WO2021018035A1 - 人源化抗il17a抗体及其应用 - Google Patents

人源化抗il17a抗体及其应用 Download PDF

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WO2021018035A1
WO2021018035A1 PCT/CN2020/104448 CN2020104448W WO2021018035A1 WO 2021018035 A1 WO2021018035 A1 WO 2021018035A1 CN 2020104448 W CN2020104448 W CN 2020104448W WO 2021018035 A1 WO2021018035 A1 WO 2021018035A1
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antibody
il17a
antigen
seq
amino acid
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English (en)
French (fr)
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谢良志
孙春昀
刘天娇
李靖
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Sinocelltech Ltd
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Sinocelltech Ltd
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Priority to BR112022001492A priority Critical patent/BR112022001492A2/pt
Priority to MX2022001068A priority patent/MX2022001068A/es
Priority to CN202080050601.7A priority patent/CN114286827B/zh
Priority to EP20846348.9A priority patent/EP4036113B1/en
Priority to PL20846348.9T priority patent/PL4036113T3/pl
Priority to KR1020227005577A priority patent/KR102815628B1/ko
Priority to RS20260008A priority patent/RS67607B1/sr
Priority to AU2020322569A priority patent/AU2020322569B2/en
Priority to JP2022505484A priority patent/JP7214921B2/ja
Application filed by Sinocelltech Ltd filed Critical Sinocelltech Ltd
Priority to MYPI2022000335A priority patent/MY205369A/en
Priority to HRP20260007TT priority patent/HRP20260007T1/hr
Priority to ES20846348T priority patent/ES3059248T3/es
Priority to CA3148491A priority patent/CA3148491C/en
Publication of WO2021018035A1 publication Critical patent/WO2021018035A1/zh
Priority to US17/584,766 priority patent/US12516113B2/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/244—Interleukins [IL]
    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6845—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a cytokine, e.g. growth factors, VEGF, TNF, a lymphokine or an interferon
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/06—Antipsoriatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • 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
    • 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
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • 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
    • C07K2317/70—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

Definitions

  • the invention belongs to the field of immunotherapy, and relates to a humanized anti-IL17A monoclonal antibody medicine and its application.
  • IL17A is also commonly referred to as IL-17, which is an inflammatory cytokine with a molecular weight of 35kD composed of 155 amino acids. It is mainly secreted by helper T cells 17.
  • CD4 + , CD8 + T, and ⁇ -T cells can also secrete IL-17.
  • the IL-17 family includes six members of IL17A, IL-17B, IL-17C, IL-17D (IL-27), IL-17E (IL-25) and IL-17F (Gu, Wu et al. 2013), of which IL17A and IL-17F are the most important components, with 55% amino acid homology, which can form homodimers or heterodimers connected by disulfide bonds (Dubin and Kolls 2009).
  • IL17A/A or IL-17A/F dimer binds to a variety of IL-17RA-expressing cells in the IL-17 receptor family, such as macrophages, dendritic cells, hematopoietic cells, osteoblasts, fibroblasts, etc. Later, it can activate NF ⁇ B, C/EBPs, MAPK and other signals in the recipient cells, induce these cells to secrete inflammatory factors and chemokines (IL-6, IL-8, CXCL1, etc.), recruit neutrophils, and mediate The occurrence of inflammation (Mitra, Raychaudhuri et al. 2014).
  • IL-17 pathway The expression level of IL-17 in the patient's serum was significantly up-regulated (Marinoni, Ceribelli et al. 2014), resulting in a persistent inflammatory response.
  • IL-17 can also directly act on endothelial cells, epithelial cells, fibroblasts and keratinocytes in the skin, increasing the release of various inflammatory factors, and forming sick skin (Mitra, Raychaudhuri et al. 2014, Brembilla, Senra et al. 2018). Therefore, blocking the IL-17 pathway provides the feasibility to inhibit the disease process of autoimmune diseases.
  • the humanized monoclonal antibody that binds to IL17A in the present invention can specifically bind to IL17A protein with high affinity, has a strong ability to block the binding of IL17A, IL17A/F and the receptor IL17RA, and is comparable to similar drugs.
  • the antibody Compared with, it has superior or equivalent blocking function of IL17A and IL17A/F to induce the secretion of inflammatory cytokines in human epidermal fibroblasts HFF; in the mouse model of psoriasis, the antibody also showed significantly better than similar drugs In vivo pharmacodynamic activity, the PASI score was significantly reduced after administration; in the cynomolgus monkey pharmacokinetic test, the humanized antibody showed superior pharmacokinetics, including fast absorption by subcutaneous injection, long half-life, and better drug exposure, etc. Features, laying the foundation for a longer administration cycle. It is intended for the treatment of psoriasis including but not limited to.
  • the present invention meets the above-mentioned needs by developing IL17A binding antibodies with new structures.
  • the present invention discloses a humanized monoclonal antibody that binds IL17A which can specifically bind IL17A protein with high affinity and has a strong ability to block the binding of IL17A, IL17A/F and receptor IL17RA.
  • the humanized monoclonal antibody of the present invention can be used for psoriasis.
  • the present invention provides an isolated anti-IL17A antibody or antigen-binding fragment thereof, which comprises a heavy chain CDR1 domain having the amino acid sequence shown in SEQ ID NO: 13 and an amino acid sequence shown in SEQ ID NO: 14
  • the anti-IL17A antibody or antigen-binding fragment thereof comprises an amino acid sequence such as SEQ ID NO: 22 or at least 90%, 92%, 95%, 98%, or 99% sequence of SEQ ID NO: 22.
  • the anti-IL17A antibody or antigen-binding fragment thereof is a humanized antibody or a chimeric antibody.
  • the anti-IL17A antibody further comprises a heavy chain constant region and a light chain constant region, preferably the heavy chain constant region is the same as the amino acid of the human IgG1 heavy chain constant region with the amino acid sequence of SEQ ID NO: 24 Sequence or an amino acid sequence with at least 90%, 92%, 95%, 98% or 99% sequence identity with SEQ ID NO: 24, and/or the light chain constant region is an amino acid sequence of SEQ ID NO: 25
  • the anti-IL17A antibody further comprises a signal peptide connected to the variable region of the heavy chain and/or a signal peptide connected to the variable region of the light chain, preferably the signal connected to the variable region of the heavy chain
  • a peptide is an amino acid sequence with the amino acid sequence of SEQ ID NO: 20 or an amino acid sequence with at least 90%, 92%, 95%, 98%, or 99% sequence identity with SEQ ID NO: 20, and/or the linkage
  • the signal peptide to the light chain variable region is the amino acid sequence of SEQ ID NO: 21 or an amino acid sequence that has at least 90%, 92%, 95%, 98%, or 99% sequence identity with SEQ ID NO: 21 .
  • the anti-IL17A antibody or antigen-binding fragment thereof is an IgG antibody, preferably an IgG1 antibody.
  • the anti-IL17A antibody or antigen-binding fragment thereof is a monoclonal antibody.
  • the binding affinity K D of the anti-IL17A antibody or its antigen-binding fragment to the recombinant human IL17A protein is 0.1-10E-11M, preferably 0.5-5E-11M, more preferably 2.88E-11M.
  • the binding affinity K D of the anti-IL17A antibody or antigen-binding fragment thereof to the recombinant human IL17A/F protein is 0.1-10E-10M, preferably 0.5-5E-10M, more preferably 5.37E-10M.
  • the antigen-binding fragment is Fv, Fab, Fab', Fab'-SH, F(ab')2, Fd fragment, Fd' fragment, single chain antibody molecule or single domain antibody;
  • the antibody molecule is preferably scFv, di-scFv, tri-scFv, diabody or scFab.
  • the present invention provides an antibody-drug conjugate comprising the anti-IL17A antibody or antigen-binding fragment thereof according to the present invention and another therapeutic agent, preferably the anti-IL17A antibody or antigen-binding fragment thereof The fragment and the additional therapeutic agent are connected by a linker.
  • the present invention provides a nucleic acid that encodes the anti-IL17A antibody or antigen-binding fragment thereof of the present invention.
  • the nucleic acid comprises the heavy chain variable region nucleotide sequence shown in SEQ ID NO: 30 and/or the light chain variable region nucleotide sequence shown in SEQ ID NO: 31.
  • the present invention provides an expression vector comprising the nucleic acid according to the present invention.
  • the present invention provides a host cell comprising the nucleic acid of the present invention or the expression vector of the present invention.
  • the present invention provides a method for producing the anti-IL17A antibody or antigen-binding fragment thereof according to the present invention, which comprises culturing the host cell according to the present invention under conditions suitable for antibody expression, And recover the expressed antibody from the culture medium.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the anti-IL17A antibody or antigen-binding fragment thereof according to the present invention or the antibody-drug conjugate according to the present invention or Nucleic acid or expression vector according to the present invention, and pharmaceutically acceptable carrier.
  • the present invention provides an anti-IL17A antibody or antigen-binding fragment thereof according to the present invention or an antibody-drug conjugate according to the present invention or a pharmaceutical composition according to the present invention for the treatment of silver Scourgery.
  • the present invention provides a method for treating psoriasis, which comprises administering to a subject in need a therapeutically effective amount of the anti-IL17A antibody or antigen-binding fragment thereof according to the present invention or according to the present invention.
  • the antibody-drug conjugate or the pharmaceutical composition according to the present invention thereby treating psoriasis.
  • the present invention provides the anti-IL17A antibody or antigen-binding fragment thereof according to the present invention or the antibody-drug conjugate according to the present invention or the pharmaceutical composition according to the present invention in preparation for treatment Use in psoriasis drugs.
  • the present invention provides a pharmaceutical combination comprising the anti-IL17A antibody or antigen-binding fragment thereof according to the present invention or the antibody-drug conjugate according to the present invention or the drug according to the present invention The composition and one or more additional therapeutic agents.
  • the present invention provides a kit comprising the anti-IL17A antibody or antigen-binding fragment thereof according to the present invention or the antibody-drug conjugate according to the present invention or the drug combination according to the present invention
  • the substance preferably, further comprises a device for administration.
  • Figure 1 shows that IL17A-M069 blocks IL17A-induced IL-6 secretion by HFF cells.
  • Figure 2 shows the binding of IL17A-H069 to recombinant human IL17A protein.
  • Figure 3 shows the binding of IL17A-H069 to recombinant human IL17A/F protein.
  • Figure 4 shows the species cross-binding between IL17A-H069 and mouse IL17A protein.
  • Figure 5 shows that IL17A-H069 blocks the binding of IL17RA to IL17A protein.
  • Figure 6 shows that IL17A-H069 blocks the binding of IL17RA to IL17A/F protein.
  • Figure 7 shows that IL17A-H069 blocks IL17A-mediated secretion of IL-6 by HFF cells.
  • Figure 8 shows the effect of IL17A-H069 on the psoriasis model index PASI in a mouse psoriasis model.
  • Figure 9 shows a single subcutaneous injection of IL17A-H069 drug concentration-time curve in cynomolgus monkeys.
  • Various aspects of the present invention relate to isolated anti-IL17A antibodies or antigen-binding fragments thereof, antibody-drug conjugates comprising the antibodies or antigen-binding fragments thereof, nucleic acids and expression vectors encoding the antibodies or antigen-binding fragments thereof, and containing the nucleic acids Or a host cell expressing a vector, a method for producing the anti-IL17A antibody or an antigen-binding fragment thereof, a pharmaceutical composition comprising the anti-IL17A antibody or an antigen-binding fragment thereof, and a method for treating psoriasis using the anti-IL17A antibody or an antigen-binding fragment thereof .
  • antibody means an immunoglobulin molecule, and refers to any form of antibody that exhibits the desired biological activity. Including but not limited to monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies and multispecific antibodies (such as bispecific antibodies), and even antibody fragments.
  • the full-length antibody structure preferably contains 4 polypeptide chains, usually 2 heavy (H) chains and 2 light (L) chains connected to each other by disulfide bonds. Each heavy chain contains a heavy chain variable region and a heavy chain constant region. Each light chain contains a light chain variable region and a light chain constant region. In addition to this typical full-length antibody structure, its structure also includes other derivative forms.
  • the heavy chain variable region and light chain variable region can be further subdivided into more conservative regions (called framework regions (FR)) and hypervariable regions interspersed (called complementarity determining regions (CDR)).
  • framework regions FR
  • CDR complementarity determining regions
  • CDR complementarity determining region
  • CDR1, CDR2, and CDR3 refers to the amino acid residues of the variable region of an antibody, the presence of which is necessary for antigen binding.
  • Each variable region usually has 3 CDR regions identified as CDR1, CDR2, and CDR3.
  • Each complementarity determining region may contain amino acid residues from the “complementarity determining region” defined by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. 1991 )) and/or those residues from the "hypervariable loop” (Chothia and Lesk; J Mol Biol 196:901-917 (1987)).
  • framework or "FR” residues are those variable region residues other than the CDR residues as defined herein.
  • Each heavy chain variable region and light chain variable region usually contains 3 CDRs and up to 4 FRs.
  • the CDRs and FRs are arranged in the following order from the amino terminal to the carboxy terminal, for example: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • CDR complementarity determining region
  • FR framework region
  • constant region refers to such amino acid sequences on the light chain and heavy chain of an antibody that do not directly participate in the binding of the antibody to the antigen, but exhibit various effector functions, such as antibody-dependent cytotoxicity.
  • the heavy chain of an antibody can be divided into five categories: ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ .
  • ⁇ When it forms a complete antibody with the light chain, it can be divided into five categories: IgA , IgD, IgE, IgG and IgM, several of these classes can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA and IgA2.
  • the light chain of an antibody can be classified into ⁇ and ⁇ .
  • an "antigen-binding fragment of an antibody” includes a portion of a complete antibody molecule that retains at least some of the binding specificity of the parent antibody, and usually includes at least a portion of the antigen-binding region or variable region (eg, one or more CDRs) of the parent antibody.
  • antigen-binding fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, Fd fragment, Fd' fragment, single-chain antibody molecules (e.g., scFv, di-scFv, or tri-scFv , Diabody or scFab), single domain antibody.
  • antibody fragment refers to an incomplete antibody molecule that retains at least some biological properties of the parent antibody, and examples thereof include, but are not limited to, Fc fragments in addition to those mentioned in the above-mentioned "antigen-binding fragments".
  • antibody-drug conjugate refers to a binding protein such as an antibody or antigen-binding fragment thereof chemically linked to one or more chemical drugs (also referred to herein as agents), which may optionally Ground is a therapeutic or cytotoxic agent.
  • the ADC includes an antibody, cytotoxic or therapeutic drug, and a linker that enables the drug to be linked or conjugated to the antibody.
  • ADCs usually have any value of 1 to 8 drugs conjugated to antibodies, including 2, 4, 6, or 8 drug-loading substances.
