Composition of anti-MASP2 antibody and medical application

Abstract

The invention relates to a composition of an anti-MASP2 antibody and a medical application of the composition. Specifically, the present disclosure relates to a pharmaceutical composition of an anti-MASP2 antibody or an antigen-binding fragment thereof, the pharmaceutical composition comprising an anti-MASP2 antibody or an antigen-binding fragment thereof and a buffering agent.

Description

Composition of anti-MASP2 antibodies and medical use thereof Technical Field

[0001] 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. Background Art

[0002] The complement system is a protein found in human and animal serum, tissue fluid, and on cell membranes. Upon activation, it becomes biologically active and mediates immune and inflammatory responses. The complement system is composed of nearly 40 components, most of which are glycoproteins, including C1q, C1r, C1s, C2-C9, factor D, factor B, as well as 10 regulatory proteins and 10 complement receptors. Complement is widely involved in the body's defense against microorganisms and immune regulation, and also in damaging responses in immunopathology. Complement is a crucial effector and effector amplification system of innate immunity.

[0003] The complement system activation process manifests as a cascade of serine protease reactions and is divided into three types: the classical pathway, the alternative pathway, and the lectin pathway, ultimately mediating the activation of the terminal pathway, marked by the formation of the membrane-permeabilizing complex. In healthy individuals, the alternative pathway maintains low-level activation to monitor for invading pathogens. Healthy cells also suppress complement attack by expressing complement regulatory proteins such as CD55 and CD59. All three pathways are typically activated on the surfaces of apoptotic cells and microorganisms. In the classical pathway, antibodies (IgG1, IgG, IgG3, IgG4, or IgM) bind to antigens to form immune complexes. Recognition by C1q, C1s, and C1r activates C2 and C4, forming C4bC2a (i.e., C3 convertase), which ultimately promotes the formation of the membrane-permeabilizing complex composed of C5-C9. The alternative pathway is an activation pathway that directly initiates with the spontaneous hydrolysis of C3. Under stimulation by activating substances such as bacterial cell wall components LPS, polysaccharides such as zymosan, peptidoglycan, and teichoic acid, Factor D hydrolyzes C3-bound Factor B to form C3bBb3, which then completes a chain reaction involving components C5 to C9. The lectin pathway involves mannan-binding lectin (MBL) or fibrin (FCN) in plasma directly recognizing sugar structures with terminal glycosylation groups such as mannose, N-acetylglucosamine, N-acetylglucosamine, and fucose on the surfaces of various pathogenic microorganisms, subsequently activating the classical complement pathway.

[0004] Clinical trials and research evidence indicate that abnormal activation of the complement system is associated with acute sepsis, ischemia-reperfusion-related stroke, myocardial infarction, and transplant rejection; as well as chronic autoimmune diseases such as arthritis, age-related macular degeneration, microvascular thrombosis, chronic kidney disease, and hemolytic diseases. Among the currently under-developed complement-targeting drugs, the C5 antibody eculizumab was approved for marketing in 2007 and is primarily used to treat hemolytic diseases such as paronychia nodosum (PNH), aHUS, and myasthenia gravis. Other anti-MASP2 antibodies targeting the lectin pathway, as well as drugs targeting Factor D, Factor B, and C3 in the alternative pathway, are also in Phase II and III clinical trials. The safety profile of complement-targeting drugs is generally safe and tolerable. Given that the complement system is involved in regulating B cell development and T cell activation, mice deficient in complement factor genes exhibit reproductive and embryonic developmental defects and are at increased risk of infection. Therefore, developing drugs targeting specific upstream targets in the complement pathway can minimize the side effects of overall complement pathway inhibition while maintaining efficacy.

[0005] The MASP2 protein is a core hydrolase in the lectin pathway. It consists of an N-terminal CUB domain responsible for binding to MBL, an EGF domain, a CCP domain that binds to downstream substrates C4 and C2, and a C-terminal enzymatic domain, SP. The MBL-MASP complex binds to pathogen surface carbohydrate structures, leading to independent activation of MASP-1 and MASP-2. Activated MASP2 exerts its SP activity, cleaving C4 and C2 to form the C3 convertase C4b2a, ultimately activating the complement system mediated by the lectin pathway. It has been implicated in IgA nephropathy, stroke, and myocardial ischemia. In mouse models of stroke (MCAO) and acute myocardial infarction, MASP2 deficiency significantly reduces infarct size. MBL-MASP2 primarily recognizes IgA lacking galactose modification. Activation of the LP pathway in the glomerular mesangium promotes cytokine secretion, ultimately leading to damage to renal tubular epithelial cells and podocytes and abnormal renal function.

[0006] Omeros' MASP2 monoclonal antibody, narsoplimab (OMS721), primarily inhibits lectin-mediated complement system activation and is being developed for the treatment of a variety of inflammatory 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. TMA studies are in pre-registration status in the United States, while HUS and IgA nephropathy studies are in Phase III clinical trials, and lupus nephritis, membranous glomerulonephritis, and glomerulonephritis studies are in Phase II clinical trials.

[0007] Given the important role of MASP2 in the lectin pathway, providing new anti-MASP2 antibodies to inhibit MASP2-dependent complement activation and treat diseases caused by abnormal complement system remains an urgent problem to be solved in this field.

[0008] The applicant's patent application WO2022228364 provides an anti-MASP2 antibody with a new structure, which has good inhibitory activity against MASP2-dependent complement activation.

[0009] Antibody drugs are an important class of biopharmaceuticals. Their large molecular weight and complex structure make them susceptible to physical or chemical degradation and aggregation during production, storage, and use, leading to reduced activity or even ineffectiveness. Therefore, developing excellent antibody preparations is crucial.

[0010] Summary of the Invention

[0011] The present disclosure provides a pharmaceutical composition comprising an anti-MASP2 antibody or an antigen-binding fragment thereof, wherein the composition has excellent stability.

[0012] The present disclosure provides a pharmaceutical composition comprising an anti-MASP2 antibody or antigen-binding fragment thereof and a buffer, wherein the buffer is selected from tris (hydroxymethylaminomethane) buffer, acetate buffer, succinate buffer, phosphate buffer, histidine salt buffer, acetate buffer, citrate buffer, tartrate buffer, fumarate buffer, and glycylglycine buffer. In certain embodiments, the buffer is selected from phosphate buffer and histidine salt buffer. In certain embodiments, 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-hydrochloric acid buffer. In certain embodiments, the buffer is a histidine-histidine hydrochloride buffer.

[0013] In certain embodiments, the pharmaceutical composition as described in any of the above items, wherein 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. In certain embodiments, the pH of the buffer is 6.0-7.5. In certain embodiments, the pH of the pharmaceutical composition has a difference of no more than ±0.5 compared to the pH of the buffer it contains.

[0014] In certain embodiments, 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 therebetween.

[0015] In certain embodiments, the pharmaceutical composition as described in any of the above, wherein 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 therebetween. In certain embodiments, the buffer concentration is about 10mM.

[0016] In certain embodiments, the pharmaceutical composition as described in any of the above items further comprises an adjuvant. In certain embodiments, the adjuvant is selected from one or more of an amino acid or a salt thereof, a sugar, a polyol, and a salt. In certain embodiments, the adjuvant is selected from one or more of an amino acid or a salt thereof, a sugar, and a salt. In certain embodiments, the adjuvant is a salt. In certain embodiments, the adjuvant is an amino acid or a salt thereof. In certain embodiments, the adjuvant is a sugar and an amino acid or a salt thereof. In certain embodiments, the amino acid is selected from one or more of proline, aspartic acid, glutamic acid, lysine, arginine, glycine, and histidine. In certain embodiments, the sugar is selected from one or more of glucose, sucrose, maltose, and trehalose. In certain embodiments, the polyol is selected from one or more of mannitol and sorbitol. In certain embodiments, the salt is selected from sodium chloride or a salt of an amino acid, for example, sodium chloride. In certain embodiments, the adjuvant is selected from one or more of sodium chloride, sucrose, and arginine or a salt thereof. In certain embodiments, the adjuvant is sodium chloride. In certain embodiments, the adjuvant is arginine or a salt thereof, for example, arginine hydrochloride. In certain embodiments, the excipients are sucrose and arginine or a salt thereof, such as sucrose and arginine hydrochloride.

[0017] In certain embodiments, 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, 1 0-40mg / 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 include about 1 mg / mL, about 2 mg / mL, about 3 mg / mL, about 4 mg / mL, about 5 mg / mL, about 6 mg / mL, about 7 mg / mL, about 7.2 mg / mL, about 7.4 mg / mL, about 7.6 mg / mL, about 7.8 mg / mL, about 8 mg / mL, about 8.2 mg / mL, about 8.4 mg / mL, about 8.6 mg / mL, about 8.8 mg / mL, about 9 mg / mL, about 10 mg / mL, about 11 mg / mL, about 12 mg / mL, about 13 mg / mL mL, about 14 mg / mL, about 15 mg / mL, about 16 mg / mL, about 17 mg / mL, about 18 mg / mL, about 19 mg / mL, about 20 mg / mL, about 21 mg / mL, about 22 mg / mL, about 23 mg / mL, about 24 mg / mL, about 25 mg / mL, about 26 mg / mL, about 27 mg / mL, about 28 mg / mL, about 29 mg / mL, about 30 mg / mL, about 31 mg / mL, about 32 mg / mL, about 33 mg / mL, about 34 mg / mL, about 35 mg / mL , about 36 mg / mL, about 37 mg / mL, about 38 mg / mL, about 39 mg / mL, about 40 mg / mL, about 41 mg / mL, about 42 mg / mL, about 43 mg / mL, about 44 mg / mL, about 45 mg / mL, about 46 mg / mL, about 47 mg / mL, about 48 mg / mL, about 49 mg / mL, about 50 mg / mL, about 55 mg / mL, about 60 mg / mL, about 65 mg / mL, about 70 mg / mL, about 75 mg / mL, about 80 mg / mL, or any value therebetween, or any value therebetween.In certain embodiments, the concentration of the adjuvant is about 8.2 mg / mL, about 15 mg / mL, about 28 mg / mL, about 30 mg / mL, about 40 mg / mL, about 45 mg / mL and about 75 mg / mL. In certain embodiments, the concentration of the adjuvant is about 5-10 mg / 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.

[0018] In certain embodiments, the pharmaceutical composition as described in any one of the above is an isotonic preparation. In some embodiments, the osmotic pressure of the pharmaceutical composition is 260-380mOsm, for example 270-360mOsm, 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, about 310mOsm, about 320mOsm, about 330mOsm, about 340mOsm, about 350mOsm, about 360mOsm, about 370mOsm, about 380mOsm, about 390mOsm, about 400mOsm, about 410mOsm, about 420mOsm, about 430mOsm, about 440mOsm, about 450mOsm, about 460mOsm, about 470mOsm, about 480mOsm, about 490mOsm, about 500mOsm, about 510mOsm, about 520mOsm, about 530mOsm, about 540mOsm, about 550mOsm In some embodiments, the pharmaceutical composition has an osmotic pressure of about 302 mOsm, about 304 mOsm, about 306 mOsm, about 308 mOsm, about 310 mOsm, about 312 mOsm, about 314 mOsm, about 316 mOsm, about 318 mOsm, about 320 mOsm, about 322 mOsm, about 324 mOsm, about 326 mOsm, about 328 mOsm, about 330 mOsm, about 335 mOsm, about 340 mOsm, about 345 mOsm, about 350 mOsm, about 355 mOsm, about 360 mOsm, or any value therebetween. In some embodiments, the pharmaceutical composition has an osmotic pressure of 270-360 mOsm.

[0019] In certain embodiments, the pharmaceutical composition of the present disclosure further comprises a surfactant. In certain embodiments, the surfactant is an ionic or nonionic surfactant. In certain embodiments, the surfactant is selected from polysorbate, polyhydroxyalkylene, Triton, sodium dodecyl sulfate, sodium lauryl sulfate, sodium octyl glucoside, lauryl-sulfobetaine, myristyl-sulfobetaine, linoleyl-sulfobetaine, stearyl-sulfobetaine, lauryl-sarcosine, myristyl-sarcosine, linoleyl-sarcosine, stearyl-sarcosine, linoleyl-betaine, myristyl-betaine, cetyl-betaine, lauroyl One or more of the copolymer of amidopropyl-betaine, cocamidopropyl-betaine, linoleamidopropyl-betaine, myristamidopropyl-betaine, palmitamidopropyl-betaine, isostearamidopropyl-betaine, myristamidopropyl-dimethylamine, palmitamidopropyl-dimethylamine, isostearamidopropyl-dimethylamine, methyl cocoyl sodium, methyl oleyl taurate sodium, polyethylene glycol, polypropylene glycol and ethylene and propylene glycol. In certain embodiments, described surfactant is polysorbate 20. In certain embodiments, described surfactant is polysorbate 80 or polysorbate 20, for example, polysorbate 80.

[0020] In certain embodiments, the surfactant concentration in the pharmaceutical composition is 0.01-2 mg / mL, for example, 0.05-1 mg / mL, 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. In certain embodiments, 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.

[0021] In certain embodiments, 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. L, about 50 mg / mL, about 55 mg / mL, about 60 mg / mL, about 65 mg / mL, about 70 mg / mL, about 75 mg / mL, about 80 mg / mL, about 85 mg / mL, about 90 mg / mL, about 95 mg / mL, about 100 mg / mL, about 105 mg / mL, about 110 mg / mL, about 115 mg / mL, about 120 mg / mL, about 125 mg / mL, about 130 In some embodiments, 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 140 mg / mL, about 145 mg / mL, about 150 mg / mL, about 155 mg / mL, about 160 mg / mL, about 165 mg / mL, about 170 mg / mL, about 175 mg / mL, about 180 mg / mL, about 185 mg / mL, about 190 mg / mL, about 195 mg / mL, about 200 mg / mL, or any value therebetween. In certain embodiments, 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. In certain embodiments, the concentration of the anti-MASP2 antibody or antigen-binding fragment thereof 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.

