WO2023098785A1

WO2023098785A1 - Anti-4-1bb antibody and use thereof

Info

Publication number: WO2023098785A1
Application number: PCT/CN2022/135784
Authority: WO
Inventors: 周进伟; 刘丹丹; 刘辉; 赵文亭; 林志妙; 吴纯; 陈素芬; 周昌全; 冯辉; 姚盛
Original assignee: 上海君实生物医药科技股份有限公司; 苏州君盟生物医药科技有限公司
Priority date: 2021-12-01
Filing date: 2022-12-01
Publication date: 2023-06-08
Also published as: CN116199778A

Abstract

Provided are an antibody capable of specifically binding to 4-1 BB or an antigen binding fragment thereof, and a pharmaceutical composition comprising same. Also provided are a polynucleotide molecule encoding the antibody or the antigen binding fragment thereof, an expression vector for expressing the antibody or the antigen binding fragment thereof, a host cell, and a use of the antibody or the antigen binding fragment thereof.

Description

Anti-4-1BB antibody and use thereof

This application claims the priority of the Chinese patent application with the application number 202111455972.6 filed on December 01, 2021, entitled "anti-4-1BB antibody and its use", the entire content of which is incorporated by reference in this application .

technical field

The application belongs to the field of biomedicine, and in particular relates to an antibody capable of specifically binding to 4-1BB or an antigen-binding fragment thereof and uses thereof.

Background technique

Tumor immunotherapy is a treatment that restores or enhances the body's immune system's natural defenses against tumors. Such treatments typically target 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. Over the past few decades, the use of monoclonal antibodies (MAbs) to treat cancer has achieved great success, and many of them have been approved for cancer treatment or clinical trials.

4-1BB (CD137/TNFRSF9), a transmembrane protein of the tumor necrosis factor receptor superfamily (TNFRS), is expressed on antigen-primed T cells but not on resting T cells. Human 4-1BB is a 255 amino acid protein comprising 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 as a monomer or dimer on the cell surface, and after binding to its ligand (4-1BBL) it undergoes signal transduction through trimerization.

In addition, 4-1BB is known to be present in dendritic cells (DCs), natural killer cells (NKs), activated CD4 ⁺ and CD8 ⁺ T lymphocytes, eosinophils, natural killer T cells (NKT) and mast cells expression, but myeloid-derived suppressor cells (MDSCs) do not express this molecule on their surface. Anti-4-1BB antibodies have the ability to activate cytotoxic T cells and increase interferon-γ (IFN-γ) production, showing great potential in anti-cancer.

Currently, several 4-1BB-related drug research projects are underway. The reported 4-1BB antibody sequence binds to 4-1BB and activates the 4-1BB signaling pathway, or overactivation leads to the enrichment of CD8 ⁺ T cells in the liver and causes hepatotoxicity, or good safety but insufficient activation leads to tumor killing effect bad. Therefore, there is an urgent need to develop novel 4-1BB antibody drugs with better activity and lower toxicity.

Contents of the invention

The purpose of the present application is to provide an antibody capable of specifically binding to 4-1BB (i.e., an anti-4-1BB antibody) or an antigen-binding fragment thereof, which can be used as an independent therapy or in combination with other therapies/or other anticancer agents. in the treatment of, for example, cancer.

The first aspect of the present application provides an antibody or an antigen-binding fragment thereof capable of specifically binding to 4-1BB, which comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3;

Wherein according to Kabat, IMGT, Chothia, AbM, Contact or North numbering system, the aminoacid sequence of described HCDR1, HCDR2 and HCDR3 is HCDR1, HCDR2 and HCDR3 of heavy chain variable region as shown in SEQ ID NO:13 or 15, And the amino acid sequence of the LCDR1, LCDR2 and LCDR3 is LCDR1, LCDR2 and LCDR3 of the light chain variable region shown in SEQ ID NO: 14 or 16, and in the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 At least one of can be substituted with a variant having 1, 2 or 3 amino acid differences therefrom.

In some embodiments, the antibody or antigen-binding fragment thereof capable of specifically binding to 4-1BB comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3;

Wherein according to Kabat, IMGT, Chothia, AbM, Contact or North numbering system, the aminoacid sequence of described HCDR1, HCDR2 and HCDR3 is HCDR1, HCDR2 and HCDR3 of heavy chain variable region as shown in SEQ ID NO:13, and said The amino acid sequence of LCDR1, LCDR2 and LCDR3 is LCDR1, LCDR2 and LCDR3 of the light chain variable region shown in SEQ ID NO: 14; or

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

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 The amino acid sequence shown in NO:7, SEQ ID NO:8 and SEQ ID NO:9;

Said LCDR1, LCDR2 and LCDR3 respectively have the amino acid sequence shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, or respectively have such as SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:11 and SEQ The amino acid sequence shown in ID NO:12;

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

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

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: 10, SEQ ID NO: Amino acid sequences shown in ID NO:11 and SEQ ID NO:12.

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

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

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

a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 13, and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 14; or

The amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 15, and the amino acid sequence of the light chain variable region shown in SEQ ID NO: 16.

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, and the human heavy chain constant region is selected from the constant heavy chain region of human IgG1, IgG2, IgG3 or IgG4. Region, preferably the heavy chain constant region of human IgG4 or the heavy chain constant region of human IgG4 with S228P amino acid substitution; the human light chain constant region is selected from the light chain constant region of lambda light chain or kappa light chain.

A heavy compound 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: 17 chain, and have 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: 18 light chain; or

A heavy compound 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 chain, and have 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 the light chain;

a heavy chain having an amino acid sequence as set forth in SEQ ID NO: 17, and a light chain having an amino acid sequence as set forth in SEQ ID NO: 18; or

The amino acid sequence is the heavy chain shown in SEQ ID NO:19, and the amino acid sequence is the light chain shown in SEQ ID NO:20.

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 a Fab, Fab', F(ab')2, Fv, scFv, and sdAb .

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

(I) the antibody or antigen-binding fragment thereof provided in the first aspect of the present application binds to the same epitope of human 4-1BB protein, or completely overlaps or partially overlaps;

(II) Compete with the antibody or antigen-binding fragment thereof provided in the first aspect of the present application for binding to the epitope of human 4-1BB protein.

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

The fourth aspect of the present application provides an expression vector comprising the polynucleotide molecule provided in the third aspect of the present application, preferably, the expression vector is a eukaryotic expression vector.

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 Mammalian cells are preferred.

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.

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 the method provided in the fifth aspect of the present application expressing the antibody or antigen-binding fragment thereof in a host cell, and recovering the expressed antibody or antigen-binding fragment thereof from the host cell.

The seventh aspect of the present application provides a pharmaceutical composition comprising 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 antibody or antigen-binding fragment thereof provided in the second aspect of the present application. The polynucleotide molecule provided in the third aspect 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.

The eighth aspect of the application provides the antibody or antigen-binding fragment thereof in the first aspect of the application or the isolated antibody or antigen-binding fragment thereof in the second aspect of the application or the polynucleotide molecule provided in the third aspect of the application or the antibody or antigen-binding fragment thereof in the third aspect of the application Use of the expression vector provided in the fourth aspect 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 medicament for treating or preventing 4-1BB-mediated diseases.

The ninth aspect of the application provides the antibody or antigen-binding fragment thereof in the first aspect of the application or the isolated antibody or antigen-binding fragment thereof in the second aspect of the application or the polynucleotide molecule provided in the third aspect of the application or the antibody or antigen-binding fragment thereof in the third aspect of the application Use of the expression vector provided in the fourth aspect 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.

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

In some embodiments, the 4-1BB-mediated disease is cancer; preferably, the cancer is selected from: melanoma, breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, kidney cancer, lung cancer, liver cancer, At least one of 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.

The eleventh aspect of the present application provides a kit comprising 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 antibody or antigen-binding fragment thereof provided in the second aspect of the present application The polynucleotide molecule provided in the third aspect 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.

The twelfth aspect of the present application provides a method for detecting 4-1BB using 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, wherein This includes contacting the antibody or antigen-binding fragment thereof with a sample, detecting the binding of the antibody or antigen-binding fragment thereof to 4-1BB, and optionally quantifying the binding.

The antibody or its antigen-binding fragment that can specifically bind to 4-1BB provided by this application can specifically bind to human 4-1BB, has low liver toxicity and obvious tumor inhibitory effect, and can be used to treat 4-1BB-mediated diseases, such as cancer.

Description of drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present application and the prior art, the following briefly introduces the accompanying drawings required in the embodiments and the prior art. Obviously, the accompanying drawings in the following description are only the present invention. For some embodiments of the application, those skilled in the art can also obtain other drawings based on these drawings without creative work.

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.

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 mouse 4-1BB.

Figure 3 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 and related controls to Jurkat NFKB 4-1BB cells.

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 and related controls blocking the binding of 4-1BBL to Jurkat NFKB 4-1BB cells.

Figure 5 shows the anti-4-1BB antibodies JS011-phage 3 and JS011-phage 6 prepared in Example 2 of the present application and related controls in the luciferase reporter gene system (CHO FCGR2B 4G6/Jurkat NFKB 4-1BB double cells) activity test results.

