CD3 and CD19 targeting bispecific antibody and application thereof

Abstract

The invention provides a bispecific antibody targeting CD3 and CD19 and application thereof, the bispecific antibody comprises: a first binding region having CD3 binding activity; and a second binding region, wherein the second binding region has CD19 binding activity. The bispecific antibody provided by the invention is extremely weak in binding with T cells, strong in binding with tumor cells, relatively high in anti-cancer activity, low in risk of causing cytokine storm, and good in safety, and has an important application value for drug development and tumor prevention and / or treatment.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a bispecific antibody targeting CD3 and CD19 and its applications. Background Technology

[0002] Cancer is a disease that affects human health and survival. In recent years, immunotherapy, including tumor-targeting antibodies, immune checkpoint antibodies, and bispecific antibodies, has become a new hotspot and a source of new hope in the fight against cancer. Immunotherapy, represented by PD-1 / L1, has shown great potential. However, even with PD-1 / L1 therapy, which currently has the broadest approved indications, its overall response rate still needs improvement, and many more patients cannot benefit from it. T cells recognize neoantigens, which are antigens of tumor gene mutations, through their surface T cell receptors (TCRs). Some tumors have low gene mutation frequencies and few types of neoantigens, and are called "cold tumors." Current immune checkpoint therapies, such as PD-1 / L1 therapy, achieve their anti-cancer purpose by restoring the function of T cells themselves. However, in "cold tumors," T cells cannot effectively recognize the tumor, making immune checkpoint therapy ineffective against "cold tumors."

[0003] CD3-based bispecific antibodies (hereinafter referred to as "CD3 bispecific antibodies") recruit T cells to the tumor site, bridging T cells and the tumor, promoting T cell activation and tumor killing. These bispecific antibodies do not require neoantigens and can guide T cells to kill "cold tumors." After binding to T cells and tumor cells, CD3 bispecific antibodies trigger strong activation signals, thus to some extent "ignoring" the inhibitory signals of immune checkpoint molecules. However, CD3 bispecific antibodies also promote the production of pro-inflammatory cytokines, such as TNFα and IL-6, triggering a cytokine storm and excessive immune responses. Therefore, CD3-based bispecific antibodies have promising clinical applications, but their safety needs further improvement.

[0004] In healthy individuals, CD19 is expressed on the surface of normal B cells. In patients with autoimmune diseases, CD19 is expressed on the surface of pathogenic memory B cells, and in patients with myeloma, CD19 is expressed on the surface of tumor cells. Targeting CD19 has significant clinical value in both cancer treatment and the treatment of autoimmune diseases.

[0005] One approach to addressing the safety concerns of bispecific antibodies is to increase their affinity for tumor targets while simultaneously weakening their binding to T cells. This allows for greater distribution of the bispecific antibody drug to the tumor site, increasing local drug concentration, reducing peripheral drug concentration, and minimizing off-target toxicity. Therefore, there is an urgent clinical need to develop bispecific antibodies with improved safety and higher clinical value. Summary of the Invention

[0006] The present invention aims to solve at least one of the technical problems existing in the prior art.

[0007] Therefore, in a first aspect, the present invention provides a bispecific antibody. According to an embodiment of the present invention, the bispecific antibody comprises: a first binding region having CD3 binding activity; and a second binding region having CD19 binding activity. The bispecific antibody of the present invention binds very weakly to T cells and strongly to tumor cells, exhibiting high anticancer activity and a low risk of triggering a cytokine storm, demonstrating good safety and significant application value for drug development and tumor prevention and / or treatment.

[0008] According to embodiments of the present invention, the above-mentioned bispecific antibody may further include at least one of the following additional technical features: According to an embodiment of the present invention, the first bonding region includes a first heavy chain variable region and a first light chain variable region, the first heavy chain variable region and the first light chain variable region being connected. According to an embodiment of the present invention, the first binding region includes a CDR selected from at least one of the following: heavy chain variable region CDR: amino acid sequence of SEQ ID NO:1~3 or its conservative modified form; light chain variable region CDR: amino acid sequence of SEQ ID NO:4~6 or its conservative modified form.

[0009] According to an embodiment of the present invention, the first bonding region includes: The heavy chain variable region CDR1 having the amino acid sequence shown in SEQ ID NO:1 or an amino acid sequence of its conserved modified form; The heavy chain variable region CDR2 having the amino acid sequence shown in SEQ ID NO:2 or an amino acid sequence of its conserved modified form; The heavy chain variable region CDR3 having the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence of its conserved modified form; The light chain variable region CDR1 having the amino acid sequence shown in SEQ ID NO:4 or an amino acid sequence of its conserved modified form; The light chain variable region CDR2 having the amino acid sequence shown in SEQ ID NO:5 or its conserved modified form; and The light chain variable region CDR3 having the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence of its conserved modified form.

[0010] According to an embodiment of the present invention, the first bonding region includes a first heavy chain frame region and / or a first light chain frame region.

[0011] According to an embodiment of the present invention, at least a portion of the first heavy chain framework region and / or the first light chain framework region is derived from a human antibody.

[0012] According to an embodiment of the present invention, the first heavy chain variable region has an amino acid sequence as shown in SEQ ID NO:20 or an amino acid sequence having at least 90% identity with it; and / or the first light chain variable region has an amino acid sequence as shown in SEQ ID NO:21 or an amino acid sequence having at least 90% identity with it.

[0013] According to an embodiment of the present invention, the second bonding region includes a second bonding region 1 and a second bonding region 2, the second bonding region 1 includes a second heavy chain variable region 1 and a second light chain variable region 1, and the second bonding region 2 includes a second heavy chain variable region 2 and a second light chain variable region 2.

[0014] According to an embodiment of the present invention, the second binding region 1 and the second binding region 2 include a CDR selected from at least one of the following: heavy chain variable region CDR: amino acid sequence of SEQ ID NO: 7-9 or its conserved modified form; light chain variable region CDR: amino acid sequence of SEQ ID NO: 10-12 or its conserved modified form.

[0015] According to an embodiment of the present invention, the second bonding region 1 and the second bonding region 2 include: The heavy chain variable region CDR1 having the amino acid sequence shown in SEQ ID NO:7 or an amino acid sequence of its conserved modified form; The heavy chain variable region CDR2 having the amino acid sequence shown in SEQ ID NO:8 or an amino acid sequence of its conserved modified form; The heavy chain variable region CDR3 having the amino acid sequence shown in SEQ ID NO:9 or an amino acid sequence of its conserved modified form; The light chain variable region CDR1 having the amino acid sequence shown in SEQ ID NO:10 or an amino acid sequence of its conserved modified form; The light chain variable region CDR2 having the amino acid sequence shown in SEQ ID NO:11 or its conserved modified form; and The light chain variable region CDR3 having the amino acid sequence shown in SEQ ID NO:12 or its conserved modified form.

[0016] According to an embodiment of the present invention, the second bonding region includes a second heavy chain frame region and / or a second light chain frame region.

[0017] According to an embodiment of the present invention, at least a portion of the second heavy chain framework region and / or the second light chain framework region is derived from a human antibody.

[0018] According to an embodiment of the present invention, the second heavy chain variable regions 1 and 2 have an amino acid sequence as shown in SEQ ID NO:16 or an amino acid sequence having at least 90% identity with it; and / or the second light chain variable regions 1 and 2 have an amino acid sequence as shown in SEQ ID NO:17 or an amino acid sequence having at least 90% identity with it.

[0019] According to an embodiment of the present invention, the second binding region 1 and the second binding region 2 are Fab fragments. Those skilled in the art will understand that the second binding region 1 and the second binding region 2 can be scFab fragments, as long as it does not affect the function of the bispecific antibody.

[0020] According to an embodiment of the present invention, the second binding region 1 further includes a first CL segment and a first CH1 segment, wherein the second heavy chain variable region 1 is connected to the first CH1 segment, and the second light chain variable region 1 is connected to the first CL segment.

