Antibodies that bind to TIGIT and their uses

Abstract

The present invention provides an isolated monoclonal antibody that specifically binds to human TIGIT. Also provided are nucleic acid molecules encoding the antibody, as well as expression vectors, host cells, and methods for expressing the antibody. The present invention also provides immunoconjugates, bispecific molecules, chimeric antigen receptors, oncolytic viruses, and pharmaceutical compositions comprising the antibody, as well as methods of treatment using the antibodies of the present invention.

Description

Technical Field

[0001] The present invention relates to an antibody specifically binding to human TIGIT, its preparation and uses, especially its use in treating TIGIT-related diseases, such as cancer and infectious diseases. Background Art

[0002] The immune system can help the body resist cancer. For example, dendritic cells can phagocytose tumor-associated antigens released by cancer cells and present them to T cells. The activated T cells reach the tumor site to kill cancer cells. However, malignant tumor cells themselves have evolved various escape mechanisms to avoid recognition and killing by the immune system. The most important one is to utilize inhibitory immune checkpoints. The inhibitory immune checkpoints expressed on the surface of immune cells bind to their ligands expressed on tumor cells, which can inhibit the functions of immune T cells, etc., and weaken or inhibit the clearance and killing of tumors by immune cells. For example, tumor cells highly express PD-L1 and PD-L2, which bind to PD-1 on the surface of T cells, triggering apoptosis of T cells. Another example is that tumor cells highly express CD47, which binds to signal regulatory protein α (SIRPa) on the surface of macrophages, inhibiting the phagocytosis of tumor cells by macrophages. Thus, in the tumor microenvironment, infiltrating immune lymphocytes face great immunosuppressive pressure and gradually become exhausted. Immunotherapies targeting inhibitory immune checkpoints such as PD-1 and CTLA-4 have shown beneficial effects in clinical trials / treatments of various tumors. And more and more studies have shown that the functions of most inhibitory immune checkpoints are non-redundant, and blocking multiple checkpoint pathways can achieve better anti-tumor effects. Therefore, the research has gradually expanded to other immune checkpoints such as TIM-3, LAG-3 and TIGIT (Le Mercier I et al., (2015) Front Immunol. 6:418).

[0003] TIGIT is a member of the poliovirus receptor (PVR) / nectin family, full name T cell immunoglobulin and ITIM domain, also known as Vsig9 (V-set and immunoglobulin domain-containing protein 9), Vstm3 (V-set and transmembrane domain-containing protein 3), or WUCAM (Washington University cell adhesion molecule). The TIGIT gene is located on human chromosome 16 and encodes a type I transmembrane protein consisting of 244 amino acids. The extracellular region of the human TIGIT molecule is 141 amino acids long and has one immunoglobulin V-like domain; the transmembrane region is 23 amino acids; the cytoplasmic region is short and contains two inhibitory motifs, namely immunoreceptor tyrosine-based inhibitory motif (ITIM) and immunoglobulin tail tyrosine (ITT)-like motif. The extracellular immunoglobulin V-like domain of TIGIT is very similar in sequence to this domain of other PVR / nectin family members, including DNAX accessory molecule-1 (DNAM-1), CD226, CD133, CD155, CD111, CD112, CD96, etc.

[0004] TIGIT is expressed on activated CD8 + T cells and CD4 + T cells, NK cells, regulatory T cells (Tregs), and follicular helper T cells. It competes with the costimulatory receptor CD226 for binding to CD155 (PVR) expressed on tumor cells and antigen-presenting cells, and its binding affinity to CD155 is much higher than the CD266-CD155 binding affinity. The relationship between TIGIT and CD226 is similar to that between CTLA-4 and CD28. CD226 is expressed on naive and resting T cells, while TIGIT is rapidly expressed upon antigen stimulation or other inflammatory stimuli. The counterbalance between the two affects the function of effector T cells. For example, the binding of TIGIT to CD155 blocks the T cell receptor pathway and inhibits CD4 +T cells secrete pro-inflammatory factors (Shibuya K et al., (1999) Immunity 11: 615-623; Lozano E et al., (2013) J Immunol 191: 3673-3680). Approximately 20-90% of resting NK cells express TIGIT, and the expression level increases after acute or chronic viral infection or cancer. The binding of TIGIT to CD115 mainly triggers the inhibitory signaling pathway of human NK cells via the ITT-like motif, inhibiting the ability of NK cells to recognize tumor cells and secrete interferon (IFN)-α (Holder KA, Grant MD. (2020) Front Cell Infect Microbiol. 10: 175; Stanietsky N et al., (2009) Proc Natl Acad Sci USA 106: 17858-17863; Liu S et al., (2013) Cell Death Differ 20: 456-464). TIGIT also binds to CD112 (also known as PVRL2 / nectin-2) and PVRL3, but the binding affinity is much weaker.

[0005] Studies have shown that TIGIT inhibits innate and adaptive immunity through multiple pathways. First, TIGIT binds to CD155 on dendritic cells, making dendritic cells adopt a tolerogenic phenotype, reducing the secretion of interleukin (IL)-12 and increasing the secretion of IL-10, thereby inhibiting the upregulation of antigen presentation-related molecules and the secretion of T cell proliferation and effector cytokines (Yu X et al., (2009) Nat. Immunol. 10: 48-57). Second, TIGIT has a direct inhibitory effect on immune cells. For example, the TIGIT pathway can weaken the activation signal driven by the T cell receptor, inhibiting the proliferation and function of T cells, and CD8 + tumor-infiltrating cells with high expression of TIGIT significantly have a poorer ability to secrete pro-inflammatory cytokines and impaired degranulation ability (Kurtulus S et al., (2015) J. Clin. Invest. 125: 4053-4062). NK cells are the most important immune cells in the initial stage of the body's elimination of cancer, and the expression of TIGIT is negatively correlated with the ability of NK cells to secrete IFN-γ and kill CD155 + cells. TIGIT +NK cells are more susceptible to MDSC than TIGIT-NK cells, and TIGIT blockade can restart the effector function of NK cells against cancer (Wang F et al., (2015) Eur. J. Immunol. 45: 2886-2897; Manieri NA et al., (2017) Trends Immunol. 38(1): 20-28; Zhang Q et al., (2018) Nat. Immunol. 19: 723-732). Again, TIGIT is constitutively highly expressed on most Tregs. TIGIT + Tregs suppress the responses of pro-inflammatory Th1 and Th7 T cells and produce IL-10 and fibrinogen-like protein 2 to inhibit T cell function, with stronger inhibitory effects than TIGIT-Tregs (Joller N., et al., (2014) Immunity 40: 569-581; Kurtulus S et al., (2015) ibid.).

[0006] Antibodies targeting TIGIT have been developed and are being clinically tested in various types of cancer. For example, Etigilimab (OMP-313M32) developed by Oncomed has currently initiated Phase 1 clinical trials, either as a single agent or in combination with nivolumab (a PD-1 antibody), in multiple solid tumors, including colorectal cancer, endometrial cancer, and pancreatic cancer, etc., to detect its safety, efficacy, pharmacokinetics, and dosage exploration. The results of the Phase 1 trial showed that the drug was safely tolerated at a dose of 20 mg / kg. Another TIGIT-blocking antibody, Tiragolumab, is an antibody drug developed by Roche. Clinical trials of this antibody drug showed that when combined with the PD-L1 antibody drug atezolizumab, it can have a synergistic effect in various types of tumors, especially in the treatment of non-small cell lung cancer, with significant results. Other TIGIT antibodies, including BMS-986207 (Bristol-Myers Squibb), BGB-A1217 (BeiGene), AB154 (Arcus biosciences), etc., are also in clinical trials either as a single agent or in combination with other anti-tumor drugs, covering various solid tumors, including multiple myeloma and melanoma (Chauvin J, Zarour HM., (2020) Journal for ImmunoTherapy of Cancer 8:e000957). Studies have also shown that the constant region of TIGIT antibodies can bind to myeloid cell FcγR to activate myeloid cells, enhance antigen presentation ability and the secretion of cytokines such as IL-23 and TNF-α, and trigger the secretion of granzyme B and perforin in T cells (Han JH et al., (2020) Front Immunol. 11:573405).

[0007] In chronic viral infections, the function of effector cells is also severely impaired. After blocking the TIGIT pathway with TIGIT antibodies, CD8 + T cells can regain antiviral activity. It has been reported that the increased expression of TIGIT on NK cells is associated with the progression of the HIV-1 disease course, and whether blocking the TIGIT pathway can restart the killing ability of NK cells against the virus remains to be verified (Holder KA, Grant MD. (2020) Ibid.; Yin X et al., (2018) Front. Immunol. 9:2341).

[0008] Based on the important role of TIGIT antibodies in regulating the function of the immune system, there is still a need in the art for more TIGIT antibodies with improved drug characteristics. Summary of the Invention

[0009] The present application provides an isolated monoclonal antibody, for example, a murine, human, chimeric or humanized monoclonal antibody that binds to TIGIT (e.g., human TIGIT and monkey TIGIT), which has comparable or better human / monkey TIGIT binding activity, comparable TIGIT-PVR blocking activity, comparable or stronger T cell activation ability, comparable or stronger ability to induce ADCC against TIGIT-positive cells, and comparable or better in vivo anti-tumor activity compared to prior art antibodies such as Tiragolumab or Etigilimab.

[0010] The antibodies of the present application can be used in a variety of applications, including the treatment of TIGIT-related diseases and the like.

[0011] Thus, in one aspect, the present application relates to an isolated monoclonal antibody (e.g., a humanized antibody), or an antigen-binding portion thereof, that binds to TIGIT and may comprise i) a heavy chain variable region that contains a VH CDR1 region, a VH CDR2 region, and a VH CDR3 region, wherein the VH CDR1 region, the VH CDR2 region, and the VH CDR3 region may respectively comprise the amino acid sequences as shown in (1) SEQ ID NOs: 1, 2, and 3; or (2) SEQ ID NOs: 7, 8, and 9, or amino acid sequences having at least 80%, 85%, 90%, 95%, 98%, or 99% identity to the above sequences; and / or ii) a light chain variable region that contains a VL CDR1 region, a VL CDR2 region, and a VL CDR3 region, wherein the VL CDR1 region, the VL CDR2 region, and the VL CDR3 region may respectively comprise the amino acid sequences as shown in (1) SEQ ID NOs: 4, 5, and 6; or (2) SEQ ID NOs: 10, 11, and 12, or amino acid sequences having at least 80%, 85%, 90%, 95%, 98%, or 99% identity to the above sequences.

[0012] The isolated monoclonal antibody or antigen-binding portion thereof of the present application may comprise a heavy chain variable region and a light chain variable region, wherein the VH CDR1 region, the VH CDR2 region, the VH CDR3 region, the VL CDR1 region, the VL CDR2 region, and the VL CDR3 region may respectively comprise the amino acid sequences as shown in (1) SEQ ID NOs: 1, 2, 3, 4, 5, and 6; or (2) SEQ ID NOs: 7, 8, 9, 10, 11, and 12, or amino acid sequences having at least 80%, 85%, 90%, 95%, 98%, or 99% identity to the above sequences.

[0013] The heavy chain variable region of the isolated monoclonal antibody or antigen-binding portion thereof of the present application may comprise an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to any of SEQ ID NOs: 13-23.

[0014] The light chain variable region of the isolated monoclonal antibody or antigen-binding portion thereof of the present application may comprise an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to any of SEQ ID NOs: 24-32.

[0015] The heavy chain variable region and the light chain variable region of the isolated monoclonal antibody or antigen-binding portion thereof of the present application may respectively comprise the amino acid sequences shown in (1) SEQ ID NOs: 13 and 24; (2) SEQ ID NOs: 14 and 25; (3) SEQ ID NOs: 15 and 26; (4) SEQ ID NOs: 15 and 27; (5) SEQ ID NOs: 15 and 28; (6) SEQ ID NOs: 16 and 26; (7) SEQ ID NOs: 16 and 27; (8) SEQ ID NOs: 16 and 28; (9) SEQ ID NOs: 17 and 26; (10) SEQ ID NOs: 17 and 27; (11) SEQ ID NOs: 17 and 28; (12) SEQ ID NOs: 18 and 26; (13) SEQ ID NOs: 18 and 27; (14) SEQ ID NOs: 18 and 28; (15) SEQ ID NOs: 19 and 29; (16) SEQ ID NOs: 20 and 30; (17) SEQ ID NOs: 21 and 31; (18) SEQ ID NOs: 21 and 32; (19) SEQ ID NOs: 22 and 31; (20) SEQ ID NOs: 22 and 32; (21) SEQ ID NOs: 23 and 31; or (22) SEQ ID NOs: 23 and 32; or amino acid sequences having at least 80%, 85%, 90%, 95%, 98% or 99% identity to the above.

[0016] In one embodiment, the isolated monoclonal antibody or antigen-binding portion thereof of the present application may comprise a heavy chain constant region and / or a light chain constant region, wherein the heavy chain constant region may be an IgG1, IgG2, IgG3 or IgG4 heavy chain constant region, particularly a heavy chain constant region having FcγR binding ability, such as the human IgG1 constant region having the amino acid sequence of SEQ ID NO: 33, or a functional fragment thereof. The light chain constant region may be a κ constant region, such as the human κ constant region having the amino acid sequence of SEQ ID NO: 34, or a functional fragment thereof. Among them, the N-terminus of the heavy chain constant region is connected to the C-terminus of the heavy chain variable region, and the N-terminus of the light chain constant region is connected to the C-terminus of the light chain variable region.

[0017] In some embodiments, the antibody of the present application comprises two heavy chains and two light chains, or is composed of two heavy chains and two light chains, wherein each heavy chain comprises the above-mentioned heavy chain constant region sequence, heavy chain variable region sequence or CDR sequence, and each light chain comprises the above-mentioned light chain constant region sequence, light chain variable region sequence or CDR sequence. The antibody of the present application may be a full-length antibody such as IgG4, IgG1, or IgG2. In some embodiments, the antibody of the present application may be a single-chain antibody, or composed of antibody fragments, such as Fab or F(ab′)2 fragments.

[0018] The antibody or antigen-binding portion thereof of the present application is an antagonistic TIGIT antibody or antigen-binding portion thereof, which can bind to human / monkey TIGIT, block TIGIT-PVR binding, promote T cell activation, induce ADCC against TIGIT-positive cells, and have an in vivo anti-tumor effect.

[0019] The present application also provides an immunoconjugate containing the antibody or antigen-binding portion thereof of the present application, wherein the antibody or antigen-binding portion thereof is linked to a therapeutic agent such as a cytotoxin or an anti-cancer agent. The present application also provides a bispecific molecule containing the antibody or antigen-binding portion thereof of the present application, wherein the antibody or antigen-binding portion thereof is linked to a second functional group, such as a second antibody, and the second functional group has a binding specificity different from that of the antibody or binding portion of the present application. On the other hand, the antibody or antigen-binding portion thereof of the present application may be a part of a chimeric antigen receptor (CAR) or a genetically engineered T cell receptor (TCR). The present application also provides immune cells having the above-mentioned CAR and / or TCR, including T cells, NK cells, etc. The antibody or antigen-binding portion thereof of the present application may also be encoded by or carried by an oncolytic virus.

[0020] This application also includes nucleic acid molecules encoding the antibodies or antigen-binding portions thereof of this application, expression vectors containing such nucleic acids, and host cells containing such expression vectors. This application also provides a method for preparing TIGIT antibodies using host cells containing the above-mentioned expression vectors, including: (i) expressing the antibodies in the host cells, and (ii) isolating the antibodies from the host cells or their cultures.

[0021] This application also provides pharmaceutical compositions, which contain the antibodies or antigen-binding portions thereof, immunoconjugates, bispecific molecules, immune cells, oncolytic viruses, nucleic acid molecules, expression vectors or host cells of this application, and pharmaceutically acceptable carriers.

[0022] In one aspect, this application provides a method for enhancing the immune response in a subject, including administering an effective amount of the pharmaceutical composition to a subject in need thereof. Enhancing the immune response includes activating T cells.

[0023] In one aspect, this application provides a method for treating or alleviating cancer diseases in a subject, including administering a therapeutically effective amount of the pharmaceutical composition of this application to the subject. The cancer can be a solid tumor, including but not limited to, liver cancer, rectal cancer, endometrial cancer, pancreatic cancer, non-small cell lung cancer, multiple myeloma, and melanoma. In some embodiments, at least one other anti-cancer antibody can be administered together with the antibody or antigen-binding portion thereof of this application, such as PD-1 antibody, PD-L1 antibody, STAT3 antibody, and ROR1 antibody, TIM-3 antibody, and / or CTLA-4 antibody, especially the PD-L1 antibody. In another embodiment, the antibody or antigen-binding portion thereof of this application is administered together with cytokines (such as IL-2 and / or IL-21) or co-stimulatory antibodies (such as CD137 antibody and / or GITR antibody). In another embodiment, the antibody or antigen-binding portion thereof of this application can be administered together with a chemotherapeutic agent, which can be a cytotoxic agent. The antibody of this application can be, for example, a murine, human, chimeric, or humanized antibody.

