Bispecific antibodies and their use

Abstract

The application provides a kind of targeting CD27 and Nectin-4 dual-specific antibody and its use.The antibody includes: the first antigen binding region, the first antigen binding region has CD27 binding activity;Second antigen binding region, the second antigen binding region has Nectin-4 binding activity, first antigen binding region is selected from CD27 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 CD27 and Nectin-4, which can simultaneously bind to CD27 and Nectin-4, and can effectively mediate CD27-expressing T cells to kill Nectin-4-expressing tumor cells.

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

[0007] CD27 belongs to one of the members of the NGFR / TNFR gene superfamily, and is expressed in activated T cells, B cell subsets, and some NK cells. Nectin-4, also known as poliovirus receptor-like 4 (PVRL4), is a type I transmembrane protein, which is lowly expressed in normal human tissues (such as skin, bladder, salivary gland, esophagus, breast and stomach), but 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 monoclonal antibodies to target and deliver chemotherapeutic drugs 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 after 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 CD27 binding activity; a second antigen binding region having Nectin-4 binding activity. The antibody according to the embodiments of the present application can simultaneously bind to CD27 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 the embodiments of the present application can encode an antibody that can simultaneously target and bind CD27 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 the 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 the 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 CD27 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 CD27 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 CD27 protein and / or Nectin-4 protein, and can effectively identify CD27 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 CD27 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 CD27 and / or Nectin-4. According to embodiments of the present application, the antibody or the kit of the present application can bind to CD27 protein and / or Nectin-4 protein, and can effectively identify CD27 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 2This is a graph showing the detection results of the binding ability of the CD27×Nectin-4 bispecific antibody to CHO-K1-CD27 cells according to Example 2 of the present invention;

[0022] Figure 3 This is a graph showing the detection results of the binding ability of the CD27×Nectin-4 bispecific antibody to human peripheral blood CD8+ T cells according to Example 3 of the present invention.

[0023] Figure 4 This is a graph showing the detection results of the binding ability of the CD27×Nectin-4 bispecific antibody to CHO-K1-Nectin-4 cells according to Example 4 of the present invention.

[0024] Figure 5 This is a graph showing the detection results of the binding ability of the recombinant bispecific antibody to human SK-BR-3 cells according to Example 5 of the present invention;

[0025] Figure 6 This is a graph showing the detection results of CD27×Nectin-4 bispecific antibody activation of Jurkat-NFAT-lucia-CD27 reporter cells according to Example 6 of the present invention;

[0026] Figure 7 This is a graph showing the detection results of the recombinant bispecific antibody promoting the killing of A375-Nectin-4 tumor cells by PBMCs according to Example 7 of the present invention. Detailed Implementation

[0027] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0028] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Further, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0029] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0030] For the purposes of the present invention, certain technical and scientific terms are specifically defined below. Unless specifically defined herein, all other technical and scientific terms used in the present document have the meanings that are commonly understood by one of ordinary skill in the art in the field of the present invention. Abbreviations for amino acid residues are in accord with standard three letter and / or one letter codes common in the art.

[0031] In the present invention, unless otherwise explicitly specified and limited, the term "connected" should be interpreted broadly, for example, it can be directly connected or indirectly connected through an intermediate medium, it 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 invention can be understood according to the specific circumstances.

[0032] In the present invention, 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. In the present invention, "bispecific antibody", "bispecific antibody molecule" and "recombinant antibody" can be used interchangeably. 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.

[0033] In the present invention, 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 (i.e. Fc fragment), which facilitates the occlusion of heavy chain and forms heterodimer, for example, in the present invention, the mutation of amino acids in the CH3 domain of human IgG1-Fc (one chain is T366S, L368A, Y407V, Y349C mutation, i.e. "hole"; the other chain is T366W, S354C mutation, i.e. "knob") is realized.

[0034] In the present invention, the amino acid number of the IgG1 Fc part is numbered according to the EU numbering system, for example, the 366th position refers to the 366th position numbered according to the EU numbering system; "T366S" refers to the replacement of threonine at the 366th position numbered according to the EU numbering system with serine; "L368A" refers to the replacement of leucine at the 368th position numbered according to the EU numbering system with alanine.