[0021] According to an embodiment of the present invention, the second binding region 1 has the amino acid sequence shown in SEQ ID NO:18 or an amino acid sequence having at least 90% identity with it, and the amino acid sequence shown in SEQ ID NO:19 or an amino acid sequence having at least 90% identity with it.

[0022] According to an embodiment of the present invention, the second binding region 2 further includes a second CL segment and a second CH1 segment, wherein the second heavy chain variable region 2 is connected to the second CH1 segment, and the second light chain variable region 2 is connected to the second CL segment.

[0023] According to an embodiment of the present invention, the second binding region 2 has the amino acid sequence shown in SEQ ID NO:18 or an amino acid sequence having at least 90% identity with it, and the amino acid sequence shown in SEQ ID NO:19 or an amino acid sequence having at least 90% identity with it.

[0024] According to an embodiment of the present invention, the second bonding region 1 is connected by covalent bonds, and / or the second bonding region 2 is connected by covalent bonds.

[0025] According to an embodiment of the present invention, the covalent bond is a disulfide bond.

[0026] According to an embodiment of the present invention, the bispecific antibody further includes a first Fc region and / or a second Fc region.

[0027] According to an embodiment of the present invention, the first Fc region and the second Fc region are connected by a knock-into-hole structure.

[0028] According to an embodiment of the present invention, the second bonding region 1 is connected to the first bonding region, and the first bonding region is connected to the first Fc region.

[0029] According to an embodiment of the present invention, the second bonding region 2 is also connected to the second Fc region.

[0030] According to an embodiment of the present invention, in the first bonding region, the C end of the first light chain variable region is connected to the N end of the first heavy chain variable region, or the N end of the first light chain variable region is connected to the C end of the first heavy chain variable region.

[0031] According to an embodiment of the present invention, in the second binding region 1, the C end of the second light chain variable region 1 is connected to the N end of the first CL segment, and the C end of the second heavy chain variable region 1 is connected to the N end of the first CH1 segment; or in the second binding region 1, the C end of the second light chain variable region 1 is connected to the N end of the first CH1 segment, and the C end of the second heavy chain variable region 1 is connected to the N end of the first CL segment.

[0032] According to an embodiment of the present invention, in the second binding region 2, the C-end of the second light chain variable region 2 is connected to the N-end of the second CH1 segment, and the C-end of the second heavy chain variable region 2 is connected to the N-end of the second CL segment; or in the second binding region 2, the C-end of the second light chain variable region 2 is connected to the N-end of the second CL segment, and the C-end of the second heavy chain variable region 2 is connected to the N-end of the second CH1 segment.

[0033] According to an embodiment of the present invention, the C-terminus of the second light chain variable region 1 is connected to the N-terminus of the first CL segment; and / or The C-terminus of the second heavy chain variable region 1 is connected to the N-terminus of the first CH1 segment, the C-terminus of the first CH1 segment is connected to the N-terminus of the first heavy chain variable region, the C-terminus of the first heavy chain variable region is connected to the N-terminus of the first light chain variable region, and the C-terminus of the first light chain variable region is connected to the N-terminus of the first Fc region; and / or The C-terminus of the second heavy chain variable region 2 is connected to the N-terminus of the second CH1 segment, and the C-terminus of the second CH1 segment is connected to the second Fc region; and / or The C-terminus of the second light chain variable region 2 is connected to the N-terminus of the second CL segment.

[0034] According to an embodiment of the present invention, the first binding region further includes a first linker peptide, wherein the C-terminus of the first light chain variable region is connected to the N-terminus of the first linker peptide, and the C-terminus of the first linker peptide is connected to the N-terminus of the first heavy chain variable region; or, the N-terminus of the first light chain variable region is connected to the C-terminus of the first linker peptide, and the N-terminus of the first linker peptide is connected to the C-terminus of the first heavy chain variable region.

[0035] According to an embodiment of the present invention, the first binding region is a single-chain antibody.

[0036] According to an embodiment of the present invention, the first binding region has the amino acid sequence shown in SEQ ID NO:22 or an amino acid sequence having at least 90% identity with it.

[0037] According to an embodiment of the present invention, the first binding region and the second binding region 1 are connected by a second linker peptide.

[0038] According to an embodiment of the present invention, the C-terminus of the second light chain variable region 1 is connected to the N-terminus of the first CL segment; and / or The C-terminus of the second heavy chain variable region 1 is connected to the N-terminus of the first CH1 fragment; the C-terminus of the first CH1 fragment is connected to the N-terminus of the second linker peptide; the C-terminus of the second linker peptide is connected to the N-terminus of the first heavy chain variable region; the C-terminus of the first heavy chain variable region is connected to the N-terminus of the first linker peptide; the C-terminus of the first linker peptide is connected to the N-terminus of the first light chain variable region; and the C-terminus of the first light chain variable region is connected to the N-terminus of the first Fc region; and / or The C-terminus of the second heavy chain variable region 2 is connected to the N-terminus of the second CH1 region, and the C-terminus of the second CH1 region is connected to the N-terminus of the second Fc region; and / or The C-terminus of the second light chain variable region 2 is connected to the N-terminus of the second CL segment.

[0039] According to an embodiment of the present invention, the first binding region and the first Fc region are connected by a third linker peptide. Those skilled in the art will understand that the first binding region and the first Fc region can be indirectly linked by the linker peptide, or they can be directly linked, as long as the linker peptide does not affect the function of the bispecific antibody.

[0040] According to an embodiment of the present invention, the C-terminus of the second light chain variable region 1 is connected to the N-terminus of the first CL segment; and / or The C-terminus of the second heavy chain variable region 1 is connected to the N-terminus of the first CH1 fragment; the C-terminus of the first CH1 fragment is connected to the N-terminus of the second linker peptide; the C-terminus of the second linker peptide is connected to the N-terminus of the first heavy chain variable region; the C-terminus of the first heavy chain variable region is connected to the N-terminus of the first linker peptide; the C-terminus of the first linker peptide is connected to the N-terminus of the first light chain variable region; the C-terminus of the first light chain variable region is connected to the N-terminus of the third linker peptide; and the C-terminus of the third linker peptide is connected to the N-terminus of the first Fc region; and / or The C-terminus of the second heavy chain variable region 2 is connected to the N-terminus of the second CH1 region, and the C-terminus of the second CH1 region is connected to the N-terminus of the second Fc region; and / or The C-terminus of the second light chain variable region 2 is connected to the N-terminus of the second CL segment.

[0041] According to an embodiment of the present invention, the first Fc region has at least one of the following mutation sites relative to the amino acid sequence of the Fc fragment of human wild-type IgG1: L234A, L235A, S354C, and T366W.

[0042] Optionally, the second Fc region has at least one of the following mutation sites relative to the amino acid sequence of the Fc fragment of human wild-type IgG1: L234A, L235A, Y349C, T366S, L368A, and Y407V.

[0043] According to an embodiment of the present invention, the first Fc region has the amino acid sequence shown in SEQ ID NO:24.

[0044] According to an embodiment of the present invention, the second Fc region has the amino acid sequence shown in SEQ ID NO:25.

[0045] According to an embodiment of the present invention, the linker peptide is a flexible linker peptide.

[0046] According to an embodiment of the present invention, the amino acid sequence of the first linker peptide is (GGGGS)n, where n is an integer greater than or equal to 1, preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12, more preferably 1, 2 or 3.

[0047] According to an embodiment of the present invention, the amino acid sequence of the second linker peptide is shown in SEQ ID NO:27.

[0048] According to an embodiment of the present invention, the bispecific antibody comprises the amino acid sequence shown in any one of SEQ ID NO: 13, 14, and 15.

