Bispecific antibodies and their applications

Abstract

The present invention provides a bispecific antibody targeting CD3 and CD79b and its use. The antibody comprises a first antigen-binding region with CD3 binding activity and a second antigen-binding region with CD79b binding activity. The first antigen-binding region is selected from a CD3 single-chain antibody, and the second antigen-binding region comprises a CD79b heavy chain and a CD79b light chain. This antibody can simultaneously bind to CD79b-positive tumor cells and T cells, effectively mediating T cell killing of tumor cells and effectively treating cancer.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine. Specifically, the present invention relates to a bispecific antibody and its application. More specifically, the present invention relates to an antibody, a nucleic acid molecule, an expression vector, a recombinant cell, a pharmaceutical composition and a kit and their use. Background Art

[0002] Cancer is a serious threat to human health and life. In recent years, the incidence and mortality rates of cancer have continued to rise globally. Currently available cancer treatments include surgical resection, radiotherapy, chemotherapy, small molecule targeted therapy, antibody targeted therapy, and macromolecular immunotherapy. However, these approaches are only effective in some cancer patients, and cancer remains a major challenge for human health and life.

[0003] Non-Hodgkin's lymphoma (NHL) is a general term for a group of malignant tumors of the lymphohematopoietic system, of which B-cell NHL accounts for 70-85%. Although NHL is a highly curable cancer, its metastatic and recurrent nature poses significant challenges.

[0004] Multiple myeloma (MM) is the second most common hematological malignancy after non-Hodgkin's lymphoma. Despite significant progress in chemotherapy, proteasome inhibitors, immunomodulatory agents such as thalidomide derivatives, and CD38-targeted antibodies, nearly all patients eventually relapse.

[0005] 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 build a bridge between target cells (tumor cells) and effector cells (immune cells), generating effector functions that target and kill tumor cells.

[0006] Therefore, there is an urgent need to develop a bispecific antibody for targeted killing of tumor cells. Summary of the Invention

[0007] The present invention aims to, at least to some extent, address at least one of the technical problems existing in the prior art. To this end, the present invention provides a bispecific antibody targeting CD3 and CD79b. This bispecific antibody can simultaneously bind to CD3 and CD79b and effectively mediate the killing of CD79b-expressing cells by CD3-expressing T cells.

[0008] The present invention is accomplished based on the following findings of the inventors:

[0009] The CD3 molecule connects to the T cell antigen receptor (TCR) through a salt bridge, forming a TCR-CD3 complex that participates in T cell signal transduction and antigen recognition. CD79b is a type I transmembrane protein, a B cell surface antigen, and a component of the BCR. CD79b is abnormally expressed in over 90% of B cell lymphomas.

[0010] Currently, only one CD79b-targeting drug has been approved by the FDA: Polivy. Polivy is an antibody-drug conjugate consisting of a CD79b monoclonal antibody conjugated to the anti-mitotic agent MMAE. Essentially, it utilizes the CD79b antibody to deliver the chemotherapy drug MMAE to tumor cells. MMAE acts on both the antibody-targeted tumor cells and bystander tumor cells, inhibiting microtubule aggregation and thereby achieving its anti-cancer effect.

[0011] However, in one aspect of the present invention, an antibody is provided. According to an embodiment of the present invention, the antibody comprises: a first antigen-binding region having CD3 binding activity; and a second antigen-binding region having CD79b binding activity. The antibody according to an embodiment of the present invention can bind to both CD3 and CD79b simultaneously, thereby effectively mediating the killing effect of T cells on cells expressing CD79b (e.g., tumor cells), and in particular has a strong tumor-suppressing effect, which can effectively treat cancer.

[0012] In another aspect of the present invention, a nucleic acid molecule is provided. According to an embodiment of the present invention, the nucleic acid molecule encodes the aforementioned antibody. According to an embodiment of the present invention, the nucleic acid molecule can encode an antibody that can simultaneously target and bind to CD3 and CD79b.

[0013] In yet another aspect, the present invention provides an expression vector. According to an embodiment of the present invention, the expression vector carries the aforementioned nucleic acid molecule. After the expression vector according to the embodiment of the present invention is introduced into a suitable recipient cell, it can effectively express the aforementioned antibody under the mediation of a regulatory system, so as to obtain the antibody in large quantities.

[0014] In yet another aspect, the present invention provides a method for preparing the aforementioned antibody. According to an embodiment of the present invention, the method comprises: introducing the aforementioned expression vector into cells; and culturing the cells under conditions suitable for protein expression and secretion to obtain the antibody. The method according to an embodiment of the present invention can effectively obtain the aforementioned antibody and has advantages such as a simple preparation method.

[0015] In yet another aspect, the present invention provides a recombinant cell. According to an embodiment of the present invention, the recombinant cell carries the aforementioned nucleic acid molecule, or the aforementioned expression vector, or expresses the aforementioned antibody. The recombinant cell is obtained by transfecting or transforming the aforementioned expression vector, and under appropriate conditions, the recombinant cell can efficiently express the aforementioned antibody that can simultaneously target and bind to CD3 and CD79b.

[0016] In another aspect of the present invention, the present invention provides a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition comprises: the aforementioned antibody, the aforementioned nucleic acid molecule, the aforementioned expression vector, or the aforementioned recombinant cell. The pharmaceutical composition according to an embodiment of the present invention can simultaneously target antibodies that bind to CD3 and CD79b, can effectively mediate the killing effect of T cells on cells expressing CD79b (such as tumor cells), and in particular has a strong tumor inhibitory effect, which can effectively treat cancer.

[0017] In yet another aspect, the present invention provides a kit. According to an embodiment of the present invention, the kit includes: the aforementioned antibody, the aforementioned nucleic acid molecule, the aforementioned expression vector, or the aforementioned recombinant cell. The kit according to an embodiment of the present invention can bind to the CD3 protein and / or the CD79b protein and can effectively identify the CD3 protein and / or the CD79b protein.

[0018] In another aspect of the present invention, the present invention 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 an embodiment of the present invention, the antibody or pharmaceutical composition of the present invention can simultaneously target antibodies that bind to CD3 and CD79b, can effectively mediate the killing effect of T cells on tumor cells expressing CD79b, has a strong tumor inhibitory effect, and can effectively treat cancer.

