WO2019015282A1 - 靶向于白介素17a的抗体、其制备方法和应用 - Google Patents

靶向于白介素17a的抗体、其制备方法和应用 Download PDF

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WO2019015282A1
WO2019015282A1 PCT/CN2018/073458 CN2018073458W WO2019015282A1 WO 2019015282 A1 WO2019015282 A1 WO 2019015282A1 CN 2018073458 W CN2018073458 W CN 2018073458W WO 2019015282 A1 WO2019015282 A1 WO 2019015282A1
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antibody
variable region
chain variable
heavy chain
light chain
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PCT/CN2018/073458
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English (en)
French (fr)
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朱向阳
蔡明清
于海佳
贾慧峰
俞玲
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华博生物医药技术（上海）有限公司
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Priority to US16/461,128 priority Critical patent/US10981983B2/en
Priority to JP2019528444A priority patent/JP6865826B2/ja
Priority to EP18835131.6A priority patent/EP3656789A4/en
Publication of WO2019015282A1 publication Critical patent/WO2019015282A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/244—Interleukins [IL]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • G01N33/6869—Interleukin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G01N2333/54—Interleukins [IL]

Definitions

  • the present invention relates to the field of medicine, and in particular to an antibody targeted to interleukin 17A (also referred to as IL-17), a preparation method thereof and use thereof.
  • IL-17 interleukin 17A
  • IL-17A IL-17
  • IL-17B IL-17B
  • IL-17C IL-17D
  • IL-17E IL-25
  • IL-17F interleukin-17 cytokines
  • IL-17A was originally found to be secreted by activated CD4 + T cells. This type of characteristic T cell subset that secretes IL-17A is called Th17 cell.
  • Th17 cell cytotoxic CD8 + T cells (Tc17), ⁇ T cells, natural killer T cells (NKT-17), and B cells can also express IL-17A under specific conditions.
  • Innate immune cells, including monocytes, neutrophils, natural killer cells, and lymphoid tissue-like (Lti-like) cells can also produce IL-17A.
  • B cells can also produce IL-17A.
  • Some non-immune cells can also produce IL-17A under stress. Since Th17 cells are the most widely distributed in the body and have a wide range of effects in the inflammatory response, it is generally considered to be the main source of IL-17A.
  • the congenital cells that produce IL-17A mainly participate in the host's anti-infective immune response as the early defense cells of the body.
  • IL-17A is a homodimer that is linked by a disulfide bond from two chains of 155 amino acids with a molecular weight of 35 kDa.
  • the structure of IL-17 consists of a signal peptide (AA) consisting of 23 amino acids and a region of 123 amino acid chains.
  • the receptor for IL-17 binding to the type I cell surface is called IL-17R, and there are at least three of them: IL-17RA, IL-17RB and IL-17RC.
  • IL-17A and IL-17F bind to IL-17RA and IL-17RC receptor complexes in the form of homodimers or heterodimers to transduce signals and participate in autoimmune diseases, multiple inflammatory responses, and The host is resistant to an immune response.
  • IL-17C binds to IL-17RA and IL-17RE receptor complexes to activate downstream signaling and promotes anti-infective immunity, autoimmune diseases and inflammatory responses.
  • IL-17B was found to bind IL-17RB, but its downstream signal remains unclear.
  • IL-17RB also forms a receptor complex with IL-17RA to mediate IL-17E causing a type II immune response.
  • Il-17E has also been reported to promote apoptosis in tumor cells.
  • the receptor and downstream signals of IL-17D and the ligand and downstream signals of the orphan receptor IL-17RD are still unclear.
  • IL-17A primarily induces signal activation of non-hematopoietic-derived cells including epithelial cells and stromal cells. IL-17A induces the expression of a variety of inflammatory factors and chemokines to promote the recruitment of a variety of immune cells, thereby promoting autoimmune diseases. Studies have found that IL-17A and IL-17F, and their major secretory T cell subsets, Th17 cells also play important roles in a variety of autoimmune diseases, including autoimmune diseases such as rheumatoid arthritis (rheumatoid).
  • RA Arthritis, RA and multiple sclerosis (MS), as well as inflammatory boweldise (IBD), psoriasis, systemic lupus erythematosus (SLE), and type 1 diabetes ( Type1diabetes, T1D).
  • IBD inflammatory boweldise
  • SLE systemic lupus erythematosus
  • T1D Type1diabetes
  • IL-17A and IL-17F exert their functions of promoting inflammatory response mainly by inducing target cells to express various inflammatory factors and chemokines.
  • IL-17A binds to the cell surface receptor IL-17RA and recruits IL-17RC to form a heterodimer that mediates downstream signaling pathways.
  • IL-17 binds to its receptor and activates TRAF6 (TNF-receptor associated factor 6).
  • TRAF6 TRAF6 (TNF-receptor associated factor 6).
  • IL-17 shares the same transcriptional pathway as IL-1 and TNF, which activates NF-kB and three MAP (mitogen-activated protein) enzymes, including ERK1, ERK2, JNK, and p38. These pathways are found in both synovial and chondrocytes.
  • a heavy chain variable region of an antibody comprising the following three complementarity determining region CDRs:
  • the amino acid sequence of any one of the above amino acid sequences further comprises at least one of optionally adding, deleting, modifying and/or substituting (eg, 1-3, preferably 1-2, more preferably 1) amino acid and a derivative sequence capable of retaining IL-17A binding affinity.
  • the heavy chain variable region further comprises a FR region of a human source or a FR region of a murine source.
  • the heavy chain variable region has the amino acid sequence set forth in SEQ ID NO: 1.
  • the heavy chain variable region has the amino acid sequence set forth in SEQ ID NO: 5.
  • a heavy chain of an antibody having a heavy chain variable region according to the first aspect of the invention.
  • the heavy chain of the antibody further comprises a heavy chain constant region.
  • the heavy chain constant region is of human, murine or rabbit origin.
  • a light chain variable region of an antibody comprising the following three complementarity determining region CDRs:
  • amino acid sequence is the CDR2' of KVS.
  • the amino acid sequence of any one of the above amino acid sequences further comprises at least one of optionally adding, deleting, modifying and/or substituting (eg, 1-3, preferably 1-2, more preferably 1) amino acid and a derivative sequence capable of retaining IL-17A binding affinity.
  • the light chain variable region further comprises a human FR region or a murine FR region.
  • the light chain variable region has the amino acid sequence set forth in SEQ ID NO: 2.
  • the light chain variable region has the amino acid sequence set forth in SEQ ID NO: 6.
  • a light chain of an antibody having a light chain variable region according to the third aspect of the invention.
  • the light chain of the antibody further comprises a light chain constant region.
  • the light chain constant region is of human, murine or rabbit origin.
  • an antibody having:
  • the antibody has: a heavy chain according to the second aspect of the invention; and/or a light chain according to the fourth aspect of the invention.
  • the antibody of the EC affinity human IL-17A protein (preferably wild type) 50 of 5-50ng / ml.
  • the antibody is selected from the group consisting of an animal-derived antibody, a chimeric antibody, a humanized antibody, or a combination thereof.
  • the antibody is a diabody, or a single chain antibody.
  • the antibody is a monoclonal antibody.
  • the antibody is a partially or fully humanized monoclonal antibody.
  • the heavy chain variable region sequence of the antibody is as set forth in SEQ ID NO.: 1 or 5; and/or
  • the light chain variable region sequence of the antibody is set forth in SEQ ID NO.: 2 or 6.
  • the heavy chain variable region sequence of the antibody is set forth in SEQ ID NO.: 1; and the light chain variable region sequence of the antibody is set forth in SEQ ID NO.: 2.
  • the heavy chain variable region sequence of the antibody is set forth in SEQ ID NO.: 5; and the light chain variable region sequence of the antibody is set forth in SEQ ID NO.: 6.
  • the antibody is in the form of a drug conjugate.
  • a recombinant protein having:
  • the tag sequence comprises a 6His tag.
  • the recombinant protein comprises a fusion protein.
  • the recombinant protein is a monomer, a dimer, or a multimer.
  • a CAR construct wherein the scFV segment of the monoclonal antibody antigen-binding region of the CAR construct is a binding region that specifically binds to IL-17A, and
  • the scFv has a heavy chain variable region according to the first aspect of the invention and a light chain variable region according to the third aspect of the invention.
  • a recombinant immune cell characterized in that said immune cell expresses an exogenous CAR construct according to the seventh aspect of the invention.
  • the immune cells are selected from the group consisting of NK cells, T cells.
  • the immune cells are from a human or non-human mammal (e.g., a mouse).
  • the invention provides an antibody drug conjugate, characterized in that the antibody drug conjugate comprises:
  • a coupling moiety coupled to the antibody moiety being selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof.
  • the antibody moiety is coupled to the coupling moiety via a chemical bond or linker.
  • an active ingredient selected from the group consisting of the heavy chain variable region according to the first aspect of the invention, the weight according to the second aspect of the invention a light chain variable region according to the third aspect of the invention, a light chain according to the fourth aspect of the invention, or an antibody according to the fifth aspect of the invention, the recombinant according to the sixth aspect of the invention A protein, an immune cell according to the eighth aspect of the invention, the antibody drug conjugate according to the ninth aspect of the invention, or a combination thereof, wherein the active ingredient is used for (a) preparing a detection reagent or A kit; and/or (b) a medicament for the prevention and/or treatment of an IL-17A-related disease.
  • the active ingredient is used to prevent and/or treat an IL-17A related disease.
  • the IL-17A-associated disease is selected from the group consisting of inflammation, autoimmune disease, or a combination thereof; preferably an autoimmune disease.
  • the disease is selected from the group consisting of psoriasis, psoriatic arthritis, ankylosing spondylitis, multiple sclerosis, inflammatory arthritis, or a combination thereof, preferably inflammatory arthritis.
  • the inflammatory arthritis is selected from the group consisting of osteoarthritis, rheumatoid arthritis, rheumatoid arthritis, or a combination thereof, preferably rheumatoid arthritis.
  • the antibody is in the form of a drug conjugate (ADC).
  • ADC drug conjugate
  • the detection reagent or kit is for diagnosing an IL-17A related disease.
  • the detection reagent or kit is for detecting IL-17A protein in a sample.
  • the detection reagent is a test piece.
  • a pharmaceutical composition comprising:
  • an active ingredient the active ingredient being selected from the group consisting of a heavy chain variable region according to the first aspect of the invention, a heavy chain according to the second aspect of the invention, according to the third aspect of the invention a light chain variable region, a light chain according to the fourth aspect of the invention, or an antibody according to the fifth aspect of the invention, the recombinant protein according to the sixth aspect of the invention, according to the eighth aspect of the invention Immunocyte, an antibody drug conjugate according to the ninth aspect of the invention, or a combination thereof;
  • the pharmaceutical composition is a liquid formulation.
  • the pharmaceutical composition is an injection.
  • a polynucleotide is provided, the polynucleotide encoding a polypeptide selected from the group consisting of:
  • the invention provides a vector comprising the polynucleotide of the twelfth aspect of the invention.
  • the vector comprises: a bacterial plasmid, a bacteriophage, a yeast plasmid, a plant cell virus, a mammalian cell virus such as an adenovirus, a retrovirus, or other vector.
  • a genetically engineered host cell comprising the vector or genome according to the thirteenth aspect of the present invention, which is integrated with the twelfth aspect of the present invention, is provided Polynucleotide.
  • a method of detecting IL-17A protein in a sample (including a diagnostic or non-diagnostic) in vitro comprising the steps of:
  • a test panel comprising: a substrate (support plate) and a test strip, the test strip comprising the antibody according to the fifth aspect of the invention or The immunoconjugate of the ninth aspect of the invention.
  • a kit of the present invention characterized in that the kit comprises:
  • a first container comprising the antibody of the fifth aspect of the invention.
  • the kit contains the test plate according to the sixteenth aspect of the invention.
  • a method for preparing a recombinant polypeptide comprising:
  • a method of the present invention comprising: administering an antibody according to the fifth aspect of the present invention, an antibody of the antibody, to a subject in need thereof a drug conjugate, or a CAR-T cell expressing the antibody, or a combination thereof.