[0035] In the present context, the term "expression vector" generally refers to a nucleic acid molecule capable of self-replication in a suitable host, which transfers nucleic acid molecule inserted into it 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 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.

[0036] In the present context, 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 eukaryotic cell into which a recombinant expression vector can be introduced. The term "transformed" or "transfected" used herein means the introduction of nucleic acid (e.g., 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 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.

[0037] In the present context, 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.

[0038] In the present context, the term "pharmaceutically acceptable" means that a substance or composition must be chemically and / or toxicologically compatible with the other ingredients constituting a formulation and / or a mammal treated therewith. Preferably, the "pharmaceutically acceptable" of the present application means approved by a federal regulatory agency or a national government or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeia for use in animals, particularly humans.

[0039] In the present context, the term "pharmaceutically acceptable excipient" includes any solvent, solid or other liquid excipient, etc., suitable for the intended target dosage form. Except insofar as any conventional excipient is incompatible with the compounds of the application, such as by producing any adverse biological effect or otherwise interacting in a deleterious manner with any other component(s) of a pharmaceutically acceptable composition, their use is contemplated to be within the scope of this application.

[0040] In the present context, the term "administration" refers to the introduction of a predetermined amount of a substance into a patient by some suitable means. The recombinant antibodies or pharmaceutical compositions of the present application can be administered by any common route, so long as it reaches the intended tissue. Various modes of administration are contemplated, including intraperitoneal, intravenous, intramuscular, subcutaneous, etc., although the present application is not limited to these exemplified modes of administration. Preferably, the compositions of the present application are administered intravenously or subcutaneously.

[0041] In the present context, the term "treatment" refers to any action providing the desired pharmacological and / or physiologic effect. The effect can be prophylactic in terms of completely or partially preventing a disease or symptom thereof and / or therapeutic in terms of a partial or complete cure of 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 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. "Treatment" as used herein covers any action providing a pharmacological and / or physiologic effect in an individual, including but not limited to the administration of a pharmaceutical comprising a compound as described herein to an individual in need thereof.

[0042] It is noted that, with respect to the nucleic acids referred to in the specification and claims, the skilled person will understand that, in fact, either one of the complementary strands, or both, are intended. For convenience, in the specification and claims, although in most cases only one strand is given, the complementary strand is also disclosed. Further, the nucleic acid sequences in the present application include either the DNA form or the RNA form, and the disclosure of one means the disclosure of the other.

[0043] 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 below, respectively.

[0044] Antibody

[0045] In one aspect of the present application, the present application provides an antibody. According to an embodiment of the present application, the antibody comprises: a first antigen binding region having CD27 binding activity; and a second antigen binding region having Nectin-4 binding activity. The antibody according to the embodiment of the present application can bind to CD27 and Nectin-4 at the same time, thereby effectively mediating the killing of a cell (e.g., a tumor cell) expressing Nectin-4 by a T cell, in particular, mediating the killing of a tumor cell by a T cell, having a strong tumor inhibition effect, and being effective in treating cancer.

[0046] According to an embodiment of the present application, the first antigen binding region comprises a first heavy chain variable region and a first light chain variable region, and a C-terminus of the first heavy chain variable region is connected to an N-terminus of the first light chain variable region or an N-terminus of the first heavy chain variable region is connected to a C-terminus of the first light chain variable region.

[0047] According to an embodiment of the present application, a C-terminus of the first heavy chain variable region is connected to an N-terminus of the first light chain variable region.

[0048] According to an embodiment of the present application, the first antigen binding region further comprises a first connecting peptide, a C-terminus of the first heavy chain variable region is connected to an N-terminus of the first connecting peptide, and a C-terminus of the first connecting peptide is connected to an N-terminus of the first light chain variable region or an C-terminus of the first light chain variable region is connected to an N-terminus of the first connecting peptide, and a C-terminus of the first connecting peptide is connected to an N-terminus of the first heavy chain variable region.

