Multispecific binding molecule and use thereof

Abstract

Provided in the present disclosure is a multispecific binding molecule, relating to the technical field of biology. The multispecific binding molecule can simultaneously bind to a plurality of tumor-associated antigens, and can improve the effect of tumor treatment.

Description

Multispecific binding molecules and uses thereof

[0001] Priority claim

[0002] This application claims priority to Chinese Patent Application No. 202411680990.8, filed on November 21, 2024, entitled “Multispecific binding molecules and uses thereof”, the entire contents of which are incorporated herein by reference. TECHNICAL FIELD

[0003] The present disclosure relates to the field of biotechnology, and in particular to a multispecific binding molecule and uses thereof. BACKGROUND

[0004] The following discussion merely provides background information related to the present disclosure and may not necessarily constitute prior art.

[0005] Immunotherapy is a promising approach for cancer treatment. Immunotherapy drugs, represented by PD-1 / PDL1 antibodies, have shown real potential for “cure” in various cancers, with good safety.

[0006] PD-L1, which stands for Programmed Death Ligand 1, is a molecule of great significance in the field of tumor immunotherapy. PDL1 is usually overexpressed on the surface of various tumor cells, and by binding to the PD1 receptor on the surface of immune cells, it inhibits the activity of immune cells, thereby helping tumor cells evade the surveillance and attack of the immune system. In recent years, immunotherapy drugs targeting PDL1 have achieved remarkable results. For example, drugs such as Pembrolizumab and Atezolizumab block the interaction between PD-L1 and PD1, reactivating the immune system's killing effect on tumor cells, significantly improving the survival rate and quality of life of patients with various tumors.

[0007] 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 and its ligand PVRL2 (CD112) are highly expressed in various tumors and are associated with poor prognosis. TIGIT transmits inhibitory signals by binding to its ligand, 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.

[0008] PVRIG (poliovirus receptor-related immunoglobulin domain-containing), also known as CD112R, was identified as a novel inhibitory receptor in 2016. Its ligand is PVRL2 (poliovirus receptor-related 2), also known as CD112. On the one hand, PVRIG can compete with the activating receptor CD226 on immune cells for binding to the ligand PVRL2 with higher affinity, thereby weakening the activation of immune cells. On the other hand, after binding to the ligand, PVRIG transmits inhibitory signals into the cell through intracellular inhibitory motifs, thereby inhibiting the activity of immune cells.

[0009] PVRIG is expressed on activated lymphocytes, especially highly on exhausted CD8+ T cells and NK cells. Its expression is tumor-specific; analysis of tumor and normal tissues from different cancer patients revealed that PVRIG expression on tumor-infiltrating lymphocytes was significantly higher than that on lymphocytes in peripheral normal tissues of the same patient. Furthermore, PVRIG expression correlated with TIGIT (T-cell immunoreceptor with Ig and ITIM domains) and PD-1 (Programmed cell death protein 1). High PVRIG expression is associated with poor prognosis in cancer patients.

[0010] The ligand PVRL2 of PVRIG is primarily expressed on tumor cells, macrophages, and activated dendritic cells (DCs). Its expression exhibits tumor tissue specificity. Analysis of tumor tissues and normal tissues from different cancer patients revealed that PVRL2 expression on tumor-infiltrating lymphocytes was significantly higher than in peripheral normal tissues of the same patient. PVRL2 expression also demonstrates tumor tissue specificity, and high ligand expression is associated with poor prognosis in cancer patients.

[0011] PVRIG, along with TIGIT and PD-1, is significantly upregulated after T cell activation, and can synergistically exert an inhibitory effect on T cells. Blocking PVRIG with antibodies can activate CD4+ / CD8+ T cells and NK cells. Targeting tumor-infiltrating lymphocytes from cancer patients, PVRIG antibodies can significantly synergistically activate T cells with TIGIT and PD-1 / PDL1 antibodies, achieving a synergistic effect greater than the sum of its parts (1+1+1>3).

[0012] Clinical trials have shown that only 20%–40% of patients benefit from PD-1 immunotherapy. To address this unmet clinical need, multispecific antibodies targeting PVRIG, TIGIT, and PDL1 simultaneously, or simultaneously targeting PVRIG, TIGIT, and PDL1, can be developed for patients who do not respond well to current immunotherapies. PVRIG, TIGIT, and PD-1 all exert their inhibitory effects on immune cells by inhibiting the activating receptor CD226. Releasing the inhibition of one of these receptors alone has limited activation of immune cells, which may explain the poor efficacy of current single-target immunotherapies. Simultaneous targeting of multiple immune checkpoints can exert a synergistic effect, achieving significant activation of immune cells to eliminate tumor cells. Furthermore, multiple immune checkpoints have tumor-targeting properties, allowing multispecific antibodies to accumulate more extensively in the tumor microenvironment and reduce toxicity to peripheral normal tissues. Multispecific antibodies can significantly enhance immune cell activation compared to monoclonal antibodies while reducing toxicity to peripheral normal tissues, which is of great significance for improving the treatment efficacy of malignant tumors. Summary of the Invention

[0013] The purpose of this disclosure is to provide multispecific binding molecules and their applications.

[0014] To solve the above-mentioned technical problems, the present disclosure adopts the following technical solution:

[0015] In a first aspect, a multispecific binding molecule is provided, wherein the multispecific binding molecule binds to at least two different antigenic epitopes; the multispecific binding molecule contains a PVRIG antigen-binding domain and a PD-L1 antigen-binding domain.

[0016] Secondly, it provides a multi-specific binding molecule.

[0017] The multispecific binding molecule binds to at least two different antigenic epitopes, and the multispecific binding molecule is selected from any one of structures (A) to (G):

[0018] (A) contains a first polypeptide chain, which has the following structure:

[0019] [First VD] - [Connector] a1 -[Second VD]-Fc-[Connector] a2 -[Third VD];

[0020] At least one domain of [first VD], [second VD], and [third VD] specifically binds to PVRIG, TIGIT, or PD-L1;

[0021] (B) Contains a first polypeptide chain, which has the following structure:

[0022] [First VD] - [Connector] b1 -[Second VD]-[Connector] b2 -[Third VD]-Fc;

[0023] At least one of the three domains in the first VD, second VD, and third VD specifically binds to PVRIG;

[0024] (C) Contains a first polypeptide chain and a second polypeptide chain:

[0025] The first polypeptide chain contains the following structure:

[0026] [VH]-[CH1]-[Fc]-[connector] c1 -[First VD];

[0027] The second polypeptide chain contains the following structure:

[0028] [Second VD] - [Connector] c2 -[VL]-[CL];

[0029] The combination of [VH] and [VL] specifically binds to PVRIG;

[0030] Alternatively, one of the [first VD] and [second VD] specifically binds to PVRIG;

[0031] (D) Contains a first polypeptide chain and a second polypeptide chain:

[0032] The first polypeptide chain contains the following structure:

[0033] [First VD] - [Connector] d1 -[VH]-[CH1]-[Fc]-[connector] d2-[Second VD];

[0034] The second polypeptide chain contains the following structure:

[0035] [VL]-[CL];

[0036] The combination of [VH] and [VL] specifically binds to PVRIG, TIGIT, or PD1;

[0037] Furthermore, the [first VD] specifically binds to TIGIT or PVRIG; and the [second VD] specifically binds to PD-1, PD-L1, or TIGIT;

[0038] (E) Contains a first polypeptide chain and a second polypeptide chain:

[0039] The first polypeptide chain contains the following structure:

[0040] [VH]-[CH1]-[Fc]-[connector] e1 -[First VD];

[0041] The second polypeptide chain contains the following structure:

[0042] [VL]-[CL]-[Connector] e2 -[Second VD];

[0043] The combination of [VH] and [VL] specifically binds to PVRIG;

[0044] Alternatively, one of the [first VD] and [second VD] specifically binds to PVRIG;

[0045] (F) contains a first polypeptide chain and a second polypeptide chain:

[0046] The first polypeptide chain contains the following structure:

[0047] [First VD] - [Connector] f1 -[Second VD]-[Connector] f2 -[VH]-[CH1]-[Fc];

[0048] The second polypeptide chain contains the following structure:

[0049] [VL]-[CL];

[0050] [VH] and [VL] combination specifically binds to PVRIG; [first VD] specifically binds to PD-L1; [second VD] specifically binds to TIGIT; or,

[0051] [VH] and [VL] combination specifically binds to TIGIT; [first VD] specifically binds to PD-L1; [second VD] specifically binds to PVRIG; or,

[0052] The combination of [VH] and [VL] specifically binds to PD-1; [first VD] specifically binds to PVRIG; [second VD] specifically binds to TIGIT;

[0053] (G) contains a first polypeptide chain and a second polypeptide chain:

[0054] The first polypeptide chain contains the following structure:

[0055] [VH]-[CH1]-[Fc]-[connector]g1-[first VD]-[connector] g2 -[Second VD];

[0056] The second polypeptide chain contains the following structure:

[0057] [VL]-[CL];

[0058] [VH] and [VL] specifically bind to PVRIG; or, one of [first VD] and [second VD] specifically binds to PVRIG;

[0059] [First VD], [Second VD], and [Third VD] represent single variable domains; [VH] represents the heavy chain variable domain VH, and [VL] represents the light chain variable domain VL; the domains in the first and second polypeptide chains are arranged in order from the N-terminus to the C-terminus; a1, a2, b1, b2, c1, c2, d1, d2, e1, e2, f1, f2, g1, and g2 are all independent positive integers ≥ 0.

[0060] Thirdly, a biomaterial is provided, said biomaterial comprising any one of the following (i) to (iii):

[0061] (i) A nucleic acid molecule that encodes the multispecific binding molecule described in the first or second aspect;

[0062] (ii) A vector, characterized in that the vector carries the nucleic acid molecule described in (i);

[0063] (iii) A recombinant cell comprising the nucleic acid molecule described in (i), or the vector described in (ii), or expressing the multispecific binding molecule described in the first or second aspect.

[0064] Fourthly, the use of the multispecific binding molecule described in the first or second aspect, or the biomaterial described in the third aspect, in any of the following:

[0065] (I) To prepare products for the treatment, prevention or relief of tumor-related diseases, symptoms or conditions;

[0066] (II) Treatment, prevention or relief of tumor-related diseases, symptoms or conditions;

[0067] (III) Activate immune cells in the tumor immune microenvironment;

[0068] (IV) Increases the secretion of IFN-γ and / or pro-inflammatory cytokines in the subjects.

[0069] Fifthly, a pharmaceutical composition is provided, the pharmaceutical composition comprising the multispecific binding molecule described in the first or second aspect, or the biological material described in the third aspect.

[0070] 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 multispecific binding molecule of the first or second aspect, or the biological material of the third aspect, or the pharmaceutical composition of the fifth aspect.

[0071] In a seventh aspect, a method for preparing the multispecific binding molecule described in the first or second aspect is provided, the method comprising culturing recombinant cells as described in the fifth or third aspect, and then isolating and purifying the multispecific binding molecule. Attached Figure Description

[0072] To more clearly illustrate the technical solutions in the specific embodiments of this disclosure or 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 this disclosure. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0073] Figure 1 is a schematic diagram of the structure of the multispecific binding molecule R3876;

[0074] Figure 2 is a schematic diagram of the structure of the multispecific binding molecule R3877;

[0075] Figure 3 is a schematic diagram of the structure of the multispecific binding molecule R3878;

[0076] Figure 4 is a schematic diagram of the structure of the multispecific binding molecule R4038;

[0077] Figure 5 is a schematic diagram of the structure of the multispecific binding molecule R4039;

[0078] Figure 6 is a schematic diagram of the structure of the multispecific binding molecule R4040;

[0079] Figure 7 is a schematic diagram of the structure of the multispecific binding molecule R4041;

[0080] Figure 8 is a schematic diagram of the structure of the multispecific binding molecule R4042;

[0081] Figure 9 is a schematic diagram of the structure of the multispecific binding molecule R3884;

[0082] Figure 10 is a schematic diagram of the structure of the multispecific binding molecule R3885;

[0083] Figure 11 is a schematic diagram of the structure of the multispecific binding molecule R3886;

[0084] Figure 12 is a schematic diagram of the structure of the multispecific binding molecule R3887;

[0085] Figure 13 is a schematic diagram of the structure of the multispecific binding molecule R3888;

[0086] Figure 14 is a schematic diagram of the structure of the multispecific binding molecule R3889;

[0087] Figure 15 is a schematic diagram of the structure of the multispecific binding molecule R3890;

[0088] Figure 16 is a schematic diagram of the structure of the multispecific binding molecule R3891;

[0089] Figure 17 shows the results of the binding activity detection of PVRIG nanobody R3242 and 293T-hPVRIG in Example 2;

[0090] Figure 18 shows the results of the binding activity detection of PVRIG nanobody R3242 and 293T-cynoPVRIG in Example 2;

[0091] Figure 19 shows the detection results of the activity of PVRIG nanobody R3242 in activating T cells by the luciferase reporter gene method in Example 2;

[0092] Figure 20 shows the detection results of the activity of humanized PVRIG nanobody activated T cells by the luciferase reporter gene method in Example 3.

[0093] Figure 21 shows the results of the binding activity detection of PVRIG nanobody and 293T-hPVRIG in Example 3;

[0094] Figure 22 shows the results of the binding activity detection of PVRIG nanobody and 293T-cynoPVRIG in Example 3;

[0095] Figure 23 shows the results of the binding activity detection of PD-L1 nanobody R3377 and CHO-hPD-L1 in Example 5;

[0096] Figure 24 shows the results of the binding activity detection of PD-L1 nanobody R3377 and CHO-cynoPD-L1 in Example 5;

[0097] Figure 25 shows the detection results of the activity of PD-L1 nanobody R3377 in T cells activated by the luciferase reporter gene method in Example 5;

[0098] Figure 26 shows the detection results of the activity of humanized PD-L1 nanobody activated T cells by the luciferase reporter gene method in Example 6;

[0099] Figure 27 shows the results of the binding activity detection of PD-L1 nanobody and CHO-hPD-L1 in Example 6;

[0100] Figure 28 shows the results of the binding activity detection of PD-L1 nanobody and CHO-cynoPD-L1 in Example 6;

[0101] Figure 29 shows the results of the binding activity detection of TIGIT nanobody R3179 and CHO-hTIGIT in Example 8;

[0102] Figure 30 shows the results of the binding activity detection of TIGIT nanobody R3179 and CHO-cynoTIGIT in Example 8;

[0103] Figure 31 shows the detection results of the activity of TIGIT nanobody R3179 in T cells activated by the luciferase reporter gene method in Example 8;

[0104] Figure 32 shows the detection results of the activity of humanized TIGIT nanobody activated T cells by the luciferase reporter gene method in Example 9;

[0105] Figure 33 shows the results of the TIGIT nanobody binding activity assay with CHO-hTIGIT in Example 9;

[0106] Figure 34 shows the results of the TIGIT nanobody binding activity assay with CHO-cynoTIGIT in Example 9;

[0107] Figure 35 shows the detection results of PVRIG conventional antibody-activated T cell activity using the luciferase reporter gene method in Example 10;

[0108] Figure 36 shows the results of the binding activity detection of conventional PVRIG antibody and 293T-hPVRIG in Example 10;

[0109] Figure 37 shows the results of the binding activity assay between the conventional PVRIG antibody and 293T-cynoPVRIG in Example 10;

[0110] Figure 38 shows the results of the detection of the binding activity of trispecific antibodies R3876, R3877 and R3878 to CHO-OKT3-PVRL2-PVR-PD-L1 cells by the luciferase reporter gene method in Example 14.

[0111] Figure 39 shows the results of detecting the binding activity of trispecific antibodies R4038-R4042 to CHO-OKT3-PVRL2-PVR-PD-L1 cells using the luciferase reporter gene method in Example 14.

[0112] Figure 40 shows the results of detecting the binding activity of trispecific antibodies R3884-R3887 to CHO-OKT3-PVRL2-PVR-PD-L1 cells using the luciferase reporter gene method in Example 14.

[0113] Figure 41 shows the results of detecting the binding activity of trispecific antibodies R3888-R3891 and positive antibodies with Jurkat-PVRIG-TIGIT-PD1 cells using the luciferase reporter gene method in Example 14.

[0114] Figure 42 shows the amount of IFN-γ secreted in the culture supernatant of CMV+CD8+T cells after treatment with trispecific antibodies (R3876, R3884, R3885, R3888 and R3890) in Example 15.

[0115] Figure 43 shows the amount of IFN-γ secreted in the culture supernatant of CMV+CD8+T cells after treatment with the three specific antibodies (R3876, R4039, R3890 and R3891) in Example 15.

[0116] Figure 44 shows the amount of IFN-γ secreted in the mixed lymphocyte culture supernatant after treatment of mDCs and induced T cells with trispecific antibodies (R3876, R3884, R3885, R3888, R3890 and R3891) in Example 16.

[0117] Figure 45 shows the amount of IFN-γ secreted in the mixed lymphocyte culture supernatant after treatment of mDCs and induced T cells with trispecific antibodies (R3891, R3889R and control antibody) in Example 16.

[0118] Figure 46 shows the change in body weight of tumor model mice over time after treatment with the trispecific antibodies (R3876, R4039, R3885, R3890, and R3891) in Example 17.