  • Figure 1 shows the effect of dexamethasone and humanized anti-IL-17A antibody on imiquimod-induced psoriasis in mice.
  • the present inventors have unexpectedly obtained an anti-IL-17A monoclonal antibody having extremely excellent affinity and specificity, and a humanized antibody obtained based on the antibody, through extensive and intensive research.
  • Antibodies of the invention bind with high specificity can be IL-17A antigen, which has high affinity (ELISA assay which EC 50 of about 15.4ng / ml), and significantly inhibit the binding of IL-17A and IL-17 receptor, and There are no visible side effects to the mammal itself.
  • the present invention has been completed on this basis.
  • conjugate refers to a soluble receptor or fragment thereof or analog thereof, or an antibody or fragment thereof, or an analog thereof, that is capable of binding to a target.
  • IL-17A conjugate refers to an antibody or a fragment thereof or an analog thereof which specifically recognizes IL-17A and binds to IL-17A.
  • administering and “treating” mean that the exogenous drug, therapeutic, diagnostic, or composition is applied to an animal, human, subject, cell, tissue, organ, or biological fluid.
  • administering and “treatment” can refer to treatment, pharmacokinetics, diagnostics, research, and experimental methods.
  • the treatment of the cells includes contact of the reagents with the cells, contact of the reagents with the fluid, and contact of the fluid with the cells.
  • administering also mean in vitro and ex vivo treatment by reagents, diagnostics, binding compositions or by another cell.
  • Treatment when applied to a human, animal or research subject, refers to therapeutic treatment, prophylactic or preventive measures, research and diagnosis; includes IL-17A conjugates with humans or animals, subjects, cells, tissues, Contact with physiological compartments or physiological fluids.
  • treating refers to the administration of a therapeutic agent for internal or external use to a patient, comprising any of the IL-17A conjugates of the invention, and compositions thereof, which have one or more symptoms of the disease, and are known Therapeutic agents have a therapeutic effect on these symptoms.
  • the patient is administered in an amount (a therapeutically effective amount) of a therapeutic agent effective to alleviate the symptoms of one or more diseases.
  • the term “optional” or “optionally” means that the subsequently described event or circumstance may occur but does not necessarily have to occur.
  • “optionally comprising 1-3 antibody heavy chain variable regions” means that the antibody heavy chain variable region of a particular sequence may have, but is not required to be, one, two or three.
  • antibody refers to an immunoglobulin which is a tetrapeptide chain structure formed by the joining of two identical heavy chains and two identical light chains through interchain disulfide bonds.
  • the immunoglobulin heavy chain constant region has different amino acid composition and arrangement order, so its antigenicity is also different. Accordingly, immunoglobulins can be classified into five classes, or different types of immunoglobulins, namely, IgM, IgD, IgG, IgA, and IgE, and the heavy chain constant regions corresponding to different classes of immunoglobulins are respectively referred to as ⁇ . , ⁇ , ⁇ , ⁇ , and ⁇ .
  • IgG stands for the most important class of immunoglobulins and can be divided into four subclasses due to differences in chemical structure and biological function: IgG1, IgG2, IgG3 and IgG4.
  • Light chains are divided into kappa or lambda chains by the constant region.
  • the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to those skilled in the art.
  • variable region The sequence of about 110 amino acids near the N-terminus of the antibody heavy and light chains varies greatly, being the variable region (V region); the remaining amino acid sequence near the C-terminus is relatively stable and is a constant region (C region).
  • the variable region includes three hypervariable regions (HVR) and four relatively conserved FR regions (FR). The amino acid sequences of the four FRs are relatively conservative and are not directly involved in the binding reaction.
  • the three hypervariable regions determine the specificity of the antibody, also known as the complementarity determining region (CDR).
  • Each of the light chain variable region (LCVR) and the heavy chain variable region (HCVR) consists of three CDR regions and four FR regions, and the order from the amino terminus to the carboxy terminus is FR1, CDR1, FR2, CDR2. FR3, CDR3, FR4.
  • the three CDR regions of the light chain i.e., the light chain hypervariable region (LCDR), refer to LCDR1, LCDR2, and LCDR3;
  • the three CDR regions of the heavy chain, the heavy chain hypervariable region (HCDR) refer to HCDR1, HCDR2, and HCDR3.
  • the CDR amino acid residues of the LCVR and HCVR regions of the antibodies or antigen-binding fragments of the invention conform to known Kabat numbering rules (LCDR1-3, HCDR2-3) in number and position, or in accordance with the kabat and chothia numbering rules (HCDR1) ).
  • the four FR regions in the native heavy and light chain variable regions are generally in a beta-sheet configuration, joined by three CDRs forming a linker, and in some cases may form a partial beta sheet structure.
  • the CDRs in each chain are closely joined together by the FR region and together with the CDRs of the other chain form the antigen binding site of the antibody.
  • the amino acid sequence of the same type of antibody can be compared to determine which amino acids constitute the FR or CDR regions.
  • the constant regions are not directly involved in the binding of the antibody to the antigen, but they exhibit different effector functions, such as antibody-dependent cytotoxicity of the participating antibodies.
  • the term "antigen-binding fragment” refers to a Fab fragment having an antigen-binding activity, a Fab' fragment, a F(ab')2 fragment, or a single Fv fragment.
  • the Fv antibody contains the antibody heavy chain variable region, the light chain variable region, but no constant region, and has the smallest antibody fragment of the entire antigen binding site.
  • Fv antibodies also comprise a polypeptide linker between the VH and VL domains and are capable of forming the desired structure for antigen binding.
  • antigenic determinant refers to a three-dimensional spatial site that is discrete on an antigen and that is recognized by an antibody or antigen-binding fragment of the invention.
  • the present invention encompasses not only intact antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by antibodies with other sequences. Accordingly, the invention also includes fragments, derivatives and analogs of the antibodies.
  • antibodies include murine, chimeric, humanized or fully human antibodies prepared by techniques well known to those skilled in the art.
  • Recombinant antibodies such as chimeric and humanized monoclonal antibodies, including human and non-human portions, can be prepared using recombinant DNA techniques well known in the art.
  • the term "monoclonal antibody” refers to an antibody that is secreted from a clone of a single cell source. Monoclonal antibodies are highly specific and target a single epitope.
  • the cell may be a eukaryotic, prokaryotic or phage clonal cell line.
  • chimeric antibody is obtained by splicing a V region gene of a murine antibody with a C region gene of a human antibody into a chimeric gene, and then inserting the vector into an antibody molecule expressed by the host cell. It not only retains the high specificity and affinity of the parental mouse antibody, but also enables its human Fc segment to effectively mediate biological effects.
  • humanized antibody is a variable region engineered form of a murine antibody of the invention having a CDR region derived from (or substantially derived from) a non-human antibody, preferably a mouse monoclonal antibody. And FR regions and constant regions derived substantially from human antibody sequences; the CDR region sequences of the murine antibodies are grafted onto different types of human germline antibody framework sequences. Because CDR sequences are responsible for most of the antibody-antigen interactions, recombinant antibodies that mimic the properties of a particular naturally occurring antibody can be expressed by constructing an expression vector.
  • the antibody may be monospecific, bispecific, trispecific, or more multiple specificity.
  • the antibody of the present invention further includes a conservative variant thereof, which means that there are up to 10, preferably up to 8, more preferably up to 5, optimally compared to the amino acid sequence of the antibody of the present invention. Up to 3 amino acids are replaced by amino acids of similar or similar nature to form a polypeptide. These conservative variant polypeptides are preferably produced by amino acid substitution according to Table A.
  • IL-17A generally refers to native or recombinant human IL-17A, as well as non-human homologs of human IL-17A. Unless otherwise indicated, the molar concentration of IL-17A was calculated using the molecular weight of the homodimer of IL-17A (eg, 30 KDa for human IL-17A).
  • human IL-17A (huIL-17A) refers to the mature form of human IL-17A protein accession numbers NP-002180 and AAT22064 (ie residues 24-155), as well as its natural variants and polymorphisms. Sex.
  • the present invention provides a highly specific and high affinity antibody against IL-17A comprising a heavy chain and a light chain, the heavy chain comprising a heavy chain variable region (VH) amino acid sequence, the light chain comprising a light chain Variable region (VL) amino acid sequence.
  • VH heavy chain variable region
  • VL light chain Variable region
  • the respective CDRs of the heavy chain variable region (VH) amino acid sequence and the light chain variable region (VL) amino acid sequence are selected from the group consisting of:
  • the sequence formed by adding, deleting, modifying and/or substituting at least one amino acid sequence preferably has a homology of at least 80%, preferably at least 85%, more preferably at least 90. %, optimally at least 95% of the amino acid sequence.
  • the antibody of the present invention may be a double-stranded or single-chain antibody, and may be selected from an animal-derived antibody, a chimeric antibody, a humanized antibody, more preferably a humanized antibody, a human-animal chimeric antibody, and more preferably a whole human. Sourced antibodies.
  • the antibody derivative of the present invention may be a single chain antibody, and/or an antibody fragment such as Fab, Fab', (Fab') 2 or other known antibody derivatives in the field, and IgA, IgD, IgE. Any one or more of IgG and IgM antibodies or antibodies of other subtypes.
  • the animal is preferably a mammal, such as a mouse.
  • the antibody of the invention may be a murine antibody, chimeric antibody, humanized antibody, CDR grafted and/or modified antibody that targets human IL-17A.
  • any one or more of the above SEQ ID No.: 7, 8 and 9 or which have been added, deleted, modified and/or substituted for at least one amino acid have IL-
  • the 17A binding affinity sequence is located in the CDR region of the heavy chain variable region (VH).
  • VL light chain variable region
  • VH CDR1, CDR2, CDR3 are each independently selected from any one or more of SEQ ID No.: 7, 8, and 9, or they are added, deleted, modified And/or a sequence having IL-17A binding affinity substituted for at least one amino acid;
  • VL CDR1, CDR2, CDR3 are each independently selected from any one of SEQ ID No.: 10, amino acid sequence: KVS and SEQ ID No.: Or a sequence of several sequences, or a sequence having IL-17A binding affinity for addition, deletion, modification and/or substitution of at least one amino acid.
  • the number of amino acids added, deleted, modified and/or substituted is preferably not more than 40%, more preferably not more than 35%, more preferably 1-33% of the total amino acid number of the initial amino acid sequence. More preferably, it is 5-30%, more preferably 10-25%, and still more preferably 15-20%.
  • the number of amino acids added, deleted, modified and/or substituted is usually 1, 2, 3, 4 or 5, preferably 1-3, more preferably 1-2. The best is 1.
  • any method suitable for the production of monoclonal antibodies can be used to produce the anti-IL-17A antibodies of the invention.
  • an animal can be immunized with a linked or naturally occurring IL-17A homodimer or a fragment thereof. Suitable immunization methods can be used, including adjuvants, immunostimulants, repeated booster immunizations, and one or more routes can be used.
  • IL-17 can be used as an immunogen (antigen) for the production of a non-human antibody specific for IL-17A, and the biological activity of the antibody is screened.
  • the stimulating immunogen can be a full length mature human IL-17A, including a native homodimer, or a peptide containing a single/multiple epitope.
  • the immunogen can be used alone or in combination with one or more immunogenic enhancers known in the art.
  • the immunogen can be purified from natural sources or produced in genetically modified cells.
  • the DNA encoding the immunogen can be genomic or non-genomic (e.g., cDNA) in its source.
  • the DNA encoding the immunogen can be expressed using a suitable genetic vector including, but not limited to, an adenoviral vector, an adeno-associated viral vector, a baculovirus vector, a material, and a non-viral vector.
  • a suitable genetic vector including, but not limited to, an adenoviral vector, an adeno-associated viral vector, a baculovirus vector, a material, and a non-viral vector.
  • Example 1 An exemplary method of producing an anti-human IL-17A antibody of the invention is described in Example 1.
  • the humanized antibody can be selected from any class of immunoglobulins, including IgM, IgD, IgG, IgA, and IgE.
  • the antibody is an IgG antibody, and an IgG1 subtype is used. Optimization of the necessary constant domain sequences is readily achieved by screening antibodies using the biological assays described in the Examples below to produce the desired biological activity.
