WO2024088293A1 - 一种抗masp2抗体的组合物及医药用途 - Google Patents

一种抗masp2抗体的组合物及医药用途 Download PDF

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Publication number
WO2024088293A1
WO2024088293A1 PCT/CN2023/126384 CN2023126384W WO2024088293A1 WO 2024088293 A1 WO2024088293 A1 WO 2024088293A1 CN 2023126384 W CN2023126384 W CN 2023126384W WO 2024088293 A1 WO2024088293 A1 WO 2024088293A1
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WIPO (PCT)
Prior art keywords
histidine
antibody
antigen
masp2
binding fragment
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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PCT/CN2023/126384
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English (en)
French (fr)
Inventor
杨喜琴
杨震
葛凌霄
王宏伟
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Suzhou Suncadia Biopharmaceuticals Co Ltd
Jiangsu Hengrui Pharmaceutical Co Ltd
Original Assignee
Suzhou Suncadia Biopharmaceuticals Co Ltd
Jiangsu Hengrui Pharmaceutical Co Ltd
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Application filed by Suzhou Suncadia Biopharmaceuticals Co Ltd, Jiangsu Hengrui Pharmaceutical Co Ltd filed Critical Suzhou Suncadia Biopharmaceuticals Co Ltd
Priority to CN202380071634.3A priority Critical patent/CN120051300A/zh
Publication of WO2024088293A1 publication Critical patent/WO2024088293A1/zh
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys

