Development and application of a complement inhibitor

Abstract

The present disclosure relates to the development and application of a complement inhibitor, which is an antibody or antigen-binding portion thereof that binds to MASP-2. The disclosed antibody binds to MASP-2 with high affinity and specifically recognizes the enzymatically active domains CCP1-CCP2-SP of human, monkey, mouse, and rat MASP-2. It effectively inhibits the production of complement components C4b and C3 convertase C4b2b at low concentrations, with blocking activity 50-200 times higher than that of known antibodies. Furthermore, the MASP-2 antibody is well tolerated, exhibits no toxicity, and exhibits superior efficacy and safety compared to similar antibodies.

Description

[0001] This application is a divisional application. The application date of the original application is December 16, 2020, the application number is 2020114857928, and the name of the invention is "Development and Application of a Complement Inhibitor". Technical Field

[0002] The present disclosure relates to a complement inhibitor and its use, in particular to a MASP-2 inhibitory antibody and its use. Background Art

[0003] Mannose-binding lectin-associated serine protease 2 (MASP-2) belongs to the serine protease family and is a key enzyme in the complement lectin activation pathway. MASP-2 contains six domains: the N-terminal CUB1, EGF, CUB2, CCP1, CCP2, and the C-terminal SP enzymatic domain, with a molecular weight of approximately 75 kDa. MASP-2 in the blood binds to the lectin pathway recognition molecules mannose-binding lectin (MBL), collectin 11 (CL-K1), and ficolins (ficolin-1, ficolin-2, and ficolin-3) through the three N-terminal domains CUB1-EGF-CUB2 to form a complex. When MBL, CL-K1, or ficolin binds to pathogen-associated molecular patterns (PAMPs) such as D-mannose, N-acetyl-D-glucosamine, or acetyl groups, MASP-1 and MASP-2 are sequentially activated. Activated MASP-2 cleaves C4 and C2. The resulting fragment, C4b, interacts with C2b to produce C3 convertase (C4b2b). C3 convertase (C4b2b) activates C3, leading to the production of C5 convertase (C4b2b3b) and the formation of the membrane attack complex (C5b-9), which can cause microbial lysis. The activated forms of C3 and C4 (C3b and C4b) are covalently deposited on foreign target surfaces. These are recognized by complement receptors on various phagocytes, leading to the phagocytosis of the corresponding pathogens. The lectin pathway plays a crucial role in the body's innate immunity against pathogens such as bacteria, yeast, and viruses.

[0004] Under certain pathological conditions, when cells rupture after damage, incompletely glycosylated glycoproteins (containing high-mannose "precursor" glycan clusters) are released. These are recognized with high affinity by MBL, initiating complement activation (Maynard et al., J. Biol. Chem. 257:3788-3794, 1982). Similarly, immune complexes formed by IgA antibodies with defective galactose modification are deposited in the lining of glomerular blood vessels and other sites, activating the complement lectin pathway through MBL-MASP-2, triggering an inflammatory response. Granzymes released by overactivated inflammatory cells can damage nearby tissues and organs. Furthermore, activated complement components deposited on nearby cells can lead to the formation of complement membrane attack complexes and cell lysis. The complement system is implicated in the pathogenesis of numerous acute and chronic diseases, including IgA nephropathy (IgAN), ischemia-reperfusion injury, transplant rejection, rheumatoid arthritis, myocardial infarction, post-stroke vascular remodeling, ARDS, septic shock, capillary leakage after thermal burns, inflammation after cardiopulmonary bypass, multiple sclerosis, myasthenia gravis, and Alzheimer's disease. Recent studies have shown that the novel coronavirus (COVID-19) infection-induced damage to various tissues, including the lungs and kidneys, is associated with the complement lectin pathway (Magro C et al. Transl Res. 2020 Jun;220:1-13), and that inhibiting MASP-2 activity can significantly reduce the mortality rate of severe COVID-19 patients (Rambaldi A et al. Immunobiology. 2020 Aug 9:15-2001).

[0005] MASP-2 is the only protease in the lectin pathway that can cleave C4. Its concentration in healthy human blood is much lower than that of MASP-1 and other complement components in the lectin pathway. Kidmose R et al. (PNAS September 18, 2012 109(38)15425-15430) showed that MASP-2 binds to C4 through its CCP2 domain, causing conformational changes and exposing the cleavage site. Mutations in the CCP2 domain can completely inhibit the activity of MASP-2. Summary of the Invention

[0006] The present disclosure relates to anti-MASP-2 inhibitory antibodies for inhibiting the adverse effects of MASP-2-dependent complement activation.

[0007] In one aspect, the disclosure provides an antibody, or antigen-binding portion thereof, that binds MASP-2.

[0008] In one aspect, the disclosure provides a bispecific antibody, or an antigen-binding portion thereof.

[0009] In one aspect, the disclosure provides nucleic acids encoding the aforementioned antibodies, or antigen-binding portions thereof, or bispecific antibodies, or antigen-binding portions thereof, that bind MASP-2.

[0010] In one aspect, the present disclosure provides a vector comprising the aforementioned nucleic acid.

[0011] In one aspect, the present disclosure provides a cell comprising the aforementioned vector.

[0012] In one aspect, the present disclosure provides an antibody or antigen-binding portion thereof, encoding nucleic acid, vector, cell, composition or kit comprising the aforementioned.

[0013] In one aspect, the present disclosure provides a method of treating a disease associated with MASP-2-dependent complement activation, comprising the step of administering to the subject a therapeutically effective amount of the antibody or antigen-binding fragment thereof, nucleic acid, vector, cell and / or composition of any of the aforementioned aspects.

[0014] In one aspect, the present disclosure provides use of an antibody or antigen-binding fragment thereof, nucleic acid, vector, cell and / or pharmaceutical composition of any of the foregoing aspects in the preparation of a medicament or kit for treating a disease associated with MASP-2-dependent complement activation in a subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Shown are the SDS-PAGE results of MASP-2D4-6 recombinant protein.

[0016] Figure 2 Inhibition of C4b deposition by hybridoma supernatants is shown.

[0017] Figure 3 Inhibition of C4b deposition by hybridoma clone chimeric antibodies is shown.

[0018] Figure 4 Inhibition of C3b deposition by hybridoma clone chimeric antibodies is shown.

[0019] Figure 5 ELISA testing of the binding of candidate chimeric antibodies to MASP-2D4-6 of different species is shown.

[0020] Figure 6 Inhibition of C4b deposition by phage display library Fab candidate clones is shown.

[0021] Figure 7 Shown is the inhibition of C4b deposition by purified Fab candidate clones from the phage display library.

[0022] Figure 8Figure 2 shows the inhibition of C4b deposition by mutations in the 28O14 clone. Ser102 represents the wild-type antibody, in which position 102 is serine. Gly102, Tyr102, Ala102, Glu102, Gln102, Val102, Leu102, and His102 represent mutant antibodies, in which position 102 is mutated from serine to glycine, tyrosine, alanine, glutamic acid, glutamine, valine, leucine, and histidine, respectively.

[0023] Figure 9A , B shows inhibition of C4b deposition by humanized MASP-2 antibodies.

[0024] Figure 10A , B, C show inhibition of C3b deposition by humanized MASP-2 antibodies.

[0025] Figure 11 ELISA testing of binding of humanized MASP-2 antibodies to antigens from different species is shown.

[0026] Figure 12 The efficacy of humanized MASP-2 antibodies in a mouse hemolytic uremic syndrome model is shown. DETAILED DESCRIPTION

[0027] I. Definition

[0028] In this disclosure, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are those widely used in the respective fields and are common procedures. To facilitate a better understanding of this disclosure, definitions and explanations of relevant terms are provided below.

[0029] Disclosed herein are antibodies (e.g., monoclonal antibodies) that specifically bind to MASP-2, and antigen-binding fragments thereof. In specific aspects, provided herein are monoclonal anti-MASP-2 antibodies that specifically bind to MASP-2, wherein the anti-MASP-2 antibodies comprise variants of a parent antibody. In specific aspects, provided herein are antibodies that specifically bind to MASP-2 (e.g., human MASP-2). In particular aspects, provided herein are anti-MASP-2 antibodies that comprise modifications in one or more amino acid residues (e.g., 5-13 amino acid substitutions in the framework regions of the heavy chain variable region) that retain affinity for the antigen compared to the parent antibody without the modification. The term "MASP-2" refers to any MASP-2 molecule known to those skilled in the art. For example, the MASP-2 can be from a mammal, such as a human.

[0030] As used herein, the term "MASP-2-dependent complement activation" includes MASP-2-dependent activation of the lectin pathway, which occurs under physiological conditions (i.e., in the presence of Ca 2+ ), leading to the formation of the lectin pathway C3 convertase C4b2a, and following the accumulation of the C3 cleavage product C3b, leading to the C5 convertase C42a(C3b)n.

[0031] As used herein, the term "MASP-2 inhibitory antibody" refers to any anti-MASP-2 antibody, or MASP-2 binding fragment thereof, that binds to or directly interacts with MASP-2 and effectively inhibits MASP-2-dependent complement activation. The MASP-2 inhibitory antibodies used in the disclosed methods can reduce MASP-2-dependent complement activation by greater than 20%, for example, greater than 30%, or greater than 40%, or greater than 50%, or greater than 60%, or greater than 70%, or greater than 80%, or greater than 90%, or greater than 95%.

[0032] The term "lectin pathway" refers to complement activation through the specific binding of serum and non-serum carbohydrate-binding proteins, including mannan-binding lectin (MBL) and ficolins.

[0033] As used herein and unless otherwise indicated, the term "about" or "approximately" means within plus or minus 10% of a given value or range. Where an integer is required, the term means within plus or minus 10% of a given value or range, rounded up or down to the nearest integer.

[0034] With respect to antibody chain polypeptide sequences, the phrase "substantially identical" is understood to mean antibody chains that exhibit at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference polypeptide sequence. With respect to nucleic acid sequences, the phrase is understood to mean nucleotide sequences that exhibit at least greater than 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference nucleic acid sequence.

[0035] Sequence "identity" or "identity" has a meaning recognized in the art, and the percentage of sequence identity between two nucleic acid or polypeptide molecules or regions can be calculated using published techniques. Sequence identity can be measured along the entire length of a polynucleotide or polypeptide or along a region of the molecule (see, for example: Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). While there are many methods to measure the identity between two polynucleotides or polypeptides, the term "identity" is well known to those of skill in the art (Carrillo, H. & Lipman, D., SIAM J Applied Math 48: 1073 (1988)).

[0036] "Substitution" variants are variants in which at least one amino acid residue in the native sequence has been removed and a different amino acid has been inserted in the same position. The substitutions can be single, where only one amino acid is substituted in the molecule, or multiple, where two or more amino acids are substituted in the same molecule. Multiple substitutions can be at consecutive sites. Likewise, a single amino acid can be substituted by multiple residues, where such variants include both substitutions and insertions. "Insertion" variants are variants in which one or more amino acids are inserted adjacent to an amino acid at a specific position in a native sequence. Adjacent to an amino acid means linked to the α-carboxyl or α-amino functional group of that amino acid. "Deletion" variants are variants in which one or more amino acids in the native amino acid sequence have been removed. Typically, deletion variants have one or two amino acids deleted in a specific region of the molecule.

[0037] With respect to the variable domains of antibodies, the term "variable" refers to those parts of the related molecule that have extensive sequence differences between antibodies and are used for specific recognition and binding of a particular antibody to its specific target. However, the variability is not evenly distributed throughout the variable domain of an antibody. The variability is concentrated in three segments called complementarity determining regions (CDRs; i.e., CDR1, CDR2, and CDR3) or hypervariable regions, which are located in the variable domains of both the light and heavy chains. The more highly conserved portions of the variable domains are called framework (FR) regions or framework sequences. Each variable domain of a native heavy and light chain includes four FR regions, which primarily adopt a β-sheet configuration, connected by three CDRs, which form loops that connect and, in some cases, form part of the β-sheet structure. The CDRs of each chain are typically linked adjacently by the FR regions and, with the help of the CDRs from the other chain, contribute to the formation of the antibody target binding site (epitope or determinant) (see Kabat et al., Sequences of Proteins of Immunological Interest, National Institute of Health, Bethesda, MD (1987)). As used herein, the numbering of immunoglobulin amino acid residues is based on the immunoglobulin amino acid residue numbering system of Kabat et al., unless otherwise indicated. A CDR may have the ability to specifically bind to a cognate epitope.

[0038] As used herein, an "antibody fragment" or "antigen-binding fragment" of an antibody refers to any portion of a full-length antibody that is less than full-length but contains at least a portion of the variable region of the antibody that binds to an antigen (e.g., one or more CDRs and / or one or more antibody binding sites) and thus retains binding specificity and at least part of the specific binding ability of the full-length antibody. Thus, an antigen-binding fragment refers to an antibody fragment that contains an antigen-binding portion that binds to the same antigen as the antibody from which the antibody fragment was derived. Antibody fragments include antibody derivatives produced by enzymatic treatment of full-length antibodies, as well as synthetically produced derivatives, such as recombinantly produced derivatives. Antibodies include antibody fragments. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, single-chain Fv (scFv), Fv, dsFv, diabodies, Fd and Fd' fragments, and other fragments, including modified fragments (see, e.g., Methods in Molecular Biology, Vol 207: Recombinant Antibodies for Cancer Therapy Methods and Protocols (2003); Chapter 1; p 3-25, Kipriyanov). The fragments may comprise multiple chains linked together, for example, by disulfide bonds and / or by peptide linkers. Antibody fragments generally contain at least or about 50 amino acids, and typically at least or about 200 amino acids. Antigen-binding fragments include any antibody fragment that, when inserted into an antibody framework (e.g., by replacing the corresponding region), obtains immunospecific binding (i.e., exhibits at least or at least about 10 7 -10 8 "Functional fragments" or "analogs of anti-MASP-2 antibodies" are fragments or analogs that prevent or substantially reduce the ability of the receptor to bind to its ligand or initiate signal transduction. As used herein, functional fragments are generally synonymous with "antibody fragments" and, with respect to antibodies, may refer to fragments that prevent or substantially reduce the ability of the receptor to bind to its ligand or initiate signal transduction, such as Fv, Fab, F(ab')2, etc. "Fv" fragments are dimers (V) formed by non-covalent association of the variable domains of one heavy chain and one light chain. H -V L In this configuration, the three CDRs of each variable domain interact to determine the V H -V L The target binding site on the surface of the dimer is the same as that of an intact antibody. The six CDRs collectively confer target binding specificity to the intact antibody. However, even a single variable domain (or half of an Fv comprising only three target-specific CDRs) can still have the ability to recognize and bind to a target.

