Anti-tigit antibodies and their use as therapeutics and diagnostics

Abstract

To provide antibodies that specifically bind to TIGIT (T cell immune receptor with Ig and ITIM domains).SOLUTION: Disclosed is an antibody or antigen binding fragment thereof capable of binding human TIGIT, the antibody comprising: (a) a heavy variable region (VH) comprising one, two or three CDR amino acid sequences selected from specified amino acid sequences; and / or (b) a light variable region (VL) comprising one, two or three CDR amino acid sequences selected from other specified amino acid sequences.SELECTED DRAWING: None

Description

[Technical field]

[0001] Related Applications This application claims priority to patent application number PCT / CN2017 / 120392, filed December 30, 2017, the entire contents of which are incorporated herein by reference for all purposes.

[0002] FIELD OF THEINVENTION This application relates to antibodies that specifically bind to TIGIT (T cell immunoreceptor with Ig and ITIM domains), and uses thereof. [Background technology]

[0003] TIGIT (T cell immunoreceptor with Ig and ITIM domains) is a type 1 transmembrane protein and a member of the CD28 family of proteins that play a key role in inhibiting T cell and NK cell-mediated functional activity in antitumor immunity (Boles KS, et al., 2009 Eur J Immunol, 39:695-703; Stanietsky N, et al., 2009 PNAS 106:17858-63; Yu X, et al. 2009 Nat. Immunol, 10:48-57).

[0004] The gene and cDNA encoding TIGIT have been cloned and characterized in mouse and human. Full-length human TIGIT has a sequence of 244 amino acids in length (SEQ ID NO: 1), the first 21 amino acids of which are a signal peptide. The amino acid sequence of mature human TIGIT contains 223 amino acid (aa) residues (NCBI accession number: NM_173799). The extracellular domain (ECD) of mature human TIGIT consists of 120 amino acid residues (SEQ ID NO:2: corresponding to amino acids 22-141 of SEQ ID NO:1), a V-type Ig-like domain (corresponding to amino acids 39-127 of SEQ ID NO:1), followed by a 21 amino acid transmembrane sequence, an 82 amino acid cytoplasmic domain, and an immunoreceptor tyrosine-based inhibitory motif (ITIM) (Yu X, et al. 2009 Nat. Immunol, 10:48-57; Stengel KF, et al. 2012 PNAS 109:5399-04). Within the ECD, human TIGIT shares 59% and 87% amino acid sequence identity with mouse and cynomolgus monkeys, respectively.

[0005] TIGIT is expressed on T cells (activated T cells, memory T cells, regulatory T (Treg) cells, and follicular T helper (Tfh) cells) and on NK cells (Boles KS, et al., 2009 Eur J Immunol, 39:695-703; Joller N, et al., 2014 Immunity 40:569-81; Levin SD, et al., 2011 Eur J Immunol, 41:902-15; Stanietsky N, et al., 2009 PNAS 106:17858-63; Yu X, et al. 2009 Nat. Immunol, 10:48-57).

[0006] So far, two TIGIT ligands have been identified: CD155 (also known as poliovirus receptor or PVR) and CD112 (also known as poliovirus receptor-related 2, PVRL2 or nectin-2). These ligands are expressed primarily on APCs (dendritic cells, macrophages, etc.) and on tumor cells (Casado JG, et al., 2009 Cancer Immunol Immunother 58:1517-26;Levin SD, et al., 2011 Eur J Immunol, 41:902-15;Mendelsohn CL et al., 1989 56:855-65;Stanietsky N, et al., 2009 PNAS 106:17858-63;Yu X, et al. 2009 Nat. Immunol, 10:48-57). As an immune "checkpoint" molecule, TIGIT induces inhibitory signaling in immune cells when bound by its ligands CD155 and CD112. The binding affinity of TIGIT to CD155 (Kd: approx. 1 nM) is much higher than that to CD112. Whether the TIGIT:CD112 interaction is functionally related to mediating inhibitory signals remains to be determined. The costimulatory receptor CD226 (DNAM-1) binds the same ligand with lower affinity (Kd: approx. 100 nM) but provides a positive signal (Bottino C, et al., 2003 J Exp Med 198:557-67). In addition, the "TIGIT-like" receptor CD96 (Tactile) also plays a similar inhibitory role in the same pathway (Chan CJ, et al., 2014 Nat. Immunol 15:431-8).

[0007] TIGIT can inhibit immune responses through different mechanisms. First, the interaction of TIGIT with PVR on dendritic cells (DCs) provides “reverse signaling” in DCs, leading to upregulation of IL-10 and reduced IL-12 secretion, thereby suppressing T cell activation (Yu X, et al. Nat Immunol. 2009 10:48-57). Second, TIGIT binds to CD155 with higher affinity, thereby dislodging and competing for the DNAM-1-CD155 interaction. Third, direct ligation of TIGIT on T cells can downregulate TCR-mediated activation, and subsequent proliferation and association of TIGIT on NK cells can block NK cell cytotoxicity (Joller N, et al. 2011 186: 1338-42;Stanietsky N, et al., 2009 PNAS 106:17858-63). Fourth, TIGIT expression on Treg cells is associated with a highly activated suppressive phenotype in tumor tissues, and TIGIT signaling in Treg cells may favor Treg cell stability (Joller N, et al. Immunity 2014 40:569-81; Kurtulus S, et al. J Clin Invest. 2015 125: 4053-4062).

[0008] TIGIT possesses immunoglobulin tail tyrosine-based (ITT)-like motifs followed by immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in its cytoplasmic tail (Yu X, et al. Nat Immunol. 2009 10:48-57;Engels N, et al. Curr Opin Immunol 2011 23: 324-329). These motifs can mediate the recruitment of the phosphatases SHIP-1 and β-arrestin 2 (Li M, et al. J Biol Chem. 2014 289:17647-17657;Liu S, et al. Cell death and differentiation 2013 20: 456-464), thereby providing a mechanism by which TIGIT provides an intrinsically inhibitory signal to attenuate activating signals.

[0009] Upregulation of TIGIT expression in tumor-infiltrating lymphocytes (TILs) and peripheral blood mononuclear cells (PBMCs) has been reported in many types of cancer, including lung cancer (Tassi, et al., Cancer Res. 2017 77: 851-861), esophageal cancer (Xie J, et al., Oncotarget 2016 7:63669-63678), breast cancer (Gil Del Alcazar CR, et al. 2017 Cancer Discov.), acute myeloid leukemia (AML) (Kong Y et al., Clin Cancer Res. 2016 22:3057-66), and melanoma (Chauvin JM, et al., J Clin Invest. 2015 125:2046-2058). Increased expression of TIGIT in AML is associated with poor prognosis of patient survival (Kong Y et al., Clin Cancer Res. 2016 22:3057-66). Upregulation of TIGIT signaling plays an important role in immune tolerance not only to cancer but also to chronic viral infections. During HIV infection, expression of TIGIT on T cells was significantly higher and positively correlated with viral load and disease progression (Chew GM, et al., 2016 PLoS Pathog. 12:e1005349). Furthermore, blocking TIGIT receptor alone or in combination with other blockades can rescue functionally "exhausted" T cells both in vitro and in vivo (Chauvin JM, et al., J Clin Invest. 2015 125:2046-2058; Chew GM, et al., 2016 PLoS Pathog. 12:e1005349; Johnston RJ, et al. Cancer Cell 2014 26:923-937). In the case of cancer and viral infection, activation of TIGIT signaling promotes immune cell dysfunction, leading to cancer growth or viral infection spread. Inhibition of TIGIT-mediated inhibitory signaling by therapeutic agents can restore the functional activity of immune cells, including T cells, NK cells, and dendritic cells (DCs), thus enhancing immunity against cancer or chronic viral infection.

