CXCL6-targeting blocking antibody and application thereof in preparation of antitumor drugs

Abstract

The invention relates to the technical field of biological medicines, and discloses a blocking antibody targeting CXCL6 and an application of the blocking antibody in preparation of antitumor drugs. The antibody or antigen-binding fragment thereof is capable of binding specifically to human CXCL6 protein with high affinity (e.g., Kd < = 1 nM). According to the invention, the specificity and high affinity of the antibody are verified by Western Blot, immunofluorescence, immunohistochemistry and surface plasmon resonance (SPR) technologies. Functional experiments show that the antibody can effectively block CXCL6-induced neutrophil chemotaxis, including in Transwell, a three-dimensional gel model and a zebra fish living body model. Besides, in a mouse MC38 colon cancer model, the antibody shows remarkable anti-tumor activity and can inhibit tumor growth driven by CXCL6. The invention also provides a pharmaceutical composition containing the antibody, and application of the antibody in preparation of drugs for preventing or treating diseases (such as inflammatory diseases, autoimmune diseases and cancers) related to abnormal expression or activity of CXCL6.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to blocking antibodies targeting CXCL6 and their application in the preparation of antitumor drugs. Background Technology

[0002] Chemokine ligand 6 (CXCL6), also known as granulocyte chemoattractant protein-2 (GCP-2), is an important member of the CXC chemokine family. It plays a central role in physiological and pathological processes such as neutrophil chemotaxis, activation, and angiogenesis by binding to and activating G protein-coupled receptors CXCR1 and CXCR2. Under physiological conditions, CXCL6 participates in tissue damage repair and host defense. However, its abnormally high expression is closely related to the occurrence, development, angiogenesis, invasion, metastasis, and formation of an immunosuppressive microenvironment in various chronic inflammatory diseases (such as rheumatoid arthritis and atherosclerosis) and malignant tumors (such as colon cancer, liver cancer, and lung cancer). CXCL6 secreted by tumor cells or stromal cells can recruit large numbers of tumor-associated neutrophils with pro-tumor phenotypes, thereby promoting tumor progression.

[0003] Currently, intervention strategies targeting the CXCL6-CXCR1 / 2 axis mainly focus on developing small molecule receptor antagonists (such as CXCR2 antagonists) or using broad-spectrum chemokine inhibitors. However, small molecule antagonists often face problems such as off-target effects and poor pharmacokinetic properties, while broad-spectrum inhibitors lack specificity and may interfere with normal immune function. To date, no highly specific, high-affinity blocking monoclonal antibody targeting CXCL6 has been successfully developed and entered clinical trials globally. Therefore, developing a novel therapeutic antibody that can precisely block the binding of CXCL6 to its receptor has significant implications and promising applications for the treatment of inflammatory diseases and tumors. Summary of the Invention

[0004] To address the problems mentioned in the background section, the present invention is implemented through the following technical solution:

[0005] A blocking antibody targeting CXCL6, wherein the blocking antibody is an isolated monoclonal antibody or its antigen-binding fragment, the antibody or its antigen-binding fragment being able to specifically bind to human CXCL6 protein and inhibit CXCL6-induced neutrophil chemotaxis.

[0006] Furthermore, the binding dissociation constant Kd between the antibody or its antigen-binding fragment and the human CXCL6 protein is less than or equal to 1 nM, preferably less than or equal to 0.5 nM, and more preferably less than or equal to 0.22 nM.

[0007] Furthermore, the antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 1, or a sequence having at least 90%, 95%, 98%, or 99% identity with it; and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 2, or a sequence having at least 90%, 95%, 98%, or 99% identity with it.

[0008] An isolated nucleic acid molecule that encodes the heavy chain variable region and / or light chain variable region of the monoclonal antibody or its antigen-binding fragment.

[0009] An expression vector comprising the aforementioned nucleic acid molecule.

[0010] A host cell comprising the expression vector or the nucleic acid molecule described herein.

[0011] A pharmaceutical composition comprising a therapeutically effective amount of the said monoclonal antibody or its antigen-binding fragment, and a pharmaceutically acceptable carrier or excipient.

