Bispecific antibodies and their use

Abstract

The present application provides a kind of dual-specificity antibody targeting CD3 and SLAMF7 and its use.The antibody includes: the first antigen binding region, the first antigen binding region has CD3 binding activity;Second antigen binding region, the second antigen binding region has SLAMF7 binding activity, the first antigen binding region is selected from CD3 single-chain antibody, and the second antigen binding region includes a SLAMF7 heavy chain and a SLAMF7 light chain.The antibody can be combined with SLAMF7 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] Multiple myeloma (MM) is the second most common hematological malignancy after non-Hodgkin's lymphoma. Despite great progress in chemotherapy, proteasome inhibitors, immunomodulators thalidomide derivatives, and CD38-targeted antibodies, almost all patients eventually relapse.

[0004] 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.

[0005] Therefore, there is an urgent need for a bispecific antibody for directed killing of tumor cells. SUMMARY

[0006] 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 CD3 and SLAMF7, which can simultaneously bind to CD3 and SLAMF7, and can effectively mediate CD3-expressing T cells to kill SLAMF7-expressing cells.

[0007] The present application is based on the following findings of the inventors:

[0008] The CD3 molecule is connected to the T cell receptor (TCR) through a salt bridge to form a TCR-CD3 complex, which participates in T cell signal transduction and T cell recognition of antigens. SLAMF7, also known as lymphocyte activation signal molecule member 7, is a type I transmembrane protein. SLAMF7 is lowly expressed on the surface of various immune cells such as some activated T, B cell subsets, dendritic cells, monocyte / macrophage cells, and plasma cells, but is highly expressed in myeloma cells.

[0009] Currently, only one SLAMF7 targeting drug, Empliciti, has been approved by FDA. Empliciti is a SLAMF7 monoclonal antibody, which has two main mechanisms of action: binding to the surface of SLAMF7 on NK cells and activating NK cells; and binding to the surface of SLAMF7 on tumor cells, using ADCC effect to mediate ADCC effector cells (NK cells, macrophages, etc.) to kill tumors.

[0010] However, 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 CD3 binding activity; and a second antigen binding region having SLAMF7 binding activity. The antibody according to embodiments of the present application can simultaneously bind to CD3 and SLAMF7, thereby effectively mediating the killing of T cells to cells expressing SLAMF7 (such as tumor cells), especially having a strong tumor inhibition effect, which can effectively treat cancer.

[0011] In another aspect of the present application, a nucleic acid molecule is provided. 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 to CD3 and SLAMF7.

[0012] In yet another aspect of the present application, an expression vector is provided. 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 achieve the expression of the aforementioned antibody under the mediation of a regulatory system after being introduced into a suitable recipient cell, so as to obtain a large amount of the antibody.

[0013] In yet another aspect of the present application, a method for preparing the aforementioned antibody is provided. 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.

[0014] 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 expression vector, and the recombinant cell can efficiently express the aforementioned antibody that can simultaneously target and bind to CD3 and SLAMF7 under suitable conditions.

[0015] 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 CD3 and SLAMF7, and can effectively mediate the killing of tumor cells expressing SLAMF7 by T cells, especially has a strong tumor inhibition effect, and can effectively treat cancer.

[0016] 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 CD3 protein and / or SLAMF7 protein, and can effectively identify CD3 protein and / or SLAMF7 protein.

[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, 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 CD3 and SLAMF7, and can effectively mediate the killing of tumor cells expressing SLAMF7 by T cells, especially has a strong tumor inhibition effect, and can effectively treat cancer.

[0018] 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 CD3 and / or SLAMF7. According to embodiments of the present application, the antibody or the kit of the present application can bind to CD3 protein and / or SLAMF7 protein, and can effectively identify CD3 protein and / or SLAMF7 protein.

[0019] Additional aspects and advantages of the present application will be in part apparent and in part pointed out hereinafter. BRIEF DESCRIPTION OF DRAWINGS

[0020] 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.

