Novel compositions and methods for treatment of immune related diseases

Abstract

To provide compositions for the diagnosis and treatment of immune related diseases and methods of using those compositions.SOLUTION: A method of determining whether a test immune cell is an activated or normal Treg, memory T cell, NK cell, or TFh cell, the method comprising assessing the level of expression of TIGIT in the test immune cell and comparing the level of expression of TIGIT in the test immune cell to the level of expression of TIGIT in a known activated or normal Treg, memory T cell, NK cell, or TFh cell, or comparing the level of expression of TIGIT in the test immune cell to one or more known standard TIGIT expression values.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to compositions and methods useful for the diagnosis and treatment of immune-related diseases.

Background Art

[0002] Immune-related and inflammatory diseases are, in normal physiological functions, manifestations or consequences of fairly complex, often multiply interrelated biological pathways that are important for initiating repair from an attack or injury and initiating innate and acquired defenses against foreign organisms in response to an attack or injury. Disease or pathology occurs when these normal physiological pathways, either directly related to the intensity of the response or as a result of abnormal regulation or excessive stimulation, cause further attacks or injuries as a reaction against self or as a combination of these. The development of these diseases often involves multiple-step pathways and often a number of different biological systems / pathways, but intervention at key points in one or more of these pathways may have an ameliorating or therapeutic effect. Therapeutic intervention occurs either by antagonizing harmful processes / pathways or by stimulating beneficial processes / pathways.

[0003] Many immune-related diseases are known and have been extensively studied. Such diseases include immune-mediated inflammatory diseases, non-immune-mediated inflammatory diseases, infectious diseases, immunodeficiency disorders, abnormal proliferation, and the like. T lymphocytes (T cells) are an important component of the mammalian immune response. T cells recognize antigens associated with self-molecules encoded by genes within the major histocompatibility complex (MHC). Antigens can be presented together with MHC molecules on the surface of antigen-presenting cells, virus-infected cells, cancer cells, grafts, etc. The T cell system serves to eliminate these modified cells that pose a threat to the health of the host mammal. T cells include helper T cells and cytotoxic T cells. Helper T cells proliferate extensively following recognition of the antigen-MHC complex on antigen-presenting cells. Helper T cells also secrete various cytokines, such as lymphokines, which play a central role in the activation of B cells, cytotoxic T cells, and various other cells contributing to the immune response. Another subcategory of helper T cells is follicular helper T cells (TFh) (see Vineusa et al., Nat. Rev. Immunol. 5: 853-865 (2005) for an overview). Detectable by the characteristic expression of CXC-chemokine receptor 5 (Schaerli et al., J. Exp. Med. 192: 1553-62 (2000)), these cells have been shown to produce IL-10 and probably IL-21. TFh cells assist germinal center B cells, particularly in supporting B cell survival and proliferation and strongly inducing antibody production during co-culture with B cells. They have also been implicated in the induction of immune tolerance.

[0004] Regulatory T cells (T regis a subset of helper T cells that plays an important role in inhibiting autoreactive immune responses and is often found at sites of chronic inflammation such as tumor tissues (Wang, H.Y. & Wang, R.F., Curr Opin Immunol 19, 217-23 (2007)). Tregs are phenotypically defined by high cell surface expression of CD25, CLTA4, GITR, and neuropilin-1 (Read, S., Malmstrom, V. & Powrie, F., J Exp Med 192, 295-302 (2000); Sakaguchi, S., et al., J Immunol 155, 1151-64 (1995); Takahashi, T. et al., J Exp Med 192, 303-10 (2000); McHugh, R.S. et al., Immunity 16, 311-23 (2002); Bruder, D. et al., Eur J Immunol 34, 623-30 (2004)), and are under the control of the transcription factor FOXP3 (Hori, S., Nomura, T. & Sakaguchi, S., Science 299, 1057-61 (2003)). Tregs exert their suppressive function on activated T cells through contact-dependent mechanisms and cytokine production (Fehervari, Z. & Sakaguchi, Curr Opin Immunol 16, 203-8 (2004)). Also, T regs regulates the immune response by direct interaction with ligands on dendritic cells (DCs), such as the interaction between B7 molecules and CTLA4 on DCs that induces the induction of indoleamine 2,3-dioxygenase (IDO) (Fallarino, F. et al., Nat Immunol 4, 1206-12 (2003)), and CD40L ligation (Serra, P. et al., Immunity 19, 877-89 (2003)). DCs are specialized antigen-presenting cells that can induce immunity or tolerance to self or non-self antigens. DC-expanded T regssuppresses alloreactive responses in vitro (Yamazaki, S. et al., Proc Natl Acad Sci U S A 103, 2758-63 (2006); Ahn, J.S., Krishnadas, D.K. & Agrawal, Int Immunol 19, 227-37 (2007)), and when adoptively transferred, appropriate T regs prevents diabetes in NOD.scid mice (Tarbell, K.V. et al., J Exp Med 199, 1467-77 (2004)), or experimentally induces asthma (Lewkowich, I.P. et al. J Exp Med 202, 1549-61 (2005)). In addition, specific interactions between ligands on DCs and Tregs can abrogate inhibitory functions such as the engagement of GITR in mice (Shimizu, J., et al., Nat Immunol 3, 135-42 (2002)), T reg is suggested to have a pleiotropic role in DCs during the regulation of T cell function.

[0005] The molecules CTLA4 and GITR are representatives of ligands defined within CD28-B7, and costimulatory / costimulatory molecules of the TNF superfamily, respectively (Greenwald, R.J., et al., Annu Rev Immunol 23, 515-48 (2005)). These molecules are highly expressed on Tregs, but are generally upregulated in activated T cells. T regTo search for novel costimulatory molecules expressed in cells, a search was conducted to identify genes specifically expressed in T cells that possess both an Ig domain and an immunoreceptor tyrosine-based activation or inhibitory (ITAM / ITIM) motif (Abbas, A.R. et al., Genes Immun 6, 319-31 (2005)). Through the intersection of these two genome-wide bioinformatics search strategies, a novel cell surface-binding protein with an IgV domain, a transmembrane domain, and a protein encoding two putative immunoreceptor tyrosine inhibitory motifs was identified (see U.S. Patent Publication US20040121370, which is incorporated herein by reference). A protein designated as TIGIT (T cell-Ig and ITIM domain) was shown to be expressed on T cells, particularly T reg and memory cell subsets as well as NK cells. There is a need for novel therapeutic methods and treatments for immunological disorders, particularly autoimmune disorders. In this specification, the applicant has identified TIGIT binding partners and provides novel compositions, detection methods, and treatment methods for immunological disorders regulated by the elucidated TIGIT action on TIGIT interaction with these binding partners, T cell maturation, and activity.

Summary of the Invention

[0006] The present invention relates to compositions and methods useful for the diagnosis and treatment of immune-related diseases in mammals including humans. The present invention is based on the identification of proteins involved in the negative regulation of the proliferation and function of certain immune cells. Immune-related diseases can be treated by suppressing or enhancing the immune response. Molecules that enhance the immune response stimulate or potentiate the immune response to an antigen. Molecules that stimulate the immune response can be used therapeutically when benefit is obtained by enhancing the immune response. Alternatively, molecules that suppress the immune response or attenuate or reduce the immune response to an antigen (e.g., neutralizing antibodies) can be used therapeutically when benefit is obtained by attenuating the immune response (e.g., inflammation). As used herein, Applicants have shown that the TIGIT (short for "T cell-Ig and ITIM domain") protein specifically binds to the poliovirus receptor (PVR, also known as CD155) and other members of a newly elucidated protein family species, and that this TIGIT-PVR interaction negatively regulates T cell activation and proliferation. Accordingly, TIGIT polypeptides, agonists and antagonists thereof, as well as PVR polypeptides, agonists and antagonists thereof, are useful for the preparation of medicaments and drugs for the treatment of immune-related diseases and inflammatory diseases. The present invention also provides methods for treating immune-related diseases and inflammatory diseases, as well as methods and compositions for detecting and evaluating the status of immune-related diseases and inflammatory diseases.

[0007] In one embodiment, the present invention provides an isolated polypeptide comprising an amino acid sequence comprising one or more of the following amino acids: alanine at the amino acid position corresponding to amino acid position 67 of human TIGIT, glycine at the amino acid position corresponding to amino acid position 74 of human TIGIT, proline at the amino acid position corresponding to amino acid position 114 of human TIGIT, and glycine at the amino acid position corresponding to amino acid position 116 of human TIGIT. In one aspect, the polypeptide is not PVR, PVRL1, PVRL2, PVRL3, PVRL4, TIGIT, CD96 or CD226. In other aspects, the polypeptide further comprises one or more of the following amino acids: an amino acid selected from valine, isoleucine and leucine at the amino acid position corresponding to amino acid position 54 of human TIGIT, an amino acid selected from serine and threonine at the amino acid position corresponding to amino acid position 55 of human TIGIT, glutamine at the amino acid position corresponding to amino acid position 56 of human TIGIT, threonine at the amino acid position corresponding to amino acid position 112 of human TIGIT, and an amino acid selected from phenylalanine and tyrosine at the amino acid position corresponding to amino acid position 113 of human TIGIT. In other aspects, the polypeptide a. an amino acid selected from valine and isoleucine at amino acid position 54 - an amino acid selected from serine and threonine at amino acid position 55 - glutamine at amino acid position 56, b. alanine at position 67 - any amino acid at each of amino acid positions 68 - 73 - glycine at amino acid position 74, and, c. threonine at amino acid position 112 - an amino acid selected from phenylalanine and tyrosine at amino acid position 113 - proline at amino acid position 114 - any amino acid at amino acid position 115 - glycine at amino acid position 116, further comprises one or more structural sub - motifs selected from the above, where the numbering of the amino acid positions corresponds to the amino acid positions of human TIGIT, provided that the absolute numbering of the amino acids of the polypeptide may be different.

[0008] In other embodiments, the present invention provides a method for determining whether a test polypeptide is a member of a polypeptide of the TLP family, the method comprising aligning the amino acid sequence of the test polypeptide with the amino acid sequence of one or more members of the polypeptide of the TLP family, and assessing the presence or absence of one or more of alanine at the amino acid position corresponding to amino acid position 67 of human TIGIT, glycine at the amino acid position corresponding to amino acid position 74 of human TIGIT, proline at the amino acid position corresponding to amino acid position 114 of human TIGIT, and glycine at the amino acid position corresponding to amino acid position 116 of human TIGIT in the amino acid sequence of the test polypeptide. In other embodiments, the present invention provides a method for identifying one or more members of the TLP protein family by identifying proteins in one or more sequence databases having an amino acid sequence comprising at least one amino acid selected from alanine at the amino acid position corresponding to amino acid position 67 of human TIGIT, glycine at the amino acid position corresponding to amino acid position 74 of human TIGIT, proline at the amino acid position corresponding to amino acid position 114 of human TIGIT, and glycine at the amino acid position corresponding to amino acid position 116 of human TIGIT.

[0009] In other embodiments, the invention provides an isolated agent that specifically interacts with one or more conserved or substantially conserved regions of a TLP family member. In one aspect, the agent is an antagonist of the expression and / or activity of a TLP family member. In other aspects, the antagonist is selected from small molecule inhibitors, inhibitory antibodies or antigen-binding fragments thereof, aptamers, inhibitory nucleic acids, and inhibitory polypeptides. In other aspects, the agent is an agonist of the expression and / or activity of a TLP family member. In other aspects, the agent is selected from agonistic antibodies or antigen-binding fragments thereof, agonistic peptides, and small molecules or proteins that activate the binding of TIGIT to intracellular signaling of PVR and / or TIGIT mediated by PVR. In other embodiments, the invention provides a method for identifying or detecting one or more TLP family members by contacting a polypeptide of a putative TLP family member with at least one of the above agents and determining the binding of at least one agent to the putative TLP family member.

[0010] In other embodiments, the invention provides a method for determining whether a test immune cell is an activated or normal T reg , memory T cell, NK cell, or T Fh cell, the method comprising evaluating the expression level of TIGIT in the test immune cell and comparing it with the expression level of TIGIT in known activated or normal T reg , memory T cell, NK cell, or T Fh cell, or comparing the expression level of TIGIT in the test immune cell with one or more known reference TIGIT expression values. In other embodiments, the invention provides a method for modulating the function and / or activity of the immune system, comprising modulating the binding of TIGIT to one or more of PVR, PVRL3, and PVRL2.

[0011] In other embodiments, the present invention provides an anti-TIGIT antibody or a fragment thereof comprising at least one HVR comprising an amino acid sequence selected from the amino acid sequences shown in SEQ ID NOs: 23 to 28. In other embodiments, the present invention provides an anti-TIGIT antibody or a fragment thereof comprising at least one HVR comprising an amino acid sequence selected from the amino acid sequences shown in SEQ ID NOs: 31 to 36. In other embodiments, the present invention provides an anti-TIGIT antibody or a fragment thereof wherein the antibody light chain comprises the amino acid sequence shown in SEQ ID NO: 21. In other embodiments, the present invention provides an anti-TIGIT antibody or a fragment thereof wherein the antibody light chain comprises the amino acid sequence shown in SEQ ID NO: 29. In other embodiments, the present invention provides an anti-TIGIT antibody or a fragment thereof wherein the antibody heavy chain comprises the amino acid sequence shown in SEQ ID NO: 22 or a part thereof. In other embodiments, the present invention provides an anti-TIGIT antibody or a fragment thereof wherein the antibody heavy chain comprises the amino acid sequence shown in SEQ ID NO: 30 or a part thereof. In other embodiments, the present invention provides an anti-TIGIT antibody or a fragment thereof wherein the antibody light chain comprises the amino acid sequence shown in SEQ ID NO: 21 or a part thereof and the antibody heavy chain comprises the amino acid sequence shown in SEQ ID NO: 22 or a part thereof. In other embodiments, the present invention provides an anti-TIGIT antibody or a fragment thereof wherein the antibody light chain comprises the amino acid sequence shown in SEQ ID NO: 29 or a part thereof and the antibody heavy chain comprises the amino acid sequence shown in SEQ ID NO: 30 or a part thereof. In other embodiments, the present invention provides an anti-TIGIT antibody or a fragment thereof encoded by the nucleotide sequence shown in SEQ ID NO: 50 or a part thereof. In other embodiments, the present invention provides an anti-TIGIT antibody or a fragment thereof encoded by the nucleotide sequence shown in SEQ ID NO: 51 or a part thereof. In one aspect, the antibody or antigen-binding fragment thereof of the present invention is selected from a humanized antibody, a chimeric antibody, a bispecific antibody, a heteroconjugate antibody, and an immunotoxin.

[0012] In other aspects, at least one HVR of the present invention is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the HVR shown in any of SEQ ID NOs: 23-28. In other aspects, at least one HVR of the present invention is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the HVR shown in any of SEQ ID NOs: 31-36. In other aspects, the light chain of the antibody or antigen-binding fragment of the present invention comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 21. In other aspects, the light chain of the antibody or antigen-binding fragment of the present invention comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 29. In other aspects, the heavy chain of the antibody or antigen-binding fragment of the present invention comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 22. In other aspects, the heavy chain of the antibody or antigen-binding fragment of the present invention comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 30. In other aspects, the antibody or antigen-binding fragment of the present invention comprises a light chain comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 21, and a heavy chain comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 22. In other aspects, the antibody or antigen-binding fragment of the present invention comprises a light chain comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 29, and a heavy chain comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 30.

[0013] In other embodiments, the present invention provides a method for modulating CD226-PVR interaction and / or CD96-PVR interaction, comprising administering in vivo or in vitro at least one of TIGIT, an agonist of TIGIT expression and / or activity, or an antagonist of TIGIT expression and / or activity. In one aspect, TIGIT or an agonist of TIGIT expression and / or activity is administered, and the CD226-PVR interaction and / or CD96-PVR interaction is inhibited or blocked. In other aspects, an antagonist of TIGIT expression and / or activity is administered, and the CD226-PVR interaction and / or CD96-PVR interaction is stimulated.

[0014] In other embodiments, the present invention provides a method for modulating immune cell function and / or activity by modulating the expression and / or activity of TIGIT and / or PVR, or by modulating intracellular signaling mediated by TIGIT that binds to PVR. In one aspect, the modulation is by treating cells in vitro or in vivo with TIGIT, an agonist of TIGIT expression and / or activity, an agonist of PVR expression and / or activity, or by stimulating intracellular signaling mediated by TIGIT that binds to PVR, to reduce or inhibit the proliferation of one or more immune cells or the release of pro-inflammatory cytokines by one or more immune cells. In other aspects, the modulation is by treating cells in vitro or in vivo with an antagonist of TIGIT expression and / or activity, an antagonist of PVR expression and / or activity, or by inhibiting intracellular signaling mediated by TIGIT that binds to PVR, to increase or stimulate the proliferation of one or more immune cells or the release of pro-inflammatory cytokines by one or more immune cells.

[0015] In other embodiments, the present invention provides a method for inhibiting an immune response by administering, in vitro or in vivo, TIGIT, an agonist of the expression and / or activity of TIGIT, an agonist of the expression and / or activity of PVR, or by stimulating intracellular signaling mediated by TIGIT that binds to PVR. In other embodiments, the present invention provides a method for enhancing or stimulating an immune response by administering, in vitro or in vivo, an antagonist of the expression and / or activity of TIGIT, an antagonist of the expression and / or activity of PVR, or by inhibiting intracellular signaling mediated by TIGIT that binds to PVR. In other embodiments, the present invention provides a method for regulating the type and / or amount of cytokine production from immune cells by regulating the expression and / or activity of TIGIT or PVR in vitro or in vivo. In one aspect, pro-inflammatory cytokine production is stimulated and / or enhanced by administering an antagonist of the expression and / or activity of TIGIT, an antagonist of the expression and / or activity of PVR, or by inhibiting intracellular signaling mediated by TIGIT that binds to PVR. In other aspects, pro-inflammatory cytokine production is inhibited by administering an agonist of the expression and / or activity of TIGIT, an agonist of the expression and / or activity of PVR, or by stimulating intracellular signaling mediated by TIGIT that binds to PVR.

[0016] In other embodiments, the present invention provides a method for stimulating intracellular signaling via the ERK pathway and / or ERK phosphorylation in one or more immune cells, comprising treating the one or more immune cells with TIGIT, an agonist of the expression and / or activity of TIGIT, or an agonist of the expression and / or activity of PVR.

[0017] In another embodiment, the present invention provides a method for diagnosing an immune-related disease associated with an abnormal immune cell response in a subject, the method comprising evaluating the expression and / or activity of TIGIT in a sample from the subject, and comparing the expression and / or activity of TIGIT with a control amount of the expression and / or activity of TIGIT and / or the amount of the expression and / or activity of TIGIT in a sample from a normal subject. In one aspect, the immune-related disease is selected from psoriasis, arthritis, inflammatory bowel disease or cancer. In another aspect, the cancer is breast cancer. In another embodiment, the present invention provides a method for evaluating the severity of an immune-related disease associated with an abnormal immune cell response in a subject, the method comprising evaluating the expression and / or activity of TIGIT in a sample from the subject, and comparing the expression and / or activity of TIGIT with a control amount of the expression and / or activity of TIGIT and / or the amount of the expression and / or activity of TIGIT in a sample from a normal subject. In one aspect, the immune-related disease is selected from psoriasis, arthritis, inflammatory bowel disease or cancer. In another aspect, the cancer is breast cancer. In another embodiment, the present invention provides a method for preventing an immune-related disease associated with an abnormal immune cell response in a subject, the method comprising modulating the expression and / or activity of TIGIT in the subject. In one aspect, the immune-related disease is selected from psoriasis, arthritis, inflammatory bowel disease or cancer. In another aspect, the cancer is breast cancer. In another embodiment, the present invention provides a method for treating or reducing the severity of an immune-related disease associated with an abnormal immune cell response in a subject, the method comprising modulating the expression and / or activity of TIGIT in the subject. In one aspect, the immune-related disease is selected from psoriasis, arthritis, inflammatory bowel disease or cancer. In another aspect, the cancer is breast cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0018]

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Mode for Carrying Out the Invention

[0019] TIGIT has been identified as a predicted modulator of immune function (see, e.g., U.S. Patent Publication No. US20040121370, which is incorporated herein by reference). In this specification, Applicants show that TIGIT is a member of a newly described family of immune-related proteins including the poliovirus receptor (PVR, also known as nectin-5 or CD155), PVR-like proteins 1-4 (PVRL1-4), CD96, and CD226. Applicants show conserved structural elements of this new family whose members have roles in immune regulation and function, and provide methods for identifying additional family members.

[0020] The applicant shows that TIGIT binds strongly to PVR and binds to PVRL3 (also known as nectin-3 or CD113) and PVRL2 (also known as nectin-2 or CD112) with a small Kd. PVR is a cell surface receptor that is highly expressed on dendritic cells (DCs), as well as FDCs, fibroblasts, endothelial cells, and some tumor cells (Sakisaka, T. & Takai, Y., Curr Opin Cell Biol 16, 513-21 (2004); Fuchs, A. & Colonna, M., Semin Cancer Biol 16, 359-66 (2006)). The applicant shows by mRNA and FACS analysis that TIGIT is expressed mainly on various activated T cells, particularly regulatory T cells (Tregs), memory T cells, NK cells, and follicular T helper cells (Tfhs). The tests described herein show the interaction between PVR and TIGIT on DCs, suggesting that this binding interaction regulates DC function, particularly cytokine production. Human DCs bound to TIGIT secreted high levels of IL-10 and low levels of pro-inflammatory cytokines (e.g., IL-12p40 and IL-12p70). When TIGIT bound to immature T cells (evaluated using a TIGIT fusion construct), T cell activation and proliferation were inhibited. In particular, since this inhibition was reversed in the presence of an ERK inhibitor, it is suggested that ERK activation may be an important step in the function of TIGIT that regulates DC activity. The applicant shows herein that TIGIT+ T cells suppress the proliferation of antigen-presenting cells when present in a mixed population of immune cells, not only with other TIGIT-T cells, and that the suppression observed when a blocking anti-TIGIT antibody is included in the mixture is greatly reduced, indicating that TIGIT itself is responsible for this suppressive effect.

[0021] As shown herein, TIGIT is expressed at increased levels in arthritis, psoriasis, inflammatory bowel disease and breast cancer tissues compared to normal control tissues. Further, Applicants directly demonstrate the ability of TIGIT to regulate the immune response by showing that the TIGIT fusion protein inhibits in vitro human T cell responses and mouse T cell activation in a delayed type hypersensitivity in vivo assay. Since TIGIT significantly altered mature DCs and only slightly altered immature DCs, once the DCs become fully activated antigen presenting cells, it is suggested that the TIGIT-PVR interaction is important for fine-tuning the regulatory immune response. The experiments presented herein suggest a mechanism by which TIGIT inhibits T cell activation by inducing IL-10 in DCs through an inhibitory feedback loop. Accordingly, the present invention further provides novel methods of modulating immune function by modulating a specific subset of cytokines or a specific subset of immune cells. These and other aspects of the invention are described in more detail below.

[0022] I. Definitions The terms "TIGIT polypeptide", "TIGIT protein" and "TIGIT" are used interchangeably herein and refer to a specific polypeptide sequence as described herein. The TIGIT polypeptides described herein may be isolated from a variety of sources such as human or non-human organism tissues, or may be prepared by recombinant or synthetic methods. In one embodiment, the TIGIT polypeptide has the amino acids shown in any of SEQ ID NOs: 1-4. All disclosures herein regarding "TIGIT polypeptide" refer to each polypeptide individually and collectively. For example, descriptions such as the preparation of ~, the purification of ~, the derivatives of ~, the formation of antibodies to or against ~, the administration of ~, the composition containing ~, the treatment of diseases having ~, etc. are separately relevant to each polypeptide of the present invention. Also, the terms "TIGIT polypeptide", "TIGIT protein" or "TIGIT" include variants of the TIGIT polypeptides disclosed herein or known in the art.

[0023] The "native sequence TIGIT polypeptide" includes a polypeptide having the same amino acid sequence as the corresponding TIGIT polypeptide of natural origin. Such native sequence TIGIT polypeptides can be isolated from nature or produced by recombinant or synthetic means. The term "native sequence TIGIT polypeptide" specifically includes naturally occurring cleavage or secreted forms (e.g., extracellular domain sequences) of a particular TIGIT polypeptide, naturally occurring variant forms (e.g., alternatively spliced forms), and naturally occurring allelic variants of the polypeptide. In various embodiments of the invention, the native sequence TIGIT polypeptides disclosed herein are mature or full-length native sequence polypeptides containing the full-length amino acid sequence. However, while the TIGIT polypeptides disclosed in the relevant figures are shown to start with the methionine residue designated as amino acid position 1 in the figure, it is also contemplated and possible that other methionine residues located either upstream or downstream of amino acid position 1 in the figure could be used as the starting amino acid residue of the TIGIT polypeptide.

[0024] The TIGIT polypeptide "extracellular domain" or "ECD" refers to a form of the TIGIT polypeptide that substantially lacks transmembrane and cytoplasmic domains. Typically, TIGIT polypeptide ECDs have less than 1% of their transmembrane and / or cytoplasmic domains, preferably less than 0.5% of such domains. It will be understood that any transmembrane domains identified for the TIGIT polypeptides of the present invention are identified according to criteria routinely used in the art to identify that type of hydrophobic domain. The exact boundaries of the transmembrane domains may vary, but are likely to not exceed about 5 amino acids from either end of any of the domains identified herein. Thus, the TIGIT polypeptide extracellular domain may, in some cases, include up to about 5 amino acids beyond either end of the transmembrane domain / extracellular domain boundary, and such polypeptides with or without an attached signal peptide and the nucleic acids encoding them are contemplated in the present invention. In one embodiment, the TIGIT ECD encompasses amino acids 1-139 of the human TIGIT protein shown in SEQ ID NO:1.

[0025] The approximate positions of the "signal peptides" of the various TIGIT polypeptides disclosed herein can be identified using methods known in the art. For example, the signal sequence of the human TIGIT polypeptide shown in SEQ ID NO:1 is predicted to span amino acids 1-15 (see, e.g., U.S. Patent Publication 20040121370). However, while the C-terminal boundary of the signal peptide may vary, it is highly likely that it differs by approximately 5 or less at either end of the C-terminal boundary of the signal peptide initially identified herein, and at this time the C-terminal boundary of the signal peptide may be identified according to criteria that are relevantly used in the art to identify types of amino acid sequence elements (e.g., Nielsen et al., Prot. Eng. 10: 1-6 (1997) and von Heinje et al., Nucl. Acids. Res. 14: 4683-4690 (1986)). Further, in some cases, cleavage of the signal sequence from the secreted polypeptide is not completely uniform, resulting in two or more secreted moieties. Mature peptides in which the signal peptide is cleaved within approximately 5 amino acids at either end of the C-terminal boundary of the signal peptide identified herein and polynucleotides encoding the same are contemplated by the present invention.

[0026] The "TIGIT polypeptide variant" means an active TIGIT polypeptide having at least about 80% amino acid sequence identity with the full-length native sequence TIGIT polypeptide disclosed herein, the TIGIT polypeptide sequence lacking the signal peptide disclosed herein, the extracellular domain of the TIGIT polypeptide disclosed herein with or without the signal peptide, or other fragments of the full-length TIGIT polypeptide sequence as defined above or below. Such TIGIT polypeptide variants include, for example, TIGIT polypeptides having one or more amino acid residues added or deleted at the N- or C-terminus of the full-length native amino acid sequence. Generally, the TIGIT polypeptide variant has at least about 80% amino acid sequence identity, or at least about 81% amino acid sequence identity, or at least about 82% amino acid sequence identity, or at least about 83% amino acid sequence identity, or at least about 84% amino acid sequence identity, or at least about 85% amino acid sequence identity, or at least about 86% amino acid sequence identity, or at least about 87% amino acid sequence identity, or at least about 88% amino acid sequence identity, or at least about 89% amino acid sequence identity, or at least about 90% amino acid sequence identity, or at least about 91% amino acid sequence identity, or at least about 92% amino acid sequence identity, or at least about 93% amino acid sequence identity, or at least about 94% amino acid sequence identity, or at least about 95% amino acid sequence identity, or at least about 96% amino acid sequence identity, or at least about 97% amino acid sequence identity, or at least about 98% amino acid sequence identity, and, or at least about 99% amino acid sequence identity with the full-length native sequence TIGIT polypeptide disclosed herein, the TIGIT polypeptide sequence lacking the signal peptide disclosed herein, the extracellular domain of the TIGIT polypeptide disclosed herein with or without the signal peptide, or other specifically identified fragments of the full-length TIGIT polypeptide sequence.Typically, the TIGIT variant polypeptide is at least about 10 amino acids in length, or at least about 20 amino acids in length, or at least about 30 amino acids in length, or at least about 40 amino acids in length, or at least about 50 amino acids in length, or at least about 60 amino acids in length, or at least about 70 amino acids in length, or at least about 80 amino acids in length, or at least about 90 amino acids in length, or at least about 100 amino acids in length, or at least about 150 amino acids in length, or at least about 200 amino acids in length, or at least about 300 amino acids in length, or more.

[0027] As used herein, the "percent (%) amino acid sequence identity" identified herein for a TIGIT polypeptide is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues of the TIGIT polypeptide, with the sequences aligned and gaps introduced if necessary to obtain the maximum percent sequence identity, and no conservative substitutions being considered part of the sequence identity. Alignments for the purpose of determining percent amino acid sequence identity can be achieved by using a variety of methods within the skill of the art, such as publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. One of ordinary skill in the art can determine appropriate parameters for measuring alignment, including any algorithm necessary to achieve the maximum alignment for the full lengths of the sequences being compared. However, for the purposes herein, the % amino acid sequence identity values are obtained by using the sequence comparison program ALIGN-2, for which the complete source code is publicly available. The ALIGN-2 sequence comparison computer program was made by Genentech, Inc., and the source code was filed with the U.S. Copyright Office, Washington, D.C., 20559, together with user documentation, and is registered under U.S. Copyright Registration No. TXU510087. ALIGN-2 is also publicly available from Genentech, Inc., South San Francisco, California. The ALIGN-2 program is compiled for use on a UNIX® operating system, preferably Digital UNIX® V4.0D. All sequence comparison parameters are set and not varied by the ALIGN-2 program.