[0049] In a second aspect, the present invention provides a nucleic acid molecule. According to an embodiment of the invention, the nucleic acid molecule encodes the bispecific antibody described in the first aspect. The nucleic acid molecule according to embodiments of the present invention can effectively encode the aforementioned bispecific antibody.

[0050] According to an embodiment of the present invention, the nucleic acid molecule is DNA.

[0051] It should be noted that those skilled in the art will understand that the nucleic acid molecules mentioned herein actually include any one or both of the complementary double strands. For convenience, although only one strand is given in most cases in this specification and claims, the other complementary strand is also disclosed. Furthermore, the nucleic acid sequences in this application include DNA or RNA forms; disclosure of one implies that the other is also disclosed.

[0052] In a third aspect, the present invention provides an expression vector. According to an embodiment of the invention, the vector carries the nucleic acid molecule described in the second aspect. When the nucleic acid molecule is ligated to the vector, the nucleic acid molecule can be directly or indirectly linked to control elements on the vector, as long as these control elements can control the translation and expression of the nucleic acid molecule. These control elements can be directly derived from the vector itself or are exogenous, i.e., not derived from the vector itself. Of course, the nucleic acid molecule and control elements need to be operably linked. In this context, "operably linked" means ligating a foreign gene to the vector so that control elements within the vector, such as transcriptional control sequences and translational control sequences, can perform their intended function of regulating the transcription and translation of the foreign gene. Commonly used vectors include plasmids, bacteriophages, etc. After the expression vector according to some specific embodiments of the present invention is introduced into suitable recipient cells, the aforementioned bispecific antibody expression can be effectively achieved under the mediation of a regulatory system, thereby enabling the large-scale in vitro production of bispecific antibodies.

[0053] According to an embodiment of the present invention, the expression vector is a eukaryotic expression vector or a prokaryotic expression vector.

[0054] According to an embodiment of the present invention, the expression vector is a plasmid expression vector.

[0055] In a fourth aspect, the present invention provides a recombinant cell. According to an embodiment of the invention, the recombinant cell carries the nucleic acid molecule described in the second aspect or expresses the bispecific antibody described in the first aspect. Using this recombinant cell, under suitable conditions, the aforementioned bispecific antibody can be effectively expressed intracellularly.

[0056] It should be noted that the "suitable conditions" mentioned in this application specification refer to conditions suitable for the expression of the bispecific antibody described in this invention. Those skilled in the art will readily understand that suitable conditions for the expression of the bispecific 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, and suitable cell culture time. The term "suitable conditions" is not particularly limited, and those skilled in the art can optimize the optimal conditions for the expression of the bispecific antibody based on the specific environment of their laboratory.

[0057] According to an embodiment of the present invention, the recombinant cells are obtained by introducing the expression vector described in the third aspect into a host cell.

[0058] According to an embodiment of the present invention, the recombinant cells are eukaryotic cells.

[0059] According to an embodiment of the present invention, the recombinant cell is a mammalian cell.

[0060] In a fifth aspect, the present invention provides a pharmaceutical composition. According to embodiments of the invention, it comprises at least one of the following: The bispecific antibody described in the first aspect; The nucleic acid molecules described in the second aspect; The expression carrier described in the third aspect; or The recombinant cells described in the fourth aspect.

[0061] According to an embodiment of the present invention, the pharmaceutical composition further comprises pharmaceutically acceptable excipients.

[0062] The pharmaceutical compositions of this invention can be administered by any acceptable method of administration. The pharmaceutical compositions of this invention can be formulated into solid, semi-solid, liquid, or gaseous forms, such as injections or lyophilized powders, and current methods for preparing these dosage forms are known or readily apparent to those skilled in the art. Typical routes of administration of such pharmaceutical compositions include, but are not limited to, oral, topical, transdermal, inhalation, parenteral, sublingual, oral, rectal, vaginal, and intranasal routes. The term parenteral, as used herein, includes subcutaneous injection, intravenous, intramuscular, intradermal, intrasternal injection, or infusion techniques. The pharmaceutical compositions of this invention are formulated to allow the bioactive components contained therein to be bioavailable after administration to a subject.

[0063] In a sixth aspect, the present invention provides the use of the bispecific antibody of the first aspect, the nucleic acid molecule of the second aspect, the expression vector of the third aspect, the recombinant cell of the fourth aspect, or the pharmaceutical composition of the fifth aspect in the preparation of a medicament for the prevention and / or treatment of tumors and / or autoimmune diseases.

[0064] According to an embodiment of the present invention, the tumor includes at least one selected from lymphoma, myeloma, colorectal cancer, lung cancer, pancreatic cancer, renal cell carcinoma, prostate cancer, and melanoma.

[0065] According to embodiments of the present invention, the autoimmune disease includes at least one of the following: systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, dermatomyositis, autoimmune hemolytic anemia, thyroid autoimmune disease, ulcerative colitis, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, pulmonary hemorrhage nephritis syndrome, pemphigus vulgaris, bullous pemphigoid, primary biliary cirrhosis, multiple cerebral sclerosis, and acute idiopathic polyneuritis.

[0066] In a seventh aspect, the present invention provides a kit. According to an embodiment of the invention, the kit comprises: the bispecific antibody described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, or the recombinant cells described in the fourth aspect. As is known prior, the aforementioned bispecific antibody can specifically bind to CD3 and / or CD19. Furthermore, under suitable conditions, the nucleic acid molecule, expression vector, or recombinant cells can all express the bispecific antibody. Further, the kit containing the above substances can effectively bind to CD3 and / or CD19, and can be used for the effective detection of CD3 and / or CD19. The kit can be used for scientific research, such as for qualitative or quantitative detection of CD3 and / or CD19 in biological samples, and can also be used to determine the state of an object, such as determining whether the CD3 and / or CD19 level of the object is too high or too low than normal after obtaining the object's CD3 and / or CD19 level. The biological sample can be at least one of tissues, cells, blood, plasma, serum, sweat, feces, and urine.

[0067] In an eighth aspect, the present invention provides the use of the bispecific antibody described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, or the recombinant cell described in the fourth aspect in the preparation of a kit for detecting CD3 and / or CD19. As previously known, the aforementioned bispecific antibody can specifically bind to CD3 and / or CD19. Furthermore, under suitable conditions, the nucleic acid molecule, expression vector, or recombinant cell can all express the bispecific antibody. Further, the kit containing the above substances can effectively bind to CD3 and / or CD19 and can be used for the effective detection of CD3 and / or CD19. The kit can be used in scientific research, such as for qualitative or quantitative detection of CD3 and / or CD19 in biological samples, and can also be used to determine the state of an object, such as determining whether the CD3 and / or CD19 level of the object is too high or too low than normal after obtaining the object's CD3 and / or CD19 level. The biological sample can be at least one of tissues, cells, blood, plasma, serum, sweat, feces, and urine.

[0068] In a ninth aspect, the present invention provides a method for treating and / or preventing tumors and / or autoimmune diseases. According to an embodiment of the invention, the method comprises administering to a subject a pharmaceutically acceptable amount of the aforementioned bispecific antibody or pharmaceutical composition. The method of the present invention is effective in treating or preventing CD3 and / or CD19-mediated diseases, such as tumors and autoimmune diseases.

[0069] The effective amount of the bispecific antibody or pharmaceutical composition described in this invention can vary depending on the administration method and the severity of the disease to be treated. A preferred effective amount can be determined by those skilled in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to: pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, and half-life; the severity of the disease to be treated, the subject's weight, the subject's immune status, and the route of administration. For example, due to the urgency of the treatment condition, several separate doses may be administered daily, or the dose may be reduced proportionally.

[0070] The bispecific antibody or pharmaceutical composition of the present invention can be incorporated into a drug suitable for parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). These drugs can be prepared in various forms, such as liquid, semi-solid, and solid dosage forms, including but not limited to liquid solutions (e.g., injection solutions and infusion solutions) or lyophilized powders. Typical drugs are in the form of injection solutions or infusion solutions. The aforementioned bispecific antibody or pharmaceutical composition can be administered by intravenous infusion or injection, or by intramuscular or subcutaneous injection.

