Anti-IL-8 monoclonal antibody as well as variable region sequence and application thereof

Abstract

The invention belongs to the technical field of biology, provides an anti-IL-8 monoclonal antibody, a variable region sequence of the anti-IL-8 monoclonal antibody and application of the anti-IL-8 monoclonal antibody, and discloses a monoclonal antibody with neutralizing interleukin-8 (IL-8) biological activity and the variable region sequence of the monoclonal antibody. The preparation method comprises the following steps: by taking IL-8 as an immune source and a mouse as an immune object, carrying out immune injection on the mouse, taking the spleen of the immune mouse to prepare a suspension, and carrying out cell fusion on the suspension and myeloma cells to obtain a hybridoma cell strain capable of expressing the anti-IL-8 monoclonal antibody so as to prepare the anti-IL-8 monoclonal antibody; according to the invention, a variable region amino acid sequence of the anti-IL-8 monoclonal antibody is cloned at the same time; the anti-IL-8 monoclonal antibody disclosed by the invention can be specifically combined with IL-8, so that the anti-IL-8 monoclonal antibody can be used for treating diseases related to abnormal or excessive or out-of-control expression and secretion of the IL-8.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, in particular to an anti-IL-8 monoclonal antibody, a variable region sequence thereof and an application thereof. Background Art

[0002] Interleukin-8 (IL-8) is a protein molecule called chemokine in cytokines. In the large family of chemokines, they can be divided into CXC chemokines and CC chemokines according to the position of the cysteine ​​at the N-terminus in their molecular structure. IL-8 belongs to the CXC chemokine with a molecular weight of about 8,000Da. IL-8 is a strong chemotactic and activating factor for neutrophils. It can strongly gather and activate neutrophils. It has a wide range of biological activities in many physiological or pathological processes and plays a key role in the inflammatory process. IL-8 can be produced and secreted by a variety of cells involved in inflammatory reactions, such as macrophages, neutrophils, lymphocytes, endothelial cells, keratinocytes, etc. It then acts on a variety of inflammatory cells such as endothelial cells, neutrophils and T lymphocytes, thereby forming an inflammatory response. Many human diseases are related to abnormal or excessive or uncontrolled expression and secretion of IL-8 in the body.

[0003] Antibodies are a type of protein molecule produced by B lymphocytes, consisting of two identical heavy chains and two identical light chains. The light chain and heavy chain are each composed of a variable region and a constant region. The specific binding of antibodies to the corresponding antigen is determined by the amino acid sequence of the variable region. The monoclonal antibody (McAb) technology was first created in 1975. Monoclonal antibodies with specific binding activity to the corresponding antigen can be obtained by immunizing mice with antigens and then screening with hybridoma technology.

[0004] The purpose of the present invention is to reapply for the expired Chinese patent with publication number CN1309738C. Summary of the invention

[0005] The first object of the present invention is to provide an anti-IL-8 monoclonal antibody.

[0006] The second object of the present invention is to provide an amino acid sequence of the variable region of an anti-IL-8 monoclonal antibody.

[0007] The third object of the present invention is to provide the use of anti-IL-8 monoclonal antibodies.

[0008] The present invention uses IL-8 as an immune source and mice as immune objects. The mice are injected with immune drugs, and the spleens of the immune mice are taken to prepare a suspension. The spleen suspension is fused with myeloma cells to obtain a hybridoma cell line that can express anti-IL-8 monoclonal antibodies. The anti-IL-8 monoclonal antibodies are prepared by screening and purification.

[0009] The amino acid sequences of the variable regions of the heavy chain and light chain of the anti-IL-8 monoclonal antibody cloned in the present invention are shown in SEQ ID No. 1 and 2, respectively.

[0010] The anti-IL-8 monoclonal antibody of the present invention can specifically bind to IL-8, and thus can be used to treat diseases associated with abnormal, excessive, or uncontrolled expression and secretion of IL-8.

[0011] Compared with the prior art, the present invention has the following beneficial effects:

[0012] The present invention has proved through experiments that anti-IL-8 monoclonal antibodies have obvious therapeutic effects on diseases related to abnormal or excessive or uncontrolled expression and secretion of IL-8, such as psoriasis, eczema, tumors, severe acute respiratory syndrome (SARS), etc., and have high specificity, so they have good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 It is the electrophoresis diagram of 5'RACE products, where No. 1 is the light chain result, No. 2 is the heavy chain result, and M is DL2000Marker (molecular weight from top to bottom is 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp);

[0014] Figure 2 This is the silver-stained electrophoresis diagram of anti-IL-8 chimeric antibody, where 1 is the protein marker; 2 is the hybridoma antibody; and 3 is the chimeric antibody. DETAILED DESCRIPTION