[0028] In the context where ALIGN-2 is used for amino acid sequence comparison, the percent amino acid sequence identity of a given amino acid sequence A to, or with respect to, a given amino acid sequence B (or, alternatively, it can be said that a given amino acid sequence B has, or contains, a certain percent amino acid sequence identity with, or to, a given amino acid sequence A) is calculated as follows: 100 times the fraction X / Y Here, X is the number of amino acid residues that match as identical by the alignment of A and B in the sequence alignment program ALIGN-2, and Y is the total number of amino acid residues in B. It will be understood that when the length of amino acid sequence A is different from the length of amino acid sequence B, the % amino acid sequence identity of A to B will be different from the % amino acid sequence identity of B to A. As an example of the calculation of % amino acid sequence identity using this method, Tables 1 and 2 show the method for calculating the % amino acid sequence identity of an amino acid sequence referred to as "comparative protein" to an amino acid sequence referred to as "TIGIT". "TIGIT" represents the amino acid sequence of a hypothetical TIGIT polypeptide of interest, "comparative protein" represents the amino acid sequence of the polypeptide to which the "TIGIT" polypeptide of interest is being compared, and each of "X", "Y", and "Z" represents a different hypothetical amino acid residue.

[0029] Table 1 Target protein XXXXXXXXXXXXXXX (length = 15 amino acids) Comparative protein XXXXXYYYYYYY (length = 12 amino acids) % Amino acid sequence identity = (Number of amino acid residues that match identically between the two polypeptide sequences determined by ALIGN-2) ÷ (Total number of amino acid residues in the target protein) = 5 ÷ 15 = 33.3% Table 2 Target protein XXXXXXXXXX (length = 10 amino acids) Comparative protein XXXXXYYYYYYZZYZ (length = 15 amino acids) % Amino acid sequence identity = (Number of amino acid residues that match identically between the two polypeptide sequences determined by ALIGN-2) ÷ (Total number of amino acid residues in the target protein) = 5 ÷ 10 = 50%

[0030] Unless otherwise specified, all % amino acid sequence identity values herein are obtained using the ALIGN-2 sequence comparison computer program as described in the previous section and Tables 1 and 2. However, the % amino acid sequence identity values may also be determined using the WU-BLAST-2 computer program (Altschul et al., Methods in Enzymology 266:460-480 (1996)). Further, most of the WU-BLAST-2 search parameters are set to their default values. The parameters that are not set to the default values, i.e., the adjustable parameters, are set to the following values: overlap span = 1, overlap fraction = 0.125, word threshold (T) = 11, and scoring matrix = BLOSUM62. When WU-BLAST-2 is used, the % amino acid sequence identity value is determined by dividing (a) the number of identical amino acid residues determined by WU-BLAST-2 between the amino acid sequence of a TIGIT polypeptide being targeted that has a sequence derived from a native TIGIT polypeptide and the amino acid sequence of the comparative amino acid sequence being targeted (i.e., the sequence that the TIGIT polypeptide being targeted may be compared to, which may be a TIGIT polypeptide variant) by (b) the total number of residues of the TIGIT polypeptide being targeted. For example, in the expression "a polypeptide comprising an amino acid sequence A having at least 80% amino acid sequence identity to amino acid sequence B", amino acid sequence A is the comparative amino acid sequence being targeted, and amino acid sequence B is the amino acid sequence of the TIGIT polypeptide being targeted.

[0031] Alternatively, the % amino acid sequence identity may be determined using the sequence comparison program NCBI-BLAST2 (Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997)). The NCBI-BLAST2 sequence comparison program can be downloaded from http: / / www.ncbi.nlm.nih.gov or obtained in another way from the National Institutes of Health, Bethesda, Maryland. NCBI-BLAST2 uses several search parameters, all of which are set to their default values, for example, unmask=yes, strand=all, expected occurrences=10, minimum low complexity length=15 / 5, multi-pass e-value=0.01, multi-pass constant=25, final gap alignment drop-off=25, and scoring matrix=BLOSUM62. In the situation where NCBI-BLAST2 is used for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A with or to a given amino acid sequence B (or it can also be said as a given amino acid sequence A having or containing a certain % amino acid sequence identity with or to a given amino acid sequence B) is calculated as follows: 100 times the fraction X / Y Here, X is the number of amino acid residues whose scores are agreed to be identical by the alignment of A and B by the sequence alignment program NCBI-BLAST2, and Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A is different from the length of amino acid sequence B, the % amino acid sequence identity of A to B will be different from the % amino acid sequence identity of B to A.

[0032] The terms "TIGIT polynucleotide" and "TIGIT nucleotide" are used interchangeably herein and refer to a specific polynucleotide sequence encoding a TIGIT polypeptide. These polynucleotides may include DNA or RNA or both DNA and RNA. The TIGIT polynucleotides described herein may be isolated from various sources such as human tissues or tissues from non-human organisms, or may be prepared by recombinant or synthetic methods. All disclosures herein regarding "TIGIT polynucleotide" refer to each polynucleotide individually and collectively. For example, descriptions such as the preparation of ~, the purification of ~, the derivatives of ~, the administration of ~, the composition containing ~, the treatment of diseases having ~, etc. are separately relevant to each polypeptide of the present invention. Also, the terms "TIGIT polynucleotide" and "TIGIT nucleotide sequence" include variants of the TIGIT polynucleotides disclosed herein "Naturally occurring TIGIT polynucleotide" includes polynucleotides having the same nucleic acid sequence as the corresponding TIGIT polynucleotide of natural origin. Such naturally occurring TIGIT polynucleotides may be isolated from nature or may be produced by recombinant or synthetic means. The term "naturally occurring TIGIT polynucleotide" particularly includes polynucleotides encoding naturally occurring cleavage or secreted forms (e.g., extracellular domain sequences) of a particular TIGIT polypeptide, naturally occurring variant forms (e.g., alternatively spliced forms), and naturally occurring allelic variants of the polypeptide. In various embodiments of the present invention, the naturally occurring TIGIT polynucleotides disclosed herein are mature or full-length naturally occurring polynucleotides containing the full-length nucleic acid sequence.

[0033] The term "TIGIT variant polynucleotide" or "TIGIT variant nucleic acid sequence" means a nucleic acid molecule that encodes an active TIGIT polypeptide, as defined below, and has at least about 80% nucleic acid sequence identity with a nucleic acid sequence encoding the full-length native sequence TIGIT polypeptide disclosed herein, the full-length native sequence TIGIT polypeptide sequence lacking the signal peptide disclosed herein, the extracellular domain of the TIGIT polypeptide disclosed herein with or without the signal peptide, or any other fragment of the full-length TIGIT polypeptide sequence. Typically, a TIGIT variant polynucleotide has at least about 80% nucleic acid sequence identity, or at least about 81% nucleic acid sequence identity, or at least about 82% nucleic acid sequence identity, or at least about 83% nucleic acid sequence identity, or at least about 84% nucleic acid sequence identity, or at least about 85% nucleic acid sequence identity, or at least about 86% nucleic acid sequence identity, or at least about 87% nucleic acid sequence identity, or at least about 88% nucleic acid sequence identity, or at least about 89% nucleic acid sequence identity, or at least about 90% nucleic acid sequence identity, or at least about 91% nucleic acid sequence identity, or at least about 92% nucleic acid sequence identity, or at least about 93% nucleic acid sequence identity, or at least about 94% nucleic acid sequence identity, or at least about 95% nucleic acid sequence identity, or at least about 96% nucleic acid sequence identity, or at least about 97% nucleic acid sequence identity, or at least about 98% nucleic acid sequence identity, and, or at least about 99% nucleic acid sequence identity with a nucleic acid sequence encoding the full-length native sequence TIGIT polypeptide disclosed herein, the full-length native sequence TIGIT polypeptide sequence lacking the signal peptide disclosed herein, the extracellular domain of the TIGIT polypeptide disclosed herein with or without the signal peptide, or any other fragment of the full-length TIGIT polypeptide sequence. The variant does not include the native nucleotide sequence.

[0034] Typically, a TIGIT variant polynucleotide is at least about 30 nucleotides in length, or at least about 60 nucleotides in length, or at least about 90 nucleotides in length, or at least about 120 nucleotides in length, or at least about 150 nucleotides in length, or at least about 180 nucleotides in length, or at least about 210 nucleotides in length, or at least about 240 nucleotides in length, or at least about 270 nucleotides in length, or at least about 300 nucleotides in length, or at least about 450 nucleotides in length, or at least about 600 nucleotides in length, or at least about 900 nucleotides in length, or more.

[0035] "Percent (%) nucleic acid sequence identity" to the TIGIT-encoding nucleic acid sequences identified herein is defined as the percentage of nucleotides in a candidate sequence that are identical to the nucleotides of the subject TIGIT nucleic acid sequence, after aligning the sequences and introducing gaps if necessary to obtain the maximum percent sequence identity. Alignments for the purpose of determining percent nucleic acid sequence identity can be achieved by using various methods known to those of skill in the art, such as publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. The ALIGN-2 sequence comparison computer program was made by Genentech, Inc., and the source code was submitted with user documentation to the U.S. Copyright Office, Washington, D.C., 20559 and is registered under U.S. Copyright Registration No. TXU510087. ALIGN-2 is preferably available from Genentech, Inc., South San Francisco, California and may be compiled from publicly available source code. The ALIGN-2 program is compiled for use on a UNIX® operating system, preferably Digital UNIX® V4.0D. All sequence comparison parameters are set and not varied by the ALIGN-2 program.

[0036] In the situation where ALIGN-2 is used for nucleic acid sequence comparison, the % nucleic acid sequence identity of a given nucleic acid sequence C with, or to, a given nucleic acid sequence D (or it can also be said as a given nucleic acid sequence C having or containing a certain % nucleic acid sequence identity with, or to, a given amino acid sequence D) is calculated as follows: 100 times the fraction W / Z Here, W is the number of nucleic acid residues with scores that match as identical by the alignment of C and D by the sequence alignment program ALIGN-2, and Z is the total number of nucleic acid residues of D. When the length of nucleic acid sequence C is different from the length of amino acid sequence D, it will be understood that the % nucleic acid sequence identity of C to D is different from the % nucleic acid sequence identity of D to C. As an example of the calculation of % nucleic acid sequence identity using this method, Tables 3 and 4 show the calculation methods of the % nucleic acid sequence identity of nucleic acid sequences referred to as "comparative DNA" to a nucleic acid sequence referred to as "TIGIT-DNA". "TIGIT-DNA" represents a hypothetical TIGIT-coding nucleic acid sequence of interest, "comparative DNA" represents the nucleic acid sequence to which the "TIGIT-DNA" nucleic acid molecule of interest is being compared, and each of "N", "L", and "V" represents different hypothetical amino acid residues.

[0037] Table 3 Target DNA NNNNNNNNNNNNNN (length = 14 nucleotides) Comparative DNA NNNNNNLLLLLLLLLL (length = 16 nucleotides) % nucleic acid sequence identity = (The number of nucleotides that match identically between the two nucleic acid sequences determined by ALIGN-2) ÷ (The total number of nucleotides of the target DNA) = 6 ÷ 14 = 42.9% Table 4 Target DNA NNNNNNNNNNNN (length = 12 nucleotides) Comparative DNA NNNNLLLVV (length = 9 nucleotides) % nucleic acid sequence identity = (The number of nucleotides that identically match between two nucleic acid sequences determined by ALIGN-2)÷(The total number of nucleotides of the target DNA)=4÷12 = 33.3%

[0038] Unless otherwise specified, all % nucleic acid sequence identity values herein are obtained using the ALIGN-2 sequence comparison computer program as shown in the immediately preceding paragraph and Tables 3 and 4. However, the % nucleic acid sequence identity value may be determined using the WU-BLAST-2 computer program (Altschul et al., Methods in Enzymology 266:460-480 (1996)). Further, most of the WU-BLAST-2 search parameters are set to their initial values. The parameters that are not set to the initial values, i.e., the adjustable parameters, are set to the following values: overlap span = 1, overlap fraction = 0.125, word threshold (T) = 11, and scoring matrix = BLOSUM62. When WU-BLAST-2 is used, the % nucleic acid sequence identity value is determined by the quotient of (a) the number of identical nucleic acid residues determined by WU-BLAST-2 between the nucleic acid sequence of a TIGIT polypeptide-encoding nucleic acid molecule having a sequence derived from a native sequence TIGIT polypeptide-encoding nucleic acid and the nucleic acid sequence of a comparative nucleic acid sequence to be targeted (i.e., a sequence that the TIGIT polypeptide-encoding nucleic acid molecule to be targeted may be compared to, which may be a TIGIT polypeptide variant), divided by (b) the total number of nucleotides of the TIGIT polypeptide-encoding nucleic acid molecule to be targeted. For example, in the expression "a polypeptide comprising a nucleic acid sequence A having at least 80% nucleic acid sequence identity to nucleic acid sequence B", nucleic acid sequence A is the comparative nucleic acid sequence to be targeted, and nucleic acid sequence B is the nucleic acid sequence of the TIGIT polypeptide-encoding nucleic acid molecule to be targeted.

[0039] Alternatively, the % nucleic acid sequence identity may be determined using the sequence comparison program NCBI-BLAST2 (Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997)). The NCBI-BLAST2 sequence comparison program can be downloaded from http: / / www.ncbi.nlm.nih.gov or obtained from the National Institutes of Health, Bethesda, Maryland, in another way. NCBI-BLAST2 uses several search parameters, all of which are set to their default values, for example, unmask=yes, strand=all, expected occurrences=10, minimum low complexity length=15 / 5, multi-pass e-value=0.01, multi-pass constant=25, final gap alignment drop-off=25, and scoring matrix=BLOSUM62.

[0040] In the situation where NCBI-BLAST2 is used for nucleic acid sequence comparison, the % nucleic acid sequence identity of a given nucleic acid sequence C with, to, or against a given nucleic acid sequence D (or it can also be said as a given nucleic acid sequence C having or containing a certain % nucleic acid sequence identity with, to, or against a given nucleic acid sequence D) is calculated as follows: 100 times the fraction W / Z where W is the number of nucleic acid residues with a score that is identical as determined by the alignment of C and D by the sequence alignment program NCBI-BLAST2, and Z is the total number of nucleic acid residues in D. It will be understood that if the length of nucleic acid sequence C is different from the length of nucleic acid sequence D, the % nucleic acid sequence identity of C to D will be different from the % nucleic acid sequence identity of D to C. In other embodiments, the TIGIT variant polypeptide nucleotide encodes an active TIGIT polypeptide and preferably hybridizes under stringent hybridization and washing conditions to a nucleotide sequence encoding the full-length TIGIT polypeptide disclosed herein. The TIGIT variant polypeptide may be encoded by the TIGIT variant polynucleotide.

[0041] As used herein to describe the various polypeptides disclosed herein, "isolated" means a polypeptide that has been identified, separated and / or recovered from the components of its natural environment. Contaminating components of its natural environment are substances that typically interfere with the diagnostic or therapeutic use of the polypeptide and include enzymes, hormones, and other protein-like or non-protein-like solutes. In preferred embodiments, the polypeptide is purified to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or preferably silver staining, or (1) to such an extent as to obtain an N-terminal or internal amino acid sequence of at least 15 residues by using a spinning cup sequenator. An isolated polypeptide includes the protein of interest within recombinant cells because at least one component of the polypeptide's natural environment is absent. However, typically, an isolated polypeptide is prepared by at least one purification step. An "isolated" nucleic acid encoding a TIGIT polypeptide is a nucleic acid molecule that has been identified and separated from at least one contaminating nucleic acid molecule that is normally associated with the natural source of the nucleic acid encoding the polypeptide. An isolated nucleic acid molecule encoding a polypeptide is other than in its natural form or setting. Thus, an isolated nucleic acid molecule encoding a polypeptide is distinguished from the particular nucleic acid molecule encoding the polypeptide that exists in natural cells. However, an isolated nucleic acid molecule encoding a polypeptide includes, for example, a nucleic acid molecule encoding a polypeptide that is included in a cell that normally expresses the polypeptide where the nucleic acid molecule is in a chromosomal location different from that of the nucleic acid molecule of the natural cell.

[0042] The expression "control sequence" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. For example, suitable control sequences for prokaryotes include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals and enhancers. A nucleic acid is "operably linked" when it is in a functional relationship with another nucleic acid sequence. For example, a DNA of a presequence or a secretory leader is operably linked to the DNA of a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned to facilitate translation. Generally, "operably linked" means that the linked DNA sequences are contiguous and, in the case of a secretory leader, contiguous and in the reading phase. However, an enhancer does not necessarily have to be contiguous. The linkage is achieved by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers are used according to conventional methods.

[0043] The term "antibody" is used in the broadest sense and includes, for example, a single anti-TIGIT monoclonal antibody or an antibody that specifically binds any other polypeptide described herein (including agonists, antagonists, and neutralizing antibodies), an anti-TIGIT antibody or antibody composition having multi-epitope specificity, a single-chain anti-TIGIT antibody or other antibody, and fragments of an anti-TIGIT antibody or other antibody (see below). As used herein, the term "monoclonal antibody" refers to a substantially homogeneous population of antibodies, i.e., a population of antibodies wherein the individual antibodies that make up the population are identical except for naturally occurring mutations that may be present in minor amounts. The "stringency" of a hybridization reaction is an empirical calculation readily determined by one of ordinary skill in the art and generally depends on probe length, wash temperature, and salt concentration. Generally, as the probe length increases, the temperature for proper annealing increases, and as the probe length decreases, the temperature decreases. Hybridization generally depends on the ability of denatured DNA to reanneal when complementary strands are present in an environment near but below their melting point. As the degree of desired homology between the probe and the sequence capable of hybridizing to the probe increases, the relative temperature that can be used increases. As a result, a higher relative temperature makes the reaction conditions more stringent, while a lower temperature decreases stringency. Further, stringency is inversely proportional to salt concentration. For further details and explanation of the stringency of hybridization reactions, see Ausubel et al., Current protocols in Molecular Biology, Wiley Interscience Publishers, (1995).

[0044] The "stringency condition" or "high stringency condition" defined herein refers to those using low ionic strength and high temperature for washing, such as those using 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% sodium dodecyl sulfate at 50 °C; those using a denaturing agent such as formamide during hybridization, such as those with 50% (v / v) formamide, 0.1% bovine serum albumin / 0.1% ficoll / 0.1% polyvinylpyrrolidone / 50 mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride and 75 mM sodium citrate added at 42 °C; or those using 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution, sonicated salmon sperm DNA (50 μg / ml), 0.1% SDS, and 10% dextran sulfate at 42 °C, washed in 0.2×SSC (sodium chloride / sodium citrate) at 42 °C and in 50% formamide at 55 °C, and then identified by using a high stringency wash consisting of 0.1×SSC containing EDTA at 55 °C.

[0045] "Moderate stringency conditions" are defined as described in Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989, and include the use of washing solutions and hybridization conditions (e.g., temperature, ionic strength, and % SDS) that are less stringent than those described above. Moderate stringency conditions involve an overnight incubation at 37 °C in a solution containing 20% formamide, 5× SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10% dextran sulfate, and 20 mg / mL denatured, sheared salmon sperm DNA, followed by filter washing in 1× SSC at 37 - 50 °C. Those skilled in the art will know how to adjust the temperature, ionic strength, etc. as appropriate to accommodate factors such as probe length.

[0046] As used herein, the term "epitope tag" refers to a chimeric polypeptide comprising a polypeptide of interest (as a non-limiting example, a TIGIT polypeptide) fused to a "tag polypeptide". The tag polypeptide has a sufficient number of residues to provide an epitope against which an antibody can be produced, or an epitope that can be identified by some other reagent, but is short enough so that its length does not inhibit the activity of the polypeptide to which it is fused. Further, the tag polypeptide is preferably quite unique so that the antibody does not substantially cross-react with other epitopes. Suitable tag polypeptides generally have at least 6 amino acid residues, usually about 8 - about 50 amino acid residues (preferably about 10 - about 20 residues). As used herein, the term "immunoadhesin" refers to an antibody-like molecule that combines the binding specificity of a heterologous protein (the "adhesin") with an immunoglobulin constant domain. Structurally, an immunoadhesin comprises a fusion of an amino acid sequence having the desired binding specificity and that is outside the antigen recognition and binding site of the antibody (i.e., "heterologous") with an immunoglobulin constant domain sequence. The adhesin portion of the immunoadhesin molecule is typically an adjacent amino acid sequence that includes at least the binding site of a receptor or ligand. The immunoglobulin constant domain sequence of the immunoadhesin can be obtained from any immunoglobulin, such as IgG-1, IgG-2, IgG-3 or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE, IgD or IgM.

[0047] As used herein, "active" or "activity" with respect to the purposes herein means a form of a polypeptide (such as a TIGIT polypeptide, by way of non-limiting example) that retains the biological and / or immunological activity (in the previous example, TIGIT activity) of the polypeptide in its native or naturally occurring form, where "biological" activity means a biological function (inhibitory or stimulatory) caused by a native or naturally occurring polypeptide other than the ability to induce the production of an antibody against an antigenic epitope retained by the native or naturally occurring polypeptide, and "immunological" activity means the ability to induce the production of an antibody against an antigenic epitope (in the previous example, the TIGIT antigenic epitope) retained by the native or naturally occurring polypeptide.

[0048] The term "aptamer" refers to a nucleic acid molecule that can bind to a target molecule such as a polypeptide. For example, the aptamers of the present invention can specifically bind to a TIGIT polypeptide or a molecule within a signaling pathway that regulates the expression of TIGIT. The generation and therapeutic use of aptamers are well established in the art. For example, for the treatment of age-related macular degeneration, reference is made to U.S. Patent No. 5,475,096 and the therapeutic efficacy of Macugen® (Eyetech, New York). The term "antagonist" is used in the broadest sense and refers to any molecule that blocks, inhibits, or neutralizes the biological activity of the natural polypeptides disclosed herein. Similarly, the term "agonist" is used in the broadest sense and refers to any molecule that mimics the biological activity of the natural polypeptides disclosed herein. Suitable agonist or antagonist molecules include, in particular, agonist or antagonist antibodies or antibody fragments, fragments or amino acid sequence variants of natural polypeptides, peptides, antisense oligonucleotides, small organic molecules, and the like. Methods for identifying agonists or antagonists of a polypeptide may include contacting the polypeptide with a candidate antagonist or agonist and measuring a change in one or more biological activities normally associated with the polypeptide.

[0049] The terms "TIGIT antagonist" and "antagonist of TIGIT activity or TIGIT expression" are used interchangeably and refer to a compound that interferes with the normal function of TIGIT by reducing or inhibiting or blocking the transcription or translation of TIGIT-encoding nucleic acid and / or inhibiting the activity of the TIGIT polypeptide. Examples of TIGIT antagonists include, but are not limited to, antisense polynucleotides, interfering RNAs, catalytic RNAs, RNA-DNA chimeras, TIGIT-specific aptamers, anti-TIGIT antibodies, TIGIT-binding fragments of anti-TIGIT antibodies, TIGIT-binding small molecules, TIGIT-binding peptides, and other polypeptides that specifically bind to TIGIT (including, but not limited to, TIGIT-binding fragments of one or more TIGIT ligands, optionally fused to one or more other domains in some cases) and cause a reduction or cessation of TIGIT activity or expression through the interaction of the TIGIT antagonist with TIGIT. It is understood by those skilled in the art that in some cases, a TIGIT antagonist may neutralize one TIGIT activity without acting on other TIGIT activities. For example, a desirable TIGIT antagonist for use in a method herein is a TIGIT antagonist that neutralizes TIGIT activity in response to one of the PVR interaction, PVRL3 interaction, or PVRL2 interaction, for example, without affecting or with minimal effect on any other TIGIT interaction.

[0050] The terms "PVR antagonist" and "antagonist of PVR activity or PVR expression" are used interchangeably and refer to a compound that interferes with the normal function of PVR by reducing or inhibiting or blocking the transcription or translation of PVR-encoding nucleic acid and / or the activity of the PVR polypeptide. Examples of PVR antagonists include, but are not limited to, antisense polynucleotides, interfering RNAs, catalytic RNAs, RNA-DNA chimeras, PVR-specific aptamers, anti-PVR antibodies, PVR-binding fragments of anti-PVR antibodies, PVR-binding small molecules, PVR-binding peptides, and other polypeptides that specifically bind to PVR (including, but not limited to, PVR-binding fragments of one or more PVR ligands, optionally fused to one or more other domains in some cases) and cause a reduction or cessation of PVR activity or expression by interaction of the PVR antagonist with PVR. It is understood by those skilled in the art that in some cases, a PVR antagonist may neutralize one PVR activity without acting on other PVR activities. For example, a desirable PVR antagonist for use in a method herein is a PVR antagonist that neutralizes PVR activity in response to TIGIT interaction without affecting the interaction of PVR-CD96 and / or PVR-CD226.

[0051] The terms "TIGIT agonist" and "agonist of TIGIT activity or TIGIT expression" are used interchangeably and refer to a compound that enhances or stimulates the normal function of TIGIT by increasing the transcription or translation of TIGIT-encoding nucleic acid and / or by interfering with or blocking the activity of a molecule that inhibits TIGIT expression or TIGIT activity and / or by enhancing (including, but not limited to, enhancing the stability of TIGIT or enhancing the binding of TIGIT to one or more target ligands) the normal activity of TIGIT. For example, a TIGIT agonist may be selected from antibodies, antigen-binding fragments, aptamers, interfering RNAs, small molecules, peptides, antisense molecules, and other binding polypeptides. In other examples, a TIGIT agonist may be a polynucleotide selected from aptamers, interfering RNAs, or antisense molecules that interfere with the transcription and / or translation of a TIGIT inhibitory molecule. It is understood by those skilled in the art that, in some cases, a TIGIT agonist may agonize one TIGIT activity without affecting other TIGIT activities. For example, a desirable TIGIT agonist for use in a method herein may be a TIGIT agonist that agonizes TIGIT activity in response to one of a PVR interaction, a PVRL3 interaction, or a PVRL2 interaction without affecting or with minimal effect on any other TIGIT interaction.

[0052] The terms "PVR agonist" and "agonist of PVR activity or PVR expression" are used interchangeably and refer to a compound that enhances or stimulates the normal function of PVR by increasing the transcription or translation of PVR-encoding nucleic acid and / or by interfering with or blocking the activity of a molecule that inhibits PVR expression or PVR activity and / or by enhancing (including, but not limited to, enhancing the stability of PVR or enhancing the binding of PVR to one or more target ligands) the normal PVR activity. For example, a PVR agonist may be selected from antibodies, antigen-binding fragments, aptamers, interfering RNAs, small molecules, peptides, antisense molecules, and other binding polypeptides. In other examples, a PVR agonist may be a polynucleotide selected from aptamers, interfering RNAs, or antisense molecules that interfere with the transcription and / or translation of a PVR inhibitory molecule. It is understood by those skilled in the art that in some cases, a PVR agonist may agonize one PVR activity without affecting other PVR activities. For example, a desirable PVR agonist for use in a method herein may agonize PVR activity in response to TIGIT interaction or mimic TIGIT when interfering with PVR with little or no effect on, for example, the binding interaction of PVR-CD96 to PVR-CD226.

[0053] "Treatment" means both curative treatment, prophylactic therapy and preventive therapy, and the patient can be prevented or reduced (decreased) from the targeted pathological condition or disease. Those in need of treatment include those already suffering from the disease, as well as those susceptible to the disease or those in whom the disease should be prevented. "Chronic" administration means administering the drug in a continuous manner, different from the acute mode, and maintaining the initial therapeutic effect (activity) over a long period of time. "Intermittent" administration is a process that is not continuous without interruption, but rather is essentially periodic. "Mammal" for purposes of treatment means any animal classified as a mammal, including humans, domestic and agricultural animals, zoo, sports, or pet animals such as dogs, cats, cows, horses, sheep, pigs, rabbits, etc. Preferably, the mammal is a human. "Administered in combination with" one or more therapeutic agents includes simultaneous (concurrent) and sequential administration in any order.

[0054] As used herein, "carrier" includes a pharmaceutically acceptable carrier, excipient, or stabilizer and is non-toxic to the cells or mammals to which they are exposed at the dosages and concentrations used. Physiologically acceptable carriers are often aqueous pH buffer solutions. Examples of physiologically acceptable carriers are buffers of phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophobic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextran; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as TWEEN (trade name), polyethylene glycol (PEG), and PLURONICS (trade name).

[0055] "Antibody fragment" includes a portion of the parent antibody, preferably the antigen-binding or variable region of the parent antibody. Examples of antibody fragments include Fab, Fab’, F(ab’)2, and Fv fragments; diabodies; linear antibodies (Zapata et al., Protein Eng. 8(10):1057-1062

[1995] ); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. Papain digestion of an antibody produces two identical antibody-binding fragments called "Fab" fragments, each of which has a single antigen-binding site, and the remainder is named the "Fc" fragment to reflect its ability to crystallize readily. Pepsin treatment yields the F(ab’)2 fragment, which has two antigen-binding sites and can cross-link antigens. "Fv" is the smallest antibody fragment that contains a complete antigen recognition and binding site. This region consists of a dimer of the variable regions of one heavy chain and one light chain that are closely non-covalently associated. In this arrangement, the three CDRs of each domain interact to determine the antigen-binding site on the surface of the V H -V L dimer. Properly, six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of the Fv containing only the three CDRs specific for the antigen), although having a lower affinity than the entire binding site, has the ability to recognize and bind the antigen.

