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CN-122011197-A - Bispecific antibody targeting CD3 and CD19 and application thereof

CN122011197ACN 122011197 ACN122011197 ACN 122011197ACN-122011197-A

Abstract

The invention provides a bispecific antibody targeting CD3 and CD19 and application thereof, wherein the bispecific antibody comprises a first binding region, the first binding region has CD3 binding activity, and a second binding region, and the second binding region has CD19 binding activity. The bispecific antibody provided by the invention has the advantages of extremely weak binding with T cells, strong binding with tumor cells, higher anticancer activity, low risk of inducing cytokine storm, good safety and important application value for drug development and tumor prevention and/or treatment.

Inventors

  • CAO GUOSHUAI
  • LI YANGYANG
  • WU YUWEI

Assignees

  • 合肥综合性国家科学中心大健康研究院

Dates

Publication Date
20260512
Application Date
20251212

Claims (13)

  1. 1. A bispecific antibody is provided, which is useful for the treatment of cancer, characterized by comprising the following steps: a first binding region having CD3 binding activity, and A second binding region, said second binding region having CD19 binding activity.
  2. 2. The bispecific antibody of claim 1, wherein the first binding region comprises 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 linked; optionally, the first binding region comprises a CDR selected from at least one of: The heavy chain variable region CDR is the amino acid sequence of SEQ ID NO 1-3 or a conservatively modified form thereof; the light chain variable region CDR is the amino acid sequence of SEQ ID NO 4-6 or a conservatively modified form thereof; Optionally, the first binding region comprises: a heavy chain variable region CDR1 having the amino acid sequence shown in SEQ ID NO. 1 or a conservatively modified version thereof; a heavy chain variable region CDR2 having the amino acid sequence shown in SEQ ID NO. 2 or a conservatively modified version thereof; a heavy chain variable region CDR3 having the amino acid sequence depicted in SEQ ID No. 3 or a conservatively modified version thereof; a light chain variable region CDR1 having the amino acid sequence shown in SEQ ID NO. 4 or a conservatively modified version thereof; A light chain variable region CDR2 having the amino acid sequence shown in SEQ ID NO.5 or a conservatively modified version thereof, and A light chain variable region CDR3 having the amino acid sequence depicted in SEQ ID No. 6 or a conservatively modified version thereof; Optionally, the first binding region comprises a first heavy chain framework region and/or a first light chain framework 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 thereto, and/or The first light chain variable region has an amino acid sequence as set forth in SEQ ID NO. 21 or an amino acid sequence having at least 90% identity thereto; Optionally, the second binding region comprises a second binding region 1 and a second binding region 2, the second binding region 1 comprising a second heavy chain variable region 1 and a second light chain variable region 1, the second binding region 2 comprising 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 comprise CDRs selected from at least one of the following: the heavy chain variable region CDR is the amino acid sequence of SEQ ID NO 7-9 or a conservatively modified form thereof; The light chain variable region CDR is the amino acid sequence of SEQ ID NO 10-12 or a conservatively modified form thereof; Optionally, the second binding region 1 and the second binding region 2 comprise: A heavy chain variable region CDR1 having the amino acid sequence shown in SEQ ID NO. 7 or a conservatively modified version thereof; a heavy chain variable region CDR2 having the amino acid sequence shown in SEQ ID NO. 8 or a conservatively modified version thereof; a heavy chain variable region CDR3 having the amino acid sequence depicted in SEQ ID No. 9 or a conservatively modified version thereof; a light chain variable region CDR1 having the amino acid sequence shown in SEQ ID NO. 10 or a conservatively modified version thereof; A light chain variable region CDR2 having the amino acid sequence shown in SEQ ID NO. 11 or a conservatively modified version thereof, and A light chain variable region CDR3 having the amino acid sequence depicted in SEQ ID No. 12 or a conservatively modified version thereof; Optionally, the second binding region comprises a second heavy chain framework region and/or a second light chain framework 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 thereto, and/or The second light chain variable regions 1 and 2 have the amino acid sequence shown as SEQ ID NO. 17 or an amino acid sequence having at least 90% identity thereto; optionally, the second binding region 1 and the second binding region 2 are Fab fragments; Optionally, the second binding region 1 further comprises a first CL fragment and a first CH1 fragment, wherein the second heavy chain variable region 1 is linked to the first CH1 