Definitions

  • the present disclosure relates to the field of pharmaceutical preparations, and in particular to a pharmaceutical composition comprising an anti-MASP2 antibody or an antigen-binding fragment thereof, and pharmaceutical uses thereof.
  • the complement system is a protein present in human and animal serum, tissue fluid and cell membrane surface. It has biological activity after activation and can mediate immune and inflammatory responses.
  • the complement system is composed of nearly 40 components, most of which are glycoproteins, including C1q, C1r, C1s, C2-C9, D factor, B factor, 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 also in the damaging response of immunopathology. 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 enzymatic reactions of serine proteases, which are divided into three types: the classical activation pathway, the alternative pathway, and the lectin pathway, which ultimately mediate the activation of the terminal pathway marked by the formation of a membrane-breaking complex.
  • the alternative pathway maintains a low level of activation for a long time to monitor the invasion of pathogenic microorganisms.
  • Healthy cells also inhibit the attack of the complement system on them by expressing complement regulatory proteins such as CD55 and CD59.
  • the 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 immune complexes with antigen structures and are recognized by C1q, C1s, and C1r, activating C2 and C4 to form C4bC2a (that is, C3 convertase), which ultimately promotes the formation of a membrane-breaking complex composed of C5-C9.
  • the alternative pathway is an activation pathway that starts directly from the spontaneous hydrolysis of C3.
  • lectin pathway is a direct recognition of mannose, N-acetylmannose, N-acetylglucosamine, fucose, and other sugar structures on the surface of various pathogenic microorganisms by mannan-binding lectin (MBL) or fibrin (FCN) in plasma, followed by activation of the classical complement pathway.
  • MBL mannan-binding lectin
  • FCN fibrin
  • the safety of targeting the complement system is generally safe and tolerable. Considering that the complement system is involved in regulating the development of B cells and the activation of T cells, mice with complement factor gene deletions have defects in reproductive capacity or embryonic development, and have an increased risk of infection. Therefore, the development of drugs targeting specific upstream targets of the complement pathway can ensure the efficacy of the drug. At the same time, it reduces the side effects of overall inhibition of the complement pathway.
  • MASP2 protein is the core hydrolase of the lectin pathway. It consists of CUB at the N-terminus responsible for binding to MBL, the EGF domain, the CCP domain that binds to the downstream substrates C4 and C2, and the SP domain at the C-terminus.
  • the MBL-MASP complex binds to the sugar structure on the surface of the pathogen, causing MASP-1 and MASP-2 to be activated independently.
  • the activated MASP2 exerts its SP activity, cleaves C4 and C2 to form the C3 convertase C4b2a, and can eventually activate the complement system mediated by the lectin pathway. It has been confirmed to be related to IgA nephropathy, stroke, and myocardial ischemia.
  • MASP2 deficiency can significantly reduce the infarct area.
  • MBL-MASP2 mainly recognizes IgA with missing galactose modification.
  • the activation of the LP pathway promotes the secretion of cytokines, ultimately leading to damage to tubular epithelial cells and podocytes, and abnormal kidney function.
  • Omeros' MASP2 monoclonal antibody narsoplimab (OMS721) is developed to treat a variety of inflammatory-related diseases, including thrombotic microangiopathy (TMA), IgA nephropathy, hemolytic uremic syndrome (HUS), lupus nephritis, membranous glomerulonephritis, glomerulonephritis, age-related macular degeneration, reperfusion injury, myocardial infarction, diabetic neuropathy, stroke, and graft-versus-host disease, mainly by inhibiting lectin-mediated complement system activation.
  • TMA thrombotic microangiopathy
  • HUS hemolytic uremic syndrome
  • lupus nephritis membranous glomerulonephritis
  • glomerulonephritis age-related macular degeneration
  • reperfusion injury myocardial infarction
  • diabetic neuropathy diabetic neuropathy
  • stroke graft-versus
  • TMA TMA-associated glomerulonephritis
  • HUS and IgA nephropathy in clinical phase III
  • lupus nephritis, membranous glomerulonephritis, and glomerulonephritis is in clinical phase II.
  • the applicant's patent application WO2022228364 provides an anti-MASP2 antibody with a new structure, which has good inhibitory activity on MASP2-dependent complement activation.
  • Antibody drugs are an important class of biological drugs. They have large molecular weight and complex structure. They are easily affected by physical or chemical factors during production, storage and use, causing degradation and aggregation, resulting in reduced activity or even failure. Therefore, it is very important to develop excellent antibody preparations.
  • the present disclosure provides a pharmaceutical composition comprising an anti-MASP2 antibody or an antigen-binding fragment thereof, wherein the composition has excellent stability.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising an anti-MASP2 antibody or an antigen-binding fragment thereof and a buffer
  • the buffer is selected from a tris (hydroxymethylaminomethane) buffer, an acetate buffer, a succinate buffer, a phosphate buffer, a histidine salt buffer, an acetate buffer, a citrate buffer, a tartrate buffer, a fumarate buffer, and a glycylglycine buffer.
  • the buffer is selected from a phosphate buffer and a histidine salt buffer.
  • the phosphate buffer is selected from sodium dihydrogen phosphate-disodium hydrogen phosphate; the histidine salt buffer is selected from histidine-hydrochloric acid buffer or histidine-acetate buffer. In certain embodiments, the buffer is a histidine salt. Buffer. In certain embodiments, the buffer is a histidine-HCl buffer. In certain embodiments, the buffer is a histidine-histidine HCl buffer.
  • the pH of the buffer is 5.0-8.5, for example, 5.5-8.0, 6.0-8.0, 7.0-8.0, 6.0-7.0, 5.5-7.5, 6.5-8.0, 6.4-6.8, non-limiting examples include about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5 or any value between any two numbers.
  • the pH of the buffer is 6.0-7.5.
  • the pH of the pharmaceutical composition has a difference of no more than ⁇ 0.5 compared to the pH of the buffer it contains.
  • the pharmaceutical composition as described in any of the above items has a pH of 5.0-8.5, for example, about 5.5-8.0, 6.0-8.0, 7.0-8.0, 6.0-7.0, 5.5-7.5, 6.5-8.0, 6.4-6.8, and non-limiting examples include about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, or any value in between any two numbers.
  • the buffer concentration is 0.1-50mM, for example, 1-30mM, 5-20mM, 8-12mM
  • non-limiting examples include about 5mM, about 6mM, about 7mM, about 8mM, about 9mM, about 10mM, about 11mM, about 12mM, about 13mM, about 14mM, about 15mM, about 16mM, about 17mM, about 18mM, about 19mM, about 20mM or any value between any two numbers.
  • the buffer concentration is about 10mM.
  • the pharmaceutical composition as described in any one of the above further comprises an adjuvant.
  • the adjuvant is selected from one or more of amino acids or their salts, sugars, polyols and salts.
  • the adjuvant is selected from one or more of amino acids or their salts, sugars and salts.
  • the adjuvant is salt.
  • the adjuvant is amino acids or their salts.
  • the adjuvant is sugar and amino acids or their salts.
  • the amino acids are selected from one or more of proline, aspartic acid, glutamic acid, lysine, arginine, glycine and histidine.
  • the sugar is selected from one or more of glucose, sucrose, maltose and trehalose.
  • the polyol is selected from one or more of mannitol and sorbitol.
  • the salt is selected from sodium chloride or a salt of an amino acid, such as sodium chloride.
  • the adjuvant is selected from one or more of sodium chloride, sucrose and arginine or its salt.
  • the adjuvant is sodium chloride.
  • the adjuvant is arginine or its salt, such as arginine hydrochloride.
  • the auxiliary materials are sucrose and arginine or a salt thereof, such as sucrose and arginine hydrochloride.
  • the concentration of the excipient is 0.1-100 mg/mL, for example, 0.5-100 mg/mL, 0.5-80 mg/mL, 1-80 mg/mL, 1-70 mg/mL, 1-60 mg/mL, 1-50 mg/mL, 1-40 mg/mL, 1-30 mg/mL, 1-20 mg/mL, 5-80 mg/mL, 5-70mg/mL, 5-60mg/mL, 5-50mg/mL, 5-40mg/mL, 5-30mg/mL, 5-20mg/mL, 10-70mg/mL, 10-60mg/mL, 10-50mg/mL, 10-40mg/mL, 10-30mg/mL, 10-20mg/mL, 20-60mg/mL, 20-50mg/mL, 20-40mg/mL, 20-30mg/mL, 30-50mg/mL, 20-40mg/mL, 8- 9 mg/mL, non-limiting examples
  • the concentration of the adjuvant is about 8.2mg/mL, about 15mg/mL, about 28mg/mL, about 30mg/mL, about 40mg/mL, about 45mg/mL and about 75mg/mL. In certain embodiments, the concentration of the adjuvant is about 5-10mg/mL. In certain embodiments, the adjuvant is about 8.2 mg/mL sodium chloride. In certain embodiments, the adjuvant is about 15 mg/mL arginine hydrochloride. In certain embodiments, the adjuvant is about 15 mg/mL arginine hydrochloride and 40 mg/mL sucrose.
  • the pharmaceutical composition as described in any one of the above is an isotonic preparation.
  • the osmotic pressure of the pharmaceutical composition is 260-380mOsm, such as 270-360mOsm, and non-limiting examples include about 270mOsm, about 272mOsm, about 274mOsm, about 276mOsm, about 278mOsm, about 280mOsm, about 282mOsm, about 284mOsm, about 286mOsm, about 288mOsm, about 290mOsm, about 292mOsm, about 294mOsm, about 296mOsm, about 298mOsm, about 300mOsm, In some embodiments, the osmotic pressure of the pharmaceutical composition is 270-360 mOsm.
  • the pharmaceutical composition of the present disclosure further comprises a surfactant.
  • the surfactant is an ionic or nonionic surfactant.
  • the surfactant is selected from polysorbate, polyhydroxyalkylene, Triton, sodium dodecyl sulfate, sodium lauryl sulfate, octyl
  • the surfactant is sodium succinoside, lauryl-sulfobetaine, myristyl-sulfobetaine, linoleyl-sulfobetaine, stearyl-sulfobetaine, lauryl-sarcosine, myristyl-sarcosine, linoleyl-sarcosine, stearyl-sarcosine, linoleyl-betaine, myristyl-betaine, cetyl-betaine, lauramidopropyl-betaine, cocamidopropyl-betaine,
  • the surfactant concentration described in the pharmaceutical composition is 0.01-2 mg/mL, for example, 0.05-1 mg/mL, and non-limiting examples include about 0.05 mg/mL, about 0.1 mg/mL, about 0.15 mg/mL, about 0.2 mg/mL, about 0.25 mg/mL, about 0.3 mg/mL, about 0.35 mg/mL, about 0.4 mg/mL, about 0.45 mg/mL, about 0.5 mg/mL, about 0.55 mg/mL, about 0.6 mg/mL, about 0.65 mg/mL, about 0.7 mg/mL, about 0.75 mg/mL, about 0.8 mg/mL or any value between any two numbers.
  • the surfactant concentration is 0.1-0.4 mg/mL. In certain embodiments, the surfactant concentration is about 0.1 mg/mL, about 0.2 mg/mL, about 0.3 mg/mL or about 0.4 mg/mL.
  • the pharmaceutical composition of any of the above items, wherein the concentration of the anti-MASP2 antibody or antigen-binding fragment thereof is 0.1-500 mg/mL, such as 0.1-300 mg/mL, 5-300 mg/mL, 5-150 mg/mL, 20-200 mg/mL, 50-250 mg/mL, 80-120 mg/mL, 70-130 mg/mL, 60-140 mg/mL, 90-200 mg/mL, 90-165 mg/mL, 50-150 mg/mL, non-limiting examples include about 5 mg/mL, about 6 mg/mL, about 7 mg/mL, about 8 mg/mL, about 9 mg/mL, about 10 mg/mL, about 15 mg/mL, about 20 mg/mL, about 25 mg/mL, about 30 mg/mL, about 35 mg/mL, about 40 mg/mL, about 45 mg/mL.
  • the concentration of the anti-MASP2 antibody or antigen-binding fragment thereof is about 10 mg/mL, about 50 mg/mL, about 90 mg/mL, about 100 mg/mL, or about 110 mg/mL, about 135 mg/mL, about 150 mg/mL, about 165 mg/mL, or about 200 mg/mL. In certain embodiments, the concentration of the anti-MASP2 antibody or antigen-binding fragment thereof is about 90-110 mg/mL or 90-165 mg/mL.
  • the concentration of the anti-MASP2 antibody or antigen-binding fragment thereof is about 100 mg/mL, about 50 mg/mL, about 90 mg/mL, about 100 mg/mL, about 110 mg/mL, about 135 mg/mL, about 150 mg/mL, about 165 mg/mL, or about 200 mg/mL.
  • the concentration is about 90 mg/mL, about 100 mg/mL, about 110 mg/mL, about 135 mg/mL, about 150 mg/mL, about 165 mg/mL or about 200 mg/mL.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising an anti-MASP2 antibody or an antigen-binding fragment thereof (e.g., 77H11 (H3L1) whose heavy chain and light chain sequences are SEQ ID NO: 24 and 25), which comprises or is any one of the following 1) to 21):
  • an anti-MASP2 antibody or an antigen-binding fragment thereof e.g., 77H11 (H3L1) whose heavy chain and light chain sequences are SEQ ID NO: 24 and 25
  • Sodium chloride, mannitol, and/or amino acids e.g., proline
  • Histidine-HCl buffer e.g., histidine-histidine HCl buffer
  • Polysorbates e.g. polysorbate 80
  • histidine-HCl buffer e.g. histidine-histidine HCl buffer
  • polysorbate 80 0.01-1 mg/mL polysorbate (e.g. polysorbate 80);
  • histidine-HCl buffer e.g. histidine-histidine HCl buffer
  • polysorbate e.g., polysorbate 80
  • histidine-HCl buffer e.g. histidine-histidine HCl buffer
  • polysorbate e.g., polysorbate 80
  • histidine-HCl buffer e.g. histidine-histidine HCl buffer
  • polysorbate e.g., polysorbate 80
  • histidine-HCl buffer e.g. histidine-histidine HCl buffer
  • polysorbate e.g., polysorbate 80
  • histidine-HCl buffer e.g., histidine-histidine HCl buffer
  • polysorbate e.g., polysorbate 80
  • pH 6.1-7.1 e.g., about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0;
  • histidine-HCl buffer e.g., histidine-histidine HCl buffer
  • polysorbate e.g., polysorbate 80
  • Histidine-HCl buffer e.g., histidine-histidine HCl buffer
  • pH 5.5-8.0 e.g., 5.5-7.5, 6.0-7.5, 6.1-7.1, about 6.4, about 6.6, or about 6.8;
  • Sodium chloride, or sucrose and arginine or a salt thereof e.g. arginine hydrochloride
  • Sodium chloride, or sucrose and arginine or a salt thereof e.g. arginine hydrochloride
  • Histidine-HCl buffer e.g., histidine-histidine HCl buffer
  • Polysorbates e.g. polysorbate 80
  • pH 5.5-8.0 e.g., 5.5-7.5, 6.0-7.5, 6.1-7.1, about 6.4, about 6.6, or about 6.8;
  • arginine or a salt thereof e.g. arginine hydrochloride
  • polysorbate e.g., polysorbate 80
  • arginine or a salt thereof e.g. arginine hydrochloride
  • polysorbate e.g., polysorbate 80
  • arginine or its salt e.g. arginine hydrochloride
  • polysorbate e.g., polysorbate 80
  • arginine or its salt e.g. arginine hydrochloride
  • polysorbate e.g., polysorbate 80
  • arginine or a salt thereof e.g., arginine hydrochloride
  • polysorbate e.g., polysorbate 80
  • arginine or a salt thereof e.g., arginine hydrochloride
  • polysorbate e.g., polysorbate 80
  • arginine or a salt thereof e.g., arginine hydrochloride
  • polysorbate 80 e.g., polysorbate 80
  • arginine or a salt thereof e.g., arginine hydrochloride
  • polysorbate e.g., polysorbate 80
  • composition described in any one of the above 1) to 21) further comprises water for injection;
  • the pharmaceutical composition described in any one of the above 1) to 22) further comprises a pH adjuster, such as hydrochloric acid and/or sodium hydroxide.
  • a pH adjuster such as hydrochloric acid and/or sodium hydroxide.
  • the present disclosure provides a pharmaceutical composition
  • an anti-MASP2 antibody or an antigen-binding fragment thereof e.g., 77H11 (H3L1) whose heavy chain and light chain sequences are SEQ ID NO: 24 and 25, comprising any one of the following 1) to 9):
  • histidine-HCl buffer e.g. histidine-histidine HCl buffer
  • polysorbate e.g., polysorbate 80
  • pH is 5.5-7.5.
  • histidine-HCl buffer e.g. histidine-histidine HCl buffer
  • polysorbate e.g., polysorbate 80
  • pH is 6.0-7.5.
  • histidine-HCl buffer e.g., histidine-histidine HCl buffer
  • polysorbate e.g., polysorbate 80
  • polysorbate 80 e.g., about 0.1, about 0.2, about 0.4 mg/mL
  • the pH is about 6.1-7.1 (e.g., about 6.4, about 6.5, about 6.6, about 6.7, about 6.8).
  • histidine-HCl buffer e.g. histidine-histidine HCl buffer
  • polysorbate e.g., polysorbate 80
  • pH is 5.5-7.5.
  • histidine-HCl buffer e.g. histidine-histidine HCl buffer
  • polysorbate e.g., polysorbate 80
  • pH is 6.0-7.5.
  • histidine-HCl buffer e.g., histidine-histidine HCl buffer
  • polysorbate e.g., polysorbate 80
  • polysorbate 80 e.g., about 0.1, about 0.2, about 0.4 mg/mL
  • the pH is about 6.1-7.1 (e.g., about 6.4, about 6.5, about 6.6, about 6.7, about 6.8).
  • histidine-HCl buffer e.g. histidine-histidine HCl buffer
  • polysorbate e.g., polysorbate 80
  • pH is 5.5-7.5.
  • histidine-HCl buffer e.g. histidine-histidine HCl buffer
  • polysorbate e.g., polysorbate 80
  • pH is 6.0-7.5.
  • histidine-HCl buffer e.g., histidine-histidine HCl buffer
  • polysorbate e.g., polysorbate 80
  • polysorbate 80 e.g., about 0.1, about 0.2, about 0.4 mg/mL
  • the pH is about 6.1-7.1 (e.g., about 6.4, about 6.5, about 6.6, about 6.7, about 6.8).
  • composition described in any one of the above 1) to 10) further comprises water for injection;
  • the pharmaceutical composition described in any one of the above 1) to 11) further comprises a pH adjuster, such as hydrochloric acid and/or sodium hydroxide.
  • a pH adjuster such as hydrochloric acid and/or sodium hydroxide.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising an anti-MASP2 antibody or an antigen-binding fragment thereof (e.g., 77H11 (H3L1) whose heavy chain and light chain sequences are SEQ ID NO: 24 and 25), which comprises or is any one of the following groups 1) to 20):
  • an anti-MASP2 antibody or an antigen-binding fragment thereof e.g., 77H11 (H3L1) whose heavy chain and light chain sequences are SEQ ID NO: 24 and 25
  • the histidine-HCl buffer is histidine-histidine-HCl buffer.
  • the present disclosure also provides a method for preparing the aforementioned pharmaceutical composition, comprising the step of mixing an anti-MASP2 antibody or an antigen-binding fragment thereof with a buffer.
  • the buffer is a histidine-HCl buffer (eg, histidine-histidine HCl).
  • the pharmaceutical composition as described in any one of the above items is a liquid preparation.
  • the solvent of the liquid preparation is water, physiological saline or glucose solution.
  • the present disclosure also provides a lyophilized preparation, characterized in that the lyophilized preparation can form any of the above pharmaceutical compositions after being reconstituted.
  • the present disclosure also provides a lyophilized preparation, which is obtained by freeze-drying the pharmaceutical composition as described above.
  • the present disclosure provides a reconstitution solution, wherein the reconstitution solution is prepared by reconstitution of the aforementioned lyophilized preparation.
  • the reconstitution solution is selected from but not limited to water for injection, physiological saline or glucose solution.
  • the present disclosure also provides a product, which includes a container, wherein the container is filled with the aforementioned pharmaceutical composition, the aforementioned lyophilized preparation, or the aforementioned reconstituted solution.
  • the container is a neutral borosilicate glass tube injection bottle.
  • the product includes a drug instruction sheet.
  • the present disclosure is a pharmaceutical composition as described in any one of the above items, wherein the anti-MASP2 antibody and antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein:
  • the VH comprises HCDR1, HCDR2, HCDR3 in VH as shown in SEQ ID NO: 7, and/or the VL comprises LCDR1, LCDR2, LCDR3 in VL as shown in SEQ ID NO: 8.
  • 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, are defined according to the Kabat numbering system.
  • the anti-MASP2 antibodies and antigen-binding fragments thereof comprise:
  • the VH comprises HCDR1, HCDR2, HCDR3 in VH as shown in SEQ ID NO: 7, and/or the VL comprises LCDR1, LCDR2, LCDR3 in VL as shown in SEQ ID NO: 8;
  • the anti-MASP2 antibody and antigen-binding fragment thereof comprises 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 NO: 9, 10, 11, respectively, and/or the VL comprises LCDR1, LCDR2, LCDR3 as shown in SEQ ID NO: 12, 13, 14, respectively.
  • the anti-MASP2 antibody or antigen-binding fragment thereof is a murine antibody, a chimeric antibody, a humanized antibody, a fully human antibody or a fragment thereof.
  • the heavy chain framework region of the humanized antibody or antigen-binding fragment thereof described above is derived from IGKV3-21*01 or IGKV4-30-4*01; and/or, the light chain framework region is derived from IGKV1-33*01 or IGKV1-27*01.
  • the anti-MASP2 antibody or antigen-binding fragment thereof wherein:
  • amino acid sequence of VH is shown in one of SEQ ID NOs: 7, 17, 18, 19, 20, and/or the amino acid sequence of VL is shown in one of SEQ ID NOs: 8, 121, 22;
  • the anti-MASP2 antibody or antigen-binding fragment thereof wherein:
  • VH and VL respectively comprise or consist of the amino acid sequences shown in SEQ ID NO: 7 and 8;
  • VH and VL respectively comprise or consist of the amino acid sequences shown in SEQ ID NO: 17 and 21;
  • VH and VL respectively comprise or consist of the amino acid sequences shown in SEQ ID NOs: 17 and 22;
  • VH and VL respectively comprise or consist of the amino acid sequences shown in SEQ ID NO: 18 and 21;
  • VH and VL respectively comprise or consist of the amino acid sequences shown in SEQ ID NOs: 18 and 22;
  • VH and VL respectively comprise or consist of the amino acid sequences shown in SEQ ID NO: 19 and 21;
  • VH and VL respectively comprise or consist of the amino acid sequences shown in SEQ ID NOs: 19 and 22;
  • VH and VL respectively comprise or consist of the amino acid sequences shown in SEQ ID NOs: 20 and 21;
  • VH and VL respectively comprise or consist of the amino acid sequences shown in SEQ ID NOs: 20 and 22;
  • an anti-MASP2 antibody or antigen-binding fragment thereof comprising a variant VH, VL having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with the VH, VL of any of groups a) to i) above.
  • the anti-MASP2 antibody or antigen-binding fragment thereof is an IgG antibody or antigen-binding fragment thereof, such as an IgG1, IgG2, IgG4 antibody or antigen-binding fragment thereof, and more preferably an IgG4 antibody or antigen-binding fragment thereof in which the Fc has any one or more mutations of S228P, F234A and L235A.
  • the above mutations are all EU numbering.
  • the anti-MASP2 antibody or antigen-binding fragment thereof further comprises a human immunoglobulin Fc region; for example, the Fc region is a human IgG1, IgG2, or IgG4 Fc region. In certain embodiments, the Fc region may have a mutation that reduces ADCC function.
  • examples of mutations are L234A/L235A on IgG1, V234A/G237A/P238S/H268A/V309L/A330S/P331S on IgG2, F234A/L235A on IgG4, S228P/F234A/L235A on IgG4, N297A on IgG1, IgG2, IgG3, or IgG4, V234A/G237A on IgG2, K214T/E233P/L234V/L235A/G236 deletion/A327G/P331S on IgG1.
  • Hybrid IgG2/4 Fc domains can also be used, such as 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 the anti-MASP2 antibody described in the present disclosure is a Fab, Fv, sFv, Fab', F(ab') 2 , linear antibody, single-chain antibody, scFv, sdAb, sdFv, nanobody, peptibody, domain antibody and multispecific antibody (bispecific antibody, diabody, triabody and tetrabody, tandem di-scFv, tandem tri-scFv), for example, specifically a scFv, Fv, Fab or Fab' fragment.
  • the full-length amino acid sequence of the heavy chain of the anti-MASP2 antibody or antigen-binding fragment thereof described in the present disclosure is as shown in SEQ ID NO: 24, or is at least 80%, at least 90% or at least 95% identical thereto; the full-length amino acid sequence of the light chain is as shown in SEQ ID NO: 25, or is at least 80%, at least 90% or at least 95% identical thereto.
  • the heavy chain variable region of the anti-MASP2 antibody or antigen-binding fragment thereof has 0 to 10 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid changes; the light chain variable region has 0 to 10 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid changes.
  • the amino acid changes are conservative replacements, substitutions or modifications, and/or deletions or additions that do not affect the function.
  • the anti-MASP2 antibodies selectively inhibit MASP2 complement activation, leaving the Clq-dependent complement activation system functionally intact.
  • the present disclosure provides the use and method of the aforementioned pharmaceutical composition, the pharmaceutical composition prepared by the aforementioned method, the aforementioned lyophilized preparation or the aforementioned reconstituted solution in preventing and/or treating a disease, wherein the disease may be related to or unrelated to the complement signaling pathway (e.g., MASP2).
  • the disease is IgA nephropathy or paroxysmal nocturnal hemoglobinuria (PNH).
  • the present disclosure also provides the aforementioned pharmaceutical composition, lyophilized preparation, reconstituted solution or product as a drug for preventing and/or treating a disease.
  • the present disclosure provides a method for treating or preventing a disease, comprising administering a therapeutically or preventively effective amount of the aforementioned pharmaceutical composition, the pharmaceutical composition prepared by the aforementioned method, the aforementioned lyophilized preparation, or the aforementioned reconstituted solution to a subject in need thereof.
  • MASP-2-dependent complement activation has been implicated as 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 disorders, pulmonary conditions, extracorporeal reperfusion procedures, skeletal muscle conditions, renal conditions, skin conditions, organ or tissue transplantation, nervous system disorders or injuries, blood disorders, genitourinary conditions, diabetes, chemotherapy or radiation therapy, malignancies, endocrine disorders, coagulation disorders, or ophthalmic conditions.
  • the aforementioned pharmaceutical composition, the pharmaceutical composition prepared by the aforementioned method, the aforementioned lyophilized formulation or the aforementioned reconstituted solution disclosed herein are provided as methods for treating the above diseases and conditions, and related pharmaceutical uses.
