New regulations for anti-MASP-2 antibodies

Abstract

Anti-MASP-2 antibodies or antigen-binding fragments thereof, isolated polynucleotides encoding same, pharmaceutical compositions containing same, and uses thereof are provided.

Description

[Technical field]

[0001]

[0001] The present disclosure relates generally to novel anti-MASP-2 antibodies and uses thereof. [Background technology]

[0002]

[0002] The complement system comprises a complex array of enzymatic and nonenzymatic proteins that are essential for the operation of innate as well as adaptive immune defenses. There are three pathways that initiate complement activation: the classical pathway, the mannan-binding lectin (MBL) pathway, and the alternative pathway. These pathways depend on different molecules for their initiation, while converging to generate the same set of effector molecules, such as the membrane attack complex (MAC). All three pathways are important parts of innate immunity and play different roles in defending against various infections. MBL is structurally related to the complement C1 subcomponent C1q and appears to activate the complement system through related serine proteases known as MASPs, which are similar to C1r and C1s of the classical pathway. MBL binds to specific carbohydrate structures found on the surface of various microorganisms, including bacteria, yeasts, parasitic protozoans and viruses, and exerts antibacterial activity through killing mediated by terminal lytic complement components or by promoting phagocytosis (S Thiel et al., Nature, 386(6624):506-510 (1997); Noris M, et al. 2013. JM.).

[0003]

[0003] MASP-2 (MBL-associated serine protease 2) is involved in the complement system and shows striking homology with MASP-1 and two C1q-related serine proteases C1r and C1s. Upon lectin recognition and binding to a pathogen, the protozyme form of MASP-2 is cleaved between the CCP2 and SP domains (cleaved between conserved R444 and I445) and converted into an active form consisting of two polypeptide chains (heavy / A chain and light / B chain) linked by a disulfide bond (C434-C552) (ABW Boldt et al., Human Immunology, 72(9):753-760 (2011)). When MBL binds to pathogens, MASP-2 is activated to cleave complement components C4 and C2 into C4a, C4b, C2a, and C2b, resulting in the generation of the convertase C4bC2b of C3, which is then converted into C3b by C4bC2b, and finally forms the membrane attack complex (MAC) after C5 is converted into C5b by C3b. The activation of C3 ultimately leads to the formation of MAC, which then initiates a series of cascade activation processes of the downstream complement system that activates the innate immune response. In fact, C4b can activate the generation of C4d. C4d deposition may be a marker of complement activation. It has been shown that C4d positive staining is an independent risk factor for the development of ESRD in IgAN (see Clin J Am Soc Nephrol 9:897-904, 2014) and that C4d stained kidneys have a significantly shorter survival time in IgAN patients than kidneys without C4d staining. C4D staining has also been detected in kidneys from both lupus nephritis and membranous glomerulonephritis patients. MASP-2 has been identified as a promising target for the treatment of autoimmune diseases.

[0004] In addition to its essential role in immune defense, the complement system contributes to tissue damage in many clinical conditions. There remains a need for therapeutically effective complement inhibitors, such as novel MASP-2 antibodies, with particularly advantageous high binding affinity and specificity to prevent side effects. Summary of the Invention [Problem to be solved by the invention]

[0005]

[0004] Throughout this disclosure, the articles "a," "an," and "the" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an antibody" means one antibody or more than one antibody.

[0006]

[0005] The complement system functions as a natural immunity that can eliminate pathogenic microorganisms, and its serine protease enzymes activate each other in a strictly ordered manner. There are numerous diseases associated with complement dysfunction, such as thrombotic microangiopathy (TMA), atypical hemolytic uraemic syndrome (aHUS), hematopoietic transplant-associated thrombotic microangiopathy (TA-TMA), lupus nephritis, and IgA nephropathy. Most patients with these diseases have been found to have tissue damage due to uncontrolled complement activation and chronic attack of complement on endothelial cells. MASP-2 antibodies that can block complement activation via the MBL pathway may be a possible therapeutic approach, which is also the mechanism of action for OMS-721 from Omeros, which is in ongoing clinical trials.

[0007]

[0006] The present disclosure provides novel monoclonal anti-MASP-2 antibodies, their amino acid and nucleotide sequences, and their uses. [Means for solving the problem]

[0008] In one aspect, the present disclosure provides an isolated antibody or antigen-binding fragment thereof that specifically binds to MASP-2, the antibody or antigen-binding fragment having the following characteristics: a) has no cross-reactivity with mouse or rat; b) has a longer serum half-life in monkeys compared to OMS721; c) has no cross-reactivity with C1s, C1r, MASP1 or MASP3; d) it can selectively block complement activation of the MBL pathway; e) 27.8nM or less (or 25nM, 20nM, 15nM, 10nM, 9nM, 8nM, 7nM, 6nM, 5nM, 4nM, 3nM, 2nM, 1nM, 0.9nM, 0.8nM, 0.7nM, 0.6nM, 0.5nM, 0.4nM, 0.3nM, 0.2nM, 0.1nM) when measured by biolayer interference , 0.09nM, 0.08nM, 0.07nM, 0.06nM, 0.05nM, 0.04nM, 0.03nM, 0.02nM, 0.01nM, 0.009nM, 0.008nM, 0.007nM, 0.006nM, 0.005nM, 0.004nM, 0.003nM, 0.002nM, or 0.001nM or less) D can specifically bind to human MASP-2 at a value f) capable of blocking complement C3 activation with an IC50 of 0.08 μg / mL or less (or 0.07 μg / mL, 0.06 μg / mL, 0.05 μg / mL, 0.04 μg / mL, 0.03 μg / mL, 0.02 μg / mL, or 0.01 μg / mL or less) in 1% human serum as measured by an ELISA assay, or with an IC50 of 0.20 μg / mL or less (or 0.15 μg / mL, 0.10 μg / mL, 0.09 μg / mL, 0.08 μg / mL, 0.07 μg / mL, 0.06 μg / mL, 0.05 μg / mL, 0.04 μg / mL, 0.03 μg / mL, 0.02 μg / mL, or 0.01 μg / mL or less) in 10% human serum as measured by an ELISA assay; g) capable of blocking complement C3 activation in 50% human serum; h) an IC50 value of 0.11 μg / mL or less (or 0.10 μg / mL, 0.09 μg / mL, 0.08 μg / mL, 0.07 μg / mL, 0.06 μg / mL, 0.05 μg / mL, 0.04 μg / mL, 0.03 μg / mL, 0.02 μg / mL or 0.01 μg / mL or less) when measured in 2% human serum by ELISA assay, or 0.69 μg / mL or less (or 0.65 μg / mL, 0.6 μg / mL, 0.6 μg / mL or less) when measured in 10% human serum by ELISA assay; 0.06μg / mL, 0.05μg / mL, 0.04μg / mL, 0.03μg / mL, 0.02μg / mL or 0.01μg / mL); i) capable of blocking MAC formation with an IC50 value of 0.27 μg / mL or less (or 0.25 μg / mL, or 0.2 μg / mL, 0.15 μg / mL, 0.1 μg / mL, 0.09 μg / mL, 0.08 μg / mL, 0.07 μg / mL, 0.06 μg / mL, 0.05 μg / mL, 0.04 μg / mL, 0.03 μg / mL, 0.02 μg / mL, or 0.01 μg / mL or less) as measured in 2% human serum by ELISA assay; The present invention provides an isolated antibody or antigen-binding fragment thereof, which exhibits at least one of the following:

[0009] In one aspect, the present disclosure provides an isolated antibody or antigen-binding fragment thereof that specifically binds to MASP-2, comprising: Heavy chain CDR1, WIFPGSX, containing the amino acid sequence of DYYIN (SEQ ID NO: 1) 1 SX 2 YX 3 X 4 X 5 X 6 FX 7 X 8and a heavy chain CDR2 comprising the amino acid sequence of (SEQ ID NO:2) 9 A heavy chain CDR3 comprising the amino acid sequence of Y (SEQ ID NO:3); and / or A light chain CDR1 having the amino acid sequence of KSSQSLLYSNGKTYLN (SEQ ID NO: 4), a light chain CDR2 having the amino acid sequence of LVSKLDS (SEQ ID NO: 5), and VQX 10 Light chain CDR3 comprising the amino acid sequence of THFPFT (SEQ ID NO: 6) Includes; where X 1 is E, D or G, and X 2 is A or P, and X 3 is H or Y, and X 4 is S or N, and X 5 is E or Q, and X 6 is K or N, and X 7 is K or Q, and X 8 is A or G, and X 9 is A or P, and X 10 is V or G, An isolated antibody or antigen-binding fragment thereof is provided.

[0010] In certain embodiments, the antibody or antigen-binding fragment thereof provided herein: a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:1, and / or a heavy chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10; and / or a heavy chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 11 and SEQ ID NO: 12, and / or a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:4, and / or a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:5, and / or A light chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 13 and SEQ ID NO: 14. Includes.

[0011] In one aspect, the present disclosure provides an isolated antibody or antigen-binding fragment thereof that specifically binds to MASP-2, comprising: a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:1, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:7, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:11; or a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:1, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:9, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:12; or a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:1, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:10, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:11; or heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:1, heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:8, and heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:11 The present invention provides an isolated antibody or antigen-binding fragment thereof comprising:

[0012] In certain embodiments, the antibody or antigen-binding fragment thereof provided herein comprises: a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:4, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:5, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:13; or A light chain CDR1 comprising the amino acid sequence of SEQ ID NO:4, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:5, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:14. Further includes:

[0013] In certain embodiments, the antibody or antigen-binding fragment thereof provided herein comprises: a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:1, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:7, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:11, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:4, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:5, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:13; or a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:1, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:9, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:12, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:4, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:5, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:13; or a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:1, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:10, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:11, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:4, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:5, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:14; or heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:1, heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:8, and heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:11, light chain CDR1 comprising the amino acid sequence of SEQ ID NO:4, light chain CDR2 comprising the amino acid sequence of SEQ ID NO:5, and light chain CDR3 comprising the amino acid sequence of SEQ ID NO:13 Includes.

[0014] In certain embodiments, the antibody or antigen-binding fragment thereof provided herein comprises: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 15, or a sequence having at least 80% sequence identity thereto; a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 17, or a sequence having at least 80% sequence identity thereto; a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 18, or a sequence having at least 80% sequence identity thereto; a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 20, or a sequence having at least 80% sequence identity thereto; a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 22, or a sequence having at least 80% sequence identity thereto; a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 24, or a sequence having at least 80% sequence identity thereto; or A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 26, or a sequence having at least 80% sequence identity thereto. Includes.

[0015] In certain embodiments, the antibody or antigen-binding fragment thereof provided herein comprises: a light chain variable region comprising the amino acid sequence of SEQ ID NO: 16, or a sequence having at least 80% sequence identity thereto; a light chain variable region comprising the amino acid sequence of SEQ ID NO: 19, or a sequence having at least 80% sequence identity thereto; a light chain variable region comprising the amino acid sequence of SEQ ID NO: 28, or a sequence having at least 80% sequence identity thereto; or A light chain variable region comprising the amino acid sequence of SEQ ID NO: 30, or a sequence having at least 80% sequence identity thereto. Includes.

[0016]

[0015] In certain embodiments, the antibody or antigen-binding fragment thereof provided herein comprises a heavy chain variable region comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, or SEQ ID NO:26, and / or a light chain variable region comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:28, or SEQ ID NO:30.

[0017] In certain embodiments, the antibody or antigen-binding fragment thereof provided herein comprises: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 15, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 16; a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:17, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:16; a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:18, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:19; a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:20, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:28; a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:20, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:30; a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:22, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:28; a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:22, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:30; a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:24, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:28; a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:24, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:30; a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:26, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:28; or A heavy chain variable region comprising the amino acid sequence of SEQ ID NO:26, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:30. Includes.

[0018]

[0017] In certain embodiments, the antibody or antigen-binding fragment thereof provided herein further comprises one or more amino acid residue mutations while still retaining binding specificity to human MASP-2. In certain embodiments, at least one of the mutations is a conservative substitution, or all of the mutations are conservative substitutions. In certain embodiments, at least one of the mutations is in one or more CDR sequences and / or one or more non-CDR sequences of the heavy chain variable region or the light chain variable region.

[0019]

[0018] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein further comprise an immunoglobulin constant region, optionally comprising a heavy chain constant region and / or a light chain constant region of an IgG. In certain embodiments, the constant region comprises a mouse constant region, a rabbit constant region, or a human constant region, optionally comprising a human IgG1, IgG2, IgG3, or IgG4 constant region. In certain embodiments, the heavy chain constant region comprises one or more amino acid substitutions at amino acid residues 252, 254, or 256 compared to a wild-type human IgG constant region, optionally wherein the amino acid substitution at amino acid residue 252 is a substitution with tyrosine, the amino acid substitution at amino acid residue 254 is a substitution with threonine, and the amino acid substitution at amino acid residue 256 is a substitution with glutamic acid. In certain embodiments, the heavy chain constant region comprises a sequence with at least 80% sequence identity thereto, except that amino acid residue 252 is substituted with tyrosine, amino acid residue 254 is substituted with threonine, and amino acid residue 256 is substituted with glutamic acid.

[0020]

[0019] In certain embodiments, the antibody or antigen-binding fragment thereof provided herein is a monoclonal antibody, a bispecific antibody, a multispecific antibody, a recombinant antibody, a chimeric antibody, a humanized antibody, a labeled antibody, a bivalent antibody, an anti-idiotypic antibody, a fusion protein, a dimerized or polymerized antibody, or a modified antibody (e.g., a glycosylated antibody).

[0021] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein are diabodies, Fab, Fab', F(ab') 2 , Fd, Fv fragment, disulfide stabilized Fv fragment (dsFv), (dsFv) 2 , bispecific dsFv (dsFv-dsFv'), disulfide stabilized diabodies (ds diabodies), single chain antibody molecules (scFv), scFv dimers (bivalent diabodies), multispecific antibodies, camelized single domain antibodies, nanobodies, domain antibodies, or bivalent domain antibodies.

[0022]

[0021] In certain embodiments, the antibody or antigen-binding fragment provided herein specifically binds to MASP-2 and has no detectable cross-reactivity with C1s, C1r, MASP1 or MASP3.

[0023]

[0022] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein are linked to one or more conjugate moieties.

[0024] In certain embodiments, the conjugate moiety comprises a clearance modifier, a chemotherapeutic agent, a toxin, a radioisotope, a lanthanide, a luminescent label, a fluorescent label, an enzyme substrate label, or a therapeutic agent.

[0025]

[0024] In one aspect, the present disclosure further provides a monoclonal antibody or antigen-binding fragment thereof that competes with the antibody or antigen-binding fragment thereof provided herein for binding to MASP-2.

[0026]

[0025] In one aspect, the present disclosure further provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof provided herein and a pharma- ceutically acceptable carrier.

[0027]

[0026] In one aspect, the present disclosure further provides an isolated polynucleotide encoding an antibody or antigen-binding fragment thereof provided herein.

[0028]

[0027] In one aspect, the present disclosure further provides a vector comprising an isolated polynucleotide provided herein.

[0029]

[0028] In one aspect, the present disclosure further provides a host cell comprising the vector provided herein.

[0030]

[0029] In one aspect, the present disclosure further provides a method for expressing an antibody or antigen-binding fragment thereof provided herein, the method comprising culturing a host cell provided herein under conditions in which a polynucleotide provided herein is expressed.

[0031]

[0030] In one aspect, the present disclosure further provides a method for inhibiting MASP-2-dependent complement activation in a subject in need of inhibition of MASP-2-dependent complement activation, the method comprising the step of administering a therapeutically effective amount of an antibody or antigen-binding fragment thereof provided herein or a pharmaceutical composition provided herein to the subject, thereby inhibiting MASP-2-dependent complement activation in the subject.

[0032]

[0031] In one aspect, the present disclosure further provides a method for treating a disease or condition in a subject that would benefit from inhibition of MASP-2-dependent complement activation, the method comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof provided herein or a pharmaceutical composition provided herein.

[0033]

[0032] In one aspect, the present disclosure further provides a method for reducing serum C4 levels in a subject, the method comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof provided herein, or a pharmaceutical composition provided herein, thereby reducing serum C4 levels in the subject.

[0034]

[0033] In one aspect, the present disclosure further provides a method for treating a disease or condition in a subject that would benefit from a reduction in serum C4 levels, or for treating or preventing a condition or disease associated with abnormal serum C4 levels, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof provided herein, or a pharmaceutical composition provided herein.

[0035]

[0034] In certain embodiments, the disease or condition is an autoimmune disease, a vascular condition, ischemia-reperfusion injury, atherosclerosis, inflammation, a pulmonary condition, an extracorporeal reperfusion procedure, a skeletal muscle condition, a renal condition, a skin condition, an organ or tissue transplant procedure, a nervous system disorder or injury, a blood disorder, a genitourinary condition, complications associated with non-obese diabetes or type 1 or type 2 diabetes, cancer, an endocrine disorder, or an ophthalmological condition.

[0036] In one particular embodiment, Autoimmune diseases include thrombotic microangiopathy (TMA), atypical hemolytic uraemic syndrome (aHUS), hematopoietic transplant-associated thrombotic microangiopathy (TA-TMA), lupus nephritis, systemic lupus erythematosus (SLE), and IgA nephropathy. Vascular conditions include cardiovascular conditions, cerebrovascular conditions, peripheral (e.g., musculoskeletal) vascular conditions, renal vascular conditions, mesenteric / intestinal vascular conditions, revascularization of grafts and / or regrafts, vasculitis, Henoch-Schönlein purpura nephritis, systemic lupus erythematosus-associated vasculitis, rheumatoid arthritis-associated vasculitis, immune complex vasculitis, Takayasu's disease, dilated cardiomyopathy, diabetic vasculopathy, Kawasaki disease (arteritis), venous gas embolism (VGE), and restenosis after stent placement, rotational atherectomy, and percutaneous transluminal coronary angioplasty (PTCA); Ischemia-reperfusion injury includes ischemia-reperfusion injury associated with aortic aneurysm repair, cardiopulmonary bypass, organ transplants and / or vascular reanastomosis along with limb / digit reimplantation, stroke, myocardial infarction, and shock and / or hemodynamic resuscitation following surgical procedures; Inflammation includes inflammatory gastrointestinal disorders including pancreatitis, Crohn's disease, ulcerative colitis, irritable bowel syndrome and diverticulitis; Pulmonary conditions include acute respiratory distress syndrome, transfusion-associated acute lung injury, ischemia / reperfusion acute lung injury, chronic obstructive pulmonary disease, asthma, Wegener's granulomatosis, anti-glomerular basement membrane disease (Goodpasture's disease), meconium aspiration syndrome, bronchiolitis obliterans syndrome, idiopathic pulmonary fibrosis, acute lung injury secondary to burn injury, noncardiogenic pulmonary edema, transfusion-associated respiratory depression, emphysema, cystic fibrosis, SARS-CoV, MERS-CoV and SARS-CoV-2 (Covid-19) related conditions, Extracorporeal reperfusion procedures include hemodialysis, plasmapheresis, leukopheresis, extracorporeal membrane oxygenation (ECMO), heparin-induced extracorporeal membrane oxygenation (HELP), and cardiopulmonary bypass (CPB). Skeletal muscle conditions include osteoarthritis, rheumatoid arthritis, juvenile rheumatoid arthritis, gout, neuropathic arthropathy, psoriatic arthritis, spondyloarthropathy, crystal arthropathy, and systemic lupus erythematosus (SLE); Renal conditions include mesangial proliferative glomerulonephritis, membranous glomerulonephritis, membranoproliferative glomerulonephritis (mesangial capillary glomerulonephritis), acute post-infectious glomerulonephritis (post-streptococcal glomerulonephritis), cryoglobulinemic glomerulonephritis, lupus nephritis, Henoch-Schönlein purpura nephritis, and IgA nephropathy. Skin conditions include psoriasis, autoimmune blistering dermatoses, eosinophilic spongiosis, bullous pemphigoid, epidermolysis bullosa acquisita (EBA), herpes gestationis, thermal burns and chemical burns; Organ or tissue transplantation procedures include organ allografts, organ xenografts, and organ and tissue grafts; Nervous system disorders or injuries include multiple sclerosis, myasthenia gravis, Huntington's disease, amyotrophic lateral sclerosis, Guillain-Barre syndrome, reperfusion after stroke, degenerative discs, brain trauma, Parkinson's disease, Alzheimer's disease, Miller-Fisher syndrome, brain trauma and / or cerebral hemorrhage, demyelination and meningitis; Blood disorders include sepsis, severe sepsis, septic shock, acute respiratory distress syndrome resulting from sepsis, systemic inflammatory response syndrome, hemorrhagic shock, hemolytic anemia, autoimmune thrombotic thrombocytopenic purpura, and hemolytic uremic syndrome; Genitourinary conditions include painful bladder disease, sensory bladder disease, non-bacterial chronic cystitis, interstitial cystitis, infertility, placental insufficiency and miscarriage and pre-eclampsia; Endocrine disorders include Hashimoto's thyroiditis, stress, anxiety, and hormonal disorders involving the controlled release of prolactin, growth factors or other insulin-like growth factors and adrenocorticotropin from the pituitary gland. Ophthalmologic conditions include age-related macular degeneration.

