Monoclonal antibody combined with human IL-8 antigen and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to a monoclonal antibody combined with a human IL-8 antigen and application of the monoclonal antibody. A heavy chain CDR region of the monoclonal antibody comprises a CDR1 of which the amino acid sequence is as shown in SEQ ID NO.5, a CDR2 of which the amino acid sequence is as shown in SEQ ID NO.6 and a CDR3 of which the amino acid sequence is as shown in SEQ ID NO.7; a light chain CDR region of the monoclonal antibody comprises a CDR4 of which the amino acid sequence is shown as SEQ ID NO.8, a CDR5 of which the amino acid sequence is shown as SEQ ID NO.9 and a CDR6 of which the amino acid sequence is shown as SEQ ID NO.10. The monoclonal antibody can specifically bind to human IL-8, and has good affinity. In-vitro experiments prove that the compound can be used for diagnosing and treating IL-8-mediated related diseases.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a monoclonal antibody binding to human IL-8 antigen and application thereof. Background Art

[0002] Interleukin-8 (IL-8), also known as CXCL8, belongs to the CXC subtype and is the earliest discovered chemotactic cytokine. IL8 is a protein with a very small molecular weight of about 8KD. Mature IL8 includes six forms: 79aa, 72aa, 71aa, 70aa and 69aa, of which 72aa is the main one. IL-8 of different molecular weights has different abilities to induce neutrophil chemotaxis and degranulation. IL-8 containing 72 amino acids has the strongest activity, which is usually referred to as mature IL-8. IL-8 is divided into two subgroups, α and β. The initial translation product of IL-8 is 99 amino acids, which is cleaved into 72 amino acids in monocytes and macrophages, and the molecular weight is 8KDa active substance. IL-8 is mainly secreted by monocytes and macrophages, and its function is to recruit and activate neutrophils, promote inflammatory response and cell killing. In addition, tumor cells, fibroblasts, endothelial cells, etc., under the action of pro-inflammatory factors such as IL-1β, tumor necrosis factor α, and lipopolysaccharide, affect the proliferation, invasion, metastasis of tumor cells and the formation of tumor blood vessels through autocrine or paracrine IL-8.

[0003] IL-8 exerts its effects by binding to IL-8 receptors. IL-8 has two receptors, CXCR1 (IL-8RA) and CXCR2 (IL-8RB). These two receptors have 77% homology and are both G protein-coupled receptors. Because of the structural differences at the N-terminus, the two receptors have different affinities with IL-8, and CXCR1 has a higher affinity with IL-8. In normal tissue cells, CXCR1 first binds to granulocyte chemoattractant protein 2 (GCP-2) to form a complex, and then binds to IL-8 to exert its effects; CXCR2 has a lower affinity with IL-8. In addition to IL-8, CXCR2 can also bind to multiple ligands such as CXCL1, 2, 3, 5, and 7. After IL-8 stimulates CXCR1 and / or CXCR2 receptors and activates heterotrimeric small G proteins, IL-8 signaling promotes the activation of the main effector (PI3-K) or phospholipase C, thereby promoting the activation of Akt, PKC, calcium mobilization and / or MAPK signaling cascades.

[0004] IL-8 is a multifunctional factor, and its biological function is not species-specific. Early on, it was discovered that neutrophils are its target cells. It can specifically attract neutrophils into inflammatory tissues, promote their degranulation, produce superoxide anions, and cause respiratory bursts; activate inflammatory cells; promote acute phase protein synthesis; cause fever, participate in inflammatory pathological damage; promote the release of inflammatory mediators; promote fibroblast proliferation-chronic inflammation, so it plays an important role in inflammatory response. With the deepening of research, it was found that IL-8 can act on different cells. It can promote the process of inflammatory response, stimulate the formation of blood vessels, promote mitosis, regulate host immune function, etc. It is closely related to the occurrence and development of various inflammatory diseases, tumors, and immune diseases.