  • the above-mentioned diseases and conditions are diseases associated with MASP-2-dependent complement activation.
  • the above diseases and conditions are microvascular endothelial cell damage and/or thrombosis.
  • the above diseases and conditions are selected from: 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), and in certain specific embodiments, the disease is associated with MASP-2-dependent complement activation.
  • TMA thrombotic microangiopathy
  • HUS hemolytic uremic syndrome
  • membranous glomerulonephritis glomerulonephritis
  • the anti-MASP-2 antibodies of the present disclosure are anti-MASP-2 antibodies described in International Patent Application WO2022228364, the entire contents of which are incorporated herein by reference in their entirety.
  • MASP-2-dependent complement activation includes MASP-2-dependent activation of the lectin pathway, which occurs under physiological conditions (i.e., in the presence of Ca++) and results in the formation of the lectin pathway C3 convertase C4b2a and, following accumulation of the C3 cleavage product C3b, the C5 convertase C42a(C3b)n.
  • the “classical pathway” is triggered by the binding of antibodies to foreign particles and requires the binding of recognition molecules.
  • the “alternative pathway” refers to complement activation, which is triggered, for example, by zymosan from the cell walls of fungi and yeast, 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 which is traditionally believed to arise from spontaneous proteolysis of complement factor C3 to produce C3b.
  • LPS lipopolysaccharide
  • the "lectin pathway” refers to complement activation, which occurs via specific binding of serum and non-serum sugar-binding proteins, including mannan-binding lectin (MBL), CL-11, and ficolins (H-ficolin, M-ficolin, or L-ficolin).
  • Antibody is used in the broadest sense, covering various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies; monospecific antibodies, multispecific antibodies (such as bispecific antibodies), full-length antibodies and antibody fragments (or antigen-binding fragments, or antigen-binding portions), as long as they exhibit the desired antigen-binding activity.
  • Antibodies may refer to immunoglobulins, which are tetrapeptide chains consisting of two identical heavy chains and two identical light chains connected by interchain disulfide bonds. The amino acid composition and arrangement order of the constant region of the immunoglobulin heavy chain are different, so their antigenicity is also different.
  • immunoglobulins can be divided into five categories, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA and IgE, and their corresponding heavy chains are ⁇ chain, ⁇ chain, ⁇ chain, ⁇ chain and ⁇ chain, respectively.
  • the same class of Ig can be divided into different subclasses according to the difference in the amino acid composition of its hinge region and the number and position of the heavy chain disulfide bonds, such as IgG can be divided into IgG1, IgG2, IgG3, and IgG4.
  • Light chains are divided into ⁇ chains or ⁇ chains according to the differences in the constant region.
  • Each of the five types of Ig can have a kappa chain or a lambda chain.
  • the sequences of about 110 amino acids near the N-terminus of the antibody heavy chain and light chain vary greatly, which is the variable region (V region); the remaining amino acid sequences near the C-terminus are relatively stable, which is the constant region (C region).
  • the variable region includes three hypervariable regions (CDRs) and four relatively conservative framework regions (FRs). The three hypervariable regions determine the specificity of the antibody, also known as the complementarity determining regions (CDRs).
  • Each light chain variable region (VL) and heavy chain variable region (VH) consists of three CDR regions and four FR regions, arranged in the order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the three CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.
  • the deterministic depiction of CDRs and the 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 the various 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, the Chothia numbering system, the AbM numbering system, the IMGT numbering system, contact definitions, conformational definitions.
  • the Kabat numbering system is a standard for numbering residues in antibodies and is often used to identify CDR regions (see, e.g., 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 position of certain structural loop regions. (See, e.g., 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 that model antibody structure (see, e.g., Martin et al., 1989, Proc Natl Acad Sci (USA), 86:9268-9272; "AbMTM, A Computer Program for Modeling Variable Regions of Antibodies," Oxford, UK; Oxford Molecular, Ltd.
  • the AbM numbering system uses a combination of knowledge databases and ab initio methods to model the tertiary structure of antibodies from the basic sequence (see Samudrala et al., 1999, "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach" in PROTEINS, Structure, Function and Genetics Suppl., 3: 194-198 for those described).
  • CDR may refer to a CDR defined by any method known in the art (including a combination of methods).
  • the CDR amino acid residues of the VL and VH regions of the antibodies or antigen-binding fragments of the present disclosure conform in number and position to the well-known Kabat or AbM numbering system.
  • “Monoclonal antibody” or “monoantibody” refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.
  • the modifier "monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies used in accordance with the present disclosure may be prepared by the hybridoma method first described by Kohler and Milstein, 1975, Nature 256:495, or may be prepared by recombinant DNA methods as described, for example, in U.S. Patent No. 4,816,567.
  • monoclonal antibodies may also be isolated from a phage library generated using the techniques described in McCafferty et al., 1990, Nature 348:552-554.
  • Fully human antibodies or “recombinant fully human antibodies” include fully human antibodies prepared, expressed, created or isolated by recombinant methods, and the techniques and methods involved are well known in the art, such as:
  • Antibodies prepared, expressed, created or isolated by methods such as splicing human immunoglobulin gene sequences to other DNA sequences.
  • Such recombinant fully human antibodies contain variable and constant regions that utilize specific human germline immunoglobulin sequences encoded by the germline genes, but also include subsequent rearrangements and mutations that occur, such as during antibody maturation.
  • murine antibody in the present disclosure refers to a monoclonal antibody against human MASP2 or its epitope prepared according to the knowledge and skills in the art. A hybridoma expressing an antibody having a desired sequence or functional property.
  • the murine anti-human anti-MASP2 antibody or antigen-binding fragment thereof may further comprise a light chain constant region of a murine ⁇ , ⁇ chain or a variant thereof, or further comprise a heavy chain constant region of a murine IgG1, IgG2, IgG3 or IgG4 or a variant thereof.
  • Fully human antibody includes antibodies with 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 (such as mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations 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 transplanted onto human framework sequences (i.e., "humanized antibodies”).
  • humanized antibody also known as CDR-grafted antibody, refers to an antibody produced by transplanting non-human CDR sequences into the human antibody variable region framework. This can overcome the strong immune response induced by chimeric antibodies due to the large amount of non-human protein components they carry. In order to avoid a decrease in activity while reducing immunogenicity, the variable region of the fully human antibody can be subjected to minimal reverse mutations to maintain activity.
  • chimeric antibody refers to an antibody formed by fusing the variable region of an antibody of a first species with the constant region of an antibody of a second species, which can reduce the immune response induced by the antibody of the first species.
  • a hybridoma that secretes mouse-specific monoclonal antibodies is selected, and then the variable region gene is cloned from the mouse hybridoma cells, and then the constant region gene of the fully human antibody is cloned as needed, and the mouse variable region gene and the human constant region gene are connected into a chimeric gene and inserted into a human vector, and finally the chimeric antibody molecule is expressed in a eukaryotic or prokaryotic industrial system.
  • the constant region of the fully human antibody can be selected from the heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or a variant thereof, preferably comprising a heavy chain constant region of human IgG2 or IgG4, or using IgG1 that is free of ADCC (antibody-dependent cell-mediated cytotoxicity) toxicity after amino acid mutation.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • Antigen-binding fragments include: single-chain antibodies (i.e., full-length heavy and light chains); Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, Fab-Fv, Fab-dsFv, single domain antibodies (e.g., 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, e.g., Holliger and Hudson, 2005, Nature Biotech.
  • Fab-Fv format 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 multispecifics such as bispecifics or may be monospecifics (see, e.g., WO92/22583 and WO05/113605), an example of the latter being Tri-Fab (or TFM) described in WO 92/22583.
  • binding to MASP2 refers to being able to interact with MASP2 or its epitope.
  • MASP2 or its epitope may be of human origin.
  • antigen binding site refers to a discrete three-dimensional site on an antigen that is recognized by the antibody or antigen-binding fragment of the present disclosure.
  • Antigen refers to a molecule used to immunize an immunocompetent vertebrate to generate antibodies that recognize the antigen, or to screen an expression library (e.g., a phage, yeast, or ribosome display library, among others).
  • an expression library e.g., a phage, yeast, or ribosome display library, among others.
  • antigens are defined more broadly to include target molecules that are specifically recognized by antibodies, as well as to include a portion or mimetic of a molecule used in an immunization process for generating antibodies or in a library screening process for selecting antibodies.
  • monomers and multimers e.g., dimers, trimers, etc.
  • antigens For antibodies that bind to human MASP2 of the present disclosure, monomers and multimers (e.g., dimers, trimers, etc.) of human MASP2, as well as truncated variants and other variants of human MASP2 are referred to as antigens.
  • epitope refers to a site on an antigen that binds to an immunoglobulin or antibody.
  • An epitope can be formed by adjacent amino acids, or non-adjacent amino acids juxtaposed by tertiary folding of a protein. Epitopes formed by adjacent amino acids are usually retained after exposure to a denaturing solvent, while epitopes formed by tertiary folding are usually lost after treatment with a denaturing solvent.
  • An epitope usually includes at least 3-15 amino acids in a unique spatial conformation. Methods for determining what epitope is bound by a given antibody are well known in the art, including immunoblotting and immunoprecipitation detection analysis, etc. Methods for determining the spatial conformation of an epitope include techniques in the art and the techniques described in the present disclosure, such as X-ray crystallography and two-dimensional nuclear magnetic resonance, etc.
  • Specific binding and “selective binding” refer to the binding of an antibody to an epitope on a predetermined antigen.
  • the antibody binds to the predetermined antigen or its epitope with an equilibrium dissociation constant ( KD ) of approximately less than 10-7 M or even less when measured in an instrument by surface plasmon resonance (SPR) technology, and its affinity for binding to the predetermined antigen or its epitope is at least twice its affinity for binding to nonspecific antigens (such as BSA, etc.) other than the predetermined antigen (or its epitope) or closely related antigens.
  • KD equilibrium dissociation constant
  • SPR surface plasmon resonance
  • the term "antibody that recognizes an antigen” can be used interchangeably with the term “specifically binding antibody” in the present disclosure.
  • Binding affinity or “affinity” is used in the present disclosure as a measure of the strength of a non-covalent interaction between two molecules (e.g., 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, the surface plasmon resonance (SPR) method (Biacore).
  • SPR surface plasmon resonance
  • the rate constants corresponding to the association and dissociation of a monovalent complex 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 for assessing the binding ability of an antibody to a target antigen are known in the art, including, 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 evaluated by standard assays known in the art, such as surface plasmon resonance (SPR), such as by using a Biacore TM system or KinExA. Binding affinities associated with different molecular interactions can be compared by comparing the K values of the individual antibody/antigen complexes. The specificity of an interaction can be evaluated by determining and comparing the KD value for an interaction of interest (e.g., a specific interaction between an antibody and an antigen) to the KD value for a non-interest interaction (e.g., a control antibody known not to bind MASP2).
  • SPR surface plasmon resonance
  • Binding affinities associated with different molecular interactions can be compared by comparing the K values of the individual antibody/antigen complexes.
  • the specificity of an interaction can be evaluated by determining and comparing the KD value for an interaction of interest (e.g., a specific interaction between an antibody and an antigen) to the KD value for
  • Constant substitution refers to substitution with another amino acid residue having a property similar to the original amino acid residue.
  • lysine, arginine and histidine have similar properties in that they have basic side chains
  • aspartic acid and glutamic acid have similar properties in that they have acidic side chains.
  • glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine and tryptophan have similar properties in that they have uncharged polar side chains
  • alanine, valine, leucine, threonine, isoleucine, proline, phenylalanine and methionine have similar properties in that they have non-polar side chains.
  • tyrosine, phenylalanine, tryptophan and histidine have similar properties in that they have aromatic side chains. Therefore, it will be apparent to those skilled in the art that even when replacing an amino acid residue in a group showing similar properties as described above, it will not show a specific change in properties.
  • Cross-reactivity refers to the ability of an antibody of the disclosure to bind to MASP2 from a different species.
  • an antibody of the disclosure that binds to human MASP2 may also bind to MASP2 from another species.
  • Cross-reactivity is measured by detecting specific reactivity with purified antigen in binding assays (e.g., SPR and ELISA), or binding or functional interaction with cells that physiologically express MASP2.
  • binding assays e.g., SPR and ELISA
  • Methods for determining cross-reactivity include standard binding assays as described in the disclosure, such as surface plasmon resonance analysis, or flow cytometry.
  • Inhibition or blocking 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 in the absence of inhibition or blocking. Inhibition and blocking are also intended to include any measurable reduction in MASP2 binding affinity when contacted with an anti-MASP2 antibody compared to MASP2 not contacted with the anti-MASP2 antibody.
  • Inhibiting growth (eg, involving cells) is intended to include any measurable decrease 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 using conventional methods.
  • Antigen-binding fragments can also be prepared using conventional methods.
  • the antibodies or antigen-binding fragments described in the present disclosure are genetically engineered to add one or more human FR regions to the non-human CDR regions. Human FR germline sequences can be obtained from the ImMunoGeneTics (IMGT) website.
  • the engineered antibodies or antigen-binding fragments disclosed herein can be prepared and purified by 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 expression systems can lead to glycosylation of antibodies, especially at the highly conserved N-terminus of the Fc region.
  • Stable clones are obtained by expressing antibodies that specifically bind to human antigens. Positive clones are expanded in serum-free culture medium in a bioreactor to produce antibodies.
  • the culture fluid that secretes antibodies can be purified and collected by conventional techniques.
  • the antibodies can be filtered and concentrated by conventional methods. Soluble mixed
  • the resulting product should be immediately frozen at -70°C or freeze-dried.
  • Antibodies can be competitively screened for binding to the same epitope using conventional techniques known to those skilled in the art. For example, competition and cross-competition studies can be performed to obtain antibodies that compete or cross-compete with each other for binding to the antigen. High-throughput methods for obtaining antibodies that bind to the same epitope based on their cross-competition are described in International Patent Publication WO03/48731. Thus, antibodies and antigen-binding fragments thereof that compete with the antibody molecules of the present disclosure for binding to the same epitope on MASP2 can be obtained using conventional techniques known to those skilled in the art.
  • administering when applied to an animal, a human, an experimental subject, a cell, a tissue, an organ, or a biological fluid, refers to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with an animal, a human, a subject, a cell, a tissue, an organ, or a biological fluid.
  • administering may refer to, for example, treatment, pharmacokinetics, diagnosis, research, and experimental procedures. Treatment of cells includes contact of an agent with a cell, and contact of an agent with a fluid, wherein the fluid is in contact with the cell.
  • administering also mean in vitro and ex vivo treatment of, for example, a cell, by an agent, a diagnosis, a combination composition, or by another cell.
  • Treatment when applied to humans, veterinary medicine, or a research subject, refers to therapeutic treatment, prophylactic or preventative 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 or conjugates disclosed herein, to a subject who has, is suspected of having, or is prone to having one or more diseases or symptoms thereof, and the therapeutic agent is known to have a therapeutic effect on these symptoms.
  • the therapeutic agent is administered in an amount effective to alleviate one or more disease symptoms in the treated subject or population, whether by inducing regression of such symptoms or inhibiting the development of such symptoms to any clinically measurable degree.
  • the amount of a therapeutic agent that is effective to alleviate any specific disease symptom may vary according to a variety of factors, such as the disease state, age, and weight of the subject, and the ability of the drug to produce the desired therapeutic effect in the subject. Whether the disease symptoms have been alleviated can be evaluated by any clinical detection method commonly used by doctors or other professional health care personnel to evaluate the severity or progression of the symptoms.
  • the embodiments of the present disclosure may not be effective in alleviating the symptoms of the target disease in a subject, they should alleviate the symptoms of the target disease in a statistically significant number of subjects as determined by any statistical test known in the art, such as Student's t-test, chi-square test, U test according to Mann and Whitney, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test.
  • any statistical test known in the art such as Student's t-test, chi-square test, U test according to Mann and Whitney, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test.
  • an "effective amount” includes an amount sufficient to improve or prevent the symptoms or conditions of a 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 to be treated, the subject's overall health, the method, route and dosage of administration, and the severity of side effects.
  • An effective amount can be the maximum dose or dosage 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. Similarity. When a position in two compared sequences is occupied by the same nucleotide or amino acid monomer subunit, for example, if every position of two DNA molecules is occupied by the same nucleotide, then the molecules are homologous at that position. The percent homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared x 100%. For example, when the sequences are optimally aligned, if 6 out of 10 positions in the two sequences are matched or homologous, then the two sequences are 60% homologous. In general, comparison is made when the two sequences are aligned to obtain the maximum percent homology.
  • Cell Cell
  • cell line cell line
  • cell culture all such designations include progeny. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or unintentional mutations. Mutant progeny that have the same function or biological activity as screened for in the originally 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 does not react with the subject's immune system. Examples include, but are not limited to, any standard pharmaceutical carrier, such as phosphate-buffered saline solution, 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.
  • PBS phosphate-buffered saline
  • Compositions containing such carriers are formulated by well-known conventional methods (see, e.g., Remington's Pharmaceutical Sciences, 18th ed., A. Gennaro, ed., Mack Publishing Co., Easton, PA, 1990; and R Remington, The Science and Practice of Pharmacy 20th ed. Mack Publishing, 2000).
  • MASP2 binding protein or "MASP2 binding molecule” disclosed herein is to be interpreted as broadly including the anti-MASP2 antibody or antigen-binding fragment thereof disclosed herein, and any protein that can bind to MASP2 is within the scope of the term.
  • the MASP2 binding protein (or binding molecule) may contain one or more effector molecules, for example, in a conjugated or fused manner.
  • effector molecule includes, for example, anti-tumor agents, drugs, toxins, biologically active proteins (such as enzymes), other antibodies or antibody fragments, synthetic or naturally occurring polymers, nucleic acids and fragments thereof such as DNA, RNA and fragments thereof, radionuclides (especially radioiodides), radioisotopes, chelated metals, nanoparticles and reporter groups (such as fluorescent compounds), or compounds that can be detected by NMR or ESR spectroscopy.
  • anti-tumor agents drugs, toxins, biologically active proteins (such as enzymes), other antibodies or antibody fragments, synthetic or naturally occurring polymers, nucleic acids and fragments thereof such as DNA, RNA and fragments thereof, radionuclides (especially radioiodides), radioisotopes, chelated metals, nanoparticles and reporter groups (such as fluorescent compounds), or compounds that can be detected by NMR or ESR spectroscopy.
  • 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 a branched polysaccharide or an unbranched polysaccharide, such as a homo- or hetero-polysaccharide.
  • Specific optional substituents that may be present on the above-mentioned synthetic polymers include one or more hydroxyl, methyl or methoxy groups.
  • polystyrene resin examples include optionally substituted linear or branched poly(ethylene glycol), poly(propylene glycol), poly(vinyl alcohol) or derivatives thereof, particularly optionally substituted poly(ethylene glycol) such as methoxypoly(ethylene glycol) or derivatives thereof.
  • polystyrene glycol examples include lactose, amylose, dextran, glycogen or derivatives thereof.
  • the polymer is albumin or a fragment thereof, such as human serum albumin or a fragment thereof.
  • the conjugation of the polymer to the anti-MASP2 antibody or antigen-binding fragment thereof of the present disclosure can be achieved by conventional methods.
  • “About” means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. In the context of a particular determination, result, or embodiment, unless otherwise expressly stated in the examples or elsewhere in the specification, "about” means a range within ⁇ 5% of a given value.
  • Buffer refers to a buffer that tolerates pH changes through the action of its acid-base conjugate components.
  • buffers that control pH in an appropriate range include Tris, acetate, succinate, gluconate, histidine, oxalate, lactate, phosphate, citrate, tartrate, fumarate, glycylglycine, and other organic acid buffers.
  • Hetidine salt buffer is a buffer containing histidine ions.
  • histidine salt buffers include histidine-hydrochloride, histidine-acetate, histidine-phosphate, histidine-sulfate and the like, preferably histidine-hydrochloride buffer or histidine-acetate buffer, histidine-acetate buffer is prepared from histidine and acetic acid, histidine-hydrochloride buffer is prepared from histidine and histidine hydrochloride, or histidine and hydrochloric acid.
  • Phosphate buffer is a buffer including phosphate ions.
  • phosphate buffers include disodium hydrogen phosphate-sodium dihydrogen phosphate, disodium hydrogen phosphate-potassium dihydrogen phosphate, disodium hydrogen phosphate-citric acid, and the like.
  • the phosphate buffer is disodium hydrogen phosphate-sodium dihydrogen phosphate.
  • Tris buffer is a buffer containing tris (Tris).
  • Tris buffer include Tris-hydrochloric acid buffer (Tris-HCl), Tris-acetic acid buffer (Tris-AA), Tris-succinic acid buffer (Tris-SA), or Tris-citrate buffer (Tris-CA).
  • citrate buffer is a buffer including citrate ions.
