Anti-ELR + CXC chemokine antibody and preparation method and application thereof

Abstract

The invention provides an anti-ELR + CXC chemotactic factor antibody or an antigen binding fragment thereof as well as a preparation method and application of the anti-ELR + CXC chemotactic factor antibody. The ELR + CXC chemotactic factor antibody or the antigen binding fragment thereof. The ELR + CXC chemotactic factor is selected from one or more of CXCL1, CXCL2, CXCL3 and CXCL8. According to the application, the antibody which is specifically combined with the ELR + CXC chemotactic factor strongly related to the disease is screened out, the antibody can block a signal channel of the ELR + CXC chemotactic factor, ERK phosphorylation and human neutrophil chemotactic activity are blocked, and a new feasible scheme is provided for treatment of inflammation, immune diseases and cancers.

Description

TECHNICAL FIELD

[0001] The present application relates to the treatment of ELR + The present application relates to the treatment of ELR + The present application relates to the treatment of ELR BACKGROUND

[0002] ELR + The CXC chemokine family includes CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, CXCL8, which share a specific amino acid sequence: glutamic acid-leucine-arginine, abbreviated as ELR motif.

[0003] ELR + The CXC chemokine family includes CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, CXCL8, which share a specific amino acid sequence: glutamic acid-leucine-arginine, abbreviated as ELR motif. +CXC chemokines, through interaction with G protein-coupled receptor CXCR2 (CXCL5 / 6 / 8 can also act with G protein-coupled receptor CXCR1), open downstream signaling pathways such as ERK phosphorylation, etc. (S. Thompson et al., "Nitration of chemokine CXCL8 acts as a natural mechanism to limit acute inflammation," (in eng), Cell Mol Life Sci, vol. 80, no. 1, p. 35, Jan 9 2023; S. L. Zhou et al., "CXCL5 contributes to tumor metastasis and recurrence of intrahepatic cholangiocarcinoma by recruiting infiltrative intratumoral neutrophils," (in eng), Carcinogenesis, vol. 35, no. 3, pp. 597-605, Mar 2014), exercise many cell functions including adhesion, invasion and spread, affect the chemotaxis of immune cells, affect innate immunity and acquired immunity, etc. (W. Zhang et al., "CXCL5 / CXCR2 axis in tumor microenvironment as potential diagnostic biomarker and therapeutic target," (in eng), Cancer Commun (Lond), vol. 40, no. 2-3, pp. 69-80, Mar 2020; C. E. Hughes and R. J. B. Nibbs, "A guide to chemokines and their receptors," (in eng), Febs j, vol. 285, no. 16, pp. 2944-2971, Aug 2018). ELR + CXC chemokines not only play an important role in a variety of chronic inflammatory diseases such as chronic obstructive pulmonary disease, asthma and inflammatory bowel disease, etc., but also can affect the occurrence and development of cancer through tumor immunity.

[0004] In the tissues of a variety of inflammatory and immune diseases, the expression of different ELR + CXC chemokines is increased. Studies have shown that in the nasal polyp tissues of Chinese patients with eosinophilic and non-eosinophilic CRSwNP, MPO +CXCL1 + , MPO + CXCL2 + and MPO + CXCL8 + cells (H. Wang et al., "The activation and function of IL-36gamma in neutrophilic inflammation in chronic rhinosinusitis," J Allergy Clin Immunol, vol. 141, no. 5, pp. 1646-1658, May 2018) and also CXCL6 and CXCL8 expression was significantly increased in nasal polyp tissues with high neutrophil infiltration in patients with chronic rhinosinusitis with nasal polyps (C. Rudack, F. Sachse, and J. Alberty, "Primary role of growth-related oncogene-alpha and granulocyte chemotactic protein-2 as neutrophil chemoattractants in chronic rhinosinusitis," Clin Exp Allergy, vol. 36, no. 6, pp. 748-59, Jun 2006; J. W. Ruan et al., "Characterizing the Neutrophilic Inflammation in Chronic Rhinosinusitis With Nasal Polyps," Front Cell Dev Biol, vol. 9, p.

[0005] CXCL1 and CXCL8 expression was detected significantly elevated in bronchial alveolar lavage fluid of asthma patients (K. Hosoki et al., “Analysis of a Panel of 48 Cytokines in BAL Fluids Specifically Identifies IL-8 Levels as the Only Cytokine that Distinguishes Controlled Asthma from Uncontrolled Asthma, and Correlates Inversely with FEV1,” PLoS One, vol. 10, no. 5, p. e0126035, 2015); CXCL1 and CXCL8 expression was detected significantly elevated in sputum and serum samples of chronic obstructive pulmonary disease patients (S. L. Traves, S. V. Culpitt, R. E. Russell, P. J. Barnes, and L. E. Donnelly, “Increased levels of the chemokines GROalpha and MCP-1 in sputum samples from patients with COPD,” (in eng) Thorax, vol. 57, no. 7, pp. 590-5, Jul 2002; J. Zhang and C. Bai, “The Significance of Serum Interleukin-8 in Acute Exacerbations of Chronic Obstructive Pulmonary Disease,” (in eng) Tanaffos, vol. 17, no. 1, pp. 13-21, Jan 2018); CXCL1 expression was also detected significantly elevated in serum of type I diabetes patients (K. Takahashi et al., “Serum CXCL1 concentrations are elevated in type 1 diabetes mellitus, possibly reflecting activity of anti-islet autoimmune activity,” Diabetes Metab Res Rev, vol. 27, no. 8, pp. 830-3, Nov 2011).

[0006] In addition in cancer, ELR +CXC chemokines, especially CXCL1, CXCL2, CXCL6, CXCL8, etc., are co-expressed in head and neck squamous cell carcinoma, pancreatic cancer, colon cancer, etc., and have certain correlation (Y. Li et al., "Analysis of the Prognosis and Therapeutic Value of the CXC Chemokine Family in Head and Neck Squamous Cell Carcinoma," (in eng), Front Oncol, vol. 10, p. 570736, 2020; Y. Jing, F. Wang, K. Zhang, and Z. Chen,

[0007] "Comprehensive analysis of prognostic value and immune infiltration of CXC chemokines in pancreatic cancer," (in eng), BMC Med Genomics, vol. 15, no. 1, p. 96, Apr 25 2022; X. Yang et al., "Comprehensive analysis of the prognosis and immune infiltration for CXC chemokines in colorectal cancer," (in eng), Aging (Albany NY), vol. 13, no. 13, pp. 17548-17567, Jul 7 2021). Recent studies using single-cell sequencing have revealed the immune microenvironment heterogeneity of liver cancer patients, and found that in the immune suppressive TIME-ISM liver cancer samples enriched with myeloid cells, neutrophils have higher enrichment, and ELR + CXC chemokines are closely related to the poor prognosis of TIME-ISM liver cancer patients (R. Xue et al., "Liver tumour immune microenvironment subtypes and neutrophil heterogeneity," (in eng), Nature, vol. 612, no. 7938, pp. 141-147, Dec 2022).