[0021] Figure 1 is a structural schematic diagram of a bispecific antibody according to Embodiment 1 of the present application;

[0022] Figure 2 is a detection result diagram of the CD3 x SLAMF7 bispecific antibody according to Embodiment 2 of the present application and the binding ability of CD3E&D protein.

[0023] Figure 3 is a graph showing the results of detecting the binding ability of the CD3 x SLAMF7 bispecific antibody according to Example 3 of the present application to Jurkat T cells;

[0024] Figure 4 is a graph showing the results of detecting the binding ability of the CD3 x SLAMF7 bispecific antibody according to Example 4 of the present application to human peripheral blood CD8+ T cells;

[0025] Figure 5 is a graph showing the results of detecting the binding ability of the CD3 x SLAMF7 bispecific antibody according to Example 5 of the present application to CHO-K1-SLAMF7 cells;

[0026] Figure 6 is a graph showing the results of detecting the binding ability of the CD3 x SLAMF7 bispecific antibody according to Example 6 of the present application to human multiple myeloma U266 tumor cells;

[0027] Figure 7 is a graph showing the results of detecting the activation of Jurkat-NFAT-lucia reporter cells by the CD3 x SLAMF7 bispecific antibody according to Example 7 of the present application;

[0028] Figure 8 is a graph showing the results of detecting the promotion of PBMC killing of A375-SLAMF7 tumor cells by the recombinant bispecific antibody according to Example 8 of the present application. DETAILED DESCRIPTION

[0029] Embodiments of the present application are described in detail below. The embodiments described below are examples only and are not to be construed as limiting the present application.

[0030] Furthermore, the terms "first", "second", etc. are used herein only to describe the different features and do not imply or suggest relative importance or a number of the features indicated. Thus, the features defined with "first", "second", etc. can include one or more of the features explicitly or implicitly. Further, in the description of the present application, the meaning of "a plurality of" is two or more, unless otherwise specified.

[0031] The endpoints of the ranges and any values disclosed herein are not limited to the precise values stated. The ranges or values should be construed to be approximations that allow for significant variation. Various ranges of values that resolve from the disclosure herein are considered within the scope of the disclosure.

[0032] 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.

[0033] In the present invention, unless otherwise explicitly specified and limited, the term "connected" should be understood broadly, for example, it can be directly connected, or indirectly connected through an intermediate medium, or 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.

[0034] 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" and "bispecific antibody molecule" 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.

[0035] 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 or Fc fragment, which facilitates the occlusion of heavy chain and forms heterodimer, for example, in the present invention, the amino acids in the CH3 domain of human IgG1-Fc are mutated (one chain is T366S, L368A, Y407V, Y349C mutation, i.e. "hole"; the other chain is T366W, S354C mutation, i.e. "knob").

[0036] 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.

[0037] 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.

[0038] 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 a 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., 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 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.

[0039] 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.

[0040] 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 being 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.

[0041] 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.

[0042] 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.

[0043] In the present context, the term "treatment" refers to any action that causes 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 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 that renders a drug or compound useful for the treatment, cure, relief, amelioration or prevention of a disease in an individual, and includes the administration of a drug or compound to an individual in need thereof for the treatment, cure, relief, amelioration or prevention of a disease in the individual.

[0044] 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 of the complementary strands, or both, are included. For convenience, in the specification and claims, although in most cases only one strand is given, the complementary strand is in fact 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.

[0045] 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.

[0046] Antibody

[0047] In one aspect of the present application, an antibody is provided. According to an embodiment of the present application, the antibody comprises: a first antigen binding region having CD3 binding activity; and a second antigen binding region having SLAMF7 binding activity. The antibody according to the embodiment of the present application can bind to CD3 and SLAMF7 at the same time, thereby effectively mediating the killing of a cell (e.g., a tumor cell) expressing SLAMF7 by T cells, especially having a strong tumor inhibition effect, and can effectively treat cancer. Moreover, the antibody of the present application can effectively activate T cells, on the one hand, the T cells have stronger proliferation ability after activation, and have stronger anti-tumor potential; on the other hand, the number of tumor-infiltrating T cells is much larger than that of NK cells activated by SLAMF7 monoclonal antibodies, and therefore, the antibody of the present application has stronger potential for anti-cancer.