[0119] Figure 47 shows the curves of tumor volume change over time in mouse tumor model mice after treatment with the trispecific antibodies (R3876, R4039, R3885, R3890, and R3891) in Example 17.

[0120] Figure 48 shows the results of the binding activity assay of the trispecific antibody R3891 to CHO-hPD1 cells in Example 18;

[0121] Figure 49 shows the results of the binding activity assay of the trispecific antibody R3891 to CHO-hTIGIT cells in Example 18;

[0122] Figure 50 shows the results of the binding activity assay of the trispecific antibody R3891 to 293T-hPVRIG cells in Example 18;

[0123] Figure 51 shows the change in body weight of tumor model mice over time after treatment with the trispecific antibodies (R3890 and R3891) in Example 19.

[0124] Figure 52 shows the curves of tumor volume change over time in mouse tumor model mice after treatment with trispecific antibodies (R3890 and R3891) in Example 19. Detailed Implementation

[0125] The technical solutions of this disclosure will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of this disclosure, not all embodiments. Based on the embodiments of this disclosure, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this disclosure.

[0126] Terminology Explanation:

[0127] In this text, 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.

[0128] 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.

[0129] In this article, the term "PD-L1" or "PDL1" refers to Programmed Death Ligand 1, also known as cluster of differentiation 274 (CD274). PD-L1 protein is widely expressed on activated T cells, B cells, and macrophages. The interaction between PD-L1 and PD-1 protein on T cells inhibits T cell activation and induces T cell apoptosis, playing a negative regulatory role in the immune response. PD-L1 is encoded by the CD274 gene, belongs to the B7 family, and is a type I transmembrane protein.

[0130] In this article, the term "PD-1" or "PD1" refers to Programmed Cell Death Protein 1, an immune checkpoint molecule primarily expressed on the surface of activated T cells, B cells, natural killer (NK) cells, and other types of immune cells. PD-1 is a key molecule in the negative regulation of the immune system, modulating immune responses by binding to its ligands PD-L1 and PD-L2 (Programmed Cell Death-Ligand 2).

[0131] In this article, the term "PVRIG" refers to the poliovirus receptor-related immunoglobulin domain-containing protein, belonging to the PVR (poliovirus receptor) family, also known as CD112R. The ligand for PVRIG is PVRL2 (poliovirus receptor-related 2), also known as CD112. PVRIG has an extracellular IgV domain and a longer intracellular region containing an ITIM domain, which is involved in regulating immune signaling. PVRIG is primarily expressed on the surface of T cells and NK cells, and exerts its inhibitory function after binding to its ligand.

[0132] 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.

[0133] Unless otherwise stated, multispecific binding antigens (e.g., PD-L1, PD-1, PVRIG, and TIGIT) include human antigens, subtypes, and species homologs expressed naturally in cells or by genetically transfected cells. The term also includes artificially modified antigens, including but not limited to mutated, truncated, or fused peptides or proteins with other domains, while retaining the necessary antigenic epitopes for antibody binding.

[0134] In this document, the term "multispecific binding molecule" refers to any molecule capable of binding to at least two different antigenic epitopes. Multispecific binding molecules may include antibodies, their antigen-binding fragments, CDR transplantation molecules, or conjugates thereof as defined in this disclosure.

[0135] In this paper, a "domain" refers to a specific region within 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 polypeptide 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. 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 losing the function of the rest of the molecule and / or the domain itself.

[0136] In this document, the term "antigen-binding domain" refers to a domain in a molecule used for binding a target antigen. The "multispecific binding molecule" of this disclosure includes one or more target antigen-binding domains. The target antigen-binding domain can be a variable domain (variable region) of an antibody or a variant thereof, such as a nanobody (VHH), a heavy chain variable region (VH) or a light chain variable region (VL) of an antibody, or a variant thereof. In some embodiments, the number of antigen-binding domains binding the same antigen epitope in a multispecific binding molecule may include 1, 2, 3, 4, or more. The number of antigen-binding domains binding different antigen epitopes may be the same or different. The amino acid sequences between multiple antigen-binding domains binding the same antigen epitope may be the same or different, for example, having different CDRs, or having the same CDR but different FRs. When the amino acid sequence of an antigen-binding domain is described herein, it indicates that at least one antigen-binding domain in the multispecific binding molecule has that amino acid sequence, without requiring all antigen-binding domains targeting the same antigen epitope to have exactly the same amino acid sequence. In some alternative embodiments, the antigen-binding domains that bind to the same antigen epitope in a multispecific binding molecule have the same amino acid sequence.

[0137] 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.

[0138] 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, and 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).

[0139] The "variable region" or "variable domain" of an antibody refers to the domain at the amino terminus of the antibody's heavy or light chain that recognizes and binds to antigens. The composition and arrangement of the amino acids in this region determine the antibody's specificity in recognizing antigens. The heavy chain variable region can be called "VH," and the light chain variable region can be called "VL." The variable region contains antigen-binding sites. Both the heavy and light chain variable regions consist of three complementarity-determining regions (CDRs) (also known as hypervariable regions) connected by four framework regions (FRs). The extent of the backbone region and CDRs has been precisely defined, for example, in Kabat (see Sequences of Proteins of Immunological Interest, E. Kabat et al.) and Chothia. Any CDR determination method well-known in the art, including combinations of methods, can identify CDRs of variable domains. CDRs in each chain are held together closely by FRs to form variable regions. Typically, the variable regions VL / VH of the heavy and light chains can be obtained by linking the following numbered CDRs with FRs in the following combination: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0140] 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).

[0141] In this paper, the term "single-variable domain (VD)" refers to the smallest functional unit constituting the antigen-binding site of an antibody molecule. A single-variable domain can form a variable domain (which can be a heavy-chain or light-chain domain, including VH, VHH, or VL domains) that serves as a functional antigen-binding site without interacting with other variable domains (e.g., without the required VH / VL interaction between the VH and VL domains of a conventional tetrachain monoclonal antibody). The term "single-variable domain" is also used in this paper as "VD." Examples of "single-variable domains" include nanobodies (including VHH, humanized VHH, and / or camelified VH, such as camelified human VH), shark-derived single-domain antibody IgNARs, and (single-domain) antibodies (such as dAbs) that are VH domains or derived from VH domains. TM ) and antibodies (such as dAbs) that are structurally derived from or derived from the VL domain (single-domain) TM Immunoglobulins with a single variable domain based on and / or derived from a heavy chain variable domain (such as a VH or VHH domain) are generally preferred. A specific example of a single variable domain is the “VHH domain” (or simply “VHH”) as defined below.

[0142] In this paper, the terms "nanobody" and "VHH" refer to heavy chain antibodies lacking the light chain (e.g., derived from camels or sharks), and single-domain antibodies obtained by cloning their variable regions. These are the smallest functional antigen-binding fragments, 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. "Nanobody" is also called heavy chain single-domain antibody, VHH, VHH domain, VHH antibody fragment, or VHH antibody. VHH specifically binds to epitopes without the need for other antigen-binding domains (however, in conventional four-chain antibodies, the epitope is recognized by both the VL and VH domains). The VHH domain is a small, stable, and highly efficient antigen-recognizing unit formed by a single immunoglobulin domain. In this article, the "VHH domain" includes, but is not limited to, naturally occurring antibodies produced by camelids, antibodies produced by camelids that have been humanized, antibodies produced by non-camelids that have been camelified, and VHH domains obtained through screening using bacterial display technology.

[0143] It should be noted that, in order to distinguish the variable region of heavy chain antibodies that have lost the light chain from the variable region of the heavy chain in a typical four-chain antibody (two heavy (H) chains and two light (L) chains), the variable region of heavy chain antibodies that have lost the light chain is denoted as VHH, and the variable region of the heavy chain in a four-chain antibody is denoted as VH.

[0144] In this document, the term "antigen-binding fragment" refers to a substance containing all or part of the antibody's CDR (Cellular Dependent Antibody), 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 are capable of binding to the same antigen as the parent antibody.

[0145] 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.

[0146] In this article, the terms "Fc", "Fc region" and similar terms can be used interchangeably. Fc is part of the antibody (IgG), located at the C-terminus of the IgG molecule, and contains two structural domains, CH2 and CH3.

[0147] 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.

[0148] In this paper, the term "full-length heavy chain" refers to a heavy chain in an antibody that meets the above definition and has a complete or substantially complete structure, i.e., a peptide chain containing the antibody heavy chain variable region (VH) and constant regions (CH1, CH2, and CH3). "Substantially complete" can refer to a full-length heavy chain, or it can be a peptide chain that retains the basic heavy chain function after modification of the antibody heavy chain, such as a peptide chain with some amino acid residues deleted and / or mutated.

[0149] In this paper, the term "full-length light chain" refers to a light chain in an antibody that meets the above definition and has a complete or substantially complete structure, i.e., a peptide chain containing the variable region (VL) and constant region (CL) of the antibody heavy chain. "Substantially complete" can refer to a full-length light chain, or it can be a peptide chain in which the antibody light chain has been modified to retain the basic heavy chain function, such as a peptide chain with some amino acid residues deleted and / or mutated.

[0150] 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.

[0151] 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.

[0152] 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.

[0153] The binding of the multispecific binding molecules provided in this disclosure to the target antigen can also be referred to as the "half-maximum effective concentration (EC50)," which means the concentration of the drug or antibody that achieves 50% of the maximum biological effect after a specific exposure time. Generally, the smaller the EC50, the better the affinity, indicating that it can bind to the target protein at a lower concentration. The EC50 value can be determined by 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).

[0154] 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).

[0155] 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.

[0156] 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.

[0157] 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).

[0158] 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).

[0159] In this paper, the term "conjugate" refers to a compound formed by the linkage of at least two molecules.

[0160] 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.

[0161] 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.

[0162] 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.

[0163] 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).

[0164] In this document, the term "recombinant cell" refers to a cell into which exogenous polynucleotides and / or vectors may be introduced, or have been introduced. The exogenous polynucleotides may or may not be integrated into the genome of the "recombinant cell." Vectors may be introduced into the cell to construct recombinant cells, which are then used to express antibodies or antigen-binding fragments provided in this disclosure. The recombinant cells can be cultured to obtain the corresponding antibodies. Recombinant cells may 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, fungi (e.g., yeast), insect cells, or mammalian cells (e.g., CHO cells, HEK293 cells, or BHK cells).

[0165] 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 binding to antigens.

[0166] 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.

[0167] 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.

[0168] In this document, the term "diagnosis" refers to the identification of a pathological state, disease, or condition, such as the identification of a tumor-related disease, or the identification of a subject with a tumor-related disease who may benefit from a specific treatment regimen. In some embodiments, diagnosis includes identifying abnormal levels or activity of tumor-related proteins (such as tumor surface antigens or immune checkpoints). In some embodiments, diagnosis refers to identifying cancer in a subject.

[0169] 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.

[0170] 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.

[0171] 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.

[0172] Structure of multispecific binding molecules - Multispecific binding molecules that specifically bind to PVRIG / PD-L1:

[0173] In a first aspect, a multispecific binding molecule is provided, which binds to at least two different antigenic epitopes and specifically binds to PVRIG and PD-L1, wherein the multispecific binding molecule contains a PVRIG antigen-binding domain and a PD-L1 antigen-binding domain.

[0174] In an optional embodiment, the multispecific binding molecule binds to three different antigenic epitopes and also contains a third antigen-binding domain, which does not bind to the antigenic epitopes bound by the PVRIG antigen-binding domain, nor to the antigenic epitopes bound by the PD-L1 antigen-binding domain.

[0175] In an optional embodiment, at least one antigen-binding domain of the multispecific binding molecule is a single variable domain (VD), for example, but not limited to 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 antigen-binding domains being single variable domains.

[0176] In an optional implementation, the antigen-binding domains that bind to the same epitope are all single variable domains.

[0177] In an optional implementation, the antigen-binding domains in the multispecific binding molecule are all single variable domains.

[0178] In some optional embodiments, the PVRIG antigen-binding domain is a single variable domain that specifically binds PVRIG, the PD-L1 antigen-binding domain is a single variable domain that specifically binds PD-L1, and the third antigen-binding domain is a single variable domain that binds a third antigen; the multispecific binding molecule further contains an Fc, and at least one antigen-binding domain is attached to the N-terminus of the Fc. More specific embodiments are as follows:

[0179] In an optional implementation, two or three antigen-binding domains are connected in series at the N-terminus of Fc.

[0180] In an optional implementation, at least one antigen-binding domain is attached to the C-terminus of Fc.

[0181] In an optional implementation, two antigen-binding domains are connected in series at the N-terminus of Fc, and one antigen-binding domain is connected to the C-terminus of Fc.

[0182] In an optional implementation, the antigen-binding domain attached to the C-terminus of Fc is a PD-L1 antigen-binding domain or a third antigen-binding domain.

[0183] In an optional implementation, the antigen-binding domain adjacent to the N-terminus of Fc is a PD-L1 antigen-binding domain or a third antigen-binding domain.

[0184] In an optional embodiment, the multispecific binding molecule contains at least one polypeptide chain, wherein the polypeptide chain contains, from the N-terminus to the C-terminus, a PVRIG antigen-binding domain, a third antigen-binding domain, and an Fc and PD-L1 antigen-binding domain, respectively.

[0185] In an optional embodiment, the multispecific binding molecule contains at least one polypeptide chain, wherein the polypeptide chain contains, from the N-terminus to the C-terminus, a PVRIG antigen-binding domain, a PD-L1 antigen-binding domain, an Fc domain, and a third antigen-binding domain.

[0186] In an optional embodiment, the multispecific binding molecule contains at least one polypeptide chain, wherein the polypeptide chain contains, from the N-terminus to the C-terminus, a PVRIG antigen-binding domain, a third antigen-binding domain, a PD-L1 antigen-binding domain, and an Fc domain.

[0187] In an optional embodiment, the multispecific binding molecule contains two polypeptide chains.

[0188] In some alternative embodiments, at least one antigen-binding domain contains a VH and a VL for binding the antigen; that is, at least one antigen-binding domain in the multispecific molecule is formed by the association of VH and VL. More specific embodiments are as follows:

[0189] In an optional embodiment, the multispecific binding molecule contains a first polypeptide chain and a second polypeptide chain; the first polypeptide chain contains a full-length heavy chain, and the second polypeptide chain contains a full-length light chain; the VH in the full-length heavy chain and the VL in the full-length light chain constitute a PVRIG antigen-binding domain.

[0190] In an optional embodiment, the PD-L1 antigen-binding domain contains a single variable domain that specifically binds PD-L1, and the third antigen-binding domain contains a single variable domain that binds a third antigen.

[0191] In optional embodiments, the PD-L1 antigen-binding domain is attached to the Fc end of the full-length heavy chain, preferably to the C-terminus of the Fc end of the full-length heavy chain; the third antigen-binding domain is attached to the N-terminus or C-terminus of the full-length light chain; or, the third antigen-binding domain is attached to the N-terminus of the full-length heavy chain. Alternatively, in optional embodiments, the third antigen-binding domain and the PD-L1 antigen-binding domain are connected in series to the N-terminus or C-terminus of the full-length heavy chain; further optionally, the third antigen-binding domain is adjacent to the full-length heavy chain.

[0192] In an optional embodiment, the multispecific binding molecule contains a first polypeptide chain and a second polypeptide chain; the first polypeptide chain contains a full-length heavy chain, and the second polypeptide chain contains a full-length light chain; the VH in the full-length heavy chain and the VL in the full-length light chain constitute a third antigen-binding domain.

[0193] In an optional embodiment, the PD-L1 antigen-binding domain contains a single variable domain that specifically binds PD-L1, and the PVRIG antigen-binding domain contains a single variable domain that specifically binds PVRIG.

[0194] In optional embodiments, the PD-L1 antigen-binding domain is attached to the Fc end of the full-length heavy chain, preferably to the C-terminus of the Fc end of the full-length heavy chain; the PVRIG antigen-binding domain is attached to the N-terminus of the full-length light chain; or, the PVRIG antigen-binding domain is attached to the N-terminus of the full-length heavy chain. Alternatively, in optional embodiments, the PVRIG antigen-binding domain and the PD-L1 antigen-binding domain are connected in series to the N-terminus or C-terminus of the full-length heavy chain; further optionally, the PVRIG antigen-binding domain is adjacent to the full-length heavy chain.

[0195] In an optional embodiment, the multispecific binding molecule in the first aspect contains two first polypeptide chains; and / or, the multispecific binding molecule contains two second polypeptide chains. It is understood that the antigen-binding domain forms part of the first or second polypeptide chain by being linked to a full-length heavy chain or a full-length light chain.

[0196] In an optional embodiment, the multispecific binding molecule in the first aspect contains two first polypeptide chains and two second polypeptide chains.

[0197] In an optional embodiment, the multispecific binding molecule in the first aspect contains two first polypeptide chains and two second polypeptide chains, and the multispecific binding molecule has a symmetrical structure; the full-length heavy chain in the first polypeptide chain and the full-length light chain in the second polypeptide chain constitute a complete conventional antibody structure.

[0198] In an optional implementation, the “connection” between the domains in the multispecific binding molecule in the first aspect can be a direct connection or a connection through a linker.

