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CN-122011183-A - Blocking antibody of targeting CXCL6 and application thereof in preparation of antitumor drugs

CN122011183ACN 122011183 ACN122011183 ACN 122011183ACN-122011183-A

Abstract

The invention relates to the technical field of biological medicines, and discloses a blocking antibody targeting CXCL6 and application thereof in preparation of antitumor drugs. The antibodies or antigen binding fragments thereof are capable of specifically binding to human CXCL6 protein with high affinity (e.g., kd≤1 nM). The invention verifies the specificity and high affinity of the antibody by Western Blot, immunofluorescence, immunohistochemistry and Surface Plasmon Resonance (SPR) technology. Functional experiments show that the antibody can effectively block CXCL 6-induced neutrophil chemotaxis, including in a Transwell, a three-dimensional gel model and a zebra fish living model. In addition, in the mouse MC38 colon cancer model, the antibody exhibited significant anti-tumor activity, capable of inhibiting CXCL 6-driven tumor growth. The invention also provides a pharmaceutical composition comprising the antibody, and the use of the antibody in the manufacture of a medicament for preventing or treating a disease associated with aberrant expression or activity of CXCL6 (e.g., inflammatory disease, autoimmune disease, and cancer).

Inventors

  • ZHANG BIN
  • HE BAOYU
  • BIE QINGLI
  • ZHAO ROU
  • XUN QINGQING
  • LIU JILAN
  • YANG DONG
  • ZHANG BAOGUI

Assignees

  • 济宁医学院附属医院

Dates

Publication Date
20260512
Application Date
20260226

Claims (10)

  1. 1. A blocking antibody that targets CXCL6, which blocking antibody is an isolated monoclonal antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof is capable of specifically binding to human CXCL6 protein and is capable of inhibiting CXCL 6-induced neutrophil chemotaxis.
  2. 2. The blocking antibody targeting CXCL6 according to claim 1, wherein the antibody or antigen binding fragment thereof has a binding dissociation constant Kd of less than or equal to 1 nM, preferably less than or equal to 0.5 nM, more preferably less than or equal to 0.22 nM, to the human CXCL6 protein.
  3. 3. The blocking antibody targeting CXCL6 of claim 1 or 2, wherein said antibody comprises a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID No. 1, or a sequence having at least 90%, 95%, 98% or 99% identity thereto, and a light chain variable region comprising an amino acid sequence as set forth in SEQ ID No. 2, or a sequence having at least 90%, 95%, 98% or 99% identity thereto.
  4. 4. An isolated nucleic acid molecule encoding the heavy chain variable region and/or the light chain variable region of the monoclonal antibody or antigen binding fragment thereof of claim 3.
  5. 5. A pharmaceutical composition comprising a therapeutically effective amount of the CXCL 6-targeting blocking antibody of claim 1, and a pharmaceutically acceptable carrier or excipient.
  6. 6. Use of the blocking antibody targeting CXCL6 of claim 1 or the pharmaceutical composition of claim 5 in the manufacture of an anti-tumor medicament for the prevention or treatment of a disease associated with abnormal expression or activity of CXCL 6.
  7. 7. The use according to claim 6, wherein the disease is selected from inflammatory diseases, autoimmune diseases or cancer, preferably the cancer is colon cancer, liver cancer or lung cancer.
  8. 8. The use of claim 7, wherein the mechanism of action of the medicament comprises inhibiting CXCL 6-induced recruitment of neutrophils to the focal site.
  9. 9. A method of making the monoclonal antibody of claim 1, comprising: (a) Immunizing a non-human animal with an immunogen comprising human CXCL6 protein or an immunogenic fragment thereof; (b) Isolating antibody-producing cells from the immunized animal; (c) Fusing the antibody-producing cells with myeloma cells to form hybridoma cells; (d) Screening for hybridoma cells that produce a monoclonal antibody that specifically binds CXCL6 and inhibits neutrophil chemotaxis induced thereby; (e) Purifying the monoclonal antibody from the hybridoma cell culture supernatant, or producing the monoclonal antibody by recombinant techniques.
  10. 10. A method of non-therapeutically inhibiting CXCL6 bioactivity in vitro comprising contacting a CXCL 6-containing system with an effective amount of the monoclonal antibody or antigen-binding fragment thereof of claim 1 to inhibit CXCL 6-induced neutrophil chemotaxis.

