CXCL12-targeting monoclonal neutralizing antibody and use thereof in treatment of CXCL12 axis-related disease

Abstract

The present application relates to the technical field of monoclonal antibodies, and provides a CXCL12-targeting monoclonal neutralizing antibody and a use thereof in the treatment of a CXCL12 axis-related disease. The CXCL12-targeting monoclonal neutralizing antibody (antibody 176 or antibody 174) in the present application is a multi-species (human, murine, and monkey) reactive CXCL12 neutralizing antibody, has three characteristics, i.e., high affinity, low immunogenicity, and cross-species reactivity, and can be used for the development of a CXCL12-targeting drug.

Description

Monoclonal neutralizing antibodies targeting CXCL12 and their application in the treatment of CXCL12 axis-related diseases

[0001] This application claims priority to Chinese Patent Application No. CN202510310463.6, filed on March 17, 2025, entitled "Monoclonal Neutralizing Antibody Targeting CXCL12 and Its Application in the Treatment of CXCL12 Axis-Related Diseases", the entire contents of which are incorporated herein by reference. Technical Field

[0002] This application relates to the field of monoclonal antibody technology, and in particular to monoclonal neutralizing antibodies targeting CXCL12 and their application in the treatment of CXCL12 axis-related diseases. Background Technology

[0003] CXCL12, also known as matrix-derived factor SDF-1, is a chemokine of the CXC family that binds to two G protein-coupled receptors (CXCR4 and CXCR7). The CXCL12 / CXCR4 / CXCR7 signaling axis plays a crucial role in tumor growth, metastasis, and treatment resistance, making its blocking a hot research topic in the treatment of malignant tumors. After binding to CXCL12, the G protein-coupled receptors CXCR4 / CXCR7 activate downstream signaling pathways such as MARK, AKT, and PI3K, collectively promoting tumor proliferation and metastasis. In breast cancer, CXCL12 mediates the directed metastasis of tumor cells to CXCL12-rich organs (such as the lungs and bone) by activating CXCR4, and promotes the colonization of circulating tumor cells (CTCs). In liver cancer, the CXCL12-CXCR4 interaction significantly induces the secretion of vascular endothelial growth factor (VEGF), driving abnormal tumor angiogenesis and the formation of an immunosuppressive microenvironment. Studies have shown that high activity of the CXCL12 signaling axis is significantly positively correlated with breast cancer liver metastasis and the risk of postoperative recurrence in liver cancer patients. [1] Therefore, targeting and blocking the CXCL12 / CXCR4 / 7 signaling axis has become a key strategy for inhibiting tumor progression.

[0004] Furthermore, the CXCL12 / CXCR4 signaling axis, as one of the core regulatory pathways of the chemokine system, has been widely demonstrated in recent years to participate in the pathological processes of multi-system diseases, possessing significant potential as a therapeutic target. In the field of fibrotic diseases, CXCL12, by binding to the CXCR4 receptor, can significantly activate the proliferation, migration, and extracellular matrix secretion of fibroblasts. This mechanism plays a key driving role in the fibrotic process of organs such as the heart, lungs, liver, kidneys, and skin, suggesting that targeted intervention of this signaling axis may be a new strategy to reverse organ fibrosis. In cardiovascular diseases, the abnormal interaction between platelet-derived CXCL12 and CXCR4 has been shown to participate in the molecular basis of arterial thrombosis by mediating platelet activation and aggregation, providing a new direction for the precise intervention of thrombotic diseases. In addition, this signaling axis exhibits multiple pathological effects in chronic joint inflammation: it not only promotes abnormal synovial angiogenesis but also exacerbates joint damage through pathways such as chemotaxis of T lymphocytes, activation of osteoclasts, and induction of chondrocyte necrosis. The latest research also found that abnormal activation of the CXCL12 / CXCR4 signaling pathway is significantly correlated with hair follicle cycle regulation disorders, and may become a novel molecular intervention target for hair loss diseases. [2] These findings collectively reveal the network-like regulatory role of the CXCL12 / CXCR4 signaling axis in major diseases such as fibrosis, thrombosis, chronic inflammation, and tissue regeneration disorders, highlighting its clinical value as a multi-indication therapeutic target.

[0005] Current treatments targeting this pathway include small molecule inhibitors (such as AMD3100) and antagonistic peptides, but these suffer from non-specific binding (such as the off-target effect of AMD3100 on CXCR7) and short half-lives, easily leading to systemic side effects (such as bone marrow suppression and immune disorders). In contrast, monoclonal antibodies, due to their high specificity and long half-life, can precisely neutralize CXCL12 and reduce systemic toxicity, making them a more ideal treatment option. However, the development of antibody drugs needs to address two core issues: 1. The rationality of humanization design: Traditional murine antibodies, after humanization, are prone to immunogenicity risks due to residual murine sequences in the frame region (FR), and may disrupt antigen-binding epitopes (such as the conformational stability of the CDR region); 2. Insufficient cross-species reactivity: Preclinical studies need to validate efficacy in multiple animal models (mice, non-human primates), but most antibodies cannot achieve multi-model compatibility due to differences in epitopes between target species.

[0006] References:

[0007] 1.YangY,Li J,Lei W,Wang H,NiY,LiuY,YanH,TianY,Wang Z,Yang Z,Yang S,YangY,Wang Q.CXCL12-CXCR4 / CXCR7 Axis in Cancer: from Mechanisms to Clinical Applications.Int J Biol Sci.2023Jun 26;19(11):3341-3359.doi:10.7150 / ijbs.82317.

[0008] 2. ZhengM, Oh SH, ChoiN, ChoiYJ, Kim J, Sung JH. CXCL12 inhibits hair growth through CXCR4. BiomedPharmacother.2022Jun;150:112996.doi:10.1016 / j.biopha.2022.112996. Summary of the Invention

[0009] To address the aforementioned issues, this application provides a monoclonal neutralizing antibody targeting CXCL12 and its application in the treatment of CXCL12 axis-related diseases. The monoclonal neutralizing antibody targeting CXCL12 provided in this application overcomes the dual bottlenecks of species-specific limitations and humanization design defects in traditional technologies. It possesses three major characteristics: high affinity, low immunogenicity, and cross-species reactivity, allowing for a seamless transition from preclinical animal experiments to human trials, significantly accelerating the development of CXCL12-targeted drugs.

[0010] To achieve the above objectives, this application provides the following technical solution:

[0011] This application provides a monoclonal neutralizing antibody targeting CXCL12, comprising antibody 176 and / or antibody 174; the heavy chain complementarity-determining region (CDR) of antibody 176 includes HCDR1-6, HCDR2-6, and HCDR3-6, and the light chain CDR includes LCDR1-6, LCDR2-6, and LCDR3-6; the heavy chain CDR of antibody 174 includes HCDR1-4, HCDR2-4, and HCDR3-4, and the light chain CDR includes LCDR1-4, LCDR2-4, and LCDR3-4; the amino acid sequence of HCDR1-6 is as shown in SEQ ID NO.1 or has at least 85% sequence identity with SEQ ID NO.1; the amino acid sequence of HCDR2-6 is as shown in SEQ ID NO.2 or has at least 85% sequence identity with SEQ ID NO.2; the amino acid sequence of HCDR3-6 is as shown in SEQ ID NO.3 or has at least 85% sequence identity with SEQ ID NO. NO.3 has at least 85% sequence identity; the amino acid sequences of LCDR1-6 are as shown in SEQ ID NO.4 or have at least 85% sequence identity with SEQ ID NO.4; the amino acid sequences of LCDR2-6 are as shown in SEQ ID NO.5 or have at least 85% sequence identity with SEQ ID NO.5; the amino acid sequences of LCDR3-6 are as shown in SEQ ID NO.6 or have at least 85% sequence identity with SEQ ID NO.6; the amino acid sequences of HCDR1-4 are as shown in SEQ ID NO.7 or have at least 85% sequence identity with SEQ ID NO.7; the amino acid sequences of HCDR2-4 are as shown in SEQ ID NO.8 or have at least 85% sequence identity with SEQ ID NO.8; the amino acid sequences of HCDR3-4 are as shown in SEQ ID NO.9 or have at least 85% sequence identity with SEQ ID NO.9; the amino acid sequences of LCDR1-4 are as shown in SEQ ID NO.10 or have at least 85% sequence identity with SEQ ID NO.9. NO.10 has at least 85% sequence identity; the amino acid sequences of LCDR2-4 are as shown in SEQ ID NO.11 or have at least 85% sequence identity with SEQ ID NO.11; the amino acid sequences of LCDR3-4 are as shown in SEQ ID NO.12 or have at least 85% sequence identity with SEQ ID NO.12.

