Anti-4-1BB antibody and its uses

Abstract

The present application provides an antibody or an antigen-binding fragment thereof that can specifically bind to 4-1BB, and a pharmaceutical composition containing the same. The present application also provides a polynucleotide molecule encoding the antibody or the antigen-binding fragment thereof, an expression vector and a host cell for expressing the antibody or the antigen-binding fragment thereof, and uses of the antibody or the antigen-binding fragment thereof of the present application.

Description

[0001] This application claims the priority of a Chinese patent application with the application number 202111455972.6 and the invention title "Anti-4-1BB Antibody and Its Use", which was filed with the Chinese Patent Office on December 1, 2021. The entire content thereof is incorporated herein by reference. Technical Field

[0002] This application belongs to the field of biomedicine, and particularly relates to an antibody or its antigen-binding fragment that can specifically bind to 4-1BB and its use. Background Art

[0003] Cancer immunotherapy is a treatment method that restores or enhances the natural defense against tumors through the human immune system. This treatment usually targets specific biomolecules on the surface of cancer cells, such as tumor-associated antigens (TAAs). Antitumor activity is achieved by directing the host immune system to TAAs, thereby establishing or inducing an adaptive immune response against cancer cells. In the past few decades, the treatment of cancer using monoclonal antibodies (MAbs) has achieved great success, and many of them have been approved for cancer treatment or clinical trials.

[0004] 4-1BB (CD137 / TNFRSF9) is a transmembrane protein of the tumor necrosis factor receptor superfamily (TNFRS), which is expressed on antigen-primed T cells but not on resting T cells. Human 4-1BB is a protein with 255 amino acids, including a signal sequence (amino acid residues 1-17), an extracellular domain (169 amino acids), a transmembrane region (27 amino acids), and an intracellular domain (42 amino acids). 4-1BB is expressed on the cell surface as a monomer or dimer, and signal transduction occurs through trimerization after binding to its ligand (4-1BBL).

[0005] In addition, 4-1BB is known to be expressed in dendritic cells (DCs), natural killer cells (NKs), activated CD4 + and CD8 + T lymphocytes, eosinophils, natural killer T cells (NKTs), and mast cells, but not expressed on the surface of myeloid-derived suppressor cells (MDSCs). Anti-4-1BB antibodies have the ability to activate cytotoxic T cells and increase the production of interferon-γ (IFN-γ), showing great potential in anti-cancer.

[0006] Currently, multiple 4-1BB-related drug research projects are underway. The reported 4-1BB antibody sequences, in binding to 4-1BB and activating the 4-1BB signaling pathway, either overactivate, leading to CD8 in the liver +T cell enrichment causes hepatotoxicity, or has good safety but insufficient activation, resulting in poor tumor killing effect. Therefore, there is an urgent need to develop new 4-1BB antibody drugs with better activity and lower toxic and side effects. Summary of the Invention

[0007] The object of the present application is to provide an antibody (i.e., anti-4-1BB antibody) or its antigen-binding fragment that can specifically bind to 4-1BB, which can be used as an independent therapy or in combination with other therapies / other anti-cancer agents for the treatment of diseases such as cancer.

[0008] In a first aspect of the present application, there is provided an antibody or its antigen-binding fragment that can specifically bind to 4-1BB, which comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3;

[0009] Wherein, according to the Kabat, IMGT, Chothia, AbM, Contact or North numbering system, the amino acid sequences of the HCDR1, HCDR2, and HCDR3 are the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region shown in SEQ ID NO: 13 or 15, and the amino acid sequences of the LCDR1, LCDR2, and LCDR3 are the LCDR1, LCDR2, and LCDR3 of the light chain variable region shown in SEQ ID NO: 14 or 16, and at least one of the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 can be replaced with a variant having 1, 2, or 3 amino acid differences therefrom.

[0010] In some embodiments, the antibody or its antigen-binding fragment that can specifically bind to 4-1BB comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3;

[0011] Wherein, according to the Kabat, IMGT, Chothia, AbM, Contact or North numbering system, the amino acid sequences of the HCDR1, HCDR2, and HCDR3 are the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region shown in SEQ ID NO: 13, and the amino acid sequences of the LCDR1, LCDR2, and LCDR3 are the LCDR1, LCDR2, and LCDR3 of the light chain variable region shown in SEQ ID NO: 14; or

[0012] The amino acid sequences of the HCDR1, HCDR2, and HCDR3 are the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region as shown in SEQ ID NO: 15, and the amino acid sequences of the LCDR1, LCDR2, and LCDR3 are the LCDR1, LCDR2, and LCDR3 of the light chain variable region as shown in SEQ ID NO: 16.

[0013] In some embodiments, according to the Kabat numbering system, the HCDR1, HCDR2, and HCDR3 respectively have the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, or respectively have the amino acid sequences shown in SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9;

[0014] The LCDR1, LCDR2, and LCDR3 respectively have the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, or respectively have the amino acid sequences shown in SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12;

[0015] Wherein, at least one of the SEQ ID NO: 1-12 can be replaced with a variant having 1, 2, or 3 amino acid differences therefrom.

[0016] In some embodiments, according to the Kabat numbering system, the HCDR1, HCDR2, and HCDR3 respectively have the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, and the LCDR1, LCDR2, and LCDR3 respectively have the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6; or

[0017] The HCDR1, HCDR2, and HCDR3 respectively have the amino acid sequences shown in SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, and the LCDR1, LCDR2, and LCDR3 respectively have the amino acid sequences shown in SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12.

[0018] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0019] A heavy chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 13, and a light chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 14; or

[0020] A heavy chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 15, and a light chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 16;

[0021] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0022] A heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 13, and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 14; or

[0023] A heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 15, and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 16.

[0024] In some embodiments, the antibody or its antigen-binding fragment further comprises a human heavy chain constant region and a human light chain constant region. The human heavy chain constant region is selected from the heavy chain constant regions of human IgG1, IgG2, IgG3 or IgG4, preferably the heavy chain constant region of human IgG4 or the heavy chain constant region of human IgG4 having the S228P amino acid substitution; the human light chain constant region is selected from the light chain constant regions of the λ light chain or the κ light chain.

[0025] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0026] A heavy chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 17, and a light chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 18; or

[0027] A heavy chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:19, and a light chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:20;

[0028] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0029] A heavy chain with an amino acid sequence as shown in SEQ ID NO:17, and a light chain with an amino acid sequence as shown in SEQ ID NO:18; or

[0030] A heavy chain with an amino acid sequence as shown in SEQ ID NO:19, and a light chain with an amino acid sequence as shown in SEQ ID NO:20.

[0031] In some embodiments, the antibody comprises at least one of a monoclonal antibody and a multispecific antibody, and the antigen-binding fragment comprises at least one of Fab, Fab', F(ab')2, Fv, scFv and sdAb.

[0032] The second aspect of the present application provides an isolated antibody or its antigen-binding fragment, which has at least one of the following characteristics:

[0033] (Ⅰ) Binding to the same epitope of human 4-1BB protein as the antibody or its antigen-binding fragment provided in the first aspect of the present application, or a completely overlapping or partially overlapping epitope;

[0034] (Ⅱ) Competing with the antibody or its antigen-binding fragment provided in the first aspect of the present application for binding to the epitope of human 4-1BB protein.

[0035] The third aspect of the present application provides a polynucleotide molecule, which comprises at least one of a nucleotide sequence encoding the antibody or its antigen-binding fragment provided in the first aspect of the present application, or a nucleotide sequence encoding the isolated antibody or its antigen-binding fragment provided in the second aspect of the present application, or its complementary sequence.

[0036] The fourth aspect of the present application provides an expression vector, which comprises the polynucleotide molecule provided in the third aspect of the present application. Preferably, the expression vector is a eukaryotic expression vector.

[0037] The fifth aspect of the present application provides a host cell, which comprises the polynucleotide molecule provided in the third aspect of the present application, or the expression vector provided in the fourth aspect of the present application. Preferably, the host cell is a eukaryotic cell, more preferably a mammalian cell.

[0038] In some embodiments, the host cell is used to express the antibody or antigen-binding fragment thereof of the first aspect of the present application or the isolated antibody or antigen-binding fragment thereof of the second aspect of the present application.

[0039] The sixth aspect of the present application provides a method for preparing the antibody or antigen-binding fragment thereof of the first aspect of the present application or the isolated antibody or antigen-binding fragment thereof of the second aspect of the present application, which includes expressing the antibody or antigen-binding fragment thereof in the host cell provided in the fifth aspect of the present application and recovering the expressed antibody or antigen-binding fragment thereof from the host cell.

[0040] The seventh aspect of the present application provides a pharmaceutical composition, which comprises the antibody or antigen-binding fragment thereof provided in the first aspect of the present application or the isolated antibody or antigen-binding fragment thereof provided in the second aspect of the present application or the polynucleotide molecule provided in the third aspect of the present application or the expression vector provided in the fourth aspect of the present application or the host cell provided in the fifth aspect of the present application, and a pharmaceutically acceptable carrier or excipient.

[0041] The eighth aspect of the present application provides the use of the antibody or antigen-binding fragment thereof of the first aspect of the present application or the isolated antibody or antigen-binding fragment thereof of the second aspect of the present application or the polynucleotide molecule provided in the third aspect of the present application or the expression vector provided in the fourth aspect of the present application or the host cell provided in the fifth aspect of the present application or the pharmaceutical composition provided in the seventh aspect of the present application in the preparation of a drug for treating or preventing 4-1BB-mediated diseases.

[0042] The ninth aspect of the present application provides the use of the antibody or antigen-binding fragment thereof of the first aspect of the present application or the isolated antibody or antigen-binding fragment thereof of the second aspect of the present application or the polynucleotide molecule provided in the third aspect of the present application or the expression vector provided in the fourth aspect of the present application or the host cell provided in the fifth aspect of the present application or the pharmaceutical composition provided in the seventh aspect of the present application in treating or preventing 4-1BB-mediated diseases.

[0043] The tenth aspect of the present application provides a method for treating or preventing a 4-1BB-mediated disease or disorder, which includes administering to a subject in need the antibody or antigen-binding fragment thereof provided in the first aspect of the present application or the isolated antibody or antigen-binding fragment thereof provided in the second aspect of the present application or the polynucleotide molecule provided in the third aspect of the present application or the expression vector provided in the fourth aspect of the present application or the host cell provided in the fifth aspect of the present application or the pharmaceutical composition provided in the seventh aspect of the present application.

[0044] In some embodiments, the 4-1BB-mediated disease is cancer; preferably, the cancer is selected from at least one of melanoma, breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, kidney cancer, lung cancer, liver cancer, gastric cancer, colorectal cancer, bladder cancer, head and neck cancer, thyroid cancer, esophageal cancer, cervical cancer, sarcoma, multiple myeloma, leukemia, lymphoma, gallbladder cancer, and glioblastoma.

[0045] The eleventh aspect of the present application provides a kit, which includes the antibody or its antigen-binding fragment provided in the first aspect of the present application, or the isolated antibody or its antigen-binding fragment provided in the second aspect of the present application, or the polynucleotide molecule provided in the third aspect of the present application, or the expression vector provided in the fourth aspect of the present application, or the host cell provided in the fifth aspect of the present application, or the pharmaceutical composition provided in the seventh aspect of the present application.