  • citrate buffers include citric acid-sodium citrate, citric acid-potassium citrate, citric acid-calcium citrate, citric acid-magnesium citrate, and the like.
  • a preferred citrate buffer is citric acid-sodium citrate.
  • succinate buffer is a buffer comprising succinate ions.
  • succinate buffers include succinic acid-succinic acid sodium salt, succinic acid-succinic acid potassium salt, succinic acid-succinic acid calcium salt, etc.
  • a preferred succinic acid buffer is succinic acid-succinic acid sodium salt.
  • the succinic acid-succinic acid sodium salt can be prepared from succinic acid and sodium hydroxide, or from succinic acid and succinic acid sodium salt.
  • Acetate buffer is a buffer including acetate ions.
  • acetate buffers include acetate-sodium acetate, histidine-histidine acetate, acetate-potassium acetate, acetate-calcium acetate, acetate-magnesium acetate, etc.
  • a preferred acetate buffer is acetate-sodium acetate.
  • “Pharmaceutical composition” means a mixture containing one or more antibodies described herein and other chemical components, such as physiologically/pharmaceutically acceptable carriers and excipients.
  • the purpose of a pharmaceutical composition is to maintain the stability of the active ingredient, promote administration to an organism, and facilitate the absorption of the active ingredient to exert biological activity.
  • composition and “formulation” are not mutually exclusive.
  • compositions described in the present disclosure are in the form of solutions, and unless otherwise specified, the solvent therein is water.
  • the composition is isotonic.
  • "Isotonic" means that the preparation has substantially the same osmotic pressure as human blood. Isotonicity can be determined by methods known in the art, for example, by using a vapor pressure or ice-type osmometer.
  • the osmotic pressure of the pharmaceutical composition is preferably controlled at 260-380 mOsm, for example, the osmotic pressure of the pharmaceutical preparation is controlled at 270-360 mOsm.
  • “Lyophilized preparation” refers to a pharmaceutical composition in liquid or solution form or a preparation or pharmaceutical composition obtained after a liquid or solution preparation has been subjected to a vacuum freeze-drying step.
  • An antibody "retains its physical stability" in a pharmaceutical formulation if it shows no significant increase in aggregation, precipitation, and/or denaturation as measured after visual inspection of color and/or clarity, or by UV light scattering, size exclusion chromatography (SEC), and dynamic light scattering (DLS). Changes in protein conformation can be assessed by fluorescence spectroscopy, which determines protein tertiary structure, and by FTIR spectroscopy, which determines protein secondary structure.
  • An antibody "retains its chemical stability" in a pharmaceutical formulation if it shows no significant chemical changes. Chemical stability can be assessed by detecting and quantifying chemically altered forms of the protein.
  • Degradation processes that often change the chemical structure of a protein include hydrolysis or truncation (assessed by methods such as size exclusion chromatography and CE-SDS), oxidation (assessed by methods such as peptide mapping in combination with mass spectrometry or MALDI/TOF/MS), deamidation (assessed by methods such as ion exchange chromatography, capillary isoelectric focusing, peptide mapping, isoaspartate measurement), and isomerization (assessed by measuring isoaspartate content, peptide mapping, etc.).
  • An antibody "retains its biological activity" in a pharmaceutical formulation if the biological activity of the antibody at a given time is within a predetermined range of the biological activity exhibited when the pharmaceutical formulation is prepared.
  • administering refers to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with an animal, human, subject, cell, tissue, organ, or biological fluid.
  • administering may refer to, for example, treatment, pharmacokinetics, diagnosis, research, and experimental procedures.
  • Treatment of cells includes contact of an agent with a cell, and contact of an agent with a fluid, wherein the fluid is in contact with the cell.
  • administering also mean in vitro and ex vivo treatment of, for example, a cell, by an agent, a diagnosis, a combination composition, or by another cell.
  • Treatment as it applies to humans, veterinary medicine, or research subjects, refers to therapeutic treatment, prophylactic or preventative measures, research and diagnostic applications.
  • Treatment means administering an internal or external therapeutic agent, such as a pharmaceutical composition comprising any of the present disclosure, to a patient who has one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect.
  • the therapeutic agent is administered in an amount effective to alleviate one or more symptoms of the disease in the patient or population being treated, to induce regression of such symptoms or to inhibit the development of such symptoms to any clinically measurable degree.
  • the amount of a therapeutic agent effective to alleviate any specific disease symptom may vary according to a variety of factors, such as the disease state, age, and weight of the patient, and the ability of the drug to produce the desired therapeutic effect in the patient.
  • Whether the disease symptoms have been alleviated can be evaluated by any clinical detection method commonly used by doctors or other professional health care personnel to evaluate the severity or progress of the symptoms.
  • the embodiments of the present disclosure e.g., treatment methods or products
  • SEC monomer content percentage A monomer/A total * 100% (A monomer is the peak area of the main peak monomer in the sample, and A total is the sum of all peak areas.)
  • a method of electrophoresis in which the gel is moved into a capillary tube as a supporting medium and the samples are separated according to their molecular weight at a certain voltage.
  • Non-reduced CE purity percentage A main peak / A total * 100% (A main peak is the peak area of the main peak in the sample, and A total is the sum of all peak areas.
  • main peak content percentage main peak area/total area*100% (the total area is the sum of the areas of the acidic peak, main peak and basic peak).
  • the instrument used for icIEF measurement is manufactured by Protein Simple, model is Muarice.
  • the freezing point method is used to determine osmotic pressure. It is based on the fact that the freezing point depression value is proportional to the molar concentration of the solution. It uses a highly sensitive temperature sensing element to measure the freezing point of the solution and convert the electricity into osmotic pressure. Instrument manufacturer: Loser, model OM819.
  • Protein concentration determination instrument UV-visible spectrophotometer, model: Nano Drop 2000, optical path length: 1 mm.
  • Tm Melting temperature
  • Tg aggregation temperature
  • Tm is the temperature at which 50% of the protein components are denatured during the heating process
  • Tagg is the temperature at which the protein aggregates during the heating process.
  • Measurement instrument High-throughput dynamic and static light scattering instrument, the instrument manufacturer is WYATT, model DynaPro plate Reader III
  • Step 1 Take a certain amount of purified anti-MASP2 antibody solution, perform solvent replacement (preferably ultrafiltration) with a buffer that does not contain antibodies, replace at least 6 times the volume through an ultrafiltration membrane, and continue to concentrate the antibody to a certain concentration. Add a certain volume of other auxiliary material mother solution, dilute with a buffer, make the antibody and each auxiliary material reach the required concentration, and mix well. After filtering the stock solution, take a sample for central control to detect bacterial endotoxins and microbial limits. Sterilize and filter the stock solution through a 0.22 ⁇ m filter element, and collect the filtrate.
  • solvent replacement preferably ultrafiltration
  • Step 2 Adjust the filling volume (target filling volume 1.70 mL), select medium borosilicate glass tube injection bottles (European anti-jump stopper) for filling, take samples at the beginning, middle and end of filling to detect the filling volume difference, and add butyl bromide coated rubber stoppers for injection.
  • target filling volume 1.70 mL
  • select medium borosilicate glass tube injection bottles European anti-jump stopper
  • Step 3 Turn on the capping machine, add aluminum caps, and start capping.
  • Step 4 Visual inspection to confirm that the product has no defects such as inaccurate filling quantity and poor appearance.
  • Figure 1A shows the activity test results of the anti-MASP2 antibody 77H11 disclosed in the present invention in the human 1% serum lectin pathway, using the hIgG4 isotype control (Isotype control) as the negative control and OMS721 as the positive control.
  • Figure 2A shows the results of the activity inhibition test of the 77H11 humanized antibody disclosed herein in 90% human serum lectin pathway, using hIgG4 as the isotype control (Isotype control) as the negative control and OMS721 as the positive control.
  • Figures 3A and 3B are graphs showing the inhibitory effect of 77H11 (H3L1) on the lectin pathway in 90% human serum, 90% monkey serum, and 90% mouse serum, respectively.
  • the hIgG4 isotype control (Isotype control) was used as the negative control, and OMS721 was used as the positive control.
  • FIG. 4A and FIG. 4B are graphs showing the results of detecting the inhibition of lectin pathway by 77H11 (H3L1) and OMS721 at doses of 3 mg/kg and 10 mg/kg in cynomolgus monkeys.
  • MASP2A human MASP2A
  • S632A mouse MASP2A
  • S633A monkey MASP2A
  • MASP2 (S632A) or MASP2 (S633A) is an active mutant form of MASP2 (J Biol Chem. 2013; 288(13): 8922-8934), the purpose of which is to reduce protein self-catalysis and improve protein stability.
  • the signal peptide was replaced, and the original signal peptide MRLLTLLGLLCGSVA (SEQ ID NO: 26) (see Uniprot website) was replaced with MEFGLSWLFLVAILKGVQC (SEQ ID NO: 27), and the above-mentioned 663rd or 662nd position is the position number based on natural counting in the original sequence.
  • the cell line was cultured, the supernatant was collected, and each recombinant protein was purified by affinity chromatography.
  • the amino acid sequence is as follows:
  • human MASP2 CCP1 human MASP2 CCP1-CCP2-SP
  • human MASP2 CCP2-SP human MASP2 CCP2-SP
  • CCP1-CCP2-SP (S633A) and CCP2-SP (S633A) were prepared by transforming the plasmid into E. coli BL21 strain, isolating and purifying the protein, and denaturing and renaturing
  • CCP1 was prepared by transiently transfecting CHO cells, expressing and isolating the protein.
  • the amino acid sequences of each MASP2 protein fragment are as follows:
  • Mouse immunization and phage display were used to screen and prepare MASP2-specific antibodies.
  • the human MASP2A protein prepared in Example 1 was used as an antigen to immunize balb/c mice, and the mice with the highest titer were selected for hybridoma cell fusion.
  • Hybridoma cell fusion and culture Take 3 culture dishes (10 cm) of myeloma cells passaged 24 hours ago, wash them once with RPMI-1640 medium without HEPES, resuspend them, and count them to 2-4 ⁇ 10 7.
  • mice immunized 72 hours later were killed, the spleens were aseptically removed, washed with RPMI-1640 medium without HEPES, the spleen cells were shredded, ground, blown away, filtered, centrifuged at 1000 rpm, 5 min, resuspended, and counted to 1-2 ⁇ 10 8.
  • the myeloma cells and spleen cells were mixed, centrifuged at 1000 rpm, 5 min, the supernatant was discarded, the precipitate was loosened, and preheated in a 40°C water bath. 1 mL of PEG preheated to 40°C was added dropwise to the centrifuge tube within 60 seconds, stirring gently while adding.
  • HAT-OPI medium RPMI-1640 medium containing 20% FBS, containing 1 ⁇ HAT and 1 ⁇ OPI
  • HAT-OPI RPMI-1640 medium containing 20% FBS, containing 1 ⁇ HAT and 1 ⁇ OPI
  • the ELISA detection method is as follows: 1ug/mL antigen was coated overnight at 4°C, 50 ⁇ L per well. Wash the plate 3 times with PBS, and then block it with PBS containing 3% BSA at room temperature for 1h. Wash the plate 3 times with PBST, add hybridoma cell supernatant, and incubate at room temperature for 1h. Wash the plate 3 times with PBST, and then wash it 3 times with PBS, and then add secondary antibody (1:2000; invitrogen, goat anti-mouse IgG (H+L) secondary antibody, 31430) and incubate at room temperature for 1h. Wash the plate 3 times with PBST, and then wash it 3 times with PBS, and then add TMB substrate and incubate at room temperature for 10min, and then terminate the reaction to detect the signal value (450nm light absorption value).
  • the human MASP2A antigen was coated on an immunoadsorption plate and incubated with a phage antibody synthesis library for solid phase screening. After three rounds of panning, human MASP2 and monkey MASP2 positive phages were obtained.
  • the method for phage display screening of positive clones is as follows: 4 mL MASP2A (5 ng/ ⁇ L concentration) was coated on the immunotube and incubated at 4°C overnight. The fully human phage library was blocked with 5% BSA/PBS at room temperature for 1 hour. The phage library was transferred to the immunotube coated with MASP2A, rotated at room temperature for 1 hour, washed 5 times with PBS, eluted with 1000 ⁇ L TEA, and neutralized with 400 ⁇ L Tris-HCl (pH 7.4). Infect 10 mL TG1 (OD value to 0.4), 37°C, 40 min. Measure output, apply Amp+ plate, 30°C overnight.
  • Centrifuge at 4000rpm for 30min collect the supernatant, add 10mL PEG/NaCl, precipitate on ice for 1h, centrifuge at 4000rpm for 30min, add 1mL PBS to the precipitate and resuspend. Centrifuge at 13000rpm for 3min, remove the precipitate. Resuspend the phage with PBS and enter the next round of selection. Inoculate the monoclonal clone to a 96-well plate, 37°C for 3h, 220rpm. Add 1mM IPTG to induce overnight at 30°C. At the same time, coat MASP2 on a 96-well plate at a concentration of 2ng/ ⁇ L, 50 ⁇ L per well, overnight at 4°C.
  • VH heavy chain variable region
  • VL light chain variable region
  • the obtained anti-MASP2 antibody was functionally verified to inhibit the activity of lectin pathway in vitro using 1% human serum.
  • the functional activity of the antibody in 1% human serum was determined by coating a 384-well plate with 50 ⁇ g/mL mannan solution at 25 ⁇ L/well and incubating overnight at 4°C. Wash the plate 4 times with 50 ⁇ L/well TBST, block the plate with 50 ⁇ L/well 3% BSA blocking solution (solvent is TBS), and incubate at room temperature for 2 hours. Wash the plate 4 times with 50 ⁇ L/well TBST. Mix the anti-MASP2 antibody of the corresponding concentration with 1% human serum, incubate at 4°C for 1 hour, add the mixture to the aforementioned 384-well plate at 15 ⁇ L/well, and incubate at 37°C for 1 hour.
  • wash the plate 4 times with 50 ⁇ L/well TBST add the primary antibody (biotin-human Anti-C4c, Agrisera #IMSO1-031-305) diluted with 0.5% BSA, 15 ⁇ L/well, centrifuge at 800 g/min, and incubate at room temperature for 1 hour. Wash the plate 4 times with 50 ⁇ L/well TBST, add 15 ⁇ L/well of secondary antibody (SA-HRP) diluted 1:5000 with 0.5% BSA solution, incubate at room temperature for 30 min. Wash the plate 4 times with 50 ⁇ L/well TBST, add 45 ⁇ L/well TMB colorimetric solution, incubate at room temperature for 15 min in the dark, and finally add 45 ⁇ L/well stop solution to detect OD450 value.
  • SA-HRP secondary antibody
  • the positive control antibody OMS721 was synthesized according to WO2012151481A, and the sequence is as follows:
  • the results are shown in Figure 1A and Table 2, which show that 77H11 can significantly inhibit the lectin pathway, which is better than the positive control OMS721.
  • the antibody 77H11 here is a full-length anti-MASP2 antibody constructed by connecting the antibody heavy chain variable region to the human IgG4 heavy chain Fc region, and the human IgG4 heavy chain Fc is shown in SEQ ID NO: 19.
  • the hIgG4 used is the isotype control (Isotype control).
  • the anti-MASP2 antibody 77H11 was selected for humanization and then its activity was identified in human serum.
  • the humanized template for the light chain of 77H11 is the human germline gene IGKV1-27*01, and the humanized template for the heavy chain is the human germline gene IGKV4-30-4*01.
  • Several back mutations were performed on both chains. Eight molecules were obtained, and their humanized sequences are shown below (underlined heavy chain or light chain CDR):
  • the antibodies in Table 3 are all full-length anti-MASP2 antibodies constructed by connecting the antibody heavy chain variable region to the human IgG4 heavy chain Fc region.
  • the heavy chain Fc region includes a hinge region, which is a human IgG4 Fc region with an S228P mutation, and the sequence is shown in SEQ ID NO: 23.
  • the S228P is based on EU numbering.
  • anti-MASP2 antibody heavy chain full length and light chain full length are shown below:
  • the antibody was expressed and purified according to conventional methods, and the full-length antibody disclosed in the present invention was obtained after testing.
  • the humanized antibodies were tested for their effects on the activity of the lectin pathway in 90% human serum, which is closer to in vivo conditions.
  • the functional activity of the antibodies in 90% human serum was identified by coating a 384-well plate with 25 ⁇ L/well of sodium carbonate-sodium bicarbonate buffer (pH>9) containing 5 ⁇ g/mL mannan solution, and incubating overnight at 4°C. Wash three times with 50 ⁇ L/well of TBST with 5 mM Ca 2+ . Block at room temperature for 1.5 to 2 hours with 50 ⁇ L/well of 3% BSA blocking solution (solvent is TBS, with 5 mM Ca 2+ added).
  • the humanized molecule of 77H11 can significantly inhibit the activity of the lectin pathway in 90% of human serum, and its efficacy is better than that of OMS721.
  • the 77H11 series antibodies here are all full-length anti-MASP2 antibodies constructed by connecting the heavy chain variable region of the antibody to the human IgG4 heavy chain Fc region, and the human IgG4 heavy chain Fc is shown in SEQ ID NO: 53.
  • the hIgG4 used is the isotype control (Isotype control).
  • the binding properties of anti-MASP2 antibodies were detected using the Biacore method.
  • the affinity of the 77H11 (H3L1) antibody molecule to human MASP2, mouse MASP2, and monkey MASP2 proteins was determined using a Biacore 8K (GE) instrument, using an isotype IgG antibody as a negative control.
  • the antibody was captured using an anti-human Fc IgG capture chip, and then different concentrations of antigen were used as the mobile phase for detection. Finally, the curve was fitted in a 1:1 mode to obtain the affinity value. The results are shown in Table 5.
  • anti-MASP2 antibodies The inhibitory effects of anti-MASP2 antibodies on the activity of lectin pathway in 90% serum of human, monkey and mouse were detected respectively.
  • the method for identifying the functional activity of the antibody in 90% human, monkey, and mouse serum is as follows: Sodium carbonate-sodium bicarbonate buffer (pH>9) containing 5 ⁇ g/mL mannan solution is coated on a 384-well plate at 25 ⁇ L/well and incubated overnight at 4°C. Wash three times with 50 ⁇ L/well of TBST with 5mM Ca 2+ added. Block with 50 ⁇ L/well of 3% BSA blocking solution (solvent is TBS, add 5mM Ca 2+ ) at room temperature for 1.5-2h. Wash three times with 50 ⁇ L/well of TBST, wash once with 50 ⁇ L/well of TBS, and wash once with 50 ⁇ L/well of VBS.
  • SA-HRP secondary antibody
  • 77H11 (H3L1) and OMS721 were injected into crab-eating macaques at doses of 3 mg/kg and 10 mg/kg, respectively, by intravenous push. Blood was collected at different times to collect serum. The lectin pathway activity of crab-eating macaque serum collected at different time points was detected. The specific blood collection time was 0 hour, 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: collect blood samples from peripheral veins and inject them directly into blank blood collection tubes. The blood sample is allowed to stand for 15-60 minutes until the blood coagulates, and then centrifuged at 4°C, 2500g for 10 minutes. The supernatant is the serum.
  • test drugs 77H11 (H3L1) and OMS721 were injected intravenously, and the dosage of each drug was 3 mg/kg or 10 mg/kg.
  • the blood was collected at 15 min, 1 h, 4 h, 8 h, 24 h, 48 h, 72 h, 96 h, 168 h, 240 h, 336 h, 408 h, 504 h, and 672 h after administration.
  • 0.3 mL of whole blood was collected each time without adding anticoagulant. After blood collection, 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.
  • the use and welfare of experimental animals in this disclosure are carried out in accordance with the regulations of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC).
  • AALAC Association for Assessment and Accreditation of Laboratory Animal Care
  • the health status and mortality of animals are monitored daily, and routine inspections include observing the effects of test substances and drugs on the daily behavior of animals, such as behavioral activities, weight changes, physical signs, etc.
  • a 10 mM phosphate buffer system was selected, and 12 different pH values were designed as follows: 4.6, 5.0, 5.4, 5.8, 6.2, 6.6, 7.0, 7.4, 7.8, 8.2, 8.6, and 9.0, to prepare an anti-MASP2 antibody (the antibody number is 77H11 (H3L1), the heavy chain and the light chain are as shown in SEQ ID NO: 24 and 25, prepared in Example 4, the same below) with a concentration of 10.0 mg/mL.
  • the melting temperature (Tm) of the sample was measured, and the thermal stability of the antibody under different pH systems was investigated.
  • the aggregation temperature (Tagg) and particle size of the sample were measured, and the colloidal stability of the antibody under different pH systems was investigated. Sequence numbers 1) to 12) are as follows:
  • the anti-MASP2 antibody had the highest Tm value in the pH 7.0 system, followed by the pH 6.6 system, indicating that the antibody had good thermal stability under the conditions of pH 6.6-7.0; the anti-MASP2 antibody had the highest Tagg value in the pH 6.6 system, followed by the pH 7.0 system.
  • the particle size was smaller in the pH 6.6 system, and the particle size distribution was more uniform, indicating that the antibody had good colloidal stability under the conditions of pH 6.6-7.0. Therefore, the anti-MASP2 antibody had good thermal stability and colloidal stability under the conditions of pH 6.6-7.0.
  • a 10 mM sodium dihydrogen phosphate-disodium hydrogen phosphate, 10 mM histidine-histidine hydrochloride buffer system was selected to prepare an antibody preparation with a pH of 6.6 and an anti-MASP2 antibody content of 50 mg/mL.
  • the buffer system pH 6.6
  • Sequence numbers 1) to 2) are as follows:
  • a 10 mM sodium dihydrogen phosphate-disodium hydrogen phosphate, 10 mM histidine-histidine hydrochloride buffer system was selected to prepare an antibody preparation with a pH of 7.0 and an anti-MASP2 antibody content of 50 mg/mL, and samples were taken for high temperature 40°C stability study.
  • Serial numbers 1) to 2) are as follows:
  • the anti-MASP2 antibody had higher Tm value and Tagg value in the 10mM histidine-histidine hydrochloride pH 7.0 buffer system, better appearance, smaller particle size, and relatively lower aggregate content. Therefore, the anti-MASP2 antibody had better stability in the 10mM histidine-histidine hydrochloride pH 7.0 buffer system.
  • Sucrose (the sucrose content was selected to be 75 mg/mL) was selected as an auxiliary material, and a 10 mM histidine-histidine hydrochloride pH 6.8 buffer system was used to prepare antibody preparations containing 100 mg/mL anti-MASP2 antibody, 75 mg/mL sucrose, and different polysorbate 80 concentrations (0.1 mg/mL, 0.4 mg/mL), and the protein stability was investigated under shaking (200 rpm, room temperature) and freeze-thaw (-35°C/room temperature) conditions.
  • Serial numbers 1) to 2) are as follows:
  • the concentration range of polysorbate 80 in the anti-MASP2 antibody preparation was set at 0.1 mg/mL to 0.4 mg/mL.
  • a 10 mM histidine-histidine hydrochloride pH 6.8 buffer system was selected to prepare antibody preparations containing 100 mg/mL anti-MASP2 antibody, different types of excipients (75 mg/mL sucrose, 45 mg/mL mannitol, 8.2 mg/mL sodium chloride, 28 mg/mL proline), and 0.2 mg/mL polysorbate 80, and the protein stability at high temperature 40°C was investigated.
  • Serial numbers 1) to 4) are as follows:
  • a 10 mM histidine-histidine hydrochloride buffer system was selected to prepare antibody preparations containing 8.2 mg/mL sodium chloride, 0.2 mg/mL polysorbate 80, 110 mg/mL anti-MASP2 antibody, and different pH values (pH 6.4, pH 6.6, and pH 6.8), and the stability of the antibody preparations at high temperature of 40°C was investigated.
  • Sequence numbers 1) to 3) are as follows:
  • a 10 mM histidine-histidine hydrochloride pH 6.6 buffer system was selected to prepare an anti-MASP2 antibody with a protein concentration of 200 mg/mL, different types of excipients (40 mg/mL sucrose + 15 mg/mL arginine hydrochloride, 30 mg/mL arginine hydrochloride, 8.0 mg/mL sodium chloride), and 0.4 mg/mL polysorbate 80.
  • the protein stability under freezing conditions was investigated. Sequence numbers 1) to 3) are as follows:
  • a 165 mg/mL anti-MASP2 antibody was prepared to investigate the protein stability under conditions of high temperature (40°C), shaking (200 rpm, room temperature), illumination (5000 ⁇ 500lx), and freeze-thaw (-35°C/room temperature).
  • the preparation is as in No. 1):
  • the iCIEF purity and SEC purity results of the preparation with a protein concentration of 150 mg/mL (10 mM histidine-histidine hydrochloride, 0.4 mg/mL polysorbate 80, 40 mg/mL sucrose + 15 mg/mL arginine hydrochloride, protein concentration of 150 mg/mL, pH 6.6) at high temperature (40°C, 2 weeks) showed that the preparation had good stability.
  • the present invention provides an anti-MASP2 antibody pharmaceutical preparation with a formulation of "90-165 mg/mL anti-MASP2 antibody, about 8.2 mg/mL sodium chloride or 40 mg/mL sucrose, 15 mg/mL arginine hydrochloride, 0.1-0.4 mg/mL polysorbate 80, about 10 mM histidine-histidine hydrochloride, pH 6.4-6.8", including but not limited to:

