CN112250764B - Monoclonal antibody for resisting interleukin 17A, coding gene and application thereof - Google Patents

Abstract

The invention discloses a fully human anti-IL-17A monoclonal antibody, a coding gene and application thereof, belonging to the field of medicine. The IL-17A monoclonal antibody of the invention can be combined with IL-17A antigen with high specificity, has high affinity and low immunogenicity, has good effect of inhibiting inflammatory cytokines secreted by various cell lines induced by IL-17A, such as IL-6, and can be applied to treating various autoimmune and inflammatory diseases mediated by immunity, such as psoriasis, rheumatoid arthritis, forced spondylitis, multiple sclerosis, Systemic Lupus Erythematosus (SLE), lupus nephritis, infectious granuloma, cystic fibrosis or cancer.

Description

Monoclonal antibody for resisting interleukin 17A, coding gene and application thereof

Technical Field

The invention belongs to the field of medicine, and particularly relates to an anti-interleukin 17A monoclonal antibody, and a coding gene and application thereof.

Background

Interleukin 17 (IL-17) is one of more than 30 interleukins that have been discovered, and to date, six members of the IL-17 family have been discovered, IL-17A (IL-17), IL-17B, IL-17C, IL-17D, IL-17E (IL-25), and IL-17F. These IL-17 cytokines can bind to corresponding receptors and mediate different inflammatory responses.

The IL-17A single chain is composed of a signal peptide (AA) consisting of 23 amino acids and a 132 amino acid chain region, and mature IL-17A is generally secreted and exists in a homodimer form and is sometimes connected with IL-17F to form heterodimer IL-17 AF.

IL-17A is predominantly activated by a class of CD known as helper T cell 17(Th17) ⁴⁺ T cells produce and act by binding to a complex of IL-17RA and IL-17 RC. IL-17A can also be synthesized and secreted by other immune cells such as gamma delta T cells, Lymphoid Tissue inducing cells (Lymphoid Tissue induced cells), ILCs (lnnate Lymphoid cells), and natural killer T cells, and some other non-immune cells such as intestinal Pan cells and intestinal epithelial cells can also produce IL-17A under stress conditions. Since Th17 cells are most widely distributed in vivo and have a broad role in inflammatory responses, Th17 cells are generally considered to be the major source of IL-17A.

Dyssecretion of IL-17 causes a number of inflammatory diseases. For example, IL-17A acts on macrophages and DC cells, induces the high expression of IL-1, IL-6, TNF and CRP, causes inflammatory reaction, and participates in the pathological process of the silver shoulder disease and the transplant rejection; IL-17A acts on fibroblasts, induces high expression of IL-6, chemokines, growth factors and MMP, causes matrix destruction, and participates in pathological processes of multiple sclerosis and Crohn's disease; IL-17A acts on endothelial cells, induces high expression of IL-6, MMP and blood coagulation factors, leads to blood vessel activation, and participates in pathological processes of thrombus and atherosclerosis; IL-17A acts on osteoblasts and chondrocytes, induces RANKL, MMP and osteoclast generation, causes bone erosion and cartilage damage, and is involved in pathological processes of rheumatoid arthritis and periodontal disease.

Antibodies targeting IL-17A can effectively alleviate the symptoms of autoimmune disease by inhibiting the IL-17A-IL-17RA/C signaling pathway. IL-17A related antibody drugs have been approved for marketing, secukinumab developed by Nowa and Ixekizumab developed by Eli Lilly, approved indications including moderate to severe plaque psoriasis and ankylosing spondylitis. The development of anti-IL-17 antibodies with different structures, better curative effects, wider indications and other different characteristics has urgent need and important significance for treating autoimmune-related diseases such as psoriasis, rheumatoid arthritis, multiple sclerosis and the like and other diseases related to IL-17.

Disclosure of Invention

In order to solve at least one of the above technical problems, the present invention aims to provide a fully human anti-IL-17A monoclonal antibody, a coding gene thereof, a preparation method and an application thereof.

Antibodies comprise four peptide chain immunoglobulin molecules, two heavy (H) chains (about 50-70kDa in length) and two light (L) chains (about 25kDa in length) interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region. Each light chain consists of a light chain variable region (VL) and a light chain constant region. The VH and VL regions can be further subdivided into Complementarity Determining Regions (CDRs) with high variability and regions that are spaced apart to be more conserved, called Framework Regions (FRs). Each VH or VL region is formed by, in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 are composed of 3 CDRs and 4 FRs arranged from amino terminus to carboxy terminus. The antigen binding properties of an antibody can be determined by complementarity determining regions (CDR1, CDR2, CDR3) located in the variable regions of the heavy and light chains, and the amino acid sequences of the 4 FRs are relatively conserved and do not directly participate in the binding reaction. These CDRs form a loop structure, and the β -sheets formed by the FRs between them are spatially close to each other, and the CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen binding site of the antibody. It is possible to determine which amino acids constitute the FR or CDR regions by comparing the amino acid sequences of antibodies of the same type.

In one aspect, the present invention provides a monoclonal antibody VH chain against interleukin 17A, whose variable region sequence includes an amino acid sequence selected from the group consisting of: 10, positions 1-127 of SEQ ID NO; 12 from position 1 to 124 of SEQ ID NO; positions 1-122 of SEQ ID NO. 14.

In some embodiments of the invention, the

In a second aspect, the present invention provides a monoclonal antibody VL chain against interleukin 17A, the variable region sequence of which comprises an amino acid sequence selected from the group consisting of: position 143-250 in SEQ ID NO: 10; position 140-245 in SEQ ID NO 12; position 138 and 243 in SEQ ID NO: 14.

In a third aspect of the present invention, there is provided a monoclonal antibody against interleukin 17A, wherein the VH chain variable region sequence comprises an amino acid sequence selected from the group consisting of: 10, positions 1-127 of SEQ ID NO; 12 from position 1 to 124 of SEQ ID NO; positions 1-122 of SEQ ID NO. 14.

In a fourth aspect, the present invention provides an anti-interleukin 17A monoclonal antibody, wherein the VL chain variable region sequence comprises an amino acid sequence selected from the group consisting of: position 143-250 in SEQ ID NO: 10; position 140-245 in SEQ ID NO 12; position 138 and 243 in SEQ ID NO: 14.