[0049] According to an embodiment of the present application, a C-terminus of the first heavy chain variable region is connected to an N-terminus of the first connecting peptide, and a C-terminus of the first connecting peptide is connected to an N-terminus of the first light chain variable region.

[0050] According to an embodiment of the present application, the first heavy chain variable region comprises CDR sequences shown in any one of SEQ ID NOs: 1 to 3, or the first light chain variable region comprises CDR sequences shown in any one of SEQ ID NOs: 4 to 6. In this way, the first antigen binding region can effectively bind to a CD27 protein.

[0051] GFTFSSYD (SEQ ID NO: 1).

[0052] IWYDGSNK (SEQ ID NO: 2).

[0053] ARGSGNWGFFDY (SEQ ID NO: 3).

[0054] QGISRW (SEQ ID NO: 4).

[0055] AAS (SEQ ID NO: 5).

[0056] QQYNTYPRT (SEQ ID NO: 6).

[0057] 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.

[0058] 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.

[0059] According to an embodiment of the present application, the first connecting peptide has an amino acid sequence as shown in SEQ ID NO: 7.

[0060] GGGGSGGGGSGGGGS (SEQ ID NO: 7).

[0061] 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.

[0062] It should be noted that "Fc peptide segment", "Fc fragment" or "Fc" in this text refers to a peptide segment comprising a hinge region, a CH2 region and a CH3 region, for example, the wild-type IgG1 Fc fragment, the first FC peptide segment and the second FC peptide segment mentioned in this application.

[0063] For example, the amino acid sequence of human wild-type IgG1 Fc (including hinge-CH2-CH3) is as follows:

[0064] PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 30).

[0065] 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.

[0066] According to an embodiment of the present application, the first hinge region is a hinge region fragment of human, primate or murine wild-type IgG1.

[0067] According to an embodiment of the present application, 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 a CH2 region fragment of human wild-type IgG1.

[0068] 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 IgG1.

[0069] According to an embodiment of the present application, the first antigen binding region further comprises a second connecting peptide.

[0070] 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.

[0071] 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 CD27 protein can be further increased.

[0072] GGGGS (SEQ ID NO: 8).

[0073] According to an embodiment of the present application, the first antigen binding region comprises an amino acid sequence as shown in SEQ ID NO: 9.

[0074] 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 (also referred to as a CD27 single chain antibody), in which 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 second connecting peptide, and the single chain antibody has an amino acid sequence as shown in SEQ ID NO: 9.

[0075] QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYDMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGSGNWGFFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQGISRWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNTYPRTFGQGTKVEIKGGGGS (SEQ ID NO: 9).

[0076] According to embodiments of the present application, the first FC peptide segment has an amino acid sequence as set forth in SEQ ID NO: 10.

[0077] PKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 10).

[0078] According to embodiments of the present application, the first antigen binding region has an amino acid sequence as set forth in SEQ ID NO: 11.

[0079] QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYDMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGSGNWGFFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQGISRWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNTYPRTFGQGTKVEIKGGGGSPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 11).

[0080] 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.

[0081] 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.

[0082] According to an embodiment of the present application, the CH1 region is a CH1 region of human, primate or murine wild-type IgG1.

[0083] 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.

[0084] 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.

[0085] 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.

[0086] 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.

[0087] According to embodiments of the present application, the CL region is a human, primate or murine wild-type CL region.

[0088] For example, the amino acid sequence of human wild-type CL region is as follows:

[0089] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 31).

[0090] 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.

[0091] GFTFSSYN (SEQ ID NO: 12).

[0092] ISSSSSTI (SEQ ID NO: 13).

[0093] ARAYYYGMDV (SEQ ID NO: 14).

[0094] QGISGW (SEQ ID NO: 15).

[0095] AAS (SEQ ID NO: 16).

[0096] QQANSFPPT (SEQ ID NO: 17).

[0097] 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.

[0098] 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.

[0099] 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.

