Therapeutic antibodies that bind to the serine protease domain of MASP-2 and uses thereof

Abstract

Provided is an isolated monoclonal antibody and its antigen-binding fragment that specifically binds to an epitope in the serine protease domain of human MASP-2. In some embodiments, the antibody or its antigen-binding fragment inhibits lectin pathway complement activation. Also provided is a polynucleotide that encodes the monoclonal antibody or its antigen-binding fragment of the present disclosure, and a cloning vector or expression cassette that includes such a polynucleotide. Further provided is a method for inhibiting lectin pathway complement activation and a method for treating diseases and disorders of the lectin pathway. TIFF2024546109000142.tif97161

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to antibodies that bind to the serine protease domain of MASP-2, as well as related compositions and methods.

[0002] SEQUENCE LISTING STATEMENT The sequence listing associated with this application is provided in XML format in lieu of a hard copy and is hereby incorporated by reference. The name of the XML file in which the sequence listing is stored is MP10329PCT.XML. This XML file is 89KB, was created on November 21, 2022, and has been submitted through the Patent Center together with the filing of this specification. [Background technology]

[0003] background The complement system underpins the innate host defense against pathogens and other acute insults (M.K. Liszewski and J.P. Atkinson, in Fundamental Immunology, ed. W.E. Paul, 3rd ed., Raven Press, Ltd., New York, 1993), and also plays a role in immune surveillance against cancer (P. Macor, et al., Front. Immunol., 9:2203, 2018). The complement system involves more than 30 fluid-phase and membrane-bound proteins (S. Meyer, et al., mAbs, 6:1133, 2014). Most of them are regulatory proteins that orchestrate a cascade of highly regulated activation events. The complement system rapidly responds to molecular stress signals through a cascade of sequential proteolytic reactions initiated by the binding of pattern recognition receptors (PRRs) to unique structures on damaged cells, biomaterial surfaces, or invading microorganisms (Reis et al., Nat. Rev. Immunol., 18:5, 2018). Activation of the complement cascade induces diverse immune effector functions, such as cell lysis, phagocytosis, chemotaxis, and immune activation (S. Meyer, et al., 2014). Furthermore, the complement system also acts as a bridge between the innate immune response and the subsequent activation of adaptive immunity. In addition to its anti-infective properties, the complement system is also involved in the clearance of immune complexes and apoptotic cells, tissue regeneration, mobilization of hematopoietic progenitor cells, and angiogenesis (TM Pierpont et al., Front. Oncol., 8:163, 2018).

[0004] The complement system can be activated by three distinct pathways: the classical pathway, the alternative pathway and the lectin pathway. See Figure 1.

[0005] The classical pathway (CP) is primarily initiated by antibody-antigen complexes. Antibodies of subclasses IgM and IgG bind to antigens on the surface of pathogens or target cells and recruit the C1 complex. The C1 complex is composed of the multimolecular recognition subcomponent C1q (composed of six heterotrimers of C1qA, B and C chains) and C1q-associated serine proteases, namely C1r and C1s. Upon binding of C1q to the Fc region of either antigen-bound IgM or at least two IgG antibodies bound to the respective antigens, the serine protease C1r is autoactivated to its enzymatically active form and subsequently cleaves and activates its substrate C1s. Once activated, C1s cleaves C4 into its fragments C4a and C4b. C2 binds to C4b to generate the C4bC2 complex. In the second cleavage step, C1s cleaves C2 in the C4bC2 complex to release C2b, forming the complement C3 convertase complex C4bC2a, the so-called C3 convertase, which cleaves the abundant plasma complement component C3 into C3a and C3b.

[0006] The lectin pathway is triggered by the binding of pattern recognition molecules, such as mannose-binding lectin (MBL), ficolins, or collectin-11 and collectin-10, to pathogen-associated molecular patterns (PAMPs) or to apoptotic or modified host cells. These recognition molecules form complexes with the MBL-associated serine proteases MASP-1 and MASP-2, which activate them upon binding to their cognate ligands. Activated MASP-2 cleaves C2 bound to C4 and C4b to form C3 convertase (C4bC2a).

[0007] The alternative pathway is initiated by spontaneous hydrolysis ("tickover") of C3 to C3(HO), which binds to factor B (fB). The enzymatic activity of another highly specific serine protease, called complement factor D, is required to convert the resulting C3(HO)fB complex into the enzymatically active C3 convertase. The availability of enzymatically active factor D is thought to be the limiting factor in the alternative pathway amplification loop, and its availability requires the action of another enzyme, MASP-3, which is required for the conversion of pro-factor D (proCFD) to its active form, mature factor D (matCFD, or simply CFD) (Dobo et al., 2016). CFD activates fB bound to C3(HO) to Ba and Bb. Bb is also a serine protease and is involved in the formation of the alternative pathway C3 convertase C3(H2O)Bb, which cleaves C3 into C3a and C3b. By this mechanism, the alternative pathway is constitutively active at a low level. An AP amplification loop is formed when newly generated C3b, formed by C3(H2O)Bb or by the lectin and classical pathway C3 convertases C4bC2a, binds to a target surface such as a bacterial cell and sequesters fB to form the C3bfB complex, which is cleaved by CFD to create the alternative pathway C3 convertase complex C3bBb. This convertase can be further stabilized by properdin, which prevents the breakdown of this complex and the conversion of C3b by factor I and cofactors.

[0008] These three pathways converge at the step of C3 activation. The C3 cleavage fragment C3a is an anaphylatoxin that promotes inflammation. C3b acts as an opsonin by covalently binding to the surface of target cells via its thioester bond, marking them for circulating complement receptor (CR)-displaying effector cells, such as natural killer (NK) cells and macrophages, which contribute to complement-dependent cellular cytotoxicity (CDCC) and complement-dependent cellular phagocytosis (CDCP), respectively. C3b also binds to C3 convertases (either C4bC2a or C3bBb) to form C5 convertases (C4bC2a(C3b)n or C3bBb(C3b)n, respectively), which lead to MAC formation and subsequent CDC. Furthermore, the cell-associated cleavage fragments of C3b, iC3b and C3dg, can promote adaptive immune responses through complement receptor-mediated cytotoxicity (CDCC and CDCP) and B cell activation (MC Carroll, Nat. Immunol., 5:981, 2004).

[0009] Formation of C5 convertase leads to cleavage of C5 into C5a and C5b. C5a is another anaphylatoxin. C5b recruits C6-9 to form the membrane attack complex (MAC or C5b-9 complex). The MAC complex triggers pore formation (so-called complement-dependent cytotoxicity, CDC) that leads to membrane destruction and cytolysis of the target cell. Although direct cytolysis by MAC formation has traditionally been recognized as the terminal effector mechanism of the complement system, C3b-mediated opsonization and proinflammatory signaling, as well as the anaphylatoxin function of C3a, are believed to play important roles in mediating complement-dependent inflammatory pathology.

[0010] Although complement activation provides a valuable first line defense against potential pathogens, complement activation, which promotes a protective immune response, can also be a potential threat to the host (KR Karli, et al., Springer Semin. Immunopathol. 15:417 431, 1994; BPM Organ, Eur. J. Clinical Investig. 24:219 228, 1994). For example, C3 and C5 proteolytic products recruit and activate neutrophils. Activated neutrophils, although essential for host defense, can indiscriminately release destructive enzymes and cause organ damage. In addition, complement activation can cause the deposition of lytic complement components not only on microbial targets but also on nearby host cells, resulting in host cell lysis.

[0011] The complement system has been implicated in the pathogenesis of numerous acute and chronic disease states, including myocardial infarction, stroke, acute respiratory distress syndrome (ARDS), diabetes-related complications, ischemia-reperfusion injury, inflammatory gastrointestinal disorders, septic shock, capillary leakage after burn injury, graft-versus-host disease, ophthalmic disorders, inflammation after cardiopulmonary bypass, transplant rejection, thrombotic microangiopathy (TMA), kidney disease, rheumatoid arthritis, multiple sclerosis, myasthenia gravis, and Alzheimer's disease. Complement activation is believed to be a major pathomechanism of these and other diseases, making it an effective point of focus for clinical management in these disease states. Increasing recognition of the importance of complement-mediated tissue injury in various disease states has highlighted the need for effective complement inhibitors. To date, only three complement-targeting drugs have been approved for human use: eculizumab (Soliris®), ravulizumab (Ultomiris), both of which are antibodies against C5, and the C3 inhibitor pegcetacoplan (Empaveli). However, C5 and C3 are "downstream" effector molecules in the complement system, and neither blocking C5 nor inhibiting C3 will prevent activation of the complement system. Therefore, inhibitors of early steps in complement activation would have a significant advantage over "downstream" complement inhibitors. Summary of the Invention

[0012] overview This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify key features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.

[0013] In one aspect, the present disclosure provides an isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds to MASP-2. In some embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof specifically binds to an epitope within the serine protease domain of human MASP-2. In some embodiments, the epitope is the amino acid sequence of human MASP-2. Located within TIFF2024546109000002.tif4128. In some embodiments, the antibody or antigen-binding fragment thereof inhibits lectin pathway complement activation. In some embodiments, the antibody or antigen-binding fragment thereof competes with C4 binding to MASP-2.

[0014] Also provided herein are polynucleotides encoding the monoclonal antibodies, or antigen-binding fragments thereof, of the present disclosure, and cloning vectors or expression cassettes comprising such polynucleotides.

[0015] Further provided herein are host cells expressing the monoclonal antibodies or antigen-binding fragments thereof of the present disclosure, and methods of producing the monoclonal antibodies or antigen-binding fragments thereof of the present disclosure, comprising culturing the host cells under conditions that allow for expression of the antibody or antigen-binding fragment thereof, and isolating the antibody or antigen-binding fragment thereof.

[0016] Also provided herein is a method of inhibiting lectin pathway complement activation in a mammal, comprising administering to a mammalian subject in need thereof an amount of a composition comprising a monoclonal antibody or antigen-binding fragment thereof of the present disclosure in an amount sufficient to inhibit lectin pathway complement activation in said mammal. In some embodiments, the monoclonal antibody or antigen-binding fragment thereof of the present disclosure may be used to treat a subject suffering from or at risk of a disease or disorder associated with the lectin pathway. In some embodiments, the disease or disorder associated with the lectin pathway is selected from thrombotic microangiopathy (TMA), kidney disease, inflammatory responses resulting from tissue or organ transplantation, ischemia-reperfusion injury, complications associated with diabetes, cardiovascular disease or disorder, inflammatory gastrointestinal disorder, pulmonary disorder, ophthalmologic disease or disorder, disseminated intravascular coagulation, graft-versus-host disease, veno-occlusive disease, and diffuse alveolar hemorrhage. [Brief description of the drawings]

[0017] The foregoing aspects and many of the attendant advantages of this invention will become more readily appreciated as they become better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings as hereinafter described.

[0018] [Figure 1] FIG. 1 is a schematic diagram of the complement system. [Diagram 2] FIG. 2 is a diagram illustrating the domain structure of human MASP-2 protein. [Diagram 3] Figure 3 is an amino acid sequence alignment of the VH and VL regions of the anti-MASP-2 inhibitory antibodies (mouse parent) mAb OMS850, mAb OMS860 and mAb OMS870 described in Example 2. [Figure 4] FIG. 4 graphically illustrates the binding of human MASP-2 (CCP1-CCP2-SP fragment) by mAb OMS850, mAb OMS860, mAb OMS870 and mAb OMS858, as described in Example 3. [Figure 5A]FIG. 5A graphically illustrates the concentration-dependent inhibition of C3b adhesion in human serum by mAb OMS850, mAb OMS860 and mAb OMS870, as described in Example 3. [Figure 5B] FIG. 5B graphically illustrates the concentration-dependent inhibition of C3b adhesion in cynomolgus monkey serum by mAb OMS850, mAb OMS860, and mAb OMS870, as described in Example 3. [Figure 5C] FIG. 5C graphically illustrates the concentration-dependent inhibition of C3b adhesion in rat serum by mAb OMS850, mAb OMS860, and mAb OMS870, as described in Example 3. [Figure 5D] FIG. 5D graphically illustrates the concentration-dependent inhibition of C3b adhesion in mouse serum by mAb OMS850, mAb OMS860, and mAb OMS870, as described in Example 3. [Figure 6] FIG. 6 graphically illustrates the concentration-dependent inhibition of C4b adhesion in 50% human serum by mAb OMS850, mAb OMS860 and mAb OMS870, as described in Example 3. [Figure 7] FIG. 7 graphically illustrates the concentration-dependent inhibition of C3b adhesion in human serum of mAb OMS852 with various p53 substitutions, as described in Example 4. [Figure 8A] FIG. 8A graphically illustrates the concentration-dependent inhibition of C3b adhesion in human serum of several candidate humanized versions of mAb OMS850, as described in Example 4. [Figure 8B] FIG. 8B graphically illustrates the concentration-dependent inhibition of C3b adhesion in human serum of several candidate humanized versions of mAb OMS850, as described in Example 4. [Figure 9A] Figure 9A graphically illustrates the levels of MAC adhesion in the presence of various concentrations of anti-MASP-2 antibody OMS858 under classical pathway-specific assay conditions described in Example 5. [Figure 9B]Figure 9B graphically illustrates the levels of MAC adhesion in the presence of various concentrations of anti-MASP-2 antibody OMS858 under lectin pathway-specific assay conditions described in Example 5. [Figure 9C] Figure 9C graphically illustrates the levels of MAC adhesion in the presence of various concentrations of anti-MASP-2 antibody OMS858 under alternative pathway-specific assay conditions described in Example 5. [Figure 10] Figure 10 shows the results of binding studies in a Biolayer Interferometry (BLI) measurement instrument (OCTET) using biotinylated OMS858 and a panel of other MASP-2 antibodies, as described in Example 6. [Figure 11] FIG. 11 is a schematic diagram showing the arrangement of the MASP-2 serine protease domain and Fab mAb OMS858 and the contacts between them, as described in Example 9. [Figure 12] The left side of Figure 12 is a schematic diagram showing the MASP-2 epitope bound by mAb OMS858, which includes the following residues in the SP domain of MASP-2: ASP496, LYS503, SER506, PRO507, HIS508 and TRP513, and the right side is a schematic diagram showing the paratope of mAb OMS858 that binds to the MASP-2 epitope, which includes two corresponding connected patches contributed by the heavy and light chains, specifically heavy chain residues HIS33, ASP50, ASP52, ASP55, GLU57, HIS59, and light chain residues TYR31, ARG30 and TRP90, as described in Example 9. [Figure 13] FIG. 13 illustrates the interaction between the mAb OMS858 paratope and the MASP-2 epitope as calculated by LigPlot+ software, as described in Example 9. [Figure 14A] Figure 14A illustrates the three-dimensional structure of MASP-2 epitopes, including ASP496, LYS503, SER506, PRO507, HIS508 and TRP513, bound by mAb OMS858, as described in Example 9. [Figure 14B] Figure 14B illustrates the three-dimensional structure of the mAb OMS858 paratope, which includes HIS33, ASP50, ASP52, ASP55, GLU57, HIS59 of the heavy chain, and ARG30, TYR31 and TRP90 of the light chain, to which MASP-2 binds, as described in Example 9. [Figure 15] Figure 15 illustrates the contacts between the MASP-2 epitope DIRMGTLKRLSPHYTQAW (SEQ ID NO:6) and the heavy and light chain variable regions of mAb OMS858, as described in Example 9. [Figure 16] Figure 16 is an amino acid alignment of the MASP-2 serine protease (SP) domains of human MASP-2 (aa445-686 of SEQ ID NO:1), cynomolgus monkey MASP-2 (aa445-686 of SEQ ID NO:4), dog MASP-2 (aa445-686 of SEQ ID NO:5), mouse MASP-2 (aa444-685 of SEQ ID NO:2), and rat MASP-2 (aa444-685 of SEQ ID NO:3) as described in Example 10, showing that HIS508 of human MASP-2 is conserved in cynomolgus monkeys, mice, and rats, but not in dogs. [Figure 17A] FIG. 17A graphically illustrates lectin pathway activity over time in cynomolgus monkeys following intravenous administration of 1.5 mg / kg OMS856, as described in Example 11. [Figure 17B] FIG. 17B graphically illustrates lectin pathway activity over time in cynomolgus monkeys following intravenous administration of 1.5 mg / kg OMS858, as described in Example 11. [Figure 18A] FIG. 18A graphically illustrates lectin pathway activity over time in cynomolgus monkeys following subcutaneous administration of 1.5 mg / kg OMS856, as described in Example 11. [Figure 18B] FIG. 18B graphically illustrates lectin pathway activity over time in cynomolgus monkeys following subcutaneous administration of 1.5 mg / kg OMS858, as described in Example 11. [Figure 19] FIG. 19 graphically illustrates the time to occlusion (TOC) in mice administered a range of doses of OMS858, as described in Example 12. [Figure 20A] Figure 20A graphically illustrates the levels of MAC adhesion in the presence of various concentrations of anti-MASP-2 antibody OMS858 under classical pathway-specific assay conditions described in Example 13. [Figure 20B] Figure 20B graphically illustrates the levels of MAC adhesion in the presence of various concentrations of anti-MASP-2 antibody OMS858 under lectin pathway-specific assay conditions described in Example 13. [Figure 20C] Figure 20C graphically illustrates the levels of MAC adhesion in the presence of various concentrations of anti-MASP-2 antibody OMS858 under alternative pathway-specific assay conditions described in Example 13. [Figure 21] FIG. 21 graphically illustrates the pharmacodynamics of OMS858 inhibition of lectin pathway activity in cynomolgus monkeys following exposure to 0.1, 0.3, 1 or 3 mg / kg SC OMS858 or 1 mg / kg IV OMS585, as described in Example 16. [Figure 22] FIG. 22 graphically illustrates the relationship between OMS858 serum concentrations and PD response data (i.e., lectin pathway inhibition) in cynomolgus monkeys, as described in Example 16. [Figure 23] Figure 23 graphically illustrates the pharmacokinetic parameters of OMS858 in mice and cynomolgus monkeys following exposure to 0.1, 0.3, 1 or 3 mg / kg SC OMS858 or 1 mg / kg IV OMS585, as described in Example 17. Shown are plots of mean OMS858 serum concentrations over time following a single IV or SC dose to mice (left panel) and monkeys (right panel). [Figure 24]FIG. 24 graphically illustrates the pharmacodynamics of OMS858 inhibition of lectin pathway activity in mice following exposure to 0.1, 0.3, 1 or 3 mg / kg SC OMS858 or 1 mg / kg IV OMS585, as described in Example 16. [Diagram 25] Figure 25 graphically illustrates the pharmacokinetics (PK) of OMS858 in healthy human subjects. Subjects were administered a single IV dose of OMS858 at 0.01 mg / kg (cohort 1), 0.03 mg / kg (cohort 2), 0.1 mg / kg (cohort 3) or 0.3 mg / ml (cohort 4). Concentrations of OMS858 detected in serum samples from subjects at various time points post-dose are shown. The dotted line indicates the lower limit of quantification of the assay used, which is 70 ng / mL. [Figure 26] Figure 26 graphically illustrates the pharmacodynamics (PD) of OMS858 in healthy human subjects. Subjects were administered a single IV dose of OMS858 at 0.01 mg / kg (cohort 1), 0.03 mg / kg (cohort 2), 0.1 mg / kg (cohort 3) or 0.3 mg / kg (cohort 4). The level of inhibition of the complement lectin pathway, as measured by C4 adhesion, at various time points after administration is shown. Measurements for subjects receiving placebo are also shown. [Figure 27] Figure 27 graphically illustrates the PK vs. PD relationship for OMS858. The level of lectin pathway inhibition at various OMS858 levels is shown for subjects receiving a single IV dose of OMS858 at 0.01 mg / kg (cohort 1), 0.03 mg / kg (cohort 2) or 0.1 mg / kg (cohort 3). The calculated EC50 is 170 ng / mL and the calculated EC90 is 400 ng / mL. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] Detailed Description I. Definition Unless specifically defined herein, all terms used herein have the same meaning as would be understood by a person skilled in the art of the present invention. The following definitions are provided to clarify the terms used in the specification and claims to describe the present invention. Further definitions are set forth throughout this disclosure.

[0020] Any concentration range, percentage range, ratio range, or integer range herein shall be understood to include any integer value within the recited range, and fractions thereof, as appropriate (e.g., tenths and hundredths of an integer), unless otherwise indicated or otherwise clear from the context. Any numerical range recited herein for any physical characteristic, such as polymer subunits, size, or thickness, shall be understood to include any integer within the recited range, and fractions thereof, as appropriate, unless otherwise indicated or otherwise clear from the context. As used herein, the term "about" shall specify that the range or value given may vary by ±10% of the range or value recited, unless otherwise indicated.

[0021] As used herein, the terms "a," "an," and "the" should be understood to refer to one or more of the referred components. The use of alternatives (e.g., "or") should be understood to mean one or both of the alternatives, or any combination thereof. As used herein, the terms "comprise," "have," and "include" are used synonymously, and these terms and variations thereof should be construed as open-ended.

[0022] "Optional" or "optionally" means that the subsequently described element, component, event or circumstance may be present or occur, or may not be present or occur, and that the description encompasses the presence or occurrence of the element, component, event or circumstance as well as its absence or not occurring.

[0023] It should be understood that each individual construct or group of constructs derived from the various combinations of structures and subunits described herein is disclosed by this application to the same extent as if each construct or group of constructs was described individually, and thus selection of a particular structure or particular subunit is within the scope of this disclosure.

[0024] The term "consisting essentially of" is not equivalent to "comprising" and refers to a specified material or step in a claim or a basic characteristic of the claimed subject matter. For example, a domain, region, or module (e.g., a binding domain) of a protein or protein "consists essentially of" a particular amino acid sequence if the amino acid sequence of the domain, region, module, or protein includes extensions, deletions, mutations, or combinations thereof (e.g., amino- or carboxy-terminal amino acids or amino acids between domains) that collectively occupy at most 20% (e.g., at most 15%, 10%, 8%, 6%, 5%, 4%, 3%, 2%, or 1%) of the length of the domain, region, module, or protein and do not substantially affect (i.e., do not reduce) the activity of the domain, region, module, or protein (e.g., the target binding affinity of a binding protein) by more than 50%, e.g., by more than 40%, 30%, 25%, 20%, 15%, 10%, 5%, or 1%).

[0025] As used herein, the terms "treat", "treatment" or "ameliorate" refer to the medical management of a disease, disorder or condition of interest. In general, an appropriate dose or treatment regimen comprising an antibody or antigen-binding fragment thereof of the present disclosure is administered in an amount sufficient to elicit a therapeutic or prophylactic benefit. Therapeutic or prophylactic / preventative benefits include improved clinical outcome, reduction or alleviation of symptoms associated with the disease, reduction in the occurrence of symptoms, improved quality of life, prolonged disease-free state, reduction in the extent of disease, stabilization of the disease state, delay or prevention of disease progression, remission, survival, extended survival, or any combination thereof.

[0026] A "therapeutically effective amount" or "effective amount" of an antibody or antigen-binding fragment thereof, polynucleotide, vector, host cell, or composition of the present disclosure refers to an amount of a composition or molecule sufficient to provide a statistically significant therapeutic benefit, including improved clinical outcome, reduction or alleviation of symptoms associated with a disease, reduction in the occurrence of symptoms, improved quality of life, prolonged disease-free state, reduction in the extent of disease, stabilization of a disease state, delayed disease progression, remission, survival, or prolonged survival. When referring to an individual active ingredient administered alone, a therapeutically effective amount refers to the effect of that ingredient or the effect of cells expressing that ingredient alone. When referring to a combination, a therapeutically effective amount refers to the combined amount of active ingredients, or of the combined complementary active ingredients and cells expressing the active ingredients, that results in a therapeutic effect, whether administered sequentially, sequentially, or simultaneously.

[0027] As used herein, a "subject" encompasses any mammal, including, but not limited to, humans, non-human primates, dogs, cats, horses, sheep, goats, cows, rabbits, pigs, and rodents. Subjects may be male or female, and subjects may be of any suitable age, including infants, juveniles, adolescents, adults, or geriatric subjects.

[0028] As used herein, "amino acid" refers to natural and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to natural amino acids. Natural amino acids are those encoded by the genetic code, as well as those that are later modified, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a natural amino acid, i.e., an α carbon bonded to a hydrogen, a carboxyl group, an amino group, and an R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a natural amino acid. Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that function similarly to a naturally occurring amino acid.

[0029] As used herein, a "mutation" refers to a change in the sequence of a nucleic acid molecule or polypeptide molecule when compared to a reference or wild-type nucleic acid molecule or polypeptide molecule, respectively. Mutations can result in several different types of changes in the sequence, including nucleotide or amino acid substitutions, insertions or deletions.

[0030] In the broadest sense, the natural amino acids can be grouped based on the chemical characteristics of each amino acid's side chain: "hydrophobic" amino acid means any of the following: Ile, Leu, Met, Phe, Trp, Tyr, Val, Ala, Cys, or Pro; "hydrophilic" amino acid means any of the following: Gly, Asn, Gln, Ser, Thr, Asp, Glu, Lys, Arg, or His.

[0031] "Conservative substitutions" refer to amino acid substitutions that do not significantly affect or alter the binding characteristics of a particular protein. Generally, conservative substitutions are those in which the substituted amino acid residue is replaced with an amino acid residue having a similar side chain. Conservative substitutions include those found in one of the following groups: Group 1: alanine (Ala or A), glycine (Gly or G), serine (Ser or S), threonine (Thr or T); Group 2: aspartic acid (Asp or D), glutamic acid (Glu or Z); Group 3: asparagine (Asn or N), glutamine (Gln or Q); Group 4: arginine (Arg or R), lysine (Lys or K), histidine (His or H); Group 5: isoleucine (Ile or I), leucine (Leu or L), methionine (Met or M), valine (Val or V); and Group 6: phenylalanine (Phe or F), tyrosine (Tyr or Y), tryptophan (Trp or W). Additionally or alternatively, amino acids can be grouped into conservative substitution groups according to similar function, chemical structure, or composition (e.g., acidic, basic, aliphatic, aromatic, or sulfur-containing). For example, where substitution is of interest, the aliphatic group can include Gly, Ala, Val, Leu, and Ile. Other conservative substitution groups include sulfur-containing: Met and cysteine ​​(Cys or C); acidic: Asp, Glu, Asn, and Gln; small aliphatic, non-polar or weakly polar residues: Ala, Ser, Thr, Pro, and Gly; polar, negatively charged residues and their amides: Asp, Asn, Glu, and Gln; polar, positively charged residues: His, Arg, and Lys; large aliphatic, non-polar residues: Met, Leu, Ile, Val, and Cys; and large aromatic residues: Phe, Tyr, and Trp. Further information can be found in Creighton (1984) Proteins, WH Freeman and Company.

