Therapeutic antibody that binds to the serine protease domain of MASP-2 and its use

Abstract

It provides an inhibitor of complement activation. [Solution] An isolated monoclonal antibody and its antigen-binding fragment are provided that specifically bind to an epitope within the serine protease domain of human MASP-2. In some embodiments, the antibody or its antigen-binding fragment inhibits lectin pathway complement activation. Polynucleotides encoding the monoclonal antibody or its antigen-binding fragment, and cloning vectors or expression cassettes containing such polynucleotides are also provided. Methods for inhibiting lectin pathway complement activation and methods for treating diseases and disorders of the lectin pathway are further provided.

Description

[Technical Field]

[0001] Field of Invention The present invention relates to an antibody that binds to the serine protease domain of MASP-2, as well as related compositions and methods.

[0002] Statement regarding sequence listings The sequence listing relating to this application is provided in XML format instead of as a hard copy and is incorporated herein by reference. The XML file containing the sequence listing is named MP10329PCT.XML. This XML file is 89KB in size, was created on November 21, 2022, and submitted via the Patent Center along with the filing of this specification. [Background technology]

[0003] background The complement system supports innate host defense against pathogens and other acute invaders (MKLiszewski and JPAtkinson, 1993, in "Fundamental Immunology" (3rd edition, Raven Press, Ltd., New York) edited by W. Paul), 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 liquid-phase and membrane-bound proteins (S. Meyer, et al., mAbs, 6:1133, 2014). The majority of these are regulatory proteins that coordinate a cascade of highly controlled activation events. The complement system responds rapidly to molecular stress signals via 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, including cell lysis, phagocytosis, chemotaxis, and immune activation (S. Meyer, et al., 2014). Furthermore, the complement system also acts as a bridge between innate immune responses and subsequent adaptive immune activation. In addition to its anti-infective properties, the complement system is involved in the clearance of immune complexes and apoptotic cells, tissue regeneration, recruitment of hematopoietic progenitor cells, and angiogenesis (TMPierpont 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, recruiting the C1 complex. The C1 complex consists of a multimolecular recognition subcomponent C1q (composed of six heterotrimers: A, B, and C chains) and C1q-associated serine proteases, namely C1r and C1s. When C1q binds to the Fc region of either an antigen-bound IgM or at least two IgG antibodies bound to their respective antigens, the serine protease C1r is self-activated into 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 form the C4bC2 complex. In the second cleavage stage, C1s cleave C2 within the C4bC2 complex, releasing C2b to form the complement C3-converting enzyme complex C4bC2a, also known as C3-converting enzyme, which then 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 lectins (MBLs), ficolin, or collectin-11 and collectin-10 to pathogen-associated molecular patterns (PAMPs) or apoptotic or modified host cells. These recognition molecules form complexes with MBL-associated serine proteases MASP-1 and MASP-2, and activate them upon binding to their respective cognitive ligands. Activated MASP-2 cleaves C2 bound to C4 and C4b to form C3 convertase (C4bC2a).

[0007] The alternative pathway is initiated by the spontaneous hydrolysis of C3 to C3(H2O) ("tickover"), where C3(H2O) binds to factor B (fB). The resulting C3(H2O)fB complex requires the enzymatic activity of another highly specific serine protease called complement factor D to convert it to an enzymatically active C3 convertase. The availability of enzymatically active factor D is considered a limiting factor in the alternative pathway amplification loop; for factor D to become available, the action of another enzyme, MASP-3, is required for pro-Factor D (proCFD) to be converted to its active form, mature factor D (matCFD, or simply CFD) (Dobo et al., 2016). CFD activates the fB bound to C3(H2O) 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. Due to this mechanism, the alternative pathway is constitutively low in activity. The newly generated C3b, formed by C3(H2O)Bb or by the lectin pathway and classical pathway C3 convertase C4bC2a, binds to the target surface, such as bacterial cells, and sequesters fB to form the C3bfB complex. This complex is then cleaved by CFD to produce the alternative pathway C3 convertase complex C3bBb, forming an AP amplification loop. This convertase can be further stabilized by propagin, which prevents the breakdown of this complex and the conversion of C3b by factor I and cofactors.

[0008] These three pathways merge at the C3 activation stage. The C3 cleavage fragment C3a is an anaphylatoxin that promotes inflammation. C3b functions as an opsonin by covalently binding to the surface of target cells via its thioester bond, marking them for circulating complement receptor (CR) display effector cells, such as natural killer (NK) cells and macrophages, which contribute to complement-dependent cell-mediated cytotoxicity (CDCC) and complement-dependent cell 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-bound degradation fragments of C3b, iC3b and C3dg, can promote complement receptor-mediated cytotoxicity (CDCC and CDCP) and adaptive immune responses through B cell activation (MC Carroll, Nat. Immunol., 5:981, 2004).

[0009] The formation of C5 convertase leads to the cleavage of C5 into C5a and C5b. C5a is another anaphylatoxin. C5b recruits C6-9 to form a membrane invasion complex (MAC or C5b-9 complex). The MAC complex causes pore formation (so-called complement-dependent cell injury, CDC), which results in membrane disruption and cytolysis of target cells. While direct cytolysis by MAC formation has traditionally been recognized as a terminal effector mechanism of the complement system, C3b-mediated opsonization and pro-inflammatory signaling, as well as the anaphylatoxin function of C3a, are thought to play important roles in mediating complement-dependent inflammatory lesions.

[0010] While complement activation provides a valuable first line of defense against potential pathogens, the activation of complement, which promotes a protective immune response, can also be a potential threat to the host (KRKalli, et al., Springer Semin. Immunopathol. 15:417 431, 1994; BPMorgan, Eur. J. Clinical Investig. 24:219 228, 1994). For example, C3 and C5 protein degradation products recruit and activate neutrophils. Although activated neutrophils are essential for host defense, they can indiscriminately release destructive enzymes, potentially causing organ damage. In addition, complement activation can lead to host cell lysis by causing soluble complement components to adhere not only to microbial targets but also to nearby host cells.

[0011] The complement system is linked to the pathogenesis of numerous acute and chronic disease conditions, including myocardial infarction, stroke, acute respiratory distress syndrome (ARDS), complications associated with diabetes, ischemia-reperfusion injury, inflammatory bowel disorders, septic shock, capillary leakage after burns, 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 considered a major pathogenesis of these and other diseases and represents a key point for clinical management in these disease conditions. The increasing recognition of the importance of complement-mediated tissue injury in various disease conditions highlights the need for effective complement inhibitors. To date, only three complement-targeted drugs have been approved for human use: eculizumab (Soliris®), ravulizumab (Ultomiris) (both 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 inhibits complement system activation. Therefore, inhibitors targeting the early stages of complement activation would likely have considerable advantages over "downstream" complement inhibitors. [Overview of the project]

[0012] Summary This summary is provided to introduce, in a simplified form, a selection of concepts that are further detailed in the following 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 an antigen-binding fragment thereof that specifically binds to MASP-2. In some embodiments, the isolated monoclonal antibody or an antigen-binding fragment thereof specifically binds to an epitope within the serine protease domain of human MASP-2. In some embodiments, the epitope is located within amino acid TIFF2026123079000002.tif4128. In some embodiments, the antibody or an antigen-binding fragment thereof inhibits lectin pathway complement activation. In some embodiments, the antibody or an 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 containing such polynucleotides.

[0015] Also provided herein are host cells expressing the monoclonal antibodies or antigen-binding fragments thereof of the present disclosure, and a method for producing the monoclonal antibodies or antigen-binding fragments thereof of the present disclosure, the method comprising culturing the host cells under conditions permitting expression of the antibody or an antigen-binding fragment thereof and isolating the antibody or an antigen-binding fragment thereof.

[0016] This specification also provides a method for inhibiting lectin pathway complement activation in mammals, comprising the step of administering to a mammalian subject in need of such inhibition a certain amount of a composition comprising a monoclonal antibody or antigen-binding fragment of the Disclosure in an amount sufficient to inhibit lectin pathway complement activation in the mammal. In some embodiments, the monoclonal antibody or antigen-binding fragment of the Disclosure may be used to treat subjects suffering from or at risk of lectin pathway-related diseases or disorders. In some embodiments, lectin pathway-related diseases or disorders are selected from thrombotic microangiopathy (TMA), kidney disease, inflammatory reactions resulting from tissue or organ transplantation, ischemia-reperfusion injury, complications associated with diabetes, cardiovascular diseases or disorders, inflammatory gastrointestinal disorders, lung disorders, ophthalmic diseases or disorders, disseminated intravascular coagulation, graft-versus-host disease, veno-occlusive diseases, and diffuse alveolar hemorrhage. [Brief explanation of the drawing]

[0017] By referring to the detailed explanations provided below and interpreting them in conjunction with the accompanying drawings described below, many of the aforementioned aspects and associated advantages of the present invention will be better understood and more easily recognized.

[0018] [Figure 1] Figure 1 is a schematic diagram of the complementary system. [Figure 2] Figure 2 is a diagram illustrating the domain structure of the human MASP-2 protein. [Figure 3] Figure 3 shows the 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] Figure 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]Figure 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] Figure 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] Figure 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] Figure 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] Figure 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] Figure 7 graphically illustrates the concentration-dependent inhibition of C3b adhesion in human serum by mAb OMS852 with various P53 substitutions, as described in Example 4. [Figure 8A] Figure 8A graphically illustrates the concentration-dependent inhibition of C3b adhesion in human serum by several candidate humanized versions of mAb OMS850, as described in Example 4. [Figure 8B] Figure 8B graphically illustrates the concentration-dependent inhibition of C3b adhesion in human serum by several candidate humanized versions of mAb OMS850, as described in Example 4. [Figure 9A] Figure 9A graphically illustrates the level of MAC adhesion in the presence of various concentrations of the anti-MASP-2 antibody OMS858 under classical pathway-specific assay conditions, as described in Example 5. [Figure 9B]Figure 9B graphically illustrates the level of MAC adhesion in the presence of various concentrations of the anti-MASP-2 antibody OMS858 under the lectin pathway-specific assay conditions described in Example 5. [Figure 9C] Figure 9C graphically illustrates the level of MAC adhesion in the presence of various concentrations of the anti-MASP-2 antibody OMS858 under the alternative pathway-specific assay conditions described in Example 5. [Figure 10] Figure 10 shows the results of the binding test using a biolayer interferometry (BLI) instrument (OCTET) with a panel of biotinylated OMS858 and other MASP-2 antibodies, as described in Example 6. [Figure 11] Figure 11 is a schematic diagram showing the arrangement of the MASP-2 serine protease domain and Fab mAb OMS858, and the contact between them, as described in Example 9. [Figure 12] The left side of Figure 12 is a schematic diagram showing the MASP-2 epitope to which mAb OMS858 binds, and this epitope contains the following residues in the SP domain of MASP-2: ASP496, LYS503, SER506, PRO507, HIS508, and TRP513. The right side is a schematic diagram showing the paratope of mAb OMS858 that binds to the MASP-2 epitope, and this paratope contains two connected corresponding patches to which the heavy chain and light chain contribute, specifically the heavy chain residues HIS33, ASP50, ASP52, ASP55, GLU57, HIS59, and the light chain residues TYR31, ARG30, and TRP90, as described in Example 9. [Figure 13] Figure 13 illustrates the interaction between the mAb OMS858 paratope and the MASP-2 epitope, as calculated by the LigPlot+ software, as described in Example 9. [Figure 14A] Figure 14A illustrates the three-dimensional structure of the MASP-2 epitope, including ASP496, LYS503, SER506, PRO507, HIS508, and TRP513 to which mAb OMS858 binds, as described in Example 9. [Figure 14B] Figure 14B illustrates the three-dimensional structure of the mAb OMS858 paratope, which includes the heavy chains HIS33, ASP50, ASP52, ASP55, GLU57, and HIS59, as well as the light chains ARG30, TYR31, and TRP90, to which MASP-2 is bound, as described in Example 9. [Figure 15] Figure 15 illustrates the contact between the MASP-2 epitope DIRMGTLKRLSPHYTQAW (SEQ ID NO:6) and the heavy-chain variable region and light-chain variable region of mAb OMS858, as described in Example 9. [Figure 16] Figure 16 shows the 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), canine 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. This indicates that HIS508 of human MASP-2 is conserved in cynomolgus monkeys, mice, and rats, but not in dogs. [Figure 17A] Figure 17A graphically illustrates the lectin pathway activity over time in cynomolgus monkeys after intravenous administration of 1.5 mg / kg of OMS856, as described in Example 11. [Figure 17B] Figure 17B graphically illustrates the lectin pathway activity over time in cynomolgus monkeys after intravenous administration of 1.5 mg / kg of OMS858, as described in Example 11. [Figure 18A] Figure 18A graphically illustrates the lectin pathway activity over time in cynomolgus monkeys after subcutaneous administration of 1.5 mg / kg of OMS856, as described in Example 11. [Figure 18B] Figure 18B graphically illustrates the lectin pathway activity over time in cynomolgus monkeys after subcutaneous administration of 1.5 mg / kg of OMS858, as described in Example 11. [Figure 19] Figure 19 graphically illustrates the time to occlusion (TOC) in mice administered OMS858 within a certain dose range, as described in Example 12. [Figure 20A] Figure 20A graphically illustrates the level of MAC adhesion in the presence of various concentrations of the anti-MASP-2 antibody OMS858 under classical pathway-specific assay conditions, as described in Example 13. [Figure 20B] Figure 20B graphically illustrates the level of MAC adhesion in the presence of various concentrations of the anti-MASP-2 antibody OMS858 under the lectin pathway-specific assay conditions described in Example 13. [Figure 20C] Figure 20C graphically illustrates the level of MAC adhesion in the presence of various concentrations of the anti-MASP-2 antibody OMS858 under alternative pathway-specific assay conditions, as described in Example 13. [Figure 21] Figure 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] Figure 22 graphically illustrates the relationship between OMS858 serum concentration 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. Plots of the mean serum concentrations of OMS858 over time after a single IV or SC administration to mice (left) and monkeys (right) are shown. [Figure 24]Figure 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. [Figure 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). The concentrations of OMS858 detected in serum samples from subjects at various time points after administration are shown. The dotted line indicates the lower limit of quantification for the assay used, which is 70 ng / mL. [Figure 26] Figure 26 graphically illustrates the pharmacodynamics (PD) of OMS858 in healthy human subjects. Subjects received 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 levels of complement lectin pathway inhibition, measured by C4 attachment, are shown at various time points after administration. Measurement results for subjects who received placebo are also shown. [Figure 27] Figure 27 graphically illustrates the relationship between PK and PD for OMS858. The levels of lectin pathway inhibition at various OMS858 levels are shown for subjects who received 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 was 170 ng / mL, and the calculated EC90 was 400 ng / mL. [Modes for carrying out the invention]

[0019] Detailed explanation I. Definition Unless specifically defined herein, all terms used herein have the same meaning as those understood by those 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 provided throughout this disclosure.

[0020] In this specification, any concentration range, percentage range, ratio range, or integer range shall be understood to include any integer value within the listed range and, where appropriate, fractions thereof (e.g., one-tenth and one-hundredth of an integer), unless otherwise indicated or evident from the context. Any numerical range listed herein with respect to any physical characteristics such as polymer subunits, size, or thickness shall be understood to include any integer within the listed range and, where appropriate, fractions thereof, unless otherwise indicated or evident from the context. Where used herein, the term “approximately” shall indicate that the given range or value may vary by ±10% from the indicated range or value unless otherwise indicated.

[0021] As used herein, the terms “a,” “an,” and “the” should be understood to refer to one or more referent components. The use of alternatives (e.g., “or”) should be understood to mean one or both of those alternatives, or any combination thereof. As used herein, the terms “encompass,” “have,” and “include” are synonymous, and these terms and their variants should be construed as non-restrictive.

[0022] "Optional" or "at will" means that the elements, components, events, or situations described thereafter may or may not exist or occur, and that such descriptions encompass both the cases in which such elements, components, events, or situations exist or occur, and the cases in which they do not exist or occur.

[0023] Individual structures or groups of structures resulting from various combinations of the structures and subunits described herein should be understood to be disclosed in the same degree as if each structure or group of structures were described individually. Therefore, the selection of a particular structure or particular subunit is within the scope of this disclosure.

[0024] The term “essentially consisting of” is not equivalent to “containing,” and refers to any material or process specified in the claim, or the subject described in the claim, that does not substantially affect the fundamental characteristics of the claim. For example, a domain, region, or module of a protein (e.g., a binding domain) or a protein “essentially consisting of” a particular amino acid sequence if that amino acid sequence includes elongations, deletions, mutations, or combinations thereof (e.g., amino acids at the amino or carboxyl end, or amino acids between domains), and such elongations, deletions, mutations, or combinations thereof, as a whole, account for a maximum of 20% (e.g., a maximum of 15%, 10%, 8%, 6%, 5%, 4%, 3%, 2%, or 1%) of the length of that domain, region, module, or protein, and does not substantially affect the activity of that domain, region, module, or protein (e.g., the target binding affinity of the binding protein) (i.e., does not reduce the activity by more than 50%, for example, more than 40%, 30%, 25%, 20%, 15%, 10%, 5%, or 1%).

[0025] As used herein, the terms “to treat,” “treatment,” or “to induce remission” refer to the medical management of the disease, disorder, or condition of interest. Generally, an appropriate dose or treatment regimen containing the antibody or its antigen-binding fragment of the Disclosure is administered in an amount sufficient to produce a therapeutic or preventive benefit. Therapeutic or preventive / preventive benefits include improvements in clinical outcomes, reduction or mitigation of disease-related symptoms, decreased symptom incidence, improved quality of life, extended disease-free status, reduction in disease severity, stabilization of the disease state, delayed or prevention of disease progression, remission, survival, extended survival time, or any combination thereof.

[0026] In this disclosure, “therapeutic effective dose” or “effective dose” of an antibody or its antigen-binding fragment, polynucleotide, vector, host cell, or composition means an amount of composition or molecule sufficient to produce a statistically significant therapeutic effect, including improvement of clinical outcomes, reduction or mitigation of disease-related symptoms, reduction of symptom onset, improvement of quality of life, extension of disease-free status, reduction of disease severity, stabilization of disease status, delay of disease progression, remission, survival, or extension of survival time. When referring to an individual active ingredient administered alone, the therapeutic effective dose means the effect of that ingredient or the effect of cells expressing only that ingredient. When referring to a combination, the therapeutic effective dose means the combined amount of the active ingredient, or the combined co-active ingredient and cells expressing the active ingredient, that produces a therapeutic effect, whether administered sequentially, sequentially, or simultaneously.