[0046] The twelfth aspect of the present application provides a method for detecting 4-1BB using the antibody or its antigen-binding fragment provided in the first aspect of the present application or the isolated antibody or its antigen-binding fragment provided in the second aspect of the present application, which includes contacting the antibody or its antigen-binding fragment with a sample, detecting the conjugate formed by the antibody or its antigen-binding fragment and 4-1BB, and optionally quantitatively determining the conjugate.

[0047] The antibody or its antigen-binding fragment provided by the present application that can specifically bind to 4-1BB can specifically bind to human 4-1BB, has low hepatotoxicity and obvious tumor suppression effects, and can be used to treat 4-1BB-mediated diseases, such as cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] To more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only one embodiment of the present application. For those of ordinary skill in the art, other embodiments can also be obtained based on these drawings.

[0049] Figure 1 Shows the test results of the binding ability of the anti-4-1BB antibodies JS011-phage 3 and JS011-phage 6 prepared in Example 2 of the present application to human 4-1BB.

[0050] Figure 2 Shows the test results of the binding ability of the anti-4-1BB antibodies JS011-phage 3 and JS011-phage 6 prepared in Example 2 of the present application to murine 4-1BB.

[0051] Figure 3Shows the test results of the binding ability of anti-4-1BB antibodies JS011-phage 3 and JS011-phage 6 prepared in Example 2 of the present application, as well as related controls, to Jurkat NFKB 4-1BB cells.

[0052] Figure 4 Shows the test results of the anti-4-1BB antibodies JS011-phage 3 and JS011-phage 6 prepared in Example 2 of the present application, as well as related controls, in blocking the binding of 4-1BBL to Jurkat NFKB 4-1BB cells.

[0053] Figure 5 Shows the activity detection results of the anti-4-1BB antibodies JS011-phage 3 and JS011-phage 6 prepared in Example 2 of the present application, as well as related controls, in the luciferase reporter gene system (CHO FCGR2B 4G6 / Jurkat NFKB 4-1BB dual cells).

[0054] Figure 6 Shows the activity detection results of the anti-4-1BB antibodies JS011-phage 3 and JS011-phage 6 prepared in Example 2 of the present application, as well as related controls, in the luciferase reporter gene system (THP-1 / Jurkat NFKB 4-1BB dual cells).

[0055] Figure 7 Shows the activity detection results of the anti-4-1BB antibodies JS011-phage 3 and JS011-phage 6 prepared in Example 2 of the present application, as well as related controls, in the luciferase reporter gene system (Raji / Jurkat NFKB 4-1BB dual cells).

[0056] Figure 8 Shows the activity detection results of the anti-4-1BB antibodies JS011-phage 3 and JS011-phage 6 prepared in Example 2 of the present application, as well as related controls, in the luciferase reporter gene system (Raji / Jurkat NFKB dual cells).

[0057] Figures 9A to 9F Respectively shows the changes in the release amounts of cytokines IL-2, IL-4, IL-6, IL-10, TNFα, and IFNγ induced by the anti-4-1BB antibodies JS011-phage 3 and JS011-phage 6 prepared in Example 2 of the present application, as well as related controls.

[0058] Figure 10Shows the tumor growth inhibitory effects of the anti-4-1BB antibodies JS011-phage 3 and JS011-phage 6 prepared in Example 2 of the present application and related controls.

[0059] Figure 11 Shows the effects of the anti-4-1BB antibodies JS011-phage 3 and JS011-phage 6 prepared in Example 2 of the present application and related controls on the spleen weight of tumor-bearing mice on the 14th day after administration.

[0060] Figure 12A Shows the effects of the anti-4-1BB antibodies JS011-phage 3 and JS011-phage 6 prepared in Example 2 of the present application and related controls on the total lymphocyte count in the liver per unit weight of tumor-bearing mice on the 14th day after administration.

[0061] Figures 12B to 12E Respectively show the effects of the anti-4-1BB antibodies JS011-phage 3 and JS011-phage 6 prepared in Example 2 of the present application and related controls on CD3 + CD4 + T cell ratio, CD3 + CD8 + T cell ratio, CD3 + CD8 + CD11c + T cell ratio, and CD3 - NK1.1 + NK cell ratio on the 14th day after administration in tumor-bearing mice. Detailed implementation mode

[0062] Definition

[0063] Unless otherwise specified, the implementation of the present application will adopt conventional techniques in molecular biology (including recombinant technology), microbiology, cell biology, biochemistry, and immunology, which are all within the scope of those skilled in the art.

[0064] To make the present application easier to understand, certain scientific and technical terms are specifically defined as follows. Unless otherwise clearly defined in other parts of this article, the scientific and technical terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present application belongs. Regarding the definitions and terms in this field, professionals can specifically refer to Current Protocols in Molecular Biology (Ausubel). The abbreviations of amino acid residues are the standard 3-letter and / or 1-letter codes used in this field to refer to one of the 20 common L-amino acids. The singular forms used in this article (including the claims) include their corresponding plural forms, unless otherwise clearly specified in the text.

[0065] As used herein, the term "about", when used in connection with a numerical value, means a numerical value that encompasses a range that is 5% less than the specified numerical value at the lower limit and 5% greater than the specified numerical value at the upper limit, including but not limited to ±5%, ±2%, ±1%, and ±0.1%.

[0066] The term "and / or" shall be understood to mean any one of the alternatives or a combination of any two or more of the alternatives.

[0067] The terms "4-1BB", "4-1BB receptor", "4-1BB protein", "CD137", or "tumor necrosis factor receptor superfamily member 9 (TNFRSF9)" are members of the tumor necrosis factor (TNF) receptor superfamily and are activation-induced T cell costimulatory molecules. In the present application, the term "4-1BB" may include human 4-1BB, and its variants, subtypes, homologs, orthologs, and paralogs. Thus, the antibodies or antigen-binding portions thereof provided in the present application may also bind to 4-1BB from species other than human, such as cynomolgus monkey 4-1BB or mouse 4-1BB. For example, in some cases, an antibody specific for the human 4-1BB protein may cross-react with 4-1BB from species other than human. In other embodiments, an antibody specific for the human 4-1BB protein may be fully specific for the human 4-1BB protein and may exhibit species or other types of cross-reactivity, or may cross-react with 4-1BB from some other species but not all other species, e.g., cross-react with cynomolgus monkey 4-1BB but not with mouse 4-1BB. The term "human 4-1BB" may refer to 4-1BB having the complete amino acid sequence of human 4-1BB with NCBI accession number NP_001552.2; the sequence of "human 4-1BB" may also be different from the amino acid sequence of human 4-1BB with NCBI accession number NP_001552.2, e.g., may have conservative mutations in non-conserved regions and have substantially the same biological function as human 4-1BB with NCBI accession number NP_001552.2. The term "mouse 4-1BB" may refer to 4-1BB having the complete amino acid sequence of mouse 4-1BB with NCBI accession number NP_033430.1 or NP_035742.1. The term "cynomolgus monkey 4-1BB" may refer to 4-1BB having the complete amino acid sequence of cynomolgus monkey 4-1BB with Genbank accession number NP_001253057.1.

[0068] The term "percent amino acid sequence identity" or simply "identity" is defined as the percentage of amino acid residues in a candidate amino acid sequence that are identical to the same amino acid residues in a reference amino acid sequence after aligning the amino acid sequences (and introducing gaps if necessary) to obtain the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Various methods in the art can be used for sequence alignment to determine percent amino acid sequence identity, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for the alignment, including any algorithms required to obtain the maximum alignment over the full length of the sequences being compared.

[0069] The term "immune response" refers to the selective impairment, destruction or elimination from the human body of invading pathogens, pathogen-infected cells or tissues, cancer cells, or in the case of autoimmunity or pathological inflammation, the impairment, destruction or elimination of normal human cells or tissues, through the action of, for example, lymphocytes, antigen-presenting cells, phagocytes, granulocytes and soluble macromolecules produced by the above cells or the liver (including antibodies, cytokines and complement).

[0070] The term "signal transduction pathway" or "signal transduction activity" refers to a biochemical causal relationship typically initiated by protein-protein interactions such as the binding of a growth factor to a receptor, which results in the transfer of a signal from one part of a cell to another part of the cell. Generally, the transfer involves the specific phosphorylation of one or more tyrosine, serine or threonine residues on one or more of the proteins in the series of reactions that cause signal transduction. The penultimate process typically involves nuclear events, which result in changes in gene expression.

[0071] The terms "activity" or "biological activity" or the terms "biological property" or "biological characteristic" are used interchangeably in this application and include, but are not limited to, epitope or antigen affinity, specificity, the ability to neutralize or antagonize 4-1BB activity in vivo or in vitro, the half-inhibitory concentration (IC 50) The in vivo stability of the antibody and the immunogenicity of the antibody, etc. Other identifiable biological properties or characteristics of antibodies known in the art include, for example, cross-reactivity (such as cross-reactivity with non-human homologues of the targeted peptide, or cross-reactivity with other proteins or tissues), and the ability to maintain high levels of protein expression in mammalian cells. The aforementioned properties or characteristics are observed, measured or evaluated using techniques known in the art, including but not limited to enzyme-linked immunosorbent assay (ELISA), fluorescence-activated cell sorting (FACS) or surface plasmon resonance analysis such as BIACORE, any in vitro or in vivo neutralization assay, receptor binding assay, cytokine or growth factor production and / or secretion assay, signal transduction assay, and immunohistochemical analysis of tissue sections from different sources (including human, primate or any other source), etc.

[0072] The term "antibody" refers to any form of antibody having the desired biological activity. Thus, it is used in the broadest sense and specifically includes, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (such as bispecific antibodies), humanized antibodies, fully human antibodies, chimeric antibodies, and camelized single-domain antibodies, etc. It is known that the basic antibody structural unit consists of a tetramer, each tetramer comprising two pairs of identical polypeptide chains, and each polypeptide chain pair has one "light" chain (L, about 25 kDa) and one "heavy" chain (H, about 50 - 70 kDa). The amino-terminal portion or fragment of each chain may include a variable region of about 100 - 110 or more amino acids mainly responsible for antigen recognition. The carboxyl-terminal portion or fragment of each chain may define a constant region mainly responsible for effector functions. Human light chains are generally classified as κ light chains and λ light chains. In addition, human heavy chains are generally classified into five classes, namely μ, δ, γ, α or ε, and the isotypes of antibodies are defined as IgM, IgD, IgG, IgA and IgE respectively according to the different heavy chains. See generally Chapter 7 of Fundamental Immunology (edited by Paul, W., 2nd edition. Raven Press, N.Y. (1989)).

[0073] As used herein, the term "isolated antibody" refers to an antibody that is substantially free of other cellular components associated with its natural state, such as nucleic acids, proteins, lipids, sugars, or other substances such as cell debris and growth medium. It is understood that the isolated antibody is in a substantially purified state, preferably the isolated antibody is in a homogeneous state, and the isolated antibody can be a dry or aqueous solution. Analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography are generally used to determine the purity and homogeneity of the antibody. The term "isolated" does not mean the complete absence of the above substances or the absence of water, buffer, or salt, unless they are present in amounts that significantly interfere with the experimental or therapeutic use of the antibody of the present application.