[0032] As used herein, "protein" or "peptide" or "polypeptide" refers to a polymer of amino acid residues. Protein applies to both naturally occurring amino acid polymers and to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of the corresponding naturally occurring amino acids, as well as to non-naturally occurring amino acid polymers. Variants of the proteins, peptides and polypeptides of the present disclosure are also contemplated. In certain embodiments, variant proteins, peptides and polypeptides comprise or consist of an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identical to the amino acid sequence of a given or reference amino acid sequence described herein.

[0033] "Nucleic acid molecule" or "oligonucleotide" or "polynucleotide" or "polynucleic acid" refers to an oligomeric or polymeric compound comprising covalently linked nucleotides that may be composed of natural subunits (e.g., purine or pyrimidine bases) or non-natural subunits (e.g., morpholine rings). Purine bases include adenine, guanine, hypoxanthine and xanthine, while pyrimidine bases include uracil, thymine and cytosine. Nucleic acid molecules include polyribonucleic acid (RNA), including, for example, mRNA, microRNA, siRNA, viral genomic RNA, and synthetic RNA, and polydeoxyribonucleic acid (DNA), including, for example, cDNA, genomic DNA, and synthetic DNA. Both RNA and DNA can be single-stranded or double-stranded. If single-stranded, the nucleic acid molecule can be the coding strand or the non-coding strand (antisense strand). A nucleic acid molecule that codes for an amino acid sequence includes all nucleotide sequences that code for the same amino acid sequence. Some versions of the nucleotide sequence may also include introns, provided that the introns can be removed by co-transcriptional or post-transcriptional mechanisms. In other words, different nucleotide sequences may code for the same amino acid sequence as a result of redundancy or degeneracy in the genetic code, or due to splicing.

[0034] Variants of the nucleic acid molecules of the present disclosure are also contemplated. A variant nucleic acid molecule is at least 70%, 75%, 80%, 85%, 90%, and preferably 95%, 96%, 97%, 98%, 99%, or 99.9% identical to a nucleic acid molecule of a given or reference polynucleotide described herein, or hybridizes to the polynucleotide under stringent hybridization conditions of 0.015 M sodium chloride, 0.0015 M sodium citrate at about 65-68° C., or 0.015 M sodium chloride, 0.0015 M sodium citrate, and 50% formamide at about 42° C. A variant nucleic acid molecule retains the ability to encode a binding domain thereof that has a functionality described herein, such as binding a target molecule.

[0035] "Percent sequence identity" refers to the relationship between two or more sequences, as determined by comparing sequences. A preferred method for determining sequence identity is designed to give the best match between the sequences being compared. For example, sequences are aligned for optimal comparison (e.g., gaps can be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment). In addition, non-homologous sequences can be ignored for comparison. Percent sequence identity referred to herein is calculated over the entire length of the reference sequence, unless otherwise indicated. Methods for determining sequence identity and sequence similarity can be found in publicly available computer programs. Sequence alignment and percent identity calculations can be performed using BLAST programs (e.g., BLAST2.0, BLASTP, BLASTN, or BLASTX), or Megalign (DNASTAR) software. The mathematical algorithm used in the BLAST program can be found in Altschul et al., Nucleic Acids Res. 25:3389-3402, 1997. Appropriate parameters for measuring alignment can be determined by known methods, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared.

[0036] The term "isolated" means that the material is removed from its original environment (e.g., the natural environment if it occurs in nature). For example, a naturally occurring nucleic acid or naturally occurring polypeptide present in a living animal is not isolated, but the same nucleic acid or polypeptide separated from some or all of the coexisting materials in a natural system is isolated. Such a nucleic acid may be part of a vector and / or such a nucleic acid or polypeptide may be part of a composition (e.g., a cell lysate), but is still isolated in that such a vector or composition is not part of the natural environment for the nucleic acid or polypeptide. "Isolated" can also describe, in some embodiments, an antibody, antigen-binding fragment, polynucleotide, vector, host cell, or composition that is outside the human body.

[0037] The term "gene" means a segment of DNA or RNA involved in producing a polypeptide chain, and the term, in certain contexts, encompasses the regions preceding and following the coding region (e.g., the 5' untranslated region (UTR) and the 3' UTR) as well as the intervening sequences (introns) between individual coding segments (exons).

[0038] "Functional variant" refers to a polypeptide or polynucleotide that is structurally similar or substantially structurally similar to a parent or reference compound of the present disclosure, but differs slightly in composition (e.g., one or more bases, atoms or functional groups are different or added or removed) such that the polypeptide or encoded polypeptide can perform at least one function of the parent polypeptide with at least 50% efficiency, preferably at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 100% activity level of the parent polypeptide, or at an activity level higher than the activity level of the parent polypeptide. In other words, a functional variant of a polypeptide of the disclosure or of the encoded polypeptide has "similar binding," "similar affinity," or "similar activity" if the functional variant exhibits improved performance or has no more than a 50% reduction in performance compared to a parent or reference polypeptide in a selected assay, such as an assay for measuring enzymatic activity or binding affinity.

[0039] As used herein, a "functional portion" or "functional fragment" refers to a polypeptide or polynucleotide that comprises only a domain, portion or fragment of a parent or reference compound, where the polypeptide or encoded polypeptide retains at least 50% of the activity associated with the domain, portion or fragment of the parent or reference compound, preferably at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 100% or a higher activity level than the activity level of the parent polypeptide, or confers a biological benefit (e.g., an effector function). A "functional portion" or "functional fragment" of a polypeptide or encoded polypeptide of the present disclosure has "similar binding" or "similar activity" if the functional portion or fragment exhibits improved performance or has no more than a 50% reduction in performance compared to a parent or reference polypeptide in a chosen assay (for affinity, the reduction is preferably no more than 20% or 10%, or no more than 1 log difference compared to the parent or reference).

[0040] As used herein, the terms "engineered," "recombinant," or "non-naturally occurring" refer to an organism, microorganism, cell, protein, polypeptide, nucleic acid molecule, or vector that contains at least one genetic modification or has been modified by the introduction of a foreign or heterologous nucleic acid molecule, where such modification or modification is introduced by genetic engineering (i.e., human intervention). Genetic modifications include, for example, modifications that introduce expressible nucleic acid molecules that code for functional RNA, proteins, fusion proteins, or enzymes, or modifications that introduce additions, deletions, substitutions, or other functional disruptions of the genetic material of a cell. Further modifications include, for example, non-coding control regions, where modifications alter the expression of a polynucleotide, gene, or operon.

[0041] As used herein, "heterologous" or "non-endogenous" or "foreign" refers to any gene, protein, compound, nucleic acid molecule, or activity that is not native to a host cell or subject, or any gene, protein, compound, nucleic acid molecule, or activity that is native to a host cell or subject that has been modified. Heterologous, non-endogenous, or foreign includes genes, proteins, compounds, or nucleic acid molecules that have been mutated or otherwise modified such that the structure, activity, or both are different between the native gene, protein, compound, or nucleic acid molecule and the modified gene, protein, compound, or nucleic acid molecule. In certain embodiments, a heterologous, non-endogenous, or foreign gene, protein, or nucleic acid molecule (e.g., a receptor, ligand, etc.) is not endogenous to a host cell or subject, and a nucleic acid encoding such a gene, protein, or nucleic acid molecule may be added to the host cell by conjugation, transformation, transfection, electroporation, etc., where the added nucleic acid molecule may be integrated into the host cell genome or may exist as extrachromosomal genetic material (e.g., as a plasmid or other self-replicating vector). "Homologous" or "homolog" refers to a gene, protein, compound, nucleic acid molecule, or activity found in or derived from a given host cell, species, or strain. For example, a heterologous or foreign polynucleotide or gene encoding a polypeptide may be homologous to a native polynucleotide or gene and may encode a homologous polypeptide or activity, but the polynucleotide or polypeptide may have an altered structure, sequence, expression level, or any combination thereof. The non-endogenous polynucleotide or gene and the encoded polypeptide or activity may be derived from the same species, a different species, or a combination thereof.

[0042] In certain embodiments, a nucleic acid molecule or a portion thereof native to a host cell will be considered heterologous to the host cell if it has been modified or mutated. Alternatively, a nucleic acid molecule native to a host cell may be considered heterologous if it has been modified with a heterologous expression control sequence or with an endogenous expression control sequence that is not normally associated with the nucleic acid molecule native to the host cell. In addition, the term "heterologous" may refer to a biological activity that is different, modified, or not endogenous to the host cell. As described herein, multiple heterologous nucleic acid molecules can be introduced into a host cell as separate nucleic acid molecules, as multiple individually controlled genes, as polycistronic nucleic acid molecules, as a single nucleic acid molecule encoding an antibody or antigen-binding fragment (or other polypeptide), or any combination thereof.

[0043] As used herein, the term "endogenous" or "native" refers to a polynucleotide, gene, protein, compound, molecule, or activity that is normally present in a host cell or subject.

[0044] The term "expression" as used herein refers to the process by which a polypeptide is produced based on a coding sequence of a nucleic acid molecule, e.g., a gene. This process can include transcription, post-transcriptional control, post-transcriptional modification, translation, post-translational control, post-translational modification, or any combination thereof. The nucleic acid molecule to be expressed is typically operably linked to an expression control sequence (e.g., a promoter).

[0045] The term "operably linked" refers to the association of two or more nucleic acid molecules on a single nucleic acid fragment such that the function of one is affected by the other. For example, a promoter is operably linked to a coding sequence if it can affect the expression of that coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter). "Unlinked" means that the associated genetic elements are not closely related to each other so that the function of one does not affect the other.

[0046] As described herein, multiple heterologous nucleic acid molecules can be introduced into a host cell as separate nucleic acid molecules, as multiple individually regulated genes, as polycistronic nucleic acid molecules, as a single nucleic acid molecule encoding a protein (e.g., the heavy chain of an antibody), or any combination thereof. When two or more heterologous nucleic acid molecules are introduced into a host cell, it is understood that the two or more heterologous nucleic acid molecules can be introduced as a single nucleic acid molecule (e.g., on a single vector), on separate vectors, integrated into a single site or multiple sites of a host chromosome, or any combination thereof. The number of heterologous nucleic acid molecules or protein activities referred to refers to the number of different encoding nucleic acid molecules or the number of different protein activities, and not the number of independent nucleic acid molecules introduced into the host cell.

[0047] The term "construct" refers to any polynucleotide (or, if the context makes clear, a fusion protein of the present disclosure) that contains a recombinant nucleic acid molecule. The (polynucleotide) construct may be present in a vector (e.g., bacterial vector, viral vector) or integrated into a genome. A "vector" is a nucleic acid molecule capable of transporting another nucleic acid molecule. A vector may be, for example, a plasmid, a cosmid, a virus, an RNA vector, or a linear or circular DNA or RNA molecule, which may include chromosomal, non-chromosomal, semisynthetic, or synthetic nucleic acid molecules. Vectors of the present disclosure also include transposon systems (e.g., Sleeping Beauty, see, e.g., Geurts et al., Mol. Ther. 8:108, 2003; Mates et al., Nat. Genet. 41:753, 2009). Exemplary vectors are those capable of autonomous replication (episomal vectors), those capable of delivering polynucleotides to a cellular genome (e.g., viral vectors), or those capable of expressing the nucleic acid molecule to which they are linked (expression vectors).

[0048] As used herein, "expression vector", "cloning vector" or "vector" refers to a DNA construct containing a nucleic acid molecule operably linked to suitable control sequences capable of achieving expression of the nucleic acid molecule in a suitable host. Such control sequences typically include a promoter to achieve transcription, an optional operator sequence to control such transcription, a sequence encoding a suitable mRNA ribosomal binding site, and sequences that control the termination of transcription and translation. A vector can be a plasmid, a phage particle, a virus, or simply a potential genomic insert. Once transformed into a suitable host, the vector can replicate and function independently of the host genome, or in some cases, can integrate into the genome itself or deliver a polynucleotide contained in the vector to the genome without vector sequences. As used herein, "plasmid", "expression plasmid", "virus" and "vector" are often used interchangeably.

[0049] The term "introduced" in the context of inserting a nucleic acid molecule into a cell means "transfection," "transformation," or "transduction," and includes reference to the incorporation of a nucleic acid molecule into a eukaryotic or prokaryotic cell, where the nucleic acid molecule may be integrated into the genome of the cell (e.g., chromosome, plasmid, plastid, or mitochondrial DNA), converted into an autonomous replicon, or expressed transiently (e.g., transfected mRNA).

[0050] In certain embodiments, the polynucleotide of the present disclosure may be operably linked to certain elements of a vector. For example, polynucleotide sequences required to affect the expression and processing of the ligated coding sequence may be operably linked. Expression control sequences may include appropriate transcription initiation, termination, promoter, and enhancer sequences, efficient RNA processing signals, such as polyadenylation signals, sequences that stabilize cytoplasmic mRNA, sequences that increase translation efficiency (i.e., Kozak consensus sequences), sequences that increase protein stability, and sequences that enhance protein secretion in some cases. Expression control sequences may be operably linked if they are contiguous with the gene of interest. Expression control sequences that act in trans or at a distance to control the gene of interest may also be considered to be operably linked.

[0051] In certain embodiments, the vector comprises a plasmid vector or a viral vector (e.g., lentivirus vector or gamma-retrovirus vector). Viral vectors include retrovirus, adenovirus, parvovirus (e.g., adeno-associated virus), coronavirus, negative-stranded RNA virus, such as orthomyxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies and vesicular stomatitis virus), paramyxovirus (e.g., measles and Sendai), positive-stranded RNA virus, such as picornavirus and alphavirus, and double-stranded DNA virus, such as adenovirus, herpesvirus (e.g., herpes simplex virus type 1 and type 2, Epstein-Barr virus, cytomegalovirus), and poxvirus (e.g., vaccinia, fowlpox, and canarypox). Other viruses include, for example, Norwalk virus, togavirus, flavivirus, reovirus, papovavirus, hepadnavirus, and hepatitis virus. Examples of retroviruses include avian leukosis sarcoma, mammalian type C, B, D viruses, the HTLV-BLV complex, lentiviruses, and spumaviruses (Coffin, JM, Retroviridae: The viruses and their replication, in Fundamental Virology, 3rd ed., BN Fields et al., ed., Lippincott-Raven Publishers, Philadelphia, 1996).Methods of using retroviral and lentiviral viral vectors and packaging cells to transduce mammalian host cells with viral particles containing transgenes are known in the art and have been previously described, for example, in U.S. Patent No. 8,119,772; Walchli et al., PLoS One 6:327930, 2011; Zhao et al., J.Immunol.174:4415, 2005; Engels et al., Hum.Gene Ther.14:1155, 2003; Frecha et al., Mol.Ther.18:1748, 2010; and Verhoeyen et al., Methods Mol.Biol.506:97, 2009. Retroviral and lentiviral vector constructs and expression systems are also commercially available. Other viral vectors, including DNA viral vectors, such as adenovirus-based vectors and adeno-associated virus (AAV)-based vectors, herpes simplex virus (HSV)-derived vectors, such as amplicon vectors, replication-deficient HSV and attenuated HSV, can also be used for polynucleotide delivery (Krisky et al., Gene Ther. 5:1517, 1998).

[0052] Other vectors that can be used in the compositions and methods of the present disclosure include those derived from baculoviruses and alphaviruses (Jolly, D J. 1999. "Emerging Viral Vectors," in "The Development of Human Gene Therapy," ed. Friedmann T., New York: Cold Spring Harbor Lab, pp. 209-40), or plasmid vectors (e.g., Sleeping Beauty or other transposon vectors).

[0053] When the viral vector genome contains multiple polynucleotides to be expressed as separate transcripts in a host cell, the viral vector may also contain additional sequences between the two (or more) transcripts that allow bicistronic or polycistronic expression. Examples of such sequences used in viral vectors include an internal ribosome entry site (IRES), a furin cleavage site, a viral 2A peptide, or any combination thereof.

[0054] Plasmid vectors are also known in the art, including DNA-based plasmid vectors for expressing one or more proteins in vitro or for direct administration to a subject.Such vectors may contain bacterial origin of replication, viral origin of replication, genes encoding components required for plasmid replication, and / or one or more selection markers.Such vectors may also contain additional sequences that allow bicistronic or polycistronic expression.

[0055] As used herein, the term "host" refers to a cell or microorganism that is the target of genetic modification with a heterologous nucleic acid molecule to produce a polypeptide of interest (e.g., an antibody of the present disclosure).

[0056] A host cell may include any individual cell or cell culture that can incorporate a vector or nucleic acid or express a protein. The term also includes the progeny of the host cell, whether genetically or phenotypically the same or different. Suitable host cells depend on the vector, and may include mammalian cells, animal cells, human cells, monkey cells, insect cells, yeast cells, and bacterial cells. These cells may be induced to incorporate vectors or other materials by transformation using viral vectors, calcium phosphate precipitation, DEAE-dextran, electroporation, microinjection, or other methods. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual 2d ed. (Cold Spring Harbor Laboratory, 1989).

[0057] As used herein, "antigen" refers to an immunogenic molecule that elicits an immune response. This immune response may involve antibody production, activation of specific immune-competent cells, activation of complement, antibody-dependent cellular cytotoxicity, or any combination thereof. An antigen (immunogenic molecule) may be, for example, a peptide, glycopeptide, polypeptide, glycopolypeptide, polynucleotide, polysaccharide, lipid, etc. It is clear that an antigen may be synthesized, recombinantly produced, or derived from a biological sample. Exemplary biological samples that may contain one or more antigens include tissue samples, fecal samples, cells, biological fluids, or combinations thereof. An antigen may be expressed by a cell that has been modified or genetically engineered to express the antigen. An antigen may also be present in or on an infectious agent, e.g., present in a virion, or expressed or presented on the surface of a cell infected with an infectious agent.

[0058] The term "epitope" or "antigenic epitope" includes any molecule, structure, amino acid sequence, protein determinant that is recognized and specifically bound by a cognate binding molecule, such as an immunoglobulin, or other binding molecule, domain, or protein. Epitope determinants generally contain chemically active surface groups of molecules, such as amino acids or sugar side chains, and can have specific three-dimensional structural characteristics and specific charge characteristics. When the antigen is or comprises a peptide or protein, the epitope can be composed of consecutive amino acids (e.g., linear epitopes), or amino acids from different parts or regions of the protein that become adjacent due to protein folding (e.g., discontinuous or conformational epitopes), or non-contiguous amino acids that are adjacent regardless of protein folding.

[0059] The term "antibody" refers to an immunoglobulin molecule consisting of one or more polypeptides that specifically bind to an antigen through at least one epitope recognition site. For example, the term "antibody" includes an intact antibody, which includes at least two heavy chains and two light chains connected by disulfide bonds, as well as any antigen-binding portion or fragment of an intact antibody, such as an scFv, Fab or Fab'2 fragment, that has or retains the ability to bind to the antigen target molecule recognized by the intact antibody. The term also includes full-length or fragments of antibodies of any class or subclass, including IgG and its subclasses (e.g., IgG1, IgG2, IgG3 and IgG4), IgM, IgE, IgA, and IgD.

[0060] The term "antibody" is used herein in the broadest sense and includes antibodies and antibody fragments thereof derived from any antibody-producing mammal (e.g., mouse, rat, rabbit, and primate, including human), or derived from hybridoma, phage selection, recombinant expression, or transgenic animals (or other methods of producing antibodies or antibody fragments). The term "antibody" is not intended to be limited as to the source of the antibody or the manner in which it is made (e.g., by hybridoma, phage selection, recombinant expression, transgenic animals, peptide synthesis, etc.). Exemplary antibodies include polyclonal, monoclonal and recombinant antibodies, multispecific antibodies (e.g., bispecific antibodies), humanized antibodies, fully human antibodies, murine antibodies, chimeric, mouse-human, mouse-primate, primate-human monoclonal antibodies, and anti-idiotypic antibodies, and exemplary antibodies can be any intact molecule or fragment thereof. As used herein, the term "antibody" includes not only intact polyclonal or monoclonal antibodies, but also fragments thereof (e.g., dAb, Fab, Fab', F(ab')2, Fv), single chain (ScFv), synthetic variants thereof, naturally occurring variants, fusion proteins comprising an antibody portion with an antigen-binding fragment of the required specificity, humanized antibodies, chimeric antibodies, and any other modified configuration of immunoglobulin molecule containing an antigen-binding site or fragment (epitope recognition site) of the required specificity. The term also includes engineered and / or otherwise modified forms of immunoglobulins, including antigen-binding fragments thereof, such as intrabodies, peptibodies, diabodies, triabodies, tetrabodies, tandem di-scFv, tandem tri-scFv, etc.

[0061] The terms "VH" and "VL" refer to the variable binding regions from antibody heavy and light chains, respectively. VL can be a kappa class chain or a lambda class chain. The variable binding region comprises discrete, well-defined subregions known as complementarity determining regions (CDRs) and framework regions (FRs). CDRs are located within the hypervariable region (HVR) of an antibody and generally refer to the sequence of amino acids within the antibody variable region that, taken as a whole, confers antigen specificity and / or binding affinity to the antibody. Consecutive CDRs (i.e., CDR1 and CDR2 and CDR2 and CDR3) are separated from each other in the primary structure by framework regions.

[0062] As used herein, a "chimeric antibody" is a recombinant protein that contains variable domains and complementarity determining regions derived from a non-human species (e.g., rodent), while the remainder of the antibody molecule is derived from a human antibody. In some embodiments, a chimeric antibody is composed of an antigen-binding fragment of one antibody operatively linked or otherwise fused to a heterologous Fc portion of a different antibody. For example, a mouse-human chimeric antibody may contain an antigen-binding fragment of a mouse antibody fused to an Fc portion derived from a human antibody. In some embodiments, the heterologous Fc domain may be derived from a different Ig class than the parent antibody, such as IgA (including subclasses IgA1 and IgA2), IgD, IgE, IgG (including subclasses IgG1, IgG2, IgG3, and IgG4), and IgM.

[0063] As used herein, a "humanized antibody" is a molecule having an antigen-binding site derived from an immunoglobulin of a non-human species, with the remainder of the immunoglobulin structure based on the structure and / or sequence of a human immunoglobulin, typically prepared using recombinant techniques. Humanized antibodies differ from chimeric antibodies in that typically only the CDRs from the non-human species are used and grafted onto appropriate framework regions in a human variable domain. The antigen-binding site may be wild-type or modified by one or more amino acid substitutions. In some embodiments, a humanized antibody retains all CDR sequences (e.g., a humanized mouse antibody containing all six CDRs from a mouse antibody). In another embodiment, a humanized antibody has one or more CDRs (one, two, three, four, five, six) that are altered compared to the original antibody, also referred to as one or more CDRs "derived from" one or more CDRs from the original antibody.

[0064] As used herein, the term "antibody fragment" refers to a portion derived from or related to a full-length antibody, generally including the antigen-binding or variable region thereof. Specific examples of antibody fragments include Fab, Fab', F(ab)2, F(ab')2 and Fv fragments, scFv fragments, diabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.

[0065] As used herein, the term "antigen-binding fragment" refers to a polypeptide fragment containing at least one CDR of an immunoglobulin heavy and / or light chain that specifically binds to the antigen against which the antibody was raised. An antigen-binding fragment may contain one, two, three, four, five, or all six CDRs of the VH and VL sequences from an antibody.

[0066] "Fab" (fragment antigen binding) is the portion of an antibody that binds an antigen and comprises the variable region and the CH1 of a heavy chain linked to a light chain by an interchain disulfide bond. Each Fab fragment is monovalent with respect to antigen binding; that is, each Fab fragment has a single antigen-binding site. Pepsin treatment of an antibody yields one large F(ab')2 fragment, which roughly corresponds to two disulfide-linked Fab fragments with bivalent antigen-binding activity and still has the ability to cross-link antigen. Both Fab and F(ab')2 are examples of "antigen-binding fragments". Fab' fragments differ from Fab fragments in that they have several additional residues at the carboxy terminus of the CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine ​​residues of the constant domains bear a free thiol group. F(ab')2 antibody fragments are often produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0067] Fab fragments may be joined, for example, by a peptide linker, to form a single chain Fab, also referred to herein as "scFab." In these embodiments, the interchain disulfide bonds present in native Fab may not be present, and the linker serves to link or connect, in whole or in part, the Fab fragments into a single polypeptide chain. A Fab fragment from a heavy chain (e.g., comprising, consisting of, or consisting essentially of VH+CH1, or "Fd") and a Fab fragment from a light chain (e.g., comprising, consisting of, or consisting essentially of VL+CL) may be linked in any configuration to form an scFab. For example, scFabs may be arranged from N-terminus to C-terminus as follows: (heavy chain Fab fragment-linker-light chain Fab fragment) or (light chain Fab fragment-linker-heavy chain Fab fragment).

[0068] "Fv" is a small antibody fragment that contains a complete antigen recognition and binding site. This fragment generally consists of a dimer of one heavy-chain variable region domain and one light-chain variable region domain in tight, non-covalent association. However, even a single variable domain (i.e., half of an Fv containing only the three CDRs specific for an antigen) has the ability to recognize and bind to an antigen, although typically with a lower affinity than the entire binding site.