[0071] According to an embodiment of the present invention, the tumor includes at least one selected from lymphoma, myeloma, colorectal cancer, lung cancer, pancreatic cancer, renal cell carcinoma, prostate cancer, and melanoma.

[0072] According to embodiments of the present invention, the autoimmune disease includes at least one of the following: systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, dermatomyositis, autoimmune hemolytic anemia, thyroid autoimmune disease, ulcerative colitis, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, pulmonary hemorrhage nephritis syndrome, pemphigus vulgaris, bullous pemphigoid, primary biliary cirrhosis, multiple cerebral sclerosis, and acute idiopathic polyneuritis.

[0073] According to an embodiment of the present invention, the method is administered via subcutaneous injection or intravenous injection.

[0074] In a tenth aspect of the invention, the invention provides the use of the aforementioned bispecific antibody or the aforementioned pharmaceutical composition in the prevention and / or treatment of tumors and / or autoimmune diseases.

[0075] According to an embodiment of the present invention, the tumor includes at least one selected from lymphoma, myeloma, rectal cancer, lung cancer, pancreatic cancer, renal cell carcinoma, prostate cancer, and melanoma.

[0076] According to embodiments of the present invention, the autoimmune disease includes at least one of the following: systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, dermatomyositis, autoimmune hemolytic anemia, thyroid autoimmune disease, ulcerative colitis, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, pulmonary hemorrhage nephritis syndrome, pemphigus vulgaris, bullous pemphigoid, primary biliary cirrhosis, multiple cerebral sclerosis, and acute idiopathic polyneuritis.

[0077] In an eleventh aspect of the present invention, the present invention provides the aforementioned bispecific antibody or the aforementioned pharmaceutical composition for the prevention and / or treatment of tumors and / or autoimmune diseases.

[0078] According to an embodiment of the present invention, the tumor includes at least one selected from lymphoma, myeloma, colorectal cancer, lung cancer, pancreatic cancer, renal cell carcinoma, prostate cancer, and melanoma.

[0079] In a twelfth aspect, the present invention provides a method for detecting CD3 and / or CD19, the method comprising: contacting a sample to be tested with the aforementioned bispecific antibody or the aforementioned kit to form an immune complex.

[0080] According to an embodiment of the present invention, based on the signal of the immune complex, it is determined whether the sample to be tested contains CD3 and / or CD19, or the content of CD3 and / or CD19.

[0081] According to an embodiment of the present invention, the immune complex further includes a second antibody, which binds to the bispecific antibody.

[0082] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0083] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0084] Figure 1 This diagram shows a schematic representation of the CD3×CD19 bispecific antibody according to an embodiment of the present invention. Figure 2 This image shows the SPR results of the CD3×CD19 bispecific antibody binding to CD3 and CD19 proteins according to an embodiment of the present invention. Figure 3 This image shows the flow cytometry results of the binding of the CD3×CD19 bispecific antibody to Namalwa lymphoma cells according to an embodiment of the present invention. Figure 4 This image shows the flow cytometry results of the binding of the CD3×CD19 bispecific antibody to CD8 T cells according to an embodiment of the present invention. Figure 5 This image shows the flow cytometry results of CD3×CD19 antibody promoting the expression of the activation marker CD69 in CD4 T cells and CD8 T cells, according to an embodiment of the present invention. Figure 6 The figure shows the results of CD3×CD19 antibody promoting the secretion of pro-inflammatory cytokines IL-6 and TNFα by PBMCs under conditions without tumor cells, according to an embodiment of the present invention. Figure 7The figure shows the results of CD3×CD19 antibody promoting the secretion of pro-inflammatory cytokines IL-6 and TNFα by PBMCs under conditions with tumor cells, according to an embodiment of the present invention. Figure 8 This figure shows the results of CD3×CD19 antibody promoting PBMC killing of Namalwa lymphoma cells according to an embodiment of the present invention. Detailed Implementation

[0085] Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.

[0086] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0087] In this document, the terms “comprising” or “including” are open-ended expressions, meaning they include the contents specified in this invention but do not exclude other aspects.

[0088] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0089] In this article, the terms "bispecific antibody" and "double antibody" are synonymous, both referring to antibodies that can recognize two antigenic epitopes. This is a broad interpretation, and the specific structure is not limited, as long as it can recognize two antigenic epitopes. For example, such as Figure 1 The antibody shown.

[0090] In this paper, the term "knob into hole structure" refers to the formation of a button (hole) mutation in the CH3 region of the constant region of the antibody heavy chain, which facilitates heavy chain interlocking and the formation of a heterodimer. For example, this can be achieved by mutating the amino acids in the CH3 domain of the constant region of the human IgG1 heavy chain (T366S, L368A, Y407V, Y349C mutation in one chain, i.e., "hole"; and T366W, S354C mutation in the other chain, i.e., "knob").

[0091] In this paper, the term "fragment" refers to a target protein or polypeptide, and a target protein or polypeptide that has been truncated at an N-terminus (N-terminus) or C-terminus (C-terminus), and / or has an internal deletion.

[0092] In this paper, the terms “identity,” “homology,” or “similarity” are used to describe the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences relative to a reference sequence, determined by conventional methods, for example, see Ausubel et al., eds. (1995), Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN procedure (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research Institute)). Foundation, Washington, DC). Numerous algorithms exist for aligning sequences and determining sequence identity, including: the homology alignment algorithm of Needleman et al. (1970) J. Mol. Biol. 48: 443; the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2: 482; the similarity search method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 85: 2444; the Smith-Waterman algorithm (Meth. Mol. Biol. 70: 173-187 (1997); and the BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J. Mol. Biol. 215: 403-410). Computer programs utilizing these algorithms are also available, including but not limited to: ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul...). See, Meth.Enzym., 266:460-480 (1996); or GAP, BESTFIT, BLAST Altschul, etc., above, FASTA, and TFASTA, available in Genetics Computing Group (GCG) package, version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0093] In this paper, the term "at least 90% identity" means at least 90% identity with each reference sequence, which may be 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%.

[0094] In this document, the amino acid numbering of the IgG1 Fc portion is based on the EU numbering system. For example, position 366 refers to position 366 according to the EU numbering system; "T366W" means that threonine at position 366 according to the EU numbering system is replaced by tryptophan; and "L368A" means that leucine at position 368 according to the EU numbering system is replaced by alanine.

[0095] In this document, the term "expression vector" generally refers to a nucleic acid molecule capable of self-replication within a suitable host, transferring the inserted nucleic acid molecule to host cells and / or between host cells. The expression vector may include vectors primarily for inserting DNA or RNA into cells, vectors primarily for replicating DNA or RNA, and expression vectors primarily for transcription and / or translation of DNA or RNA. The expression vector also includes vectors having multiple of the aforementioned functions. The expression vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, by culturing suitable host cells containing the expression vector, the expression vector can produce the desired expression product.

[0096] In this document, the term "recombinant cell" generally refers to a cell in which the genetic material of a host cell is modified or recombined using genetic engineering or cell fusion techniques to obtain a unique trait with stable inheritance. The term "host cell" refers to a prokaryotic 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 acids (e.g., vectors) into cells using various techniques known in the art. Suitable host cells can be transformed or transfected with the DNA sequences of this invention and can be used for the expression and / or secretion of target proteins. Examples of suitable host cells that can be used in this invention include immortalized hybridoma cells, NS / O myeloma cells, 293 cells, Chinese hamster ovary (CHO) cells, HeLa cells, Cap cells (cells derived from human amniotic fluid), and CoS cells.

