Methods of treating cancer using anti-TIGIT antibodies in combination with anti-PD1 antibodies

Abstract

Methods are provided for treating cancer or augmenting, enhancing or stimulating an immune response using antibodies that specifically bind to TIGIT (T cell immunoreceptor with Ig and ITIM domains, WUCAM or Vstm3) and antigen-binding fragments thereof, in combination with anti-PD1 antibodies.

Description

[Technical field]

[0001] The present application relates to an antibody that specifically binds to TIGIT (T cell immunoreceptor with Ig and ITIM domains) in combination with an anti-PD1 antibody for the treatment of cancer. [Background technology]

[0002] TIGIT (T cell immunoglobulin and ITIM domain) is a type I transmembrane protein and a member of the CD28 family of proteins that play an important 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).

[0003] 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), where the first 21 amino acids constitute 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), and contains 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, and an 82 amino acid cytoplasmic domain containing 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 only 59% and 87% aa sequence identity with mouse and cynomolgus monkey, respectively.

[0004] TIGIT is expressed on T cells (including activated T cells, memory T cells, regulatory T (Treg) cells, and follicular T helper (Tfh) cells) and 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).

[0005] So far, two TIGIT ligands have been identified: CD155 (also known as poliovirus receptor or PVR) and CD112 (poliovirus receptor-related 2, PVRL2, also known as nectin-2). These ligands are mainly expressed on APCs (such as dendritic cells and macrophages) and 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 initiates inhibitory signaling in immune cells upon binding to its ligands CD155 and CD112. The binding affinity of TIGIT for CD155 (Kd: approx. 1 nM) is much higher than that for CD112, and it remains to be elucidated whether the TIGIT:CD112 interaction is functionally relevant in mediating inhibitory signals. The costimulatory receptor CD226 (DNAM-1) binds the same ligand with lower affinity (Kd: approx. 100 nM) but transmits positive signals (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).

[0006] TIGIT can inhibit immune responses through various mechanisms. First, the interaction between TIGIT and PVR on dendritic cells (DCs) can result in "reverse signaling" in DCs, leading to upregulation of IL-10 and reduced IL-12 secretion, thereby inhibiting T cell activation (Yu X, et al. Nat Immunol. 2009 10:48-57). Second, TIGIT binds to CD155 with higher affinity, thereby competing with DNAM-1-CD155 interaction. Third, direct ligation of TIGIT on T cells can downregulate TCR-mediated activation, and the subsequent proliferation and engagement 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 Tregs is associated with a highly activated and suppressive phenotype in tumor tissues, and TIGIT signaling in Tregs may be favorable for Treg stability (Joller N, et al. Immunity 2014 40:569-81; Kurtulus S, et al. J Clin Invest. 2015 125:4053-4062).

[0007] TIGIT possesses an immunoglobulin tail tyrosine (ITT)-like motif followed by an immunoreceptor tyrosine-based inhibitory motif (ITIM) 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), thus providing a mechanism by which TIGIT may intrinsically transmit inhibitory signals to attenuate activating signals.

[0008] Upregulation of TIGIT expression in tumor infiltrating lymphocytes (TILs) and peripheral blood mononuclear cells (PBMCs) has been reported in many types of cancer, such as lung (Tassi, et al., Cancer Res. 2017 77:851-861), esophagus (Xie J, et al., Oncotarget 2016 7:63669-63678), breast (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 outcomes (Kong Y et al., Clin Cancer Res.2016 22:3057-66). Upregulation of TIGIT signaling plays an important role not only in immune tolerance to cancer but also in 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 may restore the functional activity of immune cells such as T cells, NK cells, and dendritic cells (DCs), thereby enhancing immunity against cancer or chronic viral infection.

[0009] Monoclonal antibodies targeting either PD1 or PDL1 can block this interaction and boost the immune response against cancer cells. These antibodies have been shown to be useful in the treatment of several types of cancer, including melanoma of the skin, non-small cell lung cancer (NSCLC), kidney cancer, bladder cancer, head and neck cancer, and Hodgkin's lymphoma. Cancer cells in most patients who do not respond to single-agent checkpoint inhibitors escape through innate mechanisms that allow cancer cells to grow and survive. As a result, the disease progresses at a rate consistent with its natural history. However, unlike intrinsic resistance, late relapses have emerged in patients who previously had clinical responses after long-term follow-up in clinical trials, suggesting the emergence of acquired resistance (Jenkins et al.,Br. J.Cancer 118,9-16 2018).

[0010] Therefore, the combination of anti-TIGIT and anti-PD1 antibodies may rescue immune cells from tolerance and induce efficient immune responses in the treatment of cancer or chronic viral infections. Summary of the Invention [Means for solving the problem]

[0011] The present disclosure relates to methods of treating cancer by administering an anti-TIGIT antibody in combination with an anti-PD1 antibody.

[0012] In certain embodiments, the anti-TIGIT antibody or antigen-binding fragment 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 or 13, or a variant thereof comprising one or more conservative substitutions, e.g., one or two conservative substitutions, in the amino acid sequence of SEQ ID NO: 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 NO: 6, 7 or 8, or a variant thereof comprising one or more conservative substitutions, e.g., one or two conservative substitutions, in the amino acid sequence of SEQ ID NO: 6, 7, or 8.

[0013] In a more specific embodiment, the anti-TIGIT antibody or antigen-binding fragment thereof has 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 anti-TIGIT 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, 19, or a 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, 19. In one embodiment, the sequence difference is in the framework region. 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 or 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, or 24, or a 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 difference is in the framework region. 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, or 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 1×10 -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 the IgG1, IgG2, IgG3 or IgG4 subclass or a variant thereof, and a light chain constant region of the kappa or lambda 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] A method for treating cancer, comprising administering to a subject an effective amount of an anti-TIGIT antibody or antigen-binding fragment thereof in combination with an anti-PD1 antibody or antigen-binding fragment thereof.

[0019] Binds specifically to human TIGIT and: (i) a heavy chain variable region comprising (a) an HCDR (heavy chain complementarity determining region) 1 of SEQ ID NO: 3, (b) an HCDR2 of SEQ ID NO: 4, and (c) an HCDR3 of SEQ ID NO: 5; and (d) an LCDR (light chain complementarity determining region) 1 of SEQ ID NO: 6, (e) an LCDR2 of SEQ ID NO: 7, and (f) an LCDR3 of SEQ ID NO: 8; or (ii) a heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 3, (b) an HCDR2 of SEQ ID NO: 13, and (c) an HCDR3 of SEQ ID NO: 5; and a light chain variable region comprising (d) an LCDR1 of SEQ ID NO: 6, (e) an LCDR2 of SEQ ID NO: 7, and (f) an LCDR3 of SEQ ID NO: 8, in combination with an anti-PD1 antibody.

[0020] The TIGIT antibody or antigen-binding fragment thereof is (i) a heavy chain variable region (VH) comprising SEQ ID NO: 19, and a light chain variable region (VL) comprising SEQ ID NO: 21; (ii) a heavy chain variable region (VH) comprising SEQ ID NO: 14, and a light chain variable region (VL) comprising SEQ ID NO: 16; or (iii) a heavy chain variable region (VH) comprising SEQ ID NO: 9, and a light chain variable region (VL) comprising SEQ ID NO: 11.

[0021] The anti-PD1 antibody comprises an antibody or antigen-binding fragment thereof that specifically binds to human PD1, and A method comprising: (a) a heavy chain variable region comprising an HCDR1 of SEQ ID NO: 25, (b) an HCDR2 of SEQ ID NO: 26, and (c) an HCDR3 of SEQ ID NO: 27; and (d) a light chain variable region comprising an LCDR1 of SEQ ID NO: 28, (e) an LCDR2 of SEQ ID NO: 29, and (f) an LCDR3 of SEQ ID NO: 30.

[0022] The method, wherein the anti-PD1 antibody or antigen-binding fragment thereof specifically binds to human PD1 and comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 32 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 33.

[0023] The method, wherein the anti-PD1 antibody comprises an IgG4 constant domain comprising SEQ ID NO:35.

[0024] The method, wherein the anti-TIGIT antibody is an antibody fragment selected from the group consisting of Fab, Fab'-SH, Fv, scFv, and (Fab')2 fragments.

[0025] The method, wherein the anti-PD1 antibody is an antibody fragment selected from the group consisting of Fab, Fab'-SH, Fv, scFv, and (Fab')2 fragments.

[0026] The method, wherein the cancer is selected from the group consisting of breast cancer, colon cancer, pancreatic cancer, head and neck cancer, gastric cancer, renal cancer, liver cancer, small cell lung cancer, non-small cell lung cancer, ovarian cancer, skin cancer, mesothelioma, lymphoma, leukemia, myeloma, and sarcoma.

[0027] The method, wherein the small cell lung cancer is limited-stage small cell lung cancer.

[0028] The method, wherein the cancer is non-small cell lung cancer.

[0029] The method, wherein said head and neck cancer is nasopharyngeal carcinoma.

[0030] The method, wherein the esophageal cancer is esophageal squamous cell carcinoma (ESCC).

[0031] The method, wherein the cancer is uterine cancer.

[0032] The method, wherein said gastric cancer is gastric cancer or gastroesophageal junction cancer.

[0033] The method, wherein the cervical cancer is recurrent or metastatic cervical cancer.

[0034] The method, wherein the skin cancer is basal cell carcinoma.

[0035] The method, wherein the cancer is pancreatic cancer.

[0036] The method further comprising administering chemotherapy.

[0037] The method, wherein said chemotherapy is chemoradiotherapy.

[0038] The method, wherein the anti-PD1 antibody is administered at 200 mg every three weeks.

[0039] The method, wherein the anti-TIGIT antibody is administered in the range of 50 mg to 900 mg.

[0040] The method, wherein the anti-TIGIT antibody is administered at 50 mg every three weeks.

[0041] The method, wherein the anti-TIGIT antibody is administered at 150 mg every three weeks.

[0042] The method, wherein the anti-TIGIT antibody is administered at 450 mg every three weeks.

[0043] The method, wherein the anti-TIGIT antibody is administered at 900 mg every three weeks.

[0044] The method, wherein the anti-TIGIT antibody is administered at 1800 mg every three weeks. [Brief description of the drawings]

[0045] [Figure 1] Schematic diagram of TIGIT-mIgG2a (top) and TIGIT-huIgG1 (bottom). TIGIT ECD: TIGIT extracellular domain. N: N-terminus. C: C-terminus. [Figure 2A] Phylogenetic tree of anti-TIGIT antibody Vh (A) and Vk (B) regions. Vh and Vk sequences of candidate anti-TIGIT antibodies were aligned using Megalign software from DNASTAR. Sequence homology is displayed in a phylogenetic tree. [Figure 2B] Phylogenetic tree of anti-TIGIT antibody Vh (A) and Vk (B) regions. Vh and Vk sequences of candidate anti-TIGIT antibodies were aligned using Megalign software from DNASTAR. Sequence homology is displayed in a phylogenetic tree. [Diagram 3]Affinity measurement of purified mouse anti-TIGIT antibody by surface plasmon resonance (SPR). [Figure 4] Measurement of TIGIT binding by flow cytometry. [Diagram 5] (A) Schematic diagram showing inhibition of TIGIT-ligand interaction by anti-TIGIT mAb. (B) Binding of soluble TIGIT (TIGIT-huIgG1 fusion protein) to TIGIT ligand-expressing HEK293 cells (HEK293 / PVR or HEK293 / PVR-L2) was measured 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. Human CMV peptide (NLVPMVATV, 495-503)-sensitized HLA-A2.1+ PBMCs (4 × 104) 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 measured by ELISA. All conditions were performed in triplicate. Results are shown as mean ± SD. [Figure 7] Anti-TIGIT mAb promotes NK cell-mediated cytotoxicity. (A) Expression of TIGIT and DNAM-1 in engineered NK92MI / TIGIT-DNAM-1 stable cell lines. (B) 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 measured by lactate dehydrogenase (LDH) release assay as described in Example 8. Results are shown as 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 measure the effect of bulk human IgG on trogocytosis. Surface expression of TIGIT receptor was determined by staining with SA-APC (Biolegend). MFI was measured by flow cytometry. All data points were in duplicate. Results are presented as mean ± SD. [Figure 9A] ADCC effect of anti-TIGIT mAb on human peripheral blood mononuclear cells (PBMCs) is shown. TIGIT expression on PHA-stimulated PBMCs from healthy donors was measured by flow cytometry. CD4+ (CD4+Foxp3-), CD8+ T effector and regulatory T cells (Treg, CD4+Foxp3+) all expressed significant levels (18-41%) of TIGIT. Data shown are representative results from three healthy donors. [Figure 9B] ADCC assays were performed using the CD16+ human NK cell line NK92MI / CD16V as effector cells and PHA-stimulated PBMC as target cells in the presence of TIGIT mAb (30 μg / mL) or control antibodies (OKT3 at 5 μg / mL as positive control and huIgG at 30 μg / ml as negative control) for 42 h. The percentages of CD3+, CD8+ T cells and Tregs were measured 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 source of complement. After 3 days of co-culture of preactivated PBMC with anti-TIGIT mAb (0.01-100 μg / ml) in a final concentration of 15% autologous serum, the percentage of CDC (y-axis) was measured by cell titer glow 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. [Figure 11] The decrease in serum concentrations of hu1217-2-2 following intravenous infusion of 50 mg to 900 mg is shown. [Figure 12] 1 shows that hu1217-2-2 promotes IFN-γ secretion in vitro as a single agent or in combination with BGB-A317. [Figure 13] We demonstrate that hu1217-2-2 can reduce tumor growth as a single agent in a mouse glioma model. [Figure 14] Shows efficacy of hu1217-2-2 in combination with anti-PD1 antibody in a TIGIT knock-in mouse model of MC colon cancer. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0046] definition Conservative amino acid substitutions of amino acids are generally known in the art and are illustratively shown 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. [Table 1]

[0047] Unless specifically defined elsewhere herein, 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.

[0048] As used in this specification, including the appended claims, singular words such as "a," "an," and "this," "the," etc. include their corresponding plural references unless the context clearly dictates otherwise.