[0015] The following embodiments of the present invention are described in further detail in conjunction with the accompanying drawings and examples. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0016] The following embodiments of the present invention are described in further detail in conjunction with the accompanying drawings and examples. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0017] Example 1: Preparation of anti-IL-8 monoclonal antibody hybridoma cells

[0018] 1. Immune source

[0019] The recombinant human monocyte-derived IL-8 expressed by E. coli is composed of 72 amino acids and has a molecular weight of 8.0 KDa. It has stronger biological activity than the endothelial cell-derived IL-8 composed of 77 amino acids. This recombinant IL-8 is produced by PeproTech Inc. in the United States. Its purity is >98% as determined by N-terminal analysis and SDS-PAGE silver staining. In the chemotaxis assay, it was shown that this recombinant IL-8 has a strong chemotaxis effect on human neutrophils.

[0020] 2. Immunization of animals

[0021] Balb / c mice were supplied by Charles River Laboratories, Inc. Canada. All Balb / c mice used for immunization were standardized disease-free, healthy purebred mice that met the standards required by the US FDA and Canadian HPB.

[0022] 3. Animal immunization procedures

[0023] Day 1: 200 μl of antigen solution (20 μg IL-8 / 200 μl PBS) was added with 200 μl Freund's complete adjuvant (Sigma, Catalogue number F5881) to prepare 400 μl antigen emulsion, which was injected into multiple subcutaneous points on the back and soles of the mice.

[0024] Day 27: 200 μl of antigen solution (20 μg IL-8 / 200 μl PBS) plus 200 μl of incomplete Freund's adjuvant (Sigma, Catalogue number F5506) were injected into the abdominal cavity and sole.

[0025] Day 86: Injection dosage and method are the same as Day 27.

[0026] Day 118: Injection dosage and method are the same as Day 27.

[0027] Day 179: 20 μg / 450 μl PBS antigen solution was injected intraperitoneally.

[0028] Day 183: The spleens of the immunized mice were removed and spleen cell suspensions were prepared for cell fusion.

[0029] 4. Cell fusion:

[0030] (1) Myeloma cells

[0031] SP2 / 0-Ag14 mouse myeloma cell line comes from ATCC (ATCC CRL-1581) in the United States. It does not synthesize or secrete any mouse immunoglobulin heavy chain and light chain. This cell is resistant to 8-azaguanine and cannot survive for a long time in HAT (hypoxanthine, aminopurine and thymidine) culture medium (GIBCO, Cat. No. 21060-017). SP2 / 0-Ag14 has been widely used as a fusion cell partner in the preparation of hybridomas that secrete monoclonal antibodies.

[0032] (2) Integration

[0033] A. SP2 / 0-Ag14 myeloma cells in the logarithmic growth phase were washed twice with serum-free RPMI 1640 culture medium (GIBCO, Cat. No. 11875), and counted for later use.

[0034] B. Take the spleen cell suspension of immune mice prepared under sterile conditions, wash it twice with serum-free RPMI1640 culture medium, count it and set it aside.

[0035] C. Spleen cells and SP2 / 0-Ag14 myeloma cells were mixed at a ratio of 5:1, centrifuged (1500 rpm) for 7 minutes, and the supernatant was discarded. Then, 1 ml of PEG (PEG4000, MW3,000-4,000, GIBCO, BRL Cat. No.14030-019) was slowly added within 1 minute, and the mixture was allowed to stand for 1.5 minutes. Then, 5 ml of serum-free RPMI 1640 culture medium was added within 2.5 minutes, and the mixture was allowed to stand for 5 minutes. Finally, 5 ml of serum-free culture medium was added, and the mixture was centrifuged (1000 rpm) for 5 minutes. The supernatant was discarded, and conventional 1640 culture medium (15% fetal bovine serum, FBS GIBCO, BRL Cat. No.16000-044) was added to prepare a cell suspension.

[0036] D. Transfer the above fusion cell suspension into the wells of a 96-well culture plate, add 2 drops of cell suspension to each well, and place in a 37°C, 5% CO2 incubator; after culturing for 24 hours, add 2 drops of 2x HAT culture medium to each well, and continue to culture in a 37°C, 5% CO2 incubator. Change the HAT culture medium every three days, and change to HT culture medium (GIBCO, Cat. No. 11067-030) after 7-10 days. After 12 days of culture, begin to detect the supernatant of the cloned cell wells using an enzyme-linked immunosorbent assay (ELISA) to screen out clones that are positive for anti-IL-8 antibodies.

