WO2019228266A1 - 抗白细胞介素-17a抗体、其药物组合物及其用途 - Google Patents

抗白细胞介素-17a抗体、其药物组合物及其用途 Download PDF

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WO2019228266A1
WO2019228266A1 PCT/CN2019/088344 CN2019088344W WO2019228266A1 WO 2019228266 A1 WO2019228266 A1 WO 2019228266A1 CN 2019088344 W CN2019088344 W CN 2019088344W WO 2019228266 A1 WO2019228266 A1 WO 2019228266A1
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seq
monoclonal antibody
variable region
antigen
amino acid
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PCT/CN2019/088344
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English (en)
French (fr)
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李百勇
夏瑜
王忠民
张鹏
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中山康方生物医药有限公司
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Priority to CA3099530A priority Critical patent/CA3099530A1/en
Priority to KR1020207037874A priority patent/KR20210018336A/ko
Priority to MX2020012847A priority patent/MX2020012847A/es
Priority to US17/055,252 priority patent/US20210122815A1/en
Priority to SG11202011262YA priority patent/SG11202011262YA/en
Priority to JP2020566731A priority patent/JP7538721B2/ja
Priority to IL279015A priority patent/IL279015B2/en
Priority to EP19811445.6A priority patent/EP3805264A4/en
Application filed by 中山康方生物医药有限公司 filed Critical 中山康方生物医药有限公司
Priority to CN201980036614.6A priority patent/CN112424224B/zh
Priority to BR112020024512-7A priority patent/BR112020024512A2/pt
Priority to AU2019276486A priority patent/AU2019276486A1/en
Priority to EA202092844A priority patent/EA202092844A1/ru
Publication of WO2019228266A1 publication Critical patent/WO2019228266A1/zh
Priority to PH12020551978A priority patent/PH12020551978A1/en

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Definitions

  • the invention belongs to the field of molecular immunology, and relates to an anti-interleukin-17A antibody, a pharmaceutical composition thereof and use thereof. Specifically, the present invention relates to a monoclonal antibody against interleukin-17A.
  • Interleukin-17A is a member of the IL-17 cytokine family. There are 6 members in this family, namely IL-17A (IL-17A was first discovered and also (Referred to as IL-17), IL-17B, IL-17C, IL-17D, IL-17E (also named IL-25), and IL-17F (Shi Peiqing et al., Chinese Journal of Cell Biology 33: 345-357 ( 2011)).
  • IL-17F has about 50% homology with IL-17A, and its coding gene is located in the same segment of the chromosome 6p12 (Gaffen et al., Nat Rev Immunol 9: 556-67 (2009)).
  • IL-17A and IL-17F can exist as homodimers such as IL-17A / IL-17A and IL-17F / IL-17F, and can also be IL-17A / IL-17F.
  • IL-17A and IL-17F exert biological effects through binding to receptors (Wright et al., J. Immunol 181: 2799-805 (2008)).
  • the IL-17 receptor (IL-17R) family consists of five members, namely IL-17RA, IL-17RB, IL-17RC, IL-17RD, and IL-17RE.
  • the members of the IL-17 receptor family can be combined into different receptor complexes.
  • IL-17RA the largest molecule found in this family to date, is a universal subunit that transmits signals to at least four ligands. Main biological effects (Gaffen et al., Nat Rev Immunol 9: 556-67 (2009)).
  • the IL-17RA and IL-17RC complex mediates the cell's response to IL-17A and IL-17F (Toy et al., J. Immunol 177: 36-9 (2006)).
  • IL-17A is more critical than IL-17F in the autoimmune inflammatory response.
  • the key reason is that IL17RA has a hundred times greater affinity for IL-17A than IL-17F (Ely et al., Nat Immunol 101245-51 (2009)). Cells The response to IL-17A is 10 times stronger than its response to IL-17F (Dubin et al. Immunity 30: 9-11 (2009)).
  • Using an antibody against IL-17A or an antibody against IL-17 receptor to block the binding of IL-17A to its receptor can block the biological effects of IL-17A.
  • IL-17A plays an important role in several autoimmune diseases (such as psoriasis, psoriatic arthritis, rheumatoid arthritis, ankylosing spondylitis, systemic lupus erythematosus, etc.).
  • the anti-IL-17A monoclonal antibody Secukinumab is approved by the FDA and the European Union for the treatment of moderate to severe plaque psoriasis, psoriatic arthritis, and ankylosing spondylitis.
  • Psoriasis also known as psoriasis, is a chronic autoimmune skin disease.
  • the histological characteristics of the skin are epidermal keratinocyte hyperproliferation, angiogenesis, and dendritic cells, macrophages, neutrophils, and T cell infiltration. (Nestle et al., Engl J Med, 361: 496-509 (2009)).
  • Psoriasis disease manifests in various ways. Among them, plaque psoriasis is the most common type, accounting for more than 90% of all patients with psoriasis.
  • Psoriatic arthritis (PsA) is a special type of psoriasis.
  • psoriasis rash causes pain, swelling, tenderness, stiffness, and dyskinesia in the joints and surrounding soft tissues.
  • Some patients may have ⁇ ⁇ Arthritis and (or) spondylitis, prolonged course, easy to relapse, late-stage joint stiffness, leading to disability.
  • the presence of psoriasis is an important difference between psoriatic arthritis and other inflammatory joint diseases, and the severity of skin lesions is not directly related to the degree of arthritis (Tan Zhen et al., Chinese Journal of Rheumatology 20 :: 354-357 ( 2016)).
  • IL-17A The expression of IL-17A is significantly increased in psoriatic lesion skin tissues, and this increase is closely related to psoriasis disease activity (Johansen et al., Brit J Dermatol, 160: 319-24 (2009); Lowes et al. J Invest Dermatol 128: 1207-11 (2008)).
  • the anti-IL-17A monoclonal antibody Secukinumab has shown excellent efficacy, which can significantly alleviate the disease activity of patients with psoriasis and reduce the area of psoriasis plaques (Langley et al., N.
  • Secukinumab can also significantly reduce arthritis symptoms and significantly improve joint function in patients with psoriatic arthritis (Gott Kunststoff et al., J. Drugs Dermatol, 14823-33 (2015)) .
  • Secukinumab is approved by the FDA for the treatment of moderate to severe plaque psoriasis and psoriatic arthritis.
  • rheumatoid arthritis rheumatoid arthritis
  • RA rheumatoid arthritis
  • IL-17A can cause both inflammation and bone damage in rheumatoid arthritis.
  • IL-17A is highly expressed in rheumatoid synovial monocytes in patients with rheumatoid arthritis (Sarkar et al., Clin Exp Immunol. 177: 652-61 (2014)), cells
  • IL-17A can stimulate bone resorption and collagen destruction (Kitami et al.
  • IL-17A can induce cartilage, synovial cells, macrophages and osteoblasts to secrete and exert pro-inflammatory cytokines such as TNFa, IL-1b and IL-6. These pro-inflammatory factors cause sudden onset of rheumatoid arthritis and can pass IL-17A-induced IL-6 maintains the number of TH17 cells, thereby forming a positive feedback to play a synergistic role to amplify its inflammatory effects, establishing a chronic inflammatory state (Ogura et al., Immunity 29: 628-36 (2008)). Antagonizing IL-17A can effectively alleviate rheumatoid arthritis symptoms.
  • IL-17A In a mouse model of collagen-induced arthritis, neutralizing IL-17A or its receptors can resolve the symptoms of rheumatoid arthritis (Chao et al., Autoimmunity. 2011 May; 44 (3): 243-52); IL- 17 defects protect host mice from collagen-induced arthritis (Nakae et al., J. Immunol, 171: 6173-7 (2003)), while overexpression of IL-17A can aggravate the condition (Lubberts et al., Inflamm Res 51: 102-4 (2002)).
  • Ankylosing spondylitis is a chronic autoimmune disease.
  • the early pathological features of AS are acute or chronic inflammation at the bone attachment points of sacroiliac joints, tendons, and ligaments, and later progress to intervertebral disc inflammation and small arthritis; and all patients with AS have bone mineral density decline.
  • Studies have shown that the number of Th17 cells and the concentration of IL-17A that secrete IL-17A in peripheral blood of AS patients are significantly higher than those of healthy people (Gracey et al., Arthritis Rheumatol. 68: 679-89. (2016)).
  • IL-17A can activate a variety of cells such as macrophages, dendritic cells, endothelial cells, fibroblasts, chondrocytes, and osteoblasts. These cells will generate a large number of inflammatory destructive factors (Ogura et al., Immunity 29: 628-36 (2008)).
  • IL-17A induces osteoblasts to express nuclear factor ⁇ b receptor activating factor ligand (RANKL), activates osteoclasts, induces bone resorption, accumulates bone loss cumulatively, and directly or indirectly leads to bone mass Destruction (Gaffen, Curr Rheumatol Rep 11: 365-370 (2009)).
  • RNKL nuclear factor ⁇ b receptor activating factor ligand
  • Secukinumab In AS patients, the anti-IL-17A monoclonal antibody Secukinumab has shown excellent efficacy, which can significantly reduce the symptoms and signs of ankylosing spondylitis (Baeten et al., N. Engl. J Med, 373: 2534-48 (2015) ). Based on this, Secukinumab is approved by the FDA for the treatment of ankylosing spondylitis.
  • Systemic lupus erythematosus (Systemic lupus erythematosus) is an autoimmune disease that affects multiple systems. Antibodies against autoantigens appear in patients' bodies, which directly or indirectly attack various tissues or organs in the body. The most commonly affected sites include Skin, joints and kidneys. Studies have shown that IL-17A plays a role in SLE. The proportion of IL-17A-producing cells in peripheral blood of patients with SLE is increased, and the level of IL-17A in patients' serum is abnormally high (Chen et al., J. Clin Immunol, 30: 221-5 (2010)).
  • IL-17A can cooperate with BAFF (B-cell activation factor) to protect B cells from apoptosis, thereby increasing the number of cells producing autoantibodies (Onishi et al., Immunology 129: 311-21 (2010)).
  • the inventors After intensive research and creative labor, the inventors used mammalian cell expression systems to express recombinant IL-17A (24-155) as antigens to immunize mice. A large number of hybrids were obtained by fusion of mouse spleen cells and myeloma cells. Tumor cell sample. The inventors obtained the following two hybridoma cell lines by screening a large number of samples:
  • Hybridoma cell line LT006 (IL17A-13E9), which was deposited at the China Type Culture Collection (CCTCC) on September 12, 2017 under CCTCC NO: C2017102;
  • hybridoma cell line LT007 (IL17A-2G2), which was deposited at the China Type Culture Collection (CCTCC) on September 12, 2017 under CCTCC NO: C2017165.
  • the hybridoma cell line LT006 can secrete and produce a specific monoclonal antibody (named 13E9) that specifically binds to IL-17A, and the 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 the monoclonal antibody can very effectively block the binding of IL-17A to IL-17RA;
  • 2G2 a specific monoclonal antibody
  • the inventors creatively prepared humanized antibodies against IL-17A (named 13E9H1L1, 13E9, H2L2, 13E9, H3L2; and 2G2, H1L1, 2G2, H2L2, 2G2, H3L3), which can effectively bind human IL -17A, blocking the binding of IL-17A to the IL-17A receptor and inhibiting the activation of downstream signaling pathways of the IL-17A receptor;
  • the antibody of the present invention has the potential for preparing medicines for preventing and treating autoimmune diseases such as psoriasis, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, and systemic lupus erythematosus.
  • One aspect of the invention relates to a monoclonal antibody or an antigen-binding fragment thereof, wherein:
  • the heavy chain variable region (VH) of the monoclonal antibody includes: HCDR1-HCDR3 with amino acid sequences as shown in SEQ ID NOs: 31-33 or HCDR1- with amino acid sequences as shown in SEQ ID NOs: 37-39, respectively HCDR3, and
  • the light chain variable region (VL) of the monoclonal antibody includes: LCDR1-LCDR3 with amino acid sequences shown in SEQ ID NOs: 34-36 or LCDR1-with amino acid sequences shown in SEQ ID NOs: 40-42, respectively LCDR3.
  • variable regions of the light and heavy chains determine the binding of the antigen; the variable region of each chain contains three hypervariable regions, called complementarity determining regions (CDRs) (the CDRs of the heavy chain (H) include HCDR1, HCDR2, HCDR3
  • CDRs complementarity determining regions
  • the CDR of the light chain (L) contains LCDR1, LCDR2, LCDR3; it is named by Kabat et al., See Sequences of Proteins of Immunological Interface, Fifth Edition (1991), Volumes 1-3, NIH Public Publication 91-3242, Bethesda Md ).
  • amino acid sequence of the CDR region of the monoclonal antibody sequence in (1) to (2) above is analyzed by technical means well known to those skilled in the art, for example, through the VBASE2 database:
  • the antibodies 13E9, 13E9, H1L1, 13E9, H2L2, and 13E9 H3L2 in the present invention have the same CDRs:
  • amino acid sequences of the three CDR regions of the heavy chain variable region are as follows:
  • HCDR1 SYSFTSDYA (SEQ ID NO: 31),
  • HCDR2 ITYSGVT (SEQ ID NO: 32),
  • HCDR3 ARADYDSYYTMDY (SEQ ID NO: 33);
  • 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).
  • the antibodies 2G2, 2G2, H1L1, 2G2, H2L2, 2G2 and H3L3 of the present invention have the same CDRs:
  • 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);
  • 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).
  • the monoclonal antibody or antigen-binding fragment thereof wherein,
  • the heavy chain variable region (VH) of the monoclonal antibody comprises: HCDR1-HCDR3, the amino acid sequences of which are shown in SEQ ID NOs: 31-33, and
  • the light chain variable region (VL) of the monoclonal antibody includes: LCDR1-LCDR3 with amino acid sequences as shown in SEQ ID NOs: 34-36, respectively.
  • the monoclonal antibody or antigen-binding fragment thereof wherein,
  • the heavy chain variable region (VH) of the monoclonal antibody comprises: HCDR1-HCDR3, the amino acid sequences of which are shown in SEQ ID NOs: 37-39, and
  • the light chain variable region (VL) of the monoclonal antibody includes: LCDR1-LCDR3 with amino acid sequences shown in SEQ ID NOs: 40-42, respectively.
  • the monoclonal antibody or antigen-binding fragment thereof wherein,
  • amino acid sequence of the variable region of the heavy chain 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,
  • amino acid sequence of the light chain variable region is selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 18, SEQ ID NO: 22, SEQ ID NO: 26, and SEQ ID NO: 30.
  • the monoclonal antibody or antigen-binding fragment thereof wherein,
  • amino acid sequence of the heavy chain variable region is selected from the group consisting of SEQ ID NO: 2, SEQ ID: NO: 6, SEQ ID: NO: 10, 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, SEQ ID NO: 12;
  • the amino acid sequence of the heavy chain variable region is selected from SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 24, and SEQ ID NO: 28, and the amino acid sequence of the light chain variable region is selected from SEQ ID NO: 18, SEQ ID NO: 22, SEQ ID NO: 26, and SEQ ID NO: 30.
  • the monoclonal antibody or antigen-binding fragment thereof, wherein the heavy chain variable region and light chain variable region are selected from the following (1)-(8) Any of:
  • a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 4;
  • a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 8;
  • a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 12;
  • a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 12;
  • a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 18;
  • a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 22;
  • a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 26;
  • the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 30.
  • the monoclonal antibody or antigen-binding fragment thereof wherein the monoclonal antibody or antigen-binding fragment thereof is selected from the group consisting of Fab, Fab ', F (ab') 2 , Fd, Fv, dAb, complementarity determining region fragment, single chain antibody, humanized antibody, chimeric antibody or diabody.
  • the monoclonal antibody or antigen-binding fragment thereof wherein the monoclonal antibody is less than about 100 nM, such as less than about 10 nM, 5 nM, 4 nM, 3 nM,
  • An EC 50 of 2.5 nM, 2 nM or less binds to the IL-17A protein; preferably, the EC 50 is measured by a competitive ELISA method.
  • the monoclonal antibody or antigen-binding fragment thereof wherein the monoclonal antibody is less than about 10 -5 M, such as less than about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or less K D binding protein IL-17A; preferably, the K D measured by meter obtained Fortebio molecular interactions.
  • the monoclonal antibody or antigen-binding fragment thereof wherein the monoclonal antibody is less than about 100 nM, for example, less than about 10 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6nM, 0.5nM, 0.4nM, 0.3nM, 0.2nM, 0.1nM EC 50 or less IL-17A binding protein; in particular, the EC 50 measured by indirect ELISA.
  • the monoclonal antibody includes a non-CDR region, and the non-CDR region is from a species that is not a murine, such as from a human antibody.
  • the constant region of the immunoglobulin is humanized.
  • the constant region of the heavy chain uses Ig gamma-1, the chain region, ACCESSION: P01857; the constant region of the light chain uses Ig. kappa chain C region, ACCESSION: P01834.
  • the monoclonal antibody or antigen-binding fragment thereof wherein:
  • the monoclonal antibody is a monoclonal antibody produced by a hybridoma cell line LT006, which is deposited with the China Type Culture Collection (CCTCC) under the accession number CCTCC NO: C2017102; or
  • the monoclonal antibody is a monoclonal antibody produced by a hybridoma cell line LT007, and the hybridoma cell line LT007 is deposited with the China Type Culture Collection (CCTCC) under the accession number CCTCC NO: C2017165.
  • CTCC China Type Culture Collection
  • the monoclonal antibody or antigen-binding fragment thereof is used to prevent and / or treat tumors or autoimmune diseases, or to diagnose autoimmune diseases; preferably
  • the autoimmune disease is selected from the group consisting of psoriasis, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, and systemic lupus erythematosus; preferably, the psoriasis is moderate to severe plaque Sexual psoriasis.
  • the monoclonal antibody or antigen-binding fragment thereof is used for:
  • Modulate eg, down-regulate
  • IL-17A activity or levels e.g., down-regulate
  • Another aspect of the invention relates to an isolated nucleic acid molecule comprising a nucleic acid sequence encoding a heavy chain variable region and a light chain variable region of a monoclonal antibody according to any one of the present invention.
  • the isolated nucleic acid molecule comprises a nucleic acid sequence selected from any one of (1) to (8) as follows:
  • SEQ ID NO: 1 SEQ ID NO: 3;
  • a recombinant vector comprising the isolated nucleic acid molecule of the present invention; preferably, the recombinant vector is a recombinant expression vector, such as a recombinant prokaryotic expression vector or a recombinant eukaryotic expression vector.
