WO2023138638A1

WO2023138638A1 - Bispecific antigen-binding protein against tigit and pd-l1 and use thereof

Info

Publication number: WO2023138638A1
Application number: PCT/CN2023/073040
Authority: WO
Inventors: 钟阵威; 宁亚楠; 李霄培; 张梦瑶; 邓奇; 陈晓锐; 左栋梁; 何晓文
Original assignee: 原启生物科技(上海)有限责任公司
Priority date: 2022-01-24
Filing date: 2023-01-19
Publication date: 2023-07-27

Abstract

The present application relates to an isolated antigen-binding protein, comprising a first antigen-binding domain and a second antigen-binding domain. The first antigen-binding domain can specifically bind PD-L1; the second antigen-binding domain can specifically bind TIGIT; the first antigen-binding domain contains at least one CDR in a heavy chain variable region VH; and the VH contains an amino acid sequence as represented by SEQ ID NO: 21.

Description

Anti-TIGIT and PD-L1 bispecific antigen-binding protein and use thereof

technical field

This application relates to the field of biomedicine, in particular to a bispecific antigen-binding protein targeting TIGIT and PD-L1 and its application.

Background technique

Immune checkpoint T cell immunoglobulin and immunoreceptor tyrosine inhibitory motif domain (TIGIT, also known as Vstm3, WUCAM) is a type I transmembrane protein. There are two ligands for TIGIT, CD155 (also known as poliovirus receptor or PVR) and CD112 (also known as poliovirus receptor-related ligand 2, PVRL2, cohesin-2).

TIGIT competes with the activating receptor CD226 to bind to the ligand CD155, transmits inhibitory signals, inhibits excessive cell activation and inhibits the secretion of pro-inflammatory cytokines (Manieri, Trends Immunol, 2017, 38(1):20-8). TIGIT is mainly expressed on regulatory T cells (Treg), activated T cells and natural killer cells (NK) (YU, Nat Immunol, 2009, 10(1):48-57), which can directly inhibit the proliferation, killing, degranulation and cytokine secretion of T cells and NK cells, and can also inhibit T cell activity by promoting DC cells to secrete IL-10 (Nicholas, Trends Immunol.2017, 38(1) :20-28). In addition, TIGIT expressed on Treg can maintain the immunosuppressive function of Treg (Manieri, Trends Immunol, 2017, 38(1):20-8). TIGIT is significantly up-regulated in a variety of tumor cells, infiltrating lymphocytes and peripheral blood mononuclear cells, and its ligands CD155 and CD112 are also widely expressed in a variety of tumor cells, suggesting that TIGIT may play a key role in the process of tumor immune escape (Tassi, Cancer Res.2017,77:851-861.Kong,Clin Cancer Res,2016,22:3057-66. Immunol Immunother, 2009,58(9):1517-26.).

At present, the known anti-TIGIT antibodies still have defects such as low specificity and limited lethality. Therefore, there is an urgent need to develop new anti-TIGIT antigen-binding proteins with high affinity and strong specificity for TIGIT protein.

Contents of the invention

The present application provides an isolated antigen-binding protein, which has one or more of the following properties: (1) capable of specifically binding to TIGIT protein; (2) capable of binding to human TIGIT and cynomolgus monkey TIGIT protein; (3) capable of blocking the interaction between cell surface TIGIT protein and CD155; (4) having good thermal stability; and (5) capable of inhibiting tumor growth and/or proliferation.

In one aspect, the application provides an isolated antigen-binding protein, which includes: a first antigen-binding domain and a second antigen-binding domain, the first antigen-binding domain can specifically bind PD-L1, and the second antigen-binding domain can specifically bind TIGIT, the first antigen-binding domain comprises at least one CDR in the heavy chain variable region VH, and the VH comprises the amino acid sequence shown in SEQ ID NO:21.

In certain embodiments, the first antigen binding domain of the antigen binding protein comprises HCDR3, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 17.

In certain embodiments, the first antigen binding domain of the antigen binding protein comprises HCDR2, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 16.

In certain embodiments, the first antigen binding domain of the antigen binding protein comprises HCDR1, and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 15.

In certain embodiments, the first antigen binding domain of the antigen binding protein comprises VH, and the VH comprises the amino acid sequence shown in SEQ ID NO:21.

In certain embodiments, the first antigen-binding domain of the antigen-binding protein comprises H-FR1, the C-terminus of the H-FR1 is directly or indirectly connected to the N-terminus of the HCDR1, and the H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 18.

In certain embodiments, the first antigen-binding domain of the antigen-binding protein comprises H-FR2, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:19.

In certain embodiments, the first antigen-binding domain of the antigen-binding protein comprises H-FR3, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:20.

In certain embodiments, the first antigen-binding domain of the antigen-binding protein comprises H-FR4, the N-terminus of the H-FR4 is connected to the C-terminus of the HCDR3, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:10.

In certain embodiments, the first antigen binding domain of the antigen binding protein comprises LCDR3, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:26.

In certain embodiments, the first antigen binding domain of the antigen binding protein comprises LCDR2, and the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:25.

In certain embodiments, the first antigen binding domain of the antigen binding protein comprises LCDR1, and the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:24.

In certain embodiments, said first antigen binding domain of said antigen binding protein comprises L-FR1, said L-FR1 The C-terminal of the L-FR1 is directly or indirectly connected to the N-terminal of the LCDR1, and the L-FR1 comprises the amino acid sequence shown in SEQ ID NO:27.

In certain embodiments, the first antigen-binding domain of the antigen-binding protein comprises L-FR2, the L-FR2 is located between the LCDR1 and the LCDR2, and the L-FR2 comprises the amino acid sequence shown in SEQ ID NO:28.

In certain embodiments, the first antigen-binding domain of the antigen-binding protein comprises L-FR3, the L-FR3 is located between the LCDR2 and the LCDR3, and the L-FR3 comprises the amino acid sequence shown in SEQ ID NO:29.

In certain embodiments, the first antigen-binding domain of the antigen-binding protein comprises L-FR4, the N-terminus of the L-FR4 is connected to the C-terminus of the LCDR3, and the L-FR4 comprises the amino acid sequence shown in SEQ ID NO:30.

In certain embodiments, the first antigen binding domain of the antigen binding protein comprises a VL, and the VL comprises the amino acid sequence shown in SEQ ID NO:31.

In certain embodiments, said first antigen binding domain of said antigen binding protein comprises an antibody heavy chain constant region derived from IgG.

In certain embodiments, said first antigen binding domain of said antigen binding protein comprises an antibody heavy chain constant region derived from human IgG.

In certain embodiments, said first antigen binding domain of said antigen binding protein comprises an antibody heavy chain constant region derived from human IgGl.

In certain embodiments, the first antigen binding domain of the antigen binding protein comprises an antibody heavy chain constant region comprising the amino acid sequence shown in SEQ ID NO:22.

In certain embodiments, the first antigen binding domain of the antigen binding protein comprises a heavy chain, and the heavy chain comprises the amino acid sequence shown in SEQ ID NO:23.

In certain embodiments, said first antigen binding domain of said antigen binding protein comprises an antibody light chain constant region derived from Igκ.

In certain embodiments, the first antigen binding domain of the antigen binding protein comprises an antibody light chain constant region, and the light chain constant region comprises the amino acid sequence shown in SEQ ID NO:32.

In certain embodiments, the first antigen binding domain of the antigen binding protein comprises a light chain, and the light chain comprises the amino acid sequence shown in SEQ ID NO:31.

In certain embodiments, said first antigen binding domain of said antigen binding protein comprises an antibody or antigen binding fragment thereof.

In certain embodiments, the antigen-binding fragment comprises Fab, Fab', F(ab) ₂ , Fv fragment, F(ab') ₂ , scFv, di-scFv, VHH and/or dAb.

In certain embodiments, the antibody is selected from the group consisting of monoclonal antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.

In certain embodiments, said first antigen binding domain of said antigen binding protein is a monoclonal antibody.

In certain embodiments, the second antigen binding domain of the antigen binding protein comprises at least one CDR in the variable region VH of an antibody heavy chain, and the VH comprises the amino acid sequence shown in SEQ ID NO:42.

In certain embodiments, the second antigen binding domain of the antigen binding protein comprises HCDR3, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:6.

In certain embodiments, the second antigen binding domain of the antigen binding protein comprises HCDR2, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:41.

In certain embodiments, the second antigen binding domain of the antigen binding protein comprises HCDR2, and the HCDR2 comprises the amino acid sequence shown in any one of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5.

In certain embodiments, the second antigen binding domain of the antigen binding protein comprises HCDR1, and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:1.

In some embodiments, the second antigen-binding domain of the antigen-binding protein comprises H-FR1, the C-terminus of the H-FR1 is directly or indirectly connected to the N-terminus of the HCDR1, and the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:7.

In certain embodiments, the second antigen-binding domain of the antigen-binding protein comprises H-FR2, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:8.

In certain embodiments, the second antigen-binding domain of the antigen-binding protein comprises H-FR3, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:9.

In certain embodiments, the second antigen-binding domain of the antigen-binding protein comprises H-FR4, the N-terminus of the H-FR4 is connected to the C-terminus of the HCDR3, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:10.

In certain embodiments, the second antigen binding domain of the antigen binding protein comprises VH, and the VH comprises the amino acid sequence shown in SEQ ID NO:42.

In certain embodiments, said second antigen binding domain of said antigen binding protein comprises VH, and said VH comprises Contains the amino acid sequence shown in any one of SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14.

In certain embodiments, said second antigen binding domain of said antigen binding protein comprises an antibody or antigen binding fragment thereof.

In certain embodiments, wherein the antibody is selected from the group consisting of monoclonal antibodies, chimeric antibodies, humanized antibodies and fully human antibodies.

In certain embodiments, said second antigen binding domain of said antigen binding protein is VHH.

In certain embodiments, the VHH comprises the amino acid sequence shown in SEQ ID NO:42.

In certain embodiments, the VHH comprises the amino acid sequence shown in any one of SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14.

In certain embodiments, said first antigen binding domain and said second antigen binding domain of said antigen binding protein are directly linked.

In certain embodiments, said first antigen binding domain and said second antigen binding domain of said antigen binding protein are indirectly linked.

In certain embodiments, said second antigen binding domain of said antigen binding protein is directly or indirectly linked to the light chain of said first antigen binding domain.

In certain embodiments, the C-terminus of the VHH of the second antigen-binding domain of the antigen-binding protein is directly or indirectly linked to the N-terminus of the light chain of the first antigen-binding domain.

In certain embodiments, the N-terminus of the VHH of the second antigen-binding domain of the antigen-binding protein is directly or indirectly linked to the C-terminus of the light chain of the first antigen-binding domain.

