WO2023030480A1 - 抗il-17a抗体及其用途 - Google Patents

抗il-17a抗体及其用途 Download PDF

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WO2023030480A1
WO2023030480A1 PCT/CN2022/116677 CN2022116677W WO2023030480A1 WO 2023030480 A1 WO2023030480 A1 WO 2023030480A1 CN 2022116677 W CN2022116677 W CN 2022116677W WO 2023030480 A1 WO2023030480 A1 WO 2023030480A1
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seq
amino acid
acid sequence
antibody
antigen
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PCT/CN2022/116677
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French (fr)
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刘婵娟
郎国竣
张文海
周蕴华
胡宇豪
闫闰
孙兴鲁
雷攀
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三优生物医药(上海)有限公司
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    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention belongs to the field of biomedicine. Specifically, the present invention relates to an antibody specifically binding to IL-17A or an antigen-binding fragment thereof, a preparation method and use thereof.
  • the IL-17 (Interleukin-17) family of cytokines contains 6 members, namely IL-17A, IL-17B, IL-17C, IL-17D, IL-17E and IL-17F; the most representative of the IL-17 family The most important member is IL-17A.
  • IL-17A was first discovered in T cell products in 1993 and is expressed in various T cells.
  • the human IL-17A gene contains 155 amino acids, has a signal peptide of 19 amino acids and a mature region of 132 amino acids, and has a relative molecular mass of 17kDa. The source dimer exists (Gaffen Sarah L, Nat. Rev. Immunol., 2009, 9:556-67).
  • IL-17A When the human body is violated or injured, lymphocytes that migrate to the infected or injured part of the body will secrete IL-17A.
  • IL-17A can induce the expression of inflammatory factors and chemokines, thereby recruiting more immune cells to reach the inflammatory site and aggravate the inflammatory response; on the other hand, IL-17A can also induce the expression of some tissue repair-related factors, thereby accelerating the Body recovery.
  • IL-17A plays a role in expanding the immune defense response and protecting the body in the process of host anti-infection and tissue repair, IL-17A is highly expressed in many autoimmune disease patients. Excessively high IL-17A levels can induce the expression of many inflammatory factors, which can worsen the development of autoimmune diseases.
  • PASI skin lesion area and severity index
  • PASI 75, PASI 90 and PASI 100 are important indicators of treatment success.
  • PASI 75 of these three monoclonal antibodies in the treatment of moderate to severe plaque psoriasis can reach more than 80%, the therapeutic effect of PASI 100 is not more than 40% (April W. Armstrong et al., JAMA. 2020; 323(19):1945-1960). Therefore, in order to further improve the therapeutic effect on diseases such as psoriasis, it is necessary to continue to develop new drugs targeting IL-17A with better therapeutic effect.
  • the invention provides an anti-IL-17A antibody or antigen-binding fragment thereof comprising a single variable domain of an immunoglobulin, said single variable domain being of the first family, the second family or the third family Family of single variable domains.
  • the first family single variable domain comprises:
  • CDR1 which comprises the amino acid sequence of SEQ ID NO:68:
  • CDR2 which comprises the amino acid sequence of SEQ ID NO:69:
  • CDR3 which comprises the amino acid sequence of SEQ ID NO:70:
  • the first family single variable domain comprises:
  • CDR1 comprising the amino acid sequence of SEQ ID NO: 17, 38, 20, 23 or 35;
  • CDR2 comprising the amino acid sequence of SEQ ID NO: 18, 39, 21, 24 or 36;
  • CDR3 comprising the amino acid sequence of SEQ ID NO: 19, 40, 22, 25 or 37.
  • the first family single variable domain comprises:
  • CDR1 which comprises the amino acid sequence of SEQ ID NO:17
  • CDR2 which comprises the amino acid sequence of SEQ ID NO:18
  • CDR3 which comprises the amino acid sequence of SEQ ID NO:19
  • CDR1 which comprises the amino acid sequence of SEQ ID NO:38
  • CDR2 which comprises the amino acid sequence of SEQ ID NO:39
  • CDR3 which comprises the amino acid sequence of SEQ ID NO:40;
  • CDR1 which comprises the amino acid sequence of SEQ ID NO:20
  • CDR2 which comprises the amino acid sequence of SEQ ID NO:21
  • CDR3 which comprises the amino acid sequence of SEQ ID NO:22;
  • CDR1 which comprises the amino acid sequence of SEQ ID NO:23
  • CDR2 which comprises the amino acid sequence of SEQ ID NO:24
  • CDR3 which comprises the amino acid sequence of SEQ ID NO:25;
  • CDR1 which comprises the amino acid sequence of SEQ ID NO:35
  • CDR2 which comprises the amino acid sequence of SEQ ID NO:36
  • CDR3 which comprises the amino acid sequence of SEQ ID NO:37 or a variant thereof.
  • the second family single variable domain comprises:
  • CDR1 which comprises the amino acid sequence of SEQ ID NO:71:
  • CDR2 which comprises the amino acid sequence of SEQ ID NO:72:
  • CDR3 comprising the amino acid sequence of SEQ ID NO: 28, 31, 34 or 43.
  • the second family single variable domain comprises:
  • CDR1 comprising the amino acid sequence of SEQ ID NO: 26, 29, 32 or 41;
  • CDR2 comprising the amino acid sequence of SEQ ID NO: 27, 30, 33 or 42;
  • CDR3 comprising the amino acid sequence of SEQ ID NO: 28, 31, 34 or 43.
  • the second family single variable domain comprises:
  • CDR1 which comprises the amino acid sequence of SEQ ID NO:26
  • CDR2 which comprises the amino acid sequence of SEQ ID NO:27
  • CDR3 which comprises the amino acid sequence of SEQ ID NO:28;
  • CDR1 which comprises the amino acid sequence of SEQ ID NO:29
  • CDR2 which comprises the amino acid sequence of SEQ ID NO:30
  • CDR3 which comprises the amino acid sequence of SEQ ID NO:31
  • CDR1 which comprises the amino acid sequence of SEQ ID NO:32
  • CDR2 which comprises the amino acid sequence of SEQ ID NO:33
  • CDR3 which comprises the amino acid sequence of SEQ ID NO:34;
  • CDR1 which comprises the amino acid sequence of SEQ ID NO:41
  • CDR2 which comprises the amino acid sequence of SEQ ID NO:42
  • CDR3 which comprises the amino acid sequence of SEQ ID NO:43.
  • the third family single variable domain comprises:
  • CDR1 which comprises the amino acid sequence of SEQ ID NO:73:
  • Xaa35 is T or N;
  • Xaa36 is D or Y;
  • Xaa37 is D or Y;
  • Xaa38 is A or G;
  • CDR2 which comprises the amino acid sequence of SEQ ID NO:74:
  • Xaa39 is S or T
  • CDR3 comprising the amino acid sequence of SEQ ID NO: 46, 49 or 52.
  • the third family single variable domain comprises:
  • CDR1 comprising the amino acid sequence of SEQ ID NO: 44, 47 or 50;
  • CDR2 comprising the amino acid sequence of SEQ ID NO: 45, 48 or 51;
  • CDR3 comprising the amino acid sequence of SEQ ID NO: 46, 49 or 52.
  • the third family single variable domain comprises:
  • CDR1 which comprises the amino acid sequence of SEQ ID NO:44
  • CDR2 which comprises the amino acid sequence of SEQ ID NO:45
  • CDR3 which comprises the amino acid sequence of SEQ ID NO:46
  • CDR1 which comprises the amino acid sequence of SEQ ID NO:47
  • CDR2 which comprises the amino acid sequence of SEQ ID NO:48
  • CDR3 which comprises the amino acid sequence of SEQ ID NO:49
  • CDR1 which comprises the amino acid sequence of SEQ ID NO:50
  • CDR2 which comprises the amino acid sequence of SEQ ID NO:51
  • CDR3 which comprises the amino acid sequence of SEQ ID NO:52.
  • said single variable domain comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:5, SEQ ID NO:12, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 , SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16.
  • the single variable domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO:65, SEQ ID NO:67 and SEQ ID NO:66.
  • the anti-IL-17A antibody or antigen-binding fragment thereof is conjugated to at least one detectable label.
  • the present invention also provides a multispecific antibody comprising a first antigen-binding portion that binds IL-17A and a second antigen-binding portion that binds a second antigen, wherein the first antigen-binding portion comprises the present Invented anti-IL-17A antibodies or antigen-binding fragments thereof.
  • the invention provides a polynucleotide encoding an anti-IL-17A antibody of the invention or an antigen-binding fragment thereof.
  • the present invention also provides an expression vector comprising the polynucleotide of the present invention.
  • the present invention also provides a host cell comprising the polynucleotide or expression vector of the present invention.
  • the present invention also provides a method for preparing the anti-IL-17A antibody or antigen-binding fragment thereof of the present invention, which comprises culturing the host cell of the present invention under suitable conditions to express the antibody or antigen-binding fragment thereof, and obtaining from the host cell or culture thereof to isolate the antibody or antigen-binding fragment thereof.
  • the present invention also provides a pharmaceutical composition, which comprises the anti-IL-17A antibody or antigen-binding fragment thereof of the present invention, and a pharmaceutically acceptable carrier.
  • the present invention also provides the use of the anti-IL-17A antibody or antigen-binding fragment thereof or the pharmaceutical composition of the present invention in the preparation of a medicament for treating IL-17A-mediated diseases.
  • the present invention also provides a method for detecting the presence of IL-17A in a sample or determining its expression level, comprising:
  • Figures 1A-1C show the binding activity of anti-IL-17A nanobody to recombinant antigen IL-17A-His.
  • Figures 2A-2C show the blocking activity of anti-IL-17A Nanobodies on IL-17A and IL-17RA.
  • 3A-3B show the SDS-PAGE images of anti-IL-17A heavy chain antibody under non-denaturing and denaturing conditions.
  • 4A-4C show the binding activity of anti-IL-17A heavy chain antibody to recombinant antigen IL-17A.
  • Figures 5A-5C show the IL-17A/IL-17RA blocking activity of anti-IL-17A heavy chain antibodies.
  • Figures 6A-6K show the ability of anti-IL-17A heavy chain antibodies to neutralize IL-17A-induced IL-6 secretion from HeLa cells.
  • 7A-7B show the binding activity of anti-IL-17A humanized antibody to recombinant antigen IL-17A.
  • immunoglobulin includes any immunoglobulin class (e.g., IgG, IgM, IgD, IgE, IgA, and IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass (e.g., , IgG2a and IgG2b).
  • immunoglobulin class e.g., IgG, IgM, IgD, IgE, IgA, and IgY
  • any class e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2
  • subclass e.g., IgG2a and IgG2b.
  • antibody refers to an immunoglobulin or fragment thereof that specifically binds an antigenic epitope through at least one antigen binding site. As used herein, the definition of antibody encompasses antigen-binding fragments.
  • the term “antibody” includes multispecific antibodies (eg, bispecific antibodies), human antibodies, non-human antibodies, humanized antibodies, chimeric antibodies, heavy chain antibodies, single domain antibodies, and antigen-binding fragments. Antibodies can be synthetic (eg, produced by chemical or biological conjugation), enzymatically processed, or recombinantly produced.
  • antigen-binding fragment refers to a portion of a full-length antibody that is less than full-length, but comprises at least part of the variable region of a full-length antibody (e.g., comprising one or more CDRs and/or one or more antigen-binding site), and thus retain at least part of the full-length antibody's ability to specifically bind the antigen.
  • immunoglobulin single variable domain or “single variable domain” refers to a single variable region (variable domain) having antigen binding activity. Different from the functional antigen-binding unit composed of a pair of VH and VL in conventional antibodies, a single variable domain can form a functional antigen-binding unit by itself.
  • Single variable domains can be derived from naturally occurring antibodies without light chains, such as the variable domain of heavy chain of heavy-chain antibodies (VHH) of camelids (such as llamas and alpacas)
  • VHH variable chain of heavy-chain antibodies
  • the single variable domain (IgNAR variable single-domain, VNAR) of neoantigen receptors of sharks and sharks can also be screened from full-length antibodies, such as light chain variable domains and heavy chains with antigen-binding activity in human antibodies. chain variable domain.
  • VHH usually contains three highly variable "complementarity determining regions (CDR)" and four relatively conserved “framework regions (FR)", and from N-terminus to C-terminus in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3 -Sequential connection of FR4.
  • CDR complementarity determining regions
  • FR framework regions
  • single domain antibody or “Nanobody” refers to an antibody comprising a single immunoglobulin variable domain as the functional antigen-binding unit. Unlike full-length antibodies, which typically contain two heavy and two light chains, single-domain antibodies typically contain a single peptide chain consisting of a single variable domain, with a molecular weight of only around 15 kDa.
  • heavy-chain-only antibody and “heavy-chain antibody” are used interchangeably and in their broadest sense to refer to the absence of Conventional antibody light chain antibody comprising only one VHH and heavy chain constant region (eg Fc fragment) without CH1.
  • CDRs of the anti-IL-17A antibody of the present invention are defined according to the AbM numbering system, but those skilled in the art should understand that multiple CDR numbering systems can also be used to define the same variable region, such as Chothia, Kabat and IMGT.
  • CDR boundaries defined by different numbering systems may vary, CDRs corresponding to the same numbering system represent effective antigen-binding sites capable of binding antigenic epitopes.
  • a description of the CDR numbering system can be found, for example, in the Kabat numbering system : Kabat, EA et al.
  • framework region and “framework region” are used interchangeably.
  • framework region refers to those amino acid residues in an antibody variable region other than the CDR sequences as defined above.
  • Fc fragment generally refers to papain-digested crystallizable fragments of conventional antibodies or heavy chain antibodies.
  • the Fc fragment of IgG and heavy chain antibodies may comprise part of the hinge region, CH2 and CH3.
  • the Fc fragment may comprise at least part of the hinge region (eg all or part of the hinge region), CH2 and CH3.
  • Hinge region generally refers to the part between CH1 and CH2 in conventional antibodies, the part between VHH and CH2 in heavy chain antibodies, or their functional equivalents, such as the hinge region in T cell receptor (TCR).
  • TCR T cell receptor
  • the hinge region may be the entire hinge region or a portion thereof.
  • chimeric antibody refers to an antibody in which a portion (e.g., CDRs, FRs, variable regions, constant regions, or combinations thereof) is identical or homologous to the corresponding sequence in an antibody derived from a particular species, and the remaining Portions are identical or homologous to corresponding sequences in antibodies derived from another species.
  • chimeric antibodies comprise variable regions derived from a non-human species (eg, a camelid, eg, camel and alpaca) and constant regions derived from a different species (eg, human).
  • Chimeric antibodies can also refer to multispecific antibodies that have specificities for at least two different antigens. Chimeric antibodies can be produced by antibody engineering.
  • chimeric antibodies can be produced by recombinant DNA techniques (see, e.g., Sambrook, J., et al. (1989). Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y).
  • humanized antibody refers to an antibody in which a non-human antibody has been modified to increase sequence homology to a human antibody.
  • Humanized antibodies generally retain the antigen-binding ability of the non-human antibody from which they were derived and are less immunogenic in humans.
  • Humanized antibodies can be obtained by antibody engineering of any non-human species antibody or an antibody comprising sequences derived from a non-human species (eg, chimeric antibodies). The techniques for obtaining humanized antibodies from non-human antibodies are well known to those skilled in the art. For example, CDR sequences of non-human antibodies (such as camel antibodies) can be grafted into the framework regions of human antibodies.
  • the key amino acid residues of the framework sequence of the non-human antibody can be retained in the framework region of the human antibody, that is, " Back mutation” (see, e.g., Morrison et al. (1984) Proc. Natl. Acad. Sci. 81(21):6851-6855; Neuberger et al. (1984) Nature 312:604-608).
  • percentage (%) sequence identity and “sequence identity” of amino acid sequences have definitions recognized in the art, which refer to two sequences determined by sequence alignment (for example, by manual inspection or known algorithms). The percentage of identity between polypeptide sequences. It can be determined using methods known to those skilled in the art, for example using publicly available computer software such as BLAST, BLAST-2, Clustal Omega and FASTA software.
  • an amino acid sequence "derived from” or “derived from” a reference amino acid sequence is identical or homologous to part or all of the reference amino acid sequence.
  • the amino acid sequence of a heavy chain constant region derived from a human immunoglobulin may be at least 80%, at least 85%, at least 90%, at least 91% identical to the wild-type sequence of the human immunoglobulin heavy chain constant region from which it is derived , at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity.
  • Non-critical regions in the polypeptide can be modified, such as one or more amino acid substitutions, additions and/or deletions, without changing the function of the polypeptide.
  • modified polypeptides relative to a reference amino acid sequence eg, single variable domain
  • a variant of a reference amino acid sequence may have at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% of the sequence from which it is derived (i.e., the reference amino acid sequence).
  • substitutions are conservative substitutions. It will be understood by those skilled in the art that amino acids in non-essential regions of polypeptides may be substituted with suitable conservative amino acids and generally do not alter their biological activity (see, e.g., Watson et al., Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin/Cummings Pub.co., p.224). Suitable conservative substitutions are well known to those skilled in the art.
  • amino acid substitutions are non-conservative substitutions.
  • amino acid mutations or modifications can be made to the polypeptide to change its properties, such as changing the type of antibody glycosylation modification, changing the ability to form interchain disulfide bonds, or providing activity for the preparation of antibody conjugates. group.
  • Antibodies or antigen-binding fragments thereof comprising such amino acid mutations or modifications are also encompassed within the scope of antibodies or antigen-binding fragments thereof of the present invention.
  • Affinity or "binding affinity” is a measure of the strength of non-covalent binding between an antibody and an antigen.
  • the magnitude of "affinity” can usually be reported as the equilibrium dissociation constant, KD .
  • Affinity can be determined using conventional techniques known in the art, such as biomembrane interferometry (for example, the Octet Fortebio detection system can be used), radioimmunoassay, surface plasmon resonance, enzyme-linked immunoassay (ELISA) or flow cytometry (FACS )wait.
  • the KD value between the specifically bound antibody and the antigen is at least about 10 ⁇ 6 M to at least about 10 ⁇ 9 M or lower, such as at least about 10 ⁇ 6 M, at least about 10 ⁇ 7 M, at least about 10 -8 M, at least about 10 -9 M, at least about 10 -10 M or lower.
  • the anti-IL-17A antibody or antigen-binding fragment thereof of the present invention binds human IL-17A with high affinity, for example, 5 ⁇ 10 ⁇ 10 M or lower, 1 ⁇ 10 ⁇ 10 M or lower, A KD of 5 ⁇ 10 ⁇ 11 M or lower, 2 ⁇ 10 ⁇ 11 M or lower binds human IL-17A.
  • the anti-IL-17A antibody or antigen-binding fragment, multispecific antibody, or polynucleotide encoding the same of the invention may be isolated.
  • isolated means that a substance (such as a polynucleotide or polypeptide) is separated from its source or environment in which it exists, ie does not substantially contain any other components.
  • polynucleotide and “nucleic acid” are used interchangeably to denote an oligomer or polymer comprising at least two linked nucleotides or nucleotide derivatives, which may typically include deoxyribonucleic acid ( DNA) and ribonucleic acid (RNA).
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • RNA and/or polypeptides As used herein, the term “expression” refers to the production of RNA and/or polypeptides.
  • a "vector” is a medium for introducing exogenous polynucleotides into host cells, and when the vector is transformed into an appropriate host cell, the exogenous polynucleotides are amplified or expressed.