[0039] As used herein, the term "bispecific" (Bispecific antibody, BsAb) refers to an antibody and / or antigen-binding molecule that can specifically bind to two different antigenic determinants. Typically, a bispecific antibody and / or antigen-binding molecule comprises two antigen-binding sites, each of which is specific for a different antigenic determinant. In certain embodiments, the bispecific antibody and / or antigen-binding molecule can simultaneously bind to two antigenic determinants, particularly two antigenic determinants expressed on two different cells.

[0040] As used herein, "monoclonal antibody" refers to a population of identical antibodies, meaning that each individual antibody molecule in the monoclonal antibody population is identical to other antibody molecules. This characteristic is in contrast to the characteristic of a polyclonal population of antibodies, which comprises antibodies with a variety of different sequences. Monoclonal antibodies can be prepared by many well-known methods (Smith et al. (2004) J. Clin. Pathol. 57, 912-917; and Nelson et al., J Clin Pathol (2000), 53, 111-117). For example, monoclonal antibodies can be prepared by immortalized B cells, for example, by fusion with myeloma cells to produce hybridoma cell lines or by infecting B cells with viruses such as EBV. Recombinant technology can also be used to prepare antibodies from a clonal population of host cells in vitro by transforming host cells with a plasmid carrying an artificial sequence of nucleotides encoding the antibody.

[0041] As used herein, the term "hybridoma" or "hybridoma cell" refers to a cell or cell line (usually a myeloma or lymphoma cell) produced by the fusion of an antibody-producing lymphocyte and a non-antibody-producing cancer cell. As known to those of ordinary skill in the art, hybridomas can proliferate and continuously produce a specific monoclonal antibody. Methods for producing hybridomas are known in the art (see, for example, Harlow & Lane, 1988). When referring to the term "hybridoma" or "hybridoma cell," it also includes subclones and progeny cells of the hybridoma.

[0042] As used herein, a full-length antibody is an antibody having two full-length heavy chains (e.g., VH-CH1-CH2-CH3 or VH-CH1-CH2-CH3-CH4) and two full-length light chains (VL-CL) and a hinge region, such as antibodies naturally produced by antibody-secreting B cells and antibodies with the same domains produced synthetically.

[0043] The term "chimeric antibody" refers to antibodies in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as antibodies in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody.

[0044] "Humanized" antibodies refer to non-human (e.g., mouse) antibody forms that are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin. Preferably, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementarity determining region (CDR) of the recipient antibody are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity.

[0045] In addition, in humanization, it is also possible to mutate the amino acid residues in the CDR1, CDR2 and / or CDR3 regions of VH and / or VL to thereby improve one or more binding properties (e.g., affinity) of the antibody. For example, PCR-mediated mutations can be used to introduce mutations, and their effects on antibody binding or other functional properties can be evaluated using in vitro or in vivo tests as described herein. Typically, conservative mutations are introduced. Such mutations can be amino acid substitutions, additions, or deletions. In addition, the mutations in the CDRs are typically no more than one or two. Therefore, the humanized antibodies described herein also encompass antibodies comprising one or two amino acid mutations in the CDRs.

[0046] As used herein, the term "CDR" refers to a complementarity-determining region, and each heavy chain and light chain of a known antibody molecule has three CDRs. CDRs are also referred to as hypervariable regions and are present in the variable regions of each heavy and light chain of an antibody, with very high variability sites in the primary structure of the CDRs. In this specification, the CDRs of the heavy chain are represented by CDR1, CDR2, and CDR3 at the amino terminus of the amino terminal sequence of the heavy chain, and the CDRs of the light chain are represented by CDR1, CDR2, and CDR3 at the amino terminus of the amino terminal sequence of the light chain. These sites are adjacent to each other in the tertiary structure and determine the specificity of the antigen to which the antibody binds.

[0047] As used herein, the term "epitope" refers to any antigenic determinant on an antigen to which the paratope of an antibody binds. Epitopic determinants typically comprise chemically active surface patterns of molecules, such as amino acids or sugar side chains, and typically have specific three-dimensional structural characteristics as well as specific charge characteristics.

[0048] As used herein, "specifically binds" or "immunospecifically binds" with respect to an antibody or antigen-binding fragment thereof are used interchangeably herein and refer to the ability of an antibody or antigen-binding fragment to form one or more non-covalent bonds with a cognate antigen through non-covalent interactions between the antibody combining sites of the antibody and the antigen. The antigen may be an isolated antigen or present on a tumor cell. Typically, an antibody that immunospecifically binds (or specifically binds) to an antigen is present at a level of about 1 × 10 7 M -1 or 1x10 8 M -1 or greater affinity constant Ka (or 1x10 -7 M or 1×10 -8 The affinity constant can be determined by standard kinetic methods of antibody reactions, such as immunoassays, surface plasmon resonance (SPR) (Rich and Myszka (2000) Curr. Opin. Biotechnol 11:54; Englebienne (1998) Analyst. 123:1599), isothermal titration calorimetry (ITC), or other kinetic interaction assays known in the art (see, e.g., Paul, ed., Fundamental Immunology, 2nd ed., Raven Press, New York, pages 332-336 (1989); see also U.S. Pat. No. 7,229,619 for a description of exemplary SPR and ITC methods for calculating the binding affinity of an antibody). Instruments and methods for real-time detection and monitoring of binding rates are known and commercially available (see, BiaCore 2000, Biacore AB, Upsala, Sweden and GE Healthcare Life Sciences; Malmqvist (2000) Biochem. Soc. Trans. 27:335).

[0049] As used herein, the terms "polynucleotide" and "nucleic acid molecule" refer to an oligomer or polymer comprising at least two linked nucleotides or nucleotide derivatives, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), typically linked together by a phosphodiester bond. As used herein, the term "nucleic acid molecule" is intended to include DNA molecules and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded and can be cDNA.

[0050] As used herein, isolated nucleic acid molecules are nucleic acid molecules isolated from other nucleic acid molecules present in the natural origin of nucleic acid molecules. " Isolated " nucleic acid molecules such as cDNA molecules can be substantially free of other cellular materials or culture medium when prepared by recombinant technology, or substantially free of chemical precursors or other chemical compositions when chemosynthesis. The exemplary isolated nucleic acid molecules provided herein comprise the isolated nucleic acid molecules of the antibody or Fab provided by coding.

[0051] As used herein, "operably linked" with respect to nucleic acid sequences, regions, elements, or domains means that the nucleic acid regions are functionally related to each other. For example, a promoter can be operably linked to a nucleic acid encoding a polypeptide such that the promoter regulates or mediates transcription of the nucleic acid.

[0052] Also provided are "conservative sequence modifications" of the sequences described in the sequence listings described herein, i.e., nucleotide and amino acid sequence modifications that do not eliminate binding of the antibody encoded by the nucleotide sequence or containing the amino acid sequence to the antigen. These conservative sequence modifications include conservative nucleotide and amino acid substitutions and nucleotide and amino acid additions and deletions. For example, modifications can be introduced into the sequence listings described herein by standard techniques known in the art (e.g., site-directed mutagenesis and PCR-mediated mutagenesis). Conservative sequence modifications include conservative amino acid substitutions, in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains are defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with beta-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino acid residue in an anti-MASP-2 antibody is preferably replaced with another amino acid residue from the same side chain family. Methods for identifying conservative nucleotide and amino acid substitutions that do not eliminate antigen binding are well known in the art (e.g., see Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10): 879-884 (1999); Burks et al., Proc. Natl. Acad. Sci. USA 94: 412-417 (1997)).

[0053] Alternatively, in another embodiment, mutations can be introduced randomly along all or a portion of an anti-MASP-2 antibody coding sequence by, for example, saturation mutagenesis, and the resultant modified anti-MASP-2 antibodies can be screened for improved binding activity.

[0054] As used herein, "expression" refers to the process of producing a polypeptide through transcription and translation of a polynucleotide. The expression level of a polypeptide can be assessed using any method known in the art, including, for example, methods for measuring the amount of polypeptide produced by a host cell. Such methods may include, but are not limited to, quantification of polypeptides in cell lysates by ELISA, gel electrophoresis followed by Coomassie blue staining, Lowry protein assay, and Bradford protein assay.

[0055] As used herein, a "host cell" is a cell that is used to receive, maintain, replicate, and amplify a vector. Host cells can also be used to express polypeptides encoded by the vector. When the host cell divides, the nucleic acid contained in the vector replicates, thereby amplifying the nucleic acid. The host cell can be a eukaryotic cell or a prokaryotic cell. Suitable host cells include, but are not limited to, CHO cells, various COS cells, HeLa cells, and HEK cells, such as HEK 293 cells.

[0056] As used herein, a "vector" is a replicable nucleic acid that, when transformed into an appropriate host cell, can express one or more heterologous proteins from the vector. Vectors include those into which nucleic acids encoding polypeptides or fragments thereof can be introduced, typically by restriction digestion and ligation. Vectors also include those that contain nucleic acids encoding polypeptides. Vectors are used to introduce nucleic acids encoding polypeptides into host cells for the purpose of amplifying nucleic acids or for expressing / displaying polypeptides encoded by nucleic acids. Vectors are typically kept episomal, but can be designed to integrate genes or portions thereof into chromosomes of the genome. Artificial chromosome vectors, such as yeast artificial vectors and mammalian artificial chromosomes, are also contemplated. The selection and use of such vectors are well known to those skilled in the art.

[0057] As used herein, vectors also include “viral vectors” or “viral vectors.” Viral vectors are engineered viruses that are operably linked to exogenous genes to transfer (as vehicles or shuttles) the exogenous genes into cells.

[0058] As used herein, "expression vector" includes a vector capable of expressing DNA that is operably linked to a regulatory sequence such as a promoter region that can affect the expression of such DNA fragments. Such additional fragments may include promoter and terminator sequences, and may optionally include one or more origins of replication, one or more selection markers, enhancers, polyadenylation signals, etc. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both. Therefore, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, phage, recombinant virus or other vector, which, when introduced into an appropriate host cell, results in the expression of the cloned DNA. Suitable expression vectors are well known to those skilled in the art and include expression vectors that are replicable in eukaryotic cells and / or prokaryotic cells, as well as expression vectors that remain episomal or that are integrated into the host cell genome.

[0059] As used herein, "treating" an individual suffering from a disease or condition means that the individual's symptoms are partially or completely alleviated, or remain unchanged after treatment. Thus, treatment includes prevention, treatment, and / or cure. Prevention refers to preventing the underlying disease and / or preventing the worsening of symptoms or progression of the disease. Treatment also includes any pharmaceutical use of any of the antibodies or antigen-binding fragments thereof provided, as well as the compositions provided herein.

[0060] As used herein, "therapeutic effect" refers to an effect resulting from treatment of a subject that alters, typically ameliorates or improves the symptoms of a disease or condition, or cures the disease or condition.

[0061] As used herein, "therapeutically effective amount" or "therapeutically effective dose" refers to an amount of a substance, compound, material, or composition comprising a compound that is at least sufficient to produce a therapeutic effect after administration to a subject. Thus, it is the amount necessary to prevent, cure, ameliorate, arrest, or partially arrest the symptoms of a disease or condition.

[0062] As used herein, a "prophylactically effective amount" or "prophylactically effective dose" refers to an amount of a substance, compound, material, or composition comprising a compound that, when administered to a subject, will have the desired prophylactic effect, e.g., preventing or delaying the onset or recurrence of a disease or symptom, or reducing the likelihood of the onset or recurrence of a disease or symptom. A fully prophylactically effective dose need not occur by administering one dose, and may occur only after administering a series of doses. Thus, a prophylactically effective amount may be administered in one or more administrations.

[0063] As used herein, the term "patient" or "subject" refers to a mammal, such as a human.

[0064] II. Detailed description of specific implementation plan

[0065] In one aspect, the present disclosure provides an antibody, or antigen-binding portion thereof, that binds to MASP-2 and comprises a heavy chain CDR selected from the group consisting of the amino acid sequences of SEQ ID NOs. 7, 8, 9, 17, 18, 26, 27, 28, 36, 37, 38, 46, 47, 60, 65, 66, 67, 79, 85, 86, 94, 95, 96, 104, 105, 113, 114, 126, 127, 128, 136, 137, 138, 141, 149, 150, 151, 165, 183, 192, or any variant thereof, and / or a heavy chain CDR selected from the group consisting of the amino acid sequences of SEQ ID NOs. 168, 173, 174, 201, 202, 207, 216, or any variant thereof.

[0066] The antibody or antigen-binding portion thereof according to the preceding aspect comprises a heavy chain CDR1 selected from the group consisting of amino acid sequences of SEQ ID NO. 7, 26, 36, 65, 94, 126, 136, 149 or any variant thereof, a heavy chain CDR2 selected from the group consisting of amino acid sequences of SEQ ID NO. 8, 17, 27, 37, 46, 66, 85, 95, 104, 113, 127, 137, 150, 165, 183, 192 or any variant thereof, a heavy chain CDR3 selected from the group consisting of amino acid sequences of SEQ ID NO. 9, 18, 28, 38, 47, 60, 67, 79, 86, 96, 105, 114, 128, 138, 141, 151 or any variant thereof; and / or a heavy chain CDR3 selected from the group consisting of amino acid sequences of SEQ ID NO. NO.12, 21, 31, 41, 50, 70, 99, 108, 117, 123, 131, 144, 154, 161, 201, 216 or any variant thereof, a light chain CDR2 selected from the amino acid sequence of SEQ ID NO.13, 22, 32, 42, 51, 57, 71, 100, 109, 118, 132, 145, 155, 162, 168, 173, 202, 207 or any variant thereof, and a light chain CDR3 selected from the amino acid sequence of SEQ ID NO.14, 23, 33, 43, 52, 72, 82, 91, 101, 110, 133, 146, 156, 174 or any variant thereof.

