Development and application of complement inhibitor

Abstract

The invention relates to development and application of a complement inhibitor. The complement inhibitor is an antibody which binds to MASP-2 or an antigen binding part thereof. The antibody disclosed by the invention can be combined with MASP-2 with high affinity, can specifically recognize enzyme activity domains CCP1-CCP2-SP of human, monkey, mouse and rat MASP-2, can effectively inhibit generation of complement components C4b and C3 invertase C4b2b under a low-concentration condition, has blocking activity 50-200 times higher than that of a known antibody, has good tolerance, does not have any toxic reaction, and can be used for preparing the MASP-2 antibody. The curative effect and the safety are better than those of similar antibodies.

Description

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

[0002] The present disclosure relates to a complement inhibitor and its application, particularly a MASP-2 inhibitory antibody and its application. 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 lectin activation pathway of complement. MASP-2 contains six domains, namely 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 and forms a complex with the lectin pathway recognition molecules mannose-binding lectin (MBL), collectin-11 (CL-K1), and ficolin (Ficolin-1, Ficolin-2, and Ficolin-3) through the three domains of N-terminal CUB1-EGF-CUB2. 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. The activated MASP-2 cleaves C4 and C2, and the fragments C4b and C2b formed by cleavage interact to generate 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) that can cause microbial lysis. The activated forms of C3 and C4 (C3b and C4b) are covalently deposited on the surface of exogenous targets, which are recognized by complement receptors on various phagocytes, and phagocytosis of the corresponding pathogens occurs. The lectin pathway plays an important role in the innate immunity of the body against pathogenic microorganisms such as bacteria, yeasts, and viruses.

[0004] Under certain pathological conditions, after cells are damaged and ruptured, the incompletely glycosylated glycoproteins (containing high-mannose "precursor" glycan clusters) inside them are released, which can be recognized by MBL with high affinity and initiate 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 on the inner wall of glomerular blood vessels and other places, activating the complement lectin pathway through MBL-MASP-2, causing an inflammatory response. Granzyme released by over-activated inflammatory cells may cause damage to nearby tissues and organs. In addition, activated complement components deposited on nearby cells can lead to the production of the complement membrane attack complex and cell lysis. The complement system is related to the pathogenesis of a variety of acute or chronic disease conditions, such as IgA nephropathy (IgAN), ischemia-reperfusion injury, transplant rejection, rheumatoid arthritis, myocardial infarction, vascular reconstruction after stroke, ARDS, septic shock, capillary leakage after thermal burns, inflammation after cardiopulmonary bypass, multiple sclerosis, myasthenia gravis, and Alzheimer's disease, etc. Recently, studies have shown that the tissue damage in the lungs, kidneys and other organs caused by the infection of the novel coronavirus COVID-2019 is related to the complement lectin pathway (Magro C et al Transl Res. 2020 Jun; 220:1-13), and inhibiting the activity of MASP-2 can greatly reduce the mortality of severe COVID-2019 patients (Rambaldi A et al Immunobiology. 2020 Aug 9:152001).

[0005] MASP-2 is the only protease in the lectin pathway that can cleave C4, and its concentration in the blood of healthy people 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 the CCP2 domain, causing the latter to undergo allosteric changes and expose the cleavage site, and 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 present disclosure provides an antibody that binds to MASP-2 or an antigen-binding portion thereof.

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

[0009] In one aspect, the present disclosure provides a nucleic acid encoding the aforementioned antibody that binds to MASP-2, or an antigen-binding portion thereof, or a bispecific antibody, or an antigen-binding portion thereof.

[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 an antigen-binding portion thereof, an encoding nucleic acid, a vector, a cell, a composition, or a kit as described above.

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

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

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

[0016] Figure 2 The inhibition of C4b deposition by the hybridoma supernatant is shown.

[0017] Figure 3 The inhibition of C4b deposition by the chimeric antibody of the hybridoma clone is shown.

[0018] Figure 4 The inhibition of C3b deposition by the chimeric antibody of the hybridoma clone is shown.

[0019] Figure 5 The binding of the candidate chimeric antibody to MASP-2D4-6 of different species detected by ELISA is shown.

[0020] Figure 6 The inhibition of C4b deposition by the Fab candidate clone of the phage display library is shown.

[0021] Figure 7 The inhibition of C4b deposition after purification of the Fab candidate clone of the phage library display is shown.

[0022] Figure 8Shows the inhibition of C4b deposition after 28O14 cloning mutation. Among them, Ser102 represents the wild-type antibody, and its 102nd position is serine. Gly102, Tyr102, Ala102, Glu102, Gln102, Val102, Leu102 and His102 represent mutant antibodies, and the 102nd position of which is mutated from serine to glycine, tyrosine, alanine, glutamic acid, glutamine, valine, leucine and histidine respectively.

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

[0024] Figure 10A Figures B and C show the inhibition of C3b deposition by the humanized MASP-2 antibody.

[0025] Figure 11 Shows the binding of the humanized MASP-2 antibody to antigens of different species detected by ELISA.

[0026] Figure 12 Shows the efficacy of the humanized MASP-2 antibody in a mouse model of hemolytic uremic syndrome. Detailed implementation mode

[0027] I. Definitions

[0028] In the present disclosure, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. And, the terms and laboratory operation steps related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are all terms and conventional steps widely used in the corresponding fields. At the same time, in order to better understand the present disclosure, the definitions and explanations of related terms are provided below.

[0029] Provided herein are antibodies (e.g., monoclonal antibodies) that specifically bind to MASP-2 and antigen-binding fragments thereof. In a specific aspect, provided herein is a monoclonal anti-MASP-2 antibody that specifically binds to MASP-2, wherein the anti-MASP-2 antibody comprises a variant of a parental antibody. In a specific aspect, provided herein are antibodies that specifically bind to MASP-2 (e.g., human MASP-2). In a particular aspect, provided herein are anti-MASP-2 antibodies comprising modifications in one or more amino acid residues (e.g., 5-13 amino acid substitutions in the framework region of the heavy chain variable region), which maintain affinity for the antigen compared to the parental antibody without such modifications. The term "MASP-2" refers to any MASP-2 molecule known to those skilled in the art. For example, the MASP-2 may be from a mammal, such as the MASP-2 may be from 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+ , resulting in the formation of the lectin pathway C3 convertase C4b2a and, after accumulation of the C3 cleavage product C3b, 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 or directly interacts with MASP-2 and effectively inhibits MASP-2-dependent complement activation. A MASP-2 inhibitory antibody for use in the methods of the present disclosure can reduce MASP-2-dependent complement activation by greater than 20%, such as 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 that occurs through specific binding of serum and non-serum carbohydrate-binding proteins, including mannan-binding lectin (MBL) and ficolin.

[0033] As used herein and unless otherwise specified, the term "about" or "approximately" means within plus or minus 10% of a given value or range. In instances 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" can be understood to mean an antibody chain that exhibits 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 term can be understood to mean a nucleotide sequence that exhibits at least greater than 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher sequence identity to a reference nucleic acid sequence.

[0035] The term "sequence identity" or "identity" has the meaning recognized in the art, and the percentage of sequence identity between two nucleic acid or polypeptide molecules or regions can be calculated using publicly available techniques. Sequence identity can be measured along the full length of a polynucleotide or polypeptide or along a region of the molecule (see, e.g., Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H.G., 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). Although there are many methods for measuring 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] A "substitution" variant is a variant in which at least one amino acid residue in the native sequence has been removed and a different amino acid inserted in its place. The substitution can be single, where only one amino acid in the molecule is replaced; or multiple, where two or more amino acids in the same molecule are replaced. Multiple substitutions can be at contiguous sites. Also, one amino acid can be replaced by multiple residues, where such variants include both substitutions and insertions. An "insertion" variant is a variant in which one or more amino acids are inserted adjacent to an amino acid at a particular position in a native sequence. Adjacent amino acid means linked to the α-carboxyl or α-amino functional group of the amino acid. A "deletion" variant is a variant in which one or more amino acids have been removed from the native amino acid sequence. Typically, deletion variants have one or two amino acids deleted in a particular region of their molecule.