The fifth aspect of the present invention provides an anti-interleukin 17A monoclonal antibody, comprising an amino acid sequence selected from the group consisting of: 10, positions 1-127 of SEQ ID NO; 12 from position 1 to 124 of SEQ ID NO; 14, and an amino acid sequence selected from the group consisting of SEQ ID NO: position 143-250 in SEQ ID NO: 10; position 140-245 in SEQ ID NO 12; position 138 and 243 in SEQ ID NO: 14.

In the present invention, the Fc region of the monoclonal antibody is a constant region of human IgG.

The sixth aspect of the present invention provides a gene encoding a VH chain of a monoclonal antibody against interleukin 17A, comprising a nucleotide sequence selected from the group consisting of: 1-381 of SEQ ID NO 9; position 1-372 in SEQ ID NO 11; position 1-366 of SEQ ID NO. 13.

The seventh aspect of the present invention provides a gene encoding a VL chain of a monoclonal antibody against interleukin 17A, which comprises a nucleotide sequence selected from the group consisting of: position 427-750 in SEQ ID NO 9; position 418-735 of SEQ ID NO: 11; position 412-729 of SEQ ID NO: 13.

In an eighth aspect, the present invention provides a gene encoding a monoclonal antibody against interleukin 17A, the sequence encoding a VH chain comprising a nucleotide sequence selected from the group consisting of: 1-381 of SEQ ID NO 9; position 1-372 in SEQ ID NO 11; position 1-366 of SEQ ID NO 13; the VL chain encoding sequence comprises a nucleotide sequence selected from the group consisting of: position 427-750 in SEQ ID NO 9; position 418-735 of SEQ ID NO: 11; position 412-729 of SEQ ID NO: 13.

The invention also provides a DNA molecule for coding the monoclonal antibody or the fragment thereof. The full-length nucleotide sequence or a fragment thereof of the monoclonal antibody of the present invention can be obtained by PCR amplification, recombinant methods, or synthetic methods. Alternatively, the coding sequences for the light and heavy chains may be fused together to form a single chain antibody.

Once the sequence of interest has been obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.

In addition, the sequence can be synthesized by artificial synthesis, especially when the fragment length is short. Generally, fragments with long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them. The DNA sequence may then be introduced into various existing DNA molecules (or vectors, for example) and cells known in the art.

The invention also relates to a vector comprising a suitable DNA sequence as described above and a suitable promoter or control sequence. These vectors may be used to transform an appropriate host cell so that it can express the protein.

The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Preferred animal cells include (but are not limited to): CH0-S, CH0-Kl, HEK-293 cells.

The steps described in the present invention for transforming a host cell with a recombinant DNA can be performed using techniques well known in the art. The obtained transformant can be cultured by a conventional method, and the transformant expresses the polypeptide encoded by the gene of the present invention. Depending on the host cell used, it is cultured in a conventional medium under suitable conditions.

Generally, the transformed host cell is cultured under conditions suitable for expression of the antibody of the invention, and the recombinant polypeptide may be expressed intracellularly, or on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (such as salting-out method), centrifugation, cell lysis by osmosis, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, High Performance Liquid Chromatography (HPLC), and other various liquid chromatography, and combinations thereof.

The resulting monoclonal antibodies can be identified by conventional means. For example, the binding specificity of a monoclonal antibody can be determined by immunoprecipitation or by an in vitro binding assay, such as Radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

In a ninth aspect, the invention provides the use of a monoclonal antibody VH chain according to the first aspect of the invention or a monoclonal antibody VL chain according to the second aspect of the invention or a monoclonal antibody according to any one of the third to fifth aspects of the invention in the manufacture of a medicament for the treatment of an IL-17A associated disease.

In a tenth aspect, the present invention provides a pharmaceutical composition for treating an IL-17A related disease, comprising a monoclonal antibody VH chain according to the first aspect of the present invention or a monoclonal antibody VL chain according to the second aspect of the present invention or a monoclonal antibody according to any one of the third to fifth aspects of the present invention, and a pharmaceutically acceptable carrier.

Generally, these materials will be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally from about 5 to about 8, preferably from about 6 to about 8, although the pH will vary depending on the nature of the material being formulated and the condition being treated. The formulated pharmaceutical compositions may be administered by conventional routes including, but not limited to: intramuscular, intravenous, or topical administration.

The pharmaceutical composition of the present invention contains a safe and effective amount of the antibody of the present invention and a pharmaceutically acceptable carrier or excipient. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should be compatible with the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections, solutions are preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount, for example from about 1 microgram per kilogram of body weight to about 5 milligrams per kilogram of body weight per day. In addition, the polypeptides of the invention may also be used with other therapeutic agents.

Where a pharmaceutical composition is used, a safe and effective amount of the pharmaceutical composition is administered to the mammal, wherein the safe and effective amount is generally at least about 10 micrograms/kg body weight, and in most cases does not exceed about 50 mg/kg body weight, preferably the dose is from about 10 micrograms/kg body weight to about 20 mg/kg body weight. Of course, the particular dosage will depend upon such factors as the route of administration, the health of the patient, and the like, and is within the skill of the skilled practitioner.

In some embodiments of the invention, the pharmaceutical composition further comprises an additional therapeutic agent.

In some embodiments of the invention, the gene therapy agent may be another anti-human interleukin 17A antibody

In the present invention, the IL-17A associated diseases include inflammatory diseases, autoimmune diseases and the like, including but not limited to psoriasis, psoriatic arthritis, ankylosing spondylitis, multiple sclerosis, inflammatory bowel diseases (such as crohn's disease, ulcerative colitis and the like), osteoarthritis, rheumatoid arthritis or osteoporosis, inflammatory fibrosis (such as scleroderma, pulmonary fibrosis and cirrhosis), and asthma (including allergic asthma).

The invention has the advantages of

Compared with the prior art, the invention has the following beneficial effects:

the IL-17A monoclonal antibody of the invention can be combined with IL-17A antigen with high specificity, has high affinity and low immunogenicity, has good effect of inhibiting inflammatory cytokines secreted by various cell lines induced by IL-17A, such as IL-6, and can be applied to treating various autoimmune and inflammatory diseases mediated by immunity, such as psoriasis, rheumatoid arthritis, forced spondylitis, multiple sclerosis, Systemic Lupus Erythematosus (SLE), lupus nephritis, infectious granuloma, cystic fibrosis or cancer.

