Bispecific antibodies and their applications

Abstract

The present application provides a multispecific antibody comprising a first targeting moiety capable of specifically binding to a TIGIT protein and a second targeting moiety capable of binding to a tumor-associated antigen, wherein the multispecific antibody is capable of treating tumors. The present application also provides a use of the multispecific antibody as a pharmaceutical.

Description

[Technical Field]

[0001] This application relates to the field of biopharmaceuticals, specifically to an anti-PD-L1 / TIGIT bispecific antibody and its applications. [Background technology]

[0002] Tumors are classified into benign and malignant tumors based on the cellular characteristics of the neoplasms and the degree of harm they cause to the body. Malignant tumors are a major threat to human health in modern society, and have the second highest mortality rate. Conventional treatments for solid tumors include surgery, chemotherapy, radiation therapy, molecular targeted therapy, and immunotherapy. Many patients with solid tumors are discovered at an advanced stage, missing the opportunity for surgical treatment. Furthermore, most of these patients are frail and often cannot tolerate the severe side effects of radiation therapy and chemotherapy. Biologic therapies, particularly cancer immunotherapies including immune checkpoint antibodies such as PD-1, are becoming increasingly widely used due to their low toxicity and high efficacy, and may become the only option, especially for patients with metastatic, recurrent, or refractory cancers or advanced solid tumors that are refractory to existing standard chemotherapy.

[0003] Most currently available immune checkpoint antibody drugs are monoclonal antibodies, and many of them are targeted at specific targets. However, patients receiving monoclonal antibody therapy may develop drug resistance or unresponsiveness. Furthermore, since many diseases are influenced by multiple factors in the body, such as differences in signaling pathways and differences in cytokine and receptor regulatory mechanisms, single-target immunotherapy is insufficient to destroy tumor cells. Therefore, the development of multispecific antibodies, such as bispecific antibodies (BsAbs), has the potential to solve the problems of monoclonal antibody drugs, such as off-target toxicity, low response rates, and drug resistance, and to expand the beneficiary population of immunotherapy.

[0004] While bispecific antibodies are gaining attention in the development of antibody drugs, their development is extremely difficult due to numerous challenges, including preclinical evaluation models, low expression levels, poor stability, complex processes, and significant quality control variability. Therefore, there is an urgent need in this field for the development of antitumor drugs that are highly specific, highly effective, and easy to prepare. [Overview of the Initiative]

[0005] This invention provides a multispecific antibody having one or more of the following properties: (1) it can specifically bind to human PD-L1 and human TIGIT with high affinity; (2) it can bind to PD-L1 protein and TIGIT protein simultaneously; (3) it can maintain molecular stability without altering the binding structure of the multispecific antibody and the binding target; (4) the purity of the multispecific antibody is 90% or higher under initial culture conditions; (5) it can maintain the binding affinity of the binding target and exert a synergistic effect of dual targeting compared to individual anti-PD-L1 antibodies (such as HB0023) and anti-TIGIT antibodies (such as HB0030); and (6) it can effectively kill tumor cells such as solid tumors (especially tumors that highly express PD-L1). The method for preparing the multispecific antibody of this invention is simple, easy to operate, and has good potential for future applications.

[0006] In one embodiment, the present application provides a multispecific antibody comprising a first target region capable of specifically binding to a TIGIT protein, wherein the first target region comprises an isolated antigen-binding protein, the isolated antigen-binding protein comprises a heavy chain variable region VH, the VH comprises HCDR1, HCDR2, and HCDR3, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:3, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:4 or 42, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:5 or 43.

[0007] In one embodiment, the multispecific antibody includes a light chain variable region VL, the VL includes LCDR1, LCDR2, and LCDR3, of which LCDR1 includes the amino acid sequence shown in SEQ ID NO: 6 or 52, LCDR2 includes the amino acid sequence shown in SEQ ID NO: 7, and LCDR3 includes the amino acid sequence shown in SEQ ID NO: 8 or 53.

[0008] In one embodiment, the multispecific antibody includes a heavy chain variable region VH, of which VH includes an amino acid sequence shown in any one of SEQ ID NO: 110, 1, 9, 12, 14, and 32. In one embodiment, the multispecific antibody includes a light chain variable region VL, of which the VL includes an amino acid sequence shown in any one of SEQ ID NO: 111, 2, 10, 11, 13, 33, and 34. In one embodiment, the multispecific antibody further includes a second target moiety.

[0009] In one embodiment, the second target portion can specifically bind to a tumor-associated antigen.

[0010] In one embodiment, the second target moiety can specifically bind to the PD-L1 protein.

[0011] In one embodiment, the second target portion comprises an antibody or antigen-binding fragment, and the antibodies capable of binding to the PD-L1 protein include HCDR1, HCDR2, and HCDR3, of which HCDR1, HCDR2, and HCDR3 each contain the amino acid sequences shown sequentially in SEQ ID NO: 100, 101, and 102, respectively.

[0012] In one embodiment, the antibodies capable of binding to the PD-L1 protein include LCDR1, LCDR2, and LCDR3, of which LCDR1mLCDR2 and LCDR3 contain the amino acid sequences shown sequentially in SEQ ID NO: 103, 104, and 105, respectively.

[0013] In one embodiment, the multispecific antibody comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises a heavy chain variable region VH of the antibody that can bind to the PD-L1 protein, a heavy chain variable region VH that can bind to the TIGIT protein, and a light chain variable region VL that can bind to the TIGIT protein, and the second polypeptide chain comprises a light chain variable region VL of the antibody that can bind to the PD-L1 protein, or the first polypeptide chain comprises a heavy chain variable region VH of the antibody that can bind to the TIGIT protein, a heavy chain variable region VH that can bind to the PD-L1 protein, and a light chain variable region VL that can bind to the PD-L1 protein, and further, the second polypeptide chain comprises a light chain variable region VL of the antibody that can bind to the TIGIT protein.

[0014] In one embodiment, the VH of an antibody capable of binding to the TIGIT protein in the first polypeptide chain and the VL of an antibody capable of binding to the TIGIT protein constitute an scFv, or the VH of an antibody capable of binding to the PD-L1 protein in the first polypeptide chain and the VL of an antibody capable of binding to the PD-L1 protein constitute an scFv.

[0015] In one embodiment, the first polypeptide chain comprises an amino acid sequence selected from SEQ ID NO: 78-83 and 85-87.

[0016] In one embodiment, the second polypeptide chain comprises the amino acid sequence shown in SEQ ID NO: 84 or 88.

[0017] In another embodiment, the present application provides one or more isolated nucleic acid molecules encoding the multispecific antibody.

[0018] In another embodiment, the present application provides a vector comprising the nucleic acid molecule.

[0019] In another embodiment, the present application provides cells containing the nucleic acid molecule or the vector.

[0020] In another embodiment, the present application provides a method for preparing a multispecific antibody, comprising culturing the cells under conditions in which the multispecific antibody is expressed.

[0021] In another embodiment, the present application provides a pharmaceutical composition comprising the multispecific antibody, the nucleic acid molecule, the vector and / or the cell, and an optionally selected pharmaceutically acceptable adjuvant.

[0022] In another embodiment, the present invention provides uses for the multispecific antibodies, nucleic acid molecules, vectors, cells, and / or pharmaceutical compositions in the preparation of pharmaceuticals for the prevention, remission, and / or treatment of diseases or conditions.

[0023] Those skilled in the art will readily infer other aspects and advantages of the present application from the following detailed description. The following detailed description shows and describes only exemplary embodiments of the present application. As those skilled in the art will recognize, the content of the present application is such that those skilled in the art can modify the specific embodiments disclosed without departing from the spirit and scope of the invention. Accordingly, the accompanying drawings and descriptions of the present application are illustrative and not limiting. [Brief explanation of the drawing]

[0024] The specific features of the invention described herein are as stated in the claims. The features and advantages of the invention described herein can be better understood by referring to the exemplary embodiments and accompanying drawings described in detail herein. A general description of the accompanying drawings is as follows. [Figure 1] Binding activity of TIGIT chimeric antibodies to human TIGIT-expressing cells. [Figure 2]Binding activity of humanized TIGIT antibodies to cells expressing human TIGIT. [Figure 3] Inhibitory activity of a TIGIT chimeric antibody against the binding of human TIGIT to its ligand CD155. [Figure 4] Inhibitory activity of humanized TIGIT antibodies against the binding of human TIGIT to its ligand CD155. [Figure 5] In vivo efficacy studies of TIGIT antibodies in animals. [Figure 6] Comparison of the efficacy studies of TIGIT antibody and the control antibody Tiragolumab in animals. [Figures 7A-7B] Exemplary structure of the multispecific antibody described in this application. [Figure 8] SDS-PAGE electrophoresis results of the multispecific antibody TIGIT-IgG-PDL1-scFv described in this application. [Figure 9] SDS-PAGE electrophoresis results of the multispecific antibody PDL1-IgG-TIGIT-scFv described in this application. [Figure 10] The inhibitory effect of the multispecific antibody PDL1-IgG-TIGIT-scFv described in this application on PDL1. [Figure 11] The inhibitory effect of the multispecific antibody TIGIT-IgG-PDL1-scFv described in this application on TIGIT. [Figure 12] Therapeutic effect of the multispecific antibodies described in this application on a mouse tumor model. [Modes for carrying out the invention]

[0025] The embodiments of the present application will be described below using specific embodiments, but those familiar with the art will readily understand other advantages and effects of the present application as disclosed herein.

[0026] Term definition In this application, the term "TIGIT" means "Ig and ITIM domain-containing T cell immune receptor," and typically refers to immunoglobulins that are members of the PVR (poliovirus receptor) family and bind to PVR / CD155 and Nectin-2 / CD112. In this application, TIGIT can refer to TIGIT proteins derived from any vertebrate, including mammals such as primates (e.g., humans, rhesus monkeys, and cynomolgus monkeys) and rodents (e.g., mice and rats). The term includes full-length TIGIT or its fragments (such as mature fragments lacking signal peptides), untreated TIGIT, any form of TIGIT obtained by intracellular processing, and synthetic TIGIT. It also includes variants of TIGIT, such as splicing variants and allele variants. In some embodiments, TIGIT is human TIGIT, and the amino acid sequence of human TIGIT includes the amino acid sequence shown in UniProt accession number Q495A1. In some embodiments, TIGIT is cynomolgus monkey TIGIT, and the amino acid sequence of cynomolgus monkey TIGIT includes the amino acid sequence shown in UniProt accession number G7NXM4. In some embodiments, TIGIT is mouse TIGIT, and the amino acid sequence of mouse TIGIT includes the amino acid sequence shown in UniProt accession number P86176.

[0027] In this application, the term “PD-L1” generally refers to programmed cell death ligand 1, also known as B7 homolog 1, B7-H1, differentiation cluster 274, (3)274, or CD274, which, after binding to PD-1, downregulates T cell activation and cytokine secretion. “PD-L1” includes any native PD-L1 of any vertebrate origin, including mammals such as primates (e.g., humans and cynomolgus monkeys) and rodents (e.g., mice and rats). The term includes “full-length” untreated PD-L1 and any form of PD-L1 produced by cell processing. PD-L1 may exist as a transmembrane protein or a soluble protein. “PD-L1” includes intact PD-L1 and its fragments, as well as functional variants, isoforms, species congeners, derivatives, analogs, and analogs having a covalent epitope with at least one PD-L1. The basic structure of PD-L1 includes four domains: an extracellular Ig-like V domain, an Ig-like C2 domain, a transmembrane domain, and a cytoplasmic domain. An exemplary human PD-L1 amino acid sequence can be found under NCBI accession number NP_001254653 or UniProt accession number Q9NZQ7.

[0028] In this application, the term “antigen-binding protein” generally refers to a portion that binds to an antigen and optionally a scaffold or skeletal portion that allows the antigen-binding protein to adopt a conformation that facilitates the binding of the antigen to the antigen. Examples of antigen-binding proteins include, but are not limited to, antibodies, antigen-binding fragments (Fab, Fab', F(ab)2, Fv fragments, F(ab')2, scFv, di-scFv and / or dAb), immunocomplexes, multispecific antibodies (e.g., bispecific antibodies), antibody fragments, antibody derivatives, antibody analogs, or fusion proteins, as long as they exhibit the desired antigen-binding activity.

[0029] In this application, the term "Fab" generally refers to a fragment that includes a heavy chain variable structure domain and a light chain variable structure domain, and further includes a constant structure domain of the light chain and a first constant structure domain (CH1) of the heavy chain. The term "Fab'" generally refers to a fragment that differs from Fab by adding a few residues (including one or more cysteines from the hinge region of the antibody) to the carboxyl terminus of the heavy chain CH1 structure domain. The term "F(ab')2" generally refers to an antibody fragment that includes a Fab dimer, two Fab fragments linked by a disulfide bridge at the hinge region. The term "Fv" generally refers to the smallest antibody fragment that includes a complete antigen recognition, binding site. In some embodiments, it may consist of a dimer in which the heavy chain variable region and the light chain variable region are strongly non-covalently bonded. The term "dsFv" generally refers to a disulfide-stabilized Fv fragment in which the bond between a single light chain variable region and a single heavy chain variable region is a disulfide bond. The term "dAb fragment" generally refers to an antibody fragment consisting of a VH structure domain. In this application, the term "scFv" generally refers to a monovalent molecule formed by the covalent bond between the heavy chain variable structure domain and the light chain variable structure domain of an antibody via a flexible peptide linker. Here, the scFv molecule may have a general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH.

[0030] In this application, the term "antibody" is used in its broadest sense, specifically including, but not limited to, monoclonal antibodies (including full-length monoclonal antibodies consisting of two light chains and two heavy chains), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), humanized antibodies, fully human antibodies, chimeric antibodies, and camellia-modified monostructural domain antibodies. The term "antibody" generally refers to a protein or antigen-binding fragment thereof, consisting of at least two heavy chains (HC) and two light chains (LC) linked by disulfide bonds. Each heavy chain has a heavy chain variable region (VH) and a heavy chain constant region. In some naturally occurring IgG, IgD, and IgA antibodies, the heavy chain constant region contains three structural domains, CH1, CH2, and CH3. In some naturally occurring antibodies, each light chain contains a light chain variable region (VL) and a light chain constant region. The light chain constant region contains a structural domain called CL. The VH and VL regions are further subdivided into hypervariable regions called complementary determination regions (CDRs), which alternate with more conservative regions called frame regions (FRs). Each VH and VL has three CDRs and four frame regions (FRs), arranged in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxyl terminus. The variable domains of the native heavy and light chains may each contain four FR regions (H-FR1, H-FR2, H-FR3, H-FR4, L-FR1, L-FR2, L-FR3, L-FR4), mostly adopting a β-folding structure, forming loop bonds with three CDRs, and sometimes forming part of a β-folding structure. The CDRs of each chain are held in close proximity to each other via the FR regions, and together with the CDR of the other chain, they form the antigen-binding site of the antibody. The constant region of an antibody mediates the binding of immunoglobulins to various cells of the immune system (e.g., effector cells) and to host tissues and factors, including the first component (Clq) of the classical complement system. In this application, the term "variable" generally refers to the strong variability of a portion of the sequence of the variable structural domain of an antibody, thereby shaping the binding affinity and specificity of various specific antibodies to their particular antigens. However, variability is not uniformly distributed throughout the variable region of the antibody. It is concentrated in three segments of the light-chain and heavy-chain variable regions, known as complementary determination regions (CDRs) or highly variable regions (HVRs). The more conserved portion of the variable region is called the frame (FR). In the art, antibody CDRs are defined in various ways, such as the Kabat definition rule based on sequence variability (see Kabat et al., Protein Sequences in Immunology, 5th edition, National Institutes of Health, Bethesda, Maryland (1991)), the Chothia definition rule based on the location of the structural loop region (see A1-Lazikani et al., JMol Biol 273:927-48, 1997), and the IMGT scientific chart rule and IMGT definition rule based on the concept of the IMGT ontology.

