CN115368447A

CN115368447A - Bispecific antibodies and uses thereof

Info

Publication number: CN115368447A
Application number: CN202210852146.3A
Authority: CN
Inventors: 曹国帅; 成赢; 李洋洋; 武玉伟
Original assignee: Hefei Tiangang Immune Drugs Co ltd
Current assignee: Hefei Tiangang Immune Drugs Co ltd
Priority date: 2022-07-19
Filing date: 2022-07-19
Publication date: 2022-11-22

Abstract

The invention provides a bispecific antibody targeting 4-1BB and Nectin-4 and application thereof. The antibody comprises: a first antigen binding region having 4-1BB binding activity; a second antigen binding region having Nectin-4 binding activity, the first antigen binding region being selected from a 4-1BB single chain antibody, the second antigen binding region comprising a Nectin-4 heavy chain and a Nectin-4 light chain. The antibody can be combined with Nectin-4 positive tumor cells and T cells at the same time, effectively mediates the T cells to kill the tumor cells, and can effectively treat cancers.

Description

Bispecific antibodies and uses thereof

Technical Field

The invention belongs to the field of biomedicine, and particularly relates to a bispecific antibody and application thereof, and more particularly relates to an antibody, a nucleic acid molecule, an expression vector, a recombinant cell, a pharmaceutical composition, a kit and application thereof.

Background

Cancer is a disease seriously threatening the life and health of human beings, and the incidence and mortality of cancer are increasing in recent years. The existing cancer treatment means comprise surgical resection, radiotherapy, chemotherapy, small molecule targeted therapy, antibody targeted therapy, macromolecule immunotherapy and other treatment methods, but the methods only play a limited role in part of cancer patients, and the cancer still has a problem of disturbing the life health of human beings.

In recent years, bispecific antibodies have become a focus of research in immunotherapy. The bispecific antibody is an artificial antibody containing two specific antigen binding sites, and can bridge between a target cell (tumor cell) and an effector cell (immune cell) to generate an effect function of targeted killing of tumors.

Therefore, there is a need for a bispecific antibody for targeted tumor killing.

Disclosure of Invention

The present invention aims to solve at least to some extent at least one of the technical problems of the prior art. Therefore, the invention provides a bispecific antibody targeting 4-1BB and Nectin-4, which can be simultaneously combined with 4-1BB and Nectin-4 and can effectively mediate T cells expressing 4-1BB to kill cells expressing Nectin-4.

The present invention has been completed based on the following findings of the inventors:

4-1BB belongs to the tumor necrosis factor receptor superfamily (TNFRSF 9), and is mainly expressed in activated T cells. Nectin-4, collectively known as poliovirus-like receptor 4 (PVRL 4), is a type I transmembrane protein that is poorly expressed in normal human tissues (e.g., skin, bladder, salivary glands, esophagus, breast and stomach) and highly expressed in cancer cells (e.g., breast, ovarian, cervical, colorectal, esophageal, gastric, lung, liver and thyroid cancers). In addition, studies have shown that Nectin-4 plays a key role in the development, invasion and metastasis of tumors, and that over-expression of Nectin-4 may promote resistance of the body to chemotherapy.

Currently, only Padcev is approved as an antibody coupling drug for Nectin-4 targeted antibody drugs, and the Padcev consists of a targeted Nectin-4 monoclonal antibody and a potent antimitotic agent MMAE for blocking tubulin polymerization, and the monoclonal antibody is used for targeted delivery of chemotherapeutic drugs to tumors. At present, the medicine is mainly used for treating patients with locally advanced disease progression or metastatic urothelial cancer patients during or after treatment with the PD-1/PD-L1 inhibitor.

Based on this, in one aspect of the invention, the invention provides an antibody. According to an embodiment of the invention, the antibody comprises: a first antigen binding region having 4-1BB binding activity; a second antigen binding region having Nectin-4 binding activity. The antibody provided by the embodiment of the invention can be simultaneously combined with 4-1BB and Nectin-4, so that the killing effect of T cells on cells (such as tumor cells) expressing the Nectin-4 can be effectively mediated, particularly the antibody has a strong tumor inhibition effect, and the cancer can be effectively treated.

In another aspect of the invention, the invention features a nucleic acid molecule. According to an embodiment of the invention, the nucleic acid molecule encodes an antibody as described above. Nucleic acid molecules according to embodiments of the invention may encode antibodies that target binding to 4-1BB and Nectin-4 simultaneously.

In yet another aspect of the invention, the invention features an expression vector. According to an embodiment of the invention, the nucleic acid molecule described above is carried. After the expression vector according to the embodiment of the present invention is introduced into a suitable recipient cell, the aforementioned expression of the antibody can be effectively achieved under the mediation of a regulatory system, so that the antibody can be obtained in a large amount.

In yet another aspect of the invention, the invention provides a method of making the aforementioned antibody. According to an embodiment of the invention, the method comprises: introducing the aforementioned expression vector into a cell; subjecting the cells to a culture treatment under conditions suitable for protein expression and secretion so as to obtain the antibody. The method provided by the embodiment of the invention can effectively obtain the antibody and has the advantages of simple preparation method and the like.

In yet another aspect of the invention, the invention features a recombinant cell. According to an embodiment of the invention, said recombinant cell carries a nucleic acid molecule as described above, or an expression vector as described above or expresses an antibody as described above. The recombinant cell is obtained by transfecting or transforming the expression vector, and the recombinant cell can efficiently express the antibody which can be targeted and combined with 4-1BB and Nectin-4 simultaneously under appropriate conditions.

In yet another aspect of the invention, a pharmaceutical composition is provided. According to an embodiment of the invention, the pharmaceutical composition comprises: the aforementioned antibody, the aforementioned nucleic acid molecule, the aforementioned expression vector, or the aforementioned recombinant cell. The pharmaceutical composition provided by the embodiment of the invention can simultaneously target and combine the antibodies of 4-1BB and Nectin-4, can effectively mediate the killing effect of T cells on cells (such as tumor cells) expressing the Nectin-4, particularly has a strong tumor inhibition effect, and can effectively treat cancers.

In yet another aspect of the invention, the invention features a kit. According to an embodiment of the invention, the kit comprises: the aforementioned antibody, the aforementioned nucleic acid molecule, the aforementioned expression vector, or the aforementioned recombinant cell. The kit provided by the embodiment of the invention can be combined with 4-1BB protein and/or Nectin-4 protein, and can effectively identify the 4-1BB protein and/or the Nectin-4 protein.

In a further aspect of the invention, the invention proposes the use of an aforementioned antibody, an aforementioned nucleic acid molecule, an aforementioned expression vector, an aforementioned recombinant cell or an aforementioned pharmaceutical composition for the preparation of a medicament for the treatment or prevention of cancer. According to the embodiment of the invention, the antibody or the pharmaceutical composition can be simultaneously targeted and combined with the antibodies of 4-1BB and Nectin-4, can effectively mediate the killing effect of T cells on tumor cells expressing Nectin-4, has a strong tumor inhibition effect, and can effectively treat cancers.

In a further aspect of the invention, the invention proposes the use of an antibody as described above, a nucleic acid molecule as described above, an expression vector as described above or a recombinant cell as described above for the preparation of a kit for the detection of 4-1BB and/or Nectin-4. According to the embodiment of the invention, the antibody or the kit can be combined with 4-1BB protein and/or Nectin-4 protein, so that the 4-1BB protein and/or the Nectin-4 protein can be effectively identified.

Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.

Drawings

The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:

FIG. 1 is a schematic structural diagram of a bispecific antibody according to example 1 of the present invention;

FIG. 2 is a graph showing the results of measuring the binding ability of a 4-1BB XNectin-4 bispecific antibody to a 4-1BB protein according to example 2 of the present invention;

FIG. 3 is a graph showing the results of measuring the binding ability of the 4-1BB × Nectin-4 bispecific antibody according to example 3 of the present invention to CHO-K1-4-1BB cells;

FIG. 4 is a graph showing the results of measurement of the binding ability of the 4-1BB × Nectin-4 bispecific antibody to human peripheral blood CD4+ T cells in example 4 according to the present invention;

FIG. 5 is a graph showing the results of measurement of the binding ability of the 4-1BB × Nectin-4 bispecific antibody to CHO-K1-Nectin-4 cell according to example 5 of the present invention;

FIG. 6 is a graph showing the results of measurement of the binding ability of recombinant bispecific antibody according to example 6 of the present invention to human SK-BR-3 cells;

FIG. 7 is a graph showing the results of detection of 4-1BB XNectin-4 bispecific antibody activated Jurkat-NFAT-lucia-4-1BB reporter cells according to example 7 of the present invention;

FIG. 8 is a graph showing the results of the detection of the recombinant bispecific antibody promoting PBMC to kill A375-Nectin-4 tumor cells according to example 8 of the present invention.

Detailed Description

The following describes embodiments of the present invention in detail. The following examples are illustrative only and are not to be construed as limiting the invention.

Furthermore, the terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Further, in the description of the present invention, "a plurality" means two or more unless otherwise specified.

The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and these ranges or values should be understood to encompass values close to these ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.

In order that the invention may be more readily understood, certain technical and scientific terms are specifically defined below. Unless clearly defined otherwise herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. 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.

In the present invention, unless otherwise expressly stated or limited, the term "coupled" is to be construed broadly, e.g., as meaning directly coupled to or indirectly coupled through intervening media, communicating between two elements, or interacting between two elements, unless expressly stated or limited otherwise. The specific meanings of the above terms in the present invention can be understood by those skilled in the art according to specific situations.