fragment and the second light chain variable region 1 is linked to the first CL fragment; Optionally, the second binding region 1 has an amino acid sequence shown in SEQ ID NO. 18 or an amino acid sequence having at least 90% identity thereto, and an amino acid sequence shown in SEQ ID NO. 19 or an amino acid sequence having at least 90% identity thereto; Optionally, the second binding region 2 further comprises a second CL fragment and a second CH1 fragment, wherein the second heavy chain variable region 2 is linked to the second CH1 fragment and the second light chain variable region 2 is linked to the second CL fragment; Optionally, the second binding region 2 has an amino acid sequence shown in SEQ ID NO. 18 or an amino acid sequence having at least 90% identity thereto, and an amino acid sequence shown in SEQ ID NO. 19 or an amino acid sequence having at least 90% identity thereto; Optionally, the second binding region 1 is internally linked by covalent bonds, and/or the second binding region 2 is internally linked by covalent bonds; optionally, the covalent bond is a disulfide bond; Optionally, the bispecific antibody further comprises a first Fc region and/or a second Fc region; Optionally, the first Fc region and the second Fc region are linked by a knob-intoo-hole structure; Optionally, the second binding region 1 is linked to the first binding region, which is linked to the first Fc region; Optionally, the second binding region 2 is also linked to the second Fc region.
  3. 3. The bispecific antibody of claim 2, wherein in the first binding region, the C-terminus of the first light chain variable region is linked to the N-terminus of the first heavy chain variable region or the N-terminus of the first light chain variable region is linked to the C-terminus 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 linked to the N-terminus of the first CL fragment and the C-terminus of the second heavy chain variable region 1 is linked to the N-terminus of the first CH1 fragment, or In the second binding region 1, the C-terminus of the second light chain variable region 1 is linked to the N-terminus of the first CH1 fragment, and the C-terminus of the second heavy chain variable region 1 is linked to the N-terminus of the first CL fragment; Optionally, in the second binding region 2, the C-terminus of the second light chain variable region 2 is linked to the N-terminus of the second CH1 fragment, and the C-terminus of the second heavy chain variable region 2 is linked to the N-terminus of the second CL fragment, or In the second binding region 2, the C-terminus of the second light chain variable region 2 is linked to the N-terminus of the second CL fragment, and the C-terminus of the second heavy chain variable region 2 is linked to the N-terminus of the second CH1 fragment; Optionally, the C-terminus of the second light chain variable region 1 is linked to the N-terminus of the first CL fragment, and/or The C-terminus of the second heavy chain variable region 1 is linked to the N-terminus of the first CH1 fragment, the C-terminus of the first CH1 fragment is linked to the N-terminus of the first heavy chain variable region, the C-terminus of the first heavy chain variable region is linked to the N-terminus of the first light chain variable region, the C-terminus of the first light chain variable region is linked to the N-terminus of the first Fc region, and/or The C-terminus of the second heavy chain variable region 2 is linked to the N-terminus of the second CH1 fragment, the C-terminus of the second CH1 fragment is linked to the second Fc region, and/or The C-terminus of the second light chain variable region 2 is linked to the N-terminus of the second CL fragment.
  4. 4. The bispecific antibody of claim 2, wherein the first binding region further comprises a first connecting peptide, the C-terminus of the first light chain variable region is linked to the N-terminus of the first connecting peptide, the C-terminus of the first connecting peptide is linked to the N-terminus of the first heavy chain variable region, or the N-terminus of the first light chain variable region is linked to the C-terminus of the first connecting peptide, the N-terminus of the first connecting peptide is linked 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 thereto; optionally, the first binding region and the second binding region 1 are linked by a second linking peptide; Optionally, the C-terminus of the second light chain variable region 1 is linked to the N-terminus of the first CL fragment, and/or The C-terminus of the second heavy chain variable region 1 is linked to the N-terminus of the first CH1 fragment, the C-terminus of the first CH1 fragment is linked to the N-terminus of the second linker peptide, the C-terminus of the second linker peptide is linked to the N-terminus of the first heavy chain variable region, the C-terminus of the first heavy chain variable region is linked to the N-terminus of the first linker peptide, the C-terminus of the first linker peptide is linked to the N-terminus of the first light