EA046350B1 - ANTI-INTERLEUKIN-17A ANTIBODY, PHARMACEUTICAL COMPOSITION AND THEIR APPLICATION - Google Patents

Description

Field of technology to which the invention relates

The present invention relates to the field of molecular immunology and concerns an antibody to interleukin-17A, a pharmaceutical composition based on these antibodies and their use. In particular, the present invention relates to an anti-interleukin-17A monoclonal antibody.

State of the art

Interleukin-17A (abbreviated IL-17A or IL 17A) is a member of the IL-17 family of cytokines, which consists of 6 members, i.e. IL-17A (IL-17A was discovered first, also called IL-17), IL17B, IL-17C, IL-17D, IL-17E (also called IL-25), and IL-17F (Shi Peiqing et al., Chinese Journal of Cell Biology 33:345-357 (2011)). IL-17F has approximately 50% homology with IL-17A, the coding genes of which are located on the same segment of chromosome 6p12 (Gaffen et al., Nat Rev Immunol 9:556-67 (2009)). IL-17A and IL-17F can exist in the form of homodimers, such as IL-17A/IL-17A and IL-17F/IL17F, and also as an IL-17A/IL-17F heterodimer. The biological effects of IL-17A and IL-17F occur when they bind to receptors (Wright et al., J Immunol 181:2799-805 (2008)).

The IL-17 receptor (IL-17R) family consists of 5 members, i.e. IL-17RA, IL-17RB, IL-17RC, IL-17RD and IL-17RE. Members of the IL-17 receptor family can form different receptor complexes, and among them, IL-17RA, the largest molecule discovered to date in this family, is a common subunit that transmits signals for at least four ligands and mediates major biological effects ( Gaffen et al., Nat Rev Immunol 9:556-67 (2009)). The complex of IL17RA and IL-17RC mediates cellular responses to IL-17A and IL-17F (Toy et al., J Immunol 177:36-9 (2006)).

In the autoimmune inflammatory response, IL-17A plays a more important role than IL-17F due to the fact that the affinity of IL17RA for IL-17A is one hundred times greater than its affinity for IL-17F (Ely et al., Nat Immunol 10 1245-51 ( 2009)), and the cellular response to IL-17A is 10 times stronger than to IL-17F (Dubin et al., Immunity 30:9-11 (2009)). Ahtu-IL-17A antibody or anti-IL-17 receptor antibody can be used to block the binding of IL-17A to its receptor, thereby blocking the biological activity of IL-17A.

IL-17A plays an important role in several autoimmune diseases (eg, psoriasis, psoriatic arthritis, rheumatoid arthritis, ankylosing spondylitis, systemic lupus erythematosus, etc.). Ahtu-IL-17A monoclonal antibody Secukinumab is approved by the Food and Drug Administration (FDA) and the European Medicines Agency (EMA) for the treatment of moderate to severe plaque psoriasis, psoriatic arthritis and ankylosing spondylitis.

Psoriasis, also known as poriasis (scaly), is a chronic autoimmune skin disease. Histological characteristics of psoriasis skin include hyperproliferation of epidermal keratinocytes, angiogenesis, and infiltration of dendritic cells, macrophages, neutrophils, and T cells. (Nestle et al., N Engl J. Med., 361:496-509 (2009)). Psoriasis has various manifestations, of which the most common type is plaque psoriasis, which accounts for more than 90% of all psoriasis patients. Psoriatic arthritis (PsA) is a special type of psoriasis characterized by a psoriatic rash, as well as pain, swelling, tenderness, stiffness and dyskinesia of the joints and surrounding soft tissue. Some patients may experience sacroiliitis and/or spondylitis with a long course, mild recurrence and late-stage joint stiffness leading to disability. The presence of psoriasis is an important difference between psoriatic arthritis and other inflammatory joint diseases, where the severity of skin lesions is not directly related to the degree of joint inflammation (Tan Zhen et al., Chinese Journal of Rheumatology 20:354-357 (2016)).

In skin tissues affected by psoriasis, IL-17A expression is significantly increased, and this increase is closely associated with psoriatic disease activity (Johansen et al., Brit J Dermatol, 160:319-24 (2009); Lowes et al. J Invest Dermatol 128:1207-11 (2008)). The anti-III-17A monoclonal antibody Secukinumab has been shown to be very effective in patients suffering from psoriasis and can significantly reduce disease activity in such patients and reduce the area of psoriatic plaques (Langley et al., N Engl J Med, 371:326- 38 (2014)). Secukinumab can also significantly reduce arthritis symptoms and improve joint function in patients with psoriatic arthritis (Gottlieb et al., J Drugs Dermatol, 14821-33 (2015)). Secukinumab is FDA approved for the treatment of moderate to severe plaque psoriasis and psoriatic arthritis.

Rheumatoid arthritis (RA) is mainly characterized by inflammatory proliferation of synovial fibroblasts in the joints, joint and cartilage damage, and infiltration of CD4+ helper T cells and autoantibody-producing plasma cells. IL-17A may cause both inflammation and bone damage in rheumatoid arthritis. IL-17A has high expression levels in rheumatoid synovial monocytes of patients with rheumatoid arthritis compared to healthy individuals or patients with osteoarthritis (Sarkar et al., Clin Exp Immunol. 177:652-61 (2014)). Cytological studies have shown that LL-17A can stimulate bone resorption and collagen breakdown (Kitami et al., Biochimie, 92:398-404 (2010)). IL-17A can initiate the secretion of pro-inflammatory cytokines such as TNFa, LL-1b and IL-6 in cartilage, synovial cells, macrophages and osteoblasts and have biological effects. These pro-inflammatory cytokines cause

- 1 046350 sudden onset of rheumatoid arthritis and can maintain the number of TH17 cells through IL-6 induced IL-17A, thereby forming a positive feedback loop and acting synergistically to enhance their inflammatory effects and induce a chronic inflammatory state (Ogura et al., Immunity 29:628-36 (2008)). Antagonizing IL-17A can effectively alleviate the symptoms of rheumatoid arthritis. In a mouse model of collagen-induced arthritis, neutralization of IL-17A or its receptor eliminated symptoms of rheumatoid arthritis (Chao et al., Autoimmunity. 2011 May; 44(3):243-52). IL-17 deficiency protects the mouse host from collagen-induced arthritis (Nakae et al., J Immunol, 171:6173-7 (2003)), while overexpression of IL-17A can exacerbate such conditions (Lubberts et al. , Inflamm Res 51:102-4 (2002)).

Ankylosing spondylitis (AS) is a chronic autoimmune disease. Early pathological signs of AS are acute or chronic inflammation at the attachment sites of the bones of the sacroiliac joints, tendons and ligaments, as a result of which discitis and facet arthritis may develop at a later stage; and all patients with AS experience decreased bone density. Studies have shown that both the number of Th17 cells secreting IL-17A in the peripheral blood and the concentration of IL-17A in patients with AS are significantly higher than in healthy controls (Gracey et al., Arthritis Rheumatol. 68:679-89. (2016)). IL-17A can activate various cells, such as macrophages, dendritic cells, endothelial cells, fibroblasts, chondrocytes and osteoblasts, which can produce a large number of inflammatory destructive factors (Ogura et al., Immunity 29:628-36 (2008)). In bone tissue, IL-17A induces expression of receptor activator of nuclear factor-κB ligand (RANKL) in osteoblasts, activates osteoclasts, thereby inducing bone resorption, cumulatively exacerbating bone loss, and directly or indirectly causing bone destruction (Gaffen, Curr Rheumatol Rep 11: 365-370 (2009)). In patients with AS, the anti-IL17A monoclonal antibody Secukinumab has demonstrated excellent efficacy in significantly improving the symptoms and signs of ankylosing spondylitis (Baeten et al., N Engl J Med, 373:2534-48 (2015)). Based on these results, secukinumab is approved by the FDA for the treatment of ankylosing spondylitis.

Systemic lupus erythematosus (SLE) is an autoimmune disease that affects multiple systems. In particular, antibodies appear in the body of patients against their own autoantigens, which directly or indirectly attack various tissues or organs, with the skin, joints and kidneys most often affected. Studies have shown that IL-17A plays a role in SLE. The proportion of IL-17A-producing cells in the peripheral blood of SLE patients is increased, and the level of IL-17A in the serum of patients is abnormally high (Chen et al., J Clin Immunol, 30:221-5 (2010). Peripheral blood mononuclear cells patients with SLE associated with kidney damage may produce higher levels of total IgG, anti-dsDNA IgG, and IL-6 when cultured with IL-17, indicating the possible involvement of IL-17 in B cell activation (Dong et al., Chin Med J 116:543-8 (2003) It has also recently been discovered that IL-17A can interact with BAFF (B cell activating factor) to protect B cells from apoptosis, thereby increasing cell number , producing autoantibodies (Onishi et al., Immunology 129:31121 (2010)).

The essence of the invention

Through intensive research and creativity, the inventors were able to use mammalian cell expression systems to express recombinant IL-17A (24-155) as an antigen for immunization of mice, resulting in a large number of hybridoma cell samples obtained from the fusion of mouse spleen cells and myelomas. As a result of screening a large number of samples, the following two hybridoma cell lines were isolated:

hybridoma cell line LT006 (IL-17A-13E9), deposited in the China Center for Type Culture Collection (CCTCC) on September 12, 2017 with CCTCC accession number: C2017102; and hybridoma cell line LT007 (IL-17A-2G2) deposited in China Type Culture Collection Center (CCTCC) on September 12, 2017 with CCTCC accession number: C2017165.

Unexpectedly, it was discovered that:

the hybridoma cell line LT006 can secrete and produce a specific monoclonal antibody (named 13E9) that specifically binds to IL-17A, and this monoclonal antibody can very effectively block the binding of IL-17A to IL-17RA;

the hybridoma cell line LT007 can secrete and produce a specific monoclonal antibody (named 2G2) that specifically binds to IL-17A, and this monoclonal antibody can very effectively block the binding of IL-17A to IL-17RA;

In addition, humanized anti-HE-17A antibodies were obtained (named 13E9 H1L1, 13E9 H2L2, 13E9 H3L2; and 2G2 H1L1, 2G2 H2L2, 2G2 H3L3, respectively), which can effectively bind to human IL-17A and block the binding of IL-17A. 17A with IL-17A receptors and inhibit the activation of downstream IL-17A receptor signaling pathways;

- 2046350 The antibody of the present invention can be used for the manufacture of a medicament for the prevention and/or treatment of autoimmune diseases such as psoriasis, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis and systemic lupus erythematosus.

Thus, the present invention relates to the following:

in one aspect of the present invention there is provided a monoclonal antibody or an antigen binding fragment thereof, wherein the heavy chain variable region (VH) of the monoclonal antibody comprises: HCDR1-HCDR3 with the amino acid sequences set forth in SEQ ID NOs: 31-33, respectively, or HCDR1-HCDR3 with the amino acid sequences shown in SEQ ID NO: 37-39, respectively; and the light chain variable region (VL) of the monoclonal antibody comprises: LCDR1-LCDR3 with the amino acid sequences set forth in SEQ ID NOs: 34-36, respectively, or LCDR1-LCDR3 with the amino acid sequences set forth in SEQ ID NOs: 40-42, respectively .

The variable regions of the light and heavy chains are responsible for antigen binding; the variable region of each chain contains three hypervariable regions, namely complementarity determining regions (CDRs) (heavy (H) chain CDRs include HCDR1, HCDR2, HCDR3, and light (L) chain CDRs include LCDR1, LCDR2, LCDR3; defined by Kabat et al ., Sequences of Proteins of Immunological Interest, Fifth Edition (1991), Volumes 1-3, NIH Publication 91-3242, Bethesda Md.).