[0008] Neutrophils are terminally differentiated granulocytes that develop in the bone marrow and are regulated by key transcription factors such as PU.1, CCAAT / enhancer binding protein alpha (C / EBP alpha), growth factor independence 1, and C / EBP (O. Soehnlein, S. Steffens, A. Hidalgo, and C. Weber, "Neutrophils as protagonists and targets in chronic inflammation," (in eng), Nat Rev Immunol, vol. 17, no. 4, pp. 248-261, Apr 2017; S. B. Coffelt, M. D. Wellenstein, and K. E. de Visser,

[0009] "Neutrophils in cancer: neutral no more," (in eng), Nat Rev Cancer, vol. 16, no. 7, pp. 431-46, Jul 2016). Under the chemotaxis of chemokines, neutrophils can rapidly leave the circulation and reach the site of inflammation (K. Ley et al., "Neutrophils: New insights and open questions," (in eng), Sci Immunol, vol. 3, no. 30, Dec 7 2018). This process is initiated by adhesion molecules expressed on the endothelium, accompanied by neutrophil adhesion, extravasation and migration (O. Soehnlein, S. Steffens, A. Hidalgo, and C. Weber, "Neutrophils as protagonists and targets in chronic inflammation," (in eng), Nat Rev Immunol, vol. 17, no. 4, pp. 248-261, Apr 2017; K. Ley et al., "Neutrophils: New insights and open questions," (in eng), Sci Immunol, vol. 3, no. 30, Dec 7 2018). It is reported that tissue-infiltrating leukocytes and tissue structural cells (such as epithelial cells and endothelial cells) will produce neutrophil chemokines in response to inflammatory stimuli or foreign invaders (S. de Oliveira, E. E. Rosowski, and A. Huttenlocher, "Neutrophil migration in infection and wound repair: going forward in reverse," (in eng), Nat Rev Immunol, vol. 16, no. 6, pp. 378-91, May 27 2016). Neutrophils are the most abundant effector cells in the innate immune system and are the first line of defense against infection. However, in inflammatory and immune diseases, excessive recruitment and activation of neutrophils can drive chronic inflammatory responses; in the tumor immune microenvironment, excessive infiltration of suppressive immune cells (such as neutrophils) in tumor tissues also exacerbates tumor development and metastasis.

[0010] ELR - CXC chemokines with ELR + CXC chemokines belong to CXC chemokines, and are classified as ELR because they do not have the ELR motif.- CXC chemokines. ELR + CXC chemokines and ELR - The CXC chemokine family has low sequence homology (about 30% lower). ELR + The CXC chemokine family belongs to secretory proteins, and different family members have large sequence similarity span (about 30-90%), and the structural similarity between family members is higher than 75%, which gives immunity and screening specificity binding to ELR + The antibody molecule of CXC chemokine brings feasibility and difficulty. In addition, human and monkey ELR + The CXC chemokine family sequence has high similarity, and they are different from mouse ELR + The CXC chemokine family sequence has low similarity, which brings difficulty to screen molecules that cross human, mouse and monkey species, so it is difficult to obtain effective anti-ELR + The CXC chemokine antibody is difficult, and new anti-ELR + CXC chemokine antibody. SUMMARY

[0011] Based on the defects of the above prior art, the present application uses full-length ELR + CXC chemokine recombinant protein for immunization, and ELR + CXC chemokine and ELR - CXC chemokine binding screening, and the antibody is screened for blocking function in the primary screening stage. Seven ELR + CXC chemokine binding and blocking its signal pathway. These antibodies can be used for treating inflammatory and immune diseases (such as asthma, chronic obstructive pulmonary disease, chronic sinusitis, etc.) and cancer by regulating immune / tumor immunity.

[0012] Therefore, the first aspect of the present application provides an anti-ELR + CXC chemokine antibody or antigen binding fragment thereof, said ELR +the CDR1 of the heavy chain variable region of the antibody or antigen-binding fragment thereof comprises an amino acid sequence as set forth in SEQ ID NO. 7, the CDR2 of the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO. 8, the CDR3 of the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO. 9, the CDR1 of the light chain variable region of the antibody or antigen-binding fragment thereof comprises an amino acid sequence as set forth in SEQ ID NO. 10, the CDR2 of the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO. 11, the CDR3 of the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO. 12;

[0013] and / or, the CDR1 of the heavy chain variable region of the antibody or antigen-binding fragment thereof comprises an amino acid sequence as set forth in SEQ ID NO. 7, the CDR2 of the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO. 8, the CDR3 of the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO. 9, the CDR1 of the light chain variable region of the antibody or antigen-binding fragment thereof comprises an amino acid sequence as set forth in SEQ ID NO. 10, the CDR2 of the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO. 11, the CDR3 of the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO. 12;

[0014] and / or, the CDR1 of the heavy chain variable region of the antibody or antigen-binding fragment thereof comprises an amino acid sequence as set forth in SEQ ID NO. 7, the CDR2 of the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO. 8, the CDR3 of the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO. 9, the CDR1 of the light chain variable region of the antibody or antigen-binding fragment thereof comprises an amino acid sequence as set forth in SEQ ID NO. 10, the CDR2 of the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO. 11, the CDR3 of the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO. 12;

[0015] and / or the CDR1 of the heavy chain variable region of the antibody or antigen binding fragment thereof comprises an amino acid sequence as set forth in SEQ ID NO. 19, the CDR2 of the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO. 20, the CDR3 of the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO. 21, the CDR1 of the light chain variable region of the antibody or antigen binding fragment thereof comprises an amino acid sequence as set forth in SEQ ID NO. 22, the CDR2 of the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO. 23, the CDR3 of the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO. 24;

[0016] and / or the CDR1 of the heavy chain variable region of the antibody or antigen binding fragment thereof comprises an amino acid sequence as set forth in SEQ ID NO. 25, the CDR2 of the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO. 26, the CDR3 of the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO. 27, the CDR1 of the light chain variable region of the antibody or antigen binding fragment thereof comprises an amino acid sequence as set forth in SEQ ID NO. 28, the CDR2 of the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO. 29, the CDR3 of the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO. 30;

[0017] and / or the CDR1 of the heavy chain variable region of the antibody or antigen binding fragment thereof comprises an amino acid sequence as set forth in SEQ ID NO. 31, the CDR2 of the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO. 32, the CDR3 of the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO. 33, the CDR1 of the light chain variable region of the antibody or antigen binding fragment thereof comprises an amino acid sequence as set forth in SEQ ID NO. 34, the CDR2 of the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO. 35, the CDR3 of the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO. 36;

[0018] and / or the CDR1 of the heavy chain variable region of the antibody or antigen-binding fragment thereof comprises an amino acid sequence as set forth in SEQ ID NO. 37, the CDR2 of the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO. 38, the CDR3 of the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO. 39, the CDR1 of the light chain variable region of the antibody or antigen-binding fragment thereof comprises an amino acid sequence as set forth in SEQ ID NO. 40, the CDR2 of the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO. 41, and the CDR3 of the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO. 42.

[0019] The second aspect of the present application provides a polynucleotide encoding the anti-ELR + The antibody or antigen-binding fragment thereof against CXC chemokines.

[0020] The third aspect of the present application provides a vector comprising the polynucleotide of the second aspect described above.

[0021] The fourth aspect of the present application provides a host cell comprising the vector of the third aspect described above or the polynucleotide of the second aspect described above integrated in the genome.

[0022] The fifth aspect of the present application provides a hybridoma cell capable of producing the anti-ELR + The antibody or antigen-binding fragment thereof against CXC chemokines.

[0023] The sixth aspect of the present application provides a method of preparing the anti-ELR + The antibody or antigen-binding fragment thereof against CXC chemokines.

[0024] The seventh aspect of the present application provides a pharmaceutical composition or kit comprising the anti-ELR + The antibody or antigen-binding fragment thereof against CXC chemokines.

[0025] The eighth aspect of the present application provides a use of the anti-ELR + The antibody or antigen-binding fragment thereof against CXC chemokines, or the polynucleotide of the second aspect described above, or the vector of the third aspect described above, or the host cell of the fourth aspect described above, or the hybridoma cell of the fifth aspect described above, or the method of preparation of the sixth aspect described above, in the preparation of a medicament for treating ELR + The antibody or antigen-binding fragment thereof against CXC chemokines, or the pharmaceutical composition or kit of the seventh aspect described above, in the preparation of a medicament for treating ELR+ Use in products for diseases mediated by CXC chemokines.

[0026] The beneficial effects of the present application are:

[0027] The present application screens for antibodies that specifically bind to ELR + CXC chemokines that are strongly associated with diseases, but not to ELR + CXC chemokines that are not associated with diseases, and antibodies that do not bind to ELR-CXC chemokines, and are verified by phenotype screening in vitro and in vivo tests. The antibodies screened can block their signaling pathways, including blocking ERK phosphorylation and blocking the activity of human neutrophil chemotaxis. The antibodies screened by the present application can regulate immune / tumor immunity by binding to ELR + CXC chemokines for the treatment of ELR + CXC chemokines that are highly expressed in inflammatory and immune diseases (such as asthma, chronic obstructive pulmonary disease, chronic sinusitis, etc.) and cancer, providing a new feasible solution for the treatment of inflammatory and immune diseases and cancer. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 The results are shown as chimeric anti-ELR + CXC antibodies WH1001-04, WH1001-06, WH1001-20, WH1001-23, WH1001-24, WH1001-25 and WH1001-29 bind to human ELR + CXC family proteins. Among them, Figure 1 A is WH1001-04, Figure 1 B is WH1001-06, Figure 1 C is WH1001-20, Figure 1 D is WH1001-23, Figure 1 E is WH1001-24, Figure 1 F is WH1001-25, Figure 1 G is WH1001-29.