  • a further aspect of the invention relates to a host cell comprising a recombinant vector of the invention.
  • Yet another aspect of the present invention relates to a method for preparing the monoclonal antibody or antigen-binding fragment thereof according to any one of the present invention, which comprises culturing the host cell of the present invention under appropriate conditions, and from the cell culture. A step of recovering the monoclonal antibody or an antigen-binding fragment thereof.
  • hybridoma cell line selected from:
  • hybridoma cell line LT006 is deposited with the China Type Culture Collection (CCTCC) under the accession number CCTCC NO: C2017102; or
  • the hybridoma cell line LT007 was deposited at the China Type Culture Collection (CCTCC) under the accession number CCTCC NO: C2017165.
  • a further aspect of the present invention relates to a conjugate, comprising a monoclonal antibody or an antigen-binding fragment thereof and a coupling portion, wherein the monoclonal antibody is the monoclonal antibody or the monoclonal antibody according to any one of the present invention or
  • the coupling moiety is a detectable label; preferably, the detectable label is a radioisotope, a luminescent substance such as a fluorescent substance, a colored substance, or an enzyme.
  • a further aspect of the present invention relates to a kit comprising the monoclonal antibody or the antigen-binding fragment thereof according to any one of the present invention, or the conjugate of the present invention;
  • the kit further includes a second antibody that specifically recognizes the monoclonal antibody or an antigen-binding fragment thereof; optionally, the second antibody further includes a detectable label; preferably, the second antibody Detectable labels are radioisotopes, luminescent substances such as fluorescent substances, colored substances or enzymes.
  • Another aspect of the present invention relates to the use of the monoclonal antibody or antigen-binding fragment thereof according to any one of the present invention or the conjugate of the present invention in the preparation of a kit for the characterization of IL-17A Detection or quantitative detection.
  • the qualitative detection refers to detecting the presence of IL-17A in the test sample
  • the quantitative detection refers to detecting the concentration or content of IL-17A in the test sample.
  • Yet another aspect of the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the monoclonal antibody or antigen-binding fragment thereof according to any one of the present invention or the conjugate of the present invention; optionally, the pharmaceutical composition further comprises: Including pharmaceutically acceptable carriers or excipients.
  • Another aspect of the present invention relates to the use of the monoclonal antibody or the antigen-binding fragment thereof according to any one of the present invention or the conjugate of the present invention in the preparation of a medicament for preventing and / or treating a tumor or an autoimmune disease, Or use in the preparation of a medicament for diagnosing an autoimmune disease; preferably, the autoimmune disease is selected from the group consisting of psoriasis, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, and systemic erythema Lupus; Preferably, the psoriasis is moderate to severe plaque psoriasis.
  • the inventors found through animal experiments (Example 11) that 13E9H3L2 can effectively inhibit the increase in the thickness of epidermis in a C57BL / 6 mouse psoriasis model, as shown by the administration of the antibody drug 13E9H3L2 to significantly inhibit IL-17A and subcutaneously.
  • the increase in epidermal thickness of mice caused by injection is comparable to that of Secukinumab monoclonal antibody that has been marketed for the same target.
  • the autoimmune disease e.g., psoriasis such as moderate to severe plaque psoriasis, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis or system Lupus erythematosus
  • psoriasis such as moderate to severe plaque psoriasis, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis or system Lupus erythematosus
  • An increase in the expression level mentioned here means that the expression level is higher than that of a normal person and has reached a pathogenic level.
  • Another aspect of the present invention relates to the use of the monoclonal antibody or the antigen-binding fragment thereof according to any one of the present invention or the conjugate of the present invention in the preparation of the following drugs:
  • Drugs that modulate e.g., down-regulate the activity or level of IL-17A, or
  • Yet another aspect of the present invention relates to a method in vivo or in vitro, comprising administering to a cell or a subject in need thereof an effective amount of the monoclonal antibody or antigen-binding fragment thereof according to any one of the present invention or the subject Steps of the inventive conjugate, said method being selected from the following:
  • a method of modulating e.g., down-regulating the activity or level of IL-17A, or
  • the in vitro method is non-therapeutic and / or non-diagnostic.
  • Interleukin-6 (abbreviated to IL-6 or IL-6) can be used by fibroblasts, monocytes / macrophages, T lymphocytes, B lymphocytes, epithelial cells, keratinocytes, and various tumor cells Produced. Interleukin 6 is an important factor regulating immune response. IL-6 and IL-1 can synergistically promote T cell proliferation, promote B cell differentiation, and participate in the body's inflammatory response (Schoenborn et al. Advances Immunology 96: 41-101 ( 2007)). The in vitro experiment (Example 10) of the present invention shows that the anti-IL-17A antibody can significantly reduce the secretion of IL-6 and inhibit the IL-6-mediated immune response.
  • Yet another aspect of the present invention relates to a method for treating and / or preventing tumors or autoimmune diseases, comprising administering to a subject in need thereof an effective amount of the monoclonal antibody or the antigen thereof according to any one of the present invention.
  • a step of binding a fragment or a conjugate of the present invention preferably, the autoimmune disease is selected from the group consisting of psoriasis, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, systemic lupus erythematosus; preferably Preferably, the psoriasis is moderate to severe plaque psoriasis.
  • Yet another aspect of the present invention relates to a method for diagnosing an autoimmune disease, which comprises applying a test sample (such as a tissue sample, a cell sample, or a blood sample) or administering to a subject in need thereof an effective amount of any of the invention Step of the monoclonal antibody or antigen-binding fragment thereof or the conjugate of the present invention; preferably, the autoimmune disease is selected from the group consisting of psoriasis, rheumatoid arthritis, psoriatic arthritis, tonicity Spondylitis, systemic lupus erythematosus; preferably, the psoriasis is moderate to severe plaque psoriasis.
  • a test sample such as a tissue sample, a cell sample, or a blood sample
  • the autoimmune disease is selected from the group consisting of psoriasis, rheumatoid arthritis, psoriatic arthritis, tonicity Spondylitis, systemic lupus ery
  • an autoimmune disease e.g., psoriasis such as moderate to severe plaque psoriasis, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis or system is considered Lupus erythematosus
  • psoriasis e.g., psoriasis such as moderate to severe plaque psoriasis, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis or system is considered Lupus erythematosus
  • IL-17A e.g., psoriasis such as moderate to severe plaque psoriasis, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis or system is considered Lupus erythematosus
  • IL-17A e.g., psoriasis such as moderate to severe plaque psoriasis, rheum
  • the amino acid sequence of the IL-17A protein when referring to the amino acid sequence of the IL-17A protein (interleukin-17A), it includes the full length of the IL-17A protein; it also includes fusion proteins of IL-17A, such as with mouse or human IgG The Fc protein fragment (mFc or hFc) is fused.
  • mFc or hFc The Fc protein fragment
  • IL-17A protein shall include all such sequences, including natural or artificial variants thereof.
  • a sequence fragment of the IL-17A protein when describing a sequence fragment of the IL-17A protein, it also includes the corresponding sequence fragment in its natural or artificial variant.
  • the full-length sequence of IL-17A (155aa) is as follows, where the signal peptide sequence (23aa) is underlined.
  • the IL-17R is IL-17RA; its specific protein sequence is a sequence known in the prior art, and reference may be made to the sequences disclosed in the existing literature or GenBank.
  • IL-17RA CD217, NCBI Gene ID: NP_055154.3.
  • EC 50 refers to the concentration for 50% of maximal effect, and refers to the concentration that can cause 50% of the maximum effect.
  • the term “antibody” refers to an immunoglobulin molecule that generally consists of two pairs of polypeptide chains, each pair having one "light” (L) chain and one "heavy” (H) chain. .
  • the heavy chain can be understood as a polypeptide chain with a larger molecular weight in an antibody
  • the light chain refers to a polypeptide chain with a lower molecular weight in an antibody.
  • Light chains can be classified as kappa and lambda light chains.
  • Heavy chains can generally be classified as ⁇ , ⁇ , ⁇ , ⁇ , or ⁇ , and the isotype of an antibody is defined as IgM, IgD, IgG, IgA, and IgE, respectively.
  • variable and constant regions are linked by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids.
  • Each heavy chain consists of a heavy chain variable region (V H ) and a heavy chain constant region (C H ).
  • the heavy chain constant region is comprised of three domains (C H 1, C H 2 and C H 3) components.
  • Each light chain is comprised of a light chain variable region (V L) and a light chain constant region (C L) components.
  • the light chain constant region is comprised of one domain, C L composition.
  • the constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (C1q) of the classical complement system.
  • the V H and V L regions can also be subdivided into regions with high denaturation (referred to as complementarity determining regions (CDRs)) with a more conservative region called a framework region (FR) interspersed between them.
  • CDRs complementarity determining regions
  • FR framework region
  • Each V H and V L is composed of 3 CDRs and 4 FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from the amino terminal to the carboxy terminal.
  • variable regions of each heavy / light chain pair form the antibody binding site.
  • the assignment of amino acids to various regions or domains follows 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) Definition of Nature 342: 878-883.
  • the heavy chain may also contain more than 3 CDRs, such as 6, 9, or 12.
  • the heavy chain may be a ScFv having the C-terminus of the heavy chain of an IgG antibody connected to another antibody, in which case the heavy chain contains 9 CDRs.
  • antibody is not limited by any particular method of producing antibodies. For example, it includes, in particular, recombinant antibodies, monoclonal antibodies, and polyclonal antibodies.
  • the antibodies may be antibodies of different isotypes, for example, IgG (eg, IgG1, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM antibodies.
  • the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind the same antigen to which the full-length antibody binds, and / or competes with a full-length antibody Specific binding to an antigen is also referred to as the "antigen-binding moiety".
  • antigen-binding fragment of the antibody is produced either by enzymatic or chemical cleavage of the intact antibody.
  • the antigen-binding fragment includes Fab, Fab ', F (ab') 2 , Fd, Fv, dAb, and complementarity determining regions (CDRs) Fragments, single chain antibodies (e.g., scFv), chimeric antibodies, diabody, and polypeptides comprising at least a portion of an antibody sufficient to confer a polypeptide-specific antigen-binding ability.
  • CDRs complementarity determining regions
  • the term “Fd fragment” means an antibody fragment composed of V H and C H 1 domains;
  • the term “Fv fragment” means an antibody composed of V L and V H domains of one arm of an antibody Antibody fragments;
  • the term “dAb fragment” means an antibody fragment consisting of a V H domain (Ward et al., Nature 341: 544-546 (1989));
  • the term “Fab fragment” means a combination of V L , V H , C Antibody fragment consisting of L and C H 1 domains;
  • the term “F (ab ') 2 fragment” means an antibody fragment comprising two Fab fragments connected by a disulfide bridge on the hinge region.
  • the antigen-binding fragment of an antibody is a single chain antibody (e.g., scFv), where the V L and V H domains form a monovalent molecule by pairing them to enable the production of a linker for a single polypeptide chain (see, for example, Bird Et al. Science 242: 423-426 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879-5883 (1988)).
  • scFv molecules may have a general structure: NH 2 -V L -linker-V H -COOH or NH 2 -V H -linker-V L -COOH.
  • Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof.
  • a linker having an amino acid sequence (GGGGS) 4 may be used, but a variant thereof may 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 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.
  • the antigen-binding fragment of an antibody is a diabody, that is, a bivalent antibody, in which the V H and V L domains are expressed on a single polypeptide chain, but using a linker that is too short to allow two Between two domains, forcing the domain to pair with the complementary domain of another chain and creating two antigen-binding sites (see, eg, Holliger P. et al., Proc. Natl. Acad. Sci. USA 90: 6444 -6448 (1993), and Poljak RJ et al., Structure 2: 1121-1123 (1994)).
  • Antigen-binding fragments e.g., antibody fragments described above
  • Antigen-binding fragments of an antibody can be obtained from a given antibody using conventional techniques known to those skilled in the art (e.g., recombinant DNA technology or enzymatic or chemical fragmentation methods), and The manner of the antibody is the same way that the antigen-binding fragment of the antibody is specifically screened.
  • antibody As used herein, unless the context clearly indicates otherwise, when referring to the term “antibody”, it includes not only whole antibodies but also antigen-binding fragments of antibodies.
  • the terms "monoclonal antibody” and “monoclonal antibody” refer to an antibody or a fragment of an antibody from a group of highly homologous antibody molecules, that is, in addition to natural mutations that may occur spontaneously, A group of identical antibody molecules.
  • a monoclonal antibody is highly specific for a single epitope on an antigen.
  • Polyclonal antibodies are relative to monoclonal antibodies, and they usually contain at least two or more different antibodies that usually recognize different epitopes on the antigen.
  • Monoclonal antibodies can usually be obtained using hybridoma technology first reported by Kohler et al. (Nature, 256: 495, 1975), but can also be obtained using recombinant DNA technology (see, for example, U.S.P. 4,816,567).
  • chimeric antibody refers to an antibody whose light or / and heavy chain portion is derived from an antibody (which may be derived from a particular species or belong to a particular antibody class or Subclass), and another part of the light or / and heavy chain is derived from another antibody (which may be of the same or different species or belong to the same or different antibody class or subclass), but in any case, it remains Binding activity to target antigen (USP 4,816,567 to Cabilly et al .; Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851 6685 (1984)).
  • humanized antibody means that all or part of a CDR region of a human immunoglobulin (receptor antibody) is replaced by a CDR region of a non-human antibody (donor antibody).
  • the antibody or antibody fragment thereof, wherein the donor antibody may be a non-human (eg, mouse, rat, or rabbit) antibody having a desired specificity, affinity, or reactivity.
  • some amino acid residues of the framework region (FR) of the receptor antibody can also be replaced by the amino acid residues of the corresponding non-human antibody, or by the amino acid residues of other antibodies to further improve or optimize the performance of the antibody.
  • epitope refers to a site on an antigen that is specifically bound by an immunoglobulin or antibody.
  • An "epitope” is also known in the art as an "antigenic determinant".
  • An epitope or antigenic determinant usually consists of a chemically active surface group of a molecule such as an amino acid or a carbohydrate or a sugar side chain and usually has specific three-dimensional structural characteristics and specific charge characteristics.
  • epitopes typically include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or non-contiguous amino acids in a unique spatial conformation, which can be "linear "" Or “conformational.” See, for example, Epitope Mapping Protocols Methods Molecular Biology, Vol.
  • isolated refers to obtained from a natural state by artificial means. If a certain "isolated” substance or component appears in nature, it may be that the natural environment in which it is located has been changed, or the substance has been separated from the natural environment, or both. For example, a non-isolated polynucleotide or polypeptide naturally exists in a living animal, and the same high-purity identical polynucleotide or polypeptide isolated from this natural state is called isolation. of.
  • isolated or “isolated” does not exclude the mixing of artificial or synthetic substances, nor does it exclude the presence of other impurities that do not affect the activity of the substance.
  • E. coli expression system refers to an expression system composed of E. coli (strain) and a vector, wherein E. coli (strain) is derived from a commercially available strain, such as, but not limited to: GI698 , ER2566, BL21 (DE3), B834 (DE3), BLR (DE3).
  • vector refers to a nucleic acid vehicle into which a polynucleotide can be inserted.
  • a vector When a vector enables expression of a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector.
  • a vector can be introduced into a host cell by transformation, transduction, or transfection, so that the genetic material elements carried by the vector can be expressed in the host cell.
  • Vectors are well known to those skilled in the art and include, but are not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1 derived artificial chromosomes (PAC) ; Bacteriophage such as lambda phage or M13 phage and animal viruses.
  • 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 nipples Polyoma vacuole virus (such as SV40).
  • retroviruses including lentiviruses
  • adenoviruses adeno-associated viruses
  • herpes viruses such as herpes simplex virus
  • poxviruses such as herpes simplex virus
  • baculoviruses baculoviruses
  • papillomaviruses papillomaviruses
  • Polyoma vacuole virus such as SV40
  • a vector may contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences,
  • the term "host cell” refers to a cell that can be used to introduce a vector, which includes, but is not limited to, prokaryotic cells such as E. coli or subtilis, fungal cells such as yeast cells or Aspergillus, Insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblast cells, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells or human cells.
  • prokaryotic cells such as E. coli or subtilis
  • fungal cells such as yeast cells or Aspergillus
  • Insect cells such as S2 Drosophila cells or Sf9
  • animal cells such as fibroblast cells, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells or human cells.
  • K D refers to the dissociation equilibrium constant of a particular antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen.
  • antibodies have a dissociation equilibrium constant (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, or 10 -10 M or less.
  • the dissociation equilibrium constant can be measured, for example, using a Fortebio molecular interaction instrument.
  • amino acids are generally represented by single-letter and three-letter abbreviations known in the art.
  • alanine can be represented by A or Ala.
  • hybridoma and LT007 are used interchangeably, and when referring to the terms “hybridoma” and “hybridoma cell line”, they also include subclones of the hybridoma And offspring cells. For example, when referring to the hybridoma cell line LT006 or LT007, it also refers to subclones and progeny cells of the hybridoma cell line LT006 or LT007.
  • the term "pharmaceutically acceptable carrier and / or excipient” refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, It is well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th Ed. Pennsylvania: Mack Publishing Company, 1995), and includes but is not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancement Agent.
  • pH adjusting agents include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic, or non-ionic surfactants, such as Tween-80; and ionic strength enhancers include, but are not limited to, sodium chloride.
  • adjuvant refers to a non-specific immune enhancer that, when delivered into the body with or in advance of an antigen, can enhance the body's immune response to the antigen or change the type of immune response.
  • adjuvants include but not limited to aluminum adjuvants (such as aluminum hydroxide), Freund's adjuvants (such as complete Freund's adjuvant and incomplete Freund's adjuvant), Corynebacterium pumilus, lipopolysaccharide, cytokines, etc. .
  • Freund's adjuvant is currently the most commonly used adjuvant in animal experiments.
  • Aluminum hydroxide adjuvant is used more in clinical trials.
  • an effective amount refers to an amount sufficient to obtain or at least partially obtain a desired effect.
  • an effective amount for preventing a disease e.g., a disease associated with IL-17A binding to the IL-17A receptor or a disease associated with excessive IL-17A activity such as an autoimmune disease
  • an effective amount for preventing a disease is an amount sufficient to prevent, prevent, or delay a disease (e.g., IL-17A Diseases that bind to the IL-17A receptor or are associated with excessive IL-17A activity (such as autoimmune diseases)
  • the therapeutically effective amount refers to diseases that are sufficient to cure or at least partially prevent patients who already have the disease and The amount of its complications. It is well within the ability of those skilled in the art to determine such an effective amount.