[0031] IMGT stands for the International ImMunoGeneTics Information System, a global reference database for immunogenetics and immunoinformatics (http: / / www.imgt.org). IMGT specializes in human and other vertebrate immunoglobulins (IGs), antibodies, T cell receptors (TRs), major histocompatibility (MHs), and vertebrate and nonvertebrate immunoglobulin superfamilies (Igsfs), MH superfamilies (MhSfs), and immune system-associated proteins (RPIs).

[0032] In this application, the term “Kabat’s antibody numbering rules” generally refers to the numbering system of amino acid residues in the variable regions of the heavy and light chains of antibodies or in the antigen-binding regions (Kabat et al. (1971) Ann. NY Acad. Sci. 190:382-391 and Kabat, EA et al. (1991) Sequence of Proteins of Immunological Interest, 5th edition, U.S. Department of Health and Human Services, NIH Announcement No. 91-3242). For the heavy chain variable region, CDR1 is the 31st-35th amino acids of the heavy chain variable region, CDR2 is the 50th-65th amino acids of the heavy chain variable region, and CDR3 is the 95th-102nd amino acids of the heavy chain variable region. For the light chain variable region, CDR1 is the 24th-34th amino acids of the light chain variable region, CDR21 is the 50th-56th amino acids of the light chain variable region, and CDR3 is the 89th-97th amino acids.

[0033] In this application, the term “isolated” antigen-binding protein generally refers to an antigen-binding protein identified, isolated, and / or recovered from components of the environment in which it was produced (e.g., natural or recombinant). The contaminants of the environment in which they occur are typically substances that would interfere with research, diagnostic, or therapeutic use and may include enzymes, hormones, and other protein or non-protein solutes. Isolated antigen-binding proteins or antibodies are typically prepared through at least one purification step.

[0034] In this application, the term “multispecific antibody” generally refers to an antibody that recognizes the variable regions of one or more epitopes in one or more antigens. Multispecific antibodies include, but are not limited to, full-length antibodies, antibodies having two or more VL and VH domains, antibody fragments such as Fab, Fv, dsFv, scFv, biantibodies, bispecific antibodies and triantibodies, and covalent or noncovalent antibody fragments. In some embodiments, a multispecific antibody may be a “bispecific antibody” that recognizes two different epitopes in the same or different antigens.

[0035] In this application, the term "monoclonal antibody" generally refers to an antibody obtained from a substantially homogeneous group of antibodies, i.e., an individual antibody within a cluster is identical except for a few possible spontaneous variations. Monoclonal antibodies are usually highly specific to a single antigen site. Furthermore, while conventional polyclonal antibody preparations typically have different antibodies for different determinants, each monoclonal antibody is directed towards a single determinant cluster on the antigen. In addition to their specificity, monoclonal antibodies have the advantage of being able to be synthesized by hybridoma culture and not being contaminated by other immunoglobulins. The modifier "monoclonal" is interpreted as indicating the characteristics of an antibody obtained from a substantially homogeneous antibody population and does not require a specific antibody production method. For example, the monoclonal antibodies used in this application may be prepared in hybridoma cells or by recombinant DNA.

[0036] In this application, the term "chimeric antibody" generally refers to an antibody in which the variable region originates from one species and the constant region originates from another species. Generally, the variable region originates from antibodies of experimental animals such as rodents ("parental antibodies"), and the constant region originates from human antibodies. Therefore, the resulting chimeric antibody is less likely to cause a harmful immune response in human individuals than parental (e.g., mouse-derived) antibodies.

[0037] In this application, the term “humanized antibody” generally refers to an antibody in which some or all of the amino acids outside the CDR domain of a non-human antibody (e.g., a mouse antibody) are replaced with corresponding amino acids from a human immunoglobulin. Small additions, deletions, insertions, substitutions, or modifications of amino acids in the CDR region may also be acceptable, as long as the antibody still retains its ability to bind to a particular antigen. A humanized antibody may optionally include at least a portion of the constant region of a human immunoglobulin. A “humanized antibody” retains similar antigen specificity to the original antibody. The “humanized” form of a non-human (e.g., mouse) antibody can consist of a minimal configuration of a chimeric antibody derived from the sequence of a non-human immunoglobulin. In some embodiments, residues in the CDR domain of a human immunoglobulin (acceptor antibody) may be replaced with residues in the CDR domain of a non-human species (donor antibody) (e.g., mouse, rat, rabbit, or non-human primate) having the desired properties, affinity, and / or capabilities. In some embodiments, residues in the FR domain of a human immunoglobulin may be replaced with corresponding non-human residues. Furthermore, humanized antibodies may contain amino acid modifications that are not present in recipient or donor antibodies. These modifications can be made to further improve antibody performance, such as binding affinity.

[0038] In this application, "fully human antibody" generally refers to an antibody expressed in an animal by introducing the antibody-coding gene from a human into an antibody gene-deficient animal through genetic engineering. All parts of the antibody (including the variable and constant regions of the antibody) are encoded by a human-derived gene. Immunological side effects can be significantly reduced. Methods for obtaining fully human antibodies in this field include the phage display deep Ls method, the transgenic mouse method, the ribosome display deep Ls method, and the RNA-polypeptide method.

[0039] In this application, the terms “binding,” “specific binding,” or “specific to” generally refer to measurable and reproducible interactions that allow for the determination of the presence of a target in the presence of heterologous molecules (including biomolecules), such as the binding of an antigen to an antibody. For example, an antibody binds to an epitope via its antigen-binding domain, but this binding requires some complementarity between the antigen-binding domain and the epitope. For example, an antibody that specifically binds to a target (which may be an epitope) binds to this target with higher affinity, more ease, and / or for a longer period than an antibody that binds to other targets. An antibody is said to “specifically bind” to an antigen if, via its antigen-binding domain, it binds to the epitope more easily than an unrelated epitope that binds randomly. An “epitope” is a specific group of atoms (such as a sugar side chain, phosphate group, sulfonyl group, etc.) or amino acid on an antigen that binds to an antigen-binding protein (such as an antibody).

[0040] In this application, "K D The terms "KD" and "KD" are used interchangeably and generally refer to the equilibrium dissociation constant, where "KD" refers to the ratio of the dissociation rate constant (kdis, also known as "off-rate (koff)" or "kd") to the binding rate constant (kon, also known as "binding rate (kon)" or "ka"). The binding affinity of an antigen-binding protein (such as an antibody) to an antigen can be expressed using the binding rate constant (kon), the dissociation rate constant (kdis), and the equilibrium dissociation constant (KD). Methods for determining binding and dissociation rate constants are well known in the field and include, but are not limited to, biofilm interferometry (BLI), radioimmunoassay (RIA), equilibrium dialysis, surface plasmon resonance (SPR), fluorescence resonance energy transfer (FRET), co-immunoprecipitation (Co-IP), and protein chip technology. The affinity of a particular protein-protein interaction measured may differ when measured under different conditions (e.g., salinity, pH).

[0041] In this application, the term "reference antibody" generally refers to an antibody in which the antigen-binding protein described herein competes for binding to the antigen TIGIT.

[0042] In this application, the term "ADCC" or "antibody-dependent cell-mediated cytotoxicity" generally refers to a form of cytotoxicity in which a large number of secretory immunoglobulins bind to Fc receptors (FcRs) on specific cytotoxic effector cells (e.g., NK cells, neutrophils, and macrophages), and these cytotoxic effector cells specifically bind to antigen-bearing target cells, subsequently killing the target cells with cytotoxic agents. Chief cells that mediate ADCC (e.g., NK cells) express only FcγRIII, while monocytes express FcγRI, FcγRII, and FeγRIII (see Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991)). To evaluate the ADCC activity of a target molecule, in vitro and / or in vivo cytotoxicity measurements can be performed. For example, an in vitro ADCC assay can be carried out as described in U.S. Patent No. 5500362, No. 5821337, or No. 6737056 (Presta). Effector cells that can be used in such assays include PBMCs and NK cells. Alternatively / additionally, the ADCC activity of a target molecule can be evaluated in vivo in animal models, such as those disclosed in Clynes et al., PNAS (USA) 95:652-656 (1998). For example, an Fc receptor (FcR) binding assay can be performed to confirm that an antibody lacks FcγR binding (and therefore potentially lacks ADCC activity) but retains FcRn binding ability.

[0043] ADCC activity can be reduced by modifying the Fc region. In some embodiments, sites that affect binding to the Fc receptor can be removed, for example, sites that are not rescue receptor-binding sites. In some embodiments, the Fc region may be modified to remove the ADCC site. The ADCC site is known in the art, and for the ADCC site of IgG1, see, for example, Sarmay et al. (1992) Molec. Immunol. 29 (5): 633-9.

[0044] In this application, "between..." generally refers to a situation where the C-terminus of an amino acid fragment is directly or indirectly bonded to the N-terminus of a first amino acid fragment, and that N-terminus is directly or indirectly bonded to the C-terminus of a second amino acid fragment. In the light chain, for example, the N-terminus of L-FR2 is directly or indirectly bonded to the C-terminus of LCDR1, and the C-terminus of L-FR2 is directly or indirectly bonded to the N-terminus of LCDR2. For example, the N-terminus of L-FR3 is directly or indirectly bonded to the C-terminus of LCDR2, and the C-terminus of L-FR3 is directly or indirectly bonded to the N-terminus of LCDR3. In the heavy chain, for example, the N-terminus of H-FR2 is directly or indirectly bonded to the C-terminus of HCDR1, and the C-terminus of H-FR2 is directly or indirectly bonded to the N-terminus of HCDR2. For example, the N-terminus of H-FR3 is directly or indirectly linked to the C-terminus of HCDR2, and the C-terminus of H-FR3 is directly or indirectly linked to the N-terminus of HCDR3. In this application, the "first amino acid fragment" and the "second amino acid fragment" may be the same or different amino acid fragments.

[0045] In this application, the term “isolated” antigen-binding protein generally refers to an antigen-binding protein identified, isolated, and / or recovered from components of the environment in which it is produced (e.g., natural or recombinant). The contaminants of the environment in which they occur are typically substances that would interfere with research, diagnostic, or therapeutic use and may include enzymes, hormones, and other protein or non-protein solutes. Isolated antigen-binding proteins or antibodies are typically prepared through at least one purification step. The isolated antigen-binding proteins described herein generally do not bind to antigens that are not TIGIT antigens.

[0046] In this application, the terms “isolated nucleic acid molecule” or “isolated polynucleotide” refer to a genome, mRNA, cDNA, or synthetically derived DNA or RNA, or any combination thereof, that is not linked to all or part of naturally occurring polynucleotides.

[0047] In this application, the term “vector” generally refers to a nucleic acid molecule that can self-replicate within a suitable host cell and transfers an inserted nucleic acid molecule within and / or between host cells. The vector may include vectors primarily used for inserting DNA or RNA into cells, vectors primarily used for replicating DNA or RNA, and vectors primarily used for the transcription and / or translation of DNA or RNA. The vector also includes vectors having the various functions described above. The vector may also be a polynucleotide that is transcribed or translated into a polypeptide when introduced into a suitable host cell. Typically, by culturing a suitable host cell containing the vector, the vector can produce the desired expression product.

[0048] In this application, the term “cell” generally refers to individual cells, cell lines, or cell cultures that may or may have contained plasmids or vectors comprising nucleic acid molecules described herein, or that are capable of expressing antibodies or antigen-binding fragments described herein. The cells may consist of offspring of a single host cell. Due to spontaneous, accidental, or intentional mutations, the daughter cells may not necessarily be morphologically or genomically identical to the original parent cells, but it is sufficient that they are capable of expressing antibodies or antigen-binding fragments described herein. The cells are obtained by transfecting cells in vitro using vectors described herein. The cells may be prokaryotic cells (e.g., Escherichia coli) or eukaryotic cells (e.g., yeast cells, e.g., COS cells, Chinese hamster ovary (CHO) cells, HeLa cells, HEK293 cells, COS-1 cells, NS0 cells, or myeloma cells). In some embodiments, the cells may be mammalian cells. For example, the mammalian cells may be CHO-K1 cells. In this application, the term “recombinant cell” generally refers to a cell into which a recombinant expression vector has been introduced. The recombinant host cells include not only specific cells but also their offspring.

[0049] In this application, the term “pharmaceutically acceptable adjuvant” generally includes pharmaceutically acceptable vectors, excipients, or stabilizers that are nontoxic to cells or mammals to which they are exposed at the dose and concentration used. Typically, physiologically acceptable vectors are pH-buffered aqueous solutions. Examples of physiologically acceptable vectors include buffers such as phosphates, citrates, and other organic acids; antioxidants such as ascorbic acid; proteins such as low molecular weight (less than about 10 residues) peptides, serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; carbohydrates such as monosaccharides and disaccharides such as glucose, mannose, and dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol and sorbitol; salt-forming counterions such as sodium; and / or TWEENTM (Registered trademark), polyethylene glycol (PEG) and PLURONICS TM Examples include nonionic surfactants such as (registered trademark).

[0050] In this specification, the terms “administration” and “treatment” mean the application of exogenous drugs, therapeutic agents, diagnostic agents or compositions to animals, humans, subjects, cells, tissues, organs or biological fluids. The terms “administration” and “processing” may also mean therapeutic methods, pharmacokinetic methods, diagnostic methods, research methods and experimental methods. Cell processing includes contact between reagents and cells, contact between reagents and liquids, and contact between liquids and cells. The terms “administration” and “processing” may also mean by the conjugation of reagents, diagnostic agents, or compositions or by the processing of cells in vitro and independently. “Treatment” means therapeutic procedures, preventive measures, research and diagnostics, when applied to humans, animals or research subjects, and includes contact of TIGIT conjugates with humans or animals, subjects, cells, tissues, physiological compartments or physiological fluids.

[0051] In this specification, the term “treatment” means administering, orally or topically, a therapeutic agent comprising any of the TIGIT antigen-binding proteins and their compositions to a patient having one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect. The patient is usually administered a therapeutically effective dose of the therapeutic agent that is effective in relieving one or more symptoms of the disease. Desired therapeutic effects include a reduction in the rate of disease progression, improvement or alleviation of the disease state, remission, or improved prognosis. For example, an individual is successfully “treated” if one or more symptoms associated with cancer are reduced or eliminated. This includes, but is not limited to, a reduction (or destruction) of cancer cell proliferation, a reduction in disease-related symptoms, an improvement in the quality of life of the individual with the disease, a reduction in the dosage of other drugs required to treat the disease, a delay in disease progression, and / or an extension of the individual’s survival.

[0052] In this application, the term "tumor" refers to the growth and proliferation of all redundant (neoplastic) cells, whether malignant or benign, as well as all precancerous and cancerous cells and tissues.

[0053] In this specification, the terms "optionally" or "at will" mean that the events or circumstances described below may occur, but are not necessarily to occur.

[0054] In this application, the term "include" generally refers to including, encompassing, containing, or making something the subject of. In some cases, it can also mean "for" or "consisting of."

[0055] In this application, the term "approximately" usually means a variation of 0.5% to 10% above a specified value, for example, a variation within the range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%.