  • Vectors usually remain episomal, but can be designed to allow integration of a gene, or part thereof, into the chromosome of the genome.
  • the definition of vector encompasses plasmids, linearized plasmids, viral vectors, cosmids, phage vectors, phagemids, artificial chromosomes (eg, yeast artificial chromosomes and mammalian artificial chromosomes), and the like.
  • Viral vectors include, but are not limited to, retroviral vectors (including lentiviral vectors), adenoviral vectors, adeno-associated viral vectors, herpesviral vectors, poxviral vectors, baculoviral vectors, and the like.
  • expression vector refers to a vector capable of expressing a polynucleotide of interest, including DNA and RNA.
  • a polynucleotide sequence including DNA and RNA
  • a polypeptide of interest can be operably linked to regulatory sequences (such as promoters and ribosome binding sites) that can affect the expression of the polynucleotide sequence .
  • regulatory sequences may include promoter and terminator sequences, and optionally may include origins of replication, selectable markers, enhancers, polyadenylation signals, and the like.
  • Expression vectors can be plasmids, phage vectors, recombinant viruses or other vectors which, when introduced into an appropriate host cell, result in the expression of a polynucleotide of interest. Suitable expression vectors are known to those skilled in the art. Those skilled in the art can prepare the expression vector as a vector that can replicate in the host cell, remain free in the host cell, or integrate into the genome of the host cell according to the needs.
  • a "host cell” is a cell used to receive, maintain, replicate or amplify a vector. Host cells can also be used to express polynucleotides or polypeptides encoded by vectors. Host cells can be eukaryotic or prokaryotic. Prokaryotic cells such as Escherichia coli (E.coli) or Bacillus subtilis (Bacillus subtilis), fungal cells such as yeast cells or Aspergillus, insect cells (such as S2 Drosophila cells or Sf9), and animal cells (such as fibroblasts, CHO cells , COS cells, HeLa cells, NSO cells or HEK293 cells).
  • E.coli Escherichia coli
  • Bacillus subtilis Bacillus subtilis
  • fungal cells such as yeast cells or Aspergillus
  • insect cells such as S2 Drosophila cells or Sf9
  • animal cells such as fibroblasts, CHO cells , COS cells, HeLa cells, NSO cells or
  • treatment refers to the improvement of the disease/symptom, such as reducing or disappearing the disease/symptom, preventing or slowing down the occurrence, progression and/or deterioration of the disease/symptom.
  • treatment includes prophylaxis, treatment and/or cure.
  • Effective amount means an amount sufficient to reduce the severity of disease symptoms, increase the frequency and duration of asymptomatic periods of disease, or prevent injury or disability resulting from disease affliction. "Effective amount” refers to the amount required to prevent, cure, ameliorate, arrest or partially arrest a disease or symptom. Those skilled in the art can determine the effective amount according to factors such as the subject's age, physical condition, sex, severity of symptoms, specific composition or route of administration. An effective amount can be administered in one or more administrations.
  • the term "pharmaceutically acceptable carrier” refers to a carrier that is pharmacologically and/or physiologically compatible with the subject and the active ingredient, which are well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995).
  • the invention provides an anti-IL-17A antibody or antigen-binding fragment thereof comprising an immunoglobulin single variable domain that specifically binds IL-17A.
  • IL-17A refers to interleukin 17A.
  • IL-17A is a pro-inflammatory cytokine produced by various T cells.
  • IL-17A stimulates epithelial, endothelial, and fibroblasts to produce other pro-inflammatory cytokines and chemokines including, for example, IL-6, IL-8, G-CSF, and MCP-1.
  • IL-17A can be human IL-17A and non-human (eg, camel, alpaca, mouse, rat, guinea pig, rabbit, goat, sheep, cow, horse, monkey, etc.) IL-17A.
  • the IL-17A is human IL-17A.
  • the amino acid sequence of an exemplary human IL-17A is shown in SEQ ID NO: 1.
  • IL-17A can exist in the body in the form of IL-17A homodimer and IL-17A/F heterodimer (IL-17A/IL-17F).
  • an anti-IL-17A antibody or antigen-binding fragment thereof of the invention is capable of specifically binding IL-17A. In one embodiment, the anti-IL-17A antibody or antigen-binding fragment thereof is capable of neutralizing IL-17A. In some embodiments, the anti-IL-17A antibody or antigen-binding fragment thereof is capable of binding IL-17A homodimers or IL-17A/F heterodimers. In some embodiments, the anti-IL-17A antibody or antigen-binding fragment thereof is capable of blocking the interaction of IL-17A or IL-17A/F with IL-17RA.
  • an anti-IL-17A antibody or antigen-binding fragment thereof of the invention comprises a single variable domain of an immunoglobulin comprising CDR1, CDR2, and CDR3.
  • the single variable domain is a Family I, Family II or Family III single variable domain.
  • the first family single variable domain comprises:
  • CDR1 which comprises the amino acid sequence of SEQ ID NO:68:
  • CDR2 which comprises the amino acid sequence of SEQ ID NO:69:
  • CDR3 which comprises the amino acid sequence of SEQ ID NO:70:
  • the first family single variable domain comprises:
  • CDR1 comprising the amino acid sequence of SEQ ID NO: 17, 38, 20, 23 or 35;
  • CDR2 comprising the amino acid sequence of SEQ ID NO: 18, 39, 21, 24 or 36;
  • CDR3 comprising the amino acid sequence of SEQ ID NO: 19, 40, 22, 25 or 37.
  • the first family single variable domain comprises:
  • CDR1 which comprises the amino acid sequence of SEQ ID NO:17
  • CDR2 which comprises the amino acid sequence of SEQ ID NO:18
  • CDR3 which comprises the amino acid sequence of SEQ ID NO:19
  • CDR1 which comprises the amino acid sequence of SEQ ID NO:38
  • CDR2 which comprises the amino acid sequence of SEQ ID NO:39
  • CDR3 which comprises the amino acid sequence of SEQ ID NO:40;
  • CDR1 which comprises the amino acid sequence of SEQ ID NO:20
  • CDR2 which comprises the amino acid sequence of SEQ ID NO:21
  • CDR3 which comprises the amino acid sequence of SEQ ID NO:22;
  • CDR1 which comprises the amino acid sequence of SEQ ID NO:23
  • CDR2 which comprises the amino acid sequence of SEQ ID NO:24
  • CDR3 which comprises the amino acid sequence of SEQ ID NO:25;
  • CDR1 which comprises the amino acid sequence of SEQ ID NO:35
  • CDR2 which comprises the amino acid sequence of SEQ ID NO:36
  • CDR3 which comprises the amino acid sequence of SEQ ID NO:37 or a variant thereof.
  • the second family single variable domain comprises:
  • CDR1 which comprises the amino acid sequence of SEQ ID NO:71:
  • CDR2 which comprises the amino acid sequence of SEQ ID NO:72:
  • CDR3 comprising the amino acid sequence of SEQ ID NO: 28, 31, 34 or 43.
  • the second family single variable domain comprises:
  • CDR1 comprising the amino acid sequence of SEQ ID NO: 26, 29, 32 or 41;
  • CDR2 comprising the amino acid sequence of SEQ ID NO: 27, 30, 33 or 42;
  • CDR3 comprising the amino acid sequence of SEQ ID NO: 28, 31, 34 or 43.
  • the second family single variable domain comprises:
  • CDR1 which comprises the amino acid sequence of SEQ ID NO:26
  • CDR2 which comprises the amino acid sequence of SEQ ID NO:27
  • CDR3 which comprises the amino acid sequence of SEQ ID NO:28;
  • CDR1 which comprises the amino acid sequence of SEQ ID NO:29
  • CDR2 which comprises the amino acid sequence of SEQ ID NO:30
  • CDR3 which comprises the amino acid sequence of SEQ ID NO:31
  • CDR1 which comprises the amino acid sequence of SEQ ID NO:32
  • CDR2 which comprises the amino acid sequence of SEQ ID NO:33
  • CDR3 which comprises the amino acid sequence of SEQ ID NO:34;
  • CDR1 which comprises the amino acid sequence of SEQ ID NO:41
  • CDR2 which comprises the amino acid sequence of SEQ ID NO:42
  • CDR3 which comprises the amino acid sequence of SEQ ID NO:43.
  • the third family single variable domain comprises:
  • CDR1 which comprises the amino acid sequence of SEQ ID NO:73:
  • Xaa35 is T or N;
  • Xaa36 is D or Y;
  • Xaa37 is D or Y;
  • Xaa38 is A or G;
  • CDR2 which comprises the amino acid sequence of SEQ ID NO:74:
  • Xaa39 is S or T
  • CDR3 comprising the amino acid sequence of SEQ ID NO: 46, 49 or 52.
  • the single variable domain as described above has two pairs of disulfide bonds, one more pair of disulfide bonds between CDR2 and CDR3 than a conventional single variable domain, and an increase within the single variable domain.
  • a pair of disulfide bonds makes the protein less aggregated.
  • the third family single variable domain comprises:
  • CDR1 comprising the amino acid sequence of SEQ ID NO: 44, 47 or 50;
  • CDR2 comprising the amino acid sequence of SEQ ID NO: 45, 48 or 51;
  • CDR3 comprising the amino acid sequence of SEQ ID NO: 46, 49 or 52.
  • the third family single variable domain comprises:
  • CDR1 which comprises the amino acid sequence of SEQ ID NO:44
  • CDR2 which comprises the amino acid sequence of SEQ ID NO:45
  • CDR3 which comprises the amino acid sequence of SEQ ID NO:46
  • CDR1 which comprises the amino acid sequence of SEQ ID NO:47
  • CDR2 which comprises the amino acid sequence of SEQ ID NO:48
  • CDR3 which comprises the amino acid sequence of SEQ ID NO:49
  • CDR1 which comprises the amino acid sequence of SEQ ID NO:50
  • CDR2 which comprises the amino acid sequence of SEQ ID NO:51
  • CDR3 which comprises the amino acid sequence of SEQ ID NO:52.
  • a single variable domain as described above further comprises a framework region.
  • the framework regions may each independently be derived from a framework region of an immunoglobulin of any species.
  • the framework regions are derived from alpaca immunoglobulins.
  • the single variable domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:12, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:7, or variants thereof ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16; wherein, all Said variant has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, At least 98% or at least 99% sequence identity, or one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) sequence identity compared to the sequence from which it is said to be derived ) amino acid substitutions, additions and/or deletions.
  • one or more e.
  • the framework regions are derived from human immunoglobulins.
  • the single variable domain comprises a heavy chain framework region derived from a human immunoglobulin.
  • the anti-IL-17A antibodies or antigen-binding fragments thereof of the invention are humanized.
  • the framework region may comprise one or more non-human (eg camel derived) amino acid residues, for example may comprise one or more amino acid back mutations comprising the corresponding camel derived amino acid residues.
  • the single variable domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 65, SEQ ID NO: 67 and SEQ ID NO: 66; wherein the variant is associated with
  • the derived sequence has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or At least 99% sequence identity, or one or more (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid substitutions compared to the sequence from which it is said to be derived , additions and/or deletions.
  • the substitutions, additions and/or deletions do not occur in the CDR regions.
  • an anti-IL-17A antibody of the invention or an antigen-binding fragment thereof may further comprise an immunoglobulin constant region (e.g., a heavy chain constant region and/or a light chain constant region) linked to a single variable domain of the invention. area) section.
  • an immunoglobulin constant region e.g., a heavy chain constant region and/or a light chain constant region
  • said constant region portion is linked to a single variable domain of the invention via a linker.
  • Appropriate portions of the immunoglobulin constant region can be selected and optionally modified so that antibodies possess the desired properties.
  • one or more cysteine residues can be introduced or removed (for example, mutated by recombinant DNA techniques) in the hinge region to promote or weaken antibody dimerization; CH1, hinge region, CH2 and/or CH3, to prolong or reduce the serum half-life of the antibody, promote the internalization or tissue penetration ability of the antibody, have improved or weakened binding to Fc ⁇ RIIB (see, for example, WO2008150494A1), enhance or reduce antibody-dependent cell-mediated cytotoxicity ( ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC), etc. (see, for example, Caron, P.C., et al., J.Exp.Med.176:1191-1195 (1992 ) and Shopes B.J. Immunol. 148 2918-2922 (19
  • an anti-IL-17A antibody or antigen-binding fragment thereof of the invention comprises a heavy chain constant region portion linked to a single variable domain of the invention.
  • the heavy chain constant region portion may be derived from any immunoglobulin subclass or subclass, such as IgG, IgM, IgA, IgD and IgE.
  • the heavy chain constant region portion is derived from human IgG, eg IgGl, IgG2, IgG3 and IgG4.
  • the heavy chain constant region portion preferably comprises the hinge region, CH2, CH3 or combinations thereof.
  • the heavy chain constant region portion comprises an Fc fragment.
  • an anti-IL-17A antibody or antigen-binding fragment thereof of the invention further comprises an Fc fragment of an immunoglobulin.
  • the Fc fragment is the Fc fragment of human IgG1.
  • the Fc fragment comprises the amino acid sequence of SEQ ID NO:2.
  • an anti-IL-17A antibody of the invention or an antigen-binding fragment thereof comprises an immunoglobulin single variable domain comprising an antibody selected from the group consisting of SEQ ID NO:5, SEQ ID NO:12, SEQ ID NO:6 , SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:15
  • the Fc fragment of human IgG1 it comprises the amino acid sequence of SEQ ID NO:2.
  • the anti-IL-17A antibody or antigen-binding fragment thereof of the present invention comprises an amino acid sequence selected from SEQ ID NO: 53-64.
  • an anti-IL-17A antibody of the invention or an antigen-binding fragment thereof comprises an immunoglobulin single variable domain comprising a protein selected from the group consisting of SEQ ID NO:65, SEQ ID NO:67, and SEQ ID NO:66 and the Fc fragment of human IgG1 comprising the amino acid sequence of SEQ ID NO:2.
  • Other polypeptides can be used to confer advantageous properties and/or reduce undesired properties of the anti-IL-17A antibodies or antigen-binding fragments thereof of the invention, for example increasing the half-life of the anti-IL-17A antibodies or antigen-binding fragments thereof of the invention, Solubility or absorption (for example, facilitating its penetration into skin, phospholipid membranes, organelles, etc.); and/or reducing its immunogenicity, toxicity or side effects.
  • Antibodies or antigen-binding fragments comprising such polypeptides are also encompassed within the meaning of anti-IL-17A antibodies or antigen-binding fragments thereof of the present invention.
  • serum albumin or a suitable fragment thereof can be linked to the anti-IL-17A antibody or antigen-binding fragment thereof of the present invention to increase its serum half-life (see for example CN104884473A, WO2013177101A2, WO0027435A1 and WO0177137A1).
  • polypeptides can also be, for example, polypeptides for directing the expression and secretion of antibodies or antigen-binding fragments thereof from host cells, facilitating the detection and/or isolation of antibodies or antigen-binding fragments, including but not limited to signal peptides (leader sequences), promoting Lysotags, affinity tags (such as polyhistidine tags (His 6 ) or glutathione S-transferase (GST) tags), peptides containing protease cleavage sites, and reporter tags (such as fluorescent proteins).
  • Other polypeptides may also be biologically active polypeptides, such as polypeptides or proteins having therapeutic, binding or enzymatic activity.
  • Non-limiting examples of biologically active polypeptides may include, but are not limited to: protein toxins (eg, diphtheria toxin, ricin), enzymes (eg, urease, horseradish peroxidase), and cytokines.
  • protein toxins eg, diphtheria toxin, ricin
  • enzymes eg, urease, horseradish peroxidase
  • cytokines e.g, cytokines.
  • the anti-IL-17A antibody or antigen-binding fragment thereof of the present invention is preferably linked directly or via a suitable linker to any polypeptide of interest (eg, one or more of the above-mentioned polypeptides).
  • the anti-IL-17A antibody or antigen-binding fragment thereof is linked to one or more of the above-mentioned polypeptides using a peptide linker, so that the anti-IL-17A antibody or antigen-binding fragment thereof of the present invention can be expressed as a recombinant ( fusion) protein.
  • the peptide linker may comprise an amino acid sequence of any length, in particular an amino acid sequence of 1-50, preferably 1-30, eg 1-10 amino acid residues.
  • Exemplary peptide linkers can include, but are not limited to, polyglycine (G), polyalanine (A), polyserine (S), or combinations thereof, such as GGAS, GGGS, GGGSG, or (G 4 S) n , where n is 1 -30, preferably an integer of 1-10.
  • the peptide linker may also be a hinge region or a functional equivalent thereof.
  • Other suitable linkers may be organic compounds or polymers generally suitable for use in pharmaceutical proteins, including but not limited to polyethylene glycol.
  • the anti-IL-17A antibody or antigen-binding fragment immunoglobulin of the present invention may be a single domain antibody, a heavy chain antibody, a humanized antibody or a chimeric antibody.
  • the anti-IL-17A antibody or antigen-binding fragment thereof is capable of
  • the invention provides a multispecific antibody comprising a first antigen binding portion that binds IL-17A and a second antigen binding portion that binds a second antigen, wherein the first antigen binding portion comprises an antigen binding portion of the invention Anti-IL-17A antibody or antigen-binding fragment thereof.
  • multispecific antibody refers to an antibody capable of specifically binding two or more (eg, 2, 3, 4, 5 or 6) different antigenic epitopes.
  • a multispecific antibody may, for example, be a bispecific, trispecific or tetraspecific antibody capable of specifically binding 2, 3 or 4 epitopes, respectively.
  • antigenic epitope or "antigenic determinant” means a region of an antigen that specifically binds to the antigen binding site of an antibody.
  • the second antigen may be other antigen than IL-17A.
  • the second antigen can also be IL-17A, which binds to a different epitope on IL-17A than the anti-IL-17A antibody or antigen-binding fragment thereof of the present invention.
  • Multispecific antibodies may be multivalent (eg, 2, 3, 4) antibodies, ie, they have multiple antigen-binding sites.
  • Multispecific antibodies can be produced and isolated using various techniques known in the art.
  • first antigen-binding portion and second antigen-binding portion mean an amino acid sequence comprising an antigen-binding site capable of binding to an antigenic epitope, and their definitions fall within the meaning of an antibody or antigen-binding fragment .
  • the first antigen binding moiety can be any form of antibody or antigen binding fragment including, but not limited to, single variable domain and heavy chain antibodies.
  • the first antigen binding moiety comprises a single variable domain of the invention.
  • the second antigen binding moiety can be an antibody or antigen binding fragment that binds any antigen of interest.
  • the second antigen is an antigen other than IL-17A.
  • Antigens to which the second antigen binding portion can specifically bind can include pro-inflammatory cytokines and chemokines.
  • pro-inflammatory cytokine refers to a class of cytokines secreted by immune cells or other types of cells that promote inflammation.
  • the first antigen binding moiety and the second antigen binding moiety may optionally be linked by a linker (eg, as described herein).
  • the present invention provides a polynucleotide comprising an anti-IL-17A antibody or an antigen-binding fragment thereof encoding the present invention.
  • polynucleotides of the present invention can be obtained using methods known in the art.
  • polynucleotides of the invention can be isolated from phage display libraries, yeast display libraries, immunized animals (e.g., alpacas, mice, humanized mice, monkeys), immortalized cells (e.g., mouse B cell hybrids) tumor cells, EBV-mediated immortalized B cells) or chemical synthesis.
