CN116199778A

CN116199778A - Anti-4-1 BB antibodies and uses thereof

Info

Publication number: CN116199778A
Application number: CN202211543663.9A
Authority: CN
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: Shanghai Junshi Biosciences Co Ltd; Suzhou Junmeng Biosciences Co Ltd
Current assignee: Shanghai Junshi Biosciences Co Ltd; Suzhou Junmeng Biosciences Co Ltd
Priority date: 2021-12-01
Filing date: 2022-12-01
Publication date: 2023-06-02
Also published as: WO2023098785A1

Abstract

The present application provides antibodies or antigen-binding fragments thereof capable of specifically binding to 4-1BB and pharmaceutical compositions comprising the same, as well as polynucleotide molecules encoding the antibodies or antigen-binding fragments thereof, expression vectors and host cells for expressing the antibodies or antigen-binding fragments thereof, and uses of the antibodies or antigen-binding fragments thereof.

Description

Anti-4-1 BB antibodies and uses thereof

The present application claims priority from the chinese patent office, application number 202111455972.6, entitled "anti-4-1 BB antibody and use thereof," filed on month 12 and 01 of 2021, the entire contents of which are incorporated herein by reference.

Technical Field

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

Background

Tumor immunotherapy is a treatment that restores or enhances the natural defenses against tumors through the human immune system. Such treatment is typically directed to specific biomolecules, such as tumor-associated antigens (TAAs), on the surface of cancer cells. Antitumor activity is achieved by directing the host immune system to TAAs, thereby establishing or inducing an adaptive immune response against cancer cells. The use of monoclonal antibodies (MAbs) to treat cancer has met with great success over the past several decades, many of which have been approved for use in cancer treatment or clinical trials.

4-1BB (CD 137/TNFRSF 9) is a transmembrane protein of the Tumor Necrosis Factor Receptor Superfamily (TNFRS) expressed on antigen-activated T cells and not on resting T cells. Human 4-1BB is a protein having 255 amino acids, and comprises a signal sequence (amino acid residues 1-17), an extracellular domain (169 amino acids), a transmembrane region (27 amino acids), and an intracellular domain (42 amino acids). 4-1BB is expressed on the cell surface in monomeric or dimeric form, and after binding to its ligand (4-1 BBL), it undergoes signal transduction by trimerization.

Furthermore, 4-1BB is known to be found in Dendritic Cells (DC), natural killer cells (NKs), activated CD4 ⁺ And CD8 ⁺ T lymphocytes, eosinophils, natural killer T cells (NKT) and mast cells, but myeloid-derived suppressor cells (MDSCs) do not express this molecule on their surface. The anti-4-1 BB antibody has the ability to activate cytotoxic T cells and increase the production of gamma interferon (IFN-gamma), and shows great potential in anticancer.

At present, a number of 4-1BB related pharmaceutical research projects are underway. The reported 4-1BB antibody sequences are either in binding to 4-1BB and activating the 4-1BB signaling pathway, or too active resulting in CD8 in the liver ⁺ T cell enrichment causes hepatotoxicity or is safe but not activated enough to kill tumors. Therefore, there is a need to develop new drugs with better activity and lower toxic and side effects4-1BB antibody drug.

Disclosure of Invention

The object of the present application is to provide an antibody, or antigen binding fragment thereof, capable of specifically binding to 4-1BB (i.e. an anti-4-1 BB antibody), as a stand-alone therapy or in combination with other therapies and/or other anti-cancer agents, for use in the treatment of e.g. cancer.

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

wherein the amino acid sequences of the HCDR1, HCDR2 and HCDR3 are HCDR1, HCDR2 and HCDR3 of the heavy chain variable region as shown in SEQ ID NO. 13 or 15 and the amino acid sequences of the LCDR1, LCDR2 and LCDR3 are LCDR1, LCDR2 and LCDR3 of the light chain variable region as shown in SEQ ID NO. 14 or 16 according to the Kabat, IMGT, chothia, abM, contact or North numbering system, and at least one of the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 may be replaced with a variant having 1, 2 or 3 amino acid differences thereto.

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 the amino acid sequences of the HCDR1, HCDR2 and HCDR3 are HCDR1, HCDR2 and HCDR3 of the heavy chain variable region shown in SEQ ID NO. 13 and the amino acid sequences of the LCDR1, LCDR2 and LCDR3 are LCDR1, LCDR2 and LCDR3 of the light chain variable region shown in SEQ ID NO. 14 according to Kabat, IMGT, chothia, abM, contact or North numbering system; or alternatively

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

In some embodiments, according to the Kabat numbering system, the HCDR1, HCDR2, and HCDR3 have amino acid sequences shown as SEQ ID No. 1, SEQ ID No. 2, and SEQ ID No. 3, respectively, or have amino acid sequences shown as SEQ ID No. 7, SEQ ID No. 8, and SEQ ID No. 9, respectively;

the LCDR1, LCDR2 and LCDR3 respectively have amino acid sequences shown as SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6, or respectively have amino acid sequences shown as SEQ ID NO. 10, SEQ ID NO. 11 and SEQ ID NO. 12;

Wherein at least one of said SEQ ID NOS.1-12 may be replaced with a variant having 1, 2 or 3 amino acid differences therefrom.

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

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

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

a heavy chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO. 13 and a light chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO. 14; or (b)

A heavy chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO. 15 and a light chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO. 16;

a heavy chain variable region with an amino acid sequence shown as SEQ ID NO. 13, and a light chain variable region with an amino acid sequence shown as SEQ ID NO. 14; or (b)

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

In some embodiments, the antibody or antigen binding fragment thereof further comprises a human heavy chain constant region selected from the group consisting of a heavy chain constant region of human IgG1, igG2, igG3, or IgG4, preferably a heavy chain constant region of human IgG4 or a heavy chain constant region of human IgG4 with an S228P amino acid substitution; the human light chain constant region is selected from the light chain constant region of a lambda light chain or a kappa light chain.

a heavy chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO. 17 and a light chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO. 18; or (b)

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

a heavy chain with an amino acid sequence shown as SEQ ID NO. 17, and a light chain with an amino acid sequence shown as SEQ ID NO. 18; or (b)

A heavy chain with an amino acid sequence shown as SEQ ID NO. 19, and a light chain with an amino acid sequence shown as 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.

In a second aspect, the present application provides an isolated antibody or antigen binding fragment thereof having at least one of the following properties:

binding to the same, or fully overlapping or partially overlapping, epitope of human 4-1BB protein as the antibody or antigen binding fragment thereof provided in the first aspect of the present application;

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

A third aspect of the present application provides a polynucleotide molecule comprising at least one of a nucleotide sequence encoding an antibody or antigen-binding fragment thereof provided in the first aspect of the present application, or encoding an isolated antibody or antigen-binding fragment thereof provided in the second aspect of the present application, or a complement thereof.

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

In a fifth aspect the present application provides a host cell comprising a polynucleotide molecule as provided in the third aspect of the present application, or an expression vector as provided in the fourth aspect of the present application, preferably the host cell is a eukaryotic cell, more preferably a mammalian cell.

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

In a sixth aspect the present application provides a method of preparing an antibody or antigen-binding fragment thereof of the first aspect of the present application or an isolated antibody or antigen-binding fragment thereof of the second aspect of the present application, comprising expressing said antibody or antigen-binding fragment thereof in a host cell provided in the fifth aspect of the present application and recovering the expressed antibody or antigen-binding fragment thereof from said host cell.

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

In an eighth aspect the present application provides the use of an antibody or antigen-binding fragment thereof of the first aspect of the present application or an isolated antibody or antigen-binding fragment thereof of the second aspect of the present application or a polynucleotide molecule provided in the third aspect of the present application or an expression vector provided in the fourth aspect of the present application or a host cell provided in the fifth aspect of the present application or a pharmaceutical composition provided in the seventh aspect of the present application in the manufacture of a medicament for the treatment or prophylaxis of a 4-1BB mediated disease.

A ninth aspect of the present application provides the use of an antibody or antigen-binding fragment thereof of the first aspect of the present application or an isolated antibody or antigen-binding fragment thereof of the second aspect of the present application or a polynucleotide molecule provided in the third aspect of the present application or an expression vector provided in the fourth aspect of the present application or a host cell provided in the fifth aspect of the present application or a pharmaceutical composition provided in the seventh aspect of the present application for the treatment or prophylaxis of a 4-1BB mediated disease.

A tenth aspect of the present application provides a method of treating or preventing a 4-1BB mediated disease or condition comprising administering to a subject in need thereof an antibody or antigen binding fragment thereof provided in the first aspect of the present application or an isolated antibody or antigen binding fragment thereof provided in the second aspect of the present application or a polynucleotide molecule provided in the third aspect of the present application or an expression vector provided in the fourth aspect of the present application or a host cell provided in the fifth aspect of the present application or a pharmaceutical composition provided in the seventh aspect of the present application.