The invention clones and expresses anti-IL-17A antibody based on phage antibody library technology, and the antibody obtained by screening is fully humanized, thereby greatly reducing or basically eliminating the immunogenicity of the antibody. The method for directly amplifying antibody genes from human peripheral blood lymphocytes is a fundamental way for solving the problem of human antibody source which cannot be solved by hybridoma technology and the like, and compared with the method for obtaining antibody genes from hybridomas and the like, the method for preparing fully human and chimeric antibodies has advanced and simple preparation process and low price.

Drawings

Figure 1 shows the therapeutic effect of anti-IL-17A antibody 3a9 on imiquimod-induced mouse silver shoulder disease.

Detailed Description

In order to make the technical problems, technical solutions and advantageous effects solved by the present invention more apparent, the present invention is further described in detail below with reference to the following embodiments.

Examples

The following examples are used herein to demonstrate preferred embodiments of the invention. It will be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function in the invention, and thus can be considered to constitute preferred modes for its practice. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit or scope of the invention.

Unless defined otherwise, 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 invention belongs and the disclosures and references cited herein and the materials to which they refer are incorporated by reference.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

The experimental procedures in the following examples are conventional unless otherwise specified. The instruments used in the following examples are, unless otherwise specified, laboratory-standard instruments; the test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.

Example 1 construction of human Single chain antibody (scFv) Gene library

(1) Preparation of cDNA 5mL each of 1000 persons of peripheral blood was collected, mixed, and mononuclear cells were separated with a lymphocyte separation medium (Shanghai Hengxin chemical Co., Ltd.). Total mRNA of the cells was extracted from the isolated human peripheral blood lymphocytes using a kit of Invitrogen. The first strand cDNA was reverse transcribed using mRNA purification kit from GIBCO as a template. The above steps were carried out according to the instructions provided by the manufacturer.

(2) PCR amplification

Based on the sequences of the more conserved V region FR1 and FR4 segments of various human antibody gene family sequences, the following human antibody VH gene (hVH), VL gene (hVL), 5-terminal (R) and 3-terminal (F) primers were designed and synthesized. Wherein multiple degenerate sites (polysense sites) are introduced into each primer sequence in order to further enhance the versatility of the primers for gene amplification of human antibody V regions. Appropriate restriction enzyme site sequences were added according to the vector pCANTAB5E (a phagemid, produced by Pharmacia) used.

The primer sequences are as follows:

hVH-R (containing SfiI site):

5'-CTGCGGCCCAGCCGGCCATGGCCCAGGTGCAGYTRNDGSAGTCDGS-3'(SEQID NO:1)

hVH-F：

5'-TGAGGAGACGGTGACCRKKGTBCC-3'(SEQ ID NO:2)

hVL-R：

5'-GAMATYSWGMTSACBCAGTCTCC-3'(SEQ ID NO:3)

hVL-F (including Not I site):

5'-CTATGGCCGCACGTTTGATYTCCASYYKKGTCCC-3'(SEQID NO:4)

wherein the polysense position code in the primer sequence is coded according to a standard scheme published by the International Commission on Biochemistry (IUB) Nomenclature Committee (NC). Primers were synthesized by Shanghai Producer Co.

And (3) PCR reaction: and (2) performing PCR amplification by respectively using the transcription cDNA product obtained in the step (1) as a template and using a VH primer pair or a VL primer pair. To a total reaction volume of 100. mu.L, 4U Taq enzyme (from Huamei) was added under the conditions: 60s at 95 ℃, 70s at 53 ℃ and 80s at 72 ℃ for 32 cycles, and the final extension at 72 ℃ for 10 min.

(3) Recovery and purification of PCR products

After PCR was performed under the above conditions, the product was identified by electrophoresis on 0.8% agarose gel, two bands having molecular weights of about 310bp and about 300bp, respectively, were found, and the fragments were recovered by a gel recovery kit from Promega corporation, and the operation was performed according to the manufacturer's instructions.

(4) Construction of human Single chain antibody (scFv) Gene

(Gly4Ser)3 is used as a linker sequence GGGGSGGGGSGGGS (SEQ ID NO:5) to respectively connect VH and VL to form an ScFv gene with a heavy chain-linker sequence-light chain structure. The joint primer is designed and synthesized according to the joint sequence template and the 3-end and 5-end part sequences of the human antibody V region gene sequence:

Linker template DNA sequence:

5'-GGT GGA GGC GGT TCA GGC GGA GGT GGC TCT GGC GGT GGC GGA TCG-3'(SEQID NO:6)

reverse VH sequence for ScFv linker:

5'-GVA CMM YGG TCA CCG TCT CCT CAG GTGGAG GC GTT CAG G-3'(SEQID NO:7)

for ScFv linker reverse VL sequence:

5'-GGA GAC TGV GTS AKC WSR ATK TCC GAT CCG CCA CCG CCA GAG-3'(SEQ IDNO:8)

wherein, the polysense locus code in the primer sequence executes the international standard published by NC of IUB, and the primer sequence is synthesized by Shanghai's chemical company.

PCR amplification and recovery and purification were carried out using the above linker templates and primers (PCR conditions and recovery and purification methods were as described above). Linker sequences of approximately 100bp in length were isolated.

Cloning the ScFv fragment into a phagemid vector, carrying out PCR according to the conditions to obtain the ScFv fragment with SfiI and NotI enzyme cutting sites at two ends, and carrying out centrifugal column (spun-column) chromatography purification to remove unnecessary joint primers and dNTP. The purified ScFv fragment was double-digested with SfiI and NotI (both from Promega) to generate a sticky end that was ligated to vector pCANTAB5E (Pharmacia). Again, spin column chromatography purification was performed to remove small SfiI or NotI fragments that may affect their attachment to the vector.

The phagemid vector pCANTAB5E (Pharmacia) itself contains SfiI and NotI cleaved cohesive ends, and can be ligated to the ScFv fragment described above. The ScFv fragment was cloned into the corresponding site on the phagemid vector using conventional DNA ligase. The g3p gene was located adjacent to the pCANTAB5E vector, which allowed expression of the ScFv-g3p fusion gene.