[0037]

[0036] In certain embodiments, the methods provided herein further comprise administration of a second therapeutic agent.

[0038]

[0037] In certain embodiments, the subject is a human. In certain embodiments, administration is via oral, nasal, intravenous, subcutaneous, sublingual, or intramuscular administration.

[0039]

[0038] In one aspect, the present disclosure further provides the use of an antibody or antigen-binding fragment thereof provided herein in the manufacture of a medicament for treating a disease or condition associated with MASP-2-dependent complement activation in a subject.

[0040]

[0039] In one aspect, the present disclosure further provides a kit comprising an antibody or antigen-binding fragment thereof provided herein. [Brief description of the drawings]

[0041] [Figure 1]

[0040] Figure 1 shows that MASP-2 antibodies block activation of complement C3. [Diagram 2]

[0041] Figure 2 shows that MASP-2 antibodies block activation of complement C4. [Diagram 3]

[0042] FIG. 3 shows that MASP-2 antibodies block the formation of MAC. [Figure 4]

[0043] Figure 4 shows that chimeric and humanized MASP-2 antibodies block complement C3 activation. [Diagram 5]

[0044] Figure 5 shows that MASP-2 antibody 129C10-hu and OMS721-analogue block complement C4 activation in 2% human serum. [Figure 6]

[0045] Figure 6 shows that MASP-2 antibody 129C10-hu and OMS721-analogue block MAC formation in 2% human serum. [Figure 7]

[0046] Figure 7 shows that MASP-2 antibodies block C3 activation in 1% human serum. [Figure 8]

[0047] Figure 8 shows that MASP-2 antibodies block C3 activation in 10% human serum. [Figure 9]

[0048] Figure 9 shows that MASP-2 antibodies block C3 activation in 50% human serum. [Figure 10]

[0049] Figure 10 shows that MASP-2 antibodies block C4 activation in 10% human serum. [Figure 11]

[0050] Figure 11 shows the affinity kinetics of MASP-2 antibody 129C10-hu to hMASP-2. [Figure 12-1]

[0051] Figures 12A-12E show ELISA binding of MASP-2 antibody 129C10-Hu to human C1s (12A), C1r (12B), MASP1 (12C), MASP3 (12D) and MASP2 (12E). [Figure 12-2]

[0051] Figures 12A to 12E show ELISA binding of MASP-2 antibody 129C10-Hu to human C1s (12A), C1r (12B), MASP1 (12C), MASP3 (12D) and MASP2 (12E). [Figure 12-3]

[0051] Figures 12A to 12E show ELISA binding of MASP-2 antibody 129C10-Hu to human C1s (12A), C1r (12B), MASP1 (12C), MASP3 (12D) and MASP2 (12E). [Figure 13]

[0052] Figures 13A-13B show ELISA binding of MASP-2 antibody 129C10-hu (13A) and OMS721-analogue (13B) to human, cynomolgus monkey, rat and mouse MASP2. [Figure 14]

[0053] Figure 14 shows the cross-reactivity of MASP-2 antibody 129C10-hu with cynomolgus monkey MASP-2. [Figure 15]

[0054] Figure 15 shows the selectivity of MASP-2 antibody 129C10-hu in neutralizing the three complement activation pathways. [Figure 16]

[0055] Figures 16A-16B show the binding kinetic curves of MASP-2 antibodies 129C10-hu-WT (16A) and 129C10-hu-YTE (16B) to human FcRn. [Figure 17]

[0056] Figure 17 shows the pharmacokinetic (PK) results of MASP-2 antibodies 129C10-hu-WT and 129C10-hu-YTE in cynomolgus monkey serum. [Figure 18-1]

[0057] Figures 18A-18C show the PK and pharmacodynamics (PD) results of MASP-2 antibodies 129C10-hu-WT and 129C10-hu-YTE in cynomolgus monkey serum. [Figure 18-2]

[0057] Figures 18A to 18C show the PK and pharmacodynamics (PD) results of MASP-2 antibodies 129C10-hu-WT and 129C10-hu-YTE in cynomolgus monkey serum. [Figure 19]

[0058] FIG. 19 shows the reducing effect of 129C10-hu on serum C4c in monkeys after the first dose. [Figure 20]

[0059] FIG. 20 shows the reducing effect of 129C10-hu on serum C4c in monkeys after the fourth dose. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0042]

[0060] The following description of the present disclosure is intended to merely illustrate various embodiments of the present disclosure. As such, the specific modifications discussed should not be interpreted as limitations on the scope of the present disclosure. It is clear to those skilled in the art that various equivalents, variations and modifications can be made without departing from the scope of the present disclosure, and it is understood that such equivalent embodiments should be included in this specification. All references cited in this specification, including publications, patents and patent applications, are incorporated herein by reference in their entirety.

[0043]

[0061] definition

[0062] The term "antibody" as used herein includes any immunoglobulin, monoclonal, polyclonal, multivalent, bivalent, monovalent, multispecific, or bispecific antibody that binds to a specific antigen. A naturally occurring intact antibody contains two heavy (H) chains and two light (L) chains. Mammalian heavy chains are classified as alpha, delta, epsilon, gamma, and mu, and each heavy chain contains a variable region (V H ) and the first, second, and third constant regions (C H1 , C H2 , C H3 Mammalian light chains are classified as lambda or kappa, with each light chain consisting of a variable region (respectively, V for lambda light chains and V for kappa light chains). L or V for kappa light chains K ) and constant regions (C for λ light chains, respectively). L or C for kappa light chains K). Antibodies have the shape of a "Y", the stem of the Y is composed of the second and third constant regions of two heavy chains bound to each other via disulfide bonds. Each arm of the Y contains the variable and first constant regions of a single heavy chain bound to the variable and constant regions of a single light chain. The variable regions of the light and heavy chains are responsible for antigen binding. The variable regions of both chains generally contain three highly variable loops called complementarity determining regions (CDRs) (light chain CDRs including LCDR1, LCDR2, and LCDR3, heavy chain CDRs including HCDR1, HCDR2, HCDR3). The boundaries of the CDRs of the antibodies and antigen-binding fragments disclosed herein may be defined or identified by the Kabat, IMGT, Chothia, or Al-Lazikani conventions (Al-Lazikani, B., Chothia, C., Lesk, AM, J. Mol. Biol., 273(4), 927 (1997); Chothia, C. et al., J. Mol. Biol. Dec. 5; 186(3):651-63 (1985); Chothia, C. and Lesk, AM, J. Mol. Biol., 196, 901 (1987); Chothia, C. et al., Nature. Dec. 21-28; 342(6252):877-83 (1989); Kabat EA et al., National Institutes of Health, Bethesda, Md. (1991)). The three CDRs are inserted between adjacent stretches known as framework regions (FRs), which are more highly conserved than the CDRs and form a scaffold supporting the hypervariable loops. The constant regions of the heavy and light chains are not involved in antigen binding but exhibit various effector functions. Antibodies are assigned to classes based on the amino acid sequence of the constant region of their heavy chains. The five main classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of α, delta, epsilon, gamma, and μ heavy chains, respectively. Some of the main antibody classes are divided into subclasses, such as IgG1 (gamma 1 heavy chain), IgG2 (gamma 2 heavy chain), IgG3 (gamma 3 heavy chain), IgG4 (gamma 4 heavy chain), IgA1 (α1 heavy chain), or IgA2 (α2 heavy chain).

[0044]

[0063] As used herein, the term "bivalent" refers to an antibody or antigen-binding fragment having two antigen-binding sites; the term "monovalent" refers to an antibody or antigen-binding fragment having only one single antigen-binding site; and the term "multivalent" refers to an antibody or antigen-binding fragment having multiple antigen-binding sites. In some embodiments, an antibody or antigen-binding fragment thereof is bivalent.

[0045]

[0064] As used herein, a "bispecific" antibody refers to an artificial antibody that has fragments derived from two different monoclonal antibodies and can bind to two different epitopes, which may be present on the same antigen or on two different antigens.

[0046]

[0065] As used herein, the term "antigen-binding fragment" refers to an antibody fragment formed from a portion of an antibody that contains one, two, or three CDRs, or any other antibody fragment that binds to an antigen but does not contain an intact native antibody structure. Examples of antigen-binding fragments include, but are not limited to, diabodies, Fab, Fab', F(ab') 2 , Fv fragment, disulfide stabilized Fv fragment (dsFv), (dsFv) 2 Antigen-binding fragments include, but are not limited to, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabodies (ds diabodies), single-chain antibody molecules (scFv), scFv dimers (bivalent diabodies), diabodies, multispecific antibodies, camelized single-chain domain antibodies, nanobodies, domain antibodies, and bivalent domain antibodies. Antigen-binding fragments can bind to the same antigen that the parent antibody binds.

[0047]

[0066] "Fab" with respect to an antibody refers to the portion of an antibody that is composed of a single light chain (both the variable and constant regions) linked by disulfide bonds to the variable region and first constant region of a single heavy chain.

[0048]

[0067] "Fab'" refers to a Fab fragment that includes part of the hinge region.

[0049]

[0068] "F(ab') 2 " refers to a dimer of Fab'. "Fv" with respect to an antibody refers to the minimum fragment of an antibody that retains a complete antigen-binding site. The Fv fragment is composed of the variable region of a single light chain bound to the variable region of a single heavy chain.

[0050]

[0069] "dsFv" refers to a disulfide-stabilized Fv fragment, in which the linkage between the variable region of a single light chain and the variable region of a single heavy chain is a disulfide bond. In some embodiments, "(dsFv) 2 " or "(dsFv-dsFv')" refers to three peptide chains: two Vs linked by a peptide linker (e.g., a long flexible linker) and each linked via a disulfide bridge. L Two V's joined together H In some embodiments, a dsFv-dsFv' is bispecific, where each disulfide-paired heavy and light chain has a different antigen specificity.

[0051]

[0070] A "single-chain Fv antibody" or "scFv" refers to an engineered antibody composed of a light chain variable region and a heavy chain variable region connected to each other either directly or via a peptide linker sequence (Huston JS et al. Proc Natl Acad Sci USA, 85:5879 (1988)).

[0052]

[0071] "Fc" with respect to an antibody refers to that portion of an antibody that is composed of the second and third constant regions of a first heavy chain linked via disulfide bonds to the second and third constant regions of a second heavy chain. The Fc portion of an antibody is responsible for various effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), but does not function in antigen binding.

[0053]

[0072] "Single chain Fv-Fc antibody" or "scFv-Fc" refers to an engineered antibody composed of an scFv attached to the Fc region of an antibody.

[0054]

[0073] "Camelized single domain antibody", "heavy chain antibody" or "HCAb" refers to a camelized single domain antibody consisting of two V H It refers to antibodies that contain heavy chains and do not contain light chains (Riechmann L. and Muyldermans S., J Immunol Methods. Dec. 10; 231(1-2):25-38 (1999); Muyldermans S., J Biotechnol. Jun. 74(4):277-302 (2001); WO94 / 04678; WO94 / 25591; U.S. Patent No. 6,005,079). Heavy chain antibodies were originally derived from the Camelidae (camels, dromedaries and llamas). Although lacking light chains, camelized antibodies have a robust antigen-binding repertoire (Hamers-Casterman C. et al., Nature. June 3;363(6428):446-8 (1993); Nguyen VK. et al. "Heavy-chain antibodies in Camelidae; a case of evolutionary innovation", Immunogenetics. April;54(1):39-47 (2002); Nguyen VK. et al. Immunology. May;109(1):93-101 (2003)). The variable domains of heavy-chain antibodies (VHH domains) represent the smallest known antigen-binding units generated by the adaptive immune response (Koch-Nolte F. et al., FASEB J. November;21(13):3490-8. Epub 2007 June 15 (2007)).

[0055]

[0074] "Nanobody" refers to an antibody fragment that is composed of a VHH domain derived from a heavy chain antibody and two constant domains, CH2 and CH3.

[0056]

[0075] A "diabody" or "dAb" includes small antibody fragments with two antigen-binding sites, where the fragments are arranged in the same polypeptide chain. L V connected to the domain H Domain (V H -V L or V L -V H) (see, e.g., Holliger P. et al., Proc Natl Acad Sci USA. July 15; 90(14):644-8 (1993); EP404097; WO93 / 11161). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with complementary domains on another chain, thereby creating two antigen binding sites. The two antigen binding sites can target the same or different antigens (or epitopes). In certain embodiments, a "bispecific ds diabody" is a diabody that targets two different antigens (or epitopes). In certain embodiments, a "scFv dimer" is a bivalent diabody or bivalent ScFv (BsFv), which is a divalent diabody having a V H A part of V L and to form two binding sites that can target the same antigen (or epitope) or different antigens (or epitopes). H -V L The V dimerized with the moiety H -V L (linked by a peptide linker). In another embodiment, an "scFv dimer" is a bispecific diabody, which comprises H1 and V L1 V H2 and V L2 V is arranged so that they cooperate and each cooperated pair has a different antigen specificity. L1 -V H2 (linked by a peptide linker) H1 -V L2 (also linked by a peptide linker).

[0057]

[0076] A "domain antibody" refers to an antibody fragment that contains only the variable region of a heavy chain or only the variable region of a light chain. In certain embodiments, two or more V H The domains are covalently joined with a peptide linker to create a bivalent or multivalent domain antibody.H The domains can target the same or different antigens.

[0058]

[0077] The term "chimeric" as used herein refers to an antibody or antigen-binding fragment having a portion of the heavy and / or light chain derived from one species and the remaining portion of the heavy and / or light chain derived from a different species. In an illustrative example, a chimeric antibody can include a constant region derived from a human and a variable region derived from a non-human animal, such as a mouse or a rat. In some embodiments, the non-human animal is a mammal, such as a mouse, a rat, a rabbit, a goat, a sheep, a guinea pig, or a hamster.

[0059]

[0078] As used herein, the term "humanized" means that the antibody or antigen-binding fragment contains CDRs derived from a non-human animal, FR regions derived from a human, and, where applicable, constant regions derived from a human.

[0060]

[0079] As used herein, the term "MASP-2" refers to mannan-binding lectin-associated protein serine protease 2, which is a crucial member of the MBL pathway of the complement system. The amino acid and nucleic acid sequences of human, mouse and cynomolgus MASP-2 can be found in public databases such as GenBank, UniProt and Swiss-Prot. As used herein, the term MASP-2 includes proteins that include mutations of full-length wild-type MASP-2, such as point mutations, fragments, insertions, deletions and splice variants. In certain embodiments, the human MASP-2 protein includes the amino acid sequence of SEQ ID NO: 39. In certain embodiments, the mouse MASP-2 protein includes the amino acid sequence of SEQ ID NO: 40. In certain embodiments, the cynomolgus MASP-2 protein includes the amino acid sequence of SEQ ID NO: 43. In one particular embodiment, a chimeric MASP-2 protein was synthesized comprising the mouse MASP-2 CUB1-EGF-CUB2 domain (residues 20-297) and the human MASP-2 CCP1-CCP2-SP domain (residues 298-686), which has the amino acid sequence of SEQ ID NO: 42.

[0061]

[0080] As used herein, the terms "specific binding" or "specifically bind" refer to a non-random binding reaction between two molecules, such as between an antibody and an antigen. In certain embodiments, the antibodies or antigen-binding fragments provided herein have a specific binding affinity of ≦10 -6 M (e.g., ≦5×10 -8 M, ≤2×10 -8 M, ≦10 -8 M, ≤5×10 -9 M, ≤2×10 -9 M, ≦10 -9 M, ≤5×10 -10 M, ≤2×10 -10 M, ≦10 -10 M, ≤5×10 -11 M, ≤2×10 -11 M, ≦10 -11 M, ≤5×10 -12 M, ≤4×10 -12 M, ≤3×10 -12M, ≤2×10 -12 M, or ≤ 10 -12 Binding affinity (K D ) specifically binds to human MASP-2. D is the ratio of the dissociation rate to the association rate (k off / k on ), which may be determined by using any conventional method known in the art, including, but not limited to, surface plasmon resonance, microscale thermophoresis, HPLC-MS, biolayer interferometry, and flow cytometry (e.g., FACS). In certain embodiments, K D The value may be suitably determined by using the ForteBio method.

[0062]

[0081] As used herein, the ability to "block binding" or "compete for the same epitope" refers to the ability of an antibody or antigen-binding fragment to inhibit the binding interaction between two molecules (e.g., a MASP-2 protein and an anti-MASP-2 antibody) to any detectable degree. In certain embodiments, an antibody or antigen-binding fragment that blocks binding between two molecules inhibits the binding interaction between the two molecules by at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%. In certain embodiments, this inhibition may be more than 60%, more than 70%, more than 75%, more than 80%, more than 85%, or more than 90%.

[0063]

[0082] The term "epitope" as used herein refers to a specific group of atoms or amino acids on an antigen to which an antibody binds. Two antibodies can bind to the same epitope or closely related epitopes in an antigen if they exhibit competitive binding to the antigen. For example, an antibody or antigen-binding fragment can be considered to bind to the same / closely related epitope as a reference antibody if it blocks the binding of the reference antibody to the antigen (e.g., human / monkey MASP-2) by at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%.

[0064]

[0083] One of ordinary skill in the art will understand that, without undue experimentation, it is possible to determine whether a human monoclonal antibody binds to the same epitope as an antibody of the present disclosure by determining whether the monoclonal antibody prevents the antibody of the present disclosure from binding to a MASP-2 antigen polypeptide. If the test antibody competes with the antibody of the present disclosure, as indicated by reduced binding by the antibody of the present disclosure to a MASP-2 antigen polypeptide, then the two antibodies bind to the same or closely related epitope. Alternatively, if the binding of the test antibody to a MASP-2 antigen polypeptide is inhibited by the antibody of the present disclosure, then the two antibodies bind to the same or closely related epitope.

