ANTI-INTERLEUKIN-17A ANTIBODY, PHARMACEUTICAL COMPOSITION THEREOF AND USE THEREOF

Abstract

The present invention relates to the fields of treating autoimmune diseases and molecular immunology, and specifically, to an anti-IL-17A antibody, a pharmaceutical composition thereof, and use thereof. More specifically, the present invention relates to a monoclonal antibody or an antigen binding fragment thereof, wherein a heavy chain variable region of the monoclonal antibody comprises: HCDR1-HCDR3 with amino acid sequences set forth in SEQ ID NOs: 31-33 respectively, or HCDR1-HCDR3 with amino acid sequences set forth in SEQ ID NOs: 37-39 respectively, and a light chain variable region of the monoclonal antibody comprises: LCDR1-LCDR3 with amino acid sequences set forth in SEQ ID NOs: 34-36 respectively, or LCDR1-LCDR3 with amino acid sequences set forth in SEQ ID NOs: 40-42 respectively. Monoclonal antibodies of the present invention can specifically bind to IL-17A, specifically antagonize the binding of IL-17A to a ligand thereof, and inhibit activation of fibroblasts by IL-17A.

Claims

1. A monoclonal antibody or an antigen binding fragment thereof, wherein, a heavy chain variable region of the monoclonal antibody comprises: HCDR1-HCDR3 with amino acid sequences set forth in SEQ ID NOs: 31-33 respectively, or HCDR1-HCDR3 with amino acid sequences set forth in SEQ ID NOs: 37-39 respectively, and a light chain variable region of the monoclonal antibody comprises: LCDR1-LCDR3 with amino acid sequences set forth in SEQ ID NOs: 34-36 respectively, or LCDR1-LCDR3 with amino acid sequences set forth in SEQ ID NOs: 40-42 respectively.

2. The monoclonal antibody or the antigen binding fragment thereof according to claim 1, wherein, the heavy chain variable region of the monoclonal antibody comprises HCDR1-HCDR3 with amino acid sequences set forth in SEQ ID NOs: 31-33 respectively, and the light chain variable region of the monoclonal antibody comprises LCDR1-LCDR3 with amino acid sequences set forth in SEQ ID NOs: 34-36 respectively; or the heavy chain variable region of the monoclonal antibody comprises HCDR1-HCDR3 with amino acid sequences set forth in SEQ ID NOs: 37-39 respectively, and the light chain variable region of the monoclonal antibody comprises LCDR1-LCDR3 with amino acid sequences set forth in SEQ ID NOs: 40-42 respectively.

3. The monoclonal antibody or the antigen binding fragment thereof according to claim 1 or 2, wherein, the amino acid sequence of the heavy chain variable region is selected from SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 24 and SEQ ID NO: 28, and the amino acid sequence of the light chain variable region is selected from SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 18, SEQ ID NO: 22, SEQ ID NO: 26 and SEQ ID NO: 30.

4. The monoclonal antibody or the antigen binding fragment thereof according to any one of claims 1 to 3, wherein, the amino acid sequence of the heavy chain variable region is selected from SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10, and SEQ ID NO: 14, and the amino acid sequence of the light chain variable region is selected from SEQ ID NO: 4, SEQ ID NO: 8, and SEQ ID NO: 12; or the amino acid sequence of the heavy chain variable region is selected from SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 24 and SEQ ID NO: 28, and the amino acid sequence of the light chain variable region is selected from SEQ ID NO: 18, SEQ ID NO: 22, SEQ ID NO: 26 and SEQ ID NO: 30.

5. The monoclonal antibody or the antigen binding fragment thereof according to any one of claims 1 to 4, wherein the heavy chain variable region and the light chain variable region are selected from any one of (1)-(8) below: (1) a heavy chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 2, and a light chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 4; (2) a heavy chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 6, and a light chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 8; (3) a heavy chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 10, and a light chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 12; (4) a heavy chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 14, and a light chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 12; (5) a heavy chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 16, and a light chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 18; (6) a heavy chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 20, and a light chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 22; (7) a heavy chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 24, and a light chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 26; and (8) a heavy chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 28, and a light chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 30.

6. The monoclonal antibody or the antigen binding fragment thereof according to any one of claims 1 to 5, wherein the monoclonal antibody or the antigen binding fragment thereof is selected from a Fab, a Fab′, an F(ab′).sub.2, an Fd, an Fv, a dAb, a complementarity determining region fragment, a single chain antibody, a humanized antibody, a chimeric antibody, and a diabody.

7. The monoclonal antibody or the antigen binding fragment thereof according to any one of claims 1 to 6, wherein the monoclonal antibody binds to IL-17A with an EC.sub.50 of less than about 100 nM, such as less than about 10 nM, 5 nM, 4 nM, 3 nM, 2.5 nM, 2 nM, or less; preferably, the EC.sub.50 is measured by competitive ELISA.

8. The monoclonal antibody or the antigen binding fragment thereof according to any one of claims 1 to 7, wherein the monoclonal antibody comprises a non-CDR region derived from species other than murine, such as from a human antibody.

9. The monoclonal antibody or the antigen binding fragment thereof according to any one of claims 1 to 8, wherein: the monoclonal antibody is a monoclonal antibody produced by hybridoma cell line LT006, which was deposited at China Center for Type Culture Collection (CCTCC) with the accession number of CCTCC NO: C2017102; or the monoclonal antibody is a monoclonal antibody produced by hybridoma cell line LT007, which was deposited at China Center for Type Culture Collection (CCTCC) with the accession number of CCTCC NO: C2017165.

10. An isolated nucleic acid molecule, comprising a nucleic acid encoding the heavy chain variable region and the light chain variable region of the monoclonal antibody according to any one of claims 1 to 9.

11. The isolated nucleic acid molecule according to claim 10, wherein the nucleic acid encoding the heavy chain variable region and the light chain variable region is selected from any one of (1)-(8) below: (1) SEQ ID NO: 1, SEQ ID NO: 3; (2) SEQ ID NO: 5, SEQ ID NO: 7; (3) SEQ ID NO: 9, SEQ ID NO: 11; (4) SEQ ID NO: 13, SEQ ID NO: 11; (5) SEQ ID NO: 15, SEQ ID NO: 17; (6) SEQ ID NO: 19, SEQ ID NO: 21; (7) SEQ ID NO: 23, SEQ ID NO: 25; and (8) SEQ ID NO: 27, SEQ ID NO: 29.

12. A recombinant vector, comprising the isolated nucleic acid molecule according to claim 10 or 11, wherein, preferably, the recombinant vector is a recombinant expression vector, such as a recombinant prokaryotic expression vector or a recombinant eukaryotic expression vector.

13. A host cell, comprising the recombinant vector according to claim 12.

14. A method for preparing the monoclonal antibody or the antigen binding fragment thereof according to any one of claims 1 to 9, comprising cultivating the host cell according to claim 13 under appropriate conditions and isolating the monoclonal antibody or the antigen binding fragment thereof from cell cultures.

15. A hybridoma cell line selected from: hybridoma cell line LT006, which was deposited at China Center for Type Culture Collection (CCTCC) with the accession number of CCTCC NO: C2017102; and hybridoma cell line LT007, which was deposited at China Center for Type Culture Collection (CCTCC) with the accession number of CCTCC NO: C2017165.

16. A conjugate, comprising the monoclonal antibody or the antigen binding fragment thereof according to any one of claims 1 to 9, and a conjugated portion, wherein the conjugated portion is a detectable label; preferably, the detectable label is a radioactive isotope, a luminescent substance such as a fluorescent substance, a colored substance, or an enzyme.

17. A kit, comprising the monoclonal antibody or the antigen binding fragment thereof according to any one of claims 1 to 9, or the conjugate according to claim 16, wherein: preferably, the kit further comprises a second antibody which specifically recognizes the monoclonal antibody or the antigen binding fragment thereof, optionally, the second antibody further comprises a detectable label; preferably, the detectable label is a radioactive isotope, a luminescent substance such as a fluorescent substance, a colored substance, or an enzyme.

18. Use of the monoclonal antibody or the antigen binding fragment thereof according to any one of claims 1 to 9 or the conjugate according to claim 16 in preparing a kit for qualitative or quantitative detection of IL-17A.

19. A pharmaceutical composition, comprising the monoclonal antibody or the antigen binding fragment thereof according to any one of claims 1 to 9 or the conjugate according to claim 16, wherein, optionally, the pharmaceutical composition further comprises pharmaceutically acceptable carriers or excipients.

20. Use of the monoclonal antibody or the antigen binding fragment thereof according to any one of claims 1 to 9 or the conjugate according to claim 16 in preparing a drug for preventing and/or treating tumors or autoimmune diseases, or in preparing a drug for diagnosing autoimmune diseases, wherein, preferably, the autoimmune disease is selected from psoriasis, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, and systemic lupus erythematosus; preferably, the psoriasis is a moderate to severe plaque psoriasis.