[0100] EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYNMNWVRQAPGKGLEWVSYISSSSSTIYYADSVK GRFTISRDNAKNSLSLQMNSLRDEDTAVYYCARAYYYGMDVWGQGTTVTVSS (SEQ ID NO: 18).

[0101] 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.

[0102] DIQMTQSPSSVSASVGDRVTITCRASQGISGWLAWYQQKPGKAPKFLIYAASTLQSGVPSRFSGSG SGTDFTLTISSLQPEDFATYYCQQANSFPPTFGGGTKVEIK (SEQ ID NO: 19).

[0103] 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.

[0104] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 20).

[0105] 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.

[0106] According to an embodiment of the present application, the CH1 region has an amino acid sequence as shown in SEQ ID NO: 32.

[0107] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVE (SEQ ID NO: 32).

[0108] 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.

[0109] PKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 33).

[0110] According to embodiments of the present application, the first polypeptide has an amino acid sequence as set forth in SEQ ID NO: 21.

[0111] EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYNMNWVRQAPGKGLEWVSYISSSSSTIYYADSVKGRFTISRDNAKNSLSLQMNSLRDEDTAVYYCARAYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 21).

[0112] According to embodiments of the present application, the second polypeptide has an amino acid sequence as set forth in SEQ ID NO: 22.

[0113] DIQMTQSPSSVSASVGDRVTITCRASQGISGWLAWYQQKPGKAPKFLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPPTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 22).

[0114] According to embodiments of the present application, the first and second antigen binding regions are connected by a knob-into-hole structure.

[0115] According to an embodiment 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.

[0116] According to an embodiment 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.

[0117] Nucleic acid molecule, expression vector and recombinant cell

[0118] 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.

[0119] In another aspect of the present application, a nucleic acid molecule is provided. According to an embodiment of the present application, the nucleic acid molecule encodes the aforementioned antibody. The nucleic acid molecule according to an embodiment of the present application can encode an antibody that can simultaneously target and bind CD27 and Nectin-4.

[0120] According to an embodiment of the present application, the nucleic acid molecule is a DNA molecule.

[0121] In yet another aspect of the present application, an expression vector is provided. According to an embodiment of the present application, the expression vector carries the aforementioned nucleic acid molecule. After the expression vector according to an embodiment of the present application is introduced into a suitable recipient cell, the aforementioned antibody can be effectively expressed under the mediation of the regulatory system, so as to obtain a large amount of the antibody.

[0122] 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.

[0123] 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.

[0124] According to embodiments of the present application, the expression vector is a non-pathogenic viral vector.

[0125] According to embodiments of the present application, the expression vector is an adenovirus vector, a lentivirus vector or a retrovirus vector.

[0126] In 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 CD27 and Nectin-4 under suitable conditions.

[0127] 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.

[0128] It should be noted that the "suitable conditions" described in the specification of the present application refer to conditions suitable for the expression of the antibody. 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.

[0129] According to embodiments of the present application, the recombinant cell is obtained by introducing the aforementioned expression vector into a host cell.

[0130] According to an embodiment of the present application, the recombinant cell is a eukaryotic cell.

[0131] 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.

[0132] Method for preparing an antibody

[0133] 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.

[0134] According to an embodiment of the present application, the cell is a eukaryotic cell.

[0135] 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.

[0136] 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.

[0137] Pharmaceutical composition and kit

[0138] 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 the antibody that binds to CD27 and Nectin-4 at the same time, 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.

[0139] According to an embodiment of the present application, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0140] 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 the CD27 protein and / or the Nectin-4 protein, and thus effectively identify the CD27 protein and / or the Nectin-4 protein.

[0141] Use

[0142] 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 binding to CD27 and Nectin-4, can effectively mediate T cell killing of tumor cells highly expressing Nectin-4, has a strong tumor inhibition effect, and can effectively treat cancer.

[0143] According to embodiments of the present application, the cancer is selected from a cancer highly expressing Nectin-4.

[0144] According to embodiments of the present application, 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.