[0022] In certain embodiments, the present disclosure provides a pharmaceutical composition comprising an anti-MASP2 antibody or antigen-binding fragment thereof (e.g., 77H11 (H3L1) whose heavy and light chain sequences are SEQ ID NOs: 24 and 25), which comprises or is any one of the following groups 1) to 21):

[0023] 1) anti-MASP2 antibodies or antigen-binding fragments thereof;

[0024] Sodium chloride, mannitol, and / or amino acids (e.g., proline);

[0025] Histidine-HCl buffer (e.g., histidine-histidine hydrochloride buffer);

[0026] Polysorbates (e.g., polysorbate 80);

[0027] pH 5.5-8.0;

[0028] 2) 0.1-300 mg / mL of an anti-MASP2 antibody or antigen-binding fragment thereof;

[0029] 0.5-80 mg / mL sodium chloride, mannitol, and / or amino acids (e.g., proline);

[0030] 5-30 mM histidine-HCl buffer (e.g., histidine-histidine HCl buffer);

[0031] 0.01-1 mg / mL polysorbate (e.g., polysorbate 80);

[0032] pH 5.5-8.0;

[0033] 3) 0.1-300 mg / mL of an anti-MASP2 antibody or antigen-binding fragment thereof;

[0034] 2-50 mg / mL sodium chloride;

[0035] 5-20 mM histidine-HCl buffer (e.g., histidine-histidine HCl buffer);

[0036] 0.05-1 mg / mL of polysorbate (e.g., polysorbate 80);

[0037] pH 5.5-8.0;

[0038] 4) 50-150 mg / mL of an anti-MASP2 antibody or antigen-binding fragment thereof;

[0039] 2-30 mg / mL sodium chloride;

[0040] 2-20 mM histidine-HCl buffer (e.g., histidine-histidine HCl buffer);

[0041] 0.05-0.8 mg / mL of polysorbate (e.g., polysorbate 80);

[0042] pH 5.5-7.5;

[0043] 5) 80-120 mg / mL of an anti-MASP2 antibody or antigen-binding fragment thereof;

[0044] 5-20 mg / mL sodium chloride;

[0045] 5-15 mM histidine-HCl buffer (e.g., histidine-histidine HCl buffer);

[0046] 0.05-0.6 mg / mL of polysorbate (e.g., polysorbate 80);

[0047] pH 5.5-7.5 or 6.0-7.5;

[0048] 6) 90-110 mg / mL of an anti-MASP2 antibody or antigen-binding fragment thereof;

[0049] 8-9 mg / mL sodium chloride;

[0050] 8-12 mM histidine-HCl buffer (e.g., histidine-histidine HCl buffer);

[0051] 0.05-0.5 mg / mL of polysorbate (e.g., polysorbate 80);

[0052] pH 5.5-7.5 or 6.0-7.5;

[0053] 7) 90-110 mg / mL of an anti-MASP2 antibody or antigen-binding fragment thereof;

[0054] 8-9 (e.g., about 8.2) mg / mL of sodium chloride;

[0055] About 10 mM histidine-HCl buffer (e.g., histidine-histidine HCl buffer);

[0056] About 0.1-0.4 mg / mL of polysorbate (e.g., polysorbate 80);

[0057] 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);

[0058] 8) about 90 mg / mL, about 95 mg / mL, about 100 mg / mL, about 105 mg / mL, or about 110 mg / mL of an anti-MASP2 antibody or antigen-binding fragment thereof;

[0059] approximately 8.2 mg / mL of sodium chloride;

[0060] About 10 mM histidine-HCl buffer (e.g., histidine-histidine HCl buffer);

[0061] about 0.1, about 0.2, or about 0.4 mg / mL of polysorbate (e.g., polysorbate 80);

[0062] a pH of about 6.4, about 6.6, or about 6.8;

[0063] 9) anti-MASP2 antibodies or antigen-binding fragments thereof;

[0064] Histidine-HCl buffer (e.g., histidine-histidine hydrochloride buffer);

[0065] a pH of 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);

[0066] 10) anti-MASP2 antibodies or antigen-binding fragments thereof;

[0067] Sodium chloride, or sucrose and arginine or a salt thereof (e.g., arginine hydrochloride);

[0068] 11) anti-MASP2 antibodies or antigen-binding fragments thereof;

[0069] Sodium chloride, or sucrose and arginine or a salt thereof (e.g., arginine hydrochloride);

[0070] Histidine-HCl buffer (e.g., histidine-histidine hydrochloride buffer);

[0071] Polysorbates (e.g., polysorbate 80);

[0072] a pH of 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);

[0073] 12) 0.1-500 mg / mL of an anti-MASP2 antibody or antigen-binding fragment thereof;

[0074] 0.5-100 mg / mL sucrose;

[0075] 0.5-80 mg / mL arginine or a salt thereof (e.g., arginine hydrochloride);

[0076] 0.1-50 mM histidine-HCl buffer;

[0077] 0.01-1 mg / mL of polysorbate (e.g., polysorbate 80);

[0078] pH 5.5-8.0;

[0079] 13) 5-300 mg / mL of an anti-MASP2 antibody or antigen-binding fragment thereof;

[0080] 5-80 mg / mL sucrose;

[0081] 1-60 mg / mL arginine or a salt thereof (e.g., arginine hydrochloride);

[0082] 1-30 mM histidine-HCl buffer;

[0083] 0.05-0.6 mg / mL of polysorbate (e.g., polysorbate 80);

[0084] pH 5.5-7.5;

[0085] 14) 50-250 mg / mL of an anti-MASP2 antibody or antigen-binding fragment thereof;

[0086] 10-70 mg / mL sucrose;

[0087] 5-30 mg / mL arginine or a salt thereof (e.g., arginine hydrochloride);

[0088] 5-20 mM histidine-HCl buffer;

[0089] 0.05-0.5 mg / mL of polysorbate (e.g., polysorbate 80);

[0090] pH 6.0-7.5;

[0091] 15) 90-200 mg / mL of an anti-MASP2 antibody or antigen-binding fragment thereof;

[0092] 30-50 mg / mL sucrose;

[0093] 10-20 mg / mL arginine or a salt thereof (e.g., arginine hydrochloride);

[0094] 8-12 mM histidine-HCl buffer;

[0095] 0.1-0.4 mg / mL of polysorbate (e.g., polysorbate 80);

[0096] pH 6.1-7.1;

[0097] 16) about 90-200 mg / mL or about 90-165 mg / mL of an anti-MASP2 antibody or antigen-binding fragment thereof;

[0098] approximately 40 mg / mL of sucrose;

[0099] about 15 mg / mL of arginine or a salt thereof (e.g., arginine hydrochloride);

[0100] approximately 10 mM histidine-HCl buffer;

[0101] 0.1-0.4 mg / mL of polysorbate (e.g., polysorbate 80);

[0102] a pH of about 6.4, about 6.6, or about 6.8;

[0103] 17) 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 of an anti-MASP2 antibody or antigen-binding fragment thereof;

[0104] approximately 40 mg / mL of sucrose;

[0105] about 15 mg / mL of arginine or a salt thereof (e.g., arginine hydrochloride);

[0106] approximately 10 mM histidine-HCl buffer;

[0107] 0.1-0.4 mg / mL of polysorbate (e.g., polysorbate 80);

[0108] pH 6.4-6.8;

[0109] 18) about 90-200 mg / mL or about 90-165 mg / mL of an anti-MASP2 antibody or antigen-binding fragment thereof;

[0110] approximately 40 mg / mL of sucrose;

[0111] about 15 mg / mL arginine or a salt thereof (e.g., arginine hydrochloride);

[0112] approximately 10 mM histidine-HCl buffer;

[0113] about 0.1 mg / mL, about 0.2 mg / mL, or about 0.4 mg / mL of polysorbate (e.g., polysorbate 80);

[0114] pH 6.4-6.8;

[0115] 19) about 90-200 mg / mL or about 90-165 mg / mL of an anti-MASP2 antibody or antigen-binding fragment thereof;

[0116] approximately 40 mg / mL of sucrose;

[0117] about 15 mg / mL arginine or a salt thereof (e.g., arginine hydrochloride);

[0118] approximately 10 mM histidine-HCl buffer;

[0119] About 0.1-0.4 mg / mL of polysorbate (e.g., polysorbate 80);

[0120] pH 6.4-6.8;

[0121] 20) The pharmaceutical composition of any one of 1) to 19) above, wherein the arginine or its salt is arginine hydrochloride; 21) The pharmaceutical composition of any one of 1) to 20) above, wherein the polysorbate is polysorbate 80;

[0122] 22) The pharmaceutical composition according to any one of 1) to 21) above, further comprising water for injection;

[0123] 23) The pharmaceutical composition according to any one of 1) to 22) above, further comprising a pH adjuster, such as hydrochloric acid and / or sodium hydroxide.

[0124] In certain embodiments, the present disclosure provides a pharmaceutical composition comprising an anti-MASP2 antibody or antigen-binding fragment thereof (e.g., 77H11 (H3L1) whose heavy and light chain sequences are SEQ ID NOs: 24 and 25), comprising any one of the following groups 1) to 9):

[0125] 1) 50-200 mg / mL of an anti-MASP2 antibody or antigen-binding fragment thereof;

[0126] 10-100 mg / mL (e.g., 30-60, 35-55, 40-50, about 40, about 45, about 47, about 50 mg / mL) of mannitol;

[0127] 5-15 mM histidine-HCl buffer (e.g., histidine-histidine HCl buffer);

[0128] 0.05-0.6 mg / mL of polysorbate (e.g., polysorbate 80);

[0129] pH is 5.5-7.5.

[0130] 2) 80-120 mg / mL of an anti-MASP2 antibody or antigen-binding fragment thereof;

[0131] 10-100 mg / mL (e.g., 30-60, 35-55, 40-50, about 40, about 45, about 47, about 50 mg / mL) of mannitol;

[0132] 8-12 mM histidine-HCl buffer (e.g., histidine-histidine HCl buffer);

[0133] 0.05-0.5 mg / mL of polysorbate (e.g., polysorbate 80);

[0134] pH is 6.0-7.5.

[0135] 3) 90-110 mg / mL of an anti-MASP2 antibody or antigen-binding fragment thereof;

[0136] 10-100 mg / mL (e.g., 30-60, 35-55, 40-50, about 40, about 45, about 47, about 50 mg / mL) of mannitol;

[0137] About 10 mM histidine-HCl buffer (e.g., histidine-histidine HCl buffer);

[0138] about 0.1-0.4 mg / mL of polysorbate (e.g., polysorbate 80) (e.g., about 0.1, about 0.2, about 0.4 mg / mL);

[0139] 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).

[0140] 4) 50-200 mg / mL of an anti-MASP2 antibody or antigen-binding fragment thereof;

[0141] 10-60 mg / mL (e.g., 10-50, 20-40, 27-30, about 27, about 28, about 30 mg / mL) mg / mL of proline;

[0142] 5-15 mM histidine-HCl buffer (e.g., histidine-histidine HCl buffer);

[0143] 0.05-0.6 mg / mL of polysorbate (e.g., polysorbate 80);

[0144] pH is 5.5-7.5.

[0145] 5) 80-120 mg / mL of an anti-MASP2 antibody or antigen-binding fragment thereof;

[0146] 10-60 mg / mL (e.g., 10-50, 20-40, 27-30, about 27, about 28, about 30 mg / mL) mg / mL of proline;

[0147] 8-12 mM histidine-HCl buffer (e.g., histidine-histidine HCl buffer);

[0148] 0.05-0.5 mg / mL of polysorbate (e.g., polysorbate 80);

[0149] pH is 6.0-7.5.

[0150] 6) 90-110 mg / mL of an anti-MASP2 antibody or antigen-binding fragment thereof;

[0151] 10-60 mg / mL (e.g., 10-50, 20-40, 27-30, about 27, about 28, about 30 mg / mL) mg / mL of proline;

[0152] About 10 mM histidine-HCl buffer (e.g., histidine-histidine HCl buffer);

[0153] about 0.1-0.4 mg / mL of polysorbate (e.g., polysorbate 80) (e.g., about 0.1, about 0.2, about 0.4 mg / mL);

[0154] 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).

[0155] 7) 50-200 mg / mL of an anti-MASP2 antibody or antigen-binding fragment thereof;

[0156] 20-200 mg / mL (e.g., 30-150, 40-120, 50-100, 60-90, about 65, about 70, about 75, about 80, about 90 mg / mL) of sucrose;

[0157] 5-15 mM histidine-HCl buffer (e.g., histidine-histidine HCl buffer);

[0158] 0.05-0.6 mg / mL of polysorbate (e.g., polysorbate 80);

[0159] pH is 5.5-7.5.

[0160] 8) 80-120 mg / mL of an anti-MASP2 antibody or antigen-binding fragment thereof;

[0161] 20-200 mg / mL (e.g., 30-150, 40-120, 50-100, 60-90, about 65, about 70, about 75, about 80, about 90 mg / mL) of sucrose;

[0162] 8-12 mM histidine-HCl buffer (e.g., histidine-histidine HCl buffer);

[0163] 0.05-0.5 mg / mL of polysorbate (e.g., polysorbate 80);

[0164] pH is 6.0-7.5.

[0165] 9) 90-110 mg / mL of an anti-MASP2 antibody or antigen-binding fragment thereof;

[0166] 20-200 mg / mL (e.g., 30-150, 40-120, 50-100, 60-90, about 65, about 70, about 75, about 80, about 90 mg / mL) of sucrose;

[0167] About 10 mM histidine-HCl buffer (e.g., histidine-histidine HCl buffer);

[0168] about 0.1-0.4 mg / mL of polysorbate (e.g., polysorbate 80) (e.g., about 0.1, about 0.2, about 0.4 mg / mL);

[0169] 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).

[0170] 10) The pharmaceutical composition of any one of 1) to 9) above, wherein the arginine or its salt is arginine hydrochloride; 21) The pharmaceutical composition of any one of 1) to 20) above, wherein the polysorbate is polysorbate 80;

[0171] 11) The pharmaceutical composition according to any one of 1) to 10) above, further comprising water for injection;

[0172] 12) The pharmaceutical composition according to any one of the above 1) to 11) further comprises a pH adjuster, such as hydrochloric acid and / or sodium hydroxide.