[0074] The term "monoclonal antibody" refers to an antibody made from highly identical immune cells, which are all clones of a single parental cell. Monoclonal antibodies have monovalent affinity because they bind to the same epitope (the site on an antigen recognized by an antibody). The monoclonal antibodies may also include a small number of naturally occurring mutations. In contrast, the term "polyclonal antibody" binds to multiple epitopes and is generally composed of several different lineages of plasma cells (antibody-secreting immune cells), and can be understood as a mixture of multiple monoclonal antibodies. The modifier "monoclonal" should not be construed as requiring the antibody to be produced by any particular method.

[0075] The term "multispecific antibody" refers to an antibody that contains two or more antigen-binding domains and is capable of binding to two or more different epitopes (e.g., two, three, four, or more different epitopes). The epitopes to which the multispecific antibody can specifically bind can be on the same or different antigens. Examples of multispecific antibodies include "bispecific antibodies" that bind to two different epitopes.

[0076] The term "binding domain" or "antigen-binding domain" or "antigen-binding site" refers to the region in an antibody that can specifically bind to a part or all of an antigen and is complementary to a part or all of the antigen. When the antigen is large, the antibody may only bind to a specific part of the antigen, which is called an epitope. The binding domain may include the variable domains of the heavy and light chains, i.e., the heavy chain variable region VH and the light chain variable region VL, each of which includes four conserved framework regions (FR) and three complementarity-determining regions (CDR). The CDRs can vary in sequence and determine the specificity for a particular antigen.

[0077] The term "full-length" antibody refers to an immunoglobulin molecule that, when naturally occurring, contains four peptide chains: two heavy chains (approximately 50 - 70 kDa in full length) and two light chains (approximately 25 kDa in full length) that are interconnected by disulfide bonds. Each heavy chain consists of a heavy-chain variable region (abbreviated herein as VH) and a heavy-chain constant region (abbreviated herein as CH). The heavy-chain constant region consists of three domains, CH1, CH2, and CH3. Each light chain consists of a light-chain variable region (abbreviated herein as VL) and a light-chain constant region (abbreviated herein as CL). The light-chain constant region consists of one domain, CL. The VH and VL regions can be further subdivided into complementarity-determining regions (CDRs) with high variability and framework regions (FRs) with higher conservation that are spaced between the complementarity-determining regions. The domain arrangement order of each VH or VL from the amino terminus to the carboxyl terminus is FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains each contain a binding domain that interacts with an antigen. The constant region of the antibody can mediate the binding of the antibody to various cells of the host's tissue or immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0078] The term "heavy-chain constant region" or "CH" is used interchangeably in this application and contains at least three heavy-chain constant domains (CH1, CH2, and CH3). Exemplarily, human heavy-chain constant regions include γ, δ, α, ε, and μ, and each heavy-chain constant region corresponds to an antibody isotype. For example, an antibody containing a γ constant region is an IgG antibody, an antibody containing a δ constant region is an IgD antibody, an antibody containing an α constant region is an IgA antibody, an antibody containing a μ constant region is an IgM antibody, and an antibody containing an ε constant region is an IgE antibody. Certain isotypes can be further subdivided into subclasses. For example, IgG antibodies include, but are not limited to, IgG1 (containing the γ1 constant region), IgG2 (containing the γ2 constant region), IgG3 (containing the γ3 constant region), and IgG4 (containing the γ4 constant region); IgA antibodies include, but are not limited to, IgA1 (containing the α1 constant region) and IgA2 (containing the α2 constant region); IgM antibodies include, but are not limited to, IgM1 and IgM2. Isotypes can also include some modified forms, and the modifications can alter Fc function, such as enhancing or weakening effector function or enhancing or weakening its binding to Fc receptors.

[0079] The term "light-chain constant region" or "CL" is used interchangeably in this application and contains one light-chain constant domain, CL. Exemplarily, light chains can be divided into two classes, λ and κ, according to the difference in the light-chain constant region.

[0080] The term "antigen-binding fragment" of an antibody includes fragments or derivatives of an antibody, and the antibody corresponding to the "antigen-binding fragment" may be referred to as the parental antibody. An antigen-binding fragment of an antibody generally contains at least one fragment of the antigen-binding region or variable region of the parental antibody, which retains at least some of the binding specificities of the parental antibody. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and single-chain Fv fragments, diabodies, linear antibodies, domain antibodies, single-chain antibody molecules such as scFv; nanobodies formed from antibody fragments, and multispecific antibodies, etc. At the same molar concentration, the antigen-binding fragment can retain at least 10%, at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the antigen-binding activity of the parental antibody. In addition, the antigen-binding fragment of an antibody may also include conservative or non-conservative amino acid substitutions that do not significantly alter its biological activity (referred to as "conservative variants" or "functionally conservative variants" of the antibody).

[0081] The term "single-chain Fv" or "scFv" antibody refers to an antibody fragment that contains the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. The Fv polypeptide generally also contains a polypeptide linker between the VH and VL domains, which enables the scFv to form the required structure for antigen binding.

[0082] The term "domain antibody" refers to an immunofunctional immunoglobulin fragment that contains only the variable region of the heavy chain or the variable region of the light chain. In some cases, two or more VH regions are covalently linked with a peptide linker to form a bivalent domain antibody. The two VH regions of the bivalent domain antibody can target the same or different antigens.

[0083] The term "antigen" refers to a molecule or a part of a molecule that can be bound by the antibodies of the present application. An antigen can have one or more epitopes.

[0084] The term "diabody" refers to a small antibody fragment having two antigen-binding sites, the fragment containing a heavy chain variable domain (VH) linked to a light chain variable domain (VL) in the same polypeptide chain, and the two fragments are forced to pair the domain with a complementary domain in another fragment and generate two antigen-binding sites by a linker that is too short to allow pairing between the two domains in the same chain.

[0085] The terms "specifically bind" and "selectively bind" mean that an antibody binds to an epitope on a predetermined antigen. Generally, when using recombinant human 4-1BB or its epitope as an analyte and an antibody as a ligand, and measuring by surface plasmon resonance (SPR) technology in an instrument, the antibody binds with a dissociation constant of approximately less than 10 -7It binds to a predetermined antigen or its epitope with an equilibrium dissociation constant (KD) of M or less, and the affinity of the antibody for the predetermined antigen or its epitope is at least twice that of its binding to other non-specific antigens (such as BSA, etc.). The term "recognize an antigen" can be used interchangeably with the term "specifically bind" in this application.

[0086] The term "epitope" refers to the antigen region to which an antibody binds. An epitope can be formed by contiguous amino acids or by non-contiguous amino acids juxtaposed through the tertiary folding of a protein.

[0087] "Affinity" or "binding affinity" refers to the intrinsic binding affinity reflecting the interaction between members of a binding pair (such as an antigen and an antibody). Affinity can generally be represented by the equilibrium dissociation constant (KD), which is the ratio of the dissociation rate constant and the binding rate constant (kdis and kon, respectively). Affinity can be measured by common methods known in the art, for example, it can be measured using a ForteBio biomolecular interaction workstation.

[0088] The term "not bind to" a protein or a cell means not binding to the protein or the cell, or not binding to it with high affinity, that is, the KD for binding to the protein or the cell is 1.0×10 -6 M or higher, more preferably 1.0×10 -5 M or higher, more preferably 1.0×10 -4 M or higher, 1.0×10 -3 M or higher, more preferably 1.0×10 -2 M or higher.

[0089] The term "high affinity" for an IgG antibody means a KD for the antigen of 1.0×10 -6 M or lower, preferably 5.0×10 -8 M or lower, more preferably 1.0×10 -8 M or lower, 5.0×10 -9 M or lower, more preferably 1.0×10 -9 M or lower. For other antibody subtypes, "high affinity" binding may vary. For example, "high affinity" binding for the IgM subtype means a KD of 10 -6 M or lower, preferably 10 -7 M or lower, more preferably 10 -8 M or lower.

[0090] The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single-stranded or double-stranded form. Unless explicitly restricted, the term "nucleic acid" or "polynucleotide" also includes nucleic acids containing analogs of known natural nucleotides that have binding properties similar to the reference nucleic acid and are metabolized in a manner similar to the naturally occurring nucleotides (see, U.S. Patent No. 8,278,036 to Kariko et al., which discloses mRNA molecules in which uridine is replaced by pseudouridine, methods for synthesizing such mRNA molecules, and methods for in vivo delivery of therapeutic proteins). Unless otherwise indicated, a particular nucleic acid sequence also implicitly includes its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, single nucleotide polymorphisms (SNPs), and complementary sequences as well as the explicitly recited sequence.

[0091] "Construct" refers to any recombinant polynucleotide molecule (e.g., plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, phage, linear or circular single-stranded or double-stranded DNA or RNA polynucleotide molecule) that can be derived from any source, is capable of integrating with the genome or replicating autonomously, and can be operably linked to one or more polynucleotide molecules. In the present application, a construct is generally a polynucleotide molecule of the present application that is operably linked to transcriptional initiation regulatory sequences that will direct transcription of the polynucleotide molecule of the present application in a host cell. Expression of the nucleic acid of the present application can be directed using a heterologous promoter or an endogenous promoter.

[0092] "Vector" refers to any recombinant polynucleotide construct that can be used for transformation purposes (i.e., introducing heterologous DNA into a host cell). One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop to which additional DNA segments can be ligated. Another type of vector is a viral vector, which can ligate additional DNA segments to the viral genome. Some vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of the host cell after being introduced into the host cell and replicate along with the host genome.

[0093] As used herein, the term "expression vector" refers to a nucleic acid molecule that is capable of replicating and expressing a gene of interest when transformed, transfected, or transduced into a host cell. Expression vectors typically contain one or more phenotypic selection markers and an origin of replication for maintenance of the vector and amplification in the host if desired.

[0094] In the present application, unless otherwise explicitly specified, "activation", "stimulation", and "treatment" for cells or receptors may have the same meaning. For example, cells or receptors are activated, stimulated, or treated with ligands. "Ligands" include natural and synthetic ligands such as cytokines, cytokine variants or analogs, mutant proteins, and binding compounds derived from antibodies (such as antibodies and their binding fragments). "Ligands" also include small molecules such as peptide mimics of cytokines and peptide mimics of antibodies. "Activation" may refer to cell activation regulated by internal mechanisms as well as external or environmental factors. "Response" or "reaction", such as the response of cells, tissues, organs, or organisms, includes changes in biochemical or physiological behavior, such as changes in the concentration, density, adhesion, or migration of partial components within biological compartments (such as tissues, cells, organelles, etc.), the rate of gene expression, or the differentiation state, and these changes may be related to activation, stimulation, or treatment.

[0095] As used herein, the term "treatment" or "therapy" of any disease or disorder may, in one embodiment, refer to improving the disease or disorder, such as slowing, halting, or reducing the progression of the disease, or the clinical symptoms of the disease, etc.; in another embodiment, it may refer to alleviating or improving at least one physical parameter, which may not show an obvious improvement in the symptoms of the disease; in another embodiment, it may refer to modulating the disease or disorder physically (e.g., stabilization of discernible symptoms), physiologically (e.g., stabilization of physical parameters), or both. Unless explicitly described herein, methods for assessing the treatment and / or prevention of diseases are generally known in the art.

[0096] In the present application, the "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc.

[0097] The administration of "in combination" with one or more other therapeutic agents includes simultaneous administration or co-administration, or sequential administration in any order.