[0056] TIGIT antigen-binding protein In one embodiment, the present application provides an isolated antigen-binding protein in which the heavy chain variable region VH may contain at least one CDR, wherein the VH may contain an amino acid sequence shown in any of SEQ ID NO:55.

[0057] In one embodiment, the present application provides an isolated antigen-binding protein in which the heavy chain variable region VH may contain at least one CDR, wherein the VH may contain an amino acid sequence shown in any of SEQ ID NO:54.

[0058] In this application, the VH of the isolated antigen-binding protein may include HCDR1, HCDR2, and HCDR3.

[0059] The antigen-binding protein described herein may contain HCDR1, which may contain the amino acid sequence GYSITSDYA shown in SEQ ID NO:3. For example, it may be a sequence determined according to the IMGT definition rules.

[0060] The antigen-binding protein described herein may contain HCDR2, which may contain the amino acid sequence IX2X3SGX6X7 shown in SEQ ID NO: 56, where X2 may be S or T, X3 may be S or Y, X6 may be A or S, and X7 may be P or T. For example, it may be a sequence determined according to the IMGT definition rules. For example, the HCDR2 may contain the amino acid sequence shown in either SEQ ID NO: 4 or 42.

[0061] The antigen-binding protein described herein may contain HCDR2, which may contain the amino acid sequence ITSSGST shown in SEQ ID NO:4. For example, it may be a sequence determined according to the IMGT definition rules.

[0062] The antigen-binding protein described in this application may contain HCDR3, where the HCDR3 has the amino acid sequence shown in SEQ ID NO: 57: AX2LX4X5X6X7YX9X 10 AMDY may include, where X2 is R or S, X4 is D or G, X5 is F or T, X6 is D or G, X7 is N or Y, X9 is G or nonexistent, X 10 It may be G or absent. For example, it may be a sequence determined according to the IMGT definition rules. For example, the HCDR3 may contain the amino acid sequence shown in either of the SEQ ID NO: 5 and 43.

[0063] The antigen-binding protein described in this application may contain HCDR3, which may contain the amino acid sequence ARLDFGNYGGAMDY shown in SEQ ID NO: 5. For example, it may be a sequence determined according to the IMGT definition rules.

[0064] The antigen-binding protein described in the present application may include framework regions H-FR1, H-FR2, H-FR3, and H-FR4.

[0065] The antigen-binding protein described in the present application may include framework region H-FR1, and the H-FR1 has the amino acid sequence shown in SEQ ID NO: 58: X1VQLQESGPGLVKPSX 16 X 17 LSLTCTVX 25 and may include. Here, X1 is D or Q, X 16 is E or Q, X 17 is S or T, and X 25 may be S or T. For example, it may be a sequence determined according to the IMGT definition rules.

[0066] For example, the H-FR1 may include the amino acid sequence shown in any one of SEQ ID NOs: 23 and 35. The antigen-binding protein described in the present application may include framework region H-FR1, and the H-FR1 may include the amino acid sequence shown in SEQ ID NO: 23: QVQLQESGPGLVKPSETLSLTCTVS. For example, it may be a sequence determined according to the IMGT definition rules.

[0067] The antigen-binding protein described in the present application may include framework region H-FR2, and the H-FR2 has the amino acid sequence shown in SEQ ID NO: 60: WX2WIRQX7PGX 10 X 11 X 12 EWX 15 GY and may include. Here, X2 is I or N, X7 is F or P, X 10 is K or N, X 11 is G, R or K, X 12 is L or V, X 15 may be I or M. For example, it may be a sequence determined according to the IMGT definition rules. For example, the H-FR2 may include the amino acid sequence shown in any one of SEQ ID NOs: 24-26 and 36-37.

[0068] The antigen-binding protein described in this application may include a frame region H-FR2, the amino acid sequence of which is shown in SEQ ID NO: 59: WIWIRQPPGX 10 X 11 This may include LEWIGY. Here, X 10 is K or N, X 11 This may be G or K. For example, it may be a sequence determined according to the IMGT definition rules. For example, the H-FR2 may include the amino acid sequence shown in any one of SEQ ID NO: 24-26.

[0069] The antigen-binding protein described in this application may include a frame region H-FR3, the amino acid sequence shown in SEQ ID NO:61:X1YNPSLKSRX 10 X 11 X 12 X 13 X 14 DTSKNQFX 22 LX 24 LX 26 X 27 VTX 30 X 31 It may include DTATYYC, where X1 is R, S or Y, X 10 is I or V, X 11 is S or T, X 12 is F or I, X 13 is S or T, X 14 is R or V, X 22 is F or S, X 24 is K or Q, X 26 is S or T, X 27 is F or S, X 30 is A or T, X 31 This may be A or E. For example, it may be a sequence determined according to the IMGT definition rules. For example, the H-FR3 may contain the amino acid sequence shown in any one of SEQ ID NO: 27, 38, and 39.

[0070] The antigen-binding protein described in this application may include a frame region H-FR3, which may include the amino acid sequence shown in SEQ ID NO:27: YNPSLKSRVTFSVDTSKNQFSLKLSSVTAADTATYYC. For example, it may be a sequence determined according to the IMGT definition rules.

[0071] The antigen-binding protein described herein may include a frame region H-FR4, which may include the amino acid sequence shown in SEQ ID NO: 62: WGQGTX6VX8VSS, where X6 may be L or S, and X8 may be I or T. For example, it may be a sequence determined according to the IMGT definition rules. For example, the H-FR4 may include the amino acid sequence shown in any one of SEQ ID NO: 28, 40, and 41.

[0072] The antigen-binding protein described herein may include a frame region H-FR4, which may include the amino acid sequence WGQGTLVTVSS shown in SEQ ID NO:28. For example, it may be a sequence determined according to the IMGT definition rules.

[0073] The antigen-binding protein described in this application may include a heavy chain variable region VH, wherein VH is the amino acid sequence shown in SEQ ID NO: 55: X1VQLQESGPGLVKPSX 16 X 17 LSLTCTVX 25 GYSITSDYAWX 36 WIRQX 41 PGX 44 X 45 X 46 EW Test 49 GYIX 53 X 54 SGX 57 X 58 X 59 YNPSLKSRX 68 X 69 X 70 X 71 X 72 DTSKNQFX 80 LX 82LX 84 X 85 VTX 88 X 89 DTATYYCAX 98 LX 100 X 101 X 102 X 103 YX 105 X 106 AMDYWGQGTX 116 VX 118 It may contain VSS. Here, X1 is D or Q, X 16 is E or Q, X 17 is S or T, X 25 is S or T, X 36 is I or N, X 41 is F or P, X 44 is N or K, X 45 is G, K or R, X 46 is L or V, X 49 is I or M, X 53 is S or T, X 54 is S or Y, X 57 is A or S, X 58 is P or T, X 59 is R, S or Y, X 68 [[ID= 56]]is I or V, X 69 is S or T, X 70 is F or I, X 71 is S or T, X 72 is R or V, X 80 is F or S, X 82 is K or Q, X 84 is S or T, X 85 is F or S, X 88 is A or T, X 89 is A or E, X 98 is R or S, X 100 is D or G, X 101 is F or T, X 102 is D or G, X 103 is N or Y, X 105 is G or absent, X 106 is G or absent, X 116 is L or S, X 118This may be I or T. For example, it may be a sequence determined according to the IMGT definition rules. For example, the antigen-binding protein may include a heavy chain variable region VH, and VH may include an amino acid sequence shown in any one of SEQ ID NO: 1, 9, 12, 14, and 32.

[0074] The antigen-binding protein described in this application may include a heavy chain variable region VH, the amino acid sequence shown in SEQ ID NO: 54: QVQLQESGPGLVKPSETLSLTCTVSGYSITSDYAWIWIRQPPGX 44 X 45 LEWIGYITSSGSTYYNPSLKSRVTFSVDTSKNQFSLKLSSVTAADTATYYCARLDFGNYGGAMDYWGQGTLVTVSS may include, where X 44 is K or N, X 45 This may be G or K. For example, it may be a sequence determined according to the IMGT definition rules. For example, the antigen-binding protein may include a heavy chain variable region VH, and VH may include an amino acid sequence shown in any one of SEQ ID NO: 9, 12, and 14.

[0075] The antigen-binding protein described herein may include a heavy chain constant region, which may be derived from human IgG, for example, human IgG1. In some embodiments, the Fc domain of human IgG1 can be modified to achieve desired properties (e.g., ADCC KO). Such modification may be an amino acid mutation. In some embodiments, the modification of IgG1 may be L234A / L235A, that is, according to the EU number, amino acids 234 and 235 are mutated from leucine (L) to alanine (A), respectively. In some embodiments, the heavy chain constant region of the antigen-binding protein described herein may be derived from wild-type human IgG1. For example, the antigen-binding protein described herein may include a heavy chain constant region, which may contain the amino acid sequence shown in any one of SEQ ID NO: 35-36.

[0076] In this application, the isolated antigen-binding protein may contain at least one CDR in its light chain variable region (VL), and the VL may contain the amino acid sequence shown in SEQ ID NO:64.

[0077] In this application, the isolated antigen-binding protein may contain at least one CDR in its light chain variable region (VL), and the VL may contain the amino acid sequence shown in SEQ ID NO:63.

[0078] In this application, the VL of the antigen-binding protein may include LCDR1, LCDR2, and LCDR3.

[0079] The antigen-binding protein described herein may contain LCDR1, which may contain the amino acid sequence QHVSX5A shown in SEQ ID NO: 65, where X5 may be N or T. For example, it may be a sequence determined according to the IMGT definition rules. For example, LCDR1 may contain the amino acid sequence shown in either SEQ ID NO: 6 or 52.

[0080] The antigen-binding protein described herein may contain LCDR1, which may contain the amino acid sequence QHVSTA shown in SEQ ID NO:6. For example, it may be a sequence determined according to the IMGT definition rules.

[0081] The antigen-binding protein described herein may contain LCDR2, which may contain the amino acid sequence SAS shown in SEQ ID NO:7. For example, it may be a sequence determined according to the IMGT definition rules.

[0082] The antigen-binding protein described herein may contain LCDR3, which may contain the amino acid sequence QQX3YX5X6PX8T shown in SEQ ID NO:66, where X3 is H or Y, X5 is I or S, X6 is L or T, and X8 is W or Y. For example, it may be a sequence determined according to the IMGT definition rules. For example, LCDR3 may contain the amino acid sequence shown in either SEQ ID NO:8 or 53.

[0083] The antigen-binding protein described herein may contain LCDR3, which may contain the amino acid sequence QQHYITPYT shown in SEQ ID NO:8. For example, it may be a sequence determined according to the IMGT definition rules.

[0084] The antigen-binding protein described herein may include frame regions L-FR1, L-FR2, L-FR3, and L-FR4.

[0085] The antigen-binding protein described in this application may include a frame region L-FR1, where L-FR1 is the amino acid sequence shown in SEQ ID NO:68: DIX3MTQSX8X9X 10 X 11 X 12 X 13 SX 15 GDRVX 20 ITCX 24 AS may be included. Here, X3 is Q or V, X8 is H or P, X9 is K or S, X 10 is F or S, X 11 L or M, X 12 is F or S, X 13 is A or T, X 15 is I or V, X 20 is S or T, X 24 This may be K or R. For example, it may be a sequence determined according to the IMGT definition rules. For example, L-FR1 may contain the amino acid sequence shown in any one of SEQ ID NO: 15, 16, 44, and 45.

[0086] The antigen-binding protein described in this application may include a frame region L-FR1, the amino acid sequence of SEQ ID NO: 67: DIQMTQSPSSLSASVGDRVTITCX 24 It may include AS. Here, X 24 This may be K or R. For example, it may be a sequence determined according to the IMGT definition rules. For example, L-FR1 may contain the amino acid sequence shown in either SEQ ID NO: 15 or 16.

[0087] The antigen-binding protein described in this application may include a frame region L-FR2, the amino acid sequence of which is shown in SEQ ID NO:70: X1X2WYQQKPGX 10 X11PKLIX 17 This may include: where X1 is L or V, X2 is A or N, X 10 is K or Q, X 11 is A or S, and X 17 This may be H or Y. For example, it may be a sequence determined according to the IMGT definition rules. For example, the L-FR2 may contain the amino acid sequence shown in any one of SEQ ID NO: 17, 18, 46, and 47.

[0088] The antigen-binding protein described herein may include a frame region L-FR2, which may include the amino acid sequence X1X2WYQKPGKAPKLLIY shown in SEQ ID NO:69, where X1 may be L or V, and X2 may be A or N. For example, it may be a sequence determined according to the IMGT definition rules. For example, the L-FR2 may include the amino acid sequence shown in either SEQ ID NO:17 or 18.

[0089] The antigen-binding protein described in this application may include a frame region L-FR3, the amino acid sequence of SEQ ID NO:72: YX2X3X4GVPX8RFX 11 GX 13 X 14SGTDFTX 21 TIX 24 SX 26 QX 28 EDX 31 AX 33 It may include YYC, where X2 is L or R, X3 is Q or Y, X4 is S or T, X8 is D or S, X 11 is I, S or T, X 13 is R or S, X 14 is G or R, X 21 is F or L, X 24 is N or S, X 26 is L or V, X 28 is A or P, X 33 is F or L, X 14 This may be T or V. For example, it may be a sequence determined according to the IMGT definition rules. For example, the L-FR3 may contain the amino acid sequence shown in any one of SEQ ID NO: 19-21 and 48-50.

[0090] The antigen-binding protein described in this application may include the skeletal region L-FR3, the amino acid sequence of SEQ ID NO:71: YX2X3SGVPSRFSGSX 14 It may include SGTDFTLTISSLQPEDFATYYC, where X2 is L or R, X3 is Q or Y, X 14 This may be G or R. For example, it may be a sequence determined according to the IMGT definition rules. For example, L-FR3 may contain the amino acid sequence shown in any one of SEQ ID NO: 19-21.

[0091] The antigen-binding protein described herein may include a skeletal region L-FR4, which may contain the amino acid sequence FGX3GTKLEIK shown in SEQ ID NO:73, where X3 may be G or Q. For example, it may be a sequence determined according to the IMGT definition rules. For example, the L-FR4 may contain the amino acid sequence shown in either SEQ ID NO:22 or 51.

[0092] The antigen-binding protein described herein may include a frame region L-FR4, which may include the amino acid sequence FGQGTKLEIK shown in SEQ ID NO:22. For example, it may be a sequence determined according to the IMGT definition rules.

[0093] The antigen-binding protein described in this application may include a light chain variable region (VL), the VL having the amino acid sequence shown in SEQ ID NO: 64: DIX3MTQSX8X9X 10 X 11 X 12 X13SX 15 GDRVX 20 ITCX 24 ASQHVSX 31 AX 33 X 34 WYQQKPGX 42 X 43 PKLLIX 49 SASYX 54 X 55 X 56 GVPX 60 RFX 63 GX 65 X 66 SGTDFTX 73 TIX 76 SX 78 QX 80 EDX 83 AX 85 YYCQQX 91 YX 93 X 94 PX 96 TFGX 100 It may include GTKLEIK. Here, X3 is Q or V, X8 is H or P, X9 is K or S, X 10 is F or S, and X 11 L or M, X 12 is F or S, X 13 is A or T, X 15 is I or V, X 20 is S or T, X 24 is K or R, X 31 is N or T, X 33 is L or V, X 34 is A or N, X 42 is K or Q, X43 is A or S, X 49 is H or Y, X 54 L or R, X 55 is Q or Y, X 56 is S or T, X 60 is D or S, X 63 is I, S or T, X 65 is S or R, X 66 is G or R, X 73 is L or F, X 76 is S or N, X 78 is L or V, X 80 is A or P, X 83 is F or L, X 85 is T or V, X 91 is H or Y, X 93 is I or S, X 94 is L or T, X 96 is W or Y, X 100 This may be Q or G. For example, it may be a sequence determined according to the IMGT definition rules. For example, the antigen-binding protein may include a light chain variable region VL, the VL may contain an amino acid sequence shown in any of SEQ ID NO: 2, 10, 11, 13, 33, and 34.