Figure 6 shows the anti-4-1BB antibodies JS011-phage 3 and JS011-phage 6 prepared in Example 2 of the present application and related controls in the luciferase reporter gene system (THP-1/Jurkat NFKB 4-1BB double cells) activity test results.

Figure 7 shows the activity of the anti-4-1BB antibodies JS011-phage 3 and JS011-phage 6 prepared in Example 2 of the present application and related controls in the luciferase reporter gene system (Raji/Jurkat NFKB 4-1BB double cells) Test results.

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 and related controls in the luciferase reporter gene system (Raji/Jurkat NFKB double cells).

Figure 9A to Figure 9F show the cytokines IL-2, IL-4, IL-6, IL induced by the anti-4-1BB antibodies JS011-phage 3 and JS011-phage 6 prepared in Example 2 of the present application and related controls, respectively. -10. Changes in the release of TNFα and IFNγ.

Figure 10 shows the tumor growth inhibitory effect of the anti-4-1BB antibodies JS011-phage 3 and JS011-phage 6 prepared in Example 2 of the present application and related controls.

Figure 11 shows the effect 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 day 14 after administration.

Figure 12A shows the effect of anti-4-1BB antibodies JS011-phage 3 and JS011-phage 6 prepared in Example 2 of the present application and related controls on the number of total lymphocytes in the liver of tumor-bearing mice on the 14th day after administration .

Figures 12B to 12E respectively show 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 in the liver of tumor-bearing mice on the 14th day after administration. ⁺ T cell proportion, CD3 ⁺ CD8 ⁺ T cell proportion, CD3 ⁺ CD8 ⁺ CD11c ⁺ T cell proportion and CD3 ^- NK1.1 ⁺ NK cell proportion.

Detailed ways

In order to make the purpose, technical solution, and advantages of the present application clearer, the present application will be further described in detail below with reference to the accompanying drawings and examples. Apparently, the described embodiments are only some of the embodiments of this application, not all of them. Based on the embodiments in this application, all other embodiments obtained by persons of ordinary skill in the art without creative efforts fall within the protection scope of this application.

definition

The practice of this application will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art.

In order to make this application easier to understand, certain technical terms are specifically defined as follows. Unless otherwise clearly defined elsewhere herein, the scientific and technical terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the application belongs. For definitions and terms in this field, professionals can refer to Current Protocols in Molecular Biology (Ausubel). Abbreviations for amino acid residues are the standard 3-letter and/or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids. As used herein (including the claims), singular forms include their corresponding plural forms unless the context clearly dictates otherwise.

The term "about" when used in conjunction with a numerical value is meant to encompass a numerical value within a range having a lower limit of 5% less and an upper limit of 5% greater than the indicated numerical value, including but not limited to ±5%, ± 2%, ±1%, and ±0.1%.

The term "and/or" should be understood as meaning any one of the optional items or a combination of any two or more of the optional items.

The terms "4-1BB", "4-1BB receptor", "4-1BB protein", "CD137" or "tumor necrosis factor receptor superfamily member 9 (TNFRSF9)" are tumor necrosis factor (TNF) receptor superfamily A member of the T-cell family and is an activation-inducing T-cell co-stimulatory molecule. In the present application, the term "4-1BB" may include human 4-1BB, and its variants, subtypes, homologues, orthologs and paralogs, therefore, the antibody provided by the application or its The antigen binding portion may also bind 4-1BB from a species other than human, eg, cynomolgus monkey 4-1BB or mouse 4-1BB. For example, in certain instances, antibodies specific for human 4-1BB protein may cross-react with 4-1BB from species other than human. In other embodiments, antibodies specific for human 4-1BB protein may be fully specific for human 4-1BB protein and may exhibit species or other types of cross-reactivity, or may interact with antibodies from certain other species but Not all other species cross-react with 4-1BB, eg, monkey 4-1BB but not 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; -1BB differs in amino acid sequence, for example, may have conservative mutations in non-conserved regions, and has 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 4-1BB" may refer to 4-1BB having the complete amino acid sequence of cynomolgus 4-1BB with Genbank accession number NP_001253057.1.

The term "percent (%) amino acid sequence identity" or simply "identity" is defined as aligning amino acid sequences (and introducing gaps where necessary) for maximum percent sequence identity and not considering any conservative substitutions as Following a portion of sequence identity, the percentage of amino acid residues in a candidate amino acid sequence that are identical to those in a reference amino acid sequence. Alignment of sequences to determine percent amino acid sequence identity can be performed using various methods in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

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

The term "signal transduction pathway" or "signal transduction activity" refers to a biochemical causal relationship, usually initiated by protein-protein interactions, such as the binding of a growth factor to a receptor, that results in the transmission of a signal from one part of a cell to another part of the cell. part. Typically, delivery involves specific phosphorylation of one or more tyrosine, serine, or threonine residues on one or more proteins in a cascade of reactions resulting in signal transduction. The penultimate process often involves nuclear events, resulting in changes in gene expression.

The terms "activity" or "biological activity" or the terms "biological property" or "biological signature" are used interchangeably in this application and include, but are not limited to, epitope or antigen affinity, specificity, in vivo or in vitro neutralization or The ability to antagonize the activity of 4-1BB, the half-inhibitory concentration (IC ₅₀ ), the stability of the antibody in vivo, 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 with non-human homologues of the targeting peptide, or with other proteins or tissues), and retention of mammalian The ability of a cell to express a protein at a high level. The aforementioned properties or characteristics are observed, measured or evaluated using techniques well known in the art including, but not limited to, enzyme-linked immunosorbent assay (ELISA), flow cytometry sorting (FACS) or BIACORE plasma resonance analysis, Any in vitro or in vivo neutralization assays, receptor binding assays, cytokine or growth factor production and/or secretion assays, signal transduction assays, and analysis of tissue sections of different origin, including human, primate, or any other origin Immunohistochemical analysis, etc.

The term "antibody" refers to any form of antibody that possesses the desired biological activity. Accordingly, 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. The basic antibody structural unit is known to comprise tetramers, each tetramer comprising two identical pairs of polypeptide chains, each pair having a "light" chain (L, about 25 kDa) and a "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 primarily responsible for antigen recognition. The carboxy-terminal portion or fragment of each chain may define a constant region primarily responsible for effector function. Human light chains are generally classified as kappa and lambda light chains. In addition, human heavy chains are usually classified into five types: μ, δ, γ, α, or ε, and the antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE according to the heavy chains. Within light and heavy chains, the respective variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 10 more amino acids. See generally Chapter 7 of Fundamental Immunology (Paul, W. ed., 2nd ed. Raven Press, N.Y. (1989)).

With respect to an antibody, the term "isolated antibody" means that the antibody 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 can be understood that the isolated antibody is in a substantially purified state, preferably in a homogeneous state, and the isolated antibody can be in a dry or aqueous solution. Antibody purity and homogeneity can often be determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. The term "isolated" does not mean the complete absence of the above substances or the absence of water, buffers or salts, unless they are present in an amount that significantly interferes with the experimental or therapeutic application of the antibody of the present application.

The term "monoclonal antibody" is an antibody made from highly identical immune cells that are all clones of a single parental cell. Monoclonal antibodies have monovalent affinity because they bind to the same epitope (the part of the antibody that recognizes an antigen). The monoclonal antibodies may also contain minor amounts of naturally occurring mutations. In contrast, the term "polyclonal antibody" binds multiple epitopes, usually consists of several different lineages of plasma cells (antibody-secreting immune cells), and can be understood as a hybrid of multiple monoclonal antibodies. The modifier "monoclonal" should not be construed as requiring that the antibody be produced by any particular method.

The term "multispecific antibody" means comprising two or more antigen binding domains, capable of binding two or more different epitopes (e.g., two, three, four or more different epitope) antibodies. The epitopes to which specific antibodies bind can be on the same or different antigens. Examples of multispecific antibodies include "bispecific antibodies" that bind two different epitopes.

The term "binding domain" or "antigen-binding domain" or "antigen-binding site" refers to a 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 an antigen is large, antibodies may only be able to bind to a specific part of the antigen, called an epitope. The binding domain may comprise the variable domains of the heavy and light chains, namely the heavy chain variable region VH and the light chain variable region VL, each comprising four conserved framework regions (FR) and three complementarity determining regions (CDRs). ). The CDRs can vary in sequence and determine specificity for a particular antigen.

The term "full-length" antibody refers to an immunoglobulin molecule that, as it occurs in nature, comprises four peptide chains: two heavy chains (approximately 50-70 kDa in overall length) and two light chains (approximately 25 kDa in overall length) which are disulfide-bonded interconnected. Each heavy chain is composed 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 is composed 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 highly variable complementarity-determining regions (CDRs) and spaced apart from the complementarity-determining regions, more conserved framework regions (FRs). The sequence of domains from the amino terminus to the carboxyl terminus of each VH or VL 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 the antigen. The constant regions of the antibodies may mediate the binding of the antibodies to the host's tissues or to various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.