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

An eleventh aspect of the present application provides a kit comprising an antibody or antigen-binding fragment thereof as provided in the first aspect of the present application or an isolated antibody or antigen-binding fragment thereof as provided in the second aspect of the present application or a polynucleotide molecule as provided in the third aspect of the present application or an expression vector as provided in the fourth aspect of the present application or a host cell as provided in the fifth aspect of the present application or a pharmaceutical composition as provided in the seventh aspect of the present application.

A twelfth aspect of the present application provides a method of detecting 4-1BB using an antibody or antigen-binding fragment thereof provided in the first aspect of the present application or an isolated antibody or antigen-binding fragment thereof provided in the second aspect of the present application, comprising contacting the antibody or antigen-binding fragment thereof with a sample, detecting a conjugate formed by the antibody or antigen-binding fragment thereof and 4-1BB, and optionally quantifying the conjugate.

The antibody or antigen binding fragment thereof capable of specifically binding to 4-1BB provided by the application can specifically bind to human 4-1BB, has low hepatotoxicity and obvious tumor inhibition effect, and can be used for treating 4-1BB mediated diseases, such as cancers.

Drawings

In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following description will briefly introduce the drawings that are required to be used in the embodiments or the description of the prior art, it is obvious that the drawings in the following description are only one embodiment of the present application, and other embodiments may be obtained according to these drawings to those skilled in the art.

FIG. 1 shows the results of the binding capacity test of the anti-4-1 BB antibodies JS011-phage 3 and JS011-phage 6 prepared in example 2 of the present application with human 4-1 BB.

FIG. 2 shows the results of the binding capacity test of the anti-4-1 BB antibodies JS011-phage 3 and JS011-phage 6 prepared in example 2 of the present application with murine 4-1 BB.

FIG. 3 shows the results of binding capacity test of the anti-4-1 BB antibodies JS011-phage 3 and JS011-phage 6 prepared in example 2 of the present application and the related controls to Jurkat NFKB 4-1BB cells.

FIG. 4 shows the results of the test of binding of anti-4-1 BB antibodies JS011-phage 3 and JS011-phage 6 and related control blocking 4-1BBL to Jurkat NFKB 4-1BB cells prepared in example 2 of the present application.

FIG. 5 shows the results of activity assays of anti-4-1 BB 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).

FIG. 6 shows the results of activity assays of anti-4-1 BB 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 cell).

FIG. 7 shows the results of activity assays of anti-4-1 BB 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 cell).

FIG. 8 shows the results of activity assays of anti-4-1 BB 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).

FIGS. 9A to 9F show changes in the amounts of release of cytokines IL-2, IL-4, IL-6, IL-10, TNF. Alpha. And IFN. Gamma. By the anti-4-1 BB antibodies JS011-phage 3 and JS011-phage 6 prepared in example 2 of the present application, respectively, and the related controls.

FIG. 10 shows the tumor growth inhibition of anti-4-1 BB antibodies JS011-phage 3 and JS011-phage 6 prepared in example 2 of the present application, and related controls.

FIG. 11 shows the effect of anti-4-1 BB antibodies JS011-phage 3 and JS011-phage 6 prepared in example 2 of the present application and related controls on spleen weight of tumor-bearing mice at day 14 post-administration.

FIG. 12A shows the effect of anti-4-1 BB antibodies JS011-phage 3 and JS011-phage 6 prepared in example 2 of the present application and related controls on total lymphocyte number per unit weight of liver of tumor-bearing mice at day 14 post-administration.

FIGS. 12B to 12E show the anti-4-1 BB antibodies JS011-phage 3 and JS011-phage 6 prepared in example 2 of the present application, respectively, and the related controls were administered on day 14 post-administration to CD3 in the liver of tumor-bearing mice ⁺ CD4 ⁺ T cell ratio, CD3 ⁺ CD8 ⁺ T cell ratio, CD3 ⁺ CD8 ⁺ CD11c ⁺ T cell ratio and CD3 ^- NK1.1 ⁺ The influence of NK cell proportion.

Detailed Description

Definition of the definition

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

In order that the present application may be more readily understood, certain technical and scientific terms are specifically defined as follows. Unless otherwise defined explicitly herein, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. For definitions and terms in the art, the expert may refer specifically to Current Protocolsin Molecular Biology (Ausubel). The abbreviations for amino acid residues are 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), the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

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

The term "and/or" is understood to mean any one of the selectable items or a combination of any two or more of the selectable items.

The terms "4-1BB", "4-1BB receptor", "4-1BB protein", "CD137" or "tumor necrosis factor receptor superfamily member 9 (TNFRSF 9)" are members of the Tumor Necrosis Factor (TNF) receptor superfamily and are activation-induced T cell co-stimulatory molecules. In the present application, the term "4-1BB" may include human 4-1BB, as well as variants, subtypes, homologs, orthologs, and paralogs thereof, and thus, the antibodies or antigen-binding portions thereof provided herein may also bind 4-1BB from a species other than human, such as cynomolgus monkey 4-1BB or mouse 4-1BB. For example, in some cases, antibodies specific for the human 4-1BB protein may cross-react with 4-1BB from a species other than human. In other embodiments, antibodies specific for the human 4-1BB protein may be fully specific for the human 4-1BB protein and may exhibit species or other types of cross-reactivity, or may cross-react with 4-1BB from some other species but not all other species, e.g., with monkey 4-1BB, but not with mouse 4-1BB. The term "human 4-1BB" may refer to 4-1BB having the complete amino acid sequence of human 4-1BB of NCBI accession number NP-001552.2; the sequence of "human 4-1BB" may also differ from the amino acid sequence of human 4-1BB of NCBI accession No. NP 001552.2, e.g., may have conservative mutations in non-conserved regions, and have substantially the same biological function as human 4-1BB of NCBI accession No. NP 001552.2. The term "mouse 4-1BB" may refer to 4-1BB having the complete amino acid sequence of mouse 4-1BB of NCBI accession No. NP-033430.1 or NP-035742.1. The term "cynomolgus monkey 4-1BB" may refer to 4-1BB having the complete amino acid sequence of cynomolgus monkey 4-1BB with Genbank accession number NP-001253057.1.

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

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

The term "signal transduction pathway" or "signal transduction activity" refers to a biochemical causal relationship typically initiated by protein-protein interactions such as binding of growth factors to receptors, which results in the transfer of signals from one part of a cell to another part of the cell. In general, delivery involves specific phosphorylation of one or more tyrosine, serine or threonine residues on one or more proteins in a series of reactions that cause signal transduction. The penultimate process typically involves a nuclear event, resulting in a change in gene expression.

The terms "activity" or "biological activity" or the terms "biological property" or "biological feature" are used interchangeably in this application and include, but are not limited to, epitope or antigen affinity, specificity, ability to neutralize or antagonize 4-1BB activity in vivo or in vitro, semi-inhibitory concentration (IC ₅₀ ) In vivo stability of the antibody, immunogenicity of the antibody, and the like. Other identifiable biological properties or characteristics of antibodies known in the art include, for example, cross-reactivity (e.g., with non-human homologs of the targeting peptide, or with other proteins or tissues), and the ability to maintain high levels of expression of the protein in mammalian cells. The aforementioned properties or characteristics are observed, assayed or assessed using techniques well known in the art, including but not limited to enzyme-linked immunosorbent (ELISA), flow cell sorting (FACS) or BIACORE plasma resonance analysis, any in vitro or in vivo neutralization assay, receptor binding assays, cytokine or growth factor production and/or secretion assays, signal transduction assays, and various sources (including human, primate or any other source ) Immunohistochemical analysis of tissue sections, and the like.

The term "antibody" refers to any form of antibody that has the desired biological activity. Thus, it is used in the broadest sense and specifically includes, but is not limited to, monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), humanized antibodies, fully human antibodies, chimeric antibodies, and camelized single domain antibodies, and the like. The basic antibody structural unit is known to comprise tetramers, each comprising two identical pairs of polypeptide chains, each pair having one "light" chain (L, about 25 kDa) and one "heavy" chain (H, about 50-70 kDa). The amino-terminal portion or fragment of each chain may include a variable region of about 100-110 amino acids or more that is primarily responsible for antigen recognition. The carboxy-terminal portion or fragment of each strand may define a constant region primarily responsible for effector function. Human light chains are generally classified as kappa light chains and lambda light chains. Furthermore, human heavy chains are generally classified into five classes μ, δ, γ, α or ε, and the isotypes of antibodies are defined as IgM, igD, igG, igA and IgE, respectively, depending on the heavy chain. Within the light and heavy chains, the respective variable and constant regions are linked by a "J" region of about 12 or more amino acids, wherein the heavy chain further comprises a "D" region of about 10 more amino acids. See generally chapter 7 of Fundamental Immunology (Paul, W. Main, 2 nd edition, raven Press, N.Y. (1989)).

The term "isolated antibody" with respect to an 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 media. It is understood that the isolated antibody is in a substantially purified state, preferably in a homogeneous state, and may be in a dry or aqueous solution. Analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography can be used to determine the purity and homogeneity of antibodies. The term "isolated" does not mean that the above-mentioned substances are completely absent or that water, buffer or salt are absent unless they are present in amounts that significantly interfere with the experimental or therapeutic use of the antibodies of the present application.