[0056] The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. The Fab fragment differs from the Fab’ fragment by having several residues added to the carboxy terminus of the heavy chain CH1 domain that includes one or more cysteines from the antibody hinge region. Here, Fab’-SH represents Fab’ in which the cysteine residue of the constant domain has a free thiol group. The F(ab’)2 antibody fragment is initially generated as a pair of Fab’ fragments that have a hinge cysteine between them. Other chemical linkages of antibody fragments are also known. The "light chains" of antibodies (immunoglobulins) from any vertebrate species are classified into one of two distinct types called kappa and lambda based on the amino acid sequence of their constant domains.

[0057] Based on the amino acid sequence of their heavy chain constant domains, immunoglobulins can be classified into different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and some of them are further classified into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. "Single-chain Fv" or "sFv" antibody fragments are fragments of the V H and V L The Fv polypeptide preferably comprises a V domain, which enables the sFv to form the desired structure for antigen binding. H and V L For a review of sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, edited by Rosenburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994).

[0058] The term "diabodies" refers to small antibody fragments with two antigen-binding sites, which are bound to the same polypeptide chain (V H -V L ) in the light chain variable domain (V L ) bound to a heavy chain variable domain (V H (Diabody) is a type of diabody that comprises a linker that is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with the complementary domains of another chain and creating two antigen-binding sites. Diabodies are more fully described in, for example, EP 404,097; WO 93 / 11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993).

[0059] An "isolated" antibody is one that has been identified, separated and / or recovered from the components of its natural environment. The contaminating components of its natural environment are substances that interfere with the diagnostic or therapeutic use of the antibody and include enzymes, hormones, and other protein-like or non-protein-like solutes. In certain embodiments, the antibody is (1) greater than 95%, most preferably greater than 99% by weight as determined by the Lowry method, (2) purified to homogeneity by SDS-PAGE under non-reducing or reducing conditions using, for example, Coomassie blue or silver stain, or (3) sufficiently purified such that at least 15 residues of the N-terminal or internal amino acid sequence can be obtained using a spinning cup sequenator. Isolated antibodies include antibodies in insitu within recombinant cells since at least one component of the antibody's natural environment will not be present. However, typically, isolated antibodies are prepared by at least one purification step. An antibody that "specifically binds" to a particular polypeptide or to an epitope on a particular polypeptide is one that binds to that particular polypeptide or to an epitope on that particular polypeptide without substantially binding to other polypeptides or polypeptide epitopes.

[0060] As used herein, the terms "hypervariable region", "HVR" or "HV" refer to regions of the antibody variable domain that are highly variable in sequence and / or form structurally defined loops. Generally, an antibody contains six HVRs, i.e., three in VH (H1, H2, H3) and three in VL (L1, L2, L3). In native antibodies, H3 and L3 exhibit the greatest diversity among the six HVRs, and in particular, H3 appears to play a unique role in conferring good specificity to the antibody. See, e.g., Johnson and Wu (2003) in Xu et al. (2000) Immunity 13:37-45; Methods in Molecular Biology 248:1-25 (Lo, ed., Human Press, Totowa, NJ). In fact, naturally occurring camelid antibodies consisting only of heavy chains are functional and stable in the absence of light chains. Hamers-Casterman et al. (1993) Nature 363:446-448; Sheriff et al. (1996) Nature Struct. Biol. 3:733-736. Descriptions of a number of HVRs are used and included herein. Kabat complementarity determining regions (CDRs) are based on sequence variation and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). Chothia refers instead to the positions of structural loops (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). AbM HVRs represent a compromise between Kabat HVRs and Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software. "Contact" HVRs are based on analysis of available complex crystal structures. Residues from each of these HVRs are shown below. Loop Kabat AbM Chothia Contact L1 L24-L34 L24-L34 L26-L32 L30-L36 L2 L50-L56 L50-L56 L50-L52 L46-L55 L3 L89-L97 L89-L97 L91-L96 L89-L96 H1 H31-H35B H26-H35B H26-H32 H30-H35B (Kabat numbering) H1 H31-H35 H26-H35 H26-H32 H30-H35 (Chothia numbering) H2 H50-H65 H50-H58 H53-H55 H47-H58 H3 H95-H102 H95-H102 H96-H101 H93-H101

[0061] The HVRs may include "extended HVRs" such as those of VL residues 24-36 or 24-34 (L1), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3), and VH residues 26-35 (H1), 50-65 or 49-65 (H2), and 93-102, 94-102, or 95-102 (H3). The variable domain residues are numbered according to Kabat et al. supra to define each of these. "Framework" or "FR" residues are variable domain residues other than the HVRs defined herein.

[0062] The terms "Kabat numbering of variable domain residues" or "amino acid numbering as described in Kabat" and different phrasings thereof refer to the numbering system used for the light chain variable domain or the heavy chain variable domain of the antibody editing by Kabat et al. supra. Using this numbering system, the actual linear amino acid sequence may include two or three amino acids or additional amino acids corresponding to deletions or insertions within the FR or HVR of the variable domain. For example, the heavy chain variable domain may include residues inserted after heavy chain FR residue 82 (such as residues 82a, 82b, and 82c according to Kabat, etc.) and a single amino acid insertion (residue 52a according to Kabat) after residue 52 of H2. The Kabat number of a residue may be determined for an antibody by aligning the sequence of the antibody in the homologous region by the "standard" Kabat numbering sequence. The Kabat numbering system is generally used when referring to residues within the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) (e.g., Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The "EU numbering system" or "EU index" is generally used when referring to the immunoglobulin heavy chain constant region (e.g., the EU index reported in Kabat et al. supra). The "EU index in Kabat" refers to the residue numbering of human IgG1 EU antibody. Unless otherwise specified herein, a reference to a residue number within the variable domain of an antibody means a residue numbered by the Kabat numbering system. Unless otherwise specified herein, a reference to a residue number within the constant domain of an antibody means a residue numbered by the EU numbering system (see, for example, the figure regarding EU numbering in U.S. Patent Application No. 60 / 640323).

[0063] An "affinity matured" antibody is an antibody that has one or more changes in one or more of its HVRs and has an improved affinity for antigen as compared to a parental antibody that does not have such changes. In one embodiment, the affinity matured antibody has a nanomolar or even picomolar affinity for the target antigen. Affinity matured antibodies can be produced by methods known in the art. For example, Marks et al., Bio / Technology, 10:779-783 (1992), disclose affinity maturation by shuffling of VH and VL domains. Random mutagenesis of HVRs and / or framework residues is disclosed, for example, in Barbas et al. Proc Nat. Acad. Sci, USA 91:3809-3813 (1994); Schier et al. Gene 169:147-155 (1995); Yelton et al. J. Immunol. 155:1994-2004 (1995); Jackson et al., J. Immunol. 154(7): 3310-9 (1995); and Hawkins et al, J. Mol. Biol. 226:889-896 (1992).

[0064] A "blocking" antibody or "antagonist" antibody is one that inhibits or reduces the biological activity of the antigen to which it binds. Some blocking or antagonist antibodies inhibit the biological activity of the antigen substantially or completely. As used herein, an "agonist antibody" is an antibody that mimics at least one of the functional activities of a polypeptide of interest, in part or in whole. The term "label" as used herein refers to a detectable compound or composition conjugated directly or indirectly to an antibody such that an "labeled" antibody is produced. The label may be detectable itself (e.g., a radioactive or fluorescent label) or, in the case of an enzyme label, may catalyze a chemical conversion of a detectable substrate compound or composition.

[0065] "Solid phase" means a non-aqueous matrix to which the antibody of the present invention can attach. Examples of solid phases contemplated herein include, but are not limited to, those formed in whole or in part from glass (e.g., pore-controlled glass), polysaccharides (e.g., agarose), polyacrylamide, polystyrene, polyvinyl alcohol, and silicone. In certain embodiments, depending on the context, the solid phase can constitute the wells of an assay plate; in other cases, it can be a purification column (e.g., an affinity chromatography column). The term also encompasses discrete solid phases of separate particles, as described in U.S. Patent No. 4,275,149. "Liposome" is a small vesicle composed of various types of lipids, phospholipids, and / or surfactants and is useful for the transport of drugs (such as polypeptides or their antibodies described herein) to mammals. The components of liposomes are usually arranged in a bilayer format similar to the lipid arrangement of biological membranes. "Small molecule" is hereby defined as having a molecular weight of less than about 500 daltons.

[0066] The term "immune-related disease" means a disease in which a component of the mammalian immune system causes, mediates, or contributes to a pathological condition in a mammal. It also includes diseases in which stimulation or treatment of an immune response has an ameliorative effect on the progression of the disease. This term includes immune-mediated inflammatory diseases, non-immune-mediated inflammatory diseases, infectious diseases, immunodeficiency diseases, tumor formation, and the like. The term "T cell-mediated disease" means an immune-related disease in which T cells directly or indirectly mediate or contribute to a pathological condition in a mammal. T cell-mediated diseases are related to cell-mediated effects, lymphokine-mediated effects, etc., and even effects related to B cells when, for example, B cells are stimulated by lymphokines secreted by T cells.

[0067] Immune-related diseases and inflammatory diseases include immune- or T cell-mediated ones and are treatable according to the present invention. Those diseases include systemic lupus erythematosus, rheumatoid arthritis, juvenile chronic arthritis, spondyloarthropathy, systemic sclerosis (scleroderma), idiopathic inflammatory myopathies (dermatomyositis, polymyositis), Sjogren's syndrome, systemic vasculitis, sarcoidosis, autoimmune hemolytic anemia (immune pancytopenia, paroxysmal nocturnal hemoglobinuria), autoimmune thrombocytopenia (idiopathic thrombocytopenic purpura, immune-mediated thrombocytopenia), thyroiditis (Graves' disease, Hashimoto's thyroiditis, juvenile lymphocytic thyroiditis, atrophic thyroiditis), diabetes mellitus, immune kidney diseases (glomerulonephritis, tubulointerstitial nephritis), demyelinating diseases of the central and peripheral nervous systems, such as multiple sclerosis, idiopathic demyelinating polyneuropathy, or Guillain-Barré syndrome, and chronic inflammatory demyelinating polyneuropathy, hepatobiliary diseases, such as infectious hepatitis (hepatitis A, B, C, D, E and other non-hepatic viruses), autoimmune chronic active hepatitis, primary biliary cirrhosis, granulomatous hepatitis, sclerosing cholangitis, inflammatory bowel diseases (IBD) (ulcerative colitis: Crohn's disease), gluten-sensitive enteropathy, Whipple's disease, autoimmune or immune-mediated skin diseases, vesicular skin diseases, erythema multiforme exudativum, contact dermatitis, psoriasis, allergic diseases, asthma, allergic rhinitis, atopic dermatitis, food allergy, urticaria, immunological diseases of the lung, eosinophilic pneumonia, idiopathic pulmonary fibrosis, hypersensitivity interstitial pneumonia, transplant-related diseases including graft rejection and graft-versus-host disease. Infectious diseases including viral diseases such as AIDS (HIV infection), hepatitis A, B, C, D, and E, bacterial infections, fungal infections, protozoal infections, and parasitic infections can also have immune and / or inflammatory components and / or etiologies.

[0068] Some skin diseases are associated with abnormal immune responses and autoimmune conditions. Diseases such as psoriasis are characterized by skin blister formation, skin white dot formation, edema and the presence of autoantibodies that bind to skin proteins. In the present application, it is determined experimentally that TIGIT expression is upregulated in psoriatic skin compared to normal skin. Regulation of the expression and / or activity of TIGIT may be useful in treating the symptoms or underlying cause of psoriasis.

[0069] The term "inflammatory bowel disease" ("IBD") refers to a group of chronic inflammatory disorders of unknown cause that cause inflammation in the intestinal tract (intestine) and sometimes recurrent abdominal pain or diarrhea. The prevalence of IBD in the United States is estimated to be approximately 200 per 100,000 population. Patients with IBD are divided into two main groups: those with ulcerative colitis ("UC") and those with Crohn's disease ("CD"). In patients with UC, an inflammatory reaction of the colonic mucosa is mainly observed. Usually, the inflammation is uniform and continuous, and no normal mucosa is seen in the intermediate part. An inflammatory reaction due to neutrophil infiltration occurs in the crypt epithelium, submucosa, and surface mucosal cells. Usually, such a situation ultimately progresses to epithelial damage, resulting in the loss of epithelial cells and, as a result, multiple ulcerations, fibrosis, dysplasia, and longitudinal colonic atrophy occur. CD differs from UC in that the inflammation extends through all layers of the intestinal wall, and inflammation occurs not only in the lymph nodes but also in the intestinal wall. CD can occur in any part of the gastrointestinal tract from the mouth to the anus. This disease is often discontinuous, that is, the severely affected part of the intestine is separated from the area that is clearly disease-free. In CD, the intestinal wall may also thicken and lead to obstruction. In addition, fistulas and fissures are not uncommon.

[0070] Clinically, IBD is characterized by a variety of symptoms that often lead to a chronic and unpredictable course. Bleeding diarrhea and abdominal pain are often accompanied by fever and weight loss. Anemia is also common, as is severe fatigue. Joint symptoms ranging from joint pain to acute arthritis and abnormal liver function are generally considered to be associated with IBD. In addition, the risk of IBD patients developing colorectal cancer is greater than average. During an acute "flare-up" of IBD, work and other daily activities are usually impossible, and patients are often hospitalized. The cause of IBD remains unclear, but multiple factors such as genetics, infection, and immune susceptibility are thought to be involved. IBD is more common in white people, especially Ashkenazi Jews. Due to the chronic inflammatory nature of the symptoms, an intensive investigation into the possible infectious causes was carried out urgently. Although agents that stimulate acute inflammation have been found, none have been found to be the cause of chronic inflammation associated with IBD. The hypothesis that IBD is an autoimmune disease is supported by the fact that, as mentioned above, IBD has symptoms outside the intestine such as arthritis, and that positive responses to IBD are known to be seen with therapeutic agents such as adrenal glucocorticoids, cyclosporine, and azathioprine that are known to suppress the immune response. In addition, the gastrointestinal tract is continuously exposed to the possibility of antigenic substances such as dietary proteins and bacterial by-products (LPS) more than any other organ in the body.

[0071] Furthermore, in patients with severe ulcerative colitis, especially when the disease persists for several years, the risk of colorectal cancer increases significantly. Due to massive bleeding, chronic debilitating illness, perforation of the large intestine, or the risk of cancer, approximately 20 - 25% of IBD patients will ultimately require a colectomy. Surgery may also be performed when other forms of medical treatment have failed, or when the patient's health is threatened by the side effects of steroids or other drug applications. Surgery is invasive and life-changing, so it is not a very desirable treatment option and is usually a last resort. Experiments were conducted to deepen the understanding of this disease and enable treatment, and it was confirmed that TIGIT is upregulated in both CD and UC when compared to normal tissue. Modulation of the expression and / or activity of TIGIT may be useful for the treatment of one or more forms of IBD.

[0072] Rheumatoid arthritis (RA) is a chronic systemic autoimmune inflammatory disease mainly associated with the synovium of multiple joints, resulting in damage to articular cartilage. The etiology is T lymphocyte-dependent and is associated with the production of rheumatoid factor, an autoantibody against self-IgG, resulting in the formation of immune complexes that reach high levels in synovial fluid and blood. These complexes in the joints induce a marked infiltration of lymphocytes and monocytes into the synovium and subsequent marked synovial changes; the same is true in the joint space / fluid if infiltrated with similar cells by the addition of numerous neutrophils. Affected tissues are often in a symmetrical pattern and are mainly joints. However, two main forms of extra-articular disease also occur. One form is the occurrence of extra-articular disorders with typical lesions of progressive joint disease and pulmonary fibrosis, vasculitis, and skin ulcers. The second form of extra-articular disease is the so-called Felty syndrome, which occurs at the end of the RA disease process, sometimes after the joint disease has subsided, and is associated with the presence of neutropenia, thrombocytopenia, and splenomegaly. This is accompanied by vasculitis in multiple organs with the formation of infarcts, skin ulcers, and gangrene. In many cases, patients develop rheumatoid nodules in the subcutaneous tissue overlying the affected joints; the nodules have a necrotic center surrounded by a mixed inflammatory cell infiltration at the end stage. Other signs that may occur in RA include: pericarditis, pleurisy, coronary arteritis, interstitial pneumonia with pulmonary fibrosis, dry keratoconjunctivitis, and rheumatoid nodules.

[0073] Juvenile chronic arthritis is a chronic idiopathic inflammatory disease that often presents before the age of 16. Its phenotype has some similarities to RA; some patients with a positive rheumatoid factor are classified as juvenile rheumatoid-like arthritis. This disease is subdivided into three main categories: pauciarticular, polyarticular, and systemic. The arthritis is severe and typically destructive, leading to joint ankylosis and delayed growth. Other signs include chronic anterior uveitis and systemic amyloidosis.

[0074] "Effective amount" means the concentration or amount of a polypeptide and / or agonist / antagonist that causes a particular defined objective to be achieved. The "effective amount" of a polypeptide or agonist or antagonist can be determined empirically. Further, "therapeutically effective amount" means the concentration or amount of a polypeptide and / or agonist / antagonist that is effective to achieve a defined therapeutically effective amount. Also, this amount can be determined empirically. As used herein, "cytotoxic agent" means a substance that inhibits or suppresses the function of cells and / or causes cell destruction. This term means radioactive isotopes (e.g., I 131 , I 125 , Y 90 and Re 186 ), chemotherapeutic agents, and enzymatically active toxins or fragments thereof derived from bacteria, fungi, plants or animals.

[0075] "Chemotherapeutic agents" are compounds useful for the treatment of cancer. Examples of chemotherapeutic agents include adriamycin, doxorubicin, epirubicin, 5-fluorouracil, cytosine arabinoside ("Ara-C"), cyclophosphamide, thiotepa, busulfan, taxoids such as paclitaxel (Taxol, Bristol-Myers Squibb Oncology, Princeton, NJ) and docetaxel (Taxotere, Rhone-Poulenc Rorer, Antony, France), Taxotere, methotrexate, cisplatin, melphalan, vinblastine, bleomycin, etoposide, ifosfamide, mitomycin C, mitoxantrone, vincristine, vinorelbine, carboplatin, teniposide, daunomycin, carminomycin, aminopterin, dactinomycin, mitomycin, esperamicins (see U.S. Patent No. 4,675,187), melphalan, and other related nitrogen mustards. This definition also includes hormonal agents that act to regulate or inhibit the hormonal action on tumors, such as tamoxifen and onapristone.

[0076] As used herein, "growth inhibitor" refers to a compound or composition that inhibits the growth of cells that overexpress any of the genes specifically identified herein, either in vitro or in vivo. Thus, a growth inhibitor significantly reduces the percentage of such overexpressing cells in the S phase. Examples of growth inhibitors include agents that block the progression of the cell division cycle (at locations other than the S phase), such as agents that induce G1 arrest and M phase arrest. Traditional M phase blockers include vinca (vincristine and vinblastine), taxol, and topo II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. These agents that arrest G1, such as DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C, have an impact on S phase arrest. Further information can be found, for example, in The Molecular Basis of Cancer, edited by Mendelsohn and Israel, Chapter 1 (WB Saunders; Philadelphia, 1995), particularly on page 13, in the article "Cell cycle regulation, oncogene, and antineoplastic drugs" by Murakami et al.

[0077] The term "cytokine" is a general term for proteins released from one cell population that act as intercellular mediators on other cells. Some examples of such cytokines include lymphokines, monokines, and traditional polypeptide hormones. Cytokines include, for example, growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prolactin; glycoprotein hormones such as follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), and luteinizing hormone (LH); liver growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-α and -β; Müllerian inhibiting substance; mouse gonadotropin-related peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF-β; platelet growth factor; transforming growth factors (TGF) such as TGF-α or TGF-β; insulin-like growth factors-I and -II; erythropoietin (EPO); osteoinductive factor; interferons such as interferon-α, -β, and -γ; colony-stimulating factors (CSF) such as macrophage CSF (M-CSF); granulocyte macrophage CSF (GM-CSF) and granulocyte CSF (G-CSF); interleukins (IL) such as IL-1, IL-1a, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12, or IL-17; tumor necrosis factors such as TNF-α or TNF-β; and other polypeptide factors including LIF and kit ligand (KL). As used herein, the term cytokine includes proteins derived from natural sources or recombinant cell culture and biologically active equivalents of native sequence cytokines.

[0078] As used herein, the term "immunoadhesin" refers to an antibody-like molecule that combines the binding specificity of a heterologous protein (the "adhesin") with an immunoglobulin constant domain. Structurally, an immunoadhesin comprises a fusion of an amino acid sequence that has the desired binding specificity and that is heterologous (i.e., other than) to the antigen recognition and binding sites of an antibody, and an immunoglobulin constant domain sequence. The adhesin portion of the immunoadhesin molecule typically comprises an adjacent amino acid sequence that includes at least the binding site of a receptor or ligand. The immunoglobulin constant domain sequence of an immunoadhesin can be obtained from any immunoglobulin, such as IgG-1, IgG-2, IgG-3 or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE, IgD or IgM. As used herein, the term "inflammatory cell" means a cell that enhances an inflammatory response, such as a mononuclear cell, eosinophil, macrophage, and polymorphonuclear neutrophil (PMN).

[0079] II. Compositions and Methods of the Invention TIGIT has been identified as a putative modulator of immune function (see, e.g., U.S. Patent Publication No. US20040121370, which is incorporated herein by reference). As used herein, Applicants show that TIGIT is a member of a newly described family of immune-related proteins, the “TIGIT-like protein” (TLP) family, which includes the poliovirus receptor (also known as PVR, nectin-5 or CD155), PVR-like proteins 1-4 (PVRL1-4), CD96, and CD226. Applicants show conserved structural elements of this novel TLP family, members of which have roles in the regulation and function of immunity, and provide methods for identifying additional family members. PVRL1-4 and PVR share a common domain architecture (IgV-IgC-IgV), whereas CD226 and CD96 lack the membrane-proximal IgV domain. The intracellular segments of these eight proteins show limited similarity to each other outside of an afadin-binding motif shared among PVRL1-3, and PVRL4 lacks this sequence but is known to bind afadin. Based on the crystal structure of the relevant IgV domain of nectin-1 (Dong, X. et al., J Biol Chem 281, 10610-7 (2006)), the first and third motifs are predicted to be in the hairpin loops between strands B and C and between strands F and Gβ, respectively. These two loops are adjacent to each other at one end of the IgV fold. The second motif includes C' and C''β strands that are involved in forming part of the homodimer interface for nectin-1. Thus, the motifs of these sequences may function in specific homo- and heterotypic interactions observed among PVR family members.

[0080] The TLP family includes alanine 67 , glycine 74 , proline 114 and glycine 116It contains many fully conserved amino acids. Furthermore, the TLP family contains amino acids selected from valine, isoleucine and leucine at position 54, amino acids selected from serine and threonine at position 55, glutamine at position 56, threonine at position 112, and amino acids selected from phenylalanine and tyrosine at position 113, some amino acids that are substantially conserved (e.g., found in many but not all family members). Also, members of the TLP family have three structural submotifs: valine / isoleucine 54 -serine / threonine 55 -glutamine 56 ; alanine 67 -X 68-73 -glycine 74 (X is any amino acid); and, threonine 112 -phenylalanine / tyrosine 113 -proline 114 -X 115 -glycine 116 (X is any amino acid). The numbering used above is with respect to the human TIGIT protein sequence, and those skilled in the art understand that while the relative positions of these conserved residues and the motifs in different members of the TLP protein family are the same as the positions of these amino acids in the human TIGIT sequence, the absolute numbering of these residues in other TLP families may be different.

[0081] Since TLP family members identified in immune regulation and function are involved, other members of this protein family also appear to be involved in immune regulation and function. Accordingly, the present invention provides a method for determining whether a polypeptide is a member of the TLP family by aligning the polypeptide sequence with the sequences of one or more previously identified family members and evaluating the presence or absence in a protein sequence of previously identified fully conserved residues, previously identified substantially conserved residues, and / or previously identified structural submotifs. The present invention also provides a method for identifying other members of the TLP protein family by searching one or more sequence databases for proteins that contain previously identified fully conserved residues, previously identified substantially conserved residues, and / or previously identified structural submotifs in their amino acid sequences.

[0082] In addition, the identification of the TLP family by the applicant in this specification suggests the possibility that two or more members of the TLP family can be regulated in the same way by the common structural features of the TLP family. For example, if the conserved and substantially conserved amino acid residues and submotifs in each TLP family member result in a three-dimensional structure similar to these family members in one or more domains of each protein, these similar three-dimensional structures can be targeted to regulate two or more TLP family members or all TLP family members simultaneously. Accordingly, the present invention also provides an agent (a "TLP interacting agent") that specifically interacts with such conserved or substantially conserved regions of TLP family members. By using such an agent to evaluate whether a candidate protein interacts with the TLP interacting agent, one or more additional members of the TLP family may be identified. Interaction of the candidate protein with the TLP interacting agent indicates that the protein may be a TLP family member. The TLP interacting agent can modulate TLP activity. For example, the TLP interacting agent can be an antagonist of TLP activity, including but not limited to small molecule inhibitors, inhibitory antibodies or antigen-binding fragments thereof, aptamers and inhibitory peptides. In other examples, the TLP interacting agent can be an agonist of TLP activity, including but not limited to agonizing antibodies or antigen-binding fragments thereof, agonizing peptides, and small molecules that stabilize the structure of the TLP protein to facilitate TLP protein activity. The TLP interacting agent can be identified using various methods known in the art, such as the screening methods described herein.

[0083] The applicant shows by mRNA and FACS analysis that TIGIT is expressed mainly on various activated T cells, particularly regulatory T cells (T reg ) isolated from tonsil tissue, memory T cells, NK cells and follicular B cell helper T cells (T fh ). Thus, the present invention provides for selected cells to be T reg , memory T cells, NK cells or T FhProvided is a method for identifying whether a cell is a cell or not. Further, the present invention uses any purification method known in the art and / or described herein (non-limiting example: flow cytometry) to isolate T from other types of immune cells that do not express TIGIT reg , memory T cells, NK cells and T Fh cells. Also provided is a method of using TIGIT to purify cells. Further, the applicant shows that the highest TIGIT expression occurs in activated T regs among these cell populations. Thus, the present invention also provides a method for identifying whether a cell is an activated T regs cell based on the expression level of TIGIT compared to the expression level of TIGIT in one or more control samples (where the control sample may be a default value from an exemplary group of T cell subsets, or the control sample may be an activated T reg , non-activated T reg , naive T cells, memory T cells, NK cells, T Fh cells or other samples from known cell subpopulations such as other T cell groups). Also provided is a method for determining whether a certain Treg cell is activated by determining the expression level of TIGIT compared to the TIGIT expression level in one or more control activated or non-activated T reg samples, or compared to a predetermined TIGIT expression value in a known activated or non-activated T reg cell population. Further provided is a method for separately isolating activated Tregs from other T cells using any purification method known in the art and / or described herein (non-limiting example: flow cytometry) that uses the amount of TIGIT expressed in the cells to separate the cells from other cells.

[0084] The applicant demonstrates that TIGIT binds strongly to PVR and binds to PVRL3 (also known as nectin-3 or CD113) and PVRL2 (also known as nectin-2 or CD112) with a small Kd. As exemplified by the applicant, TIGIT that binds to PVR blocks the interaction between PVR and two other ligands such as CD226 and CD96, and CD226 is an inhibitor of the TIGIT-PVR interaction that is less effective than TIGIT for the PVR-CD226 interaction. The applicant produced anti-TIGIT antibodies (such as anti-TIGIT antibody 10A7 described herein) that inhibit the binding of TIGIT or TIGIT fusion proteins to PVR expressed on the cell surface. The applicant further produced other antibodies such as antibody 1F4 described herein that have epitope specificities on TIGIT different from 10A7. In particular, CD226 is not significantly expressed in T regs or T Fh or T that are cell types that highly express TIGIT.

[0085] As supported by these findings, the present invention provides agonists and antagonists of the TIGIT-PVR interaction, the TIGIT-PVRL2 interaction, and the TIGIT-PVRL3 interaction, and methods for modulating TIGIT-PVR binding, TIGIT-PVRL2 binding, and TIGIT-PVRL3 binding in vitro or in vivo using these agonists and antagonists. Also provided are methods for modulating the CD226-PVR interaction and / or the CD96-PVR interaction by administering in vitro or in vivo a TIGIT (a competitor of PVR binding) or an anti-TIGIT antibody or an antigen-binding fragment thereof. Furthermore, the present invention encompasses anti-TIGIT antibodies and fragments thereof that agonize and antagonize, particularly anti-TIGIT antibodies 10A7 and 1F4, and alternative types of antibodies containing the CDRs of anti-TIGIT antibodies 10A7 and / or 1F4.