The term "heavy chain constant region" or "CH", which is used interchangeably in this application, comprises at least three heavy chain constant domains (CH1, CH2 and CH3). Exemplary, human heavy chain constant regions include gamma, delta, alpha, epsilon, and mu, each heavy chain constant region corresponding to an antibody isotype. For example, an antibody containing a gamma constant region is an IgG antibody, an antibody containing a delta constant region is an IgD antibody, an antibody containing an alpha constant region is an IgA antibody, an antibody containing a mu constant region is an IgM antibody, and an antibody containing an epsilon 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 (comprising the γ1 constant region), IgG2 (comprising the γ2 constant region), IgG3 (comprising the γ3 constant region), and IgG4 (comprising the γ4 constant region ); IgA antibodies include but are not limited to IgA1 (comprising the α1 constant region) and IgA2 (comprising the α2 constant region); IgM antibodies include but are not limited to IgM1 and IgM2. Isoforms may also include modifications that alter Fc function, eg, enhance or reduce effector function or enhance or reduce binding to Fc receptors.

The term "light chain constant region" or "CL" is used interchangeably in this application, which comprises a light chain constant domain, CL. Exemplarily, light chains can be classified into λ and κ according to the light chain constant region.

The term "antigen-binding fragment" of an antibody includes a fragment of an antibody or a derivative of an antibody, and the antibody corresponding to the "antigen-binding fragment" can be referred to as a parent antibody. An antigen-binding fragment of an antibody typically comprises at least a fragment of the antigen-binding or variable region of the parent antibody that retains at least some of the binding specificity of the parent 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 Antibody (nanobody) and multispecific antibody, 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 parent antibody. In addition, antigen-binding fragments of antibodies may also include conservative or non-conservative amino acid substitutions that do not significantly alter their biological activity (referred to as "conservative variants" or "functionally conservative variants" of the antibody).

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

The term "domain antibody" refers to an immunologically functional immunoglobulin fragment containing only the variable region of the heavy chain or the variable region of the light chain. In certain instances, two or more VH regions are covalently linked with a peptide linker to form a bivalent domain antibody. The two VH regions of a bivalent domain antibody can target the same or different antigens.

The term "antigen" refers to a molecule or a part of a molecule capable of being bound by an antibody of the present application. An antigen may have one or more than one epitope.

The term "diabody" refers to a small antibody fragment with two antigen-binding sites, said fragment comprising a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain, The two said fragments pass a linker that is too short to allow pairing between the two domains of the same chain, forcing this domain to pair with the complementary domain in the other fragment and creating two antigen-binding sites.

The terms "specific binding" and "selective binding" refer to the binding of an antibody to a predetermined epitope on an antigen. Typically, when recombinant human 4-1BB or its epitope is used as an analyte and an antibody is ^used as a ligand, when measured in an instrument by surface plasmon resonance (SPR) technique, the antibody has A small equilibrium dissociation constant (KD) for binding to the intended antigen or its epitope, and the binding affinity of the antibody to the intended antigen or its epitope is at least twice that of its binding affinity to other non-specific antigens (such as BSA, etc.) . The term "recognizes an antigen" is used interchangeably with the term "specifically binds" in this application.

The term "epitope" refers to the region of an antigen to which an antibody binds. Epitopes can be formed from contiguous amino acids or non-contiguous amino acids juxtaposed by the tertiary folding of the protein.

"Affinity" or "binding affinity" refers to the intrinsic binding affinity that reflects the interaction between members of a binding pair (eg, antigen and antibody). Affinity can generally be expressed by the equilibrium dissociation constant (KD), which is the ratio of the dissociation rate constant and the association rate constant (kdis and kon, respectively). Affinity can be measured by common methods known in the art, for example, it can be determined using ForteBio Biological Molecular Interaction Workstation.

The term "not binding" to proteins or cells refers to not binding to proteins or cells, or not binding to them with high affinity, that is, the KD of binding proteins or cells is 1.0×10 ^-6 M or higher, more preferably 1.0×10 ^{-6 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.

The term "high affinity" for an IgG antibody means that the KD for the antigen is 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. "High affinity" binding may vary for other antibody subtypes. For example, "high affinity" binding of IgM subtype means a KD of 10 ^-6 M or lower, preferably 10 ^-7 M or lower, more preferably 10 ^-8 M or lower.

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

"Construct" refers to any recombinant polynucleotide molecule (e.g., plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, bacteriophage, linear or circular single- or double-stranded DNA or RNA polynucleotide molecule), which can be Derived from any source, capable of integrating into the genome or replicating autonomously, it may be operably linked to one or more polynucleotide molecules. In the present application, a construct is generally a polynucleotide molecule of the present application, which is operably linked to transcriptional initiation regulatory sequences, and these sequences will direct the transcription of the polynucleotide molecule of the present application in a host cell. Expression of the nucleic acids of the present application may be directed using heterologous or endogenous promoters.

"Vector" refers to any recombinant polynucleotide construct that can be used for the purpose of transformation (ie, the introduction of heterologous DNA into a host cell). One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, which can incorporate additional DNA segments into the viral genome. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (eg, bacterial vectors with a bacterial origin of replication and episomal mammalian vectors). Other vectors (eg, non-episomal mammalian vectors) are integrated into the genome of the host cell after introduction into the host cell, where they replicate together with the host genome.

The term "expression vector" as used herein refers to a nucleic acid molecule capable of replicating and expressing a gene of interest upon transformation, transfection or transduction into a host cell. Expression vectors typically contain one or more phenotypic selectable markers and an origin of replication for maintenance of the vector and, if desired, amplification within the host.

In the present application, "activation", "stimulation" and "treatment" for cells or receptors may have the same meaning, for example, cells or receptors are activated, stimulated or treated with a ligand, unless otherwise specified. "Ligand" includes natural and synthetic ligands such as cytokines, cytokine variants or analogs, muteins, and antibody-derived binding compounds (eg, antibodies and binding fragments thereof). "Ligand" also includes small molecules, such as peptidomimetics of cytokines and peptidomimetics of antibodies. "Activation" can refer to cellular activation regulated by internal mechanisms as well as external or environmental factors. "Response" or "response", such as that of a cell, tissue, organ or organism, includes changes in biochemical or physiological behavior, such as concentration, density, viscosity, Accompanied by changes in migration, gene expression rates, or differentiation status, which changes may be associated with activation, stimulation, or processing.

As used herein, the term "treatment" or "treatment" of any disease or condition may refer to improving the disease or condition in one embodiment, such as slowing down, arresting or reducing the progression of the disease, or the clinical symptoms of the disease, etc.; In one embodiment, it may refer to alleviation or improvement of at least one physical parameter that may not show improvement in overt disease symptoms; in another embodiment, it may refer to physical (e.g., discernible stabilization of symptoms), physiologically (eg, stabilization of body parameters), or both. Unless explicitly described herein, methods for assessing treatment and/or prevention of disease are generally known in the art.

In this application, the "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, eg, mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, and the like.

Administration "in conjunction with" one or more other therapeutic agents includes simultaneous or co-administration, or sequential administration in any order.

"Therapeutically effective dose", "therapeutically effective dose" and "effective dose" refer to the dose under which the anti-4-1BB antibody or antigen-binding fragment thereof of the present application is administered to cells, tissues or subjects alone or in combination with other therapeutic drugs When, it is effective to prevent or ameliorate the symptoms of at least one disease or condition, or prevent or ameliorate the development of at least one disease or condition. A therapeutically effective dose can also refer to an amount of an antibody or antigen-binding fragment thereof sufficient to result in amelioration of symptoms (eg, treatment, cure, prevention or amelioration of an associated medical condition or an increase in the rate of treatment, cure, prevention or amelioration of such a condition). When the active ingredient (such as an antibody or antigen-binding fragment thereof) is administered to a subject alone, the therapeutically effective dose refers to that ingredient alone; The combined amount of all active ingredients for a therapeutic effect. An effective amount of a therapeutic agent will result in an increase in diagnostic criteria or parameters of at least 10%; usually at least 20%; preferably at least about 30%; more preferably at least 40%, most preferably at least 50%.

"Cancer" and "cancerous" refer to a physiological condition in mammals, often characterized by unregulated cell growth, and include within this definition benign tumors, malignant cancers, and dormant tumors or micrometastases. Examples of cancer 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, icteric liver tumors, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, and All types of head and neck cancer, as well as B-cell lymphomas (eg, low-grade/follicular non-Hodgkin lymphoma (NHL), small lymphocytic (SL) NHL, intermediate-grade/follicular NHL, intermediate-grade diffuse NHL, Immunoblastic NHL, lymphoblastic NHL, small anucleated cleaved NHL, storage disease NHL, mantle cell lymphoma, AIDS-related lymphoma, and Waldenstrom's macroglobulinemia) , chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia, post-transplant lymphoproliferative disorder (PTLD), and phakomatoses , edema associated with brain tumors and abnormal vascular proliferation associated with Meigs' syndrome (Meigs).

