WO2022228364A1 - 抗masp2抗体、其抗原结合片段及医药用途 - Google Patents

抗masp2抗体、其抗原结合片段及医药用途 Download PDF

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WO2022228364A1
WO2022228364A1 PCT/CN2022/088914 CN2022088914W WO2022228364A1 WO 2022228364 A1 WO2022228364 A1 WO 2022228364A1 CN 2022088914 W CN2022088914 W CN 2022088914W WO 2022228364 A1 WO2022228364 A1 WO 2022228364A1
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seq
antibody
antigen
masp2
amino acid
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PCT/CN2022/088914
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English (en)
French (fr)
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杨晓峰
温婧
张敬杨
周彩红
廖成
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江苏恒瑞医药股份有限公司
上海盛迪医药有限公司
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Priority to KR1020237039785A priority Critical patent/KR20240000543A/ko
Priority to AU2022263683A priority patent/AU2022263683A1/en
Priority to CA3216034A priority patent/CA3216034A1/en
Priority to JP2023564459A priority patent/JP2024518724A/ja
Priority to CN202280018960.3A priority patent/CN116981477A/zh
Priority to US18/288,078 priority patent/US20240209114A1/en
Priority to BR112023020622A priority patent/BR112023020622A2/pt
Priority to EP22794834.6A priority patent/EP4331603A1/en
Publication of WO2022228364A1 publication Critical patent/WO2022228364A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • the present disclosure relates to the field of biomedicine, in particular to anti-MASP2 antibodies and antigen-binding fragments thereof, pharmaceutical compositions and related pharmaceutical uses thereof.
  • the complement system is a protein present in human and animal serum, tissue fluid and cell membrane surface. After activation, it has biological activity and can mediate immune and inflammatory responses.
  • the complement system consists of nearly 40 components, most of which are glycoproteins, including C1q, C1r, C1s, C2-C9, factor D, factor B, as well as 10 regulatory proteins and 10 complement receptors.
  • Complement is widely involved in the body's defense response to microorganisms and immune regulation, and is also involved in the damage response of immune pathology. Complement is an important effector system and effector amplification system of innate immunity.
  • the activation process of the complement system is manifested as a cascade of serine protease enzymatic hydrolysis reactions, which are divided into three types: the classical pathway, the alternative pathway, and the lectin pathway. Complex formation is marked by activation of the terminal pathway.
  • alternative pathways remain activated at low levels for long periods of time to monitor pathogenic microbial invasion. Healthy cells also inhibit the complement system from attacking them by expressing complement regulatory proteins such as CD55 and CD59.
  • Three pathways are usually activated on the surface of apoptotic cells and microorganisms.
  • the classical pathway is that antibodies (IgG1, IGG, IgG3, IgG4 or IgM) form an immune complex after the antigen structure is recognized by C1q, C1s, C1r, activate C2, C4, form C4bC2a (that is, C3 convertase), and finally promote C5 -The formation of membrane-breaking complexes composed of C9.
  • the alternative pathway is an activation pathway directly initiated by the spontaneous hydrolysis of C3. Under the stimulation of bacterial cell wall components LPS, Zymosan and other polysaccharides, peptidoglycan, teichoic acid and other activating substances, Factor D (Factor D) hydrolyzes the factor bound by C3.
  • Fractor B forms C3bBb3, which then completes the chain reaction of components from C5 to C9.
  • the lectin pathway directly recognizes mannose, N-acetylmannose, N-acetylmannose on the surface of various pathogenic microorganisms by mannan-binding lectin (MBL) or ficolin (FCN) in plasma Glucosamine, fucose, etc. are the sugar structures of terminal sugar groups, and then activate the classical pathway of complement.
  • the safety of targeting the complement system is generally safe and tolerable. Considering that the complement system is involved in the regulation of B cell development and T cell activation, complement factor gene-deficient mice have defects in reproductive ability or embryonic development, and increase the risk of infection. Therefore, the development of drugs targeting specific upstream targets of the complement pathway can reduce the side effects of the overall inhibition of the complement pathway while ensuring the efficacy of the drug.
  • MASP2 protein is the core hydrolase of the lectin pathway. It consists of the CUB and EGF domains at the N-terminus responsible for binding to MBL, the CCP domain that binds to downstream substrates C4 and C2, and the C-terminal enzyme activity domain SP.
  • the MBL-MASP complex binds to the surface carbohydrate structure of pathogens, so that MASP-1 and MASP-2 are independently activated.
  • Activated MASP2 exerts its SP activity, cleaves C4 and C2 to form C3 convertase C4b2a, and finally activates the complement system mediated by the lectin pathway. It has been confirmed that it is related to IgA nephropathy, stroke, and myocardial ischemia.
  • MASP2 deletion can significantly reduce the infarct size.
  • MBL-MASP2 mainly recognizes the galactose-modified missing IgA.
  • the activation of the LP pathway promotes the secretion of cytokines, which eventually leads to the damage of renal tubular epithelial cells and podocytes, and abnormal renal function.
  • Omeros' MASP2 monoclonal antibody narsoplimab (OMS721) is mainly developed for the treatment of various inflammatory related diseases by inhibiting lectin-mediated complement system activation, including thrombotic microangiopathy (TMA), IgA nephropathy, hemolytic uremic syndrome syndrome (HUS), lupus nephritis, membranous glomerulonephritis, glomerulonephritis, age-related macular degeneration, reperfusion injury, myocardial infarction, diabetic neuropathy, stroke, graft-versus-host disease.
  • TMA thrombotic microangiopathy
  • IgA nephropathy IgA nephropathy
  • HUS hemolytic uremic syndrome syndrome
  • lupus nephritis membranous glomerulonephritis
  • glomerulonephritis age-related macular degeneration
  • reperfusion injury myocardial infarction
  • the research on TMA is in pre-registration status in the United States
  • the research on HUS and IgA nephropathy is in clinical phase III
  • the research on lupus nephritis, membranous glomerulonephritis and glomerulonephritis is in clinical phase II.
  • the present disclosure provides anti-MASP2 antibodies and antigen-binding fragments thereof, nucleic acids encoding the same, vectors comprising the nucleic acids, host cells, pharmaceutical compositions comprising the anti-MASP2 antibodies and antigen-binding fragments thereof, and their use in therapy or therapy Methods of delaying complement system-related disorders, and uses thereof.
  • anti-MASP2 antibodies and antigen-binding fragments thereof comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein:
  • the VH comprises HCDR1, HCDR2, and HCDR3 in the VH as shown in any of SEQ ID NOs: 7, 9, and 11, and/or, the VL comprises as shown in any of SEQ ID NOs: 8, 10, and 12; Display LCDR1, LCDR2, LCDR3 in VL;
  • the VH comprises HCDR1, HCDR2, HCDR3 in the VH shown in SEQ ID NO: 13
  • the VL comprises LCDR1, LCDR2, LCDR3 in the VL shown in SEQ ID NO: 14;
  • the VH comprises HCDR1, HCDR2, HCDR3 in the VH shown in SEQ ID NO: 15, and/or, the VL comprises LCDR1, LCDR2, LCDR3 in the VL shown in SEQ ID NO: 16;
  • the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the VH and VL are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering system, and in some embodiments, the Kabat numbering system.
  • anti-MASP2 antibodies and antigen-binding fragments thereof in some embodiments, wherein,
  • the VH comprises HCDR1, HCDR2, and HCDR3 in the VH shown in SEQ ID NO: 7, and/or the VL comprises LCDR1, LCDR2, and LCDR3 in the VL shown in SEQ ID NO: 8;
  • the VH comprises HCDR1, HCDR2, HCDR3 in the VH shown in SEQ ID NO: 9, and/or the VL comprises LCDR1, LCDR2, LCDR3 in the VL shown in SEQ ID NO: 10;
  • the VH comprises HCDR1, HCDR2, and HCDR3 in the VH shown in SEQ ID NO: 11, and/or the VL comprises LCDR1, LCDR2, and LCDR3 in the VL shown in SEQ ID NO: 12.
  • anti-MASP2 antibodies and antigen-binding fragments thereof comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein:
  • the VH comprises HCDR1, HCDR2, HCDR3 as shown in SEQ ID NOs: 17, 18, 26, respectively, and/or, the VL comprises LCDR1, LCDR2, LCDR3;
  • sequence of SEQ ID NO: 17 is: SDYAWN;
  • sequence of SEQ ID NO: 18 is: YISYSGRTSYNPSLKS;
  • SEQ ID NO: 26 The sequence of SEQ ID NO: 26 is: LYAX 1 X 2 X 3 , wherein X 1 is selected from L or M, X 2 is selected from D or N, and X 3 is selected from Y or F;
  • sequence of SEQ ID NO: 20 is: KASQNVDTNVA;
  • SEQ ID NO: 27 The sequence of SEQ ID NO: 27 is: SASYRX 4 S, wherein X 4 is selected from Y or F;
  • sequence of SEQ ID NO: 22 is: QQYNSNPLT;
  • the VH comprises HCDR1, HCDR2, HCDR3 as shown in SEQ ID NOs: 28, 29, 30, respectively, and/or, the VL comprises LCDR1, LCDR2, LCDR3; or
  • the VH comprises HCDR1, HCDR2, HCDR3 as shown in SEQ ID NOs: 34, 35, 36, respectively, and/or, the VL comprises LCDR1, LCDR2, LCDR3.
  • anti-MASP2 antibodies and antigen-binding fragments thereof in some embodiments, wherein,
  • the VH comprises HCDR1, HCDR2, HCDR3 shown in SEQ ID NOs: 17, 18, 19, respectively, and/or, the VL comprises LCDR1, LCDR2, LCDR3;
  • the VH comprises HCDR1, HCDR2, HCDR3 as shown in SEQ ID NOs: 17, 18, 23, respectively, and/or, the VL comprises LCDR1, LCDR2, LCDR3; or
  • the VH comprises HCDR1, HCDR2, HCDR3 as shown in SEQ ID NOs: 17, 18, 25, respectively, and/or, and the VL comprises LCDR1, LCDR2, LCDR3.
  • variants of the above-described anti-MASP2 antibodies and antigen-binding fragments thereof are provided, wherein,
  • the VH comprises HCDR1 with up to 3, 2 or 1 amino acid change from SEQ ID NO: 17, HCDR2 with up to 3, 2 or 1 amino acid change with SEQ ID NO: 18, and HCDR2 with SEQ ID NO: 18 NO: 19 or 25 HCDR3 with up to 3, 2 or 1 amino acid change; and/or, the VL comprises LCDR1 with up to 3, 2 or 1 amino acid change from SEQ ID NO: 20, and SEQ ID NO:21 has an LCDR2 with up to 3, 2 or 1 amino acid change, and an LCDR3 with up to 3, 2 or 1 amino acid change with SEQ ID NO:22;
  • the VH comprises HCDR1 with up to 3, 2 or 1 amino acid change from SEQ ID NO: 17, HCDR2 with up to 3, 2 or 1 amino acid change with SEQ ID NO: 18, and HCDR2 with SEQ ID NO: 18 NO: 23 HCDR3 with up to 3, 2 or 1 amino acid change; and/or, the VL comprises LCDR1 with up to 3, 2 or 1 amino acid change from SEQ ID NO: 20, and SEQ ID NO: 20 NO:24 LCDR2 with up to 3, 2 or 1 amino acid change, and LCDR3 with up to 3, 2 or 1 amino acid change from SEQ ID NO:22;
  • the VH comprises HCDR1 with at most 3, 2 or 1 amino acid change from SEQ ID NO:28, HCDR2 with at most 3, 2 or 1 amino acid change from SEQ ID NO:29, and NO:30 HCDR3 with up to 3, 2 or 1 amino acid change; and/or, the VL comprises LCDR1 with up to 3, 2 or 1 amino acid change from SEQ ID NO:31, and SEQ ID NO:31 NO:32 LCDR2 with up to 3, 2 or 1 amino acid change, and LCDR3 with up to 3, 2 or 1 amino acid change with SEQ ID NO:33;
  • the VH comprises HCDR1 with at most 3, 2 or 1 amino acid change from SEQ ID NO:34, HCDR2 with at most 3, 2 or 1 amino acid change from SEQ ID NO:35, and NO: 36 HCDR3 with up to 3, 2 or 1 amino acid change; and/or, the VL comprises LCDR1 with up to 3, 2 or 1 amino acid change from SEQ ID NO: 37, and SEQ ID NO: 37 NO:38 LCDR2 with up to 3, 2 or 1 amino acid change, and LCDR3 with up to 3, 2 or 1 amino acid change from SEQ ID NO:39.
  • the above amino acid changes may be conservative substitutions, substitutions or modifications, and/or deletions, additions that do not affect function.
  • anti-MASP2 antibodies and antigen-binding fragments thereof are provided, comprising at least one or more of any of the CDRs; eg, comprising any HCDR1 of the present disclosure or one or more of any HCDR2, HCDR3; eg, comprising the present disclosure Any LCDR1 or one or more of any LCDR2, LCDR3.
  • the above-described anti-MASP2 antibody or antigen-binding fragment thereof of the present disclosure is a murine antibody, a chimeric antibody, a humanized antibody, a fully human antibody, or a fragment thereof.
  • the heavy chain framework regions of the humanized antibodies or antigen-binding fragments thereof of the present disclosure described above are derived from IGKV3-21*01 or IGKV4-30-4*01; and/or, the light chain framework regions are derived from IGKV1 -33*01 or IGKV1-27*01.
  • anti-MASP2 antibody or antigen-binding fragment thereof in some embodiments, wherein,
  • amino acid sequence of VH is shown in one of SEQ ID NOs: 7, 9, 11, 47, 48, 49, 50, and/or the amino acid sequence of VL is shown in one of SEQ ID NOs: 8, 10, 12, 51, 52 a shown;
  • amino acid sequence of VH is set forth in one of SEQ ID NOs: 13, 42, 43, 44, and/or the amino acid sequence of VL is set forth in one of SEQ ID NOs: 14, 45, 46; or
  • amino acid sequence of VH is shown in SEQ ID NO: 15
  • amino acid sequence of VL is shown in SEQ ID NO: 16.
  • anti-MASP2 antibody or antigen-binding fragment thereof in some embodiments, wherein,
  • VH and VL respectively comprise or consist of the amino acid sequences shown in SEQ ID NOs: 7 and 8;
  • VH and VL respectively comprise or consist of the amino acid sequences shown in SEQ ID NOs: 9 and 10;
  • VH and VL respectively comprise or consist of the amino acid sequences shown in SEQ ID NOs: 11 and 12;
  • VH and VL respectively comprise or consist of amino acid sequences as shown in SEQ ID NOs: 47 and 51;
  • VH and VL respectively comprise or consist of the amino acid sequences shown in SEQ ID NOs: 47 and 52;
  • VH and VL respectively comprise or consist of the amino acid sequences shown in SEQ ID NOs: 48 and 51;
  • VH and VL respectively comprise or consist of the amino acid sequences shown in SEQ ID NOs: 48 and 52;
  • VH and VL respectively comprise or consist of the amino acid sequences shown in SEQ ID NOs: 49 and 51;
  • VH and VL respectively comprise or consist of the amino acid sequences shown in SEQ ID NOs: 49 and 52;
  • VH and VL respectively comprise or consist of the amino acid sequences shown in SEQ ID NOs: 50 and 51;
  • VH and VL respectively comprise or consist of the amino acid sequences shown in SEQ ID NOs: 50 and 52;
  • VH and VL respectively comprise amino acid sequences as shown in SEQ ID NOs: 13 and 14, or consist of them;
  • VH and VL respectively comprise or consist of the amino acid sequences shown in SEQ ID NOs: 42 and 45;
  • VH and VL respectively comprise or consist of the amino acid sequences shown in SEQ ID NOs: 42 and 46;
  • VH and VL respectively comprise the amino acid sequences shown in SEQ ID NOs: 43 and 45, or consist of them;
  • VH and VL respectively comprise or consist of the amino acid sequences shown in SEQ ID NOs: 43 and 46;
  • VH and VL respectively comprise the amino acid sequences shown in SEQ ID NOs: 44 and 45, or consist of them;
  • VH and VL respectively comprise or consist of the amino acid sequences shown in SEQ ID NOs: 44 and 46; or
  • VH and VL respectively comprise or consist of the amino acid sequences shown in SEQ ID NOs: 15 and 16.
