Bispecific antibodies and their use

Abstract

The application provides a kind of targeting 4-1BB and Nectin-4 dual-specificity antibody and its use.The antibody includes: the first antigen binding region, the first antigen binding region has 4-1BB binding activity;Second antigen binding region, the second antigen binding region has Nectin-4 binding activity, first antigen binding region is selected from 4-1BB single-chain antibody, and second antigen binding region includes 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 simultaneously, effectively mediate T cell killing tumor cells, and can effectively treat cancer.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and particularly relates to a bispecific antibody and application thereof, more particularly relates to an antibody, a nucleic acid molecule, an expression vector, a recombinant cell, a pharmaceutical composition and a kit and application thereof. BACKGROUND

[0002] Cancer is a disease that seriously threatens human life and health, and in recent years, the incidence and mortality of cancer worldwide have been rising. Current cancer treatment methods include surgical resection, radiotherapy, chemotherapy, small molecule targeted therapy, antibody targeted therapy, and large molecule immunotherapy, but the above methods only play a limited role in some cancer patients, and cancer remains a problem that plagues human life and health.

[0003] In recent years, bispecific antibodies have become a research hotspot in immunotherapy. Bispecific antibodies are artificial antibodies containing two specific antigen binding sites, which can bridge target cells (tumor cells) and effector cells (immune cells) to produce directed killing of tumor cells.

[0004] Therefore, there is an urgent need for a bispecific antibody for directed killing of tumor cells. SUMMARY

[0005] The present application aims to at least partially solve at least one of the technical problems existing in the prior art. To this end, the present application provides a bispecific antibody targeting 4-1BB and Nectin-4, which can simultaneously bind to 4-1BB and Nectin-4, and can effectively mediate T cells expressing 4-1BB to kill cells expressing Nectin-4.

[0006] The present application is based on the following findings of the inventors:

[0007] 4-1BB belongs to the tumor necrosis factor receptor superfamily member (TNFRSF9) and is mainly expressed in activated T cells. Nectin-4, also known as poliovirus receptor-like 4 (PVRL4), is a type I transmembrane protein that is lowly expressed in normal human tissues (such as skin, bladder, salivary gland, esophagus, breast and stomach) and highly expressed in cancer cells (such as breast cancer, ovarian cancer, cervical cancer, colorectal cancer, esophageal cancer, gastric cancer, lung cancer, liver cancer and thyroid cancer). In addition, studies have shown that Nectin-4 plays a key role in the occurrence, invasion and metastasis of tumors, and overexpression of Nectin-4 may promote the body's resistance to chemotherapy.

[0008] At present, the antibody drug targeting Nectin-4 is only Padcev approved, Padcev is an antibody conjugate drug composed of a monoclonal antibody targeting Nectin-4 and a potent anti-mitotic agent MMAE that blocks tubulin polymerization, which uses a monoclonal antibody to target and deliver a chemotherapeutic drug to tumors. At present, the drug is mainly used for the treatment of patients with locally advanced or metastatic urothelial carcinoma who have progressed during or following treatment with PD-1 / PD-L1 inhibitors.

[0009] Based on this, in one aspect of the present application, the present application provides an antibody. According to embodiments of the present application, 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 according to embodiments of the present application can simultaneously bind to 4-1BB and Nectin-4, thereby effectively mediating the killing effect of T cells on cells expressing Nectin-4 (such as tumor cells), especially having a strong tumor inhibition effect, which can effectively treat cancer.

[0010] In another aspect of the present application, the present application provides a nucleic acid molecule. According to embodiments of the present application, the nucleic acid molecule encodes the aforementioned antibody. The nucleic acid molecule according to embodiments of the present application can encode an antibody that can simultaneously target and bind 4-1BB and Nectin-4.

[0011] In yet another aspect of the present application, the present application provides an expression vector. According to embodiments of the present application, the expression vector carries the aforementioned nucleic acid molecule. The expression vector according to embodiments of the present application can effectively realize the expression of the aforementioned antibody under the mediation of the regulation system after being introduced into a suitable recipient cell, so as to obtain a large amount of the antibody.

[0012] In yet another aspect of the present application, the present application provides a method for preparing the aforementioned antibody. According to embodiments of the present application, the method comprises: introducing the aforementioned expression vector into a cell; and culturing the cell under conditions suitable for protein expression and secretion, so as to obtain the antibody. The method according to embodiments of the present application can effectively obtain the aforementioned antibody, and has the advantages of simple preparation method, etc.

[0013] In yet another aspect of the present application, the present application provides a recombinant cell. According to embodiments of the present application, the recombinant cell carries the aforementioned nucleic acid molecule, or the aforementioned expression vector or expresses the aforementioned antibody. The aforementioned recombinant cell is obtained by transfecting or transforming the expression vector, and the recombinant cell can efficiently express the aforementioned antibody that can simultaneously target and bind 4-1BB and Nectin-4 under suitable conditions.

[0014] In yet another aspect of the present application, the present application provides a pharmaceutical composition. According to embodiments of the present application, the pharmaceutical composition comprises the aforementioned antibody, the aforementioned nucleic acid molecule, the aforementioned expression vector, or the aforementioned recombinant cell. The pharmaceutical composition according to embodiments of the present application can be an antibody that simultaneously targets 4-1BB and Nectin-4, and can effectively mediate the killing of tumor cells expressing Nectin-4 by T cells, especially has a strong tumor inhibition effect, and can effectively treat cancer.

[0015] In yet another aspect of the present application, the present application provides a kit. According to embodiments of the present application, the kit comprises the aforementioned antibody, the aforementioned nucleic acid molecule, the aforementioned expression vector, or the aforementioned recombinant cell. The kit according to embodiments of the present application can bind to 4-1BB protein and / or Nectin-4 protein, and can effectively identify 4-1BB protein and / or Nectin-4 protein.

[0016] In yet another aspect of the present application, the present application provides a use of the aforementioned antibody, the aforementioned nucleic acid molecule, the aforementioned expression vector, the aforementioned recombinant cell, or the aforementioned pharmaceutical composition in the preparation of a medicament for treating or preventing cancer. According to embodiments of the present application, the antibody or the pharmaceutical composition of the present application can be an antibody that simultaneously targets 4-1BB and Nectin-4, and can effectively mediate the killing of tumor cells expressing Nectin-4 by T cells, especially has a strong tumor inhibition effect, and can effectively treat cancer.

[0017] In yet another aspect of the present application, the present application provides a use of the aforementioned antibody, the aforementioned nucleic acid molecule, the aforementioned expression vector, or the aforementioned recombinant cell in the preparation of a kit for detecting 4-1BB and / or Nectin-4. According to embodiments of the present application, the antibody or the kit of the present application can bind to 4-1BB protein and / or Nectin-4 protein, and can effectively identify 4-1BB protein and / or Nectin-4 protein.

[0018] Additional aspects and advantages of the present application will be in part apparent and in part pointed out hereinafter. BRIEF DESCRIPTION OF DRAWINGS

[0019] The above and / or additional aspects and advantages of the present application will become apparent and be readily appreciated from the following description, including the appended drawings.

[0020] Figure 1 is a structural schematic diagram of a bispecific antibody according to Embodiment 1 of the present application;

[0021] Figure 2is a graph of the detection results of the 4-1BB x Nectin-4 bispecific antibody according to Example 2 of the present application for the binding ability to 4-1BB protein;

[0022] Figure 3 is a graph of the detection results of the 4-1BB x Nectin-4 bispecific antibody according to Example 3 of the present application for the binding ability to CHO-K1-4-1BB cells;

[0023] Figure 4 is a graph of the detection results of the 4-1BB x Nectin-4 bispecific antibody according to Example 4 of the present application for the binding ability to human peripheral blood CD4+ T cells;

[0024] Figure 5 is a graph of the detection results of the 4-1BB x Nectin-4 bispecific antibody according to Example 5 of the present application for the binding ability to CHO-K1-Nectin-4 cells;

[0025] Figure 6 is a graph of the detection results of the recombinant bispecific antibody according to Example 6 of the present application for the binding ability to human SK-BR-3 cells;

[0026] Figure 7 is a graph of the detection results of the 4-1BB x Nectin-4 bispecific antibody according to Example 7 of the present application for the activation of Jurkat-NFAT-lucia-4-1BB reporter cells;

[0027] Figure 8 is a graph of the detection results of the recombinant bispecific antibody according to Example 8 of the present application for promoting PBMC to kill A375-Nectin-4 tumor cells. DETAILED DESCRIPTION

[0028] Embodiments of the present application are described in detail below. The embodiments described below are exemplary only and are not intended to be limiting of the present application.