In certain embodiments, said second antigen binding domain of said antigen binding protein is directly or indirectly linked to the heavy chain of said first antigen binding domain.

In certain embodiments, the C-terminus of the VHH of the second antigen-binding domain of the antigen-binding protein is directly or indirectly linked to the N-terminus of the heavy chain of the first antigen-binding domain.

In certain embodiments, the N-terminus of the VHH of the second antigen-binding domain of the antigen-binding protein is directly or indirectly linked to the C-terminus of the heavy chain of the first antigen-binding domain.

In certain embodiments, the first antigen-binding domain and the second antigen-binding domain of the antigen-binding protein are Link through linker.

In certain embodiments, said linker of said antigen binding protein is a peptide linker.

In certain embodiments, said linker of said antigen binding protein comprises the amino acid sequence of (GGGGS)n, wherein n is any integer from 0-10.

On the other hand, the present application also provides a polypeptide comprising a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises a TIGIT-targeted VHH, a linker and a light chain of an antibody targeting PD-L1, and the second polypeptide chain comprises a heavy chain of an antibody targeting PD-L1.

In certain embodiments, the C-terminus of the TIGIT-targeting VHH of the polypeptide is connected to the N-terminus of the PD-L1-targeting antibody light chain through a linker.

In certain embodiments, the N-terminus of the TIGIT-targeting VHH of the polypeptide is connected to the C-terminus of the PD-L1-targeting antibody light chain through a linker.

On the other hand, the present application also provides a polypeptide comprising a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises a PD-L1-targeting antibody light chain, and the second polypeptide chain comprises a TIGIT-targeting VHH, a linker, and a PD-L1-targeting antibody heavy chain.

In certain embodiments, the C-terminus of the TIGIT-targeting VHH of the polypeptide is connected to the N-terminus of the PD-L1-targeting antibody heavy chain through a linker.

In certain embodiments, the N-terminus of the TIGIT-targeting VHH of the polypeptide is connected to the C-terminus of the PD-L1-targeting antibody heavy chain through a linker.

In certain embodiments, the polypeptide comprises two first polypeptide chains and two second polypeptide chains.

In some embodiments, the antibody heavy chain targeting PD-L1 of the polypeptide comprises HCDR1, HCDR2 and HCDR3, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:15, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:16, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:17.

In certain embodiments, the heavy chain of the antibody targeting PD-L1 of the polypeptide comprises VH, and the VH comprises the amino acid sequence shown in SEQ ID NO:21.

In some embodiments, the antibody light chain targeting PD-L1 of the polypeptide comprises LCDR1, LCDR2 and LCDR3, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:24, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:25, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:26.

In certain embodiments, the light chain of the antibody targeting PD-L1 of the polypeptide comprises a VL, and the VL comprises the amino acid sequence shown in SEQ ID NO:31.

In certain embodiments, the linker of the polypeptide comprises the amino acid sequence of (GGGGS)n, wherein n is any integer from 0-10.

In certain embodiments, the heavy chain of the antibody targeting PD-L1 of the polypeptide comprises the amino acid sequence shown in SEQ ID NO:23.

In certain embodiments, the light chain of the antibody targeting PD-L1 of the polypeptide comprises the amino acid sequence shown in SEQ ID NO:33.

In certain embodiments, said first polypeptide chain of said polypeptide comprises the amino acid sequence shown in SEQ ID NO:33.

In certain embodiments, said second polypeptide chain of said polypeptide comprises the amino acid sequence set forth in any one of SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, and SEQ ID NO:37.

On the other hand, the present application also provides one or more isolated nucleic acid molecules encoding the antigen-binding protein or the polypeptide.

On the other hand, the present application also provides a vector comprising the nucleic acid molecule.

On the other hand, the present application also provides a cell comprising the antigen-binding protein, the polypeptide, the nucleic acid molecule, or the carrier.

On the other hand, the present application also provides a method for preparing the antigen-binding protein, the method comprising culturing the cell under the condition that the antigen-binding protein is expressed.

On the other hand, the present application also provides a pharmaceutical composition, which comprises the antigen-binding protein or the polypeptide, and optionally a pharmaceutically acceptable carrier.

On the other hand, the present application also provides the use of the antigen-binding protein, the polypeptide, the nucleic acid molecule, the carrier, the cell, and/or the pharmaceutical composition in the preparation of a medicament for preventing, treating and/or alleviating diseases and/or conditions.

In certain embodiments, the diseases and/or disorders include diseases and/or disorders associated with aberrant expression of TIGIT.

In certain embodiments, the disease and/or condition comprises a tumor.

In certain embodiments, the tumor comprises a solid tumor.

In certain embodiments, the tumor comprises a non-solid tumor.

In certain embodiments, the tumor comprises colon cancer, melanoma, non-small cell lung cancer, renal cell carcinoma, and/or hepatocellular carcinoma.

On the other hand, the present application also provides a method for preventing, treating and/or alleviating diseases and/or conditions, said method comprising administering said antigen-binding protein, said polypeptide, said nucleic acid molecule, said carrier, said cell, and/or said pharmaceutical composition to a subject in need.

In certain embodiments, the disease and/or condition comprises a tumor.

In certain embodiments, the tumor comprises a solid tumor.

In certain embodiments, the tumor comprises a non-solid tumor.

On the other hand, the present application also provides a method for blocking the interaction between TIGIT protein and CD155, the method comprising administering the antigen-binding protein, the polypeptide, the nucleic acid molecule, the carrier, the cell, and/or the pharmaceutical composition to a subject in need.

In certain embodiments, the method is an in vitro method.

In certain embodiments, the method is an ex vivo method.

In certain embodiments, the methods are non-diagnostic and therapeutic.

Those skilled in the art can easily perceive other aspects and advantages of the present application from the following detailed description. In the following detailed description, only exemplary embodiments of the present application are shown and described. As those skilled in the art will appreciate, the content of the present application enables those skilled in the art to make changes to the specific embodiments which are disclosed without departing from the spirit and scope of the invention to which this application relates. Correspondingly, the drawings and descriptions in the specification of the present application are only exemplary rather than restrictive.

Description of drawings

The particular features of the invention to which this application relates are set forth in the appended claims. The features and advantages of the invention to which this application relates can be better understood with reference to the exemplary embodiments described in detail hereinafter and the accompanying drawings. A brief description of the accompanying drawings is as follows:

Figure 1 shows a schematic diagram of the structure of the antigen-binding protein described in this application.

2A-2P show the HPLC-SEC purity test of the antigen-binding protein described in this application.

Figure 3A shows the detection of the binding activity of the antigen-binding protein described in the present application to human PD-L1; Figure 3B shows the detection of the binding activity of the antigen-binding protein described in the present application to human TIGIT.

Figure 4 shows the flow cytometric detection of the binding activity of the antigen-binding protein described in this application to cynomolgus monkey TIGIT.

Figure 5 shows the flow cytometric detection of the blocking activity of the antigen binding proteins described in this application.

Figures 6A-6J show Fortebio's detection of the affinity of the antigen-binding proteins described in this application.

Figure 7A shows the ADCC activity of the antigen-binding protein described in the present application on PD-L1; Figure 7B shows the ADCC activity of the antigen-binding protein described in the present application on TIGIT.

Figures 8A-8B show the thermostability assay of the antigen binding proteins described in this application.

Figure 9 shows the results of tumor volume inhibition in mice in the in vivo efficacy test of the antigen-binding protein described in this application.

Detailed ways

The implementation of the invention of the present application will be described in the following specific examples, and those skilled in the art can easily understand other advantages and effects of the invention of the present application from the content disclosed in this specification.

Definition of Terms

In this application, the terms "TIGIT protein" or "TIGIT antigen" are used interchangeably and include any functionally active fragments, variants and homologues of TIGIT that are naturally expressed by cells or expressed on cells transfected with the TIGIT gene. In this application, TIGIT may be human TIGIT, whose accession number in UniProt/Swiss-Prot is Q495A1. For example, TIGIT can be a functionally active fragment of human TIGIT. For example, such "functionally active fragments" may include fragments that retain at least one endogenous function of a naturally occurring protein (eg, binding to an antigen binding protein described herein). For example, the "functionally active fragment" may include a domain that binds to the antigen-binding protein of the present application. In this application, TIGIT can be expressed on the surface of immune cells. For example, it can be expressed on the surface of regulatory T cells (Treg).

In this application, the term "programmed death ligand-1 (PD-L1)" is one of two cell surface glycoprotein ligands of PD-1 (the other being PD-L2), which downregulates T cell activation and cytokine secretion upon binding to PD-1. As used herein, the term "PD-L1" includes human PD-L1 (hPD-L1), variants, isoforms and species homologs of hPD-L1, and analogs that share at least one common epitope with hPD-L1. The complete hPD-L1 sequence can be found under GenBank accession number Q9NZQ7.

In the present application, the term "isolated" generally means obtained from the natural state by artificial means. If an "isolated" substance or component occurs in nature, it may be that its natural environment has been altered, the substance has been isolated from its natural environment, or both. For example, a certain unisolated polynucleotide or polypeptide naturally exists in a living animal, and the high-purity identical polynucleotide or polypeptide isolated from this natural state is called isolated. The term "isolated" does not exclude the admixture of artificial or synthetic substances, nor the presence of other impure substances which do not affect the activity of the substance.

In this application, the term "isolated antigen-binding protein" generally refers to a protein with antigen-binding ability obtained from a natural state through artificial means. The "isolated antigen binding protein" may comprise an antigen-binding moiety and, optionally, a framework or framework portion that permits the antigen-binding moiety to adopt a conformation that facilitates binding of said antigen-binding moiety to antigen. Antigen binding proteins may comprise, for example, antibody-derived protein framework regions (FR) or alternative protein framework regions or artificial framework regions with grafted CDRs or CDR derivatives. Such frameworks include, but are not limited to, antibody-derived framework regions comprising mutations introduced, eg, to stabilize the three-dimensional structure of the antigen binding protein, and fully synthetic framework regions comprising, eg, biocompatible polymers. see example Such as Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, 53(1): 121-129 (2003); Roque et al., Biotechnol. Prog. 20: 639-654 (2004). Examples of antigen binding proteins include, but are not limited to: human _antibodies , humanized antibodies; chimeric antibodies; recombinant antibodies; single chain antibodies; diabodies _; and its fragments.

In the present application, the isolated antigen binding protein may comprise more than one antigen binding domain. In the present application, the antigen binding domains may target different antigens. In the present application, the antigen binding domains may target different epitopes of the same antigen. For example, the isolated antigen binding protein can comprise a first antigen binding domain and a second antigen binding domain. For example, the first antigen-binding domain can target PD-L1 protein, for example, the second antigen-binding domain can target TIGIT protein.

