WO2021160116A1 - 抗FcRn抗体、其抗原结合片段及其医药用途 - Google Patents

抗FcRn抗体、其抗原结合片段及其医药用途 Download PDF

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WO2021160116A1
WO2021160116A1 PCT/CN2021/076211 CN2021076211W WO2021160116A1 WO 2021160116 A1 WO2021160116 A1 WO 2021160116A1 CN 2021076211 W CN2021076211 W CN 2021076211W WO 2021160116 A1 WO2021160116 A1 WO 2021160116A1
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seq
amino acid
acid sequence
antibody
fcrn
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PCT/CN2021/076211
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French (fr)
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刘潇
王雷
杜延平
申晨曦
戴宸
吴然
崔妍
胡冬梅
杨阳
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北京拓界生物医药科技有限公司
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Priority to CN202180004919.6A priority Critical patent/CN114341184B/zh
Publication of WO2021160116A1 publication Critical patent/WO2021160116A1/zh

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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
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A61P21/04—Drugs for disorders of the muscular or neuromuscular system for myasthenia gravis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/08—Antiepileptics; Anticonvulsants
    • 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
    • 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
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants

Definitions

  • This application relates to anti-FcRn antibodies, antigen-binding fragments thereof, chimeric antibodies and humanized antibodies comprising the CDR regions of the anti-FcRn antibodies, pharmaceutical compositions comprising the anti-FcRn antibodies and antigen-binding fragments thereof, and use thereof Medical use for treating or preventing autoimmune diseases and inflammatory diseases.
  • the neonatal Fc receptor is a non-covalent heterodimer, usually present in the endosomes of endothelial cells and epithelial cells. It is a class I major histocompatibility complex (MHC)-like heavy chain and soluble ⁇ 2 micro
  • MHC major histocompatibility complex
  • ⁇ 2M globulin
  • FcRn has a molecular weight of about 46kD, is composed of three heavy chain ⁇ domains ( ⁇ 1, ⁇ 2, and ⁇ 3) and a single ⁇ 2M domain. It has the characteristics of a single sugar chain, a single transmembrane, and a relatively short cytoplasmic tail. .
  • FcRn was initially recognized as playing an important role in the life of newborns. Further studies have found that FcRn can also prolong the half-life of immunoglobulin G (IgG) and albumin in adults (Ghetie et al., 1996; Junghans and Anderson, 1996; Israel et al., 1996). Under normal conditions, the half-life of most human IgG (excluding the IgG3 isotype) in the serum is about 22-23 days, which is relatively long compared to the serum half-life of other plasma proteins.
  • IgG immunoglobulin G
  • albumin albumin
  • the IgG that enters the cell through the endocytosis process can strongly bind to the FcRn in the endosome at a slightly acidic pH (about 6.0) to avoid the degrading lysosome pathway.
  • a slightly acidic pH about 6.0
  • IgG-FcRn complex circulates to the plasma membrane
  • IgG rapidly dissociates from FcRn at a slightly alkaline pH (approximately 7.4) in the bloodstream.
  • FcRn effectively rescues IgG from degradation in the lysosome, thereby prolonging the half-life of IgG (Roopenian et al., J. Immunol. 170:3528, 2003).
  • mice are engineered to knock out at least part of the genes encoding the heavy chains of ⁇ 2M and FcRn so that they do not express the protein.
  • the serum half-life and concentration of IgG were greatly reduced, which also suggested the FcRn-dependent mechanism of IgG homeostasis.
  • the production of anti-human FcRn antibodies in these FcRn knockout mice can prevent the binding of IgG to FcRn.
  • IgG to FcRn Inhibition of the binding of IgG to FcRn prevents the regeneration of IgG, which can treat autoimmune diseases caused by autoantibodies. This possibility is shown in a mouse model of autoimmune bullous skin disease (Li et al., J. Clin. Invest. 115:3440, 2005). Therefore, drugs that block or antagonize the binding of IgG to FcRn can or are promising for the treatment or prevention of IgG-mediated autoimmune diseases and inflammatory diseases.
  • autoimmune diseases attack their normal tissues, organs or other body components due to their immune system abnormalities. Almost all parts of the body, such as the nervous system, digestive system, endocrine system, skin, skeletal system, and blood vessel tissue, may have autoimmune diseases, and most of them have not yet found a specific cause. Studies have revealed that there are over-expressed IgG autoantibodies in the blood of patients with many autoimmune diseases.
  • IgG type autoantibodies including immune neutropenia, myasthenia gravis (MG), multiple sclerosis, rheumatoid arthritis, lupus, idiopathic thrombocytopenic purpura (ITP), Pemphigus vulgaris, Guillain-Barre syndrome, autoimmune encephalitis, epilepsy, lupus nephritis or membranous nephropathy or others.
  • IVIG immunoglobulin
  • IVIG Intravenous infusion of immunoglobulin
  • IVIG can be applied to some of the above diseases, but it is accompanied by medical complications such as headache, dermatitis, allergic reaction, acute renal failure, etc., is expensive and has the risk of exposure to blood products.
  • polyclonal anti-D immunoglobulins corticosteroids, immunosuppressants (including chemotherapeutics), cytokines, plasma separation, in vitro antibody adsorption (for example, using Prosorba columns), surgical interventions such as splenectomy, etc. are also used to treat autoimmune diseases, similarly, these therapies are complex, incomplete, and expensive.
  • anti-FcRn antibodies are disclosed in WO2016/142782, WO2018/083122, WO2014/019727, WO2018/229249, CN106459215B, CN104364265B, WO2009/131702, US7662928B, WO2006/118772, WO2012/167039, etc., there is still a need to develop high resistance to FcRn.
  • Anti-human FcRn antibody with affinity, low immunogenicity, and even low-dose administration.
  • the present disclosure provides an FcRn binding protein, an anti-FcRn antibody or an antigen-binding fragment thereof, which encodes a nucleic acid, a vector, a host cell, a pharmaceutical composition, a method for treating or delaying autoimmune diseases, and a detection application thereof.
  • anti-FcRn antibodies or antigen-binding fragments which comprise a heavy chain variable region (VH) and a light chain variable region (VL), wherein:
  • the HCDR1, HCDR2 and HCDR3 of the VH respectively contain HCDR1, HCDR2 and HCDR3 in SEQ ID No: 4, and the LCDR1, LCDR2 and LCDR3 of the VL respectively contain LCDR1, LCDR2 and LCDR3 in SEQ ID No: 5;
  • the HCDR1, HCDR2 and HCDR3 of the VH respectively contain HCDR1, HCDR2 and HCDR3 in SEQ ID No: 6, and the LCDR1, LCDR2 and LCDR3 of the VL respectively contain LCDR1, LCDR2 and LCDR3 in SEQ ID No: 7;
  • the HCDR1, HCDR2, and HCDR3 of the VH respectively contain HCDR1, HCDR2, and HCDR3 in SEQ ID No: 8
  • the LCDR1, LCDR2, and LCDR3 of the VL respectively contain LCDR1, LCDR2, and LCDR3 in SEQ ID No: 9;
  • the HCDR1, HCDR2 and HCDR3 of the VH respectively contain HCDR1, HCDR2 and HCDR3 in SEQ ID No: 10
  • the LCDR1, LCDR2 and LCDR3 of the VL respectively contain LCDR1, LCDR2 and LCDR3 in SEQ ID No: 11;
  • the HCDR1, HCDR2 and HCDR3 of the VH respectively contain HCDR1, HCDR2 and HCDR3 in SEQ ID No: 12, and the LCDR1, LCDR2 and LCDR3 of the VL respectively contain LCDR1, LCDR2 and LCDR3 in SEQ ID No: 13;
  • the HCDR1, HCDR2 and HCDR3 of the VH respectively contain HCDR1, HCDR2 and HCDR3 in SEQ ID No: 14, and the LCDR1, LCDR2 and LCDR3 of the VL respectively contain LCDR1, LCDR2 and LCDR3 in SEQ ID No: 15;
  • the HCDR1, HCDR2 and HCDR3 of the VH respectively contain HCDR1, HCDR2 and HCDR3 in SEQ ID No: 16, and the LCDR1, LCDR2 and LCDR3 of the VL respectively contain LCDR1, LCDR2 and LCDR3 in SEQ ID No: 17;
  • the HCDR1, HCDR2 and HCDR3 of the VH respectively contain HCDR1, HCDR2 and HCDR3 in SEQ ID No: 18, and the LCDR1, LCDR2 and LCDR3 of the VL respectively contain LCDR1, LCDR2 and LCDR3 in SEQ ID No: 19;
  • the HCDR1, HCDR2, and HCDR3 of the VH contain SEQ ID Nos: 96, 99, 103, 104, and 107, respectively, HCDR1, HCDR2, and HCDR3.
  • the LCDR1, LCDR2 and LCDR3 of the VL contain SEQ ID No: 13 LCDR1, LCDR2, LCDR3 in the middle;
  • the HCDR1, HCDR2, and HCDR3 of the VH respectively contain HCDR1, HCDR2, and HCDR3 in any of SEQ ID Nos: 89, 93, and 94
  • the LCDR1, LCDR2, and LCDR3 of the VL respectively contain LCDR1 in SEQ ID No: 76. LCDR2, LCDR3;
  • the HCDR1, HCDR2 and HCDR3 of the VH respectively contain HCDR1, HCDR2 and HCDR3 in SEQ ID No: 87
  • the LCDR1, LCDR2 and LCDR3 of the VL respectively contain LCDR1, LCDR2 and LCDR3 in SEQ ID No: 84;
  • the HCDR1, HCDR2, and HCDR3 of the VH respectively contain HCDR1, HCDR2, and HCDR3 in any of SEQ ID Nos: 87-110, and the LCDR1, LCDR2, and LCDR3 of the VL respectively contain any of SEQ ID No: 76-86.
  • CDRs are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering system; in some specific embodiments, the CDR is defined according to the Kabat numbering system; in other embodiments, the CDR is defined according to the AbM numbering system.
  • the present disclosure provides an anti-FcRn antibody or antigen-binding fragment thereof, comprising a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein:
  • the heavy chain variable region comprises a complementarity determining region (CDR) selected from:
  • HCDR1 The amino acid sequence of HCDR1 is shown in GY X 1 F X 2 (SEQ ID No: 182) or GYX 1 F X 2 X 3 X 4 X 5 IA (SEQ ID No: 173), wherein X 1 is selected from S, N, D , V, A, P, K, X 2 is selected from T, N, K, R, S, A, X 3 is selected from G, N, K, S, E, X 4 is selected from Y, H, S, N , T, X 5 are selected from W, Y, F, M; and/or
  • HCDR2 The amino acid sequence of HCDR2 is shown in X 6 IX 7 PDX 8 SNTI (SEQ ID No: 174) or X 6 IX 7 PDX 8 SNTIYSPSFRG (SEQ ID No: 175), wherein X 6 is selected from I, L, V, X 7 is selected from Y, S, T, G, A, X 8 is selected from N, R, K, A, F; and/or
  • amino acid sequence of HCDR3 is shown in FGGPTFAQWYFDY (SEQ ID No: 40);
  • the light chain variable region comprises a CDR selected from:
  • LCDR1 The amino acid sequence of LCDR1 is shown in TGSSGSIASNYVX 9 (SEQ ID No: 176), wherein X 9 is selected from Q, S, N, A, V, T; and/or
  • X 10 DNQRAS SEQ ID No: 177
  • X 10 is selected from E, S, A, W, C; and/or
  • the amino acid sequence of LCDR3 is shown in QSYDSSSHNWV (SEQ ID No: 43).
  • the amino acid sequence of HCDR1 of the anti-FcRn antibody or its antigen-binding fragment is shown in SEQ ID No: 182, the amino acid sequence of HCDR2 is shown in SEQ ID No: 175, and the amino acid sequence of HCDR3 is shown in SEQ ID No: 40; the amino acid sequence of LCDR1 is shown in SEQ ID No: 176, the amino acid sequence of LCDR2 is shown in SEQ ID No: 177, and the amino acid sequence of LCDR3 is shown in SEQ ID No: 43.
  • the described embodiment uses the Kabat numbering system.
  • the amino acid sequence of HCDR1 of the anti-FcRn antibody or its antigen-binding fragment is shown in SEQ ID No: 173, the amino acid sequence of HCDR2 is shown in SEQ ID No: 174, and the amino acid sequence of HCDR3 is shown in SEQ ID No.
  • the amino acid sequence of LCDR1 is shown in SEQ ID No: 176, the amino acid sequence of LCDR2 is shown in SEQ ID No: 177, and the amino acid sequence of LCDR3 is shown in SEQ ID No: 43.
  • the described embodiment adopts the AbM numbering system.
  • X 1 is S
  • X 2 is T
  • X 3 is G
  • X 4 is Y
  • X 5 is W.
  • X 1 is D
  • X 2 is K
  • X 3 is K
  • X 4 is S
  • X 5 is F
  • X 1 is N
  • X 2 is N
  • X 3 is K
  • X 4 is H
  • X 5 is Y.
  • X 1 is S
  • X 2 is K
  • X 3 is N
  • X 4 is Y
  • X 5 is Y.
  • X 1 is V
  • X 2 is N
  • X 3 is K
  • X 4 is Y
  • X 5 is F
  • X 1 is D
  • X 2 is T
  • X 3 is K
  • X 4 is T
  • X 5 is M.
  • X 6 is I
  • X 7 is Y
  • X 8 is N
  • X 9 is Q
  • X 10 is E.
  • X 6 is L
  • X 7 is S
  • X 8 is A
  • X 9 is S
  • X 10 is S.
  • X 6 is L
  • X 7 is A
  • X 8 is R
  • X 9 is S
  • X 10 is S.
  • X 6 is V
  • X 7 is S
  • X 8 is R
  • X 9 is S
  • X 10 is S.
  • X 6 is L
  • X 7 is T
  • X 8 is R
  • X 9 is A
  • X 10 is S.
  • X 6 is L
  • X 7 is S
  • X 8 is R
  • X 9 is Q
  • X 10 is W.
  • amino acid sequence of HCDR1 of the VH of the anti-FcRn antibody or antigen-binding fragment thereof is as SEQ ID No: 20, 26, 32, 38, 44, 50, 56, 62, 68, 129-141, 150-165.
  • amino acid sequence of HCDR2 is shown in SEQ ID No: 21, 27, 33, 39, 45, 51, 57, 63, 69, 121-128, 142-149, 170;
  • amino acid sequence of HCDR3 is shown in SEQ ID No: 22, 28, 34, 40, 46, 52, 58, and 64;
  • LCDR1 of VL The amino acid sequence of LCDR1 of VL is shown in SEQ ID No: 23, 29, 35, 41, 47, 53, 59, 65, 111, 115-119;
  • LCDR2 The amino acid sequence of LCDR2 is shown in SEQ ID No: 24, 30, 36, 42, 48, 54, 60, 66, 112, 113, 114, 116, 120;
  • LCDR3 The amino acid sequence of LCDR3 is shown in SEQ ID No: 25, 31, 37, 43, 49, 55, 61, 67.
  • the amino acid sequence of HCDR1, HCDR2, and HCDR3 of the anti-FcRn antibody or antigen-binding fragment thereof is selected from any one of (1) to (15):
  • HCDR1 shown in SEQ ID No: 68 HCDR2 shown in one of SEQ ID No: 142-149, and HCDR3 shown in SEQ ID No: 40;
  • amino acid sequence of LCDR1, LCDR2, LCDR3 is selected from any one of (1')-(19'):
  • the anti-FcRn antibody or antigen-binding fragment thereof comprises a CDR selected from any one of the following:
  • the amino acid sequence of the VH of the anti-FcRn antibody or its antigen-binding fragment is as SEQ ID No: 4, 6, 8, 10, 12, 14, 16, 18, 76-86, 166, 168, 169. Shown or have at least 80%, 90%, 95%, 96%, 97%, 98%, 99% identity therewith; and/or
  • VL amino acid sequence of VL is shown in SEQ ID No: 5, 7, 9, 11, 13, 15, 17, 19, 76-86, 167 or at least 80%, 90%, 95%, 96% , 97%, 98%, 99% identity.
  • the amino acid sequence of the VHVH of the anti-FcRn antibody or antigen-binding fragment thereof is shown in SEQ ID No: 4 or has at least 80%, 90%, 95%, 96%, 97%, 98%, 99% identity
  • the amino acid sequence of VL is shown in SEQ ID No: 5 or has at least 80%, 90%, 95%, 96%, 97%, 98%, 99% identity with it; or
  • amino acid sequence of VH is shown in SEQ ID No: 6 or has at least 80%, 90%, 95%, 96%, 97%, 98%, 99% identity with it
  • amino acid sequence of VL is shown in SEQ ID No: 7 Shown or have at least 80%, 90%, 95%, 96%, 97%, 98%, 99% identity with; or
  • amino acid sequence of VH is shown in SEQ ID No: 8 or has at least 80%, 90%, 95%, 96%, 97%, 98%, 99% identity with it
  • amino acid sequence of VL is shown in SEQ ID No: 9 Shown or have at least 80%, 90%, 95%, 96%, 97%, 98%, 99% identity with; or
  • amino acid sequence of VH is shown in SEQ ID No: 10 or has at least 80%, 90%, 95%, 96%, 97%, 98%, 99% identity with it
  • amino acid sequence of VL is shown in SEQ ID No: 11 Shown or have at least 80%, 90%, 95%, 96%, 97%, 98%, 99% identity with; or
  • amino acid sequence of VH is shown in SEQ ID No: 12 or has at least 80%, 90%, 95%, 96%, 97%, 98%, 99% identity with it
  • amino acid sequence of VL is shown in SEQ ID No: 13 Shown or have at least 80%, 90%, 95%, 96%, 97%, 98%, 99% identity with; or
  • amino acid sequence of VH is shown in SEQ ID No: 14 or has at least 80%, 90%, 95%, 96%, 97%, 98%, 99% identity with it, and the amino acid sequence of VL is shown in SEQ ID No: 15 Shown or have at least 90% identity with it; or
  • amino acid sequence of VH is shown in SEQ ID No: 16 or has at least 80%, 90%, 95%, 96%, 97%, 98%, 99% identity with it, and the amino acid sequence of VL is shown in SEQ ID No: 17 Shown or have at least 90% identity with it; or
  • amino acid sequence of VH is shown in SEQ ID No: 18 or has at least 80%, 90%, 95%, 96%, 97%, 98%, 99% identity with it
  • amino acid sequence of VL is shown in SEQ ID No: 19 Shown or have at least 80%, 90%, 95%, 96%, 97%, 98%, 99% identity with; or
  • the amino acid sequence of VH is shown in one of SEQ ID No: 96, 99, 103, 104, 107 or has at least 80%, 90%, 95%, 96%, 97%, 98%, 99% identity with it
  • the amino acid sequence of VL is shown in SEQ ID No: 13 or has at least 80%, 90%, 95%, 96%, 97%, 98%, 99% identity with it;
  • the amino acid sequence of VH is shown in one of SEQ ID No: 89, 93, 94 or has at least 80%, 90%, 95%, 96%, 97%, 98%, 99% identity with the amino acid sequence of VL As shown in SEQ ID No: 76 or have at least 80%, 90%, 95%, 96%, 97%, 98%, 99% identity with it;
  • amino acid sequence of VH is shown in SEQ ID No: 87 or has at least 80%, 90%, 95%, 96%, 97%, 98%, 99% identity with it
  • amino acid sequence of VL is shown in SEQ ID No: 84 Shown or have at least 80%, 90%, 95%, 96%, 97%, 98%, 99% identity with;
  • amino acid sequence of VH is shown in one of SEQ ID No: 87-110 or has at least 80%, 90%, 95%, 96%, 97%, 98%, 99% identity with it
  • amino acid sequence of VL is shown in SEQ ID No: One of 76-86 shows or has at least 80%, 90%, 95%, 96%, 97%, 98%, 99% identity with it.
  • the aforementioned anti-FcRn antibody or antigen-binding fragment thereof wherein:
  • the heavy chain amino acid sequence is shown in any one of SEQ ID No: 178-180, or has at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% identity with it;
  • the light chain amino acid sequence is shown in SEQ ID No: 181, or has at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% identity with it.
  • the aforementioned anti-FcRn antibody or antigen-binding fragment thereof is a murine antibody, a chimeric antibody, a fully human antibody, or a humanized antibody. It can be a full-length antibody or a fragment thereof.
  • the aforementioned anti-FcRn antibody or antigen-binding fragment thereof is a murine antibody or fragment thereof.
  • the light chain variable region includes the light chain FR region and/or the light chain constant region of the murine ⁇ , ⁇ chain or variants thereof.
  • the murine anti-FcRn antibody or antigen-binding fragment thereof comprises a heavy chain FR region and/or a heavy chain constant region of murine IgG1, IgG2, IgG3, IgG4 or a variant thereof.
  • the aforementioned anti-FcRn antibody or antigen-binding fragment thereof is a chimeric antibody or fragment thereof. It comprises the light chain FR region and/or light chain constant region of a human ⁇ , ⁇ chain or a variant thereof, and/or a heavy chain FR region and/or a heavy chain of a human IgG1, IgG2, IgG3 or IgG4 or a variant thereof Chain constant region.
  • an anti-human FcRn antibody or an antigen-binding fragment thereof as described above which is a humanized antibody or a fragment thereof, a fully human antibody or a fragment thereof.
  • the heavy chain variable region of the anti-FcRn antibody or antigen-binding fragment thereof can be connected to human CH1 or mouse CH1.
  • the anti-FcRn antibody or antigen-binding fragment thereof comprises a constant region Fc, and the Fc is IgG1, IgG2, IgG3, IgG4 or variants thereof (such as IgG4P, that is, the S241P mutant of IgG4).
  • Fc is the Fc of mouse IgG1 shown in the amino acid sequence of SEQ ID No: 75, or is the Fc of human IgG4P shown in the amino acid sequence of SEQ ID No: 172.
  • the antigen-binding fragment of the anti-FcRn antibody is Fab, Fv, sFv, Fab', F(ab') 2 , linear antibody, single-chain antibody, scFv, sdAb, sdFv, nanobody, peptide antibody peptibody, structure Domain antibodies and multispecific antibodies (bispecific antibodies, diabody, triabody and tetrabody, tandem two-scFv, tandem three-scFv), for example specifically scFv, Fv, Fab or Fab' fragments.
  • the anti-FcRn antibody or antigen-binding fragment thereof is conjugated to a polymer.
  • the polymer is selected from starch, albumin, and polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • the polymer may be PEG having a molecular weight of 5-50kDa, 5-10kDa, 5-15kDa, 10-20kDa, 10-30kDa, 20-30kDa, 10-40kDa.
  • an anti-FcRn antibody or antigen-binding fragment thereof which binds to the same epitope as the aforementioned anti-FcRn antibody or antigen-binding fragment thereof.
