CN112480259B

CN112480259B - anti-TNFR 2 antibodies and uses thereof

Info

Publication number: CN112480259B
Application number: CN202110159645.XA
Authority: CN
Inventors: 杨勇飞; 曹淑贞; 蒋雪园; 李健晖
Original assignee: Baccetus Beijing Pharmaceutical Technology Co ltd
Current assignee: DOMA Pharmaceutical Technology (Suzhou) Co.,Ltd.
Priority date: 2021-02-05
Filing date: 2021-02-05
Publication date: 2021-05-04
Anticipated expiration: 2041-02-05
Also published as: CN112480259A

Abstract

The invention discloses an anti-TNFR 2 antibody and application thereof. The antibody or the antigen binding fragment thereof can be specifically combined with human TNFR2, has high affinity and is stable. In addition, the antibody or the antigen binding fragment thereof can effectively inhibit the growth of tumors, is safe for individual administration, and can be used for treating diseases (such as tumors) related to TNFR2 signal pathways.

Description

anti-TNFR 2 antibodies and uses thereof

Technical Field

The invention relates to the technical field of biological medicines, in particular to an anti-TNFR 2 antibody and application thereof, and especially application in disease (such as tumor) treatment.

Background

TNFRSF1B (TNF receptor super family member 1 b) is a member of the TNF receptor superfamily, also known as TNFR2, and is mainly expressed in Treg cells and a variety of tumor cells in the tumor microenvironment (e.g., ovarian cancer and intestinal cancer). Its ligand TNF mainly recognizes TNFR1 for apoptosis, and relies on TNFR2 for T cell survival-promoting the transcription of pro-survival gene by NF-kB signal, thereby promoting cell proliferation and survival. Many experiments show that the blocking antibody can specifically kill Treg cells and tumor cells in a tumor microenvironment and is used for treating various tumors; the activated antibody can activate Treg cells and further inhibit Teff cells, so that autoimmune diseases are treated, and a new target is provided for immunotherapy.

Cancer is one of the diseases that currently causes the highest mortality in humans. Therefore, the development of antibody drugs capable of efficiently targeting TNFR2 signal pathways provides possibility for the treatment of various tumors and diseases related to the immune system, and has great application potential and market value.

Disclosure of Invention

The present invention provides an antibody or antigen-binding portion thereof that specifically binds TNFR2 protein.

Specifically, the TNFR2 protein is human or other primate TNFR2 protein.

In particular, the other primates mentioned above include various monkey species such as, but not limited to, a cynomolgus monkey, a macaque, a baboon, a mandrill, and the like; specifically, the macaque includes rhesus macaque, cynomolgus macaque, macaque and the like.

Specifically, the antibody or antigen-binding portion thereof can specifically bind human TNFR2 and block the interaction of human TNFR2 with TNFa.

In particular, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable region and a light chain variable region.

In particular, the heavy chain variable region comprises VHCDR1, VHCDR2 and VHCDR3, wherein:

the amino acid sequences of the VHCDR1-3 are shown as SEQ ID NO 5, SEQ ID NO 6 and SEQ ID NO 7, respectively, or the amino acid sequences of the VHCDR1-3 have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homology (numbering according to Kabat) with the amino acid sequences shown as SEQ ID NO 5, SEQ ID NO 6 and SEQ ID NO 7, respectively; or the like, or, alternatively,

the amino acid sequences of the VHCDR1-3 are shown as SEQ ID NO 11, SEQ ID NO 12 and SEQ ID NO 13, respectively, or the amino acid sequence of the VHCDR1-3 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homology (numbering according to Kabat) with the amino acid sequences shown as SEQ ID NO 11, SEQ ID NO 12 and SEQ ID NO 13, respectively; or the like, or, alternatively,

the amino acid sequence of the VHCDR1-3 is shown as SEQ ID NO 17, SEQ ID NO 18 and SEQ ID NO 19 respectively, or the amino acid sequence of the VHCDR1-3 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homology (numbering according to Chothia) with the amino acid sequences shown as SEQ ID NO 17, SEQ ID NO 18 and SEQ ID NO 19 respectively; or the like, or, alternatively,

the amino acid sequence of the VHCDR1-3 is shown as SEQ ID NO. 23, SEQ ID NO. 24 and SEQ ID NO. 25 respectively, or the amino acid sequence of the VHCDR1-3 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homology (numbering according to Chothia) with the amino acid sequences shown as SEQ ID NO. 23, SEQ ID NO. 24 and SEQ ID NO. 25 respectively;

and, the light chain variable region comprises VLCDR1, VLCDR2, and VLCDR3, wherein:

the amino acid sequences of the VLCDR1-3 are shown in SEQ ID NO 8, SEQ ID NO 9 and SEQ ID NO 10, respectively, or the amino acid sequence of the VLCDR1-3 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homology (numbering according to Kabat) with the amino acid sequences shown in SEQ ID NO 8, SEQ ID NO 9 and SEQ ID NO 10, respectively; or the like, or, alternatively,

the amino acid sequences of the VLCDR1-3 are respectively shown as SEQ ID NO. 14, SEQ ID NO. 15 and SEQ ID NO. 16, or the amino acid sequence of the VLCDR1-3 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homology (according to Kabat numbering) with the amino acid sequences shown as SEQ ID NO. 14, SEQ ID NO. 15 and SEQ ID NO. 16, respectively; or the like, or, alternatively,

the amino acid sequences of the VLCDR1-3 are respectively shown as SEQ ID NO 20, SEQ ID NO 21 and SEQ ID NO 22, or the amino acid sequence of the VLCDR1-3 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homology (numbering according to Chothia) with the amino acid sequences shown as SEQ ID NO 20, SEQ ID NO 21 and SEQ ID NO 22; or the like, or, alternatively,

the amino acid sequence of the VLCDR1-3 is shown in SEQ ID NO 26, SEQ ID NO 27 and SEQ ID NO 28 respectively, or the amino acid sequence of the VLCDR1-3 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homology (numbering according to Chothia) with the amino acid sequences shown in SEQ ID NO 26, SEQ ID NO 27 and SEQ ID NO 28 respectively.