[0094] The antigen-binding protein described in this application may include a light chain variable region VL, the amino acid sequence shown in SEQ ID NO:63:DIQMTQSPSSLSASVGDRVTITCX 24 ASQHVSTAX 33 X 34 WYQQKPGKAPKLLIYSASYX 54 X 55 SGVPSRFSGSX 66 It may include SGTDFTLTISSLQPEDFATYYCQQHYITPYTFGQGTKLEIK, where X 24 is K or R, X 33 is L or V, X 34 is A or N, X 54 L or R, X 55 is Q or Y, X 66This may be G or R. For example, it may be a sequence determined according to the IMGT definition rules. For example, the antigen-binding protein may include a light chain variable region VL, and the VL may include an amino acid sequence shown in any of SEQ ID NO: 10, 11, and 13.

[0095] The antigen-binding protein described in this application may include a light chain constant region, and the light chain constant region may include a human light chain constant region sequence. For example, it may include a human κ-light chain constant region. For example, the light chain constant region of the isolated antigen-binding protein described in this application may include the amino acid sequence shown in SEQ ID NO:31.

[0096] In this application, the isolated antigen-binding protein may include antibody heavy chain variable regions CDR-HCDR1, HCDR2, and HCDR3, wherein HCDR1 may include the amino acid sequence shown in SEQ ID NO:3, HCDR2 may include the amino acid sequence shown in SEQ ID NO:56, and HCDR3 may include the amino acid sequence shown in SEQ ID NO:57. For example, in this application, the isolated antigen-binding protein may include antibody heavy chain variable regions CDR-HCDR1, HCDR2, and HCDR3, wherein HCDR1 may include the amino acid sequence shown in SEQ ID NO:3, HCDR2 may include the amino acid sequence shown in either SEQ ID NO:4 or 42, and HCDR3 may include the amino acid sequence shown in either SEQ ID NO:5 or 43.

[0097] In this application, the isolated antigen-binding protein may include antibody heavy chain variable regions CDR-HCDR1, HCDR2, and HCDR3, and HCDR1, HCDR2, and HCDR3 may each contain the amino acid sequences shown in SEQ ID NO: 3, 4, and 5 in order.

[0098] In this application, the isolated antigen-binding protein may include antibody light chain variable regions CDR-LCDR1, LCDR2, and LCDR3, wherein LCDR1 may include the amino acid sequence shown in SEQ ID NO:65, LCDR2 may include the amino acid sequence shown in SEQ ID NO:7, and LCDR3 may include the amino acid sequence shown in SEQ ID NO:66. For example, in this application, the isolated antigen-binding protein may include antibody light chain variable regions CDR-LCDR1, LCDR2, and LCDR3, wherein LCDR1 may include the amino acid sequence shown in either SEQ ID NO:6 or 52, LCDR2 may include the amino acid sequence shown in SEQ ID NO:7, and LCDR3 may include the amino acid sequence shown in either SEQ ID NO:8 or 53.

[0099] In this application, the isolated antigen-binding protein may include antibody light chain variable regions CDR-LCDR1, LCDR2, and LCDR3, and LCDR1, LCDR2, and LCDR3 may each contain the amino acid sequences shown in SEQ ID NO: 6, 7, and 8 in order.

[0100] In this application, the isolated antigen-binding protein may include HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein HCDR1 may include the amino acid sequence shown in SEQ ID NO:3, HCDR2 may include the amino acid sequence shown in SEQ ID NO:56, HCDR3 may include the amino acid sequence shown in SEQ ID NO:57, LCDR1 may include the amino acid sequence shown in SEQ ID NO:65, LCDR2 may include the amino acid sequence shown in SEQ ID NO:7, and LCDR3 may include the amino acid sequence shown in SEQ ID NO:66.

[0101] For example, in this application, the isolated antigen-binding protein may include HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein HCDR1 may include the amino acid sequence shown in SEQ ID NO:3, HCDR2 may include the amino acid sequence shown in either SEQ ID NO:4 or 42, HCDR3 may include the amino acid sequence shown in either SEQ ID NO:5 or 43, LCDR1 may include the amino acid sequence shown in either SEQ ID NO:6 or 52, LCDR2 may include the amino acid sequence shown in SEQ ID NO:7, and LCDR3 may include the amino acid sequence shown in either SEQ ID NO:8 or 53.

[0102] In this application, the isolated antigen-binding protein may include HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, and each of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 may contain, in order, SEQ ID NO: 3, 4, 5, 6, 7, and 8 amino acid sequences.

[0103] In some embodiments, the isolated antigen-binding protein described in this application may include HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, where each of the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 may contain, in order, the amino acid sequences shown in SEQ ID NO: 3, 42, 43, 52, 7, and 53.

[0104] In this application, the isolated antigen-binding protein may include a heavy chain variable region VH and a light chain variable region VL, wherein VH may include the amino acid sequence shown in SEQ ID NO: 55, and VL may include the amino acid sequence shown in SEQ ID NO: 64.

[0105] In some embodiments, the isolated antigen-binding protein may include a heavy chain variable region VH and a light chain variable region VL, wherein VH may include an amino acid sequence shown in any one of SEQ ID NO:110, SEQ ID NO:1, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:32, and SEQ ID NO:14, and VL may include an amino acid sequence shown in any one of SEQ ID NO:111, SEQ ID NO:2, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:33, SEQ ID NO:34, and SEQ ID NO:13.

[0106] In this application, the isolated antigen-binding protein may include a heavy chain variable region VH and a light chain variable region VL, wherein VH may include the amino acid sequence shown in SEQ ID NO:54 and VL may include the amino acid sequence shown in SEQ ID NO:63.

[0107] In some embodiments, the isolated antigen-binding protein may include a heavy chain variable region VH and a light chain variable region VL, wherein VH may include an amino acid sequence shown in any one of SEQ ID NO:110, SEQ ID NO:9, SEQ ID NO:12, and SEQ ID NO:14, and VL may include an amino acid sequence shown in any one of SEQ ID NO:111, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:13.

[0108] For example, the isolated antigen-binding protein may include a heavy chain variable region VH and a light chain variable region VL, where VH may contain the amino acid sequence shown in SEQ ID NO:9 and VL may contain the amino acid sequence shown in SEQ ID NO:10.

[0109] For example, the isolated antigen-binding protein may include a heavy chain variable region VH and a light chain variable region VL, where VH may contain the amino acid sequence shown in SEQ ID NO:9 and VL may contain the amino acid sequence shown in SEQ ID NO:11.

[0110] For example, the isolated antigen-binding protein may include a heavy chain variable region VH and a light chain variable region VL, where VH may contain the amino acid sequence shown in SEQ ID NO:12 and VL may contain the amino acid sequence shown in SEQ ID NO:13.

[0111] For example, the isolated antigen-binding protein may include a heavy chain variable region VH and a light chain variable region VL, where VH may contain the amino acid sequence shown in SEQ ID NO:14 and VL may contain the amino acid sequence shown in SEQ ID NO:11.

[0112] For example, the isolated antigen-binding protein may include a heavy chain variable region VH and a light chain variable region VL, where VH may contain the amino acid sequence shown in SEQ ID NO:1 and VL may contain the amino acid sequence shown in SEQ ID NO:2.

[0113] For example, the isolated antigen-binding protein may include a heavy chain variable region VH and a light chain variable region VL, where VH may contain the amino acid sequence shown in SEQ ID NO:32 and VL may contain the amino acid sequence shown in SEQ ID NO:34.

[0114] For example, the isolated antigen-binding protein may include a heavy chain variable region VH and a light chain variable region VL, where VH may contain the amino acid sequence shown in SEQ ID NO:32 and VL may contain the amino acid sequence shown in SEQ ID NO:33.

[0115] The isolated antigen-binding protein described in this application may further include a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region may be derived from the constant region of human IgG1, and the light chain constant region may be derived from the human κ light chain constant region.

[0116] For example, the isolated antigen-binding protein may include a heavy chain variable region VH and a light chain variable region VL, where VH may include the amino acid sequence shown in SEQ ID NO:9 and VL may include the amino acid sequence shown in SEQ ID NO:10; and the isolated antigen-binding protein may also include a heavy chain constant region and a light chain variable region, where the heavy chain constant region may include the amino acid sequence shown in SEQ ID NO:29 and the light chain constant region may include the amino acid sequence shown in SEQ ID NO:31. The isolated antigen-binding protein is also called HB0030.

[0117] For example, the isolated antigen-binding protein may include a heavy chain variable region VH and a light chain variable region VL, where VH may include the amino acid sequence shown in SEQ ID NO:9 and VL may include the amino acid sequence shown in SEQ ID NO:11; and the isolated antigen-binding protein may also include a heavy chain constant region and a light chain constant region, where the heavy chain constant region may include the amino acid sequence shown in SEQ ID NO:29 and the light chain constant region may include the amino acid sequence shown in SEQ ID NO:31. The isolated antigen-binding protein is also called HB0031.

[0118] For example, the isolated antigen-binding protein may include a heavy chain variable region VH and a light chain variable region VL, where VH may include the amino acid sequence shown in SEQ ID NO:12 and VL may include the amino acid sequence shown in SEQ ID NO:13; and the isolated antigen-binding protein may also include a heavy chain constant region and a light chain constant region, where the heavy chain constant region may include the amino acid sequence shown in SEQ ID NO:29 and the light chain constant region may include the amino acid sequence shown in SEQ ID NO:31. The isolated antigen-binding protein is also called HB0032.

[0119] For example, the isolated antigen-binding protein may include a heavy chain variable region VH and a light chain variable region VL, where VH may include the amino acid sequence shown in SEQ ID NO:14 and VL may include the amino acid sequence shown in SEQ ID NO:11; and the isolated antigen-binding protein may also include a heavy chain constant region and a light chain constant region, where the heavy chain constant region may include the amino acid sequence shown in SEQ ID NO:29 and the light chain constant region may include the amino acid sequence shown in SEQ ID NO:31. The isolated antigen-binding protein is also called HB0033.

[0120] For example, the isolated antigen-binding protein may include a heavy chain variable region VH and a light chain variable region VL, where VH may include the amino acid sequence shown in SEQ ID NO:1 and VL may include the amino acid sequence shown in SEQ ID NO:2; and the isolated antigen-binding protein may also include a heavy chain constant region and a light chain constant region, where the heavy chain constant region may include the amino acid sequence shown in SEQ ID NO:30 and the light chain constant region may include the amino acid sequence shown in SEQ ID NO:31. The isolated antigen-binding protein is also called 900424.

[0121] For example, the isolated antigen-binding protein may include a heavy chain variable region VH and a light chain variable region VL, where VH may include the amino acid sequence shown in SEQ ID NO:32 and VL may include the amino acid sequence shown in SEQ ID NO:34; and the isolated antigen-binding protein may also include a heavy chain constant region and a light chain constant region, where the heavy chain constant region may include the amino acid sequence shown in SEQ ID NO:30 and the light chain constant region may include the amino acid sequence shown in SEQ ID NO:31. The isolated antigen-binding protein is also called 900423.

[0122] For example, the isolated antigen-binding protein may include a heavy chain variable region VH and a light chain variable region VL, where VH may include the amino acid sequence shown in SEQ ID NO:32 and VL may include the amino acid sequence shown in SEQ ID NO:33; and the isolated antigen-binding protein may also include a heavy chain constant region and a light chain constant region, where the heavy chain constant region may include the amino acid sequence shown in SEQ ID NO:30 and the light chain constant region may include the amino acid sequence shown in SEQ ID NO:31. The isolated antigen-binding protein is also called 900428.

[0123] In this application, the isolated antigen-binding protein can compete with the reference antibody for binding to the TIGIT protein, the reference antibody may include a heavy chain variable region and a light chain variable region, the heavy chain variable region of the reference antibody may include HCDR1, HCDR2 and HCDR3, HCDR1 may include the amino acid sequence shown in SEQ ID NO:3, HCDR2 may include the amino acid sequence shown in SEQ ID NO:56, HCDR3 may include the amino acid sequence shown in SEQ ID NO:57, the light chain variable region of the reference antibody may include LCDR1, LCDR2 and LCDR3, LCDR1 may include the amino acid sequence shown in SEQ ID NO:65, LCDR2 may include the amino acid sequence shown in SEQ ID NO:7, and LCDR3 may include the amino acid sequence shown in SEQ ID NO:66.

[0124] In this application, the isolated antigen-binding protein can compete with the reference antibody for binding to the TIGIT protein, the reference antibody may include a heavy chain variable region and a light chain variable region, the heavy chain variable region of the reference antibody may include HCDR1, HCDR2 and HCDR3, HCDR1 may include the amino acid sequence shown in SEQ ID NO:3, HCDR2 may include the amino acid sequence shown in SEQ ID NO:4, HCDR3 may include the amino acid sequence shown in SEQ ID NO:5, the light chain variable region of the reference antibody may include LCDR1, LCDR2 and LCDR3, LCDR1 may include the amino acid sequence shown in SEQ ID NO:6, LCDR2 may include the amino acid sequence shown in SEQ ID NO:7, and LCDR3 may include the amino acid sequence shown in SEQ ID NO:8.

[0125] The proteins, polypeptides, and / or amino acid sequences relating to this application should also be understood to include at least the following: variants or homologs having the same or similar function as the protein or polypeptide.

[0126] In this application, the variant may be a protein or polypeptide in which one or more amino acids are substituted, deleted, or added to the amino acid sequence of the protein and / or polypeptide (e.g., the antigen-binding protein described herein). For example, the functional variant may consist of a protein or polypeptide that already has amino acid changes due to at least one, for example, 1 to 30, 1 to 20, or 1 to 10, for example, 1, 2, 3, 4, or 5 amino acid substitutions, deletions, and / or insertions. The functional variant can substantially retain the biological properties of the protein or polypeptide before the change (substitution, deletion, addition, etc.). For example, the functional variant may retain at least 60%, 70%, 80%, 90%, or 100% of the biological activity of the protein or polypeptide before modification (e.g., antigen-binding ability). For example, the substitution may be a conservative substitution.

[0127] In this application, a portion of the amino acid sequence of the antigen-binding protein may be homologous to the corresponding amino acid sequence in a particular type of antibody, or may belong to a particular class. For example, both the variable region and the constant region of an antibody may be derived from both the variable region and the constant region of an antibody of an animal species (e.g., human). In this application, the homologue may be a protein or polypeptide having at least about 85% (e.g., at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence homology to the amino acid sequence of the protein and / or polypeptide (e.g., its isolated antigen-binding fragment as described in this application).