Analysis of the amino acid sequences of the CDR regions of the monoclonal antibody specified in paragraphs (1), (2) above is performed by technical means well known to specialists in this field, for example, using the VBASE2 database:

antibodies 13E9, 13E9 H1L1, 13E9 H2L2 and 13E9 H3L2 of the present invention have the same CDR:

The amino acid sequences of the three CDR regions of the heavy chain variable region are as follows:

HCDR1: SYSFTSDYA (SEQ GO NO: 31),

HCDR2: ITYSGVT (SEQ ID NO: 32),

HCDR3: ARADYDSYYTMDY (SEQ ID NO: 33); and the amino acid sequences of the three CDR regions of the light chain variable region are as follows:

LCDR1: QSLVHSNGNTY (SEQ ID NO: 34),

LCDR2: KVS (SEQ ID NO: 35),

LCDR3: SQSTHFWT (SEQ ID NO: 36).

Antibodies 2G2, 2G2 H1L1, 2G2 H2L2 and 2G2 H3L3 of the present invention have the same CDR:

The amino acid sequences of the three CDR regions of the heavy chain variable region are as follows:

HCDR1: SEVFPIAD (SEQ ID NO: 37),

HCDR2: ILPSFGRT (SEQ ID NO: 38),

HCDR3: ARGNYGFAY (SEQ ID NO: 39); and the amino acid sequences of the three CDR regions of the light chain variable region are as follows:

LCDR1: QSLLNSDGKTY (SEQ ID NO: 40),

LCDR2: LVS (SEQ ID NO: 41),

LCDR3: WQGSHFPQT (SEQ ID NO: 42).

In one or more embodiments, the present invention provides a monoclonal antibody or antigen binding fragment thereof, wherein the heavy chain variable region (VH) of the monoclonal antibody comprises HCDR1-HCDR3 with the amino acid sequences set forth in SEQ ID NOs: 31-33, respectively, and the variable region The light chain (VL) of the monoclonal antibody contains LCDR1-LCDR3 with the amino acid sequences shown in SEQ ID NO: 34-36, respectively.

In one or more embodiments, the present invention provides a monoclonal antibody or antigen binding fragment thereof, wherein the heavy chain variable region (VH) of the monoclonal antibody comprises HCDR1-HCDR3 with the amino acid sequences set forth in SEQ ID NOs: 37-39, respectively, and the variable region The light chain (VL) of the monoclonal antibody contains LCDR1-LCDR3 with the amino acid sequences shown in SEQ ID NO: 40-42, respectively.

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In one or more embodiments, the present invention provides a monoclonal antibody or antigen binding fragment thereof, 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: thirty.

In one or more embodiments, the present invention provides a monoclonal antibody or antigen binding fragment thereof, 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 a heavy chain variable region amino acid sequence selected from: SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 24 and SEQ ID NO: 28, and a light chain variable region amino acid sequence selected from: SEQ ID NO: 18, SEQ ID NO: 22, SEQ ID NO: 26 and SEQ ID NO: 30.

In one or more embodiments, the present invention provides a monoclonal antibody or an antigen binding fragment thereof, wherein the heavy chain variable region and the light chain variable region are selected from any of the following (1) to (8):

(1) a heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 2, and a light chain variable region containing the amino acid sequence shown in SEQ ID NO: 4;

(2) a heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 6, and a light chain variable region containing the amino acid sequence shown in SEQ ID NO: 8;

(3) a heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 10, and a light chain variable region containing the amino acid sequence shown in SEQ ID NO: 12;

(4) a heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 14 and a light chain variable region containing the amino acid sequence shown in SEQ ID NO: 12;

(5) a heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 16 and a light chain variable region containing the amino acid sequence shown in SEQ ID NO: 18;

(6) a heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 20, and a heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 22;

(7) a heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 24 and a light chain variable region containing the amino acid sequence shown in SEQ ID NO: 26; and (8) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 28 and a light chain variable region containing the amino acid sequence set forth in SEQ ID NO: 30.

In one or more embodiments, the present invention provides a monoclonal antibody or antigen binding fragment thereof, wherein the monoclonal antibody or antigen binding fragment thereof is selected from Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity determining region fragment, single chain antibody , humanized antibody, chimeric antibody and diabody.

In one or more embodiments, the present invention provides a monoclonal antibody or antigen binding fragment thereof, wherein the monoclonal antibody binds to the IL-17A protein with an EC50 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 EC50 is measured by a competitive ELISA.

In some embodiments, the present invention provides a monoclonal antibody or antigen binding fragment thereof, wherein the monoclonal antibody binds to the IL-17A protein with a K _D of less than about 10 ^-5 M, such as less than about 10 ^-6 M, 10 ^-7 M, 10 ^-8 M , 10 ^-9 M, 10 ^-10 M or less; preferably K _D is measured using a Fortebio Molecular Interaction Analysis Instrument.

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In some embodiments, the present invention provides a monoclonal antibody or antigen binding fragment thereof, wherein the monoclonal antibody binds to the IL17A protein with an EC50 of less than about 100 nM, such as less than about 10 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM or less; in particular, EC ₅₀ is measured by indirect ELISA.

In one or more embodiments of the present invention, the monoclonal antibody comprises non-CDR regions, wherein the non-CDR regions are from a non-mouse species, such as a human antibody.

In some embodiments of the present invention, the immunoglobulin constant region is humanized, for example, the heavy chain constant regions use Ig gamma-1 chain region, accession number: P01857; and in the light chain constant regions, the Ig kappa chain region is used, accession number: P01834.

In one or more embodiments, the present invention provides a monoclonal antibody or an antigen-binding fragment thereof, wherein the monoclonal antibody is produced by the hybridoma cell line LT006 deposited in the China Type Culture Collection Center (CCCC) with CCCC accession number: C2017102; or the monoclonal antibody is produced by the hybridoma cell line LT007 deposited in the China Type Culture Collection Center (CCTCC) with CCCC accession number: C2017165.

In one or more embodiments of the present invention, a monoclonal antibody or an antigen-binding fragment thereof is used for the prevention and/or treatment of tumors or autoimmune diseases or for the diagnosis of autoimmune diseases; preferably the autoimmune disease is selected from psoriasis, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis and systemic lupus erythematosus; preferably the psoriasis is moderate to severe plaque psoriasis.

In one or more embodiments of the present invention, a monoclonal antibody or antigen binding fragment thereof is used to block the binding of IL-17A to IL-17RA, regulate (eg, reduce) the activity or level of IL-17A, or suppress the expression of IL-6 in cells.

Another aspect of the present invention relates to an isolated nucleic acid molecule containing nucleotide sequences encoding a heavy chain variable region and a light chain variable region of any of the monoclonal antibodies provided in the present invention.

In one or more embodiments of the present invention, the isolated nucleic acid molecule contains nucleotide sequences selected from any of the following paragraphs (1) to (8):

(l)SEQIDNO: 1, SEQIDNO: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.

Another aspect of the present invention relates to a recombinant vector containing an isolated nucleic acid molecule of the present invention. Preferably, the recombinant vector is a recombinant expression vector, such as a prokaryotic recombinant expression vector or a eukaryotic recombinant expression vector.

Another aspect of the present invention relates to a host cell containing a recombinant vector of the present invention.

Another aspect of the present invention relates to a method for producing any of the monoclonal antibodies or antigen binding fragments thereof provided in the present invention, comprising the steps of culturing a host cell in accordance with the present invention under appropriate conditions and isolating the monoclonal antibody or antigen binding fragment thereof from the cell cultures.

Another aspect of the present invention relates to a hybridoma cell line selected from the hybridoma cell line LT006 deposited in the China Type Culture Collection Center (CCCC) with CCCC accession number: C2017102; And

- 5 046350 hybridoma cell line LT007, deposited in the China Type Culture Collection Center (CCTCC) with CCTCC accession number: C2017165.

Another aspect of the present invention relates to a conjugate comprising a monoclonal antibody or an antigen binding fragment thereof and a conjugated portion, wherein the monoclonal antibody is any of the monoclonal antibodies or antigen binding fragments thereof provided herein and 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.

Another aspect of the present invention relates to a kit containing any of the monoclonal antibodies or antigen binding fragments thereof provided in the present invention, or containing a conjugate of the present invention;

preferably, the kit further contains a second antibody that specifically recognizes the monoclonal antibody or an antigen-binding fragment thereof; the second antibody optionally further contains 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.

Another aspect of the present invention relates to the use of any of the monoclonal antibodies or antigen binding fragments thereof provided in the present invention, or a conjugate of the present invention, in the preparation of a kit for the qualitative or quantitative determination of IL-17A. Qualitative detection refers to the detection of the presence of IL17A in a test sample, and quantitative detection refers to the determination of the concentration or content of IL-17A in a test sample.

Another aspect of the present invention relates to a pharmaceutical composition containing any of the monoclonal antibodies or antigen binding fragments thereof provided in the present invention, or a conjugate of the present invention; the pharmaceutical composition optionally further contains pharmaceutically acceptable carriers or excipients.

Another aspect of the present invention relates to the use of any of the monoclonal antibodies or antigen binding fragments thereof provided in the present invention or a conjugate of the present invention in the preparation of a medicament for the prevention and/or treatment of tumors or autoimmune diseases or for use in the preparation of a medicament for the diagnosis of autoimmune diseases. diseases; preferably the autoimmune disease is selected from psoriasis, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis and systemic lupus erythematosus; preferably the psoriasis is moderate to severe plaque psoriasis.

In particular, the inventors found in animal experiments (Example 11) that 13E9H3L2 could effectively inhibit the increase in epidermal thickness in the C57BL/6 mouse model of psoriasis, where it was shown that the 13E9H3L2 antibody drug could significantly inhibit in mice IL-17A-induced increase in epidermal thickness by subcutaneous injection with efficacy equivalent to that of the commercially available monoclonal antibody drug Secukinumab used for the same purpose.

In some embodiments of the present invention, autoimmune diseases (eg, psoriasis, such as moderate to severe plaque psoriasis, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, or systemic lupus erythematosus) are caused by increased expression of IL-17A. An elevated expression level as disclosed herein refers to an expression level higher than that found in healthy individuals that has reached a pathogenic level.

Another aspect of the present invention relates to the use of any of the monoclonal antibodies or antigen binding fragments thereof provided in the present invention, or a conjugate of the present invention in the preparation of the following drugs:

drugs that block the binding of IL-17A to IL-17RA, drugs that regulate (eg, reduce) the activity or level of IL-17A, or drugs that inhibit the expression of IL-6 in cells.

Another aspect of the present invention relates to a method in vivo or in vitro, comprising the step of introducing into a cell or patient an effective amount of any of the monoclonal antibodies or antigen binding fragments thereof provided in the present invention, or a conjugate of the present invention, where the method is selected from the following:

a method of blocking the binding of IL-17A to IL-17RA, a method of regulating (eg, reducing) the activity or level of IL-17A, or a method of inhibiting the expression of IL-6 in fibroblasts.

In a particular embodiment of the present invention, the in vitro method is not therapeutic and/or diagnostic.

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Interleukin 6 (abbreviated IL-6 or IL6) can be produced by fibroblasts, monocytes/macrophages, T lymphocytes, B lymphocytes, epithelial cells, keratinocytes and various tumor cells. Interleukin 6 is an important factor regulating the immune response, and IL-6 and IL-1 can synergistically promote T cell proliferation, stimulate B cell differentiation, and participate in host inflammatory responses (Schoenborn et al. Advances in Immunology 96:41-101 (2007)). The in vitro experiment (Example 10) of the present invention shows that the anti-P.-17A antibody can significantly reduce the secretion of IL-6 and inhibit the IL-6-mediated immune response.

Another aspect of the present invention relates to a method of treating and/or preventing tumors or autoimmune diseases, where the method includes the step of administering to a patient an effective amount of any of the monoclonal antibodies or antigen binding fragments thereof provided in the present invention, or a conjugate of the present invention; preferably the autoimmune disease is selected from psoriasis, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis and systemic lupus erythematosus; preferably the psoriasis is moderate to severe plaque psoriasis.

Another aspect of the present invention relates to a method for diagnosing autoimmune diseases, where the method includes the step of applying a sample to be tested (such as a tissue sample, a cell sample, or a blood sample) or administering to a patient an effective amount of any of the monoclonal antibodies or antigen binding fragments thereof provided in the present invention , or a conjugate of the present invention; preferably the autoimmune disease is selected from psoriasis, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis and systemic lupus erythematosus; preferably the psoriasis is moderate to severe plaque psoriasis.