[0010] Thus, modulation of TIGIT signaling by antagonistic molecules may rescue immune cells from tolerance and induce efficient immune responses to eradicate tumors or chronic viral infections. Summary of the Invention [Means for solving the problem]

[0011] The present invention is based, at least in part, on the discovery of a set of monoclonal antibodies (mAbs) that inhibit TIGIT-mediated intracellular signaling in immune cells, reactivate immune cells, and enhance immunity by specifically binding to TIGIT. Thus, in a first aspect, the present application relates to anti-TIGIT antibodies and antigen-binding fragments thereof that can bind to human TIGIT (SEQ ID NO: 1). The present invention also relates to humanized versions of the anti-TIGIT mAbs of the first aspect.

[0012] In a particular embodiment, the antibody of the present application comprises a heavy chain variable region (VH) comprising one, two or three CDRs having an amino acid sequence selected from SEQ ID NOs: 3, 4, 5 and 13 or a variant thereof comprising one or more conservative substitutions (e.g., one or two conservative substitutions of the amino acid sequence of SEQ ID NOs: 3, 4, 5 or 13); and / or a light chain variable region (VL) comprising one, two or three CDRs having an amino acid sequence selected from SEQ ID NOs: 6, 7 and 8 or a variant thereof comprising one or more conservative substitutions (e.g., one or two conservative substitutions of the amino acid sequence of SEQ ID NOs: 6, 7 or 8).

[0013] In a more specific embodiment, the antibody of the present application comprises a heavy chain variable region (VH) comprising a VH-CDR1 having the amino acid sequence of SEQ ID NO: 3 or a variant thereof comprising one or more conservative substitutions (e.g., one or two conservative substitutions), a VH-CDR2 having the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 13 or a variant thereof comprising one or more conservative substitutions (e.g., one or two conservative substitutions), and a VH-CDR3 having the amino acid sequence of SEQ ID NO: 5 or a variant thereof comprising one or more conservative substitutions (e.g., one or two conservative substitutions); and / or a light chain variable region (VL) comprising a VL-CDR1 having the amino acid sequence of SEQ ID NO: 6 or a variant thereof comprising one or more conservative substitutions (e.g., one or two conservative substitutions), a VL-CDR2 having the amino acid sequence of SEQ ID NO: 7 or a variant thereof comprising one or more conservative substitutions (e.g., one or two conservative substitutions), and a VL-CDR3 having the amino acid sequence of SEQ ID NO: 8 or a variant thereof comprising one or more conservative substitutions (e.g., one or two conservative substitutions).

[0014] The antibody or antigen-binding fragment thereof of the present application can bind to human TIGIT and comprises a heavy chain variable region having an amino acid sequence selected from SEQ ID NO: 9, 14 and 19, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to SEQ ID NO: 9, 14 or 19. In one embodiment, the sequence differences are in the framework regions. In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region encoded by a nucleotide sequence selected from SEQ ID NO: 10, 15 and 20, or a variant thereof.

[0015] The antibody or antigen-binding fragment thereof of the present application can bind to human TIGIT and comprises a heavy chain variable region having an amino acid sequence selected from SEQ ID NO: 11, 16, 21 and 24, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to SEQ ID NO: 11, 16, 21 or 24. In one embodiment, the sequence differences are in the framework regions. In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region encoded by a nucleotide sequence selected from SEQ ID NO: 12, 17 and 22, or a variant thereof.

[0016] In one embodiment, the antibody or antigen-binding fragment thereof is about 1×10 -9 M ~ approx. 1×10 -12 M. For example, the antibody or antigen-binding fragment thereof can bind to human TIGIT with a Kd value of about 1×10 -9 Less than M, approx. 1×10 -10 Less than M, approx. 1×10 -11 Less than M or about 1 x 10 -12 It can bind to human TIGIT with a Kd value of less than M

[0017] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region of IgG1, IgG2, IgG3 or IgG4 subclass or a variant thereof, and a light chain constant region of κ-type or λ-type or a variant thereof. In a more specific embodiment, the Fc region of the antibody is a human IgG1 Fc or a variant thereof, e.g., the Fc region of SEQ ID NO: 18.

[0018] In one embodiment, the antibody or antigen-binding fragment thereof promotes the production of IFN-γ by antigen-specific T cells. In a more specific embodiment, the antibody or antigen-binding fragment promotes the production of IFN-γ by antigen-specific T cells in a dose-dependent manner.

[0019] In a more specific embodiment, the antibodies of the present application reduce the surface expression of TIGIT receptor via FcγR-mediated trogocytosis, in particular FcγRIIB-mediated trogocytosis.

[0020] In one embodiment, the antibodies of the present application exhibit pH-dependent antigen binding, where the antibodies exhibit stronger binding to human TIGIT at mildly acidic pH (e.g., pH 6.0) in the tumor microenvironment compared to binding to human TIGIT at physiological pH (e.g., pH 7.4). In a more specific embodiment, the antibodies of the present application exhibit (1) a pH 7.4 / pH 6.0 K of greater than 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more, as measured by surface plasmon resonance (Biacore) or similar techniques. D and / or (2) an Rmax (RU) value at pH 6.0 that is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, or 50-fold higher than the Rmax (RU) value at pH 7.4.

[0021] The anti-TIGIT mAbs disclosed herein have potential therapeutic applications in the treatment of cancer, viral infections, and controlling other human diseases that are mechanistically involved in immune tolerance or "exhaustion." Thus, in a further embodiment, the anti-TIGIT antibodies of the present application are for use in the treatment of cancer. In another specific embodiment, the anti-TIGIT antibodies of the present application are for use in the treatment of infection, infectious disease, and / or control of viral infection. In another specific embodiment, the anti-TIGIT antibodies of the present application are for use in the treatment of other human diseases associated with or caused by immune tolerance, or diseases that can be ameliorated by increasing immune cell activation.

[0022] In a further aspect, the present application relates to a composition comprising an anti-TIGIT antibody, or an antigen-binding fragment thereof, and a pharma- ceutically acceptable excipient. [Brief description of the drawings]