[0029] Figure 2 The results are shown as chimeric anti-ELR + CXC antibodies WH1001-04, WH1001-06, WH1001-20, WH1001-23, WH1001-24, WH1001-25 and WH1001-29 block the downstream ERK signaling pathway of CXCR2 overexpressed cell lines induced by human CXCL1 recombinant protein. Among them, Figure 2 A is WH1001-04, Figure 2 B is WH1001-06, Figure 2 C is WH1001-20,Figure 2 D is WH1001-23, Figure 2 E is WH1001-24, Figure 2 F is WH1001-25, Figure 2 G is WH1001-29.

[0030] Figure 3 Shown as chimeric anti-ELR + Blocking effect of CXC antibodies WH1001-04, WH1001-20, WH1001-24, WH1001-25 and WH1001-29 on the downstream ERK signaling pathway of CXCR2 overexpression cell lines induced by human CXCL2 recombinant protein. Among them, Figure 3 A is WH1001-04, Figure 3 B is WH1001-20, Figure 3 C is WH1001-24, Figure 3 D is WH1001-25, Figure 3 E is WH1001-29.

[0031] Figure 4 Shown as chimeric anti-ELR + Blocking effect of CXC antibodies WH1001-04, WH1001-06, WH1001-24 on the downstream ERK signaling pathway of CXCR2 overexpression cell lines induced by human CXCL8 recombinant protein. Among them, Figure 4 A is WH1001-04, Figure 4 B is WH1001-06, Figure 4 C is WH1001-24.

[0032] Figure 5 Shown as chimeric anti-ELR + Blocking effect of CXC antibodies WH1001-04, WH1001-06, WH1001-20, WH1001-23, WH1001-24, WH1001-25 and WH1001-29 on human neutrophil chemotaxis induced by human CXCL1 recombinant protein. Among them, Figure 5 A is WH1001-04, Figure 5 B is WH1001-06, Figure 5 C is WH1001-20, Figure 5 D is WH1001-23, Figure 5 E is WH1001-24, Figure 5 F is WH1001-25, Figure 5 G is WH1001-29.

[0033] Figure 6 Shown as chimeric anti-ELR +Blockade of human neutrophil chemotaxis induced by human CXCL2 recombinant protein by CXC antibodies WH1001-04, WH1001-20, WH1001-24, WH1001-25 and WH1001-29. Among them, Figure 6 A is WH1001-04, Figure 6 B is WH1001-20, Figure 6 C is WH1001-24, Figure 6 D is WH1001-25, Figure 6 E is WH1001-29.

[0034] Figure 7 Shown as chimeric anti-ELR + Blockade of human neutrophil chemotaxis induced by human CXCL8 recombinant protein by CXC antibodies WH1001-04, WH1001-06 and WH1001-24. Among them, Figure 7 A is WH1001-04, Figure 7 B is WH1001-06, Figure 7 C is WH1001-24.

[0035] Figure 8 Single cell sorting results shown as WH1001-23, WH1001-24 and WH1001-25 (using CXCL1 screening). DETAILED DESCRIPTION

[0036] The present application is herein described, by way of example only, with the assistance of specific details to facilitate a comprehensive understanding of certain embodiments of the application. The description of specific embodiments of the application is not intended to limit the scope of the application, as claimed, but is merely intended to describe, by way of example, particular embodiments of the application. Various modifications to these embodiments can be made by those skilled in the art without departing from the spirit and scope of the application. For the sake of brevity and clarity, the description will not list every single feature of every aspect of the application.

[0037] Before further description of the application, it should be understood that the application will be described in relation to a specific embodiment thereof and that changes can be made to the application in light thereof. In describing the application, the following terminology will be used in accordance with the definitions set out below. Also, as used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

[0038] When the embodiments give numerical ranges, it is understood that unless the embodiments expressly contradict, every numerical range's two endpoints, and any number that falls between the two endpoints, can be elected. Unless otherwise defined, all technical and scientific terms used in the present application have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Except in the Examples, or where otherwise explicitly indicated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Any method, device, material, or the like that is similar in principle to those described in the Examples and otherwise anticipated to be useful in the practice of the application can be employed as long as the device, material, or the like does not interfere with the practice of the application.

[0039] The present application is further illustrated by the following examples which in no way should be construed as limiting the scope of the present application. The experimental methods, detection methods, preparation methods disclosed in the present application employ conventional techniques of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA techniques and related arts, unless otherwise specified.

[0040] The present application employs full-length ELR + CXC chemokine recombinant proteins were used for immunization, and antibodies were screened in the primary screening stage, and ELR + CXC chemokine and ELR - CXC chemokine binding screening, 7 antibodies capable of binding to ELR + CXC chemokine specific binding antibodies, antibodies were humanized to obtain WH1001-04, WH1001-06, WH1001-20, WH1001-23, WH1001-24, WH1001-25 and WH1001-29, and the antibodies were screened for blocking function to obtain ELR + CXC chemokine (e.g. CXCL1, CXCL2, CXCL3, CXCL8) has antibodies that can bind and block its signaling pathway. These antibodies can be used to treat inflammatory and immune diseases (e.g. asthma, chronic obstructive pulmonary disease, chronic sinusitis, etc.) and cancer by regulating immune / tumor immunity.

[0041] Therefore, the present application first provides an anti-ELR + CXC chemokine antibodies or antigen-binding fragments thereof, said ELR + The CXC chemokine is selected from one or more of CXCL1, CXCL2, CXCL3, CXCL8.

[0042] The present application has screened 7 antibodies capable of binding to ELR +The murine antibodies specifically binding to CXC chemokines, the variable regions of the murine antibodies are linked to the constant regions of human IgG1 to obtain chimeric antibodies WH1001-04, WH1001-06, WH1001-20, WH1001-23, WH1001-24, WH1001-25 and WH1001-29. The antibodies obtained by the above screening can specifically bind to ELR + One or more of CXC chemokines CXCL1, CXCL2, CXCL3, CXCL8.

[0043] The antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region. The variable region includes complementarity determining regions (CDRs), which are the key regions of the antibody molecule that specifically bind to antigens. The CDR regions have a high degree of sequence diversity, enabling the antibody to recognize and bind to various different antigens. In the variable region of the antibody, there are 3 CDR regions in the light chain and the heavy chain, respectively, which are denoted as CDR1, CDR2 and CDR3. The CDR regions directly interact with specific epitopes of the antigen, and their sequence diversity determines the specificity and affinity of the antibody. In the heavy chain variable region of the antibody, the CDR regions are usually located at specific amino acid positions.

[0044] In some embodiments of the present application, the CDR1 of the heavy chain variable region of the antibody or antigen-binding fragment thereof comprises the amino acid sequence as shown in SEQ ID NO. 1, the CDR2 of the heavy chain variable region comprises the amino acid sequence as shown in SEQ ID NO. 2, the CDR3 of the heavy chain variable region comprises the amino acid sequence as shown in SEQ ID NO. 3, the CDR1 of the light chain variable region of the antibody or antigen-binding fragment thereof comprises the amino acid sequence as shown in SEQ ID NO. 4, the CDR2 of the light chain variable region comprises the amino acid sequence as shown in SEQ ID NO. 5, and the CDR3 of the light chain variable region comprises the amino acid sequence as shown in SEQ ID NO. 6.