[0067] The antibody or antigen binding portion thereof according to the previous aspect comprises a combination of heavy and light chain CDRs selected from the group consisting of:

[0068] (1) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 7, 8, and 9, respectively, and / or light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 12, 13, and 14, respectively;

[0069] (2) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 7, 17, and 18, respectively, and / or light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 21, 22, and 23, respectively;

[0070] (3) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 26, 27, and 28, respectively, and / or light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 31, 32, and 33, respectively;

[0071] (4) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 36, 37, and 38, respectively, and / or light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 41, 42, and 43, respectively;

[0072] (5) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 7, 46, and 47, respectively, and / or light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 50, 51, and 52, respectively;

[0073] (6) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 7, 17, and 18, respectively, and / or light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 21, 57, and 23, respectively;

[0074] (7) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 7, 17, and 60, respectively, and / or light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 50, 51, and 52, respectively;

[0075] (8) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 65, 66, and 67, respectively, and / or light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 70, 71, and 72, respectively;

[0076] (9) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 7, 17, and 47, respectively, and / or light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 50, 51, and 52, respectively;

[0077] (10) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 7, 17, and 79, respectively, and / or light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 50, 51, and 82, respectively;

[0078] (11) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 36, 85, and 86, respectively, and / or light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 41, 42, and 43, respectively;

[0079] (12) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 36, 85, and 86, respectively, and / or light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 41, 42, and 91, respectively;

[0080] (13) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 94, 95, and 96, respectively, and / or light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 99, 100, and 101, respectively;

[0081] (14) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 7, 104, and 105, respectively, and / or light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 108, 109, and 110, respectively;

[0082] (15) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 26, 113, and 114, respectively, and / or light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 117, 118, and 33, respectively;

[0083] (16) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 94, 95, and 96, respectively, and / or light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 123, 100, and 101, respectively;

[0084] (17) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 126, 127, and 128, respectively, and / or light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 131, 132, and 133, respectively;

[0085] (18) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 136, 137, and 138, respectively, and / or light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 131, 132, and 133, respectively;

[0086] (19) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 7, 17, and 141, respectively, and / or light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 144, 145, and 146, respectively;

[0087] (20) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 149, 150, and 151, respectively, and / or light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 154, 155, and 156, respectively;

[0088] (21) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 7, 17, and 9, respectively, and / or light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 161, 162, and 14, respectively;

[0089] (22) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 7, 165, and 9, respectively, and / or light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 144, 168, and 14, respectively;

[0090] (23) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 7, 17, and 9, respectively, and / or light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 144, 173, and 174, respectively;

[0091] (24) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 136, 183, and 138, respectively, and / or light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 216, 132, and 133, respectively;

[0092] (25) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 126, 192, and 128, respectively, and / or light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 216, 132, and 133, respectively;

[0093] (26) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 7, 17, and 141, respectively, and / or light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 201, 202, and 146, respectively;

[0094] (27) The heavy chain CDR1, CDR2 and CDR3 sequences comprised of SEQ ID NOs: 7, 17 and 141, respectively, and / or the light chain CDR1, CDR2 and CDR3 sequences comprised of SEQ ID NOs: 201, 207 and 146, respectively.

[0095] The antibody or antigen-binding portion thereof according to the preceding aspect comprises a heavy chain variable region selected from the group consisting of the amino acid sequences of SEQ ID NO. 5, 15, 24, 34, 44, 53, 58, 63, 73, 77, 83, 92, 102, 111, 119, 124, 134, 139, 147, 157, 163, 169, 175, 177, 179, 181, 184, 186, 188, 190, 193, 195, 197 or any variant thereof, and / or a heavy chain variable region selected from the group consisting of the amino acid sequences of SEQ ID NO. 10, 19, 29, 39, 48, 55, 61, 68, 75, 80, 87, 89, 97, 106, 115, 121, 129, 142, 152, 159, 166, 171, 199, 203, 205, 208, 210, 212, 214, or any variant thereof;

[0096] In some preferred embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO.5 or any variant thereof, and a light chain variable region having an amino acid sequence of SEQ ID NO.10 or any variant thereof;

[0097] In some preferred embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO. 15 or any variant thereof, and a light chain variable region having an amino acid sequence of SEQ ID NO. 19 or any variant thereof;

[0098] In some preferred embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO. 24 or any variant thereof, and a light chain variable region having an amino acid sequence of SEQ ID NO. 29 or any variant thereof;

[0099] In some preferred embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO. 34 or any variant thereof, and a light chain variable region having an amino acid sequence of SEQ ID NO. 39 or any variant thereof;

[0100] In some preferred embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO. 44 or any variant thereof, and a light chain variable region having an amino acid sequence of SEQ ID NO. 48 or any variant thereof;

[0101] In some preferred embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO.53 or any variant thereof, and a light chain variable region having an amino acid sequence of SEQ ID NO.55 or any variant thereof;

[0102] In some preferred embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO.58 or any variant thereof, and a light chain variable region having an amino acid sequence of SEQ ID NO.61 or any variant thereof;

[0103] In some preferred embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO. 63 or any variant thereof, and a light chain variable region having an amino acid sequence of SEQ ID NO. 68 or any variant thereof;

[0104] In some preferred embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO. 73 or any variant thereof, and a light chain variable region having an amino acid sequence of SEQ ID NO. 75 or any variant thereof;

[0105] In some preferred embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO. 77 or any variant thereof, and a light chain variable region having an amino acid sequence of SEQ ID NO. 80 or any variant thereof;

[0106] In some preferred embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO.83 or any variant thereof, and a light chain variable region having an amino acid sequence of SEQ ID NO.87 or any variant thereof;

[0107] In some preferred embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO.83 or any variant thereof, and a light chain variable region having an amino acid sequence of SEQ ID NO.89 or any variant thereof;

[0108] In some preferred embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO.92 or any variant thereof, and a light chain variable region having an amino acid sequence of SEQ ID NO.97 or any variant thereof;

[0109] In some preferred embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO.102 or any variant thereof, and a light chain variable region having an amino acid sequence of SEQ ID NO.106 or any variant thereof;

[0110] In some preferred embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO.111 or any variant thereof, and a light chain variable region having an amino acid sequence of SEQ ID NO.115 or any variant thereof;

[0111] In some preferred embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO.119 or any variant thereof, and a light chain variable region having an amino acid sequence of SEQ ID NO.121 or any variant thereof;

[0112] In some preferred embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO.124 or any variant thereof, and a light chain variable region having an amino acid sequence of SEQ ID NO.129 or any variant thereof;

[0113] In some preferred embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO.134 or any variant thereof, and a light chain variable region having an amino acid sequence of SEQ ID NO.129 or any variant thereof;

[0114] In some preferred embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO.139 or any variant thereof, and a light chain variable region having an amino acid sequence of SEQ ID NO.142 or any variant thereof;

[0115] In some preferred embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain variable region of the amino acid sequence SEQ ID NO.147 or any variant thereof, and a light chain variable region of the amino acid sequence SEQ ID NO.152 or any variant thereof;

[0116] In some preferred embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO.157 or any variant thereof, and a light chain variable region having an amino acid sequence of SEQ ID NO.159 or any variant thereof;

[0117] In some preferred embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO.163 or any variant thereof, and a light chain variable region having an amino acid sequence of SEQ ID NO.166 or any variant thereof;

[0118] In some preferred embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain variable region of the amino acid sequence SEQ ID NO.169 or any variant thereof, and a light chain variable region of the amino acid sequence SEQ ID NO.171 or any variant thereof;

[0119] In some preferred embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO.181 or any variant thereof, and a light chain variable region having an amino acid sequence of SEQ ID NO.214 or any variant thereof;

[0120] In some preferred embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO.184 or any variant thereof, and a light chain variable region having an amino acid sequence of SEQ ID NO.214 or any variant thereof;

[0121] In some preferred embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO.186 or any variant thereof, and a light chain variable region having an amino acid sequence of SEQ ID NO.214 or any variant thereof;

[0122] In some preferred embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO.188 or any variant thereof, and a light chain variable region having an amino acid sequence of SEQ ID NO.214 or any variant thereof;

[0123] In some preferred embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO.190 or any variant thereof, and a light chain variable region having an amino acid sequence of SEQ ID NO.214 or any variant thereof;

[0124] In some preferred embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO.193 or any variant thereof, and a light chain variable region having an amino acid sequence of SEQ ID NO.214 or any variant thereof;

[0125] In some preferred embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO.195 or any variant thereof, and a light chain variable region having an amino acid sequence of SEQ ID NO.214 or any variant thereof;

[0126] In some preferred embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO.197 or any variant thereof, and a light chain variable region having an amino acid sequence of SEQ ID NO.214 or any variant thereof;

[0127] In some preferred embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO. 175 or any variant thereof, and a light chain variable region having an amino acid sequence of SEQ ID NO. 199 or any variant thereof;

[0128] In some preferred embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO.175 or any variant thereof, and a light chain variable region having an amino acid sequence of SEQ ID NO.203 or any variant thereof;

[0129] In some preferred embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO.175 or any variant thereof, and a light chain variable region having an amino acid sequence of SEQ ID NO.205 or any variant thereof;

[0130] In some preferred embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO.177 or any variant thereof, and a light chain variable region having an amino acid sequence of SEQ ID NO.205 or any variant thereof;

[0131] In some preferred embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO. 177 or any variant thereof, and a light chain variable region having an amino acid sequence of SEQ ID NO. 199 or any variant thereof;

[0132] In some preferred embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain variable region of the amino acid sequence SEQ ID NO.177 or any variant thereof, and a light chain variable region of the amino acid sequence SEQ ID NO.203 or any variant thereof;

[0133] In some preferred embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO. 179 or any variant thereof, and a light chain variable region having an amino acid sequence of SEQ ID NO. 199 or any variant thereof;

[0134] In some preferred embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO.179 or any variant thereof, and a light chain variable region having an amino acid sequence of SEQ ID NO.203 or any variant thereof;

[0135] In some preferred embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO.179 or any variant thereof, and a light chain variable region having an amino acid sequence of SEQ ID NO.205 or any variant thereof;

[0136] In some preferred embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO.175 or any variant thereof, and a light chain variable region having an amino acid sequence of SEQ ID NO.208 or any variant thereof;

[0137] In some preferred embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO.175 or any variant thereof, and a light chain variable region having an amino acid sequence of SEQ ID NO.210 or any variant thereof;

[0138] In some preferred embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO.175 or any variant thereof, and a light chain variable region having an amino acid sequence of SEQ ID NO.212 or any variant thereof;

[0139] In some preferred embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain variable region of the amino acid sequence SEQ ID NO.177 or any variant thereof, and a light chain variable region of the amino acid sequence SEQ ID NO.208 or any variant thereof;

[0140] In some preferred embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO.177 or any variant thereof, and a light chain variable region having an amino acid sequence of SEQ ID NO.210 or any variant thereof;

[0141] In some preferred embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO.177 or any variant thereof, and a light chain variable region having an amino acid sequence of SEQ ID NO.212 or any variant thereof;

[0142] In some preferred embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain variable region of the amino acid sequence SEQ ID NO.179 or any variant thereof, and a light chain variable region of the amino acid sequence SEQ ID NO.208 or any variant thereof;

[0143] In some preferred embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO.179 or any variant thereof, and a light chain variable region having an amino acid sequence of SEQ ID NO.210 or any variant thereof;

[0144] In some preferred embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO. 179 or any variant thereof, and a light chain variable region having an amino acid sequence of SEQ ID NO. 212 or any variant thereof.

[0145] In one aspect, the disclosure provides a bispecific or multispecific molecule comprising the antibody or antigen-binding portion thereof of any of the preceding aspects.

[0146] In some preferred embodiments of the present disclosure, the antibody or antigen-binding portion thereof is humanized.

[0147] In one aspect, the disclosure provides a nucleic acid molecule encoding an antibody, or antigen-binding portion thereof, or a bispecific or multispecific molecule according to any of the preceding aspects.

[0148] In some preferred embodiments, the nucleic acid comprises an antibody heavy chain variable region nucleic acid sequence selected from SEQ ID NO. 6, 16, 25, 35, 45, 54, 59, 64, 74, 78, 84, 93, 103, 112, 120, 125, 135, 140, 148, 158, 164, 170, 176, 178, 180, 182, 185, 187, 189, 191, 194, 196, 198, or any variant thereof, and / or an antibody heavy chain variable region nucleic acid sequence selected from SEQ ID NO. The antibody light chain variable region nucleic acid sequence of ID NO. 11, 20, 30, 40, 49, 56, 62, 69, 76, 81, 88, 90, 98, 107, 116, 122, 130, 143, 153, 160, 167, 172, 200, 204, 206, 209, 211, 213, 215 or any variant thereof;

[0149] In some preferred embodiments, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO: 6 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO: 11 or any variant thereof;

[0150] In some preferred embodiments, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO: 16 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO: 20 or any variant thereof;

[0151] In some preferred embodiments, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO: 25 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO: 30 or any variant thereof;

[0152] In some preferred embodiments, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO: 35 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO: 40 or any variant thereof;

[0153] In some preferred embodiments, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO: 45 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO: 49 or any variant thereof;

[0154] In some preferred embodiments, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO: 54 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO: 56 or any variant thereof;

[0155] In some preferred embodiments, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO: 59 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO: 62 or any variant thereof;

[0156] In some preferred embodiments, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO: 64 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO: 69 or any variant thereof;

[0157] In some preferred embodiments, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO: 74 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO: 76 or any variant thereof;

[0158] In some preferred embodiments, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO: 78 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO: 81 or any variant thereof;

[0159] In some preferred embodiments, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO: 84 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO: 88 or any variant thereof;

[0160] In some preferred embodiments, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO: 84 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO: 90 or any variant thereof;

[0161] In some preferred embodiments, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO: 93 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO: 98 or any variant thereof;

[0162] In some preferred embodiments, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO: 103 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO: 107 or any variant thereof;

[0163] In some preferred embodiments, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO: 112 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO: 116 or any variant thereof;

[0164] In some preferred embodiments, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO: 120 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO: 122 or any variant thereof;

[0165] In some preferred embodiments, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO: 125 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO: 130 or any variant thereof;

[0166] In some preferred embodiments, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO: 135 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO: 130 or any variant thereof;

[0167] In some preferred embodiments, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO: 140 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO: 143 or any variant thereof;

[0168] In some preferred embodiments, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO: 148 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO: 153 or any variant thereof;

[0169] In some preferred embodiments, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO: 158 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO: 160 or any variant thereof;

[0170] In some preferred embodiments, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO: 164 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO: 167 or any variant thereof;

[0171] In some preferred embodiments, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO: 170 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO: 172 or any variant thereof;

[0172] In some preferred embodiments, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO: 182 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO: 215 or any variant thereof;

[0173] In some preferred embodiments, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO: 185 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO: 215 or any variant thereof;

[0174] In some preferred embodiments, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO: 187 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO: 215 or any variant thereof;

[0175] In some preferred embodiments, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO: 189 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO: 215 or any variant thereof;

[0176] In some preferred embodiments, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO: 191 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO: 215 or any variant thereof;

[0177] In some preferred embodiments, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO: 194 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO: 215 or any variant thereof;

[0178] In some preferred embodiments, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO: 196 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO: 215 or any variant thereof;

[0179] In some preferred embodiments, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO: 198 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO: 215 or any variant thereof;

[0180] In some preferred embodiments, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO: 176 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO: 200 or any variant thereof;

[0181] In some preferred embodiments, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO: 176 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO: 204 or any variant thereof;

[0182] In some preferred embodiments, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO: 176 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO: 206 or any variant thereof;

[0183] In some preferred embodiments, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO: 178 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO: 206 or any variant thereof;

[0184] In some preferred embodiments, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO: 178 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO: 200 or any variant thereof;

[0185] In some preferred embodiments, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO: 178 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO: 204 or any variant thereof;

[0186] In some preferred embodiments, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO: 180 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO: 200 or any variant thereof;

[0187] In some preferred embodiments, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO: 180 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO: 204 or any variant thereof;

[0188] In some preferred embodiments, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO: 180 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO: 206 or any variant thereof;

[0189] In some preferred embodiments, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO: 176 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO: 209 or any variant thereof;

[0190] In some preferred embodiments, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO: 176 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO: 211 or any variant thereof;

[0191] In some preferred embodiments, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO: 176 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO: 213 or any variant thereof;

[0192] In some preferred embodiments, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO: 178 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO: 209 or any variant thereof;

[0193] In some preferred embodiments, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO: 178 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO: 211 or any variant thereof;

[0194] In some preferred embodiments, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO: 178 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO: 213 or any variant thereof;

[0195] In some preferred embodiments, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO: 180 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO: 209 or any variant thereof;

[0196] In some preferred embodiments, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO: 180 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO: 211 or any variant thereof;

[0197] In some preferred embodiments, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO: 180 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO: 213 or any variant thereof.