[0012] The use of the CXCL6-targeting blocking antibody or the pharmaceutical composition described herein in the preparation of an antitumor drug for the prevention or treatment of diseases associated with abnormal expression or activity of CXCL6.

[0013] Furthermore, the disease is selected from inflammatory diseases, autoimmune diseases, or cancer, preferably colon cancer, liver cancer, or lung cancer.

[0014] Furthermore, the mechanism of action of the drug includes inhibiting CXCL6-induced recruitment of neutrophils to the lesion site.

[0015] A method for preparing the monoclonal antibody, comprising:

[0016] (a) Immunize non-human animals with an immunogen containing human CXCL6 protein or an immunogenic fragment thereof;

[0017] (b) Isolating antibody-producing cells from the immunized animal;

[0018] (c) The antibody-producing cells are fused with myeloma cells to form hybridoma cells;

[0019] (d) Screening for hybridoma cells that can produce monoclonal antibodies that specifically bind to CXCL6 and inhibit its induced neutrophil chemotaxis;

[0020] (e) Purify the monoclonal antibody from the hybridoma cell culture supernatant, or produce the monoclonal antibody by recombinant technology.

[0021] A method for in vitro non-therapeutic inhibition of CXCL6 biological activity includes contacting a system containing CXCL6 with an effective amount of the said monoclonal antibody or its antigen-binding fragment to inhibit CXCL6-induced neutrophil chemotaxis.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] 1. This CXCL6-targeting blocking antibody and its application in the preparation of anti-tumor drugs provide, for the first time, a highly specific, high-affinity neutralizing monoclonal antibody against the key chemokine CXCL6, filling the gap in antibody drugs targeting this target.

[0024] 2. This CXCL6-targeting blocking antibody and its application in the preparation of anti-tumor drugs: The antibody precisely blocks the binding of CXCL6 to its receptor, inhibiting downstream signaling pathways at the source, and has the advantages of a clear mechanism of action and precise target. Compared with broad-spectrum inhibitors or small molecule receptor antagonists, it has a lower potential risk of side effects.

[0025] 3. This invention conclusively demonstrates, through multi-level and multi-faceted verification from molecular, cellular, tissue, and live animal studies, that the CXCL6-targeting blocking antibody and its application in the preparation of antitumor drugs possess: ① high affinity (SPR assay Kd = 0.22 nM); ② broad cross-reactivity and specificity; ③ effective in vitro and in vivo neutrophil chemotaxis blocking function; and ④ clear in vivo antitumor activity. The solid data provide a robust experimental foundation for its clinical application.

[0026] 4. This CXCL6-targeting blocking antibody and its application in the preparation of anti-tumor drugs provide novel and promising candidate drugs and treatment strategies for treating a series of CXCL6-driven inflammatory diseases and malignant tumors. Attached Figure Description

[0027] Figure 1 This is a schematic diagram of the Western blot results of the anti-CXCL6 monoclonal antibody of the present invention in different cell lines.

[0028] Figure 2 This is a schematic diagram of the immunofluorescence localization of the anti-CXCL6 monoclonal antibody of the present invention in RAW264.7 cells.

[0029] Figure 3 This is a schematic diagram of immunohistochemical staining of human colon cancer tissue with the anti-CXCL6 monoclonal antibody of this invention.

[0030] Figure 4 This is a sensor diagram showing the SPR binding kinetics analysis of the anti-CXCL6 monoclonal antibody and recombinant human CXCL6 protein according to the present invention.

[0031] Figure 5 This is a graph showing the inhibitory effect of the anti-CXCL6 monoclonal antibody of this invention on CXCL6-induced neutrophil chemotaxis in the Transwell model.

[0032] Figure 6 This is a diagram showing the inhibitory effect of the anti-CXCL6 monoclonal antibody of this invention on neutrophil chemotaxis in a three-dimensional collagen gel model.

[0033] Figure 7 This is a graph showing the inhibitory effect of the anti-CXCL6 monoclonal antibody of this invention on CXCL6-induced neutrophil recruitment in a zebrafish in vivo model.