[0045]

[0046] In some embodiments of the present application, the CDR1 of the heavy chain variable region of the antibody or antigen-binding fragment thereof comprises the amino acid sequence as shown in SEQ ID NO. 1, the CDR2 of the heavy chain variable region comprises the amino acid sequence as shown in SEQ ID NO. 2, the CDR3 of the heavy chain variable region comprises the amino acid sequence as shown in SEQ ID NO. 3, the CDR1 of the light chain variable region of the antibody or antigen-binding fragment thereof comprises the amino acid sequence as shown in SEQ ID NO. 4, the CDR2 of the light chain variable region comprises the amino acid sequence as shown in SEQ ID NO. 5, and the CDR3 of the light chain variable region comprises the amino acid sequence as shown in SEQ ID NO. 6.

[0047]

[0048] In some embodiments of the present application, the CDR1 of the heavy chain variable region of the antibody or antigen-binding fragment thereof comprises an amino acid sequence as set forth in SEQ ID NO. 13, the CDR2 of the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO. 14, the CDR3 of the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO. 15, the CDR1 of the light chain variable region of the antibody or antigen-binding fragment thereof comprises an amino acid sequence as set forth in SEQ ID NO. 16, the CDR2 of the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO. 17, and the CDR3 of the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO. 18.

[0049]

[0050] In some embodiments of the present application, the CDR1 of the heavy chain variable region of the antibody or antigen-binding fragment thereof comprises an amino acid sequence as set forth in SEQ ID NO. 19, the CDR2 of the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO. 20, the CDR3 of the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO. 21, the CDR1 of the light chain variable region of the antibody or antigen-binding fragment thereof comprises an amino acid sequence as set forth in SEQ ID NO. 22, the CDR2 of the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO. 23, and the CDR3 of the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO. 24.

[0051]

[0052] In some embodiments of the present application, the CDR1 of the heavy chain variable region of the antibody or antigen-binding fragment thereof comprises an amino acid sequence as set forth in SEQ ID NO. 25, the CDR2 of the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO. 26, the CDR3 of the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO. 27, the CDR1 of the light chain variable region of the antibody or antigen-binding fragment thereof comprises an amino acid sequence as set forth in SEQ ID NO. 28, the CDR2 of the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO. 29, and the CDR3 of the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO. 30.

[0053]

[0054] In some embodiments of the present application, the CDR1 of the heavy chain variable region of the antibody or antigen-binding fragment thereof comprises an amino acid sequence as set forth in SEQ ID NO. 31, the CDR2 of the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO. 32, the CDR3 of the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO. 33, the CDR1 of the light chain variable region of the antibody or antigen-binding fragment thereof comprises an amino acid sequence as set forth in SEQ ID NO. 34, the CDR2 of the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO. 35, and the CDR3 of the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO. 36.

[0055]

[0056] In some embodiments of the present application, the CDR1 of the heavy chain variable region of the antibody or antigen-binding fragment thereof comprises an amino acid sequence as set forth in SEQ ID NO. 31, the CDR2 of the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO. 32, the CDR3 of the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO. 33, the CDR1 of the light chain variable region of the antibody or antigen-binding fragment thereof comprises an amino acid sequence as set forth in SEQ ID NO. 34, the CDR2 of the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO. 35, and the CDR3 of the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO. 36.

[0057]

[0058] In some embodiments of the present application, the heavy chain variable region of the antibody or antigen-binding fragment thereof comprises an amino acid sequence as set forth in SEQ ID No. 43, and the light chain variable region of the antibody or antigen-binding fragment thereof comprises an amino acid sequence as set forth in SEQ ID NO. 44.

[0059] In some embodiments of the present application, the heavy chain variable region of the antibody or antigen-binding fragment thereof comprises an amino acid sequence as set forth in SEQ ID No. 45, and the light chain variable region of the antibody or antigen-binding fragment thereof comprises an amino acid sequence as set forth in SEQ ID NO. 46.

[0060] In some embodiments of the present application, the heavy chain variable region of the antibody or antigen-binding fragment thereof comprises an amino acid sequence as set forth in SEQ ID No. 47, and the light chain variable region of the antibody or antigen-binding fragment thereof comprises an amino acid sequence as set forth in SEQ ID NO. 48.

[0061] In some embodiments of the present application, the heavy chain variable region of the antibody or antigen-binding fragment thereof comprises an amino acid sequence as set forth in SEQ ID No. 49, and the light chain variable region of the antibody or antigen-binding fragment thereof comprises an amino acid sequence as set forth in SEQ ID No. 50.

[0062] In some embodiments of the present application, the heavy chain variable region of the antibody or antigen-binding fragment thereof comprises an amino acid sequence as set forth in SEQ ID No. 51, and the light chain variable region of the antibody or antigen-binding fragment thereof comprises an amino acid sequence as set forth in SEQ ID No. 52.

[0063] In some embodiments of the present application, the heavy chain variable region of the antibody or antigen-binding fragment thereof comprises an amino acid sequence as set forth in SEQ ID No. 53, and the light chain variable region of the antibody or antigen-binding fragment thereof comprises an amino acid sequence as set forth in SEQ ID No. 54.

[0064] In some embodiments of the present application, the heavy chain variable region of the antibody or antigen-binding fragment thereof comprises an amino acid sequence as set forth in SEQ ID No. 55, and the light chain variable region of the antibody or antigen-binding fragment thereof comprises an amino acid sequence as set forth in SEQ ID No. 56.

[0065]

[0066]

[0067] In some embodiments of the present application, the anti-ELR + The antibody against CXC chemokine can be a murine antibody or a humanized antibody.

[0068] The humanized antibody can include, but is not limited to, a chimeric antibody, a modified antibody, a surface remodeled antibody, a fully humanized antibody, etc. In some embodiments of the present application, the humanized antibody can be a chimeric antibody.

[0069] In some embodiments of the present application, the heavy chain and the light chain of the antibody comprise a variable region and a constant region, the heavy chain constant region of the antibody can be IgG, IgA, IgM, IgD, IgE, and the light chain constant region of the antibody can be a kappa chain or a lambda chain. In some embodiments of the present application, the constant region of the antibody is the constant region of human antibody IgG1.

[0070] In some embodiments of the present application, the antigen-binding fragment refers to a portion of an antibody molecule that specifically binds to an antigen, and is selected from any one of scFv, BsFv, dsFv, (dsFv)2, Fab, Fab', F(ab')2, or Fv.

[0071] Based on the screening of the present application, the antibody capable of binding to ELR + Based on the antibody capable of specifically binding to the CXC chemokine and the amino acid sequence of the antibody, the present application can also provide a polynucleotide encoding the above-mentioned antibody capable of specifically binding to the ELR + The antibody capable of specifically binding to the CXC chemokine or the antigen-binding fragment thereof. The polynucleotide refers to a polymer of nucleotides usually linked from one deoxyribose or ribose to another deoxyribose or ribose, and the polynucleotide in the present application is not limited in size and can include a polynucleotide comprising a modification, particularly a modified nucleotide. In some embodiments, the polynucleotide can be RNA, DNA or cDNA, etc. The preparation method of the polynucleotide is prior art, and the conventional method can be selected according to the specific circumstances, for example, it can be prepared by automatic DNA synthesis, or it can be obtained by recombinant DNA technology, or it can be isolated from a suitable natural source.

[0072] Based on the antibody-based recombinant expression method, the present application can also provide a vector comprising the above-mentioned polynucleotide.

[0073] The "vector" in the present application refers to a polynucleotide capable of carrying at least one polynucleotide fragment, and the vector can exist in a circular or linear (linearized) form, and also includes a vector fragment, and can also be an artificial chromosome or similar individual polynucleotide comprising a sequence allowing the transfer of an exogenous nucleic acid fragment. The vector can comprise at least one expression cassette comprising a regulatory sequence for the correct expression of the polynucleotide incorporated therein. The polynucleotide to be introduced into the cell (such as a polynucleotide encoding a product of interest or a selection marker) can be inserted into the expression cassette of the vector for expression therefrom. The vector can also integrate the exogenous DNA sequence into the genome of the host cell to achieve stable expression of the exogenous gene, and usually comprises a homologous arm or a specific DNA sequence which can homologously recombine with a specific position in the genome of the host cell. The vector can be selected from a DNA vector, an RNA vector, a plasmid, a transposon vector, a CRISPR / Cas9 vector or a viral vector, etc.