[0012] Preferably, the amino acid sequence of the heavy chain variable region of the 176 antibody is as shown in SEQ ID NO.13 or has at least 85% sequence identity with SEQ ID NO.13, and the amino acid sequence of the light chain variable region of the 176 antibody is as shown in SEQ ID NO.14 or has at least 85% sequence identity with SEQ ID NO.14; the amino acid sequence of the heavy chain variable region of the 174 antibody is as shown in SEQ ID NO.15 or has at least 85% sequence identity with SEQ ID NO.15, and the amino acid sequence of the light chain variable region of the 174 antibody is as shown in SEQ ID NO.16 or has at least 85% sequence identity with SEQ ID NO.16.

[0013] Preferably, the amino acid sequence of the heavy chain of the 176 antibody is as shown in SEQ ID NO.17 or has at least 85% sequence identity with SEQ ID NO.17, and the amino acid sequence of the light chain of the 176 antibody is as shown in SEQ ID NO.18 or has at least 85% sequence identity with SEQ ID NO.18; the amino acid sequence of the heavy chain of the 174 antibody is as shown in SEQ ID NO.19 or has at least 85% sequence identity with SEQ ID NO.19, and the amino acid sequence of the light chain of the 174 antibody is as shown in SEQ ID NO.20 or has at least 85% sequence identity with SEQ ID NO.20.

[0014] This application provides the application of the monoclonal neutralizing antibody described in the above technical solution in the preparation of CXCL12 signal axis blocking agents.

[0015] Preferably, the CXCL12 signaling axis blocker comprises a product that blocks the CXCL12 / CXCR4 signaling axis and / or blocks CXCL12 recruitment of cells, said cells including Jurkat cells and / or 4T1 cells.

[0016] This application provides the application of the CXCL12-targeting monoclonal neutralizing antibody described above in the preparation of products for treating diseases related to the CXCL12 signaling axis; the diseases related to the CXCL12 signaling axis include one or more of tumors, fibrotic diseases, thrombotic diseases, chronic inflammation, and tissue regeneration disorders.

[0017] Preferably, the product includes an immunotherapy product; the tumor includes liver cancer and / or breast cancer.

[0018] This application provides an anti-tumor drug, the active ingredient of which includes the monoclonal neutralizing antibody described in the above technical solution.

[0019] Preferably, the tumor includes liver cancer and / or breast cancer.

[0020] Preferably, the dosage form of the antitumor drug includes an injection. Beneficial effects:

[0021] The CXCL12-targeting monoclonal neutralizing antibody provided in this application is a multi-species (human, mouse, monkey) reactive CXCL12 neutralizing antibody, comprising antibody 176 and / or antibody 174, and possessing high affinity (antibody 176 affinity is 1.68–1.92 × 10⁻⁶). -11 The affinity of the KD,174 antibody is 2.13–3.02 × 10⁻⁶. -11 This application utilizes a multi-species reactive, fully human neutralizing antibody development strategy based on the conserved epitope of CXCL12. It prepares human and mouse CXCL12 antigens with different tags as screening materials for neutralizing antibodies, and employs phage display technology to cross-screen human, mouse, and monkey antigens, thereby obtaining multi-species reactive CXCL12 neutralizing candidate antibodies with binding efficacy. After full-length construction of the candidate antibodies, their binding efficacy is verified using enzyme-linked immunosorbent assay (ELISA), and the binding interaction is numerically quantified using surface plasmon resonance (SPR), resulting in the selection of CXCL12 neutralizing antibodies with high binding efficacy. Finally, the CXCL12 neutralizing antibodies with high binding efficacy are further validated through molecular, cellular, and animal-level experiments to determine their neutralizing effect and antitumor activity. The CXCL12-targeting monoclonal neutralizing antibody provided in this application seamlessly bridges the translation chain from preclinical animal experiments to human trials, significantly accelerating the development of CXCL12-targeted drugs. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0023] Figure 1 shows the screening process for multi-species reactive CXCL12 neutralizing antibodies;

[0024] Figure 2 shows the antigen activity verification results; where A represents the FC(IgG)-Hu / Mus-CXCL12 antigen activity verification results; and B represents the His-Hu / Mus-CXCL12 antigen activity verification results.

[0025] Figure 3 shows the validation results of the affinity of the candidate antibodies for the CXCL12 antigen; where A represents the enzyme-linked immunosorbent assay (ELISA) validating the affinity and EC50 of the top 3 candidate antibodies (177, 176, 174) for the human CXCL12 antigen. 50 B is an enzyme-linked immunosorbent assay (ELISA) to verify the affinity and EC50 of the candidate antibody for mouse CXCL12 antigen. 50 ;

[0026] Figure 4 shows the results of reverse-phase ELISA experiments verifying the affinity efficacy of the candidate antibody for human CXCL12 antigen.

[0027] Figure 5 shows the results of reverse-phase ELISA experiments verifying the affinity efficacy of the candidate antibody for mouse CXCL12 antigen.

[0028] Figure 6 shows the ELISA evaluation results of the competitive binding of the candidate antibody to the receptor CXCR4;

[0029] Figure 7 shows the affinity of candidate CXCL12 antibody-176 for human and mouse CXCL12 antigen as determined by SPR.

[0030] Figure 8 shows the affinity of candidate CXCL12 antibody-174 for human and mouse CXCL12 antigen as determined by SPR.

[0031] Figure 9 shows the affinity of candidate antibodies 176 and 174 for human CXCL12 antigen as determined by MST.

[0032] Figure 10 shows the Transwell assay results; where A is the Transwell assay results verifying the blocking effect of the candidate CXCL12 antibody (176, 174) on the recruitment of the human T lymphocyte leukemia cell line Jurkat by the CXCL12 antigen; B is the Transwell assay results verifying the blocking effect of the candidate CXCL12 antibody (176, 174) on the recruitment of mouse breast cancer cells 4T-1 by the CXCL12 antigen.

[0033] Figure 11 shows the Transwell assay demonstrating the inhibitory effect of the candidate antibody on CXCL12-mediated invasion of liver cancer cells and on the chemotaxis of myeloid-derived immunosuppressive cells (MDSCs).

[0034] Figure 12 shows the experimental results of verifying the blocking effect of candidate CXCL12 antibodies (176, 174) on the human CXCL12-CXCR4 signaling axis based on p65 / STAT3 dual-luciferase reporter gene assay.

[0035] Figure 13 shows the experimental results of verifying the blocking effect of candidate CXCL12 antibodies (176, 174) on the mouse CXCL12-CXCR4 signaling axis based on p65 / STAT3 dual-luciferase reporter gene assay.

[0036] Figure 14 shows the experimental results of the STAT3 dual-luciferase reporter gene assay to verify the blocking effect of candidate CXCL12 antibodies (176, 174) on the CXCL12-CXCR4 signaling axis of liver cancer cells (Hepa-3B, Huh-7).

[0037] Figure 15 shows the cAMP-ELISA results of the candidate CXCL12 antibody (176, 174) treatment on hepatocellular carcinoma cells (Hepa-3B, Huh-7, Hepa1-6).

[0038] Figure 16 shows the Western Blot results (AKT, ERK1 / 2, STAT3) of the regulatory effect of candidate CXCL12 antibodies (176, 174) on phosphorylation of key proteins in the downstream signaling pathway of CXCL12-CXCR4.

