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CN-121975018-A - Trispecific antibodies and uses thereof

CN121975018ACN 121975018 ACN121975018 ACN 121975018ACN-121975018-A

Abstract

The application discloses a trispecific antibody which comprises a first antigen binding region, a second antigen binding region, a complex of IL-15 and a receptor thereof and an Fc fragment, wherein the first antigen binding region has CD16 binding activity, the second antigen binding region has HLAG binding activity, and the complex of the IL-15 and the receptor thereof in the first antigen binding region and the second antigen binding region are respectively connected with the Fc fragment directly or indirectly. The trispecific antibody of the application can simultaneously bind HLAG on tumor cells, CD16 and IL-15 receptor on NK cells, promote cell synapses between the two cells, further activate NK cells to kill HLAG positive tumor cells, and promote NK cell proliferation. Thus, the trispecific antibody of the application can kill HLAG positive tumor cells, and is effective in treating HLAG-mediated related diseases (such as cancers).

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
20260505
Application Date
20241029

Claims (13)

  1. 1. A trispecific antibody comprising a first antigen-binding region, a second antigen-binding region, a complex of IL-15 and its receptor, and an Fc fragment; The first antigen binding region has CD16 binding activity; The second antigen binding region has HLAG binding activity; The first antigen binding region, the second antigen binding region, and the complex of IL-15 and its receptor are linked directly or indirectly to the Fc fragment, respectively.
  2. 2. The trispecific antibody of claim 1, wherein the trispecific antibody is characterized by at least one of the following: i) The first antigen binding region is linked to the N-terminus or the C-terminus of the Fc fragment; ii) the first antigen binding region is selected from a scFab fragment or a scFv fragment; iii) The first antigen binding region comprises HCDRs and LCDRs therein, the HCDRs and/or LCDRs being defined by Kabat, chothia, abM, contact or IMGT; iv) 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; v) the second antigen binding region is linked to the N-terminus or C-terminus of the Fc fragment; vi) the second antigen binding region is selected from a Fab fragment; vii) including HCDRs and LCDRs in the second antigen-binding region, the HCDRs and/or LCDRs being defined by Kabat, chothia, abM, contact or IMGT; viii) the second antigen binding region comprises: HCDR1, HCDR2 and HCDR3 respectively shown as SEQ ID NO 7-9 amino acid sequence, LCDR1, LCDR2 and LCDR3 respectively shown as SEQ ID NO 10-12 amino acid sequences; ix) the complex of IL-15 with its receptor is linked to the N-terminus or the C-terminus of the Fc fragment; x) the complex of IL-15 and its receptor is a complex of IL-15 and its receptor sushi domain.
  3. 3. The trispecific antibody of claim 2, wherein the trispecific antibody is characterized by at least one of the following: a) The C end of the scFv fragment is connected with the N end of the Fc fragment; b) The second antigen binding region is linked to the N-terminus of the Fc fragment via the first antigen binding region; c) The C-terminus of the complex of IL-15 and its receptor sushi domain is linked to the N-terminus of the Fc fragment; d) The first antigen binding region has a heavy chain variable region shown as an amino acid sequence of SEQ ID NO. 13 and a light chain variable region shown as an amino acid sequence of SEQ ID NO. 14; e) The second antigen binding region has a heavy chain variable region as shown in the amino acid sequence of SEQ ID NO. 15 and a light chain variable region as shown in the amino acid sequence of SEQ ID NO. 16.
  4. 4. The trispecific antibody of claim 2, wherein the trispecific antibody is characterized by at least one of the following: a1 The C-terminal of the light chain variable region in the scFv fragment is connected with the N-terminal of the heavy chain variable region in the scFv fragment; a2 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 bovine 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; b1 The C-terminus of the CH1 fragment in said Fab fragment is linked to the N-terminus of said first antigen binding region; b2 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 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; b3 The CH1 fragment and the CL fragment in the Fab fragment are respectively and 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; b4 CH1 fragment selected from the group consisting of IgG1, igG2, igG3, igG4, igA, igM, igE, and IgD, and/or The CL fragment in the Fab fragment is selected from a kappa type or lambda type CL fragment; c1 The complex of IL-15 with its receptor is selected from the group consisting of fusion proteins of the IL-15-linker-sushi domain.