[0037] As used in reference to the variable domains of antibodies, the term "variable" refers to certain portions of the molecule where there is extensive sequence variation among antibodies and which are used for the specific recognition and binding of a particular antibody to its specific target. However, the variability is not evenly distributed throughout the entire variable domain of an antibody. It is concentrated in three segments called complementarity determining regions (CDRs; i.e., CDR1, CDR2, and CDR3) or hypervariable regions, which are located within the variable domains of both the light and heavy chains. The more conserved portions within the variable domains are called framework (FR) regions or framework sequences. Each variable domain of a native heavy or light chain includes four FR regions, which predominantly adopt a β-sheet conformation and are connected by three CDRs, which form loops that connect the β-sheet structures and in some cases form part of the β-sheet structure. The CDRs of each chain are usually connected in proximity by FR regions and, together with 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 Kabat et al. immunoglobulin amino acid residue numbering system, unless otherwise specified. A CDR can have the ability to specifically bind to an associated 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 that includes at least the portion of the variable region of the antibody that binds the antigen (e.g., one or more CDRs and / or one or more antibody-binding sites), and thus retains the binding specificity and at least a portion of the specific binding ability of the full-length antibody. Thus, an antigen-binding fragment refers to an antibody fragment that includes an antigen-binding portion that binds the same antigen as the antibody from which the antibody fragment is derived. Antibody fragments include antibody derivatives produced by enzymatic treatment of a full-length antibody, 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 include multiple chains linked together, such as by disulfide bonds and / or by peptide linkers. Antibody fragments generally comprise 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 replacement of the corresponding region), acquires the ability to bind an antigen immunospecifically (i.e., exhibits a Ka of at least or at least about 10 7 -10 8 M-1). A "functional fragment" or "analogue of an anti-MASP-2 antibody" is a fragment or analogue that can prevent or substantially reduce the ability of the receptor to bind a ligand or initiate signal transduction. As used herein, a functional fragment generally has the same meaning as an "antibody fragment" and, with respect to an antibody, can refer to a fragment that can prevent or substantially reduce the ability of the receptor to bind a ligand or initiate signal transduction, such as an Fv, Fab, F(ab')2, and the like. An "Fv" fragment is a dimer (V H -V L dimer) formed by noncovalent association of the variable domain of one heavy chain and the variable domain of one light chain. In this configuration, the three CDRs of each variable domain interact to define the target-binding site on the surface of the V H -V L dimer, as in the case of a complete antibody. The six CDRs together confer the target-binding specificity of the complete antibody. However, even a single variable domain (or one-half of an Fv that includes only 3 target-specific CDRs) can still have the ability to recognize and bind a target.

[0039] As used herein, the term "bispecific" (Bispecific antibody, BsAb) refers to an antibody and / or antigen-binding molecule that is capable of specifically binding two different antigenic determinants. Generally, 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 is capable of binding two antigenic determinants simultaneously, 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 the other antibody molecules. This property is contrary to that of a polyclonal population of antibodies, which comprises antibodies having a variety of different sequences. Monoclonal antibodies can be prepared by a number of 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 immortalizing B cells, such as by fusing them with myeloma cells to generate a hybridoma cell line or by infecting B cells with a virus such as EBV. Recombinant techniques can also be used to prepare antibodies from a clonal population of host cells in vitro by transforming the host cells with a plasmid carrying an artificial sequence 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 fusing antibody-producing lymphocytes with cancer cells that do not produce antibodies. As is known to those of ordinary skill in the art, hybridomas can proliferate and continuously supply 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 an antibody naturally produced by an antibody-secreting B cell and a synthetically produced antibody having the same domains.

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

[0044] A "humanized" antibody refers to a non-human (e.g., murine) antibody form that is a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof (such as Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of an antibody) containing minimal sequences derived from non-human immunoglobulins. Preferably, a humanized antibody is a human immunoglobulin (recipient antibody) in which the residues of the complementarity-determining regions (CDRs) of the recipient antibody are replaced by CDR residues from a non-human species (donor antibody) such as a mouse, rat, or rabbit having the desired specificity, affinity, and capacity.

[0045] In addition, in humanization, it may also be possible to mutate amino acid residues within CDR1, CDR2, and / or CDR3 of VH and / or VL, thereby improving one or more binding properties (such as affinity) of the antibody. Mutations can be introduced, for example, by PCR-mediated mutagenesis, and the effects of such mutations on antibody binding or other functional properties can be evaluated using the in vitro or in vivo assays described herein. Generally, conservative mutations are introduced. Such mutations can be amino acid substitutions, additions, or deletions. Additionally, mutations within the CDRs typically do not exceed one or two. Thus, the humanized antibodies described in the present disclosure also encompass antibodies containing 1 or 2 amino acid mutations within the CDRs.

[0046] As used herein, the term "CDR" refers to the complementarity-determining region, and it is known that each heavy and light chain of an antibody molecule has 3 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, having sites of very high variability in the primary structure of the CDRs. In the present specification, the CDRs of the heavy chain are denoted as CDR1, CDR2, CDR3 from the amino-terminal of the amino-terminal sequence of the heavy chain, and the CDRs of the light chain are denoted as CDR1, CDR2, CDR3 from the amino-terminal 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. Epitope determinants typically comprise the chemical active surface patterns of a molecule, such as amino acid or sugar side chains, and usually have specific three-dimensional structural features as well as specific charge features.

[0048] As used herein, "specifically binds" or "immunologically specifically binds" with respect to an antibody or an 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-binding site of the antibody and the antigen. The antigen may be an isolated antigen or present on a tumor cell. Generally, an antibody that immunologically specifically binds (or specifically binds) an antigen binds the antigen with an affinity constant Ka of about 1×10 7 M -1 or 1×10 8 M -1 or greater (or a dissociation constant (Kd) of 1×10 -7 M or 1×10 -8 M or lower). The affinity constant can be determined by standard kinetic methods of antibody reaction, e.g., 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. Patent No. 7,229,619, which describes exemplary SPR and ITC methods for calculating the binding affinity of an antibody). Instruments and methods for the real-time detection and monitoring of binding rates are known and commercially available (see, BiaCore 2000, Biacore AB, Uppsala, 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), which are typically linked together by phosphodiester bonds. As used herein, the term "nucleic acid molecule" is intended to include DNA molecules as well as RNA molecules. The nucleic acid molecule may be single-stranded or double-stranded and may be cDNA.

[0050] As used herein, a "separated nucleic acid molecule" is a nucleic acid molecule separated from other nucleic acid molecules present in the natural source of the nucleic acid molecule. A "separated" nucleic acid molecule such as a cDNA molecule may be substantially free of other cellular materials or culture medium when prepared by recombinant techniques, or substantially free of chemical precursors or other chemical components when chemically synthesized. Exemplary separated nucleic acid molecules provided herein include separated nucleic acid molecules encoding the provided antibodies or antigen-binding fragments.

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

[0052] "Conservative sequence modifications" of the sequences described in the sequence listing provided herein are also provided, i.e., nucleotide and amino acid sequence modifications that do not eliminate the binding of an antibody encoded by or containing an amino acid sequence to an antigen. These conservative sequence modifications include conservative nucleotide and amino acid substitutions, as well as nucleotide and amino acid additions and deletions. For example, modifications can be introduced into the sequence listing provided 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 having similar side chains are well defined in the art. These families include amino acids having basic side chains (e.g., lysine, arginine, histidine), amino acids having acidic side chains (e.g., aspartic acid, glutamic acid), amino acids having uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids having nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids having β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids having aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, predicted non-essential amino acid residues in an anti-MASP-2 antibody are preferably replaced with another amino acid residue from the same side chain family. Methods for identifying nucleotide and amino acid conservative 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] As an alternative, in another embodiment, mutations can be randomly introduced, for example, along all or a portion of the anti-MASP-2 antibody encoding sequence by saturation mutagenesis, and the resulting 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 by transcription and translation of a polynucleotide. The expression level of a polypeptide can be evaluated using any method known in the art, including, for example, methods for determining the amount of polypeptide produced from a host cell. Such methods can include, but are not limited to, quantifying the polypeptide in cell lysates by ELISA, Coomassie blue staining after gel electrophoresis, 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. A host cell can also be used to express a polypeptide encoded by the vector. When the host cell divides, the nucleic acid contained in the vector replicates, thereby amplifying the nucleic acid. A 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, HEK cells such as HEK 293 cells.

[0056] As used herein, a "vector" is a replicable nucleic acid that, when transformed into a suitable host cell, can express one or more heterologous proteins from the vector. With respect to vectors include those into which nucleic acids encoding a polypeptide or a fragment thereof can typically be introduced by restriction enzyme digestion and ligation. With respect to vectors also include those vectors that contain nucleic acids encoding a polypeptide. Vectors are used to introduce nucleic acids encoding a polypeptide into a host cell for amplifying the nucleic acid or for expressing / displaying the polypeptide encoded by the nucleic acid. Vectors generally remain free, but can be designed to integrate a gene or a portion thereof into the chromosomes of the genome. Vectors of artificial chromosomes are also contemplated, such as yeast artificial vectors and mammalian artificial chromosomes. The selection and use of such vectors are well known to those skilled in the art.

[0057] As used herein, a vector also includes a "viral vector" or "vector of a virus". A vector of a virus is an engineered virus that is operably linked to a foreign gene to transfer (as a vehicle or shuttle) the foreign gene into a cell.