[0039] Figure 17 shows the experimental results of treating breast cancer mice with the candidate CXCL12 antibody (176, 174); where A is the tumor growth curve of mice treated with the candidate CXCL12 antibody (176, 174); B is the anatomical diagram of the tumor treated with the candidate CXCL12 antibody (176, 174); and C is the tumor weight statistics of mice treated with the candidate CXCL12 antibody (176, 174).

[0040] Figure 18 shows the experimental results of treating mice with primary liver cancer with candidate CXCL12 antibodies (176, 174); where A is a dynamic growth map of tumor in vivo imaging, B is a map of liver cancer anatomical samples, C is a map of liver-to-body ratio analysis results, and D is a map of liver biochemical indicators (total bilirubin, alanine aminotransferase, aspartate aminotransferase).

[0041] * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and ns indicates P > 0.05. Detailed Implementation

[0042] This application provides a monoclonal neutralizing antibody targeting CXCL12, comprising antibody 176 and / or antibody 174; the heavy chain complementarity-determining region (CDR) of antibody 176 includes HCDR1-6, HCDR2-6, and HCDR3-6, and the light chain CDR includes LCDR1-6, LCDR2-6, and LCDR3-6; the heavy chain CDR of antibody 174 includes HCDR1-4, HCDR2-4, and HCDR3-4, and the light chain CDR includes LCDR1-4, LCDR2-4, and LCDR3-4; the amino acid sequence of HCDR1-6 is as shown in SEQ ID NO.1 or has at least 85% sequence identity with SEQ ID NO.1; the amino acid sequence of HCDR2-6 is as shown in SEQ ID NO.2 or has at least 85% sequence identity with SEQ ID NO.2; the amino acid sequence of HCDR3-6 is as shown in SEQ ID NO.3 or has at least 85% sequence identity with SEQ ID NO. NO.3 has at least 85% sequence identity; the amino acid sequences of LCDR1-6 are as shown in SEQ ID NO.4 or have at least 85% sequence identity with SEQ ID NO.4; the amino acid sequences of LCDR2-6 are as shown in SEQ ID NO.5 or have at least 85% sequence identity with SEQ ID NO.5; the amino acid sequences of LCDR3-6 are as shown in SEQ ID NO.6 or have at least 85% sequence identity with SEQ ID NO.6; the amino acid sequences of HCDR1-4 are as shown in SEQ ID NO.7 or have at least 85% sequence identity with SEQ ID NO.7; the amino acid sequences of HCDR2-4 are as shown in SEQ ID NO.8 or have at least 85% sequence identity with SEQ ID NO.8; the amino acid sequences of HCDR3-4 are as shown in SEQ ID NO.9 or have at least 85% sequence identity with SEQ ID NO.9; the amino acid sequences of LCDR1-4 are as shown in SEQ ID NO.10 or have at least 85% sequence identity with SEQ ID NO.9. NO. 10 has at least 85% sequence identity; the amino acid sequences of LCDR2-4 are as shown in SEQ ID NO. 11 or have at least 85% sequence identity with SEQ ID NO. 11; the amino acid sequences of LCDR3-4 are as shown in SEQ ID NO. 12 or have at least 85% sequence identity with SEQ ID NO. 12, as detailed below:

[0043] SEQ ID NO.1: GGTFGSYAIS;

[0044] SEQ ID NO.2: GIIPILGTTN;

[0045] SEQ ID NO.3: DTEDYYYYYMDV;

[0046] SEQ ID NO.4: RASRSISDYVN;

[0047] SEQ ID NO.5: AASSLQS;

[0048] SEQ ID NO.6: QQSYSSPLT;

[0049] SEQ ID NO.7: GYTFTSYGIS;

[0050] SEQ ID NO.8: WISAYNGNTN;

[0051] SEQ ID NO.9: DGAYYVEMIGDY;

[0052] SEQ ID NO.10: QANQDIYNSLN;

[0053] SEQ ID NO.11: DASSLVT;

[0054] SEQ ID NO.12: QQYEDLPIT.

[0055] As one embodiment, the amino acid sequences of HCDR1-6 to LCDR3-4 described in the above technical solution may be amino acid sequences that have at least 90% or 95% sequence identity with SEQ ID NO.1 to SEQ ID NO.12.

[0056] In one embodiment, the amino acid sequence of the heavy chain variable region of the 176 antibody is as shown in SEQ ID NO.13 or has at least 85% sequence identity with SEQ ID NO.13; the amino acid sequence of the light chain variable region of the 176 antibody is as shown in SEQ ID NO.14 or has at least 85% sequence identity with SEQ ID NO.14; the amino acid sequence of the heavy chain variable region of the 174 antibody is as shown in SEQ ID NO.15 or has at least 85% sequence identity with SEQ ID NO.15; and the amino acid sequence of the light chain variable region of the 174 antibody is as shown in SEQ ID NO.16 or has at least 85% sequence identity with SEQ ID NO.16, as detailed below:

[0057] SEQ ID NO.13:

[0058] SEQ ID NO.14:

[0059] SEQ ID NO.15:

[0060] SEQ ID NO.16:

[0061] In one embodiment, the amino acid sequence of the heavy chain variable region of the 176 antibody may be an amino acid sequence having at least 90% or 95% sequence identity with SEQ ID NO. 13; the amino acid sequence of the light chain variable region of the 176 antibody may be an amino acid sequence having at least 90% or 95% sequence identity with SEQ ID NO. 14; the amino acid sequence of the heavy chain variable region of the 174 antibody may be an amino acid sequence having at least 90% or 95% sequence identity with SEQ ID NO. 15; and the amino acid sequence of the light chain variable region of the 174 antibody may be an amino acid sequence having at least 90% or 95% sequence identity with SEQ ID NO. 16.

[0062] In one embodiment, the amino acid sequence of the heavy chain of the 176 antibody is as shown in SEQ ID NO.17 or has at least 85% sequence identity with SEQ ID NO.17; the amino acid sequence of the light chain of the 176 antibody is as shown in SEQ ID NO.18 or has at least 85% sequence identity with SEQ ID NO.18; the amino acid sequence of the heavy chain of the 174 antibody is as shown in SEQ ID NO.19 or has at least 85% sequence identity with SEQ ID NO.19; the amino acid sequence of the light chain of the 174 antibody is as shown in SEQ ID NO.20 or has at least 85% sequence identity with SEQ ID NO.20, as detailed below:

[0063] SEQ ID NO.17:

[0064] SEQ ID NO.18:

[0065] SEQ ID NO.19:

[0066] SEQ ID NO.20:

[0067] In one embodiment, the amino acid sequence of the heavy chain of the 176 antibody may have at least 90% or 95% sequence identity with SEQ ID NO.17, and the amino acid sequence of the light chain of the 176 antibody may have at least 90% or 95% sequence identity with SEQ ID NO.18; the amino acid sequence of the heavy chain of the 174 antibody may have at least 90% or 95% sequence identity with SEQ ID NO.19, and the amino acid sequence of the light chain of the 174 antibody may have at least 90% or 95% sequence identity with SEQ ID NO.20.

[0068] In one embodiment, the nucleotide molecule encoding the heavy chain of the 176 antibody is shown in SEQ ID NO.21, the nucleotide molecule encoding the light chain of the 176 antibody is shown in SEQ ID NO.22, the nucleotide molecule encoding the heavy chain of the 174 antibody is shown in SEQ ID NO.23, and the nucleotide molecule encoding the light chain of the 174 antibody is shown in SEQ ID NO.24, as detailed below:

[0069] SEQ ID NO.21:

[0070] SEQ ID NO.22:

[0071] SEQ ID NO.23:

[0072] SEQ ID NO.24:

[0073] In one embodiment, the 176 antibody is an IgG 1LALA subtype with an LC (Kappa) light chain; the 174 antibody is an IgG 1LALA subtype with an LC (Kappa) light chain.