[0199] In an optional implementation, when the multispecific binding molecule in the first aspect contains a single variable domain of a third antigen, the third antigen binding domain specifically binds to tumor surface antigens or immune checkpoints.

[0200] In optional embodiments, tumor surface antigens include 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, or OX40.

[0201] In optional implementations, immune checkpoints include PD-1, PD-L2, TIGIT, 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, or CEACAM1.

[0202] In an optional implementation, in the first aspect, the multispecific binding molecule specifically binds to PVRIG, PD-L1, and TIGIT.

[0203] Structure of multispecific binding molecules - Multispecific binding molecules having any one of the structures (A) to (G):

[0204] Secondly, a multispecific binding molecule is provided, said multispecific binding molecule binding to at least two different antigenic epitopes, said multispecific binding molecule being selected from any one of structures (A) to (G):

[0205] Structure (A):

[0206] It contains a first polypeptide chain and has the following structure:

[0207] [First VD] - [Connector] a1 -[Second VD]-Fc-[Connector] a2 -[Third VD];

[0208] At least one of the domains in the [first VD], [second VD], and [third VD] specifically binds to PVRIG, TIGIT, or PD-L1.

[0209] In an optional implementation, two domains in the [first VD], [second VD], and [third VD] respectively bind to two of PVRIG, TIGIT, and PD-L1, and the [first VD], [second VD], and [third VD] bind to different antigens.

[0210] In an optional implementation, the [first VD] binds to PVRIG, TIGIT, or PD-L1, the [second VD] binds to PVRIG, TIGIT, or PD-L1, and the [third VD] binds to PVRIG, TIGIT, or PD-L1, and the [first VD], [second VD], and [third VD] bind to different antigens.

[0211] In an optional implementation, [first VD] specifically binds to PVRIG.

[0212] In a further optional implementation, the [first VD] specifically binds to PVRIG, and the [second VD] specifically binds to TIGIT or PD-L1.

[0213] In a further optional implementation, the [first VD] specifically binds to PVRIG, the [second VD] specifically binds to TIGIT, and the [third VD] specifically binds to PD-L1 (refer to Figure 1); or the [first VD] specifically binds to PVRIG, the [second VD] specifically binds to PD-L1, and the [third VD] specifically binds to TIGIT (refer to Figure 2).

[0214] Structure (B):

[0215] It contains a first polypeptide chain and has the following structure:

[0216] [First VD] - [Connector] b1 -[Second VD]-[Connector] b2 -[Third VD]-Fc;

[0217] At least one of the first VD, the second VD, and the third VD specifically binds to PVRIG.

[0218] In an optional implementation, at least one of the [first VD], [second VD] and [third VD] specifically binds to TIGIT, and / or at least one of the [first VD], [second VD] and [third VD] specifically binds to PD-L1.

[0219] In an optional implementation, [first VD] specifically binds to PVRIG.

[0220] In a further optional implementation, the [first VD] specifically binds to PVRIG, and the [second VD] specifically binds to TIGIT or PD-L1.

[0221] In a further optional implementation, the [first VD] specifically binds to PVRIG, the [second VD] specifically binds to TIGIT, and the [third VD] specifically binds to PD-L1 (refer to Figure 3).

[0222] Structure (C):

[0223] Contains a first polypeptide chain and a second polypeptide chain:

[0224] The first polypeptide chain contains the following structure:

[0225] [VH]-[CH1]-[Fc]-[connector] c1 -[First VD], that is, a C-terminus of a full-length heavy chain is connected to at least one single variable structural domain;

[0226] The second polypeptide chain contains the following structure:

[0227] [Second VD] - [Connector] c2 -[VL]-[CL] means that the N-end of a full-length light chain is connected to at least one single variable structural domain;

[0228] The combination of [VH] and [VL] specifically binds to PVRIG;

[0229] Alternatively, one of the [first VD] and [second VD] specifically binds to PVRIG.

[0230] In an optional implementation, the combination of [VH] and [VL] specifically binds to PVRIG, and at least one domain of [first VD] and [second VD] specifically binds to TIGIT, and / or at least one domain of [first VD] and [second VD] specifically binds to PD-L1.

[0231] In an optional implementation, the combination of [VH] and [VL] specifically binds to PVRIG, and [first VD] specifically binds to PD-L1.

[0232] In an optional implementation, the combination of [VH] and [VL] specifically binds to PVRIG, the first VD specifically binds to PD-L1, and the second VD specifically binds to TIGIT (refer to Figure 4).

[0233] In an optional implementation, one of the domains of the [first VD] and the [second VD] specifically binds to PVRIG, and the other domain of the [first VD] and the [second VD] specifically binds to TIGIT or PD-L1.

[0234] In an optional implementation, one of the domains of [first VD] and [second VD] specifically binds to PVRIG, and the combination of [VH] and [VL] specifically binds to TIGIT or PD-L1.

[0235] In an optional implementation, [second VD] specifically binds to PVRIG.

[0236] In an optional implementation, the [second VD] specifically binds to PVRIG, and the [first VD] specifically binds to PD-L1.

[0237] In an optional implementation, [second VD] specifically binds to PVRIG, [first VD] specifically binds to PD-L1, and [VH] and [VL] combine to specifically bind to TIGIT (refer to Figure 9).

[0238] Structure (D):

[0239] Contains a first polypeptide chain and a second polypeptide chain:

[0240] The first polypeptide chain contains the following structure:

[0241] [First VD] - [Connector] d1 -[VH]-[CH1]-[Fc]-[connector] d2 -[Second VD], that is, the C-end and N-end of a full-length heavy chain are each connected to at least one single variable structural domain;

[0242] The second polypeptide chain contains the following structure:

[0243] [VL]-[CL], meaning the second polypeptide chain contains a full-length light chain;

[0244] The combination of [VH] and [VL] specifically binds to PVRIG, TIGIT, or PD1;

[0245] Furthermore, the [first VD] specifically binds to TIGIT or PVRIG; and the [second VD] specifically binds to PD-1, PD-L1, or TIGIT.

[0246] In an optional implementation, the combination of [VH] and [VL] specifically binds to PVRIG, the first VD specifically binds to TIGIT, and the second VD specifically binds to PD-L1 (refer to Figure 5).

[0247] In an optional implementation, the combination of [VH] and [VL] specifically binds to TIGIT, the first VD specifically binds to PVRIG, and the second VD specifically binds to PD-L1 (refer to Figure 10).

[0248] In an optional implementation, the combination of [VH] and [VL] specifically binds to PD1, the first VD specifically binds to PVRIG, and the second VD specifically binds to TIGIT (refer to Figure 13).

[0249] Structure (E):

[0250] Contains a first polypeptide chain and a second polypeptide chain:

[0251] The first polypeptide chain contains the following structure:

[0252] [VH]-[CH1]-[Fc]-[connector] e1 -[First VD]; that is, a C-terminus of a full-length heavy chain is connected to at least one single variable structural domain;

[0253] The second polypeptide chain contains the following structure:

[0254] [VL]-[CL]-[Connector] e2 -[Second VD]; that is, a C-terminus of a full-length light chain is connected to at least one single variable structural domain;

[0255] The combination of [VH] and [VL] specifically binds to PVRIG;

[0256] Alternatively, one of the [first VD] and [second VD] specifically binds to PVRIG.

[0257] In optional embodiments, the combination of [VH] and [VL] specifically binds to PVRIG, and at least one domain of [first VD] and [second VD] specifically binds to TIGIT, and / or, at least one domain of [first VD] and [second VD] specifically binds to PD-L1. In further optional embodiments, the combination of [VH] and [VL] specifically binds to PVRIG, [first VD] specifically binds to PD-L1, and [second VD] specifically binds to TIGIT (refer to Figure 6).

[0258] In an optional embodiment, the [second VD] specifically binds to PVRIG, and the combination of [VH] and [VL] specifically binds to TIGIT. In a further optional embodiment, the [first VD] specifically binds to PD-L1, the [second VD] specifically binds to PVRIG, and the combination of [VH] and [VL] specifically binds to TIGIT (refer to Figure 11).

[0259] In an optional embodiment, the [first VD] specifically binds to PVRIG, and the combination of [VH] and [VL] specifically binds to PD-1. In a further optional embodiment, the [first VD] specifically binds to PVRIG, the combination of [VH] and [VL] specifically binds to PD-1, and the [second VD] specifically binds to TIGIT (refer to Figure 14).

[0260] Structure (F):

[0261] Contains a first polypeptide chain and a second polypeptide chain:

[0262] The first polypeptide chain contains the following structure:

[0263] [First VD] - [Connector] f1 -[Second VD]-[Connector] f2 -[VH]-[CH1]-[Fc], that is, the N end of a full-length heavy chain is connected to at least two single variable structural domains in series;

[0264] The second polypeptide chain contains the following structure:

[0265] The combination of [VH] and [VL] specifically binds to PVRIG. In a further optional embodiment, the combination of [VH] and [VL] specifically binds to PVRIG, [first VD] specifically binds to PD-L1, and [second VD] specifically binds to TIGIT (refer to Figure 7).

[0266] Alternatively, the combination of [VH] and [VL] specifically binds to TIGIT. In a further optional embodiment, the combination of [VH] and [VL] specifically binds to TIGIT, [first VD] specifically binds to PD-L1, and [second VD] specifically binds to PVRIG (refer to Figure 12).

[0267] Alternatively, the combination of [VH] and [VL] specifically binds to PD-1. In a further optional embodiment, the combination of [VH] and [VL] specifically binds to PD-1; [first VD] specifically binds to PVRIG, and [second VD] specifically binds to TIGIT (refer to Figure 15).

[0268] Structure (G):

[0269] Contains a first polypeptide chain and a second polypeptide chain:

[0270] The first polypeptide chain contains the following structure:

[0271] [VH]-[CH1]-[Fc]-[connector] g1 -[First VD]-[Connector] g2 -[Second VD], that is, a single variable structural domain connected to the C-end of a full-length heavy chain;

[0272] The second polypeptide chain contains the following structure:

[0273] [VL]-[CL], meaning the second polypeptide chain contains a full-length light chain;

[0274] The combination of [VH] and [VL] specifically binds to PVRIG; or, one of [first VD] and [second VD] specifically binds to PVRIG.

[0275] In an optional implementation, one of the [first VD] and [second VD] specifically binds to PVRIG, and the other VD specifically binds to TIGIT.

[0276] In an optional implementation, one of [first VD] and [second VD] specifically binds to PVRIG, and the combination of [VH] and [VL] specifically binds to PD-1.

[0277] In an optional implementation, [first VD] specifically binds to TIGIT, [second VD] specifically binds to PVRIG, and [VH] and [VL] in combination specifically bind to PD-1 (refer to Figure 16).

[0278] In an optional implementation, the combination of [VH] and [VL] specifically binds to PVRIG, and at least one domain of [first VD] and [second VD] specifically binds to TIGIT, and / or at least one domain of [first VD] and [second VD] specifically binds to PD-L1.

[0279] In an optional implementation, the combination of [VH] and [VL] specifically binds to PVRIG, the first VD specifically binds to TIGIT, and the second VD specifically binds to PD-L1 (refer to Figure 8).

[0280] In the structures (A) to (G) above, [first VD], [second VD] and [third VD] represent single variable domains, respectively; [VH] represents the heavy chain variable domain VH, and [VL] represents the light chain variable domain VL; "[VH] and [VL] combine for specific binding" means that in multispecific binding molecules, [VH] and [VL] constitute an antigen-binding domain that binds to the same epitope.

[0281] In the structures (A) to (G) described above, the domains in the first and second polypeptide chains are arranged in order from the N-terminus to the C-terminus.

[0282] In an optional implementation, each structural domain in the above structures (A) to (G) is in the direction from the N end to the C end.

[0283] In the above structures (A) to (G), a1, a2, b1, b2, c1, c2, d1, d2, e1, e2, f1, f2, g1, and g2 represent the number of connectors, which are independent positive integers ≥ 0. When the value is 0, it means that there are no connectors and the two adjacent structural fields are directly connected.

[0284] In an optional implementation, a1, a2, b1, b2, c1, c2, d1, d2, e1, e2, f1, f2, g1, and g2 are each independently 0 or 1.

[0285] In the structures (A) to (G) above, the connectors can be the same or different. In this paper, "different" can mean that all elements in a set are different, or that at least one element is different from all other elements in the set.

[0286] In an optional implementation, each connector is independently selected from (G) x S) y Connector, where x is an integer selected from 1 to 5 (e.g., 1, 2, 3, 4, 5), and y is an integer selected from 1 to 6 (e.g., 1, 2, 3, 4, 5, 6). When the connector is (G x S) y When connecting elements, for example, the connecting elements shown are (G4S)2, (G4S)3, and (G4S)4.

[0287] In an optional implementation, the connector is selected from (G4S)2(GGGGSGGGGS, SEQ ID NO.16)

[0288] In an optional embodiment, each of the multispecific binding molecules selected from structures (A) to (G) independently contains two first polypeptide chains.

[0289] In an optional embodiment, the multispecific binding molecule selected from structure (A) or (B) contains two first polypeptide chains.

[0290] In an optional embodiment, each of the multispecific binding molecules selected from structures (C) to (G) independently contains two second polypeptide chains.

[0291] In an optional embodiment, each of the multispecific binding molecules selected from structures (C) to (G) independently contains two first polypeptide chains and two second polypeptide chains.

[0292] In an optional embodiment, the multispecific binding molecule selected from each of structures (A) to (G) is a symmetrical structure.

[0293] In an optional embodiment, each of the multispecific binding molecules selected from structures (C) to (G) independently contains two first polypeptide chains and two second polypeptide chains, and is a symmetrical structure. The full-length heavy chain portion of the first polypeptide chain and the full-length light chain portion of the second polypeptide chain in the multispecific binding molecule constitute a complete four-chain conventional antibody.

[0294] In an optional embodiment, the Fc in the multispecific binding molecule of the first and second aspects contains a hinge region, and the Fc is connected to the structural domain connected to its N-terminus through the hinge region.

[0295] In an optional implementation, the single variable structural domain in either the first or second aspect is VHH.

[0296] In an optional embodiment, the multispecific binding molecule contains all or part of the constant region of the antibody.

[0297] 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.

[0298] In an optional implementation, the constant region contains a sequence of part or all of the constant region of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD.

[0299] In an optional implementation, a constant region CL containing the antibody light chain is included.

[0300] In an optional embodiment, the CL in either the first or second aspect is a κ or λ type light chain CL, preferably a κ type light chain CL.

[0301] In an optional embodiment, the Fc in either the first or second aspect is derived from an IgG1, IgG2, IgG3, or IgG4 antibody.

[0302] In an optional embodiment, the Fc region in either the first or second aspect originates from the Fc region of IgG4.

[0303] In an optional implementation, the Fc region includes the Fc region of human-derived IgG4.

[0304] 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.

[0305] In an optional embodiment, the TIGIT binding molecule contains the Fc region of human-derived IgG4 and contains 228P, 234A, 235A and 447A mutations, which remove the function of the Fc receptor, allowing T cells to exert optimal activation.

[0306] Variable region sequence:

[0307] In an optional embodiment, the sequences of the antigen-binding domains of the first and second aspects are constructed according to the following optional embodiments. The CDR and FR in the variable region sequences described below are determined according to the Kabat definition, Chothia definition, AbM definition, Contact definition, or IMGT definition.

[0308] In an optional embodiment, the single variable domain of the multispecific binding molecule that binds to PVRIG is a VHH, wherein the VHH comprises complementarity-determining regions HCDR1, HCDR2, and HCDR3. HCDR1 comprises the amino acid residue AY; HCDR2 comprises the amino acid sequence shown in SEQ ID NO. 20; and HCDR3 comprises the amino acid sequence shown in SEQ ID NO. 28; or, HCDR1, HCDR2, and HCDR3 are selected from CDR1, CDR2, and CDR3 of the VHH shown in any one of SEQ ID NO. 1 to 5.

[0309] The CDR amino acid sequences defined according to different definition methods are shown in Table 1.

[0310] Table 1. CDR amino acid sequences defined by different methods of defining the single variable domain of PVRIG.

[0311] In an optional embodiment, the single variable domain of PD-L1 bound by the multispecific binding molecule is VHH, wherein VHH comprises complementarity-determining regions HCDR1, HCDR2, and HCDR3; HCDR1 comprises the amino acid residue TY; HCDR2 comprises the amino acid sequence shown in SEQ ID NO. 34; and HCDR3 comprises the amino acid sequence shown in SEQ ID NO. 42; or, HCDR1, HCDR2, and HCDR3 are selected from HCDR1, HCDR2, and HCDR3 of VHH shown in any one of SEQ ID NO. 2 and 7-10.

[0312] The CDR amino acid sequences defined according to different definition methods are shown in Table 2.

[0313] Table 2. CDR amino acid sequences defined by different definitions of the single variable domain of PD-L1.

[0314] In an optional embodiment, the single variable domain of the multispecific binding molecule binding TIGIT is a VHH, wherein the VHH comprises complementarity-determining regions HCDR1, HCDR2, and HCDR3; HCDR1 comprises the amino acid residue RY; HCDR2 comprises the amino acid sequence shown in SEQ ID NO. 48; and HCDR3 comprises the amino acid sequence shown in SEQ ID NO. 56; or, HCDR1, HCDR2, and HCDR3 are selected from the HCDR1, HCDR2, and HCDR3 of the VHH shown in any one of SEQ ID NO. 3 and 11–14. The CDR amino acid sequences defined according to different definitions are shown in Table 3.