[0027] The term "subject" as used herein includes, but is not limited to, all mammals, including, humans, non-human primates, dogs, cats, horses, sheep, goats, cattle, rabbits, pigs, and rodents. Subjects may be male or female, and may be of any appropriate age, including infants, young adults, adolescents, adults, or elderly.

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

[0029] As used herein, “mutation” refers to a change in the sequence of a nucleic acid molecule or polypeptide molecule 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 substitutions, insertions, or deletions of nucleotides or amino acids.

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

[0031] A "conservative substitution" refers to an amino acid substitution that does not significantly affect or alter the binding characteristics of a particular protein. Generally, a conservative substitution is one in which the substituted amino acid residue is replaced by an amino acid residue with 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). In addition to or instead of the above, amino acids can also be grouped into conserved substitution groups based on similar function, chemical structure, or composition (e.g., acidic, basic, aliphatic, aromatic, or sulfur-containing). For example, when substitution is the objective, the aliphatic group may include Gly, Ala, Val, Leu, and Ile. Other conserved substitution groups include: sulfur-containing: Met and cysteine ​​(Cys or C); acidic: Asp, Glu, Asn, and Gln; small aliphatic, nonpolar or weakly polar residues: Ala, Ser, Thr, Pro, and Gly; polar, positively charged residues and their amides: Asp, Asn, Glu, and Gln; polar, positively charged residues: His, Arg, and Lys; large aliphatic, nonpolar 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,” “peptide,” or “polypeptide” refers to a polymer of amino acid residues. The term “protein” applies to natural amino acid polymers, amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of corresponding natural amino acids, and non-natural amino acid polymers. Variants of the proteins, peptides, and polypeptides of this disclosure are also envisioned. In certain embodiments, variant proteins, peptides, and polypeptides contain 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 amino acid sequence or reference amino acid sequence described herein.

[0033] "Nucleic acid molecule," "oligonucleotide," "polynucleotide," or "polynucleic acid" refers to an oligomeric or polymeric compound containing covalently linked nucleotides, which may consist 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), such as mRNA, microRNA, siRNA, viral genomic RNA, and synthetic RNA, as well as polydeoxyribonucleic acid (DNA), such as cDNA, genomic DNA, and synthetic DNA. Both RNA and DNA can be single-stranded or double-stranded. In the single-stranded case, the nucleic acid molecule can be a coding strand or a non-coding strand (antisense strand). Nucleic acid molecules that code for an amino acid sequence include all nucleotide sequences that code for the same amino acid sequence. Some versions of a nucleotide sequence may also include introns, if the introns can be removed by concurrent or post-transcriptional mechanisms. In other words, as a result of redundancy or degeneracy in the genetic code, or due to splicing, different nucleotide sequences can encode the same amino acid sequence.

[0034] Variants of the nucleic acid molecules of this disclosure are also envisioned. The variant nucleic acid molecules are at least 70%, 75%, 80%, 85%, 90%, and preferably 95%, 96%, 97%, 98%, 99%, or 99.9% identical to a given polynucleotide or reference polynucleotide nucleic acid molecule described herein, or hybridize to a 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. The nucleic acid molecule variants retain the ability to encode their binding domains, which have the functionalities described herein, such as binding to a target molecule.

[0035] "Percent sequence identity" refers to the relationship between two or more sequences determined by comparing them. Preferred methods for determining sequence identity are designed to provide 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 acids or nucleic acid sequences for optimal alignment). Furthermore, non-homologous sequences may be ignored for comparison. Percent sequence identity as 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 percentage identity calculations can be performed using the BLAST program (e.g., BLAST2.0, BLASTP, BLASTN, or BLASTX) or Megalign (DNASTAR) software. The mathematical algorithms 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 necessary to achieve maximum alignment across the entire length of the sequences being compared.

[0036] The term "isolated" means that the material has been removed from its original environment (for example, its natural environment if it is naturally occurring). For example, a natural nucleic acid or natural polypeptide present in a living animal is not isolated, but the same nucleic acid or polypeptide separated from some or all of the material coexisting 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 (for example, a cell lysate), but it is still isolated in that such a vector or composition is not part of the natural environment for that nucleic acid or polypeptide. In some embodiments, "isolated" can also describe antibodies, antigen-binding fragments, polynucleotides, vectors, host cells, or compositions that are outside the human body.

[0037] The term "gene" refers to a segment of DNA or RNA involved in the production of polypeptide chains, and in certain contexts, this term encompasses the regions before and after 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" means a polypeptide or polynucleotide that is structurally similar to or substantially structurally similar to a parent compound or reference compound of the present disclosure, but with a slightly different 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 an activity level of at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% 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 the polypeptide of the present disclosure or the encoded polypeptide has “similar binding,” “similar affinity,” or “similar activity” if, in a selected assay such as an assay for measuring enzyme activity or binding affinity, the functional variant exhibits improved performance or a performance reduction of 50% or less compared to the parent polypeptide or reference polypeptide.

[0039] As used herein, “functional portion” or “functional fragment” means a polypeptide or polynucleotide comprising only a domain, portion, or fragment of a parent compound or reference compound, wherein the polypeptide or encoded polypeptide maintains at least 50% of the activity associated with the domain, portion, or fragment of the parent compound or reference compound, preferably at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 100% of the activity level of the parent polypeptide, or a higher activity level than the activity level of the parent polypeptide, or provides a biological benefit (e.g., effector function). A polypeptide of the present disclosure or a "functional portion" or "functional fragment" of an encoded polypeptide has "similar binding" or "similar activity" if, in a selected assay, the functional portion or fragment exhibits improved performance or a performance reduction of 50% or less compared to the parent polypeptide or reference polypeptide (for affinity, preferably a reduction of 20% or 10% or less, or a difference of 1 log or less compared to the parent or reference).

[0040] As used herein, the terms “manipulated,” “recombinant,” or “non-natural” refer to an organism, microorganism, cell, protein, polypeptide, nucleic acid molecule, or vector that has been modified by at least one genetic modification or by the introduction of a foreign or heterologous nucleic acid molecule, wherein such modification or alteration is introduced by genetic engineering (i.e., human intervention). Genetic modifications include, for example, modifications that introduce an expressible nucleic acid molecule encoding a functional RNA, protein, fusion protein, or enzyme, or modifications that introduce the addition, deletion, substitution, or other functional disruption of the cellular genetic material of other nucleic acid molecules. Further modifications include, for example, non-coding regions where modifications alter the expression of polynucleotides, genes, or operons.

[0041] As used herein, “heterogeneous,” “non-intrinsic,” or “exotic” means 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 and has been modified. Heterogeneous, non-intrinsic, or exotic includes genes, proteins, compounds, or nucleic acid molecules that have been mutated or otherwise modified such that their structure, activity, or both differ between the native gene, protein, compound, or nucleic acid molecule and the modified gene, protein, compound, or nucleic acid molecule. In certain embodiments, a heterogeneous, non-intrinsic, or exotic gene, protein, or nucleic acid molecule (e.g., receptor, ligand, etc.) is not endogenous to a host cell or subject, and the nucleic acid encoding such a gene, protein, or nucleic acid molecule may be added to a host cell by conjugation, transformation, transfection, electroporation, etc., where the added nucleic acid molecule may be integrated into the host cell genome or exist as extrachromosomal genetic material (e.g., as a plasmid or other self-replicating vector). "Homologous" or "homologous" refers to a gene, protein, compound, nucleic acid molecule, or activity found in or derived from a particular host cell, species, or strain. For example, a heterologous or exogenous polynucleotide or gene encoding a polypeptide may be homologous to a native polynucleotide or native gene and may encode a homologous polypeptide or activity, but that polynucleotide or polypeptide may have a modified structure, sequence, expression level, or any combination thereof. Non-endogenous polynucleotides or genes, and the polypeptides or activities they encode, may originate from the same species, different species, or a combination thereof.

[0042] In certain embodiments, a nucleic acid molecule or a portion thereof that is native to a host cell will be considered heterogeneous to that host cell if it is modified or mutated. Alternatively, a nucleic acid molecule native to a host cell may be considered heterogeneous if it is modified with heterogeneous expression regulatory sequences or with endogenous expression regulatory sequences not typically associated with the host cell's native nucleic acid molecule. In addition, the term “heterogeneous” can also refer to biological activity that is different, modified, or non-endogenous to the host cell. As described herein, multiple heterogeneous nucleic acid molecules can be introduced into a host cell as separate nucleic acid molecules, as multiple individually controlled genes, as a polycistronic nucleic acid molecule, as a single nucleic acid molecule encoding an antibody or antigen-binding fragment (or other polypeptide), or in any combination thereof.

[0043] As used herein, the terms “endogenous” or “native” refer to polynucleotides, genes, proteins, compounds, molecules, or activities that are normally present in a host cell or subject.

[0044] As used herein, the term "expression" refers to the process by which polypeptides are produced based on the coding sequence of a nucleic acid molecule, such as a gene. This process may include transcription, post-transcriptional regulation, post-transcriptional modification, translation, post-translational regulation, post-translational modification, or any combination thereof. Nucleic acid molecules to be expressed are typically functionally linked to an expression regulatory sequence (e.g., a promoter).

[0045] The term "functionally linked" refers to the relationship between two or more nucleic acid molecules on a single nucleic acid fragment, where the function of one is influenced by the other. For example, a promoter is functionally linked to a coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter) if it can influence the expression of that coding sequence. "Dislinked" means that related genetic elements are not closely related to each other, and the function of one does not influence the other.

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

[0047] The term “construct” refers to any polynucleotide (or, where contextually clear, a fusion protein) containing a recombinant nucleic acid molecule. A (polynucleotide) construct may reside in a vector (e.g., a bacterial vector, a viral vector) or be incorporated into a genome. A “vector” is a nucleic acid molecule capable of transporting another nucleic acid molecule. A vector may be a plasmid, cosmid, virus, RNA vector, or linear or circular DNA or RNA molecule, which may include, for example, chromosomes, non-chromosomal, semi-synthetic or synthetic nucleic acid molecules. Vectors in this 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 include those with autonomous replication capabilities (episome vectors), those capable of delivering polynucleotides to a cellular genome (e.g., viral vectors), or those capable of expressing nucleic acid molecules 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 functionally ligated to appropriate regulatory sequences capable of achieving expression of the nucleic acid molecule in a suitable host. Such regulatory sequences typically include a promoter for achieving transcription, an optional operator sequence for controlling such transcription, a sequence encoding a suitable mRNA-ribosome binding site, and sequences controlling the termination of transcription and translation. A vector can be a plasmid, a phage particle, a virus, or simply a potential genome insert. Upon transformation into a suitable host, a vector can replicate and function independently of the host genome, or, in some cases, be integrated into the genome itself, or deliver the polynucleotides contained in the vector into the genome without the vector sequence. As used herein, “plasmid,” “expression plasmid,” “virus,” and “vector” are often used interchangeably.

[0049] In relation to the insertion of nucleic acid molecules into cells, the term “introduced” means “transfection,” “transformation,” or “transduction,” and includes references to the incorporation of nucleic acid molecules into eukaryotic or prokaryotic cells, where the nucleic acid molecule may be incorporated into the cell’s genome (e.g., chromosomes, plasmids, plastids, or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).

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

[0051] In certain embodiments, vectors include plasmid vectors or viral vectors (e.g., lentiviral vectors or gamma-retroviral vectors). Viral vectors include retroviruses, adenoviruses, parvoviruses (e.g., adeno-associated viruses), coronaviruses, negative-strand RNA viruses (e.g., orthomyxoviruses, influenza viruses), rhabdoviruses (e.g., rabies and vesicular stomatitis viruses), paramyxoviruses (e.g., measles and Sendai viruses), positive-strand RNA viruses (e.g., picornaviruses and alphaviruses), as well as double-strand DNA viruses (e.g., adenoviruses, herpesviruses (e.g., herpes simplex virus types 1 and 2, Epstein-Barr virus, cytomegaloviruses)), and poxviruses (e.g., vaccinia, fowlpox, and canarypox). Other viruses include, for example, Norwalk virus, togavirus, flavivirus, reovirus, papovavirus, hepadnavirus, and hepatitis viruses. Examples of retroviruses include avian leukemia sarcoma, mammalian type C, type B, and type D viruses, HTLV-BLV group, lentiviruses, and spumaviruses (Coffin, JM, "Retroviridae: The viruses and their replication" in "Fundamental Virology" (3rd edition, edited by B.N. Fields et al., Lippincott-Raven Publishers, Philadelphia, 1996)).Methods 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 this disclosure include those derived from baculoviruses and alphaviruses (see Jolly, D J. 1999, "Emerging Viral Vectors," pp. 209–2040, in Friedmann T., ed., "The Development of Human Gene Therapy" (New York: Cold Spring Harbor Lab), or plasmid vectors (e.g., Sleeping Beauty or other transposon vectors).

[0053] If a viral vector genome contains multiple polynucleotides to be expressed as separate transcripts in a host cell, the viral vector may also include additional sequences between those two (or more) transcripts that enable dicistronic or polycistronic expression. Examples of such sequences used in viral vectors include intrasequence ribosome entry sites (IRESs), furin cleavage sites, viral 2A peptides, or any combination thereof.

[0054] Plasmid vectors, including DNA-based plasmid vectors for expressing one or more proteins in vitro or for direct administration to a target, are also known in the art. Such vectors may contain bacterial origins of replication, viral origins of replication, genes encoding components necessary for plasmid replication, and / or one or more select markers. Such vectors may also contain additional sequences that enable dicistronic or polycistronic expression.

[0055] As used herein, the term “host” refers to a cell or microorganism that is targeted for genetic modification by a heterologous nucleic acid molecule to produce a polypeptide of interest (e.g., an antibody in this disclosure).

[0056] Host cells can include any individual cell or cell culture capable of receiving vector or nucleic acid incorporation or expressing a protein. This term also includes offspring of host cells, whether genetically or phenotypically identical or different. Suitable host cells are vector-dependent and may include mammalian cells, animal cells, human cells, monkey cells, insect cells, yeast cells, and bacterial cells. These cells can be induced to incorporate vectors or other materials by transformation using viral vectors, calcium phosphate precipitation, DEAE-dextran, electroporation, microinjection, or other methods. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd 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, complement activation, antibody-dependent cell-mediated cytotoxicity, or any combination thereof. Antigens (immunogenic molecules) may be, for example, peptides, glycopeptides, polypeptides, glycopolypeptides, polynucleotides, polysaccharides, lipids, etc. It is evident that antigens may be synthesized, recombinantly produced, or derived from biological samples. Exemplary biological samples that may contain one or more antigens include tissue samples, fecal samples, cells, biological fluids, or combinations thereof. Antigens may be expressed by cells that have been modified or genetically engineered to express the antigen. Antigens may also be present in or on infectious agents, such as in virions, or may be expressed or presented on the surface of cells infected with infectious agents.

[0058] The term “epitope” or “antigen epitope” includes any molecule, structure, amino acid sequence, or protein determinant that is recognized and specifically bound by a cognitive-binding molecule, such as immunoglobulin, or other binding molecule, domain, or protein. Epitope determinants generally contain chemically active surface groups of a molecule, such as amino acids or sugar side chains, and may have specific three-dimensional structural and specific charge characteristics. If the antigen is a peptide or protein, or contains a peptide or protein, the epitope may consist of a sequence of amino acids (e.g., a linear epitope), or amino acids from different parts or regions of a protein that become adjacent due to protein folding (e.g., a discontinuous epitope or conformational epitope), or discontinuous amino acids that are adjacent independently of protein folding.

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

[0060] The term “antibody” is used most broadly herein and encompasses antibodies and antibody fragments derived from any antibody-producing mammal (e.g., mice, rats, rabbits, and primates, including humans), or from hybridomas, phage selection, recombinant expression, or transgenic animals (or other methods for producing antibodies or antibody fragments). The term “antibody” is not intended to be limited to the source of the antibody or the method of producing it (e.g., by hybridomas, 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, mouse antibodies, chimeric, mouse-human, mouse-primate, primate-human monoclonal antibodies, and anti-idiotype antibodies, and exemplary antibodies may be any intact molecule or fragment thereof. As used herein, the term “antibody” encompasses not only intact polyclonal or monoclonal antibodies, but also immunoglobulin molecules of any other modified configuration, including their fragments (e.g., dAb, Fab, Fab', F(ab')2, Fv), single-chain (ScFv), synthetic variants thereof, native variants thereof, fusion proteins containing an antibody moiety with an antigen-binding fragment of the desired specificity, humanized antibodies, chimeric antibodies, and antigen-binding sites or fragments (epitope recognition sites) of the desired specificity. The term also encompasses genetically engineered and / or otherwise modified forms of immunoglobulins, including their antigen-binding fragments, 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 the antibody heavy chain and antibody light chain, respectively. The VL can be a kappa-class or lambda-class chain. The variable binding region includes discontinuous, well-defined subregions known as complementarity-determining regions (CDRs) and framework regions (FRs). The CDRs are located within the hypervariable region (HVR) of the antibody and generally refer to the sequence of amino acids within the antibody variable region that, as a whole, confer the antibody's antigen specificity and / or binding affinity. Consecutive CDRs (i.e., CDR1 and CDR2, and CDR2 and CDR3) are separated from each other by framework regions in the primary structure.

[0062] As used herein, a “chimeric antibody” is a recombinant protein that contains a variable domain and a complementarity-determining region derived from a non-human species (e.g., rodents), while the rest of the antibody molecule is derived from a human antibody. In some embodiments, a chimeric antibody consists of an antigen-binding fragment of one antibody that is functionally linked to 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, “humanized antibody” refers to a molecule having an antigen-binding site derived from a non-human immunoglobulin, wherein the remaining part of the immunoglobulin structure of the molecule is based on the structure and / or sequence of a human immunoglobulin, and is generally prepared using recombinant techniques. Humanized antibodies differ from chimeric antibodies in that typically only non-human-derived CDRs are used, and these are transplanted onto a suitable framework region 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, the humanized antibody retains all CDR sequences (e.g., a humanized mouse antibody containing all six CDRs from a mouse antibody). In other embodiments, the humanized antibody has one or more modified CDRs (one, two, three, four, five, or six) compared to the original antibody, which are also called 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 relating to a full-length antibody, and generally includes its antigen-binding region or variable region. 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 chain and / or light chain that specifically binds to the antigen that produced the antibody. An antigen-binding fragment may contain one, two, three, four, five, or all six CDRs of the VH and VL sequences from a given antibody.