In this application, the terms "variable domain" and "variable region" are used interchangeably and generally refer to a portion of an antibody heavy and/or light chain. The variable domains of the heavy and light chains may be referred to as " _VH " and " _VL ", respectively (or "VH" and "VL", respectively). These domains are usually the most variable parts of an antibody (relative to other antibodies of the same class) and comprise the antigen binding site.

In the present application, the term "variable" generally means that some segments of the variable domain may have large differences in sequence between antibodies. The variable domains mediate antigen binding and determine the specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains. It is usually concentrated in three segments called hypervariable regions (CDRs or HVRs) in the light and heavy chain variable domains. The more highly conserved portions of variable domains are called the framework regions (FR). The variable domains of native heavy and light chains each comprise four FR regions, most adopting a β-sheet configuration, connected by three CDRs, which form a circular link and, in some cases, form part of the β-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions, and the CDRs from the other chain together contribute to the formation of the antibody's antigen-binding site (see Kabat et al, Sequences of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)).

In this application, the term "antibody" generally refers to an immunoglobulin or fragment or derivative thereof, encompassing any polypeptide that includes an antigen combining site, whether produced in vitro or in vivo. The term includes, but is not limited to, polyclonal, monoclonal, monospecific, multispecific, nonspecific, humanized, single chain, chimeric, synthetic, recombinant, hybrid, mutated and grafted antibodies. Unless otherwise modified by the term "intact", as in "intact antibody", for the purposes of the present invention, the term "antibody" also includes antibody fragments, such as Fab, F(ab') ₂ , Fv, scFv, Fd, dAb and other antibody fragments that retain antigen binding function (e.g., specifically bind TIGIT). Typically, such fragments will include the antigen binding domain. The basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. IgM antibodies are composed of 5 basic heterotetrameric units and another polypeptide called the J chain, and contain There are 10 antigen-binding sites, whereas IgA antibodies consist of 2-5 basic 4-chain units that can be combined and polymerized with the J chain to form multivalent assemblies. For IgG, the 4-chain unit is typically about 150,000 Daltons. Each L chain is linked to an H chain by a covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has a variable domain (VH) at the N-terminus followed by three constant domains (CH) for the alpha and gamma chains each, and four CH domains for the mu and epsilon isoforms. Each L chain has a variable domain (VL) at its N-terminus and a constant domain at its other end. VL corresponds to VH, and CL corresponds to the first constant domain (CH1) of the heavy chain. Certain amino acid residues are believed to form the interface between the light and heavy chain variable domains. VH and VL pair together to form a single antigen-binding site. For the structure and properties of different classes of antibodies see, eg, Basic and Clinical Immunology, 8th Edition, Daniel P. Sties, Abba I. Terr and Tristram G. Parsolw (eds), Appleton & Lange, Norwalk, Conn., 1994, p. 71 and chapter 6. L chains from any vertebrate species can be classified into one of two distinct types, called kappa and lambda, based on the amino acid sequence of their constant domains. Depending on the amino acid sequence of the constant domain of the heavy chain (CH), immunoglobulins can be assigned to different classes, or isotypes. There are currently five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, with heavy chains designated alpha, delta, epsilon, gamma, and mu, respectively. The gamma and alpha classes are further divided into subclasses based on relatively minor differences in CH sequence and function, eg, humans express the following subclasses: IgGl, IgG2A, IgG2B, IgG3, IgG4, IgAl and IgKl.

In this application, the term "CDR", also referred to as "complementarity determining region", generally refers to the region in the variable domain of an antibody, the sequence of which is highly variable and/or forms a structure-defining loop. Typically, antibodies comprise six CDRs; three in the VH (HCDR1, HCDR2, HCDR3), and three in the VL (LCDR1, LCDR2, LCDR3). In certain embodiments, naturally occurring camelid antibodies consisting only of heavy chains are capable of functioning and stabilizing in the absence of light chains. See, eg, Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al, Nature Struct. Biol. 3:733-736 (1996).

In this application, the term "FR" generally refers to the more highly conserved portions of antibody variable domains, known as the framework regions. Typically, the variable domains of native heavy and light chains each comprise four FR regions, four in VH (H-FR1, H-FR2, H-FR3, and H-FR4), and four in VL (L-FR1, L-FR2, L-FR3, and L-FR4). For example, the VL of an isolated antigen binding protein described herein can include the framework regions L-FR1, L-FR2, L-FR3, and L-FR4. The VH of the isolated antigen binding proteins described herein can include framework regions H-FR1, H-FR2, H-FR3, and H-FR4.

In this application, the term "antigen-binding fragment" generally refers to one or more fragments that have the ability to specifically bind an antigen (eg, TIGIT). In the present application, the antigen-binding fragment may include Fab, Fab', F(ab) ₂ , Fv fragment, F(ab') ₂ , scFv, di-scFv and/or dAb.

In this application, the term "Fab" generally refers to an antigen-binding fragment of an antibody. As mentioned above, papain can be used Digest intact antibodies. Papain digestion of antibodies yields two identical antigen-binding fragments, the "Fab" fragment, and a residual "Fc" fragment (ie, the Fc region, supra). Fab fragments may consist of a complete L chain with the variable region of a heavy chain and the first constant region ( _CH 1 ) of the H chain ( _VH ).

In this application, the term "Fab' fragment" generally refers to a monovalent antigen-binding fragment of a human monoclonal antibody, which fragment is slightly larger than a Fab fragment. For example, a Fab' fragment may include all of the light chain, all of the variable domains of the heavy chain, and all or part of the first and second constant domains of the heavy chain. For example, a Fab' fragment may also include part or all of the 220-330 amino acid residues of the heavy chain.

In this application, the term "F(ab')2" generally refers to antibody fragments produced by pepsin digestion of intact antibodies. The F(ab')2 fragment contains two Fab fragments and part of the hinge region held together by disulfide bonds. F(ab')2 fragments have bivalent antigen binding activity and are capable of cross-linking antigen.

In this application, the term "Fv fragment" generally refers to a monovalent antigen-binding fragment of a human monoclonal antibody comprising all or part of the heavy and light chain variable regions and lacking the heavy and light chain constant regions. The heavy and light chain variable regions include, for example, CDRs. For example, an Fv fragment includes all or part of the approximately 110 amino acid amino-terminal variable regions of the heavy and light chains.

In this application, the term "scFv" generally refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chain variable regions are contiguous (e.g. via a synthetic linker such as a short flexible polypeptide linker) and capable of being expressed as a single chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it was derived. Unless otherwise specified, as used in this application, a scFv may have the VL and VH variable regions described in any order (e.g., relative to the N-terminal and C-terminal of the polypeptide), and the scFv may include a VL-linker-VH or may include a VH-linker-VL.

In the present application, the term "dAb" generally refers to an antigen-binding fragment having a VH domain, a VL domain or having a VH domain or a VL domain, see for example Ward et al. WO 06/003388 and other published patent applications of Domantis Ltd.

In this application, the term "monoclonal antibody" generally refers to a preparation of antibody molecules of single molecular composition. Monoclonal antibodies are usually highly specific against a single antigenic site. Furthermore, each monoclonal antibody is directed against a single determinant on the antigen, unlike conventional polyclonal antibody preparations, which typically have different antibodies directed against different determinants. In addition to their specificity, monoclonal antibodies have the advantage that they can be synthesized by hybridoma cultures without contamination from other immunoglobulins. The modifier "monoclonal" denote the characteristics of an antibody obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring that the antibody be produced by any particular method. For example, monoclonal antibodies used herein can be produced in hybridoma cells, or can be produced by recombinant DNA methods.

In this application, the term "chimeric antibody" generally refers to an antibody in which the variable regions are derived from one species and the constant regions are derived from another species. Typically, the variable regions are derived from an antibody of a laboratory animal, such as a rodent ("parent antibody"), and the constant regions are derived from a human antibody, such that the resulting chimeric antibody is less likely to elicit an adverse immune response in a human individual than the parental (e.g., mouse-derived) antibody.

In this application, the term "humanized antibody" generally refers to an antibody in which some or all of the amino acids other than the CDR region of a non-human antibody (such as a mouse antibody) are replaced with corresponding amino acids derived from human immunoglobulins. In the CDR regions, small additions, deletions, insertions, substitutions or modifications of amino acids may also be permissible so long as they still retain the ability of the antibody to bind a particular antigen. A humanized antibody optionally will comprise at least a portion of a human immunoglobulin constant region. A "humanized antibody" retains antigen specificity similar to the original antibody. "Humanized" forms of non-human (eg, murine) antibodies may contain, at a minimum, chimeric antibodies of sequence derived from non-human immunoglobulin. In some cases, CDR region residues in a human immunoglobulin (recipient antibody) may be replaced with CDR region residues from a non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate having the desired properties, affinity and/or capabilities. In certain instances, FR region residues of the human immunoglobulin may be replaced with corresponding non-human residues. In addition, humanized antibodies can comprise amino acid modifications that are absent in the recipient antibody or in the donor antibody. These modifications may be made to further refine antibody properties, such as binding affinity.

The term "fully human antibody" generally refers to an antibody that comprises only human immunoglobulin protein sequences. Fully human antibodies may contain murine sugar chains if they are produced in mice, in mouse cells, or in hybridomas derived from mouse cells. Similarly, a "mouse antibody" or "rat antibody" refers to an antibody comprising only mouse or rat immunoglobulin sequences, respectively. Fully human antibodies can be produced in humans, in transgenic animals with human immunoglobulin germline sequences, by phage display or other molecular biology methods. Exemplary techniques that can be used to make antibodies are described in US Patents: 6,150,584, 6,458,592, 6,420,140. Other techniques, such as using libraries, are known in the art.

In this application, the term "directly connected" is opposite to the term "indirectly connected", and the term "directly connected" generally refers to a direct connection. For example, the direct connection may be a case where substances are directly connected without a spacer. The spacer may be a linker. For example, the linker can be a peptide linker. The term "indirectly linked" generally refers to the situation where substances are not directly linked. For example, the indirect connection may be through a spacer. For example, in the isolated antigen-binding protein described herein, the C-terminus of L-FR1 may be directly or indirectly linked to the N-terminus of LCDR1.

In this application, the term "isolated nucleic acid molecule" generally refers to an isolated form of nucleotides of any length, deoxyribonucleotides or ribonucleotides, or analogs isolated from their natural environment or artificially synthesized.