In some embodiments of the present invention, given that autoimmune diseases (eg, psoriasis, such as moderate to severe plaque psoriasis, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, or systemic lupus erythematosus) are caused by increased expression of I.-17A, diagnosis can be performed by determining the expression level of I.-17A using any of the monoclonal antibodies or antigen binding fragments thereof provided in the present invention, or a conjugate of the present invention. If the expression level of IL-17A is higher than in healthy individuals and has reached pathogenic levels, the diagnosis is considered positive; otherwise the diagnosis is negative.

In the present invention, unless otherwise indicated, scientific and technical terms used herein have the meanings in which they are commonly understood by those skilled in the art. In addition, the laboratory techniques of cell culture, molecular genetics, nucleic acid chemistry and immunology used herein are routine operations widely used in their respective fields. Meanwhile, for a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

As used herein, when the amino acid sequence of the IL17A (interleukin-17A) protein is mentioned, this sequence refers to the full-length IL-17A protein; as well as an IL-17A fusion protein, such as a fragment fused to the Fc fragment of mouse or human IgG (mFc or hFc). However, those skilled in the art will appreciate that the amino acid sequence of the IL17A protein may contain mutations or changes (including, without limitation, substitutions, deletions and/or additions), naturally occurring or artificially introduced, that do not affect the biological function of the protein. Therefore, in the present invention, the term IL-17A protein includes all such sequences, including natural or artificial variants thereof. In addition, when describing a sequence fragment of the I.-17A protein, the I.-17A protein also includes corresponding sequence fragments of its natural or artificial variants.

The full length sequence (155aa) of IL-17A is as follows (signal peptide sequence (23aa) is underlined):

MTPGKTSLVSLLLLLSLEAIVKAGITIPRNPGCPNSEDKNFPRTVMVNLNIHNRNT

NTNPKRSSDYYNRSTSPWNLHRNEDPERYPSVIWEAKCRHLGCINADGNVDYHMNSVP IQQEILVLRREPPHCPNSFRLEKILVSVGCTCVTPIVHHVA (SEQ ID NO: 43)

As used herein, unless otherwise stated, IL-17R is IL-17RA; its specific protein sequence is a sequence known in the prior art, which is described in an existing document or which can be found in GenBank. For example, IL-17RA (CD217, NCBI gene ID: NP_055154.3).

As used herein, the term EC ₅₀ refers to a concentration that produces 50% of the maximum effect, i.e., a concentration that can produce 50% of the maximum effect.

In the context of the present invention, the term antibody refers to an immunoglobulin molecule that typically consists of two pairs of polypeptide chains (each pair with one light (.) chain and one heavy (H) chain). In a general sense, the heavy chain can be interpreted as a polypeptide

- 7 046350 ly chain with higher molecular weight in the antibody, and light chain refers to the polypeptide chain with lower molecular weight in the antibody. Light chains are classified as k and λ light chains. Heavy chains are usually classified as μ, δ, γ, α or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA and IgE, respectively. In light chains and heavy chains, the variable region and constant region are linked by a J region of about 12 or more amino acids, and the heavy chain also includes a D region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region ( _VH ) and a heavy chain constant region (CH). The heavy chain constant region consists of 3 domains ( _CH1 , CH2 and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of a single C _L domain. The antibody constant region can mediate the binding of immunoglobulins to host tissues or factors, including the binding of various cells of the immune system (eg, effector cells) to the first component (C1q) of the classical complement system. The VH and VL regions can be further subdivided into highly variable regions (called complementarity determining regions (CDRs)), between which are distributed conserved regions called framework regions (FRs). Each VH and VL consists of 3 CDRs and 4 FRs, located from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy chain/light chain pair form the binding site of the antibody, respectively. The assignment of amino acids to each region or domain is performed in accordance with the definition of Kabat: Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD (1987 and 1991)) or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878883. In particular, the heavy chain may also contain more than 3 CDRs, for example 6, 9 or 12. For example, in the bispecific antibody of the present invention, the heavy chain may be ScFv with the C-terminus of the heavy chain of an IgG antibody linked to another antibody, and in In this case, the heavy chain contains 9 CDRs. The term antibody is not limited to any particular method of producing an antibody. For example, an antibody includes, but is not limited to, a recombinant antibody, a monoclonal antibody, or a polyclonal antibody. Antibodies can be of different isotypes, such as IgG antibodies (eg, IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM.

As used herein, the term antigen-binding fragment refers to a polypeptide containing a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen to which the full-length antibody binds and/or competes with the full-length antibody for specific binding to the antigen, and this fragment is also called antigen-binding part. See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd edition, Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. The antigen binding fragment of an antibody can be produced by recombinant DNA technology or by enzymatic or chemical digestion of intact In some cases, the antigen binding fragment includes Fab, Fab', F(ab') ₂ , Fd, Fv, dAb, complementarity determining region (CDR) fragment and single chain antibody fragment (eg, scFv), chimeric antibody, diabody and polypeptide, which contains at least a portion of an antibody sufficient to confer on the polypeptide the ability to specifically bind an antigen.

As used herein, the term Fd fragment refers to an antibody fragment consisting of the VH and CH1 domains; the term Fv fragment refers to an antibody fragment consisting of the VL and VH domains of one arm of the antibody; the term dAb fragment refers to an antibody fragment consisting of a V _H domain (Ward et al., Nature 341:544-546 (1989)); the term Fab fragment refers to an antibody fragment consisting of V _L , V _H , C _L and CH1 domains; and the term F(ab') ₂ fragment refers to an antibody fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region.

In some cases, the antigen binding fragment of an antibody is a single chain antibody (eg, scFv) in which the VL and Vh domains are paired through a linker that allows the production of a single polypeptide chain to form a monovalent molecule (see, for example, Bird et al., Science 242 :423-426 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988)). Such scFv molecules may have the general structure: NH ₂ -V _L -linker-V _H -COOH or NH ₂ -V _H -linker-V _L -COOH. A suitable prior art linker consists of the repeating amino acid sequence GGGGS or a variant thereof. For example, a linker having the amino acid sequence (GGGGS)4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers that can be used in the present invention are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56 and Roovers et al. (2001), Cancer Immunol.

In some cases, the antigen binding fragment of an antibody is a diabody, that is, a divalent antibody in which the VH and VL domains are expressed on the same polypeptide chain. However, the linker used is too short to allow pairing of two domains on the same chain,

- 8 046350 therefore the domains are forced to pair with complementary domains of the other chain, forming two antigen-binding sites (see, for example, Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:64446448 (1993) and Poljak RJ et al., Structure 2:1121–1123 (1994)).

Antigen-binding fragments (e.g., the above-mentioned antibody fragments) of antibodies can be prepared from these antibodies by conventional methods known to those skilled in the art (e.g., recombinant DNA or enzymatic or chemical digestion), and the antigen-binding antibody fragments are then tested for specificity in the same way as intact antibodies. .

As used herein, unless the context clearly indicates otherwise, the term antibody includes not only intact antibodies, but also antigen-binding fragments of antibodies.

As used herein, the terms mAb and monoclonal antibody refer to an antibody or antibody fragment derived from a group of highly homologous antibodies, i.e., a group of identical antibody molecules, excluding natural mutations that may occur spontaneously. A monoclonal antibody is highly specific for a single epitope on an antigen. A polyclonal antibody, unlike a monoclonal antibody, typically contains at least two or more different antibodies that typically recognize different epitopes on an antigen. Monoclonal antibodies can generally be produced using hybridoma technology, which was first reported by Kohler et al. (Nature, 256:495, 1975), but they can also be produced using recombinant DNA technology (for example, see US Pat. No. 4,816,567).

As used herein, the term chimeric antibody refers to an antibody, part of the light and/or heavy chain of which is derived from an antibody (which may be derived from a particular species or belong to a particular class or subclass of antibodies) and the other part of the light and/or heavy chain is derived from another antibody (which may be derived from the same or a different species or belong to the same or a different class or subclass of antibodies). But in any case, it retains binding activity to the target antigen (USP 4816567, Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851 6855 (1984)).

As used herein, the term humanized antibody refers to an antibody or antibody fragment that is produced when all or part of the CDR regions of a human immunoglobulin (receptor antibody) are replaced with the CDR regions of a non-human antibody (donor antibody), where the donor antibody may be non-human (e.g., murine, rat or rabbit) antibody with the required specificity, affinity or reactivity. In addition, certain amino acid residues in framework regions (FR) of a receptor antibody may also be replaced by amino acid residues of corresponding non-human antibodies or amino acid residues of other antibodies to further improve or optimize the performance of the antibody. For more information on humanized antibodies, see, for example, Jones et al., Nature, 321:522-525 (1986); Reichmann et al., Nature, 332:323-329 (1988); Presta, Curr. Op Struct. Biol., 2:593-596 (1992); and Clark, Immunol. Today 21:397-402 (2000).

As used herein, the term epitope refers to the region of an antigen to which an immunoglobulin or antibody specifically binds. An epitope is also called an antigenic determinant in the art. An epitope or antigenic determinant usually consists of chemically active surface groups of a molecule, such as amino acids, carbohydrates, or sugar side chains, and usually has specific three-dimensional structural characteristics and specific charge characteristics. For example, an epitope typically comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or non-sequential amino acids in a unique spatial conformation, and may be linear or conformational. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996). In a linear epitope, all points of interaction between the protein and the interacting molecule (such as an antibody) are located linearly along the primary amino acid sequence of the protein. In a conformational epitope, the interaction points are located between amino acid residues of the protein that are separated from each other.

As used herein, the term emphasized refers to being obtained by artificial means from a natural state. If some isolated substance or component appears in nature, it may be due to a change in its natural environment or it may be isolated from the environment, or perhaps both. For example, some unisolated polynucleotide or polypeptide exists naturally in a particular living animal, and the same polynucleotide or polypeptide isolated with high purity from its natural state is called an isolated polynucleotide or polypeptide. The term isolated does not exclude the presence of artificial or synthetic substances or other impurities that do not affect the activity of the substance.

As used herein, the term E. coli expression system refers to an expression system consisting of an E. coli (strain) and a vector, where the E. coli (strain) is derived from a commercially available strain such as, but not limited to, GI698, ER2566, BL21 ( DE3), B834 (DE3) and BLR (DE3).

As used herein, the term vector refers to a nucleic acid carrier into which a polynucleotide can be inserted. When a vector expresses a protein encoded

- 9 046350 of the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction, or transfection, causing elements of the genetic material carried by the vector to be expressed in the host cell. Vectors are well known to those skilled in the art, including, but not limited to, plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-based artificial chromosomes (PAC); phages such as lambda phages or M13 phages, and animal viruses, etc. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (eg, SV40). The vector may contain several expression control elements, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may further comprise an origin of replication.

As used herein, the term host cell refers to cells that can be used to administer vectors, including, but not limited to, prokaryotic cells such as E. coli or Bacillus subtilis, fungal cells such as yeast or aspergillus cells, insect cells such such as Drosophila S2 or Sf9 cells, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.

As used herein, the term K _D refers to the equilibrium dissociation constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between antibody and antigen. The lower the equilibrium dissociation constant, the stronger the antibody-antigen binding and the higher the affinity between antibody and antigen. Typically, antibodies bind to antigens with an equilibrium dissociation constant ( _KD ) of less than about 10 ^-5 M, such as less than about 10 ^-6 M, 10 ^-7 M, 10 ^-8 M, 10 ^-9 M, 10 ^-10 M or less, and the equilibrium dissociation constant can be measured, for example, using a Fortebio molecular interaction analyzer.

As used herein, the terms monoclonal antibody and mAb have the same meaning and may be used interchangeably; the terms polyclonal antibody and PcAb have the same meaning and can be used interchangeably; the terms polypeptide and protein have the same meaning and can be used interchangeably. And in the present invention, amino acids are generally represented by one-letter and three-letter abbreviations known in the art. For example, alanine can be represented by the letter A or Ala.