  • Non-limiting examples of drugs that can be included in the ADC are mitotic inhibitors, anti-tumor antibiotics, immunomodulators, vectors for gene therapy, alkylating agents, anti-IL17A agents, antimetabolites, boron-containing agents, chemotherapeutic protective agents , Hormones, antihormonal agents, corticosteroids, photoactive therapeutic agents, oligonucleotides, radionuclide agents, topoisomerase inhibitors, tyrosine kinase inhibitors and radiosensitizers.
  • chimeric antibody refers to an antibody in which a part of the heavy chain and/or light chain is derived from a specific source or species, and the remaining part is derived from a different source or species.
  • the “chimeric antibody” may also be a functional fragment as defined above.
  • Humanized antibodies are a subset of “chimeric antibodies.”
  • humanized antibody or “humanized antigen-binding fragment” is defined herein as an antibody or antibody fragment: (i) derived from a non-human source (for example, a transgenic mouse carrying a heterologous immune system) And based on human germline sequence; or (ii) the variable region is of non-human origin and the constant region is a chimeric antibody of human origin; or (iii) CDR grafted, wherein the CDR of the variable region is derived from a non-human source, and the variable One or more framework regions of the region are of human origin, and the constant region (if any) is of human origin.
  • the purpose of "humanization” is to eliminate the immunogenicity of non-human source antibodies in the human body, while retaining the greatest possible affinity.
  • a “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous antibody population, that is, the population comprising a single antibody is identical except for possible mutations (such as natural mutations) that may be present in very small amounts. Therefore, the term “monoclonal” indicates the nature of the antibody, that is, it is not a mixture of unrelated antibodies. In contrast to polyclonal antibody preparations which usually include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen. In addition to their specificity, the advantage of monoclonal antibody preparations is that they are generally not contaminated by other antibodies. The term “monoclonal” should not be understood as requiring the production of the antibody by any specific method.
  • the antibody "specifically binds" to the target antigen, such as a tumor-associated polypeptide antigen target (herein, PD-1), that is, binds to the antigen with sufficient affinity so that the antibody can be used as a therapeutic agent to target the expression of the Cells or tissues of the antigen, and have no significant cross-reactivity with other proteins or with the exception of homologs and variants (such as mutant forms, splice variants, or truncated forms of proteolysis) of the antigen target mentioned above No significant cross-reactivity of the protein.
  • PD-1 tumor-associated polypeptide antigen target
  • binding affinity refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule and its binding partner. Unless otherwise stated, "binding affinity” as used herein refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (eg, antibody and antigen).
  • KD refers to the equilibrium dissociation constant of the antibody-antigen interaction.
  • kon refers to the rate constant at which an antibody binds to an antigen.
  • the term “koff” refers to the rate constant at which the antibody dissociates from the antibody/antigen complex.
  • Binding affinity is affected by interactions between non-covalent molecules, such as hydrogen bonds, electrostatic interactions, hydrophobicity and van der Waals forces between two molecules.
  • binding affinity between the ligand and its target molecule may be affected by the presence of other molecules. Affinity can be analyzed by conventional methods known in the art, including the ELISA described herein.
  • epitope includes any protein determinant capable of specifically binding to an antibody or T cell receptor.
  • Epitope determinants usually consist of chemically active surface groups of molecules (for example, amino acids or sugar side chains, or combinations thereof), and usually have specific three-dimensional structural characteristics and specific charge characteristics.
  • isolated antibody is an antibody that has been identified and isolated from a component of the cell that expresses it. Isolated antibodies include antibodies in situ within recombinant cells where at least one component of the antibody's natural environment is absent. However, usually, the isolated antibody is prepared through at least one purification step.
  • sequence identity between two polypeptide or nucleic acid sequences means the number of identical residues between the sequences as a percentage of the total number of residues, and is calculated based on the size of the smaller of the compared molecules.
  • sequences being compared are aligned in a way that produces the largest match between the sequences, and the gaps in the alignment (if any) are resolved by a specific algorithm.
  • Preferred computer program methods for determining the identity between two sequences include, but are not limited to, the GCG program package, including GAP, BLASTP, BLASTN, and FASTA (Altschul et al., 1990, J. Mol. Biol. 215: 403-410) .
  • the above program can be publicly obtained from the International Center for Biotechnology Information (NCBI) and other sources.
  • NCBI International Center for Biotechnology Information
  • Smith Waterman algorithm can also be used to determine identity.
  • Fc receptor refers to a receptor that binds to the Fc region of an antibody.
  • Human FcR of natural sequence is preferred, and receptors ( ⁇ receptors) that bind to IgG antibodies are preferred, which include Fc ⁇ RI, Fc ⁇ RII, and Fc ⁇ RIII subtypes, and variants of these receptors.
  • Other FcRs are included in the term “FcR”.
  • the term also includes the neonatal receptor (FcRn) which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunology 117:587 (1976) and Kim et al., J. Immunology 24:249 (1994)).
  • FcRn neonatal Fc receptor
  • the neonatal Fc receptor (FcRn) plays an important role in the metabolic fate of IgG antibodies in the body. FcRn functions to rescue IgG from the lysosomal degradation pathway, thereby reducing its clearance in serum and increasing its half-life. Therefore, the in vitro FcRn binding properties/characteristics of IgG indicate its in vivo pharmacokinetic properties in the blood circulation.
  • effector functions refers to those biological activities attributable to the Fc region of an antibody, which differ by antibody isotype.
  • antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), cytokine secretion, immune complexes Mediated antigen uptake by antigen-presenting cells, down-regulation of cell surface receptors (such as B cell receptors), and B cell activation.
  • effector cells refers to leukocytes that express one or more FcRs and perform effector functions.
  • the effector cell at least expresses FcyRIII and performs ADCC effector function.
  • human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils.
  • PBMC peripheral blood mononuclear cells
  • NK natural killer cells
  • monocytes cytotoxic T cells
  • neutrophils effector cells can be isolated from natural sources, for example, blood. Effector cells are usually lymphocytes associated with the effector stage and function to produce cytokines (helper T cells), kill cells infected by pathogens (cytotoxic T cells) or secrete antibodies (differentiated B cells) .
  • Immune cells include cells that have hematopoietic origin and play a role in immune responses. Immune cells include: lymphocytes such as B cells and T cells; natural killer cells; myeloid cells such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • cytotoxic cells such as NK cells, neutrophils, and macrophages
  • the secreted Ig on the Fc ⁇ receptor enables these cytotoxic effector cells to specifically bind to the target cell carrying the antigen, and then kill the target cell using, for example, a cytotoxin.
  • an in vitro ADCC assay can be performed, for example, the in vitro ADCC assay described in U.S. Patent No. 5,500,362 or 5,821,337 or U.S. Patent No. 6,737,056 (Presta).
  • Useful effector cells for such assays include PBMC and NK cells.
  • “Complement dependent cytotoxicity” or “CDC” refers to the lysis of target cells in the presence of complement.
  • the activation of the typical complement pathway is initiated by combining the first component of the complement system (C1q) with an antibody (of the appropriate subclass) that binds to its corresponding antigen.
  • a CDC assay can be performed, such as the CDC assay described in Gazzano-Santoro et al., J. Immunol Methods 202:163 (1996).
  • polypeptide variants with an altered Fc region amino acid sequence polypeptides with a variant Fc region
  • polypeptide variants with enhanced or reduced C1q binding are described.
  • COSENTYX analogue and “Taltz analogue” refer to antibodies prepared according to the structures of COSENTYX and Taltz, respectively.
  • the present invention first uses recombinant human IL17A protein to immunize mice, and then obtains the antibody clone IL17A-M069 specifically binding to recombinant human IL17A protein through phage display library screening. Afterwards, PCR was used to insert the nucleotide sequences encoding the heavy chain and light chain variable regions of the IL17A-M069scFv antibody into the pSTEP2 vector carrying the mouse IgG1 heavy chain constant region or the mouse kappa light chain constant region nucleotide sequence, and Perform culture expression. Purification by protein A purification column to obtain high-purity mouse monoclonal antibody. ELISA test showed that the mouse-derived antibody can block the secretion of IL-6 in HFF cells induced by IL17A.
  • the light chain or heavy chain variable region of a human antibody that is closer to the mouse light chain or heavy chain variable region is selected as the template, and each of the mouse antibody light chain or heavy chain is selected as the template.
  • Three CDRs (Table 1) were inserted into the variable region of the human antibody to obtain humanized light chain variable region (VL) and heavy chain variable region (VH) sequences. Since the mouse-derived framework region has key points that are essential to support the activity of CDR, the key points are backmutated to the sequence corresponding to mouse antibodies.
  • the IL17A-H069 light chain/heavy chain expression vector was obtained by the method of full gene synthesis, transfected into CHO-K1-GS deficient cells and cultured for expression, analyzed and selected clones with high antibody expression, cultured and produced IL17A with high purity and high quality -H069 antibody.
  • the invention also relates to nucleic acid molecules encoding the antibodies of the invention or parts thereof.
  • the sequences of these nucleic acid molecules include but are not limited to SEQ ID NO: 2-7, 26-33, 36-37, 40-41, and 43.
  • nucleic acid molecules of the present invention are not limited to the sequences disclosed herein, but also include variants thereof.
  • the variants of the present invention can be described with reference to their physical characteristics in hybridization. Those skilled in the art will recognize that using nucleic acid hybridization techniques, nucleic acids can be used to identify their complements and their equivalents or homologs. It will also be recognized that hybridization can occur with less than 100% complementarity. However, considering the proper selection of conditions, hybridization techniques can be used to distinguish DNA sequences based on their structural correlation with specific probes.
  • the invention also provides a recombinant construct comprising one or more nucleotide sequences of the invention.
  • the recombinant construct of the present invention is constructed by inserting a nucleic acid molecule encoding the antibody of the present invention into a vector, such as a plasmid, phagemid, phage, or viral vector.
  • the antibody of the present invention can be prepared by recombinantly expressing nucleotide sequences encoding the light chain and the heavy chain or parts thereof in a host cell.
  • one or more recombinant expression vectors carrying the nucleotide sequence encoding the light chain and/or heavy chain or part thereof can be used to transfect the host cell so that the light chain and the heavy chain are in the Expressed in host cells.
  • Standard recombinant DNA methodology is used to prepare and/or obtain nucleic acids encoding heavy and light chains, incorporate these nucleic acids into recombinant expression vectors and introduce the vectors into host cells, such as Sambrook, Fritsch and Maniatis (eds.
  • Suitable host cells are prokaryotic cells and eukaryotic cells.
  • prokaryotic host cells are bacteria
  • examples of eukaryotic host cells are yeast, insect or mammalian cells. It should be understood that the design of the expression vector including the selection regulatory sequence is affected by many factors, such as the choice of host cell, the desired expression level of the protein, and whether the expression is constitutive or inducible.
  • a usable expression vector for bacteria By inserting the structural DNA sequence encoding the desired antibody together with suitable translation initiation and termination signals and a functional promoter into an operable reading frame, a usable expression vector for bacteria can be constructed.
  • the vector will contain one or more phenotypic selectable markers and an origin of replication to ensure the maintenance of the vector and provide amplification in the host as needed.
  • Suitable prokaryotic hosts for transformation include E. coli, Bacillus subtilis, Salmonella typhimurium, and Pseudomonas, Streptomyces, and grapes. Multiple species in the genus Staphylococcus.
  • Bacterial vectors can be, for example, phage, plasmid or phagemid based. These vectors may contain selectable markers and bacterial origins of replication, which are derived from commercially available plasmids that usually contain elements of the well-known cloning vector pBR322 (ATCC37017). After transforming an appropriate host strain and growing the host strain to an appropriate cell density, the selected promoter is de-repressed/induced by an appropriate method (for example, temperature change or chemical induction), and the cells are cultured for an additional time. The cells are usually harvested by centrifugation, broken down by physical or chemical methods, and the resulting crude extract is retained for further purification.
  • selectable markers and bacterial origins of replication which are derived from commercially available plasmids that usually contain elements of the well-known cloning vector pBR322 (ATCC37017).
  • a variety of expression vectors can be advantageously selected according to the intended use of the expressed protein. For example, when a large number of such proteins are to be produced for antibody production or for screening peptide libraries, for example, a vector that directs high-level expression of a fusion protein product that is easy to purify may be required.
  • Preferred regulatory sequences for expression in mammalian host cells include viral elements that direct high-level protein expression in mammalian cells, such as promoters and/or enhancers derived from cytomegalovirus (CMV) (e.g. CMV promoter/enhancer) Promoter), simian virus 40 (SV40) promoter and/or enhancer (e.g. SV40 promoter/enhancer), adenovirus promoter and/or enhancer (e.g. adenovirus major late promoter (AdMLP)) and Polyoma virus promoter and/or enhancer.
  • CMV cytomegalovirus
  • SV40 simian virus 40
  • AdMLP adenovirus major late promoter
  • Polyoma virus promoter and/or enhancer e.g. adenovirus major late promoter (AdMLP)
  • the recombinant expression vector may also include an origin of replication and a selection marker (see, for example, U.S. 4,399,216, U.S. 4,634,665 and U.S. 5,179,017 of Axel et al.).
  • Suitable selection markers include genes that confer resistance to drugs such as G418, hygromycin, or methotrexate to host cells into which the vector has been introduced.
  • drugs such as G418, hygromycin
  • methotrexate to host cells into which the vector has been introduced.
  • the dihydrofolate reductase (DHFR) gene confers resistance to methotrexate
  • the neo gene confers resistance to G418.
  • Transfection of the expression vector into host cells can be performed using standard techniques such as electroporation, calcium phosphate precipitation, and DEAE-dextran transfection.
  • Suitable mammalian host cells for expressing the antibodies provided herein include Chinese Hamster Ovary (CHO cells) [including dhfr-CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216 In -4220, DHFR selection markers are used, such as those described in RJ Kaufman and PASharp (1982) Mol. Biol. 159:601-621], NSO myeloma cells, COS cells, and SP2 cells.
  • the antibody of the present invention can be recovered and purified from recombinant cell culture by known methods, including but not limited to, ammonium sulfate or ethanol precipitation, acid extraction, protein A affinity chromatography, protein G affinity chromatography, anion Or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, and lectin chromatography.
  • High performance liquid chromatography (“HPLC”) can also be used for purification. See, for example, Colligan, Current Protocols in Immunology or Current Protocols in Protein Science, John Wiley&Sons, NY, NY, (1997-2001), such as Chapters 1, 4, 6, 8, 9, and 10, each of which is fully cited Include this article.
  • the maximum neutralization rate is 85.0% vs. 79.5%
  • the activity of neutralizing IL17A/F is better than Taltz at low concentration, and at high concentration Time is close to Taltz (EC 50 : 0.83 ⁇ g/mL vs. 1.10 ⁇ g/mL, maximum neutralization rate: 72.90% vs. 76.3%) (Example 5.1);
  • the antibodies of the present invention can be used to treat psoriasis.