  • immunized animals e.g., alpacas, mice, humanized mice, monkeys
  • immortalized cells e.g., mouse B cell hybrids
  • tumor cells e.g., EBV-mediated immortalized B cells
  • EBV-mediated immortalized B cells EBV-mediated immortalized B cells
  • the invention also provides a vector comprising a polynucleotide of the invention.
  • polynucleotides of the invention are cloned into expression vectors.
  • Expression vectors may further comprise additional polynucleotide sequences, such as regulatory sequences and antibiotic resistance genes.
  • Expression vectors may also contain polynucleotide sequences encoding additional polypeptides.
  • a polynucleotide of the invention may be present in one or more expression vectors.
  • the present invention also provides a host cell comprising the polynucleotide or expression vector of the present invention.
  • the polynucleotide or expression vector of the present invention can be introduced into suitable host cells by various methods known in the art. Such methods include, but are not limited to, lipofection, electroporation, viral transduction, and calcium phosphate transfection, among others.
  • host cells are used to express the anti-IL-17A antibodies or antigen-binding fragments thereof of the invention.
  • Examples of host cells include, but are not limited to, prokaryotic cells (eg bacteria such as E. coli) and eukaryotic cells (eg yeast, insect cells, mammalian cells).
  • Mammalian host cells suitable for antibody expression include, but are not limited to, myeloma cells, HeLa cells, HEK cells (e.g., HEK 293 cells), Chinese hamster ovary (CHO) cells, and other mammalian cells suitable for expressing antibodies.
  • the present invention also provides a method for preparing the anti-IL-17A antibody or antigen-binding fragment thereof of the present invention, which comprises culturing the host cell of the present invention under suitable conditions to express the anti-IL-17A antibody or antigen-binding fragment thereof of the present invention , and isolating said antibody or antigen-binding fragment thereof from a host cell or culture thereof.
  • Anti-IL-17A antibodies or antigen-binding fragments thereof of the invention can be conjugated to at least one detectable label.
  • Conjugates comprising an anti-IL-17A antibody or antigen-binding fragment thereof of the invention and a detectable label are also encompassed within the meaning of an anti-IL-17A antibody or antigen-binding fragment thereof of the invention.
  • the invention also provides an antibody conjugate comprising an anti-IL-17A antibody or antigen-binding fragment thereof of the invention conjugated to at least one detectable label.
  • conjugate refers to the linking of two or more moieties to each other by covalent or non-covalent interactions.
  • an anti-IL-17A antibody or antigen-binding fragment thereof of the invention is covalently conjugated to a detectable label.
  • the detectable label can be any label for detection, including but not limited to radioactive isotopes (such as 212 Bi, 213 Bi, 131 I, 125 I, 111 In, 177 Lu, 186 Re, 188 Re, 153 Sm, and 90 Y, etc. ), biotin, colloidal gold, chemiluminescent labels, bioluminescent labels and fluorescent groups (such as FITC, Alexa Fluor 488, Alexa Fluor 568, Alexa Fluor 555, Alexa Fluor 594, Alexa Fluor 647, Cy3, Texas Red, Cy5 and Rhodamine, etc.).
  • the detectable label is selected from radioisotopes, biotin, colloidal gold, chemiluminescent labels, bioluminescent labels, and fluorophores.
  • a detectable label can be conjugated directly or indirectly (eg, via a linker) to an anti-IL-17A antibody or antigen-binding fragment thereof of the invention.
  • Linkers may comprise reactive groups for covalent conjugation, such as amines, hydroxylamines, maleimides, carboxyls, phenyls, thiols, sulfhydryls or hydroxyls.
  • the linker is a chemical bond.
  • the linker comprises amino acids or a peptide consisting of 2-10 amino acids.
  • the present invention also provides a pharmaceutical composition, which comprises the anti-IL-17A antibody or antigen-binding fragment thereof of the present invention, and a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers may include, but are not limited to: diluents, binders and adhesives, lubricants, disintegrants, preservatives, vehicles, dispersants, glidants, sweeteners, coatings, excipients Excipients, preservatives, antioxidants (such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, Propyl gallate, ⁇ -tocopherol, citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.), solubilizers, gelling agents, softeners, solvents (for example, water, alcohol, acetic acid and syrup), buffers (e.g., phosphate buffer, histidine buffer, and a
  • the carrier may be selected from one or more of the following: sterile diluents such as water, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, Natural oils such as synthetic mono- or diglycerides, polyethylene glycol, glycerin, or other solvents; antibacterial agents such as benzyl alcohol or methylparaben; and agents for tonicity such as sodium chloride or dextrose .
  • sterile diluents such as water, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, Natural oils such as synthetic mono- or diglycerides, polyethylene glycol, glycerin, or other solvents; antibacterial agents such as benzyl alcohol or methylparaben; and agents for tonicity such as sodium chloride or dextrose .
  • the pharmaceutical compositions provided herein can be in a variety of dosage forms, including but not limited to solids, semi-solids, liquids (such as solutions, emulsions, or suspensions), powders, lotions, sprays, patches, plasters, ointments, gels, Creams, sprays, pastes, bioadhesives, or lyophilized formulations (e.g. for use after reconstitution).
  • the pharmaceutical composition of the present invention can also be formulated as microemulsions, liposomes and micelles (see eg CN107108730A and WO2016113557A1).
  • the pharmaceutical composition can be formulated in a dosage form suitable for the desired route of administration, in particular topical, dermal, parenteral (e.g. intravenous, intramuscular, intrasternal, subcutaneous, e.g. by injection or infusion) and nasal. Internal administration.
  • the pharmaceutical composition may include a skin penetration enhancer that facilitates or enhances skin penetration.
  • Skin penetration enhancers known in the art can be used, including but not limited to, for example, those described in CN 107108730 A.
  • Administration may also be facilitated using, for example, phonophoresis, sonophoresis, electroporation or the use of microneedle techniques.
  • compositions can be prepared using methods well known in the pharmaceutical art. For example, compositions intended to be administered by injection can be prepared by combining a binding molecule of the invention with water, thereby forming a solution. Surfactants can be added to facilitate the formation of a homogeneous solution or suspension. Pharmaceutical compositions may take the form of one or more dosage units.
  • the pharmaceutical composition can be presented in ampoules, disposable syringes or multiple dose vials made of glass, plastic or other materials.
  • the anti-IL-17A antibody or antigen-binding fragment thereof or antibody conjugate of the present invention can be used to detect the presence or expression level of IL-17A.
  • the anti-IL-17A antibodies or antigen-binding fragments thereof or antibody conjugates provided herein can be used in in situ, in vivo, ex vivo and in vitro detection or imaging assays.
  • the expression level of IL-17A in a sample is detected and/or cells expressing IL-17A are tracked by using an antibody or antigen-binding fragment or antibody conjugate of the invention.
  • the invention provides a method for detecting the presence or determining the expression level of IL-17A in a sample comprising:
  • the sample can be in any form, such as cells, tissues and body fluids.
  • the above detection can be performed using techniques known in the art, such techniques include but are not limited to Western blot, flow cytometry, radioimmunoassay (RIA), immunohistochemical assay (IHC) and enzyme-linked immunoassay (ELISA). ).
  • the present invention provides the use of an anti-IL-17A antibody or an antigen-binding fragment thereof in the preparation of a medicament for treating IL-17A-mediated diseases.
  • the present invention also relates to a method of treating IL-17A-mediated diseases (such as autoimmune diseases), which comprises administering the anti-IL-17A antibody or antigen-binding fragment thereof of the present invention or the present invention to a subject in need thereof.
  • IL-17A-mediated diseases such as autoimmune diseases
  • IL-17A-mediated diseases can include, for example, autoimmune diseases, inflammatory conditions, allergies and allergic conditions, hypersensitivity reactions, severe infections, and organ or tissue transplant rejection.
  • the anti-IL-17A antibody or antigen-binding fragment thereof of the present invention can be administered in combination with one or more anti-inflammatory drugs/immunosuppressants for the treatment of the above-mentioned diseases.
  • anti-inflammatory/immunosuppressive drugs see, for example, CN104884473A and WO2013177101A2.
  • the anti-IL-17A antibody or antigen-binding fragment thereof or the pharmaceutical composition of the present invention can be administered with other drugs at the same time or at different times, such as simultaneously, separately or sequentially.
  • an anti-IL-17A antibody or antigen-binding fragment thereof or a pharmaceutical composition of the invention is administered topically to the skin of a subject in need thereof.
  • pharmaceutical compositions which are able to penetrate at least the outer layer of the skin and which can thus be delivered dermally or transdermally.
  • the anti-IL-17A or antigen-binding fragment thereof or the pharmaceutical composition of the present invention can be used as lotion, spray, solution, gel, ointment, paste, plaster, patch, bioadhesive, suspension,
  • the forms of liposomes, micelles, microspheres and microemulsions see eg CN107108730A and WO2016113557A1) etc. are directly applied to diseased or healthy skin.
  • the dosage of the anti-IL-17A antibody or antigen-binding fragment thereof or the pharmaceutical composition of the present invention will vary according to specific formulations, application methods, specific locations, hosts and diseases to be treated. Other factors such as age, body weight, sex, medical history, diet, time of administration, clearance rate, responsiveness, and severity of disease should be considered.
  • the precise dosage employed in the composition will also depend on the route of administration and the severity of the disease or condition, and should be determined according to the judgment of the practitioner and each patient's circumstances. Administration can be carried out continuously or periodically within the maximum tolerated dose.
  • kits comprising the anti-IL-17A antibody or antigen-binding fragment, antibody conjugate, multispecific antibody or pharmaceutical composition of the present invention, and instructions for use.
  • Kits may also comprise suitable containers.
  • the kit further comprises a device for administration.
  • the kit will also include a label indicating the intended use and/or method of use of the kit contents.
  • label includes any written or recorded material provided on or with the kit or otherwise provided with the kit.
  • the anti-IL-17A antibody or antigen-binding fragment thereof of the present invention can at least achieve one of the following beneficial effects:
  • the anti-IL-17A antibodies or antigen-binding fragments thereof (especially nanobodies and heavy chain antibodies) of the present invention have significant advantages such as small molecular weight and strong permeability, enabling them to recognize conventional antibodies Inaccessible cryptic epitopes, easy to produce and suitable for assembly with other antibodies into multispecific and multivalent antibodies.
  • Embodiment 1 raw material preparation
  • the DNA fragment encoding human IL-17A (NCBI Gene ID:3605, SEQ ID NO:1) was synthesized by General Biotechnology Co., Ltd., and connected to the DNA encoding human IgG1 Fc (SEQ ID NO:2) by PCR amplification The 5' end of the fragment was obtained to obtain the nucleic acid encoding the recombinant antigen IL-17A-Fc. Then, the nucleic acid was cloned into the eukaryotic expression vector pcDNA3.4-TOPO (Invitrogen) by means of homologous recombination to obtain the expression vector of the recombinant antigen IL-17A-Fc. The expression vector of recombinant antigen IL-17A-Fc was transformed into Escherichia coli DH5 ⁇ and the plasmid was extracted for expression in eukaryotic system.
  • the recombinant antigen IL-17A-Fc was expressed through the Expi293 transient expression system (ThermoFisher, A14635). After 7 days of transfection, the cell expression supernatant was centrifuged at 15000g for 10min at high speed, and the resulting Fc-tagged protein expression supernatant was affinity purified with MabSelect SuRe LX (GE, 17547403), and then eluted with 100mM sodium acetate (pH 3.0). The protein was then neutralized with 1M Tris-HCl; the eluted protein was exchanged into PBS buffer through an ultrafiltration concentrator tube (Millipore, UFC901096), and frozen at -80°C until use.
  • the positive control antibody used in this application is the anti-IL-17A antibody secukinumab, which was synthesized according to the sequence disclosed in the patent application US20170355762A, and the genes containing secukinumab heavy chain (SEQ ID NO: 3) were respectively constructed by molecular cloning methods and the plasmid of secukinumab light chain (SEQ ID NO:4) gene. Refer to Example 1.1 for the remaining steps.
  • Immunization was carried out by subcutaneous injection, and a total of 2 alpacas NSY007 and NSY008 (Nanchang Dajia Technology Co., Ltd.) were immunized, and the immune antigen used was recombinant antigen IL-17A-His (Novoprotein Company, product number C774).
  • a single immunization dose was 500 ⁇ g, supplemented with CFA/IFA (complete Freund's adjuvant and Freund's incomplete adjuvant), immunized once every 2 weeks, and immunized 4 times in total.
  • the recombinant antigen IL-17A-His was diluted with PBS to a final concentration of 2 ⁇ g/mL, 30 ⁇ L of the diluted solution was added to an ELISA plate, and coated overnight at 4°C. On the day of immunopotency determination, rinse with PBST three times, then block with PBST containing 5% skimmed milk at room temperature for two hours, and then rinse with PBST three times.
  • the non-immunized negative serum and post-immunization serum were diluted with PBS, the first well was diluted 2000 times, and then the subsequent 7 wells were serially diluted by 2 times.
  • Add the diluted serum to the first ELISA plate incubate at room temperature for 1 h, wash the plate three times with PBST, add secondary antibody Anti IgG-HRP (Millipore, MAC129) at 1:7000, and incubate at room temperature for 0.5 h.
  • Ficoll-Paque density gradient separation medium (GE Company, catalog number: 17144003S) was used to separate peripheral mononuclear cells (Peripheral Blood Mononuclear Cell, PBMC), and total RNA was extracted from the isolated PBMC cells and reverse-transcribed into cDNA. Based on the situation of the VHH antibody germline gene (germline), degenerate primers were designed, amplified by PCR and recovered by agarose gel electrophoresis to obtain a DNA fragment encoding VHH-CH2. Then by the method of secondary PCR, the DNA fragment (Sabir JS et al., C R Biol. (2014) 337 (4): 244 -249).
  • the DNA fragment encoding the VHH was digested and purified and constructed into a phage display vector.
  • the vector expressing VHH was transformed into competent Escherichia coli SS320 (Lucigen, MC1061 F) by an electroporator (Bio-Rad, MicroPulser), and the transformed Escherichia coli SS320 bacterial liquid was spread on the ampicillin resistant 2-YT solid plate. Through serial dilution and plating, the size of the library library was determined to be 10 9 grades.
  • bacterium solution of the nanobody gene library to fresh 2-YT liquid medium, place it in a shaker at 37°C and 220rpm, and cultivate it to the logarithmic growth phase, and then use 50 times the number of bacteria (ie, the multiplicity of infection) (MOI) about 50) VSCM13 helper phage (purchased from Stratagene) was added to finally obtain a phage display library of camel-derived Nanobodies.
  • MOI multiplicity of infection
  • VSCM13 helper phage purchased from Stratagene
  • the magnetic bead screening method is based on biotin-labeling the recombinant antigen IL-17A-Fc, and then combining it with magnetic beads coupled with streptavidin, and incubating the magnetic beads bound to the antigen and the antibody gene phage display library The panning process of , washing and elution, so that the specific monoclonal antibody against the antigen can be enriched in large quantities.
  • biotin-labeled IL-17A-Fc is incubated with streptavidin-coupled magnetic beads, so that the biotin-labeled IL-17A-Fc binds to the magnetic beads.
  • the magnetic beads bound to IL-17A-Fc and the constructed phage library were incubated at room temperature for 2 h. After washing with PBST for 6-8 times, non-specifically adsorbed phages were removed, and Trypsin (Gibco, 25200072) was added to gently mix and react for 20 minutes to elute specifically bound antibody-displayed phages.
  • the eluted phages were used to infect logarithmic Escherichia coli SS320 and allowed to stand for 30 minutes, and then cultured at 220rpm for 1 hour; then added VSCM13 helper phage and left for 30 minutes, and continued to cultivate at 220rpm for 1 hour; And change the medium to C + /K + 2-YT medium, and the finally obtained phages continue to be used for the next round of panning.
  • the principle of immunotube screening is to coat IL-17A protein on the surface of immunotube with high adsorption force, add the phage display antibody library to the immunotube and incubate, wash and wash with the antigen protein adsorbed on the surface of the immunotube. The de-panning process will eventually enrich the specific monoclonal antibody against the antigen.
  • the specific method is as follows: in the first round of screening, add 1 mL of 30 ⁇ g/mL IL-17A-Fc to the immunotube, and coat at 4°C overnight; discard the coating solution the next day, and add 5% milk in PBS to block for 2 hours; After washing twice with PBS, add the phage library containing anti-IL-17A nanobody display and incubate for 2 h; wash to remove non-specifically bound phage, and then add 0.8mL 0.05% EDTA trypsin digestion solution to the immunotube for The phages that specifically bind to the target antigen were eluted; then, the eluted phages were used to infect logarithmic Escherichia coli SS320, and stood at 37°C for 30 minutes, then cultured at 220rpm for 1 hour, then added VSCM13 helper phages, and stood for 30 minutes.
  • Nanobody was named by the clone number, and the complementarity-determining region sequence of the anti-IL-17A Nanobody was determined by using AbM to define the CDR, see Table 2 for details.
  • Example 3 The five Nanobodies obtained in Example 3 were constructed as human IgG1 subtype to form VHH-Fc heavy chain antibodies. The specific amino acid sequences are shown in Table 3.
  • the DNA fragment encoding VHH is obtained by PCR amplification.
  • the DNA fragments encoding each VHH were respectively constructed on the transformed eukaryotic expression vector plasmid pcDNA3.4-TOPO (Invitrogen) containing the human IgG1 Fc fragment (SEQ ID NO: 2), and the complete The recombinant plasmid of the VHH-Fc (heavy chain antibody) full-length gene. Transform the recombinant plasmid into Escherichia coli DH5 ⁇ and extract the plasmid for expression in eukaryotic cell system.
  • the heavy chain antibody was expressed through the ExpiCHO transient expression system (Thermo Fisher, A29133), and the specific method was as follows: on the day of transfection, confirm that the cell density was about 7 ⁇ 10 6 to 1 ⁇ 10 7 viable cells/mL, and the cell survival rate was >98 %, at this point, adjust the cells to a final concentration of 6 ⁇ 106 cells/mL with fresh ExpiCHO expression medium pre-warmed at 37°C.
  • OptiPRO TM SFM Dilute the target plasmid with 4°C pre-cooled OptiPRO TM SFM (add 1 ⁇ g of plasmid to 1 mL of the medium), and at the same time, dilute ExpiFectamine TM CHO with OptiPRO TM SFM, then mix the two in equal volumes and gently blow and mix to prepare Form ExpiFectamine TM CHO/plasmid DNA mixture, incubate at room temperature for 1-5min, slowly add to the prepared cell suspension, and shake gently at the same time, and finally place in a cell culture shaker at 37°C, 8% CO 2 cultivated under conditions.
  • the cell culture supernatant expressing the target protein was centrifuged at 15,000 g for 10 min at high speed, and the resulting supernatant was affinity purified with MabSelect SuRe LX (GE, 17547403), and then purified with 100 mM sodium acetate (pH 3.0)
  • the target protein was eluted, then neutralized with 1M Tris-HCl, and finally the resulting protein was exchanged into PBS buffer through an ultrafiltration concentrator tube (Millipore, UFC901096).
  • Non-reducing (non-denaturing) SDS-PAGE sample preparation Add 1 ⁇ g of heavy chain antibody or quality control IPI (i.e. Ipilimumab, prepared by the method in Example 4) to 5 ⁇ SDS loading buffer solution (containing iodoacetamide at a final concentration of 40 mM), heated in a dry bath at 75°C for 10 min, cooled to room temperature, centrifuged at 12,000 rpm for 5 min, and the supernatant was taken.
