Monoclonal antibodies that bind to human il-8 antigens and uses thereof

Abstract

The application belongs to the technical field of biology and particularly relates to a kind of monoclonal antibody combined with human IL-8 antigen and application thereof.The heavy chain CDR region of the monoclonal antibody includes CDR1 with amino acid sequence as shown in SEQ ID NO.5, CDR2 with amino acid sequence as shown in SEQ ID NO.6 and CDR3 with amino acid sequence as shown in SEQ ID NO.7;The light chain CDR region of the monoclonal antibody includes CDR4 with amino acid sequence as shown in SEQ ID NO.8, CDR5 with amino acid sequence as shown in SEQ ID NO.9 and CDR6 with amino acid sequence as shown in SEQ ID NO.10.The monoclonal antibody can specifically bind to human IL-8, and has good affinity.Through in vitro experiment verification, it can be used for diagnosing and treating IL-8 mediated related diseases.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a monoclonal antibody that binds to human IL-8 antigen and its applications. Background Technology

[0002] Interleukin-8 (IL-8), also known as CXCL8, belongs to the CXC subtype and is the earliest discovered chemokine. IL-8 is a small protein with a molecular weight of approximately 8 kDa. Mature IL-8 exists in six forms: 79 aa, 72 aa, 71 aa, 70 aa, and 69 aa, with 72 aa being the most prevalent. Different molecular weights of IL-8 induce neutrophil chemotaxis and degranulation to varying degrees, with IL-8 containing 72 amino acids exhibiting the strongest activity; this is the commonly referred to mature IL-8. IL-8 is divided into two subsets: 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, resulting in an 8 kDa active substance. IL-8 is mainly secreted by monocytes and macrophages, and its function is to recruit and activate neutrophils, promoting inflammatory responses and cell killing. In addition, tumor cells, fibroblasts, endothelial cells, etc., under the influence of pro-inflammatory factors such as IL-1β, tumor necrosis factor α, and lipopolysaccharide, can affect the proliferation, invasion, metastasis, and formation of tumor blood vessels by autocrine or paracrine IL-8.

[0003] IL-8 exerts its function by binding to its receptors. There are two receptors for IL-8: CXCR1 (IL-8RA) and CXCR2 (IL-8RB). These two receptors share 77% homology and are both G protein-coupled receptors. Due to structural differences at their N-termini, the two receptors have different affinities for IL-8, with CXCR1 having a higher affinity. 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 function. CXCR2 has a lower affinity for IL-8 and can bind to various ligands besides IL-8, including CXCL1, 2, 3, 5, and 7. After IL-8 stimulates CXCR1 and / or CXCR2 receptors, it activates heterotrimeric small G proteins. IL-8 signaling promotes the activation of the major effector (PI3-K) or phospholipase C, thereby promoting the activation of the Akt, PKC, calcium mobilization, and / or MAPK signaling cascade.

[0004] IL-8 is a multifunctional factor with no species-specific biological function. Early findings indicated that neutrophils are its target cells; it specifically chemotactically attracts neutrophils into inflammatory tissues, promoting degranulation, superoxide anion production, and triggering a respiratory burst; it also activates inflammatory cells; promotes acute-phase protein synthesis; causes fever; participates in inflammatory pathological damage; promotes the release of inflammatory mediators; and promotes fibroblast proliferation—a process associated with chronic inflammation. Therefore, it plays a crucial role in inflammatory responses. Further research has revealed that IL-8 can act on different cell types, promoting the inflammatory process, stimulating angiogenesis, promoting mitosis, and regulating host immune function. It is closely related to the occurrence and development of various inflammatory diseases, tumors, and immune diseases.