[0049] The term "or" is used to mean and is used synonymously with the term "and / or," unless context clearly dictates otherwise.

[0050] Throughout this specification and the claims which follow, unless the context otherwise requires, the word "comprising" and variations such as "comprises" and "comprising" are understood to be intended to include the recited amino acid sequences, DNA sequences, steps or groups thereof, but not to exclude other amino acid sequences, DNA sequences, steps. As used herein, the term "comprising" may be substituted with the terms "containing," "including," or, in some cases, "having."

[0051] The term "TIGIT" includes various mammalian isoforms, such as human TIGIT, orthologs of human TIGIT, and analogs that contain at least one epitope within TIGIT. The amino acid sequence of TIGIT, such as human TIGIT, and the nucleotide sequence encoding it are known in the art (see Genbank AAI01289).

[0052] The terms "administration", "administering", "treating" and "treatment" as used herein, when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refer to the contact of an exogenous pharmaceutical, therapeutic, diagnostic, or composition to the animal, human, subject, cell, tissue, organ, or biological fluid. Treatment of a cell encompasses contact of a reagent with the cell as well as contact of a reagent with a fluid, where the fluid contacts the cell. The term "administration" or "treatment" also includes in vitro and ex vivo treatment, e.g., of a cell with a reagent, diagnostic, binding compound, or with another cell. The term "subject" herein refers to any organism, preferably an animal, more preferably a mammal (e.g., rat, mouse, dog, cat, rabbit), and most preferably a human.

[0053] The term "antibody" is used herein in the broadest sense and includes antibodies (including full-length monoclonal antibodies) and antibody fragments, particularly so long as they recognize an antigen (e.g., TIGIT). Antibodies are usually monospecific, but may be described as idiospecific, heterospecific, or multispecific. An antibody molecule binds to a specific antigenic determinant or epitope on an antigen by a specific binding site.

[0054] As used herein, the term "monoclonal antibody" or "mAb" or "Mab" refers to a population of substantially homogenous antibodies, i.e., the antibody molecules comprising 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 typically contain a large number of different antibodies that have different amino acid sequences in their variable domains, particularly in their complementarity determining regions (CDRs), which are often specific for different epitopes. The modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogenous 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 known to those of skill in the art. See, e.g., Kohler G et al., Nature 1975 256:495-497; U.S. Patent No. 4,376,110; 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 can be of any immunoglobulin class, including IgG, IgM, IgD, IgE, IgA, and any subclass thereof. Hybridomas producing mAbs can be cultured in vitro or in vivo. High titer mAbs may be obtained by in vivo production, in which cells from individual hybridomas are injected intraperitoneally into mice (such as freshly primed Balb / c mice) to produce ascites fluid containing high concentrations of the desired mAb. MAbs of isotype IgM or IgG may be purified from such ascites fluids or culture supernatants using column chromatography methods well known to those skilled in the art.

[0055] Generally, the basic antibody structural unit comprises a tetramer. Each tetramer comprises two identical pairs of polypeptide chains, each pair having one "light chain" (about 25 kDa) and one "heavy chain" (about 50-70 kDa). The amino-terminal portion of each chain contains a variable region of about 100-110 or more amino acids in length that is primarily responsible for antigen recognition. The carboxy-terminal portion of the heavy chain may define a constant region that is primarily responsible for effector function. Human light chains are typically classified as kappa light chains and lambda light chains. Furthermore, human heavy chains are typically classified as α, δ, ε, γ, or μ, and the antibody isotypes are defined 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, and the heavy chains also contain a "D" region of about 10 more amino acids.

[0056] The variable regions of each light / heavy 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 generally the same.

[0057] Typically, both the heavy and light chain variable domains contain three hypervariable regions, also called "complementarity determining regions (CDRs)", which are located between relatively conserved framework regions (FRs). The CDRs are usually aligned by the framework regions, allowing binding to a specific epitope. In general, both the light and heavy chain variable domains contain, in order 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). The assignment of amino acids to each domain generally 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.

[0058] The term "hypervariable region" refers to the amino acid residues of an antibody which are involved in antigen binding. The hypervariable region comprises the amino acid residues from the "CDRs" (i.e., VL-CDR1, VL-CDR2, and VL-CDR3 of the light chain variable domain and VH-CDR1, VH-CDR2, and VH-CDR3 of 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 also 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 domain residues other than the hypervariable region residues as herein defined as CDR residues.

[0059] Unless otherwise indicated, "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; bispecific antibodies; linear antibodies; single-chain antibody molecules; e.g., single-chain Fv (ScFv); nanobodies and multispecific antibodies formed from antibody fragments.

[0060] An antibody that binds with specificity to a particular target protein is also described as specifically binding to a particular target protein. This means that the antibody exhibits preferential binding to that target compared to other proteins, but this specificity does not require absolute binding specificity. An antibody is considered to be "specific" for its intended target if its binding determines the presence of the target protein in a sample without undesirable results such as false positives. An antibody or binding fragment thereof useful in the present invention will bind 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 to a non-target protein. An antibody herein is said to specifically bind to a polypeptide if it binds to a polypeptide that includes a given amino acid sequence, e.g., the amino acid sequence of the mature human TIGIT molecule, but does not bind to a protein lacking that sequence.

[0061] The terms "pH-dependent binding", "pH-dependent target binding" and "pH-dependent antigen binding" are interchangeable in the present disclosure and 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 higher binding affinity and / or binding signal to its antigen at a weakly acidic pH typically found in tumor microenvironments, e.g., pH 6.0, 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 strength of the binding signal of the antibody of the present application are well known in the art, including, but not limited to, surface plasmon resonance (Biacore) or similar techniques. More specifically, the antibodies of the present application have a K of greater than 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more at pH 7.4 / pH 6.0 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 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 acidic pH compared to physiological conditions or normal tissues (Zhang et al. Focus on molecular Imaging 2010; Tannock and Rotin et al. Cancer Res 1989). Thus, the antibody of the present application with the above pH-dependent binding is advantageous as an anti-TIGIT therapeutic agent that selectively targets TIGIT-positive lymphocytes in the tumor microenvironment and has low toxicity associated with peripheral lymphocyte activation.

[0062] As used herein, the term "human antibody" refers to an antibody that contains only human immunoglobulin protein sequences. A human antibody may contain mouse glycosylation if produced in a mouse, a mouse cell, or 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.

[0063] The term "humanized antibody" refers to forms of antibodies that contain sequences derived from non-human (e.g., murine) and human antibodies. Such antibodies contain minimal sequences derived from non-human immunoglobulins. Generally, a humanized antibody contains substantially all of at least one, and typically two, variable domains, with all or substantially all of the hypervariable loops corresponding to those of a non-human immunoglobulin, and all or substantially all of the FR regions being those of a human immunoglobulin sequence. A humanized antibody optionally also contains at least a portion of an immunoglobulin constant region (Fc), typically at least a portion of a human immunoglobulin constant region (Fc). When necessary to distinguish a humanized antibody from a parent rodent antibody, the prefixes "hum", "hu", "Hu" or "h" are added to the name of the antibody clone. Humanized forms of rodent antibodies generally contain the same CDR sequences as the parent rodent antibody, but may contain certain amino acid substitutions to increase affinity, increase the stability of the humanized antibody, or for other reasons.

[0064] The antibodies of the present application have potential therapeutic applications in the treatment of cancer. The term "cancer" or "tumor" refers to 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, colorectal cancer, leukemia, bladder cancer, bone cancer, brain cancer, endometrial cancer, head and neck cancer, lymphoma, ovarian cancer, skin cancer, thyroid tumor, or metastatic lesions of cancer.

[0065] Furthermore, the antibodies of the present application have potential therapeutic applications in the control of viral infections and other human diseases that are mechanistically involved in immune tolerance or "exhaustion." In the context of the present application, the term "exhaustion" refers to a process that leads to the exhaustion of the ability of immune cells to respond during cancer or chronic viral infections.

[0066] The term "therapeutically effective amount" as used herein refers to an amount of an antibody that, when administered to a subject for treating a disease or disorder, or at least one of the clinical symptoms of a disease or disorder, is sufficient to effect such treatment for the disease, disorder, or condition. A "therapeutically effective amount" may vary with the antibody, the disease, disorder, and / or the symptoms of the disease or disorder, the severity of the disease, disorder, and / or the symptoms of the disease or disorder, the age of the subject being treated, and / or the weight of the subject being treated. The appropriate amount in any given case may be apparent to one of ordinary skill in the art or may be determined by routine experimentation. In the case of combination therapy, a "therapeutically effective amount" refers to the total amount of active agents that constitute the combination for effective treatment of a disease, disorder, or condition.

[0067] As used herein, a "subject" is a mammal, e.g., a rodent or a primate, preferably a higher primate, e.g., a human (e.g., a patient having or at risk of having a disorder described herein).

[0068] Anti-TIGIT antibody The present disclosure provides antibodies, antigen-binding fragments that specifically bind to human TIGIT. In addition, the present disclosure provides antibodies that have desirable pharmacokinetic properties and other desirable attributes, and thus can be used to reduce the likelihood of cancer or to treat cancer. The present disclosure further provides pharmaceutical compositions comprising the antibodies, as well as methods of making and using such pharmaceutical compositions, for the prevention and treatment of cancer and related disorders.

[0069] The present disclosure provides an antibody or antigen-binding fragment thereof that specifically binds to TIGIT. The antibody or antigen-binding fragment of the present disclosure includes, but is not limited to, an antibody or antigen-binding fragment thereof that is generated as described below.

[0070] The present disclosure provides an antibody or antigen-binding fragment that specifically binds to TIGIT, wherein the antibody or antibody fragment (e.g., antigen-binding fragment) comprises a VH domain having an amino acid sequence of SEQ ID NO: 9, 14, or 19. The present disclosure also provides an antibody or antigen-binding fragment that specifically binds to TIGIT, wherein the antibody or antigen-binding fragment comprises a VH CDR having the amino acid sequence of any one of the VH CDRs provided herein. In one aspect, the present disclosure provides an antibody or antigen-binding fragment that specifically binds to TIGIT, wherein the antibody comprises (or consists of) one, two, three, or more VH CDRs having the amino acid sequence of any one of the VH CDRs provided herein.

[0071] The present disclosure provides an antibody or antigen-binding fragment that specifically binds to TIGIT, the antibody or antigen-binding fragment comprising a VL domain having the amino acid sequence of SEQ ID NO: 11, 16, or 21. The present disclosure also provides an antibody or antigen-binding fragment that specifically binds to TIGIT, the antibody or antigen-binding fragment comprising a VL CDR having the amino acid sequence of any one of the VL CDRs provided herein. In particular, the present disclosure provides an antibody or antigen-binding fragment that specifically binds to TIGIT, the antibody or antigen-binding fragment comprising (or consisting of) one, two, three or more VL CDRs having the amino acid sequence of any VL CDR of the present disclosure.

[0072] Other antibodies or antigen-binding fragments of the present disclosure include mutated amino acids in the CDR regions shown in the sequences described herein that have at least 60%, 70%, 80%, 90%, 95% or 99% percent identity with the CDR regions. In some embodiments, mutated amino acid sequences are included in which no more than 1, 2, 3, 4 or 5 amino acids are mutated in the CDR regions when compared to the CDR regions disclosed in the sequences provided.

[0073] Other antibodies of the disclosure include those in which the amino acid or the nucleic acid encoding the amino acid has been mutated; and further have at least 60%, 70%, 80%, 90%, 95% or 99% identity to the sequences set forth in Table 5. In some embodiments, it comprises a variant amino acid sequence in which no more than 1, 2, 3, 4 or 5 amino acids have been mutated in the variable region when compared to the variable region set forth in the sequences set forth herein while retaining substantially the same therapeutic activity.

[0074] Anti-PD1 antibody The present disclosure provides anti-PD1 antibodies, e.g., as found in U.S. Patent No. 8,735,553. PD1 antibodies are also provided herein, e.g., comprising a heavy chain variable region (VH) comprising complementarity determining regions (CDRs): HCDR1 set forth in SEQ ID NO:25, HCDR2 set forth in SEQ ID NO:26, and HCDR3 set forth in SEQ ID NO:27; and a light chain variable region (VL) comprising LCDR1 set forth in SEQ ID NO:28, LCDR2 set forth in SEQ ID NO:29, and LCDR3 set forth in SEQ ID NO:30. This antibody is referred to herein as "BGB-A317."

[0075] In another embodiment, the anti-PD1 antibody or antigen-binding fragment specifically binds human PD1 and comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 32 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 34. In yet another embodiment, the anti-PD1 antibody comprises an IgG4 constant domain comprising SEQ ID NO: 35.

[0076] Further changes to the FC domain framework In yet another embodiment, the Fc region is modified by replacing at least one amino acid residue with a different amino acid residue to modify the effector function of the antibody. For example, one or more amino acids may be replaced with a different amino acid residue such that the antibody has an altered affinity for an effector ligand but retains the antigen-binding ability of the parent antibody. The effector ligand for which affinity is altered can be, for example, an Fc receptor or the C1 component of complement. This approach is described, for example, in U.S. Patent Nos. 5,624,821 and 5,648,260 (both by Winter et al.).

[0077] In another embodiment, one or more amino acid residues may be replaced with one or more different amino acid residues such that the antibody has altered C1q binding and / or reduced or eliminated complement dependent cytotoxicity (CDC). This approach is described, for example, in U.S. Patent No. 6,194,551 by Idusogie et al.

[0078] In yet another embodiment, one or more amino acid residues are altered to modify the ability of the antibody to fix complement. This approach is described, for example, in PCT Publication WO94 / 29351 by Bodmer et al. In certain embodiments, one or more amino acids of an antibody or antigen-binding fragment thereof of the present disclosure are replaced with one or more allotypic amino acid residues for the IgG1 subclass and the kappa isotype. The allotypic amino acid residues include, but are not limited to, the constant regions of the heavy chains of the IgG1, IgG2, and IgG3 subclasses, as well as the constant region of the light chain of the kappa isotype as described by Jefferis et al., MAb.1:332-338 (2009).