  • any type of light chain can be used in the compounds and methods herein.
  • kappa, lambda chains or variants thereof are useful in the compounds and methods of the invention.
  • Example 4 An exemplary method of humanizing an anti-human IL-17A antibody of the invention is described in Example 4.
  • sequence of the DNA molecule of the antibody or fragment thereof of the present invention can be obtained by a conventional technique such as PCR amplification or genomic library screening.
  • the coding sequences of the light and heavy chains can also be fused together to form a single chain antibody.
  • the recombinant sequence can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it to a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.
  • synthetic sequences can be used to synthesize related sequences, especially when the fragment length is short.
  • a long sequence of fragments can be obtained by first synthesizing a plurality of small fragments and then performing the ligation.
  • the DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art.
  • the invention also relates to vectors comprising the appropriate DNA sequences described above, as well as appropriate promoters or control sequences. These vectors can be used to transform appropriate host cells to enable them to express proteins.
  • the host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • Preferred animal cells include, but are not limited to, CHO-S, CHO-K1, HEK-293 cells.
  • the step of transforming a host cell with recombinant DNA as described in the present invention can be carried out by techniques well known in the art.
  • the obtained transformant can be cultured by a conventional method, and the transformant expresses the polypeptide encoded by the gene of the present invention. Depending on the host cell used, it is cultured under appropriate conditions using a conventional medium.
  • the resulting host cells are cultured under conditions suitable for expression of the antibody of the invention.
  • immunoglobulin purification steps such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography, etc.
  • the antibodies of the present invention are purified by conventional separation and purification means well known to those skilled in the art.
  • the resulting monoclonal antibodies can be identified by conventional means.
  • the binding specificity of a monoclonal antibody can be determined by immunoprecipitation or in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunosorbent assay
  • the invention provides the use of an antibody of the invention, for example, for the preparation of a diagnostic preparation, or for the preparation of a medicament for the prevention and/or treatment of a disease associated with IL-17A.
  • the IL-17A-related diseases include inflammatory diseases, autoimmune diseases, and the like, including but not limited to psoriasis, psoriatic arthritis, ankylosing spondylitis, multiple sclerosis, inflammatory bowel disease (such as Crohn Disease, ulcerative colitis, etc.), osteoarthritis, rheumatoid arthritis (RA), rheumatoid arthritis or osteoporosis, inflammatory fibrosis (eg scleroderma, pulmonary fibrosis and sclerosis), Asthma (including allergic asthma), allergies, and cancer.
  • the composition is a pharmaceutical composition comprising the above antibody or active fragment thereof or a fusion protein thereof or an ADC thereof or a corresponding CAR-T cell, and a pharmaceutically acceptable carrier.
  • these materials can be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium wherein the pH is usually from about 5 to about 8, preferably from about 6 to about 8, although the pH may be The nature of the formulation and the condition to be treated vary.
  • the formulated pharmaceutical compositions can be administered by conventional routes including, but not limited to, intratumoral, intraperitoneal, intravenous, or topical administration.
  • the antibody of the present invention may also be a cell therapy for expression of a nucleotide sequence in a cell, for example, the antibody is used for chimeric antigen receptor T cell immunotherapy (CAR-T) and the like.
  • CAR-T chimeric antigen receptor T cell immunotherapy
  • the pharmaceutical composition of the present invention can be directly used for binding to IL-17A protein molecules, and thus can be used for the prevention and treatment of diseases associated with IL-17A.
  • other therapeutic agents can be used simultaneously.
  • the pharmaceutical composition of the present invention contains a safe and effective amount (e.g., 0.001 to 99% by weight, preferably 0.01 to 90% by weight, more preferably 0.1 to 80% by weight) of the above-mentioned monoclonal antibody (or a conjugate thereof) of the present invention and pharmacy An acceptable carrier or excipient.
  • Such carriers include, but are not limited to, saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical preparation should be matched to the mode of administration.
  • the pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants.
  • compositions such as injections and solutions are preferably prepared under sterile conditions.
  • the amount of active ingredient administered is a therapeutically effective amount, for example from about 1 microgram per kilogram body weight to about 5 milligrams per kilogram body weight per day.
  • the polypeptides of the invention may also be used with other therapeutic agents.
  • a safe and effective amount of the pharmaceutical composition is administered to the mammal, wherein the safe and effective amount is usually at least about 10 micrograms per kilogram of body weight, and in most cases no more than about 50 milligrams per kilogram of body weight.
  • the dose is from about 10 micrograms per kilogram of body weight to about 20 milligrams per kilogram of body weight.
  • specific doses should also consider factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled physician.
  • the antibodies of the invention can be used in detection applications, for example for detecting samples, to provide diagnostic information.
  • the sample (sample) used includes cells, tissue samples, and biopsy specimens.
  • biopsy shall include all types of biopsies known to those skilled in the art.
  • the biopsy used in the present invention may include a tissue sample prepared, for example, by an endoscopic method or a puncture or needle biopsy of an organ.
  • Samples used in the present invention include fixed or preserved cell or tissue samples.
  • the invention also provides a kit comprising an antibody (or a fragment thereof) of the invention, and in a preferred embodiment of the invention, the kit further comprises a container, instructions for use, a buffer, and the like.
  • the antibody of the invention may be immobilized on a test plate.
  • the antibody of the present invention has excellent biological activity and specificity and has a high affinity (the EC 50 can be as high as about 10-20 ng/ml by ELISA). In addition, it has a good binding affinity for IL-17A, and has no binding to other family members IL-17B, IL-17D, IL-17E and IL-17F, and can be used as an antibody targeting IL-17A.
  • the humanized antibody of the present invention not only has better affinity with IL-17A but also has lower immunogenicity than the murine antibody.
  • the antibody of the present invention can significantly inhibit the binding of IL-17A to the IL-17 receptor without any visible side effects to the mammal itself.
  • the antibody of the present invention has a certain affinity with IL-17A of some non-human mammals, and is convenient for testing and quality control in animal models.
  • human IL-17A protein was used as an antigen.
  • BALB/c mice were subjected to multi-subcutaneous immunization, and the serum titer of the immunized mice was monitored. After the requirement, the mouse spleen cells and myeloma were taken (Sp2/ 0) Hybridomas polyclonal cells obtained by fusion of cells and screening by HAT.
  • High specific binding polyclonal was screened by ELISA, monoclonal culture was performed, and highly specific monoclonal cells were screened by ELISA; IL-6 release assay was performed by HT1080 cells, and cell function was screened. The effect of the monoclonal cell line, and then the affinity and half-life were analyzed by the Biacore method, and finally the monoclonal cells expressing IL-17A were obtained.
  • Human IL-17A was prepared into 1 ⁇ g/ml coating solution with CBS, 50 ⁇ L/well was added to the enzyme standard plate, and coated at 2 ⁇ 8°C for more than 12 hours. The residual liquid of the plate was discarded, and 3% milk was added, 200 ⁇ L per well, room temperature. Closed for 1 hour. One well of not less than 200 ⁇ L of PBST was added to each well, and the hybridoma supernatant was diluted to 100 ⁇ g/ml, and 10 gradients were further diluted 10 times, and 100 ⁇ L/well was added to the plate.
  • hybridoma 14F10/9F6 (or an antibody produced thereof) has a very high binding activity to human IL-17a.
  • the DNA sequence encoding the variable region of the mouse antibody expressed by hybridoma 7D6/5H8 was determined based on the 5' RACE technique. Briefly, gene-specific cDNAs for heavy and light chains were prepared using the SMART 5' RACE Synthesis Kit (TAKARA, No. 634859) according to the manufacturer's instructions. The PCR product was analyzed by agarose gel electrophoresis. The variable region size of both the heavy and light chains is approximately 500 base pairs. The amplified amplified PCR product obtained by the reaction was cloned into the vector pEASY-Blunt Simple plasmid (Beijing Full Gold, No. CB111-02) and transformed into Stellar E.
  • CDR1, CDR2, and CDR3 are underlined (SEQ ID NO.: 7, 8, and 9).
  • CDR1' amino acid sequence: KVS and SEQ ID NO.: 11).
  • Chimeric heavy and light chains were constructed by ligating PCR cloned mouse 7D6/5H8 VH and VL region cDNAs to human IgGl and k constant regions, respectively.
  • the 5' and 3' ends of the mouse cDNA sequence were modified with PCR primers designed to add appropriate leader sequences to each strand and to increase restriction sites that enable cloning into the existing recombinant antibody expression vector pHB-Fc. .
  • the pHB-Fc plasmid vector was prepared as follows: the pcDNA/HA-FLAG (Accession#: FJ524378) vector was used as the starting plasmid, and the restriction region EcoRI was followed by the constant region sequence of human IgG1 or k, endonuclease HindIII.
  • the human cytomegalovirus (HCMV) promoter sequence (Accession#: X17403) was added to the front of the ampicillin tolerance gene, and the Chinese hamster glutamine synthetase gene was added in front of the HCMV promoter (Accession#: X03495). .
  • the host cell used for protein expression was CHO-K1 cells (Cat# CCL-61) purchased from ATCC. The cells were acclimated into CHO-K1 cells that were cultured in suspension in serum-free medium (EX-CELLTM 302) after a series of domestication steps. Using the cells, the constructed light and heavy chain recombinant expression plasmids were transferred into cells by electroporation. Put in the incubator for 3-5 days. The antibody concentration from the CHO-K1 transfection supernatant was measured by indirect ELISA. This shows that transfected CHO-K1 cells secrete approximately 30 mg/L of chimeric antibody.
  • Novartis humanized anti-IL-17 antibody (Novartis mAb) as a positive control was cloned according to the humanized sequence provided in US 7,807,155 B2 (AIN 457) and transiently transfected for expression.
  • the method was the same as in Example 1.
  • the chimeric antibody was used in place of the hybridoma supernatant, and the HRP-conjugated rabbit anti-human IgG Fc antibody (Luoyang Qiongtong Experimental Material Center) was used instead of HRP-conjugated goat anti-mouse IgG Fc. Recombinant human and murine IL-17A binding activity.
  • chimeric antibody capable of binding to human IL-17A may be combined with the murine IL-17A, 50 EC and calculated values were less than 40ng / ml.
  • variable chain sequences of antibodies are compared to the available sequences in the NCBI protein database, and by identification and analysis, the human framework on which the CDR-grafted heavy and light chains are suitably constructed is finally determined.
  • one preferred FR region is the humanized FR region derived from hu-VH (SEQ ID NO: 3) and the light chain is hu-VL (SEQ ID NO: 4):
  • the humanized point mutant antibody expression plasmid was amplified and constructed by PCR.
  • the humanized point mutant antibody expression plasmid was separately expressed by CHO-K1 (ATCC, NO. CCL-61) cells, and purified to obtain a humanized antibody protein.
  • a humanized antibody (designated "HB0017 antibody”) having excellent performance was obtained by ELISA, receptor binding inhibition assay, Biacore and cell activity assay.
  • VH and VL sequences of the HB0017 antibody are shown in SEQ ID NO.: 5 and 6, respectively:
  • the method was the same as in Example 1.
  • the hybridoma supernatant was replaced with the HB0017 antibody, and the HRP-conjugated rabbit anti-human IgG Fc antibody (Luoyang Qiongtong Experimental Material Center) was used instead of the HRP-conjugated goat anti-mouse IgG Fc. IL-17A binding activity.
  • the antibody of the present invention has a lower EC 50 value and a stronger binding activity to human IL-17A than the positive control antibody.
  • the antigen-antibody binding ability of the anti-IL-17A antibody to IL-17A of different species was determined by the ELSIA method.
  • the method was the same as in Example 1.
  • the hybridoma supernatant was replaced with HB0017 antibody, and the HRP-conjugated rabbit anti-human IgG Fc antibody (Luoyang Qiongtong Experimental Material Center) was used instead of HRP-conjugated goat anti-mouse IgG Fc.
  • IL-17A, cynomolgus IL-17A, mouse IL-17A and rat IL-17A binding activity was used instead of HRP-conjugated goat anti-mouse IgG Fc.