The term "monoclonal antibody" is an antibody made from highly identical immune cells, which are all clones of a single parent cell. Monoclonal antibodies have monovalent affinity because they bind to the same epitope (the site where the antibody recognizes the antigen). The monoclonal antibodies may also include minor amounts of naturally occurring mutations. In contrast, the term "polyclonal antibody" binds to multiple epitopes, typically consisting of several different plasma cell (antibody secreting immune cell) lineages, and is understood to be a hybrid of multiple monoclonal antibodies. The modifier "monoclonal" is not to be construed as requiring antibody production by any particular method.

The term "multispecific antibody" means an antibody comprising two or more antigen-binding domains capable of binding to two or more different epitopes (e.g., two, three, four, or more different epitopes). The epitopes to which the specific antibodies bind may 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 site" refers to a region in an antibody that is capable of specifically binding to and complementing a portion or all of an antigen. When the antigen is large, the antibody may only bind to a specific portion of the antigen, which portion is referred to as 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). CDRs can vary in sequence and determine specificity for a particular antigen.

The term "full length" antibody refers to an immunoglobulin molecule that when naturally occurring comprises four peptide chains: two heavy chains (full length about 50-70 kDa) and two light chains (full length about 25 kDa) are linked to each other by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH). The heavy chain constant region consists of 3 domains, CH1, CH2 and CH 3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region (abbreviated herein as CL). The light chain constant region consists of one domain CL. VH and VL regions can be further subdivided into Complementarity Determining Regions (CDRs) with high variability and Framework Regions (FR) with higher conservation that are distributed with complementarity determining regions. The domains of each VH or VL from amino terminus to carboxy terminus are arranged in the order FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The variable domains of the heavy and light chains each contain a binding domain that interacts with an antigen. The constant region of an antibody may mediate the binding of the antibody to various cells of the host's tissue or immune system (e.g., effector cells) and the first component of the classical complement system (C1 q).

The term "heavy chain constant region" or "CH" is used interchangeably herein and comprises at least three heavy chain constant domains (CH 1, CH2 and CH 3). Illustratively, the human heavy chain constant regions include gamma, delta, alpha, epsilon, and mu, each heavy chain constant region corresponding to an antibody isotype. For example, the antibody comprising a gamma constant region is an IgG antibody, the antibody comprising a delta constant region is an IgD antibody, the antibody comprising an alpha constant region is an IgA antibody, the antibody comprising a mu constant region is an IgM antibody, and the antibody comprising 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 a γ1 constant region), igG2 (comprising a γ2 constant region), igG3 (comprising a γ3 constant region), and IgG4 (comprising a γ4 constant region); igA antibodies include, but are not limited to IgA1 (comprising an α1 constant region) and IgA2 (comprising an α2 constant region); igM antibodies include, but are not limited to, igM1 and IgM2. Isoforms may also include some form of modification that may alter Fc function, e.g., enhance or attenuate effector function or enhance or attenuate binding to Fc receptors.

The term "light chain constant region" or "CL" is used interchangeably herein and comprises 1 light chain constant domain CL. Illustratively, light chains can be classified into two classes, λ and κ, depending on the constant region of the light chain.

The term "antigen-binding fragment" of an antibody includes fragments of the antibody or derivatives of the antibody, and the antibody corresponding to the "antigen-binding fragment" may be referred to as a parent antibody. The antigen-binding fragment of an antibody typically comprises at least one 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 ', F (ab') 2, and single chain Fv fragments, diabodies, linear antibodies, domain antibodies, single chain antibody molecules, e.g., scFv; nanobodies (nanobodies) and multispecific antibodies formed from antibody fragments, and the like. The antigen binding fragment is capable of retaining at least 10%, at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the antigen binding activity of the parent antibody at the same molar concentration. Furthermore, an antigen-binding fragment of an antibody may also include conservative or non-conservative amino acid substitutions that do not significantly alter its biological activity (referred to as "conservative variants" or "functional 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 the 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 that contains only heavy chain variable regions or light chain variable regions. In some cases, two or more VH regions are covalently linked to a peptide linker to form a bivalent domain antibody. The 2 VH regions of a bivalent domain antibody may target the same or different antigens.

The term "antigen" refers to a molecule or portion of a molecule that is 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 having two antigen binding sites, said fragment being comprised in the same polypeptide chain, a heavy chain variable domain (VH) linked to a light chain variable domain (VL), both said fragments forcing the domain to pair with a complementary domain in the other fragment and creating two antigen binding sites by a linker that is short enough to not allow pairing between the two domains of the same chain.

The terms "specific binding", "selective binding" refer to binding of an antibody to an epitope on a predetermined antigen. Typically, when recombinant human 4 is used-1BB or epitope thereof as analyte and antibody as ligand, the antibody being present at a level of about less than 10 when measured in an instrument by Surface Plasmon Resonance (SPR) technique ^-7 M or even smaller equilibrium dissociation constants (KD) bind to the predetermined antigen or epitope thereof and the antibody binds to the predetermined antigen or epitope thereof with at least twice as much affinity as it binds to other non-specific antigens (e.g., BSA, etc.). The term "recognition antigen" may be used interchangeably herein with the term "specific binding".

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

"affinity" or "binding affinity" refers to the inherent binding affinity that reflects the interaction between members of a binding pair (e.g., antigen and antibody). Affinity can be generally expressed by the equilibrium dissociation constant (KD), which is the ratio of the dissociation rate constant and the binding rate constant (kdis and kon, respectively). Affinity can be measured by common methods known in the art, for example, using the ForteBio biological molecular interaction workstation.

The term "non-binding" protein or cell means that it does not bind to the protein or cell, or does not bind to it with high affinity, i.e. the binding protein or cell has a KD of 1.0X10 ^-6 M or higher, more preferably 1.0X10 ^-5 M or higher, more preferably 1.0X10 ^-4 M or higher, 1.0X10 ^-3 M or higher, more preferably 1.0X10 ^-2 M or higher.

The term "high affinity" for IgG antibodies refers to a KD of 1.0X10 for antigen ^-6 M or less, preferably 5.0X10 ^-8 M or less, more preferably 1.0X10 ^-8 M or less, 5.0X10 s ^-9 M or less, more preferably 1.0X10 ^-9 M or lower. For other antibody subtypes, "high affinity" binding may vary. For example, "high affinity" binding of IgM subtype refers to KD of 10 ^-6 M or less, preferably 10 ^-7 M or less, 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-stranded or double-stranded form. Unless specifically limited, the term "nucleic acid" or "polynucleotide" also includes nucleic acids comprising known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides (see, U.S. Pat. No.8278036 to Kariko et al, which discloses mRNA molecules with uridine replaced by pseudouridine, methods of synthesizing the mRNA molecules, and methods for delivering therapeutic proteins in vivo). Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, single Nucleotide Polymorphisms (SNPs) and complementary sequences, as well as the sequence explicitly indicated.

"construct" refers to any recombinant polynucleotide molecule (e.g., plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, phage, linear or circular single-or double-stranded DNA or RNA polynucleotide molecule) that can be derived from any source, capable of integration with the genome or autonomous replication, which can be operably linked to one or more polynucleotide molecules. In this application, constructs are generally polynucleotide molecules of the present application operably linked to transcriptional initiation regulatory sequences that direct transcription of the polynucleotide molecules of the present application in a host cell. Heterologous promoters or endogenous promoters may be used to direct expression of the nucleic acids of the present application.

"vector" refers to any recombinant polynucleotide construct that can be used for transformation purposes (i.e., 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 may be ligated. Another type of vector is a viral vector, which can ligate additional DNA segments into the viral genome. Some vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and are replicated along 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 when transformed, transfected or transduced 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 amplification in the host if desired.

In this application, unless explicitly specified otherwise, "activation", "stimulation" and "treatment" for a cell or receptor may have the same meaning, e.g., activation, stimulation or treatment of a cell or receptor with a ligand. "ligand" includes natural and synthetic ligands such as cytokines, cytokine variants or analogs, muteins, and binding compounds derived from antibodies (e.g., antibodies and binding fragments thereof). "ligand" also includes small molecules such as peptidomimetics of cytokines and peptidomimetics of antibodies. "activation" may refer to cell activation mediated by internal mechanisms and external or environmental factors. A "response" or "reaction", such as a response of a cell, tissue, organ or organism, including a change in a biochemical or physiological behavior, such as a change in the concentration, density, adhesion or migration of a portion of a constituent within a biological compartment (e.g., tissue, cell, organelle, etc.), a rate of gene expression or a state of differentiation, which change may be associated with activation, stimulation or treatment.

As used herein, the term "treatment" or "treatment" of any disease or disorder may refer in one embodiment to ameliorating the disease or disorder, such as slowing, arresting or reducing the progression of the disease, or clinical symptoms of the disease, etc.; in another embodiment, may refer to alleviation or amelioration of at least one physical parameter that may not exhibit significant improvement in disease symptoms; in another embodiment, it may refer to modulating a disease or disorder physically (e.g., stabilization of a discernible symptom), physiologically (e.g., stabilization of a physical parameter), or both. Methods for assessing treatment and/or prevention of disease are generally known in the art unless explicitly described herein.