  • the present disclosure provides a VH, VL comprising at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, An anti-MASP2 antibody or antigen-binding fragment thereof of a variant VH, VL of at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • the anti-MASP2 antibody or antigen-binding fragment thereof of the present disclosure is an IgG antibody or antigen-binding fragment thereof, eg, an IgGl, IgG2, IgG4 antibody or antigen-binding fragment thereof, more eg, an Fc with S228P, F234A, and L235A Any one or more mutated IgG4 antibodies or antigen-binding fragments thereof.
  • the above mutations are all EU numbers.
  • the anti-MASP2 antibodies or antigen-binding fragments thereof of the present disclosure further comprise a human immunoglobulin Fc region; eg, the Fc region is the Fc region of human IgGl, IgG2, or IgG4. In some embodiments, the Fc region may have mutations that reduce ADCC function.
  • mutations are exemplified as L234A/L235A on IgG1, V234A/G237A/P238S/H268A/V309L/A330S/P331S on IgG2, F234A/L235A on IgG4, S228P/F234A/L235A on IgG4, IgG1 , N297A on IgG2, IgG3 or IgG4, V234A/G237A on IgG2, K214T/E233P/L234V/L235A/G236 deletion/A327G/P331A/D365E/L358M on IgG1, H268Q/V309L/A330S/P331S on IgG2, S267E/L328F on IgG1, L234F/L235E/D265A on IgG1, L234A/L235A/G237A/P238S/H268
  • Hybrid IgG2/4 Fc domains can also be used, eg, an Fc with residues 117-260 from IgG2 and residues 261-447 from IgG4.
  • the Fc region of the human IgG4 has any one or more mutations of S228P, F234A, L235A and K447A (see WO2017079112A, WO2018031400A, etc.).
  • the antigen-binding fragment of an anti-MASP2 antibody of the present disclosure is a Fab, Fv, sFv, Fab', F(ab') 2 , linear antibody, single chain antibody, scFv, sdAb, sdFv, Nanobody, peptidobody peptibodies, domain antibodies and multispecific antibodies (bispecific antibodies, diabody, triabody and tetrabody, tandem di-scFv, tandem tri-scFv), eg in particular scFv, Fv, Fab or Fab' fragments.
  • the amino acid sequence of the full-length heavy chain of the antigen-binding fragment of an anti-MASP2 antibody of the present disclosure is set forth in or at least 80%, at least 90%, or at least 95% identical to SEQ ID NO: 54; the light chain The full-length amino acid sequence is set forth in or at least 80%, at least 90%, or at least 95% identical to SEQ ID NO: 55;
  • amino acid sequence of the full length of the heavy chain is shown in SEQ ID NO: 56 or has at least 80%, at least 90% or at least 95% identity; the amino acid sequence of the full length of the light chain is shown in SEQ ID NO: 57 or with the same. are at least 80% identical, at least 90% or at least 95% identical; or
  • amino acid sequence of the full length of the heavy chain is shown in SEQ ID NO: 58 or has at least 80%, at least 90% or at least 95% identity; the amino acid sequence of the full length of the light chain is shown in SEQ ID NO: 59 or with the same. are at least 80%, at least 90% or at least 95% identical.
  • the full-length amino acid sequences of the heavy chain and the light chain are shown in SEQ ID NOs: 54 and 55, respectively; the full-length amino acid sequences of the heavy chain and the light chain are shown in SEQ ID NOs: 56 and 57, respectively; The chain and light chain full-length amino acid sequences are shown in SEQ ID NOs: 58 and 59, respectively.
  • the anti-MASP2 antibody or antigen-binding fragment thereof according to the present disclosure has 0 to 10 heavy chain variable regions (1, 2, 3, 4, 5, 6, 7, 8, 9, 10) Amino acid changes; 0 to 10 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid changes in the light chain variable region.
  • the amino acid changes are conservative substitutions, substitutions or modifications, and/or deletions, additions that do not affect function.
  • an anti-MASP2 antibody or antigen-binding fragment that binds or competes for binding to the same epitope as the aforementioned anti-MASP2 antibody or antigen-binding fragment.
  • anti-MASP2 antibodies or antigen-binding fragments that block the binding of the aforementioned anti-MASP2 antibodies or antigen-binding fragments thereof to human MASP2.
  • anti-MASP2 antibodies or antigen-binding fragments are provided whose binding to human MASP2 is blocked by the aforementioned anti-MASP2 antibodies or antigen-binding fragments thereof.
  • an anti-MASP2 antibody or antigen-binding fragment of the present disclosure has at least one of the following:
  • the antibody or antigen-binding fragment binds human MASP-2 with a KD of 10 nM or less;
  • the antibody or antigen-binding fragment binds an epitope in the CCP1 domain of MASP-2;
  • the antibody or antigen-binding fragment inhibits C3b deposition in an in vitro assay in 1% human serum with an IC 50 of 10 nM or lower, and the IC 50 can be detected by, for example, the method of Example 3 of the present disclosure;
  • the antibody or antigen-binding fragment inhibits 90% of C3b deposition in human serum with an IC 50 of 30 nM or less, which can be detected, for example, by the method of Example 6 of the present disclosure.
  • the antibody or antigen-binding fragment does not substantially inhibit the classical pathway.
  • the anti-MASP-2 antibody selectively inhibits MASP-2 complement activation, leaving the C1q-dependent complement activation system functionally intact.
  • MASP2 binding proteins or binding molecules comprising any of the anti-MASP2 antibodies or antigen-binding fragments thereof of the present disclosure described above.
  • the MASP2 binding protein or binding molecule contains one or more effector molecules selected from the group consisting of antineoplastic agents, drugs, toxins, biologically active proteins (eg, enzymes), other antibodies or antibodies Fragments, synthetic or naturally occurring polymers, nucleic acids and fragments thereof such as DNA, RNA and fragments thereof, radionuclides (eg, radioiodides), radioisotopes, chelated metals, nanoparticles, and reporter groups (eg, fluorescent compounds), or compounds detectable by NMR or ESR spectroscopic analysis.
  • the effector molecule is conjugated or fused to an anti-MASP2 antibody or antigen-binding fragment thereof of the present disclosure.
  • the present disclosure provides isolated polynucleotides encoding anti-MASP2 antibodies or antigen-binding fragments, binding proteins, or binding molecules thereof of the present disclosure.
  • the polynucleotide can be DNA or RNA.
  • the polynucleotides of the present disclosure are substantially isolated or isolated polynucleotides.
  • the present disclosure provides expression vectors containing the polynucleotides described above, or alternatively, the polynucleotides of the present disclosure may also be in the form of, be present in, and/or be part of a vector, such as a plasmid, a plasmid, Terminal plasmid, YAC or viral vector.
  • the vector may in particular be an expression vector, i.e. a vector that provides for in vitro and/or in vivo (i.e.
  • the anti-MASP2 antibody or antigen-binding fragment thereof which may be eukaryotic expression Vectors, prokaryotic expression vectors, viral vectors such as plasmids, cosmids, phages.
  • the expression vector typically comprises at least one polynucleotide of the present disclosure operably linked to one or more suitable expression control elements (eg, promoters, enhancers, terminators, etc.). The selection of such elements and their sequences for expression in a particular host is within the general knowledge of those skilled in the art. Regulatory elements and other elements useful or necessary for the expression of the PD-1 binding proteins of the present disclosure are, for example, promoters, enhancers, terminators, integration factors, selectable markers, leader sequences, reporter genes.
  • polynucleotides of the present disclosure may be prepared or obtained by known means (eg, by automated DNA synthesis and/or recombinant DNA techniques) based on information on the amino acid sequences of the polypeptides of the present disclosure, and/or may be isolated from suitable natural sources .
  • the present disclosure provides host cells expressing an anti-MASP2 antibody or antigen-binding fragment, binding protein, or binding molecule of the present disclosure, and/or containing a polynucleotide or vector of the present disclosure.
  • the host cell is a bacterial cell, a fungal cell, or a mammalian cell
  • Bacterial cells include, for example, gram-negative bacterial strains (eg, Escherichia coli, Proteus, and Pseudomonas strains) and gram-positive bacterial strains (eg, Bacillus (Bacillus), Streptomyces (Streptomyces), Staphylococcus (Staphylococcus) and Lactococcus (Lactococcus) cells).
  • Bacillus Bacillus (Bacillus), Streptomyces (Streptomyces), Staphylococcus (Staphylococcus) and Lactococcus (Lactococcus) cells.
  • Fungal cells include, for example, cells of species of Trichoderma, Neurospora, and Aspergillus; or Saccharomyces (eg, Saccharomyces cerevisiae), Schizosaccharomyces cerevisiae Genus Schizosaccharomyces (eg Schizosaccharomyces pombe), Pichia (eg Pichia pastoris and Pichia methanolica) and Hansen A cell of a species of the genus Hansenula.
  • Saccharomyces eg, Saccharomyces cerevisiae
  • Schizosaccharomyces cerevisiae Genus Schizosaccharomyces eg Schizosaccharomyces pombe
  • Pichia eg Pichia pastoris and Pichia methanolica
  • Mammalian cells include, for example, HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, and the like.
  • amphibian cells insect cells, plant cells, and any other cell in the art for expressing heterologous proteins may also be used in the present disclosure.
  • Cells of the present disclosure cannot develop into completed plants or animals.
  • the present disclosure provides methods of making the anti-MASP2 antibodies or antigen-binding fragments, binding proteins or binding molecules thereof of the present disclosure, the methods generally comprising the steps of:
  • Anti-MASP2 antibodies or antigen-binding fragments, binding proteins or binding molecules of the present disclosure can be produced intracellularly (eg, in the cytoplasm, in the periplasm, or in inclusion bodies) in cells as described above, then isolated from the host cell and optionally further purified; or it can be produced extracellularly (eg, in the medium in which the host cells are cultured), then isolated from the medium and optionally further purified.
  • polypeptides such as specific suitable expression vectors, transformation or transfection methods, selectable markers, methods of inducing protein expression, culture conditions, etc.
  • protein isolation and purification techniques suitable for use in the methods of making the proteins of the present disclosure are well known to those of skill in the art.
  • cDNA sequences encoding heavy and light chains can be cloned and recombined into expression vectors.
  • the recombinant immunoglobulin expression vector can stably transfect CHO cells. Mammalian-like expression systems result in glycosylation of the antibody, particularly 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 were expanded in serum-free medium in bioreactors for antibody production. The antibody-secreted culture medium can be purified and collected by conventional techniques. Antibodies 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 obtained product should be frozen immediately, eg -70°C, or lyophilized.
  • anti-MASP2 antibodies or antigen-binding fragments, binding proteins or binding molecules thereof of the present disclosure can also be obtained by other methods of producing proteins known in the art, such as chemical synthesis, including solid-phase or liquid-phase synthesis.
  • compositions eg, pharmaceutical compositions, comprising a therapeutically effective amount of an anti-MASP2 antibody, or antigen-binding fragment, binding protein, or binding molecule thereof, as described above, and one or more pharmaceutically acceptable carriers, dilutions agents, buffers or excipients.
  • the unit dose of the pharmaceutical composition may contain 0.01 to 99% by weight of the anti-MASP2 antibody or antigen-binding fragment thereof, or the amount of the anti-MASP2 antibody or antigen-binding fragment thereof contained in the unit dose of the pharmaceutical composition 0.1-2000 mg, in some embodiments 1-1000 mg.
  • Kit (or kit)
  • kits or kits comprising one or more containers each independently comprising any one or a combination selected from the group consisting of an anti-MASP2 antibody or antigen-binding fragment thereof, binding protein, binding molecule, A polynucleotide encoding the above protein or molecule or antibody or antigen-binding fragment.
  • diagnostic reagents comprising the above-described polynucleotides are also provided, as well as related diagnostic uses.
  • the present disclosure provides uses and methods of anti-MASP2 antibodies or antigen-binding fragments, binding proteins, binding molecules, polynucleotides, and pharmaceutical compositions of the present disclosure for preventing and/or treating diseases, which may be related to complement signaling pathway (eg MASP2) related or not.
  • diseases which may be related to complement signaling pathway (eg MASP2) related or not.
  • the disease is IgA nephropathy, paroxysmal nocturnal hemoglobinuria (PNH).
  • the present disclosure provides methods of treating or preventing a disease comprising administering to a subject in need thereof a therapeutically or prophylactically effective amount of an anti-MASP2 antibody or antigen-binding fragment, binding protein, binding molecule, polynucleotide and/or medicament thereof of the present disclosure combination.
  • the present disclosure provides methods of inhibiting MASP-2-dependent complement activation in a subject, comprising administering in said subject an effective amount (eg, an amount sufficient to inhibit MASP-2-dependent complement activation) of the present disclosure
  • an effective amount eg, an amount sufficient to inhibit MASP-2-dependent complement activation
  • MASP-2-dependent complement activation has been implicated in contributing to the pathogenesis of many acute and chronic disease states, including MASP-2-dependent complement-mediated vascular conditions, ischemia-reperfusion injury, atherosclerosis, inflammatory gastrointestinal Tract disorders, pulmonary conditions, extracorporeal reperfusion procedures, skeletal muscle conditions, renal conditions, skin conditions, organ or tissue transplantation, neurological conditions or injuries, blood disorders, genitourinary tract conditions, diabetes, chemotherapy or radiation therapy, malignancy , endocrine disorders, coagulation disorders, or ophthalmic conditions.
  • methods of treating the aforementioned diseases and conditions, and related pharmaceutical uses are provided for the anti-MASP2 antibodies or antigen-binding fragments, binding proteins, binding molecules, polynucleotides, and/or pharmaceutical compositions thereof of the present disclosure.
  • the aforementioned diseases and conditions are MASP-2-dependent complement activation-related diseases.
  • the aforementioned diseases and conditions are microvascular endothelial cell damage and/or thrombosis.
  • the aforementioned diseases and conditions are selected from the group consisting of: IgA nephropathy, paroxysmal nocturnal hemoglobinuria (PNH), lupus nephritis, thrombotic microangiopathy (TMA) (e.g., persistent TMA associated with hematopoietic stem cell transplantation ( HSCT-TMA), thrombotic thrombocytopenic purpura (TTP)), hemolytic uremic syndrome (HUS), membranous glomerulonephritis, glomerulonephritis, age-related macular degeneration, reperfusion injury, myocardial Infarction, diabetic neuropathy, stroke, graft versus host disease (GVHD), Upshaw-Schulman syndrome (USS), in some embodiments, the disease is associated with MASP-2-dependent complement activation.
  • TMA thrombotic microangiopathy
  • HUS hemolytic uremic syndrome
  • membranous glomerulonephritis glomerul
  • the present disclosure provides compositions for detecting MASP2 comprising an anti-MASP2 antibody or antigen-binding fragment thereof according to the present disclosure.
  • the present disclosure also provides methods, systems, or devices for detecting MASP2 in vivo or in vitro, comprising treating a sample with an anti-MASP2 antibody or antigen-binding fragment thereof of the present disclosure.
  • an in vitro detection method, system or device may include, for example:
  • a change eg, a statistically significant change in complex formation in a sample or subject as compared to a control sample or subject indicates the presence of MASP2 in the sample.
  • the in vivo detection method, system or device may include:
  • Detection can include determining where or when complexes are formed.
  • the MASP2 antibody is labeled with a detectable substance, and detection of the label enables detection of a substance (eg, MASP2) that binds to the MASP2 antibody.
  • Suitable detectable substances include various enzymes, prosthetic groups, fluorescent substances, luminescent substances and radioactive substances.
  • Complex formation between an antibody or antigen-binding fragment thereof that binds MASP2 and MASP2 can be detected by measuring or visualizing the antibody that binds or does not bind to MASP2.
  • Conventional detection assays can be used, eg, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or histoimmunohistochemistry.
  • the anti-MASP2 antibodies or fragments thereof of the present disclosure can be labeled with a fluorophore chromophore.
  • kits are also provided that include an anti-MASP2 antibody or antigen-binding fragment thereof, and may also include diagnostic instructions.
  • the kit may also contain at least one additional reagent, such as a label or additional diagnostic agent.
  • the antibodies can be formulated as pharmaceutical compositions.