[0069] A "single-chain Fv", also abbreviated as "sFv" or "scFv", is an antibody fragment that comprises a VH antibody domain and a VL antibody domain connected into a single polypeptide chain. The scFv polypeptide may include a polypeptide linker disposed between the VH and VL domains to link them and enable the scFv to maintain or form the desired structure for antigen binding. However, a linker is not required. Such a peptide linker can be incorporated into the fusion polypeptide using standard techniques known in the art. Additionally or alternatively, the Fv can have a disulfide bond formed between the VH and VL to stabilize the VH and VL. For a description of scFvs, see the review by Pluckthun (1994) in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore, eds., Springer-Verlag, New York, pp. 269-315. In certain embodiments, the antibody or antigen-binding fragment comprises an scFv comprising a VH domain, a VL domain, and a peptide linker linking the VH domain to the VL domain. In particular embodiments, the scFv comprises a VH domain linked to the VL domain by a peptide linker, which can be in the VH-linker-VL or VL-linker-VH orientation. Any scFv of the disclosure can be engineered such that the C-terminus of the VL domain is linked to the N-terminus of the VH domain by a short peptide sequence, or vice versa (i.e., (N)VL(C)-linker-(N)VH(C) or (N)VH(C)-linker-(N)VL(C). Alternatively, in some embodiments, a linker can be linked to the N-terminal portion or N-terminus of the VH domain, VL domain, or both.

[0070] A peptide linker sequence for use in an scFv or other fusion protein, such as a targeted complement activating molecule described herein, can be selected, for example, based on the following criteria: (1) it can adopt an extended and flexible conformation, (2) it cannot or lacks the ability to adopt a secondary structure that can interact with a functional epitope on the first and second polypeptides and / or the target molecule, and / or (3) it lacks or is relatively low in hydrophobic or charged residues that may react with the polypeptide and / or the target molecule. Other considerations for the design (e.g., length) of the linker can include the conformation or range of conformations in which the VH and VL can form a functional antigen binding site. In certain embodiments, the peptide linker sequence contains, for example, Gly, Asn, and Ser residues. Other nearly neutral amino acids, such as Thr and Ala, can also be included in the linker sequence. Other amino acid sequences that may be usefully employed as linkers include those disclosed in Maratea et al., Gene 40:39 46 (1985), Murphy et al., Proc. Natl. Acad. Sci. USA 83:8258 8262 (1986), U.S. Patent No. 4,935,233, and U.S. Patent No. 4,751,180. Any suitable linker may be used, and a suitable linker can generally be less than about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100 amino acids in length, or about 200 amino acids in length, preferably comprises a flexible structure (which can provide flexibility and room for conformational movement between the two regions, domains, motifs, fragments or modules connected by the linker), and is preferably biologically inert and / or has a low risk of immunogenicity in humans.

[0071] Antibodies can be monospecific (e.g., bind a single epitope) or multispecific (e.g., bind multiple epitopes and / or target molecules). Bispecific or multispecific antibodies or antigen-binding fragments, in some embodiments, can comprise one, two or more antigen-binding domains (e.g., a VH and a VL). There can be two or more binding domains that bind to the same epitope or different epitopes, and bispecific or multispecific antibodies or antigen-binding fragments provided herein, in some embodiments, can comprise two or more binding domains that bind to different antigens or pathogens overall.

[0072] Antibodies and antigen-binding fragments can be constructed in a variety of formats. Exemplary antibody formats are disclosed in Spiess et al., Mol. Immunol. 67(2):95 (2015) and Brinkmann and Kontermann, mAbs 9(2):182-212 (2017).These formats and methods for making them are incorporated herein by reference and include, for example, Bispecific T Cell Engagers (BiTEs), DARTs, Knobs-Into-Holes (KIH) Assembly, scFv-CH3-KIH Assembly, KIH Common Light Chain Antibody, TandAb, Triple Body, TriBi minibody, Fab-scFv, scFv-CH-CL-scFv, F(ab')2-scFv2, tetravalent HCab, intrabody, CrossMab, Dual Action Fab (DAF) (two-in-one or four-in-one), DutaMab, DT-IgG, Charge Pair, Fab-arm Exchange, SEEDbody, Triomab, LUZ-Y Assembly, Fcab, κλ-body, orthogonal Fab ... Fab), DVD-Ig (e.g., U.S. Pat. No. 8,258,268, which formats are incorporated herein by reference in their entirety), IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V,V(H)-IgG, IgG(L)-V,V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, Zybody, and DVI-IgG (Four-in-One), as well as the so-called FIT-Ig (e.g., PCT Publication No. WO 2015 / 103072, which formats are incorporated herein by reference in their entirety), the so-called WuxiBody format (e.g., PCT Publication No. WO 2015 / 103072, which formats are incorporated herein by reference in their entirety). No. 2019 / 057122, which formats are incorporated herein by reference in their entireties), and the so-called In-Elbow-Insert Ig format (IEI-Ig, e.g., PCT Publication Nos. WO 2019 / 024979 and WO 2019 / 025391, which formats are incorporated herein by reference in their entireties).

[0073] An antibody or antigen-binding fragment may comprise two or more VH domains, two or more VL domains, or both (i.e., two or more VH domains and two or more VL domains). In certain embodiments, the antigen-binding fragment comprises the format (from N-terminus to C-terminus): VH-linker-VL-linker-VH-linker-VL, where the two VH sequences may be the same or different, and the two VL sequences may be the same or different. Such linked scFvs may comprise any combination of VH and VL domains arranged to bind a given target. In formats comprising two or more VH and / or two or more VL, one, two or more different epitopes or antigens may be bound. It will be understood that formats incorporating multiple antigen-binding domains may comprise VH and / or VL sequences in any combination or orientation. For example, the antigen-binding fragment can comprise the formats VL-linker-VH-linker-VL-linker-VH, VH-linker-VL-linker-VL-linker-VH, or VL-linker-VH-linker-VH-linker-VH-linker-VL.

[0074] As used herein, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and no limitation is intended as to the source of the antibody or the manner in which it is made (e.g., by hybridoma, phage selection, recombinant expression, transgenic animals, etc.). The term "monoclonal antibody" encompasses intact and full-length monoclonal antibodies, as well as fragments thereof (e.g., Fab, Fab', F(ab')2, Fv), single chain (ScFv), variants thereof, fusion proteins containing an antigen-binding portion, humanized monoclonal antibodies, chimeric monoclonal antibodies, and immunoglobulin molecules of any other modified configuration, including antigen-binding fragments (epitope recognition sites) having the requisite specificity and binding ability for the epitope. Monoclonal antibodies may be obtained using any technique which provides for the production of antibody molecules by continuous cell lines in culture, such as the hybridoma method described in Kohler, G., et al., Nature 256:495, 1975, or may be made by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567 to Cabilly). Monoclonal antibodies may also be isolated from phage antibody libraries using the techniques described in Clackson T. et al., Nature, 352:624-628, 1991 and Marks JD, et al., J. Mol. Biol., 222:581 597, 1991. Such antibodies may belong to any immunoglobulin class, including IgG, IgM, IgE, IgA, IgD, and any subclass thereof.

[0075] Recognized immunoglobulin polypeptides include kappa and lambda light chains, and alpha, gamma (IgG1, IgG2, IgG3, IgG4), delta, epsilon and mu heavy chains, or equivalents in other species. Full-length immunoglobulin "light chains" (about 25 kDa or about 214 amino acids) contain a variable region of about 110 amino acids at the NH2-terminus and a kappa or lambda constant region at the COOH-terminus. Full-length immunoglobulin "heavy chains" (about 50 kDa or about 446 amino acids) similarly contain a variable region (about 116 amino acids) and one of the heavy chain constant regions mentioned above, e.g., gamma (about 330 amino acids).

[0076] The basic four-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. IgM antibodies differ from the above arrangement in that they consist of five basic heterotetrameric units with an additional polypeptide called the J chain, and therefore have 10 antigen-binding sites. Secretory IgA antibodies also differ from the basic structure in that they polymerize to form multivalent assemblies containing two to five basic four-chain units with the J chain. Each L chain is linked to the H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H chain and each L chain also has regularly spaced intrachain disulfide bridges. The pairing of a VH and a VL together forms a single antigen-binding site.

[0077] Each H chain has a variable domain (VH) at the N-terminus followed by three constant domains (CH1, CH2, CH3) in the case of alpha, gamma, and delta chains, and four CH domains (CH1, CH2, CH3, CH4) in the case of mu and epsilon chains.

[0078] Each L chain has a variable domain (VL) at the N-terminus followed by a constant domain (CL) at the other end. When an L chain and an H chain pair, the VL aligns with the VH and the CL aligns with the first constant domain (CH1) of the heavy chain. The L chains of any vertebrate species can be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domains (CL).

[0079] Immunoglobulins can be assigned to different classes or isotypes depending on the amino acid sequence of the constant domain (CH) of their heavy chains. There are five classes of immunoglobulins, IgA, IgD, IgE, IgG and IgM, which have heavy chains designated alpha (α), delta (δ), epsilon (ε), gamma (γ) and mu (μ), respectively. The γ and α classes are subdivided into subclasses based on subtle differences in CH sequence and function; for example, humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2.

[0080] For the structure and properties of these various classes of antibodies, see, e.g., page 71 and chapter 6 of Basic and Clinical Immunology, 8th Edition (Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.), Appleton and Lange, Norwalk, Conn., 1994).

[0081] The term "variable" refers to the fact that the sequence of certain segments of the V domain varies widely among antibodies. The V domain mediates antigen binding and defines the specificity of a particular antibody for its particular antigen. However, variability is not evenly distributed throughout the 110 amino acid span of the variable domain. Rather, the V region consists of relatively invariant stretches of 15-30 amino acids called framework regions (FRs) separated by shorter, extremely variable regions called "hypervariable regions" each 9-12 amino acids long. The variable domains of native heavy and light chains each contain four FRs that are largely in a beta-sheet configuration, with three hypervariable regions connecting the FRs. The three hypervariable regions form loops that connect and, in some cases, form part of the n-sheet structure. The hypervariable regions in each chain are held in close proximity by the FRs, and the hypervariable regions from the other chain contribute to the formation of the antigen-binding site of antibodies (see Kabat, et al., "Sequences of Proteins of Immunological Interest," 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The constant domains are not directly involved in binding of antibodies to antigens, but exhibit various effector functions.

[0082] As used herein, "effector function" refers to a biological activity attributable to the Fc region of an antibody. Examples of antibody effector functions include antibody-dependent cellular cytotoxicity (ADCC), C1q binding and complement-dependent cytotoxicity, Fc receptor binding, phagocytosis, downregulation of cell surface receptors, and participation in B-cell activation. Modifications, such as amino acid substitutions, may be made to the Fc domain to modify (e.g., enhance or reduce) one or more functions of an Fc-containing polypeptide. Such functions include, for example, Fc receptor binding, regulation of antibody half-life, ADCC function, protein A binding, protein G binding, and complement binding. Amino acid modifications that modify Fc function include, for example, T250Q / M428L, M252Y / S254T / T256E, H433K / N434F, M428L / N434S, E233P / L234V / L235A / G236Δ / A327G / A330S / P331S, E333A, S239D / A330L / I332E, P257I / Q311, K326W / E333S, S239D / I332E / G236A, N297Q, K322A, S228P, L235E / E318A / K320A / K322A, L234A / L235A, and L234A / L235A / P329G mutations. Other Fc modifications and their effects on Fc function are known in the art.

[0083] As used herein, the term "hypervariable region" refers to the amino acid residues of an antibody that are responsible for antigen binding. The hypervariable region contains several "complementarity determining regions" (CDRs). The heavy chain contains three CDR sequences (CDRH1, CDRH2 and CDRH3) and the light chain contains three CDR sequences (CDRL1, CDRL2 and CDRL3). There are various systems for identifying and numbering the amino acids that make up the CDRs. For example, the hypervariable region generally comprises CDRs at about residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable domain and at about residues 31-35 (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable domain, as numbered according to the Kabat numbering system described in Kabat, et al., "Sequences of Proteins of Immunological Interest," 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991), and / or at CDRs at about residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable domain and at about residues 31-35 (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable domain, as numbered according to the Kabat numbering system described in Chothia and and / or comprising CDRs at about residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable domain and at about residues 26-32 (H1), 52-56 (H2), and 95-102 (H3) in the heavy chain variable domain, when numbered according to the Chothia numbering system described in Lefranc, JP, et al., Nucleic Acids Res 27:209-212; Ruiz, M., et al., Nucleic Acids Res When numbered according to the IMGT numbering system described in, e.g., J. Med. 28:219-221 (2000), the CDRs are contained at approximately residues 27-38 (L1), 56-65 (L2), and 105-117 (L3) in VL, and 27-38 (H1), 56-65 (H2), and 105-117 (H3) in VH.Equivalent residue positions can be annotated and compared for different molecules using the Antigen Receptor Numbering And Receptor Classification (ANARCI) software tool (2016, Bioinformatics 15:298-300). Thus, identification of the CDRs of an exemplary variable domain (VH or VL) sequence provided herein according to one numbering scheme does not exclude antibodies containing the CDRs of the same variable domain determined using a different numbering scheme.

[0084] As used herein, "specifically binds" refers to an antibody or antigen-binding fragment that binds to an antigen with a particular affinity, but does not significantly associate or combine with any other molecules or components in a sample. Affinity can be defined as the equilibrium association constant (Ka), calculated as the ratio of k / k, with units of 1 / M, or as the equilibrium dissociation constant (K), calculated as the ratio of k / k, with units of M.

[0085] In some contexts, antibodies and antigen-binding fragments may be described in terms of their affinity and / or avidity for the antigen. Unless otherwise indicated, avidity refers to the overall binding strength of an antibody or antigen-binding fragment to an antigen, which reflects the binding affinity of the antibody or antigen-binding fragment, the binding valency (e.g., whether the antibody or antigen-binding fragment contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more binding sites), and whether there are other agents present that can affect binding, for example, (e.g., noncompetitive inhibitors of the antibody or antigen-binding fragment).

[0086] Each embodiment herein applies mutatis mutandis to every other embodiment, unless expressly stated otherwise. It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method, kit, reagent, or composition of the invention, and vice versa. Furthermore, the compositions of the invention can be used to achieve the methods of the invention.

[0087] II. Overview The present disclosure provides an antibody or antibody-binding fragment thereof that binds to human MASP-2, an activator of the lectin pathway of the complement system. Lectins such as MBL, M-ficolin, H-ficolin, L-ficolin, collectin-10 and collectin-11 are specific recognition molecules that trigger the innate complement system. The complement system also includes a terminal pathway amplification loop that amplifies lectin-initiated complement activation to increase the release of terminal complement effector molecules.

[0088] In addition to its fundamental role in immune defense, the complement system also contributes to tissue damage in many clinical conditions. Therefore, there is an urgent need to develop therapeutically effective complement inhibitors to prevent these adverse effects. The realization that the complement lectin pathway can be inhibited while leaving the classical and alternative pathways intact leads to the realization that it would be highly desirable to specifically inhibit only the complement activation system that causes a particular pathology, without completely blocking the immune defense capabilities of complement. For example, in disease states where complement activation is primarily mediated by the lectin pathway, it would be advantageous to specifically inhibit only this pathway. This would leave the complement classical pathway intact to address immune complex processing and aid in host defense against infection.

[0089] One component of the complement system that could be targeted in the development of therapeutic agents to specifically inhibit the lectin pathway is MASP-2. Of all the known protein components of the lectin-dependent complement system (e.g., MBL, H ficolin, M ficolin, L ficolin, collectin, MASP-1, MASP-2, C4, and C2), only MASP-1 and MASP-2 are both unique to the lectin pathway and are necessary for the system to function. The lectins (e.g., MBL, H ficolin, M ficolin, L ficolin, collectin-10, and collectin-11) are also unique components in the lectin pathway. However, loss of any one of the lectin components would not necessarily inhibit activation of the system due to lectin redundancy. To ensure inhibition of the lectin-dependent complement activation system, it would be necessary to inhibit all lectins. Furthermore, since MBL and the ficolins are also known to have complement-independent opsonic activity, inhibition of lectin pathway function would result in the loss of this beneficial host defense mechanism against infection. In contrast, when MASP-2 is the target of inhibition, complement-independent opsonic activity remains intact. An additional advantage of MASP-2 as a therapeutic target for inhibiting the lectin pathway is that its plasma concentration is one of the lowest of any complement protein (approximately 500 ng / ml), and therefore a correspondingly low concentration of a high affinity inhibitor of MASP-2 may be sufficient to obtain complete inhibition (Moller Kristensen, M., et al., J. Immunol Methods 282:159 167, 2003). This is in stark contrast to MASP-1, which has a plasma concentration of approximately 10,000 ng / mL, and therefore would require a much higher concentration of a high affinity inhibitor of MASP-1 to obtain complete inhibition.

[0090] III. Antibodies and Antigen-Binding Fragments Provided herein are antibodies and antigen-binding fragments that specifically bind to MASP-2. In some embodiments, the antibodies and antigen-binding fragments are isolated monoclonal antibodies or antigen-binding fragments thereof. In some embodiments, the antibodies and antigen-binding fragments thereof inhibit lectin pathway complement activation. The antibodies and antigen-binding fragments described herein may be human, humanized, chimeric or murine antibodies, or antigen-binding fragments of any of the foregoing. In addition, the antibodies and antigen-binding fragments thereof may be single-chain antibodies, ScFvs, Fab fragments, Fab' fragments, F(ab')2 fragments, monovalent antibodies lacking the hinge region, or complete antibodies. Furthermore, the antibodies and antigen-binding fragments thereof may be monovalent, bivalent or multivalent.

[0091] In some embodiments, the antibodies and antigen-binding fragments described herein comprise an immunoglobulin constant region. The antibodies and antigen-binding fragments thereof can be IgG immunoglobulins or fragments thereof. In some embodiments, the IgG immunoglobulin is an IgG1, IgG2 or IgG4 immunoglobulin.

[0092] In some embodiments, the antibodies and antigen-binding fragments thereof described herein specifically bind to an epitope located within the serine protease domain of human MASP-2. In some embodiments, the epitope is located within the amino acid sequence of human MASP-2. Located within TIFF2024546109000003.tif4128. In some embodiments, the antibodies and antigen-binding fragments thereof compete with C4 binding to MASP-2.

[0093] In one aspect, the invention provides an antibody that provides an HC-CDR1 having the sequence NXXMH, where X at position 2 is H or Y and X at position 3 is H or W; HC-CDR2, represented as TIFF2024546109000004.tif4128, wherein X at position 4 is P or A, X at position 9 is T or I, X at position 10 is H or Y, X at position 12 is I or N, and X at position 13 is E or Q; The present invention provides an isolated antibody or antigen-binding fragment thereof that binds to MASP-2, comprising (a) a heavy chain variable region comprising an LC-CDR1 represented as SEQ ID NO:64 (SASSSVXYMY), where X at position 7 is R or S, an LC-CDR2 represented as SEQ ID NO:34 (DTSNLAS), and an LC-CDR3 represented as SEQ ID NO:36 (QQWSSYPLT). In one embodiment, the HC-CDR1 of the heavy chain variable region of (a) comprises SEQ ID NO:14 (NYWMH).

[0094] In some embodiments, the HC-CDR1 of the heavy chain variable region comprises SEQ ID NO: 14 (NYWMH). In some embodiments, the HC-CDR1 of the heavy chain variable region comprises SEQ ID NO: 56 (NYHMH). In some embodiments, the HC-CDR1 of the heavy chain variable region comprises SEQ ID NO: 57 (NHHMH).

[0095] In some embodiments, the HC-CDR2 of the heavy chain variable region is In some embodiments, the HC-CDR2 of the heavy chain variable region comprises In some embodiments, the HC-CDR2 of the heavy chain variable region comprises Includes TIFF2024546109000008.tif4128.

[0096] In some embodiments, the LC-CDR1 of the light chain variable region comprises SEQ ID NO: 32 (SASSSVRYMY). In some embodiments, the LC-CDR1 of the light chain variable region comprises SEQ ID NO: 39 (SASSSVSYMY).

[0097] In some embodiments, HC-CDR1 comprises SEQ ID NO:14, SEQ ID NO:56, or SEQ ID NO:57, HC-CDR2 comprises SEQ ID NO:16 or SEQ ID NO:53, HC-CDR3 comprises SEQ ID NO:18, LC-CDR1 comprises SEQ ID NO:32, LC-CDR2 comprises SEQ ID NO:34, and LC-CDR3 comprises SEQ ID NO:36. In some embodiments, HC-CDR1 comprises SEQ ID NO:14, HC-CDR2 comprises SEQ ID NO:16 or SEQ ID NO:53, HC-CDR3 comprises SEQ ID NO:18, LC-CDR1 comprises SEQ ID NO:32, LC-CDR2 comprises SEQ ID NO:34, and LC-CDR3 comprises SEQ ID NO:36. In some embodiments, HC-CDR1 comprises SEQ ID NO:56, HC-CDR2 comprises SEQ ID NO:16 or SEQ ID NO:53, HC-CDR3 comprises SEQ ID NO:18, LC-CDR1 comprises SEQ ID NO:32, LC-CDR2 comprises SEQ ID NO:34, and LC-CDR3 comprises SEQ ID NO:36. In some embodiments, HC-CDR1 comprises SEQ ID NO:57, HC-CDR2 comprises SEQ ID NO:16 or SEQ ID NO:53, HC-CDR3 comprises SEQ ID NO:18, LC-CDR1 comprises SEQ ID NO:32, LC-CDR2 comprises SEQ ID NO:34, and LC-CDR3 comprises SEQ ID NO:36.

[0098] In some embodiments, HC-CDR1 comprises SEQ ID NO:14 (NYWM) and HC-CDR2 comprises TIFF2024546109000009.tif4128, HC-CDR3 In some embodiments, HC-CDR1 comprises SEQ ID NO: 14 (NYWM) and HC-CDR2 comprises SEQ ID NO: 36 (QQWSSYPLT). TIFF2024546109000011.tif4128, HC-CDR3 In some embodiments, HC-CDR1 comprises SEQ ID NO:56 (NYHMH) and HC-CDR2 comprises SEQ ID NO:37 (QQWSSYPLT). TIFF2024546109000013.tif4128, HC-CDR3 In some embodiments, HC-CDR1 comprises SEQ ID NO:57 (NHHMH) and HC-CDR2 comprises SEQ ID NO:38 (QQWSSYPLT). TIFF2024546109000015.tif4128, HC-CDR3 In some embodiments, HC-CDR1 comprises SEQ ID NO: 14 (NYWM) and HC-CDR2 comprises SEQ ID NO: 36 (QQWSSYPLT). TIFF2024546109000017.tif4128, in which HC-CDR3 comprises SEQ ID NO: 18 (GDITTTLRYFDV), LC-CDR1 comprises SEQ ID NO: 39 (SASSSVSYMY), LC-CDR2 comprises SEQ ID NO: 34 (DTSNLAS), and LC-CDR3 comprises SEQ ID NO: 36 (QQWSSYPLT).

[0099] In some embodiments, HC-CDR1 comprises SEQ ID NO:14, HC-CDR2 comprises SEQ ID NO:22, HC-CDR3 comprises SEQ ID NO:18, LC-CDR1 comprises SEQ ID NO:39, LC-CDR2 comprises SEQ ID NO:34, and LC-CDR3 comprises SEQ ID NO:36.

[0100] In some embodiments, HC-CDR1 comprises SEQ ID NO:25, HC-CDR2 comprises SEQ ID NO:27, HC-CDR3 comprises SEQ ID NO:29, LC-CDR1 comprises SEQ ID NO:41, LC-CDR2 comprises SEQ ID NO:43, and LC-CDR3 comprises SEQ ID NO:45.

[0101] In some embodiments, the heavy chain variable region comprises SEQ ID NO:7, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:49, or SEQ ID NO:50. In further embodiments, the heavy chain variable region is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO:7, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:49, or SEQ ID NO:50. In some embodiments, the light chain variable region comprises SEQ ID NO:10 or SEQ ID NO:47. In further embodiments, the light chain variable region is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO:10 or SEQ ID NO:47. In some embodiments, the heavy chain variable region comprises SEQ ID NO:7, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:49, or SEQ ID NO:50, and the light chain variable region comprises SEQ ID NO:10 or SEQ ID NO:47.

[0102] In some embodiments, the heavy chain variable region comprises SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:49, or SEQ ID NO:50. In further embodiments, the heavy chain variable region is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:49, or SEQ ID NO:50. In some embodiments, the light chain variable region comprises SEQ ID NO:47. In further embodiments, the light chain variable region is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO:47. In some embodiments, the heavy chain variable region comprises SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:49, or SEQ ID NO:50 and the light chain variable region comprises SEQ ID NO:47.

[0103] In some embodiments, the heavy chain variable region comprises SEQ ID NO:8. In further embodiments, the heavy chain variable region is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO:8. In some embodiments, the light chain variable region comprises SEQ ID NO:11. In further embodiments, the light chain variable region is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO:11. In some embodiments, the heavy chain variable region comprises SEQ ID NO:8 and the light chain variable region comprises SEQ ID NO:11.

[0104] In some embodiments, the heavy chain variable region comprises SEQ ID NO:9. In further embodiments, the heavy chain variable region is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO:9. In some embodiments, the light chain variable region comprises SEQ ID NO:12. In further embodiments, the light chain variable region is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO:12. In some embodiments, the heavy chain variable region comprises SEQ ID NO:9 and the light chain variable region comprises SEQ ID NO:12.

[0105] In certain embodiments, the antibody or antigen-binding fragment thereof comprises zero, one, two or three amino acid substitutions in each of the six CDRs, where one or more of these substitutions are optionally conservative substitutions and / or substitutions to germline encoded amino acids.

[0106] The sequences of the variable regions and CDRs for certain anti-MASP-2 antibodies described herein are summarized in Tables 1A, 1B and 1C below ("SIN" indicates SEQ ID NO).

[0107] Table 1A. Summary of MASP-2 antibody sequences TIFF2024546109000018.tif82135

[0108] Table 1B. Summary of sequences and CDRs of MASP-2 high affinity inhibitory antibodies TIFF2024546109000019.tif48140

[0109] Table 1C. Summary of MASP-2 antibody OMS850 and humanized and modified versions TIFF2024546109000020.tif69134

[0110] In certain embodiments, the antibodies and antigen-binding fragments described herein comprise one or more mutations in the Fc region. For example, the Fc region may comprise one or more mutations that enhance stability or effector function. In some embodiments, the Fc region comprises an S228P amino acid substitution. In some embodiments, the Fc region comprises one or more mutations that promote FcRn interaction at low pH.