[0065]

[0084] "Conservative substitution" in the context of amino acid sequence refers to replacing an amino acid residue with a different amino acid residue that has a side chain with similar physicochemical properties.For example, conservative substitution can be made between amino acid residues with hydrophobic side chains (e.g., Met, Ala, Val, Leu and Ile), between residues with neutral hydrophilic side chains (e.g., Cys, Ser, Thr, Asn and Gln), between residues with acidic side chains (e.g., Asp, Glu), between amino acids with basic side chains (e.g., His, Lys and Arg), or between residues with aromatic side chains (e.g., Trp, Tyr and Phe).As is known in the art, conservative substitution usually does not cause significant changes in the conformational structure of protein, and therefore may retain the biological activity of protein.

[0066]

[0085] As used herein, the terms "homolog" and "homologous" are used interchangeably and refer to a nucleic acid sequence (or its complementary strand) or amino acid sequence that has at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to another sequence, optionally when aligned.

[0067]

[0086] "Percent (%) sequence identity" with respect to an amino acid sequence (or nucleic acid sequence) is defined as the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical with the amino acid (or nucleic acid) residues in a reference sequence after aligning the sequences and, if necessary, introducing gaps to obtain the maximum number of identical amino acids (or nucleic acids). Conservative substitutions of amino acid residues may or may not be considered identical residues. Alignment for purposes of determining percent amino acid (or nucleic acid) sequence identity can be performed using publicly available tools such as, for example, BLASTN, BLASTp (available at the US National Center for Biotechnology Information (NCBI) website, see also Altschul SF et al., J. Mol. Biol., 215:403-410 (1990); Stephen F. et al., Nucleic Acids Res., 25:3389-3402 (1997)), ClustalW2 (available at the European Bioinformatics Institute website, see also Higgins DG et al., Methods in Enzymology, 266:383-402 (1996); Larkin MA et al., Bioinformatics (Oxford, England), 23(21):2947-8 (2007)), and ALIGN or Megalign (DNASTAR) software. One skilled in the art may use the default parameters provided by the tool or may customize the parameters as needed for the alignment, such as by selecting an appropriate algorithm.

[0068]

[0087] As used herein, "treating" or "treatment" of a condition includes preventing or alleviating the condition, slowing the rate of onset or occurrence of the condition, reducing the risk of developing the condition, preventing or delaying the onset of symptoms associated with the condition, reducing or terminating symptoms associated with the condition, causing complete or partial regression of the condition, curing the condition, or some combination thereof.

[0069]

[0088] An "isolated" material is one that has been altered by the hand of man from its natural state. An "isolated" composition or material, when present in nature, has been changed or removed from its original environment, or both. For example, a polynucleotide or polypeptide that naturally occurs in a living animal is not "isolated", but is "isolated" when the same polynucleotide or polypeptide is sufficiently separated from the coexisting materials of its natural state so that it exists in a substantially pure state. Isolated "nucleic acid" or "polynucleotide" are used interchangeably and refer to the sequence of an isolated nucleic acid molecule. In certain embodiments, an "isolated antibody or antigen-binding fragment thereof" refers to an antibody or antigen-binding fragment having a purity of at least 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% as determined by electrophoretic (e.g., SDS-PAGE, isoelectric focusing, capillary electrophoresis) or chromatographic (e.g., ion exchange chromatography or reverse phase HPLC) methods.

[0070]

[0089] The term "vector" as used herein refers to a vehicle into which a polynucleotide encoding a protein can be operably inserted to cause expression of the protein. A vector can be used to transform, transduce, or transfect a host cell to cause expression of the genetic element carried by the vector in the host cell. Examples of vectors include plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), bacteriophages such as lambda phage or M13 phage, and animal viruses. Categories of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex viruses), pox viruses, baculoviruses, papilloma viruses, and papova viruses (e.g., SV40). A vector can contain various elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selectable elements, and reporter genes. In addition, the vector can contain an origin of replication. The vector can also contain substances that aid in the entry of the vector into a cell, including but not limited to viral particles, liposomes, or protein coatings. The vector can be an expression vector or a cloning vector. The present disclosure provides a vector (e.g., an expression vector) that contains a nucleic acid sequence provided herein that encodes an antibody or antigen-binding fragment thereof, at least one promoter (e.g., SV40, CMV, EF-1α) operably linked to the nucleic acid sequence, and at least one selectable marker.Examples of vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papilloma viruses, papova viruses (e.g., SV40), lambda phage, and M13 phage, plasmids such as pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-GSeu, pALTER, pBAD, pcDNA, pCal, pL, pET, p GEMEX, pGEX, pCI, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS10, pL Examples include exA, pACT2.2, pCMV-SCRIPT.RTM., pCDM8, pCDNA1.1 / amp, pcDNA3.1, pRc / RSV, PCR2.1, pEF-1, pFB, pSG5, pXT1, pCDEF3, pSVSPORT, pEF-Bos, and the like.

[0071]

[0090] As used herein, the phrase "host cell" refers to a cell into which an exogenous polynucleotide and / or vector has been introduced.

[0072]

[0091] The complement system has been implicated in the pathogenesis of many acute and chronic diseases or conditions, including myocardial infarction, stroke, acute respiratory distress syndrome (ARDS), reperfusion injury, septic shock, capillary leakage after thermal burn, inflammation after cardiopulmonary bypass, transplant rejection, rheumatoid arthritis, multiple sclerosis, myasthenia gravis, and Alzheimer's disease. In almost all of these diseases or conditions, complement is not the cause, but it is one of several factors involved in the pathogenesis. Complement activation may be the main pathological mechanism and is an effective key point for clinical control in many of these pathologies. MASP-2 is involved in the MBL pathway, which is one of the three main complement activation pathways, and may also be involved in the pathogenesis of many diseases or conditions. In some embodiments, the disease or condition associated with MASP-2-dependent complement activation is an autoimmune disease, a vascular condition, ischemia-reperfusion injury, atherosclerosis, inflammation, a pulmonary condition, an extracorporeal reperfusion procedure, a skeletal muscle condition, a renal condition, a skin condition, an organ or tissue transplant procedure, a nervous system disorder or injury, a blood disorder, a genitourinary condition, complications associated with non-obese diabetes or type 1 or type 2 diabetes, cancer, an endocrine disorder, or an ophthalmological condition.

[0073]

[0092] The term "mannan-binding lectin" ("MBL") is equivalent to mannan-binding protein ("MBP"). The term "membrane attack complex" ("MAC", also known as C5b-9) refers to a complex of five terminal complement components (C5-C9) that inserts into and destroys cell membranes.

[0074]

[0093] As used herein, "autoimmune disease" refers to a pathophysiological condition (autoimmunity) in which immune response is directed against and damages the body's own tissue. Examples of autoimmune disease include, but are not limited to, thrombotic microangiopathy (TMA), atypical hemolytic uremic syndrome (aHUS), hematopoietic transplant-associated thrombotic microangiopathy (TA-TMA), lupus nephritis, systemic lupus erythematosus (SLE) and IgA nephropathy.

[0075]

[0094] The term "pharmaceutical acceptable" indicates that the specified carrier, vehicle, diluent, excipient and / or salt is generally chemically and / or physically compatible with the other ingredients comprising the formulation and physiologically compatible with the recipient thereof.

[0076]

[0095] Anti-MASP-2 antibody

[0096] The present disclosure provides anti-MASP-2 antibodies and antigen-binding fragments thereof comprising one or more (e.g., one, two, three, four, five, or six) CDR sequences of anti-MASP-2 antibodies selected from the group consisting of 129C10 mouse / chimera, 160D10 mouse / chimera, 125D5 mouse / chimera, 129C10 HaLa, 129C10 HaLb, 129C10 HbLa, 129C10 HbLb, 129C10 HcLa, 129C10 HcLb, 129C10 HdLa, or 129C10 HdLb.

[0077]

[0097] As used herein, "129C10 mouse / chimeric" refers to a mouse monoclonal antibody having a heavy chain variable region of SEQ ID NO:15 and a light chain variable region of SEQ ID NO:16.

[0078]

[0098] As used herein, "160D10 murine / chimeric" refers to a murine monoclonal antibody having a heavy chain variable region of SEQ ID NO:17 and a light chain variable region of SEQ ID NO:16.

[0079]

[0099] As used herein, "125D5 mouse / chimeric" refers to a murine monoclonal antibody having a heavy chain variable region of SEQ ID NO:18 and a light chain variable region of SEQ ID NO:19.

[0080] [000100] As used herein, "129C10 HaLa" or "129C10-hu" refers to a 129C10 murine / chimeric-based humanized antibody comprising the heavy chain variable region of SEQ ID NO:20 and the light chain variable region of SEQ ID NO:28.

[0081] [000101] As used herein, "129C10 HaLb" refers to a 129C10 murine / chimeric-based humanized antibody comprising a heavy chain variable region of SEQ ID NO:20 and a light chain variable region of SEQ ID NO:30.

[0082] [000102] As used herein, "129C10 HbLa" refers to a 129C10 murine / chimeric-based humanized antibody comprising the heavy chain variable region of SEQ ID NO:22 and the light chain variable region of SEQ ID NO:28.

[0083] [000103] As used herein, "129C10 HbLb" refers to a 129C10 murine / chimeric-based humanized antibody comprising the heavy chain variable region of SEQ ID NO:22 and the light chain variable region of SEQ ID NO:30.

[0084] [000104] As used herein, "129C10 HcLa" refers to a 129C10 murine / chimeric-based humanized antibody comprising the heavy chain variable region of SEQ ID NO:24 and the light chain variable region of SEQ ID NO:28.

[0085] [000105] As used herein, "129C10 HcLb" refers to a 129C10 murine / chimeric-based humanized antibody comprising the heavy chain variable region of SEQ ID NO:24 and the light chain variable region of SEQ ID NO:30.

[0086] [000106] As used herein, "129C10 HdLa" refers to a 129C10 murine / chimeric-based humanized antibody comprising the heavy chain variable region of SEQ ID NO:26 and the light chain variable region of SEQ ID NO:28.

[0087] [000107] As used herein, "129C10 HdLb" refers to a 129C10 murine / chimeric-based humanized antibody comprising the heavy chain variable region of SEQ ID NO:26 and the light chain variable region of SEQ ID NO:30.

[0088] [000108] Antibodies 129C10 HaLa, 129C10 HaLb, 129C10 HbLa, and 129C10 HbLb share the same CDRs as the 129C10 murine / chimeric antibody.

[0089] [000109] Antibodies 129C10 HcLa, 129C10 HcLb, 129C10 HdLa, and 129C10 HdLb share the same CDRs.

[0090] [000110] Table 1 shows the CDR sequences of three murine anti-MASP-2 antibodies and eight humanized antibodies.

[0091] [Table 1]

[0092] [000111] Table 2 shows the sequences of the heavy and light chain variable regions of three mouse anti-MASP-2 antibodies (Table 2-1), the heavy and light chain variable regions of the humanized 129C10 antibody (Table 2-2), and the heavy and light chain variable regions of the 129C10-hu-YTE antibody (Table 2-3).

[0093] [Table 2-1]

[0094] [Table 2-2]

[0095] [Table 2-3]

[0096] [000112] CDRs are known to be responsible for antigen binding, but it has been found that not all six CDRs are essential or invariant. In other words, it is possible to replace, change or modify one, two or three CDRs in each of the anti-MASP-2 antibodies 129C10 mouse / chimera, 160D10 mouse / chimera, 125D5 mouse / chimera, 129C10 HaLa, 129C10 HaLb, 129C10 HbLa, 129C10 HbLb, 129C10 HcLa, 129C10 HcLb, 129C10 HdLa or 129C10 HdLb, and still substantially retain the specific binding affinity to MASP-2.

[0097] [000113] In certain embodiments, the anti-MASP-2 antibodies and antigen-binding fragments provided herein comprise the heavy chain CDR3 sequence of one of the anti-MASP-2 antibodies 129C10 mouse / chimera, 160D10 mouse / chimera, 125D5 mouse / chimera, 129C10 HaLa, 129C10 HaLb, 129C10 HbLa, 129C10 HbLb, 129C10 HcLa, 129C10 HcLb, 129C10 HdLa, or 129C10 HdLb. In certain embodiments, the anti-MASP-2 antibodies and antigen-binding fragments provided herein comprise the heavy chain CDR3 sequence selected from the group consisting of SEQ ID NOs: 3, 11, and 12. The heavy chain CDR3 region is located in the center of the antigen-binding site and is therefore believed to make the most contact with the antigen and provide the most free energy for the affinity of the antibody to the antigen. Heavy chain CDR3 is also considered to be by far the most diverse CDR for the antigen binding site in terms of length, amino acid composition and conformation due to multiple diversification mechanisms (Tonegawa S. Nature. 302: 575-81). The diversity of heavy chain CDR3 is sufficient to generate most antibody specificities (Xu JL, Davis MM. Immunity. 13: 37-45) as well as desirable antigen binding affinities (Schier R, et al. J Mol Biol. 263: 551-67).

[0098] [000114] In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein comprise suitable framework region (FR) sequences, so long as the antibodies and antigen-binding fragments thereof can specifically bind to MASP-2. The CDR sequences provided in Table 1 can be grafted to any suitable FR sequence of any suitable species, such as mouse, human, rat, rabbit, among others, using suitable methods known in the art, such as recombinant techniques.

[0099] [000115] In certain embodiments, the antibodies and antigen-binding fragments provided herein are humanized. Humanized antibodies or antigen-binding fragments are desirable in terms of their reduced immunogenicity in humans. Humanized antibodies are chimeric in their variable regions because non-human CDR sequences are grafted onto human or substantially human FR sequences. Humanization of antibodies or antigen-binding fragments can be essentially performed by substituting non-human (e.g., mouse) CDR genes for the corresponding human CDR genes in human immunoglobulin genes (see, for example, Jones et al. (1986) Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-327; Verhoeyen et al. (1988) Science 239:1534-1536).

[0100] [000116] Suitable human heavy and light chain variable domains can be selected to this end using methods known in the art. In an illustrative example, a "best-fit" approach can be used, in which a non-human (e.g., rodent) antibody variable domain sequence is screened or BLASTed against a database of known human variable domain sequences, and the human sequence closest to the non-human query sequence is identified and used as a human scaffold for grafting the non-human CDR sequences (see, e.g., Sims et al. (1993) J.Immunol. 151:2296; Chothia et al. (1987) J.Mot.Biol. 196:901). Alternatively, frameworks derived from the consensus sequence of all human antibodies can be used for grafting of non-human CDRs (see, e.g., Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89:4285; Presta et al. (1993) J. Immunol., 151:2623).

[0101] [000117] In certain embodiments, the humanized antibodies or antigen-binding fragments provided herein are composed of substantially all human sequences, except for the CDR sequences, which are non-human. In some embodiments, the variable region FR, and, if present, the constant region, are derived entirely or substantially from human immunoglobulin sequences. The human FR sequences and the human constant region sequences may be derived from different human immunoglobulin genes, e.g., the FR sequences may be derived from one human antibody and the constant region may be derived from another human antibody. In some embodiments, the humanized antibodies or antigen-binding fragments comprise human heavy / light chain FR1-4.

[0102] [000118] The exemplary humanized anti-MASP-2 antibodies, 129C10 HaLa, 129C10 HaLb, 129C10 HbLa, 129C10 HbLb, 129C10 HcLa, 129C10 HcLb, 129C10 HdLa, and 129C10 HdLb, all retained specific binding affinity for MASP-2 and are at least comparable to or even better than the parent murine antibody in these respects.

[0103] [000119] In some embodiments, the FR region derived from a human may contain the same amino acid sequence as the human immunoglobulin from which it is derived. In some embodiments, one or more amino acid residues of the human FR are replaced with the corresponding residues from the parent non-human antibody. This may be desirable in certain embodiments to make the humanized antibody or fragment thereof more similar to the non-human parent antibody structure, reducing or avoiding immunogenicity, and / or improving or retaining binding activity or binding affinity.

[0104] [000120] In certain embodiments, the humanized antibody or antigen-binding fragment provided herein comprises no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue substitutions in each human FR sequence, or no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue substitutions in all FRs of the heavy or light chain variable domain. In some embodiments, such changes in amino acid residues may be present only in the heavy chain FR region, only in the light chain FR region, or in both chains. In certain embodiments, one or more amino acid residues are mutated, e.g., backmutated to the corresponding residue found in the non-human parent antibody from which the CDR sequence is derived (e.g., in the mouse framework region). The positions suitable for mutation can be selected by the skilled artisan according to principles known in the art. For example, the position of the mutation may be selected at the following locations: 1) where the residue in the framework of the human Gremlin sequence is rarely present (e.g., in less than 20% or less than 10% of the human variable region sequences); 2) where the location is directly adjacent to one or more of the three CDRs in the primary sequence of the human germline chain, because it is more likely to interact with the residues in the CDRs; or 3) where the location is close to the CDRs in the three-dimensional model, and therefore may have a good possibility of interacting with the amino acids in the CDRs. The residue at the selected location may be backmutated to the corresponding residue in the parent antibody, or to a residue that is neither the corresponding residue in the human germline sequence nor the corresponding residue in the parent antibody, but is a common residue in human sequences, i.e., occurs more frequently at that position in known human sequences that belong to the same subgroup as the human germline sequence (see U.S. Patent No. 5,693,762).

[0105] [000121] In certain embodiments, the humanized heavy chains and humanized light chains of the antibodies and antigen-binding fragments thereof provided herein are substantially non-immunogenic in humans and retain substantially the same, or even higher, affinity for MASP-2 as the parent antibody.

[0106] [000122] In certain embodiments, the humanized antibodies and antigen-binding fragments thereof provided herein comprise one or more heavy chain FR sequences of human germline framework sequences VH / 1-2, with or without back mutations, and / or one or more light chain FR sequences of human germline framework sequences VK / 2-30. Optionally, back mutations may be introduced into the human germline framework sequences. In certain embodiments, the humanized antibody 129C10 can contain one or more mutations selected from the group consisting of: R71V, A93T, V67A, M69L in the heavy chain framework sequences VH / 1-2, all based on Kabat numbering, and / or A65G, K64Q, E61Q in the heavy chain CDR2, all based on Kabat numbering. The humanized antibody 129C10 can contain one or more back mutations selected from the group consisting of: F36L and T69A in the light chain framework sequences VK / 2-30, all based on Kabat numbering.

[0107] [000123] In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein comprise a heavy chain variable domain sequence selected from the group consisting of: SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, and SEQ ID NO: 26. In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein comprise a light chain variable domain sequence selected from the group consisting of: SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 28, and SEQ ID NO: 30.

[0108] [000124] In some embodiments, the anti-MASP-2 antibodies and antigen-binding fragments provided herein comprise all or part of the heavy chain variable domain and / or all or part of the light chain variable domain. In one embodiment, the anti-MASP-2 antibodies and antigen-binding fragments provided herein are single-domain antibodies consisting of all or part of the heavy chain variable domain provided herein. Further information regarding such single-domain antibodies is available in the art (see, for example, U.S. Patent No. 6,248,516).

[0109] [000125] In certain embodiments, the anti-MASP-2 antibodies and fragments thereof provided herein further comprise an immunoglobulin constant region. In some embodiments, the immunoglobulin constant region comprises a heavy chain and / or a light chain constant region. The heavy chain constant region comprises a CH1, hinge, and / or CH2-CH3 region. In certain embodiments, the heavy chain constant region comprises an Fc region. In certain embodiments, the light chain constant region comprises a Cκ or Cλ.

[0110] [000126] In some embodiments, the anti-MASP-2 antibodies and antigen-binding fragments thereof provided herein have an immunoglobulin (Ig) constant region, optionally a human Ig constant region, optionally a human IgG constant region. In some embodiments, the anti-MASP-2 antibodies and antigen-binding fragments thereof comprise a human IgG1, IgG2, IgG3, or IgG4 constant region.