In this application, the term "vector" generally refers to a nucleic acid delivery vehicle into which a polynucleotide encoding a protein can be inserted and the protein can be expressed. The vector can be expressed by transforming, transducing or transfecting the host cell, so that the genetic material elements carried by it can be expressed in the host cell. For example, vectors may include: plasmids; phagemids; cosmids; artificial Chromosomes such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) or P1-derived artificial chromosome (PAC); bacteriophage such as lambda phage or M13 phage and animal viruses. Animal virus species used as vectors may include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (such as SV40). A vector may contain a variety of elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain an origin of replication. Vectors may also include components that facilitate their entry into cells, such as viral particles, liposomes or protein coats, but not only.

In this application, the term "cell" generally refers to a single cell, cell line, or cell culture that can be or has been the recipient of a subject's plasmid or vector, which includes a nucleic acid molecule of the invention or a vector of the invention. Cells can include progeny of a single cell. Due to natural, accidental or deliberate mutations, the progeny may not necessarily be completely identical (either in the morphology of the total DNA complement or in the genome) to the original parent cell. Cells may include cells transfected in vitro with the vectors described herein. The cells can be bacterial cells (e.g., E. coli), yeast cells, or other eukaryotic cells, such as COS cells, Chinese hamster ovary (CHO) cells, CHO-K1 cells, LNCAP cells, HeLa cells, HEK293 cells, COS-1 cells, NSO cells. In certain embodiments, the cells are mammalian cells. In certain embodiments, the mammalian cells are HEK293 cells.

In this application, the term "pharmaceutical composition" generally refers to a composition for preventing/treating a disease or condition. The pharmaceutical composition may comprise the isolated antigen binding protein described herein, the nucleic acid molecule described herein, the carrier described herein and/or the cell described herein, and optionally a pharmaceutically acceptable adjuvant. Furthermore, the pharmaceutical composition may also comprise suitable formulations of one or more (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and/or preservatives. The acceptable ingredients of the compositions are preferably nontoxic to recipients at the dosages and concentrations employed. Pharmaceutical compositions of the present invention include, but are not limited to, liquid, frozen and lyophilized compositions.

As used herein, the term "pharmaceutically acceptable carrier" generally includes pharmaceutically acceptable carriers, excipients or stabilizers that are nontoxic to cells or mammals to which they are exposed at the dosages and concentrations employed. Physiologically acceptable carriers can include, for example, buffers, antioxidants, low molecular weight (less than about 10 residues) polypeptides, proteins, hydrophilic polymers, amino acids, monosaccharides, disaccharides, and other carbohydrates, chelating agents, sugar alcohols, salt-forming counterions, such as sodium; and/or nonionic surfactants.

In this application, the term "specific binding" or "specific" generally refers to a measurable and reproducible interaction, such as the binding between a target and an antibody, that can determine the presence of a target in the presence of a heterogeneous population of molecules, including biomolecules. For example, an antibody that specifically binds a target (which may be an epitope) can be an antibody that binds that target with greater affinity, avidity, greater ease, and/or for a greater duration than it binds other targets. In some embodiments, the antibody specific Epitopes on sex-binding proteins that are conserved among proteins of different species. In certain embodiments, specific binding can include, but does not require exclusive binding.

In this application, the term "subject" generally refers to human or non-human animals, including but not limited to cats, dogs, horses, pigs, cows, sheep, rabbits, mice, rats or monkeys.

In this application, the term "tumor" generally refers to a neoplasm or solid lesion formed by abnormal cell growth. In this application, a tumor may be a solid tumor or a non-solid tumor. For example, in the present application, a tumor may be a disease and/or disorder associated with abnormal expression of TIGIT.

The term "cancer" generally refers to a disease characterized by the rapid and uncontrolled growth of abnormal cells. Cancer cells can spread to other parts of the body locally or through the bloodstream and lymphatic system. Cancer in this application includes but not limited to gastric cancer, colon cancer and the like. The terms "tumor" and "cancer" are used interchangeably herein, eg, both terms encompass solid tumors and liquid tumors, eg, diffuse or circulating tumors. As used herein, the term "cancer" or "tumor" can include premalignant as well as malignant cancers and tumors.

In this application, the protein, polypeptide and/or amino acid sequence involved should also be understood to include at least the following scope: variants or homologues having the same or similar functions as the protein or polypeptide.

In the present application, the variant may be, for example, a protein or polypeptide that undergoes substitution, deletion or addition of one or more amino acids in the amino acid sequence of the protein and/or the polypeptide (for example, an antibody or fragment thereof that specifically binds to PD-L1 and/or TIGIT protein). For example, the functional variant may comprise a protein or polypeptide having amino acid changes through at least 1, such as 1-30, 1-20 or 1-10, and such as 1, 2, 3, 4 or 5 amino acid substitutions, deletions and/or insertions. Said functional variant may substantially retain the biological properties of said protein or said polypeptide prior to alteration (eg, substitution, deletion or addition). For example, the functional variant may retain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (eg, antigen binding ability) of the protein or polypeptide prior to the alteration. For example, the substitutions may be conservative substitutions.

In the present application, the homologue may be a protein or polypeptide having at least about 85% (for example, at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or higher) sequence homology with the amino acid sequence of the protein and/or the polypeptide (for example, an antibody or fragment thereof that specifically binds to PD-L1 and/or TIGIT protein).

In this application, the homology generally refers to the similarity, similarity or association between two or more sequences. The "percentage of sequence homology" can be calculated by comparing the two sequences to be aligned within a comparison window and determining the presence of the same nucleic acid base (for example, A, T, C, G, I) or the same amino acid residue (for example, Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) in the two sequences To obtain the number of matching positions, the number of matching positions was divided by the total number of positions in the comparison window (ie, window size), and the result was multiplied by 100 to yield the percent sequence identity. Alignment for purposes of determining percent sequence homology can be accomplished in various ways known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared or over a region of sequence of interest. The homology can also be determined by the following methods: FASTA and BLAST. A description of the FASTA algorithm can be found in WRPearson and DJ Lipman, "Improved Tools for Biological Sequence Comparison", Proc. A description of the BLAST algorithm can be found in S. Altschul, W. Gish, W. Miller, EW Myers and D. Lipman, "A Basic Local Alignment Search Tool", J. Molecular Biology, 215:403-410,1990.

In this application, the term "comprises" generally refers to the meanings comprising, encompassing, comprising or encompassing. In some cases, it also means "for" and "consisting of".

In this application, the term "about" generally refers to a variation in the range of 0.5%-10% above or below the specified value, such as within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the specified value. .

Detailed description of the invention

The CDR of an antibody, also known as the complementarity determining region, is part of the variable region. The amino acid residues in this region may make contacts with the antigen or antigenic epitope. Antibody CDRs can be determined by various coding systems, such as CCG, Kabat, Chothia, IMGT, Kabat/Chothia, etc. These coding systems are known in the art, see http://www.bioinf.org.uk/abs/index.html#kabatnum for details. Those skilled in the art can use different coding systems to determine the CDR region according to the sequence and structure of the antibody. There may be differences in the CDR regions using different coding systems. In the present application, the CDR covers the CDR sequence divided according to any CDR division method; it also covers its variants, and the variant includes the amino acid sequence of the CDR being substituted, deleted and/or added with one or more amino acids. For example, 1-30, 1-20 or 1-10, and for example 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acid substitutions, deletions and/or insertions; also contemplate homologues thereof, which may have at least about 85% (e.g., at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, Amino acid sequences having about 96%, about 97%, about 98%, about 99% or more) sequence homology.

In this application, for example, the CDR may be divided according to the Kabat method.

first antigen binding domain

In the present application, the isolated antigen-binding protein may comprise a first antigen-binding domain capable of specifically binding to PD-L1 or a functionally active fragment thereof.

In the present application, the first antigen-binding domain may comprise an antibody or an antigen-binding fragment thereof or a variant thereof.

In the present application, the antibody may be selected from the following groups: monoclonal antibody, single chain antibody, chimeric antibody, humanized antibody and fully human antibody.

In the present application, the antigen-binding fragment can be selected from the following group: Fab, Fab', Fv fragment, F(ab)' ₂ , scFv, di-scFv and/or dAb.

In the present application, the first antigen-binding domain may comprise at least one CDR in the heavy chain variable region VH, and the VH may comprise the amino acid sequence shown in SEQ ID NO:21.

In the present application, the first antigen-binding domain of the antigen-binding protein may comprise HCDR3, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:17.

In the present application, the first antigen-binding domain of the antigen-binding protein may comprise HCDR2, and the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:16.

In the present application, the first antigen-binding domain of the antigen-binding protein may comprise HCDR1, and the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:15.

In the present application, the first antigen-binding domain of the antigen-binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:15, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:16, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:17.

In the present application, the first antigen-binding domain of the antigen-binding protein may comprise VH, and the VH comprises the amino acid sequence shown in SEQ ID NO:21.

In the present application, the first antigen-binding domain of the antigen-binding protein may comprise H-FR1, the C-terminus of the H-FR1 is directly or indirectly connected to the N-terminus of the HCDR1, and the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 18.

In the present application, the first antigen-binding domain of the antigen-binding protein may comprise H-FR2, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:19.

In the present application, the first antigen-binding domain of the antigen-binding protein comprises H-FR3, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:20.

In the present application, the first antigen-binding domain of the antigen-binding protein comprises H-FR4, and the N of the H-FR4 The end is connected to the C-terminus of the HCDR3, and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:10.

In this application, the first antigen-binding domain of the antigen binding protein may contain H-FR1, H-FR2, H-FR3, and H-FR4. The H-FR1 may contain the amino acid sequence shown in SEQ ID NO: 18. The H-FR3 may contain the amino acid sequence shown in the SEQ ID NO: 20, and the H-FR4 can contain the amino acid sequence shown in the SEQ ID NO: 10.

In the present application, the first antigen-binding domain of the antigen-binding protein may comprise VH, and the VH may comprise the amino acid sequence shown in SEQ ID NO:21.

In the present application, the first antigen-binding domain of the antigen-binding protein may comprise LCDR3, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:26.

In the present application, the first antigen-binding domain of the antigen-binding protein may comprise LCDR2, and the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:25.

In the present application, the first antigen-binding domain of the antigen-binding protein may comprise LCDR1, and the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO:24.

In the present application, the first antigen-binding domain of the antigen-binding protein may comprise LCDR1, LCDR2 and LCDR3, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO:24, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:25, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:26.

In the present application, the first antigen-binding domain of the antigen-binding protein may comprise L-FR1, the C-terminus of the L-FR1 is directly or indirectly connected to the N-terminus of the LCDR1, and the L-FR1 may comprise the amino acid sequence shown in SEQ ID NO:27.

In the present application, the first antigen-binding domain of the antigen-binding protein may comprise L-FR2, the L-FR2 is located between the LCDR1 and the LCDR2, and the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO:28.

In the present application, the first antigen-binding domain of the antigen-binding protein may comprise L-FR3, the L-FR3 is located between the LCDR2 and the LCDR3, and the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO:29.