[0079] In another embodiment, the Fc region is modified to increase the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and / or to increase the affinity of the antibody for the Fcγ receptor by modifying one or more amino acids. This approach is described, for example, in PCT Publication WO00 / 42072 by Presta. Furthermore, the binding sites for FcγRI, FcγRII, FcγRIII and FcRn on human IgG1 have been mapped and variants with improved binding have been described (see Shields et al., J. Biol. Chem. 276:6591-6604, 2001).

[0080] In yet another embodiment, the glycosylation of the antibody is modified. For example, an aglycosylated antibody can be made (i.e., the antibody lacks or has reduced glycosylation). The glycosylation can be altered to, for example, increase the affinity of the antibody for an "antigen". Such carbohydrate modifications can be accomplished, for example, by altering one or more sites of glycosylation within the antibody sequence. For example, one or more amino acid substitutions can be made that result in the elimination of one or more variable region framework glycosylation sites, thereby eliminating glycosylation at that site. Such aglycosylation can increase the affinity of the antibody for the antigen. Such approaches are described, for example, in U.S. Patent Nos. 5,714,350 and 6,350,861 by Co et al.

[0081] Additionally or alternatively, antibodies can be made with altered types of glycosylation, such as hypofucosylated antibodies with reduced amounts of fucosyl residues or antibodies with increased bisecting GlcNac structures. Such altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies. Such carbohydrate modifications can be achieved, for example, by expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells to express recombinant antibodies, thereby producing antibodies with altered glycosylation. For example, Hang et al., European Patent No. 1,176,195, describes a cell line with a functionally disrupted FUT8 gene that encodes a fucosyltransferase, such that antibodies expressed in such cell lines exhibit hypofucosylation. PCT Publication WO 03 / 035835 by Presta describes a variant CHO cell line, Lecl3 cells, that has a reduced ability to attach fucose to Asn(297)-linked carbohydrates, which also results in hypofucosylation of antibodies expressed in the host cells (see also Shields et al., (2002) J. Biol. Chem. 277:26733-26740). PCT Publication WO 99 / 54342 by Umana et al. describes cell lines engineered to express glycoprotein-modifying glycosyltransferases (e.g., beta(1,4)-N-acetylglucosaminyltransferase III (GnTIII) such that antibodies expressed in the engineered cell lines exhibit increased bisecting GlcNac structures, resulting in increased ADCC activity of the antibodies (see also Umana et al., Nat. Biotech. 17:176-180, 1999).

[0082] In another aspect, when reduced ADCC is desired, human antibody subclass IgG4 has been shown in many previous reports to have only moderate ADCC and little CDC effector function (Moore GL, et al. 2010 MAbs, 2:181-189). On the other hand, native IgG4 has been found to be less stable in acidic buffer or stress conditions such as elevated temperature (Angal, S. 1993 Mol Immunol, 30:105-108; Dall'Acqua, W. et al, 1998 Biochemistry, 37:9266-9273; Aalberse et al. 2002 Immunol, 105:9-19). Reduced ADCC can be achieved by operably binding the antibody to an engineered IgG4 with a combination of modifications that reduce or abolish FcγR binding or C1q binding activity, thereby reducing or eliminating ADCC and CDC effector function. Considering the physicochemical properties of antibodies as biological drugs, one of the more undesirable intrinsic properties of IgG4 is the dynamic separation of its two heavy chains in solution to form half-antibodies, which led to bispecific antibodies being generated in vivo via a process called "Fab arm exchange" (Van der Neut Kolfschoten M, et al. 2007 Science, 317: 1554-157). Mutation of serine to proline at position 228 (EU numbering system) appeared inhibitory to IgG4 heavy chain separation (Angal, S. 1993 Mol Immunol, 30: 105-108; Aalberse et al. 2002 Immunol, 105: 9-19).Some of the amino acid residues in the hinge and gamma Fc regions have been reported to affect antibody interaction with Fcγ receptors (Chappel SM, et al. 1991 Proc. Natl. Acad. Sci. USA, 88:9036-9040; Mukherjee, J. et al., 1995 FASEB J, 9:115-119; Armour, KL et al. 1999 Eur J Immunol, 29:2613-2624; Clynes, RA et al, 2000 Nature Medicine, 6:443-446; Arnold JN, 2007 Annu Rev immunol, 25:21-50). Furthermore, some IgG4 isoforms occurring rarely in the human population may also induce different physicochemical properties (Brusco, A. et al. 1998 Eur J Immunogenet, 25:349-55; Aalberse et al. 2002 Immunol, 105:9-19). To generate TIGIT antibodies with low ADCC and CDC instability, it is possible to modify the hinge and Fc regions of human IgG4 and introduce a number of modifications. These modified IgG4 Fc molecules can be found in SEQ ID NOs: 83-88, disclosed in U.S. Pat. No. 8,735,553.

[0083] Antibody production Anti-TIGIT antibodies and antigen-binding fragments thereof may be produced by any means known in the art, including but not limited to recombinant expression, chemical synthesis, and enzymatic digestion of antibody tetramers, whereas full-length monoclonal antibodies may be obtained, for example, by hybridoma or recombinant production. Recombinant expression may be derived from any suitable host cell known in the art, for example, mammalian host cells, bacterial host cells, yeast host cells, insect host cells, etc.

[0084] The disclosure further provides polynucleotides encoding the antibodies described herein, e.g., polynucleotides encoding heavy or light chain variable regions or segments comprising the complementarity determining regions described herein. In some embodiments, the polynucleotide encoding the heavy chain variable region has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity to a polynucleotide encoding a polypeptide of SEQ ID NO: 9, 14, or 19. In some embodiments, the polynucleotide encoding the light chain variable region has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity to a polynucleotide encoding a polypeptide of SEQ ID NO: 11, 16, or 21.

[0085] The polynucleotides of the present disclosure may encode the variable region sequences of an anti-TIGIT antibody. They may also encode both the variable and constant regions of the antibody. Some of the polynucleotide sequences encode polypeptides that include both the heavy and light chain variable regions of one of the exemplified anti-TIGIT antibodies. Some other polynucleotides encode two polypeptide segments that are substantially identical to the heavy and light chain variable regions, respectively, of one of the mouse antibodies.

[0086] In the present disclosure, expression vectors and host cells for producing anti-TIGIT antibodies are also provided. The choice of expression vector depends on the host cell in which the vector is intended to be expressed. Typically, the expression vector contains a promoter and other regulatory sequences (e.g., enhancers) operably linked to the polynucleotide encoding the anti-TIGIT antibody chain or antigen-binding fragment. In some embodiments, an inducible promoter is used to prevent expression of the inserted sequence except when under the control of an inducing condition. Inducible promoters include, for example, arabinose, lacZ, metallothionein promoter, or heat shock promoter. Cultures of transformed organisms can be grown under non-inducing conditions without biasing the population in favor of coding sequences whose expression products are better tolerated by the host cell. In addition to promoters, other regulatory elements may be required or desired for efficient expression of anti-TIGIT antibodies or antigen-binding fragments. These elements typically include an ATG initiation codon and adjacent ribosome binding sites or other sequences. In addition, the efficiency of expression can be increased by including enhancers appropriate for the cell system being used (see, e.g., Scharf et al., Results Probl. Cell Differ. 20:125, 1994; and Bittner et al., Meth. Enzymol., 153:516, 1987). For example, the SV40 enhancer or CMV enhancer can be used to increase expression in mammalian host cells.

[0087] Host cells for carrying and expressing anti-TIGIT antibody chains may be prokaryotic or eukaryotic. E. coli is one prokaryotic host useful for cloning and expressing the polynucleotides of the present disclosure. Other microbial hosts suitable for use include bacilli such as Bacillus subtilis, and other Enterobacteriaceae such as Salmonella, Serratia, and various Pseudomonas species. In these prokaryotic hosts, expression vectors may be made, which typically contain expression control sequences compatible with the host cell (e.g., origin of replication). In addition, there will be a large number of different well-known promoters, such as the lactose promoter system, the tryptophan (trp) promoter system, the beta-lactamase promoter system, or a promoter system from phage lambda. The promoters typically control expression, optionally with an operator sequence, and have ribosome binding site sequences, etc., for initiating and completing transcription and translation. Other microbes, such as yeast, may also be used to express anti-TIGIT polypeptides. Insect cells in combination with baculovirus vectors may also be used.

[0088] In other aspects, mammalian host cells are used to express and produce the anti-TIGIT polypeptides of the present disclosure. For example, they may be either hybridoma cell lines expressing endogenous immunoglobulin genes, or mammalian cell lines with exogenous expression vectors. These include any normal mortal or normal or abnormal immortal animal or human cells. For example, a number of suitable host cell lines capable of secreting intact immunoglobulins have been developed, including CHO cell lines, various COS cell lines, HEK293 cells, bone marrow cell lines, transformed B cells, and hybridomas. The use of mammalian tissue cell cultures to express polypeptides is generally discussed in, for example, Winnacker, From Genes to Clones, VCH Publishers, NY, NY, 1987. Expression vectors for mammalian host cells may include expression control sequences such as origins of replication, promoters, and enhancers (see, e.g., Queen et al., Immunol. Rev. 89:49-68, 1986), and necessary processing information sites such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription terminator sequences. These expression vectors usually contain promoters derived from mammalian genes or mammalian viruses. Suitable promoters may be constitutive, cell type specific, stage specific, and / or modulatable or regulatable. Useful promoters include, but are not limited to, metallothionein promoter, constitutive adenovirus major late promoter, dexamethasone inducible MMTV promoter, SV40 promoter, MRP poly III promoter, constitutive MPSV promoter, tetracycline inducible CMV promoter (such as human immediate early CMV promoter), constitutive CMV promoter, and promoter-enhancer combinations known in the art.

[0089] Detection and Diagnostic Methods The antibody or antigen-binding fragment of the present disclosure is useful in various applications, including, but not limited to, a method for detecting TIGT. In one embodiment, the antibody or antigen-binding fragment is useful for detecting the presence of TIGIT in a biological sample. The term "detecting" as used herein includes quantitative or qualitative detection. In certain embodiments, the biological sample includes cells or tissues. In other embodiments, such tissues include normal tissues and / or cancerous tissues that express TIGIT at a high level compared to other tissues.

[0090] In one aspect, the present disclosure provides a method for detecting the presence of TIGIT in a biological sample.In a particular aspect, the method comprises contacting a biological sample with an anti-TIGIT antibody under conditions that allow the antibody to bind to the antigen, and detecting whether a complex is formed between the antibody and the antigen.The biological sample includes, but is not limited to, a urine or blood sample.

[0091] Also included is the method for diagnosing the disorder associated with the expression of TIGIT.In a particular embodiment, this method comprises: contacting test cell with anti-TIGIT antibody; determining the level of expression (either quantitative or qualitative) of TIGIT in test cell by detecting the binding of anti-TIGIT antibody to TIGIT polypeptide; comparing the level of expression in test cell with the level of TIGIT expression in control cell (e.g., normal cell or non-TIGIT expressing cell of the same tissue origin as test cell), wherein the higher level of TIGIT expression in test cell compared to control cell indicates the presence of the disorder associated with the expression of TIGIT.

[0092] Treatment Method The antibody or antigen-binding fragment of the present disclosure is useful in a variety of applications, including but not limited to, methods for treating TIGIT-related disorders or diseases. In one embodiment, the TIGIT-related disorder or disease is cancer.

[0093] In one aspect, the present disclosure provides a method for treating cancer. In a particular aspect, the method comprises administering to a patient in need thereof an effective amount of an anti-TIGIT antibody or antigen-binding fragment. Cancers include, but are not limited to, breast cancer, head and neck cancer, gastric cancer, renal cancer, liver cancer, small cell lung cancer, non-small cell lung cancer, ovarian cancer, skin cancer, mesothelioma, lymphoma, leukemia, myeloma, and sarcoma.

[0094] The antibody or antigen-binding fragment of the present invention can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, as well as intralesional administration if desired for localized treatment. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, for example, injection, such as intravenous or subcutaneous injection, depending in part on whether administration is brief or chronic. Various dosing schedules are contemplated herein, including, but not limited to, single or multiple administrations at various times, bolus administration, and pulse infusion.

[0095] The antibodies or antigen-binding fragments of the invention will be formulated, dosed, and administered in a manner consistent with good medical practice. Factors to consider in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners. The antibodies are optionally, but not necessarily, formulated with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents will depend on the amount of antibody present in the formulation, the type of disorder or treatment, and other factors discussed above. These will generally be used in the same dosages and by any route of administration as described herein, or about 1-99% of the dosages described herein, or any dosage and by any route that is empirically / clinically determined to be appropriate.

[0096] For the prevention or treatment of disease, the appropriate dosage of the antibody or antigen-binding fragment of the present invention will vary depending on the type of disease being treated, the type of antibody, the severity and course of the disease, whether the antibody is administered for prophylactic or therapeutic purposes, previous therapies, the patient's clinical history and response to the antibody, and the judgment of the attending physician. The antibody is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, for example, about 1 μg / kg to 100 mg / kg of antibody may be an initial candidate dosage for administration to the patient, whether by one or more separate administrations or by continuous infusion. One typical daily dosage may range from about 1 μg / kg to 100 mg / kg or more, depending on the factors mentioned above. For repeated administration over several or more days depending on the condition, treatment will generally be sustained until a desired suppression of disease symptoms occurs. Such doses may be administered intermittently, for example every week or every three weeks (e.g. so that the patient receives from about 2 to about 20, or for example about 6, doses of the antibody). An initial higher loading dose may be administered, followed by one or more lower doses. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.