[0058] As used herein, "expression vector" includes a vector capable of expressing DNA, said DNA being operably linked to regulatory sequences capable of affecting the expression of such DNA fragments, such as a promoter region. Such additional fragments may include promoter and terminator sequences and optionally may include one or more origins of replication, one or more selectable markers, enhancers, polyadenylation signals, etc. Expression vectors generally are derived from plasmid or viral DNA or may contain elements of both. Thus, 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 a suitable 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 replicable in eukaryotic cells and / or prokaryotic cells and expression vectors that remain episomal or integrate into the host cell genome.

[0059] As used herein, "treating" an individual having a disease or medical condition means that the symptoms of the individual are partially or completely alleviated or remain unchanged after treatment. Thus, treatment includes prevention, therapy and / or cure. Prevention means preventing a potential disease and / or preventing the worsening of symptoms or the progression of the disease. Treatment also includes any pharmaceutical use of any antibody or antigen-binding fragment thereof and any composition provided herein.

[0060] As used herein, "therapeutic effect" means the effect resulting from the treatment of an individual, which modifies, usually ameliorates or improves the symptoms of a disease or medical condition, or cures the disease or medical 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, when administered to a subject, is at least sufficient to produce a therapeutic effect. Thus, it is the amount necessary to prevent, cure, ameliorate, arrest or partially arrest the symptoms of a disease or disorder.

[0062] As used herein, "preventively effective amount" or "preventively 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 preventive effect, e.g., preventing or delaying the occurrence or recurrence of a disease or symptoms, reducing the likelihood of the occurrence or recurrence of a disease or symptoms. A fully preventive effective dose need not occur by administration of a single dose and may occur only after administration of a series of doses. Thus, a preventively effective amount may be administered in one or more administrations.

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

[0064] II. DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS

[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 amino acid sequences SEQ ID NO.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 light chain CDR selected from the amino acid sequences SEQ ID NO.12, 13, 14, 21, 22, 23, 31, 32, 33, 41, 42, 43, 50, 51, 52, 57, 70, 71, 72, 82, 91, 108, 109, 110, 117, 118, 123, 131, 132, 133, 144, 145, 146, 154, 155, 156, 161, 162, 168, 173, 174, 201, 202, 207, 216 or any variant thereof.

[0066] The antibody or antigen-binding portion thereof according to the previous aspect comprises a heavy chain CDR1 selected from the amino acid sequences SEQ ID NO.7, 26, 36, 65, 94, 126, 136, 149 or any variant thereof, a heavy chain CDR2 selected from the amino acid sequences 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 amino acid sequences 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 light chain CDR1 selected from the amino acid sequences SEQ ID 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 sequences 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 sequences 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 CDR combination of heavy and light chains selected from the following:

[0068] (1) Comprising the heavy chain CDR1, CDR2, and CDR3 sequences of SEQ ID NO:7, 8, 9 respectively, and / or the light chain CDR1, CDR2, and CDR3 sequences of SEQ ID NO:12, 13, 14 respectively;

[0069] (2) Comprising the heavy chain CDR1, CDR2, and CDR3 sequences of SEQ ID NO:7, 17, 18 respectively, and / or the light chain CDR1, CDR2, and CDR3 sequences of SEQ ID NO:21, 22, 23 respectively;

[0070] (3) Comprising the heavy chain CDR1, CDR2, and CDR3 sequences of SEQ ID NO:26, 27, 28 respectively, and / or the light chain CDR1, CDR2, and CDR3 sequences of SEQ ID NO:31, 32, 33 respectively;

[0071] (4) Comprising the heavy chain CDR1, CDR2, and CDR3 sequences of SEQ ID NO:36, 37, 38 respectively, and / or the light chain CDR1, CDR2, and CDR3 sequences of SEQ ID NO:41, 42, 43 respectively;

[0072] (5) Comprising the heavy chain CDR1, CDR2, and CDR3 sequences of SEQ ID NO:7, 46, 47 respectively, and / or the light chain CDR1, CDR2, and CDR3 sequences of SEQ ID NO:50, 51, 52 respectively;

[0073] (6) Comprising the heavy chain CDR1, CDR2, and CDR3 sequences of SEQ ID NO:7, 17, 18 respectively, and / or the light chain CDR1, CDR2, and CDR3 sequences of SEQ ID NO:21, 57, 23 respectively;

[0074] (7) Comprising the heavy chain CDR1, CDR2, and CDR3 sequences of SEQ ID NO:7, 17, 60 respectively, and / or the light chain CDR1, CDR2, and CDR3 sequences of SEQ ID NO:50, 51, 52 respectively;

[0075] (8) Comprising the heavy chain CDR1, CDR2, and CDR3 sequences of SEQ ID NO:65, 66, 67 respectively, and / or the light chain CDR1, CDR2, and CDR3 sequences of SEQ ID NO:70, 71, 72 respectively;

[0076] (9) Comprising the heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:7, 17, 47 respectively, and / or the light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:50, 51, 52 respectively;

[0077] (10) Comprising the heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:7, 17, 79 respectively, and / or the light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:50, 51, 82 respectively;

[0078] (11) Comprising the heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:36, 85, 86 respectively, and / or the light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:41, 42, 43 respectively;

[0079] (12) Comprising the heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:36, 85, 86 respectively, and / or the light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:41, 42, 91 respectively;

[0080] (13) Comprising the heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:94, 95, 96 respectively, and / or the light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:99, 100, 101 respectively;

[0081] (14) Comprising the heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:7, 104, 105 respectively, and / or the light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:108, 109, 110 respectively;

[0082] (15) Comprising the heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:26, 113, 114 respectively, and / or the light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:117, 118, 33 respectively;

[0083] (16) Comprising the heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:94, 95, 96 respectively, and / or the light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:123, 100, 101 respectively;

[0084] (17) The heavy chain CDR1, CDR2 and CDR3 sequences respectively comprising SEQ ID NO: 126, 127, 128, and / or the light chain CDR1, CDR2 and CDR3 sequences respectively comprising SEQ ID NO: 131, 132, 133;

[0085] (18) The heavy chain CDR1, CDR2 and CDR3 sequences respectively comprising SEQ ID NO: 136, 137, 138, and / or the light chain CDR1, CDR2 and CDR3 sequences respectively comprising SEQ ID NO: 131, 132, 133;

[0086] (19) The heavy chain CDR1, CDR2 and CDR3 sequences respectively comprising SEQ ID NO: 7, 17, 141, and / or the light chain CDR1, CDR2 and CDR3 sequences respectively comprising SEQ ID NO: 144, 145, 146;

[0087] (20) The heavy chain CDR1, CDR2 and CDR3 sequences respectively comprising SEQ ID NO: 149, 150, 151, and / or the light chain CDR1, CDR2 and CDR3 sequences respectively comprising SEQ ID NO: 154, 155, 156;

[0088] (21) The heavy chain CDR1, CDR2 and CDR3 sequences respectively comprising SEQ ID NO: 7, 17, 9, and / or the light chain CDR1, CDR2 and CDR3 sequences respectively comprising SEQ ID NO: 161, 162, 14;

[0089] (22) The heavy chain CDR1, CDR2 and CDR3 sequences respectively comprising SEQ ID NO: 7, 165, 9, and / or the light chain CDR1, CDR2 and CDR3 sequences respectively comprising SEQ ID NO: 144, 168, 14;

[0090] (23) The heavy chain CDR1, CDR2 and CDR3 sequences respectively comprising SEQ ID NO: 7, 17, 9, and / or the light chain CDR1, CDR2 and CDR3 sequences respectively comprising SEQ ID NO: 144, 173, 174;

[0091] (24) The heavy chain CDR1, CDR2 and CDR3 sequences respectively comprising SEQ ID NO: 136, 183, 138, and / or the light chain CDR1, CDR2 and CDR3 sequences respectively comprising SEQ ID NO: 216, 132, 133;

[0092] (25) The heavy chain CDR1, CDR2, and CDR3 sequences respectively comprising SEQ ID NO: 126, 192, 128, and / or the light chain CDR1, CDR2, and CDR3 sequences respectively comprising SEQ ID NO: 216, 132, 133;

[0093] (26) The heavy chain CDR1, CDR2, and CDR3 sequences respectively comprising SEQ ID NO: 7, 17, 141, and / or the light chain CDR1, CDR2, and CDR3 sequences respectively comprising SEQ ID NO: 201, 202, 146;

[0094] (27) The heavy chain CDR1, CDR2, and CDR3 sequences respectively comprising SEQ ID NO: 7, 17, 141, and / or the light chain CDR1, CDR2, and CDR3 sequences respectively comprising SEQ ID NO: 201, 207, 146.