[0098] "Therapeutically effective amount", "therapeutically effective dose", and "effective amount" mean that at such dose, when the anti-4-1BB antibody or its antigen-binding fragment of the present application is administered alone or in combination with other therapeutic agents to cells, tissues or subjects, it can effectively prevent or improve the symptoms of at least one disease or condition, or prevent or improve the development of at least one disease or condition. The therapeutically effective dose can also refer to the amount of the antibody or its antigen-binding fragment sufficient to cause symptom improvement (such as treating, curing, preventing or improving the relevant medical condition or increasing the speed of treatment, cure, prevention or improvement of such condition). When administering the active ingredient (such as an antibody or its antigen-binding fragment) alone to an individual, the therapeutically effective dose only refers to that ingredient; when administered in combination, whether sequentially or simultaneously, the therapeutically effective dose refers to the combined amount of all active ingredients that cause a therapeutic effect. The effective amount of the therapeutic agent will result in an increase in the diagnostic criteria or parameters by at least 10%; usually at least 20%; preferably at least about 30%; more preferably at least 40%, and most preferably at least 50%.

[0099] "Cancer" and "cancerous" refer to physiological disorders in mammals that are typically characterized by unregulated cell growth, and this definition includes benign tumors, malignant cancers, as well as dormant tumors or micrometastases. Examples of cancers include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specific examples of such cancers include squamous cell carcinoma, lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma), peritoneal cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, cholangiocarcinoma, breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, and various types of head and neck cancers, as well as B-cell lymphoma (such as low-grade / follicular non-Hodgkin lymphoma (NHL), small lymphocytic (SL) NHL, intermediate / follicular NHL, intermediate diffuse NHL, immunoblastic NHL, lymphoblastic NHL, small non-cleaved cell NHL, storage disease NHL, mantle cell lymphoma, AIDS-related lymphoma, and Waldenstrom macroglobulinemia), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myelogenous leukemia, post-transplant lymphoproliferative disorder (PTLD), and abnormal vascular proliferation associated with phakomatoses, edema associated with brain tumors, and Meigs syndrome.

[0100] antibody

[0101] The present application provides an antibody or an antigen-binding fragment thereof that can specifically bind to 4-1BB, which comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3;

[0102] Wherein, according to the Kabat, IMGT, Chothia, AbM, Contact or North numbering system, the amino acid sequences of the HCDR1, HCDR2, and HCDR3 are the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region as shown in SEQ ID NO: 13 or 15, and the amino acid sequences of the LCDR1, LCDR2, and LCDR3 are the LCDR1, LCDR2, and LCDR3 of the light chain variable region as shown in SEQ ID NO: 14 or 16, and at least one of the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 can be replaced with a variant having 1, 2, or 3 amino acid differences therewith.

[0103] In some embodiments, the antibody or an antigen-binding fragment thereof that can specifically bind to 4-1BB comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3;

[0104] Wherein, according to the Kabat, IMGT, Chothia, AbM, Contact or North numbering system, the amino acid sequences of the HCDR1, HCDR2, and HCDR3 are the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region as shown in SEQ ID NO: 13, and the amino acid sequences of the LCDR1, LCDR2, and LCDR3 are the LCDR1, LCDR2, and LCDR3 of the light chain variable region as shown in SEQ ID NO: 14; or

[0105] the amino acid sequences of the HCDR1, HCDR2, and HCDR3 are the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region as shown in SEQ ID NO: 15, and the amino acid sequences of the LCDR1, LCDR2, and LCDR3 are the LCDR1, LCDR2, and LCDR3 of the light chain variable region as shown in SEQ ID NO: 16.

[0106] In some embodiments, according to the Kabat numbering system, the antibody or an antigen-binding fragment thereof that can specifically bind to 4-1BB comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3;

[0107] Among them, the HCDR1, HCDR2, and HCDR3 respectively have the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, or respectively have the amino acid sequences shown in SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9;

[0108] The LCDR1, LCDR2, and LCDR3 respectively have the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, or respectively have the amino acid sequences shown in SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12;

[0109] Among them, at least one of the SEQ ID NO:1-12 can be replaced with a variant having 1, 2, or 3 amino acid differences therefrom.

[0110] In some embodiments, according to the Kabat numbering system, the HCDR1, HCDR2, and HCDR3 respectively have the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, and the LCDR1, LCDR2, and LCDR3 respectively have the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6; or

[0111] The HCDR1, HCDR2, and HCDR3 respectively have the amino acid sequences shown in SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, and the LCDR1, LCDR2, and LCDR3 respectively have the amino acid sequences shown in SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12.

[0112] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0113] A heavy chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:13, and a light chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:14; or

[0114] A heavy chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:15, and a light chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:16;

[0115] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0116] A heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:13, and a light chain variable region with an amino acid sequence as shown in SEQ ID NO:14; or

[0117] A heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:15, and a light chain variable region with an amino acid sequence as shown in SEQ ID NO:16.

[0118] In some embodiments, the antibody or antigen-binding fragment thereof further comprises a human heavy chain constant region and a human light chain constant region, the human heavy chain constant region is selected from the heavy chain constant regions of human IgG1, IgG2, IgG3 or IgG4, preferably the heavy chain constant region of human IgG4 or the heavy chain constant region of human IgG4 having the S228P amino acid substitution; the human light chain constant region is selected from the light chain constant regions of lambda light chain or kappa light chain.

[0119] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0120] A heavy chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:17, and a light chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:18; or

[0121] A heavy chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:19, and a light chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:20;

[0122] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0123] a heavy chain having an amino acid sequence as shown in SEQ ID NO: 17 and a light chain having an amino acid sequence as shown in SEQ ID NO: 18; or

[0124] a heavy chain having an amino acid sequence as shown in SEQ ID NO: 19 and a light chain having an amino acid sequence as shown in SEQ ID NO: 20.

[0125] In some embodiments, the antibody includes at least one of a monoclonal antibody and a multispecific antibody, and the antigen-binding fragment includes at least one of Fab, Fab', F(ab')2, Fv, scFv, and sdAb.

[0126] In some embodiments, the present application provides an isolated antibody or its antigen-binding fragment that can specifically bind to 4-1BB and has at least one of the following characteristics:

[0127] (Ⅰ) binding to the same epitope of human 4-1BB protein as the antibody or its antigen-binding fragment described in the first aspect of the present application, or a completely overlapping or partially overlapping epitope;

[0128] (Ⅱ) competing with the antibody or its antigen-binding fragment described in the first aspect of the present application for binding to the epitope of human 4-1BB protein.

[0129] Among them, an epitope that completely overlaps with the antibody or its antigen-binding fragment of the present application means that the antigen epitope bound by the antibody includes the binding epitope of the antibody or its antigen-binding fragment of the present application; an epitope that partially overlaps with the antibody or its antigen-binding fragment of the present application means that a part of the antigen epitope bound by the antibody is the same as a part of the binding epitope of the antibody or its antigen-binding fragment of the present application.

[0130] For the precise amino acid sequence boundaries of the complementarity-determining regions (CDRs) of an antibody, they can be defined according to well-known methods, such as Chothia based on the three-dimensional structure of the antibody and the topology of the CDR loops (Chothia et al., Nature 342:877-883, 1989; Al-Lazikani et al., Journal of Molecular Biology, 273:927-948, 1997); or Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Edition, U.S. Department of Health and Human Services, National Institutes of Health, 1987), AbM (University of Bath), Contact (University College London), and IMGT (the international ImMunoGeneTics database, 1999 Nucleic Acids Research, 27, 209-212) based on antibody sequence variability; or the North CDR definition based on affinity propagation clustering using a large number of crystal structures. The CDRs of the antibody in the present application can have their boundaries determined by those skilled in the art according to any protocol in the art (such as the optionally defined methods above).

[0131] It should be noted that the boundaries of the CDRs of the same antibody obtained based on different definition methods may vary, that is, the CDR sequences of the variable regions of the same antibody obtained under different definition methods are different. Therefore, when an antibody is defined by the specific CDR sequences defined in the present application, the antibody also includes those whose complementarity-determining region sequences contain the CDR sequences described in the present application, but only the antibodies whose claimed CDR boundaries are different from the specific CDR boundaries defined in the present application due to the use of different CDR boundary definition methods.

[0132] Antibodies with different specificities (i.e., targeting different antigen-binding sites) have different CDRs. However, although CDRs are different between antibodies, only a limited number of amino acid positions within the CDRs are directly involved in antigen binding. The minimal overlapping region where the antibody CDR binds to the antigen is also referred to as the "minimal binding unit" for antigen binding and can be determined using at least two of the Kabat, IMGT, Chothia, AbM, Contact, and North methods. The minimal binding unit can be part of the CDR. As will be appreciated by those skilled in the art, the residues of the remainder of the CDR sequence can be determined by the structure and protein folding of the antibody. Accordingly, the present application also contemplates variants of any CDR. For example, in a variant of a CDR, the amino acid residues of the minimal binding unit can remain unchanged while the remaining CDR residues defined according to Kabat or Chothia can be replaced with conservative amino acid residues.

[0133] The humanized antibodies described in the present application can be prepared by inserting murine CDR regions into a human germline framework region using methods known in the art. See U.S. Patent Nos. 5,225,539 to Winter et al. and 5,530,101; 5,585,089; 5,693,762; and 6,180,370 to Queen et al.

[0134] In some embodiments, the amino acid differences can be caused by amino acid changes, including amino acid deletions, insertions, or substitutions. In some embodiments, the anti-4-1BB antibody or antigen-binding fragment thereof of the present application includes an antibody having an amino acid sequence that has been mutated by amino acid deletion, insertion, or substitution but still has at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the antibody or antigen-binding fragment thereof (particularly in the CDR regions described in the above sequences).

[0135] In some embodiments, one or more amino acid modifications can be introduced into the Fc region of the antibodies provided herein to generate Fc region variants. The Fc region variants can include a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) having an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0136] In some embodiments, the antibody or antigen-binding fragment thereof includes an antibody or antigen-binding fragment thereof engineered with cysteine, such as a "thioMAb", wherein one or more residues of the antibody or antigen-binding fragment thereof are replaced with cysteine residues.

[0137] In some embodiments, the antibody or its antigen-binding fragment may be further modified to contain other non-protein moieties known in the art and readily available, such as water-soluble polymers. Examples of the water-soluble polymers include, but are not limited to: polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymer, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymer, polyamino acids (homopolymers or random copolymers), and dextran or poly(N-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (such as glycerol), polyvinyl alcohol, and mixtures thereof.

[0138] antibody expression

[0139] Another aspect of the present application provides a polynucleotide molecule comprising at least one of a nucleotide sequence encoding the antibody or its antigen-binding fragment provided in the present application or its complementary sequence. The polynucleotide molecules in the present application include double-stranded or single-stranded DNA or RNA. The antibody or its antigen-binding fragment provided in the present application includes the antibody or its antigen-binding fragment of the first aspect of the present application and the isolated antibody or its antigen-binding fragment of the second aspect of the present application.

[0140] In some embodiments, the polynucleotide molecule encoding the antibody or its antigen-binding fragment of the present application may also include a polynucleotide sequence having nucleotide deletions, insertions or substitution mutations, but still having at least about 60%, 70%, 80%, 90%, 95% or 100% identity with the coding region corresponding to the CDR of the antibody or its antigen-binding fragment of the present application.

[0141] In yet another aspect, the present application provides an expression vector comprising the polynucleotide molecule of the present application. Preferably, the expression vector is a eukaryotic expression vector.