[0315] Table 3. CDR amino acid sequences defined by different definitions of the single variable domain of TIGIT.

[0316] In an optional implementation, the VHH 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.

[0317] In an optional embodiment, each VHH in the multispecific binding molecule independently contains a framework region, which is derived from at least one of camel-derived antibodies, mouse-derived antibodies, human-derived antibodies, primate-derived antibodies, or mutants thereof.

[0318] In an optional embodiment, the VHH bound to PVRIG contains frame regions HFR1, HFR2, HFR3 and HFR4; the frame regions HFR1, HFR2, HFR3 and HFR4 of the VHH bound to PVRIG are selected from the HFR1, HFR2, HFR3 and HFR4 of the VHH shown in any one of SEQ ID NO.1 to 5.

[0319] In an optional implementation, the frame regions HFR1, HFR2, HFR3 and HFR4 of the VHH combined with PVRIG are selected from HFR1, HFR2, HFR3 and HFR4 of the VHH shown in SEQ ID NO.4.

[0320] In an optional embodiment, HFR1 comprises the amino acid sequence shown in SEQ ID NO. 57, HFR2 comprises the amino acid sequence shown in SEQ ID NO. 62, HFR3 comprises the amino acid sequence shown in SEQ ID NO. 69, and HFR4 comprises the amino acid sequence shown in SEQ ID NO. 70.

[0321] Taking antibody R3665 as an example, the FR amino acid sequences defined according to different definition methods are shown in Table 4.

[0322] Table 4. FR amino acid sequences defined by different VHH definitions in PVRIG.

[0323] In an optional embodiment, at least one VHH bound to PVRIG has an amino acid sequence as shown in SEQ ID NO.4.

[0324] In an optional embodiment, the VHH bound to PD-L1 contains frame regions HFR1, HFR2, HFR3 and HFR4; the frame regions HFR1, HFR2, HFR3 and HFR4 of the VHH bound to PVRIG are selected from the HFR1, HFR2, HFR3 and HFR4 of the VHH shown in any one of SEQ ID NO.2 and 7 to 10.

[0325] In an optional embodiment, the frame regions HFR1, HFR2, HFR3, and HFR4 of the VHH bound to PVRIG are selected from HFR1, HFR2, HFR3, and HFR4 of the VHH shown in SEQ ID NO. 7. In an optional embodiment, HFR1 comprises the amino acid sequence shown in SEQ ID NO. 72, HFR2 comprises the amino acid sequence shown in SEQ ID NO. 62, HFR3 comprises the amino acid sequence shown in SEQ ID NO. 69, and HFR4 comprises the amino acid sequence shown in SEQ ID NO. 70.

[0326] Taking antibody R3677 as an example, the FR amino acid sequences defined according to different definition methods are shown in Table 5.

[0327] Table 5 shows the FR amino acid sequences defined by different definitions of VHH in PD-L1.

[0328] In an optional embodiment, at least one amino acid sequence of VHH that binds to PD-L1 is shown in SEQ ID NO.7.

[0329] In an optional embodiment, the VHH bound to TIGIT contains frame regions HFR1, HFR2, HFR3 and HFR4; the frame regions HFR1, HFR2, HFR3 and HFR4 of the VHH bound to TIGIT are selected from the HFR1, HFR2, HFR3 and HFR4 of the VHH shown in any one of SEQ ID NO.3 and 11 to 14.

[0330] In an optional implementation, the frame regions HFR1, HFR2, HFR3 and HFR4 of the VHH combined with TIGIT are selected from HFR1, HFR2, HFR3 and HFR4 of the VHH shown in SEQ ID NO.11.

[0331] In an optional embodiment, HFR1 comprises the amino acid sequence shown in SEQ ID NO.81, HFR2 comprises the amino acid sequence shown in SEQ ID NO.86, HFR3 comprises the amino acid sequence shown in SEQ ID NO.93, and HFR4 comprises the amino acid sequence shown in SEQ ID NO.70.

[0332] Taking antibody R3694 as an example, the FR amino acid sequences defined according to different definition methods are shown in Table 6.

[0333] Table 6. FR amino acid sequences defined by different VHH definitions in TIGIT.

[0334] In an optional embodiment, at least one amino acid sequence of the VHH that binds to TIGIT is shown in SEQ ID NO.11.

[0335] In an optional embodiment, the HCDR1, HCDR2, and HCDR3 of the VH that binds PVRIG in the multispecific binding molecule are selected from the HCDR1, HCDR2, and HCDR3 of any VH shown in SEQ ID NO. 94 to 99; and / or, the LCDR1, LCDR2, and LCDR3 of the VL that binds PVRIG in the multispecific binding molecule are selected from the LCDR1, LCDR2, and LCDR3 of any VL shown in SEQ ID NO. 100 to 102.

[0336] In an optional embodiment, the HCDR1, HCDR2, and HCDR3 of the VH that binds PVRIG in the multispecific binding molecule are selected from the HCDR1, HCDR2, and HCDR3 of any VH shown in SEQ ID NO. 96; and / or, the LCDR1, LCDR2, and LCDR3 of the VL that binds PVRIG in the multispecific binding molecule are selected from the LCDR1, LCDR2, and LCDR3 of the VL shown in SEQ ID NO. 101.

[0337] In an optional embodiment, the amino acid sequence of VH binding PVRIG in the multispecific binding molecule is shown in SEQ ID NO. 96; and / or, the amino acid sequence of VL binding PVRIG in the multispecific binding molecule is shown in SEQ ID NO. 101.

[0338] In an optional embodiment, the HCDR1, HCDR2, and HCDR3 of the VH that binds TIGIT in the multispecific binding molecule are selected from the HCDR1, HCDR2, and HCDR3 of the VH shown in SEQ ID NO. 103; and / or, the LCDR1, LCDR2, and LCDR3 of the VL that binds TIGIT in the multispecific binding molecule are selected from the LCDR1, LCDR2, and LCDR3 of the VL shown in SEQ ID NO. 104.

[0339] In an optional embodiment, the amino acid sequence of VH binding TIGIT in the multispecific binding molecule is shown in SEQ ID NO. 103; and / or, the amino acid sequence of VL binding TIGIT in the multispecific binding molecule is shown in SEQ ID NO. 104.

[0340] In an optional embodiment, the HCDR1, HCDR2, and HCDR3 of the VH that binds PD-1 in the multispecific binding molecule are selected from the HCDR1, HCDR2, and HCDR3 of the VH shown in SEQ ID NO. 105; and / or, the LCDR1, LCDR2, and LCDR3 of the VL that binds PD-1 in the multispecific binding molecule are selected from the LCDR1, LCDR2, and LCDR3 of the VL shown in SEQ ID NO. 106.

[0341] In an optional embodiment, the amino acid sequence of VH binding PD-1 in the multispecific binding molecule is shown in SEQ ID NO.105; and / or, the amino acid sequence of VL binding PD-1 in the multispecific binding molecule is shown in SEQ ID NO.106.

[0342] In optional embodiments, the multispecific binding molecule is selected from structure (G), with the first polypeptide chain amino acid sequence as shown in SEQ ID NO. 107 and the second polypeptide chain amino acid sequence as shown in SEQ ID NO. 108; or, the multispecific binding molecule is selected from structure (A), with the first polypeptide chain amino acid sequence as shown in SEQ ID NO. 109; or, the multispecific binding molecule is selected from structure (D), with the first polypeptide chain amino acid sequence as shown in SEQ ID NO. 110 and the second polypeptide chain amino acid sequence as shown in SEQ ID NO. 111; or, the multispecific binding molecule is selected from structure (D), with the first polypeptide chain amino acid sequence as shown in SEQ ID NO. 112 and the second polypeptide chain amino acid sequence as shown in SEQ ID NO. 113; or, the multispecific binding molecule is selected from structure (F), with the first polypeptide chain amino acid sequence as shown in SEQ ID NO. 114 and the second polypeptide chain amino acid sequence as shown in SEQ ID NO. 115; or, the multispecific binding molecule is selected from structure (C), with the first polypeptide chain amino acid sequence as shown in SEQ ID NO. 116 and the second polypeptide chain amino acid sequence as shown in SEQ ID NO. 108. As shown in NO. 117; or, the multispecific binding molecule is selected from structure (D), with the first polypeptide chain amino acid sequence as shown in SEQ ID NO. 118 and the second polypeptide chain amino acid sequence as shown in SEQ ID NO. 119; or, the multispecific binding molecule is selected from structure (E), with the first polypeptide chain amino acid sequence as shown in SEQ ID NO. 120 and the second polypeptide chain amino acid sequence as shown in SEQ ID NO. 121; or, the multispecific binding molecule is selected from structure (C), with the first polypeptide chain amino acid sequence as shown in SEQ ID NO. 122 and the second polypeptide chain amino acid sequence as shown in SEQ ID NO. 123; or, the multispecific binding molecule is selected from structure (A), with the first polypeptide chain amino acid sequence as shown in SEQ ID NO. 124; or, the multispecific binding molecule is selected from structure (A), with the first polypeptide chain amino acid sequence as shown in SEQ ID NO. 125; or, the multispecific binding molecule is selected from structure (E), with the first polypeptide chain amino acid sequence as shown in SEQ ID NO. 126 and the second polypeptide chain amino acid sequence as shown in SEQ ID NO. 125. As shown in NO. 127; or, the multispecific binding molecule is selected from structure (F), the amino acid sequence of the first polypeptide chain is shown in SEQ ID NO. 128, and the amino acid sequence of the second polypeptide chain is shown in SEQ ID NO. 129; or, the multispecific binding molecule is selected from structure (E), the amino acid sequence of the first polypeptide chain is shown in SEQ ID NO. 130, and the amino acid sequence of the second polypeptide chain is shown in SEQ ID NO.As shown in SEQ ID NO. 131; or, the multispecific binding molecule is selected from structure (F), with the first polypeptide chain amino acid sequence as shown in SEQ ID NO. 132 and the second polypeptide chain amino acid sequence as shown in SEQ ID NO. 133; or, the multispecific binding molecule is selected from structure (G), with the first polypeptide chain amino acid sequence as shown in SEQ ID NO. 134 and the second polypeptide chain amino acid sequence as shown in SEQ ID NO. 135.

[0343] The first and second aspects identified various multispecific molecules with different structures, and further humanized the antigen-binding domains of these multispecific molecules. In in vitro functional experiments using exhausted T cells, several multispecific molecules exhibited significantly superior activity compared to the positive control trispecific antibody. Furthermore, in a mouse model of reconstituted human melanoma A375-inoculated human PBMCs with low PD-L1 antibody response, their antitumor efficacy was significantly superior to the positive control trispecific antibody. In the preferred embodiment, a multispecific molecule with structure (G) that specifically binds TIGIT with [first VD], specifically binds PVRIG with [second VD], and specifically binds PD-1 with a combination of [VH] and [VL] (e.g., R3891 as given in the examples) showed a more significant improvement over PD-L1 monoclonal antibodies in in vitro functional experiments. Simultaneously, in in vivo pharmacodynamic experiments, when PD-L1 monoclonal antibody-unresponsive donors were screened to construct a mouse model of tumors, the aforementioned multispecific molecules demonstrated highly desirable antitumor activity in this model with low PD-L1 monoclonal antibody sensitivity.

[0344] Nucleic acid molecules, vectors, and recombinant cells:

[0345] Thirdly, a biomaterial is provided, said biomaterial comprising any one of the following (i) to (iii):

[0346] (i) A nucleic acid molecule that encodes the multispecific binding molecule described in the first or second aspect;

[0347] (ii) A vector, characterized in that the vector carries the nucleic acid molecule described in (i);

[0348] (iii) A recombinant cell comprising the nucleic acid molecule described in (i), or the vector described in (ii), or expressing the multispecific binding molecule described in the first or second aspect.

[0349] application:

[0350] Fourthly, the use of the multispecific binding molecule described in the first or second aspect, or the biomaterial described in the third aspect, in any of the following:

[0351] (I) To prepare products for the treatment, prevention or relief of tumor-related diseases, symptoms or conditions;

[0352] (II) Treatment, prevention or relief of tumor-related diseases, symptoms or conditions;

[0353] (III) Activate immune cells in the tumor immune microenvironment;

[0354] In an optional embodiment, the immune cells include T cells and / or NK cells;

[0355] In an optional embodiment, the T cells include CMV+CD8+ T cells or T cells induced by mature dendritic cells.

[0356] (IV) Increase the secretion of IFN-γ and / or pro-inflammatory cytokines in the subjects;

[0357] In an optional implementation, the secretion of IFN-γ by T cells in the subject is increased.

[0358] In an optional implementation, (III) and (IV) are for non-diagnostic and non-therapeutic purposes.

[0359] Fifthly, a pharmaceutical composition is provided, the pharmaceutical composition comprising the multispecific binding molecule described in the first or second aspect, or the biological material described in the third aspect.

[0360] In an optional embodiment, the pharmaceutical composition may further include a pharmaceutically acceptable carrier and / or excipient.

[0361] 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.

[0362] In an optional embodiment, the pharmaceutical composition may further include one or more active pharmaceutical ingredients having other therapeutic effects, such as: 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.

[0363] In an optional embodiment, the pharmaceutical composition is used to treat, prevent, or alleviate tumor-related diseases, symptoms, or conditions.

[0364] 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 multispecific binding molecule of the first or second aspect, or the biological material of the third aspect, or the pharmaceutical composition of the fifth aspect.

[0365] In an optional implementation, the disease, symptom, or condition includes tumor-related diseases, symptoms, or conditions.

[0366] 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.

[0367] 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.

[0368] 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.

[0369] 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.

[0370] In optional embodiments, the pharmaceutical composition described herein 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 herein may be administered in combination with a second therapeutic agent (e.g., a combination of one or more of 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).

[0371] 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.

[0372] Methods for preparing multispecific binding molecules:

[0373] A seventh aspect provides a method for preparing the multispecific binding molecule described in the first or second aspect, the method comprising culturing recombinant cells as described in the third aspect, and then isolating and purifying the multispecific binding molecule.

[0374] In an optional embodiment, the preparation method further includes converting and expressing a polynucleotide encoding the multispecific binding molecule into cells.

[0375] In an optional embodiment, the preparation method further includes synthesizing a polynucleotide containing the gene encoding the multispecific 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 multispecific binding molecule through purification.

[0376] The present disclosure is 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 disclosure in any way.

[0377] Materials preparation:

[0378] The target genes encoding human PVRIG (Uniprot database ID: Q6DKI7), monkey PVRIG (Uniprot database ID: A0A2K5WVV8), and mouse PVRIG were cloned into plasmids containing His, human Fc, or mouse Fc, respectively. These plasmids were then transfected into HEK293 cells for transient expression, thereby obtaining hPVRIG-mFc (R2248, amino acid sequence as shown in SEQ ID NO.142) and hPVRIG-hFc (R2249, amino acid sequence as shown in SEQ ID NO.143).

[0379] 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, amino acid sequence as shown in SEQ ID NO.144) and hTIGIT-mFc (number R0253-3, amino acid sequence as shown in SEQ ID NO.145).

[0380] The target gene encoding human PD-L1 (Uniprot database ID: Q9NZQ7) was cloned into a plasmid containing a mouse Fc tag, and then transfected into HEK293 cells for transient expression to obtain hPD-L1-mFc (R2492, amino acid sequence as shown in SEQ ID NO.146).

[0381] The target gene encoding human PVRIG (Uniprot database ID: Q6DKI7) was cloned into the plvx lentiviral plasmid, the lentivirus was packaged and its titer was detected, and 293T cells were infected to express the membrane protein, thereby obtaining a cell line overexpressing hPVRIG, namely 293T-hPVRIG.

[0382] The target gene encoding cynomolgus monkey PVRIG (Uniprot database ID: XP_014989941.2) was cloned into the plvx lentiviral plasmid, the lentivirus was packaged and its titer was detected, and 293T cells were infected to express the membrane protein, thereby obtaining a cell line overexpressing cynomolgus monkey PVRIG, namely 293T-cynoPVRIG.

[0383] Using the sequence encoding human PVRIG (Uniprot database ID: Q6DKI7) as a template, PVRIG mRNA was prepared by in vitro transcription, and after purification, the mRNA was encapsulated with LNP to prepare human PVRIG mRNA-LNP complex.

[0384] 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.

[0385] 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.

[0386] The target gene encoding human PD-L1 (Uniprot database ID: Q9NZQ7) was cloned into a plvx lentiviral plasmid, the lentivirus was packaged and its titer was detected, and CHO cells were infected to express membrane proteins, thereby obtaining a cell line overexpressing hPD-L1, which is named CHO-hPD-L1.

[0387] The target gene encoding cynomolgus monkey PD-L1 was cloned into the plvx lentiviral plasmid, the lentivirus was packaged and its titer was detected, and CHO cells were infected to express membrane proteins, thereby obtaining a cell line overexpressing hPD-L1, namely CHO-cynoPD-L1.

[0388] The PVRIG reporter gene system (CBP74120+CBP74121) was purchased from Nanjing Kebai Biotechnology Co., Ltd.