[0095] The antibody or antigen-binding portion thereof according to the previous aspect, which comprises a heavy chain variable region selected from the amino acid sequences 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 light chain variable region selected from the amino acid sequences 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 of the amino acid sequence SEQ ID NO. 5 or any variant thereof, and a light chain variable region of the amino acid sequence 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 of the amino acid sequence SEQ ID NO. 15 or any variant thereof, and a light chain variable region of the amino acid sequence SEQ ID NO. 19 or any variant thereof;

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

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

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

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

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

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

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

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

[0106] In some preferred embodiments, the antibody or its antigen-binding portion comprises a heavy chain variable region of amino acid sequence SEQ ID NO.83 or any variant thereof, and a light chain variable region of amino acid sequence 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 the amino acid sequence of SEQ ID NO. 83 or any variant thereof, and a light chain variable region having the 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 the amino acid sequence of SEQ ID NO. 92 or any variant thereof, and a light chain variable region having the 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 the amino acid sequence of SEQ ID NO. 102 or any variant thereof, and a light chain variable region having the 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 the amino acid sequence of SEQ ID NO. 111 or any variant thereof, and a light chain variable region having the 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 the amino acid sequence of SEQ ID NO. 119 or any variant thereof, and a light chain variable region having the 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 the amino acid sequence of SEQ ID NO. 124 or any variant thereof, and a light chain variable region having the 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 the amino acid sequence of SEQ ID NO. 134 or any variant thereof, and a light chain variable region having the 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 the amino acid sequence of SEQ ID NO. 139 or any variant thereof, and a light chain variable region having the amino acid sequence of SEQ ID NO. 142 or any variant thereof;

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

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

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

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

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

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

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

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

[0123] In some preferred embodiments, the antibody or its antigen-binding portion comprises a heavy chain variable region having the amino acid sequence SEQ ID NO. 190 or any variant thereof, and a light chain variable region having the amino acid sequence 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 the amino acid sequence SEQ ID NO.193 or any variant thereof, and a light chain variable region having the amino acid sequence 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 the amino acid sequence SEQ ID NO.195 or any variant thereof, and a light chain variable region having the amino acid sequence 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 the amino acid sequence SEQ ID NO.197 or any variant thereof, and a light chain variable region having the amino acid sequence 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 the amino acid sequence SEQ ID NO.175 or any variant thereof, and a light chain variable region having the amino acid sequence 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 the amino acid sequence SEQ ID NO.175 or any variant thereof, and a light chain variable region having the amino acid sequence 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 the amino acid sequence SEQ ID NO.175 or any variant thereof, and a light chain variable region having the amino acid sequence 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 the amino acid sequence SEQ ID NO.177 or any variant thereof, and a light chain variable region having the amino acid sequence 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 the amino acid sequence SEQ ID NO.177 or any variant thereof, and a light chain variable region having the amino acid sequence 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 having the amino acid sequence SEQ ID NO. 177 or any variant thereof, and a light chain variable region having 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 the amino acid sequence SEQ ID NO. 179 or any variant thereof, and a light chain variable region having the amino acid sequence 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 the amino acid sequence SEQ ID NO. 179 or any variant thereof, and a light chain variable region having the amino acid sequence 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 the amino acid sequence SEQ ID NO. 179 or any variant thereof, and a light chain variable region having the amino acid sequence 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 the amino acid sequence SEQ ID NO. 175 or any variant thereof, and a light chain variable region having the amino acid sequence 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 the amino acid sequence SEQ ID NO. 175 or any variant thereof, and a light chain variable region having the amino acid sequence 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 the amino acid sequence SEQ ID NO. 175 or any variant thereof, and a light chain variable region having the amino acid sequence 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 having the amino acid sequence SEQ ID NO. 177 or any variant thereof, and a light chain variable region having 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 the amino acid sequence SEQ ID NO. 177 or any variant thereof, and a light chain variable region having the amino acid sequence 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 the amino acid sequence of SEQ ID NO. 177 or any variant thereof, and a light chain variable region having the 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 having the amino acid sequence of SEQ ID NO. 179 or any variant thereof, and a light chain variable region having the amino acid sequence of 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 the amino acid sequence of SEQ ID NO. 179 or any variant thereof, and a light chain variable region having the 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 the amino acid sequence of SEQ ID NO. 179 or any variant thereof, and a light chain variable region having the amino acid sequence of SEQ ID NO. 212 or any variant thereof.

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

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

[0147] In one aspect, the present 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 foregoing aspects.

[0148] In some preferred embodiments, the nucleic acid comprises a nucleic acid sequence of an antibody heavy chain variable region 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 a nucleic acid sequence of an antibody light chain variable region selected from SEQ 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 the nucleotide sequence of the heavy chain variable region of SEQ ID NO:6 or any variant thereof, and the nucleotide sequence of the light chain variable region of SEQ ID NO:11 or any variant thereof;

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0198] In one aspect, the present disclosure provides an antibody that binds to MASP-2 or an antigen-binding portion thereof, which has at least greater than 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the antibody or antigen-binding portion thereof in any of the foregoing aspects.

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

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

[0201] In one aspect, the present disclosure provides a cell comprising the vector in any of the foregoing aspects.

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

[0203] The antibodies of the present disclosure can be used as therapeutic or diagnostic tools in various diseases in which MASP-2 is unfavorably expressed or found.

[0204] A method of treating a disease associated with MASP-2-dependent complement activation with an MASP-2 inhibitor of the present disclosure comprises the step of administering to the subject a therapeutically effective amount of the antibody or an antigen-binding fragment thereof or a nucleic acid molecule or a vector or a cell or a pharmaceutical composition in any of the foregoing aspects.

[0205] MASP-2-dependent complement activation has been indicated to contribute to the pathogenesis of many acute and chronic disease states, including MASP-2-dependent complement-mediated vascular conditions, ischemia-reperfusion injury, atherosclerosis, inflammatory gastrointestinal disorders, pulmonary conditions, in vitro reperfusion procedures, skeletal muscle conditions, renal conditions, skin conditions, organ or tissue transplantation, neurological disorders or injuries, blood disorders, urogenital conditions, diabetes, chemotherapy or radiation therapy, malignancies, endocrine disorders, coagulation disorders, or ophthalmic conditions. Accordingly, the MASP-2 inhibitory antibodies of the present disclosure can be used to treat the above diseases and conditions.

[0206] A 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 classified into those that are primarily confined to a particular organ or tissue and those that affect the entire body. Examples of organ-specific disorders (and the affected organs) include multiple sclerosis (myelin sheath covering nerve processes), type I diabetes (pancreas), Hashimoto's thyroiditis (thyroid), pernicious anemia (stomach), Addison's disease (adrenal glands), myasthenia gravis (acetylcholine receptors at the neuromuscular junction), rheumatoid arthritis (joint lining), uveitis (eye), psoriasis (skin), Guillain-Barré 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, pemphigus, bullous pemphigoid, epidermolysis bullosa, urticaria, angioedema, vasculitis, erythema, cutaneous 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, Crohn'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, such as due to programmed cell death or necrosis. The necrosis or programmed cell death can be induced by a number of factors.

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

[0212] Accordingly, the clinical condition can be ischemic / reperfusion injury, which is a result of PTCA (percutaneous transluminal coronary angioplasty) or CABG (coronary artery bypass grafting). Additionally, the clinical condition can be ischemic / reperfusion injury, which is a result of acute myocardial infarction or cerebral ischemia.

[0213] The dosage of the medicament comprising a MASP-2 inhibitor generally varies according to the age, weight, height, sex, general medical condition and medical history of the subject being treated. By way of example, a MASP-2 inhibitor, such as a MASP-2 antibody, can be administered in a dosage range of about 0.010 - 10.0 mg / kg, preferably 0.010 - 1.0 mg / kg, 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] The compositions and methods comprising a MASP-2 inhibitor can optionally include one or more additional therapeutic agents, which can enhance the activity of the MASP-2 inhibitor or provide related therapeutic functions in an additive or synergistic manner. For example, one or more MASP-2 inhibitors can be used in combination with one or more anti-inflammatory and / or analgesic agents. The amounts and selection of the additional agents can be determined to achieve the desired therapeutic outcome. Suitable anti-inflammatory and / or analgesic agents 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 can be inhibitors of COX-1, COX-2 or non-selective inhibitors of COX-1 and COX-2), lipoxygenase inhibitors; prostaglandin receptors 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 acetylcholine inhibitors; and α-adrenergic receptor agonists (including α-1, α-2 and non-selective α-1 and α-2 agonists).