[0097] In this document, the term "pharmaceutical composition" generally refers to a unit dosage form and can be prepared by any method well known in the pharmaceutical industry. All methods involve the step of combining the active ingredient with a carrier constituting one or more adjunct components. Typically, compositions are prepared by uniformly and sufficiently combining the active compound with a liquid carrier, a finely chopped solid carrier, or both.

[0098] In this document, the term "pharmaceuticalally acceptable excipient" may include any solvent, solid excipient, diluent, or other liquid excipient, etc., suitable for the specific target dosage form. The use of any conventional excipients, except those that are incompatible with the compounds of the present invention, such as any adverse biological effects or harmful interactions with any other component of the pharmaceutically acceptable composition, is also within the scope of this invention.

[0099] In this document, the term "administration" refers to the introduction of a predetermined amount of a substance into a patient in a suitable manner. The bispecific antibody or pharmaceutical composition of the present invention can be administered via any common route, as long as it can reach the intended tissue. Various routes of administration are contemplated, including peritoneal, intravenous, intramuscular, subcutaneous, etc., but the present invention is not limited to these exemplified routes of administration. Preferably, the compositions of the present invention are administered via intravenous or subcutaneous injection.

[0100] In this document, the term "treatment" refers to the administration of a drug or compound to an individual to achieve a desired pharmacological and / or physiological effect. This effect may be preventative in terms of complete or partial prevention of a disease or its symptoms, and / or therapeutic in terms of partial or complete cure of a disease and / or adverse effects caused by the disease. As used herein, "treatment" encompasses diseases in mammals, particularly humans, including: (a) prevention of disease or the onset of a condition in an individual who is susceptible but has not yet been diagnosed with the disease; (b) inhibition of disease, such as blocking disease progression; or (c) relief of disease, such as reducing symptoms associated with the disease. As used herein, "treatment" encompasses any administration of a drug or compound to an individual to treat, cure, relieve, improve, reduce, or inhibit the individual's disease, including but not limited to administration of a drug containing a compound described herein to an individual in need.

[0101] In this document, the term "antibody" is used in the broadest sense and can include full-length monoclonal antibodies, multispecific antibodies, and chimeric antibodies, with no specific structural limitations, as long as they exhibit the desired biological activity. Antibody molecules typically consist of a lighter light chain and a heavier heavy chain linked by disulfide bonds. The amino-terminal (N-terminus) amino acid sequence of the peptide chain varies considerably and is called the variable region (V-terminus); the carboxyl-terminus (C-terminus) is relatively stable and varies little, and is called the constant region (C-terminus). The V-termini of the L-chain and H-chain are referred to as VL and VH, respectively. As used herein, the terms "complementarity-determining region," "CDR," or "CDRs" refer to highly variable regions of the heavy and light chains of an immunoglobulin, specifically regions containing one or more, or even all, of the major amino acid residues that contribute to the binding affinity of the antibody or its functional fragments to the antigens or epitopes it recognizes. In specific embodiments of this disclosure, CDRs refer to highly variable regions of the heavy and light chains of the antibody.

[0102] In this document, "Fc region" generally refers to the Fc region of IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM, including CH2, CH3 regions and optionally hinge regions. Preferably, the IgG, IgA1, IgA2, IgD, IgE, or IgM are derived from mouse, human, primate, or alpaca sources.

[0103] In this document, "conservatively modified amino acid sequences" refers to amino acid modifications that do not significantly affect or alter the binding properties of antibodies containing that amino acid sequence. These modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibodies of this invention using standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions involve replacing an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been identified in the art. These families include amino acids with basic side chains (such as lysine, arginine, and histidine), amino acids with acidic side chains (such as aspartic acid and glutamic acid), amino acids with uncharged polar side chains (such as glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), amino acids with nonpolar side chains (such as alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), amino acids with β-branched side chains (such as threonine, valine, and isoleucine), and amino acids with aromatic side chains (such as tyrosine, phenylalanine, tryptophan, and histidine). Therefore, one or more amino acid residues in the CDR region of the antibody of the present invention can be replaced by other amino acid residues from the same side chain family, and the retained function of the modified antibody can be tested using the functional assay methods described herein. Preferably, the number of conservative modifications does not exceed one or two.

[0104] In this article, the term "Fab antibody" or "Fab fragment" generally refers to an antibody or fragment containing only Fab molecules, which consists of the VH and CH1 of the heavy chain and the complete light chain, linked by a disulfide bond.

[0105] In this paper, the terms "single-chain antibody" and "scFv fragment" refer to antibodies or fragments formed by linking the variable regions of the antibody heavy chain and light chain through short peptides.

[0106] The nucleic acid or amino acid sequences used in this application are shown in Table 1: Table 1:

[0107] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0108] Unless otherwise specified, the practice of this disclosure will employ conventional techniques of cell biology, molecular biology (including recombinant technologies), microbiology, biochemistry, and immunology, which are within the capabilities of those skilled in the art. This technique is well explained in the literature, such as *Molecular Cloning: A Laboratory Manual*, 2nd edition (Sambrook et al., 1989); *Oligonucleotide Synthesis* (edited by M.J. Gait, 1984); *Animal Cell Culture* (edited by R.R. Freshney, 1987); *Methods in Enzymology* (Academic Press, Inc.); *Handbook of Experimental Immunology* (edited by D.M. Weir and C.C. Blackwell); *Gene Transfer Vectors for Mammalian Cells* (edited by J.M. Miller and M.P. Calos, 1987); *Current Protocols in Molecular Biology* (edited by F.M. Mausubel et al., 1987); and *PCR: The Polymerase Chain Reaction*. The references cited in the references are: "Reaction" (Mullis et al., ed., 1994) and "Current Protocols in Immunology" (JEColigan et al., ed., 2011), each of which is explicitly incorporated herein by reference.

[0109] In this embodiment of the invention, the nucleotide sequence used to prepare the expression vector can be obtained using conventional methods or conventional software based on its amino acid sequence.

[0110] Example 1: Antibody Production The specific experimental procedures for antibody production are as follows: (1) ExpiCHO cells (purchased from Thermo Fisher) were cultured using ExpiCHO Expression Medium (purchased from Thermo Fisher) and the cell concentration was adjusted to 6×10⁻⁶. 6 / mL, to obtain ExpiCHO cell solution. (2) Add pcDNA3.4 vector (synthesized by Nanjing GenScript) containing CD19 heavy chain-CD3 antibody, CD19 antibody heavy chain and CD19 antibody light chain to 2mL OptiSFM medium (purchased from Thermo Fisher) in a ratio of 1:1:1 to obtain solution a; (3) Add 160μL ExpiFectamineCHO transfection reagent (purchased from Thermo Fisher) to 2mL OptiSFM medium (purchased from Thermo Fisher) to obtain solution b. (4) Then mix solution a and solution b to obtain transfection mixture, and add the entire transfection mixture to 50mL ExpiCHO cell solution within 5 minutes. (5) After culturing at 37℃ and 5% CO2 for 1 day, add 8mL Feed and 300μL Enhancer (purchased from Thermo Fisher), and transfer to 32℃ and 5% CO2 for 9 days to harvest the culture supernatant, of which 8mL Feed is added on the 5th day. (6) The target antibodies CD3×CD19 and SP34×CD19 were obtained from the culture supernatant using a Protein A purification column (purchased from Nanomicro). Antibody conformations are shown in [reference needed]. Figure 1 The amino acid sequences are shown in Tables 1 and 2.

[0111] Table 2

[0112] Example 2: Antibody Affinity Detection Biacore is a method for analyzing biomolecular interactions based on the principle of optical surface plasmon resonance (SPR). It can not only detect the specific binding between antigens and antibodies, but also obtain crucial data in drug development, such as the binding rate constant (ka), dissociation rate constant (kd), and equilibrium dissociation constant (KD), thereby calculating the antibody affinity. The specific operation is as follows: In the Biacore 8K (Cytiva) system, the CD3×CD19 and SP34×CD19 antibodies described in Example 1 were diluted to 10 μg / mL with run buffer (HBS-EP) and conjugated to protein A (Cytiva, 29127556) chip at a flow rate of 10 μL / min. The kinetics and affinity data of antigen-antibody binding were detected at a flow rate of 30 μL / min, with a binding time of 120 s and a dissociation time of 800 s.