[0024] In one aspect, this application provides a method for treating or alleviating infectious diseases in a subject, including administering a therapeutically effective amount of the antibody or antigen-binding portion thereof of this application to the subject. The infectious disease can be a chronic infection caused by viruses, bacteria, fungi, mycoplasmas, etc., such as a chronic infection caused by HIV. In some embodiments, at least one other anti-infective agent can be administered together with the antibody or antigen-binding portion thereof of this application, such as antiviral agents, antibacterial agents, antifungal agents, anti-mycoplasma agents, etc.

[0025] Based on the following specific descriptions and examples, other features and advantages of the current disclosure will become clearer. The specific descriptions and examples should not be construed as restrictive. The content of all documents, Genbank records, patents, and published patent applications cited in this application are expressly incorporated herein by reference.

[0026] It should be noted that in this application, especially in the claims, terms such as "comprising", "including", etc. may have the meanings given by the Chinese Patent Law; while terms such as "consisting essentially of..." have the meanings given by the Chinese Patent Law, for example, allowing the existence of elements not expressly stated, but excluding elements existing in the prior art or elements that affect the basic or new characteristics of the present invention.

[0027] Description of the Drawings

[0028] The following is given by way of example and is not intended to limit the present invention to the specific embodiments described. It can be better understood in conjunction with the drawings.

[0029] Figure 1 Shows the role of mouse TIGIT antibodies in T cell activation. Treatment with 100 μg / ml of TIGIT antibodies, including 70E11 and 149G11, increased the IFN-γ secretion of T cells after activation (A). Treatment with TIGIT antibodies 70E11 and 149G11 increased the IFN-γ secretion of T cells in a dose-dependent manner (B).

[0030] Figure 2 Shows the binding activity of chimeric TIGIT antibodies to HEK293A / human TIGIT (A), HEK293A / monkey TIGIT (B), and HEK293A / mouse TIGIT (C).

[0031] Figure 3 Shows that chimeric TIGIT antibodies 70E11 and 149G11 induce NK92 cells to produce ADCC killing effect on HEK293A / human TIGIT cells.

[0032] Figure 4 Shows that chimeric TIGIT antibodies 70E11 and 149G11 block the interaction between TIGIT and PVR.

[0033] Figure 5 Shows the binding activity of humanized 70E11 antibody to HEK293A / human TIGIT (A), HEK293A / monkey TIGIT (B), and HEK293A / mouse TIGIT (C).

[0034] Figure 6Show the binding activity of the humanized 149G11 antibody to HEK293A / human TIGIT (A), HEK293A / monkey TIGIT (B), and HEK293A / mouse TIGIT (C).

[0035] Figure 7 Show that the humanized antibody of 70E11 (A) and the humanized antibody of 149G11 (B) block the interaction between TIGIT and PVR.

[0036] Figure 8 Show that the humanized TIGIT antibody induces T cells to secrete IFN-γ in a dose-dependent manner.

[0037] Figure 9 Show that the humanized antibody of 70E11 (A) and the humanized antibody of 149G11 (B) induce NK92 cells to produce ADCC killing effect on HEK293A / human TIGIT cells.

[0038] Figure 10 Show that the humanized TIGIT antibody induces PBMC to produce ADCC killing effect on HEK293A / human TIGIT cells in a dose-dependent manner, where PBMC are from donor 1 (A), donor 2 (B), and donor 3 (C) respectively.

[0039] Figure 11 Show the average tumor volume (A) and average tumor weight (B) of C57 humanized TIGIT mice after treatment with humanized TIGIT antibodies 149G11H2L3 and 70E11H5L3, and Tiragolumab.

[0040] Figure 12 Show the average tumor volume (A) of BALB / c humanized TIGIT mice after treatment with humanized TIGIT antibodies 149G11H2L3 and 70E11H2L4, and Tiragolumab alone, or in combination with the PDL1 antibody Tecentriq. Show the average tumor volume (B) of each group of mice after treatment with the humanized antibody 149G11H2L3 alone or in combination with the Tecentriq antibody. Show the average tumor volume (C) of each group of mice after treatment with the humanized antibody 70E11H2L4 alone or in combination with the Tecentriq antibody. Show the average tumor volume (D) of each group of mice after treatment with the humanized antibody Tiragolumab alone or in combination with the Tecentriq antibody. Detailed implementation mode

[0041] For better understanding of this application, first define some terms. Other definitions are listed throughout the detailed implementation mode section.

[0042] The term "TIGIT" refers to T cell immunoglobulin and ITIM domain. The term includes variants, homologs, orthologs and paralogs. For example, an antibody specific for human TIGIT can cross-react with TIGIT proteins of another species, such as monkey, in certain cases. In other embodiments, an antibody specific for human TIGIT protein can be specific solely for human TIGIT protein without cross-reacting with TIGIT proteins of other species or other types of proteins, or can cross-react with TIGIT proteins of some other species but not all other species.

[0043] The term "human TIGIT" refers to a TIGIT protein having a human amino acid sequence, such as a TIGIT protein having the amino acid sequence with NCBI accession number NP_776160.2 (Saleh R et al., (2020) Cancer Immunol Immunother 69(10): 1989 - 1999), or a TIGIT protein encoded by nucleotides such as those shown in SEQ ID NO: 35. The term "monkey TIGIT" refers to a TIGIT protein having a monkey amino acid sequence, such as a TIGIT protein having the amino acid sequence with GenBank accession number AFH31430.1 (Zimin A.V. et al., (2014) Biol. Direct 9(1): 20), or a TIGIT protein encoded by nucleotides such as SEQ ID NO: 36. The term "mouse TIGIT" refers to a mouse amino acid TIGIT protein, such as a TIGIT protein having the amino acid sequence with NCBI accession number NP_001139797.1 (Schorer M et al., (2020) Nat Commun 11(1): 1288), or a TIGIT protein encoded by nucleotides such as SEQ ID NO: 37.

[0044] The term "antibody" as used herein is intended to include IgG, IgA, IgD, IgE and IgM full-length antibodies and any antigen-binding fragments thereof (i.e., antigen-binding portions). A full-length antibody is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains, which are linked by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated as V H ) and a heavy chain constant region. The heavy chain constant region is composed of three domains, namely C H1 , C H2 and C H3 . Each light chain is composed of a light chain variable region (abbreviated as V L ) and a light chain constant region. The light chain constant region is composed of one domain C L . V H and V LThe region can also be divided into hypervariable regions called complementarity-determining regions (CDRs), which are separated by relatively conserved framework regions (FRs). Each V H and V L is composed of three CDRs and four FRs, arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxyl terminus. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various immune system cells (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0045] As used herein, the "antigen-binding portion" (or simply "antibody portion") of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen (e.g., the TIGIT protein). It has been demonstrated that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments included in the "antigen-binding portion" of an antibody include (i) Fab fragments, monovalent fragments composed of V L , V H , C L and C H1 ; (ii) F(ab′)2 fragments, divalent fragments containing two Fab fragments linked by a hinge region disulfide bridge; (iii) Fd fragments composed of V H and C H1 ; (iv) Fv fragments composed of the antibody single arms V L and V H ; (v) dAb fragments composed of V H (Ward et al., (1989) Nature 341:544-546); (vi) isolated complementarity-determining regions (CDRs); and (vii) nanobodies, a heavy chain variable region containing a single variable domain and two constant domains. In addition, although the two domains V L and V H of the Fv fragment are encoded by different genes, they can be linked by recombinant methods via a synthetic linker that makes the two into a single protein chain, in which the V L and V H regions pair to form a monovalent molecule (called single-chain Fv (scFv); see, e.g., Bird et al., (1988) Science 242:423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). These single-chain antibodies are also intended to be included in the meaning of the term. These antibody fragments can be obtained by conventional techniques known to those skilled in the art, and the fragments can be functionally screened in the same manner as the intact antibody.

[0046] As used herein, the term "isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigen specificities. For example, an isolated antibody that specifically binds to the TIGIT protein is substantially free of antibodies that specifically bind to antigens other than the TIGIT protein. However, an isolated antibody that specifically binds to the human TIGIT protein may have cross-reactivity to other antigens, such as the TIGIT protein of other species. In addition, the isolated antibody is substantially free of other cellular materials and / or chemicals.

[0047] The term "monoclonal antibody" or "mAb" or "monoclonal antibody composition" refers to an antibody molecule preparation consisting of a single molecule. A monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope.

[0048] The term "murine antibody" refers to an antibody in which the variable region framework and CDR regions are derived from murine germline immunoglobulin sequences. In addition, if the antibody contains a constant region, it is also derived from murine germline immunoglobulin sequences. The murine antibodies of the present application may contain amino acid residues not encoded by murine germline immunoglobulin sequences, such as mutations introduced by in vitro random mutagenesis or site-directed mutagenesis or by in vivo somatic mutagenesis. However, the term "murine antibody" does not include antibodies in which CDR sequences derived from other mammalian species are inserted into the murine framework sequence.

[0049] The term "chimeric antibody" refers to an antibody obtained by combining non-human genetic material with human genetic material. Or more generally, a chimeric antibody refers to an antibody that combines genetic material of one species with genetic material of another species.

[0050] The term "humanized antibody" refers to an antibody that is derived from a non-human species but whose protein sequence has been modified to increase its similarity to antibodies naturally produced in humans.

[0051] The terms "antibody that recognizes an antigen" and "antibody specific for an antigen" are used interchangeably herein with the term "antibody that specifically binds to an antigen".

[0052] As used herein, an antibody that "specifically binds to human TIGIT" refers to an antibody that binds to human TIGIT (and possibly also to TIGIT of other non-human species) but does not substantially bind to non-TIGIT proteins. Preferably, the antibody binds to the human TIGIT protein with "high affinity", i.e., a K D value of 5.0 x 10 -8 M or less, more preferably 1.0 x 10 -9 M or less.

[0053] The term "substantially does not bind" to a protein or cell means that it does not bind to the protein or cell, or does not bind to it with high affinity, i.e., the K for binding to the protein or cell Dis 1.0x10 -6 M or more, more preferably 1.0x10 -5 M or more, more preferably 1.0x10 -4 M or more, 1.0x10 -3 M or more, more preferably 1.0x10 -2 M or more.

[0054] The term "high affinity" for IgG antibodies means that the KD for the antigen is 1.0x10 -6 M or less, preferably 5.0x10 -8 M or less, more preferably 1.0x10 -8 M or less, 5.0x10 -9 M or less, more preferably 1.0x10 -9M or less. For other antibody subtypes, "high affinity" binding may vary. For example, "high affinity" binding for the IgM subtype means that K D is 10 -6 M or less, preferably 10 -7 M or less, more preferably 10 -8 M or less.

[0055] The term "EC 50 ", also known as the half-maximal effective concentration, refers to the antibody concentration that elicits 50% of the maximal effect.

[0056] The terms "antibody-dependent cytotoxicity", "antibody-dependent cell-mediated cytotoxicity", or "ADCC" refer to cell-mediated immune defense in which immune system effector cells actively lyse target cells that have cell membrane surface antigens bound to antibodies, such as TIGIT antibodies, for example immune cells in autoimmune diseases.

[0057] The term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians, and reptiles, although mammals, such as non-human primates, sheep, dogs, cats, cows, and horses, are preferred.

[0058] The term "therapeutically effective amount" refers to the amount of the antibody of the present application sufficient to prevent or mitigate symptoms associated with a disease or disorder (such as cancer). The therapeutically effective amount is related to the disease being treated, and those skilled in the art can conveniently determine the actual effective amount.

[0059] The term "antagonistic TIGIT antibody" refers to a TIGIT antibody that can block or inhibit the TIGIT signaling pathway triggered by the interaction of TIGIT with its ligand, such as PVR. Antagonistic TIGIT antibodies can promote T cell activation, cytokine release, enhance immune effects, and can be applied in the treatment of cancer and chronic infections, etc.

[0060] Multiple aspects of the present application are described in more detail below.

[0061] The TIGIT antibody has binding specificity for human TIGIT and other beneficial functional characteristics

[0062] The antibodies of the present application can specifically bind to human and monkey TIGIT, and the binding activity is comparable to or better than that of prior art antibodies such as Tiragolumab or Etigilimab. The antibodies of the present application can also block the TIGIT-PVR binding or interaction, and the blocking activity is comparable to that of prior art antibodies such as Tiragolumab or Etigilimab.

[0063] More importantly, the antibodies of the present application have comparable or stronger T cell activation ability to prior art antibodies such as Tiragolumab or Etigilimab, and the in vivo anti-tumor activity is comparable to or even higher than that of prior art such as Tiragolumab or Etigilimab.

[0064] Preferably, the antibodies of the present application are monoclonal antibodies. In addition, the antibodies can be, for example, murine, chimeric or humanized monoclonal antibodies.

[0065] TIGIT monoclonal antibody

[0066] Exemplary antibodies of the present application are monoclonal antibodies whose structures and chemical properties are described below.

[0067] The CDRs of the heavy chain variable region and the light chain variable region of the antibody or its antigen-binding portion of the present application are determined by the Kabat numbering system, and the SEQ ID NOs of the amino acid sequences of the CDR regions are listed in Table 1. As is well known in the art, the CDRs of the heavy chain variable region and the light chain variable region can be determined by, for example, the Chothia, IMGT, AbM or Contact numbering systems / methods.

[0068] The SEQ ID NOs of the heavy chain / light chain variable region sequences of the exemplary antibodies or their antigen-binding portions in the present application are also listed in Table 1 below. Some antibodies have the same V H or V L .

[0069] The antibodies of the present application can have a heavy chain constant region. For example, it can be an IgG1 constant region, such as a human IgG1 constant region containing the amino acid sequence shown in SEQ ID NO: 33. The light chain constant region can be a κ constant region, such as a human κ constant region, which can contain the amino acid sequence shown in SEQ ID NO: 34

[0070] V of other TIGIT antibodies that bind to human TIGIT H and / or VL A sequence (or CDR sequence) can be "mixed and paired" with the V H and / or V L sequence (or CDR sequence) of the antibody of the present application. Preferably, when the V H and V L (or the CDRs therein) are mixed and paired, the V H / V L sequence in a specific V H / V H pairing can be replaced by a V H / V L sequence with a similar structure. Similarly, preferably, the V L sequence in a specific V L / V

[0071] Therefore, in one embodiment, the antibody or antigen-binding portion thereof of the present application comprises:

[0072] (a) a heavy chain variable region comprising the amino acid sequence listed in Table 1; and

[0073] (b) a light chain variable region comprising the amino acid sequence listed in Table 1, or the V L of another TIGIT antibody, wherein the antibody specifically binds to human TIGIT.

[0074] In another embodiment, the antibody or antigen-binding portion thereof of the present application comprises:

[0075] (a) CDR1, CDR2, and CDR3 of the heavy chain variable region listed in Table 1; and

[0076] (b) CDR1, CDR2, and CDR3 of the light chain variable region listed in Table 1, or the CDRs of another TIGIT antibody, wherein the antibody specifically binds to human TIGIT.

[0077] In another embodiment, the antibody or antigen-binding portion thereof of the present application comprises CDR2 of the heavy chain variable region of a TIGIT antibody and the CDRs of other antibodies that bind to human TIGIT, such as CDR1 and / or CDR3 of the heavy chain variable region, and / or CDR1, CDR2, and / or CDR3 of the light chain variable region of another TIGIT antibody.

[0078]

[0079] In addition, it is well known in the art that the CDR3 domain, independent of CDR1 and / or CDR2, can alone determine the binding specificity of an antibody for a homologous antigen, and it can be predicted that multiple antibodies with the same binding specificity can be generated based on this CDR3 sequence. See, for example, Klimka et al., British J. of Cancer 83(2):252-260(2000); Beiboer et al., J. Mol. Biol. 296:833-849(2000); Rader et al., Proc. Natl. Acad. Sci. U.S.A. 95:8910-8915(1998); Barbas et al., J. Am. Chem. Soc. 116:2161-2162(1994); Barbas et al., Proc. Natl. Acad. Sci. U.S.A. 92:2529-2533(1995); Ditzel et al., J. Immunol. 157:739-749(1996); Berezov et al., BIA journal 8: Scientific Review 8(2001); Igarashi et al., J. Biochem(Tokyo) 117:452-7(1995); Bourgeois et al., J. Virol 72:807-10(1998); Levi et al., Proc. Natl. Acad. Sci. U.S.A. 90:4374-8(1993); Polymenis and Stoller, J. Immunol. 152:5218-5329(1994) and Xu and Davis, Immunity 13:37-45(2000); U.S. Pat. Nos. 6,951,646; 6,914,128; 6,090,382; 6,818,216; 6,156,313; 6,827,925; 5,833,943; 5,762,905 and 5,760,185. All of these references are hereby incorporated by reference in their entirety.