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

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

By "therapeutically effective amount", "therapeutically effective dose" and "effective amount" is meant that the anti-4-1 BB antibody or antigen-binding fragment thereof of the present application, alone or in combination with other therapeutic agents, is effective to prevent or ameliorate symptoms of at least one disease or condition, or to prevent or ameliorate the development of at least one disease or condition, when administered to a cell, tissue or subject. A therapeutically effective dose may also refer to an amount of an antibody or antigen-binding fragment thereof sufficient to result in an improvement in symptoms (e.g., treat, cure, prevent, or ameliorate a related medical condition, or increase the rate of treatment, cure, prevention, or amelioration of such a condition). When an active ingredient (e.g., an antibody or antigen-binding fragment thereof) is administered to an individual, a therapeutically effective dose refers to that ingredient alone; when administered in combination, a therapeutically effective dose refers to the combined amount of all active ingredients that result in a therapeutic effect, whether administered sequentially or simultaneously. An effective amount of the therapeutic agent will result in an increase in the diagnostic criteria or parameter of at least 10%; typically at least 20%; preferably at least about 30%; more preferably at least 40%, most preferably at least 50%.

"cancer" and "cancerous" refer to physiological conditions in mammals that are typically characterized by unregulated cell growth, and include within this definition benign tumors, malignant cancers, and dormant tumors or micrometastases. Examples of cancers include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specific examples of such cancers include squamous cell carcinoma, lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous carcinoma), peritoneal cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, icterohepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland carcinoma, renal cancer, prostate cancer, vulval cancer, thyroid cancer, and various types of head and neck cancer, as well as B-cell lymphomas (such as low-grade/follicular non-hodgkin's lymphoma (NHL), small Lymphocytic (SL) NHL, medium-grade/follicular NHL, medium-grade diffuse NHL, immunocytogenic NHL, lymphoblastic NHL, small non-nucleated NHL, storage disease NHL, mantle cell lymphoma, AIDS-related lymphomas and Waldenstrom's macroglobulinemia), chronic Lymphocytic Leukemia (CLL), acute Lymphoblastic Leukemia (ALL), capillary leukemia (Waldenstrom's), myelogenous leukemia (mel's) and lymphomas associated with vascular proliferation (meiosis) and vascular disorders (meiosis) associated with vascular hyperplasia.

Antibodies to

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

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

wherein, the HCDR1, HCDR2 and HCDR3 respectively have amino acid sequences shown as SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3, or respectively have amino acid sequences shown as SEQ ID NO. 7, SEQ ID NO. 8 and SEQ ID NO. 9;

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

binding to the same, or fully overlapping or partially overlapping, epitope of human 4-1BB protein as the antibody or antigen binding fragment thereof described in the first aspect of the present application;

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

Wherein an epitope that fully overlaps with the binding of an antibody or antigen binding fragment thereof of the present application means that the epitope to which the antibody binds comprises the binding epitope of the antibody or antigen binding fragment thereof of the present application; an epitope that overlaps with the binding portion of an antibody or antigen binding fragment thereof of the present application means that a portion of the epitope bound by the antibody is identical to a portion of the binding epitope of an antibody or antigen binding fragment thereof of the present application.

For the precise amino acid sequence boundaries of the CDRs of an antibody, they can be defined according to well-known methods, e.g., chothia based on the three-dimensional structure of the antibody and the topology of the CDR loops (Chothia et al, nature 342:877-883, 1989; al-Lazikani et al, journal of Molecular Biology,273:927-948, 1997); or Kabat based on antibody sequence variability (Kabat et al, sequences of Proteins of Immunological Interest, 4 th edition, U.S. Pat. No. of Health and Human Services, national Institutes of Health, 1987), abM (University of Bath), contact (University College London), and IMGT (the international ImMunoGeneTics database,1999Nucleic Acids Research,27,209-212); or on the North CDR definition of a neighbor-propagated cluster (affinity propagation clustering) that utilizes a large number of crystal structures. The CDRs of the antibodies herein can be bordered by one of skill in the art according to any protocol in the art (e.g., the optional definition methods described above).

It should be noted that the boundaries of CDRs of the same antibody obtained based on different definitions may differ, i.e. the CDR sequences of the variable regions of the same antibody obtained under different definitions may differ. Thus, when an antibody is defined using a particular CDR sequence as defined herein, the antibody also includes antibodies whose complementarity determining region sequences comprise the CDRs as described herein, except that the stated CDR boundaries differ from the particular CDR boundaries as defined herein due to the use of different CDR boundary definitions.

Antibodies with different specificities (i.e., for different antigen combining sites) have different CDRs. However, although CDRs vary from antibody to antibody, only a limited number of amino acid positions within the CDRs are directly involved in antigen binding, and the minimal overlap region of an antibody CDR with antigen binding is also referred to as the "minimal binding unit" for antigen binding, and can be determined using at least two of Kabat, IMGT, chothia, abM, contact and North methods. The minimum binding unit may be part of a CDR. As will be apparent to those skilled in the art, by the structure and protein folding of the antibody, the residues of the remainder of the CDR sequence may be determined, and thus, the present application also contemplates variants of any CDR, e.g., in a variant of one CDR, the amino acid residues of the smallest binding unit may remain unchanged, while the remaining CDR residues according to the Kabat or Chothia definition may be replaced by conserved amino acid residues.

The humanized antibodies described herein may have murine CDR regions inserted into human germline framework regions using methods known in the art. See Winter et al, U.S. patent No.5225539, queen et al, U.S. patent No.5530101;5585089;5693762 and 6180370.

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

In some embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby producing an Fc region variant. An Fc region variant may include a human Fc region sequence (e.g., a human IgG1, igG2, igG3, or IgG4 Fc region) with amino acid modifications (e.g., 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, e.g., a "thiomab," in which one or more residues of the antibody or antigen-binding fragment thereof are replaced with cysteine residues.

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

Antibody expression

Another aspect of the present application provides a polynucleotide molecule comprising at least one of a nucleotide sequence encoding an antibody or antigen binding fragment thereof provided herein or a complement thereof. Polynucleotide molecules in this application include double-stranded or single-stranded DNA or RNA. Antibodies or antigen-binding fragments thereof provided herein include antibodies or antigen-binding fragments thereof of the first aspect of the application and isolated antibodies or antigen-binding fragments thereof of the second aspect of the application.

In some embodiments, a polynucleotide molecule encoding an antibody or antigen-binding fragment thereof of the present application may also include a polynucleotide sequence that has nucleotide deletions, insertions, or substitution mutations, but which still has at least about 60%, 70%, 80%, 90%, 95%, or 100% identity to the coding region corresponding to the CDRs of the antibody or antigen-binding fragment thereof of the present application.

In a further aspect, the present application provides an expression vector comprising a 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 a 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 cell is used to express an antibody or antigen-binding fragment thereof provided herein.

Mammalian host cells provided herein for expressing the antibodies or antigen binding fragments thereof of the present application include a variety of immortalized cell lines available from the American Type Culture Collection (ATCC). Exemplary, may include Chinese Hamster Ovary (CHO) cells, NS0, SP2/0 cells, heLa cells, baby Hamster Kidney (BHK) cells, monkey kidney Cells (COS), human hepatocellular carcinoma cells, a549 cells, 293T cells, and many other cell lines. Mammalian host cells may include human, mouse, rat, dog, monkey, pig, goat, cow, 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 of making an antibody or antigen-binding fragment thereof of the present application, comprising expressing the antibody or antigen-binding fragment thereof in a host cell provided herein, and recovering the expressed antibody or antigen-binding fragment thereof from the host cell.

In particular, the present application provides a method of making an antibody or antigen-binding fragment thereof of the present application, which may comprise:

introducing a polynucleotide molecule or expression vector of the present application into a host cell described herein using well-known methods, such as lipofection, electrotransfection, transformation, and the like; culturing said host cell under conditions suitable for expression of said antibody or antigen-binding fragment thereof such that said host cell expresses said antibody or antigen-binding fragment thereof; the expressed antibodies or antigen binding fragments thereof are recovered from the host cells using methods well known in the art, such as polyacrylamide gel electrophoresis.

It should be noted that, depending on the expression vector or host cell used, the conditions suitable for the expression of the antibody or antigen-binding fragment thereof may be selected by those skilled in the art according to the specific circumstances, and the present application is not limited thereto.

The host cell used in the present application for expressing the antibody or antigen binding fragment thereof may be an isolated cell, or may be a cell that is still present in the host, e.g., the antibody or antigen binding fragment thereof may be expressed using a different cell line, or the antibody or antigen binding fragment thereof may be expressed using a transgenic animal.

Antibodies or antigen binding fragments thereof expressed by different cell lines or in different transgenic animals may have different glycosylation modifications. It is noted that, regardless of the glycosylation of these antibodies, all antibodies encoded by the polynucleotide molecules provided herein, or comprising the amino acid sequences provided herein, are part of the present application. Also, in certain embodiments, the antibody or antigen-binding fragment thereof is preferably a non-fucosylated antibody or antigen-binding fragment thereof, which the inventors have found to have a more potent efficacy in vitro and in vivo than fucosylated antibodies or antigen-binding fragments thereof, and furthermore, since these different glycostructures are both normal components of native human serum IgG, 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 referred to as a pharmaceutical formulation, preparation) comprising an antibody or antigen-binding fragment thereof, a polynucleotide molecule, an expression vector or a host cell provided herein, and a pharmaceutically acceptable carrier or excipient.