[0097] Combination therapy In one embodiment, the TIGIT antibody of the present disclosure may be used in combination with other therapeutic agents, such as anti-PD1 antibodies. Other therapeutic agents that may be used with the TIGIT antibody of the present disclosure include, but are not limited to, the following: chemotherapeutic agents (e.g., paclitaxel or paclitaxel agents (e.g., Abraxane®), docetaxel, carboplatin, topotecan, cisplatin, irinotecan, doxorubicin, lenalidomide, 5-azacytidine, ifosfamide, oxaliplatin, pemetrexed disodium, cyclophosphatase ... amide, etoposide, decitabine, fludarabine, vincristine, bendamustine, chloramvir, busulfan, gemcitabine, melphalan, pentostatin, mitoxantrone, picetrexate disodium), tyrosine kinase inhibitors (e.g., EGFR inhibitors (e.g., erlotinib), multikinase inhibitors (e.g., MGCD265, RGB-286638), CD20 targeting agents (e.g., rituximab, ofatum mabs, RO5072759, LFB-R603), CD52 targeting agents (e.g., alemtuzumab), prednisolone, darbepoetin alfa, lenalidomide, Bcl-2 inhibitors (e.g., oblemesen sodium), Aurora kinase inhibitors (e.g., MLN8237, TAK-901), proteasome inhibitors (e.g., bortezomib), CD19 targeting agents (e.g., MEDI-551, MOR208), MEK inhibitors (e.g., A BT-348), JAK-2 inhibitors (e.g., INCB018424), mTOR inhibitors (e.g., temsirolimus, everolimus), BCR / ABL inhibitors (e.g., imatinib), ET-A receptor antagonists (e.g., ZD4054), TRAIL receptor 2 (TR-2) agonists (e.g., CS-1008), HGF / SF inhibitors (e.g., AMG102), EGEN-001, polo-like kinase 1 inhibitors (e.g., BI672).

[0098] The TIGIT antibody of the present disclosure may be used in combination with other therapeutic agents, such as anti-PD1 antibodies. Anti-PD1 antibodies include, but are not limited to, those disclosed in U.S. Patent No. 8,735,553. Pembrolizumab (formerly known as MK-3475) disclosed by Merck is a humanized IgG4-K immunoglobulin with a molecular weight of approximately 149 kDa that targets the PD1 receptor and inhibits the binding of the PD1 receptor ligands PD-L1 and PD-L2. Pembrolizumab has been approved for the indications of metastatic melanoma and metastatic non-small cell lung cancer (NSCLC) and is under clinical investigation for the treatment of head and neck squamous cell carcinoma (HNSCC) and refractory Hodgkin's lymphoma (cHL). Nivolumab (disclosed by Bristol-Myers Squibb) is a fully human IgG4-K monoclonal antibody. Nivolumab (clone 5C4) is disclosed in U.S. Patent No. 8,008,449 and WO 2006 / 121168. Nivolumab is approved for the treatment of melanoma, lung cancer, kidney cancer, and Hodgkin's lymphoma.

[0099] Pharmaceutical Compositions and Formulations Also provided is a composition, including pharmaceutical preparation, that comprises anti-TIGIT antibody or antigen-binding fragment, or polynucleotide that comprises sequence that encodes anti-TIGIT antibody or antigen-binding fragment.In certain embodiments, the composition comprises one or more antibodies or antigen-binding fragments that bind to TIGIT, or one or more polynucleotides that comprise sequence that encodes one or more antibodies or antigen-binding fragments that bind to TIGIT.These compositions may further comprise suitable carriers, such as pharma-ceutically acceptable excipients, including buffers, that are well known in the art.

[0100] Pharmaceutical formulations of the TIGIT antibodies or antigen-binding fragments described herein are prepared in the form of lyophilized formulations or aqueous solutions by mixing such antibodies or antigen-binding fragments having the desired degree of purity with any one or more pharma- ceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations used, and include, but are not limited to, buffers such as phosphate, citric acid, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium 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); small molecule antibacterials such as glycerol, glycerol, tert-butyl esters ... Polypeptides of about 10 or less residues; 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); and / or non-ionic surfactants, such as polyethylene glycol (PEG). Exemplary pharmacologic carriers herein further include interstitial drug dispersion agents, such as soluble neutral active hyaluronidase glycoproteins (sHASEGPs), such as human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in U.S. Patent Nos. 7,871,607 and 2006 / 0104968.In one embodiment, the sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinases.

[0101] Exemplary lyophilized antibody formulations are described in U.S. Patent No. 6,267,958. Aqueous antibody formulations include those described in U.S. Patent No. 6,171,586 and WO2006 / 044908, the latter of which includes a histidine-acetate buffer.

[0102] Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules.

[0103] Preparations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, for example, by filtration through sterile filtration membranes.

[0104] Pharmaceutical Compositions and Kits In some aspects, the present disclosure 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 any and all solvents, dispersion media, isotonic and absorption delaying agents, etc., that are physiologically compatible. The excipient may be suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, spinal, or epidermal administration (e.g., by injection or infusion).

[0105] The compositions herein may be in various forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injection and infusion solutions), dispersions or suspensions, liposomes, and suppositories. The suitable form depends on the intended mode of administration and therapeutic application. Typical suitable compositions are in the form of injection 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. EXAMPLES

[0106] Example 1: Generation of anti-TIGIT monoclonal antibodies Anti-TIGIT monoclonal antibodies (mAbs) were generated based on conventional hybridoma fusion techniques with minor modifications (de St Groth and Sheidegger, 1980 J Immunol Methods 35:1; Mechetner, 2007 Methods Mol Biol 378:1). mAbs with high binding activity in enzyme-linked immunosorbent assay (ELISA) and fluorescence-activated cell sorting (FACS) assays were selected for further characterization.

[0107] TIGIT recombinant protein for immunization and binding assays cDNA encoding full-length human TIGIT (SEQ ID NO: 1) was synthesized by and purchased from Sino Biological (Beijing, China) based on its GenBank sequence (Accession No.: NM_173799). 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 cloned into a pcDNA3.1-based expression vector (Invitrogen, Carlsbad, CA, USA) with the C-terminus fused to either the Fc domain of mouse IgG2a or the Fc domain of human IgG1 heavy chain, thereby obtaining two recombinant fusion protein expression plasmids, TIGIT-mIgG2a and TIGIT-huIgG1, 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 transfected into 293G cells (in-house developed) and cultured for 7 days in a CO2 incubator equipped with a rotary shaker. The supernatant containing the recombinant proteins was collected and removed 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 small aliquots in a -80°C freezer.

[0108] Stable cell lines To establish stable cell lines expressing full-length human TIGIT (huTIGIT) or monkey TIGIT (mkTIGIT, accession number: XM_005548101.2), the TIGIT gene (synthesized at Genescript, Nanjing, China) was cloned into the retroviral vector pFB-Neo (catalog: 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), respectively, to generate the cell lines Jurkat / huTIGIT and NK92MI / mkTIGIT. High-expressing cell lines were selected by culture in medium containing G418 and FACS binding assay.

[0109] Immunization, hybridoma fusion and cloning Balb / c mice aged 8-12 weeks (HFK BIOSCIENCE CO.,LTD,Beijing,China) were immunized intraperitoneally (ip) with 100μL of antigen mixture containing 10μg of TIGIT-mIgG2a and water-soluble adjuvant (catalog: KX0210041, KangBiQuan,Beijing,China). This 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 50μg of TIGIT-mIgG2a by intraperitoneal (ip) injection. Three days after the boost, splenocytes were isolated and fused to SP2 / 0 cells (ATCC), a mouse myeloma cell line, using standard techniques (Gefter et al., Somat Cell Genet,1977 3(2):231-6).

[0110] Assessment of TIGIT binding activity of antibodies by ELISA and FACS Supernatants of hybridoma clones were initially 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: 7076S, Cell Signaling Technology, USA) and substrate (Cat: 00-4201-56, eBioscience, USA) were used to generate a colorimetric absorbance signal at a wavelength of 450 nm, which was measured using a plate reader (SpectraMax Paradigm, Molecular Devices, USA). ELISA-positive clones were further verified by FACS using either NK92MI / huTIGIT cells or NK92mi / mkTIGIT cells as described above. TIGIT-expressing cells (10 5 Cells / well) were incubated with ELISA-positive hybridoma supernatants and subsequently bound with Alexa Fluro-647-labeled goat anti-mouse IgG antibody (catalog: A0473, Beyotime Biotechnology, China). Cell fluorescence was quantified using a flow cytometer (Guava easyCyte 8HT, Merck-Millipore, USA).

[0111] 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 section below). Antibodies with the desired functional activity were further subcloned and characterized.

[0112] Subcloning of hybridomas and adaptation to serum-free or low-serum medium After primary screening by ELISA, FACS and functional assays as described above, positive hybridoma clones were subcloned by limiting dilution. Three positive subclones were selected based on ELISA and FACS screening from each plate and characterized by functional assays. The top antibody subclones verified through functional assays were adapted for growth in CDM4MAb medium (catalog: SH30801.02, Hyclone, USA) containing 3% FBS.

[0113] Monoclonal antibody expression and purification Hybridoma cells or 293G cells transiently transfected with antibody expression plasmids (catalog no. R79007, Invitrogen) were cultured in either CDM4MAb medium (catalog: SH30801.02, Hyclone) or Freestyle™ 293 Expression Medium (catalog: 12338018, Invitrogen) and incubated at 37°C for 5-7 days in a CO2 incubator. Conditioned medium was collected by centrifugation and filtered by passing through a 0.22 μm membrane before purification. Mouse or recombinant antibody containing supernatant was applied to a Protein A column (catalog: 17127901, GE Life Sciences) and allowed to bind, following the manufacturer's guide. This procedure usually yielded antibodies with purity of more than 90%. Protein A affinity purified antibodies were dialyzed against PBS or further purified using a HiLoad16 / 60 Superdex200 column (catalog: 17531801, GE Life Sciences) to remove aggregates. Protein concentration was determined by measuring absorbance at 280 nm. The final antibody preparation was stored in aliquots in a -80°C freezer.

[0114] Example 2: Cloning and sequence analysis of TIGIT antibodies Mouse hybridoma clones were collected and total cellular RNA was prepared using the Ultrapure RNA kit (catalog: 74104, QIAGEN, Germany) based on the manufacturer's protocol. First-strand cDNA was synthesized using the cDNA synthesis kit from Invitrogen (catalog number: 18080-051), and PCR amplification of nucleotide sequences encoding the heavy chain variable region (Vh) and kappa chain variable region (Vk) of mouse mAb was performed using a PCR kit (catalog: CW0686, CWBio, Beijing, China). Oligo primers used for antibody cDNA cloning of Vh and Vk 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 (catalog: C B101-02, TransGen, China) and sequenced by Genewiz (Beijing, China). The amino acid sequences of the Vh and Vk regions were deduced from the DNA sequencing results.

[0115] The mouse mAbs were analyzed by comparing sequence homology and grouped based on sequence similarity (Figure 2A-B). Complementary determining regions (CDRs) were defined based on the Kabat (Wu and Kabat 1970 J. Exp. Med. 132:211-250) and IMGT (Lefranc 1999 Nucleic Acids Research 27:209-212) systems (by sequence annotation and internet-based sequence analysis at the IMGT website). The amino acid sequences of representative top clones mu1217 (Vh and Vk) are listed in Table 1 (SEQ ID NOs: 9 and 11). The CDR sequences of mu1217 are listed in Table 2 (SEQ ID NOs: 3-8). [Table 2] [Table 3]

[0116] Example 3: Affinity measurement of purified mouse anti-TIGIT antibody by SPR The TIGIT antibody, which has high binding activity in ELISA and FACS, as well as strong functional activity in cell-based assays (described in Examples 1 and 2), was characterized for its binding kinetics by SPR assay using a BIAcore™ T-200 (GE Life Sciences). Briefly, anti-human IgG antibody was immobilized on an activated CM5 biosensor chip (catalog number: BR100530, GE Life Sciences). Human Fc-tagged TIGIT was flowed over the chip surface and captured by the anti-human IgG antibody. Then, serial dilutions (0.12 nM to 10 nM) of purified mouse antibody were flowed over the chip surface, and the change in surface plasmon resonance signal was analyzed to determine the binding rate (k ) by using a one-to-one Langmuir binding model (BIA Evaluation Software, GE Life Sciences). on ) and dissociation rate (k off The equilibrium dissociation constant (K D ) as the ratio k off / k on The binding affinity profiles of the top mAbs, including mu1217, mu1257, mu1226, and mu242, are shown in Figure 3 and Table 3. [Table 4]

[0117] Example 4: Humanization of mouse anti-human TIGIT mAb mu1217 Humanization and Engineering of mAbs For humanization of mu1217, we performed a comparison with the IMGT human immunoglobulin gene database to search for sequences that share high homology with the cDNA sequence of the mu1217 variable region in human germline IgG genes. The human IGVH and IGVK genes, which are frequently present in the human antibody repertoire (Glanville et al., PNAS 106:20216-20221 2009) and have high homology with mu1217, were selected as templates for humanization.

[0118] Humanization was performed by CDR grafting (Methods in Molecular Biology, Vol 248: Antibody Engineering, Methods and Protocols, Humana Press), and the humanized antibody (hu1217s) was engineered in human IgG1mf format using an in-house developed expression vector. In the first round of humanization, mutations from mouse to human amino acid residues in the framework regions were guided by the simulated 3D structure, and structurally important mouse framework residues to maintain the canonical structure of the CDRs were retained in the first version of humanized antibody 1217 (hu1217-1-1, with six CDRs having amino acid sequences of SEQ ID NOs: 3, 13, 5 (heavy chain CDRs) and SEQ ID NOs: 6, 7, 8 (light chain CDRs), a heavy chain variable region having an 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 having an amino acid sequence of SEQ ID NO: 16 and encoded by the nucleotide sequence of SEQ ID NO: 17). Specifically, the CDRs of mu1217Vκ (SEQ ID NOs: 6 to 8) were cloned by substituting one mouse framework residue (V 58 The N-terminus of H-CDR2 (SEQ ID NO: 4), H-CDR1 and H-CDR3 (SEQ ID NO: 3 and 5) of mu1217Vh were grafted onto the framework of the human germline variable gene IGVκ3-15 carrying the humanized Vκ sequence Hu1217-1-1 (amino acid sequence SEQ ID NO: 16, nucleotide sequence SEQ ID NO: 17). 24 and I 37The humanized Vh sequences were grafted onto the framework of human germline variable gene IGVH3-7, in which the 1, 2, 3 and 4 residues were retained. For the hu1217 humanized variant, only the N-terminal half of Kabat H-CDR2 was grafted, since according to the simulated 3D structure, only the N-terminal half was predicted to be important for antigen binding. The amino acid sequence and nucleotide sequence of the resulting humanized Vh sequence of Hu1217-1-1 are shown in SEQ ID NO: 14 and SEQ ID NO: 15, respectively.