  5. 5. The trispecific antibody of any one of claims 2-4, wherein the trispecific antibody has at least one of the following characteristics: f) The scFv fragment has an amino acid sequence shown as SEQ ID NO. 17; g) One chain of the Fab fragment has an amino acid sequence shown as SEQ ID NO. 18, and the other chain has an amino acid sequence shown as SEQ ID NO. 19; h) The complex of the IL-15 and the receptor thereof has an amino acid sequence shown as SEQ ID NO. 20; j) The C-terminal of the CH1 fragment in the Fab fragment is connected with the N-terminal of the scFv fragment, the C-terminal of the scFv fragment is connected with the N-terminal of one chain in the Fc fragment, and the C-terminal of the complex of the IL-15 and the receptor thereof is connected with the N-terminal of the other chain in the Fc fragment; k) The two chains of the Fc fragment are connected through a knob-intoo-hole structure; l) the Fc fragment is 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 a human Fc fragment; m) the trispecific antibody further comprises a connecting peptide, wherein the first antigen-binding region and the second antigen-binding region are connected through the connecting peptide; Optionally, the 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; Optionally, the amino acid sequence of the connecting peptide is shown as SEQ ID NO. 29; 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, one chain of the Fc fragment has the amino acid sequence shown as SEQ ID NO. 21 and the other chain has the amino acid sequence shown as SEQ ID NO. 22.
  6. 6. The trispecific antibody of claim 1, wherein the trispecific antibody comprises: has a first polypeptide chain as shown in the amino acid sequence of SEQ ID NO. 23, Has a second polypeptide chain as shown in the amino acid sequence of SEQ ID NO. 24, and Has a third polypeptide chain shown as SEQ ID NO. 25 amino acid sequence.
  7. 7. A nucleic acid molecule encoding the trispecific antibody of any one of claims 1-6.
  8. 8. An expression vector comprising the nucleic acid molecule of claim 7; 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.
  9. 9. A recombinant cell carrying the nucleic acid molecule of claim 7 or the expression vector of claim 8, or expressing the trispecific antibody of any one of claims 1-6; Optionally, the recombinant cell is obtained by introducing the expression vector of claim 8 into a host cell; optionally, the recombinant cell is a prokaryotic cell or a eukaryotic cell.
  10. 10. A pharmaceutical composition comprising the trispecific antibody of any one of claims 1-6, the nucleic acid molecule of claim 7, the expression vector of claim 8 or the recombinant cell of claim 9; optionally, further comprising pharmaceutically acceptable excipients.
  11. 11. A kit comprising the trispecific antibody of any one of claims 1 to 6, the nucleic acid molecule of claim 7, the expression vector of claim 8 or the recombinant cell of claim 9.
  12. 12. Use of the multispecific antibody of any one of claims 1 to 6, the nucleic acid molecule of claim 7, the expression vector of claim 8 or the recombinant cell of claim 9 in the preparation of a kit for detecting CD16 and/or HLAG.
  13. 13. Use of the multispecific antibody of any one of claims 1-6, the nucleic acid molecule of claim 7, the expression vector of claim 8 or the recombinant cell of claim 9 or the pharmaceutical composition of claim 10 in the manufacture of a medicament for the prevention and/or treatment of a CD16 and/or HLAG-mediated related disease; Optionally, the CD16 and/or HLAG-mediated related disease includes cancer; optionally, the cancer comprises at least one of breast cancer, colorectal cancer, cervical cancer, esophageal cancer, gastric cancer, hepatocellular cancer, lung cancer, nasopharyngeal cancer, ovarian cancer, choriocarcinoma, renal cell carcinoma, glioma, thyroid cancer, cholangiocarcinoma, prostate cancer, lung cancer, liver cancer, testicular cancer, endometrial cancer, skin cancer, bladder cancer, glioma, renal cancer, oral squamous cell carcinoma, head and neck cancer, pancreatic cancer, melanoma, multiple myeloma, acute myelogenous leukemia.