  • the antibodies of the present invention can also be used to prepare drugs for treating the diseases.
  • the antibody of the present invention and at least one other agent can be prepared into a pharmaceutical composition, which includes the antibody of the present invention and one or more pharmaceutically acceptable carriers, diluents or excipients.
  • the pharmaceutical composition may include additional therapeutic agents.
  • the invention also relates to a pharmaceutical package and a kit comprising one or more containers containing the aforementioned pharmaceutical composition of the invention. It is accompanied by a reminder of the form prescribed by the government agency that regulates the production, use or sale of drugs or biological products, which reflects that the drug has been approved by the aforementioned agencies for human administration.
  • the pharmaceutical composition of the present invention can be prepared in a manner known in the art, for example, by conventional mixing, dissolving, granulating, tablet preparation, grinding, emulsifying, coating, embedding or freeze-drying methods.
  • compositions containing the compound of the present invention formulated in an acceptable carrier After the pharmaceutical composition containing the compound of the present invention formulated in an acceptable carrier has been prepared, they can be placed in an appropriate container and labeled for the treatment of the indicated condition.
  • labels would include the amount, frequency, and method of administration.
  • composition containing the antibody of the present invention is also combined with one or more other therapeutic agents, such as anti-tumor agents, wherein the resulting combination does not cause unacceptable adverse effects.
  • the present invention will be further understood with reference to the following non-limiting experimental examples.
  • the experimental methods in the following examples are conventional methods unless otherwise specified.
  • the test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent companies.
  • mice were immunized with IL17A.
  • the protein used is recombinant human IL17A protein (source: Beijing Yiqiao Shenzhou Technology Co., Ltd., Cat.10247-H07B), and the amino acid sequence of the IL17A protein (UniprotKB Q16552) is Met1-Ala155 (SEQ ID NO:1).
  • the recombinant human IL17A protein was mixed with aluminum phosphate adjuvants (one and four were added with complete Freund’s adjuvant CFA emulsified PBS, and the mixture was injected subcutaneously into mice at a dose of 50 ⁇ g/time.
  • the immunization intervals were as follows: 2 weeks, 3 weeks, 3 weeks. A total of 4 immunizations. From the second immunization, blood was collected from the intracanthal vein of the orbit for seven days after each immunization, and the solid phase was coated with recombinant human IL17A protein on the surface of the solid phase to respond to the immune response. The test was carried out. With the 8000-fold dilution titer of the immune serum OD>1.0 as the standard, after the fourth time the titer of the immune serum reached the standard, 25 ⁇ g of recombinant human IL17A protein was used for intravenous injection at an interval of 75 days. The mice were sacrificed 4 days later and taken The mouse spleens were frozen in liquid nitrogen.
  • the light and heavy chain variable regions are connected by the linker sequence (SEQ ID NO: 2), and the ligation product is then passed through the restriction endonuclease SfiI ( Fermentas, item number) was digested and linked to the phage vector pComb3x (Beijing Yiqiao Shenzhou Technology Co., Ltd., item number), and X-Blue competent (source, item number) was electrotransformed to construct a phage display scFv antibody library for immunized mice. The size of the library for. According to the phage antibody panning process, ELISA method was used to screen to obtain a phage library enriched with anti-IL17A positive antibodies (Aitken 2002).
  • Monoclonal phages were selected from the enriched library for expression, and their binding to recombinant human IL17A protein was detected by ELISA, and scFv clones that specifically bind to recombinant human IL17A were screened.
  • One clone was sequenced for its scFv antibody The nucleotide sequence is SEQ ID NO: 3, and the clone becomes IL17A-M069 after step 1.3.
  • the nucleotide sequence (SEQ ID NO: 4) of the heavy chain variable region of the scFv antibody, the heavy chain signal peptide sequence (SEQ ID NO: 43) and the mouse IgG1 heavy chain constant region (SEQ ID NO: 6) After the sequence is spliced, it is inserted into the pSTEP2 vector by Hind III and XbaI (Fermentas, article number) to obtain the complete heavy chain sequence (SEQ ID NO: 36) expression vector; similarly, the Over-lap PCR method is used.
  • the nucleotide sequence of the light chain variable region of the scFv antibody (SEQ ID NO: 5), the light chain signal peptide sequence (SEQ ID NO: 29) and the murine kappa light chain constant region (SEQ ID NO: 7) sequence were carried out.
  • IL17A can stimulate human foreskin fibroblast HFF to secrete the cytokine IL-6.
  • the anti-IL17A antibody was added to this system, and the IL-6 secretion of cells was detected to verify the neutralizing effect of IL17A antibody on IL17A activity.
  • HFF cells Inoculate HFF cells (ATCC, SCRC-1041) at a cell density of 1 ⁇ 10 4 /well, culture them overnight in DMEM medium containing 15% FBS, and add 10 ⁇ L/well of IL17A-M069 antibody and positive control Taltz in different concentrations the next day Analogs, then 10 ⁇ L/well was added with a final concentration of 50ng/mL IL17A protein, and the 96-well plate was placed in a 37°C, 5% CO 2 incubator for 48 hours. Set up detection blank well B (no cells), negative control group M'(seeding cells, no sample, plus factor) and M (seeding cells, no sample and factor).
  • the murine antibody IL17A-M069 can block the secretion of IL-6 by HFF cells induced by IL17A, and the maximum inhibitory rate and half inhibitory concentration of IL17A-M069 on IL17A are similar to those of the positive control Taltz analogs, respectively, 96.33 %, 97.35%, EC 50 is 243.3ng/mL and 246.6ng/mL, respectively. Therefore, IL17A-M069 is an antibody with good in vitro activity, which will be subsequently humanized and tested for various functions.
  • Example 3 Humanized transformation and production of IL17A antibody
  • the mouse antibody IL17A-M069 was humanized and produced.
  • the amino acid sequence of the heavy chain and light chain variable region of the IL17A-M069-scFv antibody was deduced from the nucleotide sequence of the IL17A-M069-scFv antibody determined in Example 1.3, see SEQ ID NO: 8/9.
  • Mouse antibody humanization adopts the classic humanization method CDR transplantation method (Kettleborough, Saldanha et al. 1991), and the human antibody light chain or heavy chain variable region closest to the mouse light chain or heavy chain variable region is preferred as Template (similarity>50%), insert each of the 3 CDR sequences (SEQ ID NO: 10-15) of the mouse antibody light chain or heavy chain into the variable region of the human antibody to obtain a humanized light chain Variable region (VL) and heavy chain variable region (VH) sequences.
  • VL humanized light chain Variable region
  • VH heavy chain variable region
  • the humanized template of the light chain variable region of IL17A-M069 involved is IGKV4-1*01, which has 75.2% homology with the light chain of IL17A-M069, and the heavy chain variable region of IL17A-M069 is The humanized template is IGHV1-69-2*01, which has 65.3% homology with the heavy chain of IL17A-M069.
  • the key points of the mouse-derived framework region are essential for the maintenance of the CDR structure and the binding of antigens (Saldanha, Martin et al. 1999), the key points of the humanized antibody were backmutated to the corresponding amino acids of the mouse antibody.
  • the specific positions are as follows: the 48th position of the light chain is backmutated to V, the 49th position is backmutated to D, and the 87th position is backmutated to F; the 24th position of the heavy chain is backmutated to A, and the 43rd position
  • the back mutation is H.
  • the humanized antibody IL17A-H069 was obtained by CDR humanization transplantation and framework region back mutation.
  • the heavy chain and light chain amino acid sequence is divided into (SEQ ID NO: 16/17); it contains signal peptide in the form of heavy chain and The light chain amino acid sequences are respectively (SEQ ID NO: 18/19); respectively include the heavy chain/light chain signal peptide sequence (SEQ ID NO: 20/21) connected in sequence; the humanized antibody heavy chain/light chain can be Variable region sequence (SEQ ID NO: 22/23); the constant region of the humanized antibody is the human IgG1 heavy chain constant region/human kappa light chain constant region sequence (SEQ ID NO: 24/25).
  • the above PCR product was inserted into the self-developed pGS vector (with KpnI+XbaI double enzyme digestion) by In-fusion method, and the correct plasmid was obtained by sequencing verification.
  • PCR amplifies the IL17A-H069 antibody heavy chain nucleotide sequence (SEQ ID NO: 26), which contains the heavy chain signal peptide nucleotide sequence (SEQ ID NO: 28), humanized antibody heavy
  • SEQ ID NO: 30 The nucleotide sequence of the variable region of the chain (SEQ ID NO: 30) and the nucleotide sequence of the constant region of the human IgG1 heavy chain (SEQ ID NO: 32).
  • the above PCR products were inserted into the correct containment by the In-fusion method.
  • the correct IL17A-H069 light and heavy chain expression vector was obtained through sequencing verification.
  • the expression vector is a eukaryotic cell expression vector containing GS screening gene and antibody light and heavy chain expression elements.
  • the expression vector was transfected into CHO-K1-GS deficient cells, and the IL17A-H069 highly expressing cell line was obtained by MSX screening.
  • ELISA was used to select clones with high antibody expression, and the high-expressing cell lines were screened by combining cell growth status and key quality attribute analysis results of antibody drugs.
  • the CHO cell line that produces IL17A-H069 is cultivated in a serum-free feeding suspension culture method to obtain high-purity and high-quality IL17A-H069 antibody.
  • the graphPad Prism software was used to analyze and draw a dose-effect curve to calculate the EC 50 value of the half effective concentration.
  • recombinant human IL17A/F dimer protein (source: Beijing Yiqiao Shenzhou Technology Co., Ltd., CT047-HNAE) were coated on a 96-well plate, 100 ⁇ L per well, and coated overnight at 4°C. The plate was washed the next day, and then sealed at room temperature for 1 hour, then 100 ⁇ L of 2 ⁇ g/mL SECUKINUMAB (Beijing Shenzhou Cell Engineering Co., Ltd.) and IL17A-H069 (Beijing Shenzhou Cell Engineering Co., Ltd.) were added to incubate, and then the plate was washed to remove unbound antibodies.
  • SECUKINUMAB Beijing Shenzhou Cell Engineering Co., Ltd.
  • IL17A-H069 Beijing Shenzhou Cell Engineering Co., Ltd.
  • the plate After incubation with goat anti-human IgG Fc/HRP (Beijing Yiqiao Shenzhou Technology Co., Ltd.), the plate was washed repeatedly, and the substrate color solution was added for color development. After termination, the microplate reader reads OD 450 .
  • the graphPad Prism software was used to analyze and draw a dose-effect curve, and calculate the EC 50 value of the half effective concentration.
  • the Octet Biomolecular Interaction Analysis System was used to determine IL17A-H069 (0.42nM, 0.90nM, 1.74nM, 3.47nM) at multiple concentration points, and the positive control COSENTYX (Novartis, SHM12) (0.90nM, 1.74nM, 3.47nM, 6.94) nM, 13.9nM) and biotinylated IL17A, IL17A/F protein affinity.
  • the binding affinity KD of IL17A-H069 and recombinant human IL17A protein is 2.88E-11M
  • the binding rate constant k on is 6.71E+05M -1 s -1
  • the dissociation rate constant k off is 1.93 E-05s -1
  • IL17A-H069 with recombinant human IL17A / F protein binding affinity KD value of 5.37E-10M, association rate constant k on value of 1.44E + 05M -1 s -1, the dissociation rate constant k off value of 7.72E-05s - 1 ;
  • the binding affinity KD value of COSENTYX and IL17A/F protein is 1.10E-09M, the binding rate constant k on value is 8.00E+04M -1 s -1 , the dissociation rate constant k off value is 8.79E-05s -1 from The results show that IL17A-H069 binds IL17A protein with a stronger affinity than COSENTYX.
  • the affinity of IL17A-H069 is about 3.32 times that of COSENTYX, and IL17A-H069 has a stronger binding rate, so IL17A-H069 has a stronger binding rate than COSENTYX. Strong ability to bind IL17A protein; IL17A-H069 binds IL17A/F protein with stronger affinity than COSENTYX.
  • the affinity of IL17A-H069 is about 2.05 times the affinity of COSENTYX, and IL17A-H069 has a stronger binding rate, so IL17A -H069 has a stronger ability to bind IL17A/F protein than COSENTYX.
  • OCTET detects the binding of IL17A-H069 with IL17A and IL17A/F
  • IL17A-H069 blocks the binding of IL17A, IL17A/F protein and receptor IL17RA
  • IL17A-H069 blocks the binding of IL17A protein to receptor IL17RA
  • the IL17A protein at a concentration of 0.4 ⁇ g/mL was coated on a 96-well plate, 100 ⁇ L per well, and coated overnight at 4°C. Wash the plate the next day and block it at room temperature for 1 hour, add 100 ⁇ L, 2 ⁇ g/mL IL17RA-His-biotin biotinylated protein (Beijing Yiqiao Shenzhou Technology Co., Ltd.), and then add different concentrations of IL17A-H069 (Beijing Shenzhou Cell Engineering Co., Ltd.) Company) and the positive control COSENTYX (Novartis, SHM12), Taltz (Lilly) and the negative control antibody H7N9-R1 (Beijing Shenzhou Cell Engineering Co., Ltd.) were incubated together, washed to remove unbound antibodies, and added Streptavidin/HRP (Beijing Zhongshan Jinqiao) Biotechnology Co., Ltd., SA-5004) After incubation, the
  • Inhibition rate% (OD blank- OD sample )/OD blank ⁇ 100%, where OD blank represents the OD value of the biotin-labeled receptor and no antibody group, OD sample represents the biotin-labeled receptor and antibody at the same time OD value of the group to be tested.
  • the experimental results are shown in Figure 58.
  • the biotinylated IL17RA protein can effectively bind to the coated recombinant human IL17A protein, and the IL17A-H069 antibody can inhibit the binding of IL17A protein to the receptor IL17RA, and its inhibition curve is significantly better than the positive control COSENTYX. Close to the positive control Taltz.
  • the IC 50 of IL17A-H069, COSENTYX and Taltz were 0.50 ⁇ g/mL, 2.99 ⁇ g/mL, 0.50 ⁇ g/mL, and the maximum inhibition rates were 85.4%, 73.5%, and 89.5%, respectively.
  • IL17A-H069 blocks the binding of IL17A/F protein and receptor IL17RA
  • the IL17RA-Fc protein (Beijing Yiqiao Shenzhou Technology Co., Ltd.) at a concentration of 5 ⁇ g/mL was coated on a 96-well plate, 100 ⁇ L per well, and coated overnight at 4°C.
  • Inhibition rate% (OD blank- OD sample )/OD blank ⁇ 100%, where OD blank represents the OD value of the biotin-labeled receptor and no antibody group, OD sample represents the biotin-labeled receptor and antibody at the same time OD value of the group to be tested.