Antibody

The present application provides an antibody or an antigen-binding fragment thereof capable of specifically binding to 4-1BB, which comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3;

In some embodiments, according to the Kabat numbering system, the antibody or antigen-binding fragment thereof capable of specifically binding to 4-1BB comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3;

Wherein, 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 the amino acid sequence shown in SEQ ID NO:9;

A heavy compound 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 chain, and have 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 the light chain;

In some embodiments, the application provides an isolated antibody or antigen-binding fragment thereof capable of specifically binding to 4-1BB, and has at least one of the following properties:

(I) The antibody or antigen-binding fragment thereof according to the first aspect of the present application binds to the same epitope of human 4-1BB protein, or completely overlaps or partially overlaps;

(II) Competing with the antibody or antigen-binding fragment thereof described in the first aspect of the present application for binding to the epitope of human 4-1BB protein.

Wherein, the epitope that completely overlaps with the antibody of the present application or its antigen-binding fragment indicates that the antigenic epitope bound by the antibody includes the binding epitope of the antibody of the present application or its antigen-binding fragment; The partially overlapping epitope bound by the binding fragment means that a part of the epitope bound by the antibody is the same as a part of the binding epitope of the antibody or antigen-binding fragment thereof of the present application.

The precise amino acid sequence boundaries of the complementarity determining region CDR of an antibody can be defined according to known methods, such as Chothia based on the three-dimensional structure of the antibody and the topology of the CDR loop (Chothia et al., Nature 342:877-883, 1989; Al-Lazikani et al., Journal of Molecular Biology, 273:927-948, 1997); or Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th ed., 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); or based on affinity propagation clustering using a large number of crystal structures ) North CDR definition. The boundaries of the CDRs of the antibodies in the present application can be determined by those skilled in the art according to any scheme in the art (such as the optional definition method mentioned above).

It should be noted that the boundaries of the CDRs of the same antibody obtained based on different definition methods may be different, that is, the CDR sequences of the variable region of the same antibody obtained by different definition methods are different. Therefore, when specific CDR sequences as defined in this application are used to define an antibody, said antibody also includes sequences whose CDR sequences comprise the CDRs described in this application, only because of the different ways of defining CDR boundaries. Antibodies with CDR boundaries that differ from the specific CDR boundaries defined herein.

Antibodies with different specificities (ie, directed against different antigen combining sites) have different CDRs. However, although CDRs are different from antibody to antibody, only a limited number of amino acid positions within CDRs are directly involved in antigen binding, and the smallest overlapping region of antibody CDRs and antigen binding is also called the "minimum binding unit" for antigen binding , can be determined using at least two of the Kabat, IMGT, Chothia, AbM, Contact and North methods. The smallest binding unit can be a portion of a CDR. As will be apparent to those skilled in the art, the structure and protein folding of the antibody will determine the residues of the remainder of the CDR sequence, and therefore, this application also contemplates any CDR variant, e.g., in a CDR variant, the minimum The amino acid residues of the binding unit can remain unchanged, while the remaining CDR residues according to the Kabat or Chothia definition can be replaced by conservative amino acid residues.

For the humanized antibodies described herein, murine CDR regions can be inserted into human germline framework regions using methods known in the art. See US Patent No. 5,225,539 to Winter et al. and US Patent Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et al.

In some embodiments, amino acid differences may result from amino acid changes, including amino acid deletions, insertions, or substitutions. In some embodiments, the anti-4-1BB antibodies or antigen-binding fragments thereof of the present application include amino acid deletion, insertion or substitution mutations, but are still compatible with the antibody or antigen-binding fragments thereof (especially in the above sequence) Antibodies having amino acid sequences that are at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical in the described CDR regions).

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. Fc region variants may include human Fc region sequences (eg, human IgGl, IgG2, IgG3 or IgG4 Fc regions) with amino acid modifications (eg, substitutions) at one or more amino acid positions.

In some embodiments, the antibody or antigen-binding fragment thereof comprises a cysteine-engineered antibody or antigen-binding fragment thereof, such as a "thioMAb", wherein one or more of the antibody or antigen-binding fragment thereof residues were replaced by cysteine residues.

In some embodiments, the antibodies or antigen-binding fragments thereof may be further modified to contain other non-proteinaceous moieties known in the art and readily available, such as water-soluble polymers. Examples of such water-soluble polymers include, but are not limited to: polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymer, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1 , 3-dioxane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyamino acid (homopolymer or random copolymer), and dextran or poly(N-vinylpyrrole alk) polyethylene glycol, propylene glycol homopolymer, polypropylene oxide/ethylene oxide copolymer, polyoxyethylated polyols (eg glycerol), polyvinyl alcohol, and mixtures thereof.

Antibody expression

Another aspect of the present application provides a polynucleotide molecule, which comprises at least one of the nucleotide sequence encoding the antibody or antigen-binding fragment thereof provided in the present application or its complementary sequence. A polynucleotide molecule in the present application includes double-stranded or single-stranded DNA or RNA. The antibody or antigen-binding fragment thereof provided in the present application includes the antibody or antigen-binding fragment thereof in the first aspect of the application and the isolated antibody or antigen-binding fragment thereof in the second aspect of the application.

In some embodiments, the polynucleotide molecule encoding the antibody of the present application or an antigen-binding fragment thereof may also include a CDR that has a nucleotide deletion, insertion or substitution mutation, but is still consistent with the antibody of the present application or an antigen-binding fragment thereof The corresponding coding regions are polynucleotide sequences that are at least about 60%, 70%, 80%, 90%, 95%, or 100% identical.

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.

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.

In some embodiments, the host cells are used to express the antibodies or antigen-binding fragments thereof provided herein.

The mammalian host cells provided herein 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). Exemplary, it can include Chinese hamster ovary (CHO) cells, NSO, 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. Mammalian host cells can include human, mouse, rat, dog, monkey, pig, goat, bovine, horse and hamster cells. One skilled in the art can select particularly preferred cell lines by determining which cell lines have high expression levels.

In yet another aspect, the present application provides a method for preparing the antibody or antigen-binding fragment thereof of the present application, comprising expressing the antibody or antigen-binding fragment thereof in the host cell provided in the present application, and extracting the antibody or antigen-binding fragment thereof from the host cell The expressed antibody or antigen-binding fragment thereof is recovered.

Specifically, the application provides a method for preparing the antibody or antigen-binding fragment thereof of the application may include:

The polynucleotide molecules or expression vectors of the present application are introduced into the host cells described in the present application by known methods, such as lipofection, electroporation, transformation, etc.; Cultivating the host cell under the condition of fragment expression, so that the host cell expresses the antibody or its antigen-binding fragment; using methods known in the art, such as polyacrylamide gel electrophoresis, recovering the expressed antibody from the host cell Antibodies or antigen-binding fragments thereof.

It should be noted that due to the differences in the expression vectors or host cells used, those skilled in the art can choose the conditions suitable for the expression of the antibody or its antigen-binding fragment according to the specific situation, which is not limited in this application.

The host cell used to express the antibody or its antigen-binding fragment in the present application may be an isolated cell, or a cell that still exists in the host body, for example, different cell lines may be used to express the antibody or its antigen-binding fragment , or using transgenic animals to express the antibody or antigen-binding fragment thereof.

Antibodies or antigen-binding fragments thereof expressed by different cell lines or expressed 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 the application or comprising the amino acid sequences provided by the application are part of the application. Likewise, in certain embodiments, the antibody or antigen-binding fragment thereof is preferably a non-fucosylated antibody or antigen-binding fragment thereof. And in vivo than fucosylated antibodies or their antigen-binding fragments have stronger efficacy, in addition, because these different sugar structures are normal components of natural human serum IgG, so there is no significant difference in their immunogenicity.

pharmaceutical composition or pharmaceutical preparation

In yet another aspect, the present application provides a pharmaceutical composition (or called pharmaceutical preparation, preparation), which comprises the antibody or antigen-binding fragment thereof, polynucleotide molecule, expression vector or host cell provided in the present application, and pharmaceutically acceptable carrier or excipient.

It should be understood that the antibodies or pharmaceutical compositions thereof provided herein can be administered in combination with appropriate carriers, excipients and other agents in formulations, so as to provide improved transfer, delivery, tolerance and the like.

In the present application, the active ingredients in the pharmaceutical composition exist in a form with effective biological activity, and do not contain ingredients with unacceptable toxicity to the subject of the pharmaceutical composition.

In some embodiments of the present application, the antibody or antigen-binding fragment thereof of the present application having the required purity and one or more optional pharmaceutical excipients (see Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Editor, 1980) mixed to prepare the pharmaceutical composition of the present application, preferably, the pharmaceutical composition of the present application may be in the form of an aqueous solution or a lyophilized formulation.

The pharmaceutical composition or preparation of the present application may also contain one or more other active ingredients, which can be selected according to specific indications, preferably, the active ingredients will not adversely affect each other's activities. In some embodiments, 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 effective for the intended use Quantitative combinations exist.

In yet another aspect, the present application provides a kit comprising the antibody or antigen-binding fragment thereof, polynucleotide molecule, expression vector, host cell or pharmaceutical composition of the present application.

medical use

In yet another aspect, the present application provides the antibody or its antigen-binding fragment, polynucleotide molecule, expression vector or host cell or pharmaceutical composition of the present application in preparation for the treatment or prevention of 4-1BB-mediated diseases or disorders Uses in medicine.