[0019] In yet another aspect, the present invention provides a use of the aforementioned antibody, nucleic acid molecule, expression vector, or recombinant cell in preparing a kit for detecting CD3 and / or CD79b. According to embodiments of the present invention, the antibody or kit of the present invention can bind to CD3 protein and / or CD79b protein, and can effectively identify CD3 protein and / or CD79b protein.

[0020] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments with reference to the following drawings, in which:

[0022] Figure 1 is a schematic structural diagram of the bispecific antibody according to Example 1 of the present invention;

[0023] Figure 2 3 is a graph showing the results of the detection of the binding ability of the CD3×CD79b bispecific antibody to CD3E&D protein according to Example 2 of the present invention;

[0024] Figure 3 3 is a graph showing the results of the detection of the binding ability of the CD3×CD79b bispecific antibody according to Example 3 of the present invention to Jurkat T cells;

[0025] Figure 4 4 is a graph showing the results of the detection of the binding ability of the CD3×CD79b bispecific antibody according to Example 4 of the present invention to human peripheral blood CD8+ T cells;

[0026] Figure 5 3 is a graph showing the results of the detection of the binding ability of the CD3×CD79b bispecific antibody according to Example 5 of the present invention to CHO-K1-CD79b cells;

[0027] Figure 6 This is a graph showing the detection results of activating Jurkat-NFAT-lucia reporter cells using the CD3×CD79b bispecific antibody according to Example 6 of the present invention;

[0028] Figure 7 This is a graph showing the detection results of the recombinant bispecific antibody according to Example 7 of the present invention promoting PBMC to kill A375-CD79b tumor cells. DETAILED DESCRIPTION

[0029] 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 understood as limiting the present invention.

[0030] 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 the technical features being referred to. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of such features. Furthermore, in the description of the present invention, unless otherwise specified, "plurality" means two or more.

[0031] The endpoints of the ranges and any values 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 endpoints of each range, the endpoints of each range and individual point values, and the individual point values can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0032] To facilitate understanding of the present invention, certain technical and scientific terms are defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. The abbreviations for amino acid residues are the standard three-letter and / or one-letter codes used in the art to designate one of the 20 commonly used L-amino acids.

[0033] In the present invention, unless otherwise specified or limited, the term "connected" should be understood in a broad sense. For example, it can refer to direct connection or indirect connection through an intermediate medium. It can also refer to internal communication between two elements or interaction between two elements, unless otherwise specified. Those skilled in the art will understand the specific meanings of the above terms in the present invention based on specific circumstances.

[0034] As used herein, the term "bispecific antibody" generally refers to a bispecific antibody obtained by linking two Fc fragments to peptide chains that specifically recognize two protein molecules, wherein the two Fc fragments are connected via a knob-into-hole structure. In this application, "bispecific antibody" and "bispecific antibody molecule" are used interchangeably. The peptide chain that specifically recognizes a protein molecule can be a single-chain antibody; it can also be two chains connected by a disulfide bond, for example, one chain recognizes the heavy chain variable region and CH1 region of the protein molecule, and the other chain recognizes the light chain variable region and CL region of the protein molecule.

[0035] As used herein, the term "knob into hole structure" generally refers to the formation of a knob or hole mutation in the CH3 region of the antibody heavy chain constant region or Fc fragment to facilitate heavy chain engagement to form a heterodimer. For example, in the present application, this is achieved by mutating amino acids in the CH3 domain of human IgG1-Fc (T366S, L368A, Y407V, and Y349C mutations in one chain, i.e., the "hole"; and T366W and S354C mutations in the other chain, i.e., the "knob").

[0036] Herein, the amino acid numbering of the IgG1 Fc portion is according to the EU numbering system, for example, position 366 refers to position 366 according to the EU numbering system; the "T366S" refers to the replacement of threonine at position 366 according to the EU numbering system by serine; and "L368A" refers to the replacement of leucine at position 368 according to the EU numbering system by alanine.

[0037] In this article, the term "expression vector" generally refers to a nucleic acid molecule that can be inserted into a suitable host and replicates itself, and transfers the inserted nucleic acid molecule into and / or between host cells. The expression vector may include a vector primarily used to insert DNA or RNA into a cell, a vector primarily used to replicate DNA or RNA, and a vector primarily used for expression of the transcription and / or translation of DNA or RNA. The expression vector also includes vectors with multiple of the above functions. The expression vector can be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, the expression vector can produce a desired expression product by culturing a suitable host cell containing the expression vector.

[0038] In this article, the term "recombinant cell" generally refers to the use of genetic engineering technology or cell fusion technology to modify or reorganize the genetic material of the host cell to obtain a cell with a unique characteristic of stable inheritance. Wherein, the term "host cell" refers to a prokaryotic cell or eukaryotic cell into which a recombinant expression vector can be introduced. The terms "transformed" or "transfected" as used herein refer to the introduction of nucleic acid (e.g., a vector) into a cell by various techniques known in the art. Suitable host cells can be transformed or transfected with the DNA sequence of the present invention and can be used for the expression and / or secretion of the target protein. Examples of suitable host cells that can be used for the present invention include immortalized hybridoma cells, NS / 0 myeloma cells, 293 cells, Chinese hamster ovary (CHO) cells, HeLa cells, Cap cells (cells derived from human amniotic fluid), insect cells, PER.C6 cells, and CoS cells, preferably CHO cells.

[0039] As used herein, the term "pharmaceutical composition" generally refers to unit dosage form and can be prepared by any of the methods well known in the pharmaceutical art. All methods include the step of bringing the active ingredient into association with the carrier which constitutes one or more accessory ingredients. Generally, the compositions are prepared by uniformly and thoroughly combining the active compound with liquid carriers, finely divided solid carriers, or both.

[0040] As used herein, the term "pharmaceutically acceptable" means that the substance or composition must be chemically and / or toxicologically compatible with the other ingredients of the formulation and / or the mammal to be treated therewith. Preferably, the term "pharmaceutically acceptable" as used herein means approved by federal regulatory agencies or national governments or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopeia for use in animals, particularly humans.