  • heavy chain antibody or quality control IPI i.e. Ipilimumab, prepared by the method in Example 4
  • 5 ⁇ SDS loading buffer solution containing iodoacetamide at a final concentration of 40 mM
  • Reducing (denaturing) SDS-PAGE sample preparation Add 2 ⁇ g of heavy chain antibody or quality control IPI to 5 ⁇ SDS loading buffer (containing DTT at a final concentration of 5 mM), heat in a dry bath at 100°C for 10 minutes, and cool to room temperature Afterwards, centrifuge at 12000rpm for 5min, and take the supernatant. The supernatant was added to Bis-tris 4-15% gradient gel (GentScript) for gel electrophoresis, and the protein bands were visualized by Coomassie brilliant blue staining.
  • EPSON V550 color scanner was used to scan the protein gel with chromogenic protein bands (the decolorization solution was decolorized until the gel background was transparent), and the purity of reduced and non-reduced bands was calculated by ImageJ according to the peak area normalization method.
  • the results are shown in Figure 3A-3B and Table 4:
  • the apparent relative molecular weights of the heavy chain antibody and quality control IPI non-reducing gel bands are around 80kD and 150kD, respectively, and the apparent relative molecular weight of the heavy chain antibody reducing gel bands At about 40kD, the IPIs of quality control products are about 55kD and 25kD respectively.
  • the molecular weight of the heavy chain antibody analyzed by reducing and non-reducing SDS-PAGE was in line with the expected size, and the purity was greater than 90%.
  • Material preparation 1. Mobile phase: 150mmol/L phosphate buffer, pH 7.4; 2. Sample preparation: heavy chain antibody and quality control IPI were diluted to 0.5mg/mL with mobile phase solution.
  • Agilent HPLC 1100 chromatographic column XBridge BEH SEC 3.5 ⁇ m, 7.8mm I.D. ⁇ 30cm, Waters) flow rate was set to 0.8mL/min, injection volume was 20 ⁇ L, VWD detector wavelength was 280nm and 214nm.
  • the SEC-HPLC analysis results of the heavy chain antibodies are as follows: Calculate the percentage of high molecular aggregates, antibody monomers and low molecular aggregates in the sample according to the area normalization method. The results are shown in Table 4. The monomer purity of all heavy chain antibodies is greater than 96.0 %.
  • the ELISA-based method verified the affinity activity of the heavy chain antibody to human IL-17A antigen, and the ELISA-based method also verified the effect of the heavy chain antibody blocking the binding of IL-17A and IL-17RA.
  • the binding affinity of the heavy chain antibody to the antigen human IL-17A was detected by biofilm layer interferometry (using Fortebio equipment). As a comparison, the binding affinity of the control antibody secukinumab to the antigen human IL-17A was also determined.
  • IL-17A-His as the antigen was diluted to 10 ⁇ g/mL with 10 ⁇ KB buffer (10 ⁇ PBS containing 1% BSA, 0.5% Tween 20), and the heavy chain antibody was diluted 2-fold with 10 ⁇ KB buffer, Diluted sequentially from 80nM to 1.25nM. Under dark conditions, use 10 ⁇ KB buffer to pre-wet the sensor (Anti-Penta-HIS, HIS1K, Fortebio, CA), start testing the sample plate (GreinierBio, PN655209) after at least 10 minutes, and follow the preset procedure after the test is correct. First, combine the antigen with the sensor for 120s. After the combination is completed, continue to equilibrate in 10 ⁇ KB buffer for 30s.
  • K D affinity kinetic constant
  • K on binding constant
  • K off dissociation constant
  • the test results are shown in Table 5.
  • the results showed that the binding K D of the heavy chain antibody to the recombinant antigen IL-17A-His ranged from 4.57 ⁇ 10 -10 M to 3.17 ⁇ 10 -11 M, indicating that the heavy chain antibody had higher affinity to IL-17A.
  • the heavy chain antibodies VHH1, VHH2, VHH3, VHH5, VHH6, VHH8, VHH9 and VHH11 all showed better affinity than secukinumab.
  • NA means that the antibody did not dissociate within 120s after binding, so the K d and K D values were not calculated.
  • the cell biological activity of the anti-IL-17A heavy chain antibody was detected by detecting that the anti-IL-17A heavy chain antibody inhibited IL-17A from stimulating HeLa cells to secrete IL-6.
  • the specific method is as follows:
  • HeLa cells (Cell Bank of Chinese Academy of Sciences) were revived, and the cells with good growth status after passage 2-4 times were used for the experiment. Inoculate 1 ⁇ 10 5 cells/mL HeLa cells into a new 96-well cell culture plate at 100 ⁇ L per well, and place in a 37°C cell culture incubator for overnight culture; the next day, use DMEM medium to serially dilute the cells to be tested Heavy chain antibody and positive control antibody secukinumab, add 100ng/mL IL-17A-His protein and 30ng/mL TNF- ⁇ protein (Sino Biological, 10602-H01H), add antibody and protein mixture to 96-well cell plate cultured in a cell culture incubator at 37°C for 24 hours. After the culture, the cell culture supernatant was harvested, and the IL-6 ELISA detection kit (BD, 555220) was used for detection and quantitative detection.
  • BD IL-6 ELISA detection kit
  • the framework region of Nanobodies was humanized and mutated and back-mutated to obtain heavy chain antibodies with a higher degree of humanization while maintaining the affinity of humanized antibodies for antigens .
  • the heavy chain antibodies corresponding to the clone numbers NB27A-15, 2-NB27B-19 and NB26B-12 were selected for humanization, and the antibody Compare the sequence with the human antibody germline gene database, find 1-3 germline genes with relatively high homology with each VHH sequence, and take into account the druggability of the germline gene, and select a suitable germline gene template for comparison Yes, analyze the number of non-human sequence sites in the VHH framework regions. Homology modeling is performed on VHH, and the homology modeling refers to the nanobody model of the PDB database.
  • VHHs of heavy chain antibodies VHH1, VHH2 and VHH9 were humanized to obtain the VHHs of humanized antibodies VHH1-huVH4, VHH2-huVH3 and VHH9-huVH3 respectively, and their amino acid sequences are shown in SEQ ID NO:65, 66 and 67, respectively .
  • the expression vectors of humanized antibodies VHH1-huVH4, VHH2-huVH3 and VHH9-huVH3 were constructed as described in Example 4.1.
  • the humanized antibodies VHH1-huVH4, VHH2-huVH3 and VHH9-huVH3 respectively comprise the above-mentioned VHH amino acid sequences (SEQ ID NO:65, 66 and 67) and the Fc fragment of human IgG1 (SEQ ID NO:2).
  • VHH amino acid sequences SEQ ID NO:65, 66 and 67
  • Fc fragment of human IgG1 SEQ ID NO:2
  • the affinity of the humanized antibodies VHH1-huVH4, VHH2-huVH3 and VHH9-huVH3 to the antigen IL-17A was determined by ELISA and compared with the respective parental antibodies.
  • the specific method is as follows: a 96-well plate was coated with recombinant protein IL-17A-His (2 ⁇ g/mL, 30 ⁇ L/well), and left overnight at 4°C. The next day, the 96-well plate was washed 3 times with PBST and blocked with 5% skimmed milk for 2 h; after the plate was washed 3 times with PBST, the antibodies to be tested (humanized antibodies VHH1-huVH4, VHH2-huVH3 and VHH9 -huVH3; parental heavy chain antibodies VHH1, VHH2 and VHH9; secukinumab as a positive control; ipilimumab (IPI) as a negative control) and incubated for 1 h; after that, the secondary antibody anti- human Fc-HRP (Jackson Immuno Research, 109-035-008) and incubate for 1 h; after incubation, wash the plate six times with PBST, add TMB (SurMod

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Abstract

属于生物医药领域。具体地，涉及特异性结合IL-17A的抗体或其抗原结合片段、制备方法及其用途。

Description

抗IL-17A抗体及其用途
本申请要求2021年9月3日提交的，题为“抗IL-17A抗体及其用途”的第202111031745.0号中国专利申请的优先权，该申请的内容整体援引加入本文。
技术领域
本发明属于生物医药领域。具体地，本发明涉及特异性结合IL-17A的抗体或其抗原结合片段、制备方法及其用途。
背景技术
IL-17(白介素-17)家族细胞因子包含6个成员，分别为IL-17A、IL-17B、IL-17C、IL-17D、IL-17E和IL-17F；IL-17家族中最具代表性的成员是IL-17A。IL-17A于1993年首次在T细胞产物中被发现，在各种T细胞中都有表达。人源IL-17A基因包含155个氨基酸，具有19个氨基酸的信号肽和132个氨基酸的成熟区，相对分子质量为17kDa，以IL-17A同源二聚体或IL-17A/IL-17F异源二聚体的形式存在(Gaffen Sarah L,Nat.Rev.Immunol.,2009,9:556-67)。
当人体受到侵害或者损伤，迁移到机体受感染或损伤处的淋巴细胞会分泌IL-17A。IL-17A一方面会诱导炎症因子以及趋化因子的表达，从而招募更多的免疫细胞到达炎症部位加剧炎症反应；另一方面，IL-17A还会诱导一些组织修复相关因子的表达，从而加速机体的恢复。虽然IL-17A在宿主抗感染和组织修复过程中起到扩大免疫防御反应和保护机体的作用，但是在很多自身免疫病患者体内IL-17A是高表达的。过高的IL-17A水平会诱导诸多炎症因子的表达，对自身免疫性疾病发展起到恶化作用。诸多动物模型证实，IL-17A的缺失或者IL-17A被抗体中和，可以有效地抑制多种自身免疫疾病病理程度。有证据表明以IL-17A信号通路为靶点治疗自身免疫病，包括类风湿性关节炎(RA)、银屑病、克罗恩氏病、多发性硬化症(MS)、哮喘和红斑狼疮等，均有一定的疗效。
其中，临床上对银屑病治疗效果的评价通常采用皮损面积与严重性指数PASI(psoriasis area and severity index)，并具体将PASI 75、PASI 90和PASI 100作为治疗成功的重要指标。目前，在临床上有两种针对IL-17A的单克隆抗体Secukinumab(苏金单抗)和ixekizumab以及一种针对IL-17RA的单克隆抗体brodalumab已获批用于治疗中重度斑块型银屑病。有文献报道指出，虽然这三种单克隆抗体治疗中重度斑块型银屑病的PASI 75能达到80％以上，但PASI 100的治疗效果都不超过40％(April W.Armstrong et al.,JAMA.2020；323(19):1945-1960)。因此，为了进一步提高对诸如银屑病等病症的治疗效果，有必要继续开发新的治疗效果更优的靶向IL-17A的药物。
发明内容
在一方面，本发明提供一种抗IL-17A抗体或其抗原结合片段，其包含免疫球蛋白的单可变结构域，所述单可变结构域为第一家族、第二家族或第三家族单可变结构域。
(a)第一家族单可变结构域
在一些实施方案中，所述第一家族单可变结构域包含：
1)CDR1，其包含SEQ ID NO:68的氨基酸序列：