[0111] [000127] In certain embodiments, the anti-MASP-2 antibodies and antigen-binding fragments thereof provided herein comprise a constant region of an IgG4 or IgG2 isotype with reduced or depleted effector function. The binding affinity of the antibodies and antigen-binding fragments provided herein is K D This can be expressed by the ratio of the dissociation rate to the association rate (k off / k on ) represents the antigen binding affinity (e.g., K D ) may be suitably determined using any suitable method known in the art, including, for example, biolayer interference.

[0112] [000128] In certain embodiments, the anti-MASP-2 antibodies and antigen-binding fragments thereof provided herein can specifically bind to human / cynomolgus MASP-2. For example, the DNA sequence of human / cynomolgus MASP-2 may be cloned into an expression vector and then transfected into and expressed in CHO cells, such that human / cynomolgus MASP-2 protein can be expressed on the surface of the transfected CHO cells.

[0113] [000129] In some embodiments, the anti-MASP-2 antibodies and antigen-binding fragments thereof provided herein have a potency of 3 x 10 as measured by biolayer interference. -8 M or less, 1×10 -8 M or less, 9×10 -9 M or less, 8×10 -9 M or less, 7×10 -9 M or less, 6×10 -9 M or less, 5×10 -9 M or less, 4×10 -9 M or less, 3×10 -9 M or less, 2×10 -9 M or less, 1×10 -9 M or less, 9×10 -10 M or less, 8×10 -10 M or less, 7×10 -10 M or less, 6×10 -10 M or less, 5×10 -10 M or less, 4×10 -10 M or less, 3×10 -10 M or less, 2×10 -10 M or less, 1×10 -10 M or less, 5×10 -11 M or less, 1×10 -11 M or less, 5×10 -12 M or less, 1×10 -12 Binding affinity (K D ) and is capable of specifically binding to human MASP-2 expressed on the cell surface.

[0114] [000130] Binding of antibodies to MASP-2 was measured using the "median effective concentration" (EC 50) value, which refers to the concentration of an antibody at which 50% of its maximal effect (e.g., binding or inhibition, etc.) is observed. 50 The value can be measured by methods known in the art, for example, sandwich assays such as ELISA, Western blots, flow cytometry assays, and other binding assays. In certain embodiments, the antibodies and fragments thereof provided herein specifically bind to human MASP-2 expressed on cells with an EC50 of 0.02 μg / mL or less, 0.015 μg / mL or less, 0.01 μg / mL or less, 0.005 μg / mL or less, or 0.001 μg / mL or less by ELISA assay.

[0115] [000131] Binding of an antibody to MASP-2 can also be expressed by the "half maximal inhibitory concentration" (IC50), which measures the potency of a substance in inhibiting a particular biological or biochemical function. IC50 is a quantitative measure that indicates how much of a particular inhibitor (e.g., a drug) is required to inhibit a given biological process or biological component by 50% in vitro. IC50 values ​​can be measured by methods known in the art, such as sandwich assays such as ELISA, Western blots, flow cytometry assays, and other binding assays.

[0116] [000132] In certain embodiments, the antibodies and fragments thereof provided herein can block complement C3 activation with an IC50 of 0.08 μg / mL or less (or 0.07 μg / mL, 0.06 μg / mL, 0.05 μg / mL, 0.04 μg / mL, 0.03 μg / mL, 0.02 μg / mL, or 0.01 μg / mL or less) in 1% human serum, or with an IC50 of 0.20 μg / mL or less (or 0.15 μg / mL, 0.10 μg / mL, 0.09 μg / mL, 0.08 μg / mL, 0.07 μg / mL, 0.06 μg / mL, 0.05 μg / mL, 0.04 μg / mL, 0.03 μg / mL, 0.02 μg / mL, or 0.01 μg / mL or less) in 10% human serum. In certain embodiments, the antibodies and fragments thereof provided herein are capable of blocking complement C3 activation in 50% human serum.

[0117] [000133] In certain embodiments, the antibodies and fragments thereof provided herein have an IC50 value of 0.11 μg / mL or less (or 0.10 μg / mL, 0.09 μg / mL, 0.08 μg / mL, 0.07 μg / mL, 0.06 μg / mL, 0.05 μg / mL, 0.04 μg / mL, 0.03 μg / mL, 0.02 μg / mL, or 0.01 μg / mL or less) when measured in 2% human serum, or 0.69 μg / mL or less (or 0.65 μg / mL or less) when measured in 10% human serum. , 0.6μg / mL, 0.55μg / mL, 0.5μg / mL, 0.45μg / mL, 0.4μg / mL, 0.35μg / mL, 0.3μg / mL, 0.25μg / mL or 0.2μg / mL, 0.15μg / mL, 0.1μg / mL, 0.09μg / mL, 0.08μg / mL, 0.07μg / mL, 0.06μg / mL, 0.05μg / mL, 0.04μg / mL, 0.03μg / mL, 0.02μg / mL or 0.01μg / mL or less).

[0118] [000134] In certain embodiments, the antibodies and fragments thereof provided herein can block MAC formation with an IC50 value of 0.27 μg / mL or less (or 0.25 μg / mL, or 0.2 μg / mL, 0.15 μg / mL, 0.1 μg / mL, 0.09 μg / mL, 0.08 μg / mL, 0.07 μg / mL, 0.06 μg / mL, 0.05 μg / mL, 0.04 μg / mL, 0.03 μg / mL, 0.02 μg / mL, or 0.01 μg / mL or less) as measured in 2% human serum by ELISA.

[0119] [000135] In certain embodiments, the anti-MASP-2 antibodies and antigen-binding fragments thereof provided herein bind to cynomolgus monkey MASP-2. In certain embodiments, the antibodies and antigen-binding fragments thereof bind to cynomolgus monkey MASP-2 with a binding affinity similar to that of human MASP-2. For example, the binding of each of the exemplary antibodies 129C10 HaLa, 129C10 HaLb, 129C10 HbLa, 129C10 HbLb, 129C10 HcLa, 129C10 HcLb, 129C10 HdLa, or 129C10 HdLb to cynomolgus monkey MASP-2 indicates the affinity or EC for human MASP-2. 50 Similar affinity or EC values 50 value.

[0120] [000136] In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein have an EC of 0.02 μg / mL or less, 0.015 μg / mL or less, 0.01 μg / mL or less, 0.005 μg / mL or less, 0.001 μg / mL or less, or 0.001 μg / mL or less by ELISA assay. 50 It specifically binds to cynomolgus monkey MASP-2 expressed on cells.

[0121] [000137] In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein have an EC of 0.02 μg / mL or less, 0.015 μg / mL or less, 0.01 μg / mL or less, 0.005 μg / mL or less, 0.001 μg / mL or less, or 0.001 μg / mL or less by ELISA assay. 50It specifically binds to cynomolgus monkey MASP-2 expressed on cells.

[0122] [000138] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein have a longer serum half-life in monkeys (e.g., at least 5%, 10%, 15%, 20% longer) compared to OMS721.

[0123] [000139] OMS721 (hIgG4 kappa), also called OMS721-analog, is a benchmark antibody that has the heavy and light chain variable regions of OMS721 (narsoplimab) of US Patent No. 9011860 B2, but has the constant region of human IgG4. OMS721 (hIgG4 kappa) comprises a heavy chain of SEQ ID NO:32 and a light chain of SEQ ID NO:33.

[0124] [000140] Heavy chain of OMS721 (hIgG4kappa) (SEQ ID NO:32): QVTLKESGPVLVKPTETLTLTCTVSGFSLS RGKMGVS WIRQPPGKALEWLA HIFSSDEKSYRTSLKS RLTISKDTSKNQVVLTMTTNMDPVDTATYYCAR IRRGGIDY WGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWY VDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK [000141] Light chain of OMS721 (hIgG4kappa) (SEQ ID NO:33): QPVLTQPPSLSVSPGQTASITC SGEKLGDKYAY WYQQKPGQSPVLV MYQDKQRPS GIPERFSGSNSGNTATLTISGTQAMDEADYYC QAWDSSTAV FGGGTKLTVLRTVAAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC [000142] In certain embodiments, the antibodies or antigen-binding fragments provided herein do not cross-react with mouse or rat MASP-2.

[0125] [000143] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein do not cross-react with C1s, C1r, MASP1 or MASP3.

[0126] [000144] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein can selectively block complement activation of the MBL pathway, and not involving the classical or alternative pathways.

[0127] [000145] The antibodies or antigen-binding fragments thereof provided herein can be monoclonal antibodies, bispecific antibodies, multispecific antibodies, recombinant antibodies, chimeric antibodies, humanized antibodies, labeled antibodies, bivalent antibodies, anti-idiotypic antibodies, fusion proteins, dimerized or polymerized antibodies, or modified antibodies (e.g., glycosylated antibodies). Recombinant antibodies are antibodies prepared in vitro using recombinant methods rather than in an animal.

[0128] [000146] Antibody variants [000147] The present disclosure also encompasses various types of variants of the antibodies and antigen-binding fragments thereof provided herein. In certain embodiments, the present disclosure encompasses variants of the exemplary antibodies provided herein, namely, 129C10 mouse / chimera, 160D10 mouse / chimera, 125D5 mouse / chimera, 129C10 HaLa, 129C10 HaLb, 129C10 HbLa, 129C10 HbLb, 129C10 HcLa, 129C10 HcLb, 129C10 HdLa, or 129C10 HdLb antibodies.

[0129] [000148] In certain embodiments, the antibody variant comprises one or more modifications or substitutions in one, two or three CDR sequences provided in Table 1, in the heavy or light chain variable region sequences provided in Table 2, and / or in the constant region (e.g., Fc region). Such an antibody variant retains the specific binding affinity of its parent antibody to MASP-2, but has one or more desirable properties imparted by the modifications or substitutions. For example, the antibody variant may have improved antigen binding affinity, improved glycosylation pattern, reduced risk of glycosylation, reduced deamination, reduced or depleted effector function, improved FcRn receptor binding, extended pharmacokinetic half-life, pH sensitivity, and / or suitability for conjugation (e.g., one or more introduced cysteine ​​residues).

[0130] [000149] Parent antibody sequences can be screened to identify suitable or preferred residues for modification or substitution using methods known in the art, such as "alanine scanning mutagenesis" (see, e.g., Cunningham and Wells (1989) Science, 244:1081-1085). Briefly, target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) can be identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine), and modified antibodies are generated and screened for the desired properties. If substitution at a particular amino acid position manifests the desired functional change, that position can be identified as a potential residue for modification or substitution. Potential residues can be further evaluated by substituting with different types of residues (e.g., cysteine ​​residues, positively charged residues, etc.).

[0131] [000150] Affinity variants [000151] Affinity variants can contain modifications or substitutions in one or more CDR sequences provided in Table 1, or in the heavy chain variable region sequences or light chain variable region sequences provided in Table 2. Affinity variants retain the specific binding affinity of the parent antibody to MASP-2, or even have improved specific binding affinity for MASP-2 over the parent antibody. In certain embodiments, at least one (or all) of the substitutions in the CDR sequences, FR sequences, or variable region sequences include conservative substitutions.

[0132] [000152] Those skilled in the art will understand that one or more amino acid residues may be substituted in the CDR sequences of Table 1 and the FR sequences provided herein, while the resulting antibody or antigen-binding fragment still retains or even has improved binding affinity to MASP-2. Various methods known in the art may be used to achieve this goal. For example, a library of antibody variants (e.g., Fab variants or scFv variants) may be generated and expressed using phage display technology, and then screened for binding affinity to human MASP-2. In another example, computer software may be used to virtually simulate the binding of an antibody to human MASP-2 and identify amino acid residues on the antibody that form the binding interface. Such residues may be avoided in substitutions to prevent a decrease in binding affinity, or may be targeted for substitutions that result in stronger binding.

[0133] [000153] In certain embodiments, the humanized antibodies or antigen-binding fragments provided herein comprise one or more amino acid residue substitutions in one or more CDR sequences and / or one or more FR sequences. In certain embodiments, the affinity variants comprise a total of 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or less substitutions in the CDR and / or FR sequences.

[0134] [000154] In certain embodiments, the anti-MASP-2 antibodies and antigen-binding fragments thereof comprise one, two, or three CDR sequences having at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to the CDR sequence(s) listed in Table 1, while retaining binding affinity to MASP-2 at a similar or even higher level than that of its parent antibody.

[0135] [000155] In certain embodiments, anti-MASP-2 antibodies and antigen-binding fragments thereof comprise one or more variable region sequences having at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to the variable region sequence(s) listed in SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:28, SEQ ID NO:30, while retaining a similar or even higher level of binding affinity to MASP-2 than its parent antibody. In some embodiments, a total of 1 to 10 amino acids are substituted, inserted or deleted in a sequence selected from the group consisting of SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:28, SEQ ID NO:30. In some embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (ie, within the FRs).

[0136] [000156] Glycosylation variants [000157] The anti-MASP-2 antibodies and antigen-binding fragments provided herein also encompass glycosylation variants, which can be obtained to increase or decrease the degree of glycosylation of the antibody or antigen-binding fragment.

[0137] [000158] An anti-MASP-2 antibody or antigen-binding fragment thereof may contain one or more amino acid residues having a side chain to which a carbohydrate moiety (e.g., an oligosaccharide structure) can be attached. Glycosylation of antibodies is usually either N-linked or O-linked. N-linked refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue in a tripeptide sequence, such as asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most usually serine or threonine. Removal of native glycosylation sites can be conveniently accomplished by altering the amino acid sequence, for example, to substitute one of the above tripeptide sequences (for N-linked glycosylation sites), or a serine or threonine residue (for O-linked glycosylation sites) present in the sequence. New glycosylation sites can be created in a similar manner by introducing such tripeptide sequences or serine or threonine residues.

[0138] [000159] Fc variants [000160] The anti-MASP-2 antibodies and antigen-binding fragments provided herein also include Fc variants, which contain one or more amino acid residue modifications or substitutions in the Fc region and / or hinge region.

[0139] [000161] In certain embodiments, the anti-MASP-2 antibody or antigen-binding fragment contains one or more amino acid substitutions that improve pH-dependent binding to the neonatal Fc receptor (FcRn). Such variants can have an extended pharmacokinetic half-life because they bind to FcRn at an acidic pH, allowing them to avoid degradation in lysosomes and then be translocated and released extracellularly. Methods for engineering antibodies and antigen-binding fragments thereof to improve binding affinity to FcRn are well known in the art, see, e.g., Vaughn, D. et al., Structure, 6(1):63-73, 1998; Kontermann, R. et al., Antibody Engineering, Vol. 1, Chapter 27: Engineering of the Fc region for improved PK, Springer, 2010; Yeung, Y. et al., Cancer Research, 70:3269-3277 (2010); and Hinton, P. et al., J. Immunology, 176:346-356 (2006).

[0140] [000162] In certain embodiments, the anti-MASP-2 antibody or antigen-binding fragment thereof contains one or more amino acid substitutions that enhance the binding affinity to FcRn, thereby extending the serum half-life of the antibody. For example, the heavy chain constant region contains one or more amino acid substitutions at amino acid residues 252, 254 or 256 compared to the wild-type human IgG constant region (see U.S. Patent No. 7,083,784). In certain embodiments, the amino acid substitution at amino acid residue 252 is a substitution with tyrosine, the amino acid substitution at amino acid residue 254 is a substitution with threonine, and the amino acid substitution at amino acid residue 256 is a substitution with glutamic acid (M252Y / S254T / T256E, or YTE). In certain embodiments, the anti-MASP-2 antibody or antigen-binding fragment thereof comprises a heavy chain constant region having at least 80% (or for example, at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity to the wild-type heavy chain constant region, provided that the M252Y / S254T / T256E substitutions are maintained.

[0141] [000163] In certain embodiments, the one or more amino acid substitutions are M252Y / S254T / T256E in the heavy chain of the human IgG4 Fc region. In certain embodiments, the heavy chain constant region of human IgG4 with YTE modifications has the amino acid sequence of SEQ ID NO: 35 (see Tables 2-3).

[0142] [000164] In certain embodiments, the anti-MASP-2 antibodies and antigen-binding fragments provided herein comprise a heavy chain constant region of human IgG4 and a light chain constant region of human IgG. The heavy chain constant region of human IgG4 has the amino acid sequence of SEQ ID NO: 35, and the light chain constant region has the amino acid sequence of SEQ ID NO: 34.

[0143] [000165] In certain embodiments, the anti-MASP-2 antibody or antigen-binding fragment comprises one or more amino acid substitutions in the interface of the Fc region to facilitate and / or promote heterodimerization. Such modifications include the introduction of a protrusion into the first Fc polypeptide and a recess into the second Fc polypeptide, where the protrusion can be positioned in the recess to promote the interaction of the first and second Fc polypeptides, thereby forming a heterodimer or complex. Methods for generating antibodies with such modifications are known in the art, for example, as described in U.S. Patent No. 5,731,168.

[0144] [000166]Antigen-binding fragment [000167] Also provided herein are anti-MASP-2 antigen-binding fragments. Various types of antigen-binding fragments are known in the art and can be developed based on the anti-MASP-2 antibodies provided herein, including, for example, the exemplary antibodies whose CDRs are shown in Table 1 and their various variants (e.g., affinity variants, glycosylation variants, Fc variants, etc.).

[0145] [000168] In certain embodiments, the anti-MASP-2 antigen-binding fragments provided herein include diabodies, Fab, Fab', F(ab') 2 , Fd, Fv fragment, disulfide stabilized Fv fragment (dsFv), (dsFv) 2 , bispecific dsFv (dsFv-dsFv'), disulfide stabilized diabodies (ds diabodies), single chain antibody molecules (scFv), scFv dimers (bivalent diabodies), multispecific antibodies, camelized single domain antibodies, nanobodies, domain antibodies, or bivalent domain antibodies.

[0146] [000169] A variety of techniques can be used for the production of such antigen-binding fragments. Exemplary methods include enzymatic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992); and Brennan et al., Science 229:81 (1985)), recombinant expression in host cells such as E. coli (e.g., for Fab, Fv, and ScFv antibody fragments), screening from phage display libraries as discussed above (e.g., for ScFv), and F(ab') 2 Examples of suitable techniques include the chemical coupling of two Fab'-SH fragments to form an antibody fragment (Carter et al., Bio / Technology 10:163-167 (1992)). Other techniques for the production of antibody fragments will be apparent to those skilled in the art.

[0147] [000170] In certain embodiments, the antigen-binding fragment is scFv. The generation of scFv is described, for example, in WO93 / 16185; U.S. Patent No. 5,571,894; and U.S. Patent No. 5,587,458. scFv can be fused to effector protein at either amino or carboxy terminus to produce fusion protein (see, for example, Antibody Engineering, edited by Borrebaeck).

[0148] [000171] In certain embodiments, antibodies and antigen-binding fragments thereof can be used as the basis for bispecific or multivalent antibodies.

[0149] [000172] Bispecific antibodies, multivalent antibodies [000173] In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein are bivalent, tetravalent, hexavalent, or multivalent. In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein are monospecific or bispecific.

[0150] [000174] The term "valent" as used herein refers to the presence of a specified number of antigen binding sites in a given molecule. Thus, the terms "bivalent", "tetravalent" and "hexavalent" refer to the presence of two binding sites, four binding sites and six binding sites, respectively, in an antigen-binding molecule. A bivalent molecule can be monospecific when both binding sites are for specific binding of the same antigen or epitope. Similarly, a trivalent molecule can be bispecific when, for example, two binding sites are monospecific for a first antigen (or epitope) and the third binding site is specific for a second antigen (or epitope).

[0151] [000175] In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein may be monospecific but bivalent, trivalent, or tetravalent, with at least two binding sites specific for the same antigen or epitope. This allows for stronger binding to the antigen or epitope than their monovalent counterparts in certain embodiments. In certain embodiments, in a bivalent antigen-binding moiety, the first valency of the binding site and the second valency of the binding site are structurally identical (i.e., have the same sequence) or structurally different (i.e., have different sequences, despite having the same specificity).