[0080] In another embodiment, the antibody of the present application comprises CDR2 of the heavy chain variable region of a TIGIT antibody and at least CDR3 of the heavy chain and / or light chain variable region of a TIGIT antibody, or CDR3 of the heavy chain and / or light chain variable region of another TIGIT antibody, wherein the antibody is capable of specifically binding to human TIGIT. Preferably, these antibodies (a) competitively bind to TIGIT; (b) retain functional properties; (c) bind to the same epitope; and / or (d) have a binding affinity similar to that of the TIGIT antibody of the present application. In another embodiment, the antibody may further comprise CDR2 of the light chain variable region of a TIGIT antibody, or CDR2 of the light chain variable region of another TIGIT antibody, wherein the antibody specifically binds to human TIGIT. In another embodiment, the antibody of the present application may comprise CDR1 of the heavy chain / light chain variable region of a TIGIT antibody, or CDR1 of the heavy chain and / or light chain variable region of another TIGIT antibody, wherein the antibody specifically binds to human TIGIT.

[0081] Conservative modification

[0082] In another embodiment, the antibody of the present application comprises heavy chain and / or light chain variable region sequences or CDR1, CDR2, and CDR3 sequences that have one or more conservative modifications compared to the TIGIT antibody of the present application. As is known in the art, some conservative sequence modifications do not abolish antigen-binding. See, e.g., Brummell et al., (1993) Biochem 32:1180-8; deWildt et al., (1997) Prot. Eng. 10:835-41; Komissarov et al., (1997) J. Biol. Chem. 272:26864-26870; Hall et al., (1992) J. Immunol. 149:1605-12; Kelley and O′Connell (1993) Biochem. 32:6862-35; Adib-Conquy et al., (1998) Int. Immunol. 10:341-6 and Beers et al., (2000) Clin. Can. Res. 6:2835-43.

[0083] Thus, in one embodiment, the antibody comprises a heavy chain variable region and / or a light chain variable region, and the heavy chain variable region and the light chain variable region each comprise CDR1, CDR2, and CDR3, wherein:

[0084] (a) The heavy chain variable region CDR1 comprises the sequence listed in Table 1, and / or its conservative modification; and / or

[0085] (b) The CDR1 of the heavy chain variable region comprises the sequences listed in Table 1, and / or conservative modifications thereof; and / or

[0086] (c) The CDR3 of the heavy chain variable region comprises the sequences listed in Table 1, and / or conservative modifications thereof; and / or

[0087] (d) The CDR1, and / or CDR2, and / or CDR3 of the light chain variable region comprises the sequences listed in Table 1, and / or conservative modifications thereof; and

[0088] (e) The antibody specifically binds to human TIGIT.

[0089] The antibody of the present application has one or more of the following functional characteristics, such as high affinity for human TIGIT, and the ability to induce ADCC or CDC against TIGIT-expressing cells.

[0090] In various embodiments, the antibody can be, for example, a murine, human, chimeric or humanized antibody.

[0091] As used herein, the term "conservative sequence modification" refers to amino acid modifications that do not significantly affect or alter the binding properties of the antibody. Such conservative modifications include amino acid substitutions, additions, and deletions. The modifications can be introduced into the antibodies of the present application by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are the replacement of an amino acid residue with an amino acid residue having a similar side chain. Groups of amino acid residues having similar side chains are known in the art. These groups of amino acid residues include amino acids having basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues in the CDR regions of the antibodies of the present application can be replaced with other amino acid residues from the same side chain group, and the resulting antibodies can be tested for retention of function (i.e., the functions described above) using the functional assays described herein.

[0092] Genetically modified antibody

[0093] The antibody of the present application can be prepared as a genetically modified antibody starting from an antibody having one or more V H / V L sequences of the TIGIT antibody of the present application. The antibody can be modified by modifying one or both variable regions (i.e., V H and / or VL ) residues within (e.g., in one or more CDR regions and / or one or more framework regions) are genetically modified to improve binding affinity and / or increase similarity to antibodies naturally produced in certain species. For example, the framework region is modified to provide a humanized antibody. Additionally, or alternatively, the antibody can be genetically modified by modifying residues in the constant region, e.g., altering the effector function of the antibody.

[0094] In certain embodiments, CDR grafting can be used to genetically modify the variable regions of an antibody. Antibodies interact with target antigens primarily through amino acid residues located in six heavy and light chain complementarity determining regions (CDRs). For this reason, the amino acid residues within the CDRs are more diverse between individual antibodies than the sequences outside the CDRs. Since the CDR sequences are responsible for the major antibody-antigen interactions, recombinant antibodies that mimic the properties of a specific natural antibody can be expressed by constructing expression vectors containing the CDR sequences of a specific natural antibody grafted into the framework sequences of different antibodies with different properties (Riechmann et al., (1998) Nature 332:323-327; Jones et al., (1986) Nature 321:522-525; Queen et al..(1989) Proc. Natl. Acad; U.S.A. 86:10029-10033; U.S. Pat. Nos. 5,225,539; 5,530,101; 5,585,089; 5,693,762 and 6,180,370).

[0095] Accordingly, another embodiment of the present application relates to an isolated monoclonal antibody or antigen-binding portion thereof that comprises a heavy chain variable region and / or a light chain variable region, the heavy chain variable region comprising CDR1, CDR2, and CDR3 having the sequences described above in the present application, and the light chain variable region comprising CDR1, CDR2, and CDR3 having the sequences described above in the present application. Although these antibodies comprise the V H and V L CDR sequences of the monoclonal antibodies of the present application, they can contain different framework sequences.

[0096] Such framework sequences can be obtained from publicly available DNA databases or publicly available references that include germline antibody gene sequences. For example, germline DNA sequences for human heavy and light chain variable region genes can be found in the Vbase human germline sequence database (www.mrc-cpe.cam.ac.uk / vbase) as well as Kabat et al., (1991), supra; Tomlinson et al., (1992) J. Mol. Biol. 227:776-798; and Cox et al., (1994) Eur. J. Immunol. 24:827-836. As another embodiment, germline DNA sequences for human heavy and light chain variable region genes can be obtained from the Genbank database. For example, the Genbank accession numbers for the heavy chain germline sequences in the following HCo7 HuMAb mice are 1-69 (NG--0010109, NT--024637&BC070333), 3-33 (NG--0010109&NT--024637), and 3-7 (NG--0010109&NT--024637). As another example, the Genbank accession numbers for the heavy chain germline sequences from the following Hco12 HuMAb mice are 1-69 (NG--0010109, NT--024637&BC070333), 5-51 (NG--0010109&NT--024637), 4-34 (NG--0010109&NT--024637), 3-30.3 (CAJ556644), and 3-23 (AJ406678).

[0097] The antibody protein sequence is compared to a protein sequence database by using one of the sequence similarity search methods known in the art called gapped BLAST (Altschul et al., (1997)).

[0098] Preferred framework sequences for the antibodies of the present application are those that are structurally similar to the framework sequences used in the antibodies of the present application. V H The CDR1, CDR2, and CDR3 sequences can be implanted into a framework region having the same sequence as the germline immunoglobulin gene from which the framework sequence was obtained, or the CDR sequences can be implanted into a framework region that contains one or more mutations compared to the germline sequence. For example, in some cases, it is beneficial to mutate residues in the framework region to maintain or enhance the antigen-binding of the antibody (see, e.g., U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370).

[0099] Another class of variable region modifications is to V Hand / or V L Amino acid residues in the CDR1, CDR2 and / or CDR3 regions are mutated to improve one or more binding properties (e.g., affinity) of the target antibody. Point mutations or PCR-mediated mutations can be performed to introduce the mutations, and the effects on antibody binding or other functional properties can be evaluated in in vitro or in vivo assays known in the art. Preferably, conservative modifications known in the art are introduced. The mutations can be amino acid substitutions, additions or deletions, but are preferably substitutions. In addition, usually no more than one, two, three, four or five residues in the CDR region are altered.

[0100] In addition, in another embodiment, the present application provides an isolated TIGIT monoclonal antibody or an antigen-binding portion thereof, comprising a heavy chain variable region and a light chain variable region, which comprises: (a) V H CDR1 region, comprising the sequence of the present application, or an amino acid sequence with one, two, three, four or five amino acid substitutions, deletions or additions; (b) V H CDR2 region, comprising the sequence of the present application, or an amino acid sequence with one, two, three, four or five amino acid substitutions, deletions or additions; (c) V H CDR3 region, comprising the sequence of the present application, or an amino acid sequence with one, two, three, four or five amino acid substitutions, deletions or additions; (d) V L CDR1 region, comprising the sequence of the present application, or an amino acid sequence with one, two, three, four or five amino acid substitutions, deletions or additions; (e) V L CDR2 region, comprising the sequence of the present application, or an amino acid sequence with one, two, three, four or five amino acid substitutions, deletions or additions; and (f) V L CDR3 region, comprising the sequence of the present application, or an amino acid sequence with one, two, three, four or five amino acid substitutions, deletions or additions.

[0101] The genetically engineered antibodies of the present application include those that make genetic modifications in the framework residues of V H and / or V L to alter antibody properties, for example. Generally, these framework modifications are used to reduce the immunogenicity of the antibody. For example, one approach is to "back-mutate" one or more framework residues to the corresponding germline sequence. More specifically, an antibody that has undergone somatic mutation may contain framework residues that are different from the germline sequence from which the antibody was derived. These residues can be identified by comparing the antibody framework sequence with the germline sequence from which the antibody was derived.

[0102] Another class of framework modifications involves mutating one or more residues in the framework region or even in one or more CDR regions to remove T cell epitopes and thereby reduce the potential immunogenicity of the antibody. This method is also referred to as "deimmunization" and is described in more detail in U.S. Patent Publication 20030153043.

[0103] In addition, as an alternative to modifications within the framework or CDR regions, the antibodies of the present application can be genetically engineered to include genetic modifications in the Fc region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement binding, Fc receptor binding, and / or antibody-dependent cell cytotoxicity. In addition, the antibodies of the present application can be chemically modified (e.g., one or more chemical functional groups can be attached to the antibody), or modified to alter their glycosylation to change one or more functional properties of the antibody.

[0104] In one embodiment, the hinge region of C H1 is modified, for example, to increase or decrease the number of cysteine residues in the hinge region. This method is further described in U.S. Patent 5,677,425. Modifying the cysteine residues in the hinge region of C H1 to, for example, facilitate heavy chain-light chain assembly or increase / decrease the stability of the antibody.

[0105] In another embodiment, the Fc hinge region of the antibody is mutated to reduce the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the C H2 -C H3 linker region of the Fc hinge fragment such that the antibody has a reduced SpA binding affinity relative to the native Fc-hinge domain SpA binding. This method is described in more detail in U.S. Patent 6,165,745.

[0106] In another embodiment, the glycosylation of the antibody is modified. For example, deglycosylated antibodies (i.e., antibodies lacking glycosylation) can be prepared. The glycosylation can be altered, for example, to increase the affinity of the antibody for the antigen. Such glycosylation modifications can be achieved, for example, by changing one or more glycosylation sites in the antibody sequence. For example, one or more amino acid substitutions can be made to eliminate one or more variable region framework glycosylation sites, thereby eliminating glycosylation at that position. Such deglycosylation can increase the affinity of the antibody for the antigen. See, for example, U.S. Patents 5,714,350 and 6,350,861.

[0107] In addition, antibodies with altered glycosylation patterns can be prepared, such as hypofucosylated antibodies with a reduced amount of fucose residues, or antibodies with an increased bisecting GlcNac structure. The altered glycosylation forms have been shown to increase the ADCC activity of antibodies. Such glycosylation modifications can be carried out, for example, by expressing the antibody in host cells with an altered glycosylation system. Cells with an altered glycosylation system are known in the art and can be used as host cells for expressing the recombinant antibodies of the present application to prepare antibodies with altered glycosylation. For example, the cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene FUT8 (α(1,6)-fucosyltransferase), so that the antibodies expressed in the Ms704, Ms705, and Ms709 cell lines lack fucose in their sugars. The Ms704, Ms705, and Ms709 FUT8− / − cell lines were prepared by using two replacement vectors to disrupt the FUT8 gene in CHO / DG44 cells (see US Patent Publication 20040110704 and Yamane-Ohnuki et al., (2004) Biotechnol Bioeng 87:614-22). As another example, EP 1,176,195 describes a cell line with disrupted FUT8 gene function, which encodes a fucosyltransferase, so that the antibodies expressed in this cell line exhibit hypofucosylation by reducing or eliminating the α-1,6 bond-related enzyme. EP 1,176,195 also describes a cell line with lower enzymatic activity for adding fucose to N-acetylglucosamine binding to the Fc region of the antibody, or without the activity of this enzyme, such as the rat myeloma cell line YB2 / 0 (ATCC CRL 1662). WO 03 / 035835 describes a CHO variant cell line, Lec13 cells, which have a reduced ability to add fucose to Asn(297)-related sugars, resulting in hypofucosylation of the antibodies expressed in the host cells (see Shields et al., (2002) J. Biol. Chem. 277:26733-26740). Antibodies with an altered glycosylation profile can also be prepared in eggs, as described in WO 06 / 089231. Alternatively, antibodies with an altered glycosylation profile can be prepared in plant cells such as duckweed. WO 99 / 54342 discloses a cell line genetically engineered to express a glycosyltransferase that modifies glycoproteins (e.g., β(1,4)-N-acetylglucosaminyltransferase III (GnTIII)), so that the antibodies expressed in the genetically engineered cell line exhibit an increased bisecting GlcNac structure, which causes enhanced ADCC activity of the antibodies (Umana et al., (1999) Nat. Biotech. 17:176-180).Alternatively, the fucose residues of the antibody can be removed using fucosidase, such as α-L-fucosidase to remove fucose residues from the antibody (Tarentino et al., (1975) Biochem. 14: 5516-23).

[0108] Another modification of the antibodies herein is pegylation (PEGylation). Antibodies can be PEGylated, for example, to increase the biological (e.g., serum) half-life of the antibody. To PEGylate an antibody, the antibody or a fragment thereof is typically reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions that result in attachment of one or more PEG groups to the antibody or antibody fragment. Preferably, PEGylation is carried out by an acylation reaction or an alkylation reaction with a reactive PEG molecule (or a similar reactive water-soluble polymer). The term "polyethylene glycol" as used herein includes any form of PEG used to derivatize other proteins, such as mono(C1-C 1o )alkoxy- or aryloxy polyethylene glycol or polyethylene glycol maleimide. In certain embodiments, the antibody to be PEGylated is a deglycosylated antibody. Methods for PEGylating proteins are known in the art and can be applied to the antibodies of the present application. See, for example, EPO 154 316 and EP 0 401 384.

[0109] Physical properties of the antibody

[0110] The antibodies of the present application can be characterized by their various physical properties to detect and / or distinguish their classification.

[0111] For example, the antibody can contain one or more glycosylation sites in the variable region of the light or heavy chain. These glycosylation sites may cause increased immunogenicity of the antibody or altered pK values of the antibody due to altered antigen binding (Marshall et al (1972) Annu Rev Biochem 41: 673-702; Gala and Morrison (2004) J Immunol 172: 5489-94; Wallick et al (1988) J Exp Med 168: 1099-109; Spiro (2002) Glycobiology 12: 43R-56R; Parekh et al (1985) Nature 316: 452-7; Mimura et al., (2000) Mol Immunol 37: 697-706). Glycosylation is known to occur in motifs containing the N-X-S / T sequence. In some cases, it is preferred that the TIGIT antibody does not contain variable region glycosylation. This can be achieved by selecting an antibody that does not contain a glycosylation motif in the variable region or by mutating the residues in the glycosylation region.

[0112] In a preferred embodiment, the antibody does not contain asparagine isomerization sites. Deamidation of asparagine may occur in N-G or D-G sequences, creating isoaspartic acid residues, which introduce kinks into the polypeptide chain and reduce its stability (the isoaspartic acid effect).