[0074] Based on the above-mentioned polynucleotide and vector, the present application can also provide a host cell containing the above-mentioned vector or the genome integrated with the above-mentioned polynucleotide.

[0075] The host cell can be selected from bacterial cells, fungal cells, insect cells, plant cells, mammalian cells, etc., and specifically can be selected from E. coli, Streptomyces, Salmonella typhimurium, yeast, filamentous fungi, Drosophila S2 or Sf9 cells, CHO cells, COS cells, HEK293F cells, Bowes melanoma cells, NS0 cells, BHK cells, PER.C6 cells, etc. For example, in the detailed description of the present application, the host cell is a mammalian HEK293F, and further, the host cell is a HEK293F cell into which a vector containing the above polynucleotide is introduced.

[0076] The present application also provides a hybridoma cell capable of producing the above-mentioned anti-ELR + The present application also provides a method for producing an antibody or antigen-binding fragment thereof against an ELR + The present application also provides a hybridoma cell capable of producing the above-mentioned anti-ELR

[0077] The present application also provides a method for producing an antibody or antigen-binding fragment thereof against an ELR + The present application also provides a method for producing an antibody or antigen-binding fragment thereof against an ELR

[0078] Further, the above-mentioned conditions allowing the expression of the anti-ELR + The above-mentioned conditions allowing the expression of the anti-ELR

[0079] Further, the above-mentioned effective transfection or transduction method can be selected from microinjection, gene gun, electroporation, virus-mediated transformation, electron bombardment, calcium phosphate precipitation, liposome transfection, PEI method, etc.

[0080] Further, the above-mentioned recovery of the antibody or antigen-binding fragment thereof can be selected according to the actual situation, for example, the antibody described in the present application can be purified by Protein A affinity chromatography, and glycine is used to elute the antibody protein.

[0081] The present application also provides a pharmaceutical composition or kit comprising the above-mentioned anti-ELR + The present application also provides a pharmaceutical composition or kit comprising the above-mentioned anti-ELR

[0082] The kit also includes reagents, buffers, desiccants, instructions, etc. necessary for using the kit.

[0083] The pharmaceutical composition also includes the above-mentioned anti-ELR + The antibody and the pharmaceutically acceptable carrier or excipient. Specific examples of some substances that can be used as the pharmaceutically acceptable carrier or excipient are sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium methyl cellulose, ethyl cellulose, and carboxymethyl cellulose; powdered tragacanth; malt; gelatin; talc; lubricants such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, and cocoa butter; polyhydric alcohols such as propylene glycol, glycerin, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers such as Tween; wetting agents such as sodium lauryl sulfate; coloring agents; flavoring agents; tabletting agents, stabilizers, antioxidants; preservatives; pyrogen-free water; isotonic salt solutions; and phosphate buffer solutions, etc.

[0084] ELR + CXC chemokines, by interacting with G protein-coupled receptor CXCR2, open downstream signaling pathways (such as ERK phosphorylation, etc.), thereby exerting many cell functions including adhesion, invasion, and diffusion, and affecting the chemotaxis of immune cells (such as neutrophils).

[0085] Neutrophils are the most abundant effector cells in the innate immune system and are the first line of defense against infection. However, in ELR + In diseases with high expression of CXC chemokines, neutrophils are excessively chemotactic, for example, in inflammatory and immune diseases, high expression of chemokines causes excessive recruitment and activation of neutrophils, which can drive chronic inflammatory reactions; in the tumor immune microenvironment, high expression of chemokines causes excessive infiltration of suppressive immune cells (such as neutrophils, etc.) in tumor tissues, which also exacerbates tumor development and metastasis.

[0086] The present application discloses that the antibodies screened by the research and specific to ELR + The antibodies specific to CXC chemokines have a significant blocking effect on the downstream ERK signaling pathway of CXCR2-overexpressing cell lines induced by human CXCL1, or CXCL2, CXCL3, or CXCL8 recombinant proteins, and also have a significant blocking effect on the chemotaxis of human neutrophils induced by human CXCL1, CXCL2, CXCL3, or CXCL8 recombinant proteins.

[0087] Therefore, the present application also provides the above-mentioned antibodies or antigen-binding fragments, the above-mentioned polynucleotides, the above-mentioned expression vectors, the above-mentioned host cells, the antibodies or antigen-binding fragments prepared by the above-mentioned preparation method, the above-mentioned pharmaceutical compositions, or the above-mentioned kits in the preparation of a medicament for treating ELR+ Use in products for CXC chemokine mediated diseases.

[0088] The ELR + The CXC chemokine is selected from CXCL1, CXCL2, CXCL3, CXCL8.

[0089] In the present application, the ELR + The CXC chemokine mediated diseases include inflammatory and immune diseases, cancers, all of which are ELR + The diseases with elevated expression of CXC chemokines, such as inflammatory and immune diseases include chronic obstructive pulmonary disease, asthma, inflammatory bowel disease, chronic sinusitis with nasal polyps, diabetes, etc., and cancers include liver cancer, head and neck squamous cell carcinoma, pancreatic cancer, colon cancer, etc.

[0090] The present application is further explained and described by specific examples below. The instruments, reagents, and materials used in the examples, if not specifically stated, can be obtained through conventional routes.

[0091] Example 1 Mouse anti-human ELR + Production of CXC antibody

[0092] In order to obtain the anti-human ELR + CXC antibody, Balb / c mice were immunized with human recombinant CXCL1 or human recombinant CXCL5 or human recombinant CXCL8, etc. alone and in combination with various human ELR + CXC recombinant proteins; the immunization route was intraperitoneal injection.

[0093] Mouse anti-high-throughput screening based on hybridoma fusion: after 4 immunizations, the spleen cells of the immunized mice were fused with mouse myeloma cells SP2 / 0 using electrofusion method to obtain hybridoma cells that can both express antibodies and proliferate in vitro indefinitely, and were cultured in HAT selection medium. The fused hybridoma cells were plated in 96-well cell culture plates, and through detection of the binding ability of antibodies in the supernatant to immunogen protein, positive clones of interest were selected for 2-3 rounds of subcloning.

[0094] As shown in Table 1, according to the binding ability of subcloning supernatant to different human ELR + CXC chemokines, the hybridoma subclones with strong binding ability (the larger the OD450nm value, the stronger the binding ability) to CXCL1, CXCL2, CXCL8, etc. were finally selected: WH1001-04, WH1001-06, WH1001-20, and WH1001-29.

[0095] Table 1 Subcloning supernatant binding screening (OD450nm)

[0096]

[0097]

[0098] Single cell based mouse anti high throughput screening: Mouse anti screening was performed using Berkeley Lights. Sample buffer was from kit opto-plasma B Discovery Sample Prep kit, Mouse (BLI, 750-02050) if not otherwise specified. First, splenocytes were loaded into the chamber of screening chip (BLI, 750-00060): 300g, 4C centrifuge for 5min, remove supernatant, resuspend in Load Medium, adjust density, then load into the chamber of screening chip. Then, pre-treated CXCL1 protein microspheres were loaded into the channel above the chamber of screening chip. Finally, incubate for 60min, take fluorescent image every 5min (as shown in Figure 8

[0099] Take a piece of nuclease free 96 well PCR plate (Axygen, PCR-96M2-HS-C), add 20μL Mineral Oil (Sigma, M5904) and 10μL 2*TCL buffer (Qiagen, 1070498) into each well, seal with sealing film (Thermo, AB0558), 200g centrifuge for 1min, then keep at room temperature. Take 10μL eluted positive splenocytes D65128-1217 and elute into the prepared PCR plate, seal, 1000g centrifuge for 5min, keep at -80C until amplification and sequencing.

[0100] Example 2 Mouse anti human ELR + Variable region sequence determination of CXC antibody

[0101] Centrifuge to collect hybridoma cells, add TRIzol and chloroform, shake vigorously for 15 seconds, keep at room temperature for 3 minutes, centrifuge to take water phase, add isopropanol, keep at room temperature for 10 minutes, then collect the precipitate, dry after ethanol washing to obtain RNA. Add template RNA, primers and reverse transcriptase into pre-cooled centrifuge tube, make the primers and templates correctly paired, then perform reverse transcription process to obtain cDNA. Perform PCR amplification with cDNA as template. Separate the PCR products in agarose gel electrophoresis, purify the 350bp fragment, analyze the sequence information after third party sequencing.