[0389] The target genes encoding human PVRL2 (Uniprot database ID: Q92692) and human PVR (Uniprot database ID: P15151) were cloned into the plvx lentiviral plasmid, and human PVRL2 or human PVR lentivirus was packaged and the titer was detected.

[0390] The target gene encoding human PD1 (Uniprot database ID: Q15116) was cloned into the plvx lentiviral plasmid, the lentivirus was packaged, and the titer was detected.

[0391] 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.

[0392] Jurkat-NFAT reporter gene cells were infected with human PD1 lentivirus to construct Jurkat-hPD1-NFAT cells; CHO-OKT3 cells expressing CD3 antibody OKT3 on their membrane surface were infected with human PD-L1 lentivirus to construct CHO-OKT3-PD-L1 cells. The two cell types constituted the human PD1 / PD-L1 reporter gene system.

[0393] Jurkat-NFAT reporter gene cells were infected with human PVRIG lentivirus, human TIGIT lentivirus, and human PD1 lentivirus to construct Jurkat-PVRIG-TIGIT-PD1 cells that simultaneously express human PVRIG, human TIGIT, and human PD1; CHO-OKT3 cells expressing the CD3 antibody OKT3 on their membrane surface were infected with human PVRL2 lentivirus, human PVR lentivirus, and human PD-L1 lentivirus to construct CHO-OKT3-PVRL2-PVR-PD-L1 cells; the two cell types constituted the human PVRIG-TIGIT-PD-L1 three-target reporter gene system.

[0394] A target antibody for the hIgG4 (S228P / F234A / L235A / K447A) subtype was prepared according to the sequence numbered No. 81 in international publication number WO2021180205A1. The target antibody is numbered R2243 (the amino acid sequence of VHH is shown in SEQ ID NO. 141), which is a PVRIG positive nanobody.

[0395] A targeting antibody for the hIgG4 (S228P / F234A / L235A / K447A) subtype was prepared according to the sequence number No. 34 in international publication number WO2021216468A1. The antibody is numbered R2882 (the amino acid sequence of VHH is shown in SEQ ID NO. 140) and is a TIGIT positive nanobody.

[0396] The sequence of KN035 (Envorimab), a PD-L1 antibody from KNJ Biopharma, was used to prepare a target antibody for the hIgG4 (S228P / F234A / L235A / K447A) subtype. The target antibody was numbered R2429 (the amino acid sequence of VHH is shown in SEQ ID NO.139), which is a PD-L1 positive nanobody.

[0397] Based on the sequences numbered No. 93 and No. 68 in international publication number WO2023040945A1, a target antibody for the hIgG4 (S228P / F234A / L235A / K447A) subtype was prepared. Its number is R3023 (the amino acid sequence of the heavy chain is shown in SEQ ID NO. 137, and the amino acid sequence of the light chain is shown in SEQ ID NO. 138), which is a positive trispecific antibody for PVRIG-TIGIT-PD1.

[0398] PVRIG nanobody

[0399] The relevant sequences of PVRIG nanobodies are shown in Table 7.

[0400] Table 7 PVRIG antibody sequences

[0401] Example 1: Discovery of PVRIG nanobodies

[0402] Alpacas were immunized subcutaneously at multiple sites using recombinant human PVRIG-hFc, human PVRIG-mFc, or PVRIG mRNA-LNP complexes as immunogens to induce the production of anti-human PVRIG 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 the antigen, single clones were selected for testing. Clones that specifically bind to the human PVRIG antigen 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, hIgG4 (S228P / F234A / L235A / K447A), which was then recombinantly expressed and purified to form the PVRIG nanobody R3242. The amino acid sequence of the R3242 heavy chain variable region is shown in Table 7. The CDR information of the R3242 heavy chain variable region is shown in Table 1.

[0403] Example 2: Evaluation of PVRIG nanobodies

[0404] 1. Binding activity of candidate molecules with 293T-hPVRIG cells and 293T-cynoPVRIG cells

[0405] 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; for instrumentation: resuspend in 100μL 1×PBS, and perform detection. Plot the antibody concentration logarithm on the x-axis and the 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.

[0406] The results are shown in Figures 17 and 18. The results show that the binding activity of the PVRIG nanobody R3242 to 293-hPVRIG is close to that of the positive antibody R2243, and the binding activity to 293-cynoPVRIG is significantly stronger than that of the positive antibody R2243.

[0407] 2. Reporter gene activity assay of candidate molecules

[0408] PVRIG is an inhibitory receptor expressed on T cells. When it binds to its ligand PVRL2 (CD112, Nectin-2) 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 PVRIG reporter gene activity with a large detection window was developed using Jurkat-hPVRIG-NFAT and CHO-OKT3-PVRL2 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. The results are shown in Figure 19. The results show that the PVRIG nanobody R3242 exhibited significantly stronger activity than the positive control R2243 in the PVRIG reporter gene experiment.

[0409] Example 3: Activity evaluation of humanized PVRIG nanobodies

[0410] Using R3242 as the parent sequence, three-dimensional structural homology modeling was performed on the selected sequence, and the structure was determined to be acceptable through methods such as Laplace plots. The antibody sequence was compared with the antibody GermLine database to obtain a human template with high homology (IGHV3-11*01). The CDR was transplanted into the corresponding human template. The transplanted antibody was subjected to three-dimensional structural simulation and analysis again. Residues that were embedded, those that directly interacted with the CDR region, and those that had an important influence on the conformation of the variable region were subjected to backcomplex modification. Chemically unstable amino acid residues in the CDR region were optimized to generate a series of humanized antibody sequences.

[0411] A series of humanized molecules, R3662, R3664, and R3665, of the hIgG4 (S228P / F234A / L235A / K447A) isoform were prepared. The amino acid sequences of the heavy chain variable region of the humanized PVRIG nanobodies R3662, R3664, and R3665 are shown in Table 7.

[0412] 1. Evaluation of the reporter gene activity of humanized PVRIG nanobodies

[0413] The evaluation method was the same as in Part 2 of Example 2, and the results are shown in Figure 20. R3242 was the parent molecule, and R3662, R3664, and R3665 were humanized molecules. Among the humanized molecules, R3665 showed superior reporter gene activity compared to the parent molecule R3242.

[0414] 2. Binding activity of humanized PVRIG nanobodies with 293T-hPVRIG cells and 293T-cynoPVRIG cells

[0415] The evaluation method was the same as in Part 1 of Example 2, and the results are shown in Figures 21 and 22. The binding activity of the humanized molecule R3665 to 293-hPVRIG and 293-cynoPVRIG was comparable to that of the parent molecule.

[0416] PD-L1 nanobody

[0417] The PD-L1 nanobody-related sequences are shown in Table 8.

[0418] Table 8 PD-L1 antibody sequences

[0419] Example 4: Discovery of PD-L1 Nanobodies

[0420] Alpacas were immunized subcutaneously at multiple sites using recombinant human PD-L1-mFc as an immunogen to induce the production of anti-human PD-L1 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 the human PD-L1 antigen were selected to demonstrate receptor-ligand binding blocking activity. Clones with ideal binding activity and receptor-ligand binding blocking activity were selected for sequencing, and an antibody, hIgG4 (S228P / F234A / L235A / K447A), was constructed for recombinant expression and antibody purification, resulting in the PD-L1 nanobody R3377. The amino acid sequence of the variable region of the R3377 heavy chain is shown in Table 8, and the amino acid sequence information of the CDR of the R3377 heavy chain variable region is shown in Table 2.

[0421] Example 5: Binding activity of candidate molecules with CHO-hPD-L1 and CHO-cynoPD-L1 cells

[0422] 1. FACS binding assay method: Same as Part 1 of Example 2. The results are shown in Figures 23 and 24. The binding activity of PD-L1 nanobody R3377 to CHO-hPD-L1 is similar to that of positive antibody R2429 (slightly better at EC50), and the binding activity of R3377 to CHO-cynoPD-L1 cells is comparable to that of positive antibody R2429.

[0423] 2. Reporter gene activity assay of candidate molecules

[0424] PD-1 is an inhibitory receptor expressed on T cells. When it binds to its ligand PD-L1 expressed on dendritic cells (DCs) or tumor cells, it can inhibit T cell immune activation. A method for detecting PD-L1 reporter gene activity with a large detection window was developed using Jurkat-hPD-1-NFAT and CHO-OKT3-PD-L1 cells to detect the activity of candidate antibodies in activating T cells. A nonlinear four-parameter fitting was performed, with the logarithm of antibody concentration on the x-axis and 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. The results are shown in Figure 25. The reporter gene activity of the PD-L1 nanobody R3377 was stronger than that of the positive antibody R2429.

[0425] Example 6: Activity evaluation of humanized PD-L1 nanobody

[0426] Using R3377 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 diagrams. The antibody sequences were compared with the GermLine antibody database to obtain a highly homologous human template (IGHV3-7*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.

[0427] A series of humanized molecules R3674-R3677 of the hIgG4 (S228P / F234A / L235A / K447A) isoforms were prepared. The amino acid sequences of the heavy chain variable region of the humanized PD-L1 nanobodies R3674-R3677 are shown in Table 8.

[0428] 1. Reporter gene assay evaluation of humanized PD-L1 nanobody

[0429] The evaluation method was the same as in Part 2 of Example 5, and the results are shown in Figure 26. R3377 was the parent molecule, and R3674-R3677 were humanized molecules. Among the humanized molecules, R3677 exhibited the best reporter gene activity, comparable to that of the parent molecule R3377.

[0430] 2. Binding activity of humanized PD-L1 nanobody to CHO-hPD-L1 cells and CHO-cynoPD-L1 cells

[0431] The evaluation method was the same as in Part 1 of Example 5, and the results are shown in Figures 27 and 28. The binding activity of the humanized molecule R3677 to CHO-hPD-L1 and CHO-cynoPD-L1 was superior to that of the parent molecule R3377.

[0432] TIGIT Nanobody

[0433] The relevant sequences of TIGIT nanobody are shown in Table 9.

[0434] Table 9. TIGIT Nanobody Heavy Chain Variable Region Sequence

[0435] Example 7: Discovery of TIGIT Nanobodies

[0436] 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, hIgG4 (S228P / F234A / L235A / K447A), which 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 9, and the amino acid sequence information of the CDR of the R3179 heavy chain variable region is shown in Table 3.

[0437] Example 8: Evaluation of TIGIT Nanobody

[0438] 1. Binding activity of candidate molecules with CHO-hTIGIT and CHO-cynoTIGIT cells

[0439] FACS binding assay method: Same as Part 1 of Example 2. Results are shown in Figures 29 and 30. 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.

[0440] 2. Reporter gene activity assay of candidate molecules

[0441] 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 method with a large detection window for detecting TIGIT reporter gene activity 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 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. The results, shown in Figure 31, indicate that the TIGIT nanobody R3179 showed significantly stronger activity than the positive control R2882 in the PVRIG reporter gene assay.

[0442] Example 9: Activity evaluation of humanized TIGIT nanobody

[0443] 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.

[0444] Prepare a series of humanized molecules R3693-R3696 of hIgG4 (S228P / F234A / L235A / K447A) subtypes

[0445] 1. Evaluation of the reporter gene activity of humanized TIGIT nanobodies

[0446] The evaluation method was the same as in Part 2 of Example 8, and the results are shown in Figure 32. 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.

[0447] 2. Binding activity of humanized TIGIT nanobody with CHO-hTIGIT cells and CHO-cynoTIGIT cells

[0448] The evaluation method was the same as in Part 1 of Example 8. The results are shown in Figures 33 and 34. The binding activities of the humanized molecule R3694 with CHO-hTIGIT and CHO-cynoTIGIT were similar.

[0449] Conventional antibody construction

[0450] Example 10: Preparation and evaluation of humanized PVRIG mouse-derived conventional antibodies

[0451] Antibody preparation: hIgG4 (S228P / F234A / L235A / K447A) subtype chimeric antibody R3014 was prepared using the mouse antibody sequence named CG004-1 in the patent application number 202311579153.1 (the amino acid sequence of its heavy chain variable region is shown in SEQ ID NO.94, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO.100).

[0452] QVQLQQSGAELVRPGTSVKVSCKASEYAFTNYFIEWVKQRPGQGLEWIGVINPGSGGTNYNEKFEGKATLTADKSSSTAYMQLSSSLTSEDSAVYFCTTFYFAYWGQGTLVTVSA(SEQ ID NO.94);

[0453] DIVMTQTPSFLSVSVGEKVTMSCKSSQSLLNSGNQKNYLAWYQQKPGQPPKLLIYGASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNGHSSPYTFGGGTKLEIK (SEQ ID NO. 100).

[0454] Three-dimensional structural homology modeling was performed on the 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 human template (IGKV4-1*01) with high homology. The CDR was transplanted into the corresponding human template. The transplanted antibodies were subjected to three-dimensional structural simulation and analysis again. Residues that were embedded, interacted directly with the CDR region, and had a significant impact on the conformation of the variable region were subjected to backcomplex modification. Chemically unstable amino acid residues in the CDR region were optimized to generate a series of humanized antibody sequences. A series of humanized molecules of hIgG4 (S228P / F234A / L235A / K447A) isotypes were prepared: R3942-R3950. The amino acid sequences of the heavy chain variable region of antibody R3942-R3950 are shown in Table 10, the amino acid sequences of the light chain variable region are shown in Table 11, and the combinations of the heavy chain variable region and the light chain variable region are shown in Table 12.

[0455] Table 10 Heavy chain sequences of humanized PVRIG mouse antibody

[0456] Table 11 Light chain sequence of humanized PVRIG mouse antibody

[0457] Table 12 Combinations of heavy chain and light chain variable regions in humanized PVRIG mouse antibodies

[0458] 2. Evaluation of the reporter gene activity of humanized PVRIG mouse antibody

[0459] The evaluation method was the same as in Part 2 of Example 2, and the results are shown in Figure 35. R3014 was the parent molecule, and R3942-R3950 were humanized molecules. Among the humanized molecules, R3944 showed superior reporter gene activity compared to the parent molecule R3014.

[0460] 3. Binding activity of humanized PVRIG nanobodies with 293T-hPVRIG cells and 293T-cynoPVRIG cells

[0461] The evaluation method was the same as in Part 1 of Example 2. The results are shown in Figures 36 and 37. The binding activity of the humanized molecule R3944 to 293-hPVRIG cells was close to that of the parental molecule R3014.

[0462] Example 11: Construction of TIGIT Conventional Antibody

[0463] The humanized sequence of the mouse TIGIT antibody named 3TIGIT-16 from patent publication number CN115925945A was used to construct a trispecific antibody, and antibody R2430 carrying hIgG4 (S228P / F234A / L235A / K447A) was prepared. The amino acid sequences of VH and VL of antibody R2430 are shown in Table 13.

[0464] Table 13 Sequence of Humanized TIGIT Mouse Antibody R2430

[0465] Example 12: Construction of a conventional PD-1 antibody

[0466] The humanized PD-1 antibody sequence named PD-1-112-C2 (amino acid sequence shown in Table 1) from patent publication number CN115466328A was used to construct a trispecific antibody, and antibody R4149 containing hIgG4 (S228P / F234A / L235A / K447A) was prepared. The amino acid sequences of VH and VL of antibody R4149 are shown in Table 14.

[0467] Table 14 Sequence of Humanized PD-1 Murine Antibody R4149

[0468] Construction of trispecific antibodies with different structures

[0469] Example 13: Construction of trispecific antibodies with different structures

[0470] Trispecific antibodies with different structures were prepared using the humanized PVRIG nanobody R3665 prepared in Example 3; the humanized PD-L1 nanobody R3677 prepared in Example 6; the humanized TIGIT nanobody R3694 prepared in Example 9; and the humanized PVRIG conventional antibody R3944 prepared in Example 10; the humanized TIGIT conventional antibody R2430 prepared in Example 11; and the humanized PD-1 conventional antibody R4149 prepared in Example 12.

[0471] [PVRIG-VHH] represents the VHH of the humanized PVRIG nanobody R3665 prepared in Example 3, with the amino acid sequence shown in SEQ ID NO.4;

[0472] [PD-L1-VHH] represents the VHH of the humanized PD-L1 nanobody R3677 prepared in Example 6, with the amino acid sequence shown in SEQ ID NO.7;

[0473] [TIGIT-VHH] represents the VHH of the humanized TIGIT nanobody R3694 prepared in Example 9, with the amino acid sequence shown in SEQ ID NO.11;

[0474] [Fc] represents the Fc of hIgG4 (S228P / F234A / L235A / K447A).

[0475] [PVRIG-H] represents the full-length heavy chain of the humanized PVRIG conventional antibody R3944 prepared in Example 10. [PVRIG-H] is [PVRIG-VH]-[CH1]-[Fc] from the N-terminus to the C-terminus. [PVRIG-VH] represents the VH of antibody R3944, and the amino acid sequence is shown in SEQ ID NO.96. [CH1] represents the constant region CH1 of the antibody heavy chain.

[0476] [PVRIG-L] represents the full-length light chain of the humanized PVRIG conventional antibody R3944 prepared in Example 10. [PVRIG-L] is [PVRIG-VL]-[Cκ] from the N-terminus to the C-terminus. [PVRIG-VL] represents the VL of antibody R3944, and the amino acid sequence is shown in SEQ ID NO.101. [Cκ] represents the constant region of the κ-type light chain of the antibody.