[0145] 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 CD27 and / or Nectin-4. According to embodiments of the present application, the antibody or the kit of the present application can bind to a CD27 protein and / or a Nectin-4 protein, thereby effectively identifying a CD27 protein and / or a Nectin-4 protein.

[0146] Method for treating or preventing cancer

[0147] In another aspect of the present application, the present application provides a method for preventing and / or treating cancer. According to embodiments of the present application, the method comprises administering a pharmaceutically acceptable amount of the aforementioned antibody or the aforementioned pharmaceutical composition to a subject. According to embodiments of the present application, the method can effectively prevent or treat cancer.

[0148] It should be noted that, in the present application, 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 (for example, 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 urgency of the therapeutic situation.

[0149] According to an embodiment of the present application, the cancer is selected from a group consisting of Nectin-4 high-expressing cancer.

[0150] 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.

[0151] 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 procedures described in the literature or according to the manufacturer's instructions. Unless otherwise indicated, reagents or instruments are commercially available and are conventional products.

[0152] Example 1: Preparation of bispecific antibody molecule

[0153] 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 (respectively shown as SEQ ID NO: 23, 24, and 25) is added to 2 mL of OptiSFM medium (Thermo Fisher, 12309019) to obtain solution A. Among them, the first chain encoding gene encodes the nucleotide sequence of the CD27 single-chain antibody (SEQ ID NO: 9) and the first FC peptide segment (SEQ ID NO: 10), the second chain encoding gene encodes the nucleotide sequence of the Nectin-4 second heavy chain variable region (SEQ ID NO: 18) and the second FC peptide segment (SEQ ID NO: 20), and the third chain encoding gene encodes the nucleotide sequence of the Nectin-4 second light chain variable region (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, wherein 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 CD27xNectin-4 (herein, used interchangeably with “CD27xNectin-4 antibody”, “CD27xNectin-4 bispecific antibody” and “bispecific antibody”). It is detected that the antibody CD27xNectin-4 has an amino acid sequence as shown in SEQ ID NO: 11, SEQ ID NO: 21, and SEQ ID NO: 22.

[0154] The first chain encoding gene is used to encode SEQ ID NO: 11, and the first chain encoding gene includes the nucleotide sequence as shown below:

[0155]

[0156] The second strand encoding gene for encoding SEQ ID NO: 21 comprises the nucleotide sequence as shown below:

[0157]

[0158] The third chain coding gene is used for encoding SEQ ID NO: 22, and the third chain coding gene comprises a nucleotide sequence as shown in the following:

[0159] GATATCCAAATGACCCAATCCCCCTCTTCTGTCAGCGCTTCCGTTGGCGATAGGGTAACAATCACTTGCCGCGCAAGCCAAGGAATCTCCGGCTGGCTGGCATGGTATCAGCAGAAACCAGGGAAGGCTCCAAAGTTCCTTATCTATGCCGCCAGTACACTTCAGTCTGGAGTGCCTTCTCGTTTCTCTGGTTCTGGGAGTGGCACTGACTTTACACTTACCATTAGTTCCTTGCAGCCCGAAGACTTCGCCACATATTACTGTCAGCAGGCAAATAGCTTCCCTCCTACATTCGGAGGGGGAACAAAGGTGGAAATCAAGAGGACAGTTGCCGCACCTTCCGTGTTTATCTTTCCTCCAAGCGATGAGCAGCTGAAGAGTGGGACTGCCTCAGTCGTCTGTCTGTTGAACAATTTCTATCCAAGAGAGGCCAAAGTGCAGTGGAAAGTGGACAATGCATTGCAGTCCGGAAACTCACAGGAGAGCGTGACAGAGCAGGACTCCAAAGATTCTACATACAGCCTCTCCTCCACACTGACATTGTCCAAGGCAGATTACGAGAAGCACAAAGTGTACGCTTGCGAGGTCACCCACCAGGGCCTGTCATCCCCTGTGACAAAGTCCTTCAACCGAGGCGAGTGC (SEQ ID NO: 25).