21. Use of the monoclonal antibody or the antigen binding fragment thereof according to any one of claims 1 to 9 or the conjugate according to claim 16 in preparing: a drug for blocking the binding of IL-17A to IL17RA, a drug for regulating (e.g., down-regulating) IL-17A activity or level, or a drug for inhibiting IL-6 expression in cells.

22. An in vivo or in vitro method, comprising administering to a cell or a subject in need an effective amount of the monoclonal antibody or the antigen binding fragment thereof according to any one of claims 1 to 9 or the conjugate according to claim 16, wherein the method is selected from: a method for blocking the binding of IL-17A to IL17RA, a method for regulating (e.g., down-regulating) IL-17A activity or level, or a method for inhibiting IL-6 expression in fibroblasts.

23. A method for treating and/or preventing tumors or autoimmune diseases, comprising administering to a subject in need an effective amount of the monoclonal antibody or the antigen binding fragment thereof according to any one of claims 1 to 9 or the conjugate according to claim 16, wherein, preferably, the autoimmune disease is selected from psoriasis, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, and systemic lupus erythematosus; preferably, the psoriasis is a moderate to severe plaque psoriasis.

24. The monoclonal antibody or the antigen binding fragment thereof according to any one of claims 1 to 9, which is used to prevent and/or treat tumors or autoimmune diseases, or to diagnose autoimmune diseases, wherein, preferably, the autoimmune disease is selected from psoriasis, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, and systemic lupus erythematosus; preferably, the psoriasis is a moderate to severe plaque psoriasis.

25. The monoclonal antibody or the antigen binding fragment thereof according to any one of claims 1 to 9, which is used for: blocking the binding of IL-17A to IL-17RA, regulating (e.g., down-regulating) IL-17A activity or level, or inhibiting IL-6 expression in cells.

26. A method for diagnosing autoimmune diseases, comprising administering to a sample to be tested (such as a tissue sample, a cell sample, or a blood sample) or a subject in need an effective amount of the monoclonal antibody or the antigen binding fragment thereof according to any one of claims 1 to 9 or the conjugate according to claim 16, wherein, preferably, the autoimmune disease is selected from psoriasis, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, and systemic lupus erythematosus; preferably, the psoriasis is a moderate to severe plaque psoriasis; preferably, if the measured IL-17A expression level is higher than that in healthy subjects and reaches a pathogenic level, the diagnosis result is positive; otherwise, the diagnosis result is negative.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0142] FIG. 1: SDS-PAGE detection results of the monoclonal murine antibody 13E9. The samples of the four lanes from left to right and their respective loading amounts are: antibody in non-reducing protein electrophoresis loading buffer, 1 μg; antibody in reducing protein electrophoresis loading buffer, 1 μg; Marker, 5 μL; BSA, 1 μg.

[0143] FIG. 2: SDS-PAGE detection results of the monoclonal murine antibody 2G2. The samples of the four lanes from left to right and their respective loading amounts are: antibody in non-reducing protein electrophoresis loading buffer, 1 μg; antibody in reducing protein electrophoresis loading buffer, 1 μg; Marker, 5 μL; BSA, 1 μg.

[0144] FIG. 3: SDS-PAGE detection results of monoclonal humanized antibody 13E9 H3L2. The samples of the three lanes from left to right and their respective loadings are: BSA, 1 μg; Marker, 5 μl; antibody in reducing protein electrophoresis loading buffer, 1 μg; antibody in non-reducing protein electrophoresis loading buffer, 1 μg.

[0145] FIG. 4: detection results of the kinetic characteristic parameters of antibody 13E9 HILL. In the figure, ordinate is signal value with nm as unit; abscissa is time with sec as unit.

[0146] FIG. 5: detection results of the kinetic characteristic parameters of antibody 13E9 H2L2. In the figure, ordinate is signal value with nm as unit, abscissa is time with sec as unit.

[0147] FIG. 6: detection results of the kinetic characteristic parameters of antibody 13E9 H3L2. In the figure, ordinate is signal value with nm as unit; abscissa is time with sec as unit.

[0148] FIG. 7: detection results of the kinetic characteristic parameters of antibody Secukinumab. In the figure, ordinate is signal value with nm as unit; abscissa is time with sec as unit.

[0149] FIG. 8: detection results of the kinetic characteristic parameters of antibody 2G2 H1L1. In the figure, ordinate is signal value with nm as unit; abscissa is time with sec as unit.

[0150] FIG. 9: detection results of the kinetic characteristic parameters of antibody 2G2 H2L2. In the figure, ordinate is signal value with nm as unit; abscissa is time with sec as unit.

[0151] FIG. 10: detection results of the kinetic characteristic parameters of antibody 2G2 H3L3. In the figure, ordinate is signal value with nm as unit; abscissa is time with sec as unit.

[0152] FIG. 11: detection results of the kinetic characteristic parameters of antibody Secukinumab. In the figure, ordinate is signal value with nm as unit; abscissa is time with sec as unit.

[0153] FIG. 12: detection of the binding activity of antibodies 13E9 H1L1 and 13E9 H2L2 to antigen IL17A-His with an indirect ELISA method. The marketed drug Secukinumab is used as a positive control.

[0154] FIG. 13: detection of the binding activity of antibody 13E9 H3L2 to antigen IL 7A-His by indirect ELISA method. The marketed drug Secukinumab is used as a positive control.

[0155] FIG. 14: detection of the activity of antibodies 13E9 H1L1 and 13E9 H2L2 competing with receptor IL17RA-His (biotin) for binding by competitive ELISA method. The marketed drug Secukinumab is used as a positive control.

[0156] FIG. 15: detection of the activity of antibody 13E9 H3L2 competing with receptor IL17RA-His (biotin) for binding by competitive ELISA method. The marketed drug Secukinumab is used as a positive control.

[0157] FIG. 16: detection of the binding activity of antibodies 2G2 H1L1, 2G2 H2L2 and 2G2 H3L3 to antigen IL17A-His by indirect ELISA method. The marketed drug Secukinumab is used as a positive control.

[0158] FIG. 17: detection of the activity of antibodies 2G2 H1L1, 2G2 H2L2 and 2G2 H3L3 competing with the receptor IL17A-His (biotin) for binding to antigen IL17A-His by competitive ELISA method. The marketed drug Secukinumab is used as a positive control.

[0159] FIG. 18: effect of antibodies 13E9 H1L1, 13E9 H2L2 and 13E9 H3L2 as well as 2G2 HILL, 2G2 H2L2 and 2G2 H3L3 on the secretion of cytokine IL-6 by mixed lymphocytes.

[0160] FIG. 19: effect of the antibody drug 13E9 H3L2 on epidermal thickness of the C57BL/6 mouse model with psoriasis.

[0161] Notes on the preservation of biological materials: [0162] hybridoma cell line LT006 (IL17A-13E9), which was deposited at China Center for Type Culture Collection (CCTCC) on Sep. 12, 2017 with an accession number of CCTCC NO: C2017102, at Wuhan University, Wuhan, China, 430072. [0163] hybridoma cell line LT007 (IL17A-2G2), which was deposited at China Center for Type Culture Collection (CCTCC) on Sep. 12, 2017 with an accession number of CCTCC NO: C2017165, at Wuhan University, Wuhan, China, 430072.

DETAILED DESCRIPTION

[0164] The embodiments of the present invention will be described in detail below with reference to the examples. Those skilled in the art will understand that the following examples are only used to illustrate the present invention, and should not be regarded as limiting the scope of the present invention. An example is performed according to the technologies or conditions described in the literature in the art (e.g., see, Guide to Molecular Cloning Experiments, authored by J. Sambrook et al., and translated by Huang Peitang et al., third edition, Science Press) or according to the product manual if specific technologies or conditions are not specified therein. Reagents or instruments used are all commercially available conventional products if the manufacturers thereof are not specified.

[0165] In the following examples, the BALB/C mice used were purchased from Guangdong Medical Laboratory Animal Center; the C57BL/6 mice used were from Nanjing Galaxy Biopharma Co., Ltd.; the MRC5 cells used were from Shanghai Fudan IBS Cell Center; the monoclonal antibody Secukinumab (Cosentyx®) used was purchased from Novartis Corporation.

Example 1: Preparation of Anti-IL-17A Antibodies 13E9 and 2G2

[0166] 1. Preparation of the Hybridoma Cell Lines LT006 and LT007

[0167] Antigen IL17A (24-155)-his used to generate the anti-IL-17A antibody is the fusion protein of human IL-17A (GenBank ID: Q16552) mature peptide and the His tag. Spleen cells from the immunized BALB/C mice (purchased from Guangdong Medical Laboratory Animal Center) and mouse myeloma cells were fused into hybridoma cells, and established methods (e.g., “Monoclonal Antibody Production”, in Basic Methods in Antibody Production and Characterization, Eds. G. C. Howard and D. R. Bethell, Boca Raton: CRC Press, 2000) were referred to for specific operations.