[0477] [TIGIT-H] represents the full-length heavy chain of the humanized TIGIT conventional antibody R2430 prepared in Example 11. [TIGIT-H] is [TIGIT-VH]-[CH1]-[Fc] from the N-terminus to the C-terminus. [TIGIT-VH] represents the VH of antibody R2430, and the amino acid sequence is shown in SEQ ID NO.103. [CH1] represents the constant region CH1 of the antibody heavy chain.

[0478] [TIGIT-L] represents the full-length light chain of the humanized TIGIT conventional antibody R2430 prepared in Example 11. [TIGIT-L] is [TIGIT-VL]-[Cκ] from the N-terminus to the C-terminus. [TIGIT-VL] represents the VL of antibody R2430, and the amino acid sequence is shown in SEQ ID NO.104. [Cκ] represents the constant region of the κ-type light chain of the antibody.

[0479] [PD-1-H] represents the full-length heavy chain of the humanized PD-1 conventional antibody R4149 prepared in Example 12. [PD-1-H] is [PD-1-VH]-[CH1]-[Fc] from the N-terminus to the C-terminus. [PD-1-VH] represents the VH of antibody R4149, and the amino acid sequence is shown in SEQ ID NO.105. [CH1] represents the constant region CH1 of the antibody heavy chain.

[0480] [PD-1-L] represents the full-length light chain of the humanized PD-1 conventional antibody R4149 prepared in Example 12. [PD-1-L] is [PD-1-VL]-[Cκ] from the N-terminus to the C-terminus. [PD-1-VL] represents the VL of antibody R4149. The amino acid sequence is shown in SEQ ID NO.106. [Cκ] represents the constant region of the κ-type light chain of the antibody.

[0481] The structures in each of the following peptide chains are arranged in order from the N-terminus to the C-terminus.

[0482] [Connector] is GGGGSGGGGS (SEQ ID NO.136).

[0483] 1. Nanoscale trispecific antibody:

[0484] Trispecific antibody R3876: It has the above structure (A), and its structural schematic diagram is shown in Figure 1:

[0485] It contains two symmetrical first polypeptide chains, with the following structure:

[0486] [PVRIG-VHH]-[Connector]-[TIGIT-VHH]-[Fc]-[Connector]-[PD-L1-VHH];

[0487] The amino acid sequence of the first polypeptide chain is shown in SEQ ID NO.109.

[0488] Trispecific antibody R3877: It has the above structure (A), and its structural diagram is shown in Figure 2:

[0489] It contains two symmetrical first polypeptide chains, with the following structure:

[0490] [PVRIG-VHH]-[Connector]-[PD-L1-VHH]-[Fc]-[Connector]-[TIGIT-VHH];

[0491] The amino acid sequence of the first polypeptide chain is shown in SEQ ID NO.124.

[0492] Trispecific antibody R3878: It has the above structure (B), and its structural diagram is shown in Figure 3:

[0493] It contains two symmetrical first polypeptide chains, with the following structure:

[0494] [PVRIG-VHH]-[Connector]-[TIGIT-VHH]-[Connector]-[PD-L1-VHH]-[Fc];

[0495] The amino acid sequence of the first polypeptide chain is shown in SEQ ID NO.125.

[0496] 2. Trispecific antibodies containing conventional PVRIG antibodies:

[0497] Trispecific antibody R4038: It has the above structure (C), and its structural diagram is shown in Figure 4:

[0498] It contains two symmetrical polypeptide chains: a first polypeptide chain and a second polypeptide chain.

[0499] The first polypeptide chain structure is: [PVRIG-H]-[linker]-[PD-L1-VHH];

[0500] The second polypeptide chain structure is: [TIGIT-VHH]-[linker]-[PVRIG-L];

[0501] The amino acid sequence of the first polypeptide chain is shown in SEQ ID NO.122, and the amino acid sequence of the second polypeptide chain is shown in SEQ ID NO.123.

[0502] Trispecific antibody R4039: Possesses the above structure (D), and its structural schematic diagram is shown in Figure 5:

[0503] It contains two symmetrical polypeptide chains: a first polypeptide chain and a second polypeptide chain.

[0504] The first polypeptide chain structure is: [TIGIT-VHH]-[linker]-[PVRIG-H]-[linker]-[PD-L1-VHH];

[0505] The second polypeptide chain structure is: [PVRIG-L];

[0506] The amino acid sequence of the first polypeptide chain is shown in SEQ ID NO.110, and the amino acid sequence of the second polypeptide chain is shown in SEQ ID NO.111.

[0507] Trispecific antibody R4040: It has the above structure (E), and its structural schematic diagram is shown in Figure 6:

[0508] It contains two symmetrical polypeptide chains: a first polypeptide chain and a second polypeptide chain.

[0509] The first polypeptide chain structure is: [TIGIT-VHH]-[linker]-[PVRIG-H]-[linker]-[PD-L1-VHH];

[0510] The second polypeptide chain structure is: [PVRIG-L];

[0511] The amino acid sequence of the first polypeptide chain is shown in SEQ ID NO.130, and the amino acid sequence of the second polypeptide chain is shown in SEQ ID NO.131.

[0512] Trispecific antibody R4041: It has the above structure (F), and its structural schematic diagram is shown in Figure 7:

[0513] It contains two symmetrical polypeptide chains: a first polypeptide chain and a second polypeptide chain.

[0514] The first polypeptide chain structure is: [PD-L1-VHH]-[linker]-[TIGIT-VHH]-[linker]-[PVRIG-H];

[0515] The second polypeptide chain structure is: [PVRIG-L];

[0516] The amino acid sequence of the first polypeptide chain is shown in SEQ ID NO.132, and the amino acid sequence of the second polypeptide chain is shown in SEQ ID NO.133.

[0517] Trispecific antibody R4042: It has the above structure (G), and its structural diagram is shown in Figure 8:

[0518] It contains two symmetrical polypeptide chains: a first polypeptide chain and a second polypeptide chain.

[0519] The first polypeptide chain structure is: [PVRIG-H]-[linker]-[TIGIT-VHH]-[linker]-[PD-L1-VHH];

[0520] The second polypeptide chain structure is: [PVRIG-L];

[0521] The amino acid sequence of the first polypeptide chain is shown in SEQ ID NO.134, and the amino acid sequence of the second polypeptide chain is shown in SEQ ID NO.135.

[0522] 3. Trispecific antibodies containing conventional TIGIT antibodies:

[0523] Trispecific antibody R3884: It has the above structure (C), and its structural schematic diagram is shown in Figure 9:

[0524] It contains two symmetrical polypeptide chains: a first polypeptide chain and a second polypeptide chain.

[0525] The first polypeptide chain structure is: [TIGIT-H]-[linker]-[PD-L1-VHH];

[0526] The second polypeptide chain structure is: [PVRIG-VHH]-[linker]-[TIGIT-L];

[0527] The amino acid sequence of the first polypeptide chain is shown in SEQ ID NO.116, and the amino acid sequence of the second polypeptide chain is shown in SEQ ID NO.117.

[0528] Trispecific antibody R3885: Possesses the above structure (D), and its structural schematic diagram is shown in Figure 10:

[0529] It contains two symmetrical polypeptide chains: a first polypeptide chain and a second polypeptide chain.

[0530] The first polypeptide chain structure is: [PVRIG-VHH]-[linker]-[TIGIT-H]-[linker]-[PD-L1-VHH];

[0531] The second polypeptide chain structure is: [TIGIT-L];

[0532] The amino acid sequence of the first polypeptide chain is shown in SEQ ID NO.112, and the amino acid sequence of the second polypeptide chain is shown in SEQ ID NO.113.

[0533] Trispecific antibody R3886: Possesses the above structure (E), and its structural schematic diagram is shown in Figure 11:

[0534] It contains two symmetrical polypeptide chains: a first polypeptide chain and a second polypeptide chain.

[0535] The first polypeptide chain structure is: [TIGIT-H]-[linker]-[PD-L1-VHH];

[0536] The second polypeptide chain structure is: [TIGIT-L]-[linker]-[PVRIG-VHH];

[0537] The amino acid sequence of the first polypeptide chain is shown in SEQ ID NO.126, and the amino acid sequence of the second polypeptide chain is shown in SEQ ID NO.127.

[0538] Trispecific antibody R3887: Possesses the above structure (F), and its structural schematic diagram is shown in Figure 12:

[0539] It contains two symmetrical polypeptide chains: a first polypeptide chain and a second polypeptide chain.

[0540] The first polypeptide chain structure is: [PD-L1-VHH]-[linker]-[PVRIG-VHH]-[linker]-[TIGIT-H];

[0541] The second polypeptide chain structure is: [TIGIT-L];

[0542] The amino acid sequence of the first polypeptide chain is shown in SEQ ID NO.128, and the amino acid sequence of the second polypeptide chain is shown in SEQ ID NO.129.

[0543] 4. Trispecific antibodies containing conventional PD-1 antibodies:

[0544] Trispecific antibody R3888: It has the above structure (D), and its structural diagram is shown in Figure 13:

[0545] It contains two symmetrical polypeptide chains: a first polypeptide chain and a second polypeptide chain.

[0546] It contains two symmetrical polypeptide chains: a first polypeptide chain and a second polypeptide chain.

[0547] The first polypeptide chain structure is: [PVRIG-VHH]-[linker]-[PD-1-H]-[linker]-[TIGIT-VHH];

[0548] The second polypeptide chain structure is: [PD-1-L];

[0549] The amino acid sequence of the first polypeptide chain is shown in SEQ ID NO.118, and the amino acid sequence of the second polypeptide chain is shown in SEQ ID NO.119.

[0550] Trispecific antibody R3889: Possesses the above structure (E), and its structural schematic diagram is shown in Figure 14:

[0551] It contains two symmetrical polypeptide chains: a first polypeptide chain and a second polypeptide chain.

[0552] The first polypeptide chain structure is: [PD-1-H]-[linker]-[PVRIG-VHH];

[0553] The second polypeptide chain structure is: [PD-1-L]-[linker]-[TIGIT-VHH];

[0554] The amino acid sequence of the first polypeptide chain is shown in SEQ ID NO.120, and the amino acid sequence of the second polypeptide chain is shown in SEQ ID NO.121.

[0555] Trispecific antibody R3890: Possesses the above structure (F), and its structural schematic diagram is shown in Figure 15:

[0556] It contains two symmetrical polypeptide chains: a first polypeptide chain and a second polypeptide chain.

[0557] The first polypeptide chain structure is: [PVRIG-VHH]-[linker]-[TIGIT-VHH]-[linker]-[PD-1-H];

[0558] The second polypeptide chain structure is: [PD-1-L];

[0559] The amino acid sequence of the first polypeptide chain is shown in SEQ ID NO.114, and the amino acid sequence of the second polypeptide chain is shown in SEQ ID NO.115.

[0560] Trispecific antibody R3891: Possesses the above structure (G), and its structural schematic diagram is shown in Figure 16:

[0561] It contains two symmetrical polypeptide chains: a first polypeptide chain and a second polypeptide chain.

[0562] The first polypeptide chain structure is: [PD-1-H]-[linker]-[TIGIT-VHH]-[linker]-[PVRIG-VHH];

[0563] The second polypeptide chain structure is: [PD-1-L];

[0564] The amino acid sequence of the first polypeptide chain is shown in SEQ ID NO.107, and the amino acid sequence of the second polypeptide chain is shown in SEQ ID NO.108.

[0565] Table 15. Amino acid sequences of each peptide chain of the trispecific antibody.

[0566] Evaluation of trispecific antibodies with different structures

[0567] Example 14: Evaluation of the three-target reporter gene activity of trispecific antibodies with different structures

[0568] Take appropriate amounts of Jurkat-PVRIG-TIGIT-PD1 cells and CHO-OKT3-PVRL2-PVR-PD-L1 cells, centrifuge at 300×g for 5 min, discard the supernatant, wash once with F12K+10% FBS medium, resuspend in 1640+10% FBS medium, and adjust the cell density to 8E+05 cells / mL and 4E+6 cells / mL, respectively. Add 25 μL of each cell type to each well. Add 50 μL of diluted antibody sample to each well, and incubate the cell culture plate in a CO2 incubator for 6 h. Thaw the Bright-Lumi™ firefly luciferase assay reagent in advance and equilibrate to room temperature. Remove the cell culture plate and equilibrate at room temperature for 10 min. Add 100 μL of Bright-Lumi™ firefly luciferase assay reagent to each well and incubate at room temperature for 5 min; perform chemiluminescence detection. Plot the antibody concentration (logarithm) on the x-axis and the fluorescence signal 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.

[0569] The results are shown in Figures 38-41. Among the PVRIG-TIGIT-PD-L1 humanized nano-trispecific antibodies (R3876-R3878), R3876 showed the best three-target reporter gene activity. Among the trispecific antibodies containing conventional PVRIG antibodies (R4038-R4042), R4038 and R4039 showed the best three-target reporter gene activity. Among the trispecific antibodies containing conventional TIGIT antibodies (R3884-R3887), R3884 and R3885 showed the best three-target reporter gene activity. Among the trispecific antibodies containing conventional PD-1 antibodies (R3888-R3891), R3888, R3889, R3890, and R3891 all showed better three-target reporter gene activity than the positive control R3023, which also contained PD-1 antibody.

[0570] Example 15: Evaluation of CMV+CD8+ T cell activation activity of trispecific antibodies with different structures

[0571] 1. Induction of CMV+CD8+ T cells:

[0572] Day 0 (Preparation of culture medium: 1640 + 10% FBS + 1 μg / ml PP65 + 2 ng / ml IL-2 + 10 ng / ml IL-7, resuscitate PBMCs with CMV+donor, and adjust the PBMC density to 2E6 cells / ml with culture medium;

[0573] Day 6 (Replace the medium with 1640 medium (1640 + 10% FBS) containing 100 IU / ml IL-2 at a 1:2 ratio;

[0574] Day 8, collect cells and adjust cell density to 1-1.5E6 cells / ml using 1640 medium (1640 + 10% FBS) containing 100 IU / ml IL-2;

[0575] Day 11: Collect cells, detect the percentage of CD8+ T cells, and freeze for later use.

[0576] 2. Evaluation of the activation activity of candidate molecule CMV+CD8+ T cells:

[0577] Panc 05.04 cells (human pancreatic cancer cell line) were digested with trypsin, centrifuged at 300g for 5 min, the supernatant was removed, and the cells were counted. The cells were resuspended in experimental medium containing 0.01 μg / ml CMV-PP65 and incubated at 37°C for 1 hour. CMV-PP65 was washed away with experimental medium. Panc 05.04 cells were resuspended in experimental medium and the cell density was adjusted to 4E5 cells / ml. 50 μl of the resuspended cells were added to each well of a 96-well plate. 50 μl of the diluted test antibody was added to each well of the 96-well plate containing Panc 05.04. Resuscitated and induced CMV+CD8+ T cells were prepared, counted, and the cell density was adjusted to 8E5 cells / ml (effect-to-target ratio E:T = 4:1). 100 μl of the resuspended cells were added to each well of the 96-well plate, mixed, and incubated at 37°C for 24 hours. Centrifuge 96-well plates at 300g for 5 min, collect 150 μl of supernatant from each well, and detect the IFN-γ concentration in the supernatant using the Human IFN gamma HTRF kit. The amount of IFN-γ secreted in the supernatant indicates the activity of T cells in the system; the higher the amount of IFN-γ secreted, the stronger the antibody's activation of T cells. The results are shown in Figures 42 and 43. Among the candidate antibodies, R3876, R4039, R3885, R3890, and R3891 showed ideal CMV+CD8+ T cell activation activity, which was stronger than the positive control R3023.

[0578] Example 16: Evaluation of the MLR (Mixed Lymphocyte Reaction) Activity of Trispecific Antibodies with Different Structures

[0579] 1. Induction of mature DC (dendritic cell) cells

[0580] Day 0: The isolated monocytes were resuspended in induction medium [RPMI 1640 + 10% FBS + 50 ng / mL GM-CSF + 50 ng / mL IL-4], and the density was adjusted to 5E5 / mL. The cells were then cultured at 37°C for 3 days.

[0581] Day 3: Half volume of medium was replaced (half volume of cell culture medium was centrifuged at 350×g for 5 min, the supernatant was discarded, and the medium was resuspended in an equal volume of induction medium), and then cultured at 37℃ for 3 days.

[0582] Day 6: Add LPS to the original culture medium to a final concentration of 1 μg / mL and incubate at 37°C for 24 hours.

[0583] Day 7: Collect mature DCs [mDC] and detect the expression of related biomarkers CD86 and CD83 by flow cytometry.

[0584] 2. Induction of T cells

[0585] Day 7: Resuscitate donor #2 T cells, adjust cell density to 5E5 / mL donor #2 T cells + 1E5 / mL donor #1 mDC cells, and culture in 6-well plates for 4 days. The T-mDC co-culture medium is RPMI 1640 + 10% FBS + 100U / mL IL-2.

[0586] Day 11: Change half the medium and continue culturing for 4 days.

[0587] Day 15: Complete medium replacement (centrifuge the cultured cell suspension at 350xg for 5 minutes, discard the supernatant, resuspend in new co-culture medium, and adjust the cell density to 5E5 / mL), continue culturing for 5 days.