[0029] In addition, the terms "first", "second", etc. are used only for descriptive purposes and are not to be construed as indicating or implying relative importance or a quantity of the indicated technical features. Thus, a feature defined with "first", "second", etc. can include one or more of the features implicitly or explicitly. Further, in the description of the present application, the meaning of "a plurality of" is two or more unless otherwise specified.

[0030] The endpoints of the ranges and any values disclosed herein are not limited to the precise values recited as exactly that endpoint. Any values that fall within the range of values are included in the stated range. Whenever a numerical range is indicated, it is meant to include all cited candidate values along with the range. Likewise, whenever a range of values is provided, it is intended to specifically include the minimum and maximum values recited, as well as any values in between.

[0031] For the purposes of the present application, certain technical and scientific terms are specifically defined below. Unless explicitly stated otherwise, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which the present application belongs. Abbreviations for amino acid residues are in accord with standard three letter and / or one letter codes applicable to the twenty commonly occurring L-amino acids.

[0032] In the present application, unless otherwise explicitly stated and limited, the term "connected" should be interpreted broadly, for example, can be directly connected, can be indirectly connected through an intermediate medium, can be internal connection of two elements or interaction relationship between two elements, unless otherwise explicitly limited. For those skilled in the art, the specific meaning of the above-mentioned term in the present application can be understood according to the specific circumstances.

[0033] In the present application, the term "bispecific antibody" generally refers to a peptide chain that can specifically recognize two protein molecules connected with two Fc fragments, respectively, wherein the two Fc fragments are connected by knob into hole structure. "Bispecific antibody" and "bispecific antibody molecule" can be used interchangeably in the present application. The peptide chain that can specifically recognize protein molecules can be a single chain antibody; or two chains connected by disulfide bond, for example, one chain is heavy chain variable region and CH1 region that can recognize protein molecules, and the other chain is light chain variable region and CL region that can recognize protein molecules.

[0034] In the present application, the term "knob into hole structure" generally refers to the formation of knob (Knob) hole (hole) mutation in the CH3 region of antibody heavy chain constant region or Fc fragment, which facilitates heavy chain occlusion to form heterodimer, for example, in the present application, the CH3 domain of human IgG1-Fc is mutated to achieve this.

[0035] In the present text, the amino acid numbering of the IgGl Fc part is numbered according to the EU numbering system, e.g. position 366 refers to position 366 according to the EU numbering system; "T366S" refers to the substitution of the threonine at position 366 according to the EU numbering system by a serine; "L368A" refers to the substitution of the leucine at position 368 according to the EU numbering system by an alanine.

[0036] In the present text, the term "expression vector" generally refers to a nucleic acid molecule capable of replicating itself in a suitable host, which transfers the inserted nucleic acid molecule into and / or between host cells. The expression vector can include a vector mainly for inserting DNA or RNA into a cell, a vector mainly for replicating DNA or RNA, and a vector mainly for the transcription and / or translation of expression of DNA or RNA. The expression vector also includes a vector having a plurality of the above-mentioned functions. The expression vector can be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell. Generally, the expression vector can produce a desired expression product by culturing a suitable host cell containing the expression vector.

[0037] In the present text, the term "recombinant cell" generally refers to a cell having a unique trait stably inherited by modification or recombination of the genetic material of a host cell using genetic engineering techniques or cell fusion techniques. Among them, the term "host cell" refers to a prokaryotic cell or a eukaryotic cell into which a recombinant expression vector can be introduced. The term "transformed" or "transfected" used herein means the introduction of a nucleic acid (e.g., a vector) into a cell by various techniques known in the art. A suitable host cell can be transformed or transfected with the DNA sequence of the present application, and can be used for the expression and / or secretion of a target protein. Examples of suitable host cells that can be used in the present application 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.

[0038] In the present text, the term "pharmaceutical composition" generally refers to a unit dosage form, and can be prepared by any one of the methods well known in the pharmaceutical art. All methods include the step of bringing the active ingredient into association with the carrier that constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing the active compound into association with a liquid carrier, a finely divided solid carrier, or both,.

[0039] As used herein, the term "pharmaceutically acceptable" means that the substance or composition must be chemically and / or toxicologically compatible with the other ingredients of the formulation and / or the mammal being treated with it. Preferably, the "pharmaceutically acceptable" of the present application means approved by a regulatory agency of the Federal 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.

[0040] As used herein, the term "pharmaceutically acceptable excipient" can include any solvent, solid, diluent or other liquid excipient, etc., suitable for the particular target dosage form. Except insofar as any conventional excipient is incompatible with the compound of the application, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutically acceptable composition, its use is contemplated to be within the scope of this application.

[0041] As used herein, the term "administration" refers to the introduction of a predetermined amount of a substance into a patient by some suitable means. The recombinant antibody or pharmaceutical composition of the present application can be administered by any common route, so long as it can reach the intended tissue. Various modes of administration are contemplated, including intraperitoneal, intravenous, intramuscular, subcutaneous, etc., but the present application is not limited to these exemplified modes of administration. Preferably, the composition of the present application is administered by intravenous or subcutaneous injection.

[0042] As used herein, the term "treatment" refers to obtaining a desired pharmacologic and / or physiologic effect. The effect can be prophylactic in terms of completely or partially preventing a disease or symptom thereof and / or can be therapeutic in terms of a partial or complete cure for a disease and / or adverse effect attributable to the disease. "Treatment" as used herein covers the treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease or condition from occurring in an individual which can be predisposed to the disease but has not yet developed or diagnosed with the disease; (b) inhibiting the disease, i.e., arresting its development; or (c) relieving the disease, i.e., causing the partial or complete regression of the disease or condition. As used herein, "treatment" covers any administration of a drug or compound to an individual to treat, cure, heal, alleviate, relieve, or prevent a disease in the individual, including but not limited to administering a drug comprising a compound described herein to an individual in need thereof.

[0043] It is noted that, with respect to the nucleic acids referred to in the specification and claims, the skilled artisan will understand that either strand of a duplex, or both, are actually encompassed. For convenience, in the specification and claims, only one strand is generally shown, but the other strand is understood as disclosed as well. In addition, nucleic acid sequences in the present application include DNA forms or RNA forms, and the disclosure of one means the other is disclosed as well.

[0044] The present application 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 as follows.

[0045] Antibody

[0046] In one aspect of the present application, an antibody is provided. According to embodiments of the present application, the antibody comprises: a first antigen binding region having 4-1BB binding activity; and a second antigen binding region having Nectin-4 binding activity. The antibody according to embodiments of the present application can bind to 4-1BB and Nectin-4 at the same time, thereby effectively mediating the killing of cells (e.g., tumor cells) expressing Nectin-4 by T cells, in particular, mediating the killing of tumor cells by T cells, having a strong tumor inhibition effect, and being effective in treating cancer.

[0047] According to embodiments of the present application, the first antigen binding region comprises a first heavy chain variable region and a first light chain variable region, and the C-terminus of the first heavy chain variable region is connected to the N-terminus of the first light chain variable region, or the N-terminus of the first heavy chain variable region is connected to the C-terminus of the first light chain variable region.

[0048] According to embodiments of the present application, the C-terminus of the first heavy chain variable region is connected to the N-terminus of the first light chain variable region.

[0049] According to embodiments of the present application, the first antigen binding region further comprises a first connecting peptide, the C-terminus of the first heavy chain variable region is connected to the N-terminus of the first connecting peptide, and the C-terminus of the first connecting peptide is connected to the N-terminus of the first light chain variable region, or the C-terminus of the first light chain variable region is connected to the N-terminus of the first connecting peptide, and the C-terminus of the first connecting peptide is connected to the N-terminus of the first heavy chain variable region.

[0050] According to embodiments of the present application, the C-terminus of the first heavy chain variable region is connected to the N-terminus of the first connecting peptide, and the C-terminus of the first connecting peptide is connected to the N-terminus of the first light chain variable region.

[0051] According to embodiments of the present application, the first heavy chain variable region comprises CDR sequences shown in any one of SEQ ID NOs: 1-3; or the first light chain variable region comprises CDR sequences shown in any one of SEQ ID NOs: 4-6. In this way, the first antigen binding region can effectively bind to 4-1BB protein.

[0052] GGSFSGYY (SEQ ID NO: 1).

[0053] INHGGYV (SEQ ID NO: 2).

[0054] ARDYGPGNYDWYFDL (SEQ ID NO: 3).

[0055] QSVSSY (SEQ ID NO: 4).

[0056] DAS (SEQ ID NO: 5).

[0057] QQRSNWPPALT (SEQ ID NO: 6).

[0058] According to an embodiment of the present application, the first heavy chain variable region has CDR1, CDR2, CDR3 sequences as shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 respectively.