[0142] In yet another aspect, the present application provides a host cell comprising the polynucleotide molecule or expression vector of the present application; preferably, the host cell is a eukaryotic cell, more preferably a mammalian cell.

[0143] In some embodiments, the host cell is used to express the antibody or its antigen-binding fragment provided in the present application.

[0144] The mammalian host cells provided by the present application for expressing the antibodies or antigen-binding fragments thereof of the present application include various immortalized cell lines available from the American Type Culture Collection (ATCC). Exemplarily, it may include Chinese hamster ovary (CHO) cells, NS0, SP2 / 0 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells, A549 cells, 293T cells, and many other cell lines. The mammalian host cells may include human, mouse, rat, dog, monkey, pig, goat, cow, horse, and hamster cells. Those skilled in the art can select a particularly preferred cell line by determining which cell line has a high expression level.

[0145] In yet another aspect, the present application provides a method for preparing the antibodies or antigen-binding fragments thereof of the present application, including expressing the antibodies or antigen-binding fragments thereof in the host cells provided by the present application, and recovering the expressed antibodies or antigen-binding fragments thereof from the host cells.

[0146] Specifically, the method for preparing the antibodies or antigen-binding fragments thereof of the present application provided by the present application may include:

[0147] Introducing the polynucleotide molecule or expression vector of the present application into the host cells described in the present application by well-known methods, such as liposome transfection, electroporation, transformation, etc.; culturing the host cells under conditions suitable for the expression of the antibodies or antigen-binding fragments thereof, so that the host cells express the antibodies or antigen-binding fragments thereof; recovering the expressed antibodies or antigen-binding fragments thereof from the host cells by well-known methods in the art, such as polyacrylamide gel electrophoresis.

[0148] It should be noted that due to the differences in the expression vectors or host cells used, those skilled in the art can select conditions suitable for the expression of the antibodies or antigen-binding fragments thereof according to the specific circumstances, and the present application does not make any limitations here.

[0149] The host cells for expressing the antibodies or antigen-binding fragments thereof in the present application may be isolated cells or cells still present in the host body. For example, different cell lines may be used to express the antibodies or antigen-binding fragments thereof, or transgenic animals may be used to express the antibodies or antigen-binding fragments thereof.

[0150] Antibodies or antigen-binding fragments thereof expressed by different cell lines or in different transgenic animals may have different glycosylation modifications. It should be noted that regardless of the glycosylation of these antibodies, all antibodies encoded by the polynucleotide molecules provided by this application or comprising the amino acid sequences provided by this application are part of this application. Similarly, in certain embodiments, the antibody or its antigen-binding fragment is preferably a non-fucosylated antibody or its antigen-binding fragment. The inventors have found that non-fucosylated antibodies or their antigen-binding fragments have more potent efficacy in vitro and in vivo than fucosylated antibodies or their antigen-binding fragments. In addition, since these different sugar structures are normal components of natural human serum IgG, their immunogenicity has no significant difference.

[0151] pharmaceutical composition or pharmaceutical preparation

[0152] In yet another aspect, this application provides a pharmaceutical composition (or referred to as a pharmaceutical preparation, preparation), which comprises the antibody or its antigen-binding fragment, polynucleotide molecule, expression vector or host cell provided by this application, and a pharmaceutically acceptable carrier or excipient.

[0153] It should be understood that the antibody or its pharmaceutical composition provided by this application can incorporate suitable carriers, excipients and other reagents in the preparation for co-administration, so as to provide improved transfer, delivery, tolerance performance, etc.

[0154] In this application, the active ingredient in the pharmaceutical composition exists in a biologically active and effective form, and does not contain ingredients that are unacceptably toxic to the subject of the pharmaceutical composition.

[0155] In some embodiments of this application, the pharmaceutical composition of this application can be prepared by mixing the antibody or its antigen-binding fragment of this application with the desired purity with one or more optional pharmaceutical excipients (see Remington's Pharmaceutical Sciences, 16th edition, edited by Osol, A., 1980). Preferably, the pharmaceutical composition of this application can be in the form of an aqueous solution or a freeze-dried preparation.

[0156] The pharmaceutical composition or preparation of this application may further comprise one or more other active ingredients, and the active ingredients can be selected according to specific indications. Preferably, the active ingredients do not adversely affect each other's activities. In some embodiments, the other active ingredients can be chemotherapeutic agents, immune checkpoint inhibitors, growth inhibitors, antibiotics or various known anti-tumor or anti-cancer agents, and the active ingredients can be present in a combination in an amount effective for the intended use.

[0157] In yet another aspect, the present application provides a kit, which comprises the antibody of the present application or its antigen-binding fragment, polynucleotide molecule, expression vector, host cell or pharmaceutical composition.

[0158] medical use

[0159] In yet another aspect, the present application provides the use of the antibody of the present application or its antigen-binding fragment, polynucleotide molecule, expression vector or host cell or pharmaceutical composition in the preparation of a medicament for treating or preventing 4-1BB-mediated diseases or disorders.

[0160] In yet another aspect, the present application provides the use of the antibody of the present application or its antigen-binding fragment, polynucleotide molecule, expression vector or host cell or pharmaceutical composition for treating or preventing 4-1BB-mediated diseases or disorders.

[0161] In yet another aspect, the present application provides a method for treating or preventing 4-1BB-mediated diseases or disorders, which comprises administering to a subject in need the antibody of the present application or its antigen-binding fragment, polynucleotide molecule, expression vector, host cell or pharmaceutical composition.

[0162] In some embodiments, the 4-1BB-mediated disease is cancer; preferably, the cancer is selected from at least one of melanoma, breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, kidney cancer, lung cancer, liver cancer, gastric cancer, colorectal cancer, bladder cancer, head and neck cancer, thyroid cancer, esophageal cancer, cervical cancer, sarcoma, multiple myeloma, leukemia, lymphoma, gallbladder cancer and glioblastoma.

[0163] In some embodiments, the administration modes include but are not limited to oral administration, intravenous administration, subcutaneous administration, intramuscular administration, intra-arterial administration, intra-articular administration (such as administration in arthritic joints), inhalation administration, aerosol delivery or intratumoral administration, etc.

[0164] In some embodiments, the present application further provides a combination therapy, which comprises co-administering to a subject the antibody of the present application or its antigen-binding fragment and one or more other therapies or therapeutic agents. In some embodiments, the therapies may include surgical treatment and / or radiotherapy. In some embodiments, the therapeutic agents may include PD-1 antibody, CD4 antibody and / or CTLA-4 antibody, etc.

[0165] method for diagnosis and detection

[0166] In yet another aspect, the present application provides a method for detecting 4-1BB using the antibody or antigen-binding fragment thereof of the present application, comprising contacting the antibody or antigen-binding fragment thereof with a sample, detecting the conjugate formed by the antibody or antigen-binding fragment thereof and 4-1BB, and optionally quantitatively determining the conjugate. When the term "detect" is used in the present application, it may include quantitative or qualitative detection. In some embodiments, the sample is a biological sample. In certain embodiments, the biological sample is blood, serum or other liquid samples of biological origin. In certain embodiments, the biological sample contains cells or tissues. The present application does not limit the method for detecting the conjugate formed by the antibody or antigen-binding fragment thereof and 4-1BB; the present application does not limit the method for "quantitatively determining the conjugate".

[0167] In yet another aspect, the present application provides a method for detecting 4-1BB using the antibody or antigen-binding fragment thereof of the present application, comprising contacting the antibody or antigen-binding fragment thereof with a sample, detecting the conjugate formed by the antibody or antigen-binding fragment thereof and 4-1BB, and optionally quantitatively determining the conjugate. When the term "detect" is used in the present application, it may include quantitative or qualitative detection. In some embodiments, the sample is a biological sample. In certain embodiments, the biological sample is blood, serum or other liquid samples of biological origin. In certain embodiments, the biological sample contains cells or tissues. The present application does not limit the method for detecting the conjugate formed by the antibody or antigen-binding fragment thereof and 4-1BB; the present application does not limit the method for "quantitatively determining the conjugate".

[0168] To make the objectives, technical solutions and advantages of the present application clearer and more understandable, the following examples are given with reference to the accompanying drawings to further elaborate on the present application in detail. Obviously, the described examples are only a part of the examples of the present application, rather than all of them. All other examples obtained by those of ordinary skill in the art based on the examples in the present application fall within the scope of protection of the present application.

[0169] Example 1. Screening of Anti-4-1BB Antibodies

[0170] 1.1 Establishment of a Phage Library

[0171] Mix 50 mL of peripheral blood from 40 individuals of different origins, isolate peripheral blood mononuclear cells (PBMCs), extract total RNA using Trizol, and synthesize the first strand of cDNA using a reverse transcription kit (Thermo, 18080051). Using the synthesized cDNA as a template, amplify the variable region of the heavy chain of the antibody (VH), and the variable regions of the kappa and lambda light chains of the antibody (Vκ and Vλ) by PCR. Assemble VH-CH1, Vκ-CK, and Vλ-Cλ through the first round of overlap PCR, and then assemble Fab through the second round of overlap PCR. Digest and recover the Fab fragment and ligate it into a phagemid vector. Electroporate the constructed plasmid vector into TG1 competent cells to obtain a phage display library.

[0172] 1.2 Screening of the phage library

[0173] Package the constructed phage display library into phages. In the first round of panning, add 10 13 -10 14 phages and incubate with biotinylated 4-1BB for binding. Then add streptavidin-conjugated magnetic beads to bind 4-1BB. First, wash away the unbound phages with PBST, and then elute the phages bound to 4-1BB with glycine at pH 2.0. Use the eluted phages to infect TG1 and amplify and package phages for the second round of panning. Through four rounds of panning, the phages from the fourth round infect TG1 and are plated into monoclonal colonies. Pick the monoclonal colonies into a 96-well deep plate and induce expression with IPTG (isopropyl β-D-thiogalactopyranoside). Extract the periplasmic supernatant the next day for binding ELISA analysis of 4-1BB. The positive clones are sequenced to obtain their sequences. Finally, two groups of phages are obtained, named phage3 and phage6 for further analysis. Among them, the antibody expressed by phage phage3 is named JS011-phage3; the antibody expressed by phage phage6 is named JS011-phage6.

[0174] Example 2: Molecular construction and production of anti-4-1BB antibody

[0175] Clone the coding sequences of the heavy chain constant region of IgG4 (hIgG4 CH1-CH3) and the kappa light chain constant region (human Kappa LC) from human B lymphocytes (from the Beijing Institute of Blood) and introduce them into the pTT5 plasmid to form vectors HXT4S and HXT2, respectively.

[0176] The main components of vector HXT4S successively include: BSPQI cleavage site, CMV Promoter (promoter for the expression of the target gene), AmpR (ampicillin resistance gene), pMB1ori (replication origin), ori p, signal peptide (signal peptide for the expression of the target gene), and the target gene hIgG4 CH1-CH3.

[0177] The main components of vector HXT2 successively include: BSPQI cleavage site, CMV Promoter (promoter for the expression of the target gene), AmpR (ampicillin resistance gene), pMB1ori (replication origin), ori p, signal peptide (signal peptide for the expression of the target gene), and the target gene human Kappa LC.

[0178] 2.1 Construction of the light chain expression vector of JS011-phage3

[0179] Using the plasmid extracted after culturing TG1 strain infected with phage3 as a template, forward and reverse primers were synthesized as shown in SEQ ID No:21 and SEQ ID No:22 in Table 1 below, and PCR was performed. The obtained PCR product was digested with SapI and ligated to vector HXT2 to obtain the expression vector HXT2-JS011-3-LC.