[0037] (3) Cloning of hybridomas

[0038] Using the limiting dilution method, the cell suspension is diluted to 3-10 cells / ml, and then the cell suspension is transferred to a 96-well plate, 2 drops of cell suspension are added to each well, and the plate is cultured in a 37°C 5% CO2 incubator. After that, 1 / 3 of the fresh culture medium is replaced in each well every 2 to 3 days. The second screening and cloning are carried out about 10 days later. After three consecutive subclonings, the cloning rate must be less than 66.7% each time, and the antibody positive rate must be 100% to determine that the cloned cells are monoclonal.

[0039] 5. Characteristics of monoclonal antibodies:

[0040] Immunoglobulin subtype identification:

[0041] The hybridoma cell line was identified using Mouse Typer Sub-Isotyping Kit (Cat. No. 172-2055 BIORAD) produced by BIORAD, USA. The results showed that clone 6E6 (also known as I8-60) had immunoglobulin subtype: IgG1, Kappa.

[0042] The cell line 6E6 (I8-60) was deposited in the China General Microbiology Center (CGMCC) on November 18, 2004, with the number CGMCC No.1249.

[0043] Example 2: Specificity of anti-IL-8 hybridoma monoclonal antibodies

[0044] The anti-IL-8 monoclonal antibody obtained in Example 1 was cross-reacted with other cytokines, chemotactic factors GM-CSF, TGF-β, MCAF, TNF-α, IL-7, IL-1β, b-FGF, IL-16, MCP-3, M-CSF, EGF and cytokines GROa, PF-4, ENA78, NAP-2 and GCP2 homologous to IL-8 at the same concentration (μl / ml), and the results of ELISA detection were shown in Tables 1 and 2. It can be seen that the monoclonal antibody of the present invention has no cross-reaction with other cytokines, chemotactic factors and cells homologous to IL-8, but has a high specific reaction to IL-8.

[0045] Table 1 ELISA method to detect the cross-reactivity of this antibody to various cytokines / chemotactic factors

[0046]

[0047]

[0048] Table 2 ELISA method to detect the cross-reaction of this antibody to other cytokines with structural homology to IL-8

[0049] Cytokines IL-8 GRO PF-4 ENA78 NAP-2 GCP2 0.D. value >2.0 0.097+0.008 0.113+0.04 0.073+0.009 0.031+0.004 0.088+0.007

[0050] Example 3: Ability of anti-IL-8 monoclonal antibodies to neutralize IL-8 chemotactic activity

[0051] The ability of the antibody to inhibit the chemotaxis of IL-8 was determined by using a chemotaxis assay (Ben-Baruch A, et al. Cytokine. 1997 Jan; 9(1): 37-45.) with human neutrophils (isolated from normal human peripheral blood, Liu WC, et al. Xibao Yu Fenzi Mianyixue Zazhi 1996; 12: 64-66) or a 293 cell line stably expressing the IL-8 receptor (Ben-Baruch A, et al. Cytokine. 1997 Jan; 9(1): 37-45.) as indicator cells.

[0052] The antibody was diluted to four concentrations of 50 μg / ml, 5 μg / ml, 0.5 μg / ml, and 0.05 μg / ml, respectively, and reacted with 10 ng / ml of IL-8. After 30 minutes at 37°C, each group of samples was injected into the lower well of the chemotaxis chamber. 50 μl of human neutrophils or 293 cells stably expressing IL-8 receptor (1×10 6 / ml) was placed in the upper well of the chamber. The upper and lower layers of the chamber were separated by a 10μm pore size polycarbonate filter membrane. The chamber was placed at 37℃, 5% CO 2 After incubation for 5 hours, the filter membrane was taken out and stained with Dieff-QuicK. The number of neutrophils or 293 cells on the membrane was counted and the result was expressed as the average value of three parallel wells.

[0053] Experiments have shown that when the concentration ratio of monoclonal antibody to IL-8 is 50:1, it can effectively neutralize the chemotaxis of IL-8 on neutrophils or 293 cells stably expressing IL-8 receptors.

[0054] Table 3

[0055]

[0056]

[0057] Example 4: Construction of chimeric antibody eukaryotic expression plasmid

[0058] 1. Cloning of variable region sequences of anti-IL-8 monoclonal antibody in hybridoma cell line I8-60

[0059] The variable region sequence of the functional antibody was cloned from the hybridoma cell line I8-60 that secretes anti-human IL-8 mouse monoclonal antibody using 5'RACE (Rapid Amplification of cDNA Ends) technology. The steps can be briefly described as follows: the first strand of cDNA is synthesized by an antisense gene-specific primer (GSP1). After the first strand of cDNA is purified, a synthetic homopolynucleotide anchor sequence is added to the 3' end of the cDNA using terminal deoxynucleotidy transferase (TdT). The cDNA is amplified using a second nested gene-specific primer (GSP2) and an anchor primer that can anneal to the homopolynucleotide tail.