[0168] The fusion protein IL17A-His was enzyme-digested with TEV protease and purified by column to obtain IL-17A (24-155) protein. Indirect ELISA screening was performed in coated microplates with the IL-17A (24-155) protein as the antigen to obtain hybridoma cells that secreted new antibodies specifically bound to IL-17A (24-155).

[0169] Hybridoma cells obtained by indirect ELISA screening were screened by competitive ELISA to obtain hybridoma cell lines capable of secreting a monoclonal antibody that competed with the receptor IL-17RA (CD217, NCBI Gene ID: NP_055154.3) for binding to IL-17A, and two stable hybridoma cell lines were obtained by limited dilution.

[0170] Hybridoma cell line LT006 (IL17A-13E9) was deposited at China Center for Type Culture Collection (CCTCC) on Saturday, Sep. 12, 2015 with an accession number of CCTCC NO: C2017102, at Wuhan University, Wuhan, China, 430072. The monoclonal antibody secreted by hybridoma cell line LT006 was named as 13E9.

[0171] Hybridoma cell line LT007 (IL17A-2G2) was deposited at China Center for Type Culture Collection (CCTCC) on Sep. 12, 2017 with an accession number of CCTCC NO: C2017165, at Wuhan University, Wuhan, China, 430072. The monoclonal antibody secreted by hybridoma cell line LT007 was named as 2G2.

[0172] 2. Preparation of Anti-IL-17A Antibody 13E9

[0173] The LT006 cell line prepared above was cultured (1×10.sup.5 cells per well) with hybridoma serum-free medium (containing 1% Penicillin-Streptomycin and 4% Glutamax, cultured in a cell incubator at 37° C. with 5% CO.sub.2), and the cell culture supernatant was collected when the survival rate was around 20% after 7 days of culturing, which was then subjected to high-speed centrifugation, vacuum filtration through a microporous membrane, and purification through a HiTrap protein A HP column to obtain antibody 13E9. The purified 13E9 samples were detected by SDS-PAGE electrophoresis, and the results are shown in FIG. 1.

[0174] 3. Preparation of Anti-IL-17A Antibody 2G2

[0175] The LT007 cell line prepared above was cultured (1×10.sup.5 cells per well) with hybridoma serum-free medium (containing 1% Penicillin-Streptomycin and 4% Glutamax, cultured in a cell incubator at 37° C. with 5% CO.sub.2), and the cell culture supernatant was collected when the survival rate was around 20% after 7 days of culturing, which was then subjected to high-speed centrifugation, vacuum filtration through a microporous membrane, and purification through a HiTrap protein A HP column to obtain antibody 2G2. The purified 2G2 samples were detected by SDS-PAGE electrophoresis, and the results are shown in FIG. 2.

Example 2: Sequence Analysis of Antibody 13E9

[0176] LT006 cells were cultured according to the method in step 2 of Example 1.

[0177] Using the cell/bacterial total RNA extraction kit (Tiangen, article number DP430), mRNA was extracted from the cultured LT006 cells according to the method in the kit manual.

[0178] cDNA was synthesized according to the kit manual of the Invitrogen SuperScript® II First-Strand Synthesis System for RT-PCR, and amplified by PCR.

[0179] The PCR-amplified products were directly TA cloned, and the kit manual of the pEASY-T1 Cloning Kit (Transgen CT101) was referred to for specific operations.

[0180] The TA-cloned products were directly sequenced, and the sequencing results are as follows:

TABLE-US-00006 nucleotide sequence of the heavy chain variable  region: (360 bp) (SEQ ID NO: 1) GAAGTAAAGCTGCAGGAGTCGGGACCTGGCCTGGTGAAACCTTCTCAGT CTCTGTCCCTCACCTGCACTGTCACTAGCTACTCATTCACCAGTGATTA TGCCTGGAGCTGGATCCGGCAGTTTCCAGGAATCAAACTGGAGTGGATG GGCTACATAACCTACAGTGGTGTCACTAGCTACAACCCCTCTCTCAAAA GTCGAATCTCTATCACTCGAGACACATCCAAGAACCAGTTCTTCCTACA GTTGAATTCTGTGACTACTGAGGACACGGCCACATATTACTGTGCAAGG GCAGACTATGATAGCTACTATACTATGGACTACTGGGGTCAAGGAACCT CAGTCACCGTCTCCTCA its encoded amino acid sequence: (120 aa) (SEQ ID NO: 2) EVKLQESGPGLVKPSQSLSLTCTVTSYSFTSDYAWSWIRQFPGIKLEWM GYITYSGVTSYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCAR ADYDSYYTMDYWGQGTSVTVSS nucleotide sequence of the light chain variable  region: (333 bp) (SEQ ID NO: 3) GACATCCAGCTGACTCAGTCTCCACTCTCCCTGCCTGTCAGTCTTGGAG ATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCCTTGTACACAGTAA TGGAAACACCTATTTACATTGGTACCTGCAGAAGCCAGGCCAGTCTCCA AGGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACA GGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAG AGTGGAGGCTGAGGATCTGGGAGTTTATTTCTGCTCTCAAAGTACACAT TTTTGGACGTTCGGTGGAGGCACCAAGCTGGAAATAAAA its encoded amino acid sequence: (111 aa) (SEQ ID NO: 4) DIQLTQSPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSP RLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTH FWTFGGGTKLEIK

[0181] The underlined regions are the CDR regions.

Example 3: Design, Preparation and Detection of Anti-IL-17A Humanized Antibodies 13E9 H1L1, 13E9 H2L2 and 13E9 H3L2

[0182] 1. Design of the Light Chain and Heavy Chain Sequences of Anti-IL-17A Humanized Antibodies 13E9 H1L1, 13E9 H2L2 and 13E9 H3L2

[0183] Based on the three-dimensional crystal structure of the IL-17A protein (EMBO J. (2001) 20 p: 5332-41) and the sequence of the antibody 13E9 obtained in Example 2, the antibody model was simulated by computer, and mutations were designed according to the model to obtain the variable region sequences of antibodies 13E9 H1L1, 13E9 H2L2 and 13E9 H3L2 (antibody constant region sequences are from the NCBI database, in which the heavy chain constant region is Ig gamma-1 chain C region, ACCESSION: P01857, and the light chain constant region is Ig kappa chain C region, ACCESSION: P01834).

[0184] The designed variable region sequences are as follows:

[0185] (1) heavy chain and light chain sequences of humanized monoclonal antibody 13E9 H1L 1

TABLE-US-00007 nucleotide sequence of the heavy chain variable  region: (360 bp) (SEQ ID NO: 5) GATGTGCAGCTGCAGGAAAGCGGACCAGGACTGGTGAAGCCTA GCCAGACCCTGAGCCTGACTTGCACCGTGTCCAGCTACAGCTTC ACCAGCGACTACGCTTGGTCTTGGATCAGACAGTTCCCAGGAAT TGGCCTCGAGTGGATGGGCTACATCACCTACAGCGGCGTGACC AGCTACAACCCCAGCCTGAAGAGCAGGATCACCATCAGCCGGG ACACCAGCAAGAACCAGTTCTTCCTGCAGCTGAACAGCGTGAC AGCAGCCGATACCGCAGTGTACTATTGCGCCAGGGCCGACTAC GACAGCTACTACACCATGGACTATTGGGGCCAGGGAACCAGCG TGACAGTGTCTAGC its encoded amino acid sequence: (120 aa) (SEQ ID NO: 6) DVQLQESGPGLVKPSQTLSLTCTVSSYSFTSDYAWSWIRQFPGIGL EWMGYITYSGVTSYNPSLKSRITISRDTSKNQFFLQLNSVTAADTA VYYCARADYDSYYTMDYWGQGTSVTVSS

[0186] The underlined regions are the CDR regions.

TABLE-US-00008 nucleotide sequence of the light chain variable region: (333 bp) (SEQ ID NO: 7) GATGTCGTGATGACCCAGACCCCTCTGTCTCTGCCAGTGACACT GGGACAGCAGGCTAGCATCTCTTGCAGAAGCAGCCAGAGCCTG GTGCACAGCAACGGCAACACCTACCTGCATTGGTACCTGCAGA AGCCAGGCCAGTCTCCTAGACTGCTGATCTACAAGGTGTCCAA CCGGTTCAGCGGCGTGCCAGATAGATTCAGCGGAAGCGGAAGC GGCACCGACTTCACCCTGAAGATCAGCAGAGTGGAGGCCGAGG ATCTGGGAGTGTACTTCTGCAGCCAGAGCACCCACTTTTGGACC TTCGGCGGAGGCACCAAGCTGGAGATCAAG its encoded amino acid sequence: (111 aa) (SEQ ID NO: 8) DVVMTQTPLSLPVTLGQQASISCRSSQSLVHSNGNTYLHWYLQKP GQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYF CSQSTHFWTFGGGTKLEIK

[0187] The underlined regions are the CDR regions.