Herein, the term "bispecific antibody" generally refers to a peptide chain specifically recognizing two protein molecules linked to two Fc fragments, respectively, wherein the two Fc fragments are linked via a knob-to-hole structure. "bispecific antibody" and "bispecific antibody molecule" are used interchangeably herein. The peptide chain of the specific recognition protein molecule can be a single-chain antibody; two chains linked by a disulfide bond are also possible, for example, one chain is the heavy chain variable region and the CH1 region of the recognition protein molecule and the other chain is the light chain variable region and the CL region of the recognition protein molecule.

The term "Knob-in-hole structure" as used herein generally refers to the formation of a Knob (hole) mutation in the constant region of the heavy chain of an antibody or in the CH3 region of an Fc fragment to facilitate heavy chain engagement and heterodimer formation, for example, as used herein, by mutating amino acids in the CH3 domain of human IgG1-Fc (the T366S, L368A, Y407V, Y349C mutations in one chain, i.e., "hole"; the T366W, S354C mutations in the other chain, i.e., "Knob").

Herein, the amino acid numbering of the IgG1 Fc moiety is according to EU numbering system, e.g., position 366 refers to position 366 according to EU numbering system numbering; the expression "T366S" means that threonine at position 366 according to the EU numbering system is replaced by serine; "L368A" means that leucine at position 368 is replaced with alanine by EU numbering system.

In this context, the term "expression vector" generally refers to a nucleic acid molecule capable of being inserted into a suitable host for self-replication, which transfers the inserted nucleic acid molecule into and/or between host cells. The expression vectors may include vectors primarily for the insertion of DNA or RNA into a cell, vectors primarily for the replication of DNA or RNA, and vectors primarily for the expression of transcription and/or translation of DNA or RNA. The expression vector also includes vectors having a variety of the functions described above. The expression vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, the expression vector can produce the desired expression product by culturing a suitable host cell containing the expression vector.

In this context, the term "recombinant cell" generally refers to a host cell whose genetic material has been modified or recombined by genetic engineering or cell fusion techniques to obtain a cell with a unique trait of stable inheritance. The term "host cell" refers to a prokaryotic or eukaryotic cell into which a recombinant expression vector can be introduced. The term "transformed" or "transfected" as used herein refers to the introduction of a nucleic acid (e.g., a vector) into a cell by various techniques known in the art. Suitable host cells can be transformed or transfected with the DNA sequences of the present invention and can be used for expression and/or secretion of the target protein. Examples of suitable host cells that can be used in the present invention include immortalized hybridoma cells, NS/0 myeloma cells, 293 cells, chinese Hamster Ovary (CHO) cells, heLa cells, cap cells (human amniotic fluid derived cells), insect cells, per.c6 cells and CoS cells, preferably CHO cells.

The term "pharmaceutical composition" as used herein generally refers to a unit dosage form and may be prepared by any of the methods well known in the pharmaceutical art. All methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. Generally, compositions are prepared by uniformly and sufficiently combining the active compound with a liquid carrier, a finely divided solid carrier, or both.

As used herein, the term "pharmaceutically acceptable" means that the substance or composition must be chemically and/or toxicologically compatible with the other ingredients comprising the formulation and/or the mammal being treated therewith. Preferably, the term "pharmaceutically acceptable" as used herein refers to those approved by a federal regulatory agency or a state government or listed in the U.S. pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

As used herein, the term "pharmaceutically acceptable excipient" may include any solvent, solid excipient, diluent, or other liquid excipient, etc., suitable for the particular intended dosage form. Except insofar as any conventional adjuvant is incompatible with the compounds of the invention, e.g., any adverse biological effect produced or interaction in a deleterious manner with any other component of a pharmaceutically acceptable composition, their use is contemplated by the present invention.

As used herein, the term "administering" refers to introducing a predetermined amount of a substance into a patient by some suitable means. The recombinant antibody or pharmaceutical composition of the present invention can be administered by any common route as long as it can reach the intended tissue. Various modes of administration are contemplated, including peritoneal, intravenous, intramuscular, subcutaneous, and the like, but the present invention is not limited to these exemplified modes of administration. Preferably, the compositions of the present invention are administered intravenously or subcutaneously.

As used herein, the term "treating" is intended to mean obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of complete or partial prevention of the disease or symptoms thereof, and/or may be therapeutic in terms of a partial or complete cure for the disease and/or adverse effects resulting from the disease. As used herein, "treatment" encompasses diseases in mammals, particularly humans, including: (a) Preventing the occurrence of a disease or disorder in an individual susceptible to the disease but not yet diagnosed; (b) inhibiting a disease, e.g., arresting disease progression; or (c) alleviating the disease, e.g., alleviating symptoms associated with the disease. As used herein, "treatment" encompasses any administration of a drug or compound to an individual to treat, cure, alleviate, ameliorate, reduce or inhibit a disease in the individual, including but not limited to the administration of a drug containing a compound described herein to an individual in need thereof.

It is to be noted that, for nucleic acids mentioned in the present specification and claims, a person skilled in the art will understand that any one or two of the complementary double strands are actually included. For convenience, in the present specification and claims, although only one strand is given in most cases, the other strand complementary thereto is actually disclosed. In addition, the nucleic acid sequences in the present application include either a DNA form or an RNA form, one of which is disclosed, meaning that the other is also disclosed.

The present invention provides an antibody, a nucleic acid molecule, an expression vector, a recombinant cell, a pharmaceutical composition and a kit and uses thereof, which will be described in detail below, respectively.

Antibodies

In one aspect of the invention, an antibody is provided. According to an embodiment of the invention, the antibody comprises: a first antigen binding region having 4-1BB binding activity; a second antigen binding region having Nectin-4 binding activity. The antibody provided by the embodiment of the invention can be simultaneously combined with 4-1BB and Nectin-4, so that the killing effect of T cells on cells (such as tumor cells) expressing the Nectin-4 can be effectively mediated, particularly the killing effect of the T cells on the tumor cells can be mediated, the antibody has a strong tumor inhibition effect, and the cancer can be effectively treated.

According to an embodiment of the present invention, the first antigen binding region comprises a first heavy chain variable region and a first light chain variable region, the C-terminus of the first heavy chain variable region is linked to the N-terminus of the first light chain variable region or the N-terminus of the first heavy chain variable region is linked to the C-terminus of the first light chain variable region.

According to an embodiment of the present invention, the C-terminus of the first heavy chain variable region is linked to the N-terminus of the first light chain variable region.

According to an embodiment of the present invention, the first antigen binding region further comprises a first linker peptide, the C-terminus of the first heavy chain variable region is linked to the N-terminus of the first linker peptide, the C-terminus of the first linker peptide is linked to the N-terminus of the first light chain variable region, or the C-terminus of the first light chain variable region is linked to the N-terminus of the first linker peptide, and the C-terminus of the first linker peptide is linked to the N-terminus of the first heavy chain variable region.

According to an embodiment of the present invention, the C-terminus of the first heavy chain variable region is linked to the N-terminus of the first linker peptide, and the C-terminus of the first linker peptide is linked to the N-terminus of the first light chain variable region.

According to an embodiment of the invention, the first heavy chain variable region comprises SEQ ID NO:1 to 3; or the first light chain variable region comprises SEQ ID NO:4 to 6. Thus, the first antigen binding region can effectively bind to 4-1BB protein.

GGSFSGYY(SEQ ID NO：1)。

INHGGYV(SEQ ID NO：2)。

ARDYGPGNYDWYFDL(SEQ ID NO：3)。

QSVSSY(SEQ ID NO：4)。

DAS(SEQ ID NO：5)。

QQRSNWPPALT(SEQ ID NO：6)。

According to an embodiment of the invention, the first heavy chain variable region has the amino acid sequence as set forth in SEQ ID NO: 1. SEQ ID NO: 2. the amino acid sequence of SEQ ID NO:3, CDR1, CDR2, CDR3 sequences shown in.

According to an embodiment of the invention, the first light chain variable region has the amino acid sequence as set forth in SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO:6 CDR1, CDR2 and CDR3 sequences.

According to an embodiment of the invention, the first linker peptide has the amino acid sequence as set forth in SEQ ID NO: 7.

GGGGSGGGGSGGGGS(SEQ ID NO：7)。

According to embodiments of the invention, the first antigen binding region further comprises a first FC peptide segment, the C-terminus of the first light chain variable region is linked to the N-terminus of the first FC peptide segment, or the C-terminus of the first heavy chain variable region is linked to the N-terminus of the first FC peptide segment.

It is noted that "Fc peptide fragment", "Fc fragment" or "Fc" herein refers to a peptide fragment comprising a hinge region, a CH2 region and a CH3 region, such as wild-type IgG1 Fc fragment, a first Fc peptide fragment and a second Fc peptide fragment mentioned in the present application.

For example, the amino acid sequence of human wild-type IgG1 Fc (including hinge-CH2-CH 3) is as follows:

PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:30)。

according to an embodiment of the invention, the first FC peptide fragment comprises a first hinge region, a first CH2 region and a first CH3 region.

According to an embodiment of the invention, said first hinge region is a hinge region fragment of a wild-type IgG1 of human, primate or murine origin.

According to an embodiment of the invention, the first CH2 region is a CH2 region fragment of a wild-type IgG1 of human, primate or murine origin; or the first CH2 region has an L234A and/or L235A mutation compared to a CH2 region fragment of a human wild-type IgG 1.