  • IL17A/F-biotin protein can effectively bind to the coated recombinant human IL17RA-Fc protein, and the addition of IL17A-H069 antibody can effectively inhibit the binding of IL17A/F protein to the receptor IL17RA-Fc.
  • IL17A-H069 inhibited the binding of IL17A/F protein to receptor IL17RA-Fc better than the positive control COSENTYX analogue and Taltz analogue.
  • the IC 50 of IL17A-H069, COSENTYX analog and Taltz analog were 1.02 ⁇ g/mL, 1.20, 1.35 ⁇ g/mL, and the maximum inhibition rates were 92.3%, 87.9%, 75%, respectively.
  • IL17A-H069 blocks the secretion of IL-6 in HFF cells induced by IL17A and IL17A/F
  • HFF cells Inoculate HFF cells in a 96-well plate at a density of 1 ⁇ 10 4 /well, and culture them overnight in DMEM medium containing 15% FBS. The next day, add 10 ⁇ L of IL17A-H069 (Beijing Shenzhou Cell Engineering Co., Ltd.) of different concentrations to each well.
  • IL17A-H069 Beijing Shenzhou Cell Engineering Co., Ltd.
  • IL17A-H069 and IL17A / F of activity (EC 50: 0.83 ⁇ g / mL, and the maximum rate: 72.90%) and at high concentrations Taltz (EC 50: 1.10 ⁇ g / mL , and the maximum rate: 76.3%) of the active and close, It is better than the control Taltz at low concentrations ( Figure 6D).
  • IL17A-H069 has good biological activity of neutralizing IL17A and IL17A/F.
  • Example 6 In vivo efficacy of humanized antibody in mice
  • mice Using hPBMC from 3 donors, a total of 60 B-NDG mice (Biocytogram) humanized with the immune system were obtained (20 mice per donor). Peripheral blood was collected one week later, and the proportion of human-derived cells was measured by flow cytometry. 20 mice with a human-derived cell percentage between 0.04-1.5%, 31 mice between 1.5-7% and 8 mice with greater than 7% . Mice with a reconstruction ratio of 1.5-7% were selected to establish an IMQ (imiquimod) induced psoriasis model in mice, and based on this, the efficacy of the test drug was evaluated.
  • IMQ imiquimod
  • mice in the group were grouped according to the administration. There were 5 G1 mice in the normal control group without modeling, 5 G2 mice in the psoriasis model group without administration, and the IL17A-H069 group was given to the G3 group after modeling. After modeling, 7 rats were given to the positive control Taltz group (G4 group). The mouse shaved its back hair to form an exposed area about 2cm ⁇ 3cm in size. G2, G3, and G4 groups applied 100mg IMQ cream on the back and 10mg IMQ cream on the ears every day for 10 consecutive days. The PASI scores of mice were recorded daily.
  • mice were given a score of 0-4 for erythema, scaly and back skin thickening at the skin lesions, and the three points were added to obtain the total score.
  • G3 and G4 groups were administered IMQ cream on the first day, and the dosage was 10mpk, twice a week.
  • Example 7 In vivo pharmacokinetics of humanized antibodies
  • cynomolgus monkeys were used to perform a single subcutaneous injection of IL17A-H069 antibody at a dose of 1 mg/kg. Respectively before administration, 1h, 2h, 4h, 6h, 8h, 24h, 48h after administration, 3 days, 4 days, 7 days, 10 days, 14 days, 17 days, 21 days, 24 Serum was collected on days, 28, 31, and 35, and the established ELISA method was used to detect the drug concentration of IL17A-H069 in monkey serum, and the non-compartment model (NCA) in Phoenix-WinNonlin (Pharsight) 6.4 software was used. Calculate pharmacokinetic parameters. Investigate the dynamic changes of the drug in the body after a single subcutaneous injection of IL17A-H069.
  • the IL17A-H069 drug concentration changes over time as shown in Table 6 and Figure 9. There was no significant gender difference in C max and AUC last between female and male animals (results not shown), and the half-life t 1/2 of IL17A-H069 was 353.66 h, T max value is 34h. In terms of in vivo exposure, the AUC last of IL17A-H069 is 3846.86h* ⁇ g/mL.
  • IL17A-H069 At a dose of 1 mg/kg, IL17A-H069 has a shorter T max and a longer t 1/2 , so IL17A-H06 exhibits superior pharmacokinetics, including fast subcutaneous injection absorption, long half-life, and drug exposure The characteristics of better dosage, etc., laid the foundation for a longer administration cycle.

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Abstract

提供了一种结合IL17A的人源化单克隆抗体，以及编码所述抗体的核酸序列(包括重/轻链可变区)、含有所述核酸序列的载体、药物组合物和试剂盒。所述单克隆抗体能够以高亲和力特异性的结合IL17A蛋白、具有较强的封闭IL17A、IL17A/F与受体IL17RA的结合的能力，可用于治疗银屑病等。

Description

人源化抗IL17A抗体及其应用 技术领域
本发明属于免疫治疗领域，涉及一种人源化抗IL17A单克隆抗体药物及其应用。
背景技术
IL17A通常也称作IL-17，是由155个氨基酸构成的分子量为35kD的炎性细胞因子。它主要由辅助性T细胞17分泌，除此之外，CD4 +、CD8 +T、γδ-T细胞也可分泌IL-17。IL-17家族包含IL17A、IL-17B、IL-17C、IL-17D(IL-27)、IL-17E(IL-25)及IL-17F六个成员(Gu,Wu et al.2013)，其中IL17A与IL-17F是最主要的组分，氨基酸同源性为55％，可以形成以二硫键相连的同源二聚体或异源二聚体形式(Dubin and Kolls 2009)。IL17A/A或IL-17A/F二聚体与IL-17的受体家族中多种表达IL-17RA的细胞如：巨噬细胞、树突细胞、造血细胞、成骨细胞、纤维细胞等结合后，可以激活受体细胞内的NFκB、C/EBPs、MAPK等信号，诱导这些细胞分泌炎症因子和趋化因子(IL-6、IL-8、CXCL1等)，招募中性粒细胞，介导炎症反应的发生(Mitra,Raychaudhuri et al.2014)。许多炎症相关的自身免疫疾病如银屑病、银屑病关节炎、类风湿性关节炎、强直性脊柱炎等的发生发展与IL-17通路密切相关(Wang,Suzuki et al.2017)，在病人的血清中IL-17的表达水平显著上调(Marinoni,Ceribelli et al.2014)，导致持续性的炎症反应。此外，IL-17也可直接作用于皮肤中的内皮细胞、上皮细胞、成纤维细胞和角质细胞，增加多种炎性因子的释放，形成病态的皮肤(Mitra,Raychaudhuri et al.2014,Brembilla,Senra et al.2018)。因此，阻断IL-17通路为抑制自身免疫疾病的疾病进程提供可行性。
目前已上市IL-17靶点的抗体药物有诺华公司的
Figure PCTCN2020104448-appb-000001
(US7807155B2)及礼来制药的
Figure PCTCN2020104448-appb-000002
(US7838638B2/CN101326195B)，其主要机制为结合IL17A和IL17A/F，抑制IL17A与受体(IL-17RA/C)的结合，阻断炎症因子及趋化因子的释放，有效的缓解自身性免疫疾病(Fala 2016,Liu,Lu et al.2016)。Secukinumab获批用于斑块银屑病、银屑病关节炎、强直性脊柱炎的治疗；Ixekizumab获批用于斑块银屑病及银屑病关节炎的治疗。本发明中的一种结合IL17A的人源化单克隆抗体，能够以高亲和力特异性的结合IL17A蛋白、具有较强的封闭IL17A、IL17A/F与受体IL17RA的结合的能力，与同类药物相比， 具有优效或等效阻断IL17A、IL17A/F诱导人表皮成纤维细胞HFF分泌炎性细胞因子的功能；在银屑病小鼠模型中，该抗体也显示出显著优于同类药物的体内药效活性，用药后明显降低PASI评分；在食蟹猴药代试验中，该人源化抗体展现出优越的药代动力学，包括皮下注射吸收快、半衰期长，药物暴露量较好等特点，为更长的给药周期奠定了基础。拟用于包括但不局限于银屑病的治疗。
发明内容
本发明通过开发具有新结构的IL17A结合抗体而满足了上述需要。本发明公开了一种结合IL17A的人源化单克隆抗体能够以高亲和力特异性的结合IL17A蛋白、具有较强的封闭IL17A、IL17A/F与受体IL17RA的结合的能力。与同类药物相比，具有优效或等效阻断IL17A、IL17A/F诱导人表皮成纤维细胞HFF分泌炎性细胞因子的功能；在银屑病小鼠模型中，该抗体也显示出显著优于同类药物的体内药效活性，用药明显降低PASI评分；在食蟹猴药代试验中，该人源化抗体展现出优越的药代动力学，包括皮下注射吸收快、半衰期长，药物暴露量较好等特点，为更长的给药周期奠定了基础。本发明的人源化单克隆抗体可用于银屑病。
在一个方面，本发明提供一种分离的抗IL17A抗体或其抗原结合片段，其包含具有SEQ ID NO:13所示的氨基酸序列的重链CDR1域、具有SEQ ID NO:14所示的氨基酸序列的重链CDR2域和具有SEQ ID NO:15所示的氨基酸序列的重链CDR3域的重链可变区，和具有SEQ ID NO:10所示的氨基酸序列的轻链CDR1域、具有SEQ ID NO:11所示的氨基酸序列的轻链CDR2域和具有SEQ ID NO:12所示的氨基酸序列的轻链CDR3域的轻链可变区。(小鼠抗体M069和人源化抗体H069共有的6个CDR，在小鼠和人中相同)
在一个实施方式中，所述抗IL17A抗体或其抗原结合片段包含具有如SEQ ID NO:22的氨基酸序列或与SEQ ID NO:22至少90％、92％、95％、98％或99％序列同一性的氨基酸序列的重链可变区和具有如SEQ ID NO:23的氨基酸序列或与SEQ ID NO:23具有至少90％、92％、95％、98％或99％序列同一性的氨基酸序列的轻链可变区。(人源化抗体H069的重链轻链可变区氨基酸序列)
在一个实施方式中，所述抗IL17A抗体或其抗原结合片段为人源化抗体或嵌合抗体。