[0111] In some embodiments, the antibodies and antigen-binding fragments described herein bind to the serine protease domain of human MASP-2 at less than 0.2 nM, less than 0.3 nM, less than 0.4 nM, less than 0.5 nM, less than 0.6 nM, less than 0.7 nM, less than 0.8 nM, less than 0.9 nM, less than 1.0 nM, less than 1.2 nM, less than 1.4 nM, less than 1.6 nM, less than 1.8 nM, less than 2.0 nM, less than 2.5 nM, less than 3.0 nM, less than 3.5 nM or less than 30 nM.

[0112] In some embodiments, the antibodies and antigen-binding fragments described herein inhibit the lectin pathway of complement activation. In some embodiments, the antibodies and antigen-binding fragments inhibit the lectin pathway in mammalian blood. In some embodiments, the lectin pathway inhibition comprises a reduction in the adhesion of complement components on target cells. In some embodiments, the lectin pathway inhibition comprises a reduction in C3b adhesion, C4 adhesion or MAC adhesion. In some embodiments, the lectin pathway inhibition comprises a reduction in the adhesion of complement components under lectin pathway specific assay conditions. In some embodiments, the antibodies and antigen-binding fragments described herein inhibit the lectin pathway of complement activation in mammalian blood but do not inhibit the classical pathway of complement activation in mammalian blood.

[0113] IV. Polynucleotides, Vectors and Host Cells In another aspect, the disclosure provides isolated polynucleotides encoding any of the antibodies or antigen-binding fragments thereof disclosed herein or portions thereof (e.g., CDR, VH, VL, heavy chain, or light chain). In certain embodiments, the polynucleotides are codon-optimized for expression in a host cell. Once the coding sequence is known or identified, codon optimization can be performed using known techniques and tools, such as the GenScript® OptimumGene™ tool or ThermoFisher Scientific® GeneArt GeneOptimizer™. Codon-optimized sequences include partially codon-optimized sequences, having one or more codons optimized for expression in a host cell, and fully codon-optimized sequences. It will also be understood that polynucleotides encoding antibodies and antigen-binding fragments thereof may have different nucleotide sequences while encoding the same protein due to the degeneracy of the genetic code, splicing, etc.

[0114] In some embodiments, the polynucleotide comprises a polynucleotide having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one or more of SEQ ID NOs:69-79.

[0115] In certain embodiments, a polynucleotide encoding an antibody or antigen-binding fragment thereof may be included in a polynucleotide that includes other sequences and / or features. For example, a polynucleotide may include one or more sequences useful for control or expression of the encoded protein, such as a promoter sequence, a polyadenylation sequence, a sequence encoding a signal peptide, etc. A polynucleotide may include deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

[0116] Also provided are vectors that contain or contain a polynucleotide encoding any of the antibodies or antigen-binding fragments thereof disclosed herein. Any suitable vector may be used, including viral vectors and plasmid vectors. In certain embodiments, the vector comprises a polynucleotide encoding both an antibody heavy chain or antigen-binding fragment thereof and an antibody light chain or antigen-binding fragment thereof, which together constitute a complete antibody or antigen-binding fragment thereof. The sequences encoding the antibody heavy chain or antigen-binding fragment thereof and the antibody light chain or antigen-binding fragment thereof may be contained within a single open reading frame. In this case, they may be optionally separated by a polynucleotide encoding a protease cleavage site and / or a polynucleotide encoding a self-cleaving peptide. Alternatively, the sequences encoding the antibody heavy chain or antigen-binding fragment thereof and the antibody light chain or antigen-binding fragment thereof may be contained within separate open reading frames on a single vector. In another embodiment, the sequences encoding the antibody heavy chain or antigen-binding fragment thereof and the antibody light chain or antigen-binding fragment thereof are present on two different vectors, with a first vector encoding the antibody heavy chain or fragment thereof and a second vector encoding the antibody light chain or fragment thereof.

[0117] In a further aspect, the present disclosure also provides a host cell comprising the polynucleotide or vector disclosed herein. Any suitable cell into which such polynucleotide or vector can be introduced can be used. Examples of such cells include eukaryotic cells, such as yeast cells, animal cells, insect cells, mammalian cells, and plant cells, and prokaryotic cells, such as bacterial cells, such as E. coli. In some embodiments, the host cell is a mammalian cell. In some embodiments, the host cell is an immortalized mammalian cell line. Cells suitable for use in the production and expression of polynucleotides and vectors are known in the art.

[0118] In some embodiments, cells may be transfected with the polynucleotide or vector disclosed herein. The term "transfection" includes any method known to those skilled in the art for introducing a nucleic acid molecule into a cell. Such methods include, for example, electroporation, lipofection, nanoparticle-based transfection, virus-based transfection, etc. Host cells may be stably or transiently transfected.

[0119] In some embodiments, the host cell expresses the antibody or antigen-binding fragment thereof encoded by the polynucleotide or vector. Such expression may include post-translational modifications such as removal of signal sequences, glycosylation, and other modifications. In a related aspect, the disclosure provides a method for producing the provided antibodies and antigen-binding fragments thereof, comprising culturing host cells under conditions that allow expression of the antibody or antigen-binding fragment thereof for a sufficient time, and isolating the antibody or antigen-binding fragment thereof. Methods useful for isolating and purifying recombinantly produced proteins include, for example, obtaining a supernatant from a suitable host cell that secretes the protein into the culture medium, concentrating the medium, and purifying the protein by passing the concentrate through a suitable purification matrix or series of matrices. Methods for purifying proteins are well known in the art.

[0120] V. Pharmaceutical Compositions Also provided herein are compositions that include a therapeutic agent selected from any one or more of the antibodies or antigen-binding fragments thereof, polynucleotides, vectors, or host cells disclosed herein, alone or in any combination, and may also include other selected therapeutic agents. Such compositions may further include one or more pharma- ceutically acceptable carriers, excipients, or diluents.

[0121] Pharmaceutically acceptable carriers are selected so as to be non-toxic, biocompatible, and not to adversely affect the biological activity of the therapeutic agent (and any other therapeutic agents with which it is combined). Examples of pharma-ceutically acceptable carriers for peptides are described in Yamada, U.S. Patent No. 5,211,657. The therapeutic agents described herein can be formulated into solid, semi-solid, gel, liquid, or gaseous preparations, such as tablets, capsules, powders, granules, ointments, solutions, depositories, inhalants, and injections, that allow for oral, parenteral, or surgical administration. Local administration of the composition by coating a medical device is also envisioned.

[0122] Suitable carriers for parenteral delivery by injection, infusion or irrigation and topical delivery include distilled water, physiological phosphate buffered saline, normal or lactated Ringer's solution, dextrose solution, Hank's solution, or propanediol. In addition, sterile fixed oils can be used as a solvent or suspending medium. Any biocompatible oil, including synthetic mono- or di-glycerides, can be used for this purpose. In addition, fatty acids such as oleic acid are also useful for the preparation of injectables. The carrier and the agent can be formulated as a liquid, a suspension, a polymeric or non-polymeric gel, a paste, or a salve.

[0123] The carrier may also include a delivery vehicle to sustain (i.e., extend, delay, or control) the delivery of the agent or to enhance the delivery, uptake, stability, or pharmacokinetics of the therapeutic agent. Such delivery vehicles may include, by way of non-limiting example, microparticles, microspheres, nanospheres, or nanoparticles composed of proteins, liposomes, carbohydrates, synthetic organic compounds, inorganic compounds, polymer or copolymer hydrogels, and polymeric micelles. Suitable hydrogel and micelle delivery systems include PEO:PHB:PEO copolymers and copolymer / cyclodextrin complexes as disclosed in WO 2004 / 009664 A2, and PEO and PEO / cyclodextrin complexes as disclosed in US Patent Application Publication No. 2002 / 0019369 A1. Such hydrogels may be injected locally at the intended site of action or subcutaneously or intramuscularly to form a sustained release depot.

[0124] The compositions of the present invention may be formulated for delivery by any suitable method, including, but not limited to, oral, topical, transdermal, sublingual, buccal, subcutaneous, intramuscular, intravenous, intraarterial, or as an inhalant. The compositions of the present invention may also include biocompatible excipients, such as dispersing or wetting agents, suspending agents, diluents, buffers, penetration enhancers, emulsifying agents, binders, thickening agents, flavoring agents (for oral administration).

[0125] Pharmaceutical compositions according to certain aspects of the present invention are formulated so that the active ingredients contained therein are bioavailable when the compositions are administered to a patient. The compositions to be administered to a subject may be in the form of one or more dosage units. And the therapeutic agent containers described herein may hold multiple dosage units. The actual methods of preparing such dosage forms will be known or apparent to those skilled in the art. See, for example, "Remington: The Science and Practice of Pharmacy", 20th Edition (Philadelphia College of Pharmacy and Science, 2000). The compositions to be administered will, in any case, contain an effective amount of the therapeutic agent or composition of the present disclosure for treating the disease or condition of interest, according to the teachings of this specification.

[0126] The composition may be in solid or liquid form.In some embodiments, the carrier is granular, and thus the composition is in the form of, for example, tablet or powder.The carrier may be liquid, and in this case the composition is, for example, oral oil, injectable liquid, or aerosol, which is useful for inhalation administration, etc.When intended for oral administration, the pharmaceutical composition is preferably in solid or liquid form, where semi-solid, semi-liquid, suspension and gel form are included in the form considered as solid or liquid in this specification.

[0127] As a solid composition for oral administration, pharmaceutical compositions can be formulated into powders, granules, compressed tablets, pills, capsules, chewing gum, wafers, etc. Such solid compositions will typically contain one or more inert fillers or diluents, such as sucrose, corn starch, or cellulose. In addition, one or more of the following may be present: binders, such as carboxymethylcellulose, ethylcellulose, microcrystalline cellulose, tragacanth or gelatin; excipients, such as starch, lactose or dextrin; disintegrants, such as alginic acid, sodium alginate, primogel, corn starch, etc.; lubricants, such as magnesium stearate or sterotex; glidants, such as colloidal silicon dioxide; sweeteners, such as sucrose or saccharin; flavorings, such as peppermint, peppermint, methyl salicylate, or orange flavoring; and colorings. When the composition is in the form of a capsule, for example, a gelatin capsule, it may contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol or oil.

[0128] The composition can be in the form of liquid, such as elixir, syrup, solution, emulsion or suspension.Liquid can be for oral administration or for delivery by injection, to name just two examples.When intended for oral administration, preferred compositions contain, in addition to the compound, one or more of sweeteners, preservatives, dyes / colorants and flavor enhancers.Compositions intended for administration by injection can contain one or more of surfactants, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers and isotonicity agents.

[0129] Liquid pharmaceutical compositions, whether in solution, suspension, or other similar form, may contain one or more of the following excipients: sterile diluents, such as water for injection, saline solution, preferably saline, Ringer's solution, isotonic sodium chloride, fixed oils, such as synthetic mono- or diglycerides, polyethylene glycols, glycerin, propylene glycol, and other solvents that may serve as solvents or suspending media; antibacterial agents, such as benzyl alcohol or methylparabens; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetates, citrates, or phosphates, and agents for adjusting tonicity, such as sodium chloride or dextrose. Parenteral preparations may be enclosed in glass or plastic ampoules, disposable syringes, or multiple dose vials. Saline is the preferred excipient. Injectable pharmaceutical compositions are preferably sterile.

[0130] Liquid compositions intended for either parenteral or oral administration should contain an amount of the therapeutic agent described herein so that an appropriate dosage will be obtained. The term "parenteral" includes subcutaneous, intravenous, intramuscular, intrasternal, or intraarterial injection or infusion. Typically, the therapeutic agent is at least 0.01% of the composition. When intended for oral administration, this amount can vary and be between about 0.1% and about 70% by weight of the composition. Certain oral pharmaceutical compositions contain about 4% to about 75% of the therapeutic agent.

[0131] The composition may be intended for external administration, in which case the carrier may suitably comprise a solution, emulsion, ointment or gel base. The base may, for example, comprise one or more of the following: petrolatum, lanolin, polyethylene glycol, beeswax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers. A thickener may be present in a composition for external administration. If intended for transdermal administration, the composition may comprise a transdermal patch or an iontophoresis device. The pharmaceutical composition may be intended for rectal administration in the form of, for example, a suppository that melts in the rectum and releases the drug. The composition for rectal administration may contain an oily base as a suitable non-irritating excipient. Such bases include, but are not limited to, lanolin, cocoa butter and polyethylene glycol.

[0132] The composition may contain various materials that modify the physical form of the solid or liquid dosage unit. For example, the composition may contain a material that forms a coating shell around the active ingredient. The material that forms the coating shell is typically inert and can be selected from, for example, sugar, shellac and other enteric coating agents. Alternatively, the active ingredient can be encapsulated in a gelatin capsule. The solid or liquid composition may contain an agent that binds to the therapeutic agent of the present disclosure and thereby aids in the delivery of the compound. Suitable agents that can perform this function include one or more proteins or liposomes.

[0133] The composition may consist essentially of a dosage unit that can be administered as an aerosol. The term aerosol is used to describe a variety of systems, from colloidal to systems consisting of pressurized packages. Delivery may be by liquefied or compressed gas, or by a suitable pump system that dispenses the active ingredient. The aerosol may be delivered in a single-phase, two-phase, or three-phase system to deliver the active ingredient. Delivery of the aerosol includes the necessary containers, activators, valves, subcontainers, etc., which may form a kit as a whole. Those skilled in the art may determine the preferred aerosol without undue experimentation.

[0134] It will be understood that the compositions of the present disclosure also encompass carrier molecules (e.g., lipid nanoparticles, nanoscale delivery platforms, etc.) for the polynucleotides described herein.

[0135] Pharmaceutical compositions can be prepared by methodologies well known in the pharmaceutical field. For example, compositions intended for administration by injection can be prepared by mixing a composition comprising the therapeutic agent described herein and, optionally, one or more salts, buffers and / or stabilizers with sterile distilled water to form a solution. Surfactants may be added to facilitate the formation of a homogeneous solution or suspension. Surfactants are compounds that non-covalently interact with the composition to facilitate dissolution or homogeneous suspension in aqueous delivery systems.

[0136] VI. Methods and Uses Further provided herein is a method of using an antibody or antigen-binding fragment, a nucleic acid, a vector, a cell, or a composition of the present disclosure in activating the lectin pathway of complement in a mammalian subject. In some embodiments, the method comprises administering to a mammalian subject in need thereof an antibody or antigen-binding fragment, a nucleic acid, a vector, a cell, or a composition of the present disclosure in an amount sufficient to inhibit the lectin pathway of complement activation in said mammal. In some embodiments, the method can further comprise determining that the subject suffers from a disease or disorder of the lectin pathway before administering to the subject a compound of the present disclosure. In some embodiments, the mammalian subject is a human.

[0137] In certain aspects, the antibodies or antigen-binding fragments, nucleic acids, vectors, cells, or compositions of the disclosure are provided for use in methods of treating a disease or disorder of the lectin pathway.In certain aspects, the antibodies or antigen-binding fragments, nucleic acids, vectors, cells, or compositions of the disclosure are provided for use in methods of manufacturing or preparing a medicament for treating a disease or disorder of the lectin pathway.

[0138] No. 7,919,094, U.S. Pat. No. 8,840,893, U.S. Pat. No. 8,652,477, U.S. Pat. No. 8,951,522, U.S. Pat. No. 9,011,860, U.S. Pat. No. 9,475,885, U.S. Pat. No. 9,644,035, U.S. Patent Application Publication Nos. US2013 / 0344073, US2013 / 0266560, and US No. 2015 / 0166675, No. US2017 / 0137537, No. US2017 / 0166660, No. US2017 / 0189525, No. US2017 / 0267781, No. US2017 / 0283508, No. US2017 / 0253667, No. US2018 / 0105604, As described in WO2018 / 045054, WO2018 / 071701, WO2019 / 036460, WO2019 / 246,367, WO2021 / 178902 and co-pending U.S. patent application Ser. No. 17 / 103,672 (each of which is assigned to the assignee of the present application, Omeros Corporation, and each of which is incorporated by reference herein), MASP-2-dependent complement activation has been implicated as contributing to the pathogenesis of numerous acute and chronic disease conditions. For example, as described in U.S. Pat. No. 8,951,522, the main function of the complement system, which is part of the innate immune system, is to protect the host from infectious agents, but inappropriate or excessive activation of the complement system can lead to severe diseases, such as endothelial damage in microtubule structures and thrombotic microangiopathies (TMA, including aHUS, TTP, and HUS), in which fibrin adhesion and platelet-rich thrombi lead to organ damage. The lectin pathway plays a major role in activating complement in the context of endothelial cell stress or injury, and preventing activation of MASP-2 and the lectin pathway will stop the series of enzymatic reactions that lead to the formation of the membrane attack complex, platelet activation, and leukocyte recruitment. As described in U.S. Pat. No. 8,652,477, in addition to initiating the lectin pathway, MASP-2 can also activate the coagulation system and has the ability to cleave prothrombin into thrombin.

[0139] Thus, in some embodiments, the disease or disorder of the lectin pathway is selected from the group consisting of thrombotic microangiopathy (TMA), kidney disease, inflammatory responses resulting from tissue or organ transplantation, ischemia-reperfusion injury, complications associated with diabetes, complications associated with hemodialysis, cardiovascular diseases or disorders, inflammatory gastrointestinal disorders, pulmonary disorders, ophthalmologic diseases or disorders, disseminated intravascular coagulation, graft-versus-host disease, veno-occlusive disease, and diffuse alveolar hemorrhage.

[0140] In some embodiments, a combination therapy is provided that includes one or more of the disclosed antibodies or antigen-binding fragments, nucleic acids, vectors, cells, or compositions and one or more additional therapeutic agents. It will be understood that in such a combination therapy, the one or more of the disclosed antibodies or antigen-binding fragments, nucleic acids, vectors, cells, or compositions and one or more additional therapeutic agents can be administered in any order and sequence, at any interval, or simultaneously.

[0141] In some embodiments, the disease or disorder of the lectin pathway is thrombotic microangiopathy (TMA), such as thrombotic thrombocytopenic purpura (TTP), refractory TTP, Upshaw-Schulman syndrome (USS), hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), non-factor H-dependent atypical hemolytic syndrome, aHUS secondary to infection, plasma therapy-refractory aHUS, TMA secondary to cancer, TMA secondary to chemotherapy or other anti-cancer treatment, TMA secondary to transplantation, or TMA associated with hematopoietic stem cell transplantation, TMA secondary to infection, and IgA vasculitis.

[0142] In some embodiments, the disease or disorder of the lectin pathway is graft-versus-host disease (GVHD), such as acute GVHD, chronic GVHD, or steroid-resistant GVHD. In some embodiments, the subject suffering from GVHD or at risk of developing GVHD has previously undergone, is currently undergoing, or will undergo hematopoietic stem cell transplantation.

[0143] In some embodiments, the disease or disorder of the lectin pathway is diffuse alveolar hemorrhage (DAH). In some embodiments, the subject suffering from or at risk of developing DAH has previously undergone, is currently undergoing, or is scheduled to undergo hematopoietic stem cell transplantation.

[0144] In some embodiments, the disease or disorder of the lectin pathway is capillary leak syndrome, engraftment syndrome, fluid overload or idiopathic pneumonia syndrome.In some embodiments, the subject suffering from one or more of these diseases or disorders or at risk of developing one or more of these diseases or disorders has previously undergone, is currently undergoing, or will undergo hematopoietic stem cell transplantation.

[0145] In some embodiments, the disease or disorder of the lectin pathway is veno-occlusive disease (VOD). In some embodiments, the subject suffering from or at risk of developing VOD has previously undergone, is currently undergoing, or is scheduled to undergo hematopoietic stem cell transplantation.

[0146] In some embodiments, the disease or disorder of the lectin pathway is a neurological symptom associated with graft-versus-host disease or TMA, such as asthenia, dysesthesias, limb paralysis, sensorimotor deficits, autonomic neuropathic polyneuropathy, or neurogenic bladder.

[0147] In some embodiments, the disease or disorder of the lectin pathway is a kidney disease, including, but not limited to, mesangial proliferative glomerulonephritis, membranous glomerulonephritis, membranoproliferative glomerulonephritis (mesangial capillary glomerulonephritis), acute post-infectious glomerulonephritis (post-streptococcal glomerulonephritis), C3 glomerulopathy, cryoglobulinemic glomerulonephritis, microimmune necrotizing crescentic glomerulonephritis, lupus nephritis, Henoch-Schönlein purpura nephritis, and IgA nephropathy.

[0148] In some embodiments, a disease or disorder of the lectin pathway is selected from the group consisting of renal fibrosis (e.g., tubulointerstitial fibrosis) and / or proteinuria in subjects suffering from or at risk of developing chronic kidney disease, chronic renal failure, scleroderma in the kidney (including scleroderma renal crisis), glomerular disease (e.g., focal segmental glomerulosclerosis), immune complex disorders (e.g., IgA nephropathy, membranous nephropathy), lupus nephritis, nephrotic syndrome, diabetic nephropathy, tubulointerstitial injury, and glomerulonephritis (e.g., C 3 glomerulopathy), or diseases or conditions associated with proteinuria, including, but not limited to, nephrotic syndrome, pre-eclampsia, eclampsia, toxic lesions of the kidney, amyloidosis, collagen vascular disease (e.g., systemic lupus erythematosus), dehydration, glomerular disease (e.g., membranous glomerulonephritis, membranous glomerulonephropathy, focal segmental glomerulonephritis, C3 glomerulopathy, minimal change disease, lipoid nephrosis), strenuous exercise, stress, benign orthostatic (postural) proteinuria, focal segmental glomerulosclerosis, IgA nephropathy (s nephropathy (i.e. Berger's disease), IgM nephropathy, membranoproliferative glomerulonephritis, membranous nephropathy, minimal change disease, sarcoidosis, Alport syndrome, diabetes mellitus (diabetic nephropathy), drug-induced toxicity (e.g. NSAIDS, nicotine, penicillamine, lithium carbonate, gold and other heavy metals, ACE inhibitors, antibiotics (e.g. adriamycin) or opiates (e.g. heroin) or other nephrotoxins), Fabry's disease, infections (e.g. HIV, syphilis, hepatitis A, B or C, post-streptococcal infections, urinary schistosis) These conditions include trematodiases, aminoaciduria, Fanconi syndrome, hypertensive nephrosclerosis, interstitial nephritis, sickle cell disease, hemoglobinuria, multiple myeloma, myoglobinuria, organ rejection (e.g. kidney transplant rejection), Ebola haemorrhagic fever, nail-patella syndrome, familial Mediterranean fever, HELLP syndrome, systemic lupus erythematosus, Wegener's granulomatosis, rheumatoid arthritis, glycogen storage disease type 1, Goodpasture's syndrome, Henoch-Schönlein purpura, urinary tract infection involving the kidney, Sjögren's syndrome and post-infectious glomerulonephritis.

[0149] In some embodiments, the disease or disorder of the lectin pathway is an inflammatory response resulting from tissue or solid organ transplantation, including, but not limited to, allografts or xenografts of whole organs (e.g., kidney, heart, liver, pancreas, lung, cornea, etc.) or tissue grafts (e.g., valves, tendons, bone marrow, etc.). In some embodiments, the disease or disorder of the lectin pathway is delayed organ transplant function.

[0150] In some embodiments, the disease or disorder of the lectin pathway is ischemia-reperfusion injury (I / R), including but not limited to, myocardial I / R, gastrointestinal I / R, renal I / R, and I / R following aortic aneurysm repair, I / R associated with cardiopulmonary bypass, cerebral I / R, stroke, vascular reanastomosis associated with organ transplantation or reattachment of a severed or traumatized limb or digit, revascularization of grafts and / or regrafts, and hemodynamic resuscitation following shock and / or surgery.

[0151] In some embodiments, the disease or disorder of the lectin pathway is a complication associated with non-obese diabetes (type 1 diabetes or insulin-dependent diabetes mellitus) and / or a complication associated with type 1 or type 2 (adult-onset) diabetes, such as, but not limited to, diabetic vasculopathy, diabetic neuropathy, diabetic retinopathy, or diabetic macular edema.

[0152] In some embodiments, the lectin pathway disease or disorder is a cardiovascular disease or disorder, including, but not limited to, Henoch-Schonlein purpura nephritis, systemic lupus erythematosus-associated vasculitis, vasculitis associated with rheumatoid arthritis (also called malignant rheumatoid arthritis), immune complex vasculitis, antineutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis, Takayasu's disease, dilated cardiomyopathy, diabetic vasculopathy, Kawasaki disease (arteritis), venous gas embolism (VGE), and inhibition of restenosis after stent placement, rotational atherectomy and / or percutaneous transluminal coronary angioplasty (PTCA).

[0153] In some embodiments, the disease or disorder of lectin pathway is inflammatory gastrointestinal disorder, such as, but not limited to, pancreatitis, diverticulitis, and intestinal disorders, such as Crohn's disease, ulcerative colitis, irritable bowel syndrome, and inflammatory bowel disease (IBD).In some embodiments, the disease or disorder of lectin pathway is caused or aggravated by fibrosis of the digestive tract.In some embodiments, the disease or disorder of lectin pathway is pancreatic fibrosis.

[0154] In some embodiments, the disease or disorder of the lectin pathway is a pulmonary disorder, including but not limited to, acute respiratory distress syndrome, transfusion-associated acute lung injury, ischemia / reperfusion acute lung injury, chronic obstructive pulmonary disease, asthma, Wegener's granulomatosis, antiglomerular basement membrane disease (Goodpasture's disease), meconium aspiration syndrome, aspiration pneumonia, bronchiolitis obliterans syndrome, idiopathic pulmonary fibrosis, acute lung injury secondary to burn injury, noncardiogenic pulmonary edema, transfusion-associated respiratory depression, and emphysema.

[0155] In some embodiments, the disease or disorder of the lectin pathway is extracorporeal exposure-triggered inflammatory reaction and the method includes treating a subject undergoing extracorporeal circulation procedures, including, but not limited to, hemodialysis, plasmapheresis, leukopheresis, extracorporeal membrane oxygenation (ECMO), heparin-induced extracorporeal membrane oxygenation LDL precipitation (HELP), and cardiopulmonary bypass (CPB).