[0041] As used herein, the term "pharmaceutically acceptable excipient" may include any solvent, solid excipient, diluent or other liquid excipient, etc., suitable for the specific target dosage form. Except for the scope of any conventional excipient being incompatible with the compound of the present invention, such as any adverse biological effect produced or any other component of the pharmaceutically acceptable composition interacting in a harmful manner, their use is also within the scope of the present invention.

[0042] As used herein, the term "administer" refers to the introduction of a predetermined amount of a substance into a patient by a suitable means. The recombinant antibody or pharmaceutical composition of the present invention can be administered by any common route, as long as it can reach the desired tissue. Various modes of administration are contemplated, including peritoneal, intravenous, intramuscular, subcutaneous, and the like, but the present invention is not limited to these exemplified modes of administration. Preferably, the composition of the present invention is administered by intravenous or subcutaneous injection.

[0043] As used herein, the term "treatment" refers to any process used to obtain a desired pharmacological and / or physiological effect. The effect may be preventive in terms of completely or partially preventing a disease or its symptoms, and / or therapeutic in terms of partially or completely curing a disease and / or the adverse effects caused by the disease. "Treatment" as used herein covers diseases in mammals, particularly humans, and includes: (a) preventing the occurrence of a disease or condition in individuals who are susceptible to the disease but have not yet been diagnosed with the disease; (b) inhibiting the disease, such as arresting the progression of the disease; or (c) alleviating the disease, such as alleviating the symptoms associated with the disease. "Treatment" as used herein covers any medication that administers a drug or compound to an individual to treat, cure, alleviate, improve, reduce or inhibit the individual's disease, including but not limited to administering a drug containing a compound described herein to an individual in need.

[0044] It should be noted that, for nucleic acids mentioned in the present specification and claims, those skilled in the art will understand that they actually include any one or both of the complementary double strands. For convenience, although only one strand is provided in most cases in this specification and claims, the other complementary strand is also disclosed. In addition, the nucleic acid sequences in this application include DNA or RNA forms, and disclosure of one of them means that the other is also disclosed.

[0045] The present invention provides an antibody, a nucleic acid molecule, an expression vector, a recombinant cell, a pharmaceutical composition and a kit and uses thereof, which are described in detail below.

[0046] Antibody

[0047] In one aspect of the present invention, the present invention proposes an antibody. According to an embodiment of the present invention, the antibody comprises: a first antigen binding region, wherein the first antigen binding region has CD3 binding activity; and a second antigen binding region, wherein the second antigen binding region has CD79b binding activity. The antibody according to the embodiment of the present invention can bind to CD3 and CD79b at the same time, thereby effectively mediating the killing effect of T cells on cells expressing CD79b (such as tumor cells), especially having a strong tumor inhibitory effect, and can effectively treat cancer. In addition, the inventors have also found through experiments that, compared with chemotherapy and antibody-drug conjugates, bispecific antibody drugs avoid the side effects of chemotherapy drugs on the one hand, and on the other hand, can mobilize autoimmune cells to specifically kill tumor cells, and have the potential to stimulate their own adaptive immune system to fight cancer.

[0048] According to an embodiment of the present invention, the first antigen binding region includes 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 invention, the C-terminus of the first heavy chain variable region and the N-terminus of the first light chain variable region are connected.

[0050] According to an embodiment of the present invention, the first antigen binding region further includes 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, 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 invention, 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 invention, the first heavy chain variable region comprises a CDR sequence shown in any one of SEQ ID NOs: 1 to 3, or the first light chain variable region comprises a CDR sequence shown in any one of SEQ ID NOs: 4 to 6. Thus, the first antigen-binding region can effectively bind to the 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 invention, the first heavy chain variable region has CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively.

[0060] According to an embodiment of the present invention, the first light chain variable region has CDR1, CDR2, and CDR3 sequences shown as SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively.

[0061] According to an embodiment of the present invention, 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 invention, the first antigen binding region further includes 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 the "Fc peptide segment", "Fc fragment" or "Fc" in this article refers to a peptide segment comprising a hinge region, a CH2 region and a CH3 region, such as the wild-type IgG1 Fc fragment, the first FC peptide segment and the 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 shown below:

[0066] PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:28).

[0067] According to an embodiment of the present invention, the first FC peptide segment includes a first hinge region, a first CH2 region and a first CH3 region.

[0068] According to an embodiment of the present invention, the first hinge region is a hinge region fragment of human, primate or mouse wild-type IgG1.

[0069] According to an embodiment of the present invention, the first CH2 region is a CH2 region fragment of human, primate or mouse wild-type IgG1; or the first CH2 region has L234A and / or L235A mutations compared to the CH2 region fragment of human wild-type IgG1.

[0070] According to an embodiment of the present invention, the first CH3 region has T366W and / or S354C mutations compared to the CH3 region fragment of human wild-type IgG1.

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

[0072] According to an embodiment of the present invention, 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 invention, the second connecting peptide has an amino acid sequence as shown in SEQ ID NO: 8. Thus, the binding activity of the first antigen binding region to the CD3 protein can be further increased.

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

[0075] According to an embodiment of the present invention, the first antigen binding region includes the amino acid sequence 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 present invention exist in the form of a single-chain antibody. The C-terminus of the first heavy chain variable region in the single-chain antibody (also known as the CD3 single-chain antibody) 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. The single-chain antibody has the amino acid sequence shown in SEQ ID NO: 9.

[0077] EVQLLESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSGGGG SGGGGSGGGGSELVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVLGGGGS(SEQ ID NO: 9).

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

[0079] PKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 10).

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

[0081] EVQLLESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVT VSSGGGGSGGGGSGGGGSELVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGT KLTVLGGGGSPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 11).

[0082] According to an embodiment of the present invention, the second antigen binding region includes a first polypeptide and a second polypeptide, and the first polypeptide and the second polypeptide are connected by an interchain disulfide bond; the first polypeptide includes 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, and the C-terminus of the CH1 region is connected to the N-terminus of the second FC peptide segment; the second polypeptide includes a second light chain variable region and a CL region, and 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 invention, the second FC peptide segment includes a second hinge region, a second CH2 region and a second CH3 region.

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

[0085] According to an embodiment of the present invention, the second hinge region is a hinge region fragment of human, primate or mouse wild-type IgG1.