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

In yet another aspect, the present application provides a method for treating or preventing a 4-1BB-mediated disease or disorder, comprising administering the antibody or its antigen-binding fragment, polynucleoside provided by the present application to a subject in need Acid molecule, expression vector, host cell or pharmaceutical composition.

In some embodiments, modes of administration include, but are not limited to, oral, intravenous, subcutaneous, intramuscular, intraarterial, intraarticular (e.g., in arthritic joints), inhalation drug delivery, aerosol delivery or intratumoral administration, etc.

In some embodiments, the present application also provides a combination therapy, which includes combining the antibody of the present application or an antigen-binding fragment thereof and combining one or more other therapies or therapeutic agents to a subject. In some embodiments, the therapy may include surgery and/or radiation therapy. In some embodiments, the therapeutic agent may include PD-1 antibody, CD4 antibody and/or CTLA-4 antibody, etc.

Methods for diagnosis and detection

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, and detecting the binding of the antibody or antigen-binding fragment thereof The binding of the fragment to 4-1BB, and optionally quantifying said binding. The term "detection" as used in this application 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 sample of biological origin. In certain embodiments, a biological sample comprises cells or tissues. This application does not limit the method for detecting the conjugate formed by the antibody or its antigen-binding fragment and 4-1BB; this application does not limit the method for "quantitatively determining the conjugate".

In order to make the purpose, technical solution, and advantages of the present application clearer, the present application will be further described in detail below with reference to the accompanying drawings and examples. Apparently, the described embodiments are only some of the embodiments of this application, not all of them. All other embodiments obtained by persons of ordinary skill in the art based on the embodiments in this application belong to the protection scope of this application.

Embodiment 1, the screening of anti-4-1BB antibody

1.1 Establishment of phage library

50 mL of peripheral blood from 40 different individuals were mixed together to separate peripheral blood mononuclear cells (PBMCs). Total RNA was extracted with Trizol, and the first strand of cDNA was synthesized with a reverse transcription kit (Thermo, 18080051). Using the synthesized cDNA as a template, the variable region VH of the antibody heavy chain, the variable regions Vκ and Vλ of the antibody light chain κ chain and λ chain were amplified by PCR. VH-CH1, Vκ-CK, and Vλ-Cλ were assembled through the first round of overlapping PCR (overlap PCR), and then assembled into Fab through the second round of overlapping PCR. The Fab fragments were digested and ligated into phagemid vectors. The constructed plasmid vector was electroporated into TG1 competent cells to obtain a phage display library.

1.2 Phage library screening

The constructed phage display library was packaged into phage. In the first round of panning, 10 ¹³ -10 ¹⁴ phages were selected and added to biotinylated 4-1BB for incubation and binding, and then streptavidin-coupled magnetic beads were added to bind to 4-1BB, and the non-binding phages were first washed with PBST. The phages bound to 4-1BB were then eluted with glycine at pH 2.0 to infect TG1, and the phages were amplified and packaged for the second round of panning. After four rounds of panning, the phages in the fourth round were infected with TG1 and plated into monoclonals. The monoclonals were picked and placed in 96 deep-well plates, and their expression was induced by IPTG (isopropylthiogalactopyranoside). The next day, the periplasmic cavity supernatant was extracted for 4-1BB combined enzyme-linked immunosorbent assay (ELISA) analysis. Positive clones were sequenced by sequencing. Finally, two groups of phages were obtained, which were named phage3 and phage6 for further analysis. The antibody expressed by phage phage3 was named JS011-phage3; the antibody expressed by phage phage6 was named JS011-phage6.

Example 2, Molecular Construction and Production of Anti-4-1BB Antibody

The coding sequences of IgG4 heavy chain constant region (hIgG4CH1-CH3) and kappa light chain constant region (human Kappa LC) were cloned from human B lymphocytes (from Beijing Institute of Blood) and introduced into pTT5 plasmid to form vectors HXT4S and HXT2.

The main elements of the vector HXT4S include: BSPQI restriction site, CMV Promoter (promoter for expression of the target gene), AmpR (amphicillin resistance gene), pMB1ori (origin of replication), orip, signal peptide (promoter for expression of the target gene) signal peptide) and the target gene hIgG4CH1-CH3.

The main elements of the vector HXT2 include: BSPQI restriction site, CMV Promoter (promoter for expression of the target gene), AmpR (amphicillin resistance gene), pMB1ori (origin of replication), orip, signal peptide (promoter for expression of the target gene) signal peptide) and target gene human Kappa LC.

2.1 Construction of JS011-phage3 light chain expression vector

Infect the TG1 strain with phage3, and use the plasmid extracted after shaking the bacteria as a template to synthesize head and tail primers, as shown in SEQ ID No: 21 and SEQ ID No: 22 in Table 1 below, perform PCR, and the obtained PCR product is digested and ligated by SapI to the HXT2 vector to obtain the expression vector HXT2-JS011-3-LC.

2.2 Construction of JS011-phage3 heavy chain expression vector

The TG1 strain was infected with phage3, and the plasmid extracted after shaking the bacteria was used as a template to synthesize head-to-tail primers, as shown in SEQ ID No: 23 and SEQ ID No: 24 in Table 1 below, and PCR was performed, and the obtained PCR product was digested and ligated by SapI to the HXT4S vector to obtain the expression vector HXT4S-JS011-3-HC.

2.3 Construction of JS011-phage6 light chain expression vector

The TG1 strain was infected with phage6, and the plasmid extracted after shaking the bacteria was used as a template to synthesize head-to-tail primers, as shown in SEQ ID No: 25 and SEQ ID No: 26 in Table 1 below, and PCR was performed, and the obtained PCR product was digested and ligated by SapI to the HXT2 vector to obtain the expression vector HXT2-JS011-6-LC.

2.4 Construction of JS011-phage6 heavy chain expression vector

Infect the TG1 strain with phage6, and use the plasmid extracted after shaking the bacteria as a template to synthesize head and tail primers, as shown in SEQ ID No: 23 and SEQ ID No: 24 in Table 1 below, perform PCR, and the obtained PCR product is digested and ligated by SapI to the HXT4S vector to obtain the expression vector HXT4S-JS011-6-HC.

Table 1

引物名称Primer name	引物序列Primer sequence
JS011-3 VL F BSPQI：JS011-3 VL F BSPQI:	gatcgctcttcatgtgagctcgagctgactcagccaccctca(SEQ ID No:21)gatcgctcttcatgtgagctcgagctgactcagccaccctca (SEQ ID No: 21)
JS011-3 VL R BSPQI：JS011-3 VL R BSPQI:	gatcgctcttcttcgtaggacggtcagtctggtccctccgccga(SEQ ID No:22)gatcgctcttcttcgtaggacggtcagtctggtccctccgccga (SEQ ID No: 22)
JS011-3/6 VH F BSPQIJS011-3/6 VH F BSPQI	gatcgctcttcatctcaggtgcagctggtgcagtctggggct(SEQ ID No:23)gatcgctcttcatctcaggtgcagctggtgcagtctggggct (SEQ ID No: 23)
JS011-3/6 VH R BSPQIJS011-3/6 VH R BSPQI	atgcgctcttctagctgaggagacggtgaccagggttccctgg(SEQ ID No:24)atgcgctcttctagctgaggagacggtgaccagggttccctgg (SEQ ID No: 24)
JS011-6 VL F BSPQI：JS011-6 VL F BSPQI:	atgcgctcttcatgtgagctcgccctgactcagcctccctccg(SEQ ID No:25)atgcgctcttcatgtgagctcgccctgactcagcctccctccg (SEQ ID No: 25)
JS011-6 VL R BSPQI：JS011-6 VL R BSPQI:	atgcgctcttcttcgtaggacggtcagcttggtccctccgccg(SEQ ID No:26)atgcgctcttcttcgtaggacggtcagcttggtccctccgccg (SEQ ID No: 26)

2.5 Expression and purification of JS011-phage3 and JS011-phage6 antibodies

CHO-K1 cells (ATCC, CCL-61) were acclimated and screened in suspension without serum to make the cells suitable for transient expression, named CHO-18, and used CD CHO medium (Gibco) for CHO-18 cells at 36.5°C , 120rmp, 7% CO ₂ conditions, and when the cell density reaches (2-6)×10 ⁶ /mL, CD CHO medium is used for passage expansion. The day before transfection, dilute the CHO-18 cell density to (1.5-2.0)×10 ⁶ /mL, and place it on a shaker (36.5°C, 120rmp, 7% CO ₂ ) for culture. When the cell density reaches about Transfection was carried out at 3.5×10 ⁶ /mL. When transfecting, first add one-tenth of the transfection volume of CD CHO medium to the reactor, and then add the above expression vectors HXT2-JS011-3-LC and HXT4S-JS011-3 in order of 1-2ug/mL transfection volume -HC, or HXT4S-JS011-3-HC and HXT4S-JS011-6-HC, 3-14ug/mL of PEI (polyethyleneimine), mix well and incubate at room temperature for 20-30min, then slowly add the resulting mixture Add to the pre-treated CHO-18 cells to form a transfection mixture, and mix well while adding. The transfection mixture was cultured on a shaker at 36.5° C., 120 rpm, and 7% CO ₂ . The culture period is 6-10 days after transfection, with feeding every two days.