[0180] 2.2 Construction of the heavy chain expression vector of JS011-phage3

[0181] Using the plasmid extracted after culturing TG1 strain infected with phage3 as a template, forward and reverse primers were synthesized as shown in SEQ ID No:23 and SEQ ID No:24 in Table 1 below, and PCR was performed. The obtained PCR product was digested with SapI and ligated to vector HXT4S to obtain the expression vector HXT4S-JS011-3-HC.

[0182] 2.3 Construction of the light chain expression vector of JS011-phage6

[0183] Using the plasmid extracted after culturing TG1 strain infected with phage6 as a template, forward and reverse primers were synthesized as shown in SEQ ID No:25 and SEQ ID No:26 in Table 1 below, and PCR was performed. The obtained PCR product was digested with SapI and ligated to vector HXT2 to obtain the expression vector HXT2-JS011-6-LC.

[0184] 2.4 Construction of the heavy chain expression vector of JS011-phage6

[0185] Using the plasmid extracted after culturing TG1 strain infected with phage6 as a template, forward and reverse primers were synthesized as shown in SEQ ID No:23 and SEQ ID No:24 in Table 1 below, and PCR was performed. The obtained PCR product was digested with SapI enzyme and ligated to the HXT4S vector to obtain the expression vector HXT4S-JS011-6-HC.

[0186] Table 1

[0187] primer name primer sequence JS011-3VL F BSPQI: gatcgctcttcatgtgagctcgagctgactcagccaccctca(SEQ ID No:21) JS011-3VL R BSPQI: gatcgctcttcttcgtaggacggtcagtctggtccctccgccga(SEQ ID No:22) JS011-3 / 6VH F BSPQI gatcgctcttcatctcaggtgcagctggtgcagtctggggct(SEQ ID No:23) JS011-3 / 6VH R BSPQI atgcgctcttctagctgaggagacggtgaccagggttccctgg(SEQ ID No:24) JS011-6VL F BSPQI: atgcgctcttcatgtgagctcgccctgactcagcctccctccg(SEQ ID No:25) JS011-6VL R BSPQI: atgcgctcttcttcgtaggacggtcagcttggtccctccgccg(SEQ ID No:26)

[0188] 2.5 Expression and purification of JS011-phage3 and JS011-phage6 antibodies

[0189] CHO-K1 cells (ATCC, CCL-61) were acclimated and screened in suspension serum-free conditions to make the cells suitable for transient expression, named CHO-18. CHO-18 cells were cultured using CD CHO medium (Gibco) at 36.5 °C, 120 rmp, 7% CO 2 conditions. When the cell density reached (2 - 6)×10 6 / mL, it was passaged and amplified with CD CHO medium. One day before transfection, the CHO-18 cell density was diluted to (1.5 - 2.0)×10 6 / mL and placed in a shaker (culture conditions: 36.5 °C, 120 rmp, 7% CO 2 ) for culture. The next day, when the cell density reached approximately 3.5×10 6 / mL, transfection was performed. During transfection, first add one-tenth of the transfection volume of CD CHO medium to the reactor, and then sequentially add 1 - 2 μg / mL of the transfection volume of the above expression vectors HXT2-JS011-3-LC and HXT4S-JS011-3-HC, or HXT4S-JS011-3-HC and HXT4S-JS011-6-HC, and 3 - 14 μg / mL of PEI (polyethyleneimine). After mixing, incubate at room temperature for 20 - 30 min, and then slowly add the obtained mixture to the pre-treated above-mentioned CHO-18 cells to form a transfection mixture, mixing while adding. The transfection mixture was placed in a shaker for culture, and the culture conditions were 36.5 °C, 120 rmp, 7% CO 2 . The culture period was 6 - 10 days after transfection, and feeding was performed every two days.

[0190] After the culture of the above transfection mixture was completed, the precipitate was discarded by centrifugation at 1000 rmp for 10 min, and then the cell supernatant was collected by centrifugation at 12000 rmp for 30 min and subjected to aseptic filtration. In the first step, purification was carried out using an AKTA Avant purifier. First, the mabselect sure LX column was disinfected with 0.1 M NaOH for 15 - 20 min, and then equilibrated with PBS buffer for 3 - 5 column volumes before loading the sample. After the loading was completed, elution was carried out with a sodium acetate buffer at pH 5.5, and finally the target protein was eluted with an acetic acid - sodium acetate buffer at pH 3.6; In the second step, purification was carried out using Eshmuno CPX. The equilibration buffer was a pH 5.5, 50 mM acetic acid - sodium acetate system, and the elution buffer was a pH 5.5, 50 mM acetic acid - sodium acetate + 1 M NaCl buffer system. A linear elution method was used to collect the target protein. As determined by SEC - HPLC, the monomer purity could reach over 95%, and the monoclonal antibodies JS011 - phage3 and JS011 - phage6 were obtained.

[0191] The gene sequences of antibodies JS011 - phage3 and JS011 - phage6 were obtained by gene sequencing, and then the amino acid sequences of the antibodies of the present application were obtained.

[0192] The amino acid sequence of antibody JS011 - phage3 is as follows:

[0193] Heavy chain (JS011 - phage3 - HC, italicized indicates VH, and underlined indicates HCDR1, HCDR2, and HCDR3 in turn):

[0194]

[0195] Among them, it includes the heavy chain variable region JS011 - phage3 - VH:

[0196] QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPQSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCAREGGEWLAIPFDYWGQGTLVTVSS(SEQ ID NO:13);

[0197] Among them, according to the Kabat numbering system, it includes:

[0198] HCDR1: GYYMH(SEQ ID NO:1);

[0199] HCDR2: WINPQSGGTNYAQKFQG(SEQ ID NO:2);

[0200] HCDR3: EGGEWLAIPFDY (SEQ ID NO:3);

[0201] Light chain (JS011 - phage3 - LC, with VL in italics and LCDR1, LCDR2, and LCDR3 underlined in sequence):

[0202]

[0203] Which includes the light chain variable region JS011 - phage3 - VL:

[0204] ELELTQPPSVSVSPGQTASITCSADKLGDKYASWYQQKPGQSPVLVLYEDSKRPSGIPERISGSNSGNTATLTIRGTQPMDEADYYCQTWDTNTVLFGGGTRLTVL (SEQ ID NO:14);

[0205] According to the Kabat numbering system, it includes:

[0206] LCDR1: SADKLGDKYAS (SEQ ID NO:4);

[0207] LCDR2: EDSKRPS (SEQ ID NO:5);

[0208] LCDR3: QTWDTNTVL (SEQ ID NO:6).

[0209] The amino acid sequence of antibody JS011 - phage6 is as follows:

[0210] Heavy chain (JS011 - phage6 - HC, with VH in italics and HCDR1, HCDR2, and HCDR3 underlined in sequence):

[0211]

[0212] Which includes the heavy chain variable region JS011 - phage6 - VH:

[0213] QVQLVQSGAEVKKPGASVKVSCKASGYTFTGQYIHWARQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARDKAGADYWGQGTLVTVSS (SEQ ID NO:15);

[0214] According to the Kabat numbering system, it includes:

[0215] HCDR1: GQYIH (SEQ ID NO:7);

[0216] HCDR2: WINPNSGGTNYAQKFQG (SEQ ID NO:8);

[0217] HCDR3: DKAGADY (SEQ ID NO:9);

[0218] Light chain (JS011 - phage6 - LC, italic indicates VL, underlines indicate LCDR1, LCDR2, and LCDR3 in sequence):

[0219]

[0220] Which includes the light chain variable region JS011 - phage6 - VL:

[0221] ELALTQPPSVSGSPGQSITISCTGTSSDIGGYDYVSWYQQYPGKAPKLMISGVSNRPSGVSNRFSGSKSGNTASLIISGLQAEDEGDYYCSSYTSRSTRWVFGGGTKLTVL (SEQ ID NO:16);

[0222] According to the Kabat numbering system, it includes:

[0223] LCDR1: TGTSSDIGGYDYVS (SEQ ID NO:10);

[0224] LCDR2: GVSNRPS (SEQ ID NO:11);

[0225] LCDR3: SSYTSRSTRWV (SEQ ID NO:12).

[0226] Example 3. Detection of the binding ability of the anti - 4 - 1BB antibody

[0227] 3.1 Detection of the binding ability of the candidate antibody to the 4 - 1BB protein by Elisa:

[0228] Using a microplate reader from Thermo Scientific, plates were coated with a fixed concentration of human 4-1BB with His-tag (the extracellular region sequence of 4-1BB was fused with His-tag to construct an expression plasmid, and it was expressed and purified by transient transfection of 293 cells) or murine 4-1BB with His-tag (manufacturer Sino Biological, catalog number 50811-M08H) antigen (1.0 μg / mL). After blocking with 2% BSA, serially diluted JS011 antibodies JS011-phage3 and JS011-phage6 were added (binding to human 4-1BB: starting from 1 μg / ml, 3-fold serial dilution, a total of 12 concentrations; binding to murine 4-1BB: starting from 1 μg / ml, 2.5-fold serial dilution, a total of 12 concentrations). Mouse anti-human IgG4 Fc HRP (Southern Biotech, 9200-05) diluted 5000-fold was used as the detection antibody for detection. Then, 0.1 mg / ml TMB (3,3',5,5'-tetramethylbenzidine) was used for color development, and finally, the reaction was terminated with 2 M HCl, and the plates were read at 450 nm / 620 nm. The half maximal effective concentration EC was fitted using a four-parameter logistic regression (4PL) model. 50 Anti-KLH-IgG4 (CB25772663, or the gene sequences encoding the heavy and light chains of the human IgG4 antibody against keyhole limpet hemocyanin KLH were respectively constructed into expression plasmids and expressed and purified by transient transfection of CHO-18 cells) was used as a negative control.

[0229] The binding curves of JS011-phage3 and JS011-phage6 to human 4-1BB are as Figure 1 shown, and the binding curves of JS011-phage3 and JS011-phage6 to murine 4-1BB are as Figure 2 shown. As can be seen from Figure 1 , both JS011-phage3 and JS011-phage6 can bind to human 4-1BB and their binding activities are close, with EC 50 being 1.5 ng / ml and 1.6 ng / ml respectively; as can be seen from Figure 2 , JS011-phage3 can bind to murine 4-1BB, with EC 50 being 6.1 ng / ml, and JS011-phage6 does not bind to murine 4-1BB.

[0230] Example 4: Binding of anti-4-1BB antibody to Jurkat NFKB 4-1BB cells

[0231] Jurkat NFKB 4-1BB cells (stably expressing 4-1BB, constructing an expression plasmid by fusing the NFKB luciferase reporter gene, electrotransfecting Jurkat cells, and obtaining Jurkat cells stably expressing NFKB through pressure screening, named Jurkat NFKB cells. Then constructing an expression plasmid of the full-length 4-1BB gene, electrotransferring it into Jurkat NFKB cells, and obtaining Jurkat NFKB 4-1BB stably expressing 4-1BB) were incubated with anti-4-1BB monoclonal antibodies JS011-phage3 and JS011-phage6 at different concentrations (starting concentration of 100 μg / ml, 3-fold dilution, a total of 12 concentration gradients) at 4°C for 30 min, then washed and incubated with a secondary antibody conjugated with fluorescein PE against human IgG (SouthernBiotech, Cat#2040-09) in the dark at 4°C for 30 min. Finally, cells were collected using a flow cytometer (BD CantoⅡ) to detect the fluorescent antibodies bound to the cell surface. The original data was analyzed using FlowJo to obtain the MFI value (denoted as MFI-PE), and the antibody dose-dependent binding curve was fitted using GraphPad. The results are as Figure 3 shown, and EC 50 50 was calculated, where the positive controls were Urelumab (BMS control antibody, see patent CN1867585B) and Utomilumab (Pfizer control antibody, see patent CN103221428B) antibodies; the negative control was Anti-KLH-IgG4.