[0086] According to an embodiment of the present invention, the second CH2 region is a CH2 region fragment of human, primate or mouse wild-type IgG1; or the second CH2 region has L234A and / or L235A mutations compared to the CH2 region fragment of human wild-type IgG1.

[0087] According to an embodiment of the present invention, the second CH3 region is a CH3 region fragment of human, primate or mouse wild-type IgG1.

[0088] According to an embodiment of the present invention, the second CH3 region has at least one of T366S, L368A, Y407V, and Y349C mutations compared to the CH3 region fragment of human wild-type IgG1.

[0089] According to an embodiment of the present invention, the CL region is a wild-type CL region of human, primate or mouse origin.

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

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

[0092] According to an embodiment of the present invention, the second heavy chain variable region includes a CDR sequence shown in any one of SEQ ID NOs: 12 to 14; or the second light chain variable region includes a CDR sequence shown in any one of SEQ ID NOs: 15 to 17.

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

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

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

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

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

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

[0099] According to an embodiment of the present invention, the second heavy chain variable region has CDR1, CDR2, and CDR3 sequences shown as SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, respectively.

[0100] According to an embodiment of the present invention, the second light chain variable region has CDR1, CDR2, and CDR3 sequences shown as SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17, respectively.

[0101] According to an embodiment of the present invention, the second heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 18.

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

[0103] According to an embodiment of the present invention, the second light chain variable region has the amino acid sequence shown in SEQ ID NO: 19.

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

[0105] According to an embodiment of the present invention, the CH1 region and the second FC peptide segment have an amino acid sequence as shown in SEQ ID NO: 20.

[0106] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(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 fragment composed of a CH1 region and a second FC peptide segment connected together, wherein the C-terminus of the CH1 region is connected to the N-terminus of the second FC peptide segment.

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

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

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

[0111] PKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 31).

[0112] According to an embodiment of the present invention, the first polypeptide has an amino acid sequence as shown in SEQ ID NO: 21.

[0113] EVQLVESGGGLVQPGGSLRLSCAASGYTFSSYWIEWVRQAPGKGLEWIGEILPGGGDTNYNEIFKGRATFSADTSKNTAYLQMNSLRAEDTAVYYCTRRVPIRLDYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 21).

[0114] According to an embodiment of the present invention, the second polypeptide has an amino acid sequence as shown in SEQ ID NO: 22.

[0115] DIQLTQSPSSSLSASVGDRVTITCKASQSVDYEGDSFLNWYQQKPGKAPKLLIYAASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSNEDPLTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO: 22).

[0116] According to an embodiment of the present invention, the first antigen binding region and the second antigen binding region are connected via a knob-into-hole structure.

[0117] According to an embodiment of the present invention, the knob-into-hole structure is formed by T366W and / or S354C mutations in the first CH3 region and at least one of T366S, L368A, Y407V, and Y349C mutations in the second CH3 region.

[0118] According to an embodiment of the present invention, the knob-into-hole structure is formed by mutations T366W and S354C in the first CH3 region and mutations T366S, L368A, Y407V, and Y349C in the second CH3 region. This reduces the number of first antigen-binding regions containing two first Fc peptides connected, and reduces the number of second antigen-binding regions containing two second Fc peptides connected, thereby improving the yield of the antibodies of the present invention.

[0119] Nucleic acid molecules, expression vectors and recombinant cells

[0120] In the process of preparing or obtaining these antibodies, nucleic acid molecules expressing these antibodies can be connected to different vectors and then expressed in different cells to obtain the corresponding antibodies or antigen-binding fragments thereof.

[0121] In another aspect of the present invention, a nucleic acid molecule is provided. According to an embodiment of the present invention, the nucleic acid molecule encodes the aforementioned antibody. According to an embodiment of the present invention, the nucleic acid molecule can encode an antibody that can simultaneously target and bind to CD3 and CD79b.

[0122] According to an embodiment of the present invention, the nucleic acid molecule is a DNA molecule.

[0123] In yet another aspect, the present invention provides an expression vector. According to an embodiment of the present invention, the expression vector carries the aforementioned nucleic acid molecule. After the expression vector according to the embodiment of the present invention is introduced into a suitable recipient cell, it can effectively express the aforementioned antibody under the mediation of a regulatory system, so as to obtain the antibody in large quantities.

[0124] When the nucleic acid molecule is linked to a vector, the nucleic acid molecule can be directly or indirectly linked to control elements on the vector, as long as these control elements are capable of controlling translation and expression of the nucleic acid molecule. These control elements can be derived directly from the vector itself or exogenously, i.e., not from the vector itself. Of course, it is sufficient that the nucleic acid molecule and the control elements are operably linked.

[0125] As used herein, "operably linked" refers to linking an exogenous gene to a vector so that control elements within the vector, such as transcriptional control sequences and translational control sequences, can function as intended to regulate the transcription and translation of the exogenous gene. While the polynucleotides encoding the first antigen-binding region of an antibody, the first polypeptide, and the second polypeptide can be independently inserted into different vectors, they are typically inserted into the same vector. Commonly used vectors include plasmids, bacteriophages, and the like.

[0126] According to an embodiment of the present invention, the expression vector is a non-pathogenic viral vector.

[0127] According to an embodiment of the present invention, the expression vector is an adenoviral vector, a lentiviral vector or a retroviral vector.

[0128] In yet another aspect, the present invention provides a recombinant cell. According to an embodiment of the present invention, the recombinant cell carries the aforementioned nucleic acid molecule, or the aforementioned expression vector, or expresses the aforementioned antibody. The recombinant cell is obtained by transfecting or transforming the aforementioned expression vector, and under appropriate conditions, the recombinant cell can efficiently express the aforementioned antibody that can simultaneously target and bind to CD3 and CD79b.

[0129] It should be noted that the recombinant cells of the present invention are not particularly limited and may be prokaryotic cells, eukaryotic cells, or bacteriophages. The prokaryotic cells may be Escherichia coli, Bacillus subtilis, Streptomyces, or Proteus mirabilis, among others. The eukaryotic cells may be fungi such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, or Trichoderma, insect cells such as fall armyworms, plant cells such as tobacco, or mammalian cells such as BHK cells, CHO cells, COS cells, or myeloma cells. In some embodiments, the recombinant cells of the present invention are preferably mammalian cells, including BHK cells, CHO cells, NSO cells, or COS cells, and do not include animal germ cells, fertilized eggs, or embryonic stem cells.

