Tigit-binding molecule and use thereof

Abstract

Provided in the present disclosure are a TIGIT-binding molecule and use thereof, relating to the field of biotechnology. The TIGIT-binding molecule is capable of specifically binding to TIGIT, such as human and / or cynomolgus monkey TIGIT, thereby providing a new possibility for the treatment and / or prevention of tumors.

Description

TIGIT-bound molecules and their applications

[0001] Priority Statement

[0002] This application claims priority to Chinese Patent Application No. 202411680910.9, filed on November 21, 2024, entitled "TIGIT-binding molecule and its application", the entire contents of which are incorporated herein by reference. Technical Field

[0003] This disclosure relates to the field of biotechnology, and in particular to a TIGIT-binding molecule and its applications. Background Technology

[0004] The following statements are provided only as background information in relation to the present invention and do not necessarily constitute prior art.

[0005] TIGIT (T Cell Immunoreceptor with Ig and ITIM Domains) is an emerging immune checkpoint molecule that plays a crucial role in regulating immune responses. TIGIT is primarily expressed on the surface of activated CD8+ T cells, CD4+ T cells, and natural killer (NK) cells. TIGIT ligands include CD155 (PVR), CD112 (PVRL2), and CD113, which are also shared by other receptors such as CD226, CD112R, and CD96.

[0006] TIGIT and its ligand CD112 are highly expressed in various tumors and are associated with poor prognosis. TIGIT, through ligand binding, transmits inhibitory signals, thereby suppressing the function of immune cells and affecting the intensity and duration of the immune response. Studies have found that TIGIT plays a significant role in the tumor immune microenvironment. Tumor cells can utilize the TIGIT signaling pathway to evade the immune system. In various tumor types, such as lung cancer and colorectal cancer, TIGIT expression levels are often elevated. As an immune checkpoint, TIGIT plays a crucial role in tumor immunotherapy. Developing molecules that specifically bind to TIGIT is beneficial for blocking the functions of TIGIT and its ligands in tumor treatment and facilitates further research into the mechanism of TIGIT's action.

[0007] In view of this, the present invention is hereby proposed. Summary of the Invention

[0008] The purpose of this disclosure is to provide a TIGIT-binding molecule and its applications.

[0009] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0010] In a first aspect, a TIGIT binding molecule is provided, the TIGIT binding molecule containing an antigen-binding domain, said antigen-binding domain containing complementarity-determining regions HCDR1, HCDR2 and HCDR3 of the antibody heavy chain variable region.

[0011] In an optional embodiment, the complementarity-determining region HCDR1 includes the amino acid residue RY; the complementarity-determining region HCDR2 includes the amino acid sequence shown in SEQ ID NO.11; and the complementarity-determining region HCDR3 includes the amino acid sequence shown in SEQ ID NO.19.

[0012] In an optional implementation, the complementary determination regions HCDR1, HCDR2 and HCDR3 are selected from the heavy chain variable regions HCDR1, HCDR2 and HCDR3 shown in any one of SEQ ID NO.1 to 5.

[0013] In a second aspect, an anti-TIGIT antibody or its antigen-binding fragment is provided, which competitively binds to TIGIT with the TIGIT-binding molecule of the first aspect, or the epitope of the anti-TIGIT antigen bound by the anti-TIGIT antibody or its antigen-binding fragment is the same as the epitope of the TIGIT-binding molecule-binding fragment of the first aspect.

[0014] Thirdly, a biomaterial is provided, comprising any one of the following (i) to (vi):

[0015] (i) A multispecific antibody containing at least two antigen-binding domains, wherein one antigen-binding domain is selected from the antigen-binding domain of the TIGIT binding molecule described in the first aspect, or a variable domain of the anti-TIGIT antibody or its antigen-binding fragment described in the second aspect.

[0016] (ii) A conjugate comprising connected (Ⅰ) and (Ⅱ):

[0017] (I) the TIGIT binding molecule described in the first aspect, or the anti-TIGIT antibody or its antigen-binding fragment described in the second aspect, or (i) the multispecific antibody described.

[0018] (II) A functional molecule, said functional molecule comprising a drug and / or a signaling agent;

[0019] (iii) A fusion protein comprising at least two domains, one of which contains the antigen-binding domain of the TIGIT binding molecule described in the first aspect.

[0020] Optionally, the fusion protein includes a chimeric antigen receptor, the chimeric antigen receptor including an extracellular region, the extracellular region including the antigen-binding domain of the TIGIT binding molecule described in the first aspect;

[0021] (iv) A nucleic acid molecule that encodes the TIGIT binding molecule of the first aspect, or the anti-TIGIT antibody or its antigen-binding fragment of the second aspect, or the multispecific antibody of (i), or the fusion protein of (iii);

[0022] (v) A vector carrying the nucleic acid molecule described in (iv);

[0023] (vi) A recombinant cell comprising the nucleic acid molecule described in (iv), or the vector described in (v), or expressing the TIGIT binding molecule described in the first aspect, or expressing the anti-TIGIT antibody or its antigen-binding fragment described in the second aspect, or expressing the multispecific antibody described in (i), or expressing the fusion protein described in (iii).

[0024] (vii) An engineered immune effector cell that expresses the chimeric antigen receptor described in (iii) or contains a nucleic acid molecule encoding the chimeric antigen receptor described in (iv).

[0025] Fourthly, the application of the TIGIT binding molecule described in the first aspect, or the anti-TIGIT antibody or its antigen-binding fragment described in the second aspect, or the multispecific antibody, conjugate, fusion protein, nucleic acid molecule, vector, recombinant cell or engineered immune effector cell described in the third aspect, in any of the following:

[0026] (I) Detect TIGIT or cells expressing TIGIT;

[0027] (II) Prepare products for detecting TIGIT or cells expressing TIGIT;

[0028] (III) Activate immune cells in the tumor immune microenvironment;

[0029] (IV) To prepare products for the treatment, prevention or relief of tumor-related diseases, symptoms or conditions;

[0030] (V) To treat, prevent or alleviate diseases, symptoms or conditions associated with tumors.

[0031] Fifthly, a pharmaceutical composition is provided, the pharmaceutical composition comprising the TIGIT binding molecule of the first aspect, or the anti-TIGIT antibody or its antigen-binding fragment of the second aspect, or the multispecific antibody, conjugate, fusion protein, nucleic acid molecule, carrier, recombinant cell or engineered immune effector cell of the third aspect.

[0032] A sixth aspect provides a method for treating, preventing, or alleviating a disease, symptom, or condition, the method comprising administering to a subject a therapeutically effective amount of the TIGIT-binding molecule of the first aspect, or the anti-TIGIT antibody or its antigen-binding fragment of the second aspect, or the multispecific antibody, conjugate, fusion protein, nucleic acid molecule, vector, recombinant cell, or engineered immune effector cell of the third aspect, or the pharmaceutical composition of the fifth aspect.

[0033] In a seventh aspect, a kit for detecting TIGIT is provided, the kit comprising the TIGIT binding molecule described in the first aspect, or the anti-TIGIT antibody or its antigen-binding fragment described in the second aspect.

[0034] Eighthly, a method for preparing the TIGIT binding molecule described in the first aspect is also provided, the method comprising culturing recombinant cells as described in the third aspect, and then isolating and purifying the TIGIT binding molecule. Attached Figure Description

[0035] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0036] Figure 1 shows the results of the binding activity detection of TIGIT nanobody R3179 and CHO-hTIGIT in Example 2;

[0037] Figure 2 shows the results of the binding activity detection of TIGIT nanobody R3179 and CHO-cynoTIGIT in Example 2;

[0038] Figure 3 shows the detection results of the activity of TIGIT nanobody R3179 in T cells activated by the luciferase reporter gene method in Example 2;

[0039] Figure 4 shows the detection results of the activity of humanized TIGIT nanobody activated T cells by the luciferase reporter gene method in Example 3.

[0040] Figure 5 shows the results of the TIGIT nanobody binding activity assay with CHO-hTIGIT in Example 3;

[0041] Figure 6 shows the results of the binding activity detection of TIGIT nanobody and CHO-cynoTIGIT in Example 3. Detailed Implementation

[0042] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0043] In this article, the articles “a / an,” “an,” and “the” include plural references unless the context clearly indicates otherwise. For example, “an antibody” refers to one or more antibodies.

[0044] In this document, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this disclosure, "a plurality of" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0045] In this article, the term "TIGIT" refers to T cell immunoglobulin with Ig and ITIM domains, also known as WUCAM, Vstm3, and VSIG9. It is a transmembrane glycoprotein containing an Ig-like V-type domain and an immunoreceptor tyrosine inhibitory motif (ITIM) in the cytoplasmic domain. TIGIT ligands include CD155 (PVR), CD112 (PVRL2), and CD113, and it is primarily expressed on natural killer (NK) cells, regulatory T cells (Tregs), and activated T cells.

[0046] Unless otherwise stated, TIGIT includes human TIGIT, subtypes, and species homologs expressed by cells naturally expressed or transfected with the TIGIT gene. The term also includes artificially modified TIGIT, including but not limited to mutated, truncated, or fused peptides or proteins with other domains, while retaining the necessary antigenic epitopes for antibody binding.

[0047] In this document, TIGIT-binding molecules refer to any molecule capable of binding to the TIGIT protein or its epitopes. TIGIT-binding molecules may include antibodies against TIGIT as defined herein, their antigen-binding fragments, or conjugates thereof. TIGIT-binding molecules also encompass immunoglobulin superfamily antibodies (IgSF) or CDR transplantation molecules.

[0048] In this document, a "domain" refers to a specific region in a molecule that folds into a relatively independent structural unit in three-dimensional space, possessing specific functions and stability. A domain typically consists of one or more continuous portions of a peptide chain. These portions may not be continuous in the amino acid sequence of a protein, but they are close to each other in the three-dimensional structure, folding into a relatively independent structural unit in three-dimensional space. Generally, a domain is responsible for a single functional property and, in many cases, can be added to, removed from, or transferred to other molecules without loss of the function of the rest of the molecule and / or the domain itself. In this document, an antigen-binding domain refers to a domain in a molecule used for binding a target protein. The "TIGIT-binding molecule" of this disclosure may contain at least one TIGIT-binding antigen-binding domain. The TIGIT-binding antigen-binding domain may be a variable domain (variable region) of an antibody or a variant thereof, such as the heavy chain variable region (VH) or light chain variable region (VL) of an antibody or variants thereof. The TIGIT-binding molecule contains at least one antigen-binding domain. In some embodiments, the "TIGIT-binding molecule" may contain 1, 2, 3, 4, or more TIGIT-binding antigen-binding domains. In addition to the portion for binding TIGIT, TIGIT-binding molecules may also include portions that perform other functions, such as a half-life extension portion (e.g., an immunoglobulin single variable domain binding serum albumin) and / or a fusion partner (e.g., serum albumin) and / or a conjugated polymer (e.g., PEG) and / or an Fc region.