[0048] 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 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.

[0049] According to an embodiment 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.

[0050] According to an embodiment 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.

[0051] According to an embodiment 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.

[0052] 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: 1-3; 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 CD3 protein.

[0053] GFTFNTYA (SEQ ID NO: 1).

[0054] IRSKYNNYAT (SEQ ID NO: 2).

[0055] VRHGNFGNSYVSWFAY (SEQ ID NO: 3).

[0056] TGAVTTSNY (SEQ ID NO: 4).

[0057] GTN (SEQ ID NO: 5).

[0058] ALWYSNLWV (SEQ ID NO: 6).

[0059] 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.

[0060] 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.

[0061] According to an embodiment of the present application, the first connecting peptide has an amino acid sequence as shown in SEQ ID NO: 7.

[0062] GGGGSGGGGSGGGGS (SEQ ID NO: 7).

[0063] 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.

[0064] It should be noted that "Fc peptide segment", "Fc fragment" or "Fc" in this text 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.

[0065] For example, the amino acid sequence of human wild type IgG1 Fc (including hinge-CH2-CH3) is as follows:

[0066] PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK (SEQ ID NO: 28).

[0067] 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.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] According to an embodiment of the present application, the first antigen binding region further comprises a second connecting peptide.

[0072] 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.

[0073] 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 CD3 protein can be further increased.

[0074] GGGGS (SEQ ID NO: 8).

[0075] 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.

[0076] 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 application exist in the form of a single-chain antibody, and the C-terminal of the first heavy chain variable region is connected to the N-terminal of the first connecting peptide, the C-terminal of the first connecting peptide is connected to the N-terminal of the first light chain variable region, the C-terminal of the first light chain variable region is connected to the N-terminal of the second connecting peptide, and the single-chain antibody (also known as a CD3 single-chain antibody) has an amino acid sequence shown in SEQ ID NO: 9.

[0077] EVQLLESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSELVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVLGGGGS (SEQ ID NO: 9).

[0078] According to an embodiment of the application, the first FC peptide segment has an amino acid sequence as shown in SEQ ID NO: 10.

[0079] PKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 10).

[0080] According to an embodiment of the application, the first antigen binding region has an amino acid sequence as shown in SEQ ID NO: 11.

[0081] EVQLLESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSELVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVLGGGGSPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 11).

[0082] 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.

[0083] 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.

[0084] According to an embodiment of the present application, the CH1 region is a CH1 region of human, primate or murine wild-type IgG1.

[0085] 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.

[0086] 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.

[0087] 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.

[0088] 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.

[0089] According to embodiments of the present application, the CL region is a human, primate or murine wild-type CL region.

[0090] For example, the amino acid sequence of human wild-type CL region is as follows:

[0091] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 29).

[0092] 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.

[0093] GFDFSRYW (SEQ ID NO: 12).

[0094] INPDSSTI (SEQ ID NO: 13).

[0095] ARPDGNYWYFDV (SEQ ID NO: 14).

[0096] QDVGIA (SEQ ID NO: 15).

[0097] WAS (SEQ ID NO: 16).

[0098] QQYSSYPYT (SEQ ID NO: 17).

[0099] 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.

[0100] 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.

[0101] 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.

[0102] EVQLVESGGGLVQPGGSLRLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEINPDSSTINYAPSLKDKFIISRDNAKNSLYLQMNSLRAEDTAVYYCARPDGNYWYFDVWGQGTLVTVSS (SEQ ID NO: 18).

[0103] 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.

[0104] DIQMTQSPSSLSASVGDRVTITCKASQDVGIAVAWYQQKPGKVPKLLIYWASTRHTGVPDRFSGSGSGTDFTLTISSLQPEDVATYYCQQYSSYPYTFGQGTKVEIK (SEQ ID NO: 19).

[0105] 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.

[0106] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 20).

[0107] 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.

[0108] According to an embodiment of the present application, the CH1 region has an amino acid sequence as shown in SEQ ID NO: 30.