[0160] Example 2: Identification of binding ability of bispecific antibody to CHO-K1-CD27 cells

[0161] This example uses flow cytometry to detect the binding properties of the bispecific antibody obtained in Example 1, and the binding properties of the bispecific antibody and CHO-K1-CD27 cells are judged based on the strength of the signal after the addition of the bispecific antibody. The specific experimental operation is as follows:

[0162] HEK293T cells were seeded at 5x10 5Seed cells into six-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-CD27-IRES-puro (the pLVX-EF1a-IRES-puro vector contains an insertion of the coding sequence for CD27 protein (SEQ ID NO:26) 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 CD27 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, 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-CD27 cells.

[0163] ATGGCCAGACCTCACCCTTGGTGGCTTTGTGTCTTGGGTACTTTGGTGGGCCTCTCTGCAACACCTGCTCCAAAGTCCTGCCCCGAGAGACATTATTGGGCACAAGGCAAGCTTTGTTGTCAGATGTGCGAACCTGGAACATTTCTGGTGAAAGATTGCGACCAACACAGGAAAGCTGCTCAGTGCGACCCATGTATCCCAGGAGTATCTTTCTCTCCAGACCATCATACCCGACCTCATTGCGAGAGCTGCAGGCACTGTAACAGTGGGCTGTTGGTGCGGAACTGTACAATAACAGCTAACGCCGAGTGTGCCTGTCGAAACGGATGGCAGTGCCGGGACAAAGAGTGCACAGAATGCGACCCTCTGCCCAATCCTTCTCTGACTGCACGTTCCTCCCAGGCATTGTCTCCCCACCCACAGCCAACACATCTCCCCTATGTGTCTGAGATGCTGGAAGCCAGGACCGCTGGTCATATGCAAACCCTGGCAGACTTTCGCCAGCTGCCTGCTAGGACTCTGTCTACCCATTGGCCTCCTCAAAGGAGCCTTTGCTCCAGCGATTTCATTCGCATATTGGTGATCTTTTCCGGCATGTTTCTTGTCTTTACATTGGCCGGGGCTCTCTTTCTGCATCAGCGTAGGAAATATCGGTCCAACAAGGGCGAAAGTCCCGTTGAGCCAGCTGAACCATGCCACTATAGCTGTCCAAGAGAGGAAGAGGGATCAACCATACCCATTCAGGAGGATTACAGGAAGCCCGAGCCTGCCTGTTCCCCT (SEQ ID NO: 26).

[0164] MARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRILVIFSGMFLVFTLAGALFLHQRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSP (SEQ ID NO: 27).

[0165] CHO-K1-CD27 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 CD27 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 FIG. 6, further showing that the bispecific antibody CD27 x Nectin-4 of the present application can bind to CHO-K1-CD27 cells. Figure 2

[0166] Example 3: Identification of the binding ability of bispecific antibodies to human peripheral blood CD8+ T cells

[0167] This example uses flow cytometry to detect the binding properties of the bispecific antibodies obtained in Example 1, and the signal strength after the addition of bispecific antibodies is used to judge the binding properties of bispecific antibodies and human peripheral blood CD8+ T cells. The specific experimental operation is as follows:

[0168] Human peripheral blood mononuclear cells were diluted with PBS to 5 x 10 6 ​ / mL, 90 μL / tube was added to 1.5 mL EP tube, 10 μL / tube of rat serum was added, and it was closed at 4°C for 30 min; after closing, a series of concentration gradients (0.1, 1, 10, 30, 100, 300 μg / mL) of CD27 x Nectin-4 bispecific antibody, hIgG (control IgG1, Biolegend, QA16A12) 10 μL / tube were added, and it was incubated at 4°C for 30 min, then 1 mL PBS was added to the EP tube, and it was centrifuged at 4°C, 100 x g for 5 min, and the supernatant was discarded, and the precipitate was washed with PBS once, and the supernatant was discarded after centrifugation, and the cells were resuspended with 100 μL / tube of PBS, and 1 μL / tube of Alexa-647 labeled rat anti-human Fc antibody secondary antibody (Biolegend, M1310G05) and 1 μL / tube of FITC labeled mouse anti-human CD8 antibody (Invitrogen, OKT8) were added, and it was incubated at 4°C in the dark for 30 min. Washed with PBS twice, and the supernatant was discarded after centrifugation. Resuspend the cells with 200 μL / tube of PBS, and detect them with a flow cytometer, and the specific experimental results are shown in Figure 3 Figure 6, which shows that the bispecific antibody of the present application can bind to human peripheral blood T cells.