In one embodiment of the invention, for the antibody or antigen binding portion thereof, the VHCDR1-3 of the heavy chain variable region is set forth in SEQ ID NO 5, SEQ ID NO 6 and SEQ ID NO 7, respectively, and the VLCDR1-3 of the light chain variable region is set forth in SEQ ID NO 8, SEQ ID NO 9 and SEQ ID NO 10, respectively.

In another embodiment of the invention, for the antibody or antigen binding portion thereof, the VHCDR1-3 of the heavy chain variable region is set forth in SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13, respectively, and the VLCDR1-3 of the light chain variable region is set forth in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, respectively.

More specifically, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 1, or the amino acid sequence of the heavy chain variable region comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homology with the amino acid sequence shown as SEQ ID NO. 1, and

the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 2, or the amino acid sequence of the light chain variable region comprises an amino acid sequence which has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% of homology with the amino acid sequence shown as SEQ ID NO. 2.

More specifically, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 3, or the amino acid sequence of the heavy chain variable region comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homology with the amino acid sequence shown as SEQ ID NO. 3, and

the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 4, or the amino acid sequence of the light chain variable region comprises an amino acid sequence which has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% of homology with the amino acid sequence shown as SEQ ID NO. 4.

In one embodiment of the invention, for the antibody or antigen-binding portion thereof, the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO. 1 and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO. 2.

In another embodiment of the invention, for the antibody or antigen-binding portion thereof, the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO. 3 and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO. 4.

Specifically, for the antibodies of the invention or antigen binding portions thereof, wherein the antibody or antigen binding portion thereof is a whole antibody, a multispecific antibody (e.g., bispecific antibody), a multispecific single-chain antibody (e.g., bispecific single-chain antibody), a single-chain antibody (scFv), Fab ', F (ab')₂Linear antibodies or Fv antibodies, and any polypeptide comprising an antibody binding domain or homologous antibody binding domain. The antibody binding domain may comprise, among other things, an intact heavy and/or light chain CDR, an intact heavy and/or light chain variable region of an antibody, an intact full-length heavy and/or light chain, or a single, two, three, four, five or six CDR from the antibody. Single chain antibodies comprise a heavy chain variable region and a light chain variable region.

Specifically, the antibody of the present invention, or an antigen-binding portion thereof, is obtained using a protein immunization method or a DNA immunization method; further, the protein immunization method comprises immunizing the human TNFR2 protein serving as an immunogen to obtain an antibody or an antigen-binding part thereof; the DNA immunization method comprises the step of immunizing by taking DNA plasmid for coding the human TNFR2 protein as immunogen to obtain an antibody or an antigen binding part thereof.

In another aspect of the invention, there is provided an isolated DNA encoding the antibody or antigen binding portion thereof described above.

In another aspect of the present invention, there is provided a vector comprising the DNA of any of the above.

In particular, the vector is capable of expression in vivo or in vitro or ex vivo. Further preferably, the expression vector is expressed at a high level in vivo in cells. Specifically, the expression vector is a prokaryotic expression vector, a lentiviral expression vector, a plasmid, a cosmid, a phage, a virus, and the like. More specifically, the prokaryotic expression vector is an Escherichia coli series.

In another aspect of the present invention, there is provided a cell comprising the above DNA or vector.

In particular, the cell may be eukaryotic or prokaryotic. More specifically, the cell may be a yeast cell, 293 cell, CHO cell, escherichia coli, or the like.

In another aspect of the present invention, there is provided a method for producing the above-mentioned antibody or an antigen-binding portion thereof, which comprises preparing the antibody or the antigen-binding portion thereof by a protein immunization method, a DNA immunization method, or culturing a cell comprising the above-mentioned DNA or a vector.

Specifically, the protein immunization method comprises immunizing by taking human TNFR2 protein as an immunogen to obtain an antibody or an antigen-binding part thereof; collecting spleen cells of a non-human animal immunized with TNFR2, fusing the collected spleen cells with SP2/0 cells to obtain hybridoma cells, culturing the hybridoma cells, and isolating and purifying the above antibody or an antigen-binding portion thereof. Preferably, the hybridoma cells are introduced into a non-human animal, and ascites fluid is collected from the non-human animal.

Specifically, the DNA immunization method comprises immunizing by taking a DNA plasmid for coding the human TNFR2 protein as an immunogen to obtain an antibody or an antigen-binding part thereof.

Specifically, the step of culturing cells comprises culturing cells comprising the above DNA or vector to obtain an antibody or antigen-binding fragment thereof that specifically binds TNFR 2.

In another aspect of the present invention, there is provided a method for preparing a hybridoma, which comprises immunizing a non-human animal with human TNFR2 to obtain a hybridoma, collecting spleen cells of the non-human animal after TNFR2 immunization, and fusing the collected spleen cells with SP2/0 cells to obtain a hybridoma.

In another aspect of the invention, a hybridoma cell is provided which produces the above-described antibody, or antigen-binding portion thereof.

Specifically, the human TNFR2 protein is a recombinant human TNFR2 fusion protein, and particularly, the protein also contains a human Fc tag.

Specifically, the human TNFR2 protein is shown in SEQ ID NO: 31.