[0232] As Figure 3 shown, antibodies JS011-phage3, JS011-phage6, Urelumab, and Utomilumab were all able to bind to 4-1BB on the surface of Jurkat NFKB 4-1BB cells with high affinity, and the EC 50 50 values were 3.442 μg / mL, 6.961 μg / mL, 1.532 μg / mL, and 0.01656 μg / mL, respectively.

[0233] Example 5: Anti-4-1BB antibody blocking the binding of 4-1BB ligand (4-1BBL) to Jurkat NFKB 4-1BB cells

[0234] Jurkat NFKB 4-1BB cells were incubated with different concentrations of anti-4-1BB monoclonal antibodies JS011-phage3 and JS011-phage6 (starting concentration 100 μg / ml, 3-fold dilution, a total of 12 concentration gradients) and 2 μg / ml biotinylated 4-1BBL-hFc (a ligand for 4-1BB, the extracellular region sequence of 4-1BBL was fused with the FC (hFC) sequence to construct an expression plasmid, and it was expressed and purified by transient transfection of CHO-18 cells) at 4 °C for 30 min, then washed and incubated with a fluorescein FITC-labeled secondary antibody (BioLegend, Cat#405202) at 4 °C in the dark for 30 min. Finally, cells were collected using a flow cytometer (BD Canto II) to detect the fluorescent antibody bound to the cell surface. The original data was analyzed using FlowJo to obtain the MFI value (denoted as MFI-FITC), and the antibody dose-dependent inhibition curve was fitted using GraphPad and the IC 50 results are shown in Figure 4 Figure [0000616], where the positive controls were Urelumab (BMS control antibody, see patent CN1867585B) and Utomilumab (Pfizer control antibody, see patent CN103221428B); the negative control was Anti-KLH-IgG4.

[0235] As Figure 4 shown in Figure [0000619], neither JS011-phage3, JS011-phage6 nor the Urelumab antibody could block the binding of 4-1BBL to 4-1BB on the surface of Jurkat NFKB 4-1BB cells, while Utomilumab could effectively block the binding of 4-1BBL to 4-1BB on the surface of Jurkat NFKB 4-1BB cells, and the IC 50 was 0.3431 μg / ml.

[0236] Example 6: Activity detection of anti-4-1BB antibodies in the luciferase reporter gene system (CHO FCGR2B 4G6 / Jurkat NFKB 4-1BB dual cells)

[0237] CHO FCGR2B 4G6 cells (stably highly expressing human CD32B, the CD32B gene was constructed into an expression plasmid and electrotransfected into CHO-K1 cells, and monoclonal cells stably expressing CD32B were obtained by pressure screening and named CHO FCGR2B 4G6 cells) were seeded at 3×10 4The number of cells was added to a 96-well flat-bottom white plate (Corning, Cat#3917) and incubated overnight in a 37°C incubator. The next day, the effector cells Jurkat NFKB 4-1BB (stably expressing NlucP / NFKB-RE and 4-1BB) were added to the cell plate at a cell number of 1×10 5 per well. Subsequently, the anti-4-1BB monoclonal antibodies JS011-phage3 and JS011-phage6 (starting concentration of 25 μg / ml, diluted 4-fold, a total of 9 concentration gradients) were added to the cell plate using experimental buffer (RPMI 1640 (1×) + 2% FBS) and co-incubated in a 37°C incubator for 4 - 6 hours. Finally, Nano-Glo luciferase assay reagent (Promega) was added to the cell-antibody mixture system, and the chemiluminescence signal (recorded as RLU) was detected using a multimode microplate reader (TECAN M1000 pro). The EC 50 value was calculated by fitting a four-parameter regression curve using GraphPad prism software, and the results are as shown in Figure 5 . The positive controls were Urelumab (BMS control antibody, see patent CN1867585B) and Utomilumab (Pfizer control antibody, see patent CN103221428B).

[0238] As shown in Figure 5 , JS011-phage3, JS011-phage6, Urelumab, and Utomilumab had strong T cell activation activity in the luciferase reporter gene system composed of CHO FCGR2B4G6 cells and effector cells Jurkat NFKB 4-1BB. The EC 50 values were 0.03888 μg / mL, 0.4499 μg / mL, 1.038 μg / mL, and 0.02391 μg / mL respectively. The activity of JS011-phage3 was comparable to that of Utomilumab; the activity of JS011-phage6 was comparable to that of Urelumab, and the ability of JS011-phage3 to activate T cells was stronger than that of JS011-phage6.

[0239] Example 7: Activity detection of anti-4-1BB antibody in the luciferase reporter gene system (THP-1 / Jurkat NFKB 4-1BB dual cells)

[0240] THP-1 cells (a kind of human myeloid leukemia monocyte, CD32B positive) and effector cells Jurkat NFKB 4-1BB (stably expressing NlucP / NFKB-RE and 4-1BB) were respectively added at a cell number of 5×10 4 per well and 1×105 The cells were added to a 96-well flat-bottom white plate (Corning, Cat#3917) at a certain cell count. Subsequently, the anti-4-1BB monoclonal antibodies JS011-phage3 and JS011-phage6 (initial concentration of 100 μg / ml, serially diluted 4-fold, with a total of 10 concentration gradients) were added to the cell plate using experimental buffer (RPMI 1640 (1×) + 2% FBS), and co-incubated in an incubator at 37 °C for 4 - 6 hours. Finally, Nano-Glo luciferase assay reagent (Promega) was added to the cell-antibody mixture system, and the chemiluminescence signal (recorded as RLU) was detected using a multimode microplate reader (TECAN M1000pro). A four-parameter regression curve was fitted using GraphPad prism software to calculate the EC 50 value, and the results are as Figure 6 shown. The positive controls were Urelumab (BMS control antibody, see Patent CN1867585B) and Utomilumab (Pfizer control antibody, see Patent CN103221428B).

[0241] As Figure 6 shown, JS011-phage3, JS011-phage6, Urelumab, and Utomilumab had strong T cell activation activity in the luciferase reporter gene system composed of THP-1 cells and effector cells Jurkat NFKB 4-1BB, and the EC 50 values were 0.006642 μg / mL, 0.2826 μg / mL, 218.6 μg / mL, and 0.04173 μg / mL, respectively.

[0242] Example 8: Activity detection of anti-4-1BB antibodies in the luciferase reporter gene system (Raji / Jurkat NFKB 4-1BB dual cells or Raji / Jurkat NFKB dual cells)

[0243] Raji cells (a kind of human myeloid leukemia monocyte, CD32B positive, 4-1BBL positive) were added to a 96-well flat-bottom white plate (Corning, Cat#3917) at a cell count of 5×10 4 per well, and Jurkat NFKB 4-1BB cells (stably expressing NlucP / NFKB-RE and 4-1BB) or Jurkat NFKB cells (stably expressing NlucP / NFKB-RE) were added at 1×10 5The number of cells was added to the cell plate. Subsequently, the anti-4-1BB monoclonal antibodies JS011-phage3 and JS011-phage6 (initial concentration of 100 μg / ml, serially diluted 4-fold, a total of 10 concentration gradients) were added to the cell plate with experimental buffer (RPMI 1640 (1×) + 2% FBS), and co-incubated in an incubator at 37 °C for 4 - 6 hours. Finally, Nano-Glo luciferase assay reagent (Promega) was added to the cell-antibody mixture system, and the chemiluminescence signal (denoted as RLU) was detected using a multimode microplate reader (TECAN M1000 pro). A four-parameter regression curve was fitted using GraphPad prism software to calculate the EC 50 value, and the results are shown in Figure 7 and Figure 8 respectively. Among them, the positive controls were Urelumab (BMS control antibody, see Patent CN1867585B) and Utomilumab (Pfizer control antibody, see Patent CN103221428B).

[0244] As shown in Figure 7 , JS011-phage3, JS011-phage6, and Urelumab had the activity of activating T cells in the luciferase reporter gene system composed of Raji cells and effector cells Jurkat NFKB 4-1BB (high expression of 4-1BB), and the EC 50 values were 9.446 μg / mL, 2.713 μg / mL, and 7.240 μg / mL respectively. Utomilumab showed a decrease in activity in this system due to blocking the binding effect of 4-1BBL.

[0245] As shown in Figure 8 , in the luciferase reporter gene system composed of Raji cells and effector cells Jurkat NFKB (low expression of 4-1BB), JS011-phage3 and Urelumab respectively played a synergistic role with 4-1BBL on the surface of Raji cells in activating T cells, and the EC 50 values were 9.450 μg / mL and 86.01 μg / mL respectively. JS011-phage6 and Utomilumab were inactive in this system.

[0246] Example 9: Study on cytokine storm of anti-4-1BB antibody

[0247] PBMCs from two different volunteers (Volunteer 1 ID#: LP191225, Volunteer 2 ID#: LP190812) were seeded at 3×10 per well 5The cells were added to a 96-well round bottom plate, and then the anti-4-1BB monoclonal antibodies JS011-phage3, JS011-phage6, Urelumab, and Utomilumab were added to the cell plate at a concentration of 10 μg / mL. Incubate at 37 °C and 5% CO 2 After 24 h of incubation, the supernatant was collected by centrifugation, and the release of cytokines such as IL-2, IL-4, IL-6, IL-10, TNFα, and IFNγ was detected using the BD CBA human Th1 / Th2 cytokine kitⅡ. The results are as Figures 9A to 9F shown. The positive control was the OKT3 antibody (obtained from a hybridoma deposited at the American Type Culture Collection under accession number ATCC CRL 8001); the negative control was Anti-KLH-IgG4.

[0248] As Figures 9A to 9F shown, at a concentration of 10 μg / mL, neither JS011-phage3, JS011-phage6, Urelumab, nor Utomilumab caused a cytokine storm.

[0249] Example 10: Inhibitory effect of anti-4-1BB antibody on the growth of transplanted MC38 tumors in h41BB humanized mice

[0250] 1. Test purpose

[0251] To evaluate the anti-tumor effect of the anti-4-1BB monoclonal antibody of the present application in a subcutaneous tumor model of transplanted mouse colon cancer MC38 in h41BB humanized mice.

[0252] 2. Test procedure

[0253] Female h41BB humanized mice at 6-8 weeks of age (purchased from Beijing Biocytogen Co., Ltd.) were subcutaneously inoculated with 1×10 6 MC38 cells on the right dorsal side. When the average tumor volume was approximately 90 mm 3 , appropriate animals were selected and randomly divided into 5 groups with 6 animals in each group according to tumor volume. They were respectively

[0254] 1. Normal saline control group (solvent control group);

[0255] Treatment groups:

[0256] 2. Urelumab (1 mg / kg body weight) group;

[0257] 3. Utomilumab (1 mg / kg body weight) group;

[0258] 4. JS011-phage3 (1 mg / kg body weight) group and

[0259] 5. JS011 - phage6 (1 mg / kg body weight) group.