  • the humanized monoclonal antibody HB0017 antibody of the present invention binds to mouse, macaque and rat IL-17A in addition to binding to human IL-17A, and facilitates clinical animal experiments. .
  • the antigen-antibody binding kinetics and affinity were determined using the BIACORE method.
  • a human antibody capture antibody Human Antibody Capture Antiboy
  • an anti-human capture-CM5 chip Anti-Human Capture-CM5 chip
  • the mixture was equilibrated at room temperature for 20 to 30 minutes, and the chip was loaded into the instrument. Dilute the antigen with equilibration buffer, dilute the antigen with a 10 ml initial dilution of 5 concentration gradients, and set 2 zero concentrations (ie equilibration buffer) and one replicate concentration (typically the lowest concentration repeat).
  • the antibody sample was diluted to the experimental working concentration with an equilibration buffer and sealed at 2 to 8 °C. After the sample analysis is completed, the corresponding analysis program is used to analyze the data, confirming that there is no obvious reference binding, using the kinetics, 1:1 binding model (Kinetics, 1:1 binding modle), and fitting analysis to obtain the kinetics of the sample. parameter.
  • the affinity constant (KD(M)) of human IL-17A showed that the affinity of the HB0017 antibody of the present invention was nearly an order of magnitude higher than that of the positive control antibody, and had a stronger affinity.
  • IL-17A stimulates HT1080 to produce IL-6 under the synergistic effect of TNF ⁇ or TNF ⁇ .
  • the effect of IL-17A is neutralized by the HB0017 antibody of the present invention, thereby further inhibiting the expression level of IL-6.
  • the biological activity of the antibodies of the invention is demonstrated.
  • the confluence is 80% to 90%, and the experiment can be carried out.
  • Discard the culture medium in the cell culture flask wash it once with PBS, add an appropriate amount of 0.25% trypsin to 0.02% EDTA digest, and put it into the cell culture incubator. After 1 to 2 minutes, tap the culture flask to remove the cells. The growth medium was added to prepare a single cell suspension, and the cells were centrifuged at 1000 rpm for 5 min. The growth medium was resuspended and counted.
  • the cells were diluted with a growth medium to a dilution of 2.5 ⁇ 10 5 /ml, and 50 ⁇ l/well was added to the cell culture plate, and then cultured in a cell culture incubator for 5 hours or more to adhere the cells.
  • the antibody and the positive control were diluted to 200 ⁇ g/ml with blank medium (DMEM glutamine), and 8 gradient concentrations were further diluted 3.16 times at 200 ⁇ g/ml.
  • Recombinant human IL-17A was diluted to 40 ng/ml with blank medium, and recombinant human TNF- ⁇ was diluted to 20 ng/ml with blank medium.
  • the diluted recombinant human IL-17A and recombinant human TNF- ⁇ were mixed at a volume ratio of 1:1.
  • 9 gradient concentrations of the antibody or the positive control were mixed at a volume ratio of 1:1, and incubated at 37 ° C for 1 hour.
  • the method was the same as in Example 1.
  • the hybridoma supernatant was replaced with HB0017 antibody, and the HRP-conjugated rabbit anti-human IgG Fc antibody (Luoyang Qiongtong Experimental Material Center) was used instead of HRP-conjugated goat anti-mouse IgG Fc.
  • This example was carried out using the imiquimod-induced mouse psoriasis model to verify whether the antibodies of the invention can be improved in vivo by blocking IL-17 binding to the IL-17 receptor (eg, hIL-17RA). Symptoms or related indicators of psoriasis.
  • IL-17 receptor eg, hIL-17RA
  • test method is as follows;
  • each of the above groups was intraperitoneally injected once a day on the day of grouping and on the second day of the model (day 2).
  • day 1 On the day of sensitization (day 1), each group of mice applied about 62.5 mg of imiquimod cream (5%) to the right ear and back skin for 5 consecutive days.
  • the thickness of the right ear of the mouse was measured by a spiral micrometer every day from the day of sensitization, and the thickness of the ear swelling of the mouse was calculated by using the thickness of the right ear of day 0 as a control.
  • the mice were weighed daily, and the skin scale, induration, and erythema were observed. The score was graded using a 4-level scale: 0 points, no disease; 1 point, slight; 2 points, moderate; 3 points, severe; 4 points ,very serious.

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Abstract

本发明提供了一种靶向于白介素17A(IL-17A)的抗体、其制备方法和应用。具体地，本发明提供了一种新的抗IL-17A单克隆抗体。本发明的抗体能够高特异性地结合IL-17A抗原，具有很高的亲和力及很低的免疫原性，并且用于制备预防或治疗IL-17A相关的疾病如各种炎症或自身免疫疾病的药物。

Description

靶向于白介素17A的抗体、其制备方法和应用 技术领域
本发明涉及医药领域，具体地涉及靶向于白介素17A(也称IL-17)的抗体、其制备方法和应用。
背景技术
迄今为止，IL-17家族已有6个成员被发现：IL-17A(IL-17)、IL-17B、IL-17C、IL-17D、IL-17E(IL-25)、IL-17F。这些白介素-17细胞因子可以结合到相对应的受体上，从而介导不同的炎症反应。
IL-17A最初被发现是由激活的CD4 +T细胞分泌。这一类特征性分泌IL-17A的T细胞亚群被称为Th17细胞。除了Th17细胞以外，细胞毒性CD8 +T细胞(Tc17)、γδT细胞、自然杀伤T细胞(NKT-17)和B细胞也能在特定条件下表达IL-17A。先天免疫细胞，包括单核细胞、中性粒细胞、自然杀伤细胞和淋巴组织诱导样(Lti-like)细胞也可以产生IL-17A。最近，在一项关于锥虫感染的研究中发现，B细胞也可以产生IL-17A。一些非免疫细胞，比如肠道潘氏细胞和肠上皮细胞也可以在应激情况下产生IL-17A。由于Th17细胞在体内的分布最广，而且在炎症反应中有广泛作用，所以，通常认为它还是IL-17A的主要来源细胞。而产生IL-17A的先天细胞主要作为机体的早期防御细胞参与宿主的抗感染免疫反应。
IL-17A是由155个氨基酸的两条链通过二硫键连接的同源二聚体，分子量为35kDa。IL-17的结构由23个氨基酸组成的信号肽(AA)及123个氨基酸链区域构成。
IL-17结合I型细胞表面的受体称为IL-17R，其中有至少三种：IL-17RA、IL-17RB和IL-17RC。IL-17A与IL-17F以同源二聚体或异源二聚体的形式结合IL-17RA和IL-17RC受体复合物来转导信号，并参与机体自身免疫疾病、多种炎症反应以及宿主抗感染免疫反应。IL-17C结合IL-17RA和IL-17RE受体复合物激活下游信号，促进机体抗感染免疫、自身免疫疾病和炎症反应。IL-17B被发现可以结合IL-17RB，但其下游信号仍不清楚。IL-17RB也可与IL-17RA形成受体复合物介导IL-17E引起II型免疫反应。Il-17E也被报道可以促进肿瘤细胞的凋亡。IL-17D的受体及下游信号以及孤儿受体IL-17RD的配体及下游信号目前仍不清楚。