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Abstract

本公开涉及一种抗MASP2抗体的组合物及医药用途。具体而言，本公开涉及一种抗MASP2抗体或其抗原结合片段的药物组合物，该药物组合物包含抗MASP2抗体或其抗原结合片段和缓冲剂。

Description

一种抗MASP2抗体的组合物及医药用途 技术领域
本公开涉及药物制剂领域，具体涉及一种包含抗MASP2抗体或其抗原结合片段的药物组合物，及其药物用途。
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抗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。
本申请人的专利申请WO2022228364提供了一种新结构的抗MASP2抗体，其对MASP2依赖性补体活化具有较好的抑制活性。
抗体药物是一类重要的生物药物，其分子量大，结构复杂，在生产、储存、使用过程中容易受到物理或化学方面的影响而发生降解、聚合，导致活性降低甚至失效。因此，研制优秀的抗体制剂是十分重要的。
发明内容
本公开提供了一种含抗MASP2抗体或其抗原结合片段的药物组合物，该组合物具有优异的稳定性。
本公开提供了一种药物组合物，其包含抗MASP2抗体或其抗原结合片段和缓冲剂，其中所述缓冲剂选自三羟甲基氨基甲烷(Tris)缓冲剂、醋酸盐缓冲剂、琥珀酸盐缓冲剂、磷酸盐缓冲剂、组氨酸盐缓冲剂、醋酸盐缓冲剂、枸橼酸盐缓冲剂、酒石酸盐缓冲剂、延胡索酸盐缓冲剂、甘氨酰甘氨酸缓冲剂。在某些实施方案中，其中所述缓冲剂选自磷酸盐缓冲剂、组氨酸盐缓冲剂。在某些实施方案中，所述磷酸盐缓冲剂选自磷酸二氢钠-磷酸氢二钠；组氨酸盐缓冲剂选自组氨酸-盐酸缓冲剂或组氨酸-醋酸缓冲剂。在某些实施方案中，所述缓冲剂为组氨酸盐 缓冲剂。在某些实施方案中，所述缓冲剂为组氨酸-盐酸缓冲剂。在某些实施方案中，所述缓冲剂为组氨酸-盐酸组氨酸缓冲剂。
在某些实施方案中，如上任一项所述药物组合物，其中所述缓冲剂的pH为5.0-8.5，例如为5.5-8.0、6.0-8.0、7.0-8.0、6.0-7.0、5.5-7.5、6.5-8.0、6.4-6.8，非限制性实施例包括约5.5、约5.6、约5.7、约5.8、约5.9、约6.0、约6.1、约6.2、约6.3、约6.4、约6.5、约6.6、约6.7、约6.8、约6.9、约7.0、约7.1、约7.2、约7.3、约7.4、约7.5或任意两者数之间任意值。在某些实施方案中，所述的缓冲剂的pH为6.0-7.5。在某些实施方案中，所述药物组合物的pH相比其含有的缓冲剂的pH具有不超过±0.5的差异。
在某些实施方案中，如上任一项所述药物组合物，其pH为5.0-8.5，例如为约5.5-8.0、6.0-8.0、7.0-8.0、6.0-7.0、5.5-7.5、6.5-8.0、6.4-6.8，非限制性实施例包括约5.5、约5.6、约5.7、约5.8、约5.9、约6.0、约6.1、约6.2、约6.3、约6.4、约6.5、约6.6、约6.7、约6.8、约6.9、约7.0、约7.1、约7.2、约7.3、约7.4、约7.5或任意两者数之间任意值。
在某些实施方案中，如上任一项所述药物组合物，其中所述缓冲剂浓度为0.1-50mM，例如为1-30mM、5-20mM、8-12mM，非限制性实施例包括约5mM、约6mM、约7mM、约8mM、约9mM、约10mM、约11mM、约12mM、约13mM、约14mM、约15mM、约16mM、约17mM、约18mM、约19mM、约20mM或任意两者数之间任意值。在某些实施方案中，所述缓冲剂浓度为约10mM。
在某些实施方案中，如上任一项所述的药物组合物，其进一步包含辅料。在某些实施方案中，所述辅料选自氨基酸或其盐、糖、多元醇和盐中的一种或多种。在某些实施方案中，所述辅料选自氨基酸或其盐、糖和盐中的一种或多种。在某些实施方案中，所述辅料为盐。在某些实施方案中，所述辅料为氨基酸或其盐。在某些实施方案中，所述辅料为糖和氨基酸或其盐。在某些实施方案中，所述氨基酸选自脯氨酸、天冬氨酸、谷氨酸、赖氨酸、精氨酸、甘氨酸和组氨酸中的一种或多种。在某些实施方案中，所述糖选自葡萄糖、蔗糖、麦芽糖和海藻糖中的一种或多种。在某些实施方案中，所述多元醇选自甘露醇和山梨醇中的一种或多种。在某些实施方案中，所述的盐选自氯化钠或氨基酸的盐，例如为氯化钠。在某些实施方案中，所述辅料选自氯化钠，蔗糖和精氨酸或其盐中的一种或多种。在某些实施方案中，所述辅料为氯化钠。在某些实施方案中，所述辅料为精氨酸或其盐，例如盐酸精氨酸。在某些实施方案中，所述辅料为蔗糖和精氨酸或其盐，例如蔗糖和盐酸精氨酸。
在某些实施方案中，如上任一项所述的药物组合物，所述辅料的浓度为0.1-100mg/mL，例如为0.5-100mg/mL、0.5-80mg/mL、1-80mg/mL、1-70mg/mL、1-60mg/mL、1-50mg/mL、1-40mg/mL、1-30mg/mL、1-20mg/mL、5-80 mg/mL、5-70mg/mL、5-60mg/mL、5-50mg/mL、5-40mg/mL、5-30mg/mL、5-20mg/mL、10-70mg/mL、10-60mg/mL、10-50mg/mL、10-40mg/mL、10-30mg/mL、10-20mg/mL、20-60mg/mL、20-50mg/mL、20-40mg/mL、20-30mg/mL、30-50mg/mL、20-40mg/mL、8-9mg/mL，非限制性实施例包括约1mg/mL、约2mg/mL、约3mg/mL、约4mg/mL、约5mg/mL、约6mg/mL、约7mg/mL、约7.2mg/mL、约7.4mg/mL、约7.6mg/mL、约7.8mg/mL、约8mg/mL、约8.2mg/mL、约8.4mg/mL、约8.6mg/mL、约8.8mg/mL、约9mg/mL、约10mg/mL、约11mg/mL、约12mg/mL、约13mg/mL、约14mg/mL、约15mg/mL、约16mg/mL、约17mg/mL、约18mg/mL、约19mg/mL、约20mg/mL、约21mg/mL、约22mg/mL、约23mg/mL、约24mg/mL、约25mg/mL、约26mg/mL、约27mg/mL、约28mg/mL、约29mg/mL、约30mg/mL、约31mg/mL、约32mg/mL、约33mg/mL、约34mg/mL、约35mg/mL、约36mg/mL、约37mg/mL、约38mg/mL、约39mg/mL、约40mg/mL、约41mg/mL、约42mg/mL、约43mg/mL、约44mg/mL、约45mg/mL、约46mg/mL、约47mg/mL、约48mg/mL、约49mg/mL、约50mg/mL、约55mg/mL、约60mg/mL、约65mg/mL、约70mg/mL、约75mg/mL、约80mg/mL或任意两者数之间任意值。在某些实施方案中，所述辅料的浓度为约8.2mg/mL、约15mg/mL、约28mg/mL、约30mg/mL、约40mg/mL、约45mg/mL和约75mg/mL。在某些实施方案中，所述辅料的浓度为约5-10mg/mL。在某些实施方案中，所述辅料为约8.2mg/mL氯化钠。在某些实施方案中，所述辅料为约15mg/mL盐酸精氨酸。在某些实施方案中，所述辅料为约15mg/mL盐酸精氨酸和40mg/mL蔗糖。
在某些实施方案中，如上任一项所述的药物组合物为等渗制剂。在一些实施方案中，所述药物组合物的渗透压为260-380mOsm，例如270-360mOsm，非限制性实施例包括约270mOsm、约272mOsm、约274mOsm、约276mOsm、约278mOsm、约280mOsm、约282mOsm、约284mOsm、约286mOsm、约288mOsm、约290mOsm、约292mOsm、约294mOsm、约296mOsm、约298mOsm、约300mOsm、约302mOsm、约304mOsm、约306mOsm、约308mOsm、约310mOsm、约312mOsm、约314mOsm、约316mOsm、约318mOsm、约320mOsm、约322mOsm、约324mOsm、约326mOsm、约328mOsm、约330mOsm、约335mOsm、约340mOsm、约345mOsm、约350mOsm、约355mOsm、约360mOsm或任意两者数之间任意值。在一些实施方案中，所述药物组合物的渗透压为270-360mOsm。
在某些实施方案中，本公开药物组合物还包含表面活性剂。在某些实施方案中，所述表面活性剂为离子或非离子表面活性剂。在某些实施方案中，所述表面活性剂选自聚山梨酯、聚羟亚烃、Triton、十二烷基磺酸钠、月桂基磺酸钠、辛 基糖甙钠、月桂基-磺基甜菜碱、肉豆蔻基-磺基甜菜碱、亚油基-磺基甜菜碱、硬脂基-磺基甜菜碱、月桂基-肌氨酸、肉豆蔻基-肌氨酸、亚油基-肌氨酸、硬脂基-肌氨酸、亚油基-甜菜碱、肉豆蔻基-甜菜碱、鲸蜡基-甜菜碱、月桂酰胺基丙基-甜菜碱、柯卡酰胺基丙基-甜菜碱、亚油酰胺基丙基-甜菜碱、肉豆蔻酰胺基丙基-甜菜碱、棕榈酰胺基丙基-甜菜碱、异硬脂酰胺基丙基-甜菜碱、肉豆蔻酰胺基丙基-二甲基胺、棕榈酰胺基丙基-二甲基胺、异硬脂酰胺基丙基-二甲基胺、甲基可可酰基钠、甲基油基牛磺酸钠、聚乙二醇、聚丙二醇和乙烯与丙烯二醇的共聚物中的一种或多种。在某些实施方案中，所述的表面活性剂为聚山梨醇。在某些实施方案中，所述的表面活性剂为聚山梨醇酯20或聚山梨醇酯80，例如为聚山梨醇酯80。
在某些实施方案中，药物组合物中所述的表面活性剂浓度为0.01-2mg/mL，例如为0.05-1mg/mL，非限制性实施例包括约0.05mg/mL、约0.1mg/mL、约0.15mg/mL、约0.2mg/mL、约0.25mg/mL、约0.3mg/mL、约0.35mg/mL、约0.4mg/mL、约0.45mg/mL、约0.5mg/mL、约0.55mg/mL、约0.6mg/mL、约0.65mg/mL、约0.7mg/mL、约0.75mg/mL、约0.8mg/mL或任意两者数之间任意值。在某些实施方案中，所述表面活性剂浓度为0.1-0.4mg/mL。在某些实施方案中，所述表面活性剂浓度为约0.1mg/mL、约0.2mg/mL、约0.3mg/mL或约0.4mg/mL。
在某些实施方案中，如上任一项所述的药物组合物，其中所述抗MASP2抗体或其抗原结合片段的浓度为0.1-500mg/mL，例如0.1-300mg/mL、5-300mg/mL、5-150mg/mL、20-200mg/mL、50-250mg/mL、80-120mg/mL、70-130mg/mL、60-140mg/mL、90-200mg/mL、90-165mg/mL、50-150mg/mL，非限制性实施例包括约5mg/mL、约6mg/mL、约7mg/mL、约8mg/mL、约9mg/mL、约10mg/mL、约15mg/mL、约20mg/mL、约25mg/mL、约30mg/mL、约35mg/mL、约40mg/mL、约45mg/mL、约50mg/mL、约55mg/mL、约60mg/mL、约65mg/mL、约70mg/mL、约75mg/mL、约80mg/mL、约85mg/mL、约90mg/mL、约95mg/mL、约100mg/mL、约105mg/mL、约110mg/mL、约115mg/mL、约120mg/mL、约125mg/mL、约130mg/mL、约135mg/mL、约140mg/mL、约145mg/mL、约150mg/mL、约155mg/mL、约160mg/mL、约165mg/mL、约170mg/mL、约175mg/mL、约180mg/mL、约185mg/mL、约190mg/mL、约195mg/mL、约200mg/mL或任意两者数之间任意值。在某些实施方案中，所述抗MASP2抗体或其抗原结合片段的浓度为约10mg/mL、约50mg/mL、约90mg/mL、约100mg/mL或约110mg/mL、约135mg/mL、约150mg/mL、约165mg/mL或约200mg/mL。在某些实施方案中，所述抗MASP2抗体或其抗原结合片段的浓度为约90-110mg/mL或90-165mg/mL。在某些实施方案中，所述抗MASP2抗体或其抗原结合片段的浓 度为约90mg/mL、约100mg/mL、约110mg/mL、约135mg/mL、约150mg/mL、约165mg/mL或约200mg/mL。
在某些实施方案中，本公开提供一种包含抗MASP2抗体或其抗原结合片段(例如重链、轻链序列为SEQ ID NO:24和25的77H11(H3L1))的药物组合物，其包含或为如下1)至21)任意一组：
1)抗MASP2抗体或其抗原结合片段；
氯化钠、甘露醇和/或氨基酸(例如脯氨酸)；
组氨酸-盐酸缓冲剂(例如组氨酸-盐酸组氨酸缓冲剂)；
聚山梨醇(例如聚山梨醇酯80)；
pH为5.5-8.0；
2)0.1-300mg/mL的抗MASP2抗体或其抗原结合片段；
0.5-80mg/mL氯化钠、甘露醇和/或氨基酸(例如脯氨酸)；
5-30mM组氨酸-盐酸缓冲剂(例如组氨酸-盐酸组氨酸缓冲剂)；
0.01-1mg/mL聚山梨醇(例如聚山梨醇酯80)；
pH为5.5-8.0；
3)0.1-300mg/mL的抗MASP2抗体或其抗原结合片段；
2-50mg/mL的氯化钠；
5-20mM的组氨酸-盐酸缓冲剂(例如组氨酸-盐酸组氨酸缓冲剂)；
0.05-1mg/mL的聚山梨醇(例如聚山梨醇酯80)；
pH为5.5-8.0；
4)50-150mg/mL的抗MASP2抗体或其抗原结合片段；
2-30mg/mL的氯化钠；
2-20mM的组氨酸-盐酸缓冲剂(例如组氨酸-盐酸组氨酸缓冲剂)；
0.05-0.8mg/mL的聚山梨醇(例如聚山梨醇酯80)；
pH为5.5-7.5；
5)80-120mg/mL的抗MASP2抗体或其抗原结合片段；
5-20mg/mL的氯化钠；
5-15mM的组氨酸-盐酸缓冲剂(例如组氨酸-盐酸组氨酸缓冲剂)；
0.05-0.6mg/mL的聚山梨醇(例如聚山梨醇酯80)；
pH为5.5-7.5或6.0-7.5；
6)90-110mg/mL的抗MASP2抗体或其抗原结合片段；
8-9mg/mL的氯化钠；
8-12mM的组氨酸-盐酸缓冲剂(例如组氨酸-盐酸组氨酸缓冲剂)；
0.05-0.5mg/mL的聚山梨醇(例如聚山梨醇酯80)；
pH为5.5-7.5或6.0-7.5；
7)90-110mg/mL的抗MASP2抗体或其抗原结合片段；
8-9(例如约8.2)mg/mL的氯化钠；
约10mM的组氨酸-盐酸缓冲剂(例如组氨酸-盐酸组氨酸缓冲剂)；
约0.1-0.4mg/mL的聚山梨醇(例如聚山梨醇酯80)；
pH为6.1-7.1(例如约6.4、约6.5、约6.6、约6.7、约6.8、约6.9、约7.0)；
8)约90mg/mL、约95mg/mL、约100mg/mL、约105mg/mL或约110mg/mL的抗MASP2抗体或其抗原结合片段；
约8.2mg/mL的氯化钠；
约10mM的组氨酸-盐酸缓冲剂(例如组氨酸-盐酸组氨酸缓冲剂)；
约0.1、约0.2或约0.4mg/mL的聚山梨醇(例如聚山梨醇酯80)；
pH为约6.4、约6.6或约6.8；
9)抗MASP2抗体或其抗原结合片段；
组氨酸-盐酸缓冲剂(例如组氨酸-盐酸组氨酸缓冲剂)；
pH为5.5-8.0(例如5.5-7.5、6.0-7.5、6.1-7.1、约6.4、约6.6或约6.8)；
10)抗MASP2抗体或其抗原结合片段；
氯化钠，或者蔗糖和精氨酸或其盐(例如盐酸精氨酸)；
11)抗MASP2抗体或其抗原结合片段；
氯化钠，或者蔗糖和精氨酸或其盐(例如盐酸精氨酸)；
组氨酸-盐酸缓冲剂(例如组氨酸-盐酸组氨酸缓冲剂)；
聚山梨醇(例如聚山梨醇酯80)；
pH为5.5-8.0(例如5.5-7.5、6.0-7.5、6.1-7.1、约6.4、约6.6或约6.8)；
12)0.1-500mg/mL的抗MASP2抗体或其抗原结合片段；
0.5-100mg/mL的蔗糖；
0.5-80mg/mL精氨酸或其盐(例如盐酸精氨酸)；
0.1-50mM的组氨酸-盐酸缓冲剂；
0.01-1mg/mL的聚山梨醇(例如聚山梨醇酯80)；
pH为5.5-8.0；
13)5-300mg/mL的抗MASP2抗体或其抗原结合片段；
5-80mg/mL的蔗糖；
1-60mg/mL精氨酸或其盐(例如盐酸精氨酸)；
1-30mM的组氨酸-盐酸缓冲剂；
0.05-0.6mg/mL的聚山梨醇(例如聚山梨醇酯80)；
pH为5.5-7.5；
14)50-250mg/mL的抗MASP2抗体或其抗原结合片段；
10-70mg/mL的蔗糖；
5-30mg/mL精氨酸或其盐(例如盐酸精氨酸)；
5-20mM的组氨酸-盐酸缓冲剂；
0.05-0.5mg/mL的聚山梨醇(例如聚山梨醇酯80)；
pH为6.0-7.5；
15)90-200mg/mL的抗MASP2抗体或其抗原结合片段；
30-50mg/mL的蔗糖；
10-20mg/mL精氨酸或其盐(例如盐酸精氨酸)；
8-12mM的组氨酸-盐酸缓冲剂；
0.1-0.4mg/mL的聚山梨醇(例如聚山梨醇酯80)；
pH为6.1-7.1；
16)约90-200mg/mL或约90-165mg/mL的抗MASP2抗体或其抗原结合片段；
约40mg/mL的蔗糖；
约15mg/mL的精氨酸或其盐(例如盐酸精氨酸)；
约10mM的组氨酸-盐酸缓冲剂；
0.1-0.4mg/mL的聚山梨醇(例如聚山梨醇酯80)；
pH为约6.4、约6.6或约6.8；
17)约90mg/mL、约100mg/mL、约110mg/mL、约135mg/mL、约150mg/mL、约165mg/mL或约200mg/mL的抗MASP2抗体或其抗原结合片段；
约40mg/mL的蔗糖；
约15mg/mL的精氨酸或其盐(例如盐酸精氨酸)；
约10mM的组氨酸-盐酸缓冲剂；
0.1-0.4mg/mL的聚山梨醇(例如聚山梨醇酯80)；
pH为6.4-6.8；
18)约90-200mg/mL或约90-165mg/mL的抗MASP2抗体或其抗原结合片段；
约40mg/mL的蔗糖；
约15mg/mL精氨酸或其盐(例如盐酸精氨酸)；
约10mM的组氨酸-盐酸缓冲剂；
约0.1mg/mL、约0.2mg/mL或约0.4mg/mL的聚山梨醇(例如聚山梨醇酯80)；
pH为6.4-6.8；
19)约90-200mg/mL或约90-165mg/mL的抗MASP2抗体或其抗原结合片段；
约40mg/mL的蔗糖；
约15mg/mL精氨酸或其盐(例如盐酸精氨酸)；
约10mM的组氨酸-盐酸缓冲剂；
约0.1-0.4mg/mL的聚山梨醇(例如聚山梨醇酯80)；
pH为6.4-6.8；
20)上述1)-19)中任一组所述的药物组合物，其中所述精氨酸或其盐为盐酸精氨酸；21)上述1)-20)中任一组所述的药物组合物，其中所述聚山梨醇为聚山梨醇酯80；
22)上述1)-21)中任一组所述的药物组合物，还包含注射用水；
23)上述1)-22)中任一组所述的药物组合物，还包含pH调节剂，如盐酸和/或氢氧化钠。
在某些实施方案中，本公开提供一种包含抗MASP2抗体或其抗原结合片段(例如重链、轻链序列为SEQ ID NO:24和25的77H11(H3L1))的药物组合物，其包含如下1)至9)任意一组：
1)50-200mg/mL的抗MASP2抗体或其抗原结合片段；
10-100mg/mL(例如30-60、35-55、40-50、约40、约45、约47、约50mg/mL)的甘露醇；
5-15mM的组氨酸-盐酸缓冲剂(例如组氨酸-盐酸组氨酸缓冲剂)；
0.05-0.6mg/mL的聚山梨醇(例如聚山梨醇酯80)；
pH为5.5-7.5。
2)80-120mg/mL的抗MASP2抗体或其抗原结合片段；
10-100mg/mL(例如30-60、35-55、40-50、约40、约45、约47、约50mg/mL)的甘露醇；
8-12mM的组氨酸-盐酸缓冲剂(例如组氨酸-盐酸组氨酸缓冲剂)；
0.05-0.5mg/mL的聚山梨醇(例如聚山梨醇酯80)；
pH为6.0-7.5。
3)90-110mg/mL的抗MASP2抗体或其抗原结合片段；
10-100mg/mL(例如30-60、35-55、40-50、约40、约45、约47、约50mg/mL)的甘露醇；
约10mM的组氨酸-盐酸缓冲剂(例如组氨酸-盐酸组氨酸缓冲剂)；
约0.1-0.4mg/mL的聚山梨醇(例如聚山梨醇酯80)(例如约0.1、约0.2、约0.4mg/mL)；
pH为约6.1-7.1(例如约6.4、约6.5、约6.6、约6.7、约6.8)。
4)50-200mg/mL的抗MASP2抗体或其抗原结合片段；
10-60mg/mL(例如10-50、20-40、27-30、约27、约28、约30mg/mL)mg/mL的脯氨酸；
5-15mM的组氨酸-盐酸缓冲剂(例如组氨酸-盐酸组氨酸缓冲剂)；
0.05-0.6mg/mL的聚山梨醇(例如聚山梨醇酯80)；
pH为5.5-7.5。
5)80-120mg/mL的抗MASP2抗体或其抗原结合片段；
10-60mg/mL(例如10-50、20-40、27-30、约27、约28、约30mg/mL)mg/mL的脯氨酸；
8-12mM的组氨酸-盐酸缓冲剂(例如组氨酸-盐酸组氨酸缓冲剂)；
0.05-0.5mg/mL的聚山梨醇(例如聚山梨醇酯80)；
pH为6.0-7.5。
6)90-110mg/mL的抗MASP2抗体或其抗原结合片段；
10-60mg/mL(例如10-50、20-40、27-30、约27、约28、约30mg/mL)mg/mL的脯氨酸；
约10mM的组氨酸-盐酸缓冲剂(例如组氨酸-盐酸组氨酸缓冲剂)；
约0.1-0.4mg/mL的聚山梨醇(例如聚山梨醇酯80)(例如约0.1、约0.2、约0.4mg/mL)；
pH为约6.1-7.1(例如约6.4、约6.5、约6.6、约6.7、约6.8)。
7)50-200mg/mL的抗MASP2抗体或其抗原结合片段；
20-200mg/mL(例如30-150、40-120、50-100、60-90、约65、约70、约75、约80、约90mg/mL)的蔗糖；
5-15mM的组氨酸-盐酸缓冲剂(例如组氨酸-盐酸组氨酸缓冲剂)；
0.05-0.6mg/mL的聚山梨醇(例如聚山梨醇酯80)；
pH为5.5-7.5。
8)80-120mg/mL的抗MASP2抗体或其抗原结合片段；
20-200mg/mL(例如30-150、40-120、50-100、60-90、约65、约70、约75、约80、约90mg/mL)的蔗糖；
8-12mM的组氨酸-盐酸缓冲剂(例如组氨酸-盐酸组氨酸缓冲剂)；
0.05-0.5mg/mL的聚山梨醇(例如聚山梨醇酯80)；
pH为6.0-7.5。
9)90-110mg/mL的抗MASP2抗体或其抗原结合片段；
20-200mg/mL(例如30-150、40-120、50-100、60-90、约65、约70、约75、约80、约90mg/mL)的蔗糖；
约10mM的组氨酸-盐酸缓冲剂(例如组氨酸-盐酸组氨酸缓冲剂)；
约0.1-0.4mg/mL的聚山梨醇(例如聚山梨醇酯80)(例如约0.1、约0.2、约0.4mg/mL)；
pH为约6.1-7.1(例如约6.4、约6.5、约6.6、约6.7、约6.8)。
10)上述1)-9)中任一组所述的药物组合物，其中所述精氨酸或其盐为盐酸精氨酸；21)上述1)-20)中任一组所述的药物组合物，其中所述聚山梨醇为聚山梨醇酯80；
11)上述1)-10)中任一组所述的药物组合物，还包含注射用水；
12)上述1)-11)中任一组所述的药物组合物，还包含pH调节剂，如盐酸和/或氢氧化钠。