  • the amount effective for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the general condition of the patient such as age, weight and sex, how the drug is administered, and other treatments administered concurrently and many more.
  • Monoclonal antibodies of the present invention such as 13E9, H1L1, 13E9, H2L2, 13E9, H3L2, and 2G2, H1L1, 2G2, H2L2, 2G2, H3L3 can bind specifically to IL-17A, and can effectively block IL- The binding of 17A to the IL-17A ligand specifically decreased the IL-6 secretion in IL-17A-mediated fibroblasts;
  • the monoclonal antibody of the present invention has the same effect on inhibiting the secretion of IL-6 as the effect of the same target drug, Secukinumab;
  • the monoclonal antibodies of the present invention have the potential to be used in the treatment and / or prevention of anti-autoimmune diseases such as anti-psoriasis, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis or systemic lupus erythematosus .
  • Figure 1 SDS-PAGE results of the monoclonal murine antibody 13E9.
  • the samples from the four lanes from left to right and their loading amounts are: non-reducing protein electrophoresis loading buffer sample antibody, 1 ⁇ g; reduced protein electrophoresis loading buffer sample antibody, 1 ⁇ g; Marker, 5 ⁇ l; BSA , 1 ⁇ g.
  • Figure 2 SDS-PAGE results of the monoclonal murine antibody 2G2.
  • the samples from the four lanes from left to right and their loading amounts are: non-reducing protein electrophoresis loading buffer sample antibody, 1 ⁇ g; reduced protein electrophoresis loading buffer sample antibody, 1 ⁇ g; Marker, 5 ⁇ l; BSA , 1 ⁇ g.
  • Figure 3 SDS-PAGE results of monoclonal humanized antibody 13E9 and H3L2.
  • the samples from the three lanes from left to right and their loading amounts are: BSA, 1 ⁇ g; Marker, 5 ⁇ l; reduced protein electrophoresis loading buffer sample antibody, 1 ⁇ g; non-reduced protein electrophoresis loading buffer sample antibody , 1 ⁇ g.
  • Figure 4 Detection results of kinetic characteristic parameters of antibody 13E9 and H1L1.
  • the ordinate is the signal value, the unit is nm; the abscissa is the time, the unit is sec.
  • Figure 5 Detection results of kinetic characteristic parameters of antibody 13E9 and H2L2.
  • the ordinate is the signal value, the unit is nm; the abscissa is the time, the unit is sec.
  • Figure 6 Detection results of kinetic characteristic parameters of antibody 13E9 and H3L2.
  • the ordinate is the signal value, the unit is nm; the abscissa is the time, the unit is sec.
  • Figure 7 Detection results of kinetic characteristic parameters of antibody Secukinumab.
  • the ordinate is the signal value, the unit is nm; the abscissa is the time, the unit is sec.
  • Figure 8 Detection results of kinetic characteristic parameters of antibody 2G2 and H1L1.
  • the ordinate is the signal value, the unit is nm; the abscissa is the time, the unit is sec.
  • Figure 9 Detection results of kinetic characteristic parameters of antibody 2G2 and H2L2.
  • the ordinate is the signal value, the unit is nm; the abscissa is the time, the unit is sec.
  • Figure 10 Detection results of kinetic characteristic parameters of antibody 2G2 and H3L3.
  • the ordinate is the signal value, the unit is nm; the abscissa is the time, the unit is sec.
  • Figure 11 Detection results of kinetic characteristic parameters of antibody Secukinumab.
  • the ordinate is the signal value, the unit is nm; the abscissa is the time, the unit is sec.
  • Figure 12 Detection of the binding activity of antibodies 13E9, H1L1, 13E9, and H2L2 to the antigen IL17A-His using an indirect ELISA method.
  • the marketed drug Secukinumab is used as a positive control.
  • Figure 13 Detection of the binding activity of antibody 13E9 and H3L2 to the antigen IL17A-His by indirect ELISA.
  • the marketed drug Secukinumab is used as a positive control.
  • Figure 14 The competitive binding activity of antibodies 13E9, H1L1, 13E9, and H2L2 with the receptor IL17RA-His (biotin) was detected using a competitive ELISA method.
  • the marketed drug Secukinumab is used as a positive control.
  • Figure 15 The competitive binding activity of antibody 13E9 and H3L2 with the receptor IL17RA-His (biotin) was detected by a competitive ELISA method.
  • the marketed drug Secukinumab is used as a positive control.
  • Figure 16 Detection of the binding activity of antibodies 2G2, H1L1, 2G2, H2L2, and 2G2, H3L3 to the antigen IL17A-His by indirect ELISA.
  • the marketed drug Secukinumab is used as a positive control.
  • Figure 17 Detection of antibody 2G2, H1L1, 2G2, H2L2, and 2G2 H3L3 competition with the receptor IL17RA-His (biotin) for the activity of binding to the antigen IL17A-His using a competitive ELISA method.
  • the marketed drug Secukinumab is used as a positive control.
  • Figure 18 Effects of antibodies 13E9, H1L1, 13E9, H2L2, and 13E9, H3L2, and 2G2. H1L1, 2G2, H2L2, and 2G2 H3L3 on the secretion of mixed lymphocyte cytokine IL-6.
  • Figure 19 Effect of antibody 13E9 H3L2 on epidermal thickness of a psoriasis model in C57BL / 6 mice.
  • Hybridoma cell line LT006 (IL17A-13E9), which was deposited at the China Type Culture Collection (CCTCC) on September 12, 2017, with the accession number CCTCC NO: C2017102, and its deposit address is Wuhan, Wuhan University, China. : 430072.
  • Hybridoma cell line LT007 (IL17A-2G2), which was deposited at the China Type Culture Collection (CCTCC) on September 12, 2017, with the accession number CCTCC NO: C2017165, and its deposit address is Wuhan, Wuhan University, China. : 430072.
  • the BALB / C mice used were purchased from Guangdong Medical Laboratory Animal Center; the C57BL / 6 mice used were from Nanjing Yinhe Biomedical Co., Ltd .; the MRC5 cells used were from Shanghai Fudan IBS cell resources Center; Secukinumab used Monoclonal antibodies were purchased from Novartis.
  • the antigen IL17A (24-155) -his used to prepare the anti-IL-17A antibody is a fusion protein of human IL-17A (GenBank ID: Q16552) mature peptide and his tag.
  • Spleen cells from immunized BALB / C mice (purchased from Guangdong Medical Laboratory Animal Center) and mouse myeloma cells were fused to hybridoma cells.
  • specific operations refer to established methods (for example, Stewart, SJ, "Monoclonal Antibody Production” ", Basic Methods, Characterization, Characterization, Eds. GC Howard and DR Bethell, Boca Raton: CRC Press, 2000).
  • the fusion protein IL17A-his was digested with TEV protease and purified by column to obtain IL-17A (24-155) protein.
  • the IL-17A (24-155) protein was used as an antigen-coated enzyme plate and screened by indirect ELISA to obtain hybridoma cells that secreted new antibodies that specifically bound to IL-17A (24-155).
  • hybridoma cell lines capable of secreting monoclonal antibodies that compete with the receptor IL-17RA (CD217, NCBI Gene ID: NP_055154.3) to bind to IL-17A were screened by competitive ELISA, and Two stable hybridoma cell lines were obtained by limiting dilution.
  • Hybridoma cell line LT006 (IL17A-13E9), which was deposited at the China Type Culture Collection (CCTCC) on September 12, 2015, with the accession number CCTCC NO: C2017102, and its deposit address is Wuhan, Wuhan University, China. : 430072.
  • the secreted monoclonal antibody was named 13E9.
  • Hybridoma cell line LT007 (IL17A-2G2), which was deposited at the China Type Culture Collection (CCTCC) on September 12, 2017, with the accession number CCTCC NO: C2017165, and its deposit address is Wuhan, Wuhan University, China. : 430072.
  • the secreted monoclonal antibody was named 2G2.
  • Hybridoma serum-free medium of the above-prepared LT006 cell line (1x10 5 cells per well) were cultured (Hybridoma serum-free medium containing 1% penicillin-streptomycin, 4% Glutamax, in 5% CO 2 , cultured in a 37 ° C cell incubator), cultured for 7 days until the survival rate is about 20%, the cell culture supernatant is collected, purified by high-speed centrifugation, vacuum filtration with a microporous filter, and a HiTrap protein A HP column. Obtained antibody 13E9. The purified 13E9 samples were subjected to SDS-PAGE electrophoresis detection. The results are shown in Figure 1.
  • Hybridoma serum-free medium of the above-prepared LT007 cell line (1x10 5 cells per well) were cultured (Hybridoma serum-free medium containing 1% penicillin-streptomycin, 4% Glutamax, in 5% CO 2 , cultured in a 37 ° C cell incubator), cultured for 7 days to a survival rate of about 20%, the cell culture supernatant was collected, purified by high-speed centrifugation, vacuum filtration through a microporous membrane, and a HiTrap protein A HP column. Obtained antibody 2G2. The purified 2G2 sample was subjected to SDS-PAGE electrophoresis detection, and the results are shown in FIG. 2.
  • LT006 cells were cultured according to the method in step 2 of Example 1.
  • mRNA was extracted from the cultured LT006 cells according to the method of the kit instructions.
  • the PCR amplification products were directly TA cloned.
  • pEASY-T1 Cloning Kit Transgen CT101
  • the TA cloned products were directly sequenced.
  • the sequencing results are as follows:
  • the underlined area is the CDR area.
  • Example 3 Design, preparation and detection of anti-IL-17A humanized antibodies 13E9H1L1, 13E9H2L2 and 13E9 H3L2
  • the antibody model was simulated by computer, and mutations were designed according to the model to obtain antibodies 13E9H1L1, 13E9H2L2 and 13E9 variable region sequence of H3L2 (antibody constant region sequence, from the database of NCBI, heavy chain constant region is Ig gamma-1 chain region, ACCESSION: P01857, light chain constant region is Ig kappa chain region, ACCESSION: P01834) .
  • the designed variable region sequence is as follows:
  • the underlined area is the CDR area.
  • the underlined area is the CDR area.
  • the underlined area is the CDR area.
  • the underlined area is the CDR area.
  • the underlined area is the CDR area.
  • the nucleic acid sequence of the light chain variable region is the same as the nucleic acid sequence of the light chain variable region of 13E9H2L2, as shown in SEQ ID NO: 11.
  • the heavy chain constant region uses Ig gamma-1 chain region, ACCESSION: P01857; the light chain constant region uses Ig chain region, ACCESSION: P01834.
  • 13E9H1L1 heavy chain cDNA and light chain cDNA, 13E9H2L2 heavy chain cDNA and light chain cDNA, and 13E9 H3L2 heavy chain cDNA and light chain cDNA were cloned into pUC57simple (provided by Kingsray) vectors, respectively, to obtain pUC57simple-13E9H1, pUC57simple-13E9L1, pUC57simple-13E9H2, pUC57simple-13E9L2, and pUC57simple-13E9H3, and the corresponding heavy chain fragment and the corresponding light chain fragment were subcloned into the pcDNA3.1 vector to obtain pcDNA3.1 -13E9H1, pcDNA3.1-13E9L1, pcDNA3.1-13E9H2, pcDNA3.1-13E9L2, pcDNA3.1-13E9H3 and pcDNA3.1-13E9L2 recombinant plasmids.
  • LT007 cells were cultured according to the method in step 3 of Example 1.
  • mRNA was extracted from the cultured LT007 cells according to the method of the kit instructions.
  • the PCR amplification products were directly TA cloned.
  • pEASY-T1 Cloning Kit Transgen CT101
  • the TA cloned products were directly sequenced.
  • the sequencing results are as follows:
  • the underlined area is the CDR area.
  • the underlined area is the CDR area.
  • Example 5 Design of anti-IL-17A humanized antibodies 2G2, H1L1, 2G2, H2L2, and 2G2, H3L3, Preparation and testing
  • the underlined area is the CDR area.
  • the underlined area is the CDR area.
  • the underlined area is the CDR area.
  • the underlined area is the CDR area.
  • the underlined area is the CDR area.
  • the underlined area is the CDR area.
  • the heavy chain constant region uses Ig gamma-1 chain C region, ACCESSION: P01857; the light chain constant region uses Ig kappa chain C region, ACCESSION: P01834.
  • 2G2 H1L1 heavy chain cDNA and light chain cDNA, 2G2 H2L2 heavy chain cDNA and light chain cDNA, and 2G2 H3L3 heavy chain cDNA and light chain cDNA were cloned into pUC57simple (provided by Kingsray), respectively.
  • nucleotide fragments containing the corresponding heavy chain and the nucleotide fragments containing the corresponding light chain were subcloned into the pcDNA3.1 vector to obtain the recombinant plasmids pcDNA3.1-2G2H1, pcDNA3.1-2G2L1, and pcDNA3. 1-2G2H2, pcDNA3.1-2G2L2, pcDNA3.1-2G2H3 and pcDNA3.1-2G2L3.
  • the corresponding light chain recombination plasmid is then combined with the heavy chain recombination plasmid (pcDNA3.1-2G2H1 and pcDNA3.1-2G2L1; pcDNA3.1-2G2H2 and pcDNA3.1-2G2L2; pcDNA3.1-2G2H3 and pcDNA3.1-2G2L3 )
  • the culture fluid was collected and purified to obtain humanized antibodies 2G2, H1L1, 2G2, H2L2, and 2G2, H3L3.
  • the purified samples were detected by SDS-PAGE electrophoresis.
  • Example 6 13E9, H1L1, 13E9, H2L2, and 13E9, H3L2 bound to the antigen IL-17A (24-155) protein Determination of kinetic parameters
  • the kinetic parameters of binding of humanized antibodies 13E9, H1L1, 13E9, H2L2, 13E9, and H3L2 to the antigen IL-17A were determined using a Fortebio molecular interaction instrument.
  • EDC / NHS was used to activate the AR2G sensor, and the antibody was fixed to the activated AR2G sensor by amino coupling.
  • the sensor was blocked with 1M ethanolamine and pH 8.5. After the sensor was equilibrated in PBST for 300s, the antibody immobilized on the sensor bound to the antigen IL-17A (24-155) protein (same as the antigen used in Example 1), the antigen concentration was 6.25-400 nM (double gradient dilution), The binding time was 420s, and the antigen-antibody was dissociated in PBST for 600 s .
  • the kinetic parameters of antibodies 13E9, H1L1, 13E9, H2L2, 13E9, H3L2, and Secukinumab are shown in Table 1.
  • the results of the detection of the kinetic characteristic parameters are shown in Figures 4, 5, 6, and 7, respectively.
  • Example 7 Humanized antibodies 2G2, H1L1, 2G2, H2L2, 2G2, H3L3, and antigen IL-17A (24-155) Determination of bound kinetic parameters
  • the kinetic parameters of binding of humanized antibodies 2G2, H1L1, 2G2, H2L2, 2G2, and H3L3 to the antigen IL-17A were determined using a Fortebio molecular interaction instrument.
  • EDC / NHS was used to activate the AR2G sensor, and the antibody was fixed to the activated AR2G sensor by amino coupling.
  • the sensor was blocked with 1M ethanolamine and pH 8.5.
  • the antibody immobilized on the sensor binds to the antigen IL-17A (24-155) with an antigen concentration of 6.25-400nM (double gradient dilution) and a binding time of 420s.
  • the antigen-antibody is resolved in PBST Off, time 600s.
  • the kinetic parameters of antibodies 2G2, H1L1, 2G2, H2L2, 2G2, H3L3, and Secukinumab are shown in Table 2.
  • the results of the detection of kinetic characteristic parameters are shown in Figures 8, 9, 10, and 11, respectively.
  • the indirect ELISA method was used to detect the binding activity of antibodies 13E9, H1L1, 13E9, H2L2, and 13E9, H3L2 to the antigen IL17A-His, and compared with the drug Secukinmab that has been marketed with the same target.
  • IL17A-his can be prepared by referring to the published sequence and conventional technical means in the art, or refer to the following steps:
  • IL17A-his The protein sequence of human IL-17A was searched through the NCBI protein database, and the full-length human IL-17A sequence and His * 6 purified tag sequence were fused and designed. Nanjing Kingsray was entrusted to biosynthesize the nucleic acid encoding the fusion protein. According to the standard technology introduced in the Molecular Cloning Experiment Guide (Second Edition), PCR, enzyme digestion, gel recovery, ligation transformation, colony PCR or enzyme digestion identification were used. Standard molecular cloning technology will subclon the target gene into mammalian cell expression vector, and further sequence and analyze the target gene of the recombinant expression vector.
  • a large number of endotoxin-free expression plasmids were prepared and the plasmids were transiently transfected into HEK293 cells for protein expression. After 7 days of culture, the cell culture fluid was collected and affinity purified using a Ni Sepharose column (GE). The harvested protein samples were qualified using SDS-PAGE and SEC-HPLC standard analysis techniques, and the results were acceptable.
  • ELISA Add IL17A-His to the enzyme plate, incubate at 4 ° C overnight, block with 1% BSA in PBS at 37 ° C for 2h, add antibodies, incubate at 37 ° C for 30min, add Goat Anti Human IgG (H + L), HRP (Jackson, 109-035-088) was incubated at 37 ° C for 30 min, and a color reaction was performed with TMB (Neogen, 308177) for 5 min, and the absorbance at 450 nm was detected in a microplate reader. The obtained experimental data was analyzed and processed with SoftMax Pro 6.2.1 software. The 4-parameter fitted curve was plotted and analyzed with the antibody concentration as the abscissa and the absorbance value as the ordinate.
  • a competitive ELISA method was used to detect the activity of antibodies 13E9, H1L1, 13E9, H2L2, and 13E9 H3L2 to block the binding of IL17RA-His (biotin) to the antigen IL17A-His.
  • IL17RA-His biotin
  • IL17RA-His biotin
  • the protein sequence of human IL-17RA was searched through the NCBI protein database, and the human IL-17RA extracellular region sequence and His * 6 purified tag sequence were fused and designed. Nanjing Kingsray was commissioned to biosynthesize the nucleic acid encoding the fusion protein, using the standard techniques introduced in the Molecular Cloning Experiment Guide (Second Edition), using PCR, enzyme digestion, gel recovery, ligation transformation, colony PCR or enzyme digestion identification, etc. Standard molecular cloning technology will subclon the target gene into mammalian cell expression vector, and further sequence and analyze the target gene of the recombinant expression vector.