[0049] In this document, the term "specific binding" or "specifically bound" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and an antigen. In some specific embodiments, for example, it is determined according to flow cytometry fluorescence sorting technology.

[0050] In this document, the term "antibody" includes any immunoglobulin capable of binding to a specific antigen. The term "antibody" is used in the broadest sense to encompass a wide range of antibody structures, including but not limited to monoclonal / polyclonal antibodies, monospecific / multispecific antibodies, full-length antibodies, nanobodies, and antigen-binding fragments, as long as they exhibit the desired antigen-binding activity. Typically, a natural, complete antibody contains two heavy (H) chains and two light (L) chains. Based on the presence or absence of α, δ, ε, γ, and μ heavy chains, antibodies can be classified into five main categories or isotypes: IgA, IgD, IgE, IgG, and IgM. Several major antibody categories can also be subdivided into subclasses, such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain), or IgA2 (α2 heavy chain), etc. Each heavy chain consists of a variable region (heavy chain variable region, VH) and first, second, third, and fourth (optionally) constant regions (CH1, CH2, CH3, CH4, respectively). Mammalian light chains can be divided into λ or κ, and each light chain consists of a variable region (light chain variable region, VL) and a constant region (CL). The variable regions of the light and heavy chains determine antigen binding. Each chain's variable region typically contains three hypervariable regions called "complementarity-determining regions (CDRs)," where the light chain CDRs include LCDR1, LCDR2, and LCDR3, and the heavy chain CDRs include HCDR1, HCDR2, and HCDR3. The variable regions (VH and VL) are each composed of three complementarity-determining regions connected by four framework regions (FRs). Typically, the variable regions VL / VH of the heavy and light chains are obtained by connecting the following numbered CDRs and FRs in the following combination: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. It should be noted that the heavy chain variable region in this article refers to the variable region from any structural form of antibody heavy chain, such as the heavy chain variable region of nanobodies (single-domain antibodies) and the heavy chain variable region of single-chain antibodies, and not just the heavy chain variable region of conventional antibodies.

[0051] In this article, the term "nanobody" is also referred to as "single-domain antibody," which refers to a heavy chain antibody lacking the light chain (e.g., derived from camels or sharks). The single-domain antibody obtained by cloning its variable region is the smallest functional antigen-binding fragment, with a relative molecular mass (Mr) of only about 15,000. Nanobodies are characterized by their small molecular weight, high stability, good solubility, ease of expression, and low immunogenicity.

[0052] In this document, the term "antigen-binding fragment" refers to a substance containing all or part of the antibody's CDR (Cellular Dependent Ratio), lacking at least some amino acids present in the full-length chain but still capable of specifically binding to an antigen. Such fragments are biologically active because they bind to the target antigen and can compete with other antigen-binding molecules (including intact antibodies) for binding to a given epitope. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized bifunctional antibodies (ds diabody), single-chain antibody molecules (scFv), and scFv dimers (bivalent bifunctional antibodies). The aforementioned antigen-binding fragments can bind to the same antigen as the parent antibody.

[0053] In this document, the term "anti-TIGIT antibody" refers to an antibody capable of specifically binding to TIGIT. In some specific embodiments, "anti-TIGIT antibody" refers to an antibody that specifically binds to human or monkey TIGIT.

[0054] In this paper, the term "Fab" in antibody refers to a portion of an antibody composed of a single light chain (including variable and constant regions) and a single heavy chain whose variable and first constant regions are linked by disulfide bonds. A "Fab' fragment" refers to a Fab fragment containing a portion of the hinge region. "F(ab')2" refers to a Fab' dimer. An "Fv fragment" is composed of the variable regions of a single light chain and / or a single heavy chain. A "single-chain Fv antibody" or "scFv" refers to an antibody fragment formed by the direct interconnection of light chain variable regions and heavy chain variable regions, or by linkage through peptide linker sequences. A "minimum recognition unit" refers to a structure containing only a single CDR within the variable region; although the minimum recognition unit has a small molecular weight and low affinity, it possesses the ability to bind to antigens.

[0055] The CDR boundaries of antibodies or their antigen-binding fragments described herein can be defined or identified according to the definitions of IMGT, Kabat, Chothia, AbM, and Contact. CDRs defined in other ways acceptable in the art are also within the scope of protection of this disclosure (Kaas, Q et al. IMGT unique numbering for immunoglobulin and T cell receptor constant domains and Ig superfamily C-like domains. Dev. Comp. Immunol. 29, 185-203, (2005); RM MacCallum et al., Antibody-antigen interactions: contact analysis and binding site topography J. Mol. Biol. (1996); Martin, ACR Protein sequence and structure analysis of antibody variable domains (Book chapter). In Antibody engineering lab manual Eds. Duebel, S. and Kontermann, R. (2001); Marie-Paule Lefranc et al. IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like). domains,Developmental and Comparative Immunology 27(2003)55–77).

[0056] In this paper, the terms “variant Fc region”, “Fc mutant”, “Fc region carrying mutation”, “mutant Fc region”, “Fc region variant”, “Fc variant”, “variant Fc region” and “mutant Fc region” are used interchangeably to refer to an Fc region that contains at least one amino acid modification and is distinguished from the natural sequence Fc region / wild-type Fc region.

[0057] In this article, the term "amino acid" refers to naturally occurring amino acids and synthetic amino acids, as well as amino acid analogs and amino acid mimics that function in a similar manner to naturally occurring amino acids. Naturally occurring amino acids include amino acids encoded by the genetic code and their modified forms, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Common natural amino acids include: alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine ​​(Cys; C); glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G); histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V). Amino acid analogs are compounds that have the same basic chemical structure as naturally occurring amino acids (i.e., the α-carbon bound to hydrogen, carboxyl, amino, and R groups), such as homoserine, ortholeucine, methionine sulfoxide, and methionine methylsulfonium. Amino acid analogs typically have modified R groups (e.g., ortholeucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimics are chemical compounds that have a structure different from the general chemical structure of amino acids, but function in a similar manner to naturally occurring amino acids.

[0058] In this document, the term "identity" percentage refers to the degree to which the amino acids of two polypeptides are identical at equivalent positions when two sequences are optimally aligned. The alignment of amino acid sequence identity percentages can be performed using various methods within the art, such as software well-known in the field, including BLAST, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, or CLUSTAL OMEGA. Those skilled in the art can determine appropriate parameters for the aligned sequences, including any algorithms required to achieve maximum alignment of the full length of the compared sequences.

[0059] In this document, the term "affinity" or "affinity" refers to the strength of the non-covalent interaction between an immunoglobulin molecule (i.e., an antibody) or a fragment thereof and an antigen. The strength or affinity of an immune-binding interaction can be expressed as the equilibrium dissociation constant (KD) of the interaction, where a smaller KD value indicates higher affinity. KD can be determined using any conventional method known in the art, including but not limited to the Biacore assay, the Octet method, microthermophoresis, HPLC MS, and flow cytometry fluorescence sorting.

[0060] The binding of the nanobodies, antibodies, or any antigen-binding fragments provided in this disclosure to TIGIT can also be described using the term "half-maximum effective concentration (EC50)," which refers to the concentration of the drug or antibody that achieves 50% of the maximum biological effect after a specific exposure time. Generally, a lower EC50 indicates better affinity, meaning that binding to the target protein can occur at a lower concentration. The EC50 value can be determined using binding detection methods known in the art, such as direct or indirect binding detection methods (e.g., enzyme-linked immunosorbent assay (ELISA), flow cytometry, and other binding detection methods).

[0061] In this document, the term "epitaxy" refers to any antigenic determinant on an antigen that is bound to the complementary site of an antibody. An antigenic determinant is typically a specific chemical group with a defined composition and structure. Epitopes can be linear (i.e., continuous) or conformational (i.e., consisting of spaced-apart amino acid residues, discontinuous). Epitopes define the minimum binding site of an antibody and are therefore specific targets for antibodies or their antigen-binding fragments. Epitopes can be determined by any method well known in the art, such as conventional immunoassays, antibody competitive binding assays, or X-ray crystallography or related structural assays (e.g., nuclear magnetic resonance spectroscopy).

[0062] In this article, "humanization" refers to the modification of antibodies to reduce the adverse immune responses of non-human antibodies (such as camel-derived antibodies). Humanized antibodies mainly refer to antibodies produced by modifying non-human antibodies using gene cloning and DNA recombination technologies and re-expressing them. In this humanization process, some amino acid sequences are replaced with sequences closer to human sequences, while largely retaining the affinity and specificity of the parent antibody and reducing its heterology, which is beneficial for its application in humans.

[0063] In this article, "affinity maturation" refers to the phenomenon in humoral immunity where the average affinity of antibodies produced in a secondary immune response is higher than that of the primary immune response. This process involves antibody gene rearrangement in B cells and high-frequency mutations in the hypervariable region CDR, resulting in the production of B lymphocytes that secrete antibodies with different affinities. "Affinity maturation" modification of antibodies refers to achieving antibody "affinity maturation" through mutation and substitution, thereby increasing the affinity between the antibody and the antigen.

[0064] In this article, "antibody deamidation" refers to the spontaneous chemical degradation of asparagine residues in antibody molecules under certain conditions, transforming them into aspartic acid or isoaspartic acid (isoAsp). This degradation process may affect the stability, biological activity, and therapeutic efficacy of antibodies. "Reducing antibody deamidation" refers to modifying antibodies to minimize the occurrence of the above-mentioned situation (e.g., the degradation of asparagine residues into aspartic acid or isoaspartic acid).

[0065] In this paper, "antibody isomerization" mainly refers to conformational changes in aspartic acid residues within a specific sequence, such as the conversion of L-amino acids to D-amino acids, or the conversion of trans peptide bonds to cis peptide bonds. Such changes can affect antibody stability and binding affinity to antigens. "Reducing antibody isomerization" refers to modifying antibodies to minimize the occurrence of these changes (e.g., the conversion of aspartic acid residues from L-amino acids to D-amino acids).