[0130] It should be noted that the "suitable conditions" described in this specification refer to conditions suitable for the expression of the antibodies described herein. It will be readily understood by those skilled in the art that conditions suitable for antibody expression include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy host cell status, suitable host cell density, suitable cell culture environment, and suitable cell culture time. "Suitable conditions" are not particularly limited, and those skilled in the art can optimize the most suitable conditions for the expression of the antibodies according to the specific environment of the laboratory.

[0131] According to an embodiment of the present invention, the recombinant cell is obtained by introducing the aforementioned expression vector into a host cell.

[0132] According to an embodiment of the present invention, the recombinant cell is a eukaryotic cell.

[0133] According to an embodiment of the present invention, the recombinant cells are mammalian cells. An expression vector can be introduced into a mammalian cell to construct a recombinant cell, which can then be used to express the antibody provided by the present invention. By culturing the recombinant cell, the corresponding antibody can be obtained. These useful mammalian cells can be, for example, CHO cells.

[0134] Methods for preparing antibodies

[0135] In yet another aspect, the present invention provides a method for preparing the aforementioned antibody. According to an embodiment of the present invention, the method comprises: introducing the aforementioned expression vector into cells; and culturing the cells under conditions suitable for protein expression and secretion to obtain the antibody. The method provided in accordance with an embodiment of the present invention can effectively obtain the aforementioned antibody and has advantages such as a simple preparation method.

[0136] According to an embodiment of the present invention, the cell is a eukaryotic cell.

[0137] According to an embodiment of the present invention, 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.

[0138] According to an embodiment of the present invention, the eukaryotic cells do not include animal germ cells, fertilized eggs or embryonic stem cells.

[0139] Pharmaceutical compositions and kits

[0140] In another aspect of the present invention, the present invention provides a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition comprises: the aforementioned antibody, the aforementioned nucleic acid molecule, the aforementioned expression vector, or the aforementioned recombinant cell. The pharmaceutical composition according to an embodiment of the present invention can simultaneously target antibodies that bind to CD3 and CD79b, can effectively mediate the killing effect of T cells on cells expressing CD79b (such as tumor cells), and in particular has a strong tumor inhibitory effect, which can effectively treat cancer.

[0141] According to an embodiment of the present invention, the pharmaceutical composition further includes a pharmaceutically acceptable excipient.

[0142] In another aspect of the present invention, the present invention provides a kit. According to an embodiment of the present invention, the kit includes: the aforementioned antibody, the aforementioned nucleic acid molecule, the aforementioned expression vector or the aforementioned recombinant cell. The kit according to the embodiment of the present invention can bind to the CD3 protein and / or CD79b protein, thereby effectively identifying the CD3 protein and / or CD79b protein. The kit of the present invention can be used for scientific research, such as qualitatively or quantitatively detecting CD3 and / or CD79b proteins in biological samples, and can also be used to judge the status of an individual, such as after obtaining the CD79b level of the individual, judging whether the CD79b level is too high or too low.

[0143] use

[0144] In another aspect of the present invention, the present invention 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 an embodiment of the present invention, the antibody or pharmaceutical composition of the present invention can simultaneously target antibodies that bind to CD3 and CD79b, can effectively mediate the killing effect of T cells on tumor cells that highly express CD79b, has a strong tumor inhibitory effect, and can effectively treat cancer.

[0145] According to an embodiment of the present invention, the cancer is selected from cancers that highly express CD79b.

[0146] According to an embodiment of the present invention, the cancer that highly expresses CD79b is selected from B cell tumors.

[0147] According to an embodiment of the present invention, the B cell tumor includes at least one of B cell non-Hodgkin's lymphoma, B cell multiple myeloma and B cell chronic lymphocytic leukemia.

[0148] In another aspect of the present invention, the present invention 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 CD79b. According to an embodiment of the present invention, the antibody or kit of the present invention can bind to the CD3 protein and / or CD79b protein, thereby effectively identifying the CD3 protein and / or CD79b protein. The kit of the present invention can be used for scientific research, such as qualitatively or quantitatively detecting CD3 and / or CD79b proteins in biological samples, and can also be used to judge the status of an individual, such as after obtaining the CD79b level of the individual, judging whether the CD79b level is too high or too low.

[0149] Methods for treating or preventing cancer

[0150] In another aspect, the present invention provides a method for preventing and / or treating cancer. According to an embodiment of the present invention, the method comprises administering a pharmaceutically acceptable amount of the aforementioned antibody or pharmaceutical composition to a subject. According to an embodiment of the present invention, the method can effectively prevent or treat cancer.

[0151] It should be noted that, in this article, a "pharmaceutically acceptable amount" may 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 a pharmaceutically acceptable amount can be determined by a person of ordinary skill in the art based on various factors (e.g., through clinical trials). Such factors include, but are not limited to: pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated, the patient's weight, the patient's immune status, the route of administration, etc. For example, depending on the urgency of the treatment, several divided doses may be administered daily, or the dose may be reduced proportionally.

[0152] According to an embodiment of the present invention, the cancer is selected from cancers that highly express CD79b.

[0153] According to an embodiment of the present invention, the cancer that highly expresses CD79b is selected from B cell tumors.

[0154] According to an embodiment of the present invention, the B cell tumor includes at least one of B cell non-Hodgkin's lymphoma, B cell multiple myeloma and B cell chronic lymphocytic leukemia.

[0155] Below, the scheme of the present invention will be explained in conjunction with embodiment.It will be understood by those skilled in the art that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.In the embodiment, if specific technology or conditions are not indicated, the technology or conditions described in the literature in this area or the product instructions are used.The reagents or instruments used are not indicated by the manufacturer, and are all conventional products that can be obtained by commercial purchase.