[0023] [Figure 1] Schematic diagram of TIGIT-mIgG2a (upper panel) and TIGIT-huIgG1 (lower panel). TIGIT ECD: TIGIT extracellular domain. N: N-terminus. C: C-terminus. [Figure 2A] Phylogenetic tree of anti-TIGIT antibody Vh regions. Vh sequences of candidate anti-TIGIT antibodies were aligned using Megalign software from DNASTAR. Sequence homology is shown in the phylogenetic tree. [Figure 2B] Phylogenetic tree of anti-TIGIT antibody Vκ regions. Vκ sequences of candidate anti-TIGIT antibodies were aligned using Megalign software from DNASTAR. Sequence homology is shown in the phylogenetic tree. [Diagram 3] 1 shows the affinity determination of purified murine anti-TIGIT antibodies by surface plasmon resonance (SPR). [Figure 4] Determination of TIGIT binding by flow cytometry. [Diagram 5] Figure 5A is a schematic diagram showing the inhibition of TIGIT-ligand interaction by anti-TIGIT mAb. Figure 5B shows the binding of soluble TIGIT (TIGIT-huIgG1 fusion protein) to TIGIT ligand-expressing HEK293 cells (HEK293 / PVR or HEK293 / PVR-L2) determined by flow cytometry. Blockade of TIGIT-ligand interaction was quantitatively measured by adding serially diluted anti-TIGIT antibodies. Results are shown as the mean ± SD of duplicates. [Figure 6] Activation of CMV-specific human T cells by anti-TIGIT mAb. HLA-A2.1+ PBMCs (4 × 104) sensitized with human CMV peptide (NLVPMVATV, 495–503) were stimulated overnight with CMV peptide-pulsed target cells, HCT116 cells (104), in the presence of anti-TIGIT antibody. IFN-γ in the culture supernatant was determined by ELISA. All conditions were performed in triplicate. Results are shown as mean ± SD. [Figure 7]Anti-TIGIT mAb promotes NK cell-mediated cytotoxicity. Figure 7A shows the expression of TIGIT and DNAM-1 on the engineered NK92MI / TIGIT-DNAM-1 stable cell line. In Figure 7B, the killing of NK92MI / TIGIT-DNAM-1 cells against SK-MES-1 / PVR cells in the presence of hu1217-2-2 / IgG1mf (0.007-30 μg / ml) was determined by LDH (lactate dehydrogenase) release assay as described in Example 8. The results are shown as the mean ± SD of triplicates. [Figure 8] Anti-TIGIT mAb hu1217-2-2 / IgG1wt reduces surface expression of TIGIT receptor via FcγR-mediated trogocytosis. Jurkat / TIGIT cells were incubated overnight with FcγR-expressing HEK293 cells in complete medium in the presence of biotin-labeled anti-TIGIT mAb. In some cases, 10% human AB serum was added to determine the effect of large amounts of human IgG on trogocytosis. Surface expression of TIGIT receptor was determined by staining with SA-APC (Biolegend). MFI was determined by flow cytometry. All data points were in duplicate. Results are presented as mean ± SD. [Figure 9]ADCC effect of anti-TIGIT mAb on human peripheral blood mononuclear cells (PBMCs) is shown. In Figure 9A, TIGIT expression in PHA-stimulated PBMCs from healthy donors was determined by flow cytometry. CD4+ (CD4+ Foxp3-) effector T cells, CD8+ effector T cells, and regulatory T cells (Tregs cells, CD4+ Foxp3+) all expressed significant levels (18-41%) of TIGIT. Data shown are representative results from three healthy donors. In Figure 9B, ADCC assays were performed using CD16+ human NK cell line NK92MI / CD16V as effector cells and PHA-stimulated PBMCs as target cells in the presence of TIGIT mAb (30 μg / mL) or control antibodies (OKT3 at 5 μg / ml as a positive control, and huIgG at 30 μg / ml as a negative control) for 42 h. The percentages of CD3+ T cells, CD8+ T cells and Treg cells were determined by flow cytometry. [Figure 10] Figure 1 shows the CDC effect of anti-TIGIT mAb on human PBMC. CDC assays were performed using PHA-stimulated PBMC as target cells and autologous serum as a complement source. After 3 days of co-culture of pre-activated PBMC with anti-TIGIT mAb (0.01-100 μg / ml) in autologous serum at a final concentration of 15%, the percentage of CDC (y-axis) was measured by cell-titer grow assay and calculated as described in Example 11. Data from donors A and B are shown. huIgG was used as a negative control and anti-MHC-A, B, C were used as positive controls. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] definition Conservative amino acid substitutions are generally known in the art and are exemplified in the table below. Generally, a conservative amino acid substitution means that an amino acid residue is replaced with another amino acid residue having a similar side chain.

[0025] JPEG2025024708000001.jpg141161

[0026] Unless specifically defined elsewhere in this specification, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs.

[0027] Unless otherwise expressly stated herein, singular words such as "a," "an," and "the" as used in the specification and claims also include their plural equivalents.

[0028] Unless the text of this specification clearly indicates otherwise, the term "or" is used to mean, and is used interchangeably with, the term "and / or."

[0029] Unless otherwise expressly indicated by the description herein, the term "comprise" and variations such as "comprises" and "comprising" are understood throughout the specification and claims to mean the inclusion of a recited amino acid sequence, DNA sequence, step, or group thereof, and not the exclusion of other amino acid sequences, DNA sequences, steps. As used herein, the term "comprising" can be substituted with the terms "containing," "including," or "having."

[0030] The term "TIGIT" includes various mammalian isoforms, such as human TIGIT, species homologs of human TIGIT, and analogs that contain at least one epitope within TIGIT, and the amino acid sequences of TIGIT, such as human TIGIT, and the nucleotide sequences encoding same, are known in the art.

[0031] As used herein, the terms "administration," "administering," "treating," and "treatment," when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refer to the contact of an exogenous pharmaceutical, therapeutic, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. Treatment of a cell includes contact of a reagent with the cell, and contact of a reagent with a fluid with which the cell is in contact. The term "administration" or "treatment" also includes in vitro and ex vivo treatments, for example, with a reagent, diagnostic compound, binding compound, or other cell. The term "subject" as used herein refers to any living organism, preferably an animal, more preferably a mammal (e.g., rat, mouse, dog, cat, rabbit), and most preferably a human.

[0032] Antibody or antibody molecule Disclosed herein are antibody molecules that bind to TIGIT with high affinity and specificity.

[0033] In some embodiments, the anti-TIGIT antibody binds to human TIGIT and comprises at least one, two, three, four, five or six complementarity determining regions (CDRs) comprising the amino acid sequence of SEQ ID NO: 3, 4, 5, 13, or SEQ ID NO: 6, 7, 8. In particular embodiments, the antibody of the present application comprises a heavy chain variable region (VH) comprising one, two or three CDRs having an amino acid sequence selected from SEQ ID NO: 3, 4, 5 and 13, or variants thereof comprising one or more conservative substitutions; and / or a light chain variable region (VL) comprising one, two or three CDRs having an amino acid sequence selected from SEQ ID NO: 6, 7 and 8, or variants thereof comprising one or more conservative substitutions.

[0034] In some embodiments, the anti-TIGIT antibody is an isolated antibody, a humanized antibody, a chimeric antibody, or a recombinant antibody.

[0035] In some embodiments, the anti-TIGIT antibody comprises at least one antigen-binding site, or at least a variable region, hi some embodiments, the anti-TIGIT antibody comprises an antigen-binding fragment derived from an antibody described herein.

[0036] The term "antibody" is used herein in the broadest sense and specifically includes antibodies (including full length monoclonal antibodies) and antibody fragments that recognize an antigen, such as TIGIT. Antibodies are usually monospecific, but may also be described as idiospecific, heterospecific, or multispecific. An antibody molecule binds to a specific antigenic determinant or epitope of an antigen through a specific binding site.

[0037] The terms "monoclonal antibody", "mAb" or "Mab" as used herein refer to a population of substantially homogenous antibodies, i.e., the antibody molecules in the population are identical in amino acid sequence, except for possible naturally occurring mutations that may be present in minor amounts. In contrast, conventional (polyclonal) antibody preparations usually contain a large number of different antibodies whose variable regions, and in particular their complementarity determining regions (CDRs), have different amino acid sequences, often specific for different epitopes. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogenous population of antibodies and is not to be construed as requiring production of the antibody by any particular method. Monoclonal antibodies (mAbs) can be obtained by methods well known to those skilled in the art. See, e.g., Kohler G et al., Nature 1975 256:495-497; US4376110; Ausubel FM et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY 1992; Harlow E et al., ANTIBODIES: A LABORATORY MANUAL, Cold spring Harbor Laboratory 1988; and Colligan JE et al., CURRENT PROTOCOLS IN IMMUNOLOGY 1993. The mAbs disclosed herein may be of any immunoglobulin class, including IgG, IgM, IgD, IgE, IgA, and any subclass thereof. Hybridomas producing mAbs may be cultured in vitro or in vivo. High titers of mAbs can be obtained by in vivo production, in which cells from individual hybridomas are injected intraperitoneally into mice, such as pristine-primed Balb / c mice, to produce ascites fluid containing high concentrations of the desired mAb. MAbs of isotype IgM or IgG can be purified from the ascites fluid or culture supernatants described above using column chromatography methods well known to those of skill in the art.

[0038] Generally, the basic antibody structural unit comprises a tetramer. Each tetramer comprises two identical sets of polypeptide chains, each set having one "light chain" (about 25 kDa) and one "heavy chain" (about 50-70 kDa). The amino-terminal portion of each chain comprises a variable region of about 100-110 or more amino acids essentially involved in antigen recognition. The carboxy-terminal portion of the heavy chain may define a constant region essentially involved in effector function. Human light chains are usually classified as kappa light chains and lambda light chains. Human heavy chains are further generally classified as alpha, delta, epsilon, gamma or mu, which define the antibody isotype as IgA, IgD, IgE, IgG and IgM, respectively. Within the light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also comprising a "D" region of about 10 or more amino acids.