[0034] Figure 8 This is a graph showing the anti-tumor activity of the anti-CXCL6 monoclonal antibody of this invention in a mouse MC38 colon cancer model. Detailed Implementation

[0035] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0036] The following are examples of the CXCL6-targeting blocking antibody and its application in the preparation of antitumor drugs:

[0037] A blocking antibody targeting CXCL6 is disclosed. The blocking antibody is an isolated monoclonal antibody or its antigen-binding fragment, which specifically binds to human CXCL6 protein and inhibits CXCL6-induced neutrophil chemotaxis. The binding dissociation constant Kd between the antibody or its antigen-binding fragment and the human CXCL6 protein is less than or equal to 1 nM, preferably less than or equal to 0.5 nM, and more preferably less than or equal to 0.22 nM. The antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL). The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 1, or a sequence having at least 90%, 95%, 98%, or 99% identity with it; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 2, or a sequence having at least 90%, 95%, 98%, or 99% identity with it.

[0038] An isolated nucleic acid molecule that encodes the heavy chain variable region and / or light chain variable region of the monoclonal antibody or its antigen-binding fragment.

[0039] An expression vector comprising the aforementioned nucleic acid molecule.

[0040] A host cell comprising the expression vector or the nucleic acid molecule described herein.

[0041] A pharmaceutical composition comprising a therapeutically effective amount of the said monoclonal antibody or its antigen-binding fragment, and a pharmaceutically acceptable carrier or excipient.

[0042] The use of the CXCL6-targeting blocking antibody or the pharmaceutical composition described herein in the preparation of an antitumor drug for the prevention or treatment of diseases associated with abnormal expression or activity of CXCL6. The diseases are selected from inflammatory diseases, autoimmune diseases, or cancer, preferably colorectal cancer, liver cancer, or lung cancer. The mechanism of action of the drug includes inhibiting CXCL6-induced recruitment of neutrophils to lesion sites.

[0043] A method for preparing the monoclonal antibody includes: immunizing a non-human animal with an immunogen containing human CXCL6 protein or an immunogenic fragment thereof; isolating antibody-producing cells from the immunized animal; fusing the antibody-producing cells with myeloma cells to form hybridoma cells; screening hybridoma cells capable of producing monoclonal antibodies that specifically bind to CXCL6 and inhibit its induced neutrophil chemotaxis; purifying the monoclonal antibody from the culture supernatant of the hybridoma cells, or producing the monoclonal antibody using recombinant technology.

[0044] A method for in vitro non-therapeutic inhibition of CXCL6 biological activity includes contacting a system containing CXCL6 with an effective amount of the said monoclonal antibody or its antigen-binding fragment to inhibit CXCL6-induced neutrophil chemotaxis.

[0045] Example 1: Preparation of anti-CXCL6 monoclonal antibody

[0046] Recombinant human CXCL6 protein was used as an immunogen to immunize 6-8 week old BALB / c mice. Standard hybridoma technology was employed: spleen cells from immunized mice were fused with SP2 / 0 myeloma cells, and hybridoma cells were screened using HAT selective medium. Hybridoma cell lines whose supernatants could bind to CXCL6 protein were screened using an indirect ELISA method. Positive wells were subcloned using limiting dilution to obtain a stable hybridoma cell line secreting monoclonal antibodies, named 6C12. Antibodies were mass-produced using mouse ascites preparation or in vitro serum-free culture and purified using a Protein A / G affinity chromatography column.

[0047] Example 2: Validation of antibody specificity and cross-reactivity (Western Blot)

[0048] Six cell lines—THP-1 (human monocytic leukemia cells), NK-92 (human natural killer cells), MT-4 (human T-cell leukemia cells), RAW264.7 (mouse macrophages), HEK293T (human embryonic kidney cells), and NCM460 (human normal colonic epithelial cells)—were cultured separately, and total cellular protein was extracted. When cells reached 80-90% confluence, the culture medium was discarded, and the cells were washed twice with pre-chilled PBS. 200 μL of RIPA lysis buffer containing protease and phosphatase inhibitors was added to every 10⁷ cells, and the cells were lysed on ice for 30 minutes. The cells were centrifuged at 12,000 rpm for 15 minutes at 4°C, and the supernatant was collected as total cellular protein. Protein concentration was determined using the BCA method. 30 μg of total protein was subjected to SDS-PAGE electrophoresis (12% separating gel), and then transferred to a PVDF membrane. The membrane was blocked with 5% skim milk at room temperature for 1 hour. Purified anti-CXCL6 monoclonal antibody 6C12 (1:1000 dilution) was used as the primary antibody and incubated overnight at 4°C. After washing three times with TBST, HRP-labeled secondary antibody of the corresponding species (1:5000 dilution) was added and incubated at room temperature for 1 hour. After thorough washing with TBST, the gel was developed using ECL chemiluminescent substrate, and images were acquired in a gel imaging system for Western blotting.