[0109] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVE (SEQ ID NO: 30).

[0110] According to an embodiment of the present application, the second FC peptide segment has an amino acid sequence as shown in SEQ ID NO: 31.

[0111] PKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 31).

[0112] According to embodiments of the application, the first polypeptide has an amino acid sequence of SEQ ID NO: 21.

[0113] EVQLVESGGGLVQPGGSLRLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEINPDSSTINYAPSLKDKFIISRDNAKNSLYLQMNSLRAEDTAVYYCARPDGNYWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 21).

[0114] According to embodiments of the application, the second polypeptide has an amino acid sequence of SEQ ID NO: 22.

[0115] DIQMTQSPSSLSASVGDRVTITCKASQDVGIAVAWYQQKPGKVPKLLIYWASTRHTGVPDRFSGSGSGTDFTLTISSLQPEDVATYYCQQYSSYPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 22).

[0116] According to embodiments of the application, the first and second antigen binding regions are connected by a knob-into-hole structure.

[0117] 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. 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 linkage of the first antigen binding region containing two first Fc peptide segments and the linkage 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 molecules, expression vectors and recombinant cells

[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, a nucleic acid molecule is provided. 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 CD3 and SLAMF7.

[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, an expression vector is provided. 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 aforementioned antibody can be effectively expressed under the mediation of the regulatory system, so as to obtain the antibody in large quantities.

[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 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 CD3 and SLAMF7 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 embodiments 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, for example, a CHO cell.

[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 high.

[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 binds to CD3 and SLAMF7 at the same time, and can effectively mediate the killing of a cell expressing SLAMF7 (for example, a tumor cell) by a T cell, 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 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 CD3 protein and / or SLAMF7 protein, thereby effectively identifying CD3 protein and / or SLAMF7 protein. The kit of the present application can be used for scientific research, such as qualitative or quantitative detection of CD3 and / or SLAMF7 protein in a biological sample, and can also be used for judging the state of an individual, such as judging whether the SLAMF7 level of the individual is too high or too low compared to the normal level after obtaining the SLAMF7 level of the individual.

[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 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 simultaneously target an antibody that binds to CD3 and SLAMF7, can effectively mediate the killing effect of T cells on tumor cells that highly express SLAMF7, has a strong tumor inhibitory effect, and can effectively treat cancer.

[0144] According to embodiments of the present application, the cancer is a cancer that highly expresses SLAMF7.

[0145] According to embodiments of the present application, the cancer that highly expresses SLAMF7 is multiple myeloma.

[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 preparation of a kit for detecting CD3 and / or SLAMF7. According to embodiments of the present application, the antibody or the kit of the present application can bind to CD3 protein and / or SLAMF7 protein, thereby effectively identifying CD3 protein and / or SLAMF7 protein. The kit of the present application can be used for scientific research, such as qualitative or quantitative detection of CD3 and / or SLAMF7 protein in a biological sample, and can also be used for judging the state of an individual, such as judging whether the SLAMF7 level of the individual is too high or too low compared to the normal level after obtaining the SLAMF7 level of the individual.

[0147] Method for treating or preventing cancer

[0148] In another aspect of the present application, the present application provides a method for preventing and / or treating cancer. According to an embodiment 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 an embodiment of the present application, the method is effective for preventing or treating cancer.

[0149] It is to be noted that, in the present disclosure, 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 one 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, 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 a cancer that highly expresses SLAMF7.

[0151] According to an embodiment of the present application, the cancer that highly expresses SLAMF7 is multiple myeloma.

[0152] The schemes of the present application will be explained below in connection with examples. Those skilled in the art will understand that the following examples are only for illustrating the present application, and should not be considered as limiting the scope of the present application. In the examples, the specific techniques or conditions not mentioned are performed according to the techniques or conditions described in the literature in the art or according to the product manual. The reagents or instruments not mentioned are all conventional products that can be commercially available.