[0060] Material:

[0061] -Hybridoma cell line 6E6 (I8-60) secretes anti-human IL-8 monoclonal antibody

[0062] -Escherichia coli strain Top10 (Invitrogen, Cat. No. C4040-03)

[0063] - Cloning vector pGEM-T-easy (Promega, Cat. No. A1360)

[0064] Primer design:

[0065] According to the characteristics of immunoglobulin genes, two sets of gene-specific primers GSP1 and GSP2 corresponding to the Ig and Kappa constant regions were designed. The primer sequences are as follows:

[0066] pRace-H-GSP1:GTCCACCKYGGTSYTGCTGGCYGGGTG

[0067] pRace-H-GSP2:GCACACYRCTGGACAGGGATCCAGAGTTCC

[0068] pRace-K-GSP1:ACTTGACATTGATGTCTTTG

[0069] pRace-K-GSP2:CACGACTGAGGCACCTCCAGATG

[0070] Cloning process:

[0071] 1) Synthesis of the first strand

[0072] Using total RNA as template and GSP1 as primer, the first strand of cDNA is synthesized under the action of reverse transcriptase. The specific steps are:

[0073] ① Add the following components to a 0.5 ml microcentrifuge tube:

[0074] GSP1 2.5 pmol

[0075] Total RNA 1-5 μg

[0076] Add DEPC-treated water to a total volume of 15.5 μl and mix well.

[0077] ② Denature at 70℃ for 10min, ice bath for 1min, centrifuge briefly and add the following components in sequence:

[0078]

[0079] ③Mix well, centrifuge briefly and incubate at 42℃ for 1 min.

[0080] ④ Add 1 μl SuperScriptTMII RT (Invitrogen) to the reaction system, mix gently and react at 42°C for 50 min.

[0081] ⑤After reverse transcription, place at 70℃ for 15 minutes to terminate the reaction.

[0082] ⑥ Centrifuge for 10-20 seconds and place at 37℃.

[0083] ⑦ Add 1 μl RNase mix, mix gently and place at 37℃ for 30 minutes.

[0084] ⑧Take out the reaction tube and place it on ice for later use.

[0085] 2) Purification of cDNA

[0086] QIAquick PCR purification kit from QIAGEN was used.

[0087] ① Add 5 times the volume of PB buffer in the reverse transcription system to the reverse transcription reaction tube and mix well.

[0088] ② Place the QIAquick spin column in a 2 ml collection tube, add the sample to the spin column, and centrifuge at 13,000 rpm for 60 seconds.

[0089] ③Discard the liquid, put the centrifuge column back into the original collection tube, add 0.75 ml PE buffer to the QIAquick centrifuge column, and centrifuge at 13,000 rpm for 60 seconds.

[0090] ④Discard the liquid, return the QIAquick spin column to the original collection tube, and centrifuge at 13,000 rpm for 60 seconds.

[0091] ⑤ Place the QIAquick centrifuge column in a clean 1.5ml centrifuge tube, add 30μl EB buffer to the center of the QIAquick membrane, let it stand for 1 minute, and centrifuge at 13,000rpm for 1 minute.

[0092] 3) cDNA tailing

[0093] ① Add the following components in a 0.5 ml microcentrifuge tube:

[0094]

[0095] Mix

[0096] ② Place the reaction tube at 94°C for 3 min, place on ice for 1 min, centrifuge briefly, and place on ice.

[0097] ③Add 1 μl TdT, mix well and react at 37°C for 10 min.

[0098] ④ Place the reaction tube at 65℃ for 30 min to terminate the reaction, centrifuge and place on ice for later use.

[0099] 4) PCR

[0100] Add the following components to a 0.2 ml thin-walled PCR tube:

[0101]

[0102] After mixing, place in PCR instrument for reaction.

[0103] 5) Purification of PCR products by electrophoresis

[0104] The PCR products were identified by 1.5% agarose gel electrophoresis. Figure 1 As shown: No. 1 is the light chain result, with a specific band at about 500 bp; No. 2 is the heavy chain result, with a specific band at about 500 bp;

[0105] 6) Use the pGEM-T easy kit from Promega to clone the PCR product into the pGEM-T easy vector and transform Escherichia coli Top10 (Invitrogen, Cat. No. C4040-03). The specific process is as follows:

[0106] a) The PCR product was subjected to 1.5% agarose gel electrophoresis, and a 500 bp band was recovered and finally fixed to 20 μl of sterile water;

[0107] b) In a 0.5 ml microcentrifuge tube, add the following components:

[0108]

[0109] After mixing, connect at room temperature for 2 hours or at 4°C for more than 16 hours.