IL-17A主要诱导包括上皮细胞和基质细胞在内的非造血来源细胞的信号激活。IL-17A诱导表达的多种炎症因子和趋化因子可以促进多种免疫细胞的募集，从而对自身免疫病起到促进作用。研究发现，IL-17A和IL-17F，及其主要分泌T细胞亚群Th17细胞，在多种自身免疫疾病中也发挥着重要作用，这其中包括自身免疫疾病，如类风湿性关节炎(rheumatoid arthritis,RA)和多发性硬化(multiple sclerosis,MS)，以及炎症性大肠病(inflammatory boweldisease,IBD)、银屑病(psoriasis)、***性红斑狼疮(systemic lupus  erythematosus,SLE)和I型糖尿病(type1diabetes,T1D)。
IL-17A与IL-17F主要通过诱导靶细胞表达多种炎症因子和趋化因子来发挥其促进炎症反应的功能。IL-17A与细胞表面受体IL-17RA结合，招募IL-17RC形成异源二聚体，介导下游信号通路。IL-17与其受体结合后能激活TRAF6(TNF-receptor associated factor 6)。IL-17与IL-1及TNF共用相同的转录途径，它可以激活NF-kB和3个MAP(丝裂原活化蛋白)酶，包括ERK1、ERK2、JNK、p38。这些通路在滑膜细胞和软骨细胞中都有发现。
因此，鉴于IL-17A在各类相关疾病中作用和功能，本领域仍然需要开发适于治疗患者的改善的抗IL-17特异性抗体。
发明内容
本发明目的是提供了一种抗IL-17A的抗体、其制备方法和用途。
本发明的第一方面，提供了一种抗体的重链可变区，所述的重链可变区包括以下三个互补决定区CDR：
SEQ ID NO:7所示的CDR1，
SEQ ID NO:8所示的CDR2，和
SEQ ID NO:9所示的CDR3。
在另一优选例中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个(如1-3个，较佳地1-2个，更佳地1个)氨基酸并能够保留IL-17A结合亲和力的衍生序列。
在另一优选例中，所述重链可变区还包括人源的FR区或鼠源的FR区。
在另一优选例中，所述重链可变区具有SEQ ID NO:1所示的氨基酸序列。
在另一优选例中，所述重链可变区具有SEQ ID NO:5所示的氨基酸序列。
本发明的第二方面，提供了一种抗体的重链，所述的重链具有如本发明第一方面所述的重链可变区。
在另一优选例中，所述的抗体的重链还包括重链恒定区。
在另一优选例中，所述的重链恒定区为人源、鼠源或兔源的。
本发明的第三方面，提供了一种抗体的轻链可变区，所述的轻链可变区包括以下三个互补决定区CDR：
SEQ ID NO:10所示的CDR1’，
氨基酸序列为KVS的CDR2’，和
SEQ ID NO:11所示的CDR3’。
在另一优选例中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个(如1-3个，较佳地1-2个，更佳地1个)氨基酸并能够保留IL-17A结合亲和力的衍生序列。
在另一优选例中，所述轻链可变区还包括人源的FR区或鼠源的FR区。
在另一优选例中，所述轻链可变区具有SEQ ID NO:2所示的氨基酸序列。
在另一优选例中，所述轻链可变区具有SEQ ID NO:6所示的氨基酸序列。
本发明的第四方面，提供了一种抗体的轻链，所述的轻链具有如权本发明第三方面所述的轻链可变区。
在另一优选例中，所述的抗体的轻链还包括轻链恒定区。
在另一优选例中，所述的轻链恒定区为人源、鼠源或兔源的。
本发明的第五方面，提供了一种抗体，所述抗体具有：
(1)如本发明第一方面所述的重链可变区；和/或
(2)如本发明第三方面所述的轻链可变区；
或者，所述抗体具有：如本发明第二方面所述的重链；和/或如本发明第四方面所述的轻链。
在另一优选例中，所述抗体对人IL-17A蛋白(优选野生型)的亲和力的EC 50为5-50ng/ml。
在另一优选例中，所述抗体对人IL-17A蛋白(优选野生型)的亲和力的EC 50为15.4ng/ml。
在另一优选例中，所述抗体选自：动物源抗体、嵌合抗体、人源化抗体、或其组合。
在另一优选例中，所述的抗体为双链抗体、或单链抗体。
在另一优选例中，所述的抗体为单克隆抗体。
在另一优选例中，所述的抗体是部分或全人源化的单克隆抗体。
在另一优选例中，所述抗体的重链可变区序列如SEQ ID NO.:1或5所示；和/或
所述的抗体的轻链可变区序列如SEQ ID NO.:2或6所示。
在另一优选例中，所述抗体的重链可变区序列如SEQ ID NO.:1所示；并且所述的抗体的轻链可变区序列如SEQ ID NO.:2所示。
在另一优选例中，所述抗体的重链可变区序列如SEQ ID NO.:5所示；并且所述的抗体的轻链可变区序列如SEQ ID NO.:6所示。
在另一优选例中，所述的抗体为药物偶联物形式。
本发明的第六方面，提供了一种重组蛋白，所述的重组蛋白具有：
(i)如本发明第一方面所述的重链可变区、如本发明第二方面所述的重链、如本发明第三方面所述的轻链可变区、如本发明第四方面所述的轻链、或本发明第五方面所述的抗体；以及
(ii)任选的协助表达和/或纯化的标签序列。
在另一优选例中，所述的标签序列包括6His标签。
在另一优选例中，所述的重组蛋白(或多肽)包括融合蛋白。
在另一优选例中，所述的重组蛋白为单体、二聚体、或多聚体。
本发明的第七方面，提供了一种CAR构建物，其特征在于，所述的CAR构建物的单克隆抗体抗原结合区域的scFV段为特异性结合于IL-17A的结合区，并且所述scFv具有如本发明第一方面所述的重链可变区和如本发明第三方面所述的轻链可变区。
本发明的第八方面，提供了一种重组的免疫细胞，其特征在于，所述的免疫细胞表达外源的如本发明第七方面所述的CAR构建物。
在另一优选例中，所述的免疫细胞选自下组：NK细胞、T细胞。
在另一优选例中，所述的免疫细胞来自人或非人哺乳动物(如鼠)。
本发明的第九方面，提供了一种抗体药物偶联物，其特征在于，所述的抗体药物偶联物含有：
(a)抗体部分，所述抗体部分选自下组：如本发明第一方面所述的重链可变区、如本发明第二方面所述的重链、如本发明第三方面所述的轻链可变区、如本发明第四方面所述的轻链、或本发明第五方面所述的抗体、或其组合；和
(b)与所述抗体部分偶联的偶联部分，所述偶联部分选自下组：可检测标记物、药物、毒素、细胞因子、放射性核素、酶、或其组合。
在另一优选例中，所述的抗体部分与所述的偶联部分通过化学键或接头进行偶联。
本发明的第十方面，提供了一种活性成分的用途，所述活性成分选自下组：如本发明第一方面所述的重链可变区、如本发明第二方面所述的重链、如本发明第三方面所述的轻链可变区、如本发明第四方面所述的轻链、或本发明第五方面所述的抗体、如本发明第六方面所述的重组蛋白、如本发明第八方面所述的免疫细胞、如本发明第九方面所述的抗体药物偶联物、或其组合，其特征在于，所述活性成分用于(a)制备检测试剂或试剂盒；和/或(b)制备预防和/或治疗IL-17A相关疾病的药物。
在另一优选例中，所述活性成分用于预防和/或治疗IL-17A相关疾病。
在另一优选例中，所述IL-17A相关疾病选自下组：炎症、自身免疫疾病、或其组合；优选地为自身免疫疾病。
在另一优选例中，所述的疾病选自下组：银屑病、银屑病关节炎、强直性脊柱炎、多发性硬化症、炎性关节炎、或其组合，优选地为炎性关节炎。
在另一优选例中，所述的炎性关节炎选自下组：骨关节炎、类风湿性关节炎、风湿性关节炎、或其组合，优选地为风湿性关节炎。
在另一优选例中，所述的抗体为药物偶联物(ADC)形式。
在另一优选例中，所述的检测试剂或试剂盒用于诊断IL-17A相关疾病。
在另一优选例中，所述检测试剂或试剂盒用于检测样品中IL-17A蛋白。
在另一优选例中，所述的检测试剂为检测片。
本发明的第十一方面，提供了一种药物组合物，所述的药物组合物含有：
(i)活性成分，所述活性成分选自下组：如本发明第一方面所述的重链可变区、如本发明第二方面所述的重链、如本发明第三方面所述的轻链可变区、如本发明第四方面所述 的轻链、或本发明第五方面所述的抗体、如本发明第六方面所述的重组蛋白、如本发明第八方面所述的免疫细胞、如本发明第九方面所述的抗体药物偶联物、或其组合；以及
(ii)药学上可接受的载体。
在另一优选例中，所述的药物组合物为液态制剂。
在另一优选例中，所述的药物组合物为注射剂。
本发明的第十二方面，提供了一种多核苷酸，所述的多核苷酸编码选自下组的多肽：
(1)如本发明第一方面所述的重链可变区、如本发明第二方面所述的重链、如本发明第三方面所述的轻链可变区、如本发明第四方面所述的轻链、或本发明第五方面所述的抗体；或
(2)如本发明第六方面所述的重组蛋白；
(3)如本发明第七方面所述的CAR构建物。
本发明的第十三方面，提供了一种载体，所述的载体含有如本发明第十二方面所述的多核苷酸。
在另一优选例中，所述的载体包括：细菌质粒、噬菌体、酵母质粒、植物细胞病毒、哺乳动物细胞病毒如腺病毒、逆转录病毒、或其他载体。
本发明的第十四方面，提供了一种遗传工程化的宿主细胞，所述的宿主细胞含有如本发明第十三方面所述的载体或基因组中整合有如本发明第十二方面所述的多核苷酸。
本发明的第十五方面，提供了一种体外检测(包括诊断性或非诊断性)样品中IL-17A蛋白的方法，所述方法包括步骤：
(1)在体外，将所述样品与如本发明第五方面所述的抗体接触；
(2)检测是否形成抗原-抗体复合物，其中形成复合物就表示样品中存在IL-17A蛋白。
本发明的第十六方面，提供了一种检测板，所述的检测板包括：基片(支撑板)和测试条，所述的测试条含有如本发明第五方面所述的抗体或如本发明第九方面所述的免疫偶联物。
本发明的第十七方面，提供了一种试剂盒，其特征在于，所述试剂盒中包括：
(1)第一容器，所述第一容器中含有如本发明第五方面所述的抗体；和/或
(2)第二容器，所述第二容器中含有抗如本发明第五方面所述的抗体的二抗；
或者，所述试剂盒含有如本发明第十六方面所述的检测板。
本发明的第十八方面，提供了一种重组多肽的制备方法，其特征在于，所述方法包括：
(a)在适合表达的条件下，培养如本发明第十四方面所述的宿主细胞；
(b)从培养物中分离出重组多肽，所述的重组多肽是如本发明第五方面所述的抗体或如本发明第六方面所述的重组蛋白。
本发明的第十九方面，提供了一种IL-17A相关疾病的方法，其特征在于，所述方法包括：给需要的对象施用如本发明第五方面所述的抗体、所述抗体的抗体-药物偶联物、或表达所述抗体的CAR-T细胞、或其组合。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了***和人源化抗IL-17A抗体对咪喹莫特诱导的小鼠银屑病的改善效果。
具体实施方式
本发明人通过广泛而深入的研究，经过大量筛选，意外地获得一种具有极其优异的亲和力和特异性的抗IL-17A单克隆抗体，基于该抗体而获得的人源化抗体。本发明抗体能够高特异性地结合IL-17A抗原，其具有很高的亲和力(ELISA测定其EC 50约为15.4ng/ml)，并且显著抑制IL-17A与IL-17受体的结合，而对于哺乳动物本身没有可见的毒副作用。在此基础上完成了本发明。
术语
如本文所用，术语“结合物”是指能够与靶点结合的可溶性受体或其片段或其类似物，或抗体或其片段或其类似物。本发明所述的“IL-17A结合物”，是指能特异性识别IL-17A并与IL-17A结合的抗体或其片段或其类似物。
如本文所用，术语“给予”和“处理”是指外源性药物、治疗剂、诊断剂或组合物应用于动物、人、受试者、细胞、组织、器官或生物流体。“给予”和“处理”可以指治疗、药物代谢动力学、诊断、研究和实验方法。细胞的处理包括试剂与细胞的接触、以及试剂与流体的接触、流体与细胞的接触。“给予”和“处理”还意指通过试剂、诊断、结合组合物或通过另一种细胞体外和离体处理。“处理”当应用于人、动物或研究受试者时，是指治疗处理、预防或预防性措施，研究和诊断；包括IL-17A结合物与人或动物、受试者、细胞、组织、生理区室或生理流体的接触。
如本文所用，术语“治疗”指给予患者内用或外用治疗剂，包含本发明的任何一种IL-17A结合物及其组合物，所述患者具有一种或多种疾病症状，而已知所述治疗剂对这些症状具有治疗作用。通常，以有效缓解一种或多种疾病症状的治疗剂的量(治疗有效量)给予患者。
如本文所用，术语“任选”或“任选地”意味着随后所描述的事件或情况可以发生但不是必须发生。例如，“任选包含1-3个抗体重链可变区”是指特定序列的抗体重链可变区可以有但不是必须有，可以是1个、2个或3个。
抗体
如本文所用，术语“抗体”指免疫球蛋白，是由两条相同的重链和两条相同的轻链通过 链间二硫键连接而成的四肽链结构。免疫球蛋白重链恒定区的氨基酸组成和排列顺序不同，故其抗原性也不同。据此，可将免疫球蛋白分为五类，或称为免疫球蛋白的不同种型，即IgM、IgD、IgG、IgA和IgE，对应于不同类免疫球蛋白的重链恒定区分别称为α、δ、ε、γ、和μ。IgG代表免疫球蛋白中最重要的一类，由于化学结构和生物功能差异，它又可以分为4个子类：IgG1、IgG2、IgG3和IgG4。轻链通过恒定区的不同分为κ或λ链。不同类免疫球蛋白的亚单位结构和三维构型是本领域人员所熟知的。