In another aspect of the invention, there is provided a T cell antigen receptor or CAR molecule comprising the anti-TNFR 2 antibody or antigen binding fragment thereof described above.

In another aspect of the present invention, there is provided an antibody-drug conjugate (ADC) comprising the above antibody or an antigen-binding fragment thereof covalently bound to a drug.

Specifically, the drug in the ADC may be a chemical synthetic drug, an antibiotic, or various biological drugs.

In another aspect of the invention, there is provided a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof as described above, or a T cell antigen receptor or CAR molecule, or an antibody drug conjugate as described above, of the invention, and a pharmaceutically acceptable carrier.

Specifically, the above-mentioned pharmaceutically acceptable carrier may be one or more, and includes, but is not limited to, diluents, binders, humectants, surfactants, lubricants, or disintegrants, and the like.

Specifically, the above pharmaceutical composition may be administered in a nanocarrier, a viral vector, a microcapsule, a liposome, or the like.

In particular, the above pharmaceutical compositions may also contain other active ingredients for the treatment of the same or different indications.

In another aspect of the present invention, there is provided a kit comprising the above-described antibody or antigen-binding fragment thereof of the present invention.

In another aspect of the present invention, there is provided a chip comprising the above antibody or antigen-binding fragment thereof of the present invention.

In another aspect of the present invention, there is provided an application of the above antibody or antigen binding portion thereof, the above isolated DNA, vector, cell, T cell antigen receptor or CAR molecule, antibody drug conjugate, pharmaceutical composition, kit, and chip in TNFR2 related research.

Specifically, the above applications are applications of the antibody or antigen binding portion thereof, the isolated DNA, the vector, the cell, the T cell antigen receptor or the CAR molecule, the antibody drug conjugate, and the pharmaceutical composition of the present invention in preparing drugs for preventing and/or treating diseases, and screening drugs for preventing and/or treating diseases.

Specifically, the diseases are diseases related to TNFR2 signal pathway, such as tumor, autoimmune diseases, transplant rejection and the like. Specifically, the tumor includes malignant tumors such as cancer, sarcoma, hematological malignancies, and the like. In a specific embodiment, the malignant tumor is melanoma, lymphoma, bladder cancer, non-small cell lung cancer, head and neck cancer, colon cancer. Specifically, the autoimmune diseases, such as, but not limited to, systemic lupus erythematosus, myasthenia gravis, rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, uveitis, diabetes and the like. Specifically, the Graft rejection reaction may be Graft-Versus-Host Disease (GVHD) or Host-Versus-Graft Disease (HVGD).

Specifically, the application can also be the application of the antibody or the antigen binding part thereof, the kit and the chip in the preparation of products for detecting TNFR 2.

Specifically, the detection may be qualitative detection or quantitative detection.

In another aspect of the invention, the invention provides a method of treating a disease, said method comprising the step of administering to said subject a therapeutically effective amount of an antibody or antigen-binding portion thereof, T cell antigen receptor or CAR molecule, antibody drug conjugate, pharmaceutical composition according to the invention.

In another aspect of the invention, a method of combined treatment of a malignancy in a subject is provided, comprising administering to the subject a therapeutically effective amount of the antibody, or antigen-binding portion thereof, T-cell antigen receptor or CAR molecule, antibody drug conjugate, pharmaceutical composition of the invention, and further comprising administering to the subject a therapeutically effective amount of another agent for treating a malignancy or administering another method for treating a malignancy, such as chemotherapeutic drugs, surgical treatments, radiation treatments, biological treatments, chinese traditional medicine treatments, minimally invasive treatments, and the like.

Specifically, the malignant tumor may be selected from: melanoma, lymphoma, bladder cancer, non-small cell lung cancer, head and neck cancer, colon cancer.

In another aspect of the invention, there is provided a method of inducing an immune response, said method comprising administering to a subject an antibody or antigen-binding portion thereof, a T cell antigen receptor or CAR molecule, an antibody drug conjugate, a pharmaceutical composition according to the invention.

In another aspect of the present invention, there is provided a method for detecting TNFR2, said method comprising contacting a sample to be detected with an antibody or antigen-binding fragment thereof of the present invention, and then detecting a complex formed between TNFR2 and said antibody or antigen-binding fragment thereof.

Specifically, the detection method is to detect the existence or content of TNFR 2. Wherein the presence or absence is indicated, and the content may be an expression amount, a protein concentration, or the like. In particular, the detection may be an immunohistochemical detection.

In another aspect of the present invention, there is provided a method for diagnosing a disease associated with TNFR2, said method comprising contacting a sample to be tested with the antibody or antigen-binding fragment thereof of the present invention, and then detecting a complex formed between TNFR2 and said antibody or antigen-binding fragment thereof.

The invention has the beneficial effects that: the antibody or the antigen binding fragment thereof obtained by screening can be specifically bound with human TNFR2, has high affinity and stable performance. In addition, the antibody or the antigen binding fragment thereof can effectively inhibit the growth of tumors, is safe for individual administration, and can be used for treating diseases (such as tumors) related to TNFR2 signal pathways.

Drawings

FIG. 1 shows the results of flow cytometric assays for different anti-TNFR 2 antibodies blocking the binding between human TNFR2 and TNFa, with PBS used as a negative control.

FIG. 2 is a line graph showing the change of body weight of experimental animals with time after the anti-TNFR 2 antibody treatment, and physiological saline (G1) was used as a negative control.

FIG. 3 is a line graph showing the change of tumor volume in experimental animals with time after the anti-TNFR 2 antibody treatment, and physiological saline (G1) was used as a negative control.