[0156] Example 1: Preparation of bispecific antibody molecules

[0157] In this example, bispecific antibodies were produced. The specific experimental procedures were as follows: ExpiCHO cells (purchased from Thermo Fisher) were cultured using ExpiCHO Expression Medium (purchased from Thermo Fisher, A2910001) and the cell concentration was adjusted to 6×10 6 / mL to obtain ExpiCHO cell solution. The pTT5 vector (synthesized by Suzhou Jinweizhi Company) containing three chain encoding genes (as shown in SEQ ID NO: 23, 24, and 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 (SEQ ID NO: 10), the second chain encoding gene includes a nucleotide sequence encoding the second heavy chain variable region of CD79b (SEQ ID NO: 18) and a second FC peptide (SEQ ID NO: 20), and the third chain encoding gene includes a nucleotide sequence encoding the second light chain variable region of CD79b (SEQ ID NO: 19) and the 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 entire transfection mixture was added to 50 mL of ExpiCHO cell solution within 5 minutes. After culturing at 37°C and 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 culture at 32°C and 5% CO2, with 8 mL of Feed added on the 5th day. The bispecific antibody was affinity purified from the culture supernatant using a Protein A purification column (GE) to obtain the antibody CD3×CD79b (i.e., a bispecific antibody). After testing, the antibody CD3×CD79b (herein, "CD3×CD79b antibody", "CD3×CD79b bispecific antibody" and "bispecific antibody" can be used interchangeably) has the amino acid sequences shown in SEQ ID NO: 11, SEQ ID NO: 21 and SEQ ID NO: 22. The structure of the antibody CD3×CD79b is as shown Figure 1 shown.

[0158] The first chain encoding gene (i.e., the first antigen binding region) is used to encode SEQ ID NO: 11. The first chain encoding gene includes the following nucleotide sequence:

[0159]

[0160] The second chain encoding gene (i.e., the first polypeptide) is used to encode SEQ ID NO: 21. The second chain encoding gene includes the following nucleotide sequence:

[0161]

[0162] The third chain encoding gene (i.e., the second polypeptide) is used to encode SEQ ID NO: 22. The third chain encoding gene includes the following nucleotide sequence:

[0163] (SEQ ID NO: 25).

[0164] Example 2: Identification of the binding ability of bispecific antibodies to CD3E&D proteins

[0165] ELISA experiments were used to detect the binding properties of the CD3×CD79b antibody obtained in Example 1. CD3E&D protein was coated into a 96-well plate, and the strength of the signal after the addition of the antibody was used to determine the binding properties of the antibody and CD3E&D.

[0166] CD3 E&D protein (purchased from Acro) was diluted to 2 μg / mL in PBS buffer and added to a 96-well plate at a volume of 100 μL / well. The plate was incubated at 4°C overnight. The PBS buffer was aspirated from the 96-well plate, and the plate was washed six times with PBST (pH 7.2 PBS containing 0.1% Tween 20). Blocking was performed by adding 200 μL / well of PBS / 10% BSA and incubating at 37°C for 2 hours. The blocking solution was removed, and the plate was washed six times with PBST. The CD3×CD79b antibody to be tested was diluted to the appropriate concentration in 100 μL / well of PBST / 0.05% BSA, followed by incubation at 37°C for 1 hour. Remove the reaction system, wash the plate 6 times with PBST, and dilute HRP (horseradish peroxidase) labeled rabbit anti-human IgG secondary antibody (Boster, BA1070) with 100 μL / well PBST / 0.05% BSA, and incubate at 37°C for 1 hour. After incubation, wash the plate 6 times with PBST, add 80 μL / well TMB (tetramethylbenzidine), incubate at room temperature for 3 minutes, and add 80 μL / well 4M sulfuric acid to terminate the reaction. Read the absorbance value at 450mm with a microplate reader. The specific experimental results are as follows. Figure 2 As shown, it was demonstrated that the antibodies of the present invention can bind to CD3E&D.

[0167] Example 3: Identification of the binding ability of bispecific antibodies to Jurkat T cells

[0168] This example uses flow cytometry to detect the binding properties of the bispecific antibody obtained in Example 1, and the strength of the signal after the addition of the bispecific antibody is used to determine the binding properties of the bispecific antibody and Jurkat T cells. The specific experimental procedures are as follows:

[0169] Dilute Jurkat T cells to 1 × 10 6 / mL, 90 μL / tube were added to 1.5 mL EP tubes. 10 μL / tube of mouse serum was added and the cells were blocked at 4°C for 30 min. After blocking, 10 μL / tube of a CD3×CD79b bispecific antibody and hIgG (control IgG1, Biolegend, QA16A12) were added at a concentration gradient (0.1, 1, 10, 30, 100, and 300 μg / mL) and incubated at 4°C for 30 min. After incubation, 1 mL of PBS was added to the EP tubes, and the cells were centrifuged at 100 × g for 5 min at 4°C. The supernatant was discarded and the pellet was washed once with PBS. After centrifugation, the supernatant was discarded and the cells were resuspended in 100 μL / tube of PBS. After resuspension, 1 μL / tube of Alexa-647-labeled rat anti-human Fc secondary antibody (Biolegend, M1310G05) was added and incubated at 4°C for 30 min in the dark. Wash twice with PBS, centrifuge and discard the supernatant. Resuspend the cells with 200 μL / tube PBS and detect them by flow cytometry. The specific experimental results are as follows Figure 3 As shown, it is further demonstrated that the bispecific antibody CD3×CD79b of the present invention can bind to Jurkat T cells.

[0170] Example 4: Identification of the binding ability of bispecific antibodies to human peripheral blood CD8+ T cells

[0171] This example uses flow cytometry to detect the binding properties of the bispecific antibody obtained in Example 1, and the strength of the signal after the addition of the bispecific antibody is used to determine the binding properties of the bispecific antibody to human peripheral blood CD4+ T cells. The specific experimental procedures are as follows:

[0172] Human peripheral blood mononuclear cells were diluted to 5×10 6 / mL, added to a volume of 90μL / tube in a 1.5mL EP tube, added with 10μL / tube of rat serum, and blocked at 4℃ for 30min; after the blocking, a series of concentration gradients (0.1, 1, 10, 30, 100, 300μg / mL) of CD3×CD79b bispecific antibody and hIgG (control IgG1, Biolegend, QA16A12) were added at 10μL / tube, incubated at 4℃ for 30min, and then 1mL was added to the EP tube. PBS, centrifuge at 4°C, 100×g for 5 minutes, discard the supernatant, wash the precipitate with PBS again, discard the supernatant after centrifugation, resuspend the cells with 100μL / tube PBS, add 1μL / tube Alexa-647 labeled rat anti-human Fc antibody secondary antibody (Biolegend, M1310G05) and 1μL / tube FITC labeled mouse anti-human CD8 antibody (Invitrogen, OKT8), incubate at 4°C in the dark for 30 minutes. Wash twice with PBS, centrifuge and discard the supernatant. Resuspend the cells with 200μL / tube PBS and detect by flow cytometry. The specific experimental results are as follows. Figure 4 As shown, it is shown that the bispecific antibody of the present invention can bind to human peripheral blood T cells.