抗体重链和轻链靠近N端的约110个氨基酸的序列变化很大，为可变区(V区)；靠近C端的其余氨基酸序列相对稳定，为恒定区(C区)。可变区包括3个高变区(HVR)和4个序列相对保守的FR区(FR)。4个FR的氨基酸序列相对比较保守，不直接参与结合反应。3个高变区决定抗体的特异性，又称为互补性决定区(CDR)。每条轻链可变区(LCVR)和重链可变区(HCVR)由3个CDR区和4个FR区组成，从氨基端到羧基端依次排列的顺序为FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4。轻链的3个CDR区，即轻链高变区(LCDR),指LCDR1、LCDR2和LCDR3；重链的3个CDR区，即重链高变区(HCDR),指HCDR1、HCDR2和HCDR3。发明所述的抗体或抗原结合片段的LCVR和HCVR区的CDR氨基酸残基在数量和位置符合已知的Kabat编号规则(LCDR1-3，HCDR2-3)，或者符合kabat和chothia的编号规则(HCDR1)。天然重链和轻链可变区中的四个FR区大致上呈β-折叠构型，由形成连接环的三个CDR相连，在某些情况下可形成部分β折叠结构。每条链中的CDR通过FR区紧密地靠在一起并与另一链的CDR一起形成了抗体的抗原结合部位。可以通过比较同类型的抗体的氨基酸序列来确定是哪些氨基酸构成了FR或CDR区域。恒定区不直接参与抗体与抗原的结合，但是它们表现出不同的效应功能，例如参与抗体的依赖于抗体的细胞毒性。
如本文所用，术语“抗原结合片段”，指具有抗原结合活性的Fab片段，Fab’片段，F(ab’)2片段，或单一Fv片段。Fv抗体含有抗体重链可变区、轻链可变区，但没有恒定区，并具有全部抗原结合位点的最小抗体片段。一般的，Fv抗体还包含VH和VL结构域之间的多肽接头，且能够形成抗原结合所需的结构。
如本文所用，术语“抗原决定簇”指抗原上不连续的，由本发明抗体或抗原结合片段识别的三维空间位点。
本发明不仅包括完整的抗体，还包括具有免疫活性的抗体的片段或抗体与其他序列形成的融合蛋白。因此，本发明还包括所述抗体的片段、衍生物和类似物。
在本发明中，抗体包括用本领域技术人员熟知技术所制备的鼠的、嵌合的、人源化的或者全人的抗体。重组抗体，例如嵌合的和人源化的单克隆抗体，包括人的和非人的部分，可以采用本领域熟知的DNA重组技术制备。
如本文所用，术语“单克隆抗体”指得自单个细胞来源的克隆分泌的抗体。单克隆抗体是高度特异性的，针对单个抗原表位。所述的细胞可能是真核的、原核的或噬菌体的克隆细胞株。
如本文所用，术语“嵌合抗体”是由鼠源性抗体的V区基因与人抗体的C区基因拼接为嵌合基因，然后***载体，转染宿主细胞表达的抗体分子。既保留了亲本鼠抗体的高特异性和亲和力，又使其人源Fc段能有效介导生物学效应功能。
如本文所用，术语“人源化抗体”，是本发明鼠抗的一种可变区改造形式，具有源自(或基本上源自)非人类抗体(优选小鼠单克隆抗体)的CDR区，和基本源自人源抗体序列的FR区和恒定区；即将鼠抗的CDR区序列嫁接到不同类型的人种系抗体构架序列上。因为CDR序列负责大部分的抗体-抗原相互作用，所以可以通过构建表达载体来表达模拟特定天然存在的抗体性质的重组抗体。
在本发明中，抗体可以是单特异性、双特异性、三特异性、或者更多的多重特异性。
在本发明中，本发明的抗体还包括其保守性变异体，指与本发明抗体的氨基酸序列相比，有至多10个，较佳地至多8个，更佳地至多5个，最佳地至多3个氨基酸被性质相似或相近的氨基酸所替换而形成多肽。这些保守性变异多肽最好根据表A进行氨基酸替换而产生。
表A
最初的残基 代表性的取代 优选的取代
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
抗IL-17A抗体
如本文所用，术语“IL-17A”一般是指天然的或重组的人IL-17A，以及人IL-17A的非人同源物。除非另有指示，否则使用IL-17A的同源二聚体的分子量(例如对于人IL-17A为30KDa)计算IL-17A的摩尔浓度。
如本文所用，术语“人IL-17A(huIL-17A)”是指人IL-17A蛋白登陆号NP-002180和AAT22064的成熟形式(即残基24-155)，及其天然变体和多态性。
本发明提供一种针对IL-17A的高特异性和高亲和力的抗体，其包括重链和轻链，所述重链含有重链可变区(VH)氨基酸序列，所述轻链含有轻链可变区(VL)氨基酸序列。
优选地，重链可变区(VH)氨基酸序列和轻链可变区(VL)氨基酸序列的各自CDR选自下组：
a1)SEQ ID No.:7；
a2)SEQ ID No.:8；
a3)SEQ ID No.:9；
a4)SEQ ID No.:10；
a5)_KVS；
a6)SEQ ID No.:11；
a7)上述氨基酸序列中任意一种氨基酸序列经过添加、缺失、修饰和/或取代至少一个(如1-5、1-3个，较佳地1-2个，更佳地1个)氨基酸的具有IL-17A结合亲和力的序列。
在另一优选例中，所述经过添加、缺失、修饰和/或取代至少一个氨基酸序列所形成的序列优选为同源性为至少80％，较佳地至少85％，更佳地至少为90％，最佳地至少95％的氨基酸序列。
本发明的抗体可以是双链或单链抗体，并且可以是选自动物源抗体、嵌合抗体、人源化抗体，更优选为人源化抗体、人-动物嵌合抗体，更优选为全人源化抗体。
本发明所述抗体衍生物可以是单链抗体、和/或抗体片段，如：Fab、Fab’、(Fab’) 2或该领域内其他已知的抗体衍生物等，以及IgA、IgD、IgE、IgG以及IgM抗体或其他亚型的抗体中的任意一种或几种。
其中，所述动物优选为哺乳动物，如鼠。
本发明抗体可以是靶向人IL-17A的鼠源抗体、嵌合抗体、人源化抗体、CDR嫁接和/或修饰的抗体。
在本发明的一种优选实施例中，上述SEQ ID No.:7、8和9中任意一种或几种序列、或它们经过添加、缺失、修饰和/或取代至少一个氨基酸的具有IL-17A结合亲和力的序列，位于重链可变区(VH)的CDR区。
在本发明的一种优选实施例中，上述SEQ ID No.:10、氨基酸序列:KVS和SEQ ID No.:11中任意一种或几种序列、或它们经过添加、缺失、修饰和/或取代至少一个氨基酸的具有IL-17A结合亲和力的序列，位于轻链可变区(VL)的CDR区。
在本发明的一种更优选实施例中，VH CDR1、CDR2、CDR3分别独立地选自SEQ ID No.:7、8和9中任意一种或几种序列、或它们经过添加、缺失、修饰和/或取代至少一个氨基酸的具有IL-17A结合亲和力的序列；VL CDR1、CDR2、CDR3分别独立地选自SEQ ID No.:10、氨基酸序列:KVS和SEQ ID No.:11中任意一种或几种序列、或它们经过添加、缺失、修饰和/或取代至少一个氨基酸的具有IL-17A结合亲和力的序列。
本发明上述内容中，所述添加、缺失、修饰和/或取代的氨基酸数量，优选为不超过初始氨基酸序列总氨基酸数量的40％，更优选为不超过35％，更优选为1-33％，更优选为5-30％，更优选为10-25％，更优选为15-20％。
在本发明中，所述添加、缺失、修饰和/或取代的氨基酸数量通常是1、2、3、4或5个，较佳地为1-3个，更佳地为1-2个，最佳地为1个。
抗体的制备
任何适于产生单克隆抗体的方法都可用于产生本发明的抗IL-17A抗体。例如，可以用连接或天然存在的IL-17A同源二聚体或其片段免疫动物。可以使用合适的免疫接种方法，包括佐剂、免疫刺激剂、重复加强免疫接种，可以使用一种或多种途径。
任何合适形式的IL-17都可以作为免疫原(抗原)，用于产生对IL-17A特异的非人抗体，筛选所述抗体的生物学活性。激发免疫原可以是全长的成熟人IL-17A，包括天然的同源二聚体，或含单个/多个表位的肽。免疫原可以单独使用，或与本领域已知的一种或多种免疫原性增强剂组合使用。免疫原可以由天然来源纯化，或者在遗传修饰的细胞中产生。编码免疫原的DNA在来源上可以是基因组或非基因组的(例如cDNA)。可以使用合适的遗传载体表达编码免疫原的DNA，所述载体包括但不限于腺病毒载体、腺相关病毒载体、杆状病毒载体、质料和非病毒载体。
生产本发明的抗人IL-17A抗体的示例性方法描述于实施例1。
人源化抗体可以选自任何种类的免疫球蛋白，包括IgM、IgD、IgG、IgA和IgE。在本发明中，抗体是IgG抗体，使用IgG1亚型。通过用下文实施例中描述的生物学测定筛选抗体易于实现必需恒定结构域序列的最优化，以产生所需生物学活性。
同样，任一类轻链都可以在本文的化合物和方法中使用。具体地说，κ、λ链或其变体在本发明的化合物和方法中是可以用的。
人源化本发明的抗人IL-17A抗体的示例性方法描述于实施例4。
本发明抗体或其片段的DNA分子的序列可以用常规技术，比如利用PCR扩增或基因组文库筛选等方法获得。此外，还可将轻链和重链的编码序列融合在一起，形成单链抗体。
一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。
此外，还可用人工合成的方法来合成有关序列，尤其是片段长度较短时。通常，通过先合成多个小片段，然后再进行连接可获得序列很长的片段。然后可将该DNA序列引入 本领域中已知的各种现有的DNA分子(或如载体)和细胞中。
本发明还涉及包含上述的适当DNA序列以及适当启动子或者控制序列的载体。这些载体可以用于转化适当的宿主细胞，以使其能够表达蛋白质。
宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如哺乳动物细胞。优选的动物细胞包括(但并不限于)：CHO-S、CHO-K1、HEK-293细胞。
本发明中所述的用重组DNA转化宿主细胞的步骤可用本领域熟知的技术进行。获得的转化子可用常规方法培养，转化子表达本发明的基因所编码的多肽。根据所用的宿主细胞，用常规培养基在合适的条件下培养。
通常，在适合本发明抗体表达的条件下，培养转化所得的宿主细胞。然后用常规的免疫球蛋白纯化步骤，如蛋白A-Sepharose、羟基磷灰石层析、凝胶电泳、透析、离子交换层析、疏水层析、分子筛层析或亲和层析等本领域技术人员熟知的常规分离纯化手段纯化得到本发明的抗体。
所得单克隆抗体可用常规手段来鉴定。比如，单克隆抗体的结合特异性可用免疫沉淀或体外结合试验(如放射性免疫测定(RIA)或酶联免疫吸附测定(ELISA))来测定。
应用
本发明提供了本发明抗体的用途，例如用于制备诊断制剂、或制备用于预防和/或治疗IL-17A相关的疾病的药物。所述IL-17A相关的疾病包括炎症疾病、自身免疫疾病等，包括但不限于银屑病、银屑病关节炎、强直性脊柱炎、多发性硬化症，炎性肠病(如克罗恩氏病、溃疡性结肠炎等)、骨关节炎、类风湿性关节炎(RA)、风湿性关节炎或骨质疏松症、炎性纤维化(例如硬皮病、肺纤维化和硬化)、哮喘(包括变应性哮喘)、***反应以及癌症。
药物组合物
本发明还提供了一种组合物。在优选例中，所述的组合物是药物组合物，它含有上述的抗体或其活性片段或其融合蛋白或其ADC或相应的CAR-T细胞，以及药学上可接受的载体。通常，可将这些物质配制于无毒的、惰性的和药学上可接受的水性载体介质中，其中pH通常约为5-8，较佳地pH约为6-8，尽管pH值可随被配制物质的性质以及待治疗的病症而有所变化。配制好的药物组合物可以通过常规途径进行给药，其中包括(但并不限于)：瘤内、腹膜内、静脉内、或局部给药。
本发明所述抗体也可以是由核苷酸序列在细胞内表达用于的细胞治疗，比如，所述抗体用于嵌合抗原受体T细胞免疫疗法(CAR-T)等。
本发明的药物组合物可直接用于结合IL-17A蛋白分子，因而可用于预防和治疗IL-17A相关的疾病。此外，还可同时使用其他治疗剂。
本发明的药物组合物含有安全有效量(如0.001-99wt％,较佳地0.01-90wt％，更佳地0.1-80wt％)的本发明上述的单克隆抗体(或其偶联物)以及药学上可接受的载体或赋形剂。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。药物 制剂应与给药方式相匹配。本发明的药物组合物可以被制成针剂形式，例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。药物组合物如针剂、溶液宜在无菌条件下制造。活性成分的给药量是治疗有效量，例如每天约1微克/千克体重-约5毫克/千克体重。此外，本发明的多肽还可与其他治疗剂一起使用。
使用药物组合物时，是将安全有效量的药物组合物施用于哺乳动物，其中该安全有效量通常至少约10微克/千克体重，而且在大多数情况下不超过约50毫克/千克体重，较佳地该剂量是约10微克/千克体重-约20毫克/千克体重。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。
检测用途和试剂盒
本发明的抗体可用于检测应用，例如用于检测样本，从而提供诊断信息。
本发明中，所采用的样本(样品)包括细胞、组织样本和活检标本。本发明使用的术语“活检”应包括本领域技术人员已知的所有种类的活检。因此本发明中使用的活检可以包括例如通过内窥镜方法或器官的穿刺或针刺活检制备的组织样本。
本发明中使用的样本包括固定的或保存的细胞或组织样本。
本发明还提供了一种指含有本发明的抗体(或其片段)的试剂盒，在本发明的一个优选例中，所述的试剂盒还包括容器、使用说明书、缓冲剂等。在优选例中，本发明的抗体可以固定于检测板。
本发明的主要优点
(a)本发明抗体具有优异的生物活性和特异性，并具有很高的亲和力(ELISA测定其EC 50可高达约为10-20ng/ml)。此外，对IL-17A具有良好的结合亲合力，而与其他家族成员IL-17B、IL-17D、IL-17E及IL-17F均无结合，可用做靶向IL-17A的抗体。