[0188] (2) heavy chain and light chain sequences of humanized monoclonal antibody 13E9 H2L2

TABLE-US-00009 nucleotide sequence of the heavy chain variable region: (360 bp) (SEQ ID NO: 9) GATGTGCAGCTGCAGGAAAGCGGACCAGGACTGGTGAAGCCTA GCCAGACCCTGAGCCTGACTTGCACCGTGTCCAGCTACAGCTTC ACCAGCGACTACGCTTGGTCTTGGATCAGACAGCCACCAGGAA AGGGACTCGAGTGGATCGGCTACATCACCTACAGCGGCGTGAC CAGCTACAACCCCAGCCTGAAGAGCAGGATCACCATCAGCCGG GACACCAGCAAGAACCAGTTCTTCCTGCAGCTGTCTAGCGTGA CAGCAGCCGATACCGCAGTGTACTATTGCGCCAGGGCCGACTA CGACAGCTACTACACCATGGACTATTGGGGCCAGGGAACCAGC GTGACAGTGTCTAGC its encoded amino acid sequence: (120 aa) (SEQ ID NO: 10) DVQLQESGPGLVKPSQTLSLTCTVSSYSFTSDYAWSWIRQPPGKGL EWIGYITYSGVTSYNPSLKSRITISRDTSKNQFFLQLSSVTAADTAV YYCARADYDSYYTMDYWGQGTSVTVSS

[0189] The underlined regions are the CDR regions.

TABLE-US-00010 nucleotide sequence of the light chain variable region: (333 bp) (SEQ ID NO: 11) GATGTCGTGATGACCCAGACCCCTCTGTCTCTGCCAGTGACACT GGGACAGCCAGCTAGCATCTCTTGCAGAAGCAGCCAGAGCCTG GTGCACAGCAACGGCAACACCTACCTGCATTGGTACCTGCAGA AGCCAGGCCAGTCTCCTAGACTGCTGATCTACAAGGTGTCCAA CCGGTTCAGCGGCGTGCCAGATAGATTCAGCGGAAGCGGAAGC GGCACCGACTTCACCCTGAAGATCAGCAGAGTGGAGGCCGAGG ATCTGGGAGTGTACTACTGCAGCCAGAGCACCCACTTTTGGACC TTCGGCGGAGGCACCAAGCTGGAGATCAAG its encoded amino acid sequence: (111 aa) (SEQ ID NO: 12) DVVMTQTPLSLPVTLGQPASISCRSSQSLVHSNGNTYLHWYLQKP GQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVY YCSQSTHFWTFGGGTKLEIK

[0190] The underlined regions are the CDR regions.

[0191] (3) heavy chain and light chain sequences of humanized monoclonal antibody 13E9 H3L2

TABLE-US-00011 nucleotide sequence of the heavy chain variable region: (360 bp) (SEQ ID NO: 13) GATGTGCAGCTGCAGGAAAGCGGACCAGGACTGGTGAAGCCTA GCCAGACCCTGAGCCTGACTTGCACCGTGTCCAGCTACAGCTTC ACCAGCGACTACGCTTGGTCTTGGATCAGACAGCCACCAGGAA AGGGACTCGAGTGGATCGGCTACATCACCTACAGCGGCGTGAC CAGCTACAACCCTAGCCTGAAGAGCCGCGTGACCATTAGCGTG GACACCAGCAAGAACCAGTTCTCCCTGAAGCTGAGCAGCGTGA CAGCCGCCGATACAGCAGTGTACTATTGCGCCCGGGCCGATTA CGACAGCTACTACACCATGGACTATTGGGGCCAGGGAACCAGC GTGACAGTGTCTAGC its encoded amino acid sequence: (120 aa) (SEQ ID NO: 14) DVQLQESGPGLVKPSQTLSLTCTVSSYSFTSDYAWSWIRQPPGKGL EWIGYITYSGVTSYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTA VYYCARADYDSYYTMDYWGQGTSVTVSS

[0192] The underlined regions are the CDR regions.

[0193] The nucleotide sequence of the light chain variable region is the same as the nucleotide sequence of the light chain variable region of 13E9 H2L2, as shown in SEQ ID NO: 11.

[0194] Its encoded amino acid sequence is also the same as the amino acid sequence of the light chain variable region of 13E9 H2L2, as shown in SEQ ID NO: 12.

[0195] 2. Preparation of Humanized Antibodies 13E9 H1L1, 13E9 H2L2 and 13E9 H3L2

[0196] Heavy chain constant regions all use Ig gamma-1 chain C region, ACCESSION: P01857; the light chain constant regions use Ig kappa chain C region, ACCESSION: P01834.

[0197] Heavy chain cDNA and light chain cDNA of 13E9 H1L1, heavy chain cDNA and light chain cDNA of 13E9 H2L2, and heavy chain cDNA and light chain cDNA of 13E9 H3L2 were cloned into pUC57simple (provided by Genscript) vectors, respectively, to obtain pUC57simple-13E9H1, pUC57simple-13E9L1, pUC57simple-13E9H2, pUC57simple-13E9L2 and pUC57simple-13E9H3, respectively, and fragments containing corresponding heavy chains and fragments containing corresponding light chains were subcloned into pcDNA3.1 vectors, respectively, to obtain recombinant plasmids pcDNA3.1-13E9H1, pcDNA3.1-13E9L1, pcDNA3.1-13E9H2, pcDNA3.1-13E9L2, pcDNA3.1-13E9H3 and pcDNA3.1-13E9L2. Then, the corresponding light chain recombinant plasmids and heavy chain recombinant plasmids (pcDNA3.1-13E9H1 and pcDNA3.1-13E9L1; pcDNA3.1-13E9H2 and pcDNA3.1-13E9L2; pcDNA3.1-13E9H3 and pcDNA3.1-13E9L2) were co-transfected into 293F cells, the cell culture was collected and purified to obtain humanized antibodies 13E9 H1L1, 13E9 H2L2, and 13E9 H3L2 respectively. The purified 13E9 H3L2 sample was detected by SDS-PAGE electrophoresis, and the results are shown in FIG. 3.

Example 4: Sequence Analysis of Antibody 2G2

[0198] T007 cells were cultured according to the method in step 3 of Example 1.

[0199] Using the cell/bacterial total RNA extraction kit (Tiangen, article number DP430), mRNA was extracted from the cultured LT007 cells according to the method in the kit manual.

[0200] cDNA was synthesized according to the kit manual of the Invitrogen SuperScript® III First-Strand Synthesis System for RT-PCR, and amplified by PCR.

[0201] The PCR-amplified products were directly TA cloned, and the kit manual of the pEASY-T1 Cloning Kit (Transgen CT101) was referred to for specific operations.

[0202] The TA-cloned products were directly sequenced, and the sequencing results are as follows:

TABLE-US-00012 nucleotide sequence of the heavy chain variable region: (351 bp) (SEQ ID NO: 15) GAGGTTCAGCTGGAGCAGTCTGGTTCTGAACTGAGGAGTCCTGGATCTTC AGTAAAGCTTTCATGCAAGGATTTTGATTCAGAAGTCTTCCCTATTGCTGA TATGAGTTGGGTTAGGCAGAAGCCTGGGCATGGATTTGAATGGATTGGAG ACATACTCCCAAGTTTTGGTAGAACAATCTATGGAGAGAAGTTTGAGGAC AAAGCCAAAGTGGATGCAGACACAGTGTCCAACACAGCCTACTTGGAAC TCAACAGTCTGACATCTGAGGACTCTGCTATCTACTACTGTGCAAGGGGT AACTACGGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA its encoded amino acid sequence: (117 aa) (SEQ ID NO: 16) EVQLEQSGSELRSPGSSVKLSCKDFDSEVFPIADMSWVRQKPGHGFEWIG DILPSFGRTIYGEKFEDKAKVDADTVSNTAYLELNSLTSEDSAIYYCARG NYGFAYWGQGTLVTVSA

[0203] The underlined regions are the CDR regions.

TABLE-US-00013 nucleotide sequence of the light chain variable region: (336 bp) (SEQ ID NO: 17) GATGTTTTGATGACCCAAACTCCACTCACTTTGTCGGTTATCATTGGACAA CCAGCCTCCATCTCTTGCAAGCCAAGTCAGAGCCTCTTAAATAGTGATGG AAAGACATATTTGAATTGGTTGTTGCAGAGGCCAGGCCAGTCTCCAAAGC GCCTAATCTATCTGGTGTCTAAACTGGACTCTGGAGTCCCTGACAGGTTCA CTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATCAGCAGAGTGGA GGCTGAGGATTTGGGAGTTTATTATTGCTGGCAAGGTTCACATTTTCCTCA GACGTTCGGTGGAGGCACAAAGTTGGAAATAAAA its encoded amino acid sequence: (112 aa) (SEQ ID NO: 18) DVLMTQTPLTLSVIIGQPASISCKPSQSLLNSDGKTYLNWLLQRPGQSPKR LIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGSHFPQT FGGGTKLEIK

[0204] The underlined regions are the CDR regions.