[0074] Based on a humanized antibody library of hundreds of billions, this application screened neutralizing antibodies against CXCL12 that exhibit multi-species reactivity in humans, mice, and monkeys. The screening process is shown in Figure 1. This application employs direct screening from a fully human antibody library combined with conserved epitope-directed design. Based on the high conservation of CXCL12 among humans, mice, and cynomolgus monkeys (core functional domain homology > 92%), conserved epitopes were selected as universal targets for antibody design. Human and mouse CXCL12 antigens with different labels were prepared as screening materials for neutralizing antibodies, ensuring cross-species reactivity. Direct screening from a fully human antibody library replaces traditional animal immunization protocols, directly screening high-affinity candidate antibodies from the natural human antibody library. This avoids structural deviations and affinity attenuation issues during the humanization process of traditional mouse antibodies, significantly reducing the risk of immunogenicity. By targeting conserved epitopes across species, the monoclonal neutralizing antibody targeting CXCL12 provided in this application can simultaneously validate cellular and molecular biological efficacy (human tumor cell lines, mouse xenograft models) in preclinical studies and evaluate in vivo treatment in animals (non-human primates), providing data support for the clinical application of the drug and shortening the research and development cycle.

[0075] ELISA / SPR experiments confirmed that the CXCL12-targeting monoclonal neutralizing antibody provided in this application has an affinity (KD value) of 10 for CXCL12 in humans, cynomolgus monkeys, and mice. -11 The drug exhibits nM-level binding inhibition on tumor cells expressing the CXCL12 receptor CXCR4 (such as breast cancer MDA-MB-231, MCF-7; and liver cancer cell lines Hepa-3B, Huh-7, Hepa1-6), providing cross-species consistency data support for preclinical efficacy evaluation.

[0076] CXCL12 (SDF-1α), as a key chemokine in the tumor microenvironment, drives tumor angiogenesis, metastasis, colonization, and immunosuppression by activating the CXCR4 / CXCR7 signaling axis. The results of this application demonstrate that the CXCL12-targeting monoclonal neutralizing antibody can effectively inhibit CXCL12-induced ERK phosphorylation, exhibiting significant antitumor activity in humanized CXCL12 transgenic mice. It can block the CXCL12-CXCR4 signaling pathway, inhibit CXCL12's recruitment of Treg cells, and reshape the immune microenvironment.

[0077] This application systematically overcomes the species limitations and humanization design challenges in CXCL12 antibody development by integrating target conservation analysis, fully human antibody library screening technology, and a multi-species cross-validation system. It provides a novel therapeutic tool with both high specificity and clinical translational potential for solid tumors such as breast cancer and liver cancer. Its successful implementation will fill the technological gap in existing CXCL12-targeted drugs and propel tumor microenvironment-targeted therapy into a precision medicine phase.

[0078] Based on the aforementioned advantages, this application provides the application of the monoclonal neutralizing antibody described in the above technical solution in the preparation of a CXCL12 signaling axis blocker. As one embodiment, the CXCL12 signaling axis blocker includes products that block the CXCL12 / CXCR4 signaling axis and / or block CXCL12 recruitment of cells, wherein the cells include Jurkat cells and / or 4T1 cells. The CXCL12-targeting monoclonal neutralizing antibody provided in this application can specifically target CXCL12 and thereby block the signal transduction of this axis, blocking the recruitment effect of CXCL12 on Jurkat cells and / or 4T1 cells, and can treat diseases related to the CXCL12 signaling axis, such as inhibiting tumor proliferation.

[0079] Based on multi-species screening using a library of hundreds of billions of humanized antibodies and multiple experiments including biphasic ELISA, competitive ELISA, SPR, and MST, this application confirms that the monoclonal neutralizing antibody targeting CXCL12 provided in this application has a high affinity for human (monkey) and mouse CXCL12 antigens, with affinity (SPR KD value) reaching 10 in both cases. -11 At the nM level, candidate antibodies 176 and 174 can competitively bind to CXCL12 with the receptor CXCR4; their efficient antigen-binding ability provides a prerequisite for blocking the CXCL12-CXCR4 / 7 signaling pathway.

[0080] CXCL12 (SDF-1), as a key pro-cancer chemokine in the tumor microenvironment, promotes tumor growth and metastasis by mediating the CXCL12-CXCR4 / 7 axis-related signaling pathway. This application demonstrates, through dual-luciferase reporter gene (P65, STAT3 promoter elements) experiments, cAMP-ELISA detection, phosphorylation signaling pathway (AKT, ERK1 / 2, STAT3) experiments, and recruitment experiments of tumor cells and MDSC cells, that candidate antibodies 176 and 174 can effectively block the CXCL12-CXCR4 / 7-mediated cell signaling pathway and inhibit tumor cell metastasis, providing data support for the in vivo anti-tumor effects of the drugs. Furthermore, based on humanized breast cancer tumor models and primary liver cancer tumor treatment models, the results show that CXCL12 antibodies 176 and 174 alone exhibit certain anti-tumor effects. Combining them with PD1 antibodies reveals that the combination therapy shows better anti-tumor effects compared to PD1 antibody monotherapy.

[0081] Based on a fully humanized antibody library of hundreds of billions, this application successfully screened CXCL12 antibodies with high affinity and cross-species reactivity. Preclinical studies have shown that the candidate antibodies 176 and 174 can effectively block the CXCL12-CXCR4 / 7 signaling axis, and in vivo treatment has shown significant therapeutic effects. On the one hand, the antibody blocks CXCL12 from promoting the proliferation and migration of tumor cells, and on the other hand, it improves the immunosuppressive tumor microenvironment. The candidate antibody has great potential in clinical anti-tumor therapy.

[0082] Based on the above advantages, this application provides the application of the monoclonal antibody of the above scheme in blocking the CXCL12-CXCR4 / 7 signaling axis; as one embodiment, the monoclonal antibody can efficiently bind to CXCL12, thereby reducing the phosphorylation of signaling pathways such as AKT, ERK1 / 2, and STAT3 in tumor cells, as well as the accumulation of cAMP signaling molecules; inhibiting the proliferation and migration signals of tumor cells; and can be used for CXCL12-mediated diseases, such as tumor treatment.

[0083] Based on the above advantages, this application provides the application of the monoclonal neutralizing antibody described in the above technical solution in the preparation of products for treating diseases related to the CXCL12 signaling axis; the diseases related to the CXCL12 signaling axis include one or more of tumors, fibrotic diseases, thrombotic diseases, chronic inflammation, and tissue regeneration disorders.

[0084] In one embodiment, the product includes an immunotherapy product; the tumor includes liver cancer and / or breast cancer; the immunotherapy product may be a product that inhibits tumor proliferation and / or metastasis. In another embodiment, the immunotherapy product may be an immunotherapy drug.

[0085] Based on the above advantages, this application provides an anti-tumor drug, the active ingredient of which includes the monoclonal neutralizing antibody described in the above technical solution.

[0086] In one implementation, the tumor includes liver cancer and / or breast cancer.

[0087] As one implementation method, the dosage form of the antitumor drug includes an injection.

[0088] To further illustrate this application, the monoclonal neutralizing antibody targeting CXCL12 and its application in treating CXCL12 axis-related diseases provided in this application are described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of this application.

[0089] Example 1: Preparation and purification of multi-species reactive CXCL12 antibody

[0090] This embodiment first utilizes a human phage antibody library with a capacity of hundreds of billions of cells and employs an innovative "three-species parallel screening" strategy: human / monkey (human-monkey sequence identical) and mouse CXCL12 antigens are coated onto a solid-phase carrier, and after rigorous elution and affinity maturation screening, three candidate antibodies are successfully obtained. The experimental steps include:

[0091] 1.1 Sanyou Biopharmaceutical (Shanghai) Co., Ltd. (hereinafter referred to as Sanyou Biopharmaceutical) was commissioned to prepare His and FC (IgG) tagged human CXCL12 antigen (Hu-CXCL12) and mouse CXCL12 antigen (Mus-CXCL12), including:

[0092] 1) The DNA coding sequences of recombinant human CXCL12α (gene ID: 6387 in NCBI) and mouse CXCL12α (gene ID: 20315 in NCBI) proteins were codon-optimized, and a 6His or FC tag was introduced at the N-terminus to synthesize the target fragment. The target fragment was then constructed into the eukaryotic expression vector GSV0 (Sanyou Biotechnology) using homologous recombination. Simultaneously, the target fragment was also constructed into the prokaryotic expression vector pET22b using homologous recombination.