[0119] Hu1217-1-1 was constructed as a human full-length antibody format using an in-house developed expression vector containing the constant regions of human IgG1 variants, called IgG1mf (SEQ ID NO: 18) and kappa chains, respectively, with easily adaptable 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 (catalog: 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.

[0120] Based on the hu1217-1-1 template, several single mutations were made to convert the retained mouse residues in the framework regions of Vκ to the corresponding human germline residues, including V58I and I37VVh for Vκ and T24A. The resulting hu1217-2A-1 (T24A), hu1217-2B-1 (I37V), and hu1217-1-2a (V58I) all had similar binding and functional activity against hu1217-1-1. All humanized mutations were made using primers containing mutations at specific positions and a site-directed mutagenesis kit (catalog no. FM111-02, TransGen, Beijing, China). The desired mutations were confirmed by sequence analysis. These hu1217-derived variant antibodies were tested in binding and functional assays as previously described.

[0121] The Hu1217 antibody was further engineered by introducing mutations in the CDR and framework regions to improve its molecular and biophysical properties for therapeutic use in humans. m ), surface hydrophobicity, and isoelectronic point (pI) need to be considered.

[0122] In summary, a well-designed version of humanized monoclonal antibody, hu1217-2-2 (SEQ ID NOs: 3, 5-8, 13, and 19-21), was derived from the above mutation process and characterized in detail, which showed that both hu1217-2-2 and hu1217-1-1 are highly similar in binding affinity and functional activity, such as inhibition of TIGIT-mediated downstream signaling.

[0123] For affinity measurements, the antibodies were captured by anti-human Fc surface and used in an affinity assay 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, close to that of ch1217. [Table 5] [Table 6]

[0124] All humanized antibodies presented above were also confirmed for functional activity on primary human immune cells isolated from healthy donors (described in Example 7).

[0125] Example 5: Binding activity of different versions 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 a dilution series of anti-TIGIT antibodies. Goat anti-human IgG was used as a secondary antibody to detect antibody binding to the cell surface. EC of dose-dependent binding to human native TIGIT 50 Values ​​were determined by fitting the dose-response data to a four-parameter logistic model using GraphPad Prism. As shown in Figure 4A-B and Table 6, both humanized 1217 antibodies, hu1217-1-1 and hu1217-2-2, showed good binding affinity to native TIGIT on live cells. [Table 7]

[0126] 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: approximately 1 nM), which can compete with the CD266-PVR interaction (Yu X, et al. 2009 Nat. Immunol, 10:48-57).

[0127] To confirm 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 measured by flow cytometry (Figure 5A). Blockade of TIGIT-ligand interaction was quantitatively measured by adding serially diluted anti-TIGIT antibodies. As shown in Figure 5B, hu1217-2-2 / IgG1 (a humanized version containing wild-type IgG1 Fc region and with the same VH and VL sequences as hu1217-2-2 / IgG1mf) and hu1217-2-2 / IgG1mf showed IC of 0.64 and 0.55 μg / mL, respectively. 50 can block TIGIT binding to PVR in a dose-dependent manner. Similarly, the IC of hu1217-2-2 / IgG1 and hu1217-2-2 / IgG1mf in blocking the TIGIT-PVR-L2 interaction was 50 are 0.25 and 0.18 μg / mL, respectively.

[0128] 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 occurring T cells that recognize the human CMV PP65 peptide (NLVPMVATV, 495-503, HLA-A2.1 restricted) (Boeckh et al., 2011 J Clin Invest. 121:1673-80). Briefly, HLA-A2.1 + PBMCs from healthy donors were simulated with PP65 peptide (purity >98%, synthesized by GL Biochem, Shanghai) in complete RPMI containing 10% FBS for one week. 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 with an equal number of pp65-sensitized PBMCs in 96-well plates overnight in the presence or absence of anti-TIGIT antibody or blank control (medium only). As shown in Figure 6A-B, hu1217-2-2 / IgG1 dose-dependently promoted the secretion of IFN-γ into the cell culture supernatant of pp65-specific T cells for both donors.

[0129] Example 8: Anti-TIGIT antibodies enhanced NK cell-mediated cytotoxicity TIGIT is known to be constitutively expressed at relatively high levels on 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).

[0130] To confirm whether humanized anti-TIGIT antibodies could promote NK-mediated cytotoxicity, the NK cell line NK92MI was engineered to co-express both TIGIT and DNAM-1 receptors as effector cells by retroviral transduction (NK92MI / TIGIT-DNAM-1) according to a previously described protocol (Zhang et al., 2006 Cancer Res. 66:5927-5933). The PVR-expressing lung cancer cell line SK-MES-1 / PVR was also established as a target.

[0131] The cytotoxicity of NK92MI / TIGIT-DNAM-1 cells against SK-MES-1 / PVR cells was measured by LDH release assay using the CytoTox 96 non-radioactive cytotoxicity assay kit (Promega, Madison, WI). Briefly, NK92MI / TIGIT-DNAM-1 cells (8 × 10 5 ) in the presence of anti-TIGIT Ab (0.007–30 μg / mL) on SK-MES-1 / PVR cells (2 × 10 4) in 96-well V-bottom plates for 5 h. LDH release assay. Specific lysis was determined using the following equation: percentage of specific lysis = [(experimental-effector spontaneous-target spontaneous) / (target max-target spontaneous)] × 100. Results showed that anti-TIGIT antibody hu1217-2-2 / IgG1mf enhanced NK cell killing in a dose-dependent manner (EC 50 : 0.185 μg / mL) (Figures 7A-B).

[0132] Example 9: Anti-TIGIT antibodies can reduce the surface expression of TIGIT receptor via 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 (FcγR) causes downregulation of receptors on the cell surface (Carlsten et al. 2016 Clin Cancer Res; Beum et al. 2011 J. Immunology). Thus, downregulation of target receptors by trogocytosis may cause attenuation of signal transduction. Considering these observations, it is possible that hu1217-2-2 / IgG1 is a marker for FcγR downregulation. + It is possible that trogocytosis of the TIGIT receptor can be induced in the presence of TIGIT cells, resulting in reduced surface expression. To address this possibility, Jurkat / TIGIT cells were transfected with various FcγRs (FcγRIIA H131 , FcγRIIB, FcγRIIIA V158Biotin-labeled hu1217-2-2 / IgG1wt (humanized antibody containing the same VL and VH sequences as hu1217-2-2 / IgG1mf and wild-type IgG1 Fc region) or hu1217-2-2 / IgG1mf were incubated overnight with HEK cells expressing hu1217-2-2 / IgG1wt (humanized antibody containing the same VL and VH sequences as hu1217-2-2 / IgG1mf and wild-type IgG1 Fc region). Surface expression of the TIGIT receptor was determined using SA-APC (Biolegend). As shown in Figure 8, hu1217-2-2 / IgG1, but not hu1217-2-2 / IgG1mf, caused a significant decrease in TIGIT surface expression compared to negative control human IgG-treated cells, indicating that the reduction in surface TIGIT on Jurkat / TIGIT cells is FcγR binding dependent. Furthermore, the presence of 10% human serum (containing high levels of endogenous IgG) reduced the expression of FcγRIIA receptors. H131 -or FcγRIIIA V158 - but not FcγRIIB-mediated trogocytosis of TIGIT receptor, suggesting 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 findings (Ganesan LP, et al. 2012 J Immunol 189:4981-8; Taylor RP, et al. 2015 Blood 125:762-6).

[0133] 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.

[0134] ADCC using human PBMCs as target cells TIGIT antibody is + To determine whether T cells could induce ADCC, a flow cytometry-based ADCC assay was set up. The assay effector cell line, NK92MI / CD16V cells, expressed CD16 V158TIGIT expression was upregulated in NK92MI cells (ATCC) by co-transfecting an expression plasmid 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 9A, CD4 + Effector T cells (CD3 + CD4 + Foxp3 - ), CD8 + T cells and regulatory factor T cells (CD4 + Foxp3 + All T cells, including those from 10 healthy donors, expressed significant amounts of TIGIT. These activated PBMCs (from 3 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 in the presence of TIGIT antibodies (hu1217-2-2 / IgG1mf or hu1217-2-2 / IgG1wt, 30 μg / mL) or control antibodies (positive control anti-CD3 antibody OKT3 (5 μg / ml, Biolegend) or negative control human IgG, 30 μg / mL) for 40 h. Compared to human IgG and hu1217-2-2 / IgG1mf, hu1217-2-2 / IgG1wt could moderately deplete Tregs via ADCC. However, total T cells and CD8 + No significant ADCC effect was observed in T cells (Figure 9B).

[0135] CDC using human PBMCs as target cells Whether hu1217-2-2 / IgG1mf and hu1217-2-2 / IgG1wt induce CDC was determined using preactivated human PBMCs and fresh autologous serum from healthy donors. Cell lysis by CDC was measured by Celltiter glo assay kit (Promega, Beijing, China). Briefly, PBMCs were preactivated with PHA (10 μg / mL) for 3 days and then incubated overnight at 37°C in RPMI1640 with autologous serum (15%) and anti-TIGIT or control antibodies (0.01–100 μg / mL). Cell death by CDC 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 performed using a 96-well fluorometer (PHERA Star FS, BMG LABTECH), and CDC activity was calculated from the relative fluorescence unit (RFU) readings as follows: %CDC activity=[(RFU test-RFU background) / (RFU at total cell lysis-RFU background)]×100. Experimental results demonstrated 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 ( FIG. 10 ).

[0136] Example 11: pH-dependent binding affinity of Hu1217-2-2 / IgG1 To determine whether pH affects the binding properties of hu1217-2-2 / IgG1, target binding SPR experiments were performed with 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 immobilized hu1217-2-2 / IgG1 in running buffer HBS at pH 7.4 or pH 6.0.

[0137] As shown by the results listed in Table 7 below, hu1217-2-2 / IgG1 showed higher binding affinity (KD) and stronger binding signal (Rmax) for human TIGIT at pH 6.0 (acidic pH similar to the pH of the tumor microenvironment) when compared to data obtained at pH 7.4 (physiological pH). These results indicate the potential advantage of antibodies as therapeutic agents targeting TIGIT-positive lymphocytes in the tumor environment, as hu1217-2-2 / IgG1 may more selectively target TIGIT-positive lymphocytes in the tumor microenvironment while potentially reducing potential toxicity associated with activation of peripheral lymphocytes. [Table 8]

[0138] Example 12: Toxicology of hu1217-2-2 antibody Antibody hu1217-2-2 showed comparable binding affinity in TIGIT receptor occupancy assays using CD3+ splenocytes from humanized TIGIT knock-in mice compared to CD3+ human peripheral blood mononuclear cells (EC50 of 48.8ng / ml vs. 63.2ng / ml, respectively). Furthermore, hu1217-2-2 showed significant inhibition of GL261 tumor growth in humanized TIGIT knock-in mice at doses of 0.4mg / kg or higher administered intraperitoneally weekly.

[0139] The toxicity and safety profile of hu1217-2-2 was characterized in a 4-week repeat-dose toxicity study in humanized TIGIT knock-in mice and a 13-week repeat-dose toxicity study in cynomolgus monkeys. hu1217-2-2 was also evaluated in a 4-week repeat-dose study in humanized TIGIT knock-in mice bearing subcutaneous MC-38 tumors. Cynomolgus monkeys were considered a relevant species for toxicity studies based on the target sequence homology and interspecies TIGIT binding activity of hu1217-2-2.

[0140] No obvious toxicity was observed in monkeys receiving repeated doses of hu1217-2-2 at 10, 30, or 100 mg / kg every 2 weeks for 13 weeks. The toxicokinetic profile in the monkey study indicated that systemic exposure was proportional to dose and did not appear to differ between the sexes. No accumulation was observed in monkeys over the 13-week dosing period. No immunotoxicity was evident as no changes in clinical or histopathology were observed.

[0141] In in vitro cytokine release assays following treatment of non-activated peripheral blood mononuclear cells with hu1217-2-2, no significant increase in cytokine release was observed when compared to human IgG, indicating that hu1217-2-2 is unlikely to induce acute cytokine release syndrome.

[0142] Overall, no overt toxicity was observed in the monkey toxicity studies. No unexpected tissue cross-reactivity was found in human or monkey tissues. The toxicokinetic profile showed a dose-proportional increase in systemic exposure without obvious accumulation or sex differences. The no observed adverse effect level (NOAEL) for hu1217-2-2 in a 13-week monkey toxicity study was 100 mg / kg.

[0143] Example 13: Clinical pharmacology of hu1217-2-2 A total of 11 patients were treated with hu1217-2-2 at dose levels of 50 mg (n=1), 150 mg (n=3), 450 mg (n=4), and 900 mg (n=3) in combination with BGB-A317 at a dose of 200 mg. The maximum dose was hu1217-2-2 at 1800 mg combined with BGB-A317 at 200 mg Q3W. Serum concentrations of hu1217-2-2 declined biexponentially following intravenous infusion, and hu1217-2-2 exposure (Cmax and AUC) increased approximately dose-proportionally from 50 mg to 900 mg (Figure 11).

[0144] Peripheral TIGIT receptor occupancy data were available for 11 patients treated with hu1217-2-2 at doses of 50 mg (n=1), 150 mg (n=3), 450 mg (n=4), and 900 mg (n=3). Complete TIGIT receptor occupancy (100%) was observed on CD8, CD4, NK, and Treg cells in peripheral blood at all dose levels tested, including the lowest dose of 50 mg.