[0059] According to an embodiment of the present application, the first light chain variable region has CDR1, CDR2, CDR3 sequences as shown in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 respectively.

[0060] According to an embodiment of the present application, the first connecting peptide has an amino acid sequence as shown in SEQ ID NO: 7.

[0061] GGGGSGGGGSGGGGS (SEQ ID NO: 7).

[0062] According to an embodiment of the present application, the first antigen binding region further comprises a first FC peptide segment, the C-terminus of the first light chain variable region is connected to the N-terminus of the first FC peptide segment, or the C-terminus of the first heavy chain variable region is connected to the N-terminus of the first FC peptide segment.

[0063] It should be noted that "Fc peptide segment", "Fc fragment" or "Fc" in this article refers to a peptide segment comprising hinge region, CH2 region and CH3 region, for example, wild type IgG1 Fc fragment, first FC peptide segment and second FC peptide segment mentioned in this application.

[0064] For example, the amino acid sequence of human wild type IgG1 Fc (including hinge-CH2-CH3) is as follows:

[0065] PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK (SEQ ID NO: 30).

[0066] According to an embodiment of the present application, the first FC peptide segment comprises a first hinge region, a first CH2 region and a first CH3 region.

[0067] According to an embodiment of the present application, the first hinge region is a hinge region fragment of human, primate or murine wild-type IgGl.

[0068] According to an embodiment of the present application, the first CH2 region is a CH2 region fragment of human, primate or murine wild-type IgGl; or the first CH2 region has L234A and / or L235A mutation compared to a CH2 region fragment of human wild-type IgGl.

[0069] According to an embodiment of the present application, the first CH3 region has T366W and / or S354C mutation compared to a CH3 region fragment of human wild-type IgGl.

[0070] According to an embodiment of the present application, the first antigen binding region further comprises a second connecting peptide.

[0071] According to an embodiment of the present application, the N terminus of the second connecting peptide is connected to the C terminus of the first light chain variable region, and the C terminus of the second connecting peptide is connected to the N terminus of the first FC peptide segment; or the N terminus of the second connecting peptide is connected to the C terminus of the first heavy chain variable region, and the C terminus of the second connecting peptide is connected to the N terminus of the first FC peptide segment.

[0072] According to an embodiment of the present application, the second connecting peptide has an amino acid sequence as shown in SEQ ID NO: 8. In this way, the binding activity of the first antigen binding region to the 4-1BB protein can be further increased.

[0073] GGGGS (SEQ ID NO: 8).

[0074] According to an embodiment of the present application, the first antigen binding region comprises an amino acid sequence as shown in SEQ ID NO: 13.

[0075] It should be noted that the first heavy chain variable region, the first connecting peptide, the first light chain variable region and the second connecting peptide in the present application exist in the form of a single-chain antibody, wherein the C-terminus of the first heavy chain variable region is connected to the N-terminus of the first connecting peptide, the C-terminus of the first connecting peptide is connected to the N-terminus of the first light chain variable region, the C-terminus of the first light chain variable region is connected to the N-terminus of the second connecting peptide, and the single-chain antibody (also referred to as a 4-1BB single-chain antibody) has an amino acid sequence shown in SEQ ID NO: 9.

[0076] QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQSPEKGLEWIGEINHGGYVTYNPSLESRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDYGPGNYDWYFDLWGRGTLVTVSSGGGGSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPALTFGGGTKVEIKGGGGS (SEQ ID NO: 9).

[0077] According to an embodiment of the present application, the first FC peptide segment has an amino acid sequence as shown in SEQ ID NO: 10.

[0078] PKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 10).

[0079] According to an embodiment of the present application, the first antigen binding region has an amino acid sequence as shown in SEQ ID NO: 11.

[0080] QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQSPEKGLEWIGEINHGGYVTYNPSLESRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDYGPGNYDWYFDLWGRGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPALTFGGGTKVEIKGGGGSPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 11).

[0081] According to an embodiment of the present application, the second antigen binding region comprises a first polypeptide and a second polypeptide, the first polypeptide and the second polypeptide are connected by an inter-chain disulfide bond; the first polypeptide comprises a second heavy chain variable region, a CH1 region and a second FC peptide segment, the C-terminus of the second heavy chain variable region is connected to the N-terminus of the CH1 region, the C-terminus of the CH1 region is connected to the N-terminus 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 connected to the N-terminus of the CL region.

[0082] According to an embodiment of the present application, the second FC peptide segment comprises a second hinge region, a second CH2 region and a second CH3 region.

[0083] According to an embodiment of the present application, the CH1 region is a CH1 region of human, primate or murine wild-type IgG1.

[0084] According to an embodiment of the present application, the second hinge region is a hinge region fragment of human, primate or murine wild-type IgG1.

[0085] According to embodiments of the present application, the second CH2 region is a fragment of CH2 region of human, primate or murine wild-type IgG1; or the second CH2 region has L234A and / or L235A mutation compared to a fragment of CH2 region of human wild-type IgG1.

[0086] According to embodiments of the present application, the second CH3 region is a fragment of CH3 region of human, primate or murine wild-type IgG1.

[0087] According to embodiments of the present application, the second CH3 region has at least one of T366S, L368A, Y407V, Y349C mutation compared to a fragment of CH3 region of human wild-type IgG1.

[0088] According to embodiments of the present application, the CL region is a human, primate or murine wild-type CL region.

[0089] For example, the amino acid sequence of human wild-type CL region is as follows:

[0090] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 31).

[0091] According to embodiments of the present application, the second heavy chain variable region comprises CDR sequences as shown in any one of SEQ ID NOs: 12-14; or the second light chain variable region comprises CDR sequences as shown in any one of SEQ ID NOs: 15-17.

[0092] GFTFSSYN (SEQ ID NO: 12).

[0093] ISSSSSTI (SEQ ID NO: 13).

[0094] ARAYYYGMDV (SEQ ID NO: 14).

[0095] QGISGW (SEQ ID NO: 15).

[0096] AAS (SEQ ID NO: 16).

[0097] QQANSFPPT (SEQ ID NO: 17).

[0098] According to embodiments of the application, the second heavy chain variable region has CDR1, CDR2, CDR3 sequences as set forth in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, respectively.

[0099] According to embodiments of the application, the second light chain variable region has CDR1, CDR2, CDR3 sequences as set forth in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, respectively.

[0100] According to embodiments of the application, the second heavy chain variable region has an amino acid sequence as set forth in SEQ ID NO: 18.

[0101] EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYNMNWVRQAPGKGLEWVSYISSSSSTIYYADSVKGRFTISRDNAKNSLSLQMNSLRDEDTAVYYCARAYYYGMDVWGQGTTVTVSS (SEQ ID NO: 18).

[0102] According to embodiments of the application, the second light chain variable region has an amino acid sequence as set forth in SEQ ID NO: 19.

[0103] DIQMTQSPSSVSASVGDRVTITCRASQGISGWLAWYQQKPGKAPKFLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPPTFGGGTKVEIK (SEQ ID NO: 19).

[0104] According to embodiments of the application, the CH1 region and the second FC peptide segment have an amino acid sequence as set forth in SEQ ID NO: 20.

[0105] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 20).

[0106] It should be noted that the peptide segment of the amino acid sequence shown in SEQ ID NO: 20 is a heavy chain constant region segment composed of a CH1 region and a second FC peptide segment, wherein the C-terminus of the CH1 region and the N-terminus of the second FC peptide segment are connected.

[0107] According to an embodiment of the present application, the CH1 region has an amino acid sequence as shown in SEQ ID NO: 32.

[0108] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVE (SEQ ID NO: 32).

[0109] According to an embodiment of the present application, the second FC peptide segment has an amino acid sequence as shown in SEQ ID NO: 33.

[0110] PKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 33).

[0111] According to embodiments of the present application, the first polypeptide has an amino acid sequence as set forth in SEQ ID NO: 21.

[0112] EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYNMNWVRQAPGKGLEWVSYISSSSSTIYYADSVKGRFTISRDNAKNSLSLQMNSLRDEDTAVYYCARAYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 21).

[0113] According to embodiments of the present application, the second polypeptide has an amino acid sequence as set forth in SEQ ID NO: 22.

[0114] DIQMTQSPSSVSASVGDRVTITCRASQGISGWLAWYQQKPGKAPKFLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPPTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 22).

[0115] According to embodiments of the present application, the first and second antigen binding regions are connected by a knob-into-hole structure.

[0116] According to embodiments of the present application, the knob-into-hole structure is formed by T366W and / or S354C mutation of the first CH3 domain and at least one of T366S, L368A, Y407V, Y349C mutation of the second CH3 domain.