Example 5: Design, Preparation and Detection of Anti-IL-17A Humanized Antibodies 2G2 H1L1, 2G2 H2L2 and 2G2 H3L3

[0205] (1) heavy chain and light chain sequences of humanized monoclonal antibody 2G2 H1L1 nucleotide sequence of the heavy chain variable region: (348 bp)

TABLE-US-00014 nucleotide sequence of the heavy chain variable region: (348 bp) (SEQ ID NO: 19) GTGCAGCTGGTGCAGAGCGGAAGCGAACTGAGAAAGCCAGGCTCCAG CGTGAAGCTGTCTTGCAAGGACTTCGACAGCGAGGTGTTCCCCATCGC CGATATGTCTTGGGTCCGACAGGCTCCAGGCCAGGGATTCGAGTGGAT CGGTGACATTCTGCCCAGCTTCGGAAGAACCAACTACGCCCAGAAGTT CGAGGGCAAGGCCAAGGTGGACGCAGACAAGAGCACCAACACCGCCT ACCTGGAGCTGAACAGCCTGAGAAGCGAGGACACCGCCATCTACTAT TGCGCCAGGGGCAACTACGGATTCGCCTATTGGGGCCAGGGAACACT GGTGACAGTGTCCGCC its encoded amino add sequence: (116 aa) (SEQ ID NO: 20) VQLVQSGSELRKPGSSVKLSCKDFDSEVFPIADMSWVRQAPGQGFEWIGD ILPSFGRTNYAQKFEGKAKVDADKSTNTAYLELNSLRSEDTAIYYCARGN YGFAYWGQGTLVTVSA

[0206] The underlined regions are the CDR regions.

TABLE-US-00015 nucleotide sequence of the light chain variable region: (336 bp) (SEQ ID NO: 21) GATGTCGTGATGACCCAGACCCCTCTGTCTCTGAGCGTGACACTGGGA CAGCCAGCTAGCATCAGCTGCAGAAGCAGCCAGAGCCTGCTGAACAG CGACGGCAAGACCTACCTGAATTGGCTGCTGCAGAGACCAGGCCAGT CTCCTAGAAGGCTGATCTACCTGGTGTCCAAGCTGGACAGCGGCGTGC CAGATAGATTCAGCGGAAGCGGAAGCGGCACCGACTTCACCCTGAAG ATCAGCAGAGTGGAGGCCGAGGATCTGGGAGTGTACTACTGTTGGCA GGGCAGCCACTTCCCTCAGACATTCGGCGGCGGCACAAAGCTGGAGA TCAAG its encoded amino acid sequence: (112 aa) (SEQ ID NO: 22) DVVMTQTPLSLSVTLGQPASISCRSSQSLLNSDGKTYLNWLLQRPGQSPR RLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCWQGSHFP QTFGGGTKLEIK

[0207] The underlined regions are the CDR regions.

[0208] (2) heavy chain and light chain sequences of humanized monoclonal antibody 2G2 H2L2

TABLE-US-00016 nucleotide sequence of the heavy chain variable region: (348 bp) (SEQ ID NO: 23) GTGCAGCTGGTGCAGAGCGGAGCAGAAGTGAAGAAGCCAGGCTCCAG CGTGAAGCTGTCTTGCAAGGACTTCGACAGCGAGGTGTTCCCCATCGC CGATATGTCTTGGGTCCGACAGGCTCCAGGCCAGGGATTCGAGTGGAT CGGTGACATTCTGCCCAGCTTCGGGAGAACCAATTACGCCCAGAAGTT CCAGGGCAGAGTGACCGTGACCGCAGACAAGAGCACCAACACCGCCT ACCTGGAGCTGAACAGCCTGAGGAGCGAGGATACCGCCGTGTACTATT GCGCCAGGGGCAACTACGGCTTCGCCTATTGGGGACAGGGAACACTG GTGACAGTGTCCGCC its encoded amino acid sequence: (116 aa) (SEQ ID NO: 24) VQLVQSGAEVKKPGSSVKLSCKDFDSEVFPIADMSWVRQAPGQGFEWIG DILPSFGRTNYAQKTQGRVTVTADKSTNTAYLELNSLRSEDTAVYYCARG NYGFAYWGQGTLVTVSA

[0209] The underlined regions are the CDR regions.

TABLE-US-00017 nucleotide sequence of the light chain variable region: (336 bp) (SEQ ID NO: 25) GATGTCGTGATGACCCAGACCCCTCTGTCTCTGAGCGTGACACTGGGA CAGCCAGCTAGCATCAGCTGCAGAAGCAGCCAGAGCCTGCTGAACAG CGACGGCAAGACCTACCTGAATTGGCTGCTGCAGAGACCAGGCCAGT CTCCTAGAAGGCTGATCTACCTGGTGTCCAACCTGGACAGCGGCGTGC CAGATAGATTCAGCGGAAGCGGAAGCGGCACCGACTTCACCCTGAAG ATCAGCAGAGTGGAAGCCGAGGACGTGGGAGTGTACTACTGTTGGCA GGGCAGCCACTTCCCTCAGACATTCGGCGGCGGCACAAAGCTGGAGA TCAAG its encoded amino acid sequence: (112 aa) (SEQ ID NO: 26) DVVMTQTPLSLSVTLGQPASISCRSSQSLLNSDGKTYLNWLLQRPGQSPR RLIYLVSNLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGSHFP QTFGGGTKLEIK

[0210] The underlined regions are the CDR regions.

[0211] (3) heavy chain and light chain sequences of humanized monoclonal antibody 2G2 H3L3

TABLE-US-00018 nucleotide sequence of the heavy chain variable region: (348 bp) (SEQ ID NO: 27) GTGCAGCTGGTGCAGAGCGGAGCAGAAGTGAAGAAGCCAGGCAGCAG CGTGAAGGTGTCTTGCAAGGACTTCAGCAGCGAGGTGTTCCCCATCGC CGATATGTCTTGGGTCCGACAGGCTCCAGGCCAGGGACTGGAGTGGAT CGGTGACATTCTGCCCAGCTTCGGGAGAACCAATTACGCCCAGAAGTT CCAGGGCAGAGTGACCGTGACCGCAGACAAGAGCACCAACACCGCCT ACCTGGAGCTGTCTAGCCTGAGAAGCGAGGACACCGCCGTGTACTATT GCGCCAGGGGCAACTACGGCTTCGCCTATTGGGGACAGGGAACACTG GTGACAGTGTCCGCC its encoded amino acid sequence: (116 aa) (SEQ ID NO: 28) VQLVQSGAEVKKPGSSVKVSCKDFSSEVFPIADMSWVRQAPGQGLEWIG DILPSFGRTNYAQKFQGRVTVTADKSTNTAYLELSSLRSEDTAVYYCARG NYGFAYWGQGTLVTVSA

[0212] The underlined regions are the CDR regions.

TABLE-US-00019 nucleotide sequence of the light chain variable region: (336 bp) (SEQ ID NO: 29) GATGTCGTGATGACCCAGACCCCTCTGTCTCTGAGCGTGACACTGGGA CAGCCAGCTAGCATCAGCTGCAGAAGCAGCCAGAGCCTGCTGAACAG CGACGGCAAGACCTACCTGAATTGGTTCCTGCAGAGACCAGGCCAGTC TCCTAGAAGGCTGATCTACCTGGTGTCCAACCTGGACAGCGGCGTGCC AGATAGATTCAGCGGAAGCGGAAGCGGCACCGACTTCACCCTGAAGA TCAGCAGAGTGGAAGCCGAGGACGTGGGAGTGTACTACTGTTGGCAG GGCAGCCACTTCCCTCAGACATTCGGCGGCGGCACAAAGCTGGAGAT CAAG its encoded amino acid sequence: (112 aa) (SEQ ID NO: 30) DVVMTQTPLSLSVTLGQPASISCRSSQSLLNSDGKTYLNWFLQRPGQSPR RLIYLVSNLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGSHFP QTFGGGTKLEIK

[0213] The underlined regions are the CDR regions.

[0214] 2. Preparation and SDS-PAGE Electrophoresis Detection of Humanized Antibodies 2G2 H1L1, 2G2 H2L2 and 2G2 H3L3

[0215] Heavy chain constant regions used Ig gamma-1 chain C region, ACCESSION: P01857; the light chain constant regions used Ig kappa chain C region, ACCESSION: P01834.

[0216] Heavy chain cDNA and light chain cDNA of 2G2 H1L1, heavy chain cDNA and light chain cDNA of 2G2 H2L2, and heavy chain cDNA and light chain cDNA of 2G2 H3L3 were cloned into pUC57simple (provided by Genscript) vectors, respectively, to obtain pUC57simple-2G2H1, pUC57simple-2G2L1; pUC57simple-2G2H2, pUC57simple-2G2L2; and pUC57simple-2G2H3, pUC57simple-2G2L3, respectively. Nucleotide fragments containing corresponding heavy chains and nucleotide fragments containing corresponding light chains were then subcloned into pcDNA3.1 vectors, respectively, to obtain recombinant plasmids pcDNA3.1-2G2H1, pcDNA3.1-2G2L1, pcDNA3.1-2G2H2, pcDNA3.1-2G2L2, pcDNA3.1-2G2H3 and pcDNA3.1-2G2L3. Then, the corresponding light chain recombinant plasmids and heavy chain recombinant plasmids (pcDNA3.1-2G2H1 and pcDNA3.1-2G2L1; pcDNA3.1-2G2H2 and pcDNA3.1-2G2L2; pcDNA3.1-2G2H3 and pcDNA3.1-2G2L3) were co-transfected into 293F cells, the cell culture was collected and purified to obtain humanized antibodies 2G2 H1L1, 2G2 H2L2 and 2G2 H3L3 respectively. The purified samples were detected by SDS-PAGE electrophoresis.