  • an anti-FcRn antibody or antigen-binding fragment thereof which blocks the binding of the aforementioned anti-FcRn antibody or antigen-binding fragment thereof to human FcRn.
  • an anti-FcRn antibody or antigen-binding fragment thereof whose binding to human FcRn is blocked by the aforementioned anti-FcRn antibody or antigen-binding fragment thereof.
  • the aforementioned anti-FcRn antibody or antigen-binding fragment thereof binds to human FcRn (or its epitope).
  • the aforementioned anti-FcRn antibody or antigen-binding fragment thereof blocks the binding of human IgG to human FcRn.
  • the aforementioned anti-FcRn antibody or antigen-binding fragment thereof does not bind ⁇ 2 microglobulin.
  • an anti-FcRn antibody or antigen-binding fragment thereof is provided, and the heavy chain and/or light chain of the aforementioned anti-FcRn antibody or antigen-binding fragment thereof has at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity.
  • an anti-FcRn antibody or antigen-binding fragment variant thereof is provided, and the heavy chain variable region and/or light chain variable region of the aforementioned anti-FcRn antibody or antigen-binding fragment thereof comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid changes.
  • the amino acid changes may be conservative substitutions of amino acid residues in the variable region.
  • the present disclosure also provides an FcRn binding protein, comprising the anti-FcRn antibody or antigen-binding fragment thereof provided in the present disclosure.
  • the FcRn binding protein further comprises one or more effector molecules, including but not limited to: anti-tumor agents, drugs, toxins, biologically active proteins (such as enzymes), other antibodies or antibody fragments, synthetic or naturally occurring Polymers, nucleic acids and fragments thereof such as DNA, RNA and fragments thereof, radionuclides (especially radioiodides), radioisotopes, chelated metals, nanoparticles and reporter groups (e.g. fluorescent compounds), or can be passed through NMR Or compounds detected by ESR spectroscopy.
  • effector molecules including but not limited to: anti-tumor agents, drugs, toxins, biologically active proteins (such as enzymes), other antibodies or antibody fragments, synthetic or naturally occurring Polymers, nucleic acids and fragments thereof such as DNA, RNA and fragments thereof, radionuclides (especially radioiodides), radioisotopes, chelated metals, nanoparticles and reporter groups (e.g. fluorescent compounds), or can be passed through NMR Or compounds detected by
  • the effector molecule is connected to the anti-FcRn antibody or antigen-binding fragment thereof in a conjugation manner.
  • the effector molecule is a polymer, such as an optionally substituted linear or branched polyalkylene, polyalkenylene or polyoxyalkylene polymer or branched or unbranched polysaccharide, such as homopoly Or heteropolysaccharides, such as optionally substituted linear or branched poly(ethylene glycol), poly(propylene glycol), poly(vinyl alcohol) or derivatives thereof, lactose, amylose, dextran, glycogen Or its derivatives, human serum albumin or its fragments.
  • a polymer such as an optionally substituted linear or branched polyalkylene, polyalkenylene or polyoxyalkylene polymer or branched or unbranched polysaccharide, such as homopoly Or heteropolysaccharides, such as optionally substituted linear or branched poly(ethylene glycol), poly(propylene glycol), poly(vinyl alcohol) or derivatives thereof, lactose, amylose, dextran, glycogen Or its derivative
  • the present disclosure provides isolated polynucleotides that encode the anti-FcRn antibodies or antigen-binding fragments thereof of the present disclosure.
  • the polynucleotide may be DNA or RNA.
  • the present disclosure provides an expression vector containing the polynucleotide as described above.
  • the expression vector may be a eukaryotic expression vector, a prokaryotic expression vector, a viral vector, such as a plasmid, a cosmid, or a phage.
  • the present disclosure provides a host cell transformed with an expression vector as described above, which may be a eukaryotic cell or a prokaryotic cell.
  • the host cell is a bacterium, yeast, or mammalian cell. In some specific embodiments, the host cell is Escherichia coli, Pichia pastoris, Chinese hamster ovary (CHO) cells or human embryonic kidney (HEK) 293 cells.
  • the present disclosure provides a method for preparing an anti-FcRn antibody or an antigen-binding fragment thereof, comprising: expressing the antibody, its antigen-binding fragment or complex (such as a fusion protein) in a host cell as described above, and extracting the The antibody, its antigen-binding fragment or complex (such as a fusion protein) is isolated from the host cell.
  • it can also include a purification step, for example, using a Sepharose FF column with adjusted buffer A or G to wash out non-specifically bound components, and then use a pH gradient method to elute the bound antibody. SDS-PAGE detection and collection.
  • it is filtered and concentrated by a conventional method. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves and ion exchange. The resulting product needs to be frozen immediately, such as -70°C, or lyophilized.
  • the methods for producing and purifying antibodies and antigen-binding fragments are well known and can be found in the prior art, such as Cold Spring Harbor's Antibody Experiment Technique Guide (chapters 5-8 and 15).
  • human FcRn or its fragments can be used to immunize mice, and the obtained antibody can be renatured, purified, and amino acid sequencing can be performed by conventional methods.
  • Antigen-binding fragments can also be prepared by conventional methods.
  • the antibodies or antigen-binding fragments of the invention are genetically engineered to add one or more human FR regions to the non-human CDR regions.
  • the human FR germline sequence can be obtained from the website of ImmunoGeneTics (IMGT), for example.
  • the engineered antibodies or antigen-binding fragments of the present disclosure can be prepared and purified by conventional methods.
  • the cDNA sequences encoding the heavy and light chains can be cloned and recombined into an expression vector.
  • the recombinant immunoglobulin expression vector can be stably transfected into CHO cells.
  • Mammalian expression systems can lead to glycosylation of antibodies, especially at the highly conserved N-terminus of the Fc region.
  • Stable clones are obtained by expressing antibodies that specifically bind to human antigens. Positive clones are expanded in the serum-free medium of the bioreactor to produce antibodies.
  • the antibody-secreted culture medium can be purified and collected by conventional techniques.
  • the antibody can be filtered and concentrated by conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves and ion exchange.
  • composition such as a pharmaceutical composition, which contains a therapeutically effective amount of an anti-FcRn antibody or antigen-binding fragment thereof as described above and a pharmaceutically acceptable excipient, dilution or carrier.
  • the pharmaceutical composition may contain 0.01 to 99% by weight of the anti-FcRn antibody or its antigen-binding fragment in a unit dose, or the amount of the anti-FcRn antibody or its antigen-binding fragment in a unit dose of the pharmaceutical composition. It is 0.1-2000 mg, and in some embodiments, it is 1-1000 mg.
  • the present disclosure provides the use of any one or a combination selected from the following in the preparation of a medicine: the anti-FcRn antibody or antigen-binding fragment thereof according to the present disclosure, and the pharmaceutical composition according to the present disclosure.
  • the drug is used to treat or prevent autoimmune diseases or delay the progression of autoimmune diseases.
  • the present disclosure provides a method for treating or preventing an autoimmune disease or delaying the progression of an autoimmune disease, the method comprising administering to a subject an effective amount of the anti-FcRn antibody or antigen-binding fragment thereof according to the present disclosure, or according to the present disclosure, to treat or delay the disease.
  • Disclosed pharmaceutical composition comprising administering to a subject an effective amount of the anti-FcRn antibody or antigen-binding fragment thereof according to the present disclosure, or according to the present disclosure, to treat or delay the disease.
  • anti-FcRn antibodies or antigen-binding fragments thereof and pharmaceutical compositions of the present disclosure can be applied to all autoimmune diseases mediated by IgG and FcRn.
  • autoimmune diseases include but are not limited to: acute disseminated encephalomyelitis (ADEM), acute necrotizing hemorrhagic leukoencephalitis, Addison's disease, gammaglobulinemia, alopecia areata, amyloidosis, ANCA-related Vasculitis, ankylosing spondylitis, anti-GBM/anti-TBM nephritis, antiphospholipid syndrome (APS), autoimmune angioedema, autoimmune aplastic anemia, autoimmune familial autonomic abnormalities, Autoimmune hepatitis, autoimmune hyperlipidemia, autoimmune immune deficiency, autoimmune inner ear disease (AIED), autoimmune myocarditis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune platelets Reduced purpura (ATP), autoimmune thyroid disease, autoimmune urticaria, Axonal&nal neuropathy, Barlow disease, Behcet's disease, bullous pe
  • the present disclosure provides a composition for detecting FcRn, the composition comprising the anti-FcRn antibody or antigen-binding fragment thereof according to the present disclosure.
  • the present disclosure also provides a method, system or device for detecting FcRn in vivo or in vitro, which includes treating a sample with the anti-FcRn antibody or antigen-binding fragment thereof of the present disclosure.
  • the in vitro detection method, system or device may include, for example:
  • the in vivo detection method, system or device may include:
  • Detection can include determining the location or time of formation of the complex.
  • the FcRn antibody is labeled with a detectable substance, and detection of the substance that binds to the FcRn antibody (for example, FcRn) is achieved by detecting the label.
  • Suitable detectable substances include various enzymes, prosthetic groups, fluorescent substances, luminescent substances and radioactive substances.
  • the formation of a complex between an antibody that binds to FcRn or an antigen-binding fragment thereof and FcRn can be detected by measuring or visualizing the antibody that binds or does not bind to FcRn.
  • detection assays can be used, for example, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) or tissue immunohistochemistry.
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • tissue immunohistochemistry for detection purposes, the anti-FcRn antibodies or fragments thereof of the present disclosure can be labeled with a fluorophore chromophore.
  • kits which includes an anti-FcRn antibody or an antigen-binding fragment thereof, and may also include diagnostic instructions.
  • the kit may also contain at least one additional reagent, such as a marker or additional diagnostic agent.
  • the antibody can be formulated as a pharmaceutical composition.
  • Figure 1 ELISA detection of FcRn antibody binding to human FcRn protein.
  • Figure 2A Binding detection of FcRn antibody to cells overexpressing human FcRn under pH 6.0 environment.
  • Figure 2B Binding detection of FcRn antibody to cells overexpressing human FcRn under pH 7.4 environment.
  • Figure 3 Detection of FcRn antibody blocking the binding of cells overexpressing human FcRn to hIgG.
  • Figure 4 Detection of blocking detection of FcRn antibody blocking the binding of cells overexpressing human FcRn to HSA.
  • FIG. 5A FcRn antibody (30mpk) was intravenously administered to FcRn transgenic mice, and the percentage of IVIG content was detected at different time points.
  • Figure 5B Area under the curve (AUC) of intravenously administered FcRn antibody (30 mpk) in FcRn transgenic mice.
  • Human FcRn refers to the complex between human IgG receptor ⁇ chain and ⁇ 2 microglobulin ( ⁇ 2M), also known as neonatal Fc receptor. Its amino acid sequence can be found in, for example, UniProt No. P55899, and the amino acid sequence of ⁇ 2M can be found in, for example, UniProt. No. P61769. In the context of this application, “human FcRn” also encompasses any natural variant or recombinant product.
  • Antibody is used in the broadest sense and covers various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies; monospecific antibodies, multispecific antibodies (such as bispecific antibodies), full-length antibodies and antibody fragments ( Or antigen-binding fragments, or antigen-binding portions) as long as they exhibit the desired antigen-binding activity.
  • Antibody can refer to immunoglobulin, which is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds. The amino acid composition and sequence of the constant region of the immunoglobulin heavy chain are different, so their antigenicity is also different.
  • immunoglobulins can be divided into five categories, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE.
  • the corresponding heavy chains are ⁇ chain, ⁇ chain, and ⁇ chain. , ⁇ chain and ⁇ chain.
  • the same type of Ig can be divided into different subclasses according to the amino acid composition of the hinge region and the number and position of heavy chain disulfide bonds.
  • IgG can be divided into IgG1, IgG2, IgG3, and IgG4.
  • the light chain is divided into a kappa chain or a lambda chain by the difference of the constant region.
  • Each of the five types of Ig can have a kappa chain or a lambda chain.
  • the sequence of about 110 amino acids near the N-terminus of the antibody heavy and light chains varies greatly and is the variable region (V region); the remaining amino acid sequences near the C-terminus are relatively stable and are the constant region (C region).
  • the variable region includes 3 hypervariable regions (CDR) and 4 framework regions (FR) with relatively conservative sequences. Three hypervariable regions determine the specificity of the antibody, also known as complementarity determining regions (CDR).
  • Each light chain variable region (VL) and heavy chain variable region (VH) consists of 3 CDR regions and 4 FR regions.
  • the sequence from the amino terminal to the carboxy terminal is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the 3 CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the 3 CDR regions of the heavy chain refer to HCDR1, HCDR2 and HCDR3.
  • CDR For the determination or definition of CDR, the deterministic description of CDR and the identification of residues containing the binding site of the antibody can be completed by resolving the structure of the antibody and/or resolving the structure of the antibody-ligand complex. This can be achieved by any of various techniques known to those skilled in the art, such as X-ray crystallography. A variety of analysis methods can be used to identify CDRs, including but not limited to Kabat numbering system, Chothia numbering system, AbM numbering system, IMGT numbering system, contact definition, conformational definition.
  • the Kabat numbering system is a standard for numbering residues in antibodies and is commonly used to identify CDR regions (see, for example, Johnson & Wu, 2000, Nucleic Acids Res., 28:214-8).
  • the Chothia numbering system is similar to the Kabat numbering system, but the Chothia numbering system takes into account the location of certain structural loop regions. (See, for example, Chothia et al., 1986, J. Mol. Biol., 196:901-17; Chothia et al., 1989, Nature, 342:877-83).
  • the AbM numbering system uses a computer program integration suite produced by Oxford Molecular Group for modeling antibody structures (see, for example, Martin et al., 1989, Proc Natl Acad Sci (USA), 86: 9268-9272; "AbMTM, A Computer Program for Modeling Variable Regions of Antibodies, "Oxford, UK; Oxford Molecular, Ltd).
  • the AbM numbering system uses a combination of knowledge databases and ab initio methods to model the tertiary structure of antibodies from basic sequences (see Samudrala et al., 1999, in PROTEINS, Structure, Function and Genetics Suppl., 3:194-198, "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach).
  • CDR may refer to a CDR defined by any method known in the art (including a combination of methods).
  • the number and position of the CDR amino acid residues of the VL region and VH region of the antibody or antigen-binding fragment of the present disclosure conform to the known Kabat or AbM numbering system.
  • “Monoclonal antibody” or “monoclonal antibody” refers to antibodies obtained from a substantially homogeneous antibody population, that is, each antibody contained in the population is the same except for possible naturally occurring mutations that may be present in small amounts. Monoclonal antibodies are highly specific and are directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations which usually include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.
  • the modifier "monoclonal” indicates the characteristics of an antibody as obtained from a substantially homogeneous antibody population, and is not construed as requiring the production of the antibody by any specific method.
  • monoclonal antibodies used in accordance with the present disclosure can be prepared by the hybridoma method first described by Kohler and Milstein, 1975, Nature 256:495, or can be prepared by, for example, the recombinant DNA method described in U.S. Patent No. 4,816,567.
  • monoclonal antibodies can also be isolated from the generated phage library using the technique described in McCafferty et al., 1990, Nature 348:552-554.
  • Fully human antibody or “recombinant fully human antibody” includes fully human antibodies prepared, expressed, created or isolated by recombinant methods, and the techniques and methods involved are well known in the art, such as:
  • Antibodies prepared, expressed, created or isolated by methods such as splicing human immunoglobulin gene sequences to other DNA sequences.
  • Such recombinant fully human antibodies contain variable and constant regions, which utilize specific human germline immunoglobulin sequences encoded by germline genes, but also include subsequent rearrangements and mutations that occur during antibody maturation.
  • murine antibody in the present disclosure refers to a monoclonal antibody against human FcRn or its epitope prepared according to the knowledge and skills in the art. During the preparation, the test subject is injected with the FcRn antigen, and then the hybridoma expressing the antibody with the desired sequence or functional characteristics is isolated.
  • the murine anti-human FcRn antibody or antigen-binding fragment thereof may further comprise the light chain constant region of murine kappa, lambda chains or variants thereof, or further comprise murine IgG1 , IgG2, IgG3 or IgG4 or variants of the heavy chain constant region.
  • the term “fully human antibody” includes antibodies having variable and constant regions of human germline immunoglobulin sequences.
  • the fully human antibodies of the present disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (such as mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).
  • the term “fully human antibody” does not include antibodies in which CDR sequences derived from the germline of another mammalian species (such as a mouse) have been grafted onto human framework sequences (ie, "humanized antibodies” ).
  • humanized antibody also known as CDR-grafted antibody, refers to an antibody produced by grafting non-human CDR sequences into the framework of a human antibody variable region. It can overcome the strong immune response induced by the chimeric antibody carrying a large amount of non-human protein components. In order to avoid the decrease of immunogenicity and the decrease of activity at the same time, the variable region of the fully human antibody can be subjected to minimal reverse mutations to maintain activity.
  • chimeric antibody is an antibody formed by fusing the variable region of the antibody of the first species with the constant region of the antibody of the second species, which can reduce the immune response induced by the antibody of the first species.
  • a chimeric antibody it is necessary to select a hybridoma that secretes a murine-specific monoclonal antibody, then clone the variable region gene from the mouse hybridoma cell, and then clone the constant region gene of the fully human antibody as needed.
  • the mouse variable region gene and the human constant region gene are connected to form a chimeric gene and then inserted into a human vector, and finally the chimeric antibody molecule is expressed in a eukaryotic industrial system or a prokaryotic industrial system.
  • the constant region of a fully human antibody can be selected from the heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or variants thereof, preferably comprising human IgG2 or IgG4 heavy chain constant region, or without ADCC (antibody- dependent cell-mediated cytotoxicity, antibody-dependent cell-mediated cytotoxicity) toxic IgG1.
  • Antigen-binding fragments include: single-chain antibodies (ie, full-length heavy and light chains); Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, Fab-Fv, Fab- dsFv, single domain antibody (e.g. VH or VL or VHH), scFv, bivalent or trivalent or tetravalent antibody, Bis-scFv, diabody, tribody, triabody, tetrabody and epitope binding fragments of any of the above (see For example, Holliger and Hudson, 2005, Nature Biotech. 23(9): 1126-1136; Adair and Lawson, 2005, Drug Design Reviews-Online 2(3), 209-217).
  • the Fab-Fv format was first disclosed in WO2009/040562, and the disulfide bond stabilized Fab-dsFv format was first disclosed in WO2010/035012.
  • the antigen-binding fragments of the present disclosure also include Fab and Fab' fragments described in WO2005/003169, WO2005/003170 and WO2005/003171.
  • Multivalent antibodies may comprise multispecific such as bispecific or may be monospecific (see e.g. WO92/22583 and WO05/113605), an example of the latter is the Tri-Fab described in WO 92/22583 (or TFM).
  • binding to FcRn refers to the ability to interact with FcRn or its epitope, and the FcRn or its epitope may be of human origin.
  • antigen-binding site in the present disclosure refers to a discrete three-dimensional site on an antigen that is recognized by the antibody or antigen-binding fragment of the present disclosure.
  • Antigen refers to a molecule used to immunize immunocompetent vertebrates, to generate antibodies that recognize the antigen, or to screen expression libraries (e.g., phage, yeast, or ribosome display libraries, among others).
  • antigens are defined more broadly, including target molecules specifically recognized by antibodies, and include parts or mimetics of molecules used in the immunization process for antibody production or library screening for antibody selection.
  • monomers and multimers such as dimers, trimers, etc.
  • truncated variants and other variants of human FcRn are all referred to as antigens.
  • epitope refers to a site on an antigen that binds to an immunoglobulin or antibody.
  • Epitopes can be formed by adjacent amino acids or non-adjacent amino acids that are juxtaposed by tertiary folding of the protein. Epitopes formed by adjacent amino acids are usually maintained after exposure to a denaturing solvent, while epitopes formed by tertiary folding are usually lost after treatment with the denaturing solvent. Epitopes usually include at least 3-15 amino acids in a unique spatial conformation. Methods to determine what epitope is bound by a given antibody are well known in the art, including immunoblotting and immunoprecipitation detection analysis. The method for determining the spatial conformation of an epitope includes the techniques in the art and the techniques described in the present disclosure, such as X-ray crystal analysis and two-dimensional nuclear magnetic resonance.
  • Specific binding and “selective binding” refer to the binding of an antibody to an epitope on a predetermined antigen.
  • an antibody when measured by surface plasmon resonance (SPR) technology in the instrument, the antibody balances approximately below 10 -7 M or even less.
  • the dissociation constant (K D ) binds to the predetermined antigen or its epitope, and its binding affinity to the predetermined antigen or its epitope is non-specific other than the predetermined antigen (or its epitope) or closely related antigens
  • the binding affinity of antigen (such as BSA, etc.) is at least twice.
  • the term "antibody that recognizes an antigen” can be used interchangeably with the term “antibody that specifically binds” in this disclosure.
  • Binding affinity or “affinity” is used in the present disclosure as a measure of the strength of a non-covalent interaction between two molecules (eg, an antibody or part thereof and an antigen).
  • the binding affinity between two molecules can be quantified by determining the dissociation constant (KD).
  • KD can be determined by measuring the kinetics of complex formation and dissociation using, for example, a surface plasmon resonance (SPR) method (Biacore).
  • SPR surface plasmon resonance
  • the rate constants corresponding to the association and dissociation of the monovalent complex are called the association rate constant ka (or kon) and the dissociation rate constant kd (or koff), respectively.
  • the value of the dissociation constant can be determined directly by well-known methods, and can be calculated by methods such as those described in Caceci et al. (1984, Byte 9:340-362) even for complex mixtures.
  • a double filter nitrocellulose filter binding assay such as Wong & Lohman: that disclosed in (1993, Proc.Natl.Acad.Sci.USA 90 5428-5432) to determine the K D.
  • Other standard assays for assessing the binding ability of antibodies to target antigens are known in the art and include, for example, ELISA, Western blot, RIA, and flow cytometry analysis, as well as other assays exemplified elsewhere in this disclosure.
  • the binding kinetics and binding affinity of antibodies can also be determined by standards known in the art, such as surface plasmon resonance (SPR), for example, by using the Biacore TM system or KinExA.
  • the K D value of each antibody/antigen complex can be compared to compare the binding affinities associated with different molecular interactions, for example, the comparison of the binding affinities of different antibodies for a given antigen.
  • the specificity of the interaction can be determined and compared by determining and comparing the K D value of the target interaction (for example, the specific interaction between the antibody and the antigen) and the non-target interaction (for example, a control antibody that is not known to bind FcRn). K D value is evaluated.
  • Constant substitution refers to substitution with another amino acid residue that has similar properties to the original amino acid residue.
  • lysine, arginine, and histidine have similar properties in that they have basic side chains
  • aspartic acid and glutamic acid have similar properties in that they have acidic side chains.
  • glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine and tryptophan have similar properties in that they have uncharged polar side chains
  • alanine , Valine, leucine, threonine, isoleucine, proline, phenylalanine and methionine have similar properties in that they have non-polar side chains.
  • tyrosine, phenylalanine, tryptophan and histidine have similar properties in that they have aromatic side chains. Therefore, it will be obvious to a person skilled in the art that even when an amino acid residue in a group showing similar properties as described above is substituted, it will not show a specific change in properties.
  • Cross-reactivity refers to the ability of the antibodies of the present disclosure to bind to FcRn from different species.
  • an antibody of the present disclosure that binds to human FcRn can also bind to FcRn of another species.
  • Cross-reactivity is measured by detecting specific reactivity with purified antigens in binding assays (such as SPR and ELISA), or binding or functional interaction with cells that physiologically express FcRn. Methods of determining cross-reactivity include standard binding assays as described in this disclosure, such as surface plasmon resonance analysis, or flow cytometry.
  • Inhibition or blocking are used interchangeably and encompasses both partial and complete inhibition/blocking. Inhibition/blocking of FcRn preferably reduces or alters the normal level or type of activity that occurs when FcRn binding occurs without inhibition or blocking. Inhibition and blocking are also intended to include any measurable reduction in binding affinity for FcRn when contacted with an anti-FcRn antibody compared to FcRn not contacted with an anti-FcRn antibody.
  • Inhibiting growth (e.g., referring to cells) is intended to include any measurable decrease in cell growth.
  • the methods for producing and purifying antibodies and antigen-binding fragments are well known and can be found in the prior art, such as Cold Spring Harbor's Antibody Experiment Technique Guide (chapters 5-8 and 15).
  • human FcRn or its fragments can be used to immunize mice, and the obtained antibody can be renatured, purified, and amino acid sequencing can be performed by conventional methods.
  • Antigen-binding fragments can also be prepared by conventional methods.
  • the antibodies or antigen-binding fragments described in the present disclosure are genetically engineered to add one or more human FR regions to the CDR regions of non-human origin.
  • the human FR germline sequence can be obtained from the ImmunoGeneTics (IMGT) website.
  • the engineered antibodies or antigen-binding fragments of the present disclosure can be prepared and purified by conventional methods.
  • the cDNA sequences encoding the heavy and light chains can be cloned and recombined into an expression vector.
  • the recombinant immunoglobulin expression vector can stably transfect cells.
  • Mammalian expression systems can lead to glycosylation of antibodies, especially at the highly conserved N-terminus of the Fc region.
  • Stable clones are obtained by expressing antibodies that specifically bind to human antigens. Positive clones are expanded in the serum-free medium of the bioreactor to produce antibodies.
  • the antibody-secreted culture medium can be purified and collected by conventional techniques.
  • the antibody can be filtered and concentrated by conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves and ion exchange.
  • the resulting product needs to be frozen immediately, such as -70°C, or lyophilized.
  • administering when applied to animals, humans, experimental subjects, cells, tissues, organs or biological fluids refer to exogenous drugs, therapeutic agents, diagnostic agents or compositions and animals , Human, subject, cell, tissue, organ or biological fluid contact.
  • administering can refer to, for example, treatment, pharmacokinetics, diagnosis, research, and experimental methods.
  • the treatment of cells includes contact of reagents with cells, and contact of reagents with fluids, where the fluids are in contact with cells.
  • administering “administration” and “treatment” also mean the treatment of, for example, cells by reagents, diagnostics, binding compositions, or by another cell in vitro and ex vivo.
  • Treatment when applied to human, veterinary or research subjects, refers to therapeutic treatment, preventive or preventive measures, research and diagnostic applications.
  • Treatment means administering an internal or external therapeutic agent to a subject, such as a composition comprising any one of the antibodies or antigen-binding fragments or conjugates thereof of the present disclosure, the subject has, or is suspected of being suffering from Yes, tend to suffer from one or more diseases or their symptoms, and the therapeutic agent is known to have a therapeutic effect on these symptoms.
  • the therapeutic agent is administered in the subject or population to be treated in an amount effective to alleviate one or more symptoms of the disease, whether by inducing the regression of such symptoms or inhibiting the development of such symptoms to any clinically measured extent.
  • the amount of the therapeutic agent effective to alleviate the symptoms of any particular disease can vary depending on a variety of factors, such as the subject’s disease state, age and weight, and the amount of the drug that produces the desired therapeutic effect in the subject. ability. Through any clinical testing methods commonly used by doctors or other professional health care professionals to evaluate the severity or progression of the symptoms, it can be evaluated whether the symptoms of the disease have been alleviated.
  • the embodiments of the present disclosure may be ineffective in alleviating the symptoms of the target disease in a subject, according to any statistical test methods known in the art such as Student's t test, chi-square test, and basis Mann and Whitney's U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test determined that it should reduce the symptoms of the target disease in a statistically significant number of subjects.
  • any statistical test methods known in the art such as Student's t test, chi-square test, and basis Mann and Whitney's U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test determined that it should reduce the symptoms of the target disease in a statistically significant number of subjects.
  • an “effective amount” includes an amount sufficient to ameliorate or prevent the symptoms or conditions of the medical condition.
  • An effective amount also means an amount sufficient to allow or facilitate diagnosis.
  • the effective amount for a particular subject or veterinary subject can vary depending on factors such as the condition to be treated, the subject's general health, the method of administration and dosage, and the severity of side effects.
  • the effective amount can be the maximum dose or dosing schedule that avoids significant side effects or toxic effects.
  • “Homology” or “identity” refers to the sequence similarity between two polynucleotide sequences or between two polypeptides.
  • the positions in the two comparison sequences are occupied by the same nucleotide or amino acid monomer subunit, for example, if each position of two DNA molecules is occupied by the same nucleotide, then the molecule is Homologous.
  • the percent homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared ⁇ 100%. For example, in the optimal sequence alignment, if there are 6 matches or homology in 10 positions in the two sequences, then the two sequences are 60% homologous.
  • the comparison is made when two sequences are aligned to obtain the greatest percentage of homology.
  • Cell Cell
  • cell line cell line
  • cell culture all such names include their progeny. It should also be understood that due to deliberate or unintentional mutations, all offspring cannot be exactly the same in terms of DNA content. Including mutant progeny with the same function or biological activity as screened in the original transformed cell.
  • the “FcRn-binding protein” of the present disclosure is explained to the maximum extent, including the anti-FcRn antibody or the antigen-binding fragment thereof of the present disclosure, as long as it can achieve binding to FcRn, it is within the scope of this term.
  • the FcRn binding protein may contain one or more effector molecules, for example in a conjugation manner.
  • effector molecule includes, for example, anti-tumor agents, drugs, toxins, biologically active proteins (such as enzymes), other antibodies or antibody fragments, synthetic or naturally occurring polymers, nucleic acids and fragments thereof such as DNA, RNA and fragments thereof , Radionuclides (especially radioiodides), radioisotopes, chelated metals, nanoparticles and reporter groups (e.g. fluorescent compounds), or compounds detectable by NMR or ESR spectroscopy.
  • anti-tumor agents drugs, toxins, biologically active proteins (such as enzymes), other antibodies or antibody fragments, synthetic or naturally occurring polymers, nucleic acids and fragments thereof such as DNA, RNA and fragments thereof , Radionuclides (especially radioiodides), radioisotopes, chelated metals, nanoparticles and reporter groups (e.g. fluorescent compounds), or compounds detectable by NMR or ESR spectroscopy.
  • the effector molecule when it is a polymer, it can usually be a synthetic or naturally occurring polymer, such as an optionally substituted linear or branched polyalkylene, polyalkenylene or polyoxyalkylene polymer or branched Polysaccharides or unbranched polysaccharides, such as homo- or hetero-polysaccharides.
  • Specific optional substituents that may be present on the aforementioned synthetic polymers include one or more hydroxy, methyl or methoxy groups.
  • Specific examples of synthetic polymers include optionally substituted linear or branched poly(ethylene glycol), poly(propylene glycol), poly(vinyl alcohol) or derivatives thereof, particularly optionally substituted poly(ethylene two Alcohol) such as methoxy poly(ethylene glycol) or its derivatives.
  • polymers include lactose, amylose, dextran, glycogen or derivatives thereof.
  • the polymer is albumin or a fragment thereof, such as human serum albumin or a fragment thereof.
  • the method of conjugating the polymer to the anti-FcRn antibody or antigen-binding fragment thereof of the present disclosure can be achieved by conventional methods.
  • the human FcRn (Uniprot accession number: P55899) extracellular domain- ⁇ 2M (Uniprot accession number: P61769) complex was used as the template of FcRn to design the amino acid sequence of the FcRn antigen and the detection protein (the following FcRn antigens without special instructions refer to human FcRn- ⁇ 2M complex).
  • the italicized part is the transmembrane region, and the underlined part is the intracellular region.
  • FcRn recombinant protein with His tag Equilibrate the Ni-Sepharose affinity column for 5 column volumes with 20mM phosphate buffer, pH 8.0. Centrifuge the sample at high speed to remove impurities, and combine it on the column. Wash the column with 20mM phosphate buffer until the A280 reading drops to the baseline. Use 20mM phosphate buffer, 0-500mM imidazole gradient elution, collect the protein and identify the target protein. The sample obtained in the first step of purification was changed to PBS and concentrated to a volume of 2 mL. The gel chromatography Superdex200 (GE) equilibrated with PBS was used for further purification, and the target peak fraction was collected for equipment use. After testing, a purified FcRn recombinant protein was obtained.
  • 20mM phosphate buffer pH 8.0. Centrifuge the sample at high speed to remove impurities, and combine it on the column. Wash the column with 20mM phosphate buffer until the A280 reading drops to
  • mice Take 4 SJL white mice and 5 Balb/c white mice, and immunize them with 25-50 ⁇ g antigen and adjuvant. The time is the 0th, 14th, and 35th days. On day 0, 50 ⁇ g/head of emulsified antigen was injected intraperitoneally (IP). On the 14th and 35th day, 25 ⁇ g/mouse was injected. Blood was taken on the 21st and 42nd day, and the antibody titer in the mouse serum was determined by ELISA method. After the 4-5th immunization, mice with high antibody titer in the serum and the titer tending to the plateau were selected for spleen cell fusion. Three days before the fusion of splenocytes, the immunization was boosted, and the antigen solution prepared by 50 ⁇ g/head of physiological saline was injected intraperitoneally (IP).
  • IP intraperitoneally
  • the splenic lymphocytes and the myeloma cells Sp2/0-Ag14 cells were fused using a PEG-mediated fusion step to obtain hybridoma cells.
  • the fused hybridoma cells are seeded in a 96-well plate with a number of cells per well of 1 ⁇ 10 4 -1 ⁇ 10 5 , incubated at 37°C, 5% CO 2 and supplemented with 100 ⁇ L/well of HAT complete medium, 10-14 days later.
  • ELISA test The splenic lymphocytes and the myeloma cells Sp2/0-Ag14 cells were fused using a PEG-mediated fusion step to obtain hybridoma cells.
  • the fused hybridoma cells are seeded in a 96-well plate with a number of cells per well of 1 ⁇ 10 4 -1 ⁇ 10 5 , incubated at 37°C, 5% CO 2 and supplemented with 100 ⁇ L/well of HAT complete medium, 10-14 days later.
  • ELISA test
  • the hybridoma culture supernatant was detected by the combined ELISA method. And the cell supernatant of positive wells detected by ELISA was purified, cell binding experiment and cell blocking experiment were carried out. Cells in wells that are positive for binding and blocking are expanded, cryopreserved, and sequenced in time.
  • the amino acid sequence of the antibody variable region corresponding to the selected positive clone hybri11 is shown below:
  • mice The spleen and lymph node cells of immunized mice were resuspended with Trizol reagent (1 ⁇ 10 7 cells/mL Trizol) to lyse the cells, and placed on ice for 5 min; centrifuged at 13000 rpm for 3 min, take the supernatant and discard the precipitate; add 1/5 volume Shake vigorously for 30-60s, and let stand for 2 min in an ice bath; centrifuge at 13000 rpm for 10 min, and transfer the upper aqueous layer to a new 1.5 mL tube; add an equal volume of isopropanol, mix well, and let stand at -20°C for 30 min; Centrifuge at 13000 rpm for 10 min, remove the supernatant, and save the precipitate; add pre-cooled 75% ethanol to wash the precipitate, and place it at room temperature for 5-10 minutes; add 600 ⁇ L of deionized water removed by RNase and reconstitute to obtain RNA. Reverse transcription to obtain cDNA. Construction
  • the method of purifying recombinant antibodies is: collect the expression supernatant, high-speed centrifugation to remove impurities, and filter with a 0.45 ⁇ m PVDF filter membrane, and use a Protein A or Protein G column for affinity chromatography.
  • 1 ⁇ PBS (pH 7.4) buffer system as the equilibration buffer to equilibrate the chromatography column by 3-5 times the column volume; the cell supernatant is loaded with a low flow rate and combined, and the flow rate is controlled so that the retention time is about 1 min or longer; use 1 ⁇ PBS (pH 7.4) wash the column 3-5 times the column volume until the UV absorption falls back to the baseline; use 0.1M glycine (pH 3.0) buffer for sample elution, collect the elution peak based on UV detection, and use the eluted product 1M Tris-HCl (pH 9.0) quickly adjust the pH to 5-6 for temporary storage.
  • the eluted sample is properly diluted to a conductivity of ⁇ 2mS/cm, combined with the anion column, washed with 20mM Tris-HCl pH 8.0-9.0 to remove impurities, and 20mM Tris-HCl, NaCl 0-1M gradient elution. Collect the peaks and transfer them to the PBS sub-equipment.
  • the CDR numbering system of the anti-human FcRn antibody screened in the present disclosure is the Kabat numbering system or the AbM numbering system.
  • the CDR regions of the Kabat numbering system are underlined, and the CDR regions of the AbM numbering system are italicized.
  • the sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and the underlined or italicized parts in the sequence are the CDR1, CDR2, and CDR3 sequences, respectively.
  • Table 2 summarizes the heavy chain and light chain CDR sequences of each antibody.
  • HCDR1-3 is SEQ ID No: 38-40 and LCDR1-3 is SEQ ID No: 41-43, it is Kabat numbering system; when HCDR1-3 is SEQ ID No: 68, 69, 40, when LCDR1-3 is SEQ ID No: 41-43, it is the AbM numbering system.
  • the numbering system for other antibodies is Kabat.
  • the above heavy chain variable regions are fused with the corresponding human CH1 (SEQ ID No: 70) or mouse antibody heavy chain CH1 (SEQ ID No: 71) and mouse IgG1 Fc (SEQ ID No: 75).
  • the chain variable region is fused with the human kappa (SEQ ID No: 72) or lamda constant region CL1 (SEQ ID No: 73) or CL2 (SEQ ID No: 74) to form a recombinant antibody, which is then subjected to subsequent detection.
  • the three-dimensional simulation structure of the antibody molecule and the known antigen structure (PDB ID: 4N0U Chain A&B) are combined to simulate.
  • PDB ID: 4N0U Chain A&B known antigen structure
  • three-dimensional structure and combined simulation results some key amino acid residues in the framework and CDR regions were selected, and several random mutant phage libraries were established.
  • phage library display technology functional antibodies with improved affinity were screened out.
  • the obtained antibody molecule light chain variable region and heavy chain variable region sequences are shown in Table 3 and Table 4, respectively.
  • the CDR combinations are shown in Table 5, Table 6, and Table 7, respectively.
  • the CDR numbering system of the anti-FcRn antibodies screened in Table 3 and Table 4 is Kabat numbering system (underlined) or AbM numbering system (italics).
  • the sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and the underline or italics in the sequence are the CDR1, CDR2, and CDR3 sequences, respectively.
  • the CDR numbering system in Table 5 is the AbM or Kabat numbering system
  • the CDR numbering system in Table 6 is the Kabat numbering system
  • the CDR numbering system in Table 7 is the AbM numbering system.
  • the heavy chain and light chain variable region germline genes with high homology to the selected antibodies are used as templates, and the FR and CDR regions of the original monoclonal antibody are modified to make their sequences closer to human germline genes while retaining functions .
  • the three-dimensional structure of the transplanted single-domain antibody was simulated and analyzed again, and the specific sites in the FR region that affected the structure and morphology of the CDR region were back-mutated.
  • the amino acid residues are identified and annotated by the Kabat numbering system.
  • the modified antibody has higher stability and lower immunogenicity.
  • the heavy chain template of antibody h22 is selected from the human germline gene IGHV5-51*01 in the IMGT database, and the light chain template is selected from the human germline gene IGLV6-57*02 in the IMGT database.
  • the antibody h22 was genetically modified, and its variable region sequence is shown in Table 8.
  • the CDR numbering system in Table 8 is the Kabat numbering system (shown underlined) or the AbM numbering system (shown in italics).
  • the sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and underlined or italicized are the CDR1, CDR2, and CDR3 sequences, respectively.
  • the CDR numbering system in Table 9 is the Kabat numbering system.
  • the screened antibody heavy chain variable region was fused with human CH1 and IgG4 (S241P) Fc and cloned into a mammalian cell expression vector.
  • the antibody light chain variable region is cloned into a mammalian cell expression vector containing the human antibody light chain lamda or kappa constant region.
  • IgG4 (S241P) is abbreviated as IgG4P.
  • Transfection was carried out with liposome 293FectinReagent (Invitrogen, Cat. No. 12347019) according to the ratio of 1 ⁇ g DNA/ml transfected cells, and carried out according to the reagent instructions.
  • the cell density is 1.0X10 6 cells/ml, placed in a 37 degree shaker and cultured with shaking (5% CO 2 ). On the 6th day, the cell culture solution is collected, centrifuged at 4000 rpm, and the supernatant is taken and filtered with a 0.45 ⁇ M filter membrane. .
  • the ExpiCHO Expression System (Cat.no.A29133) was used for transfection according to the ratio of 1 ⁇ g DNA/ml transfected cells, and proceeded according to the reagent instructions. Using standard methods, the cell density after transfection is 6.0X10 6 cells/ml, placed in a 37 degree shaker and cultured with shaking (8% CO 2 ), the cell culture medium is collected on the 8th day, centrifuged at 4000 rpm, the supernatant is taken and 0.45 ⁇ M is used The filter membrane is filtered.
  • the target antibody is obtained.
  • a random biotinylated human FcRn- ⁇ 2M complex (biotin-hFcRn) was used for in vitro binding detection.
  • the negative control of this test example is PBS, and the positive control uses HEK293 transiently transfected and expressed Argx-113.
  • the sequence of Argx-113 of the positive control is from sequence 2 in WO2015/100299A1.
  • Dilute NeutrAvidin Protein (Thermo Scientific, Lot: 31000) with pH 6.0 PBS buffer to 2 ⁇ g/mL, add 100 ⁇ L/well to 96-well microtiter plate (Corning, 901825), and place at 4°C overnight 16- 20 hours. After discarding the liquid, wash the plate three times with PBST (pH 6.0, 0.05% Tween-20) buffer, and add 100 ⁇ L/well of biotin-hFcRn or biotin-cynoFcRn diluted to 3 ⁇ g/mL with PBS buffer at 37°C Incubate for 1 hour in the incubator.
  • PBST pH 6.0, 0.05% Tween-20
  • the plate was washed three times with PBST buffer, 4% BSA blocking solution (300 ⁇ L/well) diluted with PBS buffer was added, and the plate was incubated for 1 hour in a 37° C. incubator for blocking. After the blocking, discard the blocking solution and wash the plate with PBST buffer 3 times, then add the FcRn antibody protein with the initial concentration of 40 ⁇ g/mL, dilute 7 gradients with PBS buffer three-fold ratio, and incubate at 37°C incubator 1 hour.
  • FcRn antibody to cell surface FcRn was tested by measuring the binding of the antibody to hFcRn on the surface of HEK293 cells stably overexpressing hFcRn mutants (human FcRn mut (L320A, L321A)). After the HEK293 cells stably overexpressing human FcRn mut (L320A, L321A) were fixed on the bottom of a 96-well plate, the strength of the signal after the addition of the antibody and the corresponding secondary antibody was used to determine the binding activity of the antibody and hFcRn overexpressing HEK293 cells.
  • the specific experimental methods are as follows:
  • Biacore GE instrument was used to determine the affinity of the humanized anti-FcRn antibody to be tested with human FcRn.
  • Biosensing chip (Cat.#29127556, GE) to affinity capture a certain amount of antibody to be tested, and then flow through a series of human FcRn antigen under a concentration gradient on the surface of the chip, using Biacore instrument (Biacore T200, GE) Real-time detection of reaction signals to obtain binding and dissociation curves. After the dissociation of each cycle is completed, the biochip is washed and regenerated with the regeneration solution configured in the human anti-capture kit or the pH 1.5 glycine-hydrochloric acid regeneration solution (Cat.#BR-1003-54, GE).
  • Biacore instrument Biacore T200, GE
  • the buffer used in the experiment is HBS-EP+10 ⁇ buffer solution (Cat.#BR-1006-69, GE), diluted to 1 ⁇ (pH 7.4) with DIWater, or diluted to 1 ⁇ and used 3M HCl Adjust the pH to 5.5 or 6.0.
  • the heavy chain and light chain mutant sequences obtained by affinity maturation screening were cross-combined, and the antibody molecules with enhanced affinity to FcRn were obtained by SPR screening.
  • Some of the molecular affinity determination results are as follows:
  • Antibody Heavy chain number Light chain number ka(1/Ms) kd(1/s) KD(M) h22 h22-VH h22-VL 3.02E+05 4.23E-03 1.40E-08 h22-AM-2 h22-AM-VH-10 h22-VL 4.66E+06 2.84E-03 6.10E-10 h22-AM-5 h22-AM-VH-13 h22-VL 3.25E+06 3.80E-03 1.17E-09 h22-AM-9 h22-AM-VH-17 h22-VL 2.46E+06 4.28E-03 1.74E-09 h22-AM-10 h22-AM-VH-18 h22-VL 2.34E+06 3.77E-03 1.61E-09
  • the results show that the modified antibody molecule maintains an affinity comparable to the parent molecule h22 while reducing immunogenicity.
  • HCDR1 as shown in GYX 1 F X 2 (SEQ ID No: 182) or GYX 1 F X 2 X 3 X 4 X 5 IA (SEQ ID No: 173), wherein X 1 can be selected from S, N, D, V , A, P, X 2 can be selected from T, N, K, R, S, A, X 3 can be selected from G, N, K, S, E, X 4 can be selected from Y, H, S, N, T, X 5 can be selected from W, Y, F, M;
  • HCDR2 as shown in X 6 IX 7 PDX 8 SNTI (SEQ ID No: 174) or X 6 IX 7 PDX 8 SNTIYSPSFRG (SEQ ID No: 175), wherein X 6 can be selected from I, L, V, X 7 Can be selected from Y, S, T, G, A, X 8 can be selected from N, R, K, A, F;
  • amino acid sequence of HCDR3 is shown in FGGPTFAQWYFDY (SEQ ID No: 40).