[0005] In recent years, more and more studies have shown that IL-8 plays an important biological function in the tumor immune microenvironment: ① IL-8 can recruit neutrophils and macrophages into the tumor microenvironment, and these cells play a key role in tumor development, including promoting angiogenesis and tumor cell proliferation; ② IL-8 can induce the formation of new blood vessels, which provide the necessary nutrients and oxygen for tumors to support their rapid growth; ③ Promote the activity of tumor stem cells: IL-8 can indirectly support the survival and proliferation of tumor stem cells by attracting specific immune cells. These cells are considered to be the main cause of tumor recurrence and metastasis. And many studies have shown that IL-8 is negatively correlated with the prognosis of cancer patients. For example, in the treatment of melanoma, high IL-8 levels are associated with shorter overall survival; in single or combined treatment with PD-1 inhibitors, patients with high IL-8 levels also have a poor prognosis. These research results suggest that IL-8 may have a negative impact on the prognosis of cancer patients by inhibiting adaptive immunity and promoting immune tolerance.

[0006] Therefore, the development of monoclonal antibodies that can specifically bind to IL-8 antigens is of great significance for the diagnosis and treatment of IL-8 related diseases. In the prior art, the CN1309738C patent discloses an anti-IL-8 monoclonal antibody, its variable region sequence and its application. The invention discloses a monoclonal antibody with neutralizing IL-8 biological activity and the variable region sequence of the monoclonal antibody. It uses IL-8 as an immune source and mice as immune objects. By immunizing mice, the spleen of the immune mice is taken to prepare a suspension, and it is fused with myeloma cells to obtain a hybridoma cell line that can express anti-IL-8 monoclonal antibodies to prepare anti-IL-8 monoclonal antibodies. The monoclonal antibody can specifically bind to IL-8. Summary of the invention

[0007] In view of this, one of the objectives of the present invention is to provide an anti-IL-8 monoclonal antibody, which can specifically bind to the target antigen IL-8, thereby providing support for the subsequent diagnosis and treatment of IL-8-mediated related diseases.

[0008] To achieve the above object, the present invention adopts the following technical solutions:

[0009] An anti-IL-8 monoclonal antibody, wherein the heavy chain CDR region of the monoclonal antibody includes CDR1 shown in SEQ ID NO.5, CDR2 shown in SEQ ID NO.6, and CDR3 shown in SEQ ID NO.7; the light chain CDR region of the monoclonal antibody includes CDR4 shown in SEQ ID NO.8, CDR5 shown in SEQ ID NO.9, and CDR6 shown in SEQ ID NO.10.

[0010] Furthermore, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO.3; the amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO.4.

[0011] Furthermore, the IL-8 is a human IL-8 antigen.

[0012] A second object of the present invention is to provide a nucleic acid molecule encoding the aforementioned monoclonal antibody.

[0013] To achieve the above object, the present invention adopts the following technical solutions:

[0014] A nucleic acid molecule encoding the aforementioned monoclonal antibody.

[0015] Furthermore, the nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody comprises the sequence shown in SEQ ID NO.1.

[0016] Furthermore, the nucleotide sequence encoding the light chain variable region of the monoclonal antibody comprises the sequence shown in SEQ ID NO.2.

[0017] A third object of the present invention is to provide a kit for detecting the expression level of IL-8.

[0018] To achieve the above object, the present invention adopts the following technical solutions:

[0019] A kit for detecting the expression level of IL-8, said kit comprising the aforementioned monoclonal antibody.

[0020] A fourth object of the present invention is to provide a use of the aforementioned monoclonal antibody in the preparation of a product for diagnosing and / or treating IL-8-mediated related diseases.

[0021] Furthermore, the related diseases include COVID-19, tumors and / or related autoimmune diseases.

[0022] More preferably, the tumor is glioblastoma.

[0023] A fifth object of the present invention is to provide a use of the aforementioned monoclonal antibody in the preparation of a reagent and / or a kit for detecting the expression level of IL-8.