[0005] In recent years, increasing research has shown that IL-8 plays an important biological role in the tumor immune microenvironment: ① IL-8 can recruit neutrophils and macrophages into the tumor microenvironment, cells that play a crucial role in tumor development, including promoting angiogenesis and tumor cell proliferation; ② IL-8 can induce the formation of new blood vessels, which provide essential nutrients and oxygen to the tumor, supporting its rapid growth; ③ It promotes the activity of tumor stem cells: IL-8 can indirectly support the survival and proliferation of tumor stem cells by attracting specific immune cells, which are considered the main cause of tumor recurrence and metastasis. Furthermore, multiple studies have demonstrated a negative correlation between IL-8 and the prognosis of cancer patients. For example, in the treatment of melanoma, high IL-8 levels are associated with shorter overall survival; patients with high IL-8 levels also have poorer prognoses in PD-1 inhibitor monotherapy or combination therapy. These findings suggest that IL-8 may negatively impact the prognosis of cancer patients by suppressing adaptive immunity and promoting immune tolerance.

[0006] Therefore, the development of monoclonal antibodies that can specifically bind to the IL-8 antigen is of great significance for the diagnosis and treatment of IL-8-related diseases. In the prior art, patent CN1309738C discloses an anti-IL-8 monoclonal antibody, its variable region sequence, and its applications. This invention discloses a monoclonal antibody with IL-8 neutralizing biological activity and its variable region sequence. Using IL-8 as an immunogen and mice as the immunization subjects, mice are immunized by injection, and the spleens of the immunized mice are used to prepare a suspension. This suspension is then fused with myeloma cells to obtain a hybridoma cell line expressing the anti-IL-8 monoclonal antibody, thus preparing the anti-IL-8 monoclonal antibody. This 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 a monoclonal antibody against IL-8, which can specifically bind to the target antigen IL-8, thereby providing support for the diagnosis and treatment of IL-8-mediated related diseases.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] A monoclonal antibody against IL-8, wherein the heavy chain CDR region of the monoclonal antibody comprises the amino acid sequences CDR1 as shown in SEQ ID NO. 5, CDR2 as shown in SEQ ID NO. 6, and CDR3 as shown in SEQ ID NO. 7; and the light chain CDR region of the monoclonal antibody comprises the amino acid sequences CDR4 as shown in SEQ ID NO. 8, CDR5 as shown in SEQ ID NO. 9, and CDR6 as 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] The second objective of this invention is to provide a nucleic acid molecule encoding the aforementioned monoclonal antibody.

[0013] To achieve the above objectives, the present invention adopts the following technical solution:

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

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

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

[0017] The third objective of this invention is to provide a kit for detecting IL-8 expression levels.

[0018] To achieve the above objectives, the present invention adopts the following technical solution:

[0019] A kit for detecting IL-8 expression levels, the kit containing the aforementioned monoclonal antibody.

[0020] The fourth objective of this invention is to provide an application of the aforementioned monoclonal antibody in the preparation of products for the diagnosis and / or treatment of IL-8-mediated related diseases.

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

[0022] More preferably, the tumor is a glioblastoma.

[0023] The fifth objective of this invention is to provide the application of the aforementioned monoclonal antibody in the preparation of reagents and / or kits for detecting IL-8 expression levels.

[0024] To achieve the above objectives, the present invention adopts the following technical solution:

[0025] The aforementioned monoclonal antibody is used in the preparation of reagents and / or kits for detecting IL-8 expression levels, which are used for early diagnosis, disease progression monitoring, and / or prognosis assessment of patients with IL-8-mediated diseases.

[0026] Further, detection can be performed using flow cytometry, immunohistochemistry, or ELISA.

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

[0028] More preferably, the tumor is a glioblastoma.

[0029] Preferably, the flow cytometry sorting is performed on GL261-mcherry-IL-8 cells.

[0030] The beneficial effects of this invention are as follows:

[0031] 1. IL-8 plays multiple biological functions in the body and is associated with various diseases. This invention develops a monoclonal antibody against human IL-8, which specifically binds to human IL-8 with good affinity. In vitro experiments have verified its potential for the diagnosis and treatment of IL-8-mediated diseases.