  • MASP-2-dependent complement activation includes MASP-2-dependent activation of the lectin pathway, which occurs under physiological conditions (ie, in the presence of Ca++), resulting in the formation of the lectin pathway C3 convertase C4b2a, and Accumulation of the C3 cleavage product C3b leads to the C5 convertase C42a(C3b)n.
  • the "classical pathway” refers to complement activation triggered by binding of antibodies to foreign particles and requiring binding of the recognition molecule C1q.
  • "Alternative pathway” refers to complement activation by, for example, zymosan from fungal and yeast cell walls, lipopolysaccharide (LPS) from the outer membrane of Gram-negative bacteria, and rabbit erythrocytes, as well as many pure polysaccharides, rabbit erythrocytes, viruses , bacteria, animal tumor cells, parasites, and damaged cells, and it has traditionally been thought to arise from the spontaneous proteolytic production of C3b by complement factor C3.
  • LPS lipopolysaccharide
  • Lectin pathway refers to complement activation, which occurs via the specific binding of serum and non-serum carbohydrate-binding proteins, including mannan-binding lectin (MBL), CL-11, and ficolin protein (H-ficolin, M-ficolin or L-ficolin).
  • MBL mannan-binding lectin
  • CL-11 mannan-binding lectin
  • ficolin protein H-ficolin, M-ficolin or L-ficolin
  • Antibody is used in the broadest sense to encompass a variety of antibody structures including, but not limited to, monoclonal antibodies, polyclonal antibodies; monospecific antibodies, multispecific antibodies (eg, 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 immunoglobulin heavy chain constant region are different, so their antigenicity is also different.
  • immunoglobulins can be divided into five classes, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE, whose corresponding heavy chains are ⁇ , ⁇ , and ⁇ chains, respectively. , alpha chains and epsilon chains.
  • the same type of Ig can be divided into different subclasses according to the difference in the amino acid composition of the hinge region and the number and position of disulfide bonds in the heavy chain.
  • IgG can be divided into IgG1, IgG2, IgG3, and IgG4.
  • Light chains are classified into kappa chains or lambda chains by the difference in the constant region.
  • Each of the five classes 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, which 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 three hypervariable regions (CDRs) and four relatively conserved framework regions (FRs). Three hypervariable regions determine the specificity of antibodies, also known as complementarity determining regions (CDRs).
  • 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 terminus to the carboxyl terminus is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the three CDR regions of the light chain are referred to as LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain are referred to as HCDR1, HCDR2, and HCDR3.
  • the definitive delineation of the CDRs and identification of residues comprising the binding site of the antibody can be accomplished by resolving the structure of the antibody and/or resolving the structure of the antibody-ligand complex. This can be accomplished by any of a variety of techniques known to those skilled in the art, such as X-ray crystallography.
  • a variety of analytical methods can be used to identify CDRs, including but not limited to the Kabat numbering system, Chothia numbering system, AbM numbering system, IMGT numbering system, contact definitions, conformational definitions.
  • the Kabat numbering system is the standard for numbering residues in antibodies and is commonly used to identify CDR regions (see, eg, 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 positions of certain structural loop regions. (See eg, Chothia et al., 1986, J. Mol. Biol., 196:901-17; Chothia et al., 1989, Nature, 342:877-83).
  • the AbM numbering system uses an integrated suite of computer programs produced by the Oxford Molecular Group for modeling antibody structures (see, eg, Martin et al., 1989, ProcNatl Acad Sci (USA), 86:9268-9272; "AbMTM, A Computer Program for ModelingVariable Regions of Antibodies," Oxford, UK; Oxford Molecular, Ltd).
  • the AbM numbering system uses a combination of knowledge databases and de novo methods to model the tertiary structure of antibodies from basic sequences (see Samudrala et al., 1999, "Ab in PROTEINS, Structure, Function and Genetics Suppl., 3: 194-198" Initio Protein Structure Prediction Using a Combined Hierarchical Approach”).
  • a CDR can refer to a CDR defined by any method known in the art, including combinations of methods.
  • the number and position of CDR amino acid residues in the VL and VH regions of the antibodies or antigen-binding fragments of the present disclosure conform to the known Kabat or AbM numbering system.
  • “Monoclonal antibody” or “monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies, ie, the individual antibodies comprised by the population are identical except for possible naturally occurring mutations that may be present in small amounts. Monoclonal antibodies are highly specific, directed against a single antigenic site. Furthermore, each monoclonal antibody is directed against a single determinant on the antigen, as opposed to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes).
  • the modifier "monoclonal” indicates a characteristic of an antibody as obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring the production of the antibody by any particular method.
  • monoclonal antibodies for use in accordance with the present disclosure can be prepared by the hybridoma method first described by Kohler and Milstein, 1975, Nature 256:495, or by recombinant DNA methods such as described in US Pat. No. 4,816,567.
  • monoclonal antibodies can also be isolated from phage libraries generated using techniques 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, involving techniques and methods well known in the art, such as:
  • an antibody isolated from a host cell transformed to express the antibody such as a transfectoma
  • Antibodies prepared, expressed, created or isolated by methods such as splicing human immunoglobulin gene sequences into other DNA sequences.
  • Such recombinant fully human antibodies contain variable and constant regions that utilize specific human germline immunoglobulin sequences encoded by germline genes, but also include subsequent rearrangements and mutations such as those that occur during antibody maturation.
  • murine antibody in the present disclosure is a monoclonal antibody directed against human MASP2 or an epitope thereof prepared according to the knowledge and skill in the art. In preparation, test subjects are injected with MASP2 antigen, and hybridomas expressing antibodies with the desired sequence or functional properties are isolated.
  • the murine anti-human MASP2 antibody or its antigen-binding fragment may further comprise a light chain constant region of a murine ⁇ , ⁇ chain or a variant thereof, or further comprise a murine IgG1 , IgG2, IgG3 or IgG4 or the heavy chain constant region of a variant thereof.
  • Fully human antibody includes antibodies having variable and constant regions of human germline immunoglobulin sequences.
  • Fully human antibodies of the present disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (eg, 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 backbone 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 human antibody variable regions. It can overcome the strong immune response induced by chimeric antibodies because they carry a large number of non-human protein components. To avoid a decrease in activity while decreasing immunogenicity, the variable regions of the fully human antibody may be subjected to minimal reverse mutations to maintain activity.
  • chimeric antibody is an antibody obtained 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 alleviate the immune response induced by the antibody of the first species.
  • a chimeric antibody we need to select a hybridoma that secretes a mouse-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 its variants, preferably comprising the heavy chain constant region of human IgG2 or IgG4, or using amino acid mutation 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 antibodies (eg VH or VL or VHH), scFv, bivalent or trivalent or tetravalent antibodies, 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).
  • Fab-Fv form was first disclosed in WO2009/040562 and its disulfide stabilized form, Fab-dsFv, was first disclosed in WO2010/035012.
  • 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, eg bispecific, or may be monospecific (see eg WO92/22583 and WO05/113605), an example of the latter being the Tri-Fab (or Tri-Fab) described in WO 92/22583 TFM).
  • binding to MASP2 refers to the ability to interact with MASP2 or an epitope thereof, which may be of human origin.
  • antigen-binding site refers to a discrete three-dimensional spatial site on an antigen that is recognized by an antibody or antigen-binding fragment of the present disclosure.
  • Antigen refers to a molecule used to immunize an immunocompetent vertebrate, to generate antibodies recognizing the antigen, or to screen expression libraries (eg, especially phage, yeast or ribosome display libraries).
  • an antigen is defined more broadly to include target molecules specifically recognized by antibodies, as well as including portions or mimetics of molecules used in immunization procedures for the production of antibodies or library screening for the selection of antibodies.
  • monomers and multimers eg, dimers, trimers, etc.
  • truncated and other variants of human MASP2 are referred to as antigens.
  • epitopes refers to the site on an antigen to which an immunoglobulin or antibody binds.
  • Epitopes can be formed by adjacent amino acids, or non-adjacent amino acids juxtaposed by tertiary folding of the protein. Epitopes formed by adjacent amino acids are typically retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents.
  • Epitopes typically include at least 3-15 amino acids in a unique spatial conformation. Methods for determining what epitopes are bound by a given antibody are well known in the art and include immunoblotting and immunoprecipitation detection assays, among others. Methods for determining the spatial conformation of epitopes include techniques in the art and those described in this disclosure, such as X-ray crystallography and two-dimensional nuclear magnetic resonance, among others.
  • Specific binding and “selective binding” refer to the binding of an antibody to a predetermined epitope on an antigen.
  • SPR surface plasmon resonance
  • the dissociation constant (K D ) binds to a predetermined antigen or its epitope, and its affinity for binding to the predetermined antigen or its epitope is nonspecific other than its binding to the predetermined antigen (or its epitope) or a closely related antigen Antigen (eg, BSA, etc.) binds with at least twice the affinity.
  • the term "antibody that recognizes an antigen” is 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 the non-covalent interaction between two molecules (eg, an antibody or portion 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, surface plasmon resonance (SPR) methods (Biacore).
  • SPR surface plasmon resonance
  • the rate constants corresponding to the association and dissociation of monovalent complexes are referred to as 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 even for complex mixtures by methods such as those described in Caceci et al. (1984, Byte 9:340-362).
  • KD can be determined using a double filtration nitrocellulose filter binding assay such as that disclosed in Wong & Lohman (1993, Proc. Natl. Acad. Sci. USA 90: 5428-5432 ).
  • Other standard assays to assess the binding ability of an antibody to a target antigen 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.
  • Binding kinetics and binding affinity of an antibody can also be determined by standards known in the art, such as surface plasmon resonance (SPR), eg, by using the Biacore TM system or KinExA. Binding affinities associated with different molecular interactions can be compared by comparing the KD values of individual antibody/antigen complexes, eg, a comparison of the binding affinities of different antibodies for a given antigen. Similarly, the specificity of an interaction can be determined and compared by determining and comparing the K of a desired interaction (eg, a specific interaction between an antibody and an antigen) to a non-target interaction (eg, a control antibody known not to bind MASP2). KD value was evaluated.
  • SPR surface plasmon resonance
  • a “conservative () substitution” refers to the substitution of another amino acid residue with 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.
  • Cross-reactivity refers to the ability of an antibody of the present disclosure to bind MASP2 from different species.
  • an antibody of the present disclosure that binds human MASP2 can also bind MASP2 of another species.
  • Cross-reactivity is measured by detecting specific reactivity with purified antigen in binding assays such as SPR and ELISA, or binding or functional interaction with cells that physiologically express MASP2.
  • Methods for determining cross-reactivity include standard binding assays as described in the present disclosure, such as surface plasmon resonance analysis, or flow cytometry.
  • Inhibit or block are used interchangeably and encompass both partial and complete inhibition/blocking. Inhibition/blocking of MASP2 preferably reduces or alters the normal level or type of activity that occurs when MASP2 binding occurs without inhibition or blockade. Inhibition and blocking are also intended to include any measurable reduction in binding affinity of MASP2 when contacted with an anti-MASP2 antibody compared to MASP2 not contacted with an anti-MASP2 antibody.
  • “Inhibition of growth” (eg, in relation to a cell) is intended to include any measurable reduction in cell growth.
  • mice can be immunized with human MASP2 or fragments thereof, and the resulting antibodies can be renatured, purified, and amino acid sequenced by conventional methods.
  • Antigen-binding fragments can likewise be prepared by conventional methods.
  • the antibodies or antigen-binding fragments of the present disclosure are genetically engineered to add one or more human FR regions to non-human CDR regions. Human FR germline sequences are available from the ImMunoGeneTics (IMGT) website.
  • the engineered antibodies or antigen-binding fragments of the present disclosure can be prepared and purified using conventional methods.
  • cDNA sequences encoding heavy and light chains can be cloned and recombined into expression vectors.
  • the recombinant immunoglobulin expression vector can stably transfect cells.
  • Mammalian-like expression systems result in glycosylation of the antibody, particularly 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 were expanded in serum-free medium in bioreactors for antibody production.
  • the antibody-secreted culture medium can be purified and collected by conventional techniques.
  • Antibodies 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 obtained product should be frozen immediately, eg -70°C, or lyophilized.
  • Antibodies can be screened competitively for binding to the same epitope using routine techniques known to those of skill in the art. For example, competition and cross-competition studies can be performed to obtain antibodies that compete with each other or cross-compete for binding to the antigen. A high-throughput method for obtaining antibodies that bind the same epitope based on their cross-competition is described in International Patent Publication WO03/48731. Thus, antibodies and antigen-binding fragments thereof that compete for binding to the same epitope on MASP2 with the antibody molecules of the present disclosure can be obtained using conventional techniques known to those of skill in the art.
  • administering when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refer to exogenous drugs, therapeutic agents, diagnostic agents, or compositions that interact with the animal. , contact of humans, subjects, cells, tissues, organs or biological fluids.
  • administering can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods.
  • Treatment of cells includes contact of reagents with cells, and contact of reagents with fluids, wherein the fluids are in contact with cells.
  • administering also mean in vitro and ex vivo treatment of, eg, cells by an agent, diagnostic, binding composition, or by another cell.
  • Treatment when applied to human, veterinary or research subjects refers to therapeutic treatment, prophylactic or preventive measures, research and diagnostic applications.
  • Treatment means administering an internal or external therapeutic agent, such as a composition comprising any of the antibodies or antigen-binding fragments thereof of the present disclosure, or conjugates thereof, to a subject who has, is suspected of having, Presence, predisposition to suffer from one or more diseases or symptoms thereof for which the therapeutic agent is known to have a therapeutic effect.
  • a therapeutic agent is administered in an amount effective to alleviate one or more symptoms of a disease in a subject or population to be treated, whether by inducing regression of such symptoms or inhibiting progression of such symptoms to any clinically measured degree.
  • the amount of a therapeutic agent effective to alleviate symptoms of any particular disease may vary depending on a variety of factors, such as the subject's disease state, age, and weight, and the level of the drug that produces the desired effect in the subject. ability. Whether symptoms of a disease have been alleviated can be assessed by any clinical test commonly used by doctors or other health care professionals to assess the severity or progression of the symptoms.
  • embodiments of the present disclosure may be ineffective in alleviating symptoms of a target disease in a subject, according to any statistical test known in the art, such as Student's t-test, chi-square test, based on Mann and Whitney's U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test determine that it should reduce target disease symptoms in a statistically significant number of subjects.
  • any statistical test known in the art such as Student's t-test, chi-square test, based on Mann and Whitney's U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test determine that it should reduce target disease symptoms 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 may vary depending on factors such as the condition being treated, the general health of the subject, the method, route and dosage of administration, and the severity of the side effects.
  • An effective amount can be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.
  • “Homology” or “identity” refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in the two compared sequences is occupied by the same nucleotide or amino acid monomer subunit, for example if every position in two DNA molecules is occupied by the same nucleotide, then the molecules are at that position homologous.
  • the percent homology between the two sequences is a function of the number of matches or homologous positions shared by the two sequences divided by the number of positions compared x 100%. For example, when sequences are optimally aligned, two sequences are 60% homologous if 6 of 10 positions in the two sequences are matched or homologous. In general, comparisons are made when the two sequences are aligned for the greatest percent homology.
  • Cell Cell
  • cell line cell line
  • cell culture all such designations include their progeny. It should also be understood that, due to deliberate or unintentional mutations, all progeny may not be exactly the same in terms of DNA content. Mutant progeny that have the same function or biological activity as screened in the original transformed cell are included.
  • a “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” includes any material that, when combined with an active ingredient, allows the ingredient to retain biological activity and not react with the subject's immune system. Examples include, but are not limited to, any standard pharmaceutical carrier, such as phosphate buffered saline, water, emulsions such as oil/water emulsions, and various types of wetting agents.
  • the diluent for aerosol or parenteral administration is phosphate buffered saline (PBS) or physiological (0.9%) saline.
  • Compositions comprising such carriers are formulated by well-known conventional methods (see, e.g., Remington's Pharmaceutical Sciences, 18th Edition, A. Gennaro, ed., Mack Publishing Co., Easton, PA, 1990; and R Remington, The Science and Practice of Pharmacy 20th edition Mack Publishing, 2000).