[0117] According to embodiments of the present application, the knob-into-hole structure is formed by T366W and S354C mutation of the first CH3 domain and T366S, L368A, Y407V, Y349C mutation of the second CH3 domain. In this way, the connection of the first antigen binding region containing two first Fc peptide segments and the connection of the second antigen binding region containing two second Fc peptide segments can be reduced, and the yield of the antibody of the present application can be improved.

[0118] Nucleic acid molecule, expression vector and recombinant cell

[0119] In the process of preparing or obtaining these antibodies, nucleic acid molecules expressing these antibodies can be used, which are linked to different vectors and then expressed in different cells to obtain the corresponding antibodies or antigen binding fragments thereof.

[0120] In another aspect of the present application, the present application provides a nucleic acid molecule. According to embodiments of the present application, the nucleic acid molecule encodes the aforementioned antibody. The nucleic acid molecule according to embodiments of the present application can encode an antibody that can simultaneously target and bind 4-1BB and Nectin-4.

[0121] According to embodiments of the present application, the nucleic acid molecule is a DNA molecule.

[0122] In yet another aspect of the present application, the present application provides an expression vector. According to embodiments of the present application, the expression vector carries the aforementioned nucleic acid molecule. After the expression vector according to embodiments of the present application is introduced into a suitable recipient cell, the expression of the aforementioned antibody can be effectively realized under the mediation of the regulation system, so as to obtain a large amount of the antibody.

[0123] In the process of linking the aforementioned nucleic acid molecule to the vector, the nucleic acid molecule can be directly or indirectly linked to the control elements on the vector, as long as these control elements can control the translation and expression of the nucleic acid molecule. Of course, these control elements can be directly from the vector itself, or can be exogenous, i.e. not from the vector itself. Of course, the nucleic acid molecule can be operably linked to the control elements.

[0124] As used herein, "operably linked" means that the exogenous gene is ligated to the vector so that the control elements within the vector, such as transcription control sequences and translation control sequences, etc., can perform their intended functions of regulating the transcription and translation of the exogenous gene. Of course, the polynucleotides encoding the first antigen binding region of the antibody, the first polypeptide and the second polypeptide can be inserted into different vectors independently, or commonly inserted into the same vector. Commonly used vectors can be plasmids, bacteriophages, etc.

[0125] According to embodiments of the present application, the expression vector is a non-pathogenic viral vector.

[0126] According to embodiments of the present application, the expression vector is an adenovirus vector, a lentivirus vector or a retrovirus vector.

[0127] In yet another aspect of the present application, a recombinant cell is provided. According to embodiments of the present application, the recombinant cell carries the aforementioned nucleic acid molecule, or the aforementioned expression vector or expresses the aforementioned antibody. The aforementioned recombinant cell is obtained by transfecting or transforming the aforementioned expression vector, and the recombinant cell can efficiently express the aforementioned antibody capable of simultaneously targeting 4-1BB and Nectin-4 under suitable conditions.

[0128] It should be noted that the recombinant cell of the present application is not particularly limited, and can be a prokaryotic cell, a eukaryotic cell or a bacteriophage. The prokaryotic cell can be Escherichia coli, Bacillus subtilis, Streptomyces or Proteus mirabilis, etc. The eukaryotic cell can be fungi including Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces, Trichoderma, etc., insect cells such as Spodoptera exigua, plant cells such as tobacco, mammalian cells such as BHK cells, CHO cells, COS cells, myeloma cells, etc. In some embodiments, the recombinant cell of the present application is preferably a mammalian cell, including BHK cells, CHO cells, NSO cells or COS cells, and does not include animal reproductive cells, fertilized eggs or embryonic stem cells.

[0129] It should be noted that "suitable conditions" in the present application refer to conditions suitable for the expression of the antibody of the present application. It is easily understood by those skilled in the art that the conditions suitable for the expression of the antibody include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy host cell state, suitable host cell density, suitable cell culture environment, suitable cell culture time. The "suitable conditions" are not particularly limited, and those skilled in the art can optimize the most suitable conditions for the expression of the antibody according to the specific environment of the laboratory.

[0130] According to embodiments of the present application, the recombinant cell is obtained by introducing the aforementioned expression vector into a host cell.

[0131] According to an embodiment of the present application, the recombinant cell is a eukaryotic cell.

[0132] According to an embodiment of the present application, the recombinant cell is a mammalian cell. An expression vector can be introduced into a mammalian cell to construct a recombinant cell, and the recombinant cell can be used to express the antibody provided by the present application. The antibody can be obtained by culturing the recombinant cell. The mammalian cell can be a CHO cell, for example.

[0133] Method for preparing an antibody

[0134] In another aspect of the present application, a method for preparing the antibody described above is provided. According to an embodiment of the present application, the method comprises introducing the expression vector described above into a cell, and culturing the cell under conditions suitable for protein expression and secretion to obtain the antibody. The method provided by the present application can effectively obtain the antibody described above, and has the advantage of simple preparation method.

[0135] According to an embodiment of the present application, the cell is a eukaryotic cell.

[0136] According to an embodiment of the present application, the eukaryotic cell is a mammalian cell. When the cell is a eukaryotic cell, such as a mammalian cell, the expression efficiency of the recombinant antibody is higher.

[0137] According to an embodiment of the present application, the eukaryotic cell does not include an animal reproductive cell, a fertilized egg or an embryonic stem cell.

[0138] Pharmaceutical composition and kit

[0139] In another aspect of the present application, a pharmaceutical composition is provided. According to an embodiment of the present application, the pharmaceutical composition comprises the antibody described above, the nucleic acid molecule described above, the expression vector described above or the recombinant cell described above. The pharmaceutical composition provided by the present application can target an antibody that simultaneously binds 4-1BB and Nectin-4, and can effectively mediate the killing of cells (e.g., tumor cells) expressing Nectin-4 by T cells, especially has a strong tumor inhibition effect, and can effectively treat cancer.

[0140] According to an embodiment of the present application, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0141] In another aspect of the present application, a kit is provided. According to an embodiment of the present application, the kit comprises the antibody described above, the nucleic acid molecule described above, the expression vector described above or the recombinant cell described above. The kit provided by the present application can bind to 4-1BB protein and / or Nectin-4 protein, thereby effectively identifying 4-1BB protein and / or Nectin-4 protein.

[0142] Use

[0143] In yet another aspect of the present application, the present application provides use of the aforementioned antibody, the aforementioned nucleic acid molecule, the aforementioned expression vector, the aforementioned recombinant cell or the aforementioned pharmaceutical composition in the manufacture of a medicament for treating or preventing cancer. According to embodiments of the present application, the antibody or the pharmaceutical composition of the present application can simultaneously target an antibody that binds 4-1BB and Nectin-4, can effectively mediate T cell killing of tumor cells that highly express Nectin-4, has a strong tumor inhibition effect, and can effectively treat cancer.

[0144] According to embodiments of the present application, the cancer is selected from a cancer that highly expresses Nectin-4.

[0145] According to embodiments of the present application, the cancer that highly expresses 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.

[0146] In yet another aspect of the present application, the present application provides use of the aforementioned antibody, the aforementioned nucleic acid molecule, the aforementioned expression vector or the aforementioned recombinant cell in the manufacture of a kit for detecting 4-1BB and / or Nectin-4. According to embodiments of the present application, the antibody or the kit of the present application can bind to a 4-1BB protein and / or a Nectin-4 protein, thereby effectively identifying a 4-1BB protein and / or a Nectin-4 protein.

[0147] Method of treating or preventing cancer

[0148] In another aspect of the present application, the present application provides a method of preventing and / or treating cancer. According to embodiments of the present application, the method comprises administering to a subject a pharmaceutically acceptable amount of the aforementioned antibody or the aforementioned pharmaceutical composition. According to embodiments of the present application, the method can effectively prevent or treat cancer.

[0149] It should be noted that, in this context, the "pharmaceutically acceptable amount" can vary depending on the mode of administration and the severity of the disease to be treated, and is preferably an effective amount. The selection of the pharmaceutically acceptable amount can be determined by a person of ordinary skill in the art according to various factors (e.g., through clinical trials). The factors include, but are not limited to, the 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, etc. For example, several divided doses can be administered daily, or the dose can be proportionally reduced, as required by the exigencies of the therapeutic situation.

[0150] According to an embodiment of the present application, the cancer is selected from a group consisting of Nectin-4 high-expressing cancer.

[0151] According to an embodiment of the present application, the Nectin-4 high-expressing cancer includes at least one of breast cancer, ovarian cancer, cervical cancer, colorectal cancer, esophageal cancer, gastric cancer, lung cancer, liver cancer, and thyroid cancer.