After the above transfection mixture was cultured, centrifuge at 1000rmp for 10min to discard the precipitate, then centrifuge at 12000rmp for 30min to collect the cell supernatant and perform sterile filtration. The first step is to purify with the AKTA Avant purification instrument. First, sterilize the packed mabselect sure LX column with 0.1M NaOH for 15-20 minutes, then equilibrate with PBS buffer for 3-5 column volumes and load the sample. After the sample is loaded, Rinse with pH 5.5 sodium acetate buffer, and finally elute the target protein with pH 3.6 acetic acid-sodium acetate buffer; the second step, use Eshmuno CPX for purification, the balance solution is pH 5.5, 50mM acetic acid-acetic acid Sodium system, the eluent is pH5.5, 50mM acetic acid-sodium acetate + 1M NaCl buffer system, the target protein is collected by linear elution, and the purity of the monomer can reach more than 95% as determined by SEC-HPLC. Antibodies JS011-phage3 and JS011-phage6 were cloned.

The gene sequences of the 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.

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

Heavy chain (JS011-phage3-HC, italics indicate VH, underline indicates HCDR1, HCDR2 and HCDR3):

These include the heavy chain variable region JS011-phage3-VH:

Among them, according to the Kabat numbering system, include:

HCDR1:GYYMH (SEQ ID NO:1);

HCDR2:WINPQSGGTNYAQKFQG (SEQ ID NO:2);

HCDR3: EGGEWLAIPFDY (SEQ ID NO: 3);

Light chain (JS011-phage3-LC, italics indicate VL, underscore indicates LCDR1, LCDR2 and LCDR3):

These include the light chain variable region JS011-phage3-VL:

Among them, according to the Kabat numbering system, include:

LCDR1: SADKLGDKYAS (SEQ ID NO: 4);

LCDR2: EDSKRPS (SEQ ID NO: 5);

LCDR3:QTWDTNTVL (SEQ ID NO:6).

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

Heavy chain (JS011-phage6-HC, italics indicate VH, underline indicates HCDR1, HCDR2 and HCDR3):

These include the heavy chain variable region JS011-phage6-VH:

Among them, according to the Kabat numbering system, include:

HCDR1:GQYIH (SEQ ID NO:7);

HCDR2:WINPNSGGTNYAQKFQG (SEQ ID NO:8);

HCDR3:DKAGADY (SEQ ID NO:9);

Light chain (JS011-phage6-LC, italics indicate VL, underscore indicates LCDR1, LCDR2 and LCDR3):

These include the light chain variable region JS011-phage6-VL:

Among them, according to the Kabat numbering system, include:

LCDR1: TGTSSDIGGYDYVS (SEQ ID NO: 10);

LCDR2: GVSNRPS (SEQ ID NO: 11);

LCDR3: SSYTSRSTRWV (SEQ ID NO: 12).

Example 3, detection of binding ability of anti-4-1BB antibody

3.1 Elisa detects the binding ability of the candidate antibody to 4-1BB protein:

Using a Thermo Scientific microplate reader, use a fixed concentration of human 4-1BB containing a His tag (the 4-1BB extracellular region sequence is fused with a His tag to construct an expression plasmid, and obtained by transient expression and purification of 293 cells) or a human 4-1BB containing a His tag Rat 4-1BB (manufacturer Sino Biological, product number 50811-M08H) antigen (1.0 μg/mL) coated plate, 2% BSA after blocking, adding serially diluted JS011 antibodies JS011-phage3 and JS011-phage6 (with human 4-1BB Binding: starting at 1 μg/ml, 3-fold serial dilution, a total of 12 concentrations; binding to mouse 4-1BB: 1 μg/ml starting, 2.5-fold serial dilution, a total of 12 concentrations); mouse anti-human IgG4 Fc HRP ( Southern Biotech, 9200-05) was diluted 5000 times as a detection antibody for detection, then 0.1mg/ml TMB (3,3',5,5'-tetramethylbenzidine) was used for color development, and finally the reaction was terminated with 2M HCl, Plates were read at 450nm/620nm. The median effect concentration _EC50 was fitted using a four-parameter logarithmic regression (4PL) model. Anti-KLH-IgG4 (CB25772663, or the gene sequence encoding anti-hemocyanin KLH human IgG4 antibody with light weight was used to construct expression plasmids, and obtained by expressing and purifying through transient CHO-18 cells) as a negative control.

The binding curves of JS011-phage3 and JS011-phage6 to human 4-1BB are shown in FIG. 1 , and the binding curves of JS011-phage3 and JS011-phage6 to mouse 4-1BB are shown in FIG. 2 . It can be seen from Figure 1 that both JS011-phage3 and JS011-phage6 can bind to human 4-1BB with similar binding activity, with _EC50 of 1.5ng/ml and 1.6ng/ml respectively; as can be seen from Figure 2, JS011-phage3 can bind to mouse 4-1BB with an EC ₅₀ of 6.1ng/ml, and JS011-phage6 does not bind to mouse 4-1BB.

Example 4: Binding of anti-4-1BB antibodies to Jurkat NFKB 4-1BB cells

Jurkat NFKB 4-1BB cells (stable expression of 4-1BB, NFKB fused with a luciferase reporter gene to construct an expression plasmid, and electrotransfected into Jurkat cells, and pressurized selection to obtain Jurkat cells stably expressing NFKB, named Jurkat NFKB cells Then construct the expression plasmid of the full-length 4-1BB gene, transfer it to Jurkat NFKB cells, and obtain Jurkat NFKB4-1BB stably expressing 4-1BB) and different concentrations of anti-4-1BB monoclonal antibody JS011-phage3 and JS011-phage6 (initial concentration 100 μg/ml, 3-fold dilution, a total of 12 concentration gradients) was incubated at 4°C for 30 min, then washed and mixed with fluorescein PE-labeled anti-human IgG secondary antibody (SouthernBiotech, Cat#2040- 09) Incubate at 4°C for 30 minutes in the dark. Finally, the cells were collected by flow cytometry (BD Canto Ⅱ), and the fluorescent antibodies bound to the cell surface were detected. Use FlowJo to analyze the raw data to obtain the MFI value (denoted as MFI-PE), and fit the antibody dose-dependent binding curve through GraphPad, the results are shown in Figure 3, and calculate the EC ₅₀ , where the positive control is Urelumab (BMS control antibody , see patent CN1867585B) and Utomilumab (Pfizer control antibody, see patent CN103221428B) antibody; the negative control is Anti-KLH-IgG4.

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

Example 5: Anti-4-1BB Antibody Blocks Binding of 4-1BB Ligand (4-1BBL) to Jurkat NFKB 4-1BB Cells

Jurkat NFKB 4-1BB cells were mixed with different concentrations of anti-4-1BB monoclonal antibodies JS011-phage3 and JS011-phage6 (initial concentration was 100 μg/ml, diluted 3 times, a total of 12 concentration gradients) and 2 μg/ml biotin Labeled 4-1BBL-hFc (for the 4-1BB ligand, the 4-1BBL extracellular region sequence was fused with the human FC (hFC) sequence to construct an expression plasmid, and purified by transient expression in CHO-18 cells) was incubated at 4°C 30min, then washed and incubated with fluorescein FITC-labeled secondary antibody (BioLegend, Cat#405202) at 4°C in the dark for 30min. Finally, the cells were collected by flow cytometry (BD Canto Ⅱ), and the fluorescent antibodies bound to the cell surface were detected. Use FlowJo to analyze the raw data to get the MFI value (denoted as MFI-FITC), and use GraphPad to fit the antibody dose-dependent inhibition curve and calculate the IC ₅₀ , the results are shown in Figure 4, where the positive control is Urelumab (BMS control antibody, See patent CN1867585B) and Utomilumab (Pfizer control antibody, see patent CN103221428B); the negative control is Anti-KLH-IgG4.

As shown in Figure 4, none of JS011-phage3, JS011-phage6, and Urelumab antibodies could block the binding of 4-1BBL to 4-1BB on the surface of Jurkat NFKB 4-1BB cells, and Utomilumab could effectively block the binding of 4-1BBL to Jurkat NFKB 4-1BB 4-1BB binding on the surface of 1BB cells with an IC ₅₀ of 0.3431 μg/ml.

Example 6: Activity detection of anti-4-1BB antibody in luciferase reporter gene system (CHO FCGR2B 4G6/Jurkat NFKB 4-1BB double cells)

CHO FCGR2B 4G6 cells (stable and high expression of human CD32B, CD32B gene expression plasmid was constructed, and electrotransfected into CHO-K1 cells, monoclonal cells stably expressing CD32B were obtained by pressurized screening, named CHO FCGR2B 4G6 cells) according to each Add 3×10 ⁴ cells into a 96-well flat-bottomed white plate (Corning, Cat#3917), and incubate overnight in a 37°C incubator. The next day, the effector cells Jurkat NFKB 4-1BB (stable expression of NlucP/NFKB-RE and 4-1BB) were added to the cell plate at 1×10 ⁵ cells per well. Afterwards, the anti-4-1BB monoclonal antibodies JS011-phage3 and JS011-phage6 (initial concentration of 25 μg/ml, 4-fold dilution, a total of 9 concentration gradients were mixed with the experimental buffer (RPMI 1640 (1×)+2% FBS) ) into the cell plate and co-incubate for 4-6 hours in a 37°C incubator. Finally, Nano-Glo luciferase detection reagent (Promega) was added to the cell-antibody mixture system, and the chemiluminescent signal (denoted as RLU) was detected with a multifunctional microplate reader (TECAN M1000pro). The four-parameter regression curve was fitted by GraphPad prism software, and the EC ₅₀ value was calculated, and the results are shown in Figure 5. Positive controls are Urelumab (BMS control antibody, see patent CN1867585B) and Utomilumab (Pfizer control antibody, see patent CN103221428B).