[0039] The variable regions of each heavy / light chain (VL / VH) pair form the antibody binding site. Thus, an intact antibody generally has two binding sites. Except for bifunctional or bispecific antibodies, the two binding sites are usually identical.

[0040] Typically, both heavy and light chain variable regions contain three hypervariable regions, also called "complementarity determining regions (CDRs)", located between relatively conserved framework regions (FRs). The CDRs are usually aligned by the framework regions and allow binding to a specific epitope. In general, both light and heavy chain variable regions contain, from the N-terminus to the C-terminus, FR-1 (or FR1), CDR-1 (or CDR1), FR-2 (FR2), CDR-2 (CDR2), FR-3 (or FR3), CDR-3 (CDR3), and FR-4 (or FR4). In general, the assignment of amino acids to each domain follows the definitions in Sequences of Proteins of Immunological Interest, Kabat, et al., National Institutes of Health, Bethesda, Md., 5th ed., NIH Publ. No. 91-3242 (1991); Kabat (1978) Adv. Prot. Chem. 32: 1-75; Kabat, et al., (1977) J. Biol. Chem. 252:6609-6616; Chothia, et al., (1987) J Mol. Biol. 196:901-917; or Chothia, et al., (1989) Nature 342:878-883.

[0041] The term "hypervariable region" refers to the amino acid residues of an antibody which are involved in antigen binding. The hypervariable region comprises amino acid residues from the "CDRs" (i.e., VL-CDR1, VL-CDR2 and VL-CDR3 in the light chain variable domain and VH-CDR1, VH-CDR2 and VH-CDR3 in the heavy chain variable domain). See Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (defining antibody CDR regions by sequence). See Chothia and Lesk (1987) J. Mol. Biol. 196: 901-917 (defining antibody CDR regions by structure). The term "framework" or "FR" residues refers to variable region residues other than the hypervariable region residues defined herein as CDR residues.

[0042] Unless otherwise specified, "antibody fragment" or "antigen-binding fragment" refers to an antigen-binding fragment of an antibody, i.e., an antibody fragment that retains the ability to specifically bind to the antigen bound by the full-length antibody, e.g., a fragment that retains one or more CDR regions. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules such as single-chain Fv (ScFv); nanobodies and multispecific antibodies formed from antibody fragments.

[0043] An antibody that binds with specificity to a particular target protein is also described as specifically binding to the particular target protein. This means that the antibody exhibits preferential binding to the target protein over other proteins, but this specificity does not require absolute binding specificity. An antibody is considered to be "specific" for its intended target if the binding of the antibody determines the presence of the target protein in a sample without producing undesirable results such as false positives. An antibody or binding fragment thereof useful in the present invention binds to a target protein with an affinity at least 2-fold, preferably at least 10-fold, more preferably at least 20-fold, and most preferably at least 100-fold, greater than its affinity for a non-target protein. An antibody herein is described as specifically binding to a polypeptide comprising a given amino acid sequence, e.g., the amino acid sequence of a mature human TIGIT molecule, if it binds to a polypeptide comprising a given amino acid sequence, e.g., the amino acid sequence of a mature human TIGIT molecule, and does not bind to a protein lacking that sequence.

[0044] The expressions "pH-dependent binding", "pH-dependent target binding", and "pH-dependent antigen binding" are used interchangeably herein to indicate that the antibody of the present application binds to its target / antigen, i.e., human TIGIT, in a pH-dependent manner. Specifically, the antibody of the present application exhibits a higher binding affinity and / or binding signal to its antigen at a mildly acidic pH (e.g., pH 6.0) typically found in tumor microenvironments, as compared to the binding affinity and / or binding signal at physiological pH (e.g., pH 7.4). Methods for determining the binding affinity and / or binding signal strength of the antibody of the present application are well known in the art and include, but are not limited to, surface plasmon resonance (Biacore) or similar techniques. More specifically, the antibody of the present application exhibits a pH 7.4 / pH 6.0 K of greater than 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more, as measured by surface plasmon resonance (Biacore) or similar techniques. DAlternatively or additionally, the antibody of the present application has an Rmax (RU) value at pH 6.0 that is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold higher than the Rmax (RU) value at pH 7.4, as measured by surface plasmon resonance (Biacore) or similar techniques. The binding affinity of the antibody can be measured at 25°C or 37°C. It has been found that the tumor microenvironment exhibits a relatively more acidic pH than physiological conditions or normal tissues (Zhang et al. Focus on molecular Imaging 2010;Tannock and Rotin et al. Cancer Res 1989). Therefore, the antibody of the present application with the above pH-dependent binding is advantageous as an anti-TIGIT therapeutic agent for targeting TIGIT-positive lymphocytes in the tumor microenvironment with selectivity and lower toxicity associated with peripheral activation of lymphocytes.

[0045] As used herein, the term "human antibody" refers to an antibody that contains only human immunoglobulin protein sequences. A human antibody may contain murine glycochains if produced in a mouse, in a mouse cell, or in a hybridoma derived from a mouse cell. Similarly, a "mouse antibody" or a "rat antibody" refers to an antibody that contains only mouse or rat immunoglobulin protein sequences, respectively.

[0046] The term "humanized antibody" refers to forms of antibodies that contain sequences from non-human (e.g., murine) antibodies and sequences from human antibodies. Such antibodies contain minimal sequences derived from non-human immunoglobulins. Generally, a humanized antibody contains substantially all of at least one, typically two, variable regions, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are from human immunoglobulin sequences. Optionally, the humanized antibody also contains at least a portion of an immunoglobulin constant region (Fc), typically at least a portion of the Fc of a human immunoglobulin. The prefix "hum", "hu", "Hu" or "h" is added to the name of the antibody clone when necessary to distinguish the humanized antibody from the parent rodent antibody. Generally, humanized forms of rodent antibodies contain identical CDR sequences to the parent rodent antibody, but may contain certain amino acid substitutions to increase affinity, to increase the stability of the humanized antibody, or for other reasons.

[0047] The antibodies of the present application have potential therapeutic applications in the treatment of cancer. As used herein, the term "cancer" or "tumor" means or describes a physiological condition in a mammal that is typically characterized by unregulated cell proliferation. Examples of cancer include, but are not limited to, lung cancer (including small cell lung cancer or non-small cell lung cancer), adrenal cancer, liver cancer, gastric cancer, cervical cancer, melanoma, kidney cancer, breast cancer, colon cancer, leukemia, bladder cancer, osteosarcoma, brain tumor, endometrial cancer, head and neck cancer, lymphoma, ovarian cancer, skin cancer, thyroid tumor, or metastatic lesions of cancer.

[0048] Furthermore, the antibodies of the present application have potential therapeutic applications for controlling viral infections and other human diseases that are mechanistically related to immune tolerance or "exhaustion." The term "exhaustion" in the present description refers to the process during long-term chronic viral infections whereby exhaustion of the capacity of immune cells against the infecting virus occurs.

[0049] Pharmaceutical Compositions and Kits In some embodiments, the present application provides compositions, e.g., pharma- ceutically acceptable compositions comprising an anti-TIGIT antibody described herein, formulated with at least one pharma- ceutically acceptable excipient. As used herein, the term "pharma- ceutically acceptable excipient" includes all physiologically compatible solvents, dispersion media, isotonic and absorption delaying agents, and the like. The excipients are suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, spinal, or transdermal administration (such as by injection or infusion).

[0050] The compositions herein may be in a variety of forms. These forms include, for example, liquid, semi-solid, and solid forms, such as liquid solutions (e.g., injectable and infusion solutions), dispersions or suspensions, liposomes, and suppositories. Suitable forms vary depending on the intended mode of administration and therapeutic application. Exemplary suitable compositions are in the form of injectable or infusion solutions. One suitable mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). In some embodiments, the antibody is administered by intravenous infusion or injection. In certain embodiments, the antibody is administered by intramuscular or subcutaneous injection.