  • ELISA Add IL17A-His to the enzyme plate, incubate at 4 ° C overnight, block with 1% BSA in PBST at 37 ° C for 2h, then add antibodies, react with the antigen antibody at room temperature for 10 minutes, and then add the receptor IL17RA-His (biotin) After mixing with the antibody volume 1: 1, incubate at 37 ° C for 30min, add SA-HRP (KPL, 14-30-00) and incubate at 37 ° C for 30min, and perform color reaction with TMB (Neogen, 308177) for 5min. The absorbance at 450nm was detected in a microplate reader. The obtained experimental data was analyzed and processed with SoftMax Pro 6.2.1 software. The 4-parameter fitted curve was plotted and analyzed with the antibody concentration as the abscissa and the absorbance value as the ordinate. The experimental results are shown in Fig. 14, Fig. 15, and Tables 5 and 6 below.
  • Example 9 ELISA method to detect the binding of antibodies 2G2, H1L1, 2G2, H2L2, and 2G2 to H3L3 and antigen active
  • Indirect ELISA was used to detect the binding activity of antibodies 2G2, H1L1, 2G2, H2L2, and 2G2, H3L3 and the antigen IL17A-His.
  • the experimental results show that the antibodies 2G2, H1L1, 2G2, H2L2, and 2G2, H3L3 can effectively bind the antigen IL17A-His, and the binding efficiency shows a dose-dependent relationship.
  • the competitive ELISA method was used to detect the activity of antibodies 2G2, H1L1, 2G2, H2L2, and 2G2, H3L3 and the receptor IL17RA-His (biotin) for binding to the antigen IL17A-His.
  • the experimental results show that the antibodies 2G2, H1L1, 2G2, H2L2, and 2G2 and H3L3 can effectively block the binding of the receptor IL17RA-His (biotin) to the antigen IL17A-His, and the blocking efficiency is dose-dependent.
  • Example 10 Mixed human embryo fibroblast response: secretion of cytokine IL-6
  • MRC 5 (purchased from the Cell Center of the Chinese Academy of Sciences) cells were seeded into a 96-well plate, 5000 cells / well, and cultured overnight. MRC cells were added with IL-17 and antibody mixture (hIgG as control) incubated at 37 ° C for 20 min, and cultured for 48 hours. After 48 hours of incubation, the cell supernatant was collected, and the amount of IL-6 (purchased from Daktronics) was detected using an ELISA kit.
  • MRC5 cells were added with 13E9, H1L1, 13E9, H2L2, 13E9, H3L2, 2G2, H1L1, 2G2, H2L2, or 2G2, H3L3 (1, 10, 100nM) and Secukinumab (1, 10, 100nM), and the secreted IL-6 test results were as follows: This is shown in Figure 18.
  • 13E9, H1L1, 13E9, H2L2, 13E9, H3L2, and 2G2 can effectively reduce the IL-6 secretion of MRC5 cells induced by IL-17. among them:
  • the 13E9 H1L1 antibody inhibited IL-6 secretion at a concentration of 100 nM better than the control antibody Secukinumab at the same dose, and the inhibitory effect of IL-6 secretion at a concentration of 10 nM was comparable to that of the control antibody Secukinumab at 100 nM;
  • 13E9 H2L2 antibody was equivalent to the same dose of the control antibody Secukinumab in inhibiting IL-6 secretion at concentrations of 1nM, 10nM and 100nM;
  • 2G2 H2L2 was equivalent to the control antibody Secukinumab in inhibiting IL-6 secretion at a concentration of 1nM and 100nM;
  • 2G2 H3L3 inhibited IL-6 secretion at a concentration of 1 nM better than the control antibody Secukinumab at the same dose.
  • the antibody of the present invention has a biological activity that is better than or at least equivalent to that of the marketed drug Secukinumab of the same target.
  • Example 11 Effect of antibody 13E9 H3L2 on epidermal thickness of a psoriasis model in C57BL / 6 mice
  • mice C57BL / 6 mice were divided into 5 groups of 8 mice each.
  • mice In the normal group, the normal back of C57BL / 6 mice was injected intradermally with saline for 25 consecutive days from day 1 to day 6;
  • mice were injected intradermally with recombinant human IL-17A at 1 to 4 days, 2 ⁇ g / 25 ⁇ l / head, and from 5 to 6 days intradermal injection of recombinant human IL-17A at 5 ⁇ g / 25 ⁇ l / head.
  • Normal group normal saline, administered at a dose of 0 mg / kg, 3 times a week for a total of 3 times;
  • Model group negative isotype control, 50mg / kg, 3 times a week;
  • Secukinumab group Secukinumab, 50 mg / kg, 3 times a week; 3 times a week;
  • Each group was administered 3 times, one day before modeling, the third day after modeling, and the sixth day after modeling, subcutaneously.

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Abstract

本发明属于自身免疫疾病治疗和分子免疫学领域，涉及一种抗IL-17A抗体、其药物组合物及其用途。具体地，本发明涉及一种单克隆抗体或其抗原结合片段，其中，所述单克隆抗体的重链可变区包含：氨基酸序列分别如SEQ ID NOs:31－33所示的HCDR1－HCDR3或氨基酸序列分别如SEQ ID NOs:37－39所示的HCDR1－HCDR3，和所述单克隆抗体的轻链可变区包含：氨基酸序列分别如SEQ ID NOs:34－36所示的LCDR1－LCDR3或氨基酸序列分别如SEQ ID NOs:40－42所示的LCDR1－LCDR3。本发明的单克隆抗体能够很好地特异性与IL-17A结合，特异拮抗IL-17A与其配体的结合，抑制IL-17A对成纤维细胞的激活。

Description

抗白细胞介素-17A抗体、其药物组合物及其用途 技术领域
本发明属于分子免疫学领域，涉及一种抗白介素-17A抗体、其药物组合物及其用途。具体地，本发明涉及一种抗白介素-17A的单克隆抗体。
背景技术
白细胞介素-17A(Interleukin 17A，缩写为IL-17A或IL 17A)是IL-17细胞因子家族的成员，该家族共有6个成员即IL-17A(IL-17A为最先发现的，也被称为IL-17)、IL-17B、IL-17C、IL-17D、IL-17E(亦命名为IL-25)和IL-17F(施沛青等人，Chinese Journal of Cell Biology 33:345-357(2011))。IL-17F与IL-17A有约50％同源性，而且其编码基因定位于染色体的同一区段6p12(Gaffen等人，Nat Rev Immunol 9:556-67(2009))。IL-17A和IL-17F可以以IL-17A/IL-17A和IL-17F/IL-17F这样的同源二聚体形式存在，也可以以IL-17A/IL-17F。IL-17A及IL-17F通过与受体结合发挥生物学效应(Wright等人，J Immunol 181:2799-805(2008))。
IL-17受体(IL-17R)家族由五个成员组成，即IL-17RA、IL-17RB、IL-17RC、IL-17RD和IL-17RE。IL-17受体家族成员之间可组合为不同的受体复合物，其中IL-17RA作为这个家族里迄今发现的最大的分子，是至少四个配体的传递信号的通用亚基，发挥了主要的生物学效应(Gaffen等人，Nat Rev Immunol 9:556-67(2009))。IL-17RA与IL-17RC复合体介导细胞对IL-17A与IL-17F的反应(Toy等人，J Immunol 177:36-9(2006))。
IL-17A比IL-17F在自身免疫炎症反应中更为关键，关键的原因为IL17RA对IL-17A的亲和力百倍于IL-17F(Ely等人，Nat Immunol 10 1245-51(2009))，细胞对IL-17A的反应强度10倍于其对IL-17F的反应(Dubin等人，Immunity 30:9-11(2009))。运用抗IL-17A的抗体或者抗IL-17受体的抗体，阻断IL-17A与其受体的结合，可以阻断IL-17A的生物学效应。
IL-17A在几种自身免疫性疾病(例如银屑病、银屑病性关节炎、类风湿关节炎、强直性脊柱炎、系统性红斑狼疮等)中发挥着重要作用。抗IL-17A单克隆抗体Secukinumab被FDA及欧盟批准用于治疗中至重度斑块性银屑病、银屑病性关节炎以及强直性脊柱炎。
银屑病又称为牛皮癣，是慢性自身免疫性皮肤病，皮肤的组织学特征是表皮角质化细胞过度增殖、血管增生以及树突状细胞、巨噬细胞、嗜中性粒细胞、T细胞浸润(Nestle 等人，N Engl J Med，361:496-509(2009))。银屑病疾病表现多样，其中其中斑块型银屑病最为常见的类型，约占所有银屑病发病患者的90％以上。银屑病性关节炎(psoriatic arthritis，PsA)是银屑病的一种特殊类型，具有银屑病皮疹并导致关节和周围软组织疼痛、肿、压痛、僵硬和运动障碍，部分患者可有骶髂关节炎和(或)脊柱炎，病程迁延、易复发、晚期可关节强直，导致残疾。存在银屑病是银屑病性关节炎与其他炎性关节病的重要区别，皮肤病变严重性和关节炎症程度无直接关系(谭震等人，中华风湿病学杂志20：:354-357(2016))。
在银屑病的病变皮肤组织中，IL-17A的表达显著增高，这种增高与银屑病疾病活动密切相关(Johansen等人，Brit J Dermatol,160:319-24(2009)；Lowes等人，J Invest Dermatol 128:1207-11(2008))。在银屑病患者中，抗IL-17A的单克隆抗体Secukinumab展现出了优异的疗效，可显著缓解银屑病患者的疾病活动，减少银屑病斑块的面积(Langley等人，N Engl J Med,371:326-38(2014))；Secukinumab还可以显著改善银屑病性关节炎患者的减轻关节炎症状，显著改善关节功能(Gottlieb等人，J Drugs Dermatol,14821-33(2015))。Secukinumab被FDA批准用于治疗中至重度斑块型银屑病以及银屑病性关节炎。
类风湿性关节炎(rheumatoid arthritis，RA)的主要特征是炎症关节内滑膜成纤维细胞增生、关节和软骨损坏，CD4+辅助性T细胞与产生自身抗体的浆细胞浸润。IL-17A在类风湿性关节炎中既能导致炎症又能造成骨损坏。相对于健康人或骨关节炎患者IL-17A在类风湿性关节炎病人的类风湿样滑膜单核细胞中高表达(Sarkar等人，Clin Exp Immunol.177:652-61(2014))，细胞学研究提示IL-17A可刺激骨吸收和胶原毁坏(Kitami等人Biochimie.92:398-404(2010))。IL-17A可以诱导软骨、滑膜细胞、巨噬细胞和骨细胞分泌TNFa、IL-1b与IL-6等促炎症细胞因子并发挥，这些促炎症因子导致类风湿性关节炎突然发作并能通过IL-17A诱导的IL-6维持TH17细胞数量，从而形成正反馈发挥协同作用放大其炎症效应，确立一种慢性炎症状态(Ogura等人，Immunity 29:628-36(2008))。拮抗IL-17A可有效地缓解类风湿性关节炎症状。在胶原诱导的关节炎小鼠模型中，中和IL-17A或其受体可消退类风湿性关节炎的症状(Chao等人，Autoimmunity.2011May；44(3):243-52)；IL-17缺陷可保护宿主小鼠免受胶原诱导的关节炎的侵害(Nakae等人，J Immunol,171:6173-7(2003))，而IL-17A过表达可加重病情(Lubberts等人，Inflamm Res 51:102-4(2002))。
强直性脊柱炎(Ankylosing spondylitis,AS)是一种慢性自身免疫性疾病。AS的早期病 理特征是骶髂关节和肌腱、韧带的骨附着点处呈急性或慢性炎症，后期则进展为椎间盘炎和椎小关节炎；且AS患者均存在骨密度下降的现象。研究显示，AS患者外周血中分泌IL-17A的Th17细胞数量及IL-17A的浓度均较健康者显著升高(Gracey等人，Arthritis Rheumatol.68:679-89.(2016))。IL-17A能激活巨噬细胞、树突状细胞、内皮细胞、成纤维细胞、软骨细胞以及成骨细胞等多种细胞，这些细胞将生成数量众多的致炎破坏性因子(Ogura等人，Immunity 29:628-36(2008))。在骨组织中，IL-17A诱导成骨细胞表达细胞核因子κb受体活化因子配体(RANKL)，激活破骨细胞，从而诱发骨吸收，累积性地加剧骨丢失，直接或间接地导致骨质破坏(Gaffen,Curr Rheumatol Rep 11:365-370(2009))。在AS患者中，抗IL-17A的单克隆抗体Secukinumab展现出了优异的疗效，可显著降低强直性脊柱炎的症状和体征(Baeten等人，N Engl J Med,373:2534-48(2015))。基于此，Secukinumab被FDA批准用于治疗强直性脊柱炎。
系统性红斑狼疮(system lupus erythematosus，SLE)是一个影响多个系统的自身免疫性疾病，患者的体内出现针对自身抗原的抗体，直接或间接攻击身体各种组织或器官，最常影响的部位包括皮肤、关节及肾脏。研究表明IL-17A在SLE中发挥作用。在SLE病人的外周血中产生IL-17A的细胞比率增加，病人血清中的IL-17A水平也异常高(Chen等人，J Clin Immunol,30:221-5(2010))。罹患SLE伴随肾损害的病人的外周血单核细胞在加入IL-17培养时会产生更多的总IgG、抗dsDNA的IgG及IL-6，这说明IL-17能参与B细胞的激活(Dong等人，Chin Med J(Engl),116:543-8(2003))。近来还发现，IL-17A可以协同BAFF(B-cell activating factor)保护B细胞免于凋亡，从而增加了产生自身抗体的细胞数量(Onishi等人，Immunology 129:311-21(2010))。
发明内容
本发明人经过深入的研究和创造性的劳动，利用哺乳动物细胞表达系统分别表达出重组的IL-17A(24-155)作为抗原免疫小鼠，经小鼠脾脏细胞与骨髓瘤细胞融合获得大量杂交瘤细胞样本。发明人通过进行对大量样本的筛选，分别得到了如下的两个杂交瘤细胞株：
杂交瘤细胞株LT006(IL17A-13E9)，其于2017年9月12日保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO:C2017102；
以及杂交瘤细胞株LT007(IL17A-2G2)，其于2017年9月12保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO:C2017165。
本发明人惊奇地发现：