Detailed Description

Unless otherwise defined herein, scientific and technical terms and their abbreviations used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art to which the invention belongs. Some of the terms and abbreviations used herein are listed below.

Antibody: antibody, Ab;

immunoglobulin: immunologlobulin, Ig;

heavy chain: heavy chain, HC;

light chain: light chain, LC;

heavy chain variable region: heavy chain variable domain, VH;

heavy chain constant region: a heavy chain constant domain, CH;

light chain variable region: light chain variable domain, VL;

light chain constant region: light chain constant domain, CL;

complementarity determining region: complementary determining region, CDR, refers to the antigen complementary binding region of an antibody;

fab fragment: an anti binding fragment, Fab;

fc fragment: fragment crystalline region, Fc;

monoclonal antibodies: monoclonal antibodies, mAbs;

bispecific antibodies: bispecific antibodies, BsAb.

As used herein, the term "antibody" refers to an immunoglobulin molecule that comprises at least one antigen recognition site and is capable of specifically binding an antigen. The term "antibody" as referred to herein is understood in its broadest sense and encompasses monoclonal antibodies, polyclonal antibodies, antibody fragments, multispecific antibodies (e.g., bispecific antibodies) comprising at least two different antigen binding domains. Antibodies also include murine, chimeric, humanized, human, and other sources of antibodies. The antibodies of the invention may be derived from immunoglobulin molecules from any animal, including but not limited to humans, non-human primates, mice or rats, and the like. The antibody may contain additional alterations such as unnatural amino acids, Fc effector function mutations and glycosylation site mutations. Antibodies also include post-translationally modified antibodies, fusion proteins comprising an antigenic determinant of an antibody, and immunoglobulin molecules comprising any other modification to the antigen recognition site, so long as the antibodies exhibit the desired biological activity.

Immunoglobulins can be classified into 5 classes according to the amino acid sequence of the antibody heavy chain constant region: IgA, IgD, IgE, IgG and IgM, which can be further divided into different subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, and the like. Light chains can be classified as either lambda or kappa chains, depending on their amino acid sequence. The antibodies of the invention may be of any class (e.g., IgA, IgD, IgE, IgG, and IgM) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, or IgA 2).

The term "antigen-binding fragment" includes, but is not limited to: a Fab fragment having VL, CL, VH and CH1 domains; a Fab' fragment, which is a Fab fragment having one or more cysteine residues at the C-terminus of the CH1 domain; an Fd fragment having VH and CH1 domains; an Fd' fragment having VH and CH1 domains and one or more cysteine residues C-terminal to the CH1 domain; fv fragments having the VL and VH domains of a single arm of an antibody; a dAb fragment consisting of a VH domain or a VL domain; an isolated CDR region; a F (ab ')2 fragment which is a bivalent fragment comprising two Fab' fragments connected by a disulfide bridge at the hinge region; single chain antibody molecules (e.g., single chain Fv; scFv); a "diabody" with two antigen-binding sites, comprising a heavy chain variable domain (VH) linked to a light chain variable domain (VL) in the same polypeptide chain; a "linear antibody" comprising a pair of tandem Fd segments (VH-CH1-VH-CH1) that together with a complementary light chain polypeptide form a pair of antigen binding regions; and modified forms of any of the foregoing which retain antigen binding activity.

As used herein, the term "CDR" refers to complementarity determining regions within an antibody variable sequence. For each variable region, there are three CDRs, called CDR1, CDR2, and CDR3, in each variable region of the heavy and light chains. The exact boundaries of these CDRs are defined differently for different systems. The systems described by Kabat et al (Kabat et al, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provide a clear residue numbering system for the variable regions of antibodies, but also provide the residue boundaries that define the three CDRs, which may be referred to as Kabat CDRs, each of which may comprise amino acid residues from a "complementarity determining region" as defined by Kabat: (Chothia & Lesk, J. mol. Biol, 196:901-, it has boundaries that overlap with the Kabat CDRs. Still other CDR boundary definitions may not strictly follow one of the above systems, but will still overlap with the Kabat CDRs, and the methods used herein may utilize CDRs defined according to any of these systems, although preferred embodiments use Kabat or Chothia defined CDRs. As used herein, "antibody variable region" refers to the portion of the light and heavy chains of an antibody molecule that includes the amino acid sequences of the complementarity determining regions (CDRs, i.e., CDR1, CDR2, and CDR3) and the Framework Region (FR). VH refers to the variable domain of the heavy chain. VL refers to the variable domain of the light chain.

The term "chimeric antibody" as used herein refers to an antibody in which the variable regions are derived from a non-human species (e.g., from a rodent) and the constant regions are derived from a different species (e.g., a human). Chimeric antibodies can be generated by antibody engineering. "antibody engineering" is a term generally used for different kinds of modifications of antibodies, and methods for antibody engineering are well known to those skilled in the art. Thus, the chimeric antibody may be a genetically or engineered recombinant antibody. Methods of generating chimeric antibodies are known to those of skill in the art, and thus, generation of chimeric antibodies can be performed by methods other than those described herein. Chimeric monoclonal antibodies for human therapeutic applications were developed to reduce the expected antibody immunogenicity of non-human antibodies (e.g., rodent antibodies). They may typically contain non-human (e.g., murine or rabbit) variable regions specific for the antigen of interest, and human constant antibody heavy and light chain domains. The term "variable region" or "variable domain" as used in the context of a chimeric antibody refers to a region comprising the CDRs and framework regions of both the heavy and light chains of an immunoglobulin, as described below.