[0093] 2) The constructed recombinant protein expression plasmid was transformed into E. coli SS320 competent cells and cultured overnight at 37°C. After single-clone selection, sequencing, and alignment, the correct strain was selected for inoculation, and plasmid was extracted using an endotoxin-free plasmid extraction kit (OMEGA, D6950-01).

[0094] 3) Protein expression: One day before transfection, at a concentration of 2.50–3.00 × 10⁻⁶. 6 Cells were passaged at a density of 4.50–5.50 × 10⁶ cells / mL and cultured overnight. On the day of transfection, cell counts were performed, and the cell density was 4.50–5.50 × 10⁶ cells / mL. 6 cells / mL, viability > 95%; preheated at 37℃ (Expi 293) TM Expression Medium, cell density adjusted to 3.00 × 10⁻⁶ 6The number of HEK-293T cells / mL was determined based on the required expression volume. The transfection reagent / plasmid DNA mixture was incubated at room temperature for 10–20 min, then added dropwise to the prepared Expi 293 cell suspension while gently shaking the suspension. The transfected cells were then placed in a 37°C shaker with 7% CO2. On the first day after transfection (18–22 h), feed was added while gently shaking the cell suspension, and the shake flask was transferred to a 7% CO2 shaker for continued culture. On days 2, 4, and 5 post-transfection, different volumes of transfected cells were randomly sampled to measure cell density and viability. Samples were collected when the viable cell density was not less than 40% of the highest cell density and the cell viability was not less than 30%. If cell viability or density was abnormal, the sampling quantity was increased to determine whether to terminate expression.

[0095] 4) Protein purification: The sample was transferred to a 50 mL centrifuge tube and centrifuged at 15000×g at 4℃ for 10 min. The cell expression supernatant was collected for protein purification to obtain recombinant CXCL12 antigen containing His and FC(IgG) tags, denoted as N-FC(IgG)-Hu-CXCL12, N-FC(IgG)-Mus-CXCL12, N-His-Hu-CXCL12 and N-His-Mus-CXCL12.

[0096] 1.2 Verification of the activity of the recombinant CXCL12 antigen prepared in step 1.1

[0097] Antigen activity was detected using enzyme-linked immunosorbent assay (ELISA): Recombinant CXCL12 antigen was diluted to 2 μg / mL with PBS buffer, and 30 μL / well was coated onto ELISA 96-well plates and incubated overnight at 4°C. The next day, the plates were washed three times with washing buffer for 10 min each time, followed by blocking buffer at room temperature for 2 h. The washing buffer was a PBS solution containing 0.01% (v / v) Tween-20, and the blocking buffer was a washing buffer containing 10 g / L bovine serum albumin.

[0098] After removing the blocking buffer, add 30 μL of serially diluted CXCL12 monoclonal antibody (Sanyou Biotechnology) to each well and incubate at room temperature (25±2℃) for 60 min. The antibody dilution buffer is a wash buffer containing 5 g / L bovine serum albumin. After six rigorous washes with PBST, add 30 μL of TMB substrate and 2M sulfuric acid stop solution sequentially. Measure the absorbance at 450 nm using a microplate reader. Analyze the absorbance using dose-dependent OD... 450 The antigen activity was assessed by the change in the value. Compared with the NC group (no antigen coating, only secondary antibody added) and the BC control group (no secondary antibody added, only blocking), the antigen activity test results are shown in Figure 2A: It can be seen that the recombinant CXCL12 antigen prepared in step 1.1 has good antigen activity and can be used for screening humanized antibody libraries.

[0099] 1.3 Phage display technology screening based on a library of hundreds of millions of humanized antibodies

[0100] Based on a human scFv phage display library with a capacity of hundreds of millions, a targeted screening strategy was employed to obtain cross-reactive neutralizing antibodies against multiple species: The recombinant CXCL12 antigen with an N-terminal IgG-Fc tag prepared in step 1.1 was diluted to 2 μg / mL with PBS buffer and then directionally immobilized onto the surface of an ELISA plate via the Fc fragment, incubated overnight at 4°C. After washing with PBST (containing 0.05% Tween-20) and blocking with 5% skim milk blocking buffer (PBSM), pre-adsorbed phage library supernatant (purchased from Sanyou Biotechnology) was added, and separate screening systems were set up for human and mouse antigen coating. Neutralizing antibodies against CXCL12 that could simultaneously bind to multiple species reactivity (human and monkey CXCL12 antigen sequences are identical) were screened.

[0101] After three rounds of rigorous elution (using acidic glycine buffer containing 0.1% Tween-20), the enriched phages were collected and used to infect TG1 *E. coli* for amplification. Finally, a TMB colorimetric reaction (OD) mediated by anti-M13 phage HRP secondary antibody (Abcam, ab305291) (1:5000 dilution) was performed. 450 Positive clones were screened by readings. Given the complete conservation of the CXCL12 protein sequence between humans and cynomolgus monkeys, candidate antibodies obtained through human-mouse cross-screening can achieve cross-reactivity with multiple species. The full-length CXCL12 neutralizing antibodies obtained from the screening were constructed to verify their binding efficacy.

[0102] 1.4 The full-length construction of candidate multi-species reactive CXCL12 antibodies was commissioned to Sanyou Biopharmaceutical (Shanghai) Co., Ltd., resulting in three candidate antibodies, denoted as Antibody 174, Antibody 176, and Antibody 177.

[0103] Example 2: Identification of multi-species reaction CXCL12 antibody

[0104] 2.1 Enzyme-linked immunosorbent assay (ELISA) to verify antibody affinity.

[0105] Antigen coating was performed on 96-well ELISA plates. The CXCL12 human / mouse antigen with the FC tag prepared in Example 1 was dissolved in PBS to obtain a protein lysate with a concentration of 2 μg / mL. 30 μL of the protein lysate was added to each well and incubated overnight at 4°C. This facilitated Fc fragment-specific binding through epitope exposure. After antigen fixation, the plates were washed three times with PBST buffer (containing 0.05% Tween-20) for 10 min each time, 200 μL / well each time, and blocked with 5% skim milk blocking buffer (PBSM) at room temperature for 2 h to eliminate non-specific binding. The CXCL12 antibody to be tested was serially diluted with 1% PBSM (6 concentration gradients, initial concentration 10 μg / mL) and incubated with the antigen at room temperature for 60 min to form immune complexes. Signal amplification was achieved using anti-human κ / λ light chain HRP secondary antibodies (Anti-Human-κ+λ-HRP, purchased from Millipore, catalog numbers AP502P and AP506P, both diluted 1:4000). The TMB chromogenic system was used, and the reaction was carried out at 37℃ in the dark for 15 min. After termination, the results were read using a 450nm wavelength microplate reader. ELISA results are shown in Figures 3A and 3B. Antibody PC was used as a positive control antibody (Sanyou Bio). The results showed that Antibody 176, Antibody 174, and Antibody 177 exhibited highly efficient neutralizing activity against human and mouse CXCL12. Antibody 176 and Antibody 174 showed stronger neutralizing effects. Antibody 176 showed strong neutralizing activity against the human and mouse CXCL12 antigen EC. 50 The concentrations were 0.022 μg / ml and 0.015 μg / ml, respectively; Antibody174 was effective against human and mouse CXCL12 antigen EC. 50 The concentrations were 0.087 μg / ml and 0.047 μg / ml, respectively. These results further confirm that the screened CXCL12 antibody exhibits multi-species reactivity while also demonstrating specific and efficient binding to CXCL12.