Xaa1-Xaa2-Xaa3-Xaa4-S-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9              (SEQ ID NO:68)
其中，
Xaa1为G或A；Xaa2为F或S；Xaa3为T或I；Xaa4为F或I；Xaa5为S、D或I；Xaa6为Y、F或H；Xaa7为A或P；Xaa8为M或I；Xaa9为S、G或A；
2)CDR2，其包含SEQ ID NO:69的氨基酸序列：
Xaa10-Xaa11-Xaa12-Xaa13-Xaa14-Xaa15-Xaa16-S-T-Xaa17        (SEQ ID NO:69)
其中，
Xaa10为T或A；Xaa11为V或I；Xaa12为E、T或不存在；Xaa13为I、S或T；Xaa14为G、A、R或N；Xaa15为G、S或V；Xaa16为S或G；Xaa17为N或D；以及
3)CDR3，其包含SEQ ID NO:70的氨基酸序列：
D-Xaa18-Xaa19-Xaa20-Y-E-Xaa21-Xaa22-D-D-Y                 (SEQ ID NO:70)
其中，
Xaa18为W、Y或G；Xaa19为K、T或R；Xaa20为W或Y；Xaa21为V、S或H；Xaa22为I或不存在。
在优选实施方案中，第一家族单可变结构域包含：
1)CDR1，其包含SEQ ID NO:17、38、20、23或35的氨基酸序列；
2)CDR2，其包含SEQ ID NO:18、39、21、24或36的氨基酸序列；以及
3)CDR3，其包含SEQ ID NO:19、40、22、25或37的氨基酸序列。
在一具体实施方案中，第一家族单可变结构域包含：
(1)CDR1，其包含SEQ ID NO:17的氨基酸序列；CDR2，其包含SEQ ID NO:18的氨基酸序列；以及CDR3，其包含SEQ ID NO:19的氨基酸序列；或者
(2)CDR1，其包含SEQ ID NO:38的氨基酸序列；CDR2，其包含SEQ ID NO:39的氨基酸序列；以及CDR3，其包含SEQ ID NO:40的氨基酸序列；或者
(3)CDR1，其包含SEQ ID NO:20的氨基酸序列；CDR2，其包含SEQ ID NO:21的氨基酸序列；以及CDR3，其包含SEQ ID NO:22的氨基酸序列；或者
(4)CDR1，其包含SEQ ID NO:23的氨基酸序列；CDR2，其包含SEQ ID NO:24的氨基酸序列；以及CDR3，其包含SEQ ID NO:25的氨基酸序列；或者
(5)CDR1，其包含SEQ ID NO:35的氨基酸序列；CDR2，其包含SEQ ID NO:36的氨基酸序列；以及CDR3，其包含SEQ ID NO:37的氨基酸序列或其变体。
(b)第二家族单可变结构域
在一些实施方案中，第二家族单可变结构域包含：
1)CDR1，其包含SEQ ID NO:71的氨基酸序列：
Xaa23-Xaa24-I-Xaa25-Xaa26-Xaa27-Xaa28-Xaa29-M-Xaa30        (SEQ ID NO:71)
其中，
Xaa23为A或G；Xaa24为S或F；Xaa25为F或I；Xaa26为N或S；Xaa27为A、I或E；Xaa28为H或Y；Xaa29为A或S；Xaa30为G或N；
2)CDR2，其包含SEQ ID NO:72的氨基酸序列：
Xaa31-I-T-Xaa32-G-G-Xaa33-T-Xaa34                          (SEQ ID NO:72)
其中，
Xaa31为A、S、T或R；Xaa32为S、Y或R；Xaa33为S、N或T；Xaa34为D或N；以及
3)CDR3，其包含SEQ ID NO:28、31、34或43的氨基酸序列。
在优选实施方案中，第二家族单可变结构域包含：
1)CDR1，其包含SEQ ID NO:26、29、32或41的氨基酸序列；
2)CDR2，其包含SEQ ID NO:27、30、33或42的氨基酸序列；以及
3)CDR3，其包含SEQ ID NO:28、31、34或43的氨基酸序列。
在一具体实施方案中，第二家族单可变结构域包含：
(1)CDR1，其包含SEQ ID NO:26的氨基酸序列；CDR2，其包含SEQ ID NO:27的氨基酸序列；以及CDR3，其包含SEQ ID NO:28的氨基酸序列；或者
(2)CDR1，其包含SEQ ID NO:29的氨基酸序列；CDR2，其包含SEQ ID NO:30的氨基酸序列；以及CDR3，其包含SEQ ID NO:31的氨基酸序列；或者
(3)CDR1，其包含SEQ ID NO:32的氨基酸序列；CDR2，其包含SEQ ID NO:33的氨基酸序列；以及CDR3，其包含SEQ ID NO:34的氨基酸序列；或者
(4)CDR1，其包含SEQ ID NO:41的氨基酸序列；CDR2，其包含SEQ ID NO:42的氨基酸序列；以及CDR3，其包含SEQ ID NO:43的氨基酸序列。
(c)第三家族单可变结构域
在一些实施方案中，第三家族单可变结构域包含：
1)CDR1，其包含SEQ ID NO:73的氨基酸序列：
G-F-Xaa35-L-D-Xaa36-Xaa37-Xaa38-I-G                        (SEQ ID NO:73)
其中，
Xaa35为T或N；Xaa36为D或Y；Xaa37为D或Y；Xaa38为A或G；
2)CDR2，其包含SEQ ID NO:74的氨基酸序列：
C-I-Xaa39-S-S-D-G-S-T-Y                                    (SEQ ID NO:74)
其中，
Xaa39为S或T；以及
3)CDR3，其包含SEQ ID NO:46、49或52的氨基酸序列。
在优选实施方案中，第三家族单可变结构域包含：
1)CDR1，其包含SEQ ID NO:44、47或50的氨基酸序列；
2)CDR2，其包含SEQ ID NO:45、48或51的氨基酸序列；以及
3)CDR3，其包含SEQ ID NO:46、49或52的氨基酸序列。
在一具体实施方案中，第三家族单可变结构域包含：
(1)CDR1，其包含SEQ ID NO:44的氨基酸序列；CDR2，其包含SEQ ID NO:45的氨基酸序列；以及CDR3，其包含SEQ ID NO:46的氨基酸序列；或者
(2)CDR1，其包含SEQ ID NO:47的氨基酸序列；CDR2，其包含SEQ ID NO:48的氨基酸序列；以及CDR3，其包含SEQ ID NO:49的氨基酸序列；或者
(3)CDR1，其包含SEQ ID NO:50的氨基酸序列；CDR2，其包含SEQ ID NO:51的氨基酸序列；以及CDR3，其包含SEQ ID NO:52的氨基酸序列。
在一实施方案中，所述单可变结构域包含选自以下的氨基酸序列：SEQ ID NO:5、SEQ ID  NO:12、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16。
在一实施方案中，所述单可变结构域包含选自以下的氨基酸序列：SEQ ID NO:65、SEQ ID NO:67和SEQ ID NO:66。
在一些实施方案中，所述抗IL-17A抗体或其抗原结合片段与至少一种可检测标记缀合。
在另一方面，本发明还提供一种多特异性抗体，其包含结合IL-17A的第一抗原结合部分以及结合第二抗原的第二抗原结合部分，其中所述第一抗原结合部分包含本发明的抗IL-17A抗体或其抗原结合片段。
在又一方面，本发明提供一种多核苷酸，其编码本发明的抗IL-17A抗体或其抗原结合片段。
本发明还提供一种表达载体，其包含本发明的多核苷酸。
在另一方面，本发明还提供一种宿主细胞，其包含本发明的多核苷酸或表达载体。
本发明还提供一种制备本发明的抗IL-17A抗体或其抗原结合片段的方法，其包括在合适条件下培养本发明的宿主细胞以表达所述抗体或其抗原结合片段，以及从宿主细胞或其培养物分离所述抗体或其抗原结合片段。
在又一方面，本发明还提供一种药物组合物，其包含本发明抗IL-17A抗体或其抗原结合片段，以及药学上可接受的载剂。
本发明还提供本发明的抗IL-17A抗体或其抗原结合片段或者药物组合物在制备用于治疗IL-17A介导的疾病的药物中的用途。
本发明还提供一种检测样品中IL-17A的存在或确定其表达水平的方法，其包括：
(a)使本发明的抗IL-17A抗体或其抗原结合片段与所述样品接触，以及
(b)检测所述抗体或其抗原结合片段与样品中IL-17A之间免疫复合物的形成或确定所述免疫复合物的量，从而检测样品中IL-17A的存在或确定样品中IL-17A的表达水平。
附图说明
图1A-1C显示了抗IL-17A纳米抗体与重组抗原IL-17A-His结合活性。
图2A-2C显示了抗IL-17A纳米抗体对IL-17A与IL-17RA的阻断活性。
图3A-3B显示了抗IL-17A重链抗体在非变性及变性条件下的SDS-PAGE图。
图4A-4C显示了抗IL-17A重链抗体与重组抗原IL-17A的结合活性。
图5A-5C显示了抗IL-17A重链抗体对IL-17A/IL-17RA的阻断活性。
图6A-6K显示了抗IL-17A重链抗体中和IL-17A诱导的HeLa细胞分泌IL-6的能力。
图7A-7B显示了抗IL-17A人源化抗体与重组抗原IL-17A的结合活性。
具体实施方式
定义
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的蛋白质和核酸化学、分子生物学、细胞和组织培养、微生物学、免疫学相关术语和实验室操作步骤均为相应领域内广泛使用的术语和常规步骤。同时，为了更好地理 解本发明，下面提供相关术语的定义和解释。
在本文中，免疫球蛋白包括任何免疫球蛋白类型(例如，IgG、IgM、IgD、IgE、IgA和IgY)、任何类别(例如IgG1、IgG2、IgG3、IgG4、IgA1和IgA2)或亚类(例如，IgG2a和IgG2b)。
如本文所用，“抗体”指免疫球蛋白或其片段，其通过至少一个抗原结合位点特异性结合抗原表位。在本文中，抗体的定义涵盖抗原结合片段。术语“抗体”包括多特异性抗体(例如双特异性抗体)、人抗体、非人抗体、人源化抗体、嵌合抗体、重链抗体、单域抗体以及抗原结合片段。抗体可以是合成的(例如通过化学偶联或生物偶联产生的)、酶促处理得到的或重组产生的。
如本文所用，“抗原结合片段”指全长抗体的部分，其少于全长，但是至少包含全长抗体的部分可变区(例如包含一个或多个CDR和/或一个或多个抗原结合位点)，并因此保留全长抗体的至少部分特异性结合抗原的能力。
如本文所用，“免疫球蛋白单可变结构域”或者“单可变结构域”是指具有抗原结合活性的单个可变区(可变结构域)。不同于常规抗体中由一对VH和VL组成功能性抗原结合单位，单可变结构域可以独自形成功能性抗原结合单位。单可变结构域可以衍生自天然存在的无轻链抗体，例如骆驼科动物(如骆驼和羊驼)的重链抗体的可变结构域(variable domain of heavy chain of heavy-chain antibody，VHH)和鲨鱼的新抗原受体的单可变结构域(IgNAR variable single-domain，VNAR)，也可以从全长抗体中筛选得到，例如人抗体中具有抗原结合活性的轻链可变结构域和重链可变结构域。VHH通常包含三个高度可变的“互补决定区(CDR)”和四个相对保守的“框架区(FR)”，并且从N端至C端以FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4的次序连接。
如本文所用，“单域抗体(sdAb)”或“纳米抗体”是指包含单个免疫球蛋白可变结构域作为功能性抗原结合单位的抗体。与通常包含两条重链和两条轻链的全长抗体不同，单域抗体通常包含由单可变结构域组成的单个肽链，分子量仅为15kDa左右。
如本文所用，术语“仅有重链的抗体(Heavy-chain-only antibody)”和“重链抗体(Heavy-chain antibody)”可互换使用，并且以其最广泛的意义存在，是指缺乏常规抗体轻链的抗体，其仅包含一个VHH以及不包含CH1的重链恒定区(例如Fc片段)。
本领域技术人员可以使用本领域熟知的方法识别CDR，例如使用Kabat、AbM、Chothia或IMGT编号法。在本文中，本发明的抗IL-17A抗体的CDR是按照AbM编号系统定义，但是本领域技术人员应当理解，对于同一个可变区还可以使用多个CDR编号系统来定义，例如Chothia、Kabat和IMGT。尽管由不同编号系统定义的CDR边界可能会不同，但是同一编号系统对应的CDR代表能够结合抗原表位的有效抗原结合位点。有关CDR编号系统的描述可以参见例如， Kabat编号系统：Kabat,E.A.et al.(1991)Sequences of Proteins of Immunological Interest,Fifth Edition,U.S.Department of Health and Human Services,NIH Publication No.91-3242； Chothia编号系统：Chothia,C.et al.(1987)J.Mol.Biol.196:901-917； IMGT编号系统：Lefranc,M.-P.,2011(6),IMGT,the International ImMunoGeneTics Information System Cold Spring Harb Protoc.和Lefranc,M.-P.et al.,Dev.Comp.Immunol.,27,55-77(2003)； AbM编号系统：Martin,A.C.R.and J.Allen(2007)“Bioinformatics tools for antibody engineering,”in S.Dübel(ed.),Handbook of Therapeutic Antibodies.Weinheim:Wiley-VCH Verlag,pp.95–118。
如本文所用，术语“框架区”和“构架区”可以互换使用。如本文中所使用的，术语“框架区”、“构架区”或“FR”残基是指抗体可变区中除了如上定义的CDR序列以外的那些氨基酸残基。
“Fc片段”一般指常规抗体或重链抗体经木瓜蛋白酶消化的可结晶片段。一般而言，IgG和重链 抗体的Fc片段可以包含部分铰链区、CH2和CH3。在本文中，Fc片段可以包含至少部分铰链区(例如铰链区的全部或部分)、CH2和CH3。“铰链区”一般指常规抗体中CH1和CH2之间的部分、重链抗体中VHH和CH2之间的部分或者其功能性等同物，例如T细胞受体(TCR)中的铰链区。铰链区可以是完整的铰链区或者其部分。本领域技术人员可以根据已知的算法和软件判断CDR、FR、VH、VL、CL、CH1、CH2、CH3和铰链区在抗体中的位置，可以应用的算法和软件的描述可以参见例如William R.Strohl,Lila M.Strohl,(2012),Antibody structure–function relationships,In Woodhead Publishing Series in Biomedicine,Therapeutic Antibody Engineering,Woodhead Publishing,pp.37-56。
如本文所用，“嵌合抗体”指这样的抗体，其中的一部分(例如CDR、FR、可变区、恒定区或其组合)与衍生自特定物种的抗体中相应序列相同或同源，剩余的部分与衍生自另一物种的抗体中相应序列相同或同源。在本发明的一些实施方案中，嵌合抗体包含衍生自非人物种(例如骆驼科动物，例如骆驼和羊驼)的可变区以及衍生自不同物种(例如人)的恒定区。嵌合抗体还可指对至少两种不同抗原具有特异性的多特异性抗体。嵌合抗体可以通过抗体工程化产生。抗体工程化的方法是本领域技术人员公知的。特别地，可以通过DNA重组技术生成嵌合抗体(例如参见Sambrook,J.,et al.(1989).Molecular cloning:a laboratory manual,2nd ed.Cold Spring Harbor Laboratory,Cold Spring Harbor,N.Y)。
如本文所用，术语“人源化抗体”是指非人抗体经修饰以增加与人抗体的序列同源性的抗体。人源化抗体通常保留其所源自的非人抗体的抗原结合能力并且对于人体具有较低的免疫原性。人源化抗体可以通过抗体工程化改造任何非人物种抗体或其中包含非人物种来源序列的抗体(例如嵌合抗体)来获得。由非人抗体获得人源化抗体的技术是本领域技术人员熟知的，例如，可以将非人抗体(例如驼源抗体)的CDR序列移植到人抗体框架区中。在某些情况下，为了保持人源化抗体的抗原结合能力和/或稳定性，可以在人抗体框架区中保留非人抗体(例如驼源抗体)框架序列的关键氨基酸残基，即进行“回复突变”(参见，例如Morrison et al.(1984)Proc.Natl.Acad.Sci.81(21):6851-6855；Neuberger et al.(1984)Nature 312:604-608)。
如本文所用，氨基酸序列的“百分比(％)序列相同性”、“序列相同性”具有本领域公认的定义，其指通过序列比对(例如通过人工检视或可公知的算法)确定的两个多肽序列之间相同的百分比。可以使用本领域技术人员已知的方法确定，例如使用可公开获得的计算机软件如BLAST、BLAST-2、Clustal Omega和FASTA软件。
在本文中，“源自”或“衍生自”参考氨基酸序列的氨基酸序列与参考氨基酸序列的部分或者全部相同或同源。例如，衍生自人免疫球蛋白的重链恒定区的氨基酸序列可以与其所源自的人免疫球蛋白重链恒定区的野生型序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或至少100％的序列相同性。
可以修饰多肽中的非关键区域(例如抗体CDR区和框架区的非关键氨基酸以及恒定区的氨基酸)，例如进行一个或多个氨基酸的取代、添加和/或缺失，而不改变多肽的功能。这类相对于参考氨基酸序列(例如单可变结构域)修饰的多肽，可以称作参考氨基酸序列的“变体”。参考氨基酸序列的变体可以与其所源自的序列(即参考氨基酸序列)具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列相同性，或者与其所源自的序列相比具有一个或多个(例如，1、2、3、4、5、6、7、8、9或10个)氨基酸的取代、添加和/或缺失。优选地，所述取代为保守取代。本领域技术人员应当理解多肽 中非关键区域中的氨基酸可以用合适的保守氨基酸取代，并且一般不改变其生物活性(参见，例如Watson et al.,Molecular Biology of the Gene,4th Edition,1987,The Benjamin/Cummings Pub.co.,p.224)。合适的保守取代是本领域技术人员熟知的。在下表中列出一些常见的氨基酸残基保守取代的非限制性实例。在某些情况下，氨基酸取代是非保守取代。本领域技术人员应当理解，可以对多肽进行氨基酸突变或修饰来改变其性能，例如改变抗体糖基化修饰的类型，改变形成链间二硫键的能力，或者为抗体缀合物的制备提供活性基团。包含这类氨基酸突变或修饰的抗体或其抗原结合片段也涵盖在本发明的抗体或其抗原结合片段的范围之内。
“亲和力”或“结合亲和力”用来衡量抗体和抗原之间通过非共价作用相互结合的强度。“亲和力”的大小通常可以报告为平衡解离常数K D。K D可以通过测量平衡缔合常数(ka)和平衡解离常数(kd)来计算：K D＝Kd/Ka。可以用本领域已知的常规技术测定亲和力，例如生物膜干涉技术(可以采用例如Octet Fortebio检测系统)、放射免疫法、表面等离子共振法、酶联免疫测定(ELISA)或流式细胞术(FACS)等。
在本文中，抗体和抗原“特异性结合”是指抗体与抗原之间以较高的亲和力相互结合。通常，特异性结合的抗体和抗原之间的K D值为至少约10 -6M到至少约10 -9M或更低，例如至少约10 -6M、至少约10 -7M、至少约10 -8M、至少约10 -9M、至少约10 -10M或更低。在一实施方案中，本发明的抗IL-17A抗体或其抗原结合片段以高亲和力结合人IL-17A，例如以5×10 -10M或更低、1×10 -10M或更低、5×10 -11M或更低、2×10 -11M或更低的K D结合人IL-17A。
本发明的抗IL-17A抗体或抗原结合片段、多特异性抗体或者编码其的多核苷酸可以是分离的。如本文所用，表述“分离的”是指物质(例如多核苷酸或多肽)与其存在的来源或环境是分离的，即基本上不包含其他任何成分。
在本文中，术语“多核苷酸”和“核酸”可以互换用于表示包含至少两个连接的核苷酸或核苷酸衍生物的寡聚体或聚合物，通常可以包括脱氧核糖核酸(DNA)和核糖核酸(RNA)。
如本文所用，术语“表达”是指产生RNA和/或多肽。
在本文中，“载体”是用于将外源多核苷酸导入宿主细胞的媒介，当载体转化入适当的宿主细胞时，外源多核苷酸得以扩增或表达。载体通常保持游离，但是可以设计为使基因或其部分整合入基因组的染色体。如本文所用，载体的定义涵盖质粒、线性化质粒、病毒载体、粘粒、噬菌体载体、噬菌粒、人工染色体(例如，酵母人工染色体和哺乳动物人工染色体)等。病毒载体包括但不限于逆转录病毒载体(包括慢病毒载体)、腺病毒载体、腺相关病毒载体、疱疹病毒载体、痘病毒载体和杆状病毒载体等。