[0173] In certain embodiments, the present disclosure provides a pharmaceutical composition comprising an anti-MASP2 antibody or antigen-binding fragment thereof (e.g., 77H11 (H3L1) whose heavy and light chain sequences are SEQ ID NOs: 24 and 25), which comprises or is any one of the following groups 1) to 20):

[0174] (1) about 90 mg / mL, about 100 mg / mL, or about 110 mg / mL of an anti-MASP2 antibody or antigen-binding fragment thereof, about 8.2 mg / mL sodium chloride, about 0.1 mg / mL polysorbate 80, and about 10 mM histidine-histidine hydrochloride, at a pH of about 6.4;

[0175] (2) about 90 mg / mL, about 100 mg / mL, or about 110 mg / mL of an anti-MASP2 antibody or antigen-binding fragment thereof, about 8.2 mg / mL sodium chloride, about 0.2 mg / mL polysorbate 80, and about 10 mM histidine-histidine hydrochloride, at a pH of about 6.4;

[0176] (3) about 90 mg / mL, about 100 mg / mL, or about 110 mg / mL of an anti-MASP2 antibody or antigen-binding fragment thereof, about 8.2 mg / mL sodium chloride, about 0.4 mg / mL polysorbate 80, and about 10 mM histidine-histidine hydrochloride, at a pH of about 6.4;

[0177] (4) about 90 mg / mL, about 100 mg / mL, or about 110 mg / mL of an anti-MASP2 antibody or antigen-binding fragment thereof, about 8.2 mg / mL sodium chloride, about 0.1 mg / mL polysorbate 80, and about 10 mM histidine-histidine hydrochloride, at a pH of about 6.6;

[0178] (5) about 90 mg / mL, about 100 mg / mL, or about 110 mg / mL of an anti-MASP2 antibody or antigen-binding fragment thereof, about 8.2 mg / mL sodium chloride, about 0.2 mg / mL polysorbate 80, and about 10 mM histidine-histidine hydrochloride, at a pH of about 6.6;

[0179] (6) about 90 mg / mL, about 100 mg / mL, or about 110 mg / mL of an anti-MASP2 antibody or antigen-binding fragment thereof, about 8.2 mg / mL sodium chloride, about 0.4 mg / mL polysorbate 80, and about 10 mM histidine-histidine hydrochloride, at a pH of about 6.6;

[0180] (7) about 90 mg / mL, about 100 mg / mL, or about 110 mg / mL of an anti-MASP2 antibody or antigen-binding fragment thereof, about 8.2 mg / mL sodium chloride, about 0.1 mg / mL polysorbate 80, and about 10 mM histidine-histidine hydrochloride, at a pH of about 6.8;

[0181] (8) about 90 mg / mL, about 100 mg / mL, or about 110 mg / mL of an anti-MASP2 antibody or antigen-binding fragment thereof, about 8.2 mg / mL sodium chloride, about 0.2 mg / mL polysorbate 80, and about 10 mM histidine-histidine hydrochloride, at a pH of about 6.8;

[0182] (9) about 90 mg / mL, about 100 mg / mL, or about 110 mg / mL of an anti-MASP2 antibody or antigen-binding fragment thereof, about 8.2 mg / mL sodium chloride, about 0.4 mg / mL polysorbate 80, and about 10 mM histidine-histidine hydrochloride, at a pH of about 6.8;

[0183] (10) about 135 mg / mL, about 150 mg / mL, about 165 mg / mL, or about 200 mg / mL of an anti-MASP2 antibody or antigen-binding fragment thereof, about 40 mg / mL sucrose, about 15 mg / mL arginine hydrochloride, about 0.1 mg / mL polysorbate 80, and about 10 mM histidine-histidine hydrochloride, at a pH of about 6.4;

[0184] (11) about 135 mg / mL, about 150 mg / mL, about 165 mg / mL, or about 200 mg / mL of an anti-MASP2 antibody or antigen-binding fragment thereof, about 40 mg / mL sucrose, about 15 mg / mL arginine hydrochloride, about 0.2 mg / mL polysorbate 80, and about 10 mM histidine-histidine hydrochloride, at a pH of about 6.4;

[0185] (12) about 135 mg / mL, about 150 mg / mL, about 165 mg / mL, or about 200 mg / mL of an anti-MASP2 antibody or antigen-binding fragment thereof, about 40 mg / mL sucrose, about 15 mg / mL arginine hydrochloride, about 0.4 mg / mL polysorbate 80, and about 10 mM histidine-histidine hydrochloride, at a pH of about 6.4;

[0186] (13) about 135 mg / mL, about 150 mg / mL, about 165 mg / mL, or about 200 mg / mL of an anti-MASP2 antibody or antigen-binding fragment thereof, about 40 mg / mL sucrose, about 15 mg / mL arginine hydrochloride, about 0.1 mg / mL polysorbate 80, and about 10 mM histidine-histidine hydrochloride, at a pH of about 6.6;

[0187] (14) about 135 mg / mL, about 150 mg / mL, about 165 mg / mL, or about 200 mg / mL of an anti-MASP2 antibody or antigen-binding fragment thereof, about 40 mg / mL sucrose, about 15 mg / mL arginine hydrochloride, about 0.2 mg / mL polysorbate 80, and about 10 mM histidine-histidine hydrochloride, at a pH of about 6.6;

[0188] (15) about 135 mg / mL, about 150 mg / mL, about 165 mg / mL, or about 200 mg / mL of an anti-MASP2 antibody or antigen-binding fragment thereof, about 40 mg / mL sucrose, about 15 mg / mL arginine hydrochloride, about 0.4 mg / mL polysorbate 80, and about 10 mM histidine-histidine hydrochloride, at a pH of about 6.6;

[0189] (16) about 135 mg / mL, about 150 mg / mL, about 165 mg / mL, or about 200 mg / mL of an anti-MASP2 antibody or antigen-binding fragment thereof, about 40 mg / mL sucrose, about 15 mg / mL arginine hydrochloride, about 0.1 mg / mL polysorbate 80, and about 10 mM histidine-histidine hydrochloride, at a pH of about 6.8;

[0190] (17) about 135 mg / mL, about 150 mg / mL, about 165 mg / mL, or about 200 mg / mL of an anti-MASP2 antibody or antigen-binding fragment thereof, about 40 mg / mL sucrose, about 15 mg / mL arginine hydrochloride, about 0.2 mg / mL polysorbate 80, and about 10 mM histidine-histidine hydrochloride, at a pH of about 6.8;

[0191] (18) about 135 mg / mL, about 150 mg / mL, about 165 mg / mL, or about 200 mg / mL of an anti-MASP2 antibody or antigen-binding fragment thereof, about 40 mg / mL sucrose, about 15 mg / mL arginine hydrochloride, about 0.4 mg / mL polysorbate 80, and about 10 mM histidine-histidine hydrochloride, at a pH of about 6.8;

[0192] (19) about 100 mg / mL anti-MASP2 antibody, about 8.2 mg / mL sodium chloride, about 0.4 mg / mL polysorbate 80, and about 10 mM histidine-histidine hydrochloride, pH about 6.6;

[0193] (20) about 150 mg / mL anti-MASP2 antibody, about 40 mg / mL sucrose, about 15 mg / mL arginine hydrochloride, about 0.4 mg / mL polysorbate 80, and about 10 mM histidine-histidine hydrochloride, pH about 6.6

[0194] (21) The pharmaceutical composition according to any one of the above (1) to (20), further comprising water for injection;

[0195] (22) The pharmaceutical composition described in any one of the above (1) to (21), further comprising a pH adjuster, such as hydrochloric acid and / or sodium hydroxide.

[0196] In certain embodiments, the histidine-HCl buffer is histidine-histidine-HCl buffer.

[0197] The present disclosure also provides a method for preparing the aforementioned pharmaceutical composition, comprising the step of mixing an anti-MASP2 antibody or antigen-binding fragment thereof with a buffer. In certain embodiments, the buffer is a histidine-HCl buffer (e.g., histidine-histidine hydrochloride).

[0198] In certain embodiments, the pharmaceutical composition as described in any one of the above items is a liquid preparation. In some embodiments, the solvent of the liquid preparation is water, normal saline or glucose solution.

[0199] The present disclosure also provides a lyophilized preparation, characterized in that the lyophilized preparation can form any of the above pharmaceutical compositions after reconstitution.

[0200] The present disclosure also provides a lyophilized preparation, which is obtained by freeze-drying the pharmaceutical composition as described above.

[0201] The present disclosure provides a reconstitution solution, wherein the reconstitution solution is prepared by reconstitution of the aforementioned lyophilized preparation. In certain embodiments, the reconstitution solution is selected from but not limited to water for injection, physiological saline or glucose solution.

[0202] The present disclosure also provides a product, comprising a container containing the aforementioned pharmaceutical composition, the aforementioned lyophilized formulation, or the aforementioned reconstituted solution. In certain embodiments, the container is a neutral borosilicate glass injection vial. In certain embodiments, the product includes a package insert.

[0203] The pharmaceutical composition of any one of the above items of the present disclosure, 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:

[0204] The VH comprises HCDR1, HCDR2, and HCDR3 of the VH as shown in SEQ ID NO: 7, and / or the VL comprises LCDR1, LCDR2, and LCDR3 of the VL as shown in SEQ ID NO: 8.

[0205] 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 systems, and in some embodiments, are defined according to the Kabat numbering system.

[0206] In certain embodiments, the anti-MASP2 antibodies and antigen-binding fragments thereof comprise:

[0207] 1-1) the VH comprises HCDR1, HCDR2, and HCDR3 of the VH as shown in SEQ ID NO: 7, and / or the VL comprises LCDR1, LCDR2, and LCDR3 of the VL as shown in SEQ ID NO: 8;

[0208] In the pharmaceutical composition of any one of the above items, the anti-MASP2 antibody and antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein

[0209] The VH comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 9, 10, and 11, respectively, and / or the VL comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NOs: 12, 13, and 14, respectively.

[0210] In certain embodiments, 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.

[0211] In certain embodiments, the heavy chain framework region of the humanized antibody or antigen-binding fragment thereof 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.

[0212] In certain embodiments, the anti-MASP2 antibody or antigen-binding fragment thereof, wherein:

[0213] The amino acid sequence of VH is shown in one of SEQ ID NOs: 7, 17, 18, 19, and 20, and / or the amino acid sequence of VL is shown in one of SEQ ID NOs: 8, 121, and 22;

[0214] In certain embodiments, the anti-MASP2 antibody or antigen-binding fragment thereof, wherein:

[0215] a) VH and VL comprise or consist of the amino acid sequences shown in SEQ ID NOs: 7 and 8, respectively;

[0216] b) VH and VL comprise or consist of the amino acid sequences shown in SEQ ID NOs: 17 and 21, respectively;

[0217] c) VH and VL comprise or consist of the amino acid sequences shown in SEQ ID NOs: 17 and 22, respectively;

[0218] d) VH and VL comprise or consist of the amino acid sequences shown in SEQ ID NOs: 18 and 21, respectively;

[0219] e) VH and VL comprise or consist of the amino acid sequences shown in SEQ ID NOs: 18 and 22, respectively;

[0220] f) VH and VL comprise or consist of the amino acid sequences shown in SEQ ID NOs: 19 and 21, respectively;

[0221] g) VH and VL comprise or consist of the amino acid sequences shown in SEQ ID NOs: 19 and 22, respectively;

[0222] h) VH and VL comprise or consist of the amino acid sequences shown in SEQ ID NOs: 20 and 21, respectively;

[0223] or

[0224] i) VH and VL comprise or consist of the amino acid sequences shown in SEQ ID NOs: 20 and 22, respectively;

[0225] Further, the present disclosure provides anti-MASP2 antibodies or antigen-binding fragments thereof comprising 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 to the VH, VL of any of groups a) to i) above.

[0226] In certain embodiments, the anti-MASP2 antibody or antigen-binding fragment thereof is an IgG antibody or antigen-binding fragment thereof, for example, an IgG1, IgG2, or IgG4 antibody or antigen-binding fragment thereof, more preferably, an IgG4 antibody or antigen-binding fragment thereof having an Fc region depleted of any one or more of S228P, F234A, and L235A mutations. The above mutations are all EU numbering.

[0227] In certain embodiments, 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. In certain specific embodiments, exemplary mutations include 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. 1A / D365E / L358M on IgG2, H268Q / V309L / A330S / P331S on IgG2, S267E / L328F on IgG1, L234F / L235E / D265A on IgG1, L234A / L235A / G237A / P238S / H268A / A330S / P331S on IgG1, S228P / F234A / L235A / G237A / P238S on IgG4, and S228P / F234A / L235A / G236 deletion / G237A / P238S on IgG4. Hybrid IgG2 / 4 Fc domains can also be used, for example, an Fc having residues 117-260 from IgG2 and residues 261-447 from IgG4. In certain specific embodiments, the Fc region of the human IgG4 has any one or more mutations of S228P, F234A, L235A and K447A (see WO2017079112A, WO2018031400A, etc.).

[0228] In certain embodiments, the antigen-binding fragments of the anti-MASP2 antibodies described herein are Fab, Fv, sFv, Fab', F(ab')2, linear antibodies, single-chain antibodies, scFv, sdAb, sdFv, nanobodies, peptibodies, domain antibodies, and multispecific antibodies (bispecific antibodies, diabodies, triabodies and tetrabodies, tandem di-scFv, tandem tri-scFv), for example, specifically scFv, Fv, Fab, or Fab' fragments.

[0229] In certain embodiments, the full-length amino acid sequence of the heavy chain of an anti-MASP2 antibody or antigen-binding fragment thereof described herein is as set forth in SEQ ID NO: 24, or is at least 80%, at least 90%, or at least 95% identical thereto; and the full-length amino acid sequence of the light chain is as set forth in SEQ ID NO: 25, or is at least 80%, at least 90%, or at least 95% identical thereto.

[0230] In certain embodiments, 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 in the heavy chain variable region and 0 to 10 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid changes in the light chain variable region. In certain specific embodiments, the amino acid changes are conservative substitutions, replacements, or modifications, and / or deletions or additions that do not affect function.

[0231] In certain embodiments, the anti-MASP2 antibodies selectively inhibit MASP2 complement activation, leaving the C1q-dependent complement activation system functionally intact.

[0232] The present disclosure provides uses and methods of the aforementioned pharmaceutical composition, the pharmaceutical composition prepared by the aforementioned method, the aforementioned lyophilized formulation, or the aforementioned reconstituted solution in preventing and / or treating diseases, wherein the diseases may be related to or unrelated to the complement signaling pathway (e.g., MASP2). In certain embodiments, the diseases are IgA nephropathy or paroxysmal nocturnal hemoglobinuria (PNH).

[0233] 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.

[0234] 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.

[0235] MASP-2-dependent complement activation has been implicated in the pathogenesis of numerous 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. In certain embodiments, methods of treating the aforementioned diseases and conditions using the aforementioned pharmaceutical compositions, pharmaceutical compositions prepared by the aforementioned methods, the aforementioned lyophilized formulations, or the aforementioned reconstituted solutions of the present disclosure, as well as related pharmaceutical uses, are provided.

[0236] In certain embodiments, the aforementioned diseases and conditions are diseases associated with MASP-2-dependent complement activation.

[0237] In certain embodiments, the aforementioned diseases and conditions are microvascular endothelial cell damage and / or thrombosis.

[0238] In certain embodiments, the above diseases and conditions are selected from the group consisting of: IgA nephropathy, paroxysmal nocturnal hemoglobinuria (PNH), lupus nephritis, thrombotic microangiopathy (TMA) (e.g., persistent TMA associated with hematopoietic stem cell transplantation (HSCT-TMA), thrombotic thrombocytopenic purpura (TTP)), hemolytic uremic syndrome (HUS), membranous glomerulonephritis, glomerulonephritis, age-related macular degeneration, reperfusion injury, myocardial infarction, diabetic neuropathy, stroke, graft-versus-host disease (GVHD), Upshaw-Schulman syndrome (USS), and in certain specific embodiments, the disease is associated with MASP-2-dependent complement activation.

[0239] In some embodiments, 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.

[0240] Definition of terms

[0241] In order to make the present disclosure more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise explicitly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the present disclosure belongs.

[0242] The three letter and one letter codes for amino acids used in this disclosure are as described in J. biol. chem, 243, p3558 (1968).

[0243] "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.