[0198] In one aspect, the disclosure provides antibodies, or antigen-binding portions thereof, that bind to MASP-2 and have at least greater than 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the antibody, or antigen-binding portion thereof, of any of the preceding aspects.

[0199] In one aspect, the present disclosure provides a nucleic acid molecule encoding an antibody or antigen-binding portion thereof according to any of the preceding aspects, or a nucleic acid molecule having at least greater than 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto.

[0200] In one aspect, the present disclosure provides a vector comprising a nucleic acid according to any of the preceding aspects.

[0201] In one aspect, the present disclosure provides a cell comprising a vector according to any of the preceding aspects.

[0202] In one aspect, the present invention discloses a composition comprising the aforementioned antibody or antigen-binding portion thereof, bispecific or multispecific molecule, nucleic acid, rights vector and / or cell.

[0203] The antibodies of the present disclosure are useful as therapeutic or diagnostic tools in a variety of diseases in which MASP-2 is detrimentally expressed or found.

[0204] Methods of treating diseases associated with MASP-2-dependent complement activation with the disclosed MASP-2 inhibitory agents include administering to the subject a therapeutically effective amount of the antibody or antigen-binding fragment thereof, nucleic acid molecule, vector, cell, or pharmaceutical composition of any of the aforementioned aspects.

[0205] MASP-2-dependent complement activation has been implicated in the pathogenesis of numerous acute and chronic disease states, including MASP-2-dependent complement-mediated vascular conditions, ischemia-reperfusion injury, atherosclerosis, inflammatory gastrointestinal disorders, pulmonary conditions, extracorporeal reperfusion procedures, skeletal muscle conditions, renal conditions, skin conditions, organ or tissue transplantation, nervous system disorders or injuries, hematological disorders, genitourinary conditions, diabetes, chemotherapy or radiation therapy, malignancies, endocrine disorders, coagulation disorders, or ophthalmic conditions. Thus, the MASP-2 inhibitory antibodies of the present disclosure may be used to treat the aforementioned diseases and conditions.

[0206] The clinical condition of a preferred embodiment of the present disclosure is a chronic inflammatory disease. The chronic inflammatory disease can be, for example, an autoimmune inflammatory condition.

[0207] Autoimmune inflammatory conditions (also referred to herein as "autoimmune disorders") can be loosely categorized into those that are primarily confined to a specific organ or tissue and those that affect the entire body. Examples of organ-specific disorders (and affected organs) include multiple sclerosis (myelin coating on nerve processes), type 1 diabetes (pancreas), Hashimoto's thyroiditis (thyroid), pernicious anemia (stomach), Addison's disease (adrenal glands), myasthenia gravis (acetylcholine receptors on neuromuscular junctions), rheumatoid arthritis (joint linings), uveitis (eyes), psoriasis (skin), Guillain-Barre syndrome (nerve cells), and Graves' disease (thyroid). Systemic autoimmune diseases include systemic lupus erythematosus, glomerulonephritis, and dermatomyositis.

[0208] In one embodiment of the present disclosure, the preferred clinical condition is selected from rheumatoid arthritis or systemic lupus erythematosus.

[0209] Other examples of autoimmune disorders include asthma, eczema, atopic dermatitis, contact dermatitis, other eczematous dermatitis, seborrheic dermatitis, rhinitis, lichen planus, pemplugus, bullous pemphigoid, epidermolysis bullosa, urticaria, angioedema, vasculitis, erythema, eosinophilia, alopecia areata, arteriosclerosis, primary biliary cirrhosis, and nephrotic syndrome. Related diseases include enteritis, such as coeliac disease, proctitis, eosinophilic gastroenteritis, mastocytosis, inflammatory bowel disease, Chrohn's disease, and ulcerative colitis, as well as food-related allergies.

[0210] In another embodiment of the present disclosure, the clinical condition is characterized by massive cell loss, for example due to programmed cell death or necrosis. Such necrosis or programmed cell death can be induced by a variety of factors.

[0211] In a preferred embodiment of the present disclosure, the clinical condition is an ischemic / reperfusion injury. Ischemia can be caused by various reasons, such as after a stroke, myocardial infarction, major surgery, or an organ transplant. Thus, the clinical condition can be an ischemic / reperfusion injury caused by, for example, a stroke, myocardial infarction, major surgery, or an organ transplant.

[0212] Thus, the clinical condition may be an ischemic / reperfusion injury that is the result of PTCA (percutaneous transluminal coronary angioplasty) or CABG (coronary artery bypass grafting). In addition, the clinical condition may be an ischemic / reperfusion injury that is the result of acute myocardial infarction or cerebral ischemia.

[0213] The dosage of a medicament comprising a MASP-2 inhibitory agent generally varies depending on the age, weight, height, sex, general condition, and medical history of the subject. For example, a MASP-2 inhibitory agent, such as a MASP-2 antibody, can be administered in a dosage range of approximately 0.010-10.0 mg / kg, preferably 0.010-1.0 mg / kg, and more preferably 0.010-0.1 mg / kg of the subject's body weight. In certain embodiments, the composition comprises a combination of an anti-MASP-2 antibody and a MASP-2 inhibitory peptide.

[0214] Compositions and methods comprising a MASP-2 inhibitory agent may optionally include one or more additional therapeutic agents that may enhance the activity of the MASP-2 inhibitory agent or provide related therapeutic functions in an additive or synergistic manner. For example, one or more MASP-2 inhibitory agents may be used in combination with one or more anti-inflammatory and / or analgesic agents. The amount and selection of additional agents may be determined to achieve the desired therapeutic outcome. Suitable anti-inflammatory and / or analgesic drugs include: 5-hydroxytryptamine receptor antagonists; 5-hydroxytryptamine receptor agonists; histamine receptor antagonists; bradykinin receptor antagonists; kallikrein inhibitors; tachykinin receptor antagonists, including neurokinin 1 and neurokinin 2 receptor subtype antagonists; calcitonin gene-related peptide (CGRP) receptor antagonists; interleukin receptor antagonists; inhibitors of enzymes active in the arachidonic acid metabolite synthesis pathway, including phospholipase inhibitors, including PLA2 isoform inhibitors and PLCγ isoform inhibitors, cyclooxygenase (COX) inhibitors (which may be COX-1, COX-2 inhibitors or non-selective COX-1 and COX-2 inhibitors), lipoxygenase inhibitors; Prostanoid receptor antagonists, including eicosanoid EP-1 and EP-4 receptor subtype antagonists and thromboxane receptor subtype antagonists; leukotriene receptor antagonists, including leukotriene B4 receptor subtype antagonists and leukotriene D4 receptor subtype antagonists; opioid receptor agonists, including μ-opioid, δ-opioid, and κ-opioid receptor subtype agonists; purine receptor agonists and antagonists, including P2x receptor antagonists and P2Y receptor agonists; adenosine triphosphate (ATP)-sensitive potassium channel openers; MAP kinase inhibitors; nicotinic acid acetylcholine inhibitors; and alpha adrenergic receptor agonists (including alpha-1, alpha-2, and non-selective alpha-1 and alpha-2 agonists).

[0215] When used to inhibit or treat restenosis, the MASP-2 inhibitory agents of the present disclosure may be co-administered in combination with one or more anti-restenotic drugs. Suitable anti-restenotic drugs include: antiplatelet agents, including thrombin inhibitors and receptor antagonists, adenosine diphosphate (ADP) receptor antagonists (also known as purinergic receptor 1 receptor antagonists), thromboxane inhibitors and receptor antagonists, and platelet membrane glycoprotein receptor antagonists; inhibitors of cell adhesion molecules, including selectin inhibitors and integrin free radicals; anti-chemotactic agents; interleukin receptor antagonists; and intracellular signal transduction inhibitors, including protein kinase C (PKC) inhibitors and protein tyrosine phosphatases, modulators of intracellular protein tyrosine kinase inhibitors, inhibitors of src homology 2 (SH2) domains, and calcium channel antagonists.

[0216] The MASP-2 inhibitory agents of the present disclosure may also be used in combination with one or more other complement inhibitors. Currently, no complement inhibitors are approved for use in humans, however, several drugs have been shown to block complement in vivo. Many of these drugs are also toxic or only partial inhibitors, limiting their use to research tools. K76COOH and nafamostat mesylate are two drugs that have shown some efficacy in transplant animal models. Studies have shown that low molecular weight heparin is also effective in modulating complement activity. We believe that these small molecule inhibitors may be useful as agents in combination with the MASP-2 inhibitory agents of the present disclosure.

[0217] Other naturally occurring complement inhibitors can be used in combination with the MASP-2 inhibitory agents of the present disclosure. Biological inhibitors of complement include soluble complement factor 1 (sCR1). This is a naturally occurring inhibitor found on the outer membrane of human cells. Other membrane inhibitors include DAF, MCP, and CD59. Recombinant forms have been assayed for anti-complement activity in vitro and in vivo. sCR1 has been shown to be effective in xenotransplantation, where the complement system (both alternative and classical) causes an overreactive rejection syndrome within minutes of perfusion of the newly transplanted organ with blood. Administration of sCR1 protects and prolongs the survival of transplanted organs, implicating the complement pathway in organ survival mechanisms.

[0218] Other complement inhibitors suitable for use in combination with the compositions of the present disclosure include, for example, the monoclonal antibody (MoAb) being developed by Alexion Pharmaceuticals, Inc. New Haven, Connecticut, and anti-properdin monoclonal antibody (MoAb).

[0219] When used to treat arthritis (e.g., osteoarthritis and rheumatoid arthritis), the MASP-2 inhibitory agents of the present disclosure may be combined with one or more chondroprotective agents, which may include one or more promoters of cartilage anabolism and / or one or more inhibitors of cartilage catabolism, including, where appropriate, simultaneous administration of both anabolic and catabolic inhibitory agents. Suitable anabolic chondroprotective agents include interleukin (IL) receptor agonists, including IL-4, IL-10, IL-13, rhIL-4, rhIL-10 and rhIL-13, and chimeric IL-4, IL-10 or IL-13; transforming growth factor beta superfamily agonists (including TGF-β, TGF-β1, TGF-β2, TGF-β3), bone morphogenic proteins including BMP-2, BMP-4, BMP-5, BMP-6, BMP-7 (OP-1) and OP-2 / BMP-8, growth differentiation factors including GDF-5, GDF-6 and GDF-7, recombinant TGF-β and BMP, and chimeric TGF-β and BMP; insulin-like growth factors, including IGF-1; and fibroblast growth factors, including bFGF. Suitable chondroprotective agents that inhibit catabolism include interleukin-1 (IL-1) receptor antagonists (IL-1ra), including soluble human IL-1 receptor (shuIL-1R), rshuIL-1R, rhIL-1ra, anti-IL1 antibodies, AF11567, and AF12198; tumor necrosis factor (TNF) receptor antagonists (TNF-α), including soluble receptors including sTNFR1 and sTNFRII, recombinant TNF soluble receptors, and chimeric TNF soluble receptors, including chimeric rhTNFR:Fc, Fc-fusion soluble receptors, and anti-TNF antibodies; cyclooxygenase- COX-2 (COX-2 specific) inhibitors; including DuP697, SC-58451, celecoxib, rofecoxib, nimesulide, diclofenac, meloxicam, piroxicam, NS-398, RS-57067, SC-57666, SC-58125, flosulide, etodolac, L-745,337, and DFU-T-614;Mitogen-activated protein kinase (MAPK) inhibitors, including inhibitors of ERKi, ERK2, SAPK1, SAPK2a, SAPK2b, SAPK2d, SAPK3, including SB 203580, SB 203580 iodine, SB202190, SB 242235, SB 220025, RWJ 67657, RWJ68354, FR 133605, L-167307, PD 98059, PD 1693 16; nuclear factor κB (NFκB) inhibitors, including caffeic acid phenethyl ester (CAPE), DM-CAPE, SN-50 peptide, hymenialdisine, and pyrrolidone dithiocarbamate; nitric oxide synthase (NOS) inhibitors, including NG-monomethyl-L-arginine, 1400W, diphenylene iodide, S-methylisothiourea, S-(aminoethyl)isothiourea, L-N6-(1-iminoethyl)lysine, 1,3-PBITU, 2-ethyl-2-thiopseudurea, aminoguanidine, Nω-nitro-L-arginine, Nω-nitro-L-arginine methyl ester; inhibitors of matrix metalloproteinases (MMPs), including MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, and MMP-1 1. Inhibitors of MMP-12, MMP-13, MMP-14, and MMP-15, including U-24522, minocycline, 4-Abz-Gly-Pro-D-Leu-D-Ala-NHOH, Ac-Arg-Cys-Gly-Val-Pro-Asp-NH2, recombinant human TIMP1, recombinant human TIMP2, and phosphoramidon; cell adhesion molecules, including integrin agonists and antagonists, including αVβ3 MoAb LM 609 and echistatin; anti-chemotactic agents, including F-Met-Leu-Phe receptor, IL-8 receptor, MCP-1 receptor, and MIP1-I / RANTES receptor; intracellular signal transduction inhibitors, including (a) protein kinase inhibitors, including (i) protein kinase C (PKC) inhibitors (isoenzymes), including calphostin. C), G-6203 and GF109203X and (ii) protein tyrosine kinase inhibitors; (b) intracellular protein tyrosine phosphatase (PTPase) regulators; and (c) SH2 domain inhibitors (src homology 2 domain).