[0113] The results are as follows Figure 2 As shown, both CD3×CD19 and the bispecific antibody SP34×CD19 targeting the same target can bind to CD3E&D proteins and CD19 proteins (both purchased from Acro). The affinity of CD3×CD19 for binding to CD3 protein is 50 times weaker than that of the bispecific antibody SP34×CD19 targeting the same target, while the affinity of the two bispecific antibodies for binding to CD19 protein is similar.

[0114] Example 3: Bispecific antibody flow cytometry combined with experiment Flow cytometry was used to detect the binding properties of the CD3×CD19 and SP34×CD19 bispecific antibodies described in Example 1. The antibodies were added to the cells, and the strength of the signal after antibody addition was used to determine the binding properties between the antibodies and the cells. The specific procedure is as follows: (1) Dilute Namalwa lymphoma cells (purchased from Procell) with PBS to 2×10⁻⁶. 6 / ml, add 100μl / tube of goat serum to a 1.5ml EP tube, add 10μl / tube of goat serum, and block at 4℃ for 30min. Add serially diluted CD3×CD19 antibody, SP34×CD19 double antibody targeting the same target, and control hIgG1 (purchased from Baiying Biotechnology), and incubate at 4℃ for 30min. Add 1ml of PBS to the EP tube, centrifuge at 3500rpm×5min at 4℃, discard the supernatant, and wash once with PBS. After centrifugation, discard the supernatant, resuspend the cells in 100μl / tube of PBS, add 1μl / tube of Alexa-647-labeled goat anti-human IgG antibody secondary antibody (purchased from Jackson Lab), and incubate at 4℃ in the dark for 30min. Wash twice with PBS, centrifuge, and discard the supernatant. Resuspend the cells in 200μl / tube of PBS and analyze by flow cytometry. The results are as follows Figure 3 As shown, both CD3×CD19 and the bispecific antibody SP34×CD19 targeting the same target can bind to CD19-positive Namalwa lymphoma cells.

[0115] (2) Dilute PBMC (purchased from Selene Biotechnology) with PBS to a concentration of 2×10⁻⁶. 6 / ml, add 100μl / tube of goat serum to a 1.5ml EP tube, add 10μl / tube of goat serum, and block at 4℃ for 30min. Add serially diluted bispecific antibody CD3×CD19, target-specific bispecific antibody SP34×CD19, and control hIgG1 (purchased from Biolegend), and incubate at 4℃ for 30min. Add 1ml of PBS to the EP tube, centrifuge at 3500rpm×5min at 4℃, discard the supernatant, and wash once with PBS. After centrifugation, discard the supernatant, resuspend the cells in 100μl / tube of PBS, add 1μl / tube of Alexa-647-labeled goat anti-human IgG antibody secondary antibody (purchased from Jacksonlab) and 0.5μl / tube of PerCP-Cy5.5-labeled anti-human CD8 antibody (purchased from Biolegend), and incubate at 4℃ in the dark for 30min. Wash twice with PBS, centrifuge, and discard the supernatant. Cells were resuspended in 200 μl / tube of PBS and analyzed by flow cytometry.

[0116] The results are as follows Figure 4 As shown, the bispecific antibody SP34×CD19 targeting the same target binds strongly to T cells, while CD3×CD19 binds very weakly to T cells.

[0117] Example 4: Bispecific antibody promotes T cell expression of activation marker CD69 The CD3×CD19 and SP34×CD19 bispecific antibodies described in Example 1 were added to the co-incubation system of PBMCs and Namalwa lymphoma cells. After 48 hours of culture, the expression of CD69 on the surface of CD4 T cells and CD8 T cells was detected by flow cytometry to determine the characteristics of T cell activation induced by the bispecific antibodies. The specific operation is as follows: (1) Namalwa lymphoma cells were diluted to 1×10⁻⁶ using complete RPMI 1640 medium. 5 Add / ml to a 96-well plate; (2) The bispecific antibody CD3×CD19, the bispecific antibody SP34×CD19 targeting the same target, the bispecific antibody Blinatumomab targeting the same target (purchased from Baiying Biotechnology), and the control hIgG1 (purchased from Baiying Biotechnology) were serially diluted using complete RPMI 1640 medium and added to a 96-well plate at 20 μl / well. (3) Dilute PBMC (purchased from Selene Biotechnology) to 1.25 × 10⁻⁶ using complete RPMI 1640 medium. 6 Add 80 μl / well to a 96-well plate; (4) Incubate the 96-well plate at 37°C in a 5% CO2 incubator for 72 h; (5) Add PerCP-Cy5.5 labeled CD8 antibody, BV605 labeled CD4 antibody, and BV421 labeled CD69 antibody (all purchased from Biolegend), and incubate at 4°C in the dark for 30 min.

[0118] (6) Wash twice with PBS, centrifuge and discard the supernatant.

[0119] (7) Resuspend the cells in 200 μl / tube of PBS and detect them using a flow cytometer.

[0120] The results are as follows Figure 5 As shown, CD3×CD19, along with the bispecific antibodies SP34×CD19 and Blinatumomab targeting the same target, can promote the activation of CD4 and CD8 T cells and the expression of CD69. SP34×CD19 and Blinatumomab have similar effects on promoting CD69 expression, and are both stronger than the CD3×CD19 bispecific antibody of this invention.

[0121] Example 5: Experiment on bispecific antibody-induced PBMC cytokine secretion The CD3×CD19 bispecific antibody described in Example 1 was added to PBMCs. After culturing for 48 hours, the culture supernatant was collected, and the cytokine content in the supernatant was detected to determine the characteristics of cytokine release induced by the bispecific antibody. The specific operation is as follows: (1) PBMC (purchased from Selene Biotechnology) was diluted to 1.25 × 10⁻⁶ using complete RPMI 1640 medium. 6 Add 80 μl / well to a 96-well plate; (2) CD3×CD19, the same target double antibody Blinatumomab, and control hIgG1 (purchased from Baiying Biotechnology) were serially diluted using complete RPMI 1640 medium and added to 96-well plates, 20 μl / well; (3) Add 100 μl of complete RPMI 1640 medium PBMC to a 96-well plate; (4) Incubate the 96-well plate at 37°C in a 5% CO2 incubator for 48 hours; (5) Centrifuge at 300g for 10 min at room temperature and collect the cell culture supernatant; (6) The cytokine content in the supernatant was detected using the CBA kit (purchased from BD).

[0122] The results are as follows Figure 6 As shown, in the absence of tumor cells, the bispecific antibody Blinatumomab targeting the same target strongly promotes the release of cytokines IL-6 and TNFα, while the SP34×CD19 bispecific antibody of this invention only induces very little cytokine release.

[0123] Example 6: Experiment on the promotion of cytokine secretion by bispecific antibodies in a co-incubation system with tumor cells. The CD3×CD19 bispecific antibody described in Example 1 was added to the co-incubation system of PBMC and Namalwa lymphoma cells. After culturing for 48 hours, the culture supernatant was collected, and the cytokine content in the supernatant was detected to determine the characteristics of cytokine release induced by the bispecific antibody.