[0153] Example 1: Preparation of bispecific antibody molecules

[0154] In this example, the production of bispecific antibodies is performed, and the specific experimental operations are as follows: ExpiCHO cells (purchased from Thermo Fisher) are cultured using ExpiCHO Expression Medium (purchased from Thermo Fisher, A2910001), and the cell concentration is adjusted to 6 x 105cells / mL. 6 / mL, to obtain a solution of ExpiCHO cells. A pTT5 vector (synthesized by Suzhou Genewiz) containing three chain-encoding genes (SEQ ID NO: 23, 24, 25, respectively) was added to 2 mL of OptiSFM medium (Thermo Fisher, 12309019) to obtain solution A. Among them, the first chain-encoding gene includes a nucleotide sequence encoding a CD3 single-chain antibody (SEQ ID NO: 9) and a first FC peptide segment (SEQ ID NO: 10), the second chain-encoding gene includes a nucleotide sequence encoding a second heavy chain variable region of SLAMF7 (SEQ ID NO: 18) and a second FC peptide segment (SEQ ID NO: 20), and the third chain-encoding gene includes a nucleotide sequence encoding a second light chain variable region of SLAMF7 (SEQ ID NO: 19) and a CL region (SEQ ID NO: 31). 160 μL of ExpiFectamine CHO transfection reagent (Thermofisher, A29130) was added to 2 mL of OptiSFM medium to obtain solution B. Then, solution A and solution B were mixed to obtain a transfection mixture, and the transfection mixture was added to 50 mL of the solution of ExpiCHO cells 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) were added, and the culture supernatant was harvested after 9 days of incubation at 32°C, 5% CO2, wherein 8 mL of Feed was added on the 5th day. The antibody CD3 x SLAMF7 (i.e., the bispecific antibody) was affinity purified from the culture supernatant using a Protein A purification column (GE). It was detected that the antibody CD3 x SLAMF7 (herein, used interchangeably with “CD3 x SLAMF7 antibody”, “CD3 x SLAMF7 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 CD3 x SLAMF7 is shown in Figure 1 .

[0155] The first chain-encoding gene (i.e., the first antigen binding region) is used to encode SEQ ID NO: 11, and the first chain-encoding gene includes a nucleotide sequence as shown 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] GATATCCAGATGACCCAATCCCCTAGCAGTCTGTCTGCCTCTGTCGGGGATAGGGTGACCATTACCTGCAAAGCCTCTCAGGATGTCGGCATTGCTGTAGCCTGGTACCAGCAGAAGCCCGGGAAGGTCCCCAAACTGTTGATCTATTGGGCTTCAACCAGACACACCGGCGTCCCTGATCGGTTTAGCGGCAGTGGAAGCGGTACCGATTTCACTCTGACTATTTCTTCTCTGCAGCCCGAAGATGTGGCAACCTATTACTGCCAGCAGTATAGTAGCTACCCATACACATTCGGCCAAGGAACCAAAGTAGAAATTAAGCGCACCGTCGCTGCCCCAAGTGTCTTCATTTTCCCTCCCTCTGACGAGCAGCTTAAAAGCGGCACAGCCTCCGTTGTATGCCTCTTGAACAATTTCTACCCACGAGAGGCAAAGGTTCAGTGGAAGGTGGACAATGCTCTTCAGAGCGGGAATAGCCAGGAGTCCGTCACAGAGCAGGACTCTAAGGACAGCACTTACTCCTTGTCCAGTACCCTGACCTTGTCTAAAGCCGATTACGAGAAGCACAAAGTTTATGCATGCGAGGTGACACACCAGGGCCTGTCCAGCCCTGTGACCAAGAGTTTTAATCGCGGCGAGTGT (SEQ ID NO: 25).

[0161] Example 2: Identification of binding ability of bispecific antibodies to CD3E & D proteins

[0162] ELISA experiments were used to detect the binding properties of the CD3 x SLAMF7 antibodies obtained in Example 1. CD3E & D proteins were coated in 96-well plates, and the strength of the signal after the addition of the antibodies was used to determine the binding properties of the antibodies to CD3E & D.