[0220] For some applications, it may be advantageous to combine a MASP-2 inhibitory agent of the present disclosure with a spasm suppressant. For example, for genitourinary applications, it may be advantageous to include at least one smooth muscle spasm suppressant and / or at least one anti-inflammatory agent, and for vascular surgery, it may be beneficial to include at least one vasospasm suppressant and / or at least one anti-inflammatory agent and / or at least one anti-restenotic agent. Suitable examples of spasm suppressants include: serotonin 2 receptor subtype antagonists; tachykinin receptor antagonists; nitric oxide donors; ATP-sensitive potassium channel openers; calcium channel antagonists; and endothelin receptor antagonists.

[0221] In one embodiment of the present disclosure, the antibody is administered in a single dose or in multiple doses.

[0222] If the antibody is administered in multiple doses according to the present disclosure, it is preferably administered in at least 3 doses, at least 4 doses, at least 5 doses, at least 6 doses, at least 7 doses, at least 8 doses, at least 9 doses or at least 10 doses and preferably up to 30 doses, 25 doses, 20 doses, 15 doses or 10 doses. The doses of the antibody are preferably administered at intervals of at least 7 days, at least 10 days, at least 14 days or at least 20 days. The doses of the antibody are preferably administered at intervals of 7 to 30 days, 10 to 20 days and preferably about 14 days.

[0223] In one aspect, the disclosure provides use of an antibody or antigen-binding fragment thereof or a nucleic acid molecule or a vector or a cell or a pharmaceutical composition according to any of the preceding aspects for the preparation of a medicament for treating a disease associated with MASP-2-dependent complement activation in a subject.

[0224] In one aspect, the present disclosure provides a kit comprising the aforementioned antibodies or antigen-binding portions thereof, bispecific or multispecific molecules, nucleic acid molecules, vectors, cells and / or compositions.

[0225] The kits disclosed herein include antibodies, fragments, homologues, derivatives thereof, and the like disclosed herein, such as conjugates that are labeled or cytotoxic, and instructions for use of the antibodies, conjugates that kill specific types of cells, and the like. The instructions may include guidance for using the antibodies, conjugates, and the like in vitro, in vivo, or ex vivo. The antibodies may be in liquid form or solid, typically lyophilized. The kit may contain other suitable reagents, such as buffers, reconstitution solutions, and other necessary components for the intended use. It is contemplated that the reagents packaged in predetermined amounts are combined with instructions for their use, such as for therapeutic use or for performing diagnostic assays. When the antibody is labeled, such as with an enzyme, the kit may include a substrate and cofactors required for the enzyme (e.g., a substrate precursor that provides a detectable chromophore or fluorophore). In addition, other additives, such as stabilizers, buffers (e.g., blocking buffer or lysis buffer), and the like may also be included. The relative amounts of the various reagents can be varied to provide concentrates of the reagent solutions, which provides user flexibility, saves space, saves reagents, and the like. These reagents may also be provided as a dry powder, usually lyophilized, including excipients which, upon dissolution, provide a solution of the reagent having the appropriate concentration.

[0226] In one aspect, the disclosure provides use of the antibodies, or antigen-binding portions thereof, bispecific or multispecific molecules, nucleic acid molecules, vectors, cells, and / or compositions described herein in the preparation of a medicament or kit for diagnosing, treating, or preventing a disease associated with MASP-2-dependent complement activation.

[0227] In addition, the antibodies disclosed herein can also be used in immunoassays, purification methods, and other methods that utilize immunoglobulins or fragments thereof. Such uses are well known in the art.

[0228] Accordingly, the present disclosure also provides compositions comprising the anti-MASP-2 antibodies or fragments thereof of the present disclosure, conveniently combined with a pharmaceutically acceptable carrier, diluent or excipient, as is conventional in the art.

[0229] The term "pharmaceutical composition" as used in this disclosure refers to a variety of preparations. The formulation containing a therapeutically effective amount of a multivalent antibody is in the form of a sterile liquid solution, liquid suspension, or lyophilized form, optionally containing a stabilizer or excipient.

[0230] It will be understood that the therapeutic agents according to the embodiments will be administered with suitable pharmaceutically acceptable carriers, excipients, and other agents incorporated into formulations to provide improved transfer, delivery, tolerability, and the like. A wide variety of suitable formulations are available in the pharmacopoeia known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences (15th ed., Mack Publishing Company, Easton, Pa. (1975)), particularly Chapter 87 of Blaug and Seymour therein. These formulations include, for example, powders, pastes, ointments, gels, waxes, oils, lipids, lipid (cationic or anionic) carriers (e.g., Lipofectin TM ), DNA conjugates, anhydrous slurries, oil-in-water and water-in-oil emulsions, polyethylene glycol emulsions (polyethylene glycol of various molecular weights), semisolid gels, and semisolid mixtures containing polyethylene glycol. Any of the foregoing mixtures may be suitable for use in the treatment or therapy according to the present disclosure, provided that the active ingredient in the formulation is not inactivated by the formulation and that the formulation is physiologically compatible and tolerates the route of administration.

[0231] In another embodiment, antibodies to MASP-2 can be used in methods known in the art relating to the localization and / or quantification of MASP-2 (e.g., for determining MASP-2 and / or the level of MASP-2 in an appropriate physiological sample, in diagnostic methods, in protein imaging, etc.). In a given embodiment, an antibody specific for MASP-2, or a derivative, fragment, analog, or homolog thereof, comprising an antigen-binding domain derived from the antibody, is used as a pharmaceutically active compound (hereinafter referred to as a "therapeutic agent").

[0232] In another embodiment, MASP-2 polypeptides can be isolated using antibodies specific for MASP-2 by standard techniques, such as immunoaffinity, chromatography, or immunoprecipitation. Antibodies to MASP-2 proteins (or fragments thereof) can be used to detect the protein in biological samples. In some embodiments, MASP-2 can be detected in biological samples as part of a clinical testing process, for example, to determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazineaminofluorescein, dansyl chloride, or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive materials include 125 I. 131 I. 35 S or 3 H.

[0233] In another embodiment, the antibodies according to the present disclosure can be used as reagents for detecting the presence of MASP-2 or a protein fragment thereof in a sample. In some embodiments, the antibody comprises a detectable label. The antibody is a polyclonal antibody, or more preferably a monoclonal antibody. The intact antibody or a fragment thereof (e.g., Fab, scFv, or F(ab')2) is used. The term "labeled" with respect to an antibody is intended to include direct labeling of the antibody by coupling (i.e., physically linking) a detectable substance to the antibody, as well as indirect labeling of the antibody by reaction with another directly labeled reagent. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody, and end-labeling of an antibody with biotin to enable detection with fluorescently labeled streptavidin. The term "biological sample" is intended to include tissues, cells, and biological fluids isolated from a subject, as well as tissues, cells, and fluids present in a subject. Thus, the term "biological sample" as used herein includes blood and blood fractions or components, including serum, plasma, or lymph. In other words, the detection methods of the embodiments can be used to detect analyte mRNA, protein, or genomic DNA in a biological sample both in vitro and in vivo. For example, in vitro detection techniques for analyte mRNA include Northern hybridization and in situ hybridization. In vitro detection techniques for analyte protein include enzyme-linked immunosorbent assay (ELISA), Western blotting, immunoprecipitation, and immunofluorescence. In vitro detection techniques for analyte genomic DNA include Southern hybridization. The process for performing immunoassays is described in, for example, "ELISA: Theory and Practice: Methods in Molecular Biology", Volume 42, J.R. Crowther (ed.) Human Press, Totowa, NJ, 1995; "Immunoassay", E. Diamandis and T. Christopoulus, Academic Press, Inc., San Diego, Calif., 1996; and "Practice and Theory of Enzyme Immunoassays", P. Tijssen, Elsevier Science Publishers, Amsterdam, 1985. In addition, in vivo detection techniques for analyte protein include introducing labeled anti-analyte protein antibodies into the subject. For example, the antibody can be labeled with a radioactive marker, and the presence and location of the radioactive marker in a subject can then be detected by standard imaging techniques.

[0234] The antibodies described herein and their derivatives, fragments, analogs and homologs can be incorporated into pharmaceutical compositions suitable for administration. The principles and considerations involved in preparing such compositions and guidance for selecting components are well known in the art, for example, see Remington's Pharmaceutical Sciences: The Science And Practice Of Pharmacy 19th Edition (Alfonso R. Gennaro et al., eds.) Mack Pub. Co., Easton, Pa.: 1995; Drug Absorption Enhancement: Concepts, Possibilities, Limitations, And Trends, Harwood Academic Publishers, Langhorne, Pa., 1994; and Peptide And Protein Drug Delivery (Advances In Parenteral Sciences, Vol. 4), 1991, M. Dekker, New York.

[0235] Such compositions typically comprise an antibody and a pharmaceutically acceptable carrier. When an antibody fragment is used, a minimal inhibitory fragment that specifically binds to the target protein binding domain may be preferred. For example, based on the variable region sequence of the antibody, a peptide molecule that retains the ability to bind to the target protein sequence can be designed. Such peptides can be chemically synthesized and / or produced by recombinant DNA technology (see, for example, Marasco et al., Proc. Natl. Acad. Sci. USA, 90:7889-7893 (1993)).

[0236] As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are compatible with drug administration. Suitable pharmaceutically acceptable carriers are described in the latest edition of Remington's Pharmaceutical Sciences, a standard reference work in this field, which is incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous carriers, such as immobilized oils, may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Except where any conventional media or agents are incompatible with the antibody, their use in the composition is contemplated.

[0237] The pharmaceutical composition of the embodiment is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, such as intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions for parenteral, intradermal, or subcutaneous administration may include the following components: a sterile diluent for injection, such as water, saline, fixed oils, polyethylene glycols, glycerol, propylene glycol, or other synthetic solvents; an antibacterial agent, such as benzyl alcohol or methyl paraben; an antioxidant, such as ascorbic acid or sodium bisulfite; a chelating agent, such as ethylenediaminetetraacetic acid (EDTA); a buffer, such as acetate, citrate, or phosphate, and an agent for regulating osmotic pressure, such as sodium chloride or dextrose. pH can be adjusted with an acid or base, such as hydrochloric acid or sodium hydroxide. Parenteral formulations can be packaged in ampoules, disposable syringes, or multiple-dose vials made of glass or plastic.

[0238] Pharmaceutical compositions suitable for injection include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable pharmaceutically acceptable carriers include physiological saline, bacteriostatic water, Cremophor EL TM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid enough to be easily injectable. It must be stable under manufacturing and storage conditions and must be able to prevent contamination by microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, a polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and a suitable mixture thereof. Suitable fluidity can be maintained, for example, by utilizing a coating such as lecithin to maintain the desired particle size in the case of a dispersion, and by utilizing a surfactant. Prevention of microbial action can be achieved by various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it will be preferred to include an isotonic agent in the composition, such as sugar, a polyol (such as mannitol, sorbitol), sodium chloride. Prolonged absorption of the composition for injection can be achieved by including an agent that delays absorption, such as aluminum monostearate and gelatin, in the composition.

[0239] As needed, sterile injectable solutions can be prepared by incorporating the antibody in the desired amount into a suitable solvent having one or a combination (as needed) of the ingredients listed above, followed by filtration sterilization. Generally, dispersions are prepared by incorporating the antibody into a sterile carrier containing an alkaline dispersion medium and the other ingredients required by those listed above. With regard to sterile powders for the preparation of sterile injectable solutions, the preparation method is vacuum drying and freeze drying to obtain a powder comprising the active ingredient and any additional desired ingredients from a sterile filtered solution of the aforementioned ingredients.

[0240] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from a pressured container or dispenser which contains a suitable propellant, such as a gas such as carbon dioxide, or a nebulizer.

[0241] Administration can also be systemically administered through mucosal or transdermal means. For transmucosal or transdermal administration, penetrants suitable for the permeability barrier are used in the formulation. Such penetrants are generally known in the art and include detergents, bile salts, and fusidic acid derivatives for transmucosal administration. Transmucosal administration can be achieved by using nasal sprays or suppositories. For transdermal administration, one or more of the antibodies can be formulated into ointments, ointments, gels, or creams as are generally known in the art.

[0242] The compounds may also be prepared for rectal delivery in the form of suppositories (eg, with conventional suppository bases such as cocoa butter or other glycerides) or retention enemas.

[0243] In one embodiment, the antibodies can be prepared with carriers that prevent them from being rapidly eliminated by the body, such as slow-release / controlled-release formulations, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations will be apparent to those skilled in the art.

[0244] It is particularly advantageous to formulate parenteral compositions in dosage unit form for ease of administration and consistency of dosage. As used herein, dosage unit form refers to physically separable units suitable as unit dosages for the subject to be treated; each unit contains a predetermined amount of one or more of the antibodies described herein calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specifications for the dosage unit form of the embodiments described are dictated by and directly dependent upon the unique characteristics of the antibody and the specific therapeutic effect to be achieved, and the limitations inherent in the art of formulating such antibodies for use in treating individuals.

[0245] The pharmaceutical compositions can be placed in a container, pack, or dispenser together with instructions for administration.

[0246] The formulations described herein may also include more than one of the antibodies described, depending on the specific condition to be treated, preferably those with complementary activities that do not negatively affect each other. Alternatively or in addition, the composition may include, for example, an agent that enhances its function, such as a cytotoxic agent, a cytokine, a chemotherapeutic agent, or a growth inhibitory agent. Such molecules are suitably present in combination in an amount effective for the intended purpose. For example, they may be present in combination in a kit or during use.

[0247] In one embodiment, one or more of the antibodies may be administered in a combination therapy, i.e., in combination with other agents, such as therapeutic agents (which can be used to treat pathological conditions or disorders, such as various forms of autoimmune diseases and inflammatory diseases). The term "combined" herein refers to administering the agents substantially synchronously, simultaneously, or sequentially. If administered sequentially, the first of the two compounds is still preferably detected at an effective concentration at the treatment site when the second compound is administered. In one instance, "combined" may also be the simultaneous inclusion of an antibody of the present disclosure and other therapeutic agents in a kit.

[0248] For example, combination therapies can comprise one or more antibodies described herein co-formulated and / or co-administered with one or more additional therapeutic agents (e.g., one or more cytokine and growth factor inhibitors, immunosuppressants, anti-inflammatory agents, metabolic inhibitors, enzyme inhibitors, and / or cytotoxins or cytostatic agents, as described in more detail below.) Such combination therapies can advantageously utilize lower dosages of the administered therapeutic agents, thereby avoiding possible toxicities or complications associated with various monotherapies.