[0128] In this application, homology is generally defined as the similarity, resemblance, or association between two or more sequences. The "sequence homology rate" can be calculated by comparing two matching sequences in a comparison window, determining the number of positions in both sequences that contain the same nucleic acid base (e.g., A, T, C, G) or the same amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Glyn, Cys, and Met), and calculating the number of matches. The number of matching positions was determined from Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, Met), divided by the total number of positions in the comparison window (window size), and multiplied by 100 to calculate the sequence homology rate. Comparisons to determine sequence homology rates can be performed in various ways known in the art using publicly available computer software, such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for comparing sequences, including any algorithm necessary to achieve the greatest possible comparison within the range of full-length sequences being compared or within a target sequence region. These homologies can also be measured by the FASTA and BLAST methods. For a description of the FASTA algorithm, see: R. Pearson and DJ Lipman, "Improved tools for biological sequence comparison," Proc. Natl. Acad. Sci., 85: 2444-2448, 1988; and DJ Lipman and WRP Pearson, "Rapid and sensitive protein similarity searches," Science, 227: 1435-1441, 1989. For a description of the BLAST algorithm, see:S. Altschul, W. Gish, W. Miller, EWMyers, and DLipman, 'A basic local comparison (alignment) search tool', Journal of Molecular Biology, 215:403-410, 1990.

[0129] The antigen-binding proteins described herein (e.g., TIGIT antibodies) are capable of specifically binding to the TIGIT antigen. "Specific binding" TIGIT antigen-binding proteins (e.g., antibodies) can typically bind to TIGIT with a KD value of approximately 1 nM or higher affinity (e.g., 1 nM, 100 pM, 10 pM, 2 pM, or 1 pM), but do not bind to other proteins lacking the TIGIT sequence. For example, antibodies against "specific binding" TIGIT do not bind to human CD226, human CD155, and human CD112. The antigen-binding proteins described herein (e.g., antibodies) specifically bind to the TIGIT antigen or its label (e.g., fluorescently labeled TIGIT antigen) and do not bind to other proteins lacking the TIGIT epitope. Whether an antigen-binding protein (e.g., antibody) binds to the TIGIT antigen can be determined using any assay known in the art. Examples of assays known in the art for determining binding affinity include surface plasmon resonance (e.g., BIACORE) or similar techniques (e.g., KinExa or OCTET). In some embodiments, the TIGIT antibodies described herein can also cross-react with monkey and / or mouse TIGIT, for example, by flow analysis techniques or enzyme-linked immunosorbent assays. In this application, “cross-reactivity” refers to the ability of an antibody to react with homologous proteins from other species.

[0130] The antigen-binding protein described herein (e.g., TIGIT antibody) inhibits the binding of TIGIT to the CD155 ligand. The inhibition test can be performed, for example, using a competitive assay in which the antigen-binding protein (e.g., TIGIT antibody), an antigen (or a cell capable of expressing the antigen), and the ligand of the antigen (or a cell expressing the ligand) are mixed and the reaction is measured based on the intensity (e.g., fluorescence intensity or concentration) of a detection marker to determine the antigen-binding protein's ability to competitively bind to the antigen's ligand. For example, the IC50 for inhibiting the binding of the TIGIT antigen to the CD155 ligand by the antigen-binding protein described herein (e.g., TIGIT antibody) is approximately 0.1 μg / ml–0.05 μg / ml when detected by flow cytometry.

[0131] multispecific antibodies In another embodiment, a multispecific antibody is provided which may include a first target moiety capable of specifically binding to the TIGIT protein. In the present application, the first target moiety of the multispecific antibody may include the isolated antigen-binding protein of the present application.

[0132] In some embodiments, the multispecific antibody may further include a second target moiety. For example, the second target moiety can specifically bind to a tumor-associated antigen. Furthermore, for example, the second target moiety can inhibit the interaction between PD-L1 and PD-1. Furthermore, for example, the second target moiety can specifically bind to the PD-L1 protein. Furthermore, for example, the second target moiety that can bind to the PD-L1 protein includes an antibody or its antigen-binding fragment. Furthermore, for example, the antigen-binding fragment that can bind to the PD-L1 protein includes Fab, Fab', Fv fragment, F(ab')2, scFv, di-scFv and / or dAb. Furthermore, for example, the antibody that can bind to the PD-L1 protein is selected from monoclonal antibodies, chimeric antibodies, humanized antibodies and fully human antibodies.

[0133] In this application, the second target portion of the multispecific antibody may include an antibody or its antigen-binding fragment that can specifically bind to the PD-L1 protein.

[0134] In this application, the antibody capable of binding to the PD-L1 protein may include HCDR3, for example, the HCDR3 may include the amino acid sequence shown in SEQ ID NO:102.

[0135] In this application, the antibody capable of binding to the PD-L1 protein may include HCDR2, for example, the HCDR2 may include the amino acid sequence shown in SEQ ID NO:101.

[0136] In this application, the antibody capable of binding to the PD-L1 protein may include HCDR1, for example, HCDR1 may include the amino acid sequence shown in SEQ ID NO:100.

[0137] In this application, antibodies capable of binding to the PD-L1 protein may include HCDR1, HCDR2, and HCDR3, for example, HCDR1, HCDR2, and HCDR3 may contain the amino acid sequences shown in SEQ ID NO: 100, 101, and 102, respectively.

[0138] In this application, the antibody capable of binding to the PD-L1 protein may include VH, for example, VH may include the amino acid sequence shown in SEQ ID NO: 106 or 108.

[0139] In this application, the antibody capable of binding to the PD-L1 protein may include LCDR3, for example, the LCDR3 may include the amino acid sequence shown in SEQ ID NO:105.

[0140] In this application, the antibody capable of binding to the PD-L1 protein may include LCDR2, for example, the LCDR2 may include the amino acid sequence shown in SEQ ID NO:104.

[0141] In this application, the antibody capable of binding to the PD-L1 protein may include LCDR1, for example, LCDR1 may include the amino acid sequence shown in SEQ ID NO:103.

[0142] In this application, antibodies capable of binding to the PD-L1 protein may include LCDR1, LCDR2, and LCDR3, for example, LCDR1, LCDR2, and LCDR3 may contain the amino acid sequences shown in SEQ ID NO: 103, 104, and 105, respectively.

[0143] In this application, the antibody capable of binding to the PD-L1 protein may include VL, for example, VL may include the amino acid sequence shown in SEQ ID NO: 107 or 109.

[0144] In this application, the multispecific antibody may include a first polypeptide chain and a second polypeptide chain.

[0145] In this application, the first polypeptide chain may include a heavy chain variable region VH of an antibody capable of binding to the PD-L1 protein, a heavy chain variable region VH capable of binding to the TIGIT protein, and a light chain variable region VL capable of binding to the TIGIT protein, and further, the second polypeptide chain may include a light chain variable region VL of an antibody capable of binding to the PD-L1 protein.

[0146] In some embodiments, the VH of the antibody capable of binding to the TIGIT protein and the VL of the antibody capable of binding to the TIGIT protein in the first polypeptide chain can constitute an scFv. In some embodiments, the VH of the antibody capable of binding to the TIGIT protein and the VL of the antibody capable of binding to the TIGIT protein can constitute an scFv via a C-peptide. For example, the C-peptide may include an amino acid sequence shown in any one of SEQ ID NO: 74-77. For example, the sequence of the scFv capable of binding to the TIGIT protein may include an amino acid sequence shown in any one of SEQ ID NO: 97-99.

[0147] In some embodiments, in the first polypeptide chain, the scFv that can bind to the TIGIT protein may be located at the N-terminus or C-terminus of the VH of the antibody that can bind to the PD-L1 protein.

[0148] For example, in the first polypeptide chain, the N-terminus of VL of an antibody capable of binding to the TIGIT protein and the C-terminus of VH of an antibody capable of binding to the PD-L1 protein can be selectively linked via a C-peptide, and the C-terminus of VL capable of binding to the TIGIT protein and the N-terminus of VH of an antibody capable of binding to the TIGIT protein can be selectively linked via a C-peptide.

[0149] Furthermore, for example, in the first polypeptide chain, the N-terminus of VH that can bind to the TIGIT protein and the C-terminus of VH of the antibody that can bind to the PD-L1 protein can be selectively linked via a C-peptide, and the C-terminus of VH that can bind to the TIGIT protein and the N-terminus of VL of the antibody that can bind to the TIGIT protein can be selectively linked via a C-peptide.

[0150] In this application, the first polypeptide chain may include a constant region, for example, a human IgG constant region, the human IgG constant region may be located at the C-terminus of VH of an antibody capable of binding to the PD-L1 protein, and further at the N-terminus of scFv of an antibody capable of binding to the TIGIT protein. In this application, the human IgG constant region may be located at the C-terminus of VH of an antibody capable of binding to the PD-L1 protein, and further at the N-terminus of VL of an antibody capable of binding to the TIGIT protein.

[0151] In some embodiments, the human IgG constant region can be directly or indirectly ligated to the N-terminus of the VL of an antibody capable of binding to the TIGIT protein, for example, via a C-peptide. In some other embodiments, the human IgG constant region may be located at the C-terminus of the VH of an antibody capable of binding to the PD-L1 protein, and further, at the N-terminus of the VH of an antibody capable of binding to the TIGIT protein. For example, the constant region may comprise the amino acid sequence shown in SEQ ID NO: 29 or 30.

[0152] In some embodiments, the human IgG constant region can be directly or indirectly ligated to the N-terminus of the VH of an antibody capable of binding to the TIGIT protein, for example, via a C-peptide. For example, the C-peptide may comprise the amino acid sequence shown in any one of SEQ ID NO: 74-77.

[0153] For example, the first polypeptide chain may contain the amino acid sequence shown in any one of SEQ ID NO:85-87.

[0154] In this application, the first polypeptide chain may include a heavy chain variable region VH of an antibody capable of binding to the PD-L1 protein, and the second polypeptide chain may further include a light chain variable region VL of an antibody capable of binding to the PD-L1 protein, a heavy chain variable region VH capable of binding to the TIGIT protein, and a light chain variable region VL capable of binding to the TIGIT protein.

[0155] In some embodiments, the VH of the antibody capable of binding to the TIGIT protein and the VL of the antibody capable of binding to the TIGIT protein in the second polypeptide chain constitute scFv. In some embodiments, the VH of the antibody capable of binding to the TIGIT protein and the VL of the antibody capable of binding to the TIGIT protein constitute scFv via a C-peptide.

[0156] In some embodiments, the scFv in the second polypeptide chain that can bind to the TIGIT protein may be located at the N-terminus or C-terminus of the VL of the antibody that can bind to the PD-L1 protein.

[0157] In this application, the first polypeptide chain comprises a heavy chain variable region VH of an antibody capable of binding to the TIGIT protein, a heavy chain variable region VH capable of binding to the PD-L1 protein, and a light chain variable region VL capable of binding to the PD-L1 protein, and further, the second polypeptide chain comprises a light chain variable region VL of an antibody capable of binding to the TIGIT protein.

[0158] In some embodiments, the VH of the antibody capable of binding to the PD-L1 protein and the VL of the antibody capable of binding to the PD-L1 protein in the first polypeptide chain constitute scFv. In some embodiments, the VH of the antibody capable of binding to the PD-L1 protein and the VL of the antibody capable of binding to the PD-L1 protein constitute scFv via a C-peptide. For example, the C-peptide may include an amino acid sequence shown in any one of SEQ ID NO: 74-77. For example, the sequence of scFv capable of binding to the PD-L1 protein may include an amino acid sequence shown in any one of SEQ ID NO: 89-96.

[0159] In some embodiments, the scFv in the first polypeptide chain that can bind to the PD-L1 protein may be located at the N-terminus or C-terminus of the VH of the antibody that can bind to the TIGIT protein.

[0160] For example, in the first polypeptide chain, the N-terminus of VL that can bind to the PD-L1 protein and the C-terminus of antibody VH that can bind to the TIGIT protein are selectively linked via a C-peptide, and the C-terminus of VL that can bind to the PD-L1 protein and the N-terminus of antibody VH that can bind to the PD-L1 protein are selectively linked via a C-peptide.

[0161] Furthermore, for example, in the first polypeptide chain, the N-terminus of VH that can bind to the PD-L1 protein and the C-terminus of antibody VH that can bind to the TIGIT protein are selectively linked via a C-peptide, and the C-terminus of VH that can bind to the PD-L1 protein and the N-terminus of antibody VL that can bind to the PD-L1 protein are selectively linked via a C-peptide.

[0162] In this application, the first polypeptide chain may include a human IgG constant region, which may be located at the C-terminus of VH of an antibody capable of binding to the TIGIT protein, and further at the N-terminus of scFv of an antibody capable of binding to the TIGIT protein. In this application, the human IgG constant region may be located at the C-terminus of VH of an antibody capable of binding to the TIGIT protein, and further at the N-terminus of VL of an antibody capable of binding to the PD-L1 protein. For example, the constant region may include the amino acid sequence shown in SEQ ID NO: 29 or 30.

[0163] In some embodiments, the human IgG constant region can be directly or indirectly ligated to the N-terminus of the VL of an antibody capable of binding to the PD-L1 protein, for example, via a C-peptide. In some other embodiments, the human IgG constant region may be located at the C-terminus of the VH of an antibody capable of binding to the TIGIT protein, and further, at the N-terminus of the VH of an antibody capable of binding to the PD-L1 protein.

[0164] In some embodiments, the human IgG constant region can be directly or indirectly ligated to the N-terminus of the VH of an antibody capable of binding to the PD-L1 protein, for example, via a C-peptide. For example, the C-peptide may comprise the amino acid sequence shown in any one of SEQ ID NO: 74-77.

[0165] For example, the first polypeptide chain may contain the amino acid sequence shown in any one of SEQ ID NO:78-83. In this application, the first polypeptide chain may include a heavy chain variable region VH of an antibody capable of binding to the TIGIT protein, and the second polypeptide chain may further include a light chain variable region VL of an antibody capable of binding to the TIGIT protein, a heavy chain variable region VH capable of binding to the PD-L1 protein, and a light chain variable region VL capable of binding to the PD-L1 protein.

[0166] In some embodiments, the VH of the antibody capable of binding to the PD-L1 protein and the VL of the antibody capable of binding to the PD-L1 protein in the second polypeptide chain constitute an scFv. In some embodiments, the VH of the antibody capable of binding to the PD-L1 protein and the VL of the antibody capable of binding to the PD-L1 protein constitute an scFv via a linker.

[0167] In some embodiments, the scFv in the second polypeptide chain that can bind to the PD-L1 protein may be located at the N-terminus or C-terminus of the VL of the antibody that can bind to the TIGIT protein.

[0168] In this application, the VH of an antibody capable of binding to the TIGIT protein (or PD-L1 protein) and the VL of an antibody capable of binding to the TIGIT protein (or PD-L1 protein) may be modified to facilitate the formation of scFv, for example, by introducing amino acid mutations without affecting the specific binding ability and binding affinity of the antibody. For example, amino acid mutations can be introduced to form disulfide bonds, making the structure of the scFv more stable. Such amino acid mutations are common in the art, for example, amino acid mutations in the skeletal region, and furthermore, mutations of the 100th amino acid Q in the light chain variable region to C, or mutations of the 44th amino acid R or G in the heavy chain variable region to C. These pre-mutation and post-mutation VH and VL are also within the scope of protection of this application.

[0169] For example, in this application, the multispecific antibody may include the structure shown in Figure 7A. Here, the first polypeptide chain includes the amino acid sequence shown in any one of SEQ ID NO: 78-83, and the second polypeptide chain may further include the amino acid sequence shown in SEQ ID NO: 84.

[0170] For example, in this application, the multispecific antibody may include the structure shown in Figure 7B. Here, the first polypeptide chain includes the amino acid sequence shown in any one of SEQ ID NO: 85-87, and the second polypeptide chain may further include the amino acid sequence shown in SEQ ID NO: 88.