[0152] The scheme of the present application will be explained below with reference to Examples. Those skilled in the art will appreciate that the following Examples are intended to be illustrative only and should not be viewed as limiting the scope of the present application. Unless otherwise indicated, in the Examples, techniques and conditions are carried out according to the techniques or conditions described in the literature or according to the manufacturer's instructions. Unless otherwise indicated, reagents or instruments not specifically noted were conventional products available commercially.

[0153] Example 1: Preparation of bispecific antibody molecule

[0154] The present example produces a bispecific antibody, and the specific experimental operation is as follows: ExpiCHO cells (purchased from Thermo Fisher) are cultured in ExpiCHO Expression Medium (purchased from Thermo Fisher, A2910001), and the cell concentration is adjusted to 6x106 / mL to obtain an ExpiCHO cell solution. The pTT5 vector (synthesized by Suzhou Jinweizhi Company) containing three chain encoding genes (shown as SEQ ID NO: 23, 24, and 25, respectively) is added to 2 mL of OptiSFM medium (Thermo Fisher, 12309019) to obtain solution A. Among them, the first chain encoding gene includes the nucleotide sequence encoding the 4-1BB single-chain antibody (SEQ ID NO: 9) and the first FC peptide segment (SEQ ID NO: 10), the second chain encoding gene includes the nucleotide sequence encoding the second heavy chain variable region of Nectin-4 (SEQ ID NO: 18) and the second FC peptide segment (SEQ ID NO: 20), and the third chain encoding gene includes the nucleotide sequence encoding the second light chain variable region of Nectin-4 (SEQ ID NO: 19) and the CL region (SEQ ID NO: 31). 160 μL of ExpiFectamine CHO transfection reagent (Thermofisher, A29130) is added to 2 mL of OptiSFM medium to obtain solution B. Then, solution A and solution B are mixed to obtain a transfection mixture, and the transfection mixture is added to 50 mL of ExpiCHO cell solution within 5 minutes. After incubation at 37°C, 5% CO2 for 1 day, 8 mL of Feed (Thermo Fisher, A29130) and 300 μL of Enhancer (Thermo Fisher, A29130) are added, and the culture is transferred to 32°C, 5% CO2 for 9 days, and then the culture supernatant is harvested. Among them, 8 mL of Feed is added on the 5th day. The bispecific antibody is affinity purified from the culture supernatant using a Protein A purification column (GE) to obtain the antibody 4-1BBxNectin-4 (i.e., a bispecific antibody). It is detected that the antibody 4-1BBxNectin-4 (herein, used interchangeably with “4-1BBxNectin-4 antibody”, “4-1BBxNectin-4 bispecific antibody” and “bispecific antibody”) has an amino acid sequence as shown in SEQ ID NO: 11, SEQ ID NO: 21 and SEQ ID NO: 22. The structure of the antibody 4-1BBxNectin-4 is shown in Figure 1 .

[0155] The first chain encoding gene (i.e., for the first antigen binding region) encodes for SEQ ID NO: 11, and includes the nucleotide sequence set forth below:

[0156]

[0157] The second chain-encoding gene (i.e., for the first polypeptide) encodes for SEQ ID NO: 21 and includes the nucleotide sequence set forth below:

[0158]

[0159] The third chain encoding gene (i.e., for the second polypeptide) encodes SEQ ID NO: 22, and includes the nucleotide sequence set forth below:

[0160] GATATCCAAATGACCCAATCCCCCTCTTCTGTCAGCGCTTCCGTTGGCGATAGGGTAACAATCACTTGCCGCGCAAGCCAAGGAATCTCCGGCTGGCTGGCATGGTATCAGCAGAAACCAGGGAAGGCTCCAAAGTTCCTTATCTATGCCGCCAGTACACTTCAGTCTGGAGTGCCTTCTCGTTTCTCTGGTTCTGGGAGTGGCACTGACTTTACACTTACCATTAGTTCCTTGCAGCCCGAAGACTTCGCCACATATTACTGTCAGCAGGCAAATAGCTTCCCTCCTACATTCGGAGGGGGAACAAAGGTGGAAATCAAGAGGACAGTTGCCGCACCTTCCGTGTTTATCTTTCCTCCAAGCGATGAGCAGCTGAAGAGTGGGACTGCCTCAGTCGTCTGTCTGTTGAACAATTTCTATCCAAGAGAGGCCAAAGTGCAGTGGAAAGTGGACAATGCATTGCAGTCCGGAAACTCACAGGAGAGCGTGACAGAGCAGGACTCCAAAGATTCTACATACAGCAGTCTCCTCCACACTGACATTGTCCAAGGCAGATTACGAGAAGCACAAAGTGTACGCTTGCGAGGTCACCCACCAGGGCCTGTCATCCCCTGTGACAAAGTCCTTCAACCGAGGCGAGTGC (SEQ ID NO: 25).

[0161] Example 2: Identification of binding ability of bispecific antibody to 4-1BB protein

[0162] ELISA experiment was used to detect the binding property of 4-1BB x Nectin-4 antibody obtained in Example 1. 4-1BB-strep tagII protein was coated in a 96-well plate, and the strength of the signal after the addition of the antibody was used to determine the binding property of the antibody and 4-1BB.

[0163] The 4-1BB-strep tagII protein (produced using standard techniques in our laboratory) was diluted to 2 μg / mL with PBS buffer and added to 100 μL / well of a 96-well plate. The plate was incubated overnight at 4°C. The PBS buffer was then removed from the 96-well plate. The plate was washed 6 times with PBST (pH 7.2 PBS containing 0.1% Tween 20) buffer, and then 200 μL / well of PBS / 10% BSA was added for blocking. The plate was incubated at 37°C for 2 hours. The blocking buffer was removed, and the plate was washed 6 times with PBST. The 4-1BB×Nectin-4 antibody was diluted to the appropriate concentration with 100 μL / well of PBST / 0.05% BSA and then incubated at 37°C for 1 hour. After removing the reaction mixture, the plate was washed 6 times with PBST. HRP (horseradish peroxidase)-labeled rabbit anti-human IgG secondary antibody (Boster Biological, BA1070) was diluted 100 μL / well with PBST / 0.05% BSA and incubated at 37°C for 1 h. After incubation, the plate was washed 6 times with PBST, and 80 μL / well of TMB (tetramethylbenzidine) was added. The plate was incubated at room temperature for 3 min, and the reaction was terminated by adding 80 μL / well of 4M sulfuric acid. The absorbance was read at 450 nm using a microplate reader. Specific experimental results are as follows: Figure 2 As shown, the antibody of the present invention can bind to 4-1BB-strep tagII.

[0164] The amino acid sequence of 4-1BB-strep tagII:

[0165] LQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQDCKQGQELTKKGCKDCCFGTFNDQKRGICRPWTNCSLDGKSVLVNGTKERDVVCGPSPADLSPGASSVTPPAPAREPGHSPQGGSAWSHPQFEK (SEQ ID NO: 34).

[0166] Example 3: Identification of the binding ability of bispecific antibodies to CHO-K1-4-1BB cells

[0167] This embodiment uses flow cytometry to detect the binding characteristics of the bispecific antibody obtained in Example 1, and the signal strength after the addition of the bispecific antibody is used to determine the binding characteristics between the bispecific antibody and CHO-K1-4-1BB cells. The specific experimental procedures are as follows:

[0168] HEK293T cells were processed at a rate of 5 × 10 5Seed cells into 6-well plates and culture overnight in DMEM medium without antibiotics. Discard the medium before transfection and add 1 mL of fresh DMEM medium without antibiotics. A mixture of pLVX-EF1a-4-1BB-IRES-puro (containing the coding sequence of 4-1BB protein (SEQ ID NO: 26) inserted between the EcoRI and BamHI restriction sites of the pLVX-EF1a-IRES-puro vector), pMD2G, and psPAX2 vector (3 μg total) was added to 200 μL of serum-free DMEM medium in a 2:1:1 ratio. Then, 12 μg of polyetherimide (PEI, Polysciences Ltd.) was added. The resulting 4-1BB protein had the amino acid sequence shown in SEQ ID NO: 27. After mixing and standing for 16 min, the entire mixture was added to a six-well plate containing HEK293T cells. After 6 h of culture, the medium was discarded, and fresh complete DMEM medium was added. 48 h after transfection, the cell culture supernatant was collected and filtered through a 0.45 μm Millipore filter to obtain the viral supernatant. All of the viral supernatant was then added to a solution containing 1×10⁻⁶ cells. 4 In a 6-well plate, polybrene (Sigma) was added to CHO-K1 cells at a final concentration of 4 μg / mL, and the cells were cultured for 12 h. The supernatant was then discarded, and fresh complete DMEM medium was added. The resulting cells were CHO-K1-4-1BB cells.