[0106] In addition, the binding specificity was further verified by reverse ELISA (antibody immobilization and addition of gradient antigens), and the results were consistent with those of the forward ELISA (Figures 4 and 5), confirming that the three candidate antibodies have the best cross-species binding affinity and stability, providing key evidence for subsequent functional studies.

[0107] The identification of Antibody 176 was commissioned to Sanyou Biotechnology, and the results are as follows: The amino acid sequence of the heavy chain of Antibody 176 is shown in SEQ ID NO.17, and the amino acid sequence of the light chain is shown in SEQ ID NO.18; the amino acid sequence of the heavy chain of Antibody 174 is shown in SEQ ID NO.19, and the amino acid sequence of the light chain is shown in SEQ ID NO.20.

[0108] 2.2 Competitive ELISA assay

[0109] To evaluate the key blocking function of candidate antibodies against the CXCL12-CXCR4 interaction, a highly sensitive competitive ELISA detection system was established in this embodiment. Recombinant human CXCL12 protein was coated onto 96-well plates, which were then blocked. At fixed concentrations, serially diluted His-tagged CXCR4 extracellular domain protein and serially diluted candidate antibodies were added simultaneously. Anti-Fc-HRP was used to quantitatively detect the antibody binding signal. The results showed that all three antibodies exhibited a dose-dependent competitive inhibitory effect (Figure 6).

[0110] 2.3 Surface plasmon resonance (SPR) and microthermophoresis (MST) techniques were used to determine the antibody binding constant.

[0111] The binding magnitude of multi-species reactive CXCL12 neutralizing antibodies to antigens was determined using surface plasmon resonance (SPR) technology. A real-time molecular interaction detection platform was constructed based on the Biacore T200 system: His-tagged human / mouse CXCL12 antigen prepared in Example 1 was immobilized by chelation using a ProteinA chip (Biacore, 29127555), and an EDTA-treated control channel was used for background subtraction. The synthesized His-tagged human and mouse CXCL12 antigen was used as the stationary phase for protein enrichment. Subsequently, the enriched channels and control channels were activated and amino-coupled. The CXCL12 antibody to be measured was used as the mobile phase. The antibody sample was serially diluted (0.78–100 nM) with HBS-EP+ buffer (10 mM HEPES, 150 mM NaCl, 0.05% Tween-20, pH 7.4) and subjected to multiple cycles of kinetic analysis at a flow rate of 30 μL / min (binding phase 180 s, dissociation phase 600 s). The affinity between the antibody and antigen was detected using a surface-reactive protein (SPR) instrument, and the affinity was scored. Simultaneously, thermophoretic changes caused by the binding of the fluorescently labeled antigen to the antibody were detected using thermophoretic kinetics (MST) to independently verify the binding affinity. The SPR results are shown in Figures 7-8, and the MST results are shown in Figure 9. The SPR results showed that CXCL12 antibodies 176 and 174 exhibited strong interactions with the human-mouse CXCL12 antigen. The interaction strength between antibody 176 and the human-mouse CXCL12 antigen was 1.68 × 10⁻⁶. -11 KD(M) and 1.92×10 -11 The interaction forces between KD(M) antibody 174 and human / mouse CXCL12 antigen were 2.13 × 10⁻⁶. -11 KD(M) and 3.02×10 -11The KD(M) values ​​all meet the standards for antibody use; the equilibrium dissociation constant (KD) of antibody 176 is 1.15 × 10⁻⁶ in MST. -1 The MSTKD value of the nM;174 antibody was 1.1 × 10⁻⁶. -1 nM. This result further validated the neutralizing effect of the screened antibodies on human and mouse CXCL12 by binding affinity assay. Antibodies 174 and 176 showed excellent binding efficacy in both techniques, and the SPR and MST results were in high agreement, confirming the reliability and accuracy of their affinity assessment. In addition, the strong affinity provides data support for blocking the binding of CXCL12 to downstream CXCR4 / 7.

[0112] Example 3: Identification of the neutralizing effect of multi-species reactive CXCL12 antibody

[0113] 3.1 Transwell assay-based antibody blockade of CXCL12 recruits Jurkat effector

[0114] CXCL12 (also known as SDF-1α) is a chemokine that mediates the directed migration of various cells by binding to its receptor CXCR4. The CXCL12 / CXCR4 axis can promote the migration, invasion, and metastasis of tumor cells (such as 4T1 mouse breast cancer cells). To investigate the effect of antibody blocking of CXCL12 on its recruitment of Jurkat cells and 4T1 cells to the underlying HUVEC cells overexpressing CXCL12, this application uses Transwell assays to study the effect of antibody blocking of CXCL12 on its recruitment effect, thereby gaining a deeper understanding of the role of CXCL12 in the cell recruitment process.

[0115] 1) Construct HUVEC cells overexpressing CXCL12 (CXCL12) + -HUVEC), as follows:

[0116] A CXCL12 overexpression lentivirus (CXCL12 gene ID: 6387 in NCBI) was constructed by GKG Gene. This CXCL12 overexpression lentivirus was then transfected into HUVEC cells (purchased from ATCC) at an MOI of 20 to obtain CXCL12. + -HUVEC cells.

[0117] 2) HUVEC cells, Jurkat cells (purchased from ATCC), and 4T1 cells (purchased from ATCC) overexpressing CXCL12 were cultured to the logarithmic growth phase at a cell density of 60%–70%. Then, Transwell chambers were placed in 24-well plates, and the experimental groups were as follows:

[0118] Control group: The upper layer consists of Jurkat cells and 4T-1 cells, and the lower layer consists of HUVEC cells;

[0119] CXCL12 group: The upper layer consists of Jurkat cells and 4T-1 cells, and the lower layer consists of CXCL12 cells. + -HUVEC cells;

[0120] CXCL12+ antibody group (CXCL12+Ab): The upper layer consists of Jurkat cells and 4T-1 cells, and the lower layer consists of CXCL12. + -HUVEC cells, and 100 ng / mL of the CXCL12 antibody from Example 2 was added to the system.

[0121] 3) Seed cells into the corresponding chambers according to their groups and incubate at 37°C in a 5% CO2 incubator for 24 hours. Then, remove the Transwell chambers, collect cells that have migrated to the submembrane side of the upper chamber, and administer anti-CD3 antibody (Poteintech, FITC-65133-FC) (1×10⁻⁶) to each chamber. 6 (cells / 5μL), anti-CD40 (poteintech, PE-65062-FC) antibody (1×10) 6 The cells were incubated at room temperature for 30 minutes with 0.2 μg of the solution, and the migration of the cells was observed and analyzed by flow cytometry. The results are shown in Figures A and B in Figure 10.

[0122] The results showed that in the control group, fewer Jurkat and 4T1 cells migrated due to the absence of CXCL12 overexpression in the lower layer. In the CXCL12 group, under the chemotactic effect of CXCL12, HUVECs overexpressing CXCL12 in the lower layer attracted Jurkat and 4T1 cells from the upper layer, resulting in a larger number of migrations. In the CXCL12+antibody group, the number of Jurkat and 4T1 cells migrated significantly due to the blocking of CXCL12 by the antibody (176, 174), indicating that antibody blocking of CXCL12 can effectively inhibit chemotactic migration, thus proving that CXCL12 plays a key role in the recruitment effect.