[0145] Example 14: hu1217-2-2 alone or in combination with BGB-A317 promotes IFN-γ secretion in a PBMC assay To determine whether the combination of hu1217-2-2 and BGB-A317 could enhance activation of primary human immune cells over monotherapy, PBMCs from healthy donors were pre-stimulated to upregulate TIGIT expression and used as effector cells. PD-L1 and T cell engager (OS8) positive A549 cells (A549 / OS8-PD-L1) were used as target cells. Pre-activated PBMCs were co-cultured with a mixture of A549 / OS8-PD-L1 and A549 / PD-L1 in the presence of the indicated concentrations of hu1217-2-2 and BGB-A317 or either antibody alone for 18 hours. IFN-γ production was measured by ELISA. IFN-γ secretion was used as a readout for T cell activation. The results showed that treatment of PBMCs with hu1217-2-2 increased IFN-γ production in a dose-dependent manner. The combination of hu1217-2-2 and BGB-A317 significantly enhanced IFN-γ production compared to the enhancement seen with hu1217-2-2 or BGB-A317 alone, indicating that combined blockade of TIGIT and PD1 can attenuate effector cell exhaustion following activation, data which are presented graphically in FIG. 12.

[0146] Example 15: hu1217-2-2 reduces tumor growth in a mouse glioma tumor model The antitumor activity of hu1217-2-2 was also tested in the GL261 mouse glioma cancer model in humanized TIGIT knock-in mice. GL261 cells (1 × 10 7) were implanted subcutaneously into humanized TIGIT knock-in mice. Three days after implantation, mice were randomly assigned to four groups and treated intraperitoneally with DPBS (vehicle) or hu1217-2-2 as indicated for 29 days. Tumor volumes were measured twice weekly. Data are presented as the mean tumor volume ± SEM of 12 animals in each group. As shown in Figure 13 and Table 8, once weekly (QW) administration of hu1217-2-2 induced dose-dependent antitumor activity. On the 29th day of treatment (7 days after the fourth dose), hu1217-2-2 induced significant TGI (56%, 94%, and 100% of TGI) at all doses tested (0.4, 2, and 10 mg / kg QW, respectively) (Table 8).

[0147] Blood samples (approximately 100 μL) were collected prior to dosing with hu1217-2-2 and at 2, 8, 24, 48, 96, and 168 hours after the first dose (three mice per time point, each mouse used no more than two time points). The concentration of hu1217-2-2 in serum was determined by ELISA assay, and the serum concentration of hu1217-2-2 at each time point was determined by fitting the dose-response data to a five-parameter logistic model. The pharmacokinetic profile of hu1217-2-2 at three doses (0.4, 2, and 10 mg / kg) was characterized for 7 days after the first injection, and a dose-dependent exposure (Cmax 1st dose and AUC0-168h) of hu1217-2-2 was observed (Table 9). The treatment did not significantly affect the body weight of the animals throughout the study. [Table 9] [Table 10]

[0148] For Table 9, blood samples (approximately 100 μL) were collected prior to dosing and 2, 8, 24, 48, 96, and 168 hours after the first dose of hu1217-2-2 (three mice per time point, each mouse used no more than two time points). The concentration of hu1217-2-2 in serum was determined by ELISA assay, and the serum concentration of hu1217-2-2 at each time point was determined by fitting the dose-response data to a five-parameter logistic model.

[0149] Example 16: Combination of hu1217-2-2 and anti-PD1 antibody in the MC38 mouse colon cancer model in a humanized TIGIT knock-in mouse model The antitumor activity of the combination of hu1217-2-2 and anti-mouse PD1 (muPD1) was examined in the MC38 mouse colon cancer model in humanized TIGIT knock-in mice. MC38 tumor cells (1 × 10 6 ) were implanted subcutaneously into humanized TIGIT knock-in mice. Seven days after implantation, mice were randomly assigned to four groups and treated with vehicle (DPBS), muPD1 (1 mg / kg intraperitoneally once every 5 days (Q5D)), hu1217-2-2 (3 mg / kg intraperitoneally Q5D), or combinations as indicated.

[0150] Tumor volumes were measured twice weekly. Data are presented as the mean tumor volume ± SEM of 10 animals. p values ​​were calculated using Student's t-test. The combination of hu1217-2-2 and muPD1 showed a higher TGI (102%) than either antibody alone (73% for muPD1 alone or 11% for hu1217-2-2 alone) (Figure 14 and Table 10). The treatment did not significantly affect the body weight of the animals throughout the study. [Table 11]

[0151] Example 17: Administration of anti-TIGIT and anti-PD1 antibodies Doses of hu1217-2-2 ranged from 50 mg to 900 mg, with up to 1800 mg once every 3 weeks, in combination with the anti-PD1 antibody BGB-A317 at 200 mg once every 3 weeks, were tested in the ongoing Ph1 / 1b study. All hu1217-2-2 dose levels tested cleared the dose-limiting toxicity (DLT) window without significant safety or tolerability events. Exposure to hu1217-2-2 increased approximately dose-proportionally. Therefore, an administered dose of 900 mg of hu1217-2-2 was selected as the recommended dose for Phase II.

[0152] At all doses tested in this study, full TIGIT receptor occupancy was observed on circulating T cells and NK cells in peripheral blood. As mentioned above, the data showed that 100% receptor occupancy was achieved at 50 mg. The 900 mg dose of hu1217-2-2 is expected to fully enhance the effective concentration and potential saturation of TIGIT receptors in tissues throughout the dosing interval.

[0153] Pharmacokinetic data for hu1217-2-2 from this study showed no significant relationship between hu1217-2-2 exposure and patient weight, therefore indicating that a fixed dose of hu1217-2-2 is optimal. The combination of hu1217-2-2 antibody and BGB-A317 was chosen to have a fixed dose of 900 mg of hu1217-2-2 and a fixed dose of 200 mg of BGB-A317 administered intravenously once every 3 weeks. The dose of BGB-A317 was selected based on comparable safety and efficacy profiles of 2-5 mg / kg in previous BGB-A317 studies.

[0154] Example 18: hu1217-2-2 antibody in combination with BGB-A317 antibody A phase 1 study was initiated to investigate the safety / tolerability, pharmacokinetics (PK), and preliminary antitumor activity of hu1217-2-2 in combination with BGB-A317, with or without chemotherapy, in patients with unresectable locally advanced or metastatic solid tumors.

[0155] Patients enrolled in this study were treated with escalating doses of hu1217-2-2 (50, 150, 450, or 900 mg) in combination with 200 mg of BGB-A317 every 3 weeks. All patients cleared the dose-limiting toxicity (DLT) period without toxicity that required patients to be removed from treatment or dose reduction. Therefore, hu1217-2-2 appears to be safe and well tolerated.

[0156] Example 19: hu1217-2-2 antibody in combination with anti-PD1 antibody (BGB-A317) in the treatment of limited-stage small cell lung cancer BGB-A317 has been tested in phase 2 trials in locally advanced or metastatic non-squamous NSCLC, squamous NSCLC, and ES-SCLC as a single agent or in combination with chemotherapy, and BGB-A317 + chemotherapy showed anticancer activity. Thirteen of 17 patients with small cell lung cancer (SCLC) responded to BGB-A317 + chemotherapy in the first-line setting. Two phase 3 trials of BGB-A317 + platinum doublet chemotherapy as first-line treatment for locally advanced and metastatic non-squamous and squamous NSCLC have shown positive results with progression-free survival as the primary endpoint. The hu1217-2-2 and BGB-A317 antibodies have non-overlapping anticancer mechanisms and may have synergistic and / or additive activity for the treatment of SCLC.

[0157] Example 20: hu1217-2-2 antibody in combination with BGB-A317 anti-PD1 antibody and chemotherapy in the treatment of limited-stage small cell lung cancer A novel immunotherapy combination, anti-PD-L1 + anti-TIGIT administered simultaneously with radiation therapy, has been evaluated in a mouse model (Grapin et al., J Immunother Cancer 2019;7:160-71). In this study, tumor response during treatment with anti-PD-L1 + anti-TIGIT + radiation therapy was compared to either monotherapy + radiation therapy or radiation therapy alone. In the CT26 model, radiation therapy was dramatically more effective when combined with anti-TIGIT and anti-PD-L1 therapy, with a complete response rate of 9 / 10 (90%) mice compared to 2 / 10 (20%), 3 / 10 (30%), and 0 / 10 (0%) responses for anti-TIGIT, anti-PD-L1, and radiation therapy alone, respectively, at 3 × 8 Gy hypofractionated radiation therapy. Complete response rates of anti-TIGIT and anti-PD-L1 in combination with 18 × 2 Gy conventionally fractionated radiotherapy were observed in 7 / 12 (58.3%) mice compared with 3 / 12 (25%), 8 / 12 (25%), 8 / 12 (66.7%), and 1 / 10 (10%) mice for anti-TIGIT, anti-PD-L1, and radiotherapy alone at the same fractionated RT. SCLC is characterized by relatively high TIGIT and PVR expression, and high PVR expression correlates with poor prognosis (Yu et al., Cancer Res. 2018;1538-7445.AM2018-3637).

[0158] The new treatment strategy, combining hu1217-2-2 and BGB-A317 with chemoradiotherapy, aims to enhance the current modest improvements in limited-stage specific SCLC, where the standard of care remains chemotherapy combined with radiation therapy. The study will combine four cycles of concurrent chemoradiotherapy with BGB-A317 administered intravenously at 200 mg once every three weeks, followed by BGB-A317 administered intravenously at 200 mg once every three weeks.

[0159] This is compared to BGB-A317, 200 mg intravenously every 3 weeks, plus hu1217-2-2, 900 mg intravenously every 3 weeks, given with concurrent chemoradiation for 4 cycles, followed by BGB-A317, 200 mg intravenously every 3 weeks, plus hu1217-2-2, 900 mg intravenously every 3 weeks.

[0160] The preferred chemotherapy regimen is cisplatin 75 mg / m2 on day 1 and etoposide 100 mg / m2 on days 1, 2, and 3 for the first four cycles. After the first cycle, dose adjustments are permitted depending on renal, hematologic, or other toxicities. Radiotherapy should be started simultaneously with C1D1 chemotherapy, and tolerable, no later than day 1 of cycle 3 where chemotherapy is administered. The recommended total dose is 45 Gy over 3 weeks, or 60–70 Gy over 6.5 weeks, at the investigator's discretion. Prophylactic cranial irradiation is permitted as a total dose of 25 Gy (25 Gy in 10 daily fractions) over 2 weeks, according to local standard of care. This regimen is described in more detail in Table 11 below. [Table 12]

[0161] Example 21: hu1217-2-2 antibody in combination with BGB-A317 anti-PD1 antibody in the treatment of non-small cell lung cancer (NSCLC) Lung cancer is the most common cancer, with approximately 2.09 million new cases diagnosed and 1.76 million deaths worldwide in 2018, representing the highest incidence and most common cancer-related mortality rate among cancers. The disease is more prevalent in men than women, accounting for 16.8% of all cancers in men and 8.8% of all cancers in women. In China, lung cancer is the leading cause of cancer-related deaths in both men and women, with an estimated 610,200 deaths and 733,300 new cases in 2015 (Chen et al., CA Cancer J Clin. 2016;66(2):115-32). Non-small cell lung cancer (NSCLC) originates from the epithelial cells of the lung and accounts for 80%-85% of all lung cancers. There are three major histological subtypes of NSCLC: adenocarcinoma, which comprises 40% of all NSCLC, squamous cell carcinoma, which comprises 25%, and large cell carcinoma, which comprises 10% of all NSCLC.

[0162] The prognosis of patients with NSCLC is relatively poor, depending largely on the stage at which the cancer is detected. Lung cancer is staged worldwide according to the tumor, lymph node, and metastasis (TNM) Classification of Malignant Tumors, Seventh Edition (Goldstraw et al., J Thorac Oncol 2007;2(8):706-14). If lung cancer is diagnosed at an early stage, it can be cured by surgery or chemoradiotherapy. Unfortunately, most cases of lung cancer are detected at a relatively late stage. Approximately one-third of patients with NSCLC present with locally advanced stage III disease, including locoregional mediastinal lymph node or organ involvement. The 5-year survival rate for patients with stage III disease ranges from 36% (stage IIIA) to 13% (stage IIIC). 55% of newly diagnosed NSCLC patients have distant metastases (stage IV). Stage IVA patients present with contralateral lung involvement, malignant pleural effusion, and malignant pericardial effusion, or metastases in a single location outside the chest, e.g., a distal lymph node or organ such as the brain, liver, or bone. Stage IVB patients present with disease that has spread to multiple locations in distal lymph nodes or organs. The 5-year survival rate for patients with stage IV NSCLC is 5% (Siegel et al., A Cancer Journal for Clinicians 2020;70(1):7-30). Patients with squamous NSCLC may receive a platinum-based doublet containing either gemcitabine, vinorelbine, or a taxane. Patients with nonsquamous NSCLC are given pemetrexed-based combination chemotherapy with carboplatin or cisplatin. Single-agent pembrolizumab was approved by the U.S. Food and Drug Administration (FDA) as first-line therapy for patients with metastatic NSCLC whose tumors express high levels of PD-L1 (tumor proportion score [TPS] ≥ 50%) based on results from the KEYNOTE 024 trial (Reck et al., N Engl J Med. 2016;375(19):1823-33).In this study, pembrolizumab demonstrated a significant improvement in 6-month OS rate (80.2% vs. 72.4% [95% CI: 0.4, 0.9]) and progression-free survival (PFS; 10.3 vs. 6 months [95% CI: 6.7, NR]) compared with platinum-based chemotherapy. Pembrolizumab in combination with pemetrexed and platinum-based therapy has also been accelerated approved by the FDA as first-line treatment for patients with stage IIIB or stage IV nonsquamous NSCLC and without EGFR or ALK genomic aberrations, based on the results of KEYNOTE 021 (Langer et al., Lancet Oncol. 2016;17(11):1497-1508).