[0156] In some embodiments, the disease or disorder of the lectin pathway is selected from inflammatory or non-inflammatory arthritis and other musculoskeletal disorders, including but not limited to osteoarthritis, rheumatoid arthritis, juvenile rheumatoid arthritis, gout, neuropathic arthropathy, psoriatic arthritis, ankylosing spondylitis or other spondyloarthropathy and crystal arthropathies, muscular dystrophies, and systemic lupus erythematosus (SLE).

[0157] In some embodiments, the disease or disorder of the lectin pathway is for the treatment of skin disorders, such as, but not limited to, psoriasis, autoimmune bullous dermatoses, eosinophilic spongiosis, bullous pemphigoid, epidermolysis bullosa acquisita, atopic dermatitis, herpes gestationis and other skin disorders, as well as thermal and chemical burns, including capillary leakage caused thereby.

[0158] In some embodiments, the lectin pathway disease or disorder is a peripheral nervous system (PNS) and / or central nervous system (CNS) disorder or injury, including, but not limited to, multiple sclerosis (MS), myasthenia gravis (MG), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), Guillain-Barre syndrome, reperfusion after stroke, degenerative discs, brain trauma, Parkinson's disease (PD), Alzheimer's disease (AD), Miller-Fisher syndrome, brain trauma and / or cerebral hemorrhage, traumatic brain injury, CNS fibrosis, demyelination, and meningitis.

[0159] In some embodiments, the disease or disorder of the lectin pathway is sepsis or a condition resulting from sepsis, such as, but not limited to, severe sepsis, septic shock, acute respiratory distress syndrome resulting from sepsis, hemolytic anemia, systemic inflammatory response syndrome, or hemorrhagic shock.

[0160] In some embodiments, the disease or disorder of the lectin pathway is a urogenital disorder, including but not limited to, bladder disease, sensory bladder disease, chronic sterile cystitis and interstitial cystitis, male and female infertility, placental dysfunction, and miscarriage and pre-eclampsia.

[0161] In some embodiments, the disease or disorder of the lectin pathway is an inflammatory response in a subject undergoing chemotherapy and / or radiation therapy, for example, but not limited to, for the treatment of a cancerous condition.

[0162] In some embodiments, the lectin pathway disease or disorder is angiogenesis-dependent cancer, including but not limited to solid tumors, blood borne tumors, high-risk carcinoid tumors, and tumor metastasis. In some embodiments, the lectin pathway disease or disorder is angiogenesis-dependent benign tumor, including but not limited to hemangioma, acoustic neuroma, neurofibroma, trachoma, carcinoid tumor, and pyogenic granuloma.

[0163] In some embodiments, the disease or disorder of the lectin pathway is an endocrine disorder, such as, but not limited to, Hashimoto's thyroiditis, stress, anxiety, and other potential hormonal disorders involving the regulated release of prolactin, growth factors or insulin-like growth factors, and adrenocorticotropic hormone from the pituitary gland.

[0164] In some embodiments, the disease or disorder of the lectin pathway is an ophthalmic disease or disorder, including but not limited to age-related macular degeneration, glaucoma and endophthalmitis.In some embodiments, the disease or disorder of the lectin pathway is an intraocular neovascular disease or condition, including but not limited to age-related macular degeneration, uveitis, ocular melanoma, corneal neovascularization, primary pterygium, HSV stromal keratitis, HSV-1-induced corneal lymphangiogenesis, proliferative diabetic retinopathy, diabetic macular edema, retinopathy of prematurity, retinal vein occlusion, corneal graft rejection, neovascular glaucoma, vitreous hemorrhage secondary to proliferative diabetic retinopathy, neuromyelitis optica, anterior subcapsular cataract, posterior capsular opacification and rubeosis.

[0165] In some embodiments, the disease or disorder of the lectin pathway is disseminated intravascular coagulation (DIC) or other complement-mediated coagulopathy, e.g., DIC secondary to sepsis, severe trauma, e.g., neurological trauma (e.g., acute head trauma, see Kumura et al, Acta Neurochirurgica 55:23-28 (1987)), infection (bacterial, viral, fungal, parasitic), cancer, obstetric complications, liver disease, severe toxic reactions {e.g., snakebite, sting, transfusion reactions), shock, heat stroke, transplant rejection, vascular aneurysm, liver failure, chemotherapy or radiation treatment for cancer, burns, or accidental radiation exposure.

[0166] In some embodiments, the disease or disorder of the lectin pathway is selected from the group consisting of acute radiation syndrome, dense deposit disease, Degos disease, fulminant antiphospholipid syndrome (CAPS), Behcet's disease, cryoglobulinemia, paroxysmal nocturnal hemoglobinuria ("PNH") and cold agglutinin disease.

[0167] In some embodiments, the disease or disorder of the lectin pathway is selected from the group consisting of aHUS, HSCT-TMA, IgAN, and lupus nephritis (LN).

[0168] In some embodiments, the lectin pathway disease or disorder is associated with fibrin-induced activation of the complement system and associated activation of the coagulation and / or contact systems. In some embodiments, the lectin pathway disease or disorder or condition is associated with complement-associated inflammation, excessive coagulation or contact system activation initiated by fibrin or activated platelets. In some embodiments, the disease or disorder of the lectin pathway is selected from the group consisting of arterial thrombosis, venous thrombosis, deep vein thrombosis, postoperative thrombosis, restenosis after coronary artery bypass grafting and / or interventional cardiovascular procedures (e.g., angioplasty or stent placement), atherosclerosis, plaque rupture, plaque instability, restenosis, hypotension, acute respiratory distress syndrome (ARDS), systemic inflammatory response syndrome (SIRS), disseminated intravascular coagulation (DIC), venous occlusive disease (VOD), thrombotic microangiopathy, lupus nephritis, superficial thrombophlebitis, factor V Leiden mutation, ischemia / reperfusion injury, human immunodeficiency virus (HIV) infection, ongoing hormone replacement therapy (HRT), Alzheimer's disease, and / or hypercoagulable states.In some embodiments, the lectin pathway, an acquired hypercoagulable state due to at least one or more of the following: ongoing treatment with a drug selected from the group consisting of 5-FU, GM-CSF, cisplatin, heparin, COX-2 inhibitors, contrast agents, corticosteroids and antipsychotics; venous congestion (immobility, surgery, etc.), antiphospholipid syndrome, cancer (promyelocytic leukemia, lung, breast, prostate, pancreatic, cervical, esophageal squamous cell carcinoma, tumors of the stomach and colon), tissue injury due to trauma or surgery, presence of a catheter in a central vein, an acquired deficiency of a protein involved in blood clot formation (e.g., protein C), paroxysmal nocturnal hemoglobinuria (PNH), elevated homocysteine ​​levels, heart failure, mechanical valve due to a genetic defect that causes or increases the risk of developing a hypercoagulable state, such as the presence of, pulmonary hypertension with in situ thrombus formation, atrial fibrillation, heparin-induced thrombocytopenia (HIT), heparin-induced thrombocytopenia and thrombosis (HITT), Kawasaki disease with in situ thrombus, in situ thrombotic Takayasu's arteritis, thrombophilia in metastatic cancer, elevated factor VIII levels, pregnancy, inflammatory bowel disease (IBD), or a genetic defect selected from the group consisting of prothrombin 20210 gene mutations, MTHFR mutations, protein C deficiency, protein S deficiency, protein A deficiency, protein Z deficiency, antithrombin deficiency, and genetic disorders resulting in thrombophilia.

[0169] In some embodiments, the disease or disorder of the lectin pathway is breast fibrosis, muscle fibrosis, retroperitoneal fibrosis, thyroid fibrosis, lymph node fibrosis, bladder fibrosis, cardiac fibrosis, liver fibrosis, arthrofibrosis, skin fibrosis, or pleural fibrosis. In some embodiments, the disease or disorder of the lectin pathway is fibrosis of musculoskeletal soft tissue structures (e.g., osteopenia associated with adhesive capsulitis, Dupuytren's contracture, myelodysplastic conditions with increased bone fibrosis, osteoporosis, myelofibrosis, or cystic fibrosis, etc.). In some embodiments, the disease or disorder of the lectin pathway is fibrosis caused by viral infection, e.g., alphavirus infection, tuberculosis infection, or influenza infection. In some embodiments, the disease or disorder of the lectin pathway is scarring associated with trauma, e.g., surgical complications (e.g., postoperative adhesions where scar tissue forms between internal organs causing contracture, pain, and sometimes infertility), chemotherapeutic drug-induced fibrosis, or scarring associated with burn injury. In some embodiments, the lectin pathway disease or disorder is genital fibrosis (eg, endometriosis and Peyronie's disease).

[0170] In some embodiments, the disease or disorder of the lectin pathway is amenable to treatment with a kallikrein inhibitor. In some embodiments, the lectin pathway is selected from the group consisting of hereditary angioedema, diabetic macular edema, and bleeding during cardiopulmonary bypass. In some embodiments, the lectin pathway is amenable to treatment with a thrombin inhibitor, such as for arterial thrombosis, venous thrombosis, pulmonary embolism, atrial fibrillation, heparin-induced thrombocytopenia, switching from one anticoagulant to another, or off-label use (maintenance) of continuous renal replacement therapy (CRRT) for extracorporeal circuit patency in critically ill patients with HIT.

[0171] In some embodiments, diseases or disorders of the lectin pathway are amenable to treatment with a Factor XII inhibitor, such as deep vein thrombosis (primary prevention and chronic treatment therapy), pulmonary embolism, non-valvular atrial fibrillation, prevention of recurrent ischemia following acute coronary syndromes in subjects with or without atrial fibrillation, end stage renal disease, cerebral ischemia, angina, or reduction or prevention of clotting associated with medical devices (e.g., valves, small bore grafts, etc.) and / or extracorporeal circuits.

[0172] In some embodiments, the disease or disorder of the lectin pathway is an acquired disease or disorder that increases the propensity for thromboembolism, such as atherosclerosis, antiphospholipid antibodies, cancer (e.g., promyelocytic leukemia, lung cancer, breast cancer, prostate cancer, pancreatic cancer, gastric cancer, and colorectal cancer), hyperhomocysteinemia, infection, tissue injury, venous congestion (e.g., surgery, orthopedic or paralytic immobility, heart failure, pregnancy, or obesity), and subjects taking estrogen-containing oral contraceptives. In some embodiments, the subject requires anticoagulant therapy, and the antibody or antigen-binding fragment thereof, polynucleotide, vector, host cell, and / or composition described herein is used as an alternative to standard anticoagulant therapy (e.g., warfarin). In some embodiments, the subject has a condition in which standard anticoagulant therapy is normally prohibited, such as CNS amyloid angiopathy. In some embodiments of the method, the antibody or antigen-binding fragment thereof, polynucleotide, vector, host cell and / or composition described herein is administered perioperatively as a bridging agent in a subject otherwise undergoing standard anticoagulant therapy. In some embodiments, the disease or disorder of the lectin pathway is sickle cell disease, a vaso-occlusive disorder involving platelet activation.

[0173] In some embodiments, the lectin pathway disease or disorder is SARS-CoV-2 infection. SARS-CoV-2 infection may result in lectin pathway disease or disorder by several different mechanisms. In addition to acute or chronic SARS-CoV-2 infection, additional chronic conditions may be triggered by past or current SARS-CoV-2 infection. Lectin pathway disease or disorder related to SARS-CoV-2 infection may result from direct interaction of MASP-2 with viral proteins, such as binding of MASP-2 to SARS-CoV-2. For example, lectin pathway disease or disorder may result from binding of MASP-2 to the S protein or N protein of SARS-CoV-2. Alternatively, some lectin pathway disease or disorder may result following interaction of lectin pathway recognition molecules with viral glycoproteins present on the viral surface or on the surface of virally infected cells. Additionally, lectin pathway disease or disorder may result from activation of the lectin pathway by DAMPs presented by cells infected with SARS-CoV-2. In some embodiments, the SARS-CoV-2 infection is an acute infection. In alternative embodiments, the SARS-CoV-2 infection is a chronic infection. In certain embodiments, the lectin pathway disease or disorder is an ongoing chronic condition caused by a previous SARS-CoV-2 infection. In some embodiments, the lectin pathway disease or disorder is acute respiratory distress syndrome. In some embodiments, the lectin pathway disease or disorder is secondary to SARS-CoV-2 infection. In some embodiments, the antibody or antigen-binding fragment thereof disclosed herein blocks the binding of MASP-2 to the S protein and / or N protein of SARS-CoV-2.

[0174] Selected examples of lectin pathway diseases and disorders are detailed further below.

[0175] Atypical hemolytic uremic syndrome (aHUS) Atypical hemolytic uremic syndrome (aHUS) is part of a group of conditions called "thrombotic microangiopathies." In atypical forms of HUS (aHUS), the disease is associated with defective complement control and can be sporadic or familial. Familial cases of aHUS are associated with mutations in genes encoding complement activation or complement control proteins, including complement factors H, I, B, membrane-cofactor protein inhibitor (CD46), and complement factor H-related protein 1 (CFHR1) and complement factor H-related protein 3 (CFHR3) (Zipfel, PF, et al., PloS Genetics 3(3):e41(2007)). The unifying feature of this diverse set of genetic mutations associated with aHUS is the propensity for enhanced complement activation at cell or tissue surfaces. A subject is at risk of developing aHUS upon the onset of at least one or more symptoms indicative of aHUS (e.g., anemia, thrombocytopenia and / or renal insufficiency) and / or the presence of thrombotic microangiopathy in a biopsy obtained from the subject. Determining whether a subject is at risk for developing aHUS includes determining whether the subject has a genetic predisposition to developing aHUS and / or determining whether the subject has a family history of aHUS, the former of which may be performed by evaluating genetic information (e.g., from a database containing the subject's genotype) or by administering to the subject at least one genetic screening test to determine the presence or absence of a genetic marker associated with aHUS, either by genomic sequencing or gene-specific analysis (e.g., PCR analysis) (i.e., by determining the presence or absence of a genetic mutation associated with aHUS in genes encoding complement factor H (CFH), factor I (CFI), factor B (CFB), membrane cofactor protein inhibitor (CD46), C3, complement factor H related protein 1 (CFHR1), or THBD (encoding the anticoagulant protein thrombomodulin) or complement factor H related protein 3 (CFHR3), or complement factor H related protein 4 (CFHR4)). Methods of genetic screening for the presence or absence of a genetic mutation associated with aHUS have been established.See, for example, Noris M et al., "Atypical Hemolytic-Uremic Syndrome," in GeneReviews™, edited by Pagon RA, Bird TD, Dolan CR et al., Seattle, WA (University of Washington, Seattle), 2007 Nov 16 [updated March 10, 2011].

[0176] Hematopoietic stem cell transplant-related TMA (HSCT-TMA) Hematopoietic stem cell transplant-associated TMA (HSCT-TMA) is a life-threatening complication triggered by endothelial injury. Although the most commonly affected organ is the kidney, HSCT-TMA is a multisystem disease involving the lungs, intestine, heart and brain. The occurrence of even mild TMA is associated with long-term renal dysfunction. The occurrence of allogeneic HSCT-associated TMA varies in frequency depending on the diagnostic criteria and the conditioning and graft-versus-host disease prophylaxis regimen, with calcineurin inhibitors being the most frequently implicated drugs (Ho VT et al., Biol Blood Marrow Transplant, ll(8):571-5, 2005).

[0177] Immunoglobulin A Nephropathy (IgAN) Immunoglobulin A nephropathy (IgAN) is an autoimmune kidney disease that results in intrarenal inflammation and kidney injury. IgAN is the most common primary glomerular disease worldwide. The annual incidence is approximately 2.5 per 100,000 people, and it is estimated that 1 in 1400 people in the United States will develop IgAN. As many as 40% of IgAN patients will develop end-stage renal disease (ESRD). Patients typically present with microscopic hematuria with mild to moderate proteinuria and various levels of renal insufficiency (Wyatt RJ,et al.,NEngl J Med 36S(25):2402-4,2013). Clinical markers such as impaired renal function, persistent hypertension and severe proteinuria (>1 g per day) are associated with poor prognosis (Goto M et al., Nephrol Dial Transplant 24(10):3068-74, 2009; Berthoux F.et al., J Am Soc Nephrol 22(4):752-61, 2011). Proteinuria is the strongest prognostic factor independent of other risk factors in several large observational studies and prospective clinical trials (Coppo R.et al., J Nephrol 18(5):503-12, 2005; Reich HN,et al., J Am Soc Nephrol 18(12):3177-83, 2007). If left untreated, it is estimated that 15-20% of patients will reach ESRD within 10 years of disease onset (D'Amico G., Am J Kidney Dis 36(2):227-37,2000). The diagnostic feature of IgAN is the predominance of IgA deposition in the glomerular mesangium, either alone or in association with IgG, IgM, or both.

[0178] Lupus Nephritis (LN) The main complication of systemic lupus erythematosus (SLE) is nephritis, also known as lupus nephritis, which is classified as a secondary form of glomerulonephritis. Up to 60% of adults with SLE have some form of kidney involvement late in the course of their disease (Koda-Kimble et al. "Koda-Kimble and Young's Applied Therapeutics: the clinical use of drugs", 10th ed., Lippincott Williams & Wilkins: pp. 792-9, 2012), with a prevalence in the United States of 20-70 per 100,000. Lupus nephritis often presents in patients with other symptoms of active SLE, including fatigue, fever, rash, arthritis, serositis, or central nervous system disease (Pisetsky DSet al., Med Clin North Am 81(1):113-28, 1997). Some patients have asymptomatic lupus nephritis, but during routine follow-up, laboratory abnormalities such as elevated serum creatinine levels, low albumin levels, or urinary protein or sediment suggest active lupus nephritis.

[0179] VI. Arrays The sequences referred to within this specification are summarized in Table 2.

[0180] (Table 2) TIFF2024546109000021.tif118166TIFF2024546109000022.tif242166TIFF2024546109000023.tif235166 TIFF2024546109000024.tif242166TIFF2024546109000025.tif245166TIFF2024546109000026.tif245166 TIFF2024546109000027.tif244166TIFF2024546109000028.tif242166TIFF2024546109000029.tif242166 TIFF2024546109000030.tif242166TIFF2024546109000031.tif242166TIFF2024546109000032.tif154166 EXAMPLES

[0181] VII. Working Examples Example 1 Generation of high-affinity anti-human MASP-2 inhibitory antibodies C57BL / 6 MASP-2 knockout mice aged 7–14 weeks were immunized with human MASP-2 CCP2 / SP polypeptide (amino acid residues 364–686 of SEQ ID NO:1) containing a C-terminal 6×His tag using the Sigma adjuvant system (Sigma-Aldrich, St. Louis, MO). 50 μg of immunogen per mouse was mixed 1:1 with RIBI adjuvant and injected intraperitoneally into the mice. 14 days later, immunized mice were boosted with an additional immunogen mixed 1:1 with RIBI adjuvant. Mice were boosted a third time 14 days later with 50 μg of immunogen mixed 1:1 in PBS. Serum samples from mice were prepared periodically from tail bleeds and tested by ELISA for the presence of antigen-specific antibodies. Mice with significant antibody titers were given a prefusion immunogen boost in PBS 4 days prior to splenocyte fusion. Three days before fusion, mice were given 50 μg of anti-CD40 agonist mAb (R&D Systems, Minneapolis, Minn.) in PBS subcutaneously at the base of the tail to increase B cell numbers.

[0182] Mice were sacrificed, and spleen cells were harvested and fused to the selected mouse myeloma cell line P3 / NSI / 1-AG4-1 (NS-1) (ATCC No. TIB18) using 50% polyethylene glycol or 50% polyethylene glycol + 10% DMSO. Hybridoma cells generated by this fusion were plated in 96-well tissue culture plates containing HAT (hypoxanthine, aminopterin, and thymidine) medium to inhibit the growth of unfused cells, myeloma hybrids, and spleen hybrids. After hybridoma selection, culture supernatants were assessed for MASP-2 binding by ELISA and for inhibition of lectin pathway activation using the C3 adhesion assay. Positive hybridomas were identified and subcloned by serial dilution. Parallel screening of hybridoma supernatants yielded 75 initial hybridomas positive for both MASP-2 binding and function (C3 adhesion assay). After further culture and retesting of these 75 initial hybridomas, 74 were confirmed to be positive for binding, while 39 were confirmed to have functional activity. These 39 hybridomas were cloned by serial dilution.

[0183] Example 2 Cloning, purification, and characterization of recombinant antibodies Heavy and light chain variable regions were cloned by RT-PCR and sequenced from 39 hybridoma clones identified as described in Example 1. Mouse-human chimeric mAbs consisting of mouse mAb variable regions fused to human IgG4 heavy chain constant region (SEQ ID NO:66) and kappa light chain constant region (SEQ ID NO:68) were produced as recombinant proteins in Expi293F cells. The IgG4 constant hinge region used (SEQ ID NO:66) contains the stabilizing S228P amino acid substitution. In some embodiments, the chimeric mAbs were fused to human IgG4 constant hinge region (SEQ ID NO:67) that contains the S228P amino acid substitution and also contains certain mutations that promote FcRn interaction at low pH.

[0184] Twenty-six clones were determined to be unique chimeric monoclonal antibodies, which were expressed in transiently transfected Expi293F cells, purified, and tested for their binding affinity to human MASP-2 and their ability to inhibit MASP-2-mediated lectin pathway activation.

[0185] To measure the binding of 26 purified recombinant MASP-2 antibodies to human MASP-2 (CCP1-CCP2-SP fragment), a solid-phase ELISA assay was performed as follows: MaxiSorp ELISA plates were coated overnight at 4°C with 1.0 μg / mL human MASP-2 (CCP1 / 2 / SP fragment) in carbonate / bicarbonate buffer. The plates were then blocked with 1% BSA / PBS, washed in PBS, and incubated for 1 h at room temperature with serial dilutions of recombinant MASP-2 mAb in blocking buffer (PBST + 0.1% BSA). The plates were washed (PBS-T, 0.05%) and detection antibody was added for 1 h at room temperature (goat anti-human IgG-HRP). After another wash (PBS-T, 0.05%), the plates were developed (5 min) with OPT EIA TMB (BD Biosciences #555214). Absorbance readings at A450 were measured using a Spectramax M5e plate reader.

[0186] Of the 26 chimeric mAbs, 22 were found to have good binding to human MASP-2 (apparent Kd range of 0.1 nM to 1 nM). Four of the 26 chimeric mAbs were found to have weak / negligible binding to human MASP-2.

[0187] To measure the ability of these 26 purified recombinant MASP-2 antibodies to block the lectin pathway of complement activation, a C3 adhesion assay was performed as follows: Mannan was diluted to a concentration of 50 μg / ml in 50 mM carbonate buffer (15 mM Na2CO3 + 35 mM NaHCO3 + 1.5 mM NaN3) pH 9.6 and coated on ELISA plates overnight at 4°C. The next day, 250 μl of 1% BSA in PBS was added to the wells and incubated at room temperature for 2 hours. The plates were washed three times with 300 μl of PBS containing 0.05% Tween-20 and stored on ice with 200 μl of PBS until the addition of samples.

[0188] Normal human serum was diluted to 1.0% in GVB / Ca / Mg buffer, and the 26 purified MASP-2 mAbs were added to this buffer at concentrations ranging from 0.00001 to 100 nM and preincubated on ice for 10 min before being added to blocked ELISA plates. Complement activation reactions were initiated by transferring the preincubation mixture to wells of mannan-coated assay plates. After 40 min of incubation at room temperature, the reaction was stopped by washing the plates three times in ELISA wash buffer. C3b attachment was detected with anti-human C3c antibody (Dako) followed by goat α-rabbit HRP (Southern Biotech). Negative control was buffer without serum (no C3 attachment), and positive control was serum without inhibitory antibody (maximum C3b attachment). The cut-off criterion was set at half the activity of irrelevant mAb and buffer alone.

[0189] Of the 26 unique chimeric mAbs identified in the screen, the three clones with the highest inhibitory LP activity in the C3b attachment assay were determined to be OMS850, OMS860, and OMS870. These three antibodies were selected for further characterization as described below.

[0190] The sequences of the heavy and light chain variable regions of clones OMS850, OMS860 and OMS870 are shown in Figure 3 ("SIN" = "SEQ ID NO:") and included below. The respective complementarity determining regions (CDRs) and framework regions (FRs) are listed in Tables 1A, 1B and 1C (above) and Tables 3-6 (below).

[0191] Listed below are the heavy chain variable region (VH) sequences of each high affinity MASP-2 inhibitory antibody. Kabat CDRs are underlined.

[0192] Heavy chain variable region: TIFF2024546109000033.tif98159

[0193] Table 3: MASP-2 antibody VH sequences (CDR and FR regions, Kabat) Mouse parent TIFF2024546109000034.tif208166

[0194] Listed below are the light chain variable region (VL) sequences of high affinity MASP-2 inhibitory antibodies. Kabat CDRs are underlined. These regions are the same whether numbered by the Kabat system or the Chothia system.

[0195] Light chain variable region: TIFF2024546109000035.tif78159

[0196] Table 4: MASP-2 antibody VL sequence (CDR and FR regions, Kabat) Mouse parent TIFF2024546109000036.tif187160

[0197] Table 5: Consensus sequences of OMS850 and OMS860 VH CDRs TIFF2024546109000037.tif231153

[0198] Table 6: Consensus sequences of OMS850 and OMS860 VL CDRs TIFF2024546109000038.tif120151

[0199] Example 3 Further characterization of candidate MASP-2 inhibitory antibodies 1. Binding to recombinant human MASP-2 A solid-phase ELISA assay was performed to measure the binding of three selected MASP-2 inhibitory antibodies to human MASP-2 (CCP1-CCP2-SP fragment) as described in Example 1. The results are shown in Figures 4A-4D and summarized in Table 7 below.

[0200] Table 7. Results of MASP-2 binding assay TIFF2024546109000039.tif31128

[0201] 2. C3b attachment assays in human, cynomolgus monkey, rat, and mouse sera The three selected MASP-2 antibodies described in Example 2 were expressed, purified, and diluted to the same stock concentration and then incubated with Ca ++ and Mg ++ The antibodies were diluted again into GVB buffer (4.0 mM barbital, 141 mM NaCl, 1.0 mM MgCl2, 2.0 mM CaCl2, 0.1% gelatin, pH 7.4) to ensure that all antibody clones had the same amount of buffer.