[0260] Administered by intraperitoneal injection, twice a week for 3 consecutive weeks, and the experiment ended 4 days after the last administration. The tumor volume and body weight were measured twice a week, and the body weight and tumor volume of the mice were recorded. At the end of the experiment, the mice were euthanized, and the tumor growth inhibition rate TGI (%) = [1 - (Ti - T0) / (Vi - V0)] × 100% was calculated. (Ti: mean tumor volume of the treatment group on the i - th day of administration, T0: mean tumor volume of the treatment group on the 0 - th day of administration; Vi: mean tumor volume of the solvent control group on the i - th day of administration, V0: mean tumor volume of the solvent control group on the 0 - th day of administration).

[0261] As Figure 10 shown, on the 20th day after administration, the average tumor volume of the normal saline control group was 2213 mm 3 , the average tumor volume of the Urelumab (BMS, see patent CN1867585B) group was 430 mm 3 , significantly inhibiting tumor growth compared with the normal saline control group, and the tumor growth inhibition rate was 83.9%; the average tumor volume of the Utomilumab (Pfizer, see patent CN103221428B) group was 1937 mm 3 , showing no obvious tumor inhibition effect, and the tumor growth inhibition rate was 13.0%; the average tumor volume of the JS011 - phage3 group was 506 mm 3 , significantly inhibiting tumor growth, and the tumor growth inhibition rate was 64.7%; the average tumor volume of the JS011 - phage6 group was 674 mm 3 , significantly inhibiting tumor growth, and the tumor growth inhibition rate was 50.6%. The results showed that in the h41BB humanized mouse transplanted with MC38 WT model, at the dose level of 1 mg / kg, both JS011 - phage3 and JS011 - phage6 showed significant tumor inhibition effects.

[0262] Example 11: Toxicity study of anti - 4 - 1BB antibody on h41BB humanized mice transplanted with MC38 tumors

[0263] 1. Test purpose

[0264] To evaluate the toxic effect of the anti - 4 - 1BB monoclonal antibody of the present application on h41BB humanized mice transplanted with subcutaneous tumors of mouse colon cancer MC38 cells.

[0265] 2. Test procedure

[0266] 6- to 8-week-old female h41BB humanized mice (purchased from Beijing Biocytogen Co., Ltd.), were subcutaneously inoculated with 1×10 6 MC38 cells on the right dorsal side. When the average tumor volume was approximately 115 mm 3 , suitable animals were selected and randomly divided into 5 groups with 6 animals in each group according to tumor volume. They were respectively

[0267] 1. Anti-KLH-IgG4 (labeled as Anti KLH hIgG4 in the figure, negative control group);

[0268] Treatment groups:

[0269] 2. JS011-phage3 (10 mg / kg body weight) group;

[0270] 3. JS011-phage6 (10 mg / kg body weight) group;

[0271] 4. Urelumab (10 mg / kg body weight) group and

[0272] 5. Utomilumab (10 mg / kg body weight) group.

[0273] Intraperitoneal injection was given 2 times a week for 3 consecutive times. The animals were euthanized 7 days after the last dose. The spleen weight of the mice was weighed. The livers of the mice were collected, and the proportions of CD3 + , CD3 + CD4 + , CD3 + CD8 + , CD3 + CD8 + CD11c + and CD3 - NK1.1 + cell populations in the livers of animals in different groups were detected by flow cytometry to observe the in vivo toxicity of anti-4-1BB monoclonal antibodies.

[0274] As Figure 11As shown, on the 14th day after administration, compared with the Anti KLH hIgG4 negative control group, the spleen weight of mice in the Urelumab (BMS, see patent CN1867585B) group increased significantly, while there were no significant changes in the spleen weights of mice in the JS011-phage3 group, JS011-phage6 group, and Utomilumab (Pfizer, see patent CN103221428B) group. This indicates that 10 mg / kg of Urelumab can cause splenomegaly in mice, while JS011-phage3 and JS011-phage6 do not cause splenomegaly in animals. This result suggests that JS011-phage3 and JS011-phage6 of the present application do not cause excessive activation of immune cells in the spleen and may have better safety in clinical practice.

[0275] As Figure 12A shown, on the 14th day after administration, the number of lymphocytes contained in the liver unit weight was analyzed. Compared with the AntiKLH hIgG4 negative control group, the number of lymphocytes in the livers of mice in the JS011-phage3 group, JS011-phage6 group, Utomilumab group, and Urelumab group increased, and the number of lymphocytes in the livers of mice in the Urelumab group was significantly higher than that in the JS011-phage3 group, JS011-phage6 group, and Utomilumab group, indicating that 10 mg / kg of Urelumab can cause a significant increase in the number of lymphocytes in the livers of mice. As Figures 12B to 12E shown, on the 14th day after administration, the changes in the proportions of different subsets of liver lymphocytes were analyzed. Compared with the Anti KLH hIgG4 negative control group, the proportions of CD3 + CD4 + T cells in the livers of mice in the JS011-phage3 group, JS011-phage6 group, Utomilumab group, and Urelumab group decreased, while the proportions of CD3 + CD8 + T cells increased and the proportion of CD3 + CD8 + T cells in the Urelumab group was significantly higher than that in the JS011-phage3 group, JS011-phage6 group, and Utomilumab group. The proportion of CD3 + CD8 + CD11c + T cells in the Urelumab group increased significantly, indicating that the proportion of over-activated CD3 + CD8 + T cells in the livers of mice in the Urelumab group increased significantly, while the CD3 -NK1.1 + The proportion of NK cells decreased, indicating that 10 mg / kg of Urelumab would cause CD3 in the mouse liver + CD8 + The proportion and number of T cells increased significantly. The reason for the increase in the number of lymphocytes in the mouse liver was that CD3 + CD8 + The absolute number of T cells increased significantly, and the absolute numbers of CD3 + CD4 + T and NK cells increased. However, due to the substantial increase in the proportion of CD3 + CD8 + T cells, the proportions of CD3 + CD4 + T and NK cells decreased. Judging from the absolute and relative values of CD3 + CD8 + T cells in the liver after administration, JS011-phage3 and JS011-phage6 were significantly superior to Urelumab, suggesting that there might be lower hepatotoxicity in clinical practice.

[0276] The above description is only a preferred embodiment of the present application and is not intended to limit the protection scope of the present application. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present application are included in the protection scope of the present application.

Claims

1. An antibody or antigen-binding fragment thereof that can specifically bind to 4-1BB, which comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3; wherein according to the Kabat, IMGT, Chothia, AbM, Contact or North numbering system, the amino acid sequences of the HCDR1, HCDR2 and HCDR3 are the HCDR1, HCDR2 and HCDR3 of the heavy chain variable region shown in SEQ ID NO: 13, and the amino acid sequences of the LCDR1, LCDR2 and LCDR3 are the LCDR1, LCDR2 and LCDR3 of the light chain variable region shown in SEQ ID NO: 14; or the amino acid sequences of the HCDR1, HCDR2 and HCDR3 are the HCDR1, HCDR2 and HCDR3 of the heavy chain variable region shown in SEQ ID NO: 15, and the amino acid sequences of the LCDR1, LCDR2 and LCDR3 are the LCDR1, LCDR2 and LCDR3 of the light chain variable region shown in SEQ ID NO:

16.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein, according to the Kabat numbering system, the amino acid sequences of the HCDR1, HCDR2 and HCDR3 are respectively shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, and the amino acid sequences of the LCDR1, LCDR2 and LCDR3 are respectively shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6; or the amino acid sequences of the HCDR1, HCDR2 and HCDR3 are respectively shown in SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9, and the amino acid sequences of the LCDR1, LCDR2 and LCDR3 are respectively shown in SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:

12.

3. The antibody or antigen-binding fragment thereof according to claim 2, which comprises: a heavy chain variable region having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 13, and a light chain variable region having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 14; or a heavy chain variable region having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 15, and a light chain variable region having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:

16.

4. The antibody or antigen-binding fragment thereof according to claim 3, wherein, the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region with the amino acid sequence shown in SEQ ID NO:13, and a light chain variable region with the amino acid sequence shown in SEQ ID NO:14; or A heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 15, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO:

16.

5. The antibody or antigen-binding fragment thereof according to claim 1, which further comprises a human heavy chain constant region and a human light chain constant region, wherein the human heavy chain constant region is selected from the heavy chain constant regions of human IgG1, IgG2, IgG3 or IgG4; and the human light chain constant region is selected from the light chain constant regions of lambda light chain or kappa light chain.

6. The antibody or antigen-binding fragment thereof according to claim 5, wherein, the human heavy chain constant region is selected from the heavy chain constant region of human IgG4 or the heavy chain constant region of human IgG4 having an S228P amino acid substitution.

7. The antibody or antigen-binding fragment thereof according to claim 1, which comprises: a heavy chain having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 17, and a light chain having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 18; or a heavy chain having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 19, and a light chain having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:

20.

8. The antibody or antigen-binding fragment thereof according to claim 7, wherein, the antibody or antigen-binding fragment comprises: a heavy chain having an amino acid sequence as shown in SEQ ID NO: 17, and a light chain having an amino acid sequence as shown in SEQ ID NO: 18; or a heavy chain having an amino acid sequence as shown in SEQ ID NO: 19, and a light chain having an amino acid sequence as shown in SEQ ID NO:

20.

9. The antibody or antigen-binding fragment thereof according to any one of claims 1-8, wherein, the antigen-binding fragment includes at least one of Fab, Fab', F(ab')2, Fv and scFv.

10. A polynucleotide molecule comprising a nucleotide sequence encoding the antibody or antigen-binding fragment thereof according to any one of claims 1-9.

11. An expression vector comprising the polynucleotide molecule according to claim 10.

12. The expression vector according to claim 11, wherein, the expression vector is a eukaryotic expression vector.

13. A host cell comprising the polynucleotide molecule according to claim 10, or comprising the expression vector according to any one of claims 11-12.

14. The host cell according to claim 13, wherein, the host cell is a eukaryotic cell.

15. The host cell according to claim 13, wherein, the host cell is a mammalian cell.

16. The host cell according to any one of claims 13-15, which is used for expressing the antibody or antigen-binding fragment thereof according to any one of claims 1-9.

17. A method for preparing an antibody or an antigen-binding fragment thereof according to any one of claims 1-9, which comprises expressing the antibody or an antigen-binding fragment thereof in a host cell according to any one of claims 13-15, and recovering the expressed antibody or an antigen-binding fragment thereof from the host cell.

18. A pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1-9, and a pharmaceutically acceptable carrier.

19. Use of an antibody or an antigen-binding fragment thereof according to any one of claims 1-9 or a pharmaceutical composition according to claim 18 in the preparation of a medicament for treating or preventing a 4-1BB-mediated disease; the 4-1BB-mediated disease is cancer; the cancer is selected from at least one of melanoma, breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, kidney cancer, lung cancer, liver cancer, gastric cancer, colorectal cancer, bladder cancer, head and neck cancer, thyroid cancer, esophageal cancer, cervical cancer, sarcoma, multiple myeloma, leukemia, lymphoma, gallbladder cancer and glioblastoma.

20. A kit comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1-9 or a pharmaceutical composition according to claim 18.

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