杂交瘤细胞株LT006能够分泌产生与IL-17A特异性结合的特异性单克隆抗体(命名为13E9)，并且该单克隆抗体能够十分有效地阻断IL-17A与IL-17RA的结合；
杂交瘤细胞株LT007能够分泌产生与IL-17A特异性结合的特异性单克隆抗体(命名为2G2)，并且该单克隆抗体能够十分有效地阻断IL-17A与IL-17RA的结合；
进一步地，本发明人创造性地制得了抗IL-17A的人源化抗体(分别命名为13E9H1L1，13E9 H2L2，13E9 H3L2；以及2G2 H1L1，2G2 H2L2，2G2 H3L3)，其均能有效地结合人IL-17A，阻断IL-17A与IL-17A受体的结合，抑制IL-17A受体下游信号通路的激活；
本发明的抗体具有用于制备防治银屑病，类风湿关节炎，银屑病性关节炎，强直性脊柱炎，系统性红斑狼疮等自身免疫性疾病的药物的潜力。
由此提供了下述发明：
本发明的一个方面涉及单克隆抗体或其抗原结合片段，其中，
所述单克隆抗体的重链可变区(VH)包含：氨基酸序列分别如SEQ ID NOs:31－33所示的HCDR1－HCDR3或氨基酸序列分别如SEQ ID NOs:37－39所示的HCDR1－HCDR3，和
所述单克隆抗体的轻链可变区(VL)包含：氨基酸序列分别如SEQ ID NOs:34－36所示的LCDR1－LCDR3或氨基酸序列分别如SEQ ID NOs:40－42所示的LCDR1－LCDR3。
轻链和重链的可变区决定抗原的结合；每条链的可变区均含有三个高变区，称互补决定区(CDR)(重链(H)的CDR包含HCDR1、HCDR2、HCDR3，轻链(L)的CDR包含LCDR1、LCDR2、LCDR3；其由Kabat等人命名，见Sequences of Proteins of Immunological Interest,Fifth Edition(1991),第1-3卷,NIH Publication 91-3242,Bethesda Md)。
通过本领域技术人员所熟知的技术手段，例如通过VBASE2数据库分析上面的(1)-(2)项中的单克隆抗体序列的CDR区的氨基酸序列：
本发明中的抗体13E9，13E9 H1L1，13E9 H2L2，13E9 H3L2具有相同的CDR：
其重链可变区的3个CDR区的氨基酸序列如下：
HCDR1:SYSFTSDYA(SEQ ID NO:31)，
HCDR2:ITYSGVT(SEQ ID NO:32)，
HCDR3:ARADYDSYYTMDY(SEQ ID NO:33)；
其轻链可变区的3个CDR区的氨基酸序列如下：
LCDR1:QSLVHSNGNTY(SEQ ID NO:34)，
LCDR2:KVS(SEQ ID NO:35)，
LCDR3:SQSTHFWT(SEQ ID NO:36)。
本发明的抗体2G2，2G2 H1L1，2G2 H2L2，2G2 H3L3具有相同的CDR：
其重链可变区的3个CDR区的氨基酸序列如下：
HCDR1:SEVFPIAD(SEQ ID NO:37)，
HCDR2:ILPSFGRT(SEQ ID NO:38)，
HCDR3:ARGNYGFAY(SEQ ID NO:39)；
其轻链可变区的3个CDR区的氨基酸序列如下：
LCDR1:QSLLNSDGKTY(SEQ ID NO:40)，
LCDR2:LVS(SEQ ID NO:41)，
LCDR3:WQGSHFPQT(SEQ ID NO:42)。
在本发明的一个或多个实施方案中，所述的单克隆抗体或其抗原结合片段，其中，
所述单克隆抗体的重链可变区(VH)包含：氨基酸序列分别如SEQ ID NOs:31－33所示的HCDR1－HCDR3，和
所述单克隆抗体的轻链可变区(VL)包含：氨基酸序列分别如SEQ ID NOs:34－36所示的LCDR1－LCDR3。
在本发明的一个或多个实施方案中，所述的单克隆抗体或其抗原结合片段，其中，
所述单克隆抗体的重链可变区(VH)包含：氨基酸序列分别如SEQ ID NOs:37－39所示的HCDR1－HCDR3，和
所述单克隆抗体的轻链可变区(VL)包含：氨基酸序列分别如SEQ ID NOs:40－42所示的LCDR1－LCDR3。
在本发明的一个或多个实施方案中，所述的单克隆抗体或其抗原结合片段，其中，
所述重链可变区的氨基酸序列选自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和SEQ ID NO:28，
并且
所述轻链可变区的氨基酸序列选自SEQ ID NO:4、SEQ ID NO:8、SEQ ID NO:12、SEQ ID NO:18、SEQ ID NO:22、SEQ ID NO:26和SEQ ID NO:30。
在本发明的一个或多个实施方案中，所述的单克隆抗体或其抗原结合片段，其中，
所述重链可变区的氨基酸序列选自SEQ ID NO:2、SEQ ID NO:6、SEQ ID NO:10、SEQ ID NO:14，并且所述轻链可变区的氨基酸序列选自SEQ ID NO:4、SEQ ID NO:8、SEQ ID NO:12；
或者
所述重链可变区的氨基酸序列选自SEQ ID NO:16、SEQ ID NO:20、SEQ ID NO:24和SEQ ID NO:28，并且所述轻链可变区的氨基酸序列选自SEQ ID NO:18、SEQ ID NO:22、SEQ ID NO:26和SEQ ID NO:30。
在本发明的一个或多个实施方案中，所述的单克隆抗体或其抗原结合片段，其中，所述重链可变区和轻链可变区选自如下的(1)-(8)中的任一项：
(1)重链可变区，其包含SEQ ID NO:2所示的氨基酸序列，和
轻链可变区，其包含SEQ ID NO:4所示的氨基酸序列；
(2)重链可变区，其包含SEQ ID NO:6所示的氨基酸序列，和
轻链可变区，其包含SEQ ID NO:8所示的氨基酸序列；
(3)重链可变区，其包含SEQ ID NO:10所示的氨基酸序列，和
轻链可变区，其包含SEQ ID NO:12所示的氨基酸序列；
(4)重链可变区，其包含SEQ ID NO:14所示的氨基酸序列，和
轻链可变区，其包含SEQ ID NO:12所示的氨基酸序列；
(5)重链可变区，其包含SEQ ID NO:16所示的氨基酸序列，和
轻链可变区，其包含SEQ ID NO:18所示的氨基酸序列；
(6)重链可变区，其包含SEQ ID NO:20所示的氨基酸序列，和
轻链可变区，其包含SEQ ID NO:22所示的氨基酸序列；
(7)重链可变区，其包含SEQ ID NO:24所示的氨基酸序列，和
轻链可变区，其包含SEQ ID NO:26所示的氨基酸序列；以及
(8)重链可变区，其包含SEQ ID NO:28所示的氨基酸序列，和
轻链可变区，其包含SEQ ID NO:30所示的氨基酸序列。
在本发明的一个或多个实施方案中，所述的单克隆抗体或其抗原结合片段，其中， 所述单克隆抗体或其抗原结合片段选自Fab、Fab'、F(ab') 2、Fd、Fv、dAb、互补决定区片段、单链抗体、人源化抗体、嵌合抗体或双抗体。
在本发明的一个或多个实施方案中，所述的单克隆抗体或其抗原结合片段，其中，其中，所述的单克隆抗体以小于大约100nM，例如小于大约10nM、5nM、4nM、3nM、2.5nM、2nM或更小的EC 50结合IL-17A蛋白；优选地，所述EC 50通过竞争ELISA方法测得。
在本发明的一些实施方式中，所述的单克隆抗体或其抗原结合片段，其中，所述的单克隆抗体以小于大约10 -5M，例如小于大约10 -6M、10 -7M、10 -8M、10 -9M或10 -10M或更小的K D结合IL-17A蛋白；优选地，所述K D通过Fortebio分子相互作用仪测得。
在本发明的一些实施方式中，所述的单克隆抗体或其抗原结合片段，其中，所述的单克隆抗体以小于大约100nM，例如小于大约10nM、1nM、0.9nM、0.8nM、0.7nM、0.6nM、0.5nM、0.4nM、0.3nM、0.2nM、0.1nM或更小的EC 50结合IL-17A蛋白；具体地，所述EC 50通过间接ELISA方法测得。
在本发明的一个或多个实施方案中，所述的单克隆抗体包括非-CDR区，且所述非-CDR区来自不是鼠类的物种，例如来自人抗体。
在本发明的一些实施方案中，所述免疫球蛋白的恒定区是人源化的，例如，重链恒定区均采用Ig gamma-1 chain C region，ACCESSION:P01857；轻链恒定区均采用Ig kappa chain C region，ACCESSION:P01834。
在本发明的一个或多个实施方案中，所述的单克隆抗体或其抗原结合片段，其中：
所述单克隆抗体是由杂交瘤细胞株LT006产生的单克隆抗体，所述杂交瘤细胞株LT006保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO:C2017102；或者
所述单克隆抗体是由杂交瘤细胞株LT007产生的单克隆抗体，所述杂交瘤细胞株LT007保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO:C2017165。
在本发明的一个或多个实施方案中，所述的单克隆抗体或其抗原结合片段，其用于预防和/或治疗肿瘤或者自身免疫性疾病，或者用于诊断自身免疫性疾病；优选地，所述自身免疫性疾病选自银屑病、类风湿关节炎、银屑病性关节炎、强直性脊柱炎、系统性红斑狼疮；优选地，所述银屑病为中度至重度斑块性银屑病。
在本发明的一个或多个实施方案中，所述的单克隆抗体或其抗原结合片段，其用于：
阻断IL-17A与IL17RA结合，
调节(例如下调)IL-17A活性或水平，或者
抑制细胞中IL-6的表达。
本发明的另一方面涉及一种分离的核酸分子，其包含编码本发明中任一项所述的单克隆抗体的重链可变区和轻链可变区的核酸序列。
在本发明的一个或多个实施方案中，所述的分离的核酸分子，其包含选自如下的(1)-(8)中任一项的核酸序列：
(1)SEQ ID NO:1，SEQ ID NO:3；
(2)SEQ ID NO:5，SEQ ID NO:7；
(3)SEQ ID NO:9，SEQ ID NO:11；
(4)SEQ ID NO:13，SEQ ID NO:11；
(5)SEQ ID NO:15，SEQ ID NO:17；
(6)SEQ ID NO:19，SEQ ID NO:21；
(7)SEQ ID NO:23，SEQ ID NO:25；
(8)SEQ ID NO:27，SEQ ID NO:29。
本发明的再一方面涉及一种重组载体，其包含本发明的分离的核酸分子；优选地，所述重组载体为重组表达载体，例如重组原核表达载体或重组真核表达载体。
本发明的再一方面涉及一种宿主细胞，其包含本发明的重组载体。
本发明的再一方面涉及一种制备本发明中任一项所述的单克隆抗体或其抗原结合片段的方法，其包括在合适的条件下培养本发明的宿主细胞，以及从细胞培养物中回收所述单克隆抗体或其抗原结合片段的步骤。
本发明的再一方面涉及选自如下的杂交瘤细胞株：
杂交瘤细胞株LT006，其保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO:C2017102；或者
杂交瘤细胞株LT007，其保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO:C2017165。
本发明的再一方面涉及一种偶联物，其包括单克隆抗体或其抗原结合片段以及偶联部分，其中，所述单克隆抗体为本发明中任一项所述的单克隆抗体或其抗原结合片段，所述偶联部分为可检测的标记物；优选地，所述可检测的标记物为放射性同位素、发光物质例如荧光物质、有色物质或酶。
本发明的再一方面涉及一种试剂盒，其包括本发明中任一项所述的单克隆抗体或其抗原结合片段，或者包括本发明的偶联物；
优选地，所述试剂盒还包括第二抗体，其特异性识别所述单克隆抗体或其抗原结合片段；任选地，所述第二抗体还包括可检测的标记物；优选地，所述可检测的标记物为放射性同位素、发光物质例如荧光物质、有色物质或酶。
本发明的再一方面涉及本发明中任一项所述的单克隆抗体或其抗原结合片段或者本发明的偶联物在制备试剂盒中的用途，所述试剂盒用于IL-17A的定性检测或定量检测。所述定性检测是指检测待测样本中是否存在IL-17A，所述定量检测是指检测待测样本中的IL-17A的浓度或者含量。
本发明的再一方面涉及一种药物组合物，其包含本发明中任一项所述的单克隆抗体或其抗原结合片段或者本发明的偶联物；可选地，所述药物组合物还包括药学上可接受的载体或赋形剂。
本发明的再一方面涉及本发明中任一项所述的单克隆抗体或其抗原结合片段或者本发明的偶联物在制备预防和/或治疗肿瘤或者自身免疫性疾病的药物中的用途，或者用于制备诊断自身免疫性疾病的药物中的用途；优选地，所述自身免疫性疾病选自银屑病、类风湿关节炎、银屑病性关节炎、强直性脊柱炎、系统性红斑狼疮；优选地，所述银屑病为中度至重度斑块性银屑病。
特别地，本发明人通过动物实验(实施例11)发现，13E9H3L2能够有效地抑制C57BL/6小鼠银屑病模型的表皮厚度的增加，表现为给予抗体药13E9H3L2可显著抑制IL-17A、皮下注射所引起的小鼠表皮厚度的增加，与已上市同靶点药物Secukinumab单抗药效相当。
在本发明的一些实施方式中，所述自身免疫性疾病(例如银屑病如中度至重度斑块 性银屑病、类风湿关节炎、银屑病性关节炎、强直性脊柱炎或系统性红斑狼疮)是由IL-17A表达水平升高所致。这里所述的表达水平升高是指表达水平高于正常人，且达到了致病的水平。
本发明的再一方面涉及本发明中任一项所述的单克隆抗体或其抗原结合片段或者本发明的偶联物在制备如下药物中的用途：
阻断IL-17A与IL-17RA结合的药物，
调节(例如下调)IL-17A活性或水平的药物，或者
抑制细胞中IL-6表达的药物。
本发明的再一方面涉及一种在体内或在体外的方法，包括给予细胞或者有需求的受试者以有效量的本发明中任一项所述的单克隆抗体或其抗原结合片段或者本发明的偶联物的步骤，所述方法选自如下：
阻断IL-17A与IL-17RA结合的方法，
调节(例如下调)IL-17A活性或水平的方法，或者
抑制成纤维细胞中IL-6表达的方法。
在本发明的一个具体的实施方案中，所述在体外的方法是非治疗目的的和/或非诊断目的的。
白细胞介素-6(interleukin 6，缩写为IL-6或IL 6)，可由纤维母细胞、单核/巨噬细胞、T淋巴细胞、B淋巴细胞、上皮细胞、角质细胞、以及多种瘤细胞所产生。白介素6是调控免疫应答的重要因子，IL-6与IL-1一起可协同地促进T细胞增殖，促使B细胞的分化，参与机体炎症反应(Schoenborn等人，Advances in Immunology 96:41-101(2007))。本发明的体外实验(实施例10)显示，抗IL-17A抗体能显著降低IL-6的分泌，抑制IL-6介导的免疫反应。
本发明的再一方面涉及一种治疗和/或预防肿瘤或者自身免疫性疾病的方法，包括给予有需求的受试者以有效量的本发明中任一项所述的单克隆抗体或其抗原结合片段或者本发明的偶联物的步骤；优选地，所述自身免疫性疾病选自银屑病、类风湿关节炎、银屑病性关节炎、强直性脊柱炎、系统性红斑狼疮；优选地，所述银屑病为中度至重度斑块性银屑病。
本发明的再一方面涉及一种诊断自身免疫性疾病的方法，包括施加待测样品(例如组织样品、细胞样品或血液样品)或者给予有需求的受试者以有效量的本发明中任一项 所述的单克隆抗体或其抗原结合片段或者本发明的偶联物的步骤；优选地，所述自身免疫性疾病选自银屑病、类风湿关节炎、银屑病性关节炎、强直性脊柱炎、系统性红斑狼疮；优选地，所述银屑病为中度至重度斑块性银屑病。
在本发明的一些实施方式中，考虑到自身免疫性疾病(例如银屑病如中度至重度斑块性银屑病、类风湿关节炎、银屑病性关节炎、强直性脊柱炎或系统性红斑狼疮)是由IL-17A表达水平升高所致，可以通过发明中任一项所述的单克隆抗体或其抗原结合片段或者本发明的偶联物检测IL-17A表达水平来进行诊断。如果IL-17A表达水平高于正常人，且达到了致病的水平，诊断为阳性结果；反之，诊断为阴性结果。
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的细胞培养、分子遗传学、核酸化学、免疫学实验室操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
如本文中所使用的，当提及IL-17A蛋白(interleukin-17A)的氨基酸序列时，其包括IL-17A蛋白的全长；还包括IL-17A的融合蛋白，例如与小鼠或人IgG的Fc蛋白片段(mFc或hFc)进行融合的片段。然而，本领域技术人员理解，在IL-17A蛋白的氨基酸序列中，可天然产生或人工引入突变或变异(包括但不限于置换，缺失和/或添加)，而不影响其生物学功能。因此，在本发明中，术语“IL-17A蛋白”应包括所有此类序列，包括其天然或人工的变体。并且，当描述IL-17A蛋白的序列片段时，其还包括其天然或人工变体中的相应序列片段。
IL-17A的全长序列(155aa)如下，其中下划线标出的为信号肽序列(23aa)。
Figure PCTCN2019088344-appb-000001
如本文中所使用的，如果没有特别说明，所述IL-17R为IL-17RA；其具体蛋白序列为现有技术中已知序列，可以参考现有文献或者GenBank中公开的序列。例如，IL-17RA(CD217,NCBI Gene ID:NP_055154.3)。
如本文中所使用的，术语EC 50是指半最大效应浓度(concentration for 50％of maximal effect)，是指能引起50％最大效应的浓度。
如本文中所使用的，术语“抗体”是指，是指通常由两对多肽链(每对具有一条“轻”(L)链和一条“重”(H)链)组成的免疫球蛋白分子。从一般意义上，重链可以理解为抗体中分子量较大的多肽链，轻链是指抗体中分子量较小的多肽链。轻链可分类为κ和λ轻链。重链通常可分类为μ、δ、γ、α或ε，并且分别将抗体的同种型定义为IgM、IgD、IgG、IgA和IgE。在轻链和重链内，可变区和恒定区通过大约12或更多个氨基酸的“J”区连接，重链还包含大约3个或更多个氨基酸的“D”区。各重链由重链可变区(V H)和重链恒定区(C H)组成。重链恒定区由3个结构域(C H1、C H2和C H3)组成。各轻链由轻链可变区(V L)和轻链恒定区(C L)组成。轻链恒定区由一个结构域C L组成。抗体的恒定区可介导免疫球蛋白与宿主组织或因子，包括免疫系统的各种细胞(例如，效应细胞)和经典补体系统的第一组分(C1q)的结合。V H和V L区还可被细分为具有高变性的区域(称为互补决定区(CDR))，其间散布有较保守的称为构架区(FR)的区域。各V H和V L由按下列顺序：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4从氨基末端至羧基末端排列的3个CDR和4个FR组成。各重链/轻链对的可变区(V H和V L)分别形成抗体结合部位。氨基酸至各区域或结构域的分配遵循Kabat Sequences of Proteins of Immunological Interest(National Institutes of Health,Bethesda,Md.(1987and 1991))，或Chothia&Lesk(1987)J.Mol.Biol.196:901-917；Chothia等人(1989)Nature 342:878-883的定义。特别地，重链还可以包含3个以上CDR，例如6、9、或12个。例如在本发明的双功能抗体中，重链可以是IgG抗体的重链的C端连接另一个抗体的ScFv，这种情况下重链含有9个CDR。术语“抗体”不受任何特定的产生抗体的方法限制。例如，其包括，特别地，重组抗体、单克隆抗体和多克隆抗体。抗体可以是不同同种型的抗体，例如，IgG(例如，IgG1，IgG2，IgG3或IgG4亚型)，IgA1，IgA2，IgD，IgE或IgM抗体。