[0152] [000176] In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein are bispecific. In some embodiments, the bispecific antibodies and antigen-binding fragments thereof provided herein have a first specificity and a second specificity for MASP-2. In some embodiments, the second specificity is for MASP-2, but to a different epitope. In some embodiments, the second specificity is for a second antigen that is different from MASP-2.

[0153] [000177] The bispecific antibodies and antigen-binding fragments provided herein can be produced using any suitable method known in the art. In a traditional approach, two immunoglobulin heavy-light chain pairs with different antigen specificities are co-expressed in a host cell, resulting in recombinant production of a bispecific antibody (see, e.g., Milstein and Cuello, Nature, 305:537 (1983)), which can then be purified by affinity chromatography.

[0154] [000178] Recombinant approaches can also be used, in which sequences encoding antibody heavy chain variable domains for the two specificities are each fused to an immunoglobulin constant domain sequence and subsequently inserted into an expression vector, which is co-transfected with an expression vector for the light chain sequence into a host cell suitable for recombinant expression of the bispecific antibody (see, e.g., WO94 / 04690; Suresh et al., Methods in Enzymology, 121:210 (1986)). Similarly, scFv dimers can also be constructed and expressed recombinantly from a host cell (see, e.g., Gruber et al., J. Immunol., 152:5368 (1994)).

[0155] [000179] In another method, the leucine zipper peptides from the Fos and Jun proteins can be linked to the Fab' portions of two different antibodies by gene fusion. The linked antibodies are reduced at the hinge region into four half antibodies (i.e., monomers) and then reoxidized to form heterodimers (Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)).

[0156] [000180] Two antigen-binding domains can also be conjugated or cross-linked to form bispecific antibodies or antigen-binding fragments. For example, one antibody can be coupled with biotin, while the other antibody can be coupled with avidin, and the strong association between biotin and avidin will combine the two antibodies together to form a bispecific antibody (see, for example, U.S. Pat. No. 4,676,980, WO91 / 00360, WO92 / 00373, and EP03089). In another example, two antibodies or antigen-binding fragments can be cross-linked by conventional methods known in the art, for example, as disclosed in U.S. Pat. No. 4,676,980.

[0157] [000181] Bispecific antigen-binding fragments can be produced from bispecific antibodies, for example, by proteolytic cleavage or by chemical cross-linking. For example, an antigen-binding fragment of an antibody (e.g., Fab') can be prepared and converted to a Fab'-thiol derivative and then mixed and reacted with another converted Fab' derivative having a different antigen specificity to form a bispecific antigen-binding fragment (see, for example, Brennan et al., Science, 229:81 (1985)).

[0158] [000182] In certain embodiments, bispecific antibodies or antigen-binding fragments can be engineered at the interface such that knob-into-hole associations can form to promote heterodimerization of two distinct antigen-binding sites. As used herein, "knobs-into-holes" refers to an interaction between two polypeptides (e.g., Fc) where one polypeptide has a protrusion (i.e., "knob") due to the presence of an amino acid residue with a bulky side chain (e.g., tyrosine or tryptophan) and the other polypeptide has a recess (i.e., "hole") with a small side chain amino acid residue (e.g., alanine or threonine), but the protrusion can be positioned in the recess to promote the interaction of the two polypeptides, thereby forming a heterodimer or complex. Methods for generating polypeptides with knobs-into-holes are known in the art, for example, as described in U.S. Pat. No. 5,731,168.

[0159] [000183] Conjugate [000184] In some embodiments, the anti-MASP-2 antibody and its antigen-binding fragment are linked to one or more conjugate moieties. A conjugate is a moiety that can be attached to an antibody or its antigen-binding fragment. It is contemplated that various conjugates can be linked to the antibodies or antigen-binding fragments provided herein (see, for example, "Conjugate Vaccines", Contributions to Microbiology and Immunology, JM Cruse and RE Lewis, Jr. (eds.), Carger Press, New York, 1989). Such conjugates can be linked to the antibody or antigen-binding fragment by covalent bonding, affinity bonding, intercalation, coordinate bonding, complex formation, association, blending, or addition, among other methods. In certain embodiments, the antibody or its antigen-binding fragment is linked to one or more conjugates via a linker. In certain embodiments, the linker is a hydrazone linker, a disulfide linker, a bifunctional linker, a dipeptide linker, a glucuronide linker, or a thioether linker.

[0160] [000185] In certain embodiments, the anti-MASP-2 antibodies and antigen-binding fragments disclosed herein may be engineered to contain specific sites outside the epitope-binding portion that can be utilized for binding to one or more conjugates. For example, such sites may contain one or more reactive amino acid residues, such as, for example, cysteine ​​or histidine residues, to facilitate covalent linkage to a conjugate.

[0161] [000186] The conjugate may be a clearance modifier, a therapeutic agent (e.g., a chemotherapeutic agent), a toxin, a radioisotope, a detectable label (e.g., a lanthanide, a luminescent label, a fluorescent label, or an enzyme substrate label), a pharmacokinetic-modifying moiety, a DNA alkylating agent, a topoisomerase inhibitor, a tubulin binding agent, or other anti-cancer drug referred to as an androgen receptor inhibitor.

[0162] [000187] Examples of detectable labels include fluorescent labels (e.g., fluorescein, rhodamine, dansyl, phycoerythrin, or Texas Red), enzyme substrate labels (e.g., horseradish peroxidase, alkaline phosphatase, luciferase, glucoamylase, lysozyme, sugar oxidase, or β-D-galactosidase), radioisotopes, other lanthanides, luminescent labels, chromophore moieties, digoxigenin, biotin / avidin, detectable DNA molecules, or gold.

[0163] [000188] Examples of radioisotopes include: 123 I, 124 I, 125 I, 131 I, 35 S, 3 H, 111 In, 112 In, 14 C. 64 Cu, 67 Cu, 86 Y, 88 Y, 90 Y, 177 Lu, 211 At, 186 Re, 188 Re, 153 Sm, 212 Bi, and 32 Examples include P. Radioisotope-labeled antibodies are useful in receptor-targeted imaging experiments.

[0164] [000189] In certain embodiments, the conjugate may be a pharmacokinetic-modifying moiety, such as PEG, which serves to extend the half-life of the antibody. Other suitable polymers include, for example, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, ethylene glycol / propylene glycol copolymers, and the like.

[0165] [000190] In certain embodiments, the conjugate can be a purification moiety such as a magnetic bead or nanoparticle.

[0166] [000191] Polynucleotides and recombinant methods [000192] The present disclosure provides isolated polynucleotides encoding anti-MASP-2 antibodies and their antigen-binding fragments. DNA encoding monoclonal antibodies can be easily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of the antibody). Encoding DNA can also be obtained by synthetic methods.

[0167] [000193] The isolated polynucleotides encoding anti-MASP-2 antibodies and their antigen-binding fragments can be inserted into vectors for further cloning (amplification of DNA) or for expression using recombinant techniques known in the art. Many vectors are available. Vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter (e.g., SV40, CMV, EF-1α), and a transcription termination sequence.

[0168] [000194] In some embodiments, vector systems include mammalian, bacterial, yeast, etc., including plasmids such as, but not limited to, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pCMV, pEGFP, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMD18-T, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS420, pLexA, pACT2.2, etc., and other laboratory and commercially available vectors. Suitable vectors can include plasmids or viral vectors (e.g., replication-defective retroviruses, adenoviruses and adeno-associated viruses).

[0169] [000195] A vector containing a polynucleotide sequence encoding an antibody or antigen-binding fragment can be introduced into a host cell for cloning or gene expression. Suitable host cells for cloning or expressing DNA in the vectors herein are the prokaryotic cells, yeast cells, or higher eukaryotic cells described above. Suitable prokaryotes for this purpose include eubacteria, such as gram-negative or gram-positive bacteria, such as Enterobacteriaceae, such as Escherichia, such as E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, such as Salmonella typhimurium, Serratia, such as Serratia marcescens, and Shigella, as well as Bacillus, such as B. subtilis and B. licheniformis, Pseudomonas, such as Pseudomonas aeruginosa, and Streptomyces.

[0170] [000196] In addition to prokaryotes, eukaryotic microbes, such as filamentous fungi or yeast, are suitable cloning or expression hosts for vectors encoding anti-MASP-2 antibodies. Saccharomyces cerevisiae (or common baker's yeast) is the most commonly used of the lower eukaryotic host microorganisms. However, several other genera, species, and strains are commonly available and useful herein, e.g., Schizosaccharomyces pombe; Kluyveromyces hosts, e.g., K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. wartsii (ATCC 56,500), K. drosophila (ATCC 36,906), K. thermotolerans, and K. marxianus; Yarrowia spp. (EP 402,226); Pichia pastoris (EP 183,070); Candida spp.; Trichoderma lesia (EP 244,234); Neurospora crassa; Schwanniomyces spp., such as Schwanniomyces oxidentalis; and filamentous fungi, such as Neurospora spp., Penicillium spp., Tolypocladium spp., and Aspergillus hosts, such as A. nidulans and A. niger.

[0171] [000197] The host cells suitable for expressing the glycosylated antibodies or antigen fragments thereof provided herein are derived from multicellular organisms. Examples of invertebrate cells include plant cells and insect cells. Numerous baculovirus strains and variants from hosts such as Fall Armyworm (caterpillar), Aedes aegypti (mosquito), Aedes japonica (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori have been identified, and corresponding insect permissive host cells. Various virus strains for transfection, such as the L-1 variant of Pseudomonas cernua NPV and the Bm-5 strain of Bombyx mori NPV, are publicly available, and such viruses can be used as viruses herein according to the present invention, particularly for transfection of Fall Armyworm cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be utilized as hosts.

[0172] [000198] However, interest has been greatest in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines are the SV40 transformed monkey kidney CV1 line (COS-7, ATCC CRL1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)), baby hamster kidney cells (BHK, ATCC CCL10); Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL34); buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumor (MMT060562, ATCC CCL51); TRI cells (Mather et al., Annals NYAcad. Sci. 383:44-68 (1982)); MRC5 cells; FS4 cells; and a human hepatocellular carcinoma line (Hep G2). In some preferred embodiments, the host cells are CHO cells.

[0173] [000199] Host cells are transformed with the above-mentioned expression vectors or cloning vectors for producing anti-MASP-2 antibodies and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying genes encoding the desired sequences. In another embodiment, antibodies may be produced by homologous recombination as known in the art.

[0174] [000200] The host cells used to produce the antibodies or antigen-binding fragments provided herein can be cultured in a variety of media. Commercially available media, such as Ham's F10 (Sigma), Minimum Essential Medium (MEM) (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium (DMEM) (Sigma), are suitable for culturing the host cells. In addition, any of the media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90 / 03430; WO 87 / 00195; or U.S. Pat. No. Re. 30,985 may be used as a culture medium for the host cells. Any of these media may be supplemented with hormones and / or other growth factors (e.g., insulin, transferrin, or epidermal growth factor), salts (e.g., sodium chloride, calcium chloride, magnesium chloride, and phosphates), buffers such as HEPES, nucleotides (e.g., adenosine and thymidine), antibiotics (e.g., GENTAMYCIN™ drug), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source, as appropriate. Any other necessary supplements may also be included at appropriate concentrations that would be known to one of skill in the art. Culture conditions such as temperature, pH, etc. are those previously used with the host cell selected for expression and will be apparent to one of skill in the art.

[0175] [000201] Anti-MASP-2 antibodies or antigen-binding fragments thereof prepared from cells can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, DEAE cellulose ion exchange chromatography, ammonium sulfate precipitation, salting out, and affinity chromatography, with affinity chromatography being the preferred purification technique.

[0176] [000202] In certain embodiments, Protein A immobilized on a solid phase is used for immunoaffinity purification of antibodies and antigen-binding fragments thereof. The suitability of Protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain present in the antibody. Protein A can be used to purify antibodies based on human gamma 1, gamma 2, or gamma 4 heavy chains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G is recommended for all mouse isotypes and human gamma 3 (Guss et al., EMBO J. 5:1567-1575 (1986)). The matrix to which the affinity ligand is attached is most often agarose, although other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. If the antibody contains a CH3 domain, Bakerbond ABX™ resin (JT Baker, Phillipsburg, NJ) is useful for purification. Other techniques for protein purification, such as fractionation on ion exchange columns, ethanol precipitation, reverse-phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE™ chromatography on anion or cation exchange resins (e.g., polyaspartic acid columns), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also available depending on the antibody to be recovered.

[0177] [000203] Following any preliminary purification steps, the mixture containing the antibody of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography, preferably performed at a low salt concentration (e.g., about 0-0.25 M salt) and using an elution buffer at a pH between about 2.5-4.5.

[0178] [000204] Pharmaceutical composition [000205] The present disclosure further provides a pharmaceutical composition comprising an anti-MASP-2 antibody or antigen-binding fragment thereof, or an antibody-drug conjugate provided herein, and one or more pharma- ceutically acceptable carriers.

[0179] [000206] Pharmaceutically acceptable carriers for use in the pharmaceutical compositions disclosed herein can include, for example, pharma- ceutically acceptable liquid, gel, or solid carriers, aqueous vehicles, non-aqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending / dispending agents, sequestering or chelating agents, diluents, adjuvants, excipients, or non-toxic auxiliary agents, other ingredients known in the art, or various combinations thereof.

[0180] [000207] Suitable ingredients can include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavorings, thickeners, colorings, emulsifiers, or stabilizers such as sugars and cyclodextrins. Suitable antioxidants can include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, thioglycerol, thioglycolic acid, thiosorbitol, butylated hydroxyanisole, butylated hydroxytoluene, and / or propyl gallate. As disclosed herein, the inclusion of one or more antioxidants, such as methionine, in compositions comprising the antibodies or antigen-binding fragments and conjugates provided herein reduces the oxidation of the antibodies or antigen-binding fragments. Such reduction in oxidation prevents or reduces the loss of binding affinity, thereby improving antibody stability and maximizing shelf life. Thus, in certain embodiments, compositions are provided that include one or more antibodies or antigen-binding fragments disclosed herein and one or more antioxidants, such as methionine. Additionally, methods are provided to prevent oxidation of the antibodies or antigen-binding fragments provided herein, extending their shelf life, and / or improving their effectiveness by combining the antibodies or antigen-binding fragments provided herein with one or more antioxidants, such as methionine.

[0181] [000208] To further illustrate, pharma- ceutically acceptable carriers include, for example, aqueous vehicles such as Sodium Chloride Injection, Ringer's Solution, Isotonic Dextrose Injection, Sterile Water Injection, or Dextrose and Lactated Ringer's Injection, non-aqueous vehicles such as fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, or peanut oil, antimicrobial agents at bacteriostatic or fungistatic concentrations, isotonic agents such as sodium chloride or dextrose, buffers such as phosphate or citrate buffers, antioxidants such as sodium bisulfate, Local anesthetics such as procaine hydrochloride, suspending and dispersing agents such as sodium carboxymethylcellulose, hydroxypropylmethylcellulose, or polyvinylpyrrolidone, emulsifying agents such as polysorbate 80 (TWEEN®-80), sequestering or chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid), ethyl alcohol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid, or lactic acid. Antimicrobial agents utilized as carriers may be added to pharmaceutical compositions in multi-dose containers, including phenol or cresol, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride, and benzethonium chloride. Suitable excipients include, for example, water, saline, dextrose, glycerol, or ethanol. Suitable nontoxic auxiliary substances can include, for example, wetting or emulsifying agents, pH buffering agents, stabilizing agents, solubility enhancers, or agents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, or cyclodextrins.

[0182] [000209] The pharmaceutical compositions can be liquid solutions, suspensions, emulsions, pills, capsules, tablets, sustained release formulations, or powders. Oral formulations can include standard carriers, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinylpyrrolidone, sodium saccharin, cellulose, magnesium carbonate, and the like.

[0183] [000210] In certain embodiments, the pharmaceutical composition is formulated into an injectable composition. The injectable pharmaceutical composition can be prepared in any conventional form, such as a liquid solution, suspension, emulsion, or a solid form suitable for producing a liquid solution, suspension, or emulsion. The preparation for injection can include a sterile and / or non-pyrogenic solution ready for injection, a sterile dry soluble, such as a lyophilized powder, ready to be combined with a solvent immediately before use, including a subcutaneous tablet, a sterile suspension ready for injection, a sterile dry insoluble, ready to be combined with a vehicle immediately before use, and a sterile and / or non-pyrogenic emulsion. The solution can be either aqueous or non-aqueous.

[0184] [000211] In certain embodiments, unit dose parenteral preparations are packaged in ampoules, vials, or syringes with needles. As is known and practiced in the art, all preparations for parenteral administration are required to be sterile and non-pyrogenic.

[0185] [000212] In certain embodiments, a sterile, freeze-dried powder is prepared by dissolving the antibody or antigen-binding fragment disclosed herein in a suitable solvent. The solvent may contain excipients to improve stability or other pharmacological components of the powder or reconstituted solution prepared from the powder. Excipients that may be used include, but are not limited to, water, dextrose, sorbital, fructose, corn syrup, xylitol, glycerin, glucose, sucrose or other suitable agents. The solvent may contain a buffer, such as citrate, sodium or potassium phosphate, or other such buffer known to those skilled in the art, in one embodiment, at about neutral pH. Subsequent sterile filtration of the solution followed by lyophilization under standard conditions known to those skilled in the art provides the desired formulation. In one embodiment, the resulting solution is to be apportioned into vials for lyophilization. Each vial may contain a single or multiple doses of an anti-MASP-2 antibody or antigen-binding fragment thereof or composition thereof. Overfilling the vial by a small amount (e.g., about 10%) beyond that needed for a dose or series of doses is permissible to facilitate accurate sampling and accurate dosing. The lyophilized powder can be stored under appropriate conditions, such as at about 4° C. to room temperature.

[0186] [000213] Reconstitution of the lyophilized powder with water for injection provides a formulation for use in parenteral administration. In one embodiment, for reconstitution, sterile water and / or non-pyrogenic water, or other liquid suitable carrier, is added to the lyophilized powder. The exact amount depends on the selected therapy being given, but may be empirically determined.

[0187] [000214] Method of Use [000215] The present disclosure also provides a method of treatment comprising: administering a therapeutically effective amount of an antibody or antigen-binding fragment provided herein to a subject in need thereof, thereby treating or preventing a disease or condition associated with MASP-2-dependent complement activation.

[0188] [000216] MASP-2 (MBL-associated serine protease 2) participates in and activates the complement system. When MBL binds to pathogens, MASP-2 is activated to cleave complement components C4 and C2 into C4a, C4b, C2a, and C2b, resulting in the generation of the convertase C4bC2b of C3, which is then converted by C4bC2b into C3b, and finally forms the membrane attack complex (MAC) after C5 is converted by C3b into C5b. The activation of C3 ultimately leads to the formation of MAC, which then initiates a series of cascade activation processes of the downstream complement system that stimulates innate immune responses.

[0189] [000217] Therefore, inhibition of MASP-2 may be useful in inhibiting MASP-2-dependent complement activation.

[0190] [000218] The present disclosure provides a method for inhibiting MASP-2-dependent complement activation in a subject in need of inhibition of MASP-2-dependent complement activation, or a method for treating or preventing a condition or disease associated with MASP-2-dependent complement activation, comprising the step of administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment provided herein.

[0191] [000219] In another aspect, a method is provided for treating a condition in a subject that would benefit from inhibition of MASP-2-dependent complement activation, the method comprising administering a therapeutically effective amount of an antibody or antigen-binding fragment thereof provided herein to a subject in need thereof.