[0086] The tests described herein demonstrate the interaction between PVR and TIGIT on DCs, suggesting that this binding interaction regulates DC function, particularly cytokine production. PVR is a cell surface receptor known to be highly expressed on dendritic cells (DCs), as well as FDCs, fibroblasts, endothelial cells, and some tumor cells (Sakisaka, T. & Takai, Y., Curr Opin Cell Biol 16, 513-21 (2004); Fuchs, A. & Colonna, M., Semin Cancer Biol 16, 359-66 (2006)). Human DCs bound to TIGIT secreted high levels of IL-10, but only slightly secreted pro-inflammatory and other cytokines (e.g., IL-12p40, IL-12p70, IL-6, IL-18, and IFNγ). TIGIT had no effect on the production of certain cytokines such as IL-23. This cytokine skew upon TIGIT binding was only observed in cells stimulated by TNFα or CD40 / LPS and not in TLR2- or Pam3CSK4-stimulated cells, suggesting that TIGIT is one means by which the immune system can finely tune DC function. When TIGIT bound to immature T cells (evaluated using a TIGIT fusion construct), T cell activation and proliferation were inhibited. However, TIGIT treatment did not affect the maturation ability of immature monocyte-derived DCs (iMDDCs) and did not directly induce the maturation of these cells. In particular, this inhibition was reversed in the presence of an ERK inhibitor, indicating that ERK activation is an important step in the function of TIGIT in regulating DC activity. Indeed, the applicant shows that when TIGIT binds to PVR, there is an increase in the phosphorylation of PVR and the phosphorylation of pERK dimers rather than pERK monomers. This was not a general effect, for example, since the p38 intracellular signaling pathway was not regulated by treatment of cells with TIGIT-Fc. The applicant shows herein that TIGIT+ T cells also suppress the proliferation of antigen-presenting cells when present in a mixed population of immune cells, not only in the presence of other TIGIT-T cells.The applicant further shows that when anti-TIGIT antibody or anti-PVR antibody is included in the experiment, the observed suppression of proliferation, regulation of DC cytokine production, and suppression of the proliferation of other immune cells are significantly reduced, indicating that the TIGIT-PVR interaction mediates the effects observed previously. In summary, the data presented by the applicant suggest that TIGIT provides a feedback mechanism for the immune system by negatively regulating the immune response.

[0087] Accordingly, the present invention provides a method for modulating the function of immune cells (such as DC) by modulating the expression and / or activity of TIGIT or PVR. For example, a method for reducing or inhibiting the proliferation of immune cells (such as DC or antigen-presenting cells) by treating the immune cells with an agonist of TIGIT, the expression and / or activity of TIGIT or an agonist of the expression and / or activity of PVR in vitro or in vivo is provided. Also, a method for increasing the proliferation of immune cells (such as DC or antigen-presenting cells) by treating the immune cells with an antagonist of the expression and / or activity of TIGIT or an antagonist of the expression and / or activity of PVR in vitro or in vivo is provided. Further, the present invention provides a method for increasing / stimulating the immune response by administering an antagonist of the expression and / or activity of TIGIT or an antagonist of the expression and / or activity of PVR. Similarly, a method for reducing / inhibiting the immune response by administering TIGIT, an agonist of the expression and / or activity of TIGIT or an agonist of the expression and / or activity of PVR is provided.

[0088] Furthermore, the present invention provides a method for regulating the type and / or amount of cytokine production from immune cells (e.g., DC) by regulating the expression and / or activity of TIGIT or PVR. Specifically, the present invention provides a method for increasing IL-10 production by immune cells, such as DC, by treating cells in vitro or in vivo with an agonist of TIGIT, an agonist of the expression and / or activity of TIGIT, or an agonist of the expression and / or activity of PVR. Also provided is a method for reducing the production and / or release of pro-inflammatory cytokines by immune cells, such as DC, by treating cells in vitro or in vivo with an agonist of TIGIT, an agonist of the expression and / or activity of TIGIT, or an agonist of the expression and / or activity of PVR. Similarly, provided is a method for reducing IL-10 production by immune cells, such as DC, by treating cells in vitro or in vivo with an antagonist of the expression and / or activity of TIGIT or an antagonist of the expression and / or activity of PVR. The present invention further provides a method for increasing the production and / or release of pro-inflammatory cytokines by immune cells, such as DC, by treating cells in vitro or in vivo with an antagonist of the expression and / or activity of TIGIT or an antagonist of the expression and / or activity of PVR. Also provided is a method for stimulating intracellular signal transduction via the ERK pathway and / or ERK phosphorylation in one or more cells by treating the cells with an agonist of TIGIT, an agonist of the expression and / or activity of TIGIT, or an agonist of the expression and / or activity of PVR. Similarly, the present invention provides a method for inhibiting or reducing intracellular signal transduction via the ERK pathway and / or ERK phosphorylation in one or more cells by treating the cells with an antagonist of the expression and / or activity of TIGIT or an antagonist of the expression and / or activity of PVR.

[0089] As shown herein, TIGIT is increased in expression in arthritis, psoriasis, inflammatory bowel disease and breast cancer tissues compared to normal control tissues. With respect to breast cancer tissues, Applicant shows that the expression of TIGIT correlates not with the tumor cells themselves, but rather with CD4+ immune cell infiltrates in the tumor. Applicant also directly demonstrates the ability of TIGIT to regulate the immune response by showing that a TIGIT fusion protein inhibited in vitro human T cell responses and mouse T cell activation in a delayed type hypersensitivity in vivo assay. Accordingly, the present invention provides a method for diagnosing a disease / disorder associated with abnormal immune cell responses in a subject by evaluating the expression and / or activity of TIGIT in a sample from the subject and comparing the expression and / or activity to a control amount of the expression and / or activity of TIGIT or the amount of the expression and / or activity of TIGIT in a sample from a normal subject. The present invention also provides a method for assessing the severity of a disease or disorder (i.e., an immune-related disease) associated with abnormal immune cell responses in a subject by evaluating the expression and / or activity of TIGIT in a sample from the subject and comparing the expression and / or activity to a control amount of the expression and / or activity of TIGIT or the amount of the expression and / or activity of TIGIT in a sample from a normal subject. Further provided is a method for preventing a disease or disorder (i.e., an immune-related disease) associated with abnormal immune cell responses by modulating the expression and / or activity of TIGIT. Still further provided is a method for treating or reducing the severity of a disease or disorder (i.e., an immune-related disease) associated with abnormal immune cell responses by modulating the expression and / or activity of TIGIT. When the negative regulatory activity of TIGIT contributes to the disease state, modulation of the expression and / or activity of TIGIT may take the form of inhibiting the activity and / or expression of TIGIT (i.e., by a TIGIT antagonist or a PVR antagonist). For example, when an increase in DC proliferation and / or an increase in DC-induced inflammatory cytokine production is desired, it is desirable to neutralize (antagonize) the expression and / or activity of TIGIT.When negative regulatory activity of TIGIT is desirable for controlling a disease state, modulation of the expression and / or activity of TIGIT may take the form of activating or increasing the expression and / or activity of TIGIT (i.e., by administering TIGIT, a TIGIT agonist, or a PVR agonist). For example, when it is desirable to reduce DC proliferation and / or reduce DC-induced inflammatory cytokine release, it is desirable to agonize the expression and / or activity of TIGIT. These and other aspects of the invention are described in more detail below.

[0090] A. Full-length TIGIT polypeptide The present invention provides an isolated nucleotide sequence encoding a polypeptide referred to herein as a TIGIT polypeptide. In particular, as further disclosed in more detail herein and in the examples, cDNAs encoding various TIGIT polypeptides have been identified and isolated. The present invention also provides other polypeptides (i.e., PVR) useful in the methods of the invention, and it will be understood by those skilled in the art that any description herein specifically describing the creation, manufacture, labeling, post-translational modification, method of use, or other aspects of the TIGIT polypeptide is also applicable to other polypeptides that are not TIGIT.

[0091] B. TIGIT polypeptide variants In addition to the full-length native sequence TIGIT polypeptides described herein, TIGIT variants can also be prepared. TIGIT variants can be prepared by introducing appropriate nucleotide changes into the TIGIT polynucleotide and / or by synthesizing the desired TIGIT polypeptide. Those skilled in the art will understand that amino acid changes, such as changes in the number or position of glycosylation sites of the polypeptide or changes in membrane anchoring properties, can alter the post-translational processes of the TIGIT polypeptide. Mutations in the native full-length sequence of TIGIT or in the various domains of the TIGIT polypeptides described herein can be made, for example, using any technique and guidance for conservative and non-conservative mutations as described in U.S. Patent No. 5,364,934. The mutations can be substitutions, deletions and / or insertions of one or more codons encoding a TIGIT polypeptide resulting in a change in the amino acid sequence of the TIGIT polypeptide as compared to the native sequence of TIGIT. In some cases, the mutation is a substitution of any other amino acid for one or more amino acids of TIGIT in one or more domains. Guidance as to which amino acid residues can be inserted, substituted or deleted without adversely affecting the desired activity is found by comparing the sequence of TIGIT to the sequences of known homologous protein molecules and minimizing amino acid sequence changes made within regions of high homology. Amino acid substitutions can result in substitution of one amino acid with another having similar structural and / or chemical properties, e.g., substitution of serine for leucine, i.e., a conservative amino acid substitution. Insertions and deletions can, in some cases, range from 1 to 5 amino acids. Permissible mutations are determined by systematically making amino acid insertions, deletions or substitutions in the sequence and testing the resulting variants for the activity presented by the full-length or mature native protein.

[0092] Also provided herein are TIGIT polypeptide fragments. Such fragments can be truncated, for example, at the N-terminus or C-terminus or can lack internal residues when compared to the full-length native protein. Certain fragments lack amino acid residues that are not essential for the desired biological activity of the TIGIT polypeptide. TIGIT fragments may be prepared by any of a number of conventional techniques. Desired peptide fragments may be chemically synthesized. Alternative methods include generating TIGIT fragments by enzymatic digestion, e.g., treating a protein with an enzyme known to cleave the protein at sites determined by specific amino acid residues, or by digesting DNA with a suitable restriction enzyme to isolate the desired fragment. Still other suitable techniques include isolating and amplifying a DNA fragment encoding the desired polypeptide fragment by polymerase chain reaction (PCR). Oligonucleotides that determine the desired termini of the DNA fragment are used as the 5' and 3' primers for the PCR. Preferably, the TIGIT polypeptide fragment shares at least one biological and / or immunological activity with the native TIGIT polypeptide disclosed herein. In certain embodiments, conservative substitutions of interest are shown in Table 5 under the heading of preferred substitutions. If such substitutions result in a change in biological activity, more substantial changes are introduced and the products are screened as exemplified substitutions in Table 5 or as further described below in the amino acid classifications.

[0093] Table 5 TIFF2025090567000001.tif89170

[0094] Substantial modifications in function and immunological identity of the polypeptide are accomplished by selecting substitutions that differ significantly in their effect while maintaining the structure of the polypeptide backbone in the area of substitution, for example, as sheet or helical conformations, the charge or hydrophobicity of the target site, or the bulk of the side chain. Naturally occurring residues can be divided into groups based on common side-chain properties: (1) Hydrophobic: norleucine, met, ala, val, leu, ile; (2) Neutral hydrophilic: cys, ser, thr; (3) Acidic: asp, glu; (4) Basic: asn, gln, his, lys, arg; (5) Residues that influence chain orientation: gly, Pro; and (6) Aromatic: trp, tyr, phe.

[0095] Non-conservative substitutions require the exchange of one member of these classifications for another. Also, residues so substituted can be introduced into conserved substitution sites, preferably the remaining (non-conserved) sites. Mutations can be made using methods known in the art such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis. Site-directed mutagenesis [Carter et al., Nucl. Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487 (1987)], cassette mutagenesis [Wells et al., Gene, 34:315 (1985)], restriction-selection mutagenesis [Wells et al., Philos. Trans. R. Soc. London Ser A, 317:415 (1986)] or other known techniques can also be performed on cloned DNA to generate mutant DNA.

[0096] Scanning amino acid analysis can also be used to identify one or more amino acids along an adjacent sequence. Preferred scanning amino acids are relatively small, neutral amino acids. Such amino acids include alanine, glycine, serine, and cysteine. Alanine is a typically preferred scanning amino acid in this group because it excludes side chains beyond the beta carbon and is less likely to change the main chain structure of the mutant [Cunningham and Wells, Science, 244:1081-1085 (1989)]. Also, alanine is typically preferred because it is the most common amino acid. Furthermore, it is often found in both buried and exposed positions [Creighton, The Proteins, (W.H. Freeman & Co., N.Y.); Chothia, J. Mol. Biol., 150:1(1976)]. If alanine substitutions do not generate a sufficient amount of mutants, isoteric amino acids can be used.

[0097] Modification of TIGIT Covalent modification of the TIGIT polypeptide is included within the scope of the present invention. One type of covalent modification is to react the target amino acid residue of the polypeptide with an organic derivatization reagent capable of reacting with a selected side chain or N- or C-terminal residue of the TIGIT polypeptide. Derivatization with a bifunctional reagent is useful, for example, for crosslinking the TIGIT polypeptide to a water-insoluble support matrix or surface used in a method for purifying anti-TIGIT antibodies, and vice versa. Commonly used crosslinking agents include, for example, 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters such as esters with 4-azidosalicylic acid, homobifunctional imide esters including disuccinimidyl esters such as 3,3'-dithiobis(succinimidyl propionate), bifunctional maleimides such as bis-N-maleimido-1,8-octane, and reagents such as methyl-3-[(p-azidophenyl)-dithio]propioimidate. Other modifications include deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, hydroxylation of proline and lysine, phosphorylation of the hydroxyl group of a seryl or threonyl residue, methylation of the α-amino group of lysine, arginine, and histidine side chains [T.E. Creighton, Proteins: Structure and Molecular TIGITperties, W.H. Freeman & Co., San Francisco, pp.79-86 (1983)], acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group.

[0098] Other types of covalent modification of TIGIT polypeptides that are included within the scope of the present invention include alterations to the native glycosylation pattern of the polypeptide. "Alterations to the native glycosylation pattern" herein means deletion of one or more carbohydrate moieties found in the native sequence TIGIT (either by removal of an existing glycosylation site or by deletion of glycosylation by chemical and / or enzymatic means), and / or addition of one or more glycosylation sites not present in the native sequence TIGIT. Further, this phrase includes qualitative changes in the glycosylation of the native protein, which also includes changes in the nature and characteristics of the various carbohydrate moieties present. Addition of glycosylation sites to a polypeptide may be accompanied by a change in the amino acid sequence. This change may be made, for example, by addition or substitution of one or more serine or threonine residues to the native sequence polypeptide (O-linked glycosylation sites). The polypeptide amino acid sequence may, in some cases, be changed at the DNA level, in particular, by mutating the DNA encoding the polypeptide at preselected bases to generate codons that translate the desired amino acids.

[0099] Other means of increasing the number of carbohydrate moieties on a polypeptide are by chemical or enzymatic attachment of glycosides to the polypeptide. Such methods are described in the art, for example, in WO87 / 05330, published September 11, 1987, and Aplin and Wriston, CRC Crit. Rev. Biochem., pp. 259-306 (1981). Removal of carbohydrate moieties present on the polypeptide can be effected chemically or enzymatically or by mutagenic substitution of the codons encoding amino acid residues targeted for glucosylation. Chemical deglycosylation techniques are known in the art and are described, for example, by Hakimuddin et al., Arch. Biochem. Biophys., 259:52 (1987) and by Edge et al., Anal. Biochem., 118: 131 (1981). Enzymatic cleavage of carbohydrate moieties on the polypeptide can be achieved by using a variety of endo- and exo-glycosidases as described by Thotakura et al., Meth. Enzymol. 138:350 (1987). Other types of covalent modification of the polypeptides disclosed herein include the attachment of the polypeptides to one of a variety of non-proteinaceous polymers such as polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylene by the methods described in U.S. Patent Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337. Moreover, the polypeptides of the invention may be modified in a way that results in the formation of chimeric molecules containing the polypeptide fused to other heterologous polypeptides or amino acid sequences.

[0100] In one embodiment, such chimeric molecules include a fusion of a tag polypeptide that provides an epitope to which an anti-tag antibody can selectively bind and a polypeptide of interest. The epitope tag is generally located at the amino or carboxyl terminus of the polypeptide of interest. The presence of such an epitope-tagged form of the polypeptide of interest can be detected using an antibody against the tag polypeptide. Also, the provision of the epitope tag enables the polypeptide of interest to be readily purified by affinity purification using an anti-tag antibody or other type of affinity matrix that binds to the epitope tag. A variety of tag polypeptides and their respective antibodies are well known in the art. Examples include poly-histidine (poly-his) or poly-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptide and its antibody 12CA5 [Field et al., Mol. Cell. Biol., 8:2159-2165 (1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto [Evan et al., Molecular and Cellular Biology, 5:3610-3616 (1985)]; and the herpes simplex virus glycoprotein D (gD) tag and its antibody [Paborsky et al., Protein Engineering, 3(6):547-553 (1990)]. Other tag polypeptides include, but are not limited to, the FLAG peptide [Hopp et al., BioTechnology, 6:1204-1210(1988)]; the KT3 epitope peptide [Martin et al., Science, 255:192-194 (1992)]; the α-tubulin epitope peptide [Skinner et al., J. Biol. Chem., 266:15163-15166 (1991)]; and the T7 gene 10 protein peptide tag [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA, 87:6393-6397(1990)]. In an alternative embodiment, the chimeric molecule may comprise a fusion with an immunoglobulin or a specific region of an immunoglobulin. For the bivalent form of the chimeric molecule (also referred to as an "immunoadhesin"), such a fusion may be the Fc region of an IgG molecule. The Ig fusion preferably comprises a solubilized (transmembrane domain deleted or inactivated) form of the polypeptide in place of at least one variable region within the Ig molecule. In one embodiment, the immunoglobulin fusion comprises the hinge, CH2 and CH3, or the hinge, CH1, CH2 and CH3 regions of an IgG1 molecule. For the production of immunoglobulin fusions, see U.S. Patent No. 5,428,130, issued June 27, 1995.

[0101] D. Preparation of Polypeptides The following description relates primarily to a method for producing a polypeptide by culturing cells transformed or transfected with a vector containing nucleic acid encoding the polypeptide of interest. Of course, it is contemplated that other methods well known in the art can be used to prepare polypeptides. For example, a polypeptide sequence, or a portion thereof, may be produced by direct peptide synthesis using solid-phase techniques [see, e.g., Stewart et al., Solid-Phase Peptide Synthesis, W.H. Freeman Co., San Francisco, Calif. (1969); Merrifield, J. Am. Chem. Soc., 85:2149-2154 (1963)]. Manual techniques or automated in vitro protein synthesis may be performed. Automated synthesis may be carried out, for example, using an Applied Biosystems peptide synthesizer (Foster City, Calif.), according to the manufacturer's instructions. The various portions of the polypeptide may be chemically synthesized separately and joined using chemical or enzymatic methods to produce the full-length polypeptide.

[0102] 1. Isolation of DNA Encoding the Polypeptide DNA encoding the polypeptide of interest can be obtained from a cDNA library prepared from a tissue that is thought to possess the polypeptide mRNA and express it at a detectable level. Thus, DNA encoding a human polypeptide can be readily obtained from a cDNA library prepared from human tissue. The polypeptide-encoding gene can also be obtained from a genomic library or by known synthetic methods (e.g., automated nucleic acid synthesis). The library can be screened with a probe (such as an antibody against the polypeptide or an oligonucleotide of at least about 20 - 80 bases) designed to identify the gene of interest or the protein encoded by that gene. Screening of a cDNA or genomic library with a selected probe can be carried out using standard procedures described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989). Another method for isolating a gene encoding a polypeptide is to use the PCR method [Sambrook et al., supra; Dieffenbach et al., PCR Primer: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1995)].

[0103] The following examples describe screening techniques for cDNA libraries. The oligonucleotide sequence selected as the probe should be of sufficient length and be sufficiently unambiguous to minimize false positives. The oligonucleotide is preferably labeled so that it is detectable upon hybridization with the DNA in the library being screened. Methods of labeling are well known in the art and 32 include the use of radiolabels such as P-labeled ATP, biotinylation, or enzyme labeling. Hybridization conditions including moderate stringency and high stringency are shown in Sambrook et al., supra. In such a library screening method, the sequences identified can be compared and aligned with well-known sequences deposited in public databases such as Genbank or personal sequence databases and made available to the public. Sequence identity (at either the amino acid or nucleic acid level) within the determined region or over the full length of the molecule can be determined using methods known in the art and described herein. Nucleic acids having protein-coding sequences are obtained by screening a cDNA or genomic library selected using the putative amino acid sequences disclosed herein for the first time and, if necessary, conventional primer extension methods such as those described by Sambrook et al., supra, to detect mRNA production intermediates and precursors that have not been reverse-transcribed into cDNA.

[0104] 2. Selection and Transformation of Host Cells The host cells are transfected or transformed with the expression or cloning vectors for polypeptide production described herein, the promoter is induced, transformants are selected, or cultured in a conventional nutrient medium appropriately modified to amplify the gene encoding the desired sequence. Culture conditions, such as medium, temperature, pH, etc., can be selected by those skilled in the art without undue experimentation. In general, the principles, protocols, and practical techniques for maximizing the productivity of cell culture can be found in Mammalian Cell Biotechnology: a Practical Approach, edited by M. Butler (IRL Press, 1991) and Sambrook et al., supra. Methods for prokaryotic and eukaryotic cell transfection, such as CaCl2, CaPO4, liposome-mediated, and electroporation, are known to those skilled in the art. Depending on the host cell used, transformation is performed using standard methods appropriate for that cell. Calcium treatment using calcium chloride or electroporation, as described in Sambrook et al. supra, is commonly used for prokaryotes. Infection with Agrobacterium tumefaciens is used for the transformation of certain plant cells, as described in Shaw et al., Gene, 23:315 (1983) and WO89 / 05859 published June 29, 1989. For mammalian cells without such cell walls, the calcium phosphate precipitation method of Graham and van der Eb, Virology, 52:456-457 (1978) is preferred. General aspects of mammalian cell host system transformation are described in U.S. Patent No. 4,399,216. Transformation into yeast is typically carried out according to the methods of Van Solingen et al., J. Bact., 130:946 (1977) and Hsiao et al., Proc. Natl. Acad. Sci. USA, 76:3829 (1979). However, other methods of introducing DNA into cells, such as nuclear microinjection, electroporation, untreated cells, or bacterial protoplast fusion using polycations such as polybrene, polyornithine, etc., can also be used. For various techniques for transforming mammalian cells, see Keown et al., Methods in Enzymology, 185:527-537 (1990) and Mansour et al., Nature, 336:348-352 (1988).

[0105] Suitable host cells for cloning or expressing DNA in the vectors described herein are prokaryotes, yeasts, or higher eukaryotic cells. Suitable prokaryotes include, but are not limited to, eubacteria, such as Gram-negative or Gram-positive organisms, including Enterobacteriaceae such as Escherichia coli. A variety of E. coli strains are publicly available, for example, E. coli strain K12 MM294 (ATCC 31,446); E. coli X1776 (ATCC 31,537); E. coli strain W3110 (ATCC 27,325) and K5772 (ATCC 53,635). Other preferred prokaryotic host cells include Enterobacteriaceae such as E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, such as Salmonella typhimurium, Serratia, such as Serratia marcescans, and Shigella, as well as Bacilli such as Bacillus subtilis and Bacillus licheniformis (e.g., Bacillus licheniformis 41P described in DD266710 issued on April 12, 1989), Pseudomonas, such as Pseudomonas aeruginosa, and Streptomyces. These examples are illustrative and not limiting. Strain W3110 is one particularly preferred host or parental host since it is a common host strain for recombinant DNA production. Preferably, the host cell secretes a minimal amount of proteolytic enzymes. For example, strain W3110 may be modified to have a genetic mutation in the gene encoding a protein foreign to the cell. Examples of such hosts include E. coli strain W3110 1A2 having the complete genotype tonA; E. coli strain W3110 9E4 having the complete genotype tonA ptr3; E. coli strain W3110 27C7 (ATCC 55,244) having the complete genotype tonA prt3 phoA E15 (argF-lac) 169 degP ompT kan r and E. coli strain W3110 having the complete genotype tonA ptr3 phoA E15 (algF-lac) 169 degP ompT rbs7 ilvG kan rEscherichia coli strain W3110 37D6 having the same; Escherichia coli strain W3110 40B4 which is the 37D6 strain having a non-kanamycin-resistant degP deletion mutation; and an Escherichia coli strain having a mutant periplasmic protease disclosed in U.S. Patent No. 4,946,783 issued on August 7, 1990. Alternatively, in vitro methods of cloning, such as PCR or other nucleic acid polymerase reactions, are preferred.

[0106] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for polypeptide-encoding vectors. Saccharomyces cerevisiae is a commonly used lower eukaryotic host microorganism. Others include Schizosaccharomyces Prombe (Beach and Nurse, Nature, 290:140

[1981] ; EP139,383 issued on May 2, 1985); Kluveromyces hosts (U.S. Patent No. 4,943,529; Fleer et al., Bio / Technology, 9:968-975 (1991)), such as Kluveromyces lactis (MW98-8C, CBS683, CBS4574; Louvencourt et al., J. Bacteriol.154(2):737-742

[1983] ), Kluveromyces fragilis (ATCC 12,424), Kluveromyces bulgaricus (ATCC 16,045), Kluveromyces wickeramii (ATCC 24,178), Kluveromyces waltii (ATCC 56,500), Kluveromyces drosophilarum (ATCC 36,906; Van den Berg et al., Bio / Technology, 8:135 (1990)), Kluveromyces thermotolerans and Kluveromyces marxianus; Yarrowia (EP402,226); Pichia pastoris (EP183,070; Sreekrishna et al., J. Basic Microbiol, 28:265-278

[1988] ); Candida; Trichoderma reesia (EP244,234); Aspergillus (Case et al., Proc. Natl. Acad. Sci.USA, 76:5259-5263

[1979] ); Schwanniomyces, such as Schwanniomyces occidentalis (EP394,538, issued October 31, 1990); and filamentous fungi, such as Neurospora, Penicillium, Tolypocladium (WO91 / 00357, issued January 10, 1991); and Aspergillus hosts, such as Aspergillus nidulans (Balance et al., Biochem. Biophys. Res. Commun., 112:284-289

[1983] ; Tilburn et al., Gene, 26:205-221

[1983] ; Yelton et al., Proc. Natl. Acad. Sci. USA, 81:1470-1474

[1984] ), and Aspergillus niger (Kelly and Hynes, EMBO J., 4:475-479

[1985] ) are included. Preferred methylotropic (C1 compound-assimilating) yeasts here include, but are not limited to, yeasts capable of growing on methanol selected from the genera consisting of Hansenula, Candida, Kloeckera, Pichia, Saccharomyces, Torulopsis, and Rhodotorula. A list of specific species that are examples of the classification of this yeast is described in C. Anthony, The Biochemistry of Methylotrophs, 269 (1982).

[0107] Host cells suitable for the expression of glycosylated polypeptides are derived from multicellular organisms. Examples of invertebrate cells include insect cells such as Drosophila S2 and Spodoptera Sf9, as well as plant cells. Examples of useful mammalian host cell lines include Chinese hamster ovary (CHO) and COS cells. More detailed examples include monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol., 36:59 (1977)); Chinese hamster ovary cells / -DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216 (1980)); mouse Sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251 (1980)); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); and mouse mammary tumor cells (MMT 060562, ATCC CCL51). The selection of appropriate host cells is within the common general knowledge of this field.

[0108] 3. Selection and Use of Replicable Vectors Nucleic acids encoding polypeptides (e.g., cDNA or genomic DNA) are inserted into replicable vectors for cloning (amplification of DNA) or expression. A variety of vectors are publicly available. Vectors can be in the form of, for example, plasmids, cosmids, virus particles, or phages. Appropriate nucleic acid sequences are inserted into the vectors by a variety of techniques. Generally, DNA is inserted into appropriate restriction endonuclease sites using techniques well known in the art. Vector components generally include, but are not limited to, one or more signal sequences, an origin of replication, one or more marker genes, enhancer elements, a promoter, and a transcription termination sequence. Standard ligation techniques known to those skilled in the art are used to prepare appropriate vectors containing one or more of these components. Polypeptides are produced not only directly by recombinant techniques, but also as fusion peptides with heterologous polypeptides that are other polypeptides having a signal sequence or a mature protein or a specific cleavage site at the N-terminus of the polypeptide. Generally, the signal sequence is a component of the vector or part of the polypeptide-encoding DNA inserted into the vector. The signal sequence may be, for example, a prokaryotic signal sequence selected from the group of alkaline phosphatase, penicillinase, lpp or heat-stable enterotoxin II leader. For yeast secretion, the signal sequence may be the yeast invertase leader, the alpha factor leader (including the Saccharomyces and Kluyveromyces alpha factor leaders, the latter being described in U.S. Patent No. 5,010,182), or the acid phosphatase leader, the Candida albicans glucoamylase leader (EP 362,179 published April 4, 1990), or the signal described in International Publication 90 / 13646 published November 15, 1990. In mammalian cell expression, mammalian signal sequences may be used for the direct secretion of proteins such as signal sequences derived from secreted polypeptides of the same or related species and viral secretion leaders.

[0109] Expression and cloning vectors both contain nucleic acid sequences that enable the replication of the vector in one or more selected host cells. Such sequences are well known for many bacteria, yeasts and viruses. The origin of replication derived from plasmid pBR322 is suitable for most Gram-negative bacteria, the 2μ plasmid origin of replication is suitable for yeast, and various viral origins of replication (SV40, polyoma, adenovirus, VSV or BPV) are useful for cloning vectors in mammalian cells. Expression and cloning vectors typically contain a selectable gene, also commonly referred to as a selectable marker. Typical selectable genes confer resistance to antibiotics or other toxins such as ampicillin, neomycin, methotrexate or tetracycline (a), complement auxotrophic deficiencies (b), or encode a protein that supplies an essential nutrient not obtainable from complex media, e.g., a gene encoding the D-alanine racemase of Bacillus (c).