如本文所用，“表达载体”指能够表达感兴趣的多核苷酸(包括DNA和RNA)的载体。例如，在表达载体中，可以将编码感兴趣多肽的多核苷酸序列(包括DNA和RNA)与能够影响多核苷酸序列表达的调控序列(如启动子和核糖体结合位点)可操作地连接。调控序列可以包含启动子和终止子序列，并且任选地可以包含复制起点、选择标记、增强子、多腺苷酸化信号等。表达载体可以是质粒、噬菌体载体、重组病毒或其他载体，当引入适当的宿主细胞时，导致感兴趣的多核苷酸的表达。合适的表达载体是本领域技术人员公知的。本领域技术人员可以根据需要将表达载体制备为在宿主细胞中可复制、在宿主细胞中保持游离或者整合入宿主细胞基因组的载体。
如本文所用，“宿主细胞”是用于接受、保持、复制或扩增载体的细胞。宿主细胞还可以用来表达多核苷酸或载体所编码的多肽。宿主细胞可以是真核细胞或原核细胞。原核细胞例如大肠杆菌(E.coli)或枯草芽孢杆菌(Bacillus subtilis)，真菌细胞例如酵母细胞或曲霉属、昆虫细胞(如S2果蝇细胞 或Sf9)以及动物细胞(如成纤维细胞、CHO细胞，COS细胞、HeLa细胞、NSO细胞或HEK293细胞)。
如本文所用，术语“治疗”指对疾病/症状的改善，例如使疾病/症状减轻或消失、防止或减缓疾病/症状的发生、进展和/或恶化。因此，治疗包括预防、治疗和/或治愈。
“有效量”是指这样的剂量，其足以使疾病症状的严重性降低，疾病无症状期的频率和持续时间增加，或者防止因疾病痛苦而引起的损伤或失能。“有效量”指防止、治愈、改善、阻滞或部分阻滞疾病或症状所需的量。本领域技术人员可以根据例如受试者的年龄、身体状况、性别、症状的严重程度、特定组合物或给药途径等因素来确定有效量。有效量可以在一次或多次施用中给予。
如本文中所使用的，术语“药学上可接受的载剂”是指在药理学和/或生理学上与受试者和活性成分相容的载剂，其是本领域公知的(参见例如Remington's Pharmaceutical Sciences.Edited by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995)。
抗IL-17A抗体或其抗原结合片段
在一总的方面，本发明提供了一种抗IL-17A抗体或其抗原结合片段，其包含特异性结合IL-17A的免疫球蛋白单可变结构域。
如本文所用，术语“IL-17A”是指白介素17A。IL-17A是一种促炎性细胞因子，产生于各种T细胞。IL-17A刺激上皮、内皮和成纤维细胞来产生其他促炎性细胞因子和趋化因子，包括例如IL-6、IL-8、G-CSF和MCP-1。IL-17A可以是人IL-17A和非人(例如骆驼、羊驼、小鼠、大鼠、豚鼠、兔、山羊、绵羊、牛、马、猴等)IL-17A。在一实施方案中，所述IL-17A为人IL-17A。示例性人IL-17A的氨基酸序列示于SEQ ID NO:1。IL-17A在体内可以以IL-17A同源二聚体和IL-17A/F异源二聚体(IL-17A/IL-17F)形式存在。
在一些实施方案中，本发明的抗IL-17A抗体或其抗原结合片段能够特异性结合IL-17A。在一实施方案中，所述抗IL-17A抗体或其抗原结合片段能够中和IL-17A。在一些实施方案中，所述抗IL-17A抗体或其抗原结合片段能够结合IL-17A同源二聚体或者IL-17A/F异源二聚体。在一些实施方案中，所述抗IL-17A抗体或其抗原结合片段能够阻断IL-17A或IL-17A/F与IL-17RA的相互作用。
在一些实施方案中，本发明的抗IL-17A抗体或其抗原结合片段包含免疫球蛋白的单可变结构域，其包含CDR1、CDR2和CDR3。在一些实施方案中，所述单可变结构域为第一家族、第二家族或第三家族单可变结构域。
第一家族单可变结构域
在一些实施方案中，第一家族单可变结构域包含：
1)CDR1，其包含SEQ ID NO:68的氨基酸序列：
Xaa1-Xaa2-Xaa3-Xaa4-S-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9              (SEQ ID NO:68)
其中，
Xaa1为G或A；Xaa2为F或S；Xaa3为T或I；Xaa4为F或I；Xaa5为S、D或I；Xaa6为Y、F或H；Xaa7为A或P；Xaa8为M或I；Xaa9为S、G或A；
2)CDR2，其包含SEQ ID NO:69的氨基酸序列：
Xaa10-Xaa11-Xaa12-Xaa13-Xaa14-Xaa15-Xaa16-S-T-Xaa17        (SEQ ID NO:69)
其中，
Xaa10为T或A；Xaa11为V或I；Xaa12为E、T或不存在；Xaa13为I、S或T；Xaa14为G、A、R或N；Xaa15为G、S或V；Xaa16为S或G；Xaa17为N或D；以及
3)CDR3，其包含SEQ ID NO:70的氨基酸序列：
D-Xaa18-Xaa19-Xaa20-Y-E-Xaa21-Xaa22-D-D-Y                 (SEQ ID NO:70)
其中，
Xaa18为W、Y或G；Xaa19为K、T或R；Xaa20为W或Y；Xaa21为V、S或H；Xaa22为I或不存在。
在优选实施方案中，第一家族单可变结构域包含：
1)CDR1，其包含SEQ ID NO:17、38、20、23或35的氨基酸序列；
2)CDR2，其包含SEQ ID NO:18、39、21、24或36的氨基酸序列；以及
3)CDR3，其包含SEQ ID NO:19、40、22、25或37的氨基酸序列。
在一具体实施方案中，第一家族单可变结构域包含：
(1)CDR1，其包含SEQ ID NO:17的氨基酸序列；CDR2，其包含SEQ ID NO:18的氨基酸序列；以及CDR3，其包含SEQ ID NO:19的氨基酸序列；或者
(2)CDR1，其包含SEQ ID NO:38的氨基酸序列；CDR2，其包含SEQ ID NO:39的氨基酸序列；以及CDR3，其包含SEQ ID NO:40的氨基酸序列；或者
(3)CDR1，其包含SEQ ID NO:20的氨基酸序列；CDR2，其包含SEQ ID NO:21的氨基酸序列；以及CDR3，其包含SEQ ID NO:22的氨基酸序列；或者
(4)CDR1，其包含SEQ ID NO:23的氨基酸序列；CDR2，其包含SEQ ID NO:24的氨基酸序列；以及CDR3，其包含SEQ ID NO:25的氨基酸序列；或者
(5)CDR1，其包含SEQ ID NO:35的氨基酸序列；CDR2，其包含SEQ ID NO:36的氨基酸序列；以及CDR3，其包含SEQ ID NO:37的氨基酸序列或其变体。
第二家族单可变结构域
在一些实施方案中，第二家族单可变结构域包含：
1)CDR1，其包含SEQ ID NO:71的氨基酸序列：
Xaa23-Xaa24-I-Xaa25-Xaa26-Xaa27-Xaa28-Xaa29-M-Xaa30        (SEQ ID NO:71)
其中，
Xaa23为A或G；Xaa24为S或F；Xaa25为F或I；Xaa26为N或S；Xaa27为A、I或E；Xaa28为H或Y；Xaa29为A或S；Xaa30为G或N；
2)CDR2，其包含SEQ ID NO:72的氨基酸序列：
Xaa31-I-T-Xaa32-G-G-Xaa33-T-Xaa34                          (SEQ ID NO:72)
其中，
Xaa31为A、S、T或R；Xaa32为S、Y或R；Xaa33为S、N或T；Xaa34为D或N；以及
3)CDR3，其包含SEQ ID NO:28、31、34或43的氨基酸序列。
在优选实施方案中，第二家族单可变结构域包含：
1)CDR1，其包含SEQ ID NO:26、29、32或41的氨基酸序列；
2)CDR2，其包含SEQ ID NO:27、30、33或42的氨基酸序列；以及
3)CDR3，其包含SEQ ID NO:28、31、34或43的氨基酸序列。
在一具体实施方案中，第二家族单可变结构域包含：
(1)CDR1，其包含SEQ ID NO:26的氨基酸序列；CDR2，其包含SEQ ID NO:27的氨基酸序列；以及CDR3，其包含SEQ ID NO:28的氨基酸序列；或者
(2)CDR1，其包含SEQ ID NO:29的氨基酸序列；CDR2，其包含SEQ ID NO:30的氨基酸序列；以及CDR3，其包含SEQ ID NO:31的氨基酸序列；或者
(3)CDR1，其包含SEQ ID NO:32的氨基酸序列；CDR2，其包含SEQ ID NO:33的氨基酸序列；以及CDR3，其包含SEQ ID NO:34的氨基酸序列；或者
(4)CDR1，其包含SEQ ID NO:41的氨基酸序列；CDR2，其包含SEQ ID NO:42的氨基酸序列；以及CDR3，其包含SEQ ID NO:43的氨基酸序列。
第三家族单可变结构域
在一些实施方案中，第三家族单可变结构域包含：
1)CDR1，其包含SEQ ID NO:73的氨基酸序列：
G-F-Xaa35-L-D-Xaa36-Xaa37-Xaa38-I-G                       (SEQ ID NO:73)
其中，
Xaa35为T或N；Xaa36为D或Y；Xaa37为D或Y；Xaa38为A或G；
2)CDR2，其包含SEQ ID NO:74的氨基酸序列：
C-I-Xaa39-S-S-D-G-S-T-Y                                   (SEQ ID NO:74)
其中，
Xaa39为S或T；以及
3)CDR3，其包含SEQ ID NO:46、49或52的氨基酸序列。
在这些实施方案中，如上所述的单可变结构域具有两对二硫键，比常规的单可变结构域多一对CDR2和CDR3之间的二硫键，单可变结构域内部增加的一对二硫键使其蛋白聚集较少。
在优选实施方案中，第三家族单可变结构域包含：
1)CDR1，其包含SEQ ID NO:44、47或50的氨基酸序列；
2)CDR2，其包含SEQ ID NO:45、48或51的氨基酸序列；以及
3)CDR3，其包含SEQ ID NO:46、49或52的氨基酸序列。
在一具体实施方案中，第三家族单可变结构域包含：
(1)CDR1，其包含SEQ ID NO:44的氨基酸序列；CDR2，其包含SEQ ID NO:45的氨基酸序列；以及CDR3，其包含SEQ ID NO:46的氨基酸序列；或者
(2)CDR1，其包含SEQ ID NO:47的氨基酸序列；CDR2，其包含SEQ ID NO:48的氨基酸序列；以及CDR3，其包含SEQ ID NO:49的氨基酸序列；或者
(3)CDR1，其包含SEQ ID NO:50的氨基酸序列；CDR2，其包含SEQ ID NO:51的氨基酸序列；以及CDR3，其包含SEQ ID NO:52的氨基酸序列。
在一些实施方案中，如上所述的单可变结构域进一步包含框架区。所述框架区可以各自独立地衍生自任何物种免疫球蛋白的框架区。
在一些实施方案中，所述框架区衍生自羊驼的免疫球蛋白。在一实施方案中，所述单可变结构域包含选自以下的氨基酸序列或其变体：SEQ ID NO:5、SEQ ID NO:12、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16；其中，所述变体与其所源自的序列具有至少 80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列相同性，或者与其所述源自的序列相比具有一个或多个(例如，1、2、3、4、5、6、7、8、9或10个)氨基酸的取代、添加和/或缺失。在一实施方案中，所述取代、添加和/或缺失不发生在CDR区。
在一些实施方案中，所述框架区衍生自人免疫球蛋白。在某些优选的实施方案中，所述单可变结构域包含衍生自人免疫球蛋白的重链框架区。因此，在某些优选的实施方案中，本发明的抗IL-17A抗体或其抗原结合片段是人源化的。所述框架区可以包含一个或多个非人源(例如，驼源)氨基酸残基，例如可以包含一或多个氨基酸回复突变，在这些回复突变中包含相应的驼源氨基酸残基。在一实施方案中，所述单可变结构域包含选自以下的氨基酸序列或其变体：SEQ ID NO:65、SEQ ID NO:67和SEQ ID NO:66；其中，所述变体与其所源自的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列相同性，或者与其所述源自的序列相比具有一个或多个(例如，1、2、3、4、5、6、7、8、9或10个)氨基酸的取代、添加和/或缺失。在一实施方案中，所述取代、添加和/或缺失不发生在CDR区。
在一些实施方案中，本发明的抗IL-17A抗体或其抗原结合片段可以进一步包含与本发明的单可变结构域连接的免疫球蛋白恒定区(例如重链恒定区和/或轻链恒定区)部分。任选地，所述恒定区部分与本发明的单可变结构域通过接头连接。
可以选择合适的免疫球蛋白恒定区(特别是人免疫球蛋白的重链恒定区)部分，并且任选地进行修饰，以获得具有期望的特性的抗体。例如可以在铰链区中引入或移除(例如通过DNA重组技术进行突变)一个或多个半胱氨酸残基以促进或减弱抗体的二聚化；可以修饰CH1、铰链区、CH2和/或CH3，以延长或降低抗体的血清半衰期、促进抗体的内化或组织渗透能力、具有改善或削弱的与FcγRIIB的结合(参见例如WO2008150494A1)、增强或减弱抗体依赖的细胞介导的细胞毒性作用(ADCC)、抗体依赖的细胞介导的吞噬作用(ADCP)以及补体依赖的细胞毒性作用(CDC)等(参见例如Caron,P.C.,et al.,J.Exp.Med.176:1191-1195(1992)以及Shopes B.J.Immunol.148 2918-2922(1992))。
在一些实施方案中，本发明的抗IL-17A抗体或其抗原结合片段包含与本发明的单可变结构域连接的重链恒定区部分。重链恒定区部分可以衍生自任何免疫球蛋白亚型或亚类，例如IgG、IgM、IgA、IgD和IgE。优选地，重链恒定区部分衍生自人IgG，例如IgG1、IgG2、IgG3和IgG4。重链恒定区部分优选包含铰链区、CH2、CH3或其组合。在更优选的实施方案中，重链恒定区部分包含Fc片段。
在一些实施方案中，本发明的抗IL-17A抗体或其抗原结合片段进一步包含免疫球蛋白的Fc片段。优选地，所述Fc片段为人IgG1的Fc片段。在一实施方案中，所述Fc片段包含SEQ ID NO:2的氨基酸序列。
在一些实施方案中，本发明的抗IL-17A抗体或其抗原结合片段包含免疫球蛋白单可变结构域，其包含选自SEQ ID NO:5、SEQ ID NO:12、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15和SEQ ID NO:16的氨基酸序列；以及人IgG1的Fc片段，其包含SEQ ID NO:2的氨基酸序列。在一具体实施方案中，本发明的抗IL-17A抗体或其抗原结合片段包含选自SEQ ID NO:53-64的氨基酸序列。
在一些实施方案中，本发明的抗IL-17A抗体或其抗原结合片段包含免疫球蛋白单可变结构域， 其包含选自SEQ ID NO:65、SEQ ID NO:67和SEQ ID NO:66的氨基酸序列；以及人IgG1的Fc片段，其包含SEQ ID NO:2的氨基酸序列。
此外，本领域技术人员能够根据需要选择可以与本发明的本发明的抗IL-17抗体或其抗原结合片段(例如单可变结构域或重链抗体)连接的任何其他多肽。其他多肽可以用于赋予本发明的抗IL-17A抗体或其抗原结合片段的有利的特性和/或减少不希望的特性，例如增加本发明的抗IL-17A抗体或其抗原结合片段的半衰期、溶解性或者吸收(例如促进其穿透皮肤、磷脂膜、细胞器等)；和/或降低其免疫原性、毒性或者副作用。包含这类多肽的抗体或抗原结合片段也涵盖在本发明的抗IL-17A抗体或其抗原结合片段的含义内。
例如，可以将血清白蛋白或其合适的片段与本发明的抗IL-17A抗体或其抗原结合片段连接以增加其血清半衰期(参见例如CN104884473A、WO2013177101A2、WO0027435A1和WO0177137A1)。其他多肽还可以是例如，用于指导抗体或其抗原结合片段从宿主细胞表达和分泌、促进抗体或抗原结合片段的检测和/或分离的多肽，包括但不限于信号肽(前导序列)、促溶标签、亲和标签(例如聚组氨酸标签(His 6)或谷胱甘肽S-转移酶(GST)标签)、包含蛋白酶切割位点的多肽和报告标签(例如荧光蛋白)。其他多肽还可以是生物活性多肽，例如具有治疗活性、结合活性或酶活性的多肽或蛋白。生物活性多肽的非限制性实例可以包括但不限于：蛋白毒素(例如白喉毒素、蓖麻毒素)、酶(例如脲酶、辣根过氧化物酶)和细胞因子。如上所述的多肽的氨基酸序列对于本领域技术人员将是清楚的。
本发明的抗IL-17A抗体或其抗原结合片段优选地与任何感兴趣的多肽(例如上述多肽的一个或多个)直接连接或通过合适的接头连接。在优选实施方案中，使用肽接头将抗IL-17A抗体或其抗原结合片段与上述多肽的一个或多个连接，从而本发明的抗IL-17A抗体或其抗原结合片段可被表达为重组(融合)蛋白。
肽接头可以包含任何长度的氨基酸序列，特别是1-50，优选1-30，例如1-10个氨基酸残基的氨基酸序列。示例性的肽接头可以包括但不限于聚甘氨酸(G)、聚丙氨酸(A)、聚丝氨酸(S)或其组合，例如GGAS、GGGS、GGGSG或者(G 4S) n，其中n为1-30、优选1-10的整数。肽接头还可以是铰链区或其功能性等同物。其它合适的接头可以是一般适合用于药用蛋白中的有机化合物或聚合物，包括但不限于聚乙二醇。
本发明的抗IL-17A抗体或其抗原结合片段免疫球蛋白可以是单域抗体、重链抗体、人源化抗体或嵌合抗体。
在一些实施方案中，所述抗IL-17A抗体或其抗原结合片段能够
1)特异性结合IL-17A；
2)中和IL-17A；
3)特异性结合IL-17A同源二聚体或者IL-17A/F异源二聚体；和/或
4)阻断IL-17A或IL-17A/F与IL-17RA的相互作用。
多特异性抗体
在一方面，本发明提供了一种多特异性抗体，其包含结合IL-17A的第一抗原结合部分以及结合第二抗原的第二抗原结合部分，其中所述第一抗原结合部分包含本发明的抗IL-17A抗体或其抗原结合片段。
如本文所用，术语“多特异性抗体”是指能够特异性结合两种或更多种(例如2、3、4、5或6种) 不同抗原表位的抗体。多特异性抗体可以例如是双特异性、三特异性或四特异性抗体，其分别能够特异性结合2、3或4种抗原表位。如本文所用，术语“抗原表位”或“抗原决定簇”表示抗原中与抗体的抗原结合位点特异性结合的区域。第二抗原可以为不同于IL-17A的其他抗原。第二抗原也可以为IL-17A，其与本发明的抗IL-17A抗体或其抗原结合片段结合IL-17A上不同的抗原表位。可以利用本领域常规的方法确定两种抗体结合的抗原表位是否相同，例如通过ELISA、流式细胞术或表面等离子共振等测定两种抗体对相同抗原表位的竞争结合。
多特异性抗体可以是多价(例如2、3、4价)抗体，即其具有多个抗原结合位点。
利用感兴趣的抗体或抗原结合片段构建多特异性抗体的方法是本领域技术人员熟知的(参见，例如WO 93/08829；Suresh et al.,(1986)Methods in Enzymology,121:210；和Traunecker et al.,(1991)EMBO,10:3655-3659)。可以利用本领域已知的各种技术产生和分离多特异性抗体。
如本文所用，“第一抗原结合部分”和“第二抗原结合部分”表示包含抗原结合位点的、能够与抗原表位结合的氨基酸序列，其定义落入抗体或抗原结合片段的含义范围内。
第一抗原结合部分可以是任何形式的抗体或抗原结合片段，包括但不限于单可变结构域和重链抗体。在一实施方案中，第一抗原结合部分包含本发明的单可变结构域。
第二抗原结合部分可以是结合任何感兴趣的抗原的抗体或抗原结合片段。在一实施方案中，第二抗原为不同于IL-17A的其他抗原。第二抗原结合部分可以特异性结合的抗原可以包括促炎性细胞因子和趋化因子。如本文所用，术语“促炎性细胞因子”指由免疫细胞或其他类型的细胞分泌的一类促进炎症的细胞因子。
第一抗原结合部分和第二抗原结合部分可以任选地通过接头(例如如本文所述)连接。
核酸、载体和宿主细胞
在另一方面，本发明提供了一种多核苷酸，其包含编码本发明的抗IL-17A抗体或其抗原结合片段。
可以利用本领域已知的方法获得本发明的多核苷酸。例如，本发明的多核苷酸可以分离自噬菌体展示文库、酵母展示文库、免疫动物(例如羊驼、小鼠、人源化小鼠、猴)、永生化的细胞(例如，小鼠B细胞杂交瘤细胞、EBV介导的永生化B细胞)或者化学合成。本发明的多核苷酸可以针对用于表达的宿主细胞进行密码子优化。
在又一方面，本发明还提供了包含本发明的多核苷酸的载体。在一些实施方案中，将本发明的多核苷酸克隆入表达载体。表达载体可以进一步包含额外的多核苷酸序列，例如调控序列和抗生素抗性基因。表达载体还可以包含编码额外的多肽的多核苷酸序列。
本发明的多核苷酸可以存在于一种或多种表达载体中。