[0113] Each antibody will have a unique isoelectric point (pI), which generally falls within the pH range of 6 - 9.5. The pI of IgG1 antibodies generally falls within the pH range of 7 - 9.5, while the pI of IgG4 antibodies generally falls within the pH range of 6 - 8. It is speculated that antibodies with a pI outside the normal range may have some unfolded structures and be unstable under in vivo conditions. Therefore, it is preferred that the pI value of the TIGIT antibody falls within the normal range. This can be achieved by selecting antibodies with a pI within the normal range or by mutating uncharged surface residues.

[0114] Nucleic acid molecule encoding the antibody of the present application

[0115] In another aspect, the present application provides nucleic acid molecules encoding the heavy chain / light chain variable regions or CDRs of the antibodies or antigen-binding portions thereof of the present application. The nucleic acid can be present in whole cells, in cell lysates, or in a partially purified or substantially pure form. The nucleic acid is "isolated" or "substantially pure" when purified from other cellular components or other contaminants such as other cellular nucleic acids or proteins by standard techniques. The nucleic acid of the present application can be, for example, DNA or RNA, and may or may not contain intron sequences. In a preferred embodiment, the nucleic acid is a cDNA molecule.

[0116] The nucleic acids of the present application can be obtained using standard molecular biology techniques. For antibodies expressed by hybridomas (e.g., hybridomas prepared from transgenic mice carrying human immunoglobulin genes), the cDNAs encoding the light and heavy chains of the antibodies prepared by the hybridomas can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from immunoglobulin gene libraries (e.g., using phage display techniques), the nucleic acids encoding such antibodies can be collected from the gene library.

[0117] Preferred nucleic acid molecules of the present application include those encoding the V H and V L sequences or CDRs of the TIGIT monoclonal antibody. Once the DNA fragments encoding V H and V L are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, such as converting the variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. In these manipulations, the encoding V H or V LA DNA fragment is operably linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. The term "operably linked" means that the two DNA fragments are joined together such that the amino acid sequences encoded by the two DNA fragments are in frame.

[0118] The isolated DNA encoding the V H region can be converted into a full-length heavy chain gene by operably linking the V H encoding DNA to another DNA molecule encoding the heavy chain constant region (C H1 , C H2 and C H3 ). The sequences of the human heavy chain constant region genes are known in the art, and DNA fragments comprising these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but is preferably an IgGl or IgG4 constant region. For the Fab fragment heavy chain gene, the DNA encoding the V H region can be operably linked to another DNA molecule encoding only the heavy chain C H1 constant region.

[0119] The isolated DNA encoding the V L region can be converted into a full-length light chain gene by operably linking the V L encoding DNA to another DNA molecule encoding the light chain constant region C L . The sequences of the human light chain constant region genes are known in the art, and DNA fragments comprising these regions can be obtained by standard PCR amplification. In a preferred embodiment, the light chain constant region can be a κ and λ constant region.

[0120] To create a scFv gene, the DNA fragments encoding V H and V L can be operably linked to another fragment encoding a flexible linker, such as the fragment encoding the amino acid sequence (Gly4-Ser)3, such that the V H and V L sequences can be expressed as a continuous single-chain protein, wherein the V H and V L regions are joined by this flexible linker (see, for example, Bird et al., (1988) Science 242:423-426; Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., (1990) Nature 348:552-554).

[0121] Preparation of the monoclonal antibody of the present application

[0122] The monoclonal antibodies of the present application can be prepared using the somatic cell hybridization (hybridoma) technique of Kohler and Milstein (1975) Nature 256:495. Other embodiments for preparing monoclonal antibodies include viral or oncogenic transformation of B lymphocytes and phage display techniques. Chimeric or humanized antibodies are also well known in the art. See, for example, U.S. Patents 4,816,567; 5,225,539; 5,530,101; 5,585,089; 5,693,762 and 6,180,370.

[0123] Generation of transfected tumors for preparing the monoclonal antibody of the present application

[0124] The antibodies of the present application can also be produced, for example, in transfected host cell tumors using recombinant DNA technology in combination with gene transfection methods (e.g., Morrison, S. (1985) Science 229:1202). In one embodiment, DNA encoding partial or full-length light and heavy chains obtained by standard molecular biotechnology is inserted into one or more expression vectors such that the gene is operably linked to transcriptional and translational regulatory sequences. In this context, the term "operably linked" means that the antibody gene is linked into the vector such that the transcriptional and translational control sequences within the vector perform their intended function of regulating transcription and translation of the antibody gene.

[0125] The term "regulatory sequences" includes promoters, enhancers, and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody gene. Such regulatory sequences have been described, for example, in Goeddel (Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)). Preferred regulatory sequences for expression in mammalian host cells include viral elements that direct high-level protein expression in mammalian cells, such as promoters and / or enhancers derived from cytomegalovirus (CMV), simian virus 40 (SV40), adenovirus, such as the adenovirus major late promoter (AdMLP) and polyomavirus. Alternatively, non-viral regulatory sequences can be used, such as the ubiquitin promoter or the β-globin promoter. Additionally, regulatory elements are composed of sequences from different sources, such as the SRα promoter system, which contains sequences from the SV40 early promoter and the long terminal repeat of human T-cell leukemia virus type I (Takebe et al., (1988) Mol. Cell. Biol. 8:466-472). The expression vector and expression control sequences are selected to be compatible with the expression host cell used.

[0126] The antibody light chain gene and the antibody heavy chain gene can be inserted into the same or different expression vectors. In a preferred embodiment, the variable region is used to construct a full-length antibody gene by inserting it into an expression vector that already encodes the heavy chain constant region and the light chain constant region of the desired isotype, so that the V H is operably linked to the C H in the vector, and the V L is operably linked to the C L in the vector. Alternatively, the recombinant expression vector can encode a signal peptide that promotes the secretion of the antibody chain from the host cell. The antibody chain gene can be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin).

[0127] In addition to the antibody chain gene and regulatory sequences, the recombinant expression vectors of the present application can carry other sequences, such as sequences that regulate the replication of the vector in the host cell (e.g., an origin of replication) and selectable marker genes. Selectable marker genes can be used to select host cells that have taken up the vector (see, e.g., U.S. Patents 4,399,216; 4,634,665, and 5,179,017). For example, typically a selectable marker gene confers drug resistance to the host cells that have taken up the vector, such as G418, hygromycin, or methotrexate resistance. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for methotrexate selection / amplification in dhfr host cells) and the neo gene (for G418 selection).

[0128] For the expression of the light and heavy chains, the expression vectors encoding the heavy and light chains are transfected into host cells by standard techniques. The term "transfection" in its various forms encompasses a variety of techniques commonly used to introduce foreign DNA into prokaryotic or eukaryotic host cells, e.g., electroporation, calcium phosphate precipitation, DEAE-dextran transfection, etc. Although it is theoretically possible to express the antibodies of the present application in prokaryotic or eukaryotic host cells, it is preferred to express the antibodies in eukaryotic cells, most preferably in mammalian host cells, because eukaryotic cells, particularly mammalian cells, are more likely than prokaryotic cells to assemble and secrete properly folded and immunologically active antibodies.

[0129] Preferred mammalian host cells for expressing the recombinant antibodies of the present application include Chinese hamster ovary (CHO cells) (including dhfr-CHO cells co-administered with a DHFR selectable marker, described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77: 4216-4220, and the DHFR selectable marker is described in, for example, R.J. Kaufman and P.A. Sharp (1982) J. Mol. Biol. 159: 601-621), NSO myeloma cells, COS cells, and SP2 cells. Particularly when using NSO myeloma cells, another preferred expression system is the GS gene expression system, described in WO 87 / 04462, WO 89 / 01036, and EP 338,841. When the recombinant expression vector encoding the antibody gene is introduced into a mammalian host cell, the antibody is prepared by culturing the host cell for a period sufficient for antibody expression in the host cell, or preferably sufficient for secretion of the antibody into the culture medium in which the host cell grows. The antibody can be recovered from the culture medium using protein purification methods.

[0130] Immunoconjugate

[0131] The antibodies or antigen-binding portions thereof of the present application can be cross-linked with a therapeutic agent to form an immunoconjugate, such as an antibody-drug conjugate (ADC). Suitable therapeutic agents include cytotoxins, alkylating agents, DNA minor groove binding molecules, DNA intercalating agents, DNA cross-linking agents, histone deacetylase inhibitors, nuclear export inhibitors, proteasome inhibitors, inhibitors of topoisomerase I or II, heat shock protein inhibitors, tyrosine kinase inhibitors, antibiotics, and antimitotic agents. In an ADC, the antibody and the therapeutic agent are preferably cross-linked via a linker that is cleavable, such as a peptide linker, a disulfide linker, or a hydrazone linker. More preferably, the linker is a peptide linker, such as Val-Cit, Ala-Val, Val-Ala-Val, Lys-Lys, Ala-Asn-Val, Val-Leu-Lys, Ala-Ala-Asn, Git-Git, VaL-Lys, Lys, Cit, Ser, or Glu. The ADC can be prepared as described in U.S. Patents 7,087,600; 6,989,452; and 7,129,261; PCT Publications WO 02 / 096910; WO 07 / 038,658; WO 07 / 051,081; WO 07 / 059,404; WO 08 / 083,312; and WO 08 / 103,693; U.S. Patent Publications 20060024317; 20060004081; and 20060247295.

[0132] Bispecific molecule

[0133] On the other hand, the present application relates to bispecific molecules comprising one or more antibodies or antigen-binding portions thereof of the present application linked to at least one other functional molecule such as another peptide or protein (e.g., another antibody or receptor ligand) to generate a bispecific molecule that binds to at least two different binding sites or target molecules. The term "bispecific molecule" includes molecules having three or more specificities.

[0134] In an embodiment, in addition to Fc-binding specificity and TIGIT-binding specificity, the bispecific molecule further has a third specificity. The third specificity can be directed against PD-1, PD-L1 or CTLA-4 to enhance the immune system response. Alternatively, the third specificity can be against an enhancer factor (EF), e.g., a molecule that binds to a surface protein involved in cytotoxic activity and thus increases the immune response against target cells. For example, an enhancer factor antibody can bind to cytotoxic T cells (e.g., via CD2, CD3, CD8, CD28, CD4, CD40 or ICAM-1) or other immune cells, eliciting an enhanced immune response against target cells.

[0135] Bispecific molecules can occur in a variety of forms and sizes. At one end of the size spectrum, the bispecific molecule retains the conventional antibody form, except that it has two binding arms with different specificities instead of having two binding arms with the same specificity. At the other extreme are bispecific molecules composed of two single-chain antibody fragments (scFv) linked by a peptide chain, called a Bs(scFv)2 construct. Intermediate-sized bispecific molecules include two different F(ab) fragments linked by a peptide linker. These and other forms of bispecific molecules can be prepared by genetic engineering, somatic hybridization or chemical methods. See, e.g., Kufer et al, cited supra; Cao and Suresh, Bioconjugate Chemistry, 9(6), 635-644 (1998); and van Spriel et al., Immunology Today, 21(8), 391-397 (2000).

[0136] Chimeric antigen receptor

[0137] The present application also provides a chimeric antigen receptor comprising a TIGIT single-chain antibody scFv, the scFv comprising the heavy and light chain CDRs, or heavy and light chain variable regions, described herein.

[0138] The TIGIT chimeric antigen receptor can comprise (a) an extracellular antigen-binding domain containing a TIGIT scFv; (b) a transmembrane domain; and (c) an intracellular signal transduction domain.

[0139] Oncolytic virus encoding an antibody or carrying an antibody

[0140] Oncolytic viruses preferentially infect and kill cancer cells. The antibodies of the present invention are used in combination with oncolytic viruses. In addition, oncolytic viruses encoding the antibodies of the present invention can be introduced into the human body.

[0141] Pharmaceutical composition

[0142] In another aspect, the present application provides a pharmaceutical composition comprising one or more antibodies or antigen-binding portions thereof, antibody or antigen-binding portion-encoding vectors, immunoconjugates, immune cells, bispecific antibodies, and / or oncolytic viruses of the present application, formulated together with a pharmaceutically acceptable carrier. The composition can optionally comprise one or more other pharmaceutically active ingredients, such as another anti-tumor antibody, anti-infective antibody, or immune-enhancing antibody, or a non-antibody anti-tumor agent, anti-infective agent, or immune-enhancing agent. The pharmaceutical compositions of the present application can be used in combination with, for example, another anti-cancer agent, another anti-infective agent, or another immune-enhancing agent.

[0143] The pharmaceutical composition can comprise any number of excipients. Excipients that can be used include carriers, surfactants, thickening or emulsifying agents, solid binders, dispersing or suspending agents, solubilizing agents, coloring agents, flavoring agents, coatings, disintegrating agents, lubricants, sweetening agents, preservatives, isotonic agents, and combinations thereof. The selection and use of suitable excipients are taught in Gennaro, ed., Remington: The Science and Practice of Pharmacy, 20th Ed. (Lippincott Williams & Wilkins 2003).

[0144] Preferably, the pharmaceutical composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or bolus). Depending on the route of administration, the active ingredient can be encapsulated in materials to protect it from acids and other natural conditions that may inactivate it. "Parenteral administration" refers to a manner different from enteral and topical administration and is usually by injection, including but not limited to intravenous, intramuscular, intraarterial, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and bolus. Alternatively, the antibodies of the present application can be administered by non-parenteral routes, such as topical, epidermal, or mucosal administration, such as intranasal, oral, vaginal, rectal, sublingual, or topical application.

[0145] The pharmaceutical composition can be in the form of a sterile aqueous solution or dispersion. They can also be formulated in microemulsions, liposomes or other ordered structures suitable for high concentrations of the drug.

[0146] The amount of the active ingredient prepared as a single dosage form together with the carrier material will vary with the subject to be treated and the particular mode of administration, and will essentially be the amount of the composition that produces a therapeutic effect. Expressed as a percentage, this amount is about 0.01 - about 99% of the active ingredient in combination with a pharmaceutically acceptable carrier.

[0147] The dosing regimen is adjusted to provide the optimal desired response (e.g., a therapeutic response). For example, a rapid infusion can be administered, multiple divided doses can be administered over time, or the dose can be proportionally decreased or increased depending on the criticality of the treatment situation. Particularly advantageously, the parenteral composition is configured in dosage unit forms that are convenient to administer and have uniform dosing. A dosage unit form refers to physically discrete units suitable for single administration to a subject to be treated; each unit contains a predetermined amount of the active ingredient calculated to produce the desired therapeutic effect in association with the pharmaceutical carrier. Alternatively, the antibody can be administered in a sustained-release formulation, in which case the required frequency of administration is reduced.

[0148] For the administration of the antibody, the dose can be about 0.001 - 100 mg / kg of the host body weight. An exemplary treatment regimen involves administration once a week. The preferred dosing regimen of TIGIT in this application includes intravenous administration.

[0149] The "therapeutically effective amount" of the TIGIT antibody in this application causes a reduction in the severity of the disease symptoms, an increase in the frequency and duration of the asymptomatic period. For example, for the treatment of tumor-bearing subjects, the "therapeutically effective amount" preferably inhibits tumor growth by at least about 20%, more preferably inhibits by at least about 40%, even more preferably inhibits by at least about 60%, and even more preferably inhibits by at least about 80% compared to untreated subjects. The therapeutically effective amount of the therapeutic antibody can reduce the tumor size, or alleviate the symptoms of the subject, and the subject can be a human or another mammal.

[0150] The pharmaceutical composition can be a sustained-release reagent, including implants and microcapsule delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene-vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. See, for example, Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0151] The pharmaceutical composition can be administered via a medical device, such as (1) a needleless subcutaneous injection device (e.g., U.S. Pat. Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; and 4,596,556); (2) a microinfusion pump (U.S. Pat. No. 4,487,603); (3) a transdermal delivery device (U.S. Pat. No. 4,486,194); (4) a bolus injection device (U.S. Pat. Nos. 4,447,233 and 4,447,224); and (5) an osmotic device (U.S. Pat. Nos. 4,439,196 and 4,475,196).

[0152] In certain embodiments, the monoclonal antibody of the present application can be formulated to ensure proper in vivo distribution. For example, to ensure that the therapeutic antibody of the present application crosses the blood-brain barrier, the antibody can be formulated in liposomes, which may additionally contain targeting functional groups to enhance selective delivery to specific cells or organs. See, e.g., U.S. Pat. Nos. 4,522,811; 5,374,548; 5,416,016; and 5,399,331; V.V. Ranade (1989) J. Clin. Pharmacol. 29:685; Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153:1038; Bloeman et al., (1995) FEBS Lett. 357:140; M. Owais et al., (1995) Antimicrob. Agents Chemother. 39:180; Briscoe et al., (1995) Am. J. Physiol. 1233:134; Schreier et al., (1994) J. Biol. Chem. 269:9090; Keinanen and Laukkanen (1994) FEBS Lett. 346:123; and Killion and Fidler (1994) Immunomethods 4:273.