[0173] Example 5: Identification of the binding ability of bispecific antibodies to CHO-K1-CD79b cells

[0174] This example uses flow cytometry to detect the binding properties of the bispecific antibody obtained in Example 1, and the strength of the signal after the addition of the bispecific antibody is used to determine the binding properties of the bispecific antibody to CHO-K1-CD79b cells. The specific experimental procedures are as follows:

[0175] HEK293T cells were grown at 5×10 5Plate cells / well in a six-well plate and culture overnight in DMEM medium without the antibody. Before transfection, discard the medium and add 1 mL of fresh DMEM medium without the antibody. pLVX-EF1a-CD79b-IRES-puro (the coding sequence of CD79b protein (SEQ ID NO: 26) was inserted between the restriction 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 at a ratio of 2:1:1, and then 12 μg of polyetherimide (PEI, Polysciences Co., Ltd.) was added. The obtained CD79b protein has the amino acid sequence shown in SEQ ID NO: 27; after mixing, it was allowed to stand for 16 minutes, and then all the liquid was added to the above-mentioned six-well plate with HEK293T cells. After culturing for 6 hours, the culture medium was discarded and fresh complete DMEM medium was added for culturing. 48 hours after transfection, the cell culture supernatant was collected and filtered through a 0.45 μm filter (Millipore) to obtain the viral supernatant. All the viral supernatant was added to a plate containing 1×10 4 Add polybrene (Sigma) to a final concentration of 4 μg / mL to a 6-well plate of CHO-K1 cells and culture for 12 hours. Discard the supernatant and add fresh complete DMEM medium. The resulting cells are CHO-K1-CD79b cells.

[0176] ATGGCTAGACTTGCCTTGTCTCCAGTGCCTTCTCATTGGATGGTCGCATTGCTGCTCTTGTTGAGTGCAGAACCAGTGCCCGCAGCAAGGTCTGAGGACAGGTACCGAAATCCAAAGGGCTCTGCTTGCAGCCGGATTTGGCAGAGTCCCCGGTTCATTGCCCGGAAACGTGGCTTCACTGTTAAGATGCATTGCTATATGAATTCAGCAAGCGGGAATGTTTCCTGGCTGTGGAAGCAGGAGATGGACGAGAACCCCCAACAGCTCAAGTTGGAGAAGGGGCGCATGGAAGAGAGCCAGAATGAATCCTTGGCCACATTGACTATCCAGGGGATTAGATTCGAGGATAACGGCATTTACTTCTGTCAGCAAAAATGTAATAACACATCTGAAGTGTATCAGGGTTGTGGTACTGAGCTGCGAGTCATGGGATTCTCTACTCTCGCTCAGTTGAAGCAGCGGAACACTCTGAAGGACGGCATCATCATGATCCAGACTCTCCTTATCATTCTCTTCATTATCGTGCCCATCTTCCTGCTCCTCGACAAGGATGACTCTAAGGCCGGAATGGAAGAAGATCATACCTACGAGGGTCTTGACATCGACCAGACAGCCACCTACGAGGACATCGTAACTTTGCGGACTGGAGAAGTGAAGTGGTCAGTGGGAGAGCACCCCGGGCAGGAA(SEQ ID NO:26)。

[0177] MARLALSPVPSHWMVALLLLLSAEPVPAARSEDRYRNPKGSACSRIWQSPRFIARKRGFTVKMHCYMNSASGNVSWLWKQEMDENPQQLKLEKGRMEESQNESLATLTIQGIRFEDNGIYFCQQKCNNTSEVYQGCGTELRVMGFSTLAQLKQRNTLKDGIIMIQTLLIILFIIVPIFLLLDKDDSKAGMEEDHTYEGLDIDQTATYEDIVTLRTGEVKWSVGEHPGQE(SEQ ID NO:27)。

[0178] CHO-K1-CD79b cells were diluted to 1×10 6 1 mL of PBS was added to the EP tube at a volume of 90 μL / tube. 10 μL / tube of rat serum was added and the cells were blocked at 4°C for 30 min. A series of concentration gradients (0.1, 1, 10, 30, 100, and 300 μg / mL) of CD3×CD79b bispecific antibody and hIgG (control IgG1, Biolegend, QA16A12) were added at 10 μL / tube and incubated at 4°C for 30 min. After incubation, 1 mL of PBS was added to the EP tube and the cells were centrifuged at 100 × g for 5 min at 4°C. The supernatant was discarded and the pellet was washed once with PBS. After centrifugation, the supernatant was discarded and the cells were resuspended in 100 μL / tube of PBS. After resuspension, 1 μL / tube of Alexa-647-labeled rat anti-human Fc secondary antibody (Biolegend, M1310G05) was added and incubated at 4°C for 30 min in the dark. Wash twice with PBS, centrifuge and discard the supernatant. Resuspend the cells with 200 μL / tube PBS and detect them by flow cytometry. The specific experimental results are as follows Figure 5 As shown, it is further demonstrated that the bispecific antibody CD3×CD79b of the present invention can bind to CHO-K1-CD79b cells.

[0179] Example 6: Identification of Bispecific Antibodies Promoting Activation of Jurkat-NFAT-Lucia Reporter Cells

[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 CD79b on the surface of target cells and CD3 on the surface of effector cells to promote T cell activation. The strength of the relative chemiluminescence signal (RLU) is used to determine the ability of the bispecific antibody to bridge target cells and T cells, thereby activating T cells.

[0181] (1) The CHO-K1-CD79b cells obtained in Example 5 were diluted to 1×10 5 / mL, added to a 96-well plate, with an addition volume of 100 μL / well.