[0051] The term "therapeutically effective amount" as used herein means an amount of an antibody sufficient to be effective in treating a disease, disorder, or condition when administered to a subject to treat at least one of the clinical symptoms of the disease or disorder. The "therapeutically effective amount" may vary depending on the antibody, the disease, disorder, and / or symptoms of the disease or disorder, the severity of the disease or disorder and / or symptoms of the disease or disorder, the age of the subject to be treated, and / or the weight of the subject to be treated. The appropriate amount in a given case will be apparent to one skilled in the art or can be determined by routine experimentation. In the case of combination therapy, the "therapeutically effective amount" means the total amount of active ingredients contained in the combination for effective treatment of the disease, disorder, or condition.

[0052] As used herein, a "subject" is a mammal, such as a rodent or a primate, preferably a higher primate, such as a human (e.g., a patient having or suspected of having a disease described herein). EXAMPLES

[0053] Example 1 Generation of anti-TIGIT monoclonal antibody Anti-TIGIT monoclonal antibodies (mAbs) were generated based on conventional hybridoma fusion techniques (de StGroth and Sheidegger, 1980 J Immunol Methods 35:1; Mechetner, 2007 Methods Mol Biol 378:1) with minor modifications. mAbs with high binding activity in enzyme-linked immunosorbent assay (ELISA) and fluorescence-activated cell sorting (FACS) assays were selected and further characterized.

[0054] TIGIT recombinant protein for immunization and binding assays cDNA encoding full-length human TIGIT (SEQ ID NO: 1) was synthesized by Sino Biological (Beijing, China) based on the GenBank sequence (Accession No.: NM_173799) and purchased from Sino Biological. The coding region of the extracellular domain (ECD) of full-length human TIGIT, corresponding to amino acids (AA) 1-141 of SEQ ID NO: 1, was PCR amplified and C-terminally fused to the Fc domain of mouse IgG2a or the Fc domain of human IgG1 heavy chain and cloned into a pcDNA3.1-based expression vector (Invitrogen, Carlsbad, CA, USA). As a result, two recombinant fusion protein expression plasmids, TIGIT-mIgG2a and TIGIT-huIgG1, were obtained, respectively. A schematic diagram of the TIGIT fusion protein is shown in Figure 1. For the production of recombinant fusion proteins, the TIGIT-mIgG2a and TIGIT-huIgG1 plasmids were transiently introduced into 293G cells (developed in-house) and cultured for 7 days in a CO2 incubator equipped with a rotary stirrer. The supernatant containing the recombinant proteins was collected and clarified by centrifugation. TIGIT-mIgG2a and TIGIT-huIgG1 were purified using a Protein A column (Cat. No.: 17127901, GE Life Sciences). Both TIGIT-mIgG2a and TIGIT-huIgG1 proteins were dialyzed against phosphate-buffered saline (DPBS) and stored in aliquots in a -80°C freezer.

[0055] Stable expressing cell lines To establish stable cell lines expressing full-length human TIGIT (huTIGIT) or full-length monkey TIGIT (mkTIGIT, Accession No.: XM_005548101.2), the TIGIT gene (Genescript, Nanjing, China) was cloned into the retroviral vector pFB-Neo (Cat. No.: 217561, Agilent, USA). Dual-tropic retroviral vectors were generated according to a previous protocol (Zhang T, et al. 2005, Blood). The vectors containing huTIGIT and mkTIGIT were transduced into Jurkat cells and NK92MI cells (ATCC, Manassas, VA, USA) to generate Jurkat / huTIGIT and NK92MI / mkTIGIT cell lines, respectively. High-expressing cell lines were selected by culture in medium using G418 and FACS binding assay.

[0056] Immunization, hybridoma fusion and cloning Balb / c mice (HFK BIOSCIENCE, Co.Ltd., Beijing, China) aged 8-12 weeks were immunized intraperitoneally (ip) with 100 μl of antigen mixture containing 10 μg of TIGIT-mIgG2a and water-soluble adjuvant (Cat. No.: KX0210041, KangBiQuan, Beijing, China). The procedure was repeated 3 weeks later. Two weeks after the second immunization, mouse sera were evaluated for TIGIT binding by ELISA and FACS. Ten days after serum screening, mice with the highest anti-TIGIT antibody serum titers were boosted with an intraperitoneal injection of 50 μg of TIGIT-mIgG2a. Three days after the boost, spleen cells were isolated and fused to mouse myeloma cell line SP2 / 0 cells (ATCC) using standard techniques (1977 Somat Cell Genet, 3:231).

[0057] Assessment of TIGIT binding activity of antibodies by ELISA and FACS First, the supernatants of hybridoma clones were screened by ELISA as described in Methods in Molecular Biology (2007) 378:33-52 with some modifications. Briefly, TIGIT-huIgG1 protein was coated onto 96-well plates. HRP-conjugated anti-mouse IgG antibody (Cat. No.: 7076S, Cell Signaling Technology, USA) and substrate (Cat. No.: 00-4201-56, eBioscience, USA) were used to develop color absorption signals at a wavelength of 450 nm, which were measured using a plate reader (SpectraMax Paradigm, Molecular Device, USA). ELISA-positive clones were further verified by FACS using either NK92MI / huTIGIT cells or NK92mi / mkTIGIT cells as above. TIGIT-expressing cells (10 5 Cells / well) were incubated with ELISA-positive hybridoma supernatants and then bound with Alexa Fluro-647-labeled goat anti-mouse IgG antibody (Cat. No.: A0473, Beyotime Biotechonlogy, China). The fluorescence of the cells was quantified using a flow cytometer (Guava easyCyte 8HT, Merck-Millipore, USA).

[0058] Conditioned media from hybridomas that showed positive signals in both ELISA and FACS screening were subjected to functional assays to identify antibodies with good functional activity in human immune cell-based assays (see below). Antibodies with desirable functional activity were further subcloned and characterized.

[0059] Subcloning of hybridomas and adaptation to serum-free or low-serum medium After primary screening by ELISA, FACS and functional assays as above, positive hybridoma clones were subcloned by limiting dilution. Based on ELISA and FACS screening from each plate, three positive subclones were selected and characterized by functional assays. The top antibody subclones verified by functional assays were adapted for growth in CDM4MAb medium (Cat. No.: SH30801.02, Hyclone, USA) containing 3% FBS.

[0060] Monoclonal antibody expression and purification Hybridoma cells or 293G cells transiently transfected with antibody expression plasmids (Cat. No.: R79007, Invitrogen) were cultured in either CDM4MAb medium (Cat. No.: SH30801.02, Hyclone) or Freestyle™ 293 Expression Medium (Cat. No.: 12338018, Invitrogen) and incubated at 37°C for 5-7 days in a CO2 incubator. Conditioned medium was collected by centrifugation and filtered through a 0.22 μm membrane before purification. Supernatants containing murine or recombinant antibodies were applied and bound to a Protein A column (Cat. No.: 17127901, GE Life Sciences) according to the manufacturer's instructions. This procedure routinely yielded antibodies with purity greater than 90%. Protein A affinity purified antibodies were dialyzed against PBS or further purified using a HiLoad 16 / 60 Superdex200 column (Cat. No.: 17531801, GE Life Sciences) to remove aggregates. Protein concentration was determined by measuring absorbance at 280 nm. Final antibody preparations were stored in aliquots in a -80°C freezer.

[0061] Example 2 Cloning and sequence analysis of TIGIT antibodies Murine hybridoma clones were harvested and total cellular RNA was prepared using Ultrapure RNA kit (Cat. No.: 74104, QIAGEN, Germany) according to the manufacturer's protocol. First-strand cDNA was synthesized using a cDNA synthesis kit from Invitrogen (Cat. No.: 18080-051). PCR amplification of nucleotide sequences encoding the heavy chain variable region (Vh) and kappa chain variable region (Vκ) of murine mAb was performed using a PCR kit (Cat. No.: CW0686, CWBio, Beijing, China). Oligo primers used for cloning Vh and Vκ antibody cDNA were synthesized by Invitrogen (Beijing, China) based on previously reported sequences (Brocks et al. 2001 Mol Med 7:461). The PCR products were then subcloned into pEASY-Blunt cloning vector (Cat. No.: C B101-02, TransGen, China) and sequenced by Genewiz (Beijing, China). The amino acid sequences of the Vh and Vκ regions were deduced from the DNA sequencing results.