[0169] Example 4: Identification of the binding ability of bispecific antibody to CHO-K1-Nectin-4 cells

[0170] This example uses flow cytometry to detect the binding properties of the bispecific antibody obtained in Example 1, and the signal strength after the addition of the bispecific antibody is used to judge the binding properties of the bispecific antibody and CHO-K1-Nectin-4 cells. The specific experimental operation is as follows:

[0171] 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.

[0172]

[0173] MPLSLGAEMWGPEAWLLLLLLLASFTGRCPAGELETSDVVTVVLGQDAKLPCFYRGDSGEQVGQVAWARVDAGEGAQELALLHSKYGLHVSPAYEGRVEQPPPPRNPLDGSVLLRNAVQADEGEYECRVSTFPAGSFQARLRLRVLVPPLPSLNPGPALEEGQGLTLAASCTAEGSPAPSVTWDTEVKGTTSSRSFKHSRSAAVTSEFHLVPSRSMNGQPLTCVVSHPGLLQDQRITHILHVSFLAEASVRGLEDQNLWHIGREGAMLKCLSEGQPPPSYNWTRLDGPLPSGVRVDGDTLGFPPLTTEHSGIYVCHVSNEFSSRDSQVTVDVLDPQEDSGKQVDLVSASVVVVGVIAALLFCLLVVVVVLMSRYHRRKAQQMTQKYEEELTLTRENSIRRLHSHHTDPRSQPEESVGLRAEGHPDSLKDNSSCSVMSEEPEGRSYSTLTTVREIETQTELLSPGSGRAEEEEDQDEGIKQAMNHFVQENGTLRAKPTGNGIYINGRGHLV (SEQ ID NO: 29).

[0174] 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 CD27 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 4As shown, further showing that the bispecific antibody CD27 x Nectin-4 of the present application can bind to CHO-K1-Nectin-4 cells.

[0175] Example 5: Identification of the binding ability of the bispecific antibody to human breast cancer SK-BR-3 cells

[0176] 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:

[0177] Dilute human breast cancer SK-BR-3 cells with PBS to 1 x 10 6 / mL, add 90 μL / tube to a 1.5 mL EP tube, add 10 μL / tube of rat serum to it, and block at 4°C for 30 min. Add a series of concentration gradients (0.1, 1, 10, 30, 100, 300 μg / mL) of CD27 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 tube, centrifuge at 4°C and 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 it, 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 CD27 x Nectin-4 of the present application can bind to human breast cancer SK-BR-3 cells.

[0178] Example 6: Identification of the ability of the bispecific antibody to promote Jurkat-NFAT-lucia-CD27 reporter cell activation

[0179] This example uses the Jurkat-NFAT-lucia-CD27 reporter system method to identify the ability of the bispecific antibody obtained in Example 1 to cross-link Nectin-4 on the surface of target cells and CD27 on the surface of effector cells, promote T cell activation, and the relative chemiluminescence signal (RLU) is used to judge the ability of the bispecific antibody to bridge target cells and T cells, and then activate T cells.

[0180] 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-CD27-IRES-puro (the pLVX-EF1a-IRES-puro vector contains an insertion of the coding sequence for CD27 protein (SEQ ID NO:26) 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 CD27 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-CD27 cells.

[0181] (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.

[0182] (2) The CD27×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 in 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.

[0183] (3) Jurkat-NFAT-lucia-CD27 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).

[0184] (4) The reaction system obtained in step (3) was cultured at 37°C in a 5% CO2 incubator for 24 h.

[0185] (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.