[0110] Examples of suitable selectable markers for mammalian cells enable the identification of cell components capable of incorporating polypeptide-encoding nucleic acids, such as DHFR or thymidine kinase. A preferred host cell when using wild-type DHFR is a CHO cell line defective in DHFR activity, prepared and propagated as described by Urlaub et al. in Proc. Natl. Acad. Sci. USA, 77:4216 (1980). A selectable gene suitable for use in yeast is the trp1 gene present on the yeast plasmid YRp7 [Stinchcomb et al., Nature, 282:39 (1979); Kingsman et al., Gene, 7:141 (1979); Tschemper et al., Gene, 10:157 (1980)]. The trp1 gene provides a selectable marker, for example, for mutants of yeast lacking the ability to grow in tryptophan, such as ATCC number 44076 or PEP4-1 [Jones, Genetics, 85:12 (1977)]. Expression and cloning vectors usually contain a promoter that operably binds to a polypeptide-encoding nucleic acid sequence and controls mRNA synthesis. Suitable promoters recognized by various possible host cells are known. Promoters suitable for use in prokaryotic hosts include the β-lactamase and lactose promoter systems [Chang et al., Nature, 275:615 (1978); Goeddel et al., Nature, 281:544 (1979)], alkaline phosphatase, the tryptophan (trp) promoter system [Goeddel, Nucleic Acids Res., 8:4057 (1980); EP 36,776], and hybrid promoters such as the tac promoter [deBoer et al., Proc. Natl. Acad. Sci. USA, 80:21-25 (1983)]. Promoters used in bacterial systems also have a Shine-Dalgarno (S.D.) sequence operably linked to the DNA encoding the polypeptide.

[0111] Examples of suitable promoter sequences for use with yeast hosts include 3-phosphoglycerate kinase [Hitzeman et al., J. Biol. Chem., 255:2073 (1980)] or other glycolytic enzymes [Hess et al., J. Adv. Enzyme Reg., 7:149 (1968); Holland, Biochemistry, 17:4900(1987)], such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. Other yeast promoters include inducible promoters having an additional effect in which transcription is controlled by growth conditions, and there are promoter regions of alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, catabolic enzymes related to nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes governing the utilization of maltose and galactose. Vectors and promoters suitable for expression in yeast are further described in EP73,657.

[0112] Transcription of TIGIT from a vector in mammalian host cells is controlled by promoters obtained from the genomes of viruses such as polyomavirus, infectious epithelioma virus (UK2211504 published on July 5, 1989), adenovirus (e.g., adenovirus 2), bovine papillomavirus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus, and simian virus 40 (SV40), heterologous mammalian promoters such as the actin promoter or immunoglobulin promoter, and promoters obtained from heat shock promoters, as long as such promoters are compatible with the host cell line. Transcription of DNA encoding a polypeptide by higher eukaryotes can be enhanced by inserting an enhancer sequence into the vector. An enhancer is a cis-acting element of DNA, usually about 10 to 300 base pairs, that acts on a promoter to enhance its transcription. Many enhancer sequences derived from mammalian genes are currently known (globin, elastase, albumin, α-fetoprotein, and insulin). However, typically, enhancers derived from eukaryotic cell viruses will be used. Examples include the SV40 enhancer (100-270 base pairs) on the late side of the origin of replication, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the origin of replication, and the adenovirus enhancer. The enhancer can be spliced into the vector at the 5' or 3' position of the polypeptide coding sequence, but is preferably located 5' from the promoter.

[0113] In addition, expression vectors used in eukaryotic host cells (nucleated cells derived from yeast, fungi, insects, plants, animals, humans, or other multicellular organisms) also include sequences necessary for transcription termination and mRNA stabilization. Such sequences can be obtained from the normal 5', and sometimes 3', untranslated regions of eukaryotic or viral DNA or cDNA. These regions contain nucleotide segments that are transcribed as polyadenylation fragments in the untranslated portion of the mRNA encoding the polypeptide. Other methods, vectors, and host cells suitable for adaptation to the synthesis of the polypeptide of interest in recombinant vertebrate cell culture are described in Gething et al., Nature, 293:620-625 (1981); Mantei et al., Nature, 281:40-46 (1979); EP117,060; and EP117,058.

[0114] 4. Detection of Gene Amplification / Expression Gene amplification and / or expression can be measured directly in a sample based on the sequences provided herein using appropriately labeled probes, for example, by conventional Southern blotting, Northern blotting to quantify mRNA transcription [Thomas, Proc. Natl. Acad. Sci. USA, 77:5201-5205 (1980)], dot blotting (DNA analysis), or in situ hybridization. Alternatively, antibodies that can recognize specific double-stranded DNA, RNA, DNA-RNA hybrid double-stranded DNA, or DNA-protein double-stranded DNA can also be used. The antibody can then be labeled and an assay can be performed where the double-stranded DNA is bound to a surface, and as a result, the presence of the antibody bound to the double-stranded DNA at the time of formation of the double-stranded DNA on the surface can be detected. Alternatively, gene expression can also be measured by immunological methods that directly quantify the expression of the gene product, such as immunohistochemical staining of cells or tissue sections and assays of cell cultures or body fluids. Antibodies useful for immunohistochemical staining and / or assays of sample solutions can be monoclonal or polyclonal and can be prepared in any mammal. Conveniently, the antibodies can be prepared against the native sequence polypeptide, or against a synthetic peptide based on the DNA sequences provided herein, or against an exogenous sequence that encodes a specific antibody epitope and is fused to the DNA encoding the polypeptide.

[0115] 5. Purification of the Polypeptide The form of the polypeptide of interest can be recovered from the culture medium or the lysate of the host cell. If it is membrane-bound, it can be released from the membrane using a suitable washing solution (e.g., Triton-X100) or enzymatic cleavage. The cells used for the expression of the polypeptide can be disrupted by various chemical or physical means such as freeze-thaw cycles, sonication, mechanical disruption, or cell lysing agents. It is desirable to purify the polypeptide from recombinant cell proteins or polypeptides. It is purified by the following procedures, which are examples of suitable purification procedures: namely, fractionation on an ion exchange column; ethanol precipitation; reverse phase HPLC; chromatography on silica or cation exchange resin, such as DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75; protein A Sepharose column to remove contaminants such as IgG; and metal chelating column that binds the epitope-tagged form of the polypeptide. Many protein purification methods known in the art can be used, for example, those described in Deutscher, Methodes in Enzymology, 182 (1990); Scopes, Protein Purification: Principles and Practice, Springer-Verlag, New York (1982). The purification process selected depends, for example, on the production method used and particularly on the properties of the specific polypeptide being produced.

[0116] E. Tissue distribution The location of the tissue expressing the polypeptide of the present invention can be confirmed by measuring mRNA expression in various human tissues. The location of such a gene provides information on the tissues that are most susceptible to stimulation and inhibition of the activity of the polypeptide. Also, the location of the gene in a specific tissue provides a sample tissue for the activity blocking / activation assay discussed below. As described above, gene amplification or gene expression in various tissues can be measured using an appropriate labeled probe based on the sequences provided herein by conventional Southern blot, Northern blot (Thomas, Proc. Natl. Acad. Sci. USA, 77:5201-5205

[1980] ), dot blot (DNA analysis), or in situ hybridization for quantification of mRNA transcription. Alternatively, an antibody that recognizes a specific double strand including a DNA double strand, an RNA double strand, and a DNA-RNA hybrid double strand or a DNA-protein double strand may be used. Alternatively, gene expression in various tissues can also be measured by immunological methods such as immunohistological staining of tissue fragments and cell culture medium or body fluids for directly quantifying the gene product. Antibodies useful for immunohistological staining or assay of sample solutions may be monoclonal or polyclonal and are prepared from any animal. Conveniently, the antibody may be prepared against the native sequence of the polypeptide, or against a synthetic peptide based on the DNA sequence encoding the polypeptide, or against a foreign sequence fused to the DNA encoding the polypeptide and encoding a specific antibody epitope. General techniques for generating antibodies, as well as protocols for Northern blot and in situ hybridization, are provided below.

[0117] F. Study of antibody binding The activity of the polypeptides of the present invention can be further confirmed by studies of antibody binding in which the ability of anti-polypeptide antibodies to inhibit the effect of the polypeptide on tissue cells is tested. Exemplary antibodies include polyclonal, monoclonal, humanized, bispecific, and heteroconjugate antibodies, and their preparation is described below. Studies of antibody binding may be performed by known assay methods such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays. See, for example, Zola, Monoclonal Antibodies: A Manual of Techniques, pp.147-158 (CRC Press, Inc., 1987). Competitive binding assays rely on the ability of a test analyte to compete with a labeled standard for binding to a limited amount of antibody. The amount of target protein (encoded by a gene amplified in tumor cells) in the test sample is inversely proportional to the amount of standard that begins to bind to the antibody. To facilitate measurement of the amount of standard that begins to bind, the antibody is preferably immobilized before or after the competition so that the labeled standard and analyte bound to the antibody can be easily separated from the unbound standard and analyte that remain. Sandwich assays involve the use of two antibodies, each of which can bind to a different immunogenic portion, or epitope, of the protein to be detected. In a sandwich assay, the test sample analyte binds to a first antibody immobilized on a solid support, and then a second antibody binds to the analyte, thus forming an insoluble three-component complex. See, for example, U.S. Patent No. 4,376,110. The second antibody may be labeled with a detectable moiety (direct sandwich assay), or may be measured using an anti-immunoglobulin antibody labeled with a detectable moiety (indirect sandwich assay). For example, one form of a sandwich assay is an ELISA assay, in which the detectable moiety is an enzyme. For immunohistology, tumor samples may be fresh or frozen, and may be embedded in paraffin and fixed with a preservative such as formalin.

[0118] G. Cell-Based Assays Cell-based assays and animal models of immune-related diseases such as psoriasis can be used to further understand the genes and polypeptides identified herein and the relationship between the development and pathogenicity of psoriasis. Alternatively, the cDNAs described herein are transfected into cells of a cell type known to be involved in immune-related diseases, and the ability of these cDNAs to stimulate or inhibit immune-related diseases is analyzed. Suitable cells can be transfected with the desired gene and such functional activity can be monitored. Such transfected cell lines can be used to test the ability of poly- or monoclonal antibodies or antibody compositions to inhibit or stimulate psoriasis. Cells transfected with the coding sequences of the genes identified herein can further be used to identify candidate therapeutics for the treatment of immune-related diseases. Furthermore, although stable cell lines are more commonly used in the art, primary cultures derived from transgenic animals can be used in the cell-based assays herein (as described below). Techniques for deriving continuous cell lines from transgenic animals are well known in the art (see Small et al., Mol. Cell. Biol. 5, 642-648

[1985] ).

[0119] One suitable cell-based assay is the mixed lymphocyte reaction (MLR). Current Protocols in Immunology, unit3.12; edited by J E Coligan, A M Kruisbeek, D H Marglies, E M Shevach, W Strober, National Institutes of Health, John Wiley & Sons, Inc. In this assay, the ability of a test compound to stimulate or inhibit the proliferation of activated T cells is evaluated. A suspension of responder T cells is cultured with allogeneic stimulator cells, and T cell proliferation is measured by incorporation of tritiated thymidine. This assay is a general measure of T cell reactivity. Since upon activation, the majority of T cells respond to and produce IL-2, part of the difference in responsiveness in this assay reflects differences in IL-2 production by the responding cells. The results of the MLR are confirmed by standard lymphokine (IL-2) detection assays. Current Protocols in Immunology, supra, 3.15,6.3. The proliferative T cell response in the MLR assay is due to the direct mitogenic properties of the molecule assayed or to antigen-induced activation of exogenous origin. Also, the T cell stimulating activity of the polypeptide can be verified by a costimulation assay. T cell activation requires an antigen-specific signal mediated through the T cell receptor (TCR) and a costimulatory signal mediated through the interaction by the binding of a second ligand, such as the B7 (CD80, CD86) / CD28 binding interaction. CD28 crosslinking increases the secretion of lymphokines by activated T cells. T cell activation has both negative and positive controls through ligand binding with negative or positive effects. CD28 and CTLA-4 are related glycoproteins of the Ig superfamily that bind to B7. The binding of CD28 to B7 has a positive costimulatory effect on T cell activation; conversely, the binding of CTLA-4 to B7 has a T cell inactivation effect. Chambers, C.A. and Allison, J.P., Curr. Opin. Immunol. (1997) 9:396; Schwartz, R.H., Cell (1992) 71: 1065; Linsey, P.S. and Ledbetter, J.A., Annu. Rev. Immunol. (1993) 11:191; June, C.H. et al., Immunol. Today (1994) 15:321; Jenkins, M.K., Immunity (1994) 1:405. In the costimulation assay, the polypeptide is assayed for T cell costimulatory or inhibitory activity.

[0120] The direct use of a stimulatory compound as in the present invention has been confirmed in experiments involving the 4-1BB glycoprotein, a member of the tumor necrosis factor receptor family, which binds to a ligand (4-1BBL) expressed on naive T cells and transmits signals for T cell activation and growth. Alderson, M.E. et al., J. Immunol. (1994) 24:2219. The use of agonist-stimulating compounds has also been experimentally confirmed. As an example, the activation of 4-1BB by agonist anti-4-1BB antibodies in therapy promotes tumor eradication. Hellstrom, I. and Hellstrom, K.E., Crit. Rev. Immunol. (1998) 18:1. The treatment of tumors by immunoabsorbent therapy, described in more detail below, is another example of the use of the stimulating compounds of the present invention. Alternatively, the immune-stimulating or enhancing effect can also be achieved by administration of a polypeptide having the property of enhancing vascular permeability. The increased vascular permeability is beneficial for diseases that can be alleviated by local infiltration of immune cells (e.g., monocytes, eosinophils, PMN).

[0121] On the other hand, like other compounds of the present invention that are direct inhibitors of T cell proliferation / activation, inflammatory cytokine secretion, and / or vascular permeability, the TIGIT polypeptide can be directly used to suppress the immune response. These compounds are useful for reducing the degree of the immune response and for treating immune-related diseases characterized by abnormally active, unusually optimal, or autoimmune responses. The use of this compound of the present invention has been confirmed by the experiments described above in which the binding of CTLA-4 to receptor B7 inactivates T cells. The direct inhibitory compounds of the present invention function in a similar manner. The use of compounds that suppress vascular permeability can be expected to reduce inflammation. Such use is beneficial for treating symptoms associated with excessive inflammation. Similarly, for example, an antibody that binds to the TIGIT inhibitory polypeptide of the present invention and blocks the effect of the TIGIT inhibitory polypeptide can also be used to suppress the T cell-mediated immune response by generating a net inhibitory effect and releasing TIGIT to inhibit T cell proliferation / activation and / or lymphokine secretion. By blocking the inhibitory effect of this polypeptide, the immune response of a mammal is suppressed. Alternatively, inhibiting or reducing the activity and / or expression of TIGIT, or interfering with the ability of TIGIT to bind to and / or signal through PVR, is therapeutically beneficial due to a condition associated with insufficient T cell-mediated immune response and / or inflammation. Such inhibition or reduction can be provided by administration of an antagonist of TIGIT expression and / or activity and / or an antagonist of PVR expression and / or activity.

[0122] H. Animal Models Furthermore, the results of in vitro cell-based assays can be demonstrated by in vivo animal models and assays of T cell function. To further understand the role of the genes identified herein in the progression and etiology of immune-related diseases, and to test the efficacy of candidate therapeutics including antibodies and other agonists of native polypeptides including small molecule antagonists, various well-known animal models can be used. The in vivo nature of these models allows prediction of responses in human patients. Animal models of immune-related diseases include both non-recombinant and recombinant (transgenic) animals. Non-recombinant animal models include, for example, rodents such as mouse models. Such models are generated by introducing cells into syngeneic mice by standard techniques such as subcutaneous injection, tail vein injection, spleen transplantation, intraperitoneal transplantation, subrenal capsule transplantation, etc. Graft-versus-host disease occurs when immunocompetent cells are transplanted into an immunosuppressed or tolerant patient. Donor cells recognize and react to host antigens. The reactions vary from severe inflammation that can be life-threatening to mild cases such as diarrhea and weight loss. Graft-versus-host disease models provide a means of assessing T cell reactivity against MHC antigens and a minor transplantation antigen. Appropriate procedures are described in detail in Current protocols in Immunology, unit4.3. above. Animal models for skin allograft rejection are a means of testing the ability of T cells to mediate tissue destruction in vivo and are an indicator of their role in transplant rejection. In the most common and accepted models, skin grafts from the tails of mice are used. Repeated experiments have shown that skin allograft rejection is mediated by T cells, helper T cells, and killer-effector T cells, but not by antibodies. Auchincloss, H. Jr. and Sachs, D.H., Fundamental Immunology, 2nd ed., W.E. Paul ed., Raven Press, NY, 1989, 889-992. Appropriate procedures are described in detail in Current protocols in Immunology, unit 4.4, supra. Other transplant rejection models that can be used to test the compounds of the present invention are the allogeneic heart graft models described by Tanabe, M. et al., Transplantation (1994) 58:23 and Tinubu, S.A. et al., J. Immunol. (1994) 4330-4338.

[0123] Animal models of delayed hypersensitivity also provide assays of cell-mediated immune function. The detailed types of hypersensitivity reactions are T cell-mediated immune reactions characterized by inflammation that does not reach a peak until some time has elapsed after antigen loading. These reactions also cause tissue-specific autoimmune diseases such as multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE, a model of MS). Suitable procedures are described in detail in Current Protocols in Immunology, unit 4.5, supra. EAE is a T cell-mediated autoimmune disease characterized by T cell and mononuclear cell inflammation and resultant axonal demyelination of the central nervous system. EAE is generally considered to be an animal model relevant to human MS. Bolton, C., Multiple Sclerosis (1995) 1: 143. Both acute and relapsing-remitting models have been developed. Using the protocols described in Current Protocols in Immunology, units 15.1 and 15.2, the compounds of the invention can be tested for T cell stimulatory or inhibitory activity against immune demyelinating diseases. See also the model of myelin disease in which oligodendroglia or Schwann cells are transplanted into the central nervous system, described in Duncan, I.D. et al., Molec. Med. Today (1997) 554-561.

[0124] Contact hypersensitivity is a simple delayed-type hypersensitivity in vivo assay of cell-mediated immune function. In this procedure, the skin is exposed to an exogenous hapten that elicits a delayed-type hypersensitivity reaction, and the reaction is measured and quantified. Contact hypersensitivity consists of an initial sensitization phase followed by an elicitation phase. The elicitation phase occurs when T lymphocytes encounter an antigen they have previously contacted. Swelling and inflammation occur, creating an excellent model of human allergic contact dermatitis. Appropriate procedures are described in detail in Current protocols in Immunology, edited by J.E. Coligan, A.M. Kruisbeek, D.H. Marglies, E.M. Shevach, and W. Strober, John Wiley & Sons, Inc, unit 4.2. See also Grabbe, S. and Schwarz, T. Immun. Today 19(1):37-44(1998). An animal model of arthritis is collagen-induced arthritis. This model shares clinical, histological, and immunological features with human autoimmune chronic rheumatoid arthritis and is an acceptable model of human autoimmune arthritis. Mouse and rat models are characterized by synovitis, erosion of cartilage and subchondral bone. The compounds of the present invention can be tested for activity against autoimmune arthritis using the protocol described in Current Protocols in Immunology, unit 15.5, supra. See also the model using monoclonal antibodies against CD18 and VLA-4 integrins described in Issekutz, A.C. et al., Immunology 88: 569 (1996).

[0125] The collagen-induced arthritis (CIA) model has many immunological and pathological similarities to human rheumatoid arthritis (RA), including involvement of local tumor tissue compatibility, complete class II restricted T helper lymphocyte activation, and tissue lesion similarities, and is thus considered a suitable model for studying candidate drugs or biological activities in human arthritis. Features of this CIA model similar to those seen in RA patients include erosion of cartilage and bone in the joint area (seen on X-ray), proliferative synovitis, and symmetric occurrence of small and medium-sized peripheral joints of the appendages (limbs) rather than the axial or skeletal skeleton. Jamieson et al., Invest. Radiol. 20: 324-9 (1985). Furthermore, IL-1 and TN-α appear to be involved in CIA as in RA. Joosten et al., J. Immunol. 163: 5049-5055 (1999). TNF-neutralizing antibodies and TNFR:Fc have each reduced the symptoms of RA in this model (Williams et al., PNAS, 89:9784-9788 (1992); Wooley et al., J. Immunol.151: 6602-6607 (1993)). In this model of RA, type II collagen is purified from bovine articular cartilage (Miller, Biochemistry 11:4903 (1972)) and used in immunized mice (Williams et al, Proc. Natl. Acad. Sci. USA 91:2762 (1994)). The symptoms of arthritis include erythema and / or swelling of the extremities and erosion or defect of cartilage and bone as determined by histology. This widely used model is described, for example, in Holmdahl et al., APMIS 97:575 (1989), and Current Protocols in Immunology, supra, units 15.5, and Issekutz et al., Immunology, 88:569 (1996), and also in the following examples herein.

[0126] Models of asthma are described where antigen-induced airway hyperreactivity, pulmonary eosinophilia and inflammation are induced by sensitizing animals with ovalbumin and then challenging the animals with the same protein delivered by aerosol. Some animal models (guinea pigs, rats, non-human primates) exhibit symptoms similar to human atopic asthma when challenged with aerosol antigen. Mouse models possess many features of human asthma. Suitable methods for testing the activity and efficacy of the compounds of the present invention in the treatment of asthma are described in Wolyniec, W.W. et al., Am. J. Respir. Cell Mol. Biol., 18: 777(1998) and references cited therein.

[0127] Furthermore, the compounds of the present invention can be tested in animal models of psoriatic diseases. Some evidence suggests that T cells are the pathogen of psoriasis. The compounds of the present invention can be tested in the scid / scid mouse model described by Schon. M.P. et al., Nat. Med. (1997) 3:183, in which the mice exhibit histopathological skin lesions similar to psoriasis. Another suitable model is the human skin / scid mouse chimera prepared as described by Nickoloff, B.J. et al., Am. J. Path. (1995) 146:580. Recombinant (transgenic) animal models can be engineered by introducing the coding portion of the genes identified herein into the genome of the animal of interest using standard techniques for producing transgenic animals. Animals that can be provided as targets for transgenic manipulation include, but are not limited to, mice, rats, rabbits, guinea pigs, sheep, goats, pigs, and non-human primates, such as baboons, chimpanzees, and monkeys. Techniques known in the art for introducing transgenes into these animals include pronuclear microinjection (Hoppe and Wanger, U.S. Patent No. 4,873,191); retrovirus-mediated gene transfer into the germ line (e.g., Van der Putten et al., Proc. Natl. Acad. Sci. USA 82, 6148-615

[1985] ); gene targeting in embryonic stem cells (Thompson et al., Cell 56, 313-321

[1989] ); electroporation of embryos (Lo, Mol. Cel. Biol. 3, 1803-1814

[1983] ); sperm-mediated gene transfer (Lavitrano et al., Cell 57, 717-73

[1989] ). For a review, see, for example, U.S. Patent No. 4,736,866. For the purposes of the present invention, transgenic animals include those having the transgene only in a part thereof ("mosaic animals"). The transgene is incorporated as a single transgene or as a concatemer, for example in a head-to-head or head-to-tail tandem arrangement. Selective introduction of the transgene into specific cell types is also possible, for example according to the technique of Lasko et al., Proc. Natl. Acad. Sci. USA 89, 6232-636 (1992).

[0128] Expression of the transgene in transgenic animals can be monitored by standard techniques. For example, Southern blot analysis or PCR amplification is used to confirm integration of the transgene. The level of mRNA expression can then be analyzed using techniques such as in situ hybridization, Northern blot analysis, PCR, or immunohistochemistry. The animal may be further examined for signs of immune disease pathology, for example by histological examination to determine infiltration of specific cells by immune cells. Blocking experiments can also be performed in transgenic animals treated with the compound of the invention to determine the degree of stimulation or inhibition of T cell proliferation by the compound of the invention. In these experiments, a blocking antibody that binds to the polypeptide of the invention prepared as described above is administered to the animal and the effect on immune function is measured.

[0129] Alternatively, a "knockout" animal can be constructed as having a defective or modified gene encoding the polypeptide identified herein as a result of homologous recombination between the endogenous gene encoding the polypeptide identified herein and the modified genomic DNA encoding that polypeptide introduced into the embryonic cells of the animal. For example, a cDNA encoding a particular polypeptide can be used to clone the genomic DNA encoding that polypeptide by established techniques. A portion of the genomic DNA encoding a particular polypeptide can be replaced or removed by other genes, such as a gene encoding a selectable marker that can be used to monitor integration. Generally, the vector contains several kilobases of unchanged flanking DNA (both 5' and 3' ends) [see, for example, Thomas and Capecchi, Cell, 51:503 (1987) for homologous recombination vectors]. The vector is introduced into embryonic stem cells (e.g., by electroporation, etc.), and cells in which the introduced DNA has been homologously recombined with the endogenous DNA are selected [see, for example, Li et al., Cell, 69:915 (1992)]. The selected cells are then injected into the blastocysts of an animal (e.g., a mouse or rat) to form aggregate chimeras [see, for example, Bradley, Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987), pp. 113-152]. Thereafter, the chimeric embryos are transplanted into appropriate pseudopregnant female foster mothers, and after a period of time, "knockout" animals are produced. Progeny having DNA homologously recombined in embryonic cells are identified by standard techniques, and they can be used to breed animals in which all cells of the animal contain DNA homologously recombined. Knockout animals are characterized by certain pathological conditions due to the absence of the polypeptide, including, for example, tumor development, and the ability to defend against the development of such pathological conditions.

[0130] I. Immunoadjuvant Therapy In one embodiment, the immunostimulatory compounds of the present invention can be used for adjuvant therapy in tumor (cancer) treatment. It is well established that T cells recognize human tumor-specific antigens. A group of tumor antigens encoded by the MAGE, BAGE, and GAGE families of genes are silent in all adult normal tissues but are expressed in significant amounts in tumors such as melanoma, lung tumors, head and neck tumors, and bladder cancer. DeSmet. C. et al., (1996) Proc. Natl. Acad. Sci. 93:7149. Co-stimulation of T cells has been shown to induce tumor regression and anti-tumor responses both in vitro and in vivo. Melero. I. et al., Nature Medicine (1997) 3:682; Kwon. E.D. et al., Proc. Natl. Acad. Sci. USA (1997) 94:8099; Lynch. D.H. et al., Nature Medicine (1997) 3:625; Finn. O.J. and Lotze. M.T., J. Immunol. (1998) 21:114. The data presented herein show that TIGIT expression correlates with immune cell infiltration in breast cancer tumors. Also, TIGIT is shown herein to inhibit the proliferation of DCs and other immune cells and to inhibit the production of pro-inflammatory cytokines from these cells. Thus, since a decrease in T cell activity in tumors is undesirable, the expression of TIGIT in tumor-infiltrating immune cells can be abnormal. TIGIT antagonists and / or antagonists of the TIGIT-PVR signaling interaction (i.e., PVR antagonists) can be administered alone as an adjuvant or in combination with a growth regulator, a cytotoxic agent, or a chemotherapeutic agent, and stimulate T cell proliferation / activation and the anti-tumor response against tumor antigens. The growth regulator, cytotoxic agent, or chemotherapeutic agent can be administered in a conventional amount using known methods of administration. The immunostimulatory activity of the TIGIT antagonist and TIGIT activity antagonist compounds of the present invention can reduce the amount of the growth regulator, cytotoxic agent, or chemotherapeutic agent, and thus potentially reduce the toxicity to the patient.

[0131] Screening Assays for Candidate Drugs Screening assays for candidate drugs are designed to identify compounds that bind or complex with a polypeptide encoded by a gene identified herein, or a bioactive fragment thereof, or otherwise interfere with the interaction of the encoded polypeptide with other cellular proteins. Such screening assays include assays that follow high-throughput screening of chemical libraries and are particularly suitable for the identification of small molecule candidate drugs. Exemplary small molecules include synthetic organic or inorganic compounds including peptides, preferably soluble peptides, (poly)peptide-immunoglobulin fusions, and, without limitation, human antibodies and antibody fragments, as well as poly- and monoclonal antibodies and antibody fragments, single-chain antibodies, anti-idiotypic antibodies, and antibodies including chimeric or humanized variants of such antibodies or fragments. The assays are performed in a variety of formats including protein-protein binding assays, biological screening assays, immunoassays, and cell-based assays that are well-characterized in the art. All assays are common in that they require contacting these two molecules under conditions and for a sufficient time to permit the interaction of the candidate drug with the polypeptide encoded by the nucleic acid identified herein. In a binding assay, the interaction is binding and the complex formed is isolated or can be detected in the reaction mixture. In a particular embodiment, the polypeptide encoded by the gene identified herein or a candidate drug is immobilized on a solid phase, such as a microtiter plate, by covalent or non-covalent attachment. Non-covalent attachment is generally achieved by coating the solid surface with a solution of the polypeptide and allowing it to dry. Alternatively, an immobilization antibody specific for the peptide to be immobilized, such as a monoclonal antibody, can be used to attach the peptide to the solid surface. The assay is performed by adding an unimmobilized component, which may be labeled with a detectable label, to a coated surface containing an immobilized component, such as an adherent component. When the reaction is complete, unreacted components are removed, for example by washing, and the complex immobilized on the solid surface is detected. If the first unimmobilized component has a detectable label, detection of the label immobilized on the surface indicates that complex formation has occurred. If the first unimmobilized component does not have a label, complex formation can be detected, for example, by a labeled antibody that specifically binds to the immobilized complex.