在一个实施方式中，所述抗IL17A抗体进一步包含重链恒定区和轻链恒定区，优选地所述重链恒定区为与氨基酸序列为SEQ ID NO:24的人IgG1 重链恒定区的氨基酸序列或与SEQ ID NO:24具有至少90％、92％、95％、98％或99％序列同一性的氨基酸序列，和/或所述轻链恒定区为氨基酸序列为SEQ ID NO:25的人kappa轻链恒定区的氨基酸序列或与SEQ ID NO:25具有至少90％、92％、95％、98％或99％序列同一性的氨基酸序列。(人源化抗体H069的重链轻链恒定区氨基酸序列)
在一个实施方式中，所述抗IL17A抗体进一步包含连接到重链可变区的信号肽和/或连接到轻链可变区的信号肽，优选地所述连接到重链可变区的信号肽为与氨基酸序列为SEQ ID NO:20的氨基酸序列或与SEQ ID NO:20具有至少90％、92％、95％、98％或99％序列同一性的氨基酸序列，和/或所述连接到轻链可变区的信号肽为氨基酸序列为SEQ ID NO:21的氨基酸序列或与SEQ ID NO:21具有至少90％、92％、95％、98％或99％序列同一性的氨基酸序列。(人源化抗体H069的重链轻链信号肽氨基酸序列)
在一个实施方式中，所述抗IL17A抗体或其抗原结合片段为IgG抗体，优选为IgG1抗体。
在一个实施方式中，所述抗IL17A抗体或其抗原结合片段为单克隆抗体。
在一个实施方式中，所述抗IL17A抗体或其抗原结合片段与重组人IL17A蛋白的结合亲和力K D为0.1-10E-11M，优选0.5-5E-11M，更优选2.88E-11M。
在一个实施方式中，所述抗IL17A抗体或其抗原结合片段与重组人IL17A/F蛋白的结合亲和力K D为0.1-10E-10M，优选0.5-5E-10M，更优选5.37E-10M。
在一个实施方式中，所述抗原结合片段为Fv、Fab、Fab′、Fab′-SH、F(ab′)2、Fd片段、Fd'片段、单链抗体分子或单域抗体；其中单链抗体分子优选为scFv、di-scFv、tri-scFv、双体抗体或scFab。
在另一个方面，本发明提供一种抗体-药物缀合物，其包含如本发明所述的抗IL17A抗体或其抗原结合片段和另外的治疗剂，优选地所述抗IL17A抗体或其抗原结合片段和另外的治疗剂通过接头连接。
在又一个方面，本发明提供一种核酸，其编码本发明所述的抗IL17A抗体或其抗原结合片段。
在一个实施方式中，所述核酸包含如SEQ ID NO:30所示的重链可变区核苷酸序列和/或如SEQ ID NO:31所示的轻链可变区核苷酸序列。
在又一个方面，本发明提供一种表达载体，其包含如本发明所述的核酸。
在又一个方面，本发明提供一种宿主细胞，其包含本发明所述的核酸 或如本发明所述的表达载体。
在又一个方面，本发明提供一种用于产生如本发明所述的抗IL17A抗体或其抗原结合片段的方法，其包括在适合于抗体表达的条件下培养如本发明所述的宿主细胞，和从培养基中回收表达的抗体。
在又一个方面，本发明提供一种药物组合物，其包含如本发明所述的抗IL17A抗体或其抗原结合片段或如本发明所述的抗体-药物缀合物或如本发明所述的核酸或如本发明所述的表达载体，及药学上可接受的载体。
在又一个方面，本发明提供如本发明所述的抗IL17A抗体或其抗原结合片段或如本发明所述的抗体-药物缀合物或如本发明所述的药物组合物，用于治疗银屑病。
在又一个方面，本发明提供一种用于治疗银屑病方法，其包括向有需要的受试者施用治疗有效量的如本发明所述的抗IL17A抗体或其抗原结合片段或如本发明所述的抗体-药物缀合物或如本发明所述的药物组合物，从而治疗银屑病。
在又一个方面，本发明提供如本发明所述的抗IL17A抗体或其抗原结合片段或如本发明所述的抗体-药物缀合物或如本发明所述的药物组合物在制备用于治疗银屑病药物中的用途。
在又一个方面，本发明提供一种药物组合，其包含如本发明所述的抗IL17A抗体或其抗原结合片段或如本发明所述的抗体-药物缀合物或如本发明所述的药物组合物与一种或多种另外的治疗剂。
在又一个方面，本发明提供一种试剂盒，其包含如本发明所述的抗IL17A抗体或其抗原结合片段或如本发明所述的抗体-药物缀合物或如本发明述的药物组合物，优选地，还进一步包含给药的装置。
附图说明
图1显示IL17A-M069阻断IL17A诱导的HFF细胞分泌IL-6。图2显示IL17A-H069与重组人IL17A蛋白的结合。
图3显示IL17A-H069与重组人IL17A/F蛋白的结合。
图4显示IL17A-H069与小鼠IL17A蛋白的种属交叉结合。
图5显示IL17A-H069封闭IL17RA与IL17A蛋白结合。
图6显示IL17A-H069封闭IL17RA与IL17A/F蛋白结合。
图7显示IL17A-H069阻断IL17A介导的HFF细胞分泌IL-6。
图8显示小鼠银屑病模型中IL17A-H069对银屑病模型指数PASI的影响。
图9显示食蟹猴单次皮下注射IL17A-H069药物浓度-时间曲线。
具体实施方式
本发明的各个方面涉及分离的抗IL17A抗体或其抗原结合片段、包含该抗体或其抗原结合片段的抗体-药物缀合物、编码该抗体或其抗原结合片段的核酸和表达载体、包含该核酸或表达载体的宿主细胞、产生该抗IL17A抗体或其抗原结合片段的方法、包含该抗IL17A抗体或其抗原结合片段的药物组合物以及使用该抗IL17A抗体或其抗原结合片段治疗银屑病方法。
定义
除非另有说明，本文使用的所有技术和科学术语具有本发明所属的技术领域的普通技术人员通常理解的含义。为了本发明的目的，定义以下术语，以同本技术领域通常理解的含义保持一致。
当用于本文和所附权利要求书中时，单数形式“一”、“一种”、“另一”和“所述”包括复数指代对象，除非上下文明确地另有指示。
术语“抗体”意指免疫球蛋白分子，是指表现所需生物学活性的抗体的任何形式。包括但不限于单克隆抗体(包括全长单克隆抗体)、多克隆抗体和多特异性抗体(例如双特异性抗体),甚至包括抗体片段。典型地，全长抗体结构优选包含4条多肽链，通常通过二硫键相互连接的2条重(H)链和2条轻(L)链。每条重链包含重链可变区和重链恒定区。每条轻链包含轻链可变区和轻链恒定区。在此典型全长抗体结构外,其结构还包括其他衍生形式。
所述重链可变区和轻链可变区可进一步细分为更保守的区域(称为框架区(FR))和穿插其中的高变区(称为互补决定区(CDR))。
术语“互补决定区”(CDR，例如CDR1、CDR2和CDR3)是指抗体可变区的这样一些氨基酸残基，其存在对于抗原结合来说是必需的。每个可变区通常具有3个被鉴别为CDR1、CDR2和CDR3的CDR区域。每个互补决定区可包含来自如Kabat所定义的“互补决定区”的氨基酸残基(Kabat等,Sequences of Proteins of Immulological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,MD.1991))和/或来自“高变环”的那些残基(Chothia和Lesk；J Mol Biol 196:901-917(1987))。
术语“构架”或“FR”残基是如本文中所定义的CDR残基之外的那些可变区残基。
每个重链可变区和轻链可变区通常包含3个CDR和最多达4个FR，所述CDR和FR从氨基末端至羧基末端以例如以下顺序排列：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4。
给定抗体的互补性决定区(CDR)和框架区(FR)可以使用Kabat体系标识(Kabat等:Sequences of Proteins of Immunological Interest，第5版，美国卫生和公众服务部，PHS，NIH，NIH出版编号91-3242，1991)。
术语“恒定区”是指抗体的轻链和重链上的这样一些氨基酸序列，不直接参与抗体与抗原的结合，但展现出多种效应子功能，例如抗体依赖性细胞毒性。
根据其恒定区的氨基酸序列的抗原性差异，抗体的重链可以被分为α、δ、ε、γ和μ五类，当其与轻链组成完整的抗体，可被分为五类：IgA、IgD、IgE、IgG和IgM，这些类中的若干还可进一步分为亚类(同种型)，例如IgG1、IgG2、IgG3、IgG4、IgA和IgA2。基于其恒定结构域的氨基酸序列，抗体的轻链可归入κ和λ。
“抗体的抗原结合片段”包含完整抗体分子的一部分，其保留母体抗体的至少某些结合特异性，通常包括至少部分母体抗体的抗原结合区或可变区(例如一个或多个CDR)。抗原结合片段的实例包括但不限于Fv、Fab、Fab′、Fab′-SH、F(ab′)2、Fd片段、Fd'片段、单链抗体分子(例如scFv、di-scFv或tri-scFv、双体抗体或scFab)、单域抗体。
术语“抗体片段”是指保留母体抗体的至少某些生物学特性的非完整抗体分子，其实例除上述“抗原结合片段”所述及的那些之外，还包括但不限于Fc片段。
术语“抗体-药物缀合物”或“ADC”是指与一种或多种化学药物(在本文中也称为药剂)化学连接的结合蛋白如抗体或其抗原结合片段)，其可以任选地是治疗剂或细胞毒性剂。在优选的实施方案中，ADC包括抗体、细胞毒性或治疗药物，以及能够使药物与抗体连接或缀合的接头。ADC通常具有与抗体缀合的1至8个中任一值的药物，包括2、4、6或8的载药物质。可以包含在ADC中的药物的非限制性实例是有丝分裂抑制剂、抗肿瘤抗生素、免疫调节剂、用于基因治疗载体、烷化剂、抗IL17A剂、抗代谢药、含硼药剂、化疗保护剂、激素、抗激素剂、皮质类固醇、光活性治疗剂、寡核苷酸、放射性核素剂、拓扑异构酶抑制剂、酪氨酸激酶抑制剂和放射致敏剂。
术语“嵌合抗体”是指重链和/或轻链的一部分来源于特定来源或物种，而其余部分来源于不同来源或物种的抗体。“嵌合抗体”亦可以为如上定义的功能性的片段。“人源化抗体”是“嵌合抗体”的子集。
术语“人源化抗体”或“人源化抗原结合片段”在本文中被定义为这样的抗体或抗体片段：(i)来源于非人来源(例如，携带异源免疫系统的转基因小鼠)且基于人种系序列；或(ii)可变区是非人来源而恒定区是人来源 的嵌合抗体；或者(iii)CDR移植的，其中可变区的CDR来自非人来源，而可变区的一个或多个构架区为人来源的，并且恒定区(如果有的话)是人来源的。“人源化”的目的是消除非人来源抗体在人体内的免疫原性,而同时最大可能地保留亲和力。选择与非人来源抗体构架序列最相似的人构架序列为模板进行人源化改造是有利的。在某些情况下，可能需要用非人构架中相应的残基替换人类构架序列中的一个或多个氨基酸，以避免亲和性的丧失。
“单克隆抗体”是指获自基本上同质的抗体群体的抗体，即，所述包含单一抗体的群体除了可能以极少量存在的可能突变(例如天然突变)之外是相同的。因此，所述术语“单克隆”表明所述抗体的性质，即不是不相关抗体的混合物。与通常包括针对不同决定簇(表位)的不同抗体的多克隆抗体制剂相反，单克隆抗体制剂的每个单克隆抗体均针对抗原上的单独一个决定簇。除了其特异性之外，单克隆抗体制剂的优点在于它们通常不会被其他抗体污染。所述术语“单克隆”不应被理解为需要通过任何特定的方法产生所述抗体。
抗体“特异性结合”目的抗原例如肿瘤相关的多肽抗原靶(本文中，PD-1)，即以足够的亲和力结合所述抗原以使得所述抗体可用作治疗性试剂，靶向表达所述抗原的细胞或组织，并且与其他蛋白质无显著交叉反应或者与除了上文提到的抗原靶的同源体和变体(例如突变形式、剪接变体，或蛋白水解作用截短的形式)以外的蛋白质无显著交叉反应。
术语“结合亲和力”是指分子的单个结合位点与其结合伴侣之间非共价相互作用总和的强度。除非另有说明，用于本文时“结合亲和力”是指固有的结合亲和力，其反映结合对(例如抗体和抗原)的成员之间1:1的相互作用。如本文所用，术语“KD”是指抗体-抗原相互作用的平衡解离常数。如本文所用，术语“kon”是指抗体与抗原结合的速率常数。如本文所用，术语“koff”是指抗体与抗体/抗原复合物解离的速率常数。“KD”、“结合速率常数k on”和“解离速率常数k off”通常用于描述分子(例如抗体)与其结合伴侣(例如抗原)之间的亲和力，即，受体结合特定蛋白的紧密程度。结合亲和力受非共价分子间相互作用的影响，例如氢键，静电相互作用，两个分子之间的疏水和范德华力。另外，配体与其靶分子之间的结合亲和力可能受到其他分子的存在的影响。亲和力可通过本领域中已知的常规方法来分析，包括本文描述的ELISA。
术语“表位”包括能够特异性结合至抗体或T细胞受体的任何蛋白质决定簇。表位决定簇通常由分子的化学活性表面基团(例如氨基酸或糖侧链，或其组合)组成，并且通常具有特定三维结构特征以及特定的电荷特征。
术语“分离的”抗体是已经被鉴别并且从表达它的细胞的组分中分离的抗体。分离的抗体包括重组细胞内的原位抗体，所述抗体的天然环境中的至少一种组分是不存在的。然而，通常情况下，分离的抗体是通过至少一个纯化步骤进行制备。
两条多肽或核酸序列之间的“序列同一性”表示所述序列之间相同的残基的数目占残基总数的百分比，且基于比较的分子中较小者的大小来计算。在计算同一性百分数时，将正在比较的序列以产生序列之间最大匹配的方式比对，通过特定算法解决比对中的空位(如果存在的话)。确定两个序列之间同一性的优选计算机程序方法包括，但不限于，GCG程序包，包括GAP、BLASTP、BLASTN和FASTA(Altschul等人，1990，J.Mol.Biol.215：403-410)。上述程序可以公开地从国际生物技术信息中心(NCBI)和其他来源得到。熟知的Smith Waterman算法也可用于确定同一性。
术语“Fc受体”或“FcR”指与抗体Fc区结合的受体。优选天然序列的人FcR，且优选与IgG抗体结合的受体(γ受体)，其包括FcγRI、FcγRII和FcγRIII亚型，以及这些受体的变体。其它FcR均被包含在术语“FcR”中。该术语也包括新生儿受体(FcRn)其负责将母体的IgG转运至胎儿(Guyer等，免疫学杂志117：587(1976)和Kim等，免疫学杂志24：249(1994))。
术语“新生儿Fc受体”、简称“FcRn”，其结合IgG抗体Fc区。新生儿Fc受体(FcRn)在体内IgG类抗体的代谢命运中起重要作用。FcRn行使功能以从溶酶体降解途径营救IgG，从而降低其在血清中的清除率并加长半衰期。因此，IgG体外FcRn结合性质/特征指示它在血液循环中的体内药代动力学性质。
术语“效应子功能”指可归因于抗体的Fc区的那些生物学活性，其随抗体同种型而不同。抗体效应子功能的实例包括：C1q结合和依赖补体的细胞毒性(CDC)、Fc受体结合、依赖抗体的细胞毒性(ADCC)、依赖抗体的吞噬作用(ADCP)、细胞因子分泌、免疫复合物介导的抗原呈递细胞对抗原的摄取、细胞表面受体(例如B细胞受体)的下调和B细胞激活。
术语“效应细胞”指表达一种或多种FcR并行使效应子功能的白细胞。在一个方面，所述效应细胞至少表达FcγRIII并执行ADCC效应子功能。介导ADCC的人白细胞的实例包括外周血单核细胞(PBMC)、自然杀伤(NK)细胞、单核细胞、细胞毒性T细胞和嗜中性粒细胞。效应细胞可以从天然来源，例如，血液中分离。效应细胞通常是与效应子阶段相关联的淋巴细胞，并发挥作用，以产生细胞因子(辅助T细胞)、杀死被病原体感染的细胞(细胞毒性T细胞)或分泌抗体(分化的B细胞)。
"免疫细胞"包括具有造血的起源并在免疫应答中起作用的细胞。免疫细 胞包括：淋巴细胞，例如B细胞和T细胞；天然杀伤细胞；髓样细胞，例如单核细胞、巨噬细胞、嗜曙红细胞、肥大细胞、嗜碱细胞和粒细胞。