chain variable region, the C-terminus of the first light chain variable region is linked to the N-terminus of the first Fc region, and/or The C-terminus of the second heavy chain variable region 2 is linked to the N-terminus of the second CH1 region, the C-terminus of the second CH1 region is linked to the N-terminus of the second Fc region, and/or The C-terminus of the second light chain variable region 2 is linked to the N-terminus of the second CL fragment; optionally, the first binding region and the first Fc region are linked by a third linking peptide; Optionally, the C-terminus of the second light chain variable region 1 is linked to the N-terminus of the first CL fragment, and/or The C-terminus of the second heavy chain variable region 1 is linked to the N-terminus of the first CH1 fragment, the C-terminus of the first CH1 fragment is linked to the N-terminus of the second linker peptide, the C-terminus of the second linker peptide is linked to the N-terminus of the first heavy chain variable region, the C-terminus of the first heavy chain variable region is linked to the N-terminus of the first linker peptide, the C-terminus of the first linker peptide is linked to the N-terminus of the first light chain variable region, the C-terminus of the first light chain variable region is linked to the N-terminus of the third linker peptide, the C-terminus of the third linker peptide is linked to the N-terminus of the first Fc region, and/or The C-terminus of the second heavy chain variable region 2 is linked to the N-terminus of the second CH1 region, the C-terminus of the second CH1 region is linked to the N-terminus of the second Fc region, and/or The C-terminus of the second light chain variable region 2 is linked to the N-terminus of the second CL fragment.
  5. 5. The bispecific antibody of claim 2, wherein the first Fc region has at least one of the following mutation sites L234A, L235,235, 235A, S354C and T366W relative to the amino acid sequence of the Fc fragment of human wild-type IgG 1; optionally, the second Fc region has at least one of the following mutation sites L234A, L235,235A, Y349C, T366S, L A and Y407V relative to the amino acid sequence of the Fc fragment of human wild-type IgG 1; Optionally, the first Fc region has the amino acid sequence shown as SEQ ID NO. 24; Optionally, the second Fc region has the amino acid sequence shown in SEQ ID NO. 25; optionally, the connecting peptide is a flexible connecting peptide; Optionally, the amino acid sequence of the first connecting peptide is (GGGGS) n, wherein 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 connecting peptide is shown in SEQ ID NO. 27.
  6. 6. The bispecific antibody according to claim 1, characterized in that it comprises the amino acid sequence shown in any of SEQ ID NOs 13, 14, 15 or an amino acid sequence having at least 90% identity thereto.
  7. 7. A nucleic acid molecule encoding the bispecific antibody of any one of claims 1-6.
  8. 8. An expression vector carrying the nucleic acid molecule of claim 7.
  9. 9. The expression vector of claim 8, wherein the expression vector is a eukaryotic expression vector or a prokaryotic expression vector; Preferably, the expression vector is a plasmid expression vector.
  10. 10. A recombinant cell carrying the nucleic acid molecule of claim 7 or expressing the bispecific antibody of any one of claims 1-6.
  11. 11. The recombinant cell according to claim 10, wherein the recombinant cell is obtained by introducing the expression vector of claim 8 or 9 into a host cell; optionally, the recombinant cell is a eukaryotic cell; preferably, the recombinant cell is a mammalian cell.
  12. 12. A pharmaceutical composition comprising at least one of the following: The bispecific antibody of any one of claims 1-6; The nucleic acid molecule of claim 7; the expression vector of claim 8 or 9, or The recombinant cell of claim 10 or 11; Optionally, the pharmaceutical composition further comprises pharmaceutically acceptable excipients.
  13. 13. Use of a bispecific antibody according to any one of claims 1 to 6, a nucleic acid molecule according to claim 7, an expression vector according to claim 8 or 9, a recombinant cell according to claim 10 or 11 or a pharmaceutical composition according to claim 12 for the preparation of a medicament for the prevention and/or treatment of a tumor and/or an autoimmune disease; Optionally, the tumor comprises at least one selected from lymphoma, myeloma, colorectal cancer, lung cancer, pancreatic cancer, renal cell carcinoma, prostate cancer, and melanoma; Optionally, the autoimmune disease comprises at least one selected from the group consisting of systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, dermatomyositis, autoimmune hemolytic anemia, autoimmune thyroid disease, ulcerative colitis, chronic lymphocytic thyroiditis, hyperthyroidism, insulin dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, lung hemorrhagic nephritis syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple cerebral spinal sclerosis, and acute idiopathic polyneuritis.