  • MASP2-binding protein or "MASP2-binding molecule” of the present disclosure is to be interpreted to the maximum extent, including the anti-MASP2 antibody or antigen-binding fragment thereof of the present disclosure, as long as the protein capable of binding to MASP2 is within the scope of the term.
  • a MASP2 binding protein or binding molecule
  • effector molecules include, for example, antineoplastic agents, drugs, toxins, biologically active proteins (eg, 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 (eg fluorescent compounds), or compounds detectable by NMR or ESR spectroscopic analysis.
  • antineoplastic agents drugs, toxins, biologically active proteins (eg, 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 (eg fluorescent compounds), or compounds detectable by NMR or ESR spectroscopic analysis.
  • the effector molecule when it is a polymer, it can generally 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 above-described synthetic polymers include one or more hydroxy, methyl, or methoxy groups.
  • polymers include optionally substituted linear or branched poly(ethylene glycol), poly(propylene glycol), poly(vinyl alcohol) or derivatives thereof, particularly optionally substituted poly(ethylene glycol) alcohol) such as methoxypoly(ethylene glycol) or derivatives thereof.
  • Specific naturally occurring polymers include lactose, amylose, dextran, glycogen or derivatives thereof.
  • the polymer is albumin or a fragment thereof, eg, human serum albumin or a fragment thereof. Conjugation of polymers to the anti-MASP2 antibodies or antigen-binding fragments thereof of the present disclosure can be accomplished by conventional methods.
  • 1A to 1D show the activity detection results of anti-MASP2 antibodies 77H11, 29C1, 11165, 67D2, 67E8 of the present disclosure in human 1% serum lectin pathway, and the isotype control using hIgG4 is negative Control, OMS721 was a positive control.
  • Figures 2A to 2B show the results of the detection of activity inhibition of the humanized antibodies of 77H11 and 29C1 of the present disclosure in the human 90% serum lectin pathway, using hIgG4 as the isotype control (Isotype control) as the negative control, and OMS721 as the positive control.
  • Figures 3A to 3C show the results of the detection of the inhibitory effects of 29C1 (H1L1), 77H11 (H3L1) and 11165 on the lectin pathway in human 90% serum, monkey 90% serum, and mouse 90% serum, respectively.
  • Isotype control is a negative control
  • OMS721 is a positive control.
  • Fig. 4 is a graph showing the proportion of cerebral infarction area, which was a graph of the results of detecting the protective effects of 11165 and OMS721 in a mouse stroke model.
  • Figures 5A-5B are graphs showing the results of detecting the inhibition of the lectin pathway by 29C1 (H1L1), 77H11 (H3L1), 11165 and OMS721 in cynomolgus monkeys at doses of 3 mg/kg and 10 mg/kg.
  • MASP2 (S633A) or MASP2 (S633A) is an enzymatically active mutant form of MASP2 (J Biol Chem. 2013; 288(13): 8922-8934), the purpose is to reduce protein autocatalysis and improve protein stability.
  • CCP1-CCP2-SP(S633A) and CCP2-SP(S633A) are obtained by transforming plasmids into Escherichia coli BL21 strain, isolating, purifying, denaturing and renaturing the protein; CCP1 is obtained by transiently transfecting CHO cells, expressing and isolating the protein .
  • the amino acid sequence of each MASP2 protein fragment is as follows:
  • MASP2-specific antibodies were screened and prepared using both mouse immunization and phage display.
  • the human MASP2A protein prepared in Example 1 was used as the antigen to immunize balb/c mice, and the mouse with the highest titer was selected for hybridoma cell fusion.
  • Hybridoma cell fusion and culture Take 3 culture dishes (10 cm) of myeloma cells passaged 24 hours before, wash once with RPMI-1640 medium without HEPES, resuspend, and count 2-4 ⁇ 10 7 cells.
  • mice after immunization for 72 hours were sacrificed, the spleen was aseptically removed, washed with RPMI-1640 medium without HEPES, minced and ground the spleen cells, blown up, filtered, centrifuged at 1000 rpm, 5 min, resuspended, and counted as 1- 2 x 10 8 pcs.
  • the myeloma cells and spleen cells were mixed, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, the precipitate was loosened, and preheated in a 40°C water bath. Add 1 mL of PEG preheated to 40 °C into the centrifuge tube dropwise within 60 s, and stir gently while adding.
  • the ELISA detection method is as follows: 1 ug/mL of antigen was coated overnight at 4°C, 50 ⁇ L per well. The plate was washed three times with PBS, and then blocked with PBS containing 3% BSA for 1 h at room temperature. The plate was washed 3 times with PBST, and the supernatant of hybridoma cells was added for 1 h at room temperature. Washed 3 times with PBST, washed 3 times with PBS, added secondary antibody (1:2000; invitrogen, goat anti-mouse IgG (H+L) secondary antibody, 31430), room temperature for 1 h. Washed 3 times with PBST, washed 3 times with PBS, added TMB substrate, room temperature for 10 min, and then terminated the reaction to detect the signal value (450 nm light absorption value).
  • Human MASP2A antigen was coated on immunosorbent plates and incubated with phage antibody synthesis library for solid-phase screening. After three rounds of panning, human MASP2 and monkey MASP2 positive phages were obtained.
  • the method of phage display screening for positive clones is as follows: 4 mL MASP2A (5 ng/ ⁇ L concentration) was coated on the immunotube, and 4 °C overnight. Fully human phage pools were blocked with 5% BSA/PBS for 1 hour at room temperature. The phage library was transferred to an immunotube coated with MASP2A, rotated at room temperature for 1 h, washed 5 times with PBS, eluted with 1000 ⁇ L TEA, and neutralized by adding 400 ⁇ L Tris-HCl (pH 7.4). Infect 10mL of TG1 (OD value to 0.4), 37°C, 40min. To measure output, apply Amp+ plate, overnight at 30°C.
  • Scrape the bacteria inoculate the bacteria into 50 mL of 2 ⁇ TY medium (add Amp and 1% glucose) to an OD value of 0.1, grow at 37° C., 200 rpm, and grow for 80 min to an OD value of 0.4-0.6.
  • Take 10 mL add 500 ⁇ L of helper phage M13KO7, and infect at 37°C for 40 min. The supernatant was removed by centrifugation, and the pellet was resuspended in 100 mL of 2 ⁇ TY medium (plus Amp and Kana) at 30° C., 200 rpm, overnight.
  • Centrifuge at 4000rpm for 30min collect the supernatant, add 10mL of PEG/NaCl, precipitate on ice for 1h, centrifuge at 4000rpm for 30min, add 1mL of PBS to resuspend the precipitate. Centrifuge at 13,000 rpm for 3 min to remove the precipitate. The phages were resuspended in PBS and proceeded to the next round of panning. Inoculate single clones into 96-well plates, 37°C for 3h, 220rpm. Add 1 mM IPTG and induce overnight at 30°C.
  • MASP2 was coated on a 96-well plate at a concentration of 2ng/ ⁇ L, 50 ⁇ L per well, overnight at 4°C.
  • 200 ⁇ L of 2% MPBS was added to block the ELISA plate at 37°C for 1 h. Centrifuge the overnight culture at 4000 g for 10 min, and transfer the supernatant to a new 96-well plate. Wash the ELISA plate twice, add 25 ⁇ L of 2% MPBS blocking solution, then add 25 ⁇ L of culture supernatant, and mix well.
  • VH heavy chain variable region
  • VL light chain variable region
  • 77H11 and 67D2, 67E8 have the following CDRs:
  • HCDR2 YISYSGRTSYNPSLKS (SEQ ID NO: 18);
  • HCDR3 LYAX 1 X 2 X 3 (SEQ ID NO: 26), wherein X 1 is selected from L or M, X 2 is selected from D or N, and X 3 is selected from Y or F;
  • LCDR1 KASQNVDTNVA (SEQ ID NO: 20);
  • LCDR2 SASYRX 4 S (SEQ ID NO: 27), wherein X 4 is selected from Y or F;
  • LCDR3 QQYNSNPLT (SEQ ID NO: 22).
  • the obtained anti-MASP2 antibody was used for functional verification of in vitro inhibition of lectin pathway activity with 1% human serum.
  • the identification method of antibody functional activity in 1% human serum is as follows: 50 ⁇ g/mL mannan solution is coated with 25 ⁇ L/well on a 384-well plate, and incubated at 4°C overnight. Wash 4 times with 50 ⁇ L/well TBST, block with 50 ⁇ L/well 3% BSA blocking solution (solvent is TBS), and incubate at room temperature for 2 h. The plate was washed 4 times with 50 ⁇ L/well of TBST. The anti-MASP2 antibody of the corresponding concentration was mixed with 1% human serum, incubated at 4°C for 1 h, the mixture was added to the aforementioned 384-well plate at 15 ⁇ L/well, and incubated at 37°C for 1 h.
  • the positive control antibody OMS721 was synthesized according to WO2012151481A, and the sequence is as follows:
  • the results are shown in Figure 1A to Figure 1D, Tables 2 to 4, the results show that 77H11, 67D2, 67E8, 29C1 and 11165 can significantly inhibit the lectin pathway, which is better than the positive control OMS721.
  • the antibodies 77H11, 67D2, 67E8, 29C1, and 11165 here are full-length anti-MASP2 antibodies formed by linking the antibody heavy chain variable region with the human IgG4 heavy chain Fc region, and the human IgG4 heavy chain Fc is SEQ ID NO: 53 shown.
  • the hIgG4 used was the isotype control.
  • Antibody IC50 ( ⁇ g/mL) OMS721 5.278 77H11 0.630 67D2 0.559 67E8 0.738
  • Antibody IC50 ( ⁇ g/mL) OMS721 2.858 29C1 0.086
  • Antibody IC50 ( ⁇ g/mL) OMS721 0.885 11165 0.499
  • Anti-MASP2 antibodies 29C1 and 77H11 were selected for humanization and subsequent activity identification in human serum.
  • the light chain humanization template of 29C1 is human germline gene IGKV1-33*01, and the heavy chain humanization template is IGKV3-21*01. Both chains underwent several backmutations. The combination yields 6 molecules whose humanized sequences are shown below (heavy or light chain CDRs are underlined):
  • the light chain humanization template of 77H11 is human germline gene IGKV1-27*01, and the heavy chain humanization template is human germline gene IGKV4-30-4*01. Both chains underwent several backmutations.
  • the combination yields 8 molecules whose humanized sequences are shown below (heavy or light chain CDRs are underlined):
  • the antibodies in Table 5 are all full-length anti-MASP2 antibodies constructed by linking the variable region of the antibody heavy chain with the Fc region of the human IgG4 heavy chain.
  • the heavy chain Fc region includes a hinge region, which is a human IgG4 Fc region with S228P mutation, and the sequence is shown in SEQ ID NO: 53.
  • the S228P is under EU numbering.
  • anti-MASP2 antibody full-length heavy chain, full-length light chain are shown below:
  • the expression and purification of the antibody are carried out according to conventional methods, and the full-length antibody of the present disclosure is obtained after detection.
  • the above-mentioned humanized antibodies were tested for their effects on lectin pathway activity in human 90% serum, which is closer to in vivo conditions.
  • the method for identifying the functional activity of the antibody in 90% human serum is as follows: Coat 25 ⁇ L/well of sodium carbonate-sodium bicarbonate buffer (pH>9) containing 5 ⁇ g/mL mannan solution in a 384-well plate at 4°C Incubate overnight. Wash 3 times with 50 ⁇ L/well of TBST supplemented with 5 mM Ca 2+ . Use 50 ⁇ L/well of 3% BSA blocking solution (solvent is TBS, add 5 mM Ca 2+ ), and block at room temperature for 1.5-2 h.
  • both the humanized molecules of 29C1 and 77H11 significantly inhibited lectin pathway activity in 90% of human serum, and were more potent than OMS721.
  • the 29C1 and 77H11 series of antibodies here are full-length anti-MASP2 antibodies formed by linking the variable region of the heavy chain of the antibody with the Fc region of the human IgG4 heavy chain, and the human IgG4 heavy chain Fc is shown in SEQ ID NO: 53. Show.
  • the hIgG4 used was the isotype control.
  • the binding properties of anti-MASP2 antibodies were detected by the method of Biacore.
  • the affinity of the three antibody molecules 29C1 (H1L1), 77H11 (H3L1), and 11165 to human MASP2, murine MASP2, and monkey MASP2 proteins was determined with a Biacore 8K (GE) instrument, and isotype IgG antibodies were used as negative controls.
  • Anti-human Fc IgG capture chip was used to capture the antibody, and then different concentrations of antigen were used as mobile phase for detection, and finally curve fitting was performed in 1:1 mode to obtain the affinity value. The results are shown in Table 7.
  • the method for identifying the functional activity of antibodies in 90% human, monkey and mouse serum is as follows: Coat 384 wells with 25 ⁇ L/well of sodium carbonate-sodium bicarbonate buffer (pH>9) containing 5 ⁇ g/mL mannan solution. plate and incubated overnight at 4°C. Wash 3 times with 50 ⁇ L/well of TBST supplemented with 5 mM Ca 2+ . Use 50 ⁇ L/well of 3% BSA blocking solution (solvent is TBS, add 5 mM Ca 2+ ), and block at room temperature for 1.5-2 h. Wash three times with 50 ⁇ L/well of TBST, once with 50 ⁇ L/well of TBS, and once with 50 ⁇ L/well of VBS.
  • MASP2 antibody After diluting MASP2 antibody with VBS, mix it with human serum at a ratio of 1:9 (ie, 90% human serum) to obtain a mixture, incubate at 4°C for 30 min, and add 15 ⁇ L/well of the mixture to the aforementioned 384-well plate at 4°C Incubate for 1h. Wash three times with 50 ⁇ L/well of TBST, add primary antibody (Biotin-Chicken Anti-C4C, 6 ⁇ g/mL) diluted in 0.5% BSA in TBS, 15 ⁇ L/well, room temperature for 60 min.
  • primary antibody Biotin-Chicken Anti-C4C, 6 ⁇ g/mL
  • a general stroke modeling method was used. The specific method is as follows: male C57 mice (body weight 12-20 g) were injected intraperitoneally with the drug once on the 7th and 3rd days before modeling, and the drug was injected into the tail vein once ischemia for 1 h on the day of modeling (simultaneous reperfusion). 10mg/kg each time. The anesthetized mice were fixed in a supine position on a fixation plate, the neck skin was removed, and iodophor disinfection was performed. A midline incision was made in the neck to separate the right common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA).
  • CCA right common carotid artery
  • ECA external carotid artery
  • ICA internal carotid artery
  • the proximal end of the CCA and the ICA were temporarily clamped with vascular clips.
  • a small incision was made above the ECA, a silicone suture was inserted into the incision, and a surgical silk thread was gently ligated.
  • Remove the vascular clip on the ICA slowly insert the silicone suture into the CCA, and enter the ICA through the CCA until it encounters slight resistance, and then tie the surgical silk above the ECA to fix the suture firmly.
  • the vascular clip above the CCA was subsequently removed. After 1 h of ischemia, the suture was pulled out and the neck skin was sutured. The animals were returned to their cages for rearing. 48h after reperfusion, the animals were sacrificed, and the size of cerebral infarction was measured by TTC staining.
  • 77H11(H3L1), 29C1(H1L1), 11165 and OMS721 were injected into cynomolgus monkeys by intravenous injection at doses of 3 mg/kg and 10 mg/kg, respectively, and blood was collected at different times to collect serum.
  • the lectin pathway activity of cynomolgus monkey serum collected at different time points was detected.
  • the specific blood collection time is 0 hours, 15 minutes, 1 hour, 4 hours, 8 hours, 24 hours, 48 hours, 72 hours, 96 hours, 168 hours, 240 hours, 360 hours, 480 hours, 600 hours, and 720 hours.
  • the serum separation method is as follows: blood samples are collected from peripheral veins and injected directly into blank blood collection tubes. The blood samples were allowed to stand for 15-60 minutes until the blood coagulated, and then centrifuged at 2500g for 10 minutes at 4°C, and the supernatant was serum.
  • test drugs 77H11 (H3L1), 29C1 (H1L1), 11165 and OMS721 were injected intravenously, and the doses of each drug were 3 mg/kg or 10 mg/kg, respectively.