[0124] (a) Namalwa lymphoma cells were diluted to 1×10⁻⁶ using complete RPMI 1640 medium. 5 Add / ml to a 96-well plate and incubate at 37°C in a 5% CO2 incubator for 48 hours; (b) Using complete RPMI 1640 medium, the bispecific antibody CD3×CD19, the bispecific antibody Blinatumomab targeting the same target, and the control hIgG1 (purchased from Baiying Biotechnology) were serially diluted and added to 96-well plates, 20 μl / well; (c) Dilute PBMC (purchased from Selene Biotechnology) to 1.25 × 10⁻⁶ using complete RPMI 1640 medium. 6 Add 80 μl / well to a 96-well plate; (d) Incubate the 96-well plate at 37°C in a 5% CO2 incubator for 48 h; (e) Centrifuge at 300g for 10 min at room temperature and collect the cell culture supernatant; (f) The cytokine content in the supernatant was detected using the CBA kit (purchased from BD).

[0125] The results are as follows Figure 7 As shown, in the presence of tumor cells, the bispecific antibody Blinatumomab targeting the same target strongly promotes the release of cytokines IL-6 and TNFα, while the SP34×CD19 bispecific antibody of this invention only induces a very small amount of cytokine release.

[0126] Example 7: Experiment on the effect of bispecific antibody promoting tumor cell killing by PBMCs in a co-incubation system with tumor cells The ability of the CD3×CD19 bispecific antibody described in Example 1 to promote PBMC killing of tumor cells was tested.

[0127] (a) Namalwa-luciferase lymphoma cells (purchased from Procell) were diluted to 1×10⁻⁶ using complete RPMI 1640 medium. 5 Add / ml to a 96-well plate and incubate at 37°C in a 5% CO2 incubator for 48 hours; (b) Using complete RPMI 1640 medium, the bispecific antibody CD3×CD19, the bispecific antibody Blinatumomab targeting the same target, and the control hIgG1LALA (purchased from Baiying Biotechnology) were serially diluted and added to 96-well plates, 20 μl / well. (c) Dilute PBMC (purchased from Selene Biotechnology) to 1.25 × 10⁻⁶ using complete RPMI 1640 medium. 6 Add 80 μl / well to a 96-well plate; (d) Incubate the 96-well plate at 37°C in a 5% CO2 incubator for 48 h; (e) Centrifuge at 300g for 10 min at room temperature and collect the cell culture supernatant; (f) Add luciferase substrate and detect chemiluminescence.

[0128] The results are as follows Figure 8 As shown, both CD3×CD19 and the bispecific antibody Blinatumomab targeting the same target can promote the killing of CD19-positive tumor cells by PBMCs, and their maximum killing activities are similar.

[0129] In summary, the experimental results show that the bispecific antibody of this invention can bind to CD3 and CD19, thereby promoting T cell activation and cytokine secretion, effectively promoting PBMC killing of tumor cells, and exhibiting good anti-cancer activity. Furthermore, the bispecific antibody of this invention achieves extremely weak binding activity with T cells, lower secretion of pro-inflammatory cytokines, and higher safety. In conclusion, the bispecific antibody of this invention can promote immune cell anti-cancer activity, possesses good anti-cancer activity, and has higher safety, demonstrating good clinical application value and drug development value.

[0130] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0131] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A bispecific antibody, characterized in that, include: The first binding region has CD3 binding activity; as well as The second binding region has CD19 binding activity.

2. The bispecific antibody according to claim 1, characterized in that, The first binding region includes a first heavy chain variable region and a first light chain variable region, and the first heavy chain variable region and the first light chain variable region are connected. Optionally, the first binding region includes a CDR selected from at least one of the following: Heavy chain variable region (CDR): amino acid sequence of SEQ ID NO: 1-3 or its conserved modified form; Light chain variable region CDR: amino acid sequence of SEQ ID NO:4~6 or its conserved modified form; Optionally, the first bonding region includes: The heavy chain variable region CDR1 having the amino acid sequence shown in SEQ ID NO:1 or an amino acid sequence of its conserved modified form; The heavy chain variable region CDR2 having the amino acid sequence shown in SEQ ID NO:2 or an amino acid sequence of its conserved modified form; The heavy chain variable region CDR3 having the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence of its conserved modified form; The light chain variable region CDR1 having the amino acid sequence shown in SEQ ID NO:4 or an amino acid sequence of its conserved modified form; The light chain variable region CDR2 having the amino acid sequence shown in SEQ ID NO:5 or its conserved modified form; and The light chain variable region CDR3 having the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence of its conserved modified form; Optionally, the first binding region includes a first heavy chain frame region and / or a first light chain frame region; Optionally, at least a portion of the first heavy chain framework region and / or the first light chain framework region is derived from a human antibody; Optionally, the first heavy chain variable region has an amino acid sequence as shown in SEQ ID NO:20 or an amino acid sequence having at least 90% identity with it; and / or The first light chain variable region has an amino acid sequence as shown in SEQ ID NO:21 or an amino acid sequence having at least 90% identity with it; Optionally, the second binding region includes a second binding region 1 and a second binding region 2, the second binding region 1 includes a second heavy chain variable region 1 and a second light chain variable region 1, and the second binding region 2 includes a second heavy chain variable region 2 and a second light chain variable region 2. Optionally, the second binding region 1 and the second binding region 2 include a CDR selected from at least one of the following: Heavy chain variable region (CDR): amino acid sequence of SEQ ID NO: 7-9 or its conserved modified form; Light chain variable region (CDR): amino acid sequence of SEQ ID NO: 10-12 or its conserved modified form; Optionally, the second bonding region 1 and the second bonding region 2 include: The heavy chain variable region CDR1 having the amino acid sequence shown in SEQ ID NO:7 or an amino acid sequence of its conserved modified form; The heavy chain variable region CDR2 having the amino acid sequence shown in SEQ ID NO:8 or an amino acid sequence of its conserved modified form; The heavy chain variable region CDR3 having the amino acid sequence shown in SEQ ID NO:9 or an amino acid sequence of its conserved modified form; The light chain variable region CDR1 having the amino acid sequence shown in SEQ ID NO:10 or an amino acid sequence of its conserved modified form; The light chain variable region CDR2 having the amino acid sequence shown in SEQ ID NO:11 or its conserved modified form; and The light chain variable region CDR3 having the amino acid sequence shown in SEQ ID NO:12 or an amino acid sequence of its conserved modified form; Optionally, the second bonding region includes a second heavy chain frame region and / or a second light chain frame region; Optionally, at least a portion of the second heavy chain framework region and / or the second light chain framework region is derived from a human antibody; Optionally, the second heavy chain variable regions 1 and 2 have an amino acid sequence as shown in SEQ ID NO:16 or an amino acid sequence having at least 90% identity with it; and / or The second light chain variable regions 1 and 2 have an amino acid sequence as shown in SEQ ID NO:17 or an amino acid sequence that is at least 90% identical to it. Optionally, the second binding region 1 and the second binding region 2 are Fab fragments; Optionally, the second binding region 1 further includes a first CL segment and a first CH1 segment, wherein the second heavy chain variable region 1 is connected to the first CH1 segment, and the second light chain variable region 1 is connected to the first CL segment; Optionally, the second binding region 1 has the amino acid sequence shown in SEQ ID NO:18 or an amino acid sequence having at least 90% identity with it, and the amino acid sequence shown in SEQ ID NO:19 or an amino acid sequence having at least 90% identity with it. Optionally, the second binding region 2 further includes a second CL segment and a second CH1 segment, wherein the second heavy chain variable region 2 is connected to the second CH1 segment, and the second light chain variable region 2 is connected to the second CL segment; Optionally, the second binding region 2 has the amino acid sequence shown in SEQ ID NO:18 or an amino acid sequence having at least 90% identity with it, and the amino acid sequence shown in SEQ ID NO:19 or an amino acid sequence having at least 90% identity with it. Optionally, the second binding region 1 is connected by covalent bonds, and / or the second binding region 2 is connected by covalent bonds; Optionally, the covalent bond is a disulfide bond; Optionally, the bispecific antibody further includes a first Fc region and / or a second Fc region; Optionally, the first Fc region and the second Fc region are connected by a knock-into-hole structure; Optionally, the second bonding region 1 is connected to the first bonding region, and the first bonding region is connected to the first Fc region; Optionally, the second bonding region 2 is also connected to the second Fc region.