[0066] "Fab" (fragment antigen binding) is the antigen-binding portion of an antibody and includes a variable region and a heavy chain CH1 linked to the 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 divalent antigen-binding activity and still possesses the ability to crosslink antigens. Both Fab and F(ab')2 are examples of "antigen-binding fragments". The Fab' fragment differs from the Fab fragment in that it has several additional residues at the carboxyl terminus of the CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is the herein designation for Fab' in which the cysteine ​​residue of the constant domain holds a free thiol group. F(ab')2 antibody fragments are often produced as a pair of Fab' fragments with a hinge cysteine ​​between them. Other chemical couplings of antibody fragments are also known.

[0067] Fab fragments can be joined by, for example, a peptide linker to form a single-chain Fab, also referred to herein as "scFab". In these embodiments, the interchain disulfide bonds present in the native Fab may not be present, and the linker plays a role in linking or connecting the Fab fragments to a single polypeptide chain, either completely or partially. A heavy-chain derived Fab fragment (e.g., consisting of, or essentially consisting of, VH+CH1, or "Fd") and a light-chain derived Fab fragment (e.g., consisting of, or essentially consisting of, VL+CL) can be linked in any configuration to form an scFab. For example, an scFab can be configured from N-terminus to C-terminus as (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 containing a complete antigen recognition and binding site. This fragment generally consists of a dimer of one heavy chain variable domain and one light chain variable domain that are tightly associated by non-covalent bonds. However, even a single variable domain (i.e., half of the Fv containing only the three antigen-specific CDRs) has the ability to recognize and bind to an antigen, although its affinity is typically lower than that of the entire binding site.

[0069] A "single-stranded Fv," also abbreviated as "sFv" or "scFv," is an antibody fragment containing a VH antibody domain and a VL antibody domain that are linked together to form a single polypeptide chain. The scFv polypeptide may contain a polypeptide linker positioned between the VH and VL domains to link them, allowing the scFv to maintain or form a structure desirable for antigen binding. However, the linker is not necessarily required. Such peptide linkers can be incorporated into the fusion polypeptide using standard techniques well known in the art. In addition to or instead of the above, the Fv may have a disulfide bond formed between VH and VL to stabilize them. For more information on scFv, see Pluckthun's review (1994) in The Pharmacology of Monoclonal Antibodies, vol. 113, edited by Rosenburg and Moore, Springer-Verlag, New York, pp. 269-315. In certain embodiments, an antibody or antigen-binding fragment comprises an scFv including 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 may be oriented as VH-linker-VL or VL-linker-VH. Any scFv of the present disclosure may be manipulated 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 may be linked to the N-terminal portion or N-terminus of the VH domain, the VL domain, or both.

[0070] Peptide linker sequences for use in scFv or other fusion proteins, such as the targeted complement activation molecules described herein, may be selected based on the following criteria, for example: (1) the ability to adopt a long, flexible conformation; (2) the inability or lack of ability to adopt a secondary structure that can interact with the first and second polypeptides and / or functional epitopes on the target molecule; and / or (3) the absence of, or relatively few, hydrophobic or charged residues that may react with the polypeptides and / or target molecule. Other considerations regarding the design of the linker (e.g., length) include the conformation or range of conformations in which 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, may also be included in the linker sequence. Other amino acid sequences that may be useful 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 Nos. 4,935,233 and 4,751,180. Any suitable linker may be used, and a suitable linker can generally be 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 acid lengths, or less than about 200 amino acid lengths, preferably containing a flexible structure (allowing flexibility and room for conformational movement between two regions, domains, motifs, fragments, or modules connected by the linker), preferably being biologically inactive, and / or having a low risk of immunogenicity in humans.

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

[0072] Antibodies and antigen-binding fragments can be constructed in various 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 producing them are incorporated herein by reference and include, for example, bispecific T cell engagers (BiTE), DART, knob-into-hole (KIH) assemblies, scFv-CH3-KIH assemblies, KIH common light chain antibodies, 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 assemblies, Fcab, κλ body, orthogonal Fab Fab), DVD-Ig (e.g., US Patent No. 8,258,268; these 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 so-called FIT-Ig (e.g., PCT5 Publication No. WO 2015 / 103072; these formats are incorporated herein by reference in their entirety), so-called WuxiBody format (e.g., PCT Publication No. WO This includes the 2019 / 057122 format (these formats are incorporated herein by reference in their entirety), and the so-called In-Elbow-Insert Ig format (IEI-Ig, e.g., PCT publication numbers WO 2019 / 024979 and WO 2019 / 025391, these formats are incorporated herein by reference in their entirety).

[0073] An antibody or antigen-binding fragment may contain 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 may contain the format VH-linker-VL-linker-VH-linker-VL (from N-terminus to C-terminus), where the two VH sequences may be the same or different, and the two VL sequences may be the same or different. Such a concatenated scFv may contain any combination of VH and VL domains arranged to bind to a given target. In formats containing 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 contain VH sequences and / or VL sequences in any combination or orientation. For example, the antigen-binding fragment can take the following formats: VL-linker-VH-linker-VL-linker-VH, VH-linker-VL-linker-VL-linker-VH, or VL-linker-VH-linker-VH-linker-VL.

[0074] As used herein, the modifier “monoclonal” refers to an antibody characteristic of being obtained from a substantially homogeneous antibody population and is not intended to limit the source of the antibody or the method by which it was produced (e.g., by hybridoma, phage selection, recombinant expression, transgenic animals, etc.). The term “monoclonal antibody” encompasses not only intact monoclonal antibodies and full-length monoclonal antibodies, but also immunoglobulin molecules of any other modified configuration, including their fragments (e.g., Fab, Fab', F(ab')2, Fv), single-chain (ScFv), their variants, fusion proteins containing antigen-binding moieties, humanized monoclonal antibodies, chimeric monoclonal antibodies, and antigen-binding fragments (epitope recognition sites) with the required specificity and binding ability to an epitope. Monoclonal antibodies can be obtained using any technique that results in the production of antibody molecules by cultured serial passaged cell lines, such as the hybridoma method described in Kohler, G., et al., Nature 256:495, 1975, or they can be produced by recombinant DNA (see, for example, Cabilly's U.S. Patent No. 4,816,567). Monoclonal antibodies can 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, and IgD, and any subclass thereof.

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

[0076] A basic quadrivalent 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 configuration in that they consist of five basic heterotetrameric units along with an additional polypeptide called a J chain, and therefore have 10 antigen-binding sites. Secretory IgA antibodies also differ from the basic structure in that they polymerize to form a multivalent aggregate containing 2 to 5 basic quadrivalent units along with the J chain. Each L chain is linked to the H chain by one disulfide covalent bond, while two H chains are linked to each other by one or more disulfide bonds depending on the isotype of the H chains. Each H chain and each L chain also has intrachain disulfide crosslinks at regular intervals. The pairing of VH and VL forms a single antigen-binding site as a whole.

[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 light chain (L) has a variable domain (VL) at its N-terminus, followed by a constant domain (CL) at the other end. When an L chain and a heavy chain form a pair, the VL aligns with the VH, and the CL aligns with the first constant domain (CH1) of the heavy chain. Based on the amino acid sequence of their constant domains (CLs), L chains in any vertebrate species can be assigned to one of two types: kappa (κ) or lambda (λ).

[0079] Immunoglobulins can be assigned to different classes or isotypes depending on the amino acid sequence of the constant domain (CH) of their heavy chain. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, which have heavy chains called alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), respectively. The γ and α classes are further 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 information on the structures and properties of these various classes of antibodies, see, for example, page 71 and Chapter 6 of "Basic and Clinical Immunology," 8th edition (edited by Daniel P. Stites, Abba I. Terr, and Tristram G. Parslow, Appleton and Lange, Norwalk, Connecticut, 1994).

[0081] The term "variable" refers to the fact that the sequence of certain segments of the V domain differs significantly between antibodies. The V domain mediates antigen binding and defines the specificity of a particular antibody to its particular antigen. However, variability is not evenly distributed across the entire 110-amino acid interval of the variable domain. Rather, the V region consists of a relatively unchanging stretch of 15-30 amino acids called the framework region (FR), separated by shorter, extremely variable regions called "hypervariable regions" of 9-12 amino acids in length. The variable domains of the native heavy and light chains each contain four FRs, mostly in a beta-sheet configuration, with three hypervariable regions connecting these FRs. These three hypervariable regions form loops, connecting the aforementioned n-sheet structure, and in some cases forming part of it. The hypervariable regions in each chain are held in close proximity to each other by FR, and the hypervariable region from the other chain contributes to the formation of the antibody's antigen-binding site (see Kabat, et al. "Sequences of Proteins of Immunological Interest," 5th edition, Public Health Service, National Institutes of Health, Bethesda, Maryland (1991)). The constant domain does not directly participate in antibody binding to the antigen, but exhibits various effector functions.

[0082] As used herein, “effector function” refers to the biological activity resulting from the Fc region of an antibody. Examples of antibody effector functions include antibody-dependent cell-mediated cytotoxicity (ADCC), C1q binding and complement-dependent cytotoxicity, Fc receptor binding, phagocytosis, downregulation of cell surface receptors, and involvement in B cell activation. Modifications such as amino acid substitutions can 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. Examples of amino acid modifications that modify Fc function include the 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 well known in the art.

[0083] As used herein, the term “hypervariable region” refers to the amino acid residues of an antibody 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). Various systems exist for identifying and numbering the amino acids that make up the CDRs. For example, the hypervariable region generally contains CDRs around residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable domain, and around residues 31-35 (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable domain, and / or Chothia and When numbered according to the Chothia numbering system described in Lesk, J. Mol. Biol. 196:901-917 (1987), the CDR is found at approximately residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable domain, and at residues 26-32 (H1), 52-56 (H2), and 95-102 (H3) in the heavy chain variable domain, and / or 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 28:219-221(2000), CDR is found in approximately residues 27-38 (L1), 56-65 (L2), and 105-117 (L3) in VL, and in residues 27-38 (H1), 56-65 (H2), and 105-117 (H3) in VH.The Antigen Receptor Numbering and Receptor Classification (ANARCI) software tool (2016, Bioinformatics 15:298-300) can be used to annotate and compare equivalent residue positions for different molecules. Therefore, the identification of the CDR of an exemplary variable domain (VH or VL) sequence provided herein according to one numbering scheme does not exclude antibodies containing the same variable domain CDR determined using a different numbering scheme.

[0084] As used herein, “specifically binding” refers to an antibody or antigen-binding fragment that binds to an antigen with a specific affinity but does not significantly associate or combine with any other molecule or component in the sample. Affinity may be defined as the equilibrium association constant (Ka), calculated as the ratio of kon / koff with units of 1 / M, or as the equilibrium dissociation constant (Kd), calculated as the ratio of koff / kon with units of M.

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

[0086] Each embodiment described herein shall apply to any other embodiment with modifications as necessary, unless otherwise expressly stated. Any embodiment described herein may be implemented with respect to any of the methods, kits, reagents, or compositions of the present invention, and vice versa. Furthermore, the compositions of the present invention may be used to achieve the methods of the present invention.

[0087] II. Overview This disclosure provides an antibody that binds to human MASP-2, an activator of the complement system's lectin pathway, and an antibody-conjugated fragment thereof. Lectins such as MBL, M-phycoline, H-phycoline, L-phycoline, 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 in which lectins amplify complement activation, increasing 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 it is possible to inhibit the complement lectin pathway while preserving the classical and alternative pathways leads to the understanding that it would be highly desirable to specifically inhibit only the complement activation system that causes specific pathological conditions, without completely blocking the complement's immune defense capabilities. For example, in disease states where complement activation is primarily mediated by the lectin pathway, specifically inhibiting only this pathway would be advantageous. This would leave the classical complement pathway intact, allowing it to cope with immune complex processing and assist 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 unique to the lectin pathway and are necessary for this system to function. Lectins (e.g., MBL, H-ficolin, M-ficolin, L-ficolin, collectin-10, and collectin-11) are also unique components of the lectin pathway. However, the activation of this system will not necessarily be inhibited even if any one of the lectin components is lost, due to the redundancy of lectins. To ensure inhibition of the lectin-dependent complement activation system, it would be necessary to inhibit all lectins. Furthermore, since MBL and ficolins are also known to have complement-independent opsonin 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 opsonin activity remains intact. A further advantage of MASP-2 as a therapeutic target for inhibiting the lectin pathway is that its plasma concentration is among the lowest of all complement proteins (approximately 500 ng / ml), and therefore, a high-affinity inhibitor of MASP-2 may be sufficient to achieve complete inhibition at a correspondingly low concentration (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, a considerably high concentration of a high-affinity inhibitor of MASP-1 is expected to be required to achieve complete inhibition.

[0090] III. Antibodies and Antigen-Binding Fragments This specification provides antibodies and antigen-binding fragments that specifically bind to MASP-2. In some embodiments, the antibody and its antigen-binding fragment are isolated monoclonal antibodies or their antigen-binding fragments. In some embodiments, the antibody and its antigen-binding fragment inhibit lectin pathway complement activation. The antibodies and antigen-binding fragments described herein may be human antibodies, humanized antibodies, chimeric antibodies or mouse antibodies, or any of the aforementioned antigen-binding fragments. In addition, the antibody and its antigen-binding fragment may be single-chain antibodies, ScFv, Fab fragments, Fab' fragments, F(ab')2 fragments, monovalent antibodies lacking a hinge region, or complete antibodies. Furthermore, the antibody and its antigen-binding fragment may be monovalent, bivalent, or polyvalent.

[0091] In some embodiments, the antibodies and antigen-binding fragments described herein include an immunoglobulin constant region. The antibody and its antigen-binding fragment may be IgG immunoglobulin or a fragment thereof. In some embodiments, the IgG immunoglobulin is IgG1, IgG2, or IgG4 immunoglobulin.

[0092] In some embodiments, the antibodies and antigen-binding fragments described herein specifically bind to epitopes located within the serine protease domain of human MASP-2. In some embodiments, the epitopes are amino acids of human MASP-2. It is located within TIFF2026123079000003.tif4128. In some embodiments, the antibody and its antigen-binding fragment compete for C4 binding to MASP-2.

[0093] In one aspect, the present invention has (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, Shown as TIFF2026123079000004.tif4128, 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, HC-CDR2, The present invention provides an isolated antibody or antigen-binding fragment that binds to MASP-2, comprising a heavy chain variable region comprising (b) LC-CDR1, represented as SEQ ID NO:64(SASSSVXYMY), where X at position 7 is R or S, LC-CDR2, represented as SEQ ID NO:34(DTSNLAS), and 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 heavy chain variable region HC-CDR1 includes SEQ ID NO:14(NYWMH). In some embodiments, the heavy chain variable region HC-CDR1 includes SEQ ID NO:56(NYHMH). In some embodiments, the heavy chain variable region HC-CDR1 includes SEQ ID NO:57(NHHMH).

[0095] In some embodiments, the heavy chain variable region HC-CDR2 is Includes TIFF2026123079000006.tif4128. In some embodiments, the heavy chain variable region HC-CDR2 is Includes TIFF2026123079000007.tif4128. In some embodiments, the heavy chain variable region HC-CDR2 is Includes TIFF2026123079000008.tif4128.

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

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

[0098] In some embodiments, HC-CDR1 includes SEQ ID NO:14(NYWM), and HC-CDR2 is HC-CDR3 includes TIFF2026123079000009.tif4128. TIFF2026123079000010.tif4128 is included, LC-CDR1 includes SEQ ID NO:32(SASSSVRYMY), LC-CDR2 includes SEQ ID NO:34(DTSNLAS), and LC-CDR3 includes SEQ ID NO:36(QQWSSYPLT). In some embodiments, HC-CDR1 includes SEQ ID NO:14(NYWM), and HC-CDR2 is HC-CDR3 includes TIFF2026123079000011.tif4128. TIFF2026123079000012.tif4128 is included, LC-CDR1 includes SEQ ID NO:32(SASSSVRYMY), LC-CDR2 includes SEQ ID NO:34(DTSNLAS), and LC-CDR3 includes SEQ ID NO:36(QQWSSYPLT). In some embodiments, HC-CDR1 includes SEQ ID NO:56(NYHMH), and HC-CDR2 is HC-CDR3 includes TIFF2026123079000013.tif4128. TIFF2026123079000014.tif4128 is included, LC-CDR1 includes SEQ ID NO:32(SASSSVRYMY), LC-CDR2 includes SEQ ID NO:34(DTSNLAS), and LC-CDR3 includes SEQ ID NO:36(QQWSSYPLT). In some embodiments, HC-CDR1 includes SEQ ID NO:57(NHHMH), and HC-CDR2 is HC-CDR3 includes TIFF2026123079000015.tif4128. TIFF2026123079000016.tif4128 is included, LC-CDR1 includes SEQ ID NO:32(SASSSVRYMY), LC-CDR2 includes SEQ ID NO:34(DTSNLAS), and LC-CDR3 includes SEQ ID NO:36(QQWSSYPLT). In some embodiments, HC-CDR1 includes SEQ ID NO:14(NYWM), and HC-CDR2 is Includes TIFF2026123079000017.tif4128, HC-CDR3 includes SEQ ID NO:18(GDITTTLRYFDV), LC-CDR1 includes SEQ ID NO:39(SASSSVSYMY), LC-CDR2 includes SEQ ID NO:34(DTSNLAS), and LC-CDR3 includes SEQ ID NO:36(QQWSSYPLT).

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

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

[0101] In some embodiments, the heavy chain variable region includes 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 includes 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 includes 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 includes SEQ ID NO:10 or SEQ ID NO:47.

[0102] In some embodiments, the heavy chain variable region includes 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 includes 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 includes SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:49, or SEQ ID NO:50, and the light chain variable region includes SEQ ID NO:47.

[0103] In some embodiments, the heavy chain variable region includes 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 includes 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 includes SEQ ID NO:8 and the light chain variable region includes SEQ ID NO:11.

[0104] In some embodiments, the heavy chain variable region includes 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 includes 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 includes SEQ ID NO:9 and the light chain variable region includes SEQ ID NO:12.

[0105] In a certain embodiment, the antibody or its antigen-binding fragment contains 0, 1, 2, or 3 amino acid substitutions in each of the 6 CDRs, one or more of these substitutions being optional, conserved substitutions, and / or substitutions to amino acids encoded by the germline.