[0066] In this article, the term "multispecific antibody" refers to an antibody molecule that can bind to multiple (two or more) different antigenic epitopes of the same antigen or multiple (two or more) different antigens.

[0067] In this paper, the term "conjugate" refers to a compound formed by the linkage of at least two molecules.

[0068] In this document, the term "signaling substance" refers to a substance that can provide a detectable signal that can be directly observed by the naked eye or detected by conventional instruments acceptable in the art. The signaling substance can provide a signal directly, such as color (e.g., colloidal gold, colored microspheres), fluorescence (fluorescent molecules), magnetism, radiation, or luminescence; or it can provide a signal indirectly through a reaction in which the signaling substance participates, such as catalyzing a specific substrate reaction to produce any of the above signals.

[0069] In this article, "fusion protein" refers to a recombinant protein with multiple functions expressed by linking two or more protein gene fragments from different sources together using genetic engineering techniques; or a recombinant protein whose function has been improved by fusing a tag or a fusion protein that can form a multimer domain.

[0070] In this article, the "Chimeric Antigen Receptor (CAR)" is an artificial receptor that mimics the function of the TCR. It consists of an extracellular domain, a transmembrane domain, and an intracellular signaling domain connected sequentially. When the antigen (receptor) on the surface of tumor cells binds to the antibody (ligand) of the chimeric antigen receptor, the signal is transmitted into the cell through the hinge region and the transmembrane region. The intracellular signaling domain then converts the signal into an activation signal, activating effector cells. Effector cells kill tumor cells by secreting perforin or producing cytokines. At the same time, the effector cells themselves also proliferate, further amplifying the immune killing effect. The extracellular domain is generally composed of a single-chain variable fragment (scFv) of the monoclonal antibody responsible for recognizing and binding the antigen, and a hinge region that acts as a connector. The single-chain variable fragment is the antigen-binding domain of the CAR, which determines the specificity and function of the CAR-immune cell. The hinge region is the extracellular domain of the CAR, connecting the single-stranded variable fragment and the transmembrane domain. It typically maintains the stability required for robust CAR expression and activity in effector cells. Most CAR hinge regions are derived from the hinge of IgG or the extracellular region of CD8α / CD28. The type and length of the hinge region significantly influence CAR function. The transmembrane domain connects the extracellular domain of the CAR to the intracellular signal transduction domain. Commonly used transmembrane domains are derived from CD4, CD8, CD28, and CD3ζ or their derivatives. The choice of transmembrane domain affects the degree of CAR activation in cellular function. The intracellular domain consists of a co-stimulatory domain and a signal transduction domain. The co-stimulatory domain is usually derived from the CD28 receptor family (CD28, ICOS) or the tumor necrosis factor receptor family (4-1BB, OX40, CD27). The signal transduction domain is usually the T-cell receptor TCR / CD3ζ chain or the immunoglobulin Fc receptor FcεR Iγ chain. The CARs disclosed herein (including their functional portions and functional variants) can be obtained by methods known in the art, such as any suitable method for preparing peptides or proteins.

[0071] In this document, the term "engineered immune effector cell" refers to immune effector cells expressing CAR or CAR-modified immune effector cells. "Immune effector cell" generally refers to a cell that participates in an immune response, such as promoting an immune effector response. Exemplary immune effector cells include, but are not limited to, T cells (e.g., α / β T cells and γ / δ T cells), natural killer cells (NK cells), monocytes, macrophages, NKT cells (natural killer T cells), dendritic cells, granulocytes, B cells, lymphocytes, leukocytes, and / or peripheral blood mononuclear cells.

[0072] In this document, the term "nucleic acid molecule" refers to a polymeric form of nucleotides of any length, including ribonucleotides and / or deoxyribonucleotides. Examples of nucleic acid molecules include, but are not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other naturally occurring, chemically or biochemically modified, non-natural, or derived nucleotide bases. When a nucleic acid molecule encodes a protein or polypeptide, it may optionally encode either the sense or antisense strand. Nucleic acid molecules can be naturally occurring, synthetic, recombinant, or any combination thereof. The terms "nucleic acid molecule," "nucleic acid," and "polynucleotide" are used interchangeably.

[0073] In optional embodiments, the nucleic acid molecule is RNA or DNA, and can be single-stranded or double-stranded, preferably double-stranded DNA. When a nucleic acid molecule is placed in a functional relationship with another nucleic acid sequence, the nucleic acid molecule is "effectively linked." For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is effectively linked to said coding sequence. DNA is preferably used when it is ligated into a vector.

[0074] In this document, the term "vector" refers to a delivery system that can operatively insert a genetic element (such as the aforementioned nucleic acid molecule) into and express that genetic element, for example, to produce a protein, RNA, or DNA encoded by that genetic element, or to replicate the genetic element. Vectors can be used to transform, transduce, or transfect host cells, enabling the expression of the genetic element they carry within the host cells. Examples of vectors include: plasmids, episome plasmids, microcircular DNA, phage particles, cosmids, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC), bacteriophages such as λ phage or M13 phage, and animal viruses. Vectors may contain various elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, vectors may contain a replication initiation site. Vectors may also include components that facilitate their entry into the cell, including but not limited to viral particles, liposomes, or protein coats. Vectors can be expression vectors or cloning vectors. In some embodiments, the vectors (e.g., expression vectors) provided in this disclosure contain a nucleic acid sequence encoding an antibody or an antigen-binding fragment thereof as described in this disclosure, at least one promoter operatively linked to the nucleic acid sequence (e.g., SV40, CMV, EF1α), and at least one selection marker.

[0075] In this document, the terms “purified” or “isolated” associated with peptides or nucleic acids mean that the peptide or nucleic acid is not in its native medium or in its native form. Therefore, the term “isolated” includes peptides or nucleic acids removed from their original environment, such as if they are naturally occurring. Associated with nucleic acids, the terms “isolated” or “purified” indicate, for example, that the nucleic acid is not in its native genomic background (e.g., in a vector, as an expression cassette, linked to a promoter, or artificially introduced into a heterologous host cell).

[0076] In this document, the term "recombinant cell" refers to a cell into which exogenous polynucleotides and / or vectors can be or have been introduced. The exogenous polynucleotides may or may not be integrated into the genome of the "recombinant cell." Vectors can be introduced into cells to construct recombinant cells, which are then used to express antibodies or antigen-binding fragments provided in this disclosure. The corresponding antibodies can be obtained by culturing the recombinant cells. Recombinant cells can be prokaryotic or eukaryotic cells. Prokaryotic cells include, but are not limited to, *Escherichia coli*, *Bacillus*, or *Staphylococcus*. Eukaryotic cells include, but are not limited to, mammalian cells, insect cell lines, plant cells, and fungal cells. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, cattle, horse, and hamster cells, including but not limited to Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, young hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, and HEK-293 cells.Fungal cells include yeast and filamentous fungal cells, including, for example, *Pichia pastoris*, *Pichia finlandica*, *Pichia trehalophila*, *Pichia koclamae*, *Pichia membranaefaciens*, *Pichia minuta* (Ogataea minuta, *Pichia lindneri*), *Pichia puntiae*, *Pichia thermotolerans*, *Pichia salictaria*, *Pichia guercuum*, *Pichia pijperi*, *Pichia stiptis*, *Pichia methanolica*, *Pichia* genus, *Saccharomyces cerevisiae*, *Saccharomyces* genus, and *Hansenula*. The fungi include *Candida polymorpha*, *Kluyveromyces lactis*, *Candida albicans*, *Aspergillus nidulans*, *Aspergillus niger*, *Aspergillus oryzae*, *Trichoderma reesei*, *Chrysosporium lucknowense*, *Fusarium sp.*, *Fusarium gramineum*, *Fusarium venenatum*, *Physcomitrella patens*, and *Neurospora crassa*. Pichia, any Saccharomyces, Hansenula polymorpha, any Kluyveromyces, Candida albicans, any Aspergillus, Trichoderma reesei, Chrysosporium lucknowense, any Fusarium, Yarrowia lipolytica, and Neurospora crassa.

[0077] In this document, the term "pharmaceutical composition" refers to a form in which the biological activity of the active ingredient is permitted and which does not contain any additional ingredients that would have unacceptable toxicity to the subject to which the composition is administered. In some specific embodiments, the antibodies contained in or expressed in the pharmaceutical composition are capable of specifically targeting and binding to TIGIT.

[0078] In this article, "pharmaceutically acceptable carriers" can include any physiologically compatible solvent, dispersion medium, coating, antibacterial and antifungal agents, isotonic agents, and delayed absorption agents.

[0079] In this document, the terms "subject" or "patient" refer to a mammalian subject or patient. Exemplary subjects include, but are not limited to, humans, monkeys, dogs, cats, mice, rats, cattle, horses, camels, alpacas, poultry, goats, and sheep. In some embodiments, the subject is a human. In some embodiments, the subject is a person suspected of having cancer, an autoimmune disease or condition, and / or an infection.

[0080] In this document, the term "diagnosis" refers to the identification of a pathological state, disease, or condition, such as the identification of a TIGIT-related disease, or the identification of a subject with a TIGIT-related disease who may benefit from a specific treatment regimen. In some embodiments, diagnosis includes identifying an abnormal amount or activity of TIGIT. In some embodiments, diagnosis refers to identifying cancer in a subject.

[0081] In this document, the term "effective dose" refers to a therapeutic dose sufficient to reduce or improve the severity and / or duration of a condition or one or more of its symptoms; prevent disease progression; cause disease remission; prevent recurrence, development, or progression of one or more disease-related symptoms; detect disease; or enhance or improve the preventive or therapeutic effect of another therapy (e.g., a prophylactic or therapeutic agent). The therapeutically effective dose of the antibody or its antigen-binding fragment described in this disclosure depends on a variety of factors known in the art, such as weight, age, medical history, current treatment, the subject's health status and potential for cross-infection, allergies, hypersensitivity, and side effects, as well as the route of administration and the extent of tumor development. Those skilled in the art (e.g., physicians or veterinarians) may proportionally reduce or increase the dose based on these or other conditions or requirements.

[0082] In this document, the terms “treatment,” “relief,” or “improvement” are used interchangeably. These terms refer to methods of achieving beneficial or desired outcomes, including, but not limited to, treatment benefits. A “treatment benefit” means the eradication or improvement of one or more diseases, conditions, or circumstances associated with the underlying barrier being treated.