Example 2

Panning antibody library for antibodies in the phage display library obtained in example 1, antibodies with high affinity were selected by panning technique.

Native human IL-17A protein purchased from HumanZyme was used as an antigen coated ELISA plate, BSA was blocked, incubated for 2h, plate washing was followed by incubation for 2h with 50. mu.L phage antibody library (ca. 1012CFU), TBST (Tris50mmol/L, NaCl 150mmol/L, Tween-200.5%, BSA 1%, pH7.5) was washed 1 time (5 times for 2 nd round and 10 times for 5min for 3 rd round), phages were recovered with 50. mu.L of eluent, pH was adjusted to neutral with neutralization buffer, and Ecoli TGl was infected for the next round of screening. A total of 3 rounds of selection were performed to remove phagemids that did not have antigen binding ability.

Sandwich ELISA experiments were performed to determine the antigen binding activity of the antibodies. 50 μ L of 200ng/mL native human IL-17A antigen coated ELISA plate was added, BSA blocked, incubated at 37 ℃ for 1h, and double diluted phage antibody with PBST (KCl2.7mmol/L, Na2HPO410mmol/L, KH2PO41.8mol/L, NaCl 137mmol/L, Tween-200.5%, pH7.4) was added and incubated at 37 ℃ for 2 h. The plate was washed, and HRP-labeled goat anti-M13 monoclonal antibody (purchased from Pharmacia) was added thereto at 37 ℃ for 1 hour. The plate was washed with 1% Tween-20 in PBS, developed with substrate solution, and the absorbance at 595 nm was read on a plate reader. The clone with the strongest affinity is determined according to the calculated positive rate approaching 20 percent and is used for the next step of research.

The 2 selected clones with the highest affinity were infected with E.coli HB2151 cells (Pharmacia), plated, individual colonies were picked from the transformation plates, cultured overnight at 30 ℃ with 2 XYTAG (2 XYT medium containing 100mg/L ampicillin and 2% glucose), phagemid DNA was quantitatively extracted by alkaline lysis, and ScFv gene fragments were PCR-amplified and cloned into pUC19 vector for sequencing, including the expected VH, VL genes and linker sequences. The VH gene sequence is upstream of the linker and the VL gene sequence is downstream of the linker. The results were as follows:

clone 1-K4E 9:

GAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCGACTACTGGATGCACTGGGTGAGGCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGAGCAGCATCAACCAGGACGGCAGCGAGAAGGACTACGTGGGCAGGGTGAAGGGCAGGTTCACCATCAGCAGGGACAACAGCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGAGGGTGGAGGACACCGCCGTGTACTACTGCGTGAGGGACTACTACGACATCCTGACCGACTACTACATCCACTACTGGTACTTCGACCTGTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGGAGATCGTGCTGACCCAGAGCCCCGGCACCCTGAGCCTGAGCCCCGGCGAGAGGGCCACCCTGAGCTGCAGGGCCAGCCAGAGCGTGAGCAGCAGCTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCAGGCTGCTGATCTACGGCGCCAGCAGCAGGGCCACCGGCATCCCCGACAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGGCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGTACGGCAGCAGCCCCTGCACCTTCGGCCAGGGCACCAGGCTGGAGATCAAG(SEQ ID NO:9)

wherein the heavy chain variable region is represented by SEQ ID NO 9 at positions 1-381, and the light chain variable region is represented by position 427-750.

The amino acid sequence deduced from the above DNA sequence is:

EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYWMHWVRQAPGKGLEWVSSINQDGSEKDYVGRVKGRFTISRDNSKNSLYLQMNSLRVEDTAVYYCVRDYYDILTDYYIHYWYFDLWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPCTFGQGTRLEIK(SEQID NO:10)

wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO 10, positions 1-127, the light chain variable region is 143-250, and the underlined part is a linker sequence.

Clone 2-C9H 3:

CAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCGACGGCTGGATGCACTGGGTGAGGCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGAGCAGCATCAACCAGGACGGCAGCGAGAAGTACTACAACCAGAGGGTGAAGGGCAGGTTCACCATCAGCAGGGACAACAGCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGAGGGTGGAGGACACCGCCGTGTACTACTGCGTGAGGGACTACTACGACTACTTCACCGACTACCACTACTGGTACTTCGACCTGTGGGGCCAGGGCACCCTGGTGACCGTGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGGACATCCTGCTGACCCAGAGCCCCGCCATCCTGAGCGTGAGCCCCGGCGAGAGGGCCACCCTGAGCTGCAGGGCCAGCCAGAGCGTGAGCAGCAGCTACCTGGCCTGGTACCAGCAGAAGCCCCTGAGCCCCAGGCTGCTGATCTACGGCGCCAGCAGCAGGGCCACCGGCATCCCCGACAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAAGCTGGAGTGGTACGAGGCCGTGTACTACTGCCAGTACGGCAACAGCAGCCCCTGCACCTTCGGCCAGGGCACCAGGCTGGAGATCAAG(SEQ ID NO:11)

wherein the heavy chain variable region is represented by SEQ ID NO 11 at positions 1-372 and the light chain variable region is represented by sequence number 418-735.

The amino acid sequence deduced from the above DNA sequence is:

QVQLVESGGGLVQPGGSLRLSCAASGFTFSDGWMHWVRQAPGKGLEWVSSINQDGSEKYYNQRVKGRFTISRDNSKNSLYLQMNSLRVEDTAVYYCVRDYYDYFTDYHYWYFDLWGQGTLVTVSGGGGSGGGGSGGGGSDILLTQSPAILSVSPGERATLSCRASQSVSSSYLAWYQQKPLSPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISKLEWYEAVYYCQYGNSSPCTFGQGTRLEIK(SEQ ID NO:12)

wherein the amino acid sequence corresponding to the heavy chain variable region is SEQ ID NO 12, positions 1-124, the light chain variable region is 140-245, and the underlined part is a linker sequence.

