Search

CN-121991237-A - Bispecific antibodies and uses thereof

CN121991237ACN 121991237 ACN121991237 ACN 121991237ACN-121991237-A

Abstract

The application discloses a bispecific antibody and application thereof, belonging to the technical field of biological medicine. The bispecific antibody comprises one or more first antigen binding regions having NKp46 binding activity and one or more second antigen binding regions having HLAG binding activity, the first and second antigen binding regions being linked, wherein the second antigen binding regions comprise heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3, the HCDR1, HCDR2 and HCDR3 being selected from the group consisting of SEQ ID NOS: 7-9 or amino acid sequences having at least 80% homology to SEQ ID NOS: 7-9, respectively. The bispecific antibody can simultaneously target HLAG and NKp46, especially can simultaneously combine NKp46 on the surface of NK cells and HLAG on the surface of tumor cells, so as to directly activate NK cells, kill HLAG positive tumor cells, and effectively treat HLAG-mediated related diseases.

Inventors

  • LI YANGYANG
  • CAO GUOSHUAI
  • CHENG YING
  • WU YUWEI

Assignees

  • 合肥天港免疫药物有限公司

Dates

Publication Date
20260508
Application Date
20241106

Claims (14)

  1. 1.A bispecific antibody is provided, which is useful for the treatment of cancer, characterized by comprising the following steps: one or more first antigen binding regions having NKp46 binding activity; one or more second antigen binding regions having HLAG binding activities, the first and second antigen binding regions being linked; Wherein the second antigen binding region comprises heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3, the HCDR1, HCDR2 and HCDR3 being selected from the amino acid sequences shown in SEQ ID NOS.7-9, respectively.
  2. 2. The bispecific antibody of claim 1, characterized in that the bispecific antibody has at least one of the following features: i) The bispecific antibody further comprises an Fc fragment to which at least one of the first antigen-binding regions is linked and at least one of the second antigen-binding regions is linked; ii) the bispecific antibody is of symmetrical or asymmetrical structure; iii) The first antigen binding region is selected from a scFab fragment, or scFv fragment; iv) comprising HCDRs and LCDRs in the first antigen-binding region, the HCDRs and/or LCDRs being defined by Kabat, chothia, abM, contact or IMGT; v) the first antigen binding region comprises: HCDR1, HCDR2 and HCDR3 respectively shown as SEQ ID NO 1-3 amino acid sequence, LCDR1, LCDR2 and LCDR3 respectively shown as SEQ ID NO 4-6 amino acid sequences; vi) at least a portion of the framework regions of the light chain variable region and the heavy chain variable region in the scFv fragment are each independently derived from at least one of a murine antibody, a primates antibody, a bovine antibody, a equine antibody, a dairy antibody, a porcine antibody, a ovine antibody, a caprine antibody, a canine antibody, a feline antibody, a rabbit antibody, a camelid antibody, a donkey antibody, a deer antibody, a mink antibody, a chicken antibody, a duck antibody, a goose antibody, a turkey antibody, a bucket chicken antibody, or a mutant thereof, preferably at least one of a murine antibody and a human antibody; vii) at least a portion of the framework regions of the light chain variable region and the heavy chain variable region in the scFab fragment are each independently derived from at least one of a murine antibody, a primates antibody, a bovine antibody, a equine antibody, a dairy-bovine antibody, a porcine antibody, a ovine antibody, a caprine antibody, a canine antibody, a feline antibody, a rabbit antibody, a camel antibody, a donkey antibody, a deer antibody, a mink antibody, a chicken antibody, a duck antibody, a goose antibody, a turkey antibody, a bullfight antibody, or a mutant thereof, preferably at least one of a murine antibody and a human antibody; viii) said second antigen binding region is selected from Fab fragments; ix) including HCDRs and LCDRs in the second antigen-binding region, the HCDRs and/or LCDRs being defined by Kabat, chothia, abM, contact or IMGT; x) the second antigen binding region comprises: LCDR1, LCDR2 and LCDR3 respectively shown as SEQ ID NO 10-12 amino acid sequences; xi) at least a portion of the framework regions of the light chain variable region and the heavy chain variable region in the Fab fragment are each independently derived from at least one of a murine antibody, a primates antibody, a bovine antibody, a equine antibody, a dairy antibody, a porcine antibody, a ovine antibody, a caprine antibody, a canine antibody, a feline antibody, a rabbit antibody, a camel antibody, a donkey antibody, a deer antibody, a mink antibody, a chicken antibody, a duck antibody, a goose antibody, a turkey antibody, a bullfight antibody, or a mutant thereof, preferably at least one of a murine antibody and a human antibody.