[0062] Murine mAbs were analyzed by comparing sequence homology and grouping based on sequence similarity (Figure 2). Sequence annotation and internet-based sequence analysis ( http: / / www.imgt.org / IMGT_vquest / share / textes / index.html ) to define the complementarity determining regions (CDRs). The amino acid sequences of mu1217 (Vh and Vκ) of a representative top clone are shown in Table 1 (SEQ ID NOs: 9 and 11). The CDR sequences of mu1217 are shown in Table 2 (SEQ ID NOs: 3 to 8).

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[0064] JPEG2025024708000003.jpg40161

[0065] Example 3 Affinity determination of purified murine anti-TIGIT antibodies by SPR TIGIT antibodies, which have high binding activity in ELISA and FACS and strong functional activity in cell-based assays (described in Examples 1 and 2), were characterized for their binding kinetics by SPR assay using a BIAcore™ T-200 (GE Life Sciences). Briefly, anti-human IgG antibodies were immobilized on an activated CM5 biosensor chip (Cat. No.: BR100530, GE Life Sciences). Human Fc-tagged TIGIT was flowed over the chip surface and captured by the anti-human IgG antibodies. Then, serial dilutions (0.12 nM to 10 nM) of purified murine antibodies were flowed over the chip surface, and the changes in surface plasmon resonance signals were analyzed to calculate the association rate (k) and dissociation rate (k) using a one to one Langmuir binding model (BIA Evaluation Software, GE Life Sciences). The equilibrium dissociation constant (K D ) was calculated as the ratio koff / kon. The binding affinity profiles of the top mAbs, including mu1217, mu1257, mu1226 and mu242, are shown in Figure 3 and Table 3.

[0066] JPEG2025024708000004.jpg56161

[0067] Example 4 Humanization of murine anti-human TIGIT mAb mu1217 mAb humanization and design For humanization of mu1217, human germline IgG genes were searched for sequences with high homology to the cDNA sequence of mu1217 variable region by blasting the human immunoglobulin gene databases at IMGT (http: / / www.imgt.org / IMGT_vquest / share / textes / index.html) and NCBI (http: / / www.ncbi.nlm.nih.gov / igblast) websites. Human IGVH and IGVκ genes, which are present in the frequent human antibody repertoire (Glanville 2009 PNAS 106:20216-20221) and have high homology to mu1217, were selected as templates for humanization.

[0068] Humanization was performed by CDR grafting (Methods in Molecular Biology, Vol 248: Antibody Engineering, Methods and Protocols, Humana Press). A humanized antibody (hu1217) was also designed in the format of human IgG1mf using an expression vector developed in-house. In the first stage of humanization, the mutation of murine amino acid residues in the framework regions to human amino acid residues was guided by the simulated 3D structure. In the first version of humanized antibody 1217 (hu1217-1-1, which has six CDRs with amino acid sequences of SEQ ID NO:3, 13, 5 (heavy chain CDRs) and SEQ ID NO:6, 7, 8 (light chain CDRs), a heavy chain variable region with amino acid sequence of SEQ ID NO:14 and encoded by the nucleotide sequence of SEQ ID NO:15, and a light chain variable region with amino acid sequence of SEQ ID NO:16 and encoded by the nucleotide sequence of SEQ ID NO:17), structurally important murine framework residues were maintained to maintain the canonical structure of the CDRs. Specifically, the CDRs of mu1217Vκ (SEQ ID NOs: 6-8) were grafted into the framework of the human germline variable gene IGVκ3-15, retaining one murine framework residue (V58), thereby obtaining the humanized Vκ sequence of hu1121 (amino acid sequence SEQ ID NO: 16, nucleotide sequence SEQ ID NO: 17). The N-terminus of H-CDR2 (SEQ ID NO: 4), H-CDR1 (SEQ ID NO: 3) and H-CDR3 (SEQ ID NO: 5) of mu1217 Vh were grafted into the framework of two murine frameworks (T of SEQ ID NO: 10). 24 and I 37 ) residues were retained and grafted into the framework of human germline variable gene IGVH3-7. Only the N-terminal half of Kabat H-CDR2 was grafted in the humanized variant of hu1217, since only the N-terminal half of Kabat H-CDR2 was predicted to be important for antigen binding according to the simulated 3D structure. The amino acid and nucleotide sequences of the resulting humanized Vh sequence of hu1217-1-1 are shown in SEQ ID NO: 14 and SEQ ID NO: 15, respectively.

[0069] hu1217-1-1 was constructed as a human full-length antibody format using an in-house developed expression vector containing the constant region and kappa chain of a human IgG1 variant called IgG1mf (SEQ ID NO: 18), respectively, with easily adapted subcloning sites. Expression and preparation of hu1217-1-1 antibody was achieved by co-transfecting the above two constructs into 293G cells and purifying using a Protein A column (Cat. No.: 17543802, GE Life Sciences). The purified antibody was concentrated to 0.5-5 mg / mL in PBS and stored in aliquots in a -80 °C freezer.

[0070] Based on the template of hu1217-1-1, several single mutations were made to convert the conserved murine residues in the framework regions of Vκ to the corresponding human germline residues. The single mutations included V58I in Vκ and I24V and I37V in Vh. The resulting hu1217-2A-1 (T24A), hu1217-2B-1 (I37V) and hu1217-1-2a (V58I) all had similar binding and functional activities as hu1217-1-1. All humanized mutants were generated using primers containing mutations at specific positions and a site-directed mutagenesis kit (Cat. No.: FM111-02, TransGen, Beijing, China). The desired mutations were verified by sequence analysis. These hu1217-derived mutant antibodies were tested in the binding and functional assays described above.

[0071] To improve the molecular and biophysical properties for human therapeutic use, the hu1217 antibody was further engineered by introducing mutations into the CDR and framework regions. Considerations included amino acid composition, thermal stability (Tm), surface hydrophobicity, and isoelectronic point (pI) while maintaining functional activity.

[0072] Taken together, from the above mutation process, a well-designed version of humanized monoclonal antibody, hu1217-2-2 (SEQ ID NO: 3, 5-8, 13 and 19-21), was obtained and characterized in detail. The results shown for both hu1217-2-2 and hu1217-1-1 were very similar in terms of binding affinity and functional activity, such as inhibition of TIGIT-mediated downstream signaling regulation.

[0073] For affinity determination, the antibodies were captured by anti-human Fc surface and used in affinity assays based on surface plasmon resonance (SPR) technology. The results of the binding profiles of anti-TIGIT antibodies determined by SPR are summarized in Table 4. hu1217-2-2 and hu1217-1-1 showed very similar binding profiles with average dissociation constants of 0.415 nM and 0.266 nM, respectively. This binding profile is also close to that of ch1217.

[0074] JPEG2025024708000005.jpg54161

[0075] JPEG2025024708000006.jpg47161

[0076] All the above humanized antibodies were also validated for functional activity on primary human immune cells isolated from healthy donors (described in Example 7).

[0077] Example 5 Binding affinities of different forms of 1217 to native TIGIT To evaluate the binding activity of anti-TIGIT antibodies to native TIGIT on live cells, NK92mi cells were engineered to overexpress human TIGIT. Live NK92mi / TIGIT cells were seeded in 96-well plates and incubated with serial dilutions of anti-TIGIT antibodies. Goat anti-human IgG was used as a secondary antibody to detect antibody bound to the cell surface. The EC of dose-dependent binding to human native TIGIT was determined by fitting the dose-response data to a four-parameter logistic model with GraphPad Prism.50 As shown in Figure 4 and Table 6, the humanized 1217 antibodies, hu1217-1-1 and hu1217-2-2, showed good binding affinity to native TIGIT in live cells.