[0083] In this article, the terms “prevention” and “avoidance” are used interchangeably to refer to methods for obtaining beneficial or desired outcomes, including but not limited to preventive benefits. To obtain a “preventive benefit,” a drug may be given to a subject at risk of developing a specific disease, or to a subject who reports one or more physiological symptoms of a disease, even if a diagnosis of the disease may not have been made.

[0084] In a first aspect, a TIGIT binding molecule is provided, the TIGIT binding molecule containing an antigen-binding domain, wherein the antigen-binding domain of the TIGIT binding molecule contains complementarity-determining regions HCDR1, HCDR2 and HCDR3 of the heavy chain variable region.

[0085] In an optional embodiment, the complementarity-determining region HCDR1 includes the amino acid residue RY; the complementarity-determining region HCDR2 includes the amino acid sequence shown in SEQ ID NO.11; and the complementarity-determining region HCDR3 includes the amino acid sequence shown in SEQ ID NO.19.

[0086] In an optional implementation, the complementary determination regions HCDR1, HCDR2 and HCDR3 are selected from the heavy chain variable regions HCDR1, HCDR2 and HCDR3 shown in any one of SEQ ID NO.1 to 5.

[0087] In optional implementations, HCDR1, HCDR2, and HCDR3 are determined according to the Kabat definition method, Chothia definition method, AbM definition method, Contact definition method, or IMGT definition method.

[0088] In optional embodiments, the TIGIT binding molecules are defined as HCDR1, HCDR2, and HCDR3 according to the Kabat definition, Chothia definition, AbM definition, Contact definition, or IMGT definition, as shown in Table 1.

[0089] Table 1. CDR amino acid sequences of TIGIT-binding molecules defined according to different definitions.

[0090] In an optional embodiment, the antigen-binding domain contains a variable region of the antibody heavy chain, and the variable region of the antibody heavy chain contains the complementarity-determining regions HCDR1, HCDR2, and HCDR3.

[0091] In an optional embodiment, the heavy chain variable region is modified by at least one of the following: humanization, affinity maturation, removal of T cell epitopes, reduction of antibody deamidation, and reduction of antibody isomerization.

[0092] In an optional implementation, the heavy chain variable region includes a frame region.

[0093] In an optional implementation, the frame region is derived from at least one of camel-derived antibodies, mouse-derived antibodies, human-derived antibodies, primate-derived antibodies, or mutants thereof.

[0094] In an optional implementation, the frame region includes HFR1, HFR2, HFR3, and HFR4.

[0095] In an optional embodiment, the HFR1 comprises the following amino acid sequence:

[0096] Q-X1-QLVESGGG-X2-VQ-X3-G-X4-SLRLSCAAS(SEQ ID NO.66), where X1 is L or V, X2 is L or V, X3 is A or P, and X4 is D or R;

[0097] In an optional embodiment, the HFR2 comprises the following amino acid sequence:

[0098] WFRQAPGK-X5-X6-E (SEQ ID NO.67), where X5 is E or G, and X6 is H or L;

[0099] In an optional embodiment, the HFR3 comprises the following amino acid sequence:

[0100] RF-X7-ISR-X8-N-X9-KNTLYLQMN-X10-L-X11-X12-X13-DTAVYYC (SEQ ID NO.68), where X7 is I or T, X8 is V or D, X9 is P or S, X10 is N or S, X11 is K or R, X12 is P or A, and X13 is D or E;

[0101] In an optional embodiment, the HFR4 comprises the following amino acid sequence:

[0102] WGQGT-X14-VTVSS (SEQ ID NO.69), where X14 is either Q or L.

[0103] In an optional embodiment, the framework region of the TIGIT binding molecule is selected from any group of the following a to d:

[0104] a. The HFR1 comprises the amino acid sequence shown in SEQ ID NO. 20 or contains an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO. 20; and / or, the HFR2 comprises the amino acid sequence shown in SEQ ID NO. 28 or contains an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO. 28; and / or, the HFR3 comprises the amino acid sequence shown in SEQ ID NO. 43 or contains an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO. 43; and / or, the HFR4 comprises the amino acid sequence shown in SEQ ID NO. 62 or contains an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO. 62;

[0105] b. The HFR1 comprises the amino acid sequence shown in SEQ ID NO. 20 or contains an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO. 20; and / or, the HFR2 comprises the amino acid sequence shown in SEQ ID NO. 29 or contains an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO. 29; and / or, the HFR3 comprises the amino acid sequence shown in SEQ ID NO. 42 or contains an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO. 42; and / or, the HFR4 comprises the amino acid sequence shown in SEQ ID NO. 62 or contains an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO. 62.

[0106] c. The HFR1 comprises the amino acid sequence shown in SEQ ID NO. 23 or contains an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO. 23; and / or, the HFR2 comprises the amino acid sequence shown in SEQ ID NO. 32 or 36 or contains an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO. 32 or 36; and / or, the HFR3 comprises the amino acid sequence shown in SEQ ID NO. 49, 55 or 61 or contains an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO. 49, 55 or 61; and / or, the HFR4 comprises the amino acid sequence shown in SEQ ID NO. 64 or contains an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO. 64;

[0107] d. The HFR1 comprises the amino acid sequence shown in SEQ ID NO. 23 or contains an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO. 23; and / or, the HFR2 comprises the amino acid sequence shown in SEQ ID NO. 33 or 37 or contains an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO. 37; and / or, the HFR3 comprises the amino acid sequence shown in SEQ ID NO. 48, 54 or 60 or contains an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO. 48, 54 or 60; and / or, the HFR4 comprises the amino acid sequence shown in SEQ ID NO. 64 or contains an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO. 64.

[0108] In an optional embodiment, the HFR1, HFR2, HFR3 and HFR4 of the frame region are selected from the HFR1, HFR2, HFR3 and HFR4 of the heavy chain variable region shown in any one of SEQ ID NO.1 to 5.

[0109] In an optional embodiment, the HFR1, HFR2, HFR3 and HFR4 of the frame region are selected from the HFR1, HFR2, HFR3 and HFR4 of the heavy chain variable region shown in any one of SEQ ID NO.2 to 5 after humanization modification.

[0110] Table 2 shows the HFR1, HFR2, HFR3, and HFR4 of the camel-derived antibody (example antibody R3179) defined according to the Kabat, Chothia, AbM, Contact, or IMGT definitions, respectively.

[0111] Table 2 shows the FR amino acid sequences of example antibody R3179 defined according to different definitions.

[0112] The HFR1, HFR2, HFR3, and HFR4 of the heavy chain variable regions of humanized antibodies (example antibodies R3693, R3694, R3695, and R3696) defined by Kabat, Chothia, AbM, Contact, or IMGT are shown in Tables 3-6.

[0113] Table 3 shows the FR amino acid sequences of example antibody R3693 defined according to different definitions.

[0114] Table 4 shows the FR amino acid sequences of example antibody R3694 defined according to different definitions.

[0115] Table 5 shows the FR amino acid sequences of example antibody R3695 defined according to different definitions.

[0116] Table 6 shows the FR amino acid sequences of example antibody R3696 defined according to different definitions.

[0117] In an optional embodiment, the antigen-binding domain contains a heavy chain variable region having an amino acid sequence shown in any one of SEQ ID NO. 1 to 5 or a heavy chain variable region having at least 90% (e.g., 90%, 91%, or 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with an amino acid sequence shown in any one of SEQ ID NO. 1 to 5.

[0118] In an optional embodiment, the antigen-binding domain contains a heavy chain variable region having an amino acid sequence shown in any one of SEQ ID NO. 2 to 5 or a heavy chain variable region having at least 90% sequence identity with an amino acid sequence shown in any one of SEQ ID NO. 2 to 5.

[0119] In an optional embodiment, the TIGIT binding molecule further contains part or all of the sequence of the constant region of the antibody.

[0120] In an optional implementation, the constant region is derived from at least one of camel-derived antibodies, mouse-derived antibodies, human-derived antibodies, primate-derived antibodies, or mutants thereof.

[0121] In an optional embodiment, the constant region contains a portion or all of the sequence of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD. The IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD include their subclasses and mutant forms.

[0122] In an optional embodiment, the TIGIT binding molecule contains all or part of the heavy chain constant region sequence.

[0123] In an optional embodiment, the TIGIT binding molecule contains one or more of CH1, CH2, CH3 and CH4 in the heavy chain constant region.

[0124] In an optional embodiment, the TIGIT binding molecule further contains the Fc region of the antibody.

[0125] In an optional implementation, the Fc region is derived from IgG1, IgG2, IgG3 or IgG4 antibodies.

[0126] In an optional embodiment, the TIGIT binding molecule contains the Fc region of IgG4.

[0127] In an optional implementation, the Fc region includes the Fc region of human-derived IgG4.

[0128] In an optional embodiment, the Fc region of the human-derived IgG4 contains one, two, three, or four mutations selected from 228P, 234A, 235A, and 447A.

[0129] In an optional embodiment, the TIGIT binding molecule contains the Fc region of human-derived IgG4 and contains 228P, 234A, 235A and 447A mutations.

[0130] In an optional embodiment, the TIGIT binding molecule further comprises complementary determinant regions LCDR1, LCDR2, and LCDR3 of the light chain variable region.

[0131] In an optional implementation, the complementary determinant region of the variable region of the light chain of the TIGIT-binding molecule targets TIGIT.

[0132] In an optional implementation, the complementary determinant region of the light chain variable region of the TIGIT binding molecule targets other antigens, such as immune checkpoints. Exemplary immune checkpoints include, but are not limited to, PVRIG, CTLA-4, BTLA, KIR, LAG3, 10VISTA, PD-L1, PD-1, TIM3, B7H3, B7H4, PD-L2, CD80, CD86, HVEM, LLT1, GAL9, GITR, OX40, CD137, and ICOS.

[0133] In an optional embodiment, the TIGIT binding molecule contains all or part of the sequence of the light chain constant region.

[0134] In an optional implementation, the light chain constant region is a κ or λ chain.

[0135] In an optional embodiment, the TIGIT binding molecule is an antibody or its antigen-binding fragment.

[0136] In an optional embodiment, the TIGIT binding molecule is one or more of the following: full-length antibody, nanobody, F(ab')2, Fab', Fab, Fv, scFv, dsFv, and the smallest antibody recognition unit.

[0137] In an optional embodiment, the TIGIT binding molecule is a nanobody.