As shown in Figure 5, JS011-phage3, JS011-phage6, Urelumab and Utomilumab have a strong activity of activating T cells in the luciferase reporter gene system composed of CHO FCGR2B 4G6 cells and effector cells Jurkat NFKB 4-1BB, EC ₅₀ were 0.03888μg/mL, 0.4499μg/mL, 1.038μg/mL and 0.02391μg/mL respectively, JS011-phage3 and Utomilumab had similar activity; JS011-phage6 had similar activity to Urelumab, and JS011-phage3 had a stronger activation ability on T cells than JS011-phage6.

Example 7: Activity detection of anti-4-1BB antibody in luciferase reporter gene system (THP-1/Jurkat NFKB 4-1BB double cell)

THP-1 cells (a kind of human myeloid leukemia mononuclear cells, positive for CD32B) and effector cells Jurkat NFKB 4-1BB (stable expression of NlucP/NFKB-RE and 4-1BB) were divided into 5× ¹⁰⁴ cells per well Count and add 1×10 ⁵ cells per well into a 96-well flat-bottomed white plate (Corning, Cat#3917). Afterwards, the anti-4-1BB monoclonal antibodies JS011-phage3 and JS011-phage6 (initial concentration of 100 μg/ml, 4-fold dilution, a total of 10 concentration gradients were mixed with the experimental buffer (RPMI 1640 (1×)+2% FBS) ) into the cell plate and co-incubate for 4-6 hours in a 37°C incubator. Finally, Nano-Glo luciferase detection reagent (Promega) was added to the cell-antibody mixture system, and the chemiluminescent signal (denoted as RLU) was detected with a multifunctional microplate reader (TECAN M1000pro). The four-parameter regression curve was fitted by GraphPad prism software, and the EC ₅₀ value was calculated. The results are shown in Figure 6. Positive controls are Urelumab (BMS control antibody, see patent CN1867585B) and Utomilumab (Pfizer control antibody, see patent CN103221428B).

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

Example 8: Activity detection of anti-4-1BB antibody in luciferase reporter gene system (Raji/Jurkat NFKB 4-1BB double cell or Raji/Jurkat NFKB double cell)

Add Raji cells (a kind of human myeloid leukemia mononuclear cells, positive for CD32B, positive for 4-1BBL) into ⁹⁶ -well flat-bottomed white plate (Corning, Cat#3917), Jurkat NFKB 4- 1BB cells (stable expression of NlucP/NFKB-RE and 4-1BB) or Jurkat NFKB cells (stable expression of NlucP/NFKB-RE) were added to the cell plate according to the number of 1×10 ⁵ cells per well. Afterwards, the anti-4-1BB monoclonal antibodies JS011-phage3 and JS011-phage6 (initial concentration of 100 μg/ml, 4-fold dilution, a total of 10 concentration gradients were mixed with the experimental buffer (RPMI 1640 (1×)+2% FBS) ) into the cell plate and co-incubate for 4-6 hours in a 37°C incubator. Finally, Nano-Glo luciferase detection reagent (Promega) was added to the cell-antibody mixture system, and the chemiluminescent signal (denoted as RLU) was detected with a multifunctional microplate reader (TECAN M1000pro). The four-parameter regression curve was fitted by GraphPad prism software, and the EC ₅₀ value was calculated. The results are shown in Figure 7 and Figure 8, respectively. Among them, the positive controls are Urelumab (BMS control antibody, see patent CN1867585B) and Utomilumab (Pfizer control antibody, see patent CN103221428B).

As shown in Figure 7, JS011-phage3, JS011-phage6 and Urelumab have the activity of activating T cells in the luciferase reporter gene system composed of Raji cells and effector cells Jurkat NFKB 4-1BB (4-1BB high expression), EC ₅₀ are 9.446μg/mL, 2.713μg/mL and 7.240μg/mL respectively, Utomilumab has the effect of blocking 4-1BBL binding in this system and thus shows a decrease in activity.

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 interact with 4-1BBL on the surface of Raji cells to activate T cells. To the synergistic effect, the EC ₅₀ were 9.450μg/mL and 86.01μg/mL, respectively. JS011-phage6 and Utomilumab were inactive in this system.

Example 9: Anti-4-1BB Antibody Cytokine Storm Study

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

As shown in Figure 9A to Figure 9F, at a concentration of 10 μg/mL, none of JS011-phage3, JS011-phage6, Urelumab and Utomilumab caused a cytokine storm.

Example 10: Inhibitory Effect of Anti-4-1BB Antibody on the Growth of MC38 Tumor Transplanted in h41BB Humanized Mice

1. Purpose of test

Evaluate the anti-tumor effect of the anti-4-1BB monoclonal antibody of the present application in the subcutaneous tumor model of colon cancer MC38 transplanted into h41BB humanized mice.

2. Test process

6-8 week-old female h41BB humanized mice (purchased from Biocytogen Jiangsu Gene Biotechnology Co., Ltd.) were subcutaneously inoculated with 1×10 ⁶ MC38 cells on the right back. When the average tumor volume is about 90 mm ³ , select suitable animals and randomly divide them into 5 groups according to the tumor volume, with 6 animals in each group. respectively

1. Normal saline control group (solvent control group);

therapy group:

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

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

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

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

Intraperitoneal injection, 2 times a week, continuous administration for 3 weeks, 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 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 0th day of administration; Vi: mean tumor volume of the solvent control group on the i-th day of administration, V0: solvent The average tumor volume of the control group on the 0th day of administration).

As shown in Figure 10, on the 20th day after administration, the average tumor volume of the normal saline control group was 2213mm ³ , and the average tumor volume of the Urelumab (BMS, see patent CN1867585B) group was 430mm ³ , compared with the normal saline control group, Significantly inhibited tumor growth, the tumor inhibition rate was 83.9%; the average tumor volume of the Utomilumab (Pfizer, see patent CN103221428B) group was 1937mm ³ , no obvious tumor inhibition effect was seen, and the tumor inhibition rate was 13.0%; the average tumor volume of the JS011-phage3 group The tumor volume was 506mm ³ , significantly inhibited tumor growth, and the tumor inhibition rate was 64.7%; the average tumor volume of JS011-phage6 group was 674mm ³ , significantly inhibited tumor growth, and the tumor inhibition rate was 50.6%. The results showed that both JS011-phage3 and JS011-phage6 exhibited significant tumor suppressive effects at a dose level of 1 mg/kg in h41BB humanized mice transplanted with MC38WT.

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

1. Purpose of test

To evaluate the toxic effect of the anti-4-1BB monoclonal antibody of the present application in h41BB humanized mice transplanted with subcutaneous colon cancer MC38 cells in tumor-bearing mice.

2. Test process

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

1. Anti-KLH-IgG4 (marked as Anti KLH hIgG4 in the figure, negative control group);

therapy group:

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

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

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

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

Intraperitoneal injection, 2 times a week, 3 consecutive times of administration, euthanized animals 7 days after the last administration. Weigh the mouse spleen weight. The mouse liver was collected, and the ratio of CD3 ⁺ , CD3 ⁺ CD4 ⁺ , CD3 ⁺ CD8 ⁺ , CD3 ⁺ CD8 ⁺ CD11c ⁺ and CD3 ^- NK1.1 ⁺ cells in the liver of different groups of animals was detected by flow cytometry to observe the proportion of anti-4- In vivo toxicity of 1BB monoclonal antibody.

As shown in Figure 11, on the 14th day after administration, compared with the Anti KLH hIgG4 negative control group, the spleen weight of the mice in the Urelumab (BMS, see patent CN1867585B) group increased significantly, and the mice in the JS011-phage3 group, JS011-phage6 group and Utomilumab (Pfizer, refer to patent CN103221428B) group mice spleen weight no significant change. It shows that 10mg/kg Urelumab can cause spleen enlargement in mice, and JS011-phage3 and JS011-phage6 will not cause spleen enlargement in animals. The results suggest that JS011-phage3 and JS011-phage 6 of the present application will not cause excessive activation of immune cells in the spleen, and may have better safety in clinical practice.