According to an embodiment of the invention, said first CH3 region has a T366W and/or S354C mutation compared to a CH3 region fragment of a human wild type IgG 1.

According to an embodiment of the invention, the first antigen binding region further comprises a second linking peptide.

According to an embodiment of the invention, the N-terminus of the second linker peptide is linked to the C-terminus of the first light chain variable region, and the C-terminus of the second linker peptide is linked to the N-terminus of the first FC peptide fragment; or the N end of the second connecting peptide is connected with the C end of the first heavy chain variable region, and the C end of the second connecting peptide is connected with the N end of the first FC peptide segment.

According to an embodiment of the invention, the second linker peptide has the amino acid sequence as shown in SEQ ID NO: 8. Thus, the binding activity of the first antigen-binding region to the 4-1BB protein can be further increased.

GGGGS(SEQ ID NO：8)。

According to an embodiment of the invention, the first antigen binding region comprises SEQ ID NO:13, or a pharmaceutically acceptable salt thereof.

It should be noted that, in the present invention, the first heavy chain variable region, the first linker peptide, the first light chain variable region and the second linker peptide exist in the form of a single chain antibody, in which the C-terminal of the first heavy chain variable region is connected to the N-terminal of the first linker peptide, the C-terminal of the first linker peptide is connected to the N-terminal of the first light chain variable region, the C-terminal of the first light chain variable region is connected to the N-terminal of the second linker peptide, and the single chain antibody has SEQ ID NO:9, or a pharmaceutically acceptable salt thereof.

QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQSPEKGLEWIGEINHGGYVTYNPSLESRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDYGPGNYDWYFDLWGRGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPALTFGGGTKVEIKGGGGS(SEQ ID NO：9)。

According to an embodiment of the invention, the first FC peptide fragment has the amino acid sequence shown in SEQ ID No. 10.

PKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO：10)。

According to an embodiment of the invention, the first antigen binding region has the amino acid sequence shown in SEQ ID NO. 11.

QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQSPEKGLEWIGEINHGGYVTYNPSLESRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDYGPGNYDWYFDLWGRGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPALTFGGGTKVEIKGGGGSPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO：11)。

According to an embodiment of the invention, the second antigen-binding region comprises a first polypeptide and a second polypeptide, which are linked by an interchain disulfide bond; the first polypeptide comprises a second heavy chain variable region, a CH1 region and a second FC peptide segment, wherein the C end of the second heavy chain variable region is connected with the N end of the CH1 region, and the C end of the CH1 region is connected with the N end of the second FC peptide segment; the second polypeptide comprises a second light chain variable region and a CL region, wherein the C-terminus of the second light chain variable region is linked to the N-terminus of the CL region.

According to an embodiment of the invention, the second FC peptide fragment comprises a second hinge region, a second CH2 region and a second CH3 region.

According to the embodiment of the invention, the CH1 region is a CH1 region of a wild-type IgG1 of human, primate or murine origin.

According to an embodiment of the invention, said second hinge region is a hinge region fragment of a wild-type IgG1 of human, primate or murine origin.

According to the embodiment of the invention, the second CH2 region is a CH2 region fragment of wild-type IgG1 of human, primate or mouse; or the second CH2 region has an L234A and/or L235A mutation compared to a CH2 region fragment of a human wild-type IgG 1.

According to an embodiment of the invention, said second CH3 region is a CH3 region fragment of a wild-type IgG1 of human, primate or murine origin.

According to an embodiment of the invention, said second CH3 region has at least one of the T366S, L368A, Y407V, Y349C mutations compared to a CH3 region fragment of human wild type IgG 1.

According to an embodiment of the present invention, the CL region is a wild-type CL region of human, primate or murine origin.

For example, the amino acid sequence of the human wild-type CL region is shown below:

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO：31)。

according to an embodiment of the invention, the second heavy chain variable region comprises SEQ ID NO:12 to 14; or the second light chain variable region comprises SEQ ID NO:15 to 17.

GFTFSSYN(SEQ ID NO：12)。

ISSSSSTI(SEQ ID NO：13)。

ARAYYYGMDV(SEQ ID NO：14)。

QGISGW(SEQ ID NO：15)。

AAS(SEQ ID NO：16)。

QQANSFPPT(SEQ ID NO：17)。

According to an embodiment of the invention, the second heavy chain variable region has the amino acid sequence as set forth in SEQ ID NO: 12. the amino acid sequence of SEQ ID NO: 13. SEQ ID NO:14, CDR1, CDR2, CDR3 sequences.

According to an embodiment of the invention, the second light chain variable region has the amino acid sequences as set forth in SEQ ID NOs: 15. SEQ ID NO: 16. the amino acid sequence of SEQ ID NO:17, CDR1, CDR2, CDR3 sequences shown.

According to an embodiment of the invention, the second heavy chain variable region has the amino acid sequence as set forth in SEQ ID NO:18, or a pharmaceutically acceptable salt thereof.

EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYNMNWVRQAPGKGLEWVSYISSSSSTIYYADSVKGRFTISRDNAKNSLSLQMNSLRDEDTAVYYCARAYYYGMDVWGQGTTVTVSS(SEQ ID NO：18)。

According to an embodiment of the invention, the second light chain variable region has the amino acid sequence as set forth in SEQ ID NO:19, or a pharmaceutically acceptable salt thereof.

DIQMTQSPSSVSASVGDRVTITCRASQGISGWLAWYQQKPGKAPKFLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPPTFGGGTKVEIK(SEQ ID NO：19)。

According to an embodiment of the invention, the CH1 region and the second FC peptide segment have the amino acid sequences as shown in SEQ ID NO 20.

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO：20)。

It should be noted that the peptide fragment of the amino acid sequence shown in SEQ ID NO. 20 is a heavy chain constant region fragment consisting of a CH1 region linked to a second FC peptide fragment, wherein the C-terminus of the CH1 region is linked to the N-terminus of the second FC peptide fragment.

According to an embodiment of the present invention, the CH1 region has an amino acid sequence as shown in SEQ ID NO: 32.

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVE(SEQ ID NO：32)。

According to an embodiment of the invention, the second FC peptide segment has the amino acid sequence shown as SEQ ID NO. 33.

PKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO：33)。

According to an embodiment of the invention, the first polypeptide has the amino acid sequence shown as SEQ ID NO 21.

EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYNMNWVRQAPGKGLEWVSYISSSSSTIYYADSVKGRFTISRDNAKNSLSLQMNSLRDEDTAVYYCARAYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO：21)。

According to an embodiment of the invention, the second polypeptide has the amino acid sequence shown as SEQ ID NO. 22.

DIQMTQSPSSVSASVGDRVTITCRASQGISGWLAWYQQKPGKAPKFLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPPTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO：22)。

According to an embodiment of the present invention, the first antigen-binding region and the second antigen-binding region are linked by a knob-into-hole structure.

According to an embodiment of the invention, the knob-into-hole structure is formed by a mutation of T366W and/or S354C of the first CH3 region and a mutation of at least one of T366S, L368A, Y407V, Y349C of the second CH3 region.

According to an embodiment of the invention, the knob-into-hole structure is formed by mutations of T366W and S354C of the first CH3 region and of T366S, L368A, Y407V, Y349C of the second CH3 region. Thus, the association of the first antigen binding region comprising two first Fc peptide fragments and the association of the second antigen binding region comprising two second Fc peptide fragments can be reduced, which can improve the yield of the antibody of the present invention.

Nucleic acid molecules, expression vectors and recombinant cells

In the process of preparing or obtaining these antibodies, the corresponding antibodies or antigen-binding fragments thereof can be obtained by linking nucleic acid molecules expressing these antibodies to different vectors and then expressing the nucleic acid molecules in different cells.

In another aspect of the invention, the invention features a nucleic acid molecule. According to an embodiment of the invention, the nucleic acid molecule encodes an antibody as described above. Nucleic acid molecules according to embodiments of the invention may encode antibodies that can target binding to 4-1BB and Nectin-4 simultaneously.

According to an embodiment of the invention, the nucleic acid molecule is a DNA molecule.

In yet another aspect of the invention, the invention features an expression vector. According to an embodiment of the invention, the nucleic acid molecule described above is carried. After the expression vector according to the embodiment of the present invention is introduced into a suitable recipient cell, the expression of the antibody described above can be efficiently achieved under the mediation of a regulatory system, so that the antibody can be obtained in a large amount.

When the above-mentioned nucleic acid molecule is ligated to a vector, the nucleic acid molecule may be directly or indirectly ligated to control elements on the vector so long as the control elements can control translation, expression, and the like of the nucleic acid molecule. Of course, these control elements may be derived directly from the vector itself, or may be exogenous, i.e., not derived from the vector itself. Of course, the nucleic acid molecule may be operably linked to a control element.

As used herein, "operably linked" refers to the attachment of a foreign gene to a vector such that control elements within the vector, such as transcriptional and translational control sequences, and the like, are capable of performing their intended function of regulating the transcription and translation of the foreign gene. Of course, the polynucleotides encoding the first antigen-binding region of the antibody, the first polypeptide and the second polypeptide may be inserted into separate vectors, and usually into the same vector. Commonly used vectors may be, for example, plasmids, phages, etc.

According to an embodiment of the invention, the expression vector is a non-pathogenic viral vector.

According to an embodiment of the invention, the expression vector is an adenoviral vector, a lentiviral vector or a retroviral vector.