[0110] c) Transform competent E. coli cells, spread on LB plates containing ampicillin, IPTG and X-GAL, and culture at 37°C for about 12 hours.

[0111] 7) Determine the base sequence of the cloned PCR product: Select at least 5 plasmids and determine the base sequence of the PCR product contained therein.

[0112] 8) Based on the corresponding relationship between the base sequence and the amino acid coding sequence, the reading frame of the PCR product was analyzed to determine the amino acid sequence of the corresponding variable region, and the sequences of the heavy chain and light chain were shown in SEQ ID No. 1 and 2, respectively. Based on the characteristics of the immunoglobulin gene, it was verified to be an antibody sequence.

[0113] 2. Cloning of the constant regions of IgG1 and kappa chains of human antibodies

[0114] Primer design:

[0115] Using the constant region sequences of IgG1 and kappa chains of human antibodies in the NCBI database as templates, two pairs of primers were designed to clone the constant region sequences of human light chain and heavy chain, respectively. The primer sequences are as follows:

[0116]

[0117] The constant region sequences of human light chain and heavy chain were amplified by PCR, respectively, and then digested with Apal&Pstl, BsiWI&NotI, respectively, and connected to the eukaryotic expression plasmid pcDNA3.1 (Invitrogen, Cat. No. V795-20) digested with the same enzymes.

[0118] 3. Construction of expression plasmid

[0119] Using the sequence obtained by 5'RACE sequencing as a template, two pairs of primers were designed to amplify the variable region sequence and signal peptide sequence of the mouse functional antibody gene by PCR, respectively. After being digested by NheI&Apal and EcoRV&BsiWI, they were ligated to pcDNA3.1 containing the above-mentioned human antibody IgG1 and kappa chain constant regions digested with the same enzymes to obtain a chimeric antibody eukaryotic expression plasmid.

[0120] The primer sequences are as follows:

[0121]

[0122] Example 5: Chimeric Antibody Expression

[0123] CHO cells (purchased from the cell bank of the Chinese Academy of Sciences) were transfected by liposome method to obtain a stable transfected cell line. The specific method is:

[0124] 1) One day before transfection, 3×10 6 The cells were plated on a 10-cm plate and replaced with 10 ml of fresh culture medium (DMEM + 10% FBS) 3 hours before transfection;

[0125] 2) Transfection: Add 10ug heavy chain plasmid and 10ug light chain plasmid to tube A containing 1.5ml DMEM, mix gently, and place at room temperature for 5min; at the same time, add 60ul Lipofectamine2000 to tube B containing 1.5ml DMEM, mix gently, and place at room temperature for 5min. Combine the liquids in tubes A and B, mix gently, and place at room temperature for 20min. Add the DNA-Lipofectamin2000 mixture dropwise to the CHO cell culture medium. After culturing in a 37℃, CO2 incubator for 6hrs, replace the culture medium, continue culturing for 48hrs, dilute the cells 1:10, continue culturing for 10-14 days, and select single clones.

[0126] Example 6: Purification of chimeric antibodies

[0127] The chimeric antibody was purified by protein A affinity chromatography. The specific steps are as follows: (1) Wash the protein A affinity chromatography column with 3-5 column volumes of water. (2) Equilibrate the protein A affinity chromatography column with 20 mM phosphate buffer, pH 7.0. (3) Pump the monoclonal antibody sample to be purified into the protein A affinity chromatography column. (4) Wash the column with 20 mM phosphate buffer, pH 7.0 until the OD 280 <0.01. (5) Elute with 0.1M citric acid-sodium citrate buffer, pH 3.0, collect the elution peak, and neutralize the collected antibody solution with 1M Tris-HCl buffer, pH 9.0. (6) Desalt by dialysis with 10mM PBS, pH 7.2. See the electrophoresis diagram Figure 2 The purity of the purified chimeric antibody was 95.89% as determined by HPLC.

[0128] Example 7: Determination of the immunological activity of chimeric antibodies

[0129] The immunoreactivity of the chimeric antibody was measured by Elisa method and compared with the mouse hybridoma antibody. The results in Table 4 showed that there was no significant difference between the immunoreactivity of the chimeric antibody and the mouse hybridoma antibody.

[0130] Table 4 Comparison of Elisa results between chimeric antibodies and mouse hybridoma antibodies

[0131]

[0132] Elisaplate coatedwith lug / ml IL-8.

[0133] sccondantibody:goatanti-mouseproteinL-HRPconjugated

[0134] Example 8: Determination of biological function of chimeric antibodies

[0135] The microporous chamber chemotaxis assay was used to detect the inhibitory effect of the chimeric antibody on the chemotaxis activity of IL-8. The results in Table 5 showed that 1 ug / ml chimeric antibody could significantly inhibit the chemotaxis activity of IL-8, and its inhibition rate was not significantly different from that of the mouse hybridoma antibody.