[0215] When used for inhibiting or treating restenosis, the MASP-2 inhibitors of the present disclosure can be co-administered simultaneously in combination with one or more anti-restenosis drugs. Suitable anti-restenosis drugs include: antiplatelet drugs, including thrombin inhibitors and receptor antagonists, adenosine diphosphate (ADP) receptor antagonists (also known as purinergic receptor P1 receptor antagonists), thromboxane inhibitors and receptor antagonists, and platelet membrane glycoprotein receptor antagonists; inhibitors of cell adhesion molecules, including selectin inhibitors and integrin radicals; anti-chemotactic drugs; interleukin receptor antagonists; and intracellular signal transduction inhibitors, including protein kinase C (PKC) inhibitors, 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 inhibitors of the present disclosure can also be used in combination with one or more other complement inhibitors. Currently, no complement inhibitors have been approved for use in humans. However, some drugs have been shown to block complement in vivo. Many of these drugs are also toxic or are only partial inhibitors, and their use is limited to being used as research tools. K76COOH and nafamostat mesilate are two drugs that have shown some efficacy in animal models of transplantation. Studies have shown that low molecular weight heparin is also effective in modulating complement activity. We believe that these small molecule inhibitors can be used as drugs in combination with the MASP-2 inhibitors of the present disclosure.

[0217] Other naturally occurring complement inhibitors can be used in combination with the MASP-2 inhibitors of the present disclosure. Biological inhibitors of complement include soluble complement factor 1 (sCR1). This is a naturally occurring inhibitor that can be found on the outer membrane of human cells. Other membrane inhibitors include DAF, MCP, and CD59. The anti-complement activity of recombinant forms has been measured in vitro and in vivo. sCR1 has been shown to be effective in xenotransplantation, where the complement system (both alternative and classical) causes an overreaction rejection syndrome within minutes of perfusion of the newly transplanted organ with blood. The use of sCR1 protects and prolongs the survival time of the transplanted organ, indicating the complement pathway in the organ survival mechanism.

[0218] Other complement inhibitors suitable for use in combination with the compositions of the present disclosure also include, for example, the monoclonal antibody (MoAb) 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 inhibitors of the present disclosure can be used in combination with one or more chondroprotective agents, which can include one or more anabolic promoters of cartilage and / or one or more catabolic inhibitors of cartilage, appropriately including both an anabolic agent and a catabolic inhibitory agent administered simultaneously. 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 β superfamily agonists (including TGF-β, TGF-β1, TGF-β2, TGF-β3), bone morphogenetic 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 catabolic inhibitory chondroprotective agents 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 such as 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-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 pyrrolidine dithiocarbamate; nitric oxide synthase (NOS) inhibitors, including NG-monomethyl-L-arginine, 1400W, diphenyleneiodonium, S-methylisothiourea, S-(aminoethyl)isothiourea, L-N6-(1-iminoethyl)lysine, 1,3-PBITU, 2-ethyl-2-thiopseudourea, aminoguanidine, Nω-nitro-L-arginine, Nω-nitro-L-arginine methyl ester, inhibitors of matrix metalloproteinases (MMP), including inhibitors of MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, MMP-1 1, 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 αVβ3MoAb 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 the MASP-2 inhibitors of the present disclosure with spasmolytic inhibitors. For example, for urogenital applications, it may be advantageous to include at least one smooth muscle spasmolytic inhibitor and / or at least one anti-inflammatory drug, and for vascular surgery, it may be beneficial to include at least one vasospasm inhibitor and / or at least one anti-inflammatory drug and / or at least one anti-restenosis drug. Suitable examples of spasmolytic inhibitors 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 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 at most 30 doses, 25 doses, 20 doses, 15 doses or 10 doses. The doses of the antibody are preferably administered at time 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 time intervals of 7 to 30 days, 10 to 20 days and preferably about 14 days.

[0223] In one aspect, the present disclosure provides the use of the antibody or its antigen-binding fragment or nucleic acid molecule or vector or cell or pharmaceutical composition of any of the foregoing aspects in 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 foregoing antibody or its antigen-binding portion, bispecific or multispecific molecule, nucleic acid molecule, vector, cell and / or composition.

[0225] The kits described in the present disclosure include the antibodies of the present disclosure, their fragments, homologs, derivatives thereof, etc., such as labeled or cytotoxic conjugates, as well as instruction manuals for the use of the antibodies, conjugates for killing specific types of cells, etc. The instruction manual may include guidance on the use of antibodies, conjugates, etc. in vitro, in vivo or ex vivo. The antibodies may be in liquid or solid form, usually lyophilized. The kit may contain other suitable reagents, such as buffers, reconstitution solutions and other necessary components for the intended use. Combinations of reagents packaged in predetermined amounts together with instruction manuals for their use are contemplated, such as for therapeutic use or for performing diagnostic assays. When the antibody is labeled, for example, with an enzyme, then 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 buffers or lysis buffers), etc. may also be included. The relative amounts of the various reagents may be varied to provide concentrates of the reagent solutions, which provides user flexibility, space savings, reagent savings, etc. These reagents may also be provided in dry powder form, usually lyophilized, including excipients that, upon dissolution, provide a reagent solution of appropriate concentration.

[0226] In one aspect, the present disclosure provides the use of the antibodies or antigen-binding portions thereof, bispecific or multispecific molecules, nucleic acid molecules, vectors, cells, and / or compositions thereof against MASP-2 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 of the present disclosure can also be used in immunoassays, purification methods, and other methods that utilize immunoglobulins or their fragments. Such uses are well known in the art.

[0228] Accordingly, the present disclosure also provides a composition comprising an anti-MASP-2 antibody or a fragment thereof of the present disclosure, which is conveniently combined with a pharmaceutically acceptable carrier, diluent, or excipient, which is a conventional practice in the art.

[0229] As used herein, the term "pharmaceutical composition" refers to a formulation of various preparations. A formulation containing a therapeutically effective amount of a multivalent antibody is a sterile liquid solution, a liquid suspension, or a lyophilized form, optionally containing stabilizers or excipients.

[0230] It should be understood that the therapeutic agent according to the described embodiments will be administered together with a suitable pharmaceutically acceptable carrier, excipient, and other reagents incorporated into the formulation to provide improved transfer, delivery, tolerance, etc. A large number of suitable formulations can be found in the pharmacopoeias known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences (15th Edition, Mack Publishing Company, Easton, Pa. (1975)), particularly Chapter 87 by Blaug, Seymour therein. These formulations include, for example, powders, pastes, ointments, gels, waxes, oils, lipids, lipid-containing (cationic or anionic) carriers (e.g., Lipofectin TM ), DNA conjugates, anhydrous slurries, water-in-oil and oil-in-water emulsions, emulsion polyethyleneglycols (polyethyleneglycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing polyethyleneglycol. Any of the foregoing mixtures can be suitable for the treatment or therapy according to the present disclosure, provided that the active ingredient in the formulation is not inactivated by the formulation and the formulation is physiologically compatible and tolerates the route of administration.

[0231] In another embodiment, antibodies against MASP-2 can be used in methods known in the art related to the localization and / or quantification of MASP-2 (e.g., for determining the level of MASP-2 and / or MASP-2 in a suitable physiological sample, for diagnostic methods, for protein imaging, etc.). In a given embodiment, an antibody specific for MASP-2 or its derivative, fragment, analogue, or homologue, comprising an antigen-binding domain derived from an antibody, is used as a pharmacologically 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 (or fragments thereof) against the MASP-2 protein 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. Conjugating (i.e., physically linking) the antibody to a detectable substance can facilitate detection. Examples of detectable substances include various enzymes, cofactors, 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 cofactor complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, 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, an antibody according to the present disclosure can be used as a reagent 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 "label" with respect to an antibody is intended to include directly labeling the antibody by conjugating (i.e., physically linking) a detectable substance to the antibody, and indirectly labeling the antibody by reacting it with another reagent that is directly labeled. Examples of indirect labeling include using a fluorescently labeled secondary antibody to detect a primary antibody, and end-labeling an antibody with biotin so that it can be detected 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 within a subject. Thus, the term "biological sample" as used includes blood and fractions or components thereof, including serum, plasma, or lymphatic fluid. In other words, the detection methods of the described embodiments can be used to detect analyte mRNA, protein, or genomic DNA in biological samples 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 proteins include enzyme-linked immunosorbent assay (ELISA), Western blotting, immunoprecipitation, and immunofluorescence. In vitro detection techniques for analyte genomic DNA include Southern hybridization. Procedures for performing immunoassays are described, for example, in "ELISA: Theory and Practice: Methods in Molecular Biology", Volume 42, J.R. Crowther (ed.) Human Press, Totowa, N.J., 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. Additionally, in vivo detection techniques for analyte proteins include introducing a labeled anti-analyte protein antibody into a subject. For example, an antibody can be labeled with a radioactive label, and then the presence and location of the radioactive label within the subject can be detected by standard imaging techniques.

[0234] The antibodies and their derivatives, fragments, analogs, and homologs described herein can be incorporated into pharmaceutical compositions suitable for administration. The principles and considerations involved in preparing such compositions and guidelines for selecting components are well known in the art. See, for example, Remington's Pharmaceutical Sciences: The Science And Practice Of Pharmacy, 19th Edition (edited by Alfonso R. Gennaro et al.), 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, Volume 4), 1991, M. Dekker, New York.