[0202] A. C3b attachment assay in human serum Mannan was diluted to a concentration of 50 μg / ml in carbonate buffer (15 mM Na2CO3 + 35 mM NaHCO3) pH 9.6 and coated onto ELISA plates overnight at 4° C. Then, the next day, 250 μl of blocking solution, 1% BSA in PBS, was added to the wells and incubated for 2 hours at room temperature. The plates were washed three times with 300 μl PBS containing 0.05% Tween-20 (ELISA wash buffer).

[0203] Normal human serum was diluted to 1.0% in GVB / Ca / Mg buffer and pre-incubated for 15 min on ice with MASP-2 mAb clones OMS850, OMS860 and OMS870 at final concentrations ranging from 0.00001 to 100 nM. The reaction was started by transferring the pre-incubation mixture to wells of a mannan-coated and blocked assay plate, followed by incubation at room temperature for 40 min. The reaction was stopped by washing the plate three times in wash buffer, and C3b attachment was detected with anti-human C3c antibody (Dako) followed by goat α-rabbit HRP (Southern Biotech). Negative controls were serum and buffer without antibody (no C3b attachment occurs), and positive controls were serum without antibody (maximal C3b attachment). The cut-off criterion was set at half the activity of an irrelevant mAb and buffer alone.

[0204] B. C3b adhesion assay in cynomolgus monkey serum Mannan was diluted to a concentration of 50 μg / ml in carbonate buffer (15 mM Na2CO3 + 35 mM NaHCO3) pH 9.6 and coated onto ELISA plates overnight at 4° C. Then, the next day, 250 μl of blocking solution, 1% BSA in PBS, was added to the wells and incubated for 2 hours at room temperature. The plates were washed 3 times with 300 μl PBS / tween-20.

[0205] Cynomolgus serum was diluted to 0.25% in GVB / Ca / Mg buffer and pre-incubated for 15 min on ice with MASP-2 mAb clones OMS850, OMS860 and OMS870 at final concentrations ranging from 0.00001 to 100 nM. The reaction was initiated by transferring the pre-incubation mixture to wells of a mannan-coated and blocked assay plate, followed by incubation at room temperature for 40 min. The reaction was stopped by washing the plate three times with PBS / Tween-20. C3b attachment was detected with an anti-human C3c antibody (Dako) followed by goat α-rabbit HRP (Southern Biotech). Negative controls were serum and buffer without antibody (no C3b attachment) and positive controls were serum without antibody (maximal C3b attachment). The cut-off criterion was set at half the activity of an irrelevant mAb and buffer alone.

[0206] C. C3 adhesion assay in rat serum Mannan was diluted to a concentration of 50 μg / ml in carbonate buffer (15 mM Na2CO3 + 35 mM NaHCO3 + 1.5 mM NaN3) pH 9.6 and coated onto ELISA plates overnight at 4°C. Then, the next day, 250 μl of 1% BSA blocking solution was added to the wells and incubated for 2 hours at room temperature. The plates were washed 3 times with 300 μl of PBS / tween-20.

[0207] Rat serum was diluted to 0.3% in GVB / Ca / Mg buffer and pre-incubated for 15 min on ice with MASP-2 mAb clones OMS850, OMS860 and OMS870 at final concentrations ranging from 0.00001 to 100 nM. The reaction was started by transferring the pre-incubation mixture to wells of a mannan-coated and blocked assay plate, followed by incubation at room temperature for 40 min. The reaction was stopped by washing the plate three times with wash buffer. C3b attachment was detected with anti-human C3c antibody (Dako) followed by goat α-rabbit HRP (Southern Biotech). Negative controls were serum and buffer without antibody (no C3b attachment) and positive controls were serum without antibody (maximal C3b attachment). The cut-off criterion was set at half the activity of an irrelevant mAb and buffer alone.

[0208] D. C3b adhesion assay in mouse serum Mannan was diluted to a concentration of 50 μg / mL in carbonate buffer (15 mM Na2CO3 + 35 mM NaHCO3 + 1.5 mM NaN3) pH 9.6 and coated onto ELISA plates overnight at 4° C. Then, the next day, 250 μl of blocking solution, 1% BSA in PBS, was added to the wells and incubated for 2 hours at room temperature. The plates were washed 3 times with 300 μl PBS / tween-20.

[0209] Mouse serum was diluted to 1.0% in GVB / Ca / Mg buffer and pre-incubated for 15 min on ice with MASP-2 mAb clones OMS850, OMS860 and OMS870 at final concentrations ranging from 0.00001 to 100 nM. The reaction was started by transferring the pre-incubation mixture to wells of a mannan-coated and blocked assay plate, followed by incubation at room temperature for 40 min. The reaction was stopped by transferring the plate to an ice bath. C3b attachment was detected with anti-human C3c antibody (Dako) followed by goat α-rabbit HRP (Southern Biotech). Negative controls were serum and buffer without antibody (no C3b attachment) and positive controls were serum without antibody (maximal C3b attachment). The cut-off criterion was set at half the activity of an irrelevant mAb and buffer alone.

[0210] The results are shown in Figures 5A, 5B, 5C and 5D and summarized below in Table 8. The data point labeled "Buffer" in Figures 5A-5D is the buffer only negative control.

[0211] Table 8. Results of C3b adhesion assay TIFF2024546109000040.tif45128

[0212] 3. C4b attachment assay in 50% human serum Mannan was diluted to a concentration of 50 μg / ml in carbonate buffer (15 mM Na2CO3 + 35 mM NaHCO3 + 1.5 mM NaN3) pH 9.6 and coated onto ELISA plates overnight at 4° C. Then, the next day, 250 μl of blocking solution, 1% BSA in PBS, was added to the wells and incubated for 2 hours at room temperature. The plates were washed 3 times with 300 μl PBS / tween-20.

[0213] Normal human serum was diluted to 50.0% in PBS and MASP-2 mAb clones OMS850, OMS860 and OMS870 were added to this buffer at final concentrations ranging from 0.0001 to 100 nM and pre-incubated for 15 min on ice. The reaction was started by transferring the pre-incubation mixture to a mannan-coated and blocked ELISA plate, followed by incubation at 4 °C for 7 min. C4b attachment was detected with an anti-human C4c antibody (Dako) followed by goat α-rabbit HRP (Southern Biotech). The negative control was buffer without serum and antibody (=no C4b attachment). Background was determined in wells containing only buffer. The cut-off criterion was set at half the activity of an irrelevant mAb and buffer alone.

[0214] The results are shown in Figure 6 and listed below in Table 9. The data point marked "Buffer" in Figure 6 is the buffer only negative control.

[0215] Table 9. Results of C4b adhesion assay TIFF2024546109000041.tif38128

[0216] Given the good potency in humans, mice, rats and cynomolgus monkeys described above, antibody OMS850 was selected for humanization and further optimization.

[0217] Example 4 Production of humanized and variants of MASP-2 mAb OMS850 Prior to humanization, the anti-MASP-2 inhibitory mAb OMS850 was analyzed for post-translational modifications. An aspartate isomerization motif "DP" was identified in TIFF2024546109000042.tif4128. Variants of OMS850 were generated by site-directed mutagenesis to modify P53 to A, N, D, L, S, and T. These variants were expressed and purified as described above. Affinity was determined by ELISA and titer was assessed in human serum by C3 attachment assay using the full IgG4 format as described above.

[0218] The results are shown in FIG. 7 and listed in Table 10 below.

[0219] Table 10. Results of C3b adhesion assay TIFF2024546109000043.tif45128

[0220] It was determined in the C3b attachment assay that OMS850 VH P53A had the best titers in human serum as measured in the C3b attachment assay, as shown in Figure 7. Therefore, this P53A substitution was incorporated in subsequent humanizations described below.

[0221] To reduce the risk of immunogenicity, the representative high affinity MASP-2 inhibitory antibody OMS850 was humanized by CDR grafting. The CDRs of mAb OMS850 were grafted into the closest consensus human framework sequence. Some of the Vernier zone residues were modified by Quickchange site-directed mutagenesis (Agilent Technologies). The resulting humanized VH and VL regions were transferred into pcDNA3.1-based human IgG4 and IgK expression constructs, and the recombinant antibodies were expressed and purified as described above. Using the method described in Example 3 above, the affinity of the humanized antibody was determined by ELISA, and the potency was evaluated by C3 adhesion assay in 1% human serum using the full IgG4 format.

[0222] The first humanized prototype was developed and named OMS852. The amino acid sequences of the OMS852 humanized heavy and light chain variable regions are listed below. The CDRs (Kabat) are underlined.

[0223] Full sequence of OMS852 VH (humanized): (SEQ ID NO:46) TIFF2024546109000044.tif18159

[0224] Complete sequence of OMS852 VL (humanized) (SEQ ID NO:47) TIFF2024546109000045.tif11159

[0225] The results of the C3b adhesion assay using various modified versions of OMS850 mAb in comparison with the OMS850 parent mAb are shown in Figures 8A and 8B and summarized in Table 11.

[0226] Table 11. Results of C3b attachment assay for humanized candidates of OMS850 TIFF2024546109000046.tif90128

[0227] Monoclonal antibody OMS852 4PA-1, which was humanized and incorporated the previously identified P53A modifications, was selected for further development and designated OMS854.

[0228] The amino acid sequences of the heavy and light chain variable regions for humanized antibody OMS854 are listed below. The CDRs (Kabat) are underlined.

[0229] Complete sequence of OMS854 VH :(SEQ ID NO:48) TIFF2024546109000047.tif18160

[0230] Complete sequence of OMS854 VL (humanized) (SEQ ID NO:47) TIFF2024546109000048.tif11159

[0231] VH CDR1 was further modified such that it contains at least one, or optionally two, or optionally three histidines, as shown in Table 12. The variant with two histidines was named OMS856. The variant with three histidines was named OMS858.

[0232] Complete sequence of OMS856 VH (SEQ ID NO:49) TIFF2024546109000049.tif18159

[0233] Complete sequence of OMS858 VH (SEQ ID NO:50) TIFF2024546109000050.tif18159

[0234] Table 12: MASP-2 antibody VH sequence (CDR and FR regions, Kabat) TIFF2024546109000051.tif97160TIFF2024546109000052.tif245160

[0235] Table 13: MASP-2 antibody VL sequence (CDR and FR regions, Kabat) TIFF2024546109000053.tif142160

[0236] SEQ ID NO:65 : Human IgG4 constant region TIFF2024546109000054.tif41159

[0237] SEQ ID NO:66 : Human IgG4 constant region with S228P mutation TIFF2024546109000055.tif41159

[0238] SEQ ID NO:67 Human IgG4 constant region with S228P mutation and a mutation (Xtend) that enhances FcRn interaction at low pH TIFF2024546109000056.tif41159

[0239] SEQ ID NO:68 : Human IgK constant region TIFF2024546109000057.tif11158

[0240] SEQ ID NO:69: DNA encoding OMS850 VH (mouse parent) TIFF2024546109000058.tif47159

[0241] SEQ ID NO:70:OMS860 VH-encoding DNA (mouse parent) TIFF2024546109000059.tif47159

[0242] SEQ ID NO:71:OMS870 VH-encoding DNA (mouse parent) TIFF2024546109000060.tif47158

[0243] SEQ ID NO:72: DNA encoding OMS850 VL (mouse parent) TIFF2024546109000061.tif40159

[0244] SEQ ID NO:73: DNA encoding OMS860 VL (mouse parent) TIFF2024546109000062.tif40159

[0245] SEQ ID NO:74: OMS870 VL-encoding DNA (mouse parent) TIFF2024546109000063.tif48158

[0246] SEQ ID NO:75:OMS852 VH-encoding DNA TIFF2024546109000064.tif47159

[0247] SEQ ID NO:76:OMS852 VL-encoding DNA TIFF2024546109000065.tif40159

[0248] SEQ ID NO:77: DNA encoding OMS854 TIFF2024546109000066.tif47159

[0249] SEQ ID NO:78: DNA encoding OMS856 TIFF2024546109000067.tif47159

[0250] SEQ ID NO:79: DNA encoding OMS858 TIFF2024546109000068.tif47159

[0251] Example 5 Antibody OMS858 specifically blocks the lectin pathway The effect of mAb OMS858 on membrane attack complex (MAC) adhesion was analyzed using pathway-specific conditions for the lectin, classical and alternative pathways. For this purpose, the Wieslab COMPL300 complement screening kit (Wieslab, Lund, Sweden) was used according to the manufacturer's instructions. For the classical pathway, complement was activated on IgM. For the lectin pathway, complement was activated on mannan and for the alternative pathway, complement was activated on LPS.

[0252] Figure 9A graphically illustrates the level of MAC adhesion in the presence or absence of anti-MASP-2 antibody OMS858 under classical pathway-specific assay conditions. Figure 9B graphically illustrates the level of MAC adhesion in the presence or absence of anti-MASP-2 antibody OMS858 under lectin pathway-specific assay conditions. Figure 9C graphically illustrates the level of MAC adhesion in the presence or absence of anti-MASP-2 antibody OMS858 under alternative pathway-specific assay conditions.

[0253] As shown in Figure 9B, mAb OMS858 inhibited lectin pathway-mediated activation of MAC adhesion with an IC of approximately 1 nM. 50 values. However, mAb OMS858 had no effect on MAC adhesion initiated by classical (FIG. 9A) or alternative (FIG. 9C) pathway-mediated activation. In FIG. 9A, an anti-C1s antibody (TNT003), known to inhibit the classical pathway, is included as a control. In FIG. 9B and FIG. 9C, an anti-factor B antibody, known to inhibit the alternative pathway, is included as a control.

[0254] Example 6 Analysis of MASP-2 binding epitopes of antibodies OMS850, OMS858, OMS860, and OMS870 Antibody OMS858 was biotinylated and its ability to simultaneously bind several different MASP-2 antibodies was tested in an Octet Biolayer Interferometry binding assay as follows.

[0255] Antibody OMS858 was biotinylated using EZ-link Sulfo-NHS-LC-Biotin (Fischer Scientific, A39257). Super Streptavidin (SSA) biosensors (Fortebio 18-5057) were hydrated in PBS for 15 min at room temperature. 3 mL of 50 nM biotinylated OMS858 was captured on the BLI chip, followed by capture of hMASP-2-CCP1-CCP2-SP. A 500 nM solution of one of the five test anti-MASP-2 antibodies was prepared in immobilization buffer (PBS, 0.02% BSA, 0.05% Tween-20, pH 7.4). The test antibodies used were OMS850, OMS860 and OMS870, whose production has been described above, and the previously identified anti-MASP-2 antibodies OMS721 and 4A8.

[0256] The first row of plate wells received 200 μL of biotinylated OMS858, and the next row of wells received 200 μL of hMASP-2-CCP1-CCP2-SP. In another row on the plate, 200 μL of 500 nM test antibody was added to each well. Controls were also run using non-biotinylated OMS858 or buffer alone in place of test antibody.

[0257] Binding assays were performed on the Octet using the following steps: baseline establishment (60 s), loading of biotinylated OMS858 (180 s), baseline establishment (60 s), loading of hMASP-2-CCP1-CCP2-SP (180 s), followed by association (300 s) and dissociation (300 s) of the test antibody.

[0258] As shown in Figure 10, antibodies OMS850, OMS860 and OMS870 were unable to bind to MASP-2 captured by OMS858, indicating that these antibodies bind to the same or partially overlapping epitopes on MASP-2. In contrast, previously identified anti-MASP-2 antibodies OMS721 and 4A8 were able to bind to MASP-2 captured by OMS858, indicating that these two antibodies bind to epitopes on MASP-2 that are distinct from OMS850, OMS860, OMS870 and OMS858.

[0259] Example 7 Crystallization of the complex between MASP-2 protein and Fab mAb OMS858 1. Preparation of MASP-2 Protein Recombinant MASP-2 protein, human mannose-binding lectin serine protease 2, based on UniProt O00187, was prepared as follows. Expression constructs for human MASP-2 CCP2-SP and CCP2-SP-6HIS were made for recombinant expression in E. coli cells. Recombinant expression and protein purification of MASP-2 as inclusion bodies in E. coli was performed according to the method described in Ambrus G.et al., 2003, with minor modifications. For the HIS-tagged version, MASP-2 protein (CCP2-SP-6HIS) was purified under denaturing conditions according to the method described in the Ni-NTA Superflow cartridge handbook (Qiagen, March 2007).

[0260] Purification of MASP-2 included extraction, unfolding, refolding and chromatography using standard methods described in Harmat et al., J. Mol. Biol. 2004;342:1533-1546 and Gal et al., J. Biol. Chem. 2005;280:33435-33444 and Ambrus et al. J Immunol. 2003 Feb 1;170(3):1374-82. After size exclusion chromatography, recombinant MASP-2 protein was concentrated to 5mg / mL to 20mg / mL in centrifugal concentrators (Amicon NMWL 10kDa). Concentrated MASP-2 protein samples were flash frozen and stored until thawed for complex formation. Purification and cleavage were monitored by SDS-PAGE stained with Commassie Blue Simply Blue™ Safe Stain (Invitrogen).

[0261] 2. Preparation of Fab mAb OMS858 Protein OMS858 Fab was generated by proteolytic cleavage of mAb OMS858 using the FAbALACTICA® Fab kit according to the manufacturer's instructions.

[0262] 3. Generation of MASP-2-Fab mAb OMS858 Complexes Fab mAb OMS858 and MASP-2 protein samples were incubated at equimolar concentrations for 1 h. Successful formation of MASP-2-Fab mAb OMS858 complexes was verified by analytical size-exclusion chromatography.

[0263] 4. Crystallization of MASP-2-Fab mAb OMS858 Complex Crystallization attempts were set up as sitting-drop vapor diffusion experiments by combining equal volumes of MASP-2-Fab mAb OMS858 complex solution and commercial crystallization formulation. MASP-2-Fab mAb OMS858 complex protein samples were used at protein concentrations between 5 and 20 mg / mL. MASP-2-Fab mAb OMS858 crystals appeared in less than 2 months in a crystallization formulation containing 100 mM sodium acetate pH 5.26, 200 mM ammonium sulfate, 10.45% PEG 2000MME. Crystals were captured in a cryogenic loop and cryoprotected with 20% glycerol in the crystallization formulation before being flash-cooled in liquid nitrogen.

[0264] Example 8 X-ray crystal analysis MASP-2-Fab mAb OMS858 complex crystals prepared as described in Example 7 were diffracted at beamlines SSRL BL9-2, BL14-1, BL12-2 and ALS Sector 5 with 1.0 angstrom wavelength X-rays, and X-ray diffraction data sets were collected using Dectris Pilatus and Eiger detectors. A summary of the crystal parameters, data collection and refinement statistics for the MASP-2-Fab mAb OMS858 complex crystals is listed in Table 14. The X-ray structure was determined by molecular replacement using part of structure 1Q3X and part of 3C08 as search models, and partially refined with Buster 2.10.2 or Refmac 5.8. The electron density was inspected with Coot (Emsley et al., 2010) and subjected to iterative model building and refinement cycles until the densities of MASP-2 and Fab mAb OMS858 were clearly discernible and sufficient R-factors were obtained. At this point, the partial refinement was deemed complete and the model was checked for ligand, solvent and protein.

[0265] Table 14. Summary of crystal parameters, data collection, and refinement statistics for MASP-2-Fab mAb OMS858 complex crystals. TIFF2024546109000069.tif173160TIFF2024546109000070.tif117160

[0266] Example 9 Crystal structure analysis The refined structures were analyzed for type and distance of protein-protein and protein-solvent interactions using LigPlot+ (Laskowski and Swindells, 2011) with parameters set to 3.35 Å for the maximum distance between hydrogen bond donors and hydrogen bond acceptors, and non-bonded contact parameters between any contacts such as hydrophobic contacts or van der Waals interactions with a maximum contact distance of 3.90 Å.

[0267] Table 15 shows the H-bonds between atoms of mAb OMS858 and atoms of MASP-2. Table 16 shows the van der Waals interactions between MASP-2 atoms and Fab mAb OMS858 atoms obtained by LigPlot+ based analysis of the crystallographic structure using the "Antibody" setting. LigPlot+ calls these "non-bonded contacts" or "hydrophobic contacts". Despite this designation, there are atom pairings that contain suspected H-bonds. Therefore, in some cases, Table 16 also includes H-bond interactions.

[0268] FIG. 11 is a schematic diagram showing the arrangement of the MASP-2 serine protease domain and Fab mAb OMS858 and the contacts between them.

[0269] Figure 12A is a schematic diagram showing the MASP-2 epitope bound by mAb OMS858, which includes the following residues in the SP domain of MASP-2: ASP496, LYS503, SER506, PRO507, HIS508 and TRP513. As shown in Figure 12A, the epitope bound by OMS858 encompasses two patches, the first of which includes ASP496 and TRP513, and the second of which includes LYS503, SER506, PRO507 and HIS508.

[0270] Figure 12B is a schematic diagram showing the paratope of mAb OMS858, which binds to the MASP-2 epitope shown in Figure 12A. As shown in Figure 12B, this paratope encompasses two corresponding patches of heavy and light chain residues, specifically heavy chain residues HIS33, ASP50, ASP52, ASP55, GLU57, HIS59, and light chain residues TYR31, ARG30, and TRP90.

[0271] It was determined that there are 3 hydrogen bonds and 36 van der Waals contacts, as follows:

[0272] Table 15. Three H-bonds between atoms of mAb OMS858 and atoms of MASP-2 TIFF2024546109000071.tif24160

[0273] Table 16: 36 van der Waals contacts between atoms of mAb OMS858 and atoms of MASP-2 TIFF2024546109000072.tif172160

[0274] Figure 13 illustrates the interaction between the paratope of mAb OMS858 and the epitope of MASP-2, calculated by LigPlot+ software using the model derived from the corresponding crystallographic MASP-2-compound co-structure and using the "Antibody" mode with settings for hydrogen bond calculation parameters (maximum distance between hydrogen bond donors and hydrogen bond acceptors of 3.35 Å, and non-bonded contact parameters between any contacts such as hydrophobic contacts or van der Waals interactions with a maximum contact distance of 3.90 Å). Hydrogen bonds and polar contacts are depicted with dashed lines and distances are given in angstroms.

[0275] Amino acid atoms that interact with compound atoms from the crystallographic data as well as compound atoms with sufficient 2fo-fc electron density are depicted. MASP-2 amino acid residue numbering (MASP-2 AA#) follows Uniprot accession code O00187, and atom numbering for amino acids (AA atoms) follows the convention established by the Protein Data Bank and corresponds to those in Tables 15 and 16.

[0276] OMS858 amino acid residue numbering is according to VH shown as SEQ ID NO:50 and VL shown as SEQ ID NO:47. The paratope of OMS858, including the heavy chain variable region (loop H1, loop H2) and the light chain variable region (loop L3, loop L1), is depicted above the dashed line in FIG. 13, and the epitope of MASP-2 is depicted below the dashed line in FIG. 13. Certain of the amino acids shown in FIG. 13 are marked with arcs with radiating lines, indicating that they have van der Waals interactions (dotted lines) with atoms of other amino acids. Hydrogen bonds and polar contacts are depicted with dashed lines, and distances are given in angstroms. Carbon atoms are shown as filled circles, nitrogen atoms are shown as hollow circles with crosses, and oxygen atoms are shown as hollow circles with crosses. The side chains of amino acids Glu57 and Arg30 of OMS858 were only partially resolved in the X-ray structure. Therefore, due to the close backbone proximity, it is likely that additional hydrogen bonds and ionic interactions exist between VH GLU57 and VL ARG30 of OMS858 and the opposing residues on MASP-2. Specifically, VL ARG30 of OMS858 forms an ionic bond to ASP496 of MASP-2, VL ARG30 of OMS858 forms a π-π stacking interaction with the aromatic moiety of TRP513 of MASP-2, and VH GLU57 of OMS858 may form an ionic or hydrogen bond with the amino group of LYS503 of MASP-2.

[0277] As shown in Figure 13, OMS858 binds to MASP-2 through three H-bonds with residues in VH: ASP52 and ASP55, which bind to LYS503 in MASP-2, and ASP50 in VL, which binds to HIS508 in MASP-2. As further shown in Figure 13, OMS858 binds to MASP-2 through van der Waals contacts between HIS33 of VH and SER506 and PRO507 of MASP-2, between ASP52, ASP55, and GLU57 of VH and LYS503 of MASP-2, between HIS59 and ASP50 of VH and HIS508 of MASP-2, between TRP90 and HIS508 of VL, between ARG30 of VL and TRP513 of MASP-2, and between TYR31 of VL and TRP513 and ASP496 of MASP-2.

[0278] In certain aspects, the MASP-2 inhibitory antibody interacts via van der Waals contacts to one, two, three, four, five or six, or all, of the following residues in MASP-2: SER506, PRO507, LYS503; HIS508; TRP513; ASP496, and combinations thereof. In certain aspects, the MASP-2 inhibitory antibody interacts via hydrogen bonds to one or two of the following residues in MASP-2: LYS503 and HIS508.

[0279] Figure 14A illustrates the three-dimensional structure of the MASP-2 epitope, including ASP496, LYS503, SER506, PRO507, HIS508 and TRP513, to which OMS858 binds. As shown in Figure 14A, The entire MASP-2 epitope, depicted as TIFF2024546109000073.tif4128, corresponds to amino acid residues 496 to 513 of human MASP-2 (SEQ ID NO:1) and is located on a single antiparallel beta strand-loop-beta strand element.

[0280] Figure 14B illustrates the three-dimensional structure of the OMS858 paratope, which includes HIS33, ASP50, ASP52, ASP55, GLU57 and HIS59 of the heavy chain, and ARG30, TYR31 and TRP90 of the light chain, to which MASP-2 binds. HIS33 is located on loop H1. ASP50, ASP52, ASP55, GLU57, HIS59 form an antiparallel beta strand-loop-beta strand element. TYR31 and ARG30 are on loop 1 of the light chain, and TRP90 is on the L3 loop of the light chain.