As shown in Figure 12A, the number of lymphocytes contained in the liver unit weight was analyzed on the 14th day after administration. Compared with the Anti KLH hIgG4 negative control group, the JS011-phage3 group, JS011-phage6 group, Utomilumab group and Urelumab group were smaller The number of lymphocytes in the mouse liver increased, and the number of lymphocytes in the mouse liver of the Urelumab group was significantly higher than that of the JS011-phage3 group, JS011-phage6 group and Utomilumab group, indicating that 10mg/kg of Urelumab would lead to a significant increase in the number of lymphocytes in the mouse liver Increase. As shown in Figure 12B to Figure 12E, the changes in the proportions of different subsets of liver lymphocytes were analyzed on the 14th day after administration. Compared with the Anti KLH hIgG4 negative control group, the JS011-phage3 group, JS011-phage6 group, Utomilumab group and The proportion of CD3 ⁺ CD4 ⁺ T cells in the liver of mice in Urelumab group decreased, while the proportion of CD3 ⁺ CD8 ⁺ T cells increased, and the proportion of CD3 ⁺ CD8 ⁺ T cells in Urelumab group was significantly higher than that in JS011-phage3 group, JS011-phage6 group and Utomilumab group, The proportion of CD3 ⁺ CD8 ⁺ CD11c ⁺ T cells in the liver of mice in the Urelumab group was significantly increased, indicating that the proportion of overactivated CD3 ⁺ CD8 ⁺ T cells in the liver of mice in the Urelumab group was significantly increased, while the proportion of CD3 ^- NK1 in the liver of mice in the Urelumab group was significantly increased. The proportion of .1 ⁺ NK cells decreased, indicating that 10mg/kg Urelumab would lead to a significant increase in the proportion of CD3 ⁺ CD8 ⁺ T cells in the liver of mice. The increase in the number of lymphocytes in the mouse liver was due to a significant increase ⁱⁿ the absolute number of CD3 ⁺ CD8 ⁺ T cells, and an increase in the absolute number of CD3 ⁺ CD4 ⁺ T ^and NK cells, but the CD3 ⁺ The proportion of 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 are significantly better than Urelumab, suggesting that there may be lower liver toxicity in clinical practice.

The above are only preferred embodiments of the application, and are not intended to limit the application. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the application shall be included in the protection of the application. within the range.

Claims

An antibody or antigen-binding fragment thereof capable of specifically binding to 4-1BB comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3;

Wherein according to Kabat, IMGT, Chothia, AbM, Contact or North numbering system, the aminoacid sequence of described HCDR1, HCDR2 and HCDR3 is HCDR1, HCDR2 and HCDR3 of heavy chain variable region as shown in SEQ ID NO:13 or 15, And the amino acid sequence of the LCDR1, LCDR2 and LCDR3 is LCDR1, LCDR2 and LCDR3 of the light chain variable region shown in SEQ ID NO: 14 or 16, and in the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 At least one of can be substituted with a variant having 1, 2 or 3 amino acid differences therefrom.
The antibody or antigen-binding fragment thereof according to claim 1, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3;

Wherein according to Kabat, IMGT, Chothia, AbM, Contact or North numbering system, the aminoacid sequence of described HCDR1, HCDR2 and HCDR3 is HCDR1, HCDR2 and HCDR3 of heavy chain variable region as shown in SEQ ID NO:13, and said The amino acid sequence of LCDR1, LCDR2 and LCDR3 is LCDR1, LCDR2 and LCDR3 of the light chain variable region shown in SEQ ID NO: 14; or

The amino acid sequence of the HCDR1, HCDR2 and HCDR3 is HCDR1, HCDR2 and HCDR3 of the heavy chain variable region shown in SEQ ID NO: 15, and the amino acid sequence of the LCDR1, LCDR2 and LCDR3 is as SEQ ID NO: 16 LCDR1, LCDR2 and LCDR3 of the light chain variable region are shown.
The antibody or antigen-binding fragment thereof according to claim 1, wherein, according to the Kabat numbering system,

The HCDR1, HCDR2 and HCDR3 have amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 respectively, or have amino acid sequences such as SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 8 and SEQ ID NO: 3 respectively. The amino acid sequence shown in ID NO:9;

Said LCDR1, LCDR2 and LCDR3 respectively have the amino acid sequence shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, or respectively have such as SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:11 and SEQ The amino acid sequence shown in ID NO:12;

Wherein, at least one of said SEQ ID NO: 1-12 can be replaced with a variant having 1, 2 or 3 amino acid differences.
The antibody or antigen-binding fragment thereof according to claim 3, wherein, 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 have the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:4 and SEQ ID NO:3 respectively. The amino acid sequence shown in ID NO:5 and SEQ ID NO:6; or

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: 10, SEQ ID NO: Amino acid sequences shown in ID NO:11 and SEQ ID NO:12.
The antibody or antigen-binding fragment thereof according to claim 4, comprising:

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

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

Preferably, the antibody or antigen-binding fragment thereof comprises:

a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 13, and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 14; or

The amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 15, and the amino acid sequence of the light chain variable region shown in SEQ ID NO: 16.
The antibody or antigen-binding fragment thereof according to any one of claims 1-5, further comprising a human heavy chain constant region and a human light chain constant region, the human heavy chain constant region being selected from the group consisting of human IgG1, IgG2, IgG3 Or the heavy chain constant region of IgG4, preferably the heavy chain constant region of human IgG4 or the heavy chain constant region of human IgG4 with S228P amino acid substitution; the human light chain constant region is selected from the constant light chain of lambda light chain or kappa light chain district.
The antibody or antigen-binding fragment thereof according to any one of claims 1-5, comprising:

A heavy compound 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: 17 chain, and have 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: 18 light chain; or

A heavy compound 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 chain, and have 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 the light chain;

Preferably, the antibody or antigen-binding fragment thereof comprises:

a heavy chain having an amino acid sequence as set forth in SEQ ID NO: 17, and a light chain having an amino acid sequence as set forth in SEQ ID NO: 18; or

The amino acid sequence is the heavy chain shown in SEQ ID NO:19, and the amino acid sequence is the light chain shown in SEQ ID NO:20.
The antibody or antigen-binding fragment thereof according to any one of claims 1-5, wherein the antibody comprises at least one of a monoclonal antibody and a multispecific antibody, and the antigen-binding fragment comprises Fab, Fab', At least one of F(ab')2, Fv, scFv and sdAb.
An isolated antibody or antigen-binding fragment thereof having at least one of the following properties:

(1) The antibody or antigen-binding fragment thereof according to any one of claims 1-8 binds to the same epitope of human 4-1BB protein, or completely overlaps or partially overlaps;

(II) Competing with the antibody or antigen-binding fragment thereof according to any one of claims 1-8 for binding to the epitope of human 4-1BB protein.
A polynucleotide molecule comprising a nucleotide sequence encoding the antibody or antigen-binding fragment thereof of any one of claims 1-8 or encoding the isolated antibody or antigen-binding fragment thereof of claim 9 or At least one of its complementary sequences.
An expression vector comprising the polynucleotide molecule according to claim 10, preferably, the expression vector is a eukaryotic expression vector.
A host cell comprising the polynucleotide molecule according to claim 10, or comprising the expression vector according to claim 11, preferably, the host cell is a eukaryotic cell, more preferably a mammalian cell.
The host cell according to claim 12 for expressing the antibody or antigen-binding fragment thereof according to any one of claims 1-8 or the isolated antibody or antigen-binding fragment thereof according to claim 9 .
A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1-8, or the isolated antibody or antigen-binding fragment thereof according to claim 9, which is included in claim 12 expressing the antibody or antigen-binding fragment thereof in a host cell, and recovering the expressed antibody or antigen-binding fragment thereof from the host cell.
A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1-8 or the isolated antibody or antigen-binding fragment thereof according to claim 9 or according to claim 10 The polynucleotide molecule or the expression vector according to claim 11 or the host cell according to claim 12, and a pharmaceutically acceptable carrier or excipient.
The antibody or antigen-binding fragment thereof according to any one of claims 1-8 or the isolated antibody or antigen-binding fragment thereof according to claim 9 or the polynucleotide molecule according to claim 10 or according to claim 9 Use of the expression vector according to claim 11 or the host cell according to claim 12 or the pharmaceutical composition according to claim 15 in the preparation of medicines for treating or preventing 4-1BB-mediated diseases; preferably , the disease mediated by 4-1BB is cancer; more preferably, the cancer is selected from: melanoma, breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, kidney cancer, lung cancer, liver cancer, gastric cancer, colorectal cancer Cancer, bladder cancer, head and neck cancer, thyroid cancer, esophageal cancer, cervical cancer, sarcoma, multiple myeloma, leukemia, lymphoma, gallbladder cancer, and glioblastoma.
A kit comprising the antibody or antigen-binding fragment thereof according to any one of claims 1-8 or the isolated antibody or antigen-binding sheet thereof according to claim 9 or the antibody or antigen-binding fragment thereof according to claim 10 The polynucleotide molecule or the expression vector according to claim 11 or the host cell according to claim 12 or the pharmaceutical composition according to claim 15.
A method of detecting 4-1BB using the antibody or antigen-binding fragment thereof according to any one of claims 1-8, or the isolated antibody or antigen-binding fragment thereof according to claim 9, comprising making the contacting the antibody or antigen-binding fragment thereof with a sample, detecting binding of the antibody or antigen-binding fragment thereof to 4-1BB, and optionally quantifying the binding.