如本文中所使用的，术语抗体的“抗原结合片段”是指包含全长抗体的片段的多肽，其保持特异性结合全长抗体所结合的相同抗原的能力，和/或与全长抗体竞争对抗原的特异性结合，其也被称为“抗原结合部分”。通常参见，Fundamental Immunology,Ch.7(Paul,W.,ed.,第2版，Raven Press,N.Y.(1989)，其以其全文通过引用合并入本文，用于所有目的。可通过重组DNA技术或通过完整抗体的酶促或化学断裂产生抗体的抗原结合片段。在一些情况下，抗原结合片段包括Fab、Fab'、F(ab') 2、Fd、Fv、dAb和互补决定区(CDR)片段、单链抗体(例如，scFv)、嵌合抗体、双抗体(diabody)和这样的多肽，其包含足以赋予多肽特异性抗原结合能力的抗体的至少一部分。
如本文中所使用的，术语“Fd片段”意指由V H和C H1结构域组成的抗体片段；术语 “Fv片段”意指由抗体的单臂的V L和V H结构域组成的抗体片段；术语“dAb片段”意指由V H结构域组成的抗体片段(Ward等人，Nature 341:544-546(1989))；术语“Fab片段”意指由V L、V H、C L和C H1结构域组成的抗体片段；术语“F(ab') 2片段”意指包含通过铰链区上的二硫桥连接的两个Fab片段的抗体片段。
在一些情况下，抗体的抗原结合片段是单链抗体(例如，scFv)，其中V L和V H结构域通过使其能够产生为单个多肽链的连接体配对形成单价分子(参见，例如,Bird等人，Science 242:423-426(1988)和Huston等人，Proc.Natl.Acad.Sci.USA 85:5879-5883(1988))。此类scFv分子可具有一般结构：NH 2-V L-接头-V H-COOH或NH 2-V H-接头-V L-COOH。合适的现有技术接头由重复的GGGGS氨基酸序列或其变体组成。例如，可使用具有氨基酸序列(GGGGS) 4的接头，但也可使用其变体(Holliger等人(1993)，Proc.Natl.Acad.Sci.USA 90:6444-6448)。可用于本发明的其他接头由Alfthan等人(1995)，Protein Eng.8:725-731，Choi等人(2001)，Eur.J.Immunol.31:94-106，Hu等人(1996)，Cancer Res.56:3055-3061，Kipriyanov等人(1999)，J.Mol.Biol.293:41-56和Roovers等人(2001)，Cancer Immunol.描述。
在一些情况下，抗体的抗原结合片段是双抗体，即，双价抗体，其中V H和V L结构域在单个多肽链上表达，但使用太短的连接体以致不允许在相同链的两个结构域之间配对，从而迫使结构域与另一条链的互补结构域配对并且产生两个抗原结合部位(参见，例如,Holliger P.等人，Proc.Natl.Acad.Sci.USA 90:6444-6448(1993)，和Poljak R.J.等人，Structure 2:1121-1123(1994))。
可使用本领域技术人员已知的常规技术(例如，重组DNA技术或酶促或化学断裂法)从给定的抗体获得抗体的抗原结合片段(例如，上述抗体片段)，并且以与用于完整抗体的方式相同的方式就特异性筛选抗体的抗原结合片段。
在本文中，除非上下文明确指出，否则当提及术语“抗体”时，其不仅包括完整抗体，而且包括抗体的抗原结合片段。
如本文中所使用的，术语“单抗”和“单克隆抗体”是指，来自一群高度同源的抗体分子中的一个抗体或抗体的一个片断，也即除可能自发出现的自然突变外，一群完全相同的抗体分子。单抗对抗原上的单一表位具有高特异性。多克隆抗体是相对于单克隆抗体而言的，其通常包含至少2种或更多种的不同抗体，这些不同的抗体通常识别抗原上的不同表位。单克隆抗体通常可采用Kohler等首次报道的杂交瘤技术获得(Nature,256:495,1975)，但也可采用重组DNA技术获得(如参见U.S.P 4,816,567)。
如本文中所使用的，术语“嵌合抗体”是指这样的抗体，其轻链或/和重链的一部分源自一个抗体(其可以源自某一特定物种或属于某一特定抗体类或亚类)，且轻链或/和重链的另一部分源自另一个抗体(其可以源自相同或不同的物种或属于相同或不同的抗体类或亚类)，但无论如何，其仍保留对目标抗原的结合活性(U.S.P 4,816,567 to Cabilly et al.；Morrison et al.,Proc.Natl.Acad.Sci.USA,81:6851 6855(1984))。
如本文中所使用的，术语“人源化抗体”是指，人源免疫球蛋白(受体抗体)的全部或部分CDR区被一非人源抗体(供体抗体)的CDR区替换后得到的抗体或抗体片段，其中的供体抗体可以是具有预期特异性、亲和性或反应性的非人源(例如，小鼠、大鼠或兔)抗体。此外，受体抗体的构架区(FR)的一些氨基酸残基也可被相应的非人源抗体的氨基酸残基替换，或被其他抗体的氨基酸残基替换，以进一步完善或优化抗体的性能。关于人源化抗体的更多详细内容，可参见例如，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)；和Clark,Immunol.Today 21:397 402(2000)。
如本文中所使用的，术语“表位”是指，抗原上被免疫球蛋白或抗体特异性结合的部位。“表位”在本领域内也称为“抗原决定簇”。表位或抗原决定簇通常由分子的化学活性表面基团例如氨基酸或碳水化合物或糖侧链组成并且通常具有特定的三维结构特征以及特定的电荷特征。例如，表位通常以独特的空间构象包括至少3，4，5，6，7，8，9，10，11，12，13，14或15个连续或非连续的氨基酸，其可以是“线性的”或“构象的”。参见，例如，Epitope Mapping Protocols in Methods in Molecular Biology，第66卷，G.E.Morris，Ed.(1996)。在线性表位中，蛋白质与相互作用分子(例如抗体)之间的所有相互作用的点沿着蛋白质的一级氨基酸序列线性存在。在构象表位中，相互作用的点跨越彼此分开的蛋白质氨基酸残基而存在。
如本文中所使用的，术语“分离的”或“被分离的”指的是，从天然状态下经人工手段获得的。如果自然界中出现某一种“分离”的物质或成分，那么可能是其所处的天然环境发生了改变，或从天然环境下分离出该物质，或二者情况均有发生。例如，某一活体动物体内天然存在某种未被分离的多聚核苷酸或多肽，而从这种天然状态下分离出来的高纯度的相同的多聚核苷酸或多肽即称之为分离的。术语“分离的”或“被分离的”不排除混有人工或合成的物质，也不排除存在不影响物质活性的其它不纯物质。
如本文中所使用的，术语“大肠杆菌表达系统”是指由大肠杆菌(菌株)与载体组成的表达系统，其中大肠杆菌(菌株)来源于市场上可得到的菌株，例如但不限于：GI698,ER2566, BL21(DE3),B834(DE3),BLR(DE3)。
如本文中所使用的，术语“载体(vector)”是指，可将多聚核苷酸插入其中的一种核酸运载工具。当载体能使插入的多核苷酸编码的蛋白获得表达时，载体称为表达载体。载体可以通过转化，转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的，包括但不限于：质粒；噬菌粒；柯斯质粒；人工染色体，例如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。可用作载体的动物病毒包括但不限于，逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。一种载体可以含有多种控制表达的元件，包括但不限于，启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。
如本文中所使用的，术语“宿主细胞”是指，可用于导入载体的细胞，其包括但不限于，如大肠杆菌或枯草菌等的原核细胞，如酵母细胞或曲霉菌等的真菌细胞，如S2果蝇细胞或Sf9等的昆虫细胞，或者如纤维原细胞，CHO细胞，COS细胞，NSO细胞，HeLa细胞，BHK细胞，HEK 293细胞或人细胞等的动物细胞。
如本文中所使用的，术语“K D”是指特定抗体-抗原相互作用的解离平衡常数，其用于描述抗体与抗原之间的结合亲和力。平衡解离常数越小，抗体-抗原结合越紧密，抗体与抗原之间的亲和力越高。通常，抗体以小于大约10 -5M，例如小于大约10 -6M、10 -7M、10 -8M、10 -9M或10 -10M或更小的解离平衡常数(K D)结合抗原，所述解离平衡常数可以通过，例如，使用Fortebio分子相互作用仪测得。
如本文中所使用的，术语“单克隆抗体”和“单抗”具有相同的含义且可互换使用；术语“多克隆抗体”和“多抗”具有相同的含义且可互换使用；术语“多肽”和“蛋白质”具有相同的含义且可互换使用。并且在本发明中，氨基酸通常用本领域公知的单字母和三字母缩写来表示。例如，丙氨酸可用A或Ala表示。
如本文中所使用的，术语“杂交瘤”和“杂交瘤细胞株”可互换使用，并且当提及术语“杂交瘤”和“杂交瘤细胞株”时，其还包括杂交瘤的亚克隆和后代细胞。例如，当提及杂交瘤细胞株LT006或LT007时，其还指杂交瘤细胞株LT006或LT007的亚克隆和后代细胞。
如本文中所使用的，术语“药学上可接受的载体和/或赋形剂”是指在药理学和/或生理学上与受试者和活性成分相容的载体和/或赋形剂，其是本领域公知的(参见例如 Remington's Pharmaceutical Sciences.Edited by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995)，并且包括但不限于：pH调节剂，表面活性剂，佐剂，离子强度增强剂。例如，pH调节剂包括但不限于磷酸盐缓冲液；表面活性剂包括但不限于阳离子，阴离子或者非离子型表面活性剂，例如Tween-80；离子强度增强剂包括但不限于氯化钠。
如本文中所使用的，术语“佐剂”是指非特异性免疫增强剂，当其与抗原一起或预先递送入机体时，其可增强机体对抗原的免疫应答或改变免疫应答类型。佐剂有很多种，包括但不限于铝佐剂(例如氢氧化铝)、弗氏佐剂(例如完全弗氏佐剂和不完全弗氏佐剂)、短小棒状杆菌、脂多糖、细胞因子等。弗氏佐剂是目前动物试验中最常用的佐剂。氢氧化铝佐剂则在临床实验中使用较多。
如本文中所使用的，术语“有效量”是指足以获得或至少部分获得期望的效果的量。例如，预防疾病(例如IL-17A与IL-17A受体结合或者IL-17A活性过高相关的疾病如自身免疫性疾病)有效量是指，足以预防，阻止，或延迟疾病(例如IL-17A与IL-17A受体结合或者IL-17A活性过高相关的疾病如自身免疫性疾病)的发生的量；治疗疾病有效量是指，足以治愈或至少部分阻止已患有疾病的患者的疾病和其并发症的量。测定这样的有效量完全在本领域技术人员的能力范围之内。例如，对于治疗用途有效的量将取决于待治疗的疾病的严重度、患者自己的免疫系统的总体状态、患者的一般情况例如年龄，体重和性别，药物的施用方式，以及同时施用的其他治疗等等。
发明的有益效果
本发明取得了下述技术效果中的至少一项：
(1)本发明的单克隆抗体例如13E9 H1L1、13E9 H2L2、13E9 H3L2，以及2G2 H1L1、2G2 H2L2、2G2 H3L3均能够很好地特异性与IL-17A结合，并且能够十分有效地阻断IL-17A与IL-17A配体的结合，特异地降低IL-17A介导的成纤维细胞中IL-6分泌；
(2)本发明的单克隆抗体特别是13E9H2L2抑制IL-6分泌的作用与同靶点已上市药物Secukinumab效果一致；
(3)本发明的单克隆抗体具有应用于治疗和/或预防抗自身免疫疾病例如抗银屑病、类风湿关节炎、银屑病性关节炎、强直性脊柱炎或系统性红斑狼疮的潜力。
附图说明
图1：单克隆鼠源抗体13E9的SDS-PAGE检测结果。从左至右的4个泳道的样品及其上样量依次为：非还原型蛋白电泳上样缓冲液样品抗体，1μg；还原型蛋白电泳上样缓冲液样品抗体，1μg；Marker，5μl；BSA，1μg。
图2：单克隆鼠源抗体2G2的SDS-PAGE检测结果。从左至右的4个泳道的样品及其上样量依次为：非还原型蛋白电泳上样缓冲液样品抗体，1μg；还原型蛋白电泳上样缓冲液样品抗体，1μg；Marker，5μl；BSA，1μg。
图3:单克隆人源化抗体13E9 H3L2的SDS-PAGE检测结果。从左至右的3个泳道的样品及其上样量依次为：BSA，1μg；Marker，5μl；还原型蛋白电泳上样缓冲液样品抗体，1μg；非还原型蛋白电泳上样缓冲液样品抗体，1μg。
图4：抗体13E9 H1L1的动力学特征参数检测结果。图中纵坐标为信号值，单位为nm；横坐标为时间，单位为sec。
图5：抗体13E9 H2L2的动力学特征参数检测结果。图中纵坐标为信号值，单位为nm；横坐标为时间，单位为sec。
图6：抗体13E9 H3L2的动力学特征参数检测结果。图中纵坐标为信号值，单位为nm；横坐标为时间，单位为sec。
图7：抗体Secukinumab的动力学特征参数检测结果。图中纵坐标为信号值，单位为nm；横坐标为时间，单位为sec。
图8：抗体2G2 H1L1的动力学特征参数检测结果。图中纵坐标为信号值，单位为nm；横坐标为时间，单位为sec。
图9：抗体2G2 H2L2的动力学特征参数检测结果。图中纵坐标为信号值，单位为nm；横坐标为时间，单位为sec。
图10：抗体2G2 H3L3的动力学特征参数检测结果。图中纵坐标为信号值，单位为nm；横坐标为时间，单位为sec。
图11：抗体Secukinumab的动力学特征参数检测结果。图中纵坐标为信号值，单位为nm；横坐标为时间，单位为sec。
图12：采用间接ELISA方法检测抗体13E9 H1L1、13E9 H2L2与抗原IL17A-His的结合活性。已上市药物Secukinumab作为阳性对照。
图13：采用间接ELISA方法检测抗体13E9 H3L2与抗原IL17A-His的结合活性。已上市药物Secukinumab作为阳性对照。
图14：采用竞争ELISA方法检测抗体13E9 H1L1、13E9 H2L2与受体 IL17RA-His(biotin)竞争结合的活性。已上市药物Secukinumab作为阳性对照。
图15：采用竞争ELISA方法检测抗体13E9 H3L2与受体IL17RA-His(biotin)竞争结合的活性。已上市药物Secukinumab作为阳性对照。
图16：采用间接ELISA方法检测抗体2G2 H1L1、2G2 H2L2和2G2 H3L3与抗原IL17A-His的结合活性。已上市药物Secukinumab作为阳性对照。
图17：采用竞争ELISA方法检测抗体2G2 H1L1、2G2 H2L2和2G2 H3L3与受体IL17RA-His(biotin)竞争结合抗原IL17A-His的活性。已上市药物Secukinumab作为阳性对照。
图18：抗体13E9 H1L1、13E9 H2L2和13E9 H3L2以及2G2 H1L1、2G2 H2L2和2G2 H3L3对混合淋巴细胞细胞因子IL-6分泌的影响。
图19：抗体药13E9 H3L2对C57BL/6小鼠银屑病模型的表皮厚度的影响。
关于生物材料保藏的说明：
杂交瘤细胞株LT006(IL17A-13E9)，其于2017年9月12日保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO:C2017102，保藏地址为中国.武汉.武汉大学，邮编：430072。
杂交瘤细胞株LT007(IL17A-2G2)，其于2017年9月12日保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO:C2017165，保藏地址为中国.武汉.武汉大学，邮编：430072。
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述。本领域技术人员将会理解，下面的实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件(例如参考J.萨姆布鲁克等著，黄培堂等译的《分子克隆实验指南》，第三版，科学出版社)或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市场购买获得的常规产品。
在下面的实施例中，使用的BALB/C小鼠购自广东省医学实验动物中心；使用的C57BL/6小鼠来源于南京银河生物医药有限公司；使用的MRC5细胞来源于上海复旦IBS细胞资源中心；使用的Secukinumab
Figure PCTCN2019088344-appb-000002
单抗购自诺华公司。
实施例1：抗IL-17A的抗体13E9和2G2的制备
1.杂交瘤细胞株LT006和LT007的制备
制备抗IL-17A抗体所用的抗原IL17A(24-155)-his是人IL-17A(GenBank ID:Q16552)成熟肽与his标签的融合蛋白。取免疫BALB/C小鼠(购自广东医学实验动物中心)的脾细胞与小鼠骨髓瘤细胞融合成杂交瘤细胞，具体操作参考目前已确立的方法(例如，Stewart,S.J.,“Monoclonal Antibody Production”,in Basic Methods in antibody Production and Characterization,Eds.G.C.Howard and D.R.Bethell,Boca Raton:CRC Press,2000)。
用TEV蛋白酶酶切融合蛋白IL17A-his，并过柱纯化获得IL-17A(24-155)蛋白。用IL-17A(24-155)蛋白作为抗原包被酶标板，进行间接ELISA法筛选，得到分泌与IL-17A(24-155)特异性结合的新的抗体的杂交瘤细胞。
对间接ELISA筛选得到的杂交瘤细胞，通过竞争ELISA筛选出能够分泌与受体IL-17RA(CD217,NCBI Gene ID:NP_055154.3)竞争结合IL-17A的单克隆抗体的杂交瘤细胞株，并经过有限稀释法得到两株稳定的杂交瘤细胞株。
杂交瘤细胞株LT006(IL17A-13E9)，其于2015年9月12日保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO:C2017102，保藏地址为中国.武汉.武汉大学，邮编：430072。其分泌的单克隆抗体命名为13E9。
杂交瘤细胞株LT007(IL17A-2G2)，其于2017年9月12日保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO:C2017165，保藏地址为中国.武汉.武汉大学，邮编：430072。其分泌的单克隆抗体命名为2G2。
2.抗IL-17A的抗体13E9的制备
用杂交瘤无血清培养基对上面制得的LT006细胞株(1x10 5细胞每孔)进行培养(杂交瘤无血清培养基，内含1％青链霉素，4％的Glutamax，于5％CO 2，37℃细胞培养箱中进行培养)，培养7天至存活率在20％左右时收集细胞培养上清，通过高速离心、微孔滤膜抽真空过滤以及HiTrap protein A HP柱进行纯化，制得抗体13E9。纯化后13E9样品进行SDS-PAGE电泳检测，结果如图1所示。
3.抗IL-17A的抗体2G2的制备
用杂交瘤无血清培养基对上面制得的LT007细胞株(1x10 5细胞每孔)进行培养(杂交瘤无血清培养基，内含1％青链霉素，4％的Glutamax，于5％CO 2，37℃细胞培养箱中进行培养)，培养7天至存活率在20％左右时收集细胞培养上清，通过高速离心、 微孔滤膜抽真空过滤以及HiTrap protein A HP柱进行纯化，制得抗体2G2。纯化后2G2样品进行SDS-PAGE电泳检测，结果如图2所示。
实施例2：抗体13E9的序列分析
参照实施例1中步骤2中的方法培养LT006细胞。
使用培养细胞细菌总RNA提取试剂盒(Tiangen，货号DP430)，按照试剂盒说明书的方法，从培养的LT006细胞中提取mRNA。
按照Invitrogen
Figure PCTCN2019088344-appb-000003
III First-Strand Synthesis System for RT-PCR试剂盒说明书合成cDNA，并进行PCR扩增。
PCR扩增产物直接进行TA克隆，具体操作参考pEASY-T1 Cloning Kit(Transgen CT101)试剂盒说明书进行。
将TA克隆的产物直接进行测序，测序结果如下：