在某些实施方案中，本公开提供一种包含抗MASP2抗体或其抗原结合片段(例如重链、轻链序列为SEQ ID NO:24和25的77H11(H3L1))的药物组合物，其包含或为如下1)至20)任意一组：
(1)约90mg/mL、约100mg/mL或约110mg/mL的抗MASP2抗体或其抗原结合片段，约8.2mg/mL氯化钠，约0.1mg/mL聚山梨醇酯80，和约10mM组氨酸-盐酸组氨酸，pH为约6.4；
(2)约90mg/mL、约100mg/mL或约110mg/mL的抗MASP2抗体或其抗原结合片段，约8.2mg/mL氯化钠，约0.2mg/mL聚山梨醇酯80，和约10mM组氨酸-盐酸组氨酸，pH为约6.4；
(3)约90mg/mL、约100mg/mL或约110mg/mL的抗MASP2抗体或其抗原结合片段，约8.2mg/mL氯化钠，约0.4mg/mL聚山梨醇酯80，和约10mM组氨酸-盐酸组氨酸，pH为约6.4；
(4)约90mg/mL、约100mg/mL或约110mg/mL的抗MASP2抗体或其抗原结合片段，约8.2mg/mL氯化钠，约0.1mg/mL聚山梨醇酯80，和约10mM组氨酸-盐酸组氨酸，pH为约6.6；
(5)约90mg/mL、约100mg/mL或约110mg/mL的抗MASP2抗体或其抗原结合片段，约8.2mg/mL氯化钠，约0.2mg/mL聚山梨醇酯80，和约10mM组氨酸-盐酸组氨酸，pH为约6.6；
(6)约90mg/mL、约100mg/mL或约110mg/mL的抗MASP2抗体或其抗原结合片段，约8.2mg/mL氯化钠，约0.4mg/mL聚山梨醇酯80，和约10mM组氨酸-盐酸组氨酸，pH为约6.6；
(7)约90mg/mL、约100mg/mL或约110mg/mL的抗MASP2抗体或其抗原结合片段，约8.2mg/mL氯化钠，约0.1mg/mL聚山梨醇酯80，和约10mM组氨酸-盐酸组氨酸，pH为约6.8；
(8)约90mg/mL、约100mg/mL或约110mg/mL的抗MASP2抗体或其抗原结合片段，约8.2mg/mL氯化钠，约0.2mg/mL聚山梨醇酯80，和约10mM组氨酸-盐酸组氨酸，pH为约6.8；
(9)约90mg/mL、约100mg/mL或约110mg/mL的抗MASP2抗体或其抗原结合片段，约8.2mg/mL氯化钠，约0.4mg/mL聚山梨醇酯80，和约10mM组氨酸-盐酸组氨酸，pH为约6.8；
(10)约135mg/mL、约150mg/mL、约165mg/mL或约200mg/mL的抗MASP2抗体或其抗原结合片段，约40mg/mL蔗糖，约15mg/mL盐酸精氨酸，约0.1mg/mL聚山梨醇酯80，和约10mM组氨酸-盐酸组氨酸，pH为约6.4；
(11)约135mg/mL、约150mg/mL、约165mg/mL或约200mg/mL的抗MASP2抗体或其抗原结合片段，约40mg/mL蔗糖，约15mg/mL盐酸精氨酸，约0.2mg/mL聚山梨醇酯80，和约10mM组氨酸-盐酸组氨酸，pH为约6.4；
(12)约135mg/mL、约150mg/mL、约165mg/mL或约200mg/mL的抗MASP2抗体或其抗原结合片段，约40mg/mL蔗糖，约15mg/mL盐酸精氨酸，约0.4mg/mL聚山梨醇酯80，和约10mM组氨酸-盐酸组氨酸，pH为约6.4；
(13)约135mg/mL、约150mg/mL、约165mg/mL或约200mg/mL的抗MASP2抗体或其抗原结合片段，约40mg/mL蔗糖，约15mg/mL盐酸精氨酸，约0.1mg/mL聚山梨醇酯80，和约10mM组氨酸-盐酸组氨酸，pH为约6.6；
(14)约135mg/mL、约150mg/mL、约165mg/mL或约200mg/mL的抗MASP2抗体或其抗原结合片段，约40mg/mL蔗糖，约15mg/mL盐酸精氨酸，约0.2mg/mL聚山梨醇酯80，和约10mM组氨酸-盐酸组氨酸，pH为约6.6；
(15)约135mg/mL、约150mg/mL、约165mg/mL或约200mg/mL的抗MASP2抗体或其抗原结合片段，约40mg/mL蔗糖，约15mg/mL盐酸精氨酸，约0.4mg/mL聚山梨醇酯80，和约10mM组氨酸-盐酸组氨酸，pH为约6.6；
(16)约135mg/mL、约150mg/mL、约165mg/mL或约200mg/mL的抗MASP2抗体或其抗原结合片段，约40mg/mL蔗糖，约15mg/mL盐酸精氨酸，约0.1mg/mL聚山梨醇酯80，和约10mM组氨酸-盐酸组氨酸，pH为约6.8；
(17)约135mg/mL、约150mg/mL、约165mg/mL或约200mg/mL的抗MASP2抗体或其抗原结合片段，约40mg/mL蔗糖，约15mg/mL盐酸精氨酸，约0.2mg/mL聚山梨醇酯80，和约10mM组氨酸-盐酸组氨酸，pH为约6.8；
(18)约135mg/mL、约150mg/mL、约165mg/mL或约200mg/mL的的抗MASP2抗体或其抗原结合片段，约40mg/mL蔗糖，约15mg/mL盐酸精氨酸，约0.4mg/mL聚山梨醇酯80，和约10mM组氨酸-盐酸组氨酸，pH为约6.8；
(19)约100mg/mL抗MASP2抗体，约8.2mg/mL氯化钠，约0.4mg/mL聚山梨醇酯80，和约10mM组氨酸-盐酸组氨酸，pH为约6.6；
(20)约150mg/mL抗MASP2抗体，约40mg/mL蔗糖，约15mg/mL盐酸精氨酸，约0.4mg/mL聚山梨醇酯80，和约10mM组氨酸-盐酸组氨酸，pH为约6.6
(21)前述(1)-(20)中任一组所述的药物组合物，还包含注射用水；
(22)前述(1)-(21)中任一组所述的药物组合物，还包含pH调节剂，如盐酸和/或氢氧化钠。
在某些实施方案中，所述组氨酸-盐酸缓冲剂为组氨酸-盐酸组氨酸缓冲剂。
本公开还提供了一种制备前述药物组合物的方法，包括将抗MASP2抗体或其抗原结合片段与缓冲剂混合的步骤。在某些实施方案中，所述缓冲剂为组氨酸-盐酸缓冲剂(例如，组氨酸-盐酸组氨酸)。
在某些实施方案中，如上任一项所述的药物组合物，所述药物组合物是液体制剂。在一些实施方案中，所述液体制剂的溶剂是水、生理盐水或葡萄糖溶液。
本公开还提供了一种冻干制剂，其特征在于所述冻干制剂复溶后可形成如上任一项所述的药物组合物。
本公开还提供了一种冻干制剂，所述的冻干制剂通过将如上任一项所述的药物组合物经冷冻干燥获得。
本公开提供了一种复溶溶液，所述复溶溶液是通过将前述冻干制剂经复溶制备获得。在某些实施方案中，所述的复溶溶液选自但不限于注射用水，生理盐水或葡萄糖溶液。
本公开还提供一种制品，其包括容器，该容器中装有如前述的药物组合物、如前述的冻干制剂或如前述的复溶溶液。在某些实施方案中，所述容器为中性硼硅玻璃管制注射剂瓶。在某些实施方案中，所述制品中包含药品说明书。
本公开如上任一项所述的药物组合物，其中所述抗MASP2抗体及其抗原结合片段包含重链可变区(VH)和轻链可变区(VL)，其中：
所述VH包含如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
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。
在某些实施方案中，所述抗MASP2抗体及其抗原结合片段包含：
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；
本公开如上任一项所述的药物组合物中，其中所述抗MASP2抗体及其抗原结合片段包含重链可变区(VH)和轻链可变区(VL)，其中
所述VH包含分别如SEQ ID NO：9、10、11所示的HCDR1、HCDR2、HCDR3，和/或，所述VL包含分别如SEQ ID NO：12、13、14所示的LCDR1、LCDR2、LCDR3。
在某些实施方案中，上述所述的抗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。
在某些实施方案中，上述所述的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。
在某些实施方案中，所述的抗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、17、18、19、20之一所示，和/或，VL的氨基酸序列如SEQ ID NO：8、121、22之一所示；
在某些具体实施方案中所述的抗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：17、21所示氨基酸序列，或由其组成；
c)VH、VL分别包含如SEQ ID NO：17、22所示氨基酸序列，或由其组成；
d)VH、VL分别包含如SEQ ID NO：18、21所示氨基酸序列，或由其组成；
e)VH、VL分别包含如SEQ ID NO：18、22所示氨基酸序列，或由其组成；
f)VH、VL分别包含如SEQ ID NO：19、21所示氨基酸序列，或由其组成；
g)VH、VL分别包含如SEQ ID NO：19、22所示氨基酸序列，或由其组成；
h)VH、VL分别包含如SEQ ID NO：20、21所示氨基酸序列，或由其组成；
或
i)VH、VL分别包含如SEQ ID NO：20、22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i)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。
在某些实施方案中，所述的抗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。
在某些实施方案中，所述的抗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。在某些实施方案中，所述Fc区可以具有突变，所述突变使得ADCC功能减少。在某些具体实施方案中，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。在某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)。
在某些实施方案中，本公开所述的抗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。
在某些实施方案中，本公开所述的抗MASP2抗体或其抗原结合片段重链全长的氨基酸序列如SEQ ID NO：24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：25所示或与之具有至少80％同一性、至少90％或至少95％同一性。
在某些实施方案中，所述的抗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。在某些具体实施方案中，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。
在某些实施方案中，所述抗MASP2抗体选择性抑制MASP2补体活化，保留C1q-依赖性补体活化系统功能完整。
本公开提供了前述药物组合物、前述方法制备获得的药物组合物、前述冻干制剂或前述复溶溶液在预防和/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。在某些实施方案中，所述疾病为IgA肾病、阵发性睡眠性血红蛋白尿症(PNH)。
本公开还提供作为预防和/或治疗疾病的药物的前述的药物组合物、冻干制剂、复溶溶液或制品。
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前述药物组合物、前述方法制备获得的药物组合物、前述冻干制剂或前述复溶溶液。
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。在某些实施方案中，提供本公开的前述药物组合物、前述方法制备获得的药物组合物、前述冻干制剂或前述复溶溶液治疗上述疾病和状况的方法，以及相关制药用途。
在某些实施方案中，上述疾病和状况为MASP-2-依赖性补体活化相关疾病。
在某些实施方案中，上述疾病和状况为微血管内皮细胞损伤和/或血栓形成。
在某些实施方案中，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)，在某些具体实施方案中，所述疾病是MASP-2-依赖性补体活化相关的。
在一些实施方案中，本公开的抗MASP-2抗体为描述于国际专利申请WO2022228364中的抗MASP-2抗体，WO2022228364的全文内容全部引入本公开。
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，所述的鼠源抗人抗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(KD)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)。KD通过方程KD＝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KD。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BiacoreTM系统或KinExA来评价。可通过比较各个抗体/抗原复合物的KD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KD值与非目的相互作用(例如已知不结合MASP2的对照抗体)的KD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。
“约”是指处于如本领域的普通技术人员所确定的特定值的可接受误差范围之内，其将部分取决于所述值是如何测量或测定的，即所述测量系统的限制。在特定测定、结果或实施方案的上下文中，除非实施例或说明书其它地方内另有明确说明，否则“约”意指给定数值±5％以内的范围。
“缓冲剂”指通过其酸-碱共轭组分的作用而耐受pH变化的缓冲剂。将pH控制在适当范围中的缓冲剂的例子包括三羟甲基氨基甲烷(Tris)、醋酸盐、琥珀酸盐、葡萄糖酸盐、组氨酸盐、草酸盐、乳酸盐、磷酸盐、枸橼酸盐、酒石酸盐、延胡索酸盐、甘氨酰甘氨酸和其它有机酸缓冲剂。
“组氨酸盐缓冲剂”是包含组氨酸根离子的缓冲剂。组氨酸盐缓冲剂的实例包括组氨酸-盐酸盐，组氨酸-醋酸盐，组氨酸-磷酸盐，组氨酸-硫酸盐等缓冲剂，优选组氨酸-盐酸盐缓冲剂或组氨酸-醋酸盐缓冲剂，组氨酸-醋酸盐缓冲剂是组氨酸与醋酸配制而成，组氨酸-盐酸盐缓冲剂是组氨酸与组氨酸盐酸盐配制而成，或组氨酸与盐酸配制而成。
“磷酸盐缓冲剂”是包括磷酸根离子的缓冲剂。磷酸盐缓冲剂的实例包括磷酸氢二钠-磷酸二氢钠、磷酸氢二钠-磷酸二氢钾、磷酸氢二钠-枸橼酸等。优选地磷酸盐缓冲剂是磷酸氢二钠-磷酸二氢钠。
“三羟甲基氨基甲烷缓冲剂”是包含三羟甲基氨基甲烷(Tris)的缓冲剂。三羟甲基氨基甲烷缓冲剂的实例包括三羟甲基氨基甲烷-盐酸缓冲剂((Tris-HCl)、三羟甲基氨基甲烷-醋酸缓冲剂(Tris-AA)、三羟甲基氨基甲烷-琥珀酸缓冲剂(Tris-SA)或三羟甲基氨基甲烷-枸橼酸缓冲剂(Tris-CA)等缓冲剂。
“枸橼酸盐缓冲剂”是包括枸橼酸根离子的缓冲剂。枸橼酸盐缓冲剂的实例包括枸橼酸-枸橼酸钠、枸橼酸-枸橼酸钾、枸橼酸-枸橼酸钙、枸橼酸-枸橼酸镁等。优选的枸橼酸盐缓冲剂是枸橼酸-枸橼酸钠。
“琥珀酸盐缓冲剂”是包括琥珀酸根离子的缓冲剂。琥珀酸盐缓冲剂的实例包括琥珀酸-琥珀酸钠盐、琥珀酸-琥珀酸钾、琥珀酸-琥珀酸钙盐等。优选的琥珀酸盐缓冲剂是琥珀酸-琥珀酸钠盐。示例性的，所述的琥珀酸-琥珀酸钠可由琥铂酸与氢氧化钠配制而成，或由琥铂酸与琥珀酸钠盐配制而成。
“醋酸盐缓冲剂”是包括醋酸根离子的缓冲剂。醋酸盐缓冲剂的实例包括醋酸-醋酸钠、组氨酸-醋酸组氨酸、醋酸-醋酸钾、醋酸-醋酸钙、醋酸-醋酸镁等。优选的醋酸盐缓冲剂是醋酸-醋酸钠。
“药物组合物”表示含有一种或多种本文所述抗体与其他化学组分的混合物，所述其他组分例如生理学/可药用的载体和赋形剂。药物组合物的目的是保持活性成分的稳定性，促进对生物体的给药，利于活性成分的吸收进而发挥生物活性。
本披露中，“药物组合物”和“制剂”并不互相排斥。
本披露中所述药物组合物的溶液形式，若无特殊说明，其中的溶剂均为水。
组合物是等渗的“等渗”表示制剂具有与人血液基本上相同的渗透压。等渗可以通过本领域公知方法测定，例如可以采用蒸气压力或冰冻型渗压计测定。当给药途径为皮下注射时，药物组合物渗透压优选控制在260-380mOsm，例如药物制剂渗透压控制在270-360mOsm。
“冻干制剂”表示液体或溶液形式的药物组合物或液体或溶液制剂经真空冷冻干燥步骤之后获得的制剂或药物组合物。
如果在目检颜色和/或澄清度后，或者通过UV光散射、尺寸排阻色谱法(SEC)和动态光散射(DLS)测得，抗体没有显示出显著的聚集增加、沉淀和/或变性，那么所述抗体在药物制剂中“保留它的物理稳定性”。蛋白构象的变化可以通过荧光光谱法(其确定蛋白三级结构)和通过FTIR光谱法(其确定蛋白二级结构)来评价。
如果抗体没有显示出显著的化学改变，那么所述抗体在药物制剂中“保留它的化学稳定性”。通过检测和定量化学上改变的形式的蛋白，可以评估化学稳定性。经常改变蛋白化学结构的降解过程包括水解或截短(通过诸如尺寸排阻色谱法和CE-SDS等方法来评价)、氧化(通过诸如与质谱法或MALDI/TOF/MS结合的肽谱法等方法来评价)、脱酰胺作用(通过诸如离子交换色谱法、毛细管等电聚焦、肽谱法、异天冬氨酸测量等方法来评价)和异构化(通过测量异天冬氨酸含量、肽谱法等来评价)。
如果抗体在给定时间的生物活性是在制备药物制剂时表现出的生物活性的预定范围内，那么所述抗体在药物制剂中“保留它的生物活性”。
“施用”、“给予”和“处理”，当其应用于动物、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。“施用”、“给予”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。“施用”、“给予”和“处理”还意指通过试剂、诊断、结合组合物或通过另一种细胞体外和离体处理例如细胞。“处理”当其应用于人、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”意指给予患者内用或外用治疗剂，例如包含本披露的任一种的药物组合物，所述患者具有一种或多种疾病症状，而已知所述治疗剂对这些症状具有治疗作用。通常，在受治疗患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，以诱导这类症状退化或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，例如患者的疾病状态、年龄和体重，以及药物在患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。尽管本披露的实施方案(例如治疗方法或制品)在缓解每个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患者中应当减轻目标疾病症状。
药物组合物制备与检测过程中使用的设备及方法如下：
SEC分子排阻色谱法：
根据凝胶孔隙的孔径大小与高分子样品分子的线团尺寸间的相对关系而对溶质进行分离的分析的方法。
SEC单体含量百分比＝A单体/A总*100％(A单体为样品中主峰单体的峰面积，A总为所有峰面积之和。)
SEC测定用仪器：安捷伦1260-Bio；色谱柱：Waters，XBrigeSEC(300×7.8mm 3.5μm)
CE毛细管凝胶电泳：
将凝胶移到毛细管中作为支持介质进行的一种电泳，并在一定的电压下根据样品分子量的大小进行分离的方法。
非还原CE纯度百分比＝A主峰/A总*100％(A主峰为样品中主峰的峰面积，A总为所有峰面积之和。
还原CE纯度百分比＝轻重链纯度之和＝CA轻链/CA总×％+CA重链/CA总×％(CA轻链为样品中轻链校正峰面积，CA重链为样品中重链校正峰面积，CA总为轻链、重链和杂质峰面积之和)
CE测定用仪器：Sciex型号PA800plus
icIEF成像毛细管等点聚焦电泳：
根据蛋白质等电点pI不同进行分离的技术。
icIEF主峰含量百分比＝主峰峰面积/总面积*100％(总面积为酸性峰、主峰和碱性峰面积之和)。
icIEF测定所用仪器厂家Protein Simple，型号Muarice。
渗透压测定：
冰点法测定渗透压，以冰点下降值与溶液的摩尔浓度成正比例关系为基础，采用高灵敏度感温元件，测定溶液结冰点，通过电量转化为渗透压。仪器厂家罗泽Loser，型号OM819.
蛋白浓度测定：
蛋白浓度测定仪器：紫外可见分光光度计，型号：Nano Drop 2000，光程为1mm。
熔解温度(Tm)和聚集温度(Tagg)：
Tm为蛋白质在升温过程中，50％组分变性时所对应的温度；Tagg为蛋白在升温过程中发生聚集时对应的温度。将样品加载到Uni管中，以25-95℃的热升温运行测定。Tm和Taag测定仪器：Uncle,仪器厂家为Unchained。
粒径(Radius)以及PD％：
采用动静态光散射技术，使用96孔板，利用抗体分子的布朗运动对抗体粒径大小及分布进行测定。测定仪器：高通量动静态光散射仪，仪器厂家为WYATT，型号DynaPro plate ReaderIII
示例性的抗体药物组合物(制剂)制备工艺：
第一步：取一定量的纯化的抗MASP2抗体溶液，用不含抗体的缓冲剂进行溶剂置换(优选超滤)，经超滤膜至少6倍体积置换，继续浓缩，将抗体浓缩到一浓度。加入一定体积的其他辅料母液，并用缓冲剂稀释，使抗体及各辅料达到需要的浓度，混匀。原液经过滤后中控取样检测细菌内毒素和微生物限度。将原液经0.22μm滤芯除菌过滤，收集滤液。
第二步：调节装量(目标装量1.70mL)，选用中硼硅玻璃管制注射剂瓶(欧式防跳塞)进行灌装，分别于灌装开始、灌装中间、灌装结束时取样检测装量差异，加注射液用溴化丁基涂层胶塞。
第三步：开启轧盖机，加铝盖，进行轧盖。
第四步：目检，确认产品无装量不准、外观不良等缺陷。打印纸盒标签，折叠纸盒，装盒，贴纸盒标签。
附图说明
图1A显示了本公开的抗MASP2抗体77H11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显示本公开的77H11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3B分别为检测77H11(H3L1)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4A至图4B为检测77H11(H3L1)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
本申请中抗MASP-2抗体的制备、纯化方法已在国际专利申请WO2022228364中记载，前述申请文件的全部内容均可引入本公开。
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:26)(参见Uniprot网站)更换为MEFGLSWLFLVAILKGVQC(SEQ ID NO:27)，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：
(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：