[0102] ​Take collected single B cells, centrifuge 200g for 30s to pellet cells to the bottom. Add 10mL of RNA clean XP beads (Beckman Coulter, A63987) per well, mix by flicking 10 times, incubate at room temperature for 20 minutes. Once incubation is complete, transfer to magnetic stand (Borhee, MAG-96-11) and allow to adsorb for 5 minutes, remove supernatant without disturbing the magnetic beads. Leave cells on the magnetic stand, add freshly prepared 80% ethanol (Sigma-Aldrich, E7023-500ML) wash, remove supernatant after 30s. Repeat wash once, leave to dry on the magnetic stand for 2 minutes at room temperature. Use opto-plasma B Discovery cDNA Synthesis kit (BLI, 750-02030) to amplify single B cell cDNA libraries. Use AMPure XP beads (Beckman Coulter, A63881) to purify cDNA, follow instructions, elute with 15mL nuclease free water. Take 1-2mL of cDNA product, use Opto Plasma B Discovery Sanger Prep Kit (BLI, 750-01004) to amplify BCR Hc / Lc cDNA. Sequence Hc / Lc by third party sequencing.

[0103] The CDR sequences of the murine monoclonal antibody were determined using the method described by Kabat et al. (Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991), pp. 647-669).

[0104] Example 3 Chimeric Anti-ELR + Recombinant expression of CXC antibodies

[0105] After the antibody gene sequence was verified, the variable region of the murine antibody was linked to the constant region of human IgG1. The expression vector pcDNA3.4 (GenScript) containing the antibody gene was transfected into mammalian cells HEK293F using the PEI method according to the method described in Knappik, A., & Brundiers, R. (2009). Recombinant antibody expression and purification. In The protein protocols handbook (pp. 1929-1943). Totowa, NJ: Humana Press. The supernatant of the mammalian cells containing the antibody clone grown in the culture bottle was harvested, purified using Protein A affinity chromatography, and the antibody protein was eluted using glycine. Chimeric antibodies WH1001-04, WH1001-06, WH1001-20, WH1001-23, WH1001-24, WH1001-25, and WH1001-29 were obtained. The CDR sequences of the chimeric antibodies were consistent with those of the murine monoclonal antibodies, and the CDR sequences of the chimeric antibodies are shown in Table 2 below.

[0106] Table 2 CDR sequences of chimeric antibodies

[0107]

[0108]

[0109] The heavy chain variable region and light chain variable region sequences of the chimeric antibodies are shown in Table 3 below.

[0110] Table 3 Heavy chain variable region and light chain variable region sequences of chimeric antibodies

[0111]

[0112]

[0113] Example 4 Chimeric anti-ELR + Evaluation of antigen binding activity of CXC antibodies

[0114] This example investigates the binding of chimeric anti-ELR + CXC antibodies WH1001-04, WH1001-06, WH1001-20, WH1001-23, WH1001-24, WH1001-25, and WH1001-29 to human ELR + CXC family proteins.

[0115] Screening for mouse antibody-binding protein levels: Human CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, and CXCL8 were plated for screening. Proteins were diluted in DPBS, and the diluted antigen (2 ng / ml) was incubated overnight at 4°C in a flat-bottomed 96-well high-absorption plate. The supernatant was removed the next day. The supernatant containing the mouse antibody was added to the plate and incubated at room temperature for 1 hour. After removing the supernatant, a secondary antibody was added and incubated at room temperature for 0.5 hours. After removing the supernatant, a two-component TMB chromogenic buffer was added. After color development, H2SO4 was added to stop the color development. The experimental plates were read using a multi-functional microplate reader. A 96-well transparent flat-bottomed plate was selected for measurement, and an OD value of 450 nm was chosen. The binding efficacy of the mouse antibody to human CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, and CXCL8 was determined based on the OD value. Data analysis was performed using GraphPad, with the logarithm of antibody concentration on the x-axis and the OD450nm value on the y-axis. The chimeric anti-ELR was determined by curve fitting. + EC50 of CXC antibody.

[0116] Chimeric anti-ELR + CXC antibodies WH1001-04, WH1001-06, WH1001-20, WH1001-23, WH1001-24, WH1001-25, and WH1001-29 are effective against human ELR. + The binding states of CXC family proteins are as follows: Figure 1 As shown in Table 4 (A-1G): WH1001-04 has a strong binding affinity to human CXCL1, CXCL2, CXCL3, and CXCL8. Figure 1 A); WH1001-06 has good binding ability to human CXCL1 and strong binding ability to human CXCL3 and CXCL8. Figure 1 B); WH1001-20 has good binding ability to human CXCL2 and strong binding ability to human CXCL1 and CXCL3. Figure 1 C); WH1001-23 has a strong binding ability to human CXCL1 and CXCL3. Figure 1 D); WH1001-24 has good binding ability to human CXCL8, and strong binding ability to human CXCL1, CXCL2, and CXCL3. Figure 1 E); WH1001-25 has good binding ability to human CXCL2 and strong binding ability to human CXCL1 and CXCL3. Figure 1 F); WH1001-29 has good binding ability to human CXCL3, and strong binding ability to human CXCL1 and CXCL2. Figure 1 G).

[0117] Table 4 Binding of antibodies to human ELR + Binding of CXC antibodies to CXC proteins

[0118]

[0119]

[0120] Example 5 Chimeric anti-ELR + Evaluation of the blocking activity of CXC antibodies on the downstream signaling pathway (EPK phosphorylation) of CXCR2 overexpressed cell lines

[0121] This example investigates the blocking effect of chimeric anti-ELR + Evaluation of the blocking effect of CXC antibodies WH1001-04, WH1001-06, WH1001-20, WH1001-23, WH1001-24, WH1001-25 and WH1001-29 on the downstream ERK signaling pathway of CXCR2 overexpressed cell lines induced by human CXCL1, CXCL2, CXCL8 recombinant proteins.

[0122] SNU-398-CXCR2 (SNU-398 cells overexpressing CXCR2, human liver cancer cells, ref. Boyles JS, Beidler CB, Strifler BA, Girard DS, Druzina Z, Durbin JD, Swearingen ML, Lee LN, Kikly K, Chintharlapalli S, Witcher DR. Discovery and characterization of a neutralizing pan-ELR+CXC chemokine monoclonal antibody. MAbs. 2020 Jan-Dec; 12(1): 1831880. doi: 10.1080 / 19420862.2020.1831880.) in logarithmic growth phase were seeded at 50000 cells / well / 100 μL in 96-well cell culture / imaging microplates (Agilent, 204626-100) after digestion, centrifugation and resuspension, and incubated at 37°C in a 5% CO2 cell incubator for 24 h. Cells were washed once with pre-warmed serum-free medium (Corning, 10-040-Cva) and then incubated with 50 μL of pre-warmed serum-free medium for 2 h. Human ELR + CXC chemokine recombinant proteins (CXCL1, CXCL2, CXCL8, etc.) or human ELR +Mixtures of CXC chemokine recombinant proteins (CXCL1, CXCL2, CXCL8, etc.) and antibodies, the chemokine and antibody mixture needs to be incubated for more than 15 min. The prepared chemokine or chemokine and antibody mixture containing serum-free medium is added to the corresponding wells of the 96-well plate within 2 min, 50 μL per well, and the cells are treated for about 10 min. Immediately after treatment, the medium is discarded, 100 μL of 4% paraformaldehyde (Shanghai Yingxuan Biotech Co., Ltd., E672002-0500) is added for fixation, and after 20 min, it is discarded, 0.2% triton X-100 (Sigma, T9284-100ML) is added to break the membrane for 20 min, and then DPBS (Corning, 21-031-CV) is washed three times, and 5% goat serum (Biyun Tian, C0265) containing DPBS is added for blocking for 1 h. Prepare 1:300 ERK1 / 2 (CST, #4696) and its phosphorylated primary antibody (CST, #4370) incubation solution (incubation solution composition: [2% goat serum + 0.1% Tween-20 (Dingguo, DH358-4) + 97.9% DPBS]: antibody = 300:1). Add 60 μL per well, incubate at room temperature for 1 h or at 4°C overnight, discard the supernatant, wash with 0.1% Tween-20 + 99.9% DPBS for 3 times, each for 5 min. Prepare 1:800 secondary antibody (licor, 926-68070; licor, 926-32211) incubation solution (incubation solution composition: [2% goat serum + 0.1% Tween-20 + 97.9% DPBS]: antibody = 800:1), add 60 μL per well, incubate at room temperature for 1 h, discard the supernatant, wash with 0.1% Tween-20 + 99.9% DPBS for 3 times, each for 5 min. Discard the supernatant completely, read the plate with the Odyssey DLx dual-color infrared laser imaging system (licor, Odyssey DLx), and then process the data.