[0024] To achieve the above object, the present invention adopts the following technical solutions:

[0025] The aforementioned monoclonal antibody is used in the preparation of a reagent and / or a kit for detecting IL-8 expression levels, wherein the reagent and / or the kit is used for early diagnosis, disease progression monitoring and / or prognosis judgment of patients with IL-8-mediated related diseases.

[0026] Further, flow cytometry, immunohistochemistry or ELISA were used for detection.

[0027] Furthermore, the related diseases include COVID-19, tumors and / or related autoimmune diseases.

[0028] More preferably, the tumor is glioblastoma.

[0029] Preferably, in the flow cytometry sorting, the detection object is GL261-mcherry-IL-8 cells.

[0030] The beneficial effects of the present invention are:

[0031] 1. IL-8 plays a variety of biological functions in the body and is associated with a variety of diseases. In view of this, the present invention has developed a monoclonal antibody against human IL-8, which can specifically bind to human IL-8 and has good affinity. After in vitro experimental verification, it can be used to diagnose and treat IL-8-mediated related diseases.

[0032] 2. The anti-IL-8 monoclonal antibody (B52 antibody) provided by the present invention can bind to GL261-mcherry-IL-8 cells and show a concentration-dependent relationship, indicating that the B52 antibody can be used for flow cytometry detection of cells expressing IL-8. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is the result of the phage trillion library screening;

[0034] Figure 2 This is the result of the initial screening of antibodies;

[0035] Figure 3This is the result of ELISA detection of B52 antibody binding to human IL-8;

[0036] Figure 4 This is the fitting curve diagram for the KD value detection of B52 antibody;

[0037] Figure 5 This is the result of IHC staining of B52 antibody in lung tissue of COVID-19 patients, among which, Figure 5 -A is the IHC staining result of the negative control. Figure 5 -B is the IHC staining result of the positive control. Figure 5 -C is the result of IHC staining with B52 antibody;

[0038] Figure 6 This is a biological result diagram of flow cytometry detection of B52 antibody binding to GL261-mcherry-IL-8 cells, where: Figure 6 -A is the flow cytometry result of negative control. Figure 6 -B is the flow cytometry test result of Isotype. Figure 6 -C is the flow cytometry result of the positive control. Figure 6 -D is the flow cytometry detection result of B52 antibody. DETAILED DESCRIPTION

[0039] The technical solution of the present invention will be further described clearly and completely in conjunction with specific embodiments below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work belong to the protection scope of the present invention.

[0040] Example 1. Antibody screening and preparation

[0041] (1) Purpose of the experiment

[0042] The constructed humanized recombinant antibody library was screened using biotinylated and non-biotinylated target antigens and overexpressing cell lines as screening materials, and positive clones binding to the target antigen IL-8 were screened by ELISA detection method.

[0043] (2) Experimental principle

[0044] The antibody sequences of the humanized recombinant antibody library are displayed on the surface of phage using phage display technology. Specific proteins, cells or peptides are used as antigens. After multiple rounds of screening, the phage-displayed antibodies that bind to the antigen are continuously enriched. Positive clones that specifically bind to the antigen are further screened by ELISA, and the antibody sequences of the positive clones are obtained by sequencing.

[0045] (3) Experimental methods

[0046] 1) Liquid phase screening

[0047] The biotin-labeled antigen is combined with the streptavidin-coupled magnetic beads, then incubated with the prepared phage, washed and eluted. After three rounds of panning, the specific monoclonal antibodies against the antigen are enriched.

[0048] 2) Solid phase screening

[0049] The antigen is coated on the surface of an immunotube with high adsorption capacity, and then the prepared phage is added to the immunotube for incubation, washing and elution. After three rounds of selection, the specific monoclonal antibodies against the antigen are enriched.

[0050] 3) Phage preparation

[0051] The obtained output collection is prepared into enriched phages through a series of steps including inoculation, auxiliary phage infection, phage expansion, phage precipitation and resuspension, and then put into the next round of screening.