[0138] In an optional embodiment, the TIGIT-binding molecule has one or more of the following properties (i) and (ii):

[0139] (i) EC50 of no more than 10 nM (e.g., no more than 10 nM, no more than 8 nM, no more than 5 nM, no more than 3 nM or no more than 2 nM) binds to human TIGIT or cynomolgus monkey TIGIT, and EC50 is determined by flow cytometry fluorescence sorting.

[0140] (ii) Activate T cells with EC50 of no more than 10 nM (e.g., no more than 10 nM, no more than 8 nM, no more than 5 nM, no more than 3 nM, no more than 2 nM or no more than 1 nM), which is determined using a luciferase reporter gene system.

[0141] The TIGIT binding molecule provided in the first aspect can specifically bind to TIGIT and TIGIT-expressing cells. In some optional embodiments, the antigen-binding domain that binds TIGIT is further humanized, providing new possibilities for the treatment and / or prevention of tumors.

[0142] In a second aspect, an anti-TIGIT antibody or its antigen-binding fragment is provided, which competitively binds to TIGIT with the TIGIT-binding molecule of the first aspect, or the epitope of the anti-TIGIT antigen bound by the anti-TIGIT antibody or its antigen-binding fragment is the same as the epitope of the TIGIT-binding molecule-binding fragment of the first aspect.

[0143] Thirdly, a biomaterial is provided, comprising any one of the following (i) to (vi):

[0144] (i) A multispecific antibody containing at least two antigen-binding domains, wherein one antigen-binding domain is selected from the antigen-binding domain of the TIGIT binding molecule described in the first aspect, or a variable domain of the anti-TIGIT antibody or its antigen-binding fragment described in the second aspect.

[0145] (ii) A conjugate comprising connected (Ⅰ) and (Ⅱ):

[0146] (I) the TIGIT binding molecule described in the first aspect, or the anti-TIGIT antibody or its antigen-binding fragment described in the second aspect, or (i) the multispecific antibody described.

[0147] (II) A functional molecule, said functional molecule comprising a drug and / or a signaling agent;

[0148] (iii) A fusion protein comprising at least two domains, one of which contains the antigen-binding domain of the TIGIT binding molecule described in the first aspect.

[0149] Optionally, the fusion protein includes a chimeric antigen receptor, the chimeric antigen receptor including an extracellular region, the extracellular region including the antigen-binding domain of the TIGIT binding molecule described in the first aspect;

[0150] (iv) A nucleic acid molecule that encodes the TIGIT binding molecule of the first aspect, or the anti-TIGIT antibody or its antigen-binding fragment of the second aspect, or the multispecific antibody of (i), or the fusion protein of (iii);

[0151] (v) A vector carrying the nucleic acid molecule described in (iv);

[0152] (vi) A recombinant cell comprising the nucleic acid molecule described in (iv), or the vector described in (v), or expressing the TIGIT binding molecule described in the first aspect, or expressing the anti-TIGIT antibody or its antigen-binding fragment described in the second aspect, or expressing the multispecific antibody described in (i), or expressing the fusion protein described in (iii).

[0153] (vii) An engineered immune effector cell that expresses the chimeric antigen receptor described in (iii) or contains a nucleic acid molecule encoding the chimeric antigen receptor described in (iv).

[0154] In an optional embodiment, the multispecific antibody further comprises at least one antigen-binding domain that specifically binds to tumor surface antigens, and / or at least one antigen-binding domain that specifically binds to immune checkpoints.

[0155] In optional embodiments, the tumor surface antigen includes, but is not limited to, one or more of MUC16, VEGFR-2 (KDR / FIK-1), K-RAS, CD2, CD3, CD19, CD20, CD21, CD22, CD30, CD32B, CD33, CD38, CD39, CD40, CD45, CD52, CD70, CD80, CD60, CD62, CD72, CD79a, CD79B, CD123, EGFR, HER2, HER3, HER4, GPC3, CLL1, TGF-β, TGF-beta RII, VEGF, GD3, CCR4, CCR5, BCMA, CTLA4, mesothelin, and OX40. In optional implementations, immune checkpoints include, but are not limited to, one or more of PD-1, PD-L1, PD-L2, PVRIG, CD47, CD27, CD28, CD40, CD122, CD137, CD94 / NKG2A, OX40, GITR, ICOS, A2AR, B7-H3, B7-H4, BTLA, LAG3, TIM-3, VISTA, GARP, PS, CSF1R, TDO, CTLA-4, IDO, KIR, GITR, TNFR, FasR / DcR, and CEACAM1.

[0156] In optional embodiments, the drug includes, but is not limited to, one or more of the following: chemotherapeutic agents, anticancer drugs, radiotherapy agents, immunomodulators, antiangiogenic agents, targeted therapy agents, cell therapy agents, gene therapy agents, hormone therapy agents, antiviral agents, antibiotics, analgesics, antioxidants, metal chelators, cytokines, toxins, radionuclides, boron atoms, anti-apoptotic agents, photosensitizing agents, and oligonucleotides.

[0157] In optional embodiments, the signaling agent includes, but is not limited to, one or more of the following: contrast agents, paramagnetic ions, metals, fluorescent labels, chemiluminescent labels, ultrasound contrast agents, enzymes, radioactive isotopes, nanoparticles, and photosensitizers.

[0158] In an optional embodiment, the fusion protein contains at least two domains, one of which contains the antigen-binding domain of the TIGIT binding molecule of the first aspect.

[0159] In an optional embodiment, the fusion protein includes a chimeric antigen receptor, the chimeric antigen receptor including an extracellular region, the extracellular region including an antigen-binding domain of the TIGIT binding molecule of the first aspect.

[0160] In an optional embodiment, the fusion protein includes an extracellular region of a chimeric antigen receptor containing a single-chain antibody scFv, wherein the heavy chain variable region has an antigen-binding domain of a first aspect of a TIGIT-binding molecule.

[0161] In an optional embodiment, the nucleic acid molecule encodes the TIGIT binding molecule described in the first aspect, or the anti-TIGIT antibody or its antigen-binding fragment described in the second aspect, or the multispecific antibody described in (i), or the fusion protein described in (iii).

[0162] In an optional embodiment, the nucleic acid molecule is an isolated nucleic acid molecule.

[0163] In an optional embodiment, the vector carries (iv) the nucleic acid molecule.

[0164] In an optional embodiment, the recombinant cell comprises the nucleic acid molecule described in (iv), or the vector described in (v), or expresses the TIGIT binding molecule described in the first aspect, or expresses the anti-TIGIT antibody or its antigen-binding fragment described in the second aspect, or expresses the multispecific antibody described in (i), or expresses the fusion protein described in (iii).

[0165] In an optional embodiment, the chimeric antigen receptor in the immune effector cell (iii) or a nucleic acid molecule encoding the chimeric antigen receptor described in (iv) is present.

[0166] Fourthly, the application of the TIGIT binding molecule described in the first aspect, or the anti-TIGIT antibody or its antigen-binding fragment described in the second aspect, or the multispecific antibody, conjugate, fusion protein, nucleic acid molecule, vector, recombinant cell or engineered immune effector cell described in the third aspect, in any of the following:

[0167] (I) Detect TIGIT or cells expressing TIGIT;

[0168] (II) Prepare products for detecting TIGIT or cells expressing TIGIT;

[0169] (III) Activate immune cells in the tumor immune microenvironment;

[0170] Optionally, the immune cells include T cells and / or NK cells;

[0171] (IV) To prepare products for the treatment, prevention or relief of tumor-related diseases, symptoms or conditions;

[0172] (V) To treat, prevent or alleviate diseases, symptoms or conditions associated with tumors.

[0173] In optional embodiments, the above applications can be based on immunoassay technology to detect TIGIT or TIGIT-expressing cells. For example, when TIGIT is an immunoconjugate, such as one linked to a fluorescent group, a fluorescence detection device can be used to locate or detect TIGIT in real time. For example, it can be used in immunoblotting, immunoprecipitation, or flow cytometry techniques that utilize the specific binding properties of TIGIT antigens and antibodies to detect TIGIT or TIGIT-expressing cells. Accordingly, in aspect (II) above, those skilled in the art can select the reagent composition in the kit according to the actual detection method, including but not limited to antagonists, anti-TIGIT antibodies or drug reference materials; protein purification columns; immunoglobulin affinity purification buffers; cell assay diluents; instructions or literature, etc. The kit can also be used to detect diseases, symptoms, or conditions related to TIGIT. In optional embodiments, the detection of TIGIT or TIGIT-expressing cells is not for diagnostic or therapeutic purposes. In optional embodiments, the purpose of activating immune cells in the tumor immune microenvironment is not for diagnostic or therapeutic purposes.

[0174] In an optional implementation, the purpose of activating immune cells in the tumor immune microenvironment is to prevent, treat, or alleviate diseases, symptoms, or conditions, including but not limited to tumors, such as solid tumors or hematologic malignancies.

[0175] Fifthly, a pharmaceutical composition is provided, the pharmaceutical composition comprising the TIGIT binding molecule of the first aspect, or the anti-TIGIT antibody or its antigen-binding fragment of the second aspect, or the multispecific antibody, conjugate, fusion protein, nucleic acid molecule, carrier, recombinant cell or engineered immune effector cell of the third aspect.

[0176] In an optional embodiment, the pharmaceutical composition may further include a pharmaceutically acceptable carrier and / or excipient.

[0177] In optional embodiments, the pharmaceutical composition further includes a pharmaceutically acceptable carrier and / or excipient. The acceptable carrier and pharmaceutically acceptable excipient may be any conventional carrier and / or excipient known in the art. Examples of carriers include, but are not limited to, any physiologically compatible solvent, dispersion medium, coating, antibacterial and antifungal agents, isotonic agents, and delayed absorption agents; examples of excipients include, but are not limited to, fillers, disintegrants, preservatives, solubilizers, and emulsifiers.

[0178] In an optional embodiment, the pharmaceutical composition may further include one or more pharmaceutically active ingredients having other therapeutic effects, such as the drugs exemplified in the fourth aspect: chemotherapeutic agents, anticancer drugs, radiotherapy agents, immunotherapy agents, antiangiogenic agents, targeted therapy agents, cell therapy agents, gene therapy agents, hormone therapy agents, antiviral agents, antibiotics, analgesics, antioxidants, metal chelators, and cytokines, or a combination of several of these.

[0179] In an optional embodiment, the pharmaceutical composition is used to treat, prevent, or alleviate tumor-related diseases, symptoms, or conditions.