It will be appreciated that the antibodies or pharmaceutical compositions thereof provided herein may incorporate suitable carriers, excipients, and other agents in a formulation for administration in combination, thereby providing improved transfer, delivery, tolerability, etc.

In this application, the active ingredient in the pharmaceutical composition is present in a form that is effective in its biological activity and does not comprise an ingredient that is unacceptably toxic to the subject of the pharmaceutical composition.

In some embodiments of the present application, the pharmaceutical compositions of the present application may be prepared by mixing an antibody or antigen binding fragment thereof of the present application having the desired purity with one or more optional pharmaceutical excipients (see Remington's Pharmaceutical Sciences, 16 th edition, osol, a. Edit, 1980), preferably the pharmaceutical compositions of the present application may be in the form of an aqueous solution or a lyophilized formulation.

The pharmaceutical compositions or formulations of the present application may also comprise one or more other active ingredients, which may be selected according to the particular indication, preferably without the active ingredients adversely affecting each other's activity. In some embodiments, the additional active ingredient may be a chemotherapeutic agent, immune checkpoint inhibitor, growth inhibitor, antibiotic, or various known anti-tumor or anti-cancer agents, which may be present in combination in an amount effective for the intended use.

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

Medical application

In yet another aspect, the present application provides the use of an antibody or antigen binding fragment thereof, a polynucleotide molecule, an expression vector or a host cell or a pharmaceutical composition of the present application in the manufacture of a medicament for the treatment or prevention of a 4-1BB mediated disease or condition.

In yet another aspect, the present application provides the use of an antibody or antigen binding fragment thereof, a polynucleotide molecule, an expression vector or a host cell or a pharmaceutical composition of the present application for the treatment or prevention of a 4-1BB mediated disease or disorder.

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

In some embodiments, the mode of administration includes, but is not limited to, oral, intravenous, subcutaneous, intramuscular, intraarterial, intra-articular (e.g., in an arthritic joint), inhalation, aerosol delivery, or intratumoral administration, and the like.

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

Method for diagnosis and detection

In yet another aspect, the present application provides a method of detecting 4-1BB using an antibody or antigen-binding fragment thereof of the present application, comprising contacting said antibody or antigen-binding fragment thereof with a sample, detecting a conjugate formed by said antibody or antigen-binding fragment thereof and 4-1BB, and optionally quantifying said conjugate. 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, the biological sample comprises a cell or tissue. The method for detecting the conjugate formed by the antibody or the antigen binding fragment thereof and 4-1BB is not limited; the method of "quantifying the conjugate" is not limited in this application.

For the purposes of making the objects, technical solutions, and advantages of the present application more apparent, the present application will be further described in detail below by referring to the accompanying drawings and examples. It will be apparent that the described embodiments are only some, but not all, of the embodiments of the present application. All other embodiments obtained by those skilled in the art based on the embodiments herein fall within the scope of the protection of the present application.

Example 1 screening of anti-4-1 BB antibodies

1.1 establishment of phage library

50mL peripheral blood from 40 individuals were pooled together, peripheral blood mononuclear cells PBMC were isolated, trizol was used to extract total RNA, and cDNA first strand was synthesized using reverse transcription kit (Thermo, 18080051). The variable regions VH, vk and vλ of the antibody heavy chain, and the variable regions κ and λ of the antibody light chain were amplified by PCR using the synthesized cDNA as a template. VH-CH1, V kappa-CK, V lambda-C lambda were assembled by first round overlap PCR (overlap PCR), and then Fab was assembled by second round overlap PCR, and the ligation into phagemid vector was recovered by cleavage of the Fab fragment. And electrically transferring the constructed plasmid vector into TG1 competent cells to obtain a phage display library.

1.2 phage library screening

Packaging the constructed phage display library into phage. First round panning selection 10 ¹³ -10 ¹⁴ The phages were added to biotinylated 4-1BB for incubation and binding, then streptavidin-conjugated magnetic beads were added to bind 4-1BB, unbound phages were washed off with PBST first, phages bound to 4-1BB were eluted with glycine at pH 2.0 for infection of TG1, and phages were amplified and packaged for a second round of panning. Through four rounds of panning, phage from the fourth round were infected with TG1 and plated as a monoclonal, and the monoclonal was picked in 96 deep well plates and expression was induced with IPTG (isopropyl thiogalactoside). The next day the periplasmic cavity supernatant was extracted for 4-1BB binding enzyme-linked immunosorbent assay (ELISA). Positive clones were sequenced to obtain their sequence. Finally obtaining two groups of phages, namely, phase 3 and phase 6, for further analysis, wherein an antibody expressed by the phage phase 3 is named JS 011-phase 3; the antibody expressed by phage6 was named JS011-phage6.

Example 2 construction and production of molecules of anti-4-1 BB antibodies

The coding sequences for the heavy chain constant region (hIgG 4 CH1-CH 3) and the Kappa light chain constant region (human Kappa LC) of IgG4 were cloned from human B lymphocytes (from Beijing blood institute) and introduced into pTT5 plasmid to form vectors HXT4S and HXT2, respectively.

The main elements of the carrier HXT4S in turn comprise: BSPQI cleavage site, CMV Promoter (Promoter of target gene expression), ampR (ampicillin resistance gene), pMB1ori (replication initiation), ori p, signal peptide (signal peptide of target gene expression) and target gene hIgG4 CH1-CH3.

The main elements of the carrier HXT2 in turn comprise: BSPQI cleavage site, CMV Promoter (Promoter of target gene expression), ampR (ampicillin resistance gene), pMB1ori (replication initiation), orip, signal peptide (signal peptide of target gene expression), and target gene human Kappa LC.

Construction of 2.1JS011-phage3 light chain expression vector

And (3) taking a plasmid extracted after the phage3 is used for infecting the TG1 strain and shaking as a template, synthesizing head and tail primers, performing PCR as shown in SEQ ID No. 21 and SEQ ID No. 22 in the following table 1, and performing enzyme digestion on the obtained PCR product by SapI and connecting the PCR product to an HXT2 vector to obtain an expression vector HXT2-JS011-3-LC.

Construction of 2.2JS011-phage3 heavy chain expression vector

And (3) taking a plasmid extracted after the strain phage3 is subjected to bacterial shaking as a template, synthesizing head and tail primers, performing PCR as shown in SEQ ID No. 23 and SEQ ID No. 24 in the following table 1, and performing enzyme digestion on the obtained PCR product by SapI and connecting the PCR product to an HXT4S vector to obtain an expression vector HXT4S-JS011-3-HC.

Construction of 2.3JS011-phage6 light chain expression vector

And (3) taking a plasmid extracted after the strain phage6 is infected with the TG1 strain and shaken as a template, synthesizing head and tail primers, performing PCR as shown in SEQ ID No. 25 and SEQ ID No. 26 in the following table 1, and performing enzyme digestion on the obtained PCR product by SapI and connecting the PCR product to an HXT2 vector to obtain an expression vector HXT2-JS011-6-LC.

Construction of 2.4JS011-phage6 heavy chain expression vector

And (3) taking a plasmid extracted after the strain phage6 is infected with the TG1 strain and shaken as a template, synthesizing head and tail primers, performing PCR as shown in SEQ ID No. 23 and SEQ ID No. 24 in the following table 1, and performing enzyme digestion on the obtained PCR product by SapI and connecting the PCR product to an HXT4S vector to obtain an expression vector HXT4S-JS011-6-HC.

TABLE 1

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

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

The CHO-K1 cells (ATCC, CCL-61) were subjected to suspension serum-free acclimation and selection to make the cells suitable for transient expression, designated as CHO-18, and the CHO-18 cells were cultured in CD CHO medium (Gibco) at 36.5℃120rmp,7% CO ₂ Culturing under the condition that the cell density reaches (2-6) ×10 ⁶ at/mL, passage amplification was performed with CD CHO medium. The day before transfection, CHO-18 cell density was diluted to (1.5-2.0). Times.10 ⁶ Per mL, shaking table (culture conditions of 36.5 ℃,120rmp,7% CO ₂ ) Culturing, and culturing until the next day cell density reaches about 3.5X10 ⁶ Transfection was performed at/mL. During transfection, a tenth of a transfection volume of a CD CHO culture medium is firstly added into a reactor, 1-2ug/mL of the transfection volume of the expression vectors HXT2-JS011-3-LC and HXT4S-JS011-3-HC or HXT4S-JS011-3-HC and HXT4S-JS011-6-HC and 3-14ug/mL of PEI (polyethylenimine) are sequentially added, the mixture is incubated for 20-30min at room temperature after uniform mixing, and then the obtained mixture is slowly added into the pretreated CHO-18 cells to form a transfection mixture which is uniformly mixed at the same time. Placing the transfection mixture into a shaking table for culturing under the conditions of 36.5 ℃,120rmp and 7 percent CO ₂ . The culture period is 6-10 days after transfection, and the feed is fed every two days.