[0052] 4) Selection set detection

[0053] The ELISA experiment was performed on the screening output set to detect the enrichment effect. After a series of steps such as coating, blocking, incubation, adding secondary antibodies, color development, termination, and detecting OD values, a standard curve was generated by serial dilutions of known standards to quantify the Fab expression supernatant. The concentration of the expression supernatant was used as the horizontal axis and the OD value was used as the vertical axis to draw a graph. The negative value was used as a reference to evaluate the enrichment degree of the Pool.

[0054] 5) Monoclonal screening

[0055] The target antigen is coated on the plate, and a series of steps are followed, including coating, blocking, incubation, adding secondary antibodies, color development, termination, and OD value detection. Positive clones are delineated according to a certain background value and sent for testing.

[0056] (4) Experimental results

[0057] The results of the phage library screening are as follows Figure 1 The results of the initial antibody screening are shown in Figure 2 As shown, multiple antibody molecules including LJH001-B52 were obtained, which had good binding to IL-8. The sequence information of B52 antibody is shown in Table 1.

[0058] Table 1. Sequence information of B52 antibody

[0059]

[0060] Example 2. Antibody function detection

[0061] (1) Antibody B52 can bind to human IL-8

[0062] Enzyme-linked immunosorbent assay was used to detect whether B52 antibody can bind to human IL-8. The experimental steps include: coating a 96-well ELISA plate with recombinant human IL-8-his protein (2 μg / ml, diluted in PBS), coating overnight at 4°C, and then adding 2% bovine serum albumin (BSA) to block at 37°C for 2 hours after washing. After the coated plate was washed with PBS-0.1% Tween20 solution, the sample B52 to be tested, the positive control, HuMax-IL8, and the negative control hIgG were added and incubated at 37°C for 2 hours; after washing with PBS-0.1% Tween20 solution, horseradish peroxidase HRP-labeled goat anti-human IgG (product of Sigma, USA) was added and incubated at 37°C for 1 hour; after being fully washed with PBS-0.1% Tween20 solution, o-phenylenediamine (OPD)-0.1% H2O2 substrate solution was added to develop color for 10-15 minutes, and then 0.1M HCl was added to terminate the reaction. Then, the OD value at 492 nm was read in a MK3-Multiskan microplate reader (product of Thermo Scientific, USA).

[0063] The test results are shown in Table 2. Figure 3 As shown, the data show that the B52 antibody can bind to human IL-8.

[0064] Table 2. ELISA test data of B52 antibody binding to human IL-8 protein (OD value)

[0065]

[0066] (2) B52 antibody affinity test

[0067] Using the affinity detection instrument Biacore T200 (Cytiva), select the probe ProteinA, dilute the antibody sample to be tested with HBS-EP (pH 7.4) to 80-120nM, set the flow rate to 10μL / min, and then bind with the ProteinA probe. Dilute the antigen IL-8 with HBS-EP (pH 7.4) to 18.75, 9.38, 4.69, 2.34, 1.17, 0.58nM, and set the flow rate to 30μL / min. Then bind with the antibody for 30sec, then bind with Kangyuan for 180sec, and dissociate for 1800sec. The entire reaction temperature is controlled at 25°C. The obtained data is fitted by software to calculate the KD value.

[0068] The fitting curve of B52 antibody KD value detection is as follows Figure 4 The results of B52 antibody affinity detection are shown in Table 3.