As used herein, the terms hybridoma and hybridoma cell line may be used interchangeably, and when the terms hybridoma and hybridoma cell line are referred to, they also include subclones and progeny cells of the hybridoma. For example, when referring to the hybridoma cell line LT006 or LT007, this also refers to subclones and progeny cells of the hybridoma cell line LT006 or LT007.

As used herein, the term pharmaceutically acceptable carrier and/or excipient refers to a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the individual and the active ingredient, and this term is well known in the art (see, e.g., Remington's Pharmaceutical Sciences, edited by Gennaro A.R., Edited by Gennaro A.R., 19th ed. Pennsylvania: Mack Publishing Company, 1995) and includes, but is not limited to, pH adjusters, surfactants, adjuvants, and ionic strength enhancers. For example, pH adjusters include, but are not limited to, phosphate buffer; surfactants include, but are not limited to, cationic, anionic or nonionic surfactants such as Tween-80; and ionic strength enhancers include, but are not limited to, sodium chloride.

As used herein, the term adjuvant refers to a non-specific immune enhancer that can enhance the body's immune response to antigens or change the type of immune response when delivered to the body along with antigens or delivered to the body in advance. There are various adjuvants including, but not limited to, aluminum adjuvant (eg, aluminum hydroxide), Freund's adjuvant (eg, complete Freund's adjuvant and incomplete Freund's adjuvant), Corynebacterium parvum lipopolysaccharide, cytokine, etc. Freund's adjuvant is the most commonly used adjuvant in animal experiments. The adjuvant aluminum hydroxide is more commonly used in clinical trials.

As used herein, the term effective amount refers to an amount sufficient to obtain, or at least partially obtain, the desired effect. For example, an effective amount for the prevention of diseases (e.g., diseases associated with binding of IL-17A to the IL-17A receptor or excessive activity of IL-17A, such as autoimmune diseases) is an amount sufficient to prevent, reverse, or delay the onset of a disease (e.g. , diseases associated with the binding of IL-17A to the IL-17A receptor or excessive activity of IL-17A, such as autoimmune diseases); a therapeutically effective amount is an amount sufficient to cure or at least partially arrest the development of

- 10 046350 disease and its complications in patients who already suffer from this disease. Those skilled in the art will be able to determine such an effective amount. For example, the amount effective for therapeutic use will depend on the severity of the disease being treated, the general condition of the patient's immune system, the general condition of the patient such as age, weight and sex, the route of administration of the drug substance, and other treatments administered concurrently, and etc.

Beneficial effects of the invention.

The present invention provides at least one of the following technical effects:

(1) all monoclonal antibodies of the present invention, such as 13E9 H1L1, 13E9 H2L2, 13E9 H3L2 and 2G2 H1L1, 2G2 H2L2, 2G2 H3L3, are capable of binding to IL-17A with high specificity and can effectively block the binding of IL-17A to IL ligand -17A, and specifically reduce the secretion of IL-6 in III-17A-mediated fibroblasts;

(2) the monoclonal antibodies of the present invention, especially 13E9 H2L2, show the same effect as the commercially available drug Secukinumab on the same target in inhibiting IL-6 secretion;

(3) the monoclonal antibodies of the present invention can potentially be used for the treatment and/or prevention of anti-autoimmune diseases such as psoriasis, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis or systemic lupus erythematosus.

Brief description of drawings

Fig. 1 - results of detection of monoclonal mouse antibody 13E9 in SDS-PAGE. The four lane samples from left to right and the corresponding input amounts are: antibody in non-reducing protein electrophoresis loading buffer, 1 μg; antibody in loading buffer for protein electrophoresis under reducing conditions, 1 μg; marker, 5 µl; BSA, 1 mcg.

Fig. 2 - results of detection of monoclonal mouse antibody 2G2 in SDS-PAGE. The four lane samples from left to right and the corresponding input amounts are: antibody in non-reducing protein electrophoresis loading buffer, 1 μg; antibody in loading buffer for protein electrophoresis under reducing conditions, 1 μg; marker, 5 µl; BSA, 1 mcg.

Fig. 3 - results of detection of monoclonal humanized antibody 13E9 H3L2 in SDS-PAGE. Samples of the three bands from left to right and corresponding amounts administered: BSA, 1 μg; marker, 5 µl; antibody in loading buffer for protein electrophoresis under non-reducing conditions, 1 μg; antibody in loading buffer for protein electrophoresis under reducing conditions, 1 μg.

Fig. 4 - results of detection of kinetic characteristic parameters of the 13E9 H1L1 antibody. In the drawing, the ordinate axis shows the signal value in nm as a unit of measurement; The x-axis represents time in seconds as a unit of measurement.

Fig. 5 - results of detection of kinetic characteristic parameters of the antibody 13E9 H2L2. In the drawing, the ordinate axis shows the signal value in nm as a unit of measurement; The x-axis represents time in seconds as a unit of measurement.

Fig. 6 - results of detection of kinetic characteristic parameters of the 13E9 H3L2 antibody. In the drawing, the ordinate axis shows the signal value in nm as a unit of measurement; The x-axis represents time in seconds as a unit of measurement.

Fig. 7 - results of detection of kinetic characteristic parameters of the antibody Secukinumab. In the drawing, the ordinate axis shows the signal value in nm as a unit of measurement; The x-axis represents time in seconds as a unit of measurement.

Fig. 8 - results of detection of kinetic characteristic parameters of the 2G2 H1L1 antibody. In the drawing, the ordinate axis shows the signal value in nm as a unit of measurement; The x-axis represents time in seconds as a unit of measurement.

Fig. 9 - results of detection of kinetic characteristic parameters of the antibody 2G2 H2L2. In the drawing, the ordinate axis shows the signal value in nm as a unit of measurement; The x-axis represents time in seconds as a unit of measurement.

Fig. 10 - results of detection of kinetic characteristic parameters of the 2G2 H3L3 antibody. In the drawing, the ordinate axis shows the signal value in nm as a unit of measurement; The x-axis represents time in seconds as a unit of measurement.

Fig. 11 - results of detection of kinetic characteristic parameters of the antibody Secukinumab. In the drawing, the ordinate axis shows the signal value in nm as a unit of measurement; The x-axis represents time in seconds as a unit of measurement.

Fig. 12 - detection of binding activity of antibodies 13E9 H1L1 and 13E9 H2L2 with the IL17A-His antigen by indirect ELISA. The commercially available drug Secukinumab is used as a positive control.

Fig. 13 - detection of binding activity of the 13E9 H3L2 antibody to the IL17A-His antigen by indirect ELISA. The commercially available drug Secukinumab is used as a positive control.

- 11 046350

Fig. 14 - detection of the activity of antibodies 13E9 H1L1 and 13E9 H2L2, competing with the receptor IL17RA-His (biotin) for binding, by competitive ELISA. The commercially available drug Secukinumab is used as a positive control.

Fig. 15 - detection of the activity of the 13E9 H3L2 antibody, which competes with the IL17RAHis receptor (biotin) for binding, by competitive ELISA. The commercially available drug Secukinumab is used as a positive control.

Fig. 16 - detection of binding activity of antibodies 2G2 H1L1, 2G2 H2L2 and 2G2 H3L3 with the IL17A-His antigen by indirect ELISA. The commercially available drug Secukinumab is used as a positive control.

Fig. 17 - detection of the activity of antibodies 2G2 H1L1, 2G2 H2L2 and 2G2 H3L3, competing with the IL17A-His receptor (biotin) for binding to the IL17A-His antigen, by competitive ELISA. The commercially available drug Secukinumab is used as a positive control.

Fig. 18 - the effect of antibodies 13E9 H1L1, 13E9 H2L2 and 13E9 H3L2, as well as 2G2 H1L1, 2G2 H2L2 and 2G2 H3L3 on the secretion of the cytokine IL-6 in the reaction of a mixed culture of lymphocytes.

Fig. 19 - the effect of a drug based on the 13E9 H3L2 antibody on the thickness of the epidermis in the C57BL/6 mouse model with psoriasis.

Note regarding preservation of biological materials:

hybridoma cell line LT006 (IL7A-13E9), deposited in the China Type Culture Collection Center (CTCC) on September 12, 2017 with CCCC accession number: C2017102 at Wuhan University, Wuhan, China 430072.

hybridoma cell line LT007 (IL17A-2G2), deposited in the China Type Culture Collection Center (CTCC) on September 12, 2017 with CCCC accession number: C2017165 at Wuhan University, Wuhan, China 430072.

Detailed description

Embodiments of the present invention are described in detail below with reference to examples. Those skilled in the art will understand that the following examples are used to illustrate the present invention only and should not be construed as limiting the scope of the present invention. The example is performed in accordance with methods or conditions described in the art (for example, see Guide to Molecular Cloning Experiments, by J. Sambrook et al., translated by Huang Peitang et al., third ed., Science Press) or in accordance with with the product manual unless specific methods or conditions are specified therein. All reagents or instruments used are commercially available conventional products unless their manufacturers are indicated.

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

Example 1. Preparation of anti-IL-17A antibodies 13E9 and 2G2.

1. Preparation of hybridoma cell lines LT006 and LT007.

The IL-17A (24-155)-His antigen used to generate the IL-anti-17A antibody is a fusion protein of the mature human IL-17A peptide (GenBank ID: Q16552) and a His tag. Fusion of spleen cells from immunized BALB/C mice (purchased from the Guangdong Animal Medical Laboratory Center) and mouse myeloma cells to produce hybridomas was performed using well-known methods (see, for example, Monoclonal Antibody Production in Basic Methods in Antibody Production and Characterization, Eds G. C. Howard and D. R. Bethell, Boca Raton: CRC Press, 2000).

The IL17A-His fusion protein was enzymatically digested with TEV protease and column purified to yield IL-17A protein (24-155). Screening using indirect ELISA was performed in microplates coated with IL-17A(24-155) protein as the antigen to obtain hybridoma cells that secrete novel antibodies that specifically bind to IL-17A(24-155).

Hybridoma cells obtained from the indirect ELISA screen were screened using a competitive ELISA to generate hybridoma cell lines capable of secreting a monoclonal antibody that competes with the IL-17RA receptor (CD217, NCBI Gene ID: NP_055154.3) for binding to IL-17A , and two stable hybridoma cell lines were obtained by serial dilution.

The hybridoma cell line LT006 (IL17A-13E9) was deposited in the China Type Culture Collection Center (CTCCC) on Saturday, September 12, 2015, under CCTCC accession number: C2017102, Wuhan University (Wuhan, China, 430072). The monoclonal antibody secreted by the hybridoma cell line LT006 is named 13E9.

The hybridoma cell line LT007 (IL17A-2G2) was deposited in the China Type Culture Collection Center (CTCCC) on September 12, 2017 under CCTCC accession number: C2017165 at Wuhan University (Wuhan, China 430072). The monoclonal antibody secreted by the hybridoma cell line LT007 is named 2G2.

- 12 046350

2. Preparation of aHmu-1L-17A antibody 13 E9.

The above LT006 cell line was cultured (1x10 cells/well) in serum-free hybridoma medium (supplemented with 1% penicillin-streptomycin and 4% glutamax, in a cell incubator at 37°C, 5% CO ₂ ), and the cell culture supernatant was collected via 7 days of culture where survival was approximately 20% and then subjected to high speed centrifugation, vacuum filtration through a microporous membrane and purification on a HiTrap HP Protein A column to yield antibody 13E9. Detection of purified 13E9 samples was performed by SDS-PAGE electrophoresis, the results of which are shown in FIG. 1.

3. Obtaining anti-P.-17A antibody 2G2.

The above LT007 cell line was cultured (1x10 cells per well) in serum-free hybridoma medium (supplemented with 1% penicillin-streptomycin and 4% glutamax, in a cell incubator at 37°C, 5% CO ₂ ), and the cell culture supernatant was collected through 7 days of culture where survival was approximately 20% and then subjected to high speed centrifugation, vacuum filtration through a microporous membrane and purification on a HiTrap HP Protein A column to yield antibody 2G2. Detection of purified 2G2 samples was performed using SDSPAGE electrophoresis, the results of which are shown in FIG. 2.