[0235] Such compositions generally comprise an antibody and a pharmaceutically acceptable carrier. When using antibody fragments, the minimal inhibitory fragment that specifically binds to the target protein binding domain may be preferred. For example, based on the antibody variable region sequence, peptide molecules can be designed that retain the ability to bind to the target protein sequence. 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 agents, and absorption delaying agents, etc., that are compatible with the administration of a drug. Suitable pharmaceutically acceptable carriers are described in the latest edition of Remington's Pharmaceutical Sciences, which is a standard reference in the art and 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 fixed oils, can also be used. The use of such media and agents for a pharmaceutically active substance is well known in the art. Except for any conventional media or agents that are incompatible with the antibody, their use in the compositions is contemplated.

[0237] The pharmaceutical compositions of the described embodiments are formulated to be compatible with their 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 solution, fixed oils, polyethylene glycols, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents, such as benzyl alcohol or methylparaben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid (EDTA); buffers, such as acetate, citrate, or phosphate, and agents for adjusting osmotic pressure, such as sodium chloride or dextrose. The pH may be adjusted with an acid or a base, such as hydrochloric acid or sodium hydroxide. Parenteral preparations may be packaged in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

[0238] Pharmaceutical compositions suitable for injection include sterile aqueous solutions (which are water-soluble here) or dispersions and sterile powders for the immediate preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable pharmaceutically acceptable carriers include saline, bacteriostatic water, Cremophor EL TM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy injection is possible. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (such as glycerin, propylene glycol, and liquid polyethylene glycol, etc.), and suitable mixtures thereof. For example, by using coatings such as lecithin, maintaining the desired particle size in the case of dispersions, and using surfactants, suitable fluidity can be maintained. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it will be preferable to include in the composition an isotonic agent, such as sugar, polyols (such as mannitol, sorbitol), sodium chloride. Prolonged absorption of injectable compositions can be achieved by including in the composition an agent that delays absorption, such as aluminum monostearate and gelatin.

[0239] Sterile injectable solutions can be prepared by incorporating the antibody in the required amount into a suitable solvent having one or a combination (as required) of the ingredients listed above, and then filtering and sterilizing. Generally, dispersions are prepared by incorporating the antibody into a sterile vehicle containing a basic dispersion medium and the required other ingredients listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the method of preparation is to obtain the powder by vacuum drying and freeze drying, which powder contains the active ingredient and any additional desired ingredients from a sterile filtered solution of these ingredients described above.

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

[0241] Systemic administration can also be effected by transmucosal or transdermal means. For transmucosal or transdermal administration, permeants suitable for the permeation barrier are used in the formulation. Such permeants are generally known in the art and include, for example, detergents, bile salts, and fusidic acid derivatives for transmucosal administration. Transmucosal administration can be effected by using nasal sprays or suppositories. For transdermal administration, one or more of the antibodies can be formulated into ointments, pastes, gels, or creams as is generally known in the art.

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

[0243] In one embodiment, the antibody can be prepared with a carrier that prevents its rapid elimination from the body, such as a sustained-release / controlled-release formulation, including implants and microencapsulation delivery systems. Biodegradable, biocompatible polymers such as ethylene-vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. The methods for preparing such formulations are obvious to those skilled in the art.

[0244] It is particularly advantageous to formulate parenteral compositions in unit dosage form for ease of administration and uniformity of dosage. As used herein, unit dosage form refers to physically discrete units suitable as unit doses for the subject to be treated; each unit contains a predetermined quantity of one or more of the antibodies calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification of the unit dosage forms of the embodiments is indicated by and directly depends on: the unique characteristics of the antibody and the specific therapeutic effect to be achieved, and the limitations inherent in the art of compounding such antibodies for the treatment of individuals.

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

[0246] The formulations described herein may also contain more than one of said antibodies, preferably those having complementary activities and no negative impact on each other, depending on the specific circumstances to be treated. Alternatively or in addition, the composition may contain, for example, reagents that enhance its function, such as cytotoxin reagents, cytokines, chemotherapeutic agents, or growth inhibitors. Such molecules are present in appropriate combination in an amount effective for the intended purpose. For example, they may be present in combination in a kit or in use.

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

[0248] For example, combination therapy may include co-formulating and / or co-administering one or more antibodies described herein with one or more additional therapeutic agents (such as one or more cytokine and growth factor inhibitors, immunosuppressants, anti-inflammatory agents, metabolic inhibitors, enzyme inhibitors, and / or cytotoxins or cell growth inhibitors, as described in more detail below). Such combination therapy may advantageously utilize lower doses of the administered therapeutic agents, thus avoiding possible toxicities or complications associated with various single therapies.

[0249] Experimental evidence of the present disclosure shows that a variety of monoclonal antibodies and their variants provided by the present disclosure that can bind to MASP-2 with high affinity and inhibit lectin-mediated complement activation can specifically recognize the enzymatic domain CCP1-CCP2-SP of human, monkey, mouse, and rat MASP-2, and can effectively inhibit the generation of C4b and the C3 convertase C4b2b under low-concentration conditions, and its blocking activity is 50-200 times higher than that of known antibodies. The results of pre-toxicity studies conducted in rhesus monkeys show that the animals are well tolerated to high doses of the novel MASP-2 antibody without any toxic reactions, and its efficacy and safety are superior to those of similar antibodies.

[0250] For the purposes of clear and concise description, 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 combinations of all or some of the described features.

[0251] Examples

[0252] Example 1. Detection of MASP-2 Activity in the Lectin Pathway

[0253] After MBL in plasma recognizes repetitive N - acetylgalactosamine or mannose structures, it can sequentially activate serine proteases MASP - 1 and MASP - 2. Activated MASP - 2 cleaves complement components C4 and C2. The formed C4b deposits on the well plate and can be recognized by anti - C4b antibody; the C3 convertase C4b2b formed by the cleavage products of C4 and C2 cleaves complement component C3, and the formed C3b deposits on the well plate and can be recognized by anti - C3b antibody. Therefore, by detecting the production of C4b or C3b in the lectin activation reaction, the inhibitory activity of MASP - 2 antibody against MASP - 2 in serum can be shown.

[0254] Determination of C4 cleavage activity

[0255] Add 20 μg / ml mannan (Sigma, M7504) to the ELISA plate and coat it overnight at 4°C. After blocking with 2% BSA at room temperature for 60 minutes, add 0.5% diluted human serum and incubate overnight at 4°C. After washing, add 50 μl / well of MASP - 2 antibody at different concentrations (starting concentration is 30 μg / ml, diluted 5 - fold in 9 gradients), pre - incubate at 4°C for 30 minutes, then add 50 μl of 2 μg / ml human C4 protein (CompTech, #A105), react at 37°C for 90 minutes. After washing the plate, add Biotin - labeled polyclonal anti - human C4b (ASSAYPRO, #11223 - 05021) diluted 1:1000, incubate at room temperature for 90 minutes, add peroxidase - streptavidin (Jackson ImmunoResearch 016 - 030 - 084) diluted 1:5000 and incubate at room temperature for 60 minutes, add TMB chromogenic solution, develop color in the dark at 37°C for about 10 minutes, terminate the reaction and read the OD value.

[0256] Determination of C3 convertase activity

[0257] Add 20 μg / ml mannan to the ELISA plate and coat it overnight at 4°C. After blocking with 2% BSA at room temperature for 60 minutes, add MASP - 2 antibody diluted with 0.5% human serum (starting concentration is 60 μg / ml, diluted 5 - fold in 9 gradients), react at 37°C for 90 minutes, add rabbit anti - human C3 / C3b / C3c polyclonal antibody (Proteintech, #21337 - 1 - AP) diluted 1:500, incubate at room temperature for 60 minutes, add HRP - labeled goat anti - rabbit secondary antibody (Jackson ImmunoResearch111 - 035 - 144) diluted 1:5000, incubate at room temperature for 60 minutes, add TMB chromogenic solution, develop color in the dark at 37°C for about 10 minutes, terminate the reaction and read the OD value.

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

[0259] The sequence encoding the enzymatic domain CCP1-CCP2-SP of human MASP-2 was cloned into a eukaryotic expression plasmid, with an N-terminal his6 tag fused for convenient purification. After 7 days of culturing HEK293E cells transfected with the plasmid, the culture supernatant of the medium was collected and purified through a nickel chelating column to obtain the recombinant human MASP-2 D4-6 protein (SEQ ID NO.1). Similarly, the recombinant proteins of cynomolgus monkey MASP-2 D4-6 (SEQ ID NO.2), mouse MASP-2 D4-6 (SEQ ID NO.3), and rat MASP-2 D4-6 (SEQ ID NO.4) were obtained respectively. Figure 1 The SDS-PAGE results of the recombinant proteins of human, cynomolgus monkey, mouse, and rat MASP-2 D4-6 are shown.