[0032] 2. The anti-IL-8 monoclonal antibody (B52 antibody) provided by the present invention can bind to GL261-mcherry-IL-8 cells in a concentration-dependent manner, indicating that the B52 antibody can be used for flow cytometry detection of IL-8-expressing cells. Attached Figure Description

[0033] Figure 1 Image showing the results of the selection of a trillion-strong phage library;

[0034] Figure 2 This is a graph showing the initial antibody screening results;

[0035] Figure 3The image shows the results of ELISA detection of the binding of B52 antibody to human IL-8.

[0036] Figure 4 The fitted curve for the KD value detection of B52 antibody;

[0037] Figure 5 Image showing the results of IHC staining for B52 antibody in lung tissue from a COVID-19 patient. Figure 5 -A shows the IHC staining results for the negative control. Figure 5 -B shows the IHC staining results for the positive control. Figure 5 -C shows the IHC staining results of B52 antibody;

[0038] Figure 6 The image shows the biological results of flow cytometry detection of the binding of B52 antibody to GL261-mcherry-IL-8 cells. Figure 6 -A shows the flow cytometry results for the negative control. Figure 6 -B is a graph showing the flow cytometry results from Isotype. Figure 6 -C represents the flow cytometry results for the positive control. Figure 6 -D is the flow cytometry result of the B52 antibody. Detailed Implementation

[0039] The technical solution of the present invention will be described more clearly and completely below with reference to specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention.

[0040] Example 1. Antibody screening and preparation

[0041] (1) Experimental Objective

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

[0043] (2) Experimental Principle

[0044] Phage display technology is used to display antibody sequences from a humanized recombinant antibody library on the surface of phages. Then, using specific proteins, cells, or peptides as antigens, phage display antibodies that bind to the antigen are continuously enriched through multiple rounds of screening. 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 separation

[0047] Biotin-labeled antigens are combined with streptavidin-conjugated magnetic beads, and then incubated, washed, and eluted with prepared bacteriophages. After three rounds of panning, specific monoclonal antibodies against the antigen are enriched.

[0048] 2) Solid-phase mass screening

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

[0050] 3) Phage preparation

[0051] The obtained output set is processed through a series of steps, including inoculation, assisted phage infection, phage amplification, phage precipitation and resuspension, to prepare enriched phages, which are then used in the next round of screening.

[0052] 4) Preliminary selection and testing

[0053] The enrichment effect was assessed using ELISA on the selected dataset. The process included plate coating, blocking, incubation, addition of secondary antibody, color development, termination, and OD value detection. A standard curve was generated using serially diluted known standards to quantify the Fab expression supernatant. The supernatant concentration was plotted on the x-axis, and the OD value on the y-axis, with negative values ​​used as a reference to evaluate the enrichment level of the pool.

[0054] 5) Initial screening of monoclonal antibodies

[0055] The method involves coating with target antigens, followed by a series of steps including coating, blocking, incubation, adding secondary antibody, color development, termination, and detection of OD values. Positive clones are identified based on a specific background value and then sent for testing.

[0056] (4) Experimental Results

[0057] Results of the trillion-phage pool selection Figure 1 As shown. The initial antibody screening results are as follows. Figure 2 As shown, several antibody molecules, including the one designated LJH001-B52, were obtained, and they exhibited good binding to IL-8. The sequence information of the B52 antibody is shown in Table 1.

[0058] Table 1. Sequence information of B52 antibody

[0059]

[0060] Example 2. Antibody Function Detection

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

[0062] Enzyme-linked immunosorbent assay (ELISA) was used to detect whether B52 antibody could bind to human IL-8. The experimental procedure included: coating a 96-well ELISA plate with recombinant human IL-8-his protein (2 μg / ml, diluted in PBS) overnight at 4°C; washing followed by blocking with 2% bovine serum albumin (BSA) at 37°C for 2 hours; washing the plate with PBS-0.1% Tween 20 solution, adding the test samples B52, positive control, HuMax-IL8, and negative control hIgG, and incubating at 37°C for 2 hours; washing with PBS-0.1% Tween 20 solution, adding horseradish peroxidase (HRP)-labeled goat anti-human IgG (Sigma-Aldrich product), and incubating at 37°C for 1 hour; washing thoroughly with PBS-0.1% Tween 20 solution, adding o-phenylenediamine (OPD)-0.1% H2O2 substrate solution for color development for 10-15 min, and then adding 0.1M HCl to terminate the reaction. The OD value at 492 nm was then read using an MK3-Multiskan microplate reader (a product of Thermo Scientific, USA).