(b)与鼠源抗体相比，本发明人源化抗体不仅具有与IL-17A更好的亲合力，而且具有更低的免疫原性。
(c)本发明抗体能显著抑制IL-17A与IL-17受体的结合，而对于哺乳动物本身没有可见的毒副作用。
(d)本发明抗体与某些非人哺乳动物的IL-17A有一定亲和力，便于在动物模型中进行测试和进行质控检测。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
实施例1抗人IL-17A的小鼠单克隆抗体——原始抗体的制备方法
1.1制备产生鼠源单克隆抗体的杂交瘤细胞
首先用人的IL-17A蛋白作为抗原，与佐剂乳化后对BALB/c小鼠进行多点皮下免疫，监测免疫小鼠血清效价，达到要求后取小鼠的脾细胞与骨髓瘤(Sp2/0)细胞进行融合，经过HAT筛选，获得的杂交瘤多克隆细胞。
1.2间接ELISA——杂交瘤细胞的筛选方法
使用ELISA检测方法筛选出高特异性结合的多克隆，进行单克隆化培养，再使用ELISA方法筛选高特异性结合的单克隆细胞株；通过HT1080细胞对IL-6释放实验，筛选具有细胞功能学效果的单克隆细胞株，然后用Biacore方法分析亲和力及半衰期，最终得到表达IL-17A的单克隆细胞。
试验材料：
重组人IL-17A，Sino Biological，12047-HNAS
试验方法：
将人IL-17A用CBS配制成1μg/ml包被液，50μL/孔加入酶标板，2～8℃包被12小时以上弃去包板残液，加入3％牛奶，每孔200μL，室温封闭1小时。每孔加入不少于200μL的PBST洗1次，将杂交瘤上清稀释至100μg/ml，再10倍稀释10个梯度，100μL/孔加入酶标板。室温孵育1小时，每孔加不少于200μL的PBST，洗涤4次后加入3％牛奶-PBST稀释25000倍的HRP偶联的羊抗鼠IgG Fc(购自Jackson公司)，100μL/孔加样。室温孵育1小时后，每孔加不少于200μL的PBST，洗涤6次，拍干。加入TMB显色液，每孔100μL。室温反应5分钟后加2M H 2SO 4终止反应，50μL/孔。将中止反应的酶标板置酶标仪上，读取450nm波长读取吸光度OD450值。
试验结果：
表1.杂交瘤对人IL-17a结合活性比较
样品名称 EC 50(ng/mL)
1B1/7A2 310.9
1B1/7C8 214.6
7D6/5H8 194.4
7D6/6B11 210.1
7D6/6G11 226.4
1B1/8E1 268.9
1B1/8E5 448.2
由表1可以看出，在众多筛选出的抗体中，杂交瘤14F10/9F6(或其产生的抗体)与人IL-17a结合活性非常较高。
实施例2抗IL-17A抗体V-基因序列克隆
基于5’RACE技术，测定编码由杂交瘤7D6/5H8表达的小鼠抗体可变区的DNA序列。 简言之，用SMART 5’RACE合成试剂盒(TAKARA，NO.634859)按厂商用法说明制备重链和轻链的基因特异性cDNA。由琼脂糖凝胶电泳分析PCR产物。重链和轻链二者的可变区大小约为500个碱基对。将反应所得的条带大小合适的扩增PCR产物克隆到载体pEASY-Blunt Simple质粒(北京全式金，No.CB111-02)中，并转化到Stellar大肠杆菌感受态细胞(TAKARA，No.636763)中。通过用通用的M13正向或反向引物的菌落PCR筛选克隆，从每个反应选择2-3个克隆用于DNA测序分析。用Expasy-翻译工具(Expasy-translate tool)(http://web.expasy.org/translate/)分析每个克隆的每个测序反应结果。测序结果显示7D6/5H8表达的抗IL17A抗体V区序列如下：
IL17-HC1       SEQ ID NO：1
Figure PCTCN2018073458-appb-000001
其中，下划线为CDR1、CDR2、CDR3(SEQ ID NO.:7、8和9)。
IL17-LC1         SEQ ID NO：2
Figure PCTCN2018073458-appb-000002
其中，下划线为CDR1’、CDR2’、CDR3’(SEQ ID NO.:10、氨基酸序列:KVS和SEQ ID NO.:11)。
表2:鼠源抗IL-17A抗体CDR序列
Figure PCTCN2018073458-appb-000003
实施例3构建和表达嵌合抗体
3.1嵌合抗体的制备
通过将PCR克隆的小鼠7D6/5H8VH和VL区cDNA分别与人IgG1和k恒定区连接，来构建嵌合重链和轻链。用PCR引物修饰小鼠cDNA序列的5’和3’端，所述引物设计成为各链增加合适的前导序列，并增加使得能克隆到现有重组抗体表达载体pHB-Fc上的限制性位点。pHB-Fc质粒载体的制法如下：以pcDNA/HA-FLAG(Accession#:FJ524378)载体为出发质粒，在内切酶EcoRI后面加上了人IgG1或k的恒定区序列，在内切酶HindIII 前面加上了人类巨细胞病毒(HCMV)促进子序列(Accession#:X17403)，在氨苄青霉素耐受基因后面、HCMV促进子前面加上了中国仓鼠谷氨酰胺合成酶基因(Accession#:X03495)。
蛋白表达所用的宿主细胞是购自ATCC公司的CHO-K1细胞(Cat#CCL-61)。该细胞经过一系列驯化步骤，驯化成可在无血清培养基(EX-CELLTM302)中进行悬浮培养的CHO-K1细胞。利用该细胞，通过电转的方法，将构建好的轻链和重链重组表达质粒转入细胞。放入培养箱培养3-5天。用间接ELISA测量来自CHO-K1转染上清液的抗体浓度。这显示转染的CHO-K1细胞分泌约30mg/L的嵌合抗体。
作为阳性对照的Novartis人源化anti-IL-17抗体(Novartis mAb)根据US 7,807,155 B2(AIN 457)提供的人源化序列进行克隆，并瞬时转染进行表达。
3.2嵌合抗体的测定
试验材料：
重组人IL-17A，Sino Biological，12047-HNAS
重组鼠IL-17A，Sino Biological，PBV10159R-010
试验方法：
方法同实施例1，用嵌合抗体代替杂交瘤上清，用HRP偶联的兔抗人IgG Fc抗体(洛阳佰奥通实验材料中心)代替HRP偶联的羊抗鼠IgG Fc，分别测定与重组人及鼠IL-17A结合活性。
试验结果：
表3.嵌合抗体对人IL-17A结合活性比较
样品 EC 50(ng/ml)
阳性对照(Novartis mAb) 34.1
阴性对照(PBS) -
嵌合抗体 38.55
表4.嵌合抗体对鼠IL-17A结合活性比较
样品 EC 50(ng/ml)
阳性对照(Novartis mAb) -
阴性对照(PBS) -
嵌合抗体 39.2
结果证明，嵌合抗体能与人的IL-17A结合，也可以与鼠的IL-17A结合，且计算的EC 50值均低于40ng/ml。
实施例4人源化抗体的制备
4.1人源化抗体的制备
抗体的人源化采用以下方法。简言之，将抗体的可变链序列与NCBI蛋白质数据库中的可用序列比较，通过鉴定和分析，最终确定了合适在其上构建CDR移植重链和轻链的 人构架。
基于后续的测试和筛选，一种优选的FR区为源自hu-VH(SEQ ID NO：3)，轻链为hu-VL(SEQ ID NO：4)的人源化FR区：
hu-VH         SEQ ID NO：3
Figure PCTCN2018073458-appb-000004
hu-VL         SEQ ID NO：4
Figure PCTCN2018073458-appb-000005
改装时，根据人抗体FR区保守的氨基酸残基以及抗体FR区中重要的氨基酸残基，设计改造位点，对嵌合抗体的重轻链的可变区分别进行人源化突变设计，利用PCR技术扩增并构建人源化点突变抗体表达质粒。将人源化点突变抗体表达质粒分别经CHO-K1(ATCC，NO.CCL-61)细胞分别表达，纯化后得到人源化抗体蛋白。利用ELISA，受体结合抑制实验，Biacore和细胞活性检测等，获得了一种性能非常优异的人源化抗体(命名为“HB0017抗体”)。
HB0017抗体的VH和VL序列分别如SEQ ID NO.:5和6所示：
IL17-HC1-2G7      SEQ ID NO：5
Figure PCTCN2018073458-appb-000006
IL17-LC1-1C2        SEQ ID NO：6
Figure PCTCN2018073458-appb-000007
实验结果表明，与鼠源抗体相比，人源化抗体HB0017具有更好的亲和力和特异性，见实施例4.2。
4.2人源化抗体的测定
试验材料：
重组人IL-17A，Sino Biological，12047-HNAS
试验方法：
方法同实施例1，用HB0017抗体代替杂交瘤上清，用HRP偶联的兔抗人IgG Fc抗体(洛阳佰奥通实验材料中心)代替HRP偶联的羊抗鼠IgG Fc，测定与重组人IL-17A结合活性。
试验结果：
表5.HB0017抗体对人IL-17A结合活性比较
样品 EC 50(ng/ml)
阳性对照(Novartis mAb) 32.9
阴性对照(PBS) -
HB0017抗体 15.4
结果证明，经过人源化改造，本发明人意外地获得了对人IL-17A结合活性不仅没有下降，反而有进一步提高的人源化抗体HB0017。EC 50与嵌合抗体相比，提高了约150％(38.55/15.4-100％＝150％)，与原鼠源抗体相比，提高了约11.6倍。相较于阳性对照抗体，本发明抗体的EC 50值更低，具有与人IL-17A更强的结合活性。
实施例5人源化单克隆抗体的不同种属免疫交叉反应
在本实施例中，采用ELSIA方法测定抗IL-17A抗体与不同种属的IL-17A的抗原-抗体结合能力。
试验材料：
重组人IL-17A，Sino Biological，12047-HNAS
重组猕猴(Rhesus)IL-17A，Sino Biological，90306-KNAB
重组小鼠IL-17A，Sino Biological，PBV10159R-010
重组大鼠IL-17A，ABGENT，PBV10154r
试验方法：
方法同实施例1，用HB0017抗体代替杂交瘤上清，用HRP偶联的兔抗人IgG Fc抗体(洛阳佰奥通实验材料中心)代替HRP偶联的羊抗鼠IgG Fc，分别测定与人IL-17A、猕猴IL-17A、小鼠IL-17A及大鼠IL-17A结合活性。
试验结果：
表6.HB0017抗体与小鼠IL-17A结合结果
样品 EC 50(ng/ml)
阴性对照(PBS) -
HB0017抗体 75.0
表7.HB0017抗体与猕猴IL-17A结合结果
样品 EC 50(ng/ml)
阴性对照(PBS) -
HB0017抗体 88.0
表8.HB0017抗体与人IL-17A结合结果
样品 EC 50(ng/ml)
阴性对照(PBS) -
HB0017抗体 15.4
表9.HB0017抗体与大鼠IL-17A结合结果
样品 EC 50(ng/ml)
阴性对照(PBS) -
HB0017抗体 240
由表6-9可以看出，本发明人源化单克隆抗体HB0017抗体除与人IL-17A有结合外，与小鼠、猕猴及大鼠IL-17A均有结合，为临床动物试验提供便利。
实施例6人源化单克隆抗体的亲和性检测
在本实施例中，使用BIACORE方法测定抗原-抗体结合动力学及亲和力。
试验材料：
重组人IL-17A，Sino Biological，12047-HNAS
氨基偶联试剂盒，GE，BR-1000-50
HBS-EP(10X)，GE，BR-1006-69
人抗体捕获试剂盒(Human Antibody Captrue Kit)，GE，BR-1008-39
试验方法：
用氨基偶联试剂盒在S系列传感器芯片(Sereis S Sensor Chip CM5)上氨基偶联固定人抗体捕获抗体(Human Antibody Capture Antiboy)，抗人捕获-CM5芯片(Anti-Human Capture-CM5芯片)。室温平衡20～30min，将芯片装入仪器。用平衡缓冲液稀释抗原，稀释抗原10nM起始稀释度5个浓度梯度，并设置2个零浓度(即平衡缓冲液)和一个重复浓度(一般为最低浓度重复)。用平衡缓冲液将抗体样品稀释至实验工作浓度，2～8℃密封备用。样品分析完成后，选用对应的分析程序对数据分析，确认无明显参照结合(reference binding)，选用动力学，1：1结合模型(Kinetics，1:1binding modle)，拟合分析获得样品的动力学参数。
试验结果：
表10.HB0017抗体与人IL-17a亲和力检测结果
样品 Ka(1/Ms) Kd(1/s) KD(M)
阳性对照(Novartis mAb) 1.46E+06 1.61E-04 1.11E-10
HB0017 4.55E+06 9.02E-05 1.98E-11
与人IL-17A的亲和力常数(KD(M))结果显示，本发明HB0017抗体的亲和力比阳性对照抗体高将近一个数量级，具有更强的亲和力。
实施例7人源化单克隆抗体的细胞水平生物活性测定
IL-17A在TNFα或TNFβ协同作用下，能刺激HT1080产生IL-6。通过本发明的HB0017抗体中和IL-17A的作用，进而抑制IL-6的表达水平。证明本发明的抗体的生物活性。