Clone 3-P4N 3:

GAGGTGCAGCTGGTGCAGAGCGGCGCCGGCCTGGTGCAGCCCGGCGGCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCGACTACTGGATGCACTGGGTGAGGCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGAGCAGCATCAACATGTACGACGGCAGCACCACCTACTACGCCCAGAGGGTGAAGGGCAGGTTCACCATCAGCAGGGACAACAGCAAGAACAGCCTGTACATGCTGAGCAACAGCGAGGTGGAGGACACCGCCGTGTACTACTGCGTGGTGAGGGACTACTACGACTACTTCACCGACTACTACATCCACTTCGACCTGTGGGGCCAGGGCACCCTGGTGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGGACATCCTGCTGACCCAGAGCCCCGCCATCCTGAGCGTGAGCCCCGGCGAGAGGGCCACCCTGAGCTGCAGGGCCAGCCAGAGCGTGAGCAGCAGCTACCTGGCCTGGTACCAGCAGAAGCCCCTGAGCCCCAGGCTGCTGATCTACGGCGCCAGCAGCAGGGCCACCGGCATCCCCGACAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAAGCTGGAGTGGTACGAGGCCGTGTACTACTGCCAGTACGGCAACAGCAGCCCCTGCACCTTCGGCCAGGGCACCAGGCTGGAGATCAAG(SEQ ID NO:13)

wherein the heavy chain variable region is represented by SEQ ID NO 13 at positions 1-366 and the light chain variable region is represented by position 412-729.

The amino acid sequence deduced from the above DNA sequence is:

EVQLVQSGAGLVQPGGSLRLSCAASGFTFSDYWMHWVRQAPGKGLEWVSSINMYDGSTTYYAQRVKGRFTISRDNSKNSLYMLSNSEVEDTAVYYCVVRDYYDYFTDYYIHFDLWGQGTLVSGGGGSGGGGSGGGGSDILLTQSPAILSVSPGERATLSCRASQSVSSSYLAWYQQKPLSPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISKLEWYEAVYYCQYGNSSPCTFGQGTRLEIK(SEQ ID NO:14)

wherein the amino acid sequence corresponding to the heavy chain variable region is SEQ ID NO. 14, positions 1-122, the light chain variable region is 138-243, and the underlined part is a linker sequence.

The recombinant phagemid strain containing scFv gene with strong affinity is inoculated in 5mL LB culture medium and cultured overnight. Added to 50mL of SBAG (SB culture broth containing 100mg/L ampicillin and 2% glucose), cultured at 30 ℃ for 1 hour with shaking, centrifuged at 5000rpm for 15min, and the supernatant was discarded. The pellet was resuspended in 50mL SBAI (SB medium containing 100mg/L ampicillin and 1mmol/L IPTG), induced for 3h at 37 ℃ and centrifuged as above. The pellet was suspended in 5mL of freshly prepared lysozyme (1g/L), 20% (w/v) sucrose, 30mmol/L LTris-Cl (pH8.0) and 1mmol/L EDTA (pH8.0), and the supernatant was centrifuged at 12000rpm for 5min in an ice bath for 10min at 4 ℃ to obtain a periplasmic fraction. It was freeze-dried and stored at-20 ℃. It was dissolved in 0.01mol/L PBS to 500. mu.L just before use.

The single-chain antibody obtained above was subjected to Wersten blot assay according to a conventional method.

Example 3

Cloning to expression vector pCEP101 containing human constant region, transforming CHO cell to design and synthesize heavy chain and light chain primers, adding KpnI/HindIII enzyme cutting site in the front of the heavy chain primer; the front end of the light chain primer is added with XhoI/BamHI cutting sites.

Each primer was designed as follows:

clone 1-K4E 9:

primer K4E 9-Hf:

5'-CAGGGTACCGAGGTGCAGCTGGTGGAGAGCG-3'(SEQ ID NO:15)

primer K4E 9-Hr:

5'-CAGAAGCTTGCTGCTCACGGTCACCAGGGTG-3'(SEQ ID NO:16)

primer K4E 9-Lf:

5'-CAGCTCGAGGAGATCGTGCTGACCCAGAGC-3'(SEQID NO:17)

primer K4E 9-Lr:

5'-CAGGGATCC CTTGATCTCCAGCCTGGTG-3'(SEQ ID NO:18)

clone 2-C9H 3:

primer C9H 3-Hf:

5'-CAGGGTACC CAGGTGCAGCTGGTGGAGAGC-3'(SEQID NO:19)

primer C9H 3-Hr:

5'-CAGAAGCTT GCTCACGGTCACCAGGGTGCC-3'(SEQ ID NO:20)

primer C9H 3-Lf:

5'-CAGCTCGAGGACATCCTGCTGACCCAGAGCC-3'(SEQ ID NO:21)

primer C9H 3-Lr:

5'-CAGGGATCCCTTGATCTCCAGCCTGGTGCCC-3'(SEQ ID NO:22)

clone 3-P4N 3:

primer P4N 3-Hf:

5'-CAGGGTACC GAGGTGCAGCTGGTGCAGAG-3'(SEQID NO:23)

primer P4N 3-Hr:

5'-CAGAAGCTT GCTCACCAGGGTGCCCTGGC-3'(SEQ ID NO:24)

primer P4N 3-Lf:

5'-CAGCTCGAGGACATCCTGCTGACCCAGAGC-3'(SEQ ID NO:25)

primer P4N 3-Lr:

5'-CAGGGATCC TGATCTCCAGCCTGGTGCCCTG-3'(SEQ ID NO:26)

PCR was carried out by the conventional method using the ScFv fragment obtained in example 2 as a template with the primers synthesized as described above, respectively, to obtain a heavy chain variable region fragment and a light chain variable region fragment having the respective restriction sites.