(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：
(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MASP2 CCP1、人MASP2 CCP1-CCP2-SP(S633A)、人MASP2 CCP2-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)：

>CCP2-SP(S633A)：
(注：灰色为633位突变为A(S633A))
                            SEQ ID NO：5
>CCP1：
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2～4×107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1～2×108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％CO2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(1:5000 in 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：
>77H11 VL：

表1.抗MASP2抗体CDR序列(Kabat编号规则)
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：
>OMS721的轻链全长：
结果参见图1A，表2，所述结果显示77H11可以显著抑制凝集素通路，优于阳性对照OMS721。这里的抗体77H11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：19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
实施例4.抗MASP2抗体的人源化改造
选择抗MASP2抗体77H11进行人源化改造，随后在人血清中进行活性鉴定。
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：
>77H11_H2：
>77H11_H3：
>77H11_H4：
>77H11_L1：
>77H11_L2：
人源化抗MASP2抗体的序列参见表3。
表3.抗MASP2抗体重连可变区、轻链可变区
表3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：23所示。所述S228P是根据EU编号。
>人IgG4 Fc(S228P)：
示例性的抗MASP2抗体重链全长、轻链全长如下所示：
>77H11(H3L1)重链全长：
>77H11(H3L1)轻链全长：
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 Ca2+的TBST洗3次。用50μL/孔的3％BSA封闭液(溶剂为TBS，加入5mM Ca2+)，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、表4所示，77H11的人源化分子能在人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4. 90％人血清条件下抗MASP2抗体对凝集素通路的抑制作用
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77H11(H3L1)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5所示。
结果显示，77H11(H3L1)对人和猴MASP2蛋白都有10-10M到10-11M的亲合力。
表5.抗MASP2抗体的亲合力测定结果(KD)
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 Ca2+的TBST洗3次。用50μL/孔的3％BSA封闭液(溶剂为TBS，加入5mM Ca2+)，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-3B和表6。结果说明，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。其中，11165、29C1(H1L1)为本申请筛选获得的另外两种抗MASP2抗体。
表6.不同物种血清条件下抗MASP2抗体对凝集素通路的抑制作用
实施例7.抗MASP2抗体的猴体内药效实验(PD)
将77H11(H3L1)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4A和4B，可以看出4种抗体都能显著抑制凝集素通路活性，77H11(H3L1)在相同剂量下比OMS721有更长久的抑制效果。其中，11165、29C1(H1L1)为本申请筛选获得的另外两种抗MASP2抗体。
实施例8.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)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T1/2及其主要参数。结果参见表7。可以看出，77H11(H3L1)半衰期显著优于 OMS721，意味着可以在体内有更长久的药效。
表7.抗MASP2抗体的猴PK参数结果
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。
实施例9.抗MASP2抗体制剂pH值筛选
选择10mM磷酸盐缓冲体系，设计如下4.6、5.0、5.4、5.8、6.2、6.6、7.0、7.4、7.8、8.2、8.6、9.0共12个不同pH，制备抗MASP2抗体(抗体编号为77H11(H3L1)，重链和轻链如SEQ ID NO：24和25所示，实施例4制备获得，以下同)浓度为10.0mg/mL的抗体制剂，测定样品的熔解温度(Tm)，考察抗体在不同pH体系下的热稳定性，测定样品的聚集温度(Tagg)及粒径，考察抗体在不同pH体系下的胶体稳定性。序号1)至12)如下：
1)10mM磷酸二氢钠-磷酸氢二钠，pH 4.6
2)10mM磷酸二氢钠-磷酸氢二钠，pH 5.0
3)10mM磷酸二氢钠-磷酸氢二钠，pH 5.4
4)10mM磷酸二氢钠-磷酸氢二钠，pH 5.8
5)10mM磷酸二氢钠-磷酸氢二钠，pH 6.2
6)10mM磷酸二氢钠-磷酸氢二钠，pH 6.6
7)10mM磷酸二氢钠-磷酸氢二钠，pH 7.0
8)10mM磷酸二氢钠-磷酸氢二钠，pH 7.4
9)10mM磷酸二氢钠-磷酸氢二钠，pH 7.8
10)10mM磷酸二氢钠-磷酸氢二钠，pH 8.2
11)10mM磷酸二氢钠-磷酸氢二钠，pH 8.6
12)10mM磷酸二氢钠-磷酸氢二钠，pH 9.0
表8.抗MASP2抗体制剂pH值筛选结果


(注：％PD表示分散系数，值越小，表明粒径分布越均一；Multimodal表示粒径分布较差，
设备无法计算％PD值。)
结果显示：抗MASP2抗体在pH 7.0体系中Tm值最高，其次是pH 6.6体系，表明该抗体在pH 6.6～7.0条件下热稳定性较好；抗MASP2抗体在pH 6.6体系中Tagg值最高，其次是pH 7.0体系，另外在pH 6.6体系中粒径较小，且粒径分布较为均一，表明该抗体在pH 6.6～7.0条件下胶体稳定性较好。因此，抗MASP2抗体在pH值为6.6～7.0条件下热稳定性和胶体稳定性均较好。
实施例10.抗MASP2抗体制剂缓冲体系(pH 6.6)筛选
选择10mM磷酸二氢钠-磷酸氢二钠、10mM组氨酸-盐酸组氨酸缓冲体系，制备pH 6.6、抗MASP2抗体含量为50mg/mL的抗体制剂，通过考察高温40℃条件下的稳定性，进行缓冲体系(pH 6.6)筛选。序号1)至2)如下：
1)10mM磷酸二氢钠-磷酸氢二钠pH 6.6
2)10mM组氨酸-盐酸组氨酸pH 6.6
表9抗MASP2抗体制剂缓冲体系(pH 6.6)筛选结果

(注：此表为抗MASP2抗体制剂缓冲体系(pH 6.6)筛选0时样品测定结果)
表10抗MASP2抗体制剂缓冲体系(pH 6.6)筛选结果

(注：W表示周；％PD表示分散系数)
结果显示:在pH6.6条件下，相比于10mM磷酸二氢钠-磷酸氢二钠缓冲体系，抗MASP2抗体在10mM组氨酸-盐酸组氨酸缓冲体系中Tm值略低，但Tagg值更高；相同考察条件下，抗MASP2抗体在10mM组氨酸-盐酸组氨酸缓冲体系中粒径更小，聚体含量更低，表明在pH6.6条件下，抗MASP2抗体在10mM组氨酸-盐酸组氨酸缓冲体系中稳定性更好。
实施例11.抗MASP2抗体制剂缓冲体系(pH 7.0)筛选
选择10mM磷酸二氢钠-磷酸氢二钠、10mM组氨酸-盐酸组氨酸缓冲体系，制备pH 7.0、抗MASP2抗体含量为50mg/mL的抗体制剂，取样品进行高温40℃稳定性研究。序号1)至2)如下：
1)10mM磷酸二氢钠-磷酸氢二钠pH 7.0
2)10mM组氨酸-盐酸组氨酸pH 7.0
表11抗MASP2抗体制剂缓冲体系(pH 7.0)筛选结果

(注：此表为抗MASP2抗体制剂缓冲体系(pH 7.0)筛选0时样品测定结果)
表12抗MASP2抗体制剂缓冲体系(pH 7.0)筛选结果

(注：W表示周；％PD表示分散系数)
结果显示:相同考察条件下，相比于10mM磷酸二氢钠-磷酸氢二钠pH 7.0缓冲体系，抗MASP2抗体在10mM组氨酸-盐酸组氨酸pH 7.0缓冲体系中Tm值及Tagg值更高，外观更好，粒径更小，聚体含量相对更低，因此抗MASP2抗体在10mM组氨酸-盐酸组氨酸pH 7.0缓冲体系中稳定性更好。
实施例12.抗MASP2抗体制剂表面活性剂浓度筛选
选择辅料蔗糖(蔗糖含量选定为75mg/mL)，10mM组氨酸-盐酸组氨酸pH6.8缓冲体系，制备含100mg/mL抗MASP2抗体、75mg/mL蔗糖、不同聚山梨醇酯80浓度(0.1mg/mL、0.4mg/mL)的抗体制剂，考察震荡(200rpm,室温)、冻融(-35℃/室温)条件下蛋白稳定性。序号1)至2)如下：
1)10mM组氨酸-盐酸组氨酸，0.1mg/mL聚山梨醇酯80，75mg/mL蔗糖，蛋白浓度100mg/mL，pH 6.8
2)10mM组氨酸-盐酸组氨酸，0.4mg/mL聚山梨醇酯80，75mg/mL蔗糖，蛋白浓度100mg/mL，pH 6.8
表13抗MASP2抗体制剂表面活性剂浓度筛选结果
表14抗MASP2抗体制剂表面活性剂浓度筛选结果

(注：h表示小时；W表示周)
表15抗MASP2抗体制剂表面活性剂浓度筛选结果

(注：h表示小时；W表示周)
结果表明：当聚山梨醇酯80浓度为0.1mg/mL-0.4mg/mL时，抗MASP2抗体制剂在震荡、冻融条件下的稳定性无明显差异，因此，抗MASP2抗体制剂中聚山梨醇酯80的浓度范围定为0.1mg/mL～0.4mg/mL。
实施例13.抗MASP2抗体制剂辅料种类筛选
选择10mM组氨酸-盐酸组氨酸pH6.8缓冲体系，制备含100mg/mL抗MASP2抗体、不同种类辅料(75mg/mL蔗糖、45mg/mL甘露醇、8.2mg/mL氯化钠、28mg/mL脯氨酸)、0.2mg/mL聚山梨醇酯80的抗体制剂，考察高温40℃条件下蛋白稳定性。序号1)至4)如下：
1)10mM组氨酸-盐酸组氨酸，0.2mg/mL聚山梨醇酯80，75mg/mL蔗糖，蛋白浓度100mg/mL，pH 6.8
2)10mM组氨酸-盐酸组氨酸，0.2mg/mL聚山梨醇酯80，45mg/mL甘露醇，蛋白浓度100mg/mL，pH 6.8
3)10mM组氨酸-盐酸组氨酸，0.2mg/mL聚山梨醇酯80，8.2mg/mL氯化钠，蛋白浓度100mg/mL，pH 6.8
4)10mM组氨酸-盐酸组氨酸，0.2mg/mL聚山梨醇酯80，28mg/mL脯氨酸，蛋白浓度100mg/mL，pH 6.8
表16抗MASP2抗体制剂辅料种类筛选结果
表17抗MASP2抗体制剂辅料种类筛选结果

(注：h表示小时；W表示周)
表18抗MASP2抗体制剂辅料种类筛选结果

(注：h表示小时；W表示周)
结果表明，相同考察条件下，抗MASP2抗体在不同辅料体系中SEC单体含量、R-CE纯度均无明显差异，但相比于其他辅料，抗MASP2抗体在含辅料氯化钠的序号3制剂中热稳定性更好，粒径相对更小、分布更均一。
实施例14.抗MASP2抗体制剂pH对稳定性的影响
选择10mM组氨酸-盐酸组氨酸缓冲体系，制备含8.2mg/mL氯化钠、0.2mg/mL聚山梨醇酯80、110mg/mL抗MASP2抗体、不同pH(pH6.4、pH6.6、pH6.8)的抗体制剂，考察高温40℃条件下抗体制剂稳定性。序号1)至3)如下：
1)10mM组氨酸-盐酸组氨酸，0.2mg/mL聚山梨醇酯80，8.2mg/mL氯化钠，蛋白浓度110mg/mL，pH6.4
2)10mM组氨酸-盐酸组氨酸，0.2mg/mL聚山梨醇酯80，8.2mg/mL氯化钠，蛋白浓度110mg/mL，pH6.6
3)10mM组氨酸-盐酸组氨酸，0.2mg/mL聚山梨醇酯80，8.2mg/mL氯化钠，蛋白浓度110mg/mL，pH6.8
表19抗MASP2抗体制剂pH对稳定性影响考察结果

(注：h表示小时；W表示周；粒径反映胶体稳定性；％PD表示分散系数)
表20抗MASP2抗体制剂pH对稳定性影响考察结果

(注：h表示小时；W表示周)
结果表明：pH6.4、pH6.6和pH6.8对抗MASP2抗体稳定性影响的差异不大，所以抗MASP2抗体制剂的pH范围为6.4～6.8。
实施例15.抗MASP2抗体制剂辅料种类筛选
选择10mM组氨酸-盐酸组氨酸pH6.6缓冲体系，制备含200mg/mL蛋白浓度的抗MASP2抗体、不同种类辅料(40mg/mL蔗糖+15mg/mL盐酸精氨酸、30mg/mL盐酸精氨酸、8.0mg/mL氯化钠)、0.4mg/mL聚山梨醇酯80的抗体制剂，考察冻存条件下蛋白稳定性。序号1)至3)如下：
1)10mM组氨酸-盐酸组氨酸，0.4mg/mL聚山梨醇酯80，8.0mg/mL氯化钠，蛋白浓度200mg/mL，pH6.6
2)10mM组氨酸-盐酸组氨酸，0.4mg/mL聚山梨醇酯80，40mg/mL蔗糖+15mg/mL盐酸精氨酸，蛋白浓度200mg/mL，pH6.6
3)10mM组氨酸-盐酸组氨酸，0.4mg/mL聚山梨醇酯80，30mg/mL盐酸精氨酸，蛋白浓度200mg/mL，pH6.6
表21抗MASP2抗体制剂辅料种类筛选结果