  3. 3. The bispecific antibody of claim 2, wherein the bispecific antibody is of symmetrical structure, the bispecific antibody having at least one of the following characteristics: a1 The bispecific antibody comprises at least two first antigen binding regions; b1 The bispecific antibody comprises at least two second antigen binding regions; c1 The first antigen-binding region is linked to the C-terminus of the Fc fragment and the second antigen-binding region is linked to the N-terminus of the Fc fragment; d1 -the first antigen binding region is selected from the group consisting of scFv fragments; e1 The bispecific antibody comprises a first linking peptide; preferably, the N-terminus of the first antigen binding region is linked to the C-terminus of the first linker peptide, which is linked to the C-terminus of the Fc fragment; Preferably, the first connecting peptide has an amino acid sequence as shown in (GGGGS) n, wherein n is an integer greater than or equal to 1, preferably 1, 2,3, 4, 5, 6, 7, 8, 9 or 10; Preferably, the first linking peptide has an amino acid sequence as shown in (GGGGS) 3 、(GGGGS) 4 、(GGGGS) 12 .
  4. 4. The bispecific antibody of claim 2, wherein the bispecific antibody is of asymmetric structure, the bispecific antibody having at least one of the following characteristics: a2 The bispecific antibody comprises a first antigen binding region; b2 The bispecific antibody comprises one or two second antigen binding regions; c2 The first antigen binding region is linked to the N-terminus of one chain in the Fc fragment and the second antigen binding region is linked to the N-terminus of the other chain in the Fc fragment; d2 The two chains of the Fc fragment are linked by a knob-intoo-hole structure.
  5. 5. The bispecific antibody of claim 4, characterized in that the bispecific antibody has at least one of the following features: e2 The first antigen binding region is an scFv fragment and the second antigen binding region is selected from two, wherein both of the second antigen binding regions are Fab fragments; f2 The first antigen binding region is an scFv fragment and the second antigen binding region is selected from one and the second antigen binding region is a Fab fragment; g2 The first antigen binding region is a scFab fragment and the second antigen binding region is selected from one, the second antigen binding region is a Fab fragment; preferably, the C-terminus of the light chain variable region in the scFv fragment is linked to the N-terminus of the heavy chain variable region in the scFv fragment; preferably, in e 2), said first antigen binding region is linked to the N-terminus of one chain in the Fc fragment, one said second antigen binding region is linked to the N-terminus of the other chain in the Fc fragment, and the other said second antigen binding region is linked to the N-terminus of said first antigen binding region; preferably, the N-terminus of the heavy chain variable region in the scFab fragment is linked to the C-terminus of the light chain variable region in the scFab fragment.
  6. 6. The bispecific antibody of any one of claims 2-5, wherein the first antigen binding region has a heavy chain variable region as shown in amino acid sequence SEQ ID No. 13 and a light chain variable region as shown in amino acid sequence SEQ ID No. 14; And/or the first antigen binding region is selected from a scFab fragment in which the CH1 fragment and the CL fragment are each independently derived from at least one of a murine antibody, a primates antibody, a bovine antibody, a equine antibody, a dairy antibody, a porcine antibody, a ovine antibody, a caprine antibody, a canine antibody, a feline antibody, a rabbit antibody, a camel antibody, a donkey antibody, a deer antibody, a mink antibody, a chicken antibody, a duck antibody, a goose antibody, a turkey antibody, a bullfight antibody, or a mutant thereof; And/or the first antigen binding region is selected from a scFab fragment, wherein the CH1 fragment in the scFab fragment is selected from a CH1 fragment of IgG1, igG2, igG3, igG4, igA, igM, igE, or IgD; And/or, the CL fragment in the scFab fragment is selected from CL fragments of kappa or lambda type; And/or the second antigen binding region has a heavy chain variable region as shown in amino acid sequence SEQ ID NO. 15 and a second light chain variable region as shown in amino acid sequence SEQ ID NO. 16; And/or the second antigen binding region is selected from a Fab fragment, wherein the CH1 fragment and the CL fragment in the Fab fragment are each independently derived from at least one of a murine antibody, a primates antibody, a bovine antibody, a equine antibody, a dairy antibody, a porcine antibody, a ovine antibody, a caprine antibody, a canine antibody, a feline antibody, a rabbit antibody, a camel antibody, a donkey antibody, a deer antibody, a mink antibody, a chicken antibody, a duck antibody, a goose antibody, a turkey antibody, a bullfight antibody, or a mutant thereof; and/or