[0163] CD3E&D protein (purchased from Acro) was diluted with PBS buffer to 2 μg / mL, and added to a 96-well plate at a volume of 100 μL / well, and placed at 4°C overnight. The PBS buffer in the 96-well plate was aspirated, the plate was washed 6 times with PBST (pH 7.2 PBS containing 0.1% Tween 20) buffer, and 200 μL / well of PBS / 10% BSA was added for blocking at 37°C for 2 h. The blocking solution was removed, the plate was washed 6 times with PBST, and the CD3xSLAMF7 antibody to be tested was diluted to the appropriate concentration with 100 μL / well of PBST / 0.05% BSA, and then incubated at 37°C for 1 h. The reaction system was removed, the plate was washed 6 times with PBST, and the HRP (horseradish peroxidase)-labeled rabbit anti-human IgG secondary antibody (Dr. D, BA1070) was diluted with 100 μL / well of PBST / 0.05% BSA, and then 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, and incubated at room temperature for 3 min, and 80 μL / well of 4M sulfuric acid was added to stop the reaction. The absorbance value was read at 450 mm with a microplate reader. The specific experimental results are shown in Table 1, which show that the antibody of the present application can bind to CD3E&D. Figure 2

[0164] Example 3: Identification of the binding ability of bispecific antibody to Jurkat T cells

[0165] 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 Jurkat T cells are judged based on the strength of the signal after the addition of the bispecific antibody. The specific experimental operation is as follows:

[0166] Jurkat T cells were diluted with PBS to 1 x 10 6 ​ / mL, 10 μL / tube mouse serum was added, and the mixture was blocked at 4°C for 30 min. After blocking, a series of concentration gradients (0.1, 1, 10, 30, 100, 300 μg / mL) of CD3xSLAMF7 bispecific antibody, hIgG (control IgG1, Biolegend, QA16A12) 10 μL / tube were added, and the mixture was incubated at 4°C for 30 min. After incubation, 1 mL PBS was added to the EP tube, and the mixture was centrifuged at 4°C and 100xg for 5 min. The supernatant was discarded, and the precipitate was washed once with PBS. After centrifugation, the supernatant was discarded, and the cells were resuspended with 100 μL / tube PBS. After resuspension, 1 μL / tube 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 mixture was washed twice with PBS, and the supernatant was discarded after centrifugation. The cells were resuspended with 200 μL / tube PBS, and the mixture was detected by flow cytometry. The specific experimental results are shown in Figure 3 Figure 6, which further shows that the bispecific antibody CD3xSLAMF7 of the present application can bind to Jurkat T cells.

[0167] Example 4: Identification of the binding ability of the bispecific antibody to human peripheral blood CD8+T cells

[0168] 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 human peripheral blood CD4+T cells are judged based on the strength of the signal after the addition of the bispecific antibody. The specific experimental operation is as follows:

[0169] Human peripheral blood mononuclear cells were diluted with PBS to 5x10 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 the end of blocking, a series of concentration gradients (0.1, 1, 10, 30, 100, 300 μg / mL) of CD3xSLAMF7 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 of PBS was added to the EP tube, and it was centrifuged at 4°C, 100xg for 5 min, 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, 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. The specific experimental results are shown in Figure 4 Figure 5, which shows that the bispecific antibody of the present application can bind to human peripheral blood T cells.

[0170] Example 5: Identification of the binding ability of the bispecific antibody to CHO-K1-SLAMF7 cells

[0171] 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-SLAMF7 cells. The specific experimental operation is as follows:

[0172] HEK293T cells were prepared according to 5x10 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-SLAMF7-IRES-puro (the pLVX-EF1a-IRES-puro vector contains an insertion of the coding sequence for SLAMF7 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 SLAMF7 protein has 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 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 designated CHO-K1-SLAMF7 cells.