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

[0107] (Table 1A) Summary of MASP-2 antibody sequences TIFF2026123079000018.tif82135

[0108] (Table 1B) Summary of sequences and CDRs of MASP-2 high affinity inhibitory antibodies TIFF2026123079000019.tif48140

[0109] (Table 1C) Summary of MASP-2 antibody OMS850 and its humanized and modified versions TIFF2026123079000020.tif69134

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

[0111] In some aspects, the antibody and antigen-binding fragments described herein contain the serine protease domain of human MASP-2 in concentrations of <0.2nM, <0.3nM, <0.4nM, <0.5nM, <0.6nM, <0.7nM, <0.8nM, <0.9nM, <1.0nM, <1.2nM, <1.4nM, <1.6nM, <1.8nM, <2.0nM, <2.5nM, <3.0nM, 3.5nM) It binds with affinity less than 4.0 nM, less than 4.5 nM, less than 5.0 nM, less than 5.5 nM, less than 6.0 nM, less than 6.5 nM, less than 7.0 nM, less than 7.5 nM, less than 8.0 nM, less than 8.5 nM, less than 9.0 nM, less than 9.5 nM, less than 10.0 nM, less than 12 nM, less than 14 nM, less than 16 nM, less than 18 nM, less than 20 nM, less than 22 nM, less than 24 nM, less than 26 nM, less than 28 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, lectin pathway inhibition includes a reduction in the adhesion of complement components to target cells. In some embodiments, lectin pathway inhibition includes a reduction in C3b adhesion, C4 adhesion, or MAC adhesion. In some embodiments, lectin pathway inhibition includes 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, this disclosure provides isolated polynucleotides encoding either the antibody disclosed herein or its antigen-binding fragment or a portion thereof (e.g., CDR, VH, VL, heavy chain, or light chain). In certain embodiments, the polynucleotides are codon-optimized for expression in host cells. 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 host cells, and fully codon-optimized sequences. It will also be understood that polynucleotides encoding antibodies and their antigen-binding fragments may have different nucleotide sequences, even while encoding the same protein, due to genetic code degeneracy, 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 with any one or more SEQ ID NO: 69-79.

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

[0116] Vectors containing or containing polynucleotides encoding either the antibody or its antigen-binding fragment disclosed herein are also provided. Any suitable vector may be used, such as viral vectors and plasmid vectors. In a certain embodiment, the vector contains polynucleotides encoding both the antibody heavy chain or its antigen-binding fragment and the antibody light chain or its antigen-binding fragment, which together constitute a complete antibody or its antigen-binding fragment. The sequences encoding the antibody heavy chain or its antigen-binding fragment and the antibody light chain or its antigen-binding fragment may be contained within a single open reading frame. In this case, they may optionally be separated by polynucleotides encoding protease cleavage sites and / or polynucleotides encoding self-cleaving peptides. Alternatively, the sequences encoding the antibody heavy chain or its antigen-binding fragment and the antibody light chain or its antigen-binding fragment may be contained within separate open reading frames on a single vector. In another embodiment, the sequences encoding the antibody heavy chain or its antigen-binding fragment and the antibody light chain or its antigen-binding fragment are present on two different vectors, such that a first vector encodes the antibody heavy chain or its fragment, and a second vector encodes the antibody light chain or its fragment.

[0117] In a further aspect, the disclosure also provides host cells containing the polynucleotides or vectors disclosed herein. Any suitable cell into which such polynucleotides or vectors can be introduced may be used. Examples of such cells include eukaryotic cells, e.g., yeast cells, animal cells, insect cells, mammalian cells, and plant cells, as well as prokaryotic cells, e.g., bacterial cells such as Escherichia coli (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 polynucleotides or vectors disclosed herein. The term “transfection” encompasses any method known to those skilled in the art for introducing nucleic acid molecules into cells. Such methods include, for example, electroporation, lipofection, nanoparticle-based transfection, and virus-based transfection. Host cells may be transfected stably or transiently.

[0119] In some embodiments, host cells express an antibody or its antigen-binding fragment encoded by a polynucleotide or vector. Such expression may include post-translational modifications such as removal of a signal sequence, glycosylation, or other modifications. In one relevant aspect, the present disclosure provides a method for producing an antibody and its antigen-binding fragment, comprising the steps of culturing host cells for a sufficient time under conditions that enable expression of the antibody or its antigen-binding fragment, and isolating the antibody or its antigen-binding fragment. 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 a culture medium, concentrating the medium, and purifying the protein by passing the concentrate through a suitable purification matrix or a series of matrices. Methods for purifying proteins are well known in the art.

[0120] V. Pharmaceutical Compositions This specification also provides compositions comprising, alone or in any combination, any one or more of the antibodies or their antigen-binding fragments, polynucleotides, vectors, or host cells disclosed herein, and which may also comprise other selected therapeutic agents. Such compositions may further comprise one or more pharmaceutically acceptable carriers, excipients, or diluents.

[0121] Pharmacoherent carriers are non-toxic, biocompatible, and selected so as not to adversely affect the biological activity of the therapeutic agent (and any other therapeutic agent combined with it). An example of a pharmaceutically acceptable carrier for peptides is described in Yamada's U.S. Patent No. 5,211,657. The therapeutic agents described herein may be formulated into solid, semi-solid, gel, liquid, or gaseous preparations, such as tablets, capsules, powders, granules, ointments, solutions, depositories, inhalants, and injections, enabling oral, parenteral, or surgical administration. Topical administration of the composition by coating medical devices is also conceivable.

[0122] Suitable carriers for parenteral delivery by injection, infusion, or irrigation, and topical delivery include distilled water, physiological phosphate-buffered saline, Ringer's solution (normal or lactated), dextrose solution, Hanks' solution, or propanediol. In addition, sterile fixative oils may be used as solvents or suspensions. Any biocompatible oil containing synthetic mono- or di-glycerides may be used for this purpose. Fatty acids such as oleic acid are also useful in the preparation of injectable preparations. The carrier and active substance may be formulated as a liquid, suspension, polymerizable or non-polymerizable gel, paste, or ointment (salve).

[0123] The carrier may also include a delivery medium to sustain (i.e., prolong, delay, or control) the delivery of the active ingredient, or to enhance the delivery, uptake, stability, or pharmacokinetics of the therapeutic ingredient. Non-limiting examples of such delivery mediums include proteins, liposomes, carbohydrates, synthetic organic compounds, inorganic compounds, polymers or copolymer hydrogels, and microparticles, microspheres, nanospheres, or nanoparticles composed of polymer micelles. Suitable hydrogel and micelle delivery systems include the PEO:PHB:PEO copolymer and copolymer / cyclodextrin complex disclosed in WO 2004 / 009664 A2, and the PEO and PEO / cyclodextrin complex disclosed in U.S. Patent Application Publication 2002 / 0019369 A1. Such hydrogels may be injected locally to 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, intra-arterial, or inhalation. The compositions of the present invention may also include biocompatible excipients, such as dispersants or wetting agents, suspending agents, diluents, buffers, osmotic enhancers, emulsifiers, binders, thickeners, and flavoring agents (for oral administration).

[0125] A pharmaceutical composition according to certain aspects of the present invention is formulated such that the active ingredient contained therein is bioavailable when the composition is administered to a patient. The composition to be administered to the subject may take the form of one or more dosing units. The container for the therapeutic agent described herein may hold multiple dosing units. Practical methods for preparing such dosage forms are known to or will be obvious 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 composition to be administered will in any case contain an effective amount of the therapeutic agent or composition disclosed herein for the treatment of the disease or condition of interest, as taught herein.

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

[0127] As solid compositions 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 gum, or gelatin; excipients, such as starch, lactose, or dextrin; disintegrants, such as alginic acid, sodium alginate, Primogel, or corn starch; lubricants, such as magnesium stearate or Sterotex; flow enhancers, such as colloidal silicon dioxide; sweeteners, such as sucrose or saccharin; flavorings, such as peppermint, methyl salicylate, or orange flavoring; and colorants. If the composition is in the form of a capsule, for example a gelatin capsule, it may contain a liquid carrier such as polyethylene glycol or oil, in addition to the materials of the type described above.

[0128] This composition may be in the form of a liquid, such as an elixir, syrup, solution, emulsion, or suspension. Two examples of liquids are that they may be for oral administration or for delivery by injection. When intended for oral administration, a preferred composition contains, in addition to the compound, one or more sweeteners, preservatives, colorants, and flavor enhancers. Compositions intended for administration by injection may contain one or more surfactants, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, and isotonic agents.

[0129] Liquid pharmaceutical compositions, whether in solution, suspension, or other similar forms, may contain one or more of the following excipients: sterile diluents, e.g., water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride; fixing oils, e.g., synthetic monoglycerides or diglycerides that can serve as solvents or suspension media, polyethylene glycol, glycerin, propylene glycol, or other solvents; antibacterial agents, e.g., benzyl alcohol or methylparaben; antioxidants, e.g., ascorbic acid or sodium bisulfite; chelating agents, e.g., ethylenediaminetetraacetic acid; buffers, e.g., acetates, citrates, or phosphates; and agents for adjusting tonicity, e.g., sodium chloride or dextrose. Parenteral preparations may be sealed in glass or plastic ampoules, disposable syringes, or multi-dose vials. Physiological saline is a preferred excipient. Injectable pharmaceutical compositions are preferably sterile.

[0130] Liquid compositions intended for either parenteral or oral administration should contain a certain amount of the therapeutic agent described herein so that an appropriate dosage is obtained. The term “parenteral” includes subcutaneous, intravenous, intramuscular, intrasternal, or intra-arterial injection or infusion. Typically, the therapeutic agent constitutes at least 0.01% of the composition. When intended for oral administration, this amount can vary, ranging from about 0.1% to about 70% of the composition's weight. Certain oral pharmaceutical compositions contain about 4% to about 75% of the therapeutic agent.

[0131] The composition may be intended for topical administration, in which case the carrier may appropriately include a solution, emulsion, ointment, or gel base. The base may include, for example, one or more of the following: petrolatum, lanolin, polyethylene glycol, beeswax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers. Thickeners may be present in compositions for topical administration. When intended for transdermal administration, the composition may include a transdermal patch or an iontophoresis device. The pharmaceutical composition may also be intended for rectal administration in the form of a suppository, for example, which dissolves in the rectum and releases the drug. Compositions 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 include a variety of materials that modify the physical form of the solid or liquid dosage unit. For example, the composition may include a material that forms a coating shell around the active ingredient. The material forming the coating shell is typically inert and may be selected from, for example, sugar, shellac, and other enteric coating agents. Alternatively, the active ingredient may be encapsulated in a gelatin capsule. The solid or liquid composition may include an active substance that binds to the therapeutic agent of the Disclosure and thereby helps deliver the compound. Suitable active substances that can perform this function include one or more proteins or liposomes.

[0133] The composition may essentially consist of dosing units that can be administered as aerosols. The term aerosol is used to describe a variety of systems, from colloidal ones to systems consisting of pressurized packages. Delivery may be by liquefied or compressed gas, or by a suitable pump system for distributing the active ingredient. Aerosols may be delivered in single-phase, two-phase, or three-phase systems to deliver the active ingredient. Aerosol delivery may involve necessary containers, activators, valves, subcontainers, etc., which together may form a kit. Those skilled in the art can determine a preferred aerosol without excessive experimentation.

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

[0135] Pharmaceutical compositions can be prepared by methodologies well known in the field of pharmacy. For example, a composition intended for administration by injection can be prepared by mixing a composition containing the therapeutic agent described herein and optionally one or more salts, buffers, and / or stabilizers with sterile distilled water to form a solution. A surfactant may be added to facilitate the formation of a homogeneous solution or suspension. The surfactant is a compound that interacts non-covalently with the composition to facilitate dissolution or homogeneous suspension in an aqueous delivery system.

[0136] VI. Method and Use This specification further provides methods for using the antibodies or antigen-binding fragments, nucleic acids, vectors, cells, or compositions of the Disclosure in activating the complement lectin pathway in mammalian subjects. In some embodiments, the method includes administering to a mammalian subject in need of such administration an amount of the antibodies or antigen-binding fragments, nucleic acids, vectors, cells, or compositions of the Disclosure sufficient to inhibit the complement activation lectin pathway in the mammal. In some embodiments, the method may further include determining whether the subject suffers from a disease or disorder of the lectin pathway before administering the compound of the Disclosure to the subject. In some embodiments, the mammalian subject is human.

[0137] In certain embodiments, antibodies or antigen-binding fragments, nucleic acids, vectors, cells, or compositions of the Disclosure are provided for use in methods for treating diseases or disorders of the lectin pathway. In certain embodiments, antibodies or antigen-binding fragments, nucleic acids, vectors, cells, or compositions of the Disclosure are provided for use in methods for manufacturing or preparing pharmaceuticals for treating diseases or disorders of the lectin pathway.

[0138] U.S. Patent Nos. 7,919,094, 8,840,893, 8,652,477, 8,951,522, 9,011,860, 9,475,885, 9,644,035, U.S. Patent Application Publication Nos. US2013 / 0344073, US2013 / 0266560, and U.S. Issues 2015 / 0166675, US2017 / 0137537, US2017 / 0166660, US2017 / 0189525, US2017 / 0267781, US2017 / 0283508, US2017 / 0253667, and US2018 / 0105604, As described in U.S. Patent Applications WO2018 / 045054, WO2018 / 071701, WO2019 / 036460, WO2019 / 246,367, WO2021 / 178902, and the concurrently continuing U.S. Patent Application No. 17 / 103,672 (each assigned to Omeros Corporation, the assignee of the present application, and incorporated herein by reference), MASP-2-dependent complement activation has been identified as a contributing factor to the pathogenesis of numerous acute and chronic disease conditions. For example, as described in U.S. Patent No. 8,951,522, the primary function of the complement system, part of the innate immune system, is to protect the host from infectious agents. However, inappropriate or excessive activation of the complement system can lead to serious diseases such as thrombotic microangiopathy (TMA, including aHUS, TTP, and HUS), where endothelial damage in microtubule structures, as well as fibrin adhesion and platelet-rich thrombi, lead to organ damage. The lectin pathway plays a major role in activating complement under conditions of endothelial cell stress or injury, and inhibiting the activation of the lectin pathway with MASP-2 would halt a series of enzymatic reactions that lead to the formation of membrane invasion complexes, platelet activation, and leukocyte recruitment. As described in U.S. Patent 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] Therefore, in some embodiments, diseases or disorders of the lectin pathway are 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, lung disorders, ophthalmic diseases or disorders, disseminated intravascular coagulation, graft-versus-host disease, venous-occlusive diseases, and diffuse alveolar hemorrhage.

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

[0141] In some embodiments, diseases or disorders of the lectin pathway include thrombotic microangiopathy (TMA), e.g., thrombotic thrombocytopenic purpura (TTP), refractory TTP, Upshaw-Schulman syndrome (USS), hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), non-H factor-dependent atypical hemolytic syndrome, aHUS secondary to infection, plasma therapy-resistant 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 lectin pathway disorder or impairment is graft-versus-host disease (GVHD), such as acute GVHD, chronic GVHD, or steroid-resistant GVHD. In some embodiments, subjects with GVHD or at risk of developing GVHD have previously undergone, are currently undergoing, or are scheduled to undergo hematopoietic stem cell transplantation.

[0143] In some embodiments, the lectin pathway disorder or impairment is diffuse alveolar hemorrhage (DAH). In some embodiments, subjects with DAH or at risk of developing DAH have previously undergone, are currently undergoing, or are scheduled to undergo hematopoietic stem cell transplantation.

[0144] In some embodiments, lectin pathway disorders or impairments include capillary leak syndrome, engraftment syndrome, fluid overload, or idiopathic pneumonia syndrome. In some embodiments, subjects with one or more of these disorders or impairments, or at risk of developing one or more of these disorders, have previously undergone, are currently undergoing, or are scheduled to undergo hematopoietic stem cell transplantation.

[0145] In some embodiments, the lectin pathway disorder or impairment is a veno-occlusive disease (VOD). In some embodiments, subjects with VOD or at risk of developing VOD have previously undergone, are currently undergoing, or are scheduled to undergo hematopoietic stem cell transplantation.

[0146] In some embodiments, lectin pathway disorders or impairments include neurological symptoms associated with graft-versus-host disease or TMA, such as asthenia, paresthesia, quadriplegia, sensorimotor deficits, autonomic polyneuropathy, or neurogenic bladder.

[0147] In some aspects, diseases or disorders of the lectin pathway include kidney diseases, for example, but not limited to, mesangial proliferative glomerulonephritis, membranous glomerulonephritis, membranoproliferative glomerulonephritis (mesangial capillary glomerulonephritis), acute post-infectious glomerulonephritis (post-streptococcal glomerulonephritis), C3 glomerulopathy, cryoglobulinemia glomerulonephritis, microimmune necrotizing crescentic glomerulonephritis, lupus nephritis, Henoch-Schönlein purpura nephritis, and IgA nephropathy.

[0148] In some embodiments, diseases or disorders of the lectin pathway include renal fibrosis (e.g., tubulointerstitial fibrosis) and / or proteinuria, chronic renal failure, scleroderma of the kidney (including scleroderma renal crisis), glomerular diseases (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., C14). 3. Glomerulosis), or diseases or conditions associated with proteinuria, for example, but not limited to, nephrotic syndrome, pre-eclampsia, eclampsia, toxic lesions of the kidneys, amyloidosis, collagen vascular disease (e.g., systemic lupus erythematosus), dehydration, glomerular disease (e.g., membranous glomerulonephritis, membranous glomerulonephropathy, focal segmental glomerulonephritis, C3 glomerulosis, minimal change disease, lipoid nephrotic syndrome), strenuous exercise, stress, benign orthostatic (postural) proteinuria, focal segmental glomerulosclerosis, IgA nephropathy ( (e.g., Berger's disease), IgM nephropathy, membranoproliferative glomerulonephritis, membranous nephropathy, minimal change syndrome, 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 opioids (e.g., heroin) or other nephrotoxins), Fabry disease, infections (e.g., HIV, syphilis, hepatitis A, B or C, post-streptococcal infection, urinary tract schistosomes) These include trematosis, aminoaciduria, Fanconi syndrome, hypertensive nephrosclerosis, interstitial nephritis, sickle cell disease, hemoglobinuria, multiple myeloma, myoglobinuria, organ rejection (e.g., kidney transplant rejection), Ebola hemorrhagic fever, onychopatella syndrome, familial Mediterranean fever, HELLP syndrome, systemic lupus erythematosus, Wegener's granulomatosis, rheumatoid arthritis, type 1 glycogen storage disease, Goodpasture syndrome, Henoch-Schönlein purpura, urinary tract infections affecting the kidneys, Sjögren's syndrome, and post-infectious glomerulonephritis.