[0588] Day 20: Change half the medium and continue culturing for 2 days.

[0589] Day 22: Collect cells to obtain induced T cells.

[0590] 3. MLR detection method

[0591] Day 22: Resuspend the revived mDCs in the detection medium [RPMI 1640 + 10% FBS], adjust the cell density to 1E5 mDCs / well, and seed the cells with 5E5 [induced-T] cells / well obtained in step 2 in a 96-well U plate. Add different concentrations of the test antibody to a total volume of 200 μL.

[0592] Day 24: Collect the supernatant and detect IFN-γ secretion in the supernatant.

[0593] The amount of IFN-γ secreted in the supernatant indicates the activity of T cells in the system; the higher the amount of IFN-γ secreted, the stronger the activation of T cells by the antibody. The results of the two batches of experiments are shown in Figures 44 and 45, respectively.

[0594] The heavy chain amino acid sequence of the positive control bispecific antibody R4441 is shown in SEQ ID NO. 147, and the light chain amino acid sequence is shown in SEQ ID NO. 148; the heavy chain amino acid sequence of the positive control bispecific antibody R4442 is shown in SEQ ID NO. 149, and the light chain amino acid sequence is shown in SEQ ID NO. 150; the light chain 1 amino acid sequence of the positive control bispecific antibody R4443 is shown in SEQ ID NO. 151, the heavy chain 1 amino acid sequence is shown in SEQ ID NO. 152, the light chain 2 amino acid sequence is shown in SEQ ID NO. 153, and the heavy chain 2 amino acid sequence is shown in SEQ ID NO. 154.

[0595] Figure 44 shows that the MLR activities of candidate antibodies R3876, R3885, R3890 and R3891 are ideal and stronger than the positive control R3023. Figure 45 shows that the MLR activities of trispecific antibodies R3891 and R3889R are stronger than the monoclonal antibody and bispecific antibody positive controls (R4441, R4442 and R4443), among which R3891 has stronger activity than R3889 and the positive control R3023.

[0596] Example 17: Evaluation of the antitumor activity of trispecific antibodies with different structures in a mouse model of tumors

[0597] This embodiment evaluates the antitumor efficacy of different structural trispecific antibodies in a human PBMC reconstituted tumor mouse model inoculated with human melanoma A375 that has a low response to PD-L1 antibody.

[0598] NOG-dKO mice, female, 6-8 weeks old, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The mice were housed in an SPF (Specific Pathogen-Free) environment, with 5 mice per IVC (Individual Ventilation Cage). All cages and water were sterilized before use.

[0599] Each mouse was injected with 6E6 human PBMCs via the tail vein. On day 7 post-injection, logarithmic growth phase A375 cells were collected and subcutaneously inoculated into NOG-dKO mice. On day 7 post-inoculation, tail blood hCD45+ was measured once. Mice were randomly assigned to groups of 8 mice each based on a two-factor randomization method using tumor volume and CD45+ ratio. An equimolar concentration dosing regimen was designed, and administration was initiated according to the following protocol.

[0600] Table 16 Dosing regimen design

[0601] Note: In Table 16, n: number of animals used; ip: intraperitoneal injection; BIW*3-4: twice a week for a total of 3-4 weeks.

[0602] After administration, mouse body weight and tumor volume were measured twice a week. Body weight results are shown in Figure 46, and tumor volume is shown in Figure 47. The trispecific antibody R3891 containing a conventional PD-1 antibody showed the strongest antitumor activity, followed by the trispecific antibody R3890 containing a conventional PD-1 antibody, both significantly stronger than the positive trispecific antibody R3023.

[0603] Example 18: Detection of the three-terminal binding activity of the trispecific antibody R3891

[0604] FACS binding assay method: CHO-hPD1, CHO-hTIGIT, and 293T-hPVRIG cells were prepared, and cell counts were performed. Subsequent steps were performed according to Part 1 of Example 2. The results are shown in Figures 48, 49, and 50. The binding activity of the three ends of the trispecific antibody R3891 was normal, and the binding activity of the PD-1 end was similar to that of the PD-1 monoclonal antibody R1152 (using the humanized PD-1 antibody sequence named PD-1-112-C2 in the patent with patent publication number CN115466328A).

[0605] Example 19: Evaluation of the antitumor activity of different types of antibodies in a mouse model of tumors

[0606] This embodiment evaluates the antitumor efficacy of different antibody forms in a mouse model of human PBMC reconstructed tumors inoculated with human melanoma A375 that has a low response to PD-L1 antibodies.

[0607] NOG-dKO mice, female, 6-8 weeks old, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The mice were housed in an SPF (Specific Pathogen-Free) environment, with 5 mice per IVC (Individual Ventilation Cage). All cages and water were sterilized before use.

[0608] Each mouse was injected intravenously with 6E6 human PBMCs. On day 7 post-injection, logarithmically growing A375 cells were collected and subcutaneously inoculated into NOG-dKO mice. On day 7 post-inoculation, tail blood hCD45+ was measured once. Mice were randomly assigned to groups of 8 mice each based on a two-factor randomization method using tumor volume and CD45+ ratio. An equimolar dosing regimen was designed, and administration was initiated according to the following protocol:

[0609] Table 17 Dosing regimen design

[0610] Note: In Table 17, n: number of animals used; ip: intraperitoneal injection; BIW*3-4: twice a week for a total of 3-4 weeks.

[0611] After drug administration began, mouse body weight and tumor volume were measured twice weekly. Body weight results are shown in Figure 51, and tumor volume is shown in Figure 52. The trispecific antibody R3891, containing a conventional PD-1 antibody, exhibited the strongest antitumor activity, followed by the trispecific antibody R3890, both significantly stronger than the positive trispecific antibody R3023 and Keytruda. The combination of the three monoclonal antibodies showed a tumor-suppressive effect comparable to R3891. There were no significant differences in mouse body weight among the groups.

[0612] The amino acid sequences of the positive control molecules in the above embodiments are shown in Table 18.

[0613] Table 18. Amino acid sequences of positive control molecules

[0614] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this disclosure, and are not intended to limit them. Although this disclosure 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 therein. Such 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 this disclosure. Industrial applicability

[0615] The multispecific binding molecule disclosed herein can bind to multiple tumor-associated antigens simultaneously, thereby enhancing the efficacy of tumor treatment.

Claims

1. A multi-specific binding molecule, characterized in that, The multispecific binding molecule binds to at least two different antigenic epitopes; the multispecific binding molecule contains a PVRIG antigen-binding domain and a PD-L1 antigen-binding domain.

2. The multispecific binding molecule according to claim 1, characterized in that, At least one antigen-binding domain is a single variable domain; Optionally, the single variable structural domain includes VHH.

3. The multispecific binding molecule according to claim 1 or 2, characterized in that, The multispecific binding molecule also contains a third antigen-binding domain, which does not bind to the antigen epitope bound by the PVRIG antigen-binding domain, nor to the antigen epitope bound by the PD-L1 antigen-binding domain. Optionally, the multispecific binding molecule further contains Fc; The PVRIG antigen-binding domain is a single variable domain that specifically binds PVRIG, the PD-L1 antigen-binding domain is a single variable domain that specifically binds PD-L1, and the third antigen-binding domain is a single variable domain that binds a third antigen; at least one antigen-binding domain is attached to the N-terminus of Fc. Optionally, two or three antigen-binding domains are tandemly connected to the N-terminus of the Fc; Optionally, at least one antigen-binding domain is attached to the C-terminus of the Fc. Optionally, two antigen-binding domains are connected in series at the N-terminus of Fc, and one antigen-binding domain is connected to the C-terminus of Fc. Optionally, the antigen-binding domain attached to the C-terminus of Fc is a PD-L1 antigen-binding domain or a third antigen-binding domain. Optionally, the antigen-binding domain adjacent to the N-terminus of Fc is a PD-L1 antigen-binding domain or a third antigen-binding domain.

4. The multi-specific binding molecule according to claim 3, characterized in that, The multispecific binding molecule contains at least one polypeptide chain, which contains, from the N-terminus to the C-terminus, a PVRIG antigen-binding domain, a third antigen-binding domain, and an Fc and PD-L1 antigen-binding domain, respectively. Alternatively, the polypeptide chain contains, from the N-terminus to the C-terminus, a PVRIG antigen-binding domain, a PD-L1 antigen-binding domain, an Fc domain, and a third antigen-binding domain. Alternatively, the polypeptide chain contains, from the N-terminus to the C-terminus, a PVRIG antigen-binding domain, a third antigen-binding domain, a PD-L1 antigen-binding domain, and an Fc domain. Optionally, the multispecific binding molecule contains two polypeptide chains; Optionally, the multispecific binding molecule has a symmetrical structure.

5. The multispecific binding molecule according to claim 1, characterized in that, At least one antigen-binding domain contains VH and VL; Optionally, the multispecific binding molecule further comprises a third antigen-binding domain, which does not bind to the antigenic epitope bound by the PVRIG antigen-binding domain, nor to the antigenic epitope bound by the PD-L1 antigen-binding domain.

6. The multispecific binding molecule according to claim 5, characterized in that, The multispecific binding molecule contains a first polypeptide chain and a second polypeptide chain; the first polypeptide chain contains a full-length heavy chain, and the second polypeptide chain contains a full-length light chain; the VH in the full-length heavy chain and the VL in the full-length light chain form a PVRIG antigen-binding domain. Optionally, the PD-L1 antigen-binding domain contains a single variable domain that specifically binds PD-L1, and the third antigen-binding domain contains a single variable domain that binds a third antigen. Optionally, the PD-L1 antigen-binding domain is attached to the Fc end of the full-length heavy chain; the third antigen-binding domain is attached to the N-terminus or C-terminus of the full-length light chain; or, the third antigen-binding domain is attached to the N-terminus of the full-length heavy chain. Optionally, the PD-L1 antigen-binding domain is attached to the C-terminus of the Fc of the full-length heavy chain; Optionally, the third antigen-binding domain and the PD-L1 antigen-binding domain are connected in series at the N-terminus or C-terminus of the full-length heavy chain; Optionally, the third antigen-binding domain is adjacent to the full-length heavy chain.

7. The multispecific binding molecule according to claim 6, characterized in that, The multispecific binding molecule contains a first polypeptide chain and a second polypeptide chain; the first polypeptide chain contains a full-length heavy chain, and the second polypeptide chain contains a full-length light chain; the VH in the full-length heavy chain and the VL in the full-length light chain form a third antigen-binding domain. Optionally, the PD-L1 antigen-binding domain contains a single variable domain that specifically binds PD-L1, and the PVRIG antigen-binding domain contains a single variable domain that specifically binds PVRIG. Optionally, the PD-L1 antigen-binding domain is attached to the Fc end of the full-length heavy chain; the PVRIG antigen-binding domain is attached to the N-terminus or C-terminus of the full-length light chain; or, the PVRIG antigen-binding domain is attached to the N-terminus of the full-length heavy chain. Optionally, the PD-L1 antigen-binding domain is attached to the C-terminus of the Fc of the full-length heavy chain; Optionally, the PVRIG antigen-binding domain and the PD-L1 antigen-binding domain are connected in series at the N-terminus of the full-length heavy chain; Optionally, the PVRIG antigen-binding domain is adjacent to the full-length heavy chain.

8. The multispecific binding molecule according to claim 6 or 7, characterized in that, The multispecific binding molecule contains two first polypeptide chains; and / or, the multispecific binding molecule contains two second polypeptide chains; Optionally, the multispecific binding molecule has a symmetrical structure.

9. The multispecific binding molecule according to any one of claims 3 to 8, characterized in that, The third antigen-binding domain specifically binds to tumor surface antigens or immune checkpoints. Optionally, tumor surface antigens include MUC16, VEGFR-2 (KDR / FIK-1), K-RAS, CD2, CD3, CD19, 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, or OX40; Optionally, immune checkpoints include PD-1, PD-L2, TIGIT, 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, or CEACAM1; Optionally, the third antigen-binding domain specifically binds to TIGIT.

10. A multi-specific binding molecule, characterized in that, The multispecific binding molecule binds to at least two different antigenic epitopes, and the multispecific binding molecule is selected from any one of structures (A) to (G): (A) contains a first polypeptide chain, which has the following structure: [First VD] - [Connector] a1 -[Second VD]-Fc-[Connector] a2 -[Third VD]; At least one domain of [first VD], [second VD], and [third VD] specifically binds to PVRIG, TIGIT, or PD-L1; (B) Contains a first polypeptide chain, which has the following structure: [First VD] - [Connector] b1 -[Second VD]-[Connector] b2 -[Third VD]-Fc; At least one of the three domains in the first VD, second VD, and third VD specifically binds to PVRIG; (C) Contains a first polypeptide chain and a second polypeptide chain: The first polypeptide chain contains the following structure: [VH]-[CH1]-[Fc]-[connector] c1 -[First VD]; The second polypeptide chain contains the following structure: [Second VD] - [Connector] c2 -[VL]-[CL]; The combination of [VH] and [VL] specifically binds to PVRIG; Alternatively, one of the [first VD] and [second VD] specifically binds to PVRIG; (D) Contains a first polypeptide chain and a second polypeptide chain: The first polypeptide chain contains the following structure: [First VD] - [Connector] d1 -[VH]-[CH1]-[Fc]-[connector] d2 -[Second VD]; The second polypeptide chain contains the following structure: [VL]-[CL]; The combination of [VH] and [VL] specifically binds to PVRIG, TIGIT, or PD1; Furthermore, the [first VD] specifically binds to TIGIT or PVRIG; and the [second VD] specifically binds to PD-1, PD-L1, or TIGIT; (E) Contains a first polypeptide chain and a second polypeptide chain: The first polypeptide chain contains the following structure: [VH]-[CH1]-[Fc]-[connector] e1 -[First VD]; The second polypeptide chain contains the following structure: [VL]-[CL]-[Connector] e2 -[Second VD]; The combination of [VH] and [VL] specifically binds to PVRIG; Alternatively, one of the [first VD] and [second VD] specifically binds to PVRIG; (F) contains a first polypeptide chain and a second polypeptide chain: The first polypeptide chain contains the following structure: [First VD] - [Connector] f1 -[Second VD]-[Connector] f2 -[VH]-[CH1]-[Fc]; The second polypeptide chain contains the following structure: [VL]-[CL]; [VH] and [VL] combination specifically binds to PVRIG; [first VD] specifically binds to PD-L1; [second VD] specifically binds to TIGIT; or, [VH] and [VL] combination specifically binds to TIGIT; [first VD] specifically binds to PD-L1; [second VD] specifically binds to PVRIG; or, The combination of [VH] and [VL] specifically binds to PD-1; [first VD] specifically binds to PVRIG; [second VD] specifically binds to TIGIT; (G) contains a first polypeptide chain and a second polypeptide chain: The first polypeptide chain contains the following structure: [VH]-[CH1]-[Fc]-[connector] g1 -[First VD]-[Connector] g2 -[Second VD]; The second polypeptide chain contains the following structure: [VL]-[CL]; [VH] and [VL] specifically bind to PVRIG; or, one of [first VD] and [second VD] specifically binds to PVRIG; [First VD], [Second VD], and [Third VD] represent single variable domains; [VH] represents the heavy chain variable domain VH, and [VL] represents the light chain variable domain VL; the domains in the first and second polypeptide chains are arranged in order from the N-terminus to the C-terminus; a1, a2, b1, b2, c1, c2, d1, d2, e1, e2, f1, f2, g1, and g2 are all independent positive integers ≥ 0.