In yet another aspect of the invention, the invention features a recombinant cell. According to an embodiment of the invention, said recombinant cell carries a nucleic acid molecule as described above, or an expression vector as described above or expresses an antibody as described above. The recombinant cell is obtained by transfecting or transforming the expression vector, and can efficiently express the antibody which can be targeted and combined with 4-1BB and Nectin-4 simultaneously under a proper condition.

It should be noted that the recombinant cell of the present invention is not particularly limited, and may be a prokaryotic cell, a eukaryotic cell or a phage. The prokaryotic cell can be escherichia coli, bacillus subtilis, streptomyces or proteus mirabilis and the like. The eukaryotic cell can be fungi such as Pichia pastoris, saccharomyces cerevisiae, schizosaccharomyces and trichoderma, insect cells such as meadow armyworm, plant cells such as tobacco, and mammalian cells such as BHK cells, CHO cells, COS cells and myeloma cells. In some embodiments, the recombinant cell of the present invention is preferably a mammalian cell, including a BHK cell, a CHO cell, an NSO cell or a COS cell, and does not include an animal germ cell, a fertilized egg or an embryonic stem cell.

The term "suitable conditions" as used herein means conditions suitable for the expression of the antibody described herein. It will be readily understood by those skilled in the art that suitable conditions for antibody expression include, but are not limited to, suitable transformation or transfection means, suitable transformation or transfection conditions, healthy host cell status, suitable host cell density, suitable cell culture environment, and suitable cell culture time. The "suitable conditions" are not particularly limited, and those skilled in the art can optimize the conditions for the expression of the antibody optimally according to the specific circumstances of the laboratory.

According to an embodiment of the present invention, the recombinant cell is obtained by introducing the aforementioned expression vector into a host cell.

According to an embodiment of the invention, the recombinant cell is a eukaryotic cell.

According to an embodiment of the invention, the recombinant cell is a mammalian cell. The expression vector can be introduced into mammalian cells, recombinant cells can be constructed, and the recombinant cells can be used for expressing the antibody provided by the invention. The recombinant cell is cultured to obtain the corresponding antibody. These usable mammalian cells may be, for example, CHO cells or the like.

Method for producing antibody

According to an embodiment of the invention, the cell is a eukaryotic cell.

According to an embodiment of the invention, the eukaryotic cell is a mammalian cell. The recombinant antibody is expressed more efficiently when the cell is a eukaryotic cell, such as a mammalian cell.

According to an embodiment of the invention, the eukaryotic cell does not comprise an animal germ cell, a fertilized egg or an embryonic stem cell.

Pharmaceutical composition and kit

According to an embodiment of the invention, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

In yet another aspect of the invention, the invention features a kit. According to an embodiment of the invention, the kit comprises: the aforementioned antibody, the aforementioned nucleic acid molecule, the aforementioned expression vector, or the aforementioned recombinant cell. The kit according to the embodiment of the invention can be combined with 4-1BB protein and/or Nectin-4 protein, so that the 4-1BB protein and/or the Nectin-4 protein can be effectively identified.

Use of

In a further aspect of the invention, the invention proposes the use of an aforementioned antibody, an aforementioned nucleic acid molecule, an aforementioned expression vector, an aforementioned recombinant cell or an aforementioned pharmaceutical composition for the preparation of a medicament for the treatment or prevention of cancer. According to the embodiment of the invention, the antibody or the pharmaceutical composition can be simultaneously targeted and combined with the antibodies of 4-1BB and Nectin-4, can effectively mediate the killing effect of T cells on tumor cells with high expression of Nectin-4, has a stronger tumor inhibition effect, and can effectively treat cancers.

According to an embodiment of the invention, the cancer is selected from a cancer highly expressing Nectin-4.

According to an embodiment of the present invention, the cancer highly expressing Nectin-4 includes at least one of breast cancer, ovarian cancer, cervical cancer, colorectal cancer, esophageal cancer, gastric cancer, lung cancer, liver cancer, and thyroid cancer.

In a further aspect of the invention, the invention proposes the use of an antibody as described above, a nucleic acid molecule as described above, an expression vector as described above or a recombinant cell as described above for the preparation of a kit for the detection of 4-1BB and/or Nectin-4. According to an embodiment of the present invention, the antibody or kit of the present invention can bind to 4-1BB protein and/or Nectin-4 protein, thereby efficiently identifying 4-1BB protein and/or Nectin-4 protein.

Methods of treating or preventing cancer

In another aspect of the present invention, the present invention provides a method for preventing and/or treating cancer. According to an embodiment of the invention, the method comprises: administering to the subject a pharmaceutically acceptable amount of the foregoing antibody or the foregoing pharmaceutical composition. According to embodiments of the present invention, the method is effective for preventing or treating cancer.

It is to be noted that, in the present context, the "pharmaceutically acceptable amount" may vary depending on the mode of administration and the severity of the disease to be treated, etc., and is preferably an effective amount. The selection of a pharmaceutically acceptable amount can be determined by one of ordinary skill in the art based on a variety of factors (e.g., by clinical trials). Such factors include, but are not limited to: pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the weight of the patient, the immune status of the patient, the route of administration, and the like. For example, divided doses may be administered several times per day, or the dose may be proportionally reduced, as urgently required by the condition being treated.

According to an embodiment of the present invention, the cancer highly expressing Nectin-4 includes at least one of breast cancer, ovarian cancer, cervical cancer, colorectal cancer, esophageal cancer, gastric cancer, lung cancer, liver cancer and thyroid cancer.

The scheme of the invention will be explained with reference to the following examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are conventional products which are commercially available, and are not indicated by manufacturers.

Example 1: preparation of bispecific antibody molecules

The production of bispecific antibodies was carried out in this example, following the following experimental procedures: expicHO cells (purchased from Thermo Fisher) were cultured in ExpicHO Expression Medium (purchased from Thermo Fisher, A2910001) and adjusted to a cell concentration of 6X 106/mL to obtain an ExpicHO cell solution. pTT5 vector (synthesized by Kinzyme, suzhou) containing three strands of coding genes (shown in SEQ ID NOS: 23, 24 and 25, respectively) was added to 2mL OptiSFM medium (Thermo Fisher, 12309019) to obtain solution A. Wherein, the first chain encoding gene comprises nucleotide sequences for encoding a 4-1BB single-chain antibody (SEQ ID NO: 9) and a first FC peptide fragment (SEQ ID NO: 10), the second chain encoding gene comprises nucleotide sequences for encoding a second heavy chain variable region (SEQ ID NO: 18) and a second FC peptide fragment (SEQ ID NO: 20) of Nectin-4, and the third chain encoding gene comprises nucleotide sequences for encoding a second light chain variable region (SEQ ID NO: 19) and a CL region (SEQ ID NO: 31) of Nectin-4. 160. Mu.L Expifeactine CHO transfection reagent (Thermofisiher, A)29130 Add to 2mL OptiSFM medium to obtain solution B. Then, solution a and solution B were mixed to obtain a transfection mixture, and the transfection mixture was added to 50mL of expichho cell solution in its entirety within 5 minutes. At 37 ℃ 5% CO ₂ After 1 day of incubation under the conditions, 8mL of Feed (Thermo Fisher, A29130), 300. Mu.L of Enhancer (Thermo Fisher, A29130) were added, and the contents were converted to 32 ℃ and 5% CO ₂ Culture supernatants were harvested after 9 days of culture under conditions, with 8mL of Feed added on day 5. The bispecific antibody was affinity-purified from the culture supernatant using Protein A purification column (GE) to obtain antibody 4-1 BB. Times.Nectin-4 (i.e., bispecific antibody). The antibody 4-1BB × Nectin-4 (used interchangeably herein with "4-1BB × Nectin-4 antibody", "4-1BB × Nectin-4 bispecific antibody" and "bispecific antibody") was determined to have the amino acid sequences shown in SEQ ID NO:11, SEQ ID NO:21 and SEQ ID NO: 22. The structure of antibody 4-1BB × Nectin-4 is shown in FIG. 1.

The first chain encoding gene (i.e., the first antigen binding region) is used to encode SEQ ID NO. 11, and comprises the nucleotide sequence shown below:

CAAGTGCAATTGCAACAGTGGGGAGCCGGACTGCTTAAACCATCAGAGACTCTGAGCCTGACATGTGCCGTGTACGGCGGATCCTTTTCCGGCTACTATTGGAGCTGGATCAGACAGTCCCCCGAAAAGGGACTCGAATGGATTGGCGAGATAAATCACGGAGGCTACGTTACTTACAATCCCTCTCTCGAGTCCCGCGTCACCATTAGTGTCGATACATCCAAAAACCAGTTTTCCCTGAAGTTGTCCTCCGTCACCGCTGCAGACACCGCAGTCTACTACTGCGCCCGGGACTATGGTCCTGGTAACTACGATTGGTACTTCGATCTGTGGGGCAGAGGCACACTGGTGACAGTGTCATCAGGTGGTGGAGGTTCTGGAGGAGGTGGATCTGGAGGGGGGGGTTCAGAGATTGTACTGACCCAGAGCCCTGCAACCCTGAGCCTCTCTCCTGGAGAACGCGCCACATTGTCCTGTCGCGCATCACAGTCAGTCTCTAGTTACTTGGCATGGTACCAACAGAAGCCAGGGCAGGCTCCACGACTGCTGATCTACGACGCCAGCAATCGAGCTACAGGTATCCCAGCTAGGTTCTCAGGTTCCGGCAGCGGAACTGACTTTACCCTGACAATTTCATCCCTGGAGCCCGAGGATTTCGCAGTCTACTATTGTCAGCAGCGCTCAAATTGGCCCCCTGCACTTACTTTCGGTGGCGGTACCAAGGTGGAAATCAAAGGCGGAGGGGGCAGCCCAAAGTCCTGTGATAAGACTCACACATGCCCCCCTTGTCCTGCACCAGAAGCTGCAGGAGGTCCTTCCGTCTTTCTGTTCCCCCCCAAGCCAAAAGACACCCTGATGATTTCCCGGACACCCGAGGTGACATGTGTCGTGGTCGATGTGTCTCATGAGGATCCTGAAGTGAAATTCAACTGGTACGTGGACGGAGTCGAGGTCCACAATGCCAAGACCAAACCTAGGGAGGAGCAGTACAATTCAACTTATCGCGTGGTTAGTGTGCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAATGCAAAGTGTCCAACAAAGCCCTGCCTGCCCCAATCGAAAAAACTATTAGTAAAGCTAAAGGTCAGCCTCGGGAACCTCAAGTCTATACTCTGCCCCCCTGCCGGGAGGAAATGACAAAAAACCAGGTGAGCTTGTGGTGTCTTGTGAAGGGCTTCTATCCTAGCGACATCGCTGTGGAGTGGGAGAGCAACGGTCAACCTGAGAATAACTATAAAACCACCCCTCCCGTGCTGGACTCTGACGGTTCCTTCTTTCTGTACTCCAAGCTGACAGTCGATAAGAGCAGATGGCAGCAGGGAAATGTCTTTTCTTGTTCCGTTATGCACGAAGCCCTGCATAACCACTACACTCAGAAGAGCCTCAGCCTGAGCCCTGGGAAA(SEQ ID NO：23)。

the second chain encoding gene (namely the first polypeptide) is used for encoding SEQ ID NO:21, and the second chain encoding gene comprises the nucleotide sequence shown as follows:

GAAGTTCAGCTCGTGGAAAGTGGAGGAGGACTGGTTCAACCTGGAGGATCACTGCGCCTCAGCTGTGCAGCATCTGGTTTCACCTTCTCTAGCTATAACATGAACTGGGTGAGACAGGCCCCTGGAAAGGGACTGGAATGGGTGTCTTACATTAGTTCTAGCTCCTCTACTATCTATTATGCCGACTCCGTTAAAGGAAGGTTCACCATCTCACGTGACAATGCCAAAAATAGCCTGTCTCTGCAGATGAATTCCCTGCGGGATGAGGATACAGCCGTGTACTATTGCGCTCGAGCCTATTATTACGGGATGGACGTCTGGGGGCAGGGTACTACTGTGACTGTGTCTTCAGCATCCACCAAGGGCCCATCTGTGTTTCCTCTGGCTCCAAGCTCCAAAAGTACCTCCGGGGGCACAGCTGCTCTGGGTTGTTTGGTCAAGGACTATTTTCCTGAGCCAGTCACAGTGTCCTGGAACTCAGGCGCCCTGACATCTGGTGTCCATACCTTTCCTGCCGTCCTCCAGAGCTCCGGACTGTACAGTCTCTCTAGTGTGGTCACAGTTCCTTCATCATCTCTCGGCACACAGACATATATTTGCAACGTGAACCACAAGCCTTCAAATACCAAGGTTGATAAGAAGGTCGAGCCCAAAAGCTGCGACAAGACTCATACTTGTCCACCTTGTCCTGCTCCAGAAGCCGCTGGAGGCCCATCTGTGTTTTTGTTCCCTCCTAAACCCAAAGACACCCTCATGATCTCACGGACTCCAGAAGTCACATGTGTCGTGGTTGATGTGTCTCACGAGGACCCAGAAGTCAAGTTTAACTGGTACGTGGACGGCGTAGAGGTGCATAACGCTAAAACTAAGCCCCGGGAGGAGCAGTACAATAGCACATATCGAGTCGTGTCAGTCCTCACTGTGCTGCATCAGGACTGGCTCAACGGCAAGGAATACAAGTGCAAAGTCTCCAATAAGGCTTTGCCTGCCCCTATCGAGAAGACAATCTCCAAGGCAAAGGGGCAACCCAGAGAACCCCAAGTGTGTACCCTGCCACCTTCCAGGGAAGAAATGACCAAGAATCAGGTGAGTTTGAGCTGCGCTGTGAAAGGATTTTACCCCTCTGACATTGCTGTCGAATGGGAGTCTAACGGTCAGCCAGAAAACAATTATAAGACAACTCCACCTGTCCTCGACTCCGACGGATCCTTCTTTCTTGTCTCTAAGCTGACAGTGGATAAATCCCGCTGGCAGCAGGGCAATGTCTTCTCATGCAGCGTTATGCATGAGGCCCTCCATAACCATTATACCCAGAAGTCCCTGAGCCTTTCACCAGGCAAG(SEQ ID NO：24)。

the third chain encoding gene (namely the second polypeptide) is used for encoding SEQ ID NO:22, and the third chain encoding gene comprises the nucleotide sequence shown as follows:

GATATCCAAATGACCCAATCCCCCTCTTCTGTCAGCGCTTCCGTTGGCGATAGGGTAACAATCACTTGCCGCGCAAGCCAAGGAATCTCCGGCTGGCTGGCATGGTATCAGCAGAAACCAGGGAAGGCTCCAAAGTTCCTTATCTATGCCGCCAGTACACTTCAGTCTGGAGTGCCTTCTCGTTTCTCTGGTTCTGGGAGTGGCACTGACTTTACACTTACCATTAGTTCCTTGCAGCCCGAAGACTTCGCCACATATTACTGTCAGCAGGCAAATAGCTTCCCTCCTACATTCGGAGGGGGAACAAAGGTGGAAATCAAGAGGACAGTTGCCGCACCTTCCGTGTTTATCTTTCCTCCAAGCGATGAGCAGCTGAAGAGTGGGACTGCCTCAGTCGTCTGTCTGTTGAACAATTTCTATCCAAGAGAGGCCAAAGTGCAGTGGAAAGTGGACAATGCATTGCAGTCCGGAAACTCACAGGAGAGCGTGACAGAGCAGGACTCCAAAGATTCTACATACAGCCTCTCCTCCACACTGACATTGTCCAAGGCAGATTACGAGAAGCACAAAGTGTACGCTTGCGAGGTCACCCACCAGGGCCTGTCATCCCCTGTGACAAAGTCCTTCAACCGAGGCGAGTGC(SEQ ID NO:25)。

example 2: identification of binding ability of bispecific antibody and 4-1BB protein

ELISA experiments were used to test the binding properties of the 4-1BB × Nectin-4 antibody obtained in example 1. 4-1BB-strep tagII protein is coated into a 96-well plate, and the strength of a signal after the antibody is added is used for judging the binding property of the antibody and 4-1 BB.

4-1BB-strep tagII protein (produced in this laboratory using conventional techniques) was diluted to 2. Mu.g/mL with PBS buffer, added to a 96-well plate in a volume of 100. Mu.L/well, and allowed to stand overnight at 4 ℃. The 96-well plate was aspirated off PBS buffer, the plate was washed 6 times with PBST (0.1% Tween 20 in PBS pH 7.2), then blocked by adding 200. Mu.L/well PBS/10% BSA and incubating at 37 ℃ for 2h. The blocking solution was removed, and after washing the plate 6 times with PBST, the 4-1BB × Nectin-4 antibody to be tested was diluted to an appropriate concentration using 100. Mu.L/well of PBST/0.05% BSA, and then incubated at 37 ℃ for 1h. The reaction system was removed and after washing the plate 6 times with PBST, HRP (horseradish peroxidase) -labeled rabbit anti-human IgG secondary antibody (Boshide, BA 1070) was diluted with PBST/0.05% BSA at 100. Mu.L/well and incubated at 37 ℃ for 1h. After the completion of incubation, the plate was washed 6 times with PBST, 80. Mu.L/well of TMB (tetramethylbenzidine) was added, the plate was incubated at room temperature for 3min, and the reaction was terminated by adding 80. Mu.L/well of 4M sulfuric acid. The absorbance was read at 450mm using a microplate reader. The specific experimental results are shown in FIG. 2, which shows that the antibody of the present invention can bind to 4-1BB-strep tagII.

4-1BB-strep tagII:

LQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQDCKQGQELTKKGCKDCCFGTFNDQKRGICRPWTNCSLDGKSVLVNGTKERDVVCGPSPADLSPGASSVTPPAPAREPGHSPQGGSAWSHPQFEK(SEQ ID NO:34)。

example 3: characterization of the binding Capacity of bispecific antibodies to CHO-K1-4-1BB cells

This example uses flow cytometry to examine the binding properties of the bispecific antibody obtained in example 1, and the signal intensity after addition of the bispecific antibody was used to determine the binding properties of the bispecific antibody and CHO-K1-4-1BB cells. The specific experimental procedures were as follows:

HEK293T cells according to 5X 10 ⁵ Cells/well were plated in six-well plates and incubated overnight in DMEM medium without double antibody. The medium was discarded before transfection and 1mL of fresh DMEM medium without double antibody was added. The coding sequence of 4-1BB protein (SEQ ID NO: 26), pMD2G and psPAX2 vector (total 3. Mu.g) were inserted between EcoRI and BamHI at the cleavage site of pLVX-EF1a-4-1BB-IRES-puro vector in the proportion of 2 ⁴ To 6-well plates of CHO-K1 cells, polybrene (Sigma) was added at a final concentration of 4. Mu.g/mL, and the cells were cultured for 12 hours. The supernatant was then discarded and fresh complete DMEM medium was added. The obtained cell is the CHO-K1-4-1BB cell.