[0281] Figure 15 shows the MASP-2 epitope 15 illustrates the contacts between TIFF2024546109000074.tif4128 and the heavy and light chain variable regions of OMS858. Specifically, as shown in FIG. 15, heavy chain variable region residues ASP55, GLU57, and ASP52 interact with LYS503 of MASP-2, and heavy chain variable region residues HIS33, HIS59, and ASP50 interact with SER506, HIS508, and PRO507 of MASP-2. As further shown in FIG. 15, light chain variable region residue TRP90 interacts with PRO507 and HIS508 of MASP-2, and light chain variable region residues ARG30 and TYR31 interact with ASP496 and TRP513 of MASP-2.

[0282] In certain aspects, the MASP-2 SP (represented as SEQ ID NO:6), which is amino acid residues 496 to 513 of SEQ ID NO:1, is TIFF2024546109000075.tif4128) interacts with OMS858 via van der Waals interactions, which involve weak, short-range electrostatic attractions between uncharged molecules resulting from the interaction of permanent or transient electric dipole moments.

[0283] As shown in Table 16, OMS858 LC ARG30 atom CB interacts with atoms CZ2 and CE2 in TRP513 of MASP-2. LC TYR31 atom CE2 interacts with atom CH2 in TRP513 of MASP-2. HC HIS59 atoms CD2, NE2, CE1, ND1 and CG interact with atom CD2 in HIS508 of MASP-2. HC ASP50 atom OD2 interacts with atom CD2 in HIS508 of MASP-2. LC TRP90 atom CH2 interacts with atom NE2 in HIS508 of MASP-2. HC HIS59 atoms CD2, CG and CB interact with atom NE2 in HIS508 of MASP-2. HC ASP50 atom OD2 interacts with atoms CD2 and CE1 in HIS508 of MASP-2. LC TRP90 atom CZ2 interacts with atom CE1 in HIS508 of MASP-2. HC HIS59 atoms CE1, ND1, CG, CB and OD2 interact with atom CE1 in HIS508 of MASP-2. HC HIS59 atoms CE1 and CG interact with atom ND1 in HIS508 of MASP-2. HC HIS59 atoms NE2, CE1 and ND1 interact with atom CG in HIS508 of MASP-2. HC HIS59 atom ND1 interacts with atom CG in HIS508 of MASP-2. HC HIS59 atom CE1 interacts with atom CB in HIS508 of MASP-2. HC ASP50 atom CG interacts with atom NE2 in HIS508 of MASP-2. HC HIS33 atoms CE1 and ND1 interact with atom CD in PRO507 of MASP-2. HC HIS33 atom CE1 interacts with atom CA in SER506 of MASP-2. HC GLU57 atom CB interacts with atom NZ in LYS503 of MASP-2. HC ASP55 atom CG interacts with atom NZ in LYS503 of MASP-2. HC ASP52 atom CB interacts with atom NZ in LYS503 of MASP-2. HC ASP55 atom OD2 interacts with atom CE in LYS503 of MASP-2. HC ASP52 atom OD2 interacts with atoms CE and CD in LYS503 of MASP-2.LC TYR 31 atom CE2 interacts with atom OD2 in ASP496 of MASP-2.

[0284] In summary, the crystal structure of the MASP-2 inhibitor antibody OMS858 indicates that the antibody targets a novel epitope in the serine protease domain of human MASP-2. The results show that the epitope-binding domain binds to TIFF2024546109000076.tif4128. This epitope-binding domain is distinct from the serine protease activity triad region (residues HIS483, ASP532, and SER633). A novel epitope in the human MASP-2 serine protease domain It is further seen that there is an overlap between TIFF2024546109000077.tif4128 and the C4 interaction site on MASP-2 that includes residues LYS503 and TRP513 (see Kidmose et al., Proc Natl Acad Sci USA 109:15425 (2012)). Thus, the LYS503 and TRP513 residues of human MASP-2 can bind and participate in the binding of both OMS858 and C4.

[0285] The configuration of mAb OMS858 and MASP-2 shown in Figures 11 to 15 follows the standard antibody-antigen recognition format and suggests a mechanism of action in which OMS858 competes with C4 for binding to MASP-2, thereby inhibiting lectin pathway activity.

[0286] Although this crystal structure analysis was performed only on the MASP-2-Fab mAb OMS858 complex, as demonstrated in Example 6, not only does the parent mAb OMS850 cross-compete with OMS858 as expected, but also mAbs OMS860 and OMS870 cross-compete with OMS858. Thus, the binding epitopes of OMS860 and OMS870 are the same as or overlap with the epitopes on the MASP-2 serine protease identified by the analysis of the MASP-2-Fab mAb OMS858 complex. In contrast, the MASP-2 inhibitory antibodies 4A8 and OMS721 do not cross-compete with OMS858 and therefore bind to distinct epitopes on MASP-2.

[0287] Example 10 Generation of canine-like human MASP-2 to analyze the role of HIS508 in OMS858 binding As described in Example 9, structural findings of the MASP-2 / OMS858 complex indicate that HIS508 is a key side chain of the binding epitope, since it interacts with both the heavy and light chains of the variable domain of OMS858 through H-bridges to ASP50 and pi interactions to TRP90 and HIS59.

[0288] Figure 16 shows an amino acid alignment of the MASP-2 serine protease (SP) domains of human MASP-2 (aa 445-686 of SEQ ID NO:1), cynomolgus monkey MASP-2 (aa 445-686 of SEQ ID NO:4), canine MASP-2 (aa 445-686 of SEQ ID NO:5), mouse MASP-2 (aa 444-685 of SEQ ID NO:2), and rat MASP-2 (aa 444-685 of SEQ ID NO:3). TIFF2024546109000078.tif136160TIFF2024546109000079.tif244159TIFF2024546109000080.tif120159

[0289] To assess the species cross-reactivity of the OMS858 antibody, recombinant MASP-2 proteins from cynomolgus monkey and mouse were produced. DNA encoding the catalytic fragment of human MASP-2 (CCP1-CCP2-SP domains) was PCR amplified using Phusion high-fidelity DNA polymerase (New England BioLabs) and cloned into the pET-17b vector (Novagen) using the In-Fusion HD cloning kit (Clontech). A 6xHis tag was attached to the C-terminus of the polypeptide for purification. Similar MASP-2 expression constructs for cynomolgus monkey and mouse were also made.

[0290] In addition, we produced a "canine-like" variant of human MASP-2. We found that OMS858 did not inhibit lectin pathway activation in dog serum (data not shown). As shown in Figure 16, canine MASP-2 and human MASP-2 differ in sequence. One such mutation is at the residue corresponding to HIS508 in human MASP-2, which is GLN in canine MASP-2. This glutamine in canine MASP-2 is predicted to interact sufficiently with only one of the two amino acids of the histidine-interacting paratope in human MASP-2, effectively weakening the interaction between canine MASP-2 and OMS858 compared to human MASP-2. To generate a canine-like variant of human MASP-2, the H508Q mutation was introduced by PCR-based site-directed mutagenesis using PfuUltra II fusion HS DNA polymerase (Agilent). Based on the crystal structure data, it was predicted that OMS858 would have a lower affinity for this MASP-2 H508Q mutation compared to wild-type human H508.

[0291] All expression constructs were transformed into BL21(DE3)pLysS E. coli (Invitrogen), and recombinant proteins were expressed according to the manufacturer's protocol and purified by immobilized metal affinity chromatography (IMAC) using a nickel column. Protein integrity and enzymatic activity of the purified proteins were assessed by SDS-PAGE and peptide cleavage assay, respectively.

[0292] Biolayer interferometry (BLI) was applied to analyze OMS858 antibody binding to several MASP-2 proteins. All measurements were performed with the Octet RED96 system (ForteBio) using PBS containing 1% BSA and 0.02% Tween 20 as assay buffer. OMS858 (50 nM) was first loaded onto an anti-human Fc capture biosensor (ForteBio). After one baseline step, the capture sensor was submerged (120 s) into wells containing various concentrations of target protein (1.6–50 nM) for the association step, and then transferred to an empty well (200 s) for the dissociation step. In all experiments, the capture sensor was also submerged into wells not containing analyte to allow for the subtraction of a single baseline to compensate for spontaneous dissociation of the captured antibody. Binding affinity (KD) was determined by analyzing the recorded binding sensorgrams with Octet data analysis software (ForteBio).

[0293] Table 17. Binding of OMS858 to MASP-2 TIFF2024546109000081.tif46129

[0294] As shown in Figure 17, human MASP-2 carrying the H508Q "dog-like" mutation reduced binding affinity by approximately 450-fold.

[0295] Example 11 Cynomolgus Monkey Study: PK / PD of OMS856 and OMS858 Naive cynomolgus monkeys (n=3) were dosed with 1.5 mg / kg OMS856 and OMS858 either iv or sc. Blood samples were collected from each animal on day -7 and then at 0.083, 1, 4, 24, 72, 168, 240, 336, 504, 672, 840 and 1008 hours post-dose and tested for the presence of lectin pathway activity and the amount of antibody administered.

[0296] Lectin pathway assays were performed in ELISA plates coated with 5 μg / ml mannan overnight at 4° C. Plates were then blocked with 1% BSA in PBS for 2 h at room temperature. Cynomolgus serum was diluted 2-fold in PBS and added to the mannan-coated wells and incubated for 14 min at 4° C. Plates were then washed 3 times in PBS / tween-20 and C4 attachment was probed by adding rabbit anti-human C4c (Dako) followed by goat α-rabbit HRP (Southern Biotech).

[0297] FIG. 17A and FIG. 17B graphically illustrate lectin pathway activity over time in cynomolgus monkeys following iv administration of 1.5 mg / kg OMS856 or OMS858, respectively.

[0298] FIG. 18A and FIG. 18B graphically illustrate lectin pathway activity over time in cynomolgus monkeys following sc administration of 1.5 mg / kg OMS856 or OMS858, respectively.

[0299] As shown in Figures 17A and 18A, cynomolgus monkeys exhibit sustained systemic lectin pathway inhibition following iv and sc administration of 1.5 mg / kg OMS856.

[0300] As shown in Figures 17B and 18B, cynomolgus monkeys exhibit sustained systemic lectin pathway inhibition following iv and sc administration of 1.5 mg / kg OMS858.

[0301] Example 12 Analysis of OMS858 in a mouse model of thrombosis Studies were performed to determine the minimal effective dose and maximal efficacy of OMS858 in a ferric chloride-induced carotid artery occlusion model in mice.

[0302] The left carotid artery of male C57B1 / 6 mice was exposed and a miniature Transonic flowmeter (0.7 mm) was placed around the vessel. After administration of the test compound, thrombus formation was induced by applying a piece of filter paper (1.5 mm x 1 mm) saturated to the limit with 3.5% FeCl3. The filter paper was placed directly on the carotid artery in contact with the adventitial surface of the vessel. After 3 min of exposure, the filter paper was removed and the vessel was washed with saline. Carotid blood flow was recorded continuously until the vessel was completely occluded or for a maximum of 45 min. Time to occlusion (TTO) was defined as the time from application of FeCl3 until blood flow fell below 0.1 mL / min for at least 30 s or until the signal amplitude was reduced enough to prevent visualization of the heartbeat with the probe. Vessels that were not occluded at the end of the 45 min observation period were recorded as having a TTO of 45 min.

[0303] Mice (n=8 per dose group) were administered OMS858 by sc injection 24 hours prior to testing at the following dosages: 1 mg / kg, 3 mg / kg, 10 mg / kg and 30 mg / kg. Acetylsalicylic acid (ASA) at 30 mg / kg administered 1 hour prior to FeCl3 challenge was used as a positive control. An irrelevant antibody was used as a negative control.

[0304] The results are shown in Table 18 and illustrated graphically in Figure 19. As shown in Table 18 and Figure 19, the maximal effect of OMS858 was comparable to ASA. As further shown in Table 18 and Figure 19, OMS858 was effective even at the lowest dose tested (1 mg / kg). TTO in vehicle-treated control mice was 14.7 ± 4.6 minutes. As expected, pretreatment with ASA significantly extended TTO to 36.4 ± 5.7 minutes, whereas pretreatment with an isotype control antibody did not significantly extend TTO (9.3 ± 1.5 minutes), confirming the suitability of this test system. Pretreatment of mice with OMS858 at doses of 1, 3, 10 and 30 mg / kg significantly extended TTO compared to the vehicle group (40.4 ± 4.6, 37.0 ± 5.2, 39.1 ± 3.9 minutes and 37.4 ± 5.0, respectively). No clear dose-response relationship was observed among the OMS858 doses evaluated, indicating that maximal pharmacological efficacy may be achieved at a dose level of 1 mg / kg SC in this mouse model.

[0305] Table 18. Time to carotid artery occlusion after FeCl3-induced vascular injury TIFF2024546109000082.tif81170*p<0.05 compared to vehicle control; p-values ​​were generated using an unpaired, one-tailed t-test; ASA=aspirin; N=number of mice / treatment group; SEM=standard error of the mean; TTO=time to occlusion.

[0306] Example 13 Effect of OMS858 on classical, lectin, and alternative pathway-induced C5b-9 activation in human serum To assess the functional selectivity of lectin pathway inhibition by OMS858, the Wieslab® Complement System Screening Kit was used. Functional activity of OMS858 was assessed by preincubating human serum samples with serial dilutions of OMS858, followed by quantification of complement activation and C5b-9 deposition under pathway-specific assay conditions.

[0307] Human serum samples diluted in pathway-specific assay buffer were preincubated with serial dilutions of OMS858 and then incubated for 1 hour at 37° C. on appropriate Wieslab® assay wells precoated with classical pathway-specific (A), lectin pathway-specific (B) or alternative pathway-specific (C) complement activators. Deposition of the complement activation end product C5b-9 (also known as MAC) was quantified using an alkaline phosphatase-conjugated antibody specific for the C5b-9 neoantigen.

[0308] As shown in Figures 20A-C, OMS858 inhibited lectin pathway-induced activation of C5b-9 with an IC50 value of 0.81 nM (121.5 ng / mL) and had no effect on classical or alternative pathway-induced C5b-9 activation at concentrations up to 500 nM.

[0309] Example 14 Further characterization of monoclonal antibody OMS858 binding to human MASP-2 Surface plasmon resonance (SPR) was used to determine the association and dissociation rate constants (k and k, respectively) for the interaction of fluid-phase zymogen and catalytically active forms of human MASP-2 with immobilized OMS858. Optimized methods and concentration series were used to characterize the interaction in detail, and the obtained rate constants were used to calculate the dissociation equilibrium constant (K) for the OMS858-MASP-2 interaction.

[0310] k for the binding of catalytically active MASP-2 to OMS858 on and k off are 3.38×10 7 M -1 s -1 and 9.71×10 -3 s -1 So, K D The k value for the binding of zymogen MASP-2 to OMS858 was 287 pM. on and k off are 2.41×10 5 M -1 s-1 and 1.72 × 10 -4 s -1 This resulted in a KD value of 715 pM. See Table 19.

[0311] Table 19. Binding of OMS858 to zymogen and active forms of human MASP-2 TIFF2024546109000083.tif23149

[0312] The binding specificity of OMS858 to MASP-2 was assessed by solid-phase enzyme-linked immunosorbent assay (ELISA). Recombinant human MASP-2 or C1r, C1s, CFD, MASP-1 and MASP-3 were immobilized on polystyrene plates and the dose-response of OMS858 binding was measured. Apparent dissociation constants (KD) were estimated by nonlinear regression using a four-parameter logistic model.

[0313] Two different lots of OMS858 were tested. The average apparent KD for OMS858 binding to human MASP-2 was 0.047 μg / mL. No significant binding to C1r, C1s, MASP-1, MASP-3 or factor D was observed at OMS858 concentrations up to 100 μg / mL, indicating that OMS858 is at least 2000-fold more selective for MASP-2 compared to the closely related serine proteases of the complement system. The selectivity is expressed as K D (Serine protease related) / Average K D (MASP-2) (see Table 20).

[0314] Table 20. Selectivity of OMS858 binding to MASP-2 compared to related serine proteases of the complement system TIFF2024546109000084.tif55170

[0315] Example 15 Characterization of the functional activity of monoclonal antibody OMS858 in various mammalian species The functional potency of OMS858 was characterized in serum from humans, cynomolgus monkeys, dogs, and rabbits, and in mouse hirudin plasma, using an enzyme-linked immunosorbent assay (ELISA) that measures lectin-dependent C4 activation in 50% serum. Functional activity of OMS858 was assessed by preincubating serum or hirudin plasma samples from each species with serial dilutions of OMS858, and then adding the mixtures to mannan-coated ELISA plate wells that drive lectin-dependent C4 activation in vitro. Lectin pathway inhibition concentration-response curves were analyzed and IC 50 The functional potency of OMS858 inhibition of MASP-2 activity was estimated by determining the α-Kα value.

[0316] In human serum, OMS858 demonstrated potent inhibition of receptor-dependent complement activation with a mean IC 50 The mean IC value of OMS858 for inhibition of lectin pathway activation measured in cynomolgus monkey serum and mouse plasma was 1.09 nM (164 ng / mL). 50 The IC values ​​were 44.1 nM (6.620 μg / mL) and 11.9 nM (1.79 μg / mL), respectively. In contrast, OMS858 did not appreciably inhibit lectin-dependent complement activation in serum from rabbits and dogs at concentrations up to 500 nM. See Table 21. Comparing the functional potency in mice and cynomolgus monkeys with that in humans, OMS858 was shown to have a functional potency in these species that was 10.9- and 40.5-fold lower, respectively, than in humans. The relative potency to humans was calculated using the IC values ​​obtained with human serum. 50 Query type IC for 50 The ratio was calculated as the ratio of the values ​​shown in Table 22.

[0317] Table 21. IC for lectin pathway inhibition by OMS858 in mouse, rabbit, dog, and cynomolgus monkey serum compared to humans. 50 value TIFF2024546109000085.tif76162

[0318] Table 22. Interspecies titers to humans TIFF2024546109000086.tif57128

[0319] Example 16 Pharmacodynamics of OMS858 in mice and cynomolgus monkeys PD activity (inhibition of lectin pathway activity) was measured following ex vivo exposure of serum to mannan-coated surfaces followed by assessment of complement activation using an electrochemiluminescence immunoassay (ECLIA). In mice, individual animals' complement activation fragment deposition is compared to the mean complement activation fragment deposition in samples / control animals collected at baseline and data are presented as percentage inhibition. In monkeys, individual C4 deposition data is compared to the baseline response of individual animals and presented as percentage inhibition.

[0320] Results for mice are shown in FIG. 24. Baseline LP activity as measured by ECLIA was highly variable. There was no detectable PD effect at 0.1 mg / kg. However, mean MASP-2 inhibition was greater than 50% by 72 hours post-dose in the 0.3 mg / kg SC dose group, 504 hours post-dose in the 1 mg / kg IV dose group, and 1008 hours post-dose in the 1 and 3 mg / kg SC dose groups. In addition to a trend for increased duration of effect with increasing dose levels, there was also a trend for increased MASP-2 inhibition at higher OMS858 concentrations. OMS858 concentrations greater than 6 μg / mL (achieved in the 1 mg / kg IV and SC dose groups and the 3 mg / kg SC dose group) consistently resulted in greater than 50% MASP-2 inhibition. These data suggest that OMS858 consistently achieves substantial long-term inhibition of MASP-2 activity in mice at dose levels of 1 mg / kg or greater.

[0321] Male cynomolgus monkeys (n=3 / group) were administered 0.1, 0.3, 1, or 3 mg / kg OMS858 by subcutaneous (SC) injection or 1 mg / kg by intravenous (IV) injection. Serum samples for determination of OMS858 concentrations and OMS858 pharmacodynamic activity were collected pre-dose and at 0.083, 1, 4, 24, 48, 72, 168, 240, 336, 504, 672, 840, 1008, 1176, 1344, 1512, 1680, 1848, and 2016 hours post-dose.

[0322] Monkey results are shown in FIG. 21. In cynomolgus monkeys administered 1 mg / kg SC OMS858, approximately 70-80% inhibition of lectin pathway activity was observed beginning 24-48 hours after OMS858 administration and lasting for approximately 336 hours after administration, after which inhibition gradually decreased over time. In cynomolgus monkeys administered 3 mg / kg SC OMS858, near complete (>95%) inhibition of lectin pathway activity was observed beginning approximately 24 hours after OMS858 administration and lasting for approximately 672 hours after administration, after which inhibition gradually decreased over time. Near complete inhibition of lectin pathway activity was also observed following IV administration of 1 mg / kg OMS858, with the duration of maximum effect and tapering over time being similar to that observed with 1 mg / kg SC.

[0323] There was a consistent relationship between serum concentration of OMS858 and PD activity in cynomolgus monkeys, with serum concentrations of OMS858 above approximately 20 μg / mL resulting in a high degree (>80%) inhibition of lectin pathway activity, whereas serum concentrations below approximately 3 μg / mL resulted in negligible inhibition. Modeling of OMS858 serum concentration and PD response data revealed an EC 50 A sigmoidal drug concentration response profile was demonstrated with values ​​(OMS858 concentrations resulting in half-maximal lectin pathway inhibition) of 0.01-0.02 (see FIG. 22).

[0324] Example 17 Pharmacokinetics of OMS858 in mice and cynomolgus monkeys The PK of OMS858 following a single SC dose of 0.1, 0.3, 1 or 3 mg / kg or a single IV bolus dose of 1 mg / kg was characterized in male CD-1 mice and male cynomolgus monkeys. Blood samples were collected in mice pre-dose and at 0.083 (IV only), 24, 48, 72, 168, 240, 336, 504, 672, 840, 1008, 1176, and 1344 hours post-dose (N=3 / group / time point; terminal sampling) and in monkeys pre-dose and at approximately 0.083, 1, 4, 24, 48, 72, 168, 240, 336, 504, 672, 840, 1008, 1176, 1344, 1512, 1680, 1848, and 2016 hours post-dose (N=3 / group / time point; sequential sampling).

[0325] A summary of the PK parameters measured after a single dose of OMS858 administered to mice and monkeys by SC or IV injection is shown in Table 23. Figure 23 shows plots of the mean OMS858 serum concentrations over time after a single IV or SC dose to mice (left panel) and monkeys (right panel).

[0326] Time to reach maximum concentration (t max The median elimination time of OMS858 after a single SC or IV dose was 24 to 168 hours in mice and 72 or 168 hours in monkeys. 1 / 2 The mean time to death was 203-409 hours in mice and 196-487 hours in monkeys. max ) and total exposure (area under the time-concentration curve extrapolated to infinity [AUC INF ]) generally increased dose-proportionally with dose. Total exposure (AUC INF ), the bioavailability was 85.7% in mice and 93.0% in monkeys.

[0327] Table 23. Pharmacokinetic parameters following a single dose of OMS858 in mice and monkeys via SC and IV injection. TIFF2024546109000087.tif123164TIFF2024546109000088.tif238164TIFF2024546109000089.tif40164a Serum parameters reported for male mice based on composite mean concentration versus time data. b Determined only for the IV group at the 1 mg / kg dose level. c Bioavailability = AUC INF SC / AUC INF IV d Mean serum parameters reported for male monkeys AUC (0~336h) = area under the time-concentration curve from 0 to 336 hours after administration. AUC (0 336h) / Dose = area under the dose-normalized time-concentration curve from 0 to 336 hours after administration; AUC (0~t) = area under the time-concentration curve from time 0 to time t (last measurement time point); AUC INF = area under the time concentration curve extrapolated to infinity; C0 = estimated concentration at time 0; C max = highest observed concentration; C max / dose = dose-normalized highest observed concentration; h = time; NA = not applicable; SC = subcutaneous; IV = intravenous; t1 / 2 = terminal half-life; t last =Last measurable time;t max = time to peak concentration; Vss = volume of distribution at steady state; Vz = volume of distribution based on terminal elimination phase.

[0328] Example 18 A Phase 1 Single-Dose Clinical Trial of OMS858 in Healthy Human Subjects A single ascending dose blinded study of intravenous (IV) and subcutaneous (SC) administration of OMS858 in healthy subjects will be performed to evaluate safety, tolerability, PK, PD and immunogenicity compared to placebo. A total of 48 human volunteers will be divided into six cohorts of eight subjects each. In each cohort, six subjects will receive OMS858 and two subjects will receive placebo. Both subjects and the medical staff administering the medication will be blinded as to which subjects will receive OMS858 and which will receive placebo. Dosing for the six cohorts will be as follows: Cohort 1: Single IV dose of 0.01 mg / kg OMS858 or placebo Cohort 2: Single IV dose of 0.03 mg / kg OMS858 or placebo Cohort 3: Single IV dose of 0.1 mg / kg OMS858 or placebo Cohort 4: Single IV dose of 0.3 mg / kg OMS858 or placebo Cohort 5: Single SC dose of 1.0 mg / kg OMS858 or placebo Cohort 6: Single SC dose of OMS858 or placebo, dosing will be determined based on data from previous cohorts.

[0329] Subjects will be healthy human males or females aged 18-60 years, weighing 50-110 kg, and with a body mass index of 18-30 kg / m2 at the time of screening. Blood samples will be collected at screening, on day -1 before dosing, immediately before dosing, and at 0.5, 12, 24, 48, 72, 96, 120, 144, and 168 hours after dosing, and at days 15, 22, 29, 57, and 85 after dosing. The primary endpoint is the evaluation of the safety and tolerability of OMS858. Secondary endpoints are characterization of the PK and PD of OMS858, as well as the evaluation of the presence of anti-drug antibodies (ADA) against OMS858 after dosing. Exploratory endpoints are analysis of the effect of OMS858 on MASP-2 levels and mannose-binding lectin (MBL) levels.

[0330] Safety and tolerability evaluations will include monitoring for adverse events, vital signs, electrocardiograms, physical examinations, examination of blood and urine samples for clinical evaluation, and other measures of clinical status. Blood and / or serum samples from subjects will be assayed for PK and PD analyses determined by C4 adhesion assay, lectin pathway inhibition as measured by MASP-2 levels, MBL levels, ADA, and other relevant biomarkers.

[0331] Interim PK and PD data for Cohort 1, Cohort 2, Cohort 3, and Cohort 4 are shown in Figures 25-26. No safety or tolerability issues were observed at the time points shown in Figures 25-26. PK measurements for OMS858 demonstrated dose-proportional exposure throughout the time points shown in Figure 25. Robust and durable PD responses for OMS858 were observed in all three cohorts, as shown in Figure 26. A favorable PK / PD relationship was observed, as shown in Figure 27, with EC 50 was 170ng / mL and EC 90 was 400ng / mL.