In the present application, the first antigen-binding domain of the antigen-binding protein may comprise L-FR4, the N-terminus of the L-FR4 is connected to the C-terminus of the LCDR3, and the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO:30.

In the present application, the first antigen binding domain of the antigen binding protein may comprise L-FR1, L-FR2, L-FR3 and L-FR4, the L-FR1 may comprise the amino acid sequence shown in SEQ ID NO:27, the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO:28, the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO:29, and the L- FR4 may comprise the amino acid sequence shown in SEQ ID NO:30.

In the present application, the first antigen-binding domain of the antigen-binding protein may comprise a VL, and the VL may comprise the amino acid sequence shown in SEQ ID NO:31.

In the present application, the first antigen-binding domain of the antigen-binding protein may comprise VH and VL, the VH may comprise the amino acid sequence shown in SEQ ID NO:21, and the VL may comprise the amino acid sequence shown in SEQ ID NO:31.

In the present application, the first antigen-binding domain of the antigen-binding protein may comprise an antibody heavy chain constant region, and the antibody heavy chain constant region may be derived from IgG.

In the present application, the first antigen-binding domain of the antigen-binding protein may comprise an antibody heavy chain constant region, and the antibody heavy chain constant region may be derived from human IgG.

In the present application, the first antigen-binding domain of the antigen-binding protein may comprise an antibody heavy chain constant region, and the antibody heavy chain constant region may be derived from human IgG1.

In the present application, the first antigen-binding domain of the antigen-binding protein may comprise an antibody heavy chain constant region, and the antibody heavy chain constant region may comprise the amino acid sequence shown in SEQ ID NO:22.

In the present application, the first antigen-binding domain of the antigen-binding protein may comprise a heavy chain, and the heavy chain may comprise the amino acid sequence shown in SEQ ID NO:23.

In the present application, the first antigen-binding domain of the antigen-binding protein may comprise an antibody light chain constant region, which may be derived from Igκ.

In the present application, the first antigen-binding domain of the antigen-binding protein may comprise an antibody light chain constant region, and the light chain constant region may comprise the amino acid sequence shown in SEQ ID NO:32.

In the present application, the first antigen-binding domain of the antigen-binding protein may comprise a light chain, and the light chain may comprise the amino acid sequence shown in SEQ ID NO:31.

In the present application, the first antigen-binding domain of the antigen-binding protein may comprise a heavy chain and a light chain, the heavy chain may comprise the amino acid sequence shown in SEQ ID NO:23, and the light chain constant region may comprise the amino acid sequence shown in SEQ ID NO:32.

In the present application, the first antigen-binding domain of the antigen-binding protein may comprise an antibody or an antigen-binding fragment thereof.

In the present application, the antigen-binding fragment may include Fab, Fab', F(ab) ₂ , Fv fragment, F(ab') ₂ , scFv, di-scFv, VHH and/or dAb.

In the present application, the antibody may be selected from the group consisting of monoclonal antibody, chimeric antibody, humanized antibody and fully human antibody.

In the present application, the first antigen-binding domain of the antigen-binding protein may be a monoclonal antibody.

second antigen binding domain

In the present application, the second antigen-binding domain of the antigen-binding protein may comprise at least one CDR in the variable region VH of an antibody heavy chain, and the VH may comprise the amino acid sequence shown in SEQ ID NO:42.

In the present application, the second antigen-binding domain of the antigen-binding protein may comprise HCDR3, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:6.

In the present application, the second antigen-binding domain of the antigen-binding protein may comprise HCDR2, and the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:41.

In the present application, the second antigen-binding domain of the antigen-binding protein may comprise HCDR2, and the HCDR2 may comprise the amino acid sequence shown in any one of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5.

In the present application, the second antigen-binding domain of the antigen-binding protein may comprise HCDR1, and the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:1.

In the present application, the second antigen-binding domain of the antigen-binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:1, the HCDR2 may comprise the amino acid sequence shown in any one of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:6.

For example, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:1, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:2, and the HCDR3 comprises SEQ ID NO:6.

For example, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:1, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:3, and the HCDR3 comprises SEQ ID NO:6.

For example, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:1, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:4, and the HCDR3 comprises SEQ ID NO:6.

For example, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:1, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:5, and the HCDR3 comprises SEQ ID NO:6.

In the present application, the second antigen-binding domain of the antigen-binding protein may comprise H-FR1, the C-terminus of the H-FR1 is directly or indirectly connected to the N-terminus of the HCDR1, and the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:7.

In the present application, the second antigen-binding domain of the antigen-binding protein may comprise H-FR2, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:8.

In the present application, the second antigen-binding domain of the antigen-binding protein may comprise H-FR3, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:9.

In the present application, the second antigen-binding domain of the antigen-binding protein may comprise H-FR4, the N-terminus of the H-FR4 is connected to the C-terminus of the HCDR3, and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:10.

In the present application, the second antigen binding domain of the antigen binding protein may comprise H-FR1, H-FR2, H-FR3 and H-FR4, the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:7, the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:8, the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:9, and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:10.

In the present application, the second antigen-binding domain of the antigen-binding protein may comprise VH, and the VH may comprise the amino acid sequence shown in SEQ ID NO:42.

In the present application, the second antigen-binding domain of the antigen-binding protein may comprise VH, and the VH may comprise the amino acid sequence shown in any one of SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14.

In the present application, the second antigen-binding domain of the antigen-binding protein may comprise an antibody or an antigen-binding fragment thereof.

In the present application, the second antigen-binding domain of the antigen-binding protein may be VHH.

In the present application, the VHH of the second antigen-binding domain may comprise the amino acid sequence shown in SEQ ID NO:42.

In the present application, the VHH of the second antigen-binding domain may comprise the amino acid sequence shown in any one of SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14.

isolated antigen binding protein

The application provides an isolated antigen binding protein comprising a first antigen binding domain and a second antigen binding domain. For example, the first antigen binding domain can specifically bind PD-L1 protein. For example, the second antigen binding domain can specifically bind a TIGIT protein.

For example, the first antigen binding domain can be a monoclonal antibody. For example, the second antigen binding domain can be a VHH.

In the present application, the first antigen-binding domain and the second antigen-binding domain may be directly or indirectly linked.

In the present application, the first antigen-binding domain may be connected to the second antigen-binding domain through a linker. For example, the linker can be a peptide linker. For example, the linker may comprise the amino acid sequence shown in any one of SEQ ID NO:38, SEQ ID NO:39 and SEQ ID NO:40.

In the present application, the second antigen-binding domain of the antigen-binding protein may be directly or indirectly linked to the light chain of the first antigen-binding domain.

In the present application, the C-terminal of the VHH of the second antigen-binding domain of the antigen-binding protein may be directly or indirectly linked to the N-terminal of the light chain of the first antigen-binding domain.

In the present application, the N-terminal of the VHH of the second antigen-binding domain of the antigen-binding protein may be directly or indirectly linked to the C-terminal of the light chain of the first antigen-binding domain.

In the present application, the second antigen-binding domain of the antigen-binding protein may be directly or indirectly linked to the heavy chain of the first antigen-binding domain.

In the present application, the C-terminal of the VHH of the second antigen-binding domain of the antigen-binding protein may be directly or indirectly linked to the N-terminal of the heavy chain of the first antigen-binding domain.

In the present application, the N-terminal of the VHH of the second antigen-binding domain of the antigen-binding protein may be directly or indirectly linked to the C-terminal of the heavy chain of the first antigen-binding domain.

In the present application, the first antigen-binding domain and the second antigen-binding domain of the antigen-binding protein may be linked by a linker.

In the present application, the linker of the antigen binding protein is a peptide linker.

In the present application, the linker of the antigen-binding protein comprises the amino acid sequence of (GGGGS)n, wherein n is any integer from 0-10. For example, said n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

polypeptide

On the other hand, the present application also provides a polypeptide, which may comprise a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises a VHH targeting TIGIT, a linker, and a light chain of an antibody targeting PD-L1, and the second polypeptide chain comprises a heavy chain of an antibody targeting PD-L1.

In the present application, the C-terminus of the TIGIT-targeting VHH of the polypeptide can be connected to the N-terminus of the PD-L1-targeting antibody light chain through a linker.

In the present application, the N-terminal of the TIGIT-targeting VHH of the polypeptide can be connected to the C-terminal of the PD-L1-targeting antibody light chain through a linker.

On the other hand, the present application also provides a polypeptide, which may comprise a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain may comprise a PD-L1-targeting antibody light chain, and the second polypeptide chain may comprise a TIGIT-targeting VHH, a linker, and a PD-L1-targeting antibody heavy chain.

In the present application, the C-terminus of the TIGIT-targeting VHH of the polypeptide can be connected to the N-terminus of the PD-L1-targeting antibody heavy chain through a linker.

In the present application, the N-terminal of the VHH targeting TIGIT of the polypeptide can be connected to the C-terminal of the heavy chain of the antibody targeting PD-L1 through a linker.

In the present application, the polypeptide may comprise two first polypeptide chains and two second polypeptide chains.

In the present application, the antibody heavy chain targeting PD-L1 of the polypeptide may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:15, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:16, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:17.

In the present application, the heavy chain of the antibody targeting PD-L1 of the polypeptide may comprise VH, and the VH may comprise the amino acid sequence shown in SEQ ID NO:21.

In the present application, the antibody light chain targeting PD-L1 of the polypeptide may include LCDR1, LCDR2 and LCDR3, the LCDR1 may include the amino acid sequence shown in SEQ ID NO:24, the LCDR2 may include the amino acid sequence shown in SEQ ID NO:25, and the LCDR3 may include the amino acid sequence shown in SEQ ID NO:26.

In the present application, the antibody light chain targeting PD-L1 of the polypeptide may comprise VL, and the VL may comprise the amino acid sequence shown in SEQ ID NO:31.

In the present application, the linker of the polypeptide comprises the amino acid sequence of (GGGGS)n, wherein n can be any integer from 0-10. For example, said n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

In the present application, the antibody heavy chain targeting PD-L1 of the polypeptide may comprise the amino acid sequence shown in SEQ ID NO:23.

In the present application, the antibody light chain targeting PD-L1 of the polypeptide may comprise the amino acid sequence shown in SEQ ID NO:33.

In the present application, the first polypeptide chain of the polypeptide may comprise the amino acid sequence shown in SEQ ID NO:33.

In the present application, the second polypeptide chain of the polypeptide may comprise the amino acid sequence shown in any one of SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36 and SEQ ID NO:37.

In the present application, the two first polypeptide chains of the isolated antigen binding protein may comprise the same amino acid sequence. In the present application, the two second polypeptide chains of the isolated antigen binding protein may comprise the same amino acid sequence.