[0153] Uses and methods of the present application

[0154] The pharmaceutical composition of the present application has a variety of in vitro and in vivo applications, relating to, for example, the treatment of cancer and infectious diseases, or more generally, for immune enhancement in cancer and infectious disease patients. The antibody can be administered to human subjects to, for example, inhibit tumor growth in vivo, reduce or eliminate pathogens.

[0155] Considering the ability of the pharmaceutical composition of the present application to inhibit the proliferation and survival of tumor cells, the present application provides a method for inhibiting the growth of tumor cells in a subject, including administering the pharmaceutical composition of the present application to the subject, whereby tumor growth in the subject is inhibited. Non-limiting examples of tumors that can be treated with the antibodies of the present application include, but are not limited to, liver cancer, rectal cancer, endometrial cancer, pancreatic cancer, non-small cell lung cancer, multiple myeloma, and melanoma, primary or metastatic. In addition, refractory or recurrent malignant tumors may be inhibitable with the antibodies of the present application.

[0156] The pharmaceutical composition of the present application can be used to reduce or eliminate pathogens, and the present application provides a method for treating infectious diseases, including administering the pharmaceutical composition of the present application to the subject. Infectious diseases can be caused by viruses, bacteria, fungi, mycoplasmas, etc., such as chronic infections caused by HIV.

[0157] These and other methods of the present application are further discussed below.

[0158] Combination therapy

[0159] The present application provides combination therapies in which the pharmaceutical composition of the present application is administered together with one or more other antibody or non-antibody therapeutic agents, which can effectively inhibit tumor growth in a subject. In one embodiment, the present application provides a method for inhibiting tumor growth in a subject, including administering a TIGIT pharmaceutical composition to the subject and one or more other antibodies, such as a PD-L1 antibody, a PD-1 antibody, and / or a CTLA-4 antibody. In certain embodiments, the subject is a human. In another aspect, the present application provides a method for treating cancer, in which the TIGIT pharmaceutical composition of the present application is administered together with a chemotherapeutic agent, which can be a cytotoxic agent. Other therapies that can be combined with the TIGIT pharmaceutical composition include, but are not limited to, administration of immunogenic agents, administration of interleukin 2 (IL-2), radiotherapy, surgery, or hormone ablation.

[0160] The present application also provides combination therapies in which the pharmaceutical composition of the present application is administered together with one or more other antibody or non-antibody therapeutic agents, which can effectively reduce or eliminate pathogens in a subject, such as viruses, bacteria, fungi, mycoplasmas. For example, the pharmaceutical composition of the present application can be used in combination with anti-infective agents, including, but not limited to, antiviral agents, antibacterial agents, antifungal agents, and anti-mycoplasma agents, etc.

[0161] The combinations of therapeutic agents discussed herein can be administered simultaneously as a single composition in a pharmaceutically acceptable carrier, or as separate compositions simultaneously, wherein each agent is in a pharmaceutically acceptable carrier. In another embodiment, the combinations of therapeutic agents can be administered sequentially.

[0162] In addition, if multiple combination therapy administrations are performed and the agents are administered in sequence, the order of sequential administration at each time point can be reversed or remain the same, and sequential administration can be combined with simultaneous administration or any combination thereof.

[0163] This application is further described by the following examples, which should not be construed as restrictive. All drawings and all references, Genebank sequences, patents, and published patent applications cited throughout this application are hereby incorporated by reference in their entirety.

[0164] Examples

[0165] Example 1 Construction of HEK293A cell lines stably expressing human, monkey or mouse TIGIT, or human PVR

[0166] HEK293A cells were used to construct cell lines stably overexpressing human, monkey or mouse TIGIT. Briefly, the cDNA sequences of human, monkey or mouse TIGIT and human PVR (amino acid sequences are shown in SEQ ID NOs: 35, 36, 37 and 38 respectively) were synthesized and digested and cloned into the pLV-EGFP(2A)-Puro vector (Beijing Yingmao Shengye Biotechnology Co., Ltd., China). The obtained pLV-EGFP(2A)-Puro-TIGIT or pLV-EGFP(2A)-Puro-PVR and the psPAX and pMD2.G plasmids were transfected into HEK293T cells (Nanjing Kebai Co., Ltd., China) by liposome transfection to produce lentivirus. The specific transfection method was exactly the same as the steps in the instruction manual of the Lipofectamine 3000 kit (Thermo Fisher Scientific, USA). Three days after transfection, the lentivirus was harvested from the cell culture medium of HEK293T cells (DMEM medium (Cat#: SH30022.01, Gibco), supplemented with 10% FBS (Cat#: FND500, Excell)). Then HEK293A cells (Nanjing Kebai Co., Ltd., China) were transfected with the lentivirus to obtain HEK293A cells stably expressing human, monkey or mouse TIGIT (designated as HEK293A / human TIGIT, HEK293A / monkey TIGIT, HEK293A / mouse TIGIT respectively), or A549 cells (Nanjing Kebai Co., Ltd., China) were transfected to obtain the cell line A549 / human PVR cells stably expressing human PVR. The transfected HEK293A cells and A549 cells were cultured in DMEM + 10% FBS medium containing 0.2 μg / ml puromycin (Cat#: A11138-03, Gibco) for 7 days. The expression of human and monkey TIGIT was analyzed by FACS using a commercially available TIGIT antibody (PE-human TIGIT antibody, Cat#: 372703, Biolegend, USA) through a flow cytometer. Similarly, the expression of mouse TIGIT was confirmed by FACS using a commercially available mouse TIGIT antibody (PE-mouse TIGIT antibody, Cat#: 622205, Biolegend, USA). The expression of human PVR was confirmed by FACS using a commercially available human PVR antibody (PE-human PVR antibody, Cat#: 566718, BD, USA).

[0167] Example 2 Preparation of hybridoma cell lines producing mouse anti-human TIGIT

[0168] Mouse anti-human TIGIT monoclonal antibody was obtained by conventional hybridoma fusion technology with a slightly modified protocol.

[0169] Immunization

[0170] Ten BALB / c mice (Vital River, China) were immunized by cross-injecting recombinant human TIGIT (ECD)-hFc protein (Cat#: 10917-H02H, Sino Biological, China) and monkey TIGIT (ECD)-hFc protein (Cat#: TIT-C5254, Sino Biological, China). The specific immunization process is shown in Table 2. The human TIGIT (ECD)-hFc protein and the monkey TIGIT (ECD)-hFc protein were emulsified by ultrasound with an equal volume of complete Freund's adjuvant (Cat#: F5881-10*10ML, Sigma, USA), incomplete Freund's adjuvant (Cat#: F5506-6*10ML, Sigma, USA) or PBS.

[0171] Table 2. Immunization protocol

[0172]

[0173] One week after each booster immunization, 50 μl of serum was taken from the tail of each mouse, and the titer was detected by ELISA. Specifically, recombinant human TIGIT (ECD)-his (Cat#: 10917-H08H, Sino Biological, China) and monkey TIGIT (ECD)-hFc (Cat#: TIT-C5254, Sino Biological, China) were used for the binding test. The titer was also tested by FACS using the HEK293A cells overexpressing human, monkey, and mouse TIGIT prepared in Example 1.

[0174] Based on the ELISA and FACS test results after the last booster immunization, 8 mice with higher serum titers were selected for the next step of hybridoma cell line preparation.

[0175] Preparation of hybridoma cell lines

[0176] The hybridoma cell line was prepared by conventional hybridoma fusion technology with a slightly modified protocol.

[0177] Four days after the last immune boost, the mice were sacrificed, spleens were removed, and single-cell suspensions were prepared in PBS. The splenocytes were washed three times with DMEM medium (Cat#: SH30243.01B, Hyclone, USA). Mouse myeloma cells SP2 / 0 (CRL-1581, ATCC, USA) in the logarithmic growth phase were mixed with the above-isolated mouse splenocytes at a ratio of 1:4 and then washed twice with DMEM. Cell fusion was carried out by PEG (Cat#: P7181, Sigma, USA) fusion. The fused cells were washed three times with DEME and resuspended in cell growth medium (RPMI 1640 (Cat#: C22400500CP, Gibco) + 10% FBS + 1X HAT (H0262, Sigma)). The cell suspension was plated in a 96-well culture plate, 200 μl per well, 5×10 4 cells per well, and the cells were placed in a humid cell culture incubator at 37 °C and 5% CO2 for 7 days. After that, the medium was replaced with fresh medium (DMEM + 10% FBS + 1X HAT). Two to three days later, the cell culture supernatant was aspirated, and hybridoma cells were screened by ELISA and FACS.

[0178] Screening of hybridoma cell lines by ELISA

[0179] Hybridoma clones that bind to human TIGIT were screened by high-throughput ELISA binding assay, and human TIGIT (ECD)-his (Cat#: 10917-H08H, Sino Biological, China) was used in the ELISA. The hybridoma clones that bind to human TIGIT were further tested for their ability to bind to cynomolgus monkey TIGIT, and cynomolgus monkey TIGIT (ECD)-hFc (Cat#: TIT-C5254, Sino Biological, China) was used as the detection antigen in the ELISA.

[0180] Eighty-five hybridoma cell lines with specific binding ability to human and cynomolgus monkey TIGIT were screened by the above ELISA.

[0181] Screening of hybridoma cell lines by FACS detection

[0182] The binding ability of the 85 screened hybridoma cell lines to human, cynomolgus monkey, or mouse TIGIT expressed on HEK293A cells was further tested using the HEK293A / human TIGIT cells, HEK293A / cynomolgus monkey TIGIT cells, and HEK293A / mouse TIGIT cells prepared in Example 1.

[0183] Based on the above FACS screening, 58 hybridoma clones with high binding affinity for HEK293A / human TIGIT cells and HEK293A / monkey TIGIT cells, but not binding to HEK-293A / mouse TIGIT cells, were obtained.

[0184] Subcloning of hybridoma cells producing TIGIT antibody

[0185] The above 58 hybridoma clones were subjected to two rounds of subcloning. During the subcloning process, multiple subclones (n>3) of each clone were selected and characterized by the above ELISA and FACS assays. The subclones obtained through this step were identified as monoclonal hybridoma cell lines. Finally, 28 subclones with high binding affinity for human and monkey TIGIT were obtained, and each subclone was derived from a different original parental clone.

[0186] Example 3 Purification of mouse TIGIT monoclonal antibody

[0187] Twenty clones with high binding affinity for human and monkey TIGIT were selected from the 28 clones obtained in Example 2 for further study. First, the monoclonal mouse antibodies of the 20 selected clones were purified. Briefly, the hybridoma cells of each subclone were grown in T175 cell culture flasks, each flask containing 100 ml of fresh serum-free hybridoma medium (Gibco, USA, Cat#: 12045-076) and 1% HT supplement (Gibco, Cat#: 11067-030). The cells were cultured in an incubator at 37°C and 5% CO2 for 10 days. The culture was collected, centrifuged at 3500 rpm for 5 minutes, and cell debris was removed by filtration through a 0.22 μm filter membrane. The monoclonal antibody was enriched and purified by a pre-equilibrated protein-A affinity column (Cat#: 17040501, GE, USA). Elution was then performed with elution buffer (20 mM citric acid, pH 3.0 - pH 3.5). After that, the antibody was stored in PBS (pH 7.0), and the antibody concentration was detected by NanoDrop.

[0188] The subtypes of the purified antibodies were determined by using a κ and λ-mouse rapid typing kit (Thermal, USA, Cat#: 26179) and a mouse monoclonal antibody typing reagent (Sigma, USA, Cat#: IS02-1KT), and the detection steps were consistent with those in the kit instructions.

[0189] Most clones, including 70E11 and 149G11, produced IgG1 / κ antibodies, while a small number of other clones produced IgG2a / κ antibodies. The expression titers of clones 70E11 and 149G11 were 1.2 and 24.2 mg / L, respectively.

[0190] Example 4 Binding of purified mouse TIGIT antibody to human and monkey TIGIT

[0191] The purified mouse TIGIT monoclonal antibody was first determined for its binding activity to recombinant human or monkey TIGIT protein by ELISA.

[0192] The ELISA plate was coated with 100 μl of 500 ng / ml human TIGIT (ECD)-his (Cat#: 10917-H08H, Sino Biological, China) overnight at 4°C. Each well was blocked with 200 μl of blocking solution (PBS + 1% BSA + 1% goat serum + 0.05% Tween 20) for 2 hours at room temperature, and then 100 μl of serially diluted TIGIT antibody (highest concentration 40 μg / ml) was added and incubated for 1 hour at room temperature. After the ELISA plate was washed 3 times with PBST (PBS + 0.05% Tween 20), 5000-fold diluted goat anti-mouse IgG-HRP (Cat#: A9309-1ml, Sigma, USA) was added and incubated for 1 hour at room temperature. The ELISA plate was developed with freshly prepared Ultra-TMB (BD, USA, Cat# no.: 555214) for 5 minutes at room temperature.

[0193] The species cross-reactivity of 20 TIGIT monoclonal antibodies to monkey TIGIT was further tested by direct ELISA. Specifically, 100 μl of 500 ng / ml monkey TIGIT (ECD)-hFc (Cat#: TIT-C5254, Sino Biological, China) was coated in a 96-well ELISA plate and co-incubated with 100 μl of serially diluted TIGIT antibody (highest concentration 40 μg / ml). Subsequently, HRP-goat anti-mouse IgG (Sigma, USA, Cat#: A9309-1ml) was used.

[0194] The TIGIT antibody Tiragolumab (prepared according to the amino acid sequence disclosed in US20170088613A1, having a human IgG1 / κ constant region, and can also be prepared with reference to the sequences INN 10644_H and INN 10644_L in http: / / www.imgt.org / 3Dstructure-DB / cgi / details.cgi?pdbcode=10644) was used as a reference.

[0195] The EC of the binding affinity of the representative antibody 50 is summarized in Table 3. The data show that the antibodies of all 20 clones bind to human and monkey TIGIT, and the antibodies of all clones have no cross-reactivity with mouse TIGIT (not shown).

[0196] Table 3. Binding affinity of representative mouse TIGIT monoclonal antibodies to human or monkey TIGIT

[0197]

[0198] Example 5 Binding of mouse TIGIT monoclonal antibody to human and monkey TIGIT expressed by HEK293A cells

[0199] To further determine whether the TIGIT antibody binds to human, monkey or mouse TIGIT expressed by HEK293A cells, FACS cell binding assays were performed using HEK293A cells stably overexpressing human, monkey or mouse TIGIT constructed in Example 1. Briefly, 10 5 HEK293A cells in 100 μl of medium were plated in a 96-well plate, and 50 μl of serially diluted TIGIT antibody was added. After incubation at 4 °C for 1 hour, the 96-well plate was washed 3 times with PBST. Then, 500-fold diluted APC-goat anti-mouse IgG (Cat#: 405308, BioLegend, USA) was added. After incubation at 4 °C for 1 hour, the 96-well plate was washed 3 times with PBS, and then the cell fluorescence was detected using a FACS detector (BD).

[0200] The EC 50 of the binding affinities of two representative antibodies are summarized in Table 4. The data show that all mouse TIGIT monoclonal antibodies exhibit high binding affinity for human and monkey TIGIT, but do not bind to mouse TIGIT.

[0201] Table 4. Binding affinities of mouse TIGIT antibodies for human, monkey and mouse TIGIT

[0202]

[0203] Example 6 Epitope competition

[0204] The antigen-binding epitope competition between antibodies was detected by competitive ELISA. Briefly, a 96-well ELISA plate was coated with 100 μl of 0.5 μg / ml human TIGIT (ECD)-His (Sino Biological, China, Cat#: 10917-H08H) overnight at 4°C. These wells were blocked with 200 μl of blocking solution (PBS + 1% BSA + 1% goat serum + 0.05% Tween 20) for 2 hours at room temperature. The Tiragolumab antibody, Hel antibody (LifeTein, USA, Cat#: LT12031), or Etigilimab (synthesized and expressed according to the amino acid sequence in Patent US20160376365A1, with the constant region being IgG1 / κ) was diluted to 5 μg / ml and added to the wells, 100 μl per well, and incubated at room temperature for 1 hour. The plate was washed 3 times with PBST, and 1 μg / ml of the purified antibody of the present application was added and incubated at room temperature for 1 hour. After the ELISA plate was washed 3 times with PBST, anti-mouse Fc-HRP (Cat#: A9309-1MC, Simga, USA) diluted 20,000-fold was added and incubated at room temperature for 1 hour. After continuing to wash 3 times with PBST wash solution, freshly prepared Ultra-TMB (Huzhou Yingchuang, China, Cat#: TMB-S-003) was used for color development at room temperature for 5 minutes, and the absorbance was read at 450 nm using a microplate reader (Thermo Multiscan FC).