[0244] The "classical pathway" refers to complement activation that is triggered by antibody binding to foreign particles and requires binding to the recognition molecule C1q. 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 is traditionally believed to arise from the spontaneous proteolysis of complement factor C3 to produce C3b. The "lectin pathway" refers to complement activation that occurs through specific binding of serum and non-serum carbohydrate-binding proteins, including mannan-binding lectin (MBL), CL-11, and ficolins (H-ficolin, M-ficolin, or L-ficolin).

[0245] "Antibody" is used in the broadest sense to encompass various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies; monospecific antibodies, multispecific antibodies (e.g., 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. An antibody may refer to an immunoglobulin, a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds. Immunoglobulins differ in their antigenicity due to the amino acid composition and arrangement order of their heavy chain constant regions. Consequently, immunoglobulins can be divided into five classes, or isotypes, namely IgM, IgD, IgG, IgA, and IgE, corresponding to μ, δ, γ, α, and ε chains, respectively. Within the same class of Ig, different subclasses can be formed based on the amino acid composition of the hinge region and the number and location of heavy chain disulfide bonds. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are classified as either κ or λ chains based on differences in their constant regions. Each of the five classes of Ig can have either kappa or lambda chains. The approximately 110 amino acids near the N-terminus of both the heavy and light chains of antibodies vary greatly in sequence and constitute the variable region (V region); the remaining amino acid sequences near the C-terminus are relatively stable and constitute the constant region (C region). The variable region consists of three hypervariable regions (CDRs) and four framework regions (FRs) whose sequences are relatively conserved. These three hypervariable regions determine the specificity of the antibody and are also known as complementarity-determining regions (CDRs). Each light chain variable region (VL) and heavy chain variable region (VH) consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDRs of the light chain are referred to as LCDR1, LCDR2, and LCDR3; the three CDRs of the heavy chain are referred to as HCDR1, HCDR2, and HCDR3.

[0246] For the determination or definition of CDRs, the definitive delineation 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, and conformational definitions.

[0247] The Kabat numbering system is a standard for numbering residues in antibodies and is commonly 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 takes into account the positions 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 for modeling antibody structure (see, e.g., Martin et al., 1989, Proc Natl Acad Sci (USA), 86:9268-9272; "AbM™, 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 primary sequence (see Samudrala et al., 1999, "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach," PROTEINS, Structure, Function and Genetics Suppl., 3: 194-198). Contact definitions are based on analysis of available complex crystal structures (see, e.g., MacCallum et al., 1996, J. Mol. Biol., 5: 732-45). In conformational definitions, CDR positions can be identified as residues that make enthalpic contributions to antigen binding (see, e.g., Makabe et al., 2008, Journal of Biological Chemistry, 283: 1156-1166). Other CDR boundary definitions may not strictly follow one of the above methods, but still overlap with at least a portion of the Kabat CDRs, although they may be shortened or lengthened based on predictions or experimental results that a particular residue or group of residues does not significantly affect antigen binding. As used in this disclosure, CDRs may refer to CDRs defined by any method known in the art, including combinations of methods.

[0248] 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.

[0249] "Monoclonal antibody" or "monoantibody" refers to an antibody obtained from a substantially homogeneous antibody population, i.e., the individual antibodies contained in the population are identical except for possible naturally occurring mutations that may be present in small amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations that 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 characteristic of an antibody as being obtained from a substantially homogeneous antibody population, and is not to be construed as requiring the antibody to be produced by any particular method. For example, the monoclonal antibodies used in accordance with the present disclosure can be prepared by the hybridoma method first described by Kohler and Milstein, 1975, Nature 256:495, or can be prepared by, for example, the recombinant DNA method described in U.S. Patent No. 4,816,567. For example, monoclonal antibodies can also be isolated from a phage library generated using the techniques described in McCafferty et al., 1990, Nature 348:552-554.

[0250] "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:

[0251] (1) Antibodies isolated from transgenic or transchromosomal animals (e.g., mice) expressing human immunoglobulin genes or hybridomas prepared therefrom;

[0252] (2) antibodies isolated from host cells transformed to express the antibody, such as transfectomas;

[0253] (3) antibodies isolated from a recombinant combinatorial fully human antibody library; and

[0254] (4) Antibodies prepared, expressed, created, or isolated by methods such as splicing human immunoglobulin gene sequences to other DNA sequences.

[0255] 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.

[0256] The term "murine antibody," as used herein, refers to a monoclonal antibody against human MASP2 or an epitope thereof, prepared according to the knowledge and skill in the art. During preparation, a test subject is injected with a MASP2 antigen, and then a hybridoma expressing an antibody with the desired sequence or functional properties is isolated. In a specific embodiment of the present disclosure, the murine anti-human, anti-MASP2 antibody or antigen-binding fragment thereof may further comprise a light chain constant region of a murine kappa or lambda chain, or variants thereof, or a heavy chain constant region of a murine IgG1, IgG2, IgG3, or IgG4, or variants thereof.

[0257] The term "fully human antibody" includes antibodies having variable and constant regions of human germline immunoglobulin sequences. The fully human antibodies of the present disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (such as mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, 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").

[0258] The term "humanized antibody," also known as a CDR-grafted antibody, refers to an antibody produced by transplanting non-human CDR sequences into the human antibody variable region framework. This overcomes the strong immune response induced by chimeric antibodies due to the large number of non-human protein components they carry. To avoid a decrease in activity along with a decrease in immunogenicity, minimal reverse mutations can be performed on the fully human antibody variable region to maintain activity.

[0259] The term "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. As an example, to establish a chimeric antibody, it is necessary to select a hybridoma that secretes a mouse-specific monoclonal antibody, then clone the variable region gene from the mouse hybridoma cell, and then clone the constant region gene of a fully human antibody as needed. The mouse variable region gene and the human constant region gene are connected to form 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 human IgG2 or IgG4 heavy chain constant region, or using an IgG1 that has no ADCC (antibody-dependent cell-mediated cytotoxicity) toxicity after amino acid mutation.

[0260] "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. 23(9): 1126-1136; Adair and Lawson, 2005, Drug Design Reviews-Online 2(3), 209-217). Methods for generating and preparing these antibody fragments are well known in the art (see, e.g., Verma et al., 1998, Journal of Immunological Methods, 216, 165-181). The 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 multiple specificities, such as bispecifics, or may be monospecific (see, for example, WO92 / 22583 and WO05 / 113605), an example of the latter being Tri-Fab (or TFM) described in WO 92 / 22583.

[0261] The term "binds to MASP2" as used herein refers to the ability to interact with MASP2 or its epitope, which may be of human origin. The term "antigen binding site" as used herein refers to a discrete three-dimensional site on an antigen that is recognized by the antibody or antigen-binding fragment of the present disclosure.

[0262] "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., particularly a phage, yeast, or ribosome display library). In the present disclosure, antigen is defined more broadly to include a target molecule specifically recognized by an antibody, as well as a portion or mimetic of a molecule used in an immunization procedure for generating antibodies or in screening libraries for selecting antibodies. 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 and other variants of human MASP2, are referred to as antigens.

[0263] The term "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 the protein. Epitopes formed by adjacent amino acids are generally retained after exposure to denaturing solvents, while epitopes formed by tertiary folding are generally lost after treatment with denaturing solvents. An epitope generally comprises at least 3-15 amino acids in a unique spatial conformation. Methods for determining which epitope is bound by a given antibody are well known in the art and include immunoblotting and immunoprecipitation assays. Methods for determining the spatial conformation of an epitope include techniques in the art and the techniques described herein, such as X-ray crystallography and two-dimensional nuclear magnetic resonance.

[0264] "Specific binding" and "selective binding" refer to the binding of an antibody to an epitope on a predetermined antigen. Generally, when human MASP2 or its epitope is used as an analyte and an antibody is used as a ligand, the antibody binds to the epitope at a specific binding rate of less than 10 -7 M or even smaller equilibrium dissociation constant (K D ) binds to a predetermined antigen or an epitope thereof, and its affinity for binding to the predetermined antigen or an epitope thereof is at least twice that of its affinity for binding to a nonspecific antigen (such as BSA, etc.) other than the predetermined antigen (or its epitope) or a closely related antigen. The term "antibody that recognizes an antigen" can be used interchangeably with the term "specifically binding antibody" in this disclosure.

[0265] "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, a surface plasmon resonance (SPR) method (Biacore). 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. K D Through equation K D = kd / ka is related to ka and kd. 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). For example, K 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). DOther standard assays for assessing the binding ability of an antibody to a target antigen are known in the art and include, for example, ELISA, Western blot, RIA, and flow cytometry analysis, as well as other assays exemplified elsewhere in this disclosure. The binding kinetics and binding affinity of an antibody can also be determined by standard assays known in the art, such as surface plasmon resonance (SPR), for example, by using Biacore TM The K of each antibody / antigen complex can be compared by comparing the K D The K values ​​can be used to compare the binding affinities associated with different molecular interactions, for example, the binding affinities of different antibodies for a given antigen. Similarly, the specificity of an interaction can be determined and compared by determining and comparing the K values ​​of the interactions of interest (e.g., the specific interaction between an antibody and an antigen). D The K values ​​were compared with the K values ​​for non-target interactions (e.g., control antibodies known not to bind MASP2). D The value is evaluated.

[0266] "Conservative substitution" refers to substitution with another amino acid residue having properties similar to the original amino acid residue. For example, lysine, arginine, and histidine have similar properties in that they have basic side chains, and aspartic acid and glutamic acid have similar properties in that they have acidic side chains. In addition, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan have similar properties in that they have uncharged polar side chains, and alanine, valine, leucine, threonine, isoleucine, proline, phenylalanine, and methionine have similar properties in that they have non-polar side chains. In addition, 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 substituting an amino acid residue in a group showing similar properties as described above, it will not show specific changes in properties.

[0267] "Cross-reactivity" refers to the ability of an antibody of the present disclosure to bind to MASP2 from a different species. For example, an antibody of the present 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. Methods for determining cross-reactivity include standard binding assays as described herein, such as surface plasmon resonance analysis, or flow cytometry.

[0268] "Inhibit" or "block" are used interchangeably and encompass both partial and complete inhibition / blocking. Inhibition / blocking of MASP2 preferably reduces or alters the normal level or type of activity that occurs when MASP2 binding occurs in the absence of inhibition or blockade. Inhibition and blocking are also intended to encompass any measurable decrease in MASP2 binding affinity when contacted with an anti-MASP2 antibody, compared to MASP2 not contacted with the anti-MASP2 antibody.

[0269] "Inhibiting growth" (eg, involving cells) is intended to include any measurable decrease in cell growth.

[0270] Methods for producing and purifying antibodies and antigen-binding fragments are well known in the art and can be found in, for example, the Cold Spring Harbor Laboratory Manual (Chapters 5-8 and 15). For example, mice can be immunized with human MASP2 or fragments thereof, and the resulting antibodies can be renatured, purified, and subjected to amino acid sequencing using conventional methods. Antigen-binding fragments can also be prepared using conventional methods. The antibodies or antigen-binding fragments described herein can be engineered to have one or more human FR regions added to the non-human CDR regions using genetic engineering methods. Human FR germline sequences can be obtained from the ImMunoGeneTics (IMGT) website.

[0271] The engineered antibodies or antigen-binding fragments disclosed herein can be prepared and purified using conventional methods. For example, cDNA sequences encoding heavy and light chains can be cloned and recombined into expression vectors. The recombinant immunoglobulin expression vector can be stably transfected into cells. Mammalian expression systems result in glycosylation of antibodies, particularly at the highly conserved N-terminus of the Fc region. Stable clones are obtained by expressing antibodies that specifically bind to human antigens. Positive clones are expanded in serum-free culture medium in a bioreactor to produce antibodies. The culture fluid that secretes antibodies can be purified and collected using conventional techniques. The antibodies can be filtered and concentrated using conventional methods. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieves and ion exchange. The resulting product must be immediately frozen, such as at -70°C, or freeze-dried.

[0272] Antibodies can be screened competitively 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 with or cross-compete 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 No. WO 03 / 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.

[0273] "Administer," "apply," and "treat" as applied to an animal, a human, a laboratory 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. "Administer," "apply," and "treat" can refer to, for example, therapeutic, pharmacokinetics, diagnostics, 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. "Administer," "apply," and "treat" also mean the in vitro and ex vivo treatment of, for example, a cell, by an agent, a diagnostic, a binding composition, or by another cell. "Treatment," as applied to a human, veterinary, or research subject, refers to therapeutic treatment, prophylactic or preventative measures, research, and diagnostic applications.

[0274] "Treatment" means administering an internal or external therapeutic agent, such as a composition comprising any of the antibodies or antigen-binding fragments thereof disclosed herein, or a conjugate thereof, to a subject who has, is suspected of having, or is predisposed to having one or more diseases or symptoms thereof, and for which the therapeutic agent is known to have a therapeutic effect. Typically, 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 effective to alleviate any specific disease symptom (also referred to as a "therapeutically effective amount") 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 test method commonly used by doctors or other health care professionals to evaluate the severity or progression of the symptoms. Although embodiments of the present disclosure (e.g., methods of treatment or articles of manufacture) may not be effective in alleviating symptoms of the target disease in a certain subject, they should alleviate 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.

[0275] An "effective amount" encompasses an amount sufficient to ameliorate or prevent the symptoms or conditions of a medical condition. An effective amount also means an amount sufficient to permit 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 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.

[0276] "Homology" or "identity" refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in the two compared sequences is occupied by the same nucleotide or amino acid monomer subunit, for example, if every position in two DNA molecules is occupied by the same nucleotide, then the molecules are 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 × 100%. For example, if 6 out of 10 positions in the two sequences match or are homologous when the sequences are optimally aligned, then the two sequences are 60% homologous. Generally, a comparison is made when the two sequences are aligned to achieve the maximum percent homology.

[0277] "Cell," "cell line," and "cell culture" are used interchangeably, and 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.

[0278] "Pharmaceutically acceptable carriers" or "pharmaceutically acceptable excipients" include 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. In some embodiments, the diluent for aerosol or parenteral administration is phosphate-buffered saline (PBS) or normal (0.9%) saline. Compositions containing such carriers are formulated by well-known conventional methods (see, for example, Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, ed., Mack Publishing Co., Easton, PA, 1990; and R Remington, The Science and Practice of Pharmacy 20th edition Mack Publishing, 2000).

[0279] "Optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs or does not occur. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that the antibody heavy chain variable region of the specified sequence may but need not be present.