[0192] [000220] In another aspect, the disclosure further provides a method of reducing serum C4 levels in a subject, the method comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof provided herein, thereby reducing serum C4 levels in the subject.

[0193] [000221] In another aspect, the disclosure further provides a method of treating a condition in a subject that would benefit from a reduction in serum C4 levels in the subject, or treating or preventing a condition or disease associated with abnormal (e.g., elevated) serum C4 levels, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof provided herein, thereby treating the condition.

[0194] [000222] In some embodiments, the condition or disease associated with MASP-2-dependent complement activation is an endothelial damage associated disease.

[0195] [000223] In some embodiments, the condition or disease associated with MASP-2-dependent complement activation is an autoantibody immune complex-induced disease.Examples of autoantibody immune complex-induced diseases include, for example, IgA nephropathy, systemic lupus erythematosus (SLE), lupus nephritis, thrombotic microangiopathy (TMA), or hematopoietic transplantation-associated thrombotic microangiopathy (TA-TMA).

[0196] [000224] In some embodiments, the MASP-2-dependent complement activation associated disease or condition is IgA nephropathy (for details, see, e.g., Drachenberg CB, et al., Kidney International Reports, 2019, 4(11); Espinosa M, et al., Clin J Am Soc Nephrol, 2014, 9; Yeo SC, et al., Pediatr Nephrol, 2018, 33), lupus nephritis (see, e.g., Gatenby, PA Autoimmunity, 1991, 11), thrombotic microangiopathy (TMA) (see, e.g., Elhadad S. et al., Clin Exp Immunol. 2021, 203(1)), systemic lupus erythematosus (SLE) (see, e.g., Walport, MJ, Davies, et al., Ann. NY Acad Sci. § 75:267-81, 1997), vasculitis (see, e.g., Moake XL., N Engl J Med., 2002, 347), Kawasaki disease (arteritis) (see, e.g., Nakamura A. et al., Clin Immunol., 2014, 153(1)), SARS-CoV, MERS-CoV and SARS-CoV-2 (Covid-19) (see, e.g., Gao T. et al., Highly pathogenic coronavirus N protein aggravates lung injury by MASP-2-mediated complement over-activation. Preprint from medRxiv, March 30, 2020).

[0197] [000225] In some embodiments, the MASP-2-dependent complement activation associated disease or condition is an autoimmune disease, a vascular condition, ischemia-reperfusion injury, atherosclerosis, inflammation, a pulmonary condition, an extracorporeal reperfusion procedure, a skeletal muscle condition, a renal condition, a skin condition, an organ or tissue transplant procedure, a nervous system disorder or injury, a blood disorder, a genitourinary condition, complications associated with non-obese diabetes or type 1 or type 2 diabetes, cancer, an endocrine disorder, or an ophthalmological condition.

[0198] [000226] In certain embodiments, the condition or disease associated with MASP-2-dependent complement activation is an autoimmune disease.

[0199] [000227] In some embodiments, the autoimmune disease comprises thrombotic microangiopathy (TMA), atypical hemolytic uraemic syndrome (aHUS), hematopoietic transplant-associated thrombotic microangiopathy (TA-TMA), lupus nephritis, systemic lupus erythematosus (SLE), and IgA nephropathy.

[0200] [000228] In some embodiments, the vascular conditions include cardiovascular conditions, cerebrovascular conditions, peripheral (e.g., musculoskeletal) vascular conditions, renal vascular conditions, mesenteric / intestinal vascular conditions, revascularization of grafts and / or regrafts, vasculitis, Henoch-Schönlein purpura nephritis, systemic lupus erythematosus-associated vasculitis, rheumatoid arthritis-associated vasculitis, immune complex vasculitis, Takayasu's disease, dilated cardiomyopathy, diabetic vasculopathy, Kawasaki disease (arteritis), venous gas embolism (VGE), and restenosis after stent placement, rotational atherectomy, and percutaneous transluminal coronary angioplasty (PTCA).

[0201] [000229] In some embodiments, ischemia-reperfusion injury includes ischemia-reperfusion injury associated with aortic aneurysm repair, cardiopulmonary bypass, organ transplants and / or vascular reanastomosis along with limb / digit reimplantation, stroke, myocardial infarction, and shock and / or hemodynamic resuscitation following surgical procedures.

[0202] [000230] In some embodiments, inflammation comprises inflammatory gastrointestinal disorders including pancreatitis, Crohn's disease, ulcerative colitis, irritable bowel syndrome and diverticulitis.

[0203] [000231] In some embodiments, the pulmonary condition comprises acute respiratory distress syndrome, transfusion-associated acute lung injury, ischemia / reperfusion acute lung injury, chronic obstructive pulmonary disease, asthma, Wegener's granulomatosis, anti-glomerular basement membrane disease (Goodpasture's disease), meconium aspiration syndrome, bronchiolitis obliterans syndrome, idiopathic pulmonary fibrosis, acute lung injury secondary to burn injury, non-cardiogenic pulmonary edema, transfusion-associated respiratory depression, emphysema, cystic fibrosis, SARS-CoV, MERS-CoV and SARS-CoV-2 (Covid-19) related conditions.

[0204] [000232] In some embodiments, extracorporeal reperfusion procedures include hemodialysis, plasmapheresis, leukopheresis, extracorporeal membrane oxygenation (ECMO), heparin-induced extracorporeal membrane oxygenation (HELP) and cardiopulmonary bypass (CPB).

[0205] [000233] In some embodiments, musculoskeletal conditions include osteoarthritis, rheumatoid arthritis, juvenile rheumatoid arthritis, gout, neuropathic arthropathy, psoriatic arthritis, spondyloarthropathy, crystal arthropathy, and systemic lupus erythematosus (SLE).

[0206] [000234] In some embodiments, the renal condition comprises mesangial proliferative glomerulonephritis, membranous glomerulonephritis, membranoproliferative glomerulonephritis (mesangial capillary glomerulonephritis), acute post-infectious glomerulonephritis (post-streptococcal glomerulonephritis), cryoglobulinemic glomerulonephritis, lupus nephritis, Henoch-Schönlein purpura nephritis, and IgA nephropathy.

[0207] [000235] In some embodiments, the skin conditions include psoriasis, autoimmune blistering dermatoses, eosinophilic spongiosis, bullous pemphigoid, epidermolysis bullosa acquisita (EBA), herpes gestationis, thermal burns, and chemical burns.

[0208] [000236] In some embodiments, organ or tissue transplant procedures include organ allotransplants, organ xenotransplants, and organ and tissue grafts.

[0209] [000237] In some embodiments, nervous system disorders or injuries include multiple sclerosis, myasthenia gravis, Huntington's disease, amyotrophic lateral sclerosis, Guillain-Barre syndrome, reperfusion after stroke, degenerative discs, brain trauma, Parkinson's disease, Alzheimer's disease, Miller-Fisher syndrome, brain trauma and / or cerebral hemorrhage, demyelination and meningitis.

[0210] [000238] In some embodiments, blood disorders include sepsis, severe sepsis, septic shock, acute respiratory distress syndrome resulting from sepsis, systemic inflammatory response syndrome, hemorrhagic shock, hemolytic anemia, autoimmune thrombotic thrombocytopenic purpura, and hemolytic uremic syndrome.

[0211] [000239] In some embodiments, genitourinary conditions include painful bladder disease, sensory bladder disease, non-bacterial chronic cystitis, interstitial cystitis, infertility, placental insufficiency and miscarriage and pre-eclampsia.

[0212] [000240] In some embodiments, endocrine disorders include Hashimoto's thyroiditis, stress, anxiety, and hormonal disorders involving the regulated release of prolactin, growth factors or other insulin-like growth factors and adrenocorticotropin from the pituitary gland.

[0213] [000241] In some embodiments, the ophthalmic condition comprises age-related macular degeneration.

[0214] [000242] In some embodiments of the method provided herein, the subject is determined to have elevated C4 serum levels or to have C4d deposits or C4d positive staining in the sample of interest.In some embodiments, the condition or disease is IgA nephropathy.C4d positive staining has been reported as an independent risk factor for the development of ESRD in IgAN.C4d can be detected by using any suitable method known in the art, such as ELISA or immunofluorescence microscopy.

[0215] [000243] The therapeutically effective amount of the antibody or antigen-binding fragment provided herein will depend on various factors known in the art, such as, for example, weight, age, past medical history, current prescription drugs, the subject's health condition and potential cross-reactions, allergies, sensitivities and adverse side effects, as well as the route of administration and the extent of disease development. The dosage may be proportionally increased or decreased by a skilled artisan (e.g., a physician or veterinarian) as indicated by these and other circumstances or requirements.

[0216] [000244] In certain embodiments, the anti-MASP-2 antibodies or antigen-binding fragments provided herein may be administered at a therapeutically effective dose of about 0.01 mg / kg to about 100 mg / kg (e.g., about 0.01 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 2 mg / kg, about 3 mg / kg, about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, about 50 mg / kg, about 55 mg / kg, about 60 mg / kg, about 65 mg / kg, about 70 mg / kg, about 75 mg / kg, about 80 mg / kg, about 85 mg / kg, about 90 mg / kg, about 95 mg / kg, or about 100 mg / kg). In certain of these embodiments, the antibody or antigen-binding fragment is administered at a dose of about 50 mg / kg or less, and in certain of these embodiments, the dose is 10 mg / kg or less, 5 mg / kg or less, 3 mg / kg or less, 1 mg / kg or less, 0.5 mg / kg or less, or 0.1 mg / kg or less. In certain embodiments, the administered dose may vary over the course of treatment. For example, in certain embodiments, the initial administered dose may be higher than subsequent administered doses. In certain embodiments, the administered dose may vary over the course of treatment depending on the subject's response.

[0217] [000245] Dosage regimens can be adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single dose can be administered or several divided doses can be administered over time.

[0218] [000246] The anti-MASP-2 antibodies and antigen-binding fragments disclosed herein may be administered by any route known in the art, such as parenteral (e.g., subcutaneous, intraperitoneal, intravenous, including intravenous infusion, intramuscular, or intradermal injection) or non-parenteral (e.g., oral, intranasal, intraocular, sublingual, rectal, or topical) routes.

[0219] [000247] In some embodiments, the anti-MASP-2 antibodies or antigen-binding fragments disclosed herein may be administered alone or in combination with one or more additional therapeutic procedures or agents. For example, the antibodies or antigen-binding fragments disclosed herein may be administered in combination with another therapeutic agent, such as an anti-autoimmune drug.

[0220] [000248] In certain of these embodiments, the anti-MASP-2 antibodies or antigen-binding fragments disclosed herein that are administered in combination with one or more additional therapeutic agents may be administered simultaneously with the one or more additional therapeutic agents, and in certain of these embodiments, the antibodies or antigen-binding fragments and the additional therapeutic agents may be administered as part of the same pharmaceutical composition. However, an anti-MASP-2 antibody or antigen-binding fragment that is administered "in combination" with another therapeutic agent may not be administered simultaneously with the agent or in the same composition as the agent. An anti-MASP-2 antibody or antigen-binding fragment that is administered before or after another agent is considered to be administered "in combination" with the agent, as the phrase "in combination" is used herein, even if the antibody or antigen-binding fragment and the second agent are administered via different routes. When possible, additional therapeutic agents administered in combination with an antibody or antigen-binding fragment disclosed herein will be administered according to a schedule listed in the product information sheet of the additional therapeutic agent, or according to the Physicians' Desk Reference 2003 (Physicians' Desk Reference, Vol. 57; Medical Economics Company; ISBN: 1563634457; Vol. 57 (November 2002)), or according to protocols well known in the art.

[0221] [000249] In some embodiments, the present disclosure also provides the use of an antibody or antigen-binding fragment thereof provided herein in the manufacture of a medicament for treating a MASP-2-dependent complement activation associated disease or condition in a subject.

[0222] [000250] The following examples are provided to better illustrate the claimed invention and should not be construed as limiting the scope of the invention. All specific compositions, materials, and methods described below are within the scope of the invention, in whole or in part. These specific compositions, materials, and methods are not intended to limit the invention, but merely exemplify specific embodiments within the scope of the invention. Those skilled in the art can develop equivalent compositions, materials, and methods without the exercise of inventive capacity and without departing from the scope of the invention. It will be understood that many modifications can be made to the procedures described herein while remaining within the scope of the invention. It is the intention of the inventors that such variations are included within the scope of the invention.

[0223] Example 1: Generation of human, mouse and cynomolgus MASP-2 antigens [000251] 1. Construction of antigens for expression [000252] In order to restrict the epitope of the antibody to the complement binding and activation domains of human MASP-2, chimeric antigens for animal immunization were designed. Coding sequences expressing mouse MASP-2 CUB1-EGF-CUB2 domain (residues 20-297 of SEQ ID NO:40) and human MASP-2 CCP1-CCP2-SP domain (residues 298-686 of SEQ ID NO:39) were synthesized. In addition, an IL-2 secretion signal peptide sequence (SEQ ID NO:41) was added at the N-terminus, and a FLAG tag sequence was added at the C-terminus. The above elements were combined into one open reading frame (ORF) to generate a chimeric antigen expression construct (SEQ ID NO:42). Other constructs expressing full-length human MASP-2 (SEQ ID NO:39), mouse MASP-2 (SEQ ID NO:40) and cynomolgus MASP-2 (SEQ ID NO:43) with a HIS tag at the C-terminus were synthetically generated. In addition, another construct expressing full-length human MASP-2 (sequence number 39) with a FLAG tag at the C-terminus was generated by PCR.

[0224] [000253] Human MASP-2 protein (SEQ ID NO:39) MRLLTLLGLLCGSVATPLGPKWPEPVFGRLASPGFPGEYANDQERRWTLTAPPGYRLRLYFTHFDLELSHLCEYDFVKLSSGAKVLATLCGQESTDTERAPGKDTFYSLGSSLDITFRSDYSNEKPFTGFEAFYAAEDIDECQVAPGEAPTCDHHCHNHLGGFYCSCRAGY VLHRNKRTCSALCSGQVFTQRSGELSSPEYPRPYPKLSSCTYSISLEEGFSVILDFVESFDVETHPETLCPYDFLKIQTDREEHGPFCGKTLPHRIETKSNTVTITFVTDESGDHTGWKIHYTSTAQPCPYPMAPPNGHVSPVQAKYILKDSFSIFCETGYELLQGHLPLKS FTAVCQKDGSWDRPMPACSIVDCGPPDDLPSGRVEYITGPGVTTYKAVIQYSCEETFYTMKVNDGKYVCEADGFWTSSKGEKSLPVCEPVCGLSARTTGGRIYGGQKAKPGDFPWQVLILGGTTAAGALLYDNWVLTAAHAVYEQKHDASALDIRMGTLKRLSPHYTQAWS EAVFIHEGYTHDAGFDNDIALIKLNNKVVINSNITPICLPRKEAESFMRTDDIGTASGWGLTQRGFLARNLMYVDIPIVDHQKCTAAYEKPPYPRGSVTANMLCAGLESGGKDSCRGDSGGALVFLDSETERWFVGGIVSWGSMNCGEAGQYGVYTKVINYIPWIENIISDF [000254] Mouse MASP-2 protein (SEQ ID NO: 40) MRLLIFLGLLWSLVATLLGSKWPEPVFGRLVSPGFPEKYADHQDRSWTLTAPPGYRLRLYFTHFDLELSYRCEYDFVKLSSGTKVLATLCGQESTDTEQAPGNDTFYSLGPSLKVTFHSDYSNEKPFTGFEAFYAAEDVDECRVSLGDSVPCDHYCHNYLGGYYCSCRAGY VLHQNKHTCSALCSQVFTGRSGYLSSPEYPQPYPKLSSCTYSIRLEDGFSVILDFVESFDVETHPEAQCPYDSLKIQTDKGEHGPFCGKTLPPRIETDSHKVTITFATDESGNHTGWKIHYTSTARPCPDPTAPPNGSISPVQAIYVLKDRFSVFCKTGFELLQGSVPLK SFTAVCQKDGSWDRPMPECSIIDCGPPDDLPNGHVDYITGPEVTTYKAVIQYSCEETFYTMSSNGKYVCEADGFWTSSKGEKLPPVCEPVCGLSTHTIGGRIVGGQPAKPGDFPWQVLLLGQTTAAAGALIHDNWVLTAAHAVYEKRMAASSLNIRMGILKRLSPHYTQAW PEEIFIHEGYTHGAGFDNDIALIKLKNKVTINGSIMPVCLPRKEAASLMRTDFTGTVAGWGLTQKGLLARNLMFVDIPIADHQKCTAVYEKLYPGVRVSANMLCAGLETGGKDSCRGDSGGALVFLDNETQRWFVGGIVSWGSINCGAADQYGVYTKVINYIPWIENIISNF [000255] IL-2 secretory signal peptide sequence (SEQ ID NO:41) MYRMQLLSCI ALSLALVTNS [000256] Chimeric MASP-2 protein (SEQ ID NO: 42) MYRMQLLSCIALSLALVTNSSKWPEPVFGRLVSPGFPEKYADHQDRSWTLTAPPGYRLRLYFTHFDLELSYRCEYDFVKLSSGTKVLATLCGQESTDTEQAPGNDTFYSLGPSLKVTFHSDYSNEKPFTGFEAFYAAEDVDECRVSLGDSVPCDHYCHNYLGGYYCSCRAGYV LHQNKHTCSALCSQVFTGRSGYLSSPEYPQPYPKLSSCTYSIRLEDGFSVILDFVESFDVETHPEAQCPYDSLKIQTDKGEHGPFCGKTLPPRIETDSHKVTITFATDESGNHTGWKIHYTSTAQPCPYPMAPPNGHVSPVQAKYILKDSFSIFCETGYELLQGHLPLKSFTA VCQKDGSWDRPMPACSIVDCGPPDDLPSGRVEYITGPGVTTYKAVIQYSCEETFYTMKVNDGKYVCEADGFWTSSKGEKSLPVCEPVCGLSARTTGGRIYGGQKAKPGDFPWQVLILGGTTAAGALLYDNWVLTAAHAVYEQKHDASALDIRMGTLKRLSPHYTQAWSEAVFIH EGYTHDAGFDNDIALIKLNNKVVINSNITPICLPRKEAESFMRTDDIGTASGWGLTQRGFLARNLMYVDIPIVDHQKCTAAYEKPPYPRGSVTANMLCAGLESGGKDSCRGDSGGALVFLDSETERWFVGGIVSWGSMNCGEAGQYGVYTKVINYIPWIENIISDFDYKDDDDK [000257] Cynomolgus MASP-2 protein (SEQ ID NO: 43) MRLLTLLGLLCGSVATPLGPKWPEPVFGRLASPGFPGEYANDQERRWTLTAPPGYRLRLYFTHFDLELSHLCEYDFVKLSSGAKVLATLCGHESTDTERAPGNDTFYSLGSSLDITFRSDYSNEKPFTGFEAFYAAEDIDECQVAPGEAPACDHHCHNHLGGFYCSCRVGY ILHRNKRTCSALCSGQVFTQRSGELSSPEYPQPYPKLSSCTYSIRLEEGFSVILDFVESFDVETHPETLCPYDFLKIQIDSEEHGPFCGKTLPRRIETKSNTVTITFVTDESGDHTGWKIHYTSTAQPCPYPMAPPNGHLSPVQAKYILKDSFSIFCEPGYELLQGHLPLKS FAAVCQKDGSWDQPMPSCSIVDCGPPDDLPSGRVEYITGPEVTTYKAVIQYSCEETFYTMKVNDGKYVCEADGFWTSSKGERSPPVCEPVCGLSARTTGGRIYGGQKAKPGDFPWQVLILGGSTAAGALLYDNWVLTAAHAIYEQKHDASSLDIRLGALKRLSPHYTQAWA EAVFIHEGYTHDAGFDNDIALIKLNNKVVINSNITPICLPRKEAESFMRTDDIGTASGWGLTQRGLLARNLMYVDIPIVDHQKCTAAYEKPPYSGGSVTANMLCAGLESGGKDSCRGDSGGALVFLDNETQRWFVGGIVSWGSMNCGEAGQYGVYTKVINYIPWIKNIISNF [000258] 2. Expression and purification of MASP-2 antigen [000259] The above MASP-2 expression constructs were transfected separately into ExpiCHO-s cells using ExpiFectamine CHO transfection kit. ExpiCHO-s cells were cultured in serum-free ExpiCHO Expression Medium. 14 days after transfection, the supernatant was collected. After centrifugation and filtration, the supernatant was loaded onto an anti-FLAG or anti-HIS column, and then purified with a GE AKTA purification system. After washing, the MASP-2 protein was eluted with citric acid (pH 3.5) for animal immunization.