[0163] In NSCLC, upregulation of TIGIT expression in tumor-infiltrating lymphocytes has been reported (Tassi et al., Cancer Res. 2017;77:851-61). Blockade of the TIGIT receptor alone, or in combination with PD1 / PD-L1 blockade, has been shown to rescue functionally "exhausted" T cells both in vitro and in vivo (Johnston et al., Cancer Cell. 2014;26:923-3; Chauvin et al., J Clin Invest. 2015;125:2046-58). In mouse models, TIGIT blockade in combination with anti-PD1 / PD-L1 antibodies showed significantly better antitumor efficacy than either monotherapy (Johnston et al., 2014 supra; Dixon et al., J Immunol. 2018;200:3000-7).

[0164] This trial will evaluate hu1217-2-2 in combination with BGB-A317 in treatment-naïve, PD-L1-selected patients with locally advanced, unresectable, or metastatic NSCLC without EGFR or ALK genomic aberrations. Based on the mechanism of action, combined blockade of TIGIT with hu1217-2-2 and PD1 with BGB-A317 is expected to improve efficacy over that observed with BGB-A317 alone. BGB-A317 was administered at 200 mg, followed by hu1217-2-2 at 900 mg on day 1 of each 21-day cycle (once every 3 weeks). For NSCLC (squamous), early readout in patients treated with this dose showed stable disease (SD) with a lesion change from baseline of -11.1.

[0165] Example 22: hu1217-2-2 antibody in combination with BGB-A317 anti-PD1 antibody in the treatment of nasopharyngeal carcinoma Nasopharyngeal carcinoma (NPC) is relatively rare worldwide, with 129,079 new cases accounting for only 0.7% of all cancers diagnosed in 2018, with over 70% of new cases occurring in East and Southeast Asia, with an age-standardized rate (global) of 3.0 per 100,000 in China. Among endemic regions, China had a higher incidence in men than women in 2015, with a ratio of approximately 2.5 to 1 (Bray et al.,CA Cancer J Clin.2018;68(6):394-424 and Chen et al.,CA Cancer J Clin.2016;66(2):115-32).

[0166] The incidence and mortality of NPC has declined in some endemic regions, likely as a result of lifestyle changes and advances in management, including improved radiotherapy techniques, more widespread application of chemotherapy, and more accurate disease staging (Lau et al., BMC Cancer 13:298, 2013; Hsu et al., Cancer Epidemiol Biomarkers Prev 15:856-861, 2006). NPC remains the leading cause of cancer death, with a global mortality rate of approximately 72,987 cases per year (Bray et al., supra). Depending on the degree of differentiation, NPC is classified into three pathological subtypes based on the World Health Organization (WHO) criteria. Differentiated tumors with surface keratin are defined as type I, while types II and III refer to nonkeratinizing differentiated and undifferentiated tumors, respectively. In 1991, types II and III were merged into a single category of nonkeratinizing carcinoma. In areas where NPC is endemic, the nonkeratinizing subtype constitutes most cases (>95%) and is always associated with Epstein-Barr virus (EBV) infection, whereas in other parts of the world type I disease is more common (Wei et al., Lancet 2005;365:2041-54; Nicholls et al., Adv Anat Path 1997;4:71-84). EBV infection has been the most widely studied etiology of NPC. Based on in situ hybridization techniques for EBV-encoded RNA, the virus is detected exclusively in all tumor cells but not in normal nasopharyngeal epithelium, suggesting that EBV activation is required for the pathogenesis of NPC (Pathmanathan et al., N Engl J Med.1995;333:Chan et al., Cancer Res 2000;60:5365-70). NPC is endemic in southern China and Southeast Asia. The standard first-line treatment is platinum-containing multi-agent chemotherapy. However, no consensus has been reached regarding treatment beyond first-line therapy. There remains an unmet clinical need for novel agents that are more effective and well-tolerated.

[0167] As mentioned above, anti-TIGIT antibodies provide a potential mechanism to rescue immune cells from the immunosuppressive tumor microenvironment, thereby inducing an efficient anti-tumor immune response. Studies have shown that the TIGIT pathway cooperates with PD1 to maximize the suppression of effector tumor-infiltrating lymphocytes (TILs) and promote resistance to anti-PD1 therapy.

[0168] Standard of care for first-line recurrent or metastatic NPC consists of platinum-containing polychemotherapies. However, for patients with platinum-refractory / recurrent or metastatic NPC, there are no standard of care options beyond first-line. Recent clinical studies of other anti-PD1 antibodies, such as nivolumab, pembrolizumab, camrelizumab, and toripalimab monotherapy, have demonstrated favorable clinical efficacy in patients with advanced NPC in the second-line or subsequent treatment settings (median ORR, 20.5%-34%; median OS, 16.5-17.4 months; median PFS, 1.9-6.5 months). Preliminary data of BGB-A317 as monotherapy in an expansion cohort of NPC patients in a previous clinical trial are also promising (median ORR: 43%; median OS, not reached; median PFS, 12.4 months). However, one of the current challenges is that PD1 blockade only benefits a subset of patients with refractory / recurrent or metastatic NPC, leaving an unmet need for more effective treatments for this patient population.

[0169] In this trial, patients will receive either BGB-A317 + hu1217-2-2 as a comparator, or BGB-A317 alone. Patients will receive 200mg BGB-A317 + 900mg hu1217-2-2 intravenously every 3 weeks until investigator-assessed disease progression per RECIST v1.1, unacceptable toxicity, or loss of clinical benefit, whichever occurs first.

[0170] During the study, tumor assessments will be performed every 6 weeks (± 7 days) for the first 24 weeks, every 9 weeks (± 7 days) for the remainder of the first year, and every 12 weeks (± 7 days) thereafter based on RECIST v1.1, regardless of dose delays to manage toxicity. Blood samples will be collected at various time points to determine the PK characteristics of the combination of BGB-A317 and hu1217-2-2. Optional blood samples will be taken (10 mL for each time point) at baseline (before dose on day 1 of cycle 1), at first tumor response (before dose on day 1 of the next cycle), and at the end of treatment (EOT) visit after disease progression. [Table 13] [Table 14]

[0171] Preliminary clinical results demonstrated that patients with nasopharyngeal carcinoma were free of progression after 36 weeks of treatment with hu1217-2-2 in combination with BGB-A317. Tumor lesion (TL) size was 31 mm at initial onset and regressed to 23 mm over the 36-week treatment period, resulting in a 25.81% reduction in tumor size from baseline and no change from nadir. [Table 15]

[0172] Example 23: hu1217-2-2 antibody in combination with BGB-A317 anti-PD1 antibody in the treatment of esophageal cancer Esophageal cancer is the seventh most common cancer and the sixth most common cause of cancer deaths worldwide. The highest incidence areas extend from northern Iran through the Central Asian republics to northern China. The most common histological type of esophageal cancer is esophageal squamous cell carcinoma (ESCC), which is more common in Eastern Europe and Asia. More than two-thirds of patients diagnosed with esophageal cancer suffer from advanced or metastatic disease, with a median survival of 8-10 months and an expected 5-year survival rate of less than 5%. These data, combined with the relative lack of effective treatments, indicate a large unmet medical need in esophageal cancer in general and in patients diagnosed with ESCC in particular.

[0173] Anti-PD1 therapy has shown superior efficacy compared with chemotherapy as second-line treatment for ESCC. The KEYNOTE-181 trial enrolled 628 patients with recurrent locally advanced or metastatic esophageal cancer who had progressed after or had progressed after one prior systemic treatment for progressive disease, and observed a significant improvement in the primary endpoint of OS in patients treated with pembrolizumab compared with chemotherapy (10.3 vs. 6.7 months, HR, 0.62; 95% CI: 0.46-0.90). The ATTRACTION-3 trial enrolled 419 patients with unresectable advanced, recurrent, or metastatic ESCC refractory or intolerant to one or more fluoropyrimidine- and platinum-based regimens, and reported a significant improvement in the primary endpoint of OS in patients treated with nivolumab compared with investigator-choice taxane chemotherapy (10.9 vs. 8.4 months, HR, 0.77; 95% CI: 0.62-0.96). An overall survival benefit was observed regardless of PD-L1 expression level.

[0174] Administration of anti-TIGIT antibodies provides a potential mechanism to rescue immune cells from the immunosuppressive tumor microenvironment, thereby inducing an efficient anti-tumor immune response. Studies have shown that the TIGIT pathway cooperates with PD1 to maximize the suppression of effector TILs and also promotes resistance to anti-PD1 therapy. As described in the above studies, blocking antibodies targeting the PD1 / PD-L1 pathway have achieved remarkable results in the treatment of ESCC. Thus, anti-TIGIT antibodies may significantly improve and / or prolong the therapeutic effect of anti-PD1 therapy in ESCC.

[0175] Patients with unresectable locally advanced, recurrent or metastatic ESCC who have failed first-line chemotherapy represent a patient population with significant unmet medical need. Therefore, this trial is designed to compare the efficacy of BGB-A317+hu1217-2-2 with the efficacy of BGB-A317 as a single agent in treating patients with PD-L1 positive and esophageal cancer. Esophageal cancer may be unresectable, locally advanced, recurrent or metastatic ESCC.

[0176] Patients will receive BGB-A317 (200 mg) + hu1217-2-2 (900 mg) intravenously once every 3 weeks until investigator assessment for RECISTv1.1, unacceptable toxicity, or patient withdrawal. Patients will receive 200 mg of BGB-A317 on day 1 of each 21-day cycle (i.e., once every 3 weeks), followed by 900 mg of hu1217-2-2 on day 1 of each 21-day cycle. [Table 16] [Table 17]

[0177] Preliminary clinical results showed that patients with esophageal cancer were progression-free after 6 weeks of administration of hu1217-2-2 in combination with BGB-A317. The size of the tumor lesion (TL) was 38 mm at first onset and regressed to 23 mm during the treatment period, resulting in a 39.47% reduction from baseline and nadir. [Table 18]

[0178] Example 24: hu1217-2-2 antibody in combination with BGB-A317 anti-PD1 antibody in the treatment of cervical cancer Cervical cancer is the fourth most common cancer among women and the fourth leading cause of cancer deaths in women, with approximately 570,000 new cases diagnosed and 311,000 deaths worldwide in 2018. The estimated age-standardized incidence rate of cervical cancer is 13.1 per 100,000 women worldwide, with incidence rates ranging from less than 2 to 75 per 100,000 women. China and India together account for more than one-third of the global burden of cervical cancer, with 106,000 cases and 48,000 deaths in China and 97,000 cases and 60,000 deaths in India (Arbyn et al., Lancet Glob Health. 2020;8:e191-e203).

[0179] It is estimated that recurrent or metastatic cervical cancer occurs in 15-61% of women, usually within the first 2 years after completing primary treatment. For patients who progress after first-line platinum-based therapy, treatment options are limited and no standard of care has been established. Single cytostatic agents have only induced limited response rates and duration. This represents a significant unmet medical need in patients with cervical and / or metastatic cervical cancer.

[0180] The most significant cause of cervical cancer is persistent papillomavirus infection. Human papillomavirus (HPV) is detected in 99% of cervical tumors, especially oncogenic subtypes such as HPV16 and 18. HPV is recognized as the most important etiological agent of cervical cancer, and thus cervical cancer is an attractive target for immunotherapy, since viral proteins are potent immune stimulators. In June 2018, pembrolizumab received accelerated approval from the US FDA for the treatment of patients with cervical cancer whose disease has progressed during or after chemotherapy and whose tumors express programmed death ligand 1 (PD-L1).

[0181] Considering the promising antitumor activity of anti-PD1 antibodies reported in this indication, and the scientific evidence that anti-TIGIT antibodies may improve the therapeutic efficacy of anti-PD1 therapy, the combination of hu1217-2-2 and BGB-A317 may provide significant clinical benefit in the treatment of cervical cancer.

[0182] The trial is to evaluate the administration of hu1217-2-2 in combination with BGB-A317 in patients with cervical cancer, metastatic cervical cancer, or previously treated recurrent cervical cancer. In the first phase of the trial, hu1217-2-2 will be administered in combination with BGB-A317 to patients with cervical cancer regardless of PD-L1 expression. Patients will be randomized in a 1:1 ratio to receive either hu1217-2-2 (900 mg every 3 weeks (Q3W)) in combination with BGB-A317 (200 mg Q3W) or BGB-A317 (200 mg Q3W) as monotherapy. After the first phase, more patients will be recruited. These patients will receive a combination of hu1217-2-2 (900 mg Q3W) and BGB-A317 (200 mg Q3W).

[0183] Example 25: hu1217-2-2 antibody in combination with BGB-A317 anti-PD1 antibody in the treatment of solid tumors hu1217-2-2 was administered in combination with BGB-A317 in a clinical trial targeting solid tumors. Table 18 shows the response of seven (7) patients who received hu1217-2-2 in combination with BGB-317 at varying doses of hu1217-2-2 while maintaining the dose of BGB-A317 at 200 mg. In the following cases, tumor lesions (TL) showed regression under combination therapy, with six patients achieving stable disease (SD) and one patient showing a partial response (PR). In general, tumors regressed or did not progress after combination treatment. For example, a partial responder gastric / gastroesophageal junction patient showed a TL size of 84 mm, but after 6 weeks of treatment, the TL size decreased to 53 mm, resulting in a 36.9% TL reduction from baseline and nadir. [Table 19-1] [Table 19-2]

[0184] Example 26: hu1217-2-2 antibody in combination with BGB-A317 anti-PD1 antibody in dose escalation A phase 1 dose escalation study was conducted in patients with advanced, metastatic, unresectable solid tumors for whom standard therapies were ineffective, intolerable, or unavailable. Patients received a 50 mg dose of hu1217-2-2 intravenously (IV) as a single agent on day 1 of cycle 1, followed by 200 mg IV of BGB-A317 on day 8 of cycle 1. If tolerated, patients received four escalating doses of hu1217-2-2 ranging from 150 to 900 mg, plus BGB-A317 200 mg continuously on day 29 and every 3 weeks (Q3W) thereafter until discontinuation. A patient with nasopharyngeal carcinoma had stable disease on a combination of hu1217-2-2 150 mg (Q3W) and BGB-A317 200 mg (Q3W) for the longest period of the study, 54 weeks. The combination of hu1217-2-2 at 450 mg (Q3W) and BGB-A317 at 200 mg (Q3W) produced stable disease in a patient with uterine cancer and another with basal cell carcinoma. At this same dose, a patient with gastric cancer showed a partial response. Finally, the combination of hu1217-2-2 at 900 mg (Q3W) and BGB-A317 at 200 mg (Q3W) produced stable disease in six patients, two with renal carcinoma, one with melanoma, one with sarcoma, one with pancreatic carcinoma, and one with salivary gland carcinoma. This dose also produced a partial response in one patient with mesothelioma.