  • the blood collection time points after administration were 15min, 1h, 4h, 8h, 24h, 48h, 72h, 96h, 168h, 240h, 336h, 408h, 504h, 672h. Each time, 0.3 mL of whole blood was taken without anticoagulant. After blood was collected, it was placed at 4 °C for 30 min, centrifuged at 1000 g for 15 min, and the supernatant was placed in an EP tube and stored at 80 °C.
  • Serum drug concentration was detected by ELISA, and the T 1/2 of the tested drug and its main parameters were calculated by matrix fitting. See Table 9 for the results. It can be seen that the half-life of 11165 is similar to that of OMS721, while the half-life of 77H11 (H3L1) and 29C1 (H1L1) is significantly better than that of OMS721, which means that it can have a longer drug effect in the body.
  • the use and welfare of laboratory animals in this disclosure is performed in accordance with the "International Association for the Assessment and Accreditation of Laboratory Animals (AAALAC)".
  • the health status and death of the animals are monitored daily. Routine inspections include observing the effects of the test substances and drugs on the animals' daily behaviors, such as behavioral activities, weight changes, and physical signs.

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Abstract

抗MASP2抗体、其抗原结合片段及医药用途；具体地，提供了抗MASP2抗体或其抗原结合片段、其药物组合物，及用于治疗IgA肾病等疾病的方法和相关制药用途。

Description

抗MASP2抗体、其抗原结合片段及医药用途
本申请要求2021年4月25日提出的中国专利申请CN202110450267.0的优先权,本申请全文引入上述中国专利申请。
技术领域
本公开涉及生物医药领域，具体涉及抗MASP2抗体及其抗原结合片段，其药物组合物和相关制药用途。
背景技术
补体系统是存在于人和动物血清、组织液和细胞膜表面的蛋白，经活化后具有生物活性，可介导免疫和炎症反应。补体系统由近40种成分组成，多数为糖蛋白，包括C1q、C1r、C1s、C2-C9、D因子、B因子，以及10种调节蛋白和10种补体受体。补体广泛参与机体对微生物的防御反应以及免疫调节，也参与免疫病理的损伤性反应。补体是固有免疫重要的效应系统和效应放大系统。
补体系统的活化过程表现为丝氨酸蛋白酶级联酶解反应，分为三种类型：经典激活通路(Classic pathway)，替代通路(Alternative pathway)，凝集素通路(Lectin pathway)，最终介导以破膜复合物形成为标志的末端通路的活化。在健康人中，替代通路长期保持低水平激活以监视病原微生物入侵。健康细胞也通过表达CD55、CD59等补体调节蛋白，抑制补体系统对其攻击。三个通路通常在凋亡细胞、微生物表面活化。经典通路是抗体(IgG1，IGG，IgG3，IgG4或者IgM)与抗原结构后形成免疫复合物被C1q、C1s，C1r识别后，激活C2、C4，形成C4bC2a(即为C3转化酶)，最终促进C5-C9组成的破膜复合物形成。替代通路是直接由C3自发水解开始的激活途径，在细菌细胞壁成分LPS、酵母聚糖Zymosan等多糖、肽聚糖、磷壁酸等激活物质刺激下，因子D(Factor D)水解C3结合的因子B(Factor B)形成C3bBb3，继而完成C5至C9各成分的连锁反应。凝集素途径由血浆中甘露聚糖结合凝集素(mannan-binding lectin,MBL)或纤维胶凝蛋白(ficolin，FCN)直接识别多种病原微生物表面的甘露糖、N-乙酰甘露糖、N-乙酰葡萄糖氨、岩藻糖等为末端糖基的糖结构，继之活化补体经典通路。
临床实验和研究证据表明，补体系统的异常活化与急性败血症、缺血再灌注相关的中风、心肌梗死、移植物排斥；与慢性自身免疫疾病如关节炎、眼科年龄相关黄斑病变、微血管血栓、慢性肾病、溶血性疾病的发生相关。目前靶向补体的在研药物中，C5抗体Eculizumab已于2007年获批上市，主要用于治疗溶血性疾病PNH、aHUS、重症肌无力等疾病。其他靶向凝集素通路的MASP2抗体，靶向替代通路的Factor D，FactorB，C3的药物也处在临床II到III期。靶向补体系统的安全性，总体是安全可耐受的。考虑到补体系统参与调控B细胞的发育、T 细胞的活化，补体因子基因缺失小鼠在繁殖能力或胚胎发育的缺陷、并有增加感染的风险。因此，开发针对特定补体通路上游靶点的药物，能够在保证药效的同时，降低补体通路整体抑制后的副作用。
MASP2蛋白是凝集素通路的核心水解酶，由N端负责与MBL结合的CUB，EGF结构域，与下游底物C4、C2结合的CCP结构域，以及C端的酶活结构域SP组成。MBL-MASP复合物与病原体表面糖结构结合，使MASP-1、MASP-2被独立地激活。活化的MASP2发挥其SP活性，裂解C4、C2形成C3转化酶C4b2a，最终能够激活凝集素通路介导的补体系统，目前已经证实与IgA肾病、中风、心肌缺血有关。在小鼠脑卒中MCAO模型中及急性心肌梗死模型中，可以看到MASP2缺失能够显著降低梗死面积。MBL-MASP2主要通过识别半乳糖修饰缺失的IgA，在肾小球系膜上，LP通路的激活促进细胞因子的分泌，最终导致肾小管上皮细胞、足细胞的损伤，肾脏功能的异常。
Omeros公司的MASP2单抗narsoplimab(OMS721)主要通过抑制凝集素介导的补体系统活化，被开发用于治疗多种炎性相关疾病，包括血栓性微血管病(TMA)、IgA肾病、溶血性尿毒综合征(HUS)、狼疮性肾炎、膜性肾小球肾炎、肾小球性肾炎、年龄相关性黄斑变性、再灌注性损伤、心肌梗死、糖尿病神经病变、中风、移植物抗宿主病。其中，TMA的研究在美国处于预注册状态，HUS、IgA肾病的研究处于临床III期，狼疮性肾炎、膜性肾小球肾炎、肾小球性肾炎的研究处于临床II期。
鉴于MASP2在凝集素通路的重要作用，提供新结构的抗MASP2抗体，抑制MASP2依赖性补体活化，治疗补体系统异常导致的疾病，仍然是本领域亟待解决的问题。
发明内容
本公开提供了抗MASP2抗体及其抗原结合片段，编码其的核酸、包含所述核酸的载体、宿主细胞、包含所述抗MASP2抗体及其抗原结合片段的药物组合物、以及其用于治疗或延缓补体系统相关疾病的方法，及其用途。
抗MASP2抗体及其抗原结合片段
本公开提供抗MASP2抗体及其抗原结合片段，其包含重链可变区(VH)和轻链可变区(VL)，其中：
1)所述VH包含如SEQ ID NO：7、9、11任一所示VH中的HCDR1、HCDR2、HCDR3，和/或，所述VL包含如SEQ ID NO：8、10、12任一所示VL中的LCDR1、LCDR2、LCDR3；
2)所述VH包含如SEQ ID NO：13所示VH中的HCDR1、HCDR2、HCDR3，和/或，所述VL包含如SEQ ID NO：14所示VL中的LCDR1、LCDR2、LCDR3；或
3)所述VH包含如SEQ ID NO：15所示VH中的HCDR1、HCDR2、HCDR3，和/或，所述VL包含如SEQ ID NO：16所示VL中的LCDR1、LCDR2、LCDR3；
所述VH和VL的HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3是根据Kabat、IMGT、Chothia、AbM或Contact编号系统定义的，一些实施方案中，是根据Kabat编号系统定义的。
一些实施方案中的所述抗MASP2抗体及其抗原结合片段，其中，
1-1)所述VH包含如SEQ ID NO：7所示VH中的HCDR1、HCDR2、HCDR3，和/或，所述VL包含如SEQ ID NO：8所示VL中的LCDR1、LCDR2、LCDR3；
1-2)所述VH包含如SEQ ID NO：9所示VH中的HCDR1、HCDR2、HCDR3，和/或，所述VL包含如SEQ ID NO：10所示VL中的LCDR1、LCDR2、LCDR3；
1-3)所述VH包含如SEQ ID NO：11所示VH中的HCDR1、HCDR2、HCDR3，和/或，所述VL包含如SEQ ID NO：12所示VL中的LCDR1、LCDR2、LCDR3。
本公开提供抗MASP2抗体及其抗原结合片段，其包含重链可变区(VH)和轻链可变区(VL)，其中：
1)所述VH包含分别如SEQ ID NO：17、18、26所示的HCDR1、HCDR2、HCDR3，和/或，所述VL包含分别如SEQ ID NO：20、27、22所示的LCDR1、LCDR2、LCDR3；
其中，SEQ ID NO：17的序列为：SDYAWN；
SEQ ID NO：18的序列为：YISYSGRTSYNPSLKS；
SEQ ID NO：26的序列为：LYAX 1X 2X 3，其中，X 1选自L或M，X 2选自D或N，X 3选自Y或F；
SEQ ID NO：20的序列为：KASQNVDTNVA；
SEQ ID NO：27的序列为：SASYRX 4S，其中，X 4选自Y或F；
SEQ ID NO：22的序列为：QQYNSNPLT；
2)所述VH包含分别如SEQ ID NO：28、29、30所示的HCDR1、HCDR2、HCDR3，和/或，所述VL包含分别如SEQ ID NO：31、32、33所示的LCDR1、LCDR2、LCDR3；或
3)所述VH包含分别如SEQ ID NO：34、35、36所示的HCDR1、HCDR2、HCDR3，和/或，所述VL包含分别如SEQ ID NO：37、38、39所示的LCDR1、LCDR2、LCDR3。
一些实施方案中的所述抗MASP2抗体及其抗原结合片段，其中，
所述VH包含分别如SEQ ID NO：17、18、19所示的HCDR1、HCDR2、HCDR3，和/或，所述VL包含分别如SEQ ID NO：20、21、22所示的LCDR1、LCDR2、LCDR3；
所述VH包含分别如SEQ ID NO：17、18、23所示的HCDR1、HCDR2、HCDR3，和/或，所述VL包含分别如SEQ ID NO：20、24、22所示的LCDR1、LCDR2、 LCDR3；或
所述VH包含分别如SEQ ID NO：17、18、25所示的HCDR1、HCDR2、HCDR3，和/或，所述VL包含分别如SEQ ID NO：20、21、22所示的LCDR1、LCDR2、LCDR3。
一些实施方案中，提供上述抗MASP2抗体及其抗原结合片段的变体，其中，
所述VH包含与SEQ ID NO：17具有至多3个、2个或1个氨基酸变化的HCDR1，与SEQ ID NO：18具有至多3个、2个或1个氨基酸变化的HCDR2，和与SEQ ID NO：19或25具有至多3个、2个或1个氨基酸变化的HCDR3；和/或，所述VL包含与SEQ ID NO：20具有至多3个、2个或1个氨基酸变化的LCDR1，与SEQ ID NO：21具有至多3个、2个或1个氨基酸变化的LCDR2，和与SEQ ID NO：22具有至多3个、2个或1个氨基酸变化的LCDR3；
所述VH包含与SEQ ID NO：17具有至多3个、2个或1个氨基酸变化的HCDR1，与SEQ ID NO：18具有至多3个、2个或1个氨基酸变化的HCDR2，和与SEQ ID NO：23具有至多3个、2个或1个氨基酸变化的HCDR3；和/或，所述VL包含与SEQ ID NO：20具有至多3个、2个或1个氨基酸变化的LCDR1，与SEQ ID NO：24具有至多3个、2个或1个氨基酸变化的LCDR2，和与SEQ ID NO：22具有至多3个、2个或1个氨基酸变化的LCDR3；
所述VH包含与SEQ ID NO：28具有至多3个、2个或1个氨基酸变化的HCDR1，与SEQ ID NO：29具有至多3个、2个或1个氨基酸变化的HCDR2，和与SEQ ID NO：30具有至多3个、2个或1个氨基酸变化的HCDR3；和/或，所述VL包含与SEQ ID NO：31具有至多3个、2个或1个氨基酸变化的LCDR1，与SEQ ID NO：32具有至多3个、2个或1个氨基酸变化的LCDR2，和与SEQ ID NO：33具有至多3个、2个或1个氨基酸变化的LCDR3；
所述VH包含与SEQ ID NO：34具有至多3个、2个或1个氨基酸变化的HCDR1，与SEQ ID NO：35具有至多3个、2个或1个氨基酸变化的HCDR2，和与SEQ ID NO：36具有至多3个、2个或1个氨基酸变化的HCDR3；和/或，所述VL包含与SEQ ID NO：37具有至多3个、2个或1个氨基酸变化的LCDR1，与SEQ ID NO：38具有至多3个、2个或1个氨基酸变化的LCDR2，和与SEQ ID NO：39具有至多3个、2个或1个氨基酸变化的LCDR3。
一些实施方案中，以上氨基酸变化可以是保守的替换、取代或修饰，和/或不影响功能的缺失、添加。
一些实施方案中，提供抗MASP2抗体及其抗原结合片段，其包含至少一个以上任意的CDR；例如，包含本公开任意的HCDR1或任意的HCDR2、HCDR3中的一个或多个；例如，包含本公开任意的LCDR1或任意的LCDR2、LCDR3中的一个或多个。
一些实施方案中，上述本公开的抗MASP2抗体或其抗原结合片段为鼠源抗 体、嵌合抗体、人源化抗体、全人抗体或其片段。
一些实施方案中，上述本公开中人源化抗体或其抗原结合片段的重链框架区源自IGKV3-21*01或IGKV4-30-4*01；和/或，轻链框架区源自IGKV1-33*01或IGKV1-27*01。
一些实施方案中的抗MASP2抗体或其抗原结合片段，其中，
VH的氨基酸序列如SEQ ID NO：7、9、11、47、48、49、50之一所示，和/或，VL的氨基酸序列如SEQ ID NO：8、10、12、51、52之一所示；
VH的氨基酸序列如SEQ ID NO：13、42、43、44之一所示，和/或，VL的氨基酸序列如SEQ ID NO：14、45、46之一所示；或
VH的氨基酸序列如SEQ ID NO：15所示，VL的氨基酸序列如SEQ ID NO：16所示。
一些具体实施方案中的抗MASP2抗体或其抗原结合片段，其中，
a)VH、VL分别包含如SEQ ID NO：7、8所示氨基酸序列，或由其组成；
b)VH、VL分别包含如SEQ ID NO：9、10所示氨基酸序列，或由其组成；
c)VH、VL分别包含如SEQ ID NO：11、12所示氨基酸序列，或由其组成；
d)VH、VL分别包含如SEQ ID NO：47、51所示氨基酸序列，或由其组成；
e)VH、VL分别包含如SEQ ID NO：47、52所示氨基酸序列，或由其组成；
f)VH、VL分别包含如SEQ ID NO：48、51所示氨基酸序列，或由其组成；
g)VH、VL分别包含如SEQ ID NO：48、52所示氨基酸序列，或由其组成；
h)VH、VL分别包含如SEQ ID NO：49、51所示氨基酸序列，或由其组成；
i)VH、VL分别包含如SEQ ID NO：49、52所示氨基酸序列，或由其组成；
j)VH、VL分别包含如SEQ ID NO：50、51所示氨基酸序列，或由其组成；
k)VH、VL分别包含如SEQ ID NO：50、52所示氨基酸序列，或由其组成；
l)VH、VL分别包含如SEQ ID NO：13、14所示氨基酸序列，或由其组成；
m)VH、VL分别包含如SEQ ID NO：42、45所示氨基酸序列，或由其组成；
n)VH、VL分别包含如SEQ ID NO：42、46所示氨基酸序列，或由其组成；
o)VH、VL分别包含如SEQ ID NO：43、45所示氨基酸序列，或由其组成；
p)VH、VL分别包含如SEQ ID NO：43、46所示氨基酸序列，或由其组成；
q)VH、VL分别包含如SEQ ID NO：44、45所示氨基酸序列，或由其组成；
r)VH、VL分别包含如SEQ ID NO：44、46所示氨基酸序列，或由其组成；或
s)VH、VL分别包含如SEQ ID NO：15、16所示氨基酸序列，或由其组成。
进一步地，本公开提供包含与上述a)至s)任一组的VH、VL具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％序列同一性的变体VH、VL的抗MASP2抗体或其抗原结合片段。
一些实施方案中，本公开的抗MASP2抗体或其抗原结合片段为IgG抗体或其抗原结合片段，例如为IgG1、IgG2、IgG4抗体或其抗原结合片段，更例如为Fc去具有S228P、F234A和L235A中任一个或多个突变的IgG4抗体或其抗原结合片段。以上突变均是EU编号。