[0049] The results are as follows Figure 1 As shown, the antibody recognized a single, clear band of approximately 8-10 kDa in THP-1 and RAW264.7 cells, consistent with the expected molecular weight of CXCL6. A weak band was observed in NK-92 and MT-4 cells, while no obvious band was observed in HEK293T and NCM460 cells, demonstrating the antibody's specificity. These results show that the antibody of this invention can specifically recognize CXCL6 protein bands from multiple cell sources, demonstrating its good cross-reactivity and broad applicability, laying a solid foundation for subsequent functional studies.

[0050] Example 3: Antibody subcellular localization verification (immunofluorescence)

[0051] Considering that CXCL6 is a secreted chemokine, its synthesis and processing mainly occur in the cytoplasm, immunofluorescence (IF) experiments were performed using mouse macrophage RAW264.7 cells. RAW264.7 cells were seeded in confocal culture dishes. After cell adhesion, they were fixed with 4% paraformaldehyde at room temperature for 15 minutes. Permeabilization was performed with 0.2% Triton X-100 for 10 minutes, followed by blocking with 3% BSA at room temperature for 30 minutes. Anti-CXCL6-mAb (1:200 dilution) was added as the primary antibody, and the cells were incubated overnight at 4°C. After washing with PBS, Alexa Fluor 594-labeled goat anti-mouse IgG secondary antibody (1:500 dilution) was added, and the cells were incubated at room temperature in the dark for 1 hour. After washing with PBS, the nuclei were stained with DAPI for 5 minutes. Finally, the cells were mounted with anti-fluorescence quenching mounting medium, and images were observed and acquired under a laser confocal microscope. The results clearly show that the CXCL6 signal recognized by the antibody of this invention is mainly located in the cytoplasmic region, which is highly consistent with the biological characteristics of CXCL6, further confirming the specificity of the antibody.

[0052] The results are as follows Figure 2 As shown, RAW264.7 cells were stained with immunofluorescence using the anti-CXCL6-mAb of this invention. Red fluorescence (Alexa Fluor 594) represents the binding signal of the anti-CXCL6-mAb, and blue fluorescence (DAPI) labels the cell nucleus. The results showed that the green fluorescence signal was widely distributed in the cytoplasm, appearing as granules or diffuse patterns, consistent with the localization characteristics of CXCL6 as a secreted protein synthesized, processed, and stored in the endoplasmic reticulum-Golgi system. No obvious green fluorescence signal was observed in the cell nucleus. This result confirms that this antibody can specifically recognize and bind to endogenous CXCL6 protein within cells.

[0053] Example 4: Validation of antibody recognition ability in pathological tissues (immunohistochemistry)

[0054] To bridge basic research with clinical application, the antibody of this invention was used to perform immunohistochemical (IHC) staining on human colon cancer tissue sections. Human colon cancer and paired adjacent normal tissue samples were collected, fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 4μm thick sections. After dewaxing and hydration, the sections underwent antigen heat retrieval (sodium citrate buffer, pH 6.0). Incubation with 3% H2O2 at room temperature for 10 minutes was performed to inactivate endogenous peroxidase. Blocking with 10% normal goat serum at room temperature for 30 minutes was performed. Anti-CXCL6-mAb (1:100 dilution) was added, and incubation was carried out overnight at 4°C. After washing with PBS, HRP-labeled secondary antibody was added, and incubation was carried out at room temperature for 30 minutes. DAB staining was performed, followed by hematoxylin counterstaining, dehydration, clearing, and mounting. The sections were observed and photographed under an optical microscope. The experiment successfully detected a positive expression signal of CXCL6 in tumor tissue, indicating that this antibody is not only suitable for laboratory cell models but also effectively applied to the detection of clinical pathological samples, possessing potential diagnostic value.