[0225] Example 2: Generation of MASP-2 antibodies [000260] 1. Immunization and Hybridoma Fusion [000261] Different strains of mice or rats were immunized with DNA expressing the above chimeric MASP-2 antigen via the Helios gene gun system (Bio-Rad). The immunized animals were boosted with recombinant MASP-2 protein every two weeks. Four days after the last boost, the animals were sacrificed for hybridoma fusion. Spleen cells were isolated and fused with SP2-0 cells via electrofusion. The resulting hybridoma cells were cultured in hypoxanthine-aminopterin-thymidine-containing DMEM medium.

[0226] [000262] 2. Hybridoma screening [000263] After 10 days of culture, hybridoma supernatants were collected for antigen binding screening. Full-length human MASP-2 protein was coated at a concentration of 0.5 μg / ml in 100 μl per well of ELISA plates. After blocking with PBS containing 1% BSA + 1% normal goat serum + 0.05% Tween® 20, hybridoma supernatants were added to the plates for 1 hour. Specific binding of hybridoma antibodies to human MASP-2 was detected using horseradish peroxidase (HRP)-conjugated anti-mouse antibodies. ELISA-binding positive clones were selected and proceeded to further activity screening.

[0227] [000264] 3. Activity Screening [000265] ELISA plates were coated with 100 μl of mannan 10 μg / ml per well overnight at 4° C. After washing three times with PBS+0.1% Tween® 20, plates were blocked for 1 hour with blocking buffer (10 mM Tris-HCl+0.1% human serum albumin+140 mM NaCl). 50 μl of hybridoma supernatant was added to assay buffer (0.1% human serum albumin+20 mM Tris-HCl+2 mM CaCl 2 +140mM NaCl + 1mM MgCl2 +0.05% Tween® 20) and then incubated on ice for 45 minutes. The blocking buffer was removed from the mannan-coated plate, and the mixture of supernatant and serum was added. The plate was incubated at 37°C for 1.5 hours. After washing three times with washing buffer, C4 activation was monitored by measuring C4b deposition. The deposited C4b was detected by HRP-conjugated anti-C4c antibody (Quidel-A211). If the activity of MASP-2 is inhibited by the antibody, less C4b is deposited on the bottom of the plate. By using this method, hybridoma antibodies with MASP-2 inhibitory activity were selected.

[0228] [000266] Positive antibodies were then subjected to subcloning and rescreened using both ELISA binding and C4 activation assays.

[0229] Example 3: Characterization of the inhibitory activity of anti-human MASP-2 antibodies.

[0230] [000267] 1. Purification of hybridoma antibodies [000268] After subcloning, positive hybridoma clones with neutralizing activity were grown in 10 cm dishes. After centrifugation and filtration, the antibody-containing supernatant was loaded onto a Protein A column and purified on a GE AKTA purification system. After washing, the antibody was eluted with citric acid (pH 3.5).

[0231] [000269] 2. Activity evaluation of purified antibodies [000270] MASP-2 is a key component of the lectin pathway, cleaving complement factors C4 and C2 to generate the C3 convertase C4bC2a. Activation of C3 ultimately leads to the formation of the membrane attack complex (MAC). To test whether an antibody that inhibits MASP-2 could reduce activation of the lectin pathway, activation of C4, C3, and MAC was evaluated in the presence of purified MASP-2 antibody.

[0232] [000271] ELISA plates were coated with 100 μl of mannan 10 μg / ml per well overnight at 4° C. After washing three times with PBS+0.1% Tween® 20, plates were blocked for 1 hour with blocking buffer (10 mM Tris-HCl+0.1% human serum albumin+140 mM NaCl). Antibodies were added in assay buffer (0.1% human serum albumin+20 mM Tris-HCl+2 mM CaCl) containing 1% human serum (Quidel, A113). 2 +140mM NaCl + 1mM MgCl 2 +0.05% Tween® 20) and incubated on ice for 45 minutes. The blocking buffer was removed from the mannan-coated plate and the antibody-sera mixture was added. The plate was incubated at 37°C for 1.5 hours. The activated complement components should deposit on the bottom of the plate, while the inactivated components remain dissolved in the buffer. After washing three times with washing buffer, the activation of complement components was monitored by HRP-conjugated complement antibodies, including anti-C3c antibody (Quidel-A205), anti-C4c antibody (Quidel-A211) and anti-MAC (SC5b-9) antibody (Quidel-A239). The detection antibodies were linked to our own HRP. MASP-2 antibodies purified from hybridoma cells blocked the activation of complement C3 (see Figure 1), complement C4 (see Figure 2) and MAC (see Figure 3) in a dose-dependent manner.

[0233] Example 4: Cloning of V genes and generation of chimeric antibodies [000272] 1. Hybridoma antibody V gene cloning and sequencing [000273] Lead antibodies with desired profiles were selected for V gene cloning. The sequences of mouse anti-human MASP-2 light and heavy chain variable regions were obtained by polymerase chain reaction (PCR) amplification technique. Total RNA was isolated from positive hybridoma cells by using MiniBest Universal RNA Extraction Kit (TaKaRa), and cDNA was synthesized by using 1st Strand cDNA Synthesis Kit (TaKaRa) with Oligo(dT) primer. The variable regions of mouse IgG genes were amplified by PCR by using different isotype primers for heavy chain variable region and kappa chain primers for light chain variable region. The PCR products were subcloned into TA cloning vector. For each variable gene construct, more than 10 single colonies were used for DNA sequencing by Synbio Technologies (Suzhou, China). The amino acid sequences of VH and Vκ were derived from the results of DNA sequencing.

[0234] [000274] 2. Construction of chimeric antibodies [000275] Three antibodies with different sequences, including 129C10, 160D10 and 125D5, were selected as lead antibodies to generate chimeric antibodies, and their sequences are listed in Table 1 and Table 2. After sequence analysis and confirmation, the cDNAs of the variable regions of heavy and light chains were synthesized and fused with the sequences of the constant regions of human IgG4 and human kappa. To enhance antibody secretion, signal peptide sequences were added at the N-terminus of heavy and light chains, respectively. The resulting chimeric antibody genes were cloned into expression vectors. Large-scale DNA was prepared by using the Plasmid Maxi-prep System from Qiagen.

[0235] [000276] 3. Expression and purification of chimeric antibodies [000277] Co-transfection of heavy and light chains was performed using ExpiFectamine™ CHO Reagent from Invitrogen according to the manufacturer's protocol. 5-6×10 6 ExpiCHO-S cells in ExpiCHO Expression Medium at 1000 cells / ml were transfected with equal amounts of heavy and light chain vector DNA at a final concentration of 0.8 μg / ml by using ExpiFectamine™ CHO Reagent. Plasmid DNA or ExpiFectamine™ CHO Reagent was diluted in cold OptiPRO™ medium and then mixed by swirling and / or inversion of the tube. The ExpiFectamine™ CHO / plasmid DNA mixture was incubated at room temperature for 1-5 minutes and then gently transferred to the shake flask containing the cells. The transfected cells were incubated at 37 °C for 1 h at 25 °C for 3 min at 37 °C for 1 h at 25 °C for 1 h at 25 °C for 3 h at 25 ... 2 The cells were incubated at 37°C in a humidified atmosphere at 4°C for 18-22 hours after transfection, and the conditioned medium was harvested on day 10. The supernatant was centrifuged at 4,000 rpm for 20 minutes and then filtered through a 0.22 μm filtration capsule to remove cell debris. The filtered supernatant was loaded onto a pre-equilibrated Protein A-affinity column. The Protein A resin was washed with equilibration buffer (PBS) and then the antibody was eluted using 25 mM citrate (pH 3.5). The purified antibody solution was adjusted to pH 6.0-7.0 by using 1 M Tris base (pH 9.0). Endotoxin was controlled to less than 1 EU / mg. Finally, the purified antibody was characterized by SDS-PAGE.

[0236] Example 5: Generation and characterization of humanized antibodies [000278] 1. Generation, expression and purification of humanized antibodies [000279] The sequences of the variable domains of the murine antibody 129C10 were used to identify the germline sequences with the highest homology to each of the murine frameworks. Computer modeling was used to design humanized variants with CDR grafting and back mutations.

[0237] [000280]129C10 [000281] Human germline framework sequences VH / 1-2 for the heavy chain and VK / 2-30 for the light chain, respectively, were used for CDR grafting.

[0238] [000282] Heavy chain (HC) variants 1, 2, 3, and 4 were obtained by directly grafting three CDRs into the germline sequence (SEQ ID NO:37) with the following mutations: R71V, A93T for HC variant 1 (SEQ ID NO:20), R71V, A93T, V67A, M69L for HC variant 2 (SEQ ID NO:22), R71V, A93T, A65G, K64Q, E61Q for HC variant 3 (SEQ ID NO:24), and R71V, A93T, V67A, M69L, A65G, K64Q, E61Q for HC variant 4 (SEQ ID NO:26). Note that there are three mutations (A65G, K64Q, E61Q) for HC variants 3 and 4 introduced into HC CDR2 to further improve the humanity of the antibody.

[0239] [000283] 129C10 HC germline sequence: [000284]VH / 1~2 (129C10-HC germline, SEQ ID NO: 37): QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCAR [000285] VH / 1-2 variant 1 (Hu129C10_Ha, SEQ ID NO: 20): QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYINWVRQAPGQGLEWMGIFPGSESAYHSEKFKARVTMTVDTSISTAYMELSRLRSDDTAVYYCTRGDRSGPFAYWGQGTLVTVSS [000286] VH / 1-2 variant 2 (Hu129C10_Hb, SEQ ID NO: 22): QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYINWVRQAPGQGLEWMGIFPGSESAYHSEKFKARATLTVDTSISTAYMELSRLRSDDTAVYYCTRGDRSGPFAYWGQGTLVTVSS [000287] VH / 1-2 variant 3 (Hu129C10_Hc, SEQ ID NO: 24): QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYINWVRQAPGQGLEWMGWIFPGSESAYHSQKFQGRVTMTVDTSISTAYMELSRLRSDDTAVYYCTRGDRSGPFAYWGQGTLVTVSS [000288] VH / 1-2 variant 4 (Hu129C10_Hd, SEQ ID NO: 26): QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYINWVRQAPGQGLEWMGWIFPGSESAYHSQKFQGRATLTVDTSISTAYMELSRLRSDDTAVYYCTRGDRSGPFAYWGQGTLVTVSS [000289] Light chain (LC) variants 1 and 2 were obtained by directly grafting the three CDRs into the germline sequence (SEQ ID NO: 38) with the addition of F36L backmutations for LC variant 1 (SEQ ID NO: 28) and F36L, T69A backmutations for LC variant 2 (SEQ ID NO: 30), respectively.

[0240] [000290] 129C10 LC germline sequence [000291] VK / 2-30 (129C10-LC-germline, SEQ ID NO:38) DVVMTQSPLSLPVTLGQPASISCRSSQSLVYSDGNTYLNWFQQRPGQSPRRLIYKVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGTHWP [000292] VK / 2-30 variant 1 (Hu129C10_La, SEQ ID NO: 28) DVVMTQSPLSLPVTLGQPASISCKSSQSLLYSNGKTYLNWLQQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCVQVTHFPFTFGQGTKLEIK [000293] VK / 2-30 variant 2 (Hu129C10_Lb, SEQ ID NO: 30) DVVMTQSPLSLPVTLGQPASISCKSSQSLLYSNGKTYLNWLQQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGADFTLKISRVEAEDVGVYYCVQVTHFPFTFGQGTKLEIK [000294] The cDNAs of the variable regions of the above heavy and light chains were synthesized and then fused with the sequences of the constant regions of human IgG4 and human kappa. The resulting antibody gene sequences were cloned into expression vectors. Large-scale DNA was prepared by using the Plasmid Maxiprep System from Qiagen, and cell transfection was performed using ExpiFectamine™ CHO Reagent from Invitrogen according to the manufacturer's protocol. The supernatant was harvested when the cell viability was above 60% and filtered through a 0.22 μm filtration capsule to remove cell debris. The filtered supernatant was then loaded onto a pre-equilibrated Protein A-affinity column. The Protein A resin was washed with equilibration buffer (PBS), and then the antibody was eluted using 25 mM citrate (pH 3.5). The purified antibody solution was adjusted to pH 6.0-7.0 by using 1 M Tris base (pH 9.0). Endotoxin was controlled to less than 1 EU / mg. Finally, the purified antibodies were characterized by SDS-PAGE.

[0241] [000295] Benchmark antibodies OMS721-analogue and 129C10-hu-YTE (Hu129C10_HaLa-hIgG4 with amino acid substitutions M252Y / S254T / T256E [YTE], see Tables 2-3) were also constructed and expression and purification procedures were the same as above.

[0242] Example 6: Blocking activity of chimeric and humanized MASP-2 antibodies [000296] Purified antibodies were serially diluted from 100 μg / ml to obtain gradient concentrations. Complement C3 activation assay was used to evaluate MASP-2 antibody blocking activity as described in Example 3. 100 μl of mannan 10 μg / ml per well was coated onto ELISA plates overnight at 4° C. Antibodies were incubated with 1% human serum (Quidel, A113) on ice for 45 minutes. After washing and blocking the plates, the antibody and serum mixture was added and incubated at 37° C. for 90 minutes. After washing, the deposited activated C3 was detected with HRP-conjugated anti-C3c antibody (Quidel-A205). Figure 4 shows the blocking activity of chimeric and humanized antibodies against complement C3 activation. Because the humanized 129C10 variant, 129C10 HaLa, showed the highest affinity and C3 blocking activity, 129C10 HaLa was selected as the lead antibody for further in vitro and in vivo studies and was designated 129C10-hu.

[0243] Example 7: Comparison of complement C4 and MAC blocking activity between lead antibody 129C10-hu and benchmark antibody OMS721-analogue [000297] 1. Comparison of activity in blocking complement C4 and MAC activation [000298] The lead MASP-2 antibody 129C10-hu was compared with OMS721-analogue in their blocking activity on complement C4 and MAC activation in 2% human serum using the assay described in Example 3 with minor modifications. 100 μl of mannan 10 μg / ml per well was coated onto ELISA plates overnight at 4° C. The antibodies were incubated with 2% human serum (Quidel, A113) on ice for 45 minutes. After washing and blocking the plates, the antibody and serum mixture was added and incubated at 37° C. for 90 minutes. After washing, the deposited activated C4 was detected with HRP-conjugated anti-C4c antibody (Quidel-A211). In the case of MAC activation, MAC activation was detected with HRP-conjugated anti-MAC (SC5b-9) antibody (Quidel-A239). As shown in Figures 5 and 6, the results demonstrated that 129C10-hu was approximately 10-fold more potent than the OMS721-analogue in blocking activation of complement C4 (IC50: 0.11 μg / mL vs. 1.70 μg / mL) and MAC (IC50: 0.27 μg / mL vs. 1.97 μg / mL).

[0244] [000299] 2. Comparison of activity at different concentrations of serum [000300] Different concentrations of human serum (1%, 10%, and 50%) were used in the complement C3 activation assay. For 1% and 10% human serum, the assay method was the same as described in Example 3. For 50% human serum, mannan was coated at a concentration of 1 μg / ml in 100 μl per well overnight at 4° C. After washing three times with PBS+0.1% Tween® 20, the plates were blocked with blocking buffer (10 mM Tris-HCl+0.1% human serum albumin+140 mM NaCl) for 1 hour. Antibodies were added in assay buffer (0.1% human serum albumin+20 mM Tris-HCl+2 mM CaCl) containing 50% human serum (Quidel, A113). 2 +140mM NaCl + 1mM MgCl 2+0.05% Tween® 20) and then incubated on ice for 45 min. Blocking buffer was removed from the mannan-coated plate and the antibody-sera mixture was added. The plate was incubated at 37°C for 30 min. After washing three times, activated C3 was detected by HRP-conjugated anti-C3c antibody (Quidel-A205). As shown in Figure 7, 129C10-hu and OMS721-analogue had similar inhibitory efficacy against C3 activation in 1% serum (IC50: 0.08 μg / mL vs. 0.10 μg / mL). Interestingly, 129C10-hu demonstrated 3-fold greater potency than OMS721-analogue at high concentrations of 10% serum (IC50: 0.20 μg / mL vs. 0.69 μg / mL) (see Figure 8). More importantly, at 50% serum concentration, 129C10-hu maintained its blocking activity against C3 activation with an IC50 of 0.05 μg / mL, while the OMS721-analogue lost its activity (see FIG. 9). With regard to blocking C4 activation, 129C10-hu also showed to be 2-fold more potent than the OMS721-analogue in 10% human serum (IC50: 0.69 μg / mL vs. 1.59 μg / mL) (see FIG. 10).

[0245] Example 8: Binding affinity of MASP-2 antibodies to human MASP-2 by biolayer interference (ForteBio) [000301] The antibody to be tested was diluted to a concentration of 100 nM in ForteBio kinetic buffer (PBS pH 7.4, 0.1% BSA + 0.002% Tween®-20). Human MASP-2 protein was diluted in kinetic buffer to obtain three concentration gradients of 100 nM, 50 nM, and 25 nM. 0 nM was used as a reference control. The antibody was immobilized on a Protein A biosensor. The baseline was detected for 60 seconds, and then the association of the antibody with MASP-2 was detected for 180 seconds to obtain the K on Factor data was obtained. The protein was then dissociated in kinetic buffer for 180 seconds and the K offFactor data was obtained. Regeneration of the biosensor was with 10 mM glycine buffer, pH 2.0. All kinetic data was collected at 30°C. Data was acquired using a ForteBio Octet RED96 and analyzed using Octet Data Analysis software. As shown in Table 3, all MASP-2 hybridoma antibodies tested exhibited a 10 -10 M to 10 -8 After humanization, the lead antibody 129C10-hu (129C10-HaLa) had high binding affinity to human MASP-2 with K values ​​ranging from 0.01 to 0.01 M. on The value 3.53E+4 and K off With values ​​<1.0E-7 (see Figure 11), <10 -12 It maintained the highest binding affinity to hMASP-2 with a KD value of M (reaching the detection limit of ForteBio) (Table 4).

[0246] [Table 3]

[0247] [Table 4]

[0248] Example 9: Binding specificity of 129C10-hu by ELISA [000302] Human C1s / C1r, MASP-1 / 3 were purchased from R&D, Cusbio, etc. Recombinant human complement components C1s / C1r, MASP-1 or MASP-3 (1 mg / ml) were coated overnight at 4°C; washed 3 times with washing buffer; blocking buffer (200 μL / well) was added for 1 h at 4°C; washed 3 times; serially diluted Ab was added for 1 h at RT; washed 3 times; mouse anti-human IgG4 HRP (1:20000) was added for 1 h at RT; washed 3 times; and detected with tetramethylbenzidine (TMB) at OD450 nm for 40 min. The EC50 of 129C10-Hu and OMS721-analog binding to C1s, C1r, MASP1, MASP2 or MASP3, respectively, are shown in Figures 12A-12E. The results showed that 129C10-Hu bound only to human MASP2, but not to human C1s, C1r, MASP1 or MASP3.