[0249] Experiments disclosed herein demonstrate that the various monoclonal antibodies and variants thereof, which bind to MASP-2 with high affinity and inhibit lectin-mediated complement activation, specifically recognize the enzymatic domains CCP1-CCP2-SP of human, monkey, mouse, and rat MASP-2, effectively inhibiting the production of C4b and C3 convertase C4b2b at low concentrations, with blocking activity 50-200 times greater than that of known antibodies. Preliminary toxicology studies conducted in rhesus monkeys demonstrated that high doses of the novel MASP-2 antibodies were well tolerated by the animals without any toxic reactions, demonstrating superior efficacy and safety compared to similar antibodies.

[0250] For purposes of clarity and conciseness, features are described herein as part of the same or separate embodiments; however, it will be understood that the scope of the present disclosure may include embodiments having a combination of all or some of the described features.

[0251] Example

[0252] Example 1. Detection of MASP-2 activity in the lectin pathway

[0253] After MBL in plasma recognizes repetitive N-galactosamine or mannose structures, it sequentially activates the serine proteases MASP-1 and MASP-2. Activated MASP-2 cleaves complement components C4 and C2, resulting in C4b that is deposited on the plate and can be recognized by anti-C4b antibodies. The C3 convertase C4b2b, formed from the cleavage products of C4 and C2, cleaves complement component C3, resulting in C3b that is deposited on the plate and can be recognized by anti-C3b antibodies. Therefore, by detecting the production of C4b or C3b during lectin activation reactions, the inhibitory activity of MASP-2 antibodies against MASP-2 in serum can be demonstrated.

[0254] C4 cleavage activity assay

[0255] 20 μg / ml mannan (Sigma, M7504) was added to the ELISA plate and coated overnight at 4°C. After blocking with 2% BSA at room temperature for 60 minutes, 0.5% diluted human serum was added and incubated overnight at 4°C. After washing, 50 μl / well of various concentrations of MASP-2 antibody (starting at 30 μg / ml, with a 5-fold serial dilution series, for a total of nine dilutions) was added. After preincubation at 4°C for 30 minutes, 50 μl of 2 μg / ml human C4 protein (CompTech, #A105) was added and incubated at 37°C for 90 minutes. After washing, a 1:1000 dilution of Biotin-labeled human C4b polyclonal antibody (ASSAYPRO, #11223-05021) was added and incubated at room temperature for 90 minutes. A 1:5000 dilution of peroxidase-streptavidin (Jackson ImmunoResearch 016-030-084) was added and incubated at room temperature for 60 minutes. TMB color development solution was added and color was developed at 37°C in the dark for about 10 minutes. The reaction was terminated and the OD value was read.

[0256] C3 convertase activity assay

[0257] 20 μg / ml mannan was added to the ELISA plate and coated overnight at 4°C. After blocking with 2% BSA at room temperature for 60 minutes, MASP-2 antibody diluted in 0.5% human serum was added (starting at 60 μg / ml, with a five-fold serial dilution series, for a total of nine dilutions) and incubated at 37°C for 90 minutes. A 1:500 rabbit anti-human C3 / C3b / C3c polyclonal antibody (Proteintech, #21337-1-AP) was added and incubated at room temperature for 60 minutes. A 1:5000 HRP-conjugated goat anti-rabbit secondary antibody (Jackson ImmunoResearch 111-035-144) was added and incubated at room temperature for 60 minutes. TMB color development solution was added and color was developed at 37°C in the dark for approximately 10 minutes. The reaction was terminated and the OD value was read.

[0258] Example 2: Preparation of MASP-2 recombinant protein

[0259] The sequence encoding the CCP1-CCP2-SP domains of human MASP-2 was cloned into a eukaryotic expression plasmid, and a His6 tag was fused to the N-terminus for easy purification. After transfection into HEK293E cells and culturing for 7 days, the culture supernatant was collected and purified via a nickel chelate column to obtain the human MASP-2D4-6 recombinant protein (SEQ ID NO. 1). Similarly, recombinant proteins of cynomolgus monkey MASP-2D4-6 (SEQ ID NO. 2), mouse MASP-2D4-6 (SEQ ID NO. 3), and rat MASP-2D4-6 (SEQ ID NO. 4) were obtained. Figure 1 Shown are SDS-PAGE results of human, cynomolgus monkey, mouse, and rat MASP-2D4-6 recombinant proteins.

[0260] Example 3. Animal Immunization and Preparation of Hybridoma Antibodies

[0261] Six-week-old BALB / c female mice were immunized subcutaneously or in the footpad with equal volumes of human or cynomolgus macaque MASP-2D4-6 recombinant protein mixed with Freund's adjuvant. Two weeks after the initial immunization, a booster immunization was performed. Once the serum titer of the immunized mice reached 1:100,000, a pulse immunization was performed. Three days later, the spleens and peripheral lymph nodes of the mice were harvested and B cells were isolated.

[0262] An appropriate number of B cells were fused with SP2 / 0 cells and selected in complete DMEM medium containing HAT. Hybridoma cells were cultured at 37°C in a 5% CO2 atmosphere for 5-7 days. Human or cynomolgus macaque MASP-2D4-6 recombinant protein (1 μg / mL) was coated and hybridoma culture supernatants were assayed by ELISA. A total of 778 monoclonal antibodies cross-recognized human or cynomolgus macaque MASP-2. The supernatant was harvested and assayed for C4 cleavage activity. 61 clones exhibited significant MASP-2 inhibitory activity (e.g., Figure 2 shown).

[0263] Based on the results of hybridoma screening, RNA from candidate hybridoma monoclonal cells was extracted using the TRNzol lysis method, and then reverse transcribed to synthesize single-stranded cDNA, which was used as a template to amplify the variable region sequence of the antibody in the hybridoma monoclonal cells.

[0264] The variable region sequences of the MASP-2 antibody are as follows:

[0265] Table 1. Variable region sequences of MASP-2 mouse antibodies

[0266]

[0267] Clone: ​​1B2

[0268] The amino acid sequence of the heavy chain VH is shown in SEQ ID NO. 5, the encoding nucleic acid is shown in SEQ ID NO. 6, and the CDR1, CDR2 and CDR3 are shown in SEQ ID NOs. 7, 8 and 9, respectively.

[0269]

[0270] Nucleic acid sequence

[0271]

[0272] The amino acid sequence of the light chain VK is shown in SEQ ID NO.10, its encoding nucleic acid is shown in SEQ ID NO.11, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NOs.12, 13 and 14, respectively.

[0273]

[0274] Nucleic acid sequence

[0275]

[0276] Clone: ​​3B9

[0277] The amino acid sequence of the heavy chain VH is shown in SEQ ID NO. 15, the encoding nucleic acid is shown in SEQ ID NO. 16, and the CDR1, CDR2 and CDR3 are shown in SEQ ID NOs. 7, 17 and 18, respectively.

[0278]

[0279] Nucleic acid sequence

[0280]

[0281] The amino acid sequence of the light chain VK is shown in SEQ ID NO.19, its encoding nucleic acid is shown in SEQ ID NO.20, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NOs.21, 22 and 23, respectively.

[0282]

[0283] Nucleic acid sequence

[0284]

[0285] Clone: ​​4F8

[0286] The amino acid sequence of the heavy chain VH is shown in SEQ ID NO. 24, the encoding nucleic acid is shown in SEQ ID NO. 25, and the CDR1, CDR2 and CDR3 are shown in SEQ ID NOs. 26, 27 and 28, respectively.

[0287]

[0288] Nucleic acid sequence

[0289]

[0290] The amino acid sequence of the light chain VK is shown in SEQ ID NO.29, its encoding nucleic acid is shown in SEQ ID NO.30, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NOs.31, 32 and 33, respectively.

[0291]

[0292] Nucleic acid sequence

[0293]

[0294] Clone: ​​5J11

[0295] The amino acid sequence of the heavy chain VH is shown in SEQ ID NO. 34, the encoding nucleic acid is shown in SEQ ID NO. 35, and the CDR1, CDR2 and CDR3 are shown in SEQ ID NOs. 36, 37 and 38, respectively.

[0296]

[0297]

[0298] Nucleic acid sequence

[0299]

[0300] The amino acid sequence of the light chain VK is shown in SEQ ID NO.39, its encoding nucleic acid is shown in SEQ ID NO.40, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NOs.41, 42 and 43, respectively.

[0301]

[0302] Nucleic acid sequence

[0303]

[0304] Clone: ​​9P13

[0305] The amino acid sequence of the heavy chain VH is shown in SEQ ID NO.44, its encoding nucleic acid is shown in SEQ ID NO.45, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NOs.7, 46 and 47, respectively.

[0306]

[0307] Nucleic acid sequence

[0308]

[0309] The amino acid sequence of the light chain VK is shown in SEQ ID NO.48, its encoding nucleic acid is shown in SEQ ID NO.49, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NOs.50, 51 and 52, respectively.

[0310]

[0311] Nucleic acid sequence

[0312]

[0313] Clone: ​​10H9

[0314] The amino acid sequence of the heavy chain VH is shown in SEQ ID NO. 53, its encoding nucleic acid is shown in SEQ ID NO. 54, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NOs. 7, 17 and 18, respectively.

[0315]

[0316] Nucleic acid sequence

[0317]

[0318] The amino acid sequence of the light chain VK is shown in SEQ ID NO.55, its encoding nucleic acid is shown in SEQ ID NO.56, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NOs.21, 57 and 23, respectively.

[0319]

[0320] Nucleic acid sequence

[0321]

[0322] Clone: ​​10L3

[0323] The amino acid sequence of the heavy chain VH is shown in SEQ ID NO. 58, its encoding nucleic acid is shown in SEQ ID NO. 59, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NOs. 7, 17 and 60, respectively.

[0324]

[0325] Nucleic acid sequence

[0326]

[0327] The amino acid sequence of the light chain VK is shown in SEQ ID NO.61, its encoding nucleic acid is shown in SEQ ID NO.62, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NOs.50, 51 and 52, respectively.

[0328]

[0329] Nucleic acid sequence

[0330]

[0331] Clone: ​​11F8

[0332] The amino acid sequence of the heavy chain VH is shown in SEQ ID NO.63, its encoding nucleic acid is shown in SEQ ID NO.64, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NOs.65, 66 and 67, respectively.

[0333]

[0334] Nucleic acid sequence

[0335]

[0336] The amino acid sequence of the light chain VK is shown in SEQ ID NO.68, its encoding nucleic acid is shown in SEQ ID NO.69, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NOs.70, 71 and 72, respectively.

[0337]

[0338] Nucleic acid sequence

[0339]

[0340] Clone: ​​11G8

[0341] The amino acid sequence of the heavy chain VH is shown in SEQ ID NO.73, its encoding nucleic acid is shown in SEQ ID NO.74, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NOs.7, 17 and 47, respectively.

[0342]

[0343] Nucleic acid sequence

[0344]

[0345] The amino acid sequence of the light chain VK is shown in SEQ ID NO.75, its encoding nucleic acid is shown in SEQ ID NO.76, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NOs.50, 51 and 52, respectively.

[0346]

[0347] Nucleic acid sequence

[0348]

[0349] Clone: ​​14K22

[0350] The amino acid sequence of the heavy chain VH is shown in SEQ ID NO. 77, its encoding nucleic acid is shown in SEQ ID NO. 78, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NOs. 7, 17 and 79, respectively.

[0351]

[0352] Nucleic acid sequence

[0353]

[0354] The amino acid sequence of the light chain VK is shown in SEQ ID NO.80, its encoding nucleic acid is shown in SEQ ID NO.81, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NOs.50, 51 and 82, respectively.

[0355]

[0356] Nucleic acid sequence

[0357]

[0358] Clone: ​​16A15

[0359] The amino acid sequence of the heavy chain VH is shown in SEQ ID NO.83, its encoding nucleic acid is shown in SEQ ID NO.84, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NOs.36, 85 and 86, respectively.

[0360]

[0361] Nucleic acid sequence

[0362]

[0363]

[0364] The amino acid sequence of the light chain VK is shown in SEQ ID NO.87, its encoding nucleic acid is shown in SEQ ID NO.88, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NOs.41, 42 and 43, respectively.

[0365]

[0366] Nucleic acid sequence

[0367]

[0368] Clone: ​​16A17

[0369] The amino acid sequence of the heavy chain VH is shown in SEQ ID NO.83, its encoding nucleic acid is shown in SEQ ID NO.84, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NOs.36, 85 and 86, respectively.

[0370]

[0371] Nucleic acid sequence

[0372]

[0373] The amino acid sequence of the light chain VK is shown in SEQ ID NO.89, its encoding nucleic acid is shown in SEQ ID NO.90, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NOs.41, 42 and 91, respectively.

[0374]

[0375] Nucleic acid sequence

[0376]

[0377] Clone: ​​20J6

[0378] The amino acid sequence of the heavy chain VH is shown in SEQ ID NO.92, the encoding nucleic acid is shown in SEQ ID NO.93, and the CDR1, CDR2 and CDR3 are shown in SEQ ID NOs.94, 95 and 96, respectively.

[0379]

[0380] Nucleic acid sequence

[0381]

[0382] The amino acid sequence of the light chain VK is shown in SEQ ID NO.97, its encoding nucleic acid is shown in SEQ ID NO.98, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NOs.99, 100 and 101, respectively.

[0383]

[0384] Nucleic acid sequence

[0385]

[0386] Clone: ​​21E1

[0387] The amino acid sequence of the heavy chain VH is shown in SEQ ID NO.102, the encoding nucleic acid is shown in SEQ ID NO.103, and the CDR1, CDR2 and CDR3 are shown in SEQ ID NOs.7, 104 and 105, respectively.

[0388]

[0389] Nucleic acid sequence

[0390]

[0391] The amino acid sequence of the light chain VK is shown in SEQ ID NO.106, its encoding nucleic acid is shown in SEQ ID NO.107, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NOs.108, 109 and 110, respectively.

[0392]

[0393] Nucleic acid sequence

[0394]

[0395] Clone: ​​24G17

[0396] The amino acid sequence of the heavy chain VH is shown in SEQ ID NO.111, the encoding nucleic acid is shown in SEQ ID NO.112, and the CDR1, CDR2 and CDR3 are shown in SEQ ID NOs.26, 113 and 114, respectively.

[0397]

[0398] Nucleic acid sequence

[0399]

[0400] The amino acid sequence of the light chain VK is shown in SEQ ID NO.115, its encoding nucleic acid is shown in SEQ ID NO.116, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NOs.117, 118 and 33, respectively.

[0401]

[0402] Nucleic acid sequence

[0403]

[0404] Clone: ​​25L15

[0405] The amino acid sequence of the heavy chain VH is shown in SEQ ID NO.119, the encoding nucleic acid is shown in SEQ ID NO.120, and the CDR1, CDR2 and CDR3 are shown in SEQ ID NOs.94, 95 and 96, respectively.