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

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

[0065]

[0066] (2) B52 antibody affinity detection

[0067] Using a Biacore T200 (Cytiva) affinity assay instrument, the probe Protein A was selected. The antibody test sample was diluted to 80-120 nM with HBS-EP (pH 7.4) at a flow rate of 10 μL / min, and then bound to the Protein A probe. The antigen IL-8 was diluted with HBS-EP (pH 7.4) to 18.75, 9.38, 4.69, 2.34, 1.17, and 0.58 nM, with a flow rate of 30 μL / min. It was then bound to the antibody for 30 seconds, followed by binding to a specific antigen for 180 seconds, and then dissociated for 1800 seconds. The entire reaction was controlled at 25℃. The obtained data were fitted using software to calculate the KD value.

[0068] The fitted curve for B52 antibody KD value detection is shown below. Figure 4 As shown in Table 3, the results of the B52 antibody affinity test are as follows.

[0069] Table 3. Results of B52 antibody affinity assay

[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 is used for immunohistochemical (IHC) detection

[0072] Frozen sections (4-8 μm) of lung tissue from COVID-19 patients were incubated at room temperature for 30 minutes, fixed with acetone at 4°C for 10 minutes, washed three times with PBS for 5 minutes each time, and incubated with hydrogen peroxide for 5-10 minutes to eliminate endogenous peroxidase activity. The sections were then rinsed with distilled water, soaked in PBS for 5 minutes, and repeated once. Next, 10% normal goat serum was added for blocking, and the sections were incubated at room temperature for 10 minutes. The serum was discarded, and 10 μg / mL of B52 antibody was added. Negative and positive controls were provided, and the sections were incubated at 37°C for 2 hours. The sections were then rinsed three times with PBS for 5 minutes each time. HRP-labeled goat anti-mouse IgG2a secondary antibody (1 μg / mL) was added, and the sections were incubated at 37°C for 30 minutes. The sections were rinsed three times with PBS for 5 minutes each time. DAB staining was performed for 10 minutes, followed by thorough rinsing with tap water, counterstaining, dehydration, clearing, and mounting.

[0073] Results of IHC staining for B52 antibody in lung tissue from COVID-19 patients are as follows: Figure 5 As shown in the figure. This result indicates that high expression of IL-8 and B52 antibodies in the lung tissue of COVID-19 patients is a potential diagnostic biomarker or potential therapeutic agent for COVID-19.

[0074] (4) B52 antibody is used for flow cytometry (FACS) detection.

[0075] Collect 10 GL261-mcherry-IL-8 cells 5 Add 200 μL of punching buffer to each well and incubate at 4°C for 30 minutes. Add the B52 antibody to be tested, positive control: Anti-human IL-8 APC (0.3 μg / mL), negative control: mouse IgG, and incubate at 37°C for 45 minutes. Wash once with 200 μL of washing buffer. Then add the enzyme-labeled secondary antibody Anti mouse IgG IF647 (1:1500), wash twice, and then perform the assay.

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

Claims

1. A monoclonal antibody against human IL-8, characterized in that, The heavy chain variable region of the monoclonal antibody comprises 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 variable region of the monoclonal antibody comprises CDR1 with an amino acid sequence as shown in SEQ ID NO. 8, CDR2 with an amino acid sequence as shown in SEQ ID NO. 9, and CDR3 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 as shown in SEQ ID NO. 3; the amino acid sequence of the light chain variable region of the monoclonal antibody is as shown in SEQ ID NO.

4.

3. A nucleic acid molecule encoding the monoclonal antibody according to any one of claims 1-2.

4. The nucleic acid molecule of claim 3, wherein, 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.

5. A kit for detecting the level of IL-8 expression, characterized in that, The kit contains the monoclonal antibody according to any one of claims 1-2.

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