[0149] In some embodiments, a disorder or impairment of the lectin pathway is an inflammatory response resulting from tissue transplantation or solid organ transplantation, such as, but not limited to, allogeneic or xenotransplantation of a whole organ (e.g., kidney, heart, liver, pancreas, lung, cornea, etc.) or tissue graft (e.g., valve, tendon, bone marrow, etc.). In some embodiments, a disorder or impairment of the lectin pathway is post-transplant organ dysfunction.

[0150] In some embodiments, diseases or disorders of the lectin pathway include ischemia-reperfusion injury (I / R), for example, but not limited to myocardial I / R, gastrointestinal I / R, renal I / R, and I / R after aortic aneurysm repair, I / R associated with cardiopulmonary bypass, cerebral I / R, stroke, vascular reanastomosis associated with organ transplantation or reattachment of amputated or traumatized limbs or fingers, vascular regeneration to grafts and / or replants, and hemodynamic resuscitation after shock and / or surgery.

[0151] In some embodiments, lectin pathway disorders or impairments include complications associated with non-obese diabetes (type 1 diabetes or insulin-dependent diabetes mellitus) and / or complications associated with type 1 or type 2 (adult-onset) diabetes, such as, but not limited to, diabetic vascular disease, diabetic neuropathy, diabetic retinopathy, or diabetic macular edema.

[0152] In some aspects, diseases or disorders of the lectin pathway include cardiovascular diseases or disorders, such as, but not limited to, Henoch-Schönlein purpura nephritis, systemic lupus erythematosus-associated vasculitis, vasculitis associated with rheumatoid arthritis (also known as malignant rheumatoid arthritis), immune complex vasculitis, anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis, Takayasu's arteritis, dilated cardiomyopathy, diabetic vascular disease, Kawasaki disease (arteritis), venous gas embolism (VGE), and inhibition of restenosis after stent placement, rotational atherosclerosis and / or percutaneous transcatheter coronary angioplasty (PTCA).

[0153] In some embodiments, lectin pathway disorders or disorders are inflammatory gastrointestinal disorders, such as pancreatitis, diverticulitis, and bowel disorders, such as Crohn's disease, ulcerative colitis, irritable bowel syndrome, and inflammatory bowel disease (IBD), though not limited to these. In some embodiments, lectin pathway disorders or disorders are caused by or exacerbated by gastrointestinal fibrosis. In some embodiments, lectin pathway disorders or disorders are pancreatic fibrosis.

[0154] In some aspects, diseases or disorders of the lectin pathway include, but are not limited to, lung disorders such as acute respiratory distress syndrome, transfusion-associated acute lung injury, ischemia / reperfusion acute lung injury, chronic obstructive pulmonary disease, asthma, Wegener's granulomatosis, anti-glomerular basement membrane antibody disease (Goodpasture's disease), meconium aspiration syndrome, aspiration pneumonia, bronchiolitis obstructive, idiopathic pulmonary fibrosis, acute lung injury secondary to burns, non-cardiogenic pulmonary edema, transfusion-associated respiratory depression, and emphysema.

[0155] In some embodiments, lectin pathway disorders or impairments are extracorporeal exposure-triggered inflammatory reactions, and the method includes treating subjects undergoing extracorporeal circulation, such as, but not limited to, hemodialysis, plasmapheresis, leukocyte ferresis, extracorporeal membrane oxygenation (ECMO), heparin-induced extracorporeal membrane oxygenation LDL precipitation (HELP), and cardiopulmonary bypass (CPB).

[0156] In some embodiments, lectin pathway disorders or impairments are selected from inflammatory or non-inflammatory arthritis and other musculoskeletal disorders, such as, but not limited to, osteoarthritis, rheumatoid arthritis, juvenile rheumatoid arthritis, gout, neuropathic arthritis, psoriatic arthritis, ankylosing spondylitis or other spondyloarthropathy and crystalline arthropathy, muscular dystrophy and systemic lupus erythematosus (SLE).

[0157] In some embodiments, diseases or disorders of the lectin pathway are for the treatment of skin disorders, such as, but not limited to, psoriasis, autoimmune bullous dermatosis, eosinophilic spongiform disease, bullous pemphigoid, acquired epidermolysis bullosa, atopic dermatitis, herpes zoster of pregnancy, and other skin disorders, as well as burns and chemical burns, such as capillary leakage resulting therefrom.

[0158] In some embodiments, diseases or disorders of the lectin pathway include disorders or injuries of the peripheral nervous system (PNS) and / or central nervous system (CNS), such as, but not limited to, multiple sclerosis (MS), myasthenia gravis (MG), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), Guillain-Barré syndrome, post-stroke reperfusion, degenerative discs, traumatic brain injury, Parkinson's disease (PD), Alzheimer's disease (AD), Miller-Fischer syndrome, traumatic brain injury and / or cerebral hemorrhage, traumatic brain injury, fibrosis of the CNS, demyelination, and meningitis.

[0159] In some embodiments, diseases or disorders of the lectin pathway include, but are not limited to, sepsis or conditions resulting from sepsis, such as severe sepsis, septic shock, acute respiratory distress syndrome resulting from sepsis, hemolytic anemia, systemic inflammatory response syndrome, or hemorrhagic shock.

[0160] In some embodiments, disorders or impairments of the lectin pathway include genitourinary disorders, such as, but not limited to, bladder disorders, sensory bladder disease, chronic sterile cystitis and interstitial cystitis, male and female infertility, placental dysfunction, and miscarriage and pre-eclampsia.

[0161] In some embodiments, lectin pathway disorders or impairments are inflammatory responses in subjects receiving chemotherapy and / or radiotherapy, for example, for the treatment of cancerous conditions.

[0162] In some embodiments, diseases or disorders of the lectin pathway are angiogenesis-dependent cancers, such as, but not limited to, solid tumors, bloodborne tumors, high-risk carcinoid tumors, and tumor metastases. In some embodiments, diseases or disorders of the lectin pathway are angiogenesis-dependent benign tumors, such as, but not limited to, hemangiomas, acoustic neuromas, neurofibromas, trachomas, carcinoid tumors, and pyogenic granulomas.

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

[0164] In some embodiments, lectin pathway diseases or disorders include ophthalmic diseases or disorders, such as but not limited to age-related macular degeneration, glaucoma, and endophthalmitis. In some embodiments, lectin pathway diseases or disorders include neovascular diseases or conditions, such as but not limited to age-related macular degeneration, uveitis, melanoma, corneal neovascularization, primary pterygium, HSV keratitis, HSV-I 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 capsule opacity, and rubeosis.

[0165] In some embodiments, lectin pathway disorders or impairments include disseminated intravascular coagulation (DIC) or other complement-mediated coagulation disorders, 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)); infections (bacteria, viruses, fungi, parasites); cancer; complications of childbirth; liver disease; severe toxic reactions (e.g., snake bites, stinging insects, transfusion reactions); shock; heatstroke; transplant rejection; hemangiomas; liver failure; cancer treatment with chemotherapy or radiotherapy; burns; or accidental radiation exposure.

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

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

[0168] In some embodiments, lectin pathway disorders or conditions are associated with fibrin-induced complement system activation and associated coagulation and / or contact system activation. In some embodiments, lectin pathway disorders or conditions are associated with complement-related inflammation initiated by fibrin or activated platelets, excessive coagulation or contact system activation. In some embodiments, lectin pathway disorders or impairments are 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), veno-occlusive disease (VOD), thrombotic microangiopathy, lupus nephritis, superficial thrombophlebitis, factor V Leiden mutation, ischemia / reperfusion injury, human immunodeficiency virus (HIV) infection, hormone replacement therapy (HRT) in progress, Alzheimer's disease and / or hypercoagulable states.In some embodiments, the lectin pathway, acquired hypercoagulable states due to at least one or more of the following: treatment during administration with drugs selected from the group consisting of 5-FU, GM-CSF, cisplatin, heparin, COX-2 inhibitors, contrast agents, corticosteroids and antipsychotics; venous congestion (e.g., immobility, surgery), antiphospholipid syndrome, cancer (promyelocytic leukemia, lung, breast, prostate, pancreatic, cervical, esophageal squamous cell carcinoma, gastric and colorectal tumors), tissue injury due to trauma or surgery, presence of a catheter in a central vein, acquired deficiency of a protein involved in blood clot formation (e.g., protein C), paroxysmal nocturnal hemoglobinuria (PNH), elevated homocysteine ​​levels, heart failure, mechanical valves Due to the presence of, pulmonary hypertension with in-situ thrombosis, atrial fibrillation, heparin-induced thrombocytopenia (HIT), heparin-induced thrombocytopenia and thrombosis (HITT), Kawasaki disease with in-situ thrombosis, in-situ thrombotakayasu arteritis, thrombotic tendency in metastatic cancer, elevated factor VIII levels, pregnancy, inflammatory bowel disease (IBD), or genetic defects 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 that cause thrombotic tendencies, or genetic defects that cause or increase the risk of developing hypercoagulable states.

[0169] In some embodiments, lectin pathway diseases or disorders are breast fibrosis, myofibrosis, retroperitoneal fibrosis, thyroid fibrosis, lymphadenopathy, bladder fibrosis, cardiac fibrosis, hepatic fibrosis, arthral fibrosis, cutaneous fibrosis, or pleural fibrosis. In some embodiments, lectin pathway diseases or disorders are 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). In some embodiments, lectin pathway diseases or disorders are fibrosis resulting from viral infections, e.g., alphavirus infection, tuberculosis infection, or influenza infection. In some embodiments, lectin pathway diseases or disorders are scarring associated with trauma, e.g., surgical complications (e.g., postoperative adhesions where scar tissue forms between viscera, causing contracture, pain, and possibly infertility), chemotherapy-induced fibrosis, or burn-related scarring. In some embodiments, diseases or disorders of the lectin pathway include fibrosis of the genital organs (e.g., endometriosis and Peyronie's disease).

[0170] In some embodiments, diseases or disorders of the lectin pathway are targets for treatment with kallikrein inhibitors. In some embodiments, the lectin pathway is selected from the group consisting of hereditary angioedema, diabetic macular edema, and bleeding in cardiopulmonary bypass. In some embodiments, the lectin pathway is targets for treatment with thrombin inhibitors, such as 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 targets for treatment with factor XII inhibitors, such as deep vein thrombosis (primary prevention and long-term treatment), pulmonary embolism, non-valvular atrial fibrillation, prevention of recurrent ischemia after acute coronary syndrome in subjects with or without atrial fibrillation, end-stage renal disease, cerebral ischemia, angina, or reduction or prevention of coagulation associated with medical devices (e.g., valves, small-bore grafts, etc.) and / or extracorporeal circulation.

[0172] In some embodiments, lectin pathway disorders or impairments are diseases or impairments selected from the group consisting of subjects with acquired diseases or impairments that increase the tendency toward 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, infections, tissue injury, venous congestion (e.g., surgical, orthopedic or paralytic immobility, heart failure, pregnancy, or obesity), and subjects taking oral contraceptives containing estrogen. In some embodiments, subjects require anticoagulant therapy, and the antibodies or antigen-binding fragments, polynucleotides, vectors, host cells, and / or compositions described herein are used as an alternative to standard anticoagulant therapy (e.g., warfarin). In some embodiments, subjects have conditions for which standard anticoagulant therapy is normally contraindicated, such as CNS amyloid angiopathy. In some embodiments of this method, the antibodies or their antigen-binding fragments, polynucleotides, vectors, host cells and / or compositions described herein are administered as bridging agents perioperatively to subjects otherwise receiving standard anticoagulant therapy. In some embodiments, the lectin pathway disorder or impairment is sickle cell disease, a vascular occlusive disorder involving platelet activation.

[0173] In some aspects, lectin pathway disorders or dysfunctions are caused by SARS-CoV-2 infection. SARS-CoV-2 infection can lead to lectin pathway disorders or dysfunctions through several different mechanisms. In addition to acute or chronic SARS-CoV-2 infection, further chronic conditions can be triggered by past or current SARS-CoV-2 infection. Lectin pathway disorders or dysfunctions associated with SARS-CoV-2 infection can result from direct interactions between MASP-2 and viral proteins, such as the binding of MASP-2 to SARS-CoV-2. For example, lectin pathway disorders or dysfunctions can result from the binding of MASP-2 to the S or N protein of SARS-CoV-2. Alternatively, some lectin pathway disorders or dysfunctions may result from interactions between lectin pathway recognition molecules and viral glycoproteins present on the viral surface or on the surface of virus-infected cells. Furthermore, lectin pathway disorders or dysfunctions can result from activation of the lectin pathway by DAMP presented by cells infected with SARS-CoV-2. In some embodiments, SARS-CoV-2 infection is an acute infection. In alternative embodiments, SARS-CoV-2 infection is a chronic infection. In certain embodiments, lectin pathway disease or disorder is a continuing chronic condition caused by a past SARS-CoV-2 infection. In some embodiments, lectin pathway disease or disorder is acute respiratory distress syndrome. In some embodiments, lectin pathway disease or disorder is secondary to SARS-CoV-2 infection. In some embodiments, the antibodies or antigen-binding fragments disclosed herein block the binding of MASP-2 to the S and / or N proteins of SARS-CoV-2.

[0174] Selected examples of lectin pathway diseases and disorders are described in further detail below.

[0175] Atypical hemolytic uremic syndrome (aHUS) Atypical hemolytic uremic syndrome (aHUS) is part of a group of conditions called "thrombotic microangiopathy." In atypical HUS (aHUS), the disorder is associated with defective complement regulation and can be sporadic or familial. Familial aHUS cases are associated with mutations in genes encoding complement activation or complement regulatory proteins, including complement factor H, factor I, factor 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)). A unifying feature of this diverse range of gene mutations associated with aHUS is a tendency towards enhanced complement activation on the cell or tissue surface. Subjects are at risk of developing aHUS at the onset of at least one symptom indicating aHUS (e.g., anemia, thrombocytopenia, and / or renal failure) and / or in the presence of thrombotic microangiopathy in a biopsy taken from the subject. Determining whether a subject is at risk of developing aHUS includes the steps of determining whether the subject has a genetic predisposition to developing aHUS and / or whether there is 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 performing at least one genetic screening test on the subject to determine the presence or absence of aHUS-related genetic markers by either genome sequencing or gene-specific analysis (e.g., PCR analysis) (i.e., by determining the presence or absence of aHUS-related genetic mutations 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 for genetic screening for the presence or absence of aHUS-related genetic mutations are established.For example, see Noris M et al., "Atypical Hemolytic-Uremic Syndrome," November 16, 2007 [updated March 10, 2011], in GeneReviews™, Seattle (Washington State) (University of Washington, Seattle), edited by Pagon RA, Bird TD, Dolan CR, et al.

[0176] Hematopoietic stem cell transplant-related TMA (HSCT-TMA) Hematopoietic stem cell transplant-associated tumorigenesis (HSCT-TMA) is a life-threatening complication triggered by endothelial damage. While the kidney is the most commonly affected organ, HSCT-TMA is a multisystem disease involving the lungs, intestines, heart, and brain. Even mild TMA is associated with long-term renal dysfunction. The incidence of allogeneic HSCT-TMA varies depending on diagnostic criteria and conditioning and graft-versus-host disease prophylaxis regimens, with calcineurin inhibitors being the most frequently associated drug (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 causes inflammation and damage to the kidneys. 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 1,400 people in the United States will develop IgAN. As many as 40% of IgAN patients develop end-stage renal disease (ESRD). Patients typically present with microscopic hematuria with mild to moderate proteinuria and varying levels of renal failure (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 multiple 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 develop ESRD within 10 years of disease onset (D'Amico G., Am J Kidney Dis 36(2):227-37, 2000). The diagnostic features of IgAN are the predominance of IgA adhesion in the glomerular mesangium, either alone or in association with IgG, IgM, or both.

[0178] Lupus nephritis (LN) The primary 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 renal involvement in the later stages of the disease (Koda-Kimble et al. "Koda-Kimble and Young's Applied Therapeutics: the clinical use of drugs," 10th edition, Lippincott Williams & Wilkins: pp. 792-79, 2012), and the prevalence in the United States is 20-70 per 100,000 people. Lupus nephritis often appears in patients with other symptoms of active SLE, including fatigue, fever, rash, arthritis, serositis, or central nervous system disorders (Pisetsky DSet al., Med Clin North Am 81(1):113-28, 1997). Some patients have asymptomatic lupus nephritis, but during regular follow-up, abnormal laboratory findings such as elevated serum creatinine levels, low albumin levels, or proteinuria or urinary sediment may suggest active lupus nephritis.

[0179] VI. Arrays Table 2 summarizes the sequences mentioned herein.

[0180] (Table 2) TIFF2026123079000021.tif118166TIFF2026123079000022.tif242166TIFF2026123079000023.tif235166 TIFF2026123079000024.tif242166TIFF2026123079000025.tif245166TIFF2026123079000026.tif245166 TIFF2026123079000027.tif244166TIFF2026123079000028.tif242166TIFF2026123079000029.tif242166 TIFF2026123079000030.tif242166TIFF2026123079000031.tif242166TIFF2026123079000032.tif154166 [Examples]

[0181] VII. Examples Example 1 Generation of high-affinity anti-human MASP-2 inhibitory antibodies Using the Sigma adjuvant system (Sigma-Aldrich, St. Louis, Missouri), 7-14 week old C57BL / 6 MASP-2 knockout mice were immunized with human MASP-2 CCP2 / SP polypeptide containing a 6×His tag at the C-terminus (amino acid residues 364-686 of SEQ ID NO:1). 50 μg of immunogen per mouse was mixed 1:1 with RIBI adjuvant and injected intraperitoneally. After 14 days, immunized mice were boosted with an additional immunogen mixed 1:1 with RIBI adjuvant. After another 14 days, mice received a third booster immunization with 50 μg of immunogen mixed 1:1 with PBS. Serum samples were regularly prepared from tail blood collection and tested for the presence of antigen-specific antibodies by ELISA. Four days before splenocyte fusion, mice with significant antibody titers received a pre-fusion immunogen booster immunization in PBS. Three days prior to fusion, 50 μg of an anti-CD40 agonist mAb (R&D Systems, Minneapolis, Minnesota) in PBS was administered subcutaneously to the base of the mouse tail to increase the number of B cells.

[0182] Mice were sacrificed, spleen cells were harvested, and fused to 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. The hybridoma cells generated by this fusion were plated into 96-well tissue culture plates containing HAT (hypoxanthine, aminopterin, and thymidine) medium to inhibit the proliferation of non-fused cells, myeloma hybrids, and spleen hybrids. After hybridoma selection, the culture supernatant was evaluated for MASP-2 binding by ELISA and for inhibition of lectin pathway activation using a C3 adhesion assay. Positive hybridomas were identified and subcloned by serial dilution. Parallel screening of hybridoma supernatant yielded 75 initial hybridomas positive for both MASP-2 binding and function (C3 adhesion assay). After further culturing and retesting of these 75 initial hybridomas, 74 were confirmed to be positive for binding, while 39 were confirmed to possess functional activity. These 39 hybridomas were cloned by serial dilution.