[0169] ATGGGTAATTCCTGTTATAACATCGTCGCTACTTTGTTGTTGGTGCTGAACTTCGAGAGGACTAGATCTCTGCAGGATCCTTGCTCCAACTGTCCAGCTGGCACCTTTTGCGATAATAACCGTAATCAGATCTGTTCTCCCTGCCCTCCAAATAGTTTCAGCTCCGCTGGTGGCCAAAGAACATGCGATATTTGTCGGCAATGCAAGGGGGTCTTTAGGACCAGGAAAGAGTGCAGTTCTACCTCCAATGCCGAATGCGATTGCACACCCGGATTCCATTGCTTGGGGGCCGGCTGCTCCATGTGCGAACAGGACTGTAAGCAGGGCCAAGAGCTTACAAAAAAGGGCTGTAAGGACTGCTGTTTTGGCACCTTTAACGACCAGAAGCGAGGCATCTGCCGCCCATGGACCAACTGTAGCTTGGATGGGAAGAGCGTCCTGGTCAATGGAACCAAGGAGCGGGACGTTGTGTGTGGCCCATCTCCAGCAGACCTGAGTCCTGGGGCTAGTTCCGTCACACCACCCGCCCCCGCTAGGGAGCCTGGCCATTCTCCTCAAATCATCTCCTTTTTCCTCGCACTCACTAGCACTGCTCTTCTCTTTCTGCTCTTTTTCCTGACCCTGCGGTTCTCTGTTGTGAAAAGAGGCCGGAAGAAACTTCTGTATATATTTAAGCAGCCATTCATGCGGCCAGTACAAACAACACAGGAGGAGGATGGATGCTCTTGTCGGTTCCCTGAGGAAGAGGAAGGAGGATGCGAGCTG (SEQ ID NO: 26).

[0170] MGNSCYNIVATLLLVLNFERTRSLQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQDCKQGQELTKKGCKDCCFGTFNDQKRGICRPWTNCSLDGKSVLVNGTKERDVVCGPSPADLSPGASSVTPPAPAREPGHSPQIISFFLALTSTALLFLLFFLTLRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 27).

[0171] The CHO-K1-4-1BB cells were diluted with PBS to 1 x 10 6 / mL, and added to 1.5 mL EP tubes at a volume of 90 μL / tube, 10 μL / tube of mouse serum was added, and blocked at 4°C for 30 min. A series of concentration gradients (0.1, 1, 10, 30, 100, 300 μg / mL) of 4-1BB x Nectin-4 bispecific antibodies, hIgG (control IgG1, Biolegend, QA16A12) 10 μL / tube were added, and incubated at 4°C for 30 min. After incubation, 1 mL of PBS was added to the EP tube, centrifuged at 100 x g at 4°C for 5 min, and the supernatant was discarded. The precipitate was washed once with PBS. After centrifugation, the supernatant was discarded, and the cells were resuspended with 100 μL / tube of PBS. After resuspension, 1 μL / tube of Alexa-647 labeled rat anti-human Fc antibody secondary antibody (Biolegend, M1310G05) was added, and incubated at 4°C in the dark for 30 min. Washed twice with PBS, and the supernatant was discarded after centrifugation. The cells were resuspended with 200 μL / tube of PBS, and detected by flow cytometry. The specific experimental results are shown in Figure 3 , which further show that the bispecific antibody 4-1BB x Nectin-4 of the application can bind to CHO-K1-4-1BB cells.

[0172] Example 4: Identification of the binding ability of the bispecific antibody to human peripheral blood CD4+ T cells

[0173] This example uses flow cytometry to detect the binding properties of the bispecific antibody obtained in Example 1, and the signal strength after addition of the bispecific antibody is used to judge the binding properties of the bispecific antibody and human peripheral blood CD4+ T cells. The specific experimental operation is as follows:

[0174] The human peripheral blood mononuclear cells were diluted with PBS to 5 x 10 6 / mL, 10 μL / tube rat serum was added, and the mixture was blocked at 4°C for 30 min; after blocking, 4-1BB x Nectin-4 bispecific antibody of a series of concentration gradients (0.1, 1, 10, 30, 100, 300 μg / mL) and hIgG (control IgG1, Biolegend, QA16A12) 10 μL / tube were added, and the mixture was incubated at 4°C for 30 min; then, 1 mL PBS was added to the EP tube, and the mixture was centrifuged at 4°C and 100 x g for 5 min, and the supernatant was discarded; the precipitate was washed with PBS once, and the supernatant was discarded after centrifugation; the precipitate was resuspended with 100 μL / tube PBS, and 1 μL / tube Alexa-647 labeled rat anti-human Fc antibody secondary antibody (Biolegend, M1310G05) and 1 μL / tube FITC labeled mouse anti-human CD4 antibody (Invitrogen, OKT4) were added, and the mixture was incubated at 4°C in the dark for 30 min; the mixture was washed with PBS twice, and the supernatant was discarded after centrifugation; the precipitate was resuspended with 200 μL / tube PBS, and the mixture was detected by flow cytometry, and the specific experimental results are shown in Figure 4 Figure 2, which shows that the bispecific antibody of the present application can bind to human peripheral blood T cells.

[0175] Example 5: Identification of the binding ability of the bispecific antibody to CHO-K1-Nectin-4 cells

[0176] In this example, the binding properties of the bispecific antibody obtained in Example 1 were detected by flow cytometry, and the binding properties of the bispecific antibody and CHO-K1-Nectin-4 cells were judged based on the strength of the signal after the addition of the bispecific antibody. The specific experimental operation is as follows:

[0177] HEK293T cells were prepared according to 5 x 10 5Seed cells into 6-well plates and culture overnight in DMEM medium without antibiotics. Discard the medium before transfection and add 1 mL of fresh DMEM medium without antibiotics. A mixture of pLVX-EF1a-Nectin-4-IRES-puro (the pLVX-EF1a-IRES-puro vector contains the coding sequence of Nectin-4 protein (SEQ ID NO:28) inserted between the EcoRI and BamHI restriction sites), pMD2G, and psPAX2 vector (3 μg total) was added to 200 μL of serum-free DMEM medium in a 2:1:1 ratio. Then, 12 μg of polyetherimide (PEI, Polysciences Ltd.) was added. The resulting Nectin-4 protein had the amino acid sequence shown in SEQ ID NO:29. After mixing and standing for 16 min, the entire mixture was added to a six-well plate containing HEK293T cells. After 6 h of culture, the medium was discarded, and fresh complete DMEM medium was added. 48 h after transfection, the cell culture supernatant was collected and filtered through a 0.45 μm Millipore filter to obtain the viral supernatant. All of the viral supernatant was then added to a solution containing 1×10⁻⁶ cells / mL of DMEM. 4 In a 6-well plate, CHO-K1 cells were incubated with Sigma polybrene at a final concentration of 4 μg / mL for 12 h. The supernatant was then discarded, and fresh complete DMEM medium was added. The resulting cells were CHO-K1-Nectin-4 cells.

[0178]

[0179] MPLSLGAEMWGPEAWLLLLLLLASFTGRCPAGELETSDVVTVVLGQDAKLPCFYRGDSGEQVGQVAWARVDAGEGAQELALLHSKYGLHVSPAYEGRVEQPPPPRNPLDGSVLLRNAVQADEGEYECRVSTFPAGSFQARLRLRVLVPPLPSLNPGPALEEGQGLTLAASCTAEGSPAPSVTWDTEVKGTTSSRSFKHSRSAAVTSEFHLVPSRSMNGQPLTCVVSHPGLLQDQRITHILHVSFLAEASVRGLEDQNLWHIGREGAMLKCLSEGQPPPSYNWTRLDGPLPSGVRVDGDTLGFPPLTTEHSGIYVCHVSNEFSSRDSQVTVDVLDPQEDSGKQVDLVSASVVVVGVIAALLFCLLVVVVVLMSRYHRRKAQQMTQKYEEELTLTRENSIRRLHSHHTDPRSQPEESVGLRAEGHPDSLKDNSSCSVMSEEPEGRSYSTLTTVREIETQTELLSPGSGRAEEEEDQDEGIKQAMNHFVQENGTLRAKPTGNGIYINGRGHLV (SEQ ID NO: 29).