“抗体依赖性细胞介导的细胞毒性”或“ADCC”是指一种细胞毒性形式，其中结合到在某些细胞毒性细胞(例如NK细胞、嗜中性粒细胞和巨噬细胞)上存在的Fcγ受体上的分泌Ig使得这些细胞毒性效应细胞能够特异性结合至承载抗原的靶细胞，随后使用例如细胞毒素杀死所述靶细胞。为了评估目的抗体的ADCC活性，可进行体外ADCC测定法，例如记载于美国专利No.5,500,362或5,821,337或美国专利No.6,737,056(Presta)中的体外ADCC测定法。用于这类测定法的有用效应细胞包括PBMC和NK细胞。
“补体依赖性细胞毒性”或“CDC”是指在补体的存在下靶细胞的裂解。典型的补体途径的活化是通过将补体系统的第一组分(C1q)与结合至其相应抗原的(适当亚类的)抗体结合来起始。为了评估补体活化，可进行CDC测定法，例如记载于Gazzano-Santoro等,J.Immunol Methods 202:163(1996)中的CDC测定法。例如在美国专利No.6,194,551B1和WO1999/51642中描述了具有改变的Fc区氨基酸序列的多肽变体(具有变体Fc区的多肽)和具有增强或降低的C1q结合的多肽变体。
术语“COSENTYX类似物”及“Taltz类似物”分别指按COSENTYX及Taltz的结构制备的抗体。
本发明的抗体的氨基酸序列
本发明首先采用重组人IL17A蛋白来免疫小鼠，然后通过噬菌体展示文库筛选获得与重组人IL17A蛋白特异性结合的抗体克隆IL17A-M069。之后采用PCR方法将编码IL17A-M069scFv抗体的重链和轻链可变区的核苷酸序列分别插入携带鼠IgG1重链恒定区或鼠kappa轻链恒定区核苷酸序列的pSTEP2载体中，并进行培养表达。采用蛋白A纯化柱进行纯化获得高纯度鼠单克隆抗体。ELISA测试表明，该小鼠源抗体能够阻断IL17A诱导的HFF细胞IL-6的分泌。
然后，采用经典的人源化方式CDR移植方法，选择与鼠轻链或重链可变区较接近的人抗体轻链或重链可变区为模板，将鼠抗体轻链或重链的各3个CDR(表1)插入到该人抗体的可变区中，获得人源化的轻链可变区(VL)和重链可变区(VH)序列。由于小鼠源框架区有关键点对于支撑CDR的活性至关重要，因此将关键位点回复突变为小鼠抗体对应的序列。通过全基因合成的方法获得IL17A-H069轻链/重链表达载体，转染CHO-K1-GS缺陷细胞并进行培养表达，分析选取抗体高表达的克隆，培养生产获得高纯度和高质量的IL17A-H069抗体。
本发明的核酸
本发明还涉及编码本发明的抗体或其部分的核酸分子。这些核酸分子的序列包括但不限于SEQ ID NO:2-7、26-33、36-37、40-41和43。
本发明的核酸分子不限于本文公开的序列，还包括其变体。本发明中变体可以参照它们在杂交中的物理特性来描述。本领域技术人员会认识到利用核酸杂交技术，核酸可用于鉴别其互补物以及其等同物或同系物。还会认识到杂交可以以低于100％互补性发生。然而，考虑到条件的适当选择，杂交技术可用于基于DNA序列与特定探针的结构相关性来区分所述DNA序列。对于这类条件的指导参见Sambrook等,Molecular Cloning:A Laboratory Manual,2nd Ed.；Cold Spring Harbor Laboratory Press,Cold Spring Harbor,N.Y.,1989和Ausubel,F.M.,Brent,R.,Kingston,R.E.,Moore,D.D.,Sedman,J.G.,Smith,J.A.,&Struhl,K.eds.(1995).Current Protocols in Molecular Biology.New York:John Wiley and Sons。
重组载体和表达
本发明还提供了包含本发明的一个或多个核苷酸序列的重组构建体。通过将编码本发明的抗体的核酸分子插入载体例如质粒、噬粒、噬菌体或病毒载体中构建本发明的重组构建体。
本发明的抗体可通过在宿主细胞中重组表达编码轻链和重链或其部分的核苷酸序列来制备。为了以重组方法表达抗体，可用携带编码轻链和/或重链或其部分的核苷酸序列的一个或多个重组表达载体转染宿主细胞，以使得所述轻链和重链在所述宿主细胞中表达。标准重组DNA方法学被用于制备和/或获得编码重链和轻链的核酸、将这些核酸纳入重组表达载体中并且将所述载体引入至宿主细胞中，例如Sambrook,Fritsch and Maniatis(eds.),Molecular Cloning；A Laboratory Manual,Second Edition,Cold Spring Harbor,N.Y.,(1989)、Ausubel,F.M.等(eds.)Current Protocols in Molecular Biology,Greene Publishing Associates,(1989)和Boss等的美国专利No.4,816,397中记载的那些。
合适的宿主细胞为原核细胞和真核细胞。原核宿主细胞的实例为细菌，真核宿主细胞的实例为酵母、昆虫或哺乳动物细胞。应理解，包括选择调节序列的表达载体的设计受到多种因素的影响，例如宿主细胞的选择、所需的蛋白质的表达水平以及表达是组成型的还是可诱导型的。
细菌表达
通过将编码所需抗体的结构DNA序列连同合适的翻译起始和终止信号以及有功能的启动子插入可操作阅读框中，来构建可用于细菌的可用表达载体。所述载体会包含一个或多个表型选择标记以及复制起点以确保维持所述载体，以及根据需要在宿主内提供扩增。用于转化的合适的原核宿主包括大肠杆菌(E.coli)、枯草芽孢杆菌(Bacillus subtilis)、鼠伤寒沙门氏菌(Salmonella typhimurium)以及假单胞菌属(Pseudomonas)、链霉菌属(Streptomyces)和葡萄球菌属(Staphylococcus)中的多个物种。
细菌载体可以是例如基于噬菌体、质粒或噬粒的。这些载体可含有选择标记和细菌复制起点，其来源于通常含有公知的克隆载体pBR322(ATCC37017)的元件的可商购的质粒。转化合适的宿主菌株并使所述宿主菌株生长至适当细胞密度之后，通过适当的方法(例如，温度变化或化学诱导)将所选择的启动子去阻遏/诱导，并且将细胞培养额外的时间。通常通过离心收获细胞，通过物理或化学方法使细胞破裂，并且保留所得的粗提取物用于进一步纯化。
在细菌系统中，根据所表达的蛋白的目的用途，可有利地选择多种表达载体。例如，当要生产大量这样的蛋白用于生产抗体或用于筛选肽文库时，例如，可能需要指导易于纯化的融合蛋白产物的高水平表达的载体。
哺乳动物表达和纯化
用于哺乳动物宿主细胞表达的优选调节序列包括在哺乳动物细胞中指导高水平蛋白表达的病毒元件，例如源于巨细胞病毒(CMV)的启动子和/或增强子(例如CMV启动子/增强子)、猿猴病毒40(SV40)的启动子和/或增强子(例如SV40启动子/增强子)、腺病毒的启动子和/或增强子(例如腺病毒主要晚期启动子(AdMLP))和多瘤病毒的启动子和/或增强子。对病毒调节元件及其序列的进一步描述参见例如，Stinski的U.S.5,168,062、Bell等的U.S.4,510,245和Schaffner等的U.S.4,968,615。重组表达载体还可以包括复制起点和选择标记(参见例如，Axel等的U.S.4,399,216、U.S.4,634,665和U.S.5,179,017)。合适的选择标记包括赋予已经引入所述载体的宿主细胞对药物例如G418、潮霉素或甲氨蝶呤的抗性的基因。例如，二氢叶酸还原酶(DHFR)基因赋予对甲氨蝶呤的抗性，而neo基因赋予对G418的抗性。
将所述表达载体至宿主细胞中的转染可以利用标准技术例如电穿孔、磷酸钙沉淀和DEAE-葡聚糖转染来进行。
用于表达本文提供的抗体的合适的哺乳动物宿主细胞包括中国仓鼠卵巢(CHO细胞)[包括dhfr-CHO细胞，记载于Urlaub和Chasin,(1980)Proc. Natl.Acad.Sci.USA 77:4216-4220中，使用DHFR选择标记，例如记载于R.J.Kaufman和P.A.Sharp(1982)Mol.Biol.159:601-621中]、NSO骨髓瘤细胞、COS细胞和SP2细胞。
本发明的抗体可通过公知方法从重组细胞培养物回收和纯化，所述公知方法包括但不限于，硫酸铵或乙醇沉淀、酸提取、蛋白A亲和层析、蛋白G亲和层析、阴离子或阳离子交换色谱法、磷酸纤维素色谱法、疏水相互作用色谱法、亲和色谱法、羟磷灰石色谱法以及凝集素色谱法。高效液相色谱法(“HPLC”)也可用于纯化。参见例如，Colligan,Current Protocols in Immunology或Current Protocols in Protein Science,John Wiley&Sons,NY,N.Y.,(1997-2001)，例如第1、4、6、8、9、10章，各自以引用的方式全文纳入本文。
本发明的抗体的特性和功能
对本发明的人源化IL17A-H069抗体进行特性分析和功能分析。分析结果表明，本发明的抗体具备以下优势：
(1)与重组人IL17A蛋白有较好的特异性结合，优于COSENTYX类似物(EC 50为46ng/mL vs.EC 50为74.8ng/mL实施例4.1.1)；
(2)与重组人IL17A/F二聚体蛋白有较好的特异性结合，优于COSENTYX类似物(EC 50为36.3ng/mL vs.EC 50为63.9ng/mL实施例4.1.2)；
(3)与重组人IL17A蛋白有良好的亲和力(远高于COSENTYX；2.88E-11M vs.9.55-11M)和结合速率(远快于COSENTYX；6.71E+05M -1s -1vs.1.78E+05M -1s -1),与重组人IL17A/F蛋白有良好的亲和力(高于COSENTYX；5.37E-10M vs.1.10-09M)和结合速率(快于COSENTYX；1.44E+05M -1s -1vs.8.00E+04M -1s -1),(实施例4.1.3)；
(4)与小鼠mIL17A蛋白无交叉结合(实施例4.1.4)；
(5)有效结合重组人IL17A蛋白，有效抑制IL17A蛋白与受体IL17RA结合，显著优于COSENTYX,接近Taltz(IC 50：0.50μg/mL vs.2.99μg/mL vs.0.50μg/mL；最大抑制率：85.4％vs.73.5％vs.89.5％(实施例4.2.1)；
(6)有效结合重组人IL17A/F二聚体蛋白，有效抑制IL17A蛋白与受体IL17RA结合，优于COSENTYX类似物及Taltz类似物(IC 50：1.02μg/mL vs.1.2μg/mL vs 1.35μg/mL；最大抑制率：92.3％、87.9％、75％)(实施例4.2.2)；
(7)阻断IL17A、IL17A/F诱导的HFF细胞IL-6的分泌；中和IL17A的活性远高于COSENTYX(EC 50为0.19μg/mL vs.0.22μg/mL，最大中和率为94.6％vs.51.6％)，中和IL17A的活性接近Taltz(EC 50为0.20μg/mL  vs.0.19μg/mL，最大中和率为90.3％vs.95.9％)；中和IL17A/F的活性也高于COSENTYX(EC 50为1.19μg/mL vs.2.25μg/mL，最大中和率为85.0％vs.79.5％)；，中和IL17A/F的活性在低浓度时优于Taltz，在高浓度时接近Taltz(EC 50：0.83μg/mL vs.1.10μg/mL，最大中和率：72.90％vs.76.3％)(实施例5.1)；
(8)在小鼠银屑病动物模型中验证，有效缓解银屑病进展，减轻症状，其药效显著优于对照Taltz(实施例6)；
(9)药代动力学上能够经皮下注射后被较快吸收、半衰期长(实施例7)。
用途
本发明的抗体可用于治疗银屑病。本发明的抗体还可用于制备治疗所述病症的药物。
药物组合物
可将本发明的抗体与至少一种其他试剂(例如稳定化合物)制备成药物组合物，其包括本发明的抗体和一种或多种药学上可接受的载体、稀释剂或赋形剂。任选地，所述药物组合物可包含另外的治疗剂。
试剂盒
本发明还涉及药物包装和包含一个或多个容器的试剂盒，所述容器含有上文提到的本发明的药物组合物。其上附有管理药物或生物制品的生产、使用或销售的政府机构规定形式提示，其反映该药物被上述机构批准用于人类给药。
制备和储存
本发明的药物组合物可以以本领域中已知的方式制备，例如通过常规的混合、溶解、造粒、锭剂制备、研磨、乳化、包裹、包埋或冻干方法。
在已经制备包含配制于可接受的载体中的本发明化合物的药物组合物之后，可以将它们放置在适当的容器中并贴上标签用于治疗所标明的病症。这类标签会包括给药的量、频率和方法。
药物组合
上述包含本发明的抗体的药物组合物还与一种或多种其他治疗剂，例如抗肿瘤剂组合，其中所得组合不会引起不可接受的不利影响。
实施例
将参照下述非限制性实验实施例进一步理解本发明。下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的试验材料，如无特殊说明，均自常规生化试剂公司购买。
实施例1：IL17A抗体的免疫及筛选
1.1小鼠的免疫
按照StGroth等人描述的方法(de StGroth and Scheidegger 1980)并加以适当修改，用IL17A对小鼠进行免疫。所用蛋白为重组人IL17A蛋白(来源：北京义翘神州科技有限公司，Cat.10247-H07B)，该IL17A蛋白(UniprotKB Q16552)的氨基酸序列为Met1-Ala155(SEQ ID NO:1)。将该重组人IL17A蛋白与磷酸铝佐剂(其中一免、四免额外添加完全弗氏佐剂CFA乳化PBS混合，混合物以50μg/次的剂量对小鼠进行多点皮下注射，免疫间隔依次为2周、3周、3周。共免疫4次。从第二次免疫起，每次免疫后七天经眼眶内眦静脉采血，用ELISA方法，在固相表面包被重组人IL17A蛋白对免疫应答进行检测。以免疫血清8000倍稀释效价OD>1.0为标准，第四次免疫血清效价达标后，间隔75天，使用25μg重组人IL17A蛋白进行静脉注射加强，4天之后处死小鼠，取小鼠的脾脏冻存于液氮中。
1.2噬菌体抗体文库构建及筛选
用TriPure Isolation Reagent试剂(来源：Roche Cat.No.11667165001)从小鼠的脾组织提取RNA，利用反转录试剂盒(Invitrogen,Cat.No.18080-051)对RNA反转录获得cDNA参考(Jones and Bendig 1991)设计2对引物扩增鼠抗体的轻链可变区，1对引物扩增重链可变区，经重叠延伸拼接PCR(Bryksin and Matsumura 2010)法将编码小鼠抗体的轻链和重链可变区核苷酸序列拼接成编码scFv的核苷酸序列，轻重链可变区通过接头linker序列(SEQ ID NO:2)进行连接，连接产物再通过限制性内切酶SfiI(Fermentas，货号)酶切连接到噬菌体载体pComb3x(北京义翘神州科技有限公司，货号)中，电转化X-Blue感受态(来源，货号)构建免疫小鼠的噬菌体展示scFv抗体库，文库的大小为。按照噬菌体抗体淘选的流程，用ELISA方法筛选获得抗IL17A阳性抗体富集的噬菌体文库(Aitken 2002)。从富集的文库中挑取单克隆噬菌体进行表达，用ELISA方法检测其与重组人IL17A蛋白的结合，筛选获得与重组人IL17A特异性结合的scFv克隆，其中的一个克隆经测序其scFv抗体的核苷酸序列为SEQ ID NO:3,该克隆经1.3步骤后成为IL17A-M069。
1.3 IL17A鼠单克隆抗体的生产