[0069] Table 3. B52 antibody affinity test results

[0070] KD(M) Ka(1 / Ms) Kd(1 / s) <![CDATA[R 2 ]]> Rmax(nm) 4.99E-10 1.02E+06 5.10E-04 0.988 0.183

[0071] (3) B52 antibody for immunohistochemistry (IHC) detection

[0072] Take frozen sections (4-8μm) of lung tissue from COVID-19 patients, place at room temperature for 30 minutes, add 4℃ acetone to fix for 10 minutes, wash three times with PBS for 5 minutes each time, and incubate with hydrogen peroxide for 5-10 minutes to eliminate the activity of endogenous peroxidase. Rinse with distilled water, soak in PBS for 5 minutes, and repeat once. Then add 10% normal goat serum to block, incubate at room temperature for 10 minutes, pour off the serum, add B52 antibody 10μg / mL, set negative and positive controls respectively, incubate at 37℃ for 2 hours, and then rinse with PBS three times for 5 minutes each time. Add HRP-labeled goat anti-mouse IgG2a secondary antibody (1μg / mL), incubate at 37℃ for 30 minutes, and rinse with PBS three times for 5 minutes each time. DAB color development for 10 minutes, then rinse with tap water thoroughly, counterstain, dehydrate, make transparent, and seal the slide.

[0073] Results of B52 antibody IHC staining of lung tissues of COVID-19 patients Figure 5 The results showed that IL-8 was highly expressed in the lung tissues of COVID-19 patients, and B52 antibodies were potential markers or potential therapeutic drugs for COVID-19 diagnosis.

[0074] (4) B52 antibody for flow cytometry (FACS)

[0075] Collect GL261-mcherry-IL-8 cells for 10 5 100 μL / well, add 200 μL punching solution and place at 4℃ for 30 minutes. Add the sample B52 antibody to be tested, positive control: Anti-human IL-8APC (0.3 μg / mL), negative control mouse IgG, incubate at 37℃ for 45 minutes. Wash once with 200 μL rinse solution. Then add enzyme-labeled secondary antibody Anti mouse IgG IF647 (1:1500), rinse twice, and detect on the machine.

[0076] The results are as follows Figure 6 As shown, B52 antibody can bind to GL261-mcherry-IL-8 cells in a concentration-dependent manner. Therefore, B52 antibody can be used for flow cytometry detection of IL-8 expressing cells.

Claims

1. An anti-IL-8 monoclonal antibody, characterized in that The heavy chain CDR region of the monoclonal antibody includes CDR1 with an amino acid sequence as shown in SEQ ID NO.5, CDR2 with an amino acid sequence as shown in SEQ ID NO.6, and CDR3 with an amino acid sequence as shown in SEQ ID NO.7; the light chain CDR region of the monoclonal antibody includes CDR4 with an amino acid sequence as shown in SEQ ID NO.8, CDR5 with an amino acid sequence as shown in SEQ ID NO.9, and CDR6 with an amino acid sequence as shown in SEQ ID NO.

10.

2. The monoclonal antibody according to claim 1, characterized in that The amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO.3; the amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO.

4.

3. The monoclonal antibody according to claim 1, characterized in that The IL-8 is a human IL-8 antigen.

4. A nucleic acid molecule encoding the monoclonal antibody according to any one of claims 1 to 3.

5. The nucleic acid molecule according to claim 4, characterized in that The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody comprises the sequence shown in SEQ ID NO.1, and the nucleotide sequence encoding the light chain variable region of the monoclonal antibody comprises the sequence shown in SEQ ID NO.

2.

6. A kit for detecting IL-8 expression level, characterized in that: The kit contains the monoclonal antibody according to any one of claims 1 to 3.

7. Use of the monoclonal antibody according to any one of claims 1 to 3 in the preparation of a product for diagnosing and / or treating IL-8-mediated related diseases.

8. The use according to claim 7, characterized in that: The related diseases include COVID-19, tumors and / or related autoimmune diseases.

9. Use of the monoclonal antibody according to any one of claims 1 to 3 in the preparation of a reagent and / or a kit for detecting the expression level of IL-8, characterized in that: The reagent and / or kit is used for early diagnosis, disease progression monitoring and / or prognosis judgment of patients with IL-8-mediated related diseases.

10. The use according to claim 9, characterized in that: Detection was performed by flow cytometry, immunohistochemistry or ELISA.

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