[0332] IX. ILLUSTRATIVE EMBODIMENTS All publications and patent applications and patents mentioned herein are hereby incorporated by reference.

[0333] While certain embodiments of the invention have been illustrated and described, it will be understood that various changes can be made therein without departing from the spirit and scope of the invention. Although the invention has been described in connection with specific embodiments, it should be understood that the invention herein should not be unduly limited to such specific embodiments. Indeed, various modifications of the described specific embodiments that are obvious to those skilled in the fields of medicine, immunology, pharmacology, or related fields are intended to be within the scope of the invention.

[0334] Thus, for clarity of disclosure, numbered paragraphs describing specific embodiments are provided below, which should not be construed as limiting the scope of the claims.

[0335] 1. An isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds to an epitope located within the serine protease domain of human MASP-2, the epitope comprising the amino acid sequence The isolated monoclonal antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof is located within TIFF2024546109000090.tif4128, and wherein the antibody or antigen-binding fragment thereof inhibits lectin pathway complement activation. 2. An isolated antibody or antigen-binding fragment thereof of paragraph 1, which competes with C4 binding to MASP-2. 3. An isolated antibody or antigen-binding fragment thereof of paragraph 1, which forms a hydrogen bond with at least one of human MASP-2 Lys503 and / or His508. 4. An isolated antibody or antigen-binding fragment thereof of paragraph 1, which forms a hydrogen bond with human MASP-2 His508. 5. An isolated antibody or antigen-binding fragment thereof of paragraph 1, which forms van der Waals contacts with one or more of the following human MASP-2 amino acids: Asp496, Lys503, Ser506, Pro507, His508, and Trp513. 6. (a) an HC-CDR1 having the sequence NXXMH, where X at position 2 is H or Y and X at position 3 is H or W; HC-CDR2, represented as TIFF2024546109000091.tif4128, in which X at position 4 is P or A, X at position 9 is T or I, X at position 10 is H or Y, X at position 12 is I or N, and X at position 13 is E or Q; HC-CDR3, shown as TIFF2024546109000092.tif4128 and a heavy chain variable region comprising (b) LC-CDR1 as depicted in SEQ ID NO:64 (SASSSVXYMY), wherein X at position 7 is R or S; LC-CDR2, represented as SEQ ID NO:34 (DTSNLAS); LC-CDR3, shown as SEQ ID NO:36 (QQWSSYPLT); A light chain variable region comprising Contains or (b) HC-CDR1 as shown in SEQ ID NO:25 (SYWMH); HC-CDR2, shown as TIFF2024546109000093.tif4128; HC-CDR3, shown as SEQ ID NO:29 (WAYDAMDY); and a heavy chain variable region comprising LC-CDR1, shown as TIFF2024546109000094.tif5128; LC-CDR2, shown as SEQ ID NO: 43 (FASNLES); LC-CDR3, shown as SEQ ID NO:45 (QQSNEDPLT); Including, The isolated antibody or antigen-binding fragment thereof of paragraph 1. 7. The isolated antibody or antigen-binding fragment thereof of paragraph 6(a), wherein HC-CDR1 comprises SEQ ID NO:14 (NYWMH). 8. The isolated antibody or antigen-binding fragment thereof of paragraph 6(a), wherein HC-CDR1 comprises SEQ ID NO:56 (NYHMH). 9. The isolated antibody or antigen-binding fragment thereof of paragraph 6(a), wherein HC-CDR1 comprises SEQ ID NO:57 (NHHMH). 10. HC-CDR2 The isolated antibody or antigen-binding fragment thereof of paragraph 6(a), comprising TIFF2024546109000095.tif4128. 11. HC-CDR2 The isolated antibody or antigen-binding fragment thereof of paragraph 6(a), comprising TIFF2024546109000096.tif4128. 12. HC-CDR2 The isolated antibody or antigen-binding fragment thereof of paragraph 6(a), comprising TIFF2024546109000097.tif4128. 13. The isolated antibody or antigen-binding fragment thereof of paragraph 6(a), wherein LC-CDR1 comprises SEQ ID NO:32 (SASSSVRYMY). 14. The isolated antibody or antigen-binding fragment thereof of paragraph 6(a), wherein LC-CDR1 comprises SEQ ID NO:39 (SASSSVSYMY). 15. HC-CDR1 comprises SEQ ID NO:14 (NYWMH), SEQ ID NO:56 (NYHMH), or SEQ ID NO:57 (NHHMH), and HC-CDR2 comprises TIFF2024546109000098.tif5163, containing HC-CDR3 The isolated antibody or antigen-binding fragment thereof of paragraph 6(a), comprising TIFF2024546109000099.tif4128, wherein LC-CDR1 comprises SEQ ID NO:32 (SASSSVRYMY), LC-CDR2 comprises SEQ ID NO:34 (DTSNLAS), and LC-CDR3 comprises SEQ ID NO:36 (QQWSSYPLT). 16. HC-CDR1 comprises SEQ ID NO:14 (NYWMH) and HC-CDR2 comprises TIFF2024546109000100.tif5163, containing HC-CDR3 The isolated antibody or antigen-binding fragment thereof of paragraph 6(a), comprising TIFF2024546109000101.tif4128, wherein LC-CDR1 comprises SEQ ID NO:32 (SASSSVRYMY), LC-CDR2 comprises SEQ ID NO:34 (DTSNLAS), and LC-CDR3 comprises SEQ ID NO:36 (QQWSSYPLT). 17. HC-CDR1 comprises SEQ ID NO:56 (NYHMH) and HC-CDR2 comprises TIFF2024546109000102.tif5164, containing HC-CDR3 The isolated antibody or antigen-binding fragment thereof of paragraph 6(a), comprising TIFF2024546109000103.tif4128, wherein LC-CDR1 comprises SEQ ID NO:32 (SASSSVRYMY), LC-CDR2 comprises SEQ ID NO:34 (DTSNLAS), and LC-CDR3 comprises SEQ ID NO:36 (QQWSSYPLT). 18. HC-CDR1 comprises SEQ ID NO:57 (NHHMH) and HC-CDR2 comprises TIFF2024546109000104.tif4163, containing HC-CDR3 The isolated antibody or antigen-binding fragment thereof of paragraph 6(a), comprising TIFF2024546109000105.tif4128, wherein LC-CDR1 comprises SEQ ID NO:32 (SASSSVRYMY), LC-CDR2 comprises SEQ ID NO:34 (DTSNLAS), and LC-CDR3 comprises SEQ ID NO:36 (QQWSSYPLT). 19. HC-CDR1 comprises SEQ ID NO:14 (NYWM) and HC-CDR2 comprises TIFF2024546109000106.tif5128, containing HC-CDR3 The isolated antibody or antigen-binding fragment thereof of paragraph 6(a), comprising TIFF2024546109000107.tif4128, wherein LC-CDR1 comprises SEQ ID NO:32 (SASSSVRYMY), LC-CDR2 comprises SEQ ID NO:34 (DTSNLAS), and LC-CDR3 comprises SEQ ID NO:36 (QQWSSYPLT). 20. HC-CDR1 comprises SEQ ID NO:14 (NYWM) and HC-CDR2 comprises TIFF2024546109000108.tif4128, containing HC-CDR3 The isolated antibody or antigen-binding fragment thereof of paragraph 6(a), comprising TIFF2024546109000109.tif4128, wherein LC-CDR1 comprises SEQ ID NO:32 (SASSSVRYMY), LC-CDR2 comprises SEQ ID NO:34 (DTSNLAS), and LC-CDR3 comprises SEQ ID NO:36 (QQWSSYPLT). 21. HC-CDR1 comprises SEQ ID NO:56 (NYHMH) and HC-CDR2 comprises TIFF2024546109000110.tif4128, containing HC-CDR3 The isolated antibody or antigen-binding fragment thereof of paragraph 6(a), comprising TIFF2024546109000111.tif5128, wherein LC-CDR1 comprises SEQ ID NO:32 (SASSSVRYMY), LC-CDR2 comprises SEQ ID NO:34 (DTSNLAS), and LC-CDR3 comprises SEQ ID NO:36 (QQWSSYPLT). 22. HC-CDR1 comprises SEQ ID NO:57 (NHHMH) and HC-CDR2 comprises TIFF2024546109000112.tif4128, containing HC-CDR3 The isolated antibody or antigen-binding fragment thereof of paragraph 6(a), comprising TIFF2024546109000113.tif4128, wherein LC-CDR1 comprises SEQ ID NO:32 (SASSSVRYMY), LC-CDR2 comprises SEQ ID NO:34 (DTSNLAS), and LC-CDR3 comprises SEQ ID NO:36 (QQWSSYPLT). 23. HC-CDR1 comprises SEQ ID NO:14 (NYWM) and HC-CDR2 comprises TIFF2024546109000114.tif4128, containing HC-CDR3 The isolated antibody or antigen-binding fragment thereof of paragraph 6(a), comprising TIFF2024546109000115.tif5128, wherein LC-CDR1 comprises SEQ ID NO:39 (SASSSVSYMY), LC-CDR2 comprises SEQ ID NO:34 (DTSNLAS), and LC-CDR3 comprises SEQ ID NO:36 (QQWSSYPLT). 24. The isolated antibody or antigen-binding fragment of paragraph 6(a), comprising a heavy chain that comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity to SEQ ID NO:7, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:49, or SEQ ID NO:50, and a light chain that comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity to SEQ ID NO:10 or SEQ ID NO:47. 25. The isolated antibody or antigen-binding fragment of paragraph 6(a), comprising a heavy chain that comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity to SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:49, or SEQ ID NO:50, and a light chain that comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity to SEQ ID NO:47. 26. The isolated antibody or antigen-binding fragment of paragraph 6(a), comprising a heavy chain that comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity to SEQ ID NO:8 and a light chain that comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity to SEQ ID NO:11. 27. The isolated antibody or antigen-binding fragment of paragraph 6(b), comprising a heavy chain that comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity to SEQ ID NO:9 and a light chain that comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity to SEQ ID NO:12. 28. Any one of the isolated antibodies or antigen-binding fragments thereof of paragraphs 1 to 27, wherein the antibody or antigen-binding fragment is selected from the group consisting of a human antibody, a humanized antibody, a chimeric antibody, a murine antibody, and an antigen-binding fragment of any of the foregoing. 29. The isolated antibody or antigen-binding fragment thereof of any one of paragraphs 1 to 27, wherein the antibody or antigen-binding fragment is selected from the group consisting of a single chain antibody, an ScFv, a Fab fragment, a Fab' fragment, an F(ab')2 fragment, a monovalent antibody lacking the hinge region, and a complete antibody. 30. The isolated antibody or antigen-binding fragment of any one of paragraphs 1-27, further comprising an immunoglobulin constant region. 31. The isolated antibody or antigen-binding fragment of any one of paragraphs 1 to 27, which is humanized. 32. The isolated antibody or antigen-binding fragment of any one of paragraphs 1 to 27, which is an IgG immunoglobulin selected from the group consisting of IgG1, IgG2, and IgG4. 33. The isolated antibody or antigen-binding fragment thereof of any one of paragraphs 1 to 27, comprising one or more mutations in the Fc region. 34. The isolated antibody or antigen-binding fragment thereof of paragraph 33, wherein the Fc region comprises a S228P amino acid substitution. 35. Any one of the isolated antibodies or antigen-binding fragments of paragraphs 1 to 34, which binds to the serine protease domain of human MASP-2 with an affinity of less than 20 nM. 36. An isolated antibody or antigen-binding fragment thereof of paragraph 35, which binds to the serine protease domain of human MASP-2 with an affinity of less than 10 nM. 37. An isolated antibody or antigen-binding fragment thereof according to any one of paragraphs 1 to 34, which inhibits the lectin pathway in mammalian blood. 38. The isolated antibody or antigen-binding fragment thereof of paragraph 37, wherein said lectin pathway inhibition comprises a decrease in C3b deposition under lectin pathway specific assay conditions. 39. The isolated antibody or antigen-binding fragment thereof of paragraph 37, wherein said lectin pathway inhibition comprises a decrease in C4 adhesion under lectin pathway-specific assay conditions. 40. The isolated antibody or antigen-binding fragment thereof of paragraph 37, wherein said lectin pathway inhibition comprises a decrease in MAC adhesion under lectin pathway specific assay conditions. 41. An isolated antibody or antigen-binding fragment thereof according to any one of paragraphs 1 to 34, which does not inhibit the classical pathway in mammalian blood. 42. A composition comprising the antibody or antigen-binding fragment thereof of any one of paragraphs 1 to 34 and a pharma- ceutically acceptable excipient. 43. The composition of paragraph 42, formulated for subcutaneous administration. 44. An isolated polynucleotide encoding the heavy chain variable region and the light chain variable region of the antibody or antigen-binding fragment thereof of any one of paragraphs 1 to 34. 45. A combination of an isolated polynucleotide encoding a heavy chain variable region of the antibody or antigen-binding fragment thereof of any one of paragraphs 1 to 34 and an isolated polynucleotide encoding a light chain variable region of the antibody or antigen-binding fragment thereof of any one of paragraphs 1 to 34. 46. ​​A cloning or expression vector comprising a polynucleotide of paragraph 44 or a combination of polynucleotides of paragraph 45. 47. A combination of a cloning vector or expression vector comprising an isolated polynucleotide encoding the heavy chain variable region of the antibody or antigen-binding fragment thereof of any one of paragraphs 1 to 34, and a cloning vector or expression vector comprising an isolated polynucleotide encoding the light chain variable region of the antibody or antigen-binding fragment thereof of any one of paragraphs 1 to 34. 48. A host cell comprising one or more of the cloning or expression vectors of paragraph 46 or paragraph 47. 49. A process for producing the antibody or antigen-binding fragment of any one of paragraphs 1 to 34, comprising culturing the host cell of paragraph 48 and isolating the antibody or antigen-binding fragment thereof. 50. A method for inhibiting lectin pathway complement activation in a mammal, comprising the step of administering to a mammalian subject in need thereof an amount of the composition of paragraph 42 comprising a high affinity MASP-2 inhibitory antibody or antigen-binding fragment thereof sufficient to inhibit lectin pathway complement activation in the mammal. 51. The method of paragraph 50, wherein the subject in need thereof is suffering from or at risk of developing a disease or disorder of the lectin pathway selected from the group consisting of thrombotic microangiopathy (TMA), kidney disease, inflammatory responses resulting from tissue or organ transplantation, ischemia-reperfusion injury, complications associated with diabetes, cardiovascular disease or disorder, inflammatory gastrointestinal disorder, pulmonary disorder, ophthalmologic disease or disorder, disseminated intravascular coagulation, graft-versus-host disease, veno-occlusive disease, and diffuse alveolar hemorrhage.

Claims

1. 1. An isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds to an epitope located within the serine protease domain of human MASP-2, wherein the epitope has the amino acid sequence wherein the antibody or antigen-binding fragment thereof inhibits lectin pathway complement activation.

2. An isolated antibody or antigen-binding fragment thereof as described in claim 1, which competes with C4 binding to MASP-2.

3. An isolated antibody or antigen-binding fragment thereof as described in claim 1, which forms a hydrogen bond with at least one of human MASP-2 Lys503 and / or His508.

4. An isolated antibody or antigen-binding fragment thereof as described in claim 1, which forms a hydrogen bond with human MASP-2 His508.

5. An isolated antibody or antigen-binding fragment thereof as described in claim 1, which forms van der Waals contacts with one or more of the following human MASP-2 amino acids: Asp496, Lys503, Ser506, Pro507, His508, and Trp513.

6. (a) an HC-CDR1 having the sequence NXXMH, where X at position 2 is H or Y and X at position 3 is H or W; where X at position 4 is P or A, X at position 9 is T or I, X at position 10 is H or Y, X at position 12 is I or N, and X at position 13 is E or Q; and and HC-CDR3, shown as and a heavy chain variable region comprising (b) LC-CDR1, as represented by SEQ ID NO:64 (SASSSVXYMY), in which X at position 7 is R or S; LC-CDR2, shown as SEQ ID NO:34 (DTSNLAS); LC-CDR3, shown as SEQ ID NO:36 (QQWSSYPLT); A light chain variable region comprising Contains or (b) HC-CDR1, shown as SEQ ID NO:25 (SYWMH); and HC-CDR2, shown as HC-CDR3, shown as SEQ ID NO:29 (WAYDAMDY); and a heavy chain variable region comprising and LC-CDR1, shown as LC-CDR2, shown as SEQ ID NO:43 (FASNLES); LC-CDR3, shown as SEQ ID NO:45 (QQSNEDPLT); Including, 2. An isolated antibody or antigen-binding fragment thereof according to claim 1.

7. The isolated antibody or antigen-binding fragment thereof of claim 6(a), wherein HC-CDR1 comprises SEQ ID NO:14 (NYWMH).

8. The isolated antibody or antigen-binding fragment thereof of claim 6(a), wherein HC-CDR1 comprises SEQ ID NO:56 (NYHMH).

9. The isolated antibody or antigen-binding fragment thereof of claim 6(a), wherein HC-CDR1 comprises SEQ ID NO:57 (NHHMH).

10. HC-CDR2 The isolated antibody or antigen-binding fragment thereof of claim 6(a), comprising:

11. HC-CDR2 The isolated antibody or antigen-binding fragment thereof of claim 6(a), comprising:

12. HC-CDR2 The isolated antibody or antigen-binding fragment thereof of claim 6(a), comprising:

13. The isolated antibody or antigen-binding fragment thereof of claim 6(a), wherein LC-CDR1 comprises SEQ ID NO:32 (SASSSVRYMY).

14. The isolated antibody or antigen-binding fragment thereof of claim 6(a), wherein LC-CDR1 comprises SEQ ID NO:39 (SASSSVSYMY).

15. HC-CDR1 comprises SEQ ID NO:14 (NYWMH), SEQ ID NO:56 (NYHMH), or SEQ ID NO:57 (NHHMH), and HC-CDR2 comprises and HC-CDR3 is LC-CDR1 comprises SEQ ID NO: 32 (SASSSVRYMY), LC-CDR2 comprises SEQ ID NO: 34 (DTSNLAS), and LC-CDR3 comprises SEQ ID NO: 36 (QQWSSYPLT), an isolated antibody or antigen-binding fragment thereof according to claim 6(a).

16. HC-CDR1 comprises SEQ ID NO:14 (NYWMH) and HC-CDR2 comprises and HC-CDR3 is LC-CDR1 comprises SEQ ID NO: 32 (SASSSVRYMY), LC-CDR2 comprises SEQ ID NO: 34 (DTSNLAS), and LC-CDR3 comprises SEQ ID NO: 36 (QQWSSYPLT), an isolated antibody or antigen-binding fragment thereof according to claim 6(a).

17. HC-CDR1 comprises SEQ ID NO:56 (NYHMH) and HC-CDR2 comprises and HC-CDR3 is LC-CDR1 comprises SEQ ID NO: 32 (SASSSVRYMY), LC-CDR2 comprises SEQ ID NO: 34 (DTSNLAS), and LC-CDR3 comprises SEQ ID NO: 36 (QQWSSYPLT), an isolated antibody or antigen-binding fragment thereof according to claim 6(a).

18. HC-CDR1 comprises SEQ ID NO:57 (NHHMH) and HC-CDR2 comprises and HC-CDR3 is LC-CDR1 comprises SEQ ID NO: 32 (SASSSVRYMY), LC-CDR2 comprises SEQ ID NO: 34 (DTSNLAS), and LC-CDR3 comprises SEQ ID NO: 36 (QQWSSYPLT), an isolated antibody or antigen-binding fragment thereof according to claim 6(a).

19. HC-CDR1 comprises SEQ ID NO:14 (NYWM) and HC-CDR2 comprises and HC-CDR3 is LC-CDR1 comprises SEQ ID NO: 32 (SASSSVRYMY), LC-CDR2 comprises SEQ ID NO: 34 (DTSNLAS), and LC-CDR3 comprises SEQ ID NO: 36 (QQWSSYPLT), an isolated antibody or antigen-binding fragment thereof according to claim 6(a).

20. HC-CDR1 comprises SEQ ID NO:14 (NYWM) and HC-CDR2 comprises and HC-CDR3 is LC-CDR1 comprises SEQ ID NO: 32 (SASSSVRYMY), LC-CDR2 comprises SEQ ID NO: 34 (DTSNLAS), and LC-CDR3 comprises SEQ ID NO: 36 (QQWSSYPLT), an isolated antibody or antigen-binding fragment thereof according to claim 6(a).

21. HC-CDR1 comprises SEQ ID NO:56 (NYHMH) and HC-CDR2 comprises and HC-CDR3 is LC-CDR1 comprises SEQ ID NO: 32 (SASSSVRYMY), LC-CDR2 comprises SEQ ID NO: 34 (DTSNLAS), and LC-CDR3 comprises SEQ ID NO: 36 (QQWSSYPLT), an isolated antibody or antigen-binding fragment thereof according to claim 6(a).

22. HC-CDR1 comprises SEQ ID NO:57 (NHHMH) and HC-CDR2 comprises and HC-CDR3 is LC-CDR1 comprises SEQ ID NO: 32 (SASSSVRYMY), LC-CDR2 comprises SEQ ID NO: 34 (DTSNLAS), and LC-CDR3 comprises SEQ ID NO: 36 (QQWSSYPLT), an isolated antibody or antigen-binding fragment thereof according to claim 6(a).

23. HC-CDR1 comprises SEQ ID NO:14 (NYWM) and HC-CDR2 comprises and HC-CDR3 is LC-CDR1 comprises SEQ ID NO: 39 (SASSSVSYMY), LC-CDR2 comprises SEQ ID NO: 34 (DTSNLAS), and LC-CDR3 comprises SEQ ID NO: 36 (QQWSSYPLT), an isolated antibody or antigen-binding fragment thereof according to claim 6(a).

24. The isolated antibody or antigen-binding fragment of claim 6(a), comprising a heavy chain that comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity to SEQ ID NO:7, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:49, or SEQ ID NO:50, and a light chain that comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity to SEQ ID NO:10 or SEQ ID NO:

47.

25. The isolated antibody or antigen-binding fragment of claim 6(a), comprising a heavy chain that comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity to SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:49, or SEQ ID NO:50, and a light chain that comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity to SEQ ID NO:

47.

26. The isolated antibody or antigen-binding fragment of claim 6(a), comprising a heavy chain that comprises at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to SEQ ID NO:8 and a light chain that comprises at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to SEQ ID NO:

11.

27. The isolated antibody or antigen-binding fragment of claim 6(b), comprising a heavy chain that comprises at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to SEQ ID NO:9 and a light chain that comprises at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to SEQ ID NO:

12.

28. 28. The isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 27, which is selected from the group consisting of a human antibody, a humanized antibody, a chimeric antibody, a murine antibody, and an antigen-binding fragment of any of the foregoing.

29. 28. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 27, wherein the antibody or antigen-binding fragment is selected from the group consisting of a single chain antibody, an ScFv, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a monovalent antibody lacking a hinge region, and a complete antibody.

30. 28. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 27, further comprising an immunoglobulin constant region.

31. 28. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 27, which is humanized.

32. 28. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 27, which is an IgG immunoglobulin selected from the group consisting of IgG1, IgG2, and IgG4.

33. 28. The isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 27, comprising one or more mutations in the Fc region.

34. 34. The isolated antibody or antigen-binding fragment thereof of claim 33, wherein the Fc region comprises a S228P amino acid substitution.

35. An isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 34, which binds to the serine protease domain of human MASP-2 with an affinity of less than 20 nM.

36. An isolated antibody or antigen-binding fragment thereof as described in claim 35, which binds to the serine protease domain of human MASP-2 with an affinity of less than 10 nM.

37. 35. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 34, which inhibits the lectin pathway in mammalian blood.

38. 38. The isolated antibody or antigen-binding fragment thereof of claim 37, wherein the lectin pathway inhibition comprises a decrease in C3b adhesion under lectin pathway specific assay conditions.

39. 38. The isolated antibody or antigen-binding fragment thereof of claim 37, wherein the lectin pathway inhibition comprises a decrease in C4 adhesion under lectin pathway specific assay conditions.

40. 38. The isolated antibody or antigen-binding fragment thereof of claim 37, wherein the lectin pathway inhibition comprises a decrease in MAC adhesion under lectin pathway specific assay conditions.

41. 35. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 34, which does not inhibit the classical pathway in mammalian blood.

42. A composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 34 and a pharma- ceutically acceptable excipient.

43. 43. The composition of claim 42, formulated for subcutaneous administration.

44. An isolated polynucleotide encoding the heavy chain variable region and the light chain variable region of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 34.

45. A combination of an isolated polynucleotide encoding the heavy chain variable region of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 34 and an isolated polynucleotide encoding the light chain variable region of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 34.

46. A cloning or expression vector comprising a polynucleotide according to claim 44 or a combination of polynucleotides according to claim 45.

47. A combination of a cloning vector or expression vector comprising an isolated polynucleotide encoding the heavy chain variable region of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 34, and a cloning vector or expression vector comprising an isolated polynucleotide encoding the light chain variable region of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 34.

48. A host cell comprising one or more of the cloning or expression vectors of claim 46 or claim 47.

49. 49. A process for producing the antibody or antigen-binding fragment of any one of claims 1 to 34, comprising culturing the host cell of claim 48 and isolating the antibody or antigen-binding fragment thereof.

50. A method for inhibiting lectin pathway complement activation in a mammal, comprising the step of administering to a mammalian subject in need thereof an amount of a composition described in claim 42 comprising a high affinity MASP-2 inhibitory antibody or its antigen-binding fragment sufficient to inhibit lectin pathway complement activation in the mammal.

51. 51. The method of claim 50, wherein the subject in need thereof is suffering from or at risk of developing a disease or disorder of the lectin pathway selected from the group consisting of thrombotic microangiopathy (TMA), kidney disease, inflammatory responses due to tissue or organ transplantation, ischemia-reperfusion injury, complications associated with diabetes, cardiovascular disease or disorder, inflammatory gastrointestinal disorder, pulmonary disorder, ophthalmologic disease or disorder, disseminated intravascular coagulation, graft-versus-host disease, veno-occlusive disease, and diffuse alveolar hemorrhage.

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