[0055] The results are as follows Figure 3 As shown in the immunohistochemical images, tumor cell cytoplasm and some tumor stromal cells (presumably tumor-infiltrating immune cells) exhibit strong positive brown staining. This result demonstrates that this antibody can effectively recognize and stain CXCL6 protein in histopathological sections.

[0056] Example 5: Antibody affinity assay (surface plasmon resonance, SPR)

[0057] The interaction between the antibody of this invention and the in vitro purified recombinant human CXCL6 protein was directly detected using surface plasmon resonance (SPR) technology. The recombinant human CXCL6 protein was immobilized on the surface of a CM5 chip using an amino-coupled method, forming a density of approximately 500 RU. The running buffer was HBS-EP+ (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% P2O, pH 7.4). The purified anti-CXCL6-mAb was injected sequentially from low to high concentrations (3.125 nM, 6.25 nM, 12.5 nM, 25 nM, 50 nM) at a flow rate of 30 μL / min, with a binding time of 120 seconds and a dissociation time of 300 seconds. After each analysis, the chip surface was regenerated with 10 mM glycine (pH 2.0). The sensor spectrum was fitted using a 1:1 Langmuir binding model, and the equilibrium dissociation constant (Kd) was calculated. Kinetic analysis showed a significant and stable binding between the two (Kd = 0.22 nM), confirming that the antibody of this invention has a high affinity for the CXCL6 target, which is a prerequisite for its functional blocking effect.

[0058] The results are as follows Figure 4As shown, the sensor map (response-time curve) of different concentrations of anti-CXCL6-mAb (colored curve, concentration increasing from bottom to top) flowing through the surface of a chip immobilized with CXCL6 protein is displayed. Arrows indicate the start and end of injection. The curve showing the relationship between the steady-state binding response value at the end of the binding phase and the antibody concentration is also shown (steady-state fitting). The fitting calculation yielded a kd of 0.22 nM for this antibody and CXCL6 protein. The results indicate that this antibody has high affinity for CXCL6 and a slow dissociation rate.

[0059] Example 6: In vitro blocking of CXCL6-induced neutrophil chemotaxis by antibody (Transwell assay)

[0060] Neutrophils were isolated from peripheral blood of healthy volunteers using density gradient centrifugation. 100 ng / mL of recombinant human CXCL6 protein (rCXCL6) was added to the lower chamber of a 24-well plate as a chemotactic agent. The upper chamber was a 3 μm Transwell chamber. The experiment consisted of three groups: ① control group (lower chamber contained only culture medium); ② CXCL6 group (lower chamber contained rCXCL6); ③ rCXCL6 + antibody group (lower chamber contained CXCL6, and anti-CXCL6-mAb (10 μg / mL) was pre-incubated with CXCL6 for 30 minutes before being added to the lower chamber, or the antibody was directly added to the cell suspension in the upper chamber). One × 10⁵ neutrophils were added to the upper chamber of each group, and chemotaxis was performed at 37°C in a 5% CO₂ incubator for 4 hours. Cells were gently wiped from the inner surface of the upper chamber membrane with a cotton swab, and cells that migrated to the lower chamber were collected and counted using trypan blue staining. The results showed that, compared with the control group, the number of migrating Neu was significantly reduced in the experimental group with the addition of CXCL6 blocking antibody (p < 0.0001), proving that the antibody can effectively block CXCL6-mediated transmembrane chemotaxis.

[0061] The results are as follows Figure 5 As shown in Figures AB: A 24-well Transwell plate was used. The lower chamber was filled with 500 μL of RPMI-1640 medium containing 10% FBS, medium containing recombinant rCXCL6 protein, and medium containing rCXCL6 + CXCL6 mAb, respectively. 1 × 10⁵ neutrophils were seeded in the upper chamber. After 4 hours of incubation, unmigrated cells in the upper chamber were wiped away with a cotton swab, and the cells that migrated to the lower chamber were fixed, stained, and counted. The bar chart shows the number of neutrophils that migrated to the lower chamber (mean ± standard deviation, n=3). Compared with the control group, CXCL6 (100 ng / mL) significantly induced neutrophil migration (p < 0.001). Pretreatment with 10 μg / mL anti-CXCL6-mAb resulted in a 95% chemotactic effect (p < 0.001 compared to the rCXCL6 group). These results demonstrate in vitro that this antibody can effectively block the chemotactic function of CXCL6.