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

[0184] Twenty-six clones were identified as unique chimeric monoclonal antibodies. These chimeric monoclonal antibodies were expressed in transiently transfected Expi293F cells, purified, and tested for their binding affinity to human MASP-2, as well as 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 fragments), a solid-phase ELISA assay was performed as follows: A MaxiSorp ELISA plate was coated overnight at 4°C with 1.0 μg / mL of human MASP-2 (CCP1 / 2 / SP fragments) in carbonic acid / bicarbonate buffer. The plate was then blocked with 1% BSA / PBS, washed in PBS, and incubated at room temperature for 1 hour with serial dilutions of recombinant MASP-2 mAb in blocking buffer (PBST + 0.1% BSA). The plate was washed (PBS-T, 0.05%) and the detection antibody (goat anti-human IgG-HRP) was added at room temperature for 1 hour. After another wash (PBS-T, 0.05%), the plate was stained with OPT EIA TMB (BD Biosciences #555214) for 5 minutes. The absorbance readings for 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 0.1nM to 1nM). Of the 26 chimeric mAbs, 4 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 complement activation lectin pathway, a C3 adhesion assay was performed as follows: Mannan was diluted to a concentration of 50 μg / ml in 50 mM carbonate (15 mM Na2CO3 + 35 mM NaHCO3 + 1.5 mM NaN3) pH 9.6 and coated onto an ELISA plate overnight at 4°C. The following 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 sample addition.

[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. The mixture was pre-incubated on ice for 10 minutes before being added to a blocked ELISA plate. The complement activation reaction was initiated by transferring the pre-incubation mixture to the wells of a mannan-coated assay plate. After incubation at room temperature for 40 minutes, the reaction was stopped by washing the plate three times in ELISA washing buffer. C3b adhesion was detected using anti-human C3c antibody (Dako) followed by goat α-rabbit HRP (Southern Biotech). Negative control was buffer without serum (no C3 adhesion), and positive control was serum without inhibitory antibody (maximum C3b adhesion). The cutoff criterion was set at half the activity of the unrelated mAbs and buffer alone.

[0189] Of the 26 unique chimeric mAbs identified in the screening, the three clones with the highest inhibitory LP activity in the C3b adhesion 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-chain and light-chain variable regions of clones OMS850, OMS860, and OMS870 are shown in Figure 3 (in Figure 3, "SIN" = "SEQ ID NO:"), and are 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] The following 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: TIFF2026123079000033.tif98159

[0193] (Table 3) MASP-2 antibody VH sequence (CDR and FR regions, Kabat) mouse parent TIFF2026123079000034.tif208166

[0194] The following 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 using the Kabat system or the Chothia system.

[0195] Light chain variable region: TIFF2026123079000035.tif78159

[0196] (Table 4) MASP-2 antibody VL sequence (CDR and FR regions, Kabat) mouse parent TIFF2026123079000036.tif187160

[0197] (Table 5) Consensus sequences of OMS850 and OMS860 VH CDRs TIFF2026123079000037.tif231153

[0198] (Table 6) Consensus sequences of OMS850 and OMS860 VL CDRs TIFF2026123079000038.tif120151

[0199] Example 3 Further characterization of candidate MASP-2 inhibitory antibodies 1. Binding to recombinant human MASP-2 As described in Example 1, 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). The results are shown in Figures 4A to 4D and summarized in Table 7 below.

[0200] (Table 7) Results of MASP-2 binding assay TIFF2026123079000039.tif31128

[0201] 2. C3b adhesion assay in human, cynomolgus monkey, rat, and mouse serum The three selected MASP-2 antibodies described in Example 2 were expressed, purified, diluted to the same stock concentration, and then Ca ++ and Mg ++ By diluting the solutions again in the contained GVB buffer (4.0 mM barbiturate, 141 mM NaCl, 1.0 mM MgCl2, 2.0 mM CaCl2, 0.1% gelatin, pH 7.4), all antibody clones were ensured to have the same amount of buffer.

[0202] A. C3b adhesion 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 an ELISA plate overnight at 4°C. The following day, 250 μl of blocking solution (1% BSA in PBS) was added to the wells and incubated at room temperature for 2 hours. The plate was washed three times with 300 μl of PBS (ELISA wash buffer) containing 0.05% Tween-20.

[0203] Normal human serum was diluted to 1.0% in GVB / Ca / Mg buffer, and MASP-2 mAb clones OMS850, OMS860, and OMS870 were added in a final concentration range of 0.00001–100 nM. The mixture was pre-incubated on ice for 15 minutes. The reaction was initiated by transferring the pre-incubation mixture to wells of an assay plate coated with mannan and blocked, and then incubated at room temperature for 40 minutes. The reaction was stopped by washing the plate three times in wash buffer, and C3b adhesion was detected by anti-human C3c antibody (Dako) followed by goat α-rabbit HRP (Southern Biotech). Negative controls were serum and buffer without antibody (no C3b adhesion occurred), and positive controls were serum without antibody (maximum C3b adhesion). The cutoff criterion was set at half the activity of the unrelated 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 an ELISA plate overnight at 4°C. The following day, 250 μl of blocking solution (1% BSA in PBS) was added to the wells and incubated at room temperature for 2 hours. The plate was washed three times with 300 μl of PBS / tween-20.

[0205] Cynomolgus monkey serum was diluted to 0.25% in GVB / Ca / Mg buffer, and MASP-2 mAb clones OMS850, OMS860, and OMS870 were added in a final concentration range of 0.00001–100 nM. The mixture was pre-incubated on ice for 15 minutes. The reaction was initiated by transferring the pre-incubation mixture to wells of an assay plate coated with mannan and blocked, and then incubated at room temperature for 40 minutes. The reaction was stopped by washing the plate three times with PBS / Tween-20. C3b adhesion was detected by anti-human C3c antibody (Dako) followed by goat α-rabbit HRP (Southern Biotech). Negative controls were serum and buffer without antibody (no C3b adhesion occurred), and positive controls were serum without antibody (maximum C3b adhesion). The cutoff criterion was set at half the activity of the unrelated 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 an ELISA plate overnight at 4°C. The following day, 250 μl of 1% BSA blocking solution was added to each well and incubated at room temperature for 2 hours. The plate was washed three times with 300 μl of PBS / tween-20.

[0207] Rat serum was diluted to 0.3% in GVB / Ca / Mg buffer, and MASP-2 mAb clones OMS850, OMS860, and OMS870 were added in a final concentration range of 0.00001–100 nM. The mixture was pre-incubated on ice for 15 minutes. The reaction was initiated by transferring the pre-incubation mixture to wells of an assay plate coated with mannan and blocked, and then incubated at room temperature for 40 minutes. The reaction was stopped by washing the plate three times with wash buffer. C3b adhesion was detected by anti-human C3c antibody (Dako) followed by goat α-rabbit HRP (Southern Biotech). Negative controls were serum and buffer without antibody (no C3b adhesion occurred), and positive controls were serum without antibody (maximum C3b adhesion). The cutoff criterion was set at half the activity of the unrelated 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. The following day, 250 μl of blocking solution (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 / tween-20.

[0209] Mouse serum was diluted to 1.0% in GVB / Ca / Mg buffer, and MASP-2 mAb clones OMS850, OMS860, and OMS870 were added in a final concentration range of 0.00001–100 nM. The mixture was pre-incubated on ice for 15 minutes. The reaction was initiated by transferring the pre-incubation mixture to wells of an assay plate coated with mannan and blocked, and then incubated at room temperature for 40 minutes. The reaction was stopped by transferring the plate to an ice bath. C3b adhesion was detected by anti-human C3c antibody (Dako) followed by goat α-rabbit HRP (Southern Biotech). Negative controls were serum and buffer without antibody (no C3b adhesion occurred), and positive controls were serum without antibody (maximum C3b adhesion). The cutoff criterion was set at half the activity of the unrelated mAb and buffer alone.

[0210] The results are shown in Figures 5A, 5B, 5C, and 5D, and summarized in Table 8 below. In Figures 5A to 5D, the data points labeled "buffer solution" represent negative controls using only the buffer solution.

[0211] (Table 8) Results of C3b adhesion assay TIFF2026123079000040.tif45128

[0212] 3. C4b adhesion 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 an ELISA plate overnight at 4°C. The following day, 250 μl of blocking solution (1% BSA in PBS) was added to the wells and incubated at room temperature for 2 hours. The plate was washed three times with 300 μl of 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 in a final concentration range of 0.0001–100 nM. The mixture was pre-incubated on ice for 15 minutes. The reaction was initiated by transferring the pre-incubation mixture to a mannan-coated and blocked ELISA plate, and then incubated at 4°C for 7 minutes. C4b adhesion was detected by anti-human C4c antibody (Dako) followed by goat α-rabbit HRP (Southern Biotech). Negative controls were serum and buffer without antibody (= no C4b adhesion). Background was determined in wells containing only buffer. The cutoff criterion was set at half the activity of the unrelated mAb and buffer alone.

[0214] The results are shown in Figure 6 and listed in Table 9 below. In Figure 6, the data points labeled "buffer solution" represent the negative control of buffer solution only.

[0215] (Table 9) Results of C4b adhesion assay TIFF2026123079000041.tif38128

[0216] Considering the favorable titers observed in humans, mice, rats, and cynomolgus monkeys as described above, the antibody OMS850 was selected for humanization and further optimization.

[0217] Example 4 Humanization and Variant Production of MASP-2 mAb OMS850 Prior to humanization, the anti-MASP-2 inhibitory mAb OMS850 was analyzed for post-translational modifications. The aspartic acid isomerization motif "DP" was identified in TIFF2026123079000042.tif4128. Variants of OMS850 were generated by designated site 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 using the complete IgG4 format as described above, and titer was evaluated by C3 adhesion assay in human serum.

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

[0219] (Table 10) Results of C3b adhesion assay TIFF2026123079000043.tif45128

[0220] As shown in Figure 7, when measured using the C3b adhesion assay, OMS850 VH P53A was determined to have the best titer in human serum. Therefore, this P53A substitution was incorporated in subsequent humanizations described below.

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

[0222] We developed the first humanized prototype and named it OMS852. The amino acid sequences of the heavy chain variable region and light chain variable region of the humanized OMS852 are listed below. CDR(Kabat) is underlined.

[0223] Complete sequence of OMS852 VH (humanized): (SEQ ID NO: 46) TIFF2026123079000044.tif18159

[0224] The complete sequence of OMS852 VL (humanized) (SEQ ID NO: 47) TIFF2026123079000045.tif11159

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

[0226] (Table 11) Results of C3b adhesion assay for humanization candidates of OMS850 TIFF2026123079000046.tif90128

[0227] The monoclonal antibody OMS852 4PA-1, which was humanized and incorporated the previously identified P53A modification, was selected for further development and named OMS854.

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

[0229] The complete sequence of OMS854 VH :(SEQ ID NO:48) TIFF2026123079000047.tif18160

[0230] The complete sequence of OMS854 VL (humanized) (SEQ ID NO: 47) TIFF2026123079000048.tif11159

[0231] As shown in Table 12, further modifications were made to VH CDR1 such that it contains at least one, or optionally two, or optionally three histidines. 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) TIFF2026123079000049.tif18159

[0233] Complete sequence of OMS858 VH (SEQ ID NO: 50) TIFF2026123079000050.tif18159

[0234] (Table 12) VH sequences of MASP-2 antibodies (CDR and FR regions, Kabat) TIFF2026123079000051.tif97160TIFF2026123079000052.tif245160

[0235] (Table 13) VL sequences of MASP-2 antibodies (CDR and FR regions, Kabat) TIFF2026123079000053.tif142160

[0236] SEQ ID NO:65 : Human IgG4 constant region TIFF2026123079000054.tif41159

[0237] SEQ ID NO:66 : Human IgG4 constant region with S228P mutation TIFF2026123079000055.tif41159

[0238] SEQ ID NO:67 : Human IgG4 constant region with S228P mutation and a mutation (Xtend) that promotes FcRn interaction at low pH TIFF2026123079000056.tif41159

[0239] SEQ ID NO:68 : Human IgK constant region TIFF2026123079000057.tif11158

[0240] SEQ ID NO:69:OMS850 VH encoding DNA (mouse parent) TIFF2026123079000058.tif47159

[0241] SEQ ID NO:70:OMS860 VH encoding DNA (mouse parent) TIFF2026123079000059.tif47159

[0242] SEQ ID NO:71:OMS870 VH encoding DNA (mouse parent) TIFF2026123079000060.tif47158

[0243] SEQ ID NO:72:OMS850 VL encoding DNA (mouse parent) TIFF2026123079000061.tif40159

[0244] SEQ ID NO:73:OMS860 VL encoding DNA (mouse parent) TIFF2026123079000062.tif40159

[0245] SEQ ID NO:74:OMS870 VL encoding DNA (mouse parent) <"0000966">TIFF2026123079000063.tif48158

[0246] SEQ ID NO:75:OMS852 DNA encoding VH TIFF2026123079000064.tif47159

[0247] SEQ ID NO:76:OMS852 DNA encoding VL TIFF2026123079000065.tif40159

[0248] SEQ ID NO:77:DNA encoding OMS854 TIFF2026123079000066.tif47159

[0249] SEQ ID NO:78:DNA encoding OMS856 TIFF2026123079000067.tif47159

[0250] SEQ ID NO:79:DNA encoding OMS858 TIFF2026123079000068.tif47159

[0251] Example 5 The antibody OMS858 specifically blocks the lectin pathway. The effects of mAb OMS858 on membrane invasion complex (MAC) adhesion were analyzed using pathway-specific conditions for the lectin pathway, classical pathway, and alternative pathway. 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 under classical pathway-specific assay conditions, in and out of the presence of the anti-MASP-2 antibody OMS858. Figure 9B graphically illustrates the level of MAC adhesion under lectin pathway-specific assay conditions, in and out of the presence of the anti-MASP-2 antibody OMS858. Figure 9C graphically illustrates the level of MAC adhesion under alternative pathway-specific assay conditions, in and out of the presence of the anti-MASP-2 antibody OMS858.

[0253] As shown in Figure 9B, mAb OMS858 mediated lectin pathway activation of MAC attachment at approximately 1 nM IC50. 50 The value blocked the pathway. However, mAb OMS858 had no effect on MAC adhesion initiated by classical pathway-mediated activation (Figure 9A) or alternative pathway-mediated activation (Figure 9C). Figure 9A includes an anti-C1s antibody (TNT003), known to inhibit the classical pathway, as a control. Figures 9B and 9C include anti-factor B antibodies, known to inhibit the alternative pathway, as controls.

[0254] Example 6 Analysis of MASP-2 binding epitopes of antibodies OMS850, OMS858, OMS860, and OMS870. The antibody OMS858 was biotinylated, and its ability to simultaneously bind to several different MASP-2 antibodies was tested using the Octet Biolayer Interference Binding Assay as follows.

[0255] The antibody OMS858 was biotinylated using EZ-link sulfo-NHS-LC-biotin (Fischer Scientific, A39257). A super streptavidin (SSA) biosensor (Fortebio 18-5057) was hydrated in PBS at room temperature for 15 minutes. 3 mL of 50 nM biotinylated OMS858 was captured on a BLI chip, followed by capture of hMASP-2-CCP1-CCP2-SP. A 500 nM solution of one of 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 was described above, and the previously identified anti-MASP-2 antibodies OMS721 and 4A8.

[0256] 200 μL of biotinylated OMS858 was added to the first row of plate wells, and 200 μL of hMASP-2-CCP1-CCP2-SP was added to the wells in the next row. In another row on the same plate, 200 μL of 500 nM test antibody was added to each well. Controls were also performed using unbiotinylated OMS858 or buffer only instead of test antibody.

[0257] The binding assay was performed in Octet using the following steps: baseline establishment (60 sec), loading with biotinylated OMS858 (180 sec), baseline establishment (60 sec), loading with hMASP-2-CCP1-CCP2-SP (180 sec), followed by association (300 sec) and dissociation (300 sec) 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, the 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 different epitopes on MASP-2 than OMS850, OMS860, OMS870, and OMS858.

[0259] Example 7 Crystallization of the complex of MASP-2 protein and Fab mAb OMS858 1. Preparation of MASP-2 protein Recombinant MASP-2 protein and human mannose-binding lectin serine protease 2 based on UniProt O00187 were prepared as follows. Expression constructs for human MASP-2 CCP2-SP and CCP2-SP-6HIS were prepared for recombinant expression in E. coli cells. Recombinant expression of MASP-2 as an inclusion body and protein purification in E. coli were performed according to the method described in Ambrus G. et al., 2003, with minor modifications. For the HIS-tagged version, the MASP-2 protein (CCP2-SP-6HIS) was purified under denaturation 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 the standard methods described in Harmat et al., J.Mol.Biol.2004;342:1533-1546, 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 from 5 mg / mL to 20 mg / mL using a centrifugal concentrator (Amicon NMWL 10 kDa). The concentrated MASP-2 protein samples were rapidly 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 prepared by subjecting mAb OMS858 to proteolytic cleavage using the FAbALACTICA® Fab kit, in accordance with the manufacturer's instructions.

[0262] 3. Generation of the MASP-2-Fab mAb OMS858 complex Fab mAb OMS858 and MASP-2 protein samples were incubated at equimolar concentrations for 1 hour. The successful formation of the MASP-2-Fab mAb OMS858 complex was verified by analytical size exclusion chromatography.

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

[0264] Example 8 X-ray crystal analysis The MASP-2-Fab mAb OMS858 composite crystal, prepared as described in Example 7, was diffracted with 1.0 angstrom X-rays at beamlines SSRL BL9-2, BL14-1, BL12-2, and ALS sector 5, and X-ray diffraction data sets were collected using Dectris Pilatus and Eiger detectors. A summary of the crystal parameters, data acquisition, and refinement statistics for the MASP-2-Fab mAb OMS858 composite crystal is shown in Table 14. The X-ray structure was determined by molecular substitution using parts of structures 1Q3X and 3C08 as exploratory models, and partially refined with Buster 2.10.2 or Refmac 5.8. Electron density was checked with Coot (Emsley et al., 2010), and the model was subjected to iterative model building and refinement cycles until the densities of MASP-2 and Fab mAb OMS858 were clearly recognizable and a sufficient R factor was obtained. At this point, we determined that partial refinement was complete and checked the model for ligands, solvents, and proteins.