[0171] nucleic acids, vectors, cells In another embodiment, the present application also provides one or more isolated nucleic acid molecules. The one or more nucleic acid molecules may encode the multispecific antibody described herein. For example, each of the one or more nucleic acid molecules may encode the complete multispecific antibody or a portion thereof (e.g., one or more of the first target moiety, the second target moiety, HCDR1-3, LCDR1-3, VL, VH, light chain, or heavy chain).

[0172] The nucleic acid molecules described herein can be isolated. For example, they can be produced or synthesized by (i) in vitro, e.g., polymerase chain reaction (PCR) amplification, (ii) clonal recombination, (iii) purification, e.g., by stepwise separation by enzymatic digestion and gel electrophoresis, or (iv) synthesis, e.g., chemical synthesis. In some embodiments, the isolated nucleic acid is a nucleic acid molecule prepared by recombinant DNA technology.

[0173] In this application, nucleic acids encoding multispecific antibodies can be prepared by various methods known in the art, including but not limited to restriction fragment manipulation or overlap extension PCR of synthetic oligonucleotides. For details, see Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; and Ausube et al., Current Protocols in Molecular Biology, Greene Publishing and Wiley-Interscience, New York, NY, 1993.

[0174] In another embodiment, the present application provides one or more vectors comprising one or more nucleic acid molecules described herein. Each vector may comprise one or more of the nucleic acid molecules. The vector may also comprise other genes, such as marker genes, that select the vector under appropriate conditions in a suitable host cell. The vector may also comprise expression regulatory elements that enable the coding region to be correctly expressed in a suitable host. Such regulatory elements are well known to those skilled in the art and may comprise, for example, promoters, ribosome binding sites, enhancers, and other regulatory elements that regulate gene transcription or mRNA translation. In some embodiments, the expression regulatory sequence is a tunable element. The specific structure of the expression regulatory sequence may vary depending on the function of the species or cell type, but may typically comprise a 5' untranscribed sequence and 5' and 3' untranslated sequences involved in transcription and translation initiation, such as TATA, capping sequences, or CAAT sequences. For example, the 5' untranscribed expression regulatory sequence may comprise a promoter region, and the promoter region may comprise a promoter sequence that functionally ligates to a nucleic acid for transcriptional regulation. The expression regulatory sequence may further include an enhancer sequence or an upstream activator sequence. In this application, preferred promoters include, for example, SP6, T3, and T7 polymerase promoters, human U6RNA promoter, CMV promoter, and its artificial heterologous promoter (e.g., CMV), where a portion of the promoter may be fused to a portion of the promoter of another cellular protein (e.g., human GAPDH, glyceraldehyde 3-phosphate dehydrogenase) gene, and these may or may not further include introns. One or more nucleic acid molecules described in this application can be operably linked to an expression regulatory element. The vector may consist of, for example, a plasmid, slime mold, virus, phage, or other vectors commonly used in, for example, genetic engineering. For example, the vector is an expression vector.

[0175] On the other hand, the present application provides a host cell which may contain one or more nucleic acid molecules and / or one or more vectors described herein. In some embodiments, each host cell may contain one or more nucleic acid molecules or vectors described herein. In some embodiments, each host cell may contain multiple (e.g., two or more) or multiple types (e.g., two or more) nucleic acid molecules or vectors described herein. For example, a vector described herein can be introduced into a host cell, eukaryotic cell such as a plant, fungal or yeast cell. A vector described herein may be introduced into the host cell by methods known in the art, such as electroporation, lipofectine transfection, or lipofectamin transfection.

[0176] Manufacturing method In another embodiment, the present application provides a method for preparing the multispecific antibody. The method may include culturing the cells under conditions in which the multispecific antibody is expressed. This can be done, for example, by using a suitable culture medium, a suitable temperature and incubation time, and is a method known to those skilled in the art.

[0177] An exemplary method for producing the anti-human TIGIT antibody of this application is described in Example 1.

[0178] Humanized antibodies can be selected from any type of immunoglobulin, including IgM, IgD, IgG, IgA, and IgE. In this application, the antibody is an IgG antibody, and the IgG1 isotype is used. Optimization of the sequence of the required constant structural domain can be achieved by screening the antibody with the biological assays described in the following examples to obtain the desired biological activity. Here again, either class of light chains can be used in the compounds and methods herein. Specifically, κ, λ chains or their variants are available in the compounds and methods herein.

[0179] An exemplary method for humanizing the anti-human TIGIT antibody of this application is described in Example 2.

[0180] The DNA molecule sequences of the multispecific antibodies or their fragments according to this invention can be obtained by conventional techniques such as amplification using PCR or genome library screening. Furthermore, single-chain antibodies can also be formed by fusing the coding sequences of the light chain and heavy chain.

[0181] Once the desired sequence is obtained, it can be acquired in large quantities by recombination. This is usually done by cloning it into a vector, transplanting it into cells, and then isolating the desired sequence from the proliferated host cells using conventional methods.

[0182] Furthermore, especially when the fragment length is short, it is possible to synthesize the sequence artificially. In many cases, very long fragments can be obtained by synthesizing several smaller fragments and ligating them. This nucleic acid molecule can then be introduced into various existing DNA molecules (or vectors, etc.) or cells known in this art.

[0183] The present invention also relates to vectors comprising a suitable nucleic acid molecule and a suitable promoter or regulatory sequence. These vectors can be used to transform suitable host cells so that they can express proteins. The host cells may be prokaryotic cells such as bacterial cells; lower eukaryotic cells such as yeast cells; or higher eukaryotic cells such as mammalian cells. Examples of animal cells include (but are not limited to) CHO-S, CHO-K1, and HEK-293 cells.

[0184] The steps described herein for transforming host cells with recombinant DNA can be carried out using techniques well known in the art. The resulting transformants can be cultured by conventional methods, and the transformants express the polypeptide encoded by the nucleic acid molecule of the present invention. Depending on the host cells used, they may be cultured under appropriate conditions using conventional media. Typically, the transformed host cells are cultured under conditions suitable for the expression of the multispecific antibodies of the present invention. The multispecific antibodies of the present invention can then be purified using conventional immunoglobulin purification steps such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography, or affinity chromatography, and conventional separation and purification methods known to those skilled in the art.

[0185] The obtained monoclonal or multispecific antibodies can be identified by conventional methods. For example, the binding specificity of monoclonal or multispecific antibodies can be measured by immunoprecipitation or in vitro binding assays (e.g., flow cytometry sorting (FACS), radioimmunoassay (RIA), or enzyme-linked immunosorbent assay (ELISA)).

[0186] Pharmaceutical composition Furthermore, the present application provides compositions. In some embodiments, the composition may be a pharmaceutical composition comprising the multispecific antibody of the present application and a pharmaceutically acceptable vector. Typically, these substances may be prepared in a non-toxic, inert, and pharmaceutically acceptable aqueous vector medium. The prepared pharmaceutical composition can be administered by conventional routes.

[0187] The pharmaceutical compositions described herein can directly bind to TIGIT protein molecules or PD-L1 protein molecules and can therefore be used for the prevention and treatment of TIGIT-related, PD-L1-related, or PD-1-related diseases. The pharmaceutical compositions described herein may contain a safe and effective amount of the antigen-binding protein described herein and a pharmaceutically acceptable adjuvant (which may include a vector or excipient). The formulations should be adapted to the dosage form.

[0188] The multispecific antibodies or pharmaceutical compositions described herein can be compounded, administered, and used in a manner consistent with good medical practice. In this context, considerations include the specific condition being treated, the specific mammal being treated, the clinical condition of the individual patient, the etiology of the condition, the site of drug delivery, the method of administration, the administration schedule, and other factors known to the practitioner.

[0189] Methods and Uses In another embodiment, the multispecific antibodies, nucleic acid molecules, vectors, cells, and / or pharmaceutical compositions are provided for use in pharmaceutical preparation. The pharmaceuticals are used for the prevention, remission, and / or treatment of diseases or conditions such as TIGIT disorder-related diseases. In some embodiments, the TIGIT disorder-related disease may be a T-cell dysfunction disorder. T-cell dysfunction is reflected in T-cell depletion, and by enhancing NK cells and activating T cells, the body's immune activity can be increased to treat, delay, or achieve remission of the disease. For example, the TIGIT disorder-related disease may be a tumor, cancer, or infection. For example, the TIGIT disorder-related disease may be a CD155-positive or PVR-positive tumor, cancer, immune disease, or infection, among others. In some embodiments, the tumor may be a solid tumor or a non-solid tumor. For example, the tumor may be colon cancer.

[0190] The antigen-binding protein of the present invention can inhibit tumor growth and / or the proliferation of tumor cells. In some embodiments, the tumor includes colorectal cancer. In some embodiments, the tumor or cancer is a tumor or cancer in which TIGIT expression is abnormal.

[0191] For example, in a mouse model of colorectal cancer, the antigen-binding protein described in this application (e.g., TIGIT antibody) can slow tumor growth.

[0192] In another embodiment, the present invention further provides a method for inhibiting the binding of CD155 to TIGIT, which includes using the multispecific antibody.

[0193] In another embodiment, the present invention further provides a method for inhibiting the binding of PD-L1 to PD-1, comprising using the multispecific antibody.

[0194] The following examples are not intended to be limited to theory, but are used solely to illustrate the proteins, preparation methods, and uses of this application, and are not intended to limit the scope of this application. Experimental methods in the following examples where specific conditions are not given generally follow conventional conditions, such as those described in Sambrook et al, Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or conditions recommended by the manufacturer. Unless otherwise specified, percentages and quantities refer to weight percentages and weight quantities. [Examples]

[0195] Example 1: Preparation of a mouse monoclonal antibody-primary antibody against human TIGIT. 1.1 Creation of hybridoma cells that produce mouse-derived monoclonal antibodies The method for producing monoclonal antibodies derived from mice uses the hybridoma production technology invented by Kohler and Milstein in 1975 (Nature, 1975, 256:495-497). After emulsifying the mouse Fc-tagged protein of human TIGIT (ACRO, # tit-h5253) and Freund's adjuvant, 5 mice of each strain of BALB / c, CD1, and C57BL / 6 were immunized subcutaneously. After three immunizations, sera were collected, and the potency by ELISA, binding activity, and functional activity by FACS were examined. The mice optimal for the fusion of spleen cells and SP2 / 0 myeloma cells were selected. After screening the hybridoma polyclonal cells with HAT, polyclonal cell lines that specifically bind to human TIGIT and can inhibit TIGIT-CD155 binding were screened for monoclonality by ELISA and FACS methods. Monoclonal cell lines that specifically bind were screened again by ELISA and FACS methods, and the binding to monkey and mouse TIGIT was tested. FACS screening of monoclonal antibodies that inhibit the binding of TIGIT-CD155 and TIGIT-CD122 was performed. Subsequently, the cell functional activity measurement and affinity (Biacore) screening of the screened monoclonal cell lines were carried out to obtain a monoclonal hybridoma cell line for human TIGIT antibody expression for sequence analysis, and the screening data are shown in Table 1.

[0196]

Table 1

[0197] As a result of high-throughput screening, a final monoclonal hybridoma cell line with high affinity for the binding activities of both human and monkey, simultaneously inhibiting two ligands of TIGIT, CD155 and CD122, and having biological activity was successfully obtained. The hybridoma cell line in Table 1 above is humanized.

[0198] Example 2: Cloning and humanization of the variable region gene sequence of an anti-TIGIT antibody. 2.1 Cloning of antibody variable region genes in hybridoma cells The cDNA sequences of the variable regions of mouse antibodies expressed by hybridoma cell lines were cloned based on the principles of TAKARA's 5'RACE technology. Briefly, using the SMARTer 5' RACE synthesis experiment kit (TAKARA, #634859), heavy chain and light chain variable region gene-specific cDNAs were synthesized according to the instructions. The 5' and 3' ends of the cDNA sequences were modified with PCR primers. These primers are designed to add appropriate read sequences to the heavy chain and light chain variable region cDNAs, respectively, enabling seamless cloning of the resulting PCR products into existing recombinant antibody expression heavy chain vectors pHB-Fc and light chain vectors pHB-Cκ. The pHB-Fc expression vector contains the gene sequence of the human IgG1 heavy chain constant region with L234A and L235A (Eu-encoded) mutations on CH2 that have an antibody ADCC knockout (KO) effect, while the pHB-Cκ vector contains the gene sequence of the human κ light chain constant region. The heavy and light chain variable region PCR amplification products were cloned into expression vectors using an in-fusion cloning reagent (TAKARA, #639650) and transformed into E. coli DH5α receptor cells (YB Biotech, #FYE607-80VL). Monoclonal colonies were selected for Sanger sequencing and analyzed to obtain antibody variable region sequences. The sequence of the variable region of the anti-TIGIT antibody expressed by B3 / 29F6 was as follows:

[0199] B3 / 29F6 VH SEQ ID NO:1 DVQLQESGPGLVKPSQSLSLTCTVTGYSITSDYAWIWIRQFPGNKVEWMGYITSSGSTSYNPSLKSRISFTRDTSKNQFFLQLTSVTTEDTATYYCARLDFGNYGGAMDYWGQGTSVTVSS B3 / 29F6 VL SEQ ID NO:2 DIVMTQSHKFMSTSIGDRVSITCKASQHVSTAVAWYQQKPGQSPKLLIYSASYRYTGVPDRFTGSRSGTDFTFTISSVQAEDLAVYYCQQHYITPYTFGGGTKLEIK The underlined parts are CDRs (defined in IMGT, with the following sequences).

[0200] [Table 2]

[0201] 2.2 Construction of an IgG1 wild-type chimeric expression vector The IgG1 wild-type chimeric antibody and the ADCC KO chimeric antibody share the same light chain vector. The difference lies in amino acids 234 and 235 of the heavy chain CH2 on the vector containing the human heavy chain constant region sequence: A234 / A235 for the ADCC KO type and L234 / L235 for the wild type. The IgG1 wild-type chimeric heavy chain expression vector is constructed in the same manner as described in 2.1. Specifically, the heavy chain variable region gene is amplified by PCR using primers containing the read sequence, and then cloned into a vector containing the human heavy chain constant region sequence (CH2 L234 / L235) using seamless ligation to complete the construction of the IgG1 wild-type chimeric expression vector.

[0202] 2.3 Expression of Chimeric Antibodies The expression vectors obtained in sections 2.1 and 2.2 were amplified in E. coli, and plasmids sufficient for transient transfection of chimeric antibodies were prepared using an endotoxin removal plasmid extraction kit (TianJian Biotech, #DP117). The host cells used for expression were CHO-S cells (Thermo Fisher, #R80007). CHO-S cells were transfected by mixing two separately prepared heavy-chain and light-chain vectors with polyetherimide (PEI, Polyscience, #24765-1) to form liposome complexes, and then culturing them in an incubator for 5-7 days. The cell culture supernatant was collected by centrifugation and purified using a protein A affinity chromatography column to obtain ADCC KO-type human mouse chimeric antibody (number 900424) and IgG1 wild-type human mouse chimeric antibody (protein number 900445).