一些实施方案中，本公开的抗MASP2抗体或其抗原结合片段还包含人免疫球蛋白Fc区；例如，所述Fc区是人IgG1、IgG2或IgG4的Fc区。一些实施方案中，所述Fc区可以具有突变，所述突变使得ADCC功能减少。一些具体实施方案中，突变示例为IgG1上的L234A/L235A，IgG2上的V234A/G237A/P238S/H268A/V309L/A330S/P331S，IgG4上的F234A/L235A，IgG4上的S228P/F234A/L235A，IgG1、IgG2、IgG3或IgG4上的N297A，IgG2上的V234A/G237A，IgG1上的K214T/E233P/L234V/L235A/G236缺失/A327G/P331A/D365E/L358M，IgG2上的H268Q/V309L/A330S/P331S，IgG1上的S267E/L328F，IgG1上的L234F/L235E/D265A，IgG1上的L234A/L235A/G237A/P238S/H268A/A330S/P331S，IgG4上的S228P/F234A/L235A/G237A/P238S，以及IgG4上的S228P/F234A/L235A/G236缺失/G237A/P238S。还可使用杂合IgG2/4Fc域，例如具有来自IgG2的残基117-260和来自IgG4的残基261-447的Fc。一些具体的实施方案中，所述人IgG4的Fc区具有S228P、F234A、L235A和K447A中任一个或多个突变(参见WO2017079112A，WO2018031400A等)。
一些实施方案中，本公开的抗MASP2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，本公开的抗MASP2抗体的抗原结合片段重链全长的氨基酸序列如SEQ ID NO：54所示或与之具有至少80％、至少90％或至少95％同一性；轻链全长的氨基酸序列如SEQ ID NO：55所示或与之具有至少80％、至少90％或至少95％同一性；
重链全长的氨基酸序列如SEQ ID NO：56所示或与之具有至少80％、至少90％或至少95％同一性；轻链全长的氨基酸序列如SEQ ID NO：57所示或与之具有至少80％同一性、至少90％或至少95％同一性；或
重链全长的氨基酸序列如SEQ ID NO：58所示或与之具有至少80％、至少90％或至少95％同一性；轻链全长的氨基酸序列如SEQ ID NO：59所示或与之具有至少80％、至少90％或至少95％同一性。
一些具体实施方案中，重链、轻链全长氨基酸序列分别如SEQ ID NO：54、55所示；重链、轻链全长氨基酸序列分别如SEQ ID NO：56、57所示；或者重链、轻链全长氨基酸序列分别如SEQ ID NO：58、59所示。
一些实施方案中，根据本公开的抗MASP2抗体或其抗原结合片段的重链可变区有0至10个(1、2、3、4、5、6、7、8、9、10个)氨基酸变化；轻链可变区有0至10个(1、2、3、4、5、6、7、8、9、10个)氨基酸变化。在一些具体实施方案中，所述氨基酸变化为保守的替换、取代或修饰，和/或不影响功能的缺失、添加。
一些实施方案中，提供抗MASP2抗体或抗原结合片段，其与前述抗MASP2抗体或抗原结合片段结合或竞争结合相同的表位。
一些实施方案中，提供抗MASP2抗体或抗原结合片段，其阻断前述抗MASP2抗体或其抗原结合片段与人MASP2的结合。
一些实施方案中，提供抗MASP2抗体或抗原结合片段，其与人MASP2的结合被前述抗MASP2抗体或其抗原结合片段阻断。
一些实施方案中，本公开抗MASP2抗体或抗原结合片段具有以下至少一项：
(i)所述抗体或抗原结合片段以10nM或更低的KD结合人MASP-2；
(ii)所述抗体或抗原结合片段结合MASP-2的CCP1结构域中的表位；
(iii)所述抗体或抗原结合片段以10nM或更低的IC 50在1％人血清中的体外测定中抑制C3b沉积，所述IC 50例如可采用本公开实施例3的方法检测获得；
(iv)所述抗体或抗原结合片段以30nM或更低的IC 50抑制90％人血清中的C3b沉积，所述IC 50例如可采用本公开实施例6的方法检测获得；和
(v)所述抗体或抗原结合片段基本上不抑制经典途径。
一些实施方案中，抗MASP-2抗体选择性抑制MASP-2补体活化，保留C1q-依赖性补体活化系统功能完整。
一些实施方案中，提供MASP2结合蛋白或结合分子，其包含上述任意的本公开的抗MASP2抗体或其抗原结合片段。
一些具体实施方案中，所述MASP2结合蛋白或结合分子中含有一个或多个效应分子，所述效应分子选自抗肿瘤剂、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(例如，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。一些具体实施方案中，所述效应分子缀合或融合于本公开的抗MASP2抗体或其抗原结合片段。
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MASP2抗体或其抗原结合片段、结合蛋白或结合分子。所述多核苷酸可以是DNA或RNA。根据本公开的一些实施方案，本公开的多核苷酸是基本上分离或经分离的多核苷酸。
本公开提供含有如上所述的多核苷酸的表达载体，或者说，本公开的多核苷酸也可呈载体形式，可存在于载体中和/或可为载体的一部分，该载体例如质粒、 粘端质粒、YAC或病毒载体。载体可尤其为表达载体，即可提供抗MASP2抗体或其抗原结合片段体外和/或体内(即在适合宿主细胞、宿主有机体和/或表达系统中)表达的载体，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。该表达载体通常包含至少一种本公开的多核苷酸，其可操作地连接至一个或多个适合的表达调控元件(例如启动子、增强子、终止子等)。针对在特定宿主中的表达对所述元件及其序列进行选择为本领域技术人员的常识。对本公开的PD-1结合蛋白的表达有用或必需的调控元件及其他元件例如为启动子、增强子、终止子、整合因子、选择标记物、前导序列、报告基因。
本公开的多核苷酸可基于本公开的多肽的氨基酸序列的信息通过已知的方式(例如通过自动DNA合成和/或重组DNA技术)制备或获得，和/或可从适合的天然来源加以分离。
宿主细胞
本公开提供表达本公开的抗MASP2抗体或其抗原结合片段、结合蛋白或结合分子，和/或含有本公开的多核苷酸或载体的宿主细胞。一些实施方案中，宿主细胞为细菌细胞、真菌细胞或哺乳动物细胞
细菌细胞例如包括革兰氏阴性细菌菌株(例如大肠杆菌(Escherichia coli)菌株、变形杆菌属(Proteus)菌株及假单胞菌属(Pseudomonas)菌株)及革兰氏阳性细菌菌株(例如芽孢杆菌属(Bacillus)菌株、链霉菌属(Streptomyces)菌株、葡萄球菌属(Staphylococcus)菌株及乳球菌属(Lactococcus)菌株)的细胞。
真菌细胞例如包括木霉属(Trichoderma)、脉孢菌属(Neurospora)及曲菌属(Aspergillus)的物种的细胞；或者包括酵母属(Saccharomyces)(例如酿酒酵母(Saccharomyces cerevisiae))、裂殖酵母属(Schizosaccharomyces)(例如粟酒裂殖酵母(Schizosaccharomyces pombe))、毕赤酵母属(Pichia)(例如巴斯德毕赤酵母(Pichia pastoris)及嗜甲醇毕赤酵母(Pichia methanolica))及汉森酵母属(Hansenula)的物种的细胞。
哺乳动物细胞例如包括例如HEK293细胞、CHO细胞、BHK细胞、HeLa细胞、COS细胞等。
然而，本公开也可使用两栖类细胞、昆虫细胞、植物细胞及本领域中用于表达异源蛋白的任何其他细胞。
本公开的细胞不能发育成完成的植株或动物个体。
生产或制备方法
本公开提供制备本公开的抗MASP2抗体或其抗原结合片段、结合蛋白或结合分子的方法，所述方法通常包含以下步骤：
-在允许表达本公开的抗MASP2抗体或其抗原结合片段、结合蛋白或结合分子的条件下，培养本公开的宿主细胞；及
-从培养物中回收由所述宿主细胞表达的目的蛋白；及
-任选的，包括进一步纯化和/或修饰本公开的目的蛋白。
本公开的抗MASP2抗体或其抗原结合片段、结合蛋白或结合分子可在如上所述细胞中以细胞内方式(例如在细胞质中、在周质中或在包涵体中)产生，接着从宿主细胞分离且任选进一步纯化；或其可以细胞外方式(例如在培养宿主细胞的培养基中)产生，接着自培养基分离且任选进一步纯化。
用于重组产生多肽的方法及试剂，例如特定适合表达载体、转化或转染方法、选择标记物、诱导蛋白表达的方法、培养条件等在本领域中是已知的。类似地，适用于制造本公开的蛋白的方法中的蛋白分离及纯化技术为本领域技术人员所公知。作为一个示例，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。得到的产物需立即冷冻，如-70℃，或者冻干。
然而，本公开的抗MASP2抗体或其抗原结合片段、结合蛋白或结合分子也可以通过本领域已知的其它产生蛋白质的方法获得，例如化学合成，包括固相或液相合成。
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MASP2抗体或其抗原结合片段、结合蛋白或结合分子，以及一种或多种药学上可接受的载体、稀释剂、缓冲剂或赋形剂。
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MASP2抗体或其抗原结合片段，或药物组合物单位剂量中含抗MASP2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。
试剂盒(或药盒)
本公开提供试剂盒或药盒，包含一个或多个容器，其各自独立地包含选自以下的任一项或其组合：本公开的抗MASP2抗体或其抗原结合片段、结合蛋白、结合分子、以上蛋白或分子或抗体或抗原结合片段的编码多核苷酸。
一些实施方案中，还提供包含上述多核苷酸的诊断试剂，以及提供相关诊断用途。
预防、治疗疾病的方法和制药用途
本公开提供了本公开的抗MASP2抗体或其抗原结合片段、结合蛋白、结合分子、多核苷酸、药物组合物在预防和/或治疗疾病中用途和方法，所述疾病可以是与补体信号通路(例如MASP2)相关或不相关的。一些实施方案中，所述疾病为IgA肾病、阵发性睡眠性血红蛋白尿症(PNH)。
本公开提供治疗或预防疾病的方法，包括向有需要的受试者施用治疗或预防有效量的本公开的抗MASP2抗体或其抗原结合片段、结合蛋白、结合分子、多核苷酸和/或药物组合物。
本公开提供在受试者中抑制MASP-2依赖的补体活化的方法，包括以在所述受试者中以有效量(例如，足够抑制MASP-2依赖的补体活化的量)施用本公开的抗MASP2抗体或其抗原结合片段、结合蛋白、结合分子、多核苷酸和/或药物组合物。
MASP-2依赖的补体活化已被指示为促进很多急性和慢性疾病状态的发病机制，包括MASP-2依赖的补体介导的血管状况、缺血再灌注损伤、动脉粥样硬化、炎性胃肠道病症、肺部状况、体外再灌注过程、骨骼肌状况、肾状况、皮肤状况、器官或组织移植、神经系统病症或损伤、血液病症、泌尿生殖道状况、糖尿病、化疗或辐射治疗、恶性肿瘤、内分泌紊乱、凝血障碍，或眼科状况。一些实施方案中，提供本公开的抗MASP2抗体或其抗原结合片段、结合蛋白、结合分子、多核苷酸和/或药物组合物治疗上述疾病和状况的方法，以及相关制药用途。
一些实施方案中，上述疾病和状况为MASP-2-依赖性补体活化相关疾病。
一些实施方案中，上述疾病和状况为微血管内皮细胞损伤和/或血栓形成。
一些实施方案中，上述疾病和状况选自：IgA肾病、阵发性睡眠性血红蛋白尿症(PNH)、狼疮性肾炎、血栓性微血管病(TMA)(例如与造血干细胞移植相关的持续性TMA(HSCT-TMA)、血栓性血小板减少性紫癜(TTP))、溶血性尿毒综合征(HUS)、膜性肾小球肾炎、肾小球性肾炎、年龄相关性黄斑变性、再灌注性损伤、心肌梗死、糖尿病神经病变、中风、移植物抗宿主病(GVHD)、Upshaw-Schulman综合征(USS)，一些具体实施方案中，所述疾病是MASP-2-依赖性补体活化相关的。
检测
本公开提供检测MASP2的组合物，所述组合物包含根据本公开的抗MASP2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MASP2的方法、系统或装置，其包括用本公开的抗MASP2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MASP2的抗体或其抗原结合片段接触；
(2)检测在结合MASP2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MASP2。
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MASP2的抗体或其抗原结合片段；和
(2)检测在结合MASP2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MASP2抗体标记，通过对所述标记检测以实现对结合MASP2抗体的物质(例如MASP2)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MASP2结合或不结合的抗体或使其可视化，检测在结合MASP2的抗体或其抗原结合片段和MASP2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MASP2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MASP2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。
术语定义
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。除非在本文中另有明确定义，本文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。
“MASP-2依赖的补体活化”包括凝集素途径的MASP-2依赖的活化，其在生理条件下发生(即，在存在Ca++的情况下)，导致凝集素途径C3转化酶C4b2a的形成，并且在C3切割产物C3b的积累后导致C5转化酶C42a(C3b)n。
“经典途径”是指由抗体与外源颗粒结合而触发的并且需要结合识别分子C1q的补体活化。"替代途径"是指补体活化，其例如通过来自真菌和酵母细胞壁的酵母聚糖、来自革兰氏阴性细菌外膜的脂多糖(LPS)和兔红细胞，以及许多纯的多糖、兔红细胞、病毒、细菌、动物肿瘤细胞、寄生虫和受损的细胞触发，并且其在传统上被认为自补体因子C3自发蛋白水解产生C3b而产生。“凝集素途径”是指补体活化，其经由血清和非-血清糖结合蛋白的特异性结合发生，所述糖结合蛋白包括甘露聚糖-结合凝集素(MBL)、CL-11和纤维胶凝蛋白(H-纤维胶凝蛋白、M-纤维胶凝蛋白或L-纤维胶凝蛋白)。
“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MASP2或其表位的单克隆抗体。制备时用MASP2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MASP2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MASP2结合”，指能与MASP2或其表位相互作用，所述MASP2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MASP2结合的抗体，人MASP2的单体和多聚体(例如二聚体、三聚体等)，以及人MASP2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MASP2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MASP2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MASP2结合的能力。例如，结合人MASP2的本公开的抗体也可以结合另一物种的MASP2。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MASP2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MASP2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MASP2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MASP2抗体接触时，与未与抗MASP2抗体接触的MASP2相比，任何可测量的MASP2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MASP2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MASP2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。
“药学可接受的载体”或“药学可接受的赋形剂”包括当与活性成分组合时，允许该成分保留生物学活性并且不与受试者的免疫系统反应的任何材料。例子包括但不限于任何标准药物载体，例如磷酸盐缓冲盐水溶液、水、乳剂如油/水乳剂、和各种类型的润湿剂。在一些实施例中，用于气雾剂或肠胃外施用的稀释剂是磷酸盐缓冲盐水(PBS)或生理(0.9％)盐水。包含此类载体的组合物通过众所周知的常规方法配制(参见例如Remington′s Pharmaceutical Sciences，第18 版，A.Gennaro，编辑，Mack PublishingCo.，Easton，PA，1990；以及R Remington，The Science and Practice of Pharmacy第20版Mack Publishing，2000)。