[0180] A sixth aspect provides a method for treating, preventing, or alleviating a disease, symptom, or condition, the method comprising administering to a subject a therapeutically effective amount of the TIGIT-binding molecule of the first aspect, or the anti-TIGIT antibody or its antigen-binding fragment of the second aspect, or the multispecific antibody, conjugate, fusion protein, nucleic acid molecule, vector, recombinant cell, or engineered immune effector cell of the third aspect, or the pharmaceutical composition of the fifth aspect.

[0181] In an optional implementation, the disease, symptom, or condition includes tumor-related diseases, symptoms, or conditions.

[0182] In optional embodiments, the treatment, prevention, or mitigation of tumor-related diseases, symptoms, or conditions in any of the above embodiments is achieved by activating immune cells in the tumor immune microenvironment, for example by activating T cells and / or NK cells.

[0183] In optional embodiments, the tumor in any of the above embodiments includes solid tumors. Exemplary solid tumors include, but are not limited to, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, endothelial sarcoma, lymphangiosarcoma, angiosarcoma, lymphangioendothelial sarcoma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, and carcinoma. The term "tumor" includes, but is not limited to, one or more of the following: bronchial cancer, medullary carcinoma, renal cell carcinoma, liver cancer, Nile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, nephroblastoma, cervical cancer, uterine cancer, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, schwannoma, meningioma, melanoma, neuroblastoma, retinoblastoma, esophageal cancer, gallbladder cancer, kidney cancer, and multiple myeloma. Preferably, the term "tumor" includes, but is not limited to, one or more of the following: pancreatic cancer, liver cancer, lung cancer, gastric cancer, esophageal cancer, head and neck squamous cell carcinoma, prostate cancer, colon cancer, breast cancer, lymphoma, gallbladder cancer, kidney cancer, leukemia, multiple myeloma, ovarian cancer, cervical cancer, and glioma.

[0184] In optional embodiments, the tumor in any of the above embodiments includes hematologic malignancies, exemplary hematologic malignancies including but not limited to one or more of the following: chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML), B-cell malignancies, lymphomas (Hodgkin's lymphoma, non-Hodgkin's lymphoma, chronic lymphocytic leukemia, acute lymphoblastic leukemia, myeloma), and myeloproliferative disorders.

[0185] In optional embodiments, the therapeutically effective dose in any of the above embodiments is between about 0.001 mg / kg and about 1000 mg / kg. In some embodiments, the dosage may vary with the course of treatment. For example, in some embodiments, the initial dosage may be higher than subsequent dosages. In some embodiments, the dosage is adjusted during the treatment process based on the subject's response.

[0186] In optional embodiments, the pharmaceutical composition described in this disclosure may be administered alone or in combination with a therapeutically effective amount of a second therapeutic agent. For example, the antibody or antigen-binding fragment disclosed in this disclosure may be administered in combination with a second therapeutic agent (e.g., a drug exemplified in the fourth aspect).

[0187] In optional embodiments, when the pharmaceutical composition described in this disclosure is used in combination with one or more other therapeutic agents, it may be administered simultaneously with the one or more other therapeutic agents. In some such embodiments, the pharmaceutical composition and the other therapeutic agent may be administered simultaneously as part of the same composition. However, a pharmaceutical composition “used in combination” with another therapeutic agent does not need to be administered simultaneously or in the same composition as that therapeutic agent. The meaning of “used in combination” in this disclosure also includes a pharmaceutical composition administered before or after another therapeutic agent, which is also considered to be “used in combination” with that therapeutic agent, i.e., the pharmaceutical composition and the second substance are administered via different routes of administration.

[0188] In a seventh aspect, a kit for detecting TIGIT is provided, the kit comprising the TIGIT binding molecule described in the first aspect, or the anti-TIGIT antibody or its antigen-binding fragment described in the second aspect.

[0189] In optional embodiments, the detection reagent or kit further contains detection reagents, including but not limited to one or more of the following: buffer reagents, salts, secondary antibodies, chromogenic substrates, blocking solutions, washing solutions, solvents, elution solutions, coupling agents, negative controls, positive controls, standards, quality control materials, and signaling substances. Those skilled in the art can formulate other reagents in the reagent or kit according to the corresponding detection methods, and this disclosure does not limit this.

[0190] Eighthly, a method for preparing the TIGIT binding molecule described in the first aspect is also provided, the method comprising culturing recombinant cells as described in the third aspect, and then isolating and purifying the TIGIT binding molecule.

[0191] In an optional embodiment, the preparation method further includes converting and expressing a polynucleotide encoding the TIGIT binding molecule into cells.

[0192] In an optional embodiment, the preparation method further includes synthesizing a polynucleotide containing the gene encoding the TIGIT binding molecule as needed, and / or preparing a suitable expression vector as needed, transforming the expression vector into the desired cells and expressing it, and obtaining the TIGIT binding molecule through purification.

[0193] Exemplary radionuclides in any of the above embodiments include, but are not limited to, those mentioned above. 110 In、 111 In、 177 Lu、 18 F, 52 Fe、 62 Cu、 64 Cu、 67 Cu、 67 Ga、 68 Ga、86 Y、 90 Y、 89 Zr、 94 mTc, 94 Tc, 99 mTc, 120 I, 123 I, 124 I, 125 I, 131 I, 154-158 Gd, 32 P, 11 C 13 N、 15 O、 186 Re、 188 Re、 51 Mn, 52 mMn, 55 Co、 72 As、 75 Br、 76 Br、 82 mRb and 83 One or more of Sr.

[0194] Exemplary paramagnetic ions in any of the above embodiments include, but are not limited to, one or more of chromium (III), manganese (II), iron (III), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III), and erbium (III).

[0195] Exemplary fluorescent markers in any of the above embodiments include, but are not limited to, Alexa 350, Alexa 405, Alexa 430, Alexa 488, Alexa 555, Alexa 647, AMCA, aminoacridine, BODIPY 630 / 650, BODIPY 650 / 665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, 5-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein, 5-carboxy-2′,4′,5′,7′-tetrachlorofluorescein, 5-carboxyfluorescein, 5-carboxyrhodamine, 6-carboxyrhodamine, 6-carboxytetramethylrhodamine, and Cascade. Blue, Cy2, Cy3, Cy5, Cy7, 6-FAM, Dansyl chloride, Fluorescein, HEX, 6-JOE, NBD (7-nitrobenzo-2-oxa-1,3-diazole), Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, Phthalic acid, Terephthalic acid, Isophthalic acid, Cresol Violet, Cresol Blue Violet, Brilliant Cresol Blue, p-Aminobenzoic acid, Erythrosine, Phthalocyanine, Azocyanine, Anthocyanin, Xanthine, Succinyl fluorescein, Rare earth metal cavitation compounds, Tribispyridyldiamine europium, europium cavitation compounds or chelates, Diamine, Dianthocyanin, La Jolla Blue dye, Allococyanin B. Phycocyanin C. Phycocyanin R. Thiamine, Phycoerythrin, Phycoerythrin R. REG. Rhodamine Green, Rhodamine Isothiocyanate, Rhodamine Red, ROX, TAMRA, TET, TRIT (Tetramethylrhodamine isothiol), Tetramethylrhodamine, and Texas Red.

[0196] Exemplary oligonucleotides in any of the above embodiments include, but are not limited to, one or more of shRNA, miRNA, and siRNA.

[0197] Exemplary anticancer drugs in any of the above embodiments include, but are not limited to, methotrexate, fluorouracil, mercaptopurine, hydroxyurea, cytarabine, nitrogen mustard, cyclophosphamide, thiotepa, cisplatin, mitomycin, bleomycin, camptothecin, podophyllotoxin, actinomycin D, doxorubicin, daunorubicin, vincristine, paclitaxel, Cephalotaxine alkaloids, and L-asparaginase. Exemplary immunomodulators in any of the above embodiments include, but are not limited to, cytokines, chemokines, stem cell growth factors, lymphotoxins, hematopoietic factors, colony-stimulating factors (CSF), interferon, erythropoietin, thrombopoietin, tumor necrosis factor (TNF), interleukin (IL), granulocyte-colony-stimulating factor (G-CSF), granulocyte-macrophage-colony-stimulating factor (GM-CSF), and one or more of stem cell growth factors.

[0198] Exemplary radionuclides in any of the above embodiments include, but are not limited to, those mentioned above. 111 In、 111 At、 177 Lu、 211 Bi、 212 Bi、 213 Bi、 211 At、 62 Cu、 67 Cu、 90 Y、 125 I, 131 I, 133 I, 32 P, 33 P, 47 Sc、 111 Ag、 67 Ga、 153 Sm、 161 Tb, 152 Dy、 166 Dy、 161 Ho、 166 Ho、 186 Re、 188 Re、 189 Re、 211 Pb, 212 Pb, 223 Ra、 225 Ac、 77 As、 89 Sr、 99 Mo、 105 Rh、 149 Pm, 169 Er、 194 Ir、 58 Co、 80 mBr、 99 mTc, 103 mRh、109 Pt, 119 Sb, 189 mOs, 192 Ir、 219 Rn、 215 Po、 221 Fr、 255 Fm、 11 C 13 N、 15 O、 75 Br、 198 Au、 199 Au、 224 Ac、 77 Br、 113 mIn, 95 Ru、 97 Ru、 103 Ru、 105 Ru、 107 Hg, 203 Hg, 121 mTe, 122 mTe, 125 mTe, 165 Tm、 167 Tm、 168 Tm、 197 Pt, 109 Pd, 142 Pr、 143 Pr、 161 Tb, 57 Co、 58 Co、 51 Cr 59 Fe、 75 Se、 201 Tl、 76 Br and 169 One or more of Yb.

[0199] The exemplary enzymes in any of the above embodiments include, but are not limited to, any one of horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate dehydrogenase.

[0200] Exemplary radioactive isotopes in any of the above embodiments include, but are not limited to, those mentioned above. 212 Bi、 131 I, 111 In、 90 Y、 186 Re、 211 At、 125 I, 188 Re、 153 Sm、 213 Bi、32 P, 94 mTc, 99 mTc, 203 Pb, 67 Ga、 68 Ga、 43 Sc、 47 Sc、 110 mIn, 97 Ru、 62 Cu、 64 Cu、 67 Cu、 68 Cu、 86 Y、 88 Y、 121 Sn、 161 Tb, 166 Ho、 105 Rh、 177 Lu、 172 Lu and 18 At least one of F.