[0203] The sequences of the chimeric antibodies VH and VH obtained according to the above method are as follows: 900423 and 900428 VH SEQ ID NO:32 DVQLQESGPGLVKPSQSLSLTCTVTGYSITSDYAWNWIRQFPGNRLEWMGYISYSGAPRYNPSLKSRISITRDTSKNQFFLQLSFVTTEDTATYYCASLGTDYYAMDYWGQGTSVIVSS 900424 VH SEQ ID NO:1 DVQLQESGPGLVKPSQSLSLTCTVTGYSITSDYAWIWIRQFPGNKVEWMGYITSSGSTSYNPSLKSRISFTRDTSKNQFFLQLTSVTTEDTATYYCARLDFGNYGGAMDYWGQGTSVTVSS 900424 VL SEQ ID NO:2 DIVMTQSHKFMSTSIGDRVSITCKASQHVSTAVAWYQQKPGQSPKLLIYSASYRYTGVPDRFTGSRSGTDFTFTISSVQAEDLAVYYCQQHYITPYTFGGGTKLEIK 900423 VL SEQ ID NO:34 DIVMTQSHKFMFTSVGDRVSITCKASQHVSNAVAWYQQKPGQSPKLLIHSASYRYTGVPDRFIGRGSGTFTFTISSVQAEDLAVYYCQQYYSLPWTFGGGTKLEIK 900428 VL SEQ ID NO:33 DIVMTQSHKFMFTSVGDRVSITCKASQHVSNAVAWYQQKPGQSPKLLIHSASYRYTGVPDRFTGRGSGTDFTFTINSVQAEDLAVYYCQQYYSLPWTFGGGTKLEIK.

[0204] Here, the amino acid sequences of the heavy chain constant regions of 900424, 900423, and 900428 are all shown in SEQ ID NO:30. The light chain constant regions are all human κ-light chain constant regions (SEQ ID NO:31).

[0205] 2.4 Humanization of mouse-derived anti-human TIGIT antibody - Method for preparing humanized antibodies The following method was used to humanize the antibody: The sequence of the antibody's variable region was compared with available sequences in the NCBI IgBlast database, and human-derived conformational regions (FR regions) suitable for constructing CDR graft heavy and light chains on top of it were identified and analyzed.

[0206] Modifications were designed according to the conserved and important amino acid residues in the FR region of the human antibody. Humanization mutations were designed for the variable regions of the heavy and light chains of the chimeric antibody, respectively, and humanization point mutation antibody expression plasmids were amplified and constructed using PCR technology. These humanization point mutation antibody expression plasmids were expressed in CHO-S cells and purified to obtain humanized antibody proteins. Receptor binding ability, functional inhibitory activity, and ADCC effect were screened using ELISA, Biacore, and flow cytometry assays, yielding four humanized anti-TIGIT antibodies with superior performance.

[0207] The VH and VL sequences of the obtained humanized anti-TIGIT antibody are shown below: 900461 and 900464 VH SEQ ID NO:9 QVQLQESGPGLVKPSETLSLTTCTVSGYSITSDYAWIWIRQPPGKGLEWIGYITSSGSTYYNPSLKSRVTFSVDTSKNQFSLKLSSVTAADTATYYCARLDFGNYGGAMDYWGQGTLVTVSS 900461 VL SEQ ID NO:10 DIQMTQSPSSLSASVGDRVTITCRASQHVSTAVNWYQQKPGKAPKLLIYSASYLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYITPYTFGQGTKLEIK 900464 and 900476 VL SEQ ID NO:11 DIQMTQSPSSLSASVGDRVTITCKASQHVSTALAWYQQKPGKAPKLLIYSASYLQSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYITPYTFGQGTKLEIK 900466 VH SEQ ID NO:12 QVQLQESGPGLVKPSETLSLTCTVSGYSITSDYAWIWIRQPPGKKLEWIGYITSSGSTYYNPSLKSRVTFSVDTSKNQFSLKLSSVTAADTATYYCARLDFGNYGGAMDYWGQGTLVTVSS 900466 VL SEQ ID NO:13 DIQMTQSPSSLSASVGDRVTITCKASQHVSTAVNWYQQKPGKAPKLLIYSASYRQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHYITPYTFGQGTKLEIK 900476VH SEQ ID NO:14 QVQLQESGPGLVKPSETLSLTCTVSGYSITSDYAWIWIRQPPGNKLEWIGYITSSGSTYYNPSLKSRVTFSVDTSKNQFSLKLSSVTAADTATYYCARLDFGNYGGAMDYWGQGTLVTVSS。

[0208] Here, the underlined parts are CDRs (defined by IMGT), and 900461, 900464, 900466, and 900476 are the four humanized antibody protein numbers for product numbers HB0030, HB0031, HB0032, and HB0033, respectively.

[0209] Here, the amino acid sequences of the heavy chain constant regions of HB0030, HB0031, HB0032, and HB0033 are all shown in SEQ ID NO:29, and the light chain constant regions are all human κ light chain constant regions (SEQ ID NO:31).

[0210] Example 3 Assay of Chimeric Antibodies and Humanized Antibodies 3.1 Measurement of binding activity of TIGIT antibodies against cells expressing human TIGIT Chimeric antibodies 900324, 900423, 900424, 900428, and humanized antibodies HB0030, HB0031, HB0032, and HB0033 were assayed for their binding activity to cells expressing human TIGIT (CHOK1-huTIGIT-2A3, Haber Bio).

[0211] All chimeric antibodies and humanized antibodies were diluted to 20 μg / ml in PBS solution containing 1% BSA (1% BSA / PBS), and 20 μL was added per well to a 96-well U plate to simultaneously establish a negative control (1% BSA / PBS only). A suspension of human TIGIT-expressing cells (CHOK1-huTIGIT-2A3, Huabo Bio) in the logarithmic growth phase was collected, the culture medium was discarded by centrifugation (1000 rpm × 5 min), and the cells were diluted to a viable cell density of 1 × 10⁶ in 1% BSA / PBS. 6 Resuspend in 1 / mL and add 20 μL (2 × 10) of anti-TIGIT antibody to a 96-well U plate. 4 Cells were added at room temperature for 30 minutes. The 96-well U-plate was resuspended in 1% BSA / PBS, centrifuged (300g x 3min) and the upper layer was discarded. After one wash, PE-sheepantihuman-Fc (Jackson ImmunoResearch, #109-115-098) 1:200 dilution was added, and the mixture was allowed to react at room temperature for 15 minutes in the dark. The 96-well U-plate was resuspended in 1% BSA / PBS, centrifuged (300g x 3min) and the upper layer was discarded. This process was repeated three times, and finally, the mixture was resuspended in 100 μL of 1% BSA / PBS per well. The fluorescence intensity of the PE channels was measured by flow cytometry (BD, #Canto II).

[0212] Figure 1 and Table 3 show the results of the chimeric antibody binding survival rate, and Figure 2 and Table 4 show the results of the humanized antibody binding survival rate. 900324 is a Genentech anti-human TIGIT antibody, tilagolumab, which has the same sequence of light and heavy chain variable regions as antibody 10A7 in patent application WO2015009856A2, transiently expressed in CHO cells. As a result, both the chimeric antibody and the humanized antibody were confirmed to have good and equivalent affinity to cells expressing human TIGIT (CHOK1-huTIGIT-2A3, Haber Bio).

[0213] [Table 3]

[0214] [Table 4]

[0215] 3.2 Test to measure the inhibition of TIGIT antibody against the binding of TIGIT antigen to TIGIT molecule (competitive method) The antigen huCD155-moFc (ACROBiosystems, #CD5-H5254) was diluted to 40 μg / ml in PBS solution containing 1% BSA. 10 μL was added to each well of a 96-well U plate and thoroughly mixed with anti-TIGIT antibody, which had been serially diluted in a 1:1 volume ratio. A positive control was also established simultaneously (only CD155-moFc was added). The suspension of logarithmically growing human TIGIT-expressing cells (CHOK1-huTIGIT-2A3, Huabo Bio) was removed by centrifugation (1000 rpm × 5 min), and the culture medium was diluted with 1% BSA / PBS to a viable cell density of 1 × 10⁶. 6 Resuspend in 1 / mL and pour 20 μL (2 × 10) into a 96-well U plate. 4Cells were added to each well, mixed with CD155-moFc and anti-TIGIT antibody at room temperature for 30 minutes, and culture was continued. The 96-well U plate was resuspended in 1% BSA / PBS, centrifuged (300g x 3min) and the upper layer was discarded. After one wash, Alexa488-gat anti-mouse-Fc (Jackson ImmunoResearch, #115-545-071) 1:300 dilution was added, and the mixture was incubated in the dark at room temperature for 15 minutes. The U-well plate was resuspended in 1% BSA / PBS, centrifuged (300g x 3min) and the upper layer was discarded. This process was repeated three times, and finally, 100 μL of 1% BSA / PBS per well was added and the mixture was resuspended. The fluorescence intensity of the FITC channel was measured by flow cytometry (BD, #Canto II).

[0216] The results of chimeric antibody binding activity are shown in Figure 3 and Table 5, and the results of humanized antibody binding activity are shown in Figure 4 and Table 6. The results clearly showed that both the chimeric antibody and the humanized antibody had equivalent inhibitory activity against human CD155 and exhibited significant inhibitory effects.

[0217] [Table 5]

[0218] [Table 6]

[0219] 3.3 Affinity testing of humanized monoclonal antibodies In this study, the dynamics and affinity of antigen-antibody binding were measured using the SPR method. A BIOCORE (GE, #Biacore 8K) was used. A Sereis S Sensor Chip Protein A chip (GE, #29-1275-56) was equilibrated at room temperature for 20-30 minutes, and the chip was set in the instrument. The antibody sample was diluted to the experimental working concentration with equilibration buffer and stored in a sealed container at 2-8°C. The antigen, histidine-tagged (His-Tag) human TIGIT protein (huTIGIT, Acro Biosystems, #TIT-H52H3), was diluted with equilibration buffer HBS-EP (10x) (GE, #BR-1006-69). The diluted antigen was subjected to a concentration gradient of 2.5-fold dilutions from 20 nM five times, with zero concentration twice (i.e., equilibration buffer) and a minimum concentration replica set. A pH 1.5 glycine solution (GE, #BR100354) was used to regenerate the chip. The samples were analyzed using a capture method multicycle kinetic program. After confirming the absence of significant reference bindings, a corresponding analysis program was selected, and the 1:1 binding model, Kinetics, was chosen and fitted to the analysis to determine the kinetic parameters of the samples.

[0220] The assay results are shown in Table 7. Based on the affinity constant (KD(M)) results from huTIGIT, the humanized monoclonal antibody HB0030 of this invention has a affinity of 10. -11 It was shown to have an affinity close to an order of magnitude, indicating an extremely strong affinity.

[0221] [Table 7]

[0222] Example 4: In vivo efficacy study in animals In this experiment, the effects of anti-human TIGIT antibodies HB0030, HB0031, HB0032, and HB0033 were investigated in BALB / c-hPD1 / hTIGIT mice, which were immunohistochemically humanized to CT26.WT colon cancer models administered subcutaneously.

[0223] In this experiment, CT26.WT cells were collected from mouse colon cancer cells, the logarithmic growth phase was isolated, the culture medium was removed, and the cells were washed twice with PBS before inoculation (CT26.WT cell viability was 98.57% before and 98.28% after cancer development). The inoculation dose was 5 × 10⁻⁶. 5 / 100μL / animal. Average tumor volume was 94.02 mm² 11 days after vaccination. 3 Once the tumor volume was reached, the mice were randomly grouped according to their tumor volume, with 8 mice assigned to each group. Grouping was defined as day D0, and administration was performed on D0, D3, D7, D10, D14, and D17. After the start of administration, tumor size was observed and mouse weight was measured on D0, D2, D4, D6, D8, D10, D13, D15, D17, D20, and D23. Tumor volume calculation method: Tumor volume (mm 3 ) = 0.5 × Tumor length × Tumor width 2 The results are shown in Figure 5.

[0224] Observing the experimental results in Figure 5, it is clear that the anti-TIGIT humanized monoclonal antibodies HB0030, HB0031, HB0032, and HB0033 of this application have a significant tumor-suppressing effect when administered alone at a dose of 10 mg / kg. Furthermore, the efficacy of HB0030 and the control antibody tilagolumab at 10 mg / kg was investigated in BALB / c-hPD1 / hTIGIT, humanized mice with an immunodetection site that were subcutaneously inoculated with the CT26.WT colorectal cancer model.

[0225] In this experiment, CT26 cells were collected from mouse colon cancer cells in the logarithmic growth phase, the culture medium was removed, and the cells were washed twice with PBS before being processed into 5 × 10⁻⁶ cells. 5 Inoculation was performed at a dose of 100 μL per cell (the survival rates of CT26 cells before and after tumor formation were 98.51% and 97.250%, respectively). The average tumor volume was 80.33 mm² 12 days after inoculation. 3Once the target was reached, the mice were randomly grouped according to tumor volume, with 6 mice assigned to each group. The grouping day was defined as day D0, and the drug was administered on D0, D3, D7, D10, D14, and D17. After the start of administration, the tumor size was observed and the mouse body weight was measured on D0, D2, D4, D6, D8, D10, D13, D15, D17, D20, and D23. Tumor volume calculation method: Tumor volume (mm 3 ) = 0.5 × Tumor length × Tumor width 2 The results are shown in Figure 6.

[0226] Observing the results in Figure 6, the present invention's anti-TIGIT antibody HB0030 showed superior tumor suppression compared to the control antibody tiragolumab.

[0227] In this study, no significant changes in body weight were observed in any of the mouse groups that received the vaccine 24 days after vaccination.

[0228] Example 5 Sequence design of heavy and light chains of bispecific antibodies Using HB0030 to construct the PDL1-TIGIT bispecific antibody, the structural mode of the bispecific antibody belongs to the Morrison mode (IgG-scFv), that is, the scFv fragment of one antibody is ligated to the C-terminus of both heavy chains of the IgG antibody, and its structure is shown in Figures 7A-7B. Here, in order to distinguish between different antibody morphologies, when TIGIT is complete IgG and PD-L1 is scFv, this bispecific antibody morphology is called TIGIT-IgG-PD-L1-scFv, and the main structural designs of its heavy and light chains are shown in Table 8 below. When PD-L1 is complete IgG and TIGIT is scFv, this bispecific antibody morphology is called PD-L1-IgG-TIGIT-scFv, and the main structural designs of its heavy and light chains are shown in Table 9 below.

[0229] [Table 8]

[0230] [Table 9]

[0231] Tables 8 and 9 show, The sequence of antibody 1:900339 is derived from protein 900339 of patent WO2020199860A1. 900339-VL refers to the light chain variable region of 900339, and 900339-VH refers to the heavy chain variable region of 900339. HB0030-VL refers to the light chain variable region of HB0030, and HB0030-VH refers to the heavy chain variable region of HB0030. 2:Q100C refers to a mutation in the light chain variable region where the 100th amino acid Q is changed to C, according to Kabat's antibody numbering rules. R44C or G44C refers to a mutation in the heavy chain variable region where the 44th amino acid R or G is changed to C, according to Kabat's antibody numbering rules. After these two amino acid mutations, a disulfide bond is formed, increasing the stability of the scFv region and enhancing the drug discovery potential of the biantibody. 3: Amino acid sequence of Linker1: (G4S)3 (SEQ ID NO: 74), Amino acid sequence of Linker2: (G4S)4 (SEQ ID NO: 75), Amino acid sequence of Linker3: (G4S)5 (SEQ ID NO: 76), Amino acid sequence of Linker4: (G4S)6 (SEQ ID NO: 77).

[0232] Example 6 Expression and purification of bispecific antibodies The expression vector obtained in Example 5 was amplified in E. coli, and plasmids sufficient for transient transfection to express bispecific antibodies were prepared using an endotoxin removal plasmid extraction kit (TianJian Biotech, #DP117). The host cells used for expression were CHO-S cells (Thermo Fisher, #R80007). CHO-S cells were transfected by mixing two separately prepared heavy-chain and light-chain vectors with polyetherimide (PEI, Polyscience, #24765-1) to form liposome complexes and culturing them in an incubator for 5-7 days. The cell culture supernatant was collected by centrifugation and purified using a protein A affinity chromatography column to obtain a series of bispecific antibodies, and the aggregation status of the proteins was further determined by molecular sieve chromatography.