Description

Blocking antibody of targeting CXCL6 and application thereof in preparation of antitumor drugs Technical Field The invention relates to the technical field of biological medicines, in particular to a blocking antibody targeting CXCL6 and application thereof in preparing an antitumor drug. Background Chemokine ligand 6 (CXCL 6), also known as granulocyte chemotactic protein-2 (GCP-2), is an important member of the CXC chemokine family. It plays a central role in chemotaxis, activation, angiogenesis and other physiological and pathological processes of neutrophils by binding to and activating the G-protein coupled receptors CXCR1 and CXCR 2. In the physiological state, CXCL6 is involved in tissue injury repair and host defense. However, its abnormally high expression is closely related to the occurrence, development, angiogenesis, invasion and metastasis of various chronic inflammatory diseases (e.g., rheumatoid arthritis, atherosclerosis) and malignant tumors (e.g., colon cancer, liver cancer, lung cancer) and the formation of immunosuppressive microenvironments. CXCL6 secreted by tumor cells or stromal cells can recruit a large number of tumor-associated neutrophils with a pro-tumor phenotype, thereby promoting tumor progression. Currently, intervention strategies against the CXCL6-CXCR1/2 axis have focused mainly on the development of small molecule receptor antagonists (such as antagonists against CXCR 2) or the use of broad-spectrum chemokine inhibitors. However, small molecule antagonists often suffer from off-target effects, poor pharmacokinetic properties, etc., whereas broad-spectrum inhibitors lack specificity and may interfere with normal immune function. To date, high-specificity, high-affinity blocking monoclonal antibodies targeting CXCL6 have not been successfully developed worldwide and entered the clinical research stage. Therefore, a novel therapeutic antibody capable of precisely blocking CXCL6 from binding with a receptor thereof is developed, and has great significance and application prospect for treating inflammatory diseases and tumors. Disclosure of Invention In order to solve the problems in the background art, the invention is realized by the following technical scheme: A blocking antibody that targets CXCL6, the blocking antibody being an isolated monoclonal antibody or antigen-binding fragment thereof, which antibody or antigen-binding fragment thereof is capable of specifically binding to human CXCL6 protein and is capable of inhibiting CXCL 6-induced neutrophil chemotaxis. Further, the antibody or antigen-binding fragment thereof has a binding dissociation constant Kd of less than or equal to 1 nM, preferably less than or equal to 0.5 nM, more preferably less than or equal to 0.22 nM, to the human CXCL6 protein. Further, the antibody comprises a heavy chain variable region (VH) comprising an amino acid sequence as set forth in SEQ ID NO. 1, or a sequence having at least 90%, 95%, 98% or 99% identity thereto, and a light chain variable region (VL) comprising an amino acid sequence as set forth in SEQ ID NO. 2, or a sequence having at least 90%, 95%, 98% or 99% identity thereto. An isolated nucleic acid molecule encoding the heavy chain variable region and/or the light chain variable region of said monoclonal antibody or antigen binding fragment thereof. An expression vector comprising said nucleic acid molecule. A host cell comprising said expression vector or said nucleic acid molecule. A pharmaceutical composition comprising a therapeutically effective amount of said monoclonal antibody or antigen-binding fragment thereof, and a pharmaceutically acceptable carrier or excipient. The use of said blocking antibody targeting CXCL6 or said pharmaceutical composition for the manufacture of an anti-tumor medicament for the prevention or treatment of a disease associated with aberrant CXCL6 expression or activity. Further, the disease is selected from inflammatory diseases, autoimmune diseases or cancers, preferably the cancer is colon cancer, liver cancer or lung cancer. Further, the mechanism of action of the drug includes inhibition of CXCL 6-induced recruitment of neutrophils to the focal site. A method of making the monoclonal antibody comprising: (a) Immunizing a non-human animal with an immunogen comprising human CXCL6 protein or an immunogenic fragment thereof; (b) Isolating antibody-producing cells from the immunized animal; (c) Fusing the antibody-producing cells with myeloma cells to form hybridoma cells; (d) Screening for hybridoma cells that produce a monoclonal antibody that specifically binds CXCL6 and inhibits neutrophil chemotaxis induced thereby; (e) Purifying the monoclonal antibody from the hybridoma cell culture supernatant, or producing the monoclonal antibody by recombinant techniques. A method of non-therapeutically inhibiting CXCL6 bioactivity in vitro comprising contacting a system comprising CXCL6 with an effective amount of said monoclonal antibody or antige