[0173]

[0174] MAGSPTCLTLIYILWQLTGSAASGPVKELVGSVGGAVTFPLKSKVKQVDSIVWTFNTTPLVTIQPEGGTIIVTQNRNRERVDFPDGGYSLKLSKLKKNDSGIYYVGIYSSSLQQPSTQEYVLHVYEHLSKPKVTMGLQSNKNGTCVTNLTCCMEHGEEDVIYTWKALGQAANESHNGSILPISWRWGESDMTFICVARNPVSRNFSSPILARKLCEGAADDPDSSMVLLCLLLVPLLLSLFVLGLFLWFLKRERQEEYIEEKKRVDICRETPNICPHSGENTEYDTIPHTNRTILKEDPANTVYSTVEIPKKMENPHSLLTMPDTPRLFAYENVI (SEQ ID NO: 27).

[0175] CHO-K1-SLAMF7 cells were diluted with PBS to 1 x 10 6 / mL, 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 CD3 x SLAMF7 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, which further shows that the bispecific antibody CD3 x SLAMF7 of the present application can bind to CHO-K1-SLAMF7 cells. Figure 5

[0176] Example 6: Identification of the binding ability of the bispecific antibody to human multiple myeloma U266 cells

[0177] ​This example uses flow cytometry to detect the binding properties of the bispecific antibody obtained in Example 1, and based on the strength of the signal after the addition of the bispecific antibody to judge the binding properties of the bispecific antibody and human multiple myeloma U266 tumor cells. The specific experimental operation is as follows:

[0178] The U266 cells were diluted with PBS to 1×10 6 / mL, 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 CD3xSLAMF7 bispecific antibody, 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, 100xg 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. 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 6 , which shows that the CD3xSLAMF7 bispecific antibody of the present application can bind to multiple myeloma U266 cells.

[0179] Example 7: Identification of bispecific antibody promoting Jurkat-NFAT-lucia reporter cell activation

[0180] This example uses the Jurkat-NFAT-lucia reporter system method to identify the ability of the bispecific antibody obtained in Example 1 to cross-link the target cell surface SLAMF7 and the effector cell surface CD3, 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.

[0181] (1) The CHO-K1-SLAMF7 cells obtained in Example 5 were diluted with complete RPMI-1640 medium to 1×10 5 / mL, added to a 96-well plate, and 100 μL / well was added.

[0182] (2) Dilute the CD3xSLAMF7 bispecific antibody obtained in Example 1 to 500 μg / mL, 100 μg / mL, 20 μg / mL, 4 μg / mL, 0.8 μg / mL, 160 ng / mL, 32 ng / mL, 6.4 ng / mL respectively with complete RPMI-1640 medium, and add to the 96-well plate containing CHO-K1-SLAMF7 cells in step (1) with a volume of 20 μL / well.

[0183] (3) Dilute Jurkat-NFAT-lucia cells (Invivogen, jktl-nfat) to 1.25x10 5 / mL with complete RPMI-1640 medium, and add to the 96-well plate containing the bispecific antibody in step (2) with a volume of 80 μL / well.

[0184] (4) Incubate the reaction system obtained in step (3) in a 37°C, 5% CO2 incubator for 24 h.

[0185] (5) Take 50 μL of the culture supernatant obtained in step (4) and add to the 96-well plate, then add luciferase substrate to the plate with a volume of 50 μL / well.

[0186] (6) Detect chemiluminescence using a multifunctional enzyme marker.

[0187] The specific experimental results are shown in Table 1, which further show that the bispecific antibody CD3xSLAMF7 of the present application can bridge target cells (i.e. CHO-K1-SLAMF7 cells) and T cells (Jurkat-NFAT-lucia cells), and promote T cell activation. Figure 7

[0188] Example 8: 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-SLAMF7 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 diluted to 5x10 5 ​Seed 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-SLAMF7-IRES-puro (the pLVX-EF1a-IRES-puro vector contains an insertion of the coding sequence for SLAMF7 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 SLAMF7 protein has 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 added to a solution containing 1 × 10⁻⁶ cells. 4 In a 6-well plate containing A375 cells, Sigma polybrene was added to 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 designated A375-SLAMF7 cells.

[0191] (1) Add complete RPMI-1640 medium to a 16-well RTCA plate at a volume of 50 μL / well and calibrate the plate.