Example 6: Measurement of Kinetic Parameters for the Binding of 13E9 H1L1, 13E9 H2L2 and 13E9 H3L2 to the Antigen IL-17A (24-155) Protein

[0217] The kinetic parameters for the binding of humanized antibodies 13E9 H1L1, 13E9 H2L2 and 13E9 H3L2 to the antigen IL-17A (24-155) were measured using a Fortebio molecular interaction instrument.

[0218] The AR2G sensor was activated by EDC/NHS, and the antibody was fixed to the activated AR2G sensor by amine coupling, and the sensor was blocked with 1 M ethanolamine (pH 8.5). After the sensor was equilibrated in PBST for 300 s, the antibody fixed on the sensor binded to the antigen IL-17A (24-155) protein (same as the antigen used in Example 1), in which the antigen concentration was 6.25-400 nM (double gradient dilution) and the binding time was 420 s, and the antigen and the antibody were dissociated in PBST for 600 s.

[0219] The kinetic parameters of antibodies 13E9 H1L1, 13E9 H2L2, 13E9 H3L2 and Secukinumab are shown in Table 1, and detection results of the kinetic characteristic parameters are shown in FIG. 4, FIG. 5, FIG. 6 and FIG. 7, respectively.

TABLE-US-00020 TABLE 1 the kinetic parameters of 13E9 humanized antibody Antibody K.sub.D (M) Kon (1/Ms) Kon error Kdis (1/s) Kdis error R.sub.max (nm) 13E9 H1L1 9.72E−10 1.34E+05 2.60E+03 1.31E−04 9.06E−06 0.0858 − 0.3326 13E9 H2L2 1.03E−09 9.86E+04 1.51E+03 1.01E−04 7.57E−06 0.0546 − 0.3049 13E9 H3L2 5.08E−10 2.90E+05 8.22E+03 1.47E−04 1.02E−05 0.1457 − 0.3546 Secukinumab 6.74E−10 9.28E+04 3.03E+03 6.26E−05 1.57E−05 0.0379 − 0.2064

[0220] KD is affinity constant; Kon is binding rate of antigen and antibody; Kdis is dissociation rate of antigen and antibody; KD=Kdis/Kon.

[0221] The results show that 13E9 H1L1, 13E9 H2L2, and 13E9 H3L2 all have good affinity to the antigen IL-17A (24-155), and the affinity is equivalent to that of the control antibody Secukinumab.

Example 7: Measurement of Kinetic Parameters for the Binding of 2G2 H1L1, 2G2 H2L2, and 2G2 H3L3 to the Antigen IL-17A (24-155)

[0222] The kinetic parameters for the binding of humanized antibodies 2G2 H1L1, 2G2 H2L2 and 2G2 H3L3 to the antigen IL-17A (24-155) were measured using a Fortebio molecular interaction instrument.

[0223] The AR2G sensor was activated by EDC/NHS, and the antibody was fixed to the activated AR2G sensor by amine coupling, and the sensor was blocked with 1 M ethanolamine (pH 8.5). After the sensor is equilibrated in PBST for 300 s, the antibody fixed on the sensor bound to the antigen IL-17A (24-155), in which the antigen concentration was 6.25-400 nM (double gradient dilution) and the binding time was 420 s, and the antigen and the antibody were dissociated in PBST for 600 s.

[0224] The kinetic parameters of antibodies 2G2 H1L1, 2G2 H2L2, 2G2 H3L3, and Secukinumab are shown in Table 2, and detection results of the kinetic characteristic parameters are shown in FIG. 8, FIG. 9, FIG. 10, and FIG. 11, respectively.

TABLE-US-00021 TABLE 2 the kinetic parameters of 2G2 H1L1, 2G2 H2L2, 2G2 H3L3 and Secukinumab Antibody K.sub.D (M) Kon(1/Ms) Kon error Kdis (1/s) Kdis error Rmax(nm) 2G2 H1L1 1.53E−09 1.46E+05 3.15E+03 2.24E−04 9.67E−06 0.0035 − 0.2192 2G2 H2L2 8.43E−10 1.42E+05 2.18E+03 1.20E−04 6.73E−06 0.0444 − 0.2732 2G2 H3L3 1.54E−09 1.98E+05 5.30E+03 3.06E−04 1.08E−05  0.011 − 0.2109 Secukinumab 1.08E−09 1.33E+05 2.38E+03 1.43E−04 8.09E−06 0.0118 − 0.2584

[0225] KD is affinity constant; Kon is binding rate of antigen and antibody; Kdis is dissociation rate of antigen and antibody; KD=Kdis/Kon.

[0226] The results show that compared with the control antibody Secukinumab, 2G2 H2L2 has a higher affinity to the antigen IL-17A (24-155); the affinity of 2G2 H1L1 and 2G2 H3L3 is equivalent to that of the control antibody Secukinumab.

Example 8: Detection of the Binding Activity of the Antibodies 13E9 H1L1, 13E9 H2L2 and 13E9 H3L2 to the Antigen with an ELISA Method

[0227] 1. The binding activity of the antibodies 13E9 H1L1, 13E9 H2L2 and 13E9 H3L2 to the antigen IL17A-His was detected with an indirect ELISA and compared with the marketed drug Secukinumab for the same target.

[0228] IL17A-His can be prepared by referring to the published sequences and conventional technical means in the art, or referring to the following steps:

[0229] Preparation of IL17A-His: the full-length protein sequence of human IL-17A was found in the NCBI protein database, and fused with His*6 purification tag. Genscript in Nanjing was entrusted to synthesize the nucleic acid encoding the fusion protein, and by referring to the standard technologies introduced in the Guide to Molecular Cloning Experiments (Second Edition) and using standard molecular cloning technologies such as PCR, enzyme digestion, gel recovery, ligation transformation, colony PCR or enzyme digestion identification, the target gene was subcloned into mammalian cell expression vectors, and the target gene with the recombinant expression vectors was further sequenced and analyzed. After the sequence was verified to be correct, a medium and large amount of endotoxin-free expression plasmids were prepared, and transiently transfected HEK293 cells for protein expression. After 7 days of culture, the cell culture fluid was collected and affinity purified using a Ni Sepharose column (GE), and the quality of the resulting protein samples was determined using SDS-PAGE and SEC-HPLC standard analysis techniques to be up to standard.

[0230] ELISA: IL17A-His was added to the microplate and incubated at 4° C. overnight; after blocking with 1% BSA in PBS at 37° C. for 2 h, antibodies were added respectively, and incubated at 37° C. for 30 min; and Goat Anti Human IgG (H+L)-HRP (Jackson, 109-035-088) was added and incubated at 37° C. for 30 min; and then the color reaction was performed with TMB (Neogen, 308177) for 5 min, and the absorbance at 450 nm was detected in a microplate reader. The obtained experimental data were analyzed and processed with SoftMax Pro 6.2.1 software, and the 4-parameter fitted curve was plotted for analysis with the antibody concentration as the abscissa and the absorbance value as the ordinate.

[0231] The experimental results are shown in FIG. 12, FIG. 13, and Table 3 and Table 4 below.

TABLE-US-00022 TABLE 3 detection results of the binding activity of 13E9 H1L1 and 13E9 H2L2 to the antigen IL17A-His Antibody concentration/ Coating antigen: IL17A-His, 1 μg/mL gradient 13E9 H1L1 13E9 H2L2 Secukinumab 1 μg/mL 2.726 2.731 2.804 2.737 2.184 2.227 1:3  2.875 2.852 2.832 2.873 2.595 2.505 1:9  2.858 2.815 2.717 2.712 2.297 2.364 1:27 2.564 2.494 2.479 2.481 2.049 2.064 1:81 1.934 1.925 1.891 1.834 1.372 1.314  1:243 1.159 1.116 1.097 1.062 0.672 0.697  1:729 0.522 0.537 0.514 0.511 0.313 0.309 0 0.047 0.048 0.048 0.048 0.046 0.046 EC.sub.50 (nM) 0.044 0.048 0.082

TABLE-US-00023 TABLE 4 detection results of the binding activity of 13E9 H3L2 to the antigen IL17A-His Coating antigen: IL17A-His: 1 μg/mL Antibody concentration/ gradient 13E9 H3L2 Secukinumab 0.5 μg/mL 3.074 3.068 2.929 2.929 1:3  3.175 3.026 2.924 2.885 1:9  2.944 2.895 2.660 2.666 1:27 2.472 2.393 1.947 1.862 1:81 1.610 1.617 1.140 1.092  1:243 0.786 0.783 0.521 0.522  1:729 0.343 0.348 0.252 0.253 0 0.091 0.084 0.087 0.087 EC.sub.50 (nM) 0.043 0.078

[0232] The experimental results show that the antibodies 13E9 HL, 13E9 H2L2 and 13E9T H3L2 all can effectively bind to the antigen IL17A-His, and their binding efficiency is dose-dependent. Under the same experimental conditions, the binding EC50 of 13E9 H1L1 is 0.044 nM, the binding EC50 of 13E9 H2L2 is 0.048 nM, and the EC50 of the marketed drug Secukinumab for the same target is 0.082 nM (FIG. 12, Table 3) under the same experimental conditions, the binding EC50 of 13E9 H3L2 is 0.043 nM, and the EC50 of the marketed drug Secukinumab for the same target is 0.078 nM (FIG. 13, Table 4).