The heavy chain variable region (positions 1-381 in SEQ ID NO:9 (the corresponding amino acid sequence is positions 1-127 in SEQ ID NO: 10), or positions 1-372 in SEQ ID NO:11 (the corresponding amino acid sequence is positions 1-124 in SEQ ID NO: 12), or positions 1-366 in SEQ ID NO:13 (the corresponding amino acid sequence is positions 1-122 in SEQ ID NO: 14)) is inserted into KpnI/HindIII site of expression vector pCEP101, and then HindIII and Bsi WI are used to insert the light chain variable region of the above antibody (position 427-750 in SEQ ID NO:9 (the corresponding amino acid sequence is positions 143-250 in SEQ ID NO: 10), or position 418-735 in SEQ ID NO:11 (the corresponding amino acid sequence is positions 140-245 in SEQ ID NO: 12), or position 412-729 in SEQ ID NO:13 (the corresponding amino acid sequence is position 138 in SEQ ID NO: 14)) into the KpnI/HindIII site of expression vector pCEP101, and HindIII are used to insert the light chain variable region of the above antibody (position 427-750 in SEQ ID NO: 9) (the corresponding amino acid sequence is position 418-735) (the sequence is position 138 in SEQ ID NO: 14) Into the XhoI/BamHI site of pCEP4 inserted with the coding sequence of the heavy chain variable region, an expression vector for human IL-17A antibody was constructed. Expression vector pCEP101 backbone plasmid pCEP4 was purchased from Invitrogen.

The expression vector with the antibody gene constructed above was transferred to E.coli Top10 strain, and then inoculated into 100 ml of LB medium for amplification, and Plasmid DNA was extracted and purified using an ultra pure Plasmid DNA Purification Kit (Ultrapure Plasmid DNA Purification Kit) from Qiagen. The purified plasmid DNA was transfected into CHO cells using a kit for liposome method of Invitrogen, and the procedures were carried out according to the manufacturer's instructions.

The transformed CHO cells were selected on selection medium for 9 consecutive weeks, and finally cultured in 96-well plates with extreme dilution, 3 consecutive times, for monoclonality.

The selected monoclonal cell lines are cultured on an RPMI 1641 culture medium, a Western blotting experiment is carried out on the supernatant, the expression intensity is judged according to a staining reaction, and 8 clones with strong expression are selected as candidate cell strains.

Example 4

Screening of clone having high expression intensity in CHO cell the high expression candidate clone obtained by the above screening was cultured in a 10mL square cell flask, and the expression level of the antibody was measured by ELISA: coating goat anti-human IgG (Fc) on ELISA plate, standing overnight at 4 deg.C, blocking with 2% BSA at 37 deg.C for 2 hr, adding culture supernatant and standard (human IgG1) to be tested, incubating at 37 deg.C for 2 hr, adding HRP-goat anti-human IgG (kappa) for binding reaction, incubating at 37 deg.C for 1 hr, adding TMB for 10 min at 37 deg.C, and adding H ₂ SO ₄ The reaction was terminated and the A450 value was measured. The expression levels of the above 8 candidate clones were determined as follows: TABLE 1 expression intensity (mg/L) of antibody genes in CHO cells

As can be seen from Table 1, the cell lines numbered 8E3, 3A9 and 7D6 had very high expression levels (140-290mg/L), and the expression level of 3A9 reached 236.1 mg/L. And selecting the clone with high expression quantity to perform large-scale cell culture and purification, and preparing the anti-IL-17A monoclonal antibody.

TABLE 1 expression intensity in CHO cells (mg/L)

Example 5

The affinity of the anti-human IL-17A monoclonal antibody 3A9 and IL-17A is detected by Biacore3000, after IL-17A with different concentrations is coated on a chip of the Biacore3000 by amino coupling, the affinity of the antibody is detected, and a positive antibody Secukinumab is used as a control. The experimental results are shown in Table 2, and the results show that the 3A9 monoclonal antibody of the invention has higher affinity with IL-17A than that of the positive antibody Secukinumab.

TABLE 2 Biacore3000 assay

Ka((1/Ms)

Antibodies	Ka(1/Ms)	Kd(1/s)	KD(pM)
				3A9	1.66×10 6	1.58×10 -5	35
Secukinumab	2.71×10 5	4.03×10 -5	89

Example 6

Adding 15ng/mL IL-17A and monoclonal antibodies with different concentrations into Hela cells with good growth state, setting four multiple holes in each group, incubating and culturing at 37 ℃ and 5% CO2 for 24 hours, collecting cell culture supernatant, detecting the content of IL-6 secreted by each group of cells by using an ELISA kit, and measuring the inhibition of the monoclonal antibodies on the IL-6 secretion of the Hela cells induced by the IL-17A. The IC50 value of 3A9 for IL-17A inhibition was 84.2ng/mL, while the IC50 value of the reference antibody Secukinumab was 133.7 ng/mL. Therefore, the 3A9 monoclonal antibody has stronger inhibiting function on IL-17A than Secukinumab.

Example 7

Detection of monoclonal antibodies blocked the binding of IL-17A to IL-17RA on cells using a competitive cell-based flow cytometry assay (FACS). 3A9 and the reference antibody Secukinumab both significantly inhibited specific binding of IL-17A to IL-17RA on cells with IC 50 of 467.6ng/mL and 580.8ng/mL, respectively, and thus, 3A9 blocked IL-17A binding to IL-17RA on cells more significantly than the reference antibody Secukinumab.

Example 8

The IL-17A can stimulate the expression and release of a plurality of epithelial cells and other cells to secrete the cytokine CXCL1, and the change of the expression level of the CXCL1 in cell supernatant can be quantitatively detected by ELISA (enzyme-linked immunosorbent assay) to judge the influence of the humanized antibody on the biological activity mediated by the IL-17A in the cells. HT-29 cells (human colorectal adenocarcinoma epithelial cells, ATCC) were maintained in culture/assay medium in tissue culture treated flasks using standard techniques. HT-29 were grown in tissue culture flasks until they reached 50-80% confluence on the day of the assay. On the day of assay, cells were washed with PBS and detached from the flask with trypsin + EDTA and made into cell suspensions. 3A9 or a dilution of the reference antibody Secukinumab (starting at 40. mu.g/mL, 3-fold concentration gradient dilution) was mixed with human IL-17A (1. mu.g/mL), plated in a 96-well plate, and incubated for 1 h. Add 100. mu.L (2X 10) to each well ⁴ One) HT-29 cell suspension, cultured at 37 ℃ for 48h in 7% CO 2. Centrifugation was carried out at 500Xg for 5min, and the culture supernatant was transferred to a new 96-well plate, and the expression of CXCL1 was detected by ELISA kit. 3A9 and a reference antibody Secukinumab can remarkably inhibit the release of CXCL from epithelial cells, and IC50 of the CXCL is 663.1ng/mL and 782.4ng/mL respectively, so that 3A9 has stronger antagonism on the release of CXCL1 from the IL-17A stimulated epithelial cells compared with the reference antibody Secukinumab.