[0280] The "MASP2 binding proteins" or "MASP2 binding molecules" disclosed herein are broadly interpreted to encompass the anti-MASP2 antibodies or antigen-binding fragments thereof disclosed herein, as well as any protein capable of binding to MASP2. For example, a MASP2 binding protein (or binding molecule) may comprise one or more effector molecules, for example, by conjugation or fusion. Such "effector molecules" include, for example, anti-tumor agents, drugs, toxins, biologically active proteins (e.g., enzymes), other antibodies or antibody fragments, synthetic or naturally occurring polymers, nucleic acids and fragments thereof, such as DNA, RNA and fragments thereof, radionuclides (particularly radioiodides), radioisotopes, chelated metals, nanoparticles, and reporter groups (e.g., fluorescent compounds), or compounds detectable by NMR or ESR spectroscopy. When the effector molecule is a polymer, it may generally be a synthetic or naturally occurring polymer, such as an optionally substituted linear or branched polyalkylene, polyalkenylene, or polyoxyalkylene polymer, or a branched or 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. Specific examples of synthetic polymers include optionally substituted linear or branched poly(ethylene glycol), poly(propylene glycol), poly(vinyl alcohol), or derivatives thereof, particularly optionally substituted poly(ethylene glycol) such as methoxypoly(ethylene glycol) or derivatives thereof. Specific naturally occurring polymers include lactose, amylose, dextran, glycogen, or derivatives thereof. In one embodiment, the polymer is albumin or a fragment thereof, such as human serum albumin or a fragment thereof. Conjugation of the polymer to the anti-MASP2 antibodies or antigen-binding fragments thereof of the present disclosure can be accomplished by conventional methods.

[0281] "About" means within an acceptable error range for the 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 expressly stated otherwise in the examples or elsewhere in the specification, "about" means a range within ±5% of the given value.

[0282] "Buffer" refers to a buffer that tolerates changes in pH through the action of its acid-base conjugate components. Examples of buffers that control pH within an appropriate range include tris (Tris), acetate, succinate, gluconate, histidine, oxalate, lactate, phosphate, citrate, tartrate, fumarate, glycylglycine, and other organic acid buffers.

[0283] A "histidine salt buffer" is a buffer containing histidine ions. Examples of histidine salt buffers include histidine hydrochloride, histidine acetate, histidine phosphate, histidine sulfate, and the like. Preferred are histidine hydrochloride buffers or histidine acetate buffers. Histidine acetate buffers are prepared from histidine and acetic acid, and histidine hydrochloride buffers are prepared from histidine and histidine hydrochloride, or histidine and hydrochloric acid.

[0284] "Phosphate buffer" is a buffer containing phosphate ions. Examples of phosphate buffers include disodium hydrogen phosphate-sodium dihydrogen phosphate, disodium hydrogen phosphate-potassium dihydrogen phosphate, disodium hydrogen phosphate-citric acid, and the like. Preferably, the phosphate buffer is disodium hydrogen phosphate-sodium dihydrogen phosphate.

[0285] "Tris buffer" is a buffer containing tris (Tris). Examples of the Tris buffer include Tris-hydrochloric acid buffer (Tris-HCl), Tris-acetic acid buffer (Tris-AA), Tris-succinic acid buffer (Tris-SA), and Tris-citrate buffer (Tris-CA).

[0286] "Citrate buffer" is a buffer comprising citrate ions. Examples of 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.

[0287] A "succinate buffer" is a buffer comprising succinate ions. Examples of succinate buffers include succinic acid-sodium succinate, succinic acid-potassium succinate, succinic acid-calcium succinate, and the like. A preferred succinate buffer is succinic acid-sodium succinate. For example, the succinic acid-sodium succinate can be prepared from succinic acid and sodium hydroxide, or from succinic acid and sodium succinate.

[0288] An "acetate buffer" is a buffer comprising acetate ions. Examples of acetate buffers include acetic acid-sodium acetate, histidine-histidine acetate, acetic acid-potassium acetate, acetic acid-calcium acetate, acetic acid-magnesium acetate, and the like. A preferred acetate buffer is acetic acid-sodium acetate.

[0289] A "pharmaceutical composition" refers to a mixture containing one or more antibodies described herein with 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, facilitate administration to an organism, and promote absorption of the active ingredient to exert its biological activity.

[0290] In the present disclosure, "pharmaceutical composition" and "formulation" are not mutually exclusive.

[0291] Unless otherwise specified, the solvent in the solution form of the pharmaceutical composition described in the present disclosure is water.

[0292] The composition is isotonic. "Isotonic" means that the formulation has substantially the same osmotic pressure as human blood. Isotonicity can be determined by methods known in the art, for example, using a vapor pressure or ice-type osmometer. When administered subcutaneously, the osmotic pressure of the pharmaceutical composition is preferably controlled within a range of 260-380 mOsm, for example, within a range of 270-360 mOsm.

[0293] "Lyophilized formulation" refers to a pharmaceutical composition in liquid or solution form or a formulation or pharmaceutical composition obtained after a liquid or solution formulation has been subjected to a vacuum freeze-drying step.

[0294] An antibody "retains its physical stability" in a pharmaceutical formulation if it shows no significant increase in aggregation, precipitation, and / or denaturation as measured by 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).

[0295] 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.).

[0296] 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.

[0297] "Administer," "give," and "treat," as they apply to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refer to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with an animal, human, subject, cell, tissue, organ, or biological fluid. "Administer," "give," and "treat" can refer to, for example, therapeutic, pharmacokinetics, diagnostics, 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. "Administer," "give," and "treat" also mean the in vitro and ex vivo treatment of, for example, a cell, by an agent, a diagnostic, a binding 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.

[0298] "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. Typically, a therapeutic agent is administered in an amount effective to alleviate one or more symptoms of a disease in a treated patient or population to induce regression of such symptoms or inhibit the development of such symptoms to any clinically measured extent. The amount of a therapeutic agent that effectively alleviates any specific disease symptom (also referred to as a "therapeutically effective amount") can vary according to a variety of factors, such as the patient's disease state, age, and weight, and the ability of the drug to produce the desired therapeutic effect in the patient. Whether the symptoms of the disease 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. Although embodiments of the present disclosure (e.g., methods of treatment or articles of manufacture) may not be effective in alleviating every symptom of the target disease, they should alleviate the target disease symptoms in a statistically significant number of patients 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.

[0299] The equipment and methods used in the preparation and testing of the pharmaceutical composition are as follows:

[0300] SEC size exclusion chromatography:

[0301] An analytical method that separates solutes based on the relative relationship between the pore size of the gel and the coil size of the polymer sample molecules.

[0302] 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.)

[0303] SEC instrument: Agilent 1260-Bio; chromatographic columns: Waters, XBrige SEC (300×7.8mm 3.5μm)

[0304] CE capillary gel electrophoresis:

[0305] A method of electrophoresis in which gel is transferred to a capillary tube as a supporting medium and samples are separated according to their molecular weight at a certain voltage.

[0306] 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.

[0307] Reducing CE purity percentage = sum of light and heavy chain purity = CA light chain / CA total × % + CA heavy chain / CA total × % (CA light chain is the light chain corrected peak area in the sample, CA heavy chain is the heavy chain corrected peak area in the sample, and CA total is the sum of the light chain, heavy chain, and impurity peak areas)

[0308] CE measurement instrument: Sciex model PA800plus

[0309] icIEF imaging capillary isoelectric focusing electrophoresis:

[0310] A technique for separating proteins based on their isoelectric points (pI).

[0311] icIEF main peak content percentage = main peak area / total area*100% (total area is the sum of the areas of acidic peak, main peak and basic peak).

[0312] The instrument used for icIEF assay was manufactured by Protein Simple, model: Muarice.

[0313] Osmolality determination:

[0314] The freezing point method determines osmotic pressure, based on the proportional relationship between freezing point depression and the molar concentration of a solution. It uses a highly sensitive temperature sensor to measure the freezing point of a solution and converts the electrical charge into osmotic pressure. Instrument manufacturer: Loser, model OM819.

[0315] Protein concentration determination:

[0316] Protein concentration was determined using a UV-visible spectrophotometer (Nano Drop 2000) with a pathlength of 1 mm.

[0317] Melting temperature (Tm) and aggregation temperature (Tagg):

[0318] Tm is the temperature at which 50% of the protein components are denatured during heating; Tagg is the temperature at which protein aggregation occurs during heating. Load the sample into a Unitube and run the measurement at a 25-95°C heating ramp. Tm and Taag measurements were performed by Uncle Instruments, manufactured by Unchained.

[0319] Particle size (Radius) and PD%:

[0320] Using dynamic and static light scattering technology, a 96-well plate was used to measure the antibody particle size and distribution using the Brownian motion of antibody molecules. Measurement instrument: High-throughput dynamic and static light scattering instrument, instrument manufacturer is WYATT, model DynaPro plate ReaderIII

[0321] Exemplary antibody pharmaceutical composition (preparation) preparation process:

[0322] Step 1: Take a certain amount of purified anti-MASP2 antibody solution and perform solvent exchange (preferably ultrafiltration) with a buffer that does not contain antibody. Displace at least six times the volume through an ultrafiltration membrane and continue concentrating the antibody to a desired concentration. Add a certain volume of the other excipient stock solutions and dilute with buffer to achieve the desired concentration of the antibody and excipients. Mix thoroughly. After filtering the stock solution, sample it for in-process testing of bacterial endotoxins and microbial limits. Sterile filter the stock solution through a 0.22μm filter cartridge and collect the filtrate.

[0323] 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 stopper for injection.

[0324] Step 3: Turn on the capping machine, add aluminum caps, and start capping.

[0325] Step 4: Visual inspection to confirm that the product has no defects such as inaccurate filling quantity and poor appearance. Print carton labels, fold cartons, pack cartons, and apply carton labels. BRIEF DESCRIPTION OF THE DRAWINGS

[0326] FIG1A shows the activity test results of the anti-MASP2 antibody 77H11 disclosed herein in the human 1% serum lectin pathway, using an hIgG4 isotype control as a negative control and OMS721 as a positive control.

[0327] FIG2A shows the results of the activity inhibition test of the 77H11 humanized antibody disclosed herein in 90% human serum lectin pathway, using hIgG4 as an isotype control (Isotype control) as a negative control and OMS721 as a positive control.

[0328] 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. An hIgG4 isotype control was used as a negative control, and OMS721 was used as a positive control.

[0329] 4A and 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. DETAILED DESCRIPTION

[0330] The present disclosure is further described below with reference to the following examples, but these examples are not intended to limit the scope of the present disclosure.

[0331] Experimental methods in the disclosed embodiments or test examples, where specific conditions are not specified, generally followed conventional conditions or those recommended by the raw material or commercial manufacturer. See Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory; and Current Methods in Molecular Biology, Ausubel et al., Greene Publishing Associates, Wiley Interscience, NY. Reagents whose sources are not specified were commercially available.

[0332] Example

[0333] The preparation and purification methods of the anti-MASP-2 antibodies in this application have been described in International Patent Application WO2022228364, and the entire contents of the aforementioned application documents can be incorporated into the present disclosure.

[0334] Example 1. Preparation of MASP2 recombinant protein

[0335] In order to prepare antigens for antibody screening, CHO-S stably transfected cell lines expressing recombinant proteins of human MASP2A (S633A), mouse MASP2A (S632A), and monkey MASP2A (S633A) were prepared. MASP2 (S632A) or MASP2 (S633A) is an enzymatic 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. In addition, in order to increase the yield during expression, 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). The above-mentioned 663rd or 662nd position are both position numbers based on natural counting in the original sequence. The cell lines were cultured, the supernatants were collected, and the recombinant proteins were purified by affinity chromatography. The amino acid sequences were as follows:

[0336] >Human MASP2A (S633A) protein:

[0337] (Note: The underline is the signal peptide, the italic is the His tag, and the gray is the mutation at position 633 to A (S633A))

[0338] SEQ ID NO: 1

[0339] Monkey MASP2A (S633A) protein:

[0340] (Note: The underline is the signal peptide, the italic is the His tag, and the gray is the mutation at position 633 to A (S633A))

[0341] SEQ ID NO: 2

[0342] > Mouse MASP2A (S632A) protein:

[0343] (Note: The underline is the signal peptide, the italic is the His tag, and the gray is the mutation at position 632 to A (S632A))

[0344] SEQ ID NO: 3

[0345] To identify the antigen-binding site of the antibody, different human MASP2 fragments were prepared, including human MASP2 CCP1, human MASP2 CCP1-CCP2-SP(S633A), and human MASP2 CCP2-SP(S633A). CCP1-CCP2-SP(S633A) and CCP2-SP(S633A) were prepared by transforming plasmids into Escherichia coli BL21 strains, isolating and purifying the proteins, and then denaturing and renaturing them. CCP1 was prepared by transiently transfecting CHO cells, expressing and isolating the proteins. The amino acid sequences of the various MASP2 protein fragments are as follows:

[0346] >CCP1-CCP2-SP(S633A):

[0347] >CCP2-SP(S633A):

[0348] (Note: Gray indicates the mutation at position 633 to A (S633A))

[0349] SEQ ID NO: 5

[0350] >CCP1:

[0351] Example 2. Screening and Preparation of Anti-MASP2 Antibodies

[0352] Mouse immunization and phage display were used to screen and produce MASP2-specific antibodies.

[0353] 1. Mouse immunization:

[0354] The human MASP2A protein prepared in Example 1 was used as an antigen to immunize BALB / c mice, and the mouse with the highest titer was selected for hybridoma cell fusion. Hybridoma cell fusion and culture: Three culture dishes (10 cm) of myeloma cells passaged 24 hours earlier were taken, washed once with RPMI-1640 medium without HEPES, resuspended, and counted at 2-4×10 7 Mice were killed 72 hours after immunization, and the spleens were removed aseptically and washed with RPMI-1640 medium without HEPES. The spleen cells were minced, ground, blown away, filtered, centrifuged at 1000 rpm for 5 min, resuspended, and counted to 1-2 × 10 8Mix myeloma cells and spleen cells, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, loosen the pellet, and preheat in a 40°C water bath. Add 1 mL of PEG preheated to 40°C dropwise to the centrifuge tube over 60 seconds, stirring gently. Gently stir for 1 minute, then add 1 mL of culture medium over 30 seconds, 3 mL of culture medium over 1 minute, and 16 mL of culture medium over 1 minute. Let stand for 10 minutes, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and resuspend the cells in HAT-OPI medium (RPMI-1640 medium with 20% FBS, containing 1× HAT and 1× OPI) and incubate at 37°C in 5% CO2. On day 5 after fusion, add 50 μL / well of RPMI-1640 medium with 20% FBS (containing 2× HAT and 1× OPI). On days 7-10 after fusion, perform a complete medium change with HAT-OPI medium. On day 10 to 14 after fusion, human MASP2 and monkey MASP2 protein ELISA tests were performed based on cell growth density to select positive clones.

[0355] The ELISA assay was performed as follows: 1 μg / mL antigen was coated overnight at 4°C, with 50 μL per well. The plate was washed three times with PBS and then blocked with PBS containing 3% BSA for 1 hour at room temperature. The plate was washed three times with PBST and then hybridoma cell supernatant was added and incubated at room temperature for 1 hour. The plate was washed three times with PBST and then three times with PBS. The secondary antibody (1:2000; Invitrogen, goat anti-mouse IgG (H+L) secondary antibody, 31430) was added and incubated at room temperature for 1 hour. The plate was washed three times with PBST and then three times with PBS. TMB substrate was added and incubated at room temperature for 10 minutes. The reaction was then terminated and the signal was measured (absorbance at 450 nm).