采用Over-lap PCR方法将scFv抗体的重链可变区的核苷酸序列(SEQ ID NO:4)、重链信号肽序列(SEQ ID NO:43)和鼠IgG1重链恒定区(SEQ ID NO:6)序列进行拼接后，通过Hind III和XbaI(Fermentas，货号)酶切插入pSTEP2载体中获得完整的重链序列(SEQ ID NO:36)表达载体；类似的，采用Over-lap PCR方法将scFv抗体的轻链可变区的核苷酸序列(SEQ ID NO:5)、轻链信号肽序列(SEQ ID NO:29)和鼠kappa轻链恒定区(SEQ ID NO:7)序列进行拼接后，通过Hind III和XbaI(Fermentas，货号)酶切插入pSTEP2载体中获得完整的轻链序列(SEQ ID NO:37)表达载体。拼接重链信号肽、重链可变区和鼠IgG1重链恒定区引物序列：
F1 AAGCTTGCCGCCACCATGGGCTGGTCCCTGATTCTGC
F2 GCTGGTCCCTGATTCTGCTGTTCCTGGTGGCTGTGGCT
F3 TTCCTGGTGGCTGTGGCTACCAGGGTGCTGAGCCA
F4 ACCAGGGTGCTGAGCCAGGCCCACCTTCAACAG
R1 TCGTTTTGGCTGAGGAGACTGTGAGAGTGGT
F5 TCTCCTCAGCCAAAACGACACCCCCATC
R2 CACTATAGAATAGGGCCCTCTA
拼接轻链信号肽、轻链可变区和鼠kappa轻链恒定区引物序列：
F6 CTGAAGCTTGCCGCCACCATGGGCTGGTCCTGTATCATCCTG
F7 GCTGGTCCTGTATCATCCTGTTCCTGGTGGCTACAGCC
F8 TTCCTGGTGGCTACAGCCACAGGAGTGCATAGCGACA
F9 ACAGGAGTGCATAGCGACATTGTGATGTCACAGTC
R3 CATCAGCCCGTTTTATTTCCAGCTTGGTCCC
F10 AAATAAAACGGGCTGATGCTGCACCAAC
R4 CACTATAGAATAGGGCCCTCTAGA
用SCD4-4-TC2培养基(北京义翘神州科技有限公司，货号)传代293E细胞(ATCC)至200mL/瓶，起始接种密度在0.3～0.4*10 6个/mL，放入温度为37℃转速为175rpm的CO 2摇床中培养，待细胞密度达到1.5～3*10 6个/mL后，按1:1混合轻重链质粒DNA，分别向培养瓶中加入100μg混合后的质粒DNA和800μL的TF2转染试剂，放入温度为37℃转速为175rpm的 摇床中继续培养至第7天收料。培养液4000rpm离心25min，收集上清，加入1/5上清体积的Stock buffer(来源：货号)。用PBS将蛋白A层析柱平衡5-10倍柱体积后，将过滤后的培养上清加入层析柱，再次平衡5-10倍柱体积后，用醋酸钠缓冲液洗脱样品。样品洗脱后用Tris中和至中性获得高纯度鼠单克隆抗体。
实施例2：IL17A鼠源抗体功能检测
2.1鼠抗体IL17A-M069阻断IL17A诱导的HFF细胞IL-6的分泌
按照(Beerli,Bauer et al.2014)所描述的在体外条件下，IL17A能够刺激人包皮成纤维细胞HFF分泌细胞因子IL-6。在此体系中加入抗IL17A抗体，通过检测细胞IL-6的分泌，验证IL17A抗体对IL17A活性的中和作用。以1×10 4/孔细胞密度接种HFF细胞(ATCC，SCRC-1041)，在含有15％FBS的DMEM培养基中培养过夜，次日10μL/孔加入不同浓度的IL17A-M069抗体及阳性对照Taltz类似物，随后10μL/孔加入终浓度为50ng/mL的IL17A蛋白,将96孔板置37℃、5％CO 2培养箱内作用48h。设置检测空白孔B(无细胞)、阴性对照组M’(接种细胞，不加样品，加因子)和M(接种细胞，不加样品及因子)。作用结束后，收取上清，利用ELISA检测IL-6的分泌。结果以IL-6的分泌量减去阴性对照组M的分泌量计算，抑制率％＝(1－样品分泌量)/阴性对照M’组分泌量×100％，采用统计软件计算标准曲线，横坐标为样品的浓度，纵坐标为IL-6分泌量。结果如图1所示，鼠抗体IL17A-M069可阻断IL17A诱导的HFF细胞分泌IL-6，且IL17A-M069对IL17A的最大抑制率、半数抑制浓度与阳性对照Taltz类似物相近，分别为96.33％、97.35％，EC 50分别为243.3ng/mL及246.6ng/mL。因此，IL17A-M069是一株体外活性较好抗体，后续对其进行人源化改造及各功能检测。
实施例3：IL17A抗体人源化改造及生产
根据实施例2中鼠抗体的功能检测结果，对鼠抗体IL17A-M069进行人源化改造及生产。
3.1鼠抗体轻链及重链的CDR序列确定
从实施例1.3中测定的IL17A-M069-scFv抗体的核苷酸序列推导出IL17A-M069-scFv抗体的重链和轻链可变区氨基酸序列，见SEQ ID NO:8/9。
参考Kabat(Abhinandan and Martin 2008,Dondelinger,Filée et al.2018)以 及IMGT编号(Lefranc 2014)方式确定抗体IL17A-M069-scFv轻链及重链各3个CDR的氨基酸序列,见表1及SEQ ID NO:10-15。上述的轻链及重链各3个CDR在后续步骤中被移植且保留在最终获得的人源化抗体IL17A-H069中,见实施例3.2和3.3。
表1.IL17A-M069轻链及重链CDR序列
Figure PCTCN2020104448-appb-000003
3.2鼠抗体人源化的CDR移植
鼠抗体人源化采用经典的人源化方式CDR移植方法(Kettleborough,Saldanha et al.1991)，首选与鼠轻链或重链可变区最接近的人抗体轻链或重链可变区作为模板(相似度>50％)，将鼠抗体轻链或重链的各3个CDR序列(SEQ ID NO:10-15)插入到该人抗体的可变区中，获得人源化的轻链可变区(VL)和重链可变区(VH)序列。所涉及的IL17A-M069的轻链可变区的人源化模板为IGKV4-1*01，该模板与IL17A-M069轻链的同源性为75.2％，IL17A-M069的重链可变区的人源化模板为IGHV1-69-2*01，该模板与IL17A-M069重链的同源性为65.3％。
3.3人源化可变区序列框架区的回复突变
由于鼠源框架区的关键点对于CDR区结构的维持及对抗原的结合至关重要(Saldanha,Martin et al.1999)，因此将人源化抗体的关键点回复突变为鼠抗体的相应氨基酸，按照Kabat编号，具体位点如下：轻链的第48位回复突变为V，第49位回复突变为D，第87位回复突变为F；重链的第24位回复突变为A，第43位回复突变为H。经CDR人源化移植和框架区回复突变获得人源化抗体IL17A-H069,其重链和轻链氨基酸序列分为(SEQ ID NO:16/17)；其含有信号肽的形式的重链和轻链氨基酸序列分别为(SEQ ID NO:18/19)；分别包含依次连接的重链/轻链信号肽序列(SEQ ID NO:20/21)；人源化抗体重链/轻链的可变区序列(SEQ ID NO:22/23)；人 源化抗体的恒定区为人IgG1重链恒定区/人kappa轻链恒定区序列(SEQ ID NO:24/25)。
3.4人源化抗体的生产
PCR扩增含有信号肽的IL17A-H069抗体轻链核苷酸序列(SEQ ID NO:27)，其中包含依次连接的轻链信号肽核苷酸序列(SEQ ID NO:29)、人源化抗体轻链可变区核苷酸序列(SEQ ID NO:31)及人kappa轻链恒定区核苷酸序列(SEQ ID NO:33)。上述PCR产物通过In-fusion方法插入自主研发的pGS载体(经KpnI+XbaI双酶切)中，通过测序验证获得正确的质粒。类似的，PCR扩增IL17A-H069抗体重链核苷酸序列(SEQ ID NO:26)，其中包含依次连接的重链信号肽核苷酸序列(SEQ ID NO:28)、人源化抗体重链可变区的核苷酸序列(SEQ ID NO:30)及人IgG1重链恒定区的核苷酸序列(SEQ ID NO:32)，上述PCR产物通过In-fusion方法插入已经构建正确的包含轻链的pGS载体(经NheI+NotI双酶切)中，通过测序验证获得正确的IL17A-H069轻重链表达载体。该表达载体为包含GS筛选基因和抗体轻、重链表达元件的真核细胞表达载体。将该表达载体转染至CHO-K1-GS缺陷的细胞中，经MSX筛选获得IL17A-H069高表达细胞株。采用ELISA检测选取抗体高表达的克隆，并结合细胞生长状态和抗体药物的关键质量属性分析结果筛选获得高表达细胞株。采用无血清加料悬浮培养的方式培养生产IL17A-H069的CHO细胞株，获得高纯度和高质量的IL17A-H069抗体。
实施例4：人源化抗体抗原结合及亲和力分析
4.1人源化抗体与IL17A蛋白结合亲和力分析
4.1.1 IL17A-H069与重组人IL17A蛋白的结合
将不同浓度的重组人IL17A蛋白(来源：北京义翘神州科技有限公司)包被于96孔板上，每孔100μL，4℃包被过夜。次日洗板，室温封闭1小时后，分别加入100μL、2μg/mL的COSENTYX类似物(北京神州细胞工程有限公司)、IL17A-H069(神州细胞工程有限公司)孵育，之后洗板去除未结合抗体，加入山羊抗人IgG Fc/HRP孵育后重复洗板，加入底物显色液进行显色，终止后酶标仪读取OD 450。以重组人IL17A蛋白包被浓度为横坐标，OD 450读数为纵坐标，利用GraphPad Prism软件分析并绘制量效曲线，计算半数有效浓度EC 50值。
结果如图2所示，COSENTYX类似物与重组人IL17A蛋白结合的EC 50为74.8ng/mL，R 2＝0.9993；IL17A-H069与重组人IL17A蛋白结合EC 50为46ng/mL，R 2＝0.9958，IL17A-H069与重组人IL17A蛋白的结合稍好于COSENTYX类似物。
4.1.2 IL17A-H069与重组人IL17A/IL17F蛋白的结合
将不同浓度的重组人IL17A/F二聚体蛋白(来源：北京义翘神州科技有限公司，CT047-HNAE)包被于96孔板上，每孔100μL，4℃包被过夜。次日洗板，室温封闭1小时后，分别加入100μL 2μg/mL的SECUKINUMAB(北京神州细胞工程有限公司)、IL17A-H069(北京神州细胞工程有限公司)孵育，之后洗板去除未结合抗体，加入山羊抗人IgG Fc/HRP(北京义翘神州科技有限公司)孵育后重复洗板，加入底物显色液进行显色，终止后酶标仪读取OD 450。以重组人IL17A/F蛋白包被浓度为横坐标，OD 450读数为纵坐标，利用GraphPad Prism软件分析并绘制量效曲线，计算半数有效浓度EC 50值。
结果显示(图3)，COSENTYX与IL17A/F蛋白结合EC 50为63.9ng/mL,R 2＝0.9999；IL17A-H069与IL17A/F蛋白结合EC 50为36.3ng/mL,R 2＝1.0，IL17A-H069与IL17A/F二聚体蛋白的结合稍好于COSENTYX类似物。
4.1.3 IL17A-H069与重组人IL17A、IL17A/F蛋白的结合亲和力
利用Octet生物分子相互作用分析系统测定多个浓度点的IL17A-H069(0.42nM、0.90nM、1.74nM、3.47nM)，阳性对照COSENTYX(诺华，SHM12)(0.90nM、1.74nM、3.47nM、6.94nM、13.9nM)与生物素化IL17A、IL17A/F蛋白的亲和力。如表2所示，IL17A-H069与重组人IL17A蛋白结合亲和力KD值为2.88E-11M，结合速率常数k on值为6.71E+05M -1s -1，解离速率常数k off值为1.93E-05s -1；COSENTYX与IL17A蛋白结合亲和力KD值为9.55E-11M，结合速率常数k on值为1.78E+05M -1s -1，解离速率常数k off值为1.70E-05s -1。IL17A-H069与重组人IL17A/F蛋白结合亲和力KD值为5.37E-10M，结合速率常数k on值为1.44E+05M -1s -1，解离速率常数k off值为7.72E-05s -1；COSENTYX与IL17A/F蛋白结合亲和力KD值为1.10E-09M，结合速率常数k on值为8.00E+04M -1s -1，解离速率常数k off值为8.79E-05s -1从结果可以看出IL17A-H069结合IL17A蛋白的亲和力强于COSENTYX结合的亲和力，IL17A-H069亲和力约为COSENTYX亲和力的3.32倍，并且IL17A-H069具有更强的结合速率，因此IL17A-H069比COSENTYX具有更强的结合IL17A蛋白的能力；IL17A-H069结合IL17A/F 蛋白的亲和力强于COSENTYX结合的亲和力，IL17A-H069亲和力约为COSENTYX亲和力的2.05倍，并且IL17A-H069具有更强的结合速率，因此IL17A-H069比COSENTYX具有更强的结合IL17A/F蛋白的能力。
表2.OCTET检测IL17A-H069与IL17A、IL17A/F结合
Figure PCTCN2020104448-appb-000004
4.1.4 IL17A-H069与小鼠IL17A蛋白的种属交叉结合
将不同浓度的重组人IL17A蛋白(北京义翘神州科技有限公司)和小鼠mIL17A蛋白(北京义翘神州科技有限公司)包被于96孔板上，每孔100μL，4℃包被过夜。次日洗板，室温封闭1小时后，分别加入100μL，2μg/mL的IL17A-H069(北京神州细胞工程有限公司)，阳性对照COSENTYX(诺华，SHM12)和阴性对照抗体H7N9-R1(北京神州细胞工程有限公司)孵育后洗板去除未结合抗体，加入山羊抗人IgG Fc/HRP(北京义翘神州科技有限公司)孵育后重复洗板，加入底物显色液进行显色，终止后酶标仪读取OD 450。结果如图4所示，IL17A-H069与小鼠mIL17A蛋白无结合。
4.2 IL17A-H069封闭IL17A、IL17A/F蛋白与受体IL17RA结合
4.2.1 IL17A-H069封闭IL17A蛋白与受体IL17RA结合
将浓度为0.4μg/mL的IL17A蛋白包被于96孔板上，每孔100μL，4℃包被过夜。次日洗板，室温封闭1小时后，加入100μL、2μg/mL IL17RA-His-biotin生物素化蛋白(北京义翘神州科技有限公司)，再加入不同浓度的IL17A-H069(北京神州细胞工程有限公司)及阳性对照COSENTYX(诺华，SHM12)、Taltz(礼来)及阴性对照抗体H7N9-R1(北京神州细胞工程有限公司)共同孵育，洗板去除未结合抗体，加入Streptavidin/HRP(北京中杉金桥生物技术有限公司，SA-5004)孵育后重复洗板，加入底物显色液进行显色，终止后酶标仪读取OD 450，每组2次平行。
以抗体浓度为横坐标，抑制率％为纵坐标，利用GraphPad Prism软件分析并绘图，计算IC 50值。抑制率％＝(OD 空白-OD 样品)/OD 空白×100％，其中OD 空白表示只加生物素标记受体不加抗体组的OD值，OD 样品表示同时加 生物素标记受体及抗体的待测组OD值。
实验结果如图58所示，生物素化的IL17RA蛋白可有效结合包被的重组人IL17A蛋白，IL17A-H069抗体可抑制IL17A蛋白与受体IL17RA的结合，其抑制曲线显著优于阳性对照COSENTYX，接近阳性对照Taltz。IL17A-H069、COSENTYX及Taltz的IC 50分别为：0.50μg/mL、2.99μg/mL、0.50μg/mL，最大抑制率分别为85.4％、73.5％、89.5％。
4.2.2 IL17A-H069封闭IL17A/F蛋白与受体IL17RA结合