[0132] If a candidate compound interacts but does not bind to a specific protein, its interaction with the receptor can be assayed by methods well known for detecting protein-protein interactions. Such assays include traditional techniques such as cross-linking, co-immunoprecipitation, and co-purification through gradient or chromatography columns. Further, protein-protein interactions can be monitored as disclosed by Chevray and Nathans [Proc. Natl. Acad. Sci. USA 89, 5789-5793 (1991)] by using yeast-based genetic systems described by Fields and co-workers etc. [Fiels and Song, Nature (London) 340, 245-246 (1989); Chien et al., Proc. Natl. Acad. Sci. USA 88, 9578-9582 (1991)]. Many transcriptional activators such as yeast GAL4 consist of two physically distinct modular domains, one acting as a DNA-binding domain and the other functioning as a transcriptional activation domain. The yeast expression system described in previous literature (commonly referred to as the "two-hybrid system") takes advantage of this property and uses two hybrid proteins, where on one hand the target protein is fused to the DNA-binding domain of GAL4, and on the other hand, the candidate activating protein is fused to the activation domain. Expression under the control of the GAL4 activation promoter of the GAL1-lacZ reporter gene depends on the reconstitution of GAL4 activity through protein-protein interaction. Colonies containing interacting polypeptides are detected by a chromogenic substance for β-galactosidase. A complete kit (MATCHMAKER (trade name)) for identifying protein-protein interactions between two specific proteins using the two-hybrid technique is commercially available from Clontech. This system can also be extended to mapping the protein domains involved in a specific protein interaction and identifying the amino acid residues important for this interaction. To find compounds that interfere with the interactions of the genes identified herein and other intracellular or extracellular components that can be tested, a reaction mixture is usually prepared to contain the gene products and intracellular or extracellular components under conditions and for a time that allow the interaction and binding of the two products. To test the ability of a test compound to inhibit binding, the reaction is carried out in the presence and absence of the test compound. Additionally, a placebo may be added to a third reaction mixture as a positive control. The presence of complex formation in the control reaction and the absence of complex formation in the reaction mixture containing no test compound indicate that the test compound interferes with the interaction between the test compound and its reaction partner.

[0133] K. Compositions and Methods for the Treatment of Immune-Related Diseases Compositions useful for the treatment of immune-related diseases include, but are not limited to, proteins, antibodies, small organic molecules, peptides, phosphopeptides, antisense and ribozyme molecules, triple helix molecules, etc., which inhibit immune functions such as T cell proliferation / activation, lymphokine release, or infiltration of immune cells. For example, antisense RNA and RNA molecules directly block mRNA translation by hybridizing to the target mRNA and preventing protein translation. When antisense DNA is used, oligodeoxyribonucleotides derived from the translation initiation site, for example, between positions -10 and +10 of the target gene nucleotide sequence, are preferred. Ribozymes are enzymatic RNA molecules that can catalyze the specific cleavage of RNA. Ribozymes act by sequence-specific hybridization to a complementary target RNA, followed by nucleotide strand cleavage. Specific ribozyme cleavage sites within potential RNA targets can be identified by known techniques. For further details, see, for example, Rossi, Current Biology 4:469-471 (1994) and PCT Publication No. WO97 / 33551 (published September 18, 1997). The nucleic acid molecules in triple helix formation used for transcription inhibition are single-stranded and consist of deoxynucleotides. The basic composition of these oligonucleotides is designed to promote triple helix formation via the Hoogsteen base pairing rule, which generally requires a size-variable stretch of purines or pyrimidines on one strand of the double helix. For further details, see, for example, PCT Publication No. WO97 / 33551, supra. These molecules can be identified by any or any combination of the above screening assays, or by other screening techniques known to those skilled in the art.

[0134] L. anti-TIGIT antibody The present invention further provides anti-TIGIT antibodies. Examples of antibodies include polyclonal, monoclonal, humanized, bispecific and heteroconjugate antibodies. Those skilled in the art will understand that the present invention provides antibodies against other polypeptides (i.e., anti-PVR antibodies), and that the specific descriptions herein regarding the production, generation, types, methods of use or other aspects of anti-TIGIT antibodies may also be applicable to antibodies specific for polypeptides other than TIGIT.

[0135] 1. Polyclonal antibody Anti-TIGIT antibodies include polyclonal antibodies. Methods for preparing polyclonal antibodies are known to those skilled in the art. In mammals, polyclonal antibodies can be generated, for example, by injecting an immunizing agent and, if desired, an adjuvant one or more times. Typically, the immunizing agent and / or adjuvant are injected into the mammal by multiple subcutaneous or intraperitoneal injections. The immunizing agent can include a TIGIT polypeptide or a fusion protein thereof. It is useful to conjugate the immunizing agent to a protein known to be immunogenic in the immunized mammal. Examples of such immunogenic proteins include, but are not limited to, keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. Examples of adjuvants that can be used include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl lipid A, synthetic trehalose dicorynomycolate). The immunization protocol will be selected by those skilled in the art without undue experimentation.

[0136] 2. Monoclonal antibodies Alternatively, the anti-TIGIT antibody may be a monoclonal antibody. Monoclonal antibodies can be prepared using the hybridoma method as described in Kohler and Milstein, Nature, 256:495 (1975). In the hybridoma method, a mouse, hamster, or other suitable host animal is typically immunized with an immunizing agent to generate antibodies that specifically bind to the immunizing agent or to induce lymphocytes capable of generating such antibodies. Alternatively, lymphocytes can also be immunized in vitro. The immunizing agent typically comprises a target TIGIT polypeptide or a fusion protein thereof. When generally human-derived cells are desired, peripheral blood lymphocytes ("PBL") are used, or spleen cells or lymph node cells are used when a non-human mammalian source is desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent such as polyethylene glycol to form hybridoma cells [Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103]. The immortalized cell line is usually a transformed mammalian cell, particularly a myeloma cell derived from a rodent, bovine, and human. Usually, a rat or mouse myeloma cell line is used. The hybridoma cells are preferably cultured in a suitable medium containing one or more substances that inhibit the survival or growth of unfused immortalized cells. For example, if the parent cells lack the enzyme hypoxanthine-guanine phosphoribosyl transferase (HGPRT or HPRT), the hybridoma medium typically contains hypoxanthine, aminopterin, and thymidine ("HAT medium"), and this substance blocks the growth of HGPRT-deficient cells.

[0137] Preferred immortalized cell lines fuse efficiently, support stable high-level antibody expression by the selected antibody-producing cells, and are sensitive to media such as HAT medium. More preferred immortalized cell lines are mouse myeloma lines, which are available, for example, from the Salk Institute Cell Distribution Center in San Diego, California, and the American Type Culture Collection in Rockville, Maryland. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies are also disclosed [Kozbor, J. Immunol., 133:3001 (1984), Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51-63]. Next, the culture medium in which the hybridoma cells are cultured is assayed for the presence of monoclonal antibodies against the polypeptide. Preferably, the binding specificity of the monoclonal antibody produced by the hybridoma cells is measured by in vitro binding assays such as immunoprecipitation or radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). Such techniques and assays are known in the art. The binding affinity of the monoclonal antibody can be measured, for example, by the Scatchard analysis method by Munson and Pollard, Anal. Biochem., 107:220 (1980).

[0138] After the desired hybridoma cells are identified, clones can be subcloned by the limiting dilution method and grown by standard methods [Goding, supra]. Suitable media for this purpose include, for example, Dulbecco's modified Eagle's medium and RPMI-1640 medium. Alternatively, the hybridoma cells can also be grown as ascites in vivo in mammals. The monoclonal antibody secreted by the subclone is isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification methods such as, for example, protein A-Sepharose method, hydroxylapatite chromatography method, gel electrophoresis method, dialysis method or affinity chromatography.

[0139] In addition, monoclonal antibodies can be produced by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567. The DNA encoding the monoclonal antibodies of the present invention can be readily isolated and sequenced using conventional methods (e.g., using oligonucleotide probes that specifically bind to genes encoding the heavy and light chains of murine antibodies). Hybridoma cells of the present invention are a preferred source of such DNA. Once isolated, the DNA can be placed into an expression vector, which is then transfected into a host cell, such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not produce immunoglobulin proteins, and monoclonal antibodies can be synthesized in the recombinant host cell. In addition, the DNA can be modified, for example, by substituting the coding sequences for human heavy and light chain constant domains for the homologous murine sequences [U.S. Patent No. 4,816,567; Morrison et al., supra], or by covalently linking a portion or all of the coding sequence of a non-immunoglobulin polypeptide to the immunoglobulin coding sequence. Such non-immunoglobulin polypeptides can be substituted for the constant domain of the antibodies of the present invention, or for the variable domain of one of the antigen-binding sites of the antibodies of the present invention, to produce chimeric bivalent antibodies. The antibody may be a monovalent antibody. Methods for preparing monovalent antibodies are well known in the art. For example, one method involves the recombinant expression of immunoglobulin light chains and modified heavy chains. The heavy chain is generally cleaved at any point in the Fc region to prevent heavy chain cross-linking. Alternatively, the relevant cysteine residues are substituted with other amino acid residues or deleted to prevent cross-linking. In vitro methods are also suitable for the preparation of monovalent antibodies. Generation of fragments of the antibody, particularly Fab fragments, by digestion of the antibody can be achieved using conventional techniques known in the art.

[0140] 3. Human and Humanized Antibodies The anti-TIGIT antibodies of the present invention further include humanized antibodies or human antibodies. The humanized form of a non-human (e.g., mouse) antibody is a chimeric immunoglobulin, immunoglobulin chain or fragment thereof (e.g., Fv, Fab, Fab’, F(ab’)2 or other antigen-binding subsequences of an antibody) that includes minimal sequences derived from a non-human immunoglobulin. A humanized antibody contains a human immunoglobulin (recipient antibody) in which the residues of the recipient's complementarity-determining regions (CDRs) are replaced by the residues of the CDRs of a non-human species (donor antibody) having the desired specificity, affinity and capacity, such as a mouse, rat or rabbit. In some instances, the Fv framework residues of the human immunoglobulin are replaced by the corresponding non-human residues. Also, a humanized antibody may contain residues not found in the recipient antibody, the transferred CDRs or the framework sequences. In general, a humanized antibody contains substantially all of at least one, typically two, variable domains in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. A humanized antibody optimally includes at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op Struct. Biol., 2:593-596 (1992)]. Methods for humanizing non-human antibodies are well known in the art. Generally, one or more amino acid residues of non-human origin are introduced into humanized antibodies. These non-human amino acid residues are often, typically, referred to as "transferred" residues obtained from the "transfer" variable domain. Humanization is essentially carried out by replacing the corresponding sequences of human antibodies with rodent CDR or CDR sequences according to the methods of Winter and co-workers [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], that is, by replacing rodent CDR or CDR sequences with the corresponding sequences of human antibodies. Thus, such "humanized" antibodies are chimeric antibodies (U.S. Patent No. 4,816,567) in which substantially less of the original human variable domain is replaced with the corresponding sequences from non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and, in some cases, some FR residues are replaced with residues from similar sites of rodent antibodies.

[0141] Alternatively, human antibodies can also be prepared using various methods known in the art including phage display libraries [Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:381 (1991)]. The methods of Cole et al. and Boerner et al. can also be utilized for the preparation of human monoclonal antibodies [Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss. p.77 (1985) and Boerner et al., J. Immunol., 147(1):86-95 (1991)]. Similarly, human antibodies can be produced by introducing the human immunoglobulin locus into transgenic animals such as mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon administration, production of human antibodies is observed that are very similar to those found in humans in all respects including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and the following scientific literature: Marks et al., Bio / Technology 10, 779-783 (1992); Lonberg et al., Nature 368 856-859 (1994); Morrison, Nature 368, 812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51 (1996); Neuberger, Nature Biotechnology 14, 826 (1996); Lonberg and Huszar, Intern. Rev. Immunol. 13 65-93 (1995). Alternatively, the antibodies are affinity matured using known selection and / or mutagenesis methods as described above. Preferred affinity matured antibodies have an affinity that is 5-fold, more preferably 10-fold, even more preferably 20 or 30-fold higher than the starting antibody (generally mouse, humanized or human) from which the matured antibody was prepared.

[0142] 4. Bispecific antibody A bispecific antibody is a monoclonal antibody, preferably a human or humanized antibody, having binding specificities for at least two different antigens. In the context of the present invention, one of the binding specificities is for TIGIT and the other is for any other antigen, preferably a cell surface protein or receptor or receptor subunit. Methods for making bispecific antibodies are well known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain / light chain pairs where the two heavy chains have different specificities [Milstein and Cuello, Nature, 305:537-539 (1983)]. To randomly pair the heavy and light chains of the immunoglobulin, these hybridomas (quadromas) generate a potential mixture of 10 different antibody molecules, only one of which has the correct bispecific structure. Purification of the correct molecule is usually achieved by an affinity chromatography step. Similar procedures are disclosed in International Publication 93 / 08829 published on May 13, 1993, and Traunecker et al., EMBO J., 10:3655-3656 (1991).

[0143] Antibody variable domains having the desired binding specificities (antibody-antigen binding sites) can be fused to immunoglobulin constant domain sequences. The fusion is preferably with an immunoglobulin heavy chain constant domain that includes at least a portion of the hinge, CH2, and CH3 regions. It is desirable for at least one of the fusions to have a first heavy chain constant region (CH1) that includes the site necessary for light chain binding. DNA encoding the immunoglobulin heavy chain fusion and, if desired, the immunoglobulin light chain are inserted into separate expression vectors and co-transfected into a suitable host organism. For further details on making bispecific antibodies, see, for example, Suresh et al., Methods in Enzymology, 121:210 (1986). According to other methods described in International Publication No. 96 / 27011, the interface between a pair of antibody molecules can be engineered to maximize the proportion of heterodimers recovered from recombinant cell culture. A preferred interface includes at least a portion of the CH3 region of the antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). A complementary "cavity" of the same or similar size as the large side chain is created at the interface of the second antibody molecule by replacing the large amino acid side chain with a smaller one (e.g., alanine or threonine). This provides a mechanism for increasing the yield of heterodimers relative to other unwanted end products such as homodimers.

[0144] Bispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab’)2 bispecific antibodies). Techniques for generating bispecific antibodies from antibody fragments have also been described in the literature. For example, chemical conjugation can be used to prepare bispecific antibodies. Brennan et al., Science, 229:81 (1985) describe procedures for proteolytically cleaving a prototype antibody to produce F(ab’)2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize adjacent dithiols and prevent intermolecular disulfide formation. The resulting Fab’ fragments are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab’-TNB derivatives is then reconverted to Fab’-thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of another Fab’-TNB derivative to form a bispecific antibody. The bispecific antibody thus produced can be used as an agent for the selective immobilization of enzymes. Fab' fragments can be directly recovered from E. coli and chemically conjugated to form bispecific antibodies. Shalaby et al., J. Exp. Med., 175:217-225 (1992) describe the production of fully humanized bispecific antibody F(ab')2 molecules. Each Fab' fragment is secreted separately from E. coli and undergoes site-directed chemical conjugation in vitro to form a bispecific antibody. The bispecific antibody thus formed can bind to normal human T cells and cells overexpressing the ErbB2 receptor, and induce the cytolytic activity of human cytotoxic lymphocytes against human breast tumor targets.

[0145] Also, various methods for directly producing and separating bispecific antibody fragments from recombinant cell culture are described. For example, bispecific antibodies are produced using leucine zippers. Kostelny et al., J. Immunol. 148(5):1547-1553 (1992). Leucine zipper peptides from Fos and Jun proteins are genetically fused to the Fab' portions of two different antibodies. Antibody homodimers are reduced at the hinge region to form monomers, which are then re-oxidized to form antibody heterodimers. This method can also be used for the production of antibody homodimers. The "diabody" technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993) provides another mechanism for producing bispecific antibody fragments. The fragment is formed by linking the light chain variable domain (V L ) to the heavy chain variable domain (V H ) with a linker that is short enough to allow pairing between two domains on the same chain. Thus, the V H and V L domains of one fragment are forced to pair with the complementary V L and V H domains of the other fragment to form two antigen-binding sites. Other strategies for producing bispecific antibody fragments by using single-chain Fv (sFv) dimers have also been reported. See Gruber et al., J. Immunol. 152:5368 (1994). Antibodies with more than two valences are also contemplated. As a non-limiting example, trispecific antibodies can be prepared. See, for example, Tutt et al., J. Immunol. 147:60 (1991). Exemplary bispecific antibodies can bind to two different epitopes of the TIGIT polypeptide provided herein. Alternatively, the arm of the anti-TIGIT polypeptide can bind to a trigger molecule on a leukocyte such as a T cell receptor molecule (e.g., CD2, CD3, CD28, or B7) or an Fc receptor for IgG (FcγR) such as FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16) so as to concentrate a cell defense mechanism on a particular TIGIT polypeptide-expressing cell. Also, bispecific antibodies can be used to localize a cytotoxic agent to a cell expressing a particular TIGIT polypeptide. These antibodies have a TIGIT-binding arm and an arm that binds to a cytotoxic agent or a radioactive chelating agent such as EOTUBE, DPTA, DOTA, or TETA. Other bispecific antibodies of interest bind the TIGIT polypeptide and further bind tissue factor (TF).

[0146] 5. Heteroconjugate antibodies Heteroconjugate antibodies also fall within the scope of the present invention. Heteroconjugate antibodies consist of two covalently linked antibodies. Such antibodies have been proposed, for example, for targeting immune system cells to unwanted cells [U.S. Patent No. 4,676,980] and for the treatment of HIV infection [International Publication Nos. 91 / 00360; 92 / 200373; European Patent 03089]. It is contemplated that such antibodies can be prepared in vitro using known methods in synthetic protein chemistry including those related to cross-linking agents. For example, immunotoxins can be made by using disulfide exchange reactions or by forming thioether bonds. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate, and those disclosed, for example, in U.S. Patent No. 4,676,980.

[0147] 6. Engineering of Effector Functions It is desirable to modify the antibodies of the present invention with respect to effector functions to, for example, improve the effectiveness of the antibodies in cancer treatment. For example, cysteine residues may be introduced into the Fc region so as to form inter-chain disulfide bonds in this region. The homodimeric antibodies thus produced may have improved internalization ability and / or increased complement-mediated cell killing and antibody-dependent cell-mediated cytotoxicity (ADCC). See Caron et al., J. Exp. Med. 176: 1191-1195 (1992) and Shopes, J. Immunol. 148: 2918-2922 (1992). Also, homodimeric antibodies with improved antitumor activity can be prepared using the heterobifunctional crosslinking described in Wolff et al., Cancer research 53: 2560-2565 (1993). Alternatively, the antibody can be engineered to have two Fc regions, thereby also improving complement lysis and ADCC capabilities. See Stevenson et al., Anti-Cancer Drug Design 3: 219-230 (1989).

[0148] 7. Immunoconjugates The present invention also relates to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, a toxin (e.g., an enzymatically active toxin or fragment thereof derived from bacteria, fungi, plants or animals), or a radioisotope (i.e., a radioactive conjugate). The present invention also provides an immunoconjugate (alternatively referred to as an "antibody-drug conjugate" or "ADC") comprising an antibody conjugated to one or more cytotoxic agents such as a chemotherapeutic agent, a drug, a growth inhibitor, a toxin (e.g., a protein toxin, an enzymatically active toxin or fragment thereof derived from bacteria, fungi, plants or animals), or a radioisotope (i.e., a radioactive conjugate).

[0149] Immunoconjugates are useful for the local delivery of cytotoxic agents, i.e., agents for killing cells or inhibiting proliferation or growth in cancer treatment (Lambert, J. (2005) Curr. Opinion in Pharmacology 5:543-549; Wu et al (2005) Nature Biotechnology 23(9): 1137-1146; Payne, G. (2003) i 3:207-212; Syrigos and Epenetos (1999) Anticancer Research 19:605-614; Niculescu-Duvaz and Springer (1997) Adv. Drug Deliv. Rev. 26: 151-172; U.S. Patent No. 4,975,278). Immunoconjugates enable targeted delivery of the drug component to tumors and intracellular accumulation therein, and the toxicity to normal cells as well as tumor cells that are to be removed by systemic administration of this unconjugated drug can reach an unacceptable level (Baldwin et al., (1986) Lancet pp. (Mar. 15, 1986):603-05; Thorpe, (1985) "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review," in Monoclonal Antibodies '84: Biological And Clinical Applications, A. Pinchera et al. (eds), pp. 475-506). Polyclonal antibodies and monoclonal antibodies have been reported to be useful in this approach (Rowland et al., (1986) Cancer Immunol. Immunother., 21:183-87). Drugs used in this method include daunomycin, doxorubicin, methotrexate and vindesine (Rowland et al., (1986), supra).Toxins used in antibody-toxin conjugates include bacterial toxins such as diphtheria toxin, geldanamycin (Mandler et al. (2000) Jour. of the Nat. Cancer Inst. 92(19):1573-1581; Mandler et al. (2000) Bioorganic & Med. Chem. Letters 10:1025-1028; Mandler et al. (2002) Bioconjugate Chem. 13:786-791), maytansinoids (EP 1391213; Liu et al., (1996) Proc. Natl. Acad. Sci. USA 93:8618-8623), and lysins such as calicheamicin (Lode et al., (1998) Cancer Res. 58:2928; Hinman et al., (1993) Cancer Res. 53:3336-3342), and plant poisons such as small molecule toxins. The toxin exerts its cytotoxic effect through functions including tubulin binding, DNA binding, or topoisomerase inhibition. Some cytotoxic agents tend to be inactive or have reduced activity when conjugated to large antibodies or protein receptor ligands.

[0150] Zevalin (registered trademark) (ibritumomab tiuxetan, Biogen / Idec) is a mouse IgG1κ monoclonal antibody against the CD20 antigen found on the cell surface of normal and malignant B lymphocytes and 111 In or 90An antibody-radioisotope conjugate in which a radioisotope of Y is bound to a thiourea linker chelating agent (Wiseman et al., (2000) Eur. Jour. Nucl. Med. 27(7):766-77; Wiseman et al., (2002) Blood 99(12):4336-42; Witzig et al., (2002) J. Clin. Oncol. 20(10):2453-63; Witzig et al., (2002) J. Clin. Oncol. 20(15):3262-69). Zevalin has activity against B-cell non-Hodgkin lymphoma (NHL), but administration causes severe and long-term cytopenia in most patients. Mylotarg (registered trademark) (gemtuzumab ozogamicin, Wyeth Pharmaceuticals), an antibody-drug conjugate consisting of a huCD33 antibody linked to calicheamicin, was approved in 2000 as an injectable for the treatment of acute myeloid leukemia (Drugs of the Future (2000) 25(7):686; U.S. Patent Nos. 4970198; 5079233; 5585089; 5606040; 5693762; 5739116; 5767285; 5773001). Cantuzumab mertansine (Immunogen, Inc.), an antibody-drug conjugate consisting of a huC242 antibody linked to the maytansinoid drug molecule DM1 via a disulfide linker SPP, is progressing to a Phase II trial for the treatment of cancers expressing CanAg, such as colorectal, pancreatic, gastric, and other cancers. MLN-2704 (Millennium Pharm., BZL Biologics, Immunogen Inc.), an antibody-drug conjugate consisting of an anti-prostate specific membrane antigen (PSMA) monoclonal antibody linked to the maytansinoid drug molecule DM1, is in the development stage for the potential treatment of prostate cancer.Auristatin peptides, auristatin E (AE) and monomethyl auristatin (MMAE), synthetic analogs of dolastatin, are conjugated to chimeric monoclonal antibodies cBR96 (specific for Lewis Y on cancer cells) and cAC10 (specific for CD30 on hematologic malignancies) (Doronina et al., (2003) Nature Biotechnology 21(7):778-784) and are in the therapeutic development stage.

[0151] In certain embodiments, the immunoconjugate comprises an antibody and a chemotherapeutic agent or other toxin. Chemotherapeutic agents useful for the generation of immune complexes (immunoconjugates) are described herein (e.g., supra). Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and tricothecene. See, for example, International Publication No. 93 / 21232, published October 28, 1993. A variety of radioactive nucleotides are available for the production of radiolabeled conjugate antibodies. Examples include, 212 Bi, 131 I, 131 In, 90 Y and 186It contains Re. The complex of an antibody and a cytotoxic agent can be prepared using various bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azide compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolylene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxin can be prepared as described in Vitetta et al., Science 238: 1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an example of a chelating agent for the conjugation of radioactive nucleotides to antibodies. See International Publication No. 94 / 11026. Conjugates of antibodies with one or more small molecule toxins such as calicheamicin, maytansinoid, dolastatin, auristatin, trichothene, and CC1065, and derivatives of these toxins having toxic activity are considered here.

[0152] a. Maytansine and maytansinoids In some embodiments, the immunoconjugate comprises an antibody (full length or fragment) conjugated to one or more maytansinoid molecules. Maytansinoids are mitotic inhibitors that act to inhibit tubulin polymerization. Maytansine was first isolated from Maytenus serrata in East Africa (U.S. Patent No. 3,896,111). Subsequently, it was discovered that certain microorganisms produce maytansinoids, such as maytansinol and C-3 maytansinol esters (U.S. Patent No. 4,151,042). Synthetic maytansinol and its derivatives and analogs are disclosed, for example, in U.S. Patent Nos. 4,137,230; 4,248,870; 4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268; 4,308,269; 4,309,428; 4,313,946; 4,315,929; 4,317,821; 4,322,348; 4,331,598; 4,361,650; 4,364,866; 4,424,219; 4,450,254; 4,362,663; and 4,371,533. The maytansinoid drug component is an attractive drug component for antibody-drug conjugates because it is (i) relatively accessible for preparation by fermentation or chemical modification, derivatization of fermentation products, (ii) derivatized according to functional groups suitable for conjugation to the antibody via a non-disulfide linker, (iii) stable in plasma, and (iv) effective against various tumor cell lines.

[0153] Immunoconjugates containing maytansinoids, methods for their preparation, and their therapeutic uses are disclosed, for example, in U.S. Patent Nos. 5,208,020 and 5,416,064, and European Patent No. 0,425,235 B1, the disclosures of which are hereby incorporated by reference. Liu et al., Proc. Natl. Acad. Sci. USA 93:8618-8623 (1996) describes an immunoconjugate containing a maytansinoid named DM1 that binds to the monoclonal antibody C242 against human colorectal cancer. The conjugate has been found to have high cytotoxicity against cultured colon cancer cells and exhibits antitumor activity in in vivo tumor growth assays. Chari et al., Cancer Research, 52:127-131 (1992) describes an immunoconjugate in which a maytansinoid is bound via a disulfide bond to the mouse antibody A7 that binds to an antigen of a human colon cancer cell line, or to another mouse monoclonal antibody TA.1 that binds to the HER-2 / neu oncogene. The cytotoxicity of the TA.1-maytansinoid conjugate was tested in vitro in the human breast cancer cell line SK-BR-3, and 5 the HER-2 surface antigen was expressed. The drug conjugate achieved a cytotoxicity similar to that of the free maytansinoid agent, and this cytotoxicity increased by increasing the number of maytansinoid molecules per antibody molecule. The A7-maytansinoid conjugate showed low systemic cytotoxicity in mice.

[0154] An antibody - maytansinoid conjugate is prepared by chemically conjugating an antibody to a maytansinoid molecule with little reduction in any of the biological activities of either the antibody or the maytansinoid molecule. See, for example, U.S. Patent No. 5,208,020, the disclosure of which is specifically incorporated by reference. While a single molecule of toxin / antibody is expected to enhance cytotoxicity in the use of naked antibodies, those with an average of 3 - 4 maytansinoid molecules conjugated per antibody molecule show efficacy such as improving cytotoxicity against target cells without adversely affecting the function or solubility of the antibody. Maytansinoids are well known in the art and can be synthesized by known techniques or isolated from natural sources. Suitable maytansinoids are disclosed, for example, in U.S. Patent No. 5,208,020, and other patents, and publications not mentioned above. Preferred maytansinoids are maytansinol and maytansinol analogs in which the aromatic ring or other positions of the maytansinol molecule are modified, such as various maytansinol esters.

[0155] There are many linking groups known in the art for making antibody - maytansinoid conjugates, including, for example, those disclosed in U.S. Patent No. 5,208,020 or European Patent No. 0425235 B1, Chari et al., Cancer Research, 52:127 - 131 (1992), and U.S. Patent Application No. 10 / 960,602 filed October 8, 2004, the disclosures of which are specifically incorporated by reference. Antibody - maytansinoid conjugates comprising the linker component SMCC can be prepared as disclosed in U.S. Patent Application No. 10 / 960,602 filed October 8, 2004. Linking groups include disulfide groups, thioether groups, acid - labile groups, photo - labile groups, peptidase - labile groups, or esterase - labile groups as disclosed in the patents mentioned above, with disulfide and thioether groups being preferred. Further linking groups are described and exemplified herein.

[0156] Conjugates of antibodies and maytansinoids can be prepared using various bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate HCL), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bisazide compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)ethylenediamine), diisocyanates (e.g., toluene-2,6-diisocyanate), and diactive fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). Particularly preferred coupling agents include N-succinimidyl-4-(2-pyridylthio)pentanoate (SPP) and N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) provided by a disulfide bond (Carlsson et al., Biochem. J. 173:723-737

[1978] ). The linker can be attached to the maytansinoid molecule at various positions depending on the type of bond. For example, an ester bond can be formed by reacting with a hydroxyl group using conventional coupling techniques. The reaction occurs at the C-3 position having a hydroxyl group, the C-14 position modified with hydroxymethyl, the C-15 position modified with a hydroxyl group, and the C-20 position having a hydroxyl group. In a preferred embodiment, the bond is formed at the C-3 position of maytan sinol or an analog of maytan sinol.

[0157] b. Auristatins and dolastatins In some embodiments, the immunoconjugate comprises an antibody conjugated to dolastatin or a dolastatin peptidyl analogue and derivative, auristatin (U.S. Patent Nos. 5,635,483; 5,780,588). Dolastatin and auristatin have been shown to interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cellular division (Woyke et al. (2001) Antimicrob. Agents and Chemother. 45(12): 3580-3584), and have anticancer activity (U.S. Patent No. 5,663,149) and antifungal activity (Pettit et al. (1998) Antimicrob. Agents Chemother. 42:2961-2965). The dolastatin or auristatin drug component can be attached to the antibody by the N (amino) terminus or C (carboxyl) terminus of the peptidyl drug molecule (WO 02 / 088172). Exemplary auristatin embodiments include the N-terminal linked monomethyl auristatin drug components DE and DF, disclosed in U.S. Patent Application No. 10 / 983,340, "Monomethylvaline Compounds Capable of Conjugation to Ligands," filed November 5, 2004. This disclosure is hereby incorporated by reference in its entirety.