本发明还提供了一种宿主细胞，其包含本发明的多核苷酸或表达载体。可以采用本领域已知的各种方法将本发明的多核苷酸或表达载体导入合适的宿主细胞中。这类方法包括但不限于脂质体转染、电穿孔、病毒转导和磷酸钙转染等。在优选的实施方案中，宿主细胞用于表达本发明的抗IL-17A抗体或其抗原结合片段。宿主细胞的实例包括但不限于原核细胞(例如细菌，例如大肠杆菌)和真核细胞(例如酵母、昆虫细胞、哺乳动物细胞)。适合于抗体表达的哺乳动物宿主细胞包括但不限于骨髓瘤细胞、HeLa细胞、HEK细胞(例如HEK 293细胞)、中国仓鼠卵巢(CHO)细胞和其他适于表达抗体的哺乳动物细胞。
本发明还提供一种制备本发明的抗IL-17A抗体或其抗原结合片段的方法，其包括在合适条件 下培养本发明的宿主细胞以表达本发明的抗IL-17A抗体或其抗原结合片段，以及从宿主细胞或其培养物分离所述抗体或其抗原结合片段。
抗体缀合物
本发明的抗IL-17A抗体或其抗原结合片段可以与至少一种可检测标记缀合。包含本发明的抗IL-17A抗体或其抗原结合片段与可检测标记的缀合物也涵盖在本发明的抗IL-17A抗体或其抗原结合片段的含义内。因此，在另一方面，本发明还提供了一种抗体缀合物，其包含与至少一种可检测标记缀合的本发明的抗IL-17A抗体或其抗原结合片段。
如本文所用，“缀合”是指两个或多个部分通过共价或非共价作用相互连接。优选地，本发明的抗IL-17A抗体或其抗原结合片段与可检测标记共价缀合。
可检测标记可以是任何用于检测的标记，包括但不限于放射性同位素(例如 212Bi、 213Bi、 131I、 125I、 111In、 177Lu、 186Re、 188Re、 153Sm和 90Y等)、生物素、胶体金、化学发光标记、生物发光标记和荧光基团(例如FITC、Alexa Fluor 488、Alexa Fluor 568、Alexa Fluor 555、Alexa Fluor 594、Alexa Fluor 647、Cy3、Texas Red、Cy5和Rhodamine等)。在一些实施方案中，所述可检测标记选自放射性同位素、生物素、胶体金、化学发光标记、生物发光标记和荧光基团。
可检测标记可以直接或间接(例如通过接头)与本发明的抗IL-17A抗体或其抗原结合片段缀合。接头可以包含用于共价缀合的活性基团，例如胺、羟胺、马来酰亚胺基、羧基、苯基、硫醇、巯基或羟基。在一实施方案中，接头为化学键。在一实施方案中，接头包含氨基酸或2-10个氨基酸组成的肽。
药物组合物
本发明还提供药物组合物，其包含本发明的抗IL-17A抗体或其抗原结合片段，以及药学上可接受的载剂。
药学上可接受的载剂可以包括但不限于：稀释剂、粘合剂和胶粘剂、润滑剂、崩解剂、防腐剂、媒介物、分散剂、助流剂、甜味剂、包衣、赋形剂、防腐剂、抗氧化剂(如抗坏血酸、盐酸半胱氨酸、硫酸氢钠、焦亚硫酸钠、亚硫酸钠、抗坏血酸棕榈酸酯、丁羟茴醚(BHA)、丁羟甲苯(BHT)、卵磷脂、没食子酸丙酯、α-生育酚、柠檬酸、乙二胺四乙酸(EDTA)、山梨糖醇、酒石酸、磷酸等)、增溶剂、胶凝剂、软化剂、溶剂(例如，水、酒精、乙酸和糖浆)、缓冲剂(例如，磷酸盐缓冲剂、组氨酸缓冲剂和乙酸盐缓冲剂)、表面活性剂(例如非离子表面活性剂，例如聚山梨酯80、聚山梨酯20、泊洛沙姆或聚乙二醇)、抗细菌剂、抗真菌剂、等渗剂(例如海藻糖、蔗糖、甘露醇、山梨醇、乳糖、葡萄糖)、吸收延迟剂、螯合剂和乳化剂。对于液体组合物，载剂可以选自以下中的一种或多种：无菌稀释剂，例如水、盐水溶液优选生理盐水、林格氏溶液(Ringer's solution)、等渗氯化钠，非发挥性油如合成的甘油单酯或甘油二酯、聚乙二醇、甘油或其他溶剂；抗菌剂，例如苄醇或对羟苯甲酸甲酯；以及用于调节张力的试剂如氯化钠或葡萄糖。
本文提供的药物组合物可以为多种剂型，包括但不限于固体、半固体、液体(例如溶液、乳液或悬浮液)、粉末、洗剂、喷剂、贴剂、膏药、软膏、凝胶、乳膏、喷雾、糊状物、生物粘合剂或冻干制剂(例如在复溶后使用)。还可以将本发明的药物组合物配制为微乳液、脂质体和胶束(参见例如CN107108730A和WO2016113557A1)。优选地，可以将药物组合物配制为适于需要的给药途径 的剂型，特别是局部、皮肤、肠胃外(例如静脉内、肌内、胸骨内、皮下，例如通过注射或输注)和鼻内给药。
在涉及通过皮肤给药的实施方案中，药物组合物可以包含有助于或提高皮肤穿透的皮肤穿透增强剂。可以使用本领域已知的皮肤穿透增强剂，包括但不限于例如CN 107108730 A描述的那些。还可以使用例如超声透入(phonophoresis)、超声促渗(sonophoresis)、电穿孔或使用微针技术促进给药。
药物组合物可以使用在药物领域中公知的方法制备。例如，可以通过将本发明的结合分子与水组合从而形成溶液来制备预期通过注射施用的组合物。可以加入表面活性剂以促进均匀溶液或悬浮液的形成。药物组合物可以采取一个或多个剂量单位的形式。可以将药物组合物制备于安瓿瓶、一次性注射器或由玻璃、塑料或其他材料制成的多剂量小药瓶中。
检测
本发明的抗IL-17A抗体或其抗原结合片段或者抗体缀合物可用于检测IL-17A的存在或其表达水平。例如，本发明提供的抗IL-17A抗体或其抗原结合片段或者抗体缀合物可以用于原位、体内、离体和体外检测或成像测定。
在一些实施方案中，通过使用本发明的抗体或抗原结合片段或者抗体缀合物检测样品中IL-17A的表达水平和/或追踪表达IL-17A的细胞。
在一方面，本发明提供了检测样品中IL-17A的存在或确定其表达水平的方法，其包括：
(a)使本发明的抗IL-17A抗体或其抗原结合片段(例如本发明的抗体缀合物)与样品接触；以及
(b)检测所述抗体或其抗原结合片段(例如本发明的抗体缀合物)与样品中IL-17A之间免疫复合物的形成或确定所述免疫复合物的量，从而检测样品中IL-17A的存在或确定样品中IL-17A的表达水平。
所述样品可以是任何形式，例如细胞、组织和体液。
以上检测可以利用本领域已知的技术来进行，这类技术包括但不限于蛋白质免疫印记、流式细胞术、放射免疫测定(RIA)、免疫组织化学测定(IHC)和酶联免疫测定(ELISA)。
治疗
本发明提供抗IL-17A抗体或其抗原结合片段在制备用于治疗IL-17A介导的疾病的药物中的用途。本发明还涉及一种治疗IL-17A介导的疾病(例如自身免疫性疾病)的方法，其包括向有需要的受试者给药本发明的抗IL-17A抗体或其抗原结合片段或者本发明的药物组合物。
在一些实施方案中，IL-17A介导的疾病可以包括例如自身免疫疾病、炎性病况、过敏和过敏病况、超敏反应、严重感染以及器官或组织移植排斥。
本发明的抗IL-17A抗体或其抗原结合片段可以与一种或多种抗炎性药物/免疫抑制剂组合施用用于治疗上述疾病。关于抗炎性/免疫抑制性药物的描述可以参见例如CN104884473A和WO2013177101A2。
本发明的抗IL-17A抗体或其抗原结合片段或者药物组合物可以与其他药物在相同时间或不同时间施用，例如同时、单独或依次。
在特定实施方案中，向有需要的受试者的皮肤局部给药本发明的抗IL-17A抗体或其抗原结合片段或者药物组合物。优选使用能够至少穿透皮肤的外层并且可以因此皮肤递送或经皮递送的药物 组合物。例如，可以将本发明的抗IL-17A或其抗原结合片段或者药物组合物以洗剂、喷雾、溶液、凝胶、软膏、糊状物、膏药、贴剂、生物粘合剂、悬浮液、脂质体、胶束、微球和微乳液(参见例如CN107108730A和WO2016113557A1)等的形式直接施用至患病的或健康的皮肤。
本发明的抗IL-17A抗体或其抗原结合片段或者药物组合物的给药剂量将会根据特定的配制物、应用方式、以及其特定的位置、宿主和被治疗的疾病而变化。应当考虑其他因素，比如年龄、体重、性别、病史、饮食、给药时间、清除率、反应灵敏度和疾病的严重程度。在组合物中采用的精确剂量还将会取决于给药途径和疾病或病症的严重性，并且应当根据从业人员的判断和每位患者的情况确定。可以在最大耐受剂量内连续地或周期地进行施用。
试剂盒
本发明还提供试剂盒，其包含本发明的抗IL-17A抗体或抗原结合片段、抗体缀合物、多特异性抗体或者药物组合物，以及使用说明。试剂盒还可以包含合适的容器。在某些实施方案中，试剂盒还包含给药的装置。通常，试剂盒还包括标签，其用于表明试剂盒内容物的预期用途和/或使用方法。术语“标签”包括在试剂盒上或与试剂盒一起提供的或以其他方式随试剂盒提供的任何书面的或记录的材料。
有益效果
本发明的抗IL-17A抗体或其抗原结合片段可以至少实现以下有益效果之一：
1)以高亲和力特异性结合IL-17A；
2)能够特异性中和IL-17A；以及
3)阻断IL-17A与IL-17RA的相互作用。
在其他方面，相比于常规抗体，本发明的抗IL-17A抗体或其抗原结合片段(特别是纳米抗体和重链抗体)具有分子量小、渗透性强等显著优点，使得其能够识别常规抗体不可及的隐蔽抗原表位，易于生产并且适合与其他抗体组装成多特异性和多价抗体。
实施例
以下实施例旨在仅对本发明进行举例说明，因此并不应被视为以任何方式限制本发明。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书进行。
实施例1原材料制备
1.1重组抗原IL-17A-Fc的制备
编码人IL-17A(NCBI Gene ID:3605，SEQ ID NO:1)的DNA片段由通用生物科技股份有限公司合成，并通过PCR扩增连接至编码人IgG1 Fc(SEQ ID NO:2)的DNA片段的5’端以获得编码重组抗原IL-17A-Fc的核酸。然后通过同源重组的方法将所述核酸克隆至真核表达载体pcDNA3.4-TOPO(Invitrogen)以获得重组抗原IL-17A-Fc的表达载体。将重组抗原IL-17A-Fc的表达载体转化到大肠杆菌DH5α中并提取质粒用于真核系统表达。
重组抗原IL-17A-Fc通过Expi293瞬转表达系统(ThermoFisher，A14635)表达。在转染7天后，将细胞表达上清于15000g高速离心10min，所得Fc标签蛋白表达上清用MabSelect SuRe LX(GE，17547403)进行亲和纯化，然后用100mM乙酸钠(pH 3.0)洗脱目的蛋白，接着用1M Tris-HCl中和； 洗脱下来的蛋白通过超滤浓缩管(Millipore，UFC901096)换液至PBS缓冲液中，于-80℃冻存待用。1.2阳性对照抗体的制备
本申请中使用的阳性对照抗体为抗IL-17A抗体苏金单抗，根据专利申请US20170355762A所披露的序列合成，通过分子克隆方法分别构建包含苏金单抗重链(SEQ ID NO:3)基因的质粒和苏金单抗轻链(SEQ ID NO:4)基因的质粒。其余步骤参考实施例1.1。
实施例2重组抗原IL-17A-His羊驼免疫与血清效价检测
2.1动物免疫
通过皮下注射的方式进行免疫，共免疫2只羊驼NSY007和NSY008(南昌大佳科技有限公司)，所使用的免疫抗原为重组抗原IL-17A-His(Novoprotein公司，货号C774)。单次免疫剂量为500μg，并辅以CFA/IFA(弗氏完全佐剂和弗式不完全佐剂)，每间隔2周免疫1次，共免疫4次。
2.2血清效价检测
在第2次、第3次和第4次免疫结束后，分别采血检测血清中靶向重组抗原IL-17A-His抗体的效价。具体检测方法如下：将重组抗原IL-17A-His用PBS稀释至终浓度2μg/mL，取30μL稀释液加入到ELISA板中，4℃包被过夜。在免疫效价测定当日用PBST润洗三遍，后用含有5％脱脂牛奶的PBST室温封闭两小时，再用PBST润洗三遍。在另外一块稀释板上将未经免疫接种的阴性血清和免疫后血清用PBS进行稀释，首孔2000倍稀释，然后后续7个孔采用2倍梯度稀释。将稀释好的血清加到第一块ELISA板中，室温下孵育1h，PBST洗板三次后，以1:7000加入二抗Anti IgG-HRP(Millipore，MAC129)，室温下孵育0.5h。孵育完成后，PBST洗板六次，加TMB(SurModics，TMBS-1000-01)显色，根据显色结果，加入2M HCl终止反应，通过酶标仪(Molecular Devices，SpecterMax 190)在OD450下读板。
结果如表1所示，4次免疫结束后，两只羊驼血清中靶向重组抗原IL-17-His的抗体的效价达到1:256K以上。
表1 IL-17A免疫羊驼血清IgG效价检测
Figure PCTCN2022116677-appb-000001
实施例3噬菌体展示文库构建和靶向IL-17A纳米抗体筛选
克隆如实施例2所述经重组抗原IL-17A-His免疫的羊驼外周血B细胞的抗体基因，构建了纳米抗体基因噬菌体展示文库，并用重组抗原IL-17A-Fc(如实施例1.1所述)作为筛选抗原对该文库进行筛选，获得了多个特异性结合IL-17A蛋白的纳米抗体。
3.1构建驼源纳米抗体的噬菌体展示文库
取Ficoll-Paque密度梯度分离液(GE公司，目录号：17144003S)分离外周单核细胞(Peripheral Blood Mononuclear Cell，PBMC)，从分离的PBMC细胞中提取总RNA并将其反转录成cDNA。基于VHH抗体胚系基因(germline)的情况设计简并引物，通过PCR扩增并用琼脂糖凝胶电泳回收PCR产物后获得编码VHH-CH2的DNA片段。然后通过二次PCR的方法，以编码VHH-CH2的DNA片段为模板扩增编码可变结构域(VHH)的DNA片段(Sabir JS et al.,C R Biol.(2014)337(4):244-249)。接着将编码VHH的DNA片段酶切纯化后构建至噬菌体展示用载体。最后，通过电转仪(Bio-Rad，MicroPulser)将表达VHH的载体转化至感受态大肠杆菌SS320(Lucigen，MC1061 F)中，并将经转化的大肠杆菌SS320菌液涂布于具有氨苄青霉素抗性的2-YT固体平板。通过梯度稀释铺板，经测定文库库容大小为10 9级。将纳米抗体基因库的菌液加入到新鲜的2-YT液体培养基中，置于37℃、220rpm的摇床中培养至对数生长期，再以50倍于细菌数的数量(即感染复数(MOI)大约50)加入VSCM13辅助噬菌体(购自Stratagene)，最终获得驼源纳米抗体的噬菌体展示文库。
3.2抗体基因噬菌体展示文库的筛选
3.2.1磁珠法筛选抗体基因噬菌体展示文库
磁珠法筛选是基于将重组抗原IL-17A-Fc进行生物素标记后，再与偶联有链霉亲和素的磁珠结合，通过将结合抗原的磁珠和抗体基因噬菌体展示文库进行孵育、洗涤和洗脱的淘选过程，由此针对抗原的特异性单克隆抗体可以大量富集。
具体方法如下：首先用生物素标记IL-17A-Fc与链霉亲和素偶联的磁珠孵育，使得生物素标记的IL-17A-Fc结合到磁珠上。将结合IL-17A-Fc的磁珠和构建的噬菌体库室温下孵育2h。经PBST洗涤6-8次后，去除非特异性吸附的噬菌体，加入Trypsin(Gibco，25200072)轻轻混匀并反应20min，以洗脱特异性结合的抗体展示噬菌体。随后，用洗脱下来的噬菌体侵染对数期的大肠杆菌SS320并静置30min，然后在220rpm条件下培养1小时；再加入VSCM13辅助噬菌体并静置30min，继续在220rpm条件下培养1h；离心并换液至C +/K +2-YT培养基中，最终得到的噬菌体继续用于下一轮的淘选。
3.2.2免疫管法筛选抗体基因噬菌体展示文库
免疫管法筛选的原理是将IL-17A蛋白包被在具有高吸附力的免疫管表面，通过将噬菌体展示抗体文库加入免疫管中并和吸附于免疫管表面的抗原蛋白进行孵育、洗涤和洗脱的淘选过程，最终将针对抗原的特异性单克隆抗体富集下来。
具体方法如下：第一轮筛选时，在免疫管中加入1mL 30μg/mL的IL-17A-Fc，4℃包被过夜；第二天弃去包被液，加入5％牛奶的PBS封闭2h；PBS润洗两次后加入含有抗IL-17A纳米抗体展示的噬菌体库，孵育2h；润洗以去除非特异性结合的噬菌体，然后向免疫管中加入0.8mL 0.05％EDTA胰酶消化液，用于洗脱特异性结合目标抗原的噬菌体；接着用洗脱下来的噬菌体侵染对数期的大肠杆菌SS320，37℃静置30min，然后220rpm条件下培养1h，再加入VSCM13辅助噬菌体，静置30min，继续在220rpm条件下培养1h；离心并换液至C +/K +2-YT培养基中，并于30℃、220rpm环境下继续培养过夜。第二天沉淀噬菌体，用于后续下一轮的筛选。
3.3单克隆的挑选
对每轮洗脱下来的磁珠法筛选获得的噬菌体池和免疫管法筛选获得的噬菌体池进行ELISA检测来评价富集的效果。针对富集较好的第三轮，挑取大量单克隆进行ELISA初筛(方法参考CN112745391A中的实施例3)。经测序分析和ELISA结合初筛获得134个序列多样性且和IL-17A抗原结合的纳米抗体，再经阻断初筛(方法参考CN112745391A中的实施例3)后选择30个纳米抗 体进行ELISA亲和与阻断复检，结果见图1A-1C和图2A-2C。最后根据亲和、阻断活性选取了12个单克隆。以克隆号对纳米抗体进行命名，采用AbM定义CDR的方式，确定了抗IL-17A纳米抗体的互补决定区序列，具体见表2。
表2抗IL-17A纳米抗体的可变区序列
Figure PCTCN2022116677-appb-000002
实施例4重链抗体的构建、表达和纯化
将实施例3中获得的5个纳米抗体构建为人IgG1亚型，形成VHH-Fc的重链抗体，具体氨基酸序列见表3。
4.1质粒构建
从筛选获得的含有候选单克隆菌株中，PCR扩增获取编码VHH的DNA片段。通过同源重组方法，将编码各个VHH的DNA片段分别构建至经过改造的含有人IgG1 Fc片段(SEQ ID NO:2)的真核表达载体质粒pcDNA3.4-TOPO(Invitrogen)上，获得包含完整的VHH-Fc(重链抗体)全长基因的重组质粒。将重组质粒转化到大肠杆菌DH5α中并提取质粒用于真核细胞系统表达。
4.2抗体的表达和纯化
重链抗体通过ExpiCHO瞬转表达系统(Thermo Fisher，A29133)表达，具体方法如下：转染当天，确认细胞密度为7×10 6至1×10 7个活细胞/mL左右，细胞存活率>98％，此时，用37℃预温的新鲜ExpiCHO表达培养基将细胞调整到终浓度为6×10 6个细胞/mL。用4℃预冷的OptiPRO TMSFM稀释目的质粒(向1mL所述培养基中加入1μg质粒)，同时，用OptiPRO TMSFM稀释ExpiFectamine TMCHO，再将两者等体积混合并轻轻吹打混匀制备成ExpiFectamine TMCHO/质粒DNA混合液，室温孵育1-5min，缓慢加入到准备好的细胞悬液中，并同时轻轻摇晃，最后置于细胞培养摇床中，在37℃、8％CO 2条件下培养。
在转染后18-22h，向培养液中添加ExpiCHO TMEnhancer和ExpiCHO TMFeed，摇瓶放置于32℃摇床和5％CO 2条件下继续培养。在转染后的第5天，添加相同体积的ExpiCHO TMFeed，缓慢加入的同时轻轻混匀细胞混悬液。在转染7-15天后，将表达有目的蛋白的细胞培养上清于15000g高速离心10min，所得上清用MabSelect SuRe LX(GE，17547403)进行亲和纯化，然后用100mM乙酸钠(pH 3.0)洗脱目的蛋白，接着用1M Tris-HCl中和，最后通过超滤浓缩管(Millipore，UFC901096)将所得蛋白换液至PBS缓冲液中。
表3抗IL-17重链抗体的氨基酸序列
Figure PCTCN2022116677-appb-000003
实施例5重链抗体的理化性质鉴定
5.1重链抗体SDS-PAGE鉴定
非还原(非变性)SDS-PAGE样品准备：将1μg的重链抗体或质控品IPI(即伊匹木单抗(Ipilimumab)，通过实施例4的方法制备获得)加入5×SDS上样缓冲液(含有终浓度为40mM的碘代乙酰胺)中，75℃干浴加热10min，冷却到室温后，12000rpm离心5min，取上清。还原(变性)SDS-PAGE样品准备：将2μg的重链抗体或质控品IPI加入5×SDS上样缓冲液(含有终浓度为5mM的DTT)中，100℃干浴加热10min，冷却到室温后，12000rpm离心5min，取上清。将上清加入Bis-tris 4-15％梯度胶(金斯瑞)进行凝胶电泳并通过考马斯亮蓝染色使蛋白条带显色。使用EPSON V550彩色扫描仪扫描带有显色蛋白条带的蛋白凝胶(脱色液脱色至凝胶背景透明)，通过ImageJ按照峰面积归一法计算还原和非还原条带纯度。
结果显示在图3A-3B和表4：重链抗体和质控品IPI非还原胶的条带的表观相对分子量分别在80kD和150kD左右，重链抗体还原胶的条带的表观相对分子量在40kD左右，质控品IPI分别是55kD左右和25kD左右。通过还原和非还原SDS-PAGE分析的重链抗体的分子量符合预期大小，并且纯度均大于90％。
5.2重链抗体的SEC-HPLC单体纯度鉴定
材料准备：1、流动相：150mmol/L磷酸缓冲液，pH 7.4；2、样品制备：重链抗体以及质控品IPI均用流动相溶液稀释到0.5mg/mL。Agilent HPLC 1100色谱柱(XBridge BEH SEC 3.5μm，7.8mm I.D.×30cm，Waters)流速设为0.8mL/min，进样体积20μL，VWD检测器波长为280nm和214nm。