Example 9

The psoriasis mouse model can be constructed by applying imiquimod on the skin of the ear back of a mouse to induce psoriasis-like pathological features, namely keratinocyte hyper-proliferation, inflammatory cell aggregation, dermal papilla vascular hyperplasia and the like. The treatment effect of the medicament on psoriasis mice is judged by taking clinical scores, ear swelling degrees and the like as indexes.

32 female mice (about 20g, C57 BL/6) were collected, depilated on the back, and sensitized three days later. Two days prior to sensitization, were randomly divided into 4 groups (8 per group): group I was a solvent control group given PBS; group II is isotype control group, and human IgGl (PBS diluted) given negative control sample is 100 mg/kg; group III was the group to which 3A9 antibody was administered, and 3A9 antibody was administered at 100 mg/kg; group IV is a positive control group and dexamethasone (PBS dilution) lmg/kg is given. The above groups were injected intraperitoneally 1 time each on the day of grouping and day 2 of molding (day 2). On the day of sensitization (day 1), each group of mice was applied approximately 62.5mg of imiquimod cream (5%) to the right ear and back skin for 5 consecutive days.

The thickness of the right ear of the mouse was measured daily with a micrometer screw from the day of sensitization, and the value of the ear swelling thickness of the mouse was calculated with day 0 of the right ear thickness as a control. Meanwhile, weighing the mice every day, observing the skin scale, shoulder, induration and erythema conditions, and grading by adopting a 4-grade grading method: 0 point, disease does not occur; 1 minute, slight; 2 points, medium; grade 3, severe; 4 points, very serious. The results are shown in FIG. 1. The result shows that compared with dexamethasone, the humanized anti-IL-17A antibody 3A9 has a remarkable improvement effect on the mouse silver shoulder disease model induced by imiquimod.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Sequence listing

<110> Shenzhen kang Rui Biotech Limited liability company

<120> monoclonal antibody for resisting interleukin 17A, coding gene and application thereof

<130> JIA-2020-1-W-030

<160> 26

<170> SIPOSequenceListing 1.0

<210> 1

<211> 46

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

ctgcggccca gccggccatg gcccaggtgc agytrndgsa gtcdgs 46

<210> 2

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

tgaggagacg gtgaccrkkg tbcc 24

<210> 3

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

gamatyswgm tsacbcagtc tcc 23

<210> 4

<211> 34

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

ctatggccgc acgtttgaty tccasyykkg tccc 34

<210> 5

<211> 15

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 6

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ggtggaggcg gttcaggcgg aggtggctct ggcggtggcg gatcg 45

<210> 7

<211> 39

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

gvacmmyggt caccgtctcc tcaggtggag gcgttcagg 39

<210> 8

<211> 42

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

ggagactgvg tsakcwsrat ktccgatccg ccaccgccag ag 42

<210> 9

<211> 750

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

gaggtgcagc tggtggagag cggcggcggc ctggtgcagc ccggcggcag cctgaggctg 60

agctgcgccg ccagcggctt caccttcagc gactactgga tgcactgggt gaggcaggcc 120

cccggcaagg gcctggagtg ggtgagcagc atcaaccagg acggcagcga gaaggactac 180

gtgggcaggg tgaagggcag gttcaccatc agcagggaca acagcaagaa cagcctgtac 240

ctgcagatga acagcctgag ggtggaggac accgccgtgt actactgcgt gagggactac 300

tacgacatcc tgaccgacta ctacatccac tactggtact tcgacctgtg gggccagggc 360

accctggtga ccgtgagcag cggtggaggc ggttcaggcg gaggtggctc tggcggtggc 420

ggatcggaga tcgtgctgac ccagagcccc ggcaccctga gcctgagccc cggcgagagg 480

gccaccctga gctgcagggc cagccagagc gtgagcagca gctacctggc ctggtaccag 540

cagaagcccg gccaggcccc caggctgctg atctacggcg ccagcagcag ggccaccggc 600

atccccgaca ggttcagcgg cagcggcagc ggcaccgact tcaccctgac catcagcagg 660

ctggagcccg aggacttcgc cgtgtactac tgccagcagt acggcagcag cccctgcacc 720

ttcggccagg gcaccaggct ggagatcaag 750

<210> 10

<211> 250

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 10

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Asn Gln Asp Gly Ser Glu Lys Asp Tyr Val Gly Arg Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Val Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Val Arg Asp Tyr Tyr Asp Ile Leu Thr Asp Tyr Tyr Ile His Tyr Trp

100 105 110

Tyr Phe Asp Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile

130 135 140

Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly Glu Arg

145 150 155 160

Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser Tyr Leu

165 170 175

Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr

180 185 190

Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly Ser

195 200 205

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro Glu

210 215 220

Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro Cys Thr

225 230 235 240

Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

245 250

<210> 11

<211> 735

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

caggtgcagc tggtggagag cggcggcggc ctggtgcagc ccggcggcag cctgaggctg 60

agctgcgccg ccagcggctt caccttcagc gacggctgga tgcactgggt gaggcaggcc 120

cccggcaagg gcctggagtg ggtgagcagc atcaaccagg acggcagcga gaagtactac 180

aaccagaggg tgaagggcag gttcaccatc agcagggaca acagcaagaa cagcctgtac 240

ctgcagatga acagcctgag ggtggaggac accgccgtgt actactgcgt gagggactac 300

tacgactact tcaccgacta ccactactgg tacttcgacc tgtggggcca gggcaccctg 360

gtgaccgtga gcggtggagg cggttcaggc ggaggtggct ctggcggtgg cggatcggac 420

atcctgctga cccagagccc cgccatcctg agcgtgagcc ccggcgagag ggccaccctg 480

agctgcaggg ccagccagag cgtgagcagc agctacctgg cctggtacca gcagaagccc 540

ctgagcccca ggctgctgat ctacggcgcc agcagcaggg ccaccggcat ccccgacagg 600

ttcagcggca gcggcagcgg caccgacttc accctgacca tcagcaagct ggagtggtac 660

gaggccgtgt actactgcca gtacggcaac agcagcccct gcaccttcgg ccagggcacc 720

aggctggaga tcaag 735

<210> 12

<211> 245

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 12

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Gly

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Asn Gln Asp Gly Ser Glu Lys Tyr Tyr Asn Gln Arg Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Val Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Val Arg Asp Tyr Tyr Asp Tyr Phe Thr Asp Tyr His Tyr Trp Tyr Phe