  • LCDR1 The amino acid sequence of LCDR1 is shown in TGSSGSIASNYVX 9 (SEQ ID No: 176), wherein X 9 can be selected from Q, S, N, A, V, T;
  • X 10 DNQRAS SEQ ID No: 177
  • X 10 can be selected from E, S, A, W, and C;
  • the amino acid sequence of LCDR3 is shown in QSYDSSSHNWV (SEQ ID No: 43).
  • the anti-human FcRn antibody was tested for the blocking of biotin-hIgG binding to FcRn in cells overexpressing hFcRn in vitro.
  • the blocking effect of anti-human FcRn antibody on the binding of IgG to FcRn was measured in HEK293 cells overexpressing hFcRn mutant (human FcRn mut (L320A, L321A)). I4506) the amount of fluorescence detection.
  • HEK293 cells ie, 293-hFcRn-mut
  • human FcRn mut L320A, L321A
  • the hFcRn mutant can better maintain cell membrane surface localization.
  • the results show that the antibody obtained in the present disclosure has a good effect of blocking the binding of IgG to FcRn at the cellular level.
  • N/A means not detected or unable to fit the specific value
  • Example 9 The blocking experiment of anti-FcRn antibody on the binding of FcRn overexpressing cells to human serum albumin (HSA)
  • this example measures the binding of the antibody to the cell surface in HEK293 cells overexpressing the hFcRn mutant Randomly detect the fluorescence of biotinylated HSA (Sigma catalog number: 126658).
  • the HEK293 cell line overexpressing the hFcRn mutant (human FcRn mut (L320A, L321A)) was used.
  • the hFcRn mutant can better maintain the cell membrane surface localization.
  • SA-FITC secondary antibody was added to incubate. The increase or decrease of the fluorescence signal of the secondary antibody was used as a measure of the effect of FcRn antibody on biotin-HSA and biotin-HSA. FcRn binding blocking effect is strong or weak.
  • the specific experimental methods are as follows:
  • test results of some antibodies are shown in Figure 4.
  • the results showed that HSA showed a blockade of HSA binding, and h22 and its genetically modified antibodies had similar effects on the binding of biotin-HSA and hFcRn to the negative control IgG, and did not affect the binding of FcRn to HSA.
  • human IVIG was injected intravenously into transgenic mice expressing human FcRn (C57BL/6-Fcgrttm1(FCGRT)/Bcgen, Biocytometer), 24 hours later, anti-FcRn antibody was injected intravenously, and blood was collected at different time points after administration. The purpose is to check whether the antibody affects the metabolism of human IgG.
  • the hFcRn transgenic mice were randomly divided into groups, three in each group, and intravenous injection of 500mpk IVIG. After 24 hours, the anti-FcRn antibody to be tested or the control human IgG1 isotype control (BioXcell catalog number: BE0297) was intravenously injected with a dose of 30mpk. Blood was collected before administration, that is, 0 hours, and 8, 24, and 48 hours after administration. The sample uses a human IgG detection kit (Cisbio) to detect the IgG concentration. The data obtained was processed with GraphPad Prism 9 and analyzed using the single-factor ANOVA method.

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Abstract

提供了一种抗FcRn抗体、其抗原结合片段及其医药用途，具体而言，提供了一种抗FcRn抗体、其抗原结合片段、及其治疗自身免疫疾病、炎性病症的医药用途。

Description

抗FcRn抗体、其抗原结合片段及其医药用途
本申请要求2020年02月10日提交的中国专利申请(申请号202010083892.1)的优先权。本申请引用上述中国专利申请的全文。
技术领域
本申请涉及抗FcRn抗体、其抗原结合片段，包含所述抗FcRn抗体CDR区的嵌合抗体、人源化抗体，包含所述抗FcRn抗体及其抗原结合片段的药物组合物，及其用于治疗或预防自身免疫疾病、炎症疾病的医药用途。
背景技术
新生儿Fc受体(FcRn)是非共价异二聚体，通常存在于内皮细胞和上皮细胞的内体中，其是I类主要组织相容性复合物(MHC)样重链与可溶性β2微球蛋白(β2M)轻链组合而成的细胞内运输整合膜Fc受体。具体地，FcRn具有约46kD的分子量，由三个重链α结构域(α1、α2和α3)和单个β2M结构域组成，具有单条糖链、单次跨膜、相对短的胞质尾的特点。
FcRn最初被认定为在新生儿生命中起到重要作用。进一步研究发现，FcRn在成人体内也起到对免疫球蛋白G(IgG)和白蛋白半衰期的延长作用(Ghetie等人，1996；Junghans和Anderson,1996；Israel等人，1996)。在正常条件下，人类大部分IgG(不包括IgG3同种型)在血清中的半寿期是约22-23天，相对于其它血浆蛋白的血清半衰期较长。对于IgG的这种较长的血清半衰期，通过内吞过程进入细胞的IgG可以在略酸性pH(约6.0)与内体中的FcRn强结合以避开降解性溶酶体途径。当IgG-FcRn复合物循环到质膜时，IgG在血流中在略碱性pH(约7.4)从FcRn快速解离。通过这种受体介导的再生机制，FcRn有效地在溶酶体中拯救IgG免于降解，从而延长IgG的半衰期(Roopenian等人，J.Immunol.170:3528,2003)。
关于FcRn与IgG稳态的相关性，小鼠实验已经证明，使用特定药物分子阻断FcRn与IgG的结合，可有效降低血液内IgG的含量(Vaccaro等,Nat Biotechnol.23:1283,2005)。此外，对小鼠进行工程化，敲除至少部分编码β2M和FcRn重链的基因，使其不表达所述蛋白质。在这些小鼠中，IgG的血清半衰期和浓度大幅度降低，这也提示了IgG稳态的FcRn依赖性机制。在这些FcRn敲除小鼠中生成抗人FcRn抗体，可以阻止IgG与FcRn的结合。对IgG与FcRn结合的抑制，阻止了IgG再生，从而可以治疗由自身抗体引起的自身免疫疾病。在自身免疫性皮肤大疱病的小鼠模型中显示了这种可能性(Li等,J.Clin.Invest.115:3440,2005)。因此，阻断或拮抗IgG与FcRn结合的药物能够或有希望用于治疗或预防IgG介导的自身免疫疾病和炎性疾病的方法。
自身免疫疾病患者因其免疫系统异常而攻击自身正常组织、器官或其他体内组分。神经系统、消化系统、内分泌系统、皮肤、骨骼系统和血管组织等几乎身体的各个部位都可能出现自身免疫疾病，大多数尚未发现具体的病因。研究揭示多种自身免疫疾病患者血液内都有过量表达的IgG型自身抗体。由IgG型自身抗 体引起的自身免疫疾病，包括免疫中性粒细胞减少症、重症肌无力(MG)、多发性硬化、类风湿性关节炎、狼疮、特发性血小板减少性紫癜(ITP)、寻常天疱疮、Guillain-Barre综合征、自身免疫脑炎、癫痫、狼疮性肾炎或膜性肾病或其它。静脉滴注免疫球蛋白(IVIG)可应用于部分上述疾病，但与之伴随头痛、皮炎、过敏反应、急性肾衰竭等医学并发症，昂贵且有暴露于血液制品的风险。此外，多克隆抗D免疫球蛋白、皮质类固醇类、免疫抑制剂(包括化疗药)、细胞因子、血浆分离、体外抗体吸附(例如，使用Prosorba柱)、手术介入如脾切除术等也用于治疗自身免疫疾病，同样，这些疗法复杂、疗效不完全且昂贵。
目前有多家跨国制药公司、生物技术公司(如Shire、UCB、Argenx)在研发针对FcRn-IgG这一机理的单克隆抗体或抗原结合蛋白，Harbour BioMed公司通过与韩国HanAll生物医药公司合作，在中国区域展开药物开发。部分在研药物分子已经进行到临床阶段，对不同自身免疫疾病的适应症展开临床研究，临床数据显示达到了不同程度上缓解疾病症状的效果。因此，需要开发有效特异性阻断IgG与FcRn结合的抗体或药物组合物，有潜力应用于多种自身免疫疾病。
虽然抗FcRn抗体在WO2016/142782、WO2018/083122、WO2014/019727、WO2018/229249、CN106459215B、CN104364265B、WO2009/131702、US7662928B、WO2006/118772、WO2012/167039等中公开，但仍需要开发对FcRn具有高亲和力、低免疫原性，甚至可以以低剂量施用的抗人FcRn抗体。
发明内容
本公开提供一种FcRn结合蛋白、抗FcRn抗体或其抗原结合片段，其编码核酸、载体、宿主细胞、药物组合物、其用于治疗或延缓自身免疫疾病的方法，及其检测用途。
抗FcRn抗体或其抗原结合片段
本公开提供抗FcRn抗体或抗原结合片段，其包含重链可变区(VH)和轻链可变区(VL)，其中：
所述VH的HCDR1、HCDR2、HCDR3分别含有SEQ ID No：4中的HCDR1、HCDR2、HCDR3，所述VL的LCDR1、LCDR2、LCDR3分别含有SEQ ID No：5中的LCDR1、LCDR2、LCDR3；
所述VH的HCDR1、HCDR2、HCDR3分别含有SEQ ID No：6中的HCDR1、HCDR2、HCDR3，所述VL的LCDR1、LCDR2、LCDR3分别含有SEQ ID No：7中的LCDR1、LCDR2、LCDR3；
所述VH的HCDR1、HCDR2、HCDR3分别含有SEQ ID No：8中的HCDR1、HCDR2、HCDR3，所述VL的LCDR1、LCDR2、LCDR3分别含有SEQ ID No：9中的LCDR1、LCDR2、LCDR3；
所述VH的HCDR1、HCDR2、HCDR3分别含有SEQ ID No：10中的HCDR1、HCDR2、HCDR3，所述VL的LCDR1、LCDR2、LCDR3分别含有SEQ ID No：11中的LCDR1、LCDR2、LCDR3；
所述VH的HCDR1、HCDR2、HCDR3分别含有SEQ ID No：12中的HCDR1、 HCDR2、HCDR3，所述VL的LCDR1、LCDR2、LCDR3分别含有SEQ ID No：13中的LCDR1、LCDR2、LCDR3；
所述VH的HCDR1、HCDR2、HCDR3分别含有SEQ ID No：14中的HCDR1、HCDR2、HCDR3，所述VL的LCDR1、LCDR2、LCDR3分别含有SEQ ID No：15中的LCDR1、LCDR2、LCDR3；
所述VH的HCDR1、HCDR2、HCDR3分别含有SEQ ID No：16中的HCDR1、HCDR2、HCDR3，所述VL的LCDR1、LCDR2、LCDR3分别含有SEQ ID No：17中的LCDR1、LCDR2、LCDR3；
所述VH的HCDR1、HCDR2、HCDR3分别含有SEQ ID No：18中的HCDR1、HCDR2、HCDR3，所述VL的LCDR1、LCDR2、LCDR3分别含有SEQ ID No：19中的LCDR1、LCDR2、LCDR3；
所述VH的HCDR1、HCDR2、HCDR3分别含有SEQ ID No：96、99、103、104、107任一中的HCDR1、HCDR2、HCDR3，所述VL的LCDR1、LCDR2、LCDR3分别含有SEQ ID No：13中的LCDR1、LCDR2、LCDR3；
所述VH的HCDR1、HCDR2、HCDR3分别含有SEQ ID No：89、93、94任一中的HCDR1、HCDR2、HCDR3，所述VL的LCDR1、LCDR2、LCDR3分别含有SEQ ID No：76中的LCDR1、LCDR2、LCDR3；
所述VH的HCDR1、HCDR2、HCDR3分别含有SEQ ID No：87中的HCDR1、HCDR2、HCDR3，所述VL的LCDR1、LCDR2、LCDR3分别含有SEQ ID No：84中的LCDR1、LCDR2、LCDR3；
所述VH的HCDR1、HCDR2、HCDR3分别含有SEQ ID No：87-110任一中的HCDR1、HCDR2、HCDR3，所述VL的LCDR1、LCDR2、LCDR3分别含有SEQ ID No：76-86任一中的LCDR1、LCDR2、LCDR3；
上述CDR是根据Kabat、IMGT、Chothia、AbM或Contact编号系统定义的；一些具体实施方案中，CDR是根据Kabat编号系统定义的；另一些实施方案中，CDR是根据AbM编号系统定义的。
本公开提供抗FcRn抗体或其抗原结合片段，包含重链可变区(VH)和/或轻链可变区(VL)，其中：
重链可变区包含选自以下的互补决定区(CDR)：
HCDR1的氨基酸序列如GY X 1F X 2(SEQ ID No：182)或GYX 1F X 2X 3X 4X 5IA(SEQ ID No：173)所示，其中，X 1选自S、N、D、V、A、P、K，X 2选自T、N、K、R、S、A，X 3选自G、N、K、S、E，X 4选自Y、H、S、N、T，X 5选自W、Y、F、M；和/或
HCDR2的氨基酸序列如X 6IX 7PDX 8SNTI(SEQ ID No：174)或X 6IX 7PDX 8SNTIYSPSFRG(SEQ ID No：175)所示，其中，X 6选自I、L、V，X 7选自Y、S、T、G、A，X 8选自N、R、K、A、F；和/或
HCDR3的氨基酸序列如FGGPTFAQWYFDY(SEQ ID No：40)所示；
轻链可变区包含选自以下CDR：
LCDR1的氨基酸序列如TGSSGSIASNYVX 9(SEQ ID No：176)所示，其中， X 9选自Q、S、N、A、V、T；和/或
LCDR2的氨基酸序列如X 10DNQRAS(SEQ ID No：177)所示，其中，X 10选自E、S、A、W、C；和/或
LCDR3的氨基酸序列如QSYDSSSHNWV(SEQ ID No：43)所示。
在一些实施方案中，抗FcRn抗体或其抗原结合片段的HCDR1的氨基酸序列如SEQ ID No：182所示，HCDR2的氨基酸序列如SEQ ID No：175所示，HCDR3的氨基酸序列如SEQ ID No：40所示；LCDR1的氨基酸序列如SEQ ID No：176所示，LCDR2的氨基酸序列如SEQ ID No：177所示，LCDR3的氨基酸序列如SEQ ID No：43所示。所述实施方案采用Kabat编号系统。
在另一些实施方案中，抗FcRn抗体或其抗原结合片段的HCDR1的氨基酸序列如SEQ ID No：173所示，HCDR2的氨基酸序列如SEQ ID No：174所示，HCDR3的氨基酸序列如SEQ ID No：40所示；LCDR1的氨基酸序列如SEQ ID No：176所示，LCDR2的氨基酸序列如SEQ ID No：177所示，LCDR3的氨基酸序列如SEQ ID No：43所示。所述实施方案采用AbM编号系统。
在一些具体实施方案中，X 1为S，X 2为T，X 3为G，X 4为Y，X 5为W。
在一些具体实施方案中，X 1为D，X 2为K，X 3为K，X 4为S，X 5为F。
在一些具体实施方案中，X 1为N，X 2为N，X 3为K，X 4为H，X 5为Y。
在一些具体实施方案中，X 1为S，X 2为K，X 3为N，X 4为Y，X 5为Y。
在一些具体实施方案中，X 1为V，X 2为N，X 3为K，X 4为Y，X 5为F。
在一些具体实施方案中，X 1为D，X 2为T，X 3为K，X 4为T，X 5为M。
在一些具体实施方案中，X 6为I，X 7为Y，X 8为N，X 9为Q，X 10为E。
在一些具体实施方案中，X 6为L，X 7为S，X 8为A，X 9为S，X 10为S。
在一些具体实施方案中，X 6为L，X 7为A，X 8为R，X 9为S，X 10为S。
在一些具体实施方案中，X 6为V，X 7为S，X 8为R，X 9为S，X 10为S。
在一些具体实施方案中，X 6为L，X 7为T，X 8为R，X 9为A，X 10为S。
在一些具体实施方案中，X 6为L，X 7为S，X 8为R，X 9为Q，X 10为W。
一些实施方案中，抗FcRn抗体或其抗原结合片段的VH的HCDR1的氨基酸序列如SEQ ID No：20、26、32、38、44、50、56、62、68、129-141、150-165之一所示；
HCDR2的氨基酸序列如SEQ ID No：21、27、33、39、45、51、57、63、69、121-128、142-149、170之一所示；
HCDR3的氨基酸序列如SEQ ID No：22、28、34、40、46、52、58、64之一所示；
VL的LCDR1的氨基酸序列如SEQ ID No：23、29、35、41、47、53、59、65、111、115-119之一所示；
LCDR2的氨基酸序列如SEQ ID No：24、30、36、42、48、54、60、66、112、113、114、116、120之一所示；
LCDR3的氨基酸序列如SEQ ID No：25、31、37、43、49、55、61、67之一所示。
一些实施方案中，抗FcRn抗体或其抗原结合片段的HCDR1、HCDR2、HCDR3的氨基酸序列选自如(1)-(15)中的任一项：
(1)如SEQ ID No：20-22所示；
(2)如SEQ ID No：26-28所示；
(3)如SEQ ID No：32-34所示；
(4)如SEQ ID No：38-40所示；
(5)如SEQ ID No：44-46所示；
(6)如SEQ ID No：50-52所示；
(7)如SEQ ID No：56-58所示；
(8)如SEQ ID No：62-64所示；
(9)如SEQ ID No：68、69、40所示；
(10)如SEQ ID No：38所示的HCDR1，如SEQ ID No：121-128之一所示的HCDR2，如SEQ ID No：40所示的HCDR3；
(11)如SEQ ID No：129-141之一所示的HCDR1，如SEQ ID No：39所示的HCDR2，如SEQ ID No：40所示的HCDR3；
(12)如SEQ ID No：68所示的HCDR1，如SEQ ID No：142-149之一所示的HCDR2，如SEQ ID No：40所示的HCDR3；
(13)如SEQ ID No：150-165之一所示的HCDR1，如SEQ ID No：69所示的HCDR2，如SEQ ID No：40所示的HCDR3；
(14)如SEQ ID No：38、170、40所示；
(15)如SEQ ID No：133、170、40所示；
和LCDR1、LCDR2、LCDR3的氨基酸序列选自如(1’)-(19’)中的任一项：
(1’)如SEQ ID No：23-25所示；
(2’)如SEQ ID No：29-31所示；
(3’)如SEQ ID No：35-37所示；
(4’)如SEQ ID No：41-43所示
(5’)如SEQ ID No：47-49所示；
(6’)如SEQ ID No：53-55所示；
(7’)如SEQ ID No：59-61所示；
(8’)如SEQ ID No：65-67所示；
(9’)如SEQ ID No：111、112、43所示；
(10’)如SEQ ID No：111、113、43所示；
(11’)如SEQ ID No：111、114、43所示；
(12’)如SEQ ID No：115、113、43所示；
(13’)如SEQ ID No：115、112、43所示；
(14’)如SEQ ID No：115、116、43所示；
(15’)如SEQ ID No：117、112、43所示；
(16’)如SEQ ID No：118、42、43所示；