[0205] Among the 20 mouse antibodies, 7 mouse antibodies, including clone 70E11 and 149G11 antibodies, showed antigen epitope competition with Tiragolumab, indicating that these antibodies bind to the same or similar antigen epitopes as Tiragolumab. No antibody showed antigen epitope competition with Etigilimab, indicating that these antibodies bind to completely different antigen epitopes from Etigilimab.

[0206] Example 7 Inhibition of human TIGIT-PVR interaction by mouse TIGIT antibody

[0207] Studies have shown that PVR is the main ligand of TIGIT. Blocking experiments in cell systems were performed by FACS to detect the blocking effect of murine TIGIT antibodies on the TIGIT-PVR interaction. A549 cells stably overexpressing human PVR prepared in Example 1 were used for blocking detection and measured by FACS. Briefly, serially diluted TIGIT antibodies were mixed and incubated with TIGIT (ECD)-hFc protein (Cat#: 10917-H02H, Sino Biological, China) at a final concentration of 5 μg / ml at 37°C for 1 hour and then added to a 96-well plate. 10 5A549 / hPVR cells were plated in 96-well plates, and 100 μl of the above mixture of antibody and fusion protein was added. After incubation at 4 °C for 1 hour, the plates were washed 3 times with PBST. Then, PE-goat anti-human IgG (Cat#: PAI-86078, Thermofisher, USA) diluted 500-fold was added. After incubation at 4 °C for 1 hour, the plates were washed 3 times with PBST, and the fluorescence intensity of the cells was detected using a FACS instrument (BD).

[0208] Data showed that among the 20 antibodies, 14 antibodies including 70E11 and 149G11 could block the interaction between TIGIT and PVR. The EC 50 values of two representative antibodies, 70E11 and 149G11, that could block the interaction between TIGIT and PVR are summarized in Table 5.

[0209] Table 5. Blocking ability of mouse TIGIT antibodies against TIGIT-PVR interaction

[0210] Antibody <![CDATA[Blocking detection EC 50 (M / L)]]> Tiragolumab 4.2E-08 70E11 4.3E-08 149G11 4.1E-08

[0211] Example 8 Promotion of T cell activation by mouse TIGIT antibody

[0212] The regulatory effect of mouse TIGIT antibodies on T cell activity was studied by a T cell activity assay.

[0213] Briefly, PBMCs were collected from healthy human donor blood samples by gradient density centrifugation and resuspended in RPMI1640 medium. CD4+ T cells were isolated from PBMCs using the Invitrogen Dynabeads non-contact human CD4+ T cell isolation kit (Cat#: 11346D, Thermal Fisher Scientific, USA). The CD4+ T cells were resuspended in RPMI complete medium (90% RPMI medium + 10% fetal bovine serum), and the density was adjusted to 1.0×10 6 / ml. Activation beads of CD3 / CD28 (Gibco, USA, Cat#: 11132D) were added to the T cells and cultured at 37 °C in 5% CO2 for 10 days to activate the T cells.

[0214] The above-cultured CD4+ T cells were collected, washed 3 times with RPIM medium, and the cell concentration was adjusted to 2×10 5cells / ml. In a 96-well cell culture plate, pre-coated overnight at 4°C with 50 μl of 0.25 μg / ml CD3 antibody (OKT3, Sino Biological, China, Cat#: GMP-10977-H001) and 50 μl of 0.25 μg / ml PVR-hFc fusion protein (Sino Biological, China, Cat: 10109-H02H). The plate was washed 3 times with PBS, and then blocked with PBS buffer containing 1% bovine serum albumin at 37°C for 90 minutes. The cell culture plate was washed 3 times with PBS, and 150 μl of the above-mentioned CD4 + T cells and 50 μl of anti-TIGIT antibody with a final concentration of 50 μg / ml or serially diluted were added, and the cells were further cultured in a 37°C cell culture incubator for 3 days. The concentration of IFN-γ was determined by ELISA (Cat#: SIF50, R&D, USA) using the manufacturer's method steps. The experiment was set with three replicates.

[0215] As Figure 1 shown in A below, 9 out of 20 tested antibodies were able to enhance the activity of T cells and increase the secretion of IFN-γ. Among them, 70E11 had the strongest activation effect, and 149G11 also had a good activation effect. And compared with the Hel control, these antibodies increased the secretion of IFN-γ by T cells in a dose-dependent manner (Figure 1, B). Among them, 70E11 had a stronger ability to activate T cells than the positive control at certain concentrations.

[0216] Example 9 Expression and purification of chimeric TIGIT antibody

[0217] 70E11 and 149G11 were selected for further study. First, the heavy chain / light chain variable region sequences of the hybridoma cells of the selected antibodies were cloned by PCR using the primers mentioned in the literature (Juste et al., (2006), Anal Biochem. 349(1): 159-61), and sequenced. The sequences are summarized in Table 1 and Table 10. An expression vector was constructed by inserting the sequences encoding the variable region and the human IgG1 / K constant region (the amino acid sequences of the heavy chain constant region and the light chain constant region are listed in SEQ ID NOs: 33 and 34 respectively) between the restriction enzyme sites XhoI / BamHI of pCDNA3.1 (Invitrogen, USA).

[0218] The above-obtained expression vector was transfected into HEK-293F cells (Cobioer, China) with PEI. Specifically, HEK-293F cells were cultured in Free Style TMCultured in 293 expression medium (Cat#: 12338-018, Gibco), and each expression vector was transfected into the cells by the method of polyethyleneimine (PEI) transfection. The ratio of DNA to PEI was 1:3, and the amount of DNA added to each milliliter of cell culture medium was 1.5 μg. The transfected HEK-293F cells were cultured in an incubator at 37 °C and 5% CO2 at a rotation speed of 120 RPM. After 10-12 days, the cell culture supernatant was collected, and the monoclonal antibody was purified according to the method steps of Example 3.

[0219] Example 10 Binding of chimeric TIGIT monoclonal antibody to human or monkey TIGIT

[0220] According to the method steps of Example 5, the binding affinities of the obtained chimeric antibodies to the HEK293A / human TIGIT cells, HEK293A / monkey TIGIT cells, and HEK293A / mouse TIGIT cells prepared in Example 1 were detected. The results are shown respectively in Figure 2 .

[0221] As Figure 2 shown, the chimeric antibodies had high binding affinities for human TIGIT ( Figure 2 , A) and monkey TIGIT ( Figure 2 , B), and did not bind to mouse TIGIT ( Figure 2 , C).

[0222] Example 11 ADCC activity of chimeric TIGIT monoclonal antibody

[0223] The antibody-dependent cell cytotoxicity effect (ADCC) of the chimeric TIGIT antibody on HEK293A / human TIGIT cells was further detected. Briefly, HEK293A / human TIGIT cells were generated by the lentiviral transfection system as shown in Example 1. Both HEK293A / human TIGIT cells and effector cells NK92MI-CD16a (Huabo Bio) were centrifuged at 1200 rpm for 5 minutes. These cells were then resuspended in ADCC detection medium (MEM medium, Gibco, Cat#: 12561-056; 1% FBS, EX-cell, Cat#: FND500; 1% BSA, VETEC, Cat#: V900933-1KG), and the cell viability was approximately 90% according to cell counting. The cell density of HEK293A / human TIGIT was adjusted to 4x10 5 / ml, and the cell density of NK92MI-CD16a was adjusted to 2x10 6 / ml, 50 μl of each of the two types of cells was added to each well of a 96-well plate (the effector-to-target ratio was 5:1). The test antibodies were diluted to different concentrations and added to each well, so that the final concentrations of the antibodies were 32000 ng / ml, 6400 ng / ml, 1280 ng / ml, 256 ng / ml, 51.2 ng / ml, 10.24 ng / ml, and 2.048 ng / ml, respectively. The samples were incubated at 37 °C for 4 hours, and then 100 μl / well of LDH chromogenic solution (Cytotoxicity Detection Kit PLUS (LDH), Roche, Cat#: 04744926001) was added. After standing at room temperature in the dark for 20 minutes, the plate was read on a MDSpectraMax i3. HEL isotype control antibody (LifeTein, LLC, Cat.#: LT12031) was used as a negative control, and Tiragolumab was used as a positive antibody control.

[0224] As Figure 3 shown, both chimeric antibodies 70E11 and 149G11 could significantly induce the killing of HEK293A / human TIGIT cells by NK92MI-CD16a, and the activity was comparable to that of the positive control.

[0225] Example 12 Blocking of the interaction between chimeric TIGIT monoclonal antibody and TIGIT-PVR

[0226] To further detect the blocking effect of the chimeric antibodies on the interaction between TIGIT protein and PVR protein, the A549 / human PVR cell line prepared in Example 1 and the experimental method in Example 7 were used. The results are as Figure 4 shown, both chimeric antibodies could significantly block the interaction between TIGIT and PVR.

[0227] Example 13 Humanization of TIGIT antibody

[0228] Based on the above related functional tests, 70E11 and 149G11 were humanized and further studied. The humanization of the mouse antibodies was carried out by the complementary determining region (CDR) grafting method (U.S. Patent 5,225,539), and the specific method is described in detail below.

[0229] To select the humanized receptor frameworks of murine antibodies 70E11 and 149G11, the variable region sequences of the light and heavy chains of 70E11 and 149G11 were aligned with the human immunoglobulin gene database on the NCBI website (http: / / www.ncbi.nlm.nih.gov / igblast / ). The human germline IGVH and IGVK with the highest homology to 70E11 and 149G11 were selected as the frameworks for humanization. For 70E11, the selected heavy-chain germline receptor sequence is human IGHV1-46*01, and the selected light-chain germline receptor sequence is human IGKV3-20*01. For 149G11, the selected heavy-chain germline receptor sequence is human IGHV1-46*01, and the selected light-chain germline receptor sequence is human IGKV4-1*01.

[0230] Three-dimensional structure simulations of the variable domains of 70E11 and 149G11 were performed to identify the key framework amino acid residues that may play important roles in maintaining the CDR loop structures, thereby designing the back mutations of the humanized antibodies.

[0231] Based on the above structural modeling, 8 potential back mutations (M70L, R72A, M48I, T74K, R38K, A40T, R67K, V68A) were identified in the heavy chain of 70E11, and 9 potential back mutations (R46K, D71S, Q43A, A44S, F72Y, L21M, S22T, I59V, D61A) were identified in the light chain. 7 potential back mutations (M48I, M70L, R72A, R87T, R38K, A40R, Q43H) were identified in the heavy chain of 149G11, and 4 potential back mutations (Y42F, V89L, I21V, P49S) were identified in the light chain. Based on these potential back mutations, humanized designs were made for the antibody framework regions.

[0232] As shown in Table 6, for the Tigit antibody 70E11, a total of 5 humanized heavy-chain variable regions and 4 light-chain variable regions were designed.

[0233] Table 6. Back Mutations Designed for Tigit Antibody 70E11

[0234]

[0235] As shown in Table 7, for the Tigit antibody 149G11, a total of 4 humanized heavy-chain variable regions and 3 light-chain variable regions were designed.

[0236] As shown in Table 1, for 70E11, a total of 13 humanized antibodies were obtained. For 149G11, a total of 7 humanized antibodies were obtained. All sequence information is summarized in Table 1 and Table 10.

[0237] Table 7. Reverse mutations designed for Tigit antibody 149G11

[0238]

[0239] Synthesize the sequences encoding the humanized heavy chain variable region plus the human IgG1 constant region, and the sequences encoding the light chain variable region plus the human κ constant region. The amino acid sequences of the heavy chain constant region and the light chain constant region are listed in SEQ ID NOs: 33 and 34 respectively, and are cloned into the GS expression vector (Invitrogen, USA) using the EcoR I / Xho I and Cla I / Hind III restriction enzyme sites respectively. All expression constructs were confirmed by sequencing. The EXPiCHO expression system (Invitrogen, USA) was transfected with the expression vector, and 20 humanized TIGIT antibodies were transiently expressed as described in Example 9.

[0240] Example 14 Identification of the antigen-binding ability of humanized TIGIT antibody to human and monkey TIGIT

[0241] According to the method steps of Example 5, the binding ability of the humanized antibody to HEK293A / human TIGIT cells, HEK293A / monkey TIGIT cells, and HEK293A / mouse TIGIT cells prepared in Example 1 was detected. The results are shown respectively in Figure 5 and Figure 6 .

[0242] Table 8. Binding affinity of TIGIT antibody for human / monkey TIGIT

[0243]

[0244] The binding affinity of the humanized TIGIT antibody for human and monkey TIGIT was also quantitatively determined by BIAcore TM 8K (GE Life Sciences, USA). Specifically, 100 - 200 RU (response units) of human TIGIT (ECD)-his protein (Sino Biological, China, Cat#: 10917-H08H) or monkey TIGIT (ECD)-hFc protein (Sino Biological, China, Cat#: TIT-C5254) was coupled to a CM5 biosensor chip (Cat#: BR-1005-30, GE Life Sciences, USA), and then the unreacted groups on the chip were blocked with 1M ethanolamine. Antibodies diluted in gradients (concentration from 0.3 μM to 10 μM) were injected into the SPR reaction solution (HBS-EP buffer, pH 7.4, Cat#: BR-1006-69, GE Life Sciences, USA) at a rate controlled at 30 μL / min. When calculating the binding ability of the antibody, the RU of the blank control well was subtracted. The association rate (ka ) Dissociation rate (k d ) was calculated using the formula of the 1:1 pairing model in the BIA evaluation software. The equilibrium dissociation constant K D was obtained by calculating k d / k a .

[0245] As Figure 5 and Figure 6 shown, the binding affinity of the humanized TIGIT antibodies was similar to their respective chimeric antibodies, that is, they had high affinity for human and monkey TIGIT, but did not bind to mouse TIGIT antigen.

[0246] The binding affinities of the humanized antibodies measured by BIAcore TM are shown in Table 8.

[0247] Example 15 Blocking of TIGIT-PVR binding by humanized TIGIT antibody

[0248] The blocking effect of the humanized antibodies on the interaction between TIGIT protein and PVR protein was further detected. The A549 / human PVR cell line prepared in Example 1 was used, and the experimental method in Example 7 was adopted for detection. The results are as Figure 7 shown, and all humanized antibodies could significantly block the interaction between TIGIT and PVR.

[0249] Example 16 Identification of the T cell activation function of humanized TIGIT antibody

[0250] The activation effect of the humanized antibodies on T cells was further detected. The experimental method in Example 8 was adopted for detection. The secretion of IFN-γ was detected by a commercial detection kit (R&D, US, Cat#: STA00C) according to the instructions.

[0251] The experimental results are as Figure 8 shown, and all the detected humanized antibodies could promote the activity of T cells and increase the secretion of IFN-γ. Among the detected antibodies, 70E11VH2VL4 and 149G11VH4VL3 had the strongest T cell activation effect, especially at low concentrations, which was higher than that of Tiragolumab.

[0252] Example 17 ADCC induction effect of humanized TIGIT antibody on TIGIT-positive cells

[0253] The ADCC killing effect of the humanized antibodies on NK92 cells against HEK293A / human TIGIT cells was further detected. The experimental method in Example 11 was adopted for detection.

[0254] The humanized TIGTIT antibody was further analyzed for its ADCC effect on HEK293A / human TIGIT cells prepared in Example 1 induced by human PBMC. Among them, HEK293A / human TIGIT cells were obtained by infection with the pLV-EGFP(2A)-Puro vector expressing GFP protein. Human PBMC were obtained by density gradient centrifugation using lymphocyte separation medium and cultured overnight in a medium (RIPM1640 + 10% FBS + 300 IU IL-2). The ADCC effect was detected using the LIVE / DEAD Dead Cell Staining Kit (Thermo Fisher, USA, Cat#: L34964). The target cells and effector cells PBMC were centrifuged at 1200 rpm for 5 minutes. The cells were resuspended in ADCC assay medium (RIPM1640 medium + 1% FBS), and according to cell counting, the cell viability was approximately 90%. The target cell density was adjusted to 4x10 5 / ml, and the PBMC cell density was adjusted to 8x10 6 / ml. 50 μl of each (effector-to-target ratio 20:1) was added to each well. Diluted various detection antibodies were added to the sample detection wells, and the final concentrations of the antibodies targeting HEK293A / human TIGIT were 2000 ng / ml, 400 ng / ml, 80 ng / ml, 16 ng / ml, 3.2 ng / ml, 0.64 ng / ml, 0.128 ng / ml, 0.0256 ng / ml, 0.00512 ng / ml, and 0.001024 ng / ml, respectively. Then the samples were incubated at 37°C for 6 hours. The mixture was washed 3 times with PBS and then incubated with LIVE / DEAD dead cell dye at 37°C for 30 minutes. The cells were washed 3 times with PBS and detected by FACS. The cell death rate of GFP-positive cells (HEK293A / human TIGIT cells) was calculated.