(注：W表示周，制剂序号1～3蛋白样品T0时SEC-单体为98.5％，SEC聚体为1.4％)
结果表明：-35℃冻存条件下，含蔗糖+盐酸精氨酸的制剂对SEC纯度保护优于L含氯化钠或盐酸精氨酸处方。
实施例16.抗MASP2抗体制剂蛋白浓度筛选
制备含165mg/mL抗MASP2抗体，考察高温(40℃)、振荡(200rpm，室温)、光照(5000±500lx)、冻融(-35℃/室温)条件下蛋白稳定性，制剂如序号1)：
1)10mM组氨酸-盐酸组氨酸，0.4mg/mL聚山梨醇酯80，40mg/mL蔗糖+15mg/mL盐酸精氨酸，蛋白浓度165mg/mL，pH6.6
表22抗MASP2抗体制剂稳定性

(注：W表示周，FVP表示无可见蛋白颗粒)
结果表明：上述含蔗糖+盐酸精氨酸，蛋白浓度165mg/mL的制剂，所有考察条件下SEC和iCIEF纯度及外观均无明显变化，稳定性良好。
另外，150mg/mL蛋白浓度的制剂(10mM组氨酸-盐酸组氨酸，0.4mg/mL聚山梨醇酯80，40mg/mL蔗糖+15mg/mL盐酸精氨酸，蛋白浓度150mg/mL，pH6.6)，在高温下(40℃，2周)iCIEF纯度和SEC纯度结果显示该制剂稳定性良好。
实施例17.可选择制剂配方
本发明提供“90～165mg/mL抗MASP2抗体，约8.2mg/mL氯化钠或40mg/mL蔗糖，15mg/mL盐酸精氨酸，0.1～0.4mg/mL聚山梨醇酯80，约10mM组氨酸-盐酸组氨酸，pH6.4～6.8”制剂配方的抗MASP2抗体药物制剂，包含但不限于：
(1)90、100或110mg/mL抗MASP2抗体，8.2mg/mL氯化钠，0.1mg/mL聚山梨醇酯80，和10mM组氨酸-盐酸组氨酸，pH6.4
(2)90、100或110mg/mL抗MASP2抗体，8.2mg/mL氯化钠，0.2mg/mL聚山梨醇酯80，和10mM组氨酸-盐酸组氨酸，pH6.4
(3)90、100或110mg/mL抗MASP2抗体，8.2mg/mL氯化钠，0.4mg/mL聚山梨醇酯80，和10mM组氨酸-盐酸组氨酸，pH6.4
(4)90、100或110mg/mL抗MASP2抗体，8.2mg/mL氯化钠，0.1mg/mL聚山梨醇酯80，和10mM组氨酸-盐酸组氨酸，pH6.6
(5)90、100或110mg/mL抗MASP2抗体，8.2mg/mL氯化钠，0.2mg/mL聚山梨醇酯80，和10mM组氨酸-盐酸组氨酸，pH6.6
(6)90、100或110mg/mL抗MASP2抗体，8.2mg/mL氯化钠，0.4mg/mL聚山梨醇酯80，和10mM组氨酸-盐酸组氨酸，pH6.6
(7)90、100或110mg/mL抗MASP2抗体，8.2mg/mL氯化钠，0.1mg/mL聚山梨醇酯80，和10mM组氨酸-盐酸组氨酸，pH6.8
(8)90、100或110mg/mL抗MASP2抗体，8.2mg/mL氯化钠，0.2mg/mL聚山梨醇酯80，和10mM组氨酸-盐酸组氨酸，pH6.8
(9)90、100或110mg/mL抗MASP2抗体，8.2mg/mL氯化钠，0.4mg/mL聚山梨醇酯80，和10mM组氨酸-盐酸组氨酸，pH6.8
(10)100mg/mL抗MASP2抗体，8.2mg/mL氯化钠，0.4mg/mL聚山梨醇酯80，和10mM组氨酸-盐酸组氨酸，pH6.6
(11)135、150或165mg/mL抗MASP2抗体，40mg/mL蔗糖，15mg/mL盐酸精氨酸，0.4mg/mL聚山梨醇酯80，和10mM组氨酸-盐酸组氨酸，pH6.6
(12)150mg/mL抗MASP2抗体，40mg/mL蔗糖，15mg/mL盐酸精氨酸，0.4mg/mL聚山梨醇酯80，和10mM组氨酸-盐酸组氨酸，pH6.6。
实验结果表明，上述制剂配方的抗MASP2抗体制剂均具有良好的稳定性，可应用于抗MASP2抗体药物的配制。
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 (20)

  1. 一种药物组合物，其包含抗MASP2抗体或其抗原结合片段和缓冲剂，其中所述抗MASP2抗体或其抗原结合片段包含重链可变区(VH)和轻链可变区(VL)，其中：
    所述VH包含如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
    其中所述HCDR1、HCDR2、HCDR3和LCDR1、LCDR2、LCDR3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；
    其中所述缓冲剂选自磷酸盐缓冲剂、组氨酸盐缓冲剂，优选为组氨酸盐缓冲剂，更优选地为组氨酸-盐酸缓冲剂或组氨酸-醋酸缓冲剂，最优选地为组氨酸-盐酸组氨酸缓冲剂。
  2. 根据权利要求1所述的药物组合物，其中所述抗MASP2抗体或其抗原结合片段中的VH和VL如下：
    所述VH包含分别如SEQ ID NO：9、10、11所示的HCDR1、HCDR2、HCDR3，和所述VL包含分别如SEQ ID NO：12、13、14所示的LCDR1、LCDR2、LCDR3。
  3. 根据权利要求1或2所述的药物组合物，其中所述抗MASP2抗体为鼠源抗体、嵌合抗体、人源化抗体、全人抗体。
  4. 根据权利要求3所述的药物组合物，其中所述抗MASP2抗体或其抗原结合片段为人源化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中任一项所述的药物组合物，其中所述的抗MASP2抗体或其抗原结合片段包含：
    所述VH包含如SEQ ID NO：17-20中任一所示或与之具有至少90％、至少95％同一性的氨基酸序列，所述VL包含如SEQ ID NO：21或22所示或与之具有至少90％、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。
  6. 根据权利要求1至5中任一项所述的药物组合物，其中抗MASP2抗体或其抗原结合片段为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FC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中任一项所述的药物组合物，其中抗MASP2抗体或其抗原结合片段包含重链和轻链，其中：
    重链包含如SEQ ID NO：24所示或与之具有至少90％、至少95％同一性的氨基酸序列；轻链包含如SEQ ID NO：25所示或与之具有至少90％、至少95％同一性的氨基酸序列。
  8. 根据权利要求1至7中任一项所述的药物组合物，其中所述缓冲剂的pH为5.0-8.5，优选为5.5-8，更优选为6.0-7.5。
  9. 根据权利要求1至8中任一项所述的药物组合物，其中所述缓冲剂浓度为1-30mM，优选为5-20mM，最优选约10mM。
  10. 根据权利要求1至9中任一项所述的药物组合物，其还包含选自氨基酸或其盐、糖、多元醇和盐中的一种或多种辅料，所述辅料优选为盐，或糖和氨基酸或其盐；更优选为氯化钠，蔗糖和精氨酸或其盐，最优选为蔗糖和盐酸精氨酸。
  11. 根据权利要求10所述的药物组合物，其中所述辅料浓度为0.1-100mg/mL，优选为0.5-80mg/mL，更优选自约8.2mg/mL、约15mg/mL、约28mg/mL、约30mg/mL、约40mg/mL、约45mg/mL和约75mg/mL。
  12. 根据权利要求1至11中任一项所述的药物组合物，其还包含表面活性剂，所述表面活性剂优选为聚山梨醇，更优选为聚山梨醇酯80。
  13. 根据权利要求12所述的药物组合物，其中所述表面活性剂浓度为0.01-2mg/mL，优选为0.05-1mg/mL，更优选为0.1-0.4mg/mL。
  14. 根据权利要求1至13中任一项所述的药物组合物，其中所述抗MASP2抗体或其抗原结合片段浓度为0.1-500mg/mL，优选为5-300mg/mL，更优选选自约90mg/mL、约100mg/mL、约110mg/mL、约135mg/mL、约150mg/mL、约165mg/mL或约200mg/mL。
  15. 一种药物组合物，其包含以下任一组：
    A)0.1-300mg/mL的如权利要求1至7中任一项所述的抗MASP2抗体或其抗原结合片段；
    0.5-80mg/mL的氯化钠；
    5-30mM的组氨酸-盐酸缓冲剂；
    0.01-1mg/mL的聚山梨醇酯；
    pH为5.5-8.0；
    B)80-120mg/mL的如权利要求1至7中任一项所述的抗MASP2抗体或其抗原结合片段；
    5-20mg/mL的氯化钠；
    5-15mM的组氨酸-盐酸缓冲剂；
    0.05-0.6mg/mL的聚山梨醇酯；
    pH为5.5-7.5；
    C)90-110mg/mL的如权利要求1至7中任一项所述的抗MASP2抗体或其抗原结合片段；
    8-9mg/mL的氯化钠；
    8-12mM的组氨酸-盐酸缓冲剂；
    0.05-0.5mg/mL的聚山梨醇酯；
    pH为6.0-7.5；
    D)90-110mg/mL的如权利要求1至7中任一项所述的抗MASP2抗体或其抗原结合片段；
    约8.2mg/mL的氯化钠；
    约10mM的组氨酸-盐酸缓冲剂；
    0.1-0.4mg/mL的聚山梨醇酯；
    pH为6.1-7.1；
    E)0.1-500mg/mL的如权利要求1至7中任一项所述的抗MASP2抗体或其抗原结合片段；
    0.5-100mg/mL的蔗糖；
    0.5-80mg/mL的精氨酸或其盐；
    0.1-50mM的组氨酸-盐酸缓冲剂；
    0.01-1mg/mL的聚山梨醇酯；
    pH为5.5-8.0；
    F)5-300mg/mL的如权利要求1至7中任一项所述的抗MASP2抗体或其抗原结合片段；
    5-80mg/mL的蔗糖；
    1-60mg/mL的精氨酸或其盐；
    1-30mM的组氨酸-盐酸缓冲剂；
    0.05-0.6mg/mL的聚山梨醇酯；
    pH为5.5-7.5；
    G)50-250mg/mL的如权利要求1至7中任一项所述的抗MASP2抗体或其抗原结合片段；
    10-70mg/mL的蔗糖；
    5-30mg/mL的精氨酸或其盐；
    5-20mM的组氨酸-盐酸缓冲剂；
    0.05-0.5mg/mL的聚山梨醇酯；
    pH为6.0-7.5；
    H)90-200mg/mL的如权利要求1至7中任一项所述的抗MASP2抗体或其抗原结合片段；
    30-50mg/mL的蔗糖；
    10-20mg/mL的精氨酸或其盐；
    8-12mM的组氨酸-盐酸缓冲剂；
    0.1-0.4mg/mL的聚山梨醇酯；
    pH为6.1-7.1；
    I)90-200mg/mL的如权利要求1至7中任一项所述的抗MASP2抗体或其抗原结合片段；
    约40mg/mL的蔗糖；
    约15mg/mL精氨酸或其盐；
    约10mM的组氨酸-盐酸缓冲剂；
    0.1-0.4mg/mL的聚山梨醇酯；
    pH为6.4-6.8。
    优选地，A)至I)任一组所述药物组合物中，其中所述精氨酸或其盐为盐酸精氨酸；
    优选地，A)至I)任一组所述药物组合物中，其中所述聚山梨醇酯为聚山梨醇酯80。
  16. 一种药物组合物，其包含以下任一组：
    (1)约90mg/mL、约100mg/mL或约110mg/mL的如权利要求1至7中任一项所述的抗MASP2抗体或其抗原结合片段，约8.2mg/mL氯化钠，约0.1mg/mL聚山梨醇酯80，和约10mM组氨酸-盐酸组氨酸，pH为约6.4；
    (2)约90mg/mL、约100mg/mL或约110mg/mL的如权利要求1至7中任一项所述的抗MASP2抗体或其抗原结合片段，约8.2mg/mL氯化钠，约0.2mg/mL聚山梨醇酯80，和约10mM组氨酸-盐酸组氨酸，pH为约6.4；
    (3)约90mg/mL、约100mg/mL或约110mg/mL的如权利要求1至7中任一项所述的抗MASP2抗体或其抗原结合片段，约8.2mg/mL氯化钠，约0.4mg/mL聚山梨醇酯80，和约10mM组氨酸-盐酸组氨酸，pH为约6.4；
    (4)约90mg/mL、约100mg/mL或约110mg/mL的如权利要求1至7中任一项所述的抗MASP2抗体或其抗原结合片段，约8.2mg/mL氯化钠，约0.1mg/mL聚山梨醇酯80，和约10mM组氨酸-盐酸组氨酸，pH为约6.6；
    (5)约90mg/mL、约100mg/mL或约110mg/mL的如权利要求1至7中任一项所述的抗MASP2抗体或其抗原结合片段，约8.2mg/mL氯化钠，约0.2mg/mL聚山梨醇酯80，和约10mM组氨酸-盐酸组氨酸，pH为约6.6；
    (6)约90mg/mL、约100mg/mL或约110mg/mL的如权利要求1至7中任一项所述的抗MASP2抗体或其抗原结合片段，约8.2mg/mL氯化钠，约0.4mg/mL聚山梨醇酯80，和约10mM组氨酸-盐酸组氨酸，pH为约6.6；
    (7)约90mg/mL、约100mg/mL或约110mg/mL的如权利要求1至7中任一项所述的抗MASP2抗体或其抗原结合片段，约8.2mg/mL氯化钠，约0.1mg/mL聚山梨醇酯80，和约10mM组氨酸-盐酸组氨酸，pH为约6.8；
    (8)约90mg/mL、约100mg/mL或约110mg/mL的如权利要求1至7中任一项所述的抗MASP2抗体或其抗原结合片段，约8.2mg/mL氯化钠，约0.2mg/mL聚山梨醇酯80，和约10mM组氨酸-盐酸组氨酸，pH为约6.8；
    (9)约90mg/mL、约100mg/mL或约110mg/mL的如权利要求1至7中任一项所述的抗MASP2抗体或其抗原结合片段，约8.2mg/mL氯化钠，约0.4mg/mL聚山梨醇酯80，和约10mM组氨酸-盐酸组氨酸，pH为约6.8；
    (10)约135mg/mL、约150mg/mL、约165mg/mL或约200mg/mL的如权利要求1至7中任一项所述的抗MASP2抗体或其抗原结合片段，约40mg/mL蔗糖，约15mg/mL盐酸精氨酸，约0.1mg/mL聚山梨醇酯80，和约10mM组氨酸-盐酸组氨酸，pH为约6.4；
    (11)约135mg/mL、约150mg/mL、约165mg/mL或约200mg/mL的如权利要求1至7中任一项所述的抗MASP2抗体或其抗原结合片段，约40mg/mL蔗糖，约15mg/mL盐酸精氨酸，约0.2mg/mL聚山梨醇酯80，和约10mM组氨酸-盐酸组氨酸，pH为约6.4；
    (12)约135mg/mL、约150mg/mL、约165mg/mL或约200mg/mL的如权利要求1至7中任一项所述的抗MASP2抗体或其抗原结合片段，约40mg/mL蔗糖，约15mg/mL盐酸精氨酸，约0.4mg/mL聚山梨醇酯80，和约10mM组氨酸-盐酸组氨酸，pH为约6.4；
    (13)约135mg/mL、约150mg/mL、约165mg/mL或约200mg/mL的如权利要求1至7中任一项所述的抗MASP2抗体或其抗原结合片段，约40mg/mL蔗糖，约15mg/mL盐酸精氨酸，约0.1mg/mL聚山梨醇酯80，和约10mM组氨酸-盐酸组氨酸，pH为约6.6；
    (14)约135mg/mL、约150mg/mL、约165mg/mL或约200mg/mL的如权利要求1至7中任一项所述的抗MASP2抗体或其抗原结合片段，约40mg/mL蔗糖，约15mg/mL盐酸精氨酸，约0.2mg/mL聚山梨醇酯80，和约10mM组氨酸-盐酸组氨酸，pH为约6.6；
    (15)约135mg/mL、约150mg/mL、约165mg/mL或约200mg/mL的如权利要求1至7中任一项所述的抗MASP2抗体或其抗原结合片段，约40mg/mL蔗糖，约15mg/mL盐酸精氨酸，约0.4mg/mL聚山梨醇酯80，和约10mM组氨酸-盐酸组氨酸，pH为约6.6；
    (16)约135mg/mL、约150mg/mL、约165mg/mL或约200mg/mL的如权利要求1至7中任一项所述的抗MASP2抗体或其抗原结合片段，约40mg/mL蔗糖，约15mg/mL盐酸精氨酸，约0.1mg/mL聚山梨醇酯80，和约10mM组氨酸-盐酸组氨酸，pH为约6.8；
    (17)约135mg/mL、约150mg/mL、约165mg/mL或约200mg/mL的如权利要求1至7中任一项所述的抗MASP2抗体或其抗原结合片段，约40mg/mL蔗糖，约15mg/mL盐酸精氨酸，约0.2mg/mL聚山梨醇酯80，和约10mM组氨酸-盐酸组氨酸，pH为约6.8；
    (18)约135mg/mL、约150mg/mL、约165mg/mL或约200mg/mL的如权利要求1至7中任一项所述的抗MASP2抗体或其抗原结合片段，约40mg/mL蔗糖，约15mg/mL盐酸精氨酸，约0.4mg/mL聚山梨醇酯80，和约10mM组氨酸-盐酸组氨酸，pH为约6.8。
  17. 一种冻干制剂，所述冻干制剂复溶后可形成权利要求1至16中任一项所述的药物组合物，或所述冻干制剂通过将权利要求1至16中任一项所述的药物组合物经冷冻干燥获得。
  18. 一种复溶溶液，其中所述复溶溶液是通过将权利要求17所述的冻干制剂经复溶制备获得。
  19. 一种制品，其包括容器，该容器中装有如权利要求1至16中任一项所述的药物组合物，权利要求17所述的冻干制剂，或权利要求18所述的复溶溶液。
  20. 权利要求1至16中任一项所述的药物组合物、权利要求17所述的冻干制剂或权利要求18的复溶溶液在制备用于治疗疾病的药物中的用途；
    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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WO2022228364A1 (zh) * 2021-04-25 2022-11-03 江苏恒瑞医药股份有限公司 抗masp2抗体、其抗原结合片段及医药用途

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WO2022228364A1 (zh) * 2021-04-25 2022-11-03 江苏恒瑞医药股份有限公司 抗masp2抗体、其抗原结合片段及医药用途

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