(17’)如SEQ ID No：41、116、43所示；
(18’)如SEQ ID No：119、120、43所示；
(19’)如SEQ ID No：119、116、43所示。
一些实施方案中，抗FcRn抗体或其抗原结合片段包含选自以下任一项的CDR：
(a)HCDR1、HCDR2、HCDR3的氨基酸序列如SEQ ID No：20-21所示，LCDR1、LCDR2、LCDR3的氨基酸序列如SEQ ID No：23-25所示；
(b)HCDR1、HCDR2、HCDR3的氨基酸序列如SEQ ID No：26-28所示，LCDR1、LCDR2、LCDR3的氨基酸序列如SEQ ID No：29-31所示；
(c)HCDR1、HCDR2、HCDR3的氨基酸序列如SEQ ID No：32-34所示，LCDR1、LCDR2、LCDR3的氨基酸序列如SEQ ID No：35-37所示；
(d)HCDR1、HCDR2、HCDR3的氨基酸序列如SEQ ID No：38-40所示，LCDR1、LCDR2、LCDR3的氨基酸序列如SEQ ID No：41-43所示；
(e)HCDR1、HCDR2、HCDR3的氨基酸序列如SEQ ID No：44-46所示，LCDR1、LCDR2、LCDR3的氨基酸序列如SEQ ID No：47-49所示；
(f)HCDR1、HCDR2、HCDR3的氨基酸序列如SEQ ID No：50-52所示，LCDR1、LCDR2、LCDR3的氨基酸序列如SEQ ID No：53-55所示；
(g)HCDR1、HCDR2、HCDR3的氨基酸序列如SEQ ID No：56-58所示，LCDR1、LCDR2、LCDR3的氨基酸序列如SEQ ID No：59-61所示；
(h)HCDR1、HCDR2、HCDR3的氨基酸序列如SEQ ID No：62-64所示，LCDR1、LCDR2、LCDR3的氨基酸序列如SEQ ID No：65-67所示；
(i)HCDR1、HCDR2、HCDR3的氨基酸序列如SEQ ID No：68、69、40所示，LCDR1、LCDR2、LCDR3的氨基酸序列如SEQ ID No：41-43所示；
(j)HCDR1、HCDR2、HCDR3的氨基酸序列如SEQ ID No：38、170、40所示，LCDR1、LCDR2、LCDR3的氨基酸序列如SEQ ID No：41-43所示；
(k)HCDR1、HCDR2、HCDR3的氨基酸序列如SEQ ID No：133、170、40所示，LCDR1、LCDR2、LCDR3的氨基酸序列如SEQ ID No：41-43所示；
(l)HCDR1、HCDR2、HCDR3的氨基酸序列如SEQ ID No：151、69、40所示，LCDR1、LCDR2、LCDR3的氨基酸序列如SEQ ID No：41-43所示；
(m)HCDR1、HCDR2、HCDR3的氨基酸序列如SEQ ID No：154、69、40所示，LCDR1、LCDR2、LCDR3的氨基酸序列如SEQ ID No：41-43所示；
(n)HCDR1、HCDR2、HCDR3的氨基酸序列如SEQ ID No：158、69、40所示，LCDR1、LCDR2、LCDR3的氨基酸序列如SEQ ID No：41-43所示；
(o)HCDR1、HCDR2、HCDR3的氨基酸序列如SEQ ID No：159、69、40所示，LCDR1、LCDR2、LCDR3的氨基酸序列如SEQ ID No：41-43所示；
(p)HCDR1、HCDR2、HCDR3的氨基酸序列如SEQ ID No：162、69、40所示，LCDR1、LCDR2、LCDR3的氨基酸序列如SEQ ID No：41-43所示；
(q)HCDR1、HCDR2、HCDR3的氨基酸序列如SEQ ID No：38、123、40所示，LCDR1、LCDR2、LCDR3的氨基酸序列如SEQ ID No：111、112、43所示；
(r)HCDR1、HCDR2、HCDR3的氨基酸序列如SEQ ID No：38、127、40 所示，LCDR1、LCDR2、LCDR3的氨基酸序列如SEQ ID No：111、112、43所示；
(s)HCDR1、HCDR2、HCDR3的氨基酸序列如SEQ ID No：38、128、40所示，LCDR1、LCDR2、LCDR3的氨基酸序列如SEQ ID No：111、112、43所示；
(t)HCDR1、HCDR2、HCDR3的氨基酸序列如SEQ ID No：38、124、40所示，LCDR1、LCDR2、LCDR3的氨基酸序列如SEQ ID No：117、112、43所示；
(u)HCDR1、HCDR2、HCDR3的氨基酸序列如SEQ ID No：38、121、40所示，LCDR1、LCDR2、LCDR3的氨基酸序列如SEQ ID No：41、116、43所示。
一些实施方案中，抗FcRn抗体或其抗原结合片段的VH的氨基酸序列如SEQ ID No：4、6、8、10、12、14、16、18、76-86、166、168、169之一所示或与之具有至少80％、90％、95％、96％、97％、98％、99％同一性；和/或
VL的氨基酸序列如SEQ ID No：5、7、9、11、13、15、17、19、76-86、167之一所示或与之具有至少80％、90％、95％、96％、97％、98％、99％同一性。
一些具体实施方案中，抗FcRn抗体或其抗原结合片段的VHVH的氨基酸序列如SEQ ID No：4所示或与之具有至少80％、90％、95％、96％、97％、98％、99％同一性，VL的氨基酸序列如SEQ ID No：5所示或与之具有至少80％、90％、95％、96％、97％、98％、99％同一性；或
VH的氨基酸序列如SEQ ID No：6所示或与之具有至少80％、90％、95％、96％、97％、98％、99％同一性，VL的氨基酸序列如SEQ ID No：7所示或与之具有至少80％、90％、95％、96％、97％、98％、99％同一性；或
VH的氨基酸序列如SEQ ID No：8所示或与之具有至少80％、90％、95％、96％、97％、98％、99％同一性，VL的氨基酸序列如SEQ ID No：9所示或与之具有至少80％、90％、95％、96％、97％、98％、99％同一性；或
VH的氨基酸序列如SEQ ID No：10所示或与之具有至少80％、90％、95％、96％、97％、98％、99％同一性，VL的氨基酸序列如SEQ ID No：11所示或与之具有至少80％、90％、95％、96％、97％、98％、99％同一性；或
VH的氨基酸序列如SEQ ID No：12所示或与之具有至少80％、90％、95％、96％、97％、98％、99％同一性，VL的氨基酸序列如SEQ ID No：13所示或与之具有至少80％、90％、95％、96％、97％、98％、99％同一性；或
VH的氨基酸序列如SEQ ID No：14所示或与之具有至少80％、90％、95％、96％、97％、98％、99％同一性，VL的氨基酸序列如SEQ ID No：15所示或与之具有至少90％同一性；或
VH的氨基酸序列如SEQ ID No：16所示或与之具有至少80％、90％、95％、96％、97％、98％、99％同一性，VL的氨基酸序列如SEQ ID No：17所示或与之具有至少90％同一性；或
VH的氨基酸序列如SEQ ID No：18所示或与之具有至少80％、90％、95％、96％、97％、98％、99％同一性，VL的氨基酸序列如SEQ ID No：19所示或与之具有至少80％、90％、95％、96％、97％、98％、99％同一性；或
VH的氨基酸序列如SEQ ID No：96、99、103、104、107之一所示或与之具有至少80％、90％、95％、96％、97％、98％、99％同一性，VL的氨基酸序列如 SEQ ID No：13所示或与之具有至少80％、90％、95％、96％、97％、98％、99％同一性；
VH的氨基酸序列如SEQ ID No：89、93、94之一所示或与之具有至少80％、90％、95％、96％、97％、98％、99％同一性，VL的氨基酸序列如SEQ ID No：76所示或与之具有至少80％、90％、95％、96％、97％、98％、99％同一性；
VH的氨基酸序列如SEQ ID No：87所示或与之具有至少80％、90％、95％、96％、97％、98％、99％同一性，VL的氨基酸序列如SEQ ID No：84所示或与之具有至少80％、90％、95％、96％、97％、98％、99％同一性；
VH的氨基酸序列如SEQ ID No：87-110之一所示或与之具有至少80％、90％、95％、96％、97％、98％、99％同一性，VL的氨基酸序列如SEQ ID No：76-86之一所示或与之具有至少80％、90％、95％、96％、97％、98％、99％同一性。
一些实施方案中，如前述的抗FcRn抗体或其抗原结合片段，其中：
重链氨基酸序列如SEQ ID No：178-180任一所示，或与之具有至少70％、80％、90％、95％、96％、97％、98％、99％同一性；
轻链氨基酸序列如SEQ ID No：181所示，或与之具有至少70％、80％、90％、95％、96％、97％、98％、99％同一性。
一些实施方案中，上述抗FcRn抗体或其抗原结合片段为鼠源抗体、嵌合抗体、全人抗体、人源化抗体。其可以为全长抗体或其片段。
一些实施方案中，上述抗FcRn抗体或其抗原结合片段为鼠源抗体或其片段。其轻链可变区包含鼠源κ、λ链或其变体的轻链FR区和/或轻链恒定区。一些具体实施方案中，所述鼠源抗FcRn抗体或其抗原结合片段包含鼠源IgG1、IgG2、IgG3、IgG4或其变体的重链FR区和/或重链恒定区。
一些实施方案中，上述抗FcRn抗体或其抗原结合片段为嵌合抗体或其片段。其包含人源κ、λ链或其变体的轻链FR区和/或轻链恒定区，和/或人源IgG1、IgG2、IgG3或IgG4或其变体的重链FR区和/或重链恒定区。
一些实施方案中，提供一种如上所述的抗人FcRn抗体或其抗原结合片段，其为人源化抗体或其片段、全人抗体或其片段。
一些具体实施方案中，所述的抗FcRn抗体或其抗原结合片段，其重链可变区可以与人源CH1或小鼠CH1连接。一些实施方案中，所述的抗FcRn抗体或其抗原结合片段，其包含恒定区Fc，所述Fc为IgG1、IgG2、IgG3、IgG4或其变体(如IgG4P，即IgG4的S241P突变体)的Fc。一些具体实施方案中，Fc为氨基酸序列为SEQ ID No：75所示的小鼠IgG1的Fc，或为氨基酸序列为SEQ ID No：172所示的人IgG4P的Fc。
一些实施方案中，抗FcRn抗体的抗原结合片段为Fab、Fv、sFv、Fab’、F(ab’) 2、线性抗体、单链抗体、scFv、sdAb、sdFv、纳米抗体、肽抗体peptibody、结构域抗体和多特异性抗体(双特异性抗体、diabody、triabody和tetrabody、串联二-scFv、串联三-scFv)，例如具体为scFv、Fv、Fab或Fab’片段。
一些实施方案中，所述的抗FcRn抗体或其抗原结合片段缀合于聚合物。一些实施方案中，聚合物选自淀粉、白蛋白和聚乙二醇(PEG)。当所述聚合物是PEG 时，可以是具有5-50kDa、5-10kDa、5-15kDa、10-20kDa、10-30kDa、20-30kDa、10-40kDa分子量的PEG。
一些实施方案中，提供一种抗FcRn抗体或其抗原结合片段，其结合与前述抗FcRn抗体或其抗原结合片段相同的表位。
一些实施方案中，提供一种抗FcRn抗体或其抗原结合片段，其阻断前述抗FcRn抗体或其抗原结合片段与人FcRn的结合。
另一些实施方案中，提供一种抗FcRn抗体或其抗原结合片段，其与人FcRn的结合被前述抗FcRn抗体或其抗原结合片段阻断。
一些实施方案中，前述抗FcRn抗体或其抗原结合片段与人FcRn(或其表位)结合。
一些实施方案中，前述抗FcRn抗体或其抗原结合片段阻断人IgG与人FcRn的结合。
一些实施方案中，前述抗FcRn抗体或其抗原结合片段不结合β2微球蛋白。
一些实施方案中，提供抗FcRn抗体或其抗原结合片段，与前述抗FcRn抗体或其抗原结合片段的重链和/或轻链具有至少70％、80％、85％、90％、95％、96％、97％、98％或99％同一性。
一些实施方案中，提供抗FcRn抗体或其抗原结合片段变体，在前述抗FcRn抗体或其抗原结合片段的重链可变区和/或轻链可变区包含0、1、2、3、4、5、6、7、8、9或10个氨基酸变化。所述氨基酸变化可以是可变区中的氨基酸残基保守性置换。
FcRn结合蛋白
本公开还提供FcRn结合蛋白，包含前述本公开提供的抗FcRn抗体或其抗原结合片段。
一些实施方案中，所述FcRn结合蛋白进一步包含一个或多个效应分子，包括但不限于：抗肿瘤剂、药物、毒素、生物活性蛋白(例如酶)、其它抗体或抗体片段、合成或天然存在的聚合物、核酸及其片段例如DNA、RNA及其片段、放射性核素(特别地放射性碘化物)、放射性同位素、螯合金属、纳米颗粒和报道基团(例如荧光化合物)、或可通过NMR或ESR光谱分析检测的化合物。
一些实施方案中，所述效应分子以缀合方式与抗FcRn抗体或其抗原结合片段连接。
一些实施方案中，效应分子是聚合物，例如任选地取代的直链或支链聚亚烷基、聚亚烯基或聚氧化亚烷基聚合物或分支多糖或未分支多糖，例如同聚或异聚多糖，例如任选地取代的直链或支链聚(乙二醇)、聚(丙二醇)、聚(乙烯醇)或其衍生物、乳糖、直链淀粉、葡聚糖、糖原或其衍生物、人血清白蛋白或其片段。
多核苷酸和载体
本公开提供经分离的多核苷酸，其编码本公开的抗FcRn抗体或其抗原结合片段。所述多核苷酸可以是DNA或RNA。
本公开提供含有如上所述的多核苷酸的表达载体，表达载体可以是真核表达 载体、原核表达载体、病毒载体，例如质粒、粘粒、噬菌体。
宿主细胞
本公开提供用如上所述的表达载体转化的宿主细胞，其可以是真核细胞、原核细胞。
一些实施方案中，所述宿主细胞为细菌、酵母菌、哺乳动物细胞。一些具体实施方案中，所述宿主细胞为大肠杆菌、毕赤酵母、中国仓鼠卵巢(CHO)细胞或人胚肾(HEK)293细胞。
制备方法
本公开提供一种用于制备抗FcRn抗体或其抗原结合片段的方法，包括：在如前所述的宿主细胞中表达该抗体、其抗原结合片段或复合物(例如融合蛋白)，并自该宿主细胞中分离该抗体、其抗原结合片段或复合物(例如融合蛋白)。可选地，还可以包含纯化步骤，例如，用含调整过的缓冲液的A或G Sepharose FF柱进行纯化，洗去非特异性结合的组分，再用PH梯度法洗脱结合的抗体，用SDS-PAGE检测，收集。可选地，用常规方法进行过滤浓缩。可溶的混合物和多聚体，也可以用常规方法去除，比如分子筛、离子交换。得到的产物需立即冷冻，如-70℃，或者冻干。
生产和纯化抗体和抗原结合片段的方法在现有技术中熟知和能找到，如冷泉港的抗体实验技术指南(5-8章和15章)。如，可以用人FcRn或其片段免疫小鼠，所得到的抗体能被复性、纯化，并且可以用常规的方法进行氨基酸测序。抗原结合片段同样可以用常规方法制备。发明所述的抗体或抗原结合片段用基因工程方法在非人源的CDR区加上一个或多个人FR区。人FR种系序列例如可以从ImMunoGeneTics(IMGT)的网站得到。
本公开工程化的抗体或抗原结合片段可用常规方法制备和纯化。比如，编码重链和轻链的cDNA序列，可以克隆并重组至表达载体。重组的免疫球蛋白表达载体可以稳定地转染CHO细胞。哺乳动物类表达系统会导致抗体的糖基化，特别是在Fc区的高度保守N端。通过表达与人源抗原特异性结合的抗体得到稳定的克隆。阳性的克隆在生物反应器的无血清培养基中扩大培养以生产抗体。分泌了抗体的培养液可以用常规技术纯化、收集。抗体可用常规方法进行过滤浓缩。可溶的混合物和多聚体，也可以用常规方法去除，比如分子筛，离子交换。
组合物
本公开提供组合物，例如药物组合物，其含有治疗有效量的如上所述的抗FcRn抗体或其抗原结合片段和可药用的赋形剂、稀释或载体。
在一些具体实施方式中，所述药物组合物单位计量中可含有0.01至99重量％的抗FcRn抗体或其抗原结合片段，或药物组合物单位剂量中含抗FcRn抗体或其抗原结合片段的量为0.1-2000mg，在一些具体实施方式中为1-1000mg。
治疗方法和制药用途
本公开提供选自以下的任一项或组合在制备药物中的用途：根据本公开的抗FcRn抗体或其抗原结合片段、根据本公开的药物组合物。一些实施方案中，所述药物用于治疗或预防自身免疫疾病或延缓自身免疫疾病进展。
本公开提供一种治疗或预防自身免疫疾病或延缓自身免疫疾病进展的方法，该方法包括给予受试者治疗或延缓疾病有效量的根据本公开的抗FcRn抗体或其抗原结合片段、或根据本公开的药物组合物。
本公开的抗FcRn抗体或其抗原结合片段、药物组合物可以应用于IgG和FcRn介导的全部自身免疫疾病。
上述自身免疫疾病包括但不限于：急性播散性脑脊髓炎(ADEM)、急性坏死性出血性白质脑炎、阿狄森病、丙种球蛋白缺乏血症、斑秃、淀粉样变性、ANCA相关性脉管炎、强直性脊柱炎、抗-GBM/抗-TBM肾炎、抗磷脂综合征(APS)、自身免疫性血管性水肿、自身免疫性再生障碍性贫血、自身免疫性家族性自主神经异常、自身免疫性肝炎、自身免疫性高脂血症、自身免疫性免疫缺乏症、自身免疫性内耳病(AIED)、自身免疫性心肌炎、自身免疫性胰腺炎、自身免疫性视网膜病变、自身免疫性血小板减少性紫癜(ATP)、自身免疫性甲状腺病、自身免疫性荨麻疹、Axonal&nal神经病、巴洛病、贝赫切特病(Behcet’sdisease)、大疱性类天疱疮、心肌病、Castleman病、乳糜泻、美洲锥虫病(Chagas disease)、慢性炎症性脱髓鞘性多发性神经病(CIDP)、慢性复发性多病灶性骨髓炎(CRMO)、变应性肉芽肿性血管炎(Churg-Strauss syndrome)、瘢痕性类天疱疮/良性粘膜类天疱疮、克罗恩病、Cogans综合征、冷凝集素疾病、先天性心脏传导阻滞、柯萨奇病毒性心肌炎、CREST病、特发性混合性冷球蛋白血症、脱髓鞘性神经病、疱疹样皮炎、皮肌炎、德维克病(视神经脊髓炎)、扩张型心肌病、盘状狼疮、Dressler综合征、子宫内膜异位症、嗜酸性血管中心性纤维变性、嗜酸性筋膜炎、结节性红斑、实验性过敏性脑脊髓炎、伊文思综合征、纤维化肺泡炎、巨细胞动脉炎(颞动脉炎)、肾小球肾炎、肺出血性肾炎综合征、肉芽肿性血管炎(GPA)参见韦格纳氏病和格雷夫斯病、格-巴二氏综合征、Hashimoto脑炎、Hashimoto甲状腺炎、溶血性贫血、亨诺-许兰氏紫癜、妊娠疱疹、低丙球蛋白血症、特发性低补体血肾小管间质性肾炎(Idiopathic hypocomplementemic tubulointestitial nephritis)、特发性血小板减少性紫癜(ITP)、IgA肾病、IgG4相关疾病、IgG4相关硬化性疾病、免疫调节性脂蛋白、炎性主动脉瘤、炎性假瘤、包涵体肌炎、胰岛素依赖性糖尿病(I型)、间质性膀胱炎、少年关节炎、青少年糖尿病、川畸综合征、Kuttner肿瘤、朗-伊二氏综合征、白细胞分裂性血管炎、扁平苔癣、硬化性苔癣、木样结膜炎、线性IgA疾病(LAD)、狼疮(SLE)、莱姆病、慢性、纵隔纤维变性、梅尼埃病、显微镜下多血管炎、米库利兹综合征、混合性结缔组织病(MCTD)、莫伦溃疡、穆-哈二氏病、多灶性纤维硬化、多发性硬化、重症肌无力、肌炎、发作性睡病、视神经脊髓炎(Devic氏)、中性粒细胞减少、眼瘢痕性类天疱疮、视神经炎、奥蒙德病(腹膜后纤维化)、复发性风湿病、PANDAS(与链球菌相关的儿童自身免疫性神经精神性障碍)、副肿瘤性小脑变性、副蛋白血症多发性神经病(Paraproteinemic polyneuropathy)、阵发性睡眠性血红蛋白尿症(PNH)、帕-罗二氏综合征、Parsonnage-Turner综合征、睫状体扁平部炎(周边葡萄膜炎)、寻常天疱疮、主动脉周围炎、动脉周围炎、周围神经病、静脉周脑脊髓炎、恶性贫血、POEMS综 合征、结节性多发性动脉炎、I、II和III型自身免疫性多腺体综合征、风湿性多肌痛、多发性肌炎、心肌梗塞后综合征、心包切开术后综合征、黄体酮皮炎、原发性胆汁性肝硬变、原发性硬化性胆管炎、银屑病、银屑病关节炎、特发性肺纤维化、坏疽性脓皮病、单纯红细胞再生障碍、雷诺现象、反射性交感神经营养障碍、莱特尔氏综合征、复发性多软骨炎、不宁腿综合征、腹膜后纤维化(奥蒙德病)、风湿热、风湿性关节炎、里德耳甲状腺炎、结节病、施密特综合征、巩膜炎、硬皮病、干燥综合征、精子和睾丸自身免疫、僵人综合征、亚急性细菌性心内膜炎(SBE)、Susac综合征、交感性眼炎、高安动脉炎、颞动脉炎/巨细胞动脉炎、血栓性血小板减少性紫癜(TTP)、托-亨二氏综合征、横贯性脊髓炎、溃疡性结肠炎、未分化结缔组织病(UCTD)、葡萄膜炎、血管炎、水疱大疱性皮肤病、白癜风、Waldenstrom巨球蛋白血症、温暖性特发性溶血性贫血和韦格纳氏肉芽肿(现在称作肉芽肿性血管炎(GPA))。
检测
本公开提供检测FcRn的组合物，所述组合物包含根据本公开的抗FcRn抗体或其抗原结合片段。本公开还提供用于体内或体外检测FcRn的方法、系统或装置，其包括用本公开的抗FcRn抗体或其抗原结合片段处理样品。
一些实施方案中，体外检测方法、系统或装置可能例如包括：
(1)使样品与结合FcRn的抗体或其抗原结合片段接触；
(2)检测在结合FcRn的抗体或其抗原结合片段和样品之间形成的复合物；和/或
(3)使参比样品(例如，对照样品)与抗体接触；和
(4)通过与参比样品比较，确定复合物形成的程度。如与对照样品或受试者中相比，样品或受试者中复合物形成的变化(例如，统计学上的显著变化)表示样品中存在FcRn。
另一些实施方案中，体内检测方法、系统或装置可以包括：
(1)向受试者施用结合FcRn的抗体或其抗原结合片段；和
(2)检测在结合FcRn的抗体或其抗原结合片段和受试者之间复合物的形成。
检测可以包括确定形成复合物的位置或时间。用可检测物质对FcRn抗体标记，通过对所述标记检测以实现对结合FcRn抗体的物质(例如FcRn)的检测。合适的可检测物质包括多种酶、辅基、荧光物质、发光物质和放射性物质。可以通过测量与FcRn结合或不结合的抗体或使其可视化，检测在结合FcRn的抗体或其抗原结合片段和FcRn之间的复合物形成。可以使用常规检测测定法，例如，酶联免疫吸附测定(ELISA)、放射免疫测定(RIA)或组织免疫组织化学。出于检测目的，本公开的抗FcRn抗体或其片段可以用荧光团发色团标记。
一些实施方案中，还提供试剂盒，所述试剂盒包含与抗FcRn抗体或其抗原结合片段，还可以包含诊断使用说明。试剂盒还可以含有至少一种额外的试剂，如标记物或额外的诊断剂。对于体内使用，抗体可以配制为药物组合物。
附图说明
图1：FcRn抗体结合人FcRn蛋白的ELISA检测。
图2A：FcRn抗体与过表达人FcRn的细胞在pH 6.0环境下的结合检测。
图2B：FcRn抗体与过表达人FcRn的细胞在pH 7.4环境下的结合检测。
图3：检测FcRn抗体阻断过表达人FcRn的细胞与hIgG的结合。
图4：检测FcRn抗体阻断过表达人FcRn的细胞与HSA的结合的阻断检测。
图5A：FcRn转基因小鼠中静脉给药FcRn抗体(30mpk)，不同时间点检测IVIG的含量百分比。
图5B：FcRn转基因小鼠中静脉给药FcRn抗体(30mpk)的曲线下面积(AUC)。
具体实施方式
术语