“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“MASP2结合蛋白”或“MASP2结合分子”以最大化解释，包含本公开的抗MASP2抗体或其抗原结合片段，只要能够实现与MASP2结合的蛋白均在该术语范围内。例如，MASP2结合蛋白(或结合分子)可以包含一个或多个效应分子，以例如缀和或融合的方式。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MASP2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。
附图说明
图1A至图1D显示了本公开的抗MASP2抗体77H11、29C1、11165、67D2、67E8在人1％血清凝集素通路中的活性检测结果图，采用hIgG4的同种型对照(Isotype control)为阴性对照，OMS721为阳性对照。
图2A至图2B显示本公开的77H11和29C1的各人源化抗体在人90％血清凝集素通路中活性抑制检测结果图，采用hIgG4为同种型对照(Isotype control)为阴性对照，OMS721为阳性对照。
图3A至图3C分别为检测29C1(H1L1)、77H11(H3L1)和11165在人90％血清、猴90％血清、小鼠90％血清中对凝集素通路的抑制作用结果图，采用hIgG4的同种型对照(Isotype control)为阴性对照，OMS721为阳性对照。
图4为显示脑梗死面积比例的图，其检测11165和OMS721在小鼠脑卒中模型中的保护作用的结果图。
图5A至图5B为检测29C1(H1L1)、77H11(H3L1)、11165和OMS721在食蟹猴体 内3mg/kg和10mg/kg剂量下对凝集素通路的抑制结果图。
具体实施方式
以下结合实施例用于进一步描述本公开，但这些实施例并非限制本公开的范围。
本公开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.MASP2重组蛋白的制备
为制备抗原以筛选抗体，制备了分别表达MASP2的人源酶活突变体人MASP2A(S633A)、鼠源酶活突变体鼠MASP2A(S632A)、猴源酶活突变体猴MASP2A(S633A)重组蛋白CHO-S稳定转染细胞株。其中，MASP2(S632A)或MASP2(S633A)是MASP2的酶活突变形式(J Biol Chem.2013；288(13):8922-8934)，目的是为了减少蛋白的自我催化，提高蛋白稳定性。并且，为了在表达过程中提高产量，更换了信号肽，将原始信号肽MRLLTLLGLLCGSVA(SEQ ID NO:60)(参见Uniprot网站)更换为MEFGLSWLFLVAILKGVQC(SEQ ID NO:61)，上述的663位或662位均为原始序列中基于自然计数的位置编号。培养所述细胞株，收集上清，用亲和层析的方法纯化获得了各重组蛋白，氨基酸序列如下：
>人MASP2A(S633A)蛋白：
Figure PCTCN2022088914-appb-000001
(注：下划线为信号肽，斜体为His标签，灰色为633位突变为A(S633A))
                                  SEQ ID NO：1
>猴MASP2A(S633A)蛋白：
Figure PCTCN2022088914-appb-000002
(注：下划线为信号肽，斜体为His标签，灰色为633位突变为A(S633A))
                              SEQ ID NO：2
>鼠MASP2A(S632A)蛋白：
Figure PCTCN2022088914-appb-000003
(注：下划线为信号肽，斜体为His标签，灰色为632位突变为A(S632A))
                                      SEQ ID NO：3
为了鉴定抗体的抗原结合位点，制备了人MASP2不同的片段，包括人MASP2CCP1、人MASP2CCP1-CCP2-SP(S633A)、人MASP2CCP2-SP(S633A)。CCP1-CCP2-SP(S633A)和CCP2-SP(S633A)是将质粒转化大肠杆菌BL21菌株，分离、纯化蛋白，变性、复性制备获得；CCP1是瞬时转染CHO细胞，表达并分离蛋白制备获得。各MASP2蛋白片段的氨基酸序列如下：
>CCP1-CCP2-SP(S633A)：
Figure PCTCN2022088914-appb-000004
                             SEQ ID NO：4
>CCP2-SP(S633A)：
Figure PCTCN2022088914-appb-000005
(注：灰色为633位突变为A(S633A))
                            SEQ ID NO：5
>CCP1：
Figure PCTCN2022088914-appb-000006
                            SEQ ID NO：6
实施例2.抗MASP2抗体的筛选和制备
使用小鼠免疫和噬菌体展示两种方法筛选和制备MASP2特异性抗体。
1、小鼠免疫：
使用实施例1中制备获得的人MASP2A蛋白作为抗原免疫balb/c小鼠，选取效价最高的小鼠进行杂交瘤细胞融合。杂交瘤细胞融合和培养：取3培养皿(10cm)24h前传代的骨髓瘤细胞，用不含HEPES的RPMI-1640培养基洗一遍，重悬，计数为2～4×10 7个。将免疫72h后的小鼠处死，无菌取脾，用不含HEPES的RPMI-1640培养基清洗，剪碎、研磨脾细胞，吹散，过滤，离心1000rpm、5min，重悬，计数为1～2×10 8个。将骨髓瘤细胞、脾细胞混匀，离心1000rpm，5min，弃上清，使沉淀松散，于40℃水浴中预热。将预热至40℃的1mL PEG于60s内滴加到离心管中，边加边轻轻搅拌。轻轻搅拌1min，于30s内加入1mL培养基，于1min内加入3mL培养基，于1min内加入16mL培养基。静置10min，离心1000rpm，5min，弃上清，用HAT-OPI培养基(含20％FBS的RPMI-1640培养基，含1×HAT和1×OPI)重悬细胞，37℃，5％CO 2孵育。融合后的第5天加入20％FBS的RPMI-1640培养基(含2×HAT和1×OPI)，50μL/孔。融合后第7天～10天，用HAT-OPI培养基进 行全换液。融合后第10～14天，根据细胞生长密度，进行人MASP2和猴MASP2蛋白ELISA检测，挑选阳性克隆。
ELISA检测方法如下：将1ug/mL的抗原4℃包被过夜，每孔50μL。PBS洗板3次，然后用含有3％BSA的PBS室温封闭1h。PBST洗板3次，加入杂交瘤细胞上清，室温1h。PBST洗3次，PBS再洗3次，加入二抗(1:2000；invitrogen，山羊抗小鼠IgG(H+L)二抗,31430)，室温1h。PBST洗3次，PBS再洗3次，加入TMB底物，室温10min，然后终止反应检测信号值(450nm光吸收值)。
2、噬菌体展示：
将人MASP2A抗原包被在免疫吸附板上，与噬菌体抗体合成库孵育进行固相筛选。经过三轮淘选，获得人MASP2和猴MASP2阳性噬菌体。
噬菌体展示筛选阳性克隆方法如下：免疫管上包被4mL MASP2A(5ng/μL浓度)，4℃过夜。将全人源噬菌体库用5％BSA/PBS室温封闭1小时。将噬菌体库转移至包被有MASP2A的免疫管中，室温旋转1h，PBS洗5次，1000μL TEA洗脱，加入400μL Tris-HCl(pH7.4)中和。感染10mL TG1(OD值至0.4)，37℃，40min。测output(产出)，涂Amp+平板，30℃过夜。刮菌，将菌接种到50mL 2×TY培养基(加Amp和1％葡萄糖)至OD值为0.1，37℃，200rpm，生长80min，至OD值为0.4-0.6。取10mL，加入500μL辅助噬菌体M13KO7，37℃感染40min。离心去上清，沉淀用100mL 2×TY培养基(加Amp和Kana)重悬，30℃，200rpm，过夜。4000rpm离心30min，收集上清，加入10mL PEG/NaCl，冰上沉淀1h，4000rpm离心30min，沉淀加入1mL PBS重悬。13000rpm离心3min，去掉沉淀。噬菌体再用PBS重悬，进入下一轮淘选。接种单克隆至96孔板，37℃3h，220rpm。加入1mM IPTG 30℃诱导过夜。同时在96孔板上包被MASP2，2ng/μL的浓度，每孔50μL，4℃过夜。第二天洗ELISA板1次后，加入200μL 2％MPBS封闭ELISA板，37℃，1h。将过夜培养的菌液，4000g离心10min，转移上清至新的96孔板中。洗ELISA板2次，加入25μL 2％MPBS封闭液，再加入25μL培养上清，混匀。25℃1hr洗ELISA板3次后，加入100μL抗Fab-HRP抗体(1:5000in 2％MPBS)，25℃1hr。洗ELISA板4次后显色。经ELISA检测，获得阳性克隆。
经抗原蛋白免疫小鼠和噬菌体展示筛选两种方法，获得阳性克隆，测序、纯化，获得抗MASP2抗体，其重链可变区(VH)和轻链可变区(VL)的氨基酸序列如下所示，下划线为重链可变区或轻链可变区的CDR：
>77H11 VH：
Figure PCTCN2022088914-appb-000007
                       SEQ ID NO：7
>77H11 VL：
Figure PCTCN2022088914-appb-000008
Figure PCTCN2022088914-appb-000009
                          SEQ ID NO：8
>67D2 VH：
Figure PCTCN2022088914-appb-000010
                          SEQ ID NO：9
>67D2 VL：
Figure PCTCN2022088914-appb-000011
                          SEQ ID NO：10
>67E8 VH：
Figure PCTCN2022088914-appb-000012
                           SEQ ID NO：11
>67E8 VL：
Figure PCTCN2022088914-appb-000013
                           SEQ ID NO：12
>29C1 VH：
Figure PCTCN2022088914-appb-000014
                           SEQ ID NO：13
>29C1 VL：
Figure PCTCN2022088914-appb-000015
                           SEQ ID NO：14
>11165 VH：
Figure PCTCN2022088914-appb-000016
                           SEQ ID NO：15
>11165 VL：
Figure PCTCN2022088914-appb-000017
                           SEQ ID NO：16
表1.抗MASP2抗体CDR序列(Kabat编号规则)
Figure PCTCN2022088914-appb-000018
77H11和67D2、67E8具有如下CDR：
HCDR1：SDYAWN(SEQ ID NO：17)；
HCDR2：YISYSGRTSYNPSLKS(SEQ ID NO：18)；
HCDR3：LYAX 1X 2X 3(SEQ ID NO：26)，其中，X 1选自L或M，X 2选自D或N，X 3选自Y或F；
LCDR1：KASQNVDTNVA(SEQ ID NO：20)；
LCDR2：SASYRX 4S(SEQ ID NO：27)，其中，X 4选自Y或F；
LCDR3：QQYNSNPLT(SEQ ID NO：22)。
实施例3.抗MASP2抗体抑制凝集素通路活性鉴定
用1％人血清对获得的抗MASP2抗体进行体外抑制凝集素通路活性的功能验证。
抗体在1％人血清中功能活性的鉴定方法为：将50μg/mL甘露聚糖溶液以25μL/孔包被384孔板，4℃过夜孵育。用50μL/孔TBST洗4次，用50μL/孔的3％BSA封闭液(溶剂为TBS)封闭，室温孵育2h。用TBST 50μL/孔洗板4遍。将相应浓度的抗MASP2抗体与1％人血清混合，4℃孵育1h，将所述混合物以15μL/孔加到前述384孔板中，37℃孵育1h。用50μL/孔的TBST洗板4遍，加入用0.5％BSA稀释的一抗(biotin-human Anti-C4c，Agrisera#IMSO1-031-305)，15μL/孔，800g/min离心后，室温孵育1h。再用50μL/孔的TBST洗板4遍，加入用0.5％BSA溶液1：5000倍稀释的二抗(SA-HRP)，15μL/孔，室温孵育30min。再用50μL/孔的TBST洗板4遍，加入45μL/孔的TMB显色液，室温避光孵育15min，最后加入45μL/孔的终止液，检测OD450值。
根据WO2012151481A合成阳性对照抗体OMS721，序列如下：
>OMS721的重链全长：
Figure PCTCN2022088914-appb-000019
                          SEQ ID NO：40
>OMS721的轻链全长：
Figure PCTCN2022088914-appb-000020
                          SEQ ID NO：41
结果参见图1A至图1D，表2至表4，所述结果显示77H11、67D2、67E8、29C1和11165都可以显著抑制凝集素通路，均优于阳性对照OMS721。这里的抗体77H11、67D2、67E8、29C1、11165均是将抗体重链可变区与人IgG4重链Fc区域连接，构造形成的全长抗MASP2抗体，所述的人IgG4重链Fc为SEQ ID NO：53所示。采用的hIgG4为同种型对照(Isotype control)。
表2.1％人血清条件下抗MASP2抗体对凝集素通路的抑制作用
抗体 IC 50(μg/mL)
OMS721 5.278
77H11 0.630
67D2 0.559
67E8 0.738
表3.1％人血清条件下抗MASP2抗体对凝集素通路的抑制作用
抗体 IC 50(μg/mL)
OMS721 2.858
29C1 0.086
表4.1％人血清条件下抗MASP2抗体对凝集素通路的抑制作用
抗体 IC 50(μg/mL)
OMS721 0.885
11165 0.499
实施例4.抗MASP2抗体的人源化改造
选择抗MASP2抗体29C1和77H11进行人源化改造，随后在人血清中进行活性鉴定。
29C1的轻链人源化模板为人胚系基因IGKV1-33*01，重链人源化模板为IGKV3-21*01。两条链都进行了若干回复突变。组合得到6个分子，其人源化序列如下所示(下划线为重链或轻链CDR)：
>29C1_H1：
Figure PCTCN2022088914-appb-000021
                        SEQ ID NO：42
>29C1_H2：
Figure PCTCN2022088914-appb-000022
                        SEQ ID NO：43
>29C1_H3：
Figure PCTCN2022088914-appb-000023
                        SEQ ID NO：44
>29C1_L1：
Figure PCTCN2022088914-appb-000024
                            SEQ ID NO：45
>29C1_L2：
Figure PCTCN2022088914-appb-000025
                            SEQ ID NO：46
77H11的轻链人源化模板为人的胚系基因IGKV1-27*01，重链人源化模板为人的胚系基因IGKV4-30-4*01。两条链都进行了若干回复突变。组合得到8个分子，其人源化序列如下所示(下划线为重链或轻链CDR)：
>77H11_H1：
Figure PCTCN2022088914-appb-000026
                            SEQ ID NO：47
>77H11_H2：
Figure PCTCN2022088914-appb-000027
                            SEQ ID NO：48
>77H11_H3：
Figure PCTCN2022088914-appb-000028
                            SEQ ID NO：49
>77H11_H4：
Figure PCTCN2022088914-appb-000029
                            SEQ ID NO：50
>77H11_L1：
Figure PCTCN2022088914-appb-000030
                            SEQ ID NO：51
>77H11_L2：
Figure PCTCN2022088914-appb-000031
                            SEQ ID NO：52
各人源化抗MASP2抗体的序列参见表5。
表5.抗MASP2抗体重连可变区、轻链可变区
Figure PCTCN2022088914-appb-000032
表5中的抗体均是将抗体重链可变区与人IgG4重链Fc区域连接，构造形成的全长抗MASP2抗体。其中重链Fc区域包括铰链(hinge)区，为带有S228P突变的人IgG4 Fc区，序列如SEQ ID NO：53所示。所述S228P是根据EU编号。
>人IgG4 Fc(S228P)：
Figure PCTCN2022088914-appb-000033
                         SEQ ID NO：53
示例性的抗MASP2抗体重链全长、轻链全长如下所示：
>29C1(H1L1)重链全长：
Figure PCTCN2022088914-appb-000034