[0255] As Figure 9 shown in 50 Table 9, all the humanized antibodies tested could induce NK92 cells to kill TIGIT-positive cells, and the EC

[0256] values of the induced killing were all lower than those of Tiragolumab and Etigilimab. 50 Summary

[0257] Antibody <![CDATA[EC 50 (M / L)]]> Antibody <![CDATA[EC 50 (M / L)]]> 149G11 VH2 VL3 7.95E-12 70E11 VH1 VL1 4.36E-12 149G11 VH4 VL3 1.77E-11 70E11 VH5 VL3 7.14E-12 149G11 VH3 VL3 1.40E-11 70E11 VH4 VL2 9.12E-12 149G11 VH2 VL2 1.62E-11 70E11 VH2 VL4 1.72E-11 149G11 VH4 VL2 1.84E-11 70E11 VH3 VL3 1.91E-11 149G11 VH3 VL2 1.30E-11 70E11 VH4 VL3 7.286E-12 Tiragolumab 4.65E-11 Etigilimab 2.36E-11 HEL /

[0258] As Figure 10As shown, all the tested humanized TIGIT antibodies could induce specific killing of HEK293A / human TIGIT cells by PBMC cells, and their induced killing effect was slightly stronger than that of Tiragolumab.

[0259] Example 18 Anti-tumor effect of humanized antibody in vivo

[0260] The in vivo antitumor effects of antibodies 70E11VH5VL3 and 149G11VH2VL3 were studied. These antibodies have human IgG1 / κ constant regions, and the animal model used was established by implanting HEPAL-6 mouse liver cancer into transgenic mice with humanized TIGIT target (GemPharmatech Co., Ltd, China). The mice were subcutaneously injected with 7×10 6 HEPAL-6 cells on day 0 and randomly divided into six groups with 8 mice in each group. The mice were intraperitoneally injected with 70E11VH5VL3, 149G11VH2VL3, Tiragolumab or PBS at 10 mg / kg (10 mg antibody / kg mouse body weight) on days 0, 4, 7, 11, 14 and 18.

[0261] The tumor size and mouse body weight were tracked over time. The long side (D) and short side (d) of the tumor were measured with vernier calipers, and the tumor volume was calculated by the formula TV = 0.5×D×d 2 The experiment was stopped before the tumor in the solvent control group reached 3.5 cm 3 One-way ANOVA was used to determine the difference in tumor volume.

[0262] The results were as Figure 11 shown. All TIGIT antibodies could significantly inhibit the growth of tumors in humanized TIGIT mice, and the in vivo antitumor effects of antibodies 149G11VH4VL3 and 70E11VH5VL3 were better than that of Tiragolumab.

[0263] Example 19 Enhancement of the anti-tumor effect of PD-L1 antibody in vivo by humanized TIGIT antibody

[0264] The in vivo antitumor synergistic effects of the above antibodies 70E11VH2VL4, 149G11VH2VL3 and the positive control antibody Tiragolumab with the PD-L1 antibody were studied. The animal model used was established by implanting CT26 mouse intestinal adenocarcinoma into transgenic BALB / C mice with humanized TIGIT target (GemPharmatech Co., Ltd, China).

[0265] The mice were subcutaneously injected with 1×10 6CT26 cells were randomly divided into groups of 8 mice each. Mice in each group were intraperitoneally injected with 70E11VH2VL4 (10 mg / kg), 149G11VH2VL3 (10 mg / kg), Tiragolumab (10 mg / kg), (10 mg / kg), and

[0266] at days 0, 4, 7, 11, 14, and 18, respectively. Tumor size and mouse body weight were tracked over time. The long side (D) and short side (d) of the tumor were measured with vernier calipers, and the tumor volume was calculated by the formula TV = 0.5 × D × d 2 The experiment was stopped before the tumor in the solvent control group reached 3.5 cm 3 One-way ANOVA was used to determine the differences in tumor volume.

[0267] The results are as Figure 12 shown. Among them, Figure 12 A summarizes the inhibitory effects of 70E11VH2VL4, 149G11VH2VL3, Tiragolumab alone, and in combination with the PD-L1 antibody on tumor growth in humanized TIGIT mice. To more clearly display the data, Figure 12 B, 12C, and 12D respectively show that there is a synergistic effect when 70E11VH2VL4, 149G11VH2VL3, and Tiragolumab are combined with the PD-L1 antibody.

[0268] Specifically, as Figure 12 shown in A, although there are large individual differences in the anti-tumor effects among individual mice, 70E11VH2VL4 and 149G11VH2VL3 can significantly inhibit the growth of mouse tumors compared with the control solvent. As Figure 12 shown in B, 12C, and 12D, when the PD-L1 antibody is combined with the TIGIT antibody, the anti-tumor effect is better than that of any one antibody alone. In addition, as Figure 12 shown in A, the anti-tumor effect of 70E11VH2VL4, whether used alone or in combination with the PD-L1 antibody, is significantly better than that of Tiragolumab, and the anti-tumor effect of 149G11VH2VL3, whether used alone or in combination with the PD-L1 antibody, is comparable to that of Tiragolumab.

[0269] The sequences mentioned in this application are summarized in Table 10.

[0270] Table 10. Sequences

[0271]

[0272]

[0273]

[0274]