将浓度为5μg/mL的IL17RA-Fc蛋白(北京义翘神州科技有限公司)包被于96孔板上，每孔100μL，4℃包被过夜。次日洗板，室温封闭1小时后，加入100μL、0.8μg/mL IL17A/F-Biotin蛋白(北京义翘神州科技有限公司)，再加入不同浓度的IL17A-H069(北京神州细胞工程有限公司)，阳性对照COSENTYX类似物(北京神州细胞工程有限公司)、Taltz类似物(北京神州细胞工程有限公司)共同孵育，洗板去除未结合抗体，加入Streptavidin/HRP(北京中杉金桥生物技术有限公司，SA-5004)孵育后重复洗板，加入底物显色液进行显色，终止后酶标仪读取OD 450。
以抗体浓度为横坐标，抑制率％为纵坐标，利用GraphPad Prism软件分析并绘图，计算IC 50值。抑制率％＝(OD 空白-OD 样品)/OD 空白×100％，其中OD 空白表示只加生物素标记受体不加抗体组的OD值，OD 样品表示同时加生物素标记受体及抗体的待测组OD值。
实验结果如图6所示，IL17A/F-biotin蛋白可有效结合包被的重组人IL17RA-Fc蛋白，加入IL17A-H069抗体可有效的抑制IL17A/F蛋白与受体IL17RA-Fc的结合。IL17A-H069对IL17A/F蛋白与受体IL17RA-Fc结合的抑制优于阳性对照COSENTYX类似物及Taltz类似物。IL17A-H069、COSENTYX类似物及Taltz类似物的IC 50分别为：1.02μg/mL、1.20、1.35μg/mL，最大抑制率分别为92.3％、87.9％、75％。
实施例5：人源化抗体的功能分析
5.1 IL17A-H069阻断IL17A、IL17A/F诱导的HFF细胞IL-6的分泌
以1×10 4/孔密度接种HFF细胞于96孔板，在含有15％FBS的DMEM培养基中培养过夜，次日每孔加入10μL不同浓度的IL17A-H069(北京神州细胞工程有限公司)、及阳性对照COSENTYX(诺华)或Taltz(礼来)抗体，随后分别每孔加入10μL终浓度为50ng/mL的IL17A蛋白(北京义翘神州科技有限公司，12047-HNAS)或终浓度为1μg/mL的IL17A/F蛋白 (北京义翘神州科技有限公司，CT047-HNAE),将96孔板置于37℃、5％CO 2的培养箱内反应48h。设置检测空白对照B(无细胞)、阴性对照组M’(接种细胞，含因子，无抗体)和M(接种细胞，无因子及抗体)。作用结束后，收取上清，利用ELISA检测细胞因子IL-6的分泌。结果以IL-6的分泌量减去阴性对照组M的分泌量计算，中和率％＝(1－样品分泌量)/阴性对照M’组分泌量×100％，采用统计软件GraphPad Prism计算标准曲线，横坐标为样品的浓度，纵坐标为中和率，回归方程为四参数方程，得到“S”型曲线，计算样品半数有效浓度EC 50。
在描述的测定中，如图7及表3所示，IL17A-H069中和IL17A的活性(EC 50：0.19μg/mL，最大中和率：94.6％)远高于阳性对照COSENTYX(EC 50：0.22μg/mL，最大中和率：51.6％)(图6A)，在与阳性对照Taltz的对比中，IL17A-H069中和IL17A的活性(EC 50：0.20μg/mL，最大中和率：90.3％)与Taltz(EC 50：0.19μg/mL，最大中和率：95.9％)的中和活性接近(图6C)；IL17A-H069(EC 50：1.19μg/mL，最大中和率：85.0％)中和IL17A/F的活性也略高于COSENTYX(EC 50：2.25μg/mL，最大中和率：79.5％)(图6B)，在与阳性对照Taltz的对比中，IL17A-H069中和IL17A/F的活性在高浓度时(EC 50：0.83μg/mL，最大中和率：72.90％)与Taltz(EC 50：1.10μg/mL，最大中和率：76.3％)的中和活性接近，在低浓度优于对照Taltz(图6D)。综上，IL17A-H069具有较好的中和IL17A及IL17A/F的生物活性。
表3.IL17A-H069阻断IL17A介导HFF细胞分泌IL6的EC 50及最大中和率
Figure PCTCN2020104448-appb-000005
实施例6：人源化抗体的小鼠体内药效
6.1 IL17A-H069在hPBMC免疫重建小鼠银屑病(PsO)模型中的体内药效
用来自3个供者的hPBMC，获得免疫系统人源化的B-NDG小鼠(百奥赛图)，共60只(每个供者20只)。一周后采集外周血，流式检测人源细胞比例，人源细胞百分比0.04-1.5％之间的小鼠20只，1.5-7％之间的小鼠31只，大于7％的小鼠8只。选取重建比例1.5-7％之间的小鼠，建立IMQ(咪喹莫特)诱导的小鼠银屑病模型，并基于此进行受试药物药效评价。
入组小鼠按照给药进行分组，未建模正常对照组G1小鼠5只，建模未给药即银屑病模型组G2小鼠5只，建模后给予IL17A-H069组即G3组7只，建模后给予阳性对照Taltz组即G4组7只。小鼠剃除其背部毛发，形成约2cm×3cm大小的暴露区域。G2、G3、G4组每天在背部涂抹100mg IMQ乳膏，耳朵涂抹10mg IMQ乳膏，连续10天。每天记录小鼠PASI评分。依据PASI评分标准，如表4所示，给予小鼠皮损处红斑、鳞屑及背部皮肤增厚程度0-4的评分，将三者积分相加得到总分。G3、G4组涂抹IMQ乳膏的第一天即开始给药，给药剂量均为10mpk，每周给药2次。
表4.PASI评分(严重度指数)标准
Figure PCTCN2020104448-appb-000006
结果如图8所示，与正常对照组小鼠相比，银屑病模型组PASI评分显著升高，反映该小鼠模型在一定程度上可以表征银屑病的特征；从第五天开始，与银屑病模型组相比IL17A-H069给药组PASI评分显著下降，并且 相比于对照Taltz，IL17A-H069具有更强的降低小鼠银屑病评分的体内药效，因此IL17A-H069能有效缓解银屑病(PsO)模型小鼠的发病程度，减轻银屑病症状。
实施例7：人源化抗体的体内药代动力学
7.1食蟹猴单次皮下注射IL17A-H069的药代动力学
本实施例采用食蟹猴，进行IL17A-H069抗体单次皮下注射给药，剂量均为1mg/kg。分别于给药前、给药当天药后1h、2h、4h、6h、8h、24h、48h以及给药后3天、4天、7天、10天、14天、17天、21天、24天、28天、31天、35天采集血清，采用已建立的ELISA方法对猴血清中IL17A-H069的药物浓度进行检测，并采用Phoenix-WinNonlin(Pharsight)6.4软件中非房室模型(NCA)计算药代动力学参数。考察单次皮下注射IL17A-H069后药物在体内的动态变化规律。
IL17A-H069药物浓度随时间变化如表6及图9所示，雌性动物和雄性动物C max、AUC last均未见明显性别差异(结果未展示)，IL17A-H069的半衰期t 1/2为353.66h，T max值为34h。在体内暴露量方面，IL17A-H069的AUC last为3846.86h*μg/mL。
在1mg/kg的给药剂量下，IL17A-H069的T max较短、t 1/2较长，因此IL17A-H06展现出优越的药代动力学，包括皮下注射吸收快、半衰期长、药物暴露量较好等特点，为更长的给药周期奠定了基础。
表6.单次皮下注射给予食蟹猴的药代动力学参数
Figure PCTCN2020104448-appb-000007
序列表
Figure PCTCN2020104448-appb-000008
Figure PCTCN2020104448-appb-000009
Figure PCTCN2020104448-appb-000010
Figure PCTCN2020104448-appb-000011
Figure PCTCN2020104448-appb-000012
Figure PCTCN2020104448-appb-000013
Figure PCTCN2020104448-appb-000014
Figure PCTCN2020104448-appb-000015
Figure PCTCN2020104448-appb-000016
Figure PCTCN2020104448-appb-000017
Figure PCTCN2020104448-appb-000018
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Claims (22)

  1. 一种分离的抗IL17A抗体或其抗原结合片段，其包含具有SEQ IDNO:13所示的氨基酸序列的重链CDR1域、具有SEQ ID NO:14所示的氨基酸序列的重链CDR2域和具有SEQ ID NO:15所示的氨基酸序列的重链CDR3域的重链可变区，和具有SEQ ID NO:10所示的氨基酸序列的轻链CDR1域、具有SEQ ID NO:11所示的氨基酸序列的轻链CDR2域和具有SEQ ID NO:12所示的氨基酸序列的轻链CDR3域的轻链可变区。
  2. 如权利要求1所述的抗IL17A抗体或其抗原结合片段，其包含具有如SEQ ID NO:22的氨基酸序列或与SEQ ID NO:22至少90％、92％、95％、98％或99％序列同一性的氨基酸序列的重链可变区和具有如SEQ ID NO:23的氨基酸序列或与SEQ ID NO:23具有至少90％、92％、95％、98％或99％序列同一性的氨基酸序列的轻链可变区。
  3. 如权利要求1所述的抗IL17A抗体或其抗原结合片段，其为人源化抗体或嵌合抗体。
  4. 如权利要求1-3中任一项所述的抗IL17A抗体或其抗原结合片段，其中所述抗体进一步包含重链恒定区和轻链恒定区，优选地所述重链恒定区为与氨基酸序列为SEQ ID NO:24的人IgG1重链恒定区的氨基酸序列或与SEQ ID NO:24具有至少90％、92％、95％、98％或99％序列同一性的氨基酸序列，和/或所述轻链恒定区为氨基酸序列为SEQ ID NO:25的人kappa轻链恒定区的氨基酸序列或与SEQ ID NO:25具有至少90％、92％、95％、98％或99％序列同一性的氨基酸序列。
  5. 如权利要求1-3中任一项所述的抗IL17A抗体或其抗原结合片段，其中所述抗体进一步包含连接到重链可变区的信号肽和/或连接到轻链可变区的信号肽，优选地所述连接到重链可变区的信号肽为氨基酸序列为SEQID NO:20的氨基酸序列或与SEQ ID NO:20具有至少90％、92％、95％、98％或99％序列同一性的氨基酸序列，和/或所述连接到轻链可变区的信号肽为氨基酸序列为SEQ ID NO:21的氨基酸序列或与SEQ ID NO:21具有至少90％、92％、95％、98％或99％序列同一性的氨基酸序列。
  6. 如权利要求1-5中任一项所述的抗IL17A抗体或其抗原结合片段，其为IgG抗体，优选为IgG1抗体。
  7. 如权利要求1-6中任一项所述的抗IL17A抗体或其抗原结合片段，其为单克隆抗体。
  8. 如权利要求1-7中任一项所述的抗IL17A抗体或其抗原结合片段，其与重组人IL17A蛋白的结合亲和力K D为0.1-10E-11M，优选0.5-5E-11M，更优选2.88E-11M。
  9. 如权利要求1-7中任一项所述的抗IL17A抗体或其抗原结合片段，其与重组人IL17A/F蛋白的结合亲和力K D为0.1-10E-10M，优选0.5-5E-10M，更优选5.37E-10M。
  10. 如权利要求1-9任一项所述的抗IL17A抗体或其抗原结合片段，其中所述抗原结合片段为Fv、Fab、Fab′、Fab′-SH、F(ab′)2、Fd片段、Fd'片段、单链抗体分子或单域抗体；其中单链抗体分子优选为scFv、di-scFv、tri-scFv、双体抗体或scFab。
  11. 一种抗体-药物缀合物，其包含如权利要求1-10任一项所述的抗IL17A抗体或其抗原结合片段和另外的治疗剂，优选地所述抗IL17A抗体或其抗原结合片段和另外的治疗剂通过接头连接。
  12. 一种核酸，其编码根据权利要求1-10任一项的抗IL17A抗体或其抗原结合片段。
  13. 如权利要求12所述的核酸，其包含如SEQ ID NO:30所示的重链可变区核苷酸序列和/或如SEQ ID NO:31所示的轻链可变区核苷酸序列。
  14. 一种表达载体，其包含如权利要求12或13所述的核酸。
  15. 一种宿主细胞，其包含如权利要求12或13所述的核酸或如权利要求14所述的表达载体。
  16. 一种用于产生如权利要求1-10任一项所述的抗IL17A抗体或其抗原结合片段的方法，其包括在适合于抗体表达的条件下培养如权利要求15所述的宿主细胞，和从培养基中回收表达的抗体。
  17. 一种药物组合物，其包含如权利要求1-10任一项所述的抗IL17A抗体或其抗原结合片段或如权利要求11所述的抗体-药物缀合物或如权利要求12-13任一项所述的核酸或如权利要求14所述的表达载体，及药学上可接受的载体。
  18. 如权利要求1-10任一项所述的抗IL17A抗体或其抗原结合片段或如权利要求11所述的抗体-药物缀合物或如权利要求17所述的药物组合物，其用于治疗银屑病。
  19. 一种用于治疗银屑病的方法，其包括向有需要的受试者施用治疗有效量的如权利要求1-10任一项所述的抗IL17A抗体或其抗原结合片段或如权利要求11所述的抗体-药物缀合物或如权利要求17所述的药物组合物，从而治疗银屑病。
  20. 如权利要求1-10任一项所述的抗IL17A抗体或其抗原结合片段或 如权利要求11所述的抗体-药物缀合物或如权利要求17所述的药物组合物在制备用于治疗银屑病的药物中的用途。
  21. 一种药物组合，其包含如权利要求1-10任一项所述的抗IL17A抗体或其抗原结合片段或如权利要求11所述的抗体-药物缀合物或如权利要求17所述的药物组合物与一种或多种另外的治疗剂。
  22. 一种试剂盒，其包含如权利要求1-10任一项所述的抗IL17A抗体或其抗原结合片段或如权利要求11所述的抗体-药物缀合物或如权利要求17所述的药物组合物，优选地，还进一步包含给药的装置。
PCT/CN2020/104448 2019-07-26 2020-07-24 人源化抗il17a抗体及其应用 Ceased WO2021018035A1 (zh)

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CN117866902A (zh) * 2024-03-12 2024-04-12 北京贝来生物科技有限公司 具有抗il-17a活性的基因修饰干细胞及其制备方法以及药物组合物
CN117866902B (zh) * 2024-03-12 2024-06-04 北京贝来生物科技有限公司 具有抗il-17a活性的基因修饰干细胞及其制备方法以及药物组合物

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CN114286827A (zh) 2022-04-05
US20220144936A1 (en) 2022-05-12
HRP20260007T1 (hr) 2026-03-13
CA3148491A1 (en) 2021-02-04
CN114286827B (zh) 2024-01-12
MX2022001068A (es) 2022-02-14
KR20220071179A (ko) 2022-05-31

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