[0136] Table 5 Comparison of chemotaxis test results between chimeric antibodies and mouse hybridoma antibodies

[0137]

[0138] Example 9 Application of anti-IL-8 monoclonal antibody

[0139] 1) Preparation of cold cream containing 0.004% anti-human interleukin-8 monoclonal antibody drug

[0140] Substrate used:

[0141]

[0142] First, mix the base evenly, heat it to 80℃, continue stirring for 0.5 hours, then cool it to 35℃, use a dropper to draw the drug solution and add it to the constantly stirred base, continue stirring for 30 minutes until it is evenly mixed to make a cold cream, which is used in the following studies on psoriasis and eczema

[0143] 2) Clinical research results on the treatment of psoriasis

[0144] Based on the results of Phase I, II, and III clinical trials and the spirit of the approval document from the Drug Evaluation Center of the State Food and Drug Administration, from August 2001 to August 2002, the Institute of Dermatology, Chinese Academy of Medical Sciences, led the team and worked with 17 central hospitals across the country to conduct Phase IV clinical trials on patients with psoriasis vulgaris and palmoplantar pustular psoriasis using cold cream prepared with the anti-IL-8 monoclonal antibody. The large-sample, multi-center, long-term, open clinical trial was used to deeply observe the efficacy and safety of the cold cream for topical treatment of psoriasis, providing reliable scientific data for its widespread clinical application.

[0145] A total of 1,431 cases of psoriasis vulgaris were enrolled in the Phase IV clinical trial. The cold cream was applied to the lesions twice a day for 8 consecutive weeks. 1,365 cases completed the 8-week treatment course. 99 cases of palmoplantar pustular psoriasis were enrolled, and 87 cases completed the 8-week treatment course. A total of 1,530 cases were enrolled in this clinical trial, and 1,452 cases completed the 8-week treatment course.

[0146] After a one-year clinical study and observation in 18 clinical dermatology medical institutions, the results showed that after 8 weeks of treatment of psoriasis vulgaris with the cold cream, the symptoms and signs observed, namely the diameter of the target lesions, erythema, skin thickness infiltration, scaling, and itching score, were significantly reduced (P values ​​were all less than 0.001). The cold cream has a strong therapeutic effect. After 8 weeks of treatment, the basic cure rate was 17.85% and the effective rate was 61.91%. Analysis of clinical data showed that the cold cream had a good effect on patients with guttate, coin-shaped, small plaque-shaped and mixed psoriasis lesions, with the guttate being the best. It is effective for both active and quiescent psoriasis, with the active stage being the best.

[0147] 226 cases of psoriasis vulgaris were cured within 8 weeks of treatment with the cold cream, and the relapse rate was only 5.8% after a further 8-week follow-up, indicating that the condition remained stable after treatment with the cold cream and was not prone to rebound and relapse.

[0148] After 8 weeks of treatment of palmoplantar pustular psoriasis, the clinically observed symptoms and signs, i.e., the target lesion diameter, the number of pustules, the degree scores of erythema, blisters, scaling, itching and pain, decreased significantly (all P values ​​were less than 0.001), and the main symptoms and signs of the disease were significantly cured. After 8 weeks of treatment, the basic cure rate of palmoplantar pustular psoriasis was 14.8%, and the effective rate was 60.5%. Therefore, the IV phase clinical trial showed that the cold cream had a good effect in treating palmoplantar pustular psoriasis.

[0149] In conclusion, the Phase IV clinical trial of cold cream prepared with anti-IL-8 monoclonal antibody for the treatment of psoriasis, combined with the results of Phase I, II, and III clinical trials, proves that the cold cream can be used externally to treat various types of progressive or quiescent psoriasis vulgaris, as well as palmoplantar pustular psoriasis. Long-term treatment is safe and effective, with high patient compliance and low relapse rate. It can be widely used in clinical practice.

[0150] 3) Treatment and research of eczema

[0151] Preliminary results of cold cream prepared with anti-IL-8 monoclonal antibody in the treatment of eczema

[0152]

[0153] 4) Research results on tumor treatment

[0154] A distinct characteristic of solid tumor growth is its strict dependence on new angiogenesis. Folkman first inferred that tumor growth is angiogenesis-dependent, and that tumor growth requires an increase in blood supply. Many papers have reported that IL-8 is present as a pro-angiogenic factor in freshly isolated human non-small cell lung cancer (NSCLC) tissues. As a member of the CXC chemokine family, IL-8 is an effective pro-angiogenic factor. In animal experiments with IL-8, immunohistochemical analysis of transplanted tumors showed that IL-8 is localized to human tumor cells. Since mice cannot produce IL-8, and anti-human IL-8 antibodies have no cross-reactivity with mouse homologous CXC chemokines, the results can prove that the reduction of tumors in SCID mice is caused by anti-human IL-8 antibodies. For NSCLC cell lines, IL-8 is not an autocrine growth factor, which further confirms that the role of anti-human IL-8 antibodies in tumor suppression is by blocking angiogenesis rather than blocking cell growth.