[0123] In addition, this embodiment also uses the Transwell experimental system to evaluate the inhibitory effects of candidate antibodies on CXCL12-mediated hepatocellular carcinoma cell invasion behavior and on the chemotaxis of myeloid-derived immunosuppressive cells (MDSCs). The specific experimental design is as follows: 100 μL of cell suspension, including hepatocellular carcinoma cell lines (Hep3B, Huh7, Hepa1-6) and MDSCs isolated from mouse bone marrow, were seeded in the upper chamber. The lower chamber was set up as follows: (1) Blank control group: only 600 μL of serum-free medium was added; (2) CXCL12 stimulation group: the lower chamber was serum-free medium containing CXCL12 (100 ng / mL); (3) Tumor conditioned medium group (MDSCs): the lower chamber was mouse hepatocellular carcinoma cells Hepa1-6 overexpressing CXCL12; (4) Antibody neutralization group: candidate antibodies were added to CXCL12 stimulation medium or tumor conditioned medium for co-incubation. By comparing the blank control group and the CXCL12 stimulation group, the promoting effect of CXCL12 on the migration / invasion ability of hepatocellular carcinoma cells can be clarified. By comparing the CXCL12 stimulation group and the antibody neutralization group, the neutralizing effect of the candidate antibody on the chemotactic activity of CXCL12 can be evaluated. The results are shown in Figure 11. The results show that CXCL12 can recruit tumor cells, suggesting that it may play a role in tumor migration or metastasis. In addition, by analyzing the migration of MDSCs in the Transwell system, the recruitment effect of tumor-derived CXCL12 on MDSCs can be further verified, as well as the potential of the candidate antibody to inhibit the invasion of MDSCs into the tumor microenvironment by blocking the CXCL12-CXCR4 axis.

[0124] 3.2 Evaluation of the neutralizing effect of dual-luciferase reporter antibody blocking the CXCL12-CXCR4 / 7 axis

[0125] To verify the antigen-blocking effect of multi-species reactivity CXCL12 antibodies at the molecular and cellular levels, a reporter gene assay based on dual luciferase was designed. Studies have shown that the binding of CXCR4 to CXCL12 activates downstream signaling pathways, ultimately leading to the activation of transcription factors p65 and STAT3, which initiate the expression of downstream genes [Guo F, Wang Y, Liu J, Mok SC, Xue F, Zhang W. CXCL12 / CXCR4: a symbiotic bridge linking cancer cells and their stromal neighbors in oncogenic communication networks. Oncogene. 2016 Feb 18; 35(7):816-26. doi:10.1038 / onc.2015.139]. Therefore, this application constructs a luciferase reporter gene assay by combining the promoter sequence of p65 and STAT3 binding with the luciferase translation sequence. Mechanistically, activation of the CXCL12-CXCR4 signaling pathway leads to the high expression of luciferase, which can then be detected by the presence of a substrate. CXCL12 antibody, in its effect of neutralizing the antigen, blocks signal activation, thereby reducing the expression of luciferase.

[0126] In addition, this application incorporates the Renaissance fluorescent plasmid (purchased from Qingke Biotechnology) as an internal control. The breast cancer cell line MCF-7, which highly expresses CXCR4 / 7, and the liver cancer cell line MHCC-97H (both cell lines purchased from ATCC) were cultured at a concentration of 1×10⁻⁶. 5 100 μL of the reporter gene plasmid (containing p65, stat3 binding sequences as promoter sequences and luciferase sequences as translation regions, purchased from Qingke Biotechnology) was seeded into 96-well plates at a concentration of 1 / mL. 50 ng of reporter gene plasmid was added to each well. The plasmid was then transfected using Lipo3000 transfection reagent. After 24 h of transfection, the plates were divided into groups. The CXCL12 antibody (1 μg / mL) from Example 2 and the human and mouse CXCL12 antigens (both 1 μg / mL, human CXCL12 antigen denoted as rh-CXCL12, mouse CXCL12 antigen denoted as rm-CXCL12) from Example 1 were premixed 1:1 in PBS for 2 h and added to 96-well plates. Control groups (rh-CXCL12, rm-CXCL12) with CXCL12 antigen but without CXCL12 antibody and blank control groups (NC) without CXCL12 antigen and CXCL12 antibody were also set up. The results of the reporter gene experiment based on dual-luciferase are shown in Figures 12 and 13.

[0127] Furthermore, this embodiment also verified the specific inhibitory function of antibodies 174 and 176 on the signaling pathway of hepatocellular carcinoma cells (CXCL12-CXCR4 / CXCR7) using two key technology platforms: a dual-luciferase reporter gene system and cAMP signal detection (cAMP-ELISA experiment). Quantitative analysis showed that at 100 ng / ml Under CXCL12 stimulation, antibody treatments with 174 and 176 inhibited luciferase activity in liver cancer cells (Hepa-3B, Huh-7, and Hepa1-6), respectively (Figure 14). CXCL12, upon binding to its receptor CXCR4, inhibits adenylate cyclase (AC) function via Gγ subunit-mediated inhibition, while froskolin, an agonist of AC, served as a control. In cAMP signal detection, a cAMP-specific ELISA kit revealed that CXCL12 stimulation reduced cAMP levels in Hep3B, Huh7, and Hepa1-6 cells (compared to the Froskolin group), while antibody treatments with 176 and 174 significantly reversed this inhibitory effect, restoring cAMP levels (Figure 15). These two experiments, from the perspectives of STAT transcriptional activation and G protein-coupled receptor signaling, jointly demonstrate that antibody 176 or antibody 174 can efficiently block the biological interaction between CXCL12 and its receptor, providing a solid mechanistic basis for subsequent in vivo studies.

[0128] In summary, the multi-species reactive CXCL12 antibodies (176, 174) exhibit highly efficient CXCL12-CXCR4 signal blocking activity. This further confirms that the antibodies can block this signal axis through their efficient neutralization, providing data support for antibody pharmacodynamic studies.

[0129] Example 4: Western Blot verification of the blocking effect of the antibody on the signaling pathway by phosphorylated proteins.

[0130] This embodiment uses Western blot technology to systematically evaluate the regulatory effect of candidate antibodies on the phosphorylation of key proteins in the downstream signaling pathway of CXCL12. The experimental design is as follows: Hep3B, Huh7, and mouse hepatocellular carcinoma Hepa1-6 cell lines were treated with CXCL12 (100 ng / ml), and 174 and 176 antibodies (100 ng / ml) were added. After extracting total protein by lysing cells, the expression levels of phosphorylated AKT (p-AKT), phosphorylated STAT3 (p-STAT3), and phosphorylated ERK1 / 2 (p-ERK1 / 2) were detected using specific antibodies. Total AKT, total ERK1 / 2, total STAT3, and GAPDH were used as internal controls for normalization. The results showed that compared with the CXCL12 stimulation group alone, the treatment groups with 174 and 176 antibodies significantly reduced the phosphorylation levels of p-AKT, p-STAT3, and p-ERK1 / 2 (Figure 16).

[0131] Example 5: Evaluation of the antitumor effect of multi-species reactive CXCL12 antibody

[0132] Evaluation of the antitumor effect of CXCL12 based on (NSG) humanized mouse model

[0133] HUVEC cells (denoted as CXCL12) were separately... - -HUVEC) and CXCL12 constructed in Example 3 + HUVEC cells and the breast cancer cell line MCF-7 were mixed at a ratio of 1:3 and seeded into the fat pads of 6-week-old female humanized mice (mice purchased from Shanghai Southern Model Biotechnology Co., Ltd.). Tumors were allowed to grow to 10 mm. 3 Treatment was initiated at that time. Tumor-bearing mice were randomly assigned to CXCL12 groups. + -HUVEC, CXCL12 - -HUVEC group, 5 mice in each group. The mice in the two groups were treated with PBS and CXCL12 antibody via tail vein, respectively.

[0134] Mice were treated with either PBS (containing 1.25 mg / mL IgG antibody (nearshore protein, C30D)) or CXCL12 antibody for 4 weeks, once a week, with each injection being 100 μL. In addition, tumor size was measured every 4 days using calipers to plot tumor growth curves. After treatment, tumor tissue was collected and statistically analyzed to evaluate the efficacy of the antibody therapy. The treatment results are shown in Figure 17.

[0135] The results showed that after 4 weeks of antibody treatment, the breast cancer tumors in mice were significantly smaller compared to the control group (PBS).

[0136] Synergistic antitumor efficacy demonstrated in a primary liver cancer model.