The term "humanized antibody" as used herein refers to a genetically engineered non-human antibody that contains human antibody constant domains and non-human variable domains modified to contain a high level of sequence homology to human variable domains. This can be achieved by grafting six nonhuman antibody CDRs, which together form the antigen binding site, onto a homologous human acceptor Framework Region (FR). To fully reconstitute the binding affinity and specificity of a parent antibody, it may be necessary to replace framework residues from the parent antibody (i.e., the non-human antibody) into human framework regions (back mutations). Thus, a humanized antibody may comprise non-human CDR sequences, primarily human framework regions, optionally comprising one or more amino acid back mutations to the non-human amino acid sequence, and fully human constant regions. Optionally, additional amino acid modifications (which are not necessarily back mutations) may be applied to obtain a humanized antibody with preferred characteristics, such as affinity and biochemical properties and/or additional amino acid mutations may be introduced in the constant region.

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical, except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigen. Furthermore, in contrast to polyclonal antibody preparations which typically include different antibodies directed against different determinants (epitopes), each antibody in a monoclonal preparation is directed against the same single determinant on the antigen. As used herein, the term "monoclonal antibody" is not limited to antibodies produced by hybridoma technology, and the modifier "monoclonal antibody" is not to be construed as requiring production of the antibody by any particular method.

"administration" as used herein includes, but is not limited to, oral, enteral, subcutaneous, intradermal, intramuscular, intraarterial, intravenous, nasal, transdermal, subconjunctival, intraperitoneal, intrabulbar, orbital, retrobulbar, retinal, choroidal, intrathecal administration and the like.

An "effective amount" as described herein refers to an amount or dose of a product described herein (preferably an anti-TNFR 2 antibody or antigen-binding fragment thereof) that provides the desired treatment after administration to a patient or organ or individual in a single or multiple doses.

As used herein, the term "chemotherapeutic agent" refers to any chemical agent that has therapeutic usefulness in treating a disease characterized by abnormal cell growth. Chemotherapeutic agents as used herein include chemical agents and biological agents. These agents function to inhibit the cellular activity on which cancer cells rely for sustained survival. Classes of chemotherapeutic agents include alkylating/alkaloid agents, antimetabolites, hormones or hormone analogs, and a wide variety of anti-neobiological agents.

"diagnosis" as used herein refers to the determination of whether a patient has suffered from a disease or condition in the past, at the time of diagnosis, or in the future, or the determination of the progression or likely progression of a disease in the future, or the assessment of a patient's response to a therapy.

As used herein, "treating" means slowing, interrupting, arresting, controlling, halting, reducing, or reversing the progression or severity of one sign, symptom, disorder, condition, or disease, but does not necessarily involve the complete elimination of all disease-related signs, symptoms, conditions, or disorders.

The "subject" according to the invention is a mammal, in particular a primate, in particular a human.

The "tumor" according to the present invention is selected from leukemia, lymphoma, ovarian cancer, breast cancer, endometrial cancer, colon cancer, rectal cancer, stomach cancer, bladder cancer, lung cancer (e.g., non-small cell lung cancer, etc.), bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, liver and bile duct cancer, esophageal cancer, kidney cancer, thyroid cancer, head and neck cancer, testicular cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, and sarcoma. Wherein the leukemia is selected from the group consisting of: acute lymphocytic (lymphoblastic) leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, multiple myeloma, plasma cell leukemia, and chronic myelogenous leukemia; the lymphoma is selected from the group consisting of: hodgkin's lymphoma and non-hodgkin's lymphoma, including B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma, T-cell lymphoma, and waldenstrom's macroglobulinemia; and the sarcoma is selected from the group consisting of: osteosarcoma, ewing's sarcoma, leiomyosarcoma, synovial sarcoma, alveolar soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma, and chondrosarcoma.

The term "cancer" as used herein refers to malignant tumors originating in epithelial tissues, and is the most common type of malignant tumor. In contrast, a malignant tumor originating in mesenchymal tissue is called "sarcoma", and a malignant tumor originating in the blood system is called "hematological malignancy".

The practice of the present invention will employ, without limitation, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology. These techniques are explained in detail in the following documents. For example: molecular Cloning A Laboratory Manual, 2nd Ed., ed. By Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (d.n. glovered., 1985); immunochemical Methods In Cell And Molecular Biology (Mayer And Walker, eds., Academic Press, London, 1987); handbook Of Experimental Immunology, Volumes V (d.m.weir and c.c.blackwell, eds., 1986).

The term "and/or" as used herein includes a list of items in the alternative as well as any number of combinations of items.

The terms "comprises" and "comprising" as used herein are intended to be open-ended terms that specify the presence of the stated elements or steps, as well as any other elements or steps that do not materially affect the technical effects specified. When used herein to describe the sequence of a protein or nucleic acid, the protein or nucleic acid may be composed of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still have the activity described herein.

The disclosures of the various publications, patents, and published patent specifications cited herein are hereby incorporated by reference in their entirety.

The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.

Example 1: antibody production

Mice were immunized by using recombinant human TNFR2 protein (hTNFR 2, SEQ ID NO: 31) or an expression plasmid encoding recombinant human TNFR2 protein. Specifically, female BALB/c mice 6-8 weeks old were immunized with human Fc-labeled human TNFR2 at a concentration of 100. mu.g/ml at 20. mu.g/mouse. The Fc-labeled human TNFR2 protein was emulsified with adjuvant and injected at four locations on the back of the mouse. For the first subcutaneous (s.c.) injection, the diluted antigen was emulsified with an equal volume of Complete Freund's Adjuvant (CFA). In a subsequent subcutaneous injection, the protein was emulsified with an equal volume of Incomplete Freund's Adjuvant (IFA). Three days after the third injection or booster immunization, blood (serum) was collected and analyzed for antibody titer using ELISA.