[0406]

[0407]

[0408] Nucleic acid sequence

[0409]

[0410] The amino acid sequence of the light chain VK is shown in SEQ ID NO.121, its encoding nucleic acid is shown in SEQ ID NO.122, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NOs.123, 100 and 101, respectively.

[0411]

[0412] Nucleic acid sequence

[0413]

[0414] Clone: ​​28O14

[0415] The amino acid sequence of the heavy chain VH is shown in SEQ ID NO.124, its encoding nucleic acid is shown in SEQ ID NO.125, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NOs.126, 127 and 128, respectively.

[0416]

[0417] Nucleic acid sequence

[0418]

[0419] The amino acid sequence of the light chain VK is shown in SEQ ID NO.129, its encoding nucleic acid is shown in SEQ ID NO.130, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NOs.131, 132 and 133, respectively.

[0420]

[0421] Nucleic acid sequence

[0422]

[0423] The light chain variable region of the above-mentioned antibody was cloned into a vector containing the human light chain constant region, and the heavy chain variable region was cloned into a vector containing the human heavy chain constant region. The plasmids were co-transfected into CHO-S cells, and the intact IgG antibody was expressed and secreted into the culture medium. After 7 days of expression at 37°C, 5% CO2, and 125 rpm, the supernatant was collected and purified using Protein A chromatography to obtain chimeric IgG. The IgG concentration was determined spectrophotometrically. The control antibody 17D20_3521N11 (OMS646) was synthesized according to the literature US20120282263.

[0424] According to the method of Example 1, the inhibitory activity of each chimeric antibody on the split C4 reaction was detected, and the OD450nm value was read. After collecting the data, the results were calculated using GraphPad Prism 5 software. Figure 3 As shown, chimeras 10L3, 3B9, 16A17, 4F18, 25L15, 28O14 and 1B2 showed significantly stronger inhibitory activity against MASP-2 than the control antibody in the C4 cleavage activity assay.

[0425] According to the method of Example 1, the C3 convertase activity inhibition test was performed on the selected advantageous chimeric antibodies, and the data were collected and the results were calculated using GraphPad Prism 5 software. Figure 4As shown, in the C3 convertase activity assay, the dominant chimeras 10L3, 3B9, 16A17, 4F18, 25L15, 28O14 and 1B2 showed extremely strong inhibitory activity, which was significantly stronger than that of the control antibody.

[0426] Affinity evaluation

[0427] A 96-well microtiter plate was coated with 1 μg / mL of human MASP-2D4-6 recombinant protein and incubated overnight at 4°C. The plate was blocked with 2% skim milk for 1 hour at room temperature. A serial dilution of MASP-2 antibodies—chimeras 10L3, 3B9, 16A17, 4F18, 25L15, 28O14, and 1B2—was added (starting at 30 μg / mL and diluted 1:4) and incubated at room temperature for 60 minutes. A 1:4000 dilution of Goat anti-human Fc-HRP was added and incubated at room temperature for 60 minutes. TMB was used for color development for 10 minutes. The reaction was terminated with concentrated sulfuric acid, and the OD at 450 nm was measured and calculated using Graph Pad Prism 5 software.

[0428] The results are as follows Figure 5 As shown, the chimeric antibody bound to human, cynomolgus monkey, rat, and mouse MASP-2 (Table 2).

[0429] Table 2 Binding of MASP-2 Antibodies to Various Genera Proteins

[0430] EC50 (nM) Human MASP-2 Cynomolgus monkey MASP-2 Rat MASP-2 Mouse MASP-2 1B2 0.015 0.014 1.439 0.947 3B9 0.025 0.038 1.624 4.393 4F18 0.004 0.003 0.127 0.287 10L3 0.022 0.016 30.19 30.90 16A17 0.014 0.014 4.296 1.973 25L15 0.012 0.008 0.021 0.023 28O14 0.033 0.043 - -

[0431] Using surface plasmon resonance (SPR / Biacore T200), 0.1 μg / ml of antibody was captured on a Protein A chip. The capture time was set to 40 seconds at a flow rate of 10 μl / min. The chip was flushed with buffer until the baseline was stable. Then, serially diluted antigens were passed through the chip at a flow rate of 30 μl / min. The association time was set to 120 seconds and the dissociation time was set to 600 seconds. The equilibrium dissociation constant (KD) was calculated using a Langmuir 1:1 kinetics model. The affinities of the candidate antibodies for various MASP-2 antigens are shown in Table 3.

[0432] Table 3 Affinity of MASP-2 Antibodies to Various Gene Proteins

[0433] KD (nM) Human MASP-2 Cynomolgus monkey MASP-2 Rat MASP-2 Mouse MASP-2 1B2 0.002 0.078 weak 632 3B9 0.067 0.051 1043 476 4F18 0.108 24.76 2471 218 10L3 0.070 1.290 weak 214 16A17 0.008 0.063 weak 487 25L15 0.120 0.920 167 499 28O14 0.090 0.010 162 767 Control Antibody 21.1 8.750 88.5 225

[0434] Example 4: Construction and screening of phage antibody libraries

[0435] The total RNA of some immunized mouse B cells obtained in Example 3 was extracted and reverse transcribed using light chain and heavy chain specific primers to obtain an antibody cDNA library. The variable region fragments of the antibody were specifically amplified using variable region primers, and cloned into a phage plasmid after enzyme digestion to construct an antibody Fab phage display library based on M13 phage with a library capacity of 1×10 10 .

[0436] The library was screened using the traditional phage panning method. MASP-2D4-6 recombinant protein was coated on the immunotube, and approximately 1×10 12 Phage were incubated and panned. After two rounds of panning, single clones were picked and inoculated into 96-well U-shaped plates for culture and induced expression. The culture supernatant was collected for ELISA detection of MASP-2 recombinant protein. A total of 494 monoclonal Fab antibodies that cross-recognized human, cynomolgus monkey, rat, and mouse MASP-2 were obtained, of which 34 clones could inhibit MASP-2-catalyzed C4 deposition ( Figure 6 ).

[0437] After the above Fab clones with inhibitory activity were re-induced to express, the Fab antibodies were purified and the inhibitory activity of the MASP-2 antibodies was tested by C4 cleavage activity assay. Figure 7 As shown, monoclonal antibodies 3H6, 14F11, 16B11, 20C8, 21A3 and 21B1 had the most significant inhibitory activity on MASP-2-catalyzed C4b deposition when the concentration was greater than 0.01 μg / ml.

[0438] Using surface plasmon resonance (SPR / Biacore T200) technology, anti-Flag antibodies were captured on a Protein A chip. 10 μg / ml of purified Flag-tagged Fab antibodies were captured on the chip surface. The capture time was set to 40 seconds at a flow rate of 10 μl / min. The chip was rinsed with buffer until the baseline was stable. Then, serially diluted MASP-2 antigens were injected as analytes to measure the affinity of the Fabs for antigens from different species. Data were fitted using Biacore T200 Evaluationsoftware 3.1 using a Langmuir 1:1 kinetics model. Table 4 shows the affinity of mAbs 16B11 and 14F11 for MASP-2 antigens from various species.

[0439] Table 4 Affinity of MASP-2 Antibodies (Fab) to Antigens

[0440]

[0441]

[0442] The variable region sequence of the Fab antibody was obtained by Sanger sequencing as follows:

[0443] Table 5. Variable region sequences of MASP-2 mouse antibodies

[0444]

[0445] Clone: ​​3H6

[0446] The amino acid sequence of the heavy chain VH is shown in SEQ ID NO.134, the encoding nucleic acid is shown in SEQ ID NO.135, and the CDR1, CDR2 and CDR3 are shown in SEQ ID NOs.136, 137 and 138, respectively.

[0447]

[0448] Nucleic acid sequence

[0449]

[0450] The amino acid sequence of the light chain VK is shown in SEQ ID NO.129, its encoding nucleic acid is shown in SEQ ID NO.130, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NOs.131, 132 and 133, respectively.

[0451]

[0452] Nucleic acid sequence

[0453]

[0454] Clone: ​​16B11

[0455] The amino acid sequence of the heavy chain VH is shown in SEQ ID NO. 139, the encoding nucleic acid is shown in SEQ ID NO. 140, and the CDR1, CDR2 and CDR3 are shown in SEQ ID NOs. 7, 17 and 141, respectively.

[0456]

[0457] Nucleic acid sequence

[0458]

[0459] The amino acid sequence of the light chain VK is shown in SEQ ID NO.142, its encoding nucleic acid is shown in SEQ ID NO.143, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NOs.144, 145 and 146, respectively.

[0460]

[0461] Nucleic acid sequence

[0462]

[0463] Clone: ​​14F11

[0464] The amino acid sequence of the heavy chain VH is shown in SEQ ID NO. 147, the encoding nucleic acid is shown in SEQ ID NO. 148, and the CDR1, CDR2 and CDR3 are shown in SEQ ID NOs. 149, 150 and 151, respectively.

[0465]

[0466] Nucleic acid sequence

[0467]

[0468] The amino acid sequence of the light chain VK is shown in SEQ ID NO.152, its encoding nucleic acid is shown in SEQ ID NO.153, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NOs.154, 155 and 156, respectively.

[0469]

[0470] Nucleic acid sequence

[0471]

[0472] Clone: ​​20C8

[0473] The amino acid sequence of the heavy chain VH is shown in SEQ ID NO. 157, the encoding nucleic acid is shown in SEQ ID NO. 158, and the CDR1, CDR2 and CDR3 are shown in SEQ ID NOs. 7, 17 and 9, respectively.

[0474]

[0475] Nucleic acid sequence

[0476]

[0477] The amino acid sequence of the light chain VK is shown in SEQ ID NO.159, its encoding nucleic acid is shown in SEQ ID NO.160, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NOs.161, 162 and 14, respectively.

[0478]

[0479] Nucleic acid sequence

[0480]

[0481]

[0482] Clone: ​​21A3

[0483] The amino acid sequence of the heavy chain VH is shown in SEQ ID NO. 163, its encoding nucleic acid is shown in SEQ ID NO. 164, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NOs. 7, 165 and 9, respectively.

[0484]

[0485] Nucleic acid sequence

[0486]

[0487] The amino acid sequence of the light chain VK is shown in SEQ ID NO.166, its encoding nucleic acid is shown in SEQ ID NO.167, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NOs.144, 168 and 14, respectively.

[0488]

[0489] Nucleic acid sequence

[0490]

[0491] Clone: ​​21B1

[0492] The amino acid sequence of the heavy chain VH is shown in SEQ ID NO. 169, the encoding nucleic acid is shown in SEQ ID NO. 170, and the CDR1, CDR2 and CDR3 are shown in SEQ ID NOs. 7, 17 and 9, respectively.

[0493]

[0494] Nucleic acid sequence

[0495]

[0496] The amino acid sequence of the light chain VK is shown in SEQ ID NO.171, its encoding nucleic acid is shown in SEQ ID NO.172, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NOs.144, 173 and 174, respectively.

[0497]

[0498] Nucleic acid sequence

[0499]

[0500] Example 5. Humanization of MASP-2 Antibodies

[0501] High-affinity and highly active mouse antibodies 28O14, 3H6, and 16B11 were selected for variable region humanization. First, the glycosylation site NYS of the 28O14 heavy chain CDR3 was cloned and site-directed mutagenesis was performed. The mutants were then expressed as Fab in E. coli. Affinity testing showed that after the glycosylation site in the 28O14 heavy chain CDR3 was removed, the SA mutant (Ala102), which mutated from serine to alanine at position 102, did not affect MASP-2 binding activity (e.g., Figure 8 shown).

[0502] Mouse antibody sequences were aligned with human germline sequences to identify human germline light chain genes with high homology, identical key amino acid sequences maintaining the core antibody structure, and high human frequency. Mouse antibody CDRs were then transplanted. Computer-generated homology modeling was performed to analyze the CDR regions and surrounding framework amino acid sequences to avoid concentrated distribution of surface charge or hydrophobic regions. The humanized designs resulted in three heavy chain and six light chain 16B11 variants, four heavy chain and one light chain 3H6 variants, four heavy chain and one light chain 28O14 variants, and three heavy chain and six light chain 16B11 variants.

[0503] The humanized sequences are as follows:

[0504] Table 6. Heavy chain variable region sequences of MASP-2 humanized antibodies

[0505]

[0506] Table 7. Light chain variable region sequences of MASP-2 humanized antibodies

[0507]

[0508] h16B11:

[0509] h16B11 H1

[0510] The amino acid sequence of h16B11 H1 is shown in SEQ ID NO. 175, the encoding nucleic acid is shown in SEQ ID NO. 176, and the CDR1, CDR2 and CDR3 are shown in SEQ ID NOs. 7, 17 and 141, respectively.

[0511]

[0512] Nucleic acid sequence

[0513]

[0514] h16B11 H2

[0515] The amino acid sequence of h16B11 H2 is shown in SEQ ID NO. 177, the encoding nucleic acid thereof is shown in SEQ ID NO. 178, and the CDR1, CDR2 and CDR3 thereof are shown in SEQ ID NOs. 7, 17 and 141, respectively.

[0516]

[0517] Nucleic acid sequence

[0518]

[0519] h16B11 H3

[0520] The amino acid sequence of h16B11 H3 is shown in SEQ ID NO. 179, the encoding nucleic acid thereof is shown in SEQ ID NO. 180, and the CDR1, CDR2 and CDR3 thereof are shown in SEQ ID NOs. 7, 17 and 141, respectively.

[0521]

[0522] Nucleic acid sequence

[0523]

[0524] h3H6:

[0525] h3H6 H1

[0526] The amino acid sequence of h3H6 H1 is shown in SEQ ID NO. 181, the encoding nucleic acid is shown in SEQ ID NO. 182, and the CDR1, CDR2 and CDR3 are shown in SEQ ID NOs. 136, 183 and 138, respectively.

[0527]

[0528] Nucleic acid sequence

[0529]

[0530] h3H6 H2

[0531] The amino acid sequence of h3H6 H2 is shown in SEQ ID NO. 184, the encoding nucleic acid is shown in SEQ ID NO. 185, and the CDR1, CDR2 and CDR3 are shown in SEQ ID NOs. 136, 183 and 138, respectively.

[0532]

[0533] Nucleic acid sequence

[0534]

[0535] h3H6 H3

[0536] The amino acid sequence of h3H6 H3 is shown in SEQ ID NO. 186, the encoding nucleic acid is shown in SEQ ID NO. 187, and the CDR1, CDR2 and CDR3 are shown in SEQ ID NOs. 136, 183 and 138, respectively.

[0537]

[0538] Nucleic acid sequence

[0539]

[0540] h3H6 H4

[0541] The amino acid sequence of h3H6 H4 is shown in SEQ ID NO. 188, the encoding nucleic acid is shown in SEQ ID NO. 189, and the CDR1, CDR2 and CDR3 are shown in SEQ ID NOs. 136, 183 and 138, respectively.