[0182] (2) The CD3×CD79b 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, using complete RPMI-1640 medium and added to the 96-well plate containing CHO-K1-CD79b cells in step (1) at a volume of 20 μL / well.

[0183] (3) Jurkat-NFAT-Lucia cells (Invivogen, jktl-nfat) were diluted to 1.25×10 5 / mL, and added to the 96-well plate containing the bispecific antibody in step (2) at a volume of 80 μL / well.

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

[0185] (5) 50 μL of the culture supernatant obtained in step (4) was aspirated and added to a 96-well plate. Luciferase substrate was then added to the plate at a volume of 50 μL / well.

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

[0187] Specific experimental results such as Figure 6 As shown, it is further shown that the bispecific antibody CD3×CD79b of the present invention can bridge target cells (ie, CHO-K1-CD79b cells) and T cells (Jurkat-NFAT-lucia cells), and promote T cell activation.

[0188] Example 7: Bispecific antibodies promote PBMC killing of tumor cells

[0189] This example tests the effect of the bispecific antibody obtained in Example 1 on the killing of A375-CD79b tumor cells by PBMC. The test is performed by constructing a reaction system of tumor cells + PBMC + bispecific antibodies at different concentrations. The specific experimental procedures are as follows:

[0190] HEK293T cells were grown at 5×10 5Plate cells / well in a six-well plate and culture overnight in DMEM medium without the antibody. Before transfection, discard the medium and add 1 mL of fresh DMEM medium without the antibody. pLVX-EF1a-CD79b-IRES-puro (the coding sequence of CD79b protein (SEQ ID NO: 26) was inserted between the restriction 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 at a ratio of 2:1:1, and then 12 μg of polyetherimide (PEI, Polysciences Co., Ltd.) was added. The obtained CD79b protein has the amino acid sequence shown in SEQ ID NO: 27; after mixing, it was allowed to stand for 16 minutes, and then all the liquid was added to the above-mentioned six-well plate with HEK293T cells. After culturing for 6 hours, the culture medium was discarded and fresh complete DMEM medium was added for culturing. 48 hours after transfection, the cell culture supernatant was collected and filtered through a 0.45 μm filter (Millipore) to obtain the viral supernatant. All the viral supernatant was added to a plate containing 1×10 4 A375 cells were cultured in a 6-well plate with polybrene (Sigma) at a final concentration of 4 μg / mL and incubated for 12 hours. The supernatant was then discarded and fresh complete DMEM medium was added. The resulting cells are A375-CD79b 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) A375-CD79b cells were diluted to 2×10 5 / mL, and added to the RTCA plate obtained in step (1) at a volume of 50 μL / well, and then the cell coefficient was detected using the xCELLigence RTCA TP device at 37°C and 5% CO2 for 24 hours;

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

[0194] (4) PBMC (Sai Li Biotechnology) were diluted to 1.25×10 6 pcs / mL, added to the RTCA plate obtained in step (3), with an addition volume of 80 μL / well;

[0195] (5) The reaction system obtained in step (4) was incubated at 37°C and 5% CO2 for 48 hours using an xCELLigence RTCA TP instrument to detect the cell coefficient.

[0196] Specific experimental results such as Figure 7 As shown, it is further shown that the bispecific antibody of the present invention can promote PBMC to kill CD79b-expressing positive tumor cells.

[0197] It can be seen from the above experimental results that the bispecific antibody obtained by the present invention 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 this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0199] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. An antibody, characterized in that include: a first antigen-binding region, wherein the first antigen-binding region has CD3 binding activity and has the amino acid sequence shown in SEQ ID NO: 11; A second antigen-binding region, wherein the second antigen-binding region has CD79b binding activity, the second antigen-binding region comprises a first polypeptide and a second polypeptide, the first polypeptide and the second polypeptide are connected by an interchain disulfide bond; the first polypeptide has the amino acid sequence as shown in SEQ ID NO: 21, and the second polypeptide has the amino acid sequence as shown 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 according to claim 2, characterized in that The nucleic acid molecule is a DNA molecule.

4. An expression vector, characterized in that Carrying the nucleic acid molecule according to any one of claims 2 to 3.

5. The expression vector according to claim 4, characterized in that The expression vector is a non-pathogenic viral vector.

6. The expression vector according to claim 5, characterized in that The expression vector is an adenoviral vector, a lentiviral vector or a retroviral vector.

7. A method for preparing the antibody according to claim 1, characterized in that: include: Introducing the expression vector according to any one of claims 4 to 6 into a cell; The cells are cultured under conditions suitable for protein expression and secretion to obtain the antibodies.

8. The method according to claim 7, characterized in that The cells are eukaryotic cells.

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

10. The recombinant cell according to claim 9, characterized in that The recombinant cell is obtained by introducing the expression vector according to any one of claims 4 to 6 into a host cell.

11. The recombinant cell according to claim 9, characterized in that The recombinant cell is a eukaryotic cell.

12. The recombinant cell according to claim 9, characterized in that The recombinant cell is a mammalian cell.

13. A pharmaceutical composition, characterized in that include: The antibody according to claim 1, the nucleic acid molecule according to any one of claims 2 to 3, the expression vector according to any one of claims 4 to 6, or the recombinant cell according to any one of claims 9 to 12.

14. The pharmaceutical composition according to claim 13, characterized in that The pharmaceutical composition further includes pharmaceutically acceptable excipients.

15. A kit, characterized in that include: The antibody according to claim 1, the nucleic acid molecule according to any one of claims 2 to 3, the expression vector according to any one of claims 4 to 6, or the recombinant cell according to any one of claims 9 to 12.

16. Use of the antibody according to claim 1, the nucleic acid molecule according to any one of claims 2 to 3, the expression vector according to any one of claims 4 to 6, the recombinant cell according to any one of claims 9 to 12, or the pharmaceutical composition according to any one of claims 13 to 14 in the preparation of a medicament for treating or preventing cancer; The cancer is selected from at least one of B-cell non-Hodgkin's lymphoma, B-cell multiple myeloma and B-cell chronic lymphocytic leukemia.

17. Use of the antibody according to claim 1, the nucleic acid molecule according to any one of claims 2 to 3, the expression vector according to any one of claims 4 to 6, or the recombinant cell according to any one of claims 9 to 12 in preparing a kit for detecting CD3 and / or CD79b.

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