Example 2. Sequence analysis of antibody 13E9.

LT006 cells were cultured according to the method described in step 2 of example 1.

Using a Cellular/Bacterial Total RNA Extraction Kit (Tiangen, part number DP430), mRNA was extracted from cultured LT006 cells according to the method described in the kit manual.

cDNA was synthesized according to the Invitrogen Superscript® III First Strand Synthesis System RT-PCR kit manual and amplified by PCR.

PCR amplified products were directly subjected to direct TA cloning and the pEASY-T1 Cloning Kit (Transgen CT101) manual was used for specific operations.

TA-cloned products were directly sequenced; The sequencing results were as follows:

nucleotide sequence of the heavy chain variable region: (360 bp)

GAAGTAAAGCTGCAGGAGTCGGGACCTGGCCTGGTGAAACCTTCTCAGTCTC

TGTCCCTCACCTGCACTGTCACTAGCTACTCATTCACCAGTGATTATGCCTGGAGCTG

GATCCGGCAGTTTCCAGGAATCAAACTGGAGTGGATGGGCTACATAACCTACAGTG GTGTCACTAGCTACAACCCCTCTCTCAAAAGTCGAATCTCTATCACTCGAGACACAT CCAAGAACCAGTTCTTCCTACAGTTGAATTCTGTGACTACTGAGGACACGGCCACAT ATTACTGTGCAAGGGCAGACTATGATAGCTACTATACTATGGACTACTGGGGTCAAG GAACCTCAGTCACCGTCTCCTCA (SEQ ID NO: 1) amino acid sequence it encodes: (120 aa)

EVKLOESGPGLVKPSOSLSLTCTVTSYSFTSDYAWSWIROFPGIKLEWMGYITYS

GVTSYNPSLKSRISITRDTSKNOFFLQLNSVTTEDTATYYCARADYDSYYTMDYWGOGT

SVTVSS (SEQ ID NO: 2) light chain variable region nucleotide sequence: (333 bp)

GACATCCAGCTGACTCAGTCTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCA

AGCCTCCATCTCTTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTA

TTTACATTGGTACCTGCAGAAGCCAGGCCAGTCTCCAAGGCTCCTGATCTACAAAGT TTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAG ATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTTCTGCT CTCAAAGTACACATTTTTGGACGTTCGGTGGAGGCACCAAGCTGGAAATAAAA (SEQ ID NO: 3) amino acid sequence encoded by it: (111 aa)

DIOLTOSPLSLPVSLGDOASISCRSSOSLVHSNGNTYLHWYLOKPGOSPRLLIYKV

SNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHFWTFGGGTKLEIK (SEQ

ID NO: 4)

CDR areas are underlined.

Example. Development, production and detection of humanized anti-III-17A antibodies 13E9 H1L1. 13E9 H2L2 and 13E9 H3L2.

1. Development of light and heavy chain sequences of humanized anti-III-17A antibodies 13E9 H1L1, 13E9 H2L2 and 13E9 H3L2.

Based on the three-dimensional crystal structure of the IL-17A protein (EMBO J. (2001) 20, p. 5332-41) and

- 13 046350 sequence of the antibody 13E9 obtained in example 2, using a computer, a model of the antibody was modeled, and mutations were calculated in accordance with the model, obtaining sequences of the variable region of antibodies 13E9 H1L1, 13E9 H2L2 and 13E9 H3L2 (sequences of the constant region of the antibody were taken from the database NCBI, in which the heavy chain constant region is the Ig gamma-1 chain C region, accession number: P01857, and the light chain constant region is the Ig kappa chain region, accession number: P01834).

The developed sequences of the variable regions are as follows:

(1) sequences of the heavy and light chains of the humanized monoclonal antibody 13E9 H1L1:

nucleotide sequence of the heavy chain variable region: (360 bp)

GATGTGCAGCTGCAGGAAAGCGGACCAGGACTGGTGAAGCCTAGCCAGACC

CTGAGCCTGACTTGCACCGTGTCCAGCTACAGCTTCACCAGCGACTACGCTTGGTCT TGGATCAGACAGTTCCCAGGAATTGGCCTCGAGTGGATGGGCTACATCACCTACAG CGGCGTGACCAGCTACAACCCCAGCCTGAAGAGCAGGATCACCATCAGCCGGGACA CCAGCAAGAACCAGTTCTTCCTGCAGCTGAACAGCGTGACAGCAGCCGATACCGCA GTGTACTATTGCGCC AGGGCCGACTACGACAGCTACTACACCATGGACTATTGGGGC CAGGGAACCAGCGTGACAGTGTCTAGC (SEQ ID NO: 5) amino acid sequence it encodes: (120 aa)

DVOLOESGPGLVKPSOTLSLTCTVSSYSFTSDYAWSWIROFPGIGLEWMGYITYS

GVTSYNPSLKSRITIISRDTSKNOFFLQLNSVT AARTA VYYCARADYDSYYTMDYWGOG

TSVTVSS (SEQ GO NO: 6)

CDR regions are underlined, nucleotide sequence of the light chain variable region: (333 bp)

GATGTCGTGATGACCCAGACCCCTCTGTCTCTGCCAGTGACACTGGGACAGC

AGGCTAGCATCTCTTGCAGAAGCAGCCAGAGCCTGGTGCACAGCAACGGCAACACC TACCTGCATTGGTACCTGCAGAAGCCAGGCCAGTCTCCTAGACTGCTGATCTACAAG GTGTCCAACCGGTTCAGCGGCGTGCCAGATAGATTCAGCGGAAGCGGAAGCGGCAC CGACTTCACCCTGAAGATCAGCAGAGTGGAGGCCGAGGATCTGGAGTGTACTTCT GCAGCCAGAGCAC CCACTTTTGGACCTTCGGCGGAGGCACCAAGCTGGAGATCAAG (SEQ ID NO: 7) amino acid sequence it encodes: (111 aa)

DVVMTOPLSLPVTLGOOASISCRSSOSLVHSNGNTYLHWYLQKPGOSPRLLIYK

VSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHFWTFGGGTKLEIK (SEQ

ID NO: 8)

CDR areas are underlined;

(2) sequences of the heavy and light chains of the humanized monoclonal antibody 13E9 H2L2:

nucleotide sequence of the heavy chain variable region: (360 bp)

GATGTGCAGCTGCAGGAAAGCGGACCAGGACTGGTGAAGCCTAGCCAGACC

CTGAGCCTGACTTGCACCGTGTCCAGCTACAGCTTCACCAGCGACTACGCTTGGTCT TGGATCAGACAGCCACCAGGAAAGGGACTCGAGTGGATCGGCTACATCACCTACAG CGGCGTGACCAGCTACAACCCCAGCCTGAAGAGCAGGATCACCATCAGCCGGGACA CCAGCAAGAACCAGTTCTTCCTGCAGCTGTCTAGCGTGACAGCAGCCGATACCGCA GTGTACTATTGCGCC AGGGCCGACTACGACAGCTACTACACCATGGACTATTGGGGC CAGGGAACCAGCGTGACAGTGTCTAGC (SEQ ID NO: 9) amino acid sequence it encodes: (120 aa)

DVQLQESGPGLVKPSQTLSLTCTVSSYSFTSDYAWSWIRQPPGKGLEWIGYITYS

GVTSYNPSLKSRITIISRDTSKNQFFLQLSSVTAADTA VYYCARADYDSYYTMDYWGOG

TSVTVSS (SEQ GO NO: 10)

CDR areas are underlined,

- 14 046350 nucleotide sequence of the light chain variable region: (333 bp)

GATGTCGTGATGACCCAGACCCCTCTGTCTCTGCCAGTGACACTGGGACAGC

CAGCTAGCATCTCTTGCAGAAGCAGCCAGAGCCTGGTGCAGCAACGGCAACACC TACCTGCATTGGTACCTGCAGAAGCCAGGCCAGTCTCCTAGACTGCTGATCTACAAG GTGTCCAACCGGTTCAGCGGCGTGCCAGATAGATTCAGCGGAAGCGGAAGCGGCAC CGACTTCACCCTGAAGATCAGCAGAGTGGAGGCCGAGGATCTGGAGTGTACTACT GCAGCCAGAGCACCC ACTTTTGGACCTTCGGCGGAGGCACCAAGCTGGAGATCAAG (SEQIDNO: 11) amino acid sequence it encodes: (111 aa)

DVVMTOPLSLPVTLGOPASISCRSSOSLVHSNGNTYLHWYLQKPGOSPRLLIYK VSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCSQSTHFWTFGGGTKLEIK (SEQ ID NO: 12)

CDR areas are underlined;

(3) sequences of the heavy and light chains of the humanized monoclonal antibody 13E9 H3L2 nucleotide sequence of the heavy chain variable region: (360 bp) GATGTGCAGCTGCAGGAAAGCGGACCAGGACTGGTGAAGCCTAGCCAGACC

CTGAGCCTGACTTGCACCGTGTCCAGCTACAGCTTCACCAGCGACTACGCTTGGTCT TGGATCAGACAGCCACCAGGAAAGGGACTCGAGTGGATCGGCTACATCACCTACAG CGGCGTGACCAGCTACAACCCTAGCCTGAAGAGCCGCGTGACCATTAGCGTGGACA CCAGCAAGAACCAGTTCTCCCTGAAGCTGAGCAGCGTGACAGCCGCCGATACAGCA GTGTACTATTGCGCCCG GGCCGATTACGACAGCTACTACACCATGGACTATTGGGGC CAGGGAACCAGCGTGACAGTGTCTAGC (SEQ ID NO: 13) amino acid sequence it encodes: (120 aa)

DVOLOESGPGLVKPSOTLSLTCTVSSYSFTSDYAWSWIROPPGKGLEWIGYITYS

GVTSYNPSLKSRVTISVDTSKNOFSLKLSSVTAADTAVYYCARADYDSYYTMDYWGOG

TSVTVSS (SEQ GO NO: 14)

CDR areas are underlined.

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

The encoded amino acid sequence of the antibody also matches the amino acid sequence of the light chain variable region of the 13E9 H2L2 antibody set forth in SEQ ID NO: 12.

2. Preparation of humanized antibodies 13E9 H1L1, 13E9 H2L2 and 13E9 H3L2.

All heavy chain constant regions contain the Ig gamma-1 chain C region, accession number: P01857; light chain constant regions contain the Ig kappa chain C region, accession number: P01834.

The 13E9 H1L1 heavy chain cDNA and light chain cDNA, the 13E9 H2L2 heavy chain cDNA and light chain cDNA, and the 13E9 H3L2 heavy chain cDNA and light chain cDNA were cloned into pUC57simple vectors (provided by Genscript), respectively, yielding pUC57simple-13E9H1, pUC57simple-13E9Ll, pUC57simple-13E9H2, pUC57simple-13E9L2 and pUC57simple-13E9H3, respectively, and fragments containing the corresponding heavy chains and fragments containing the corresponding light chains were subcloned into the pcDNA3.1 vectors, respectively, to obtain the recombinant plasmids pcDNA3.1-13E9H1, pcDNA3 .1-13E9L1, pcDNA3.113E9H2, pcDNA3.1-13E9L2, pcDNA3.1-13E9H3 and pcDNA3.1-13E9L2. The corresponding light chain recombinant plasmids and heavy chain recombinant plasmids (pcDNA3.1-13E9H1 and pcDNA3.1-13E9Ll; pcDNA3.1-13E9H2 and pcDNA3.1-13E9L2; pcDNA3.1-13E9H3 and pcDNA3.1-13E9L2) were then cotransfected into 293F cells, the cell culture was collected and purified to obtain humanized antibodies 13E9 H1L1, 13E9 H2L2 and 13E9 H3L2, respectively. Detection of purified 13E9 H3L2 sample was performed by SDS-PAGE electrophoresis, the results of which are shown in FIG. 3.

Example 4 Sequence analysis of the 2G2 antibody.

LT007 cells were cultured according to the method described in step 3 of example 1.