试验材料：
DMEM谷氨酰胺+10％灭活的胎牛血清(FBS)+1％PS(双抗)(生长培养基)
重组人IL-17A，Sino Biological，12047-HNAS
重组人TNFα，Sino Biological，10602-HNAE
Human IL-6ELISA MAX TM Deluxe Set，430506，BioLegend
试验方法：
取对数生长期HT1080细胞，汇合度为80％～90％，即可进行实验。弃去细胞培养瓶内培养液，用PBS洗1次，加入适量0.25％胰酶～0.02％EDTA消化液，放入细胞培养箱中，1～2min后，轻敲培养瓶使细胞脱落。加入生长培养基，制得单细胞悬液，1000rpm转速5min离心细胞。生长培养基重悬，计数。将细胞用生长培养基稀释稀释至2.5×10 5/ml，50μl/well加入细胞培养板后，置于细胞培养箱中培养5h以上使细胞贴壁。用空白培养基(DMEM谷氨酰胺)将抗体和阳性对照稀释至200μg/ml，以200μg/ml起始再3.16倍稀释8个梯度浓度。用空白培养基将重组人IL-17A稀释至40ng/ml，用空白培养基将重组人TNF-α稀释至20ng/ml。将稀释后的重组人IL-17A与重组人TNF-α按体积比1:1混匀。再与抗体或阳性对照的9个梯度浓度各按体积比1:1混匀，置37℃孵育1小时。
将已孵育的抗体或阳性对照的9个梯度浓度，各取50μl/孔加至96孔细胞培养板中，每个浓度做2个复孔，50μl的IL-17&TNFα混合液作阳性对照孔和50μl的空白培养基作空白对照孔，各三个复孔。然后置细胞培养板于37度、5％CO 2的条件培养过夜。
按Human IL-6ELISA MAX TM Deluxe Set Kit说明包被ELISA板，第二天取出过夜培养的96孔细胞培养板，吸取培养上清，20X稀释。按照Human IL-6ELISA MAX TM Deluxe Set说明书进行ELISA检测。
试验结果：
表11.HB0017抗体生物活性测定结果
样品 IC 50(μg/ml) IC 80(μg/ml) IC 90(μg/ml)
阳性对照(Novartis mAb) 0.52 2.11 4.80
HB0017抗体 0.09 0.13 0.17
表11结果显示，较阳性对照抗体，本发明HB0017抗体的生物学活性提高了近6倍，具有良好的生物活性。
实施例8人源化单克隆抗体的抗原结合特异性
在本实施例中，测试HB0017抗体与IL-17不同亚型的结合特异性。
试验材料：
重组人IL-17A，Sino Biological，12047-HNAS
重组人IL-17B，R&D，8129-IL
重组人IL-17D，Sino Biological，10076-H08S
重组人IL-17E，ABGENT，PBV10154r
重组人IL-17F，Sino Biological，11855-H07H-5
重组人IL-17A/F，Sino Biological，CT047-H08H-20
试验方法：
方法同实施例1，用HB0017抗体代替杂交瘤上清，用HRP偶联的兔抗人IgG Fc抗体(洛阳佰奥通实验材料中心)代替HRP偶联的羊抗鼠IgG Fc，分别测定与人IL-17A、人IL-17B、人IL-17D、人IL-17E、人IL-17F及人IL-17A/F的结合活性。
试验结果：
表12.HB0017抗体与人IL-17家族的结合特异性结果
Figure PCTCN2018073458-appb-000008
表12结果显示，HB0017抗体除与人IL-17A及IL-17A/F有结合，与其他家族成员IL-17B、IL-17D、IL-17E及IL-17F均无结合，具有良好的抗原结合特异性。
实施例9人源化单克隆抗体的体内药效检测
本实施例采用咪喹莫特诱导的小鼠银屑病模型进行，以验证本发明的抗体在体内可以是否通过阻断IL-17结合到IL-17受体(如hIL-17RA)上而改善银屑病的症状或相关指标。
试验方法如下；
取20g左右C57BL/6雌性小鼠32只，背部脱毛，三天后致敏。致敏前两天随机分成4组(每组8只)：I组为溶剂对照组，给予PBS；II组为同型对照组，给予阴性对照样品的人IgG1(PBS稀释)100mg/kg；III组为HB0017抗体给药组，给予HB0017抗体100mg/kg；IV组为阳性对照组，给予***(PBS稀释)1mg/kg。上述各组于分组当天及造模第2天(day 2)各腹腔注射药物1次。致敏当天(day 1)，各组小鼠于右耳及背部皮肤上涂抹约62.5mg咪喹莫特乳膏(5％)，连续5天。
自致敏当天起每天用螺旋测微仪测量小鼠右耳厚度，以day 0右耳厚度为对照计算小鼠耳肿胀厚度数值。同时给小鼠每天称重，观察皮肤鳞屑、硬结、红斑情况，进行评分，采用4级评分法：0分，不发病；1分，轻微；2分，中度；3分，严重；4分，非常严重。
结果如图1所示。结果表明，与***相比，本发明人源化抗IL-17A抗体HB0017对咪喹莫特诱导的小鼠银屑病模型有显著的改善效果。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种抗体的重链可变区，其特征在于，所述的重链可变区包括以下三个互补决定区CDR：
    SEQ ID NO:7所示的CDR1，
    SEQ ID NO:8所示的CDR2，和
    SEQ ID NO:9所示的CDR3。
  2. 一种抗体的重链，其特征在于，所述的重链具有如权利要求1所述的重链可变区。
  3. 一种抗体的轻链可变区，其特征在于，所述的轻链可变区包括以下三个互补决定区CDR：
    SEQ ID NO:10所示的CDR1’，
    氨基酸序列为KVS的CDR2’，和
    SEQ ID NO:11所示的CDR3’。
  4. 一种抗体的轻链，其特征在于，所述的轻链具有如权利要求3所述的轻链可变区。
  5. 一种抗体，其特征在于，所述抗体具有：
    (1)如权利要求1所述的重链可变区；和/或
    (2)如权利要求3所述的轻链可变区；
    或者，所述抗体具有：如权利要求2所述的重链；和/或如权利要求4所述的轻链。
  6. 如权利要求5所述的抗体，其特征在于，所述抗体选自：动物源抗体、嵌合抗体、人源化抗体、或其组合。
  7. 如权利要求5所述的抗体，其特征在于，所述抗体的重链可变区序列如SEQ ID NO.:1或5所示；和/或
    所述抗体的轻链可变区序列如SEQ ID NO.:2或6所示。
  8. 一种重组蛋白，其特征在于，所述的重组蛋白具有：
    (i)如权利要求1所述的重链可变区、如权利要求2所述的重链、如权利要求3所述的轻链可变区、如权利要求4所述的轻链、或如权利要求5所述的抗体；以及
    (ii)任选的协助表达和/或纯化的标签序列。
  9. 一种CAR构建物，其特征在于，所述的CAR构建物的单克隆抗体抗原结合区域的scFV段为特异性结合于IL-17A的结合区，并且所述scFv具有如权利要求1所述的重链可变区和如权利要求3所述的轻链可变区。
  10. 一种活性成分的用途，所述活性成分选自下组：如权利要求1所述的重链可变区、如权利要求2所述的重链、如权利要求3所述的轻链可变区、如权利要求4所述的轻链、或如权利要求5所述的抗体、如权利要求8所述的重组蛋白、或其组合，其特征在于，所述活性成分用于(a)制备检测试剂或试剂盒；和/或(b)制备预防和/或治疗IL-17A相关疾病的药物。
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115819579A (zh) * 2022-11-10 2023-03-21 浙江大学 全人源抗白介素17A单链抗体No.34及应用

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109929037B (zh) * 2019-04-01 2023-03-17 华博生物医药技术（上海）有限公司 针对程序性死亡配体的结合物及其应用
CN110585430B (zh) * 2019-09-29 2023-09-08 华博生物医药技术（上海）有限公司 一种人源化抗人il-17a单克隆抗体的药物组合物
CN112300285B (zh) * 2020-11-06 2022-08-02 江苏荃信生物医药股份有限公司 一种定量检测血清中抗人白介素17单克隆抗体含量的酶联免疫分析方法
CN116685605A (zh) * 2020-12-17 2023-09-01 上海华奥泰生物药业股份有限公司 靶向il-17a和il-36r的双特异性抗体及其应用
CN117106083A (zh) * 2022-03-25 2023-11-24 南京融捷康生物科技有限公司 一种抗il-17a的单域抗体及其用途
WO2024120518A1 (zh) * 2022-12-09 2024-06-13 上海华奥泰生物药业股份有限公司 Il-17a抗体制剂及其用途

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7807155B2 (en) 2004-08-05 2010-10-05 Novartis Ag IL-17 antagonistic antibodies
CN103215293A (zh) * 2006-01-27 2013-07-24 比奥根艾迪克Ma公司 Nogo受体拮抗剂
CN103717618A (zh) * 2011-05-05 2014-04-09 默克专利股份有限公司 抗il-17a，il-17f和/或il17-a/f的氨基酸序列以及包含其的多肽
CN104936981A (zh) * 2013-11-18 2015-09-23 上海恒瑞医药有限公司 Il-17a结合物及其用途
CN105073775A (zh) * 2013-02-08 2015-11-18 诺华股份有限公司 抗-il-17a抗体及其在治疗自身免疫性和炎性病症中的用途
CN106336459A (zh) * 2015-07-13 2017-01-18 三生国健药业（上海）股份有限公司 抗人白细胞介素-17a单克隆抗体、其制备方法和应用
CN106795219A (zh) * 2014-03-14 2017-05-31 Biocad股份有限公司 抗il‑17抗体及其生产和使用的方法

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2810652A3 (en) * 2009-03-05 2015-03-11 AbbVie Inc. IL-17 binding proteins
WO2011053763A2 (en) * 2009-10-30 2011-05-05 Centocor Ortho Biotech Inc. Il-17a antagonists

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7807155B2 (en) 2004-08-05 2010-10-05 Novartis Ag IL-17 antagonistic antibodies
CN103215293A (zh) * 2006-01-27 2013-07-24 比奥根艾迪克Ma公司 Nogo受体拮抗剂
CN103717618A (zh) * 2011-05-05 2014-04-09 默克专利股份有限公司 抗il-17a，il-17f和/或il17-a/f的氨基酸序列以及包含其的多肽
CN105073775A (zh) * 2013-02-08 2015-11-18 诺华股份有限公司 抗-il-17a抗体及其在治疗自身免疫性和炎性病症中的用途
CN104936981A (zh) * 2013-11-18 2015-09-23 上海恒瑞医药有限公司 Il-17a结合物及其用途
CN106795219A (zh) * 2014-03-14 2017-05-31 Biocad股份有限公司 抗il‑17抗体及其生产和使用的方法
CN106336459A (zh) * 2015-07-13 2017-01-18 三生国健药业（上海）股份有限公司 抗人白细胞介素-17a单克隆抗体、其制备方法和应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
J. SAMBROOK ET AL.: "Molecular Cloning: A Laboratory Manual", 1989, COLD SPRING HARBOR LABORATORY PRESS
See also references of EP3656789A4

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115819579A (zh) * 2022-11-10 2023-03-21 浙江大学 全人源抗白介素17A单链抗体No.34及应用
CN115819579B (zh) * 2022-11-10 2023-11-07 浙江大学 全人源抗白介素17A单链抗体No.34及应用

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