[0249] Example 10: Cross-reactivity of 129C10-hu by ELISA [000303] Rat / mouse MASP-2 was ordered from Cusbio. Human / cynomolgus MASP-2 was produced in-house. ELISA assay was performed as follows: Different species MASP2 (1 μg / ml) was coated overnight at 4° C.; washed 3 times with washing buffer; blocking buffer (200 μL / well) was added for 2 h at RT; washed 3 times; serially diluted 129C10-hu or OMS721-analogue was added for 1 h at RT as control; washed 3 times; mouse anti-human IgG4 Fc HRP (1:20000) was added for 1 h at RT; washed 3 times; detected with TMB at OD450 nm for 2 min. The EC50 of binding of 129C10-Hu and OMS721-analogue to different species MASP-2 is shown in FIG. 13A and FIG. 13B, respectively.

[0250] Example 11: Cross-reactivity of 129C10-hu to cynomolgus monkey MASP-2 using a C4 activation assay in cynomolgus monkey serum [000304] The assay method was the same as described in Example 3, except that cynomolgus serum was used. 100 μl of mannan 10 μg / ml per well was coated onto ELISA plates overnight at 4° C. Antibodies were incubated with 1% cynomolgus serum on ice for 45 min. After washing and blocking the plates, the antibody and serum mixture was added and then incubated at 37° C. for 90 min. After washing, the deposited activated C4 was detected with HRP-conjugated anti-C4c antibody (Quidel-A211). As the results shown in FIG. 14, 129C10 had an IC of 0.4973 μg / ml. 50 blocked cynomolgus C4 activation, suggesting that 129C10 was able to bind to and block the activity of cynomolgus MASP-2.

[0251] Example 12: Selectivity of 129C10 in blocking activation of the MB-lectin complement pathway [000305] There are three pathways that initiate complement activation: the classical pathway, the MB-lectin (MBL) pathway, and the alternative pathway. These pathways depend on different molecules for their initiation, but converge to generate the same set of effector molecules, such as the membrane attack complex (MAC). All three pathways are important parts of innate immunity and play different roles in defending against different infections (Noris M, et al. 2013.JM.). We next tested the selectivity of blocking the MBL pathway by 129C10-hu.

[0252] [000306] The selectivity of the lead antibody 129C10-hu was determined using the Wieslab Complement System Screening Kit (IBL America, Cat. No. COMPL 300 RUO). Plates were pre-coated with mannan as an initiator of the MBL pathway, IgM as an initiator of the classical pathway, and LPS as an initiator of the alternative pathway. 129C10-hu was serially diluted in assay buffer containing human serum (Quidel, A113) and then incubated on ice for 45 min. The antibody and serum mixture was added to the plate and incubated at 37°C for 60 min. After washing, the deposited MAC was detected with AP-conjugated anti-C5b-9 antibody. EDTA was used as a positive control to block complement activation. As shown in Figure 15, 129C10-hu only blocked complement activation initiated by the MBL pathway, but did not block the other two complement pathways, suggesting that the 129C10-hu antibody selectively blocked complement activation of the MBL pathway.

[0253] Example 13: Modification of 129C10-hu of the present disclosure to extend half-life by introducing a YTE mutation into the Fc [000307] Dall'Acqua WF et al. reported that the introduction of triple mutation M252Y / S254T / T256E (YTE) into the Fc portion of IgG can enhance its binding affinity to FcRn and extend its half-life in vivo (Dall'Acqua WF et al. 2006.JBC). Motavizumab-YTE, the first YTE-mutated IgG in humans, was demonstrated to be well tolerated in a phase I clinical trial and exhibited an extended half-life (Robbie, GJ, et al. 2013.AAC).

[0254] [000308] We introduced this M252Y / S254T / T256E (YTE) mutation into 129C10-hu to generate 129C10-hu-YTE. Its binding affinity to FcRn was evaluated using Biolayer Interference (ForteBio). 129C10-hu or 129C10-hu-YTE was diluted in ForteBio kinetic buffer (PBS pH 7.4, 0.1% BSA + 0.002% Tween®-20) to a series of concentrations of 500 nM, 167 nM, 56 nM, 19 nM and 0 nM. Human FcRn (FCGRT&B2M) protein with His tag was diluted in kinetic buffer to a concentration gradient of 100 nM. FcRn protein was immobilized on Ni-NTA biosensor. The association and dissociation kinetics were recorded and analyzed. As shown in Figures 16A and 16B and Table 5, the YTE mutation improved the binding affinity of 129C10-hu to human FcRn by 3-fold.

[0255] [Table 5]

[0256] Example 14: Pharmacokinetic (PK) / Pharmacodynamic (PD) Study of MASP-2 Antibodies 129C10-hu and 129C10-hu-YTE in Cynomolgus Monkeys [000309] Two cynomolgus monkeys per group were administered 10 mg / kg of 129C10-hu, 129C10-hu-YTE or OMS721-analog intravenously. Serum samples were collected at 0, 0.5, 2, 8, 24, 48, 72, 96, 168, 336, 504, 672, and 840 hours after injection. Serum was tested for antibody concentration and efficacy of lectin pathway activation.

[0257] [000310] Serum concentrations were measured by the developed ELISA method with a detection range of 0.625-40ng / mL. Microplate wells were precoated with human IgG-specific anti-IgG antibody [R10z8e6]. After blocking, standard samples (STD), quality control (QC) samples, matrix blank samples, and test samples were added. After washing, biotin mouse anti-human IgG4 was added to the microplate wells, followed by the addition of streptavidin labeled with HRP. TMB was added to the microplate wells. Conversion of OD values ​​of QC and test samples to concentrations was performed by comparison with a simultaneous analysis standard curve, regressed according to a four-parameter logistic model.

[0258] [000311] For lectin pathway activation potency testing, 10 μg / ml of mannan was coated onto ELISA plates. Cynomolgus serum samples were diluted to 2% with C4 activation buffer. After washing and blocking the plates, the diluted serum was added and incubated at 37° C. for 90 minutes. After washing, the deposited activated C4 was detected with HRP-conjugated anti-C4c antibody (Quidel-A211).

[0259] [000312] As shown in the PK results in Figure 17 and Table 6, the half-life of 129C10-hu in cynomolgus monkeys is 164.77 hours, which is longer than that of OMS721-analogue (130.152 hours). The YTE mutation extended the half-life of 129C10-hu to 274.404 hours. As shown in the PD results in Figure 18, the lectin pathway activation potency was inhibited to basal levels 0.5 hours after antibody administration. The inhibitory effect lasted for 2 weeks in the OMS721-analogue group, 3 weeks in the 129C10-hu group, and approximately 4 weeks in the 129C10-hu-YTE group.

[0260] [000313]

[0261] [Table 6]

[0262] [000314] In another separate study, male and female cynomolgus monkeys were assigned to five groups, two males and two females per group, and dosed with 129C10-hu 0 (vehicle control), 15, 20, and 295.8 mg / kg via subcutaneous injection or 129C10-hu 15 mg / kg via intravenous injection in a volume of 3 mL / kg for four weeks (up to five doses). The vehicle control was the drug formulation buffer.

[0263] [000315] Blood samples were collected before dosing and approximately 0.083, 2, 6, 24, 48, 96 and 168 hours after the first dose (see FIG. 19) and the fourth dose (see FIG. 20), respectively. Complement 4c (C4c) in serum was analyzed by enzyme-linked immunosorbent assay (ELISA). Briefly, mannan (Sigma) was diluted to 10 μg / ml in coating buffer. 100 μl of mannan working solution was added to each well of a 96-well plate. The plate was incubated overnight at 4° C. The plate was washed three times, then 200 μl of blocking buffer was added and incubated at room temperature for 1 hour. Monkey serum was diluted to 2% in C4 activation buffer. 100 μl of diluted serum was added to the 96-well plate and incubated at 37° C. for 70 minutes. The plate was washed three times with washing buffer. 100 μl of HRP-conjugated anti-C4c antibody (diluted 1:5000 in block buffer) was added and incubated for 1 h at room temperature. The plate was washed three times with wash buffer. 100 μl of TMB substrate solution was added to each well of the 96-well plate and incubated for 5-10 min at room temperature. 50 μl of stop solution was added to each well of the plate. The plate was read at 450 nm. Optical density (OD) values ​​at 450 nm (OD450) were expressed as individual values ​​and the average value for each time point for all animals was calculated.

[0264] [000316] A dose-dependent decrease in serum C4c was observed at ≥15 mg / kg after the first and fourth doses, with the decrease occurring 2 hours after dosing in animals dosed via SC and 0.083 hours after dosing in animals dosed via IV, and the effect persisted throughout dosing across the three dose levels by SC or IV with the greatest effect occurring 24-96 hours after the first dose, with maximum mean decreases of 88.4% in males and 92.8% in females recorded at 295.8 mg / kg SC compared to baseline values. In conclusion, 129C10-hu exerted a significant dose-dependent decreasing effect on serum C4c in monkeys after weekly SC or IV administration at 15, 60, or 295.8 mg / kg for five doses, suggesting that serum C4c could be a potential pharmacodynamic marker.

Claims

**Claim 1** An isolated antibody or antigen-binding fragment thereof that specifically binds to MASP-2, comprising: a) A heavy-chain CDR1 comprising the amino acid sequence of DYYIN (SEQ ID NO: 1), WIFPGSX １ SX ２ YX ３ X ４ X ５ X ６ FX ７ X ８ a heavy-chain CDR2 comprising the amino acid sequence of (SEQ ID NO: 2), and GDRSGPFX ９ a heavy-chain CDR3 comprising the amino acid sequence of Y (SEQ ID NO: 3); and / or b) A light chain CDR1 comprising the amino acid sequence of KSSQSLlysnGKTYLN (SEQ ID NO: 4), a light chain CDR2 comprising the amino acid sequence of LVSKLDs (SEQ ID NO: 5), and VQX １０ A light chain CDR3 comprising the amino acid sequence of THFPFT (SEQ ID NO: 6) comprising; Here, X １ is E, D, or G, and X ２ is A or P, and X ３ is H or Y, and X ４ is S or N, and X ５ is E or Q, and X ６ is K or N, and X ７ is K or Q, and X ８ is A or G, and X ９ is A or P, and X １０ is an isolated antibody or an antigen-binding fragment thereof that is V or G. **Claim 2** a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1, and / or b) a heavy chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, and / or c) a heavy chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 11 and SEQ ID NO: 12, and / or d) a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4, and / or e) a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and / or f) a light chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 13 and SEQ ID NO: 14 The antibody or antigen-binding fragment thereof according to claim 1, comprising. **Claim 3** a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 11, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 13; or b) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 12, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 13; or c) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 11, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 14; or d) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 8, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 11, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 13 The antibody or antigen-binding fragment thereof according to claim 1, comprising. **Claim 4** a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 15 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 16; b) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 17 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 16; c) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 18 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 19; d) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 20 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 28; e) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 20 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 30; f) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 22 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 28; g) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 22 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 30; h) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 24 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 28; i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 24 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 30; j) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 26 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 28; or k) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 26 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 30 The antibody or antigen-binding fragment thereof according to claim 1, comprising.

5. The antibody or antigen-binding fragment thereof according to claim 1, further comprising an immunoglobulin constant region, optionally comprising a heavy chain constant region and / or a light chain constant region of IgG.

6. The constant region comprises a murine constant region, a rabbit constant region, or a human constant region, and optionally, the constant region comprises a constant region of human IgG1, IgG2, IgG3, or IgG4, or The heavy chain constant region comprises one or more amino acid substitutions at amino acid residues 252, 254, or 256 compared to the wild-type human IgG constant region, Optionally, the amino acid substitution at amino acid residue 252 is a substitution with tyrosine, the amino acid substitution at amino acid residue 254 is a substitution with threonine, and the amino acid substitution at amino acid residue 256 is a substitution with glutamic acid. The antibody or antigen-binding fragment thereof according to claim 5.

7. a monoclonal antibody, bispecific antibody, multispecific antibody, recombinant antibody, chimeric antibody, humanized antibody, labeled antibody, bivalent antibody, anti-idiotype antibody, fusion protein, dimerized or polymerized antibody, or modified antibody (e.g., glycosylated antibody), or a diabody, Fab, Fab’, F(ab’)2, Fd, Fv fragment, disulfide-stabilized Fv fragment (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv’), disulfide-stabilized diabody (ds diabody), single-chain antibody molecule (scFv), scFv dimer (bivalent diabody), multispecific antibody, camelized single-domain antibody, nanobody, domain antibody, or bivalent domain antibody, The antibody or antigen-binding fragment thereof according to claim 1.

8. The antibody or antigen-binding fragment thereof according to claim 1, which specifically binds to MASP-2 and has no detectable cross-reactivity with C1s, C1r, MASP1 or MASP3.

9. The antibody or antigen-binding fragment thereof according to claim 1, which is linked to one or more conjugate moieties, and the conjugate moiety contains a clearance modifier, chemotherapeutic agent, toxin, radioisotope, lanthanide, luminescent label, fluorescent label, enzyme substrate label, or therapeutic agent.

10. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to claim 1 and a pharmaceutically acceptable carrier.

11. An isolated polynucleotide encoding the antibody or antigen-binding fragment thereof according to claim 1.

12. A vector comprising the isolated polynucleotide according to claim 11.

13. A host cell comprising the vector according to claim 12.

14. A method for expressing the antibody or antigen-binding fragment thereof according to claim 1, comprising culturing a host cell comprising a vector containing the polynucleotide under conditions in which the isolated polynucleotide encoding the antibody or antigen-binding fragment thereof according to claim 1 is expressed.

15. A method of inhibiting MASP-2-dependent complement activation in a subject in need thereof, treating a disease or condition in a subject that would benefit from inhibition of MASP-2-dependent complement activation, reducing the level of serum C4 in a subject, treating a disease or condition in a subject that would benefit from a decrease in serum C4 level, or treating or preventing a condition or disease associated with an abnormal serum C4 level, the method comprising administering to the subject a therapeutically effective amount of the antibody or antigen-binding fragment thereof according to claim 1 or a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof and a pharmaceutically acceptable carrier.

16. The method according to claim 15, wherein the disease or condition is an autoimmune disease, a vascular condition, ischemia-reperfusion injury, atherosclerosis, inflammation, a pulmonary condition, an extracorporeal reperfusion procedure, a skeletal muscle condition, a renal condition, a skin condition, an organ or tissue transplantation procedure, a neurological disorder or injury, a blood disorder, a urogenital condition, a complication associated with non-obese diabetes or type 1 or type 2 diabetes, cancer, an endocrine disorder, or an ophthalmic condition.

17. The autoimmune diseases include thrombotic microangiopathy (TMA), atypical hemolytic uremic syndrome (aHUS), hematopoietic transplantation-associated thrombotic microangiopathy (TA-TMA), lupus nephritis, systemic lupus erythematosus (SLE), and IgA nephropathy, The vascular conditions include cardiovascular conditions, cerebrovascular conditions, peripheral (e.g., musculoskeletal) vascular conditions, renal vascular conditions, mesenteric / intestinal vascular conditions, angiogenesis to grafts and / or re-grafts, vasculitis, Henoch-Schönlein purpura nephritis, systemic lupus erythematosus-related vasculitis, vasculitis associated with rheumatoid arthritis, immune complex vasculitis, Takayasu disease, dilated cardiomyopathy, diabetic angiopathy, Kawasaki disease (arteritis), venous gas embolism (VGE), and restenosis after stent placement, rotational atherectomy, and percutaneous transluminal coronary angioplasty (PTCA). The ischemia-reperfusion injuries include ischemia-reperfusion injuries associated with aortic aneurysm repair, cardiopulmonary bypass, organ transplantation and / or vascular reanastomosis together with limb / digit re-transplantation, stroke, myocardial infarction, and hemodynamic resuscitation after shock and / or surgical procedures. The inflammation includes inflammatory gastrointestinal disorders including pancreatitis, Crohn's disease, ulcerative colitis, irritable bowel syndrome, and diverticulitis. The state of the lung includes acute respiratory distress syndrome, transfusion-related acute lung injury, ischemia / reperfusion acute lung injury, chronic obstructive pulmonary disease, asthma, Wegener's granulomatosis, anti-glomerular basement membrane disease (Goodpasture's disease), meconium aspiration syndrome, bronchiolitis obliterans syndrome, idiopathic pulmonary fibrosis, acute lung injury secondary to burns, non-cardiogenic pulmonary edema, transfusion-related respiratory depression, emphysema, cystic fibrosis, states related to SARS-CoV, MERS-CoV and SARS-CoV-2 (COVID-19), The extracorporeal reperfusion treatment includes hemodialysis, plasmapheresis, leukapheresis, extracorporeal membrane oxygenation (ECMO), heparin-induced extracorporeal membrane oxygenation LDL precipitation method (HELP) and cardiopulmonary bypass (CPB), The skeletal muscle state includes osteoarthritis, rheumatoid arthritis, juvenile rheumatoid arthritis, gout, neuropathic arthropathy, psoriatic arthritis, spondyloarthritis, crystalline arthropathy and systemic lupus erythematosus (SLE), The renal state includes mesangial proliferative glomerulonephritis, membranous glomerulonephritis, membranoproliferative glomerulonephritis (mesangiocapillary glomerulonephritis), acute post-infectious glomerulonephritis (post-streptococcal glomerulonephritis), cryoglobulinemic glomerulonephritis, lupus nephritis, Henoch-Schönlein purpura nephritis and IgA nephropathy, The skin state includes psoriasis, autoimmune blistering skin diseases, eosinophilic spongiosis, bullous pemphigoid, acquired epidermolysis bullosa (EBA), herpes gestationis, thermal burns and chemical burns, The organ or tissue transplantation procedure includes organ allotransplantation, organ xenotransplantation and tissue grafts, The disorders or injuries of the nervous system include multiple sclerosis, myasthenia gravis, Huntington's disease, amyotrophic lateral sclerosis, Guillain-Barré syndrome, reperfusion after stroke, degenerative disc, brain trauma, Parkinson's disease, Alzheimer's disease, Miller-Fisher syndrome, brain trauma and / or cerebral hemorrhage, demyelination and meningitis, The blood disorders include sepsis, severe sepsis, septic shock, acute respiratory distress syndrome caused by sepsis, systemic inflammatory response syndrome, hemorrhagic shock, hemolytic anemia, autoimmune thrombotic thrombocytopenic purpura and hemolytic uremic syndrome, The urogenital state includes painful bladder disorders, sensory bladder disorders, non-bacterial chronic cystitis, interstitial cystitis, infertility, placental insufficiency and miscarriage and preeclampsia, The endocrine disorders include Hashimoto's thyroiditis, stress, anxiety, and hormonal disorders involving the controlled release of prolactin, growth factors or other insulin-like growth factors, and adrenocorticotropic hormone from the pituitary gland, The method according to claim 16, wherein the ophthalmic condition includes age-related macular degeneration.

18. The method according to claim 15, further comprising administration of a second therapeutic agent.

19. The method according to claim 15, wherein the subject is a human.

20. The method according to claim 15, wherein the administration is via oral, nasal, intravenous, subcutaneous, sublingual, or intramuscular administration.

21. A kit comprising the antibody according to claim 1 or an antigen-binding fragment thereof.

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