[0123] Chimeric anti-ELR + The blocking effects of CXC antibodies WH1001-04, WH1001-06, WH1001-20, WH1001-23, WH1001-24, WH1001-25, and WH1001-29 on the downstream ERK signaling pathway of human CXCL1 recombinant protein-induced CXCR2 overexpression cell lines are as follows: Figure 2 A-2G shows that under the experimental conditions, WH1001-20( Figure 2 C) has a strong blocking effect on the downstream ERK signaling pathway induced by human CXCL1; WH1001-24( Figure 2 E) and WH1001-29( Figure 2 G) have good blocking effects on the downstream ERK signaling pathway induced by human CXCL1; and WH1001-04( Figure 2A) WH1001-06 Figure 2 B) WH1001-23 Figure 2 D) WH1001-25 Figure 2 F) cannot block the downstream ERK signaling pathway induced by human CXCL1.

[0124] Chimeric anti-ELR + The blocking effect of CXC antibodies WH1001-04, WH1001-20, WH1001-24, WH1001-25 and WH1001-29 on the downstream ERK signaling pathway of CXCR2 overexpressed cell lines induced by human CXCL2 recombinant protein is shown in Figure 3 A-3E: under the experimental conditions, WH1001-04 Figure 3 A) WH1001-24 Figure 3 C) has a blocking effect on the downstream ERK signaling pathway induced by human CXCL2; WH1001-20 Figure 3 B) and WH1001-25 Figure 3 D) WH1001-29 Figure 3 E) cannot block the downstream ERK signaling pathway induced by human CXCL2.

[0125] Chimeric anti-ELR + The blocking effect of CXC antibodies WH1001-04, WH1001-06, WH1001-24 on the downstream ERK signaling pathway of CXCR2 overexpressed cell lines induced by human CXCL8 recombinant protein is shown in Figure 4 A-4C: under the experimental conditions, WH1001-04 Figure 4 A) WH1001-06 Figure 4 B) has a good blocking effect on the downstream ERK signaling pathway induced by human CXCL8, so as to inhibit the adhesion, invasion and diffusion of SNU-398 liver cancer cells; WH1001-24 Figure 4 C) cannot block the downstream ERK signaling pathway induced by human CXCL8.

[0126] Example 6 Chimeric anti-ELR + Evaluation of the blocking activity of CXC antibodies on human neutrophil chemotaxis

[0127] This example explores the chimeric anti-ELR + The blocking effect of CXC antibodies WH1001-04, WH1001-06, WH1001-20, WH1001-23, WH1001-24, WH1001-25 and WH1001-29 on human neutrophil chemotaxis induced by human CXCL1, CXCL2, CXCL8 recombinant protein.

[0128] First, human neutrophils were isolated and purified. 100 mL of anticoagulated blood was centrifuged at 800g for 10 min, and the plasma was centrifuged at 1500g for 10 min to obtain platelet-poor plasma (PPP) for later use. The blood cell pellet was resuspended with an equal volume of DPBS (Corning, 21-031-CV) after the blood cells had settled. A new 50 mL centrifuge tube was prepared by sequentially adding human peripheral blood neutrophil separation kit reagents (Solarbio, P9040-200ml / KIT) A (15 mL) and C (12 mL) slowly, followed by the DPBS-resuspended blood cells, and centrifuging at 1100g for 30 min. The cell layer between A and C was collected, washed once with 30 mL of DPBS, and centrifuged at 500g for 10 min. The cell pellet was resuspended with 2 mL of DPBS + 1 mL of PPP. A percoll (Sigma, P4937-100ML) density gradient tube was prepared by adding 1.08, 1.09, and 1.1 g / mL density layers, each 3 mL from top to bottom, to a 15 mL centrifuge tube, followed by the cell resuspension. After centrifugation at 800g for 30 min, the cells were separated into four layers, and all the intermediate layers except the top and bottom layers were collected. The collected cells were washed twice with 15 mL of DPBS and centrifuged at 500g for 10 min. A portion of the cells was stained with CD15 antibody (BD Pharmingen, 551376) for 30 min, and the purity of the purified neutrophils was identified by flow cytometry. The cells were resuspended in 1 mL of 1640 medium (Corning, 10-040-Cva) containing 2% FBS (BOCOGEN, SFBS).

[0129] Chimeric anti-ELR + Blocking experiment of CXC antibody on human CXCL1, human CXCL2, human CXCL8 recombinant protein on neutrophil chemotaxis. The extracted neutrophils were counted and prepared at a concentration of 200,000 cells / 75 μL. 75 μL of cell solution was added to the upper chamber of a transwell (Corning, 3387) with a pore size of 5 μm, and 200 μL of ELR + CXC chemokine recombinant protein (CXCL1, CXCL2, CXCL8, etc.) or ELR + Mixing of CXC chemokine recombinant protein (CXCL1, CXCL2, CXCL8, etc.) and antibody, and incubation of the mixture for at least 15 min. The upper chamber was removed, and 50 μL of the cell suspension in the lower chamber was mixed and transferred to 50 μL of medium containing 2X concentration of calcein AM (Invitrogen, C34851) (96-well plate system), and centrifuged at 500g for 10 min. The fluorescence cell count of all wells was analyzed using a cello go full field cell scanner (Nexcelom, 200-BFFL-S), and the chemotaxis ratio and inhibition rate were calculated to generate a concentration-chemotaxis ratio curve.

[0130] Chimeric anti-ELR + The blocking effect of CXC antibodies WH1001-04, WH1001-06, WH1001-20, WH1001-23, WH1001-24, WH1001-25 and WH1001-29 on human neutrophil chemotaxis induced by human CXCL1 recombinant protein is shown in Figure 5 A-5G: under the experimental conditions, WH1001-20 Figure 5 C) has a strong blocking effect on human neutrophil chemotaxis induced by human CXCL1 ; WH1001-24 Figure 5 E) has a good blocking effect on human neutrophil chemotaxis induced by human CXCL1 ; WH1001-04 Figure 5 A), WH1001-29 Figure 5 G) can block human neutrophil chemotaxis induced by human CXCL1 ; and WH1001-06 Figure 5 B), WH1001-23 Figure 5 D), WH1001-25 Figure 5 F) cannot block human neutrophil chemotaxis induced by human CXCL1.

[0131] Chimeric anti-ELR + The blocking effect of CXC antibodies WH1001-04, WH1001-20, WH1001-24, WH1001-25 and WH1001-29 on human neutrophil chemotaxis induced by human CXCL2 recombinant protein is shown in Figure 6 A-6E: under the experimental conditions, WH1001-24 Figure 6 C) has a good blocking effect on human neutrophil chemotaxis induced by human CXCL2 recombinant protein; WH1001-04 Figure 6 A) can block human neutrophil chemotaxis induced by human CXCL2 recombinant protein; WH1001-20 Figure 6 B) and WH1001-25 Figure 6 D), WH1001-29 Figure 6 E) cannot block human neutrophil chemotaxis induced by human CXCL2 recombinant protein.