After the completion of the culture of the above transfection mixture, the pellet was discarded by centrifugation at 1000rmp for 10min, and then the cell supernatant was collected by centrifugation at 12000rmp for 30min and subjected to aseptic filtration. Firstly, purifying by using an AKTA Avant purifier, sterilizing a mabselect sure LX column filled with 0.1MNaOH for 15-20min, balancing 3-5 column volumes by using PBS buffer solution, loading the sample, eluting by using pH5.5 sodium acetate buffer solution after loading the sample, and eluting target protein by using pH 3.6 acetic acid-sodium acetate buffer solution; and secondly, carrying out fine purification by using Eshmuno CPX, wherein the balance liquid is a pH5.5, a 50mM acetic acid-sodium acetate system, the eluent is a pH5.5, a 50mM acetic acid-sodium acetate+1M NaCl buffer system, collecting target proteins by adopting a linear elution mode, and obtaining monoclonal antibodies JS011-phage3 and JS011-phage6 by measuring the purity of the monomers by SEC-HPLC.

The gene sequences of antibodies JS011-phage3 and JS011-phage6 are obtained through gene sequencing, so that the amino acid sequence of the antibody is obtained.

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

heavy chain (JS 011-phage3-HC, italics for VH, underlined for HCDR1, HCDR2 and HCDR3 in that order):

the heavy chain variable region JS011-phage3-VH:

QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPQSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCAREGGEWLAIPFDYWGQGTLVTVSS(SEQ ID NO:13)；

wherein according to the Kabat numbering system, comprising:

HCDR1:GYYMH(SEQ ID NO:1)；

HCDR2:WINPQSGGTNYAQKFQG(SEQ ID NO:2)；

HCDR3:EGGEWLAIPFDY(SEQ ID NO:3)；

light chain (JS 011-phage3-LC, italics for VL, underlined for LCDR1, LCDR2 and LCDR3 in that order):

the light chain variable region JS011-phage3-VL:

ELELTQPPSVSVSPGQTASITCSADKLGDKYASWYQQKPGQSPVLVLYEDSKRPSGIPERISGSNSGNTATLTIRGTQPMDEADYYCQTWDTNTVLFGGGTRLTVL(SEQ ID NO:14)；

wherein according to the Kabat numbering system, comprising:

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 (JS 011-phage6-HC, italics for VH, underlined for HCDR1, HCDR2 and HCDR3 in this order):

the heavy chain variable region JS011-phage6-VH:

QVQLVQSGAEVKKPGASVKVSCKASGYTFTGQYIHWARQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARDKAGADYWGQGTLVTVSS(SEQ ID NO:15)；

wherein according to the Kabat numbering system, comprising:

HCDR1:GQYIH(SEQ ID NO:7)；

HCDR2:WINPNSGGTNYAQKFQG(SEQ ID NO:8)；

HCDR3:DKAGADY(SEQ ID NO:9)；

light chain (JS 011-phage6-LC, italics for VL, underlined for LCDR1, LCDR2 and LCDR3 in that order):

the light chain variable region JS011-phage6-VL:

ELALTQPPSVSGSPGQSITISCTGTSSDIGGYDYVSWYQQYPGKAPKLMISGVSNRPSGVSNRFSGSKSGNTASLIISGLQAEDEGDYYCSSYTSRSTRWVFGGGTKLTVL(SEQ ID NO:16)；

wherein according to the Kabat numbering system, comprising:

LCDR1:TGTSSDIGGYDYVS(SEQ ID NO:10)；

LCDR2:GVSNRPS(SEQ ID NO:11)；

LCDR3:SSYTSRSTRWV(SEQ ID NO:12)。

example 3 detection of binding Capacity of anti-4-1 BB antibody

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

Human 4-1BB (obtained by fusing the His tag with the extracellular region sequence of 4-1BB to construct an expression plasmid and purifying by transient 293 cell expression) or murine 4-1BB (vendor Sino Biological, cat. No. 50811-M08H) antigen (1.0. Mu.g/mL) containing His tag was coated with a fixed concentration of His tag using a Thermo Scientific enzyme-labeled instrument, 2% BSA was blockedThen, adding the JS011 antibodies JS011-phage3 and JS011-phage6 (combined with human 4-1 BB: 1 mug/ml start, 3-fold gradient dilution, total 12 concentrations; combined with mouse 4-1 BB: 1 mug/ml start, 2.5-fold gradient dilution, total 12 concentrations); the murine anti-human IgG4 Fc HRP (Southern Biotech, 9200-05) was diluted 5000-fold and used as detection antibody, followed by development with 0.1mg/ml TMB (3, 3', 5' -tetramethylbenzidine), and finally the reaction was stopped with 2M HCl and the plate read at 450nm/620 nm. Fitting of half-maximal effect concentration EC using a four-parameter logistic regression (4 PL) model ₅₀ . An Anti-KLH-IgG4 (CB 25772663, or a light-weight gene sequence of a human IgG4 antibody encoding Anti-hemocyanin KLH is respectively used for constructing expression plasmids, and the expression plasmids are obtained by transient CHO-18 cell expression and purification) is used as a negative control.

The combination curves of JS011-phage3 and JS011-phage6 and human 4-1BB are shown in figure 1, and the combination curves of JS011-phage3 and JS011-phage6 and mouse 4-1BB are shown in figure 2. As can be seen from FIG. 1, JS011-phage3 and JS011-phage6 can both bind to human 4-1BB with similar binding activity, EC ₅₀ 1.5ng/ml and 1.6ng/ml respectively; as can be seen from FIG. 2, JS011-phage3 can be combined with mouse 4-1BB, EC ₅₀ No binding of JS011-phage6 to murine 4-1BB was 6.1 ng/ml.

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

The Jurkat NFKB 4-1BB cells (stably expressing 4-1BB, fusing NFKB with a luciferase reporter gene to construct an expression plasmid, electrically transfecting Jurkat cells, obtaining the Jurkat cells stably expressing NFKB through pressure screening, named Jurkat NFKB cells, electrically transferring the 4-1BB full-length gene to Jurkat NFKB cells, obtaining the Jurkat NFKB 4-1BB stably expressing 4-1BB through pressure screening, and incubating with anti-4-1 BB monoclonal antibodies JS011-phage3 and JS011-phage6 (initial concentration is 100 mug/ml, concentration gradient is 12 times) at 4 ℃ for 30min, and then washing and incubating with a secondary antibody (southern Biotech, cat#2040-09) of fluorescein PE labeled anti-human IgG at 4 ℃ for 30min under a dark condition. Finally, the cells were collected by a flow cytometer (BD Canto II) and the fluorescent antibodies bound to the cell surface were detected. Raw data were analyzed by FlowJo to obtain MFI values (noted MFI-PE) and fitting the antibody dose-dependent binding curve by GraphPad, the results are shown in figure 3, and EC is calculated ₅₀ Wherein the positive controls are Urelumab (BMS control antibody, see patent CN 1867585B) and utomill control antibody, see patent CN 103221428B; the negative control was Anti-KLH-IgG4.

As shown in FIG. 3, JS011-phage6, urerlumab and Utomimumab antibodies were all able to bind with high affinity to 4-1BB on the cell surface of Jurkat NFKB 4-1BB, EC ₅₀ 3.442. Mu.g/mL, 6.961. Mu.g/mL, 1.532. Mu.g/mL and 0.01656. Mu.g/mL, respectively.

Example 5: anti-4-1 BB antibodies block the binding of 4-1BB ligand (4-1 BBL) to Jurkat NFKB 4-1BB cells

The Jurkat NFKB 4-1BB cells were incubated with different concentrations of anti-4-1 BB monoclonal antibodies JS011-phage3 and JS011-phage6 (initial concentration 100. Mu.g/ml, 3-fold dilution, total 12 concentration gradients) and 2. Mu.g/ml biotin-labeled 4-1BBL-hFc (4-1 BB ligand, fusion of the 4-1BBL extracellular region sequence to human FC (hFC) sequence, and purification by transient CHO-18 cell expression) at 4℃for 30min, followed by washing and incubation with fluorescein FITC-labeled secondary antibody (BioLegend, cat# 405202) at 4℃for 30min in the absence of light. Finally, the cells were collected by a flow cytometer (BD Canto II) and the fluorescent antibodies bound to the cell surface were detected. Raw data were analyzed with FlowJo to obtain MFI values (noted MFI-FITC), and antibody dose-dependent inhibition curves were fitted by GraphPad and IC calculated ₅₀ The results are shown in FIG. 4, wherein the positive controls are Urerlumab (BMS control antibody, see patent CN 1867585B) and Utomimumab (Pfizer control antibody, see patent CN 103221428B); the negative control was Anti-KLH-IgG4.

As shown in FIG. 4, the antibodies JS011-phage3, JS011-phage6, urerlumab are incapable of blocking the binding of 4-1BBL to 4-1BB on the cell surface of Jurkat NFKB4-1BB, utomimumab is capable of effectively blocking the binding of 4-1BBL to 4-1BB on the cell surface of Jurkat NFKB4-1BB, IC ₅₀ 0.3431. Mu.g/ml.