[0158] Generally, peptide-based drug components can be prepared by forming peptide bonds between two or more amino acids and / or peptide fragments. Such peptide bonds can be prepared, for example, according to liquid-phase synthesis methods well-known in the field of peptide chemistry (see E. Schroder and K. Lubke, "The Peptides", volume 1, pp 76-136, 1965, Academic Press). The auristatin / dolastatin drug components can be prepared according to the methods of U.S. Patent Nos. 5,635,483; 5,780,588; Pettit et al. (1989) J. Am. Chem. Soc. 111: 5463-5465; Pettit et al. (1998) Anti-Cancer Drug Design 13:243-277; Pettit, G.R., et al. Synthesis, 1996, 719-725; and Pettit et al. (1996) J. Chem. Soc. Perkin Trans. 1 5:859-863. See also Doronina (2003) Nat Biotechnol 21(7): 778-784; "Monomethylvaline Compounds Capable of Conjugation to Ligands", US Ser. No. 10 / 983,340 filed Nov. 5, 2004. These are hereby incorporated by reference in their entirety (e.g., disclose methods for preparing linkers and monomethylvaline compounds, such as MMAE and MMAF conjugated to linkers).

[0159] c. Calicheamicin In other embodiments, the immunoconjugate comprises an antibody conjugated to one or more calicheamicin molecules. The calicheamicin family of antibiotics can effect double-strand DNA cleavage at sub-picomolar concentrations. For the preparation of conjugates of the calicheamicin family, see U.S. Pat. Nos. 5,712,374; 5,714,586; 5,739,116; 5,767,285; 5,770,701; 5,770,710; 5,773,001; 5,877,296 (all to American Cyanamid Company). Structural analogs of calicheamicin that can be used include, but are not limited to, γ1 I , α2 I , α3 I , N-acetyl-γ1 I , PSAG and θ I 1 (Hinman et al., Cancer Research, 53:3336-3342 (1993); Lode et al., Cancer Research, 58:2925-2928 (1998); and the above-mentioned U.S. Patents to American Cyanamid). Another antitumor agent to which the antibody can bind is QFA, a folic acid antagonist. Both calicheamicin and QFA have intracellular sites of action and do not readily cross the plasma membrane. Thus, cellular uptake of these agents by antibody-mediated internalization greatly enhances the cytotoxic effect.

[0160] d. Other cytotoxic agents Other antitumor agents that can be conjugated to the antibody include BCNU, streptozocin, vincristine and 5-fluorouracil, as described in U.S. Pat. Nos. 5,053,394 and 5,770,710, a family of agents known collectively as the LL-E33288 complex, and esperamicine (U.S. Pat. No. 5,877,296). Potentially useful enzyme active poisons and fragments thereof include diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana protein (PAPI, PAPII and PAP-S), Momordica charantia inhibitor, curcin, crocin, Sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin and tricothecenes. See, for example, International Publication No. 93 / 21232 published October 28, 1993. The present invention further contemplates immunoconjugates formed between antibodies and compounds having nuclease activity (e.g., ribonucleases or DNA endonucleases such as deoxyribonucleases; DNases).

[0161] For selectively destroying tumors, the antibody may contain atoms having high radioactivity. Various radioisotopes are utilized to produce radioactively conjugated antibodies. Examples include At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 and radioisotopes of Lu. When the conjugate is used for detection, it is a radioactive atom for scintigraphy studies, e.g., tc 99m or I 123or spin labels for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron. The radiolabel or other label is introduced into the conjugate by known methods. For example, the peptide is biosynthesized or synthesized by chemical amino acid synthesis using an appropriate amino acid precursor containing fluorine-19 instead of hydrogen. Labels such as Tc 99m or I 123 Re 186 Re 188 and In 111 can be attached via the cysteine residue of the peptide. Yttrium-90 can be attached via the lysine residue. The IODOGEN method (Fraker et al. (1978) Biochem. Biophys. Res. Commun. 80:49-57) can be used for the introduction of iodine-123. Details of other methods are described in "Monoclonal Antibodies in Immunoscintigraphy" (Chatal, CRC Press 1989).

[0162] Conjugates of antibodies and cytotoxic agents can be prepared using various bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate HCL), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bisazide compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)ethylenediamine), diisocyanates (e.g., toluene-2,6-diisocyanate), and biactive fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described by Vitetta et al., Science 238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an example of a chelating agent for conjugating radioactive nucleotides to antibodies. See International Publication No. 94 / 11026. The linker may be a "cleavable linker" to facilitate release of the cytotoxic agent in the cell. For example, acid-labile linkers, peptidase-sensitive linkers, photo-labile linkers, dimethyl linkers or disulfide-containing linkers may be used (Chari et al., Cancer Research, 52:127-131 (1992); U.S. Patent No. 5,208,020). Compounds include, but are not limited to, crosslinking agents: commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, IL, U.S.A.) BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and ADCs prepared with SVSB (succinimidyl-(4-vinylsulfone)benzoate). See pages 467-498 of the 2003-2004 Applications Handbook and Catalog.

[0163] e. Preparation of Antibody-Drug Conjugates In an antibody-drug conjugate (ADC), an antibody (Ab) is conjugated via a linker (L) to one or more drug moieties (D), e.g., from about 1 to about 20 drug moieties per antibody. The ADCs of Formula I can be prepared by several means, using organic chemical reactions, conditions, and reagents known to those of skill in the art: (1) reaction of a nucleophilic group of the antibody with a bivalent linker reagent that reacts with the drug moiety D after covalent attachment to form Ab-L; and (2) reaction of a nucleophilic group of the drug moiety with a bivalent linker reagent that reacts with the nucleophilic group of the antibody after covalent attachment to form D-L. Further methods for preparing ADCs are described herein. Ab-(L-D)p I The linker may consist of one or more linker components. Exemplary linker components include 6-maleimidocaproyl ("MC"), maleimidopropanoyl ("MP"), valine-citrulline ("val-cit"), alanine-phenylalanine ("ala-phe"), p-aminobenzyl oxycarbonyl ("PAB"), N-succinimidyl 4-(2-pyridylthio) pentanoate ("SPP"), N-succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate ("SMCC"), and N-succinimidyl (4-iodo-acetyl) aminobenzoate ("SIAB"). Further linker components are known in the art, some of which are described herein. See also "Monomethylvaline Compounds Capable of Conjugation to Ligands", U.S. Application No. 10 / 983340, filed November 5, 2004. The content is incorporated herein by reference.

[0164] In some embodiments, the linker may contain amino acid residues. Exemplary amino acid linker components include dipeptides, tripeptides, tetrapeptides, or pentapeptides. Exemplary dipeptides include valine-citrulline (vc or val-cit), alanine-phenylalanine (af or ala-phe). Exemplary tripeptides include glycine-valine-citrulline (gly-val-cit) and glycine-glycine-glycine (gly-gly-gly). Amino acid residues comprising amino acid linker components include naturally occurring ones, as well as trace amounts of amino acids and non-naturally occurring amino acid analogs, such as citrulline. The amino acid linker components are configured and can be optimized for selectivity of enzymatic cleavage by enzymes, such as tumor-related proteases, cathepsin B, C, and D, or plasmin proteases.

[0165] The nucleophilic groups on the antibody include, but are not limited to, the following: (i) the N-terminal amine group, (ii) side-chain amine groups such as lysine, (iii) side-chain thiol groups such as cysteine, and (iv) the sugar hydroxyl or amino groups to which the antibody is glycosylated. Amines, thiols, and hydroxyl groups are nucleophilic and can react to form covalent bonds with electrophilic groups on the linker moiety and linker reagents: (i) active esters such as NHS esters, HOBt esters, carboxylic acids, and acid halides; (ii) alkyl and benzyl halides such as haloacetamides; (iii) aldehydes, ketones, carboxyl, and maleimide groups. Certain antibodies have reducible interchain disulfides, i.e., cysteine bridges. The antibody may be subjected to a conjugation reaction using a linker reagent by treatment with a reducing agent such as DTT (dithiothreitol). Thus, each cysteine bridge theoretically forms two reactive thiol nucleophilic groups. Additional nucleophilic groups can be introduced into the antibody by reacting lysine with 2-iminothiolane (Traut's reagent), which converts thiol to amine. Reactive thiol groups may be introduced into the antibody (or a fragment thereof) by introducing one, two, three, four, or more cysteine residues (e.g., preparing a variant antibody comprising one or more non-natural cysteine amino acid residues).

[0166] Alternatively, an antibody-drug conjugate may be generated by modifying an antibody to introduce an electrophilic moiety (capable of reacting with a linker reagent or a nucleophilic substituent on the drug). The carbohydrate of a glycosylated antibody may be oxidized, for example, using a periodate oxidizing agent, to form an aldehyde or ketone group that reacts with an amine group of the linker reagent or drug moiety. The resulting imine Schiff base groups may form stable bonds or may be reduced, for example, by a borohydride reagent that forms stable amine bonds. In one embodiment, reaction of the carbohydrate portion of a glycosylated antibody with either galactose oxidase or sodium metaperiodate may result in a carbonyl (aldehyde and ketone) group of the protein that is capable of reacting with a suitable group on the drug (Hermanson, Bioconjugate Techniques). In other embodiments, a protein containing an N-terminal serine or threonine residue reacts with sodium metaperiodate to produce an aldehyde in place of the first amino acid (Geoghegan & Stroh, (1992) Bioconjugate Chem. 3:138-146; U.S. Patent No. 5,362,852). Such aldehydes are capable of reacting with a drug moiety or a linker nucleophilic group.

[0167] Similarly, nucleophilic groups on the drug moiety include, but are not limited to: amines, thiols, hydroxyls, hydrazides, oximes, hydrazines, thiosemicarbazones, hydrazinecarboxylic acid esters, and arylhydrazide groups that are capable of reacting to form a covalent bond with an electrophilic group on the linker portion and linker reagent: (i) active esters (e.g., NHS ester, HOBt ester, formic acid, and acid halides); (ii) alkyl and benzyl halides, such as haloacetamides; (iii) aldehyde, ketone, carboxyl, and maleimide groups. Alternatively, a fusion protein containing an antibody and a cytotoxic agent is produced, for example, by recombinant techniques or peptide synthesis. The DNA lengths either contain regions encoding linker peptides that separate regions encoding two portions of the conjugate without disrupting the desired properties of the conjugate, or are adjacent to each other. In other embodiments, for use in pre-targeting tumors, an antibody is conjugated to a "receptor" (e.g., streptavidin), where the antibody-receptor conjugate is administered to a patient, followed by use of a clearing agent to remove unbound conjugate from the circulation, and administration of a "ligand" (e.g., avidin) conjugated to a cytotoxic agent (e.g., a radioactive nucleotide).

[0168] 8. Immunoliposomes Also, the antibodies disclosed herein may be prepared as immunoliposomes. Liposomes containing the antibody are prepared by methods known in the art, such as those described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with improved circulation times are disclosed in U.S. Pat. No. 5,013,556. Particularly useful liposomes are produced by the reverse phase evaporation method in a lipid composition containing phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). The liposomes are extruded through a filter of a predetermined size to produce liposomes having the desired diameter. The Fab' fragment of the antibody of the present invention can be conjugated to the liposome via a disulfide exchange reaction, as described in Martin et al., J. Biol. Chem., 257: 286-288 (1982). Chemotherapeutic agents (such as doxorubicin) are optionally included within the liposome. See Gabizon et al., J. National Cancer Inst., 81(19) 1484 (1989).

[0169] M. Pharmaceutical composition The active molecules of the present invention (e.g., TIGIT polypeptide, anti-TIGIT antibody, variants thereof, TIGIT agonist, TIGIT antagonist, PVR agonist and PVR antagonist) and other molecules identified by the screening assays disclosed above can be administered in the form of pharmaceutical compositions for the treatment of immune-related diseases. Therapeutic formulations of active molecules, such as the polypeptides or antibodies of the present invention, are prepared and preserved by mixing an active molecule having the desired degree of purity with any pharmaceutically acceptable carrier, excipient or stabilizer in the form of a lipophilic formulation or an aqueous solution (Remington's Pharmaceutical Science, 16th edition, Osol, A. Ed.

[1980] ). Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the dosages and concentrations employed, and include buffers such as phosphoric acid, citric acid, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (octadecyl dimethyl benzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol; butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol, etc.); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes) or nonionic surfactants such as TWEEN TM , pluronics TM , and nonionic surfactants such as polyethylene glycol (PEG).

[0170] Compounds identified in the screening assays disclosed herein are formulated in a similar manner using standard techniques known in the art. Lipofection or liposomes can also be used to deliver the active molecule to the cell. When antibody fragments are used, the minimal inhibitory fragment that specifically binds to the binding domain of the target protein is preferred. For example, based on the variable region sequence of the antibody, a peptide molecule that retains the ability to bind to the target protein sequence can be designed. Such peptides can be chemically synthesized or generated by recombinant DNA technology (e.g., Marasco et al., Proc. Natl. Acad. Sci. USA, 90:7889-7893

[1993] ).

[0171] The formulations herein may include one or more active compounds, preferably those having complementary hypotheses that do not adversely affect each other, if required for the particular indication to be treated. Alternatively, or in addition thereto, the composition may include a cytotoxic agent, a cytokine or a growth inhibitor. These molecules are appropriately present in a combination effective for the intended purpose. The active molecule may also be included in, for example, microcapsules prepared by coacervation techniques or interfacial polymerization, such as hydroxy methylcellulose or gelatin - microcapsules and poly(methyl methacrylate) microcapsules, respectively, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or in microemulsions. These techniques are disclosed in Remington's Pharmaceutical Science, 16th edition, Osol, A. Ed.

[1980] . Formulations used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.

[0172] A sustained-release formulation of the active molecule may be prepared. Suitable examples of the sustained-release formulation include a semipermeable matrix of a solid hydrophobic polymer containing an antibody, and this matrix is in the form of a molded article, such as a film, or a microcapsule. Examples of the sustained-release matrix include polyester hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polyactides (U.S. Patent No. 3,773,919), L-glutamic acid and γ-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, LUPRON DEPOT TM (injectable microspheres of a copolymer of lactic acid-glycolic acid and leuprolide acetate), etc., and include degradable lactic acid-glycolic acid copolymers, poly-(D)-3-hydroxybutyric acid. Polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid can release molecules over 100 days, while certain hydrogels release proteins in a shorter time. When encapsulated antibodies remain in the body for a long time, they are denatured or aggregated by exposure to moisture at 37°C, resulting in a decrease in biological activity and possible changes in immunogenicity. A reasonable approach can be to devise stabilization depending on the mechanisms involved. For example, if the aggregation mechanism is found to be intermolecular S-S bond formation through thio-disulfide exchange, stabilization can be achieved by modification of sulfhydryl residues, lyophilization from acidic solutions, control of water content, addition of appropriate additives, and development of specific polymer matrix compositions.

[0173] N. Therapeutic methods The polypeptides, antibodies and other active substances of the present invention can be used to treat various immune-related diseases and conditions, including T cell-mediated diseases, etc., characterized by infiltration of inflammatory cells into tissues, stimulation of T cell proliferation, inhibition of T cell proliferation, increase or decrease in cytokine production, and / or increase or decrease or inhibition of vascular permeability. Since the role of TIGIT in the regulation of T cell proliferation and cytokine production has been shown herein, regulation of the expression and / or activity of TIGIT may be useful for the prevention and / or treatment of these diseases. Examples of conditions or diseases that can be treated with the polypeptides, antibodies and other compounds of the present invention include, but are not limited to, systemic lupus erythematosus, rheumatoid arthritis, juvenile chronic arthritis, osteoarthritis, spondyloarthritis, systemic sclerosis (scleroderma), idiopathic inflammatory myopathy (dermatomyositis, polymyositis), Sjogren's syndrome, systemic vasculitis, sarcoidosis, autoimmune hemolytic anemia (immune hypoplastic anemia, paroxysmal nocturnal hemoglobinuria), autoimmune thrombocytopenia (idiopathic thrombocytopenic purpura, immune-mediated thrombocytopenia), thyroiditis (Graves' disease, Hashimoto's thyroiditis, juvenile lymphocytic thyroiditis, atrophic thyroiditis), diabetes mellitus, immune-mediated renal diseases (glomerulonephritis, tubulointerstitial nephritis), multiple sclerosis, idiopathic demyelinating polyneuropathy, or Guillain - Barré syndrome, and demyelinating diseases of the central and peripheral nervous systems such as chronic inflammatory demyelinating polyneuropathy, infectious hepatitis (hepatitis A, B, C, D, E and other non - liver - tropic viruses), autoimmune chronic active hepatitis, primary biliary cirrhosis, granulomatous hepatitis, and sclerosing cholangitis, inflammatory bowel diseases (ulcerative colitis; Crohn's disease), gluten - sensitive enteropathy, and Whipple's disease, vesicular skin diseases, autoimmune or immune - mediated skin diseases including erythema multiforme and contact dermatitis, psoriasis, asthma, allergic rhinitis, atopic dermatitis, food allergies and urticaria, eosinophilic pneumonia, idiopathic pulmonary fibrosis and hypersensitivity pneumonitis, and transplantation - related diseases including rejection and graft - versus - host disease.

[0174] In systemic lupus erythematosus, the central mediators of the disease are the production of auto - reactive antibodies against self - proteins / tissues and the subsequent development of immune - mediated inflammation. The antibodies mediate tissue damage either directly or indirectly. T lymphocytes have not been shown to be directly involved in tissue damage, but T lymphocytes are required for the development of auto - reactive antibodies. Thus, the development of the disease is T - lymphocyte - dependent. Multiple organs and systems including the kidney, lung, musculoskeletal, skin mucosa, eye, central nervous system, cardiovascular system, gastrointestinal tract, bone marrow and blood are clinically affected. Rheumatoid arthritis (RA) is a chronic systemic autoimmune inflammatory disease mainly associated with the synovium of multiple joints, resulting in damage to articular cartilage. The etiology is T lymphocyte-dependent, associated with the production of rheumatoid factor, an autoantibody against self-IgG, resulting in the production of immune complexes that reach high levels in synovial fluid and blood. These complexes in the joints induce a marked infiltration of lymphocytes and monocytes into the synovium and subsequent marked synovial changes; the same is true in the joint space / fluid if infiltrated with similar cells by the addition of numerous neutrophils. The tissues affected are often in a symmetrical pattern and mainly the joints. However, two main forms of extra-articular disease also occur. One form is the occurrence of extra-articular disorders with typical lesions of progressive joint disease and pulmonary fibrosis, vasculitis, and skin ulcers. The second form of extra-articular disease is the so-called Felty syndrome, which occurs at the end of the RA disease process, sometimes after the joint disease has subsided, and is associated with the presence of neutropenia, thrombocytopenia, and splenomegaly. This is accompanied by vasculitis in multiple organs with the formation of infarcts, skin ulcers, and gangrene. In many cases, patients develop rheumatoid nodules in the subcutaneous tissue overlying the affected joints; the nodules have a necrotic center surrounded by a mixed inflammatory cell infiltration in the late stage. Other signs that can occur in RA include: pericarditis, pleurisy, coronary arteritis, interstitial pneumonia with pulmonary fibrosis, dry keratoconjunctivitis, and rheumatoid nodules.

[0175] Juvenile chronic arthritis is a chronic idiopathic inflammatory disease that often presents before the age of 16. Its phenotype has some similarities to RA; some patients with a positive rheumatoid factor are classified as juvenile rheumatoid-like arthritis. This disease is subdivided into three main categories: pauarticular, polyarticular, and systemic. The arthritis is severe and typically destructive, leading to joint ankylosis and delayed growth. Other signs include chronic anterior uveitis and systemic amyloidosis. Spondyloarthritis is a group of diseases with several common clinical features and a common association with the expression of the HLA-B27 gene product. The diseases include: Bechterew's disease (ankylosing sponylitis), Reiter's syndrome (reactive arthritis), arthritis associated with inflammatory bowel disease, spondylitis associated with psoriasis, juvenile-onset spondyloarthritis, and undifferentiated spondyloarthritis. Prominent features include sacroiliitis with or without spondylitis; inflammatory asymmetric arthritis; association with HLA-B27 (a serologically defined allele at the HLA-B locus of class I MHC); eye inflammation, and the absence of autoantibodies associated with other rheumatic diseases. The cell most involved as a key to the induction of the disease is CD8 + T lymphocytes, cells that target antigens presented by class I MHC molecules. CD8 + T cells react against the class I MHC allele HLA-B27 as if it were a foreign peptide expressed by MHC class I molecules. It is hypothesized that an epitope of HLA-B27 mimics an antigenic epitope of bacteria or other microorganisms, thus inducing a reaction of CD8 + T cells. As shown herein, TIGIT is expressed on CD8 + T cells, and regulation of the expression and / or activity of TIGIT in these cells may regulate and / or prevent the symptoms of this disease.

[0176] Systemic sclerosis (scleroderma) has an unknown etiology. A prominent feature of the disease is skin induration, which is thought to be induced by an active inflammatory process. Scleroderma can be local or systemic, with common vascular lesions, and endothelial cell injury in the microvasculature is an early important event in the development of systemic sclerosis. Vascular injury can be immune-mediated. Immunological criteria are derived from the presence of mononuclear cell infiltration in skin lesions and the presence of antinuclear antibodies in many patients. In many cases, ICAM-1 is upregulated on the cell surface of fibroblasts in skin lesions, suggesting that the interaction of T cells with these cells plays a role in the etiology of the disease. Other related organs include: the gastrointestinal tract, resulting in abnormal peristalsis / motility, smooth muscle atrophy and fibrosis; the kidneys, affecting the arcuate and interlobular arteries, resulting in reduced blood flow in the renal cortex, proteinuria, azotemic hematuria and concentric subendothelial intimal proliferation leading to hypertension; skeletal muscle, atrophy, interstitial fibrosis; inflammation, the lungs, interstitial pneumonia and interstitial fibrosis; and the heart, contraction band necrosis, scar / fibrosis. Idiopathic inflammatory myopathies, including dermatomyositis, polymyositis and others, are chronic muscle inflammatory diseases with an unknown etiology that lead to muscle weakness. Muscle injury / inflammation is often asymmetric and progressive. Autoantibodies are associated with many forms. These myositis-specific autoantibodies are produced against components involved in protein synthesis, proteins and RNA and inhibit their functions.

[0177] Sjögren's syndrome is due to immune-mediated inflammation and subsequent functional destruction of the lacrimal and salivary glands. This disease may be associated with or accompanied by an inflammatory connective tissue disease. This disease is associated with the production of autoantibodies against the Ro and La antigens, both of which are small RNA-protein complexes. Lesions lead to keratoconjunctivitis sicca, xerostomia, and other signs or associations including biliary cirrhosis, peripheral or sensory neuropathy, and palpable purpura. Systemic vasculitis is a disease in which the primary lesion is inflammation, subsequently the blood vessels are damaged, and as a result, ischemia / necrosis / degeneration occurs in the tissues supplied by the affected blood vessels, and in some cases, it leads to final end-organ dysfunction. Vasculitides may also occur as sequelae or secondary lesions of other immune-inflammatory mediated diseases, such as rheumatoid arthritis, systemic sclerosis, etc., especially diseases associated with the production of immune complexes. Diseases in the primary systemic vasculitis group include: systemic necrotizing vasculitis: polyarteritis nodosa, allergic angitis and granulomatosis, polyangiitis: Wegener's granulomatosis; lymphomatoid granulomatosis; and giant cell arteritis. Other vasculitides include: mucocutaneous lymph node syndrome (MLNS or Kawasaki disease), isolated CNS vasculitis, Behet's disease, obliterative thrombangiitis (Buerger's disease) and cutaneous necrotizing venulitis. The pathogenesis of most types of the listed vasculitides is thought to be mainly due to the attachment of immunoglobulin complexes to the blood vessel wall, followed by the induction of an inflammatory reaction via ADCC, complement activation or both.

[0178] Sarcoidosis is a condition of poorly understood etiology characterized by the presence of epithelioid cell granulomas in almost all tissues in the body; lung involvement is the most common. The etiology is related to the persistence of activated macrophages and lymphocytes at the disease site, followed by the development of chronic sequelae as a result of the release of local or systemic active products released from these cell types. Autoimmune hemolytic anemias, including autoimmune hemolytic anemia, immune aplastic anemia, and paroxysmal nocturnal hemoglobinuria, are due to the production of antibodies that react with antigens expressed on the surface of red blood cells (and in some cases other blood cells including platelets as well), and are reflected in the removal of the antibody-coated cells via complement-mediated lysis and / or ADCC / Fc-receptor-mediated mechanisms.

[0179] In autoimmune thrombocytopenia, including thrombocytopenic purpura and immune-mediated thrombocytopenia in other clinical settings, platelet destruction / removal results from antibodies or complement binding to platelets, followed by removal via complement lysis, ADCC, or Fc-receptor-mediated mechanisms. Thyroiditis, including Graves' disease, Hashimoto's thyroiditis, juvenile lymphocytic thyroiditis, and atrophic thyroiditis, is the result of an autoimmune reaction against thyroid antigens, which is accompanied by the production of antibodies that are present within the thyroid and often react with thyroid-specific proteins. Natural models: rats (BUF and BB rats) and chickens (obese chicken breeds); Induced models: experimental models exist that involve immunization of animals with either thyroglobulin or thyroid microsomal antigen (thyroid peroxidase). Type 1 diabetes mellitus or insulin-dependent diabetes is the autoimmune destruction of pancreatic islet beta cells; this destruction is mediated by autoantibodies and autoreactive T cells. Also, antibodies against insulin or insulin-like receptors can create an insulin-nonresponsive phenotype.

[0180] Immune-mediated renal diseases, including glomerulonephritis and tubulointerstitial nephritis, are due to antibody or T cell-mediated injury to renal tissue, either directly as a result of the production of autoreactive antibodies or T cells against renal antigens, or indirectly as a result of the deposition of antibodies and / or immune complexes in the kidney that are reactive against other non-renal antigens. Thus, immune-mediated renal diseases can also be induced as indirect sequelae by other immune-mediated diseases that result in the formation of immune complexes. Both direct and indirect immune mechanisms result in an inflammatory reaction in renal tissue that causes lesion development, which in some cases progresses to renal insufficiency, impairing organ function. Both humoral and cellular immune mechanisms can be involved in the pathogenesis of the disorder. Demyelinating diseases of the central and peripheral nervous systems, including multiple sclerosis; idiopathic demyelinating polyneuropathy or Guillain-Barré syndrome; and chronic inflammatory demyelinating polyneuropathy, are thought to be caused by autoimmunity and result in nerve demyelination as a consequence of damage directly caused to oligodendrocytes or myelin. In MS, there is evidence suggesting that the induction and progression of the disease are T lymphocyte-dependent. Multiple sclerosis is a demyelinating disease that is T lymphocyte-dependent and has either a relapsing-remitting or a chronic-progressive course. The etiology is not well understood, but all of viral infection, genetic predisposition, environment, and autoimmunity contribute. Lesions include prominent T cell-mediated microglial infiltration and infiltrating macrophages; CD4 + T lymphocytes are the predominant cell type in the lesions. The mechanism of oligodendrocyte cell death and subsequent demyelination is not well understood, but it is thought to be driven by T lymphocytes.

[0181] Inflammatory and fibrotic lung diseases, including eosinophilic pneumonia; idiopathic pulmonary fibrosis; and hypersensitivity pneumonitis, are associated with an unregulated immune-inflammatory response. Inhibition of that response would be therapeutically beneficial. Autoimmune or immune-mediated skin diseases, including vesicular skin diseases, erythema multiforme, and contact dermatitis, are mediated by autoantibodies and their pathogenesis is T lymphocyte-dependent. Psoriasis is a T lymphocyte-mediated inflammatory disease. Lesions include infiltration of T lymphocytes, macrophages, antigen-processing cells, and certain neutrophils. Allergic diseases, including asthma; allergic rhinitis; atopic dermatitis; food allergies; and urticaria, are T lymphocyte-dependent. These diseases are mainly mediated by T lymphocyte-induced inflammation, IgE-mediated inflammation, or a combination of both. Transplantation-related diseases, including rejection and graft-versus-host disease (GVHD), are T lymphocyte-dependent and are improved by inhibiting the function of T lymphocytes.

[0182] Other diseases in which intervention in the immune and / or inflammatory response is beneficial include, but are not limited to, viral infections (including, but not limited to, AIDS, hepatitis A, B, C, D, and E, and herpes), bacterial infections, fungal infections, protozoal infections, and parasitic infections (molecules (or derivatives / agonists) that stimulate the MLR can be used therapeutically to enhance immunoreactivity against infectious agents), immunodeficiency diseases (molecules / derivatives / agonists) or iatrogenic (i.e., from chemotherapy) immunodeficiency that stimulate the MLR, are used therapeutically, genetically, acquired, or infection-induced (e.g., HIV infection) states, and abnormal proliferation. Among human cancer patients, it has been demonstrated that there are also those that generate antibodies and / or T lymphocytes that react to antigens on abnormal proliferating cells. Also, in animal models with abnormal proliferation, it has been shown that by enhancing the immune response, certain abnormal proliferations result in rejection or regression. Molecules that enhance the T lymphocyte response in the MLR have in vivo utility for enhancing the immune response against abnormal proliferation. Molecules (or small molecule agonists or antibodies that antagonistically affect the same receptor) that enhance the T lymphocyte proliferative response in the MLR can be used therapeutically for the treatment of cancer. Also, molecules that inhibit the lymphocyte response in the MLR (i.e., TIGIT) function in vivo to suppress the immune response against neoplasms during abnormal proliferation; such molecules can be expressed by the neoplastic cells themselves or their expression can be induced by neoplasms in other cells. Antagonism of such inhibitory molecules (by antibodies, small molecule antagonists, or other means) enhances immune-mediated tumor rejection. Furthermore, inhibiting molecules with pro-inflammatory properties is beneficial for the treatment of reperfusion injury; stroke; myocardial infarction; atherosclerosis; acute lung injury; hemorrhagic shock; burns; sepsis / septic shock; acute tubular necrosis; endometriosis; degenerative joint disease, and pancreatitis. The compounds of the present invention, such as polypeptides, small molecules or antibodies, are administered to mammals, preferably humans, by well-known methods, such as intravenous administration as a bolus or by continuous infusion over a predetermined period of time, intramuscular, intraperitoneal, intrathecal, subcutaneous, interarticular, intra-synovial, intrathecal, oral, topical, or inhalation (intranasal, intrapulmonary) routes, etc. Intravenous, subcutaneous or inhalation administration of polypeptides and antibodies is most commonly used.