[0201] Exemplary chemiluminescent reagents in any of the above embodiments include, but are not limited to, at least one of luminol and its derivatives, luciferin, luteolin and its derivatives, ruthenium bipyridine and its derivatives, acridine ester and its derivatives, dioxane and its derivatives, rofenine and its derivatives, and peroxazone and its derivatives. Exemplary nanoparticle markers in any of the above embodiments include, but are not limited to, any one of colloids, organic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

[0202] The exemplary colloids in any of the above embodiments include, but are not limited to, at least one of colloidal metals, dispersed dyes, dye-labeled microspheres, and latex.

[0203] In optional embodiments, exemplary colloidal metals include, but are not limited to, at least one of colloidal gold, colloidal silver, and colloidal selenium.

[0204] The present invention will be further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present invention in any way.

[0205] Materials preparation:

[0206] The target gene encoding human TIGIT (Q495A1) was cloned into plasmids containing human Fc and mouse Fc, respectively, and transfected into HEK293 cells for transient expression, thereby obtaining hTIGIT-hFc (number R2291) and hTIGIT-mFc (number R0253-3).

[0207] The target gene encoding human TIGIT (Uniprot database ID: Q495A1) was cloned into a plvx lentiviral plasmid, the lentivirus was packaged and its titer was detected, and CHO cells were infected to express the membrane protein, thereby obtaining a cell line overexpressing hTIGIT, which is CHO-hTIGIT.

[0208] The target gene encoding cynomolgus monkey TIGIT (Uniprot database ID: A0A7N9I9V4) was cloned into a plvx lentiviral plasmid, the lentivirus was packaged and its titer was detected, and CHO cells were infected to express the membrane protein, thereby obtaining a cell line overexpressing cynomolgus monkey TIGIT, namely CHO-cynoTIGIT.

[0209] Jurkat-NFAT reporter gene cells were infected with human TIGIT lentivirus to construct Jurkat-hTIGIT-NFAT cells; CHO-OKT3 cells expressing CD3 antibody OKT3 on their membrane surface were infected with human PVR lentivirus to construct CHO-OKT3-PVR cells. The two cell types constituted the human TIGIT reporter gene system.

[0210] A targeting antibody for the hIgG4 (S228P / F234A / L235A / K447A) subtype was prepared based on the sequence numbered No. 34 in international publication number WO2021216468A1. The antibody, numbered R2882 (amino acid sequence as shown in SEQ ID NO. 70), is a TIGIT-positive nanobody.

[0211] Example 1: Discovery of TIGIT Nanobodies

[0212] Alpacas were immunized subcutaneously at multiple sites using recombinant human TIGIT-hFc as an immunogen to induce the production of anti-human TIGIT antibodies. After immunization, peripheral blood was collected from the animals, and PBMCs were isolated to construct phage libraries or mammalian cell display libraries. After multiple rounds of screening or enrichment using antigens, single clones were selected for testing. Clones that specifically bind to human TIGIT antigens were selected to demonstrate receptor-ligand binding blocking activity. Clones with ideal binding activity and receptor-ligand binding blocking activity were sequenced to construct an antibody as hIgG4 (S228P / F234A / L235A / K447A). This antibody was then recombinantly expressed and purified to produce the TIGIT nanobody R3179. The amino acid sequence of the variable region of the R3179 heavy chain is shown in Table 7, and the CDR sequence information of the variable region of the TIGIT nanobody R3179 heavy chain is shown in Table 1.

[0213] Table 7. TIGIT Nanobody Heavy Chain Variable Region Sequence

[0214] Example 2: Evaluation of TIGIT Nanobody

[0215] 1. Binding activity of candidate molecules with CHO-hTIGIT and CHO-cynoTIGIT cells

[0216] FACS combined with experimental methods: Cell counting: Count the cells to be tested, centrifuge at 300g for 5 min, resuspend in FCM buffer, and adjust the cell density to 4E+06 cells / mL. Add 2E+05 cells / well to each 96-well V-plate. Dilute the sample 3-fold with buffer (3% BSA + 1×PBS). Add the sample and cells to 96-well V-plates (50 μL each) and incubate at 4°C in the dark for 30 min. Wash: Centrifuge (300g / 5 min), discard the supernatant, add 200 μL of FCM buffer, wash once, centrifuge (300g / 5 min), discard the supernatant. Add secondary antibody (APC-anti-human IgG Fc, 1:500 dilution) to 96-well V-plates (100 μL / well), incubate at 4°C in the dark for 30 min. Wash: Centrifuge (300g / 5 min), discard the supernatant, add 200 μL of FCM buffer. Wash once with buffer, centrifuge (300g / 5min), discard supernatant; resuspend in 100μL 1×PBS, and analyze. Plot the antibody concentration logarithm on the x-axis and MFI value on the y-axis, perform nonlinear four-parameter fitting, and calculate the EC50 and the difference between the upper and lower plateaus of the curve. The smaller the EC50 and the larger the difference between the upper and lower plateaus, the better the antibody activity. The results are shown in Figures 1 and 2. The binding activity of TIGIT nanobody R3179 to CHO-hTIGIT is close to that of positive antibody R2882, and the binding activity to CHO-cynoTIGIT is significantly stronger than that of positive antibody R2882.

[0217] 2. Reporter gene activity assay of candidate molecules

[0218] TIGIT is an inhibitory receptor expressed on T cells. When it binds to its ligand PVR expressed on dendritic cells (DCs) or tumor cells, it can inhibit T cell immune activation. A luciferase reporter gene (Luc) is a reporter system that uses luciferin as a substrate to detect the activity of firefly luciferase. Luciferase catalyzes the oxidation of oxyluciferin by luciferin, emitting bioluminescence during this process. A method for detecting TIGIT reporter gene activity with a large detection window was developed using Jurkat-hTIGIT-NFAT cells and CHO-OKT3-PVR cells to detect the T cell activation activity of candidate antibodies. A nonlinear four-parameter fitting was performed, with the logarithm of antibody concentration on the x-axis and the fluorescence signal value on the y-axis. The EC50 and the difference between the upper and lower plateaus of the curve were calculated. A smaller EC50 and a larger difference between the upper and lower plateaus indicate better antibody activity. As shown in Figure 3, the TIGIT nanobody R3179 exhibited significantly stronger activity than the positive control R2882 in the TIGIT reporter gene assay.

[0219] Example 3: Activity evaluation of humanized TIGIT nanobody

[0220] Using R3179 as the parent molecule, three-dimensional structural homology modeling was performed on selected sequences, and the structures were determined to be acceptable through methods such as Laplace plots. The antibody sequences were compared with the GermLine antibody database to obtain a highly homologous human template (IGHV3-30*01). The CDR was then transplanted into the corresponding human template. The transplanted antibody underwent further three-dimensional structural simulation and analysis. Complex modifications were performed on embedded residues, residues that directly interact with the CDR region, and residues that significantly influence the conformation of the variable region. Chemically unstable amino acid residues in the CDR region were optimized, resulting in a series of humanized antibody sequences.

[0221] A series of humanized molecules R3693-R3696 of the hIgG4 (S228P / F234A / L235A / K447A) isoforms were prepared. The amino acid sequences of the heavy chain variable region of the humanized TIGIT nanobodies R3693-R3696 are shown in Table 8.

[0222] Table 8. TIGIT Nanobody Heavy Chain Variable Region Sequence

[0223] 1. Evaluation of the reporter gene activity of humanized TIGIT nanobodies

[0224] The evaluation method was the same as in Part 2 of Example 2, and the results are shown in Figure 4. R3179 was the parent molecule, and R3693-R3696 were humanized molecules. Among the humanized molecules, R3694 exhibited the best reporter gene activity, which was close to that of the parent molecule R3179.

[0225] 2. Binding activity of humanized TIGIT nanobody with CHO-hTIGIT cells and CHO-cynoTIGIT cells

[0226] The evaluation method was the same as in Part 1 of Example 2. The results are shown in Figures 5 and 6. The binding activities of the humanized molecule R3694 with CHO-hTIGIT and CHO-cynoTIGIT were similar.

[0227] The amino acid sequences of the positive control molecules in the above embodiments are shown in Table 9.

[0228] Table 9. Amino acid sequences of positive control molecules

[0229] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention. Industrial applicability

[0230] The TIGIT binding molecule disclosed herein can specifically bind to TIGIT, such as binding to human and / or cynomolgus monkey TIGIT, providing new possibilities for the treatment and / or prevention of tumors.

Claims

1. A TIGIT-binding molecule, characterized in that, It contains an antigen-binding domain, wherein the antigen-binding domain contains the complementarity-determining regions HCDR1, HCDR2 and HCDR3 of the antibody heavy chain variable region; The complementarity-determining region HCDR1 includes the amino acid residue RY; the complementarity-determining region HCDR2 includes the amino acid sequence shown in SEQ ID NO.11; and the complementarity-determining region HCDR3 includes the amino acid sequence shown in SEQ ID NO.

19.

2. The TIGIT-binding molecule according to claim 1, characterized in that, According to the IMGT definition, the complementarity-determining region HCDR1 includes the amino acid sequence shown in SEQ ID NO.10; the complementarity-determining region HCDR2 includes the amino acid sequence shown in SEQ ID NO.15; and the complementarity-determining region HCDR3 includes the amino acid sequence shown in SEQ ID NO.

18.

3. A TIGIT-binding molecule, characterized in that, It contains an antigen-binding domain, wherein the antigen-binding domain contains the complementarity-determining regions HCDR1, HCDR2 and HCDR3 of the antibody heavy chain variable region; The complementary determination regions HCDR1, HCDR2 and HCDR3 are selected from the heavy chain variable regions HCDR1, HCDR2 and HCDR3 shown in any one of SEQ ID NO.1 to 5; Optionally, HCDR1, HCDR2, and HCDR3 are determined according to the Kabat definition method, Chothia definition method, AbM definition method, Contact definition method, or IMGT definition method.

4. The TIGIT-binding molecule according to any one of claims 1 to 3, characterized in that, The heavy chain variable region is modified by at least one of the following: humanization, affinity maturation, removal of T cell epitopes, reduction of antibody deamidation, and reduction of antibody isomerization.