Example 6: activity detection of anti-4-1 BB antibodies in the luciferase reporter System (CHO FCGR2B 4G6/Jurkat NFKB4-1BB double cells)

CHO FCGR2B 4G6 cells (stably high expressing human CD32B, constructing an expression plasmid from the CD32B gene, and electrotransfecting CHO-K1 cells, and pressure screening to obtain monoclonal cells stably expressing CD32B, designated CHO FCGR2B 4G6 cells) at 3×10 per well ⁴ The individual cell numbers were added to 96-well flat bottom white plates (Corning, cat#3917) and incubated overnight at 37 ℃. The following day effector cells Jurkat NFKB4-1BB (stably expressing NlucP/NFKB-RE and 4-1 BB) were grown at 1X 10 per well ⁵ The number of individual cells was added to the cell plate. The anti-4-1 BB monoclonal antibodies JS011-phage3 and JS011-phage6 (initial concentration 25. Mu.g/ml, 4-fold dilution, total 9 concentration gradients) were then added to the cell plates with assay buffer (RPMI 1640 (1X) +2% FBS) and incubated in an incubator at 37℃for 4-6 hours. Finally, a Nano-Glo luciferase detection reagent (Promega) is added into the cell antibody mixed system, and a chemiluminescent signal (marked as RLU) is detected by a multifunctional enzyme-labeled instrument (TECAN M1000 pro). EC was calculated by fitting a four-parameter regression curve with GraphPad prism software ₅₀ Values, results are shown in figure 5. Positive controls were Urelumab (BMS control antibody, see patent CN 1867585B) and utomill control antibody, see patent CN 103221428B.

As shown in FIG. 5, JS 011-phase 3, JS 011-phase 6, urerlumab and Utomimumab have strong T cell activating activity in a luciferase reporter gene system composed of CHO FCGR2B4G6 cells and effector cell Jurkat NFKB4-1BB, EC ₅₀ 0.03888 mug/mL, 0.4499 mug/mL, 1.038 mug/mL and 0.02391 mug/mL respectively, JS011-phage3 is equivalent to Utomimumab in activity; the JS011-phage6 has the same activity as Urerlumab, and the activating capacity of the JS011-phage3 to T cells is stronger than that of the JS011-phage6.

Example 7: activity detection of anti-4-1 BB antibody in luciferase reporter System (THP-1/Jurkat NFKB4-1BB double cell)

THP-1 cells (a human myeloid leukemia monocyte, CD32B positive) and effector cells Jurkat NFKB4-1BB (stably expressed NlucP/NFKB-RE and 4-1 BB) were each 5X 10 per well ⁴ Number of cells and 1X 10 per well ⁵ The individual cell numbers were added to 96-well flat bottom white plates (Corning, cat#3917). Then the sample was buffered with assay buffer (RPMI 1640 (1X) + 2%F)BS) anti-4-1 BB monoclonal antibodies JS011-phage3 and JS011-phage6 (initial concentration 100. Mu.g/ml, 4-fold dilution, total 10 concentration gradients) were added to the cell plates and incubated for 4-6 hours in an incubator at 37 ℃. Finally, a Nano-Glo luciferase detection reagent (Promega) is added into the cell antibody mixed system, and a chemiluminescent signal (marked as RLU) is detected by a multifunctional enzyme-labeled instrument (TECAN M1000 pro). EC was calculated by fitting a four-parameter regression curve with GraphPad prism software ₅₀ Values, results are shown in FIG. 6. Positive controls were Urelumab (BMS control antibody, see patent CN 1867585B) and utomill control antibody, see patent CN 103221428B.

As shown in FIG. 6, JS 011-phase 3, JS 011-phase 6, urerlumab and Utomimumab have strong T cell activating activity in a luciferase reporter gene system composed of THP-1 cells and effector cells Jurkat NFKB 4-1BB, EC ₅₀ 0.006642. Mu.g/mL, 0.2826. Mu.g/mL, 218.6. Mu.g/mL and 0.04173. Mu.g/mL, respectively.

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

Raji cells (a human myeloid leukemia monocyte, CD32B positive, 4-1BBL positive) were plated at 5X 10 per well ⁴ The number of cells was added to 96-well flat-bottomed white plates (Corning, cat#3917), jurkat NFKB 4-1BB cells (stably expressing NlucP/NFKB-RE and 4-1 BB) or Jurkat NFKB cells (stably expressing NlucP/NFKB-RE) at 1X 10 per well, respectively ⁵ The individual cell numbers were added to the cell plates. The anti-4-1 BB monoclonal antibodies JS011-phage3 and JS011-phage6 (initial concentration 100. Mu.g/ml, 4-fold dilution, total 10 concentration gradients) were then added to the cell plates with experimental buffer (RPMI 1640 (1X) +2% FBS) and incubated for 4-6 hours in an incubator at 37 ℃. Finally, a Nano-Glo luciferase detection reagent (Promega) is added into the cell antibody mixed system, and a chemiluminescent signal (marked as RLU) is detected by a multifunctional enzyme-labeled instrument (TECAN M1000 pro). EC was calculated by fitting a four-parameter regression curve with GraphPad prism software ₅₀ Values, results are shown in fig. 7 and 8, respectively. Wherein the positive control is Urerlumab (BMS control antibody, see patent CN 1867585B) and UtomimumabPfizer control antibody, see patent CN 103221428B).

As shown in FIG. 7, JS011-phage3, JS011-phage6 and Urerlumab have the activity of activating T cells in a luciferase reporter gene system formed by Raji cells and effector cells Jurkat NFKB 4-1BB (4-1 BB is highly expressed), and EC ₅₀ 9.446. Mu.g/mL, 2.713. Mu.g/mL and 7.240. Mu.g/mL, respectively, utomimumab was present in this system to block the 4-1BBL binding effect and thus showed a decrease in activity.

As shown in FIG. 8, in the luciferase reporter gene system composed of Raji cells and effector cells Jurkat NFKB (4-1 BB low expression), JS011-phage3 and Urerlumab respectively act synergistically with 4-1BBL on the surface of Raji cells to activate T cells, EC ₅₀ 9.450. Mu.g/mL and 86.01. Mu.g/mL, respectively. JS011-phage6 and Utomimumab are inactive in this system.

Example 9: anti-4-1 BB antibody cytokine storm Studies

PBMC from two different volunteers (volunteer 1ID #: LP191225, volunteer 2ID #: LP 190812) were used at 3X 10 per well ⁵ The cell number is respectively added into a 96-well round bottom plate, and then anti-4-1 BB monoclonal antibodies JS011-phage3, JS011-phage6, urerlumab and Utomimumab are added into the cell plate according to the concentration of 10 mug/mL, the temperature is 37 ℃ and the concentration of 5% CO is 5% ₂ After 24h incubation, the supernatants were collected by centrifugation and assayed for IL-2, IL-4, IL-6, IL-10, TNF. Alpha. And IFN. Gamma. Cytokine release using BD CBAhuman Th1/Th2 cytokine kit II, as shown in FIGS. 9A-9F. The positive control was OKT3 antibody (obtainable from the hybridoma deposited with the american type culture collection under accession number ATCC CRL 8001); the negative control was Anti-KLH-IgG4.

As shown in fig. 9A to 9F, none of JS011-phage3, JS011-phage6, urelumab, and utomill caused cytokine storm at the concentration of 10 μg/mL.

Example 10: inhibition of h41BB humanized mice transplanted MC38 tumor growth by anti-4-1 BB antibodies

1. Purpose of testing

The anti-4-1 BB monoclonal antibodies of the present application were evaluated for their anti-tumor effect in a h41BB humanized mice transplanted mice colon cancer MC38 subcutaneous tumor model.

2. Test procedure

6-8 week old female h41BB humanized mice (purchased from Biotechnology Co., ltd.) were inoculated subcutaneously on the right back with 1X 10 ⁶ MC38 cells. The tumor volume to be averaged is about 90mm ³ At this time, the appropriate animals were selected and randomly divided into 5 groups of 6 animals each according to tumor volume. Respectively is

1. Saline control group (solvent control group);

Treatment group:

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

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

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

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

Intraperitoneal injection was administered 2 times per week for 3 weeks, and the experiment was ended 4 days after the last administration. Tumor volume and body weight were measured 2 times per week and mice body weight and tumor volume were recorded. At the end of the experiment, mice were euthanized and tumor inhibition rates TGI (%) = [1- (Ti-T0)/(Vi-V0) ]x100% were calculated. (Ti: mean tumor volume of the treatment group on day i of administration, T0: mean tumor volume of the treatment group on day 0 of administration; vi: mean tumor volume of the solvent control group on day i of administration, V0: mean tumor volume of the solvent control group on day 0 of administration).