In another experiment, 6-8 week old female BALB/c mice were immunized by injecting an expression plasmid encoding human TNFR2 into the mice. The plasmid encoding the antigen was injected into the tibialis anterior muscle of the mice at a concentration of 1000. mu.g/. mu.l by using a gene gun at 60. mu.g per mouse (intramuscular injection; i.m. injection). At least four injections were performed with at least 14 days between each injection. Blood (serum) was collected 7 days after the last immunization and the serum was tested for antibody titer by ELISA.

Procedures to enhance immunization (either by injection of plasmids or by injection of proteins) were also performed at least fourteen days after the previous immunization. CHO cells expressing the OX40 antigen on the surface were injected intravenously into mice via the tail vein. Spleens were then collected four days after injection. Splenocytes were first selected by CD3 epsilon microbeads and anti-mouse IgM microbeads, and then fused to SP2/0 cells. The cells were then plated in 96-well plates with hypoxanthine-aminopterin-thymidine (HAT) medium.

Hybridoma supernatants from 96-well plates were initially screened using Fluorescence-Activated Cell Sorting (FACS) according to standard procedures. Chinese Hamster Ovary (CHO) cells were added to 96-well plates (2 × 104 cells per well) prior to screening. 50 μ l of the supernatant was used. The antibodies used in the experiments were:

(1) fluorescein (FITC) -conjugated affinipurf (ab)2 fragment goat anti-mouse IgG, specific for Fc γ fragment; and

(2) alexa Fluor 647 conjugated AffiniPure F (ab)2 fragment goat anti-human IgG, Fc gamma fragment specific.

Subcloning was performed using ClonePix 2. Briefly, positive wells identified in the primary screen were transferred to semi-solid medium and IgG positive clones were identified and tested. FITC anti-mouse IgG Fc antibody was used.

Will be 1 × 10⁶Each positive hybridoma cell was injected intraperitoneally into B-NDG mice (Beijing BaiOnosh chart, Beijing, China (Beijing Biocytogen, Beijing, China)). Monoclonal antibodies were produced by growing hybridoma cells intraperitoneally in mice. Hybridoma cells expand in the abdomen of mice and produce ascites fluid. Ascites fluid contains high concentrations of antibodies and can be harvested for later use.

Antibody purification was performed using a GE AKTA protein chromatography full-automatic purifier (GE Healthcare) according to the manufacturer's instructions. The anti-TNFR 2 monoclonal antibodies 10-5E6 (sometimes abbreviated as "5E 6") and 10-1H5 (sometimes abbreviated as "1H 5") of the present invention were obtained. The amino acid sequences of the heavy chain variable region (VH) and the light chain variable region (VL) of the above antibody are shown in tables 1 to 3 below.

The heavy chain CDR1, CDR2 and CDR3 of 5E6 and the light chain CDR1, CDR2 and CDR3 amino acid sequences are shown in SEQ ID NOS: 5-10 (Kabat numbering) or SEQ ID NOS: 17-22 (Chothia numbering).

The heavy chain CDR1, CDR2 and CDR3 of 1H5 and the light chain CDR1, CDR2 and CDR3 amino acid sequences are shown in SEQ ID NOS: 11-16 (Kabat numbering) or SEQ ID NOS: 23-28 (Chothia numbering).

Kabat numbering is used by default in this disclosure unless specifically noted otherwise in this disclosure.

TABLE 1 VH and VL sequences of antibodies

TABLE 2 Kabat CDR sequences

TABLE 3 Chothia CDR sequences

Example 2: in vitro detection of antibodies

Antibody blocking assay

This experiment was conducted to examine the blocking ability of the anti-TNFR 2 antibodies (10-5E 6 and 10-1H 5) prepared in example 1 against TNFR2 and TNFa. The specific experimental operations were as follows: a96-well cell culture plate was taken and CHO-S-hTNFR2 protein-expressing CHO-hTNFR 2(T) -EGFP cells (25. mu.l, 2X 10) were added to each well⁴Cells/well). The purified antibody was diluted at a concentration of 50, 5, 0.5, 0.05, 0.005. mu.g/ml, 25. mu.l was added to each well, and allowed to stand at 4 ℃ for 30 minutes.

After two washes with Phosphate Buffered Saline (PBS), 50 μ L of Biotinylated Human TNF-alpha Protein, His, Avitag (Popsbeing, Cat. TNA-H82E 3) (1 μ g/mL) was added to each well, incubated at 4 ℃ for 15 minutes, then washed 2 times with PBS, 50 μ L of 1:100 diluted Streptavidin-PE (Jackson ImmunoResearch, Cat. 109. 116. sup. quadrature. 098) was added to each well, incubated at 4 ℃ for 30 minutes, washed 2 times with PBS, 200 μ L of PBS was added to each well and flow cytometric analysis was performed. The results are shown in FIG. 1.

As can be seen from the results shown in FIG. 1, the PE label fluorescence intensity gradually decreased as the concentration of the anti-TNFR 2 antibody (10-5E 6 and 10-1H 5) increased, indicating that the anti-TNFR 2 antibody of the present invention can effectively block the binding to human TNFR2 and TNFa.

Antibody affinity detection

Subsequently, the affinity of these anti-TNFR 2 antibodies for binding to hTNFR2-His (Acro, cat # TN 2-H5227) was examined using a method of capturing antibodies using the Protein A sensor chip of Biacore T200 (Biacore).