[0233] The expression, purification, and measurement status of the bispecific antibodies are shown in Figures 8 and 9 and Table 10. According to the measurement results, the SEC purity of 900683 was good for the TIGIT-IgG-PD-L1-scFv form of the bispecific antibody, while the SEC purity of 900693 was good for the PD-L1-IgG-TIGIT-scFv form of the bispecific antibody. The reduced and unreduced SDS-PAGE figures clearly showed no degradation of either the TIGIT-IgG-PD-L1-scFv or PD-L1-IgG-TIGIT-scFv forms of the bispecific antibody, suggesting good protein stability.

[0234] [Table 10]

[0235] Example 7 Measurement of kinetic parameters of bispecific antibodies This study measured the binding dynamics and affinity of PDL1-TIGIT bispecific antibody, PDL1 parental monoclonal antibody 900339, and TIGIT parental monoclonal antibody HB0030 to the antigen using the SPR method.

[0236] The testing method is as follows. Anti-Human Capture-CM5 chips (GE, #BR-1005-30) were prepared using the coupling method of the Human Antibody Captrue Kit (GE, #BR-1008-39) and the Amino Group Coupling Kit (GE, BR-1000-50). The chips were equilibrated at room temperature for 20-30 minutes and then placed in a Biacore 8K instrument. The antigen and antibody were diluted to the test working concentration in equilibrium buffer. The antigen was diluted to 50 nM in equilibrium buffer and then further diluted threefold to create seven concentration gradients, establishing two zero concentrations (i.e., equilibrium buffer) and one repeat concentration (generally the lowest concentration repeat). The test analysis was repeated in the order of antibody, antigen, and regeneration at 10 antigen concentrations (two zero concentrations, seven gradient concentrations, and one repeat concentration), with an antigen injection flow rate of 30 μL / min, a binding time of 120 seconds, and a dissociation time of 600 seconds. After the analysis was completed, the data was analyzed using the corresponding analysis program to confirm the absence of obvious reference binding. The kinetic parameters Ka, Kd, ​​and KD values ​​of the bispecific antibody and parent monoclonal antibody were obtained using kinetics, a 1:1 binding model, and curve fitting.

[0237] The measurement results are shown in Tables 11 and 12. As a result, for the TIGIT-IgG-PD-L1-scFv bispecific antibody, the affinity of the bispecific antibody against TIGIT was equivalent to that of the parent HB0030, and the affinity of the bispecific antibody against PD-L1 was slightly higher than that of the parent 900339 monoclonal antibody. For the PD-L1-IgG-TIGIT-scFv bispecific antibody, the affinity of the bispecific antibodies 900691, 900692, and 900693 against PD-L1 was equivalent to that of the parent monoclonal antibody 900339, and the affinity of 900691, 900692, and 900693 against TIGIT was on the same order of magnitude as that of the parent monoclonal antibody HB0030.

[0238] [Table 11]

[0239] [Table 12]

[0240] Example 8 Measurement of the biological activity of bispecific antibodies 8.1 Inhibitory effect of PDL1-TIGIT bispecific antibody on PDL1 function The following is the procedure for measuring the inhibitory effect of the PDL1-TIGIT bispecific antibody on PDL1 function. The PBS solution for the bispecific antibody and the 900339 parent monoclonal antibody was diluted to 10 μg / ml, and then further diluted in nine gradients at a 2-fold ratio. Target cells Jurkat-NFAT-PD-1-5B8 overexpressing PD-1 were counted, resuspended in 5 × 10⁵ E₅ / mL, and spread in a 96-well white base plate at 30 μl / well. Effector cells CHO-K1-OS8-PD-L1-8D6 overexpressing PD-L1 were counted, resuspended in 5 × 10⁵ E₅ / mL, and added to the 96-well white base plate containing the Jurkat-NFAT-PD-1-5B8 cells at 0 μl / well. Next, diluted bispecific antibodies and parental control antibodies were sequentially added at 30 μl / well to a 96-well white base plate spread with cells, and incubated at 37°C for 6 hours. After incubation, the culture plate was equilibrated at room temperature for at least 15 minutes, then 90 μL of BIO-Glo assay was added per well, and the mixture was allowed to react at room temperature in the dark for 5 minutes. Finally, the signal intensity was measured using an MD i3x microplate reader.

[0241] The measurement results for the PD-L1-IgG-TIGIT-scFv bispecific antibodies are shown in Figure 10 and Table 13. As a result, the inhibitory effects on PD-L1 function were equivalent between the bispecific antibodies 900691, 900692, and 900693 and the PD-L1 parent monoclonal antibody 900339.

[0242] [Table 13]

[0243] 8.2 Inhibitory effect of PDL1-TIGIT bispecific antibody on TIGIT function The following is the procedure for measuring the inhibitory effect of the PDL1-TIGIT bispecific antibody on TIGIT function. The PBS solution for the bispecific antibody and the HB0030 parent monoclonal antibody was diluted to 10 μg / ml, and then further diluted in nine gradients at a 2-fold ratio. Target cells Jurkat-TIGIT-22G8 overexpressing TIGIT were counted, resuspended in 4 × 10⁶ E⁶ / mL, and spread in a 96-well white base plate at 50 μl / well. Next, the diluted bispecific antibody and parent control antibody were sequentially added in 50 μl / well to the 96-well white base plate with the spread cells, and incubated at 37°C for 10 minutes. Then, 50 μl of PHA at a concentration of 20 μg / ml and 50 μl of CD155 at a concentration of 8 ug / ml were added, mixed well, and cultured in an incubator for 24 hours. After incubation, the supernatant was removed by centrifugation at 1500 rpm for 5 minutes, and the IL-2 content was measured using an ELISA kit.

[0244] The measurement results for the TIGIT-IgG-PD-L1-scFv bispecific antibody are shown in Figure 11 and Table 14. As a result, the bispecific antibody in the TIGIT-IgG-PD-L1-scFv form did not show a significant difference in inhibitory effect on TIGIT function compared to the TIGIT parent monoclonal antibody HB0030.

[0245] [Table 14]

[0246] Example 9: Measurement of thermal stability of PDL1-TIGIT bispecific antibody The procedure for testing the thermal stability of PDL1-TIGIT bispecific antibodies is as follows: Using a protein stability analyzer (UNcle, UNCHAINED LABS, US), the melting temperature (Tm) and agglutination temperature (Tagg) of bispecific antibodies 900691, 900692, and 900693 in PD-L1-IgG-TIGIT-scFv form were measured. The heating range for Tm and Tag was 25°C to 95°C, and the heating rate was 0.3°C.

[0247] The thermal stability measurements of the PD-L1-IgG-TIGIT-scFv bispecific antibodies are shown in Table 15. As a result, the Tm and Tag values, which are thermal stability data for the PD-L1-IgG-TIGIT-scFv bispecific antibodies 900691, 900692, and 900693, were all within the range of 65°C to 70°C, suggesting that the three bispecific antibodies exhibit good thermal stability.

[0248] [Table 15]

[0249] Example 10: Measurement of the in vivo tumor therapeutic effect of a bispecific antibody. This study constructed an MC38-hPD-L1 colon cancer animal model using humanized B-hPD-1 / hPD-L1 / hTIGIT mice and measured the therapeutic effect of the test antibody. First, an MC38-hPD-L1 colon cancer model was constructed using humanized B-hPD-1 / hPD-L1 / hTIGIT mice. Tumor-forming mice were divided into seven test groups, with an average of six mice per group, based on tumor volume and body weight. The antibody was administered intraperitoneally twice a week for a total of six times. The specific administration plan is shown in Table 16 below.

[0250] [Table 16]

[0251] Measurement of tumor volume and tumor weight: After group assignment, tumor volume was measured twice a week. After the completion of the study, the tumors of the mice were removed and weighed to calculate the tumor growth inhibition rate (TGITV), which was then statistically analyzed to evaluate the antitumor effect of the test drug on the model mice.

[0252] Body weight and general clinical observation: Body weight was measured twice a week during the study period, and general clinical observation was performed once a day during the adaptation feeding period and the study period to evaluate the safety and tolerance of the test drug in the model mice.

[0253] As a result, the bispecific antibodies 900693 / 900692 / 900691 described in this application suppressed the increase in tumor volume and tumor weight in MC38-hPD-L1 colon cancer mice and demonstrated a certain level of safety and tolerance.

[0254] Example 11: Measurement of the in vivo tumor therapeutic effect of a bispecific antibody. This study evaluated the in vivo tumor therapeutic effect of a bispecific antibody using a humanized mouse model, BALB / C-hPD1 / hPDL1 / hTIGIT, which was subcutaneously inoculated with mouse colorectal cancer cells CT26-hPDL1.

[0255] Mouse colorectal cancer CT26-hPDL1 cells were inoculated subcutaneously into the right back of female humanized BALB / C-hPD1 / hPDL1 / hTIGIT mice. The average tumor growth in the tumor-bearing mice was 70.03 mm. 3 When tumor volume reached a certain level, the mice were randomly divided into four groups based on tumor volume. Six mice were assigned to each group: 900201 (control antibody, 3 mg / kg), 900339 (hPDL1 antibody, 3 mg / kg), HB0030 (TIGIT antibody, 3 mg / kg), and 900693 (bispecific antibody, 4.1 mg / kg). After grouping, each group of mice received intraperitoneal injection twice a week for a total of six times over three weeks. Tumor volume and body weight were measured weekly, and the relationship between the changes in body weight and tumor volume of the tumor-bearing mice and the time of administration was recorded.

[0256] The results are shown in Figure 12 (where G1-G4 represent the results of using 900201, 900458, HB0030, and 900693, respectively). According to the results in Figure 12, the bispecific antibody of this application showed a significant antitumor effect in humanized mice BALB / C-hPD1 / hPDL1 / hTIGIT subcutaneously inoculated with mouse colorectal cancer cells CT26-hPDL1, and was superior to 900458 and HB0030, suggesting potential clinical efficacy.

Claims

1. It is a multispecific antibody, It comprises a first target region that can specifically bind to the TIGIT protein and a second target region that can specifically bind to the PD-L1 protein. The first target region capable of binding to the TIGIT protein comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise amino acid sequences selected from the following group: (1) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 3, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 4, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 5, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 6, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 7, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 8; and (2) The HCDR1 contains the amino acid sequence shown in SEQ ID NO: 3, the HCDR2 contains the amino acid sequence shown in SEQ ID NO: 42, the HCDR3 contains the amino acid sequence shown in SEQ ID NO: 43, the LCDR1 contains the amino acid sequence shown in SEQ ID NO: 52, the LCDR2 contains the amino acid sequence shown in SEQ ID NO: 7, and the LCDR3 contains the amino acid sequence shown in SEQ ID NO:

53. and, The second target moiety capable of binding to the PD-L1 protein comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 102, HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 101, HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 100, LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 105, LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 104, and LCDR1 comprises the amino acid sequence shown in SEQ ID NO:

103. Multispecific antibodies.

2. The first target region capable of binding to the TIGIT protein comprises a heavy chain variable region VH and a light chain variable region VL, and the first target region capable of binding to the TIGIT protein comprises any set of amino acid sequences selected from the following group: (1) VH: SEQ ID NO: 9 and VL: SEQ ID NO: 10; (2) VH: SEQ ID NO: 9 and VL: SEQ ID NO: 11; (3) VH: SEQ ID NO: 12 and VL: SEQ ID NO: 13; (4) VH: SEQ ID NO: 14 and VL: SEQ ID NO: 11; (5) VH: SEQ ID NO: 1 and VL: SEQ ID NO: 2; (6) VH: SEQ ID NO: 32 and VL: SEQ ID NO: 34; (7) VH: SEQ ID NO: 32 and VL: SEQ ID NO: 33; and (8) VH: SEQ ID NO: 110 and VL: SEQ ID NO: 111 A multispecific antibody according to claim 1, characterized in that

3. The second target region capable of binding to the PD-L1 protein comprises a heavy chain variable region VH and a light chain variable region VL, and the second target region capable of binding to the PD-L1 protein comprises any set of amino acid sequences selected from the following group: (1) VH: SEQ ID NO: 106 and VL: SEQ ID NO: 107; and (2) VH: SEQ ID NO: 108 and VL: SEQ ID NO: 109 A multispecific antibody according to claim 1, characterized in that...

4. A multispecific antibody according to claim 1, comprising a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises a heavy chain variable region VH of an antibody capable of binding to the PD-L1 protein, a heavy chain variable region VH capable of binding to the TIGIT protein, and a light chain variable region VL capable of binding to the TIGIT protein, and the second polypeptide chain comprises a light chain variable region VL of an antibody capable of binding to the PD-L1 protein.

5. The multispecific antibody according to claim 4, characterized in that, in the first polypeptide chain, VH of the antibody capable of binding to the TIGIT protein and VL of the antibody capable of binding to the TIGIT protein constitute scFv.

6. The multispecific antibody according to claim 5, characterized in that the scFv sequence includes the amino acid sequence shown in any one of SEQ ID NO: 97-99.

7. The multispecific antibody according to claim 4, characterized in that the first polypeptide chain is selected from any one of the amino acid sequences of SEQ ID NO: 85-87.

8. The multispecific antibody according to claim 4, characterized in that the second polypeptide chain contains the amino acid sequence shown in SEQ ID NO:

88.

9. The multispecific antibody according to claim 1, comprising a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises a heavy chain variable region VH of an antibody capable of binding to the TIGIT protein, a heavy chain variable region VH capable of binding to the PD-L1 protein, and a light chain variable region VL capable of binding to the PD-L1 protein, and the second polypeptide chain comprises a light chain variable region VL of an antibody capable of binding to the TIGIT protein.

10. The multispecific antibody according to claim 9, characterized in that, in the first polypeptide chain, VH of the antibody capable of binding to the PD-L1 protein and VL of the antibody capable of binding to the PD-L1 protein constitute scFv.

11. The multispecific antibody according to claim 10, characterized in that the scFv sequence includes the amino acid sequence shown in any one of SEQ ID NO: 89-96.

12. The multispecific antibody according to claim 9, characterized in that the first polypeptide chain is selected from any one of the amino acid sequences: SEQ ID NO: 78-83.

13. The multispecific antibody according to claim 9, characterized in that the second polypeptide chain contains the amino acid sequence shown in SEQ ID NO:

84.

14. One or more nucleic acid molecules characterized by encoding a multispecific antibody as described in any one of claims 1 to 13.

15. A vector comprising the nucleic acid molecule described in claim 14.

16. A cell comprising the nucleic acid molecule described in claim 14 or the vector described in claim 15.

17. A pharmaceutical composition comprising a multispecific antibody according to any one of claims 1 to 13, a nucleic acid molecule according to claim 14, a vector according to claim 15, and / or a cell according to claim 16.

18. A pharmaceutical composition according to claim 17, used for the prevention, remission, and / or treatment of a disease or symptom.

19. The pharmaceutical composition according to claim 18, characterized in that the disease or symptom includes TIGIT-related diseases.

20. The pharmaceutical composition according to claim 19, characterized in that the TIGIT-related disease is a T-cell dysfunction disorder.

21. The pharmaceutical composition according to claim 18, characterized in that the disease or symptom is colon cancer.

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