3. The bispecific antibody according to claim 2, characterized in that, In the first bonding region, the C end of the first light chain variable region is connected to the N end of the first heavy chain variable region, or the N end of the first light chain variable region is connected to the C end of the first heavy chain variable region. Optionally, in the second binding region 1, the C-terminus of the second light chain variable region 1 is connected to the N-terminus of the first CL segment, and the C-terminus of the second heavy chain variable region 1 is connected to the N-terminus of the first CH1 segment; or In the second binding region 1, the C-terminus of the second light chain variable region 1 is connected to the N-terminus of the first CH1 segment, and the C-terminus of the second heavy chain variable region 1 is connected to the N-terminus of the first CL segment. Optionally, in the second binding region 2, the C-terminus of the second light chain variable region 2 is connected to the N-terminus of the second CH1 segment, and the C-terminus of the second heavy chain variable region 2 is connected to the N-terminus of the second CL segment; or In the second binding region 2, the C-terminus of the second light chain variable region 2 is connected to the N-terminus of the second CL segment, and the C-terminus of the second heavy chain variable region 2 is connected to the N-terminus of the second CH1 segment. Optionally, the C-terminus of the second light chain variable region 1 is connected to the N-terminus of the first CL segment; and / or The C-terminus of the second heavy chain variable region 1 is connected to the N-terminus of the first CH1 segment, the C-terminus of the first CH1 segment is connected to the N-terminus of the first heavy chain variable region, the C-terminus of the first heavy chain variable region is connected to the N-terminus of the first light chain variable region, and the C-terminus of the first light chain variable region is connected to the N-terminus of the first Fc region; and / or The C-terminus of the second heavy chain variable region 2 is connected to the N-terminus of the second CH1 segment, and the C-terminus of the second CH1 segment is connected to the second Fc region; and / or The C-terminus of the second light chain variable region 2 is connected to the N-terminus of the second CL segment.

4. The bispecific antibody according to claim 2, characterized in that, The first binding region further includes a first linker peptide, wherein the C-terminus of the first light chain variable region is connected to the N-terminus of the first linker peptide, and the C-terminus of the first linker peptide is connected to the N-terminus of the first heavy chain variable region; or, the N-terminus of the first light chain variable region is connected to the C-terminus of the first linker peptide, and the N-terminus of the first linker peptide is connected to the C-terminus of the first heavy chain variable region. Optionally, the first binding region is a single-chain antibody; Optionally, the first binding region has the amino acid sequence shown in SEQ ID NO:22 or an amino acid sequence having at least 90% identity with it; Optionally, the first binding region and the second binding region 1 are connected by a second linker peptide; Optionally, the C-terminus of the second light chain variable region 1 is connected to the N-terminus of the first CL segment; and / or The C-terminus of the second heavy chain variable region 1 is connected to the N-terminus of the first CH1 fragment; the C-terminus of the first CH1 fragment is connected to the N-terminus of the second linker peptide; the C-terminus of the second linker peptide is connected to the N-terminus of the first heavy chain variable region; the C-terminus of the first heavy chain variable region is connected to the N-terminus of the first linker peptide; the C-terminus of the first linker peptide is connected to the N-terminus of the first light chain variable region; and the C-terminus of the first light chain variable region is connected to the N-terminus of the first Fc region; and / or The C-terminus of the second heavy chain variable region 2 is connected to the N-terminus of the second CH1 region, and the C-terminus of the second CH1 region is connected to the N-terminus of the second Fc region; and / or The C-terminus of the second light chain variable region 2 is connected to the N-terminus of the second CL segment; Optionally, the first binding region and the first Fc region are connected by a third linker peptide; Optionally, the C-terminus of the second light chain variable region 1 is connected to the N-terminus of the first CL segment; and / or The C-terminus of the second heavy chain variable region 1 is connected to the N-terminus of the first CH1 fragment; the C-terminus of the first CH1 fragment is connected to the N-terminus of the second linker peptide; the C-terminus of the second linker peptide is connected to the N-terminus of the first heavy chain variable region; the C-terminus of the first heavy chain variable region is connected to the N-terminus of the first linker peptide; the C-terminus of the first linker peptide is connected to the N-terminus of the first light chain variable region; the C-terminus of the first light chain variable region is connected to the N-terminus of the third linker peptide; and the C-terminus of the third linker peptide is connected to the N-terminus of the first Fc region; and / or The C-terminus of the second heavy chain variable region 2 is connected to the N-terminus of the second CH1 region, and the C-terminus of the second CH1 region is connected to the N-terminus of the second Fc region; and / or The C-terminus of the second light chain variable region 2 is connected to the N-terminus of the second CL segment.

5. The bispecific antibody according to claim 2, characterized in that, The first Fc region, relative to the amino acid sequence of the Fc fragment of human wild-type IgG1, has at least one of the following mutation sites: L234A, L235A, S354C, and T366W; Optionally, the second Fc region has at least one of the following mutation sites relative to the amino acid sequence of the Fc fragment of human wild-type IgG1: L234A, L235A, Y349C, T366S, L368A, and Y407V. Optionally, the first Fc region has the amino acid sequence shown in SEQ ID NO:24; Optionally, the second Fc region has the amino acid sequence shown in SEQ ID NO:25; Optionally, the linker peptide is a flexible linker peptide; Optionally, the amino acid sequence of the first linker peptide is (GGGGS)n, where n is an integer greater than or equal to 1, preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12, more preferably 1, 2 or 3; Optionally, the amino acid sequence of the second linker peptide is shown in SEQ ID NO:

27.

6. The bispecific antibody according to claim 1, characterized in that, Includes the amino acid sequence shown in any one of SEQ ID NO:13, 14, 15, or an amino acid sequence that is at least 90% identical to it.

7. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the bispecific antibody as described in any one of claims 1 to 6.

8. An expression carrier, characterized in that, Carrying the nucleic acid molecule as described in claim 7.

9. The expression vector according to claim 8, characterized in that, The expression vector is a eukaryotic expression vector or a prokaryotic expression vector; Preferably, the expression vector is a plasmid expression vector.

10. A recombinant cell, characterized in that, The recombinant cells carry the nucleic acid molecule of claim 7 or express the bispecific antibody of any one of claims 1 to 6.

11. The recombinant cell according to claim 10, characterized in that, The recombinant cells are obtained by introducing the expression vector of claim 8 or 9 into host cells; Optionally, the recombinant cells are eukaryotic cells; Preferably, the recombinant cells are mammalian cells.

12. A pharmaceutical composition, characterized in that, Includes at least one of the following: The bispecific antibody according to any one of claims 1 to 6; The nucleic acid molecule according to claim 7; The expression vector as described in claim 8 or 9; or The recombinant cells according to claim 10 or 11; Optionally, the pharmaceutical composition further comprises pharmaceutically acceptable excipients.

13. Use of the bispecific antibody according to any one of claims 1 to 6, the nucleic acid molecule according to claim 7, the expression vector according to claim 8 or 9, the recombinant cell according to claim 10 or 11, or the pharmaceutical composition according to claim 12 in the preparation of a medicament for the prevention and / or treatment of tumors and / or autoimmune diseases; Optionally, the tumor includes at least one selected from lymphoma, myeloma, colorectal cancer, lung cancer, pancreatic cancer, renal cell carcinoma, prostate cancer, and melanoma; Optionally, the autoimmune disease includes at least one of the following: systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, dermatomyositis, autoimmune hemolytic anemia, thyroid autoimmune disease, ulcerative colitis, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, pulmonary hemorrhage nephritis syndrome, pemphigus vulgaris, bullous pemphigoid, primary biliary cirrhosis, multiple cerebral sclerosis, and acute idiopathic polyneuritis.

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