ATGGGTAATTCCTGTTATAACATCGTCGCTACTTTGTTGTTGGTGCTGAACTTCGAGAGGACTAGATCTCTGCAGGATCCTTGCTCCAACTGTCCAGCTGGCACCTTTTGCGATAATAACCGTAATCAGATCTGTTCTCCCTGCCCTCCAAATAGTTTCAGCTCCGCTGGTGGCCAAAGAACATGCGATATTTGTCGGCAATGCAAGGGGGTCTTTAGGACCAGGAAAGAGTGCAGTTCTACCTCCAATGCCGAATGCGATTGCACACCCGGATTCCATTGCTTGGGGGCCGGCTGCTCCATGTGCGAACAGGACTGTAAGCAGGGCCAAGAGCTTACAAAAAAGGGCTGTAAGGACTGCTGTTTTGGCACCTTTAACGACCAGAAGCGAGGCATCTGCCGCCCATGGACCAACTGTAGCTTGGATGGGAAGAGCGTCCTGGTCAATGGAACCAAGGAGCGGGACGTTGTGTGTGGCCCATCTCCAGCAGACCTGAGTCCTGGGGCTAGTTCCGTCACACCACCCGCCCCCGCTAGGGAGCCTGGCCATTCTCCTCAAATCATCTCCTTTTTCCTCGCACTCACTAGCACTGCTCTTCTCTTTCTGCTCTTTTTCCTGACCCTGCGGTTCTCTGTTGTGAAAAGAGGCCGGAAGAAACTTCTGTATATATTTAAGCAGCCATTCATGCGGCCAGTACAAACAACACAGGAGGAGGATGGATGCTCTTGTCGGTTCCCTGAGGAAGAGGAAGGAGGATGCGAGCTG(SEQ ID NO:26)。

MGNSCYNIVATLLLVLNFERTRSLQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQDCKQGQELTKKGCKDCCFGTFNDQKRGICRPWTNCSLDGKSVLVNGTKERDVVCGPSPADLSPGASSVTPPAPAREPGHSPQIISFFLALTSTALLFLLFFLTLRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL(SEQ ID NO:27)。

CHO-K1-4-1BB cells were diluted to 1X 10 with PBS ⁶ To 1.5mL of EP tube was added 90. Mu.L/tube, 10. Mu.L/tube of mouse serum was added, and the mixture was blocked at 4 ℃ for 30min. A series of concentration gradients (0.1, 1, 10, 30, 100, 300. Mu.g/mL) of 4-1BB × Nectin-4 bispecific antibody, hIgG (control IgG1, biolegend, QA16A 12) 10. Mu.L/tube were added and incubated at 4 ℃ for 30min. After the incubation, 1mL of PBS was added to the EP tube, centrifuged at 100 Xg for 5min at 4 ℃ and the supernatant was discarded, and the precipitate was washed with PBS. After centrifugation, the supernatant was discarded, and the cells were resuspended in 100. Mu.L/tube of PBS, and after resuspension, a 1. Mu.L/tube of Alexa-647-labeled secondary rat anti-human Fc antibody (Biolegend, M1310G 05) was added thereto, and incubated at 4 ℃ for 30min in the absence of light. Washed twice with PBS, centrifuged and discarded the supernatant. The cells were resuspended in 200. Mu.L/tube PBS and assayed by flow cytometry, and the results are shown in FIG. 3, which further shows that the bispecific antibody 4-1BB × Nectin-4 of the present invention is able to bind to CHO-K1-4-1BB cells.

Example 4: characterization of the binding Capacity of bispecific antibodies to human peripheral blood CD4+ T cells

This example uses flow cytometry to examine the binding properties of the bispecific antibody obtained in example 1 and uses the intensity of the signal after the addition of the bispecific antibody to determine the binding properties of the bispecific antibody and human peripheral blood CD4+ T cells. The specific experimental procedures were as follows:

human peripheral blood mononuclear cells were diluted 5X 10 with PBS ⁶ mL, added to a 1.5mL EP tube in a volume of 90 μ L/tube, to which 10 μ L/tube rat serum was added, blocked at 4 ℃ for 30min; after blocking, a series of concentration gradients (0.1, 1, 10, 30, 100, 300. Mu.g/mL) of 4-1BB × Nectin-4 bispecific antibody, hIgG (control IgG1, biolegend, QA16A 12) 10. Mu.L/tube were added, incubated at 4 ℃ for 30min, 1mL of PBS was added to the EP tube, centrifuged at 4 ℃ and 100 × G for 5min, the supernatant was discarded, the precipitate was washed with PBS, the supernatant was discarded after centrifugation, the cells were resuspended in 100. Mu.L/tube of PBS, 1. Mu.L/tube of Alexa-647 labeled secondary rat anti-human Fc antibody (Biolegend, M1310G 05) and 1. Mu.L/tube of FITC labeled mouse anti-human CD4 antibody (Invitrogen, OKT 4) were added thereto, and incubated at 4 ℃ for 30min in the dark. Washed twice with PBS, centrifuged and the supernatant discarded. The cells were resuspended in 200. Mu.L/tube PBS and assayed by flow cytometry, and the results are shown in FIG. 4, which illustrates that the bispecific antibody of the present invention is capable of binding to human peripheral blood T cells.

Example 5: characterization of the binding Capacity of bispecific antibodies to CHO-K1-Nectin-4 cells

This example uses flow cytometry to examine the binding properties of the bispecific antibody obtained in example 1 and uses the intensity of the signal after the addition of the bispecific antibody to determine the binding properties of the bispecific antibody and CHO-K1-Nectin-4 cells. The specific experimental procedures were as follows:

HEK293T cells according to 5X 10 ⁵ Cells/well were plated in six-well plates and incubated overnight in DMEM medium without double antibody. Media was discarded before transfection and 1mL of fresh DMEM media without double antibody was added. The coding sequence for the Nectin-4 protein (SEQ ID NO: 28) was inserted between EcoRI and BamHI in pLVX-EF1a-Nectin-4-IRES-puro (pLVX-EF 1a-IRES-puro vector), pMD2G, psPAX2 vector (total 3. Mu.g) in 200. Mu.L of serum-free DMEM medium in the ratio 2Company), the obtained Nectin-4 protein has an amino acid sequence shown as SEQ ID NO. 29; after mixing, the mixture was allowed to stand for 16min, and then the whole liquid was added to the above six-well plate in which HEK293T cells were spread. After 6h of culture, the medium was discarded and fresh complete DMEM medium was added for culture. 48h after transfection, cell culture supernatants were harvested and passed through 0.45 μm filters (Millipore) to give viral supernatants. Adding all the virus supernatant to the solution containing 1 × 10 ⁴ To a 6-well plate of CHO-K1 cells, polybrene (Sigma) was added at a final concentration of 4. Mu.g/mL, and the cells were cultured for 12 hours. The supernatant was then discarded and fresh complete DMEM medium was added. The obtained cell is the CHO-K1-Nectin-4 cell.

ATGCCACTTAGCTTGGGAGCTGAAATGTGGGGTCCAGAAGCTTGGCTCCTCCTTTTGCTCTTGTTGGCCTCTTTTACAGGCCGTTGCCCTGCTGGAGAACTGGAAACTAGTGATGTGGTGACCGTGGTGCTGGGACAGGACGCAAAGCTCCCCTGCTTTTACAGAGGTGATTCTGGTGAGCAGGTTGGCCAGGTTGCATGGGCTAGAGTCGACGCAGGTGAGGGCGCACAGGAGTTGGCACTGCTTCATTCCAAGTACGGCCTGCACGTTTCTCCAGCTTATGAAGGCAGAGTGGAACAGCCACCACCACCTCGTAACCCACTGGATGGCAGCGTGCTGCTGAGGAACGCTGTGCAGGCTGACGAGGGCGAATATGAGTGCCGCGTGAGCACATTCCCCGCTGGTAGCTTTCAGGCACGACTGAGATTGAGGGTGCTGGTCCCACCCCTCCCATCTCTTAATCCTGGCCCTGCCTTGGAAGAAGGGCAGGGACTTACACTGGCTGCTAGCTGCACAGCTGAGGGGAGTCCAGCACCTTCAGTGACTTGGGATACCGAGGTTAAAGGGACTACCAGTTCTCGAAGTTTCAAGCATTCCCGATCTGCTGCCGTGACTTCAGAGTTCCATCTGGTTCCCAGCAGGAGCATGAATGGACAGCCACTGACATGTGTGGTTTCCCACCCTGGCCTGCTCCAGGATCAGAGGATTACCCACATACTTCACGTCTCTTTTCTGGCAGAGGCATCTGTGAGAGGTCTGGAGGACCAGAATTTGTGGCATATCGGCCGAGAGGGCGCAATGCTTAAATGTCTCAGCGAAGGACAGCCACCACCAAGCTACAATTGGACTAGACTGGATGGACCACTGCCAAGCGGAGTGCGTGTCGATGGTGATACACTCGGCTTTCCCCCACTGACTACAGAGCATAGCGGCATATACGTGTGTCATGTTTCTAACGAATTTTCTAGCCGGGACTCTCAGGTGACCGTGGATGTGCTCGACCCACAAGAGGACTCCGGCAAGCAAGTCGATCTGGTTAGTGCCTCTGTGGTGGTCGTGGGGGTGATTGCAGCTCTTCTGTTTTGTTTGCTGGTCGTTGTGGTAGTGCTGATGAGCCGCTATCACCGTCGAAAGGCACAGCAAATGACTCAGAAGTATGAGGAAGAACTGACTCTCACTCGTGAGAACTCAATCAGGCGCCTGCACAGCCATCACACAGACCCAAGGAGCCAACCTGAGGAGAGCGTTGGTCTTCGAGCCGAGGGACATCCTGATTCACTCAAAGACAACAGCTCATGCTCCGTCATGTCAGAGGAGCCTGAAGGACGCTCCTATTCCACATTGACCACCGTGCGAGAAATTGAAACACAGACTGAGCTCCTGTCTCCTGGCAGCGGACGTGCAGAGGAAGAGGAGGATCAGGATGAGGGCATTAAGCAGGCCATGAACCATTTTGTTCAAGAGAATGGAACCCTGCGAGCTAAGCCTACCGGCAATGGCATATACATCAATGGACGAGGCCACCTCGTC(SEQ ID NO:28)。