The specific experimental operations were as follows: anti-TNFR 2 antibodies 10-5E6 and 10-1H5 were injected into a sensor chip (10. mu.l/min, 50 s) and the protein was captured at 45-100 RU. Then, hTNFR2-His protein is injected into a sensing chip (30 μ l/min, 100-. The chip was regenerated with glycine (30. mu.l/min, 10-30 s) at pH2.0 and the assay results were read. Kinetic association rates (kon) and Kinetic dissociation rates (koff) were measured by the Bioacore T200 evaluation software for 1: the 1 langmuir binding model was fit ((Karlsson, r. Roos, h. fagerstat, l. Petersson, b., 1994. Methods Enzymology 6.99-110). affinity rate constant KD = koff/kon, and the antibody affinity assay fit results are shown in table 4.

TABLE 4 anti-TNFR 2 antibody affinity assays

Example 3: in vivo drug efficacy testing of antibodies

To examine the in vivo efficacy of the anti-TNFR 2 antibody (10-5E 6 and 10-1H 5) prepared in example 1, a TNFR2 gene-humanized mouse was used to prepare a tumor animal model. The mouse expresses a human-mouse chimeric TNFR2 protein (SEQ ID NO: 29), wherein the extracellular region of the murine TNFR2 protein is replaced by a humanized sequence: amino acids 33-260 of murine TNFR2 protein (SEQ ID NO: 30) were replaced with amino acids 33-259 of human TNFR2 protein (SEQ ID NO: 31). The B-hTNFR2 mouse model provides a new detection method for the preclinical animal experiments of TNFR2 monoclonal antibody drugs, and greatly improves the predictability of the clinical experiments (see PCT application No. PCT/CN2020/113618 and Chinese application No. 202010922139.7, which are incorporated by reference in their entirety).

The preparation process of the tumor animal model is as follows: murine MC-38 cells (colon cancer cells) were inoculated in B-hTNFR2 mice by subcutaneous injection. When the tumor volume reaches 150 +/-50 mm³Thereafter, the mice were randomly divided into an anti-TNFR 2 antibody administration group and a control group (physiological saline) to be subjected to a tumor suppression drug effect test. The administration is by intraperitoneal injection. Mice body weight and tumor volume were measured periodically 2 times per week. Tumor volume (mm)³) =0.5x long diameter x short diameter². After inoculation, the tumor volume of a single mouse reaches 3000mm³An euthanasia end test was performed.

Tumor growth inhibition ratio (TGI) calculation formula: TGI (%) = [1- (Ti-T0)/(Vi-V0) ] × 100, where Ti is the mean tumor volume on day i of the treatment group; t0 is the mean tumor volume at day 0 in the treated group; vi is the mean tumor volume on day i of the control group; v0 is the mean tumor volume at day 0 of the control group. Statistical analysis was performed using the T-test. When TGI% is greater than 60%, it indicates a significant inhibitory effect on tumor growth. P <0.05 indicates that the statistical results are significantly different.

5-9 week old B-hTNFR2 mice were inoculated with MC38 cells (5X 10)⁵One) until the tumor volume reaches 150 +/-50 mm³The latter were randomly divided into 3 groups of 6 individuals each. The administration group was treated with anti-TNFR 2 antibodies 10-5E6 and 10-1H5 by intraperitoneal injection at a dose of 10mg/kg, and the control group was injected with physiological saline. The administration was once every 3 days for 6 times. Body weight and tumor volume of mice were measured 2 times a week, and the experiment was terminated after 5 weeks. As shown in fig. 2, the average body weight of the control and administered mice was steadily increased throughout the experimental period, and there was no significant difference between the groups, indicating that the anti-TNFR 2 antibody did not cause significant toxicity to the mice after administration. As shown in the results in fig. 3 (tumor volume data 35 days after grouping), the growth of tumors was inhibited to a different extent in the administration group compared to the control group. Table 5 below shows the TGI% results for each group.

TABLE 5 tumor growth inhibition Rate

The above results indicate that the anti-TNFR 2 antibodies 10-5E6 and 10-1H5 of the present invention both showed tumor-inhibiting effects, with 10-5E6 having the best tumor-inhibiting effect.

The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.

It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.

In addition, any combination of the various embodiments of the present invention is also possible, and the same should be considered as the disclosure of the present invention as long as it does not depart from the spirit of the present invention.

Sequence listing

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Glu Gln Asn Arg Ile Cys Thr Cys Arg Pro Gly Trp Tyr Cys Ala Leu

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Ser Lys Gln Glu Gly Cys Arg Leu Cys Ala Pro Leu Arg Lys Cys Arg

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Phe Leu Leu Pro Met Gly Pro Ser Pro Pro Ala Glu Gly Ser Thr Gly

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Lys Lys Pro Ser Cys Leu Gln Arg Asp Ala Lys Val Pro His Val Pro

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Met Ala Glu Ala Gln Gly Phe Gln Glu Ala Arg Ala Ser Ser Arg Ile

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Phe Asp Gln Ile Ala Val Lys Val Ala

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Lys His Phe Cys Asn Lys Thr Ser Asp Thr Val Cys Ala Asp Cys Glu

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Arg Ser Gln Pro Leu Asp Gln Glu Pro Gly Pro Ser Gln Thr Pro Ser

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Ile Leu Thr Ser Leu Gly Ser Thr Pro Ile Ile Glu Gln Ser Thr Lys

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Gly Gly Ile Ser Leu Pro Ile Gly Leu Ile Val Gly Val Thr Ser Leu

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Gly Leu Leu Met Leu Gly Leu Val Asn Cys Ile Ile Leu Val Gln Arg

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Lys Lys Lys Pro Ser Cys Leu Gln Arg Asp Ala Lys Val Pro His Val