Using a Cellular/Bacterial Total RNA Extraction Kit (Tiangen, part number DP430), mRNA was extracted from cultured LT007 cells according to the method described in the kit manual.

cDNA was synthesized according to the Invitrogen Superscript® III First Strand Synthesis System RT-PCR kit manual and amplified by PCR.

PCR amplified products were directly subjected to direct TA cloning and the pEASY-T1 Cloning Kit (Transgen CT101) manual was used for specific operations.

- 15 046350

TA-cloned products were directly sequenced; The sequencing results were as follows:

nucleotide sequence of the heavy chain variable region: (351 bp)

GAGGTTCAGCTGGAGCAGTCTGGTTCTGAACTGAGGAGTCCTGGATCTTCAG TAAAGCTTTCATGCAAGGATTTTGATTCAGAAGTCTTCCCTATTGCTGATATGAGTTG GGTTAGGCAGAAGCCTGGGCATGGATTTGAATGGATTGGAGACATACTCCCAAGTT TTGGTAGAACAATCTATGGAGAGAAGTTTGAGGACAAAGCCAAAGTGGATGCAGAC ACAGTGTCCAACACAGC CTACTTGGAACTCAACAGTCTGACATCTGAGGACTCTGCT ATCTACTACTGTGCAAGGGGTAACTACGGGTTTGCTTACTGGGGCCAAGGGACTCTG GTCACTGTCTCTGCA (SEQ ID NO: 15) amino acid sequence it encodes: (117 aa)

EVOLEOSGSELRSPGSSVKLSCKDFDSEVFPIADMSWVROKPGHGFEWIGDILPSF GRTIYGEKFEDKAKVDADTVSNTAYLELNSLTSEDSAIYYCARGNYGFAYWGOGTLVT VSA (SEQ ID NO: 16) CDR regions are underlined, nucleotide sequence of light chain variable region: (336 bp)

GATGTTTTGATGACCCAAACTCCACTCACTTTGTCGGTTATCATTGGACAACC AGCCTCCATCTCTTGCAAGCCAAGTCAGAGCCTCTTAAATAGTGATGGAAAGACATA TTTGAATTGGTTGTTGCAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGT GTCTAAACTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAG ATTTCACACTGAAAATCAG CAGAGTGGAGGCTGAGGATTTGGGAGTTTATTATTGCT GGCAAGGTTCACATTTTCCTCAGACGTTCGGTGGAGGCACAAAGTTGGAAATAAAA (SEQ ID NO: 17) amino acid sequence it encodes: (112 aa)

DVLMTOTPLTLSVIIGOPASISCKPSOSLLNSDGKTYLNWLLORRPGOSPKRLIYLV

SKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGSHFPQTFGGGTKLEIK (SEQ ID NO: 18)

CDR areas are underlined.

Example 5. Development, production and detection of humanized api-11.-17A antibodies 2G2 H1L1. 2G2 H2L2 and 2G2 H3L3.

(1) sequences of the heavy and light chains of the humanized monoclonal antibody 2G2 H1L1:

nucleotide sequence of the heavy chain variable region: (348 bp)

GTGCAGCTGGTGCAGAGCGGAAGCGAACTGAGAAAGCCAGGCTCCAGCGTG AAGCTGTCTTGCAAGGACTTCGACAGCGAGGTGTTCCCCATCGCCGATATGTCTTGG GTCCGACAGGCTCCAGGCCAGGGATTCGAGTGGATCGGTGACATTCTGCCCAGCTTC GGAAGAACCAACTACGCCCAGAAGTTCGAGGGCAAGGCCAAGGTGGACGCAGACA AGAGCACCAACACCGCCTACCTG GAGCTGAACAGCCTGAGAAGCGAGGACACCGCC ATCTACTATTGCGCCAGGGCAACTACGGATTCGCCTATTGGGGCCAGGGAACACT GGTGACAGTGTCCGCC (SEQ ID NO: 19) amino acid sequence it encodes: (116 aa)

VOLVQSGSELRKPGSSVKLSCKDFDSEVFPIADMSWVROAPGOGFEWIGDILPSF GRTNYAQKFEGKAKVDADKSTNTAYLELNSLRSEDTAIYYCARGNYGFAYWGOGTLV TVSA (SEQ ID NO: 20) CDR regions are underlined, nucleotide sequence of light chain variable region: (336 bp)

GATGTCGTGATGACCCAGACCCCTCTGTCTCTGAGCGTGACACTGGGACAGC CAGCTAGCATCAGCTGCAGAAGCAGCCAGAGCCTGCTGAACAGCGACGGCAAGACC TACCTGAATTGGCTGCTGCAGAGACCAGGCCAGTCTCCTAGAAGGCTGATCTACCTG GTGTCCAAGCTGGACAGCGGCGTGCCAGATAGATTCAGCGGAAGCGGAAGCGGCAC CGACTTCACCCCTGAAGATCA GCAGAGTGGAGGCCGAGGATCTGGGAGTGTACTACT GTTGGCAGGGCAGCCACTTCCCTCAGACATTCGGCGGCGGCACAAAGCTGGAGATC AAG (SEQ ID NO: 21)

- 16 046350 amino acid sequence encoded by it: (112 aa)

DVVMTOTPLSLSVTLGOPASISCRSSOSLLNSDGKTYLNWLLQRPGOSPRRLIYL VSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCWOGSHFPQTFGGGTKLEIK (SEQ ID NO: 22) CDR regions are underlined.

(2) Sequences of the heavy and light chains of the humanized monoclonal antibody 2G2 H2L2:

nucleotide sequence of the heavy chain variable region: (348 bp)

GTGCAGCTGGTGCAGAGCGGAGCAGAAGTGAAGAAGCCAGGCTCCAGCGTG AAGCTGTCTTGCAAGGACTTCGACAGCGAGGTGTTCCCCATCGCCGATATGTCTTGG GTCCGACAGGCTCCAGGCCAGGGATTCGAGTGGATCGGTGACATTCTGCCCAGCTTC GGGAGAACCAATTACGCCCAGAAGTTCCAGGGCAGAGTGACCGTGACCGCAGACAA GAGCACCAACACCGCCTAC CTGGAGCTGAACAGCCTGAGGAGCGAGGATACCGCCG TGTACTATTGCGCCAGGGCAACTACGGCTTCGCCTATTGGGGACAGGGAACACTG GTGACAGTGTCCGCC (SEQ ID NO: 23) amino acid sequence it encodes: (116 aa)

VOLVOSGAEVKKPGSSVKLSCKDFDSEVFPIADMSWVROAPGOGFEWIGDILPSF GRTNYAOKFOGRVTVTADKSTNTAYLELNSLRSEDTAVYYCARGNYGFAYWGOGTLV TVSA (SEQ ID NO: 24) CDR regions are underlined, nucleotide sequence of light chain variable region: (336 bp)

GATGTCGTGATGACCCAGACCCCTCTGTCTCTGAGCGTGACACTGGGACAGC CAGCTAGCATCAGCTGCAGAAGCAGCCAGAGCCTGCTGAACAGCGACGGCAAGACC TACCTGAATTGGCTGCTGCAGAGACCAGGCCAGTCTCCTAGAAGGCTGATCTACCTG GTGTCCAACCTGGACAGCGGCGTGCCAGATAGATTCAGCGGAAGCGGAAGCGGCAC CGACTTCACCCCTGAAGATCA GCAGAGTGGAAGCCGAGGACGTGGGAGTGTACTACT GTTGGCAGGGCAGCCACTTCCCTCAGACATTCGGCGGCGGCACAAAGCTGGAGATC AAG (SEQ ID NO: 25) amino acid sequence it encodes: (112 aa)

DVVMTOTPLSLSVTLGOPASISCRSSOSLLNSDGKTYLNWLLQRPGOSPRRLIYL VSNLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWOGSHFPQTFGGGTKLEIK (SEQ ID NO: 26)

CDR areas are underlined;

(3) Sequences of the heavy and light chains of the humanized monoclonal antibody 2G2 H3L3:

nucleotide sequence of the heavy chain variable region: (348 bp)

GTGCAGCTGGTGCAGAGCGGAGCAGAAGTGAAGAAGCCAGGCAGCAGCGTG AAGGTGTCTTGCAAGGACTTCAGCAGCGAGGTGTTCCCCATCGCCGATATGTCTTGG GTCCGACAGGCTCCAGGCCAGGACTGGAGTGGATCGGTGACATTCTGCCCAGCTT CGGGAGAACCAATTACGCCCAGAAGTTCCAGGGCAGAGTGACCGTGACCGCAGACA AGAGCACCAACACCGCCTACCTG GAGCTGTCTAGCCTGAGAAGCGAGGACACCGCC GTGTACTATTGCGCCAGGGCAACTACGGCTTCGCCTATTGGGGACAGGGAACACT GGTGACAGTGTCCGCC (SEQ ID NO: 27) amino acid sequence it encodes: (116 aa)

VOLVOSGAEVKKPGSSVKVSCKDFSSEVFPIADMSWVROAPGOGLEWIGDILPSF GRTNYAOKFOGRVTVTADKSTNTAYLELSSLRSEDTAVYYCARGNYGFAYWGOGTLV TVSA (SEQ ID NO: 28)

CDR areas are underlined,

- 17 046350 nucleotide sequence of the light chain variable region: (336 bp)

GATGTCGTGATGACCCAGACCCCTCTGTCTCTGAGCGTGACACTGGGACAGC

CAGCTAGCATCAGCTGCAGAAGCAGCCAGAGCCTGCTGAACAGCGACGGCAAGACC TACCTGAATTGGTTCCTGCAGAGACCAGGCCAGTCTCCTAGAAGGCTGATCTACCTG GTGTCCAACCTGGACAGCGGCGTGCCAGATAGATTCAGCGGAAGCGGAAGCGGCAC CGACTTCACCCTGAAGATCAGCAGAGTGGAAGCCGAGGACGTGGGAGTGTACTACT GTTGGCAGGGCAGCC ACTTCCCTCAGACATTCGGCGGCGGCACAAAAGCTGGAGATC

AAG (SEQ ID NO: 29) amino acid sequence it encodes: (116 aa)

DVVMTOTPLSLSVTLGOPASISCRSSOSLLNSDGKTYLNWFLQRPGOSPRRLIYL

VSNLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWOGSHFPOTFGGGTKLEIK (SEQ ID NO: 30)

CDR areas are underlined.

2. Preparation and detection by SDS-PAGE electrophoresis of humanized antibodies 2G2 H1L1, 2G2 H2L2 and 2G2 H3L3.

For the heavy chain constant regions, Ig gamma 1 chain C region was used, accession number: P01857; for light chain constant regions, the Ig kappa chain C region was used, accession number: P01834.

H1L1 heavy chain cDNA and light chain cDNA 2G2, H2L2 heavy chain cDNA and light chain cDNA 2G2, and H3L3 heavy chain cDNA and light chain 2G2 cDNA were cloned into pUC57simple vectors (provided by Genscript), respectively, yielding pUC57simple-2G2H1, pUC57simple-2G2L1 ; pUC57simple-2G2H2, pUC57simple-2G2L2; and pUC57simple-2G2H3, pUC57simple-2G2L3, respectively. Then, the nucleotide fragments containing the corresponding heavy chains and the nucleotide fragments containing the corresponding light chains were subcloned into the pcDNA3.1 vectors, respectively, to obtain the recombinant plasmids pcDNA3.1-2G2H1, pcDNA3.1-2G2L1, pcDNA3.1-2G2H2, pcDNA3 .1-2G2L2, pcDNA3.1-2G2H3 and pcDNA3.1-2G2L3. Thereafter, the corresponding recombinant light chain plasmids and recombinant heavy chain plasmids (pcDNA3.1-2G2H1 and pcDNA3.12G2L1; pcDNA3.1-2G2H2 and pcDNA3.1-2G2L2; pcDNA3.1-2G2H3 and pcDNA3.1-2G2L3) were cotransfected into the cells 293F, the cell culture was harvested and purified to obtain humanized antibodies 2G2 H1L1, 2G2 H2L2 and 2G2 H3L3, respectively. Purified samples were detected using SDSPAGE electrophoresis.