[0078] JPEG2025024708000007.jpg47161

[0079] Example 6 Anti-TIGIT antibodies block the interaction of TIGIT with its ligands PVR and PVR-L2 TIGIT binds to PVR with high affinity (Kd: ∼1 nM), thereby competing with the CD266-PVR interaction (Yu et al., 2009).

[0080] To determine whether anti-TIGIT antibodies can block TIGIT-PVR and TIGIT-PVR-L2 interactions, HEK293 cells were engineered to express high levels of PVR or PVR-L2. The resulting cell lines were named HEK293 / PVR and HEK293 / PVR-L2, respectively. Binding of soluble TIGIT (TIGIT-mIgG2a fusion protein) to PVR or PVR-L2 was determined by flow cytometry (Figure 5A). Blockade of TIGIT-ligand interaction was quantitatively measured by adding serially diluted anti-TIGIT antibodies. As shown in FIG. 5B, hu1217-2-2 / IgG1 (a humanized version containing a wild-type IgG1 Fc region and the same VH and VL sequences as hu1217-2-2 / IgG1mf) and hu1217-2-2 / IgG1mf had IC 50 The IC of hu1217-2-2 / IgG1 and hu1217-2-2 / IgG1mf in blocking the TIGIT-PVR-L2 interaction was 0.01, 0.01, and 0.01, respectively. 50 are 0.25 μg / mL and 0.18 μg / mL, respectively.

[0081] Example 7 Activation of CMV-specific human T cells by anti-TIGIT antibodies The functional activity of the TIGIT antibody was further evaluated using naturally derived T cells that recognize the human CMV PP65 peptide (NLVPMVATV, 495-503, HLA-A2.1-restricted) (Boeckh M, Boeckh M and Geballe AP, 2011 J Clin Invest. 121:1673-80). Briefly, TIGIT antibodies were incubated with PP65 peptide (>98% purity, synthesized by GL Biochem, Shanghai) in complete RPMI with 10% FBS for 1 week, and then transfected with HLA-A2.1 + PBMCs from healthy donors were simulated. PP65-primed PBMCs were used as effector cells. Prior to the assay, target cells, HCT116 cells (HLA-A2.1 + , 10 4 ) were pulsed with pp65 peptide (5 μg / mL) for 30 min and co-cultured overnight with an equal number of pp65-sensitized PBMCs in 96-well plates in the presence or absence of anti-TIGIT antibody or blank control (medium only). As shown in Figure 6, hu1217-2-2 / IgG1 promoted pp65-specific T cells to secrete IFN-γ into cell culture supernatants in a dose-dependent manner in both donors.

[0082] Example 8 Anti-TIGIT antibody enhanced NK cell-mediated cytotoxicity TIGIT is known to be constitutively expressed at relatively high levels in natural killer (NK) cells, and the interaction between TIGIT and its ligands inhibits NK cell-mediated cytotoxicity (Wang F, et al. 2015 Eur. J. Immunology 45:2886-97;Stanietsky N et al., 2009 Proc Natl Acad Sci USA 106:17858-63).

[0083] To confirm whether humanized anti-TIGIT antibodies can promote NK cell-mediated cytotoxicity, we engineered the NK cell line NK92MI (NK92MI / TIGIT-DNAM-1) to co-express both TIGIT and DNAM-1 receptors as effector cells by retroviral transduction according to the previously described protocol (Zhang et al., 2006 Cancer Res. 66: 5927-5933). Similarly, we established the PVR-expressing lung cancer cell line SK-MES-1 / PVR as a target.

[0084] The cytotoxicity of NK-MI / TIGIT-DNAM-1 cells against SK-MES-1 / PVR cells was determined by LDH release assay using the CytoTox96 non-radioactive cytotoxicity assay kit (Promega, Madison, WI). Briefly, NK92MI / TIGIT-DNAM-1 cells (8 × 10 5 ) into SK-MES-1 / PVR cells (2 × 10 4 ) were co-cultured. Specific lysis in the LDH release assay was determined using the following formula: Percentage of specific lysis = [(experimental - effector spontaneous - target spontaneous) / (target maximum - target spontaneous)] x 100 The results showed that the anti-TIGIT antibody hu1217-2-2 / IgG1mf increased NK cell death in a dose-dependent manner (EC 50 : 0.185 μg / mL) (Figure 7).

[0085] Example 9 Anti-TIGIT antibodies can reduce the surface expression of TIGIT receptors through FcγR-mediated trogocytosis Trogocytosis is a phenomenon in which cell surface molecules are transferred from donor cells to acceptor cells (Joly E, et al. 2003 Nat. Immunol;Machlenkin A, et al. 2008 Cancer Res.;Beum PV et al. 2008 J. Immunol;Rossi EA, et al. 2013 Blood). Antibody-induced trogocytosis via Fcγ receptors (FcγRs) causes down-modulation of receptors on the cell surface (Carlsten M, et al. 2016 Clin Cancer Res;Beum PV, et al. 2011 J. Immunology). Thus, down-regulation of target receptors by trogocytosis can cause reduced signaling. Considering these observations, hu1217-2-2 / IgG1 is a potential candidate for the antibody-induced trogocytosis of FcγRs. + It is possible that in the presence of TIGIT cells, trogocytosis of the TIGIT receptor is induced, resulting in lower surface expression. To address this possibility, we investigated the expression of various FcγRs (FcγRIIA H131 , FcγRIIB, FcγRIIIA V158 Jurkat / TIGIT cells were cultured overnight with HEK cells expressing hu1217-2-2 / IgG1 (hu1217-2-2 / IgG1mf and a humanized antibody containing the same VL and VH as the wild-type IgG1 Fc region) or hu1217-2-2 / IgG1mf. Surface expression of the TIGIT receptor was determined by SA-APC (Biolegend). As shown in Figure 8, hu1217-2-2 / IgG1, but not hu1217-2-2 / IgG1mf, significantly reduces TIGIT surface expression compared to the negative control human IgG-treated cells. This indicates that the reduction of surface TIGIT in Jurkat / TIGIT cells is dependent on FcγR binding. In addition, the presence of 10% human serum (containing high levels of endogenous IgG) suppressed the FcγRIIA binding of the TIGIT receptor. H131 Mediated trogocytosis or FcγRIIIA V158Although FcγRIIB can partially reduce TIGIT-mediated trogocytosis, it cannot reduce FcγRIIB-mediated trogocytosis. This suggests that FcγRIIB may play an important role in reducing TIGIT surface expression by anti-TIGIT mAb (e.g., hu1217-2-2 / IgG1wt) in vivo. These observations are consistent with previous studies (Ganesan LP, et al. 2012 J Immunol 189:4981-8; Taylor RP, et al. 2015 Blood 125:762-6).

[0086] Example 10 ADCC and CDC effector functions of anti-TIGIT antibodies The ability of anti-TIGIT antibodies to induce ADCC and CDC in human primary PBMCs was determined using the in vitro assays described below.

[0087] ADCC using human PBMCs as target cells TIGIT antibody is + To determine whether or not the antibody can induce ADCC of T cells, a flow cytometry-based ADCC assay was set up. V158 The assay effector cell line, NK92MI / CD16V cells, was generated from NK92MI cells (ATCC) by co-transduction of expression plasmids containing FcRγ cDNA (V158 allele) and FcRγ cDNA. Human PBMCs from healthy donors were stimulated with PHA (1 μg / ml) to upregulate TIGIT expression. As shown in Figure 9, CD4 + Effector (CD3 + CD4 + Foxp3 - )T cells, CD8 + T cells, and regulatory T cells (CD4 + Foxp3 +) expressed high amounts of TIGIT. These activated PBMCs (from three healthy donors) were used as target cells. Fluorescent dye CFSE-labeled NK92MI / CD16V cells (5 × 10 4 ) were co-cultured with the same number of target cells. Compared with human IgG and hu1217-2-2 / IgG1mf, hu1217-2-2 / IgG1wt can induce a modest reduction in Treg cells via ADCC. However, the reduction in total T cells and CD8 + No significant ADCC effect was observed in T cells (Figure 9).