Nucleic acid molecules, vectors, cells and pharmaceutical compositions

In another aspect, the application provides one or more nucleic acid molecules that encode the isolated antigen binding proteins described herein. For example, it may be produced or synthesized by (i) amplified in vitro, such as by polymerase chain reaction (PCR) amplification, (ii) recombinantly produced by cloning, (iii) purified, such as by enzymatic cleavage and fractionation by gel electrophoresis, or (iv) synthesized, such as by chemical synthesis.

In another aspect, the present application provides a vector, which may comprise the nucleic acid molecule described in the present application. In addition, other genes may be included in the vector, such as marker genes that allow selection of the vector in appropriate host cells and under appropriate conditions. In addition, the vector may also contain expression control elements that permit proper expression of the coding region in an appropriate host. Such control elements are well known to those skilled in the art, and may include, for example, promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation, and the like. The vector can be expressed by transforming, transducing or transfecting the host cell so that the genetic material elements it carries can be expressed in the host cell. Such vectors may include, for example, plasmids, cosmids, viruses, phages, or other vectors commonly used in, for example, genetic engineering. For example, the vector is an expression vector. In addition, the vector may also include components that facilitate its entry into cells, such as, but not exclusively, viral particles, liposomes or protein coats.

In another aspect, the present application provides a cell, which may comprise the nucleic acid molecule or the vector described in the present application. In certain embodiments, each or each host cell may comprise one or more of the nucleic acid molecules or vectors described herein. In certain embodiments, each or each host cell may comprise a plurality (eg, 2 or more) or a plurality (eg, 2 or more) of the nucleic acid molecules or vectors described herein. For example, the vectors described herein can be introduced into the host cells, such as eukaryotic cells, such as cells from plants, fungal or yeast cells, and the like. In certain embodiments, the cells can be bacterial cells (eg, E. coli), yeast cells, or other eukaryotic cells. The vectors described herein can be introduced into the host cells by methods known in the art.

On the other hand, the present application also provides a pharmaceutical composition, which may comprise the isolated antigen-binding protein described in the present application, the polypeptide molecule described in the present application, the nucleic acid molecule described in the present application, the carrier described in the present application and/or the cell described in the present application, and optionally a pharmaceutically acceptable carrier.

In certain embodiments, the pharmaceutical composition may also comprise suitable formulations of one or more (pharmaceutically effective) adjuvants, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and/or preservatives. The acceptable ingredients of the compositions are preferably nontoxic to recipients at the dosages and concentrations employed. Pharmaceutical compositions of the present invention include, but are not limited to, liquid, frozen and lyophilized compositions.

In certain embodiments, the pharmaceutical compositions may also contain more than one active compound, generally those with complementary activities that do not adversely affect each other. The type and effective amount of such drugs may depend, for example, on the formulation The amount and type of antagonist present, as well as the clinical parameters of the subjects.

In certain embodiments, the pharmaceutically acceptable carrier may include any and all solvents, dispersion media, coatings, isotonic agents and absorption delaying agents compatible with pharmaceutical administration, and are generally safe and non-toxic.

In certain embodiments, the pharmaceutical composition may comprise parenteral, transdermal, intracavity, intraarterial, intrathecal and/or intranasal administration or direct injection into tissue. For example, the pharmaceutical composition can be administered to a patient or subject by infusion or injection. In certain embodiments, the administration of the pharmaceutical composition can be performed by different means, such as intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration. In certain embodiments, the pharmaceutical composition can be administered without interruption. Such uninterrupted (or continuous) administration can be achieved by a small pump system worn by the patient to measure the influx of the therapeutic agent into the patient, as described in WO2015/036583.

Preparation

In another aspect, the present application provides a method for preparing the antigen-binding protein. The method may comprise culturing the host cell described herein under conditions such that the antigen binding protein is expressed. For example, by using appropriate medium, appropriate temperature and incubation time, etc., these methods are understood by those of ordinary skill in the art.

method and use

On the other hand, the present application also provides the use of the isolated antigen-binding protein, the polypeptide, the nucleic acid molecule, the carrier, the cell and/or the pharmaceutical composition in the preparation of a medicament for the prevention, alleviation and/or treatment of diseases and/or conditions.

On the other hand, the present application also provides a method for preventing, alleviating or treating a disease and/or disorder, the method may comprise administering the isolated antigen-binding protein, the polypeptide, the nucleic acid molecule, the carrier, the cell and/or the pharmaceutical composition described in the present application to a subject in need. In this application, the administration can be carried out in different ways, such as intravenous, intratumoral, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.

On the other hand, the isolated antigen-binding protein, the polypeptide, the nucleic acid molecule, the carrier, the cell and/or the pharmaceutical composition described in this application can be used to prevent, alleviate or treat diseases and/or conditions.

In the present application, the diseases and/or disorders may include diseases and/or disorders associated with abnormal expression of TIGIT.

In the present application, the diseases and/or conditions may include tumors.

In the present application, the tumor may include solid tumors.

In the present application, the tumor may include non-solid tumors.

In the present application, the tumor may include colon cancer, melanoma, non-small cell lung cancer, renal cell carcinoma and/or hepatocellular carcinoma.

On the other hand, the application also provides a method for blocking the interaction between TIGIT protein and CD155, the method It may comprise administering said antigen binding protein, said polypeptide, said nucleic acid molecule, said carrier, said cell, and/or said pharmaceutical composition to a subject in need thereof.

In the present application, the method may be an in vitro method.

In the present application, the method may be an ex vivo method.

In the present application, the method may be a method for non-diagnostic and therapeutic purposes.

Without intending to be limited by any theory, the following examples are only for explaining the antigen-binding protein of the present application, the preparation method and application, etc., and are not intended to limit the scope of the invention of the present application.

Example

Example 1 Design, Molecular Cloning, Expression and Purification of Bispecific Antibody

The antibody targeting PD-L1 is IgG, while the antibody targeting TIGIT is nanobody. Since both are immune checkpoints, in order to achieve the best blocking effect, when designing bispecific antibodies, the bivalent binding activity similar to that of the parental antibody is retained. In addition, considering that the biological activity of TIGIT monoclonal antibody is largely dependent on the elimination of regulatory T cells in the tumor microenvironment, the Fc of IgG1 is used to retain its complete ADCC activity. The specific design is shown in Figure 1. The nanobody is connected to the N-terminal and C-terminal of the heavy and light chains, respectively, where the flexible linker is (G4S)n, where n=0, 1, 2 or 3, and the bispecific antibody is named BiPT-m, m=2-25. Through conventional molecular cloning methods, the nucleotide sequences encoding peptide chains 1 and 2 are loaded into the eukaryotic expression vector pCMV, and then the vectors expressing peptide chains 1 and 2 are transfected into EXPI293 at a ratio of 2:3, and placed in a cell culture incubator. After culturing for 5 days, the supernatant was collected and purified with protein A (GenScript, catalog number: L00695-80). The amino acid sequence of the BiPT-m is shown in Table 1.

Table 1 Amino acid sequence of BiPT-m

Example 2 Detecting the Purity of Bispecific Antibodies by HPLC-SEC

Turn on the high performance liquid chromatograph (Agilent Technologies 1200Series), first use purified water to remove the air in the system pipeline at a flow rate of 2ml/min, rinse for 30min, then connect SET-C SEC-300 (Sepax, 2F36102) to the instrument, first wash the chromatographic column with purified water at a flow rate of 0.5ml/min for 1h, and then wash the chromatographic column with PBS at a flow rate of 1ml/min for 1h, and wash the sample to be tested Dilute to 1 mg/ml, take 100 μL of Marker and the diluted sample and add them to the sampling tank tube respectively, set up the instrument, set the marker sample volume to 6 μl with a flow rate of 1 ml/min, run for 20 min, set the diluted sample volume to be tested at 10 μL with a flow rate of 1 ml/min, and run for 20 min, after the sample detection is completed, rinse the column with purified water at a flow rate of 1 ml/min for 1 hour, and then use 20% ethanol to rinse the column at a flow rate of 0.5 ml/min. After h is finished, export the spectrum and turn off the instrument. The results are shown in Figure 2A-P. Linking the nanobody to the light chain will cause problems in IgG heavy and light chain pairing, and some bispecific antibodies will lose the light chain during the purification process, regardless of the length of the flexible linker. And linking the nanobody to the heavy chain can get 100% pure bis-antibody.

Example 3 Detection of binding activity of bispecific antibody to human PD-L1 and TIGIT by flow cytometry

After digesting and centrifuging normally cultured 293T-human TIGIT and 293T-human PD-L1 cells, wash with 0.1% BSA Resuspend in PBS, take a 96-well pointed-bottom plate, add 4E4 cells to each well, 30 μl per well. The corresponding antibody BiPT-18-25 was diluted to 200nM, and then 11 gradients were diluted by 3 times, and no antibody was added to the last gradient. Take 30 μl of antibody solution, mix it with the cell suspension, and incubate at 4°C for 1 hour. The cells were washed twice with PBS containing 0.1% BSA, centrifuged at 500 g for 5 min each time, and the liquid in the wells was shaken dry. Anti-human IgG Fc-650 secondary antibody (abcam, product number: ab98593) was diluted 1:200 times with PBS containing 0.1% BSA, added 30 μl to each well, mixed with cells, and incubated at 4°C for 30 min. Wash the cells 3 times with PBS containing 0.1% BSA, 500g each time, centrifuge for 5min, and finally resuspend the cells in 30μl PBS containing 0.1%BSA, put them on the machine, and use Graphpad to make a four-parameter fitting curve after reading the data, and calculate EC50. Wherein, the VHH sequence of hTIGI7.11E is shown in SEQ ID NO:12, the VH sequence of YN035 is shown in SEQ ID NO:21, and the VL sequence of YN035 is shown in SEQ ID NO:31. The results of the binding activity test are shown in Figure 3. When the Nanobody is attached to the N-terminus of the heavy chain, its binding activity is not affected. The activities of all bi-antibodies binding to PD-L1 remained consistent.