[0356] 2. Phage display:

[0357] Human MASP2A antigen was coated onto immunosorbent plates and incubated with a synthetic phage antibody library for solid-phase screening. After three rounds of panning, human MASP2 and monkey MASP2-positive phage were obtained.

[0358] Phage display screening of positive clones was performed as follows: 4 mL of MASP2A (5 ng / μL) was coated onto an immunotube 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 a MASP2A-coated immunotube, rotated at room temperature for 1 hour, washed five times with PBS, eluted with 1000 μL of TEA, and neutralized with 400 μL of Tris-HCl (pH 7.4). The cells were infected with 10 mL of TG1 (OD value to 0.4) at 37°C for 40 minutes. The output was measured, and the cells were plated on Amp+ plates at 30°C overnight. The cells were scraped and inoculated into 50 mL of 2×TY medium (supplemented with Amp and 1% glucose) to an OD value of 0.1. The cells were grown at 37°C at 200 rpm for 80 minutes to an OD value of 0.4-0.6. Take 10 mL of the solution, add 500 μL of helper phage M13KO7, and infect at 37°C for 40 minutes. Centrifuge and remove the supernatant. Resuspend the pellet in 100 mL of 2×TY medium (with Amp and Kana) and incubate at 30°C, 200 rpm, overnight. Centrifuge at 4000 rpm for 30 minutes, collect the supernatant, add 10 mL of PEG / NaCl, and precipitate on ice for 1 hour. Centrifuge at 4000 rpm for 30 minutes, and resuspend the pellet in 1 mL of PBS. Centrifuge at 13000 rpm for 3 minutes, and discard the pellet. Resuspend the phage in PBS and proceed to the next round of panning. Inoculate a single colony into a 96-well plate and incubate at 37°C, 220 rpm, for 3 hours. Add 1 mM IPTG and induce overnight at 30°C. Simultaneously, coat the 96-well plate with MASP2 at a concentration of 2 ng / μL, 50 μL per well, and incubate overnight at 4°C. The next day, wash the ELISA plate once and block it with 200 μL of 2% MPBS in 37°C for 1 hour. Centrifuge the overnight culture at 4000 g for 10 minutes and transfer the supernatant to a new 96-well plate. Wash the plate twice, add 25 μL of 2% MPBS blocking buffer, then add 25 μL of culture supernatant and mix thoroughly. Wash the plate three times at 25°C for 1 hour and add 100 μL of anti-Fab-HRP antibody (1:5000 in 2% MPBS) at 25°C for 1 hour. Wash the plate four times and develop the color. Positive clones were identified by ELISA.

[0359] Positive clones were obtained by immunizing mice with antigen proteins and by phage display screening. These clones were sequenced and purified to obtain anti-MASP2 antibodies. The amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) are shown below. The CDRs of the heavy chain variable region or light chain variable region are underlined:

[0360] >77H11 VH:

[0361] >77H11 VL:

[0362] Table 1. Anti-MASP2 Antibody CDR Sequences (Kabat Numbering Convention)

[0363] Example 3. Identification of anti-MASP2 antibody inhibition of lectin pathway activity

[0364] The obtained anti-MASP2 antibodies were functionally verified to inhibit lectin pathway activity in vitro using 1% human serum.

[0365] Antibody functional activity in 1% human serum was assessed by coating 384-well plates with 25 μL / well of a 50 μg / mL mannan solution and incubating overnight at 4°C. Plates were washed four times with 50 μL / well of TBST, blocked with 50 μL / well of 3% BSA blocking buffer (in TBS), and incubated for 2 hours at room temperature. Plates were washed four times with 50 μL / well of TBST. Anti-MASP2 antibodies of the appropriate concentration were mixed with 1% human serum and incubated at 4°C for 1 hour. The mixture was added to the 384-well plates at 15 μL / well and incubated at 37°C for 1 hour. Plates were washed four times with 50 μL / well of TBST, and primary antibody (biotin-human Anti-C4c, Agrisera #IMSO1-031-305) diluted in 0.5% BSA was added. After centrifugation at 15 μL / well at 800 g / min, the plates were incubated for 1 hour at room temperature. Wash the plate four times with 50 μL / well of TBST, add 15 μL / well of secondary antibody (SA-HRP) diluted 1:5000 in 0.5% BSA solution, and incubate at room temperature for 30 minutes. Wash the plate four times with 50 μL / well of TBST, add 45 μL / well of TMB colorimetric solution, incubate at room temperature in the dark for 15 minutes, and finally add 45 μL / well of stop solution and measure the OD450 value.

[0366] The positive control antibody OMS721 was synthesized according to WO2012151481A, and the sequence is as follows:

[0367] >Full length of heavy chain of OMS721:

[0368] >Full length of light chain of OMS721:

[0369] The results, shown in Figure 1A and Table 2, demonstrate that 77H11 significantly inhibits the lectin pathway, outperforming the positive control, OMS721. Antibody 77H11 is a full-length anti-MASP2 antibody constructed by linking the heavy chain variable region to the Fc region of a human IgG4 heavy chain. The human IgG4 heavy chain Fc region is represented by SEQ ID NO: 19. hIgG4 was used as an isotype control.

[0370] Table 2. Inhibitory effect of anti-MASP2 antibodies on the lectin pathway in the presence of 1% human serum

[0371] Example 4. Humanization of anti-MASP2 antibodies

[0372] The anti-MASP2 antibody 77H11 was selected for humanization and subsequently assayed for activity in human serum.

[0373] The humanized template for the 77H11 light chain was the human germline gene IGKV1-27*01, and the humanized template for the heavy chain was the human germline gene IGKV4-30-4*01. Several back mutations were performed on both chains. Eight molecules were generated, and their humanized sequences are shown below (heavy or light chain CDRs are underlined):

[0374] >77H11_H1:

[0375] >77H11_H2:

[0376] >77H11_H3:

[0377] >77H11_H4:

[0378] >77H11_L1:

[0379] >77H11_L2:

[0380] See Table 3 for the sequences of humanized anti-MASP2 antibodies.

[0381] Table 3. Anti-MASP2 Antibody Reconnected Variable Regions, Light Chain Variable Regions

[0382] The antibodies listed in Table 3 are all full-length anti-MASP2 antibodies constructed by linking the antibody heavy chain variable region to the human IgG4 heavy chain Fc region. The heavy chain Fc region, including the hinge region, is a human IgG4 Fc region with an S228P mutation, the sequence of which is shown in SEQ ID NO: 23. S228P is based on EU numbering.

[0383] >Human IgG4 Fc(S228P):

[0384] Exemplary anti-MASP2 antibody full-length heavy chains and full-length light chains are shown below:

[0385] >77H11(H3L1) full-length heavy chain:

[0386] >77H11 (H3L1) full length light chain:

[0387] The antibody was expressed and purified according to conventional methods, and the full-length antibody disclosed herein was obtained after testing.

[0388] The humanized antibodies were tested for their effects on lectin pathway activity in 90% human serum, which is closer to in vivo conditions. The functional activity of the antibodies in 90% human serum was determined by coating 384-well plates with 25 μL / well of sodium carbonate-sodium bicarbonate buffer (pH>9) containing 5 μg / mL mannan solution and incubating overnight at 4°C. 50 μL / well of 5 mM CaCl2 was added. 2+ Wash 3 times with TBST. Use 50 μL / well of 3% BSA blocking solution (solvent is TBS, add 5mM Ca 2+ ) and blocked at room temperature for 1.5-2 hours. Wash three times with 50 μL / well TBST, once with 50 μL / well TBS, and once with 50 μL / well VBS. MASP2 antibody was diluted with VBS and mixed with human serum at a ratio of 1:9 (i.e., 90% human serum) to obtain a mixture. The mixture was incubated at 4°C for 30 minutes. 15 μL / well of this mixture was added to the 384-well plate described above and incubated at 4°C for 1 hour. Wash three times with 50 μL / well TBST, and add 15 μL / well of the primary antibody (Biotin-Chicken Anti-C4C, 6 μg / mL) diluted in 0.5% BSA in TBS for 60 minutes at room temperature. Wash three times with 50 μL / well TBST, and add 15 μL / well of the secondary antibody (SA-HRP) diluted 1:5000 in 0.5% BSA solution for 30 minutes at room temperature. The plate was then washed three times with 50 μL / well TBST, and 45 μL / well TMB colorimetric solution was added. The plate was incubated at room temperature in the dark for 15 min, and finally 45 μL / well stop solution was added to detect the OD450 value.

[0389] As shown in Figure 2A and Table 4, the humanized 77H11 molecule significantly inhibited lectin pathway activity in 90% human serum, with superior potency compared to OMS721. The 77H11 series of antibodies are full-length anti-MASP2 antibodies constructed by linking the heavy chain variable region of an antibody to the Fc region of a human IgG4 heavy chain. The human IgG4 heavy chain Fc region is represented by SEQ ID NO: 53. hIgG4 was used as an isotype control.

[0390] Table 4. Inhibitory effect of anti-MASP2 antibodies on the lectin pathway in the presence of 90% human serum

[0391] Example 5. Avidity Determination of Anti-MASP2 Antibodies

[0392] Biacore was used to examine the binding properties of anti-MASP2 antibodies. The affinity of the 77H11 (H3L1) antibody to human, mouse, and monkey MASP2 proteins was determined using a Biacore 8K (GE) instrument. Isotype IgG antibodies served as negative controls. Antibodies were captured using an anti-human Fc IgG capture chip. Different concentrations of antigen were used as the mobile phase for detection. Finally, a 1:1 curve fit was performed to obtain affinity values. The results are shown in Table 5.

[0393] The results showed that 77H11 (H3L1) has 10 -10 M to 10 -11 Affinity of M.

[0394] Table 5. Avidity determination results (KD) of anti-MASP2 antibodies

[0395] Example 6. In vitro inhibition of lectin pathway activity in different species by anti-MASP2 antibodies

[0396] The inhibitory effect of anti-MASP2 antibodies on the activity of the lectin pathway in 90% serum of humans, monkeys and mice was detected respectively.

[0397] The method for identifying the functional activity of the antibody in 90% human, monkey, and mouse serum is as follows: 25 μL / well of sodium carbonate-sodium bicarbonate buffer (pH>9) containing 5 μg / mL mannan solution was coated on a 384-well plate and incubated overnight at 4°C. 50 μL / well of 5 mM Ca 2+ Wash 3 times with TBST. Use 50 μL / well of 3% BSA blocking solution (solvent is TBS, add 5mM Ca 2+) and blocked at room temperature for 1.5-2 hours. Wash three times with 50 μL / well TBST, once with 50 μL / well TBS, and once with 50 μL / well VBS. MASP2 antibody was diluted with VBS and mixed with human serum at a ratio of 1:9 (i.e., 90% human serum) to obtain a mixture. The mixture was incubated at 4°C for 30 minutes. 15 μL / well of this mixture was added to the 384-well plate described above and incubated at 4°C for 1 hour. Wash three times with 50 μL / well TBST, and add 15 μL / well of the primary antibody (Biotin-Chicken Anti-C4C, 6 μg / mL) diluted in 0.5% BSA in TBS for 60 minutes at room temperature. Wash three times with 50 μL / well TBST, and add 15 μL / well of the secondary antibody (SA-HRP) diluted 1:5000 in 0.5% BSA solution for 30 minutes at room temperature. The plate was then washed three times with 50 μL / well TBST, and 45 μL / well TMB colorimetric solution was added. The plate was incubated at room temperature in the dark for 15 min, and finally 45 μL / well stop solution was added to detect the OD450 value.

[0398] The results are shown in Figures 3A-3B and Table 6. The results demonstrate that 77H11 (H3L1) exhibits superior inhibitory activity against the lectin pathway in human serum compared to OMS721, using an IgG isotype antibody as a negative control. 11165 and 29C1 (H1L1) are two other anti-MASP2 antibodies screened in this application.

[0399] Table 6. Inhibitory effect of anti-MASP2 antibodies on the lectin pathway under serum conditions of different species

[0400] Example 7. In vivo pharmacodynamics study of anti-MASP2 antibodies in monkeys (PD)

[0401] 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 and serum was collected. 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 as follows: blood samples are collected from peripheral veins and injected directly into blank blood collection tubes. The blood samples are 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.

[0402] The results are shown in Figures 4A and 4B. It can be seen that all four antibodies significantly inhibited lectin pathway activity, with 77H11 (H3L1) exhibiting a longer-lasting inhibitory effect than OMS721 at the same dose. 11165 and 29C1 (H1L1) are two other anti-MASP2 antibodies screened in this application.

[0403] Example 8. Pharmacokinetics (PK) of anti-MASP2 antibodies in monkeys

[0404] Six healthy male crab-eating macaques weighing 2-5 kg ​​were selected. They had not previously received macromolecular drugs and were injected intravenously with the test drugs 77H11 (H3L1) and OMS721, with each drug administered at a dose of 3 mg / kg or 10 mg / kg. Blood was collected at 15 minutes, 1 hour, 4 hours, 8 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 168 hours, 240 hours, 336 hours, 408 hours, 504 hours, and 672 hours 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 minutes, centrifuged at 1000g for 15 minutes, and the supernatant was placed in an EP tube and stored at 80°C.

[0405] The blood drug concentration in serum was detected by ELISA method, and the T 1 / 2 The results are shown in Table 7. As can be seen, the half-life of 77H11 (H3L1) is significantly better than that of OMS721, which means that it can have a longer-lasting effect in the body.

[0406] Table 7. Monkey PK parameter results for anti-MASP2 antibodies

[0407] The use and welfare of experimental animals in this disclosure were conducted in accordance with the guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). The animals were monitored daily for health and mortality. Routine examinations included observing the effects of test substances and drugs on the animals' daily behaviors, such as activity, weight, and physical appearance.

[0408] Example 9. pH screening of anti-MASP2 antibody formulations

[0409] A 10 mM phosphate buffer system was selected, and 12 different pH values ​​were designed: 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. Anti-MASP2 antibody preparations (antibody number 77H11 (H3L1), heavy and light chains as shown in SEQ ID NOs: 24 and 25, prepared in Example 4, the same below) were prepared at a concentration of 10.0 mg / mL. The melting temperature (Tm) of the samples was measured, and the thermal stability of the antibody at different pH values ​​was investigated. The aggregation temperature (Tg) and particle size of the samples were also measured, and the colloidal stability of the antibody at different pH values ​​was investigated. Sequence numbers 1) to 12) are as follows:

[0410] 1) 10 mM sodium dihydrogen phosphate-disodium hydrogen phosphate, pH 4.6

[0411] 2) 10 mM sodium dihydrogen phosphate-disodium hydrogen phosphate, pH 5.0

[0412] 3) 10 mM sodium dihydrogen phosphate-disodium hydrogen phosphate, pH 5.4

[0413] 4) 10 mM sodium dihydrogen phosphate-disodium hydrogen phosphate, pH 5.8

[0414] 5) 10 mM sodium dihydrogen phosphate-disodium hydrogen phosphate, pH 6.2

[0415] 6) 10 mM sodium dihydrogen phosphate-disodium hydrogen phosphate, pH 6.6

[0416] 7) 10 mM sodium dihydrogen phosphate-disodium hydrogen phosphate, pH 7.0

[0417] 8) 10 mM sodium dihydrogen phosphate-disodium hydrogen phosphate, pH 7.4

[0418] 9) 10 mM sodium dihydrogen phosphate-disodium hydrogen phosphate, pH 7.8

[0419] 10) 10 mM sodium dihydrogen phosphate-disodium hydrogen phosphate, pH 8.2

[0420] 11) 10 mM sodium dihydrogen phosphate-disodium hydrogen phosphate, pH 8.6

[0421] 12) 10 mM sodium dihydrogen phosphate-disodium hydrogen phosphate, pH 9.0

[0422] Table 8. pH screening results of anti-MASP2 antibody formulations (Note: %PD represents the dispersion coefficient. The smaller the value, the more uniform the particle size distribution. Multimodal indicates a poor particle size distribution, and the equipment cannot calculate the %PD value.)