[0088] CDC using human PBMCs as target cells We determined whether hu1217-2-2 / IgG1mf and hu1217-2-2 / IgG1wt induce CDC by using preactivated human PBMCs and fresh autologous serum from healthy donors. CDC-induced cell lysis was determined with a Celltiter glo assay kit (Promega, Beijing, China). Briefly, PBMCs were preactivated with PHA (10 μg / mL) for 3 days and incubated overnight at 37°C in RPMI1640 and autologous serum (15%) and anti-TIGIT antibody or control antibody (0.01-100 μg / mL). CDC-induced cell death was assessed by the decrease in ATP released from viable cells after cell lysis at the end of the reaction. Anti-MHC-I A, B, and C were used as positive controls. Fluorescence readings were taken using a 96-well fluorometer (PHERA Star FS, BMG LABTECH), and CDC activity was calculated from the relative fluorescence unit (RFU) readings by the following formula: %CDC activity=[(RFU test-RFU background) / (RFU in total cell lysis-RFU background)]×100 The results of that experiment showed that both hu1217-2-2 / IgG1mf and hu1217-2-2 / IgG1wt had no detectable CDC in PBMCs isolated from two different donors. In contrast, the positive control antibody, anti-MHC-I, induced significant CDC activity (Figure 10).

[0089] Example 11 pH-dependent binding affinity of Hu1217-2-2 / IgG1 To investigate whether pH affects the binding properties of hu1217-2-2 / IgG1, target binding SPR studies were performed in running buffers of pH 7.4 and pH 6.0 for comparison. Antibody hu1217-2-2 / IgG1 was immobilized on a CM5 chip (GE). Serial dilutions of TIGIT-his were run over the immobilized hu1217-2-2 / IgG1 in running buffer HBS at pH 7.4 or pH 6.0.

[0090] As shown by the results in Table 7 below, hu1217-2-2 / IgG1 exhibits a higher binding affinity (K ) for human TIGIT at pH 6.0 (an acidic pH equivalent to the pH of the tumor microenvironment) compared to data obtained at pH 7.4 (physiological pH). D ) and stronger binding signals (Rmax). These results indicate that hu1217-2-2 / IgG1 can more selectively target TIGIT-positive lymphocytes in the tumor microenvironment with less potential toxicity associated with activation of peripheral lymphocytes, demonstrating the potential advantages of antibodies as therapeutic agents targeting TIGIT-positive lymphocytes in the tumor environment.

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Claims

1. (a) a heavy chain variable region (VH) comprising one, two or three CDR amino acid sequences selected from SEQ ID NOs: 3, 4, 5 and 13; and / or (b) a light chain variable region (VL) comprising one, two or three CDR amino acid sequences selected from SEQ ID NOs: 6, 7 and 8; An antibody or antigen-binding fragment thereof capable of binding to human TIGIT, comprising:

2. (a) a heavy chain variable region (VH) comprising a VH-CDR1 amino acid sequence of SEQ ID NO: 3, a VH-CDR2 amino acid sequence of SEQ ID NO: 4, and a VH-CDR3 amino acid sequence of SEQ ID NO: 5, and a light chain variable region (VL) comprising a VL-CDR1 amino acid sequence of SEQ ID NO: 6, a VL-CDR2 amino acid sequence of SEQ ID NO: 7, and a VL-CDR3 amino acid sequence of SEQ ID NO: 8; or (b) a heavy chain variable region (VH) comprising the VH-CDR1 amino acid sequence of SEQ ID NO: 3, the VH-CDR2 amino acid sequence of SEQ ID NO: 13, and the VH-CDR3 amino acid sequence of SEQ ID NO: 5, and a light chain variable region (VL) comprising the VL-CDR1 amino acid sequence of SEQ ID NO: 6, the VL-CDR2 amino acid sequence of SEQ ID NO: 7, and the VL-CDR3 amino acid sequence of SEQ ID NO:

8. The antibody or antigen-binding fragment thereof of claim 1 .

3. The antibody or antigen-binding fragment thereof of claim 1 , wherein the antibody is a humanized antibody molecule.

4. The antibody or antigen-binding fragment thereof of claim 1, comprising a heavy chain variable region having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 9, 14 or 19.

5. The antibody or antigen-binding fragment thereof of claim 1, comprising a light chain variable region having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 11, 16 or 21.

6. (a) a heavy chain variable region having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:9, and a light chain variable region having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:11; (b) a heavy chain variable region having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:9, and a light chain variable region having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:16; (c) a heavy chain variable region having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:9, and a light chain variable region having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:21; (d) a heavy chain variable region having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 14, and a light chain variable region having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 11; (e) a heavy chain variable region having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 14, and a light chain variable region having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 16; (f) a heavy chain variable region having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 14, and a light chain variable region having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 21; (g) a heavy chain variable region having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 19, and a light chain variable region having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 11; (h) a heavy chain variable region having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 19, and a light chain variable region having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 16; or (i) a heavy chain variable region having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 19, and a light chain variable region having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 21; The antibody or antigen-binding fragment thereof of claim 1 .

7. (a) a heavy chain variable region having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:9, and a light chain variable region having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:11; (b) a heavy chain variable region having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 14, and a light chain variable region having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 16; or (c) a heavy chain variable region having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 19, and a light chain variable region having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 21; The antibody or antigen-binding fragment thereof of claim 1 .

8. (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:9, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:11; (b) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 14, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 16; or (c) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 19, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 21; The antibody or antigen-binding fragment thereof of claim 1 .

9. The antibody or antigen-binding fragment thereof of any one of claims 1 to 8, wherein the antibody comprises one or more of (a) to (c): (a) a heavy chain having a threonine to alanine mutation at position 24 of SEQ ID NO: 14; (b) a heavy chain having an isoleucine to valine mutation at position 37 of SEQ ID NO:14; (c) a light chain having a valine to isoleucine mutation at position 58 of SEQ ID NO:

16.

10. The antigen-binding fragment is Fab, F(ab') ２ 2. The antibody or antigen-binding fragment thereof of claim 1, which is a single-chain Fv or a single-chain Fv (ScFv).

11. The antibody or antigen-binding fragment thereof according to claim 1, comprising a heavy chain constant region or a variant thereof of the IgG1, IgG2, IgG3 or IgG4 subclass, and a light chain constant region or a variant thereof of the κ or λ type.

12. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 11 and a pharma- ceutically acceptable excipient.

13. 13. The pharmaceutical composition of claim 12, further comprising a second therapeutic agent.

14. A method of stimulating an immune response in a subject, comprising administering to a subject in need thereof an antibody or antigen-binding fragment thereof according to any one of claims 1 to 11 in an amount effective to stimulate an immune response.

15. A method for treating cancer or tumor, comprising administering to a subject in need thereof an antibody or antigen-binding fragment thereof according to any one of claims 1 to 11 in an amount effective for treating the cancer or tumor.

16. 16. The method of claim 15, wherein the antibody or antigen-binding fragment thereof is administered in combination with a second therapeutic agent or treatment, the second therapeutic agent or treatment being selected from chemotherapy, targeted therapy, oncolytic drugs, cytotoxic agents, immunotherapy, cytokines, surgical treatment, radiation treatment, activators of costimulatory molecules, inhibitors of inhibitory molecules, vaccines, or cellular immunotherapy.

17. A method of treating an infectious disease comprising administering to a subject in need thereof an antibody of claim 1 in an amount effective to treat the infectious disease.

18. 18. The method of claim 17, wherein the infectious disease is a viral infection.

19. Use of an anti-TIGIT antibody molecule according to any one of claims 1 to 11 in the manufacture of a medicament for the treatment of cancer, tumor or infectious disease.

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