11. The multispecific binding molecule according to claim 10, characterized in that, The multispecific binding molecule is selected from any one of the following structures I to X: I: The multispecific binding molecule is selected from structure (G), one of [first VD] and [second VD] specifically binds PVRIG, and the other VD specifically binds TIGIT; Optionally, one of [first VD] and [second VD] specifically binds to PVRIG, and the combination of [VH] and [VL] specifically binds to PD-1; Optionally, [first VD] specifically binds to TIGIT, [second VD] specifically binds to PVRIG, and [VH] and [VL] in combination specifically bind to PD-1; II: The multispecific binding molecules are selected from the combination of structure (G), [VH] and [VL] to specifically bind PVRIG; Furthermore, at least one domain of [first VD] and [second VD] specifically binds to TIGIT, and / or, at least one domain of [first VD] and [second VD] specifically binds to PD-L1; Optionally, the combination of [VH] and [VL] specifically binds to PVRIG, [first VD] specifically binds to TIGIT, and [second VD] specifically binds to PD-L1; III: The multispecific binding molecule is selected from structure (A); and there are two domains in [first VD], [second VD] and [third VD] that bind two of PVRIG, TIGIT and PD-L1 respectively, and [first VD], [second VD] and [third VD] bind different antigens; Alternatively, [first VD] binds to PVRIG, TIGIT, or PD-L1, [second VD] binds to PVRIG, TIGIT, or PD-L1, and [third VD] binds to PVRIG, TIGIT, or PD-L1, and [first VD], [second VD], and [third VD] bind to different antigens; Optionally, [first VD] specifically binds to PVRIG; Optionally, the [first VD] specifically binds to PVRIG, and the [second VD] specifically binds to TIGIT or PD-L1; Optionally, the [first VD] specifically binds to PVRIG, the [second VD] specifically binds to TIGIT, and the [third VD] specifically binds to PD-L1; Optionally, the [first VD] specifically binds to PVRIG, the [second VD] specifically binds to PD-L1, and the [third VD] specifically binds to TIGIT; IV: The multispecific binding molecule is selected from structure (B); and at least one domain of [first VD], [second VD] and [third VD] specifically binds TIGIT, and / or at least one domain of [first VD], [second VD] and [third VD] specifically binds PD-L1; Optionally, [first VD] specifically binds to PVRIG; Optionally, the [first VD] specifically binds to PVRIG, and the [second VD] specifically binds to TIGIT or PD-L1; Optionally, the [first VD] specifically binds to PVRIG, the [second VD] specifically binds to TIGIT, and the [third VD] specifically binds to PD-L1; V: The multispecific binding molecules are selected from the structure (C), [VH] and [VL] combination specifically binding PVRIG; Furthermore, at least one domain of [first VD] and [second VD] specifically binds to TIGIT, and / or, at least one domain of [first VD] and [second VD] specifically binds to PD-L1. Optionally, [first VD] specifically binds to PD-L1. Optionally, the [first VD] specifically binds to PD-L1, and the [second VD] specifically binds to TIGIT; VI: The multispecific binding molecule is selected from structure (C); and one domain of [first VD] and [second VD] specifically binds PVRIG, and the other domain of [first VD] and [second VD] specifically binds TIGIT or PD-L1; And / or, one of the domains of [first VD] and [second VD] specifically binds to PVRIG, and the combination of [VH] and [VL] specifically binds to TIGIT or PD-L1; Optionally, [second VD] specifically binds to PVRIG; Optionally, the [second VD] specifically binds to PVRIG, and the [first VD] specifically binds to PD-L1; Optionally, [second VD] specifically binds to PVRIG, [first VD] specifically binds to PD-L1, and [VH] and [VL] combination specifically binds to TIGIT; VII: The multispecific binding molecules are selected from structure (D); and the combination of [VH] and [VL] specifically binds to PVRIG, [first VD] specifically binds to TIGIT, and [second VD] specifically binds to PD-L1; Alternatively, the combination of [VH] and [VL] specifically binds to TIGIT, [first VD] specifically binds to PVRIG, and [second VD] specifically binds to PD-L1; Alternatively, the combination of [VH] and [VL] specifically binds to PD1, [first VD] specifically binds to PVRIG, and [second VD] specifically binds to TIGIT; VIII: The multispecific binding molecules are selected from the combination of structure (E), [VH] and [VL] specifically binding to PVRIG; Furthermore, at least one domain of [first VD] and [second VD] specifically binds to TIGIT, and / or, at least one domain of [first VD] and [second VD] specifically binds to PD-L1; Optionally, the combination of [VH] and [VL] specifically binds to PVRIG, [first VD] specifically binds to PD-L1, and [second VD] specifically binds to TIGIT; IX: The multispecific binding molecules are selected from structure (E), [second VD] specifically binds PVRIG, and [VH] and [VL] combination specifically binds TIGIT; Optionally, [first VD] specifically binds to PD-L1, [second VD] specifically binds to PVRIG, and [VH] and [VL] in combination specifically bind to TIGIT; X: The multispecific binding molecules are selected from structure (E), [first VD] specifically binds to PVRIG, and [VH] and [VL] combination specifically bind to PD-1; Optionally, [first VD] specifically binds to PVRIG, [VH] and [VL] combination specifically binds to PD-1, and [second VD] specifically binds to TIGIT.

12. The multispecific binding molecule according to claim 10 or 11, characterized in that, Each of the multispecific binding molecules selected from structures (A) to (G) contains two independent first polypeptide chains; and / or, each of the multispecific binding molecules selected from structures (C) to (G) contains two independent second polypeptide chains; Optionally, the multispecific binding molecule selected from structure (A) or (B) contains two first polypeptide chains; Optionally, each of the multispecific binding molecules selected from structures (C) to (G) independently contains two first polypeptide chains and two second polypeptide chains; Optionally, the multispecific binding molecules selected from each of structures (A) to (G) are symmetrical structures.

13. The multispecific binding molecule according to any one of claims 1 to 12, characterized in that, (1) The single variable domain of the multispecific binding molecule that binds to PVRIG is VHH, wherein VHH contains complementarity-determining regions HCDR1, HCDR2 and HCDR3; HCDR1 includes amino acid residue AY; HCDR2 includes the amino acid sequence shown in SEQ ID NO.20; and HCDR3 includes the amino acid sequence shown in SEQ ID NO.

28. Alternatively, HCDR1, HCDR2 and HCDR3 are selected from HCDR1, HCDR2 and HCDR3 of VHH shown in any one of SEQ ID NO.1 to 5; Optionally, (2) the single variable domain of the multispecific binding molecule binding PD-L1 is VHH, wherein VHH contains complementarity-determining regions HCDR1, HCDR2 and HCDR3; HCDR1 includes the amino acid residue TY; HCDR2 includes the amino acid sequence shown in SEQ ID NO.34; and HCDR3 includes the amino acid sequence shown in SEQ ID NO.

42. Alternatively, HCDR1, HCDR2 and HCDR3 are selected from HCDR1, HCDR2 and HCDR3 of VHH shown in any one of SEQ ID NO.2 and 7 to 10; Optionally, (3) the single variable domain of the multispecific binding molecule binding TIGIT is VHH, wherein VHH contains complementarity-determining regions HCDR1, HCDR2 and HCDR3; HCDR1 includes the amino acid residue RY; HCDR2 includes the amino acid sequence shown in SEQ ID NO.48; and HCDR3 includes the amino acid sequence shown in SEQ ID NO.

56. Alternatively, HCDR1, HCDR2 and HCDR3 are selected from HCDR1, HCDR2 and HCDR3 of VHH shown in any one of SEQ ID NO.3 and 11 to 14.

14. The multispecific binding molecule according to claim 13, characterized in that, The VHH 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; Optionally, each VHH in the multispecific binding molecule independently contains a framework region, which is derived from at least one of camel-derived antibodies, mouse-derived antibodies, human-derived antibodies, primate-derived antibodies, or mutants thereof.

15. The multispecific binding molecule according to claim 14, characterized in that, (4) The VHH bound to PVRIG contains frame regions HFR1, HFR2, HFR3 and HFR4; the frame regions HFR1, HFR2, HFR3 and HFR4 of the VHH bound to PVRIG are selected from the HFR1, HFR2, HFR3 and HFR4 of the VHH shown in any one of SEQ ID NO.1 to 5. Optionally, the frame regions HFR1, HFR2, HFR3 and HFR4 of the VHH combined with PVRIG are selected from HFR1, HFR2, HFR3 and HFR4 of the VHH shown in SEQ ID NO.4; Optionally, the HFR1 comprises the amino acid sequence shown in SEQ ID NO. 57, the HFR2 comprises the amino acid sequence shown in SEQ ID NO. 62, the HFR3 comprises the amino acid sequence shown in SEQ ID NO. 69, and the HFR4 comprises the amino acid sequence shown in SEQ ID NO.

70. Optionally, (5) the VHH combined with PD-L1 contains frame regions HFR1, HFR2, HFR3 and HFR4; The frame regions HFR1, HFR2, HFR3 and HFR4 of the VHH combined with PVRIG are selected from the HFR1, HFR2, HFR3 and HFR4 of the VHH shown in any one of SEQ ID NO.2 and 7 to 10; Optionally, the frame regions HFR1, HFR2, HFR3 and HFR4 of the VHH combined with PVRIG are selected from HFR1, HFR2, HFR3 and HFR4 of the VHH shown in SEQ ID NO.7; Optionally, the HFR1 comprises the amino acid sequence shown in SEQ ID NO.72, the HFR2 comprises the amino acid sequence shown in SEQ ID NO.62, the HFR3 comprises the amino acid sequence shown in SEQ ID NO.69, and the HFR4 comprises the amino acid sequence shown in SEQ ID NO.

70. Optionally, (6) the VHH combined with TIGIT contains frame regions HFR1, HFR2, HFR3 and HFR4; The frame regions HFR1, HFR2, HFR3 and HFR4 of the VHH combined with TIGIT are selected from the HFR1, HFR2, HFR3 and HFR4 of the VHH shown in any one of SEQ ID NO.3 and 11 to 14; Optionally, the frame regions HFR1, HFR2, HFR3 and HFR4 of the VHH combined with TIGIT are selected from HFR1, HFR2, HFR3 and HFR4 of the VHH shown in SEQ ID NO.11; Optionally, the HFR1 comprises the amino acid sequence shown in SEQ ID NO.81, the HFR2 comprises the amino acid sequence shown in SEQ ID NO.86, the HFR3 comprises the amino acid sequence shown in SEQ ID NO.93, and the HFR4 comprises the amino acid sequence shown in SEQ ID NO.

70.

16. The multispecific binding molecule according to claim 15, characterized in that, At least one amino acid sequence of VHH bound to PVRIG is shown in SEQ ID NO.4; And / or, at least one amino acid sequence of a VHH that binds to PD-L1 is shown in SEQ ID NO.7; And / or, at least one amino acid sequence of a VHH bound to TIGIT is shown in SEQ ID NO.

11.

17. The multispecific binding molecule according to any one of claims 1 to 16, characterized in that, The HCDR1, HCDR2, and HCDR3 of the VH that binds PVRIG in the multispecific binding molecule are selected from the HCDR1, HCDR2, and HCDR3 of any VH shown in SEQ ID NO. 94 to 99; and / or, the LCDR1, LCDR2, and LCDR3 of the VL that binds PVRIG in the multispecific binding molecule are selected from the LCDR1, LCDR2, and LCDR3 of any VL shown in SEQ ID NO. 100 to 102; Optionally, the HCDR1, HCDR2, and HCDR3 of the VH that binds PVRIG in the multispecific binding molecule are selected from the HCDR1, HCDR2, and HCDR3 of any VH shown in SEQ ID NO. 96; and / or, the LCDR1, LCDR2, and LCDR3 of the VL that binds PVRIG in the multispecific binding molecule are selected from the LCDR1, LCDR2, and LCDR3 of the VL shown in SEQ ID NO. 101; Optionally, the amino acid sequence of VH binding PVRIG in the multispecific binding molecule is shown in SEQ ID NO. 96; and / or, the amino acid sequence of VL binding PVRIG in the multispecific binding molecule is shown in SEQ ID NO.

101.

18. The multispecific binding molecule according to any one of claims 1 to 17, characterized in that, The HCDR1, HCDR2, and HCDR3 of the VH that binds TIGIT in the multispecific binding molecule are selected from the HCDR1, HCDR2, and HCDR3 of the VH shown in SEQ ID NO. 103; and / or, the LCDR1, LCDR2, and LCDR3 of the VL that binds TIGIT in the multispecific binding molecule are selected from the LCDR1, LCDR2, and LCDR3 of the VL shown in SEQ ID NO. 104; Optionally, the amino acid sequence of VH binding TIGIT in the multispecific binding molecule is shown in SEQ ID NO. 103; and / or, the amino acid sequence of VL binding TIGIT in the multispecific binding molecule is shown in SEQ ID NO.

104.

19. The multispecific binding molecule according to any one of claims 1 to 18, characterized in that, The HCDR1, HCDR2, and HCDR3 of the VH that binds PD-1 in the multispecific binding molecule are selected from the HCDR1, HCDR2, and HCDR3 of the VH shown in SEQ ID NO. 105; and / or, the LCDR1, LCDR2, and LCDR3 of the VL that binds PD-1 in the multispecific binding molecule are selected from the LCDR1, LCDR2, and LCDR3 of the VL shown in SEQ ID NO. 106; Optionally, the amino acid sequence of VH binding PD-1 in the multispecific binding molecule is shown in SEQ ID NO.105; and / or, the amino acid sequence of VL binding PD-1 in the multispecific binding molecule is shown in SEQ ID NO.

106.

20. The multispecific binding molecule according to any one of claims 1 to 19, characterized in that, The multispecific binding molecule contains all or part 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 a κ or λ light chain; Optionally, the Fc of the multispecific binding molecule is derived from the Fc of an IgG1, IgG2, IgG, or IgG4 antibody; Optionally, the Fc contains the Fc region of IgG4; Optionally, the Fc contains the Fc region of human IgG4 and contains one, two, three or four mutations of 228P, 234A, 235A and 447A.

21. The multispecific binding molecule according to any one of claims 1 to 20, characterized in that, The multispecific binding molecule is selected from structure (G), the amino acid sequence of the first polypeptide chain is shown in SEQ ID NO.107, and the amino acid sequence of the second polypeptide chain is shown in SEQ ID NO.108; Alternatively, the multispecific binding molecule is selected from structure (A), and the amino acid sequence of the first polypeptide chain is shown in SEQ ID NO.109; Alternatively, the multispecific binding molecule is selected from structure (D), the amino acid sequence of the first polypeptide chain is shown in SEQ ID NO.110, and the amino acid sequence of the second polypeptide chain is shown in SEQ ID NO.111; Alternatively, the multispecific binding molecule is selected from structure (D), the amino acid sequence of the first polypeptide chain is shown in SEQ ID NO.112, and the amino acid sequence of the second polypeptide chain is shown in SEQ ID NO.113; Alternatively, the multispecific binding molecule is selected from structure (F), the amino acid sequence of the first polypeptide chain is shown in SEQ ID NO.114, and the amino acid sequence of the second polypeptide chain is shown in SEQ ID NO.115; Alternatively, the multispecific binding molecule is selected from structure (C), the amino acid sequence of the first polypeptide chain is shown in SEQ ID NO.116, and the amino acid sequence of the second polypeptide chain is shown in SEQ ID NO.117; Alternatively, the multispecific binding molecule is selected from structure (D), the amino acid sequence of the first polypeptide chain is shown in SEQ ID NO.118, and the amino acid sequence of the second polypeptide chain is shown in SEQ ID NO.119; Alternatively, the multispecific binding molecule is selected from structure (E), the amino acid sequence of the first polypeptide chain is shown in SEQ ID NO.120, and the amino acid sequence of the second polypeptide chain is shown in SEQ ID NO.121; Alternatively, the multispecific binding molecule is selected from structure (C), the amino acid sequence of the first polypeptide chain is shown in SEQ ID NO.122, and the amino acid sequence of the second polypeptide chain is shown in SEQ ID NO.123; Alternatively, the multispecific binding molecule is selected from structure (A), and the amino acid sequence of the first polypeptide chain is shown in SEQ ID NO.124; Alternatively, the multispecific binding molecule is selected from structure (A), and the amino acid sequence of the first polypeptide chain is shown in SEQ ID NO.125; Alternatively, the multispecific binding molecule is selected from structure (E), the amino acid sequence of the first polypeptide chain is shown in SEQ ID NO.126, and the amino acid sequence of the second polypeptide chain is shown in SEQ ID NO.127; Alternatively, the multispecific binding molecule is selected from structure (F), the amino acid sequence of the first polypeptide chain is shown in SEQ ID NO.128, and the amino acid sequence of the second polypeptide chain is shown in SEQ ID NO.129; Alternatively, the multispecific binding molecule is selected from structure (E), the amino acid sequence of the first polypeptide chain is shown in SEQ ID NO.130, and the amino acid sequence of the second polypeptide chain is shown in SEQ ID NO.131; Alternatively, the multispecific binding molecule is selected from structure (F), the amino acid sequence of the first polypeptide chain is shown in SEQ ID NO.132, and the amino acid sequence of the second polypeptide chain is shown in SEQ ID NO.133; Alternatively, the multispecific binding molecule is selected from structure (G), with the first polypeptide chain amino acid sequence as shown in SEQ ID NO.134 and the second polypeptide chain amino acid sequence as shown in SEQ ID NO.

135.

22. A biomaterial, characterized in that, The biomaterial includes any one of the following (i) to (iii): (i) A nucleic acid molecule, said nucleic acid molecule encoding the multispecific binding molecule according to any one of claims 1 to 21; (ii) A vector carrying the nucleic acid molecule described in (i); (iii) A recombinant cell comprising the nucleic acid molecule described in (i), or the vector described in (ii), or expressing the multispecific binding molecule described in any one of claims 1 to 21.

23. The use of the multispecific binding molecule according to any one of claims 1 to 21, or the biomaterial according to claim 22, in any one of the following: (I) To prepare products for the treatment, prevention or relief of tumor-related diseases, symptoms or conditions; (II) Treatment, prevention or relief of tumor-related diseases, symptoms or conditions; (III) Activate immune cells in the tumor immune microenvironment; (IV) Increases the secretion of IFN-γ and / or pro-inflammatory cytokines in the subjects.

24. A pharmaceutical composition, characterized in that, The pharmaceutical composition contains the multispecific binding molecule as described in any one of claims 1 to 21, or the biomaterial as described in claim 22.

25. A method for treating, preventing, or alleviating a disease, symptom, or condition, characterized in that, The method includes administering to a subject a therapeutically effective amount of the multispecific binding molecule of any one of claims 1 to 21, or the biological material of claim 22, or the pharmaceutical composition of claim 24.

26. A method for preparing the multispecific binding molecule according to any one of claims 1 to 21, characterized in that, The method includes culturing the recombinant cells as described in claim 22, and then isolating and purifying the multispecific binding molecule.

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