重链可变区的核酸序列：(360bp)
Figure PCTCN2019088344-appb-000004
其编码的氨基酸序列：(120aa)
Figure PCTCN2019088344-appb-000005
轻链可变区的核酸序列：(333bp)
Figure PCTCN2019088344-appb-000006
Figure PCTCN2019088344-appb-000007
其编码的氨基酸序列：(111aa)
Figure PCTCN2019088344-appb-000008
下划线标注区域为CDR区域。
实施例3：抗IL-17A的人源化抗体13E9H1L1、13E9H2L2和13E9  H3L2的设计、 制备和检测
1.抗IL-17A人源化抗体13E9H1L1、13E9H2L2和13E9 H3L2的轻链和重链序列的设计
根据IL-17A蛋白的三维晶体结构(EMBO J.(2001)20p:5332-41)以及实施例2获得的抗体13E9的序列，通过计算机模拟抗体模型，根据模型设计突变，得到抗体13E9H1L1、13E9H2L2和13E9 H3L2的可变区序列(抗体恒定区序列，来自NCBI的数据库，重链恒定区为Ig gamma-1 chain C region，ACCESSION:P01857，轻链恒定区为Ig kappa chain C region，ACCESSION:P01834)。
设计的可变区序列如下：
(1)人源化单克隆抗体13E9H1L1的重链和轻链序列
重链可变区的核酸序列：(360bp)
Figure PCTCN2019088344-appb-000009
其编码的氨基酸序列：(120aa)
Figure PCTCN2019088344-appb-000010
下划线标注区域为CDR区域。
轻链可变区的核酸序列：(333bp)
Figure PCTCN2019088344-appb-000011
其编码的氨基酸序列：(111aa)
Figure PCTCN2019088344-appb-000012
下划线标注区域为CDR区域。
(2)人源化单克隆抗体13E9H2L2的重链和轻链序列
重链可变区的核酸序列：(360bp)
Figure PCTCN2019088344-appb-000013
其编码的氨基酸序列：(120aa)
Figure PCTCN2019088344-appb-000014
下划线标注区域为CDR区域。
轻链可变区的核酸序列：(333bp)
Figure PCTCN2019088344-appb-000015
其编码的氨基酸序列：(111aa)
Figure PCTCN2019088344-appb-000016
下划线标注区域为CDR区域。
(3)人源化单克隆抗体13E9H3L2的重链和轻链序列
重链可变区的核酸序列：(360bp)
Figure PCTCN2019088344-appb-000017
其编码的氨基酸序列：(120aa)
Figure PCTCN2019088344-appb-000018
下划线标注区域为CDR区域。
轻链可变区的核酸序列与13E9H2L2的轻链可变区的核酸序列相同，如SEQ ID NO:11所示。
其编码的氨基酸序列亦与13E9H2L2的轻链可变区的氨基酸序列相同，如SEQ ID NO:12所示。
2.人源化抗体13E9H1L1、13E9H2L2和13E9 H3L2的制备
重链恒定区均采用Ig gamma-1 chain C region，ACCESSION:P01857；轻链恒定区均采用Ig kappa chain C region，ACCESSION:P01834。
将13E9H1L1重链cDNA和轻链的cDNA、13E9H2L2的重链cDNA和轻链的cDNA、以及13E9 H3L2重链cDNA和轻链的cDNA，分别克隆到pUC57simple(金斯瑞公司提供)载体中，分别获得pUC57simple-13E9H1、pUC57simple-13E9L1、pUC57simple-13E9H2、pUC57simple-13E9L2和pUC57simple-13E9H3，并将含有相应的重链的片段和含有相应的轻链的片段分别亚克隆到pcDNA3.1载体中获得pcDNA3.1-13E9H1、pcDNA3.1-13E9L1、pcDNA3.1-13E9H2、pcDNA3.1-13E9L2、pcDNA3.1-13E9H3以及pcDNA3.1-13E9L2重组质粒。之后将相应的轻链重组质粒与重链重组质粒一起(pcDNA3.1-13E9H1与pcDNA3.1-13E9L1；pcDNA3.1-13E9H2与pcDNA3.1-13E9L2；pcDNA3.1-13E9H3与pcDNA3.1-13E9L2)分别转染293F细胞后收集培养液进行纯化获得人源化抗体13E9H1L1、13E9H2L2和13E9 H3L2。纯化后13E9H3L2样品进行SDS-PAGE电泳检测，结果如图3所示。
实施例4：抗体2G2的序列分析
参照实施例1中步骤3中的方法培养LT007细胞。
使用培养细胞细菌总RNA提取试剂盒(Tiangen，货号DP430)，按照试剂盒说明书的方法，从培养的LT007细胞中提取mRNA。
按照Invitrogen
Figure PCTCN2019088344-appb-000019
III First-Strand Synthesis System for RT-PCR试剂盒说明书合成cDNA，并进行PCR扩增。
PCR扩增产物直接进行TA克隆，具体操作参考pEASY-T1 Cloning Kit(Transgen CT101)试剂盒说明书进行。
将TA克隆的产物直接进行测序，测序结果如下：
重链可变区的核酸序列：(351bp)
Figure PCTCN2019088344-appb-000020
其编码的氨基酸序列：(117aa)
Figure PCTCN2019088344-appb-000021
下划线标注区域为CDR区域。
轻链可变区的核酸序列：(336bp)
Figure PCTCN2019088344-appb-000022
其编码的氨基酸序列：(112aa)
Figure PCTCN2019088344-appb-000023
Figure PCTCN2019088344-appb-000024
下划线标注区域为CDR区域。
实施例5：抗IL-17A的人源化抗体2G2 H1L1、2G2 H2L2和2G2 H3L3的设计、 制备和检测
(1)人源化单克隆抗体2G2 H1L1的重链和轻链序列
重链可变区的核酸序列：(348bp)
Figure PCTCN2019088344-appb-000025
其编码的氨基酸序列：(116aa)
Figure PCTCN2019088344-appb-000026
下划线标注区域为CDR区域。
轻链可变区的核酸序列：(336bp)
Figure PCTCN2019088344-appb-000027
其编码的氨基酸序列：(112aa)
Figure PCTCN2019088344-appb-000028
Figure PCTCN2019088344-appb-000029
下划线标注区域为CDR区域。
(2)人源化单克隆抗体2G2 H2L2的重链和轻链序列
重链可变区的核酸序列：(348bp)
Figure PCTCN2019088344-appb-000030
其编码的氨基酸序列：(116aa)
Figure PCTCN2019088344-appb-000031
下划线标注区域为CDR区域。
轻链可变区的核酸序列：(336bp)
Figure PCTCN2019088344-appb-000032
其编码的氨基酸序列：(112aa)
Figure PCTCN2019088344-appb-000033
Figure PCTCN2019088344-appb-000034
下划线标注区域为CDR区域。
(3)人源化单克隆抗体2G2 H3L3的重链和轻链序列
重链可变区的核酸序列：(348bp)
Figure PCTCN2019088344-appb-000035
其编码的氨基酸序列：(116aa)
Figure PCTCN2019088344-appb-000036
下划线标注区域为CDR区域。
轻链可变区的核酸序列：(336bp)
Figure PCTCN2019088344-appb-000037
其编码的氨基酸序列：(112aa)
Figure PCTCN2019088344-appb-000038
下划线标注区域为CDR区域。
2.人源化抗体2G2 H1L1，2G2 H2L2，2G2 H3L3的制备和SDS-PAGE电泳检测
重链恒定区采用Ig gamma-1chain C region，ACCESSION:P01857；轻链恒定区采用Ig kappa chain C region，ACCESSION:P01834。
将2G2 H1L1重链cDNA和轻链的cDNA、2G2 H2L2的重链cDNA和轻链的cDNA、以及2G2 H3L3重链cDNA和轻链的cDNA，分别克隆到pUC57simple(金斯瑞公司提供)载体中，分别获得pUC57simple-2G2H1、pUC57simple-2G2L1；pUC57simple-2G2H2、pUC57simple-2G2L2；和pUC57simple-2G2H3、pUC57simple-2G2L3。后将将含有相应的重链的核苷酸片段和含有相应的轻链的核苷酸片段分别亚克隆到pcDNA3.1载体中获得重组质粒pcDNA3.1-2G2H1、pcDNA3.1-2G2L1、pcDNA3.1-2G2H2、pcDNA3.1-2G2L2、pcDNA3.1-2G2H3和pcDNA3.1-2G2L3。后将相应的轻链重组质粒与重链重组质粒一起(pcDNA3.1-2G2H1和pcDNA3.1-2G2L1；pcDNA3.1-2G2H2和pcDNA3.1-2G2L2；pcDNA3.1-2G2H3和pcDNA3.1-2G2L3)分别转染293F细胞后收集培养液进行纯化获得人源化抗体2G2 H1L1、2G2 H2L2和2G2 H3L3。纯化后的样品进行SDS-PAGE电泳检测。
实施例6：13E9 H1L1、13E9 H2L2、13E9 H3L2与抗原IL-17A(24-155)蛋白结合的 动力学参数的测定
使用Fortebio分子相互作用仪测定人源化抗体13E9 H1L1、13E9 H2L2、13E9 H3L2与抗原IL-17A(24-155)结合的动力学参数。
使用EDC/NHS激活AR2G传感器，采用氨基偶联的方式把抗体固定到激活的AR2G传感器上，使用1M乙醇胺，pH8.5封闭传感器。传感器在PBST中平衡300s后，固定在传感器上的抗体与抗原IL-17A(24-155)蛋白(与实施例1使用的抗原相同)结合，抗原浓度为6.25-400nM(两倍梯度稀释)，结合时间为420s，抗原抗体在PBST中解离，时间600 s。
抗体13E9 H1L1、13E9 H2L2、13E9 H3L2和Secukinumab的动力学参数见表1，动力学特征参数检测结果分别如图4、图5、图6和图7所示。
表1：13E9人源化抗体动力学参数
抗体名称 K D(M) Kon(1/Ms) kon误差 kdis(1/s) kdis误差 R max(nm)
13E9 H1L1 9.72E-10 1.34E+05 2.60E+03 1.31E-04 9.06E-06 0.0858-0.3326
13E9 H2L2 1.03E-09 9.86E+04 1.51E+03 1.01E-04 7.57E-06 0.0546-0.3049
13E9 H3L2 5.08E-10 2.90E+05 8.22E+03 1.47E-04 1.02E-05 0.1457-0.3546
Secukinumab 6.74E-10 9.28E+04 3.03E+03 6.26E-05 1.57E-05 0.0379-0.2064
K D为亲和力常数；kon为抗原抗体结合速率；kdis为抗原抗体解离速率；K D＝kdis/kon。
结果表明，13E9 H1L1、13E9 H2L2、13E9 H3L2均与抗原IL-17A(24-155)有较好的亲和力，且亲和力与对照抗体Secukinumab相当。
实施例7：人源化抗体2G2 H1L1、2G2 H2L2、2G2 H3L3与抗原IL-17A(24-155) 结合的动力学参数的测定
使用Fortebio分子相互作用仪测定人源化抗体2G2 H1L1、2G2 H2L2、2G2 H3L3与抗原IL-17A(24-155)结合的动力学参数。
使用EDC/NHS激活AR2G传感器，采用氨基偶联的方式把抗体固定到激活的AR2G传感器上，使用1M乙醇胺，pH8.5封闭传感器。传感器在PBST中平衡300s后，固定在传感器上的抗体与抗原IL-17A(24-155)结合，抗原浓度为6.25-400nM(两倍梯度稀释)，结合时间为420s，抗原抗体在PBST中解离，时间600s。
抗体2G2 H1L1、2G2 H2L2、2G2 H3L3和Secukinumab的动力学参数见表2，动力学特征参数检测结果分别如图8、图9、图10和图11所示。
表2：2G2 H1L1、2G2 H2L2、2G2 H3L3和Secukinumab的动力学参数
抗体名称 K D(M) kon(1/Ms) kon误差 kdis(1/s) kdis误差 Rmax(nm)
2G2 H1L1 1.53E-09 1.46E+05 3.15E+03 2.24E-04 9.67E-06 0.0035-0.2192
2G2 H2L2 8.43E-10 1.42E+05 2.18E+03 1.20E-04 6.73E-06 0.0444-0.2732
2G2 H3L3 1.54E-09 1.98E+05 5.30E+03 3.06E-04 1.08E-05 0.011-0.2109
Secukinumab 1.08E-09 1.33E+05 2.38E+03 1.43E-04 8.09E-06 0.0118-0.2584
K D为亲和力常数；kon为抗原抗体结合速率；kdis为抗原抗体解离速率；K D＝kdis/kon。
结果表明，与对照抗体Secukinumab相比，2G2 H2L2与抗原IL-17A(24-155)具有更高亲和力；2G2 H1L1、2G2 H3L3亲和力与对照抗体Secukinumab相当。
实施例8：ELISA方法检测抗体13E9 H1L1、13E9 H2L2、13E9 H3L2与抗原的结 合活性
1.采用间接ELISA方法检测抗体13E9 H1L1、13E9 H2L2和13E9 H3L2与抗原IL17A-His的结合活性，并与同靶点已上市药物Secukinmab进行比较。
IL17A-his可以参照公开序列和本领域常规技术手段制备，也可以参考下面的步骤：
IL17A-his制备：通过NCBI蛋白质数据库查找人IL-17A的蛋白质序列，将人IL-17A全长序列与His*6纯化标签序列进行融合设计。委托南京金斯瑞生物合成编码该融合蛋白的核酸，参照《分子克隆实验指南(第二版)》介绍的标准技术，采用PCR、酶切、胶回收、连接转化、菌落PCR或酶切鉴定等标准的分子克隆技术将目的基因亚克隆到哺乳动物细胞表达载体，并进一步对重组表达载体的目的基因进行测序分析。测序验证正确后，中大量制备去内毒素级别的表达质粒并将质粒瞬时转染HEK293细胞进行蛋白表达。培养7天后收集细胞培养液，采用Ni Sepharose柱(GE)进行亲和纯化，收获的蛋白样品使用SDS-PAGE和SEC-HPLC标准分析技术对其进行质量鉴定，结果合格。
ELISA：酶标板中加入IL17A-His孵育，4℃过夜，用1％BSA in PBS于37℃封闭2h后，分别加入抗体，37℃孵育30min，加入Goat Anti Human IgG(H+L)，HRP(Jackson，109-035-088)于37℃孵育30min，用TMB(Neogen，308177)进行显色反应5min，并在酶标仪中检测450nm波长吸光度。得到的实验数据用SoftMax Pro 6.2.1软件对数据进行分析处理，以抗体浓度为横坐标，吸光度值为纵坐标进行4-parameter拟合曲线作图分析。
实验结果如图12、图13以及下面的表3和表4所示。
表3：13E9 H1L1、13E9 H2L2与抗原IL17A-His的结合活性检测结果
Figure PCTCN2019088344-appb-000039
Figure PCTCN2019088344-appb-000040
表4：13E9 H3L2与抗原IL17A-His的结合活性检测结果
Figure PCTCN2019088344-appb-000041
实验结果显示，抗体13E9 H1L1、13E9 H2L2和13E9 H3L2均能有效地结合抗原IL17A-His，其结合效率呈剂量依赖关系。在相同实验条件下，13E9 H1L1的结合EC 50为0.044nM，13E9 H2L2的结合EC 50为0.048nM，同靶点已上市药物Secukinumab的EC 50为0.082nM(图12，表3)；在相同实验条件下，13E9 H3L2的结合EC 50为0.043nM，同靶点已上市药物Secukinumab的EC 50为0.078(图13，表4)。
以上实验结果表明，在相同实验条件的情况下13E9 H1L1、13E9 H2L2和13E9 H3L2的EC 50值均小于同靶点阳性对照药物Secukinumab，表明13E9 H1L1、13E9 H2L2和13E9 H3L2的与IL17A-His的结合活性优于已上市同靶点对照药物Secukinumab。
2.采用竞争ELISA方法检测抗体13E9 H1L1、13E9 H2L2、13E9 H3L2阻断IL17RA-His(biotin)与抗原IL17A-His结合的活性。
IL17RA-His(biotin)可以参照公开序列和本领域常规技术手段制备，也可以参考下面的步骤：
IL17RA-His(biotin)制备：通过NCBI蛋白质数据库查找人IL-17RA的蛋白质序列，将人IL-17RA胞外区序列与His*6纯化标签序列进行融合设计。委托南京金斯瑞生物合成编码该融合蛋白的核酸，参照《分子克隆实验指南(第二版)》介绍的标准技术， 采用PCR、酶切、胶回收、连接转化、菌落PCR或酶切鉴定等标准的分子克隆技术将目的基因亚克隆到哺乳动物细胞表达载体，并进一步对重组表达载体的目的基因进行测序分析。测序验证正确后，中大量制备去内毒素级别的表达质粒并将质粒瞬时转染HEK293细胞进行蛋白表达。培养7天后收集细胞培养液，采用Ni Sepharose柱(GE)进行亲和纯化，收获的蛋白样品使用SDS-PAGE和SEC-HPLC标准分析技术对其进行质量鉴定，结果合格。蛋白样品质量鉴定完毕后，使用Thermo scientific的商业化试剂盒
Figure PCTCN2019088344-appb-000042
Sulfo-NHS-LC-Biotinylation Kit标记获得生物素化偶联的人IL-17RA-His蛋白样品，具体制备方法参照该试剂盒的说明书进行。
ELISA：酶标板中加入IL17A-His孵育，4℃过夜，用1％BSA in PBST于37℃封闭2h后，分别加入抗体，抗原抗体室温反应10分钟，再加入受体IL17RA-His(biotin)，与抗体体积1:1混匀后于37℃孵育30min，加入SA-HRP(KPL,14-30-00)于37℃孵育30min，用TMB(Neogen，308177)进行显色反应5min，并在酶标仪中检测450nm波长吸光度。得到的实验数据用SoftMax Pro 6.2.1软件对数据进行分析处理，以抗体浓度为横坐标，吸光度值为纵坐标进行4-parameter拟合曲线作图分析。实验结果如图14、图15以及下面的表5和表6所示。
表5：13E9 H1L1、13E9 H2L2与受体IL17RA-His(biotin)竞争结合抗原IL-17A-His的活性检测结果
Figure PCTCN2019088344-appb-000043
表6：13E9 H3L2与受体IL17RA-His(biotin)竞争结合抗原IL17A-His的活性检测结果
Figure PCTCN2019088344-appb-000044