重链抗体的SEC-HPLC分析结果如下：按照面积归一法计算样品中高分子聚集物，抗体单体和低分子聚集物百分比，结果显示在表4，所有重链抗体的单体纯度均大于96.0％。
表4重链抗体理化数据
Figure PCTCN2022116677-appb-000004
实施例6基于ELISA方法测定重链抗体的亲和与阻断活性
基于ELISA的方法验证了重链抗体对人IL-17A抗原的亲和活性，还基于ELISA的方法验证了重链抗体阻断IL-17A和IL-17RA的结合的效果。
6.1基于ELISA检测重链抗体对人IL-17A的亲和活性
用IL-17A-Fc(2μg/mL、30μL/孔)包被96孔板，4℃过夜；次日，将96孔板用PBST洗3次后用5％脱脂牛奶封闭2h；用PBST洗板3次后，加入梯度稀释的待测抗体(重链抗体VHH1、VHH2、VHH3、VHH8和VHH9；伊匹木单抗(IPI)作为阴性对照；苏金单抗作为阳性对照)或者仅PBS(空白对照)并孵育1h；之后，用PBST清洗3次后加入二抗：对于重链抗体，anti-VHH-HRP(金斯瑞生物科技，A01861)；对于苏金单抗和伊匹木单抗，anti-kappa-HRP(Millipore，A18853)，并孵育1h；孵育完成后，PBST洗板六次，加TMB(SurModics，TMBS-1000-01)显色；根据显色结果，加入2M HCl终止反应，通过酶标仪(Molecular Devices，SpecterMax 190)在OD450下读板。
结果显示在图4A-4C中，结果表明，所有重链抗体都对抗原人IL-17A具有高的亲和活性。因重链抗体与对照抗体使用二抗不同，EC 50不具有可比较性。
6.2基于ELISA检测重链抗体阻断IL-17A和IL-17RA的结合的效果
用4μg/mL、30μL/孔的人IL-17RA-His蛋白(Sino Biological，货号为10895-H08H)包被96孔板，4℃过夜；次日，将96孔板用PBST洗3次后用5％脱脂牛奶封闭2h；然后将分别梯度稀释的重链抗体或阳性对照抗体苏金单抗(PBS作为空白对照)与生物素标记的IL-17A-Fc(1.5μg/mL)提前预混0.5h，在封闭完成并洗板结束后加至96孔板中，孵育1h；之后，用PBST清洗3次后加入NeutrAvidin-HRP(Therofisher，31001)并孵育1h；孵育完成后，PBST洗板六次，加TMB(SurModics，TMBS-1000-01)显色；根据显色结果，加入2M HCl终止反应，通过酶标仪(Molecular Devices，SpecterMax 190)在OD450下读板。结果显示在图5A-5C中。结果表明，所有重链抗体都具有较好的阻断IL-17A与IL-17RA结合的效果：重链抗体VHH3、VHH4、VHH5、VHH6、VHH8、VHH10、VHH11、VHH12和VHH13的阻断效果与苏金单抗相当；重链抗体VHH1、VHH2和VHH9的阻断效果显著优于苏金单抗，其中，VHH1的IC 50＝7.183nM、VHH2的IC 50＝7.387nM、VHH9的IC 50＝5.766nM，苏金单抗的IC 50＝10.81nM。
实施例7通过生物膜层干涉技术测定重链抗体的亲和力
通过生物膜层干涉技术(使用Fortebio设备)检测重链抗体对抗原人IL-17A的结合亲和力。作为比较，还测定了对照抗体苏金单抗对抗原人IL-17A的结合亲和力。
作为抗原的IL-17A-His以10×KB缓冲液(含有1％BSA、0.5％Tween 20的10×PBS)稀释成10μg/mL，重链抗体以10×KB缓冲液进行2倍梯度稀释，依次从80nM稀释至1.25nM。避光条件下，采用10×KB缓冲液预湿传感器(Anti-Penta-HIS，HIS1K，Fortebio，CA)，至少10min后开始测试样品板(GreinierBio，PN655209)，测试无误后按预设程序进行。首先将抗原和传感器进行结合120s，结合完毕在10×KB缓冲液中继续平衡30s后，将结合有抗原的传感器转移至不同浓度抗体稀释液中结合120s，待信号稳定后，再转移到10×KB缓冲液中，解离时间为120s，最后通过不同浓度抗体的结合解离数据拟合得到K D(亲和力动力学常数)、K on(结合常数)和K off(解离常数)，K on可写成K a，K off可写成K d。
检测结果详见表5。结果显示，重链抗体与重组抗原IL-17A-His结合的K D在4.57×10 -10M-3.17×10 -11M之间，表明重链抗体对IL-17A均具有较高的亲和力。其中，重链抗体VHH1、VHH2、VHH3、VHH5、VHH6、VHH8、VHH9和VHH11都表现出优于苏金单抗的亲和力。
表5抗体亲和力测定数据表
抗体名称 K D(M) K a(1/Ms) K d(1/s)
Secukinumab 2.51E-10 5.31E+05 1.33E-04
VHH1 1.72E-10 8.62E+05 1.48E-04
VHH2 1.53E-10 9.49E+05 1.46E-04
VHH3 1.01E-10 7.42E+05 7.48E-05
VHH4 4.57E-10 8.36E+05 3.82E-04
VHH5 3.17E-11 1.56E+05 4.95E-06
VHH6 2.09E-10 1.49E+06 3.13E-04
VHH8 1.22E-11 2.81E+05 3.42E-06
VHH9 2.37E-11 2.63E+06 6.25E-05
VHH10 NA 1.37E+05 NA
VHH11 1.35E-10 4.66E+05 6.28E-05
VHH12 NA 9.36E+04 NA
VHH13 NA 3.41E+05 NA
备注：NA表示抗体结合后在120s内未解离下来，因此未计算出K d及K D值。
实施例8重链抗体中和活性测定
通过检测抗IL-17A重链抗体抑制IL-17A刺激HeLa细胞分泌IL-6来检测抗IL-17A重链抗体的细胞生物学活性。具体方法如下：
将HeLa细胞(中科院细胞库)复苏，传代2-4次后且生长状态良好的细胞用于实验。将1×10 5个细胞/mL的HeLa细胞以每孔100μL接种至新的96孔细胞培养板中，置于37℃细胞培养箱中过夜培养；第二天，使用DMEM培养基梯度稀释待测重链抗体和阳性对照抗体苏金单抗，加入100ng/mL IL-17A-His蛋白和30ng/mL TNF-α蛋白(Sino Biological，10602-H01H)，将抗体和蛋白混合物加入至96孔细胞板中，置于37℃细胞培养箱中培养24h。培养结束后，收获细胞培养上清，利用IL-6ELISA检测试剂盒(BD，555220)进行检测定量检测。
检测结果如图6A-6K所示，结果显示，添加重链抗体或苏金单抗的HeLa细胞中，IL-17A和TNF-α诱导的IL-6的分泌被显著抑制，表明重链抗体均具有较好的IL-17A中和活性：其中， VHH1、VHH2、VHH3、VHH4、VHH5、VHH9、VHH11、VHH12和VHH13的中和活性均优于苏金单抗；VHH6和VHH8的中和活性与苏金单抗相当。
实施例9纳米抗体人源化改造
为降低驼源纳米抗体可能引起的免疫原性，将纳米抗体的框架区进行人源化突变并进行回复突变以获得人源化程度较高的重链抗体同时保持人源化抗体对抗原的亲和力。
9.1纳米抗体人源化改造过程
选择克隆编号NB27A-15、2-NB27B-19和NB26B-12对应的重链抗体(对应的VHH氨基酸序列分别为重链抗体VHH1、VHH2和VHH9的VHH氨基酸序列)进行人源化改造，将抗体序列和人源抗体胚系基因数据库进行比对，找到和各VHH序列同源性比较高的1-3条胚系基因，同时兼顾胚系基因的成药性，选择合适的胚系基因模板进行比对，分析VHH框架区中非人源序列位点的数量。对VHH进行同源建模，同源建模参考PDB数据库的纳米抗体模型。结合VHH的模拟结构模型和非人源位点情况，进行组合回复突变设计(同时避免引入潜在翻译后修饰位点)，设计了不同程度人源化的序列。重链抗体VHH1、VHH2和VHH9的VHH经人源化后分别获得人源化抗体VHH1-huVH4、VHH2-huVH3和VHH9-huVH3的VHH，其氨基酸序列分别示于SEQ ID NO:65、66和67。
如实施例4.1所述构建人源化抗体VHH1-huVH4、VHH2-huVH3和VHH9-huVH3的表达载体。人源化抗体VHH1-huVH4、VHH2-huVH3和VHH9-huVH3分别包含如上所述的VHH氨基酸序列(SEQ ID NO:65、66和67)以及人IgG1的Fc片段(SEQ ID NO:2)。人源化抗体的表达与纯化参见实施例4.2。
9.2人源化抗体的亲和活性检测(ELISA)
通过ELISA测定人源化抗体VHH1-huVH4、VHH2-huVH3和VHH9-huVH3对抗原IL-17A的亲和力，并与各自的亲本抗体进行比较。
具体方法如下：用重组蛋白IL-17A-His(2μg/mL、30μL/孔)包被96孔板，4℃过夜。次日，将96孔板用PBST洗3次后用5％脱脂牛奶封闭2h；用PBST洗板3次后，加入梯度稀释的待测抗体(人源化抗体VHH1-huVH4、VHH2-huVH3和VHH9-huVH3；亲本重链抗体VHH1、VHH2和VHH9；苏金单抗作为阳性对照；伊匹木单抗(IPI)作为阴性对照)并孵育1h；之后，用PBST清洗3次后加入二抗anti-human Fc-HRP(Jackson Immuno Research，109-035-008)并孵育1h；孵育完成后，PBST洗板六次，加TMB(SurModics，TMBS-1000-01)显色；根据显色结果，加入2M HCl终止反应，通过酶标仪(Molecular Devices，SpecterMax 190)在OD450下读板。
结果显示在图7A-7B中，结果表明，人源化抗体VHH1-huVH4、VHH2-huVH3和VHH9-huVH3对抗原IL-17A的亲和力与各自的亲本重链抗体VHH1、VHH2和VHH9相当，并且都显著优于阳性对照抗体苏金单抗。
尽管本发明的具体实施方式已经得到详细的描述，但本领域技术人员将理解：根据已经公布的所有教导，可以对细节进行各种修改和变动，并且这些改变均在本发明的保护范围之内。本发明的保护范围由所附权利要求及其任何等同物给出。
序列表
Figure PCTCN2022116677-appb-000005
Figure PCTCN2022116677-appb-000006
Figure PCTCN2022116677-appb-000007
Figure PCTCN2022116677-appb-000008
Figure PCTCN2022116677-appb-000009
Figure PCTCN2022116677-appb-000010

Claims (17)

  1. 一种抗IL-17A抗体或其抗原结合片段，其包含免疫球蛋白的单可变结构域，所述单可变结构域包含选自以下的CDR1、CDR2和CDR3：
    (a)CDR1，其包含SEQ ID NO:68的氨基酸序列：
    Xaa1-Xaa2-Xaa3-Xaa4-S-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9    (SEQ ID NO:68)
    其中，
    Xaa1为G或A；Xaa2为F或S；Xaa3为T或I；Xaa4为F或I；Xaa5为S、D或I；Xaa6为Y、F或H；Xaa7为A或P；Xaa8为M或I；Xaa9为S、G或A；
    CDR2，其包含SEQ ID NO:69的氨基酸序列：
    Xaa10-Xaa11-Xaa12-Xaa13-Xaa14-Xaa15-Xaa16-S-T-Xaa17    (SEQ ID NO:69)
    其中，
    Xaa10为T或A；Xaa11为V或I；Xaa12为E、T或不存在；Xaa13为I、S或T；Xaa14为G、A、R或N；Xaa15为G、S或V；Xaa16为S或G；Xaa17为N或D；以及
    CDR3，其包含SEQ ID NO:70的氨基酸序列：
    D-Xaa18-Xaa19-Xaa20-Y-E-Xaa21-Xaa22-D-D-Y    (SEQ ID NO:70)
    其中，
    Xaa18为W、Y或G；Xaa19为K、T或R；Xaa20为W或Y；Xaa21为V、S或H；Xaa22为I或不存在；
    (b)CDR1，其包含SEQ ID NO:71的氨基酸序列：
    Xaa23-Xaa24-I-Xaa25-Xaa26-Xaa27-Xaa28-Xaa29-M-Xaa30    (SEQ ID NO:71)
    其中，
    Xaa23为A或G；Xaa24为S或F；Xaa25为F或I；Xaa26为N或S；Xaa27为A、I或E；Xaa28为H或Y；Xaa29为A或S；Xaa30为G或N；
    CDR2，其包含SEQ ID NO:72的氨基酸序列：
    Xaa31-I-T-Xaa32-G-G-Xaa33-T-Xaa34    (SEQ ID NO:72)
    其中，
    Xaa31为A、S、T或R；Xaa32为S、Y或R；Xaa33为S、N或T；Xaa34为D或N；以及
    CDR3，其包含SEQ ID NO:28、31、34或43的氨基酸序列；
    (c)CDR1，其包含SEQ ID NO:73的氨基酸序列：
    G-F-Xaa35-L-D-Xaa36-Xaa37-Xaa38-I-G    (SEQ ID NO:73)
    其中，
    Xaa35为T或N；Xaa36为D或Y；Xaa37为D或Y；Xaa38为A或G；
    CDR2，其包含SEQ ID NO:74的氨基酸序列：
    C-I-Xaa39-S-S-D-G-S-T-Y    (SEQ ID NO:74)
    其中，
    Xaa39为S或T；以及
    CDR3，其包含SEQ ID NO:46、49或52的氨基酸序列。
  2. 权利要求1的抗体或其抗原结合片段，其中所述单可变结构域包含：
    1)CDR1，其包含SEQ ID NO:20、17、38、23或35的氨基酸序列；
    2)CDR2，其包含SEQ ID NO:21、18、39、24或36的氨基酸序列；以及
    3)CDR3，其包含SEQ ID NO:22、19、40、25或37的氨基酸序列；
    或者
    1)CDR1，其包含SEQ ID NO:26、29、32或41的氨基酸序列；
    2)CDR2，其包含SEQ ID NO:27、30、33或42的氨基酸序列；以及
    3)CDR3，其包含SEQ ID NO:28、31、34或43的氨基酸序列；
    或者
    1)CDR1，其包含SEQ ID NO:44、47或50的氨基酸序列；
    2)CDR2，其包含SEQ ID NO:45、48或51的氨基酸序列；以及
    3)CDR3，其包含SEQ ID NO:46、49或52的氨基酸序列。
  3. 权利要求1或2的抗体或其抗原结合片段，其中所述单可变结构域包含选自以下的CDR1、CDR2和CDR3：
    (a-1)CDR1，其包含SEQ ID NO:20的氨基酸序列；CDR2，其包含SEQ ID NO:21的氨基酸序列；以及CDR3，其包含SEQ ID NO:22的氨基酸序列；
    (a-2)CDR1，其包含SEQ ID NO:17的氨基酸序列；CDR2，其包含SEQ ID NO:18的氨基酸序列；以及CDR3，其包含SEQ ID NO:19的氨基酸序列；
    (a-3)CDR1，其包含SEQ ID NO:38的氨基酸序列；CDR2，其包含SEQ ID NO:39的氨基酸序列；以及CDR3，其包含SEQ ID NO:40的氨基酸序列；
    (a-4)CDR1，其包含SEQ ID NO:23的氨基酸序列；CDR2，其包含SEQ ID NO:24的氨基酸序列；以及CDR3，其包含SEQ ID NO:25的氨基酸序列；
    (a-5)CDR1，其包含SEQ ID NO:35的氨基酸序列；CDR2，其包含SEQ ID NO:36的氨基酸序列；以及CDR3，其包含SEQ ID NO:37的氨基酸序列；
    (b-1)CDR1，其包含SEQ ID NO:26的氨基酸序列；CDR2，其包含SEQ ID NO:27的氨基酸序列；以及CDR3，其包含SEQ ID NO:28的氨基酸序列；
    (b-2)CDR1，其包含SEQ ID NO:29的氨基酸序列；CDR2，其包含SEQ ID NO:30的氨基酸序列；以及CDR3，其包含SEQ ID NO:31的氨基酸序列；
    (b-3)CDR1，其包含SEQ ID NO:32的氨基酸序列；CDR2，其包含SEQ ID NO:33的氨基酸序列；以及CDR3，其包含SEQ ID NO:34的氨基酸序列；
    (b-4)CDR1，其包含SEQ ID NO:41的氨基酸序列；CDR2，其包含SEQ ID NO:42的氨基酸序列；以及CDR3，其包含SEQ ID NO:43的氨基酸序列；
    (c-1)CDR1，其包含SEQ ID NO:44的氨基酸序列；CDR2，其包含SEQ ID NO:45的氨基酸序列；以及CDR3，其包含SEQ ID NO:46的氨基酸序列；
    (c-2)CDR1，其包含SEQ ID NO:47的氨基酸序列；CDR2，其包含SEQ ID NO:48的氨基酸序列；以及CDR3，其包含SEQ ID NO:49的氨基酸序列；
    (c-3)CDR1，其包含SEQ ID NO:50的氨基酸序列；CDR2，其包含SEQ ID NO:51的氨基酸序列；以及CDR3，其包含SEQ ID NO:52的氨基酸序列。
  4. 权利要求1-3中任一项的抗体或其抗原结合片段，其中所述单可变结构域包含选自以下的氨基酸序列或其变体：SEQ ID NO:6、SEQ ID NO:5、SEQ ID NO:12、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16；
    其中，所述变体与其所源自的序列相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列相同性。
  5. 权利要求1-3中任一项的抗体或其抗原结合片段，其中所述单可变结构域包含选自以下的氨基酸序列或其变体：SEQ ID NO:66、SEQ ID NO:65和SEQ ID NO:67；
    其中，所述变体与其所源自的序列相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列相同性。
  6. 权利要求1-5中任一项的抗体或其抗原结合片段，其为单域抗体、重链抗体、人源化抗体或嵌合抗体。
  7. 权利要求1-6中任一项的抗体或其抗原结合片段，其进一步包含免疫球蛋白的Fc片段；优选地，所述Fc片段为人IgG1的Fc片段；更优选地，所述Fc片段包含SEQ ID NO:2的氨基酸序列。
  8. 权利要求1-7中任一项的抗体或其抗原结合片段，其能够
    1)特异性结合IL-17A；
    2)中和IL-17A；
    3)特异性结合IL-17A同源二聚体或者IL-17A/F异源二聚体；和/或
    4)阻断IL-17A或IL-17A/F与IL-17RA的相互作用。
  9. 权利要求1-8中任一项的抗体或其抗原结合片段，其与至少一种可检测标记缀合；优选地，所述可检测标记选自放射性同位素、生物素、胶体金、化学发光标记、生物发光标记和荧光基团。
  10. 一种多特异性抗体，其包含结合IL-17A的第一抗原结合部分以及结合第二抗原的第二抗原结合部分，其中所述第一抗原结合部分包含权利要求1-9中任一项的抗体或其抗原结合片段。
  11. 一种多核苷酸，其编码权利要求1-8中任一项的抗体或其抗原结合片段。
  12. 一种表达载体，其包含权利要求11的多核苷酸。
  13. 一种宿主细胞，其包含权利要求11的多核苷酸或权利要求12的表达载体。
  14. 一种制备权利要求1-8中任一项的抗IL-17A抗体或其抗原结合片段的方法，其包括在合适条件下培养权利要求13的宿主细胞以表达所述抗体或其抗原结合片段，以及从宿主细胞或其培养物分离所述抗体或其抗原结合片段。
  15. 一种药物组合物，其包含权利要求1-9中任一项的抗体或其抗原结合片段，以及药学上 可接受的载剂。
  16. 权利要求1-9中任一项的抗体或其抗原结合片段或者权利要求15的药物组合物在制备用于治疗IL-17A介导的疾病的药物中的用途。
  17. 一种检测样品中IL-17A的存在或确定其表达水平的方法，其包括：
    (a)使权利要求1-9中任一项的抗体或其抗原结合片段与所述样品接触，以及
    (b)检测所述抗体或其抗原结合片段与样品中IL-17A之间免疫复合物的形成或确定所述免疫复合物的量，从而检测样品中IL-17A的存在或确定样品中IL-17A的表达水平。
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