Description

Bispecific antibody targeting CD3 and CD19 and application thereof Technical Field The invention relates to the technical field of biological medicine, in particular to a bispecific antibody targeting CD3 and CD19 and application thereof. Background Cancer is a disease affecting human health and survival and development, and in recent years, immunotherapy including tumor-targeting antibodies, immune checkpoint antibodies, bispecific antibodies, and the like has become a new hot spot and a new hope for anticancer. Immunotherapy represented by PD-1/L1 has demonstrated great potential, but even with the currently most widely approved PD-1/L1 therapies, the overall response rate needs to be improved, and more patients cannot benefit from it. T cells recognize neo-antigens (TCR) through their surface T cell receptors, i.e. antigens mutated in tumor genes, while partial tumors have low frequency of gene mutation and a small number of neo-antigen species, called "cold tumors". Current immune checkpoint therapies, such as PD-1/L1 therapies, achieve anti-cancer goals by restoring T cell self function, whereas in "cold tumors" T cells cannot effectively recognize the tumor, resulting in immune checkpoint therapies that are ineffective against "cold tumors. Bispecific antibodies based on CD3 (hereinafter referred to as "CD3 bispecific antibodies") promote T cell activation and killing of tumors by recruiting T cells to tumor sites, bridging T cells and tumors, and these bispecific antibodies do not require neoantigens and can guide T cells to kill "cold tumors". CD3 bispecific antibodies trigger strong activation signals after binding to T cells and tumor cells, and thus also "disregard" the inhibitory signals of immune checkpoint molecules to some extent, but CD3 bispecific antibodies also promote pro-inflammatory cytokine production, such as TNFa, IL-6, etc., triggering cytokine storms and excessive immune responses. Therefore, the bispecific antibody based on CD3 has good application prospect in clinic, but the safety is still to be further improved. In healthy humans, CD19 is expressed on the surface of normal B cells, in autoimmune disease patients, CD19 is expressed on the surface of pathogenic memory B cells, and in myeloma patients, CD19 is expressed on the surface of tumor cells. CD 19-targeted therapies are of great clinical value in both tumor therapy and autoimmune disease therapy. One way to solve the safety problem of bispecific antibodies is to increase the binding affinity of the bispecific antibody to the tumor target and to weaken its binding to T cells, thereby allowing the bispecific antibody drug to be distributed more locally to the tumor, increasing the local drug concentration of the tumor, decreasing the peripheral drug concentration, and reducing the off-target toxicity. Therefore, there is a need to develop bispecific antibodies with better safety and higher clinical application value in clinic. Disclosure of Invention The present invention aims to solve at least one of the technical problems existing in the prior art to at least some extent. Thus, in a first aspect of the invention, the invention provides a bispecific antibody. According to an embodiment of the 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 provided by the invention has the advantages of extremely weak binding with T cells, strong binding with tumor cells, higher anticancer activity, low risk of inducing cytokine storm, good safety and important application value for drug development and tumor prevention and/or treatment. According to an embodiment of the present invention, the above bispecific antibody may further comprise at least one of the following additional technical features: According to an embodiment of the invention, the first binding region comprises 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 linked According to an embodiment of the invention, the first binding region comprises a CDR selected from at least one of the amino acid sequences of SEQ ID NOS 1-3 or conservatively modified forms thereof and the amino acid sequences of the light chain variable region CDR SEQ ID NOS 4-6 or conservatively modified forms thereof. According to an embodiment of the invention, the first binding region comprises: a heavy chain variable region CDR1 having the amino acid sequence shown in SEQ ID NO. 1 or a conservatively modified version thereof; a heavy chain variable region CDR2 having the amino acid sequence shown in SEQ ID NO. 2 or a conservatively modified version thereof; a heavy chain variable region CDR3 having the amino acid sequence depicted in SEQ ID No. 3 or a conservatively modified version thereof; a light chain variable region CDR1 having the amino acid sequence shown