[0183] In immunoadjuvant therapy, other dosing regimens, such as the administration of anti-cancer agents, may be combined with the administration of the proteins, antibodies or compounds of the present invention. For example, a patient being treated with an immunoadjuvant of the present invention may receive an anti-cancer agent (chemotherapeutic agent) or radiation therapy. Methods of preparation and dosing schedules for such chemotherapeutic agents are used according to the manufacturer's instructions or are determined empirically by a skilled practitioner. Methods of preparation and dosing schedules for such chemotherapy are also described in Chemotherapy Service Ed. M.C. Perry, Williams & Wilkins, Baltimore, MD (1992). Chemotherapeutic agents may be administered prior to, subsequent to, or simultaneously with the administration of the immunoadjuvant. In addition, anti-estrogen compounds such as tamoxifen or anti-progesterone such as onapristone (see European Patent 616812) may be administered at doses known for those molecules.

[0184] Also, it is also preferable to administer an antibody against an antigen associated with another immune disease or a tumor, such as, but not limited to, an antibody that binds to CD20, CD11a, CD18, ErbB2, EGFR, ErbB3, ErbB4, or vascular endothelial growth factor (VEGF). Alternatively, or additionally, two or more antibodies that bind to the same antigen or two or more different antigens disclosed herein may be co-administered to a patient. Often, it is also beneficial to administer one or more cytokines to a patient. For example, in one embodiment, the TIGIT polypeptide is co-administered with a growth inhibitor. For example, first the growth inhibitor is administered, followed by the TIGIT polypeptide. However, co-administration or administration first is also conceivable. The appropriate dose for the growth inhibitor is the amount currently used, but can be reduced by the combined (synergistic) effect of the growth inhibitor and, for example, the TIGIT polypeptide.

[0185] The appropriate dose of the compound of the present invention for the treatment or reduction of severe immune-related diseases depends on the type of disease being treated as defined above, the severity and course of the disease, whether the agent is administered for prophylactic or therapeutic purposes, previous treatment, the patient's clinical history and response to the compound, and the discretion of the attending physician. The compound can be appropriately administered to the patient once or over a series of treatments. For example, depending on the type and severity of the disease, a polypeptide or antibody in the range of about 1 μg / kg to 15 mg / kg (e.g., 0.1 to 20 mg / kg) is a first candidate dose for administration to a patient, whether by one or more separate administrations or continuous infusion, for example. A typical daily dose will be in the range of about 1 μg / kg to 100 mg / kg or more, depending on the factors described above. For repeated administration over several days or more, treatment is continued, depending on the condition, until the desired suppression of the symptoms of the disease appears. However, other dosing regimens may also be useful. The progress of this treatment is easily monitored by conventional techniques and assays.

[0186] O. Manufactured article In other embodiments of the present invention, there are provided articles of manufacture comprising a substance useful for the diagnosis or treatment of the above-mentioned diseases (for example, those containing a TIGIT molecule, a TIGIT agonist, a TIGIT antagonist, a PVR agonist or a PVR antagonist). The article of manufacture comprises a container and instructions for use. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The container may be formed from various materials such as glass or plastic. The container may contain a composition effective for diagnosing and treating the condition and may have a sterile access port (for example, the container may be an intravenous solution bag or vial having a stopper penetrable by a hypodermic needle). The active agent in the composition is usually the polypeptide or antibody of the present invention. The instructions for use or label on or attached to the container indicate that the composition is to be used for the diagnosis or treatment of the selected condition. The article of manufacture may further comprise a second container containing a pharmaceutically acceptable buffer such as phosphate buffered saline, Ringer's solution and dextrose solution. Further, other materials desirable from a commercial and user perspective may be included, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

[0187] P. Diagnosis and prediction of immune-related diseases Cell surface proteins such as proteins (i.e., TIGIT) overexpressed in certain immune-related diseases are excellent regulatory targets for candidate drugs or disease treatment. The same protein, together with the secreted protein encoded by the gene amplified in immune-related diseases, finds additional uses in the diagnosis and prediction of these diseases. For example, antibodies against the protein products of genes amplified in rheumatoid arthritis or other immune-related diseases can be used for diagnosis or prediction. For example, an antibody containing an antibody fragment can be used for qualitative or quantitative detection of the expression of a protein encoded by an amplified or overexpressed gene (the "marker gene product"). The antibody is preferably detectable, for example, equipped with a fluorescent label, and the binding can be monitored by light microscopy, flow cytometry, fluorometry, or other techniques known in the art. These techniques are particularly suitable when the overexpressed gene encodes a cell surface protein. Such binding assays are essentially carried out as described above. Insight detection of an antibody that binds to a marker gene product can be carried out, for example, by immunofluorescence or immunoelectron microscopy. For this purpose, a histological sample is taken from a patient, and a labeled antibody is applied thereto, preferably by overlaying the antibody on the biological sample. This technique also makes it possible to determine the distribution of the marker gene product in the tissue being tested. It will be apparent to those skilled in the art that a wide range of histological methods are readily available for insight detection. Other techniques are also well known in the art, such as, for example, a fluorescence-activated cell sorter (FACS). The following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way.

Examples

[0188] Example 1: Other features of TIGIT TIGIT has been previously identified in a genome-wide search strategy targeting genes specifically expressed by immune cells that have a domain structure consisting of an extracellular Ig domain, a type 1 transmembrane region, and one or more intracellular immunoreceptor tyrosine-based activation or inhibition (ITAM / ITIM) motifs (see, e.g., U.S. Patent Publication No. US20040121370, which is hereby incorporated by reference in its entirety; Abbas, A.R. et al. Genes Immun 6, 319-31 (2005); Burshtyn, D.N. et al., J Biol Chem 272, 13066-72 (1997); Kashiwada, M. et al., J Immunol 167, 6382-7 (2001)). The sequence of human TIGIT and homologs in mouse (submitted to Genbank), rhesus monkey (Genbank accession number XP_001107698), and dog (Genbank accession number XP_545108) are shown in FIG. 1. To further elucidate the role of TIGIT in immune function, a homology search was performed showing that the TIGIT Ig domain is similar to the N-terminal IgV domains of the poliovirus receptor (PVR) protein and PVR-like proteins 1-4 (PVRL1-4), as well as the N-terminal IgV domains of CD96 and CD226 (see FIGS. 2A-2B). Alignment of these proteins showed that highly conserved residues that define the canonical IgV domain are conserved in TIGIT, further suggesting that these eight proteins may comprise a related subset of the Ig family. The conserved V-frame residues have been shown to be important for constructing the V-frame hold (Wiesmann, C. & de Vos, A.M. Cell Mol Life Sci 58, 748-59 (2001)). Three submotifs (V / I 54 -S / T 55 -Q 56 ), (A 67 -X(6)-G 74 ) and (T 112 -F / Y 113 -P 114 -X-G 116Four fully conserved residues (A 67 , G 74 , P 114 and G 116 ), and five conserved residues (V / I / L 54 , S / T 55 , Q 56 , T 112 and F / Y 113 ) were identified to be conserved among eight proteins near the V-frame shift. In the case of TIGIT, these sub-motifs appear to be conserved across species (see Figure 1) and are not present in other currently shown IgV domain-containing proteins. These conserved residues may define a class of PVR-like proteins including PVR, PVR-like proteins 1-4, CD96, CD226, and TIGIT.

[0189] Whereas PVRL1-4 and PVR share a common domain structure (IgV-IgC-IgV), CD226 and CD96 lack the membrane-proximal IgV domain. TIGIT is the most efficient family member consisting of a single IgV domain. The intracellular segments of these eight proteins show limited similarity to each other outside of the afadin-binding motif shared among PVRL1-3. Based on the crystal structure of the relevant IgV domain of NECL-1 (Dong, X. et al., J Biol Chem 281, 10610-7 (2006)), the first and third motifs are predicted to be in the hairpin loops between B and C and between F and Gβ-strands, respectively. These two loops are adjacent to each other at one end of the IgV hold. The second motif includes the C' and C''β-strands that are involved in forming part of the homodimer interface for NECL-1. Thus, the sequence motifs observed in the TIGIT / PVR family may function in the specific homo- and hetero-type interactions observed among PVR family members. PVR has already been characterized as a nectin-like protein, but the above sequence analysis suggests that rather PVR may be considered a PVR family member, and certain nectins (i.e., PVRL1-4) are classified as having branched off from the PVR family.

[0190] Example 2: Identification of PVR Ligands To search for proteins that bind to immobilized TIGIT, substances that could be TIGIT binding partners were identified by screening a large library of secreted proteins. Briefly, the Fc fusion of TIGIT (TIGIT-Fc) was constructed by cloning amino acids 1-138 of human TIGIT into a vector immediately before the Fc region of human IgG1 (TIGIT-Fc). Also, two mutations were introduced into the Fc tail of TIGIT-Fc at D256A and N297A using standard site-directed mutagenesis techniques to construct an alternative version of TIGIT-Fc in which FcγR binding was disrupted (TIGIT-Fc-DANA). The resulting fusion proteins were transiently expressed in CHO cells and purified using standard affinity chromatography techniques. A library of individual secreted proteins fused to hexahistidine or an Fc tag was screened for binding to TIGIT-Fc using the Octet system (ForteBio). Proteins were tested for binding in HBS-P (10 mM Hepes, pH 7.4; 0.15 M NaCl; 0.005% Surfactant P20). TIGIT-Fc or a control Fc fusion protein was flowed over an anti-human Fc biosensor to saturation. The biosensor was washed in buffer (30 seconds), placed in wells containing 5 μg / ml of protein for 3 minutes, and washed again for 30 seconds. The sensor was reloaded and washed after each of two binding cycles. Binding was shown as an increase in response level greater than 0.2 nm, and specificity was determined by comparison to a control Fc fusion protein. A single protein that bound TIGIT was identified among more than 1000 proteins analyzed. As shown in Figure 3, the TIGIT-Fc fusion protein immobilized on the anti-human Fc biosensor specifically interacted with the PVR-Fc fusion protein. The specificity of this interaction was further supported by the fact that TIGIT did not specifically interact with any other protein in the library and that a biosensor loaded with other Ig domain-containing proteins did not induce a response to PVR.

[0191] Since it is already known that PVR, PVRL1-4, CD96, and CD226 interact with each other (He, Y. et al., J Virol 77, 4827-35 (2003); Satoh-Horikawa, K. et al., J Biol Chem 275, 10291-9 (2000); Bottino, C. et al. J Exp Med 198, 557-67 (2003); Fuchs, A. et al., J Immunol 172, 3994-8 (2004); Reymond, N. et al., J Exp Med 199, 1331-41 (2004)), the interaction of TIGIT with each of these proteins was evaluated using the biosensor system described above. As described above for TIGIT-Fc, Fc fusion proteins were constructed and purified for each of the proteins tested. Specifically, amino acids 1-343 of poliovirus receptor-like protein 1 (PVRL1), amino acids 1-360 of poliovirus receptor-like protein 2 (PVRL2), amino acids 1-411 of poliovirus receptor-like protein 3 (PVRL3), amino acids 1-349 of poliovirus receptor-like protein 4 (PVRL4), amino acids 1-259 of CD226, or amino acids 1-500 of CD96 were fused immediately prior to the Fc region of human IgG1. The resulting Fc fusion proteins were tested for binding to TIGIT-Fc. PVR-Fc, PVRL3-Fc, and PVRL2-Fc bound TIGIT-Fc, whereas CD226-Fc, CD96-Fc, PVRL1-Fc, and PVRL4-Fc did not bind TIGIT-Fc (Figure 4A). Among the three observed conjugates, PVR-Fc showed the greatest binding to TIGIT-Fc, followed by PVRL3-Fc, and the lowest binding amount among the three to TIGIT-Fc was observed for PVRL2-Fc.

[0192] In addition, FACS analysis was performed to evaluate the binding of PVR family member Fc fusions constructed on CHO cells expressing TIGIT. The Fc fusion proteins were biotinylated by amine conjugation using PBS containing NHS-PEO4-biotin (Pierce). Biotin-ligand binding was detected using phycoerythrin-conjugated streptavidin (Caltag). The mouse monoclonal antibody against the gD tag (Genentech) was conjugated to AlexaFluor647 (Invitrogen). The antibody was conjugated to the appropriate fluorescent label using standard techniques. Cells were stained according to the manufacturer's instructions. Prior to staining, cells were blocked with appropriate serum or purified IgG. Acquisition was performed on a FACSCalibur (BD Biosciences) and analysis was performed using JoFlo software (Tree Star, Inc.). Forward and side scatter live cells were gated. The results are shown in FIGS. 4B-1 to 4B-6, indicating that the binding patterns observed in the artificial biosensor assay were the same as those observed in the physiological environment of the cell surface.

[0193] To determine the strength of the PVR-TIGIT, PVRL2-TIGIT and PVRL3-TIGIT binding interactions, direct radioligand binding assays were performed using CHO cells stably transfected with these proteins. For cell surface expression, the full-length DNAs of TIGIT, PVR, PVRL2, PVRL3, CD226 and CD96 were cloned immediately downstream of the gD signal sequence (MGGTAARLGAVILFVVIVGLHGVRG (SEQ ID NO: 19)) and the gD tag (KYALADASLKMADPNRFRGKDLPVL (SEQ ID NO: 20)) in a vector. The plasmid was transfected into CHO cells using Lipofectamine LTX (Invitrogen). Expression of the gD-tagged proteins was examined by flow cytometry using an Alexa-647 anti-gD conjugate. Stably transfected cell lines were sorted twice by FACS for purification prior to use. The Fc-fusion proteins constructed as described above were iodinated using the Iodogen method (125 I) Using iodinated ligands at 0.1 - 3 nM, binding assays were performed using three stable transfectants. The iodinated protein was incubated with 1×10 5 -2×10 5 cells at 4°C for 4 hours in the presence of serial dilutions of unlabeled competing protein (25 pM - 5 μM). The cell suspension was harvested onto nitrocellulose membranes (Millipore) and washed thoroughly. The dried filters were counted and Scatchard analysis was performed using NewLigand 1.05 software (Genentech) to determine the binding affinity (Kd).

[0194] Figures 5A and 5B show the binding of radiolabeled TIGIT-Fc protein to PVR-expressing CHO cells. The mean Kd for the TIGIT-Fc - PVR interaction for four experiments was 3.15 nM. Table 6 shows the results for all analytes in tabular form. Table 6. Cell binding of PVR family proteins. Receptors were expressed on CHO cells and all ligands were -Fc constructs. After gating on receptor-positive cells, the MFI was determined by flow cytometry with biotinylated Fc-ligands. Binding affinity (Kd) was determined by competitive radioligand binding assay. Kd is shown in (nM) and is the mean of at least three independent assays except where marked with (*). TIFF2025090567000002.tif39170++++ MFI>5000 +++ MFI = 1000 - 4999 ++ MFI = 100 - 999 + MFI < 100 - No binding & Specific binding but Kd is unclear * Mean of two assays

[0195] The interaction of TIGIT with PVP showed the highest affinity (Kd = 1 - 3 nM), while the affinity of TIGIT for binding to PVRL3 was approximately one tenth to one thirtieth (Kd = 38.9 nM) (see Table 6). Since the curve fitting in the radioligand assay was poor, the binding constant of the PVRL2 - TIGIT interaction could not be determined. Nevertheless, specific binding was observed, which was consistent with the above - mentioned FACS data showing moderate binding of PVRL2 - Fc to CHO - TIGIT, further strengthening the finding that the binding between PVRL2 and TIGIT is a low - affinity interaction. The iodinated Fc - fusion protein (ligand) bound to receptor - expressing CHO cells at the indicated concentrations and competed with 10 - fold serial dilutions of CD226 - Fc (8 μM for CHO - TIGIT; 5 μM for CHO - PVR), TIGIT - Fc (2 μM for CHO - PVR; 6 μM for CHO - CD226 and CHO - CD96). Nonspecific binding was determined using 2000 - fold excess unlabeled ligand and subtracted from the total binding. The competition assay showed that TIGIT effectively blocked the interaction of PVR with other coreceptors CD226 and CD96, while CD226 was a weak inhibitor of the TIGIT - PVR interaction (Figure 6). This data was consistent with the observed high affinity of the PVR - TIGIT interaction (1 - 3 nM) compared to the PVR - CD226 interaction (Tahara - Hanaoka, S. et al. Int Immunol 16, 533 - 8 (2004), approximately 115 nM). A direct competition assay with CD96 could not be performed due to low expression of the protein, but TIGIT completely inhibited PVR binding to CD96 - expressing CHO cells. The aforementioned competition assays showed that TIGIT, CD226, and CD96 share a common binding site or overlapping binding sites on PVR. This finding was supported by the observation that the anti - PVR antibody D171, which binds to the N - terminal IgV domain of PVR, blocks the binding of TIGIT and CD226 to PVR (Figure 7).

[0196] Example 3: Expression of TIGIT and PVR (A) Expression of TIGIT and PVR in resting and activated immune cells The relative distribution and expression of TIGIT and PVR on immune cells were evaluated as an indicator of the role of these two molecules in normal immune function and compared with the expression of CD226, a molecule that is already known to interact with PVR in vivo and is shown in Example 2. Previous studies have shown that the expression of TIGIT was specific to T and NK cells across multiple immune cell types and tissue arrays (Abbas, A.R. et al., Genes Immun 6, 319-31 (2005)). The expression of TIGIT in various immune cells and ex vivo tissues, and after activation, was further analyzed. As shown in FIGS. 8A and 8B, TIGIT was most strongly expressed in regulatory T cells (T reg ), and also highly expressed in NK cells and T fh cells from human tonsil tissue. TIGIT was expressed to a lesser extent in unstimulated NK cells, activated and resting memory T cells, CD8 + T cells, and Th2 and Th1 cells. This data correlates with the data shown in US Patent Publication 20040121370, which shows that TIGIT is significantly overexpressed in isolated CD4 + T cells activated by anti-CD3 / ICAM-1 and anti-CD3 / anti-CD28 compared to isolated resting CD4 + T cells. In contrast, PVR has been reported to be expressed in endothelial cells, fibroblasts, osteoclasts, follicular dendritic cells, dendritic cells, and tumor cells (Sakisaka, T. & Takai, Y., Curr Opin Cell Biol 16, 513-21 (2004); Fuchs, A. & Colonna, M., Semin Cancer Biol 16, 359-66 (2006)). This data emphasizes that TIGIT is associated with T cells that produce regulatory cytokines that can suppress the immune response.

[0197] Furthermore, complementary flow cytometry analysis was performed using the same method as described in Example 2. Human ex vivo T cells were tested for surface TIGIT expression after activation using a hamster anti-mouse TIGIT antibody (10A7) that cross-reacts with human TIGIT and blocks the interaction of TIGIT with PVR (see Figure 9). The anti-TIGIT antibody was produced by immunizing hamsters with mouse TIGIT-Fc fusion protein and obtaining hamster anti-mouse antibodies therefrom using standard techniques. Two antibodies, 10A7 and 1F4, also specifically bound to human TIGIT (data not shown) and were used in further experiments. In particular, 10A7 and 1F4 bind to different epitopes on human TIGIT. This is supported by the fact that the binding of 1F4 to TIGIT does not block the binding of 10A7 to TIGIT on the surface of 293 cells expressing TIGIT (data not shown). The amino acid sequences of the light and heavy chains of 10A7 were determined using standard techniques. The light chain sequence of this antibody is DIVMTQSPSSLAVSPGEKVTMTC KSSQSLYYSGVKENLLA WYQQKPGQS PKLLIYY ASIRFT GVPDRFTGSGSGTDYTLTITSVQAEDMGQYFC QQGINNPLT FGDGTKLEIKR (SEQ ID NO: 21), and the heavy chain sequence of this antibody is EVQLVESGGGLTQPGKSLKLSCEAS GFTFSSFTMH WVRQSPGK...

Claims

1. Test immune cells are activated or normal T ｒｅｇ , memory T cells, NK cells or T Ｆｈ The method for determining whether a test immune cell is an activated or normal T cell comprises assessing the expression level of TIGIT in the test immune cell and ｒｅｇ , memory T cells, NK cells or T Ｆｈ and comparing the expression level of TIGIT in the test immune cell to one or more known reference TIGIT expression values.

2. An isolated polypeptide comprising an amino acid sequence including one or more of: alanine at amino acid position corresponding to amino acid position 67 of human TIGIT, glycine at amino acid position corresponding to amino acid position 74 of human TIGIT, proline at amino acid position corresponding to amino acid position 114 of human TIGIT, and glycine at amino acid position corresponding to amino acid position 116 of human TIGIT.

3. 3. The polypeptide of claim 2, wherein the polypeptide is not PVR, PVRL1, PVRL2, PVRL3, PVRL4, TIGIT, CD96 or CD226.

4. The polypeptide of claim 2 or 3, further comprising one or more of an amino acid selected from valine, isoleucine and leucine at an amino acid position corresponding to amino acid position 54 of human TIGIT, an amino acid selected from serine and threonine at an amino acid position corresponding to amino acid position 55 of human TIGIT, a glutamine at an amino acid position corresponding to amino acid position 56 of human TIGIT, a threonine at an amino acid position corresponding to amino acid position 112 of human TIGIT, and an amino acid selected from phenylalanine and tyrosine at an amino acid position corresponding to amino acid position 113 of human TIGIT.

5. The polypeptide is a. an amino acid selected from valine and isoleucine at amino acid position 54, an amino acid selected from serine and threonine at amino acid position 55, and a glutamine at amino acid position 56; b. an alanine at position 67, any amino acid at each of amino acid positions 68-73, and glycine at amino acid position 74; and c. a threonine at amino acid position 112, an amino acid selected from phenylalanine and tyrosine at amino acid position 113, a proline at amino acid position 114, any amino acid at amino acid position 115, and glycine at amino acid position 116.

4. The polypeptide of claim 2 or 3, further comprising one or more structural submotifs selected from the following: wherein the amino acid position numbers correspond to the amino acid positions in human TIGIT.

6. A method for determining whether a test polypeptide is a member of the TLP family of polypeptides, comprising aligning the amino acid sequence of the test polypeptide with the amino acid sequence of one or more members of the TLP family of polypeptides, and evaluating the presence or absence of any of the amino acids set forth in claim 2 in the amino acid sequence of the test polypeptide.

7. 3. A method of identifying one or more members of the TLP protein family by identifying proteins in one or more sequence databases having an amino acid sequence that includes at least one of the amino acids set forth in claim 2.

8. An isolated agent that specifically interacts with one or more conserved or substantially conserved regions of the TLP family members.

9. The agent of claim 8, wherein the agent is an antagonist of the expression and / or activity of a TLP family member.

10. The agent of claim 9, wherein the antagonist is selected from a small molecule inhibitor, an inhibitory antibody or antigen-binding fragment thereof, an aptamer, an inhibitory nucleic acid, and an inhibitory polypeptide.

11. The agent of claim 8, wherein the agent is an agonist of expression and / or activity of a TLP family member.

12. The agent of claim 11, wherein the agent is selected from an agonistic antibody or antigen-binding fragment thereof, an agonistic peptide, and a small molecule or protein that activates TIGIT binding to PVR-mediated PVR and / or TIGIT intracellular signaling.

13. 10. A method for identifying or detecting one or more TLP family members by contacting a putative TLP family member polypeptide with an agent of claim 8 and determining binding of the agent to the putative TLP family member.

14. A method of modulating immune system function and / or activity comprising modulating binding of TIGIT to one or more of PVR, PVRL3 and PVRL2.

15. An anti-TIGIT antibody or a fragment thereof, comprising at least one HVR comprising an amino acid sequence selected from the amino acid sequences shown in SEQ ID NOs: 23 to 28 or SEQ ID NOs: 31 to 36.

16. The anti-TIGIT antibody or its antigen-binding fragment according to claim 15, wherein the antibody light chain comprises the amino acid sequence shown in SEQ ID NO: 21 or 29.

17. The anti-TIGIT antibody or its antigen-binding fragment according to claim 15, wherein the antibody heavy chain comprises the amino acid sequence shown in SEQ ID NO: 22 or 30.

18. The anti-TIGIT antibody or its antigen-binding fragment according to claim 15, wherein the antibody light chain comprises the amino acid sequence shown in SEQ ID NO: 21 or 29, and the antibody heavy chain comprises the amino acid sequence shown in SEQ ID NO: 22 or 30.

19. The anti-TIGIT antibody or antigen-binding fragment thereof of claim 15, wherein the antibody is selected from a humanized antibody, a chimeric antibody, a bispecific antibody, a heteroconjugate antibody, and an immunotoxin.

20. The anti-TIGIT antibody or antigen-binding fragment thereof according to claim 15, wherein at least one HVR is at least 90% identical to an HVR set forth in any one of SEQ ID NOs: 23 to 28 or 31 to 36.

21. 18. An anti-TIGIT antibody or fragment thereof according to claim 16 or 17, wherein the light chain and / or the heavy chain comprises an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 21 or 29 or 22 or 30, respectively.

22. A method for modulating CD226-PVR interaction and / or CD96-PVR interaction, comprising administering, in vivo or in vitro, at least one of TIGIT, an agonist of TIGIT expression and / or activity, or an antagonist of TIGIT expression and / or activity.

23. The method of claim 22, wherein TIGIT or an agonist of TIGIT expression and / or activity is administered to inhibit CD226-PVR interaction and / or CD96-PVR interaction.

24. The method of claim 22, wherein an antagonist of TIGIT expression and / or activity is administered to stimulate CD226-PVR interaction and / or CD96-PVR interaction.

25. A method for modulating immune cell function and / or activity by modulating expression and / or activity of TIGIT and / or PVR, or by modulating intracellular signaling mediated by TIGIT binding to PVR.

26. The method of claim 25, wherein the modulation is reducing or inhibiting the proliferation of one or more immune cells or the release of proinflammatory cytokines by one or more immune cells by treating cells in vitro or in vivo with TIGIT, an agonist of TIGIT expression and / or activity, an agonist of PVR expression and / or activity, or by stimulating intracellular signaling mediated by TIGIT binding to PVR.

27. The method of claim 25, wherein the modulation is increasing or stimulating the proliferation of one or more immune cells or the release of proinflammatory cytokines by one or more immune cells by treating cells in vitro or in vivo with an antagonist of TIGIT expression and / or activity, an antagonist of PVR expression and / or activity, or by inhibiting intracellular signaling mediated by TIGIT binding to PVR.

28. A method for inhibiting an immune response in vitro or in vivo by administering TIGIT, an agonist of TIGIT expression and / or activity, an agonist of PVR expression and / or activity, or by stimulating intracellular signaling mediated by TIGIT binding to PVR.

29. A method for enhancing or stimulating an immune response in vitro or in vivo by administering an antagonist of TIGIT expression and / or activity, an antagonist of PVR expression and / or activity, or by inhibiting intracellular signaling mediated by TIGIT binding to PVR.

30. A method for modulating the type and / or amount of cytokine production from immune cells by modulating the expression and / or activity of TIGIT or PVR in vitro or in vivo.

31. The method of claim 30, wherein proinflammatory cytokine production is stimulated and / or enhanced by administering an antagonist of TIGIT expression and / or activity, an antagonist of PVR expression and / or activity, or by inhibiting intracellular signaling mediated by TIGIT binding to PVR.

32. The method of claim 30, wherein proinflammatory cytokine production is inhibited by administering an agonist of TIGIT expression and / or activity, an agonist of PVR expression and / or activity, or by stimulating intracellular signaling mediated by TIGIT binding to PVR.

33. A method for stimulating intracellular signaling and / or ERK phosphorylation via the ERK pathway in one or more immune cells, comprising treating one or more immune cells with TIGIT, an agonist of TIGIT expression and / or activity, or an agonist of PVR expression and / or activity.

34. A method for diagnosing an immune-related disease associated with an abnormal immune cell response in a subject, comprising assessing the expression and / or activity of TIGIT in a sample from the subject and comparing the expression and / or activity of TIGIT with a control amount of TIGIT expression and / or activity or the amount of TIGIT expression and / or activity in a sample from a normal subject.

35. A method for assessing the severity of an immune-related disease associated with an abnormal immune cell response in a subject, comprising assessing the expression and / or activity of TIGIT in a sample from the subject and comparing the expression and / or activity of TIGIT with a control amount of TIGIT expression and / or activity or the amount of TIGIT expression and / or activity in a sample from a normal subject.

36. A method for preventing an immune-related disease associated with an abnormal immune cell response in a subject, comprising regulating the expression and / or activity of TIGIT in the subject.

37. A method for treating or reducing the severity of an immune-related disease associated with an aberrant immune cell response in a subject, comprising modulating the expression and / or activity of TIGIT in the subject.

38. 38. The method of any one of claims 34 to 37, wherein the immune-related disease is selected from psoriasis, arthritis, inflammatory bowel disease or cancer.

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