[0132] Chimeric anti-ELR + The blocking effect of CXC antibodies WH1001-04, WH1001-06, WH1001-24 on human neutrophil chemotaxis induced by human CXCL8 recombinant protein is shown in Figure 7 A-7C: under the experimental conditions, WH1001-04 Figure 7A) have a strong blocking effect on human CXCL8-induced human neutrophil chemotaxis; WH1001-06 Figure 7 B) have a good blocking effect on human CXCL8-induced human neutrophil chemotaxis; WH1001-24 Figure 7 C) cannot block human CXCL8-induced human neutrophil chemotaxis.

[0133] When a numerical range is given in the examples, it is understood that, unless otherwise stated by the present application, both the end points of each numerical range and any number between the two end points can be selected. Unless otherwise defined, all technical and scientific terms used in the present application have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, devices, materials and the like described in the examples, any methods, devices, and materials similar or equivalent to those described in the examples of the present application can be used in the practice of the present application, using the present state of the art knowledge, in light of the teachings of the present application.

Claims

1. An anti-ELR + Antibodies to CXC chemokines or their antigen-binding fragments, the ELR + The CXC chemokine is selected from one or more of CXCL1, CXCL2, CXCL3, and CXCL8; the CDR1 of the heavy chain variable region of the antibody or its antigen-binding fragment includes the amino acid sequence shown in SEQ ID NO.1, the CDR2 of the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO.2, and the CDR3 of the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO.3; the CDR1 of the light chain variable region of the antibody or its antigen-binding fragment includes the amino acid sequence shown in SEQ ID NO.4, the CDR2 of the light chain variable region includes the amino acid sequence shown in SEQ ID NO.5, and the CDR3 of the light chain variable region includes the amino acid sequence shown in SEQ ID NO.

6. And / or, the CDR1 of the heavy chain variable region of the antibody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO.7, the CDR2 of the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO.8, the CDR3 of the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO.9, the CDR1 of the light chain variable region of the antibody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO.10, the CDR2 of the light chain variable region comprises the amino acid sequence shown in SEQ ID NO.11, and the CDR3 of the light chain variable region comprises the amino acid sequence shown in SEQ ID NO.12; And / or, the CDR1 of the heavy chain variable region of the antibody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO.13, the CDR2 of the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO.14, the CDR3 of the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO.15, the CDR1 of the light chain variable region of the antibody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO.16, the CDR2 of the light chain variable region comprises the amino acid sequence shown in SEQ ID NO.17, and the CDR3 of the light chain variable region comprises the amino acid sequence shown in SEQ ID NO.18; And / or, the CDR1 of the heavy chain variable region of the antibody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO.19, the CDR2 of the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO.20, and the CDR3 of the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO.21; the CDR1 of the light chain variable region of the antibody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO.22, the CDR2 of the light chain variable region comprises the amino acid sequence shown in SEQ ID NO.23, and the CDR3 of the light chain variable region comprises the amino acid sequence shown in SEQ ID NO.24; And / or, the CDR1 of the heavy chain variable region of the antibody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO.25, the CDR2 of the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO.26, the CDR3 of the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO.27, the CDR1 of the light chain variable region of the antibody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO.28, the CDR2 of the light chain variable region comprises the amino acid sequence shown in SEQ ID NO.29, and the CDR3 of the light chain variable region comprises the amino acid sequence shown in SEQ ID NO.30; And / or, the CDR1 of the heavy chain variable region of the antibody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO.31, the CDR2 of the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO.32, the CDR3 of the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO.33, the CDR1 of the light chain variable region of the antibody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO.34, the CDR2 of the light chain variable region comprises the amino acid sequence shown in SEQ ID NO.35, and the CDR3 of the light chain variable region comprises the amino acid sequence shown in SEQ ID NO.36; And / or, the CDR1 of the heavy chain variable region of the antibody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO. 37, the CDR2 of the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO. 38, and the CDR3 of the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO. 39; the CDR1 of the light chain variable region of the antibody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO. 40, the CDR2 of the light chain variable region comprises the amino acid sequence shown in SEQ ID NO. 41, and the CDR3 of the light chain variable region comprises the amino acid sequence shown in SEQ ID NO.

42.

2. The anti-ELR method according to claim 1 + An antibody or antigen-binding fragment of CXC chemokine, characterized in that, The heavy chain variable region of the antibody or its antigen-binding fragment includes the amino acid sequence shown in SEQ ID No. 43, and the light chain variable region of the antibody or its antigen-binding fragment includes the amino acid sequence shown in SEQ ID No. 44; And / or, the heavy chain variable region of the antibody or its antigen-binding fragment includes the amino acid sequence shown in SEQ ID No. 45, and the light chain variable region of the antibody or its antigen-binding fragment includes the amino acid sequence shown in SEQ ID No. 46; And / or, the heavy chain variable region of the antibody or its antigen-binding fragment includes the amino acid sequence shown in SEQ ID No. 47, and the light chain variable region of the antibody or its antigen-binding fragment includes the amino acid sequence shown in SEQ ID No. 48; And / or, the heavy chain variable region of the antibody or its antigen-binding fragment includes the amino acid sequence shown in SEQ ID No. 49, and the light chain variable region of the antibody or its antigen-binding fragment includes the amino acid sequence shown in SEQ ID No. 50; And / or, the heavy chain variable region of the antibody or its antigen-binding fragment includes the amino acid sequence shown in SEQ ID No. 51, and the light chain variable region of the antibody or its antigen-binding fragment includes the amino acid sequence shown in SEQ ID No. 52; And / or, the heavy chain variable region of the antibody or its antigen-binding fragment includes the amino acid sequence shown in SEQ ID No. 53, and the light chain variable region of the antibody or its antigen-binding fragment includes the amino acid sequence shown in SEQ ID No. 54; And / or, the heavy chain variable region of the antibody or its antigen-binding fragment includes the amino acid sequence shown in SEQ ID No. 55, and the light chain variable region of the antibody or its antigen-binding fragment includes the amino acid sequence shown in SEQ ID No.

56.

3. The anti-ELR method according to claim 1 + An antibody or antigen-binding fragment of CXC chemokine, characterized in that, The anti-ELR + The antibody against CXC chemokine is a murine antibody or a humanized antibody, preferably a chimeric antibody; And / or, the antigen-binding fragment is selected from any one of scFv, BsFv, dsFv, (dsFv)2, Fab, Fab', F(ab')2 or Fv.

4. A polynucleotide encoding the anti-ELR as described in any one of claims 1-3 + Antibodies against CXC chemokines or their antigen-binding fragments.

5. A vector comprising the polynucleotide of claim 4.

6. A host cell containing the vector of claim 5 or the polynucleotide of claim 4 integrated into the genome.

7. A hybridoma cell capable of producing the anti-ELR as described in any one of claims 1-3 + Antibodies against CXC chemokines or their antigen-binding fragments.

8. An anti-ELR method as described in any one of claims 1 to 3 + A method for preparing an antibody or antigen-binding fragment of CXC chemokine, comprising culturing the host cell of claim 6 or the hybridoma cell of claim 7 under conditions allowing expression of the antibody or antigen-binding fragment, and recovering the antibody or antigen-binding fragment from the culture.

9. A pharmaceutical composition or kit comprising the anti-ELR as described in any one of claims 1-3 + Antibodies against CXC chemokines or their antigen-binding fragments.

10. The anti-ELR according to any one of claims 1-3 + Antibody to CXC chemokine or its antigen-binding fragment, or the polynucleotide of claim 4, or the vector of claim 5, or the host cell of claim 6, or the hybridoma cell of claim 7, or the anti-ELR prepared by the preparation method of claim 8. + An antibody or antigen-binding fragment of CXC chemokine, or the pharmaceutical composition or kit of claim 9, in the preparation of a treatment for ELR + Use in products for CXC chemokine-mediated diseases; preferably, the ELR + CXC chemokine-mediated diseases include inflammatory and immune diseases or cancer, more preferably, the inflammatory and immune diseases are selected from chronic obstructive pulmonary disease, asthma, inflammatory bowel disease, chronic sinusitis with nasal polyps, and diabetes; the cancers are selected from liver cancer, head and neck squamous cell carcinoma, pancreatic cancer, and colon cancer.

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