[0062] Example 7: Blocking of neutrophil chemotaxis by antibodies in a three-dimensional environment (collagen gel migration assay)

[0063] A neutral mixture containing 1.5 mg / mL collagen I and 100 ng / mL rCXCL6 was prepared. 10 μg / mL anti-CXCL6-mAb was added to the experimental group mixture, while no antibody was added to the control group. Polymerization was carried out at 37°C for 30 minutes to form a gel. A culture medium containing 1% agarose was prepared, and after the agar solidified, human neutrophils (1×10⁶ / mL) pre-labeled with Calcein-AM green fluorescent dye were seeded at one end of the gel. After incubation at 37°C and 5% carbon dioxide for 4 hours, the entire migration area was automatically scanned using an inverted fluorescence microscope, and the number of migrating neutrophils was analyzed using ImageJ software. The results again confirmed that the antibody of this invention can significantly inhibit the directional migration ability of neutrophils in the three-dimensional matrix (p < 0.0001).

[0064] The results are as follows Figure 6 As shown in Figure A: Representative cell migration trajectories. In gels containing only CXCL6, neutrophils exhibited clear directional migration towards the rCXCL6 concentration gradient (longer trajectories with concentrated direction). In gels containing both rCXCL6 and CXCL6-mAb, cell migration trajectories were significantly shortened and the direction was random. Figure B: Quantitative analysis. Compared to the CXCL6 group, the number of migrating neutrophils in the antibody-treated group was significantly reduced (p < 0.0001). This experiment confirmed the blocking effect of the invented antibody on neutrophil chemotaxis in a model simulating the three-dimensional structure of tissue.

[0065] Example 8: Antibody inhibits neutrophil recruitment in an in vivo model (zebrafish model)

[0066] To verify the antibody's effectiveness in a more physiological in vivo environment, a transgenic zebrafish model (lyz:dsRed, where neutrophils express red fluorescent protein) was used. Western blot experiments have shown that RAW264.7 cells express more CXCL6 protein, while HEK293T cells express very little CXCL6. Therefore, HEK293T or RAW264.7 cells expressing green fluorescent protein (GFP) were microinjected into the yolk sac of juvenile zebrafish. Subsequently, the CXCL6 blocking antibody of this invention or control IgG was added to the system. Six hours after injection, the yolk sac region was observed using a fluorescence microscope. Quantitative analysis showed that the number of red neutrophils recruited by RAW264.7 cells in the CXCL6 blocking antibody treatment group was significantly lower than that in the control group (p < 0.0001).

[0067] The results are as follows Figure 7 As shown, lyz:dsRed transgenic zebrafish larvae (whose neutrophils express red fluorescent protein) were selected 2 days after fertilization. Approximately 100 HEK293T cells or RAW264.7 cells (containing GFP green fluorescent protein) were injected into the zebrafish yolk sac using a microinjection system. Six hours after injection, the yolk sac was observed and photographed under a fluorescence microscope, and the number of recruited red neutrophils within the yolk sac was quantitatively analyzed using ZEN software.

[0068] Example 9: Validation of the in vivo antitumor activity of the antibody (mouse tumor model)

[0069] A tumor model was established by subcutaneously inoculating MC38 colon cancer cells into C57BL / 6 mice. After the tumor volume reached approximately 100 mm³, the mice were randomly divided into three groups of eight mice each: ① Vehicle + IgG Ab group: administered an equal volume of PBS and isotype control IgG antibody; ② rCXCL6 + IgG Ab group: administered recombinant CXCL6 protein and isotype control IgG antibody; ③ rCXCL6 + CXCL6 Ab group: administered recombinant CXCL6 protein and the CXCL6 antibody of this invention.

[0070] Tumor volume was recorded every 3 days, and the results were as follows: Figure 8 As shown, compared with the Vehicle + IgG Ab group, tumor growth was significantly accelerated in the rCXCL6 + IgG Ab group, indicating that exogenous CXCL6 promotes tumor growth. However, after administration of the CXCL6 antibody of this invention (rCXCL6 + CXCL6 Ab group), tumor growth was significantly inhibited, and its tumor volume was not significantly different from that of the Vehicle + IgG Ab group, indicating that the CXCL6 antibody can effectively neutralize the pro-tumor effect of CXCL6 and has clear anti-tumor activity.