the second antigen binding region is selected from a Fab fragment, wherein the CH1 fragment in the Fab fragment is selected from a CH1 fragment of IgG1, igG2, igG3, igG4, igA, igM, igE, or IgD; And/or, the CL fragment in the Fab fragment is selected from CL fragments of kappa or lambda type; And/or the Fc fragment is derived from at least one of a murine antibody, a primates antibody, a bovine antibody, a equine antibody, a dairy cow antibody, a porcine antibody, a ovine antibody, a caprine antibody, a canine antibody, a feline antibody, a rabbit antibody, a camel antibody, a donkey antibody, a deer antibody, a mink antibody, a chicken antibody, a duck antibody, a goose antibody, a turkey antibody, a bullfight antibody, or a mutant thereof, preferably a human Fc fragment; Optionally, the first antigen binding region is selected from the group consisting of scFab fragments having the amino acid sequence set forth in SEQ ID No. 17; Optionally, the first antigen binding region is selected from the group consisting of an scFv fragment having the amino acid sequence shown in SEQ ID No. 18; Optionally, the second antigen binding region is selected from a Fab fragment, one chain of which has the amino acid sequence shown in SEQ ID NO. 19 and the other chain has the amino acid sequence shown in SEQ ID NO. 20; Optionally, the Fc fragment is derived from an Fc fragment of IgG1, igG2, igG3, igG4, igA, igM, igE, or IgD; Optionally, the Fc fragment is a human IgG1 Fc fragment or mutant thereof; optionally, the bispecific antibody is of symmetrical structure, and the mutant of the human IgG1 Fc fragment has L234A and L235A mutations compared to the wild type human IgG1 Fc fragment.
  7. 7. The bispecific antibody of claim 1, wherein the bispecific antibody comprises: Having a first polypeptide chain as shown in the amino acid sequence of SEQ ID NO. 20 and a second polypeptide chain as shown in the amino acid sequence of SEQ ID NO. 21, or Has a first polypeptide chain shown as the amino acid sequence of SEQ ID NO. 20, a second polypeptide chain shown as the amino acid sequence of SEQ ID NO. 22, a third polypeptide chain shown as the amino acid sequence of SEQ ID NO. 23, and a fourth polypeptide chain shown as the amino acid sequence of SEQ ID NO. 20, or Has a first polypeptide chain shown as the amino acid sequence of SEQ ID NO. 20, a second polypeptide chain shown as the amino acid sequence of SEQ ID NO. 22, and a third polypeptide chain shown as the amino acid sequence of SEQ ID NO. 24, or Has a first polypeptide chain shown as SEQ ID NO. 20, a second polypeptide chain shown as SEQ ID NO. 22, and a third polypeptide chain shown as SEQ ID NO. 25.
  8. 8. A nucleic acid molecule encoding the bispecific antibody of any one of claims 1-7.
  9. 9. An expression vector comprising the nucleic acid molecule of claim 8; optionally, the expression vector is a eukaryotic vector or a prokaryotic vector; optionally, the expression vector comprises at least one selected from the group consisting of a plasmid vector, an adenovirus vector, a lentiviral vector, and an adeno-associated virus vector.
  10. 10. A recombinant cell carrying the nucleic acid molecule of claim 8 or the expression vector of claim 9, or expressing the bispecific antibody of any one of claims 1-7; Optionally, the recombinant cell is obtained by introducing the expression vector of claim 9 into a host cell; Optionally, the recombinant cell is a prokaryotic cell or a eukaryotic cell.
  11. 11. A pharmaceutical composition comprising the multispecific antibody of any one of claims 1-7, the nucleic acid molecule of claim 8, the expression vector of claim 9, or the recombinant cell of claim 10; optionally, further comprising pharmaceutically acceptable excipients.
  12. 12. A kit comprising the multispecific antibody of any one of claims 1-7, the nucleic acid molecule of claim 8, the expression vector of claim 9, or the recombinant cell of claim 10.
  13. 13. Use of the bispecific antibody of any one of claims 1-7, the nucleic acid molecule of claim 8, the expression vector of claim 9 or the recombinant cell of claim 10 in the preparation of a kit for detecting NKp46 and/or HLAG.
  14. 14. Use of a bispecific antibody according to any one of claims 1 to 7, a nucleic acid molecule according to claim 8, an expression vector according to claim 9 or a recombinant cell according to claim 10 or a pharmaceutical composition according to claim 11 for the preparation of a medicament for the prevention and/or treatment of HLAG-mediated related diseases; optionally, the HLAG-mediated related diseases include cancer; Optionally, the cancer comprises at least one of choriocarcinoma, esophageal cancer, head and neck cancer, glioma, thyroid cancer, lung cancer, colorectal cancer, gastric cancer, liver cancer, cholangiocarcinoma, breast cancer, ovarian cancer, endometrial cancer, renal cancer, prostate cancer, bladder cancer, pancreatic cancer, melanoma, multiple myeloma, and acute myelogenous leukemia.