为了更容易理解本公开，以下具体定义了某些技术和科学术语。除显而易见在本公开件中的它处另有明确定义，否则本公开使用的所有其它技术和科学术语都具有本公开所属领域的一般技术人员通常理解的含义。
本公开所用氨基酸三字母代码和单字母代码如J.Biol.Chem，243，p3558(1968)中所述。
“人FcRn”指人IgG受体α链与β2微球蛋白(β2M)之间的复合物，也称为新生儿Fc受体，其氨基酸序列见于例如UniProt号P55899，β2M的氨基酸序列见于例如UniProt号P61769。在本申请上下文中，“人FcRn”也涵盖任何天然变体或重组产物。
“抗体”以最广义使用，涵盖各种抗体结构，包括但不限于单克隆抗体，多克隆抗体；单特异性抗体，多特异性抗体(例如双特异性抗体)，全长抗体和抗体片段(或抗原结合片段，或抗原结合部分)，只要它们展现出期望的抗原结合活性。抗体可以指免疫球蛋白，是由两条相同的重链和两条相同的轻链通过链间二硫键连接而成的四肽链结构。免疫球蛋白重链恒定区的氨基酸组成和排列顺序不同，故其抗原性也不同。据此，可将免疫球蛋白分为五类，或称为免疫球蛋白的同种型，即IgM、IgD、IgG、IgA和IgE，其相应的重链分别为μ链、δ链、γ链、α链和ε链。同一类Ig根据其铰链区氨基酸组成和重链二硫键的数目和位置的差别，又可分为不同的亚类，如IgG可分为IgG1、IgG2、IgG3、IgG4。轻链通过恒定区的不同分为κ链或λ链。五类Ig中第每类Ig都可以有κ链或λ链。抗体重链和轻链靠近N端的约110个氨基酸的序列变化很大，为可变区(V区)；靠近C端的其余氨基酸序列相对稳定，为恒定区(C区)。可变区包括3个高变区(CDR)和4个序列相对保守的骨架区(FR)。3个高变区决定抗体的特异性，又称为互补性决定区(CDR)。每条轻链可变区(VL)和重链可变区(VH)由3个CDR区4个FR区组成，从氨基端到羧基端依次排列的顺序为：FR1，CDR1，FR2，CDR2，FR3，CDR3，FR4。轻链的3个CDR区指LCDR1，LCDR2，和LCDR3；重链的3个CDR区指HCDR1，HCDR2和HCDR3。
对于CDR的确定或定义，能够通过分辨抗体的结构和/或分辨抗体-配体复合物的结构来完成CDR的确定性描绘和包含抗体的结合位点的残基的鉴定。这可通 过本领域技术人员已知的各种技术中的任一种，例如X射线晶体学来实现。多种分析方法可用于鉴定CDR，包括但不限于Kabat编号系统、Chothia编号系统、AbM编号系统、IMGT编号系统、接触定义、构象定义。
Kabat编号系统是用于编号抗体中残基的标准并且通常用于鉴定CDR区域(参见例如Johnson&Wu，2000，Nucleic Acids Res.，28：214-8)。Chothia编号系统与Kabat编号系统类似，但Chothia编号系统考虑了某些结构环区域的位置。(参见例如Chothia等，1986，J.Mol.Biol.，196：901-17；Chothia等人，1989，Nature，342：877-83)。AbM编号系统使用建模抗体结构的由Oxford Molecular Group生产的计算机程序集成套件(参见例如Martin等，1989，ProcNatl Acad Sci(USA)，86：9268-9272；“AbMTM，A Computer Program for ModelingVariable Regions of Antibodies，”Oxford，UK；Oxford Molecular，Ltd)。AbM编号系统使用知识数据库和从头开始方法的组合，从基本序列建模抗体的三级结构(参见Samudrala等，1999，在PROTEINS，Structure，Function and Genetics Suppl.，3：194-198中的“Ab Initio Protein Structure Prediction Using a Combined HierarchicalApproach”描述的那些)。接触定义基于可用复杂晶体结构的分析(参见例如MacCallum等，1996，J.Mol.Biol.，5：732-45)。构象定义中，CDR的位置可鉴定为对抗原结合做出焓贡献的残基(参见例如Makabe等，2008，Journal ofBiological Chemistry，283：1156-1166)。另外其它的CDR边界定义可能不严格遵循上述方法之一，但仍然与Kabat CDR的至少一部分重叠，尽管根据特定残基或残基组不显著影响抗原结合的预测或实验结果，它们可缩短或延长。如本公开使用的，CDR可指通过本领域已知的任何方法(包括方法的组合)定义的CDR。
本公开的抗体或抗原结合片段的VL区和VH区的CDR氨基酸残基在数量和位置符合已知的Kabat或AbM编号系统。
“单克隆抗体”或“单抗”指从基本上同质的抗体群体获得的抗体，即除了可能以少量存在的可能天然存在的突变之外，群体包含的各个抗体是相同的。单克隆抗体是高度特异性的，针对单个抗原位点。此外，与通常包括针对不同决定簇(表位)的不同抗体的多克隆抗体制剂相反，每种单克隆抗体针对抗原上的单个决定簇。修饰语“单克隆”指示如从基本上同质的抗体群体获得的抗体的特征，并且不被解释为需要通过任何特定方法产生抗体。例如，根据本公开使用的单克隆抗体可通过首先由Kohler和Milstein，1975，Nature256：495描述的杂交瘤方法来制备，或者可通过例如美国专利号4,816,567中所述的重组DNA方法来制备。例如，单克隆抗体也可从使用McCafferty等，1990，Nature 348：552-554中描述的技术，从所生成的噬菌体文库中分离。
“全人抗体”或“重组全人抗体”包括通过重组方法制备、表达、创建或分离的全人抗体，所涉及的技术和方法在本领域中是熟知的，诸如：
(1)从人免疫球蛋白基因的转基因、转染色体动物(例如小鼠)或由其制备的杂交瘤中分离的抗体；
(2)从经转化以表达抗体的宿主细胞如转染瘤中分离的抗体；
(3)从重组组合全人抗体文库中分离的抗体；以及
(4)通过将人免疫球蛋白基因序列剪接到其他DNA序列等方法制备、表达、创建或分离的抗体。
此类重组全人抗体包含可变区和恒定区，这些区域利用特定的由种系基因编码的人种系免疫球蛋白序列，但也包括随后诸如在抗体成熟过程中发生的重排和突变。
术语“鼠源抗体”在本公开中为根据本领域知识和技能制备的针对人FcRn或其表位的单克隆抗体。制备时用FcRn抗原注射试验对象，然后分离表达具有所需序列或功能特性的抗体的杂交瘤。在本公开一个具体的实施方案中，所述的鼠源抗人FcRn抗体或其抗原结合片段，可进一步包含鼠源κ、λ链或其变体的轻链恒定区，或进一步包含鼠源IgG1、IgG2、IgG3或IgG4或其变体的重链恒定区。
术语“全人抗体”包括具有人种系免疫球蛋白序列的可变和恒定区的抗体。本公开的全人抗体可包括不由人种系免疫球蛋白序列编码的氨基酸残基(如通过体外随机或位点特异性诱变或通过体内体细胞突变所引入的突变)。然而，术语“全人抗体”不包括这样的抗体，即其中已将衍生自另一种哺乳动物物种(诸如小鼠)种系的CDR序列移植到人骨架序列上(即“人源化抗体”)。
术语“人源化抗体(humanized antibody)”，也称为CDR移植抗体(CDR-grafted antibody)，是指将非人CDR序列移植到人的抗体可变区框架中产生的抗体。可以克服嵌合抗体由于携带大量非人蛋白成分，从而诱导的强烈的免疫应答反应。为避免在免疫原性下降的同时引起活性的下降，可对所述的全人抗体可变区可进行最少反向突变，以保持活性。
术语“嵌合抗体(chimeric antibody)”，是将第一物种抗体的可变区与第二物种抗体的恒定区融合而成的抗体，可以减轻第一物种抗体诱发的免疫应答反应。作为一个示例，建立嵌合抗体，要选建立分泌鼠源性特异性单抗的杂交瘤，然后从小鼠杂交瘤细胞中克隆可变区基因，再要据需要克隆全人抗体的恒定区基因，将小鼠可变区基因与人恒定区基因连接成嵌合基因后插入人载体中，最后在真核工业系统或原核工业系统中表达嵌合抗体分子。全人抗体的恒定区可选自人源IgG1、IgG2、IgG3或IgG4或其变体的重链恒定区，优选包含人源IgG2或IgG4重链恒定区，或者使用氨基酸突变后无ADCC(antibody-dependent cell-mediated cytotoxicity，抗体依赖的细胞介导的细胞毒作用)毒性的IgG1。
“抗原结合片段”包括：单链抗体(即全长重链和轻链)；Fab、修饰的Fab、Fab’、修饰的Fab’、F(ab’)2、Fv、Fab-Fv、Fab-dsFv、单结构域抗体(例如VH或VL或VHH)、scFv、二价或三价或四价抗体、Bis-scFv、diabody、tribody、triabody、tetrabody和上述任意一种的表位结合片段(参见例如Holliger and Hudson,2005,Nature Biotech.23(9):1126-1136；Adair and Lawson,2005,Drug Design Reviews-Online 2(3),209-217)。产生和制备这些抗体片段的方法在本领域是公知的(参见例如Verma等人,1998,Journal ofImmunological Methods,216,165-181)。Fab-Fv形式首先公开于WO2009/040562，其二硫键稳定化形式Fab-dsFv首先公开于WO2010/035012。本公开的抗原结合片段还包括描述于WO2005/003169、WO2005/003170和WO2005/003171中的Fab和Fab’片段。多价抗体可包含多特异 性例如双特异性或可以是单特异性的(参见例如WO92/22583和WO05/113605)，后者的一个示例是描述于WO 92/22583中的Tri-Fab(或TFM)。
本公开的术语“与FcRn结合”，指能与FcRn或其表位相互作用，所述FcRn或其表位可以是人源的。本公开的术语“抗原结合位点”指抗原上不连续的，由本公开抗体或抗原结合片段识别的三维空间位点。
“抗原”指用于免疫接种免疫活性的脊椎动物的分子，以产生识别抗原的抗体，或筛选表达文库(例如尤其是噬菌体、酵母或核糖体展示文库)。在本公开中，抗原被更广义地定义，包括由抗体特异性识别的靶分子，以及包括用于产生抗体的免疫接种过程或用于选择抗体的文库筛选中使用的分子的一部分或模拟物。对于本公开的与人FcRn结合的抗体，人FcRn的单体和多聚体(例如二聚体、三聚体等)，以及人FcRn的截短变体和其它变体均被称为抗原。
术语“表位”是指抗原上与免疫球蛋白或抗体结合的位点。表位可以由相邻的氨基酸、或通过蛋白质的三级折叠而并列的不相邻的氨基酸形成。由相邻的氨基酸形成的表位通常在暴露于变性溶剂后保持，而通过三级折叠形成的表位通常在变性溶剂处理后丧失。表位通常以独特的空间构象包括至少3-15个氨基酸。确定什么表位由给定的抗体结合的方法在本领域中是熟知的，包括免疫印迹和免疫沉淀检测分析等。确定表位的空间构象的方法包括本领域中的技术和本公开所述的技术，例如X射线晶体分析法和二维核磁共振等。
“特异性结合”、“选择性结合”是指抗体与预定的抗原上的表位结合。通常，当使用人FcRn或其表位作为分析物并使用抗体作为配体，在仪器中通过表面等离子体共振(SPR)技术测定时，抗体以大约低于10 -7M或甚至更小的平衡解离常数(K D)与预定的抗原或其表位结合，并且其与预定抗原或其表位结合的亲和力是其与预定抗原(或其表位)或紧密相关的抗原之外的非特异性抗原(如BSA等)结合的亲和力的至少两倍。术语“识别抗原的抗体”在本公开中可以与术语“特异性结合的抗体”互换使用。
“结合亲和力”或“亲和力”在本公开中用作两个分子(例如抗体或其部分与抗原)之间的非共价相互作用的强度量度。两个分子之间的结合亲和力可通过确定解离常数(KD)来量化。可通过使用例如表面等离子共振(SPR)方法(Biacore)测量复合物形成和解离的动力学来确定KD。对应于单价复合物的结合和解离的速率常数分别被称为结合速率常数ka(或kon)和解离速率常数kd(或koff)。K D通过方程K D＝kd/ka与ka和kd有关。解离常数的值可通过众所周知的方法直接确定，并且可通过方法例如Caceci等人(1984，Byte 9：340-362)中所述的那些甚至对于复杂混合物进行计算。例如，可使用双重过滤硝化纤维素滤器结合测定如Wong&Lohman(1993，Proc.Natl.Acad.Sci.USA 90：5428-5432)中公开的那种来确定K D。评估抗体针对靶抗原的结合能力的其它标准测定是本领域已知的，包括例如ELISA、蛋白质印迹、RIA和流式细胞术分析、以及本公开其它地方例举的其它测定。抗体的结合动力学和结合亲和力也可通过本领域已知的标准测定，例如表面等离子共振(SPR)，例如通过使用Biacore TM系统或KinExA来评价。可通过比较各个抗体/抗原复合物的K D值来比较与不同分子相互作用相关的结合亲和 力，例如，不同抗体对于给定抗原的结合亲和力的比较。类似地，相互作用的特异性可通过确定和比较目的相互作用(例如抗体和抗原之间的特异性相互作用)的K D值与非目的相互作用(例如已知不结合FcRn的对照抗体)的K D值进行评价。
“保守性置换”指置换为具有与原始氨基酸残基相似的特性的另一个氨基酸残基。例如，赖氨酸、精氨酸和组氨酸具有相似的特性，在于它们具有碱性侧链，并且天冬氨酸和谷氨酸具有相似的特性，在于它们具有酸性侧链。此外，甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸和色氨酸具有相似的特性，在于它们具有不带电荷极性侧链，并且丙氨酸、缬氨酸、亮氨酸、苏氨酸、异亮氨酸、脯氨酸、苯丙氨酸和甲硫氨酸具有相似的特性，在于它们具有非极性侧链。另外，酪氨酸、苯丙氨酸、色氨酸和组氨酸具有相似的特性，在于它们具有芳族侧链。因此，本领域技术人员将显而易见，甚至当置换如上文所述的显示相似特性的组中的氨基酸残基时，它将不显示特性的特定变化。
“交叉反应”是指本公开的抗体与来自不同物种的FcRn结合的能力。例如，结合人FcRn的本公开的抗体也可以结合另一物种的FcRn。交叉反应性是通过在结合测定(例如SPR和ELISA)中检测与纯化抗原的特异性反应性，或与生理表达FcRn的细胞的结合或功能性相互作用来测量。确定交叉反应性的方法包括如本公开所述的标准结合测定，例如表面等离子体共振分析，或流式细胞术。
“抑制”或“阻断”可互换使用，并涵盖部分和完全抑制/阻断这两者。对FcRn的抑制/阻断优选地降低或改变无抑制或阻断的情况下发生FcRn结合时出现活性的正常水平或类型。抑制和阻断也旨在包括与抗FcRn抗体接触时，与未与抗FcRn抗体接触的FcRn相比，任何可测量的FcRn结合亲和力降低。
“抑制生长”(例如涉及细胞)旨在包括细胞生长任何可测量的降低。
生产和纯化抗体和抗原结合片段的方法在现有技术中熟知和能找到，如冷泉港的抗体实验技术指南(5-8章和15章)。如，可以用人FcRn或其片段免疫小鼠，所得到的抗体能被复性、纯化，并且可以用常规的方法进行氨基酸测序。抗原结合片段同样可以用常规方法制备。本公开所述的抗体或抗原结合片段用基因工程方法在非人源的CDR区加上一个或多个人FR区。人FR种系序列可以从ImMunoGeneTics(IMGT)网站得到。
本公开工程化的抗体或抗原结合片段可用常规方法制备和纯化。比如，编码重链和轻链的cDNA序列，可以克隆并重组至表达载体。重组的免疫球蛋白表达载体可以稳定地转染细胞。哺乳动物类表达系统会导致抗体的糖基化，特别是在Fc区的高度保守N端。通过表达与人源抗原特异性结合的抗体得到稳定的克隆。阳性的克隆在生物反应器的无血清培养基中扩大培养以生产抗体。分泌了抗体的培养液可以用常规技术纯化、收集。抗体可用常规方法进行过滤浓缩。可溶的混合物和多聚体，也可以用常规方法去除，比如分子筛，离子交换。得到的产物需立即冷冻，如-70℃，或者冻干。
可使用本领域技术人员已知的常规技术，就与相同表位的结合竞争性筛选抗体。例如，可进行竞争和交叉竞争研究，以获得彼此竞争或交叉竞争与抗原结合的抗体。基于它们的交叉竞争来获得结合相同表位的抗体的高通量方法描述于国 际专利公开WO03/48731中。因此，可使用本领域技术人员已知的常规技术，获得与本公开的抗体分子竞争结合FcRn上的相同表位的抗体及其抗原结合片段。
“给予”、“施用”和“处理”当应用于动物、人、实验受试者、细胞、组织、器官或生物流体时，是指外源性药物、治疗剂、诊断剂或组合物与动物、人、受试者、细胞、组织、器官或生物流体的接触。“给予”、“施用”和“处理”可以指例如治疗、药物代谢动力学、诊断、研究和实验方法。细胞的处理包括试剂与细胞的接触，以及试剂与流体的接触，其中所述流体与细胞接触。“给予”、“施用”和“处理”还意指通过试剂、诊断、结合组合物或通过另一种细胞体外和离体处理例如细胞。“处理”当应用于人、兽医学或研究受试者时，是指治疗处理、预防或预防性措施，研究和诊断应用。
“治疗”意指给予受试者内用或外用治疗剂，诸如包含本公开的任一种抗体或其抗原结合片段或其偶联物的组合物，所述受试者已经患有、疑似患有、倾向于患有一种或多种疾病或其症状，而已知所述治疗剂对这些症状具有治疗作用。通常，在受治疗受试者或群体中以有效缓解一种或多种疾病症状的量给予治疗剂，无论是通过诱导这类症状退化还是抑制这类症状发展到任何临床右测量的程度。有效缓解任何具体疾病症状的治疗剂的量(也称作“治疗有效量”)可根据多种因素变化，例如受试者的疾病状态、年龄和体重，以及药物在受试者产生需要疗效的能力。通过医生或其它专业卫生保健人士通常用于评价该症状的严重性或进展状况的任何临床检测方法，可评价疾病症状是否已被减轻。尽本公开的实施方案(例如治疗方法或制品)在缓解某个受试者中目标疾病症状方面可能无效，但是根据本领域已知的任何统计学检验方法如Student t检验、卡方检验、依据Mann和Whitney的U检验、Kruskal-Wallis检验(H检验)、Jonckheere-Terpstra检验和Wilcoxon检验确定，其在统计学显著数目的受试者中应当减轻目标疾病症状。
“有效量”包含足以改善或预防医学病症的症状或病症的量。有效量还意指足以允许或促进诊断的量。用于特定受试者或兽医学受试者的有效量可依据以下因素而变化：如待治疗的病症、受试者的总体健康情况、给药的方法途径和剂量以及副作用严重性。有效量可以是避免显著副作用或毒性作用的最大剂量或给药方案。
“同源性”或“同一性”是指两个多核苷酸序列之间或两个多肽之间的序列相似性。当两个比较序列中的位置均被相同核苷酸或氨基酸单体亚基占据时，例如如果两个DNA分子的每一个位置都被相同核苷酸占据时，那么所述分子在该位置是同源的。两个序列之间的同源性百分率是两个序列共有的匹配或同源位置数除以比较的位置数×100％的函数。例如，在序列最佳比对时，如果两个序列中的10个位置有6个匹配或同源，那么两个序列为60％同源。一般而言，当比对两个序列而得到最大的同源性百分率时进行比较。
“细胞”、“细胞系”和“细胞培养物”可互换使用，并且所有这类名称都包括其后代。还应当理解的是，由于故意或非有意的突变，所有后代在DNA含量方面不可能精确相同。包括具有与最初转化细胞中筛选的相同的功能或生物学活性的突变后代。
“任选”或“任选地”意味着随后所描述地事件或环境可以但不必发生，该说明包 括该事件或环境发生或不发生地场合。例如，“任选包含1-3个抗体重链可变区”意味着特定序列的抗体重链可变区可以但不必须存在。
本公开的“FcRn结合蛋白”以最大化解释，包含本公开的抗FcRn抗体或其抗原结合片段，只要能够实现与FcRn结合的均在该术语范围内。例如，FcRn结合蛋白可以包含一个或多个效应分子，以例如缀和的方式。所述“效应分子”包括：例如抗肿瘤剂、药物、毒素、生物活性蛋白(例如酶)、其它抗体或抗体片段、合成或天然存在的聚合物、核酸及其片段例如DNA、RNA及其片段、放射性核素(特别地放射性碘化物)、放射性同位素、螯合金属、纳米颗粒和报道基团(例如荧光化合物)、或可通过NMR或ESR光谱分析检测的化合物。当效应分子是聚合物时，其通常可以是合成或天然存在的聚合物，例如任选地取代的直链或支链聚亚烷基、聚亚烯基或聚氧化亚烷基聚合物或分支多糖或未分支多糖，例如同聚或异聚多糖。可存在于上述合成聚合物上的具体的任选取代基包括一个或多个羟基、甲基或甲氧基。合成聚合物的具体示例包括任选地取代的直链或支链聚(乙二醇)、聚(丙二醇)、聚(乙烯醇)或其衍生物，特别地任选地取代的聚(乙二醇)例如甲氧基聚(乙二醇)或其衍生物。具体的天然存在的聚合物包括乳糖、直链淀粉、葡聚糖、糖原或其衍生物。在一个实施方案中，聚合物是白蛋白或其片段，例如人血清白蛋白或其片段。聚合物与本公开抗FcRn抗体或其抗原结合片段的缀和方式可以通过常规方法实现。
实施例
以下结合实施例用于进一步描述，但这些实施例并非限制的范围。
实施例或测试例中未注明具体条件的实验方法，通常按照常规条件，或按照原料或商品制造厂商所建议的条件。参见Sambrook等，分子克隆，实验室手册，冷泉港实验室；当代分子生物学方法，Ausubel等著，Greene出版协会，Wiley Interscience，NY。未注明具体来源的试剂，为市场购买的常规试剂。
实施例1.人FcRn抗原及检测用蛋白的制备
1、蛋白设计及表达
以人FcRn(Uniprot登录号:P55899)胞外区-β2M(Uniprot登录号:P61769)复合物作为FcRn的模板，设计FcRn抗原及检测用蛋白的氨基酸序列(以下FcRn抗原未特殊说明的均指人FcRn-β2M复合物)。
>人FcRn胞外区：
Figure PCTCN2021076211-appb-000001
>带His标签的人β2M：
Figure PCTCN2021076211-appb-000002
Figure PCTCN2021076211-appb-000003
(注：斜体部分为His标签)。
全长人FcRn在细胞膜表面会发生内吞作用，无法进行正常的细胞结合与阻断实验。因此，制备人FcRn的突变体人FcRn mut(L320A，L321A)胞外区与跨膜区，与人β2M共同转染，此突变体可较好的维持FcRn-β2M复合物在细胞膜表面的定位，以用于构建检测用稳定细胞株。
>人FcRn mut(L320A，L321A)：
Figure PCTCN2021076211-appb-000004
(注：斜体部分为跨膜区，下划线部分为胞内区)。
2、FcRn重组蛋白纯化
对带His标签的FcRn重组蛋白的纯化。用20mM磷酸缓冲液，pH 8.0平衡Ni-Sepharose亲和柱5个柱体积。将样品高速离心去除杂质，上柱结合。用20mM磷酸缓冲液冲洗柱子直至A280读数降至基线，用20mM磷酸缓冲液，0-500mM咪唑梯度洗脱，收集蛋白并鉴定目的蛋白。将第一步纯化得到样品换液至PBS并浓缩体积至2mL，利用PBS平衡好的凝胶层析Superdex200(GE)进一步纯化，收集目的峰分装备用。经检测，获得了纯化的FcRn重组蛋白。
实施例2.抗人FcRn鼠源单克隆抗体的筛选
实验步骤如下：
1)小鼠免疫和血清效价检测
取4只SJL白小鼠，5只Balb/c白小鼠，用25-50μg抗原与佐剂混合免疫。时间为第0、14、35天。第0天腹膜内(IP)注射50μg/只的乳化后抗原。第14、35天注射25μg/只。于第21、42天取血，用ELISA方法确定小鼠血清中的抗体滴度。在第4-5次免疫以后，选择血清中抗体滴度高并且滴度趋于平台的小鼠进行脾细胞融合。在进行脾细胞融合前3天加强免疫，腹膜内(IP)注射50μg/只的生理盐水配制的抗原溶液。