[0265] (Table 14) Summary of crystal parameters, data acquisition, and refined statistics for the MASP-2-Fab mAb OMS858 composite crystal. TIFF2026123079000069.tif173160TIFF2026123079000070.tif117160

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

[0267] Table 15 shows the hydrogen bonds between atoms of mAb OMS858 and 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 crystallographic structures using the “antibody” setting. LigPlot+ calls these “unbonded contacts” or “hydrophobic contacts”. Despite this naming, there are atom pairings that involve suspected hydrogen bonds. Therefore, in some cases, Table 16 also includes hydrogen bond interactions.

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

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

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

[0271] As follows, it was determined that there are 3 hydrogen bonds and 36 van der Waals contacts.

[0272] (Table 15) Three H bonds between atoms of mAb OMS858 and MASP-2 TIFF2026123079000071.tif24160

[0273] (Table 16) 36 van der Waals contacts between atoms of mAb OMS858 and atoms of MASP-2 TIFF2026123079000072.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 “antibody” mode with settings for hydrogen bond calculation parameters (maximum distance of 3.35 Å between hydrogen bond donors and hydrogen bond acceptors, and unbonded contact parameters between hydrophobic contacts or any contacts such as van der Waals interactions with a maximum contact distance of 3.90 Å), using a model derived from the corresponding crystallographic MASP-2-compound costructure. Hydrogen bonds and polar contacts are drawn with dashed lines, and distances are given in angstroms.

[0275] Crystallographic data depict the amino acid atoms that interact with compound atoms, as well as compound atoms with sufficient 2fo-fc electron density. MASP-2 amino acid residue numbering (MASP-2 AA#) follows Uniprot accession code O00187, and atomic numbering for amino acids (AA atoms) follows the practice established by the Protein Data Bank, corresponding to those in Tables 15 and 16.

[0276] The amino acid residue numbering for OMS858 follows VH, indicated as SEQ ID NO: 50, and VL, indicated as SEQ ID NO: 47. The paratopes of OMS858, including the heavy chain variable regions (loop H1, loop H2) and light chain variable regions (loop L3, loop L1), are drawn above the discontinuous lines in Figure 13, while the epitopes of MASP-2 are drawn below the discontinuous lines in Figure 13. Certain amino acids shown in Figure 13 are denoted by arcs with radial lines, indicating that they have van der Waals interactions (dotted lines) with atoms of other amino acids. Hydrogen bonds and polar contacts are denoted by dashed lines, and distances are given in angstroms. Carbon atoms are shown as filled circles, nitrogen atoms as hollow circles with crosses, and oxygen atoms as hollow circles with crosses. The side chains of amino acids Glu57 and Arg30 of OMS858 were only partially elucidated by X-ray structure. Therefore, given the proximity of the backbones, it is highly probable that further hydrogen bonds and ionic interactions exist between the VH GLU57 and VL ARG30 of OMS858 and the corresponding residues on MASP-2. Specifically, the VL ARG30 of OMS858 may form an ionic bond to ASP496 of MASP-2, the VL ARG30 of OMS858 may form a π-π stacking interaction with the aromatic moiety of TRP513 of MASP-2, and the 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 via three H bonds: ASP52 and ASP55 in VH that bind to LYS503 in MASP-2, and ASP50 in VL that binds to HIS508 in MASP-2. Further as shown in Figure 13, OMS858 also binds to MASP-2 via van der Waals contacts between HIS33 in VH and SER506 and PRO507 in MASP-2, between ASP52, ASP55 and GLU57 in VH and LYS503 in MASP-2, between HIS59 and ASP50 in VH and HIS508 in MASP-2, between TRP90 in VL and HIS508, between ARG30 in VL and TRP513 in MASP-2, and between TYR31 in VL and TRP513 and ASP496 in MASP-2.

[0278] In certain contexts, MASP-2 inhibitory antibodies interact via van der Waals contact with one, two, three, four, five, six, or all of the following residues of MASP-2: SER506, PRO507, LYS503; HIS508; TRP513; ASP496 and combinations thereof. In certain contexts, MASP-2 inhibitory antibodies interact via hydrogen bonding with one or two of the following residues of 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, represented as TIFF2026123079000073.tif4128, corresponds to amino acid residues 496-513 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, including the heavy chain HIS33, ASP50, ASP52, ASP55, GLU57, and HIS59, and the light chain ARG30, TYR31, and TRP90, to which MASP-2 is bound. HIS33 is located on loop H1. ASP50, ASP52, ASP55, GLU57, and 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 loop L3 of the light chain.

[0281] Figure 15 shows the MASP-2 epitope. The contact between TIFF2026123079000074.tif4128 and the heavy chain variable regions and light chain variable regions of OMS858 is illustrated. Specifically, as shown in Figure 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. Further shown in Figure 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 situations, MASP-2 SP (SEQ ID NO:6) is represented by amino acid residues 496-513 of SEQ ID NO:1. TIFF2026123079000075.tif4128) interacts with OMS858 via van der Waals interactions. Van der Waals interactions involve weak, near-field electrostatic attraction between uncharged molecules arising from the interaction of persistent or transient electric dipole moments.

[0283] As shown in Table 16, the OMS858 LC ARG30 atom CB interacts with atoms CZ2 and CE2 in TRP513 of MASP-2. The LC TYR31 atom CE2 interacts with atom CH2 in TRP513 of MASP-2. The HC HIS59 atoms CD2, NE2, CE1, ND1, and CG interact with atom CD2 in HIS508 of MASP-2. The HC ASP50 atom OD2 interacts with atom CD2 in HIS508 of MASP-2. The LC TRP90 atom CH2 interacts with atom NE2 in HIS508 of MASP-2. The HC HIS59 atoms CD2, CG, and CB interact with atom NE2 in HIS508 of MASP-2. The 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.The LC TYR 31 atom CE2 interacts with the atom OD2 in ASP496 of MASP-2.

[0284] In summary, crystal structure analysis of the MASP-2 inhibitor antibody OMS858 revealed that this antibody contains a novel epitope in the human MASP-2 serine protease domain. This indicates binding to TIFF2026123079000076.tif4128. This epitope-binding domain is distinct from the serine protease activity triad region (residues HIS483, ASP532, and SER633). This represents a novel epitope in the human MASP-2 serine protease domain. It is also evident that there is an overlap between TIFF2026123079000077.tif4128 and the C4 interaction site on MASP-2 containing residues LYS503 and TRP513 (see Kidmose et al., Proc Natl Acad Sci USA 109:15425 (2012)). Therefore, the LYS503 and TRP513 residues of human MASP-2 can bind and participate in the binding of both OMS858 and C4.

[0285] The arrangement of mAb OMS858 and MASP-2 shown in Figures 11-15 follows that of a standard antibody-antigen recognition format, suggesting a mechanism of action in which lectin pathway activity is inhibited by OMS858 competing with C4 for binding to MASP-2.

[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 mAbs OMS860 and OMS870 also cross-compete with OMS858. Therefore, 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 Preparation of canine-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, because 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 the amino acid alignment of the MASP-2 serine protease (SP) domains of human MASP-2 (aa445-686, SEQ ID NO:1), cynomolgus monkey MASP-2 (aa445-686, SEQ ID NO:4), canine MASP-2 (aa445-686, SEQ ID NO:5), mouse MASP-2 (aa444-685, SEQ ID NO:2), and rat MASP-2 (aa444-685, SEQ ID NO:3). TIFF2026123079000078.tif136160TIFF2026123079000079.tif244159TIFF2026123079000080.tif120159

[0289] To evaluate the species cross-reactivity of the OMS858 antibody, recombinant MASP-2 protein was produced from cynomolgus monkeys and mice. DNA encoding the catalytic fragment (CCP1-CCP2-SP domain) of human MASP-2 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 6×His tag was attached to the C-terminus of the polypeptide for purification. Similar MASP-2 expression constructs were also prepared from cynomolgus monkeys and mice.

[0290] In addition, we produced a "canine-like" variant of human MASP-2. We found that OMS858 does not inhibit lectin pathway activation in canine serum (data omitted). As shown in Figure 16, the sequences of canine and human MASP-2 differ. 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 expected to sufficiently interact with only one of the two amino acids in the paratope that histidine interacts with in human MASP-2, effectively weakening the interaction between canine MASP-2 and OMS858 compared to human MASP-2. To produce a canine-like variant of human MASP-2, we introduced the H508Q mutation using PfuUltra II fusion HS DNA polymerase (Agilent) via PCR-based designated-site mutagenesis. Based on 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 Escherichia coli (Invitrogen), and the 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 enzyme activity of the purified proteins were evaluated by SDS-PAGE and peptide cleavage assay, respectively.

[0292] Biolayer interferometry (BLI) was used to analyze the binding of OMS858 antibodies to multiple MASP-2 proteins. All measurements were performed using the Octet RED96 system (ForteBio) with PBS containing 1% BSA and 0.02% Tween 20 as the assay buffer. OMS858 (50 nM) was first loaded onto an anti-human Fc capture biosensor (ForteBio). After a one-step baseline, the capture sensor was immersed in wells containing various concentrations of target protein (1.6–50 nM) for the association step (120 seconds), and then transferred to an empty well for the dissociation step (200 seconds). In all experiments, the capture sensor was also immersed in a well without the analyte to allow for a single-reference subtraction to compensate for the 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 TIFF2026123079000081.tif46129

[0294] As shown in Figure 17, human MASP-2 with the H508Q "canine-like" mutation reduced its binding affinity to approximately 1 / 450th.

[0295] Example 11 Cynomolgus monkey test: PK / PD of OMS856 and OMS858 Naive cynomolgus monkeys (n=3) were administered 1.5 mg / kg of OMS856 and OMS858 either intravenously or via IV or SC. Blood samples were collected from each animal at day 7 and then at 0.083, 1, 4, 24, 72, 168, 240, 336, 504, 672, 840, and 1008 hours after administration, and tested for the presence of lectin pathway activity and the amount of antibody administered.

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

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

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

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

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

[0301] Example 12 Analysis of OMS858 in a mouse model of thrombosis In a mouse model of ferric chloride-induced carotid artery occlusion, we conducted studies to determine the minimum effective dose and maximum efficacy of OMS858.

[0302] The left carotid artery of male C57B1 / 6 mice was exposed, and a small Transonic flowmeter (0.7 mm) was attached around the vessel. After administration of the test compound, thrombus formation was induced by applying a piece of filter paper (1.5 mm × 1 mm) soaked to its maximum limit with 3.5% FeCl3. The filter paper was placed directly on the carotid artery so as to be in contact with the outer surface of the vessel. After 3 minutes of exposure, the filter paper was removed, and the vessel was washed with saline. Carotid blood flow was continuously recorded until the vessel was completely occluded or for a maximum of 45 minutes. Time to occlusion (TTO) was defined as the time from the application of FeCl3 until the blood flow decreased to less than 0.1 mL / min for at least 30 seconds, or until the signal amplitude decreased to the point where it prevented visualization of the heartbeat with the probe. Vessels that were not occluded at the end of the 45-minute observation period were recorded as having a 45-minute TTO.

[0303] OMS858 was administered to mice (n=8 per dose group) by sc injection 24 hours prior to the study at the following doses: 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 the FeCl3 challenge, was used as a positive control. An unrelated 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 maximum effect of OMS858 was comparable to that of ASA. As further shown in Table 18 and Figure 19, OMS858 was effective even at the lowest test dose (1 mg / kg). The time to death (TTO) in the media-treated control mice was 14.7 ± 4.6 minutes. As expected, pretreatment with ASA significantly prolonged TTO to 36.4 ± 5.7 minutes, while pretreatment with the isotype control antibody did not significantly prolong 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 prolonged TTO compared to the media group (40.4 ± 4.6, 37.0 ± 5.2, 39.1 ± 3.9 minutes, and 37.4 ± 5.0 minutes, respectively). Since no clear dose-response relationship was observed among the evaluated OMS858 doses, this suggests that the maximum pharmacological effect can 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 TIFF2026123079000082.tif81170* p<0.05 compared to media control; p-values ​​were generated using an independent one-sided 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 C5b-9 activation induced by classical, lectin, and alternative pathways in human serum. To evaluate the functional selectivity of OMS858 in inhibiting the lectin pathway, the Wieslab® complement system screening kit was used. Human serum samples were pre-incubated with serially diluted OMS858, and then the functional activity of OMS858 was evaluated by quantifying complement activation and C5b-9 adhesion under pathway-specific assay conditions.

[0307] A human serum sample diluted in a pathway-specific assay buffer was pre-incubated with serial dilutions of OMS858 and then incubated at 37 °C for 1 h on appropriate Wieslab® assay wells pre-coated with a classical pathway-specific (A), lectin pathway-specific (B) or alternative pathway-specific (C) complement activator. The attachment of the final complement activation product C5b-9 (also called MAC) was quantified using an alkaline phosphatase-conjugated antibody specific for the C5b-9 neoantigen.

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

[0309] Example 14 Further Characterization of the Binding of Monoclonal Antibody OMS858 to Human MASP-2 Using surface plasmon resonance (SPR), the association rate constant and dissociation rate constant (kon and koff, respectively) were determined for the interaction between fluid-phasezymogen and catalytically active human MASP-2 and immobilized OMS858. An optimized method and concentration series were used for the detailed characterization of this interaction, and the dissociation equilibrium constant (KD) for the OMS858-MASP-2 interaction was calculated using the obtained rate constants.

[0310] k for the binding of catalytically active MASP-2 to OMS858 on ​​​​​​​​​​​​​​​​​​​​​​​-1 and 1.72 × 10 -4 s -1 Therefore, the KD value was 715 pM. See Table 19.

[0311] (Table 19) Binding of OMS858 to zymogenized and active human MASP-2 TIFF2026123079000083.tif23149

[0312] The binding specificity of OMS858 to MASP-2 was evaluated 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. The apparent dissociation constant (KD) was estimated by nonlinear regression using a four-variable logistic model.

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

[0314] (Table 20) Selectivity of OMS858 binding to MASP-2 compared with complement system-related serine proteases TIFF2026123079000084.tif55170

[0315] Example 15 Characterization of the functional activity of the monoclonal antibody OMS858 in various mammalian species. The functional titer of OMS858 in serum from humans, cynomolgus monkeys, dogs, rabbits, and mouse hirudin plasma was characterized using an enzyme-linked immunosorbent assay (ELISA) to measure lectin-dependent C4 activation in 50% serum. The functional activity of OMS858 was evaluated by pre-incubating serially diluted OMS858 samples from each species with the mixture, and then adding the mixture to mannan-coated ELISA plate wells that drive lectin-dependent C4 activation in vitro. Lectin pathway inhibition concentration-response curves were analyzed, and IC50 was determined. 50 By determining the value, we estimated the functional potency of MASP-2 activity in inhibiting OMS858.

[0316] In human serum, OMS858 showed potent inhibition of rectification-dependent complement activation, and average IC50. 50 The value was 1.09 nM (164 ng / mL). Mean IC50 of OMS858 for inhibition of lectin pathway activation measured in cynomolgus monkey serum and mouse plasma. 50 The values ​​were 44.1 nM (6.620 μg / mL) and 11.9 nM (1.79 μg / mL), respectively. In contrast, OMS858 did not clearly inhibit lectin-dependent complement activation in rabbit and dog serum at concentrations up to 500 nM. See Table 21. By comparing the functional titers of mice and cynomolgus monkeys with those of humans, OMS858 was shown to have functional titers of 1 / 10.9 and 1 / 40.5, respectively, compared to humans in these species. Relative titers to humans were obtained using IC25 in human serum. 50 IC for query types 50 The values ​​were calculated as a ratio. See Table 22.

[0317] (Table 21) IC on lectin pathway inhibition by OMS858 in mouse, rabbit, dog, and cynomolgus monkey serum compared to human. 50 value TIFF2026123079000085.tif76162

[0318] (Table 22) Interspecies titers in humans TIFF2026123079000086.tif57128

[0319] Example 16 Pharmacodynamics of OMS858 in mice and cynomolgus monkeys After ex vivo exposure of mannan-coated surfaces with serum, complement activation was subsequently evaluated using electrochemiluminescence immunoassay (ECLIA) to measure PD activity (inhibition of lectin pathway activity). In mice, the adhesion of complement-activating fragments in individual animals was compared to the mean adhesion of complement-activating fragments in sample / control animals collected at baseline, and the data were presented as an inhibition percentage. In monkeys, individual C4 adhesion data were compared to the baseline response of individual animals, and the data were presented as an inhibition percentage.

[0320] The results for mice are shown in Figure 24. There was considerable variability in baseline LP activity as measured by ECLIA. No detectable PD effect was observed at 0.1 mg / kg. However, mean MASP-2 inhibition exceeded 50% up to 72 hours post-administration in the 0.3 mg / kg SC dose group, up to 504 hours post-administration in the 1 mg / kg IV dose group, and up to 1008 hours post-administration in the 1 and 3 mg / kg SC dose groups. In addition to a tendency for the duration of effect to increase with increasing dose levels, higher OMS858 concentrations also tended to increase MASP-2 inhibition. OMS858 concentrations above 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 MASP-2 inhibition exceeding 50%. 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 higher.

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

[0322] The results for monkeys are shown in Figure 21. In cynomolgus monkeys administered 1 mg / kg SC of OMS858, approximately 70-80% inhibition of lectin pathway activity was observed, starting 24-48 hours after OMS858 administration and lasting for approximately 336 hours post-administration, after which the inhibition gradually decreased over time. In cynomolgus monkeys administered 3 mg / kg SC of OMS858, nearly complete (>95%) inhibition of lectin pathway activity was observed, starting approximately 24 hours after OMS858 administration and lasting for approximately 672 hours post-administration, after which the inhibition gradually decreased over time. Nearly complete inhibition of lectin pathway activity was also observed after IV administration of 1 mg / kg OMS858, and the duration of the maximum effect and the gradual decrease over time were similar to those observed with 1 mg / kg SC.

[0323] A consistent relationship was observed between serum OMS858 concentration and PD activity in cynomolgus monkeys. Serum concentrations of OMS858 above approximately 20 μg / mL resulted in significant (>80%) inhibition of lectin pathway activity, while serum concentrations below approximately 3 μg / mL resulted in only slight inhibition. Modeling of OMS858 serum concentration and PD response data suggests that approximately 9 μg / mL of EC288 was associated with PD activity. 50 A sigmoid-shaped drug concentration response profile was shown with a value (OMS858 concentration that resulted in maximum half-life lectin pathway inhibition). See Figure 22.