[0542]

[0543] Nucleic acid sequence

[0544]

[0545] h28O14:

[0546] h28O14 H1

[0547] The amino acid sequence of h28O14 H1 is shown in SEQ ID NO. 190, the encoding nucleic acid thereof is shown in SEQ ID NO. 191, and the CDR1, CDR2 and CDR3 thereof are shown in SEQ ID NOs. 126, 192 and 128, respectively.

[0548]

[0549] Nucleic acid sequence

[0550]

[0551] h28O14 H2

[0552] The amino acid sequence of h28O14 H2 is shown in SEQ ID NO. 193, the encoding nucleic acid thereof is shown in SEQ ID NO. 194, and the CDR1, CDR2 and CDR3 thereof are shown in SEQ ID NOs. 126, 192 and 128, respectively.

[0553]

[0554] Nucleic acid sequence

[0555]

[0556] h28O14 H3

[0557] The amino acid sequence of h28O14 H3 is shown in SEQ ID NO. 195, the encoding nucleic acid thereof is shown in SEQ ID NO. 196, and the CDR1, CDR2 and CDR3 thereof are shown in SEQ ID NOs. 126, 192 and 128, respectively.

[0558]

[0559] Nucleic acid sequence

[0560]

[0561] h28O14 H4

[0562] The amino acid sequence of h28O14 H4 is shown in SEQ ID NO. 197, the encoding nucleic acid thereof is shown in SEQ ID NO. 198, and the CDR1, CDR2 and CDR3 thereof are shown in SEQ ID NOs. 126, 192 and 128, respectively.

[0563]

[0564] Nucleic acid sequence

[0565]

[0566] h16B11:

[0567] h16B11 K1

[0568] The amino acid sequence of h16B11 K1 is shown in SEQ ID NO. 199, the encoding nucleic acid thereof is shown in SEQ ID NO. 200, and the CDR1, CDR2 and CDR3 thereof are shown in SEQ ID NOs. 201, 202 and 146, respectively.

[0569]

[0570] Nucleic acid sequence

[0571]

[0572] h16B11 K2

[0573] The amino acid sequence of h16B11 K2 is shown in SEQ ID NO. 203, the encoding nucleic acid thereof is shown in SEQ ID NO. 204, and the CDR1, CDR2 and CDR3 thereof are shown in SEQ ID NOs. 201, 202 and 146, respectively.

[0574]

[0575] Nucleic acid sequence

[0576]

[0577] h16B11 K3

[0578] The amino acid sequence of h16B11 K3 is shown in SEQ ID NO. 205, the encoding nucleic acid thereof is shown in SEQ ID NO. 206, and the CDR1, CDR2 and CDR3 thereof are shown in SEQ ID NOs. 201, 207 and 146, respectively.

[0579]

[0580] Nucleic acid sequence

[0581]

[0582] h16B11 K4

[0583] The amino acid sequence of h16B11 K4 is shown in SEQ ID NO. 208, the encoding nucleic acid thereof is shown in SEQ ID NO. 209, and the CDR1, CDR2 and CDR3 thereof are shown in SEQ ID NOs. 201, 202 and 146, respectively.

[0584]

[0585] Nucleic acid sequence

[0586]

[0587] h16B11 K5

[0588] The amino acid sequence of h16B11 K5 is shown in SEQ ID NO. 210, the encoding nucleic acid thereof is shown in SEQ ID NO. 211, and the CDR1, CDR2 and CDR3 thereof are shown in SEQ ID NOs. 201, 202 and 146, respectively.

[0589]

[0590] Nucleic acid sequence

[0591]

[0592] h16B11 K6

[0593] The amino acid sequence of h16B11 K6 is shown in SEQ ID NO. 212, the encoding nucleic acid thereof is shown in SEQ ID NO. 213, and the CDR1, CDR2 and CDR3 thereof are shown in SEQ ID NOs. 201, 207 and 146, respectively.

[0594]

[0595] Nucleic acid sequence

[0596]

[0597] h28O14:

[0598] h28O14 K1

[0599] The amino acid sequence of h28O14 K1 is shown in SEQ ID NO. 214, the encoding nucleic acid thereof is shown in SEQ ID NO. 215, and the CDR1, CDR2 and CDR3 thereof are shown in SEQ ID NOs. 216, 132 and 133, respectively.

[0600]

[0601] Nucleic acid sequence

[0602]

[0603] h3H6 K:

[0604] h3H6 K1

[0605] The amino acid sequence of h3H6 K1 is shown in SEQ ID NO. 214, the encoding nucleic acid is shown in SEQ ID NO. 215, and the CDR1, CDR2 and CDR3 are shown in SEQ ID NOs. 216, 132 and 133, respectively.

[0606]

[0607] Nucleic acid sequence

[0608]

[0609] After full sequence synthesis of the light and heavy chains, respectively, they were cloned into eukaryotic expression vectors containing the antibody kappa chain constant region Ckappa or the human IgG4 constant region CH1-CH3. After combining and pairing the light and heavy chain plasmids, they were transfected into CHO-S cells and expressed at 37°C for 5-6 days. The culture supernatant was collected and purified using a Protein A column.

[0610] Example 6: MASP-2 Inhibitory Activity of Humanized Antibodies

[0611] C4 cleavage activity assay

[0612] According to the C4 cleavage assay described in Example 1, 50 μl of MASP-2 antibody at different concentrations (starting at 30 μg / ml, 5-fold serial dilutions, a total of 9 serial dilutions) was added to the reaction wells and incubated at 4°C for 30 minutes. 50 μl of 2 μg / ml purified C4 protein (CompTech) was added and the reaction was incubated at 37°C for 90 minutes. C4c antibody was then added to detect C4b deposition. Figure 9A 、 9B As shown in Table 8, all humanized antibodies can significantly inhibit the activity of MASP-2.

[0613] Table 8. Inhibitory activity of MASP-2 humanized antibodies against C4b deposition

[0614] Humanized antibodies Heavy chain variable region Light chain variable region IC50(nM) h3H6 H1K1 h3H6 H1 h3H6 K1 0.014 h3H6 H2K1 h3H6 H2 h3H6 K1 0.086 h3H6 H3K1 h3H6 H3 h3H6 K1 0.017 h3H6 H4K1 h3H6 H4 h3H6 K1 0.090 h28O14H1K1 h28O14 H1 h28O14 K1 0.083 h28O14H2K1 h28O14 H2 h28O14 K1 0.324 h28O14H3K1 h28O14 H3 h28O14 K1 0.064 h28O14H4K1 h28O14 H4 h28O14 K1 0.338 h16B11H1K1 h16B11 H1 h16B11 K1 0.020 h16B11H1K2 h16B11 H1 h16B11 K2 0.051 h16B11H1K3 h16B11 H1 h16B11 K3 0.021 h16B11H2K1 h16B11 H2 h16B11 K1 0.020 h16B11H2K2 h16B11 H2 h16B11 K2 0.032 h16B11H2K3 h16B11 H2 h16B11 K3 0.012 h16B11H3K1 h16B11 H3 h16B11 K1 0.098 h16B11H3K2 h16B11 H3 h16B11 K2 1.691 h16B11H3K3 h16B11 H3 h16B11 K3 0.440 Control Antibody 0.909

[0615] C3 convertase activity assay

[0616] According to the C3 convertase activity assay described in Example 1, different concentrations of MASP-2 antibody (starting at 30 μg / ml, 5-fold serial dilution, 9 serial dilutions in total) were added to the reaction wells at a rate of 50 μl per well and reacted at 37°C for 90 minutes. C3b antibody was added at a dilution ratio of 1:500 to detect C3b deposition. Figure 10A 、 10B As shown in Figures 10C and 10C and Table 9, each humanized antibody can inhibit the deposition of C3b.

[0617] Table 9. Inhibitory activity of MASP-2 humanized antibodies against C3b deposition

[0618] Humanized antibodies Heavy chain variable region Light chain variable region IC50(nM) h3H6 H1K1 h3H6 H1 h3H6 K1 0.030 h3H6 H2K1 h3H6 H2 h3H6 K1 0.151 h3H6 H3K1 h3H6 H3 h3H6 K1 0.034 h3H6 H4K1 h3H6 H4 h3H6 K1 0.151 h28O14H1K1 h28O14 H1 h28O14 K1 0.146 h28O14H2K1 h28O14 H2 h28O14 K1 0.483 h28O14H3K1 h28O14 H3 h28O14 K1 0.161 h28O14H4K1 h28O14 H4 h28O14 K1 0.506 h16B11H1K1 h16B11 H1 h16B11 K1 0.014 h16B11H1K2 h16B11 H1 h16B11 K2 0.040 h16B11H1K3 h16B11 H1 h16B11 K3 0.023 h16B11H2K3 h16B11 H2 h16B11 K3 0.018 h16B11H2K1 h16B11 H2 h16B11 K1 0.017 h16B11H2K2 h16B11 H2 h16B11 K2 0.028 h16B11H3K1 h16B11 H3 h16B11 K1 0.060 h16B11H3K2 h16B11 H3 h16B11 K2 0.399 h16B11H3K3 h16B11 H3 h16B11 K3 0.112 h16B11H1K4 h16B11 H1 h16B11 K4 0.015 h16B11H1K5 h16B11 H1 h16B11 K5 0.024 h16B11H1K6 h16B11 H1 h16B11 K6 0.018 h16B11H2K4 h16B11 H2 h16B11 K4 0.015 h16B11H2K5 h16B11 H2 h16B11 K5 0.032 h16B11H2K6 h16B11 H2 h16B11 K6 0.018 h16B11H3K4 h16B11 H3 h16B11 K4 0.052 h16B11H3K5 h16B11 H3 h16B11 K5 0.440 h16B11H3K6 h16B11 H3 h16B11 K6 0.071 Control Antibody 3.692

[0619] Example 7. Affinity of MASP-2 Humanized Antibodies

[0620] (1) ELISA test of the affinity of humanized MASP-2 antibodies to various antigens

[0621] A 96-well microtiter plate was coated with 1 μg / mL of human MASP-2D4-6 recombinant protein and incubated overnight at 4°C. The plate was blocked with 2% skim milk for 1 hour at room temperature. A serial dilution of MASP-2 antibody (starting at 30 μg / mL, 1:4 dilution) was added and incubated for 60 minutes at room temperature. A 1:4000 dilution of goat anti-human Fc-HRP was added and incubated for 60 minutes at room temperature. TMB was used for color development for 10 minutes. The reaction was terminated with concentrated sulfuric acid, and the OD at 450 nm was measured. Results were calculated using Graph Pad Prism 5 software.

[0622] The results are as follows Figure 11 As shown, the humanized MASP-2 antibody binds to human, cynomolgus monkey, rat, and mouse MASP-2 with comparable affinities.

[0623] (2) BIAcore detection of affinity of humanized antibodies to human and mouse MASP-2

[0624] 0.1 μg / ml of antibody was captured on the Protein A chip surface with a capture time of 40 seconds and a flow rate of 10 μl / min. The chip was flushed with buffer until the baseline was stable. A gradient of antigen dilutions was then passed through the chip at a flow rate of 30 μl / min. The association time was set to 120 seconds and the dissociation time to 600 seconds. The equilibrium dissociation constant (KD) was obtained by fitting. The affinity values ​​are shown in Table 10.

[0625] Table 10. Affinity of MASP-2 Antibodies to MASP-2

[0626]

[0627] Example 8: Shiga toxin 2-induced hemolytic uremic syndrome model in mice

[0628] Using the STX2 cDNA plasmid (Sino Biological, Cat. No. EG40019-G) as a template, the STX2A (Arg23-Lys319) and STX2B (Ala20-Asp89) gene fragments of E. coli O157:H7 were PCR amplified and cloned into prokaryotic expression plasmids. The twin strep tag was fused to the N-terminus of the STX2A protein for purification, and the enterokinase cleavage site DDDDK was added between the two. The constructed STX2 expression plasmid was transformed into TG1 Escherichia coli competent cells, and single colonies were picked and cultured in bacterial culture medium. IPTG was added for induction overnight, and the cells were collected. PPB and 5 mM MgSO4 solution were added successively to lyse the bacterial cell wall. The protein expression solution in the periplasmic cavity was collected by centrifugation, and the Shiga toxin STX2 protein was purified using Strep-TactinX (Sino HaiGene, Catalog No.: C0402). EK enterokinase was added to the protein collection solution, and the cells were incubated at 25°C overnight to remove the twin strep tag.

[0629] Female C57 BL / 6 mice weighing approximately 18 g were divided into four MASP-2 antibody dose groups (0 mg / kg, 3 mg / kg, 10 mg / kg, and 30 mg / kg), with seven mice in each group. Two hours before administration, the anti-MASP-2 antibody h16B11 H2K4 (100 μl) was injected into the tail vein. LPS (5 μg / mouse, L6136-Sigma) and STX2 (10 ng / mouse) were then injected intraperitoneally. Survival of the mice was monitored starting 60 hours after administration, and mice were sacrificed when they reached the coma stage of HUS pathology. The results are shown in Figure 1. Figure 12 As shown, anti-MASP-2 antibodies can significantly prolong the survival time of mice in a dose-related manner.

Claims

1. An antibody or antigen-binding portion thereof that binds to MASP 2, comprising the heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 126, 192, and 128, respectively, and the light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 216, 132, and 133, respectively.

2. The antibody or antigen-binding portion thereof according to claim 1, comprising a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 190, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO:

214.

3. A bispecific molecule comprising the MASP 2-binding antibody of claim 1 or 2, or an antigen-binding portion thereof.

4. A multispecific molecule comprising the MASP 2-binding antibody of claim 1 or 2, or an antigen-binding portion thereof.

5. A nucleic acid encoding the antibody or antigen-binding portion thereof of claim 1 or 2, the bispecific molecule of claim 3, or the multispecific molecule of claim 4.

6. The nucleic acid according to claim 5, comprising a heavy chain variable region nucleotide sequence as shown in SEQ ID NO: 191, and a light chain variable region nucleotide sequence as shown in SEQ ID NO:

215.

7. A vector comprising the nucleic acid according to claim 5 or 6.

8. A cell comprising the nucleic acid of claim 5 or 6 or the vector of claim 7.

9. A composition comprising the antibody or antigen-binding portion thereof of claim 1 or 2, the bispecific molecule of claim 3, the multispecific molecule of claim 4, the nucleic acid of claim 5 or 6, the vector of claim 7, and / or the cell of claim 8.

10. A kit comprising the antibody or antigen-binding portion thereof of claim 1 or 2, the bispecific molecule of claim 3, the multispecific molecule of claim 4, the nucleic acid of claim 5 or 6, the vector of claim 7, the cell of claim 8, and / or the composition of claim 9.

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