As shown in FIG. 10, on day 20 after administration, the mean tumor volume of the saline control group was 2213mm ³ The average tumor volume of the Urilumab (BMS, see patent CN 1867585B) group was 430mm ³ Compared with a normal saline control group, the tumor growth is obviously inhibited, and the tumor inhibition rate is 83.9%; the average tumor volume of the Utomimumab (Pfizer, see patent CN 103221428B) group was 1937mm ³ No obvious tumor inhibition effect is seen, and the tumor inhibition rate is 13.0%; the average tumor volume of JS011-phage3 group is 506mm ³ Remarkably inhibit the growth of tumor with a tumor inhibition rate of 64.7%; the average tumor volume of JS011-phage6 group is 674mm ³ Remarkably inhibit the growth of tumor, and the tumor inhibition rate is 50.6%. The results indicate that the humanization of h41BBIn the mouse transplanted MC38 WT model, at a dosage level of 1mg/kg, JS011-phage3 and JS011-phage6 both show remarkable tumor inhibition.

Example 11: toxicity study of anti-4-1 BB antibody on h41BB humanized mouse transplanted MC38 tumor-bearing mice

1. Purpose of testing

The toxicity of the anti-4-1 BB monoclonal antibody in the application was evaluated in mice transplanted with h41BB humanized mice, colon cancer MC38 cell subcutaneous tumor-bearing mice.

2. Test procedure

6-8 week old female h41BB humanized mice (purchased from Biotechnology Co., ltd.) were inoculated subcutaneously on the right back with 1X 10 ⁶ MC38 cells. The tumor volume to be averaged was about 115mm ³ At this time, the appropriate animals were selected and randomly divided into 5 groups of 6 animals each according to tumor volume. Respectively is

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

treatment group:

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

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

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

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

Intraperitoneal injection was given 2 times per week, 3 times continuously, and animals were euthanized 7 days after the last administration. The spleen of the mice was weighed. Mouse livers were collected and CD3 was detected in the livers of different groups of animals by flow cytometry ⁺ 、CD3 ⁺ CD4 ⁺ 、CD3 ⁺ CD8 ⁺ 、CD3 ⁺ CD8 ⁺ CD11c ⁺ CD3 ^- NK1.1 ⁺ Cell ratio to observe in vivo toxicity of the anti-4-1 BB monoclonal antibody.

As shown in fig. 11, on day 14 after dosing, the spleen weights of the mice in the Urelumab (BMS, see patent CN 1867585B), JS011-phage3, JS011-phage6, and Utomilumab (Pfizer, see patent CN 103221428B) groups were significantly increased compared to the Anti KLH igg4 negative control group. Indicating that 10mg/kg Urerlumab can cause splenomegaly of mice, and JS011-phage3 and JS011-phage6 can not cause splenomegaly of animals. The result suggests that JS011-phage3 and JS011-phage6 of the application cannot cause excessive activation of immune cells in spleen, and can have better safety in clinic.

As shown in fig. 12A, the number of lymphocytes contained in the liver unit weight was analyzed on day 14 after administration, and the number of lymphocytes in the mouse livers of JS011-phage3 group, JS011-phage6 group, utomilumab group and Urelumab group was increased compared to the Anti-KLH igg4 negative control group, and the number of lymphocytes in the mouse livers of the Urelumab group was significantly higher than those of the JS011-phage3 group, JS011-phage6 group and Utomilumab group, indicating that 10mg/kg of Urelumab resulted in a significant increase in the number of lymphocytes in the mouse livers. As shown in fig. 12B to 12E, changes in the proportions of different sub-populations of liver lymphocytes were analyzed on day 14 after dosing, and CD3 in the livers of mice of JS011-phage3 group, JS011-phage6 group, utomilumab group and Urelumab group, compared to the Anti-KLH igg4 negative control group ⁺ CD4 ⁺ T cell ratio is reduced, CD3 ⁺ CD8 ⁺ T cell proportion was elevated and Urerlumab group CD3 ⁺ CD8 ⁺ The T cell proportion is significantly higher than that of CD3 in the liver of mice of JS011-phage3 group, JS011-phage6 group and Utomimumab group, urerlumab group ⁺ CD8 ⁺ CD11c ⁺ T cell proportion was significantly increased, indicating overactivated CD3 in liver of Urerlumab mice ⁺ CD8 ⁺ T cell fraction increased significantly, while Urerlumab group mice had CD3 in liver ^- NK1.1 ⁺ NK cell proportion of (B) was decreased, indicating that 10mg/kg Urilumab resulted in CD3 in mouse liver ⁺ CD8 ⁺ The proportional number of T cells increases significantly. The reason for the increased lymphocyte number in the liver of mice is CD3 ⁺ CD8 ⁺ Absolute number of T cells increases significantly, CD3 ⁺ CD4 ⁺ Absolute numbers of T and NK cells increased, but due to CD3 ⁺ CD8 ⁺ The large increase in T cell proportion leads to CD3 ⁺ CD4 ⁺ T and NK cell ratios decreased. From post-administration CD3 ⁺ CD8 ⁺ The JS011-phage3 and JS011-phage6 are obviously excellent from the view of the absolute value and the relative value of the T cells in the liverIn Urerlumab, it was suggested that there may be lower hepatotoxicity in the clinic.

The foregoing description is only of the preferred embodiments of the present application and is not intended to limit the scope of the present application. Any modifications, equivalent substitutions, improvements, etc. that are within the spirit and principles of the present application are intended to be included within the scope of the present application.

Claims

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

2. The antibody or antigen-binding fragment thereof of claim 1, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3;

3. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody is according to the Kabat numbering system,

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

4. An antibody or antigen binding fragment thereof according to claim 3, wherein the antibody is according to the Kabat numbering system,

the HCDR1, HCDR2 and HCDR3 respectively have amino acid sequences shown as SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3, and the LCDR1, LCDR2 and LCDR3 respectively have amino acid sequences shown as SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6; or alternatively

5. The antibody or antigen-binding fragment thereof of claim 4, comprising:

preferably, the antibody or antigen binding fragment thereof comprises:

6. The antibody or antigen binding fragment thereof of any one of claims 1-5, further comprising a human heavy chain constant region selected from the group consisting of a heavy chain constant region of human IgG1, igG2, igG3, or IgG4, preferably a heavy chain constant region of human IgG4 or a heavy chain constant region of human IgG4 with an S228P amino acid substitution; the human light chain constant region is selected from the light chain constant region of a lambda light chain or a kappa light chain.

7. The antibody or antigen-binding fragment thereof of any one of claims 1-5, comprising:

Preferably, the antibody or antigen binding fragment thereof comprises:

8. The antibody or antigen-binding fragment thereof of 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 at least one of a Fab, fab ', F (ab') 2, fv, scFv, and sdAb.

9. An isolated antibody or antigen binding fragment thereof having at least one of the following properties:

binding to the same, or fully overlapping or partially overlapping epitopes of human 4-1BB protein as the antibody or antigen binding fragment thereof of any one of claims 1-8;

(ii) competes with the antibody or antigen-binding fragment thereof of any one of claims 1-8 for binding to an epitope of human 4-1BB protein.

10. A polynucleotide molecule comprising at least one of 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 a complement thereof.

11. An expression vector comprising the polynucleotide molecule of claim 10, preferably the expression vector is a eukaryotic expression vector.

12. A host cell comprising the polynucleotide molecule of claim 10, or comprising the expression vector of claim 11, preferably the host cell is a eukaryotic cell, more preferably a mammalian cell.

13. A host cell according to claim 12 for expressing an antibody or antigen-binding fragment thereof according to any one of claims 1-8 or an isolated antibody or antigen-binding fragment thereof according to claim 9.

14. A method of making an antibody or antigen-binding fragment thereof according to any one of claims 1-8, or an isolated antibody or antigen-binding fragment thereof according to claim 9, comprising expressing the antibody or antigen-binding fragment thereof in the host cell of claim 12, and recovering the expressed antibody or antigen-binding fragment thereof from the host cell.

15. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1-8 or an isolated antibody or antigen-binding fragment thereof according to claim 9 or a polynucleotide molecule according to claim 10 or an expression vector according to claim 11 or a host cell according to claim 12, and a pharmaceutically acceptable carrier or excipient.

16. Use of 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 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 manufacture of a medicament for the treatment or prevention of a 4-1BB mediated disease; preferably, the 4-1 BB-mediated disease is cancer; more preferably, the cancer is selected from: at least one of melanoma, breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, kidney cancer, lung cancer, liver cancer, gastric cancer, colorectal cancer, bladder cancer, head and neck cancer, thyroid cancer, esophageal cancer, cervical cancer, sarcoma, multiple myeloma, leukemia, lymphoma, gall bladder cancer, and glioblastoma.

17. A kit comprising an antibody or antigen-binding fragment thereof according to any one of claims 1-8 or an isolated antibody or antigen-binding fragment thereof according to claim 9 or a polynucleotide molecule according to claim 10 or an expression vector according to claim 11 or a host cell according to claim 12 or a pharmaceutical composition according to claim 15.

18. A method of detecting 4-1BB using the antibody or antigen-binding fragment thereof of any one of claims 1-8, or the isolated antibody or antigen-binding fragment thereof of claim 9, comprising contacting the antibody or antigen-binding fragment thereof with a sample, detecting a conjugate formed by the antibody or antigen-binding fragment thereof and 4-1BB, and optionally quantifying the conjugate.