Description

Bispecific antibodies and uses thereof Technical Field The application belongs to the technical field of biological medicines, and particularly relates to a bispecific antibody and application thereof, in particular to a bispecific antibody for resisting NKp46 and HLAG and application thereof. Background Bispecific antibodies are antibodies that can specifically bind to two antigenic sites simultaneously. HLAG is a non-classical MHC class I molecule, which is mainly expressed in placental tissue, and is not expressed or is very expressed in normal tissues. However, HLAG exhibits a highly expressed state in many tumor types, such as breast cancer, colorectal cancer, cervical cancer, endometrial cancer, esophageal cancer, gastric cancer, hepatocellular carcinoma, lung cancer, nasopharyngeal cancer, ovarian cancer, and renal cell carcinoma, and is inversely related to the prognosis of a patient. Thus HLAG is a very desirable tumor target. But most antibodies lack truly specific HLA-G binding properties due to the high polymorphism and high homology of the HLA family. NK cells are a very important class of natural immune cells capable of recognizing and killing tumor cells infected or malignant by viruses. NKp46 is an important activating receptor on the surface of NK cells, and crosslinking NKp46 can promote NK cell activation and degranulation, thereby realizing killing of tumor cells. Currently, bispecific antibodies against HLAG and NK cells are few and the binding specificity is low. Therefore, the development of a dual antibody specifically targeting HLAG and NK cells is of great clinical significance. Disclosure of Invention The present application aims to solve at least one of the technical problems existing in the prior art to at least some extent. To this end, the present application provides a bispecific antibody that can simultaneously target HLAG and NKp46, in particular, NKp46 capable of simultaneously binding to the surface of NK cells and HLAG on the surface of tumor cells, thereby directly activating NK cells and killing HLAG positive tumor cells. In a first aspect of the application, the application proposes a bispecific antibody. According to an embodiment of the application, the bispecific antibody comprises one or more first antigen binding regions having NKp46 binding activity, one or more second antigen binding regions having HLAG binding activity, the first and second antigen binding regions being linked, wherein the second antigen binding regions comprise heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3, the HCDR1, HCDR2 and HCDR3 being selected from the group consisting of SEQ ID NOs 7-9 or amino acid sequences having at least 80% homology with SEQ ID NOs 7-9, respectively. The bispecific antibody of the application can simultaneously target HLAG and NKp46, especially can simultaneously bind NKp46 on the surface of NK cells and HLAG on the surface of tumor cells, thereby directly activating NK cells and killing HLAG positive tumor cells. In a second aspect of the application, the application provides a nucleic acid molecule. According to an embodiment of the application, the nucleic acid molecule encodes the bispecific antibody of the first aspect. Nucleic acid molecules according to embodiments of the application may encode bispecific antibodies that can target NKp46 and HLAG simultaneously. In a third aspect of the application, the application provides an expression vector. According to an embodiment of the application, the expression vector comprises the nucleic acid molecule according to the second aspect. Thus, the bispecific antibody of the first aspect can be efficiently expressed by using the constructed expression vector. In a fourth aspect of the application, the application provides a recombinant cell. According to an embodiment of the application, the recombinant cell carries the nucleic acid molecule according to the second aspect or the expression vector according to the third aspect, or expresses the bispecific antibody according to the first aspect. According to an embodiment of the present application, the recombinant cell may be obtained by transfecting or transforming the expression vector of the third aspect, and the bispecific antibody of the first aspect may be efficiently expressed under suitable conditions. In a fifth aspect of the application, the application provides a pharmaceutical composition. According to an embodiment of the application, the pharmaceutical composition comprises a bispecific antibody according to the first aspect, a nucleic acid molecule according to the second aspect, an expression according to the third aspect or a recombinant cell according to the fourth aspect. From the foregoing, it can be seen that the bispecific antibody according to the first aspect or the bispecific antibody prepared by using the nucleic acid molecule according to the second aspect, the expression according to the third aspect or the recombin