2)脾细胞融合
采用PEG介导的融合步骤将脾淋巴细胞与骨髓瘤细胞Sp2/0-Ag14细胞进行融合得到杂交瘤细胞。融合好的杂交瘤细胞以每孔细胞数1×10 4-1×10 5种于96孔板中，37℃，5％CO 2孵育并补充HAT完全培养基100μL/孔，10-14天后进行ELISA检测。
3)杂交瘤细胞筛选
根据杂交瘤细胞生长密度，用结合ELISA方法进行杂交瘤培养上清检测。并 将结合ELISA检测的阳性孔细胞上清进行纯化、细胞结合实验和细胞阻断实验。结合和阻断均为阳性的孔细胞及时进行扩增冻存保种和测序。
所筛选到的阳性克隆hybri11对应的抗体可变区氨基酸序列如下所示：
>hybri11-VH氨基酸序列：
Figure PCTCN2021076211-appb-000005
>hybri11-VL氨基酸序列：
Figure PCTCN2021076211-appb-000006
4)小鼠免疫库构建与筛选
免疫小鼠脾脏及淋巴结细胞中加入Trizol试剂重悬(1×10 7个细胞/mL Trizol)，以裂解细胞，冰上放置5min；13000rpm离心3min，取上清，弃沉淀；加入1/5体积的氯仿，剧烈震荡30-60s，冰浴静置2min；13000rpm离心10min，吸取上层水相层至新的1.5mL管中；加入等体积的异丙醇，混匀，-20℃静置30min；13000rpm离心10min，去掉上清，保留沉淀；加入预冷的75％乙醇洗涤沉淀，室温放置5-10min；加入RNA酶去除的去离子水600μL，复溶，得到RNA，逆转录得到cDNA，进行单链抗体噬菌体文库的构建。
通过小鼠免疫单链抗体噬菌体库的筛选来获得与FcRn具有高亲和力的抗体。用10μg随机生物素化的FcRn蛋白结合1mg Dynabeads MyOne链霉亲和素T1，室温1小时。PBST(0.05％Tween-20)洗3遍，将人白蛋白(Sigma,126658)溶于1×PBS(pH 6.0)，终浓度1mg/mL作为封闭剂，加入体系，室温封闭1小时。加入用2％的牛奶室温封闭1小时的全人源单链抗体噬菌体展示文库，在室温下作用1小时。PBST(0.05％Tween-20)，pH 7.4溶液洗8遍，去除不结合的噬菌体，白蛋白及与之结合的噬菌体。先用100μg/mL与IgG竞争性结合FcRn抗原的蛋白0.5mL洗脱，再用1mg/mL的0.5mL的胰蛋白酶将与FcRn特异性结合的剩余噬菌体洗脱，分别感染处于对数期生长的大肠杆菌TG1，产生并纯化噬菌体用于下一轮筛选。相同筛选过程重复2-3轮后，阳性的克隆被富集。
从筛选富集的克隆中挑取380(4×96孔板)个单克隆菌落包装成单链抗体噬菌体，用于噬菌体ELISA测试。ELISA板上分别包被2μg/mL的FcRn的蛋白，4度放置过夜，PBST(0.05％Tween-20)洗3遍，用2％脱脂牛奶室温封闭1个小时，PBST(0.05％Tween-20)洗3遍后，加入封闭液稀释的噬菌体上清，室温反应1小时，PBST(0.05％Tween-20)洗6遍，加入抗M13HRP(义翘神州，11973-MM05T-H)，室温反应1小时，PBST(0.05％Tween-20)洗3遍，加入100μL TMB显色底物，并用100μL 1M硫酸终止反应，用SpectraMax M5酶标仪在450nm处读取吸收值检测。将ELISA结合测试中OD450值大于0.5的克隆进行测序，得到58个特异性序列。
实施例3.全人源单链抗体噬菌体库筛选特异性结合人FcRn的阳性抗体
通过全人源单链抗体噬菌体库的筛选来获得与FcRn具有高亲和力的抗体。
用10μg随机生物素化的FcRn蛋白结合1mg Dynabeads MyOne链霉亲和素T1，室温1小时。PBST(0.05％Tween-20)洗3遍，将人白蛋白(Sigma,126658)溶于1×PBS(pH 6.0)，终浓度1mg/mL作为封闭剂，加入体系，室温封闭1小时。加入用2％的牛奶室温封闭1小时的全人源单链抗体噬菌体展示文库，在室温下作用1小时。PBST(0.05％Tween-20)，pH 7.4溶液洗8遍，去除不结合的噬菌体，白蛋白及与之结合的噬菌体。先用100μg/mL与IgG竞争性结合FcRn抗原的蛋白0.5mL洗脱，再用1mg/mL的0.5mL的Trypsin将与FcRn特异性结合的剩余噬菌体洗脱，分别感染处于对数期生长的大肠杆菌TG1，产生并纯化噬菌体用于下一轮筛选。相同筛选过程重复2-3轮后，阳性的克隆被富集。
从筛选富集的克隆中挑取2185(33×96)个单克隆菌落包装成单链抗体噬菌体，用于噬菌体ELISA测试。ELISA板上分别包被2μg/mL的FcRn的蛋白，4度放置过夜，PBST(0.05％Tween-20)洗3遍，用2％脱脂牛奶室温封闭1个小时，PBST(0.05％Tween-20)洗3遍后，加入封闭液稀释的噬菌体上清，室温反应1小时，PBST(0.05％Tween-20)洗6遍，加入抗M13HRP(义翘神州，11973-MM05T-H)，室温反应1小时，PBST(0.05％Tween-20)洗3遍，加入100μL TMB显色底物，并用100μL 1M硫酸终止反应，用SpectraMax M5酶标仪在450nm处读取吸收值检测。将ELISA结合测试中OD450值大于0.5的克隆进行测序，得到43个特异性序列。
实施例4.构建完整抗人FcRn单克隆抗体
从实施例2小鼠免疫噬菌体文库筛选得到的58个特异性序列中挑选25个，从实施例3人源噬菌体文库筛选得到的43个特异性序列中挑选39个，构建完整重组抗体。
纯化重组抗体的方法为：收集表达上清，高速离心去除杂质，并用0.45μm的PVDF滤膜过滤，用Protein A或Protein G柱进行亲和层析。用1×PBS(pH 7.4)缓冲体系作为平衡缓冲液对层析柱平衡3-5倍柱体积；细胞上清利用低流速上样结合，控制流速使保留时间约1min或更长时间；利用1×PBS(pH 7.4)洗涤层析柱3-5倍柱体积至紫外吸收回落至基线；利用0.1M甘氨酸(pH 3.0)缓冲液进行样品洗脱，根据紫外检测收集洗脱峰，洗脱产物利用1M Tris-HCl(pH 9.0)快速调节pH至5-6暂存。洗脱样品适当稀释至电导<2mS/cm，与阴离子柱结合，用20mM Tris-HCl pH 8.0-9.0冲洗去除杂质，20mM Tris-HCl、NaCl 0-1M梯度洗脱。收集峰换液至PBS分装备用。
对于获得的抗体，通过ELISA结合实验、ForteBio蛋白相互作用实验和细胞水平上与IgG竞争实验，确定其中7个抗体结合力强并能抑制FcRn与IgG的相互作用。
其完整可变区序列如表1所示：
表1.抗人FcRn单克隆抗体序列
Figure PCTCN2021076211-appb-000007
Figure PCTCN2021076211-appb-000008
本公开筛选获得的抗人FcRn抗体的CDR编号系统为Kabat编号系统或AbM编号系统。Kabat编号系统CDR区用下划线表示，AbM编号系统CDR区用斜体表示。以上序列中，顺序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4，序列中下划线或斜体部分分别为CDR1、CDR2、CDR3序列。
各抗体的重链及轻链CDR序列总结如表2。
表2.重链及轻链CDR序列
Figure PCTCN2021076211-appb-000009
Figure PCTCN2021076211-appb-000010
表2中，h22的编号系统有两种，当HCDR1-3为SEQ ID No：38-40、LCDR1-3为SEQ ID No：41-43时，为Kabat编号系统；当HCDR1-3为SEQ ID No：68、69、40，LCDR1-3为SEQ ID No：41-43时，为AbM编号系统。其他抗体的编号系统均为Kabat。
以上各重链可变区与对应的人源CH1(SEQ ID No：70)或小鼠抗体重链CH1(SEQ ID No：71)和小鼠IgG1的Fc(SEQ ID No：75)融合，轻链可变区与人源kappa(SEQ ID No：72)或lamda的恒定区CL1(SEQ ID No：73)或CL2(SEQ ID No：74)融合，组成重组抗体，再进行后续检测。
>全人抗体重链CH1：
Figure PCTCN2021076211-appb-000011
>小鼠抗体重链CH1：
Figure PCTCN2021076211-appb-000012
>全人抗体轻链Cκ：
Figure PCTCN2021076211-appb-000013
>全人抗体轻链CL1：
Figure PCTCN2021076211-appb-000014
>全人抗体轻链CL2：
Figure PCTCN2021076211-appb-000015
>小鼠IgG1Fc：
Figure PCTCN2021076211-appb-000016
实施例5.抗人FcRn单克隆抗体的基因改造
1、抗FcRn单克隆抗体h22的亲和力成熟
将抗体分子进行三维模拟结构与已知抗原结构(PDB ID:4N0U Chain A&B)进行结合模拟。参考人种系基因突变热点，三维结构与结合模拟结果，选定框架区和CDR区部分关键氨基酸残基，建立若干随机突变噬菌体文库。利用噬菌体文库展示技术，筛选出亲和力有所提高的功能性抗体。对不同文库获得新氨基酸残 基进行组合与验证，获得亲和力与功能均有提高的功能性抗体。得到的抗体分子轻链可变区和重链可变区序列分别如表3、表4所示。CDR组合分别如表5、表6、表7所示。
表3.抗人FcRn单克隆抗体h22的亲和力成熟后的轻链可变区序列
Figure PCTCN2021076211-appb-000017
Figure PCTCN2021076211-appb-000018
表4.抗人FcRn单克隆抗体h22的亲和力成熟后的重链可变区序列
Figure PCTCN2021076211-appb-000019
Figure PCTCN2021076211-appb-000020
Figure PCTCN2021076211-appb-000021
表3、表4筛选获得的抗FcRn抗体的CDR编号系统为Kabat编号系统(下划线)或AbM编号系统(斜体)。以上序列中，顺序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4，序列中下划线或斜体分别为CDR1、CDR2、CDR3序列。
表5.抗人FcRn单克隆抗体h22的亲和力成熟后的轻链CDR序列
Figure PCTCN2021076211-appb-000022
Figure PCTCN2021076211-appb-000023
表6.抗人FcRn单克隆抗体h22的亲和力成熟后的重链CDR序列(Kabat编号系统)
Figure PCTCN2021076211-appb-000024
Figure PCTCN2021076211-appb-000025
Figure PCTCN2021076211-appb-000026
Figure PCTCN2021076211-appb-000027
表7.抗人FcRn单克隆抗体h22的亲和力成熟后的重链CDR序列(AbM编号系统)
Figure PCTCN2021076211-appb-000028
Figure PCTCN2021076211-appb-000029
Figure PCTCN2021076211-appb-000030
表5的CDR编号系统为AbM或Kabat编号系统，表6的CDR编号系统为Kabat编号系统，表7的CDR编号系统为AbM编号系统。
2、抗人FcRn单克隆抗体的人源化改造(Germlining)
通过对选定的全人源特异性抗体分子进行三维结构同源建模，结合与V-base人种系序列数据库、IMGT人类抗体重链可变区种系基因数据库进行比对的结果，挑选与筛选出来的抗体同源性高的重链和轻链可变区种系基因作为模板，将原单克隆抗体FR区与CDR区进行改造，在保留功能的同时使其序列更接近人种系基因。对移植后的单域抗体再次进行三维结构模拟并分析，将FR区中影响CDR区结构形态的特定位点进行回复突变。其中氨基酸残基由Kabat编号系统确定并注释。改造后的抗体具有更高的稳定性，更低的免疫原性。
以抗人FcRn单克隆抗体h22为例的种系基因架构选择：
经分析，抗体h22的重链模板选用IMGT数据库中人源种系基因IGHV5-51*01，轻链模板选用IMGT数据库中人源种系基因IGLV6-57*02。
对抗体h22进行基因改造，其可变区序列如表8。
表8.种系基因改造结合亲和力成熟改造后的h22单克隆抗体分子序列
Figure PCTCN2021076211-appb-000031
表8的CDR编号系统为Kabat编号系统(下划线所示)或AbM编号系统(斜体所示)。以上序列中，顺序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4，下划线或斜体分别为CDR1、CDR2、CDR3序列。
经分析，经人源化改造(Germlining)后，有部分抗体的CDR发生变化，如表9所示。
表9.种系基因改造结合亲和力成熟改造后的单克隆抗体分子CDR序列
Figure PCTCN2021076211-appb-000032
表9的CDR编号系统为Kabat编号系统。
实施例6.抗人FcRn抗体的制备
将筛选得到抗体重链可变区与人CH1和IgG4(S241P)Fc融合并克隆到哺乳动物细胞表达载体。将抗体轻链可变区克隆到含有人源抗体轻链lamda或kappa恒定区的哺乳动物细胞表达载体。IgG4(S241P)简称为IgG4P。
>人CH1序列：
Figure PCTCN2021076211-appb-000033
>人IgG4(S241P)序列：
Figure PCTCN2021076211-appb-000034
将所述载体转染至HEK293E细胞或expiCHO细胞，方法分别如下：
转染至HEK293E细胞：
按照1μg DNA/ml转染细胞的量比采用脂质体293FectinReagent(Invitrogen，Cat.No.12347019)进行转染，按试剂说明书进行。转染后细胞密度为1.0X10 6细胞/ml,放置于37度摇床震荡培养(5％CO 2)，第6天收取细胞培养液，4000rpm离心，取上清并使用0.45μM的滤膜过滤。
转染至expiCHO细胞，方法：
按照1μg DNA/ml转染细胞的量比使用ExpiCHO Expression System(Cat.no.A29133)进行转染，按试剂说明书进行。采用标准方法，转染后细胞密度为6.0X10 6细胞/ml,放置于37度摇床震荡培养(8％CO 2)，第8天收取细胞培养液，4000rpm离心，取上清并使用0.45μM的滤膜过滤。
经检测，获得目标抗体。
实施例7.抗人FcRn抗体与抗原的亲和力检测
1、抗FcRn抗体结合FcRn的ELISA实验
为检测筛选到的FcRn抗体对于人FcRn蛋白的体外结合能力，随机生物素化的人FcRn-β2M复合物(生物素-hFcRn)被用于进行体外结合检测。本测试例的阴性对照为PBS，阳性对照使用HEK293瞬时转染表达的Argx-113。阳性对照的Argx-113的序列来自WO2015/100299A1中的序列2。
用pH 6.0的PBS缓冲液将NeutrAvidin Protein(Thermo Scientific,Lot:31000)稀释至2μg/mL，以100μL/孔的体积加入96孔酶标板(Corning，901825)中，于4℃放置过夜16-20小时。弃去液体后，用PBST(pH 6.0，0.05％Tween-20)缓冲液洗板三次后，加入100μL/孔用PBS缓冲液稀释至3μg/mL的生物素-hFcRn或生物素-cynoFcRn，37℃孵育箱孵育1小时。弃去液体后，用PBST缓冲液洗板三次后，加入用PBS缓冲液稀释的4％BSA封闭液(300μL/孔)，37℃孵育箱孵育1小时进行封闭。封闭结束后，弃去封闭液，并用PBST缓冲液洗板3次后，加入初始浓度为40μg/mL的FcRn抗体蛋白，用PBS缓冲液三倍比稀释7个梯度，置于37℃孵育箱孵育1小时。孵育结束后，弃去酶标板中的反应液，用PBST洗板6次，每孔加入100μL HRP标记的抗鼠Fc的二抗(Abcam ab197780,1:5000稀释)，37℃孵育1小时。用PBST洗板6次后，加入100μL TMB显色底物，室温孵育3-5min，加入50μL 1M硫酸终止反应，用SpectraMax M5酶标仪在450nm处读取吸收值，计算抗体对抗原的结合EC 50值。部分抗体的EC 50结果见图1与表10。
表10.抗人FcRn抗体与FcRn抗原的结合力EC 50
抗体编号 结合人FcRn的EC 50(μg/mL)
h30 0.54
h33 0.42
m14 0.15
hybri11 0.15
阳性对照Argx-113 0.90
阴性对照PBS N/A
2、抗人FcRn抗体与FcRn过表达HEK293细胞的结合实验
FcRn抗体与细胞表面FcRn的结合能力通过测量抗体与稳定过表达hFcRn突变体(人FcRn mut(L320A，L321A))的HEK293细胞表面上的hFcRn结合实验检测。将稳定过表达人FcRn mut(L320A，L321A)的HEK293细胞固定于96孔板底后，抗体及相应二抗加入后信号的强弱被用于判断抗体和hFcRn过表达HEK293细胞的结合活性。具体实验方法如下：
将稳定转染人FcRn mut(L320A，L321A)的HEK293细胞用PBS清洗两次后，在U型孔96孔板中每孔加入5×10 5细胞。离心并去上清后，每孔加入100μL以样品稀释液稀释的FcRn抗体(样品稀释液为PBS中加入1％BSA)，在冰上孵育40分钟。以200μL样品稀释液清洗两次后，每孔加入100μL以样品稀释液稀释的抗体Fc相应二抗(小鼠Fc:Thermo Fisher,Cat No.A16085,1:500；人Fc:Jackson Immuno Research,Cat No.109-095-008,1:200)，在冰上孵育40分钟。以200μL样品稀释液清洗两次后，每孔加入200μL样品稀释液，用流式细胞仪(BD Biosciences, BD Accuri C6)与FL1通道测量荧光值，并使用Flowjo软件对数据进行分析，用Prism6软件对分析结果进行绘制。阴性对照为PBS，阳性对照使用HEK293瞬时转染表达的Argx-113。部分抗体测试结果见下图2A、图2B及表11。
结果显示，本公开的抗体在中性条件(pH 7.4，图2B)和酸性条件(pH 6.0，图2A)下，细胞水平上与FcRn均有很好的结合活性，亲和力优于阳性对照Argx-113。
表11.抗人FcRn抗体与过表达FcRn的细胞的结合力
Figure PCTCN2021076211-appb-000035
3、Biacore测定
用Biacore，GE仪器测定待测人源化抗FcRn抗体与人FcRn的亲和力。
用Protein A生物传感芯片(Cat.#29127556,GE)亲和捕获一定量的待测抗体，然后于芯片表面流经一系列浓度梯度下的人FcRn抗原，利用Biacore仪器(Biacore T200，GE)实时检测反应信号从而获得结合和解离曲线。在每个循环解离完成后，用人抗捕获试剂盒里配置的再生溶液或pH 1.5的甘氨酸-盐酸再生溶液(Cat.#BR-1003-54,GE)将生物芯片洗净再生。实验中用到的缓冲液为HBS-EP+10×缓冲溶液(Cat.#BR-1006-69，GE)，用D.I.Water稀释至1×(pH为7.4)，或稀释至1×并用3M HCl调节pH至5.5或6.0。
实验得到的数据用BIAevaluation version 4.1，GE软件以(1:1)Langmuir模型进行拟合，得出亲和力数值，结果见表12、表13。
结果显示，抗体h22及其基因改造分子在中性条件(pH 7.4)和酸性条件(pH6.0或5.5)下均与FcRn有很好的结合活性，酸性环境下亲和力略高于中性环境。
将亲和力成熟筛选得到的重链轻链突变体序列进行交叉组合，SPR筛选得到与FcRn亲和力增强的抗体分子。部分分子亲和力测定结果如下：
表12.部分抗FcRn亲和力成熟抗体的亲和力测定(pH 5.5)
抗体 重链编号 轻链编号 ka(1/Ms) kd(1/s) KD(M)
h22 h22-VH h22-VL 3.02E+05 4.23E-03 1.40E-08
h22-AM-2 h22-AM-VH-10 h22-VL 4.66E+06 2.84E-03 6.10E-10
h22-AM-5 h22-AM-VH-13 h22-VL 3.25E+06 3.80E-03 1.17E-09
h22-AM-9 h22-AM-VH-17 h22-VL 2.46E+06 4.28E-03 1.74E-09
h22-AM-10 h22-AM-VH-18 h22-VL 2.34E+06 3.77E-03 1.61E-09
h22-AM-13 h22-AM-VH-21 h22-VL 2.89E+06 3.26E-03 1.13E-09
h22-AM-C2 h22-AM-VH-3 h22-AM-VL-1 3.34E+05 3.15E-03 9.45E-09
h22-AM-G2 h22-AM-VH-7 h22-AM-VL-1 4.52E+05 2.19E-03 4.85E-09
h22-AM-H2 h22-AM-VH-8 h22-AM-VL-1 9.51E+04 3.25E-03 3.42E-08
h22-AM-D8 h22-AM-VH-4 h22-AM-VL-7 3.72E+05 1.83E-03 4.93E-09
h22-AM-A10 h22-AM-VH-1 h22-AM-VL-9 3.11E+05 3.03E-03 9.75E-09
结合人源化设计和亲和力成熟，得到的分子在酸性和中性条件下进行亲和力测定，如下。
表13.抗FcRn抗体的亲和力测定
Figure PCTCN2021076211-appb-000036
结果显示，改造后的抗体分子，在降低免疫原性的同时，维持了与亲本分子h22相当的亲和力。
经鉴定，上述维持抗体亲和力、降低免疫原性的重要氨基酸残基包括X 1-X 10，具体如下：
HCDR1：如GYX 1F X 2(SEQ ID No：182)或GYX 1F X 2X 3X 4X 5IA(SEQ ID No：173)所示，其中，X 1可以选自S、N、D、V、A、P，X 2可以选自T、N、K、R、S、A，X 3可以选自G、N、K、S、E，X 4可以选自Y、H、S、N、T，X 5可以选自W、Y、F、M；
HCDR2：如X 6IX 7PDX 8SNTI(SEQ ID No：174)或X 6IX 7PDX 8SNTIYSPSFRG(SEQ ID No：175)所示，其中，X 6可以选自I、L、V，X 7可以选自Y、S、T、G、A，X 8可以选自N、R、K、A、F；
HCDR3的氨基酸序列如FGGPTFAQWYFDY(SEQ ID No：40)所示。
LCDR1的氨基酸序列如TGSSGSIASNYVX 9(SEQ ID No：176)所示，其中，X 9可以选自Q、S、N、A、V、T；