Description

Trispecific antibodies and uses thereof Technical Field The application belongs to the technical field of biological medicines, and particularly relates to a trispecific antibody and application thereof. Background 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. Therefore, based on HLAG targets, it is necessary to explore related multispecific antibodies. 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. Therefore, the application provides a trispecific antibody which can simultaneously bind HLAG on tumor cells, CD16 on NK cells and IL-15 receptors, promote cell synapses between the two cells, further activate NK cells to kill HLAG positive tumor cells, and promote NK cell proliferation. The present application has been completed based on the following findings by the inventors: Natural Killer (NK) cells are a very important class of natural immune cells that are capable of recognizing and killing tumor cells that are infected or malignant by viruses. CD16 is an important activating receptor on the surface of NK cells, and crosslinking CD16 can cause ADCC. Although antibodies developed based on CD16 can well promote NK cells to kill tumor cells, their effect is limited because NK cells cannot survive for a long period of time. Human cytokines IL-15 are normally secreted by myeloid cells and by some epithelial cells, IL-15 can interact with the IL-15 receptors on the surfaces of dendritic cells, CD 8T cells and NK cells, thereby promoting activation and proliferation of these cells and secretion of the corresponding cytokines. IL-15 receptors have 3 chains, respectively the alpha, beta and gamma chains, wherein the IL-15 alpha chain is unique to IL-15 and the receptor beta chain is identical to the IL-2 receptor beta chain, and IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21 receptors all have identical gamma chains. IL-15 is typically bound to the IL-15Rα chain and then to the β and γ chains to form a polymer. Trispecific antibodies targeting both CD16 and IL-15 receptors on HLAG, NK cells have not been reported at present. Based on this, in a first aspect of the application, the application proposes a trispecific antibody. According to an embodiment of the application, the trispecific antibody comprises a first antigen-binding region, a second antigen-binding region, a complex of IL-15 and its receptor, and an Fc fragment, wherein the first antigen-binding region has CD16 binding activity, the second antigen-binding region has HLAG binding activity, and the complexes of the first antigen-binding region, the second antigen-binding region, and the IL-15 and its receptor are respectively linked directly or indirectly to the Fc fragment. The trispecific antibody of the application can simultaneously bind HLAG on tumor cells, CD16 and IL-15 receptor on NK cells, promote cell synapses between the two cells, further activate NK cells to kill HLAG positive tumor cells, and promote NK cell proliferation. Thus, the trispecific antibody of the application can kill HLAG positive tumor cells, and is effective in treating HLAG-mediated related diseases (such as cancers). According to an embodiment of the application, the trispecific antibody has at least one of the following features: i) The first antigen binding region is linked to the N-terminus or the C-terminus of the Fc fragment; ii) the first antigen binding region is selected from a scFab fragment or a scFv fragment; iii) The first antigen binding region comprises HCDRs and LCDRs therein, the HCDRs and/or LCDRs being defined by Kabat, chothia, abM, contact or IMGT; iv) 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; v) the second antigen binding region is linked to the N-terminus or C-terminus of the Fc fragment; vi) the second antigen binding region is selected from a Fab fragment; vii) including HCDRs and LCDRs in the second antigen-binding region, the HCDRs and/or LCDRs being defined by Kabat, chothia, abM, contact or IMGT; viii) the second antigen binding region comprises: HCDR1, HCDR2 and HCDR3 respectively shown as SEQ ID NO 7-9 amino acid sequence, LCDR1, LCDR2 and LCDR3 respectively shown as SEQ ID NO 10-12 amino acid sequences; ix) the complex of IL-15 with its receptor is linked to the N-terminus or the C-terminus of the Fc