[0137] In this embodiment, a C57 / BL6 mouse model of primary liver cancer was successfully constructed by high-pressure tail vein injection of plasmid. Seven days after modeling, the animals were randomly divided into six groups based on in vivo imaging fluorescence data: IgG control, PD-1 monoclonal antibody, 174 antibody, 176 antibody, 174 combined with PD-1, and 176 combined with PD-1. In vivo imaging dynamic monitoring showed (Figure 18A) that all treatment groups had certain anti-tumor effects. Among them, PD-1 monoclonal antibody (125 μg / animal, twice a week) significantly inhibited tumor growth compared with the control group, while the dual antibody regimen of CXCL12 antibody (125 μg / animal) combined with PD-1 antibody (100 μg / animal) further enhanced the efficacy compared with PD-1 monoclonal antibody, showing a synergistic effect. The liver samples at the experimental endpoint were visually displayed (Figure 18B). The liver of the control group was significantly enlarged and covered with white nodules, showing typical tumor burden characteristics. The liver morphology of all treatment groups improved, with the combined treatment group showing the most significant recovery. Analysis of the liver weight to body weight ratio (Figure 18, C) showed that the control group had the highest liver weight ratio, while the combined treatment group had the lowest, exhibiting a superior tumor-suppressive synergistic effect compared to the PD-1 monoclonal antibody group. Serum liver function indicators (total bilirubin, alanine aminotransferase, and aspartate aminotransferase) (Figure 18, D) also showed that the combined treatment group had the lowest values ​​for each indicator, further suggesting that combined treatment can more effectively reduce liver function damage caused by liver cancer. In summary, CXCL12 antibody alone can inhibit liver cancer development, and its combination with PD-1 antibody significantly enhances the latter's efficacy, exerting a synergistic tumor-suppressive effect. In conclusion, the CXCL12 antibody screened in this application can exert an antigen-neutralizing effect, exhibiting effective anti-tumor effects by blocking the CXCL12 / CXCR4 / 7 signaling axis, providing technical support for the treatment of various tumors.

[0138] In summary, the CXCL12-targeting monoclonal neutralizing antibody provided in this application is a multi-species (human, mouse, monkey) reactive CXCL12 neutralizing antibody with three major characteristics: high affinity, low immunogenicity, and cross-species reactivity.

[0139] Although the above embodiments have provided a detailed description of this application, they are only some embodiments of this application, not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of this application.

Claims

1. A monoclonal neutralizing antibody targeting CXCL12, characterized in that, The antibody includes 176 antibody and / or 174 antibody; the heavy chain complementarity-determining region of the 176 antibody includes HCDR1-6, HCDR2-6 and HCDR3-6, and the light chain complementarity-determining region of the 176 antibody includes LCDR1-6, LCDR2-6 and LCDR3-6; the heavy chain complementarity-determining region of the 174 antibody includes HCDR1-4, HCDR2-4 and HCDR3-4, and the light chain complementarity-determining region of the 174 antibody includes LCDR1-4, LCDR2-4 and LCDR3-4. The amino acid sequence of HCDR1-6 is as shown in SEQ ID NO.1 or has at least 85% sequence identity with SEQ ID NO.1; The amino acid sequence of HCDR2-6 is as shown in SEQ ID NO.2 or has at least 85% sequence identity with SEQ ID NO.2; The amino acid sequence of HCDR3-6 is as shown in SEQ ID NO.3 or has at least 85% sequence identity with SEQ ID NO.3; The amino acid sequences of LCDR1-6 are as shown in SEQ ID NO.4 or have at least 85% sequence identity with SEQ ID NO.4; The amino acid sequence of LCDR2-6 is as shown in SEQ ID NO.5 or has at least 85% sequence identity with SEQ ID NO.5; The amino acid sequence of LCDR3-6 is as shown in SEQ ID NO.6 or has at least 85% sequence identity with SEQ ID NO.6; The amino acid sequence of HCDR1-4 is as shown in SEQ ID NO.7 or has at least 85% sequence identity with SEQ ID NO.7; The amino acid sequence of HCDR2-4 is as shown in SEQ ID NO.8 or has at least 85% sequence identity with SEQ ID NO.8; The amino acid sequence of HCDR3-4 is as shown in SEQ ID NO.9 or has at least 85% sequence identity with SEQ ID NO.9; The amino acid sequences of LCDR1-4 are as shown in SEQ ID NO.10 or have at least 85% sequence identity with SEQ ID NO.10; The amino acid sequence of LCDR2-4 is as shown in SEQ ID NO.11 or has at least 85% sequence identity with SEQ ID NO.11; The amino acid sequence of LCDR3-4 is as shown in SEQ ID NO.12 or has at least 85% sequence identity with SEQ ID NO.

12.

2. The monoclonal neutralizing antibody according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the 176 antibody is as shown in SEQ ID NO.13 or has at least 85% sequence identity with SEQ ID NO.13; the amino acid sequence of the light chain variable region of the 176 antibody is as shown in SEQ ID NO.14 or has at least 85% sequence identity with SEQ ID NO.14; the amino acid sequence of the heavy chain variable region of the 174 antibody is as shown in SEQ ID NO.15 or has at least 85% sequence identity with SEQ ID NO.15; the amino acid sequence of the light chain variable region of the 174 antibody is as shown in SEQ ID NO.16 or has at least 85% sequence identity with SEQ ID NO.

16.

3. The monoclonal neutralizing antibody according to claim 1 or 2, characterized in that, The amino acid sequence of the heavy chain of the 176 antibody is as shown in SEQ ID NO.17 or has at least 85% sequence identity with SEQ ID NO.17; the amino acid sequence of the light chain of the 176 antibody is as shown in SEQ ID NO.18 or has at least 85% sequence identity with SEQ ID NO.18; the amino acid sequence of the heavy chain of the 174 antibody is as shown in SEQ ID NO.19 or has at least 85% sequence identity with SEQ ID NO.19; the amino acid sequence of the light chain of the 174 antibody is as shown in SEQ ID NO.20 or has at least 85% sequence identity with SEQ ID NO.

20.

4. The use of the monoclonal neutralizing antibody according to any one of claims 1 to 3 in the preparation of CXCL12 signaling axis blockers.

5. The application according to claim 4, characterized in that, The CXCL12 signaling axis blocker includes products that block the CXCL12 / CXCR4 signaling axis and / or block CXCL12 recruitment of cells, including Jurkat cells and / or 4T1 cells.

6. The use of the CXCL12-targeting monoclonal neutralizing antibody according to any one of claims 1 to 3 in the preparation of a product for treating diseases related to the CXCL12 signaling axis; wherein the diseases related to the CXCL12 signaling axis include one or more of tumors, fibrotic diseases, thrombotic diseases, chronic inflammation, and tissue regeneration disorders.

7. The application according to claim 6, characterized in that, The products include immunotherapy products; the tumors include liver cancer and / or breast cancer.

8. The use of the monoclonal neutralizing antibody targeting CXCL12 according to any one of claims 1 to 3 in the treatment of diseases related to the CXCL12 signaling axis.

9. The application according to claim 8, characterized in that, The diseases associated with the CXCL12 signal axis include one or more of the following: tumors, fibrotic diseases, thrombotic diseases, chronic inflammation, and tissue regeneration disorders.

10. The application according to claim 9, characterized in that, The tumors include liver cancer and / or breast cancer.

11. A method for treating diseases related to the CXCL12 signal axis, characterized in that, include: The monoclonal neutralizing antibody targeting CXCL12 as described in any one of claims 1 to 3 is injected into the body to be treated.

12. The method according to claim 11, characterized in that, The injection includes intravenous injection.

13. The method according to claim 11, characterized in that, The diseases associated with the CXCL12 signal axis include one or more of the following: tumors, fibrotic diseases, thrombotic diseases, chronic inflammation, and tissue regeneration disorders.

14. The method of claim 13, wherein, The tumors include liver cancer and / or breast cancer.

15. An antitumor drug, characterized in that, The active ingredient includes the monoclonal neutralizing antibody as described in any one of claims 1 to 3.

16. The antitumor drug according to claim 15, characterized in that, The dosage forms of the antitumor drugs include injections.

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