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

[0261] Six-week-old female BALB / c mice were selected and immunized subcutaneously or via footpad with an equal volume mixture of recombinant human or cynomolgus monkey MASP-2 D4-6 protein and Freund's adjuvant. Two weeks after the primary immunization, a booster immunization was carried out. When the serum titer of the immunized mice reached 1:100000, a boost immunization was performed. Three days later, the spleens and peripheral lymph nodes of the mice were taken, and B cells were isolated.

[0262] An appropriate amount of B cells and SP2 / 0 cells were fused and screened in a complete DMEM medium containing HAT. The hybridoma cells were cultured under the conditions of 5% CO2 at 37 °C for 5 - 7 days. The recombinant human or cynomolgus monkey MASP-2 D4-6 protein (1 μg / mL) was coated, and the culture supernatant of the hybridoma was detected by ELISA. A total of 778 monoclonal antibodies could cross-recognize human or cynomolgus monkey MASP-2. The supernatant was harvested and assayed for C4 cleavage activity, among which 61 clones had significant MASP-2 inhibitory activity (as Figure 2 shown).

[0263] According to the results of hybridoma screening, RNA of the candidate hybridoma monoclonal cells was extracted by the TRNzol lysis method, then reverse transcribed into single-stranded cDNA, and the variable region sequences of the antibodies in the hybridoma monoclonal cells were amplified using this as a template.

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

[0265] Table 1: Variable Region Sequences of Murine MASP-2 Antibodies

[0266]

[0267] Clone: 1B2

[0268] The amino acid sequence of the heavy chain VH is shown in SEQ ID NO.5, its encoding nucleic acid is shown in SEQ ID NO.6, and its CDR1, CDR2, and CDR3 are shown in SEQ ID NO.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 NO.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, its encoding nucleic acid is shown in SEQ ID NO.16, and its CDR1, CDR2, and CDR3 are shown in SEQ ID NO.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 NO.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, its encoding nucleic acid is shown in SEQ ID NO.25, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NO.26, 27, 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 NO.31, 32, 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, its encoding nucleic acid is shown in SEQ ID NO.35, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NO.36, 37, 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 NO.41, 42, 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 NO. 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 NO. 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 NO. 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 NO. 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 NO.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 NO.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 NO.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 NO.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 NO.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 NO.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 NO.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 NO.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 NO.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 NO.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 NO.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 NO.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, its encoding nucleic acid is shown in SEQ ID NO.93, and its CDR1, CDR2, and CDR3 are shown in SEQ ID NO.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 NO.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, its encoding nucleic acid is shown in SEQ ID NO.103, and its CDR1, CDR2, and CDR3 are shown in SEQ ID NO.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 NO.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, its encoding nucleic acid is shown in SEQ ID NO.112, and its CDR1, CDR2, and CDR3 are shown in SEQ ID NO.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 NO.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, its encoding nucleic acid is shown in SEQ ID NO.120, and its CDR1, CDR2, and CDR3 are shown in SEQ ID NO.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 NO.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 NO.126, 127, 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 NO.131, 132, 133 respectively.

[0420]

[0421] Nucleic acid sequence

[0422]

[0423] The variable region of the light chain of the above antibody was cloned into a vector containing the human light chain constant region, and the variable region of the heavy chain was cloned into a vector containing the human heavy chain constant region. The plasmid was co-transfected into CHO-S cells, and the complete IgG antibody was expressed and secreted into the culture medium. After expression at 37°C, 5% CO2, 125 rpm for 7 days, the supernatant was collected, and the chimeric IgG was purified by Protein A chromatography. The concentration of IgG was determined by spectrophotometry. 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 against C4 cleavage reaction was detected, the OD450nm value was read, and the results were calculated using GraphPad Prism 5 software after collecting the data. The results are as Figure 3 shown. In the C4 cleavage activity assay, the inhibitory activities of chimeras 10L3, 3B9, 16A17, 4F18, 25L15, 28O14 and 1B2 against MASP-2 were significantly stronger than those of the control antibody.

[0425] According to the method of Example 1, an inhibitory test of C3 convertase activity was carried out on the selected dominant chimeric antibodies, and the results were calculated using GraphPad Prism 5 software after collecting the data. The results are as 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, significantly stronger than the control antibody.

[0426] Affinity evaluation

[0427] Coat a 96-well ELISA plate with 1 μg / mL of human MASP-2 D4-6 recombinant protein and incubate overnight at 4°C. Block with 2% non-fat milk at room temperature for 1 hour. Add serially diluted MASP-2 antibodies - chimeras 10L3, 3B9, 16A17, 4F18, 25L15, 28O14, and 1B2 (starting concentration 30 μg / ml, diluted 1:4 serially), incubate at room temperature for 60 minutes, add Goat anti-human Fc-HRP diluted at a ratio of 1:4000, and incubate at room temperature for 60 minutes. Develop color with TMB for 10 minutes, add concentrated sulfuric acid to terminate the reaction, measure OD450nm, and calculate the results using Graph Pad Prism 5 software.

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

[0429] Table 2 Binding of MASP-2 antibodies to proteins of various species

[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 technology (SPR / Biacore T200), capture 0.1 μg / ml of the antibody onto the surface of a Protein A chip, set the capture time to 40 seconds, the flow rate to 10 μl / min, rinse the chip with buffer until the baseline is stable, then flow serially diluted antigen over the chip at a flow rate of 30 μl / min, set the binding time to 120 seconds, the dissociation time to 600 seconds, and fit with the Langmuir 1:1 kinetics model to obtain the equilibrium dissociation constant (KD). The affinities of the candidate antibodies for MASP-2 antigens of various species are shown in Table 3.

[0432] Table 3 Affinities of MASP-2 antibodies for proteins of various species

[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 a phage antibody library

[0435] Take a part of the immune mouse B cells obtained in Example 3, extract total RNA, and reverse transcribe to obtain an antibody cDNA library using light and heavy chain specific primers respectively. Specifically amplify the antibody variable region fragment with variable region primers, digest with enzymes and clone it into a phagemid to construct an M13 phage-based antibody Fab phage display library with a library capacity of 1×10 10 .

[0436] Use the traditional phage panning method for library screening. Coat the MASP-2D4-6 recombinant protein on an Immuno tube, and add about 1×10 12 phages for incubation and panning in each screening reaction. After 2 rounds of panning, pick monoclonal clones, inoculate them into a 96-well U-shaped plate for culture and induction of expression, and take the culture supernatant for ELISA detection of the MASP-2 recombinant protein. A total of 494 monoclonal Fab antibodies that can cross-recognize human, cynomolgus monkey, rat, and mouse MASP-2 were obtained, and 34 clones of them can inhibit the C4 deposition catalyzed by MASP-2 ( Figure 6 ).

[0437] After re-inducing the expression of the above Fab clones with inhibitory activity, purify the Fab antibody and detect the inhibitory activity of the MASP-2 antibody by measuring the C4 cleavage activity. The results are as Figure 7 shown. Among them, when the concentration of monoclonal antibodies 3H6, 14F11, 16B11, 20C8, 21A3, and 21B1 is greater than 0.01 μg / ml, the inhibitory activity on the deposition of C4b catalyzed by MASP-2 is the most significant.

[0438] Adopt surface plasmon resonance technology (SPR / Biacore T200), use a Protein A chip to capture the Anti-Flag antibody, capture 10 μg / ml of the purified Fab antibody with a Flag tag on the chip surface, set the capture time to 40 seconds, the flow rate to 10 μl / min, rinse the chip with buffer until the baseline is stable, and then inject the gradient-diluted MASP-2 antigen as the analyte in turn to measure the affinity between the Fab and antigens of different species. The data is fitted in the Langmuir 1:1 kinetics mode through the Biacore T200 Evaluation software 3.1. Table 4 shows the affinities of monoclonal antibodies 16B11 and 14F11 with MASP-2 antigens of various species.

[0439] Table 4 Affinities of MASP-2 Antibodies (Fab) with Antigens

[0440]

[0441]

[0442] The variable region sequences of the Fab antibodies were obtained by Sanger sequencing as follows:

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

[0444]

[0445] Clone: 3H6

[0446] The amino acid sequence of the heavy chain VH is shown in SEQ ID NO.134, its encoding nucleic acid is shown in SEQ ID NO.135, and its CDR1, CDR2, and CDR3 are shown in SEQ ID NO.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 NO.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, its encoding nucleic acid is shown in SEQ ID NO.140, and its CDR1, CDR2, and CDR3 are shown in SEQ ID NO.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 NO.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, its encoding nucleic acid is shown in SEQ ID NO.148, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NO.149, 150, 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 NO.154, 155, 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, its encoding nucleic acid is shown in SEQ ID NO.158, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NO.7, 17, 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 NO.161, 162, 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 NO.7, 165, 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 NO.144, 168, 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, its encoding nucleic acid is shown in SEQ ID NO.170, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NO.7, 17, 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 NO.144, 173, 174 respectively.