[0233] The above experimental results show that under the same experimental conditions, the EC.sub.50 values of 13E9 H1L1, 13E9 H2L2 and 13E9 H3L2 are all smaller than those of the positive control drug Secukinumab for the same target, indicating that the binding activity of 13E9 H1L1, 13E9 H2L2 and 13E9 H3L2 to IL17A-His is better than that of the marketed control drug Secukinumab for the same target.

[0234] 2. The Activity of Antibodies 13E9 H1L1, 13E9 H2L2 and 13E9 H3L2 Blocking the Binding of IL17RA-his (Biotin) to the Antigen IL17A-his is Detected with a Competitive ELISA.

[0235] IL17RA-His (biotin) can be prepared by referring to the published sequences and conventional technical means in the art, or referring to the following steps:

[0236] Preparation of IL17RA-His (biotin): the extracellular domain sequence of human IL-17RA was found in the NCBI protein database, and fused with His*6 purification tag. Genscript in Nanjing was entrusted to synthesize the nucleic acid encoding the fusion protein, and by referring to the standard technologies introduced in the Guide to Molecular Cloning Experiments (Second Edition) and using standard molecular cloning technologies such as PCR, enzyme digestion, gel recovery, ligation transformation, colony PCR or enzyme digestion identification, the target gene was subcloned into mammalian cell expression vectors, and the target gene with the recombinant expression vectors was further sequenced and analyzed. After the sequence was verified to be correct, a medium and large amount of endotoxin-free expression plasmids were prepared, and transiently transfected HEK293 cells for protein expression. After 7 days of culture, the cell culture was collected and affinity purified using a Ni Sepharose column (GE), and the quality of the resulting protein samples was determined using SDS-PAGE and SEC-HPLC standard analysis techniques to be up to standard. After the quality determination was complete, the biotinylated human IL-17RA-His protein samples were labeled and obtained with the commercial kit EZ-Link® Sulfo-NHS-LC-Biotinylation of Thermo scientific, and the specific preparation method was performed according to the manual of the kit.

[0237] ELISA: IL17A-His was added to the microplate and incubated at 4° C. overnight; after blocking with 1% BSA in PBST at 37° C. for 2 h, antibodies are added respectively, and the antigen and the antibody reacted at room temperature for 10 min; then the receptor IL17RA-His (biotin) was added which was mixed well with the antibody at a volume ration of 1:1 and incubated at 37° C. for 30 min: and SA-HRP (KPL, 14-30-00) was added and incubated at 37° C. for 30 min; and then the color reaction was performed with TMB (Neogen, 308177) for 5 min, and the absorbance at 450 nm was detected in a microplate reader. The obtained experimental data were analyzed and processed with SoftMax Pro 6.2.1 software, and the 4-parameter fitted curve was plotted for analysis with the antibody concentration as the abscissa and the absorbance value as the ordinate. The experimental results are shown in FIG. 14, FIG. 15, and Table 5 and Table 6 below.

TABLE-US-00024 TABLE 5 detection results of the activity of 13E9 H1L1and 13E9 H2L2 competing with the receptor IL17RA-His (biotin) for binding to the antigen IL17A-His Antibody concentration/ Antigen coating: IL17A-His (20150213) 0.5 μg/mL gradient 13E9 H1L1 13E9 H2L2 Secukinumab 10 μg/mL 0.273 0.249 0.274 0.290 0.402 0.392 1:3  0.299 0.220 0.313 0.339 0.469 0.502 1:9  0.418 0.379 0.431 0.379 0.642 0.708 1:27 0.673 0.610 0.646 0.636 0.842 0.855 1:81 0.986 1.008 1.011 1.069 1.169 1.201  1:243 1.342 1.362 1.428 1.331 1.418 1.468  1:729 1.363 1.375 1.447 1.519 1.467 1.657 0 1.495 1.406 1.429 1.561 1.610 1.495 Receptor IL17RA-His(bio) 0.1 μg/mL EC.sub.50(nM) 1.437 1.281 1.807

TABLE-US-00025 TABLE 6 detection results of the activity of 13E9 H3L2 competing with the receptor IL17RA-His (biotin) for binding to the antigen IL17A-His Antigen coating: IL17A-His 0.5 μg/mL Antibody concentration/ gradient 13E9 H3L2 Secukinumab 10 μg/mL 0.193 0.182 0.263 0.282 1:3  0.231 0.206 0.318 0.327 1:9  0.265 0.267 0.419 0.462 1:27 0.424 0.415 0.630 0.663 1:81 0.725 0.627 0.813 0.859  1:243 1.069 1.018 1.120 1.182  1:729 1.218 1.095 1.133 1.250 0 1.184 1.334 1.182 1.232 Receptor IL17RA-His(bio) 0.1 μg/mL EC.sub.50 (nM) 0.805 1.580

[0238] The experimental results show that the antibodies 13E9 H1L1, 13E9 H2L2 and 13E9 H3L2 all can effectively block the binding of the receptor IL7RA-His (biotin) to the antigen IL17A-His, and the blocking efficiency is dose-dependent. Under the same experimental conditions, the EC50 of 13E9 H1L1 competing with IL17AR-His (biotin) for binding to IL17A-His is 1.437 nM, the EC50 of 13E9 H2L2 competing with IL17AR-His (biotin) for binding to IL17A-His is 1.281 nM, and the EC50 of the positive control drug Secukinumab for the same target competing with IL17AR-His (biotin) for binding to IL17A-His is 1.807 nM (Table 5, FIG. 14); the EC50 of 13E9 H3L2 competing with IL17AR-His (biotin) for binding to IL17A-His is 0.805 nM, and the EC50 of the marketed drug Secukinumab for the same target competing with IL17AR-His (biotin) for binding to IL17A-His is 1.580 nM (Table 6, FIG. 15).

[0239] The above experimental results show that under the same experimental conditions, the EC.sub.50 values of 13E9 H1L1, 13E9 H2L2 and 13E9 H3L2 competing with IL17AR-His (biotin) for binding to IL17A-His are all smaller than those of the marketed control drug Secukinumab for the same target, indicating the activity of 13E9 H1L1, 13E9 H2L2 and 13E9 H3L2 competing with IL17AR-His (biotin) for binding to IL17A-His is better than that of the marketed drug Secukinumab for the same target.

Example 9: Detection of the Binding Activity of the Antibodies 2G2 H1L1, 2G2 H2L2 and 2G2 H3L3 to the Antigen with an ELISA Method

[0240] 1. The Binding Activity of the Antibodies 2G2 H1L1, 2G2 H2L2 and 2G2 H3L3 to the Antigen IL17A-his is Detected with an Indirect ELISA.

[0241] Experimental steps: IL17A-His was added to the microplate and incubated at 4° C. overnight; after blocking with 1% BSA in PBS at 37° C. for 2 h, antibodies were added respectively, and incubated at 37° C. for 30 min; and Goat Anti Human IgG (H+L)-HRP (Jackson, 109-035-088) was added and incubated at 37° C. for 30 min; and then the color reaction was performed with TMB (Neogen, 308177) for 5 min, and the absorbance at 450 nm was detected in a microplate reader. The obtained experimental data were analyzed and processed with SoftMax Pro 6.2.1 software, and the 4-parameter fitted curve was plotted for analysis with the antibody concentration as the abscissa and the absorbance value as the ordinate.

[0242] The experimental results are shown in FIG. 16 and Table 7 below. Wherein, the binding EC.sub.50 of 2G2 H1L1 is 0.177 nM, the binding EC.sub.50 of 2G2 H2L2 is 0.372 nM, and the binding so of 2G2 H3L3 is 0.421 nM.