[0275] Although the present invention has been described in connection with one or more embodiments, it should be understood that the present invention is not limited to these embodiments, and the above description is intended to cover all other alternative forms, modifications, and equivalents included within the spirit and scope of the appended claims. All documents cited herein are hereby incorporated by reference in their entirety. Sequence Listing <110> Beijing Mabworks Biotech Co., Ltd. Beijing Huafang Mabworks Biopharmaceutical Co., Ltd. <120> Antibodies Binding to TIGIT and Their Uses <130> 55556 00053 <160> 38 <170> PatentIn version 3.5 <210> 1 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> VH-CDR1 of Mouse, Chimeric, and Humanized 70E11 Antibodies <400> 1 Ser Tyr Asn Val His 1 5 <210> 2 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> VH-CDR2 of Mouse, Chimeric, and Humanized 70E11 Antibodies <400> 2 Thr Ile Tyr Pro Gly Asn Leu Ala Thr Ser Tyr Asn Gln Lys Phe Lys 1 5 10 15 Gly <210> 3 <211> 6 <212> PRT <213> Artificial sequence <220> <223> VH-CDR3 of mouse, chimeric and humanized 70E11 antibody <400> 3 Ser Gly Thr Met Asp Tyr 1 5 <210> 4 <211> 12 <212> PRT <213> Artificial sequence <220> <223> VL-CDR1 of mouse, chimeric and humanized 70E11 antibody <400> 4 Arg Ala Ser Ser Ser Ile Ser Ser Thr Tyr Leu His 1 5 10 <210> 5 <211> 7 <212> PRT <213> Artificial sequence <220> <223> VL-CDR2 of mouse, chimeric and humanized 70E11 antibody <400> 5 Asn Thr Gln Asn Leu Ala Ser 1 5 <210> 6 <211> 10 <212> PRT <213> Artificial sequence <220> <223> VL-CDR3 of mouse, chimeric and humanized 70E11 antibody <400> 6 Gln Gln Phe Gly Gly Tyr Pro Leu Ile Thr 1 5 10 <210> 7 <211> 5 <212> PRT <213> Artificial sequence <220> <223> VH - CDR1 of mouse, chimeric, and humanized 149G11 antibody <400> 7 Asn Tyr Trp Ile His 1 5 <210> 8 <211> 17 <212> PRT <213> Artificial sequence <220> <223> VH - CDR2 of mouse, chimeric, and humanized 149G11 antibody <400> 8 Asp Ile Tyr Pro Gly Gly Arg Tyr Ser Asn Tyr Asn Glu Lys Phe Arg 1 5 10 15 Gly <210> 9 <211> 8 <212> PRT <213> Artificial sequence <220> <223> VH - CDR3 of mouse, chimeric, and humanized 149G11 antibody <400> 9 Tyr Tyr Glu Ser Ala Met Asp Phe 1 5 <210> 10 <211> 17 <212> PRT <213> Artificial sequence <220> <223> VL - CDR1 of mouse, chimeric, and humanized 149G11 antibody <400> 10 Lys Ser Ser Gln Asn Leu Leu Tyr Asn Ser Asn Gln Lys Ser Tyr Leu 1 5 10 15 Ala <210> 11 <211> 7 <212> PRT <213> Artificial sequence <220> <223> VL-CDR2 of mouse, chimeric, and humanized 149G11 antibody <400> 11 Trp Ala Ser Thr Arg Glu Ser 1 5 <210> 12 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> VL-CDR3 of mouse, chimeric, and humanized 149G11 antibody <400> 12 Gln Gln Tyr Tyr Asn Tyr Pro Phe Thr 1 5 <210> 13 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> VH of mouse and chimeric 70E11 antibody <400> 13 Gln Val Gln Leu Gln Gln Pro Gly Thr Asp Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Arg Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Asn Val His Trp Val Lys Gln Thr Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Thr Ile Tyr Pro Gly Asn Leu Ala Thr Ser Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Thr Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Gly Thr Met Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr 100 105 110 Val Ser Ser 115 <210> 14 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> VH of Humanized Antibody 70E11-VH1VL1 <400> 14 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Asn Val His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Thr Ile Tyr Pro Gly Asn Leu Ala Thr Ser Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Gly Thr Met Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr 100 105 110 Val Ser Ser 115 <210> 15 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> VH of humanized antibodies 70E11-VH2VL2, 70E11-VH2VL3 and 70E11-VH2VL4 <400> 15 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Asn Val His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Thr Ile Tyr Pro Gly Asn Leu Ala Thr Ser Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Ala Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Gly Thr Met Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr 100 105 110 Val Ser Ser 115 <210> 16 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> VH of humanized antibodies 70E11-VH3VL2, 70E11-VH3VL3 and 70E11-VH3VL4 <400> 16 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Asn Val His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Thr Ile Tyr Pro Gly Asn Leu Ala Thr Ser Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Gly Thr Met Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr 100 105 110 Val Ser Ser 115 <210> 17 <211> 115 <212> PRT <213> Artificial sequence <220> <223> VH of humanized antibodies 70E11-VH4VL2, 70E11-VH4VL3 and 70E11-VH4VL4 <400> 17 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Asn Val His Trp Val Lys Gln Thr Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Thr Ile Tyr Pro Gly Asn Leu Ala Thr Ser Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Gly Thr Met Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr 100 105 110 Val Ser Ser 115 <210> 18 <211> 115 <212> PRT <213> Artificial sequence <220> <223> VH of humanized antibodies 70E11-VH5VL2, 70E11-VH5VL3, and 70E11-VH5VL4 <400> 18 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Asn Val His Trp Val Lys Gln Thr Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Thr Ile Tyr Pro Gly Asn Leu Ala Thr Ser Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Gly Thr Met Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr 100 105 110 Val Ser Ser 115 <210> 19 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> VH of mouse and chimeric 149G11 antibodies <400> 19 Gln Val Gln Leu Gln Gln Ser Gly Thr Glu Leu Val Arg Pro Gly Thr 1 5 10 15 Ser Val Lys Met Ser Cys Arg Thr Ala Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Trp Ile His Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp Ile 35 40 45 Gly Asp Ile Tyr Pro Gly Gly Arg Tyr Ser Asn Tyr Asn Glu Lys Phe 50 55 60 Arg Gly Lys Ala Thr Leu Thr Ala Asp Thr Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Ile Tyr Tyr Cys 85 90 95 Ala Arg Tyr Tyr Glu Ser Ala Met Asp Phe Trp Gly Gln Gly Thr Ser 100 105 110 Val Thr Val Ser Ser 115 <210> 20 <211> 117 <212> PRT <213> Synthetic Sequence <220> <223> VH of Humanized Antibody 149G11-VH1VL1 <400> 20 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Trp Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Asp Ile Tyr Pro Gly Gly Arg Tyr Ser Asn Tyr Asn Glu Lys Phe 50 55 60 Arg Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Tyr Glu Ser Ala Met Asp Phe Trp Gly Gln Gly Thr Thr 100 105 110 Val Thr Val Ser Ser 115 <210> 21 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> VH of Humanized Antibodies 149G11-VH2VL2 and 149G11-VH2VL3 <400> 21 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Trp Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asp Ile Tyr Pro Gly Gly Arg Tyr Ser Asn Tyr Asn Glu Lys Phe 50 55 60 Arg Gly Arg Val Thr Leu Thr Ala Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Tyr Glu Ser Ala Met Asp Phe Trp Gly Gln Gly Thr Thr 100 105 110 Val Thr Val Ser Ser 115 <210> 22 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> VH of Humanized Antibodies 149G11-VH3VL2 and 149G11-VH3VL3 <400> 22 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Trp Ile His Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asp Ile Tyr Pro Gly Gly Arg Tyr Ser Asn Tyr Asn Glu Lys Phe 50 55 60 Arg Gly Arg Val Thr Leu Thr Ala Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Tyr Glu Ser Ala Met Asp Phe Trp Gly Gln Gly Thr Thr 100 105 110 Val Thr Val Ser Ser 115 <210> 23 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Humanized antibody and VH of 149G11-VH4VL3 <400> 23 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Trp Ile His Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp Ile 35 40 45 Gly Asp Ile Tyr Pro Gly Gly Arg Tyr Ser Asn Tyr Asn Glu Lys Phe 50 55 60 Arg Gly Arg Val Thr Leu Thr Ala Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Tyr Glu Ser Ala Met Asp Phe Trp Gly Gln Gly Thr Thr 100 105 110 Val Thr Val Ser Ser 115 <210> 24 <211> 109 <212> PRT <213> Artificial Sequence <220> <223> VL of mouse and chimeric 70E11 antibody <400> 24 Glu Asn Val Val Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Ile Ser Ser Thr 20 25 30 Tyr Leu His Trp Tyr Gln Gln Lys Ser Gly Ala Ser Pro Lys Leu Trp 35 40 45 Ile Tyr Asn Thr Gln Asn Leu Ala Ser Gly Val Pro Ala Arg Ile Ser 50 55 60 Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Val Glu 65 70 75 80 Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Phe Gly Gly Tyr Pro 85 90 95 Leu Ile Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 <210> 25 <211> 109 <212> PRT <213> Artificial Sequence <220> <223> VL of humanized antibody 70E11-VH1VL1 <400> 25 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser Ile Ser Ser Thr 20 25 30 Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Asn Thr Gln Asn Leu Ala Ser Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Gly Gly Tyr Pro 85 90 95 Leu Ile Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 <210> 26 <211> 109 <212> PRT <213> Artificial Sequence <220> <223> Of humanized antibodies 70E11-VH2VL2, 70E11-VH3VL2, 70E11-VH4VL2 and 70E11-VH5VL2 VL <400> 26 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser Ile Ser Ser Thr 20 25 30 Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Asn Thr Gln Asn Leu Ala Ser Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Ser Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Gly Gly Tyr Pro 85 90 95 Leu Ile Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 <210> 27 <211> 109 <212> PRT <213> Artificial sequence <220> <223> Of the humanized antibodies 70E11-VH2VL3, 70E11-VH3VL3, 70E11-VH4VL3 and 70E11-VH5VL3 VL <400> 27 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser Ile Ser Ser Thr 20 25 30 Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Ala Ser Pro Lys Leu Leu 35 40 45 Ile Tyr Asn Thr Gln Asn Leu Ala Ser Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Ser Tyr Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Gly Gly Tyr Pro 85 90 95 Leu Ile Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 <210> 28 <211> 109 <212> PRT <213> Artificial sequence <220> <223> VL of humanized antibodies 70E11-VH2VL4, 70E11-VH3VL4, 70E11-VH4VL4 and 70E11-VH5VL4 VL <400> 28 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Met Thr Cys Arg Ala Ser Ser Ser Ile Ser Ser Thr 20 25 30 Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Ala Ser Pro Lys Leu Leu 35 40 45 Ile Tyr Asn Thr Gln Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Ser Tyr Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Gly Gly Tyr Pro 85 90 95 Leu Ile Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 <210> 29 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> VL of mouse and chimeric 149G11 antibodies <400> 29 Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Val Gly 1 5 10 15 Glu Lys Val Thr Val Asn Cys Lys Ser Ser Gln Asn Leu Leu Tyr Asn 20 25 30 Ser Asn Gln Lys Ser Tyr Leu Ala Trp Phe Gln Gln Lys Pro Gly Gln 35 40 45 Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Lys Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln 85 90 95 Tyr Tyr Asn Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile 100 105 110 Lys <210> 30 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> VL of Humanized Antibody 149G11-VH1VL1 <400> 30 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Asn Leu Leu Tyr Asn 20 25 30 Ser Asn Gln Lys Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln 85 90 95 Tyr Tyr Asn Tyr Pro Phe Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu 100 105 110 Lys <210> 31 <211> 113 <212> PRT <213> Synthetic Sequence <220> <223> VL of Humanized Antibodies 149G11-VH2VL2, 149G11-VH3VL2 and 149G11-VH4VL2 <400> 31 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Asn Leu Leu Tyr Asn 20 25 30 Ser Asn Gln Lys Ser Tyr Leu Ala Trp Phe Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln 85 90 95 Tyr Tyr Asn Tyr Pro Phe Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu 100 105 110 Lys <210> 32 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> VL of humanized antibodies 149G11-VH2VL3, 149G11-VH3VL3 and 149G11-VH4VL3 <400> 32 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Val Asn Cys Lys Ser Ser Gln Asn Leu Leu Tyr Asn 20 25 30 Ser Asn Gln Lys Ser Tyr Leu Ala Trp Phe Gln Gln Lys Pro Gly Gln 35 40 45 Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln 85 90 95 Tyr Tyr Asn Tyr Pro Phe Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu 100 105 110 Lys <210> 33 <211> 330 <212> PRT <213> Artificial Sequence <220> <223> Human IgG1 Heavy Chain Constant Region <400> 33 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 34 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Human κ Light Chain Constant Region <400> 34 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 35 <211> 244 <212> PRT <213> Homo sapiens <400> 35 Met Arg Trp Cys Leu Leu Leu Ile Trp Ala Gln Gly Leu Arg Gln Ala 1 5 10 15 Pro Leu Ala Ser Gly Met Met Thr Gly Thr Ile Glu Thr Thr Gly Asn 20 25 30 Ile Ser Ala Glu Lys Gly Gly Ser Ile Ile Leu Gln Cys His Leu Ser 35 40 45 Ser Thr Thr Ala Gln Val Thr Gln Val Asn Trp Glu Gln Gln Asp Gln 50 55 60 Leu Leu Ala Ile Cys Asn Ala Asp Leu Gly Trp His Ile Ser Pro Ser 65 70 75 80 Phe Lys Asp Arg Val Ala Pro Gly Pro Gly Leu Gly Leu Thr Leu Gln 85 90 95 Ser Leu Thr Val Asn Asp Thr Gly Glu Tyr Phe Cys Ile Tyr His Thr 100 105 110 Tyr Pro Asp Gly Thr Tyr Thr Gly Arg Ile Phe Leu Glu Val Leu Glu 115 120 125 Ser Ser Val Ala Glu His Gly Ala Arg Phe Gln Ile Pro Leu Leu Gly 130 135 140 Ala Met Ala Ala Thr Leu Val Val Ile Cys Thr Ala Val Ile Val Val 145 150 155 160 Val Ala Leu Thr Arg Lys Lys Lys Ala Leu Arg Ile His Ser Val Glu 165 170 175 Gly Asp Leu Arg Arg Lys Ser Ala Gly Gln Glu Glu Trp Ser Pro Ser 180 185 190 Ala Pro Ser Pro Pro Gly Ser Cys Val Gln Ala Glu Ala Ala Pro Ala 195 200 205 Gly Leu Cys Gly Glu Gln Arg Gly Glu Asp Cys Ala Glu Leu His Asp 210 215 220 Tyr Phe Asn Val Leu Ser Tyr Arg Ser Leu Gly Asn Cys Ser Phe Phe 225 230 235 240 Thr Glu Thr Gly <210> 36 <211> 312 <212> PRT <213> Cynomolgus monkey (Macaca fascicularis) <400> 36 Met Ala Phe Leu Val Ala Pro Pro Met Gln Phe Val Tyr Leu Leu Lys 1 5 10 15 Thr Leu Cys Val Phe Asn Met Val Phe Ala Lys Pro Gly Phe Ser Glu 20 25 30 Thr Val Phe Ser His Arg Leu Ser Phe Thr Val Leu Ser Ala Val Gly 35 40 45 Tyr Phe Arg Trp Gln Lys Arg Pro His Leu Leu Pro Val Ser Pro Leu 50 55 60 Gly Arg Ser Met Arg Trp Cys Leu Phe Leu Ile Trp Ala Gln Gly Leu 65 70 75 80 Arg Gln Ala Pro Leu Ala Ser Gly Met Met Thr Gly Thr Ile Glu Thr 85 90 95 Thr Gly Asn Ile Ser Ala Lys Lys Gly Gly Ser Val Ile Leu Gln Cys 100 105 110 His Leu Ser Ser Thr Met Ala Gln Val Thr Gln Val Asn Trp Glu Gln 115 120 125 His Asp His Ser Leu Leu Ala Ile Arg Asn Ala Glu Leu Gly Trp His 130 135 140 Ile Tyr Pro Ala Phe Lys Asp Arg Val Ala Pro Gly Pro Gly Leu Gly 145 150 155 160 Leu Thr Leu Gln Ser Leu Thr Met Asn Asp Thr Gly Glu Tyr Phe Cys 165 170 175 Thr Tyr His Thr Tyr Pro Asp Gly Thr Tyr Arg Gly Arg Ile Phe Leu 180 185 190 Glu Val Leu Glu Ser Ser Val Ala Glu His Ser Ala Arg Phe Gln Ile 195 200 205 Pro Leu Leu Gly Ala Met Ala Met Met Leu Val Val Ile Cys Ile Ala 210 215 220 Val Ile Val Val Val Val Leu Ala Arg Lys Lys Lys Ser Leu Arg Ile 225 230 235 240 His Ser Val Glu Ser Gly Leu Gln Arg Lys Ser Thr Gly Gln Glu Glu 245 250 255 Gln Ile Pro Ser Ala Pro Ser Pro Pro Gly Ser Cys Val Gln Ala Glu 260 265 270 Ala Ala Pro Ala Gly Leu Cys Gly Glu Gln Gln Gly Asp Asp Cys Ala 275 280 285 Glu Leu His Asp Tyr Phe Asn Val Leu Ser Tyr Arg Ser Leu Gly Ser 290 295 300 Cys Ser Phe Phe Thr Glu Thr Gly 305 310 <210> 37 <211> 241 <212> PRT <213> Mouse (Mus musculus) <400> 37 Met His Gly Trp Leu Leu Leu Val Trp Val Gln Gly Leu Ile Gln Ala 1 5 10 15 Ala Phe Leu Ala Thr Gly Ala Thr Ala Gly Thr Ile Asp Thr Lys Arg 20 25 30 Asn Ile Ser Ala Glu Glu Gly Gly Ser Val Ile Leu Gln Cys His Phe 35 40 45 Ser Ser Asp Thr Ala Glu Val Thr Gln Val Asp Trp Lys Gln Gln Asp 50 55 60 Gln Leu Leu Ala Ile Tyr Ser Val Asp Leu Gly Trp His Val Ala Ser 65 70 75 80 Val Phe Ser Asp Arg Val Val Pro Gly Pro Ser Leu Gly Leu Thr Phe 85 90 95 Gln Ser Leu Thr Met Asn Asp Thr Gly Glu Tyr Phe Cys Thr Tyr His 100 105 110 Thr Tyr Pro Gly Gly Ile Tyr Lys Gly Arg Ile Phe Leu Lys Val Gln 115 120 125 Glu Ser Ser Val Ala Gln Phe Gln Thr Ala Pro Leu Gly Gly Thr Met 130 135 140 Ala Ala Val Leu Gly Leu Ile Cys Leu Met Val Thr Gly Val Thr Val 145 150 155 160 Leu Ala Arg Lys Lys Ser Ile Arg Met His Ser Ile Glu Ser Gly Leu 165 170 175 Gly Arg Thr Glu Ala Glu Pro Gln Glu Trp Asn Leu Arg Ser Leu Ser 180 185 190 Ser Pro Gly Ser Pro Val Gln Thr Gln Thr Ala Pro Ala Gly Pro Cys 195 200 205 Gly Glu Gln Ala Glu Asp Asp Tyr Ala Asp Pro Gln Glu Tyr Phe Asn 210 215 220 Val Leu Ser Tyr Arg Ser Leu Glu Ser Phe Ile Ala Val Ser Lys Thr 225 230 235 240 Gly <210> 38 <211> 417 <212> PRT <213> Homo sapiens <400> 38 Met Ala Arg Ala Met Ala Ala Ala Trp Pro Leu Leu Leu Val Ala Leu 1 5 10 15 Leu Val Leu Ser Trp Pro Pro Pro Gly Thr Gly Asp Val Val Val Gln 20 25 30 Ala Pro Thr Gln Val Pro Gly Phe Leu Gly Asp Ser Val Thr Leu Pro 35 40 45 Cys Tyr Leu Gln Val Pro Asn Met Glu Val Thr His Val Ser Gln Leu 50 55 60 Thr Trp Ala Arg His Gly Glu Ser Gly Ser Met Ala Val Phe His Gln 65 70 75 80 Thr Gln Gly Pro Ser Tyr Ser Glu Ser Lys Arg Leu Glu Phe Val Ala 85 90 95 Ala Arg Leu Gly Ala Glu Leu Arg Asn Ala Ser Leu Arg Met Phe Gly 100 105 110 Leu Arg Val Glu Asp Glu Gly Asn Tyr Thr Cys Leu Phe Val Thr Phe 115 120 125 Pro Gln Gly Ser Arg Ser Val Asp Ile Trp Leu Arg Val Leu Ala Lys 130 135 140 Pro Gln Asn Thr Ala Glu Val Gln Lys Val Gln Leu Thr Gly Glu Pro 145 150 155 160 Val Pro Met Ala Arg Cys Val Ser Thr Gly Gly Arg Pro Pro Ala Gln 165 170 175 Ile Thr Trp His Ser Asp Leu Gly Gly Met Pro Asn Thr Ser Gln Val 180 185 190 Pro Gly Phe Leu Ser Gly Thr Val Thr Val Thr Ser Leu Trp Ile Leu 195 200 205 Val Pro Ser Ser Gln Val Asp Gly Lys Asn Val Thr Cys Lys Val Glu 210 215 220 His Glu Ser Phe Glu Lys Pro Gln Leu Leu Thr Val Asn Leu Thr Val 225 230 235 240 Tyr Tyr Pro Pro Glu Val Ser Ile Ser Gly Tyr Asp Asn Asn Trp Tyr 245 250 255 Leu Gly Gln Asn Glu Ala Thr Leu Thr Cys Asp Ala Arg Ser Asn Pro 260 265 270 Glu Pro Thr Gly Tyr Asn Trp Ser Thr Thr Met Gly Pro Leu Pro Pro 275 280 285 Phe Ala Val Ala Gln Gly Ala Gln Leu Leu Ile Arg Pro Val Asp Lys 290 295 300 Pro Ile Asn Thr Thr Leu Ile Cys Asn Val Thr Asn Ala Leu Gly Ala 305 310 315 320 Arg Gln Ala Glu Leu Thr Val Gln Val Lys Glu Gly Pro Pro Ser Glu 325 330 335 His Ser Gly Ile Ser Arg Asn Ala Ile Ile Phe Leu Val Leu Gly Ile 340 345 350 Leu Val Phe Leu Ile Leu Leu Gly Ile Gly Ile Tyr Phe Tyr Trp Ser 355 360 365 Lys Cys Ser Arg Glu Val Leu Trp His Cys His Leu Cys Pro Ser Ser 370 375 380 Thr Glu His Ala Ser Ala Ser Ala Asn Gly His Val Ser Tyr Ser Ala 385 390 395 400 Val Ser Arg Glu Asn Ser Ser Ser Gln Asp Pro Gln Thr Glu Gly Thr 405 410 415 Arg

Claims

1. A isolated monoclonal antibody, or an antigen-binding portion thereof, which is capable of binding to TIGIT, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a VH CDR1 region, a VH CDR2 region and a VH CDR3 region, and the light chain variable region comprises a VL CDR1 region, a VL CDR2 region and a VL CDR3 region, and wherein the amino acid sequences of the VH CDR1 region, VH CDR2 region, VH CDR3 region, VL CDR1 region, VL CDR2 region and VL CDR3 region are respectively as shown in (1) SEQ ID NOs: 1, 2, 3, 4, 5 and 6; or (2) SEQ ID NOs: 7, 8, 9, 10, 11 and 12.

2. The isolated monoclonal antibody or an antigen-binding portion thereof according to claim 1, wherein the heavy chain variable region comprises an amino acid sequence shown in any one of SEQ ID NOs: 13 - 23.

3. The isolated monoclonal antibody or antigen-binding portion thereof according to claim 1, wherein, The light chain variable region comprises an amino acid sequence shown in any one of SEQ ID NOs: 24 - 32.

4. The isolated monoclonal antibody or an antigen-binding portion thereof according to claim 2, wherein the heavy chain variable region and the light chain variable region respectively comprise (1) SEQ ID NOs: 13 and 24; (2) SEQ ID NOs: 14 and 25; (3) SEQ ID NOs: 15 and 26; (4) SEQ ID NOs: 15 and 27; (5) SEQ ID NOs: 15 and 28; (6) SEQ ID NOs: 16 and 26; (7) SEQ ID NOs: 16 and 27; (8) SEQ ID NOs: 16 and 28; (9) SEQ ID NOs: 17 and 26; (10) SEQ ID NOs: 17 and 27; (11) SEQ ID NOs: 17 and 28; (12) SEQ ID NOs: 18 and 26; (13) SEQ ID NOs: 18 and 27; (14) SEQ ID NOs: 18 and 28; (15) SEQ ID NOs: 19 and 29; (16) SEQ ID NOs: 20 and 30; (17) SEQ ID NOs: 21 and 31; (18) SEQ ID NOs: 21 and 32; (19) SEQ ID NOs: 22 and 31; (20) SEQ ID NOs: 22 and 32; (21) SEQ ID NOs: 23 and 31; or (22) SEQ ID NOs: 23 and 32.

5. The isolated monoclonal antibody or an antigen-binding portion thereof according to claim 1, further comprising a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region comprises an amino acid sequence shown in SEQ ID NO: 33, and the light chain constant region comprises an amino acid sequence shown in SEQ ID NO:

34.

6. The isolated monoclonal antibody or antigen-binding portion thereof according to claim 1, which (a) binds to human TIGIT; (b) binds to cynomolgus TIGIT; (c) does not bind to murine TIGIT; (d) blocks the TIGIT-PVR interaction; (e) promotes T cell activation; (f) induces ADCC of immune cells against TIGIT-positive cells; (g) has an in vivo anti-tumor effect; and / or (h) has a synergistic anti-tumor effect with a PD-L1 antibody.

7. The isolated monoclonal antibody or antigen-binding portion thereof according to claim 1, which is a murine, chimeric or humanized antibody or antigen-binding portion thereof.

8. A bispecific molecule comprising the isolated monoclonal antibody or antigen-binding portion thereof according to any one of claims 1-7.

9. A nucleic acid encoding the isolated monoclonal antibody or antigen-binding portion thereof according to any one of claims 1-7.

10. An expression vector comprising the nucleic acid according to claim 9.

11. A host cell comprising the expression vector according to claim 10.

12. A pharmaceutical composition comprising the isolated monoclonal antibody or antigen-binding portion thereof according to any one of claims 1-7, and a pharmaceutically acceptable carrier.

13. The composition according to claim 12, further comprising another anti-tumor agent, or an anti-infective agent.

14. The composition according to claim 13, wherein the other anti-tumor agent is a PD-1 antibody, a PD-L1 antibody, or a CTLA-4 antibody.

15. Use of the isolated monoclonal antibody or antigen-binding portion thereof according to any one of claims 1-7, or the composition according to claim 12 or 13, in the preparation of a medicament for treating a tumor, wherein the tumor is liver cancer, rectal cancer, endometrial cancer, pancreatic cancer, non-small cell lung cancer, multiple myeloma or melanoma.

16. Use of the isolated monoclonal antibody or antigen-binding portion thereof according to any one of claims 1-7, or the composition according to claim 12 or 13, in the preparation of a medicament for treating a tumor, wherein the tumor is intestinal adenocarcinoma.

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