Example 4 Detection of binding activity of bispecific antibody to TIGIT of cynomolgus monkey by flow cytometry

Digest and centrifuge normally cultured 293T-cynomolgus TIGIT cells, resuspend with PBS containing 0.1% BSA, take 96-well tip-bottom plate, add 4E4 cells to each well, 30 μl per well. The corresponding antibody BiPT-18-25 was diluted to 200nM, and then 11 gradients were diluted by 3 times, and no antibody was added to the last gradient. Take 30 μl of antibody solution, mix it with the cell suspension, and incubate at 4°C for 1 hour. The cells were washed twice with PBS containing 0.1% BSA, centrifuged at 500 g for 5 min each time, and the liquid in the wells was shaken dry. Anti-human IgG Fc-650 secondary antibody (abcam, product number: ab98593) was diluted 1:200 times with PBS containing 0.1% BSA, added 30 μl to each well, mixed with cells, and incubated at 4°C for 30 min. Wash the cells 3 times with PBS containing 0.1% BSA, 500g each time, centrifuge for 5min, and finally resuspend the cells in 30μl PBS containing 0.1%BSA, put them on the machine, and use Graphpad to make a four-parameter fitting curve after reading the data, and calculate EC50. The results are shown in Figure 4. When the Nanobody is attached to the N-terminal of the heavy chain, the TIGIT binding activity of the Nanobody is not affected.

Example 5 Flow cytometric detection of bispecific antibody BiPT-22-25 blocking activity

复苏293T-hTIGIT细胞传代；收获细胞，计数后调整细胞密度为1×10 ⁶ /ml，30μl/well(3×10 ⁴ /well)；hTIGI7.11E，YN035，BiPT22-25双抗，阳性抗体Tiragolumab(VH的氨基酸序列如SEQ ID NO:63所示，VL的氨基酸序列如SEQ ID NO:64所示)以800nM为最高浓度，3倍比，11个梯度，设PBS对照，15μl/well，之后加入12ug/ml的Biotin-CD155，15μl/well，混匀后4℃孵育1小时；含0.1％BSA的PBS洗涤二次，500g，5min，4℃，甩干；1：500稀释Streptavidin,Alexa Fluor ^TM APC conjugate，30ul/well，混匀后4℃孵育30分钟；含0.1％BSA的PBS洗涤二次，500g，5min，4℃，甩干；30μl/well重悬，上高通量流式检测仪读值，采用Graphpad做四参数拟合曲线。 The results are shown in Figure 5, hTIGI7.11E, BiPT-22, BiPT-23, BiPT-24 and BiPT- The IC50 of 25 is about 2-3 times better than tiragolumab.

Example 6 Fortebio detects the affinity of BiPT-22-25

Dilute BiPT-22-25 to 100nM and add antibody to 384-well plate. Dilute human TIGIT-His antigen (Beijing Baipu Saisi Biotechnology Co., Ltd., TIT-H52H5-100ug) and PD-L1-His protein to 100nM, 2-fold dilution, a total of 7 gradients, and add to a 384-well plate. Use the AHC probe (Sartorius, catalog number: 18-5060), set the binding time to 180 sec, the dissociation time to 300 sec, the baseline to 60 sec, and the regeneration cycle three times, 5 sec each. Afterwards, software was used to fit the binding-dissociation curve to calculate the affinity of the antibody. The results are shown in Figure 6A-J. All bispecific antibodies BiPT-22, BiPT-23, BiPT-24 and BiPT-25 retained the affinity of the parental antibody.

Example 7 Detection of ADCC activity of BiPT-18-25

The target cells are: 293T-human TIGIT cells, which are 293T cell lines that can stably express human TIGIT and luciferase luciferase established in the laboratory; target cells are counted and centrifuged and then resuspended in "DMEM+10% FBS" medium, with a cell density of 2E5/ml, 50 μl/well, that is, 5000 target cells per well; a frozen PBMC, 8E7cells, was shipped from the company's warehouse; washed 3 times with PBS, 500g/5min, 400g/5min, 300g/5min, centrifuge at room temperature. After counting; the effector cells were also resuspended in the medium of "DMEM+10% FBS", the cell density of 1.1E7/ml, 50ul/well, that is, 1.75E5/well target cells, the target cell ratio E:T=35:1; the antibodies were: hTIGIT7.11E, IgG1-FC, BiPTs, the initial working concentration of the antibody was 100nM, 8-fold gradient dilution, a total of 7 concentration gradients were formed, 50ul/well; , effector cells and antibodies were added together into a 96-well cell culture plate with a white background that is opaque, and a separate well of target cells was set as a control. After culturing in a 37°C incubator for 48 hours, the luciferase content in the plate was detected with a Tecan microplate reader; cleavage percentage (%)=(single target well-experimental well)/target single well×100. The results are shown in Figure 7. For PD-L1, the ADCC activity of all biantibodies was consistent with that of the parental antibody; as for TIGIT, when the Nanobody was linked to the N-terminus of the heavy chain, the ADCC activity was less affected.

Example 8 Flow cytometric detection of thermal stability of BiPT-18-25

100nM antibodies YN035, hTIGI7.11E, BiPT-22, BiPT-23, BiPT-24 and BiPT-25 were incubated at 20°C, 30°C, 40°C, 50°C, 60°C, 70°C, 80°C, 90°C for 1 hour, respectively. Then the normally cultured 293T-human TIGIT cells and 293T-human PD-L1 cells were digested and centrifuged, and then resuspended in PBS containing 0.1% BSA. Take a 96-well sharp-bottom plate and add 4E4 cells to each well, 30 μl per well. Dilute the antibody incubated at the corresponding temperature to 50nM. Take 30 μl of antibody solution, mix it with the cell suspension, and incubate at 4°C for 1 hour in duplicate wells. The cells were washed twice with PBS containing 0.1% BSA, centrifuged at 500 g for 5 min each time, and the liquid in the wells was shaken dry. Dilute the anti-human IgG Fc-650 secondary antibody (abcam, product number: ab98593) with PBS containing 0.1% BSA at 1:200 times, add 30 μl to each well and mix with the cells, Incubate at 4°C for 30 min. The cells were washed 3 times with PBS containing 0.1% BSA, 500 g each time, centrifuged for 5 min, and finally the cells were resuspended with 30 μl of PBS containing 0.1% BSA, put on the machine, and read the data. The results are shown in Figure 8. All the double antibodies can still maintain high binding activity after incubation at 60°C for 1 hour, and have good thermal stability.

Example 9 Verifying the in vivo efficacy of BiPT-23

MC38 cells were inoculated subcutaneously on the right side of C57BL/6-hTIGIT humanized mice at a concentration of 1×10 ⁶ cells/0.1 mL. When the tumors grew to about 150 mm ³ , they were selected according to the tumor volume and randomly divided into groups. The route of administration for all groups was intraperitoneal injection, administered once every three days, and administered continuously for 6 times. The experiment was terminated 11 days after the last administration. During the period of administration and observation, the body weight and tumor volume of the mice were measured 3 times a week, and the measured values were recorded, and the tumor volume inhibition rate (TGITV%) was calculated. At the end of the experiment, the animals were euthanized. The results are shown in Figure 9. On the 30th day, compared with IgG1-Fc and Tiragolumab in the control group, BiPT-23 at low, medium and high concentrations could significantly inhibit tumor growth.