[0423] The results showed that the anti-MASP2 antibody had the highest Tm value at pH 7.0, followed by pH 6.6, indicating that the antibody had good thermal stability at pH 6.6-7.0. The anti-MASP2 antibody also had the highest Tagg value at pH 6.6, followed by pH 7.0. Furthermore, the particle size was smaller and the particle size distribution was more uniform at pH 6.6, indicating that the antibody had good colloidal stability at pH 6.6-7.0. Therefore, the anti-MASP2 antibody had good thermal and colloidal stability at pH 6.6-7.0.

[0424] Example 10. Screening of anti-MASP2 antibody preparation buffer system (pH 6.6)

[0425] A 10 mM sodium dihydrogen phosphate-disodium hydrogen phosphate, 10 mM histidine-histidine hydrochloride buffer system was used to prepare an antibody formulation at pH 6.6 with an anti-MASP2 antibody content of 50 mg / mL. Buffer system (pH 6.6) screening was conducted by examining stability at 40°C. Sequence numbers 1) to 2) are as follows:

[0426] 1) 10 mM sodium dihydrogen phosphate-sodium hydrogen phosphate pH 6.6

[0427] 2) 10 mM histidine-histidine hydrochloride pH 6.6

[0428] Table 9 Anti-MASP2 Antibody Preparation Buffer System (pH 6.6) Screening Results (Note: This table shows the sample measurement results of the anti-MASP2 antibody preparation buffer system (pH 6.6) at time 0 screening)

[0429] Table 10 Anti-MASP2 Antibody Preparation Buffer System (pH 6.6) Screening Results (Note: W stands for week; %PD stands for dispersion coefficient)

[0430] The results showed that at pH 6.6, the Tm value of the anti-MASP2 antibody in the 10 mM histidine-histidine hydrochloride buffer system was slightly lower than that in the 10 mM sodium dihydrogen phosphate-disodium hydrogen phosphate buffer system, but the Tagg value was higher; under the same experimental conditions, the anti-MASP2 antibody had a smaller particle size and lower aggregate content in the 10 mM histidine-histidine hydrochloride buffer system, indicating that at pH 6.6, the anti-MASP2 antibody had better stability in the 10 mM histidine-histidine hydrochloride buffer system.

[0431] Example 11. Screening of anti-MASP2 antibody preparation buffer system (pH 7.0)

[0432] A 10 mM sodium dihydrogen phosphate-disodium hydrogen phosphate, 10 mM histidine-histidine hydrochloride buffer system was used to prepare an antibody formulation at pH 7.0 with an anti-MASP2 antibody content of 50 mg / mL. Samples were then taken for high temperature stability studies at 40°C. Sequence numbers 1) to 2) are as follows:

[0433] 1) 10 mM sodium dihydrogen phosphate-sodium hydrogen phosphate pH 7.0

[0434] 2) 10 mM histidine-histidine hydrochloride pH 7.0

[0435] Table 11 Anti-MASP2 Antibody Preparation Buffer System (pH 7.0) Screening Results (Note: This table shows the sample measurement results of the anti-MASP2 antibody preparation buffer system (pH 7.0) at time 0 screening)

[0436] Table 12 Anti-MASP2 Antibody Preparation Buffer System (pH 7.0) Screening Results (Note: W stands for week; %PD stands for dispersion coefficient)

[0437] The results showed that under the same experimental conditions, compared with the 10 mM sodium dihydrogen phosphate-disodium hydrogen phosphate pH 7.0 buffer system, the anti-MASP2 antibody in the 10 mM histidine-histidine hydrochloride pH 7.0 buffer system had higher Tm and Tagg values, better appearance, smaller particle size, and relatively lower aggregate content. Therefore, the anti-MASP2 antibody was more stable in the 10 mM histidine-histidine hydrochloride pH 7.0 buffer system.

[0438] Example 12. Screening of surfactant concentrations for anti-MASP2 antibody formulations

[0439] Sucrose (75 mg / mL) was used as the excipient, and a 10 mM histidine-histidine hydrochloride pH 6.8 buffer system was used to prepare antibody formulations containing 100 mg / mL anti-MASP2 antibody, 75 mg / mL sucrose, and different polysorbate 80 concentrations (0.1 mg / mL and 0.4 mg / mL). Protein stability was evaluated under shaking (200 rpm, room temperature) and freeze-thaw (-35°C / room temperature) conditions. Sequence numbers 1) to 2) are as follows:

[0440] 1) 10 mM histidine-histidine hydrochloride, 0.1 mg / mL polysorbate 80, 75 mg / mL sucrose, protein concentration 100 mg / mL, pH 6.8

[0441] 2) 10 mM histidine-histidine hydrochloride, 0.4 mg / mL polysorbate 80, 75 mg / mL sucrose, protein concentration 100 mg / mL, pH 6.8

[0442] Table 13 Anti-MASP2 Antibody Formulation Surfactant Concentration Screening Results

[0443] Table 14 Anti-MASP2 Antibody Formulation Surfactant Concentration Screening Results (Note: h stands for hour; W stands for week)

[0444] Table 15 Anti-MASP2 Antibody Formulation Surfactant Concentration Screening Results (Note: h stands for hour; W stands for week)

[0445] The results showed that when the polysorbate 80 concentration was 0.1 mg / mL-0.4 mg / mL, there was no significant difference in the stability of the anti-MASP2 antibody preparation under shaking and freeze-thaw conditions. Therefore, the concentration range of polysorbate 80 in the anti-MASP2 antibody preparation was set at 0.1 mg / mL to 0.4 mg / mL.

[0446] Example 13. Screening of excipients for anti-MASP2 antibody formulations

[0447] Antibody formulations containing 100 mg / mL anti-MASP2 antibody, various 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 were prepared using a 10 mM histidine-histidine hydrochloride pH 6.8 buffer system. Protein stability was evaluated at 40°C. Sequence numbers 1) to 4) are as follows:

[0448] 1) 10 mM histidine-histidine hydrochloride, 0.2 mg / mL polysorbate 80, 75 mg / mL sucrose, protein concentration 100 mg / mL, pH 6.8

[0449] 2) 10 mM histidine-histidine hydrochloride, 0.2 mg / mL polysorbate 80, 45 mg / mL mannitol, protein concentration 100 mg / mL, pH 6.8

[0450] 3) 10 mM histidine-histidine hydrochloride, 0.2 mg / mL polysorbate 80, 8.2 mg / mL sodium chloride, protein concentration 100 mg / mL, pH 6.8

[0451] 4) 10 mM histidine-histidine hydrochloride, 0.2 mg / mL polysorbate 80, 28 mg / mL proline, protein concentration 100 mg / mL, pH 6.8

[0452] Table 16 Results of screening of excipient types for anti-MASP2 antibody preparations

[0453] Table 17 Results of screening of excipient types for anti-MASP2 antibody preparations (Note: h stands for hour; W stands for week)

[0454] Table 18 Screening results of excipient types for anti-MASP2 antibody preparations (Note: h stands for hour; W stands for week)

[0455] The results showed that under the same experimental conditions, there was no significant difference in the SEC monomer content and R-CE purity of the anti-MASP2 antibody in different excipient systems. However, compared with other excipients, the anti-MASP2 antibody in the No. 3 preparation containing the excipient sodium chloride had better thermal stability, relatively smaller particle size, and more uniform distribution.

[0456] Example 14. Effect of pH on Stability of Anti-MASP2 Antibody Formulations

[0457] A 10 mM histidine-histidine hydrochloride buffer system was used to prepare antibody formulations containing 8.2 mg / mL sodium chloride, 0.2 mg / mL polysorbate 80, and 110 mg / mL anti-MASP2 antibody at different pH values ​​(pH 6.4, pH 6.6, and pH 6.8). The stability of the antibody formulations was investigated at 40°C. Sequence numbers 1) to 3) are as follows:

[0458] 1) 10 mM histidine-histidine hydrochloride, 0.2 mg / mL polysorbate 80, 8.2 mg / mL sodium chloride, protein concentration 110 mg / mL, pH 6.4

[0459] 2) 10 mM histidine-histidine hydrochloride, 0.2 mg / mL polysorbate 80, 8.2 mg / mL sodium chloride, protein concentration 110 mg / mL, pH 6.6

[0460] 3) 10 mM histidine-histidine hydrochloride, 0.2 mg / mL polysorbate 80, 8.2 mg / mL sodium chloride, protein concentration 110 mg / mL, pH 6.8

[0461] Table 19 Results of investigation on the effect of pH on the stability of anti-MASP2 antibody formulations (Note: h stands for hours; W stands for weeks; particle size reflects colloidal stability; %PD stands for dispersion coefficient)

[0462] Table 20 Results of investigation on the effect of pH on the stability of anti-MASP2 antibody formulations (Note: h stands for hour; W stands for week)

[0463] The results showed that the effects of pH 6.4, pH 6.6 and pH 6.8 on the stability of anti-MASP2 antibodies were not significantly different, so the pH range of the anti-MASP2 antibody preparation was 6.4-6.8.

[0464] Example 15. Screening of excipients for anti-MASP2 antibody formulations

[0465] Antibody formulations containing 200 mg / mL anti-MASP2 antibody, different 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 were prepared using a 10 mM histidine-histidine hydrochloride pH 6.6 buffer system. Protein stability under frozen storage conditions was investigated. Sequence numbers 1) to 3) are as follows:

[0466] 1) 10 mM histidine-histidine hydrochloride, 0.4 mg / mL polysorbate 80, 8.0 mg / mL sodium chloride, protein concentration 200 mg / mL, pH 6.6

[0467] 2) 10 mM histidine-histidine hydrochloride, 0.4 mg / mL polysorbate 80, 40 mg / mL sucrose + 15 mg / mL arginine hydrochloride, protein concentration 200 mg / mL, pH 6.6

[0468] 3) 10 mM histidine-histidine hydrochloride, 0.4 mg / mL polysorbate 80, 30 mg / mL arginine hydrochloride, protein concentration 200 mg / mL, pH 6.6

[0469] Table 21 Screening results of excipient types for anti-MASP2 antibody preparations (Note: W stands for week. For protein samples 1 to 3, the SEC monomer content at T0 was 98.5%, and the SEC aggregate content was 1.4%)

[0470] The results showed that under -35℃ freezing conditions, the preparation containing sucrose + arginine hydrochloride had better SEC purity protection than the preparation containing sodium chloride or arginine hydrochloride.

[0471] Example 16. Screening of protein concentration of anti-MASP2 antibody preparations

[0472] A 165 mg / mL anti-MASP2 antibody was prepared and the protein stability was investigated under conditions of high temperature (40°C), shaking (200 rpm, room temperature), illumination (5000 ± 500 lx), and freeze-thaw (-35°C / room temperature). The formulation is as in SEQ ID 1):

[0473] 1) 10 mM histidine-histidine hydrochloride, 0.4 mg / mL polysorbate 80, 40 mg / mL sucrose + 15 mg / mL arginine hydrochloride, protein concentration 165 mg / mL, pH 6.6

[0474] Table 22 Anti-MASP2 Antibody Formulation Stability (Note: W stands for week, FVP stands for no visible protein particles)

[0475] The results showed that the above-mentioned preparation containing sucrose + arginine hydrochloride and a protein concentration of 165 mg / mL had no significant changes in purity and appearance under all the conditions investigated by SEC and iCIEF, and had good stability.

[0476] In addition, 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.

[0477] Example 17. Optional formulations

[0478] The present invention provides an anti-MASP2 antibody pharmaceutical formulation having a formulation of "90-165 mg / mL anti-MASP2 antibody, approximately 8.2 mg / mL sodium chloride or 40 mg / mL sucrose, 15 mg / mL arginine hydrochloride, 0.1-0.4 mg / mL polysorbate 80, approximately 10 mM histidine-histidine hydrochloride, pH 6.4-6.8", including but not limited to:

[0479] (1) 90, 100, or 110 mg / mL anti-MASP2 antibody, 8.2 mg / mL sodium chloride, 0.1 mg / mL polysorbate 80, and 10 mM histidine-histidine hydrochloride, pH 6.4

[0480] (2) 90, 100, or 110 mg / mL anti-MASP2 antibody, 8.2 mg / mL sodium chloride, 0.2 mg / mL polysorbate 80, and 10 mM histidine-histidine hydrochloride, pH 6.4

[0481] (3) 90, 100, or 110 mg / mL anti-MASP2 antibody, 8.2 mg / mL sodium chloride, 0.4 mg / mL polysorbate 80, and 10 mM histidine-histidine hydrochloride, pH 6.4

[0482] (4) 90, 100, or 110 mg / mL anti-MASP2 antibody, 8.2 mg / mL sodium chloride, 0.1 mg / mL polysorbate 80, and 10 mM histidine-histidine hydrochloride, pH 6.6

[0483] (5) 90, 100, or 110 mg / mL anti-MASP2 antibody, 8.2 mg / mL sodium chloride, 0.2 mg / mL polysorbate 80, and 10 mM histidine-histidine hydrochloride, pH 6.6

[0484] (6) 90, 100, or 110 mg / mL anti-MASP2 antibody, 8.2 mg / mL sodium chloride, 0.4 mg / mL polysorbate 80, and 10 mM histidine-histidine hydrochloride, pH 6.6

[0485] (7) 90, 100, or 110 mg / mL anti-MASP2 antibody, 8.2 mg / mL sodium chloride, 0.1 mg / mL polysorbate 80, and 10 mM histidine-histidine hydrochloride, pH 6.8

[0486] (8) 90, 100, or 110 mg / mL anti-MASP2 antibody, 8.2 mg / mL sodium chloride, 0.2 mg / mL polysorbate 80, and 10 mM histidine-histidine hydrochloride, pH 6.8

[0487] (9) 90, 100, or 110 mg / mL anti-MASP2 antibody, 8.2 mg / mL sodium chloride, 0.4 mg / mL polysorbate 80, and 10 mM histidine-histidine hydrochloride, pH 6.8

[0488] (10) 100 mg / mL anti-MASP2 antibody, 8.2 mg / mL sodium chloride, 0.4 mg / mL polysorbate 80, and 10 mM histidine-histidine hydrochloride, pH 6.6

[0489] (11) 135, 150, or 165 mg / mL anti-MASP2 antibody, 40 mg / mL sucrose, 15 mg / mL arginine hydrochloride, 0.4 mg / mL polysorbate 80, and 10 mM histidine-histidine hydrochloride, pH 6.6

[0490] (12) 150 mg / mL anti-MASP2 antibody, 40 mg / mL sucrose, 15 mg / mL arginine hydrochloride, 0.4 mg / mL polysorbate 80, and 10 mM histidine-histidine hydrochloride, pH 6.6.