Example 6. Measurement of kinetic parameters of binding of 13E9 H1L1, 13E9 H2L2 and 13E9 H3L2 to the IL-17A (24-155) antigen protein.

Binding kinetics of humanized antibodies 13E9 H1L1, 13E9 H2L2 and 13E9 H3L2 to IL-17A (24-155) antigen were measured using a Fortebio molecular interaction assay.

The AR2G sensor was activated using EDC/NHS, and antibody was immobilized onto the activated AR2G sensor via amine coupling, then the sensor was blocked with 1 M ethanolamine (pH 8.5). After equilibrating the sensor in PBST for 300 s, binding of the antibody immobilized on the sensor to the IL-17A (24-155) antigen protein (same as the antigen used in Example 1) was performed at an antigen concentration of 6.25-400 nM (double gradient dilution), binding time was 420 s, followed by dissociation of the antigen-antibody complex in PBST for 600 s.

The kinetic parameters of antibodies 13E9 H1L1, 13E9 H2L2, 13E9 H3L2 and Secukinumab are shown in table. 1, and the results of determining the kinetic characteristics are shown in Fig. 4, 5, 6 and 7, respectively.

Table 1

Kinetic parameters of the humanized antibody 13E9

Antibody _KD (M) Cop (1/Ms) Cop error Kdis (1/s) Kdis error _Rmax (nm) 13E9H1L1 9J2E-10 1.34E+05 2.60E+03 1.31E-04 9.06E-06 0.0858-0.3326 13E9 H2L2 1, OZE-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 1D7E-04 1.02E-05 0.1457-0.3546 Sekhu throw mab 6.74E-10 9.28E+04 3.03E+03 6.26E-05 1.57E-05 0.0379-0.2064

KD - affinity constant; Kon is the rate of binding of antigen to antibody; Kdis - rate of dissociation of antigen and antibody; K _D =Kon/Kdis.

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

Example 7. Measurement of kinetic parameters of binding of 2G2 H1L1, 2G2 H2L2 and 2G2 H3L3 to the IL-17A (24-155) antigen.

Binding kinetics of humanized antibodies 2G2 H1L1, 2G2 H2L2 and 2G2 H3L3 to IL-17A (24-155) antigen were measured using a Fortebio molecular interaction assay.

The AR2G sensor was activated using EDC/NHS, and antibody was immobilized onto the activated AR2G sensor via amine coupling, and the sensor was blocked with 1 M ethanolamine (pH 8.5). After equilibrating the sensor in PBST for 300 s, the antibody immobilized on the sensor was bound to the IL-17A (24-155) antigen at an antigen concentration of 6.25-400 nM (double gradient dilution), and the binding time was 420 s, then dissociation of the antigen-antibody complex was performed in PBST for 600 s.

The kinetic parameters of antibodies 2G2 H1L1, 2G2 H2L2, 2G2 H3L3 and Secukinumab are shown in table. 2, and the results of determining the kinetic characteristics are shown in FIG. 8, 9, 10 and 11, respectively.

table 2

Kinetic parameters of 2G2 H1L1, 2G2 H2L2, 2G2 H3L3 and secukinumab

Antibody _KD (M) Cop(1/Ms) Cop, mistake Kdis (1/s) Kdis error Kshah(nm) 2G2 H1L1 1.53E-09 1D6E+05 ZD5E+OZ 2.24E-04 9.67E-06 0.0035-0.2192 2G2 H2L2 8DZE-10 1D2E+05 2D8E+03 1D0E-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,ЗЗЭ+05 2.38E+03 1DZE-04 8.09E-06 0.0118-0.2584

K _D - affinity constant; Kon is the rate of binding of antigen to antibody; Kdis - rate of dissociation of antigen and antibody; K _D =Kon/Kdis.

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

Example 8. Determination of the binding activity of antibodies 13E9 H1LL1, 13E9 H2L2 and 13E9 H3L2 to antigen by ELISA.

1. The binding activity of antibodies 13E9 H1L1, 13E9 H2L2 and 13E9 H3L2 to the IL17A-His antigen was determined using an indirect ELISA and compared with the commercially available drug Secukinumab, used for the same purpose.

IL17A-His can be obtained using published sequences and conventional techniques or using the following steps.

Preparation of IL17A-His: The full-length human IL-17A protein sequence was obtained from the NCBI Protein Database and fused to the His*6 tag used for purification. The synthesis of the nucleic acid encoding the fusion protein was carried out at Genscript in Nanjing and using standard methods described in the Guide to Molecular Cloning Experiments (second edition) and standard molecular cloning methods such as PCR, enzymatic digestion, gel extraction, ligation, colony PCR, or enzymatic digestion identification, the target gene was subcloned into expression vectors in mammalian cells, and then the target gene with recombinant expression vectors was sequenced and analyzed. After sequence verification, medium to high endotoxin-free expression plasmids were prepared and transiently transfected into HEK293 cells for protein expression. After 7 days, the culture liquid was collected and purified by affinity chromatography on a Ni-Sepharose (GE) column, and the quality of the resulting protein samples was determined by standard analytical methods SDS-PAGE and SEC-HPLC for compliance with the standard.

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 accordingly and incubated at 37°C for 30 min; goat anti-human IgG(H+L)-HRP antibodies (Jackson, 109-035088) were added and incubated at 37°C for 30 min; and then a color reaction was performed with TMB (Neogen, 308177) for 5 min, and the absorbance was determined on a microplate reader at 450 nm. The obtained experimental data were analyzed and processed using SoftMax Pro 6.2.1 software, then a curve approximated by a 4-parameter function was plotted for analysis, plotting the antibody concentration on the abscissa axis, and the absorption value on the ordinate axis.

- 19 046350

The experimental results are shown in Fig. 12, 13, and in table. 3 and 4 below.

Table 3

Results of detection of binding activity of 13E9 H1L1 and 13E9 H2L2 with IL17A-His antigen

Antibody concentration/gradient Coating antigen: IL17A-His, 1 µg/ml 13E9H1L1 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 ₅₀ (nM) 0.044 0.048 0.082

Table 4

Results of detecting the binding activity of 13E9 H3L2 with the IL17A-His antigen

Antibody concentration/gradient Coating Antigen: IL17A-His: 1 µg/ml 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 ₅₀ (nM) 0.043 0.078

The experimental results show that the antibodies 13E9 H1L1, 13E9 H2L2 and 13E9 H3L2 can effectively bind to the IL17A-His antigen, and their binding efficiency is dose dependent. Under the same experimental conditions, the EC50 value of 13E9 H1L1 binding was 0.044 nM, the EC50 value of 13E9 H2L2 binding was 0.048 nM, and the _EC50 value of the commercially available drug Secukinumab was 0.082 nM (Fig. 12, Table 3); under the same experimental conditions, the EC ₅₀ value of 13E9 H3L2 binding was 0.043 nM, and the EC ₅₀ value of the commercially available drug Secukinumab was 0.078 nM (Fig. 13, Table 4).

The above experimental results show that under the same experimental conditions, the EC50 values of antibodies 13E9 H1L1, 13E9 H2L2 and 13E9 H3L2 are less than the same target binding value observed for the positive control drug Secukinumab, indicating improved binding activity of antibodies 13E9 H1L1, 13E9 H2L2 and 13E9 H3L2 to IL17AHis compared with commercially available Secukinumab used as a control.

2. The activity of antibodies 13E9 H1L1, 13E9 H2L2 and 13E9 H3L2, which block the binding of IL17RA-His (biotin) to the IL17A-His antigen, was detected using a competitive ELISA.

IL17RA-His (biotin) can be obtained using published sequences and conventional techniques or by performing the following steps.

Preparation of IL17RA-His (biotin): The human IL17RA extracellular domain sequence was obtained from the NCBI Protein Database and fused to the His*6 tag used for purification. Synthesis of the nucleic acid encoding the fusion protein was performed at Genscript in Nanjing and using standard methods described in the Guide to Molecular Cloning Experiments (second edition) and standard molecular cloning methods such as PCR, enzymatic digestion, gel extraction, ligation, colony PCR, or enzymatic digestion identification, subcloned the target gene into expression vectors in mammalian cells, then the target gene with recombinant expression vectors was sequenced and analyzed. After checking the correctness of the sequence, medium and large quantities of endotoxin-free expression plasmids were prepared and cells were transiently transfected

- 20 046350

HEK293 for protein expression. After 7 days, the culture liquid was collected and purified by affinity chromatography on a Ni-Sepharose (GE) column, and the quality of the resulting protein samples was determined by standard SDS-PAGE and SEC-HPLC analytical methods for compliance with the standard. Once quality determination was completed, biotinylated human IL-17RA-His protein samples were labeled using the commercial EZ-Lmk®Sulfo-NHS-LC-Biotmylation kit from Thermo Scientific and the specific preparation method was performed according to the manufacturer's instructions.

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 hours, antibodies were added accordingly, and antigen-antibody interaction was allowed at room temperature for 10 minutes; then the IL17RA-His receptor (biotin) was added, mixed well with the antibody in a volume ratio of 1:1 and incubated at 37°C for 30 min; SA-HRP (KPL, 14-30-00) was added and incubated at 37°C for 30 min; and then performed a color reaction with TMB (Neogen, 308177) for 5 min, after which the absorbance was determined on a microplate reader at 450 nm. The obtained experimental data were analyzed and processed using SoftMax Pro 6.2.1 software, then a curve approximated by a 4-parameter function was plotted for analysis, plotting the antibody concentration on the abscissa axis and the absorption value on the ordinate axis. The experimental results are shown in Fig. 14, 15 and in table. 5 and 6 below.

Table 5

Results of detection of the activity of 13E9 H1L1 and 13E9 H2L2, competing with the IL17RA-His receptor (biotin) for binding to the IL17A-His antigen

Antibody concentration/gradient Coating antigen: IL17A-His (20150213) 0.5 μg/ml 13E9H1L1 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 ESbo(nM) 1.437 1.281 1,807

Table 6

Results of detecting the activity of 13E9 H3L2, which competes with the IL17RA-His receptor (biotin) for binding to the IL17A-His antigen

Antibody concentration/gradient Coating antigen: IL17A-His 0.5 µg/ml 13E9 H3L2 Secukinumab 10µg/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 ESbo(nM) 0.805 1,580

The experimental results show that the antibodies 13E9 H1L1, 13E9 H2L2 and 13E9 H3L2 can effectively block the binding of the IL17RA-His receptor (biotin) to the IL17A-His antigen, and the blocking efficiency is dose dependent. Under the same experimental conditions, the EC value is ₅₀ 13E9

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H1L1 competing with IL17AR-His (biotin) for binding to IL17A-His was 1.437 nM, EC50 value of 13E9 H2L2 competing with IL17AR-His (biotin) for binding to IL17A-His was 1.281 nM, and EC50 value of positive control the drug Secukinumab, which competes with IL17ARHis (biotin) for binding to the same target, IL17A-His, was 1.807 nM (Table 5, Fig. 14); the EC50 value of 13E9 H3L2 competing with IL17AR-His (biotin) for binding to IL17A-His was 0.805 nM, and the _EC50 value of the commercially available drug Secukinumab competing with IL17AR-His (biotin) for binding to the same target, IL17A- His was 1,580 nM (Table 6, Fig. 15).

The above experimental results show that under the same experimental conditions, all EC ₅₀ values of antibodies 13E9 H1L1, 13E9 H2L2 and 13E9 H3L2 competing with IL17AR-His (biotin) for binding to IL17A-His are less than those of the commercially available drug Secukinumab used as a control, indicating improved activity of antibodies 13E9 H1L1, 13E9 H2L2 and 13E9 H3L2, which compete with IL17AR-His (biotin) for binding to IL17A-His, compared to the commercially available drug Secukinumab.

Example 9. Determination by ELISA of the binding activity of antibodies 2G2 H1L1, 2G2 H2L2 and 2G2 H3L3 to antigen.

1. The binding activity of antibodies 2G2 H1L1, 2G2 H2L2 and 2G2 H3L3 to the IL17A-His antigen was determined by indirect ELISA.