100 105 110

Asp Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser Gly Gly Gly Gly

115 120 125

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Leu Leu Thr

130 135 140

Gln Ser Pro Ala Ile Leu Ser Val Ser Pro Gly Glu Arg Ala Thr Leu

145 150 155 160

Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser Tyr Leu Ala Trp Tyr

165 170 175

Gln Gln Lys Pro Leu Ser Pro Arg Leu Leu Ile Tyr Gly Ala Ser Ser

180 185 190

Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr

195 200 205

Asp Phe Thr Leu Thr Ile Ser Lys Leu Glu Trp Tyr Glu Ala Val Tyr

210 215 220

Tyr Cys Gln Tyr Gly Asn Ser Ser Pro Cys Thr Phe Gly Gln Gly Thr

225 230 235 240

Arg Leu Glu Ile Lys

245

<210> 13

<211> 729

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

gaggtgcagc tggtgcagag cggcgccggc ctggtgcagc ccggcggcag cctgaggctg 60

agctgcgccg ccagcggctt caccttcagc gactactgga tgcactgggt gaggcaggcc 120

cccggcaagg gcctggagtg ggtgagcagc atcaacatgt acgacggcag caccacctac 180

tacgcccaga gggtgaaggg caggttcacc atcagcaggg acaacagcaa gaacagcctg 240

tacatgctga gcaacagcga ggtggaggac accgccgtgt actactgcgt ggtgagggac 300

tactacgact acttcaccga ctactacatc cacttcgacc tgtggggcca gggcaccctg 360

gtgagcggtg gaggcggttc aggcggaggt ggctctggcg gtggcggatc ggacatcctg 420

ctgacccaga gccccgccat cctgagcgtg agccccggcg agagggccac cctgagctgc 480

agggccagcc agagcgtgag cagcagctac ctggcctggt accagcagaa gcccctgagc 540

cccaggctgc tgatctacgg cgccagcagc agggccaccg gcatccccga caggttcagc 600

ggcagcggca gcggcaccga cttcaccctg accatcagca agctggagtg gtacgaggcc 660

gtgtactact gccagtacgg caacagcagc ccctgcacct tcggccaggg caccaggctg 720

gagatcaag 729

<210> 14

<211> 243

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 14

Glu Val Gln Leu Val Gln Ser Gly Ala Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Asn Met Tyr Asp Gly Ser Thr Thr Tyr Tyr Ala Gln Arg

50 55 60

Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Ser Leu

65 70 75 80

Tyr Met Leu Ser Asn Ser Glu Val Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Val Val Arg Asp Tyr Tyr Asp Tyr Phe Thr Asp Tyr Tyr Ile His Phe

100 105 110

Asp Leu Trp Gly Gln Gly Thr Leu Val Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Leu Leu Thr Gln Ser

130 135 140

Pro Ala Ile Leu Ser Val Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys

145 150 155 160

Arg Ala Ser Gln Ser Val Ser Ser Ser Tyr Leu Ala Trp Tyr Gln Gln

165 170 175

Lys Pro Leu Ser Pro Arg Leu Leu Ile Tyr Gly Ala Ser Ser Arg Ala

180 185 190

Thr Gly Ile Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe

195 200 205

Thr Leu Thr Ile Ser Lys Leu Glu Trp Tyr Glu Ala Val Tyr Tyr Cys

210 215 220

Gln Tyr Gly Asn Ser Ser Pro Cys Thr Phe Gly Gln Gly Thr Arg Leu

225 230 235 240

Glu Ile Lys

<210> 15

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

cagggtaccg aggtgcagct ggtggagagc g 31

<210> 16

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

cagaagcttg ctgctcacgg tcaccagggt g 31

<210> 17

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

cagctcgagg agatcgtgct gacccagagc 30

<210> 18

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

cagggatccc ttgatctcca gcctggtg 28

<210> 19

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

cagggtaccc aggtgcagct ggtggagagc 30

<210> 20

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

cagaagcttg ctcacggtca ccagggtgcc 30

<210> 21

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

cagctcgagg acatcctgct gacccagagc c 31

<210> 22

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

cagggatccc ttgatctcca gcctggtgcc c 31

<210> 23

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

cagggtaccg aggtgcagct ggtgcagag 29

<210> 24

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

cagaagcttg ctcaccaggg tgccctggc 29

<210> 25

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

cagctcgagg acatcctgct gacccagagc 30

<210> 26

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

cagggatcct gatctccagc ctggtgccct g 31

Claims (4)

1. The monoclonal antibody against interleukin 17A, characterized in that the CDR sequences of the variable region sequence of the VH of the monoclonal antibody consist of the amino acid sequences of the following groups respectively: 10, positions 1-127 of SEQ ID NO; 12 from position 1 to 124 of SEQ ID NO; 14, and the CDR sequences of the variable region sequence of the monoclonal antibody VL respectively consist of the amino acid sequences of the following groups: position 143-250 in SEQ ID NO: 10; position 140-245 in SEQ ID NO 12; position 138 and 243 in SEQ ID NO: 14.

2. A gene encoding the anti-interleukin 17A monoclonal antibody of claim 1, characterized in that it comprises the nucleotide sequence of the following group: 1-381 of SEQ ID NO 9; position 1-372 in SEQ ID NO 11; position 1-366 of SEQ ID NO 13; position 427-750 in SEQ ID NO 9; position 418-735 of SEQ ID NO: 11; position 412-729 of SEQ ID NO: 13.

3. Use of a monoclonal antibody according to claim 1 in the manufacture of a medicament for the treatment of an IL-17A associated disease, said IL-17A associated disease being psoriasis.

4. A pharmaceutical composition for treating an IL-17A related disease, comprising the monoclonal antibody of claim 1, and a pharmaceutically acceptable carrier.

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