[0491] The experimental results show that the anti-MASP2 antibody preparations of the above formulations all have good stability and can be used in the preparation of anti-MASP2 antibody drugs.

[0492] Although specific embodiments of the present disclosure are described above, those skilled in the art will appreciate that these are merely examples and that various changes or modifications may be made to these embodiments without departing from the principles and essence of the present disclosure.

Claims

1. A pharmaceutical composition comprising an anti-MASP2 antibody or antigen-binding fragment thereof and a buffer, wherein the anti-MASP2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein: The VH comprises HCDR1, HCDR2, and HCDR3 in VH as shown in SEQ ID NO: 7, and the VL comprises LCDR1, LCDR2, and LCDR3 in VL as shown in SEQ ID NO:

8. wherein the HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3 are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering systems, preferably according to the Kabat numbering system; The buffer is selected from phosphate buffer and histidine salt buffer, preferably histidine salt buffer, more preferably histidine-hydrochloric acid buffer or histidine-acetate buffer, most preferably histidine-histidine hydrochloride buffer.

2. The pharmaceutical composition of claim 1, wherein the VH and VL of the anti-MASP2 antibody or antigen-binding fragment thereof are as follows: The VH comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 9, 10, and 11, respectively, and the VL comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NOs: 12, 13, and 14, respectively.

3. The pharmaceutical composition according to claim 1 or 2, wherein the anti-MASP2 antibody is a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

4. The pharmaceutical composition of claim 3, wherein the anti-MASP2 antibody or antigen-binding fragment thereof is a humanized antibody or antigen-binding fragment thereof, wherein: The heavy chain framework region of the humanized antibody or antigen-binding fragment thereof 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.

5. The pharmaceutical composition of any one of claims 1 to 4, wherein the anti-MASP2 antibody or antigen-binding fragment thereof comprises: The VH comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 17-20, or at least 90%, at least 95% identical thereto, and the VL comprises an amino acid sequence as set forth in SEQ ID NOs: 21 or 22, or at least 90%, at least 95% identical thereto; The VH comprises an amino acid sequence as shown in SEQ ID NO: 7, or at least 90% or at least 95% identical thereto, and the VL comprises an amino acid sequence as shown in SEQ ID NO: 8, or at least 90% or at least 95% identical thereto.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the anti-MASP2 antibody or antigen-binding fragment thereof is an IgG antibody or antigen-binding fragment thereof, preferably an IgG1, IgG2, or IgG4 antibody or antigen-binding fragment thereof, more preferably F C The invention relates to an IgG4 antibody or an antigen-binding fragment thereof having any one or more mutations among S228P, F234A and L235A.

7. The pharmaceutical composition of any one of claims 1 to 6, wherein the anti-MASP2 antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, wherein: The heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 24, or at least 90%, at least 95% identical thereto; the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 25, or at least 90%, at least 95% identical thereto.

8. The pharmaceutical composition according to any one of claims 1 to 7, wherein the pH of the buffer is 5.0-8.5, preferably 5.5-8, more preferably 6.0-7.

5.

9. The pharmaceutical composition according to any one of claims 1 to 8, wherein the buffer concentration is 1-30 mM, preferably 5-20 mM, most preferably about 10 mM.

10. The pharmaceutical composition according to any one of claims 1 to 9, further comprising one or more excipients selected from amino acids or salts thereof, sugars, polyols and salts, wherein the excipients are preferably salts, or sugars and amino acids or salts thereof; more preferably sodium chloride, sucrose and arginine or salts thereof, and most preferably sucrose and arginine hydrochloride.

11. The pharmaceutical composition of claim 10, wherein the excipient concentration is 0.1-100 mg / mL, preferably 0.5-80 mg / mL, more preferably about 8.2 mg / mL, about 15 mg / mL, about 28 mg / mL, about 30 mg / mL, about 40 mg / mL, about 45 mg / mL and about 75 mg / mL. 12 . The pharmaceutical composition according to claim 1 , further comprising a surfactant, preferably polysorbate, more preferably polysorbate 80.

13. The pharmaceutical composition according to claim 12, wherein the surfactant concentration is 0.01-2 mg / mL, preferably 0.05-1 mg / mL, more preferably 0.1-0.4 mg / mL.

14. The pharmaceutical composition of any one of claims 1 to 13, wherein the anti-MASP2 antibody or antigen-binding fragment thereof is present at a concentration of 0.1-500 mg / mL, preferably 5-300 mg / mL, more preferably selected from 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.

15. A pharmaceutical composition comprising any one of the following: A) 0.1-300 mg / mL of an anti-MASP2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 7; 0.5-80 mg / mL sodium chloride; 5-30 mM histidine-HCl buffer; 0.01-1 mg / mL polysorbate; pH 5.5-8.0; B) 80-120 mg / mL of the anti-MASP2 antibody or antigen-binding fragment thereof of any one of claims 1 to 7; 5-20 mg / mL sodium chloride; 5-15 mM histidine-HCl buffer; 0.05-0.6 mg / mL polysorbate; pH 5.5-7.5; C) 90-110 mg / mL of the anti-MASP2 antibody or antigen-binding fragment thereof of any one of claims 1 to 7; 8-9 mg / mL sodium chloride; 8-12 mM histidine-HCl buffer; 0.05-0.5 mg / mL polysorbate; pH 6.0-7.5; D) 90-110 mg / mL of the anti-MASP2 antibody or antigen-binding fragment thereof of any one of claims 1 to 7; approximately 8.2 mg / mL of sodium chloride; approximately 10 mM histidine-HCl buffer; 0.1-0.4 mg / mL polysorbate; pH 6.1-7.1; E) 0.1-500 mg / mL of the anti-MASP2 antibody or antigen-binding fragment thereof of any one of claims 1 to 7; 0.5-100 mg / mL sucrose; 0.5-80 mg / mL of arginine or its salt; 0.1-50 mM histidine-HCl buffer; 0.01-1 mg / mL polysorbate; pH 5.5-8.0; F) 5-300 mg / mL of the anti-MASP2 antibody or antigen-binding fragment thereof of any one of claims 1 to 7; 5-80 mg / mL sucrose; 1-60 mg / mL of arginine or its salt; 1-30 mM histidine-HCl buffer; 0.05-0.6 mg / mL polysorbate; pH 5.5-7.5; G) 50-250 mg / mL of the anti-MASP2 antibody or antigen-binding fragment thereof of any one of claims 1 to 7; 10-70 mg / mL sucrose; 5-30 mg / mL of arginine or its salt; 5-20 mM histidine-HCl buffer; 0.05-0.5 mg / mL polysorbate; pH 6.0-7.5; H) 90-200 mg / mL of the anti-MASP2 antibody or antigen-binding fragment thereof of any one of claims 1 to 7; 30-50 mg / mL sucrose; 10-20 mg / mL of arginine or its salt; 8-12 mM histidine-HCl buffer; 0.1-0.4 mg / mL polysorbate; pH 6.1-7.1; 1) 90-200 mg / mL of an anti-MASP2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 7; approximately 40 mg / mL of sucrose; about 15 mg / mL arginine or a salt thereof; approximately 10 mM histidine-HCl buffer; 0.1-0.4 mg / mL polysorbate; pH is 6.4-6.

8. Preferably, in any one of the pharmaceutical compositions of groups A) to I), the arginine or its salt is arginine hydrochloride; Preferably, in any one of the pharmaceutical compositions of groups A) to I), the polysorbate is polysorbate 80.

16. A pharmaceutical composition comprising any one of the following: (1) about 90 mg / mL, about 100 mg / mL, or about 110 mg / mL of an anti-MASP2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, about 8.2 mg / mL sodium chloride, about 0.1 mg / mL polysorbate 80, and about 10 mM histidine-histidine hydrochloride, at a pH of about 6.4; (2) about 90 mg / mL, about 100 mg / mL, or about 110 mg / mL of an anti-MASP2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, about 8.2 mg / mL sodium chloride, about 0.2 mg / mL polysorbate 80, and about 10 mM histidine-histidine hydrochloride, at a pH of about 6.4; (3) about 90 mg / mL, about 100 mg / mL, or about 110 mg / mL of an anti-MASP2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, about 8.2 mg / mL sodium chloride, about 0.4 mg / mL polysorbate 80, and about 10 mM histidine-histidine hydrochloride, at a pH of about 6.4; (4) about 90 mg / mL, about 100 mg / mL, or about 110 mg / mL of an anti-MASP2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, about 8.2 mg / mL sodium chloride, about 0.1 mg / mL polysorbate 80, and about 10 mM histidine-histidine hydrochloride, at a pH of about 6.6; (5) about 90 mg / mL, about 100 mg / mL, or about 110 mg / mL of an anti-MASP2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, about 8.2 mg / mL sodium chloride, about 0.2 mg / mL polysorbate 80, and about 10 mM histidine-histidine hydrochloride, at a pH of about 6.6; (6) about 90 mg / mL, about 100 mg / mL, or about 110 mg / mL of an anti-MASP2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, about 8.2 mg / mL sodium chloride, about 0.4 mg / mL polysorbate 80, and about 10 mM histidine-histidine hydrochloride, at a pH of about 6.6; (7) about 90 mg / mL, about 100 mg / mL, or about 110 mg / mL of an anti-MASP2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, about 8.2 mg / mL sodium chloride, about 0.1 mg / mL polysorbate 80, and about 10 mM histidine-histidine hydrochloride, at a pH of about 6.8; (8) about 90 mg / mL, about 100 mg / mL, or about 110 mg / mL of an anti-MASP2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, about 8.2 mg / mL sodium chloride, about 0.2 mg / mL polysorbate 80, and about 10 mM histidine-histidine hydrochloride, at a pH of about 6.8; (9) about 90 mg / mL, about 100 mg / mL, or about 110 mg / mL of an anti-MASP2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, about 8.2 mg / mL sodium chloride, about 0.4 mg / mL polysorbate 80, and about 10 mM histidine-histidine hydrochloride, at a pH of about 6.8; (10) about 135 mg / mL, about 150 mg / mL, about 165 mg / mL, or about 200 mg / mL of an anti-MASP2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, about 40 mg / mL sucrose, about 15 mg / mL arginine hydrochloride, about 0.1 mg / mL polysorbate 80, and about 10 mM histidine-histidine hydrochloride, at a pH of about 6.4; (11) about 135 mg / mL, about 150 mg / mL, about 165 mg / mL, or about 200 mg / mL of an anti-MASP2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, about 40 mg / mL sucrose, about 15 mg / mL arginine hydrochloride, about 0.2 mg / mL polysorbate 80, and about 10 mM histidine-histidine hydrochloride, at a pH of about 6.4; (12) about 135 mg / mL, about 150 mg / mL, about 165 mg / mL, or about 200 mg / mL of an anti-MASP2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, about 40 mg / mL sucrose, about 15 mg / mL arginine hydrochloride, about 0.4 mg / mL polysorbate 80, and about 10 mM histidine-histidine hydrochloride, at a pH of about 6.4; (13) about 135 mg / mL, about 150 mg / mL, about 165 mg / mL, or about 200 mg / mL of an anti-MASP2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, about 40 mg / mL sucrose, about 15 mg / mL arginine hydrochloride, about 0.1 mg / mL polysorbate 80, and about 10 mM histidine-histidine hydrochloride, at a pH of about 6.6; (14) about 135 mg / mL, about 150 mg / mL, about 165 mg / mL, or about 200 mg / mL of an anti-MASP2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, about 40 mg / mL sucrose, about 15 mg / mL arginine hydrochloride, about 0.2 mg / mL polysorbate 80, and about 10 mM histidine-histidine hydrochloride, at a pH of about 6.6; (15) about 135 mg / mL, about 150 mg / mL, about 165 mg / mL, or about 200 mg / mL of an anti-MASP2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, about 40 mg / mL sucrose, about 15 mg / mL arginine hydrochloride, about 0.4 mg / mL polysorbate 80, and about 10 mM histidine-histidine hydrochloride, at a pH of about 6.6; (16) about 135 mg / mL, about 150 mg / mL, about 165 mg / mL, or about 200 mg / mL of an anti-MASP2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, about 40 mg / mL sucrose, about 15 mg / mL arginine hydrochloride, about 0.1 mg / mL polysorbate 80, and about 10 mM histidine-histidine hydrochloride, at a pH of about 6.8; (17) about 135 mg / mL, about 150 mg / mL, about 165 mg / mL, or about 200 mg / mL of an anti-MASP2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, about 40 mg / mL sucrose, about 15 mg / mL arginine hydrochloride, about 0.2 mg / mL polysorbate 80, and about 10 mM histidine-histidine hydrochloride, at a pH of about 6.8; (18) about 135 mg / mL, about 150 mg / mL, about 165 mg / mL, or about 200 mg / mL of an anti-MASP2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, about 40 mg / mL sucrose, about 15 mg / mL arginine hydrochloride, about 0.4 mg / mL polysorbate 80, and about 10 mM histidine-histidine hydrochloride, at a pH of about 6.

8.

17. A lyophilized preparation, which can form the pharmaceutical composition of any one of claims 1 to 16 after being reconstituted, or which is obtained by freeze-drying the pharmaceutical composition of any one of claims 1 to 16.

18. A reconstituted solution, wherein the reconstituted solution is obtained by reconstituted the lyophilized preparation according to claim 17.

19. A product comprising a container containing the pharmaceutical composition of any one of claims 1 to 16, the lyophilized formulation of claim 17, or the reconstituted solution of claim 18.

20. Use of the pharmaceutical composition according to any one of claims 1 to 16, the lyophilized preparation according to claim 17, or the reconstituted solution according to claim 18 in the preparation of a medicament for treating a disease; Preferably, the disease is a MASP-2-dependent complement activation-related disease; Preferably, the disease is selected from the group consisting of: IgA nephropathy, paroxysmal nocturnal hemoglobinuria (PNH), lupus nephritis, thrombotic microangiopathy (TMA), hemolytic uremic syndrome (HUS), membranous glomerulonephritis, glomerulonephritis, age-related macular degeneration, reperfusion injury, myocardial infarction, diabetic neuropathy, stroke, graft-versus-host disease (GVHD), and Upshaw-Schulman syndrome (USS).

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