LCDR2的氨基酸序列如X 10DNQRAS(SEQ ID No：177)所示，其中，X 10可以选自E、S、A、W、C；
LCDR3的氨基酸序列如QSYDSSSHNWV(SEQ ID No：43)所示。
部分抗体的全长序列如下：
>h22-0011的重链全长：
Figure PCTCN2021076211-appb-000037
Figure PCTCN2021076211-appb-000038
>h22-0014的重链全长：
Figure PCTCN2021076211-appb-000039
>h22-0015的重链全长：
Figure PCTCN2021076211-appb-000040
>h22-0011/0014/0015的轻链全长：
Figure PCTCN2021076211-appb-000041
实施例8.抗人FcRn抗体阻断IgG与FcRn结合的检测实验
本实施例对抗人FcRn抗体在体外过表达hFcRn细胞中对生物素-hIgG结合FcRn的阻断进行检测。
抗人FcRn抗体对IgG与FcRn结合的阻断效果通过在过表达hFcRn突变体(人FcRn mut(L320A，L321A))的HEK293细胞中测量抗体降低细胞表面结合随机生物素化人IgG(Sigma货号:I4506)的荧光量的方式检测。本实验使用过表达人FcRn mut(L320A，L321A)的HEK293细胞(即293-hFcRn-mut)，该hFcRn突变体可 较好地维持细胞膜表面定位。将表达hFcRn突变的细胞以稀释好的不同浓度FcRn抗体孵育后，再先后加入生物素-hIgG及SA-FITC二抗孵育，以二抗荧光信号的减弱程度作为衡量FcRn抗体对于生物素-hIgG与FcRn结合阻断作用的强弱。具体实验方法如下：
将293-hFcRn-mut细胞用pH 6.0 PBS清洗两次后，在U型孔96孔板中每孔加入5×10 5细胞。离心并去上清后，每孔加入100μL以样品稀释液稀释的FcRn抗体(样品稀释液为pH 6.0 PBS中加入1％BSA)，在冰上孵育40分钟。以200μL样品稀释液清洗两次后，每孔加入100μL以样品稀释液稀释的生物素-hIgG(终浓度100μg/mL)，在冰上孵育40分钟。以200μL样品稀释液清洗两次后，每孔加入100μL以样品稀释液稀释的链霉亲和素-FITC二抗(eBioscience，Cat No.11-4317-87)，稀释比例1:200，在冰上孵育40分钟。以200μL样品稀释液清洗两次后，每孔加入200μL样品稀释液，用流式细胞仪(BD Biosciences,BD Accuri C6)与FL1通道测量荧光值，并使用Flowjo软件对数据进行分析，用Prism6软件对分析结果进行绘制。部分抗体的抑制作用测试结果见图3及表14、表15。
结果显示，本公开获得的抗体在细胞水平上有良好的阻断IgG与FcRn结合的效果。
表14.抗人FcRn抗体对FcRn过表达细胞与hIgG结合的阻断实验
候选抗体 阻断活性IC 50(nM)
hybri11 8.39
h3 N/A
h4 3.15
h22 3.73
h33 33.55
h35 8.60
m14 27.15
阳性对照(Argx-113) 9.77
阴性对照mIgG N/A
(注：N/A代表未检测到或无法拟合出具体数值)
表15.抗人FcRn抗体对FcRn过表达细胞与hIgG结合的阻断实验
抗体 阻断活性IC 50(nM)
h22 13.0
h22-AM-5 6.22
h22-0011 12.6
h22-0014 12.8
h22-0015 11.4
实施例9.抗FcRn抗体对FcRn过表达细胞与人血清白蛋白(HSA)结合的阻断实验
IgG和HSA均通过与FcRn结合实现体内循环，但是结合的FcRn表位不同。 只阻断FcRn与IgG结合而不影响FcRn与HSA结合的抗体能够特异性地降低病理性IgG的体内循环而不影响HSA，从而具有更好的特异性与安全性。
为检测抗FcRn抗体特异性结合FcRn-IgG结合表位，并检测是否抗FcRn抗体同时不影响FcRn-HSA的结合，本实施例通过在过表达hFcRn突变体的HEK293细胞中测量抗体对细胞表面结合随机生物素化HSA(Sigma货号:126658)的荧光量的方式检测。
本实验使用过表达hFcRn突变体(人FcRn mut(L320A，L321A))的HEK293细胞株，该hFcRn突变体可较好地维持细胞膜表面定位。将表达hFcRn突变的细胞以稀释好的FcRn抗体及生物素-HSA共同孵育后，再加入SA-FITC二抗孵育，以二抗荧光信号的增强或减弱程度作为衡量FcRn抗体对于生物素-HSA与FcRn结合阻断作用的强弱。具体实验方法如下：
将293-hFcRn-mut细胞用pH 6.0 PBS清洗两次后，在U型孔96孔板中每孔加入5×10 5细胞。离心并去上清后，每孔加入100μL以样品稀释液稀释的FcRn抗体(终浓度200μg/mL)和生物素-HSA(终浓度250μg/mL)混合物，在冰上孵育40分钟。以200μL样品稀释液清洗两次后，每孔加入100μL以样品稀释液稀释的链霉亲和素-FITC二抗(eBioscience，Cat No.11-4317-87)，稀释比例1:200，在冰上孵育40分钟。以200μL样品稀释液清洗两次后，每孔加入200μL样品稀释液，用流式细胞仪(BD Biosciences,BD Accuri C6)与FL1通道测量荧光值，并使用Flowjo软件对数据进行分析，用Prism6软件对分析结果进行绘制。将未加抗体的对照组作为零点，若抗体使平均荧光强度降低则抗体阻断生物素-HSA与FcRn结合。此实验阳性对照为HSA(Sigma货号:126658)，阴性对照为人IgG(Sigma货号:I4506)。
部分抗体的测试结果见图4。结果显示，HSA展现出了对HSA结合的阻断，而h22及其基因改造获得抗体对生物素-HSA与hFcRn结合的影响与阴性对照IgG类似，不影响FcRn与HSA的结合。
实施例10.在人FcRn转基因小鼠中检验抗FcRn抗体的作用
本实验将人IVIG静脉注射至表达人FcRn的转基因小鼠(C57BL/6-Fcgrttm1(FCGRT)/Bcgen，百奥赛图)中，24小时后静脉注射抗FcRn抗体，给药后不同时间点采血，目的是检查抗体是否影响人IgG的代谢。
将hFcRn转基因小鼠随机分组，每组三只，静脉注射500mpk IVIG。24小时后，静脉注射待检测抗FcRn抗体或对照组人IgG1同种型对照(BioXcell货号:BE0297)，剂量30mpk。给药前，即0小时，及给药后8，24，48小时采血。样品采用人IgG检测试剂盒(Cisbio)检测IgG浓度。所得数据用GraphPad Prism 9处理，使用单因素ANOVA方法分析。与阴性对照hIgG1同种型相比，抗体h22-0011、h22-0014、h22-0015均显著加快hIgG在hFcRn转基因鼠体内的代谢。分析曲线下面积(AUC)得到候选抗体h22-0011、h22-0014、h22-0015对比hIgG1同种型能够显著降低人IgG在小鼠体内的潴留。由以上数据得出结论候选抗体能够通过阻断FcRn的结合使体内的IgG分解代谢增加。部分测试结果见图5A、图 5B。
虽然以上描述了本公开的具体实施方案，但是本领域的技术人员应当理解，这些仅是举例说明，在不背离本公开的原理和实质的前提下，可以对这些实施方案做出多种变更或修改。

Claims (22)

  1. 抗FcRn抗体或其抗原结合片段，包含重链可变区(VH)和轻链可变区(VL)，其中：
    所述VH包含选自以下的HCDR1-3：
    HCDR1的氨基酸序列如GYX 1F X 2(SEQ ID No：182)或GYX 1F X 2 X 3 X 4 X 5IA(SEQ ID No：173)所示，
    其中，
    X 1选自S、N、D、V、A、P、K，
    X 2选自T、N、K、R、S、A，
    X 3选自G、N、K、S、E，
    X 4选自Y、H、S、N、T，
    X 5选自W、Y、F、M；
    HCDR2的氨基酸序列如X 6IX 7PDX 8SNTI(SEQ ID No：174)或X 6IX 7PDX 8SNTIYSPSFRG(SEQ ID No：175)所示，
    其中，
    X 6选自I、L、V，
    X 7选自Y、S、T、G、A，
    X 8选自N、R、K、A、F；
    HCDR3的氨基酸序列如FGGPTFAQWYFDY(SEQ ID No：40)所示；
    所述VL包含选自以下的LCDR1-3：
    LCDR1的氨基酸序列如TGSSGSIASNYVX 9(SEQ ID No：176)所示，
    其中，X 9选自Q、S、N、A、V、T；
    LCDR2的氨基酸序列如X 10DNQRAS(SEQ ID No：177)所示，
    其中，X 10选自E、S、A、W、C；
    LCDR3的氨基酸序列如QSYDSSSHNWV(SEQ ID No：43)所示。
  2. 如权利要求1所述的抗FcRn抗体或其抗原结合片段，其中，
    VH包含如SEQ ID No：12、87-110、169之一所示的VH的HCDR1、HCDR2、HCDR3，
    VL包含如SEQ ID No：13、76-86、167之一所示的VL的LCDR1、LCDR2、LCDR3；
    优选地，
    VH包含分别如SEQ ID No：38-40所示的HCDR1、HCDR2、HCDR3，
    VL包含分别如SEQ ID No：41-43所示的LCDR1、LCDR2、LCDR3；或
    VH包含分别如SEQ ID No：68、69、40所示的HCDR1、HCDR2、HCDR3，
    VL包含分别如SEQ ID No：41-43所示的LCDR1、LCDR2、LCDR3；或
    VH包含分别如SEQ ID No：133、170、40所示的HCDR1、HCDR2、HCDR3，
    VL包含分别如SEQ ID No：41-43所示的LCDR1、LCDR2、LCDR3。
  3. 如权利要求1-2任一项所述的抗FcRn抗体或其抗原结合片段，其中，
    VH如SEQ ID No：12、87-110、169之一所示或与之具有至少90％同一性，
    VL如SEQ ID No：13、76-86、167之一所示或与之具有至少90％同一性；
    优选地，
    VH如SEQ ID No：169所示或与之具有至少90％同一性，
    VL如SEQ ID No：167所示或与之具有至少90％同一性。
  4. 如权利要求1-3任一项所述的抗FcRn抗体或其抗原结合片段，其中：
    重链如SEQ ID No：180所示或与之具有至少80％同一性；
    轻链如SEQ ID No：181所示或与之具有至少80％同一性。
  5. 抗FcRn抗体或其抗原结合片段，包含VH和VL，其中：
    所述VH包含选自以下的HCDR1-3：
    HCDR1的氨基酸序列如SEQ ID No：20、26、32、38、44、50、56、62、68、129-141、150-165之一所示；
    HCDR2的氨基酸序列如SEQ ID No：21、27、33、39、45、51、57、63、69、121-128、142-149、170之一所示；
    HCDR3的氨基酸序列如SEQ ID No：22、28、34、40、46、52、58、64之一所示；
    所述VL包含选自以下的LCDR1-3：
    LCDR1的氨基酸序列如SEQ ID No：23、29、35、41、47、53、59、65、111、115-119之一所示；
    LCDR2的氨基酸序列如SEQ ID No：24、30、36、42、48、54、60、66、112、113、114、116、120之一所示；
    LCDR3的氨基酸序列如SEQ ID No：25、31、37、43、49、55、61、67之一所示。
  6. 如权利要求5所述的抗FcRn抗体或其抗原结合片段，其中：
    (a)VH包含分别如SEQ ID No：20-21所示的HCDR1、HCDR2、HCDR3，VL包含分别如SEQ ID No：23-25所示的LCDR1、LCDR2、LCDR3；
    (b)VH包含分别如SEQ ID No：26-28所示的HCDR1、HCDR2、HCDR3，VL包含分别如SEQ ID No：29-31所示的LCDR1、LCDR2、LCDR3；
    (c)VH包含分别如SEQ ID No：32-34所示的HCDR1、HCDR2、HCDR3，VL包含分别如SEQ ID No：35-37所示的LCDR1、LCDR2、LCDR3；
    (d)VH包含分别如SEQ ID No：38-40所示的HCDR1、HCDR2、HCDR3，VL包含分别如SEQ ID No：41-43所示的LCDR1、LCDR2、LCDR3；
    (e)VH包含分别如SEQ ID No：44-46所示的HCDR1、HCDR2、HCDR3，VL包含分别如SEQ ID No：47-49所示的LCDR1、LCDR2、LCDR3；
    (f)VH包含分别如SEQ ID No：50-52所示的HCDR1、HCDR2、HCDR3，VL包含分别如SEQ ID No：53-55所示的LCDR1、LCDR2、LCDR3；
    (g)VH包含分别如SEQ ID No：56-58所示的HCDR1、HCDR2、HCDR3，VL包含分别如SEQ ID No：59-61所示的LCDR1、LCDR2、LCDR3；
    (h)VH包含分别如SEQ ID No：62-64所示的HCDR1、HCDR2、HCDR3，VL包含分别如SEQ ID No：65-67所示的LCDR1、LCDR2、LCDR3；
    (i)VH包含分别如SEQ ID No：68、69、40所示的HCDR1、HCDR2、HCDR3，VL包含分别如SEQ ID No：41-43所示的LCDR1、LCDR2、LCDR3；
    (j)VH包含分别如SEQ ID No：38、170、40所示的HCDR1、HCDR2、HCDR3，VL包含分别如SEQ ID No：41-43所示的LCDR1、LCDR2、LCDR3；
    (k)VH包含分别如SEQ ID No：133、170、40所示的HCDR1、HCDR2、HCDR3，VL包含分别如SEQ ID No：41-43所示的LCDR1、LCDR2、LCDR3；
    (l)VH包含分别如SEQ ID No：151、69、40所示的HCDR1、HCDR2、HCDR3，VL包含分别如SEQ ID No：41-43所示的LCDR1、LCDR2、LCDR3；
    (m)VH包含分别如SEQ ID No：154、69、40所示的HCDR1、HCDR2、HCDR3，VL包含分别如SEQ ID No：41-43所示的LCDR1、LCDR2、LCDR3；
    (n)VH包含分别如SEQ ID No：158、69、40所示的HCDR1、HCDR2、HCDR3，VL包含分别如SEQ ID No：41-43所示的LCDR1、LCDR2、LCDR3；
    (o)VH包含分别SEQ ID No：159、69、40所示的HCDR1、HCDR2、HCDR3，VL包含分别如SEQ ID No：41-43所示的LCDR1、LCDR2、LCDR3；
    (p)VH包含分别如SEQ ID No：162、69、40所示的HCDR1、HCDR2、HCDR3，VL包含分别如SEQ ID No：41-43所示的LCDR1、LCDR2、LCDR3；
    (q)VH包含分别如SEQ ID No：38、123、40所示的HCDR1、HCDR2、HCDR3，VL包含分别如SEQ ID No：111、112、43所示的LCDR1、LCDR2、LCDR3；
    (r)VH包含分别如SEQ ID No：38、127、40所示的HCDR1、HCDR2、HCDR3，VL包含分别如SEQ ID No：111、112、43所示的LCDR1、LCDR2、LCDR3；
    (s)VH包含分别如SEQ ID No：38、128、40所示的HCDR1、HCDR2、HCDR3，VL包含分别如SEQ ID No：111、112、43所示的LCDR1、LCDR2、LCDR3；
    (t)VH包含分别如SEQ ID No：38、124、40所示的HCDR1、HCDR2、HCDR3，VL包含分别如SEQ ID No：117、112、43所示的LCDR1、LCDR2、LCDR3；
    (u)VH包含分别如SEQ ID No：38、121、40所示的HCDR1、HCDR2、HCDR3，VL包含分别如SEQ ID No：41、116、43所示的LCDR1、LCDR2、LCDR3。
  7. 如权利要求5-6任一项所述的抗FcRn抗体或其抗原结合片段，其中：
    VH的氨基酸序列如SEQ ID No：4、6、8、10、12、14、16、18、87-110、166、168、169之一所示或与之具有至少90％同一性；和
    VL的氨基酸序列如SEQ ID No：5、7、9、11、13、15、17、19、76-86、167之一所示或与之具有至少90％同一性。
  8. 如权利要求7所述的抗FcRn抗体或其抗原结合片段，其中：
    VH的氨基酸序列如SEQ ID No：4所示或与之具有至少90％同一性，VL的 氨基酸序列如SEQ ID No：5所示或与之具有至少90％同一性；
    VH的氨基酸序列如SEQ ID No：6所示或与之具有至少90％同一性，VL的氨基酸序列如SEQ ID No：7所示或与之具有至少90％同一性；
    VH的氨基酸序列如SEQ ID No：8所示或与之具有至少90％同一性，VL的氨基酸序列如SEQ ID No：9所示或与之具有至少90％同一性；
    VH的氨基酸序列如SEQ ID No：10所示或与之具有至少90％同一性，VL的氨基酸序列如SEQ ID No：11所示或与之具有至少90％同一性；
    VH的氨基酸序列如SEQ ID No：12所示或与之具有至少90％同一性，VL的氨基酸序列如SEQ ID No：13所示或与之具有至少90％同一性；
    VH的氨基酸序列如SEQ ID No：14所示或与之具有至少90％同一性，VL的氨基酸序列如SEQ ID No：15所示或与之具有至少90％同一性；
    VH的氨基酸序列如SEQ ID No：16所示或与之具有至少90％同一性，VL的氨基酸序列如SEQ ID No：17所示或与之具有至少90％同一性；
    VH的氨基酸序列如SEQ ID No：18所示或与之具有至少90％同一性，VL的氨基酸序列如SEQ ID No：19所示或与之具有至少90％同一性；
    VH的氨基酸序列如SEQ ID No：96、99、103、104、107之一所示或与之具有至少90％同一性，VL的氨基酸序列如SEQ ID No：13所示或与之具有至少90％同一性；
    VH的氨基酸序列如SEQ ID No：89、93、94之一所示或与之具有至少90％同一性，VL的氨基酸序列如SEQ ID No：76所示或与之具有至少90％同一性；
    VH的氨基酸序列如SEQ ID No：87所示或与之具有至少90％同一性，VL的氨基酸序列如SEQ ID No：84所示或与之具有至少90％同一性；或
    VH的氨基酸序列如SEQ ID No：87-110之一所示或与之具有至少90％同一性，VL的氨基酸序列如SEQ ID No：76-86之一所示或与之具有至少90％同一性。
  9. 如权利要求5-8任一项所述的抗FcRn抗体或其抗原结合片段，其中：
    重链氨基酸序列如SEQ ID No：178-180任一所示，或与之具有至少80％同一性；轻链氨基酸序列如SEQ ID No：181所示，或与之具有至少80％同一性。
  10. 如权利要求1-9任一项所述的抗FcRn抗体或其抗原结合片段，其为鼠源抗体、嵌合抗体、全人抗体、人源化抗体或其片段。
  11. 如权利要求1-10任一项所述的抗FcRn抗体或其抗原结合片段，其为IgG抗体或其抗原结合片段，优选为IgG1、IgG4抗体或其抗原结合片段。
  12. 如权利要求1-10任一项所述的抗FcRn抗体或其抗原结合片段，所述抗原结合片段为scFv、Fv、Fab或Fab’片段。
  13. 如权利要求1-12任一项所述的抗FcRn抗体或其抗原结合片段，其阻断人IgG与人FcRn的结合。
  14. 如权利要求1-13任一项所述的抗FcRn抗体或其抗原结合片段，其不结合β2微球蛋白。
  15. FcRn结合蛋白，其含有权利要求1-14任一项所述的抗FcRn抗体或其抗原结合片段。
  16. 经分离的多核苷酸，其编码权利要求1-15任一项所述的抗FcRn抗体或其抗原结合片段。
  17. 载体，其含有如权利要求16所述经分离的多核苷酸。
  18. 宿主细胞，其包含权利要求17所述的载体或权利要求16所述经分离的多核苷酸。
  19. 制备抗FcRn抗体或其抗原结合片段的方法，包括：
    在权利要求18所述的宿主细胞中表达抗FcRn抗体或其抗原结合片段，以及
    从所述宿主细胞中分离所述抗FcRn抗体或其抗原结合片段。
  20. 药物组合物，其含有权利要求1-14任一项所述的抗FcRn抗体或其抗原结合片段、权利要求15所述的FcRn结合蛋白或权利要求16所述经分离的多核苷酸；以及，可药用的赋形剂、稀释剂或载体。
  21. 选自以下的任一项或其任意组合在制备药物或药物组合物中的用途：
    权利要求1-14任一项所述的抗FcRn抗体或其抗原结合片段、权利要求15所述的FcRn结合蛋白或权利要求16所述经分离的多核苷酸，其中：
    优选地，所述药物或药物组合物用于治疗自身免疫疾病或延缓自身免疫疾病进展；
    更优选地，所述自身免疫疾病选自：免疫中性粒细胞减少症、Guillain-Barre综合征、癫痫、自身免疫脑炎、Isaac综合征、痣综合征、寻常天疱疮、落叶型天疱疮、大疱性类天疱疮、获得性大疱性表皮松解症、妊娠性类天疱疮、粘膜类天疱疮、抗磷脂综合征、自身免疫贫血、自身免疫Grave病、Goodpasture综合征、重症肌无力、多发性硬化、类风湿性关节炎、狼疮、特发性血小板减少性紫癜、狼疮性肾炎和膜性肾病。
  22. 治疗自身免疫疾病或延缓自身免疫疾病进展的方法，所述方法包括：
    向受试者施用治疗有效量或延缓疾病有效量的权利要求1-14任一项所述的抗FcRn抗体或其抗原结合片段、权利要求15所述的FcRn结合蛋白或权利要求16所述经分离的多核苷酸，
    优选地，所述自身免疫疾病选自：免疫中性粒细胞减少症、Guillain-Barre综 合征、癫痫、自身免疫脑炎、Isaac综合征、痣综合征、寻常天疱疮、落叶型天疱疮、大疱性类天疱疮、获得性大疱性表皮松解症、妊娠性类天疱疮、粘膜类天疱疮、抗磷脂综合征、自身免疫贫血、自身免疫Grave病、Goodpasture综合征、重症肌无力、多发性硬化、类风湿性关节炎、狼疮、特发性血小板减少性紫癜、狼疮性肾炎和膜性肾病。
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