[0192] (2) Dilute A375-SLAMF7 cells to 2×10⁻⁶ using complete RPMI-1640 medium. 5 / mL, added to the RTCA plate obtained in step (1) at a volume of 50μL / well, and then the cell coefficient was detected for 24h at 37℃ and 5% CO2 using the xCELLigenceRTCA TP device;

[0193] (3) Dilute the bispecific antibody obtained in Example 1 to a series of concentration gradients (0.32, 1.6, 8, 40, 200, 1000 ng / mL) with complete RPMI-1640 medium and add it to the RTCA plate obtained in step (2) at a volume of 20 μL / well.

[0194] (4) Dilute PBMC (SaiLi Biotechnology) to 1.25 × 10⁻⁶ using complete RPMI-1640 medium. 6 Add 80 μL / well to the RTCA plate obtained in step (3);

[0195] (5) The reaction system obtained in step (4) was detected by xCELLigence RTCA TP device at 37℃, 5% CO2 for 48h.

[0196] The specific experimental results are shown in Figure 8 Further, it is shown that the bispecific antibody of the present application can promote PBMC to kill SLAMF7 positive tumor cells.

[0197] From the above experimental results, it can be seen that the bispecific antibody obtained by 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 present specification, the description of the terms "one embodiment", "some embodiments", "an example", "a specific example", or "some examples" and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the present 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 appropriate manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in the present specification and the features of the different embodiments or examples can be combined and combined by those skilled in the art without contradiction.

[0199] Although the embodiments of the present application have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present application, and those skilled in the art can make changes, modifications, replacements and variations to the above embodiments within the scope of the present application.

Claims

1. An antibody, characterized in that, Comprising: a first antigen binding region having CD3 binding activity, the first antigen binding region having an amino acid sequence as set forth in SEQ ID NO: 11; a second antigen binding region having SLAMF7 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. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the antibody of claim 1.

3. The nucleic acid molecule of claim 2, wherein, The nucleic acid molecule is a DNA molecule.

4. An expression vector, characterized by, Carrying the nucleic acid molecule of any one of claims 2-3.

5. The expression vector of claim 4, wherein, The expression vector is a non-pathogenic viral vector.

6. The expression vector of claim 5, wherein, The expression vector is an adenoviral vector, a lentiviral vector or a retroviral vector.

7. A method of producing the antibody of claim 1, wherein, Comprising: introducing the expression vector of any one of claims 4-6 into a cell; culturing the cell under conditions suitable for protein expression and secretion so as to obtain the antibody.

8. The method of claim 7, wherein, The cell is a eukaryotic cell.

9. A recombinant cell, characterized in that, The recombinant cell carries the nucleic acid molecule of any one of claims 2-3, or the expression vector of any one of claims 4-6 or expresses the antibody of claim 1.

10. The recombinant cell of claim 9, wherein, The recombinant cell is obtained by introducing the expression vector of any one of claims 4-6 into a host cell.

11. The recombinant cell of claim 9, wherein, The recombinant cell is a eukaryotic cell.

12. The recombinant cell of claim 9, wherein, The recombinant cell is a mammalian cell.

13. A pharmaceutical composition, characterized by, Comprising: the antibody of claim 1, the nucleic acid molecule of any one of claims 2-3, the expression vector of any one of claims 4-6 or the recombinant cell of any one of claims 9-12.

14. The pharmaceutical composition of claim 13, wherein, The pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

15. A kit comprising, Comprising: the antibody of claim 1, the nucleic acid molecule of any one of claims 2-3, the expression vector of any one of claims 4-6 or the recombinant cell of any one of claims 9-12.

16. Use of the antibody of claim 1, the nucleic acid molecule of any one of claims 2-3, the expression vector of any one of claims 4-6, the recombinant cell of any one of claims 9-12 or the pharmaceutical composition of any one of claims 13-14 in the manufacture of a medicament for the treatment or prevention of multiple myeloma.

17. Use of the antibody of claim 1, the nucleic acid molecule of any one of claims 2-3, the expression vector of any one of claims 4-6 or the recombinant cell of any one of claims 9-12 in the manufacture of a kit for the detection of CD3 and / or SLAMF7.

Images