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 accordingly and incubated at 37°C for 30 min; goat anti-human IgG (H+L)-HRP (Jackson, 109-035-088) was added and incubated at 37°C for 30 min; then a color reaction was performed with TMB (Neogen, 308177) for 5 min, and the absorbance at 450 nm was determined using a microplate reader. The obtained experimental data were analyzed and processed using SoftMax Pro 6.2.1 software, then a curve approximated by a 4-parameter function was plotted for analysis, plotting the antibody concentration on the abscissa axis and the absorption value on the ordinate axis.

The experimental results are shown in Fig. 16 and in table. 7 below. The EC ₅₀ value of 2G2 H1L1 binding was 0.177 nM, the EC ₅₀ value of 2G2 H2L2 binding was 0.372 nM, and the EC ₅₀ value of 2G2 H3L3 binding was 0.421 nM.

Table 7

Results of detection of binding of 2G2 H1L1, 2G2 H2L2 and 2G2 H3L3 to the IL17 A-His antigen

Antibody concentration/gradient Coating antigen: IL17A-His, 1 µg/ml 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 ESbo(nM) 0.177 0.372 0.421 0.090

The experimental results show that the antibodies 2G2 H1L1, 2G2 H2L2 and 2G2 H3L3 can effectively bind to the IL17A-His antigen, and their binding efficiency is dose dependent.

2. The activity of antibodies 2G2 H1L1, 2G2 H2L2 and 2G2 H3L3, which compete with the IL17A-His receptor (biotin) for binding to the IL17A-His antigen, was detected using a competitive ELISA.

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 hours, antibodies were added accordingly, and antigen-antibody interaction was allowed at room temperature for 10 minutes; then the IL17RA-His receptor (biotin) was added, mixed well with the antibody in a volume ratio of 1:1 and incubated at 37°C for 30 min; SA-HRP (KPL, 14-30-00) was added and incubated at 37°C for 30 min; then a color reaction was performed with TMB (Neogen, 308177) for 5 min, and the absorbance at 450 nm was determined using a microplate reader. Expe received

- 22 046350 The experimental data were analyzed and processed using SoftMax Pro 6.2.1 software, then a curve approximated by a 4-parameter function was plotted for analysis, plotting the antibody concentration on the abscissa axis and the absorbance value on the ordinate axis.

The experimental results are shown in Fig. 17 and in table. 8 below. The EC50 value of 2G2 H1L1 blocking was 2.264 nM, the EC50 value of 2G2 H2L2 blocking was 5.408 nM, and the EC50 value of 2G2 H3L3 blocking was 5.911 nM (Fig. 17, Table 8).

Table 8

Results of detection of the activity of 2G2 H1L1, 2G2 H2L2 and 2G2 H3L3, competing with the IL17RA-His receptor (biotin) for binding to the IL17A-His antigen

Antibody concentration/gradient Coating antigen: IL17A-His 0.5 µg/ml 2G2H1L1 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 ₅₀ (nM) 2,264 5,408 5,911 2,749

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

Example 10. Reaction of a mixed culture of human embryonic fibroblasts: secretion of the cytokine IL-6.

MRC 5 cells (purchased from the Cell Center of the Chinese Academy of Sciences) were plated in a 96-well plate at a concentration of 5000 cells/well and cultured overnight. A mixture of IL-17 and antibody (hIgG as control) was incubated at 37°C for 20 minutes, added to MRC 5 cells and cultured for 48 hours. After 48 h of culture, the cell supernatant was collected, and the amount of secreted IL-6 was determined using an ELISA kit (purchased from Dakewe Corporation).

MRC 5 cells 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. The detection results of secreted IL-6 are shown in FIG. 18.

From fig. 18 shows that 13E9 H1L1, 13E9 H2L2, 13E9 H3L2 and 2G2 H2L2 can effectively reduce the secretion of IL-6 in MRC 5 cells induced by IL-17, while:

the effect of the 13E9 H1L1 antibody on inhibiting IL-6 secretion at a concentration of 100 nM is greater 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 the effect of the control antibody Secukinumab at a concentration of 100 nM;

the effect of the 13E9 H2L2 antibody on inhibition of IL-6 secretion at concentrations of 1 nM, 10 nM and 100 nM is equivalent to the effect of the control antibody Secukinumab at the same doses;

the inhibitory effects of the 13E9 H3L2 antibody on IL-6 secretion at concentrations of 10 nM and 100 nM are greater than those of the control antibody Secukinumab at the same doses, and the effect at a concentration of 1 nM is equivalent to the effect of the control antibody Secukinumab;

the effect of the 2G2 H2L2 antibody on inhibiting IL-6 secretion at concentrations of 1 nM and 100 nM is equivalent to that of the control antibody Secukinumab;

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

The above results indicate that in an in vitro mixed culture reaction of human embryonic fibroblasts, the biological activity of the antibodies of the present invention in blocking IL-17A-mediated IL-6 secretion is superior to or at least equivalent to that of the commercially available drug Secukinumab.

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Claims (24)

Example 11. Effect of a drug based on the 13E9 H3L2 antibody on the thickness of the epidermis in a model of psoriasis in C57BL/6 mice. C57BL/6 mice were divided into 5 groups of 8 mice in each group. 1. A monoclonal antibody or an antigen-binding fragment thereof that binds to IL17A, wherein the heavy chain variable region of the monoclonal antibody comprises HCDR1-HCDR3 with the amino acid sequences of SEQ ID NO: 31-33, respectively; and the light chain variable region of the monoclonal antibody comprises LCDR1-LCDR3 with the amino acid sequences of SEQ ID NO: 34-36, respectively. (1) Simulation: normal group: C57BL/6 mice were intradermally injected with saline into the smooth skin area on the back for 6 consecutive days (days 1 to 6) at 25 μl/mouse; the remaining groups of mice were intradermally injected with recombinant human IL-17A from days 1 to 4 at 2 μg/25 μl/mouse, and recombinant human IL-17A was intradermally injected from days 5 to 6 at 5 μg/25 μl/mouse. 2. The monoclonal antibody or antigen binding fragment thereof according to claim 1, wherein 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 light chain variable region selected from SEQ ID NO: 4, SEQ ID NO: 8 and SEQ ID NO: 12. (2) Division into groups and introduction: normal group: saline solution, dose 0 mg/kg 3 times a week, 3 times in total; model group: negative isotype control, dose of 50 mg/kg 3 times a week, 3 times in total; secukinumab group: Secukinumab is administered at a dose of 50 mg/kg 3 times a week, 3 times in total; high dose group 13E9 H3L2: administration of a dose of 50 mg/kg 3 times a week, 3 times in total; low dose group 13E9 H3L2: administration of a dose of 10 mg/kg 3 times a week, 3 times in total. In each group, subcutaneous administration was performed 3 times, namely 1 day before the simulation, on the 3rd day of simulation and on the 6th day of simulation, respectively. On the 7th day, the skin at the injection sites on the back of the mice was fixed, pathological sections were subsequently obtained, and the thickness of the epidermis was measured. The experimental results are shown in Fig. 19. The results show that statistically, the epidermal thickness in the secukinumab group (50 mg/kg), high dose 13E9 H3L2 group (50 mg/kg) and low dose 13E9 H3L2 group (10 mg/kg) was significantly less than that of the model group ( P<0.01). According to the results obtained, the 13E9 H3L2 antibody (50 mg/kg) has a statistically significant inhibitory effect on epidermal thickness in the psoriasis model in C57BL/6 mice and is as effective as secukinumab (50 mg/kg); the inhibitory effect of 13E9 H3L2 (10 mg/kg) on epidermal thickness is also statistically significant. Although specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and substitutions are possible in accordance with the disclosure, all of which will be within the scope of the present invention. The entire scope of the present invention is defined by the appended claims and any equivalents thereof. CLAIM 3. A monoclonal antibody or an antigen-binding fragment thereof according to claim 1 or 2, wherein the heavy chain variable region contains the amino acid sequence of SEQ ID NO: 2 and the light chain variable region contains the amino acid sequence of SEQ ID NO: 4. 4. A monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the heavy chain variable region contains the amino acid sequence of SEQ ID NO: 6 and the light chain variable region contains the amino acid sequence of SEQ ID NO: 8. 5. A monoclonal antibody or an antigen-binding fragment thereof according to claim 1 or 2, wherein the heavy chain variable region contains the amino acid sequence of SEQ ID NO: 10 and the light chain variable region contains the amino acid sequence of SEQ ID NO: 12. 6. A monoclonal antibody or an antigen-binding fragment thereof according to claim 1 or 2, wherein the heavy chain variable region contains the amino acid sequence of SEQ ID NO: 14, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 12. 7. A monoclonal antibody or an antigen-binding fragment thereof according to any one of claims 1 to 6, where the monoclonal antibody or an antigen-binding fragment thereof is selected from Fab, Fab', F(ab') ₂ , Fd, Fv, dAb, single chain antibody, humanized antibody, chimeric antibody and diabody. 8. A monoclonal antibody or antigen binding fragment thereof according to any one of claims 1 to 7, wherein the monoclonal antibody binds to IL-17A with an EC ₅₀ of less than about 100 nM. - 24 046350 9. A monoclonal antibody or antigen binding fragment thereof according to any one of claims 1 to 8, wherein the monoclonal antibody comprises a non-CDR region derived from a non-mouse species. 10. A monoclonal antibody or an antigen-binding fragment thereof according to any one of claims 1 to 9, where the monoclonal antibody is a monoclonal antibody produced by the hybridoma cell line LT006 deposited in the China Type Culture Collection Center (CCCC) under CCCC accession number: C2017102. 11. An isolated nucleic acid molecule comprising a nucleic acid encoding a heavy chain variable region and a light chain variable region of a monoclonal antibody or an antigen binding fragment thereof according to any one of claims 1 to 9. 12. The isolated nucleic acid molecule according to claim 11, wherein the nucleic acid encoding the heavy chain variable region contains the sequence of SEQ ID NO: 1 and the nucleic acid encoding the light chain variable region contains SEQ ID NO: 3. 13. The isolated nucleic acid molecule according to claim 11, wherein the nucleic acid encoding the heavy chain variable region contains the sequence of SEQ ID NO: 5 and the nucleic acid encoding the light chain variable region contains the sequence SEQ ID NO: 7. 14. The isolated nucleic acid molecule according to claim 11, wherein the nucleic acid encoding the heavy chain variable region contains the sequence of SEQ ID NO: 9 and the nucleic acid encoding the light chain variable region contains the sequence SEQ ID NO: 11. 15. The isolated nucleic acid molecule according to claim 11, wherein the nucleic acid encoding the heavy chain variable region contains the sequence of SEQ ID NO: 13 and the nucleic acid encoding the heavy chain variable region contains the sequence SEQ ID NO: 11. 16. A recombinant vector containing an isolated nucleic acid molecule according to any one of claims 11-15. 17. A host cell containing the recombinant vector according to claim 16. 18. A method for producing a monoclonal antibody or an antigen-binding fragment thereof according to any one of claims 1 to 9, comprising culturing a host cell according to claim 17 under appropriate conditions and isolating the monoclonal antibody or an antigen-binding fragment thereof from the cell culture. 19. A conjugate containing a monoclonal antibody or an antigen-binding fragment thereof according to any one of claims 1 to 9 and a detectable label. 20. A set of reagents for quantitative and qualitative detection of IL-17A, containing a monoclonal antibody or its antigen-binding fragment according to any one of claims 1-9. 21. A pharmaceutical composition containing a monoclonal antibody or an antigen-binding fragment thereof according to any one of claims 1 to 9 and at least one pharmaceutically acceptable carrier or excipient. 22. Use of a monoclonal antibody or an antigen-binding fragment thereof according to any one of claims 1 to 9 for the preparation of a medicament for treating an autoimmune disease in a patient, wherein the monoclonal antibody or an antigen-binding fragment thereof is intended to be administered to the patient in at least an effective amount. 23. Use according to claim 22, wherein the autoimmune disease is selected from psoriasis, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis and systemic lupus erythematosus. 24. Use according to claim 22, wherein the autoimmune disease is moderate to severe plaque psoriasis. -