[0180] CHO-K1-Nectin-4 cells were diluted to 1 x 10 6 / mL with PBS, added to 1.5 mL EP tubes at a volume of 90 μL / tube, 10 μL / tube of rat serum was added, and blocked at 4°C for 30 min. A series of concentration gradients (0.1, 1, 10, 30, 100, 300 μg / mL) of 4-1BB x Nectin-4 bispecific antibodies, hIgG (control IgG1, Biolegend, QA16A12) 10 μL / tube were added, respectively, and incubated at 4°C for 30 min. After incubation, 1 mL of PBS was added to the EP tube, centrifuged at 4°C and 100 x g for 5 min, and the supernatant was discarded. The precipitate was washed once with PBS. After centrifugation, the supernatant was discarded, and the cells were resuspended with 100 μL / tube of PBS. After resuspension, 1 μL / tube of Alexa-647 labeled rat anti-human Fc antibody secondary antibody (Biolegend, M1310G05) was added, and the mixture was incubated at 4°C in the dark for 30 min. The cells were washed twice with PBS, and the supernatant was discarded after centrifugation. The cells were resuspended with 200 μL / tube of PBS, and detected by flow cytometry. The specific experimental results are shown in FIG. 6. Figure 5As shown, further showing that the bispecific antibody 4-1BB x Nectin-4 of the present application can bind to CHO-K1-Nectin-4 cells.

[0181] Example 6: Identification of the binding ability of the bispecific antibody to human breast cancer SK-BR-3 cells

[0182] This example uses flow cytometry to detect the binding properties of the bispecific antibody obtained in Example 1, and the signal intensity after the addition of the bispecific antibody is used to judge the binding properties of the bispecific antibody and human breast cancer SK-BR-3 cells. The specific experimental operation is as follows:

[0183] Dilute human breast cancer SK-BR-3 cells with PBS to 1 x 10 6 / mL, add to 1.5 mL EP tubes at a volume of 90 μL / tube, add 10 μL / tube of rat serum to each, and block at 4°C for 30 min. Add a series of concentration gradients (0.1, 1, 10, 30, 100, 300 μg / mL) of 4-1BB x Nectin-4 bispecific antibody, hIgG (control IgG1, Biolegend, QA16A12) 10 μL / tube, respectively, and incubate at 4°C for 30 min. After incubation, add 1 mL of PBS to the EP tubes, centrifuge at 4°C, 100 x g for 5 min, discard the supernatant, and wash the precipitate with PBS once. After centrifugation, discard the supernatant, resuspend the cells with 100 μL / tube of PBS, and after resuspension, add 1 μL / tube of Alexa-647 labeled rat anti-human Fc antibody secondary antibody (Biolegend, M1310G05) to each, and incubate at 4°C in the dark for 30 min. Wash twice with PBS, and after centrifugation, discard the supernatant. Resuspend the cells with 200 μL / tube of PBS, and detect with a flow cytometer. The specific experimental results are shown in Figure 6 As shown, further showing that the bispecific antibody 4-1BB x Nectin-4 of the present application can bind to human breast cancer SK-BR-3 cells.

[0184] Example 7: Identification of the ability of the bispecific antibody to promote Jurkat-NFAT-lucia-4-1BB reporter cell activation

[0185] This example uses the Jurkat-NFAT-lucia-4-1BB reporter system method to identify the ability of the bispecific antibody obtained in Example 1 to crosslink Nectin-4 on the surface of target cells and 4-1BB on the surface of effector cells, and promote T cell activation. The relative chemiluminescence signal (RLU) is used to judge the ability of the bispecific antibody to bridge target cells and T cells, and further activate T cells.

[0186] HEK293T cells were plated at 5 x 10 5Seed cells into 6-well plates and culture overnight in DMEM medium without antibiotics. Discard the medium before transfection and add 1 mL of fresh DMEM medium without antibiotics. A mixture of pLVX-EF1a-4-1BB-IRES-puro (containing the coding sequence of 4-1BB protein (SEQ ID NO: 26) inserted between the EcoRI and BamHI restriction sites of the pLVX-EF1a-IRES-puro vector), pMD2G, and psPAX2 vector (3 μg total) was added to 200 μL of serum-free DMEM medium in a 2:1:1 ratio. Then, 12 μg of polyetherimide (PEI, Polysciences Ltd.) was added. The resulting 4-1BB protein had the amino acid sequence shown in SEQ ID NO: 27. After mixing and standing for 16 min, the entire mixture was added to a six-well plate containing HEK293T cells. After 6 h of culture, the medium was discarded, and fresh complete DMEM medium was added. 48 h after transfection, the cell culture supernatant was collected and filtered through a 0.45 μm Millipore filter to obtain the viral supernatant. All of the viral supernatant was then added to a solution containing 1×10⁻⁶ cells. 4 Jurkat-NFAT-lucia cells were placed in 6-well plates with Sigma polybrene at a final concentration of 4 μg / mL and cultured for 12 h. The supernatant was then discarded, and fresh complete DMEM medium was added. The resulting cells were Jurkat-NFAT-lucia-4-1BB cells.

[0187] (1) The CHO-K1-Nectin-4 cells obtained in Example 5 were diluted to 1×10⁻⁶ using complete RPMI-1640 medium. 5 Add 100 μL / well to a 96-well plate.

[0188] (2) The 4-1BB×Nectin-4 bispecific antibody obtained in Example 1 was diluted to 500 μg / mL, 100 μg / mL, 20 μg / mL, 4 μg / mL, 0.8 μg / mL, 160 ng / mL, 32 ng / mL and 6.4 ng / mL respectively with complete RPMI-1640 medium and added to the 96-well plate containing CHO-K1-Nectin-4 cells in step (1) at a volume of 20 μL / well.

[0189] (3) Jurkat-NFAT-lucia-4-1BB cells were diluted to 1.25 × 10⁻⁶ using complete RPMI-1640 medium. 5 Add 80 μL / well to the 96-well plate containing the bispecific antibody in step (2).

[0190] (4) The reaction system obtained in step (3) was cultured at 37°C in a 5% CO2 incubator for 24 h.

[0191] (5) 50 μL of the culture supernatant obtained in step (4) was taken and added to a 96-well plate, then luciferase substrate was added to the plate, with a volume of 50 μL / well.

[0192] (6) Chemiluminescence was detected using a multifunctional enzyme label meter.

[0193] Specific experimental results are shown in Table 1, which further show that the bispecific antibody 4-1BB x Nectin-4 of the present application can bridge target cells (CHO-K1-Nectin-4 cells) and T cells (Jurkat-NFAT-lucia-4-1BB cells), and promote T cell activation. Figure 7

[0194] Example 8: Bispecific antibody promotes PBMC killing of tumor cells

[0195] This example detects the effect of the bispecific antibody obtained in Example 1 on the killing of A375-Nectin-4 tumor cells by PBMC, which is detected by constructing a reaction system of the tumor cells + PBMC + different concentrations of bispecific antibody, and the specific experimental operation is as follows:

[0196] HEK293T cells were plated in a six-well plate at a density of 5 x 10 5 The cells were discarded, and 1 mL of fresh DMEM medium without double antibodies was added. The pLVX-EF1a-Nectin-4-IRES-puro (the coding sequence of Nectin-4 protein (SEQ ID NO: 28) was inserted between the enzyme digestion sites EcoRI and BamHI of the pLVX-EF1a-IRES-puro vector, pMD2G, and psPAX2 vector (3 μg in total) were added to 200 μL of serum-free DMEM medium in a ratio of 2:1:1, followed by the addition of 12 μg of polyetherimide (PEI, Polysciences Inc.), and the obtained Nectin-4 protein had an amino acid sequence as shown in SEQ ID NO: 29; after mixing, the mixture was allowed to stand for 16 min, and then the entire liquid was added to the six-well plate containing the HEK293T cells. After 6 h of culture, the culture medium was discarded, and fresh complete DMEM medium was added for culture. After 48 h of transfection, the cell culture supernatant was collected and filtered through a 0.45 μm filter (Millipore) to obtain the virus supernatant. The virus supernatant was added to the six-well plate containing 1 x 10 4 ​A375 cells in 6-well plates were added with polybrene (Sigma) at a final concentration of 4 μg / mL and incubated for 12 h. Then the supernatant was discarded and fresh complete DMEM medium was added. The resulting cells were A375-Nectin-4 cells.

[0197] (1) A 16-well RTCA plate was added with complete RPMI-1640 medium at a volume of 50 μL / well for calibration;

[0198] (2) A375-Nectin-4 cells were diluted with complete RPMI-1640 medium to a concentration of 2 x 10 5 / mL, and added to the RTCA plate obtained in step (1) at a volume of 50 μL / well, and then detected using an xCELLigence RTCA TP device at 37 °C and 5% CO2 for 24 h;

[0199] (3) The bispecific antibody obtained in Example 1 was diluted with complete RPMI-1640 medium to a series of concentration gradients (0.32, 1.6, 8, 40, 200, 1000 ng / mL), and added to the RTCA plate obtained in step (2) at a volume of 20 μL / well;

[0200] (4) PBMC (Selleck) were diluted with complete RPMI-1640 medium to a concentration of 1.25 x 10 6 / mL, and added to the RTCA plate obtained in step (3) at a volume of 80 μL / well;

[0201] (5) The reaction system obtained in step (4) was detected using an xCELLigence RTCA TP device at 37 °C and 5% CO2 for 48 h.