实验结果显示，抗体13E9 H1L1、13E9 H2L2和13E9 H3L2均能有效地阻断受体IL17RA-His(biotin)与抗原IL17A-His的结合，阻断效率呈现剂量依赖关系。在相同实验条件下，13E9 H1L1与IL17AR-His(biotin)竞争结合IL17A-His的EC50为1.437nM，13E9 H2L2与IL17AR-His(biotin)竞争结合IL17A-His的EC 50为1.281nM，同靶点阳性对照药物Secukinumab与IL17AR-His(biotin)竞争结合IL17A-His的EC 50为1.807nM(表5，图14)；13E9 H3L2与IL17AR-His(biotin)竞争结合IL17A-His的EC 50为0.805nM，同靶点已上市药物Secukinumab与IL17AR-His(biotin)竞争结合IL17A-His的EC 50为1.580nM(表6，图15)。
以上实验结果表明，在相同实验条件的情况下13E9 H1L1、13E9 H2L2和13E9 H3L2与IL17AR-His(biotin)竞争结合IL17A-His的EC 50值均小于同靶点已上市对照药物Secukinumab，提示13E9 H1L1、13E9 H2L2和13E9 H3L2与IL17AR-His(biotin)竞争结合IL17A-His的活性优于同靶点已上市药物Secukinumab。
实施例9：ELISA方法检测抗体2G2 H1L1、2G2 H2L2和2G2 H3L3与抗原的结合 活性
1.采用间接ELISA方法检测抗体2G2 H1L1、2G2 H2L2和2G2 H3L3与抗原 IL17A-His的结合活性。
实验步骤：酶标板中加入IL17A-His孵育，4℃过夜，用1％BSA in PBS于37℃封闭2h后，分别加入抗体，37℃孵育30min，加入Goat Anti Human IgG(H+L)，HRP(Jackson，109-035-088)于37℃孵育30min，用TMB(Neogen，308177)进行显色反应5min，并在酶标仪中检测450nm波长吸光度。得到的实验数据采用SoftMax Pro 6.2.1软件进行分析处理，以抗体浓度为横坐标，吸光度值为纵坐标进行4-parameter拟合曲线作图分析。
实验结果如图16和下面的表7所示。其中，2G2 H1L1的结合EC 50为0.177nM，2G2H2L2的结合EC 50为0.372nM，2G2 H3L3的结合EC 50为0.421nM。
表7：2G2 H1L1、2G2 H2L2、2G2 H3L3与抗原IL17A-His的结合检测结果
Figure PCTCN2019088344-appb-000045
实验结果表明，抗体2G2 H1L1、2G2 H2L2和2G2 H3L3均能有效地结合抗原IL17A-His，结合效率呈现剂量依赖关系。
2.采用竞争ELISA方法检测抗体2G2 H1L1、2G2 H2L2和2G2 H3L3与受体IL17RA-His(biotin)竞争结合抗原IL17A-His的活性。
实验步骤：酶标板中加入IL17A-His孵育，4℃过夜，用1％BSA in PBST于37℃封闭2h后，分别加入抗体，抗原抗体室温反应10分钟，再加入受体IL17RA-His(biotin)，与抗体体积1:1混匀后于37℃孵育30min，加入SA-HRP(KPL,14-30-00)于37℃孵育30min，用TMB(Neogen，308177)进行显色反应5min，并在酶标仪中检测450nm波 长吸光度。得到的实验数据用SoftMax Pro 6.2.1软件对数据进行分析处理，以抗体浓度为横坐标，吸光度值为纵坐标进行4-parameter拟合曲线作图分析。
实验结果如图17和下面的表8所示。其中，2G2 H1L1的阻断EC 50为2.264nM，2G2H2L2的阻断EC 50为5.408nM，2G2 H3L3的阻断EC 50为5.911nM(图17，表8)。
表8：2G2 H1L1、2G2 H2L2、2G2 H3L3与受体IL17RA-His(biotin)竞争结合抗原IL17A-His的活性检测结果
Figure PCTCN2019088344-appb-000046
实验结果表明，抗体2G2 H1L1、2G2 H2L2和2G2 H3L3均能有效地阻断受体IL17RA-His(biotin)与抗原IL17A-His的结合，阻断效率呈现剂量依赖关系。
实施例10：混合人胚胎成纤维细胞反应：细胞因子IL-6的分泌
MRC 5(购自中国科学院细胞中心)细胞接种到96孔板，5000cell/孔，培养过夜。MRC 5细胞中加入在37摄氏度下孵育20min的IL-17和抗体混合物后(hIgG做为对照)，培养48小时。48小时培养结束后后收集细胞上清液，采用ELISA试剂盒检测IL-6(购自达科为公司)的分泌量。
MRC 5细胞分别加入13E9 H1L1、13E9 H2L2、13E9 H3L2、2G2 H1L1、2G2 H2L2或2G2 H3L3(1，10，100nM)以及Secukinumab(1，10，100nM)混合培养后，分泌的IL-6检测结果如图18所示。
由图18可见，13E9 H1L1、13E9 H2L2、13E9 H3L2和2G2 H2L2均能有效地降低MRC 5细胞在IL-17诱导下的IL-6的分泌。其中：
13E9 H1L1抗体在浓度为100nM时抑制IL-6分泌的效果优于同剂量对照抗体Secukinumab，浓度为10nM时对IL-6分泌的抑制效果与对照抗体Secukinumab在100nM时相当；
13E9 H2L2抗体在浓度为1nM、10nM以及100nM水平时均与同剂量对照抗体Secukinumab抑制IL-6分泌的效果相当；
13E9 H3L2抗体对IL-6分泌的抑制效果在浓度为10nM和100nM时均优于同剂量对照抗体Secukinumab，在1nM水平与对照抗体Secukinumab相当；
2G2 H2L2在浓度为1nM、100nM时均与对照抗体Secukinumab抑制IL-6分泌的效果相当；
2G2 H3L3在浓度为1nM时抑制IL-6分泌的效果优于同剂量对照抗体Secukinumab。
以上结果表明，在体外混合人胚胎成纤维细胞反应中，本发明的抗体阻断IL-17A介导的IL-6分泌的生活学活性优于或至少相当于同靶点已上市药物Secukinumab。
实施例11：抗体药13E9 H3L2对C57BL/6小鼠银屑病模型的表皮厚度的影响
C57BL/6小鼠，分为5组，每组8只。
(1)造模：
正常组，在C57BL/6小鼠光滑的背部第1天至第6天连续6天皮内注射生理盐水，25μl/只；
其余各组小鼠第1天至第4天皮内注射重组人IL-17A，2μg/25μl/只，第5天至第6天皮内注射重组人IL-17A 5μg/25μl/只。
(2)具体分组和给药情况：
正常组：生理盐水，给药剂量0mg/kg，一周3次共给药3次；
模型组：阴性同型对照，给药剂量50mg/kg，一周3次共给药3次；
Secukinumab组：Secukinumab，给药剂量50mg/kg，一周3次共给药3次；
13E9 H3L2高剂量组：给药剂量50mg/kg，一周3次共给药3次；
13E9 H3L2低剂量组：给药剂量10mg/kg，一周3次共给药3次。
每组均给药3次，分别是在造模前1天、造模第3天、造模第6天，皮下给药。
造模第7天固定小鼠背部注射部位皮肤制作病理切片并测量表皮厚度。
实验结果如图19所示。
结果显示，Secukinumab组(50mg/kg)和13E9 H3L2高剂量组(50mg/kg)、13E9 H3L2低剂量组(10mg/kg)的表皮厚度在统计学上均显著小于模型组(P<0.01)。
结果表明，抗体药13E9 H3L2(50mg/kg)在C57BL/6小鼠银屑病模型中显示有统计学显著意义的表皮厚度的抑制作用，与Secukinumab(50mg/kg)药效一致；抗体药13E9 H3L2(10mg/kg)的表皮厚度的抑制作用在统计学上也有显著差异。
尽管本发明的具体实施方式已经得到详细的描述，本领域技术人员将会理解。根据已经公开的所有教导，可以对那些细节进行各种修改和替换，这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。

Claims (26)

  1. 单克隆抗体或其抗原结合片段，其中，
    所述单克隆抗体的重链可变区包含：氨基酸序列分别如SEQ ID NOs:31－33所示的HCDR1－HCDR3或氨基酸序列分别如SEQ ID NOs:37－39所示的HCDR1－HCDR3，和
    所述单克隆抗体的轻链可变区包含：氨基酸序列分别如SEQ ID NOs:34－36所示的LCDR1－LCDR3或氨基酸序列分别如SEQ ID NOs:40－42所示的LCDR1－LCDR3。
  2. 根据权利要求1所述的单克隆抗体或其抗原结合片段，其中，
    所述单克隆抗体的重链可变区包含氨基酸序列分别如SEQ ID NOs:31－33所示的HCDR1－HCDR3，并且所述单克隆抗体的轻链可变区包含氨基酸序列分别如SEQ ID NOs:34－36所示的LCDR1－LCDR3；
    或者
    所述单克隆抗体的重链可变区包含氨基酸序列分别如SEQ ID NOs:37－39所示的HCDR1－HCDR3并且所述单克隆抗体的轻链可变区包含氨基酸序列分别如SEQ ID NOs:40－42所示的LCDR1－LCDR3。
  3. 根据权利要求1或2所述的单克隆抗体或其抗原结合片段，其中，
    所述重链可变区的氨基酸序列选自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和SEQ ID NO:28，
    并且
    所述轻链可变区的氨基酸序列选自SEQ ID NO:4、SEQ ID NO:8、SEQ ID NO:12、SEQ ID NO:18、SEQ ID NO:22、SEQ ID NO:26和SEQ ID NO:30。
  4. 根据权利要求1至3中任一权利要求所述的单克隆抗体或其抗原结合片段，其中，
    所述重链可变区的氨基酸序列选自SEQ ID NO:2、SEQ ID NO:6、SEQ ID NO:10、SEQ ID NO:14，并且所述轻链可变区的氨基酸序列选自SEQ ID NO:4、SEQ ID NO:8、SEQ ID NO:12；
    或者
    所述重链可变区的氨基酸序列选自SEQ ID NO:16、SEQ ID NO:20、SEQ ID NO:24和SEQ ID NO:28，并且所述轻链可变区的氨基酸序列选自SEQ ID NO:18、SEQ ID NO:22、SEQ ID NO:26和SEQ ID NO:30。
  5. 根据权利要求1至4中任一权利要求所述的单克隆抗体或其抗原结合片段，其中，所述重链可变区和轻链可变区选自如下的(1)-(8)中的任一项：
    (1)重链可变区，其包含SEQ ID NO:2所示的氨基酸序列，和
    轻链可变区，其包含SEQ ID NO:4所示的氨基酸序列；
    (2)重链可变区，其包含SEQ ID NO:6所示的氨基酸序列，和
    轻链可变区，其包含SEQ ID NO:8所示的氨基酸序列；
    (3)重链可变区，其包含SEQ ID NO:10所示的氨基酸序列，和
    轻链可变区，其包含SEQ ID NO:12所示的氨基酸序列；
    (4)重链可变区，其包含SEQ ID NO:14所示的氨基酸序列，和
    轻链可变区，其包含SEQ ID NO:12所示的氨基酸序列；
    (5)重链可变区，其包含SEQ ID NO:16所示的氨基酸序列，和
    轻链可变区，其包含SEQ ID NO:18所示的氨基酸序列；
    (6)重链可变区，其包含SEQ ID NO:20所示的氨基酸序列，和
    轻链可变区，其包含SEQ ID NO:22所示的氨基酸序列；
    (7)重链可变区，其包含SEQ ID NO:24所示的氨基酸序列，和
    轻链可变区，其包含SEQ ID NO:26所示的氨基酸序列；以及
    (8)重链可变区，其包含SEQ ID NO:28所示的氨基酸序列，和
    轻链可变区，其包含SEQ ID NO:30所示的氨基酸序列。
  6. 根据权利要求1至5中任一权利要求所述的单克隆抗体或其抗原结合片段，其中，所述单克隆抗体或其抗原结合片段选自Fab、Fab'、F(ab') 2、Fd、Fv、dAb、互补决定区片段、单链抗体、人源化抗体、嵌合抗体或双抗体。
  7. 根据权利要求1至6中任一权利要求所述的单克隆抗体或其抗原结合片段，其中， 其中，所述的单克隆抗体以小于大约100nM，例如小于大约10nM、5nM、4nM、3nM、2.5nM、2nM或更小的EC 50结合IL-17A蛋白；优选地，所述EC 50通过竞争ELISA方法测得。
  8. 根据权利要求1至7中任一权利要求所述的单克隆抗体或其抗原结合片段，其中，所述的单克隆抗体包括非-CDR区，且所述非-CDR区来自不是鼠类的物种，例如来自人抗体。
  9. 根据权利要求1至8中任一权利要求所述的单克隆抗体或其抗原结合片段，其中：
    所述单克隆抗体是由杂交瘤细胞株LT006产生的单克隆抗体，所述杂交瘤细胞株LT006保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO:C2017102；或者
    所述单克隆抗体是由杂交瘤细胞株LT007产生的单克隆抗体，所述杂交瘤细胞株LT007保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO:C2017165。
  10. 分离的核酸分子，其包含编码权利要求1至9中任一权利要求所述的单克隆抗体的重链可变区和轻链可变区的核酸。
  11. 根据权利要求10所述的分离的核酸分子，其中，编码重链可变区和轻链可变区的核酸选自如下的(1)-(8)中的任一项：
    (1)SEQ ID NO:1，SEQ ID NO:3；
    (2)SEQ ID NO:5，SEQ ID NO:7；
    (3)SEQ ID NO:9，SEQ ID NO:11；
    (4)SEQ ID NO:13，SEQ ID NO:11；
    (5)SEQ ID NO:15，SEQ ID NO:17；
    (6)SEQ ID NO:19，SEQ ID NO:21；
    (7)SEQ ID NO:23，SEQ ID NO:25；
    (8)SEQ ID NO:27，SEQ ID NO:29。
  12. 一种重组载体，其包含权利要求10或11所述的分离的核酸分子；优选地，所述重组载体为重组表达载体，例如重组原核表达载体或重组真核表达载体。
  13. 一种宿主细胞，其包含权利要求12所述的重组载体。
  14. 制备权利要求1至9中任一权利要求所述的单克隆抗体或其抗原结合片段的方法，其包括在合适的条件下培养权利要求13的宿主细胞，以及从细胞培养物中回收所述单克隆抗体或其抗原结合片段的步骤。
  15. 选自如下的杂交瘤细胞株：
    杂交瘤细胞株LT006，其保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO:C2017102；或者
    杂交瘤细胞株LT007，其保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO:C2017165。
  16. 一种偶联物，其包括单克隆抗体或其抗原结合片段以及偶联部分，其中，所述单克隆抗体为权利要求1至9中任一权利要求所述的单克隆抗体或其抗原结合片段，所述偶联部分为可检测的标记物；优选地，所述可检测的标记物为放射性同位素、发光物质例如荧光物质、有色物质或酶。
  17. 一种试剂盒，其包括权利要求1至9中任一权利要求所述的单克隆抗体或其抗原结合片段，或者包括权利要求16所述的偶联物；
    优选地，所述试剂盒还包括第二抗体，其特异性识别所述单克隆抗体或其抗原结合片段；任选地，所述第二抗体还包括可检测的标记物；优选地，所述可检测的标记物为放射性同位素、发光物质例如荧光物质、有色物质或酶。
  18. 权利要求1至9中任一权利要求所述的单克隆抗体或其抗原结合片段或者权利要求16所述的偶联物在制备试剂盒中的用途，所述试剂盒用于IL-17A的定性检测或定量检测。
  19. 一种药物组合物，其包含权利要求1至9中任一权利要求所述的单克隆抗体或其抗原结合片段或者权利要求16所述的偶联物；可选地，所述药物组合物还包括药学上可接受的载体或赋形剂。
  20. 权利要求1至9中任一权利要求所述的单克隆抗体或其抗原结合片段或者权利要求16所述的偶联物在制备预防和/或治疗肿瘤或者自身免疫性疾病的药物中的用途，或者用于制备诊断自身免疫性疾病的药物中的用途；优选地，所述自身免疫性疾病选自银屑病、类风湿关节炎、银屑病性关节炎、强直性脊柱炎和系统性红斑狼疮；优选地，所述银屑病为中度至重度斑块性银屑病。
  21. 权利要求1至9中任一权利要求所述的单克隆抗体或其抗原结合片段或者权利要求16所述的偶联物在制备如下药物中的用途：
    阻断IL-17A与IL17RA结合的药物，
    调节(例如下调)IL-17A活性或水平的药物，或者
    抑制细胞中IL-6表达的药物。
  22. 一种在体内或在体外的方法，包括给予细胞或者有需求的受试者以有效量的权利要求1至9中任一权利要求所述的单克隆抗体或其抗原结合片段或者权利要求16所述的偶联物的步骤，所述方法选自如下：
    阻断IL-17A与IL17RA结合的方法，
    调节(例如下调)IL-17A活性或水平的方法，或者
    抑制成纤维细胞中IL-6表达的方法。
  23. 一种治疗和/或预防肿瘤或者自身免疫性疾病的方法，包括给予有需求的受试者以有效量的权利要求1至9中任一权利要求所述的单克隆抗体或其抗原结合片段或者权利要求16所述的偶联物的步骤；优选地，所述自身免疫性疾病选自银屑病、类风湿关节炎、银屑病性关节炎、强直性脊柱炎、系统性红斑狼疮；优选地，所述银屑病为中度至重度斑块性银屑病。
  24. 根据权利要求1至9中任一权利要求所述的单克隆抗体或其抗原结合片段，其用于预防和/或治疗肿瘤或者自身免疫性疾病，或者用于诊断自身免疫性疾病；优选地，所述自身免疫性疾病选自银屑病、类风湿关节炎、银屑病性关节炎、强直性脊柱炎、系统性红斑狼疮；优选地，所述银屑病为中度至重度斑块性银屑病。
  25. 根据权利要求1至9中任一权利要求所述的单克隆抗体或其抗原结合片段，其用于：
    阻断IL-17A与IL17RA结合，
    调节(例如下调)IL-17A活性或水平，或者
    抑制细胞中IL-6的表达。
  26. 一种诊断自身免疫性疾病的方法，包括施加待测样品(例如组织样品、细胞样品或血液样品)或者给予有需求的受试者以有效量的权利要求1至9中任一权利要求所述的单克隆抗体或其抗原结合片段或者权利要求16所述的偶联物的步骤；优选地，所述自身免疫性疾病选自银屑病、类风湿关节炎、银屑病性关节炎、强直性脊柱炎、系统性红斑狼疮；优选地，所述银屑病为中度至重度斑块性银屑病；
    优选地，如果被测的IL-17A表达水平高于正常人，且达到了致病的水平，诊断为阳性结果；反之，诊断为阴性结果。
PCT/CN2019/088344 2018-05-30 2019-05-24 抗白细胞介素-17a抗体、其药物组合物及其用途 WO2019228266A1 (zh)

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CN114685668A (zh) * 2020-12-28 2022-07-01 石药集团巨石生物制药有限公司 一种人gpc3单克隆抗体及其缀合物
CN114685668B (zh) * 2020-12-28 2023-10-13 石药集团巨石生物制药有限公司 一种人gpc3单克隆抗体及其缀合物
CN117843778A (zh) * 2023-12-21 2024-04-09 北京贝来药业有限公司 针对白介素家族成员的新型纳米抗体及其产品和应用

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