEQ ID NO: 44).

7. An isolated antibody or antigen-binding fragment thereof that binds to TIGIT (a human T-cell immunoreceptor having Ig and ITIM domains), wherein the antibody heavy chain variable domain is derived from the human V-region germline sequence V4-39, V4-61 or V1-69.

8. 8. The isolated antibody or fragment of claim 7, wherein the antibody heavy and light chain variable domains are derived from the germline sequence combinations of human heavy and light chain V regions V4-39 / VA27, V4-61 / VL6, V4-39 / VL6 or V1-69 / VL15.

9. 9. The isolated antibody or fragment of claim 1, which substantially inhibits the binding of human TIGIT to human PVR / CD155.

10. The antibody has a K of 2 nM or less with human TIGIT as measured by BIACORE® SPR analysis. Ｄ 9. The isolated antibody or fragment of claim 1 , which binds to

11. 9. The isolated antibody or fragment of any one of claims 1 to 8, wherein the antibody binds to both human and cynomolgus monkey TIGIT.

12. 12. The isolated antibody or fragment of any one of claims 1 to 11, wherein the antibody is not mAb 10A7 or 1F4 of U.S. Patent Application No. 2009 / 0258013.

13. 13. The isolated antibody or fragment of claim 12, which does not bind to the same epitope on huTIGIT as mAb 10A7 or 1F4 of U.S. Patent Application No. 2009 / 0258013, and further does not compete with mAb 10A7 or 1F4 of U.S. Patent Application No. 2009 / 0258013 for binding to TIGIT.

14. i) a CDRH1 comprising the sequence of SEQ ID NO: 14, 20, 26 or 32; i) a CDRH2 comprising the sequence of SEQ ID NO: 15, 21, 27 or 33; and i) a CDRH3 comprising the sequence of SEQ ID NO: 16, 22, 28 or 34 a) a heavy chain variable domain comprising: i) CDRL1 comprising the sequence of SEQ ID NO: 17, 23, 29 or 35; i) a CDRL2 comprising the sequence of SEQ ID NO: 18, 24, 30 or 36; and i) a CDRL3 comprising the sequence of SEQ ID NO: 19, 25, 31 or 37 b) a light chain variable domain comprising 1. An isolated antibody or antigen-binding fragment thereof that binds to TIGIT (a human T cell immunoreceptor having Ig and ITIM domains), consisting essentially of:

15. one or more heavy chains and one or more light chains, wherein a) the heavy chain comprises a heavy chain variable region having at least 80% sequence identity to the sequence of SEQ ID NO: 2, 3, 4, 5, 7, 8, 10, or 12; 15. The isolated antibody or fragment of claim 14, wherein a) the light chain comprises a light chain variable region having at least 80% sequence identity to the sequence of SEQ ID NO: 6, 9, 11 or 13.

16. i) SEQ ID NOs: 14 to 19; ii) SEQ ID NOs: 20 to 25; iii) SEQ ID NOs: 26 to 31, and iv) SEQ ID NOs: 32 to 37 15. The isolated antibody or fragment of claim 14, comprising heavy and light chain variable domains comprising CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 sequences selected from the group consisting of:

17. 17. The isolated antibody of any one of claims 1 to 16, which is a human IgG1 antibody or a variant thereof with increased effector function.

18. 17. The isolated antibody of any one of claims 11 to 16, which is a human IgGl Fc variant with reduced or eliminated effector function.

19. 19. The isolated antibody or fragment of claim 18, comprising the following mutations according to EU numbering: L234A, L235E, G237A, A330S and P331S (SEQ ID NO: 48).

20. A nucleic acid encoding the heavy and / or light chain variable region of an antibody or fragment of any one of claims 1 to 19.

21. 21. An expression vector comprising the nucleic acid of claim 20.

22. A host cell transformed with the expression vector of claim 21.

23. A method for producing an anti-TIGIT antibody or antigen-binding fragment thereof, comprising culturing the host cell of claim 22 under conditions that allow production of the antibody or fragment, and purifying the antibody from the cell.

24. 20. A method of enhancing an antigen-specific T cell response in a subject in need thereof, comprising contacting T cells with the antibody or fragment of any one of claims 1 to 19, such that the antigen-specific T cell response is enhanced.

25. 25. The method of claim 24, wherein the subject has a tumor or a chronic viral infection and the immune response to the tumor or viral infection is enhanced.

26. 20. A method for reducing or depleting regulatory T cells in a tumor in a subject in need thereof, comprising administering an effective amount of the antibody or fragment of any one of claims 1 to 17, such that the number of T regulatory cells in the tumor is reduced.

27. 20. A method for treating cancer, comprising administering to a subject in need thereof a therapeutically effective amount of the antibody or fragment of any one of claims 1 to 19.

28. 28. The method of claim 27, wherein the cancer is selected from the group consisting of bladder cancer, breast cancer, uterine / cervical cancer, ovarian cancer, prostate cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, pancreatic cancer, colorectal cancer, colon cancer, renal cancer, head and neck cancer, lung cancer, stomach cancer, germ cell cancer, bone cancer, liver cancer, thyroid cancer, skin cancer, neoplasms of the central nervous system, lymphoma, leukemia, myeloma, sarcoma, and virus-associated cancer.

29. 28. The method of claim 27, wherein the cancer is metastatic cancer, refractory cancer, or recurrent cancer.

30. 30. The method of any one of claims 24 to 29, further comprising administering one or more additional therapeutic agents selected from the group consisting of an anti-PD-1 antibody, an anti-LAG-3 antibody, an anti-CTLA-4 antibody, or an anti-PD-L1 antibody.

31. 31. The method of claim 30, wherein the additional therapeutic agent is an anti-PD-1 antibody.

32. 31. The method of claim 30, wherein the additional therapeutic agent is an anti-PD-L1 antibody.

33. A bispecific antibody comprising a first antigen-binding domain and a second antigen-binding domain, a) a first antigen-binding domain derived from an anti-huTIGIT antibody according to any one of claims 1 to 16; b) A bispecific antibody, wherein the second antigen-binding domain is derived from an antibody selected from the group consisting of an anti-PD-1 antibody, an anti-LAG-3 antibody, an anti-CTLA-4 antibody, and an anti-PD-L1 antibody.

34. 34. The bispecific antibody of claim 33, wherein the second binding domain is derived from an anti-PD-1 antibody.

35. The bispecific antibody of claim 33, wherein the second binding domain is derived from an anti-PD-L1 antibody.

36. A method for detecting the presence of TIGIT in a sample, comprising contacting the sample with an antibody or antigen-binding fragment thereof described in any one of claims 1 to 16 under conditions that allow the formation of a complex between the antibody or antigen-binding fragment thereof and TIGIT, and detecting the formation of the complex.

37. If the tumor is: a) High levels of infiltrating TIGIT ＋ T cells and / or NK cells, b) increased expression of PVR and / or Nectin-2 in tumor cells or tumor-infiltrating myeloid cells; and c) Fusobacterium nucleatum infection 10. A method of treating cancer comprising administering to a subject having a tumor an antagonist anti-TIGIT antibody or antigen-binding fragment thereof if, and only if, the tumor comprises one or more of:

38. If the tumor is: a) High levels of infiltrating TIGIT ＋ T cells and / or NK cells, b) increased expression of PVR and / or Nectin-2 in tumor cells or tumor-infiltrating myeloid cells, and c) Fusobacterium nucleatum infection 20. A method of treating cancer, comprising administering to a subject having a tumor an antagonist anti-TIGIT antibody or antigen-binding fragment thereof described in any one of claims 1 to 19, if and only if the tumor comprises one or more of:

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