[0071] like Figure 8 As shown, Vehicle + IgG Ab: control group; rCXCL6 + IgG Ab: CXCL6 stimulation + isotype control antibody group; rCXCL6 + CXCL6 Ab: CXCL6 stimulation + CXCL6 antibody group. Data are expressed as mean ± standard deviation.

[0072] Example 10: Preparation of the pharmaceutical composition

[0073] The purified anti-CXCL6 monoclonal antibody 6C12 (the active ingredient) was mixed with a pharmaceutically acceptable carrier to prepare a formulation suitable for intravenous injection. For example, the antibody was dissolved in a buffer containing 10 mM histidine, 5% sucrose, and pH 6.0, and the antibody concentration was adjusted to 10 mg / mL. The solution was then sterilized by passing it through a 0.22 μm filter and dispensed into sterile vials.

[0074] In summary, through multi-level and multi-angle experiments from molecules and cells to live animals, this invention systematically demonstrates that the developed CXCL6 antibody not only has high specificity and high affinity, but also effectively blocks the core biological function of CXCL6—neutrophil chemotaxis, and exhibits significant anti-tumor activity in in vivo experiments, providing a solid basis for the application of this antibody in the field of tumor treatment.

[0075] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A blocking antibody targeting CXCL6, wherein the blocking antibody is an isolated monoclonal antibody or its antigen-binding fragment, characterized in that, The antibody or its antigen-binding fragment can specifically bind to human CXCL6 protein and inhibit CXCL6-induced neutrophil chemotaxis.

2. The blocking antibody targeting CXCL6 according to claim 1, characterized in that, The binding dissociation constant Kd of the antibody or its antigen-binding fragment to the human CXCL6 protein is less than or equal to 1 nM, preferably less than or equal to 0.5 nM, and more preferably less than or equal to 0.22 nM.

3. The blocking antibody targeting CXCL6 according to claim 1 or 2, characterized in that, The antibody comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 1, or a sequence having at least 90%, 95%, 98%, or 99% identity with it. The light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 2, or a sequence having at least 90%, 95%, 98%, or 99% identity with it.

4. An isolated nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the heavy chain variable region and / or light chain variable region of the monoclonal antibody or its antigen-binding fragment as described in claim 3.

5. A pharmaceutical composition, characterized in that, The invention comprises a therapeutically effective amount of the CXCL6-targeting blocking antibody of claim 1, and a pharmaceutically acceptable carrier or excipient.

6. Use of the CXCL6-targeting blocking antibody of claim 1 or the pharmaceutical composition of claim 5 in the preparation of an antitumor drug, wherein the drug is used to prevent or treat diseases associated with abnormal expression or activity of CXCL6.

7. The use according to claim 6, characterized in that, The disease is selected from inflammatory diseases, autoimmune diseases, or cancer, preferably colon cancer, liver cancer, or lung cancer.

8. The use according to claim 7, characterized in that, The drug's mechanism of action includes inhibiting CXCL6-induced recruitment of neutrophils to the lesion site.

9. A method for preparing the monoclonal antibody according to claim 1, characterized in that, include: (a) Immunize non-human animals with an immunogen containing human CXCL6 protein or an immunogenic fragment thereof; (b) Isolating antibody-producing cells from the immunized animal; (c) The antibody-producing cells are fused with myeloma cells to form hybridoma cells; (d) Screening for hybridoma cells that can produce monoclonal antibodies that specifically bind to CXCL6 and inhibit its induced neutrophil chemotaxis; (e) Purify the monoclonal antibody from the hybridoma cell culture supernatant, or produce the monoclonal antibody by recombinant technology.

10. A method for non-therapeutic inhibition of CXCL6 bioactivity in vitro, characterized in that, This includes contacting a system containing CXCL6 with an effective amount of the monoclonal antibody of claim 1 or its antigen-binding fragment to inhibit CXCL6-induced neutrophil chemotaxis.

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