Figure PCTCN2022088914-appb-000035
                        SEQ ID NO：54
>29C1(H1L1)轻链全长：
Figure PCTCN2022088914-appb-000036
                        SEQ ID NO：55
>77H11(H3L1)重链全长：
Figure PCTCN2022088914-appb-000037
                       SEQ ID NO：56
>77H11(H3L1)轻链全长：
Figure PCTCN2022088914-appb-000038
                       SEQ ID NO：57
以及，11165全长序列如下。
>11165重链全长：
Figure PCTCN2022088914-appb-000039
                        SEQ ID NO：58
>11165轻链全长：
Figure PCTCN2022088914-appb-000040
                      SEQ ID NO：59
按常规方法进行抗体的表达和纯化，经检测，得到本公开的全长抗体。
将上述人源化抗体在更接近体内条件的人90％血清中检测其对凝集素通路活性的影响。抗体在90％人血清中功能活性的鉴定方法为：将含有5μg/mL甘露聚糖溶液的碳酸钠-碳酸氢钠缓冲液(pH>9)，以25μL/孔包被384孔板，4℃过夜孵育。用50μL/孔的加入5mM Ca 2+的TBST洗3次。用50μL/孔的3％BSA封闭液(溶剂为TBS，加入5mM Ca 2+)，室温封闭1.5～2h。用50μL/孔的TBST洗3次，50μL/孔的TBS洗1次，50μL/孔的VBS洗1次。用VBS稀释MASP2抗体后，与人血清以1:9的比例混合(即90％人血清)获得混合物，4℃孵育30min，将所述混合物以15μL/孔加到前述384孔板中，4℃孵育1h。用50μL/孔的TBST洗3遍，加入0.5％BSA的TBS稀释的一抗(Biotin-Chicken Anti-C4C，6μg/mL)，15μL/孔，室温60min。用50μL/孔的TBST洗3遍，加入用0.5％BSA溶液1：5000倍稀释的二抗(SA-HRP)，15μL/孔，室温孵育30min。再用50μL/孔的TBST洗板3遍，加入45μL/孔的TMB显色液，室温避光孵育15min，最后加入45μL/孔的终止液，检测OD450值。
如图2A和2B、表6所示，29C1和77H11的人源化分子均能在人90％血清中显著抑制凝集素通路活性，并且效力优于OMS721。这里的29C1、77H11系列抗体均是将抗体的重链可变区与人IgG4重链Fc区域连接，构造形成的全长抗MASP2抗体，所述的人IgG4重链Fc为SEQ ID NO：53所示。采用的hIgG4为同种型对照(Isotype control)。
表6.90％人血清条件下抗MASP2抗体对凝集素通路的抑制作用
Figure PCTCN2022088914-appb-000041
实施例5.抗MASP2抗体的亲合力测定
用Biacore的方法来检测抗MASP2抗体的结合特性。用Biacore 8K(GE)仪器测定了29C1(H1L1)，77H11(H3L1)，11165三个抗体分子和人MASP2、鼠MASP2、猴MASP2蛋白的亲合力，使用同种型IgG抗体作为阴性对照。使用抗人Fc IgG捕获芯片捕获抗体，而后将不同浓度的抗原作为流动相进行检测，最后按1:1模式进行曲线拟合，得到亲合力数值。结果如表7所示。
结果显示，11165、29C1(H1L1)、77H11(H3L1)对人和猴MASP2蛋白都有10 -10M到10 -11M的亲合力，11165对鼠MASP2也有10 -10M级别的亲合力。
表7.抗MASP2抗体的亲合力测定结果(KD)
Figure PCTCN2022088914-appb-000042
实施例6.抗MASP2抗体在体外抑制不同物种凝集素通路活性检测
分别检测抗MASP2抗体在人、猴、鼠90％血清中对凝集素通路的活性抑制情况。
抗体在90％人、猴、鼠血清中功能活性的鉴定方法为：将含有5μg/mL甘露聚糖溶液的碳酸钠-碳酸氢钠缓冲液(pH>9)，以25μL/孔包被384孔板，4℃过夜孵育。用50μL/孔的加入5mM Ca 2+的TBST洗3次。用50μL/孔的3％BSA封闭液(溶剂为TBS，加入5mM Ca 2+)，室温封闭1.5～2h。用50μL/孔的TBST洗3次，50μL/孔的TBS洗1次，50μL/孔的VBS洗1次。用VBS稀释MASP2抗体后，与人血清以1:9的比例混合(即90％人血清)获得混合物，4℃孵育30min，将所述混合物以15μL/孔加到前述384孔板中，4℃孵育1h。用50μL/孔的TBST洗3遍，加入0.5％BSA的TBS稀释的一抗(Biotin-Chicken Anti-C4C，6μg/mL)，15μL/孔，室温60min。用50μL/孔的TBST洗3遍，加入用0.5％BSA溶液1：5000倍稀释的二抗(SA-HRP)，15μL/孔，室温孵育30min。再用50μL/孔的TBST洗板3遍，加入45μL/孔的TMB显色液，室温避光孵育15min，最后加入45μL/孔的终止液，检测OD450值。
结果参见图3A-3C和表8。结果说明，11165、29C1(H1L1)、77H11(H3L1)在人血清中对凝集素通路的抑制活性优于OMS721，使用同种型IgG抗体作为阴性对照。
表8.不同物种血清条件下抗MASP2抗体对凝集素通路的抑制作用
Figure PCTCN2022088914-appb-000043
实施例7.抗MASP2抗体在脑卒中动物模型中的功能验证
采用通用的脑卒中造模方法。具体方法为：将雄性C57小鼠(体重12-20g)在造模前第7天和第3天腹腔注射1次药物，造模当日缺血1h(再灌注同时)尾静脉注射药物1次，每次10mg/kg。将麻醉后的小鼠仰卧位固定于固定板上，去除颈部皮肤，碘伏消毒。颈部正中切口，分离右侧颈总动脉(CCA)、颈外动脉(ECA)、颈内动脉(ICA)。用血管夹暂时夹闭CCA近心端和ICA。在ECA上面备一条6-0手术丝线，然后用电凝笔烧断ECA。在ECA上面切一小口，将硅胶线栓插入切口，用手术丝线轻轻结扎。撤去ICA上面的血管夹，将硅胶线栓缓慢插入CCA，经CCA进入ICA，直至插到遇到轻微阻力，然后扎紧ECA上面的手术丝线，将线栓固定牢固。随后撤去CCA上面的血管夹。缺血1h后，拔出线栓，缝合颈部皮肤。将动物放回笼中饲养。再灌注48h时，处死动物，TTC染色进行脑梗死大小计量。
结果参见图4，结果显示，11165与OMS721均可显著抑制小鼠脑梗死面积，使用同种型IgG为阴性对照。
实施例8.抗MASP2抗体的猴体内药效实验(PD)
将77H11(H3L1)、29C1(H1L1)、11165和OMS721分别通过静脉推注的方式以3mg/kg和10mg/kg的剂量打入食蟹猴体内，在不同的时间采血，收集血清。检测不同时间点采集的食蟹猴血清的凝集素通路活性。具体采血时间为0小时，15分钟，1小时，4小时，8小时，24小时，48小时，72小时，96小时，168小时，240小时，360小时，480小时，600小时，720小时。血清分离方法为：从外周静脉采集血样，直接注入到空白采血管。血样静置15-60分钟至血液凝固后，在4℃，2500g离心10min，上清即为血清。
结果参见图5A和5B，可以看出4种抗体都能显著抑制凝集素通路活性，其中77H11(H3L1)和29C1(H1L1)在相同剂量下比OMS721有更长久的抑制效果。
实施例9.抗MASP2抗体的猴体内药代动力学实验(PK)
取健康雄性食蟹猴6只，之前没有接受过大分子药物，体重2～5kg。实验当天，静脉推注受试药物77H11(H3L1)、29C1(H1L1)、11165和OMS721，每种药的给药剂量都分别为3mg/kg或10mg/kg。给药后采血时间点为15min，1h，4h，8h，24h，48h，72h，96h，168h，240h，336h，408h，504h，672h。每次取全血0.3mL，不加抗凝剂，取血后在4℃放置30min，1000g离心15min，取上清置于EP管中，80℃保存。
用ELISA方法检测血清中的血药浓度，采用矩阵拟合的方式计算受试药物的T 1/2及其主要参数。结果参见表9。可以看出，11165的半衰期与OMS721相近，而77H11(H3L1)、29C1(H1L1)的半衰期显著优于OMS721，意味着可以在体内有更长久的药效。
表9.抗MASP2抗体的猴PK参数结果
Figure PCTCN2022088914-appb-000044
本公开中实验动物的使用及福利遵照“国际实验动物评估和认可委员会(AAALAC)”的规定执行。每天监测动物的健康状况及死亡情况，例行检查包括观察受试物和药物对动物日常行为表现的影响如行为活动，体重变化，外观体征等。
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 (16)

  1. 抗MASP2抗体或其抗原结合片段，其包含重链可变区(VH)和轻链可变区(VL)，其中：
    1)所述VH包含分别如SEQ ID NO：17、18、26所示的HCDR1、HCDR2、HCDR3，所述VL包含分别如SEQ ID NO：20、27、22所示的LCDR1、LCDR2、LCDR3；
    2)所述VH包含分别如SEQ ID NO：28、29、30所示的HCDR1、HCDR2、HCDR3，和所述VL包含分别如SEQ ID NO：31、32、33所示的LCDR1、LCDR2、LCDR3；或
    3)所述VH包含分别如SEQ ID NO：34、35、36所示的HCDR1、HCDR2、HCDR3，和所述VL包含分别如SEQ ID NO：37、38、39所示的LCDR1、LCDR2、LCDR3；
    优选地，
    所述VH包含分别如SEQ ID NO：17、18、19所示的HCDR1、HCDR2、HCDR3，和所述VL包含分别如SEQ ID NO：20、21、22所示的LCDR1、LCDR2、LCDR3；
    所述VH包含分别如SEQ ID NO：17、18、23所示的HCDR1、HCDR2、HCDR3，和所述VL包含分别如SEQ ID NO：20、24、22所示的LCDR1、LCDR2、LCDR3；或
    所述VH包含分别如SEQ ID NO：17、18、25所示的HCDR1、HCDR2、HCDR3，和所述VL包含分别如SEQ ID NO：20、21、22所示的LCDR1、LCDR2、LCDR3。
  2. 抗MASP2抗体或其抗原结合片段，其包含重链可变区(VH)和轻链可变区(VL)，其中：
    所述VH包含如SEQ ID NO：7所示VH中的HCDR1、HCDR2、HCDR3，VL包含如SEQ ID NO：8所示VL中的LCDR1、LCDR2、LCDR3；
    所述VH包含如SEQ ID NO：9所示VH中的HCDR1、HCDR2、HCDR3，VL包含如SEQ ID NO：10所示VL中的LCDR1、LCDR2、LCDR3；
    所述VH包含如SEQ ID NO：11所示VH中的HCDR1、HCDR2、HCDR3，VL包含如SEQ ID NO：12所示VL中的LCDR1、LCDR2、LCDR3；
    所述VH包含如SEQ ID NO：13所示VH中的HCDR1、HCDR2、HCDR3，VL包含如SEQ ID NO：14所示VL中的LCDR1、LCDR2、LCDR3；或
    所述VH包含如SEQ ID NO：15所示VH中的HCDR1、HCDR2、HCDR3，VL包含如SEQ ID NO：16所示VL中的LCDR1、LCDR2、LCDR3；
    其中所述HCDR1-3和LCDR1-3是根据Kabat、IMGT、Chothia、AbM或Contact编号系统定义的，优选是根据Kabat编号系统定义的。
  3. 根据权利要求1或2所述的抗MASP2抗体或其抗原结合片段，其为鼠源抗体、嵌合抗体、人源化抗体、全人抗体或其片段。
  4. 根据权利要求3所述的抗MASP2抗体或其抗原结合片段，其中：
    所述人源化抗体或其抗原结合片段的重链框架区源自IGKV3-21*01或IGKV4-30-4*01；和/或，轻链框架区源自IGKV1-33*01或IGKV1-27*01。
  5. 根据权利要求1至4中任一项所述的抗MASP2抗体或其抗原结合片段，其中：
    所述VH包含如SEQ ID NO：47-50中任一所示或与之具有至少90％或至少95％同一性的氨基酸序列，所述VL包含如SEQ ID NO：51或52所示或与之具有至少90％或至少95％同一性的氨基酸序列；
    所述VH包含如SEQ ID NO：7所示或与之具有至少90％或至少95％同一性的氨基酸序列，所述VL包含如SEQ ID NO：8所示或与之具有至少90％或至少95％同一性的氨基酸序列；
    所述VH包含如SEQ ID NO：9所示或与之具有至少90％或至少95％同一性的氨基酸序列，所述VL包含如SEQ ID NO：10所示或与之具有至少90％或至少95％同一性的氨基酸序列；
    所述VH包含如SEQ ID NO：11所示或与之具有至少90％或至少95％同一性的氨基酸序列，所述VL包含如SEQ ID NO：12所示或与之具有至少90％或至少95％同一性的氨基酸序列；
    所述VH包含如SEQ ID NO：42-44任一所示或与之具有至少90％或至少95％同一性的氨基酸序列，所述VL包含如SEQ ID NO：45或46所示或与之具有至少90％或至少95％同一性的氨基酸序列；
    所述VH包含如SEQ ID NO：13所示或与之具有至少90％或至少95％同一性的氨基酸序列，所述VL包含如SEQ ID NO：14所示或与之具有至少90％或至少95％同一性的氨基酸序列；或
    所述VH包含如SEQ ID NO：15所示或与之具有至少90％或至少95％同一性的氨基酸序列，所述VL包含如SEQ ID NO：16所示或与之具有至少90％或至少95％同一性的氨基酸序列。
  6. 根据权利要求1至5中任一项所述的抗MASP2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、IgG2、IgG4抗体或其抗原结合片段，更优选为F C区具有S228P、F234A和L235A中任一个或多个突变的IgG4抗体或其抗原结合片段。
  7. 根据权利要求1至6中任一项所述的抗MASP2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。
  8. 根据权利要求1至7中任一项所述的抗MASP2抗体或其抗原结合片段，其中：
    1)重链包含如SEQ ID NO：56所示或与之具有至少90％或至少95％同一性的氨基酸序列；轻链包含如SEQ ID NO：57所示或与之具有至少90％或至少95％同一性的氨基酸序列；
    2)重链包含如SEQ ID NO：54所示或与之具有至少90％或至少95％同一性的氨基酸序列；轻链包含如SEQ ID NO：55所示或与之具有至少90％或至少95％同一性的氨基酸序列；
    3)重链包含如SEQ ID NO：58所示或与之具有至少90％或至少95％同一性的氨基酸序列；轻链包含如SEQ ID NO：59所示或与之具有至少90％或至少95％同一性的氨基酸序列。
  9. 如权利要求1至8中任一项所述的抗MASP2抗体或其抗原结合片段，其具有以下至少一项：
    (i)所述抗体或抗原结合片段以10nM或更低的KD结合人MASP-2；
    (ii)所述抗体或抗原结合片段结合MASP-2的CCP1结构域中的表位；
    (iii)所述抗体或抗原结合片段以10nM或更低的IC 50在1％人血清中的体外测定中抑制C3b沉积；
    (iv)所述抗体或抗原结合片段以30nM或更低的IC 50抑制90％人血清中的C3b沉积；和
    (v)所述抗体或抗原结合片段基本上不抑制经典途径。
  10. 经分离的多核苷酸，其编码权利要求1至9中任一项所述的抗MASP2抗体或其抗原结合片段。
  11. 表达载体，其包含权利要求10所述的多核苷酸。
  12. 宿主细胞，其包含权利要求11所述的表达载体。
  13. 制备如权利要求1至9中任一项所述的抗MASP2抗体或其抗原结合片段的方法，包括：
    在权利要求12所述的宿主细胞中表达权利要求11所述的表达载体，以及从所述宿主细胞中分离表达的抗MASP2抗体或其抗原结合片段。
  14. 药物组合物，其含有权利要求1至9中任一项所述的抗MASP2抗体或其抗原结合片段，以及一种或多种可药用的赋形剂、稀释剂或载体。
  15. 权利要求1至9中任一项所述的抗MASP2抗体或其抗原结合片段在制备药物或药物组合物中的用途，所述药物或药物组合物用于治疗疾病；
    优选地，所述疾病为MASP-2依赖性补体活化相关疾病；
    优选地，所述疾病选自：IgA肾病、阵发性睡眠性血红蛋白尿症(PNH)、狼疮性肾炎、血栓性微血管病(TMA)、溶血性尿毒综合征(HUS)、膜性肾小球肾炎、肾小球性肾炎、年龄相关性黄斑变性、再灌注性损伤、心肌梗死、糖尿病神经病变、中风、移植物抗宿主病(GVHD)、Upshaw-Schulman综合征(USS)。
  16. 治疗疾病的方法，所述方法包括：
    向有需要的受试者施用治疗有效量的权利要求1至9中任一项所述的抗MASP2抗体或其抗原结合片段，或权利要求14所述的药物组合物；
    优选地，所述疾病为MASP-2依赖性补体活化相关疾病；
    优选地，所述疾病选自：IgA肾病、阵发性睡眠性血红蛋白尿症(PNH)、狼疮性肾炎、血栓性微血管病(TMA)、溶血性尿毒综合征(HUS)、膜性肾小球肾炎、肾小球性肾炎、年龄相关性黄斑变性、再灌注性损伤、心肌梗死、糖尿病神经病变、中风、移植物抗宿主病(GVHD)、Upshaw-Schulman综合征(USS)。
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