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Pro Asp Glu Lys Ser Gln Asp Ala Val Gly Leu Glu Gln Gln His Leu

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Ser Gln Ala Ser Ala Thr Val Gly Asp Pro Asp Ala Lys Pro Ser Ala

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Trp Phe Asp Gln Ile Ala Val Lys Val Ala

465 470

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Met Ala Pro Val Ala Val Trp Ala Ala Leu Ala Val Gly Leu Glu Leu

1 5 10 15

Trp Ala Ala Ala His Ala Leu Pro Ala Gln Val Ala Phe Thr Pro Tyr

20 25 30

Ala Pro Glu Pro Gly Ser Thr Cys Arg Leu Arg Glu Tyr Tyr Asp Gln

35 40 45

Thr Ala Gln Met Cys Cys Ser Lys Cys Ser Pro Gly Gln His Ala Lys

50 55 60

Val Phe Cys Thr Lys Thr Ser Asp Thr Val Cys Asp Ser Cys Glu Asp

65 70 75 80

Ser Thr Tyr Thr Gln Leu Trp Asn Trp Val Pro Glu Cys Leu Ser Cys

85 90 95

Gly Ser Arg Cys Ser Ser Asp Gln Val Glu Thr Gln Ala Cys Thr Arg

100 105 110

Glu Gln Asn Arg Ile Cys Thr Cys Arg Pro Gly Trp Tyr Cys Ala Leu

115 120 125

Ser Lys Gln Glu Gly Cys Arg Leu Cys Ala Pro Leu Arg Lys Cys Arg

130 135 140

Pro Gly Phe Gly Val Ala Arg Pro Gly Thr Glu Thr Ser Asp Val Val

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Asn Ala Ser Met Asp Ala Val Cys Thr Ser Thr Ser Pro Thr Arg Ser

195 200 205

Met Ala Pro Gly Ala Val His Leu Pro Gln Pro Val Ser Thr Arg Ser

210 215 220

Gln His Thr Gln Pro Thr Pro Glu Pro Ser Thr Ala Pro Ser Thr Ser

225 230 235 240

Phe Leu Leu Pro Met Gly Pro Ser Pro Pro Ala Glu Gly Ser Thr Gly

245 250 255

Asp Phe Ala Leu Pro Val Gly Leu Ile Val Gly Val Thr Ala Leu Gly

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Lys Lys Pro Leu Cys Leu Gln Arg Glu Ala Lys Val Pro His Leu Pro

290 295 300

Ala Asp Lys Ala Arg Gly Thr Gln Gly Pro Glu Gln Gln His Leu Leu

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Ile Thr Ala Pro Ser Ser Ser Ser Ser Ser Leu Glu Ser Ser Ala Ser

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Ala Leu Asp Arg Arg Ala Pro Thr Arg Asn Gln Pro Gln Ala Pro Gly

340 345 350

Val Glu Ala Ser Gly Ala Gly Glu Ala Arg Ala Ser Thr Gly Ser Ser

355 360 365

Asp Ser Ser Pro Gly Gly His Gly Thr Gln Val Asn Val Thr Cys Ile

370 375 380

Val Asn Val Cys Ser Ser Ser Asp His Ser Ser Gln Cys Ser Ser Gln

385 390 395 400

Ala Ser Ser Thr Met Gly Asp Thr Asp Ser Ser Pro Ser Glu Ser Pro

405 410 415

Lys Asp Glu Gln Val Pro Phe Ser Lys Glu Glu Cys Ala Phe Arg Ser

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Gln Leu Glu Thr Pro Glu Thr Leu Leu Gly Ser Thr Glu Glu Lys Pro

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Leu Pro Leu Gly Val Pro Asp Ala Gly Met Lys Pro Ser

450 455 460

Claims

1. An antibody or antigen-binding portion thereof that specifically binds a TNFR2 protein, wherein the antibody or antigen-binding portion thereof comprises a heavy chain variable region comprising VHCDR1, VHCDR2 and VHCDR3 and a light chain variable region comprising VLCDR1, VLCDR2 and VLCDR3, wherein,

the amino acid sequences of the VHCDR1-3 are respectively shown as SEQ ID NO 5, SEQ ID NO 6 and SEQ ID NO 7, and the amino acid sequences of the VLCDR1-3 are respectively shown as SEQ ID NO 8, SEQ ID NO 9 and SEQ ID NO 10.

2. The antibody or antigen-binding portion thereof of claim 1, wherein the amino acid sequence of the heavy chain variable region is set forth in SEQ ID No. 1 and the amino acid sequence of the light chain variable region is set forth in SEQ ID No. 2.

3. The antibody or antigen-binding portion thereof of claim 1 or 2, wherein the antibody or antigen-binding fragment thereof specifically binds human TNFR2 and blocks the interaction of human TNFR2 with TNFa.

4. An isolated DNA encoding the antibody or antigen binding portion thereof of any one of claims 1-3.

5. A vector comprising the DNA of claim 4.

6. A cell comprising the DNA of claim 4 or the vector of claim 5.

7. A method of producing the antibody or antigen-binding portion thereof of any one of claims 1-3, comprising the step of culturing the cell of claim 6.

8. An antibody drug conjugate comprising the antibody or antigen-binding fragment thereof of any one of claims 1-3 covalently bound to a drug.

9. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-3, or the antibody drug conjugate of claim 8, and a pharmaceutically acceptable carrier.

10. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, the antibody drug conjugate according to claim 8, or the pharmaceutical composition according to claim 9 for the preparation of a medicament for the prevention and/or treatment of a disease, for screening a medicament for the prevention and/or treatment of a disease, wherein the disease is a disease associated with the TNFR2 signaling pathway.