MPLSLGAEMWGPEAWLLLLLLLASFTGRCPAGELETSDVVTVVLGQDAKLPCFYRGDSGEQVGQVAWARVDAGEGAQELALLHSKYGLHVSPAYEGRVEQPPPPRNPLDGSVLLRNAVQADEGEYECRVSTFPAGSFQARLRLRVLVPPLPSLNPGPALEEGQGLTLAASCTAEGSPAPSVTWDTEVKGTTSSRSFKHSRSAAVTSEFHLVPSRSMNGQPLTCVVSHPGLLQDQRITHILHVSFLAEASVRGLEDQNLWHIGREGAMLKCLSEGQPPPSYNWTRLDGPLPSGVRVDGDTLGFPPLTTEHSGIYVCHVSNEFSSRDSQVTVDVLDPQEDSGKQVDLVSASVVVVGVIAALLFCLLVVVVVLMSRYHRRKAQQMTQKYEEELTLTRENSIRRLHSHHTDPRSQPEESVGLRAEGHPDSLKDNSSCSVMSEEPEGRSYSTLTTVREIETQTELLSPGSGRAEEEEDQDEGIKQAMNHFVQENGTLRAKPTGNGIYINGRGHLV(SEQ ID NO:29)。

CHO-K1-Nectin-4 cells were diluted to 1X 10 with PBS ⁶ PermL, in a volume of 90. Mu.L/tube in a 1.5mL EP tube, 10. Mu.L/tube rat serum was added thereto, and the tube was sealed at 4 ℃ for 30min. A series of concentration gradients (0.1, 1, 10, 30, 100, 300. Mu.g/mL) of 4-1BB × Nectin-4 bispecific antibody, hIgG (control IgG1, biolegend, QA16A 12) 10. Mu.L/tube were added and incubated at 4 ℃ for 30min. After the incubation, 1mL of PBS was added to the EP tube, centrifuged at 100 Xg for 5min at 4 ℃ and the supernatant was discarded, and the precipitate was washed with PBS. After centrifugation, the supernatant was discarded, and the cells were resuspended in 100. Mu.L/tube of PBS, and after resuspension, a 1. Mu.L/tube of Alexa-647-labeled secondary rat anti-human Fc antibody (Biolegend, M1310G 05) was added thereto, and incubated at 4 ℃ for 30min in the absence of light. Washed twice with PBS, centrifuged and the supernatant discarded. The cells were resuspended in 200. Mu.L/tube PBS and assayed by flow cytometry, and the results are shown in FIG. 5, which further shows that the bispecific antibody 4-1BB × Nectin-4 of the present invention can bind to CHO-K1-Nectin-4 cells.

Example 6: identification of the binding Capacity of bispecific antibodies to human Breast cancer SK-BR-3 cells

This example uses flow cytometry to examine the binding properties of the bispecific antibody obtained in example 1 and uses the intensity of the signal after the addition of the bispecific antibody to determine the binding properties of the bispecific antibody and human breast cancer SK-BR-3 cells. The specific experimental procedures were as follows:

human milk with PBSAdenocarcinoma SK-BR-3 cells diluted to 1X 10 ⁶ To 1.5mL of EP tube was added 90. Mu.L/tube, 10. Mu.L/tube of rat serum was added, and the mixture was blocked at 4 ℃ for 30min. A series of concentration gradients (0.1, 1, 10, 30, 100, 300. Mu.g/mL) of 4-1BB XNectin-4 bispecific antibody, hIgG (control IgG1, biolegend, QA16A 12) 10. Mu.L/tube were added and incubated at 4 ℃ for 30min. After the incubation, 1mL of PBS was added to the EP tube, centrifuged at 100 Xg for 5min at 4 ℃ and the supernatant was discarded, and the precipitate was washed with PBS. After centrifugation, the supernatant was discarded, and the cells were resuspended in 100. Mu.L/tube of PBS, and after resuspension, a 1. Mu.L/tube of Alexa-647-labeled secondary rat anti-human Fc antibody (Biolegend, M1310G 05) was added thereto, and incubated at 4 ℃ for 30min in the absence of light. Washed twice with PBS, centrifuged and the supernatant discarded. The cells were resuspended in 200. Mu.L/tube PBS and detected by flow cytometry, and the specific experimental results are shown in FIG. 6, which further shows that the bispecific antibody 4-1BB × Nectin-4 of the present invention can bind to human breast cancer SK-BR-3 cells.

Example 7: characterization of bispecific antibodies to promote activation of Jurkat-NFAT-lucia-4-1BB reporter cells

This example identifies the ability of the bispecific antibody obtained in example 1 to cross-link Nectin-4 on the surface of target cell and 4-1BB on the surface of effector cell, and to promote T cell activation, using Jurkat-NFAT-lucia-4-1BB reporter method, and the relative intensity of the chemiluminescent signal (RLU) to determine the ability of the bispecific antibody to cross-link target cell and T cell, and thus to activate T cell.

HEK293T cells according to 5X 10 ⁵ Cells/well were plated in six-well plates and incubated overnight in DMEM medium without double antibody. The medium was discarded before transfection and 1mL of fresh DMEM medium without double antibody was added. The coding sequence of 4-1BB protein (SEQ ID NO: 26), pMD2G, and psPAX2 vector (total 3. Mu.g) were inserted between EcoRI and BamHI at the cleavage site of pLVX-EF1a-IRES-puro vector in a ratio of 2The medium was discarded, and fresh complete DMEM medium was added for culture. After 48h of transfection, the cell culture supernatant was collected and passed through a 0.45 μm filter (Millipore), to obtain a virus supernatant. Adding all the virus supernatant to the solution containing 1 × 10 ⁴ Jurkat-NFAT-lucia cells were cultured in 6-well plates for 12 hours with the addition of polybrene (Sigma) at a final concentration of 4. Mu.g/mL. The supernatant was then discarded and fresh complete DMEM medium was added. The resulting cells were Jurkat-NFAT-lucia-4-1BB cells.

(1) CHO-K1-Nectin-4 cells obtained in example 5 were diluted to 1X 10 with complete RPMI-1640 medium ⁵ PermL, added to a 96-well plate in a volume of 100. Mu.L/well.

(2) The 4-1BB XNectin-4 bispecific antibody obtained in example 1 was diluted to 500. Mu.g/mL, 100. Mu.g/mL, 20. Mu.g/mL, 4. Mu.g/mL, 0.8. Mu.g/mL, 160ng/mL, 32ng/mL, and 6.4ng/mL using complete RPMI-1640 medium, and added to the 96-well plate containing CHO-K1-Nectin-4 cells in step (1) in a volume of 20. Mu.L/well.

(3) Jurkat-NFAT-lucia-4-1BB cells were diluted to 1.25X 10 with complete RPMI-1640 medium ⁵ and/mL, adding the mixture into a 96-well plate containing the bispecific antibody in the step (2), and adding the mixture into the plate in a volume of 80 mu L/well.

(4) The reaction system obtained in step (3) was subjected to 5% CO at 37 ℃ C ₂ Culturing in an incubator for 24h.

(5) 50. Mu.L of the culture supernatant obtained in step (4) was aspirated and added to a 96-well plate, and then luciferase substrate was added to the plate in a volume of 50. Mu.L/well.

(6) Chemiluminescence was detected using a multifunctional microplate reader.

The specific experimental results are shown in FIG. 7, and further show that the bispecific antibody 4-1BB XNectin-4 of the present invention can bridge target cells (CHO-K1-Nectin-4 cells) and T cells (Jurkat-NFAT-lucia-4-1 BB cells) and promote T cell activation.

Example 8: bispecific antibody for promoting PBMC to kill tumor cells

In this example, the effect of the bispecific antibody obtained in example 1 on killing A375-Nectin-4 tumor cells by PBMC was examined by constructing a reaction system of the tumor cells + PBMC + bispecific antibody at different concentrations, and the following specific experimental procedures were performed:

HEK293T cells according to 5X 10 ⁵ Cells/well were plated in six-well plates and cultured overnight in DMEM medium without double antibody. Media was discarded before transfection and 1mL of fresh DMEM media without double antibody was added. The coding sequence of Nectin-4 protein (SEQ ID NO: 28), pMD2G and psPAX2 vector (total 3. Mu.g) were inserted between EcoRI and BamHI sites of pLVX-EF1a-Nectin-4-IRES-puro vector at a ratio of 2 ⁴ A375 cells were plated in 6-well plates and incubated for 12h with polybrene (Sigma) at a final concentration of 4. Mu.g/mL. The supernatant was then discarded and fresh complete DMEM medium was added. The obtained cells are A375-Nectin-4 cells.