Claims

An isolated antigen-binding protein, comprising: a first antigen-binding domain and a second antigen-binding domain, the first antigen-binding domain can specifically bind PD-L1, the second antigen-binding domain can specifically bind TIGIT, the first antigen-binding domain comprises at least one CDR in the heavy chain variable region VH, and the VH comprises the amino acid sequence shown in SEQ ID NO:21.
The antigen binding protein according to claim 1, wherein the first antigen binding domain comprises HCDR3, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:17.
The antigen-binding protein according to any one of claims 1-2, wherein the first antigen-binding domain comprises HCDR2, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:16.
The antigen-binding protein according to any one of claims 1-3, wherein the first antigen-binding domain comprises HCDR1, and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:15.
The antigen-binding protein according to claim 4, wherein the first antigen-binding domain comprises H-FR1, the C-terminus of the H-FR1 is directly or indirectly linked to the N-terminus of the HCDR1, and the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:18.
The antigen-binding protein according to any one of claims 4-5, wherein the first antigen-binding domain comprises H-FR2, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:19.
The antigen-binding protein according to any one of claims 3-6, wherein the first antigen-binding domain comprises H-FR3, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:20.
The antigen-binding protein according to any one of claims 2-7, wherein the first antigen-binding domain comprises H-FR4, the N-terminus of the H-FR4 is connected to the C-terminus of the HCDR3, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:10.
The antigen-binding protein according to any one of claims 1-8, wherein the first antigen-binding domain comprises VH, and the VH comprises the amino acid sequence shown in SEQ ID NO:21.
The antigen-binding protein according to any one of claims 1-9, wherein the first antigen-binding domain comprises LCDR3, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:26.
The antigen-binding protein according to any one of claims 1-10, wherein the first antigen-binding domain comprises LCDR2, and the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:25.
The antigen-binding protein according to any one of claims 1-11, wherein the first antigen-binding domain comprises LCDR1, and the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:24.
The antigen binding protein according to claim 12, wherein said first antigen binding domain comprises L-FR1, said L- The C-terminal of FR1 is directly or indirectly connected to the N-terminal of LCDR1, and the L-FR1 comprises the amino acid sequence shown in SEQ ID NO:27.
The antigen-binding protein according to any one of claims 12-13, wherein the first antigen-binding domain comprises L-FR2, the L-FR2 is located between the LCDR1 and the LCDR2, and the L-FR2 comprises the amino acid sequence shown in SEQ ID NO:28.
The antigen-binding protein according to any one of claims 11-14, wherein the first antigen-binding domain comprises L-FR3, the L-FR3 is located between the LCDR2 and the LCDR3, and the L-FR3 comprises the amino acid sequence shown in SEQ ID NO:29.
The antigen-binding protein according to any one of claims 10-15, wherein the first antigen-binding domain comprises L-FR4, the N-terminus of the L-FR4 is connected to the C-terminus of the LCDR3, and the L-FR4 comprises the amino acid sequence shown in SEQ ID NO:30.
The antigen-binding protein according to any one of claims 1-16, wherein the first antigen-binding domain comprises a VL, and the VL comprises the amino acid sequence shown in SEQ ID NO:31.
The antigen binding protein according to any one of claims 1-17, wherein said first antigen binding domain comprises an antibody heavy chain constant region derived from IgG.
The antigen binding protein according to any one of claims 1-18, wherein said first antigen binding domain comprises an antibody heavy chain constant region derived from human IgG.
The antigen binding protein according to any one of claims 1-19, wherein said first antigen binding domain comprises an antibody heavy chain constant region derived from human IgG1.
The antigen-binding protein according to any one of claims 1-20, wherein the first antigen-binding domain comprises an antibody heavy chain constant region, and the antibody heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO:22.
The antigen binding protein according to any one of claims 1-21, wherein the first antigen binding domain comprises a heavy chain, and the heavy chain comprises the amino acid sequence shown in SEQ ID NO:23.
The antigen binding protein of any one of claims 1-22, wherein the first antigen binding domain comprises an antibody light chain constant region derived from Igκ.
The antigen-binding protein according to any one of claims 1-23, wherein the first antigen-binding domain comprises an antibody light chain constant region, and the light chain constant region comprises the amino acid sequence shown in SEQ ID NO:32.
The antigen binding protein according to any one of claims 1-24, wherein the first antigen binding domain comprises a light chain, and the light chain comprises the amino acid sequence shown in SEQ ID NO:31.
The antigen binding protein of any one of claims 1-25, wherein the first antigen binding domain comprises an antibody or antigen binding fragment thereof.
The antigen binding protein of claim 26, wherein the antigen binding fragment comprises Fab, Fab', F(ab) 2 , Fv fragment, F(ab') 2 , scFv, di-scFv, VHH and/or dAb.
The antigen binding protein according to any one of claims 26-27, wherein said antibody is selected from the group consisting of monoclonal antibodies, chimeric antibodies, humanized antibodies and fully human antibodies.
The antigen binding protein of any one of claims 1-28, wherein the first antigen binding domain is a monoclonal antibody.
The antigen-binding protein according to any one of claims 1-29, wherein the second antigen-binding domain comprises at least one CDR in the variable region VH of an antibody heavy chain, and the VH comprises the amino acid sequence shown in SEQ ID NO:42.
The antigen-binding protein according to any one of claims 1-30, wherein the second antigen-binding domain comprises HCDR3, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:6.
The antigen-binding protein according to any one of claims 1-31, wherein the second antigen-binding domain comprises HCDR2, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:41.
The antigen-binding protein according to any one of claims 1-32, wherein the second antigen-binding domain comprises HCDR2, and the HCDR2 comprises the amino acid sequence shown in any one of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5.
The antigen binding protein according to any one of claims 1-33, wherein the second antigen binding domain comprises HCDR1, and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:1.
The antigen-binding protein according to claim 34, wherein the second antigen-binding domain comprises H-FR1, the C-terminus of the H-FR1 is directly or indirectly connected to the N-terminus of the HCDR1, and the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:7.
The antigen-binding protein according to any one of claims 34-35, wherein the second antigen-binding domain comprises H-FR2, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:8.
The antigen binding protein according to any one of claims 32-36, wherein the second antigen binding domain comprises H-FR3, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:9.
The antigen-binding protein according to any one of claims 31-37, wherein the second antigen-binding domain comprises H-FR4, the N-terminus of the H-FR4 is connected to the C-terminus of the HCDR3, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:10.
The antigen binding protein according to any one of claims 1-38, wherein the second antigen binding domain comprises VH, and the VH comprises the amino acid sequence shown in SEQ ID NO:42.
The antigen-binding protein according to any one of claims 1-39, wherein the second antigen-binding domain comprises VH, and the VH comprises the amino acid sequence shown in any one of SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14.
The antigen binding protein of any one of claims 1-40, wherein the second antigen binding domain comprises an antibody or antigen binding fragment thereof.
The antigen binding protein according to claim 41, wherein said antigen binding fragment comprises Fab, Fab', F(ab) 2 , Fv fragment, F(ab') 2 , scFv, di-scFv, VHH and/or dAb.
The antigen binding protein according to any one of claims 41-42, wherein said antibody is selected from the group consisting of monoclonal antibodies, chimeric antibodies, humanized antibodies and fully human antibodies.
The antigen binding protein of any one of claims 1-43, wherein the second antigen binding domain is VHH.
The antigen binding protein according to claim 44, wherein said VHH comprises the amino acid sequence shown in SEQ ID NO:42.
The antigen binding protein according to claim 45, wherein the VHH comprises the amino acid sequence shown in any one of SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14.
The antigen binding protein of any one of claims 1-46, wherein the first antigen binding domain and the second antigen binding domain are directly linked.
The antigen binding protein of any one of claims 1-46, wherein the first antigen binding domain and the second antigen binding domain are indirectly linked.
The antigen binding protein according to any one of claims 1-48, wherein the second antigen binding domain is directly or indirectly linked to the light chain of the first antigen binding domain.
The antigen binding protein according to any one of claim 49, wherein the C-terminus of the VHH of the second antigen-binding domain is directly or indirectly linked to the N-terminus of the light chain of the first antigen-binding domain.
The antigen binding protein according to any one of claim 49, wherein the N-terminus of the VHH of the second antigen-binding domain is directly or indirectly linked to the C-terminus of the light chain of the first antigen-binding domain.
The antigen binding protein according to any one of claims 1-48, wherein the second antigen binding domain is directly or indirectly linked to the heavy chain of the first antigen binding domain.
The antigen binding protein according to claim 52, wherein the C-terminus of the VHH of the second antigen-binding domain is directly or indirectly linked to the N-terminus of the heavy chain of the first antigen-binding domain.
The antigen binding protein according to claim 52, wherein the N-terminus of the VHH of the second antigen-binding domain is directly or indirectly linked to the C-terminus of the heavy chain of the first antigen-binding domain.
The antigen binding protein according to any one of claims 1-54, wherein the second antigen binding domain is associated with the first The light chains of the antigen binding domains are directly or indirectly linked.
The antigen binding protein according to any one of claims 1-55, wherein the first antigen binding domain and the second antigen binding domain are linked by a linker.
The antigen binding protein of claim 56, wherein the linker is a peptide linker.
The antigen binding protein according to any one of claims 56-57, wherein the linker comprises the amino acid sequence of (GGGGS)n, wherein n is any integer from 0-10.
A polypeptide comprising a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises a VHH targeting TIGIT, a linker and an antibody light chain targeting PD-L1, and the second polypeptide chain comprises an antibody heavy chain targeting PD-L1.
The polypeptide according to claim 59, wherein the C-terminus of the VHH targeting TIGIT is connected to the N-terminus of the light chain of the antibody targeting PD-L1 through a linker.
The polypeptide according to claim 59, wherein the N-terminal of the VHH targeting TIGIT is connected to the C-terminal of the light chain of the antibody targeting PD-L1 through a linker.
A polypeptide comprising a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises a PD-L1-targeting antibody light chain, and the second polypeptide chain comprises a TIGIT-targeting VHH, a linker, and a PD-L1-targeting antibody heavy chain.
The polypeptide according to claim 62, wherein the C-terminus of the VHH targeting TIGIT is connected to the N-terminus of the heavy chain of the antibody targeting PD-L1 through a linker.
The polypeptide according to claim 62, wherein the N-terminal of the VHH targeting TIGIT is connected to the C-terminal of the heavy chain of the antibody targeting PD-L1 through a linker.
The polypeptide according to any one of claims 59-64, comprising two first polypeptide chains and two second polypeptide chains.
The polypeptide according to any one of claims 59-65, wherein the antibody heavy chain targeting PD-L1 comprises HCDR1, HCDR2 and HCDR3, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:15, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:16, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:17.
The polypeptide according to any one of claims 59-66, wherein the heavy chain of the antibody targeting PD-L1 comprises VH, and the VH comprises the amino acid sequence shown in SEQ ID NO:21.
The polypeptide according to any one of claims 59-67, wherein the antibody light chain targeting PD-L1 comprises LCDR1, LCDR2 and LCDR3, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:24, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:25, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:26.
The polypeptide according to any one of claims 59-68, wherein the antibody light chain targeting PD-L1 comprises VL, And the VL comprises the amino acid sequence shown in SEQ ID NO:31.
The polypeptide according to any one of claims 59-69, wherein the linker comprises the amino acid sequence of (GGGGS)n, wherein n is any integer from 0-10.
The polypeptide according to any one of claims 59-70, wherein the heavy chain of the antibody targeting PD-L1 comprises the amino acid sequence shown in SEQ ID NO:23.
The polypeptide according to any one of claims 59-71, wherein the antibody light chain targeting PD-L1 comprises the amino acid sequence shown in SEQ ID NO:33.
The polypeptide according to any one of claims 59-72, wherein the first polypeptide chain comprises the amino acid sequence shown in SEQ ID NO:33.
The polypeptide according to any one of claims 59-73, wherein the second polypeptide chain comprises the amino acid sequence shown in any one of SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36 and SEQ ID NO:37.
One or more isolated nucleic acid molecules encoding the antigen binding protein of any one of claims 1-58 or the polypeptide of any one of claims 59-74.
A vector comprising the nucleic acid molecule of claim 75.
A cell comprising the antigen binding protein of any one of claims 1-58, the polypeptide of any one of 59-74, the nucleic acid molecule of claim 75, or the vector of claim 76.
A method of producing the antigen-binding protein of any one of claims 1-58, said method comprising culturing the cell of claim 77 under conditions such that said antigen-binding protein is expressed.
A pharmaceutical composition comprising the antigen-binding protein of any one of claims 1-58 or the polypeptide of any one of claims 59-74, and optionally a pharmaceutically acceptable carrier.
Use of the antigen-binding protein according to any one of claims 1-58, the polypeptide according to any one of claims 59-74, the nucleic acid molecule according to claim 75, the carrier according to claim 76, the cell according to claim 77, and/or the use of the pharmaceutical composition according to claim 79 in the preparation of a medicament for preventing, treating and/or alleviating diseases and/or conditions.
The use according to claim 80, wherein the diseases and/or disorders include diseases and/or disorders associated with abnormal expression of TIGIT.
Use according to any one of claims 80-81, wherein the disease and/or condition comprises a tumor.
The use according to claim 82, wherein said tumor comprises a solid tumor.
The use according to claim 82, wherein the tumor comprises a non-solid tumor.
The use according to any one of claims 82-84, wherein the tumor comprises colon cancer, melanoma, non-small cell lung cancer, renal cell carcinoma and/or hepatocellular carcinoma.
A method for preventing, treating and/or alleviating diseases and/or disorders, said method comprising administering to a subject in need the antigen-binding protein of any one of claims 1-58, the polypeptide of any one of claims 59-74, the nucleic acid molecule of claim 75, the carrier of claim 76, the cell of claim 77, and/or the pharmaceutical composition of claim 79.
The method according to claim 86, wherein the disease and/or condition comprises a disease and/or condition associated with abnormal expression of TIGIT.
The method according to any one of claims 86-87, wherein the disease and/or condition comprises a tumor.
The method of claim 88, wherein the tumor comprises a solid tumor.
The method of claim 88, wherein the tumor comprises a non-solid tumor.
The method according to any one of claims 88-90, wherein the tumor comprises colon cancer, melanoma, non-small cell lung cancer, renal cell carcinoma and/or hepatocellular carcinoma.
A method for blocking the interaction between TIGIT protein and CD155, the method comprising administering to a subject in need the antigen-binding protein of any one of claims 1-58, the polypeptide of any one of claims 59-74, the nucleic acid molecule of claim 75, the carrier of claim 76, the cell of claim 77, and/or the pharmaceutical composition of claim 79.