TABLE-US-00026 TABLE 7 detection results of the binding of 2G2 H1L1, 2G2 H2L2 and 2G2 H3L3 to the antigen IL17A-His Antibody concentration/ Antigen coating: IL17A-His, 1 μg/mL gradient 2G2 H1L1 2G2 H2L2 2G2 H3L3 Secukinumab 1 μg/mL 3.220 3.135 3.030 2.960 2.485 2.384 3.448 3.448 1:3  3.284 3.149 2.899 2.886 2.311 2.314 3.477 3.478 1:9  2.820 2.747 2.199 2.126 1.693 1.703 3.320 3.331 1:27 2.020 1.969 1.348 1.328 0.982 1.002 2.768 2.764 1:81 1.113 1.075 0.638 0.624 0.439 0.440 1.761 1.689  1:243 0.469 0.426 0.261 0.350 0.188 0.186 0.807 0.806  1:729 0.224 0.188 0.131 0.148 0.113 0.102 0.337 0.326 0 0.061 0.057 0.058 0.068 0.063 0.060 0.062 0.060 EC.sub.50 (nM) 0.177 0.372 0.421 0.090

[0243] The experimental results show that the antibodies 2G2 H1L 1, 2G2 H2L2 and 2G2 H3L3 all can effectively bind to the antigen IL17A-His, and their binding efficiency is dose-dependent.

[0244] 2. The Activity of Antibodies 2G2 H1L1, 2G2 H2L2 and 2G2 H3L3 Competing with the Receptor IL17A-His (Biotin) for Binding to the Antigen IL17A-his was Detected by Competitive ELISA.

[0245] Experimental steps: IL17A-His was added to the microplate and incubated at 4° C. overnight; after blocking with 1% BSA in PBST at 37° C. for 2 h, antibodies were added respectively, and the antigen and the antibody reacted at room temperature for 10 min; then the receptor IL17RA-His (biotin) was added which is mixed well with the antibody at a volume ration of 1:1 and incubated at 37° C. for 30 min; and SA-HRP (KPL, 14-30-00) was added and incubated at 37° C. for 30 min: and then the color reaction was performed with TMB (Neogen, 308177) for 5 min, and the absorbance at 450 nm was detected in a microplate reader. The obtained experimental data were analyzed and processed with SoftMax Pro 6.2.1 software, and the 4-parameter fitted curve was plotted for analysis with the antibody concentration as the abscissa and the absorbance value as the ordinate.

[0246] The experimental results are shown in FIG. 17 and Table 8below. Wherein, the blocking EC.sub.50 of 2G2 H1L1 is 2.264 nM, the blocking EC.sub.50 of 2G2 H2L2 is 5.408 nM, and the blocking EC.sub.50 of 2G2 H3L3 is 5.911 nM (FIG. 17, Table 8).

TABLE-US-00027 TABLE 8 detection results of the activity of 2G2 H1L1, 2G2 H2L2 and 2G2 H3L3 competing with the receptor IL17RA-His (biotin) for binding to the antigen IL17A-His Antibody concentration/ Antigen coating: IL17A-His 0.5 μg/mL gradient 2G2 H1L1 2G2 H2L2 2G2 H3L3 Secukinumab 10 μg/mL 0.285 0.272 0.337 0.366 0.680 0.718 0.247 0.267 1:3  0.417 0.485 0.500 0.623 0.971 0.876 0.333 0.361 1:9  0.506 0.502 0.785 0.751 0.971 0.954 0.478 0.507 1:27 0.891 0.763 1.087 1.038 1.242 1.178 0.758 0.784 1:81 1.162 1.167 1.286 1.309 1.246 1.312 1.088 1.135  1:243 1.231 1.281 1.423 1.402 1.398 1.466 1.272 1.184  1:729 1.453 1.472 1.464 1.596 1.607 1.466 1.392 1.324 0 1.455 1.425 1.494 1.424 1.470 1.340 1.170 1.230 Receptor IL17RA-His (biotin) :0.1 μg/mL EC.sub.50 (nM) 2.264 5.408 5.911 2.749

[0247] The experimental results show that the antibodies 2G2 H1L1, 2G2 H2L2, and 2G2 H3L3 all can effectively block the binding of the receptor IL17RA-His (biotin) to the antigen IL17A-His, and the blocking efficiency is dose-dependent.

Example 10: Mixed Human Embryonic Fibroblast Reaction: Secretion of Cytokine IL-6

[0248] MRC 5 (purchased from the Cell Center of the Chinese Academy of Sciences) cells were plated into a 96-well plate with 5000 cells/well and cultured overnight. A mixture of IL-17 and antibody (hIgG as a control) incubated at 37° C. for 20 min was added to the MRC 5cells and cultured for 48 h. After 48 h of culturing, the cell supernatant was collected, and the amount of IL-6 secreted was detected by an ELISA kit (purchased from Dakewe Corporation).

[0249] MRC 5cells were mixed and cultured with 13E9 H1L1, 13E9 H2L2, 13E9 H3L2, 2G2 H1L1, 2G2 H2L2 or 2G2 H3L3 (1 nM, 10 nM, 100 nM) and Secukinumab (1 nM, 10 nM, 100 nM), respectively, and the detection results of secreted IL-6 are shown in FIG. 18.

[0250] It can be seen from FIG. 18 that 13E9 H1L1, 13E9 H2L2, 13E9 H3L2, and 2G2 H2L2 all can effectively reduce the IL-6 secretion of MRC 5 cells induced by IL-17, wherein:

[0251] the effect of 13E9 H1L1 antibody on inhibiting IL-6 secretion at a concentration of 100 nM is better than that of the control antibody Secukinumab at the same dose, and the inhibitory effect on IL-6 secretion at a concentration of 10 nM is equivalent to that of the control antibody Secukinumab at a concentration of 100 nM;

[0252] the effects of 13E9 H2L2 antibody on inhibiting IL-6 secretion at concentrations of 1 nM, 10 nM and 100 nM all are equivalent to that of the control antibody Secukinumab at the same dose;

[0253] the inhibitory effects of 13E9 H3L2 antibody on IL-6 secretion at concentrations of 10 nM and 100 nM all are better than that of the control antibody Secukinumab at the same dose, and the effect at a concentration of 1 nM is equivalent to that of the control antibody Secukinumab;

[0254] the effects of 2G2 H2L2 antibody on inhibiting IL-6 secretion at concentrations of 1 nM and 100 nM all are equivalent to that of the control antibody Secukinumab;

[0255] the effect of 2G2 H3L3 on inhibiting IL-6 secretion at a concentration of 1 nM is better than that of the control antibody Secukinumab at the same dose.

[0256] The above results show that in a mixed human embryonic fibroblast reaction in vitro, the biological activity of the antibodies of the present invention in blocking the IL-17A-mediated secretion of IL-6 is better than or at least equivalent to that of the marketed drug Secukinumab for the same target.

Example 11: Effect of the Antibody Drug 13E9 H3L2 on Epidermal Thickness of the C57BL/6 Mouse Psoriasis Model

[0257] C57BL/6 mice were divided into 5 groups with 8 mice in each.

[0258] (1) Modeling: [0259] normal group, C57BL/6 mice were injected intradermally with normal saline on the smooth back for 6 consecutive days from day 1 to day 6, 25 μL/mouse; [0260] the remaining groups of mice were injected intradermally with a recombinant human IL-17A from day 1 to day 4, 2 μg/25 L/mouse, and were injected intradermally with the recombinant human IL-17A from day 5 to day 6, 5 μg/25 μL/mouse.

[0261] (2) Specific grouping and administration: [0262] normal group: normal saline, administered at a dose of 0 mg/kg, 3 times a week for a total of 3 times; [0263] model group: negative isotype control, administered at a dose of 50 mg/kg, 3 times a week for a total of 3 times: [0264] Secukinumab group: Secukinumab, administered at a dose of 50 mg/kg, 3 times a week for a total of 3 times; [0265] 13E9 H3L2 high dose group: administered at a dose of 50 mg/kg, 3 times a week for a total of 3 times; [0266] 13E9 H3L2 low dose group: administered at a dose of 10 mg/kg, 3 times a week for a total of 3 times.

[0267] Each group was administered subcutaneously 3 times, namely 1 day before the modeling, modeling day 3 and modeling day 6, respectively.

[0268] On day 7, the skins of the injection sites on the backs of the mice were fixed to make pathological sections and measure the epidermal thickness.

[0269] The experimental results are shown in FIG. 19.

[0270] The results show that statistically, the epidermal thickness of the Secukinumab group (50 mg/kg), the 13E9 H3L2 high dose group (50 mg/kg) and the 13E9 H3L2 low dose group (10 mg/kg) was significantly smaller than that of the model group (P<0.01).

[0271] The results show that the antibody 13E9 H3L2 (50 mg/kg) shows statistically significant inhibitory effect on epidermal thickness in a C57BL/6 mouse psoriasis model, and has the same efficacy as Secukinumab (50 mg/kg); the inhibitory effect of 13E9 H3L2 (10 mg/kg) on epidermal thickness is also statistically significant.

[0272] Although specific embodiments of the present invention have been described in detail, those skilled in the art will understand. Various modifications and substitutions can be made to those details according to all the teachings that have been disclosed, and these changes are all within the protection scope of the present invention. The full scope of the present invention is given by the appended claims and any equivalent thereof.