[0155] 1. Materials:

[0156] 1. Cell line A549 was maintained in DMEM containing 10% calf serum.

[0157] 2. SCID mice: 8-week-old female mice.

[0158] 3. Anti-human IL-8 antibody.

[0159] (II) Methods:

[0160] i) SCID mouse transplant tumor experiment: A549 cells in the active proliferation stage were obtained and the cell concentration was adjusted to 4×10 7 / ml, mixed with Matrigel (BD, USA) at a ratio of 1:1, and 0.1ml of this A549 cell mixture was subcutaneously injected into each mouse. On the second day of inoculation, the mice were randomly divided into a control group and a 125μg anti-IL-8 antibody treatment group, with 4 mice in each group. Treatment plan: intraperitoneal injection of 200μl of normal saline or anti-human IL-8 antibody, every Monday, Wednesday, and Friday. The experiment was stopped after three weeks.

[0161] ii) Isolation of mouse tumor tissue: Mice were killed by cervical dislocation, and tumor tissue was carefully removed from the subcutaneous tissue of the mouse, weighed, and quickly frozen in a -70°C refrigerator.

[0162] ii) RNA extraction and cDNA synthesis Tissue blocks with a diameter of 5 mm were taken and total RNA was extracted using Trizol reagent (Shanghai Shenneng Gaming Co., Ltd.). The obtained RNA (2 μg) was used to synthesize cDNA (reaction volume 20 μl) using the Revertaid First-Strand cDNA Synthesis Kit (MBI Ferments, Lithuania).

[0163] iv) Real-time quantitative PCR detection Quantitative PCR detection was performed on a LightCycler PCR instrument with a reaction volume of 25 μl, containing 2 μl of cDNA, 5 μl of 5× Buffer, and 250 mM MgCl 2 0.5μl, 10mM dNTP 0.75μl, Taq HS 0.25μl, 100μM primer 0.1μl each, 50μM probe 0.1μl. The reaction conditions are: 50℃180s, 95℃300s, then 95℃15s, 60℃60s, 40 cycles. The results of IL-8, VEGF, CD31 and GAPDH mRNA detection are given by computer software automatically calculated according to the standard curve and expressed as copy number / reaction. Each cDNA sample was detected under the same reaction conditions, and the expression values ​​of IL-8, VEGF and CD31 were expressed as (copy number × 103) / GAPDH copy number.

[0164] (III) Results:

[0165] 1. Effect of anti-human IL-8 antibody on tumor size of SCID mice inoculated with different tumor cells three weeks after administration As can be seen from Table 6, three weeks after administration, the tumors inoculated with A549 were significantly reduced, and the tumor inhibition rate of anti-human IL-8 antibody was 75.62%.

[0166] Table 6 Effect of anti-human IL-8 antibody on tumor size in SCID mice inoculated with tumor cells after three weeks

[0167] A549 Control group 100.9±11.6 Drug group 24.6±12.2

[0168] Note: Unit: mg

[0169] 2. Real-time quantitative PCR analysis of CD31, IL-8 and VEGF gene expression in different tumor tissues The expression values ​​of IL-8, VEGF and CD31 genes in different tumor tissues were analyzed by quantitative PCR method and compared. As can be seen from Table 7, the amount of IL-8 gene expression in the solid tumor formed by A549 is very high. After treatment with anti-human IL-8 antibody, the expression of IL-8 in A549 tumors was significantly reduced.

[0170] Table 7 Gene expression in tumors inoculated with different cells

[0171]

[0172] Note: Unit: (copy number × 10) / GAPDH copy number

[0173] 4) Research results on the treatment of SARS:

[0174] 1. In order to explore whether the excessive expression and secretion of chemokines (inflammatory factors) in the infected parts of the body caused by viral infection in SARS patients is the cause of the severe inflammatory response in lung tissue. We conducted quantitative detection of chemokines such as IL-8 and MCAF in the serum of SARS patients by Peking Union Medical College Hospital and China-Japan Friendship Hospital. The results are as follows:

[0175] Table 8 Test results of Peking Union Medical College Hospital (unit: pg / ml)

[0176]

[0177] The concentrations of IL-8, MCAF, IL-6, and TNF-α in acute-stage SARS patients were significantly higher than those in normal controls (P<0.01). In the recovery stage, the IL-8 level dropped to the level of normal controls (P>0.05). However, the MCAF level continued to be higher than that of normal controls (P<0.01), indicating that the changes in IL-8 and MCAF were more obvious than those in IL-6 and TNF-α.