[0202] Specific experimental results are shown in Table 1, which further show that the bispecific antibody of the present application can promote PBMC to kill Nectin-4 positive tumor cells. Figure 8

[0203] From the above experimental results, it can be seen that the bispecific antibody obtained in the present application can bind to T cells and tumor cells, bridge T cells and tumor cells, and promote T cells to kill tumor cells.

[0204] ​In the description of the specification, the description of the terms "one embodiment", "some embodiments", "an example", "a specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are contained in at least one embodiment or example of the present application. In the specification, the illustrative description of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any suitable manner in any one or more embodiments or examples. In addition, the person skilled in the art can combine and combine the different embodiments or examples described in the specification and the features of the different embodiments or examples without contradiction.

[0205] Although the embodiments of the present application have been shown and described above, it is understood that the above-described embodiments are exemplary and are not to be construed as limiting the present application, and those skilled in the art can make changes, modifications, replacements and variations to the above-described embodiments within the scope of the present application.

Claims

1. An antibody, characterized in that, Comprising: a first antigen binding region having 4-1BB binding activity, the first antigen binding region comprising a first heavy chain variable region, a first light chain variable region, a first connecting peptide, a first FC peptide segment; wherein: the C-terminus of the first heavy chain variable region is connected to the N-terminus of the first connecting peptide, the C-terminus of the first connecting peptide is connected to the N-terminus of the first light chain variable region, and the C-terminus of the first light chain variable region is connected to the N-terminus of the first FC peptide segment; the first heavy chain variable region has CDR1, CDR2, CDR3 sequences as shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, respectively; the first light chain variable region has CDR1, CDR2, CDR3 sequences as shown in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, respectively; a second antigen binding region having Nectin-4 binding activity, the second antigen binding region comprising a first polypeptide and a second polypeptide, the first polypeptide and the second polypeptide being connected by an inter-chain disulfide bond; the first polypeptide comprising a second heavy chain variable region, a CH1 region, and a second FC peptide segment, the C-terminus of the second heavy chain variable region being connected to the N-terminus of the CH1 region, and the C-terminus of the CH1 region being connected to the N-terminus of the second FC peptide segment; the second polypeptide comprising a second light chain variable region and a CL region, the C-terminus of the second light chain variable region being connected to the N-terminus of the CL region; the second heavy chain variable region has CDR1, CDR2, CDR3 sequences as shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, respectively; the second light chain variable region has CDR1, CDR2, CDR3 sequences as shown in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, respectively; the first antigen binding region has an amino acid sequence as shown in SEQ ID NO: 11; the first polypeptide has an amino acid sequence as shown in SEQ ID NO: 21; the second polypeptide has an amino acid sequence as shown in SEQ ID NO:

22.

2. The antibody of claim 1, wherein the first connecting peptide has an amino acid sequence as shown in SEQ ID NO:

7.

3. The antibody of claim 1, wherein the first FC peptide segment comprises a first hinge region, a first CH2 region, and a first CH3 region.

4. The antibody of claim 3, wherein the first hinge region is a hinge region fragment of human, primate, or murine wild-type IgG1.

5. The antibody of claim 3, wherein the first CH2 region is a CH2 region fragment of human, primate, or murine wild-type IgG1; or the first CH2 region has L234A and / or L235A mutation compared to the CH2 region fragment of human wild-type IgG1.

6. The antibody of claim 3, wherein the first CH3 region has T366W and / or S354C mutation compared to the CH3 region fragment of human wild-type IgG1.

7. The antibody of claim 1, wherein the first antigen binding region further comprises a second connecting peptide.

8. The antibody of claim 7, wherein the N-terminus of the second connecting peptide is connected to the C-terminus of the first light chain variable region, and the C-terminus of the second connecting peptide is connected to the N-terminus of the first FC peptide segment.

9. The antibody of claim 7, wherein the second connecting peptide has an amino acid sequence as set forth in SEQ ID NO:

8.

10. The antibody of claim 1, wherein the first FC peptide segment has an amino acid sequence as set forth in SEQ ID NO:

10.

11. The antibody of claim 3, wherein the second FC peptide segment comprises a second hinge region, a second CH2 region and a second CH3 region.

12. The antibody of claim 1, wherein the CH1 region is a CH1 region of a human, primate or murine wild-type IgG1.

13. The antibody of claim 11, wherein the second hinge region is a hinge region fragment of a human, primate or murine wild-type IgG1.

14. The antibody of claim 11, wherein the second CH2 region is a CH2 region fragment of a human, primate or murine wild-type IgG1; or the second CH2 region has L234A and / or L235A mutation compared to a CH2 region fragment of a human wild-type IgG1.

15. The antibody of claim 11, wherein the second CH3 region is a CH3 region fragment of a human, primate or murine wild-type IgG1.

16. The antibody of claim 11, wherein the second CH3 region has at least one of T366S, L368A, Y407V, Y349C mutations compared to a CH3 region fragment of a human wild-type IgG1.

17. The antibody of claim 1, wherein the CL region is a wild-type CL region of a human, primate or murine.

18. The antibody of claim 1, wherein the second heavy chain variable region has an amino acid sequence as set forth in SEQ ID NO:

18.

19. The antibody of claim 1, wherein the second light chain variable region has an amino acid sequence as set forth in SEQ ID NO:

19.

20. The antibody of claim 1, wherein the CH1 region and the second FC peptide segment have an amino acid sequence as set forth in SEQ ID NO:

20.

21. The antibody of claim 11, wherein the first antigen binding region and the second antigen binding region are connected by a knob-into-hole structure.

22. The antibody of claim 21, wherein the knob-into-hole structure is formed by T366W and / or S354C mutation of the first CH3 region and at least one of T366S, L368A, Y407V, Y349C mutation of the second CH3 region.

23. A nucleic acid molecule, characterized in that, the nucleic acid molecule encodes the antibody of any one of claims 1-22.

24. The nucleic acid molecule of claim 23, wherein, the nucleic acid molecule is a DNA molecule.

25. An expression vector comprising the nucleic acid of claim 24. carrying the nucleic acid molecule of any one of claims 23-24.

26. The expression vector of claim 25, wherein, the expression vector is a non-pathogenic viral vector.

27. The expression vector of claim 25, wherein the expression vector is an adenoviral vector, a lentiviral vector or a retroviral vector.

28. A method of producing an antibody according to any one of claims 1 to 22, characterized by, comprising: introducing the expression vector of any one of claims 25-27 into a cell; culturing the cell under conditions suitable for protein expression and secretion so as to obtain the antibody.

29. The method of claim 28, wherein, the cell is a eukaryotic cell.

30. A recombinant cell, wherein, the recombinant cell carries the nucleic acid molecule of any one of claims 23-24, or the expression vector of any one of claims 25-27 or expresses the antibody of any one of claims 1-22.

31. The recombinant cell of claim 30, wherein, the recombinant cell is obtained by introducing the expression vector of any one of claims 25-27 into a host cell.

32. The recombinant cell of claim 30, wherein, the recombinant cell is a eukaryotic cell.

33. The recombinant cell of claim 30, wherein, the recombinant cell is a mammalian cell.

34. A pharmaceutical composition comprising, comprising: The antibody of any one of claims 1 to 22, the nucleic acid molecule of any one of claims 23 to 24, the expression vector of any one of claims 25 to 27, or the recombinant cell of any one of claims 30 to 33.

35. The pharmaceutical composition of claim 34, wherein, The pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

36. A kit comprising, Comprising: The antibody of any one of claims 1 to 22, the nucleic acid molecule of any one of claims 23 to 24, the expression vector of any one of claims 25 to 27, or the recombinant cell of any one of claims 30 to 33.

37. Use of the antibody of any one of claims 1 to 22, the nucleic acid molecule of any one of claims 23 to 24, the expression vector of any one of claims 25 to 27, the recombinant cell of any one of claims 30 to 33, or the pharmaceutical composition of claim 34 or 35 for the manufacture of a medicament for the treatment or prevention of breast cancer.

38. Use of the antibody of any one of claims 1 to 22, the nucleic acid molecule of any one of claims 23 to 24, the expression vector of any one of claims 25 to 27, or the recombinant cell of any one of claims 30 to 33 for the manufacture of a kit for the detection of 4-1BB and / or Nectin-4.

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