[0324] Example 17 Pharmacokinetics of OMS858 in mice and cynomolgus monkeys The pharmacokinetic (PK) of OMS858 after a single dose of 0.1, 0.3, 1, or 3 mg / kg of SC, 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 before administration and at 0.083 (IV only), 24, 48, 72, 168, 240, 336, 504, 672, 840, 1008, 1176, and 1344 hours after administration (N=3 / group / time point; terminal sample collection). In monkeys, samples were collected before administration 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 after administration (N=3 / group / time point; sequential sample collection).

[0325] Table 23 summarizes the PK parameters measured after a single dose of OMS858 in mice and monkeys via SC or IV injection. Figure 23 shows plots of mean OMS858 serum concentrations over time after a single IV or SC administration in mice (left) and monkeys (right).

[0326] The time (t) to reach the maximum concentration after a single SC administration. max The median time to efflux was 24–168 hours post-administration in mice and 72 or 168 hours post-administration in monkeys. Efflux of OMS858 after single SC or IV administration. 1 / 2 In mice, the incubation period was 203–409 hours, and in monkeys, it was 196–487 hours. In mice and monkeys, the maximum observed concentration (C) was max ) and total exposure (area under the time-concentration curve extrapolated to infinity [AUC INF The total exposure (AUC) after a single SC administration of 1 mg / kg OMS858 generally increased proportionally with the dose. INF The bioavailability determined using ) was 85.7% in mice and 93.0% in monkeys.

[0327] (Table 23) Pharmacokinetic parameters of OMS858 after single administration to mice and monkeys by SC and IV injection Serum parameters reported for male mice based on composite mean concentration versus time data. TIFF2026123079000087.tif123164TIFF2026123079000088.tif238164TIFF2026123079000089.tif40164a b. The determination was made only for group IV at a dose level of 1 mg / kg. 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 hours 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 0 hours to t hours (the last measurement time); 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 reach maximum concentration; Vss =Distribution volume in steady state; Vz =Distribution volume based on terminal efflux phase.

[0328] Example 18 Phase 1 single-dose clinical trial of OMS858 in healthy human subjects To evaluate safety, tolerability, PK, PD, and immunogenicity compared to placebo, a single-dose, dose-escalating, blind trial of intravenous (IV) and subcutaneous (SC) administration of OMS858 will be conducted in healthy subjects. 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. Neither the subjects nor the medical staff administering the medication will know which subjects will receive OMS858 and which will receive placebo. The medication distribution for the six cohorts is as follows: • Cohort 1: Single IV dose of OMS858 at 0.01 mg / kg 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: 0.3 mg / kg OMS858 or placebo, single IV dose. • Cohort 5: Single SC dose of 1.0 mg / kg OMS858 or placebo • Cohort 6: Single SC dose of OMS858 or placebo; dosage will be determined based on data from previous cohorts.

[0329] Participants are 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, 1 day before administration, immediately before administration, and at 0.5, 12, 24, 48, 72, 96, 120, 144, and 168 hours after administration, as well as at 15, 22, 29, 57, and 85 days after administration. The primary endpoint is the assessment of the safety and tolerability of OMS858. Secondary endpoints include characterization of the PK and PD of OMS858, and assessment of the presence of anti-drug antibodies (ADAs) against OMS858 after administration. Exploratory endpoints include the analysis of the effects of OMS858 on MASP-2 levels and mannose-binding lectin (MBL) levels.

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

[0331] Preliminary PK and PD data for cohorts 1, 2, 3, and 4 are shown in Figures 25 and 26. No safety or tolerability issues were observed at the time points shown in Figures 25 and 26. PK measurements for OMS858 showed dose-proportional exposure throughout the time points shown in Figure 25. As shown in Figure 26, a robust and sustained PD response for OMS858 was observed in all three cohorts. A favorable PK / PD relationship was observed as shown in Figure 27, and based on the data from cohorts 1-3, EC 50 It is 170 ng / mL and EC 90 The level was 400 ng / mL.

[0332] IX. Exemplary Examples All publications, patent applications, and patents referenced herein are incorporated herein by reference.

[0333] While certain aspects of the present invention have been illustrated and described, it will be understood that various modifications can be made without departing from the gist and scope of the invention. Although the present invention has been described in terms of specific aspects, it should be understood that the invention as described herein should not be unreasonably limited to such specific aspects. In fact, various modifications of the described specific aspects that are obvious to those skilled in the art in the fields of medicine, immunology, pharmacology, or related fields shall be considered to be within the scope of the present invention.

[0334] Therefore, for the sake of clear disclosure, the following numbered paragraphs describe specific embodiments, but these should not be construed as limiting the scope of the claims.

[0335] 1. An isolated monoclonal antibody or its antigen-binding fragment that specifically binds to an epitope located within the serine protease domain of human MASP-2, wherein the epitope is an amino acid The isolated monoclonal antibody or its antigen-binding fragment, located within TIFF2026123079000090.tif4128, wherein the antibody or its antigen-binding fragment inhibits lectin pathway complement activation. 2. The isolated antibody or its antigen-binding fragment from paragraph 1 that competes for C4 binding to MASP-2. 3. The isolated antibody or antigen-binding fragment of paragraph 1 that forms a hydrogen bond with at least one of human MASP-2 Lys503 and / or His508. 4. The isolated antibody or antigen-binding fragment from paragraph 1 that forms a hydrogen bond with human MASP-2 His508. 5. The isolated antibody or antigen-binding fragment from paragraph 1 that forms van der Waals contact with one or more of the following human MASP-2 amino acids: Asp496, Lys503, Ser506, Pro507, His508, and Trp513. 6. (a) HC-CDR1 having the sequence NXXMH, where X at position 2 is H or Y, and X at position 3 is H or W, Shown as TIFF2026123079000091.tif4128, 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, HC-CDR2, HC-CDR3, shown as TIFF2026123079000092.tif4128 Heavy chain variable region including, (b) SEQ ID NO: 64 (SASSSVXYMY), where X at position 7 is R or S, LC-CDR1, LC-CDR2, indicated as SEQ ID NO:34(DTSNLAS), LC-CDR3, indicated as SEQ ID NO:36(QQWSSYPLT) Light chain variable region including Includes, or (b) HC-CDR1, indicated as SEQ ID NO:25(SYWMH), HC-CDR2, which is shown as TIFF2026123079000093.tif4128, HC-CDR3, which is shown as SEQ ID NO:29(WAYDAMDY) Heavy chain variable region including, LC-CDR1, which is shown as TIFF2026123079000094.tif5128, LC-CDR2, indicated as SEQ ID NO:43(FASNLES), LC-CDR3, indicated as SEQ ID NO:45(QQSNEDPLT) including, The isolated antibody or its antigen-binding fragment from paragraph 1. 7. The isolated antibody or antigen-binding fragment of paragraph 6(a) containing HC-CDR1 SEQ ID NO:14(NYWMH). 8. The isolated antibody or antigen-binding fragment of paragraph 6(a) containing HC-CDR1 SEQ ID NO:56(NYHMH). 9. The isolated antibody or antigen-binding fragment of paragraph 6(a) containing HC-CDR1 SEQ ID NO:57(NHHMH). 10. HC-CDR2 The isolated antibody or its antigen-binding fragment from paragraph 6(a), including TIFF2026123079000095.tif4128. 11. HC-CDR2 The isolated antibody or its antigen-binding fragment from paragraph 6(a), including TIFF2026123079000096.tif4128. 12. HC-CDR2 The isolated antibody or its antigen-binding fragment from paragraph 6(a), including TIFF2026123079000097.tif4128. 13. The isolated antibody or antigen-binding fragment of paragraph 6(a) in which LC-CDR1 contains SEQ ID NO:32(SASSSVRYMY). 14. The isolated antibody or antigen-binding fragment of paragraph 6(a) in which LC-CDR1 contains SEQ ID NO:39(SASSSVSYMY). 15. HC-CDR1 contains SEQ ID NO:14(NYWMH), SEQ ID NO:56(NYHMH), or SEQ ID NO:57(NHHMH), and HC-CDR2 is HC-CDR3 includes TIFF2026123079000098.tif5163 The isolated antibody or antigen-binding fragment of paragraph 6(a), comprising TIFF2026123079000099.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 contains SEQ ID NO:14(NYWMH), and HC-CDR2 contains HC-CDR3 includes TIFF2026123079000100.tif5163 The isolated antibody or antigen-binding fragment of paragraph 6(a), comprising TIFF2026123079000101.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 contains SEQ ID NO: 56 (NYHMH), and HC-CDR2 contains HC-CDR3 includes TIFF2026123079000102.tif5164 The isolated antibody or antigen-conjugated fragment of paragraph 6(a), comprising TIFF2026123079000103.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 contains SEQ ID NO: 57 (NHHMH), and HC-CDR2 contains HC-CDR3 includes TIFF2026123079000104.tif4163 The isolated antibody or antigen-binding fragment of paragraph 6(a), comprising TIFF2026123079000105.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 contains SEQ ID NO:14(NYWM) and HC-CDR2 contains HC-CDR3 includes TIFF2026123079000106.tif5128 The isolated antibody or antigen-binding fragment of paragraph 6(a), comprising TIFF2026123079000107.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 contains SEQ ID NO:14(NYWM), and HC-CDR2 contains HC-CDR3 includes TIFF2026123079000108.tif4128 The isolated antibody or antigen-binding fragment of paragraph 6(a), comprising TIFF2026123079000109.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 contains SEQ ID NO: 56 (NYHMH), and HC-CDR2 contains HC-CDR3 includes TIFF2026123079000110.tif4128 The isolated antibody or antigen-binding fragment of paragraph 6(a), comprising TIFF2026123079000111.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 contains SEQ ID NO: 57 (NHHMH), and HC-CDR2 contains HC-CDR3 includes TIFF2026123079000112.tif4128 The isolated antibody or antigen-binding fragment of paragraph 6(a), comprising TIFF2026123079000113.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 contains SEQ ID NO:14(NYWM), and HC-CDR2 contains HC-CDR3 includes TIFF2026123079000114.tif4128 The isolated antibody or antigen-binding fragment of paragraph 6(a), comprising TIFF2026123079000115.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. An isolated antibody or antigen-binding fragment from paragraph 6(a), comprising a heavy chain having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with 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 having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with SEQ ID NO:10 or SEQ ID NO:47. 25. An isolated antibody or antigen-binding fragment from paragraph 6(a), comprising a heavy chain having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:49, or SEQ ID NO:50, and a light chain having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with SEQ ID NO:47. 26. An isolated antibody or antigen-binding fragment from paragraph 6(a), comprising a heavy chain having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with SEQ ID NO:8, and a light chain having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with SEQ ID NO:11. 27. An isolated antibody or antigen-binding fragment from paragraph 6(b), comprising a heavy chain having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with SEQ ID NO:9, and a light chain having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with SEQ ID NO:12. 28. One isolated antibody or antigen-binding fragment thereof, selected from the group consisting of human antibodies, humanized antibodies, chimeric antibodies, mouse antibodies, and any of the antigen-binding fragments described above, as specified in paragraphs 1 to 27. 29. One isolated antibody or antigen-binding fragment thereof, selected from the group consisting of single-chain antibodies, ScFv, Fab fragments, Fab' fragments, F(ab')2 fragments, monovalent antibodies lacking a hinge region, and complete antibodies, as described in paragraphs 1 to 27. 30. One isolated antibody or antigen-binding fragment from paragraphs 1-27, further comprising an immunoglobulin constant region. 31. A humanized isolated antibody or its antigen-binding fragment from any of paragraphs 1-27. 32. An isolated antibody or its antigen-binding fragment, selected from any of paragraphs 1-27, which is an IgG immunoglobulin selected from the group consisting of IgG1, IgG2, and IgG4. 33. An isolated antibody or antigen-binding fragment from any of paragraphs 1-27, containing one or more mutations in the Fc region. 34. The isolated antibody or antigen-binding fragment of paragraph 33, wherein the Fc region contains the S228P amino acid substitution. 35. One isolated antibody or antigen-binding fragment from paragraphs 1-34 that binds to the serine protease domain of human MASP-2 with an affinity of less than 20 nM. 36. The isolated antibody or antigen-binding fragment of paragraph 35 that binds to the serine protease domain of human MASP-2 with an affinity of less than 10 nM. 37. One of the isolated antibodies or antigen-binding fragments from paragraphs 1-34 that inhibit the lectin pathway in mammalian blood. 38. The lectin pathway inhibition comprises a reduction in C3b adhesion under lectin pathway-specific assay conditions, wherein the isolated antibody or antigen-binding fragment of paragraph 37. 39. The lectin pathway inhibition comprises a reduction in C4 adhesion under lectin pathway-specific assay conditions, wherein the isolated antibody or antigen-binding fragment of paragraph 37. 40. The lectin pathway inhibition includes a reduction in MAC adhesion under lectin pathway-specific assay conditions, wherein the isolated antibody or its antigen-binding fragment from paragraph 37. 41. One isolated antibody or antigen-binding fragment from paragraphs 1-34 that does not inhibit the classical pathway in mammalian blood. 42. A composition comprising one antibody or antigen-binding fragment from any of paragraphs 1 to 34 and a pharmaceutically acceptable excipient. 43. The composition of paragraph 42, formulated for subcutaneous administration. 44. Isolated polynucleotides encoding the heavy chain variable region and light chain variable region of any one antibody or antigen-binding fragment from paragraphs 1-34. 45. A combination of an isolated polynucleotide encoding the heavy chain variable region of one antibody or its antigen-binding fragment from paragraphs 1 to 34, and an isolated polynucleotide encoding the light chain variable region of one antibody or its antigen-binding fragment from paragraphs 1 to 34. 46. ​​A cloning vector or expression vector containing the polynucleotides of paragraph 44 or a combination of polynucleotides of paragraph 45. 47. A combination of a cloning vector or expression vector containing an isolated polynucleotide encoding the heavy chain variable region of one antibody or its antigen-binding fragment from paragraphs 1 to 34, and a cloning vector or expression vector containing an isolated polynucleotide encoding the light chain variable region of one antibody or its antigen-binding fragment from paragraphs 1 to 34. 48. A host cell containing one or more cloning or expression vectors according to paragraph 46 or paragraph 47. 49. A process for producing one antibody or antigen-binding fragment from any of paragraphs 1 to 34, comprising the steps of culturing the host cells of paragraph 48 and isolating the antibody or its antigen-binding fragment. 50. A method for inhibiting lectin pathway complement activation in a mammal, comprising the step of administering to a mammal subject in need of such inhibition a certain amount of the composition of paragraph 42, which comprises a high-affinity MASP-2 inhibitory antibody or an 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 has or is at risk of developing a disease or disorder of the lectin pathway selected from the group consisting of thrombotic microangiopathy (TMA), kidney disease, inflammatory response resulting from tissue or organ transplantation, ischemia-reperfusion injury, complications associated with diabetes, cardiovascular disease or disorder, inflammatory gastrointestinal disorder, pulmonary disorder, ophthalmic disease or disorder, disseminated intravascular coagulation, graft-versus-host disease, veno-occlusive disease, and diffuse alveolar hemorrhage.

Claims

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

2. An isolated antibody or its antigen-binding fragment according to claim 1, which competes with C4 binding to MASP-2.

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

4. An isolated antibody or its antigen-binding fragment according to claim 1, which forms a hydrogen bond with human MASP-2 His508.

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

6. (a) HC-CDR1 having the sequence NXXMH, where X at position 2 is H or Y, and X at position 3 is H or W, This is shown as follows, 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, HC-CDR2, HC-CDR3 and Heavy chain variable region including, (b) Indicated as SEQ ID NO: 64 (SASSSVXYMY), where X at position 7 is R or S, LC-CDR1, LC-CDR2, indicated as SEQ ID NO:34 (DTSNLAS), LC-CDR3, indicated as SEQ ID NO:36 (QQWSSYPLT) Light chain variable region including Includes, or (b) HC-CDR1, indicated as SEQ ID NO:25 (SYWMH), HC-CDR2, which is shown as, HC-CDR3, indicated as SEQ ID NO:29 (WAYDAMDY) Heavy chain variable region including, LC-CDR1, which is shown as, LC-CDR2, indicated as SEQ ID NO:43 (FASNLES), LC-CDR3, indicated as SEQ ID NO:45 (QQSNEDPLT) including, The isolated antibody or its antigen-binding fragment according to claim 1.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

24. An isolated antibody or antigen-binding fragment according to claim 6(a), comprising a heavy chain having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with 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 having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with SEQ ID NO:10 or SEQ ID NO:

47.

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

47.

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

11.

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

12.

28. An isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 27, selected from the group consisting of human antibodies, humanized antibodies, chimeric antibodies, mouse antibodies, and any of the aforementioned antigen-binding fragments.

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

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

31. A humanized isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 27.

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

33. An 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. The isolated antibody or antigen-binding fragment according to claim 33, wherein the Fc region contains an S228P amino acid substitution.

35. An isolated antibody or its antigen-binding fragment 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 its antigen-binding fragment according to claim 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 claims 1 to 34, which inhibits the lectin pathway in the blood of a mammal.

38. The isolated antibody or its antigen-binding fragment according to claim 37, wherein the lectin pathway inhibition includes a reduction in C3b adhesion under lectin pathway-specific assay conditions.

39. The isolated antibody or its antigen-binding fragment according to claim 37, wherein the lectin pathway inhibition includes a reduction in C4 adhesion under lectin pathway-specific assay conditions.

40. The isolated antibody or its antigen-binding fragment according to claim 37, wherein the lectin pathway inhibition includes a reduction in MAC adhesion under lectin pathway-specific assay conditions.

41. An isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 34, which does not inhibit the classical pathway in the blood of a mammal.

42. A composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 34 and a pharmaceutically acceptable excipient.

43. The composition according to claim 42, formulated for subcutaneous administration.

44. Isolated polynucleotides encoding the heavy chain variable region and the light chain variable region of an antibody or antigen-binding fragment according to any one of claims 1 to 34.

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

46. A cloning vector or expression vector comprising the polynucleotide described in claim 44 or the combination of polynucleotides described in claim 45.

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

48. A host cell comprising one or more cloning vectors or expression vectors according to claim 46 or claim 47.

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

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

51. The method according to claim 50, wherein the subject requiring such treatment 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 response resulting from tissue or organ transplantation, ischemia-reperfusion injury, complications associated with diabetes, cardiovascular disease or disorder, inflammatory gastrointestinal disorder, lung disorder, ophthalmic disease or disorder, disseminated intravascular coagulation, graft-versus-host disease, veno-occlusive disease, and diffuse alveolar hemorrhage.

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