[0497]

[0498] Nucleic acid sequence

[0499]

[0500] Example 5, Humanization of MASP-2 Antibody

[0501] Mouse anti-28O14, 3H6 and 16B11 with high affinity and strong activity were selected for humanization of variable region sequences. First, the glycosylation site NYS of the heavy chain CDR3 of clone 28O14 was cloned and site-directed mutagenesis was performed. The mutant was expressed as Fab in E.coli. Prokaryotic expression. Affinity detection showed that after the removal of the glycosylation site of the heavy chain CDR3 of 28O14, the S-A mutant (Ala102) with serine mutated to alanine at position 102 did not affect the MASP-2 binding activity (as Figure 8 shown).

[0502] The mouse antibody sequences were aligned with the human antibody germline sequences to find the human germline light chain genes with good homology, the same key amino acid sequences that maintain the core of the antibody structure, and high frequency in the human body, and the mouse antibody CDR transplantation was performed; homologous modeling was carried out by computer to analyze the CDR region and its surrounding framework amino acid sequences to avoid the concentrated distribution of molecular surface charge or hydrophobic regions. A total of 3 heavy chains and 6 light chain 16B11 variants, 4 heavy chains and 1 light chain 3H6 variants, 4 heavy chains and 1 light chain 28O14 variants, and 3 heavy chains and 6 light chain 16B11 variants were obtained by humanization design.

[0503] The humanized sequences are as follows:

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

[0505]

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

[0507]

[0508] h16B11:

[0509] h16B11 H1

[0510] The amino acid sequence of h16B11 H1 is shown in SEQ ID NO.175, its encoding nucleic acid is shown in SEQ ID NO.176, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NO.7, 17, 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, its encoding nucleic acid is shown in SEQ ID NO.178, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NO.7, 17, 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, its encoding nucleic acid is shown in SEQ ID NO.180, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NO.7, 17, 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, its encoding nucleic acid is shown in SEQ ID NO.182, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NO.136, 183, 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, its encoding nucleic acid is shown in SEQ ID NO.185, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NO.136, 183, 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, its encoding nucleic acid is shown in SEQ ID NO.187, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NO.136, 183, 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, its encoding nucleic acid is shown in SEQ ID NO.189, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NO.136, 183, 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, its encoding nucleic acid is shown in SEQ ID NO.191, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NO.126, 192, 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, its encoding nucleic acid is shown in SEQ ID NO.194, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NO.126, 192, 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, its encoding nucleic acid is shown in SEQ ID NO.196, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NO.126, 192, 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, its encoding nucleic acid is shown in SEQ ID NO.198, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NO.126, 192, 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, its encoding nucleic acid is shown in SEQ ID NO.200, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NO.201, 202, 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, its encoding nucleic acid is shown in SEQ ID NO.204, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NO.201, 202, 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, its encoding nucleic acid is shown in SEQ ID NO.206, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NO.201, 207, 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, its encoding nucleic acid is shown in SEQ ID NO.209, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NO.201, 202, 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, its encoding nucleic acid is shown in SEQ ID NO.211, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NO.201, 202, 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, its encoding nucleic acid is shown in SEQ ID NO.213, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NO.201, 207, 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, its encoding nucleic acid is shown in SEQ ID NO.215, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NO.216, 132, 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, its encoding nucleic acid is shown in SEQ ID NO.215, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NO.216, 132, 133 respectively.

[0606]

[0607] Nucleic acid sequence

[0608]

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

[0610] Example 6. MASP-2 inhibitory activity of humanized antibody

[0611] Determination of C4 cleavage activity

[0612] According to the C4 cleavage assay method in Example 1, different concentrations of MASP-2 antibody (starting concentration 30 μg / ml, 5-fold serial dilution, a total of 9 dilutions) were added to the reaction wells at 50 μl per well and reacted at 4°C for 30 minutes. 50 μl of 2 μg / ml C4 pure protein (CompTech) was added and reacted in a 37°C incubator for 90 minutes. C4c antibody was added to detect C4b deposition. From Figure 9A 、 9B and Table 8, it can be seen that the humanized antibodies can significantly inhibit MASP-2 activity.

[0613] Table 8. Inhibitory activity of MASP-2 humanized antibody on C4b deposition

[0614] Humanized antibody 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] Determination of C3 convertase activity

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

[0617] Table 9. Inhibitory Activity of Humanized MASP-2 Antibody against C3b Deposition

[0618] Humanized antibody 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 Humanized MASP-2 Antibody

[0620] (1) Detection of Affinity of Humanized MASP-2 Antibody against Antigens of Various Species by ELISA

[0621] Coat a 96-well ELISA plate with 1 μg / mL of human MASP-2 D4-6 recombinant protein and incubate overnight at 4°C. Block with 2% skim milk at room temperature for 1 hour. Add serially diluted MASP-2 antibody (starting concentration 30 μg / ml, diluted 1:4 serially), incubate at room temperature for 60 minutes. Add goat anti-human Fc-HRP diluted at a ratio of 1:4000, incubate at room temperature for 60 minutes. Develop color with TMB for 10 minutes, add concentrated sulfuric acid to terminate the reaction, measure OD450nm, and calculate the results using Graph Pad Prism 5 software.

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

[0623] (2) Detection of Affinity of Humanized Antibody against Human and Mouse MASP-2 by BIAcore

[0624] Capture 0.1 μg / ml of the antibody on the surface of a Protein A chip, set the capture time to 40 seconds, the flow rate to 10 μl / min. Wash the chip with buffer until the baseline is stable. Then flow the serially diluted antigen over the chip at a flow rate of 30 μl / min, set the binding time to 120 seconds, the dissociation time to 600 seconds, and fit to obtain the equilibrium dissociation constant (KD). The numerical values of the affinity determination are shown in Table 10.

[0625] Table 10. Affinity of MASP-2 Antibody and MASP-2

[0626]

[0627] Example 8. Shiga Toxin 2-Induced Hemolytic Uremic Syndrome Model in Mice

[0628] Using the cDNA plasmid of STX2 (Sino Biological, catalog number: EG40019-G) as a template, the gene fragments of STX2A (Arg23-Lys319) and STX2B (Ala20-Asp89) of E. coli O157:H7 were amplified by PCR respectively and cloned into a prokaryotic expression plasmid. The N-terminus of the STX2A protein was fused with a twin strep tag for purification, and a enterokinase cleavage site DDDDK was added between the two. The constructed STX2 expression plasmid was transformed into TG1 E. coli competent cells, single colonies were picked into a bacterial medium, induced with IPTG and cultured overnight, the bacteria were collected, PPB and 5 mM MgSO4 solution were added successively to lyse the bacterial cell wall, and the protein expression solution in the periplasmic cavity was collected by centrifugation. The Shiga toxin STX2 protein was purified by Strep-TactinX (Sino HaiGene, catalog number: C0402), and EK enterokinase was added to the protein collection solution and incubated at 25 °C overnight to remove the twin strep tag.

[0629] Female C57 BL / 6 mice weighing about 18 g were selected and divided into 4 dose groups of 0 mg / kg, 3 mg / kg, 10 mg / kg and 30 mg / kg of anti-MASP-2 antibody, with 7 mice in each group. Anti-MASP-2 antibody h16B11 H2K4 (100 μl) was injected into the tail vein 2 hours in advance, and then LPS (5 μg / mouse, L6136-Sigma) and STX2 (10 ng / mouse) were injected into the peritoneal cavity. The survival rate of the mice was monitored 60 hours after administration, and the mice were sacrificed when they reached the lethargic stage of HUS pathology. The results are as Figure 12 shown that anti-MASP-2 antibody can significantly prolong the survival time of mice, and there is a dose correlation.

Claims

1. An antibody or antigen-binding portion thereof that binds to MASP-2, which comprises heavy chain CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO: 126, 192, and 128 respectively, and light chain CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO: 216, 132, and 133 respectively.

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

214.

3. A bispecific or multispecific molecule, which comprises the antibody or antigen-binding portion thereof that binds to MASP-2 according to claim 1 or 2.

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

5. The nucleic acid according to claim 4, wherein the nucleic acid comprises 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.

6. A vector comprising the nucleic acid according to claim 4 or 5.

7. A cell comprising the nucleic acid according to claim 4 or 5, or the vector according to claim 6.

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

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

10. Use of the antibody or antigen-binding portion thereof according to claim 1 or 2, the bispecific or multispecific molecule according to claim 3, the nucleic acid according to claim 4 or 5, the vector according to claim 6, the cell according to claim 7, and / or the composition according to claim 8 in the preparation of a medicament or kit for diagnosing, treating, or preventing a disease associated with MASP-2-dependent complement activation, wherein the disease associated with MASP-2-dependent complement activation is an autoimmune inflammatory condition or an autoimmune disease.

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