[0185] Example 27: hu1217-2-2 antibody in combination with BGB-A317 and BAT1706 antibodies for the treatment of hepatocellular carcinoma BAT1706 is a biological drug similar to bevacizumab injection (bevacizumab injection, trade name Avastin®). Bevacizumab is a recombinant humanized immunoglobulin G1 (IgG1) monoclonal antibody that binds to human vascular endothelial growth factor (VEGF) with high affinity. Bevacizumab can selectively bind to all human VEGF A isoforms with high affinity and blocks the binding of VEGF to VEGF receptor 1 (VEGFR-1) and VEGF receptor 2 (VEGFR-2), thereby neutralizing the biological activity of VEGF. Bevacizumab is currently the only anti-tumor angiogenesis drug with proven clinical efficacy in extending overall survival and progression-free survival in cancer patients. Several randomized controlled phase II and phase III studies have shown that treatment with bevacizumab in combination with standard chemotherapy results in statistically significant improvements in overall survival, progression-free survival, and overall remission rate compared to the use of standard chemotherapy alone.

[0186] The results of a comparative in vitro pharmacodynamic study showed that both BAT1706 and bevacizumab exhibited specific binding to VEGF, consistent with literature reports. The biological activity of BAT1706 was measured using a human umbilical vein endothelial cell (HUVEC) proliferation inhibition assay. The results demonstrated that BAT1706 exhibited a dose-dependent relationship with HUVEC proliferation similar to that of bevacizumab, and both exhibited potent neutralizing effects against VEGF. The biological activity of BAT1706 was found to be comparable to that of bevacizumab within the defined range of (1.0±0.2)×104U / mg.

[0187] The antitumor effects of BAT1706 and bevacizumab were compared in several human cancer models (NSCLC, ovarian cancer, and rhabdomyosarcoma). NCI-H358 human NSCLC cells were subcutaneously inoculated into nonobese diabetic / severe combined immunodeficiency (NOD / SCID) female mice to establish human NSCLC xenograft models. Animals were divided into five groups, each consisting of eight female mice: Vehicle control group Bevacizumab 5.0mg / kg group Bevacizumab 0.5mg / kg group BAT1706 5.0mg / kg group BAT1706 0.5mg / kg group. The mean tumor volume was 128 mm 3 The tumors were allowed to grow until 100%. Treatments were then administered by tail vein injection on days 1, 3, and 5 of each week for the following 3 weeks. Efficacy was measured by tumor weight, tumor volume, and relative tumor growth inhibition (TGI). Tumor latency was also observed but not assessed for statistical significance. In this study, the target tumor weights used to evaluate tumor latency compared to the vehicle control group were the same (600 mm3) for BAT1706 and bevacizumab. The mean tumor volumes of the BAT1706 5 mg / kg and bevacizumab 5 mg / kg groups, but not the BAT1706 0.5 mg / kg and bevacizumab 0.5 mg / kg groups, showed statistically significant differences from the vehicle control group 1 day after the end of treatment. The tumor weight data were consistent with the tumor volume data. Of note, the tumor volume and tumor weight results for the paired doses of BAT1706 and bevacizumab (0.5 or 5 mg / kg) were not statistically different. The relative TGI(%) of BAT1706 at 5 mg / kg and bevacizumab at 5 mg / kg was 94% and 93%, respectively. The relative TGI(%) of BAT1706 at 0.5 mg / kg and bevacizumab at 0.5 mg / kg was 67% and 63%, respectively. In summary, BAT1706 showed similar efficacy to bevacizumab in a dose-dependent manner.

[0188] Similar results were seen in SK-OV-3 human ovarian cancer cell xenograft mouse models and 673 human rhabdomyosarcoma cell xenograft mouse models, with BAT1706 demonstrating efficacy similar to bevacizumab in a dose-dependent manner. There were no deaths or signs of severe toxicity in all treatment arms of both studies, and both antibodies were well tolerated throughout treatment.

[0189] A Phase I study conducted in New Zealand evaluated the safety of BAT1706 and compared it with EU-bevacizumab and US-bevacizumab in healthy subjects. A total of 125 healthy subjects received a single dose of 1 mg / kg of one of three investigational drugs, BAT1706, EU-bevacizumab, or US-bevacizumab, administered as a 90-minute IV infusion, and the results of the study revealed that a single IV infusion of BAT1706 was safe and well tolerated, with mild injection-site reactions.

[0190] Another Phase I study (BAT1706-002-CR) conducted in China also evaluated the safety of BAT1706 and compared it with bevacizumab (Europe) in healthy subjects. A total of 80 healthy subjects received a single dose of 1 mg / kg of the investigational drug (39 subjects in the BAT1706 group and 41 subjects in the bevacizumab group). The study revealed that both drugs exhibited good safety and tolerability profiles. No positive anti-drug antibody results were reported in any subjects in either the New Zealand or Chinese studies.

[0191] Hepatocellular carcinoma (HCC) is a major global health problem, accounting for 85-90% of all reported liver cancer cases (HCC is a term often used interchangeably) (El-Serag et al.,Gastroenterology 2007;132(7):2557-76). According to the World Health Organization's GLOBOCAN 2012 database, liver cancer was the sixth most common type of cancer that year, with 782,000 new cases occurring worldwide; liver cancer is also the second leading cause of cancer-related mortality, responsible for an estimated 746,000 deaths (Torre et al.,CA Cancer J Clin. 2015;65(2):87-108). Most HCC cases (>80%) occur in East Asia and sub-Saharan Africa, with a typical incidence rate of >20 per 100,000. China alone accounts for approximately 50% of new HCC cases and HCC-related deaths worldwide (Torre, supra). Southern European countries such as Spain, Italy, and Greece tend to have relatively moderate incidence of HCC (approximately 10-20 per 100,000), while North and South America, Northern Europe, and Oceania have relatively low incidence of HCC (less than 5 per 100,000) (El-Serag et al., Gastroenterology. 2012;142(6):1264-73).

[0192] As previously mentioned, anti-PD-1 / PD-L1 inhibitor monotherapy has shown clinical benefit in previously treated HCC (Qin et al., Lancet Oncol. 2020;21(4):571-80). However, no significant improvement has been shown compared with sorafenib in first-line HCC patients (Yau et al., Ann Oncol. 2019;30,(Suppl 5);v874-v75). Considering the promising antitumor activity of anti-PD-1 antibodies reported in HCC and that TIGIT may improve the therapeutic efficacy of anti-PD-1 therapy, the combination of hu1217-2-2 and BGB-A317 may provide significant clinical benefit in this indication.

[0193] Single-agent activity of bevacizumab has been observed in patients with HCC (Boige et al., Oncologist. 2012;17:1063-72; Siegel J Clin Oncol 2008;26:2992-8). Bevacizumab, in combination with PD-L1 inhibitors, has shown immunomodulatory effects in other tumor types, with promising clinical benefits and a favorable safety profile (Wallin et al., Nat Commun. 2016;7:12624). Positive results from the IMbrave150 and Orient32 studies demonstrate synergistic effects of combining anti-PD-1 / PD-L1 inhibitors with antiangiogenic agents.

[0194] In a study investigating the efficacy and safety of hu1217-2-2 in combination with BGB-A317+BAT1706 as first-line treatment of patients with advanced HCC, the study will enroll approximately 90 patients randomized in a 2:1 ratio to one of two treatment arms. Group A (n=60): hu1217-2-2 900 mg intravenously once every 3 weeks (21-day cycle) + BGB-A317 200 mg intravenously once every 3 weeks (21-day cycle) + BAT1706 15 mg / kg intravenously once every 3 weeks (21-day cycle). Arm B (n=30): BGB-A317 200 mg intravenously once every 3 weeks (21-day cycle) + BAT1706 15 mg / kg intravenously once every 3 weeks (21-day cycle).

[0195] As disclosed above, targeting TIGIT provides a potential mechanism for rescuing immune cells from the immunosuppressive tumor microenvironment, thereby inducing an efficient antitumor immune response. Studies have shown that the TIGIT pathway cooperates with PD-1 to maximize the suppression of effector tumor infiltrating lymphocytes (TILs), as well as promote resistance to anti-PD-1 therapy. Antibodies targeting the PD-1 / PD-L1 pathway have been successful in treating HCC, so treatment with hu1217-2-2 in combination with BGB-A317 and BAT1706 may significantly improve the treatment of HCC.

Claims

1. Use of an anti-TIGIT antibody or an antigen-binding fragment thereof in combination with an anti-PD1 antibody or an antigen-binding fragment thereof in the preparation of a medicament for treating cancer.

2. 2. The use according to claim 1, wherein the anti-PD1 antibody or antigen-binding fragment thereof is capable of binding to human TIGIT and comprises one of the following: (i) a heavy chain variable region comprising (a) an HCDR (heavy chain complementarity determining region) 1 of SEQ ID NO: 3, (b) an HCDR2 of SEQ ID NO: 13, and (c) an HCDR3 of SEQ ID NO: 5; and (d) an LCDR (light chain complementarity determining region) 1 of SEQ ID NO: 6, (e) an LCDR2 of SEQ ID NO: 7, and (f) an LCDR3 of SEQ ID NO: 8; or (ii) a heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO:3, (b) an HCDR2 of SEQ ID NO:4, and (c) an HCDR3 of SEQ ID NO:5; and (d) an LCDR1 of SEQ ID NO:6, (e) an LCDR2 of SEQ ID NO:7, and (f) an LCDR3 of SEQ ID NO:8; Including, use.

3. The anti-TIGIT antibody or antigen-binding fragment thereof is (i) a heavy chain variable region (VH) comprising SEQ ID NO: 19, and a light chain variable region (VL) comprising SEQ ID NO: 21; (ii) a heavy chain variable region (VH) comprising SEQ ID NO: 14, and a light chain variable region (VL) comprising SEQ ID NO: 16; or (iii) a heavy chain variable region (VH) comprising SEQ ID NO: 9, and a light chain variable region (VL) comprising SEQ ID NO: 11; The use according to claim 2, comprising:

4. The anti-PD1 antibody or antigen-binding fragment thereof is capable of binding to human PD1 and has the following characteristics: (a) a heavy chain variable region comprising an HCDR1 of SEQ ID NO:25, (b) an HCDR2 of SEQ ID NO:26, and (c) an HCDR3 of SEQ ID NO:27; and (d) an LCDR1 of SEQ ID NO:28, (e) an LCDR2 of SEQ ID NO:29, and (f) an LCDR3 of SEQ ID NO:

30. The use according to claim 1, comprising:

5. The use of claim 4, wherein the anti-PD1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 32 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:

34.

6. The use of claim 4 or 5, wherein the anti-PD1 antibody or antigen-binding fragment thereof comprises an IgG4 constant domain comprising SEQ ID NO:

35.

7. The use according to claim 1, wherein the anti-TIGIT antibody or antigen-binding fragment thereof is an antibody fragment selected from the group consisting of Fab, Fab'-SH, Fv, scFv, and (Fab')2 fragments.

8. The anti-PD1 antibody or antigen-binding fragment thereof is selected from the group consisting of Fab, Fab'-SH, Fv, scFv, and (Fab') 2 The use according to claim 1 , wherein the antibody is an antibody fragment selected from the group consisting of antibody fragments.

9. The use of claim 1, further in combination with an anti-VEGF antibody or an antigen-binding fragment thereof.

10. The use according to claim 9, wherein the anti-VEGF antibody is bevacizumab or BAT1706.

11. 10. The use according to claim 1 or claim 9, wherein the cancer is selected from the group consisting of breast cancer, colon cancer, pancreatic cancer, head and neck cancer, gastric cancer, renal cancer, liver cancer, small cell lung cancer, non-small cell lung cancer, esophageal cancer, ovarian cancer, uterine cancer, cervical cancer, skin cancer, mesothelioma, lymphoma, leukemia, myeloma and sarcoma.

12. The use according to claim 11, wherein the small cell lung cancer is limited-stage small cell lung cancer.

13. The use according to claim 11, wherein the cancer is non-small cell lung cancer (NSCLC).

14. The use according to claim 11, wherein the head and neck cancer is nasopharyngeal carcinoma.

15. The use according to claim 11, wherein the esophageal cancer is esophageal squamous cell carcinoma (ESCC).

16. The use according to claim 11, wherein the cancer is uterine cancer.

17. The use according to claim 11, wherein the gastric cancer is gastric cancer or gastroesophageal junction cancer.

18. The use according to claim 11, wherein the cervical cancer is recurrent or metastatic cervical cancer.

19. The use according to claim 11, wherein the skin cancer is basal cell carcinoma.

20. The use according to claim 11, wherein the cancer is pancreatic cancer.

21. The use according to claim 11, wherein the renal cancer is hepatocellular carcinoma.

22. 2. The use according to claim 1 further in combination with chemotherapy.

23. 23. The use according to claim 22, wherein the chemotherapy is chemoradiotherapy.

24. 2. The use of claim 1, wherein the anti-PD1 antibody or antigen-binding fragment thereof is administered at 200 mg every three weeks.

25. The use of claim 1, wherein the anti-TIGIT antibody or antigen-binding fragment thereof is administered in the range of 50 mg to 900 mg.

26. The use of claim 1, wherein the anti-TIGIT antibody or antigen-binding fragment thereof is administered at 50 mg every three weeks.

27. The use of claim 1, wherein the anti-TIGIT antibody or antigen-binding fragment thereof is administered at 150 mg every three weeks.

28. The use of claim 1, wherein the anti-TIGIT antibody or antigen-binding fragment thereof is administered at 450 mg every three weeks.

29. The use of claim 1, wherein the anti-TIGIT antibody or antigen-binding fragment thereof is administered at 900 mg every three weeks.

Images