5. The TIGIT-binding molecule according to any one of claims 1 to 4, characterized in that, The heavy chain variable region contains a frame region; Optionally, the frame region is derived from at least one of camel-derived antibodies, mouse-derived antibodies, human-derived antibodies, primate-derived antibodies, or mutants thereof; Optionally, the frame region includes HFR1, HFR2, HFR3 and HFR4; The HFR1 comprises the following amino acid sequence: Q-X1-QLVESGGG-X2-VQ-X3-G-X4-SLRLSCAAS(SEQ ID NO.66), where X1 is L or V, X2 is L or V, X3 is A or P, and X4 is D or R; And / or, the HFR2 comprises the following amino acid sequence: WFRQAPGK-X5-X6-E (SEQ ID NO.67), where X5 is E or G, and X6 is H or L; And / or, the HFR3 comprises the following amino acid sequence: RF-X7-ISR-X8-N-X9-KNTLYLQMN-X10-L-X11-X12-X13-DTAVYYC (SEQ ID NO.68), where X7 is I or T, X8 is V or D, X9 is P or S, X10 is N or S, X11 is K or R, X12 is P or A, and X13 is D or E; And / or, the HFR4 comprises the following amino acid sequence: WGQGT-X14-VTVSS (SEQ ID NO.69), where X14 is either Q or L; Optionally, the framework region of the TIGIT binding molecule is selected from any group of the following a to d: a. The HFR1 comprises the amino acid sequence shown in SEQ ID NO. 20 or contains an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO. 20; and / or, the HFR2 comprises the amino acid sequence shown in SEQ ID NO. 28 or contains an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO. 28; and / or, the HFR3 comprises the amino acid sequence shown in SEQ ID NO. 43 or contains an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO. 43; and / or, the HFR4 comprises the amino acid sequence shown in SEQ ID NO. 62 or contains an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO. 62; b. The HFR1 comprises the amino acid sequence shown in SEQ ID NO. 20 or contains an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO. 20; and / or, the HFR2 comprises the amino acid sequence shown in SEQ ID NO. 29 or contains an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO. 29; and / or, the HFR3 comprises the amino acid sequence shown in SEQ ID NO. 42 or contains an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO. 42; and / or, the HFR4 comprises the amino acid sequence shown in SEQ ID NO. 62 or contains an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO.

62. c. The HFR1 comprises the amino acid sequence shown in SEQ ID NO. 23 or contains an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO. 23; and / or, the HFR2 comprises the amino acid sequence shown in SEQ ID NO. 32 or 36 or contains an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO. 32 or 36; and / or, the HFR3 comprises the amino acid sequence shown in SEQ ID NO. 49, 55 or 61 or contains an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO. 49, 55 or 61; and / or, the HFR4 comprises the amino acid sequence shown in SEQ ID NO. 64 or contains an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO. 64; d. The HFR1 comprises the amino acid sequence shown in SEQ ID NO. 23 or contains an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO. 23; and / or, the HFR2 comprises the amino acid sequence shown in SEQ ID NO. 33 or 37 or contains an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO. 37; and / or, the HFR3 comprises the amino acid sequence shown in SEQ ID NO. 48, 54 or 60 or contains an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO. 48, 54 or 60; and / or, the HFR4 comprises the amino acid sequence shown in SEQ ID NO. 64 or contains an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO.

64.

6. The TIGIT-binding molecule according to any one of claims 1 to 5, characterized in that, The HFR1, HFR2, HFR3 and HFR4 of the frame region are selected from the HFR1, HFR2, HFR3 and HFR4 of the heavy chain variable region shown in any one of SEQ ID NO.1 to 5; Optionally, the HFR1, HFR2, HFR3 and HFR4 of the frame region are selected from the HFR1, HFR2, HFR3 and HFR4 of the heavy chain variable region shown in any one of SEQ ID NO.2 to 5; Optionally, HFR1, HFR2, HFR3 and HFR4 are determined according to the Kabat definition method, Chothia definition method, AbM definition method, Contact definition method or IMGT definition method.

7. A TIGIT-binding molecule, characterized in that, The antigen-binding domain contains a heavy chain variable region having an amino acid sequence shown in any one of SEQ ID NO. 1 to 5 or contains a heavy chain variable region having at least 90% sequence identity with an amino acid sequence shown in any one of SEQ ID NO. 1 to 5; Optionally, the antigen-binding domain contains a heavy chain variable region having an amino acid sequence shown in any one of SEQ ID NO. 2 to 5, or contains a heavy chain variable region having at least 90% sequence identity with an amino acid sequence shown in any one of SEQ ID NO. 2 to 5.

8. The TIGIT-binding molecule according to any one of claims 1 to 7, characterized in that, The TIGIT binding molecule also contains part or all of the sequence of the constant region of the antibody; Optionally, the constant region is derived from at least one of camel-derived antibodies, mouse-derived antibodies, human-derived antibodies, primate-derived antibodies, or mutants thereof; Optionally, the constant region contains a portion or all of the sequence of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD. Optionally, the constant region contains one or more of the heavy chain constant regions CH1, CH2, CH3 and CH4; Optionally, the TIGIT binding molecule further contains the Fc region of the antibody; Optionally, the Fc region is derived from IgG1, IgG2, IgG3 or IgG4 antibodies; Optionally, the TIGIT binding molecule contains the Fc region of IgG4; Optionally, the Fc region includes the Fc region of human-derived IgG4; Optionally, the Fc region of the human-derived IgG4 contains one, two, three, or four mutations from 228P, 234A, 235A, and 447A.

9. The TIGIT-binding molecule according to any one of claims 1 to 8, characterized in that, The TIGIT binding molecule is an antibody or its antigen-binding fragment; Optionally, the TIGIT binding molecule is one or more of the following: full-length antibody, nanobody, F(ab')2, Fab', Fab, Fv, scFv, dsFv, and the smallest antibody recognition unit; Optionally, the TIGIT binding molecule is a nanobody.

10. The TIGIT-binding molecule according to any one of claims 1 to 9, characterized in that, It has one or more of the following properties (i) and (ii): (i) EC50 at a concentration not exceeding 10 nM was used to bind human TIGIT or cynomolgus monkey TIGIT. EC50 was measured by flow cytometry fluorescence sorting. (ii) T cells were activated with EC50 of no more than 10 nM, which was determined using a luciferase reporter gene system.

11. An anti-TIGIT antibody or its antigen-binding fragment, characterized in that, The TIGIT binding molecule of any one of claims 1 to 10 competitively binds to TIGIT, or the epitope of the TIGIT binding antigen is the same as the epitope of the binding fragment of the TIGIT binding molecule of any one of claims 1 to 10.

12. A biomaterial, characterized in that, The biomaterial includes any one of the following (i) to (vi): (i) A multispecific antibody containing at least two antigen-binding domains, wherein one of the antigen-binding domains is selected from the antigen-binding domain of the TIGIT binding molecule according to any one of claims 1 to 10, or a variable domain of the anti-TIGIT antibody or its antigen-binding fragment according to claim 11. (ii) A conjugate comprising connected (Ⅰ) and (Ⅱ): (I) the TIGIT binding molecule according to any one of claims 1 to 10, or the anti-TIGIT antibody or its antigen-binding fragment according to claim 11, or (i) the multispecific antibody; (II) A functional molecule, said functional molecule comprising a drug and / or a signaling agent; (iii) A fusion protein comprising at least two domains, one of which comprises the antigen-binding domain of the TIGIT binding molecule according to any one of claims 1 to 10. Optionally, the fusion protein includes a chimeric antigen receptor, the chimeric antigen receptor including an extracellular region, the extracellular region including the antigen-binding domain of the TIGIT binding molecule according to any one of claims 1 to 10; (iv) A nucleic acid molecule that encodes a TIGIT binding molecule according to any one of claims 1 to 10, or an anti-TIGIT antibody or an antigen-binding fragment thereof according to claim 11, or a multispecific antibody according to (i), or a fusion protein according to (iii); (v) A vector carrying the nucleic acid molecule described in (iv); (vi) A recombinant cell comprising the nucleic acid molecule of (iv), or the vector of (v), or expressing the TIGIT binding molecule of any one of claims 1 to 10, or expressing the anti-TIGIT antibody of claim 11 or its antigen-binding fragment, or expressing the multispecific antibody of (i), or expressing the fusion protein of (iii). (vii) An engineered immune effector cell that expresses the chimeric antigen receptor described in (iii) or contains a nucleic acid molecule encoding the chimeric antigen receptor described in (iv).

13. The use of the TIGIT binding molecule according to any one of claims 1 to 10, or the anti-TIGIT antibody or its antigen-binding fragment according to claim 11, or the multispecific antibody according to (i), or the conjugate according to (ii), or the fusion protein according to (iii), or the nucleic acid molecule according to (iv), or the vector according to (v), or the recombinant cell according to (vi), or the engineered immune effector cell according to (vii) in any one of the following: (I) Detect TIGIT or cells expressing TIGIT; (II) Prepare products for detecting TIGIT or cells expressing TIGIT; (III) Activate immune cells in the tumor immune microenvironment; Optionally, the immune cells include T cells and / or NK cells; (IV) To prepare products for the treatment, prevention or relief of tumor-related diseases, symptoms or conditions; (V) To treat, prevent or alleviate diseases, symptoms or conditions associated with tumors.

14. A pharmaceutical composition, characterized in that, The pharmaceutical composition contains the TIGIT binding molecule according to any one of claims 1 to 10, or the anti-TIGIT antibody or its antigen-binding fragment according to claim 11, or the multispecific antibody according to (i), or the conjugate according to (ii), or the fusion protein according to (iii), or the nucleic acid molecule according to (iv), or the carrier according to (v), or the recombinant cell according to (vi), or the engineered immune effector cell according to (vii). Optionally, the pharmaceutical composition may further include a pharmaceutically acceptable carrier and / or excipient.

15. A method for treating, preventing, or alleviating a disease, symptom, or condition, characterized in that, The method comprises administering to a subject a therapeutically effective amount of the TIGIT binding molecule of any one of claims 1 to 10, or the anti-TIGIT antibody or its antigen-binding fragment of claim 11, or the multispecific antibody of (i), or the conjugate of (ii), or the fusion protein of (iii), or the nucleic acid molecule of (iv), or the carrier of (v), or the recombinant cell of (vi), or the engineered immune effector cell of (vii), or the pharmaceutical composition of claim 14; Optionally, the disease, symptom, or condition includes tumor-related diseases, symptoms, or conditions.

16. A kit for detecting TIGIT, characterized in that, It comprises the TIGIT binding molecule as described in any one of claims 1 to 10, or the anti-TIGIT antibody or its antigen-binding fragment as described in claim 11.

17. A method for preparing the TIGIT-binding molecule according to any one of claims 1 to 10, characterized in that, The method includes culturing the recombinant cells as described in claim 12, and then isolating and purifying the TIGIT-binding molecule.

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