(1) Adding complete RPMI-1640 culture medium into a 16-well RTCA plate according to the volume of 50 mu L/well, and performing on-machine calibration;

(2) A375-Nectin-4 cells were diluted to 2X 10 with complete RPMI-1640 medium ⁵ mL, added to the RTCA plate obtained in step (1) in a volume of 50. Mu.L/well, respectively, and then CO 5% ₂ Detecting the cell coefficient for 24h by using an xCELLigence RTCA TP device under the condition;

(3) The bispecific antibody obtained in example 1 was diluted to a series of concentration gradients (0.32, 1.6, 8, 40, 200, 1000 ng/mL) with complete RPMI-1640 medium, added to the RTCA plates obtained in step (2) in a volume of 20 μ L/well;

(4) PBMC (Seawamori L.Chiense organisms) were diluted to 1.25X 10 with complete RPMI-1640 medium ⁶ Adding the obtained product into the RTCA plate obtained in the step (3) in a volume of 80 mu L/well;

(5) Reacting the reaction system obtained in the step (4)At 37 ℃ C, 5% CO ₂ The cell coefficient was measured using an xcelgene RTCA TP device for 48h.

Specific experimental results are shown in fig. 8, and further show that the bispecific antibody of the invention can promote PBMC to kill and express Nectin-4 positive tumor cells.

The experimental results show that the bispecific antibody obtained by the invention can be combined with T cells and tumor cells, bridge the T cells and the tumor cells, and promote the T cells to kill the tumor cells.

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.

Claims

1. An antibody, comprising:

a first antigen binding region having 4-1BB binding activity;

a second antigen binding region having Nectin-4 binding activity.

2. The antibody of claim 1, wherein the first antigen binding region comprises a first heavy chain variable region and a first light chain variable region, wherein the C-terminus of the first heavy chain variable region is linked to the N-terminus of the first light chain variable region or the N-terminus of the first heavy chain variable region is linked to the C-terminus of the first light chain variable region;

optionally, the first antigen-binding region further comprises a first linker peptide, the C-terminus of the first heavy chain variable region is linked to the N-terminus of the first linker peptide, the C-terminus of the first linker peptide is linked to the N-terminus of the first light chain variable region, or the C-terminus of the first light chain variable region is linked to the N-terminus of the first linker peptide, the C-terminus of the first linker peptide is linked to the N-terminus of the first heavy chain variable region;

optionally, the first heavy chain variable region comprises SEQ ID NO:1 to 3; or

The first light chain variable region comprises SEQ ID NO:4 to 6;

optionally, the first heavy chain variable region has the amino acid sequence set forth in SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO:3, CDR1, CDR2, CDR3 sequences;

optionally, the first light chain variable region has the amino acid sequence set forth in SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO:6, CDR1, CDR2, CDR3 sequences shown in;

optionally, the first linker peptide has the amino acid sequence as set forth in SEQ ID NO: 7.

3. The antibody of claim 1, wherein the first antigen binding region further comprises a first FC peptide fragment, wherein the C-terminus of the first light chain variable region is linked to the N-terminus of the first FC peptide fragment, or wherein the C-terminus of the first heavy chain variable region is linked to the N-terminus of the first FC peptide fragment;

optionally, the first FC peptide fragment comprises a first hinge region, a first CH2 region, and a first CH3 region;

optionally, the first hinge region is a hinge region fragment of a wild-type IgG1 of human, primate or murine origin;

optionally, the first CH2 region is a CH2 region fragment of a wild-type IgG1 of human, primate or murine origin; or alternatively

The first CH2 region has L234A and/or L235A mutations compared to a CH2 region fragment of a human wild-type IgG 1;

optionally, the first CH3 region has a T366W and/or S354C mutation compared to a CH3 region fragment of a human wild type IgG 1;

optionally, the first antigen-binding region further comprises a second linking peptide;

optionally, the N-terminus of the second linker peptide is linked to the C-terminus of the first light chain variable region, and the C-terminus of the second linker peptide is linked to the N-terminus of the first FC peptide fragment; or

The N end of the second connecting peptide is connected with the C end of the first heavy chain variable region, and the C end of the second connecting peptide is connected with the N end of the first FC peptide segment;

optionally, the second linking peptide has the amino acid sequence as set forth in SEQ ID NO: 8;

optionally, the first antigen binding region comprises SEQ ID NO: 9;

optionally, the first FC peptide fragment has an amino acid sequence as shown in SEQ ID No. 10;

optionally, the first antigen binding region has the amino acid sequence shown in SEQ ID NO. 11.

4. The antibody of claim 1, wherein said second antigen-binding region comprises a first polypeptide and a second polypeptide, said first and second polypeptides being linked by an interchain disulfide bond;

the first polypeptide comprises a second heavy chain variable region, a CH1 region and a second FC peptide segment, wherein the C end of the second heavy chain variable region is connected with the N end of the CH1 region, and the C end of the CH1 region is connected with the N end of the second FC peptide segment;

the second polypeptide comprises a second light chain variable region and a CL region, the C-terminus of the second light chain variable region is linked to the N-terminus of the CL region;

optionally, the second FC peptide fragment comprises a second hinge region, a second CH2 region, and a second CH3 region;

optionally, the CH1 region is a CH1 region of a wild-type IgG1 of human, primate or murine origin;

optionally, the second hinge region is a hinge region fragment of a wild-type IgG1 of human, primate or murine origin;

optionally, the second CH2 region is a CH2 region fragment of a wild-type IgG1 of human, primate or murine origin; or

The second CH2 region has L234A and/or L235A mutations compared to a CH2 region fragment of a human wild-type IgG 1;

optionally, the second CH3 region is a CH3 region fragment of a wild-type IgG1 of human, primate or murine origin;

optionally, the second CH3 region has at least one of T366S, L368A, Y407V, Y349C mutations compared to a CH3 region fragment of human wild type IgG 1;

optionally, the CL region is a wild-type CL region of human, primate, or murine origin;

optionally, the second heavy chain variable region comprises SEQ ID NO:12 to 14; or

The second light chain variable region comprises SEQ ID NO:15 to 17;

optionally, the second heavy chain variable region has the amino acid sequences as set forth in SEQ ID NOs: 12. SEQ ID NO: 13. SEQ ID NO:14, CDR1, CDR2, CDR3 sequences;

optionally, the second light chain variable region has the amino acid sequences as set forth in SEQ ID NOs: 15. SEQ ID NO: 16. the amino acid sequence of SEQ ID NO:17, CDR1, CDR2, CDR3 sequences shown;

optionally, the second heavy chain variable region has the amino acid sequence as set forth in SEQ ID NO: 18;

optionally, the second light chain variable region has the amino acid sequence as set forth in SEQ ID NO: 19;

optionally, the CH1 region and the second FC peptide segment have amino acid sequences as shown in SEQ ID NO. 20;

optionally, the first polypeptide has an amino acid sequence shown as SEQ ID NO. 21;

optionally, the second polypeptide has an amino acid sequence as shown in SEQ ID NO. 22.

5. The antibody of claim 3 or 4, wherein the first antigen binding region and the second antigen binding region are linked by a knob-into-hole structure;

optionally, the knob-into-hole structure is formed by a mutation of T366W and/or S354C of the first CH3 region and a mutation of at least one of T366S, L368A, Y407V, Y349C of the second CH3 region.

6. A nucleic acid molecule encoding the antibody of any one of claims 1 to 5;

optionally, the nucleic acid molecule is a DNA molecule.

7. An expression vector carrying the nucleic acid molecule of claim 6;

optionally, the expression vector is a non-pathogenic viral vector;

optionally, the expression vector is an adenoviral vector, a lentiviral vector or a retroviral vector.

8. A method of producing the antibody of any one of claims 1 to 5, comprising:

introducing the expression vector of claim 7 into a cell;

subjecting said cells to a culture treatment under conditions suitable for protein expression and secretion so as to obtain said antibody;

optionally, the cell is a eukaryotic cell.

9. A recombinant cell carrying the nucleic acid molecule of claim 6, or the expression vector of claim 7, or expressing the antibody of any one of claims 1 to 5;

optionally, the recombinant cell is obtained by introducing the expression vector of claim 7 into a host cell;

optionally, the recombinant cell is a eukaryotic cell;

preferably, the recombinant cell is a mammalian cell.

10. A pharmaceutical composition, comprising:

an antibody according to any one of claims 1 to 5, a nucleic acid molecule according to claim 6, an expression vector according to claim 7 or a recombinant cell according to claim 9;

optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

11. A kit, comprising:

the antibody of any one of claims 1 to 5, the nucleic acid molecule of claim 6, the expression vector of claim 7 or the recombinant cell of claim 9.

12. Use of the antibody of any one of claims 1 to 5, the nucleic acid molecule of claim 6, the expression vector of claim 7, the recombinant cell of claim 9, or the pharmaceutical composition of claim 10 in the manufacture of a medicament for treating or preventing cancer;

optionally, the cancer is selected from a cancer that is high expressing Nectin-4;

optionally, the cancer highly expressing Nectin-4 is breast cancer, ovarian cancer, cervical cancer, colorectal cancer, esophageal cancer, gastric cancer, lung cancer, liver cancer or thyroid cancer.

13. Use of the antibody of any one of claims 1 to 5, the nucleic acid molecule of claim 6, the expression vector of claim 7 or the recombinant cell of claim 9 in the preparation of a kit for the detection of 4-1BB and/or Nectin-4.