[0178] Table 9 Test results of China-Japan Friendship Hospital (unit: pg / ml)

[0179]

[0180] 1. Serological tests of SARS patients showed that the levels of IL-8 and MCAF in patients in the acute phase were significantly increased, and the levels of IL-8 in the recovery phase dropped to the level of normal people. This indicates that the severe inflammatory response caused by SARS virus infection is likely related to the overexpression and secretion of chemokines, and the degree of lesions in infected lung tissue and possible sequelae may also be related to the presence of IL-8 and MCAF.

[0181] 2. In order to further study the pathogenesis and pathological mechanism of SARS patients from the perspective of tissue pathology, it is still necessary to analyze autopsy lung tissue specimens using immunohistochemistry. Preliminary tests have shown that in SARS patients and ARDS (acute respiratory failure syndrome) patients, lung inflammation is indeed related to the overexpression and secretion of IL-8 and MCAF. Therefore, the use of high-titer neutralizing anti-IL-8 and anti-MCAF monoclonal antibodies to remove overexpressed chemokines and relieve the strong inflammatory response caused by them may be a very effective and feasible treatment for treating SARS patients, reducing the damage to SARS patients' lung tissues, and reducing the mortality rate of SARS patients.

[0182] Although only examples of anti-IL-8 monoclonal antibodies containing heavy chain and light chain variable regions shown in SEQ ID NOs: 1 and 2 are listed above, it is obvious that a person skilled in the art can use the heavy chain and light chain variable regions for other molecules, such as whole immunoglobulin molecules, antibody fragments Fab or (Fab')2 or single-chain antibodies, fusions with other components, etc. through the disclosure of the present invention, without the need for creative labor, and therefore it will not be repeated here.

[0183]

[0184]

Claims

1. An anti-IL-8 monoclonal antibody, characterized in that: The monoclonal antibody has a heavy chain represented by SEQ ID No. 1 and a variable region amino acid sequence of a light chain represented by SEQ ID No.

2.

2. An anti-IL-8 monoclonal antibody as claimed in claim 1, wherein the anti-IL-8 monoclonal antibody is an antibody specific for interleukin-8, which can specifically bind to IL-8 and neutralize its biological activity.

3. A variable region sequence of an anti-IL-8 monoclonal antibody, characterized in that: The variable region sequence has the variable region amino acid sequence of the heavy chain shown in SEQ ID No. 1 and the light chain shown in SEQ ID No.

2.

4. The variable region sequence of an anti-IL-8 monoclonal antibody according to claim 3, characterized in that: The amino acid sequence of the heavy chain variable region is: GSTGDQTVKESGGRLVTPGTPLTLTCTVSGFSLNNYAMSWVRQAPGKGLEYIGVIGSDGIPYYATW AKGRFAISKTSTTVDLKITSPTTEDTATYFCASGYVGDDRYNIWGPGTLVTVSS; The amino acid sequence of the light chain variable region is: GSTGDADGVMTQTPSPVSAAVGGTVSISCQSSPSVYNNNYLSWYQQKPGQPPKLLIYAASTLASGVPSR FKGSGSGTQFTLTISDVQCDDAATYYCAGAYSNDSDDGFGGGTKVEIK.

5. The anti-IL-8 monoclonal antibody according to claim 1, characterized in that: The preparation process of the anti-IL-8 monoclonal antibody is as follows: IL-8 is used as an immune source and mice are used as immune objects, mice are injected with immune injections, spleens of the immune mice are taken to prepare a suspension, the spleen suspension is fused with myeloma cells to obtain a hybridoma cell line that can express the anti-IL-8 monoclonal antibody, and the anti-IL-8 monoclonal antibody is prepared by screening and purification.

6. Use of the anti-IL-8 monoclonal antibody according to claim 1 for preparing a drug for treating diseases associated with abnormal, excessive or uncontrolled expression and secretion of IL-8.

7. The use of the anti-IL-8 monoclonal antibody according to claim 6, characterized in that: The disease is psoriasis.

8. The use of the anti-IL-8 monoclonal antibody according to claim 6, characterized in that: The disease is eczema.

9. The use of the anti-IL-8 monoclonal antibody according to claim 6, characterized in that: The disease is a tumor.

10. The use of the anti-IL-8 monoclonal antibody according to claim 6, characterized in that: The disease is SARS.

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