[0186] (6) Chemiluminescence was detected using a multifunctional enzyme label meter.

[0187] Specific experimental results are shown in Table 1, which further show that the bispecific antibody CD27 x Nectin-4 of the present application can bridge target cells (CHO-K1-Nectin-4 cells) and T cells (Jurkat-NFAT-lucia-CD27 cells), and promote T cell activation. Figure 6

[0188] Example 7: Bispecific antibody promotes PBMC killing of tumor cells

[0189] This example detects the effect of the bispecific antibody obtained in Example 1 on the killing of A375-Nectin-4 tumor cells by PBMC, by constructing a reaction system of the tumor cells + PBMC + different concentrations of bispecific antibody, and the specific experimental operation is as follows:

[0190] 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.), to obtain Nectin-4 protein having 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.

[0191] (1) A 16-well RTCA plate was added with complete RPMI-1640 medium at a volume of 50 μL / well for calibration;

[0192] (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;

[0193] (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;

[0194] (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;

[0195] (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.

[0196] 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 7

[0197] 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.

[0198] ​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.

[0199] 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. a first antigen binding region having CD27 binding activity, the first antigen binding region having an amino acid sequence as set forth in SEQ ID NO: 11; b. 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 linked by an inter-chain disulfide bond; the first polypeptide having an amino acid sequence as set forth in SEQ ID NO: 21; the second polypeptide having an amino acid sequence as set forth in SEQ ID NO:

22.

2. The antibody of claim 1, wherein the first antigen binding region and the second antigen binding region are connected by a knob-into-hole structure.

3. A nucleic acid molecule, characterized in that, the nucleic acid molecule encodes the antibody of any one of claims 1-2.

4. The nucleic acid molecule of claim 3, wherein, the nucleic acid molecule is a DNA molecule.

5. An expression vector, characterized by, carrying the nucleic acid molecule of any one of claims 3-4.

6. The expression vector of claim 5, wherein, the expression vector is a non-pathogenic viral vector.

7. The expression vector of claim 5, wherein, the expression vector is an adenoviral vector or a retroviral vector.

8. The expression vector of claim 5, wherein, the expression vector is a lentiviral vector.

9. A method of producing the antibody according to any one of claims 1 to 2, characterized by, Comprising: introducing the expression vector of any one of claims 5-8 into a cell; culturing the cell under conditions suitable for protein expression and secretion, so as to obtain the antibody.

10. The method of claim 9, wherein, the cell is a eukaryotic cell.

11. A recombinant cell, wherein, the recombinant cell carries the nucleic acid molecule of any one of claims 3-4, or the expression vector of any one of claims 5-8, or expresses the antibody of any one of claims 1-2.

12. The recombinant cell of claim 11, wherein, the recombinant cell is obtained by introducing the expression vector of any one of claims 5-8 into a host cell.

13. The recombinant cell of claim 11, wherein, the recombinant cell is a eukaryotic cell.

14. The recombinant cell of claim 11, wherein, the recombinant cell is a mammalian cell.

15. A pharmaceutical composition comprising, Comprising: the antibody of any one of claims 1-2, the nucleic acid molecule of any one of claims 3-4, the expression vector of any one of claims 5-8, or the recombinant cell of any one of claims 11-14.

16. A kit comprising, Comprising: the antibody of any one of claims 1-2, the nucleic acid molecule of any one of claims 3-4, the expression vector of any one of claims 5-8, or the recombinant cell of any one of claims 11-14.

17. Use of the antibody of any one of claims 1-2, the nucleic acid molecule of any one of claims 3-4, the expression vector of any one of claims 5-8, the recombinant cell of any one of claims 11-14, or the pharmaceutical composition of claim 15, in the manufacture of a medicament for treating cancer; the cancer is selected from breast cancer.

18. Use of the antibody of any one of claims 1-2, the nucleic acid molecule of any one of claims 3-4, the expression vector of any one of claims 5-8, or the recombinant cell of any one of claims 11-14, in the manufacture of a kit for detecting CD27 and / or Nectin-4.

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